Oxidative Damage in Pea Plants Exposed to Water Deficit or Paraquat1

The application of a moderate water deficit (water potential of −1.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (−1.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, β-carotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. PQ decreased markedly ascorbate, NADPH, ascorbate peroxidase, and chloroplastic Fe-superoxide dismutase activity, and caused major increases in oxidized glutathione, NAD+, NADH, and catalytic Fe. It is concluded that, in cv Lincoln, the increase in catalytic Fe and the lowering of antioxidant protection may be involved in the oxidative damage caused by severe water deficit and PQ, but not necessarily in the incipient stress induced by moderate water deficit. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Aluminum Induces Oxidative Stress Genes in Arabidopsis thaliana1

Changes in gene expression induced by toxic levels of Al were characterized to investigate the nature of Al stress. A cDNA library was constructed from Arabidopsis thaliana seedlings treated with Al for 2 h. We identified five cDNA clones that showed a transient induction of their mRNA levels, four cDNA clones that showed a longer induction period, and two down-regulated genes. Expression of the four long-term-induced genes remained at elevated levels for at least 48 h. The genes encoded peroxidase, glutathione-S-transferase, blue copper-binding protein, and a protein homologous to the reticuline:oxygen oxidoreductase enzyme. Three of these genes are known to be induced by oxidative stresses and the fourth is induced by pathogen treatment. Another oxidative stress gene, superoxide dismutase, and a gene for Bowman-Birk protease inhibitor were also induced by Al in A. thaliana. These results suggested that Al treatment of Arabidopsis induces oxidative stress. In confirmation of this hypothesis, three of four genes induced by Al stress in A. thaliana were also shown to be induced by ozone. Our results demonstrate that oxidative stress is an important component of the plant's reaction to toxic levels of Al.

Hydrogen peroxide-mediated neuronal cell death induced by an endogenous neurotoxin, 3-hydroxykynurenine.

3-Hydroxykynurenine (3-HK) is a tryptophan metabolite whose level in the brain is markedly elevated under several pathological conditions, including Huntington disease and human immunodeficiency virus infection. Here we demonstrate that micromolar concentrations (1-100 microM) of 3-HK cause cell death in primary neuronal cultures prepared from rat striatum. The neurotoxicity of 3-HK was blocked by catalase and desferrioxamine but not by superoxide dismutase, indicating that the generation of hydrogen peroxide and hydroxyl radical is involved in the toxicity. Measurement of peroxide levels revealed that 3-HK caused intracellular accumulation of peroxide, which was largely attenuated by application of catalase. The peroxide accumulation and cell death caused by 1-10 microM 3-HK were also blocked by pretreatment with allopurinol or oxypurinol, suggesting that endogenous xanthine oxidase activity is involved in exacerbation of 3-HK neurotoxicity. Furthermore, NADPH diaphorase-containing neurons were spared from toxicity of these concentrations of 3-HK, a finding reminiscent of the pathological characteristics of several neurodegenerative disorders such as Huntington disease. These results suggest that 3-HK at pathologically relevant concentrations renders neuronal cells subject to oxidative stress leading to cell death, and therefore that this endogenous compound should be regarded as an important factor in pathogenesis of neurodegenerative disorders.

Intrinsic compressibility and volume compression in solvated proteins by molecular dynamics simulation at high pressure.

Constant pressure and temperature molecular dynamics techniques have been employed to investigate the changes in structure and volumes of two globular proteins, superoxide dismutase and lysozyme, under pressure. Compression (the relative changes in the proteins' volumes), computed with the Voronoi technique, is closely related with the so-called protein intrinsic compressibility, estimated by sound velocity measurements. In particular, compression computed with Voronoi volumes predicts, in agreement with experimental estimates, a negative bound water contribution to the apparent protein compression. While the use of van der Waals and molecular volumes underestimates the intrinsic compressibilities of proteins, Voronoi volumes produce results closer to experimental estimates. Remarkably, for two globular proteins of very different secondary structures, we compute identical (within statistical error) protein intrinsic compressions, as predicted by recent experimental studies. Changes in the protein interatomic distances under compression are also investigated. It is found that, on average, short distances compress less than longer ones. This nonuniform contraction underlines the peculiar nature of the structural changes due to pressure in contrast with temperature effects, which instead produce spatially uniform changes in proteins. The structural effects observed in the simulations at high pressure can explain protein compressibility measurements carried out by fluorimetric and hole burning techniques. Finally, the calculation of the proteins static structure factor shows significant shifts in the peaks at short wavenumber as pressure changes. These effects might provide an alternative way to obtain information concerning compressibilities of selected protein regions.

Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Circulating plasma xanthine oxidase contributes to vascular dysfunction in hypercholesterolemic rabbits.

Reactive oxygen species play a central role in vascular inflammation and atherogenesis, with enhanced superoxide (O2.-) production contributing significantly to impairment of nitric oxide (.NO)-dependent relaxation of vessels from cholesterol-fed rabbits. We investigated potential sources of O2.- production, which contribute to this loss of endothelium-dependent vascular responses. The vasorelaxation elicited by acetylcholine (ACh) in phenylephrine-contracted, aortic ring segments was impaired by cholesterol feeding. Pretreatment of aortic vessels with either heparin, which competes with xanthine oxidase (XO) for binding to sulfated glycosaminoglycans, or the XO inhibitor allopurinol resulted in a partial restoration (36-40% at 1 muM ACh) of ACh-dependent relaxation. Furthermore, O2.(-)-dependent lucigenin chemiluminescence, measured in intact ring segments from hypercholesterolemic rabbits, was decreased by addition of heparin, allopurinol or a chimeric, heparin-binding superoxide dismutase. XO activity was elevated more than two-fold in plasma of hypercholesterolemic rabbits. Incubation of vascular rings from rabbits on a normal diet with purified XO (10 milliunits/ml) also impaired .NO-dependent relaxation but only in the presence of purine substrate. As with vessels from hypercholesterolemic rabbits, this effect was prevented by heparin and allopurinol treatment. We hypothesize that increases in plasma cholesterol induce the release of XO into the circulation, where it binds to endothelial cell glycosaminoglycans. Only in hypercholesterolemic vessels is sufficient substrate available to sustain the production of O2.- and impair NO-dependent vasorelaxation. Chronically, the continued production of peroxynitrite, (ONOO-) which the simultaneous generation of NO and O2.- implies, may irreversibly impair vessel function.

Immunotargeting of antioxidant enzyme to the pulmonary endothelium.

Oxidative injury to the pulmonary endothelium has pathological significance for a spectrum of diseases. Administration of antioxidant enzymes, superoxide dismutase (SOD) and catalase (Cat), has been proposed as a method to protect endothelium. However, neither these enzymes nor their derivatives possess specific affinity to endothelium and do not accumulate in the lung. Previously we have described a monoclonal antibody to angiotensin-converting enzyme (ACE) that accumulates selectively in the lung after systemic injection in rats, hamsters, cats, monkeys, and humans. In the present work we describe a system for selective intrapulmonary delivery of CuZn-SOD and Cat conjugated with biotinylated anti-ACE antibody mAb 9B9 (b-mAb 9B9) by a streptavidin (SA)-biotin bridge. Both enzymes biotinylated with biotin ester at biotin/enzyme ratio 20 retain enzymatic activity and bind SA without loss of activity. We have constructed tri-molecular heteropolymer complexes consisting of b-mAb 9B9, SA, and biotinylated SOD or biotinylated Cat and have studied biodistribution and pulmonary uptake of these complexes in the rat after i.v. injection. Biodistribution of biotinylated enzymes was similar to that of nonmodified enzymes. Binding of SA markedly prolonged lifetime of biotinylated enzymes in the circulation. In contrast to enzymes conjugated with nonspecific IgG, other enzyme derivatives, and nonmodified enzymes, biotinylated enzymes conjugated with b-mAb 9B9 accumulated specifically in the rat lung (9% of injected SOD/g of lung tissue and 7.5% of injected Cat/g of lung tissue). Pulmonary uptake of nonmodified enzymes or derivatives with nonspecific IgG did not exceed 0.5% of injected dose/g. Both SOD and Cat conjugated with b-mAb 9B9 were retained in the rat lung for at least several hours. Trichloracetic acid-precipitable radiolabeled Cat was associated with microsomal and plasma membrane fractions of the lung tissue homogenate. Thus, modification of antioxidant enzymes with biotin and SA-mediated conjugation with b-mAb 9B9 prolongs the circulation of enzymes resulting in selective accumulation in the lung and intracellular delivery of enzymes to the pulmonary endothelium. These results provide the background for an approach to provide protection of pulmonary endothelium against oxidative insults.

Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae.

The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxidant genes catalase, superoxide dismutase, and cytochrome c peroxidase were more sensitive to the lethal effect of heat than isogenic wild-type cells. Overexpression of catalase and superoxide dismutase genes caused an increase in thermotolerance. Anaerobic conditions caused a 500- to 20,000-fold increase in thermotolerance. The thermotolerance of cells in anaerobic conditions was immediately abolished upon oxygen exposure. HSP104 is not responsible for the increased resistance of anaerobically grown cells. The thermotolerance of anaerobically grown cells is not due to expression of heat-shock proteins. By using an oxidation-dependent fluorescent molecular probe a 2- to 3-fold increase in fluorescence was found upon heating. Thus, we conclude that oxidative stress is involved in heat-induced cell death.

On signal sequence polymorphisms and diseases of distribution.

We report a previously unappreciated property of the signals that target organelle-specific proteins to their subcellular sites of action. Such targeting sequences are shown to be polymorphic. We discovered this polymorphism when we cloned the mitochondrial manganese-containing superoxide dismutase from cell lines of normal individuals and patients with genetic diseases of premature aging and compared their sequences to each other and to those previously reported. The polymorphism consists of a single nucleotide change in the region of the DNA that encodes the signal sequence such that either an alanine or valine is present. Subsequently, eight cell lines were analyzed and all three possible combinations of the two signal sequences were observed. Such signal sequence polymorphisms could result in diseases of distribution, where essential proteins are not properly targeted, thereby leading to absolute or relative deficiencies of critical enzymes within specific cellular compartments. Progeria and related syndromes may be diseases of distribution.

Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.

The estrogen receptor locus is associated with a major gene influencing litter size in pigs.

Identification of individual major genes affecting quantitative traits in livestock species has been limited to date. By using a candidate gene approach and a divergent breed cross involving the Chinese Meishan pig, we have shown that a specific allele of the estrogen receptor (ER) locus is associated with increased litter size. Female pigs from synthetic lines with a 50% Meishan background that were homozygous for this beneficial allele produced 2.3 more pigs in first parities and 1.5 more pigs averaged over all parities than females from the same synthetic lines and homozygous for the undesirable allele. This beneficial ER allele was also found in pigs with Large White breed ancestory. Analysis of females with Large White breed background showed an advantage for females homozygous for the beneficial allele as compared to females homozygous for the other allele of more than 1 total pig born. Analyses of growth performance test records detected no significant unfavorable associations of the beneficial allele with growth and developmental traits. Mapping of the ER gene demonstrated that the closest known genes or markers were 3 centimorgans from ER. To our knowledge, one of these, superoxide dismutase gene (SOD2), was mapped for the first time in the pig. Analysis of ER and these linked markers indicated that ER is the best predictor of litter size differences. Introgression of the beneficial allele into commercial pig breeding lines, in which the allele was not present, and marker-assisted selection for the beneficial allele in lines with Meishan and Large White background have begun.

Muscle-specific expression of Drosophila hsp70 in response to aging and oxidative stress.

Induction of Drosophila hsp70 protein was detected during aging in flight muscle and leg muscle in the absence of heat shock, using an hsp70-specific monoclonal antibody, and in transgenic flies containing hsp70-beta-galactosidase fusion protein reporter constructs. While hsp70 and reporter proteins were induced during aging, hsp70 message levels were not, indicating that aging-specific induction is primarily posttranscriptional. In contrast, hsp22 and hsp23 were found to be induced during aging at the RNA level and with a broader tissue distribution. The same muscle-specific hsp70 reporter expression pattern was observed in young flies mutant for catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6). In catalase (cat) hypomorphic lines where flies survived to older ages, the time course of hsp70 reporter expression during aging was accelerated, and the initial and ultimate levels of expression were increased. The hsp70 reporter was also induced in young flies mutant for copper/zinc superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1). Taken together, the results suggest that aging-specific hsp70 expression may be a result of oxidative damage.

Dietary lipids and calorie restriction affect mammary tumor incidence and gene expression in mouse mammary tumor virus/v-Ha-ras transgenic mice.

We have studied the effects of food restriction (FR) and substitution of fish oil (FO; omega 3) for corn oil (CO; omega 6) on breast tumor incidence and survival in mouse mammary tumor virus/v-Ha-ras transgenic (Onco) mice. The diets were as follows: group 1, 5% (wt/wt) CO fed ad libitum (AL); group 2, 5% CO, restricted calories (40% fewer calories than AL; FR); group 3, 20% CO fed AL; and group 4, 20% FO fed AL. After 3 years, 40% of FR Onco (group 2) mice were alive, whereas there were no survivors in the other three groups. Similarly, tumor incidence was reduced to 27% (5 out of 18) in FR animals (group 2), whereas it was 83% (11 out of 13) in group 1 mice, 89% (16 out of 18) in group 3 mice, and 71% (10 out of 14) in group 4 mice. These protective effects of FR on survival and tumor incidence were paralleled by higher expression of the tumor suppressor gene p53 (wild type) and free-radical scavenging enzymes (catalase and superoxide dismutase) in breast tumors. Immunoblotting showed less ras gene product, p21, and increased p53 levels in the tumors of FR mice. In addition, FR decreased RNA levels of c-erbB-2, interleukin 6, and the transgene v-Ha-ras in tumors. In contrast, analysis of hepatic mRNA from tumor-bearing FR mice revealed higher expression of catalase, glutathione peroxidase, and superoxide dismutase. Survival and tumor incidence were not influenced significantly by dietary supplementation with FO in place of CO. Taken together, our studies suggest that moderate restriction of energy intake significantly inhibited the development of mammary tumors and altered expression of cytokines, oncogenes, and free-radical scavenging enzymes.

The ATX1 gene of Saccharomyces cerevisiae encodes a small metal homeostasis factor that protects cells against reactive oxygen toxicity.

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as superoxide dismutase (SOD) and catalase. Here we describe the isolation and characterization of another gene in the yeast Saccharomyces cerevisiae that plays a critical role in detoxification of reactive oxygen species. This gene, named ATX1, was originally isolated by its ability to suppress oxygen toxicity in yeast lacking SOD. ATX1 encodes a 8.2-kDa polypeptide exhibiting significant similarity and identity to various bacterial metal transporters. Potential ATX1 homologues were also identified in multicellular eukaryotes, including the plants Arabidopsis thaliana and Oryza sativa and the nematode Caenorhabditis elegans. In yeast cells, ATX1 evidently acts in the transport and/or partitioning of copper, and this role in copper homeostasis appears to be directly relevant to the ATX1 suppression of oxygen toxicity: ATX1 was incapable of compensating for SOD when cells were depleted of exogenous copper. Strains containing a deletion in the chromosomal ATX1 locus were generated. Loss of ATX1 function rendered both mutant and wild-type SOD strains hypersensitive toward paraquat (a generator of superoxide anion) and was also associated with an increased sensitivity toward hydrogen peroxide. Hence, ATX1 protects cells against the toxicity of both superoxide anion and hydrogen peroxide.

Use of a New Tetrazolium-Based Assay to Study the Production of Superoxide Radicals by Tobacco Cell Cultures Challenged with Avirulent Zoospores of Phytophthora parasitica var nicotianae1

The relationship between the production of reactive oxygen species and the hypersensitive response (HR) of tobacco (Nicotiana tabacum L.) toward an incompatible race of the Oomycete Phytophthora parasitica var nicotianae has been investigated. A new assay for superoxide radical (O2−) production based on reduction of the tetrazolium dye sodium,3′-(1-[phenylamino-carbonyl]-3,4-tetrazolium)-bis(4-methoxy-6-nitro) benzene-sulfonic acid hydrate (XTT) has enabled the quantitative estimation of perhydroxyl/superoxide radical acid-base pair (HO2·/O2−) production during the resistant response. Tobacco suspension cells were inoculated with zoospores from compatible or incompatible races of the pathogen. Subsequent HO2·/O2− production was monitored by following the formation of XTT formazan. In the incompatible interaction only, HO2·/O2− was produced in a minor burst between 0 and 2 h and then in a major burst between 8 and 10 h postinoculation. During this second burst, rates of XTT reduction equivalent to a radical flux of 9.9 × 10−15 mol min−1 cell−1 were observed. The HO2·/O2− scavengers O2− dismutase and Mn(III)desferal each inhibited dye reduction. An HR was observed in challenged, resistant cells immediately following the second burst of radical production. Both scavengers inhibited the HR when added prior to the occurrence of either radical burst, indicating that O2− production is a necessary precursor to the HR.