Yeast lacking superoxide dismutase(s) show elevated levels of "free iron" as measured by whole cell electron paramagnetic resonance

A current hypothesis explaining the toxicity of superoxide anion in vivo is that it oxidizes exposed [4Fe-4S] clusters in certain vulnerable enzymes causing release of iron and enzyme inactivation. The resulting increased levels of "free iron" catalyze deleterious oxidative reactions in the cell. In this study, we used low temperature Fe(III) electron paramagnetic resonance (EPR) spectroscopy to monitor iron status in whole cells of the unicellular eukaryote, Saccharomyces cerevisiae. The experimental protocol involved treatment of the cells with desferrioxamine, a cell-permeant, Fe(III)-specific chelator, to promote oxidation of all of the "free iron" to the Fe(III) state wherein it is EPR-detectable. Using this method, a small amount of EPR-detectable iron was detected in the wild-type strain, whereas significantly elevated levels were found in strains lacking CuZn-superoxide dismutase (CuZn-SOD) (sod1 delta), Mn-SOD (sod2 delta), or both SODs, throughout their growth but particularly in stationary phase. The accumulation was suppressed by expression of wild-type human CuZn-SOD (in the sod1 delta mutant), by pmr1, a genetic suppressor of the sod delta mutant phenotype (in the sod1 delta sod2 delta double knockout strain), and by anaerobic growth. In wild-type cells, an increase in the EPR-detectable iron pool could be induced by treatment with paraquat, a redox-cycling drug that generates superoxide. Cells that were not pretreated with desferrioxamine had Fe(III) EPR signals that were equally as strong as those from treated cells, indicating that "free iron" accumulated in the ferric form in our strains in vivo. Our results indicate a relationship between superoxide stress and iron handling and support the above hypothesis for superoxide-related oxidative damage.     

Yeast frataxin mutants display decreased superoxide dismutase activity crucial to promote protein oxidative damage

Iron overload is involved in several pathological conditions, including Friedreich ataxia, a disease caused by decreased expression of the mitochondrial protein frataxin. In a previous study, we identified 14 proteins selectively oxidized in yeast cells lacking Yfh1, the yeast frataxin homolog. Most of these were magnesium-binding proteins. Decreased Mn-SOD activity, oxidative damage to CuZn-SOD, and increased levels of chelatable iron were also observed in this model. This study explores the relationship between low SOD activity, the presence of chelatable iron, and protein damage. We observed that addition of copper and manganese to the culture medium restored SOD activity and prevented both oxidative damage and inactivation of magnesium-binding proteins. This protection was compartment specific: recovery of mitochondrial enzymes required the addition of manganese, whereas cytosolic enzymes were recovered by adding copper. Copper treatment also decreased Δyfh1 sensitivity to menadione. Finally, a Δsod1 mutant showed high levels of chelatable iron and inactivation of magnesium-binding enzymes. These results suggest that reduced superoxide dismutase activity contributes to the toxic effects of iron overloading. This would also apply to pathologies involving iron accumulation.     

A compensatory increase in trehalose synthesis in response to desiccation stress in Saccharomyces cerevisiae cells lacking the heat shock protein Hsp12p

The effect of HSP12 deletion on the response of yeast to desiccation was investigated. The Deltahsp12 strain was found to be more desiccation tolerant than the wild-type strain. Furthermore, the increased intracellular trehalose levels in the Deltahsp12 strain suggested that this strain compensated for the lack of Hsp12p synthesis by increasing trehalose synthesis, which facilitated increased desiccation tolerance. Results obtained from flow cytometry using the membrane exclusion dye propidium iodide suggested that Hsp12p helped maintain plasma membrane integrity during desiccation. Analysis of the oxidative loads experienced by the wild-type and Deltahsp12 strains showed that during mid-exponential phase, the increased trehalose levels present in the Deltahsp12 cells resulted in increased protection of these cells against reactive oxygen species compared with wild-type cells. During stationary phase, lower levels of reactive oxygen species reduction by reduced glutathione was enhanced in the wild-type strain, which displayed lower intracellular trehalose concentrations. Comparison of the tolerance of the wild-type and Deltahsp12 strains with applied oxidative stress showed that the Deltahsp12 strain was more tolerant to exogenously applied H2O2, which we attributed to the higher intracellular trehalose concentration. Flow cytometry demonstrated that Hsp12p played a role in maintaining plasma membrane integrity during applied oxidative stress.     

G209A mutant alpha synuclein expression specifically enhances dopamine induced oxidative damage

Alpha synuclein protein may play an important role in familial and sporadic Parkinson's disease pathology. We have induced G209A mutant or wild-type alpha-synuclein expression in stable HEK293 cell models to determine if this influences markers of oxidative stress and damage under normal conditions or in the presence of dopamine or paraquat. Induced wild-type or mutant alpha-synuclein expression alone had no effect upon levels of oxidative stress or damage, as measured by glutathione levels or aconitase activity. Both wild-type and mutant alpha-synuclein expression decreased the oxidative damage induced by paraquat, although the protection was less marked with mutant alpha-synuclein expression. This suggests that alpha-synuclein expression may either have anti-oxidant properties or may upregulate cellular antioxidant levels, a function that was diminished by the G209A mutation. However, mutant but not wild-type alpha-synuclein expression specifically enhanced dopamine associated oxidative damage. Non-expressing cells treated with reserpine to inhibit the vesicular monoamine compartmentalisation produced similar results. However, consistent with the hypothesis that mutant alpha-synuclein disrupts vesicular dopamine compartmentalization, this effect was diminished in cells expressing mutant alpha-synuclein. This may result in increased dopamine metabolism and cause selective oxidative damage to dopaminergic cells.     

Differential effects of mitochondrial heat shock protein 60 and related molecular chaperones to prevent intracellular beta-amyloid-induced inhibition of complex IV and limit apoptosis

Defects in mitochondrial oxidative metabolism, in particular decreased activity of cytochrome c oxidase, have been reported in Alzheimer disease tissue and in cultured cells that overexpress amyloid precursor protein. Mitochondrial dysfunction contributes to neurodegeneration in Alzheimer disease partly through formation of reactive oxygen species and the release of sequestered molecules that initiate programmed cell death pathways. The heat shock proteins (HSP) are cytoprotective against a number of stressors, including accumulations of misfolded proteins and reactive oxygen species. We reported on the property of Hsp70 to protect cultured neurons from cell death caused by intraneuronal beta-amyloid. Here we demonstrate that Hsp60, Hsp70, and Hsp90 both alone and in combination provide differential protection against intracellular beta-amyloid stress through the maintenance of mitochondrial oxidative phosphorylation and functionality of tricarboxylic acid cycle enzymes. Notably, beta-amyloid was found to selectively inhibit complex IV activity, an effect selectively neutralized by Hsp60. The combined effect of HSPs was to reduce the free radical burden, preserve ATP generation, decrease cytochrome c release, and prevent caspase-9 activation, all important mediators of beta-amyloid-induced neuronal dysfunction and death.     

Free Radical Inactivation of Rabbit Muscle Creatine Kinase: Catalysis by Physiological and Hydrolyzed ICRF-187 (ICRF-198) Iron Chelates

Creatine kinase is a sulfhydryl containing enzyme that is particularly susceptible to oxidative inactivation. This enzyme is potentially vulnerable to inactivation under conditions when it would be used as a diagnostic marker of tissue damage such as during cardiac ischemia/reperfusion or other oxidative tissue injury. Oxidative stress in tissues can induce the release of iron from its storage proteins, making it an available catalyst for free radical reactions. Although creatine kinase inactivation in a heart reperfusion model has been documented, the mechanism has not been fully described, particularly with regard to the role of iron. We have investigated the inactivation of rabbit muscle creatine kinase by hydrogen peroxide and by xanthine oxidase generated superoxide or Adriamycin radicals in the presence of iron catalysts. As shown previously, creatine kinase was inactivated by hydrogen peroxide. Ferrous iron enhanced the inactivation. In addition, micromolar levels of iron and iron chelates that were reduced and recycled by superoxide or Adriamycin radicals were effective catalysts of creatine kinase inactivation. Of the physiological iron chelates studied, Fe(ATP) was an especially effective catalyst of inactivation by what appeared to be a site-localized reaction. Fe(ICRF-198), a non-physiological chelate of interest because of its putative role in alleviating Adriamycin-induced cardiotoxicity, also catalyzed the inactivation. Scavenger studies implicated hydroxyl radical as the oxidant involved in iron-dependent creatine kinase inactivation. Loss of protein thiols accompanied loss of creatine kinase activity. Reduced glutathione (GSH) provided marked protection from oxidative inactivation, suggesting that enzyme inactivation under physiological conditions would occur only after GSH depletion.     

Commentary: The Role of Iron-Sulfur Clusters in in vivo Hydroxyl Radical Production

The in vivo production of HO- requires iron ions, H₂O₂ and O₂- or other oxidants but probably does not occur through the Haber-Weiss reaction. Instead oxidants, such as O₂-, increase free iron by releasing Fe(II) from the iron-sulfur clusters of dehydratases and by interfering with the iron-sulfur clusters reassembly. Fe(II) then reduces H₂O₂, and in turn Fe(III) and the oxidized cluster are re-reduced by cellular reductants such as NADPH and glutathione. In this way, SOD cooperates with cellular reductants in keeping the iron-sulfur clusters intact and the rate of HO- production to a minimum.

O₂- and other oxidants can release iron from Fe(II)-containing enzymes as well as copper from thionein. The released Fe(III) and Cu(II) are then reduced to Fe(II) and Cu(I) and can then participate in the Fenton reaction.

In mammalian cells oxidants are able to convert cytosolic aconitase into active IRE-BP, which increases the “free” iron concentration intracellularly both by decreasing the biosynthesis of ferritin and increasing biosynthesis of transferrin receptors.

The biological role of the soxRS regulon of Escherichia coli, which is involved in the adaptation toward oxidative stress, is presumably to counteract the oxidative inactivation of the iron clusters and the subsequent release of iron with consequent increased rate of production of HO.     

DNA base modifications and membrane damage in cultured mammalian cells treated with iron ions

We investigated DNA base damage in mammalian cells exposed to exogenous iron ions in culture. Murine hybridoma cells were treated with Fe(II) ions at concentrations of 10 μM, 100 μM, and 1 mM. Chromatin was isolated from treated and control cells and analyzed by gas chromatography/mass spectrometry for DNA base damage. Ten modified DNA bases were identified in both Fe(II)-treated and control cells. The quantification of modified bases was achieved by isotope-dilution mass spectrometry. In Fe(II)-treated cells, the amounts of modified bases were increased significantly above the background levels found in control cells. Dimethyl sulfoxide at concentrations up to 1 M in the culture medium did not significantly inhibit the formation of modified DNA bases. A mathematical simulation used to evaluate the plausibility of DNA damage upon Fe(II) treatment predicted a dose-dependent response, which agreed with the experimental results. In addition, Fe(II) treatment of cells increased the cell membrane permeability and caused production of lipid peroxides. The nature of DNA base lesions suggests the involvement of the hydroxyl radical in their formation. The failure of dimethyl sulfoxide to inhibit their formation indicates a site-specific mechanism for DNA damage with involvement of DNA-bound metal ions. Fe(II) treatment of cells may increase the intracellular iron ion concentration and/or cause oxidative stress releasing metal ions from their storage sites with subsequent binding to DNA. Identified DNA base lesions may be promutagenic and play a role in pathologic processes associated with iron ions.     

Lethal oxidative damage and mutagenesis are generated by iron in delta fur mutants of Escherichia coli: protective role of superoxide dismutase.

The Escherichia coli Fur protein, with its iron(II) cofactor, represses iron assimilation and manganese superoxide dismutase (MnSOD) genes, thus coupling iron metabolism to protection against oxygen toxicity. Iron assimilation is triggered by iron starvation in wild-type cells and is constitutive in fur mutants. We show that iron metabolism deregulation in fur mutants produces an iron overload, leading to oxidative stress and DNA damage including lethal and mutagenic lesions. fur recA mutants were not viable under aerobic conditions and died after a shift from anaerobiosis to aerobiosis. Reduction of the intracellular iron concentration by an iron chelator (ferrozine), by inhibition of ferric iron transport (tonB mutants), or by overexpression of the iron storage ferritin H-like (FTN) protein eliminated oxygen sensitivity. Hydroxyl radical scavengers dimethyl sulfoxide and thiourea also provided protection. Functional recombinational repair was necessary for protection, but SOS induction was not involved. Oxygen-dependent spontaneous mutagenesis was significantly increased in fur mutants. Similarly, SOD deficiency rendered sodA sodB recA mutants nonviable under aerobic conditions. Lethality was suppressed by tonB mutations but not by iron chelation or overexpression of FTN. Thus, superoxide-mediated iron reduction was responsible for oxygen sensitivity. Furthermore, overexpression of SOD partially protected fur recA mutants. We propose that a transient iron overload, which could potentially generate oxidative stress, occurs in wild-type cells on return to normal growth conditions following iron starvation, with the coupling between iron and MnSOD regulation helping the cells cope.     

Does vitamin C act as a pro-oxidant under physiological conditions?

Vitamin C readily scavenges reactive oxygen and nitrogen species and may thereby prevent oxidative damage to important biological macromolecules such as DNA, lipids, and proteins. Vitamin C also reduces redox active transition metal ions in the active sites of specific biosynthetic enzymes. The interaction of vitamin C with 'free', catalytically active metal ions could contribute to oxidative damage through the production of hydroxyl and alkoxyl radicals; whether these mechanisms occur in vivo, however, is uncertain. To examine this issue, we reviewed studies that investigated the role of vitamin C, both in the presence and absence of metal ions, in oxidative DNA, lipid, and protein damage. We found compelling evidence for antioxidant protection of lipids by vitamin C in biological fluids, animals, and humans, both with and without iron cosupplementation. Although the data on protein oxidation in humans are sparse and inconclusive, the available data in animals consistently show an antioxidant role of vitamin C. The data on vitamin C and DNA oxidation in vivo are inconsistent and conflicting, but some of the discrepancies can be explained by flaws in experimental design and methodology. These and other important issues discussed here need to be addressed in future studies of the role of vitamin C in oxidative damage.     

Oxidative damage to specific proteins in replicative and chronological-aged Saccharomyces cerevisiae: common targets and prevention by calorie restriction

Oxidative modifications of cellular components have been described as one of the main contributions to aged phenotype. In Saccharomyces cerevisiae, two distinct life spans can be considered, replicative and chronological. The relationship between both aging models is still not clear despite suggestions that these phenomena may be related. In this work, we show that replicative and chronological-aged yeast cells are affected by an oxidative stress situation demonstrated by increased protein carbonylation when compared with young cells. The data on the identification of these oxidatively modified proteins gives clues to better understand cellular dysfunction that occurs during aging. Strikingly, although in both aging models metabolic differences are important, major targets are almost the same. Common targets include stress resistance proteins (Hsp60 and Hsp70) and enzymes involved in glucose metabolism such as enolase, glyceraldehydes-3-P dehydrogenase, fructose-1,6-biphosphate aldolase, pyruvate decarboxylase, and alcohol dehydrogenase. In both aging models, calorie restriction results in decreased damage to these proteins. In addition, chronological-aged cells grown under glucose restriction displayed lowered levels of lipid peroxidation product lipofuscin. Intracellular iron concentration is kept almost unchanged, whereas in non-restricted cells, the values increase up 4-5 times. The pro-oxidant effects of such increased iron concentration would account for the damage observed. Also, calorie-restricted cells show undamaged catalase, which clearly appears carbonylated in cells grown at a high glucose concentration. These results may explain lengthening of the viability of chronological-aged cells and could have an important role in replicative life span extension by calorie restriction.     

Analysis of oxidative events induced by expanded polyglutamine huntingtin exon 1 that are differentially restored by expression of heat shock proteins or treatment with an antioxidant

We recently reported that the transient expression of polyglutamine tracts of various size in exon 1 of the huntingtin polypeptide (httEx1) generated abnormally high levels of intracellular reactive oxygen species that directly contributed to cell death. Here, we compared the protection generated by heat shock proteins to that provided by the antioxidant agent N-acetyl-L-cysteine. In cells expressing httEx1 with 72 glutamine repeats (httEx1-72Q), the overexpression of Hsp27 or Hsp70 plus Hdj-1(Hsp40) or treatment of the cells with N-acetyl-L-cysteine inhibited not only mitochondrial membrane potential disruption but also the increase in reactive oxygen species, nitric oxide and protein oxidation. However, only heat shock proteins and not N-acetyl-L-cysteine reduced the size of the inclusion bodies formed by httEx1-72Q. In cells expressing httEx1 polypeptide with 103 glutamine repeats (httEx1-103Q), heat shock proteins neither decreased oxidative damage nor reduced the size of the inclusions. In contrast, N-acetyl-L-cysteine still efficiently decreased the oxidative damage induced by httEx1-103Q polypeptide without altering the inclusions. N-Acetyl-L-cysteine was inactive with regard to proteasome inhibition, whereas heat shock proteins partially restored the caspase-like activity of this protease. These observations suggest some relationships between the presence of inclusion bodies and the oxidative damage induced by httEx1-polyQ.     

How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress

We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.     

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

Characterization of iron-sulfur protein assembly in isolated mitochondria. A requirement for ATP,NADH, and reduced iron

To study the biochemical requirements for maturation of iron-sulfur (Fe/S) proteins, we have reconstituted the process in vitro using detergent extracts from Saccharomyces cerevisiae mitochondria. Efficient assembly of biotin synthase as a model Fe/S protein required anaerobic conditions, dithiothreitol, cysteine, ATP, and NADH. Cysteine is utilized by the cysteine desulfurase Nfs1p to release sulfan sulfur; ATP presumably reflects the function of the Hsp70 family chaperone Ssq1p; and NADH is used for reduction of the ferredoxin Yah1p involved in Fe/S protein biogenesis. Hence, our assay system faithfully reproduces the in vivo pathway. We have further investigated the involvement of various mitochondrial proteins suspected to participate in Fe/S protein biogenesis. In mitochondrial extracts depleted in Isa1p, Fe/S protein formation was severely decreased. A similar strong decline was observed with extracts from Delta yfh1 mitochondria, indicating that both Isa1p and the yeast frataxin homologue, Yfh1p, are crucial for biogenesis of mitochondrial Fe/S proteins. Conversely, the activities of mitochondrial extracts from Delta nfu1 cells were only moderately reduced, suggesting a dispensable role for Nfu1p. Finally, iron utilized for Fe/S protein formation was imported into the matrix of intact mitochondria in ferrous form in a membrane potential-dependent transport step. Our results represent the first in vitro reconstitution of the entire pathway of Fe/S protein maturation.     

Robust Glyoxalase activity of Hsp31, a ThiJ/DJ-1/PfpI Family Member Protein,Is Critical for Oxidative Stress Resistance in Saccharomyces cerevisiae

Methylglyoxal (MG) is a reactive metabolic intermediate generated during various cellular biochemical reactions, including glycolysis. The accumulation of MG indiscriminately modifies proteins, including important cellular antioxidant machinery, leading to severe oxidative stress, which is implicated in multiple neurodegenerative disorders, aging, and cardiac disorders. Although cells possess efficient glyoxalase systems for detoxification, their functions are largely dependent on the glutathione cofactor, the availability of which is self-limiting under oxidative stress. Thus, higher organisms require alternate modes of reducing the MG-mediated toxicity and maintaining redox balance. In this report, we demonstrate that Hsp31 protein, a member of the ThiJ/DJ-1/PfpI family in Saccharomyces cerevisiae, plays an indispensable role in regulating redox homeostasis. Our results show that Hsp31 possesses robust glutathione-independent methylglyoxalase activity and suppresses MG-mediated toxicity and ROS levels as compared with another paralog, Hsp34. On the other hand, glyoxalase-defective mutants of Hsp31 were found highly compromised in regulating the ROS levels. Additionally, Hsp31 maintains cellular glutathione and NADPH levels, thus conferring protection against oxidative stress, and Hsp31 relocalizes to mitochondria to provide cytoprotection to the organelle under oxidative stress conditions. Importantly, human DJ-1, which is implicated in the familial form of Parkinson disease, complements the function of Hsp31 by suppressing methylglyoxal and oxidative stress, thus signifying the importance of these proteins in the maintenance of ROS homeostasis across phylogeny.     

Fatty acid-mediated intracellular iron translocation: a synergistic mechanism of oxidative injury

Fatty acid has been reported to be associated with cardiovascular diseases and cancer, but the possible mechanism remains unclear. Here, we reported a novel mechanism for the permissive role of fatty acid on iron intracellular translocation and subsequent oxidative injury. In vitro study from endothelial cells showed that iron alone had little effect, whereas in combination with PA (palmitic acid), iron-mediated toxicity was markedly potentiated, as reflected in mitochondrial dysfunction, cell death, apoptosis, and DNA mutation. We also showed that PA not only facilitated iron translocation into cells through a transferrin-receptor (TfR)-independent mechanism, but also translocated iron into mitochondria; the subsequent intracellular iron overload resulted in reactive oxygen species (ROS) overgeneration and lipid oxidation. Further investigation revealed that PA-facilitated iron translocation is due to Fe/PA-mediated extracellular oxidative stress and the subsequent membrane damage with increased membrane permeability. Fe/PA-mediated toxic effects were reduced in rho0 cells lacking mitochondrial DNA or by antioxidant enzyme SOD, especially mitochondrially localized MnSOD, suggesting a permissive role of PA for iron deposition on the vascular wall and its subsequent toxicity via mitochondrial oxidative stress. This observation was confirmed in vivo in mice, wherein higher vascular iron deposition and accompanying superoxide release were observed in the presence of a high-fat diet with iron administration.     

Agrobacterium tumefaciens fur has important physiological roles in iron and manganese homeostasis, the oxidative stress response, and full virulence

In Agrobacterium tumefaciens, the balance between acquiring enough iron and avoiding iron-induced toxicity is regulated in part by Fur (ferric uptake regulator). A fur mutant was constructed to address the physiological role of the regulator. Atypically, the mutant did not show alterations in the levels of siderophore biosynthesis and the expression of iron transport genes. However, the fur mutant was more sensitive than the wild type to an iron chelator, 2,2'-dipyridyl, and was also more resistant to an iron-activated antibiotic, streptonigrin, suggesting that Fur has a role in regulating iron concentrations. A. tumefaciens sitA, the periplasmic binding protein of a putative ABC-type iron and manganese transport system (sitABCD), was strongly repressed by Mn(2+) and, to a lesser extent, by Fe(2+), and this regulation was Fur dependent. Moreover, the fur mutant was more sensitive to manganese than the wild type. This was consistent with the fact that the fur mutant showed constitutive up-expression of the manganese uptake sit operon. Fur(At) showed a regulatory role under iron-limiting conditions. Furthermore, Fur has a role in determining oxidative resistance levels. The fur mutant was hypersensitive to hydrogen peroxide and had reduced catalase activity. The virulence assay showed that the fur mutant had a reduced ability to cause tumors on tobacco leaves compared to wild-type NTL4.     

Antioxidant responses of Chilo suppressalis (Lepidoptera: Pyralidae) larvae exposed to thermal stress

High temperatures cause a variety of physiological stress responses in insects, including increased generation of reactive oxygen species (ROS), which can cause oxidative damage. This study investigated the effects of thermal stress on ROS generation, the expression of heat shock protein 70 (Hsp70) at the mRNA and protein levels, the activity of antioxidant enzymes (SOD, CAT), and apoptosis in hemocytes of Chilo suppressalis larvae. Results indicated that thermal stress significantly elevated the level of ROS and antioxidant enzyme activity in C. suppressalis larvae. Real-time quantitative PCR showed that hsp70 gene expression was induced by heat stress. Flow cytometric results revealed that the expression profile of Hsp70 at the protein level was in agreement with that at the mRNA level. The expression of Hsp70 at both the mRNA and protein levels reached a maximum at 36 °C in larval hemocytes. Exposure to tested temperatures did not cause any significant change in the rate of apoptosis in larval hemocytes. These results suggest that thermal stress leads to oxidative stress and that antioxidant enzymes and the Hsp70 play an important role in reducing oxidative damage in C. suppressalis larvae.