Extracellular Superoxide Dismutase Protects Histoplasma Yeast Cells from Host-Derived Oxidative Stress

In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3) and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs) and cytokine-activated macrophages triggers production of reactive oxygen species (ROS). Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.     

Superoxide Dismutase in Plants

Superoxide dismutases (SODs) are metal-containing enzymes that catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined where it plays a major role in the defense against toxic-reduced oxygen species, which are generated as byproducts of many biological oxidations. The generation of oxygen radicals can be further exacerbated during environmental adversity and consequently SOD has been proposed to be important for plant stress tolerance. In plants, three forms of the enzyme exist, as classified by their active site metal ion: copper/zinc, manganese, and iron forms. The distribution of these enzymes has been studied both at the subcellular level and at the phylogenic level. It is only in plants that all three different types of SOD coexist. Their occurrence in the different subcellular compartments of plant cells allows a study of their molecular evolution and the possibility of understanding why three functionally equivalent but structurally different types of SOD have been maintained. Several cDNA sequences that encode the different SODs have recently become available, and the use of molecular techniques have greatly increased our knowledge about this enzyme system and about oxidative stress in plants in general, such that now is an appropriate time to review our current knowledge.     

Overexpression of Extracellular Superoxide Dismutase Protects against Brain Injury Induced by Chronic Hypoxia

Extracellular superoxide dismutase (EC-SOD) is an isoform of SOD normally found both intra- and extra-cellularly and accounting for most SOD activity in blood vessels. Here we explored the role of EC-SOD in protecting against brain damage induced by chronic hypoxia. EC-SOD Transgenic mice, were exposed to hypoxia (FiO2.1%) for 10 days (H-KI) and compared to transgenic animals housed in room air (RA-KI), wild type animals exposed to hypoxia (H-WT or wild type mice housed in room air (RA-WT). Overall brain metabolism evaluated by positron emission tomography (PET) showed that H-WT mice had significantly higher uptake of ¹⁸FDG in the brain particularly the hippocampus, hypothalamus, and cerebellum. H-KI mice had comparable uptake to the RA-KI and RA-WT groups. To investigate the functional state of the hippocampus, electrophysiological techniques in ex vivo hippocampal slices were performed and showed that H-KI had normal synaptic plasticity, whereas H-WT were severely affected. Markers of oxidative stress, GFAP, IBA1, MIF, and pAMPK showed similar values in the H-KI and RA-WT groups, but were significantly increased in the H-WT group. Caspase-3 assay and histopathological studies showed significant apoptosis/cell damage in the H-WT group, but no significant difference in the H-KI group compared to the RA groups. The data suggest that EC-SOD has potential prophylactic and therapeutic roles in diseases with compromised brain oxygenation.     

植物维生素C过氧化物酶研究进展

维生素C过氧化物酶(ascorbate peroxidase,APX)是植物体内的重要酶系,是植物AsA-GSH氧化还原途径的重要组分,是清除H2O2(特别是叶绿体中的H2O2)的关键酶.本文综述了维生素C过氧化物酶表达调控方面的研究进展,包括逆境(干旱胁迫、空气污染、微量元素缺乏、离子胁迫、过度光强、照射以及盐胁迫等)与APX的表达调控、植物细胞程序性死亡(PCD)与APX的表达调控、植物生长发育与APX的表达调控、植物进化与APX表达调控等.植物体内的APX基因包括基质和类囊体两类,不同的APX基因序列存在一定差异,本文还综述了这两类APX基因在植物方面的分离和克隆进展情况,同时对APX基因的遗传转化进行了简要回顾,最后指出了APX今后的研究方向.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因甘薯的耐旱性

水分胁迫使转铜／锌超氧化物歧化酶基因（Cu／Zn SOD）和抗坏血酸过氧化物酶基因（APX）甘薯及未转基因甘薯中超氧阴离子（O2^-）、过氧化氢（H2O2）、丙二醛（MDA）含量和细胞膜相对透性增加,在相同条件下以上指标均为转基因甘薯低于未转基因甘薯;而叶片含水量、净光合速率（Pn）和气孔导度（Gs）均下降,SOD和APX酶活性随胁迫程度的加重先增大后减小,胞间CO2浓度（Ci）则先减小后增大,在相同条件下转基因甘薯中以上指标均高于未转基因甘薯。这些结果表明：转入Cu／Zn SOD和APX基因使转基因甘薯清除活性氧的能力增强,在水分胁迫下能保持较高的叶片含水量和Pn,耐旱性得到提高。     

Induced Activity of Superoxide Dismutase and Peroxidase of in vitro Plants by Low Concentrations of Ethanol

A rapid micropropagation protocol was established for Aloe vera L. var. chinensis (Haw.) Berger, Chinese Aloe. The effects of three factors, namely BA, NAA and sucrose, on bud initiation were evaluated by L₉ (3⁴) orthogonal design. The variance analysis of the experimental results showed that the actions of the three factors were all considerable. Among the three factors, sucrose was the most important for bud initiation followed by BA, and NAA had the weakest effect. The best medium for bud initiation was semi-solid MS supplemented with 2.0 mg l^–1 BA, 0.3 mg l^–1 NAA, 30 g l^–1 sucrose and 0.6 g l^–1 PVP (pH 5.8), on which Chinese aloe could multiply 15 times in 4 weeks. Some shoots rooted spontaneously on 1/2 strength MS medium, but the rooting percentage was improved in the presence of 0.2 mg l^–1 NAA. Rooted plantlets were acclimatized to greenhouse conditions. The young plantlets from tissue culture were transplanted successfully. In vitro propagation can be a useful tool in the conservation of this endangered medicinal species.     

植物抗坏血酸过氧化物酶研究进展(综述)

本文从酶的作用机制、酶学特征、分子生物学等方面综述植物抗坏血酸过氧化物酶（APX）的研究进展。     

Cytosolic Superoxide Dismutase (SOD1) Is Critical for Tolerating the Oxidative Stress of Zinc Deficiency in Yeast

Zinc deficiency causes oxidative stress in many organisms including the yeast Saccharomyces cerevisiae. Previous studies of this yeast indicated that the Tsa1 peroxiredoxin is required for optimal growth in low zinc because of its role in degrading H₂O₂. In this report, we assessed the importance of other antioxidant genes to zinc-limited growth. Our results indicated that the cytosolic superoxide dismutase Sod1 is also critical for growth under zinc-limiting conditions. We also found that Ccs1, the copper-delivering chaperone required for Sod1 activity is essential for optimal zinc-limited growth. To our knowledge, this is the first demonstration of the important roles these proteins play under this condition. It has been proposed previously that a loss of Sod1 activity due to inefficient metallation is one source of reactive oxygen species (ROS) under zinc-limiting conditions. Consistent with this hypothesis, we found that both the level and activity of Sod1 is diminished in zinc-deficient cells. However, under conditions in which Sod1 was overexpressed in zinc-limited cells and activity was restored, we observed no decrease in ROS levels. Thus, these data indicate that while Sod1 activity is critical for low zinc growth, diminished Sod1 activity is not a major source of the elevated ROS observed under these conditions.     

Ascorbate Peroxidase from Leishmania major Controls the Virulence of Infective Stage of Promastigotes by Regulating Oxidative Stress

Background

Peroxidase represents a heterogeneous group of distinct enzyme family that plays extremely diverse biological functions. Ascorbate peroxidase from Leishmania major (LmAPX) has been shown to be central to the redox defense system of Leishmania. To investigate further its exact physiological role in Leishmania, we attempted to create LmAPX -knockout mutants by gene replacement in L. major strains.

Methodology/Principal Findings

The null mutant cell culture contains a higher percentage of metacyclic and apoptotic cells compared to both wild type and LmAPX overexpressing cells. Flowcytometric analysis reveals the presence of a higher concentration of intracellular H₂O₂, indicative of increased oxidative stress in parasites lacking LmAPX. IC₅₀ value for exogenously added H₂O₂ shows that deletion of LmAPX in L. major renders the cell more susceptible to H₂O₂. Real time PCR studies demonstrate an elevated mRNA level of non-selenium glutathione peroxidase in LmAPX null mutant cell line, suggesting that these enzymes were induced to compensate the LmAPX enzyme. The null mutant cells exhibit hypervirulence after infection with macrophages as well as inoculation into BALB/c mice; in contrast, overexpressing cells show avirulence.

Conclusions/Significance

Collectively, these data provide strong evidence that LmAPX is an important factor for controlling parasite differentiation and survival within macrophages.     

Coimmobilization of Superoxide Dismutase,Catalase, and Peroxidase

Various orders of sequential coimmobilization of superoxide dismutase (SOD), catalase, and horseradish peroxidase (HRP) were tested in order to prepare a multienzyme antioxidant complex of these enzymes. Simultaneous coimmobilization of catalase with a preliminarily cross-linked complex between SOD and HRP was found to be the optimum procedure. The catalytic enzyme activity and working stability of catalase was tested kinetically in the multienzyme complexes prepared by different methods. The effects of ascorbic acid, glutathione, and ethanol on the kinetic parameters of catalase were studied. A possible scheme of H₂O₂ degradation catalyzed by coimmobilized SOD, catalase, and HRP in the presence of reducing substrates is suggested.     

Malondialdehyde,Bcl-2, Superoxide Dismutase and Glutathione Peroxidase may Mediate the Association of Sonic Hedgehog Protein and Oxidative Stress in Autism

Sonic hedgehog signaling and brain-derived neurotrophic factor play a neuro-protective role against oxidative stress in autism. Sonic hedgehog also increases Bcl-2 expression and the activities of superoxide dismutase and glutathione peroxidase. The level or activity of Bcl-2, brain-derived neurotrophic factor, and the activities of superoxide dismutase and glutathione peroxidase are decreased in autism. Sonic hedgehog also decreases the production of malondialdehyde that its level is high in autism. Therefore, it is supposed that sonic hedgehog may be associated with oxidative stress in autism through other pathways too.     

Induction of Superoxide Dismutase by Molecular Oxygen

Oxygen induces superoxide dismutase in Streptococcus faecalis and in Escherichia coli B. S. faecalis grown under 20 atm of O(2) had 16 times more of this enzyme than did anaerobically grown cells. In the case of E. coli, changing the conditions of growth from anaerobic to 5 atm of O(2) caused a 25-fold increase in the level of superoxide dismutase. Induction of this enzyme was a response to O(2) rather than to pressure, since 20 atm of N(2) was without effect. Induction of superoxide dismutase was a rapid process, and half of the maximal level was reached within 90 min after N(2)-grown cells of S. faecalis were exposed to 20 atm of O(2) at 37 C. S. faecalis did not contain perceptible levels of catalase under any of the growth conditions investigated by Stanier, Doudoroff, and Adelberg (23), and the concentration of catalase in E. coli was not affected by the presence of O(2) during growth. S. faecalis, which had been grown under 100% O(2) and which therefore contained an elevated level of superoxide dismutase, was more resistant of 46 atm of O(2) than were cells which had been grown under N(2). E. coli grown under N(2) contained as much superoxide dismutase as did S. faecalis grown under 1 atm of O(2). The E. coli which had been grown under N(2) was as resistant to the deleterious effects of 50 atm of O(2) as was S. faecalis which had been grown under 1 atm of O(2). These results are consistent with the proposal that the peroxide radical is an important agent of the toxicity of oxygen and that superoxide dismutase may be a component of the systems which have been evolved to deal with this potential toxicity.     

Separate Assays Specific for Ascorbate Peroxidase and Guaiacol Peroxidase and for the Chloroplastic and Cytosolic Isozymes of Ascorbate Peroxidase in Plants

Ascorbate (AsA) peroxidase can be inactivated both by p-chloromercuribenzoateand by the depletion of AsA but guaiacol peroxidases, such ashorseradish peroxidase, cannot. The cytosolic isozymes of AsAperoxidase are less sensitive to depletion of AsA than the chloroplasticisozymes, which include stromal [Chen and Asada (1989) PlantCell Physiol. 30: 987] and thyla-koid-bound [Miyake and Asada(1992) Plant Cell Physiol. 33: 541] enzymes. Exploring theseproperties, we established simple methods for separate assaysof AsA peroxidase and guaiacol peroxidase and of the three isozymesof AsA peroxidase in plant extracts. These methods were usedto characterize the guaiacol peroxidases and isozymes of AsAperoxidase in plants and algae. (Received October 20, 1993; Accepted February 7, 1994)     

DNA-Triggered Aggregation of Copper,Zinc Superoxide Dismutase in the Presence of Ascorbate

The oxidative damage hypothesis proposed for the function gain of copper, zinc superoxide dismutase (SOD1) maintains that both mutant and wild-type (WT) SOD1 catalyze reactions with abnormal substrates that damage cellular components critical for viability of the affected cells. However, whether the oxidative damage of SOD1 is involved in the formation of aggregates rich in SOD1 or not remains elusive. Here, we sought to explore the oxidative aggregation of WT SOD1 exposed to environments containing both ascorbate (Asc) and DNA under neutral conditions. The results showed that the WT SOD1 protein was oxidized in the presence of Asc. The oxidation results in the higher affinity of the modified protein for DNA than that of the unmodified protein. The oxidized SOD1 was observed to be more prone to aggregation than the WT SOD1, and the addition of DNA can significantly accelerate the oxidative aggregation. Moreover, a reasonable relationship can be found between the oxidation, increased hydrophobicity, and aggregation of SOD1 in the presence of DNA. The crucial step in aggregation is neutralization of the positive charges on some SOD1 surfaces by DNA binding. This study might be crucial for understanding molecular forces driving the protein aggregation.     

Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H₂O₂ or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca²⁺ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H₂O₂-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H₂O₂- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H₂O₂ or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.In multicellular organisms, mitochondria are the major physiological source of reactive oxygen species (ROS) within cells and also are important checkpoints for the control of programmed cell death (27). There are increasing numbers of reports that describe apoptosis- or programmed cell death-like processes in unicellular organisms also, such as trypanosomatids (4, 60), bacteria (20, 25), yeasts (34), and Plasmodium (3). Among the kinetoplastid parasites, Trypanosoma and Leishmania are the most carefully studied genera where apoptotic features are well established (49). Several reports have shown that mitochondrial dysfunction or an imbalance of antioxidant homeostasis causes an increase in mitochondrion-generated ROS, which include H₂O₂, superoxide radical anions, singlet oxygen, and hydroxyl radicals. These species have all been implicated in apoptosis (16, 26, 28, 41). Increasing evidence has been presented to support that ROS homeostasis regulates two major types of important physiological processes and exerts diverse functions within cells. One type of function includes damage or oxidation of cellular macromolecules (DNA, proteins, and lipids), which can lead to necrotic cell death or protein modification (7). The second type of function includes the activation of cellular signaling cascades that regulate proliferation, detoxification, DNA repair, or apoptosis (11). The detoxification of toxic mitochondrial ROS in cells occurs through a variety of cellular antioxidant enzymes, such as superoxide dismutase, which detoxifies cells from superoxide released into the mitochondrial matrix, and several other antioxidant proteins, such as catalase, glutathione (GSH) peroxidase, and peroxiredoxins, which are known to catalyze further degradation of H₂O₂ (44). During its life cycle, the Leishmania sp. encounters a pool of ROS that is generated either by its own physiological processes or as a result of host immune reaction and drug metabolism. However, unlike most eukaryotes, Leishmania lacks catalase- and selenium-containing GSH peroxidases, enzymes that play a front-line role in detoxifying ROS. Hence, the mechanism by which it resists the toxic effects of H₂O₂ remains poorly understood.Recently, we cloned, expressed and characterized the unusual heme-containing ascorbate peroxidase from Leishmania major (LmAPX) and observed that the expression of LmAPX is increased when Leishmania cells are treated with exogenous H₂O₂ (1, 18). This enzyme is a functional hybrid between cytochrome c peroxidase and APX, owing to its ability to use both ascorbate and cytochrome c as reducing electron donors (58). Colocalization studies by confocal microscopy, submitochondrial fractionation analysis of the isolated mitochondria, and subsequent Western blot analysis with anti-LmAPX antibody have confirmed that the mature enzyme is present in intermembrane space side of the inner membrane. It has also been shown that overexpression of LmAPX causes a decrease in the mitochondrial ROS burden, an increase in tolerance to H₂O₂, and protection against cardiolipin oxidation under oxidative stress (18). Although previous studies have shown that Leishmania species use superoxide dismutase (23), peroxiredoxins (8), intracellular thiols (14), lipophosphoglycan (13), trypanothione (5), HSP 70 (a heat shock protein) (36), tryparedoxin peroxidase (29), and APX (18) for detoxification of ROS, it is still unclear how the antioxidants protect against oxidative stress-induced apoptotic events in the unicellular organism Leishmania.Since the LmAPX protein is localized in the mitochondria, we hypothesized that it would be a key protein for the maintenance of mitochondrial functions due to its antioxidant properties via its ROS-scavenging function (18). To test this hypothesis, we overexpressed LmAPX in Leishmania major cells and investigated whether overexpression of LmAPX can confer resistance to oxidant-mediated mitochondrial damage as well as oxidative stress-induced cell death. In this study, we provide evidence that the overexpression of LmAPX in Leishmania cells can indeed protect against camptothecin (CPT) or H₂O₂-mediated mitochondrial damage as measured by various parameters, including disruption of mitochondrial membrane potential (Δψ_m), decrease of ATP production, and cytochrome c and endonuclease G release from mitochondria. Cells overexpressing LmAPX were also protected against oxidative stress-induced protein carbonylation, DNA fragmentation, and apoptosis. To the best of our knowledge, this is the first report of a mitochondrial hemeperoxidase that controls the ROS-induced mitochondrial death pathway.     

Bursopentin (BP5) Protects Dendritic Cells from Lipopolysaccharide-Induced Oxidative Stress for Immunosuppression

Dendritic cells (DCs) play a vital role in the regulation of immune-mediated inflammatory diseases. Thus, DCs have been regarded as a major target for the development of immunomodulators. However, oxidative stress could disturb inflammatory regulation in DCs. Here, we examined the effect of bursopentine (BP5), a novel pentapeptide isolated from chicken bursa of fabricius, on the protection of DCs against oxidative stress for immunosuppression. BP5 showed potent protective effects against the lipopolysaccharide (LPS)-induced oxidative stress in DCs, including nitric oxide, reactive oxygen species and lipid peroxidation. Furthermore, BP5 elevated the level of cellular reductive status through increasing the reduced glutathione (GSH) and the GSH/GSSG ratio. Concomitant with these, the activities of several antioxidative redox enzymes, including glutathione peroxidase (GPx), catalase (CAT) and superoxide dismutase (SOD), were obviously enhanced. BP5 also suppressed submucosal DC maturation in the LPS-stimulated intestinal epithelial cells (ECs)/DCs coculture system. Finally, we found that heme oxygenase 1 (HO-1) was remarkably upregulated by BP5 in the LPS-induced DCs, and played an important role in the suppression of oxidative stress and DC maturation. These results suggested that BP5 could protect the LPS-activated DCs against oxidative stress and have potential applications in DC-related inflammatory responses.     

Inhibition of Cell Growth by Overexpression of Manganese Superoxide Dismutase (MnSOD) in Human Pancreatic Carcinoma

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res.63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.     

Structural Evidence for a Copper-Bound Carbonate Intermediate in the Peroxidase and Dismutase Activities of Superoxide Dismutase

Copper-zinc superoxide dismutase (SOD) is of fundamental importance to our understanding of oxidative damage. Its primary function is catalysing the dismutation of superoxide to O₂ and H₂O₂. SOD also reacts with H₂O₂, leading to the formation of a strong copper-bound oxidant species that can either inactivate the enzyme or oxidise other substrates. In the presence of bicarbonate (or CO₂) and H₂O₂, this peroxidase activity is enhanced and produces the carbonate radical. This freely diffusible reactive oxygen species is proposed as the agent for oxidation of large substrates that are too bulky to enter the active site. Here, we provide direct structural evidence, from a 2.15 Å resolution crystal structure, of (bi)carbonate captured at the active site of reduced SOD, consistent with the view that a bound carbonate intermediate could be formed, producing a diffusible carbonate radical upon reoxidation of copper. The bound carbonate blocks direct access of substrates to Cu(I), suggesting that an adjunct to the accepted mechanism of SOD catalysed dismutation of superoxide operates, with Cu(I) oxidation by superoxide being driven via a proton-coupled electron transfer mechanism involving the bound carbonate rather than the solvent. Carbonate is captured in a different site when SOD is oxidised, being located in the active site channel adjacent to the catalytically important Arg143. This is the probable route of diffusion from the active site following reoxidation of the copper. In this position, the carbonate is poised for re-entry into the active site and binding to the reduced copper.