Increased Tolerance to Mn Deficiency in Transgenic Tobacco Overproducing Superoxide Dismutase

The effect of Mn deficiency on plant growth and activities ofsuperoxide dismutase (SOD) was studied in hydroponically-grownseedlings of transgenic tobacco (Nicotiana tabacum L.) engineeredto overexpress FeSOD in chloroplasts or MnSOD in chloroplastsor mitochondria. In comparison to the non-transgenic parentalline, the activity of MnSOD in the lines overproducing MnSODwas 1.6-fold greater, and the activity of FeSOD in the FeSOD-overproducinglines was 3.2-fold greater, regardless of the Mn treatment (deficientor sufficient). The MnSOD activities decreased due to Mn deficiency,while activities of FeSOD and Cu/ZnSOD remained unaffected 25d after transplanting (DAT). With an increased duration of theMn deficiency stress (45 DAT), FeSOD activity decreased, andthat of MnSOD continued to decrease, while Cu/ZnSOD activitysimultaneously increased. Under Mn sufficiency, non-transgenicparental plants had greater shoot biomass than the transgenics;however, when subjected to Mn deficiency stress, non-transgenicparents suffered a proportionally greater growth reduction thantransgenic lines. Thus, overproduction of MnSOD in chloroplastsmay provide protection from oxidative stress caused by Mn deficiency.Copyright 1999 Annals of Botany Company Manganese deficiency, Nicotiana tabacum, superoxide dismutase (SOD), transgenic tobacco.     

Molecular Interaction Mechanism between 2-Mercaptobenzimidazole and Copper-Zinc Superoxide Dismutase

2-Mercaptobenzimidazole (MBI) is widely utilized as a corrosion inhibitor, copper-plating brightener and rubber accelerator. The residue of MBI in the environment is potentially harmful. In the present work, the toxic interaction of MBI with the important antioxidant enzyme copper-zinc superoxide dismutase (Cu/ZnSOD) was investigated using spectroscopic and molecular docking methods. MBI can interact with Cu/ZnSOD to form an MBI-Cu/ZnSOD complex. The binding constant, number of binding sites and thermodynamic parameters were measured, which indicated that MBI could spontaneously bind with Cu/ZnSOD with one binding site through hydrogen bonds and van der Waals forces. MBI bound into the Cu/ZnSOD interface of two subdomains, which caused some microenvironmental and secondary structure changes of Cu/ZnSOD and further resulted in the inhibition of Cu/ZnSOD activity. This work provides direct evidence at a molecular level to show that exposure to MBI could induce changes in the structure and function of the enzyme Cu/ZnSOD. The estimated methods in this work may be applied to probe molecular interactions of biomacromolecules and other pollutants and drugs.     

超氧物歧化酶活性与小麦对HSO3^—,NO2^—抗性之间的关系

用在培养液中加低浓度NO_2~-,HSO_3~-,Mn~(2 )及向地上部喷洒氯霉素等四种方法促使小麦苗超氧物歧化酶(SOD)活性增加。SOD活性提高后,植株叶片对高浓度NO_2~-,HSO_3~-的抗性增强,表现在因NO_2~-,HSO_3~-害而产生的膜脂过氧化产物乙烷减少,叶绿素分解减轻。低浓度NO_2~-处理后的麦苗,不仅提高了对高浓度NO_2~-的抗性,同时对HSO_3~-的抗性也增强;同样,低浓度HSO_3~-处理也同时促进了麦苗对HSO_3~-和NO_2~-的抗性,即可交叉地提高。这些结果直接说明SOD和抗性有关。同时也为SO_2/HSO_3~-,NO_2/NO_2~-害的自由基学说提供了一个佐证。     

Oxygen Toxicity and the Superoxide Dismutase

Oxygen caused an increase in the amount of superoxide dismutase in Escherichia coli B but not in Bacillus subtilis. E. coli B cells, induced by growth under 100% O(2), were much more resistant to the lethal effects of 20 atm of O(2) than were cells which contained the low uninduced level of this enzyme. In contrast, B. subtilis, which could not respond to O(2) by increasing its content of superoxide dismutase, remained equally sensitive to hyperbaric O(2) whether grown under 100% O(2) or areobically. The catalase in these organisms exhibited a reciprocal response to oxygen. Thus, the catalase of E. coli B was not induced by O(2), whereas that of B. subtilis was so induced. These results are consistent with the view that superoxide dismutase is an important component of the defenses of these organisms against the toxicity of oxygen, whereas their catalases are of secondary importance in this respect. The ability of streptonigrin to generate O(2) (-), by a cycle of reduction followed by spontaneous reoxidation, has been verified in vitro. It is further observed that E. coli B which contain the high induced level of superoxide dismutase were more resistant to the lethality of this antibiotic, in the presence of oxygen, than were E. coli B which contained the low uninduced level of this enzyme. This difference between induced and uninduced cells was eliminated by the removal of O(2). These results are consistent with the proposal that the enhanced lethality of streptonigrin under aerobic conditions may relate to its in vivo generation of O(2) (-) by a cycle of reduction and spontaneous reoxidation. In toto, these observations lend support to the hypothesis that O(2) (-) is an important agent of oxygen toxicity and that superoxide dismutase functions to blunt the threat posed by this reactive radical.     

Interleukin-6 Deficiency Does Not Affect Motor Neuron Disease Caused by Superoxide Dismutase 1 Mutation

MethodsMice with mutant SOD1 (G93A) transgene, a model for familial ALS, were used in this study. The expression of the major inflammatory cytokines, IL-6, IL-1β and TNF-α, in spinal cords of these SOD1 transgenic (TG) mice were assessed by real time PCR. Mice were then crossed with IL-6(-/-) mice to generate SOD1TG/IL-6(-/-) mice. SOD1 TG/IL-6(-/-) mice (n = 17) were compared with SOD1 TG/IL-6(+/-) mice (n = 18), SOD1 TG/IL-6(+/+) mice (n = 11), WT mice (n = 15), IL-6(+/-) mice (n = 5) and IL-6(-/-) mice (n = 8), with respect to neurological disease severity score, body weight and the survival. We also histologically compared the motor neuron loss in lumber spinal cords and the atrophy of hamstring muscles between these mouse groups.ResultsLevels of IL-6, IL-1β and TNF-α in spinal cords of SOD1 TG mice was increased compared to WT mice. However, SOD1 TG/IL-6(-/-) mice exhibited weight loss, deterioration in motor function and shortened lifespan (167.55 ± 11.52 days), similarly to SOD1 TG /IL-6(+/+) mice (164.31±12.16 days). Motor neuron numbers and IL-1β and TNF-α levels in spinal cords were not significantly different in SOD1 TG /IL-6(-/-) mice and SOD1 TG /IL-6 (+/+) mice.ConclusionThese results provide compelling preclinical evidence indicating that IL-6 does not directly contribute to motor neuron disease caused by SOD1 mutations.     

Association between Gene Polymorphism of Manganese Superoxide Dismutase and Prostate Cancer Risk

Manganese superoxide dismutase (MnSOD) is the most effective antioxidant enzyme in mitochondria and protects cells from reactive oxygen species‐induced oxidative damage. The aim of this study was to investigate the association between MnSOD Ala‐9Val gene polymorphism and prostate cancer (PCa) risk in Turkish men with prostate cancer. 33 patients with PCa and 81 control individuals were included in the study. We observed an association between MnSOD Ala/Ala frequency and a higher PCa risk. In addition, we found that the increased risk of early‐onset PCa (under age of 65) in the men homozygous for Ala allele was higher than the men homozygous for Val allele. However, we determined that MnSOD Ala‐9Val genotype was not associated with the aggressiveness of the disease. The results of our study suggest that MnSOD Ala/Ala genotype may influence on early‐onset of PCa patients, but no effect on subsequent development of the disease in Turkish men. However, our study has a limitation that is small numbers of individuals for cases and controls. Therefore, the presented study limited our statistical power to fully investigate the gene polymorphism on cancer risk. © 2013 Wiley Periodicals, Inc. J BiochemMol Toxicol 27:213‐218, 2013; View this article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21472     

Assay and Inhibitors of Spinach Superoxide Dismutase

Allosamidin, a product of Streptomyces sp. No 1713, inhibited Bombyx mori chitinase specifically in a competitive way with a Ki o f about 0.1 μm. The effect of allosamidin on chitinases from r Streptomyces griseus and Serratia marcescens was weaker, about 1/500 that on B. mori chitinase. Allosamidin did not inhibit yam chitinase, lysozymes of hen egg-white or human urine, or B. mori α-N-acetyl-d-glucosaminidase. The results suggest that allosamidin is a specific inhibitor of the insect chitinase.     

Human Bcl-2 Reverses Survival Defects in Yeast Lacking Superoxide Dismutase and Delays Death of Wild-Type Yeast

We expressed the human anti-apoptotic protein, Bcl-2, in Saccharomyces cerevisiae to investigate its effects on antioxidant protection and stationary phase survival. Yeast lacking copper-zinc superoxide dismutase (sod1Δ) show a profound defect in entry into and survival during stationary phase even under conditions optimal for survival of wild-type strains (incubation in water after stationary phase is reached). Expression of Bcl-2 in the sod1Δ strain caused a large improvement in viability at entry into stationary phase, as well as increased resistance to 100% oxygen and increased catalase activity. In addition, Bcl-2 expression reduced mutation frequency in both wild-type and sod1Δ strains. In another set of experiments, wild-type yeast incubated in expired minimal medium instead of water lost viability quickly; expression of Bcl-2 significantly delayed this stationary phase death. Our results demonstrate that Bcl-2 has activities in yeast that are similar to activities it is known to possess in mammalian cells: (a) stimulation of antioxidant protection and (b) delay of processes leading to cell death.     

Mechanism and Atomic Structure of Superoxide Dismutase

The active site Cu ion in Cu,Zn superoxide dismutase is alternately oxidized and reduced during the enzymatic dismutation of superoxide to hydrogen peroxide and molecular oxygen. For oxidized Cu,Zn superoxide dismutase, an atomic structure has been determined for the human enzyme at 2.5 A resolution. The resolution of the bovine enzyme structure has been extended to 1.8 A. Atomic resolution data has been, collected for reduced and inhibitor-bound Cu,Zn superoxide dismutases. and the interpretation of the' electron density difference maps is in progress. The geometry and molecular surfaces of the active sites in these structures, together with biochemical data, suggest a specific model for the enzyme mechanism. Similarities in the active site geometry of the Mn and Fe superoxide dismutases with the Cu.Zn enzyme suggest that dismutation in these enzymes may follow a similar mechanism.     

Developmental Analysis of Steady-State Levels of Cu/Zn and Mn Superoxide Dismutase mRNAs in Maize Tissues

The superoxide dismutase (Sod) steady-state mRNA levels in maizeseedlings and developing kernels were examined by RNA blot analysis,using Cu/Zn and Mn cDNA probes encoding the cytosolic and mitochondrialSOD isozymes, respectively. The cytosolic (Cu/Zn) Sod steady-statemRNA levels remained relatively constant for the various tissuesexamined. In contrast, the mRNA levels of the mitochondrial(Mn) SOD-3 isozyme increased in postgerminative scutella. Thesteady-state mRNA of the Atp2 gene, F₁ ATPase ß subunit,was compared to the Sod3 (Mn) mRNA levels. Results of this comparativestudy suggest that the steady-state levels of mRNAs transcribedby nuclear genes encoding essential mitochondrial proteins areindependently regulated throughout development. (Received April 2, 1990; Accepted September 10, 1990)     

Advances in the Understanding of the Structure-Function Relationship in Cu,Zn Superoxide Dismutase

The structure-function relationship in Cu,Zn superoxide dismutase has been partially elucidated by the combined use of many spectroscopic techniques (electronic spectroscopy, circular dichroism. EPR and NMR) and site-directed mutagenesis techniques. The comparison of the spectroscopic and catalytic properties of various mutants, in which some active site residues have been substituted through site-directed mutagenesis, allowed us to establish that the activity is in general more sensitive to electrostatic effects rather than to steric effects or changes in the copper hydration or coordination geometry.     

Superoxide Dismutase and Oxygen Toxicity in a Eukaryote

Saccharomyces cerevisiae var. ellipsoideus contained 6.5 times more superoxide dismutase and 2.3 times more catalase when grown under 100% O(2) than when grown anaerobically. Growth under oxygen caused equal increases in both the cyanide-sensitive and the cyanide-insensitive superoxide dismutases of this organism. Experience with other eukaryotes has shown that cyanide sensitivity is a property of the cupro-zinc superoxide dismutase of the cytosol, whereas cyanide insensitivity is a property of the corresponding mangani-enzyme found in mitochondria. Cu(2+), which has been shown to increase the radioresistance of yeast, also caused an increase of both of the superoxide dismutases of S. cerevisiae. Yeast which had been grown under 1 atm of O(2) were more resistant toward the lethal effects of 20 atm of O(2) than were yeast which had been grown in the absence of O(2). Escherichia coli K-12 his(-) responded to growth under 1 atm of O(2) by increasing its content of catalase and of peroxidase, but not of superoxide dismutase. This contrasts with E. coli B, which was previously shown to respond to O(2) by a striking increase in superoxide dismutase. E. coli K-12 his(-) did not gain resistance toward 20 atm of O(2) because of having been grown under 1 atm of O(2). Once again, this contrasts with the behavior of E. coli B. These data indicate that, in both prokaryotes and in eukaryotes, superoxide dismutase is an important component of the defenses against oxygen toxicity.     

Reduction of Cold Injury by Superoxide Dismutase and Catalase

The pathophysiology of cold injury was examined by cooling a hind leg of an anesthetized New Zealand white rabbit. A flow probe and a thermocouple were placed in the leg to be cooled to monitor the blood flow and tissue temperature. After baseline measurements, the leg was cooled with a freezing mixture up to 0°C. which was followed by rewarming. The other leg served as control. In the experimental group, liposome-bound superoxide dismutase and catalase were infused through the femoral vein 15 minutes prior to putting the freezing mixture on the leg. Salicylic acid was injected through the femoral vein at the end of some experiments to assay hydroxy radical (OH). Our results demonstrated reduction of local blood flow in cold-exposed leg, indicating development of ischemia. Creatine kinase and lactage dehydrogenase were increased during rewarming in conjunction with hydroxyl radical formation, phospholipid breakdown, and lipid peroxidation. Treatment with superoxide dismutase and catalase reduced OH formation, prevented phospholipid degradation, and decreased creatine kinase. lactate dehydrogenase. and malonaldehyde formation. These results indicate that rewarming of cooled tissue is associated with “rewarming injury” similar to “reperfusion injury”, and that oxygen-derived free radicals play a signidcant role in the pathophysiology of such injury.     

Changes in Expression of Manganese Superoxide Dismutase,Copper and Zinc Superoxide Dismutase and Catalase in Brachionus calyciflorus during the Aging Process

Rotifers are useful model organisms for aging research, owing to their small body size (0.1–1 mm), short lifespan (6–14 days) and the relative easy in which aging and senescence phenotypes can be measured. Recent studies have shown that antioxidants can extend the lifespan of rotifers. In this paper, we analyzed changes in the mRNA expression level of genes encoding the antioxidants manganese superoxide dismutase (MnSOD), copper and zinc SOD (CuZnSOD) and catalase (CAT) during rotifer aging to clarify the function of these enzymes in this process. We also investigated the effects of common life-prolonging methods [dietary restriction (DR) and resveratrol] on the mRNA expression level of these genes. The results showed that the mRNA expression level of MnSOD decreased with aging, whereas that of CuZnSOD increased. The mRNA expression of CAT did not change significantly. This suggests that the ability to eliminate reactive oxygen species (ROS) in the mitochondria reduces with aging, thus aggravating the damaging effect of ROS on the mitochondria. DR significantly increased the mRNA expression level of MnSOD, CuZnSOD and CAT, which might explain why DR is able to extend rotifer lifespan. Although resveratrol also increased the mRNA expression level of MnSOD, it had significant inhibitory effects on the mRNA expression of CuZnSOD and CAT. In short, mRNA expression levels of CAT, MnSOD and CuZnSOD are likely to reflect the ability of mitochondria to eliminate ROS and delay the aging process.     

鸭血超氧化物歧化酶(SOD)的纯化及性质研究

超氧化物歧化酶(E、C、1.15.1.1)是催化超氧阴离子起歧化反应的金属酶类,血红细胞中的SOD属Cu Zn—SOD。本文详细报道了从鸭血分离纯化SOD的改进方法(同时用猪血作对比研究)。设计了热变性、(NH_4)_2SO_4分级监析、低浓乙醇—氯仿短时去除残留血红蛋白、凝胶层析四步纯化方案,获得高产率电泳纯SOD。用紫外扫描,PAGE电泳后考马斯亮蓝和SOD活性杂色,SDS—电泳,HPLC分离和各电泳带组分的N—端测定等技术检测纯度,并进行性质研究。证明所得SOD是均一的;N—端为Ala;分子量和亚分子量各为32,359和16,500dt;并与猪血SOD对比研究其某些性质;对HPLC出现的多个活力峰及PAGE电泳显现的多条活性带进行了讨论。     

Catalase and Superoxide Dismutase of Root-Colonizing Saprophytic Fluorescent Pseudomonads

Root-colonizing, saprophytic fluorescent pseudomonads of the Pseudomonas putida-P. fluorescens group express similar levels of catalase and superoxide dismutase activities during growth on a sucrose- and amino acid-rich medium. Increased specific activities of catalase but not superoxide dismutase were observed during growth of these bacteria on components washed from root surfaces. The specific activities of both enzymes were also regulated during contact of these bacteria with intact bean roots. Increased superoxide dismutase and decreased catalase activities were observed rapidly, by 10 min upon inoculation of cells onto intact bean roots. Catalase specific activity increased with time to peak at 12 h before declining. By 48 h, the cells displayed this low catalase but maintained high superoxide dismutase specific activities. Catalase with a low specific activity and a high superoxide dismutase activity also were present in extracts of cells obtained from 7-day-old roots colonized from inoculum applied to seed. This specific activity of superoxide dismutase of root-contacted cells was about fourfold-higher in comparison to cells grown on rich medium, whereas the specific activity for catalase was reduced about fivefold. A single catalase isozyme, isozyme A, and one isozyme of superoxide dismutase, isozyme 1, were detected during growth of the bacteria on root surface components and during exposure of cells to intact bean roots for 1 h. An additional catalase, isozyme B, was detected from bacteria after exposure to the intact bean roots for 12 h. Catalase isozyme A and superoxide dismutase isozyme 1 were located in the cytoplasm and catalase band B was located in the membrane of P. putida.     

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.     

Superoxide Dismutase from Lens esculenta: Purification and Properties

Superoxide dismutase has been purified to homogeneity from Lens esculenta cotyledons and shoots. The two forms appeared to be identical. The purified enzyme contained two electrophoretically distinct bands. It contained two ions of Cu and two ions of Zn. Gel filtration experiments indicate a molecular weight of about 33,000. The spectrum of ultraviolet and visible regions and electron paramagnetic resonance were similar to those of Cu-Zn mammalian superoxide dismutase.