Heparin, A Potent Releasing Agent of Extracellular Superoxide Dismutase (EC-SOD C), Suppresses Ischaemic Paw Oedema in Mice

Heparin (2,000 U/kg, i.v.) increases the plasma superoxide dismutase (SOD) activity by 2-3 times after 5min. followed by a gradual decrease. A high dose of heparin (4 × 10³ and 10 × 10³U/kg) exhibits a lower increase in the release of SOD. Ischaemic paw oedema in mice was suppressed by various types of SOD and heparin also suppresses this oedema. The dose-dependent curve of heparin of oedema suppression corresponds well with the increased plasma level of SOD. Inducibility with heparin, slow clearance from the bloodstream and blocking of oedema suppression by the copper chelator, diethyldithiocarbamate (DDC), suggest that the oedema suppressing SOD was the extracellular (EC)-SOD C. Other anticoagulants such as citrate and EDTA had no effect. Chondroitin sulphate A and C or carrageenan exhibited weak suppression. A complex of EC-SOD C and heparin appears not to bind to the endothelium in contrast to the injected free EC-SOD C. When heparin is re-injected, more than 1 week was required to get the same degree of oedema suppression. This indicates the necessity of newly synthesized enzyme. A biological role for heparin-induced release of plasma SOD is demonstrated for the first time in this investigation.     

小胸鳖甲胞外铜锌超氧化物歧化酶重组蛋白的原核表达及多克隆抗体制备

超氧化物歧化酶(SOD)是清除生物体内超氧阴离子自由基的主要抗氧化酶家族。基于原核表达系统,成功表达了拟步甲科Tenebrionidae小胸鳖甲Micordera punctipennis胞外铜锌SOD的重组蛋白(本文定义为Trx-His-MpecCu/Zn-SOD)。经Ni 2+亲和层析法纯化重组蛋白后,研究了重组蛋白的部分酶学性质。通过足垫加皮下注射法3次免疫小鼠后,分别用ELISA和Western blot的方法检测抗体效价和抗体特异性。结果表明,重组蛋白主要以包涵体形式存在,纯化后的重组蛋白浓度为1.33 mg·mL^-1,酶活力为27.52 U·mg^-1。Trx-His-MpecCu/Zn-SOD在25~45℃具有比较稳定的酶活性,在35℃最高,同时表现出比较广泛的酸碱耐受性(pH3~12),最适pH为9.0,表明重组蛋白的酶活性相对比较稳定。蛋白免疫法制备的鼠抗MpecCu/Zn-SOD多克隆抗体滴度高于1∶819 200。Western blot结果显示,该抗体能免疫结合重组蛋白Trx-His-MpecCu/Zn-SOD和小胸鳖甲体内天然MpecCu/Zn-SOD,但不能与黄粉虫Tenebrio molitor的总蛋白结合,说明制备的抗体效价较高且特异性较好。本研究结果为小胸鳖甲ecCu/Zn-SOD功能的深入研究奠定了基础。     

Role of Superoxide Dismutase in Ischemic Brain Injury: A Study Using SOD-1 Transgenic Mice

1. Nitric oxide radicals (NO) play an important role in the pathophysiology of focal cerebral ischemia.2. Vascular NO can reduce ischemic brain injury by increasing CBF, whereas neuronal NO may mediate neurotoxicity following brain ischemia, mainly by its reaction with superoxide to generate peroxynitrite.3. These findings could contribute to a strategy for the treatment of cerebral ischemia.     

Suppressions of Ischemic Paw Oedema in Mice, Rats and Guinea Pigs by Superoxide Dismutases from Different Sources

Various sources of superoxide dismutases (SOD) suppressed ischaemic paw oedemata (tourniquet poditis) of mice, rats and guinea pigs with different potencies. Intravenous (i.v.) dosing of mouse Cu, Zn-SOD had no effect on mouse ischaemic oedema, yet rat and guinea pig Cu, Zn-SOD suppressed ischaemic oedemata of rats and guinea pigs. Homologous SOD was anti-inflammatory at least in these two models. Guinea pig SOD was one of the most potent in all models, but showed a very narrow range of effective dose. This bell-shape suppressive pattern was ameliorated by concomitant catalase injection. Bovine and human Cu, Zn-SOD had a rather broad range of effective dose. Bacterial Mn-SODS were suppressive in mice, as well as the oxygen radical scavenger MK-447 and cytochrome c. Dexamethasone was effective only when administered more than 3 hrs in advance. As ischaemic paw oedema of mice was not sensitive to cyclooxy-genase and lipoxygenase inhibitors, this model could serve for screening new types of anti-inflammatory or anti-ischaemic drugs.     

Suppressions of Ischemic Paw Oedema in Mice,Rats and Guinea Pigs by Superoxide Dismutases from Different Sources

Various sources of superoxide dismutases (SOD) suppressed ischaemic paw oedemata (tourniquet poditis) of mice, rats and guinea pigs with different potencies. Intravenous (i.v.) dosing of mouse Cu, Zn-SOD had no effect on mouse ischaemic oedema, yet rat and guinea pig Cu, Zn-SOD suppressed ischaemic oedemata of rats and guinea pigs. Homologous SOD was anti-inflammatory at least in these two models. Guinea pig SOD was one of the most potent in all models, but showed a very narrow range of effective dose. This bell-shape suppressive pattern was ameliorated by concomitant catalase injection. Bovine and human Cu, Zn-SOD had a rather broad range of effective dose. Bacterial Mn-SODS were suppressive in mice, as well as the oxygen radical scavenger MK-447 and cytochrome c. Dexamethasone was effective only when administered more than 3 hrs in advance. As ischaemic paw oedema of mice was not sensitive to cyclooxy-genase and lipoxygenase inhibitors, this model could serve for screening new types of anti-inflammatory or anti-ischaemic drugs.     

Superoxide Dismutase, Catalase, and Glutathione Peroxidase Activities in Copper/Zinc-Superoxide Dismutase Transgenic Mice

Copper/zinc-superoxide dismutase (CuZn-SOD) transgenic mice overexpress the gene for human CuZn-SOD. To assess the effects of the overexpression of CuZn-SOD on the brain scavenging systems, we have measured the activities of manganese-SOD (Mn-SOD), catalase, and glutathione peroxidase (GSH-Px) in various regions of the mouse brain. In nontransgenic mice, cytosolic CuZn-SOD activity was highest in the caudate-putamen complex; this was followed by the brainstem and the hippocampus. The lowest activity was observed in the cerebellum. In transgenic mice, there were significant increases of cytosolic CuZn-SOD activity in all of these regions, with ratios varying from a twofold increase in the brainstem to 3.42-fold in the cerebellum in comparison with nontransgenic mice. Particulate Mn-SOD was similarly distributed in all brain regions, and its levels also were significantly increased in superoxide dismutase (SOD)-transgenic mice. In the brains of nontransgenic mice, cytosolic catalase activity was similar in all brain regions except the cortex, which showed less than 50% of the activity observed in the other regions. In transgenic mice, cytosolic catalase activity was significantly increased, with the cortex showing the greatest changes (133%) in comparison with nontransgenic mice. The smallest increases were observed in the hippocampus (34%). In contrast to what was observed for SOD and catalase, there were no significant changes in cytosolic GSH-Px activity in any of the brain regions examined. The present results indicate that, in addition to displaying marked increases in the levels of brain CuZn-SOD activity, SOD-transgenic mice also exhibit increases in other enzymes that scavenge oxygen-based radicals.(ABSTRACT TRUNCATED AT 250 WORDS)     

CuZn-Superoxide Dismutase, Extracellular Superoxide Dismutase, and Glutathione Peroxidase in Blood from Individuals Homozygous for Asp90Ala CuZn-Superoxide Dismutase Mutation

Abstract: The Asp⁹⁰Ala CuZn-superoxide dismutase mutation is associated with amyotrophic lateral sclerosis (ALS) in both homo- and heterozygous form. We analyzed antioxidant enzymes in blood from 44 individuals homozygous and 114 individuals heterozygous for the Asp⁹⁰Ala mutation as well as 66 blood relatives carrying the wild-type allele only. Erythrocyte CuZn-superoxide dismutase activity was reduced by 9% in the homozygous individuals, confirming previous findings on a smaller cohort. The specific activity of Asp⁹⁰Ala mutant CuZn-superoxide dismutase in erythrocytes was equal to that of isolated mutant enzyme and slightly higher than that of isolated wild-type enzyme. There was no evidence for the presence of inactive mutant molecules in erythrocytes, and the lower activity is due to the occurrence of fewer active molecules. There were no significant differences between the groups in plasma extracellular superoxide dismutase content, and the erythrocyte glutathione peroxidase activities were virtually identical. Also, there were no differences in these parameters between homozygous individuals with or without ALS. There was no evidence for any association with ALS of a polymorphic extracellular superoxide dismutase mutation, Arg²¹³Gly. The absence of response of the blood antioxidant enzymes to the Asp⁹⁰Ala CuZn-superoxide dismutase mutation does not support the theory that the ALS-linked CuZn-superoxide dismutase mutations cause disease by increased oxidant stress.     

Cloning,Purification, and Characterization of Recombinant Human Extracellular Superoxide Dismutase in SF9 Insect Cells

A balance between production and degradation of reactive oxygen species (ROS) is critical for maintaining cellular homeostasis. Increased levels of ROS during oxidative stress are associated with disease conditions. Antioxidant enzymes, such as extracellular superoxide dismutase (EC-SOD), in the extracellular matrix (ECM) neutralize the toxicity of superoxide. Recent studies have emphasized the importance of EC-SOD in protecting the brain, lungs, and other tissues from oxidative stress. Therefore, EC-SOD would be an excellent therapeutic drug for treatment of diseases caused by oxidative stress. We cloned both the full length (residues 1–240) and truncated (residues 19–240) forms of human EC-SOD (hEC-SOD) into the donor plasmid pFastBacHTb. After transposition, the bacmid was transfected into the Sf9-baculovirus expression system and the expressed hEC-SOD purified using FLAG-tag. Western blot analysis revealed that hEC-SOD is present both as a monomer (33 kDa) and a dimer (66 kDa), as detected by the FLAG antibody. A water-soluble tetrazolium (WST-1) assay showed that both full length and truncated hEC-SOD proteins were enzymatically active. We showed that a potent superoxide dismutase inhibitor, diethyldithiocarbamate (DDC), inhibits hEC-SOD activity.     

Expression of Human Cu-Zn Superoxide Dismutase Gene in Transgenic Mice: Model for Gene Dosage Effect in Down Syndrome

It was suggested that increased Cu-Zn superoxide dismutase (SOD-I) might be involved in the various biological abnormalities found in Down's syndrome (DS) such as premature aging and Alzheimer-type neurological lesions. As a model system for testing this hypothesis we have developed two strains of transgenic mice carrying only one copy of the human SOD-I gene. In the first strain (TGI). no expression has been found by northern blot analysis. The second strain (TG2) exhibited human SOD-I mRNA and increased SOD-l activity in the brain (1.93 fold), in the heart (I.69 fold), thymus (I.49 fold) and to a lesser extent in muscle (1.25 fold), liver (1.19 fold), kidney (1.18 fold), spleen (1.35 fold), lung (1.26 fold) and erythrocytes (1.09 fold). In this strain, increased SOD-1 activity in the brain did not induce modifications in the seleno-dependent glutathione peroxidase. glutathione reductase and glutathione S-transferase activities. In brain homogenates, we have focused our studies on Tau proteins which are known to be the major antigenic components of paired helical filaments (P11F). both in DS and Alzheimer's disease. Our results suggested that, in our experimental conditions, the overexpression of SOD-I did not induce the modifications of Tau proteins similar to those seen during neurofibrillary degeneration.     

3-Nitropropionic Acid Neurotoxicity Is Attenuated in Copper/Zinc Superoxide Dismutase Transgenic Mice

Abstract: The mitochondrial toxin 3-nitropropionic acid (3-NP) produces selective striatal lesions in both experimental animals and humans. The pathogenesis of the lesions involves secondary excitotoxicity that may then lead to free radical generation. To test this further we examined the effects of 3-NP in both transgenic (Tg) mice that carry the complete sequence for the human copper/zinc superoxide dismutase (SOD) gene as well as non-Tg littermate controls. The Tg-SOD mice showed a pronounced attenuation of Nissl-stained striatal lesions compared with non-Tg mice. Systemic administration of 3-NP resulted in production of hydroxyl free radicals as assessed by the conversion of salicylate to 2,3- and 2,5-dihydroxybenzoic acid. This production was attenuated significantly in Tg-SOD mice. In a similar way, 3-NP produced significant increases in 3-nitrotyrosine/tyrosine, a marker for peroxynitrite-mediated damage, which were significantly attenuated in Tg-SOD mice. These results support that oxygen free radicals and peroxynitrite play an important role in the pathogenesis of 3-NP neurotoxicity.     

Expression of Human Cu-Zn Superoxide Dismutase Gene in Transgenic Mice: Model for Gene Dosage Effect in Down Syndrome

It was suggested that increased Cu-Zn superoxide dismutase (SOD-I) might be involved in the various biological abnormalities found in Down's syndrome (DS) such as premature aging and Alzheimer-type neurological lesions. As a model system for testing this hypothesis we have developed two strains of transgenic mice carrying only one copy of the human SOD-I gene. In the first strain (TGI). no expression has been found by northern blot analysis. The second strain (TG2) exhibited human SOD-I mRNA and increased SOD-l activity in the brain (1.93 fold), in the heart (I.69 fold), thymus (I.49 fold) and to a lesser extent in muscle (1.25 fold), liver (1.19 fold), kidney (1.18 fold), spleen (1.35 fold), lung (1.26 fold) and erythrocytes (1.09 fold). In this strain, increased SOD-1 activity in the brain did not induce modifications in the seleno-dependent glutathione peroxidase. glutathione reductase and glutathione S-transferase activities. In brain homogenates, we have focused our studies on Tau proteins which are known to be the major antigenic components of paired helical filaments (P11F). both in DS and Alzheimer's disease. Our results suggested that, in our experimental conditions, the overexpression of SOD-I did not induce the modifications of Tau proteins similar to those seen during neurofibrillary degeneration.     

Purification, N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Purification,N-Terminal Amino Acid Sequence and Partial Characterization of A Cu,Zn Superoxide Dismutase From the Pathogenic Fungus Aspergillusfumiga Tus

A superoxide dismutase (SOD) has been purified to homogeneity from the fungal pathogen Aspergillus fumigatus using a combination of cell homogenization, isoelectric focusing and gel filtration FPLC. The N-terminal amino acid sequence of the purified enzyme demonstrated substantial homology to known Cu, Zn superoxide dismutases for a range of organisms, including Neurospora crassa and Saccharomyces cerevisiae. The enzyme subunit has a pl of 5.9, a relative molecular mass of 19 kDa and a spectral absorbance maximum of 550nm. The non reduced enzyme has a relative molecular mass of 95 kDa. The enzyme remained active after prolonged incubation at 70°C and was pH insensitive in the range 7-11. Potassium cyanide and diethyldithiocarbamate, known Cu, Zn SOD inhibitors, caused inhibition of the purified enzyme at working concentrations of 0.25 mM, whilst sodium azide and o-phenanthroline demonstrated inhibition at higher concentrations (10-30 mM). SOD activity was also detectable in culture filtrate of A. fumigatus. This enzyme may have a potential role as a virulence factor in the avoidance of neutrophil and phagocyte oxidative burst killing mechanisms.     

Purification of an Iron-Containing Superoxide Dismutase from A Citrus Plant,Citrus Limonum R

A cyanide-insensitive superoxide dismutase was purified to apparent homogeneity from lemon leaves (Citrus limonum R). The enzyme was isolated from leaf extracts by ammonium sulfate salting-out. and ion-exchange, gel filtration and hydroxylapatite column chromatography. The purified Fe-SOD had a specific activity of about 1.500 U/mg and represents approximately 1.6% of the total soluble protein in lemon leaf extracts. A molecular weight of 47,500 was determined for the enzyme. Analytical gel electro-focusing of the purified preparation revealed the presence of two isozymes with pl values of 5.13 and 4.98. Metal analysis showed the presence of I g-atom of iron and 0.5g-atom of manganese per mol of enzyme. The visible and UV absorption spectra of the Citrus enzyme were similar to those reported for other iron-containing SODS from different origins. The significance of the presence of Fe-SOD in higher plants is briefly discussed.     

Catalase and Superoxide Dismutase: Distribution, Properties, and Physiological Role in Cells of Strict Anaerobes

This review considers the distribution of the main enzymes of antioxidative defense, superoxide dismutase (SOD) and catalase, in various groups of strictly anaerobic microorganisms: bacteria of the genus Clostridium, Bacteroides, sulfate-reducing and acetogenic bacteria, methanogenic archaea, etc. Molecular and biochemical properties of purified Fe-containing SODs, cambialistic SODs, and heme catalases are presented. The physiological role and origin of the enzymes of antioxidative defense in strict anaerobes are discussed. Physiological responses (induction of SOD and catalase) to factors provoking oxidative stress in the cells of strict anaerobes able to maintain viability under aerobic conditions are also considered.     

Overexpression of Glia Maturation Factor in C6 Cells Promotes Differentiation and Activates Superoxide Dismutase

In order to evaluate the intracellular function of glia maturation factor (GMF), we overexpressed GMF in C6 rat glioma cells using two methods: stable transfection using the pcDNA3 plasmid, and transient transfection using replication-defective human adenovirus. With both methods, C6 cells transfected with GMF and overexpressing the protein exhibit a lower saturation density in culture compared to non-transfected or vector alone controls. Transfected cells also exhibit morphological differentiation as shown by the outgrowth of cell processes. When inoculated into nude mice, transfected cells are less tumorigenic than controls, and express the mature astrocytic marker glial fibrillary acidic protein. In tissue culture, transfected cells show a 3.5-fold increase in CuZn-dependent superoxide dismutase (CuZnSOD) activity. Western blot analysis reveals a 3.5-fold increase in CuZnSOD protein, suggesting an induction of the enzyme. In view of recent findings that reactive oxygen species (ROS) and the antioxidant enzymes are intricately involved in key physiologic processes such as proliferation, differentiation and apoptosis, the study raises the possibility that CuZnSOD may be a mediator of GMF function.     

A bioluminescent transgenic mouse model: Real-time in vivo imaging of antioxidant EC-SOD gene expression and regulation by interferon gamma

Extracellular superoxide dismutase (EC-SOD) is the main antioxidant enzyme in the extracellular matrix. We developed transgenic mice to analyze the EC-SOD promoter activity in vivo in real time and to identify the important cis-elements flanking the 5′ region of the murine EC-SOD gene. Using this model, we demonstrated that luciferase reporter activity correlates closely with endogenous EC-SOD expression, although several interesting differences were also observed. Specifically, luciferase activity was detected at the highest levels in testes, aorta and perirenal fat. Reporter expression was regulated by interferon gamma, a finding that is in agreement with published endogenous EC-SOD gene expression studies. Thus, the 5′-flanking region of mouse EC-SOD gene is responsible, at least in part, for cell specific and inducible expression.     

靶向抑制铜伴侣蛋白CCS逆转卵巢癌C13*细胞的顺铂耐药性

顺铂（cisplatin）,即二胺二氯铂/diaminedichloroplatinum（DDP）,是治疗卵巢癌最有效的化疗药物;然而,耐药性是限制顺铂临床治疗效果的最重要因素。目前,卵巢癌对顺铂的耐药机制仍不十分清楚。超氧化物歧化酶1铜伴侣蛋白（copper chaperone for superoxide dismutase 1, CCS）介导Cu2+特异性传递给超氧化物歧化酶1（superoxide dismutase1, SOD1）,为维持细胞增殖和生存所必需;相反,抑制CCS可减缓肿瘤细胞增殖。本研究旨在证明,AMPK依赖的CCS表达与卵巢癌的顺铂耐药有关。实时定量PCR及免疫印迹结果显示,与顺铂敏感细胞株OV2008比较,顺铂耐药细胞株C13*中的CCS mRNA和蛋白质表达水平明显上调。shRNA靶向沉默CCS或采用抑制剂DC_AC50抑制CCS后,可明显增强顺铂对C13*细胞增殖的抑制作用,提示CCS高表达与顺铂耐药相关,而抑制CCS可逆转顺铂的耐药性。同样,免疫印迹结果证明,CCS在A549、H1944等6种不同肺癌细胞中的表达水平高低与顺铂敏感性密切相关。采用siRNA干扰CCS在A549细胞中的高表达,或在CCS低表达的H1944细胞中过表达CCS,可明显增加A549或减弱H1944细胞对顺铂的敏感性,进一步证明CCS表达与顺铂耐药性相关。此外,采用AMPK抑制剂化合物C阻断AMPKα Thr172磷酸化（激活）,即抑制AMPK信号通路,可明显抑制CCS在C13*细胞中的表达,提高其对顺铂的敏感性。以上研究结果提示,AMPK信号通路依赖的CCS表达参与肿瘤的顺铂耐药机制,而抑制CCS逆转顺铂耐药。本文的结果还提示,CCS有望成为克服顺铂耐药的潜在靶点。     

Superoxide Dismutase and Unribosylated Cytokinins Interact in the Control of NAD(P)H-Peroxidation

NAD(P)H is rapidly oxidized in the presence of peroxidase, a substituted monophenol such as p-coumaric acid, and Mn²⁺ ions. As recently reported by Miller (1985), this NAD(P)H oxidation at the expense of molecular oxygen is inhibited by submicromolar concentrations of Cu²⁺ ions. This inhibition by cupric ions is counteracted by micromolar concentrations of cytokinins. We now show that NAD(P)H-oxidation by the above system is under the control of superoxide dismutase, where in the absence of cytokinin-copper, superoxide dismutase stimulates NAD(P)H-oxidation. In the presence of unribosylated cytokinins and copper, however, superoxide dismutase acts as an inhibitor. Thus cytokinins and superoxide dismutase may interact in the control of the redox state of plant cells.