Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Metabolic analysis of the synthesis of high levels of intracellular human SOD in Saccharomyces cerevisiae rhSOD 2060 411 SGA122

The synthesis of human superoxide dismutase (SOD) in batch cultures of a Saccharomyces cerevisiae strain using a glucose-limited minimal medium was studied through metabolic flux analysis. A stoichiometric model was built, which included 78 reactions, according to metabolic pathways operative in these strains during respirofermentative and oxidative metabolism. It allowed calculation of the distribution of metabolic fluxes during diauxic growth on glucose and ethanol. Fermentation profiles and metabolic fluxes were analyzed at different phases of diauxic growth for the recombinant strain (P+) and for its wild type (P-). The synthesis of SOD by the strain P+ resulted in a decrease in specific growth rate of 34 and 54% (growth on glucose and ethanol respectively) in comparison to the wild type. Both strains exhibited similar flux of glucose consumption and ethanol synthesis but important differences in carbon distribution with biomass/substrate yields and ATP production 50% higher in P-. A higher contribution of fermentative metabolism, with 64% of the energy produced at the phosphorylation level, was observed during SOD production. The flux of precursors to amino acids and nucleotides was higher in the recombinant strain, in agreement with the higher total RNA and protein levels. Lower specific growth rates in strain P+ appear to be related to the decrease in the rate of synthesis of nonrecombinant protein, as well as a decrease in the activities of the pentose phosphate (PP) pathway and TCA cycle. A very different way of entry into the stationary phase was observed for each strain: in the wild-type strain most metabolic fluxes decreased and fluxes related to energy reserve synthesis increased, while in the P+ strain the flux of 22 reactions (including PP pathway and amino acids biosynthesis) related to SOD production increased their fluxes. Changes in SOD production rates at different physiological states appear to be related to the differences in building blocks availability between respirofermentative and oxidative metabolism. Using the present expression system, ideal conditions for SOD synthesis are represented by either active growth during respirofermentative metabolism or transition from a growing to a nongrowing state. An increase in SOD flux could be achieved using an expression system nonassociated to growth and potentially eliminating part of the metabolic burden.     

大鼠多囊卵巢颗粒细胞凋亡时MDA含量及SOD活性的变化

目的和方法以大鼠多囊卵巢（PCO）为动物模型,观察了PCO大鼠卵泡颗粒细胞凋亡的发生率,并检测了颗粒细胞凋亡时丙二醛（MDA）的含量及超氧化物歧化酶（SOD）活性的变化。结果①PCO颗粒细胞凋亡发生率明显高于正常（P<0．001）;②PCO颗粒细胞发生凋亡时,细胞内MDA含量增加而SOD活性降低。结论大鼠PCO颗粒细胞的凋亡可能与细胞内MDA含量增加和SOD活性降低有关。     

Improved Human Sperm Recovery Using Superoxide Dismutase and Catalase Supplementation in Semen Cryopreservation Procedure

The aim of this work was to evaluate the effects of ROS scavenger supplementation in human semen samples undergoing cryopreservation procedures.After screening out andrological pathologies, we selected 25 male partners of infertile couples with the following semen profile: volume >/= 2.0 ml, normal viscosity, sperm count >/=20 x 10(6)/ml, straight progressive motility (classes 1 and 2) >/= 40% (Mazzilli, Rossi, Delfino and Nofroni (1999) Andrologia 31: 187-194), atypical forms SOD) was added to the second, 200 U/ml of catalase to the third and both SOD (100 U/ml) and catalase (100 U/ml) were added to the fourth aliquot. Each aliquot was mixed (v/v) with TEST yolk buffer freezing medium (Irvine Scientific) and then frozen at -196 degrees C. The percent recovery of progressive motile and swollen spermatozoa was evaluated after thawing.No significant variation in the recovery of progressive motility was seen in the aliquots with added SOD or catalase alone, compared to the control group. On the other hand, a significant improvement in sperm parameter recovery was seen in the aliquot with both SOD and catalase supplementation; perhaps because of their combined and simultaneous action on superoxide anion and hydrogen peroxide. These results suggest that, in some selected cases, SOD and catalase supplementation can contribute greatly to the prevention of sperm membrane lipid peroxidation by ROS and thus allow good sperm parameter recovery after freezing-thawing procedures.     

Manganese supplementation relieves the phenotypic deficits seen in superoxide-dismutase-null Escherichia coli

Escherichia coli, lacking cytoplasmic superoxide dismutases, exhibits a variety of oxygen-dependent phenotypic deficits. Enrichment of the growth medium with Mn(II) relieved those deficits. Extracts of cells grown on Mn(II)-rich medium exhibited superoxide dismutase-like activity that was due partially to low-molecular-weight and partially to high-molecular-weight complexes. The high-molecular-weight activity was sensitive to proteolysis. Hence this activity is likely associated with low-affinity binding of Mn to proteins.     

Molecular evolution and structure--function relationships of the superoxide dismutase gene families in angiosperms and their relationship to other eukaryotic and prokaryotic superoxide dismutases

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.     

X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration

Mutations in the superoxide dismutase 1 (SOD1) gene cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS). An apoptotic process including caspase-1 and -3 has been shown to participate in the pathogenesis of FALS transgenic (Tg) mouse model. Here we report that IAP proteins, potent inhibitors of apoptosis, are involved in the FALS Tg mouse pathologic process. The levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein were significantly decreased in the spinal cord of symptomatic G93A-SOD1 Tg mice compared with littermates. In contrast, the levels of cIAP-1 mRNA and protein were increased in symptomatic G93A-SOD1 Tg mice, whereas the levels of cIAP-2 mRNA and protein were unchanged. In situ hybridization showed that the expression of XIAP was remarkably reduced in the motor neurons of Tg mice, and the expression of cIAP-1 was strongly increased in the reactive astrocytes of Tg mice. Overexpression of XIAP markedly inhibited the cell death and caspase-3 activity in the neuro2a cells expressing mutant SOD1. Deletional mutant analysis revealed that the N-terminal domain of XIAP, the BIR1-2 domains, was essential for this inhibitory activity. These results suggest that XIAP plays a role in the apoptotic mechanism in the progression of disease in mutant SOD1 Tg mice and holds therapeutic possibilities for FALS.     

Dietary Zn deficiency does not influence systemic blood pressure and vascular nitric oxide signaling in normotensive rats

Because zinc (Zn) is an important component for cell protection against certain oxygen species, it has been suggested that Zn deficiency impairs the potent oxidant defense capacity, which is constitutively provided in the vascular system. However, the influence of dietary Zn deficiency on systemic blood pressure and vascular system is controversial and unclear. We therefore examine the effect of dietary Zn deficiency on systemic blood pressure, a potent superoxide scavenger, aortic Cu/Zn superoxide dismutase (SOD) activity, a most representative synthase of the endothelium-derived relaxing factor, and aortic endothelial nitric oxide synthase (eNOS) expression. Furthermore, the direct effects of intravenous administration of NOS inhibitor, N ^ω-nitro-l-arginine methyl ester (l-NAME), and a SOD mimetic compound, tempol, in normotensives were tested in Wistar-Kyoto (WKY) rats. A Zn-deficient diet (4 wk) contributed to growth retardation, the decrease in thymus weight, and the lower levels of serum Zn compared with the standard diet group. However, no significant difference in conscious systolic and diastolic blood pressure was found in the Zn-deficiency group. The administration of l-NAME caused an increase in the mean arterial pressure (MAP) levels in the two groups of rats and the involvement of the vasodilator nitric oxide (NO) in the regulation of systemic BP in the normotensive state. On the other hand, administration of the superoxide scavenger, tempol, led to a decrease in MAP levels in the two groups of rats, indicating the participation of the oxygen free radical, superoxide, in the maintenance of the systemic BP in a normotensive state. There were no significant differences between the Zn-deficient diet group and the standard diet group in the normotensive state. eNOS expression and Cu/Zn SOD activity in the aorta were also intact in Zn-deficient normotensive rats. These findings suggest that the 4 wk of Zn deficiency was inadequate to alter systemic blood pressure and focal NO signaling in the normotensive state. Long-term Zn deficiency affects the neuronal, immune, and hematopoietic systems, which contribute to systemic and/or local circulation. However, Zn deficiency alone does not cause hypertension and local vascular dysfunction in the normotensive state.     

Oxidative stress in Caenorhabditis elegans: protective effects of superoxide dismutase/catalase mimetics

The lifespan of Caenorhabditis elegans can be extended by the administration of synthetic superoxide dismutase/catalase mimetics (SCMs) without any effects on development or fertility. Here we demonstrate that the mimetics, Euk-134 and Euk-8, confer resistance to the oxidative stress-inducing agent, paraquat and to thermal stress. The protective effects of the compounds are apparent with treatments either during development or during adulthood and are independent of an insulin/IGF-I-like signalling pathway also known to affect thermal and oxidative stress resistance. Worms exposed to the compounds do not induce a cellular stress response and no detrimental effects are observed.     

The relationship between total sialic acid levels and antioxidant status in the tadpoles of Bufo viridis and Rana ridibunda ridibunda

Total sialic acid levels (TSA), antioxidant enzymes activities such as superoxide dismutase (SOD), catalase (CAT) and lipid peroxidation (LPO) levels were investigated during the developmental period in tadpoles of the predominantly terrestrial amphibian B. viridis and the predominantly aquatic amphibian R. r. ridibunda. Maximum TSA levels were observed in B. viridis and R. r. ridibunda at the fifth and third week of their development, respectively. SOD and CAT activity variations during development in B. viridis were higher than in R. r. ridibunda. Although SOD activity in B. viridis was higher than R. r. ridibunda at the eighth week, SOD activity increased 19.2-fold in R. r. ridibunda and 10.4-fold in B. viridis between the first and eighth week. CAT activity in R. r. ridibunda did not significantly change (p>0.001) until the fifth week then increased, whereas in B. viridis CAT increased after the third week. In contrast to the rise in the antioxidant enzyme activities, LPO levels tended to decrease during the developmental period. Levels of LPO showed a similar trend until the third week for both species. The minimum LPO levels in B. viridis and R. r. ridibunda were 23+/-1.2 and 146+/-7.3 nmol MDA g(-1) tissue, at the eighth week, respectively. While decreasing LPO levels correlated with increasing antioxidant enzyme activities, TSA tended to decrease after reaching a maximum point.