Zinc Superoxide Dismutase Research Articles

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

BackgroundAmyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1.Methodology/Principal FindingsAs revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn2-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu2+ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu2+: a moderate-affinity site (106 M-1) and a high-affinity site (108 M-1). Furthermore, Cu2+ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form.Conclusions/SignificanceWe demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu2+ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.

The characterization, expression and activity analysis of superoxide dismutases (SODs) from Procambarus clarkii

The superoxide dismutases (SODs) are important antioxidant enzymes to remove excess biologically reactive oxygen intermediates, and thus avoid adverse conditions. In this study, an extracellular copper–zinc superoxide dismutase (ecCuZnSOD) gene and a mitochondrial manganese superoxide dismutase (mtMnSOD) gene were cloned from hemocytes of the freshwater crayfish, Procambarus clarkii. The open reading frame (ORF) of ecCuZnSOD is 612bp encoding a 204 amino acid (aa) protein with a 24 aa signal peptide, whereas the ORF of mtMnSOD is 654bp and encodes a 218 aa protein with a 20 aa mitochondrial-targeting sequence in the N-terminus. P. clarkii ecCuZnSOD and mtMnSOD proteins showed high similarity with ecCuZnSODs and mtMnSODs from other crustaceans, respectively. Both ecCuZnSOD and mtMnSOD of P. clarkii were highly expressed in hepatopancreas, hemocytes and gill; lower expression levels were seen in intestine, stomach, nerve, heart and muscle. The mRNA expressions of three SODs of P. clarkii (ecCuZnSOD, mtMnSOD and cytosolic MnSOD (cytMnSOD)) were studied after Spiroplasma eriocheiris and Aeromonas hydrophila stimulations. After S. eriocheiris challenge, the mRNAs of three SODs in hemocytes and gill were up-regulated, and ecCuZnSOD also increased in the hepatopancreas. However, the mtMnSOD and cytosolic MnSOD in the hepatopancreas were down-regulated. After A. hydrophila challenge, the mRNA expressions of three SODs in hepatopancreas and hemocytes were up-regulated and down-regulated in gill. The total SOD activity and CuZnSOD activity were increased compared to the control group with both S. eriocheiris and A. hydrophila challenges. The results may indicate that the SODs of P. clarkii have important roles in innate immune responses against S. eriocheiris and A. hydrophila.

A Novel Superoxide Dismutase from Cicer arietinum L. Seedlings: Isolation, Purification and Characterization

Superoxide dismutase is an important enzyme with various therapeutic applications. Search of a new source of superoxide dismutase with novel properties has significant importance. The current work reports purification of a novel superoxide dismutase enzyme with unique characteristics. A copper zinc superoxide dismutase (Cu-Zn SOD) was purified and characterized from Cicer arietinum L. seedlings germinated under aluminium (Al+3) stress. The specific activity of purified protein was 158 units/mg with 28 fold purification. The superoxide dismutase is a homodimeric protein with approx subunit molecular weight of 33.27 kDa. The enzyme is identified as Cu-Zn category of superoxide dismutase, reflected by H2O2 induced inhibition of in-gel activity and presence of quantifiable copper and zinc ions. The optimum pH range for purified Cu-Zn SOD activity was observed within 6.5-8.5 (highest at pH 8.0) and the pH stability was in the range of 6.0-8.5. The enzyme was more stable at low temperature (below 30°C) and the Km of purified Cu-Zn SOD for riboflavin as substrate was 10.16 ± 2.5 µM. The N-terminal amino acid sequence showed homology at conserved residues with other plant Cu-Zn SODs.

Fullerenol C60(OH)24 nanoparticles decrease relaxing effects of dimethyl sulfoxide on rat uterus spontaneous contraction

Dimethyl sulfoxide (DMSO) is a widely used solvent and cryoprotectant that can cause impaired blood flow, reduction in intracranial pressure, tissue edema, inflammatory reactions, inhibition of vascular smooth muscle cell migration and proliferation, processes which can lead to atherosclerosis of the coronary, peripheral and cerebral circulation. Although the adverse effects are rare when DMSO is administered in clinically established concentrations, there is no safe antagonist for an overdose. In this work, we treated isolated spontaneous and calcium-induced contractile active rat uteri (Wistar, virgo intacta), with DMSO and fullerenol C60(OH)24 nanoparticle (FNP) in DMSO. FNP is a water-soluble derivative of fullerene C60. Its size is a 1.1 nm in diameter and is a very promising candidate for a drug carrier in nanomedicine. FNP also displays free radical scavenging activity. DMSO decreased both spontaneous and calcium-induced contractions. In contrast, FNP only decreased spontaneous contraction. FNP decreased copper–zinc superoxide dismutase activity and prevented the DMSO-induced increase in glutathione reductase activity. Atomic force microscopy detected that FNP aggregated with calcium ions. Our results indicate that FNP has properties that make it a good candidate to be a modulator of DMSO activity which could minimize side effects of the latter.

Effect of Hyperoxia on Resuscitation of Experimental Combined Traumatic Brain Injury and Hemorrhagic Shock in Mice

Hypotension and hypoxemia worsen traumatic brain injury outcomes. Hyperoxic resuscitation is controversial. The authors proposed that hyperoxia would improve hemodynamics and neuronal survival by augmenting oxygen delivery despite increased oxidative stress and neuroinflammation in experimental combined controlled cortical impact plus hemorrhagic shock in mice. Adult C57BL6 mice received controlled cortical impact followed by 35 min of hemorrhagic shock (mean arterial pressure, 25-27 mmHg). The resuscitation phase consisted of lactated Ringer's boluses titrated to mean arterial pressure greater than 70 mmHg. Definitive care included returning shed blood. Either oxygen or room air was administered during the resuscitation phases. Brain tissue levels of oxidative stress and inflammatory markers were measured at 24 h and hippocampal neuronal survival was quantified at 7 days. Hyperoxia markedly increased brain tissue oxygen tension approximately four- to fivefold (n = 8) and reduced resuscitation fluid requirements approximately 15% (n = 53; both P < 0.05). Systemic and cerebral physiologic variables were not significantly affected by hyperoxia. Hippocampal neuron survival was approximately 40% greater with oxygen versus room air (n = 18, P = 0.03). However, ascorbate depletion doubled with oxygen versus room air (n = 11, P < 0.05). Brain tissue cytokines and chemokines were increased approximately 2- to 20-fold (n = 10) after combined controlled cortical impact injury plus hemorrhagic shock, whereas hyperoxia shifted cytokines toward a proinflammatory profile. Hyperoxic resuscitation of cortical impact plus hemorrhagic shock reduced fluid requirements and increased brain tissue oxygen tension and hippocampal neuronal survival but exacerbated ascorbate depletion and neuroinflammation. The benefits of enhanced oxygen delivery during resuscitation of traumatic brain injury may outweigh detrimental increases in oxidative stress and neuroinflammation.

Low-Dose Angiotensin II Infusion Restores Vascular Function in Cerebral Arteries of High Salt-Fed Rats by Increasing Copper/Zinc Superoxide Dimutase Expression

This study examined the vasoprotective role of circulating angiotensin II (ANG II) levels in the cerebral circulation of high salt (HS)-fed (SS.BN-(D13hmgc41-13hmgc23)/Mcwi) (Ren1-BN) congenic rats, which carry a normally functioning renin allele from the Brown Norway (BN) rat on the Dahl salt-sensitive genetic background. Ren1-BN rats were placed on an HS (4.0% NaCl) diet for 3 days. The vasodilator response to acetylcholine (ACh; 10(-10) - 10(-6) mol/L) was assessed in isolated middle cerebral arteries (MCAs), and Western blots were performed to assess the expression of the antioxidant enzymes copper (Cu)/zinc (Zn) superoxide dismutase (SOD) and manganese (Mn) SOD in cerebral resistance vessels. A separate group of HS-fed animals were infused with either a subpressor dose of ANG II (100ng/kg/min) or saline vehicle via osmotic minipump for 3 days. HS diet eliminated acetylcholine (ACh)-induced dilation in the MCAs of the congenic rats. Western blot analysis of antioxidant enzymes showed that Cu/Zn SOD and Mn SOD expression were significantly reduced in the cerebral resistance arteries of the HS-fed rats compared with control animals fed a normal salt diet. Infusion of ANG II restored the vasodilator response to ACh in the MCAs and increased Cu/Zn SOD (but not Mn SOD) expression compared with saline-infused animals. These results indicate that prevention of salt-induced ANG II suppression prevents vascular dysfunction in the cerebral circulation by preventing the downregulation of Cu/Zn SOD and vascular oxidant stress that normally occurs with HS diet.

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.

Effect of dietary copper on the growth performance, non-specific immunity and resistance to Aeromonas hydrophila of juvenile Chinese mitten crab, Eriocheir sinensis

An 8-week feeding trial was conducted to determine the dietary copper (Cu) on growth performance and immune responses of juvenile Chinese mitten crab Eriocheir sinensis. Six semi-purified diets with six copper levels (1.88, 11.85, 20.78, 40.34, 79.56 and 381.2 mg kg−1 diet) of CuSO4·5H2O were fed to E. sinensis (0.45 ± 0.01 g). Each diet was fed to the crab in five replicates. The crab fed diets with 20.78 and 40.34 mg Cu kg−1 diet had significantly greater weight gain and hemolymph oxyhemocyanin content than those fed diets with 1.88 and 381.2 mg Cu kg−1 diet. Survival rates of crab were not significantly different between all treatment groups. The activities of copper–zinc superoxide dismutase (Cu–Zn SOD), phenoloxidase (PO), and total hemocyte count (THC) significantly increased when the supplementation of dietary copper reached 20.78–40.34 mg Cu kg−1 diets. In the bacteria challenge experiment with Aeromonas hydrophila, survival rates significantly increased and reached a plateau when the dietary copper increased from 1.88 to 40.34 mg kg−1, whereas significantly decreased when the dietary copper increased from 40.34 to 381.2 mg kg−1. This study indicates that the level of dietary copper is important in regulating growth and immune response in crab.

Copper–zinc superoxide dismutase-deficient mice show increased susceptibility to experimental autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein 35–55

In this report, we have addressed the role of copper–zinc superoxide dismutase (SOD1) deficiency in the mediation of central nervous system autoimmunity. We demonstrate that SOD1-deficient C57Bl/6 mice develop more severe autoimmune encephalomyelitis induced with myelin oligodendrocyte glycoprotein (MOG) 35–55, compared with wild type mice. This alteration in the disease phenotype was not due to aberrant expansion of MOG-specific T cells nor their ability to produce inflammatory cytokines; rather lymphocytes generated in SOD1-deficient mice were more prone to spontaneous cell death when compared with their wild type littermate controls. The data point to a role for SOD1 in the maintenance of self-tolerance leading to the suppression of autoimmune responses.

Superoxide Dismutase Gene Polymorphisms in Patients with Age-related Cataract

ABSTRACTBackground: Functional polymorphisms in genes encoding antioxidant enzymes may result in reduced enzyme activity and increased levels of reactive oxygen species, such as superoxide radicals, which in turn may contribute to increased risk of age-related disorders. Copper–zinc superoxide dismutases, SOD-1 and SOD-3, and manganese superoxide dismutase, SOD-2, are enzymes involved in the protection against oxidative stress and detoxification of superoxide. In this study, we investigated a number of disease-associated single nucleotide polymorphisms (SNPs) of SOD1, SOD2 and SOD3, in patients with age-related cataract.Materials and methods: The study included an Estonian sample of 492 patients with age-related cataract, subgrouped into nuclear, cortical, posterior subcapsular and mixed cataract, and 185 controls. Twelve SNPs in SOD1, SOD2 and SOD3 were genotyped using TaqMan Allelic Discrimination. Haplotype analysis was performed on the SNPs in SOD2.Results: None of the studied SNPs showed an association with risk of cataract. These results were consistent after adding known risk factors (age, sex and smoking) as covariates in the multivariate analyses and after stratification by cataract subtype. Analysis of SOD2 haplotypes did not show any associations with risk of cataract.Conclusions: If genetic variation in genes encoding SOD-1, SOD-2 and SOD-3 contributes to cataract formation, there is no major contribution of the SNPs analyzed in the present study.

Optimized Methods for In Vitro and In Vivo Anti-Inflammatory Assays and Its Applications in Herbal and Synthetic Drug Analysis

Inflammatory diseases including, different types of rheumatic diseases are the major problems associated with the presently available non-steroidal anti-inflammatory agents. The numbers of plant derived drugs have been screened for their anti-inflammatory and anti-arthritic activity. Drug development in the recent times often relies on use of natural and synthetic drugs, which are promising candidates as therapeutic agents for prevention of diseases and disorders. These drugs possess different chemical structures, with wide range of therapeutic activities. The mechanism of Inflammation mainly involve in development of serious diseases, such as cancer, rheumatoid arthritis, sprains, bronchitis, muscle pains, chronic inflammatory bowel disease, persistent asthma, and liver fibrosis. Development of inflammatory events basically related to various chemicals, such as glucocorticoids (GCs) and mometasone furoate (MF); endogenous factors such as tumor necrosis factor alpha (TNF-α); enzymes and proteins such as copper and zinc-superoxide dismutase (SOD), proinflammatory peptide substance (PPS), RGD peptides, interleukin-4 (IL-4), IL-10, interferon-γ (IFN-γ), COX, LOX, cytokines such as interleukin-1 (IL-1); reactive oxygen species (ROS), nitric oxide (NO) and prostaglandin E2; as well as pro-inflammatory cells such as T and NK cells are well known to have an important role. Based on these correlations, numerous assays were used for inflammatory mechanism research, which was described in this paper.

A Study of Oxidative Stress Biomarkers and Effect of Oral Antioxidant Supplementation in Severe Acute Malnutrition

Malnutrition represents one of the most severe health problems in India. Free radicals play an important role in immunological response, which induces the oxidative surplus in severe acute malnutrition. Severe dietary deficiency of nutrients leads to increased oxidative stress in cellular compartments. The goal of this study was to inspect impact of oxidative stress in the form of serum malondialdehyde as product of lipid peroxidation, vitamin E, zinc and erythrocyte superoxide dismutase in patients with severe acute malnutrition. Sixty severe acute malnutrition patients were studied before and after supplementation of antioxidants for one month, and their status were compared with those of 60 age and sex matched healthy controls. The level of serum MDA was analyzed by the Kei Satoh method, serum vitamin E concentration was measured by Baker and Frank Method, serum zinc was measured by using Atomic Absorption Spectrophotometer (AAS) and erythrocyte superoxide dismutase was measured by Kajari Das Method. Significantly increased levels of serum malondialdehyde (p<0.001) were found in the patients as compared to those in controls, and significant depletions were found in the levels of serum vitamin E, zinc and erythrocyte superoxide dismutase in patients with severe acute malnutrition as compared to those in controls. After supplementation of antioxidants for one month, the levels of malondialdehyde were found to be decreased significantly (p<0.001) and zinc and erythrocyte superoxide dismutase capacity levels were increased significantly (p<0.05). Also, there was a non-significant (p>0.05) increase in vitamin E levels as compared to those before supplementation results. Harsh deficiency of various nutrients in severe acute malnutrition leads to generation of heavy oxidative stress. These effects may be minimized with supplementation of antioxidants.

Protection of HeLa cells against ROS stress by CuZnSOD mimic system.

A new copper zinc complex (CZpbi), [(bipyridinyl)2Cu-μ-pbi-Zn(pbi)](ClO4)2, pbi = 2-(2-pyridyl)-benzimidazole, was synthesized to mimic copper zinc superoxide dismutase (CuZnSOD). CZpbi was then encapsulated onto a dye (fluorescein isothiocyanate, FITC) functionalized mesoporous silica nanoparticles (MSN) where MSN serves as a nanofactory platform for delivering the catalytic action of the center copper complex. We examined the inhibition of oxidative damage of HeLa cells in the presence of paraquat (PQ, 1,1'-dimethyl-4,4'-bipyridylium dichloride, an oxidative stressor). We further studied the cell viability and cell cycle process under the influence of paraquat stress in the presence of the SOD-mimic FITC-MSN-CZpbi material by flow cytometry and western blot test. We found the nano-sized FITC-MSN is an excellent nano-carrier for delivering the mimic SOD complexes to HeLa cells. Here the reactants are already inside the cell and the result is a promotion of cell function. We present the methodology of encapsulating bio-inspired SOD-mimic in FITC-MSN to achieve a better drug delivery vehicle and catalytic (detoxification) activity. The potency and the stability of SOD-mimic FITC-MSN-CZpbi may provide a new avenue in the future development of artificial antioxidant enzymes.

Optimized Methods for In Vitro and In Vivo Anti-Inflammatory Assays and Its Applications in Herbal and Synthetic Drug Analysis

Inflammatory diseases including, different types of rheumatic diseases are the major problems associated with the presently available non-steroidal anti-inflammatory agents. The numbers of plant derived drugs have been screened for their anti-inflammatory and anti-arthritic activity. Drug development in the recent times often relies on use of natural and synthetic drugs, which are promising candidates as therapeutic agents for prevention of diseases and disorders. These drugs possess different chemical structures, with wide range of therapeutic activities. The mechanism of Inflammation mainly involve in development of serious diseases, such as cancer, rheumatoid arthritis, sprains, bronchitis, muscle pains, chronic inflammatory bowel disease, persistent asthma, and liver fibrosis. Development of inflammatory events basically related to various chemicals, such as glucocorticoids (GCs) and mometasone furoate (MF); endogenous factors such as tumor necrosis factor alpha (TNF-α); enzymes and proteins such as copper and zinc-superoxide dismutase (SOD), proinflammatory peptide substance (PPS), RGD peptides, interleukin-4 (IL-4), IL-10, interferon- λ (IFN- λ), COX, LOX, cytokines such as interleukin-1 (IL-1); reactive oxygen species (ROS), nitric oxide (NO) and prostaglandin E2; as well as pro-inflammatory cells such as T and NK cells are well known to have an important role. Based on these correlations, numerous assays were used for inflammatory mechanism research, which was described in this paper. Keywords: Herbal drugs, Synthetic drugs, Anti-Inflammatory assays

Virtual electrochemical nitric oxide analyzer using copper, zinc superoxide dismutase immobilized on carbon nanotubes in polypyrrole matrix

In this work, we have designed and developed a novel and cost effective virtual electrochemical analyzer for the measurement of NO in exhaled breath and from hydrogen peroxide stimulated endothelial cells using home-made potentiostat. Here, data acquisition system (NI MyDAQ) was used to acquire the data from the electrochemical oxidation of NO mediated by copper, zinc superoxide dismutase (Cu,ZnSOD). The electrochemical control programs (graphical user-interface software) were developed using LabVIEW 10.0 to sweep the potential, acquire the current response and process the acquired current signal. The Cu,ZnSOD (SOD1) immobilized on the carbon nanotubes in polypyrrole modified platinum electrode was used as the NO biosensor. The electrochemical behavior of the SOD1 modified electrode exhibited the characteristic quasi-reversible redox peak at the potential, +0.06V vs. Ag/AgCl. The biological interferences were eliminated by nafion coated SOD1 electrode and then NO was measured selectively. Further, this biosensor showed a wide linear range of response over the concentration of NO from 0.1μM to 1mM with a detection limit of 0.1μM and high sensitivity of 1.1μAμM−1. The electroanalytical results obtained here using the developed virtual electrochemical instrument were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.

Overexpression of a protein disulfide isomerase-like protein from Methanothermobacter thermoautotrophicum enhances mercury tolerance in transgenic rice

MTH1745, from thermophilic archaea Methanothermobacter thermoautotrophicum, is a protein disulfide isomerase-like protein (PDIL) with a chaperone function and disulfide isomerase activity. Mercuric cations have a high affinity for sulfhydryl groups and consequently inhibit plant growth. Disulfide compounds (e.g., copper–zinc superoxide dismutase, Cu/Zn SOD) and sulfhydryl compounds (e.g., glutathione, phytochelatins, and metallothioneins) play important roles in mercury (Hg) response. To study the relationship between Hg detoxification and PDILs, we overexpressed MTH1745 in Oryza sativa L. cv. Nipponbare by Agrobacterium-mediated transformation. The transgenic rice seedlings displayed Hg tolerance with obvious phenotypes and more effective photosynthesis compared to wild-type plants. Furthermore, lower levels of superoxide anion radicals, hydrogen peroxide, and malondialdehyde were observed in leaves or roots of transgenic plants. Antioxidant enzyme activities of superoxide dismutase and peroxidase were notably higher in transgenic seedlings under different concentrations of mercuric chloride. Moreover, increased content of non-protein thiols, reduced glutathione (GSH), and GSH/GSSG (GSSG, oxidized glutathione) ratio were also observed in the detoxification of Hg. These results indicated that heterologous expression of a PDIL from extremophiles in rice could protect the synthesis, increase stability of proteins, and enhance Hg tolerance in rice.

Impaired enzymatic antioxidant defense in erythrocytes of women with general and abdominal obesity

The aim of this study was to investigate erythrocyte antioxidant enzyme activities in general and abdominal obese women. In this study, 160 women 20-45 years old were randomly selected. General information data were gathered from each sample using questionnaires and face-to-face interviews. Activities of erythrocyte antioxidant enzymes were also evaluated by measuring activities of copper zinc-superoxide dismutase (CuZn-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT) in selected subjects. Mean activity of CuZn-SOD was significantly lower in overweight and obese groups compared to normal weight group (721 ± 81 vs. 987 ± 84, p < 0.01 and 638 ± 74 vs. 987 ± 84, p < 0.001, respectively). Furthermore, both erythrocyte GSH-Px (97.6 ± 45.2 vs. 148.7 ± 54.2, p < 0.01) and CAT (184.2 ± 37.8 vs. 316.7 ± 65.8, p < 0.01) activities was significantly lower in obese women compared to normal weight women. In addition, women with abdominal obesity had lower erythrocyte CuZn-SOD (632 ± 76 vs. 871 ± 84, p < 0.001), GSH-Px (103.6 ± 52.8 vs. 154.7 ± 62.4, p < 0.01) and CAT (216.6 ± 76.4 vs. 298.3 ± 71.1, p < 0.05) activities compared to women with normal body fat distribution. Moreover, multiple linear regression analysis suggests higher prediction of waist circumference (i.e. higher R(2)) on antioxidant enzyme activities in relation to other adiposity indicators. Our results provides further evidence suggesting that obesity and, especially, abdominal adiposity associated with decreased antioxidant enzyme activities which in turn, may contribute to oxidative stress related to obesity.

Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein

Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP. Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser(1177) in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH(4)) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH(4) and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH(4) bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91(phox) and SODs, thereby reducing production of superoxide anion in the aortas. Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production.

Uncoupling of eNOS causes superoxide anion production and impairs NO signaling in the cerebral microvessels of hph‐1 mice

In this study, we used the GTP cyclohydrolase I-deficient mice, i.e., hyperphenylalaninemic (hph-1) mice, to test the hypothesis that the loss of tetrahydrobiopterin (BH(4)) in cerebral microvessels causes endothelial nitric oxide synthase (eNOS) uncoupling, resulting in increased superoxide anion production and inhibition of endothelial nitric oxide signaling. Both homozygous mutant (hph-1(-/-)) and heterozygous mutant (hph-1(+/-) mice) demonstrated reduction in GTP cyclohydrolase I activity and reduced bioavailability of BH(4). In the cerebral microvessels of hph-1(+/-) and hph-1(-/-) mice, increased superoxide anion production was inhibited by supplementation of BH(4) or NOS inhibitor- L- N(G) -nitro arginine-methyl ester, indicative of eNOS uncoupling. Expression of 3-nitrotyrosine was significantly increased, whereas NO production and cGMP levels were significantly reduced. Expressions of antioxidant enzymes namely copper and zinc superoxide dismutase, manganese superoxide dismutase, and catalase were not affected by uncoupling of eNOS. Reduced levels of BH(4), increased superoxide anion production, as well as inhibition of NO signaling were not different between the microvessels of male and female mice. The results of our study are the first to demonstrate that, regardless of gender, reduced BH(4) bioavailability causes eNOS uncoupling, increases superoxide anion production, inhibits eNOS/cGMP signaling, and imposes significant oxidative stress in the cerebral microvasculature.

Molecular characterization and expression analysis of extracellular copper/zinc superoxide dismutase (ecCuZnSOD) from oriental river prawn, Macrobrachium nipponense

Superoxide dismutase (SOD, EC 1.15.1.1) is an enzyme catalyzing the dismutation of the superoxide anion into molecular oxygen and hydrogen peroxide. In the present study, the full length cDNA of extracellular copper–zinc superoxide dismutase (MnECSOD) from Macrobrachium nipponense was cloned (GenBank accession no. JX045662). Analysis of the nucleotide sequence revealed that the MnECSOD cDNA consisted of 817bp with an open reading frame (ORF) of 597bp encoding a polypeptide of 198 amino acids with a putative signal peptide of 19 amino acids. The mature protein had a predicted molecular weight of 18.71kDa with an estimated pI of 4.87. Four copper binding sites, 4 zinc binding sites and 2 cysteines involved in the formation of the disulfide bridge were conserved in the protein. Sequence comparison showed that the MnECSOD protein had 86% and 55% similarity to that of freshwater prawn Macrobrachium rosenbergii and white shrimp Litopenaeus vannamei, respectively. After challenge with Aeromonas hydrophila, the expression of MnECSOD mRNA in the hepatopancreas was increased responsively. After a drastic decrease at 12h, the expression level increased again by 6.75-fold compared to that in the control group at 48-h post-infection. These results may indicate that MnECSOD was involved in the innate immune responses of M. nipponense.