Zinc Superoxide Dismutase Research Articles

Changes in Copper, Zinc, and Malondialdehyde Levels and Superoxide Dismutase Activities in Pre-Eclamptic Pregnancies.

BackgroundPreeclampsia (PE) is a hypertensive disorder that occurs in 2% to 8% of pregnancies. Although numerous studies have investigated the etiology and pathophysiology of preeclampsia, the precise pathological mechanisms remain poorly understood. Hence, in the present study malondialdehyde (MDA) levels and SOD expression, and Cu and Zn concentrations and ratios were correlated with birth weights in pregnant women with and without PE, and in non-pregnant females of reproductive age.Material/MethodsMalondialdehyde (MDA) levels and superoxide dismutase (SOD) activities were determined spectrophotometrically, and Cu and Zn levels were determined using atomic absorption spectrometry in serum from 42 non-pregnant women (NP), 40 healthy pregnant women (HP), and 38 pre-eclamptic pregnant (PE) women. Subsequently, Cu/Zn ratios were calculated and associations with birth weights were analyzed using Spearman correlations.ResultsCu, Zn, and MDA levels and Cu/Zn ratios were significantly higher in the PE group than in the HP and NP groups, and were significantly higher in the HP than in the NP group (p<0.001 and p<0.001; respectively). In contrast, serum Zn and SOD levels were significantly lower in the PE group than in HP and NP groups, and were significantly lower in the HP group than in the NP group (p<0.001 and p<0.001; respectively). However, only Cu and Zn levels were significantly associated with fetal birth weights (r=−0.433, p<0.001).ConclusionsSerum Cu/Zn ratios may reflect vascular complications of PE, and the ensuing increases in lipid peroxidation may play important pathogenic roles.

288 INFLUENCE OF MELATONIN ON IN VITRO MATURATION OF BOVINE OOCYTES

The present study evaluated the effects of melatonin (MEL) during IVM of bovine cumulus-oocyte complexes (COC). The COC were cultured in droplets (25–30/100 µL) under mineral oil at 38.5°C and 5% CO2 in air. Medium (TCM199 + 0.1% polyvinyl alcohol, 0.25 mM sodium pyruvate, and 25 µg mL–1 of gentamycin) was supplemented with FSH (0.5 µg mL–1), MEL (10–9 and 10–6 M), or no hormones (control). In experiment 1, oocytes were assessed for nuclear maturation rates (6, 12, 18, and 24 h of IVM). In experiment 2, relative abundance of antioxidant enzymes copper, zinc superoxide dismutases (CuZnSOD), manganese superoxide dismutases (MnSOD), and glutathione peroxidase (GPx) was evaluated in oocytes and cumulus cells (0 and 24 h) by real time PCR. The immature group was the reference and endogenous controls were actin β and glyceraldehyde 3-phosphate dehydrogenase. In experiment 3, nuclear fragmentation in cumulus was assessed by TUNEL and flow cytometry (24 h). In experiment 4, embryo development after in vitro fertilization and culture was analysed (cleavage rates D2, blastocysts Day 8, and hatching Day 10). The control group was matured in complete IVM medium (10% FCS, 0.5 µg mL–1 of FSH, 50 µg mL–1 of LH, and 1 µg mL–1 of oestradiol). The MEL and FSH groups were the same. Data (3–4 replicates) were analysed by Chi-square (experiment 1; GraphPad Prism) or ANOVA and Tukey test (SAS, 1995) and 5% significance. At 6 h of IVM, all oocytes were at germinal vesicle stage. At 12 h, hormone groups had similar metaphase I rates (71–81%, P &gt; 0.05) and were superior to control (54%, P &lt; 0.05). At 18 h, metaphase II (MII) rates were similar (57–74%, P &gt; 0.05). After 24 h, MEL (10–9 and 10–6 M) was similar to control (51, 67, and 69% MII, respectively, P &gt; 0.05) and FSH had the highest MII rates (90%, P &gt; 0.05). The 10–6 M MEL was similar to FSH (P &gt; 0.05). Antioxidant enzymes were unaffected in oocytes (P &gt; 0.05%). The CuZnSOD transcripts increased in cumulus after IVM with 10–6 M MEL compared to immature cells (3.5 and 1.0, respectively, P &lt; 0.05). Control, FSH, and 10–9 M MEL did not differ (2.1–2.5, P &gt; 0.05) or increased relative to immature cells (P &lt; 0.05), but were inferior to 10–6 M MEL (P &lt; 0.05). The MnSOD relative abundance was similar for immature, control, and 10–9 M MEL (1.0–3.0, P &gt; 0.05). The 10–6 M MEL increased MnSOD relative to immature cells (3.0 and 1.0, respectively, P &lt; 0.05), but was similar to control and 10–9 M MEL (P &gt; 0.05). The FSH showed the highest levels (9.1, P &lt; 0.05); GPx4 transcripts were not affected (P &gt; 0.05). Nuclear fragmentation in cumulus was not influenced (33.4–41.5/10 000 cells; P &gt; 0.05). Embryo development rates were similar for all groups (cleavage: 82–87%, blastocysts: 49–54, hatching: 91–96%, P &gt; 0.05). In conclusion, MEL during IVM stimulates meiosis resumption at rates similar to FSH and embryo development similar to FSH and complete IVM medium. The MEL increased CuZnSOD expression in cumulus, but no parallel effect was observed on nuclear fragmentation.

Erythropoietin prevents endothelial dysfunction in GTP-cyclohydrolase I-deficient hph1 mice.

: In this study, we used the mutant hph1 mouse model, which has deficiency in GTP-cyclohydrolase I (GTPCH I) activity, to test the hypothesis that erythropoietin (EPO) protects aortic wall from oxidative stress induced by uncoupling of endothelial nitric oxide synthase (eNOS). Both GTPCH I activity and tetrahydrobiopterin (BH4) levels were reduced in hph1 mice, whereas 7,8-dihydrobiopterin (7,8-BH2) levels were significantly increased. Furthermore, BH4 deficiency caused increased production of superoxide anion and hydrogen peroxide in the aorta thus resulting in impairment of endothelium-dependent relaxations to acetylcholine. Treatment of hph1 mice with recombinant human EPO (1000 U/kg, subcutaneously for 3 days) significantly decreased superoxide anion production by eNOS and improved BH4 to 7,8-BH2 ratio in aortas. EPO also significantly decreased production of hydrogen peroxide and improved endothelium-dependent relaxations in aortas of hph1 mice. In addition, EPO treatment increased protein expressions of copper-/zinc-superoxide dismutase, manganese-superoxide dismutase, and catalase in the aorta of hph1 mice. Our findings demonstrate that treatment with EPO prevented oxidative stress and endothelial dysfunction caused by eNOS uncoupling. Increased vascular expressions of antioxidants seem to be an important molecular mechanism underlying vascular protection by EPO during chronic BH4 deficiency.

A deficiency or an excess of dietary threonine level affects weight gain, enzyme activity, immune response and immune-related gene expression in juvenile blunt snout bream (Megalobrama amblycephala)

A feeding trial was conducted to investigate the impacts of deficient and excess dietary threonine levels on weight gain, plasma enzymes activities, immune responses and expressions of immune-related genes in the intestine of juvenile blunt snout bream. Triplicate groups of fish (initial weight 3.01 ± 0.01 g, 30 fish per tank) were fed with deficient (0.58%), optimum (1.58%) and excess (2.58%) threonine level diets to near satiation four times a day for 9 weeks. A mixture of l-amino acids was supplemented to simulate the whole body amino acid pattern of blunt snout bream, except for threonine. The results showed that both deficiency and excess threonine level diets significantly (P < 0.05) reduced the weight gain of blunt snout bream. Excess dietary threonine level triggered plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities (P < 0.05); whereas superoxide dismutase (SOD) activity was not significantly influenced by imbalanced-dietary threonine level (P > 0.05). Plasma complement component 3 (C3) and component 4 (C4) concentrations were significantly depressed by the deficiency of dietary threonine (P < 0.05). Dietary threonine regulated the target of rapamycin (TOR), eukaryotic translation initiation factor 4E-binding protein 2 (4E-BP2), tumour necrosis factor alpha (TNF-α) and copper–zinc superoxide dismutase (Cu/Zn-SOD) gene expressions in the intestine of blunt snout bream, which may go further to explain the adverse effects of a deficient and/or an excess dietary threonine level on growth, immunity and health of fish. Furthermore, the present study also suggests that an optimum dietary threonine could play an important role in improving growth, enhancing immune function and maintaining health of fish.

Copper–zinc superoxide dismutase-mediated redox regulation of bortezomib resistance in multiple myeloma

Multiple myeloma (MM) is an incurable B-cell malignancy. The proteasome inhibitor bortezomib (BTZ) is a frontline MM drug; however, intrinsic or acquired resistance to BTZ remains a clinical hurdle. As BTZ induces oxidative stress in MM cells, we queried if altered redox homeostasis promotes BTZ resistance. In primary human MM samples, increased gene expression of copper–zinc superoxide dismutase (CuZnSOD or SOD1) correlated with cancer progression, high-risk disease, and adverse overall and event-free survival outcomes. As an in vitro model, human MM cell lines (MM.1S, 8226, U266) and the BTZ-resistant (BR) lines (MM.1SBR, 8226BR) were utilized to determine the role of antioxidants in intrinsic or acquired BTZ-resistance. An up-regulation of CuZnSOD, glutathione peroxidase-1 (GPx-1), and glutathione (GSH) were associated with BTZ resistance and attenuated prooxidant production by BTZ. Enforced overexpression of SOD1 induced BTZ resistance and pharmacological inhibition of CuZnSOD with disulfiram (DSF) augmented BTZ cytotoxicity in both BTZ-sensitive and BTZ-resistant cell lines. Our data validates CuZnSOD as a novel therapeutic target in MM. We propose DSF as an adjuvant to BTZ in MM that is expected to overcome intrinsic and acquired BTZ resistance as well as augment BTZ cytotoxicity.

Effect of including torsional parameters for histidine-metal interactions in classical force fields for metalloproteins.

Classical force-field parameters of the metal site of metalloproteins usually comprise only the partial charges of the involved atoms, as well as the bond-stretching and bending parameters of the metal-ligand interactions. Although for certain metal ligands such as histidine residues, the torsional motions at the metal site play an important role for the dynamics of the protein, no such terms have been considered to be crucial in the parametrization of the force fields, and they have therefore been omitted in the parametrization. In this work, we have optimized AMBER-compatible force-field parameters for the reduced state of the metal site of copper, zinc superoxide dismutase (SOD1) and assessed the effect of including torsional parameters for the histidine-metal interactions in molecular dynamics simulations. On the basis of the obtained results, we recommend that torsion parameters of the metal site are included when processes at the metal site are investigated or when free-energy calculations are performed. As the torsion parameters mainly affect the structure of the metal site, other kinds of structural studies can be performed without considering the torsional parameters of the metal site.

Oenanthe javanica extract increases immunoreactivities of antioxidant enzymes in the rat kidney

Background Oenanthe javanica is an aquatic perennial herb originated from East Asia. Nowadays, the effects of Oenanthe javanica have been proven in various disease models. Studies regarding the antioxidant effect of Oenanthe javanica in the kidney are still unclear. Methods This study was therefore performed to investigate the effect of the Oenanthe javanica extract (OJE) in the rat kidney using immunohistochemistry for antioxidant enzymes, copper, zinc-superoxide dismutase (SOD1), manganese superoxide dismutase (SOD2), catalase (CAT) and glutathione peroxidase (GPx). Sprague-Dawley rats were randomly assigned to three groups: (1) normal diet fed-group (normal-group), (2) diet containing ascorbic acid (AA)-fed group (AAgroup) as a positive control, (3) diet containing OJE-fed group (OJE-group). AA and OJE were supplied during 28 days. Results The side-effects were not observed in all the groups. Immunoreactivities of SOD1, SOD2, CAT and GPx were easily detected in the distal tubules of the kidney, and their immunoreactivities in the AA- and OJE-groups were increased to about 1.4–1.5 times and 2 times, respectively, compared with those in the normal-group. Conclusion OJE significantly increased expressions of SOD1 &amp; 2, CAT and GPx immunoreactivities in the distal tubules of the rat kidney, and this finding suggests that significant enhancements of endogenous enzymatic antioxidants by OJE treatment may be a legitimate strategy for decreasing oxidative stresses in the kidney.

The megavirus chilensis Cu,Zn-superoxide dismutase: the first viral structure of a typical cellular copper chaperone-independent hyperstable dimeric enzyme.

Giant viruses able to replicate in Acanthamoeba castellanii penetrate their host through phagocytosis. After capsid opening, a fusion between the internal membranes of the virion and the phagocytic vacuole triggers the transfer in the cytoplasm of the viral DNA together with the DNA repair enzymes and the transcription machinery present in the particles. In addition, the proteome analysis of purified mimivirus virions revealed the presence of many enzymes meant to resist oxidative stress and conserved in the Mimiviridae. Megavirus chilensis encodes a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD), an enzyme known to detoxify reactive oxygen species released in the course of host defense reactions. While it was thought that the metal ions are required for the formation of the active-site lid and dimer stabilization, megavirus chilensis SOD forms a very stable metal-free dimer. We used electron paramagnetic resonance (EPR) analysis and activity measurements to show that the supplementation of the bacterial culture with copper and zinc during the recombinant expression of Mg277 is sufficient to restore a fully active holoenzyme. These results demonstrate that the viral enzyme's activation is independent of a chaperone both for disulfide bridge formation and for copper incorporation and suggest that its assembly may not be as regulated as that of its cellular counterparts. A SOD protein is encoded by a variety of DNA viruses but is absent from mimivirus. As in poxviruses, the enzyme might be dispensable when the virus infects Acanthamoeba cells but may allow megavirus chilensis to infect a broad range of eukaryotic hosts. Mimiviridae are giant viruses encoding more than 1,000 proteins. The virion particles are loaded with proteins used by the virus to resist the vacuole's oxidative stress. The megavirus chilensis virion contains a predicted copper, zinc superoxide dismutase (Cu,Zn-SOD). The corresponding gene is present in some megavirus chilensis relatives but is absent from mimivirus. This first crystallographic structure of a viral Cu,Zn-SOD highlights the features that it has in common with and its differences from cellular SODs. It corresponds to a very stable dimer of the apo form of the enzyme. We demonstrate that upon supplementation of the growth medium with Cu and Zn, the recombinant protein is fully active, suggesting that the virus's SOD activation is independent of a copper chaperone for SOD generally used by eukaryotic SODs.

Probing the interactions between carboxylated multi-walled carbon nanotubes and copper-zinc superoxide dismutase at a molecular level.

In order to evaluate the toxicity of multi-walled carbon nanotubes (MWCNTs-COOH) at a molecular level, the effect of MWCNTs-COOH on antioxidant enzyme copper-zinc superoxide dismutase (Cu/ZnSOD) was investigated using fluorescence spectroscopy, UV/vis absorption spectroscopy, circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC). By deducting the inner filter effect (IFE), the fluorescence emission spectra and synchronous fluorescence spectra indicated that there were interactions between MWCNTs-COOH and Cu/ZnSOD. Moreover, the microenvironment of the amino acid residues in the enzyme was changed slightly. The UV/vis absorption and CD spectroscopic results showed appreciable conformational changes in Cu/ZnSOD. However, the results of a Cu/ZnSOD activity determination did not show any significant difference. In other words, MWCNTs-COOH has no significant effect on enzyme activity. The ITC results showed that the binding of MWCNTs-COOH to Cu/ZnSOD was a weak endothermic process, indicating that the predominant force of the binding was hydrophobic interaction. Moreover, it was essential to consider the IFE in fluorescence assays, which might affect the accuracy and precision of the results. The above results are helpful in evaluating the oxidative stress induced by MWCNTs-COOH in vivo.

High-protein diet induces oxidative stress in rat brain: protective action of high-intensity exercise against lipid peroxidation.

It is well established that soy protein diets as well as aerobic exercise could promote antioxidant capacity and consequently reduce free radicals overproduction on brain. However, little is know regarding to the high-protein diets and high intensity exercise on oxidative stress production. The aim of this study was to analyse the effects of high-protein diets and high-intensity exercise (HIE) on brain oxidative stress markers. A total of 40 male Wistar rats were randomly distributed in 4 experimental groups (n=10): normal-protein or high-protein diets with or without HIE for an experimental period of 12 weeks. Main oxidative damage markers in brain such as thiobarbituric acid-reactive substances (TBARs) and protein carbonyl content (PCC) were assessed. In addition, brain manganese superoxide dismutase (Mn-SOD), cooper/ zinc superoxide dismutase (CuZn-SOD) and catalase (CAT) antioxidant enzymes activity, and protein level of Nuclear factor erythroid 2 related factor 2 (Nrf2) were measured. Brain TBARs, PCC, tSOD, Mn-SOD, CuZn-SOD and CAT levels were higher in the high-protein compared to the normal-protein groups (all, p.

NiCl2-down-regulated antioxidant enzyme mRNA expression causes oxidative damage in the broiler(')s kidney.

The kidney serves as a major organ of nickel (Ni) excretion and is a target organ for acute Ni toxicity due to Ni accumulation. There are no studies on the Ni or Ni compound-regulated antioxidant enzyme mRNA expression in animals and human beings at present. This study was conducted to investigate the pathway of nickel chloride (NiCl2)-caused renal oxidative damage by the methods of biochemistry, quantitative real-time polymerase chain reaction, and enzyme-linked immunosorbent assay. Two hundred and eighty one-day-old broilers were randomly divided into four groups and fed on a control diet and three experimental diets supplemented with 300, 600, and 900 mg/kg of NiCl2 for 42 days. Dietary NiCl2 elevated the malondialdehyde (MDA), nitric oxide (NO), 8-hydroxy-2'-deoxyguanosine (8-OHdG) contents, and reduced the ability to inhibit hydroxy radical in the NiCl2-treated groups. Also, the renal inducible nitric oxide synthase (iNOS) activity and mRNA expression levels were increased. The total antioxidant (T-AOC) and activities of antioxidant enzymes including copper zinc superoxide dismutase (CuZn-SOD), manganese superoxide dismutase (Mn-SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), and glutathione-s-transferase (GST) were decreased, and the glutathione (GSH) contents as well were decreased in the kidney. Concurrently, the renal CuZn-SOD, Mn-SOD, CAT, GSH-Px, GST, and GR mRNA expression levels were decreased. The above-mentioned results showed that dietary NiCl2 in excess of 300 mg/kg caused renal oxidative damage by reducing mRNA expression levels and activities of antioxidant enzymes, and then enhancing free radicals generation, lipid peroxidation, and DNA oxidation.

Perinatal exposure to lead: reduction in alterations of brain mitochondrial antioxidant system with calcium supplement.

Lead (Pb) is a potent neurotoxicant that causes several neurochemical and behavioral alterations. Previous studies showed that the gestational and lactational exposure to Pb reduces the cholinergic and aminergic systems, and behavior of rats. The present study was designed to examine the protective effects of calcium supplementation against Pb-induced oxidative stress in cerebellum and hippocampus of brain at postnatal day (PND) 21, PND 28, PND 35, and PND 60. Pregnant rats were exposed to 0.2 % Pb (Pb acetate in drinking water) from gestational day 6 (GD 6) and pups were exposed through maternal milk till weaning (PND 21). We found that the activity of serum ceruloplasmin oxidase (Cp), mitochondrial manganese superoxide dismutase (Mn-SOD), copper zinc superoxide dismutase (Cu/Zn-SOD), glutathione peroxidase (GPx), catalase (CAT), and xanthine oxidase (XO) enzyme activities were decreased, whereas the malondialdehyde (MDA) levels increased in the cerebellum and hippocampus of Pb-exposed rats. These changes were more prominent at PND 35 and greater in hippocampus compared to cerebellum. Among the enzyme activities, Mn-SOD and Cu/Zn-SOD showed maximum decrease compared to GPx, CAT, XO, and Cp. Furthermore, 0.02 % calcium supplementation together with 0.2 % Pb significantly reversed the Pb-induced alterations in the enzyme activities, and MDA levels. In conclusion, these data suggest that early life exposure to Pb induce alterations in the mitochondrial antioxidant system of brain regions which remain for long even after Pb exposure has stopped. Calcium supplementation may potentially be beneficial in treating Pb toxicity in the developing rat brain.

Lymphocyte Levels of Redox-Sensitive Transcription Factors and Antioxidative Enzymes as Indicators of Pro-Oxidative State in Depressive Patients

Background: Oxidative stress is reliably observed in major depressive disorder (MDD). However, molecular data on the principal cellular redox-sensitive transcriptional factors and the levels of their downstream-regulated antioxidant enzymes in MDD are scarce. Methods: In the peripheral blood mononuclear cells (PBMC) of subjects with a current episode of MDD (n = 30) and healthy controls (n = 35), we investigated alterations in the levels of redox-sensing nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein, its inhibitor Keap1, and nuclear factor-κB (NF-κB), along with their cognate downstream effectors, the antioxidant enzymes (AOEs): manganese and copper zinc superoxide dismutase (MnSOD and CuZnSOD, respectively), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GLR). Results: MDD subjects exhibited higher levels of Nrf2 and its regulator Keap1, as well as NF-κB in the cytoplasm of PBMC compared to controls. This state was further reflected by increased levels of MnSOD, CuZnSOD and CAT proteins and by the lack of correlation between MnSOD and CAT, which could indicate impaired oxidative detoxification capacity in MDD patients. Moreover, increased levels of MnSOD, CuZnSOD and CAT in MDD patients positively correlated with levels of Nrf2, while increased levels of SODs were also positively related to NF-κB. There were no differences regarding the levels of GPx and GLR proteins, but the ratio of GLR/GPx was reduced, suggesting diminished capacity of GPx in antioxidative defence in PBMC of MDD subjects. Conclusion: These data provide evidence that MDD is characterized by up-regulation of redox-sensitive transcriptional factors (Nrf2 and NF-κB) and AOEs (MnSOD, CuZnSOD and CAT), indicating pro-oxidative state in the PBMC of MDD patients.

Suppressing mutation-induced protein aggregation in mammalian cells by mutating residues significantly displaced upon the original mutation

Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.

The lack of CuZnSOD leads to impaired neurotransmitter release, neuromuscular junction destabilization and reduced muscle strength in mice.

Elevated reactive oxygen species (ROS) production and ROS-dependent protein damage is a common observation in the pathogenesis of many muscle wasting disorders, including sarcopenia. However, the contribution of elevated ROS levels to –a breakdown in neuromuscular communication and muscle atrophy remains unknown. In this study, we examined a copper zinc superoxide dismutase [CuZnSOD (Sod1)] knockout mouse (Sod1 −/−), a mouse model of elevated oxidative stress that exhibits accelerated loss of muscle mass, which recapitulates many phenotypes of sarcopenia as early as 5 months of age. We found that young adult Sod1 −/− mice display a considerable reduction in hind limb skeletal muscle mass and strength when compared to age-matched wild-type mice. These changes are accompanied by gross alterations in neuromuscular junction (NMJ) morphology, including reduced occupancy of the motor endplates by axons, terminal sprouting and axon thinning and irregular swelling. Surprisingly however, the average density of acetylcholine receptors in endplates is preserved. Using in vivo electromyography and ex vivo electrophysiological studies of hind limb muscles in Sod1 −/− mice, we found that motor axons innervating the extensor digitorum longus (EDL) and gastrocnemius muscles release fewer synaptic vesicles upon nerve stimulation. Recordings from individually identified EDL NMJs show that reductions in neurotransmitter release are apparent in the Sod1 −/− mice even when endplates are close to fully innervated. However, electrophysiological properties, such as input resistance, resting membrane potential and spontaneous neurotransmitter release kinetics (but not frequency) are similar between EDL muscles of Sod1 −/− and wild-type mice. Administration of the potassium channel blocker 3,4-diaminopyridine, which broadens the presynaptic action potential, improves both neurotransmitter release and muscle strength. Together, these results suggest that ROS-associated motor nerve terminal dysfunction is a contributor to the observed muscle changes in Sod1 −/− mice.

Alteration of copper–zinc superoxide dismutase 1 expression by influenza A virus is correlated with virus replication

Viruses have evolved mechanisms designated to potentiate virus replication by modulating the physiological condition of host cells. The generation of reactive oxygen species (ROS) during infection with influenza virus A (IAV) is a well-established mechanism in animals, but little is known about the generation of ROS in in vitro cell culture models and about its role in virus replication. We show here that IAV H1N1 infected human alveolar cells increased superoxide anion level mainly by suppressing the copper–zinc superoxide dismutase 1 (SOD1) gene, and that the SOD1-controlled generation of ROS was tightly correlated with virus replication. The transcription factor Sp1, which is a major element of the proximal region of the sod1 promoter, was slightly downregulated at the transcriptional level during IAV infection, and subsequently modulated by post-translational control. A gradual reduction of whole Sp1 was largely responsible for the repression of sod1 transcription with increasing time post-infection, and their rescue by the proteasome inhibitor, MG132, proved the involvement of proteasomal degradation in Sp1 regulation during IAV infection. Furthermore, we observed that expression of viral polymerase PB1 was inversely proportional to SOD1 level. The antioxidant N-acetyl-cysteine (NAC) neutralized IAV-mediated oxidative stress, and either NAC treatment or sod1 transfection considerably diminished viral polymerase activity. These data indicate that IAV-induced SOD1 repression, which may cause impaired redox balance in host cells, can be attributed, at least in part, to enhance viral replication.

Copper, zinc superoxide dismutase of Curcuma aromatica is a kinetically stable protein

This study details on cloning and characterization of Cu,Zn superoxide dismutase (Ca–Cu,Zn SOD) from a medicinally important plant species Curcuma aromatica. Ca–Cu,Zn SOD was 692bp with an open reading frame of 459bp. Expression of the gene in Escherichia coli cells followed by purification yielded the enzyme with Km of 0.047±0.008μM and Vmax of 1250±24units/mg of protein. The enzyme functioned (i) across a temperature range of −10 to +80°C with temperature optima at 20°C; and (ii) at pH range of 6–9 with optimum activity at pH 7.8. Ca–Cu,Zn SOD retained 50% of the maximum activity after autoclaving, and was stable at a wide storage pH ranging from 3 to 10. The enzyme tolerated varying concentrations of denaturating agent, reductants, inhibitors, trypsin, was fairly resistant to inactivation at 80°C for 180min (kd, 6.54±0.17×10−3 min−1; t1/2, 106.07±2.68min), and had midpoint of thermal transition (Tm) of 70.45°C. The results suggested Ca–Cu,Zn SOD to be a kinetically stable protein that could be used for various industrial applications.

Blood Volatile Organic Compounds as Potential Biomarkers for Amyotrophic Lateral Sclerosis: an Animal Study in the SOD1 G93A Mouse

Amyotrophic lateral sclerosis (ALS) is a rapid progressive motor neuron disease. Currently, there are no specific or reliable biomarkers for the diagnosis of this disease, and there are no effective medical treatments. The early diagnosis and treatment of this disease has the potential to prolong the survival of ALS patients, but typically, approximately 1 year passes between the onset of symptoms and the diagnosis of this disease. Therefore, there is an urgent need to find specific biomarkers to enable early diagnosis and therapeutic intervention in this disease. Analyzing the volatile organic compounds (VOCs) present in the blood and exhaled breath is a useful and convenient approach for investigating potential biomarkers. In this study, we examined the VOCs present in blood samples from copper zinc superoxide dismutase 1 (SOD1) glycine to alanine mutation at position 93 (G93A) mice to determine whether a specific biomarker pattern exists in these transgenic mice. Blood samples from ALS mice and their age-matched littermates were analyzed using gas chromatography-mass spectrometry. A total of 12 independent compounds associated with oxidative stress were identified at the early stage of disease. The data show that there is a specific pattern of blood VOCs in ALS mice that could potentially be used as biomarkers that could improve the diagnosis of this disease. Furthermore, these compounds could also potentially be used to monitor the response to neuroprotective agents and to help us better understand the underlying mechanisms of ALS.

Insights into SOD1-linked amyotrophic lateral sclerosis from NMR studies of Ni(2+)- and other metal-ion-substituted wild-type copper-zinc superoxide dismutases.

The dimeric Cu-Zn superoxide dismutase (SOD1) is a particularly interesting system for biological inorganic chemical studies because substitutions of the native Cu and/or Zn ions by a nonnative metal ion cause minimal structural changes and result in high enzymatic activity for those derivatives with Cu remaining in the Cu site. The pioneering NMR studies of the magnetically coupled derivative Cu2Co2SOD1 by Ivano Bertini and coworkers are of particular importance in this regard. In addition to Co(2+), Ni(2+) is a versatile metal ion for substitution into SOD1, showing very little disturbance of the structure in Cu2Ni2SOD1 and acting as a very good mimic of the native Cu ion in Ni2Zn2SOD1. The NMR studies presented here were inspired by and are indebted to Ivano Bertini's paramagnetic NMR pursuits of metalloproteins. We report Ni(2+) binding to apo wild-type SOD1 and a time-dependent Ni(2+) migration from the Zn site to the Cu site, and the preparation and characterization of Ni2Ni2SOD1, which shows coordination properties similar to those of Cu2Cu2SOD1, namely, an anion-binding property different from that of the wild type and a possibly broken bridging His. Mutations in the human SOD1 gene can cause familial amyotrophic lateral sclerosis (ALS), and mutant SOD1 proteins with significantly altered metal-binding behaviors are implicated in causing the disease. We conclude by discussing the effects of the ALS mutations on the remarkable stabilities and metal-binding properties of wild-type SOD1 proteins and the implications concerning the causes of SOD1-linked ALS.

Response to “Does Angiotensin-Dependent Superoxide Production Help to Prevent Salt-Induced Endothelial Dysfunction in 2 Kidney–1 Clip Hypertensive Rats?”

To the Editor: We recently reported the surprising finding that the salt-induced endothelial dysfunction and downregulation of copper (Cu)/zinc (Zn) superoxide dismutase (SOD) that occurred in cerebral arteries of sham-operated animals were absent in 2 kidney–1 clip (2K1C) hypertensive rats, which exhibit prolonged elevations of plasma angiotensin II (ANG II) levels, and that AT1 receptor blockade with losartan eliminated endothelium-dependent vasodilation in 2K1C rats.1 We concluded that the elevated ANG II levels in the 2K1C rats played a key role in protecting against salt-induced vascular dysfunction and suppression of Cu/Zn SOD by upregulating antioxidant defense mechanisms in the arteries. In a letter to the editor entitled “Could AT1 Receptor Activation Increase Antioxidant Defense to Prevent Salt-Induced Vascular Dysfunction of 2 Kidney-1 Clip Hypertensive Rats?”, Dr Valdir A. Braga stated that it is difficult to draw conclusions about the vascular actions of ANG II without looking at oxidant enzymes and downstream production of reactive oxygen species. Braga cites a list of well-known and indisputable findings showing that ANG II, acting via AT1 receptors, activates NADPH oxidase to produce superoxide (O2 −) radicals and that ANG II has been linked to oxidative stress in the context of vascular dysfunction and hypertension. Based on those conclusions, Braga suggests that the upregulation of Cu/Zn SOD occurs in response to increases in O2 − rather than a possible antioxidant effect of ANG II. In our estimation, Braga’s conclusion that O2 − production in response to the elevated ANG II levels in the 2K1C rats may play an important role in preventing salt-induced endothelial dysfunction through upregulation of Cu/Zn SOD is quite plausible and could provide an explanation for the surprising findings in our study (i.e., the “antioxidant effect” of ANG II to upregulate Cu/Zn SOD in 2K1C rats). In support of that hypothesis, Papaiahgari et al.2 reported that O2 − produced by NADPH oxidase was essential to activate the ERK 1/2 pathway in C10 alveolar epithelial cells exposed to hyperoxia and that this led to upregulation of the nuclear transcription factor Nrf2, which upregulates multiple antioxidant and cell protective enzymes, including Cu/Zn SOD. Consistent with those findings, McEwen et al.3 showed that the protective effect of low-dose ANG II infusion to restore vascular relaxation mechanisms in cerebral arteries of salt-fed Sprague-Dawley rats is eliminated by AT1 receptor blockade and by inhibiting the ERK 1/2 pathway. Further emphasizing the importance of physiological levels of ANG II in maintaining normal vascular function is our recent study,4 which showed that diet-induced obesity, which is known to elevate plasma ANG II levels, restores normal endothelial function in a model of chronically low levels of plasma ANG II, the Dahl salt-sensitive rat.The precise mechanisms by which O2 − radicals formed by NADPH oxidase in response to elevated ANG II levels contribute to maintenance of antioxidant defense mechanisms (including Cu/Zn SOD) in 2K1C hypertensive rats remain to be determined. However, investigation of the relationship between ANG II levels, vascular O2 −, and activation of the Nrf2 antioxidant defense system appears to be a promising avenue of investigation.