Copper-zinc Superoxide Dismutase Research Articles

Dynamic behavior of Yarrowia lipolytica to well-controlled pH and oxygen perturbations: dependence of the stress response on the culture mode

The developing central nervous system is particularly vulnerable to environmental contaminants such as non-dioxin-like polychlorinated biphenyls (NDL-PCBs). This study investigated the potential oxidative effects in mice pups exposed via lactation to the sum of the six indicator NDL-PCBs (∑6 NDL-PCBs) at 0, 1, 10 and 100 ng/kg per 14 days, constituting levels below the guidance values fixed by French food safety agencies for humans at 10 ng/kg body weight per day. For this purpose, the oxidative status was assessed by flow cytometry via dichloro-dihydro-fluorescein diacetate in the cerebellum of juvenile male offspring mice during brain growth spurt [postnatal day (PND) 14]. No significant differences were found in the levels of reactive oxygen species in the cerebellar neurons or glial cells (astrocytes, oligodendrocytes and microglia) of lactationally exposed male mice at PND 14 (p > 0.05). Concordantly, oxidative-stress related gene expression was measured by qPCR for catalase, copper zinc superoxide dismutase 1, glyoxalase 1, glutathione peroxidase 1, and glutathione reductase 1, in the cerebellum at PND 14 appeared unaffected, as also verified at the protein level by immunoblots. Moreover, transcriptomic data from our previous work have not shown differences in the mRNA expressions of genes belonging to GO terms involved in oxidative stress in neurons of male mice exposed to ∑6 NDL-PCBs compared to controls; except for glyoxalase 1 which was downregulated in neurons isolated from exposed group compared to controls. Our findings suggest that lactational exposure to NDL-PCBs at environmental relevant concentrations may not cause significant oxidative effect on juvenile cerebellum.

A novel 10-base pair insertion mutation in exon 5 of the SOD1 gene in a Chinese family with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an adult-onset, progressive, fatal neurodegenerative disease. Several genes are associated with ALS. Copper-zinc superoxide dismutase 1 (SOD1) is one of the most commonly mutated genes in ALS, and more than 160 mutations in SOD1 have been reported. We reported a novel heterozygous insertion mutation that led to a frameshift and a premature termination at position 136 in exon 5 of the SOD1 gene (c.392_393insGCAAAGGTGG; p.N132Qfs*5) in a Chinese familial ALS pedigree. This mutation in the pedigree demonstrated an autosomal dominant pattern of inheritance and a phenotype characterized by an early onset (approximately 34 years old) with a relatively rapid course (approximately 2 years) and limb onset with respiratory involvement. The clinical feature of the p.N132Qfs*5 mutation was nearly identical to a previously reported mutation (Gly127insTGGG). Experiments in G127X mice demonstrated that the G127X mutation was pathogenic. SOD1 activity in the p.N132Qfs*5 mutation carriers in the family decreased significantly compared with normal family members. In conclusion, we identified a novel SOD1 mutation in an ALS family, which is an important addition to the catalog of SOD1 mutations in ALS.

Differences in seminal plasma and spermatozoa antioxidative systems and seminal plasma lipid and protein levels among boar breeds and hybrid genetic traits

The objectives of this study were to determine the influence of breed and hybrid genetic traits of boars on lipid and protein concentrations and antioxidative system variables in seminal plasma (SP) and spermatozoa and their correlations with semen quality variables. Semen samples from 27 boars: Swedish Landraces (SL), German Landraces (GL), Large Whites (LW), Pietrains (P) and Pig Improvement Company hybrids (PIC-hybrid), aged from 1.5 to 3 years old, were collected. SP was spectrophotometrically analyzed to determine total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triacylglycerol (TAG), total protein (TP), albumin, and zinc concentrations. The antioxidative system in SP and spermatozoa was established spectrophotometrically by determining total antioxidative status (TAS), total superoxide dismutase (TSOD) and glutathione peroxidase (GSH-Px) parameters, as well as copper-zinc superoxide dismutase (CuZnSOD) and manganese superoxide dismutase (MnSOD) activity in spermatozoa. The hybrid boars had higher (P<0.05) SP concentrations of: TC, LDL-C and TAG than P and GL; HDL-C than P, GL and SL; and TP than P and LW. PIC-hybrid had lower values (P<0.05) in spermatozoa of: TAS and CuZnSOD than SL; TSOD and GSH-Px than SL and P; and MnSOD than SL and LW. Differences in SP and spermatozoa antioxidative system variables and the significant differences in SP protein and lipid variables exist among boars of different breeds and hybrid. Novel data and observed differences in semen variables among boar breeds and hybrids and their correlations with semen quality parameters in this study could contribute to better assessment of boar semen quality.

Age Impaired endothelium-dependent vasodilation is improved by resveratrol in rat mesenteric arteries.

[Purpose]To determine whether resveratrol improves the adverse effects age on vascular function in mesenteric arteries (MAs), and diminishes the hyperactivity in adrenal gland with age.[Methods]Male F344 x Brown Norway rats were assigned to 6-month control (YC), 6-month resveratrol (YR), 24-month control (OC) and 24-month resveratrol (OR). Resveratrol (15 mg/kg) was provided to resveratrol groups in drinking water for 14 days.[Results]Concentration response curves to phenylephrine (PE, 10-9-10-5M), acetylcholine (Ach, 10-9-10-5M) and resveratrol (10-8-10-4M) were evaluated in pressurized isolated MAs. The Ach concentration-response curve was right shifted with maximal response diminished in OC compared with YC rats. These effects were reversed by resveratrol treatment. The resveratrol-mediated relaxant responses were unchanged with age or resveratrol suggesting an endothelium-independent mechanism. Resveratrol tended to increase endothelial nitric oxide synthase; caused no effect on copper-zinc superoxide dismutase; and normalized the age-related elevatation in DβH and NPY levels in adrenal medulla, two indicators of sympathetic activity[Conclusion]These data indicate that resveratrol reverses age-related dysfunction in endothelium-dependent vasodilation in MAs and partially reverses hyperactivity of adrenomedullary function with age. This treatment may have a therapeuticpotential in the treatment of cardiovascular diseases or hypertension in the elderly.

Influence of different periods of the year and age on the parameters of antioxidative status and oxidative stress in the blood serum of breeding bulls

The sources of variations that may cause physiological differences between blood serum biochemistry parameters of bulls have not been investigated in detail. Aim of the present study was to establish influence of different periods of the year and the age of breeding bulls on parameters of antioxidative status and oxidative stress in their serum and to correlate these monitored variables. Research was performed on two groups, each comprising 9 Simmental bulls: a younger group (YB) (aged 2–4 years) and older one (OB) (aged 5–10 years). Blood samples for biochemical analyses were collected from jugular vein in cold (CP) and warm periods (WP) of the year. Reduced glutathione (GSH), uric acid (UA), total protein (TP), albumin (ALB), thiobarbituric acid reactive substance (TBARS), and protein carbonyl content (PCC) serum concentration were determined, as well as activities of selenium-dependent glutathione peroxidase (Se-GSH-Px), total superoxide dismutase (TSOD), manganese superoxide dismutase (MnSOD), copper–zinc superoxide dismutase (CuZnSOD) and catalase (CAT). Serum values of SeGSH-Px, MnSOD, UA and TP in OB were significantly higher compared to those in YB during CP of the year. Significantly higher PCC concentration in serum of YB and OB were established in CP of the year than in WP. TBARS serum concentration in YB was significantly higher in comparison to that in OB during CP of the year. It can be concluded that both OB and YB show a great sensitivity to climate condition alterations during CP in comparison to WP of the year and that YB show even greater sensitivity.

Disease Mechanisms in ALS: Misfolded SOD1 Transferred Through Exosome-Dependent and Exosome-Independent Pathways.

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neuromuscular degenerative disorder with a poorly defined etiology. ALS patients experience motor weakness, which starts focally and spreads throughout the nervous system, culminating in paralysis and death within a few years of diagnosis. While the vast majority of clinical ALS is sporadic with no known cause, mutations in human copper-zinc superoxide dismutase 1 (SOD1) cause about 20% of inherited cases of ALS. ALS with SOD1 mutations is caused by a toxic gain of function associated with the propensity of mutant SOD1 to misfold, presenting a non-native structure. The mechanisms responsible for the progressive spreading of ALS pathology have been the focus of intense study. We have shown that misfolded SOD1 protein can seed misfolding and aggregation of endogenous wild-type SOD1 similar to amyloid-β and prion protein seeding. Our recent observations demonstrate a transfer of the misfolded SOD1 species from cell to cell, modeling the intercellular transmission of disease through the neuroaxis. We have shown that both mutant and misfolded wild-type SOD1 can traverse cell-to-cell, either as protein aggregates that are released from dying cells and taken up by neighboring cells via macropinocytosis, or in association with vesicles which are released into the extracellular environment. Furthermore, once misfolding of wild-type SOD1 has been initiated in a human cell culture, it can induce misfolding in naïve cell cultures over multiple passages of media transfer long after the initial misfolding template is degraded. Herein we review the data on mechanisms of intercellular transmission of misfolded SOD1.

A wheat superoxide dismutase gene TaSOD2 enhances salt resistance through modulating redox homeostasis by promoting NADPH oxidase activity.

Superoxide dismutase (SOD) is believed to enhance abiotic stress resistance by converting superoxide radical (O2 (-)) to H2O2 to lower ROS level and maintain redox homeostasis. ROS level is controlled via biphasic machinery of ROS production and scavenging. However, whether the role of SOD in abiotic stress resistance is achieved through influencing the biophasic machinery is not well documented. Here, we identified a wheat copper-zinc (Cu/Zn) SOD gene, TaSOD2, who was responsive to NaCl and H2O2. TaSOD2 overexpression in wheat and Arabidopsis elevated SOD activities, and enhanced the resistance to salt and oxidative stress. TaSOD2 overexpression reduced H2O2 level but accelerated O2 (-) accumulation. Further, it improved the activities of H2O2 metabolic enzymes, elevated the activity of O2 (-) producer NADPH oxidase (NOX), and promoted the transcription of NOX encoding genes. The inhibition of NOX activity and the mutation of NOX encoding genes both abolished the salt resistance of TaSOD2 overexpression lines. These data indicate that Cu/Zn SOD enhances salt resistance, which is accomplished through modulating redox homeostasis via promoting NOX activity.

Critical Nucleus Structure and Aggregation Mechanism of the C-terminal Fragment of Copper-Zinc Superoxide Dismutase Protein.

The aggregation of the copper-zinc superoxide dismutase (SOD1) protein is linked to familial amyotrophic lateral sclerosis, a progressive neurodegenerative disease. A recent experimental study has shown that the (147)GVIGIAQ(153) SOD1 C-terminal segment not only forms amyloid fibrils in isolation but also accelerates the aggregation of full-length SOD1, while substitution of isoleucine at site 149 by proline blocks its fibril formation. Amyloid formation is a nucleation-polymerization process. In this study, we investigated the oligomerization and the nucleus structure of this heptapeptide. By performing extensive replica-exchange molecular dynamics (REMD) simulations and conventional MD simulations, we found that the GVIGIAQ hexamers can adopt highly ordered bilayer β-sheets and β-barrels. In contrast, substitution of I149 by proline significantly reduces the β-sheet probability and results in the disappearance of bilayer β-sheet structures and the increase of disordered hexamers. We identified mixed parallel-antiparallel bilayer β-sheets in both REMD and conventional MD simulations and provided the conformational transition from the experimentally observed parallel bilayer sheets to the mixed parallel-antiparallel bilayer β-sheets. Our simulations suggest that the critical nucleus consists of six peptide chains and two additional peptide chains strongly stabilize this critical nucleus. The stabilized octamer is able to recruit additional random peptides into the β-sheet. Therefore, our simulations provide insights into the critical nucleus formation and the smallest stable nucleus of the (147)GVIGIAQ(153) peptide.

Oligomeric Structures of an Aggregation-Triggering Fragment of SOD1 Protein

Abstract: Amyloid fibril formation of copper-zinc superoxide dismutase (SOD1) is linked to familial amyotrophic lateral sclerosis (fALS), a progressive neurodegenerative disease. A recent experimental study has shown that 147GVIGIAQ153 peptide of the SOD1 C-terminal segment can form amyloid fibril and also accelerate the aggregation of full-length SOD1, while substitution of isoleucine at site 149 by proline blocks fibril formation of this peptide. However, the oligomeric structure and aggregation mechanism of this fragment remain elusive. In this study, we have investigated the hexameric structures of GVIGIAQ and its I149P variant by performing extensive atomistic explicit-solvent replica exchange molecular dynamics (REMD) simulations. We found that the GVIGIAQ hexamers adopt various β-sheet-rich conformations, including disordered aggregates and, to a much lesser extent, ordered fibril-like bilayer β-sheets and β-barrels. Structural analysis shows that four-stranded monolayer β-sheets are the smallest β-sheets that can form both bilayer β-sheets and β-barrels. In contrast, substitution of I149 by proline significantly reduces the β-sheet probability of all residues, especially those close to I149 in amino acid sequence, thus resulting in the vanish of bilayer β-sheets, a decrease of β-barrels, and a dramatic increase of disordered aggregates. The lack of bilayer β-sheets provides an explanation for the experimental observation of no fibril formation for I149P variant. MD simulation results on the structural stability of different sizes of experimental bilayer β-sheet fibril models further support the importance of I149 on fibril formation and also suggest that the bilayer β-sheet consisting of eight chains might be the smallest nucleus for 147GVIGIAQ153 fibril formation.

Acute exercise stress promotes Ref1/Nrf2 signalling and increases mitochondrial antioxidant activity in skeletal muscle.

What is the central question of this study? How does acute exercise affect the redox effector factor-1 (Ref1) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling and its association with mitochondrial H(2)O(2) production and antioxidant capacity? What is the main finding and its importance? Ref1/Nrf2 signalling in skeletal muscles was activated by acute exercise, and this activation was correlated with increased mitochondrial H(2)O(2) content and antioxidant capacity (reduced glutathione and manganese superoxide dismutase). The finding indicates that the oxidative stress induced by acute exercise in skeletal muscle mitochondria is associated with the upregulation of Ref1/Nrf2 signalling and enhancement of antioxidant defense pathways. This mechanism may play a role in preventing cellular oxidative stress resistance during acute exercise. The molecular mechanism of exercise-induced oxidative stress and adaptive activation of antioxidant responses in skeletal muscle has not been fully elucidated. This study aimed to investigate the effect of acute exercise on redox effector factor-1 (Ref1) and nuclear factor erythroid 2-related factor 2 (Nrf2) signalling and associations with mitochondrial H(2)O(2) production and antioxidant mechanisms in skeletal muscles. Groups of male ICR/CD-1 mice were subjected to an acute exercise bout of different durations (45, 90, 120 or 150 min). Muscle tissues (gastrocnemius and quadriceps femoris) were harvested after exercise to measure mitochondrial manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities, reduced glutathione (GSH) content and expression of Ref1/Nrf2 genes and Ref1/Nrf2 proteins. The acute exercise increased oxidative stress and activated Ref1/Nrf2 signalling in a time-dependent manner, with a linear correlation between the mitochondrial H(2)O(2) content and Ref1/Nrf2 expressions. The GSH content and MnSOD activity were also significantly increased, but CuZnSOD activity was not significantly affected. The findings indicate that the H(2)O(2) production induced by acute exercise in skeletal muscle mitochondria in the mouse is closely associated with upregulation of the Ref1/Nrf2 signalling pathway and enhancement of antioxidant defense components, including GSH and MnSOD. Activation of Ref1/Nrf2/antioxidant defense pathways may play a role in preventing cellular oxidative stress resistance during acute exercise.

Poor maternal nutrition and accelerated postnatal growth induces an accelerated aging phenotype and oxidative stress in skeletal muscle of male rats.

ABSTRACT‘Developmental programming’, which occurs as a consequence of suboptimal in utero and early environments, can be associated with metabolic dysfunction in later life, including an increased incidence of cardiovascular disease and type 2 diabetes, and predisposition of older men to sarcopenia. However, the molecular mechanisms underpinning these associations are poorly understood. Many conditions associated with developmental programming are also known to be associated with the aging process. We therefore utilized our well-established rat model of low birth weight and accelerated postnatal catch-up growth (termed ‘recuperated’) in this study to establish the effects of suboptimal maternal nutrition on age-associated factors in skeletal muscle. We demonstrated accelerated telomere shortening (a robust marker of cellular aging) as evidenced by a reduced frequency of long telomeres (48.5-8.6 kb) and an increased frequency of short telomeres (4.2-1.3 kb) in vastus lateralis muscle from aged recuperated offspring compared to controls. This was associated with increased protein expression of the DNA-damage-repair marker 8-oxoguanine-glycosylase (OGG1) in recuperated offspring. Recuperated animals also demonstrated an oxidative stress phenotype, with decreased citrate synthase activity, increased electron-transport-complex activities of complex I, complex II-III and complex IV (all markers of functional mitochondria), and increased xanthine oxidase (XO), p67phox and nuclear-factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Recuperated offspring also demonstrated increased antioxidant defense capacity, with increased protein expression of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), catalase and heme oxygenase-1 (HO1), all of which are known targets of NF-κB and can be upregulated as a consequence of oxidative stress. Recuperated offspring also had a pro-inflammatory phenotype, as evidenced by increased tumor necrosis factor-α (TNFα) and interleukin-1β (IL1β) protein levels. Taken together, we demonstrate, for the first time to our knowledge, an accelerated aging phenotype in skeletal muscle in the context of developmental programming. These findings may pave the way for suitable interventions in at-risk populations.

Contribution of CD3 &amp;lt;i&amp;gt;ε&amp;lt;/i&amp;gt; Epitope and Oxidative Type of Copper-Zinc Superoxide Dismutase to the Degeneration Processes of Cerebellar Purkinje Cells in Patients with Multiple System Atrophy-Cerebellar Type (MSA-C: Olivopontocerebellar Atrophy, OPCA): An Immunohistochemical Study

Objective: This study aimed to investigate the contribution of CD3 epsilon (e) epitope and oxidative type of copper-zinc superoxide dismutase to the degeneration processes of cerebellar Purkinje cells in patients with Multiple System Atrophy-Cerebellar type (MSA-C). Methods: This retrospective study was carried out on autopsy specimens of 17 patients with sporadic MSA-C and 10 normal individuals. Paraffin sections of autopsied cerebella and pontes were immunostained with polyclonal antibodies against CD3 e epitope and oxidative modification to cysteine sulfonic acid of cys111 in human copper-zinc superoxide dismutase (Ox-SOD1). With respect to the areas of CD3-e-epitope expression, the immunohistochemical study and the quantitative statistical analysis between the areas of CD3-e-epitope expression in the surviving Purkinje cells of MSA-C patients and their disease duration were performed. Results: The cell bodies and dendritic arborization including primary, secondary, and tertiary dendrites of normal Purkinje cells were intensely immunostained by the antibody against CD3 e epitope. Both the immunohistochemical study and the quantitative statistical analysis revealed that the areas positive for CD3 e epitope disappeared in the order from tertiary dendrites, secondary dendrites, primary dendrites toward the cell bodies, along with the disease progression. In addition, Glial Cytoplasmic Inclusions (GCIs) and Neuronal Cytoplasmic Inclusions (NCIs) were strongly positive for CD3 e epitope. The surviving Purkinje cells in MSA-C showed immunostaining by the anti-Ox-SOD1 antibody, although normal Purkinje cells did not. Conclusion: Based on the oxidative stress that the surviving Purkinje cells in MSA-C express Ox-SOD1, the functions of morphogenesis and morphological maintenance related to CD3-e-epitope expression of the MSA-C Purkinje cells are impaired from the peripheral dendrites toward the cell bodies as the center of the Purkinje cell system. In addition, GCIs and NCIs that are pathological hallmarks of MSA also intensely express CD3 e epitope.

Electron transfer mechanism of catalytic superoxide dismutation via Cu(ii/i) complexes: evidence of cupric-superoxo/-hydroperoxo species.

To understand the electron transfer mechanisms (outer versus inner sphere) of catalytic superoxide dismutation via a Cu(ii/i) redox couple such as occur in the enzyme copper-zinc superoxide dismutase, the Cu(ii/i) complexes [(L1)2Cu](ClO4)2·CH3CN, (1·CH3CN) and [(L1)2Cu](ClO4), (2) supported by a bis-N2Sthioether ligand, 2-pyridyl-N-(2'-methylthiophenyl)methyleneimine (L1) have been synthesized and structurally characterised. Both 1 and 2 display the same cyclic voltammogram (CV) featuring a quasireversible response at E1/2 = +0.33 V vs. SCE that falls in the SOD potential window of -0.04 V to +0.99 V. These complexes catalytically dismutate superoxide radicals at 298 K in aqueous medium (the IC50 for 1 is 2.15 μM). Electronic absorption spectra (233 K and 298 K), FTIR, ESI mass spectra, CV (233 K and 298 K) and DFT calculations collectively indicate formation of [(L1)2Cu(O2˙(-))](+), [(L1)2Cu(O2(2-))] and [(L1)2Cu(OOH(-))](+) species and help to elucidate the electron transfer mechanism for the SOD function of 1 and 2. Once O2˙(-) binds to Cu(II) (evident at 233 K), the first step of the catalytic cycle (Cu(II) + O2˙(-)→ Cu(I) + O2) does not follow but the second step (Cu(I) + O2˙(-) + 2H(+)→ H2O2 + Cu(II)) does follow. Therefore, the catalytic disproportionation of superoxide radicals via1 and 2 at 298 K indicates that the first and second steps of the catalytic cycle proceed through outer and inner sphere electron transfer mechanisms, respectively. Feasibility of the first step to occur in pure aprotic solvent (where 18-crown-6-ether is used to solubilise KO2) was tested and also supports the same notion of the electron transfer mechanisms as stated above.

No evidence for oxidative stress in the cerebellar tissues or cells of juvenile male mice exposed via lactation to the 6 non-dioxin-like PCBs at levels below the regulatory safe limits for humans.

The developing central nervous system is particularly vulnerable to environmental contaminants such as non-dioxin-like polychlorinated biphenyls (NDL-PCBs). This study investigated the potential oxidative effects in mice pups exposed via lactation to the sum of the six indicator NDL-PCBs (∑6 NDL-PCBs) at 0, 1, 10 and 100 ng/kg per 14 days, constituting levels below the guidance values fixed by French food safety agencies for humans at 10 ng/kg body weight per day. For this purpose, the oxidative status was assessed by flow cytometry via dichloro-dihydro-fluorescein diacetate in the cerebellum of juvenile male offspring mice during brain growth spurt [postnatal day (PND) 14]. No significant differences were found in the levels of reactive oxygen species in the cerebellar neurons or glial cells (astrocytes, oligodendrocytes and microglia) of lactationally exposed male mice at PND 14 (p>0.05). Concordantly, oxidative-stress related gene expression was measured by qPCR for catalase, copper zinc superoxide dismutase 1, glyoxalase 1, glutathione peroxidase 1, and glutathione reductase 1, in the cerebellum at PND 14 appeared unaffected, as also verified at the protein level by immunoblots. Moreover, transcriptomic data from our previous work have not shown differences in the mRNA expressions of genes belonging to GO terms involved in oxidative stress in neurons of male mice exposed to ∑6 NDL-PCBs compared to controls; except for glyoxalase 1 which was downregulated in neurons isolated from exposed group compared to controls. Our findings suggest that lactational exposure to NDL-PCBs at environmental relevant concentrations may not cause significant oxidative effect on juvenile cerebellum.

Dietary copper requirement of juvenile Russian sturgeon Acipenser gueldenstaedtii

To quantify the dietary copper (Cu) requirement of Russian sturgeon Acipenser gueldenstaedtii, copper sulfate (CuSO4·5H2O) was added into the basal semi-purified diet at seven levels 0, 1, 2, 4, 6, 8 and 16mgCukg−1 diet yielding the actual dietary values of 0.3, 1.5, 2.4, 4.4, 6.2, 8.3 and 16.1mgCukg−1 diet, respectively. Each diet was fed to sturgeon (11.84±0.07g) in triplicate in a flow-through system for 8weeks. The Cu concentration in the rearing water was 0.8–1.2μgL−1 during the trial period. The weight gain rate (WGR) and feed efficiency were significantly higher in fish fed 2.4 to 8.3mgCukg−1 than those fed 0.3, 1.5 or 16.1mgCukg−1. The highest WGR was found in fish fed 6.2mgCukg−1. The whole-body Cu concentrations were highest in fish fed 6.2 to 16.1mgCukg−1. The liver Cu concentration was highest in fish fed 16.1mgCukg−1 diet and lowest in fish fed diets Cu levels range from 0.3 to 4.4mgCukg−1. Hepatic copper–zinc superoxide dismutase (Cu–Zn SOD) activities, total antioxidant capacity and serum ceruloplasmin activity were significantly higher in fish fed 6.2 and 8.3mgCukg−1 diet than those in other treatments. The lowest hepatic malondialdehyde values were observed in fish fed 6.2 and 8.3mgCukg−1. No significant differences were in survival, hepatosomatic index, viscerosomatic index and condition factor among all the treatments. There were also no significant differences in whole body moisture, crude protein, crude lipid and ash between all the treatments. Analysis of broken-line regression based on the WGR, whole-body Cu concentration, hepatic Cu–Zn SOD and serum ceruloplasmin activity of the fish fed different diets indicates that the minimum Cu concentration in the semi-purified diet for juvenile Russian sturgeon (12–70g) is at 7–8mgkg−1. Statement of relevanceThe dietary copper (Cu) requirement of juvenile Russian sturgeon was investigated. Dietary Cu deficiency and over-loading significantly reduced growth, feed utilization and antioxidant capacity. The results suggest that the appropriate dietary Cu content is considerable in Russian sturgeon farming. Minimum dietary Cu level for this species is recommend as 7–8mgCukg−1 in this study. These information will be useful in developing a practical diet for Russian sturgeon culture.

Effects of maternal dietary manganese and incubation temperature on hatchability, antioxidant status, and expression of heat shock proteins in chick embryos.

To investigate whether supplementing manganese (Mn) to the maternal diet could reduce the deleterious effect of heat stress on the developing embryo, the hatchability, antioxidant status, and expression of heat shock proteins (HSP) were evaluated in chick embryos under normal and high incubation temperatures. A completely randomized design ( = 6) with 2 maternal dietary Mn treatments (unsupplemented control basal diet versus the basal diet + 120 mg Mn/kg as inorganic Mn) × 2 incubation temperatures (normal, 37.8°C, versus high, 39.0°C) was used. High incubation temperature did not affect ( > 0.19) hatchability and embryo mortality and development but did increase ( < 0.05) activities of heart manganese superoxide dismutase (MnSOD) and liver copper zinc superoxide dismutase and liver MnSOD mRNA and protein levels in embryos. High incubation temperature also decreased ( < 0.003) HSP70 protein level in the heart but had no effects ( > 0.07) in the liver of embryos. Maternal diet with Mn supplementation not only increased ( < 0.05) the hatchability and Mn content ( < 0.001) in the yolk and embryonic tissues and the activity of MnSOD in the heart ( < 0.004) as well as relative liver weight ( < 0.05) under normal incubation temperature but also decreased ( ≤ 0.05) embryo mortality and HSP90 mRNA level in the liver and heart of embryos. Furthermore, under high incubation temperature, maternal diet Mn supplementation increased ( < 0.002) MnSOD protein expression in the liver of embryos but had no effect ( > 0.43) under normal incubation temperature. These results indicated that high incubation temperature induced self-protective responses of chick embryos with a modification of antioxidant status and a depression of HSP70 protein level. Maternal dietary supplementation of Mn could improve the hatchability as well as antioxidant ability to protect against heat challenge in embryos during incubation.

The Disulfide Bond, but Not Zinc or Dimerization, Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of post-translational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57-Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.

Mahogunin ring finger 1 confers cytoprotection against mutant SOD1 aggresomes and is defective in an ALS mouse model

Proteotoxicity of misfolded, disease-causing proteins is deeply implicated in the pathomechanisms for neurodegenerative diseases including copper-zinc superoxide dismutase (SOD1)-linked amyotrophic lateral sclerosis (ALS). However, the precise cellular quality control (QC) mechanisms against aggregation of misfolded mutant SOD1 proteins remain elusive. Here, we found that the Mahogunin ring finger-1 (MGRN1) E3 ubiquitin ligase, which catalyzes mono-ubiquitination to the substrate, was dysregulated in the cellular and mouse models of ALS and that it preferentially interacted with various mutant forms of SOD1. Intriguingly, the motor neurons of presymptomatic ALS mice have diminished MGRN1 cytoplasmic distribution. MGRN1 was partially recruited to mutant SOD1 inclusions where they were positive for p62 and Lamp2. Moreover, overexpression of MGRN1 reduced mutant SOD1 aggregation and alleviated its proteotoxic effects on cells. Taken together, our findings suggest that MGRN1 contributes to the clearance of toxic mutant SOD1 inclusions likely through autophagic pathway, and, most likely, the sequestration of MGRN1 sensitizes motor neurons to degeneration in the ALS mouse model. Furthermore, the present study identifies the MGRN1-mediated protein QC mechanism as a novel therapeutic target in neurodegenerative diseases.

Clones of FeSOD, MDHAR, DHAR Genes from White Clover and Gene Expression Analysis of ROS-Scavenging Enzymes during Abiotic Stress and Hormone Treatments.

Increased transcriptional levels of genes encoding antioxidant enzymes play important protective roles in coping with excessive accumulation of reactive oxygen species (ROS) in plants exposed to various abiotic stresses. To fully elucidate different evolutions and functions of ROS-scavenging enzymatic genes, we isolated iron superoxide dismutase (FeSOD), dehydroascorbate reductase (DHAR) and monodehydroascorbate reductase (MDHAR) from white clover for the first time and subsequently tested dynamic expression profiles of these genes together with previously identified other antioxidant enzyme genes including copper zinc superoxide dismutase (Cu/ZnSOD), manganese superoxide dismutase (MnSOD), glutathione reductase (GR), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) in response to cold, drought, salinity, cadmium stress and exogenous abscisic acid (ABA) or spermidine (Spd) treatment. The cloned fragments of FeSOD, DHAR and MDHAR genes were 630, 471 and 669 bp nucleotide sequences encoding 210, 157 and 223 amino acids, respectively. Phylogenetic analysis indicated that both amino acid and nucleotide sequences of these three genes are highly conservative. In addition, the analysis of genes expression showed the transcription of GR, POD, MDHAR, DHAR and Cu/ZnSOD were rapidly activated with relatively high abundance during cold stress. Differently, CAT, APX, FeSOD, Cu/ZnSOD and MnSOD exhibited more abundant transcripts compared to others under drought stress. Under salt stress, CAT was induced preferentially (3–12 h) compared to GR which was induced later (12–72 h). Cadmium stress mainly up-regulated Cu/ZnSOD, DHAR and MDHAR. Interestingly, most of genes expression induced by ABA or Spd happened prior to various abiotic stresses. The particular expression patterns and different response time of these genes indicated that white clover differentially activates genes encoding antioxidant enzymes to mitigate the damage of ROS during various environmental stresses.

Effect of dietary manganese on antioxidant status and expression levels of heat-shock proteins and factors in tissues of laying broiler breeders under normal and high environmental temperatures.

To investigate the effect of Mn on antioxidant status and expression levels of heat-shock proteins/factors in tissues of laying broiler breeders subjected to heat challenge, we used a completely randomised design (n 6) with a factorial arrangement of 2 environmental temperatures (normal, 21 (sem 1)°C and high, 32 (sem 1)°C)×3 dietary Mn treatments (an Mn-unsupplemented basal diet (CON), or a basal diet supplemented with 120 mg Mn/kg diet as inorganic Mn sulphate (iMn) or organic Mn proteinate (oMn)). There were no interactions (P>0·10) between environmental temperature and dietary Mn in all of the measured indices. High temperature decreased (P<0·003) Mn content, and also tended (P=0·07) to decrease copper zinc superoxide dismutase (CuZnSOD) activity in the liver and heart. However, an increased manganese superoxide dismutase (MnSOD) activity (P<0·05) and a slight increase of malondialdehyde level (P=0·06) were detected in breast muscle. Up-regulated (P<0·05) expression levels of heat-shock factor 1 (HSF1) and HSF3 mRNA and heat-shock protein 70 (HSP70) mRNA and protein were found in all three tissues. Broiler breeders fed either iMn or oMn had higher tissue Mn content (P<0·0001), heart MnSOD and CuZnSOD activities (P<0·01) and breast muscle MnSOD protein levels (P<0·05), and lower (P<0·05) breast muscle HSP70 mRNA and protein levels than those fed CON. Broiler breeders fed oMn had higher (P<0·03) bone Mn content than those fed iMn. These results indicate that high temperature decreases Mn retention and increases HSP70 and HSF1, HSF3 expression levels in tissues of laying broiler breeders. Furthermore, dietary supplementation with Mn in either source may enhance heart antioxidant ability and inhibit the expression of HSP70 in breast muscle. Finally, the organic Mn appears to be more available than inorganic Mn for bone in laying broiler breeders regardless of environmental temperatures.