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Sample records for superoxide dismutase-1 sod1

  1. Superoxide dismutase 1 and tgSOD1 mouse spinal cord seed fibrils, suggesting a propagative cell death mechanism in amyotrophic lateral sclerosis.

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    Ruth Chia

    Full Text Available BACKGROUND: Amyotrophic lateral sclerosis (ALS is a neurodegenerative disease that specifically affects motor neurons and leads to a progressive and ultimately fatal loss of function, resulting in death typically within 3 to 5 years of diagnosis. The disease starts with a focal centre of weakness, such as one limb, and appears to spread to other parts of the body. Mutations in superoxide dismutase 1 (SOD1 are known to cause disease and it is generally accepted they lead to pathology not by loss of enzymatic activity but by gain of some unknown toxic function(s. Although different mutations lead to varying tendencies of SOD1 to aggregate, we suggest abnormal proteins share a common misfolding pathway that leads to the formation of amyloid fibrils. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that misfolding of superoxide dismutase 1 leads to the formation of amyloid fibrils associated with seeding activity, which can accelerate the formation of new fibrils in an autocatalytic cascade. The time limiting event is nucleation to form a stable protein "seed" before a rapid linear polymerisation results in amyloid fibrils analogous to other protein misfolding disorders. This phenomenon was not confined to fibrils of recombinant protein as here we show, for the first time, that spinal cord homogenates obtained from a transgenic mouse model that overexpresses mutant human superoxide dismutase 1 (the TgSOD1(G93A mouse also contain amyloid seeds that accelerate the formation of new fibrils in both wildtype and mutant SOD1 protein in vitro. CONCLUSIONS/SIGNIFICANCE: These findings provide new insights into ALS disease mechanism and in particular a mechanism that could account for the spread of pathology throughout the nervous system. This model of disease spread, which has analogies to other protein misfolding disorders such as prion disease, also suggests it may be possible to design assays for therapeutics that can inhibit fibril propagation and

  2. The effects of 2% rebamipide ophthalmic solution on the tear functions and ocular surface of the superoxide dismutase-1 (sod1) knockout mice.

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    Ohguchi, Takeshi; Kojima, Takashi; Ibrahim, Osama M; Nagata, Taeko; Shimizu, Takahiko; Shirasawa, Takuji; Kawakita, Tetsuya; Satake, Yoshiyuki; Tsubota, Kazuo; Shimazaki, Jun; Ishida, Susumu

    2013-11-21

    To investigate the efficacy of 2% rebamipide ophthalmic solution on the tear functions and ocular surface status of the superoxide dismutase-1(Sod1(-/-)) mice. Two percent Rebamipide ophthalmic solution was applied to 40-week-old male Sod1(-/-) and wild-type (WT) mice four times a day for 2 weeks. We examined the cytokine concentrations in the tear fluid (by CytoBead assay), tear film break-up time, amount of tear production, and expressions of mucins 1, 4, and 5AC, by RT-PCR. We also performed vital staining of the ocular surface, PAS staining for muc5AC, and immunohistochemical stainings for 4-hydroxy-2-nonenal (4-HNE), 8-hydroxy-2'-deoxyguanosine (8-OHdG), in the conjunctiva to compare the results before and after rebamipide instillations. The tear functions and ocular surface epithelial damage scores were significantly worse in the Sod1(-/-) than in the WT mice. Application of 2% rebamipide for 2 weeks significantly improved the tear film break-up time, the amount of tear production, and the corneal epithelial damage scores, which also significantly increased the conjunctival goblet cell density and muc5 mRNA expression, in the Sod1(-/-) mice. The mean IL-6, IL-17, TNF-α, and IFN-γ levels in the tear fluid were reduced significantly along with a significant decrease in the density of cells positive for 4-HNE and 8-OHdG in the conjunctiva. Two percent Rebamipide ophthalmic solution significantly improved the tear stability and corneal epithelial damage, and enhanced the expression of muc5 mRNA on the ocular surface. We also observed anti-inflammatory effects in the tear film together with antioxidative effects in the conjunctiva, suggesting the efficacy of rebamipide in age-related dry eye disease attributable to SOD1 knockout.

  3. Mutant superoxide dismutase 1 (SOD1), a cause of amyotrophic lateral sclerosis, disrupts the recruitment of SMN, the spinal muscular atrophy protein to nuclear Cajal bodies.

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    Kariya, Shingo; Re, Diane B; Jacquier, Arnaud; Nelson, Katelyn; Przedborski, Serge; Monani, Umrao R

    2012-08-01

    Spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS) are among the most common motor neuron diseases to afflict the human population. A deficiency of the survival of motor neuron (SMN) protein causes SMA and is also reported to be an exacerbating factor in the development of ALS. However, pathways linking the two diseases have yet to be defined and it is not clear precisely how the pathology of ALS is aggravated by reduced SMN or whether mutant proteins underlying familial forms of ALS interfere with SMN-related biochemical pathways to exacerbate the neurodegenerative process. In this study, we show that mutant superoxide dismutase-1 (SOD1), a cause of familial ALS, profoundly alters the sub-cellular localization of the SMN protein, preventing the formation of nuclear 'gems' by disrupting the recruitment of the protein to Cajal bodies. Overexpressing the SMN protein in mutant SOD1 mice, a model of familial ALS, alleviates this phenomenon, most likely in a cell-autonomous manner, and significantly mitigates the loss of motor neurons in the spinal cord and in culture dishes. In the mice, the onset of the neuromuscular phenotype is delayed and motor function enhanced, suggestive of a therapeutic benefit for ALS patients treated with agents that augment the SMN protein. Nevertheless, this finding is tempered by an inability to prolong survival, a limitation most likely imposed by the inexorable denervation that characterizes ALS and eventually disrupts the neuromuscular synapses even in the presence of increased SMN.

  4. Enhancing NAD+ Salvage Pathway Reverts the Toxicity of Primary Astrocytes Expressing Amyotrophic Lateral Sclerosis-linked Mutant Superoxide Dismutase 1 (SOD1).

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    Harlan, Benjamin A; Pehar, Mariana; Sharma, Deep R; Beeson, Gyda; Beeson, Craig C; Vargas, Marcelo R

    2016-05-13

    Nicotinamide adenine dinucleotide (NAD(+)) participates in redox reactions and NAD(+)-dependent signaling pathways. Although the redox reactions are critical for efficient mitochondrial metabolism, they are not accompanied by any net consumption of the nucleotide. On the contrary, NAD(+)-dependent signaling processes lead to its degradation. Three distinct families of enzymes consume NAD(+) as substrate: poly(ADP-ribose) polymerases, ADP-ribosyl cyclases (CD38 and CD157), and sirtuins (SIRT1-7). Because all of the above enzymes generate nicotinamide as a byproduct, mammalian cells have evolved an NAD(+) salvage pathway capable of resynthesizing NAD(+) from nicotinamide. Overexpression of the rate-limiting enzyme in this pathway, nicotinamide phosphoribosyltransferase, increases total and mitochondrial NAD(+) levels in astrocytes. Moreover, targeting nicotinamide phosphoribosyltransferase to the mitochondria also enhances NAD(+) salvage pathway in astrocytes. Supplementation with the NAD(+) precursors nicotinamide mononucleotide and nicotinamide riboside also increases NAD(+) levels in astrocytes. Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons in the spinal cord, brain stem, and motor cortex. Superoxide dismutase 1 (SOD1) mutations account for up to 20% of familial ALS and 1-2% of apparently sporadic ALS cases. Primary astrocytes isolated from mutant human superoxide dismutase 1-overexpressing mice as well as human post-mortem ALS spinal cord-derived astrocytes induce motor neuron death in co-culture. Increasing total and mitochondrial NAD(+) content in ALS astrocytes increases oxidative stress resistance and reverts their toxicity toward co-cultured motor neurons. Taken together, our results suggest that enhancing the NAD(+) salvage pathway in astrocytes could be a potential therapeutic target to prevent astrocyte-mediated motor neuron death in ALS.

  5. Modification of superoxide dismutase 1 (SOD1 properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS.

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    James C Stevens

    Full Text Available BACKGROUND: Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS, many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts. METHODOLOGY/PRINCIPAL FINDINGS: Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.

  6. Modification of Superoxide Dismutase 1 (SOD1) Properties by a GFP Tag – Implications for Research into Amyotrophic Lateral Sclerosis (ALS)

    OpenAIRE

    Stevens, James C.; Ruth Chia; Hendriks, William T.; Virginie Bros-Facer; Jan van Minnen; Joanne E Martin; Jackson, Graham S.; Linda Greensmith; Giampietro Schiavo; Elizabeth M. C. Fisher

    2010-01-01

    BACKGROUND: Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whet...

  7. Computing Stability Effects of Mutations in Human Superoxide Dismutase 1

    DEFF Research Database (Denmark)

    Kepp, Kasper Planeta

    2014-01-01

    Protein stability is affected in several diseases and is of substantial interest in efforts to correlate genotypes to phenotypes. Superoxide dismutase 1 (SOD1) is a suitable test case for such correlations due to its abundance, stability, available crystal structures and thermochemical data...

  8. Glial nuclear aggregates of superoxide dismutase-1 are regularly present in patients with amyotrophic lateral sclerosis

    OpenAIRE

    2011-01-01

    The most common cause of amyotrophic lateral sclerosis (ALS) is mutations in superoxide dismutase-1 (SOD1). Since there is evidence for the involvement of non-neuronal cells in ALS, we searched for signs of SOD1 abnormalities focusing on glia. Spinal cords from nine ALS patients carrying SOD1 mutations, 51 patients with sporadic or familial ALS who lacked such mutations, and 46 controls were examined by immunohistochemistry. A set of anti-peptide antibodies with specificity for misfolded SOD1...

  9. Superoxide Dismutase 1 Nanozyme for Treatment of Eye Inflammation

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    Olga A. Kost

    2016-01-01

    Full Text Available Use of antioxidants to mitigate oxidative stress during ocular inflammatory diseases has shown therapeutic potential. This work examines a nanoscale therapeutic modality for the eye on the base of antioxidant enzyme, superoxide dismutase 1 (SOD1, termed “nanozyme.” The nanozyme is produced by electrostatic coupling of the SOD1 with a cationic block copolymer, poly(L-lysine-poly(ethyleneglycol, followed by covalent cross-linking of the complexes with 3,3′-dithiobis(sulfosuccinimidylpropionate sodium salt. The ability of SOD1 nanozyme as well as the native SOD1 to reduce inflammatory processes in the eye was examined in vivo in rabbits with immunogenic uveitis. Results suggested that topical instillations of both enzyme forms demonstrated anti-inflammatory activity; however, the nanozyme was much more effective compared to the free enzyme in decreasing uveitis manifestations. In particular, we noted statistically significant differences in such inflammatory signs in the eye as the intensities of corneal and iris edema, hyperemia of conjunctiva, lens opacity, fibrin clots, and the protein content in aqueous humor. Clinical findings were confirmed by histological data. Thus, SOD1-containing nanozyme is potentially useful therapeutic agent for the treatment of ocular inflammatory disorders.

  10. Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts.

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    Turner, Bradley J; Lopes, Elizabeth C; Cheema, Surindar S

    2004-04-01

    High molecular weight detergent-insoluble complexes of superoxide dismutase 1 (SOD1) enzyme are a biochemical abnormality associated with mutant SOD1-linked familial amyotrophic lateral sclerosis (FALS). In the present study, SOD1 protein from spinal cords of transgenic FALS mice was fractionated according to solubility in saline, zwitterionic, non-ionic or anionic detergents. Both endogenous mouse SOD1 and mutant human SOD1 were least soluble in SDS, followed by NP-40 and CHAPS, with an eight-fold greater detergent resistance of mutant protein overall. Importantly, high molecular weight mutant SOD1 complexes were isolated with SDS-extraction only. To reproduce SOD1 aggregate pathology in vitro, primary fibroblasts were isolated and cultured from neonatal transgenic FALS mice. Fibroblasts expressed abundant mutant SOD1 without spontaneous aggregation over time with passage. Proteasomal inhibition of cultures using lactacystin induced dose-dependent aggregation and increased the SDS-insoluble fraction of mutant SOD1, but not endogenous SOD1. In contrast, paraquat-mediated superoxide stress in fibroblasts promoted aggregation of endogenous SOD1, but not mutant SOD1. Treatment of cultures with peroxynitrite or the copper chelator diethyldithiocarbamate (DDC) alone did not modulate aggregation. However, DDC inhibited lactacystin-induced mutant SOD1 aggregation in transgenic fibroblasts, while exogenous copper slightly augmented aggregation. These data suggest that SOD1 aggregates may derive from proteasomal or oxidation-mediated oligomerisation pathways from mutant and endogenous subunits respectively. Furthermore, these pathways may be affected by copper availability. We propose that non-neural cultures such as these transgenic fibroblasts with inducible SOD1 aggregation may be useful for rapid screening of compounds with anti-aggregation potential in FALS.

  11. Localization and distribution of superoxide dismutase-1 in the neural tube morphogenesis of chick embryo.

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    Dhage, Prajakta A; Kamble, Lekha K; Bhargava, Shobha Y

    2017-02-01

    Superoxide dismutase 1 (SOD- 1) is an antioxidant enzyme that regulates the levels of Reactive oxygen species (ROS) by catalyzing the conversion of superoxide radical into hydrogen peroxide (H2O2) and oxygen. ROS are known to play a significant role in various cellular processes, via redox modification of a variety of molecules that participate in signaling pathways involved in this processes. As the levels of ROS in cells are controlled by the levels of antioxidant enzymes, thus SOD-1 may be indirectly involved in regulating different cellular processes by maintaining the required levels of H2O2. Therefore, in the present study we have investigated the possible involvement of SOD- 1 in the neurulation during the development of chick embryo. During gastrulation, SOD- 1 immunoreactivity was observed throughout the ectoderm and cauda mesoderm areas, however, its presence during neurulation was restricted to certain areas of neural tube particularly in the dorsal neural tube where neural tube closure takes place. Assaying enzyme activity revealed a significant increase in the SOD activity during neurulation. Further, inhibition of SOD- 1 by Diethyldithiocarbamate (DDC) induced abnormalities in the development of the neural tube. SOD- 1 inhibition specifically affected the closure of neural tube in the anterior region. Thus, here we report the presence of SOD- 1 mainly in the ectoderm and tissues of ectodermal origin during gastrulation to neurulation which suggests that it may be involved in the regulating the cellular processes during neural tube morphogenesis.

  12. Effects of histone acetylation on superoxide dismutase 1 gene expression in the pathogenesis of senile cataract

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    Rong, Xianfang; Qiu, Xiaodi; Jiang, Yongxiang; Li, Dan; Xu, Jie; Zhang, Yinglei; Lu, Yi

    2016-01-01

    Histone acetylation plays key roles in gene expression, but its effects on superoxide dismutase 1 (SOD1) expression in senile cataract remains unknown. To address this problem, the study was to investigate the influence of histone acetylation on SOD1 expression and its effects in the pathogenesis of senile cataract. Senile cataract was classified into three types—nuclear cataract (NC), cortical cataract (CC), and posterior subcapsular cataract (SC)—using the Lens Opacities Classification System III. In senile cataracts, SOD1 expression decreased significantly. Both H3 and H4 were deacetylated at −600 bp of the SOD1 promoter of cataract lenses, and hypoacetylated at −1500, −1200, and −900 bp. In hypoacetylated histones, the hypoacetylation pattern differed among the cataracts. In vitro, anacardic acid (AA) significantly reduced H3 and H4 acetylation at the SOD1 promoter, decreased protein expression, and induced cataract formation in rabbits. AA also inhibited HLEC viability and increased cell apoptosis. In contrast, trichostatin A (TSA) was able to efficaciously stop AA’s effects on both rabbit lenses and HLECs. Decreased histone acetylation at the SOD1 promoter is associated with declined SOD1 expression in senile cataracts. Histone acetylation plays an essential role in the regulation of SOD1 expression and in the pathogenesis of senile cataracts. PMID:27703255

  13. Brain beta-amyloid accumulation in transgenic mice expressing mutant superoxide dismutase 1.

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    Turner, Bradley J; Li, Qiao-Xin; Laughton, Katrina M; Masters, Colin L; Lopes, Elizabeth C; Atkin, Julie D; Cheema, Surindar S

    2004-12-01

    Oxidative stress is implicated in both the deposition and pathogenesis of beta-amyloid (Abeta) protein in Alzheimer's disease (AD). Accordingly, overexpression of the antioxidant enzyme superoxide dismutase 1 (SOD1) in neuronal cells and transgenic AD mice reduces Abeta toxicity and accumulation. In contrast, mutations in SOD1 associated with amyotrophic lateral sclerosis (ALS) confer enhanced pro-oxidative enzyme activities. We therefore examined whether ALS-linked mutant SOD1 overexpression in motor neuronal cells or transgenic ALS mice modulates Abeta toxicity or its accumulation in the brain. Aggregated, but not freshly solubilised, substrate-bound Abeta peptides induced degenerative morphology and cytotoxicity in motor neuron-like NSC-34 cells. Transfection of NSC-34 cells with human wild-type SOD1 attenuated Abeta-induced toxicity, however this neuroprotective effect was also observed for ALS-linked mutant SOD1. Analysis of the cerebral cortex, brainstem, cerebellum and olfactory bulb from transgenic SOD1G93A mice using enzyme-linked immunosorbent assay of acid-guanidine extracts revealed age-dependent elevations in Abeta levels, although not significantly different from wild-type mouse brain. In addition, brain amyloid protein precursor (APP) levels remained unaltered as a consequence of mutant SOD1 expression. We therefore conclude that mutant SOD1 overexpression promotes neither Abeta toxicity nor brain accumulation in these ALS models.

  14. Superoxide Dismutase1 Levels in North Indian Population with Age-Related Macular Degeneration

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    Akshay Anand

    2013-01-01

    Full Text Available Aim. The aim of the study was to estimate the levels of superoxide dismutase1 (SOD1 in patients of age-related macular degeneration (AMD and examine the role of oxidative stress, smoking, hypertension, and other factors involved in the pathogenesis of AMD. Methods. 115 AMD patients and 61 healthy controls were recruited for this study. Serum SOD1 levels were determined by ELISA and were correlated to various risk factors. Logistic regression model of authenticity, by considering SOD1 as independent variable, has been developed along with ROC curve. Results. The SOD1 levels were significantly higher in AMD patients as compared to those of the controls. The difference was not significant for wet and dry AMD. However, the difference was significant between wet AMD subtypes. Nonsignificance of the Hosmer-Lemeshow goodness of fit statistic (χ2=10.516, df=8, P=0.231 indicates the appropriateness of logistic regression model to predict AMD. Conclusion. Oxidative stress in AMD patients may mount compensatory response resulting in increased levels of SOD1 in AMD patients. To predict the risk of AMD on the basis of SOD1, a logistic regression model shows authenticity of 78%, and area under the ROC curve (0.827, P=.0001 with less standard error of 0.033 coupled with 95% confidence interval of 0.762–0.891 further validates the model.

  15. Superoxide dismutase 1 is positively selected to minimize protein aggregation in great apes.

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    Dasmeh, Pouria; Kepp, Kasper P

    2017-08-01

    Positive (adaptive) selection has recently been implied in human superoxide dismutase 1 (SOD1), a highly abundant antioxidant protein with energy signaling and antiaging functions, one of very few examples of direct selection on a human protein product (exon); the molecular drivers of this selection are unknown. We mapped 30 extant SOD1 sequences to the recently established mammalian species tree and inferred ancestors, key substitutions, and signatures of selection during the protein's evolution. We detected elevated substitution rates leading to great apes (Hominidae) at ~1 per 2 million years, significantly higher than in other primates and rodents, although these paradoxically generally evolve much faster. The high evolutionary rate was partly due to relaxation of some selection pressures and partly to distinct positive selection of SOD1 in great apes. We then show that higher stability and net charge and changes at the dimer interface were selectively introduced upon separation from old world monkeys and lesser apes (gibbons). Consequently, human, chimpanzee and gorilla SOD1s have a net charge of -6 at physiological pH, whereas the closely related gibbons and macaques have -3. These features consistently point towards selection against the malicious aggregation effects of elevated SOD1 levels in long-living great apes. The findings mirror the impact of human SOD1 mutations that reduce net charge and/or stability and cause ALS, a motor neuron disease characterized by oxidative stress and SOD1 aggregates and triggered by aging. Our study thus marks an example of direct selection for a particular chemical phenotype (high net charge and stability) in a single human protein with possible implications for the evolution of aging.

  16. Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation.

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    Ann-Sofi Johansson

    Full Text Available Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1, associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS. The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+ ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+ homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+ affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+. Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.

  17. Intrinsic properties of lumbar motor neurones in the adult G127insTGGG superoxide dismutase-1 mutant mouse in vivo: evidence for increased persistent inward currents

    DEFF Research Database (Denmark)

    Meehan, Claire Francesca; Moldovan, Mihai; Marklund, Stefan L.

    2010-01-01

    Aim: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a preferential loss of motoneurones. Previous publications using in vitro neonatal preparations suggest an increased excitability of motoneurones in various superoxide dismutase-1 (SOD1) mutant mice...

  18. Senescence marker protein-30/superoxide dismutase 1 double knockout mice exhibit increased oxidative stress and hepatic steatosis

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    Yoshitaka Kondo

    2014-01-01

    Full Text Available Superoxide dismutase 1 (SOD1 is an antioxidant enzyme that converts superoxide anion radicals into hydrogen peroxide and molecular oxygen. The senescence marker protein-30 (SMP30 is a gluconolactonase that functions as an antioxidant protein in mammals due to its involvement in ascorbic acid (AA biosynthesis. SMP30 also participates in Ca2+ efflux by activating the calmodulin-dependent Ca2+-pump. To reveal the role of oxidative stress in lipid metabolism defects occurring in non-alcoholic fatty liver disease pathogenesis, we generated SMP30/SOD1-double knockout (SMP30/SOD1-DKO mice and investigated their survival curves, plasma and hepatic lipid profiles, amounts of hepatic oxidative stress, and hepatic protein levels expressed by genes related to lipid metabolism. While SMP30/SOD1-DKO pups had no growth retardation by 14 days of age, they did have low plasma and hepatic AA levels. Thereafter, 39% and 53% of male and female pups died by 15–24 and 89 days of age, respectively. Compared to wild type, SMP30-KO and SOD1-KO mice, by 14 days SMP30/SOD1-DKO mice exhibited: (1 higher plasma levels of triglyceride and aspartate aminotransferase; (2 severe accumulation of hepatic triglyceride and total cholesterol; (3 higher levels of superoxide anion radicals and thiobarbituric acid reactive substances in livers; and (4 decreased mRNA and protein levels of Apolipoprotein B (ApoB in livers – ApoB is an essential component of VLDL secretion. These results suggest that high levels of oxidative stress due to concomitant deficiency of SMP30 and/or AA, and SOD1 cause abnormal plasma lipid metabolism, hepatic lipid accumulation and premature death resulting from impaired VLDL secretion.

  19. Disulfide scrambling in superoxide dismutase 1 reduces its cytotoxic effect in cultured cells and promotes protein aggregation.

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    Lina Leinartaitė

    Full Text Available Mutations in the gene coding for superoxide dismutase 1 (SOD1 are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS. These mutations are believed to result in a "gain of toxic function", leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn(2+ site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn(2+. This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.

  20. Superoxide Dismutase 1 Loss Disturbs Intracellular Redox Signaling, Resulting in Global Age-Related Pathological Changes

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    Kenji Watanabe

    2014-01-01

    Full Text Available Aging is characterized by increased oxidative stress, chronic inflammation, and organ dysfunction, which occur in a progressive and irreversible manner. Superoxide dismutase (SOD serves as a major antioxidant and neutralizes superoxide radicals throughout the body. In vivo studies have demonstrated that copper/zinc superoxide dismutase-deficient (Sod1−/− mice show various aging-like pathologies, accompanied by augmentation of oxidative damage in organs. We found that antioxidant treatment significantly attenuated the age-related tissue changes and oxidative damage-associated p53 upregulation in Sod1−/− mice. This review will focus on various age-related pathologies caused by the loss of Sod1 and will discuss the molecular mechanisms underlying the pathogenesis in Sod1−/− mice.

  1. Conjugates of Superoxide Dismutase 1 with Amphiphilic Poly(2-oxazoline) Block Copolymers for Enhanced Brain Delivery: Synthesis, Characterization and Evaluation in Vitro and in Vivo

    KAUST Repository

    Tong, Jing

    2013-01-07

    Superoxide dismutase 1 (SOD1) efficiently catalyzes dismutation of superoxide, but its poor delivery to the target sites in the body, such as brain, hinders its use as a therapeutic agent for superoxide-associated disorders. Here to enhance the delivery of SOD1 across the blood-brain barrier (BBB) and in neurons the enzyme was conjugated with poly(2-oxazoline) (POx) block copolymers, P(MeOx-b-BuOx) or P(EtOx-b-BuOx), composed of (1) hydrophilic 2-methyl-2-oxazoline (MeOx) or 2-ethyl-2-oxazoline (EtOx) and (2) hydrophobic 2-butyl-2-oxazoline (BuOx) repeating units. The conjugates contained from 2 to 3 POx chains joining the protein amino groups via cleavable -(ss)- or noncleavable -(cc)- linkers at the BuOx block terminus. They retained 30% to 50% of initial SOD1 activity, were conformationally and thermally stable, and assembled in 8 or 20 nm aggregates in aqueous solution. They had little if any toxicity to CATH.a neurons and displayed enhanced uptake in these neurons as compared to native or PEGylated SOD1. Of the two conjugates, SOD1-(cc)-P(MeOx-b-BuOx) and SOD1-(cc)-P(EtOx-b-BuOx), compared, the latter was entering cells 4 to 7 times faster and at 6 h colocalized predominantly with endoplasmic reticulum (41 ± 3%) and mitochondria (21 ± 2%). Colocalization with endocytosis markers and pathway inhibition assays suggested that it was internalized through lipid raft/caveolae, also employed by the P(EtOx-b-BuOx) copolymer. The SOD activity in cell lysates and ability to attenuate angiotensin II (Ang II)-induced superoxide in live cells were increased for this conjugate compared to SOD1 and PEG-SOD1. Studies in mice showed that SOD1-POx had ca. 1.75 times longer half-life in blood than native SOD1 (28.4 vs 15.9 min) and after iv administration penetrated the BBB significantly faster than albumin to accumulate in brain parenchyma. The conjugate maintained high stability both in serum and in brain (77% vs 84% at 1 h postinjection). Its amount taken up by the brain

  2. Different human copper-zinc superoxide dismutase mutants, SOD1G93A and SOD1H46R, exert distinct harmful effects on gross phenotype in mice.

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    Lei Pan

    Full Text Available Amyotrophic lateral sclerosis (ALS is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1, as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1(G93A mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1(G93A and SOD1(H46R mice on two different genetic backgrounds; C57BL/6N (B6 and FVB/N (FVB. Copy number of the transgene and their expression between SOD1(G93A and SOD1(H46R lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1(G93A mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1(G93A mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1(H46R, but not SOD1(G93A, mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1(G93A- and SOD1(H46R-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender

  3. Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

    Institute of Scientific and Technical Information of China (English)

    Guisheng Chen; Xu Peng; Shugui Shi; Lusi Li; Kangning Chen; Ju Hu; Zhenhua Zhou; Jun Wu; Gaoxing Luo; Shunzong Yuan

    2011-01-01

    The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBC1D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor α2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.

  4. Mesenchymal Stem Cell Therapy Protects Lungs from Radiation-Induced Endothelial Cell Loss by Restoring Superoxide Dismutase 1 Expression.

    Science.gov (United States)

    Klein, Diana; Steens, Jennifer; Wiesemann, Alina; Schulz, Florian; Kaschani, Farnusch; Röck, Katharina; Yamaguchi, Masahiro; Wirsdörfer, Florian; Kaiser, Markus; Fischer, Jens W; Stuschke, Martin; Jendrossek, Verena

    2017-04-10

    Radiation-induced normal tissue toxicity is closely linked to endothelial cell (EC) damage and dysfunction (acute effects). However, the underlying mechanisms of radiation-induced adverse late effects with respect to the vascular compartment remain elusive, and no causative radioprotective treatment is available to date. The importance of injury to EC for radiation-induced late toxicity in lungs after whole thorax irradiation (WTI) was investigated using a mouse model of radiation-induced pneumopathy. We show that WTI induces EC loss as long-term complication, which is accompanied by the development of fibrosis. Adoptive transfer of mesenchymal stem cells (MSCs) either derived from bone marrow or aorta (vascular wall-resident MSCs) in the early phase after irradiation limited the radiation-induced EC loss and fibrosis progression. Furthermore, MSC-derived culture supernatants rescued the radiation-induced reduction in viability and long-term survival of cultured lung EC. We further identified the antioxidant enzyme superoxide dismutase 1 (SOD1) as a MSC-secreted factor. Importantly, MSC treatment restored the radiation-induced reduction of SOD1 levels after WTI. A similar protective effect was achieved by using the SOD-mimetic EUK134, suggesting that MSC-derived SOD1 is involved in the protective action of MSC, presumably through paracrine signaling. In this study, we explored the therapeutic potential of MSC therapy to prevent radiation-induced EC loss (late effect) and identified the protective mechanisms of MSC action. Adoptive transfer of MSCs early after irradiation counteracts radiation-induced vascular damage and EC loss as late adverse effects. The high activity of vascular wall-derived MSCs for radioprotection may be due to their tissue-specific action. Antioxid. Redox Signal. 26, 563-582.

  5. The Impact of Superoxide Dismutase-1 Genetic Variation on Cardiovascular and All-Cause Mortality in a Prospective Cohort Study: The Yamagata (Takahata) Study

    Science.gov (United States)

    Otaki, Yoichiro; Watanabe, Tetsu; Nishiyama, Satoshi; Takahashi, Hiroki; Arimoto, Takanori; Shishido, Tetsuro; Miyamoto, Takuya; Konta, Tsuneo; Shibata, Yoko; Sato, Hidenori; Kawasaki, Ryo; Daimon, Makoto; Ueno, Yoshiyuki; Kato, Takeo; Kayama, Takamasa; Kubota, Isao

    2016-01-01

    Background Oxidative stress is a major cause of cardiovascular disease. Superoxide dismutase-1 (SOD1) is an antioxidant that protects against oxidative stress. Deoxyribonucleic acid (DNA) variations such as single nucleotide polymorphism (SNP) or haplotypes within the SOD gene are reportedly associated with the development of cardiovascular disease. However, it remains to be determined whether SOD1 variability is associated with cardiovascular or all-cause mortality in the general population. Methods and Results This prospective cohort study included 2799 subjects who participated in a community-based health study with a 10-year follow-up. We genotyped 639 SNPs and found the association of SNP rs1041740 and rs17880487 within a SOD1 gene with cardiovascular mortality. There were 193 deaths during the follow-up period including 57 cardiovascular deaths. Multivariate Cox proportional hazard regression analysis revealed that the homozygous T-allele of rs1041740 was associated with all-cause and cardiovascular deaths after adjusting for confounding factors. The net reclassification index was significantly improved by adding rs1041740 as a cardiovascular risk factor. On the other hand, cardiovascular death was not observed in homozygous T-allele carriers of rs17880487. Haplotype analysis identified the haplotype with T-allele of rs1041740 and that with T-allele of rs17880487 as increasing and decreasing susceptibility for cardiovascular mortality, and it had complementary SNP sequences. Conclusion Variation in the SOD1 gene was associated with cardiovascular deaths in the general population. PMID:27755600

  6. Pyrimethamine significantly lowers cerebrospinal fluid Cu/Zn superoxide dismutase in amyotrophic lateral sclerosis patients with SOD1 mutations.

    Science.gov (United States)

    Lange, Dale J; Shahbazi, Mona; Silani, Vincenzo; Ludolph, Albert C; Weishaupt, Jochen H; Ajroud-Driss, Senda; Fields, Kara G; Remanan, Rahul; Appel, Stanley H; Morelli, Claudia; Doretti, Alberto; Maderna, Luca; Messina, Stefano; Weiland, Ulrike; Marklund, Stefan L; Andersen, Peter M

    2017-06-01

    Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). A multicenter (5 sites), open-label, 9-month-duration, dose-ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale-Revised, and McGill Quality of Life Single-Item Scale were measured at screening, visit 6, and visit 9. We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p < 0.001) with a mean reduction of 13.5% (95% confidence interval [CI] = 8.4-18.5) and at visit 9 (p < 0.001) with a mean reduction of 10.5% (95% CI = 5.2-15.8). Pyrimethamine is safe and well tolerated in ALS. Pyrimethamine is capable of producing a significant reduction in total CSF SOD1 protein content in patients with ALS caused by different SOD1 mutations. Further long-term studies are warranted to assess clinical efficacy. Ann Neurol 2017;81:837-848. © 2017 The Authors. Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

  7. Reactive oxygen species on bone mineral density and mechanics in Cu,Zn superoxide dismutase (Sod1) knockout mice

    Energy Technology Data Exchange (ETDEWEB)

    Smietana, Michael J. [Biomedical Engineering, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (United States); Arruda, Ellen M. [Biomedical Engineering, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (United States); Mechanical Engineering, University of Michigan, 2250 GG Brown, 2350 Hayward, Ann Arbor, MI 48109 (United States); Program in Macromolecular Science and Engineering, University of Michigan, 2250 GG Brown, 2350 Hayward, Ann Arbor, MI 48109 (United States); Faulkner, John A.; Brooks, Susan V. [Biomedical Engineering, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (United States); Molecular and Integrative Physiology, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (United States); Larkin, Lisa M., E-mail: llarkin@umich.edu [Biomedical Engineering, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (United States); Molecular and Integrative Physiology, University of Michigan, 2025 BSRB, 109 Zina Pitcher Place, Ann Arbor, MI 48109-2200 (United States)

    2010-12-03

    Research highlights: {yields} Reactive oxygen species (ROS) are considered to be a factor in the onset of a number of age-associated conditions, including loss of BMD. {yields} Cu,Zn-superoxide dismutase (Sod1) deficient mice have increased ROS, reduced bone mineral density, decreased bending stiffness, and decreased strength compared to WT controls. {yields} Increased ROS caused by the deficiency of Sod1, may be responsible for the changes in BMD and bone mechanics and therefore represent an appropriate model for studying mechanisms of age-associated bone loss. -- Abstract: Reactive oxygen species (ROS) play a role in a number of degenerative conditions including osteoporosis. Mice deficient in Cu,Zn-superoxide dismutase (Sod1) (Sod1{sup -/-} mice) have elevated oxidative stress and decreased muscle mass and strength compared to wild-type mice (WT) and appear to have an accelerated muscular aging phenotype. Thus, Sod1{sup -/-} mice may be a good model for evaluating the effects of free radical generation on diseases associated with aging. In this experiment, we tested the hypothesis that the structural integrity of bone as measured by bending stiffness (EI; N/mm{sup 2}) and strength (MPa) is diminished in Sod1{sup -/-} compared to WT mice. Femurs were obtained from male and female WT and Sod1{sup -/-} mice at 8 months of age and three-point bending tests were used to determine bending stiffness and strength. Bones were also analyzed for bone mineral density (BMD; mg/cc) using micro-computed tomography. Femurs were approximately equal in length across all groups, and there were no significant differences in BMD or EI with respect to gender in either genotype. Although male and female mice demonstrated similar properties within each genotype, Sod1{sup -/-} mice exhibited lower BMD and EI of femurs from both males and females compared with gender matched WT mice. Strength of femurs was also lower in Sod1{sup -/-} mice compared to WT as well as between genders. These

  8. A Central Role for JNK/AP-1 Pathway in the Pro-Oxidant Effect of Pyrrolidine Dithiocarbamate through Superoxide Dismutase 1 Gene Repression and Reactive Oxygen Species Generation in Hematopoietic Human Cancer Cell Line U937.

    Science.gov (United States)

    Riera, Humberto; Afonso, Valéry; Collin, Pascal; Lomri, Abderrahim

    2015-01-01

    Pyrrolidine dithiocarbamate (PDTC) known as antioxidant and specific inhibitor of NF-κB was also described as pro-oxidant by inducing cell death and reactive oxygen species (ROS) accumulation in cancer. However, the mechanism by which PDTC indices its pro-oxidant effect is unknown. Therefore, we aimed to evaluate the effect of PDTC on the human Cu/Zn superoxide dismutase 1 (SOD1) gene transcription in hematopoietic human cancer cell line U937. We herein show for the first time that PDTC decreases SOD1 transcripts, protein and promoter activity. Furthermore, SOD1 repression by PDTC was associated with an increase in oxidative stress as evidenced by ROS production. Electrophoretic mobility-shift assays (EMSA) show that PDTC increased binding of activating protein-1 (AP-1) in dose dependent-manner suggesting that the MAPkinase up-stream of AP-1 is involved. Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription. In contrast, in the presence of JNK inhibitor (SP600125), p65 induced a marked increase of SOD1 promoter, suggesting that JNK pathway is up-stream of NF-κB signaling and controls negatively its activity. Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production. Finally, PDTC represses SOD1 in U937 cells through JNK/c-Jun phosphorylation. Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

  9. A Central Role for JNK/AP-1 Pathway in the Pro-Oxidant Effect of Pyrrolidine Dithiocarbamate through Superoxide Dismutase 1 Gene Repression and Reactive Oxygen Species Generation in Hematopoietic Human Cancer Cell Line U937.

    Directory of Open Access Journals (Sweden)

    Humberto Riera

    Full Text Available Pyrrolidine dithiocarbamate (PDTC known as antioxidant and specific inhibitor of NF-κB was also described as pro-oxidant by inducing cell death and reactive oxygen species (ROS accumulation in cancer. However, the mechanism by which PDTC indices its pro-oxidant effect is unknown. Therefore, we aimed to evaluate the effect of PDTC on the human Cu/Zn superoxide dismutase 1 (SOD1 gene transcription in hematopoietic human cancer cell line U937. We herein show for the first time that PDTC decreases SOD1 transcripts, protein and promoter activity. Furthermore, SOD1 repression by PDTC was associated with an increase in oxidative stress as evidenced by ROS production. Electrophoretic mobility-shift assays (EMSA show that PDTC increased binding of activating protein-1 (AP-1 in dose dependent-manner suggesting that the MAPkinase up-stream of AP-1 is involved. Ectopic NF-κB p65 subunit overexpression had no effect on SOD1 transcription. In contrast, in the presence of JNK inhibitor (SP600125, p65 induced a marked increase of SOD1 promoter, suggesting that JNK pathway is up-stream of NF-κB signaling and controls negatively its activity. Indeed, using JNK deficient cells, PDTC effect was not observed nether on SOD1 transcription or enzymatic activity, nor on ROS production. Finally, PDTC represses SOD1 in U937 cells through JNK/c-Jun phosphorylation. Taken together, these results suggest that PDTC acts as pro-oxidant compound in JNK/AP-1 dependent-manner by repressing the superoxide dismutase 1 gene leading to intracellular ROS accumulation.

  10. Superoxide Dismutase 1 Regulation of CXCR4-Mediated Signaling in Prostate Cancer Cells is Dependent on Cellular Oxidative State

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    Brent Young

    2015-11-01

    Full Text Available Background/Aims: CXCL12, acting via one of its G protein-coupled receptors, CXCR4, is a chemoattractant for a broad range of cell types, including several types of cancer cells. Elevated expression of CXCR4, and its ligand CXCL12, play important roles in promoting cancer metastasis. Cancer cells have the potential for rapid and unlimited growth in an area that may have restricted blood supply, as oxidative stress is a common feature of solid tumors. Recent studies have reported that enhanced expression of cytosolic superoxide dismutase (SOD1, a critical enzyme responsible for regulation of superoxide radicals, may increase the aggressive and invasive potential of malignant cells in some cancers. Methods: We used a variety of biochemical approaches and a prostate cancer cell line to study the effects of SOD1 on CXCR4 signaling. Results: Here, we report a direct interaction between SOD1 and CXCR4. We showed that SOD1 interacts directly with the first intracellular loop (ICL1 of CXCR4 and that the CXCL12/CXCR4-mediated regulation of AKT activation, apoptosis and cell migration in prostate cancer (PCa cells is differentially modulated under normal versus hypoxic conditions when SOD1 is present. Conclusions: This study highlights a potential new regulatory mechanism by which a sensor of the oxidative environment could directly regulate signal transduction of a receptor involved in cancer cell survival and migration.

  11. Observation of c.260A > G mutation in superoxide dismutase 1 that causes p.Asn86Ser in Iranian amyotrophic lateral sclerosis patient and absence of genotype/phenotype correlation

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    Marzieh Khani

    2015-10-01

    Full Text Available Background: Amyotrophic lateral sclerosis (ALS is the most common motor neuron disorder in European populations. ALS can be sporadic ALS (SALS or familial ALS (FALS. Among 20 known ALS genes, mutations in C9orf72 and superoxide dismutase 1 (SOD1 are the most common genetic causes of the disease. Whereas C9orf72 mutations are more common in Western populations, the contribution of SOD1 to ALS in Iran is more than C9orf72. At present, a clear genotype/phenotype correlation for ALS has not been identified. We aimed to perform mutation screening of SOD1 in a newly identified Iranian FALS patient and to assess whether a genotype/phenotype correlation for the identified mutation exists.Methods: The five exons of SOD1 and flanking intronic sequences of a FALS proband were screened for mutations by direct sequencing. The clinical features of the proband were assessed by a neuromuscular specialist (SN. The phenotypic presentations were compared to previously reported patients with the same mutation.Results: Heterozygous c.260A > G mutation in SOD1 that causes Asn86Ser was identified in the proband. Age at onset was 34 years and site of the first presentation was in the lower extremities. Comparisons of clinical features of different ALS patients with the same mutation evidenced variable presentations.Conclusion: The c.260A > G mutation in SOD1 that causes Asn86Ser appears to cause ALS with variable clinical presentations.

  12. Superoxide dismutase 1 is positively selected to minimize protein aggregation in great apes

    DEFF Research Database (Denmark)

    Dasmeh, Pouria; Kepp, Kasper Planeta

    2017-01-01

    of this selection are unknown. We mapped 30 extant SOD1 sequences to the recently established mammalian species tree and inferred ancestors, key substitutions, and signatures of selection during the protein's evolution. We detected elevated substitution rates leading to great apes (Hominidae) at ~1 per 2 million...

  13. Curcumin binds to the pre-fibrillar aggregates of Cu/Zn superoxide dismutase (SOD1) and alters its amyloidogenic pathway resulting in reduced cytotoxicity.

    Science.gov (United States)

    Bhatia, Nidhi K; Srivastava, Ankit; Katyal, Nidhi; Jain, Nidhi; Khan, M Ashhar I; Kundu, Bishwajit; Deep, Shashank

    2015-05-01

    Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons. Unfortunately, effective therapeutics against this disease is still not available. Almost 20% of familial ALS (fALS) is suggested to be associated with pathological deposition of superoxide dismutase (SOD1). Evidences suggest that SOD1-containing pathological inclusions in ALS exhibit amyloid like properties. An effective strategy to combat ALS may be to inhibit amyloid formation of SOD1 using small molecules. In the present study, we observed the fibrillation of one of the premature forms of SOD1 (SOD1 with reduced disulfide) in the presence of curcumin. Using ThT binding assay, AFM, TEM images and FTIR, we demonstrate that curcumin inhibits the DTT-induced fibrillation of SOD1 and favors the formation of smaller and disordered aggregates of SOD1. The enhancement in curcumin fluorescence on the addition of oligomers and pre-fibrillar aggregates of SOD1 suggests binding of these species to curcumin. Docking studies indicate that putative binding site of curcumin may be the amyloidogenic regions of SOD1. Further, there is a significant increase in SOD1 mediated toxicity in the regime of pre-fibrillar and fibrillar aggregates which is not evident in curcumin containing samples. All these data suggest that curcumin reduces toxicity by binding to the amyloidogenic regions of the species on the aggregation pathway and blocking the formation of the toxic species. Nanoparticles of curcumin with higher aqueous solubility show similar aggregation control as that of curcumin bulk. This suggests a potential role for curcumin in the treatment of ALS.

  14. Superoxide dismutase 1 and glutathione peroxidase 1 are involved in the protective effect of sulodexide on vascular endothelial cells exposed to oxygen-glucose deprivation.

    Science.gov (United States)

    Gabryel, Bożena; Jarząbek, Karolina; Machnik, Grzegorz; Adamczyk, Jakub; Belowski, Dariusz; Obuchowicz, Ewa; Urbanek, Tomasz

    2016-01-01

    Sulodexide (SDX) is widely used in the treatment of both arterial and venous thrombotic disorders. In addition to its recognized antithrombotic action, SDX has endothelial protective potential, which is independent of the coagulation/fibrinolysis system. However, the detailed molecular mechanisms of the endothelioprotective action of the drug are still unresolved. The aim of the present study was to determine whether treatment with SDX at concentrations of 0.125-0.5 lipase releasing unit (LRU)/ml have on the expression and activity of antioxidant enzymes in ischemic endothelial cells and how these effects might be related to the antiapoptotic properties of SDX. In the present study, human umbilical vein endothelial cells (HUVECs) were subjected to ischemia-simulating conditions (combined oxygen and glucose deprivation, OGD) for 6h to determine the protective effects of SDX. SDX (0.25 and 0.5LRU/ml) in OGD significantly increased the cell viability and prevented mitochondrial depolarization in the HUVECs. Moreover, SDX protected the HUVECs against OGD-induced apoptosis. At concentrations of 0.25 and 0.5LRU/ml, the drug increased both superoxide dismutase 1 (SOD1) and glutathione peroxidase 1 (GPx1) mRNA/protein expression together with a significant attenuation of oxidative stress in ischemic HUVECs. Our findings also demonstrate that an increase in both SOD and GPx activity is involved in the protective effect of SDX on ischemic endothelial cells. Altogether, these results suggest that SDX has a positive effect on ischemia-induced endothelial damage because of its antioxidant and antiapoptotic properties.

  15. Muscle cells and motoneurons differentially remove mutant SOD1 causing familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Onesto, Elisa; Rusmini, Paola; Crippa, Valeria; Ferri, Nicola; Zito, Arianna; Galbiati, Mariarita; Poletti, Angelo

    2011-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

  16. Increased expression and local accumulation of the Prion Protein, Alzheimer Aβ peptides, superoxide dismutase 1, and Nitric oxide synthases 1 & 2 in muscle in a rabbit model of diabetes

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    Bitel Claudine L

    2010-09-01

    Full Text Available Abstract Background Muscle disease associated with different etiologies has been shown to produce localized accumulations of amyloid and oxidative stress-related proteins that are more commonly associated with neurodegeneration in the brain. In this study we examined changes in muscle tissue in a classic model of diabetes and hyperglycemia in rabbits to determine if similar dysregulation of Alzheimer Aβ peptides, the prion protein (PrP, and superoxide dismutase 1 (SOD1, as well as nitric oxide synthases is produced in muscle in diabetic animals. This wild-type rabbit model includes systemic physiological expression of human-like Alzheimer precursor proteins and Aβ peptides that are considered key in Alzheimer protein studies. Results Diabetes was produced in rabbits by injection of the toxic glucose analogue alloxan, which selectively enters pancreatic beta cells and irreversibly decreases insulin production, similar to streptozotocin. Quadriceps muscle from rabbits 16 wks after onset of diabetes and hyperglycemia were analyzed with biochemical and in situ methods. Immunoblots of whole muscle protein samples demonstrated increased PrP, SOD1, as well as neuronal and inducible Nitric oxide synthases (NOS1 and NOS2 in diabetic muscle. In contrast, we detected little change in Alzheimer Aβ precursor protein expression, or BACE1 and Presenilin 1 levels. However, Aβ peptides measured by ELISA increased several fold in diabetic muscle, suggesting a key role for Aβ cleavage in muscle similar to Alzheimer neurodegeneration in this diabetes model. Histological changes in diabetic muscle included localized accumulations of PrP, Aβ, NOS1 and 2, and SOD1, and evidence of increased central nuclei and cell infiltration. Conclusions The present study provides evidence that several classic amyloid and oxidative stress-related disease proteins coordinately increase in overall expression and form localized accumulations in diabetic muscle. The present study

  17. Autophagic induction of amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase 1 G93A mutant in NSC34 cells

    Institute of Scientific and Technical Information of China (English)

    Yanming Wei

    2014-01-01

    Previous studies have conifrmed that the beclin 1 complex plays a key role in the initial stage of autophagy and deregulated autophagy might involve in amyotrophic lateral sclerosis. However, the mechanism underlying altered autophagy associated with the beclin 1 complex remains un-clear. In this study, we transfected the Cu/Zn superoxide dismutase 1 G93A mutant protein into the motor neuron-like cell line NSC34 cultured in vitro. Western blotting and co-immunopre-cipitation showed that the Cu/Zn superoxide dismutase 1 G93A mutant enhanced the turnover of autophagic marker microtubule-associated protein light chain 3II (LC3II) and stimulated the conversion of EGFP-LC3I to EGFP-LC3II, but had little inlfuence on the binding capacity of the autophagy modulators ATG14L, rubicon, UVRAG, and hVps34 to beclin 1 during auto-phagosome formation. These results suggest that the amyotrophic lateral sclerosis-linked Cu/Zn superoxide dismutase 1 G93A mutant can upregulate autophagic activity in NSC34 cells, but that this does not markedly affect beclin 1 complex components.

  18. SOD1 Lysine 123 Acetylation in the Adult Central Nervous System

    Science.gov (United States)

    Kaliszewski, Michael; Kennedy, Austin K.; Blaes, Shelby L.; Shaffer, Robert S.; Knott, Andrew B.; Song, Wenjun; Hauser, Henry A.; Bossy, Blaise; Huang, Ting-Ting; Bossy-Wetzel, Ella

    2016-01-01

    Superoxide dismutase 1 (SOD1) knockout (Sod1−/−) mice exhibit an accelerated aging phenotype. In humans, SOD1 mutations are linked to familial amyotrophic lateral sclerosis (ALS), and post-translational modification (PTM) of wild-type SOD1 has been associated with sporadic ALS. Reversible acetylation regulates many enzymes and proteomic studies have identified SOD1 acetylation at lysine 123 (K123). The function and distribution of K123-acetylated SOD1 (Ac-K123 SOD1) in the nervous system is unknown. Here, we generated polyclonal rabbit antibodies against Ac-K123 SOD1. Sod1 deletion in Sod1−/− mice, K123 mutation or preabsorption with Ac-K123 peptide all abolished antibody binding. Using immunohistochemistry, we assessed Ac-K123 SOD1 distribution in the normal adult mouse nervous system. In the cerebellum, Ac-K123 SOD1 staining was prominent in cell bodies of the granular cell layer (GCL) and Purkinje cell dendrites and interneurons of the molecular cell layer. In the hippocampus, Ac-K123 SOD1 staining was strong in the fimbria, subiculum, pyramidal cells and Schaffer collateral fibers of the cornus ammonis field 1 (CA1) region and granule and neuronal progenitor cells of the dentate gyrus. In addition, labeling was observed in the choroid plexus (CP) and the ependyma of the brain ventricles and central canal of the spinal cord. In the olfactory bulb, Ac-K123 SOD1 staining was prominent in axons of sensory neurons, in cell bodies of interneurons and neurites of the mitral and tufted cells. In the retina, labeling was strong in the retinal ganglion cell layer (RGCL) and axons of retinal ganglion cells (RGCs), the inner nuclear layer (INL) and cone photoreceptors of the outer nuclear layer (ONL). In summary, our findings describe Ac-K123 SOD1 distribution to distinct regions and cell types of the normal nervous system. PMID:28066183

  19. SOD1 Lysine 123 Acetylation in the Adult Central Nervous System

    Directory of Open Access Journals (Sweden)

    Michael Kaliszewski

    2016-12-01

    Full Text Available Superoxide dismutase 1 (SOD1 knockout (Sod1-/- mice exhibit an accelerated aging phenotype. In humans, SOD1 mutations are linked to familial amyotrophic lateral sclerosis (ALS, and post-translational modification (PTM of wild-type SOD1 has been associated with sporadic ALS. Reversible acetylation regulates many enzymes and proteomic studies have identified SOD1 acetylation at lysine 123 (K123. The function and distribution of K123-acetylated SOD1 (Ac-K123 SOD1 in the nervous system is unknown. Here, we generated polyclonal rabbit antibodies against Ac-K123 SOD1. Sod1 deletion in Sod1-/- mice, K123 mutation, or preabsorption with Ac-K123 peptide all abolished antibody binding. Using immunohistochemistry, we assessed Ac-K123 SOD1 distribution in the normal adult mouse nervous system. In the cerebellum, Ac-K123 SOD1 staining was prominent in cell bodies of the granular cell layer and Purkinje cell dendrites and interneurons of the molecular cell layer. In the hippocampus, Ac-K123 SOD1 staining was strong in the fimbria, subiculum, pyramidal cells, and Schaffer collateral fibers of the cornus ammonis (CA1 region and granule and neuronal progenitor cells of the dentate gyrus. In addition, labeling was observed in the choroid plexus and the ependyma of the brain ventricles and central canal of the spinal cord. In the olfactory bulb, Ac-K123 SOD1 staining was prominent in axons of sensory neurons, in cell bodies of interneurons, and neurites of the mitral and tufted cells. In the retina, labeling was strong in the retinal ganglion cell layer and axons of retinal ganglion cells, the inner nuclear layer, and cone photoreceptors of the outer nuclear layer. In summary, our findings describe Ac-K123 SOD1 distribution to distinct regions and cell types of the normal nervous system.

  20. Phenotype of transgenic mice carrying a very low copy number of the mutant human G93A superoxide dismutase-1 gene associated with amyotrophic lateral sclerosis.

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    Jeffrey S Deitch

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease of the motor neuron. While most cases of ALS are sporadic, 10% are familial (FALS with 20% of FALS caused by a mutation in the gene that codes for the enzyme Cu/Zn superoxide dismutase (SOD1. There is variability in sporadic ALS as well as FALS where even within the same family some siblings with the same mutation do not manifest disease. A transgenic (Tg mouse model of FALS containing 25 copies of the mutant human SOD1 gene demonstrates motor neuron pathology and progressive weakness similar to ALS patients, leading to death at approximately 130 days. The onset of symptoms and survival of these transgenic mice are directly related to the number of copies of the mutant gene. We report the phenotype of a very low expressing (VLE G93A SOD1 Tg carrying only 4 copies of the mutant G93ASOD1 gene. While weakness can start at 9 months, only 74% of mice 18 months or older demonstrate disease. The VLE mice show decreased motor neurons compared to wild-type mice as well as increased cytoplasmic translocation of TDP-43. In contrast to the standard G93A SOD1 Tg mouse which always develops motor weakness leading to death, not all VLE animals manifested clinical disease or shortened life span. In fact, approximately 20% of mice older than 24 months had no motor symptoms and only 18% of VLE mice older than 22 months reached end stage. Given the variable penetrance of clinical phenotype, prolonged survival, and protracted loss of motor neurons the VLE mouse provides a new tool that closely mimics human ALS. This tool will allow the study of pathologic events over time as well as the study of genetic and environmental modifiers that may not be causative, but can exacerbate or accelerate motor neuron disease.

  1. Apparent anticipation in SOD1 familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Vucic, Steve; Nicholson, Garth A; Chio, Adriano; Kiernan, Matthew C

    2013-09-01

    Although anticipation has been previously reported in the copper/zinc superoxide dismutase-1 (SOD1) associated familial amyotrophic lateral sclerosis (FALS), some have argued that this may represent ascertainment bias. Consequently, the aim of the present study was to determine whether anticipation was a feature in SOD1 FALS. From a cohort of 112 individuals, the clinical and genetic history of 34 SOD1 patients was assessed. Clinical history was collected with the age of death and disease duration determined in successive generations (generation 1, grandparent; generation 2, parents; generation 3, children), from five large SOD1 families. Results showed that the age of death was significantly less in generation 3 (40.1 ± 2.8 years) compared to generation 2 (46.2 ± 2.0 years, p < 0.05) and generation 1 (56.7 ± 4.5 years, p < 0.01). Furthermore, disease duration was longer in generation 1 (18.4 ± 3.7 months) compared to the disease duration in generation 2 (12.6 ± 2.6 months) and generation 3 (12.3 ± 1.9 months, p = 0.08). Positive intergenerational differences were evident in 92% of parent-offspring transmissions in the present SOD1 FALS cohort (c(2) = 70.6, p < 0.001). In conclusion, the present study supports anticipation as a phenomenon in FALS, possibly due to co-inheritance of modifier genes.

  2. Accumulation of SOD1 mutants in postnatal motoneurons does not cause motoneuron pathology or motoneuron disease.

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    Lino, Maria Maddalena; Schneider, Corinna; Caroni, Pico

    2002-06-15

    Transgenic mice expressing high levels of familial amyotrophic lateral sclerosis (FALS)-associated mutant superoxide dismutase 1 (SOD1) under the control of a human SOD1 minigene (hMg) accumulate mutant protein ubiquitously and develop motoneuron disease. However, restricted expression of SOD1 mutants in neurons apparently does not cause motor impairments in mice. Here, we investigated the possible pathogenic roles of mutant SOD1 accumulation in motoneurons. First, we used a Thy1 expression cassette to drive high constitutive expression of transgene in postnatal mouse neurons, including upper and lower motoneurons. Second, we expressed human (h) SOD1(G93A) and hSOD1(G85R) as transgenes (i.e., two SOD1 mutants with aggressive pathogenic properties in inducing FALS). Third, in addition to clinical signs of disease, we monitored early signs of disease onset and pathogenesis, including muscle innervation, astrogliosis in the spinal cord, and accumulation of ubiquitinated deposits in motoneurons and astrocytes. We report that high-level expression and accumulation of the mutant proteins in neurons failed to produce any detectable sign of pathology or disease in these transgenic mice. Crossing hMg-SOD1(G93A) mice (Gurney et al., 1994) with Thy1-SOD1(G93A) mice produced double-transgenic mice with spinal cord SOD1(G93A) levels that were approximately twofold higher than in the hMg-SOD1(G93A) single transgenics but did not affect the onset or progression of pathology or motoneuron disease. The accumulation of mutant SOD1 in postnatal motoneurons is thus not sufficient and probably also not critical to induce or accelerate motoneuron disease in FALS mice. The pathogenic process in FALS may involve non-neuronal cells, and selective vulnerability of motoneurons to this process may lead to motoneuron pathology and disease.

  3. Mutant copper-zinc superoxide dismutase (SOD1) induces protein secretion pathway alterations and exosome release in astrocytes: implications for disease spreading and motor neuron pathology in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Basso, Manuela; Pozzi, Silvia; Tortarolo, Massimo; Fiordaliso, Fabio; Bisighini, Cinzia; Pasetto, Laura; Spaltro, Gabriella; Lidonnici, Dario; Gensano, Francesco; Battaglia, Elisa; Bendotti, Caterina; Bonetto, Valentina

    2013-05-31

    Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.

  4. Mice overexpressing both non-mutated human SOD1 and mutated SOD1G93A genes: a competent experimental model for studying iron metabolism in amyotrophic lateral sclerosis

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    Anna eGajowiak

    2016-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disease characterized by degeneration and loss of motor neurons in the spinal cord, brainstem and motor cortex. Up to 10% of ALS cases are inherited (familial, fALS and associated with mutations, frequently in the superoxide dismutase 1 (SOD1 gene. Rodent transgenic models of ALS are often used to elucidate a complex pathogenesis of this disease. Of importance, both ALS patients and animals carrying mutated human SOD1 gene show symptoms of oxidative stress and iron metabolism misregulation. The aim of our study was to characterize changes in iron metabolism in one of the most commonly used models of ALS – transgenic mice overexpressing human mutated SOD1G93A gene. We analyzed the expression of iron-related genes in asymptomatic, 2-month old and symptomatic, 4-month old SOD1G93A mice. In parallel, respective age-matched mice overexpressing human non-mutated SOD1 transgene and control mice were analyzed. We demonstrate that the overexpression of both SOD1 and SOD1G93A genes account for a substantial increase in SOD1 protein levels and activity in selected tissues and that not all the changes in iron metabolism genes expression are specific for the overexpression of the mutated form of SOD1.

  5. A botanical containing freeze dried açai pulp promotes healthy aging and reduces oxidative damage in sod1 knockdown flies

    OpenAIRE

    Laslo, Mara; Sun, Xiaoping; Hsiao, Cheng-Te; Wu, Wells W; Shen, Rong-Fong; Zou, Sige

    2012-01-01

    Superoxide dismutase 1 (SOD1), a critical enzyme against oxidative stress, is implicated in aging and degenerative diseases. We previously showed that a nutraceutical containing freeze-dried açai pulp promotes survival of flies fed a high-fat diet or sod1 knockdown flies fed a standard diet. Here, we investigated the effect of açai supplementation initiated at the early or late young adulthood on lifespan, physiological function, and oxidative damage in sod1 knockdown flies. We found that Aça...

  6. Comparative Analyses of Cu-Zn Superoxide Dismutase (SOD1) and Thioredoxin Reductase (TrxR) at the mRNA Level between Apis mellifera L. and Apis cerana F. (Hymenoptera: Apidae) Under Stress Conditions.

    Science.gov (United States)

    Koo, Hyun-Na; Lee, Soon-Gyu; Yun, Seung-Hwan; Kim, Hyun Kyung; Choi, Yong Soo; Kim, Gil-Hah

    2016-01-01

    This study compared stress-induced expression of Cu-Zn superoxide dismutase (SOD1) and thioredoxin reductase (TrxR) genes in the European honeybee Apis mellifera L. and Asian honeybee Apis cerana F. Expression of both SOD1 and TrxR rapidly increased up to 5 h after exposure to cold (4 °C) or heat (37 °C) treatment and then gradually decreased, with a stronger effect induced by cold stress in A. mellifera compared with A. cerana. Injection of stress-inducing substances (methyl viologen, [MV] and H2O2) also increased SOD1 and TrxR expression in both A. mellifera and A. cerana, and this effect was more pronounced with MV than H2O2. Additionally, we heterologously expressed the A. mellifera and A. cerana SOD1 and TrxR proteins in an Escherichia coli expression system, and detection by SDS-PAGE, confirmed by Western blotting using anti-His tag antibodies, revealed bands at 16 and 60 kDa, respectively. Our results show that the expression patterns of SOD1 and TrxR differ between A. mellifera and A. cerana under conditions of low or high temperature as well as oxidative stress.

  7. Prognostic role of ‘prion-like propagation’ in SOD1-linked familial ALS: an alternative view

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    Keizo eSugaya

    2014-10-01

    Full Text Available ‘Prion-like propagation’ has recently been proposed for disease spread in Cu/Zn superoxide dismutase 1 (SOD1-linked familial amyotrophic lateral sclerosis (ALS. Pathological SOD1 conformers are presumed to propagate via cell-to-cell transmission. In this model, the risk-based kinetics of neuronal cell loss over time appears to be represented by a sigmoidal function that reflects the kinetics of intercellular transmission. Here, we describe an alternative view of prion-like propagation in SOD1-linked ALS−its relation to disease prognosis under the protective-aggregation hypothesis. Nucleation-dependent polymerization has been widely accepted as the molecular mechanism of prion propagation. If toxic species of misfolded SOD1, as soluble oligomers, are formed as on-pathway intermediates of nucleation-dependent polymerization, further fibril extension via sequential addition of monomeric mutant SOD1 would be protective against neurodegeneration. This is because the concentration of unfolded mutant SOD1 monomers, which serve as precursor of nucleation and toxic species of mutant SOD1, would decline in proportion to the extent of aggregation. The nucleation process requires that native conformers exist in an unfolded state that may result from escaping the cellular protein quality control machinery. However, prion-like propagation−SOD1 aggregated form self-propagates by imposing its altered conformation on normal SOD1−appears to antagonize the protective role of aggregate growth. The cross-seeding reaction with normal SOD1 would lead to a failure to reduce the concentration of unfolded mutant SOD1 monomers, resulting in continuous nucleation and subsequent generation of toxic species, and influence disease prognosis. In this alternative view, the kinetics of neuronal loss appears to be represented by an exponential function, with decreasing risk reflecting the protective role of aggregate and the potential for cross-seeding reactions between

  8. Mia40 and MINOS act in parallel with Ccs1 in the biogenesis of mitochondrial Sod1.

    Science.gov (United States)

    Varabyova, Aksana; Topf, Ulrike; Kwiatkowska, Paulina; Wrobel, Lidia; Kaus-Drobek, Magdalena; Chacinska, Agnieszka

    2013-10-01

    Superoxide dismutase 1 (Sod1) is a major superoxide-scavenging enzyme in the eukaryotic cell, and is localized in the cytosol and intermembrane space of mitochondria. Sod1 requires its specific chaperone Ccs1 and disulfide bond formation in order to be retained in the intermembrane space. Our study identified a pool of Sod1 that is present in the reduced state in mitochondria that lack Ccs1. We created yeast mutants with mutations in highly conserved amino acid residues corresponding to human mutations that cause amyotrophic lateral sclerosis, and found that some of the mutant proteins were present in the reduced state. These mutant variants of Sod1 were efficiently localized in mitochondria. Localization of the reduced, Ccs1-independent forms of Sod1 relied on Mia40, an essential component of the mitochondrial intermembrane space import and assembly pathway that is responsible for the biogenesis of intermembrane space proteins. Furthermore, the mitochondrial inner membrane organizing system (MINOS), which is responsible for mitochondrial membrane architecture, differentially modulated the presence of reduced Sod1 in mitochondria. Thus, we identified novel mitochondrial players that are possibly involved in pathological conditions caused by changes in the biogenesis of Sod1. © 2013 FEBS.

  9. TDP-43 identified from a genome wide RNAi screen for SOD1 regulators.

    Directory of Open Access Journals (Sweden)

    Balajee R Somalinga

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a late-onset, progressive neurodegenerative disease affecting motor neurons in the brain stem and spinal cord leading to loss of voluntary muscular function and ultimately, death due to respiratory failure. A subset of ALS cases are familial and associated with mutations in superoxide dismutase 1 (SOD1 that destabilize the protein and predispose it to aggregation. In spite of the fact that sporadic and familial forms of ALS share many common patho-physiological features, the mechanistic relationship between SOD1-associated and sporadic forms of the disease if any, is not well understood. To better understand any molecular connections, a cell-based protein folding assay was employed to screen a whole genome RNAi library for genes that regulate levels of soluble SOD1. Statistically significant hits that modulate SOD1 levels, when analyzed by pathway analysis revealed a highly ranked network containing TAR DNA binging protein (TDP-43, a major component of aggregates characteristic of sporadic ALS. Biochemical experiments confirmed the action of TDP-43 on SOD1. These results highlight an unexpected relationship between TDP-43 and SOD1 which may have implications in disease pathogenesis.

  10. Is SOD1 loss of function involved in amyotrophic lateral sclerosis?

    Science.gov (United States)

    Saccon, Rachele A; Bunton-Stasyshyn, Rosie K A; Fisher, Elizabeth M C; Fratta, Pietro

    2013-08-01

    Mutations in the gene superoxide dismutase 1 (SOD1) are causative for familial forms of the neurodegenerative disease amyotrophic lateral sclerosis. When the first SOD1 mutations were identified they were postulated to give rise to amyotrophic lateral sclerosis through a loss of function mechanism, but experimental data soon showed that the disease arises from a--still unknown--toxic gain of function, and the possibility that loss of function plays a role in amyotrophic lateral sclerosis pathogenesis was abandoned. Although loss of function is not causative for amyotrophic lateral sclerosis, here we re-examine two decades of evidence regarding whether loss of function may play a modifying role in SOD1-amyotrophic lateral sclerosis. From analysing published data from patients with SOD1-amyotrophic lateral sclerosis, we find a marked loss of SOD1 enzyme activity arising from almost all mutations. We continue to examine functional data from all Sod1 knockout mice and we find obvious detrimental effects within the nervous system with, interestingly, some specificity for the motor system. Here, we bring together historical and recent experimental findings to conclude that there is a possibility that SOD1 loss of function may play a modifying role in amyotrophic lateral sclerosis. This likelihood has implications for some current therapies aimed at knocking down the level of mutant protein in patients with SOD1-amyotrophic lateral sclerosis. Finally, the wide-ranging phenotypes that result from loss of function indicate that SOD1 gene sequences should be screened in diseases other than amyotrophic lateral sclerosis.

  11. 丙泊酚对过氧化氢诱导入脐静脉内皮细胞超氧化物歧化酶1表达的影响%Effects of propofol on expression of superoxide dismutase-1 in hydrogen dioxide mediated human umbilical vein endothelial cells

    Institute of Scientific and Technical Information of China (English)

    杨艳琴; 唐靖; 古妙宁

    2011-01-01

    目的 探讨丙泊酚对过氧化氢(H2O2)诱导入脐静脉内皮细胞(human umbilical vein endothelial cells,HUVEC)超氧化物歧化酶1(superoxide dismutase-1,SOD1)表达的影响.方法 将体外培养24孔板中的HUVEC分为3部分:第1部分(分4组,每组6例):①正常对照组(C组);②H2O2刺激组(H组):H2O2 200 μmol/L;③丙泊酚组(P组):丙泊酚100μmol/L;④丙泊酚加H2O2刺激组(P+H组);第2部分(分5组,每组6例):分别给予丙泊酚12.5、25、50、100、200 μmol/L预处理30 min后,再给予H2O2 200 μmol/L.第3部分(分5组,每组6例):丙泊酚最佳浓度预处理30 min后,再给予H2O2 200μmol/L,分别培养6、12、24、36、48 h,采用Western blot观察HUVEC在H2O2刺激下,丙泊酚对HUVEC中SOD1表达影响的量效与时效关系.将96孔板中的HUVEC分为3组(每组5例):①空白对照组(C组);②H2O2刺激组(H组);③丙泊酚+H2O2组(P+H组),采用MTT法检测丙泊酚对HUVEC存活率的影响.结果 H2O2(200 μmol/L)刺激HUVEC后,能明显抑制HUVEC中SOD1的表达.丙泊酚预处理后,能部分逆转H2O2对HUVEC中SOD1的抑制作用.丙泊酚浓度为100μmol/L,H2O2刺激时间为24 h时,逆转效应达高峰.结论 丙泊酚可通过上调SOD1表达而减轻H2O2诱导HUVEC氧化应激损伤.%Objective To investigate the effects of propofol on the expression of superoxide dismutase-1 (SOD1 ) in hydrogen dioxide (H2O2)-mediated human umbilical vein endothelia cells (HUVEC). Methods The HUVEC cultured in 24-well plate was with propofol 12.5, 25, 50, 100 and 200 μmol/L respectively for 30 min before H2O2 sitimulating HUVEC. Part 3 (divided into five groups, n=6): the optimal concentration of propofol was used for pretreatment for 30 min before H2O2 sitimulated HUVEC,HUVEC was cultured for 6, 12, 24, 36 and 48 h accordingly. Western blot was used to determine the dose-depedent and time-dependent effect of propofol on the expression of SOD1 in HUVEC. The HUVEC which cultured in 96-well plate was divided into

  12. Accumulation of Misfolded SOD1 in Dorsal Root Ganglion Degenerating Proprioceptive Sensory Neurons of Transgenic Mice with Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Javier Sábado

    2014-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is an adult-onset progressive neurodegenerative disease affecting upper and lower motoneurons (MNs. Although the motor phenotype is a hallmark for ALS, there is increasing evidence that systems other than the efferent MN system can be involved. Mutations of superoxide dismutase 1 (SOD1 gene cause a proportion of familial forms of this disease. Misfolding and aggregation of mutant SOD1 exert neurotoxicity in a noncell autonomous manner, as evidenced in studies using transgenic mouse models. Here, we used the SOD1G93A mouse model for ALS to detect, by means of conformational-specific anti-SOD1 antibodies, whether misfolded SOD1-mediated neurotoxicity extended to neuronal types other than MNs. We report that large dorsal root ganglion (DRG proprioceptive neurons accumulate misfolded SOD1 and suffer a degenerative process involving the inflammatory recruitment of macrophagic cells. Degenerating sensory axons were also detected in association with activated microglial cells in the spinal cord dorsal horn of diseased animals. As large proprioceptive DRG neurons project monosynaptically to ventral horn MNs, we hypothesise that a prion-like mechanism may be responsible for the transsynaptic propagation of SOD1 misfolding from ventral horn MNs to DRG sensory neurons.

  13. Inhibition of fast axonal transport by pathogenic SOD1 involves activation of p38 MAP kinase.

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    Gerardo A Morfini

    Full Text Available Dying-back degeneration of motor neuron axons represents an established feature of familial amyotrophic lateral sclerosis (FALS associated with superoxide dismutase 1 (SOD1 mutations, but axon-autonomous effects of pathogenic SOD1 remained undefined. Characteristics of motor neurons affected in FALS include abnormal kinase activation, aberrant neurofilament phosphorylation, and fast axonal transport (FAT deficits, but functional relationships among these pathogenic events were unclear. Experiments in isolated squid axoplasm reveal that FALS-related SOD1 mutant polypeptides inhibit FAT through a mechanism involving a p38 mitogen activated protein kinase pathway. Mutant SOD1 activated neuronal p38 in mouse spinal cord, neuroblastoma cells and squid axoplasm. Active p38 MAP kinase phosphorylated kinesin-1, and this phosphorylation event inhibited kinesin-1. Finally, vesicle motility assays revealed previously unrecognized, isoform-specific effects of p38 on FAT. Axon-autonomous activation of the p38 pathway represents a novel gain of toxic function for FALS-linked SOD1 proteins consistent with the dying-back pattern of neurodegeneration characteristic of ALS.

  14. Association of Superoxide dismutases (SOD1 and SOD2) and Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus.

    Science.gov (United States)

    Vats, P; Sagar, N; Singh, T P; Banerjee, M

    2015-01-01

    Type 2 diabetes mellitus (T2DM) is a metabolic disorder resulting from oxidative stress (OS), the root cause of insulin resistance, β-cell dysfunction, and impaired glucose tolerance. Antioxidant enzymes play key roles in cellular defense and can be used as important biomarkers for T2DM. The present study was undertaken to evaluate three genetic polymorphisms viz. SOD1 + 35A/C, SOD2 + 47C/T, and GPx + 599C/T in 207 T2DM cases and 210 healthy controls from North India. DNA was extracted from blood samples and genotyping was done by PCR-RFLP. Genotypic/allelic frequencies and haplotype/gene-gene interaction analysis were performed using SPSS (version 15.0) and SHEsis (v. online). Except age, all other biochemical parameters showed highly significant association in T2DM cases (P 2.434). Genotype/allele frequencies of GPx1 + 599C/T and carriage rate showed no association although the odds ratio of GPx1 'C' allele indicated a 1.362 times higher risk of T2DM. SOD2 'CT' and GPx1 'CC' genotypes showed maximum association with biochemical parameters. Haplotype/gene-gene interaction analysis in controls and cases showed that SOD2 + 47C/T and GPx1 + 599C/T were in linkage disequilibrium (D: 0.168; r(2): 0.10) and individuals with this combination had a 1.273 times higher risk [OR; CI (95%)] of developing T2DM. Thus, we conclude that it is essential to assess the combinatorial association of gene variants with T2DM in order to identify risk haplotypes in a population.

  15. Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.

    Science.gov (United States)

    Foust, Kevin D; Salazar, Desirée L; Likhite, Shibi; Ferraiuolo, Laura; Ditsworth, Dara; Ilieva, Hristelina; Meyer, Kathrin; Schmelzer, Leah; Braun, Lyndsey; Cleveland, Don W; Kaspar, Brian K

    2013-12-01

    Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

  16. Amyotrophic lateral sclerosis-associated mutant SOD1 inhibits anterograde axonal transport of mitochondria by reducing Miro1 levels.

    Science.gov (United States)

    Moller, Annekathrin; Bauer, Claudia S; Cohen, Rebecca N; Webster, Christopher P; De Vos, Kurt J

    2017-09-14

    Defective axonal transport is an early neuropathological feature of amyotrophic lateral sclerosis (ALS). We have previously shown that ALS-associated mutations in Cu/Zn superoxide dismutase 1 (SOD1) impair axonal transport of mitochondria in motor neurons isolated from SOD1 G93A transgenic mice and in ALS mutant SOD1 transfected cortical neurons, but the underlying mechanisms remained unresolved.The outer mitochondrial membrane protein mitochondrial Rho GTPase 1 (Miro1) is a master regulator of mitochondrial axonal transport in response to cytosolic calcium (Ca2+) levels ([Ca2+]c) and mitochondrial damage. Ca2+ binding to Miro1 halts mitochondrial transport by modifying its interaction with kinesin-1 whereas mitochondrial damage induces Phosphatase and Tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and Parkin-dependent degradation of Miro1 and consequently stops transport.To identify the mechanism underlying impaired axonal transport of mitochondria in SOD1-related ALS we investigated [Ca2+]c and Miro1 levels in ALS mutant SOD1 expressing neurons. We found that expression of ALS mutant SOD1 reduced the level of endogenous Miro1 but did not affect [Ca2+]c. ALS mutant SOD1 induced reductions in Miro1 levels were Parkin dependent. Moreover, both overexpression of Miro1 and ablation of PINK1 rescued the mitochondrial axonal transport deficit in ALS mutant SOD1-expressing cortical and motor neurons.Together these results provide evidence that ALS mutant SOD1 inhibits axonal transport of mitochondria by inducing PINK1/Parkin-dependent Miro1 degradation. © The Author 2017. Published by Oxford University Press.

  17. Nitric oxide inhibits neointimal hyperplasia following vascular injury via differential, cell-specific modulation of SOD-1 in the arterial wall.

    Science.gov (United States)

    Bahnson, Edward S M; Koo, Nathaniel; Cantu-Medellin, Nadiezhda; Tsui, Aaron Y; Havelka, George E; Vercammen, Janet M; Jiang, Qun; Kelley, Eric E; Kibbe, Melina R

    2015-01-30

    Superoxide (O2(•-)) promotes neointimal hyperplasia following arterial injury. Conversely, nitric oxide ((•)NO) inhibits neointimal hyperplasia through various cell-specific mechanisms, including redox regulation. What remains unclear is whether (•)NO exerts cell-specific regulation of the vascular redox environment following arterial injury to inhibit neointimal hyperplasia. Therefore, the aim of the present study was to assess whether (•)NO exerts cell-specific, differential modulation of O2(•-) levels throughout the arterial wall, establish the mechanism of such modulation, and determine if it regulates (•)NO-dependent inhibition of neointimal hyperplasia. In vivo, (•)NO increased superoxide dismutase-1 (SOD-1) levels following carotid artery balloon injury in a rat model. In vitro, (•)NO increased SOD-1 levels in vascular smooth muscle cells (VSMC), but had no effect on SOD-1 in endothelial cells or adventitial fibroblasts. This SOD-1 increase was associated with an increase in sod1 gene expression, increase in SOD-1 activity, and decrease in O2(•-) levels. Lastly, to determine the role of SOD-1 in (•)NO-mediated inhibition of neointimal hyperplasia, we performed the femoral artery wire injury model in wild type and SOD-1 knockout (KO) mice, with and without (•)NO. Interestingly, (•)NO inhibited neointimal hyperplasia only in wild type mice, with no effect in SOD-1 KO mice. In conclusion, these data show the cell-specific modulation of O2(•-) by (•)NO through regulation of SOD-1 in the vasculature, highlighting its importance on the inhibition of neointimal hyperplasia. These results also shed light into the mechanism of (•)NO-dependent redox balance, and suggest a novel VSMC redox target to prevent neointimal hyperplasia.

  18. Analysis of SOD1 mutations in a Chinese population with amyotrophic lateral sclerosis: a case-control study and literature review

    Science.gov (United States)

    Wei, QianQian; Zhou, QingQing; Chen, YongPing; Ou, RuWei; Cao, Bei; Xu, YaQian; Yang, Jing; Shang, Hui-Fang

    2017-01-01

    Although the copper/zinc superoxide dismutase-1 (SOD1) gene has been identified in both familial ALS (FALS) and sporadic ALS (SALS), it has rarely been studied in Chinese patients with ALS, and there are few studies with large samples. This study sought to assess the prevalence of SOD1 mutations in Chinese ALS patients. We screened a cohort of 499 ALS patients (487 SALS and 12 FALS) from the Department of Neurology at the West China Hospital of Sichuan University and analyzed all coding exons of SOD1 by Sanger sequencing. In addition, we reviewed the mutation frequencies of common ALS causative genes in Chinese populations. Eight missense mutations in SOD1 were found in 8 ALS individuals: two novel mutations (p.G73D and p.V120F) and six previously reported mutations. The frequencies of SOD1 mutations were 1.03% (5/487) in SALS and 25% (3/12) in FALS from Southwest China. A literature review indicated that the mutation rates of major ALS causative genes were 53.55% in FALS and 6.29% in SALS. In Chinese SALS and FALS, the highest mutation frequency was in the SOD1 gene. Our results suggest that SOD1 mutation is the most common cause of ALS in Chinese populations and that the mutation spectrum of ALS varies among different ethnic populations. PMID:28291249

  19. SOD1 targeted to the mitochondrial intermembrane space prevents motor neuropathy in the Sod1 knockout mouse.

    Science.gov (United States)

    Fischer, Lindsey R; Igoudjil, Anissa; Magrané, Jordi; Li, Yingjie; Hansen, Jason M; Manfredi, Giovanni; Glass, Jonathan D

    2011-01-01

    Motor axon degeneration is a critical but poorly understood event leading to weakness and muscle atrophy in motor neuron diseases. Here, we investigated oxidative stress-mediated axonal degeneration in mice lacking the antioxidant enzyme, Cu,Zn superoxide dismutase (SOD1). We demonstrate a progressive motor axonopathy in these mice and show that Sod1(-/-) primary motor neurons extend short axons in vitro with reduced mitochondrial density. Sod1(-/-) neurons also show oxidation of mitochondrial--but not cytosolic--thioredoxin, suggesting that loss of SOD1 causes preferential oxidative stress in mitochondria, a primary source of superoxide in cells. SOD1 is widely regarded as the cytosolic isoform of superoxide dismutase, but is also found in the mitochondrial intermembrane space. The functional significance of SOD1 in the intermembrane space is unknown. We used a transgenic approach to express SOD1 exclusively in the intermembrane space and found that mitochondrial SOD1 is sufficient to prevent biochemical and morphological defects in the Sod1(-/-) model, and to rescue the motor phenotype of these mice when followed to 12 months of age. These results suggest that SOD1 in the mitochondrial intermembrane space is fundamental for motor axon maintenance, and implicate oxidative damage initiated at mitochondrial sites in the pathogenesis of motor axon degeneration.

  20. Low autophagy capacity implicated in motor system vulnerability to mutant superoxide dismutase

    OpenAIRE

    2016-01-01

    Introduction The motor system is selectively vulnerable to mutations in the ubiquitously expressed aggregation-prone enzyme superoxide dismutase-1 (SOD1). Results Autophagy clears aggregates, and factors involved in the process were analyzed in multiple areas of the CNS from human control subjects (n = 10) and amyotrophic lateral sclerosis (ALS) patients (n = 18) with or without SOD1 mutations. In control subjects, the key regulatory protein Beclin 1 and downstream factors were remarkably sca...

  1. Role of mitochondria in mutant SOD1 linked amyotrophic lateral sclerosis.

    Science.gov (United States)

    Tan, Wenzhi; Pasinelli, Piera; Trotti, Davide

    2014-08-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an adult onset characterized by loss of both upper and lower motor neurons. In ~10% of cases, patients developed ALS with an apparent genetic linkage (familial ALS or fALS). Approximately 20% of fALS displays mutations in the SOD1 gene encoding superoxide dismutase 1. There are many proposed cellular and molecular mechanisms among which, mitochondrial dysfunctions occur early, prior to symptoms occurrence. In this review, we modeled the effect of mutant SOD1 protein via the formation of a toxic complex with Bcl2 on mitochondrial bioenergetics. Furthermore, we discuss that the shutdown of ATP permeation through mitochondrial outer membrane could lead to both respiration inhibition and temporary mitochondrial hyperpolarization. Moreover, we reviewed mitochondrial calcium signaling, oxidative stress, fission and fusion, autophagy and apoptosis in mutant SOD1-linked ALS. Functional defects in mitochondria appear early before symptoms are manifested in ALS. Therefore, mitochondrial dysfunction is a promising therapeutic target in ALS.

  2. Mutational screening of VSX1, SPARC, SOD1, LOX, and TIMP3 in keratoconus

    Science.gov (United States)

    De Bonis, Patrizia; Laborante, Antonio; Pizzicoli, Costantina; Stallone, Raffaella; Barbano, Raffaela; Longo, Costanza; Mazzilli, Emilio; Zelante, Leopoldo

    2011-01-01

    Purpose To evaluate the involvement of Visual System Homeobox 1 (VSX1), Secreted Protein Acidic and Rich in Cysteine (SPARC), Superoxide Dismutase 1 (SOD1), Lysyl Oxidase (LOX), and Tissue Inhibitor of Metalloproteinase 3 (TIMP3) in sporadic and familial keratoconus. Methods Mutational analysis of the five genes was performed by sequencing and fragment analysis in a large cohort of 302 Italian patients, with a diagnosis of keratoconus based on clinical examination and corneal topography. The variants identified in VSX1 and SPARC were also assessed in the available relatives of the probands. Results A novel mutation p.G239R and previously reported mutations were found in VSX1. Novel and already reported variants were identified in SPARC and SOD1, whose pathogenic significance has not been established. No pathogenic variants have been identified in LOX and TIMP3. Conclusions Molecular analysis of the five genes in a cohort of 225 sporadic and 77 familial keratoconus cases confirms the possible pathogenic role of VSX1 though in a small number of patients; a possible involvement of LOX and TIMP3 could be excluded; and the role played by SOD1 and SPARC in determining the disease as not been definitively clarified. Further studies are required to identify other important genetic factors involved in the pathogenesis and progression of the disease that in the authors’ opinion, and according with several authors, should be considered as a complex disease. PMID:21976959

  3. Differential Motor Neuron Impairment and Axonal Regeneration in Sporadic and Familiar Amyotrophic Lateral Sclerosis with SOD-1 Mutations: Lessons from Neurophysiology

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    Tommaso Bocci

    2011-12-01

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a degenerative disorder of the motor system. About 10% of cases are familial and 20% of these families have point mutations in the Cu/Zn superoxide dismutase 1 (SOD-1 gene. SOD-1 catalyses the superoxide radical (O−2 into hydrogen peroxide and molecular oxygen. The clinical neurophysiology in ALS plays a fundamental role in differential diagnosis between the familial and sporadic forms and in the assessment of its severity and progression. Sixty ALS patients (34 males; 26 females were enrolled in the study and examined basally (T0 and every 4 months (T1, T2, and T3. Fifteen of these patients are SOD-1 symptomatic mutation carriers (nine males, six females. We used Macro-EMG and Motor Unit Number Estimation (MUNE in order to evaluate the neuronal loss and the re-innervation process at the onset of disease and during follow-up period. Results and Discussion: SOD-1 mutation carriers have a higher number of motor units at the moment of diagnosis when compared with the sporadic form, despite a more dramatic drop in later stages. Moreover, in familiar SOD-1 ALS there is not a specific time interval in which the axonal regeneration can balance the neuronal damage. Taken together, these results strengthen the idea of a different pathogenetic mechanism at the base of sALS and fALS.

  4. Nodularin Exposure Induces SOD1 Phosphorylation and Disrupts SOD1 Co-localization with Actin Filaments

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    Kari E. Fladmark

    2012-12-01

    Full Text Available Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS, which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1, without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized.

  5. Four familial ALS pedigrees discordant for two SOD1 mutations: Are all SOD1 mutations pathogenic?

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    Felbecker, Ansgar; Camu, William; Valdmanis, Paul; Sperfeld, Anne-Dorte; Waibel, Stefan; Steinbach, Peter; Rouleau, Guy A.; Ludolph, Albert C.; Andersen, Peter Munch

    2010-01-01

    Abstract Background: One-hundred and fiftythree mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been claimed to be associated with amyotrophic lateral sclerosis (ALS) in familial and sporadic ALS in an autosomal dominant or autosomal recessive pattern with complete or reduced penetrance. We now report four ALS pedigrees from Finland, France, Germany and Sweden with either the D90A and E100K SOD1 mutations in some but not all affected members. After re-collecting of DNA...

  6. Adaptive immune neuroprotection in G93A-SOD1 amyotrophic lateral sclerosis mice.

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    Rebecca Banerjee

    Full Text Available BACKGROUND: Innate neuroimmune dysfunction is a pathobiological feature of amyotrophic lateral sclerosis (ALS. However, links, if any, between disease and adaptive immunity are poorly understood. Thus, the role of T cell immunity in disease was investigated in human G93A superoxide dismutase 1 (SOD1 transgenic (Tg mice and subsequently in ALS patients. METHODS AND FINDINGS: Quantitative and qualitative immune deficits in lymphoid cell and T cell function were seen in G93A-SOD1 Tg mice. Spleens of Tg animals showed reductions in size, weight, lymphocyte numbers, and morphological deficits at terminal stages of disease compared to their wild-type (Wt littermates. Spleen sizes and weights of pre-symptomatic Tg mice were unchanged, but deficits were readily seen in T cell proliferation coincident with increased annexin-V associated apoptosis and necrosis of lymphocytes. These lymphoid deficits paralleled failure of Copolymer-1 (COP-1 immunization to affect longevity. In addition, among CD4(+ T cells in ALS patients, levels of CD45RA(+ (naïve T cells were diminished, while CD45RO(+ (memory T cells were increased compared to age-matched caregivers. In attempts to correct mutant SOD1 associated immune deficits, we reconstituted SOD1 Tg mice with unfractionated naïve lymphocytes or anti-CD3 activated CD4(+CD25(+ T regulatory cells (Treg or CD4(+CD25(- T effector cells (Teff from Wt donor mice. While naive lymphocytes failed to enhance survival, both polyclonal-activated Treg and Teff subsets delayed loss of motor function and extended survival; however, only Treg delayed neurological symptom onset, whereas Teff increased latency between disease onset and entry into late stage. CONCLUSIONS: A profound and progressive immunodeficiency is operative in G93A-SOD1 mice and is linked to T cell dysfunction and the failure to elicit COP-1 neuroprotective immune responses. In preliminary studies T cell deficits were also observed in human ALS. These findings

  7. The effects of wild-type and mutant SOD1 on smooth muscle contraction

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    Nikolić-Kokić Aleksandra

    2015-01-01

    Full Text Available In this work we compared the mutated liver copper zinc-containing superoxide dismutase (SOD1 protein G93A of the transgenic rat model of familial amyotrophic lateral sclerosis (FALS, to wild-type (WT rat SOD1. We examined their enzymatic activities and effects on isometric contractions of uteri of healthy virgin rats. G93A SOD1 showed a slightly higher activity than WT SOD1 and, in contrast to WT SOD1, G93A SOD1 did not induce smooth muscle relaxation. This result indicates that effects on smooth muscles are not related to SOD1 enzyme activity and suggest that heterodimers of G93A SOD1 form an ion-conducting pore that diminishes the relaxatory effects of SOD1. We propose that this type of pathogenic feedback affects neurons in FALS. [Projekat Ministarstva nauke Republike Srbije, br. 173014 i br. 175083

  8. Increased Zn/Glutathione Levels and Higher Superoxide Dismutase-1 Activity as Biomarkers of Oxidative Stress in Women with Long-Term Dental Amalgam Fillings: Correlation between Mercury/Aluminium Levels (in Hair) and Antioxidant Systems in Plasma

    Science.gov (United States)

    Cabaña-Muñoz, María Eugenia; Parmigiani-Izquierdo, José María; Bravo-González, Luis Alberto; Kyung, Hee-Moon; Merino, José Joaquín

    2015-01-01

    Background The induction of oxidative stress by Hg can affect antioxidant enzymes. However, epidemiological studies have failed to establish clear association between dental fillings presence and health problems. Objectives To determine whether heavy metals (in hair), antioxidant enzymes (SOD-1) and glutathione levels could be affected by the chronic presence of heavy metals in women who had dental amalgam fillings. Materials and Methods 55 hair samples (42 females with amalgam fillings and 13 female control subjects) were obtained. All subjects (mean age 44 years) who had dental amalgam filling for more than 10 years (average 15 years). Certain metals were quantified by ICP-MS (Mass Spectrophotometry) in hair (μg/g: Al, Hg, Ba, Ag, Sb, As, Be, Bi, Cd, Pb, Pt, Tl, Th, U, Ni, Sn, Ti) and SOD-1 and Glutathione (reduced form) levels in plasma. Data were compared with controls without amalgams, and analyzed to identify any significant relation between metals and the total number of amalgam fillings, comparing those with four or less (n = 27) with those with more than four (n = 15). As no significant differences were detected, the two groups were pooled (Amlgam; n = 42). Findings Hg, Ag, Al and Ba were higher in the amalgam group but without significant differences for most of the heavy metals analyzed. Increased SOD-1 activity and glutathione levels (reduced form) were observed in the amalgam group. Aluminum (Al) correlated with glutathione levels while Hg levels correlated with SOD-1. The observed Al/glutathione and Hg/SOD-1 correlation could be adaptive responses against the chronic presence of mercury. Conclusions Hg, Ag, Al and Ba levels increased in women who had dental amalgam fillings for long periods. Al correlated with glutathione, and Hg with SOD-1. SOD-1 may be a possible biomarker for assessing chronic Hg toxicity. PMID:26076368

  9. Increased Zn/Glutathione Levels and Higher Superoxide Dismutase-1 Activity as Biomarkers of Oxidative Stress in Women with Long-Term Dental Amalgam Fillings: Correlation between Mercury/Aluminium Levels (in Hair and Antioxidant Systems in Plasma.

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    María Eugenia Cabaña-Muñoz

    Full Text Available The induction of oxidative stress by Hg can affect antioxidant enzymes. However, epidemiological studies have failed to establish clear association between dental fillings presence and health problems.To determine whether heavy metals (in hair, antioxidant enzymes (SOD-1 and glutathione levels could be affected by the chronic presence of heavy metals in women who had dental amalgam fillings.55 hair samples (42 females with amalgam fillings and 13 female control subjects were obtained. All subjects (mean age 44 years who had dental amalgam filling for more than 10 years (average 15 years. Certain metals were quantified by ICP-MS (Mass Spectrophotometry in hair (μg/g: Al, Hg, Ba, Ag, Sb, As, Be, Bi, Cd, Pb, Pt, Tl, Th, U, Ni, Sn, Ti and SOD-1 and Glutathione (reduced form levels in plasma. Data were compared with controls without amalgams, and analyzed to identify any significant relation between metals and the total number of amalgam fillings, comparing those with four or less (n = 27 with those with more than four (n = 15. As no significant differences were detected, the two groups were pooled (Amlgam; n = 42.Hg, Ag, Al and Ba were higher in the amalgam group but without significant differences for most of the heavy metals analyzed. Increased SOD-1 activity and glutathione levels (reduced form were observed in the amalgam group. Aluminum (Al correlated with glutathione levels while Hg levels correlated with SOD-1. The observed Al/glutathione and Hg/SOD-1 correlation could be adaptive responses against the chronic presence of mercury.Hg, Ag, Al and Ba levels increased in women who had dental amalgam fillings for long periods. Al correlated with glutathione, and Hg with SOD-1. SOD-1 may be a possible biomarker for assessing chronic Hg toxicity.

  10. Collagen peptide and vitamin C additively attenuate age-related skin atrophy in Sod1-deficient mice.

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    Shibuya, Shuichi; Ozawa, Yusuke; Toda, Toshihiko; Watanabe, Kenji; Tometsuka, Chisa; Ogura, Takayuki; Koyama, Yoh-ichi; Shimizu, Takahiko

    2014-01-01

    Age-related skin thinning is correlated with a decrease in the content of collagen in the skin. Accumulating evidence suggests that collagen peptide (CP) and vitamin C (VC) transcriptionally upregulate type I collagen in vivo. However, the additive effects of CP and VC on age-related skin changes remain unclear. We herein demonstrate that CP and a VC derivative additively corrected age-related skin thinning via reduced oxidative damage in superoxide dismutase 1 (Sod1)-deficient mice. Co-treatment with these compounds significantly normalized the altered gene expression of Col1a1, Has2, and Ci1, a proton-coupled oligopeptide transporter, in Sod1(-/-) skin. The in vitro analyses further revealed that collagen oligopeptide, a digestive product of ingested CP, significantly promoted the bioactivity of the VC derivative with respect to the migration and proliferation of Sod1(-/-) fibroblasts. These findings suggest that combined treatment with CP and VC is effective in cases of age-related skin pathology.

  11. Chronic infusion of SOD1(G93A) astrocyte-secreted factors induces spinal motoneuron degeneration and neuromuscular dysfunction in healthy rats.

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    Ramírez-Jarquín, Uri N; Rojas, Fabiola; van Zundert, Brigitte; Tapia, Ricardo

    2017-01-27

    Amyotrophic lateral sclerosis is a fatal neurodegenerative disease and studies in vitro show that motoneuron degeneration is triggered by non-cell-autonomous mechanisms. However, whether soluble toxic factor(s) released by mutant superoxide dismutase 1 (SOD1) expressing astrocytes induces death of motoneurons and leads to motor dysfunction in vivo is not known. To directly test this, healthy adult rats were treated with conditioned media derived from primary mouse astrocytes (ACM) that express human (h) SOD1(G93A) (ACM-hG93A) via chronic osmotic pump infusion in the lumbar spinal cord. Controls included ACM derived from transgenic mice expressing hSOD1(WT) (ACM-hWT) or non-transgenic mouse SOD1(WT) (ACM-WT) astrocytes. Rats chronically infused with ACM-hG93A started to develop motor dysfunction at 8 days, as measured by rotarod performance. Additionally, immunohistochemical analyses at day 16 revealed reactive astrogliosis and significant loss of motoneurons in the ventral horn of the infused region. Controls did not show significant motor behavior alterations or neuronal damage. Thus, we demonstrate that factors released in vitro from astrocytes derived from ALS mice cause spinal motoneuron death and consequent neuromuscular dysfunction in vivo.

  12. Hexokinase I N-terminal based peptide prevents the VDAC1-SOD1 G93A interaction and re-establishes ALS cell viability

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    Magrì, Andrea; Belfiore, Ramona; Reina, Simona; Tomasello, Marianna Flora; Di Rosa, Maria Carmela; Guarino, Francesca; Leggio, Loredana; De Pinto, Vito; Messina, Angela

    2016-01-01

    Superoxide Dismutase 1 mutants associate with 20–25% of familial Amyotrophic Lateral Sclerosis (ALS) cases, producing toxic aggregates on mitochondria, notably in spinal cord. The Voltage Dependent Anion Channel isoform 1 (VDAC1) in the outer mitochondrial membrane is a docking site for SOD1 G93A mutant in ALS mice and the physiological receptor of Hexokinase I (HK1), which is poorly expressed in mouse spinal cord. Our results demonstrate that HK1 competes with SOD1 G93A for binding VDAC1, suggesting that in ALS spinal cord the available HK1-binding sites could be used by SOD1 mutants for docking mitochondria, producing thus organelle dysfunction. We tested this model by studying the action of a HK1-N-terminal based peptide (NHK1). This NHK1 peptide specifically interacts with VDAC1, inhibits the SOD1 G93A binding to mitochondria and restores the viability of ALS model NSC34 cells. Altogether, our results suggest that NHK1 peptide could be developed as a therapeutic tool in ALS, predicting an effective role also in other proteinopathies. PMID:27721436

  13. A comprehensive assessment of the SOD1G93A low-copy transgenic mouse, which models human amyotrophic lateral sclerosis

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    Abraham Acevedo-Arozena

    2011-09-01

    Amyotrophic lateral sclerosis (ALS is a progressive neurodegenerative disorder that results in the death of motor neurons in the brain and spinal cord. The disorder generally strikes in mid-life, relentlessly leading to paralysis and death, typically 3–5 years after diagnosis. No effective treatments are available. Up to 10% of ALS is familial, usually autosomal dominant. Several causative genes are known and, of these, mutant superoxide dismutase 1 (SOD1 is by far the most frequently found, accounting for up to 20% of familial ALS. A range of human mutant SOD1 transgenic mouse strains has been produced, and these largely successfully model the human disease. Of these, the most widely used is the SOD1 mouse, which expresses a human SOD1 transgene with a causative G93A mutation. This mouse model is excellent for many purposes but carries up to 25 copies of the transgene and produces a great excess of SOD1 protein, which might affect our interpretation of disease processes. A variant of this strain carries a deletion of the transgene array such that the copy number is dropped to eight to ten mutant SOD1 genes. This ‘deleted’ ‘low-copy’ mouse undergoes a slower course of disease, over many months. Here we have carried out a comprehensive analysis of phenotype, including nerve and muscle physiology and histology, to add to our knowledge of this ‘deleted’ strain and give baseline data for future studies. We find differences in phenotype that arise from genetic background and sex, and we quantify the loss of nerve and muscle function over time. The slowly progressive pathology observed in this mouse strain could provide us with a more appropriate model for studying early-stage pathological processes in ALS and aid the development of therapies for early-stage treatments.

  14. The anabolic/androgenic steroid nandrolone exacerbates gene expression modifications induced by mutant SOD1 in muscles of mice models of amyotrophic lateral sclerosis.

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    Galbiati, Mariarita; Onesto, Elisa; Zito, Arianna; Crippa, Valeria; Rusmini, Paola; Mariotti, Raffaella; Bentivoglio, Marina; Bendotti, Caterina; Poletti, Angelo

    2012-02-01

    Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)β1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFβ1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS.

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    Wang, Jiou; Farr, George W; Zeiss, Caroline J; Rodriguez-Gil, Diego J; Wilson, Jean H; Furtak, Krystyna; Rutkowski, D Thomas; Kaufman, Randal J; Ruse, Cristian I; Yates, John R; Perrin, Steve; Feany, Mel B; Horwich, Arthur L

    2009-02-03

    Recent studies suggest that superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis results from destabilization and misfolding of mutant forms of this abundant cytosolic enzyme. Here, we have tracked the expression and fate of a misfolding-prone human SOD1, G85R, fused to YFP, in a line of transgenic G85R SOD1-YFP mice. These mice, but not wild-type human SOD1-YFP transgenics, developed lethal paralyzing motor symptoms at 9 months. In situ RNA hybridization of spinal cords revealed predominant expression in motor neurons in spinal cord gray matter in all transgenic animals. Concordantly, G85R SOD-YFP was diffusely fluorescent in motor neurons of animals at 1 and 6 months of age, but at the time of symptoms, punctate aggregates were observed in cell bodies and processes. Biochemical analyses of spinal cord soluble extracts indicated that G85R SOD-YFP behaved as a misfolded monomer at all ages. It became progressively insoluble at 6 and 9 months of age, associated with presence of soluble oligomers observable by gel filtration. Immunoaffinity capture and mass spectrometry revealed association of G85R SOD-YFP, but not WT SOD-YFP, with the cytosolic chaperone Hsc70 at all ages. In addition, 3 Hsp110's, nucleotide exchange factors for Hsp70s, were captured at 6 and 9 months. Despite such chaperone interactions, G85R SOD-YFP formed insoluble inclusions at late times, containing predominantly intermediate filament proteins. We conclude that motor neurons, initially "compensated" to maintain the misfolded protein in a soluble state, become progressively unable to do so.

  16. Mutant SOD1 mediated pathogenesis of Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Kaur, Simran J; McKeown, Stephanie R; Rashid, Shazia

    2016-02-15

    Amyotrophic lateral sclerosis (ALS) is a neural disorder that causes death of the motor neurons in the brain and spinal cord; this affects the voluntary muscles and gradually leads to paralysis of the whole body. Most ALS cases are sporadic, though about 5-10% are familial. ALS is caused by multiple factors including mutation in any one of a number of specific genes, one of the most frequently affected is superoxide dismutase (SOD) 1. Alterations in SOD 1 have been linked with several variants of familial ALS. SOD 1 is a powerful antioxidant enzyme that protects cells from the damaging effects of superoxide radicals. The enzyme binds both copper and zinc ions that are directly involved in the deactivation of toxic superoxide radicals. Mutated SOD1 gene can acquire both gain and loss of function mutations. The most commonly identified mutations in SOD1 that affect protein activity are D90A, A4V and G93A. Deleterious mutations have been shown to modify SOD1 activity, which leads to the accumulation of highly toxic hydroxyl radicals. Accumulation of these free radicals causes degradation of both nuclear and mitochondrial DNA and protein misfolding, features which can be used as pathological indicators associated with ALS. Numerous clinical trials have been carried out over last few years with limited success. In some patients advanced techniques like gene and stem cell therapy have been trialed. However no definitive treatment option can provide a cure and currently ALS is managed by drugs and other supportive therapies. Consequently there is a need to identify new approaches for treatment of this ultimately fatal disease.

  17. Selective knockdown of mutant SOD1 in Schwann cells ameliorates disease in G85R mutant SOD1 transgenic mice.

    Science.gov (United States)

    Wang, Lijun; Pytel, Peter; Feltri, M Laura; Wrabetz, Lawrence; Roos, Raymond P

    2012-10-01

    Mutants of superoxide dismutase type 1 (mtSOD1) that have full dismutase activity (e.g., G37R) as well as none (e.g., G85R) cause familial amyotrophic lateral sclerosis (FALS), indicating that mtSOD1-induced FALS results from a toxicity rather than loss in SOD1 enzymatic activity. Still, it has remained unclear whether mtSOD1 dismutase activity can influence disease. A previous study demonstrated that Cre-mediated knockdown of G37R expression in Schwann cells (SCs) of G37R transgenic mice shortened the late phase of disease and survival. These results suggested that the neuroprotective effect of G37R expressed in SCs was greater than its toxicity, presumably because its dismutase activity counteracted reactive oxygen species (ROS). In order to further investigate this, we knocked down G85R in SCs by crossing G85R(flox) mice with myelin-protein-zero (P(0)):Cre mice, which express Cre recombinase in SCs. Knockdown of G85R in SCs of G85R mice delayed disease onset and extended survival indicating that G85R expression in SCs is neurotoxic. These results demonstrate differences in the effect on disease of dismutase active vs. inactive mtSOD1 suggesting that both a loss as well as gain in function of mtSOD1 influence FALS pathogenesis. The results suggest that mtSOD1-induced FALS treatment may have to be adjusted depending on the cell type targeted and particular mtSOD1 involved.

  18. Early onset of severe familial amyotrophic lateral sclerosis with a SOD-1 mutation: potential impact of CNTF as a candidate modifier gene.

    Science.gov (United States)

    Giess, Ralf; Holtmann, Bettina; Braga, Massimiliano; Grimm, Tiemo; Müller-Myhsok, Bertram; Toyka, Klaus V; Sendtner, Michael

    2002-05-01

    Mutations in the copper/zinc superoxide dismutase 1 (SOD-1) gene are found in approximately 20% of patients with familial amyotrophic lateral sclerosis (FALS), or amyotrophic lateral sclerosis 1. Here we describe a 25-year-old male patient who died from FALS after a rapid disease course of 11 mo. Sequencing of the SOD-1 gene revealed a heterozygous T-->G exchange at position 1513 within exon 5, coding for a V-->G substitution at position 148 of the mature protein. Genetic analysis of this family revealed the same mutation in both his healthy 35-year-old sister and his mother, who did not develop the disease before age 54 years. Screening for candidate modifier genes that might be responsible for the early onset and severe course of the disease in the 25-year-old patient revealed an additional homozygous mutation of the CNTF gene not found in his yet unaffected sister. hSOD-1G93A mice were crossbred with CNTF(-/-) mice and were investigated with respect to disease onset and duration, to test the hypothesis that CNTF acts as a candidate modifier gene in FALS with mutations in the SOD-1 gene. Such hSOD-1G93A/CNTF-deficient mice develop motoneuron disease at a significantly earlier stage than hSOD-1G93A/CNTF-wild-type mice. Linkage analysis revealed that the SOD-1 gene was solely responsible for the disease. However, disease onset as a quantitative trait was regulated by the allelic constitution at the CNTF locus. In addition, patients with sporadic amyotrophic lateral sclerosis who had a homozygous CNTF gene defect showed significantly earlier disease onset but did not show a significant difference in disease duration. Thus, we conclude that CNTF acts as a modifier gene that leads to early onset of disease in patients with FALS who have SOD-1 mutations, in patients with sporadic amyotrophic lateral sclerosis, and in the hSOD-1G93A mouse model.

  19. SOD1 Transcriptional and Posttranscriptional Regulation and Its Potential Implications in ALS

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    Pamela Milani

    2011-01-01

    Full Text Available Copper-zinc superoxide dismutase (SOD1 is a detoxifying enzyme localized in the cytosol, nucleus, peroxisomes, and mitochondria. The discovery that mutations in SOD1 gene cause a subset of familial amyotrophic lateral sclerosis (FALS has attracted great attention, and studies to date have been mainly focused on discovering mutations in the coding region and investigation at protein level. Considering that changes in SOD1 mRNA levels have been associated with sporadic ALS (SALS, a molecular understanding of the processes involved in the regulation of SOD1 gene expression could not only unravel novel regulatory pathways that may govern cellular phenotypes and changes in diseases but also might reveal therapeutic targets and treatments. This review seeks to provide an overview of SOD1 gene structure and of the processes through which SOD1 transcription is controlled. Furthermore, we emphasize the importance to focus future researches on investigating posttranscriptional mechanisms and their relevance to ALS.

  20. Preservation of neuromuscular function in symptomatic SOD1-G93A mice by peripheral infusion of methylene blue.

    Science.gov (United States)

    Talbot, Janet D; Barrett, John N; Nonner, Doris; Zhang, Zhongsheng; Wicomb, Kyle; Barrett, Ellen F

    2016-11-01

    In mutant superoxide dismutase 1 (SOD1) mouse models of familial amyotrophic lateral sclerosis (fALS) some of the earliest signs of morphological and functional damage occur in the motor nerve terminals that innervate fast limb muscles. This study tested whether localized peripheral application of a protective drug could effectively preserve neuromuscular junctions in late-stage disease. Methylene blue (MB), which has mitochondria-protective properties, was infused via an osmotic pump into the anterior muscle compartment of one hind limb of late pre- symptomatic SOD1-G93A mice for ≥3weeks. When mice reached end-stage disease, peak twitch and tetanic contractions evoked by stimulation of the muscle nerve were measured in two anterior compartment muscles (tibialis anterior [TA] and extensor digitorum longus [EDL], both predominantly fast muscles). With 400μM MB in the infusion reservoir, muscles on the MB-infused side exhibited on average a ~100% increase in nerve-evoked contractile force compared to muscles on the contralateral non-infused side (p<0.01 for both twitch and tetanus in EDL and TA). Pairwise comparisons of endplate innervation also revealed a beneficial effect of MB infusion, with an average of 65% of endplates innervated in infused EDL, compared to only 35% on the non-infused side (p<0.01). Results suggested that MB's protective effects required an extracellular [MB] of ~1μM, were initiated peripherally (no evidence of retrograde transport into the spinal cord), and involved MB's reduced form. Thus peripherally-initiated actions of MB can help preserve neuromuscular structure and function in SOD1-G93A mice, even at late stages of disease. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. Seeking homeostasis: Temporal trends in respiration, oxidation, and calcium in SOD1 G93A Amyotrophic Lateral Sclerosis mice

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    Cameron W Irvin

    2015-07-01

    Full Text Available Impairments in mitochondria, oxidative regulation, and calcium homeostasis have been well documented in numerous amyotrophic lateral sclerosis (ALS experimental models, especially in the superoxide dismutase 1 glycine 93 to alanine (SOD1 G93A transgenic mouse. However, the timing of these deficiencies has been debatable. In a systematic review of 45 articles, we examine experimental measurements of cellular respiration, mitochondrial mechanisms, oxidative markers, and calcium regulation. We evaluate the quantitative magnitude and statistical temporal trend of these aggregated assessments in high transgene copy SOD1 G93A mice compared to wild type mice. Analysis of overall trends reveals cellular respiration, intracellular ATP, and corresponding mitochondrial elements (Cox, cytochrome c, complex I, enzyme activity are depressed for the entire lifespan of the SOD1 G93A mouse. Oxidant markers (H2O2, 8OH2’dG, MDA are initially similar to wild type but are double that of wild type by the time of symptom onset despite early post-natal elevation of protective heat shock proteins. All aspects of calcium regulation show early disturbances, although a notable and likely compensatory convergence to near wild type levels appears to occur between 40-80 days (pre-onset, followed by a post-onset elevation in intracellular calcium. The identified temporal trends and compensatory fluctuations provide evidence that the cause of ALS may lay within failed homeostatic regulation, itself, rather than any one particular perturbing event or cellular mechanism. We discuss the vulnerabilities of motoneurons to regulatory instability and possible hypotheses regarding failed regulation and its potential treatment in ALS.

  2. Structures of the G85R Variant of SOD1 in Familial Amyotrophic Lateral Sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Xiaohang; Antonyuk, Svetlana V.; Seetharaman, Sai V.; Whitson, Lisa J.; Taylor, Alexander B.; Holloway, Stephen P.; Strange, Richard W.; Doucette, Peter A.; Valentine, Joan Selverstone; Tiwari, Ashutosh; Hayward, Lawrence J.; Padua, Shelby; Cohlberg, Jeffrey A.; Hasnain, S. Samar; Hart, P. John (Texas-HSC); (Cal. State); (UMASS, MED); (UCLA); (Daresbury)

    2008-07-21

    Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.

  3. HDAC6 regulates mutant SOD1 aggregation through two SMIR motifs and tubulin acetylation.

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    Gal, Jozsef; Chen, Jing; Barnett, Kelly R; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-05-24

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.

  4. HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation*

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    Gal, Jozsef; Chen, Jing; Barnett, Kelly R.; Yang, Liuqing; Brumley, Erin; Zhu, Haining

    2013-01-01

    Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS. PMID:23580651

  5. Degeneration of axons in spinal white matter in G93A mSOD1 mouse characterized by NFL and α-internexin immunoreactivity.

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    King, Anna E; Blizzard, Catherine A; Southam, Katherine A; Vickers, James C; Dickson, Tracey C

    2012-07-17

    Axonal degeneration is a prominent feature of amyotrophic lateral sclerosis (ALS) both in lower motor nerves as well as descending white matter axons in the spinal cord of human patients. Although the pathology of lower motor axonal degeneration has been described in both human ALS and related transgenic animal models, few studies have examined the pathological features of descending axon degeneration, particularly in mouse models of ALS. We have examined the degeneration of white matter tracts in the G93A mutant superoxide dismutase-1 (mSOD1+) mouse spinal cord white matter from 12 weeks of age to end-stage disease. In a G93A mSOD1 mouse model where green fluorescent protein was expressed in neurons (mSOD1+/GFP+), degeneration of white matter tracts was present from the ventral to dorsolateral funiculi. This pattern of axonal pathology occurred from 16 weeks of age. However, the dorsal funiculus, the site of the major corticospinal tract in mice, showed relatively less degeneration. Immunohistochemical analysis demonstrated that the neurofilament light chain (NFL) and neuronal intermediate filament protein alpha-internexin accumulated in axon swellings in the spinal white matter. Increased levels of alpha-internexin protein, in mSOD1+ mouse spinal cord tissue, were demonstrated by Western blotting. In contrast, degenerating axons did not show obvious accumulations of neurofilament medium and heavy chain proteins (NFM and NFH). These data suggest that white matter degeneration in this mouse model of ALS is widespread and involves a specific molecular signature, particularly the accumulation of NFL and alpha-internexin proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

  6. Amyotrophic lateral sclerosis linked to a novel SOD1 mutation with muscle mitochondrial dysfunction.

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    Corti, Stefania; Donadoni, Chiara; Ronchi, Dario; Bordoni, Andreina; Fortunato, Francesco; Santoro, Domenico; Del Bo, Roberto; Lucchini, Valeria; Crugnola, Veronica; Papadimitriou, Dimitra; Salani, Sabrina; Moggio, Maurizio; Bresolin, Nereo; Comi, Giacomo P

    2009-01-15

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disorder. Mutations in Cu,Zn superoxide dismutase (SOD1) cause approximately 20% of familial ALS. One of the possible mechanisms whereby they induce disease is mitochondrial dysfunction in motor neurons. Here we describe a patient with ALS and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation. Direct sequencing of SOD1 gene revealed a heterozygous mutation in codon 22 substituting a highly conserved amino acid, from glutamine to arginine (Q22R). Muscle biopsy showed a neurogenic pattern associated with cytochrome c oxidase (COX) deficiency in several muscle fibers. Western blot analysis demonstrated a reduction in SOD1 content in the cytoplasmic and mitochondrial fractions. These results suggest that a minute quantity of mutant SOD1 protein contributes to a mitochondrial toxicity also in muscle tissue.

  7. Exosomes from NSC-34 Cells Transfected with hSOD1-G93A Are Enriched in miR-124 and Drive Alterations in Microglia Phenotype

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    Sara Pinto

    2017-05-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal adult-onset neurodegenerative disorder affecting motor neurons (MNs. Evidences indicate that ALS is a non-cell autonomous disease in which glial cells participate in both disease onset and progression. Exosomal transfer of mutant copper-zinc superoxide dismutase 1 (mSOD1 from cell-to-cell was suggested to contribute to disease dissemination. Data from our group and others showed that exosomes from activated cells contain inflammatory-related microRNAs (inflamma-miRNAs that recapitulate the donor cell. While glia-derived exosomes and their effects in neurons have been addressed by several studies, only a few investigated the influence of motor neuron (MN-derived exosomes in other cell function, the aim of the present study. We assessed a set of inflamma-miRs in NSC-34 MN-like cells transfected with mutant SOD1(G93A and extended the study into their derived exosomes (mSOD1 exosomes. Then, the effects produced by mSOD1 exosomes in the activation and polarization of the recipient N9 microglial cells were investigated. Exosomes in coculture with N9 microglia and NSC-34 cells [either transfected with either wild-type (wt human SOD1 or mutant SOD1(G93A] showed to be transferred into N9 cells. Increased miR-124 expression was found in mSOD1 NSC-34 cells and in their derived exosomes. Incubation of mSOD1 exosomes with N9 cells determined a sustained 50% reduction in the cell phagocytic ability. It also caused a persistent NF-kB activation and an acute generation of NO, MMP-2, and MMP-9 activation, as well as upregulation of IL-1β, TNF-α, MHC-II, and iNOS gene expression, suggestive of induced M1 polarization. Marked elevation of IL-10, Arginase 1, TREM2, RAGE, and TLR4 mRNA levels, together with increased miR-124, miR-146a, and miR-155, at 24 h incubation, suggest the switch to mixed M1 and M2 subpopulations in the exosome-treated N9 microglial cells. Exosomes from mSOD1 NSC-34 MNs also enhanced the number

  8. Novel antibodies reveal inclusions containing non-native SOD1 in sporadic ALS patients.

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    Karin Forsberg

    Full Text Available Mutations in CuZn-superoxide dismutase (SOD1 cause amyotrophic lateral sclerosis (ALS and are found in 6% of ALS patients. Non-native and aggregation-prone forms of mutant SOD1s are thought to trigger the disease. Two sets of novel antibodies, raised in rabbits and chicken, against peptides spaced along the human SOD1 sequence, were by enzyme-linked immunosorbent assay and an immunocapture method shown to be specific for denatured SOD1. These were used to examine SOD1 in spinal cords of ALS patients lacking mutations in the enzyme. Small granular SOD1-immunoreactive inclusions were found in spinal motoneurons of all 37 sporadic and familial ALS patients studied, but only sparsely in 3 of 28 neurodegenerative and 2 of 19 non-neurological control patients. The granular inclusions were by confocal microscopy found to partly colocalize with markers for lysosomes but not with inclusions containing TAR DNA binding protein-43, ubiquitin or markers for endoplasmic reticulum, autophagosomes or mitochondria. Granular inclusions were also found in carriers of SOD1 mutations and in spinobulbar muscular atrophy (SBMA patients and they were the major type of inclusion detected in ALS patients homozygous for the wild type-like D90A mutation. The findings suggest that SOD1 may be involved in ALS pathogenesis in patients lacking mutations in the enzyme.

  9. Evidence of a genomic insertion in intron 2 of SOD1 causing allelic drop-out during routine diagnostic testing for canine degenerative myelopathy.

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    Turba, M E; Loechel, R; Rombolà, E; Gandini, G; Gentilini, F

    2017-06-01

    Degenerative myelopathy is a severe and progressive neurodegenerative disease and, in the majority of breeds, is associated with the c.118G>A substitution in exon 2 of the canine superoxide dismutase 1 (SOD1) gene. Our laboratories have been engaged in determining the cause of many discordant findings between the parental and the offspring genotypes found by different laboratories. In both cases, the discordant findings refer to actual heterozygous dogs that had been typed as homozygous for the variant allele. To that aim, the genomic context of the causative variant was investigated in two Hovawart dogs. An insertion of 54 nucleotides composed of a poly-T stretch and 15 nucleotides containing the duplication of the exon 2-intron 2 junction was found. The insertion was responsible for the partial mismatch of the reverse primer used for a direct sequencing assay. The mismatch hampered the amplification of the corresponding allele and caused an evident drop-out effect. The insertion is in complete linkage disequilibrium with the c.118G allele. The allele containing the insertion was highly prevalent in Hovawart dogs, accounting for the 26.6% of allele frequency. The insertion was also found in other unrelated breeds such as Rough Collies and Standard Poodles. In conclusion, the study illustrates the importance of correctly designing the primers to avoid inaccurate genotyping of the degenerative myelopathy causative variant in exon 2 of the SOD1 gene. © 2016 Stichting International Foundation for Animal Genetics.

  10. Non-invasive assessment of animal exercise stress: real-time PCR of GLUT4, COX2, SOD1 and HSP70 in avalanche military dog saliva.

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    Diverio, S; Guelfi, G; Barbato, O; Di Mari, W; Egidi, M G; Santoro, M M

    2015-01-01

    Exercise has been shown to increase mRNA expression of a growing number of genes. The aim of this study was to assess if mRNA expression of the metabolism- and oxidative stress-related genes GLUT4 (glucose transporter 4), COX2 (cyclooxygenase 2), SOD1 (superoxide dismutase 1) and HSP70 (heat shock protein 70) in saliva changes following acute exercise stress in dogs. For this purpose, 12 avalanche dogs of the Italian Military Force Guardia di Finanza were monitored during simulation of a search for a buried person in an artificial avalanche area. Rectal temperature (RT) and saliva samples were collected the day before the trial (T0), immediately after the descent from a helicopter at the onset of a simulated avalanche search and rescue operation (T1), after the discovery of the buried person (T2) and 2 h later (T3). Expressions of GLUT4, SOD1, COX2 and HSP70 were measured by real-time PCR. The simulated avalanche search and rescue operation was shown to exert a significant effect on RT, as well as on the expression of all metabolism- and oxidative stress-related genes investigated, which peaked at T2. The observed expression patterns indicate an acute exercise stress-induced upregulation, as confirmed by the reductions in expression at T3. Moreover, our findings indicate that saliva is useful for assessing metabolism- and oxidative stress-related genes without the need for restraint, which could affect working dog performance.

  11. The human G93A-SOD1 mutation in a pre-symptomatic rat model of amyotrophic lateral sclerosis increases the vulnerability to a mild spinal cord compression

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    Priestley John V

    2010-11-01

    Full Text Available Abstract Background Traumatic injuries can undermine neurological functions and act as risk factors for the development of irreversible and fatal neurodegenerative disorders like amyotrophic lateral sclerosis (ALS. In this study, we have investigated how a mutation of the superoxide dismutase 1 (SOD1 gene, linked to the development of ALS, modifies the acute response to a gentle mechanical compression of the spinal cord. In a 7-day post-injury time period, we have performed a comparative ontological analysis of the gene expression profiles of injured spinal cords obtained from pre-symptomatic rats over-expressing the G93A-SOD1 gene mutation and from wild type (WT littermates. Results The steady post-injury functional recovery observed in WT rats was accompanied by the early activation at the epicenter of injury of several growth-promoting signals and by the down-regulation of intermediate neurofilaments and of genes involved in the regulation of ion currents at the 7 day post-injury time point. The poor functional recovery observed in G93A-SOD1 transgenic animals was accompanied by the induction of fewer pro-survival signals, by an early activation of inflammatory markers, of several pro-apoptotic genes involved in cytochrome-C release and by the persistent up-regulation of the heavy neurofilament subunits and of genes involved in membrane excitability. These molecular changes occurred along with a pronounced atrophy of spinal cord motor neurones in the G93A-SOD1 rats compared to WT littermates after compression injury. Conclusions In an experimental paradigm of mild mechanical trauma which causes no major tissue damage, the G93A-SOD1 gene mutation alters the balance between pro-apoptotic and pro-survival molecular signals in the spinal cord tissue from the pre-symptomatic rat, leading to a premature activation of molecular pathways implicated in the natural development of ALS.

  12. An emerging role for misfolded wild-type SOD1 in sporadic ALS pathogenesis

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    Melissa S Rotunno

    2013-12-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder that targets motor neurons, leading to paralysis and death within a few years of disease onset. While several genes have been linked to the inheritable, or familial, form of ALS, much less is known about the cause(s of sporadic ALS, which accounts for approximately 90% of ALS cases. Due to the clinical similarities between familial and sporadic ALS, it is plausible that both forms of the disease converge on a common pathway and, therefore, involve common factors. Recent evidence suggests the Cu,Zn-superoxide dismutase (SOD1 protein to be one such factor that is common to both sporadic and familial ALS. In 1993, mutations were uncovered in SOD1 that represent the first known genetic cause of familial ALS. While the exact mechanism of mutant-SOD1 toxicity is still not known today, most evidence points to a gain of toxic function that stems, at least in part, from the propensity of this protein to misfold. In the wild-type SOD1 protein, non-genetic perturbations such as metal depletion, disruption of the quaternary structure, and oxidation, can also induce SOD1 to misfold. In fact, these aforementioned post-translational modifications cause wild-type SOD1 to adopt a toxic conformation that is similar to familial ALS-linked SOD1 variants. These observations, together with the detection of misfolded wild-type SOD1 within human post-mortem sporadic ALS samples, have been used to support the controversial hypothesis that misfolded forms of wild-type SOD1 contribute to sporadic ALS pathogenesis. In this review, we present data from the literature that both support and contradict this hypothesis. We also discuss SOD1 as a potential therapeutic target for both familial and sporadic ALS.

  13. In vivo pathogenic role of mutant SOD1 localized in the mitochondrial intermembrane space.

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    Igoudjil, Anissa; Magrané, Jordi; Fischer, Lindsey R; Kim, Hyun Jeong; Hervias, Isabel; Dumont, Magali; Cortez, Czrina; Glass, Jonathan D; Starkov, Anatoly A; Manfredi, Giovanni

    2011-11-02

    Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes a complex array of pathological events, through toxic gain of function mechanisms, leading to selective motor neuron degeneration. Mitochondrial dysfunction is among the well established toxic effects of mutant SOD1, but its mechanisms are just starting to be elucidated. A portion of mutant SOD1 is localized in mitochondria, where it accumulates mostly on the outer membrane and inside the intermembrane space (IMS). Evidence in cultured cells suggests that mutant SOD1 in the IMS causes mitochondrial dysfunction and compromises cell viability. Therefore, to test its pathogenic role in vivo we generated transgenic mice expressing G93A mutant or wild-type (WT) human SOD1 targeted selectively to the mitochondrial IMS (mito-SOD1). We show that mito-SOD1 is correctly localized in the IMS, where it oligomerizes and acquires enzymatic activity. Mito-G93ASOD1 mice, but not mito-WTSOD1 mice, develop a progressive disease characterized by body weight loss, muscle weakness, brain atrophy, and motor impairment, which is more severe in females. These symptoms are associated with reduced spinal motor neuron counts and impaired mitochondrial bioenergetics, characterized by decreased cytochrome oxidase activity and defective calcium handling. However, there is no evidence of muscle denervation, a cardinal pathological feature of ALS. Together, our findings indicate that mutant SOD1 in the mitochondrial IMS causes mitochondrial dysfunction and neurodegeneration, but per se it is not sufficient to cause a full-fledged ALS phenotype, which requires the participation of mutant SOD1 localized in other cellular compartments.

  14. Riluzole But Not Melatonin Ameliorates Acute Motor Neuron Degeneration and Moderately Inhibits SOD1-Mediated Excitotoxicity Induced Disrupted Mitochondrial Ca2+ Signaling in Amyotrophic Lateral Sclerosis

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    Jaiswal, Manoj Kumar

    2017-01-01

    Selective motoneurons (MNs) degeneration in the brain stem, hypoglossal motoneurons (HMNs), and the spinal cord resulting in patients paralysis and eventual death are prominent features of amyotrophic lateral sclerosis (ALS). Previous studies have suggested that mitochondrial respiratory impairment, low Ca2+ buffering and homeostasis and excitotoxicity are the pathological phenotypes found in mice, and cell culture models of familial ALS (fALS) linked with Cu/Zn-superoxide dismutase 1 (SOD1) mutation. In our study, we aimed to understand the impact of riluzole and melatonin on excitotoxicity, neuronal protection and Ca2+ signaling in individual HMNs ex vivo in symptomatic adult ALS mouse brain stem slice preparations and in WT and SOD1-G93A transfected SH-SY5Y neuroblastoma cell line using fluorescence microscopy, calcium imaging with high speed charged coupled device camera, together with immunohistochemistry, cell survival assay and histology. In our experiments, riluzole but not melatonin ameliorates MNs degeneration and moderately inhibit excitotoxicity and cell death in SH-SY5YWT or SH-SY5YG93A cell lines induced by complex IV blocker sodium azide. In brain stem slice preparations, riluzole significantly inhibit HMNs cell death induced by inhibiting the mitochondrial electron transport chain by Na-azide. In the HMNs of brainstem slice prepared from adult (14–15 weeks) WT, and corresponding symptomatic SOD1G93A mice, we measured the effect of riluzole and melatonin on [Ca2+]i using fura-2 AM ratiometric calcium imaging in individual MNs. Riluzole caused a significant decrease in [Ca2+]i transients and reversibly inhibited [Ca2+]i transients in Fura-2 AM loaded HMNs exposed to Na-azide in adult symptomatic SOD1G93A mice. On the contrary, melatonin failed to show similar effects in the HMNs of WT and SOD1G93A mice. Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence, an indicator of mitochondrial metabolism and health in MNs, showed enhanced

  15. Molecular chaperone mediated late-stage neuroprotection in the SOD1(G93A mouse model of amyotrophic lateral sclerosis.

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    Sergey S Novoselov

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons in the spinal cord, brain stem, and motor cortex. Mutations in superoxide dismutase (SOD1 are associated with familial ALS and lead to SOD1 protein misfolding and aggregation. Here we show that the molecular chaperone, HSJ1 (DNAJB2, mutations in which cause distal hereditary motor neuropathy, can reduce mutant SOD1 aggregation and improve motor neuron survival in mutant SOD1 models of ALS. Overexpression of human HSJ1a (hHSJ1a in vivo in motor neurons of SOD1(G93A transgenic mice ameliorated disease. In particular, there was a significant improvement in muscle force, increased motor unit number and enhanced motor neuron survival. hHSJ1a was present in a complex with SOD1(G93A and led to reduced SOD1 aggregation at late stages of disease progression. We also observed altered ubiquitin immunoreactivity in the double transgenic animals, suggesting that ubiquitin modification might be important for the observed improvements. In a cell model of SOD1(G93A aggregation, HSJ1a preferentially bound to mutant SOD1, enhanced SOD1 ubiquitylation and reduced SOD1 aggregation in a J-domain and ubiquitin interaction motif (UIM dependent manner. Collectively, the data suggest that HSJ1a acts on mutant SOD1 through a combination of chaperone, co-chaperone and pro-ubiquitylation activity. These results show that targeting SOD1 protein misfolding and aggregation in vivo can be neuroprotective and suggest that manipulation of DnaJ molecular chaperones might be useful in the treatment of ALS.

  16. Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset.

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    Christine Vande Velde

    Full Text Available Mutations in superoxide dismutase (SOD1 are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS.

  17. Mutant SOD1 inhibits ER-Golgi transport in amyotrophic lateral sclerosis.

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    Atkin, Julie D; Farg, Manal A; Soo, Kai Ying; Walker, Adam K; Halloran, Mark; Turner, Bradley J; Nagley, Phillip; Horne, Malcolm K

    2014-04-01

    Cu/Zn-superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER-Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER-Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER-Golgi transport by over-expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER-Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

  18. Cytoplasmic reactive oxygen species and SOD1 regulate bone mass during mechanical unloading.

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    Morikawa, Daichi; Nojiri, Hidetoshi; Saita, Yoshitomo; Kobayashi, Keiji; Watanabe, Kenji; Ozawa, Yusuke; Koike, Masato; Asou, Yoshinori; Takaku, Tomoiku; Kaneko, Kazuo; Shimizu, Takahiko

    2013-11-01

    Oxidative stress contributes to the pathogenesis of age-related diseases as well as bone fragility. Our previous study demonstrated that copper/zinc superoxide dismutase (Sod1)-deficient mice exhibit the induction of intracellular reactive oxygen species (ROS) and bone fragility resulting from low-turnover bone loss and impaired collagen cross-linking (Nojiri et al. J Bone Miner Res. 2011;26:2682-94). Mechanical stress also plays an important role in the maintenance of homeostasis in bone tissue. However, the molecular links between oxidative and mechanical stresses in bone tissue have not been fully elucidated. We herein report that mechanical unloading significantly increased intracellular ROS production and the specific upregulation of Sod1 in bone tissue in a tail-suspension experiment. We also reveal that Sod1 loss exacerbated bone loss via reduced osteoblastic abilities during mechanical unloading. Interestingly, we found that the administration of an antioxidant, vitamin C, significantly attenuated bone loss during unloading. These results indicate that mechanical unloading, in part, regulates bone mass via intracellular ROS generation and the Sod1 expression, suggesting that activating Sod1 may be a preventive strategy for ameliorating mechanical unloading-induced bone loss.

  19. Vacuolization correlates with spin-spin relaxation time in motor brainstem nuclei and behavioural tests in the transgenic G93A-SOD1 mouse model of ALS.

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    Bucher, Selina; Braunstein, Kerstin E; Niessen, Heiko G; Kaulisch, Thomas; Neumaier, Michael; Boeckers, Tobias M; Stiller, Detlef; Ludolph, Albert C

    2007-10-01

    In recent years, magnetic resonance imaging (MRI) has emerged as a preferred tool for the diagnosis of amyotrophic lateral sclerosis (ALS) in humans. A widely used animal model for human ALS is the G93A-superoxide dismutase 1 (G93A-SOD1) transgenic mouse model. However, the mechanisms for the selective degeneration of motor neurons in the brainstem and spinal cord are still uncertain. In our study, we applied MRI at 4.7 Tesla to non-invasively evaluate pathological alterations in the brainstem of this animal model and to follow the progression of the disease. Extending previous investigation, we used the relaxation parameter T(2) as a suitable measure for the progression of ALS, and evaluated the potential agreement with histological evaluation and behavioural data of open-field tests. In the brainstem of G93A-SOD1 mice, T(2) values were significantly increased in the motor nuclei Nc. V, Nc. VII and Nc. XII, as early as Day 80, i.e. before the average disease onset at about Day 90. Moreover, this increase is associated with a progressive development of vacuoles in the brainstem motor nuclei and a significantly decreased performance in behavioural tests. Overall, MRI is a very sensitive tool to obtain correlates for neuronal degeneration in vivo. Furthermore, MRI enables us to investigate a follow up at different time points of the disease. These advantages are especially useful for therapeutic studies with respect to survival rates of motor neurons using mouse models. Finally, our data suggest that MRI does not only resemble the findings of behavioural tests, but is potentially superior to behavioural studies.

  20. Cell-penetrating superoxide dismutase attenuates oxidative stress-induced senescence by regulating the p53-p21Cip1 pathway and restores osteoblastic differentiation in human dental pulp stem cells

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    Park YJ

    2012-09-01

    Full Text Available Yoon Jung Choi,1,* Jue Yeon Lee,2,* Chong Pyoung Chung,2 Yoon Jeong Park,1,21Craniomaxillofacial Reconstructive Sciences, Dental Research Institute, School of Dentistry, Seoul National University, Seoul, Republic of Korea; 2Research Institute, Nano Intelligent Biomedical Engineering, Seoul, Republic of Korea*These authors contributed equally to this workBackground: Human dental pulp stem cells (DPSCs have potential applications in tissue regeneration because of their convenient cell harvesting procedures and multipotent capacity. However, the tissue regenerative potential of DPSCs is known to be negatively regulated by aging in long-term culture and under oxidative stress. With an aim of reducing cellular senescence and oxidative stress in DPSCs, an intracellular delivery system for superoxide dismutase 1 (SOD1 was developed. We conjugated SOD1 with a cell-penetrating peptide known as low-molecular weight protamine (LMWP, and investigated the effect of LMWP-SOD1 conjugates on hydrogen peroxide-induced cellular senescence and osteoblastic differentiation.Results: LMWP-SOD1 significantly attenuated enlarged and flattened cell morphology and increased senescence-associated β-galactosidase activity. Under the same conditions, LMWP-SOD1 abolished activation of the cell cycle regulator proteins, p53 and p21Cip1, induced by hydrogen peroxide. In addition, LMWP-SOD1 reversed the inhibition of osteoblastic differentiation and downregulation of osteogenic gene markers induced by hydrogen peroxide. However, LMWP-SOD1 could not reverse the decrease in odontogenesis caused by hydrogen peroxide.Conclusion: Overall, cell-penetrating LMWP-SOD1 conjugates are effective for attenuation of cellular senescence and reversal of osteoblastic differentiation of DPSCs caused by oxidative stress inhibition. This result suggests potential application in the field of antiaging and tissue engineering to overcome the limitations of senescent stem cells.Keywords: superoxide

  1. Soluble RAGE Treatment Delays Progression of Amyotrophic Lateral Sclerosis in SOD1 Mice.

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    Juranek, Judyta K; Daffu, Gurdip K; Geddis, Matthew S; Li, Huilin; Rosario, Rosa; Kaplan, Benjamin J; Kelly, Lauren; Schmidt, Ann Marie

    2016-01-01

    The etiology of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder characterized by progressive muscle weakness and spasticity, remains largely unknown. Approximately 5-10% of cases are familial, and of those, 15-20% are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1). Mutations of the SOD1 gene interrupt cellular homeostasis and contribute to cellular toxicity evoked by the presence of altered SOD1, along with other toxic species, such as advanced glycation end products (AGEs). AGEs trigger activation of their chief cell surface receptor, RAGE (receptor for advanced glycation end products), and induce RAGE-dependent cellular stress and inflammation in neurons, thereby affecting their function and leading to apoptosis. Here, we show for the first time that the expression of RAGE is higher in the SOD1 transgenic mouse model of ALS vs. wild-type mouse spinal cord. We tested whether pharmacological blockade of RAGE may delay the onset and progression of disease in this mouse model. Our findings reveal that treatment of SOD1 transgenic mice with soluble RAGE (sRAGE), a natural competitor of RAGE that sequesters RAGE ligands and blocks their interaction with cell surface RAGE, significantly delays the progression of ALS and prolongs life span compared to vehicle treatment. We demonstrate that in sRAGE-treated SOD1 transgenic animals at the final stage of the disease, a significantly higher number of neurons and lower number of astrocytes is detectable in the spinal cord. We conclude that RAGE antagonism may provide a novel therapeutic strategy for ALS intervention.

  2. Soluble RAGE Treatment Delays Progression of Amyotrophic Lateral Sclerosis in SOD1 Mice

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    Judyta K Juranek

    2016-05-01

    Full Text Available The etiology of amyotrophic lateral sclerosis (ALS, a fatal motor neuron disorder characterized by progressive muscle weakness and spasticity, remains largely unknown. Approximately 5-10% of cases are familial, and of those, 15-20% are associated with mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1. Mutations of the SOD1 gene interrupt cellular homeostasis and contribute to cellular toxicity evoked by the presence of altered SOD1, along with other toxic species, such as advanced glycation end products (AGEs. AGEs trigger activation of their chief cell surface receptor, RAGE (receptor for advanced glycation end products, and induce RAGE-dependent cellular stress and inflammation in neurons, thereby affecting their function and leading to apoptosis. Here, we show for the first time that the expression of RAGE is higher in the SOD1 transgenic mouse model of ALS versus wild-type mouse spinal cord. We tested whether pharmacological blockade of RAGE may delay the onset and progression of disease in this mouse model. Our findings reveal that treatment of SOD1 transgenic mice with soluble RAGE (sRAGE, a natural competitor of RAGE that sequesters RAGE ligands and blocks their interaction with cell surface RAGE, significantly delays the progression of ALS and prolongs life span compared to vehicle treatment. We demonstrate that in sRAGE-treated SOD1 transgenic animals at the final stage of the disease, a significantly higher number of neurons and lower number of astrocytes is detectable in the spinal cord. We conclude that RAGE antagonism may provide a novel therapeutic strategy for ALS intervention.

  3. Tar DNA binding protein of 43 kDa (TDP-43), 14-3-3 proteins and copper/zinc superoxide dismutase (SOD1) interact to modulate NFL mRNA stability. Implications for altered RNA processing in amyotrophic lateral sclerosis (ALS).

    Science.gov (United States)

    Volkening, Kathryn; Leystra-Lantz, Cheryl; Yang, Wenchang; Jaffee, Howard; Strong, Michael J

    2009-12-11

    Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease characterized by progressive motor neuron degeneration in association with neurofilament (NF) aggregate formation. This process is accompanied by an alteration in the stoichiometry of NF subunit protein expression such that the steady state levels of the low molecular weight NF (NFL) mRNA levels are selectively suppressed. We have previously shown that each of TDP-43, 14-3-3 and mutant SOD1 can function as NFL mRNA 3'UTR binding proteins that directly affect the stability of NFL transcripts. In this study, we demonstrate that the interaction of TDP-43 with the NFL mRNA 3' UTR involves ribonucleotide (UG) motifs present on stem loops of the 3'UTR as well as the RRM1 and RRM2 motifs of TDP-43. Ex vivo, TDP-43, 14-3-3 and SOD1 proteins interact to modulate NFL mRNA stability, although in vivo, only TDP-43 and either mutant or wild-type SOD1 co-localize in ALS motor neurons. TDP-43 was observed to co-localize to RNA transport granules (Staufen immunoreactive) in both control and ALS spinal motor neurons. In contrast, both stress granules (TIA-1 immunoreactive) and processing bodies (P-bodies; XRN-1 immunoreactive) were more prevalent in ALS motor neurons than in controls and demonstrated strong co-localization with TDP-43. Using RNA-IP-PCR, we further demonstrate that NFL mRNA is preferentially sequestered to both stress granules and P-bodies in ALS. These data suggest that NFL mRNA processing is fundamentally altered in ALS spinal motor neurons to favour compartmentalization within both stress granules and P-bodies, and that TDP-43 plays a fundamental role in this process.

  4. An inactivating mutation in the SOD 1 gene causes familial amyotrophic lateral sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Pramatarova, A.; Rouleau, G.A. [Montreal General Hospital Research Institute (Canada); Goto, J. [Univ. of Tokyo (Japan)] [and others

    1994-09-01

    Amyotrophic lateral sclerosis (ALS) is characterized by highly selective death of large motor neurons in the cerebral cortex and spinal cord. The familial form of ALS (FALS) accounts for approximately 10% of the cases and is transmitted in an autosomal dominant manner. Recently the defective gene causing chromosome 21-linked FALS was shown to be the Cu/Zn superoxide dismutase (SOD 1). However, the precise mechanism of neurotoxicity seen in FALS with SOD 1 mutations is still unknown. Until now all SOD 1 mutations reported were single base pair substitutions (missense). We have identified a nonsense mutation in exon 5 of the SOD 1 gene in a FALS kindred. This two base pair deletion provokes a frameshift and a predicted premature truncation of the protein. The region affected has a very important structural and functional role: it contains part of the active loop and is involved in dimer contact. We would predict that the loss of these structures would impair the functioning of the enzyme.

  5. Structural and biophysical properties of metal-free pathogenic SOD1 mutants A4V and G93A

    Energy Technology Data Exchange (ETDEWEB)

    Galaleldeen, Ahmad; Strange, Richard W.; Whitson, Lisa J.; Antonyuk, Svetlana V.; Narayana, Narendra; Taylor, Alexander B.; Schuermann, Jonathan P.; Holloway, Stephen P.; Hasnain, S.Samar; Hart, P. John; (Texas-HSC); (Liverpool)

    2010-07-19

    Amyotrophic lateral sclerosis (ALS) is a fatal, progressive neurodegenerative disease characterized by the destruction of motor neurons in the spinal cord and brain. A subset of ALS cases are linked to dominant mutations in copper-zinc superoxide dismutase (SOD1). The pathogenic SOD1 variants A4V and G93A have been the foci of multiple studies aimed at understanding the molecular basis for SOD1-linked ALS. The A4V variant is responsible for the majority of familial ALS cases in North America, causing rapidly progressing paralysis once symptoms begin and the G93A SOD1 variant is overexpressed in often studied murine models of the disease. Here we report the three-dimensional structures of metal-free A4V and of metal-bound and metal-free G93A SOD1. In the metal-free structures, the metal-binding loop elements are observed to be severely disordered, suggesting that these variants may share mechanisms of aggregation proposed previously for other pathogenic SOD1 proteins.

  6. SOD1 Overexpression Preserves Baroreflex Control of Heart Rate with an Increase of Aortic Depressor Nerve Function

    Science.gov (United States)

    Hatcher, Jeffrey; Gu, He; Cheng, Zixi (Jack)

    2016-01-01

    Overproduction of reactive oxygen species (ROS), such as the superoxide radical (O2∙−), is associated with diseases which compromise cardiac autonomic function. Overexpression of SOD1 may offer protection against ROS damage to the cardiac autonomic nervous system, but reductions of O2∙− may interfere with normal cellular functions. We have selected the C57B6SJL-Tg (SOD1)2 Gur/J mouse as a model to determine whether SOD1 overexpression alters cardiac autonomic function, as measured by baroreflex sensitivity (BRS) and aortic depressor nerve (ADN) recordings, as well as evaluation of baseline heart rate (HR) and mean arterial pressure (MAP). Under isoflurane anesthesia, C57 wild-type and SOD1 mice were catheterized with an arterial pressure transducer and measurements of HR and MAP were taken. After establishing a baseline, hypotension and hypertension were induced by injection of sodium nitroprusside (SNP) and phenylephrine (PE), respectively, and ΔHR versus ΔMAP were recorded as a measure of baroreflex sensitivity (BRS). SNP and PE treatment were administered sequentially after a recovery period to measure arterial baroreceptor activation by recording aortic depressor nerve activity. Our findings show that overexpression of SOD1 in C57B6SJL-Tg (SOD1)2 Gur/J mouse preserved the normal HR, MAP, and BRS but enhanced aortic depressor nerve function. PMID:26823951

  7. Solution oxygen-17 NMR application for observing a peroxidized cysteine residue in oxidized human SOD1

    Science.gov (United States)

    Fujiwara, Noriko; Yoshihara, Daisaku; Sakiyama, Haruhiko; Eguchi, Hironobu; Suzuki, Keiichiro

    2016-12-01

    NMR active nuclei, 1H, 13C and 15N, are usually used for determination of protein structure. However, solution 17O-NMR application to proteins is extremely limited although oxygen is an essential element in biomolecules. Proteins are oxidized through cysteine residues by two types of oxidation. One is reversible oxidation such as disulphide bonding (Cys-S-S-Cys) and the other is irreversible oxidation to cysteine sulfinic acid (Cys-SO 2H) and cysteine sulfonic acid (Cys-SO 3H). Copper,Zinc-superoxide dismutase (SOD1) is a key enzyme in the protection of cells from the superoxide anion radical. The SH group at Cys 111 residue in human SOD1 is selectively oxidized to -SO 2H and -SO 3H with atmospheric oxygen, and this oxidized human SOD1 is also suggested to play an important role in the pathophysiology of various neurodegenerative diseases, probably mainly via protein aggregation. Therefore, information on the structural and the dynamics of the oxidized cysteine residue would be crucial for the understanding of protein aggregation mechanism. Although the -SO 3H group on proteins cannot be directly detected by conventional NMR techniques, we successfully performed the site-specific 17O-labeling of Cys 111 in SOD1 using ^{17}it {O}2 gas and the 17O-NMR analysis for the first time. We observed clear 17O signal derived from a protein molecule and show that 17O-NMR is a sensitive probe for studying the structure and dynamics of the 17O-labeled protein molecule. This novel and unique strategy can have great impact on many research fields in biology and chemistry.

  8. Overexpression of human SOD1 improves survival of mice susceptible to endotoxic shock

    Directory of Open Access Journals (Sweden)

    Charchaflieh J

    2012-07-01

    Full Text Available Jean Charchaflieh,1,2 Georges I Labaze,1 Pulsar Li,1 Holly Van Remmen,3 Haekyung Lee,1 Helen Stutz,1 Arlan Richardson,3 Asher Emanuel,1 Ming Zhang1,41Department of Anesthesiology, State University of New York (SUNY Downstate Medical Center, New York, NY, USA; 2Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USA; 3Barshop Center for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA; 4Department of Cell Biology, State University of New York (SUNY Downstate Medical Center, New York, NY, USABackground: Protective effects of the antioxidant enzyme Cu-Zn superoxide dismutase (SOD1 against endotoxic shock have not been demonstrated in animal models. We used a murine model to investigate whether overexpression of SOD1 protects against endotoxic shock, and whether the genetic background of SOD1 affects its effective protective effects and susceptibility to endotoxic shock.Methods: Transgenic (tg mice overexpressing human SOD1 and control mice were divided into four groups based on their genetic background: (1 tg mice with mixed genetic background (tg-JAX; (2 wild-type (WT littermates of tg-JAX strain (WT-JAX; (3 tg mice with C57BL/6J background (tg-TX; (4 WT littermates of tg-TX strain (WT-TX. Activity of SOD1 in the intestine, heart, and liver of tg and control mice was confirmed using a polyacrylamide activity gel. Endotoxic shock was induced by intraperitoneal injection of lipopolysaccharide. Survival rates over 120 hours (mean, 95% confidence interval were analyzed using Kaplan–Meier survival curves.Results: Human SOD1 enzymatic activities were significantly higher in the intestine, heart, and liver of both tg strains (tg-JAX and tg-TX compared with their WT littermates (WT-JAX and WT-TX, respectively. Interestingly, the endogenous SOD1 activities in tg-JAX mice were decreased compared with their WT littermates (WT-JAX, but such aberrant changes were not

  9. Screening of drugs inhibiting in vitro oligomerization of Cu/Zn-superoxide dismutase with a mutation causing amyotrophic lateral sclerosis

    OpenAIRE

    Itsuki Anzai; Keisuke Toichi; Eiichi Tokuda; Atsushi Mukaiyama; Shuji Akiyama; Yoshiaki Furukawa

    2016-01-01

    Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS). A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers ab...

  10. 50-bp Ins/Del polymorphism of SOD1 is associated with increased risk of cardiovascular disease.

    Science.gov (United States)

    Eskandari-Nasab, Ebrahim; Kharazi-Nejad, Eslam; Nakhaee, Alireza; Afzali, Masoumeh; Tabatabaei, Seyed Payman; Tirgar-Fakheri, Kourosh; Hashemi, Mohammad

    2014-01-01

    Compelling evidence suggests that the oxidative stress plays a key role in the pathophysiology of cardiovascular disease (CVD). Superoxide dismutase (SOD) enzymes play a major role in detoxification of reactive oxygen species and protection against oxidative stress. We examined the possible association between a 50-bp insertion/deletion in the SOD1 promoter 1684-bp upstream of the SOD1 ATG with CVD in an Iranian population. A total of 400 individuals including 200 CVD patients and 200 healthy subjects from the same ethnic background as the control group were participated in this study. Genomic DNA from all subjects was screened for the 50-bp SOD1 promoter deletion using a polymerase chain reaction (PCR) assay. Our finding showed an association between SOD1 DEL/DEL (9% vs. 2.5%) and INS/DEL genotypes and risk of CVD and these genotypes increased the susceptibility to CVD (OR=2.096, 95% CI: 1.336-3.286, P= 0.001 for the INS/DEL genotype; OR=4.811, 95% CI: 1.734-13.346, P= 0.003 for the DEL/DEL genotype). Additionally, the DEL allele of the SOD1 variation was found to be more prevalent in the CVD patients with the frequency of 26.3% and 13.5% in cases and controls, respectively, and this difference reached statistical significance (OR=2.281, 95% CI: 1.586-3.279, P= 0.001).  The analysis of SOD1 genotypes according to patients' characteristics revealed that the SOD1 Ins/del and Del/Del genotypes were more prevalent in CVD patients with a history of CVD or hypertension or DM (PIns/Ins genotype carriers had no history of these diseases. Overall, our results demonstrated that SOD1 50-bp Del/Del and Ins/Del genotypes, as well as Del, allele, were associated with an increased risk of CVD.

  11. Mitochondria-targeted catalase reverts the neurotoxicity of hSOD1G⁹³A astrocytes without extending the survival of ALS-linked mutant hSOD1 mice.

    Directory of Open Access Journals (Sweden)

    Mariana Pehar

    Full Text Available Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1 cause familial forms of amyotrophic lateral sclerosis (ALS, a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1G93A astrocyte cultures. It also reverted the toxicity of hSOD1G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage

  12. Fipronil induced oxidative stress involves alterations in SOD1 and catalase gene expression in male mice liver: Protection by vitamins E and C.

    Science.gov (United States)

    Badgujar, Prarabdh C; Chandratre, Gauri A; Pawar, Nitin N; Telang, A G; Kurade, N P

    2016-09-01

    In the present investigation, hepatic oxidative stress induced by fipronil was evaluated in male mice. We also investigated whether pretreatment with antioxidant vitamins E and C could protect mice against these effects. Several studies conducted in cell lines have shown fipronil as a potent oxidant; however, no information is available regarding its oxidative stress inducing potential in an animal model. Out of 8 mice groups, fipronil was administered to three groups at low, medium, and high dose based on its oral LD50 (2.5, 5, and 10 mg/kg). All three doses of fipronil caused a significant increase in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) level with concomitant increase in the absolute and relative weight of liver. High dose of fipronil caused significant down-regulation in the hepatic mRNA expression of superoxide dismutase 1 (SOD1) and catalase (0.412 ± 0.01 and 0.376 ± 0.05-fold, respectively) as well as an increase in the lipid peroxidation (LPO). Also, decrease in the activity of antioxidant enzymes; SOD, catalase, and glutathione-S-transferase (GST) and the content of nonantioxidant enzymes; glutathione and total thiol were recorded. Histopathological examination of liver revealed dose dependant changes such as severe fatty degeneration and vacuolation leading to hepatocellular necrosis. Prior administration of vitamin E or vitamin C against fipronil high dose caused decrease in lipid peroxidation and increased activity of antioxidant enzymes. Severe reduction observed in functional activities of antioxidant enzymes was aptly substantiated by down-regulation seen in their relative mRNA expression. Thus results of the present study imply that liver is an important target organ for fipronil and similar to in vitro reports, it induces oxidative stress in the mice liver, which in turn could be responsible for its hepatotoxic nature. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1147-1158, 2016. © 2015

  13. Notch pathway is activated in cell culture and mouse models of mutant SOD1-related familial amyotrophic lateral sclerosis, with suppression of its activation as an additional mechanism of neuroprotection for lithium and valproate.

    Science.gov (United States)

    Wang, S-Y; Ren, M; Jiang, H-Z; Wang, J; Jiang, H-Q; Yin, X; Qi, Y; Wang, X-D; Dong, G-T; Wang, T-H; Yang, Y-Q; Feng, H-L

    2015-08-20

    Amyotrophic lateral sclerosis (ALS) is an idiopathic and lethal neurodegenerative disease that currently has no effective treatment. A recent study found that the Notch signaling pathway was up-regulated in a TAR DNA-binding protein-43 (TDP-43) Drosophila model of ALS. Notch signaling acts as a master regulator in the central nervous system. However, the mechanisms by which Notch participates in the pathogenesis of ALS have not been completely elucidated. Recent studies have shown that the mood stabilizers lithium and valproic acid (VPA) are able to regulate Notch signaling. Our study sought to confirm the relationship between the Notch pathway and ALS and whether the Notch pathway contributes to the neuroprotective effects of lithium and VPA in ALS. We found that the Notch pathway was activated in in vitro and in vivo models of ALS, and suppression of Notch activation with a Notch signaling inhibitor, N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT) and Notch1 siRNA significantly reduced neuronal apoptotic signaling, as evidenced by the up-regulation of Bcl-2 as well as the down-regulation of Bax and cytochrome c. We also found that lithium and VPA suppressed the Notch activation associated with the superoxide dismutase-1 (SOD1) mutation, and the combination of lithium and VPA produced a more robust effect than either agent alone. Our findings indicate that the Notch pathway plays a critical role in ALS, and the neuroprotective effects of lithium and VPA against mutant SOD1-mediated neuronal damage are at least partially dependent on their suppression of Notch activation.

  14. Carbonic Anhydrase I Is Recognized by an SOD1 Antibody upon Biotinylation of Human Spinal Cord Extracts

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    Robert Bowser

    2010-10-01

    Full Text Available We recently reported the presence of a novel 32 kDa protein immunoreactive to a copper, zinc superoxide dismutase (SOD1 antibody within the spinal cord of patients with amyotrophic lateral sclerosis (ALS. This unique protein species was generated by biotinylation of spinal cord tissue extracts to detect conformational changes of SOD1 specific to ALS patients. To further characterize this protein, we enriched the protein by column chromatography and determined its protein identity by mass spectrometry. The protein that gave rise to the 32 kDa species upon biotinylation was identified as carbonic anhydrase I (CA I. Biotinylation of CA I from ALS spinal cord resulted in the generation of a novel epitope recognized by the SOD1 antibody. This epitope could also be generated by biotinylation of extracts from cultured cells expressing human CA I. Peptide competition assays identified the amino acid sequence in carbonic anhydrase I responsible for binding the SOD1 antibody. We conclude that chemical modifications used to identify pathogenic protein conformations can lead to the identification of unanticipated proteins that may participate in disease pathogenesis.

  15. Rapid Progression of Sporadic ALS in a Patient Carrying SOD1 p.Gly13Arg Mutation

    Science.gov (United States)

    Kim, Myung-Jin; Bae, Jae-Han; Kim, Jeong-Min; Kim, Hye Ryoun; Yoon, Byung-Nam; Sung, Jung-Joon

    2016-01-01

    Amyotrophic lateral sclerosis (ALS), the most common adult onset motor neuron disease, is pathologically characterized by progressive loss of the upper and lower motor neurons. Mutations in the Cu/Zn superoxide dismutase gene (SOD1) account for about 20% of familial ALS cases and a small percentage of sporadic ALS (SALS) cases, and have revealed a validated genotype-phenotype correlation. Herein, we report a p.Gly13Arg mutation in SOD1 exon 1 in a patient with SALS who presented with a rapidly progressive course, predominantly affecting the lower motor neurons. A 48-year-old man presented with progressive weakness and muscle atrophy of the left upper and lower limbs, followed by muscle fasciculation and cramping. The clinical features of the patient were clearly suggestive of ALS, and implied a sporadic form with rapid progression, predominantly affecting the lower motor neurons. Sequencing of the SOD1 gene by PCR revealed a missense mutation of G to C (c.37G>C) in exon 1, and amino acid substitution of glycine by arginine (p.Gly13Arg). This is the first case identifying the p.Gly13Arg mutation of SOD1 in the Korean population, and clinical assessments of this patient revealed a different phenotype compared with other cases. PMID:28035186

  16. Unraveling the complexity of amyotrophic lateral sclerosis: recent advances from the transgenic mutant SOD1 mice.

    Science.gov (United States)

    Peviani, M; Caron, I; Pizzasegola, C; Gensano, F; Tortarolo, M; Bendotti, C

    2010-08-01

    Amyotrophic Lateral Sclerosis (ALS), which accounts for the majority of motor neuron disorders, is a progressive and fatal neurodegenerative disease leading to complete paralysis of skeletal muscles and premature death usually from respiratory failure. About 10% of all ALS cases are inherited, with the responsible gene having been identified in approximately 25% of these individuals. Mutations in the copper-zinc superoxide dismutase (SOD1) gene were the first to be recognized nearly twenty years ago, and since then different animal models, in particular transgenic rodents, have been developed. They replicate many of the clinical, neuropathological and molecular features of ALS patients and have contributed significantly to our understanding of the pathogenic mechanisms of this disease. Although results obtained so far with mutant SOD1 mice have not translated into effective therapies in ALS patients, these models still represent the only experimentally accessible system to study multiple aspects of disease pathogenesis and to provide proof-of-principle for the development of new therapeutic strategies. This review will examine the most recent discoveries obtained from these animal models in an attempt to elucidate the complex mechanisms of the disease. In particular it will focus on the contribution of multiple cell types in governing the disease development and progression.

  17. The combined transduction of copper, zinc-superoxide dismutase and catalase mediated by cell-penetrating peptide, PEP-1, to protect myocardium from ischemia-reperfusion injury

    Directory of Open Access Journals (Sweden)

    Guo Ling-Yun

    2011-05-01

    Full Text Available Abstract Background Our previous studies indicate that either PEP-1-superoxide dismutase 1 (SOD1 or PEP-1-catalase (CAT fusion proteins protects myocardium from ischemia-reperfusion-induced injury in rats. The aim of this study is to explore whether combined use of PEP-1-SOD1 and PEP-1-CAT enhances their protective effects. Methods SOD1, PEP-1-SOD1, CAT or PEP-1-CAT fusion proteins were prepared and purified by genetic engineering. In vitro and in vivo effects of these proteins on cell apoptosis and the protection of myocardium after ischemia-reperfusion injury were measured. Embryo cardiac myocyte H9c2 cells were used for the in vitro studies. In vitro cellular injury was determined by the expression of lactate dehydrogenase (LDH. Cell apoptosis was quantitatively assessed with Annexin V and PI double staining by Flow cytometry. In vivo, rat left anterior descending coronary artery (LAD was ligated for one hour followed by two hours of reperfusion. Hemodynamics was then measured. Myocardial infarct size was evaluated by TTC staining. Serum levels of myocardial markers, creatine kinase-MB (CK-MB and cTnT were quantified by ELISA. Bcl-2 and Bax expression in left ventricle myocardium were analyzed by western blot. Results In vitro, PEP-1-SOD1 or PEP-1-CAT inhibited LDH release and apoptosis rate of H9c2 cells. Combined transduction of PEP-1-SOD1 and PEP-1-CAT, however, further reduced the LDH level and apoptosis rate. In vivo, combined usage of PEP-1-SOD1 and PEP-1-CAT produced a greater effect than individual proteins on the reduction of CK-MB, cTnT, apoptosis rate, lipoxidation end product malondialdehyde, and the infarct size of myocardium. Functionally, the combination of these two proteins further increased left ventricle systolic pressure, but decreased left ventricle end-diastolic pressure. Conclusion This study provided a basis for the treatment or prevention of myocardial ischemia-reperfusion injury with the combined usage of PEP-1-SOD1

  18. ROLE OF COPPER,ZINC-SUPEROXIDE DISMUTASE IN CATALYZING NITROTYROSINE FORMATION IN MURINE LIVER

    Science.gov (United States)

    The solely known function of Cu,Zn-superoxide dismutase (SOD1) is to catalyze the dismutation of superoxide anion into hydrogen peroxide. Our objective was to determine if SOD1 catalyzed murine liver protein nitration induced by acetaminophen (APAP) and lipopolysaccharide (LPS). Liver and plasma ...

  19. Superoxide Dismutase Protects Cells from DNA Damage Induced by Trivalent Methylated Arsenicals

    Science.gov (United States)

    Superoxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide. Heterozygous mice of strain B6; 129S7-Sod1(tm1Leb)/J were obtained from Jackson Laboratories and bred to produce offspring that were heterozygous (+/Sod1(tm1Leb)), homozygous wild-type (+/+), ...

  20. Molecular cloning and biochemical characterization of iron superoxide dismutase from the rodent malaria parasite Plasmodium vinckei.

    Science.gov (United States)

    Prakash, Kirtika; Goyal, Manish; Soni, Awakash; Siddiqui, Arif Jamal; Bhardwaj, Jyoti; Puri, Sunil K

    2014-12-01

    Plasmodium parasite utilizes superoxide dismutase family proteins to limit the toxicity of reactive oxygen species, such as produced through hemoglobin degradation. These proteins play an important role in parasite survival during intra-erythrocytic phase. We have identified, and biochemically characterized a putative iron dependent superoxide dismutase from rodent malaria parasite Plasmodium vinckei (PvSOD1). The recombinant PvSOD1 protein was purified to homogeneity through a combination of affinity and gel filtration chromatography. Crosslinking, Native-PAGE and FPLC gel filtration analyses documented that PvSOD1 exists as a dimer in solution, a common feature shared by other Fe-SODs. PvSOD1 is cytosolic in localization and its expression is comparatively higher during trophozoite as compared to that of ring and schizont stages. Enzymatic activity of recombinant PvSOD1 was validated using conventional zymogram analyses and xanthine-xanthine oxidase system. Under optimal conditions, PvSOD1 was highly active and catalyzed the dismutation of superoxide radicals. Furthermore, PvSOD1 showed activity over a broad range of pH and temperature. Inhibition studies suggested that PvSOD1 was inactivated by hydrogen peroxide, and peroxynitrite, but not by cyanide and azide. Since, PvSOD1 plays a central role in oxidative defense mechanism, therefore, characterization of PvSOD1 will be exploited in the screening of new superoxide dismutase inhibitors for their antimalarial activity. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  1. Screening of Drugs Inhibiting In vitro Oligomerization of Cu/Zn-Superoxide Dismutase with a Mutation Causing Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Anzai, Itsuki; Toichi, Keisuke; Tokuda, Eiichi; Mukaiyama, Atsushi; Akiyama, Shuji; Furukawa, Yoshiaki

    2016-01-01

    Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS). A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.

  2. PEP-1-SOD1对离体缺血再灌注损伤大鼠心肌Bax、Bcl-2蛋白表达的影响%Effects of PEP-1-SOD1 on Expression of Bax,Bcl-2 of Myocardium in Rats Induced with Ischemia-Reperfusion Injury ex vivo

    Institute of Scientific and Technical Information of China (English)

    柯尊平; 王家宁; 王磊; 唐俊明; 杨建业; 黄永章; 张宏考

    2011-01-01

    Objective To investigate the effects of PEP-1 mediated human Cu,Zn superoxide dismutase on the myocardium apoptosis induced by ischemia-reperfusion injury(IRI) in rats ex vivo.Methods The MIRI rats model were prepared with Langendorff perfusion system ex vivo.The model rats were randomly divided into control group ,SOD1 pretreated group,PEP1-SOD1 pretreated with 25,50,100 μmol/L groups.The myocardium apoptosis and expression of Bax, Bcl-2 were determined with TUNEL method and immunofluorescencein method after reperfusion.Results Compared with control and SOD1 groups,the apoptosis indexes(AI) were significantly decreased,the expression of Bcl-2 were upregulated,the expression of Bax were downregulated in all PEP-1-SOD1 groups( all P < 0.01 ).Conclusion The fusion protein PEP-1-SOD1 could inhibit the apoptosis of myocardium in MIRI rats,which may be related to its upregulation of Bax expression and downregulation of Bcl-2 expression.%目的:研究细胞穿透肽PEP-1介导人铜锌-超氧化物歧化酶(cu,Zn-SOD,SOD1)对大鼠离体心肌缺血再灌注损伤(MIRI)细胞凋亡的影响.方法:采用Langendorff灌流系统对离体大鼠心脏进行停灌-复灌建立心肌缺血再灌注损伤模型.大鼠随机分为对照组、SODI蛋白预处理组,25、50、100μmol/L PEP-1-SOD1蛋白预处理组.复灌结束后,TUNEL法检测心肌细胞凋亡,免疫荧光法检测心肌组织Bax、Bel-2蛋白表达.结果:与对照组及SOD1组相比,各PEP-1-SOD1组心肌细胞凋亡指数(AI)显著下降,Bcl-2蛋白表达升高,Bax蛋白表达显著减少(P<0.01).结论:PEP-1-SOD1融合蛋白可抑制离体心脏缺血再灌注损伤大鼠心肌细胞凋亡,其机制可能与上调Bax表达及下调Bcl-2表达有关.

  3. Acquisition of pro-oxidant activity of fALS-linked SOD1 mutants as revealed using circular dichroism and UV-resonance Raman spectroscopy

    Science.gov (United States)

    Fujimaki, Nobuhiro; Nishiya, Ken; Miura, Takashi; Nakabayashi, Takakazu

    2016-11-01

    The acquisition of pro-oxidant activity of the mutated form of human Cu, Zn-superoxide dismutase (SOD1) has been investigated to clarify the relationship between mutations in SOD1 and the pathogenesis of amyotrophic lateral sclerosis (ALS). Ala4 → Val (A4V) and Gly93 → Ala (G93A) mutants, which are representative ALS-linked SOD1 mutants, have been found to exhibit both the denaturation and the gain of pro-oxidant activity after incubation in the apo-form at a physiological condition of 37 °C and pH 7.4 and the rebinding of Cu2+. These characteristics are similar to those previously reported for the His43 → Arg (H43R) mutant. UV-resonance Raman spectra indicated that the coordination structure of the Cu-binding site catalyzing the oxidation reaction is the same among the denatured A4V, G93A, and H43R. Since wild-type SOD1 does not exhibit the denaturation in its apo-form at 37 °C and pH 7.4, the instability of the protein structure due to mutation can be considered as a significant factor that induces the denaturation and the subsequent pro-oxidant activity.

  4. Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

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    Yoshiaki Furukawa

    2012-01-01

    Full Text Available Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1 gene cause a familial form of amyotrophic lateral sclerosis (ALS. While it remains controversial how SOD1 mutations lead to onset and progression of the disease, many in vitro and in vivo studies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations in sod1 gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

  5. Secretion expression of SOD1 and its overlapping function with GSH in brewing yeast strain for better flavor and anti-aging ability.

    Science.gov (United States)

    Wang, Zhaoyue; Bai, Xuejing; He, Xiuping; Zhang, Borun

    2014-09-01

    Superoxide dismutase (SOD) is a significant antioxidant, but unlike glutathione (GSH), SOD cannot be secreted into beer by yeast cells during fermentation, this directly leads to the limited application of SOD in beer anti-aging. In this investigation, we constructed the SOD1 secretion cassette in which strong promoter PGK1p and the sequence of secreting signal factor from Saccharomyces cerevisiae were both harbored to the upstream of coding sequence of SOD1 gene, as a result, the obtained strains carrying this cassette successfully realized the secretion of SOD1. In order to overcome the limitation of previous genetic modification on yeast strains, one new comprehensive strategy was adopted targeting the suitable homologous sites by gene deletion and SOD1 + GSH1 co-overexpression, and the new strain ST31 (Δadh2::SOD1 + Δilv2::GSH1) was constructed. The results of the pilot-scale fermentation showed that the diacetyl content of ST31 was lower by 42 % than that of the host, and the acetaldehyde content decreased by 29 %, the GSH content in the fermenting liquor of ST31 increased by 29 % compared with the host. Both SOD activity test and the positive and negative staining assay after native PAGE indicated that the secreted active SOD in the fermenting liquor of ST31 was mainly a dimer with the size of 32,500 Da. The anti-aging indexes such as the thiobarbituric acid and the resistance staling value further proved that the flavor stability of the beer brewed with strain ST31 was not only better than that of the original strain, but also better than that of the previous engineering strains. The multi-modification and comprehensive improvement of the beer yeast strain would greatly enhance beer quality than ever, and the self-cloning strain would be attractive to the public due to its bio-safety.

  6. Inclusions of amyotrophic lateral sclerosis-linked superoxide dismutase in ventral horns, liver, and kidney

    DEFF Research Database (Denmark)

    Jonsson, P.A.; Bergemalm, D.; Andersen, P.M.

    2008-01-01

    Mutant superoxide dismutases type 1 (SOD1s) cause amyotrophic lateral sclerosis by an unidentified toxic property. In a patient carrying the G127X truncation mutation, minute amounts of SOD1 were found in ventral horns using a mutant-specific antibody. Still, both absolute levels and ratios versus...

  7. Cu,Zn Superoxide Dismutase Maturation and Activity Are Regulated by COMMD1

    NARCIS (Netherlands)

    Vonk, Willianne I. M.; Wijmenga, Cisca; Berger, Ruud; van de Sluis, Bart; Klomp, Leo W. J.

    2010-01-01

    The maturation and activation of the anti-oxidant Cu, Zn superoxide dismutase (SOD1) are highly regulated processes that require several post-translational modifications. The maturation of SOD1 is initiated by incorporation of zinc and copper ions followed by disulfide oxidation leading to the forma

  8. Destabilizing protein polymorphisms in the genetic background direct phenotypic expression of mutant SOD1 toxicity.

    Directory of Open Access Journals (Sweden)

    Tali Gidalevitz

    2009-03-01

    Full Text Available Genetic background exerts a strong modulatory effect on the toxicity of aggregation-prone proteins in conformational diseases. In addition to influencing the misfolding and aggregation behavior of the mutant proteins, polymorphisms in putative modifier genes may affect the molecular processes leading to the disease phenotype. Mutations in SOD1 in a subset of familial amyotrophic lateral sclerosis (ALS cases confer dominant but clinically variable toxicity, thought to be mediated by misfolding and aggregation of mutant SOD1 protein. While the mechanism of toxicity remains unknown, both the nature of the SOD1 mutation and the genetic background in which it is expressed appear important. To address this, we established a Caenorhabditis elegans model to systematically examine the aggregation behavior and genetic interactions of mutant forms of SOD1. Expression of three structurally distinct SOD1 mutants in C. elegans muscle cells resulted in the appearance of heterogeneous populations of aggregates and was associated with only mild cellular dysfunction. However, introduction of destabilizing temperature-sensitive mutations into the genetic background strongly enhanced the toxicity of SOD1 mutants, resulting in exposure of several deleterious phenotypes at permissive conditions in a manner dependent on the specific SOD1 mutation. The nature of the observed phenotype was dependent on the temperature-sensitive mutation present, while its penetrance reflected the specific combination of temperature-sensitive and SOD1 mutations. Thus, the specific toxic phenotypes of conformational disease may not be simply due to misfolding/aggregation toxicity of the causative mutant proteins, but may be defined by their genetic interactions with cellular pathways harboring mildly destabilizing missense alleles.

  9. Neural stem cells genetically modified to overexpress cu/zn-superoxide dismutase enhance amelioration of ischemic stroke in mice.

    Science.gov (United States)

    Sakata, Hiroyuki; Niizuma, Kuniyasu; Wakai, Takuma; Narasimhan, Purnima; Maier, Carolina M; Chan, Pak H

    2012-09-01

    The harsh host brain microenvironment caused by production of reactive oxygen species after ischemic reperfusion injury offers a significant challenge to survival of transplanted neural stem cells (NSCs) after ischemic stroke. Copper/zinc-superoxide dismutase (SOD1) is a specific antioxidant enzyme that counteracts superoxide anions. We have investigated whether genetic manipulation to overexpress SOD1 enhances survival of grafted stem cells and accelerates amelioration of ischemic stroke. NSCs genetically modified to overexpress or downexpress SOD1 were administered intracerebrally 2 days after transient middle cerebral artery occlusion. Histological and behavioral tests were examined from Days 0 to 28 after stroke. Overexpression of SOD1 suppressed production of superoxide anions after ischemic reperfusion injury and reduced NSC death after transplantation. In contrast, downexpression of SOD1 promoted superoxide generation and increased oxidative stress-mediated NSC death. Transplantation of SOD1-overexpressing NSCs enhanced angiogenesis in the ischemic border zone through upregulation of vascular endothelial growth factor. Moreover, grafted SOD1-overexpressing NSCs reduced infarct size and improved behavioral performance compared with NSCs that were not genetically modified. Our findings reveal a strong involvement of SOD1 expression in NSC survival after ischemic reperfusion injury. We propose that conferring antioxidant properties on NSCs by genetic manipulation of SOD1 is a potential approach for enhancing the effectiveness of cell transplantation therapy in ischemic stroke.

  10. Sod1 deficiency reduces incubation time in mouse models of prion disease.

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    Shaheen Akhtar

    Full Text Available Prion infections, causing neurodegenerative conditions such as Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep and BSE in cattle are characterised by prolonged and variable incubation periods that are faithfully reproduced in mouse models. Incubation time is partly determined by genetic factors including polymorphisms in the prion protein gene. Quantitative trait loci studies in mice and human genome-wide association studies have confirmed that multiple genes are involved. Candidate gene approaches have also been used and identified App, Il1-r1 and Sod1 as affecting incubation times. In this study we looked for an association between App, Il1-r1 and Sod1 representative SNPs and prion disease incubation time in the Northport heterogeneous stock of mice inoculated with the Chandler/RML prion strain. No association was seen with App, however, significant associations were seen with Il1-r1 (P = 0.02 and Sod1 (P<0.0001 suggesting that polymorphisms at these loci contribute to the natural variation observed in incubation time. Furthermore, following challenge with Chandler/RML, ME7 and MRC2 prion strains, Sod1 deficient mice showed highly significant reductions in incubation time of 20, 13 and 24%, respectively. No differences were detected in Sod1 expression or activity. Our data confirm the protective role of endogenous Sod1 in prion disease.

  11. Genetic analysis and SOD1 mutation screening in Iranian amyotrophic lateral sclerosis patients.

    Science.gov (United States)

    Alavi, Afagh; Nafissi, Shahriar; Rohani, Mohammad; Zamani, Babak; Sedighi, Behnaz; Shamshiri, Hosein; Fan, Jian-Bing; Ronaghi, Mostafa; Elahi, Elahe

    2013-05-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, and the most common in European populations. Results of genetic analysis and mutation screening of SOD1 in a cohort of 60 Iranian ALS patients are here reported. Initially, linkage analysis in 4 families identified a disease-linked locus that included the known ALS gene, SOD1. Screening of SOD1 identified homozygous p.Asp90Ala causing mutations in all the linked families. Haplotype analysis suggests that the p.Asp90Ala alleles in the Iranian patients might share a common founder with the renowned Scandinavian recessive p.Asp90Ala allele. Subsequent screening in all the patients resulted in identification of 3 other mutations in SOD1, including p.Leu84Phe in the homozygous state. Phenotypic features of the mutation-bearing patients are presented. SOD1 mutations were found in 11.7% of the cohort, 38.5% of the familial ALS probands, and 4.25% of the sporadic ALS cases. SOD1 mutations contribute significantly to ALS among Iranians.

  12. Oregano Essential Oil Induces SOD1 and GSH Expression through Nrf2 Activation and Alleviates Hydrogen Peroxide-Induced Oxidative Damage in IPEC-J2 Cells

    Science.gov (United States)

    Zou, Yi; Wang, Jun; Peng, Jian

    2016-01-01

    Oregano essential oil (OEO) has long been used to improve the health of animals, particularly their intestinal health. The health benefits of OEO are generally attributed to antioxidative actions, but the mechanisms remain unclear. Here, we investigate the antioxidative effects of OEO and their underlying molecular mechanisms in porcine small intestinal epithelial (IPEC-J2) cells. We found that OEO treatment prior to hydrogen peroxide (H2O2) exposure increased cell viability and prevented lactate dehydrogenase (LDH) release into the medium. H2O2-induced reactive oxygen species (ROS) and malondialdehyde (MDA) were remarkably suppressed by OEO. OEO dose-dependently increased mRNA and protein levels of the nuclear factor-erythroid 2-related factor-2 (Nrf2) target genes Cu/Zn-superoxide dismutase (SOD1) and g-glutamylcysteine ligase (GCLC, GLCM), as well as intracellular concentrations of SOD1 and glutathione. OEO also increased intranuclear expression of Nrf2 and the activity of an antioxidant response element reporter plasmid in IPEC-J2 cells. The OEO-induced expression of Nrf2-regulated genes and increased SOD1 and glutathione concentrations in IPEC-J2 cells were reduced by Nrf2 small interfering (si) RNAs, counteracting the protective effects of OEO against oxidative stress in IPEC-J2 cells. Our results suggest that OEO protects against H2O2-induced IPEC-J2 cell damage by inducing Nrf2 and related antioxidant enzymes. PMID:28105249

  13. Characterization of the caspase cascade in a cell culture model of SOD1-related familial amyotrophic lateral sclerosis: expression, activation and therapeutic effects of inhibition.

    Science.gov (United States)

    Sathasivam, S; Grierson, A J; Shaw, P J

    2005-10-01

    There is increasing evidence that apoptosis or a similar programmed cell death pathway is the mechanism of cell death responsible for motor neurone degeneration in amyotrophic lateral sclerosis. Knowledge of the relative importance of different caspases in the cell death process is at present incomplete. In addition, there is little information on the critical point of the death pathway when the process of dying becomes irreversible. In this study, using the well-established NSC34 motor neurone-like cell line stably transfected with empty vector, normal or mutant human Cu-Zn superoxide dismutase (SOD1), we have characterized the activation of the caspase cascade in detail, revealing that the activation of caspases-9, -3 and -8 are important in motor neurone death and that the presence of mutant SOD1 causes increased activation of components of the apoptotic cascade under both basal culture conditions and following oxidative stress induced by serum withdrawal. Activation of the caspases identified in the cellular model has been confirmed in the G93A SOD1 transgenic mice. Furthermore, investigation of the effects of anti-apoptotic neuroprotective agents including specific caspase inhibitors, minocycline and nifedipine, have supported the importance of the mitochondrion-dependent apoptotic pathway in the death process and revealed that the upstream caspase cascade needs to be inhibited if useful neuro-protection is to be achieved.

  14. Immunohistochemical identification and quantitative analysis of cytoplasmic Cu/Zn superoxide dismutase in mouse organogenesis

    Science.gov (United States)

    Yon, Jung-Min; Baek, In-Jeoung; Lee, Se-Ra; Kim, Mi-Ra; Lee, Beom Jun; Yun, Young Won

    2008-01-01

    Cytoplasmic Cu/Zn superoxide dismutase (SOD1) is an antioxidant enzyme that converts superoxide to hydrogen peroxide in cells. Its spatial distribution matches that of superoxide production, allowing it to protect cells from oxidative stress. SOD1 deficiencies result in embryonic lethality and a wide range of pathologies in mice, but little is known about normal SOD1 protein expression in developing embryos. In this study, the expression pattern of SOD1 was investigated in post-implantation mouse embryos and extraembryonic tissues, including placenta, using Western blotting and immunohistochemical analyses. SOD1 was detected in embryos and extraembryonic tissues from embryonic day (ED) 8.5 to 18.5. The signal in embryos was observed at the lowest level on ED 9.5-11.5, and the highest level on ED 17.5-18.5, while levels remained constant in the surrounding extraembryonic tissues during all developmental stages examined. Immunohistochemical analysis of SOD1 expression on ED 13.5-18.5 revealed its ubiquitous distribution throughout developing organs. In particular, high levels of SOD1 expression were observed in the ependymal epithelium of the choroid plexus, ganglia, sensory cells of the olfactory and vestibulocochlear epithelia, blood cells and vessels, hepatocytes and hematopoietic cells of the liver, lymph nodes, osteogenic tissues, and skin. Thus, SOD1 is highly expressed at late stages of embryonic development in a cell- and tissue-specific manner, and can function as an important antioxidant enzyme during organogenesis in mouse embryos. PMID:18716442

  15. SOD isoforms play no role in lifespan in ad lib or dietary restricted conditions, but mutational inactivation of SOD-1 reduces life extension by cold.

    Science.gov (United States)

    Yen, Kelvin; Patel, Harshil B; Lublin, Alex L; Mobbs, Charles V

    2009-03-01

    The free radical theory of aging is one of the most prominent theories of aging and senescence, but has yet to be definitively proven. If free radicals are the cause of senescence, then the cellular anti-oxidant system should play a large role in lifespan determination. Because superoxide dismutase (SOD) plays a central role in detoxifying superoxide radicals, we have examined the effects of mutational inactivation of each isoform of sod on normal lifespan and lifespan extension by dietary restriction (DR) or cold-/hypothermic-induced longevity (CHIL). We find no significant decrease in lifespan for control worms or worms undergoing DR when sod isoforms are knocked-out even though sod mutational inactivation produces hypersensitivity to paraquat. In contrast, sod-1 inactivation significantly reduces lifespan extension by CHIL, suggesting that CHIL requires a specific genetic program beyond simple reduction in metabolic rate. Furthermore, CHIL paradoxically increases lifespan while reducing resistance to oxidative stress, further disassociating oxidative stress resistance and lifespan.

  16. Differential gene expression in the axotomized facial motor nucleus of presymptomatic SOD1 mice.

    Science.gov (United States)

    Mesnard, Nichole A; Sanders, Virginia M; Jones, Kathryn J

    2011-12-01

    Previously, we compared molecular profiles of one population of wild-type (WT) mouse facial motoneurons (FMNs) surviving with FMNs undergoing significant cell death after axotomy. Regardless of their ultimate fate, injured FMNs respond with a vigorous pro-survival/regenerative molecular response. In contrast, the neuropil surrounding the two different injured FMN populations contained distinct molecular differences that support a causative role for glial and/or immune-derived molecules in directing contrasting responses of the same cell types to the same injury. In the current investigation, we utilized the facial nerve axotomy model and a presymptomatic amyotrophic lateral sclerosis (ALS) mouse (SOD1) model to experimentally mimic the axonal die-back process observed in ALS pathogenesis without the confounding variable of disease onset. Presymptomatic SOD1 mice had a significant decrease in FMN survival compared with WT, which suggests an increased susceptibility to axotomy. Laser microdissection was used to accurately collect uninjured and axotomized facial motor nuclei of WT and presymptomatic SOD1 mice for mRNA expression pattern analyses of pro-survival/pro-regeneration genes, neuropil-specific genes, and genes involved in or responsive to the interaction of FMNs and non-neuronal cells. Axotomized presymptomatic SOD1 FMNs displayed a dynamic pro-survival/regenerative response to axotomy, similar to WT, despite increased cell death. However, significant differences were revealed when the axotomy-induced gene expression response of presymptomatic SOD1 neuropil was compared with WT. We propose that the increased susceptibility of presymptomatic SOD1 FMNs to axotomy-induced cell death and, by extrapolation, disease progression, is not intrinsic to the motoneuron, but rather involves a dysregulated response by non-neuronal cells in the surrounding neuropil.

  17. Apparent segregation distortion for the SOD1 mutation in amyotrophic lateral sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Rimmer, J.B.; Pericak-Vance, M.A. [Duke Univ. Medical Center, Durham, NC (United States); Hentati, A. [Northwestern Univ., Chicago, IL (United States)] [and others

    1994-09-01

    Amyotrophic lateral sclerosis (ALS) is a devastating, progressive neurodegenerative disorder with a short duration form onset to death. Approximately 15% of all ALS cases are familial. Of this a subset of families ({approximately}20%) are caused by mutations in the SOD1 gene on chromosome 21. The recent identification of SOD1 as the causative factor in a subset of families has enabled us to classify at-risk as well as symptomatic SOD1 mutation carriers in completed sibships. Our investigations suggest that the transmission of the SOD1 mutation from parent to child occurs substantially more often than 50% of the time. At the present time we have examined this phenomena in 17 SOD1/ALS families with mutations in exons 1 (N=6 families), 2 (N=2), 4 (N=8) and 5 (N=1). In fully ascertained sibships from a mutation-carrying parent, there were 318 offspring available for mutation evaluation. Of these, 195 were found to have the SOD1 mutation, while 123 were without the mutation. These data result in a segregation ratio of 0.61 [chi-square = 16.3, P<0.0001]. Analysis of the individual exon mutation types indicated that the majority of the distortion was occurring in the exon 4 mutation families [chi-square=13.4, p<0.001 vs. non-4, chi-square=4.97, p<0.05]. These findings are of interest in light of the recent report of meiotic drive at the myotonic dystrophy locus, a CTG repeat expansion, variable onset, neurological disorder on chromosome 19. Additional SOD1/ALS mutation families are presently under study and these data will be similarily evaluated. Future studies include the genotyping of human sperm specimens for SOD1 mutation-bearing males. The possibility that over 66% of children of a mutation carrier could inherit the mutation preferentially would dramatically alter the counseling risk in such families. These studies provide further evidence of the occurrence of segregation distortion in humans.

  18. Copper and Zinc Metallation Status of Copper Zinc Superoxide Dismutase form Amyotrophic Lateral Sclerosis Transgenic Mice

    Energy Technology Data Exchange (ETDEWEB)

    Lelie, H.L.; Miller, L.; Liba, A.; Bourassa, M.W.; Chattopadhyay, M.; Chan, P.K.; Gralla, E.B.; Borchelt, D.R.; et al

    2010-09-24

    Mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1) cause one form of familial amyotrophic lateral sclerosis (ALS), and metals are suspected to play a pivotal role in ALS pathology. To learn more about metals in ALS, we determined the metallation states of human wild-type or mutant (G37R, G93A, and H46R/H48Q) SOD1 proteins from SOD1-ALS transgenic mice spinal cords. SOD1 was gently extracted from spinal cord and separated into insoluble (aggregated) and soluble (supernatant) fractions, and then metallation states were determined by HPLC inductively coupled plasma MS. Insoluble SOD1-rich fractions were not enriched in copper and zinc. However, the soluble mutant and WT SOD1s were highly metallated except for the metal-binding-region mutant H46R/H48Q, which did not bind any copper. Due to the stability conferred by high metallation of G37R and G93A, it is unlikely that these soluble SOD1s are prone to aggregation in vivo, supporting the hypothesis that immature nascent SOD1 is the substrate for aggregation. We also investigated the effect of SOD1 overexpression and disease on metal homeostasis in spinal cord cross-sections of SOD1-ALS mice using synchrotron-based x-ray fluorescence microscopy. In each mouse genotype, except for the H46R/H48Q mouse, we found a redistribution of copper between gray and white matters correlated to areas of high SOD1. Interestingly, a disease-specific increase of zinc was observed in the white matter for all mutant SOD1 mice. Together these data provide a picture of copper and zinc in the cell as well as highlight the importance of these metals in understanding SOD1-ALS pathology.

  19. Addition of exogenous SOD1 aggregates causes TDP-43 mislocalisation and aggregation.

    Science.gov (United States)

    Zeineddine, Rafaa; Farrawell, Natalie E; Lambert-Smith, Isabella A; Yerbury, Justin J

    2017-05-30

    ALS is characterised by a focal onset of motor neuron loss, followed by contiguous outward spreading of pathology throughout the nervous system, resulting in paralysis and death generally within a few years after diagnosis. The aberrant release and uptake of toxic proteins including SOD1 and TDP-43 and their subsequent propagation, accumulation and deposition in motor neurons may explain such a pattern of pathology. Previous work has suggested that the internalization of aggregates triggers stress granule formation. Given the close association of stress granules and TDP-43, we wondered whether internalisation of SOD1 aggregates stimulated TDP-43 cytosolic aggregate structures. Addition of recombinant mutant G93A SOD1 aggregates to NSC-34 cells was found to trigger a rapid shift of TDP-43 to the cytoplasm where it was still accumulated after 48 h. In addition, SOD1 aggregates also triggered cleavage of TDP-43 into fragments including a 25 kDa fragment. Collectively, this study suggests a role for protein aggregate uptake in TDP-43 pathology.

  20. SOD1 aggregation in astrocytes following ischemia/reperfusion injury: a role of NO-mediated S-nitrosylation of protein disulfide isomerase (PDI

    Directory of Open Access Journals (Sweden)

    Chen Xueping

    2012-10-01

    Full Text Available Abstract Background Ubiquitinated-protein aggregates are implicated in cerebral ischemia/reperfusion injury. The very presence of these ubiquitinated-protein aggregates is abnormal and seems to be disease-related. However, it is not clear what leads to aggregate formation and whether the aggregations represent a reaction to aggregate-mediated neurodegeneration. Methods To study the nitrosative stress-induced protein aggregation in cerebral ischemia/reperfusion injury, we used primary astrocyte cultures as a cell model, and systematically examined their iNOS expression and consequent NO generation following oxygen glucose deprivation and reperfusion. The expression of protein disulfide isomerase (PDI and copper-zinc superoxide dismutase (SOD1 were also examined, and the biochemical interaction between PDI and SOD1 was determined by immunoprecipitation. In addition, the levels of S-nitrosylated PDI in cultured astrocytes after oxygen glucose deprivation and reperfusion treatment were measured using the biotin-switch assay. The formation of ubiquitinated-protein aggregates was detected by immunoblot and immunofluorescence staining. Results Our data showed that the up-regulation of iNOS expression after oxygen glucose deprivation and reperfusion treatment led to excessive NO generation. Up-regulation of PDI and SOD1 was also identified in cultured astrocytes following oxygen glucose deprivation and reperfusion, and these two proteins were found to bind to each other. Furthermore, the increased nitrosative stress due to ischemia/reperfusion injury was highly associated with NO-induced S-nitrosylation of PDI, and this S-nitrosylation of PDI was correlated with the formation of ubiquitinated-protein aggregates; the levels of S-nitrosylated PDI increased in parallel with the formation of aggregates. When NO generation was pharmacologically inhibited by iNOS specific inhibitor 1400W, S-nitrosylation of PDI was significantly blocked. In addition, the

  1. Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca2+ transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Tobias eMühling

    2014-11-01

    Full Text Available Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS, a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs from a common ALS mouse model, endstage superoxide dismutase SOD1G93A transgenic mice, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT positive hMNs from wildtype (WT and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1 and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na2+/Ca2+exchanger NCX1 was also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a novel neuroprotective strategy for ALS.

  2. The carbonate radical anion-induced covalent aggregation of human copper, zinc superoxide dismutase, and alpha-synuclein: intermediacy of tryptophan- and tyrosine-derived oxidation products.

    Science.gov (United States)

    Zhang, Hao; Andrekopoulos, Christopher; Joseph, Joy; Crow, John; Kalyanaraman, B

    2004-06-01

    In this review, we describe the free radical mechanism of covalent aggregation of human copper, zinc superoxide dismutase (hSOD1). Bicarbonate anion (HCO3-) enhances the covalent aggregation of hSOD1 mediated by the SOD1 peroxidase-dependent formation of carbonate radical anion (CO3*-), a potent and selective oxidant. This species presumably diffuses out the active site of hSOD1 and reacts with tryptophan residue located on the surface of hSOD1. The oxidative degradation of tryptophan to kynurenine and N-formyl kynurenine results in the covalent crosslinking and aggregation of hSOD1. Implications of oxidant-mediated aggregation of hSOD1 in the increased cytotoxicity of motor neurons in amyotrophic lateral sclerosis are discussed.

  3. Loss of metal ions, disulfide reduction and mutations related to familial ALS promote formation of amyloid-like aggregates from superoxide dismutase.

    Directory of Open Access Journals (Sweden)

    Zeynep A Oztug Durer

    Full Text Available Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1 are one of the causes of familial amyotrophic lateral sclerosis (FALS. Fibrillar inclusions containing SOD1 and SOD1 inclusions that bind the amyloid-specific dye thioflavin S have been found in neurons of transgenic mice expressing mutant SOD1. Therefore, the formation of amyloid fibrils from human SOD1 was investigated. When agitated at acidic pH in the presence of low concentrations of guanidine or acetonitrile, metalated SOD1 formed fibrillar material which bound both thioflavin T and Congo red and had circular dichroism and infrared spectra characteristic of amyloid. While metalated SOD1 did not form amyloid-like aggregates at neutral pH, either removing metals from SOD1 with its intramolecular disulfide bond intact or reducing the intramolecular disulfide bond of metalated SOD1 was sufficient to promote formation of these aggregates. SOD1 formed amyloid-like aggregates both with and without intermolecular disulfide bonds, depending on the incubation conditions, and a mutant SOD1 lacking free sulfhydryl groups (AS-SOD1 formed amyloid-like aggregates at neutral pH under reducing conditions. ALS mutations enhanced the ability of disulfide-reduced SOD1 to form amyloid-like aggregates, and apo-AS-SOD1 formed amyloid-like aggregates at pH 7 only when an ALS mutation was also present. These results indicate that some mutations related to ALS promote formation of amyloid-like aggregates by facilitating the loss of metals and/or by making the intramolecular disulfide bond more susceptible to reduction, thus allowing the conversion of SOD1 to a form that aggregates to form resembling amyloid. Furthermore, the occurrence of amyloid-like aggregates per se does not depend on forming intermolecular disulfide bonds, and multiple forms of such aggregates can be produced from SOD1.

  4. SO(d,1)-invariant Yang-Baxter operators and the dS/CFT correspondence

    CERN Document Server

    Hollands, Stefan

    2016-01-01

    We propose a model for the dS/CFT correspondence. The model is constructed in terms of a "Yang-Baxter operator" $R$ for unitary representations of the deSitter group $SO(d,1)$. This $R$-operator is shown to satisfy the Yang-Baxter equation, unitarity, as well as certain analyticity relations, including in particular a crossing symmetry. With the aid of this operator we construct: a) A chiral (light-ray) conformal quantum field theory whose internal degrees of freedom transform under the given unitary representation of $SO(d,1)$. By analogy with the $O(N)$ non-linear sigma model, this chiral CFT can be viewed as propagating in a deSitter spacetime. b) A (non-unitary) Euclidean conformal quantum field theory on ${\\mathbb R}^{d-1}$, where $SO(d,1)$ now acts by conformal transformations in (Euclidean) spacetime. These two theories can be viewed as dual to each other if we interpret ${\\mathbb R}^{d-1}$ as conformal infinity of deSitter spacetime. Our constructions use semi-local generator fields defined in terms o...

  5. The effects of superoxide dismutase knockout on the oxidative stress parameters and survival of mouse erythrocytes.

    Science.gov (United States)

    Grzelak, Agnieszka; Kruszewski, Marcin; Macierzyńska, Ewa; Piotrowski, Łukasz; Pułaski, Łukasz; Rychlik, Błazej; Bartosz, Grzegorz

    2009-01-01

    The erythrocytes of 12-month old Sod1 (-/-) mice showed an increased level of reactive oxygen species (ROS), as estimated by the degree of dihydroethidine and dihydrorhodamine oxidation, and the increased level of Heinz bodies. No indices of severe oxidative stress were found in the red blood cells and blood plasma of Sod1 (-/-) mice as judged from the lack of significant changes in the levels of erythrocyte and plasma glutathione, plasma protein thiol and carbonyl groups and thiobarbituric-acid reactive substances in the blood plasma. However, a decreased erythrocyte lifespan, increased reticulocyte count and splenomegaly were noted, indicating the importance of superoxide dismutase for maintaining erythrocyte viability. The levels of erythrocyte ROS and Heinz bodies and the reticulocyte count were indistinguishable in Sod1 (+/+) and Sod1 (+/-) mice, suggesting that a superoxide dismutase activity decrease to half of its normal value may be sufficient to secure the protective effects of the enzyme.

  6. Cu,Zn-superoxide dismutase without Zn is folded but catalytically inactive.

    Science.gov (United States)

    Nedd, Sean; Redler, Rachel L; Proctor, Elizabeth A; Dokholyan, Nikolay V; Alexandrova, Anastassia N

    2014-12-12

    Amyotrophic lateral sclerosis has been linked to the gain of aberrant function of superoxide dismutase, Cu,Zn-SOD1 upon protein misfolding. The mechanism of SOD1 misfolding is thought to involve mutations leading to the loss of Zn, followed by protein unfolding and aggregation. We show that the removal of Zn from SOD1 may not lead to an immediate unfolding but immediately deactivates the enzyme through a combination of subtle structural and electronic effects. Using quantum mechanics/discrete molecular dynamics, we showed that both Zn-less wild-type (WT)-SOD1 and its D124N mutant that does not bind Zn have at least metastable folded states. In those states, the reduction potential of Cu increases, leading to the presence of detectable amounts of Cu(I) instead of Cu(II) in the active site, as confirmed experimentally. The Cu(I) protein cannot participate in the catalytic Cu(I)-Cu(II) cycle. However, even without the full reduction to Cu(I), the Cu site in the Zn-less variants of SOD1 is shown to be catalytically incompetent: unable to bind superoxide in a way comparable to the WT-SOD1. The changes are more radical and different in the D124N Zn-less mutant than in the Zn-less WT-SOD1, suggesting D124N being perhaps not the most adequate model for Zn-less SOD1. Overall, Zn in SOD1 appears to be influencing the Cu site directly by adjusting its reduction potential and geometry. Thus, the role of Zn in SOD1 is not just structural, as was previously thought; it is a vital part of the catalytic machinery.

  7. Acrolein-Induced Oxidative Stress and Cell Death Exhibiting Features of Apoptosis in the Yeast Saccharomyces cerevisiae Deficient in SOD1.

    Science.gov (United States)

    Kwolek-Mirek, Magdalena; Zadrąg-Tęcza, Renata; Bednarska, Sabina; Bartosz, Grzegorz

    2015-04-01

    The yeast Saccharomyces cerevisiae is a useful eukaryotic model to study the toxicity of acrolein, an important environmental toxin and endogenous product of lipid peroxidation. The study was aimed at elucidation of the cytotoxic effect of acrolein on the yeast deficient in SOD1, Cu, Zn-superoxide dismutase which is hypersensitive to aldehydes. Acrolein generated within the cell from its precursor allyl alcohol caused growth arrest and cell death of the yeast cells. The growth inhibition involved an increase in production of reactive oxygen species and high level of protein carbonylation. DNA condensation and fragmentation, exposition of phosphatidylserine at the cell surface as well as decreased dynamic of actin microfilaments and mitochondria disintegration point to the induction of apoptotic-type cell death besides necrotic cell death.

  8. Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Furukawa, Yoshiaki; Anzai, Itsuki; Akiyama, Shuji; Imai, Mizue; Cruz, Fatima Joy C; Saio, Tomohide; Nagasawa, Kenichi; Nomura, Takao; Ishimori, Koichiro

    2016-02-19

    Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Genetic biomarkers for ALS disease in transgenic SOD1(G93A mice.

    Directory of Open Access Journals (Sweden)

    Ana C Calvo

    Full Text Available The pathophysiological mechanisms of both familial and sporadic Amyotrophic Lateral Sclerosis (ALS are unknown, although growing evidence suggests that skeletal muscle tissue is a primary target of ALS toxicity. Skeletal muscle biopsies were performed on transgenic SOD1(G93A mice, a mouse model of ALS, to determine genetic biomarkers of disease longevity. Mice were anesthetized with isoflurane, and three biopsy samples were obtained per animal at the three main stages of the disease. Transcriptional expression levels of seventeen genes, Ankrd1, Calm1, Col19a1, Fbxo32, Gsr, Impa1, Mef2c, Mt2, Myf5, Myod1, Myog, Nnt, Nogo A, Pax7, Rrad, Sln and Snx10, were tested in each muscle biopsy sample. Total RNA was extracted using TRIzol Reagent according to the manufacturer's protocol, and variations in gene expression were assayed by real-time PCR for all of the samples. The Pearson correlation coefficient was used to determine the linear correlation between transcriptional expression levels throughout disease progression and longevity. Consistent with the results obtained from total skeletal muscle of transgenic SOD1(G93A mice and 74-day-old denervated mice, five genes (Mef2c, Gsr, Col19a1, Calm1 and Snx10 could be considered potential genetic biomarkers of longevity in transgenic SOD1(G93A mice. These results are important because they may lead to the exploration of previously unexamined tissues in the search for new disease biomarkers and even to the application of these findings in human studies.

  10. Free Superoxide is an Intermediate in the Production of H2O2 by Copper(I)-Aβ Peptide and O2.

    Science.gov (United States)

    Reybier, Karine; Ayala, Sara; Alies, Bruno; Rodrigues, João V; Bustos Rodriguez, Susana; La Penna, Giovanni; Collin, Fabrice; Gomes, Cláudio M; Hureau, Christelle; Faller, Peter

    2016-01-18

    Oxidative stress is considered as an important factor and an early event in the etiology of Alzheimer's disease (AD). Cu bound to the peptide amyloid-β (Aβ) is found in AD brains, and Cu-Aβ could contribute to this oxidative stress, as it is able to produce in vitro H2O2 and HO˙ in the presence of oxygen and biological reducing agents such as ascorbate. The mechanism of Cu-Aβ-catalyzed H2O2 production is however not known, although it was proposed that H2O2 is directly formed from O2 via a 2-electron process. Here, we implement an electrochemical setup and use the specificity of superoxide dismutase-1 (SOD1) to show, for the first time, that H2O2 production by Cu-Aβ in the presence of ascorbate occurs mainly via a free O2˙(-) intermediate. This finding radically changes the view on the catalytic mechanism of H2O2 production by Cu-Aβ, and opens the possibility that Cu-Aβ-catalyzed O2˙(-) contributes to oxidative stress in AD, and hence may be of interest.

  11. SOD1 Mutations Targeting Surface Hydrogen Bonds Promote Amyotrophic Lateral Sclerosis without Reducing Apo-state Stability*

    OpenAIRE

    Byström, Roberth; Andersen, Peter M.; Gröbner, Gerhard; Oliveberg, Mikael

    2010-01-01

    In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival...

  12. Superoxide dismutase type 1 in monocytes of chronic kidney disease patients

    DEFF Research Database (Denmark)

    Scholze, Alexandra; Krueger, Katharina; Diedrich, Madeleine

    2011-01-01

    We analyzed proteomic profiles in monocytes of chronic kidney disease (CKD) patients and healthy control subjects. Two-dimensional electrophoresis (2-DE) and silver staining indicated differences in protein pattern. Among the analyzed proteins, superoxide dismutase type 1 (SOD1), which was identi......We analyzed proteomic profiles in monocytes of chronic kidney disease (CKD) patients and healthy control subjects. Two-dimensional electrophoresis (2-DE) and silver staining indicated differences in protein pattern. Among the analyzed proteins, superoxide dismutase type 1 (SOD1), which...

  13. Redox environment is an intracellular factor to operate distinct pathways for aggregation of Cu,Zn-superoxide dismutase in amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Yoshiaki eFurukawa

    2013-11-01

    Full Text Available Dominant mutations in Cu,Zn-superoxide dismutase (SOD1 cause a familial form of amyotrophic lateral sclerosis (fALS. Misfolding and aggregation of mutant SOD1 proteins are a pathological hallmark of SOD1-related fALS cases; however, the molecular mechanism of SOD1 aggregation remains controversial. Here, I have used E. coli as a model organism and shown multiple distinct pathways of SOD1 aggregation that are dependent upon its thiol-disulfide status. Overexpression of fALS-mutant SOD1s in the cytoplasm of E. coli BL21 and SHuffleTM, where redox environment is reducing and oxidizing, respectively, resulted in the formation of insoluble aggregates with notable differences; a disulfide bond of SOD1 was completely reduced in BL21 or abnormally formed between SOD1 molecules in SHuffleTM. Depending upon intracellular redox environment, therefore, mutant SOD1 is considered to misfold/aggregate through distinct pathways, which would be relevant in description of the pathological heterogeneity of SOD1-related fALS cases.

  14. Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis.

    Science.gov (United States)

    Iñarrea, Pedro; Moini, Hadi; Han, Derick; Rettori, Daniel; Aguiló, Ignacio; Alava, Maria Angeles; Iturralde, María; Cadenas, Enrique

    2007-07-01

    IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.

  15. Abnormal mitochondrial transport and morphology are common pathological denominators in SOD1 and TDP43 ALS mouse models.

    Science.gov (United States)

    Magrané, Jordi; Cortez, Czrina; Gan, Wen-Biao; Manfredi, Giovanni

    2014-03-15

    Neuronal mitochondrial morphology abnormalities occur in models of familial amyotrophic lateral sclerosis (ALS) associated with SOD1 and TDP43 mutations. These abnormalities have been linked to mitochondrial axonal transport defects, but the temporal and spatial relationship between mitochondrial morphology and transport alterations in these two distinct genetic forms of ALS has not been investigated in vivo. To address this question, we crossed SOD1 (wild-type SOD1(WT) and mutant SOD1(G93A)) or TDP43 (mutant TDP43(A315T)) transgenic mice with mice expressing the fluorescent protein Dendra targeted to mitochondria in neurons (mitoDendra). At different time points during the disease course, we studied mitochondrial transport in the intact sciatic nerve of living mice and analyzed axonal mitochondrial morphology at multiple sites, spanning from the spinal cord to the motor terminals. Defects of retrograde mitochondrial transport were detected at 45 days of age, before the onset of symptoms, in SOD1(G93A) and TDP43(A315T) mice, but not in SOD1(WT). At later disease stages, also anterograde mitochondrial transport was affected in both mutant mouse lines. In SOD1(G93A) mice, mitochondrial morphological abnormalities were apparent at 15 days of age, thus preceding transport abnormalities. Conversely, in TDP43(A315T) mice, morphological abnormalities appeared after the onset of transport defects. Taken together, these findings demonstrate that neuronal mitochondrial transport and morphology abnormalities occur in vivo and that they are common denominators of different genetic forms of the ALS. At the same time, differences in the temporal and spatial manifestation of mitochondrial abnormalities between the two mouse models of familial ALS imply that different molecular mechanisms may be involved.

  16. Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fALS-Associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer.

    Science.gov (United States)

    Broom, Helen R; Vassall, Kenrick A; Rumfeldt, Jessica A O; Doyle, Colleen M; Tong, Ming Sze; Bonner, Julia M; Meiering, Elizabeth M

    2016-01-26

    Many proteins are naturally homooligomers, homodimers most frequently. The overall stability of oligomeric proteins may be described in terms of the stability of the constituent monomers and the stability of their association; together, these stabilities determine the populations of different monomer and associated species, which generally have different roles in the function or dysfunction of the protein. Here we show how a new combined calorimetry approach, using isothermal titration calorimetry to define monomer association energetics together with differential scanning calorimetry to measure total energetics of oligomer unfolding, can be used to analyze homodimeric unmetalated (apo) superoxide dismutase (SOD1) and determine the effects on the stability of structurally diverse mutations associated with amyotrophic lateral sclerosis (ALS). Despite being located throughout the protein, all mutations studied weaken the dimer interface, while concomitantly either decreasing or increasing the marginal stability of the monomer. Analysis of the populations of dimer, monomer, and unfolded monomer under physiological conditions of temperature, pH, and protein concentration shows that all mutations promote the formation of folded monomers. These findings may help rationalize the key roles proposed for monomer forms of SOD1 in neurotoxic aggregation in ALS, as well as roles for other forms of SOD1. Thus, the results obtained here provide a valuable approach for the quantitative analysis of homooligomeric protein stabilities, which can be used to elucidate the natural and aberrant roles of different forms of these proteins and to improve methods for predicting protein stabilities.

  17. Exercise training improves relaxation response and SOD-1 expression in aortic and mesenteric rings from high caloric diet-fed rats

    Directory of Open Access Journals (Sweden)

    Antunes Edson

    2008-05-01

    Full Text Available Abstract Background Obesity has been associated with a variety of disease such as type II diabetes mellitus, arterial hypertension and atherosclerosis. Evidences have shown that exercise training promotes beneficial effects on these disorders, but the underlying mechanisms are not fully understood. The aim of this study was to investigate whether physical preconditioning prevents the deleterious effect of high caloric diet in vascular reactivity of rat aortic and mesenteric rings. Methods Male Wistar rats were divided into sedentary (SD; trained (TR; sedentary diet (SDD and trained diet (TRD groups. Run training (RT was performed in sessions of 60 min, 5 days/week for 12 weeks (70–80% VO2max. Triglycerides, glucose, insulin and nitrite/nitrate concentrations (NOx- were measured. Concentration-response curves to acetylcholine (ACh and sodium nitroprusside (SNP were obtained. Expression of Cu/Zn superoxide dismutase (SOD-1 was assessed by Western blotting. Results High caloric diet increased triglycerides concentration (SDD: 216 ± 25 mg/dl and exercise training restored to the baseline value (TRD: 89 ± 9 mg/dl. Physical preconditioning significantly reduced insulin levels in both groups (TR: 0.54 ± 0.1 and TRD: 1.24 ± 0.3 ng/ml as compared to sedentary animals (SD: 0.87 ± 0.1 and SDD: 2.57 ± 0.3 ng/ml. On the other hand, glucose concentration was slightly increased by high caloric diet, and RT did not modify this parameter (SD: 126 ± 6; TR: 140 ± 8; SDD: 156 ± 8 and TRD 153 ± 9 mg/dl. Neither high caloric diet nor RT modified NOx- levels (SD: 27 ± 4; TR: 28 ± 6; SDD: 27 ± 3 and TRD: 30 ± 2 μM. Functional assays showed that high caloric diet impaired the relaxing response to ACh in mesenteric (about 13%, but not in aortic rings. RT improved the relaxing responses to ACh either in aortic (28%, for TR and 16%, to TRD groups or mesenteric rings (10%, for TR and 17%, to TRD groups that was accompanied by up-regulation of SOD-1

  18. Folding of Cu, Zn superoxide dismutase and Familial Amyotrophic Lateral Sclerosis

    CERN Document Server

    Khare, S D; Dokholyan, N V; Khare, Sagar D.; Ding, Feng; Dokholyan, Nikolay V.

    2003-01-01

    Cu,Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1...

  19. PTCA和BMS置人术对冠心病患者血液中ET、SOD-1和TNF的影响及临床意义

    Institute of Scientific and Technical Information of China (English)

    周军; 王腾; 秦牧; 胡河; 曹红; 唐其柱

    2012-01-01

    首要病症.作为治疗CHD的现代综合治疗手段的经皮冠状动脉腔内成形术(Percutaneous Transluminal Coronary Angioplasty,PTCA)和裸金属支架(Bare-mental Stent,BMS)置人术开创了CHD诊断和防治的新纪元[2].但PTCA所致血管再狭窄(Restenosis,RS)和BMS置入术引发的亚急性血栓形成和支架内再狭窄( In-stent Restenosis,ISR)[3],使其在治疗CHD过程中面临着长期挑战.因此,及早检测PTCA和BMS置人术后血液中一些特异性物质水平变化,对制定预防和治疗CHD措施显得尤其重要[4].已有研究[5,6]表明,内皮素( Endothelin,ET)、超氧化物歧化酶(Superoxide Dismutase,SOD)或肿瘤坏死因子(Tumor Necrosis Factor,TNF)的合成、分泌异常参与了CHD的始动环节和发病过程,所以研究PTCA或BMS是否引起ET、SOD和TNF水平变化,对于认识CHD患者介入治疗后的病理生理过程及预后评估有重要价值.本研究通过对CHD患者PTCA或BMS置入术前后血浆ET、血清SOD-1和TNF水平的检测,评价PTCA或BMS对这三种活性物质水平的影响及其临床意义.

  20. The effects of Brazilian green propolis that contains flavonols against mutant copper-zinc superoxide dismutase-mediated toxicity.

    Science.gov (United States)

    Ueda, Tomoyuki; Inden, Masatoshi; Shirai, Katsuhiro; Sekine, Shin-Ichiro; Masaki, Yuji; Kurita, Hisaka; Ichihara, Kenji; Inuzuka, Takashi; Hozumi, Isao

    2017-06-06

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective and progressive loss of motor neurons. The purpose of this study was to clarify effects of brazilian green propolis and the active ingredient against ALS-associated mutant copper-zinc superoxide dismutase (SOD1)-mediated toxicity. Ethanol extract of brazilian green propolis (EBGP) protected N2a cells against mutant SOD1-induced neurotoxicity and reduced aggregated mutant SOD1 by induction of autophagy. Kaempferide and kaempferol, the active ingredients of EBGP, also inhibited mutant SOD1-induced cell death and reduced the intracellular mutant SOD1 aggregates. Both kaempferide and kaempferol significantly suppressed mutant SOD1-induced superoxide in mitochondria. Western blot analysis showed that kaempferol potentially induced autophagy via the AMP-activated protein kinase (AMPK) - the mammalian target of rapamycin (mTOR) pathway. These results suggest that EBGP containing the active ingredient against mutant SOD1-mediated toxicity is a promising medicine or health food for prevention and treatment of ALS.

  1. Transient structural distortion of metal-free Cu/Zn superoxide dismutase triggers aberrant oligomerization

    DEFF Research Database (Denmark)

    Teilum, Kaare; Smith, Melanie H; Schulz, Eike

    2009-01-01

    of the excited-state structure that forms intermolecular contacts in the earliest nonnative dimer/oligomer. The conformational transition that triggers oligomerization is a common feature of WT SOD1 and ALS-associated mutants that have widely different physicochemical properties. But compared with WT SOD1......, the mutants have enhanced structural distortions in their excited states, and in some cases slightly higher excited-state populations and lower kinetic barriers, implying increased susceptibility to oligomerization. Our results provide a unified picture that highlights both (i) a common denominator among......Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to the misfolding of Cu/Zn superoxide dismutase (SOD1). ALS-related defects in SOD1 result in a gain of toxic function that coincides with aberrant oligomerization. The structural events triggering oligomerization have...

  2. TDP-43 or FUS-induced misfolded human wild-type SOD1 can propagate intercellularly in a prion-like fashion.

    Science.gov (United States)

    Pokrishevsky, Edward; Grad, Leslie I; Cashman, Neil R

    2016-03-01

    Amyotrophic lateral sclerosis (ALS), which appears to spread through the neuroaxis in a spatiotemporally restricted manner, is linked to heritable mutations in genes encoding SOD1, TDP-43, FUS, C9ORF72, or can occur sporadically without recognized genetic mutations. Misfolded human wild-type (HuWt) SOD1 has been detected in both familial and sporadic ALS patients, despite mutations in SOD1 accounting for only 2% of total cases. We previously showed that accumulation of pathological TDP-43 or FUS coexist with misfolded HuWtSOD1 in patient motor neurons, and can trigger its misfolding in cultured cells. Here, we used immunocytochemistry and immunoprecipitation to demonstrate that TDP-43 or FUS-induced misfolded HuWtSOD1 can propagate from cell-to-cell via conditioned media, and seed cytotoxic misfolding of endogenous HuWtSOD1 in the recipient cells in a prion-like fashion. Knockdown of SOD1 using siRNA in recipient cells, or incubation of conditioned media with misfolded SOD1-specific antibodies, inhibits intercellular transmission, indicating that HuWtSOD1 is an obligate seed and substrate of propagated misfolding. In this system, intercellular spread of SOD1 misfolding is not accompanied by transmission of TDP-43 or FUS pathology. Our findings argue that pathological TDP-43 and FUS may exert motor neuron pathology in ALS through the initiation of propagated misfolding of SOD1.

  3. Motor neuron-astrocyte interactions and levels of Cu,Zn superoxide dismutase in sporadic amyotrophic lateral sclerosis.

    Science.gov (United States)

    O'Reilly, S A; Roedica, J; Nagy, D; Hallewell, R A; Alderson, K; Marklund, S L; Kuby, J; Kushner, P D

    1995-02-01

    Copper, zinc superoxide dismutase (SOD1) is involved in neutralizing free radicals within cells, and mutant forms of the enzyme have recently been shown to occur in about 20% of familial cases of amyotrophic lateral sclerosis (ALS). To explore the mechanism of SOD1 involvement in ALS, we have analyzed SOD1 in sporadic ALS using activity assays and immunocyto-chemistry. Analyses of SOD1 activity in washed erythrocytes revealed no difference between 13 ALS cases and 4 controls. Spinal cord sections from 6 ALS cases, 1 primary lateral sclerosis (PLS) case, and 1 control case were stained using three different antibodies to SOD1. Since astrocytes are closely associated with motor neurons, antibodies to glial fibrillary acidic protein (GFAP) and vimentin were used as independent monitors of astrocytes. The principal findings from localizations are: (1) normal motor neurons do not have higher levels of SOD1 than other neurons, (2) there was no detectable difference in SOD1 levels in motor neurons of ALS cases and controls, (3) ALS spinal cord displayed a reduction or absence of SOD1-reactive astrocytes compared to the control and PLS cases, and (4) examination of GFAP-stained sections and morphometry showed that the normal close association between astrocytic processes and motor neuron somata was decreased in the ALS and PLS cases. These results indicate the disease mechanism in sporadic ALS may involve alterations in spinal cord astrocytes.

  4. Roles of zinc and copper in modulating the oxidative refolding of bovine copper, zinc superoxide dismutase.

    Science.gov (United States)

    Li, Hong-Tao; Jiao, Ming; Chen, Jie; Liang, Yi

    2010-03-15

    The structural integrity of the ubiquitous enzyme copper, zinc superoxide dismutase (SOD1) depends critically on the correct coordination of zinc and copper. We investigate here the roles of the stoichiometric zinc and copper ions in modulating the oxidative refolding of reduced, denatured bovine erythrocyte SOD1 at physiological pH and room temperature. Fluorescence experiment results showed that the oxidative refolding of the demetalated SOD1 (apo-SOD1) is biphasic, and the addition of stoichiometric Zn(2+) into the refolding buffer remarkably accelerates both the fast phase and the slow phase of the oxidative refolding, compared with without Zn(2+). Aggregation of apo-SOD1 in the presence of stoichiometric Zn(2+) is remarkably slower than that in the absence of Zn(2+). In contrast, the effects of stoichiometric Cu(2+) on both the rates of the oxidative refolding and the aggregation of apo-SOD1 are not remarkable. Experiments of resistance to proteinase K showed that apo-SOD1 forms a conformation with low-level proteinase K resistance during refolding and stoichiometric Cu(2+) has no obvious effect on the resistance to proteinase K. In contrast, when the refolding buffer contains stoichiometric zinc, SOD1 forms a compact conformation with high-level proteinase K resistance during refolding. Our data here demonstrated that stoichiometric zinc plays an important role in the oxidative refolding of low micromolar bovine SOD1 by accelerating the oxidative refolding, suppressing the aggregation during refolding, and helping the protein to form a compact conformation with high protease resistance activity.

  5. Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

    2015-07-03

    In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.

  6. Molecular Cloning and Expression of Sequence Variants of Manganese Superoxide Dismutase Genes from Wheat

    Science.gov (United States)

    Reactive oxygen species (ROS) are very harmful to living organisms due to the potential oxidation of membrane lipids, DNA, proteins, and carbohydrates. Transformed E.coli strain QC 871, superoxide dismutase (SOD) double-mutant, with three sequence variant MnSOD1, MnSOD2, and MnSOD3 manganese supero...

  7. Inhibitors of SOD1 Interaction as an Approach to Slow the Progressive Spread of ALS Symptoms

    Science.gov (United States)

    2016-07-01

    formation of dimers (as depicted by Fig. 1), rather than non-specific aggregate formation . a. Use size-exclusion and gel electrophoresis chromatography...structure and thos that block dimer formation . Successfully achieving both of our Aims over the course of the two-year program has the potential to...chromatography (SEC) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) split-luciferase complementation assay (SLCA) superoxide-1

  8. The cytoplasmic Cu,Zn superoxide dismutase of saccharomyces cerevisiae is required for resistance to freeze-thaw stress. Generation of free radicals during freezing and thawing

    DEFF Research Database (Denmark)

    Park, J I; Grant, C M; Davies, Michael Jonathan

    1998-01-01

    The involvement of oxidative stress in freeze-thaw injury to yeast cells was analyzed using mutants defective in a range of antioxidant functions, including Cu,Zn superoxide dismutase (encoded by SOD1), Mn superoxide dismutase (SOD2), catalase A, catalase T, glutathione reductase, gamma-glutamylc...

  9. Comparative Magnetic Resonance Imaging and Histopathological Correlates in Two SOD1 Transgenic Mouse Models of Amyotrophic Lateral Sclerosis.

    Directory of Open Access Journals (Sweden)

    Ilaria Caron

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a progressive and fatal disease due to motoneuron degeneration. Magnetic resonance imaging (MRI is becoming a promising non-invasive approach to monitor the disease course but a direct correlation with neuropathology is not feasible in human. Therefore in this study we aimed to examine MRI changes in relation to histopathology in two mouse models of ALS (C57BL6/J and 129S2/SvHsd SOD1G93A mice with different disease onset and progression. A longitudinal in vivo analysis of T2 maps, compared to ex vivo histological changes, was performed on cranial motor nuclei. An increased T2 value was associated with a significant tissue vacuolization that occurred prior to motoneuron loss in the cranial nuclei of C57 SOD1G93A mice. Conversely, in 129Sv SOD1G93A mice, which exhibit a more severe phenotype, MRI detected a milder increase of T2 value, associated with a milder vacuolization. This suggests that alteration within brainstem nuclei is not predictive of a more severe phenotype in the SOD1G93A mouse model. Using an ex vivo paradigm, Diffusion Tensor Imaging was also applied to study white matter spinal cord degeneration. In contrast to degeneration of cranial nuclei, alterations in white matter and axons loss reflected the different disease phenotype of SOD1G93A mice. The correspondence between MRI and histology further highlights the potential of MRI to monitor progressive motoneuron and axonal degeneration non-invasively in vivo. The identification of prognostic markers of the disease nevertheless requires validation in multiple models of ALS to ensure that these are not merely model-specific. Eventually this approach has the potential to lead to the development of robust and validated non-invasive imaging biomarkers in ALS patients, which may help to monitor the efficacy of therapies.

  10. SOD1 mutations targeting surface hydrogen bonds promote amyotrophic lateral sclerosis without reducing apo-state stability.

    Science.gov (United States)

    Byström, Roberth; Andersen, Peter M; Gröbner, Gerhard; Oliveberg, Mikael

    2010-06-18

    In good accord with the protein aggregation hypothesis for neurodegenerative diseases, ALS-associated SOD1 mutations are found to reduce structural stability or net repulsive charge. Moreover there are weak indications that the ALS disease progression rate is correlated with the degree of mutational impact on the apoSOD1 structure. A bottleneck for obtaining more conclusive information about these structure-disease relationships, however, is the large intrinsic variability in patient survival times and insufficient disease statistics for the majority of ALS-provoking mutations. As an alternative test of the structure-disease relationship we focus here on the SOD1 mutations that appear to be outliers in the data set. The results identify several ALS-provoking mutations whose only effect on apoSOD1 is the elimination or introduction of a single charge, i.e. D76V/Y, D101N, and N139D/K. The thermodynamic stability and folding behavior of these mutants are indistinguishable from the wild-type control. Moreover, D101N is an outlier in the plot of stability loss versus patient survival time by having rapid disease progression. Common to the identified mutations is that they truncate conserved salt-links and/or H-bond networks in the functional loops IV or VII. The results show that the local impact of ALS-associated mutations on the SOD1 molecule can sometimes overrun their global effects on apo-state stability and net repulsive charge, and point at the analysis of property outliers as an efficient strategy for mapping out new ALS-provoking features.

  11. Cysteine 111 affects aggregation and cytotoxicity of mutant Cu,Zn-superoxide dismutase associated with familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Cozzolino, Mauro; Amori, Ilaria; Pesaresi, Maria Grazia; Ferri, Alberto; Nencini, Monica; Carrì, Maria Teresa

    2008-01-11

    Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process.

  12. A Second Superoxide Dismutase Gene in the Medfly, Ceratitis Capitata

    Science.gov (United States)

    Banks, G. K.; Robinson, A. S.; Kwiatowski, J.; Ayala, F. J.; Scott, M. J.; Kriticou, D.

    1995-01-01

    We report the first case of two Cu/Zn Sod genes (ccSod1 and ccSod2) that have been cloned and sequenced from an insect, the medfly, Ceratitis capitata. Biochemical evidence suggested the presence of two Sod genes in the medfly. The two genes are isolated using different molecular strategies: ccSod1 via cross-hybridization to a genomic library using a heterologous probe and ccSod2 from cDNA using a homologous probe generated by PCR. Sequence analysis shows that ccSod1 and ccSod2 are different genes. The inferred amino sequences show that all essential residues of the active site are strictly conserved, which suggests both genes encode functional Cu/Zn superoxide dismutase (SOD). Phylogenetic analysis by the maximum parsimony method with bootstrap resampling of previously known Cu/Zn SOD reveals two monophyletic groups, vertebrates and insects. The position of ccSOD2 in this phylogeny is undefined with respect to dipteran ccSOD1, vertebrate, plant, fungal, and extracellular Cu/Zn SOD, which suggests that the duplication detected in Ceratitis is ancient, perhaps as old as the origins of the arthropod phylum in the Cambrian more than 500 million years ago. In situ hybridization to polytene chromosomes places the genes on different chromosomes, which is consistent with an ancient gene duplication. PMID:7498747

  13. Peripheral motor axons of SOD1(G127X) mutant mice are susceptible to activity-dependent degeneration

    DEFF Research Database (Denmark)

    Alvarez Herrero, Susana; Calin, A; Graffmo, K S

    2013-01-01

    Motor neuron disorders may be associated with mitochondrial dysfunction, and repetitive electrical impulse conduction during energy restriction has been found to cause neuronal degeneration. The aim of this study was to investigate the vulnerability of motor axons of a presymptomatic late......-onset, fast-progression SOD1(G127X) mouse model of amyotrophic lateral sclerosis to long-lasting, high-frequency repetitive activity. Tibial nerves were stimulated at ankle in 7 to 8-month-old SOD1(G127X) mice when they were clinically indistinguishable from wild-type (WT) mice. The evoked compound muscle...... action potentials and ascending compound nerve action potentials were recorded from plantar muscles and from the sciatic nerve, respectively. Repetitive stimulation (RS) was carried out in interrupted trains of 200-Hz for 3h. During the stimulation-sequence there was progressive conduction failure in WT...

  14. Superoxide dismutase overexpression protects against glucocorticoid-induced depressive-like behavioral phenotypes in mice.

    Science.gov (United States)

    Uchihara, Yuki; Tanaka, Ken-ichiro; Asano, Teita; Tamura, Fumiya; Mizushima, Tohru

    2016-01-22

    In the stress response, activation of the hypothalamic-pituitary-adrenal axis, and particularly the release of glucocorticoids, plays a critical role. However, dysregulation of this system and sustained high plasma levels of glucocorticoids can result in depression. Recent studies have suggested the involvement of reactive oxygen species (ROS), such as superoxide anion, in depression. However, direct evidence for a role of ROS in the pathogenesis of this disorder is lacking. In this study, using transgenic mice expressing human Cu/Zn-superoxide dismutase (SOD1), an enzyme that catalyzes the dismutation of superoxide anions, we examined the effect of SOD1 overexpression on depressive-like behavioral phenotypes in mice. Depressive-like behaviors were induced by daily subcutaneous administration of the glucocorticoid corticosterone for 4 weeks, and was monitored with the social interaction test, the sucrose preference test and the forced swim test. These tests revealed that transgenic mice overexpressing SOD1 are more resistant to glucocorticoid-induced depressive-like behavioral disorders than wild-type animals. Furthermore, compared with wild-type mice, transgenic mice showed a reduction in the number of 8-hydroxy-2'-deoxyguanosine (a marker of oxidative stress)-positive cells in the hippocampal CA3 region following corticosterone administration. These results suggest that overexpression of SOD1 protects mice against glucocorticoid-induced depressive-like behaviors by decreasing cellular ROS levels.

  15. Measuring Neuromuscular Junction Functionality in the SOD1(G93A) Animal Model of Amyotrophic Lateral Sclerosis.

    Science.gov (United States)

    Rizzuto, Emanuele; Pisu, Simona; Musarò, Antonio; Del Prete, Zaccaria

    2015-09-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to motor neuron degeneration, alteration in neuromuscular junctions (NMJs), muscle atrophy, and paralysis. To investigate the NMJ functionality in ALS we tested, in vitro, two innervated muscle types excised from SOD1(G93A) transgenic mice at the end-stage of the disease: the Soleus, a postural muscle almost completely paralyzed at that stage, and the diaphragm, which, on the contrary, is functional until death. To this aim we employed an experimental protocol that combined two types of electrical stimulation: the direct stimulation and the stimulation through the nerve. The technique we applied allowed us to determine the relevance of NMJ functionality separately from muscle contractile properties in SOD1(G93A) animal model. Functional measurements revealed that the muscle contractility of transgenic diaphragms is almost unaltered in comparison to control muscles, while transgenic Soleus muscles were severely compromised. In contrast, when stimulated via the nerve, both transgenic muscle types showed a strong decrease of the contraction force, a slowing down of the kinetic parameters, as well as alterations in the neurotransmission failure parameter. All together, these results confirm a severely impaired functionality in the SOD1(G93A) neuromuscular junctions.

  16. Androgens affect muscle, motor neuron, and survival in a mouse model of SOD1-related amyotrophic lateral sclerosis.

    Science.gov (United States)

    Aggarwal, Tanya; Polanco, Maria J; Scaramuzzino, Chiara; Rocchi, Anna; Milioto, Carmelo; Emionite, Laura; Ognio, Emanuela; Sambataro, Fabio; Galbiati, Mariarita; Poletti, Angelo; Pennuto, Maria

    2014-08-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective loss of upper and lower motor neurons and skeletal muscle atrophy. Epidemiologic and experimental evidence suggest the involvement of androgens in ALS pathogenesis, but the mechanism through which androgens modify the ALS phenotype is unknown. Here, we show that androgen ablation by surgical castration extends survival and disease duration of a transgenic mouse model of ALS expressing mutant human SOD1 (hSOD1-G93A). Furthermore, long-term treatment of orchiectomized hSOD1-G93A mice with nandrolone decanoate (ND), an anabolic androgenic steroid, worsened disease manifestations. ND treatment induced muscle fiber hypertrophy but caused motor neuron death. ND negatively affected survival, thereby dissociating skeletal muscle pathology from life span in this ALS mouse model. Interestingly, orchiectomy decreased androgen receptor levels in the spinal cord and muscle, whereas ND treatment had the opposite effect. Notably, stimulation with ND promoted the recruitment of endogenous androgen receptor into biochemical complexes that were insoluble in sodium dodecyl sulfate, a finding consistent with protein aggregation. Overall, our results shed light on the role of androgens as modifiers of ALS pathogenesis via dysregulation of androgen receptor homeostasis. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Golgi fragmentation precedes neuromuscular denervation and is associated with endosome abnormalities in SOD1-ALS mouse motor neurons

    Science.gov (United States)

    2014-01-01

    Background Fragmentation of stacked cisterns of the Golgi apparatus into dispersed smaller elements is a feature associated with degeneration of neurons in amyotrophic lateral sclerosis (ALS) and some other neurodegenerative disorders. However, the role of Golgi fragmentation in motor neuron degeneration is not well understood. Results Here we use a SOD1-ALS mouse model (low-copy Gurney G93A-SOD1 mouse) to show that motor neurons with Golgi fragmentation are retrogradely labeled by intramuscularly injected CTB (beta subunit of cholera toxin), indicating that Golgi fragmentation precedes neuromuscular denervation and axon retraction. We further show that Golgi fragmentation may occur in the absence of and precede two other pathological markers, i.e. somatodendritic SOD1 inclusions, and the induction of ATF3 expression. In addition, we show that Golgi fragmentation is associated with an altered dendritic organization of the Golgi apparatus, does not depend on intact apoptotic machinery, and is facilitated in transgenic mice with impaired retrograde dynein-dependent transport (BICD2-N mice). A connection to altered dynein-dependent transport also is suggested by reduced expression of endosomal markers in neurons with Golgi fragmentation, which also occurs in neurons with impaired dynein function. Conclusions Together the data indicate that Golgi fragmentation is a very early event in the pathological cascade in ALS that is associated with altered organization of intracellular trafficking. PMID:24708899

  18. Aberrant association of misfolded SOD1 with Na(+)/K(+)ATPase-α3 impairs its activity and contributes to motor neuron vulnerability in ALS.

    Science.gov (United States)

    Ruegsegger, Céline; Maharjan, Niran; Goswami, Anand; Filézac de L'Etang, Audrey; Weis, Joachim; Troost, Dirk; Heller, Manfred; Gut, Heinz; Saxena, Smita

    2016-03-01

    Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na(+)/K(+)ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na(+)/K(+)ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na(+)/K(+)ATPase-α3 onto a 10 amino acid stretch that is unique to Na(+)/K(+)ATPase-α3 but not found in the closely related Na(+)/K(+)ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na(+)/K(+)ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na(+)/K(+)ATPase-α3 restored Na(+)/K(+)ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na(+)/K(+)ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.

  19. Comparison of dendritic calcium transients in juvenile wild type and SOD1G93A mouse lumbar motoneurons

    Directory of Open Access Journals (Sweden)

    Katharina Ann Quinlan

    2015-04-01

    Full Text Available Previous studies of spinal motoneurons in the SOD1 mouse model of amyotrophic lateral sclerosis have shown alterations long before disease onset, including increased dendritic branching, increased persistent Na+ and Ca2+ currents, and impaired axonal transport. In this study dendritic Ca2+ entry was investigated using 2 photon excitation fluorescence microscopy and whole-cell patch-clamp of juvenile (P4-11 motoneurons. Neurons were filled with both Ca2+ Green-1 and Texas Red dextrans, and line scans performed throughout. Steps were taken to account for different sources of variability, including 1 dye filling and laser penetration, 2 dendritic anatomy, and 3 the time elapsed from the start of recording. First, Ca2+ Green-1 fluorescence was normalized by Texas Red; next, neurons were reconstructed so anatomy could be evaluated; finally, time was recorded. Customized software detected the largest Ca2+ transients (area under the curve from each line scan and matched it with parameters above. Overall, larger dendritic diameter and shorter path distance from the soma were significant predictors of larger transients, while time was not significant up to 2 hours (data thereafter was dropped. However, Ca2+ transients showed additional variability. Controlling for previous factors, significant variation was found between Ca2+ signals from different processes of the same neuron in 3/7 neurons. This could reflect differential expression of Ca2+ channels, local neuromodulation or other variations. Finally, Ca2+ transients in SOD1G93A motoneurons were significantly smaller than in non-transgenic motoneurons. In conclusion, motoneuron processes show highly variable Ca2+ transients, but these transients are smaller overall SOD1G93A motoneurons.

  20. Neuroprotective effect of bexarotene in the SOD1G93A mouse model of amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Javier eRiancho

    2015-07-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a neurodegenerative disease characterized by progressive weakness and muscle atrophy related to the loss of upper and lower motor neurons (MNs without a curative treatment. There is experimental evidence suggesting that retinoids may be involved in ALS pathogenesis. Bexarotene (Bxt is a retinoid-X receptor agonist used in the treatment of cutaneous lymphoma with a favourable safety profile whose effects have been recently investigated in other neurodegenerative diseases. In this study, we analyze the potential therapeutic effect of Bxt in the SOD1G93A mouse model of ALS.Mice were treated with Bxt or vehicle five times per week from day 60 onwards. Survival, weight and neuromuscular function studies together with histological and biochemical analyses were performed. Bxt significantly delayed motor function deterioration, ameliorated the loss of body weight and extended mice survival up to 30% of the symptomatic period. Histological analyses of the lumbosacral spinal cord revealed that Bxt markedly delayed the early motor-neuron degeneration occurring at presymptomatic stages in ALS-transgenic mice. Bxt treatment contributed to preserve the MN homeostasis in the SOD1G93A mice. Particularly, it reduced the neuronal loss and the chromatolytic response, induced nucleolar hypertrophy, decreased the formation of ubiquitylated inclusions and modulated the lysosomal response. As an agonist of the retinoic-X receptor pathway (RXR, Bxt notably increased the nuclear expression of the RXRα throughout transcriptionally active euchromatin domains. Bxt also contributed to protect the MN environment by reducing reactive astrogliosis and preserving perisomatic synapsis. Overall, these neuroprotective effects suggest that treatment with Bxt could be useful in ALS, particularly in those cases related to SOD1 mutations.

  1. Increased anxiety-like behavior and selective learning impairments are concomitant to loss of hippocampal interneurons in the presymptomatic SOD1(G93A) ALS mouse model.

    Science.gov (United States)

    Quarta, Eros; Bravi, Riccardo; Scambi, Ilaria; Mariotti, Raffaella; Minciacchi, Diego

    2015-08-01

    Amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease primarily characterized by motor neuron death, causes damages beyond motor-related areas. In particular, cognitive impairments and hippocampal damage have been reported in ALS patients. We investigated spatial navigation learning and hippocampal interneurons in a mutant SOD1(G93A) mouse (mSOD1) model of ALS. Behavioral tests were performed by using presymptomatic mSOD1 mice. General motor activity was comparable to that of wild-type mice in the open-field test, in which, however mSOD1 exhibited increased anxiety-like behavior. In the Barnes maze test, mSOD1 mice displayed a delay in learning, outperformed wild-type mice during the first probe trial, and exhibited impaired long-term memory. Stereological counts of parvalbumin-positive interneurons, which are crucial for hippocampal physiology and known to be altered in other central nervous system regions of mSOD1 mice, were also performed. At postnatal day (P) 56, the population of parvalbumin-positive interneurons in mSOD1 mice was already reduced in CA1 and in CA3, and at P90 the reduction extended to the dentate gyrus. Loss of parvalbumin-positive hippocampal interneurons occurred mostly during the presymptomatic stage. Western blot analysis showed that hippocampal parvalbumin expression levels were already reduced in mSOD1 mice at P56. The hippocampal alterations in mSOD1 mice could at least partly account for the increased anxiety-like behavior and deficits in spatial navigation learning. Our study provides evidence for cognitive alterations and damage to the γ-aminobutyric acid (GABA)ergic system in the hippocampus of murine ALS, thereby revealing selective deficits antecedent to the onset of motor symptoms.

  2. Gain of interaction of ALS-linked G93A superoxide dismutase with cytosolic malate dehydrogenase.

    Science.gov (United States)

    Mali, Yael; Zisapels, Nava

    2008-10-01

    Protein interactions of the Amyotrophic Lateral Sclerosis (ALS)-linked copper-zinc superoxide dismutase (hSOD1) G93A mutation were studied using a fluorescence resonance energy transfer (FRET) based screening system. The FRET results confirmed by pull-down immunoprecipitation indicated "gain-of-interaction" of the G93A-hSOD1 mutant with cytosolic malate dehydrogenase (cytMDH)-a key enzyme in the malate-aspartate shuttle which is vital to neurons. Furthermore, cytMDH mRNA expression was upregulated in G93A-hSOD1 expressing cells but endogenous cytMDH enzymatic activity was not enhanced, not even with exogenously added-on enzyme. Consistent with inhibition of the malate-aspartate shuttle, G93A-hSOD1 had lower malate and higher lactate levels compared to non-induced or Wild-Type-hSOD1 expressing cells. Mitochondrial NADH/NAD+ ratio is also elevated. Malate-aspartate shuttle dysfunction may explain the damage to neurons and the vulnerability to impairments of glycolytic pathways in ALS and provide a new target for the development of potential therapies.

  3. Tempol moderately extends survival in a hSOD1(G93A ALS rat model by inhibiting neuronal cell loss, oxidative damage and levels of non-native hSOD1(G93A forms.

    Directory of Open Access Journals (Sweden)

    Edlaine Linares

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1(G93A . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.

  4. Molecular and biochemical characterization of a unique mutation in CCS, the human copper chaperone to superoxide dismutase

    DEFF Research Database (Denmark)

    Huppke, Peter; Brendel, Cornelia; Korenke, Georg Christoph

    2012-01-01

    support the pathogenicity of the mutation. Expression of CCS was reduced and binding of CCS to SOD1 impaired. As a result, this mutation causes reduced SOD1 activity and may impair other mechanisms important for normal Cu homeostasis. CCS-Arg163Trp represents the primary example of a human mutation...... chaperone mutations have been described to date. We describe a child from a consanguineous family who inherited homozygous mutations in the SLC33A1, encoding an acetyl CoA transporter, and in CCS, encoding the Cu chaperone for superoxide dismutase. The CCS mutation, p.Arg163Trp, predicts substitution...

  5. EGFR inhibitor erlotinib delays disease progression but does not extend survival in the SOD1 mouse model of ALS.

    Directory of Open Access Journals (Sweden)

    Claire E Le Pichon

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease that causes progressive paralysis due to motor neuron death. Several lines of published evidence suggested that inhibition of epidermal growth factor receptor (EGFR signaling might protect neurons from degeneration. To test this hypothesis in vivo, we treated the SOD1 transgenic mouse model of ALS with erlotinib, an EGFR inhibitor clinically approved for oncology indications. Although erlotinib failed to extend ALS mouse survival it did provide a modest but significant delay in the onset of multiple behavioral measures of disease progression. However, given the lack of protection of motor neuron synapses and the lack of survival extension, the small benefits observed after erlotinib treatment appear purely symptomatic, with no modification of disease course.

  6. Depressed excitability and ion currents linked to slow exocytotic fusion pore in chromaffin cells of the SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Calvo-Gallardo, Enrique; de Pascual, Ricardo; Fernández-Morales, José-Carlos; Arranz-Tagarro, Juan-Alberto; Maroto, Marcos; Nanclares, Carmen; Gandía, Luis; de Diego, Antonio M G; Padín, Juan-Fernando; García, Antonio G

    2015-01-01

    Altered synaptic transmission with excess glutamate release has been implicated in the loss of motoneurons occurring in amyotrophic lateral sclerosis (ALS). Hyperexcitability or hypoexcitability of motoneurons from mice carrying the ALS mutation SOD1(G93A) (mSOD1) has also been reported. Here we have investigated the excitability, the ion currents, and the kinetics of the exocytotic fusion pore in chromaffin cells from postnatal day 90 to postnatal day 130 mSOD1 mice, when motor deficits are already established. With respect to wild-type (WT), mSOD1 chromaffin cells had a decrease in the following parameters: 95% in spontaneous action potentials, 70% in nicotinic current for acetylcholine (ACh), 35% in Na(+) current, 40% in Ca(2+)-dependent K(+) current, and 53% in voltage-dependent K(+) current. Ca(2+) current was increased by 37%, but the ACh-evoked elevation of cytosolic Ca(2+) was unchanged. Single exocytotic spike events triggered by ACh had the following differences (mSOD1 vs. WT): 36% lower rise rate, 60% higher decay time, 51% higher half-width, 13% lower amplitude, and 61% higher quantal size. The expression of the α3-subtype of nicotinic receptors and proteins of the exocytotic machinery was unchanged in the brain and adrenal medulla of mSOD1, with respect to WT mice. A slower fusion pore opening, expansion, and closure are likely linked to the pronounced reduction in cell excitability and in the ion currents driving action potentials in mSOD1, compared with WT chromaffin cells.

  7. Ceruloplasmin copper induces oxidant damage by a redox process utilizing cell-derived superoxide as reductant

    Science.gov (United States)

    Mukhopadhyay, C. K.; Fox, P. L.

    1998-01-01

    Oxidative damage by transition metals bound to proteins may be an important pathogenic mechanism. Ceruloplasmin (Cp) is a Cu-containing plasma protein thought to be involved in oxidative modification of lipoproteins. We have previously shown that Cp increased cell-mediated low-density lipoprotein (LDL) oxidation by a process requiring cell-derived superoxide, but the underlying chemical mechanism(s) is (are) unknown. We now show that superoxide reduction of Cp Cu is a critical reaction in cellular LDL oxidation. By bathocuproine disulfonate (BCS) binding and by superoxide utilization, we showed that exogenous superoxide reduces a single Cp Cu atom, the same Cu required for LDL oxidation. The Cu atom remained bound to Cp during the redox cycle. Three avenues of evidence showed that vascular cells reduce Cp Cu by a superoxide-dependent process. The 2-fold higher rate of Cp Cu reduction by smooth muscle cells (SMC) compared to endothelial cells (EC) was consistent with their relative rates of superoxide release. Furthermore, Cp Cu reduction by cells was blocked by Cu,Zn superoxide dismutase (SOD1). Finally, the level of superoxide produced by EC and SMC was sufficient to cause the amount of Cu reduction observed. An important role of Cp Cu reduction in LDL oxidation was suggested by results showing that SOD1 inhibited Cp Cu reduction and LDL oxidation by SMC with equal potency, while tumor necrosis factor-alpha stimulated both processes. In summary, these results show that superoxide is a critical cellular reductant of divalent transition metals involved in oxidation, and that protein-bound Cu is a substrate for this reaction. The role of these mechanisms in oxidative processes in vivo has yet to be defined.

  8. Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction

    Directory of Open Access Journals (Sweden)

    Erin L. Foresman

    2013-01-01

    Full Text Available Superoxide (O2•− contributes to the development of cardiovascular disease. Generation of O2•− occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1 or extracellular SOD (SOD3 to blood vessels would differentially protect against O2•−-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad containing the gene for Escherichia coli β-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24 h following gene transfer. Histochemical staining for β-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05, whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control but not AdSOD1 (34±4%. We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2•− production in vascular disease.

  9. P2X7 antagonism using Brilliant Blue G reduces body weight loss and prolongs survival in female SOD1G93A amyotrophic lateral sclerosis mice

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    Rachael Bartlett

    2017-03-01

    Full Text Available Background Amyotrophic lateral sclerosis (ALS is a rapidly progressive neurodegenerative disease characterised by the accumulation of aggregated proteins, microglia activation and motor neuron loss. The mechanisms underlying neurodegeneration and disease progression in ALS are unknown, but the ATP-gated P2X7 receptor channel is implicated in this disease. Therefore, the current study aimed to examine P2X7 in the context of neurodegeneration, and investigate whether the P2X7 antagonist, Brilliant Blue G (BBG, could alter disease progression in a murine model of ALS. Methods Human SOD1G93A transgenic mice, which normally develop ALS, were injected with BBG or saline, three times per week, from pre-onset of clinical disease (62–64 days of age until end-stage. During the course of treatment mice were assessed for weight, clinical score and survival, and motor coordination, which was assessed by rotarod performance. Various parameters from end-stage mice were assessed as follows. Motor neuron loss and microgliosis were assessed by immunohistochemistry. Relative amounts of lumbar spinal cord SOD1 and P2X7 were quantified by immunoblotting. Serum monocyte chemoattractant protein-1 was measured by ELISA. Splenic leukocyte populations were assessed by flow cytometry. Relative expression of splenic and hepatic P2X7 mRNA was measured by quantitative real-time PCR. Lumbar spinal cord SOD1 and P2X7 were also quantified by immunoblotting in untreated female SOD1G93A mice during the course of disease. Results BBG treatment reduced body weight loss in SOD1G93A mice of combined sex, but had no effect on clinical score, survival or motor coordination. BBG treatment reduced body weight loss in female, but not male, SOD1G93A mice. BBG treatment also prolonged survival in female, but not male, SOD1G93A mice, extending the mean survival time by 4.3% in female mice compared to female mice treated with saline. BBG treatment had no effect on clinical score or motor

  10. P2X7 antagonism using Brilliant Blue G reduces body weight loss and prolongs survival in female SOD1G93A amyotrophic lateral sclerosis mice

    Science.gov (United States)

    Bartlett, Rachael; Sluyter, Vanessa; Watson, Debbie

    2017-01-01

    Background Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterised by the accumulation of aggregated proteins, microglia activation and motor neuron loss. The mechanisms underlying neurodegeneration and disease progression in ALS are unknown, but the ATP-gated P2X7 receptor channel is implicated in this disease. Therefore, the current study aimed to examine P2X7 in the context of neurodegeneration, and investigate whether the P2X7 antagonist, Brilliant Blue G (BBG), could alter disease progression in a murine model of ALS. Methods Human SOD1G93A transgenic mice, which normally develop ALS, were injected with BBG or saline, three times per week, from pre-onset of clinical disease (62–64 days of age) until end-stage. During the course of treatment mice were assessed for weight, clinical score and survival, and motor coordination, which was assessed by rotarod performance. Various parameters from end-stage mice were assessed as follows. Motor neuron loss and microgliosis were assessed by immunohistochemistry. Relative amounts of lumbar spinal cord SOD1 and P2X7 were quantified by immunoblotting. Serum monocyte chemoattractant protein-1 was measured by ELISA. Splenic leukocyte populations were assessed by flow cytometry. Relative expression of splenic and hepatic P2X7 mRNA was measured by quantitative real-time PCR. Lumbar spinal cord SOD1 and P2X7 were also quantified by immunoblotting in untreated female SOD1G93A mice during the course of disease. Results BBG treatment reduced body weight loss in SOD1G93A mice of combined sex, but had no effect on clinical score, survival or motor coordination. BBG treatment reduced body weight loss in female, but not male, SOD1G93A mice. BBG treatment also prolonged survival in female, but not male, SOD1G93A mice, extending the mean survival time by 4.3% in female mice compared to female mice treated with saline. BBG treatment had no effect on clinical score or motor coordination in

  11. Age-associated intracellular superoxide dismutase deficiency potentiates dermal fibroblast dysfunction during wound healing.

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    Fujiwara, Toshihiro; Dohi, Teruyuki; Maan, Zeshaan N; Rustad, Kristine C; Kwon, Sun Hyung; Padmanabhan, Jagannath; Whittam, Alexander J; Suga, Hirotaka; Duscher, Dominik; Rodrigues, Melanie; Gurtner, Geoffrey C

    2017-07-04

    Reactive oxygen species (ROS) impair wound healing through destructive oxidation of intracellular proteins, lipids and nucleic acids. Intracellular superoxide dismutase (SOD1) regulates ROS levels and plays a critical role in tissue homoeostasis. Recent evidence suggests that age-associated wound healing impairments may partially result from decreased SOD1 expression. We investigated the mechanistic basis by which increased oxidative stress links to age-associated impaired wound healing. Fibroblasts were isolated from unwounded skin of young and aged mice, and myofibroblast differentiation was assessed by measuring α-smooth muscle actin and collagen gel contraction. Excisional wounds were created on young and aged mice to study the healing rate, ROS levels and SOD1 expression. A mechanistic link between oxidative stress and fibroblast function was explored by assessing the TGF-β1 signalling pathway components in young and aged mice. Age-related wounds displayed reduced myofibroblast differentiation and delayed wound healing, consistent with a decrease in the in vitro capacity for fibroblast-myofibroblast transition following oxidative stress. Young fibroblasts with normal SOD1 expression exhibited increased phosphorylation of ERK in response to elevated ROS. In contrast, aged fibroblasts with reduced SOD1 expression displayed a reduced capacity to modulate intracellular ROS. Collectively, age-associated wound healing impairments are associated with fibroblast dysfunction that is likely the result of decreased SOD1 expression and subsequent dysregulation of intracellular ROS. Strategies targeting these mechanisms may suggest a new therapeutic approach in the treatment of chronic non-healing wounds in the aged population. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis

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    Pramatarova, A.; Han, F.Y.; Rouleau, G.A. [McGill Univ., Montreal (Canada); Figlewicz, D.A. [Univ. of Rochester Medical Center, NY (United States); Ceballos-Picot, I.; Nicole, A.; Meininger, V. [Centre de Diagnostic, Paris (France); Grown, R.H. [Massachusetts General Hospital, Charlestown, MA (United States)

    1995-03-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Although most cases of ALS are sporadic, {approximately}10% are inherited as an autosomal dominant trait. Mutations in the CU/An superoxide dismutase gene (SOD 1) are responsible for a fraction of familial ALS (FALS). Screening our FALS kindreds by SSCP, we have identified mutations in 15 families, of which 9 have not been previously reported. Two of the new mutations alter amino acids that have never been implicated in FALS. One of them affects a highly conserved amino acid involved in dimer contact, and the other one affects the active-site loop of the enzyme. These two mutations reduce significantly SOD 1 enzyme activity in lymphoblasts. Our results suggest that SOD 1 mutations are responsible for {>=}13% of FALS cases. 16 refs., 2 figs., 2 tabs.

  13. Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease

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    Zippelius Annette

    2009-06-01

    Full Text Available Abstract Background Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disorder characterized by the selective loss of motor neurons (MN in the brain stem and spinal cord. Intracellular disruptions of cytosolic and mitochondrial calcium have been associated with selective MN degeneration, but the underlying mechanisms are not well understood. The present evidence supports a hypothesis that mitochondria are a target of mutant SOD1-mediated toxicity in familial amyotrophic lateral sclerosis (fALS and intracellular alterations of cytosolic and mitochondrial calcium might aggravate the course of this neurodegenerative disease. In this study, we used a fluorescence charged cool device (CCD imaging system to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations in individual cells in an established cellular model of ALS. Results To gain insights into the molecular mechanisms of SOD1G93A associated motor neuron disease, we simultaneously monitored cytosolic and mitochondrial calcium concentrations in individual cells. Voltage – dependent cytosolic Ca2+ elevations and mitochondria – controlled calcium release mechanisms were monitored after loading cells with fluorescent dyes fura-2 and rhod-2. Interestingly, comparable voltage-dependent cytosolic Ca2+ elevations in WT (SH-SY5YWT and G93A (SH-SY5YG93A expressing cells were observed. In contrast, mitochondrial intracellular Ca2+ release responses evoked by bath application of the mitochondrial toxin FCCP were significantly smaller in G93A expressing cells, suggesting impaired calcium stores. Pharmacological experiments further supported the concept that the presence of G93A severely disrupts mitochondrial Ca2+ regulation. Conclusion In this study, by fluorescence measurement of cytosolic calcium and using simultaneous [Ca2+]i and [Ca2+]mito measurements, we are able to separate and simultaneously monitor cytosolic and mitochondrial calcium concentrations

  14. Cloning,characterization and expression analysis of two superoxide dismutase (SOD) genes in wheat (Triticum aestivum L.)

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Superoxide dismutases (SODs) play an important role in catalyzing the conversion of 02-to H2O2,which can reduce the amount of harmful reactive oxygen specie (ROS) generated by the adverse environments,and alleviate the damage to plants.As one class of SODs,CuZnSODs have vital functions in preventing the ROS-generated cell damage and the death in aerobically growing organisms.In this study,two novel CuZnSOD genes in wheat,referred to TaSODI.1 and TaSOD1.2 were identified,cloned and characterized.TaSOD1.1 and TaSOD1.2 were 780 bp and 1121 bp,respectively,predicting all to encode 201 amino acids.A 45-aa length of transit peptide at the N-terminal and a 79-aa conserved CuZn-SOD domain were respectively located in TaSOD1.1 and TaSOD1.2.Phylogenetic analysis indicated that the query SODs,most of CuZnSODs,could be classified into four subgroups.Compared with the control (CK),the abundance of TaSOD1.1 transcripts did not change under drought,salt,low and high temperature conditions,but the TaSOD1.2 transcripts were strongly induced by the above abiotic stresses,which was in accordance with the elevated SOD activities in leaves in the above stress treatments to some extent,suggesting its involvement in the plant's acclimation and tolerance to the above abiotic stresses by possibly reducing the amount of the harmful ROS from enhancement of the SOD activity.

  15. Rab1-dependent ER-Golgi transport dysfunction is a common pathogenic mechanism in SOD1, TDP-43 and FUS-associated ALS.

    Science.gov (United States)

    Soo, Kai Y; Halloran, Mark; Sundaramoorthy, Vinod; Parakh, Sonam; Toth, Reka P; Southam, Katherine A; McLean, Catriona A; Lock, Peter; King, Anna; Farg, Manal A; Atkin, Julie D

    2015-11-01

    Several diverse proteins are linked genetically/pathologically to neurodegeneration in amyotrophic lateral sclerosis (ALS) including SOD1, TDP-43 and FUS. Using a variety of cellular and biochemical techniques, we demonstrate that ALS-associated mutant TDP-43, FUS and SOD1 inhibit protein transport between the endoplasmic reticulum (ER) and Golgi apparatus in neuronal cells. ER-Golgi transport was also inhibited in embryonic cortical and motor neurons obtained from a widely used animal model (SOD1(G93A) mice), validating this mechanism as an early event in disease. Each protein inhibited transport by distinct mechanisms, but each process was dependent on Rab1. Mutant TDP-43 and mutant FUS both inhibited the incorporation of secretory protein cargo into COPII vesicles as they bud from the ER, and inhibited transport from ER to the ER-Golgi intermediate (ERGIC) compartment. TDP-43 was detected on the cytoplasmic face of the ER membrane, whereas FUS was present within the ER, suggesting that transport is inhibited from the cytoplasm by mutant TDP-43, and from the ER by mutant FUS. In contrast, mutant SOD1 destabilised microtubules and inhibited transport from the ERGIC compartment to Golgi, but not from ER to ERGIC. Rab1 performs multiple roles in ER-Golgi transport, and over-expression of Rab1 restored ER-Golgi transport, and prevented ER stress, mSOD1 inclusion formation and induction of apoptosis, in cells expressing mutant TDP-43, FUS or SOD1. Rab1 also co-localised extensively with mutant TDP-43, FUS and SOD1 in neuronal cells, and Rab1 formed inclusions in motor neurons of spinal cords from sporadic ALS patients, which were positive for ubiquitinated TDP-43, implying that Rab1 is misfolded and dysfunctional in sporadic disease. These results demonstrate that ALS-mutant forms of TDP-43, FUS, and SOD1 all perturb protein transport in the early secretory pathway, between ER and Golgi compartments. These data also imply that restoring Rab1-mediated ER

  16. Presymptomatic Treatment with Acetylcholinesterase Antisense Oligonucleotides Prolongs Survival in ALS (G93A-SOD1 Mice

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    Gotkine Marc

    2013-01-01

    Full Text Available Objective. Previous research suggests that acetylcholinesterase (AChE may be involved in ALS pathogenesis. AChE enzyme inhibitors can upregulate AChE transcription which in certain contexts can have deleterious (noncatalytic effects, making them theoretically harmful in ALS, whilst AChE antisense-oligonucleotides (mEN101, which downregulate AChE may be beneficial. Our aim was to investigate whether downregulation of AChE using mEN101 is beneficial in an ALS mouse model. Methods. ALS (G93A-SOD1 mice received saline, mEN101, inverse-EN101, or neostigmine. Treatments were administered from 5 weeks. Disease-onset and survival were recorded. Additional mice were sacrificed for pathological analysis at 15 weeks of age. In a follow-up experiment treatment was started at the symptomatic stage at a higher dose. Results. mEN101 given at the presymptomatic (but not symptomatic stage prolonged survival and attenuated motor-neuron loss in ALS mice. In contrast, neostigmine exacerbated the clinical parameters. Conclusions. These results suggest that AChE may be involved in ALS pathogenesis. The accelerated disease course with neostigmine suggests that any beneficial effects of mEN101 occur through a non-catalytic rather than cholinergic mechanism.

  17. The rho kinase inhibitor Y-27632 improves motor performance in male SOD1-G93A mice

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    Rene eGünther

    2014-10-01

    Full Text Available Disease progression in amyotrophic lateral sclerosis (ALS is characterized by degeneration of motoneurons and their axons which results in a progressive muscle weakness and ultimately death from respiratory failure. The only approved drug, riluzole, lacks clinical efficacy so that more potent treatment options are needed. We have identified rho kinase (ROCK as a target, which can be manipulated to beneficially influence disease progression in models of ALS. Here, we examined the therapeutic potential of the ROCK inhibitor Y-27632 in both an in vitro and in an in vivo paradigm of motoneuron disease. Application of Y-27632 to primary motoneurons in vitro increased survival and promoted neurite outgrowth. In vivo, SOD1G93A mice were orally treated with 2 or 30 mg/kg body weight of Y-27632. The 2 mg/kg group did not benefit from Y-27632 treatment, whereas treatment with 30 mg/kg resulted in improved motor function in male mice. Female mice showed only limited improvement and overall survival was not modified in both 2 and 30 mg/kg Y-27632 groups. In conclusion, we provide evidence that inhibition of ROCK by Y-27632 is neuroprotective in vitro but has limited beneficial effects in vivo being restricted to male mice. Therefore, the evaluation of ROCK inhibitors in preclinical models of ALS should always take gender differences into account.

  18. Intrathecal Delivery of ssAAV9-DAO Extends Survival in SOD1(G93A) ALS Mice.

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    Wang, Wan; Duan, Weisong; Wang, Ying; Wen, Di; Liu, Yakun; Li, Zhongyao; Hu, Haojie; Cui, Hongying; Cui, Can; Lin, Huiqian; Li, Chunyan

    2016-12-26

    Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible neurodegenerative disease that leads to progressive paralysis and inevitable death 3-5 years after diagnosis. The mechanisms underlying this process remain unknown, but new evidence indicates that accumulating levels of D-serine result from the downregulation of D-amino acid oxidase (DAO) and that this is a novel mechanism that leads to motoneuronal death in ALS via N-methyl-D-aspartate receptor-mediated cell toxicity. Here, we explored a new therapeutic approach to ALS by overexpressing DAO in the lumbar region of the mouse spinal cord using a single stranded adeno-associated virus serotype 9 (ssAAV9) vector. A single intrathecal injection of ssAAV9-DAO was made in SOD1(G93A) mice, a well-established mouse model of ALS. Treatment resulted in moderate expression of exogenous DAO in motorneurons in the lumbar spinal cord, reduced immunoreactivity of D-serine, alleviated motoneuronal loss and glial activation, and extended survival. The potential mechanisms underlying these effects were associated with the down-regulation of NF-κB and the restoration of the phosphorylation of Akt. In conclusion, administering ssAAV9-DAO may be an effective complementary approach to gene therapy to extend lifespans in symptomatic ALS.

  19. Melatonin and steroid hormones activate intermembrane Cu,Zn-superoxide dismutase by means of mitochondrial cytochrome P450.

    Science.gov (United States)

    Iñarrea, Pedro; Casanova, Alvaro; Alava, Maria Angeles; Iturralde, María; Cadenas, Enrique

    2011-06-01

    Melatonin and steroid hormones are cytochrome P450 (CYP or P450; EC 1.14.14.1) substrates that have antioxidant properties and mitochondrial protective activities. The mitochondrial intermembrane space (IMS) Cu,Zn-superoxide dismutase (SOD1) is activated after oxidative modification of its critical thiol moieties by superoxide anion (O₂(•-)). This study was aimed at investigating the potential association between the hormonal protective antioxidant actions in mitochondria and the regulation of IMS SOD1 activity. Melatonin, testosterone, dihydrotestosterone, estradiol, and vitamin D induced a sustained activation over time of SOD1 in intact mitochondria, showing a bell-shaped enzyme activation dose response with a threshold at 50nM and a maximum effect at 1μM concentration. Enzyme activation was not affected by furafylline, but it was inhibited by omeprazole, ketoconazole, and tiron, thereby supporting the occurrence of a mitochondrial P450 activity and O₂(•-) requirements. Mitochondrial P450-dependent activation of IMS SOD1 prevented O₂(•-)-induced loss of aconitase activity in intact mitochondria respiring in State 3. Optimal protection of aconitase activity was observed at 0.1μM P450 substrate concentration, evidencing a likely oxidative effect on the mitochondrial matrix by higher substrate concentrations. Likewise, enzyme activation mediated by mitochondrial P450 activity delayed CaCl₂-induced loss of transmembrane potential and decreased cytochrome c release. Omeprazole and ketoconazole abrogated both protecting mitochondrial functions promoted by melatonin and steroid hormones.

  20. The effects of bee venom acupuncture on the central nervous system and muscle in an animal hSOD1G93A mutant.

    Science.gov (United States)

    Cai, MuDan; Choi, Sun-Mi; Yang, Eun Jin

    2015-03-13

    Amyotrophic lateral sclerosis (ALS) is caused by the degeneration of lower and upper motor neurons, leading to muscle paralysis and respiratory failure. However, there is no effective drug or therapy to treat ALS. Complementary and alternative medicine (CAM), including acupuncture, pharmacopuncture, herbal medicine, and massage is popular due to the significant limitations of conventional therapy. Bee venom acupuncture (BVA), also known as one of pharmacopunctures, has been used in Oriental medicine to treat inflammatory diseases. The purpose of this study is to investigate the effect of BVA on the central nervous system (CNS) and muscle in symptomatic hSOD1G93A transgenic mice, an animal model of ALS. Our findings show that BVA at ST36 enhanced motor function and decreased motor neuron death in the spinal cord compared to that observed in hSOD1G93A transgenic mice injected intraperitoneally (i.p.) with BV. Furthermore, BV treatment at ST36 eliminated signaling downstream of inflammatory proteins such as TLR4 in the spinal cords of symptomatic hSOD1G93A transgenic mice. However, i.p. treatment with BV reduced the levels of TNF-α and Bcl-2 expression in the muscle hSOD1G93A transgenic mice. Taken together, our findings suggest that BV pharmacopuncture into certain acupoints may act as a chemical stimulant to activate those acupoints and subsequently engage the endogenous immune modulatory system in the CNS in an animal model of ALS.

  1. The Effects of Bee Venom Acupuncture on the Central Nervous System and Muscle in an Animal hSOD1G93A Mutant

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    MuDan Cai

    2015-03-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is caused by the degeneration of lower and upper motor neurons, leading to muscle paralysis and respiratory failure. However, there is no effective drug or therapy to treat ALS. Complementary and alternative medicine (CAM, including acupuncture, pharmacopuncture, herbal medicine, and massage is popular due to the significant limitations of conventional therapy. Bee venom acupuncture (BVA, also known as one of pharmacopunctures, has been used in Oriental medicine to treat inflammatory diseases. The purpose of this study is to investigate the effect of BVA on the central nervous system (CNS and muscle in symptomatic hSOD1G93A transgenic mice, an animal model of ALS. Our findings show that BVA at ST36 enhanced motor function and decreased motor neuron death in the spinal cord compared to that observed in hSOD1G93A transgenic mice injected intraperitoneally (i.p. with BV. Furthermore, BV treatment at ST36 eliminated signaling downstream of inflammatory proteins such as TLR4 in the spinal cords of symptomatic hSOD1G93A transgenic mice. However, i.p. treatment with BV reduced the levels of TNF-α and Bcl-2 expression in the muscle hSOD1G93A transgenic mice. Taken together, our findings suggest that BV pharmacopuncture into certain acupoints may act as a chemical stimulant to activate those acupoints and subsequently engage the endogenous immune modulatory system in the CNS in an animal model of ALS.

  2. Evidence of compromised blood-spinal cord barrier in early and late symptomatic SOD1 mice modeling ALS.

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    Svitlana Garbuzova-Davis

    Full Text Available BACKGROUND: The blood-brain barrier (BBB, blood-spinal cord barrier (BSCB, and blood-cerebrospinal fluid barrier (BCSFB control cerebral/spinal cord homeostasis by selective transport of molecules and cells from the systemic compartment. In the spinal cord and brain of both ALS patients and animal models, infiltration of T-cell lymphocytes, monocyte-derived macrophages and dendritic cells, and IgG deposits have been observed that may have a critical role in motor neuron damage. Additionally, increased levels of albumin and IgG have been found in the cerebrospinal fluid in ALS patients. These findings suggest altered barrier permeability in ALS. Recently, we showed disruption of the BBB and BSCB in areas of motor neuron degeneration in the brain and spinal cord in G93A SOD1 mice modeling ALS at both early and late stages of disease using electron microscopy. Examination of capillary ultrastructure revealed endothelial cell degeneration, which, along with astrocyte alteration, compromised the BBB and BSCB. However, the effect of these alterations upon barrier function in ALS is still unclear. The aim of this study was to determine the functional competence of the BSCB in G93A mice at different stages of disease. METHODOLOGY/PRINCIPAL FINDINGS: Evans Blue (EB dye was intravenously injected into ALS mice at early or late stage disease. Vascular leakage and the condition of basement membranes, endothelial cells, and astrocytes were investigated in cervical and lumbar spinal cords using immunohistochemistry. Results showed EB leakage in spinal cord microvessels from all G93A mice, indicating dysfunction in endothelia and basement membranes and confirming our previous ultrastructural findings on BSCB disruption. Additionally, downregulation of Glut-1 and CD146 expressions in the endothelial cells of the BSCB were found which may relate to vascular leakage. CONCLUSIONS/SIGNIFICANCE: Results suggest that the BSCB is compromised in areas of motor neuron

  3. Early gene expression changes in spinal cord from SOD1G93A Amyotrophic Lateral Sclerosis animal model

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    Gabriela Pintar Oliveira

    2013-11-01

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is an adult-onset and fast progression neurodegenerative disease that leads to the loss of motor neurons. Mechanisms of selective motor neuron loss in ALS are unknown. The early events occurring in the spinal cord that may contribute to motor neuron death are not described, neither astrocytes participation in the pre-symptomatic phases of the disease. In order to identify ALS early events, we performed a microarray analysis employing a whole mouse genome platform to evaluate the gene expression pattern of lumbar spinal cords of transgenic SOD1G93A mice and their littermate controls at pre-symptomatic ages of 40 and 80 days. Differentially expressed genes were identified by means of the Bioconductor packages Agi4x44Preprocess and limma. FunNet web based tool was used for analysis of over-represented pathways. Furthermore, immunolabeled astrocytes from 40 and 80 days old mice were submitted to laser microdissection and RNA was extracted for evaluation of a selected gene by qPCR. Statistical analysis has pointed to 492 differentially expressed genes (155 up and 337 down regulated in 40 days and 1105 (433 up and 672 down in 80 days old ALS mice. KEGG analysis demonstrated the over-represented pathways tight junction, antigen processing and presentation, oxidative phosphorylation, endocytosis, chemokine signaling pathway, ubiquitin mediated proteolysis and glutamatergic synapse at both pre-symptomatic ages. Ube2i gene expression was evaluated in astrocytes from both transgenic ages, being up regulated in 40 and 80 days astrocytes enriched samples. Our data points to important early molecular events occurring in pre-symptomatic phases of ALS in mouse model. Early SUMOylation process linked to astrocytes might account to non autonomous cell toxicity in ALS. Further studies on the signaling pathways presented here may provide new insights to better understand the events triggering motor neuron death in this devastating

  4. Alterations in the motor neuron-Renshaw cell circuit in the Sod1G93A mouse model

    Science.gov (United States)

    Wootz, Hanna; FitzSimons-Kantamneni, Eileen; Larhammar, Martin; Rotterman, Travis M.; Enjin, Anders; Patra, Kalicharan; Andre, Elodie; van Zundert, Brigitte; Kullander, Klas; Alvarez, Francisco J.

    2012-01-01

    Motor neurons become hyperexcitable during progression of amyotrophic lateral sclerosis (ALS). This abnormal firing behavior has been explained by changes in their membrane properties, but more recently it has been suggested that changes in premotor circuits may also contribute to this abnormal activity. The specific circuits that may be altered during development of ALS have not been investigated. Here we examined the Renshaw cell recurrent circuit that exerts inhibitory feedback control on motor neuron firing. Using two markers for Renshaw cells (calbindin and Chrna2 , cholinergic nicotinic receptor subunit alpha2), two general markers for motor neurons (NeuN and VAChT, vesicular acethylcholine transporter ) and two markers for fast motor neurons (Chondrolectin and Calca, calcitonin-related polypeptide alpha), we analyzed the survival and connectivity of these cells during disease progression in the Sod1G93A mouse model. Most calbindin-immunoreactive (IR) Renshaw cells survive to end-stage but downregulate postsynaptic Chrna2 in presymptomatic animals. In motor neurons, some markers are downregulated early (NeuN, VAChT, Chondrolectin) and others at end-stage(Calca). Early downregulation of presynaptic VAChT and Chrna2 was correlated with disconnection from Renshaw cells as well as major structural abnormalities of motor axon synapses inside the spinal cord. Renshaw cell synapses on motor neurons underwent more complex changes, including transitional sprouting preferentially over remaining NeuN-IR motor neurons. We conclude that the loss of presynaptic motor axon input on Renshaw cells occurs at early stages of ALS and disconnects the recurrent inhibitory circuit, presumably resulting in a diminished control of motor neuron firing. PMID:23172249

  5. The effect of SOD1 mutation on cellular bioenergetic profile and viability in response to oxidative stress and influence of mutation-type.

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    Katie Richardson

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disorder characterized by the progressive degeneration of motor neurons. Substantial evidence implicates oxidative stress and mitochondrial dysfunction as early events in disease progression. Our aim was to ascertain whether mutation of the SOD1 protein increases metabolic functional susceptibility to oxidative stress. Here we used a motor neuron-like cell line (NSC34 stably transfected with various human mutant SOD1 transgenes (G93A, G37R, H48Q to investigate the impact of oxidative stress on cell viability and metabolic function within intact cells. NSC34 cells expressing mutant SOD1 showed a dose dependent reduction in cell viability when exposed to oxidative stress induced by hydrogen peroxide, with variation between mutations. The G93A transfectants showed greater cell death and LDH release compared to cells transfected with the other SOD1 mutations, and H48Q showed an accelerated decline at later time points. Differences in mitochondrial bioenergetics, including mitochondrial respiration, coupling efficiency and proton leak, were identified between the mutations, consistent with the differences observed in viability. NSC34 cells expressing G93A SOD1 displayed reduced coupled respiration and mitochondrial membrane potential compared to controls. Furthermore, the G93A mutation had significantly increased metabolic susceptibility to oxidative stress, with hydrogen peroxide increasing ROS production, reducing both cellular oxygen consumption and glycolytic flux in the cell. This study highlights bioenergetic defects within a cellular model of ALS and suggests that oxidative stress is not only detrimental to oxygen consumption but also glycolytic flux, which could lead to an energy deficit in the cell.

  6. Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

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    Muid, Khandaker Ashfaqul; Karakaya, Hüseyin Çaglar; Koc, Ahmet, E-mail: ahmetkoc@iyte.edu.tr

    2014-02-07

    Highlights: • Aging process increases ROS accumulation. • Aging process increases DNA damage levels. • Absence of SOD activity does not cause DNA damage in young cells. • Absence of SOD activity accelerate aging and increase oxidative DNA damages during the aging process. - Abstract: Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.

  7. Atomic-resolution monitoring of protein maturation in live human cells by NMR

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    Banci, Lucia; Barbieri, Letizia; Luchinat, Enrico; Secci, Erica; Zhao, Yuguang; Aricescu, A. Radu

    2014-01-01

    We used NMR directly in live human cells to describe the complete post-translational maturation process of human superoxide dismutase 1 (SOD1). We could follow, at atomic resolution, zinc binding, homodimer formation and copper uptake, and discover that copper chaperone for SOD1 (CCS) oxidation of the SOD1 intrasubunit disulfide bond occurs through both copper-dependent and independent mechanisms. Our approach represents a new strategy for structural investigation of endogeneously expressed proteins within a physiological (cellular) environment. PMID:23455544

  8. Superoxide Dismutase and Catalase Genotypes in Pediatric Migraine Patients.

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    Saygi, Semra; Erol, İlknur; Alehan, Füsun; Yalçın, Yaprak Yılmaz; Kubat, Gözde; Ataç, Fatma Belgin

    2015-10-01

    This study compared superoxide dismutase (SOD) and catalase (CAT) alleles in 97 consecutive children and adolescents with migraine to 96 healthy children and adolescents. Isolated genomic DNA was used as a template for SOD1 (35 A/C), SOD2 16 C/T, and CAT2 [(-262 C/T) and (-21 A/T)] allele genotyping. The SOD2 16 C/T genotype and C allele frequency differed significantly between controls and migraine (P = .047; P = .038). CAT -21 AA genotype and A allele frequency were significantly higher in both migraine with aura patients (P = .013; P = .004) and migraine without aura patients (P = .003; P = .001) compared to controls. To our knowledge, this is the first demonstration of differences in SOD and CAT genotypes between pediatric migraine patients and age-matched controls. Further studies on the functional implications of these genetic variants on neural antioxidant capacity and the use of antioxidant modulators for migraine treatment are warranted.

  9. Post activation depression of the Ia EPSP in motoneurones is reduced in both aged mice and in the G127X SOD1 model of Amyotrophic lateral sclerosis

    DEFF Research Database (Denmark)

    Hedegaard, Anne; Lehnhoff, Janna; Moldovan, Mihai

    2014-01-01

    D in both normal aging and in the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). We used both wild type (WT) C57BL/6J mice and the G127X SOD1 transgenic model of ALS (Jonsson et al 2004)Mice were anaesthetized with Hypnorm (0.315mg/mL fentanyl-citrate +10mg/mL fluanisone), Midazolam (5mg...

  10. n-butylidenephthalide treatment prolongs life span and attenuates motor neuron loss in SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

    Science.gov (United States)

    Zhou, Qin-Ming; Zhang, Jing-Jing; Li, Song; Chen, Sheng; Le, Wei-Dong

    2017-05-01

    To evaluate the therapeutic effects of n-butylidenephthalide (BP) in SOD1(G93A) mouse model of amyotrophic lateral sclerosis and explore the possible mechanisms. The SOD1(G93A) mice were treated by oral administration of BP (q.d., 400 mg/kg d) starting from 60 days of age and continuing until death. The rotarod test was performed to assess the disease onset. The expression levels of apoptosis-related proteins, inflammatory molecules, and autophagy-associated proteins were determined. The number of apoptotic motor neurons and the extent of microglial and astroglial activation were also assessed in the lumbar spinal cords of BP-treated mice. Grip strength test, hematoxylin-eosin staining, nicotinamide adenine dinucleotide hydrogen staining, and malondialdehyde assay were conducted to evaluate the muscle function and pathology. Although BP treatment did not delay the disease onset, it prolonged the life span and thereafter extended the disease duration in SOD1(G93A) mouse model of ALS. BP treatment also reduced the motor neuron loss through inhibiting apoptosis. We further demonstrated that the neuroprotective effects of BP might be resulted from the inhibition of inflammatory, oxidative stress, and autophagy. Our study suggests that BP may be a promising candidate for the treatment of ALS. © 2017 John Wiley & Sons Ltd.

  11. Administration of 4-(α-L-Rhamnosyloxy-benzyl Isothiocyanate Delays Disease Phenotype in SOD1G93A Rats: A Transgenic Model of Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Maria Galuppo

    2015-01-01

    Full Text Available 4-(α-L-Rhamnosyloxy-benzyl glucosinolate (glucomoringin, GMG is a compound found in Moringa oleifera seeds. Myrosinase-catalyzed hydrolysis at neutral pH of GMG releases the biologically active compound 4-(α-L-rhamnosyloxy-benzyl isothiocyanate (GMG-ITC. The present study was designed to test the potential therapeutic effectiveness of GMG-ITC to counteract the amyotrophic lateral sclerosis (ALS using SOD1tg rats, which physiologically develops SOD1G93A at about 16 weeks of life, and can be considered a genetic model of disease. Rats were treated once a day with GMG (10 mg/Kg bioactivated with myrosinase (20 µL/rat via intraperitoneal (i.p. injection for two weeks before disease onset and the treatment was prolonged for further two weeks before the sacrifice. Immune-inflammatory markers as well as apoptotic pathway were investigated to establish whether GMG-ITC could represent a new promising tool in clinical practice to prevent ALS. Achieved data display clear differences in molecular and biological profiles between treated and untreated SOD1tg rats leading to guessing that GMG-ITC can interfere with the pathophysiological mechanisms at the basis of ALS development. Therefore, GMG-ITC produced from myrosinase-catalyzed hydrolysis of pure GMG could be a candidate for further studies aimed to assess its possible use in clinical practice for the prevention or to slow down this disease.

  12. An ALS-linked mutant SOD1 produces a locomotor defect associated with aggregation and synaptic dysfunction when expressed in neurons of Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Jiou Wang

    2009-01-01

    Full Text Available The nature of toxic effects exerted on neurons by misfolded proteins, occurring in a number of neurodegenerative diseases, is poorly understood. One approach to this problem is to measure effects when such proteins are expressed in heterologous neurons. We report on effects of an ALS-associated, misfolding-prone mutant human SOD1, G85R, when expressed in the neurons of Caenorhabditis elegans. Stable mutant transgenic animals, but not wild-type human SOD1 transgenics, exhibited a strong locomotor defect associated with the presence, specifically in mutant animals, of both soluble oligomers and insoluble aggregates of G85R protein. A whole-genome RNAi screen identified chaperones and other components whose deficiency increased aggregation and further diminished locomotion. The nature of the locomotor defect was investigated. Mutant animals were resistant to paralysis by the cholinesterase inhibitor aldicarb, while exhibiting normal sensitivity to the cholinergic agonist levamisole and normal muscle morphology. When fluorescently labeled presynaptic components were examined in the dorsal nerve cord, decreased numbers of puncta corresponding to neuromuscular junctions were observed in mutant animals and brightness was also diminished. At the EM level, mutant animals exhibited a reduced number of synaptic vesicles. Neurotoxicity in this system thus appears to be mediated by misfolded SOD1 and is exerted on synaptic vesicle biogenesis and/or trafficking.

  13. Cu, Zn-Superoxide Dismutase Increases the Therapeutic Potential of Adipose-derived Mesenchymal Stem Cells by Maintaining Antioxidant Enzyme Levels.

    Science.gov (United States)

    Yoo, Dae Young; Kim, Dae Won; Chung, Jin Young; Jung, Hyo Young; Kim, Jong Whi; Yoon, Yeo Sung; Hwang, In Koo; Choi, Jung Hoon; Choi, Goang-Min; Choi, Soo Young; Moon, Seung Myung

    2016-12-01

    In the present study, we investigated the ability of Cu, Zn-superoxide dismutase (SOD1) to improve the therapeutic potential of adipose tissue-derived mesenchymal stem cells (Ad-MSCs) against ischemic damage in the spinal cord. Animals were divided into four groups: the control group, vehicle (PEP-1 peptide and artificial cerebrospinal fluid)-treated group, Ad-MSC alone group, and Ad-MSC-treated group with PEP-1-SOD1. The abdominal aorta of the rabbit was occluded for 30 min in the subrenal region to induce ischemic damage, and immediately after reperfusion, artificial cerebrospinal fluid or Ad-MSCs (2 × 10(5)) were administered intrathecally. In addition, PEP-1 or 0.5 mg/kg PEP-1-SOD1 was administered intraperitoneally to the Ad-MSC-treated rabbits. Motor behaviors and NeuN-immunoreactive neurons were significantly decreased in the vehicle-treated group after ischemia/reperfusion. Administration of Ad-MSCs significantly ameliorated the changes in motor behavior and NeuN-immunoreactive neuronal survival. In addition, the combination of PEP-1-SOD1 and Ad-MSCs further increased the ameliorative effects of Ad-MSCs in the spinal cord after ischemia. Furthermore, the administration of Ad-MSCs with PEP-1-SOD1 decreased lipid peroxidation and maintained levels of antioxidants such as SOD1 and glutathione peroxidase compared to the Ad-MSC alone group. These results suggest that combination therapy using Ad-MSCs and PEP-1-SOD1 strongly protects neurons from ischemic damage by modulating the balance of lipid peroxidation and antioxidants.

  14. Dose response and time course studies on superoxide dismutase as a urinary biomarker of carbon tetrachloride-induced hepatic injury in the Hanover Wistar rat.

    Science.gov (United States)

    Smyth, Rosemary; Munday, Michael R; York, Malcolm J; Clarke, Christopher J; Dare, Theo; Turton, John A

    2009-10-01

    Previous studies have shown that the enzyme copper/zinc superoxide dismutase (SOD-1) is increased in the urine of rats with carbon tetrachloride (CCl(4))-induced hepatotoxicity. The present experiments aimed to investigate further the usefulness of urinary SOD-1 as a non-invasive biomarker of liver injury. Two investigations were carried out, a dose response study and a time course study. In the dose response study, rats were given a single dose of CCl(4) at 0 (control), 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.80 ml/kg and urine samples collected from 12 to 36 h postdosing. In the time course study, rats were dosed at 0.80 ml/kg CCl(4) and urine sampled at 4, 12, 24 and 36 h postdosing. In both studies, the presence of SOD-1 in the urine was confirmed by Western blotting with an SOD-1 antibody. In the dose response study, serum SOD activity was elevated in all CCl(4)-treated animals and urinary SOD-1 activity was increased 2.2 times at the lowest dose (0.10 ml/kg) and 60.4 times at the highest CCl(4) dose level (0.80 ml/kg). In the time course study, urinary SOD-1 was first detected in samples collected from 4 to 12 h postdosing. We conclude that urinary SOD-1 has potential as a sensitive non-invasive biomarker of CCl(4)-induced hepatocellular injury.

  15. Synthesis of calcium superoxide

    Science.gov (United States)

    Rewick, R. T.; Blucher, W. G.; Estacio, P. L.

    1972-01-01

    Efforts to prepare Ca(O2) sub 2 from reactions of calcium compounds with 100% O3 and with O(D-1) atoms generated by photolysis of O3 at 2537 A are described. Samples of Ca(OH) sub 2, CaO, CaO2, Ca metal, and mixtures containing suspected impurities to promote reaction have been treated with excess O3 under static and flow conditions in the presence and absence of UV irradiation. Studies with KO2 suggest that the superoxide anion is stable to radiation at 2537 A but reacts with oxygen atoms generated by the photolysis of O3 to form KO3. Calcium superoxide is expected to behave in an analogous.

  16. Superoxide dismutases and glutaredoxins have a distinct role in the response of Candida albicans to oxidative stress generated by the chemical compounds menadione and diamide

    Directory of Open Access Journals (Sweden)

    Guilherme Maranhão Chaves

    2012-12-01

    Full Text Available To cope with oxidative stress, Candida albicans possesses several enzymes involved in a number of biological processes, including superoxide dismutases (Sods and glutaredoxins (Grxs. The resistance of C. albicans to reactive oxygen species is thought to act as a virulence factor. Genes such as SOD1 and GRX2, which encode for a Sod and Grx, respectively, in C. albicans are widely recognised to be important for pathogenesis. We generated a double mutant, Δgrx2/sod1, for both genes. This strain is very defective in hyphae formation and is susceptible to killing by neutrophils. When exposed to two compounds that generate reactive oxygen species, the double null mutant was susceptible to menadione and resistant to diamide. The reintegration of the SOD1 gene in the null mutant led to recovery in resistance to menadione, whereas reintegration of the GRX2 gene made the null mutant sensitive to diamide. Despite having two different roles in the responses to oxidative stress generated by chemical compounds, GRX2 and SOD1 are important for C. albicans pathogenesis because the double mutant Δgrx2/sod1 was very susceptible to neutrophil killing and was defective in hyphae formation in addition to having a lower virulence in an animal model of systemic infection.

  17. A specific superoxide dismutase mutation is on the same genetic background in sporadic and familial cases of amyotrophic lateral sclerosis

    Energy Technology Data Exchange (ETDEWEB)

    Hayward, C.; Brock, D.J.H. [Univ. of Edinburgh (United Kingdom); Swingler, R.J. [Dundee Royal Infirmary (United Kingdom)] [and others

    1996-11-01

    Amyotrophic lateral sclerosis (ALS) is a degenerative disease of motor neurons, causing progressive muscular atrophy, weakness, and death from respiratory failure, often within 2-3 years. Although most cases are sporadic, some 5%-10% are inherited as autosomal dominants with age-dependent penetrance. An ALS locus has been mapped to chromosome 21q, and causative mutations identified in the Cu/Zn superoxide dismutase (SOD1) gene. A majority of SOD1 mutations have been found in cases with a clear family history of ALS. However, we and others have also described SOD1 mutations in patients where the disease appears to be sporadic. This is especially true for the missense mutation in codon 113 of the SOD1 gene, which substitutes threonine for isoleucine (I113T). One explanation for this finding is that this codon is a mutational hot spot with sporadic cases representing new mutations. Another is that the inherited nature of the cases is disguised by the reduced penetrance of this specific mutation. We have now shown that each of six unrelated cases of I113T mutation that we have collected in the Scottish population occurs on the same genetic background. Association analysis of multiple flanking loci on chromosome 21q supports the conclusion of a founder effect, with the original mutational event occurring {ge}10 generations ago. 12 refs., 1 fig., 1 tab.

  18. SITR1 expression and the effects of resveratral on SIRT1 in the motor cortex and lumber spinal cord of SOD1G93A mice%SOD1-G93A小鼠中枢神经系统SIRT1的变化及白藜芦醇对其的影响

    Institute of Scientific and Technical Information of China (English)

    于莎莎; 吴红然; 李园园; 杜娟; 宋学琴

    2015-01-01

    目的 观察SOD1-G93A小鼠运动皮质和腰髓中SIRT1的变化以及白藜芦醇对SIRT1的影响.方法 利用SOD1-G93A小鼠及其野生型小鼠作为实验对象,分别检测随着疾病进展小鼠神经系统中SIRT1表达的动态变化及给予白藜芦醇后SIRT1的变化.结果 随着ALS疾病进展,症状早期及终末期SIRT1表达增多,而给予白藜芦醇后,SIRT1表达无明显变化,给予溶剂后SIRT1表达增多.结论 随着疾病进展,SOD1-G9A小鼠运动皮质与腰髓中SIRT1的表达逐渐增多;白藜芦醇对SIRT1未起到激活作用,白藜芦醇在SOD1-G93A小鼠模型中有无有益作用尚不能定论.

  19. Optimised and rapid pre-clinical screening in the SOD1(G93A transgenic mouse model of amyotrophic lateral sclerosis (ALS.

    Directory of Open Access Journals (Sweden)

    Richard J Mead

    Full Text Available The human SOD1(G93A transgenic mouse has been used extensively since its development in 1994 as a model for amyotrophic lateral sclerosis (ALS. In that time, a great many insights into the toxicity of mutant SOD1 have been gained using this and other mutant SOD transgenic mouse models. They all demonstrate a selective toxicity towards motor neurons and in some cases features of the pathology seen in the human disease. These models have two major drawbacks. Firstly the generation of robust preclinical data in these models has been highlighted as an area for concern. Secondly, the amount of time required for a single preclinical experiment in these models (3-4 months is a hurdle to the development of new therapies. We have developed an inbred C57BL/6 mouse line from the original mixed background (SJLxC57BL/6 SOD1(G93A transgenic line and show here that the disease course is remarkably consistent and much less prone to background noise, enabling reduced numbers of mice for testing of therapeutics. Secondly we have identified very early readouts showing a large decline in motor function compared to normal mice. This loss of motor function has allowed us to develop an early, sensitive and rapid screening protocol for the initial phases of denervation of muscle fibers, observed in this model. We describe multiple, quantitative readouts of motor function that can be used to interrogate this early mechanism. Such an approach will increase throughput for reduced costs, whilst reducing the severity of the experimental procedures involved.

  20. Increased Orexin Expression Promotes Sleep/Wake Disturbances in the SOD1-G93A Mouse Model of Amyotrophic Lateral Sclerosis

    Institute of Scientific and Technical Information of China (English)

    Rong Liu; Zhao-Fu Sheng; Bing Cai; Yong-He Zhang; Dong-Sheng Fan

    2015-01-01

    Background:Sleep/wake disturbances in patients with amyotrophic lateral sclerosis (ALS) are well-documented,however,no animal or mechanistic studies on these disturbances exist.Orexin is a crucial neurotransmitter in promoting wakefulness in sleep/wake regulation,and may play an important role in sleep disturbances in ALS.In this study,we used SOD1-G93A transgenic mice as an ALS mouse model to investigate the sleep/wake disturbances and their possible mechanisms in ALS.Methods:Electroencephalogram/electromyogram recordings were performed in SOD1-G93A transgenic mice and their littermate control mice at the ages of 90 and 120 days,and the samples obtained from these groups were subjected to quantitative reverse transcriptase-polymerase chain reaction,western blotting,and enzyme-linked immunosorbent assay.Results:For the first time in SOD1-G93A transgenic mice,we observed significantly increased wakefulness,reduced sleep time,and up-regulated orexins (prepro-orexin,orexin A and B) at both 90 and 120 days.Correlation analysis confirmed moderate to high correlations between sleep/wake time (total sleep time,wakefulness time,rapid eye movement [REM] sleep time,non-REM sleep time,and deep sleep time) and increase in orexins (prepro-orexin,orexin A and B).Conclusion:Sleep/wake disturbances occur before disease onset in this ALS mouse model.Increased orexins may promote wakefulness and result in these disturbances before and after disease onset,thus making them potential therapeutic targets for amelioration of sleep disturbances in ALS.Further studies are required to elucidate the underlying mechanisms in the future.

  1. Rho Kinase Inhibition with Fasudil in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis—Symptomatic Treatment Potential after Disease Onset

    Science.gov (United States)

    Günther, René; Balck, Alexander; Koch, Jan C.; Nientiedt, Tobias; Sereda, Michael; Bähr, Mathias; Lingor, Paul; Tönges, Lars

    2017-01-01

    Despite an improved understanding of the genetic background and the pathomechanisms of amyotrophic lateral sclerosis (ALS) no novel disease-modifying therapies have been successfully implemented in clinical routine. Riluzole still remains the only clinically approved substance in human ALS treatment with limited efficacy. We have previously identified pharmacological rho kinase (ROCK) inhibitors as orally applicable substances in SOD1.G93A transgenic ALS mice (SOD1G93A), which are able to extend survival time and improve motor function after presymptomatic treatment. Here, we have evaluated the therapeutic effect of the orally administered ROCK inhibitor Fasudil starting at a symptomatic disease stage, more realistically reflecting the clinical situation. Oral Fasudil treatment was initiated at a symptomatic stage at 80 days of life (d80) with 30 or 100 mg/kg body weight in both female and male mice. While baseline neurological scoring and survival were not influenced, Fasudil significantly improved motor behavior in male mice. Spinal cord pathology of motoneurons (MN) and infiltrating microglial cells (MG) at disease end-stage were not significantly modified. Although treatment after symptom onset was less potent than treatment in asymptomatic animals, our study shows the therapeutic benefits of this well-tolerated substance, which is already in clinical use for other indications. PMID:28197100

  2. An astrocyte regenerative response from vimentin-containing cells in the spinal cord of amyotrophic lateral sclerosis's disease-like transgenic (G93A SOD1) mice.

    Science.gov (United States)

    Zhou, Yiyi; Lu, Yi; Fang, Xin; Zhang, Jie; Li, Jiao; Li, Shujuan; Deng, Xia; Yu, Yaqing; Xu, Renshi

    2015-01-01

    The reason for regeneration in the adult spinal cord during motor neuron degeneration in amyotrophic lateral sclerosis (ALS) remains largely unknown. To this end, we studied the alteration of vimentin (a neural precursor cells marker in CNS)-containing cells (VCCs) in spinal cord during different stages of ALS used C57BL/6J G93A SOD1 transgenic mice mimicking ALS. Results showed that VCCs were mostly distributed in the ependymal zone (EZ) surrounding the central canal of spinal cord in SOD1 wild type mice; a few of VCCs were sparsely distributed in other regions. However, the number of VCCs significantly increased in the spinal cord during the onset and progression stages of ALS. They were extensively distributed in the EZ, the anterior, the lateral and the posterior horn of grey matter, particularly in the posterior horn region at the progression stage. A majority of VCCs in the anterior, the lateral and the posterior horn of grey matter (outside of EZ) generated astrocytes, but no neurons, oligodendrocytes and microgliocytes. Our results suggested that there was a potential astrocyte regenerative response to motor neuron degeneration in motor neurons-degenerated regions in the adult spinal cord during the onset and progression stages of ALS-like disease. The regenerative responses in the adult spinal cord of ALS-like mice may be a potential pathway in attempting to repair the degenerated motor neurons and restore the dysfunctional neural circuitry.

  3. Genetic Correction of SOD1 Mutant iPSCs Reveals ERK and JNK Activated AP1 as a Driver of Neurodegeneration in Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Akshay Bhinge

    2017-04-01

    Full Text Available Although mutations in several genes with diverse functions have been known to cause amyotrophic lateral sclerosis (ALS, it is unknown to what extent causal mutations impinge on common pathways that drive motor neuron (MN-specific neurodegeneration. In this study, we combined induced pluripotent stem cells-based disease modeling with genome engineering and deep RNA sequencing to identify pathways dysregulated by mutant SOD1 in human MNs. Gene expression profiling and pathway analysis followed by pharmacological screening identified activated ERK and JNK signaling as key drivers of neurodegeneration in mutant SOD1 MNs. The AP1 complex member JUN, an ERK/JNK downstream target, was observed to be highly expressed in MNs compared with non-MNs, providing a mechanistic insight into the specific degeneration of MNs. Importantly, investigations of mutant FUS MNs identified activated p38 and ERK, indicating that network perturbations induced by ALS-causing mutations converge partly on a few specific pathways that are drug responsive and provide immense therapeutic potential.

  4. Models of Superoxide Dismutases

    Energy Technology Data Exchange (ETDEWEB)

    Cabelli, Diane E.; Riley, Dennis; Rodriguez, Jorge A.; Valentine, Joan Selverstone; Zhu, Haining

    1998-05-20

    In this review we have focused much of our discussion on the mechanistic details of how the native enzymes function and how mechanistic developments/insights with synthetic small molecule complexes possessing SOD activity have influenced our understanding of the electron transfer processes involved with the natural enzymes. A few overriding themes have emerged. Clearly, the SOD enzymes operate at near diffusion controlled rates and to achieve such catalytic turnover activity, several important physical principles must be operative. Such fast electron transfer processes requires a role for protons; i.e., proton-coupled electron transfer (''H-atom transfer'') solves the dilemma of charge separation developing in the transition state for the electron transfer step. Additionally, outer-sphere electron transfer is likely a most important pathway for manganese and iron dismutases. This situation arises because the ligand exchange rates on these two ions in water never exceed {approx}10{sup +7} s{sup -1}; consequently, 10{sup +9} catalytic rates require more subtle mechanistic insights. In contrast, copper complexes can achieve diffusion controlled (>10{sup +9}) exchange rates in water; thus inner-sphere electron transfer processes are more likely to be operative in the Cu/Zn enzymes. Recent studies have continued to expand our understanding of the mechanism of action of this most important class of redox active enzymes, the superoxide dismutases, which have been critical in the successful adaptation of life on this planet to an oxygen-based metabolism. The design of SOD mimic drugs, synthetic models compounds that incorporate this superoxide dismutase catalytic activity and are capable of functioning in vivo, offers clear potential benefits in the control of diseases, ranging from the control of neurodegenerative conditions, such as Parkinson's or Alzheimer's disease, to cancer.

  5. INaP selective inhibition reverts precocious inter- and motorneurons hyperexcitability in the Sod1-G93R zebrafish ALS model

    Science.gov (United States)

    Benedetti, Lorena; Ghilardi, Anna; Rottoli, Elsa; De Maglie, Marcella; Prosperi, Laura; Perego, Carla; Baruscotti, Mirko; Bucchi, Annalisa; Del Giacco, Luca; Francolini, Maura

    2016-01-01

    The pathogenic role of SOD1 mutations in amyotrophic lateral sclerosis (ALS) was investigated using a zebrafish disease model stably expressing the ALS-linked G93R mutation. In addition to the main pathological features of ALS shown by adult fish, we found remarkably precocious alterations in the development of motor nerve circuitry and embryo behavior, and suggest that these alterations are prompted by interneuron and motor neuron hyperexcitability triggered by anomalies in the persistent pacemaker sodium current INaP. The riluzole-induced modulation of INaP reduced spinal neuron excitability, reverted the behavioral phenotypes and improved the deficits in motor nerve circuitry development, thus shedding new light on the use of riluzole in the management of ALS. Our findings provide a valid phenotype-based tool for unbiased in vivo drug screening that can be used to develop new therapies. PMID:27079797

  6. Unlike physical exercise, modified environment increases the lifespan of SOD1G93A mice however both conditions induce cellular changes.

    Directory of Open Access Journals (Sweden)

    Yannick N Gerber

    Full Text Available BACKGROUND: Amyotrophic lateral sclerosis (ALS is characterized by a gradual muscular paralysis resulting from progressive motoneurons death. ALS etiology remains unknown although it has been demonstrated to be a multifactorial disease involving several cellular partners. There is currently no effective treatment. Even if the effect of exercise is under investigation for many years, whether physical exercise is beneficial or harmful is still under debate. METHODS AND FINDINGS: We investigated the effect of three different intensities of running exercises on the survival of SOD1(G93A mice. At the early-symptomatic stage (P60, males were isolated and randomly assigned to 5 conditions: 2 sedentary groups ("sedentary" and "sedentary treadmill" placed on the inert treadmill, and 3 different training intensity groups (5 cm/s, 10 cm/s and 21 cm/s; 15 min/day, 5days/week. We first demonstrated that an appropriate "control" of the environment is of the utmost importance since comparison of the two sedentary groups evidenced an 11.6% increase in survival in the "sedentary treadmill" group. Moreover, we showed by immunohistochemistry that this increased lifespan is accompanied with motoneurons survival and increased glial reactivity in the spinal cord. In a second step, we showed that when compared with the proper control, all three running-based training did not modify lifespan of the animals, but result in motoneurons preservation and changes in glial cells activation. CONCLUSIONS/SIGNIFICANCE: We demonstrate that increase in survival induced by a slight daily modification of the environment is associated with motoneurons preservation and strong glial modifications in the lumbar spinal cord of SOD1(G93A. Using the appropriate control, we then demonstrate that all running intensities have no effect on the survival of ALS mice but induce cellular modifications. Our results highlight the critical importance of the control of the environment in ALS studies

  7. Mutant glycyl-tRNA synthetase (Gars ameliorates SOD1(G93A motor neuron degeneration phenotype but has little affect on Loa dynein heavy chain mutant mice.

    Directory of Open Access Journals (Sweden)

    Gareth T Banks

    Full Text Available BACKGROUND: In humans, mutations in the enzyme glycyl-tRNA synthetase (GARS cause motor and sensory axon loss in the peripheral nervous system, and clinical phenotypes ranging from Charcot-Marie-Tooth neuropathy to a severe infantile form of spinal muscular atrophy. GARS is ubiquitously expressed and may have functions in addition to its canonical role in protein synthesis through catalyzing the addition of glycine to cognate tRNAs. METHODOLOGY/PRINCIPAL FINDINGS: We have recently described a new mouse model with a point mutation in the Gars gene resulting in a cysteine to arginine change at residue 201. Heterozygous Gars(C201R/+ mice have locomotor and sensory deficits. In an investigation of genetic mutations that lead to death of motor and sensory neurons, we have crossed the Gars(C201R/+ mice to two other mutants: the TgSOD1(G93A model of human amyotrophic lateral sclerosis and the Legs at odd angles mouse (Dync1h1(Loa which has a defect in the heavy chain of the dynein complex. We found the Dync1h1(Loa/+;Gars(C201R/+ double heterozygous mice are more impaired than either parent, and this is may be an additive effect of both mutations. Surprisingly, the Gars(C201R mutation significantly delayed disease onset in the SOD1(G93A;Gars(C201R/+ double heterozygous mutant mice and increased lifespan by 29% on the genetic background investigated. CONCLUSIONS/SIGNIFICANCE: These findings raise intriguing possibilities for the study of pathogenetic mechanisms in all three mouse mutant strains.

  8. Intra-bone marrow-bone marrow transplantation slows disease progression and prolongs survival in G93A mutant SOD1 transgenic mice, an animal model mouse for amyotrophic lateral sclerosis.

    Science.gov (United States)

    Ohnishi, Shizuo; Ito, Hidefumi; Suzuki, Yasuhiro; Adachi, Yasushi; Wate, Reika; Zhang, Jianhua; Nakano, Satoshi; Kusaka, Hirofumi; Ikehara, Susumu

    2009-11-03

    It has been reported that bone marrow transplantation (BMT) has clinical effects on not only hematopoietic diseases and autoimmune diseases but also solid malignant tumors and metabolic diseases. We have found that intra-bone marrow-bone marrow transplantation (IBM-BMT) is superior to conventional intravenous BMT, since IBM-BMT enables rapid recovery of donor hematopoiesis and reduces the extent of graft-versus-host disease (GVHD). In this experiment, we examined the effects of IBM-BMT on symptomatic G93A mutant SOD1 transgenic mice (mSOD1 Tg mice), a model mouse line for amyotrophic lateral sclerosis (ALS). Symptomatic mSOD1 Tg mice (12 weeks old) were irradiated with 6Gyx2 at a 4-hour interval, one day before IBM-BMT. The mice were transplanted with bone marrow cells (BMCs) from 12-wk-old eGFP-transgenic C57BL/6 mice (eGFP Tg mice) or BMCs from 12-wk-old mSOD1 Tg mice. The ALS model mice transplanted with BMCs from eGFP Tg mice showed longer survival and slower disease progression than those transplanted with BMCs from mSOD1 Tg mice or untreated mSOD1 Tg mice. There was a significantly high number of eGFP(+) cells in the anterior horn of the spinal cord of the mSOD1 Tg mice transplanted with BMCs of eGFP Tg mice, some of which expressed Iba-1, a marker of microglia, although they did not differentiate into neural cells. These results suggest that the replacement with normal hematopoietic cells improved the neural cell environment, thereby slowing the progression of the disease.

  9. Ionol (BHT) produces superoxide anion.

    Science.gov (United States)

    Smirnova, E G; Lyubimov, Yu I; Malinina, T G; Lyubimova, E Yu; Alexandrushkina, N I; Vanyushin, B F; Kolesova, G M; Yaguzhinsky, L S

    2002-11-01

    In aqueous medium etiolated wheat seedlings release superoxide anion (O2*-). Interaction of a synthetic antioxidant, butylated hydroxytoluene (BHT, ionol), with oxygen in the aqueous medium is accompanied by O2*- formation. This suggests that under certain conditions BHT behaves as a prooxidant. A natural antioxidant, superoxide dismutase (SOD), and also a wound healing preparation, emulsified denatured placenta (EDP), do not exhibit the prooxidant properties. In contrast to BHT, they reduce O2*- production by the etiolated wheat seedling system.

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

    Science.gov (United States)

    Madasamy, Thangamuthu; Pandiaraj, Manickam; Balamurugan, Murugesan; Karnewar, Santosh; Benjamin, Alby Robson; Venkatesh, Krishna Arun; Vairamani, Kanagavel; Kotamraju, Srigiridhar; Karunakaran, Chandran

    2012-10-15

    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, +0.06 V 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 0.1 μM to 1 mM with a detection limit of 0.1 μM and high sensitivity of 1.1 μA μM(-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.

  11. Neuroprotective effects of the Sigma-1 receptor (S1R) agonist PRE-084, in a mouse model of motor neuron disease not linked to SOD1 mutation.

    Science.gov (United States)

    Peviani, Marco; Salvaneschi, Eleonora; Bontempi, Leonardo; Petese, Alessandro; Manzo, Antonio; Rossi, Daniela; Salmona, Mario; Collina, Simona; Bigini, Paolo; Curti, Daniela

    2014-02-01

    The identification of novel molecular targets crucially involved in motor neuron degeneration/survival is a necessary step for the development of hopefully more effective therapeutic strategies for amyotrophic lateral sclerosis (ALS) patients. In this view, S1R, an endoplasmic reticulum (ER)-resident receptor with chaperone-like activity, has recently attracted great interest. S1R is involved in several processes leading to acute and chronic neurodegeneration, including ALS pathology. Treatment with the S1R agonist PRE-084 improves locomotor function and motor neuron survival in presymptomatic and early symptomatic mutant SOD1-G93A ALS mice. Here, we tested the efficacy of PRE-084 in a model of spontaneous motor neuron degeneration, the wobbler mouse (wr) as a proof of concept that S1R may be regarded as a key therapeutic target also for ALS cases not linked to SOD1 mutation. Increased staining for S1R was detectable in morphologically spared cervical spinal cord motor neurons of wr mice both at early (6th week) and late (12th week) phases of clinical progression. S1R signal was also detectable in hypertrophic astrocytes and reactive microglia of wr mice. Chronic treatment with PRE-084 (three times a week, for 8weeks), starting at symptom onset, significantly increased the levels of BDNF in the gray matter, improved motor neuron survival and ameliorated paw abnormality and grip strength performance. In addition, the treatment significantly reduced the number of reactive astrocytes whereas, that of CD11b+ microglial cells was increased. A deeper evaluation of microglial markers revealed significant increased number of cells positive for the pan-macrophage marker CD68 and of CD206+ cells, involved in tissue restoration, in the white matter of PRE-084-treated mice. The mRNA levels of TNF-α and IL-1β were not affected by PRE-084 treatment. Thus, our results support pharmacological manipulation of S1R as a promising strategy to cure ALS and point to increased

  12. Spinal cord pathology is ameliorated by P2X7 antagonism in a SOD1-mutant mouse model of amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Savina Apolloni

    2014-09-01

    Full Text Available In recent years there has been an increasing awareness of the role of P2X7, a receptor for extracellular ATP, in modulating physiopathological mechanisms in the central nervous system. In particular, P2X7 has been shown to be implicated in neuropsychiatry, chronic pain, neurodegeneration and neuroinflammation. Remarkably, P2X7 has also been shown to be a ‘gene modifier’ in amyotrophic lateral sclerosis (ALS: the receptor is upregulated in spinal cord microglia in human and rat at advanced stages of the disease; in vitro, activation of P2X7 exacerbates pro-inflammatory responses in microglia that have an ALS phenotype, as well as toxicity towards neuronal cells. Despite this detrimental in vitro role of P2X7, in SOD1-G93A mice lacking P2X7, the clinical onset of ALS was significantly accelerated and disease progression worsened, thus indicating that the receptor might have some beneficial effects, at least at certain stages of disease. In order to clarify this dual action of P2X7 in ALS pathogenesis, in the present work we used the antagonist Brilliant Blue G (BBG, a blood-brain barrier permeable and safe drug that has already been proven to reduce neuroinflammation in traumatic brain injury, cerebral ischemia-reperfusion, neuropathic pain and experimental autoimmune encephalitis. We tested BBG in the SOD1-G93A ALS mouse model at asymptomatic, pre-symptomatic and late pre-symptomatic phases of disease. BBG at late pre-onset significantly enhanced motor neuron survival and reduced microgliosis in lumbar spinal cord, modulating inflammatory markers such as NF-κB, NADPH oxidase 2, interleukin-1β, interleukin-10 and brain-derived neurotrophic factor. This was accompanied by delayed onset and improved general conditions and motor performance, in both male and female mice, although survival appeared unaffected. Our results prove the twofold role of P2X7 in the course of ALS and establish that P2X7 modulation might represent a promising

  13. Nitric oxide alterations following acute ductal constriction in the fetal lamb: a role for superoxide.

    Science.gov (United States)

    Hsu, Jong-Hau; Oishi, Peter; Wiseman, Dean A; Hou, Yali; Chikovani, Omar; Datar, Sanjeev; Sajti, Eniko; Johengen, Michael J; Harmon, Cynthia; Black, Stephen M; Fineman, Jeffrey R

    2010-06-01

    Acute partial compression of the fetal ductus arteriosus (DA) results in an initial abrupt increase in pulmonary blood flow (PBF), which is followed by a significant reduction in PBF to baseline values over the ensuing 2-4 h. We have previously demonstrated that this potent vasoconstricting response is due, in part, to an endothelin-1 (ET-1)-mediated decrease in nitric oxide synthase (NOS) activity. In addition, in vitro data demonstrate that ET-1 increases superoxide levels in pulmonary arterial smooth muscle cells and that oxidative stress alters NOS activity. Therefore, the objectives of this study were to determine the potential role of superoxide in the alterations of hemodynamics and NOS activity following acute ductal constriction in the late-gestation fetal lamb. Eighteen anesthetized near-term fetal lambs were instrumented, and a lung biopsy was performed. After a 48-h recovery, acute constriction of the DA was performed by inflating a vascular occluder. Polyethylene glycol-superoxide dismutase (PEG-SOD; 1,000-1,500 units/kg, n = 7) or PEG-alone (vehicle control group, n = 5) was injected into the pulmonary artery before ductal constriction. Six animals had a sham operation. In PEG-alone-treated lambs, acute ductal constriction rapidly decreased pulmonary vascular resistance (PVR) by 88%. However, by 4 h, PVR returned to preconstriction baseline. This vasoconstriction was associated with an increase in lung superoxide levels (82%), a decrease in total NOS activity (50%), and an increase in P-eNOS-Thr495 (52%) (P < 0.05). PEG-SOD prevented the increase of superoxide after ductal constriction, attenuated the vasoconstriction, preserved NOS activity, and increased P-eNOS Ser1177 (307%, P < 0.05). Sham procedure induced no changes. These data suggest that an acute decrease in NOS activity that is mediated, in part, by increased superoxide levels, and alterations in the phosphorylation status of the endothelial NOS isoform, underlie the pulmonary vascular

  14. Association between single nucleotide polymorphisms in the antioxidant genes CAT, GR and SOD1, erythrocyte enzyme activities, dietary and life style factors and breast cancer risk in a Danish, prospective cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Vogel, Ulla; Dragsted, Lars Ove

    2017-01-01

    Exposure to estrogens and alcohol consumption - the two only well-established risk factors for breast cancer - are capable of causing oxidative stress, which has been linked to progression of breast cancer. Here, five functional polymorphisms in the antioxidant genes SOD1, CAT and GSR were...... showed that genetically determined variations in the antioxidant enzyme activities of SOD1, CAT and GSR were not associated with risk of breast cancer per se. However, intake of alcohol, fruit and vegetables, and smoking status interacted with some of the polymorphisms in relation to breast cancer risk...

  15. Systemic injection of AAV9-GDNF provides modest functional improvements in the SOD1(G93A) ALS rat but has adverse side effects.

    Science.gov (United States)

    Thomsen, G M; Alkaslasi, M; Vit, J-P; Lawless, G; Godoy, M; Gowing, G; Shelest, O; Svendsen, C N

    2017-03-09

    Injecting proteins into the central nervous system that stimulate neuronal growth can lead to beneficial effects in animal models of disease. In particular, glial cell line-derived neurotrophic factor (GDNF) has shown promise in animal and cell models of Parkinson's disease, Huntington's disease and amyotrophic lateral sclerosis (ALS). Here, systemic AAV9-GDNF was delivered via tail vein injections to young rats to determine whether this could be a safe and functional strategy to treat the SOD1(G93A) rat model of ALS and, therefore, translated to a therapy for ALS patients. We found that GDNF administration in this manner resulted in modest functional improvement, whereby grip strength was maintained for longer and the onset of forelimb paralysis was delayed compared to non-treated rats. This did not, however, translate into an extension in survival. In addition, ALS rats receiving GDNF exhibited slower weight gain, reduced activity levels and decreased working memory. Collectively, these results confirm that caution should be applied when applying growth factors such as GDNF systemically to multiple tissues.Gene Therapy advance online publication, 9 March 2017; doi:10.1038/gt.2017.9.

  16. Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1G93A mouse model of Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    Chrystian Junqueira Alves

    2015-09-01

    Full Text Available Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS. Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown. Sciatic nerves of presymptomatic (60-day-old SOD1G93A mice were submitted to a high-density oligonucleotide microarray analysis. DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways. The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1 and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3. Schwann cells were enriched from the sciatic nerve of presymptomatic mice using flow cytometry cell sorting. qPCR showed the up regulated (Ngfr, Cdnkn1b, E2f1, Traf2 and Erbb3, H2afx, Cdkn1a, Hspa1, Prdx, Mapk10 and down-regulated (Foxo3, Mtor genes in the enriched Schwann cells. In conclusion, molecular analyses in the presymptomatic sciatic nerve demonstrated the involvement of death, oxidative stress, and mitochondrial pathways in the Schwann cell non-autonomous mechanisms in the early stages of ALS.

  17. Quantitative assessment of hsp70, IL-1β and TNF-α in the spinal cord of dogs with E40K SOD1-associated degenerative myelopathy.

    Science.gov (United States)

    Lovett, M C; Coates, J R; Shu, Y; Oglesbee, M J; Fenner, W; Moore, S A

    2014-05-01

    Inflammation is involved in the pathogenesis of many neurodegenerative diseases. Canine degenerative myelopathy (DM) is a progressive adult-onset neurodegenerative disease commonly associated with an E40K missense mutation in the SOD1 gene. DM has many similarities to some familial forms of human amyotrophic lateral sclerosis (ALS) and may serve as an important disease model for therapy development. Pro-inflammatory mediators such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α and heat shock protein (hsp) 70 play a role in the pathogenesis of ALS. The focus of the current work was to determine whether an inflammatory phenotype is present in canine DM as defined by IL-1β, TNF-α, and hsp70 responses in cerebrospinal fluid (CSF) and spinal cord tissue. Concentrations of hsp70, IL-1β and TNF-α were below the limits of detection by ELISA in the CSF of both normal and DM-affected dogs. Immunohistochemical staining for hsp70 was significantly increased in ependymal cells lining the spinal cord central canal of DM-affected dogs (P = 0.003). This was not associated with increased IL-1β or TNF-α staining, but was associated with increased CD18 staining in the gray matter of DM-affected dogs. These results suggest that hsp70 in spinal cord tissue is a potential inflammatory signature in canine DM.

  18. Calretinin and Neuropeptide Y interneurons are differentially altered in the motor cortex of the SOD1G93A mouse model of ALS

    Science.gov (United States)

    Clark, Rosemary M.; Blizzard, Catherine A.; Young, Kaylene M.; King, Anna E.; Dickson, Tracey C.

    2017-01-01

    Increasing evidence indicates an excitatory/inhibitory imbalance may have a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Impaired inhibitory circuitry is consistently reported in the motor cortex of both familial and sporadic patients, closely associated with cortical hyperexcitability and ALS onset. Inhibitory network dysfunction is presumably mediated by intra-cortical inhibitory interneurons, however, the exact cell types responsible are yet to be identified. In this study we demonstrate dynamic changes in the number of calretinin- (CR) and neuropeptide Y-expressing (NPY) interneurons in the motor cortex of the familial hSOD1G93A ALS mouse model, suggesting their potential involvement in motor neuron circuitry defects. We show that the density of NPY-populations is significantly decreased by ~17% at symptom onset (8 weeks), and by end-stage disease (20 weeks) is significantly increased by ~30%. Conversely, the density of CR-populations is progressively reduced during later symptomatic stages (~31%) to end-stage (~36%), while CR-expressing interneurons also show alteration of neurite branching patterns at symptom onset. We conclude that a differential capacity for interneurons exists in the ALS motor cortex, which may not be a static phenomenon, but involves early dynamic changes throughout disease, implicating specific inhibitory circuitry. PMID:28294153

  19. Administration of Recombinant Heat Shock Protein 70 Delays Peripheral Muscle Denervation in the SOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis

    Directory of Open Access Journals (Sweden)

    David J. Gifondorwa

    2012-01-01

    Full Text Available A prominent clinical feature of ALS is muscle weakness due to dysfunction, denervation and degeneration of motoneurons (MNs. While MN degeneration is a late stage event in the ALS mouse model, muscle denervation occurs significantly earlier in the disease. Strategies to prevent this early denervation may improve quality of life by maintaining muscle control and slowing disease progression. The precise cause of MN dysfunction and denervation is not known, but several mechanisms have been proposed that involve potentially toxic intra- and extracellular changes. Many cells confront these changes by mounting a stress response that includes increased expression of heat shock protein 70 (Hsp70. MNs do not upregulate Hsp70, and this may result in a potentially increased vulnerability. We previously reported that recombinant human hsp70 (rhHsp70 injections delayed symptom onset and increased lifespan in SOD1G93A mice. The exogenous rhHsp70 was localized to the muscle and not to spinal cord or brain suggesting it modulates peripheral pathophysiology. In the current study, we focused on earlier administration of Hsp70 and its effect on initial muscle denervation. Injections of the protein appeared to arrest denervation with preserved large myelinated peripheral axons, and reduced glial activation.

  20. Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.

    Science.gov (United States)

    Doyle, Colleen M; Rumfeldt, Jessica A; Broom, Helen R; Sekhar, Ashok; Kay, Lewis E; Meiering, Elizabeth M

    2016-03-08

    The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.

  1. Bacteriocuprein superoxide dismutases in pseudomonads

    Energy Technology Data Exchange (ETDEWEB)

    Steinman, H.M.

    1985-06-01

    Two new instances of the rare bacteriocuprein form of superoxide dismutase have been discovered in Pseudomonas diminuta and P. maltophilia. Each species contains a manganese superoxide dismutase as well. Eight other strains of Pseudomonas and Xanthomonas spp. lacked bacteriocupreins and contained either a manganese or an iron superoxide dismutase. Native molecular weights and isoelectric points were determined for all these bacterial dismutases. A monospecific polyclonal antibody was prepared against the bacteriocuprein from Photobacterium leiognathi; it was not cross-reactive with the bacteriocuprein from either Pseudomonas strain. Bacteriocupreins have previously been identified in only two procaryotes, P. leiognathi and Caulobacter crescentus. The discovery of the Pseudomonas bacteriocupreins reveals a broader distribution, raising the possibility that bacteriocupreins are a continuous line of descent among procryotes and not isolated evolutionary occurrences, as previous data suggested.

  2. A seeded propagation of Cu,Zn-superoxide dismutase aggregates in amyotrophic lateral sclerosis

    Directory of Open Access Journals (Sweden)

    Mariko eOgawa

    2014-03-01

    Full Text Available Abnormal accumulation of protein inclusions in motor neurons has been known as a major pathological change in amyotrophic lateral sclerosis (ALS. Increasing numbers of proteins including mutant Cu,Zn-superoxide dismutase (SOD1 have been identified as constituents of pathological inclusions in a form of insoluble fibrillar aggregates. Notably, protein fibrillar aggregates exhibit a self-perpetuating property, which can convert a soluble native protein into insoluble fibrillar aggregates. Such seeding reaction of protein fibrils can accelerate the aggregation significantly and would contribute to the spread of inclusion pathologies from an affected cell to its neighboring cells in neurodegenerative diseases. In ALS, a pathological change first occurs at the site of disease onset and then propagates throughout the affected tissues in a time-dependent manner; therefore, it can be assumed that seeded aggregation may be the key factor of disease progression in ALS. In this mini review, we will briefly summarize recent studies on possible roles of a seeded aggregation of SOD1 in pathomechanism of ALS.

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

    Directory of Open Access Journals (Sweden)

    Kelley Salem

    2015-04-01

    Full Text Available 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.

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

    Science.gov (United States)

    Salem, Kelley; McCormick, Michael L; Wendlandt, Erik; Zhan, Fenghuang; Goel, Apollina

    2015-01-01

    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.

  5. A seeded propagation of Cu, Zn-superoxide dismutase aggregates in amyotrophic lateral sclerosis

    Science.gov (United States)

    Ogawa, Mariko; Furukawa, Yoshiaki

    2014-01-01

    Abnormal accumulation of protein inclusions in motor neurons has been known as a major pathological change in amyotrophic lateral sclerosis (ALS). Increasing numbers of proteins including mutant Cu, Zn-superoxide dismutase (SOD1) have been identified as constituents of pathological inclusions in a form of insoluble fibrillar aggregates. Notably, protein fibrillar aggregates exhibit a self-perpetuating property, which can convert a soluble native protein into insoluble fibrillar aggregates. Such “seeding reaction” of protein fibrils can accelerate the aggregation significantly and would contribute to the spread of inclusion pathologies from an affected cell to its neighboring cells in neurodegenerative diseases. In ALS, a pathological change first occurs at the site of disease onset and then propagates throughout the affected tissues in a time-dependent manner; therefore, it can be assumed that seeded aggregation may be the key factor of disease progression in ALS. In this mini review, we will briefly summarize recent studies on possible roles of a seeded aggregation of SOD1 in pathomechanism of ALS. PMID:24672430

  6. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

    Science.gov (United States)

    2017-01-01

    Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1) can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1) and three mutant SOD1s (mhSOD1s), in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S) increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S) decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

  7. Production of Human Cu,Zn SOD with Higher Activity and Lower Toxicity in E. coli via Mutation of Free Cysteine Residues

    Directory of Open Access Journals (Sweden)

    Kun Zhang

    2017-01-01

    Full Text Available Although, as an antioxidant enzyme, human Cu,Zn superoxide dismutase 1 (hSOD1 can mitigate damage to cell components caused by free radicals generated by aerobic metabolism, large-scale manufacturing and clinical use of hSOD1 are still limited by the challenge of rapid and inexpensive production of high-quality eukaryotic hSOD1 in recombinant forms. We have demonstrated previously that it is a promising strategy to increase the expression levels of soluble hSOD1 so as to increase hSOD1 yields in E. coli. In this study, a wild-type hSOD1 (wtSOD1 and three mutant SOD1s (mhSOD1s, in which free cysteines were substituted with serine, were constructed and their expression in soluble form was measured. Results show that the substitution of Cys111 (mhSOD1/C111S increased the expression of soluble hSOD1 in E. coli whereas substitution of the internal Cys6 (mhSOD1/C6S decreased it. Besides, raised levels of soluble expression led to an increase in hSOD1 yields. In addition, mhSOD1/C111S expressed at a higher soluble level showed lower toxicity and stronger whitening and antiradiation activities than those of wtSOD1. Taken together, our data demonstrate that C111S mutation in hSOD1 is an effective strategy to develop new SOD1-associated reagents and that mhSOD1/C111S is a satisfactory candidate for large-scale production.

  8. Association between single nucleotide polymorphisms in the antioxidant genes CAT, GR and SOD1, erythrocyte enzyme activities, dietary and life style factors and breast cancer risk in a Danish, prospective cohort study

    DEFF Research Database (Denmark)

    Kopp, Tine Iskov; Vogel, Ulla; Dragsted, Lars Ove

    2017-01-01

    Exposure to estrogens and alcohol consumption - the two only well-established risk factors for breast cancer - are capable of causing oxidative stress, which has been linked to progression of breast cancer. Here, five functional polymorphisms in the antioxidant genes SOD1, CAT and GSR were invest...

  9. Postactivation depression of the Ia EPSP in motoneurons is reduced in both the G127X SOD1 model of amyotrophic lateral sclerosis and in aged mice.

    Science.gov (United States)

    Hedegaard, A; Lehnhoff, J; Moldovan, M; Grøndahl, L; Petersen, N C; Meehan, C F

    2015-08-01

    Postactivation depression (PActD) of Ia afferent excitatory postsynaptic potentials (EPSPs) in spinal motoneurons results in a long-lasting depression of the stretch reflex. This phenomenon (PActD) is of clinical interest as it has been shown to be reduced in a number of spastic disorders. Using in vivo intracellular recordings of Ia EPSPs in adult mice, we demonstrate that PActD in adult (100-220 days old) C57BL/6J mice is both qualitatively and quantitatively similar to that which has been observed in larger animals with respect to both the magnitude (with ∼20% depression of EPSPs at 0.5 ms after a train of stimuli) and the time course (returning to almost normal amplitudes by 5 ms after the train). This validates the use of mouse models to study PActD. Changes in such excitatory inputs to spinal motoneurons may have important implications for hyperreflexia and/or glutamate-induced excitotoxicity in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). With the use of the G127X SOD1 mutant mouse, an ALS model with a prolonged asymptomatic phase and fulminant symptom onset, we observed that PActD is significantly reduced at both presymptomatic (16% depression) and symptomatic (17.3% depression) time points compared with aged-matched controls (22.4% depression). The PActD reduction was not markedly altered by symptom onset. Comparing these PActD changes at the EPSP with the known effect of the depression on the monosynaptic reflex, we conclude that this is likely to have a much larger effect on the reflex itself (a 20-40% difference). Nevertheless, it should also be accounted that in aged (580 day old) C57BL/6J mice there was also a reduction in PActD although, aging is not usually associated with spasticity.

  10. Molecular signatures of amyotrophic lateral sclerosis disease progression in hind and forelimb muscles of an SOD1(G93A) mouse model.

    Science.gov (United States)

    Capitanio, Daniele; Vasso, Michele; Ratti, Antonia; Grignaschi, Giuliano; Volta, Manuela; Moriggi, Manuela; Daleno, Cristina; Bendotti, Caterina; Silani, Vincenzo; Gelfi, Cecilia

    2012-11-15

    This study utilized proteomics, biochemical and enzymatic assays, and bioinformatics tools that characterize protein alterations in hindlimb (gastrocnemius) and forelimb (triceps) muscles in an amyotrophic lateral sclerosis (ALS) (SOD1(G93A)) mouse model. The aim of this study was to identify the key molecular signatures involved in disease progression. Both muscle types have in common an early down-regulation of complex I. In the hindlimb, early increases in oxidative metabolism are associated with uncoupling of the respiratory chain, an imbalance of NADH/NAD(+), and an increase in reactive oxygen species (ROS) production. The NADH overflow due to complex I inactivation induces TCA flux perturbations, leading to citrate production, triggering fatty acid synthase (FAS), and lipid peroxidation. These early metabolic changes in the hindlimb followed by sustained and comparatively higher metabolic and cytoskeletal derangements over time precede and may catalyze the progressive muscle wasting in this muscle at the late stage. By contrast, in the forelimb, there is an early down-regulation of complexes I and II that is associated with the reduction of oxidative metabolism, which promotes metabolic homeostasis that is accompanied by a greater cytoskeletal stabilization response. However, these early compensatory systems diminish by a later time point. The identification of potential early- and late-stage disease molecular signatures in an ALS model: muscle albumin, complex I, complex II, citrate synthase, FAS, and phosphoinositide 3-kinase functions as diagnostic markers and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α), Sema-3A, and Rho-associated protein kinase 1 (ROCK1) play the role of disease progression markers. The differing pattern of cellular metabolism and cytoskeletal derangements in the hind and forelimb identifies the potential dysmetabolism/hypermetabolism molecular signatures associated with disease progression, which may serve as

  11. Economical synthesis of potassium superoxide

    Science.gov (United States)

    Bell, A. T.; Sadhukhan, P.

    1979-01-01

    High-frequency discharge in oxygen can be used to prepare superoxides of alkali and alkaline-earth metals. Since no direct-current discharge at the electrodes is present, no sputtering can contaminate the product, hence a high conversion efficiency.

  12. Change of the expeession of uncoupling protein-3 in SOD1-G93A transgenicm mice skeleton%SOD1转基因小鼠骨骼肌中解偶联蛋白-3表达水平变化及其意义

    Institute of Scientific and Technical Information of China (English)

    房娉平; 孔勇; 宋学琴; 霍浩然

    2014-01-01

    目的 通过观察SOD1转基因小鼠骨骼肌中解偶联蛋白-3(UCP-3)表达水平的变化,探讨其在肌萎缩侧索硬化(ALS)疾病发生发展过程中的作用及意义.方法 随机选取不同时期SOD1转基因模型雌性小鼠18只作为实验组,并选取非转基因同窝对照雌性小鼠18只为对照组,分别在生长不同阶段取两组小鼠骨骼肌为标本,应用Western blot及RT-PCR检测小鼠骨骼肌内UCP-3蛋白及其mRNA的表达水平变化,并对所得数据进行统计学分析.结果 通过实验所得两组数据比较发现,实验组较对照组症状期、终末期UCP-3蛋白表达及mRNA表达均明显升高,且UCP-3蛋白表达与疾病发展呈时间相关趋势,差异有统计学意义(P<0.05).结论 本实验结果表明随着ALS疾病进展UCP-3蛋白及mRNA表达水平均明显升高,证实UCP-3可能通过参与机体抗氧化及代谢过程在ALS疾病发生发展中发挥重要作用.

  13. ENVIRONMENTAL EFFECTS ON SUPEROXIDE DISMUTASE AND CATALASE ACTIVITY AND EXPRESSION IN HONEY BEE.

    Science.gov (United States)

    Nikolić, Tatjana V; Purać, Jelena; Orčić, Snežana; Kojić, Danijela; Vujanović, Dragana; Stanimirović, Zoran; Gržetić, Ivan; Ilijević, Konstantin; Šikoparija, Branko; Blagojević, Duško P

    2015-12-01

    Understanding the cellular stress response in honey bees will significantly contribute to their conservation. The aim of this study was to analyze the response of the antioxidative enzymes superoxide dismutase and catalase in honey bees related to the presence of toxic metals in different habitats. Three locations were selected: (i) Tunovo on the mountain Golija, as control area, without industry and large human impact, (ii) Belgrade as urban area, and (iii) Zajača, as mining and industrial zone. Our results showed that the concentrations of lead (Pb) in whole body of bees vary according to habitat, but there was very significant increase of Pb in bees from investigated industrial area. Bees from urban and industrial area had increased expression of both Sod1 and Cat genes, suggesting adaptation to increased oxidative stress. However, in spite increased gene expression, the enzyme activity of catalase was lower in bees from industrial area suggesting inhibitory effect of Pb on catalase.

  14. Shared Molecular Mechanisms in Alzheimer's Disease and Amyotrophic Lateral Sclerosis: Neurofilament-Dependent Transport of sAPP, FUS, TDP-43 and SOD1, with Endoplasmic Reticulum-Like Tubules.

    Science.gov (United States)

    Muresan, Virgil; Ladescu Muresan, Zoia

    2016-01-01

    Amyotrophic lateral sclerosis (ALS), a debilitating neurodegenerative disorder of the motor neurons, leads to the disorganization of the neurofilament (NF) cytoskeleton and - ultimately - the deterioration of the neuromuscular junction. Some familial cases of ALS are caused by mutated FUS, TDP-43 or SOD1; it is thought that the mutated proteins inflict pathology either by gain or loss of function. The proper function of the neuromuscular junction requires sAPP, a soluble proteolytic fragment of the amyloid-β precursor protein (APP) - a transmembrane protein implicated in the pathology of Alzheimer's disease (AD). Whether sAPP, FUS, TDP-43 and SOD1 are mechanistically linked in a common pathway deregulated in both AD and ALS is not known. We show that sAPP, TDP-43, FUS and SOD1 are transported to neurite terminals by a mechanism that involves endoplasmic reticulum (ER)-like tubules and requires peripherin NFs. The transport of these proteins, and the translocation of the ER protein reticulon 4 (Rtn4) into neurites was studied in CAD cells, a brainstem-derived neuronal cell line highly relevant to AD and ALS. We show that a significant fraction of sAPP is generated in the soma and accumulates in a juxtanuclear ER subdomain. In neurites, sAPP localizes to Rtn4-positive ER-like tubules that extend from the soma into the growth cone and colocalizes with peripherin NFs. Knocking down peripherin disrupts the NF network and diminishes the accumulation of sAPP, TDP-43, FUS, SOD1 and Rtn4 at terminals. We propose that the impediment of a common, ER-mediated mechanism of transport of sAPP, TDP-43, FUS and SOD1, caused by a disrupted NF network, could be part of the mechanisms leading to AD and ALS. © 2015 S. Karger AG, Basel.

  15. SOD1 Gene +35A/C (exon3/intron3) Polymorphism in Type 2 Diabetes Mellitus among South Indian Population

    OpenAIRE

    Nithya, K.; Angeline, T.; Isabel, W.; A J Asirvatham

    2016-01-01

    Superoxide dismutase is an antioxidant enzyme that is involved in defence mechanisms against oxidative stress. Cu/Zn SOD is a variant that is located in exon3/intron3 boundary. The aim of the present study was to investigate whether the Cu/Zn SOD (+35A/C) gene polymorphism is associated with the susceptibility to type 2 diabetes mellitus among south Indian population. The study included patients with type 2 diabetes mellitus (n = 100) and healthy controls (n = 75). DNA was isolated from the b...

  16. Free copper, ferroxidase and SOD1 activities, lipid peroxidation and NO(x) content in the CSF. A different marker profile in four neurodegenerative diseases.

    Science.gov (United States)

    Boll, Marie-Catherine; Alcaraz-Zubeldia, Mireya; Montes, Sergio; Rios, Camilo

    2008-09-01

    The understanding of oxidative damage in different neurodegenerative diseases could enhance therapeutic strategies. Our objective was to quantify lipoperoxidation and other oxidative products as well as the activity of antioxidant enzymes and cofactors in cerebrospinal fluid (CSF) samples. We recorded data from all new patients with a diagnosis of either one of the four most frequent neurodegenerative diseases: Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD) and lateral amyotrophic sclerosis (ALS). The sum of nitrites and nitrates as end products of nitric oxide (NO) were increased in the four degenerative diseases and fluorescent lipoperoxidation products in three (excepting ALS). A decreased Cu/Zn-dependent superoxide dismutase (SOD) activity characterized the four diseases. A significantly decreased ferroxidase activity was found in PD, HD and AD, agreeing with findings of iron deposition in these entities, while free copper was found to be increased in CSF and appeared to be a good biomarker of PD.

  17. Lack of TNF-alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression.

    Science.gov (United States)

    Tortarolo, Massimo; Vallarola, Antonio; Lidonnici, Dario; Battaglia, Elisa; Gensano, Francesco; Spaltro, Gabriella; Fiordaliso, Fabio; Corbelli, Alessandro; Garetto, Stefano; Martini, Elisa; Pasetto, Laura; Kallikourdis, Marinos; Bonetto, Valentina; Bendotti, Caterina

    2015-10-01

    Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies.

  18. A caspase-3-cleaved fragment of the glial glutamate transporter EAAT2 is sumoylated and targeted to promyelocytic leukemia nuclear bodies in mutant SOD1-linked amyotrophic lateral sclerosis.

    Science.gov (United States)

    Gibb, Stuart L; Boston-Howes, William; Lavina, Zeno S; Gustincich, Stefano; Brown, Robert H; Pasinelli, Piera; Trotti, Davide

    2007-11-02

    EAAT2 (excitatory amino acid transporter 2) is a high affinity, Na+-dependent glutamate transporter of glial origin that is essential for the clearance of synaptically released glutamate and prevention of excitotoxicity. During the course of human amyotrophic lateral sclerosis (ALS) and in a transgenic mutant SOD1 mouse model of the disease, expression and activity of EAAT2 is remarkably reduced. We previously showed that some of the mutant SOD1 proteins exposed to oxidative stress inhibit EAAT2 by triggering caspase-3 cleavage of EAAT2 at a single defined locus. This gives rise to two fragments that we termed truncated EAAT2 and COOH terminus of EAAT2 (CTE). In this study, we report that analysis of spinal cord homogenates prepared from mutant G93A-SOD1 mice reveals CTE to be of a higher molecular weight than expected because it is conjugated with SUMO-1. The sumoylated CTE fragment (CTE-SUMO-1) accumulates in the spinal cord of these mice as early as presymptomatic stage (70 days of age) and not in other central nervous system areas unaffected by the disease. The presence and accumulation of CTE-SUMO-1 is specific to ALS mice, since it does not occur in the R6/2 mouse model for Huntington disease. Furthermore, using an astroglial cell line, primary culture of astrocytes, and tissue samples from G93A-SOD1 mice, we show that CTE-SUMO-1 is targeted to promyelocytic leukemia nuclear bodies. Since one of the proposed functions of promyelocytic leukemia nuclear bodies is regulation of gene transcription, we suggest a possible novel mechanism by which the glial glutamate transporter EAAT2 could contribute to the pathology of ALS.

  19. Superoxide and its metabolism during germination and axis growth of Vigna radiata (L.) Wilczek seeds.

    Science.gov (United States)

    Singh, Khangembam Lenin; Chaudhuri, Abira; Kar, Rup Kumar

    2014-01-01

    Involvement of reactive oxygen species in regulation of plant growth and development is recently being demonstrated with various results depending on the experimental system and plant species. Role of superoxide and its metabolism in germination and axis growth was investigated in case of Vigna radiata seeds, a non-endospermous leguminous species having epigeal germination, by studying the effect of different reactive oxygen species (ROS) inhibitors, distribution of O2(•)- and H2O2 and ROS enzyme profile in axes. Germination percentage and axis growth were determined under treatment with ROS inhibitors and scavengers. Localization of O2(•)- and H2O2 was done using nitroblue tetrazolium (NBT) and 3,3',5,5'-tetramethyl benzidine dihydrochloride hydrate (TMB), respectively. Apoplastic level of O2(•)- was monitored by spectrophotometric analysis of bathing medium of axes. Profiles of NADPH oxidase and superoxide dismutase (SOD) were studied by in-gel assay. Germination was retarded by treatments affecting ROS level except H2O2 scavengers, while axis growth was retarded by all. Superoxide synthesis inhibitor and scavenger prevented H2O2 accumulation in axes in later phase as revealed from TMB staining. Activity of Cu/Zn SOD1 was initially high and declined thereafter. Superoxide being produced in apoplast possibly by NADPH oxidase activity is further metabolized to (•)OH via H2O2. Germination process depends possibly on (•)OH production in the axes. Post-germinative axis growth requires O2(•)- while the differentiating zone of axis (radicle) requires H2O2 for cell wall stiffening.

  20. Role of Lipid Peroxidation Products, Plasma Total Antioxidant Status, and Cu-, Zn-Superoxide Dismutase Activity as Biomarkers of Oxidative Stress in Elderly Prediabetics

    Directory of Open Access Journals (Sweden)

    Sylwia Dzięgielewska-Gęsiak

    2014-01-01

    Full Text Available The relationship between hyperglycemia and oxidative stress in diabetes is well known, but the influence of metabolic disturbances recognized as prediabetes, in elderly patients especially, awaits for an explanation. Methods. 52 elderly persons (65 years old and older with no acute or severe chronic disorders were assessed: waist circumference (WC, body mass index (BMI, percentage of body fat (FAT, and arterial blood pressure. During an oral glucose tolerance test (OGTT fasting (0′ and 120-minute (120′ glycemia and insulinemia were determined, and type 2 diabetics (n=6 were excluded. Subjects were tested for glycated hemoglobin HbA1c, plasma lipids, total antioxidant status (TAS, thiobarbituric acid-reacting substances (TBARS, and activity of erythrocyte superoxide dismutase (SOD-1. According to OGTT results, patients were classified as normoglycemics, (NGT, n=18 and prediabetics, (PRE, n=28. Results. Both groups did not differ with their lipids, FAT, and TBARS. PRE group had higher WC (P<0.002 and BMI (P<0.002. Lower SOD-1 activity (P<0.04 and TAS status (P<0.04 were found in PRE versus NGT group. Significance. In elderly prediabetics, SOD-1 and TAS seem to reflect the first symptoms of oxidative stress, while TBARS are later biomarkers of oxidative stress.

  1. A correlation of reactive oxygen species accumulation by depletion of superoxide dismutases with age-dependent impairment in the nervous system and muscles of Drosophila adults.

    Science.gov (United States)

    Oka, Saori; Hirai, Jun; Yasukawa, Takashi; Nakahara, Yasuyuki; Inoue, Yoshihiro H

    2015-08-01

    The theory that accumulation of reactive oxygen species (ROS) in internal organs is a major promoter of aging has been considered negatively. However, it is still controversial whether overexpression of superoxide dismutases (SODs), which remove ROS, extends the lifespan in Drosophila adults. We examined whether ROS accumulation by depletion of Cu/Zn-SOD (SOD1) or Mn-SOD (SOD2) influenced age-related impairment of the nervous system and muscles in Drosophila. We confirmed the efficient depletion of Sod1 and Sod2 through RNAi and ROS accumulation by monitoring of ROS-inducible gene expression. Both RNAi flies displayed accelerated impairment of locomotor activity with age and shortened lifespan. Similarly, adults with nervous system-specific depletion of Sod1 or Sod2 also showed reduced lifespan. We then found an accelerated loss of dopaminergic neurons in the flies with suppressed SOD expression. A half-dose reduction of three pro-apoptotic genes resulted in a significant suppression of the neuronal loss, suggesting that apoptosis was involved in the neuronal loss caused by SOD silencing. In addition, depletion of Sod1 or Sod2 in musculature is also associated with enhancement of age-related locomotion impairment. In indirect flight muscles from SOD-depleted adults, abnormal protein aggregates containing poly-ubiquitin accumulated at an early adult stage and continued to increase as the flies aged. Most of these protein aggregates were observed between myofibril layers. Moreover, immuno-electron microscopy indicated that the aggregates were predominantly localized in damaged mitochondria. These findings suggest that muscular and neuronal ROS accumulation may have a significant effect on age-dependent impairment of the Drosophila adults.

  2. SOD1 Gene +35A/C (exon3/intron3 Polymorphism in Type 2 Diabetes Mellitus among South Indian Population

    Directory of Open Access Journals (Sweden)

    K. Nithya

    2016-01-01

    Full Text Available Superoxide dismutase is an antioxidant enzyme that is involved in defence mechanisms against oxidative stress. Cu/Zn SOD is a variant that is located in exon3/intron3 boundary. The aim of the present study was to investigate whether the Cu/Zn SOD (+35A/C gene polymorphism is associated with the susceptibility to type 2 diabetes mellitus among south Indian population. The study included patients with type 2 diabetes mellitus (n=100 and healthy controls (n=75. DNA was isolated from the blood and genotyping of Cu/Zn SOD gene polymorphism was done by polymerase chain reaction based restriction fragment length polymorphism method. Occurrence of different genotypes and normal (A and mutant (C allele frequencies were determined. The frequency of the three genotypes of the total subjects was as follows: homozygous wild-type A/A (95%, heterozygous genotype A/C (3%, and homozygous mutant C/C (2%. The mutant (C allele and the mutant genotypes (AC/CC were found to be completely absent among the patients with type 2 diabetes mellitus. Absence of mutant genotype (CC shows that the Cu/Zn SOD gene polymorphism may not be associated with the susceptibility to type 2 diabetes mellitus among south Indian population.

  3. SOD1 Gene +35A/C (exon3/intron3) Polymorphism in Type 2 Diabetes Mellitus among South Indian Population.

    Science.gov (United States)

    Nithya, K; Angeline, T; Isabel, W; Asirvatham, A J

    2016-01-01

    Superoxide dismutase is an antioxidant enzyme that is involved in defence mechanisms against oxidative stress. Cu/Zn SOD is a variant that is located in exon3/intron3 boundary. The aim of the present study was to investigate whether the Cu/Zn SOD (+35A/C) gene polymorphism is associated with the susceptibility to type 2 diabetes mellitus among south Indian population. The study included patients with type 2 diabetes mellitus (n = 100) and healthy controls (n = 75). DNA was isolated from the blood and genotyping of Cu/Zn SOD gene polymorphism was done by polymerase chain reaction based restriction fragment length polymorphism method. Occurrence of different genotypes and normal (A) and mutant (C) allele frequencies were determined. The frequency of the three genotypes of the total subjects was as follows: homozygous wild-type A/A (95%), heterozygous genotype A/C (3%), and homozygous mutant C/C (2%). The mutant (C) allele and the mutant genotypes (AC/CC) were found to be completely absent among the patients with type 2 diabetes mellitus. Absence of mutant genotype (CC) shows that the Cu/Zn SOD gene polymorphism may not be associated with the susceptibility to type 2 diabetes mellitus among south Indian population.

  4. Role of antioxidant enzymes in redox regulation of N-methyl-D-aspartate receptor function and memory in middle-aged rats.

    Science.gov (United States)

    Lee, Wei-Hua; Kumar, Ashok; Rani, Asha; Foster, Thomas C

    2014-06-01

    Overexpression of superoxide dismutase 1 (SOD1) in the hippocampus results in age-dependent impaired cognition and altered synaptic plasticity suggesting a possible model for examining the role of oxidative stress in senescent neurophysiology. However, it is unclear if SOD1 overexpression involves an altered redox environment and a decrease in N-methyl-D-aspartate receptor (NMDAR) synaptic function reported for aging animals. Viral vectors were used to express SOD1 and green fluorescent protein (SOD1 + GFP), SOD1 and catalase (SOD1 + CAT), or GFP alone in the hippocampus of middle-aged (17 months) male Fischer 344 rats. We confirm that SOD1 + GFP and SOD1 + CAT reduced lipid peroxidation indicating superoxide metabolites were primarily responsible for lipid peroxidation. SOD1 + GFP impaired learning, decreased glutathione peroxidase activity, decreased glutathione levels, decreased NMDAR-mediated synaptic responses, and impaired long-term potentiation. Co-expression of SOD1 + CAT rescued the effects of SOD1 expression on learning, redox measures, and synaptic function suggesting the effects were mediated by excess hydrogen peroxide. Application of the reducing agent dithiolthreitol to hippocampal slices increased the NMDAR-mediated component of the synaptic response in SOD1 + GFP animals relative to animals that overexpress SOD1 + CAT indicating that the effect of antioxidant enzyme expression on NMDAR function was because of a shift in the redox environment. The results suggest that overexpression of neuronal SOD1 and CAT in middle age may provide a model for examining the role of oxidative stress in senescent physiology and the progression of age-related neurodegenerative diseases.

  5. Ursodeoxycholic acid and superoxide anion

    Institute of Scientific and Technical Information of China (English)

    Predrag Ljubuncic; Omar Abu-Salach; Arieh Bomzon

    2005-01-01

    AIM: To investigate the ability of ursodeoxycholic acid (UDCA) to scavenge superoxide anion (O2-).METHODS: We assessed the ability of UDCA to scavenge (O2-) generated by xanthine-xanthine oxidase (X-XO) in a cell-free system and its effect on the rate of O2--induced ascorbic acid (AA) oxidation in hepatic post-mitochondrial supernatants.RESULTS: UDCA at a concentration as high as 1 mmol/Ldid not impair the ability of the X-XO system to generate O2-, but could scavenge O2- at concentrations of 0.5 and 1 mmol/L, and decrease the rate of AA oxidation at a concentration of 100 μmol/L.CONCLUSION: UDCA can scavenge O2-, an action that may be beneficial to patients with primary biliary cirrhosis.

  6. Induction of oxidative stress and inhibition of superoxide dismutase expression in rat cerebral cortex and cerebellum by PTU-induced hypothyroidism and its reversal by curcumin.

    Science.gov (United States)

    Jena, Srikanta; Anand, Chinmay; Chainy, Gagan Bihari Nityananda; Dandapat, Jagneshwar

    2012-08-01

    The present study was carried out to elucidate the effectiveness of curcumin in ameliorating the expression of superoxide dismutase (SOD) in cerebral cortex and cerebellum of rat brain under 6-propyl-2-thiouracil (PTU)-induced hypothyroidism. Induction of hypothyroidism in adult rats by PTU resulted in augmentation of lipid peroxidation (LPx), an index of oxidative stress in cerebellum but not in cerebral cortex. Curcumin-supplementation to PTU-treated (hypothyroid) rats showed significant reduction in the level of LPx in both the regions of brain. The decreased translated products (SOD1 and SOD2) and the unchanged activity of SOD in cerebral cortex of PTU-treated rats were increased on supplementation of curcumin to the hypothyroid rats. Declined translated products of SOD1 and SOD2 in cerebellum of PTU-treated rats were alleviated on administration of curcumin to hypothyroid rats. On the other hand, the decreased activity of SOD in cerebellum of PTU-treated rats was further declined on administration of curcumin to the hypothyroid rats. Results of the present investigation indicate that curcumin differentially modulates the expression of superoxide dismutase in rat brain cortex and cerebellum under PTU-induced hypothyroidism.

  7. Extracellular superoxide dismutase protects Histoplasma yeast cells from host-derived oxidative stress.

    Directory of Open Access Journals (Sweden)

    Brian H Youseff

    Full Text Available In order to establish infections within the mammalian host, pathogens must protect themselves against toxic reactive oxygen species produced by phagocytes of the immune system. The fungal pathogen Histoplasma capsulatum infects both neutrophils and macrophages but the mechanisms enabling Histoplasma yeasts to survive in these phagocytes have not been fully elucidated. We show that Histoplasma yeasts produce a superoxide dismutase (Sod3 and direct it to the extracellular environment via N-terminal and C-terminal signals which promote its secretion and association with the yeast cell surface. This localization permits Sod3 to protect yeasts specifically from exogenous superoxide whereas amelioration of endogenous reactive oxygen depends on intracellular dismutases such as Sod1. While infection of resting macrophages by Histoplasma does not stimulate the phagocyte oxidative burst, interaction with polymorphonuclear leukocytes (PMNs and cytokine-activated macrophages triggers production of reactive oxygen species (ROS. Histoplasma yeasts producing Sod3 survive co-incubation with these phagocytes but yeasts lacking Sod3 are rapidly eliminated through oxidative killing similar to the effect of phagocytes on Candida albicans yeasts. The protection provided by Sod3 against host-derived ROS extends in vivo. Without Sod3, Histoplasma yeasts are attenuated in their ability to establish respiratory infections and are rapidly cleared with the onset of adaptive immunity. The virulence of Sod3-deficient yeasts is restored in murine hosts unable to produce superoxide due to loss of the NADPH-oxidase function. These results demonstrate that phagocyte-produced ROS contributes to the immune response to Histoplasma and that Sod3 facilitates Histoplasma pathogenesis by detoxifying host-derived reactive oxygen thereby enabling Histoplasma survival.

  8. Superoxide dismutases in chronic gastritis.

    Science.gov (United States)

    Švagelj, Dražen; Terzić, Velimir; Dovhanj, Jasna; Švagelj, Marija; Cvrković, Mirta; Švagelj, Ivan

    2016-04-01

    Human gastric diseases have shown significant changes in the activity and expression of superoxide dismutase (SOD) isoforms. The aim of this study was to detect Mn-SOD activity and expression in the tissue of gastric mucosa, primarily in chronic gastritis (immunohistochemical Helicobacter pylori-negative gastritis, without other pathohistological changes) and to evaluate their possible connection with pathohistological diagnosis. We examined 51 consecutive outpatients undergoing endoscopy for upper gastrointestinal symptoms. Patients were classified based on their histopathological examinations and divided into three groups: 51 patients (archive samples between 2004-2009) with chronic immunohistochemical Helicobacter pylori-negative gastritis (mononuclear cells infiltration were graded as absent, moderate, severe) divided into three groups. Severity of gastritis was graded according to the updated Sydney system. Gastric tissue samples were used to determine the expression of Mn-SOD with anti-Mn-SOD Ab immunohistochemically. The Mn-SOD expression was more frequently present in specimens with severe and moderate inflammation of gastric mucosa than in those with normal mucosa. In patients with normal histological finding, positive immunoreactivity of Mn-SOD was not found. Our results determine the changes in Mn-SOD expression occurring in the normal gastric mucosa that had undergone changes in the intensity of chronic inflammatory infiltrates in the lamina propria. © 2016 APMIS. Published by John Wiley & Sons Ltd.

  9. Effects of dl-3n-butyphthalide on behaviour of different sex hSOD1G93A transgenic mice%丁苯酞对不同性别hSOD1G93A转基因小鼠行为学的影响

    Institute of Scientific and Technical Information of China (English)

    张红丽; 刘珊; 阴均涛; 闫欣; 王亚飞; 刘亚玲

    2012-01-01

    AIM To observe the effect on behavior when dl-3n-butyphthalide ( NBP) exposure in hSODlG93A transgenic mice with different sex. METHODS Twenty four hSODlG93A mice for amyotrophic lateral sclerosis model were divided into control group and NBP group randomly. Every group had 6 males and 6 females. Corn oil 10 mL·kg-1·d-1 and NBP 180 mg·kg-1·d-1 were administered through oral gavages respectively. The behavior manifestations of the mice were recorded. RESULTS At 90 d, there was no significant difference between the control and NBP group ( P > 0.05) . At 120 d, there were significant differences between male mice and female mice on rotating time and grip strength in the control group (P 0.05) , while the step length of female mice showed remarkably increase in the NBP group (P < 0.05) . The lifespan of males in the NBP group was remarkably lengthened compared with the control group (138.5 ± 3.4 vs. 119.3 ± 3.2, P = 0.002) , the lifespan of females in both groups had no obvious difference ( 134.5 ± 3.5 vs. 132.0 ±2.4, P = 0.573) . CONCLUSION NBP can remarkably increase male ALS model mice ability of movement coordination and muscle strength, delay the disease progress and extend the lifespan.%目的 观察丁苯酞对不同性别hSOD1G93A转基因小鼠行为学的影响.方法 肌萎缩侧索硬化(ALS)模型hSOD1G93A转基因小鼠24只,随机分为对照组和丁苯酞组,每组雄雌各6只,分别灌胃给予玉米油10 mL·kg-1·d-1和丁苯酞180 mg·kg-1·d-1,记录雄、雌小鼠的行为学表现.结果 90 d时,对照组与丁苯酞组相比均无显著差异(P>0.05).120 d时,对照组雄性小鼠与雌性小鼠相比,旋转时间和后肢抓力均显著减小(均P< 0.05),后肢步长无显著差异(P=0.857);丁苯酞组雄性小鼠与对照组雄性小鼠相比旋转时间、后肢抓力和后肢步长均显著增加(均P< 0.05),2组雌性小鼠间比较,旋转时间和后肢抓力无显著差异(均P> 0.05),但丁苯酞组雌性小

  10. Superoxide-dependent hydroxylation by myeloperoxidase.

    Science.gov (United States)

    Kettle, A J; Winterbourn, C C

    1994-06-24

    When stimulated, neutrophils undergo a respiratory burst converting oxygen to superoxide. Although superoxide is critical for microbial killing by phagocytic cells, the precise role it plays has yet to be established. It has been proposed to optimize their production of hypochlorous acid and to be required for the generation of hydroxyl radicals. Superoxide is also involved in the hydroxylation of salicylate by neutrophils. However, the mechanism of this reaction is unknown. We found that neutrophils stimulated with opsonized zymosan hydroxylated salicylate to produce mainly 2,5-dihydroxybenzoate. Its formation was dependent on superoxide and a heme protein but was independent of hydrogen peroxide and hydroxyl radicals. Production of 2,5-dihydroxybenzoate was enhanced by methionine, which scavenges hypochlorous acid. Neutrophils from an individual with myeloperoxidase deficiency hydroxylated salicylate at only 13% of the level of control cells. Purified human myeloperoxidase and xanthine oxidase plus hypoxanthine hydroxylated salicylate to produce 2,5-dihydroxybenzoate. As with neutrophils, the reaction required superoxide but not hydrogen peroxide and was unaffected by hydroxyl radical scavengers. Thus, myeloperoxidase catalyzes superoxide-dependent hydroxylation. This newly recognized reaction may be relevant to the in vivo functions of superoxide and myeloperoxidase.

  11. Biological Superoxide In Manganese Oxide Formation

    Science.gov (United States)

    Hansel, C.; Learman, D.; Zeiner, C.; Santelli, C. M.

    2011-12-01

    Manganese (Mn) oxides are among the strongest sorbents and oxidants within the environment, controlling the fate and transport of numerous elements and the degradation of recalcitrant carbon. Both bacteria and fungi mediate the oxidation of Mn(II) to Mn(III/IV) oxides but the genetic and biochemical mechanisms responsible remain poorly understood. Furthermore, the physiological basis for microbial Mn(II) oxidation remains an enigma. We have recently reported that a common marine bacterium (Roseobacter sp. AzwK-3b) oxidizes Mn(II) via reaction with extracellular superoxide (O2-) produced during exponential growth. Here we expand this superoxide-mediated Mn(II) oxidation pathway to fungi, introducing a surprising homology between prokaryotic and eukaryotic metal redox processes. For instance, Stibella aciculosa, a common soil Ascomycete filamentous fungus, precipitates Mn oxides at the base of asexual reproductive structures (synnemata) used to support conidia (Figure 1). This distribution is a consequence of localized production of superoxide (and it's dismutation product hydrogen peroxide, H2O2), leading to abiotic oxidation of Mn(II) by superoxide. Disruption of NADPH oxidase activity using the oxidoreductase inhibitor DPI leads to diminished cell differentiation and subsequent Mn(II) oxidation inhibition. Addition of Cu(II) (an effective superoxide scavenger) leads to a concentration dependent decrease in Mn oxide formation. We predict that due to the widespread production of extracellular superoxide within the fungal and likely bacterial kingdoms, biological superoxide may be an important contributor to the cycling of Mn, as well as other metals (e.g., Hg, Fe). Current and future explorations of the genes and proteins involved in superoxide production and Mn(II) oxidation will ideally lend insight into the physiological and biochemical basis for these processes.

  12. Differential production of superoxide by neuronal mitochondria

    Directory of Open Access Journals (Sweden)

    Levin Leonard A

    2008-01-01

    Full Text Available Abstract Background Mitochondrial DNA (mtDNA mutations, which are present in all mitochondria-containing cells, paradoxically cause tissue-specific disease. For example, Leber's hereditary optic neuropathy (LHON results from one of three point mutations mtDNA coding for complex I components, but is only manifested in retinal ganglion cells (RGCs, a central neuron contained within the retina. Given that RGCs use superoxide for intracellular signaling after axotomy, and that LHON mutations increase superoxide levels in non-RGC transmitochondrial cybrids, we hypothesized that RGCs regulate superoxide levels differently than other neuronal cells. To study this, we compared superoxide production and mitochondrial electron transport chain (METC components in isolated RGC mitochondria to mitochondria isolated from cerebral cortex and neuroblastoma SK-N-AS cells. Results In the presence of the complex I substrate glutamate/malate or the complex II substrate succinate, the rate of superoxide production in RGC-5 cells was significantly lower than cerebral or neuroblastoma cells. Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A. Immunoblotting and real-time quantitative PCR of METC components demonstrated different patterns of expression among the three different sources of neuronal mitochondria. Conclusion RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components. Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

  13. The Copper Metabolism MURR1 domain protein 1 (COMMD1 modulates the aggregation of misfolded protein species in a client-specific manner.

    Directory of Open Access Journals (Sweden)

    Willianne I M Vonk

    Full Text Available The Copper Metabolism MURR1 domain protein 1 (COMMD1 is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α. Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1 and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS, cause misfolding and aggregation of the mutant SOD1 (mSOD1 protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  14. The Copper Metabolism MURR1 domain protein 1 (COMMD1) modulates the aggregation of misfolded protein species in a client-specific manner.

    Science.gov (United States)

    Vonk, Willianne I M; Kakkar, Vaishali; Bartuzi, Paulina; Jaarsma, Dick; Berger, Ruud; Hofker, Marten H; Klomp, Leo W J; Wijmenga, Cisca; Kampinga, Harm H; van de Sluis, Bart

    2014-01-01

    The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson's disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington's disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.

  15. Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration.

    Science.gov (United States)

    Holzmeister, Christian; Gaupels, Frank; Geerlof, Arie; Sarioglu, Hakan; Sattler, Michael; Durner, Jörg; Lindermayr, Christian

    2015-02-01

    Despite the importance of superoxide dismutases (SODs) in the plant antioxidant defence system little is known about their regulation by post-translational modifications. Here, we investigated the in vitro effects of nitric oxide derivatives on the seven SOD isoforms of Arabidopsis thaliana. S-nitrosoglutathione, which causes S-nitrosylation of cysteine residues, did not influence SOD activities. By contrast, peroxynitrite inhibited the mitochondrial manganese SOD1 (MSD1), peroxisomal copper/zinc SOD3 (CSD3), and chloroplastic iron SOD3 (FSD3), but no other SODs. MSD1 was inhibited by up to 90% but CSD3 and FSD3 only by a maximum of 30%. Down-regulation of these SOD isoforms correlated with tyrosine (Tyr) nitration and both could be prevented by the peroxynitrite scavenger urate. Site-directed mutagenesis revealed that-amongst the 10 Tyr residues present in MSD1-Tyr63 was the main target responsible for nitration and inactivation of the enzyme. Tyr63 is located nearby the active centre at a distance of only 5.26 Å indicating that nitration could affect accessibility of the substrate binding pocket. The corresponding Tyr34 of human manganese SOD is also nitrated, suggesting that this might be an evolutionarily conserved mechanism for regulation of manganese SODs. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  16. Antioxidants Improve the Phenotypes of Dilated Cardiomyopathy and Muscle Fatigue in Mitochondrial Superoxide Dismutase-Deficient Mice

    Directory of Open Access Journals (Sweden)

    Takahiko Shimizu

    2013-01-01

    Full Text Available Redox imbalance elevates the reactive oxygen species (ROS level in cells and promotes age-related diseases. Superoxide dismutases (SODs are antioxidative enzymes that catalyze the degradation of ROS. There are three SOD isoforms: SOD1/CuZn-SOD, SOD2/Mn-SOD, and SOD3/EC-SOD. SOD2, which is localized in the mitochondria, is an essential enzyme required for mouse survival, and systemic knockout causes neonatal lethality in mice. To investigate the physiological function of SOD2 in adult mice, we generated a conditional Sod2 knockout mouse using a Cre-loxP system. When Sod2 was specifically deleted in the heart and muscle, all mice exhibited dilated cardiomyopathy (DCM and died by six months of age. On the other hand, when Sod2 was specifically deleted in the skeletal muscle, mice showed severe exercise disturbance without morphological abnormalities. These provide useful model of DCM and muscle fatigue. In this review, we summarize the impact of antioxidants, which were able to regulate mitochondrial superoxide generation and improve the phenotypes of the DCM and the muscle fatigue in mice.

  17. The fibrate decreases radiation sensitivity via peroxisome proliferator-activated receptor {alpha}-mediated superoxide dismutase induction in HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xianguang; An, Zhengzhe; Song, Hye Jin; Kim, Won Dong; Park, Woo Yoon [Chungbuk National University College of Medicine, Cheongju (Korea, Republic of); Jang, Seong Soon [The Catholic University of Korea College of Medicine, Seoul (Korea, Republic of); Yu, Jae Ran [Konkuk University College of Medicine, Chungju (Korea, Republic of)

    2012-06-15

    The fibrates are ligands for peroxisome proliferator-activated receptor (PPAR) {alpha} and used clinically as hypolipidemic drugs. The fibrates are known to cause peroxisome proliferation, enhance superoxide dismutase (SOD) expression and catalase activity. The antioxidant actions of the fibrates may modify radiation sensitivity. Here, we investigated the change of the radiation sensitivity in two cervix cancer cell lines in combination with fenofi brate (FF). Activity and protein expression of SOD were measured according to the concentration of FF. The mRNA expressions were measured by using real time reverse-transcription polymerase chain reaction. Combined cytotoxic effect of FF and radiation was measured by using clonogenic assay. In HeLa cells total SOD activity was increased with increasing FF doses up to 30 {mu}M. In the other hand, the catalase activity was increased a little. As with activity the protein expression of SOD1 and SOD2 was increased with increasing doses of FF. The mRNAs of SOD1, SOD2, PPAR{alpha} and PPAR{gamma} were increased with increasing doses of FF. The reactive oxygen species (ROS) produced by radiation was decreased by preincubation with FF. The surviving fractions (SF) by combining FF and radiation was higher than those of radiation alone. In Me180 cells SOD and catalase activity were not increased with FF. Also, the mRNAs of SOD1, SOD2, and PPAR{alpha} were not increased with FF. However, the mRNA of PPAR{gamma} was increased with FF. FF can reduce radiation sensitivity by ROS scavenging via SOD induction in HeLa. SOD induction by FF is related with PPAR{alpha}.

  18. Process for the preparation of calcium superoxide

    Science.gov (United States)

    Ballou, E. V.; Wood, P. C.; Wydeven, T. J.; Spitze, L. A. (Inventor)

    1978-01-01

    Calcium superoxide is prepared in high yields by spreading a quantity of calcium peroxide diperoxyhydrate on the surface of a container, positioning said container in a vacuum chamber on a support structure through which a coolant fluid can be circulated, partially evacuating said vacuum chamber, allowing the temperature of the diperoxyhydrate to reach the range of about 0 to about 40 C; maintaining the temperature selected for a period of time sufficient to complete the disproproriation of the diperoxyhydrate to calcium superoxide, calcium hydroxide, oxygen, and water; constantly and systematically removing the water as it is formed by sweeping the reacting material with a current of dry inert gas and/or by condensation of said water on a cold surface; backfilling the chamber with a dry inert gas; and finally, recovering the calcium superoxide produced.

  19. Effects of PEP-1 Superoxide Dismutase on B-cell lymphoma-2 in Rats after Cerebral Ischemia and Reperfusion%PEP-1超氧化物歧化酶对大鼠脑缺血再灌注后Bcl-2的影响

    Institute of Scientific and Technical Information of China (English)

    汪健; 周发明; 陈涛; 席刚明; 邓晓玲; 赵斌

    2011-01-01

    Objective: To observe the cell penetrating peptide-copper, zinc superoxide dismutase (PEP-1-SOD1) preconditioning on focal cerebral ischemia-reperfusion injury in the brain and its protective mechanism to improve. Methods: Suture technique in rats 6h after focal cerebral ischemia-reperfusion injury model, the neural behavior scores, and by HE histological changes of nerve cell damage,immune staining B-cell lymphoma gene-2 (B-cell lymphoma-2, Bcl-2) protein expression. Results:Saline control group (ischemia -reperfusion group or model group) neurological disorders were significantly higher than sham operation group (P<0.05), compared with model group, PEP-1-SOD1 pretreatment could reduce the neurological deficits score (P<0.05); light microscope, the sham operation group of neurons was normal, PEP-1-SOD1 pretreatrnent group and the ischemia reperfusion group had different levels of ischemia-reperfusion injury, PEP-1-SOD1 pretreatment group than in the ischemia-reperfusion group of light damage;sham group the expression of Bcl-2 protein in very weak, ischemia-reperfnsion group and PEP-1-SOD1 pretreatment group 6h after cerebral isehemia-reperfusion in the ischemic penumbra appears around the Bcl-2 protein expression, 24 h reached a peak, 48h expression started to decrease; compared with the sham group, PEP-1-SOD1 pretreatment group and the ischemia-reperfusion group Bcl-2 protein positive cells was significantly increased (P<0.05); compared with the ischemia-reperfusion group, PEP-1-SOD1 pretreatment group Bcl-2 protein positive cells was significantly increased (P <0.05). Conclusions: PEP-1-SOD1 on focal cerebral ischemia-reperfusion injury had a protective effect,PEP-1-SOD 1 could increase the expression of Bcl-2 proteins play a role in cerebral protection.%目的:观察细胞穿透肤-铜,锌超氧化物歧化酶(PEP-1-SOD1)预处理对大鼠局灶性脑缺血再灌注损伤的改善作用及其脑保护机制.方法:线栓法

  20. Superoxide anion production and superoxide dismutase and catalase activities in Coxiella burnetii.

    OpenAIRE

    Akporiaye, E T; Baca, O G

    1983-01-01

    Coxiella burnetii was examined for superoxide anion (O2-) production and superoxide dismutase and catalase activities. The organism generated O2- at pH 4.5 but not at pH 7.4. The rickettsia displayed superoxide dismutase activity distinguishable from that of the host cell (L-929 mouse fibroblast). Catalase activity was maximal at pH 7.0 and diminished at pH 4.5. These enzymes may account, in part, for the ability of this obligate intracellular parasite to survive within phagocytes.

  1. Magnetoreception through Cryptochrome may involve superoxide

    DEFF Research Database (Denmark)

    Solov'yov, Ilia; Schulten, Klaus

    2009-01-01

    pair-based reaction in the photoreceptor cryptochrome that reduces the protein's flavin group from its signaling state FADH$^bullet$ to the inactive state FADH$^–$ (which reacts to the likewise inactive FAD) by means of the superoxide radical, O2$^$. We argue that the spin dynamics in the suggested...

  2. Manganese superoxide dismutase and breast cancer recurrence

    DEFF Research Database (Denmark)

    Cronin-Fenton, Deirdre P; Christensen, Mariann; Lash, Timothy L

    2014-01-01

    BACKGROUND: Manganese superoxide dismutase (MnSOD) inhibits oxidative damage and cancer therapy effectiveness. A polymorphism in its encoding gene (SOD2: Val16Ala rs4880) may confer poorer breast cancer survival, but data are inconsistent. We examined the association of SOD2 genotype and breast...

  3. In vitro inhibition of superoxide anion production and superoxide dismutase activity by zinc in human spermatozoa.

    Science.gov (United States)

    Gavella, M; Lipovac, V; Vucić, M; Sverko, V

    1999-08-01

    The in vitro effect of zinc on superoxide anion (O2-) generation and on SOD-like activity in spermatozoa of infertile men was investigated. The formation of superoxide anion was stimulated by NADPH and the level of superoxide anion was measured by the reduction of ferricytochrome c. Both Percoll-isolated (n = 14) and washed spermatozoa (n = 14) exposed to 1 mmol/L zinc (60 min, 37 degrees C), released less (p zinc-untreated spermatozoa. These results implicate a possible role for zinc as a scavenger of excessive superoxide anions produced by defective spermatozoa in semen after ejaculation. Additionally, zinc was found to dose-dependently inhibit superoxide dismutase (SOD)-like activity of spermatozoa in vitro. The inhibition of SOD-like activity by an equal concentration of zinc (1 mmol/L) was less pronounced in oligospermic (p zinc to inhibit SOD-like activity may be relevant to the physiological function of spermatozoa in fertilization. The evidence that zinc may elicit an inhibition of both superoxide anion production and SOD-like activity in human spermatozoa, indicate the existence of novel, zinc-related mechanism(s) involved in the oxidative events occurring after ejaculation, with a possible modulatory effect on germ cell function.

  4. Role of extracellular superoxide dismutase in hypertension.

    Science.gov (United States)

    Gongora, Maria Carolina; Qin, Zhenyu; Laude, Karine; Kim, Ha Won; McCann, Louise; Folz, J Rodney; Dikalov, Sergey; Fukai, Tohru; Harrison, David G

    2006-09-01

    We previously found that angiotensin II-induced hypertension increases vascular extracellular superoxide dismutase (ecSOD), and proposed that this is a compensatory mechanism that blunts the hypertensive response and preserves endothelium-dependent vasodilatation. To test this hypothesis, we studied ecSOD-deficient mice. ecSOD(-/-) and C57Blk/6 mice had similar blood pressure at baseline; however, the hypertension caused by angiotensin II was greater in ecSOD(-/-) compared with wild-type mice (168 versus 147 mm Hg, respectively; P<0.01). In keeping with this, angiotensin II increased superoxide and reduced endothelium-dependent vasodilatation in small mesenteric arterioles to a greater extent in ecSOD(-/-) than in wild-type mice. In contrast to these findings in resistance vessels, angiotensin II paradoxically improved endothelium-dependent vasodilatation, reduced intracellular and extracellular superoxide, and increased NO production in aortas of ecSOD(-/-) mice. Whereas aortic expression of endothelial NO synthase, Cu/ZnSOD, and MnSOD were not altered in ecSOD(-/-) mice, the activity of Cu/ZnSOD was increased by 80% after angiotensin II infusion. This was associated with a concomitant increase in expression of the copper chaperone for Cu/ZnSOD in the aorta but not in the mesenteric arteries. Moreover, the angiotensin II-induced increase in aortic reduced nicotinamide-adenine dinucleotide phosphate oxidase activity was diminished in ecSOD(-/-) mice as compared with controls. Thus, during angiotensin II infusion, ecSOD reduces hypertension, minimizes vascular superoxide production, and preserves endothelial function in resistance arterioles. We also identified novel compensatory mechanisms involving upregulation of copper chaperone for Cu/ZnSOD, increased Cu/ZnSOD activity, and decreased reduced nicotinamide-adenine dinucleotide phosphate oxidase activity in larger vessels. These compensatory mechanisms preserve large vessel function when ecSOD is absent in

  5. Extracellular superoxide dismutase of boar seminal plasma.

    Science.gov (United States)

    Kowalowka, M; Wysocki, P; Fraser, L; Strzezek, J

    2008-08-01

    Superoxide dismutase (SOD) is an enzymatic component of the antioxidant defense system that protects spermatozoa by catalysing the dismutation of superoxide anions to hydrogen peroxide and oxygen. Age and season effects on SOD activity in the seminal plasma were measured in boars at the onset of 8 months through a 35-month period. It was found that age-related changes in SOD activity in the seminal plasma were markedly higher in boars less than 2 years of age. However, it appeared that SOD activity was established at the early sexual maturity age (8-12 months). There were variations in SOD activity throughout the season, being significantly higher in spring and autumn than in summer. A secretory extracellular form of SOD (EC-SOD) was purified to homogeneity (350-fold) from boar seminal plasma, using a three-step purification protocol (affinity chromatography followed by ion exchange and ceramic hydroxyapatite chromatography). The molecular properties and specificity of SOD (molecular mass, isoelectric point, optimum pH, thermostability and susceptibility to inhibitors) confirmed that the purified enzyme is an extracellular form of Cu/Zn-superoxide dismutase occurring in boar seminal plasma. The results of this study indicate that EC-SOD is an important antioxidant enzyme of boar seminal plasma, which plays an important physiological role in counteracting oxidative stress in spermatozoa.

  6. Scavenging of superoxide anions by lecithinized superoxide dismutase in HL-60 cells.

    Science.gov (United States)

    Ishihara, Tsutomu; Shibui, Misaki; Hoshi, Takaya; Mizushima, Tohru

    2016-01-01

    Superoxide dismutase covalently bound to four lecithin molecules (PC-SOD) has been found to have beneficial therapeutic effects in animal models of various diseases. However, the mechanism underlying these improved therapeutic effects has not yet been elucidated. It has previously been shown that PC-SOD localizes on the plasma membrane and in the lysosomes of cells. In this study, we evaluated the superoxide anion-scavenging activity of PC-SOD in HL-60 human promyelocytic leukemia cells. Compared to SOD, PC-SOD had only 17% scavenging activity in cell-free systems. Nevertheless, by analyzing enzyme activities in cell suspensions containing PC-SOD or SOD, PC-SOD and SOD showed almost equal activity for scavenging extracellular superoxide anions produced by HL-60 cells. Furthermore, the activity for scavenging extracellular superoxide anions increased with increased amount of PC-SOD on the plasma membrane. Moreover, PC-SOD exhibited no obvious inhibitory effect on the scavenging of intracellular superoxide anions. These results suggested that the association of PC-SOD with the plasma membrane plays a key role in its beneficial therapeutic effects. Thus, this finding may provide a rationale for selecting target diseases for PC-SOD treatment.

  7. Differential effects of superoxide dismutase and superoxide dismutase/catalase mimetics on human breast cancer cells.

    Science.gov (United States)

    Shah, Manisha H; Liu, Guei-Sheung; Thompson, Erik W; Dusting, Gregory J; Peshavariya, Hitesh M

    2015-04-01

    Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide (H2O2) have been implicated in development and progression of breast cancer. In the present study, we have evaluated the effects of the superoxide dismutase (SOD) mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 on superoxide and H2O2 formation as well as proliferation, adhesion, and migration of MCF-7 and MDA-MB-231 cells. Superoxide and H2O2 production was examined using dihydroethidium and Amplex red assays, respectively. Cell viability and adhesion were measured using a tetrazolium-based MTT assay. Cell proliferation was determined using trypan blue assay. Cell cycle progression was analyzed using flow cytometry. Clonal expansion of a single cell was performed using a colony formation assay. Cell migration was measured using transwell migration assay. Dual luciferase assay was used to determine NF-κB reporter activity. EUK 134 effectively reduced both superoxide and H2O2, whereas MnTmPyP removed superoxide but enhanced H2O2 formation. EUK 134 effectively attenuated viability, proliferation, clonal expansion, adhesion, and migration of MCF-7 and MDA-MB-231 cells. In contrast, MnTmPyP only reduced clonal expansion of MCF-7 and MDA-MB-231 cells but had no effect on adhesion and cell cycle progression. Tumor necrosis factor-alpha-induced NF-κB activity was reduced by EUK 134, whereas MnTmPyP enhanced this activity. These data indicate that the SOD mimetic MnTmPyP and the SOD/catalase mimetic EUK 134 exert differential effects on breast cancer cell growth. Inhibition of H2O2 signaling using EUK 134-like compound might be a promising approach to breast cancer therapy.

  8. The small heat shock protein B8 (HspB8) promotes autophagic removal of misfolded proteins involved in amyotrophic lateral sclerosis (ALS).

    Science.gov (United States)

    Crippa, Valeria; Sau, Daniela; Rusmini, Paola; Boncoraglio, Alessandra; Onesto, Elisa; Bolzoni, Elena; Galbiati, Mariarita; Fontana, Elena; Marino, Marianna; Carra, Serena; Bendotti, Caterina; De Biasi, Silvia; Poletti, Angelo

    2010-09-01

    Several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), are characterized by the presence of misfolded proteins, thought to trigger neurotoxicity. Some familial forms of ALS (fALS), clinically indistinguishable from sporadic ALS (sALS), are linked to superoxide dismutase 1 (SOD1) gene mutations. It has been shown that the mutant SOD1 misfolds, forms insoluble aggregates and impairs the proteasome. Using transgenic G93A-SOD1 mice, we found that spinal cord motor neurons, accumulating mutant SOD1 also over-express the small heat shock protein HspB8. Using motor neuronal fALS models, we demonstrated that HspB8 decreases aggregation and increases mutant SOD1 solubility and clearance, without affecting wild-type SOD1 turnover. Notably, HspB8 acts on mutant SOD1 even when the proteasome activity is specifically blocked. The pharmacological blockage of autophagy resulted in a dramatic increase of mutant SOD1 aggregates. Immunoprecipitation studies, performed during autophagic flux blockage, demonstrated that mutant SOD1 interacts with the HspB8/Bag3/Hsc70/CHIP multiheteromeric complex, known to selectively activate autophagic removal of misfolded proteins. Thus, HspB8 increases mutant SOD1 clearance via autophagy. Autophagy activation was also observed in lumbar spinal cord of transgenic G93A-SOD1 mice since several autophago-lysosomal structures were present in affected surviving motor neurons. Finally, we extended our observation to a different ALS model and demonstrated that HspB8 exerts similar effects on a truncated version of TDP-43, another protein involved both in fALS and in sALS. Overall, these results indicate that the pharmacological modulation of HspB8 expression in motor neurons may have important implications to unravel the molecular mechanisms involved both in fALS and in sALS.

  9. Subsarcolemmal and interfibrillar mitochondria display distinct superoxide production profiles.

    Science.gov (United States)

    Crochemore, C; Mekki, M; Corbière, C; Karoui, A; Noël, R; Vendeville, C; Vaugeois, J-M; Monteil, C

    2015-03-01

    Cardiac subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) subpopulations display distinct biochemical, morphological, and functional characteristics. Moreover, they appear to be differently influenced during cardiac pathologies or toxic injuries. Although mitochondrial reactive oxygen species seem to play a critical role in cardiac function and diseases, limited information exists about the superoxide production characteristics of these mitochondrial subpopulations. In this work, using direct measurement of superoxide by electron paramagnetic resonance, we showed that differences in superoxide production profiles were present between cardiac IFM and SSM, in terms of intensity and major sites of superoxide generation. In SSM incubated with glutamate plus malate as substrates, the total observed superoxide levels were significantly higher than those observed with IFM, with an important contribution of the NADH-oxidizing site of complex I (site If) and the quinol-oxidizing site of complex III (site IIIQ0). In both IFM and SSM, succinate leads to similar rates of total superoxide levels with a substantial role for contribution of reverse electron transfer. Finally, using two spin probes with different membrane permeabilities, our data on complex III showed direct intra- and extra-mitochondrial superoxide release whereas complex I- and II-dependent superoxide were exclusively released inside the mitochondria, confirming previous studies. Feasibility of this approach to measure intra- and extra-mitochondrial superoxide levels and to characterize distinct superoxide production profiles of cardiac IFM and SSM has been demonstrated.

  10. Combined effects of temperature and copper ion concentration on the superoxide dismutase activity inCrassostrea ariakensis

    Institute of Scientific and Technical Information of China (English)

    WANG Hui; YANG Hongshuai; LIU Jiahui; LI Yanhong; LIU Zhigang

    2016-01-01

    Superoxide dismutase (SOD) is a crucial antioxidant enzyme playing the first defense line in antioxidant pathways against reactive oxygen species in various organisms including marine invertebrates. There exist mainly two specific forms, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2), in eukaryotes. SODs are known to be concurrently modulated by a variety of environmental stressors. By using central composite experimental design and response surface method, the joint effects of water temperature (18–34°C) and copper ion concentration (0.1–1.5 mg/L) on the total SOD activity in the digestive gland ofCrassostrea ariakensis were studied. The results showed that the linear effect of temperature was highly significant (P0.05), while the quadratic effect of copper ion concentration was highly significant (P0.05); the effect of temperature was greater than that of copper ion concentration. The model equation of digestive gland SOD enzyme activity towards the two factors of interest was established, withR2 and predictiveR2 as high as 0.961 6 and 0.820 7, respectively, suggesting that the goodness-of-fit to experimental data be very satisfactory, and could be applied to prediction of digestive gland SOD activity in C. ariakensis under the conditions of the experiment. Our results would be conducive to addressing the health of aquatic animals and/or to detecting environmental problems by taking SOD as a potential bioindicator.

  11. Superoxide reduction by a superoxide reductase lacking the highly conserved lysine residue.

    Science.gov (United States)

    Pinto, Ana F; Romão, Célia V; Pinto, Liliana C; Huber, Harald; Saraiva, Lígia M; Todorovic, Smilja; Cabelli, Diane; Teixeira, Miguel

    2015-01-01

    Superoxide reductases (SORs) are the most recently identified superoxide detoxification systems, being found in microorganisms from the three domains of life. These enzymes are characterized by a catalytic mononuclear iron site, with one cysteine and four histidine ligands of the ferrous active form. A lysine residue in the -EKHVP- motif, located close to the active site, has been considered to be essential for the enzyme function, by contributing to the positive surface patch that attracts the superoxide anion and by controlling the chemistry of the catalytic mechanism through a hydrogen bond network. However, we show here that this residue is substituted by non-equivalent amino acids in several putative SORs from Archaea and unicellular Eukarya. In this work, we focus on mechanistic and spectroscopic studies of one of these less common enzymes, the SOR from the hyperthermophilic Crenarchaeon Ignicoccus hospitalis. We employ pulse radiolysis fast kinetics and spectroscopic approaches to study the wild-type enzyme (-E23T24HVP-), and two mutants, T24K and E23A, the later mimicking enzymes lacking both the lysine and glutamate (a ferric ion ligand) of the motif. The efficiency of the wild-type protein and mutants in reducing superoxide is comparable to other SORs, revealing the robustness of these enzymes to single mutations.

  12. Secondary analysis of publicly available data reveals superoxide and oxygen radical pathways are enriched for associations between type 2 diabetes and low-frequency variants.

    Science.gov (United States)

    Yazdanpanah, Mojgan; Chen, Chuhua; Graham, Jinko

    2013-11-01

    Genome-wide association studies explain at most 5%-10% of the heritable components of type 2 diabetes. Some of the "missing type 2 diabetes heritability" could be explained by low-frequency variants. We examined the associations between low-frequency variants and type 2 diabetes, using data from 2538 diabetic and 2977 nondiabetic subjects in the publicly available database of Genotypes and Phenotypes. We applied two approaches. First, we combined information from all low-frequency (1%-5%) variants at a locus in a gene-centric analysis of associations with diabetes. Next, we searched for gene ontology (GO) biological processes that were enriched for gene-centric associations, after correcting for multiple testing to control the false discovery rate (FDR). We found three GO biological processes that were significantly enriched for associations to diabetes: "response to superoxide" (FDR-adjusted p=2.7×10(-3)), "response to oxygen radical" (FDR-adjusted p=2.7×10(-3)), and "heart contraction" (FDR-adjusted p=2.6×10(-2)). There were three genes that contributed to "response to superoxide" and "oxygen radical" pathways, including the SOD1 gene. Gene-centric tests of association with low-frequency variants, followed by analysis to evaluate which biological pathways are enriched for these associations has the potential to recover, at least some proportion of, the "missing heritability" of type 2 diabetes.

  13. A mitochondrial superoxide theory for oxidative stress diseases and aging.

    Science.gov (United States)

    Indo, Hiroko P; Yen, Hsiu-Chuan; Nakanishi, Ikuo; Matsumoto, Ken-Ichiro; Tamura, Masato; Nagano, Yumiko; Matsui, Hirofumi; Gusev, Oleg; Cornette, Richard; Okuda, Takashi; Minamiyama, Yukiko; Ichikawa, Hiroshi; Suenaga, Shigeaki; Oki, Misato; Sato, Tsuyoshi; Ozawa, Toshihiko; Clair, Daret K St; Majima, Hideyuki J

    2015-01-01

    Fridovich identified CuZnSOD in 1969 and manganese superoxide dismutase (MnSOD) in 1973, and proposed "the Superoxide Theory," which postulates that superoxide (O2 (•-)) is the origin of most reactive oxygen species (ROS) and that it undergoes a chain reaction in a cell, playing a central role in the ROS producing system. Increased oxidative stress on an organism causes damage to cells, the smallest constituent unit of an organism, which can lead to the onset of a variety of chronic diseases, such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis and other neurological diseases caused by abnormalities in biological defenses or increased intracellular reactive oxygen levels. Oxidative stress also plays a role in aging. Antioxidant systems, including non-enzyme low-molecular-weight antioxidants (such as, vitamins A, C and E, polyphenols, glutathione, and coenzyme Q10) and antioxidant enzymes, fight against oxidants in cells. Superoxide is considered to be a major factor in oxidant toxicity, and mitochondrial MnSOD enzymes constitute an essential defense against superoxide. Mitochondria are the major source of superoxide. The reaction of superoxide generated from mitochondria with nitric oxide is faster than SOD catalyzed reaction, and produces peroxynitrite. Thus, based on research conducted after Fridovich's seminal studies, we now propose a modified superoxide theory; i.e., superoxide is the origin of reactive oxygen and nitrogen species (RONS) and, as such, causes various redox related diseases and aging.

  14. Host genetic variations in glutathione-S-transferases, superoxide dismutases and catalase genes influence susceptibility to malaria infection in an Indian population.

    Science.gov (United States)

    Fernandes, Rayzel C; Hasan, Marriyah; Gupta, Himanshu; Geetha, K; Rai, Padmalatha S; Hande, Manjunath H; D'Souza, Sydney C; Adhikari, Prabha; Brand, Angela; Satyamoorthy, Kapaettu

    2015-06-01

    Antioxidant enzymes can contribute to disease susceptibility or determine response to therapy in individuals with malaria. Genetic variations due to polymorphisms in host genes encoding antioxidant enzymes such as glutathione S-transferases-theta, mu, pi (GSTT, GSTM, GSTP), superoxide dismutases (SOD) and catalase (CAT), may therefore, influence inter-individual response to malaria pathology and propensity of infection caused by Plasmodium vivax (Pv) and Plasmodium falciparum (Pf). Therefore, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing, we investigated the association of deletions of GSTT1 and GSTM1, single nucleotide polymorphisms (SNPs) of GSTP1 (rs1695), SOD1 (rs2234694), SOD2 (rs4880, rs1141718), SOD3 (rs2536512) and CAT (rs1001179) in individuals infected with Pf (n = 100) and Pv (n = 100) against healthy controls (n = 150). Our data suggest a significant role for GSTM1 deletions in complicated Pv (p = 0.0007) malaria with ODDs ratio 3.8 [with 95 % confidence interval (CI) 1.9-7.4]. The results also indicated that polymorphisms present in GSTP1, SOD1 and CAT genes may be associated with malaria susceptibility (p < 0.05), whereas SOD3 polymorphism may play a role in malarial resistance (p < 0.05). In addition, we observed significant SNP-SNP interactions with synergistic genetic effects in SOD2, SOD3 and CAT genes for Pv and in SOD2 and SOD3 genes for Pf. In conclusion, our results provide convincing evidence for a relationship between polymorphisms in host antioxidant enzymes and susceptibility to malaria infection.

  15. Revisiting the reactions of superoxide with glutathione and other thiols.

    Science.gov (United States)

    Winterbourn, Christine C

    2016-04-01

    The reaction between GSH and superoxide has long been of interest in the free radical biology. Early studies were confusing, as some reports suggested that the reaction could be a major pathway for superoxide removal whereas others questioned whether it happened at all. Further research by several investigators, including Helmut Sies, was required to clarify this complex reaction. We now know that superoxide does react with GSH, but the reaction is relatively slow and occurs mostly by a chain reaction that consumes oxygen and regenerates superoxide. Most of the GSH is converted to GSSG, with a small amount of sulfonic acid. As shown by Sies and colleagues, singlet oxygen is a by-product. Although removal of superoxide by GSH may be a minor pathway, GSH and superoxide have a strong physiological connection. GSH is an efficient free radical scavenger, and when it does so, thiyl radicals are generated. These further react to generate superoxide. Therefore, radical scavenging by GSH and other thiols is a source of superoxide and hydrogen peroxide, and to be an antioxidant pathway, there must be efficient removal of these species.

  16. Titanium dioxide nanoparticles enhance production of superoxide anion and alter the antioxidant system in human osteoblast cells

    Directory of Open Access Journals (Sweden)

    Niska K

    2015-02-01

    Full Text Available Karolina Niska,1 Katarzyna Pyszka,1 Cecylia Tukaj,2 Michal Wozniak,1 Marek Witold Radomski,3–5 Iwona Inkielewicz-Stepniak1 1Department of Medical Chemistry, 2Department of Electron Microscopy, Medical University of Gdansk, Gdansk, Poland; 3School of Pharmacy and Pharmaceutical Sciences, Trinity Biomedical Sciences Institute, The University of Dublin Trinity College, Dublin, Ireland; 4Kardio-Med Silesia, 5Silesian Medical University, Zabrze, Poland Abstract: Titanium dioxide (TiO2 nanoparticles (NPs are manufactured worldwide for a variety of engineering and bioengineering applications. TiO2NPs are frequently used as a material for orthopedic implants. However, to the best of our knowledge, the biocompatibility of TiO2NPs and their effects on osteoblast cells, which are responsible for the growth and remodeling of the human skeleton, have not been thoroughly investigated. In the research reported here, we studied the effects of exposing hFOB 1.19 human osteoblast cells to TiO2NPs (5–15 nm for 24 and 48 hours. Cell viability, alkaline phosphatase (ALP activity, cellular uptake of NPs, cell morphology, superoxide anion (O2•- generation, superoxide dismutase (SOD activity and protein level, sirtuin 3 (SIR3 protein level, correlation between manganese (Mn SOD and SIR, total antioxidant capacity, and malondialdehyde were measured following exposure of hFOB 1.19 cells to TiO2NPs. Exposure of hFOB 1.19 cells to TiO2NPs resulted in: (1 cellular uptake of NPs; (2 increased cytotoxicity and cell death in a time- and concentration-dependent manner; (3 ultrastructure changes; (4 decreased SOD and ALP activity; (5 decreased protein levels of SOD1, SOD2, and SIR3; (6 decreased total antioxidant capacity; (7 increased O2•- generation; and (8 enhanced lipid peroxidation (malondialdehyde level. The linear relationship between the protein level of MnSOD and SIR3 and between O2•- content and SIR3 protein level was observed. Importantly, the cytotoxic

  17. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    Science.gov (United States)

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  18. Manganese Superoxide Dismutase: Guardian of the Powerhouse

    Directory of Open Access Journals (Sweden)

    Daret K. St. Clair

    2011-10-01

    Full Text Available The mitochondrion is vital for many metabolic pathways in the cell, contributing all or important constituent enzymes for diverse functions such as β-oxidation of fatty acids, the urea cycle, the citric acid cycle, and ATP synthesis. The mitochondrion is also a major site of reactive oxygen species (ROS production in the cell. Aberrant production of mitochondrial ROS can have dramatic effects on cellular function, in part, due to oxidative modification of key metabolic proteins localized in the mitochondrion. The cell is equipped with myriad antioxidant enzyme systems to combat deleterious ROS production in mitochondria, with the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD acting as the chief ROS scavenging enzyme in the cell. Factors that affect the expression and/or the activity of MnSOD, resulting in diminished antioxidant capacity of the cell, can have extraordinary consequences on the overall health of the cell by altering mitochondrial metabolic function, leading to the development and progression of numerous diseases. A better understanding of the mechanisms by which MnSOD protects cells from the harmful effects of overproduction of ROS, in particular, the effects of ROS on mitochondrial metabolic enzymes, may contribute to the development of novel treatments for various diseases in which ROS are an important component.

  19. Study of superoxide dismutase's expression in the colon produced by azoxymethane and inositol hexaphosfate's paper, in mice Estudo da expressão de superóxido dismutase no cólon produzida por azoximetano e o papel do inositol hexafosfato, em ratos

    Directory of Open Access Journals (Sweden)

    Eva Glória Abrão Siufi do Amaral

    2006-01-01

    Full Text Available PURPOSE: To investigate the expression of superoxide dismutase (SOD, with use of antioxidant inositol hexaphosfate, in the presence of the carcinogen azoxymethane, in FCA of colon rats. METHODS: Wistar rats (n=48 were distributed in four groups of 12 mice. Divided in control (n=12; with azoxymethane administration AOM (n=12; administration of IP6 (n=12 and with administration of IP6/AOM (n=12. The subcutaneous administration of azoxymethane happened in the week 3 and 4 of the experiment, in dose 20mg/Kg, weekly; and administration of IP6 to 1% in water of drinking for 6 weeks in the group 3 and 4. The identification of the expression SOD-1 was accomplished through the quantification imunohistochemistry by the image processing attended by computer in crypts and focus of aberrant crypts in right colon. RESULTS: The group control presented expression of SOD1, on average 16,0%; group AOM, 26,7%; group IP6, 16,9%; group IP6/AOM, 20,9%. Variance analysis among the groups, was calculated 0,0078. CONCLUSION: The azoxymethane increase expression SOD1, while inositol hexaphosphate decreases in a significant way the expression of SOD1 promoted by the administration of the carcinogen azoxymethane.OBJETIVO: Investigar a expressão de superóxido dismutase, com uso de antioxidante inositol hexafosfato, na presença do carcinógeno azoximetano em FCA de cólon de ratos. MÉTODOS: Quarenta e oito ratos Wistar, distribuídos em 4 grupos, divididos em controle (n=12; com administração de azoximetano AOM (n=12; administração de IP6 (n=12 e com administração de IP6/AOM (n=12. A administração subcutânea de azoximetano aconteceu na semana 3 e 4 do experimento, em dose 20mg/Kg, semanal; e administração de IP6 a 1% em água de beber durante 6 semanas no grupo 3 e 4. A identificação da expressão SOD1 foi realizada através da quantificação imunohistoquimíca pelo processamento de imagem assistida por computador em criptas e focos de cripta aberrante em c

  20. Corticosteroids increase superoxide anion production by rat liver microsomes.

    Science.gov (United States)

    Nelson, D H; Ruhmann-Wennhold, A

    1975-01-01

    Superoxide anion production by liver microsomes from intact, adrenalectomized, and cortisoltreated adrenalectomized rats has been determined. The amount formed was roughly proportionate to the amount of cortisol given, and a similar response was seen in the activity of NADPH-cytochrome c reductase. The amount of measurable superoxide anion was markedly reduced by the addition of superoxide dismutase. The increased production of this potent free radical with cortisol therapy suggests that its formation may contribute to some of the harmful effects of corticosteroids given in more than physiologic amounts. PMID:239969

  1. Mitochondrial Flashes: Dump Superoxide and Dance with Protons Now.

    Science.gov (United States)

    Demaurex, Nicolas; Schwarzländer, Markus

    2016-09-20

    Transient changes in the physiology of individual mitochondria have recently drawn much interest. The use of a circular permuted yellow fluorescent protein (cpYFP) to monitor mitochondrial flashes and their interpretation as superoxide bursts has added confusion, however. Reviewing mitochondrial flashes in this Forum, Wang et al. again deem cpYFP to be a specific and reversible superoxide indicator, dismissing evidence that purified cpYFP is insensitive to superoxide. This interpretation lacks reproducible evidence and conflicts with the parsimony principle. We offer a constructive, transparent pathway to reach definitive clarification of contradictory reports. Antioxid. Redox Signal. 25, 550-551.

  2. Sirt1在肌萎缩侧索硬化模型小鼠脊髓内表达水平的研究%Expression of Sirt1 in spinal cord of SOD1G93A transgenic mice——an in vivo model of amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    宋林; 乐卫东

    2013-01-01

    Objective To study the expression of Sirt1,which is a mammalian homolog of yeast nicotinamide adenine dinucleotide-dependent deacetylase silent information regulator 2 (Sir2),in the spinal cord of SOD1G93A transgenic mice——a model of amyotrophic lateral sclerosis (ALS).Methods To explore the level of Sirt1 in different age groups of SOD1G93A transgenic mice.Twelve transgenic mice were divided into four groups:immature stage group (aged 30 days),presymptomatic stage group (aged 60 days),early-symptomatic stage group (aged 90 days) and late-symptomatic stage group (aged 120 days) (n=3 in each group).They were sacrificed to collect the spinal cord tissues for real-time PCR,Western blot and immunohistochemistry (IHC) assay.Twelve age-matched wild-type mice were used as control.Results Increased expression of Sirt1 was found in the spinal cord of symptomatic SOD1G93A transgenic mice,and no significant difference of Sirt1 expression was found between immature and pre-symptomatic mice and their wild-type controls.Conclusions Sirt1 level was increased as a protective response to the neurodegenerative process.These findings suggest a role of Sirt1 in the motor function of ALS,however,the mechanism and functional implication of increased Sirt1 need to be further elucidated.%目的:研究各年龄段肌萎缩侧索硬化(ALS)模型小鼠脊髓中Sirt1 mRNA和蛋白的表达水平.方法:以SOD1G93A转基因小鼠作为ALS的模型小鼠,以同龄野生型小鼠作为对照,分别于幼年期(30日龄)、疾病前期(60日龄)、疾病初期(90日龄)和疾病晚期(120日龄)取小鼠的脊髓组织,用实时定量PCR反应、蛋白质印迹(Western blot)和免疫组织化学方法检测Sirt1的表达水平.结果:SOD1G93A小鼠在疾病发生前其脊髓内Sirt1 mRNA和蛋白的表达水平与野生型小鼠没有显著差异(P>0.05),而在疾病初期分别增加了82.20%和40.39%(均P<0.01),至疾病晚期分别增加了195.33%和70.09%(均P<0.001).结论:SOD

  3. An altered antioxidant balance occurs in Down syndrome fetal organs: implications for the "gene dosage effect" hypothesis.

    Science.gov (United States)

    de Haan, J B; Susil, B; Pritchard, M; Kola, I

    2003-01-01

    Down syndrome (DS) is the congenital birth defect responsible for the greatest number of individuals with mental retardation. It arises due to trisomy of human chromosome 21 (HSA21) or part thereof. To date there have been limited studies of HSA21 gene expression in trisomy 21 conceptuses. In this study we investigate the expression of the HSA21 antioxidant gene, Cu/Zn-superoxide dismutase-1 (SOD1) in various organs of control and DS aborted conceptuses. We show that SOD1 mRNA levels are elevated in DS brain, lung, heart and thymus. DS livers show decreased SOD1 mRNA expression compared with controls. Since non-HSA21 antioxidant genes are reported to be concomitantly upregulated in certain DS tissues, we examined the expression of glutathione peroxidase-1 (GPX1) in control and DS fetal organs. Interestingly, GPX1 expression was unchanged in the majority of DS organs and decreased in DS livers. We examined the SOD1 to GPX1 mRNA ratio in individual organs, as both enzymes form part of the body's defense against oxidative stress, and because a disproportionate increase of SOD1 to GPX1 results in noxious hydroxyl radical damage. All organs investigated show an approximately 2-fold increase in the SOD1 to GPX1 mRNA ratio. We propose that it is the altered antioxidant ratio that contributes to certain aspects of the DS phenotype.

  4. Requirements for superoxide-dependent tyrosine hydroperoxide formation in peptides

    DEFF Research Database (Denmark)

    Winterbourn, Christine C; Parsons-Mair, Helena N; Gebicki, Silvia

    2004-01-01

    Superoxide reacts rapidly with other radicals, but these reactions have received little attention in the context of oxidative stress. For tyrosyl radicals, reaction with superoxide is 3-fold faster than dimerization, and forms the addition product tyrosine hydroperoxide. We have explored structural...... requirements for hydroperoxide formation using tyrosine analogues and di- and tri-peptides. Superoxide and phenoxyl radicals were generated using xanthine oxidase, peroxidase and the respective tyrosine derivative, or by gamma-radiation. Peroxides were measured using FeSO4/Xylenol Orange. Tyrosine and tyramine...... losses, indicated that, in the absence of a free amino group, reaction with superoxide resulted primarily in restitution of the parent compound. With dipeptides, hydroperoxides were formed only on N-terminal tyrosines. However, adjacent lysines promoted hydroperoxide formation, as did addition of free...

  5. Water stress induces overexpression of superoxide dismutases that ...

    African Journals Online (AJOL)

    SERVER

    2007-09-05

    Sep 5, 2007 ... aim of this study was to determine the effect of water stress on superoxide ... In the same time, photosynthesis characteristics were deter- ... tion rate per reaction centre. ..... Factors affecting the enhancement of oxidative stress.

  6. Oxidative stress and superoxide dismutase activity in brain of rats ...

    African Journals Online (AJOL)

    JTEkanem

    effect of superoxide dismutase (SOD) activity in brain homogenates of Wistar rats. Oxidative stress measured as ... SOD is an important enzyme family in living cells for maintaining ..... one unit of activity with oxidation rate of organic substrate in.

  7. Male-specific differences in proliferation, neurogenesis, and sensitivity to oxidative stress in neural progenitor cells derived from a rat model of ALS.

    Directory of Open Access Journals (Sweden)

    Ruojia Li

    Full Text Available Amyotrophic Lateral Sclerosis (ALS is a fatal neurodegenerative disease characterized by progressive motor dysfunction and the loss of large motor neurons in the spinal cord and brain stem. A clear genetic link to point mutations in the superoxide dismutase 1 (SOD1 gene has been shown in a small group of familial ALS patients. The exact etiology of ALS is still uncertain, but males have consistently been shown to be at a higher risk for the disease than females. Here we present male-specific effects of the mutant SOD1 transgene on proliferation, neurogenesis, and sensitivity to oxidative stress in rat neural progenitor cells (rNPCs. E14 pups were bred using SOD1(G93A transgenic male rats and wild-type female rats. The spinal cord and cortex tissues were collected, genotyped by PCR using primers for the SOD1(G93A transgene or the male-specific Sry gene, and cultured as neurospheres. The number of dividing cells was higher in male rNPCs compared to female rNPCs. However, SOD1(G93A over-expression significantly reduced cell proliferation in male cells but not female cells. Similarly, male rNPCs produced more neurons compared to female rNPCs, but SOD1(G93A over-expression significantly reduced the number of neurons produced in male cells. Finally we asked whether sex and SOD1(G93A transgenes affected sensitivity to oxidative stress. There was no sex-based difference in cell viability after treatment with hydrogen peroxide or 3-morpholinosydnonimine, a free radical-generating agent. However, increased cytotoxicity by SOD1(G93A over-expression occurred, especially in male rNPCs. These results provide essential information on how the mutant SOD1 gene and sexual dimorphism are involved in ALS disease progression.

  8. Suppressors of superoxide production from mitochondrial complex III.

    Science.gov (United States)

    Orr, Adam L; Vargas, Leonardo; Turk, Carolina N; Baaten, Janine E; Matzen, Jason T; Dardov, Victoria J; Attle, Stephen J; Li, Jing; Quackenbush, Douglas C; Goncalves, Renata L S; Perevoshchikova, Irina V; Petrassi, H Michael; Meeusen, Shelly L; Ainscow, Edward K; Brand, Martin D

    2015-11-01

    Mitochondrial electron transport drives ATP synthesis but also generates reactive oxygen species, which are both cellular signals and damaging oxidants. Superoxide production by respiratory complex III is implicated in diverse signaling events and pathologies, but its role remains controversial. Using high-throughput screening, we identified compounds that selectively eliminate superoxide production by complex III without altering oxidative phosphorylation; they modulate retrograde signaling including cellular responses to hypoxic and oxidative stress.

  9. Superoxide dismutase phenotypes in duodenal ulcers: A genetic marker?

    Directory of Open Access Journals (Sweden)

    Sulekha S

    2006-01-01

    Full Text Available Background:Cu-Zn superoxide dismutases are antioxidative defensive enzymes that catalyze the reduction of superoxide anions to hydrogen peroxide. Aim:The study focuses on the association of electromorph of superoxide dismutase with duodenal ulcers, which result due to an imbalance between aggressive and defensive factors. Materials and Methods:Endoscopically confirmed 210 duodenal ulcer patients and 185 healthy individuals for comparative analysis were considered for the present study. Phenotyping of superoxide dismutase was carried out by subjecting the RBC membranes to polyacrylamide gel electrophoresis, using appropriate staining protocols. Results:Statistical analysis of SOD phenotypes revealed a significant increase of SOD AFNx012 allele and Superoxide dismutases (SOD 2-2 phenotype in duodenal ulcer group. Among these individuals, a predominance of Helicobacter pylori infection was observed. The increased preponderance of homozygotes can be explained on the basis of reduced and altered enzyme activity, which may lead to disturbance in homeostasis of antioxidant/oxidant culminating in high lipid peroxidative gastric mucosal tissue damage and ulceration. No variation in the distribution of SOD phenotypes with respect to Helicobacter pylori indicates the role of Mn-SOD rather than Cu-Zn SOD in the Helicobacter pylori infected cases as reported earlier. Conclusions:Superoxide dismutase as a genetic marker / gene modifier, encoding for an antioxidant enzyme in maintaining tissue homeostasis of the gastric mucosa is discussed.

  10. Clastogenic Factors as Potential Biomarkers of Increased Superoxide Production

    Directory of Open Access Journals (Sweden)

    Ingrid Emerit

    2007-01-01

    Full Text Available The formation of clastogenic factors (CF and their damaging effects are mediated by superoxide, since superoxide dismutase is regularly protective. CF are produced via superoxide and stimulate the production of superoxide by monocytes and neutrophils. This results in a selfsustaining and longlasting process of clastogenesis, which may exceed the DNA repair system and ultimately lead to cancer (Emerit, 1994. An increased cancer risk is indeed observed in conditions accompanied by CF formation. These include irradiated persons, patients with chronic inflammatory diseases, HIV-infected persons and the chromosomal breakage syndromes ataxia telangiectasia, Bloom’s syndrome and Fanconi’s anemia. Biochemical analysis has identifi ed lipid peroxidation products, arachidonic acid metabolites, nucleotides of inosine and cytokines, in particular tumor necrosis factor alpha, as the clastogenic and also superoxide stimulating components of CF. Due to their chromosome damaging effects, these oxidants can be detected with classical cytogenetic techniques. Their synergistic action renders the CF-test particularly sensitive for the detection of a pro-oxidant state. Correlations were observed between CF and other biomarkers of oxidative stress such as decreases in total plasma thiols or increases in TBARS or chemiluminescence. Correlations between CF and disease activity, between CF and radiation exposure, suggest the study of CF for monitoring these conditions. CF may also be useful as biochemical markers and intermediate endpoints for the evaluation of promising antioxidant drugs. CF formation represents a link between chronic inflammation and carcinogenesis. Prophylactic use of superoxide scavengers as anticarcinogens is therefore suggested.

  11. Production of superoxide and activity of superoxide dismutase in rabbit epididymal spermatozoa.

    Science.gov (United States)

    Holland, M K; Alvarez, J G; Storey, B T

    1982-12-01

    Mature rabbit spermatozoa from the cauda epididymidis suspended in potassium Tris phosphate buffer at 24 degrees C produced O2.-, as measured by reduction of acetylated ferricytochrome c, with an intrinsic rate of 0.20 nmol/min per 10(8) cells. This rate increased to 1.80 nmol/min per 10(8) cells in the presence of 10 mM cyanide. These spermatozoa contain 2.8 units per 10(8) cells of superoxide dismutase activity, 95% of which is sensitive, and 5% of which is insensitive, to cyanide inhibition. These activities correspond to the cytosolic Cu-Zn form and the mitochondrial Mn form of the dismutase, respectively. Only the cyanide-sensitive form is released from the sperm on hypo-osmotic treatment or sonication. Hypo-osmotically treated rabbit epididymal spermatozoa produced O2.- with an intrinsic rate of 0.24 nmol/min per 10(8) cells, which increased to 0.58 nmol/min per 10(8) cells in the presence of 10 mM cyanide. Both intact and hypo-osmotically treated cells react with O2.- in a second order reaction as inferred from the hyperbolic dependence on cell concentration of O2.- production rate in both the absence and presence of cyanide. The second order rate constant for this reaction with intact cells, kS, was calculated to be 22.9 X 10(-8) (cells/ml)-1 min-1 in its absence. For hypo-osmotically treated cells, the values of kS were 10.8 X 10(-8) (cells/ml)-1 min-1 and 8.2 X 10(-8) (cells/ml) -1 min-1, respectively. Since hypo-osmotically treated cells have lost much of their plasma membrane, the lower value of kS for the treated cells implies that this membrane is one site of reaction of O2.- with the cells. The increase in kS in the presence of cyanide, which inhibits superoxide dismutase and so increases O2.- production, suggests that the cells become more reactive with O2.- as its production rate increase, as would be expected for the occurrence of radical chain oxidation. This in turn suggests that superoxide dismutase plays a major role in protecting rabbit sperm

  12. Skeletal muscle contractions induce acute changes in cytosolic superoxide, but slower responses in mitochondrial superoxide and cellular hydrogen peroxide.

    Science.gov (United States)

    Pearson, Timothy; Kabayo, Tabitha; Ng, Rainer; Chamberlain, Jeffrey; McArdle, Anne; Jackson, Malcolm J

    2014-01-01

    Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

  13. Superoxide dismutase analog (TEMPOL: 4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) treatment restores erectile function in diabetes-induced impotence

    Science.gov (United States)

    Kawakami, Toshifumi; Urakami, Shinji; Hirata, Hiroshi; Tanaka, Yuichiro; Nakajima, Koichi; Enokida, Hideki; Shiina, Hiroaki; Ogishima, Tatsuya; Tokizane, Takashi; Kawamoto, Ken; Miura, Kazukiyo; Ishii, Nobuhisa; Dahiya, Rajvir

    2014-01-01

    We hypothesized that administration of the superoxide dismutase (SOD) mimetic Tempol (4-hydroxy-2, 2, 6, 6-tetramethylpiperidine 1-oxyl) may reverse diabetes induced ED(erectile dysfunction). To test this hypothesis, ROS related genes (SOD1, SOD2, GPx1, CAT, NOS2, NOS3), erectile functional studies, and immunohistochemical analysis were performed in diabetic rats treated with or without Tempol. Thirty Sprague-Dawley (3–4 months old) rats were divided into 3 groups (n=10 each), 20 with diabetes (diabetic control and Tempol treatment) and 10 healthy controls. Twelve weeks after induction of diabetes by streptozotocin and Tempol treatment, all groups underwent in vivo cavernous nerve stimulation. Rat crura were harvested and expression of antioxidative defense enzymes examined by semi-quantitative RT-PCR. To confirm the RT-PCR results, we performed immunohistochemistry (IHC) for catalase (CAT) and iNOS (NOS2). Nitration of tyrosine groups in proteins was also examined by IHC. Mean intracavernous pressure in the diabetic group was significantly lower than in healthy controls (p<0.001) and was reversed by Tempol treatment (p<0.0108). NOS2 protein expression was significantly increased in diabetic animals compared to healthy controls and Tempol restored NOS2 protein level. Nitrotyrosine was also higher in diabetic animals and though Tempol treatment decreased its formation, it remained higher than that found in healthy controls. This study suggests that Tempol treatment increased erectile function through modulating oxidative stress related genes in diabetic rats. This is the first report about the relationship between diabetes induced erectile dysfunction and oxidative stress, and anti-oxidative therapy using the superoxide dismutase mimetic, Tempol to restore erectile function. PMID:19554009

  14. Superoxide production in aprotic interior of chloroplast thylakoids.

    Science.gov (United States)

    Takahashi, M; Asada, K

    1988-12-01

    The site of superoxide production in spinach thylakoids was found to be the aprotic interior of the thylakoid membranes near the P700 chlorophyll a protein at the reaction center of photosystem I complexes. This conclusion was drawn from the following findings. (i) Cytochrome c reduction by illuminated thylakoids, which was confirmed to be superoxide dependent by the failure of this reaction to occur in anaerobiosis, was completely inhibited by a dibutyl catechol, but partially inhibited by a hydrophilic disulfonated derivative. (ii) P700 chlorophyll a proteins were preferentially iodinated by lactoperoxidase by the use of hydrogen peroxide that was derived from the disproportionation of superoxides in illuminated thylakoids. (iii) Hydrogen peroxide production and oxygen uptake were induced by ammonium chloride, a proton conductor that can permeate through thylakoid membranes, but whole superoxide in the bulk solution was oxidized back to molecular oxygen by cytochrome c. The effective concentration of ammonium chloride decreased to one-sixtieth of the original, when an ammonium ion ionophore, nonactin, was added. Thus, the weak acid allowed superoxide to yield hydrogen peroxide disproportionately in the thylakoid membrane interior.

  15. Familial Amyotrophic Lateral Sclerosis-associated Mutations Decrease the Thermal Stability of Distinctly Metallated Species of Human Copper/Zinc Superoxide Dismutase

    National Research Council Canada - National Science Library

    Jorge A. Rodriguez; Joan S. Valentine; Daryl K. Eggers; James A. Roe; Ashutosh Tiwari; Robert H. Brown, Jr; Lawrence J. Hayward

    2002-01-01

    ...) associated with familial amyotrophic lateral sclerosis (FALS). Multiple endothermic unfolding transitions were observed by differential scanning calorimetry for partially metallated SOD1 enzymes isolated from a baculovirus system...

  16. Bleaching of the red anthocyanin induced by superoxide radical.

    Science.gov (United States)

    Yamasaki, H; Uefuji, H; Sakihama, Y

    1996-08-01

    Red anthocyanin prepared from petals of Hibiscus rosa-sinensis L. was photobleached in the EDTA-riboflavin system. The rate of bleaching monitored at 565 nm depended on the light intensity and EDTA concentrations. Anaerobic conditions and/or addition of superoxide dismutase prevented the bleaching of anthocyanin, whereas mannitol and catalase did not. A similar bleaching was observed under dark conditions in the xanthine-xanthine oxidase system. The results indicate that anthocyanin is bleached by the nonenzymatic reaction with the superoxide radical and suggest that the pigment can function as an antioxidant. The antioxidative efficiency of cyanidin to superoxide was 10-fold higher than that of cyanidin-3-sophoroside as a Hibiscus anthocyanin.

  17. Ferric human neuroglobin scavenges superoxide to form oxy adduct.

    Science.gov (United States)

    Yamashita, Taku; Hafsi, Leila; Masuda, Eri; Tsujino, Hirofumi; Uno, Tadayuki

    2014-01-01

    Neuroglobin (Ngb) is the third member of the vertebrate globin family, and the structure was solved as a typical globin fold with a b-type heme. Although it has been proposed that Ngb could be involved in neuroprotection against oxidative stress, the protective mechanism has not been fully identified yet. In order to clarify functions under hypoxic condition, in this study, we focused on the scavenger activity of human Ngb (hNgb) against superoxide. The activity of hNgb for superoxide was evaluated to be 7.4 µM for IC50, the half maximal inhibitory concentration. The result indicates that hNgb can be an anti-oxidant, and the value was almost the same as that of ascorbic acid. In addition, we characterized oxidation states of a heme iron in superoxide-treated hNgb with spectroscopic measurements. Superoxide-treated hNgb in the ferric form was readily converted to the oxygenated ferrous form, and the result suggested that ferric hNgb could scavenge superoxide by change of an oxidation state in a heme iron. Moreover, mutational experiments were performed, and the each variant mutated at 46 and 55 positions suggested a disulfide bond between Cys46 and Cys55 could be essential to be sensors for oxidative stress with the direct binding of superoxide. As a consequence, we concluded that redox changes of the heme iron and the disulfide bond could regulate neuroprotective functions of hNgb, and it suggests that hNgb can afford protection against hypoxic and ischemic stress in the brain.

  18. Constraints on superoxide mediated formation of manganese oxides

    Directory of Open Access Journals (Sweden)

    Deric R. Learman

    2013-09-01

    Full Text Available Manganese (Mn oxides are among the most reactive sorbents and oxidants within the environment, where they play a central role in the cycling of nutrients, metals, and carbon. Recent discoveries have identified superoxide (O2- (both of biogenic and abiogenic origin as an effective oxidant of Mn(II leading to the formation of Mn oxides. Here we examined the conditions under which abiotically produced superoxide led to oxidative precipitation of Mn and the solid-phases produced. Oxidized Mn, as both aqueous Mn(III and Mn(III/IV oxides, was only observed in the presence of active catalase, indicating that hydrogen peroxide, a product of the reaction of O2- with Mn(II, inhibits the oxidation process presumably through the reduction of Mn(III. Citrate and pyrophosphate increased the yield of oxidized Mn but decreased the amount of Mn oxide produced via formation of Mn(III-ligand complexes. While complexing ligands played a role in stabilizing Mn(III, they did not eliminate the inhibition of net Mn(III formation by H2O2. The Mn oxides precipitated were highly disordered colloidal hexagonal birnessite, similar to those produced by biotically generated superoxide. Yet, in contrast to the large particulate Mn oxides formed by biogenic superoxide, abiotic Mn oxides did not ripen to larger, more crystalline phases. This suggests that the deposition of crystalline Mn oxides within the environment requires a biological, or at least organic, influence. This work provides the first direct evidence that, under conditions relevant to natural waters, oxidation of Mn(II by superoxide can occur and lead to formation of Mn oxides. For organisms that oxidize Mn(II by producing superoxide, these findings may also point to other microbially mediated processes, in particular enzymatic hydrogen peroxide degradation and/or production of organic ligand metabolites, that allow for Mn oxide formation.

  19. Adenosine monophosphate-activated protein kinase activation enhances embryonic neural stem cell apoptosis in a mouse model of amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    Yanling Sui; Zichun Zhao; Rong Liu; Bin Cai; Dongsheng Fan

    2014-01-01

    Alterations in embryonic neural stem cells play crucial roles in the pathogenesis of amyotrophic lateral sclerosis. We hypothesized that embryonic neural stem cells from SOD1G93A individuals might be more susceptible to oxidative injury, resulting in a propensity for neurodegeneration at later stages. In this study, embryonic neural stem cells obtained from human superoxide dis-mutase 1 mutant (SOD1G93A) and wild-type (SOD1WT) mouse models were exposed to H2O2. We assayed cell viability with mitochondrial succinic dehydrogenase colorimetric reagent, and measured cell apoptosis by lfow cytometry. Moreover, we evaluated the expression of the adenos-ine monophosphate-activated protein kinase (AMPK)α-subunit, paired box 3 (Pax3) protein, and p53 in western blot analyses. Compared with SOD1WT cells, SOD1G93A embryonic neural stem cells were more likely to undergo H2O2-induced apoptosis. Phosphorylation of AMPKαin SOD1G93A cells was higher than that in SOD1WT cells. Pax3 expression was inversely correlated with the phosphorylation levels of AMPKα. p53 protein levels were also correlated with AMPKαphosphorylation levels. Compound C, an inhibitor of AMPKα, attenuated the effects of H2O2. These results suggest that embryonic neural stem cells from SOD1G93A mice are more susceptible to apoptosis in the presence of oxidative stress compared with those from wild-type controls, and the effects are mainly mediated by Pax3 and p53 in the AMPKα pathway.

  20. A novel murrel Channa striatus mitochondrial manganese superoxide dismutase: gene silencing, SOD activity, superoxide anion production and expression.

    Science.gov (United States)

    Arockiaraj, Jesu; Palanisamy, Rajesh; Bhatt, Prasanth; Kumaresan, Venkatesh; Gnanam, Annie J; Pasupuleti, Mukesh; Kasi, Marimuthu

    2014-12-01

    We have reported the molecular characterization including gene silencing, superoxide activity, superoxide anion production, gene expression and molecular characterization of a mitochondrial manganese superoxide dismutase (mMnSOD) from striped murrel Channa striatus (named as CsmMnSOD). The CsmMnSOD polypeptide contains 225 amino acids with a molecular weight of 25 kDa and a theoretical isoelectric point of 8.3. In the N-terminal region, CsmMnSOD carries a mitochondrial targeting sequence and a superoxide dismutases (SOD) Fe domain (28-109), and in C-terminal region, it carries another SOD Fe domain (114-220). The CsmMnSOD protein sequence shared significant similarity with its homolog of MnSOD from rock bream Oplegnathus fasciatus (96%). The phylogenetic analysis showed that the CsmMnSOD fell in the clade of fish mMnSOD group. The monomeric structure of CsmMnSOD possesses 9 α-helices (52.4%), 3 β-sheets (8.8%) and 38.8% random coils. The highest gene expression was noticed in liver, and its expression was inducted with fungal (Aphanomyces invadans) and bacterial (Aeromonas hydrophila) infections. The gene silencing results show that the fish that received dsRNA exhibited significant (P superoxide anion production was determined by calculating the granular blood cell count during infection in murrel. It shows that the infection influenced the superoxide radical production which plays a major role in killing the pathogens. Overall, this study indicated the defense potentiality of CsmMnSOD; however, further research is necessary to explore its capability at protein level.

  1. Quercetin inhibits degranulation and superoxide generation in PMA stimulated neutrophils

    OpenAIRE

    2012-01-01

    Activated neutrophils represent the main source of myeloperoxidase (MPO), superoxide (SO) and subsequently derived oxygen metabolites. They have important microbicidal activities, however in inflammatory conditions they may secondarily attack surrounding tissues. Overproduction of reactive oxygen species, prolonged or excessive liberation of MPO and other effective yet also toxic substances from neutrophils may participate in disturbed apoptosis, intensify the inflammatory processes and resul...

  2. High glucose impairs superoxide production from isolated blood neutrophils

    DEFF Research Database (Denmark)

    Perner, A; Nielsen, S E; Rask-Madsen, J

    2003-01-01

    Superoxide (O(2)(-)), a key antimicrobial agent in phagocytes, is produced by the activity of NADPH oxidase. High glucose concentrations may, however, impair the production of O(2)(-) through inhibition of glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the formation of NADPH. This study...

  3. Cu/Zn superoxide dismutases in developing cotton fibers

    Science.gov (United States)

    Hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) are important signaling molecules in diverse physiological processes. Previously, we discovered superoxide dismutase (SOD) activity in extracellular protein preparations from fiber-bearing cotton (Gossypium hirsutum L.) seeds. We sho...

  4. Superoxide dismutase in the marine sponge Cliona celata

    NARCIS (Netherlands)

    Marques, D.; Esteves, A.I.; Almeida, M.; Xavier, J.; Humanes, M.

    2008-01-01

    The aim of this work is to investigate the activity of the antioxidant enzyme superoxide dismutase in the cosmopolitan sponge Cliona celata (Grant, 1826), since this enzyme has been described as a useful biomarker for marine pollution in other marine invertebrates. The quantification of the catalyti

  5. Superoxide dismutase in the marine sponge Cliona celata

    NARCIS (Netherlands)

    Marques, D.; Esteves, A.I.; Almeida, M.; Xavier, J.; Humanes, M.

    2008-01-01

    The aim of this work is to investigate the activity of the antioxidant enzyme superoxide dismutase in the cosmopolitan sponge Cliona celata (Grant, 1826), since this enzyme has been described as a useful biomarker for marine pollution in other marine invertebrates. The quantification of the

  6. Effect of Low Level Cadmium Exposure on Superoxide Dismutase ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research January 2016; 15 (1): 115-119. ISSN: 1596-5996 ... Results: The data revealed a significant (p < 0.05) decrease in organ weight of the exposed rats, and with the highest ... system such as superoxide dismutase. Antioxidants ... the guiding principles of laboratory animal care.

  7. Effect of yogic exercise on superoxide dismutase levels in diabetics

    Directory of Open Access Journals (Sweden)

    Mahapure Hemant

    2008-01-01

    Full Text Available Context: Reactive oxygen species are known to aggravate disease progression. To counteract their harmful effects, the body produces various antioxidant enzymes, viz , superoxide dismutase, glutathione reductase etc. Literature reviews revealed that exercises help to enhance antioxidant enzyme systems; hence, yogic exercises may be useful to combat various diseases. Aims: This study aims to record the efficacy of yoga on superoxide dismutase, glycosylated hemoglobin (Hb and fasting blood glucose levels in diabetics. Settings and Design: Forty diabetics aged 40-55 years were assigned to experimental (30 and control (10 groups. The experimental subjects underwent a Yoga program comprising of various Asanas (isometric type exercises and Pranayamas (breathing exercises along with regular anti-diabetic therapy whereas the control group received anti-diabetic therapy only. Methods and Material: Heparinized blood samples were used to determine erythrocyte superoxide dismutase (SOD activity and glycosylated Hb levels and fasting blood specimens collected in fluoride Vacutainers were used for assessing blood glucose. Statistical analysis used: Data were analyzed by using 2 x 2 x 3 Factorial ANOVA followed by Scheffe′s posthoc test. Results: The results revealed that Yogic exercise enhanced the levels of Superoxide dismutase and reduced glycosylated Hb and glucose levels in the experimental group as compared to the control group. Conclusion: The findings conclude that Yogic exercises have enhanced the antioxidant defence mechanism in diabetics by reducing oxidative stress.

  8. Novel mechanisms for superoxide-scavenging activity of human manganese superoxide dismutase determined by the K68 key acetylation site.

    Science.gov (United States)

    Lu, Jiaqi; Cheng, Kuoyuan; Zhang, Bo; Xu, Huan; Cao, Yuanzhao; Guo, Fei; Feng, Xudong; Xia, Qing

    2015-08-01

    Superoxide is the primary reactive oxygen species generated in the mitochondria. Manganese superoxide dismutase (SOD2) is the major enzymatic superoxide scavenger present in the mitochondrial matrix and one of the most crucial reactive oxygen species-scavenging enzymes in the cell. SOD2 is activated by sirtuin 3 (SIRT3) through NAD(+)-dependent deacetylation. However, the exact acetylation sites of SOD2 are ambiguous and the mechanisms underlying the deacetylation-mediated SOD2 activation largely remain unknown. We are the first to characterize SOD2 mutants of the acetylation sites by investigating the relative enzymatic activity, structures, and electrostatic potential of SOD2 in this study. These SOD2 mutations affected the superoxide-scavenging activity in vitro and in HEK293T cells. The lysine 68 (K68) site is the most important acetylation site contributing to SOD2 activation and plays a role in cell survival after paraquat treatment. The molecular basis underlying the regulation of SOD2 activity by K68 was investigated in detail. Molecular dynamics simulations revealed that K68 mutations induced a conformational shift of residues located in the active center of SOD2 and altered the charge distribution on the SOD2 surface. Thus, the entry of the superoxide anion into the coordinated core of SOD2 was inhibited. Our results provide a novel mechanistic insight, whereby SOD2 acetylation affects the structure and charge distribution of SOD2, its tetramerization, and p53-SOD2 interactions of SOD2 in the mitochondria, which may play a role in nuclear-mitochondrial communication during aging.

  9. Carbon dioxide suppresses macrophage superoxide anion production independent of extracellular pH and mitochondrial activity

    NARCIS (Netherlands)

    Kuebler, Joachim F.; Kos, Marcin; Jesch, NataLie K.; Metzelder, Martin L.; van der Zee, David C.; Bax, Klaas M.; Vieten, Gertrud; Ure, Benno M.

    2007-01-01

    Background: Superoxide anions released by activated inacrophages during surgery are considered to be responsible for local cellular damage. Application of CO2 prieumoperitoneum during laparoscopy affects superoxide anion release, but the underlying mechanism remains unclear and the data reported are

  10. Measurement of Antioxidant Activity Towards Superoxide in Natural Waters.

    Directory of Open Access Journals (Sweden)

    D. Whitney King

    2016-11-01

    Full Text Available Antioxidants are a class of molecules that provide a protective function against reactive oxygen species (ROS in biological systems by out competing physiologically important molecules for ROS oxidation. In natural waters, the reactivity of antioxidants gives an estimate of oxidative stress and may determine the reactivity and distribution of reactive oxidants. We present an analytical method to measure antioxidant activity in natural waters through the competition between ascorbic acid, an antioxidant, and MCLA, a chemiluminescent probe for superoxide. A numerical kinetic model of the analytical method has been developed to optimize analytical performance. Measurements of antioxidant concentrations in pure and seawater are possible with detection limits below 0.1 nM. Surface seawater samples collected at solar noon contained over 0.4 nM of antioxidants and exhibited first-order decay with a half-life of 3-7 minutes, consistent with a reactive species capable of scavenging photochemically produced superoxide.

  11. [Generation of superoxides during the interaction of melanins with oxygen].

    Science.gov (United States)

    Lapina, V A; Dontsov, A E; Ostrovskiĭ, M A

    1984-10-01

    The rate of nitroblue tetrazolium (NBT) reduction by dihydroxyphenylalanine-melanin, pheomelanin and retinal pigment epithelium melanosomes under aerobic conditions (pH 7.4) is low both in the dark and upon illumination, but increases drastically in the presence of cetyltrimethylammonium bromide (CTAB). Under these conditions, the light insignificantly stimulates NBT reduction (1.3-fold). The reaction is effectively inhibited by superoxide dismutase. This suggests that superoxide anions (O2-. are formed as intermediate reaction products in the course of NBT reduction by melanins. At alkaline values of pH (greater than or equal to 9.0), the O2-.-dependent reduction of NBT can also take place in the absence of CTAB. In contrast with oxidation of photoreduced riboflavin, the melanin oxidation by O2 cannot induce lipid peroxidation. It is concluded that O2-. generation via melanin oxidation of melanosomes occurs only under non-physiological conditions and can hardly take place in vivo.

  12. ACTIVITY OF SUPEROXIDE DISMUTASE ENZYME IN YEAST SACCHAROMYCES CEREVISIAE

    Directory of Open Access Journals (Sweden)

    Blažena Lavová

    2014-02-01

    Full Text Available Reactive oxygen species (ROS with reactive nitrogen species (RNS are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD is to accelerate the dismutation of the toxic superoxide radical, produced during oxidative energy processes, to hydrogen peroxide and molecular oxygen. In this study, SOD activity of three yeast strains Saccharomyces cerevisiae was determined. It was found that SOD activity was the highest (23.7 U.mg-1 protein in strain 612 after 28 hours of cultivation. The lowest SOD activity from all tested strains was found after 56 hours of cultivation of strain Gyöng (0.7 U.mg-1 protein.

  13. Pulse radiolysis studies on superoxide reductase from Treponema pallidum

    CERN Document Server

    Nivière, V; Fontecave, M; Houée-Levin, C

    2015-01-01

    Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2*- to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi-molecular reaction of the ferrous center with O2, with a rate constant of 6 x 10 (8) M(-1) s(-1). A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s(-1). This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron-peroxide species.

  14. ACTIVITY OF SUPEROXIDE DISMUTASE ENZYME IN YEAST SACCHAROMYCES CEREVISIAE

    OpenAIRE

    2014-01-01

    Reactive oxygen species (ROS) with reactive nitrogen species (RNS) are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD) is to accelerate the dismutation of the toxic su...

  15. The Superoxide Reductase from the Early Diverging Eukaryote Giardia Intestinalis

    Energy Technology Data Exchange (ETDEWEB)

    Cabelli, D.E.; Testa, F.; Mastronicola, D.; Bordi, E.; Pucillo, L.P.; Sarti, P.; Saraiva, L.M.; Giuffre, A.; Teixeira, M.

    2011-10-15

    Unlike superoxide dismutases (SODs), superoxidereductases (SORs) eliminate superoxide anion (O{sub 2}{sup {sm_bullet}-}) not through its dismutation, but via reduction to hydrogen peroxide (H{sub 2}O{sub 2}) in the presence of an electron donor. The microaerobic protist Giardia intestinalis, responsible for a common intestinal disease in humans, though lacking SOD and other canonical reactive oxygen species-detoxifying systems, is among the very few eukaryotes encoding a SOR yet identified. In this study, the recombinant SOR from Giardia (SOR{sub Gi}) was purified and characterized by pulse radiolysis and stopped-flow spectrophotometry. The protein, isolated in the reduced state, after oxidation by superoxide or hexachloroiridate(IV), yields a resting species (T{sub final}) with Fe{sup 3+} ligated to glutamate or hydroxide depending on pH (apparent pK{sub a} = 8.7). Although showing negligible SOD activity, reduced SOR{sub Gi} reacts with O{sub 2}{sup {sm_bullet}-} with a pH-independent second-order rate constant k{sub 1} = 1.0 x 10{sup 9} M{sup -1} s{sup -1} and yields the ferric-(hydro)peroxo intermediate T{sub 1}; this in turn rapidly decays to the T{sub final} state with pH-dependent rates, without populating other detectable intermediates. Immunoblotting assays show that SOR{sub Gi} is expressed in the disease-causing trophozoite of Giardia. We propose that the superoxide-scavenging activity of SOR in Giardia may promote the survival of this air-sensitive parasite in the fairly aerobic proximal human small intestine during infection.

  16. High glucose impairs superoxide production from isolated blood neutrophils

    DEFF Research Database (Denmark)

    Perner, A; Nielsen, S E; Rask-Madsen, J

    2003-01-01

    Superoxide (O(2)(-)), a key antimicrobial agent in phagocytes, is produced by the activity of NADPH oxidase. High glucose concentrations may, however, impair the production of O(2)(-) through inhibition of glucose-6-phosphate dehydrogenase (G6PD), which catalyzes the formation of NADPH. This stud...... measured the acute effects of high glucose or the G6PD inhibitor dehydroepiandrosterone (DHEA) on the production of O(2)(-) from isolated human neutrophils....

  17. Levels of Malondialdehyde and Superoxide Dismutase in Subclinical Hyperthyroidism

    OpenAIRE

    2005-01-01

    We aimed to determine whether patients with subclinical hyperthyroidism (SH) are subject to oxidative stress. Twenty-two women and 8 men having endogenous subclinical hyperthyroidism for a duration of at least 6 months, and 21 women and 9 men healthy controls were included in this study. We measured the level of plasma malondialdehyde, as one of the lipid peroxidation markers, and the activity of erythrocyte superoxide dismutase, which is an antioxidant enzyme. The activity of erythrocyte sup...

  18. Impact of Thyroid Dysfunction on Antioxidant Capacity, Superoxide Dismutase

    Directory of Open Access Journals (Sweden)

    Mehdi Hedayati

    2014-01-01

    Full Text Available Background: In hypothyroidism and hyperthyroidism, disturbance of oxidant/antioxidant balance leads to reactive oxygen species (ROS generation. The aim of this study is assaying total antioxidant capacity and superoxide dismutase and catalase activities in patients with hypo-and hyperthyroidism in order to control the progression of its pathology and health care. Materials and Methods: This case-control study was performed on 85 patients with hypothyroidism, 66 patients with hyperthyroidism and 74 normal individuals as control that referred to the clinic of the Research Institute for Endocrine Sciences of Shahid-Beheshti University in year 2010. Serum enzymatic activity of catalase, superoxide dismutase and total antioxidant capacity was measured in the fasting state. Data was described as mean±SD and data means of the two groups was compared by independent t-test. Data was analyzed by SPSS-18 application. Results: The total antioxidant capacity in individuals with hyperthyroidism decreased compared to healthy controls, but individuals with hypothyroidism compared to the healthy control group showed no significant difference. Catalase and superoxide dismutase activity in hypo-and hyperthyroidism were significantly increased compared with healthy controls (p=0.005. Conclusion: Decreasing of antioxidant capacity in hyperthyroid patients is probably because of increased production of free radicals. There was not observed significant difference in total antioxidant capacity in hypothyroid patients. Also in hypo-and hyperthyroidism patients, increasing of enzymes activity is probably due to increasing of the production of ROS.

  19. Superoxide Radical Formation in Isolated PMN from Experimental Vaginal Trichomoniasis

    Directory of Open Access Journals (Sweden)

    Z Valadkhani

    2006-06-01

    Full Text Available Trichomoniasis, the most widespread sexually transmitted disease is caused by Trichomonas vaginalis. This parasite is site specific for the genitourinary tract and recruitment of macrophages as well as polymorphonuclear nutrophils (PMN to the site of infection is the first line of defense as a component of non-specific resistance and immunity. In this study, BALB/c mice were infected with 10 isolates from symptomatic and 10 from asymptomatic patients. Then PMN from vaginal washes, vaginal tissue and blood of infected mice was isolated and the rate of superoxide formation by intact stimulated PMN was measured. Results showed that, mice infected with symptomatic isolates indicated significant increase in polymorphs with increase in days of infection as compared with mice infected with asymptomatic isolate and control (uninfected animals. Vaginal tissue cells generated maximal amount of superoxide in symptomatic isolates infected animals (5.17 ± 0.36 as compared to asymptomatic isolates (4.54 ± 0.43, which brings out the maximal abnormality in PMN in this localized area. The amount of superoxide radicals generated by cells of vaginal washes and blood of symptomatic isolate infected mice 4.29 ± 0.25 and 2.16 ± 0.35 was less than the asymptomatic isolate (4.94 ± 0.49 and 3.18 ± 0.26, respectively. This study indicates that super oxide radical generation may play role in establishing the infection.

  20. Copper complexes of bioactive ligands with superoxide dismutase activity.

    Science.gov (United States)

    Khalid, Huma; Hanif, Muhammad; Hashmi, Muhammad Ali; Mahmood, Tariq; Ayub, Khurshid; Monim-Ul-Mehboob, Muhammad

    2013-11-01

    Free radicals or reactive oxygen species (ROS) are highly toxic and their damaging effects result in a variety of detrimental health issues such as neurodegenerative, cardiovascular and age-related diseases. Human body has evolved an effective defense system including superoxide dismutase (SOD) and catalase against the toxicity of these free radicals. SOD is a metalloenzyme and it acts as an excellent antioxidant to protect the body from superoxide radicals that are generated in the biological system. However, the clinical use of SOD is limited due to its short in vivo life span, and its large size that hampered its penetration across the cell membranes. Pharmaceuticals that provide ROS scavenging systems are the most effective when the production of ROS exceeds the scavenging capacity of endogenous SOD as a result of aging or pathological processes. Inspired by the Nature, scientists have designed metal-based mimics of the superoxide dismutase. This review focuses on different copper complexes that are developed from bioactive ligands and mimic the protecting action of the SOD.

  1. A new method to prevent degradation of lithium-oxygen batteries: reduction of superoxide by viologen.

    Science.gov (United States)

    Yang, L; Frith, J T; Garcia-Araez, N; Owen, J R

    2015-01-31

    Lithium-oxygen battery development is hampered by degradation reactions initiated by superoxide, which is formed in the pathway of oxygen reduction to peroxide. This work demonstrates that the superoxide lifetime is drastically decreased upon addition of ethyl viologen, which catalyses the reduction of superoxide to peroxide.

  2. Lecithinized copper,zinc-superoxide dismutase as a protector against doxorubicin-induced cardiotoxicity in mice.

    NARCIS (Netherlands)

    Hartog, den GJ; Haenen, GR; Boven, E.; Vijgh, van der WJ

    2004-01-01

    Production of superoxide radicals from doxorubicin is widely accepted to be the cause of the cardiotoxicity induced by this antitumor agent. Pretreatment with superoxide dismutase could improve the therapeutic application. Aim of the present study was to determine whether lecithinized superoxide

  3. Reactions of superoxide dismutases with HS(-)/H2S and superoxide radical anion: An in vitro EPR study.

    Science.gov (United States)

    Bolić, Bojana; Mijušković, Ana; Popović-Bijelić, Ana; Nikolić-Kokić, Aleksandra; Spasić, Snežana; Blagojević, Duško; Spasić, Mihajlo B; Spasojević, Ivan

    2015-12-01

    Interactions of hydrogen sulfide (HS(-)/H2S), a reducing signaling species, with superoxide dimutases (SOD) are poorly understood. We applied low-T EPR spectroscopy to examine the effects of HS(-)/H2S and superoxide radical anion O2.- on metallocenters of FeSOD, MnSOD, and CuZnSOD. HS(-)/H2S did not affect FeSOD, whereas active centers of MnSOD and CuZnSOD were open to this agent. Cu(2+) was reduced to Cu(1+), while manganese appears to be released from MnSOD active center. Untreated and O2.- treated FeSOD and MnSOD predominantly show 5 d-electron systems, i.e. Fe(3+) and Mn(2+). Our study provides new details on the mechanisms of (patho)physiological effects of HS(-)/H2S.

  4. Intrinsic disorder in proteins involved in amyotrophic lateral sclerosis.

    Science.gov (United States)

    Santamaria, Nikolas; Alhothali, Marwa; Alfonso, Maria Harreguy; Breydo, Leonid; Uversky, Vladimir N

    2017-04-01

    Five structurally and functionally different proteins, an enzyme superoxide dismutase 1 (SOD1), a TAR-DNA binding protein-43 (TDP-43), an RNA-binding protein FUS, a cofilin-binding protein C9orf72, and polypeptides generated as a result of its intronic hexanucleotide expansions, and to lesser degree actin-binding profilin-1 (PFN1), are considered to be the major drivers of amyotrophic lateral sclerosis. One of the features common to these proteins is the presence of significant levels of intrinsic disorder. The goal of this study is to consider these neurodegeneration-related proteins from the intrinsic disorder perspective. To this end, we employed a broad set of computational tools for intrinsic disorder analysis and conducted intensive literature search to gain information on the structural peculiarities of SOD1, TDP-43, FUS, C9orf72, and PFN1 and their intrinsic disorder predispositions, and the roles of intrinsic disorder in their normal and pathological functions.

  5. Astrocytes from familial and sporadic ALS patients are toxic to motor neurons.

    Science.gov (United States)

    Haidet-Phillips, Amanda M; Hester, Mark E; Miranda, Carlos J; Meyer, Kathrin; Braun, Lyndsey; Frakes, Ashley; Song, SungWon; Likhite, Shibi; Murtha, Matthew J; Foust, Kevin D; Rao, Meghan; Eagle, Amy; Kammesheidt, Anja; Christensen, Ashley; Mendell, Jerry R; Burghes, Arthur H M; Kaspar, Brian K

    2011-08-10

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for 90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non-cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.

  6. Role of superoxide dismutase enzymes and ascorbate in protection of nitrergic relaxation against superoxide anions in mouse duodenum

    Institute of Scientific and Technical Information of China (English)

    M Ata SECILMIS; Olcay Ergurhan KIROGLU; Nuran OGULENER

    2008-01-01

    Aim: The aim of this study was to investigate whether superoxide dismutase (SOD) enzymes and ascorbate play a role in the protection of the nitrergic relax-ation against superoxide anion inhibition in the mouse duodenum. Methods: The effects of exogenous SOD, N,N'-bis(salicylidene) ethylenediamine chlo-ride (EUK-8; a synthetic cell-permeable mimetic of the manganese SOD [Mn SOD] and ascorbate on relaxant responses induced by nitrergic nerve stimulation), exogenous nitric oxide (NO), and nitroglycerin were investigated in isolated mouse duodenum tissues. Results: Diethyidithiocarbamate (DETCA) inhibited the relaxation to exogenous NO and nitroglycerin, but not relaxation to electri-cal field stimulation (EFS). SOD and ascorbate partially prevented the inhibi-tory effect of DETCA on relaxation to NO, abut not to nitroglycerin. The DETCA-induced inhibition on nitroglycerin was prevented by ELrK-8. Hemoglobin, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolinel-oxyl-3-oxide, and hydroxo-cobalamin inhibited the relaxation to NO, but not to EFS and nitroglycerin in the presence of DETCA. Pyrogallol and hydroquinone inhibited the relaxation to NO, but not to EFS and nitroglycerin. This inhibition was prevented by exog-enous SOD and ascorbate, but was not prevented by EUK-8. Pyrogallol and hy-droquinone did not inhibit the EFS-induced relaxation in the presence of DETCA. Duroquinone and 6-anilino-5.8-quinolinedione inhibited the relaxation to EFS, NO, and nitroglycerin, and this inhibition was prevented by EUK-8. Conclusion: These results suggest that the nitrergic neurotransmission in the mouse duode-num is protected by endogenous tissue antioxidants against superoxide anions, and Mn SOD, in addition to copper/zinc SOD, can protect NO from attack from superoxide anion generators intracellularly. Also, the possibility that the endog-enous neurotransmitter may not be the free NO but a NO-containing or NO-generating molecule in the mouse duodenum remains open.

  7. 草鱼线粒体型超氧化物歧化酶的生化遗传特性%Genetic and biochemical characteristics of mitochondrial superoxide dismutase in grass carp Ctenopharyngodon idellus

    Institute of Scientific and Technical Information of China (English)

    颜勤; 罗琛

    2004-01-01

    超氧化物歧化酶(SOD)是一种对生物细胞保护至关重要、在进化上比较保守的酶.因此,超氧化物歧化酶作为分子钟或分子标记已被广泛应用于生物进化研究、群体遗传结构分析以及品系鉴定.但鱼类SOD的生物化学和遗传学特性都尚未进行过系统和深入的研究.为使这一重要的分子标记能更好地应用于鱼类遗传育种、种质资源保护以及进化研究,本实验采用聚丙烯酰胺梯度凝胶垂直电泳法,研究了草鱼线粒体型超氧化物歧化酶(fm-SOD)的同功酶形式,生化遗传表型、亚基组成以及金属类型.实验结果表明,草鱼fm-SOD有三种不同的同功酶形式;按从正极到负极的排列分别命名为fm-SOD 1,fm-SOD 2,fm-SOD 3.这三种不同的fm-SOD在草鱼群体中可构成3种不同的生化遗传学表型:表型1个体只含有迁移率最快的fm-SOD 1同功酶;表型3个体只含有迁移率最慢的fm-SOD 3同功酶;而表型2个体中含有所有三种不同形式的同功酶.在野生草鱼群体中,存在所有三种表现型;而在基因纯合型的雌核发育草鱼群体中只检测到表型1和表型3.野生草鱼群体中三种表现型的个体数之比符合一对等位基因分离的1:2:1孟德尔遗传分离比例.由这些实验结果得出以下结论:(1)草鱼fm-SOD是由细胞核DNA上的基因所编码而不是由线粒体DNA上的基因所编码的;(2)草鱼fm-SOD由单个基因座位控制;(3)至少存在两个有较明显差异等位基因;(4)草鱼fm-SOD由两个亚基组成.此外,草鱼fm-SOD对15%乙醇/25%氯仿昆合物敏感而对过氧化氢具有一定的抗性,表明它为锰型的SOD(Mn-SOD)[动物学报50(3):389-394,2004].%Superoxide dismutase is an evolutionarily conserved enzyme with an important role in protecting organisms and cells from the damages caused by oxygen free radicals. As a molecular clock and genetic marker, superoxide dismutase has been widely used in evolutionary studies

  8. Detoxification of superoxide without production of H2O2: antioxidant activity of superoxide reductase complexed with ferrocyanide

    CERN Document Server

    Molina-Heredia, Fernando P; Berthomieu, Catherine; Touati, Danièle; Tremey, Emilie; Favaudon, Vincent; Adam, Virgile; Nivière, Vincent

    2015-01-01

    The superoxide radical O(2)(-.) is a toxic by-product of oxygen metabolism. Two O(2)(-.) detoxifying enzymes have been described so far, superoxide dismutase and superoxide reductase (SOR), both forming H2O2 as a reaction product. Recently, the SOR active site, a ferrous iron in a [Fe(2+) (N-His)(4) (S-Cys)] pentacoordination, was shown to have the ability to form a complex with the organometallic compound ferrocyanide. Here, we have investigated in detail the reactivity of the SOR-ferrocyanide complex with O(2)(-.) by pulse and gamma-ray radiolysis, infrared, and UV-visible spectroscopies. The complex reacts very efficiently with O(2)(-.). However, the presence of the ferrocyanide adduct markedly modifies the reaction mechanism of SOR, with the formation of transient intermediates different from those observed for SOR alone. A one-electron redox chemistry appears to be carried out by the ferrocyanide moiety of the complex, whereas the SOR iron site remains in the reduced state. Surprisingly, the toxic H2O2 s...

  9. Hollow-fiber blood-dialysis membranes: superoxide generation, permeation, and dismutation measured by chemiluminescence.

    Science.gov (United States)

    Yamamoto, Ken-ichiro; Kobayashi, Kazuyoshi; Endo, Kosuke; Miyasaka, Takehiro; Mochizuki, Seiichi; Kohori, Fukashi; Sakai, Kiyotaka

    2005-01-01

    The interaction of blood with a material surface results in activation of the body's humoral immune system and the generation of reactive oxygen species (ROS). It has recently become clear that ROS are central to the pathology of many diseases. In this study, we evaluated the superoxide generation, permeation, and dismutation in hollow-fiber dialysis membranes by using 2-methyl-6-p-methoxyphenylethynyl-imidazopyrazinone (MPEC) as a superoxide-reactive chemiluminescence producer and an optical fiber probe to detect the resulting chemiluminescence in the hollow fiber lumen. We measured the superoxide generated when bovine blood leukocytes were brought into contact with dialysis membranes. Superoxide permeation was determined by measuring MPEC chemiluminescence in the hollow fiber lumen using an optical fiber probe. Additionally, superoxide dismutation was evaluated by examining the difference in superoxide permeability for membranes with and without vitamin E coating. Superoxide generation varies for different membrane materials, depending on the membrane's biocompatibility. Superoxide permeability depends on the diffusive permeability of membranes. No marked decrease in superoxide permeability was observed among membrane materials. The superoxide permeability of vitamin E-coated membrane was smaller than that of uncoated membrane. The antioxidant property of vitamin E-coated membranes is hence effective in causing superoxide dismutation.

  10. Mechanical overloading causes mitochondrial superoxide and SOD2 imbalance in chondrocytes resulting in cartilage degeneration.

    Science.gov (United States)

    Koike, Masato; Nojiri, Hidetoshi; Ozawa, Yusuke; Watanabe, Kenji; Muramatsu, Yuta; Kaneko, Haruka; Morikawa, Daichi; Kobayashi, Keiji; Saita, Yoshitomo; Sasho, Takahisa; Shirasawa, Takuji; Yokote, Koutaro; Kaneko, Kazuo; Shimizu, Takahiko

    2015-06-25

    Mechanical stress and aging are major risk factors of cartilage degeneration. Human studies have previously reported that oxidative damage increased, while SOD2 protein was reciprocally downregulated in osteoarthritic degenerated cartilage. However, it remains unclear whether mitochondrial superoxide imbalance in chondrocytes causes cartilage degeneration. We herein demonstrate that mechanical loading promoted mitochondrial superoxide generation and selective Sod2 downregulation in chondrocytes in vivo and that mitochondrial superoxide inducer also downregulated Sod2 expression in chondrocytes in vitro. A genetically manipulated model revealed that Sod2 deficiency in chondrocytes also resulted in mitochondrial superoxide overproduction and dysfunction, thus leading to cartilage degeneration. Intra-articular injection of a permeable antioxidant effectively suppressed the mechanical loading-induced mitochondrial superoxide generation and cartilage degeneration in mice. Our findings demonstrate that mitochondrial superoxide plays a pivotal role in the development and progression of osteoarthritis, and the mitochondrial superoxide balance may therefore be a promising target for the treatment of cartilage degeneration.

  11. Healing of colonic ischemic anastomoses in the rat: role of superoxide radicals.

    Science.gov (United States)

    Garcia, J G; Criado, F J; Persona, M A; Alonso, A G

    1998-07-01

    The aim of this study was to evaluate the role of superoxide radicals in the healing of ischemic colonic anastomoses in the rat. Adult male Wistar rats were used in a factorial design with two factors (normal or ischemic colonic anastomoses) each having two levels (treatment with saline or allopurinol). Colonic anastomoses were performed either in normal or previously devascularized colons (ischemic anastomoses) at identical locations, using the same technique. On the fourth postoperative day, animals were killed, and specimens were taken for determinations. Ischemic anastomoses displayed significant increases in superoxide radical (assayed as superoxide anion), superoxide dismutase, and glutathione peroxidase concentrations. Bursting strength and hydroxyproline levels were also significantly lower in these anastomoses. Allopurinol administration elicited a significant decrease in superoxide anions and raised both bursting strength and hydroxyproline levels only in ischemic anastomoses. Superoxide radicals are involved in the delay in healing of ischemic anastomoses. Allopurinol lowers superoxide anion production and has beneficial effects on the cicatrization of ischemic anastomoses.

  12. Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage and is associated with inhibition of estrogen-induced breast cancer

    Science.gov (United States)

    Bhat, Hari K.

    2012-01-01

    Epidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Oxidative stress has been implicated in this carcinogenic process. We have recently demonstrated that antioxidants vitamin C or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the mechanism of antioxidant-mediated prevention of breast cancer. Female August Copenhagen Irish (ACI) rats were treated with E2, vitamin C, vitamin C + E2, BHA and BHA + E2 for up to 8 months. Superoxide dismutase 3 (SOD3) was suppressed in E2-exposed mammary tissues and in mammary tumors of rats treated with E2. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. 8-Hydroxydeoxyguanosine (8-OHdG) levels determined as a marker of oxidative DNA damage were higher in E2-exposed mammary tissues and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissues and in MCF-10A cells. Increased DNA damage, colony and mammosphere formation, and migration in SOD3 knocked down MCF-10A cells, and nuclear translocation of SOD3 in vitamin C-treated mammary tissues and in MCF-10A cells suggest protective role of SOD3 against DNA damage and mammary carcinogenesis. Our studies further demonstrate that SOD3, but not SOD2 and SOD1, is induced by antioxidants and is regulated through NRF2. SOD3 may thus be an important gene in defense against oxidative stress and in the prevention of estrogen-mediated breast cancer. PMID:23027624

  13. Analysis of DHE-derived oxidation products by HPLC in the assessment of superoxide production and NADPH oxidase activity in vascular systems.

    Science.gov (United States)

    Fernandes, Denise C; Wosniak, João; Pescatore, Luciana A; Bertoline, Maria A; Liberman, Marcel; Laurindo, Francisco R M; Santos, Célio X C

    2007-01-01

    Dihydroethidium (DHE) is a widely used sensitive superoxide (O2(*-)) probe. However, DHE oxidation yields at least two fluorescent products, 2-hydroxyethidium (EOH), known to be more specific for O2(*-), and the less-specific product ethidium. We validated HPLC methods to allow quantification of DHE products in usual vascular experimental situations. Studies in vitro showed that xanthine/xanthine oxidase, and to a lesser degree peroxynitrite/carbon dioxide system led to EOH and ethidium formation. Peroxidase/H2O2 but not H2O2 alone yielded ethidium as the main product. In vascular smooth muscle cells incubated with ANG II (100 nM, 4 h), we showed a 60% increase in EOH/DHE ratio, prevented by PEG-SOD or SOD1 overexpression. We further validated a novel DHE-based NADPH oxidase assay in vascular smooth muscle cell membrane fractions, showing that EOH was uniquely increased after ANG II. This assay was also adapted to a fluorescence microplate reader, providing results in line with HPLC results. In injured artery slices, shown to exhibit increased DHE-derived fluorescence at microscopy, there was approximately 1.5- to 2-fold increase in EOH/DHE and ethidium/DHE ratios after injury, and PEG-SOD inhibited only EOH formation. We found that the amount of ethidium product and EOH/ethidium ratios are influenced by factors such as cell density and ambient light. In addition, we indirectly disclosed potential roles of heme groups and peroxidase activity in ethidium generation. Thus HPLC analysis of DHE-derived oxidation products can improve assessment of O2(*-) production or NADPH oxidase activity in many vascular experimental studies.

  14. Enhancement of Superoxide Dismutase and Catalase Activities and Salt Tolerance of Euhalophyte Suaeda salsa L.by Mycorrhizal Fungus Glomus mosseae

    Institute of Scientific and Technical Information of China (English)

    LI Tao1; LIU Run-Jin; HE Xin-Hua; WANG Bao-Shan

    2012-01-01

    Arbuscular mycorrhizal (AM)-mediated plant physiological activities could contribute to plant salt tolerance.However,the biochemical mechanism by which AM fungi enhance salt tolerance of halophytic plants is unclear.A pot experiment was conducted to determine whether salt tolerance of the C3 halophyte Suaeda salsa was enhanced by the AM fungus Glomus mosseae.When 60-day-old S.salsa seedlings were subjected to 400 mmol L-1 NaC1 stress for 35 days,plant height,number of leaves and branches,shoot and root biomass,and root length of G.mosseae-colonized seedlings were significantly greater than those of the nonmycorrizal seedlings.Leaf superoxide dismutase (SOD) activity at all sampling times (weekly for 35 days after salt stress was initiated) and leaf catalase (CAT) activity at 2 and 3 weeks after salt stress was initiated were also significantly enhanced in G.mosseae-colonized S.salsa seedlings,while the content of leaf malondialdehyde (MDA),a product of membrane lipid peroxidation,was significantly reduced,indicating an alleviation of oxidative damage.The corresponding leaf isoenzymes of SOD (Fe-SOD,Cu/Zn-SOD1,and Cu/Zn-SOD2) and CAT (CAT1 and CAT2) were also significantly increased in the mycorrhizal seedlings after 14 days of 400 mmol L-1 NaC1 stress.Our results suggested that G.mosseae increased salt tolerance by increasing SOD and CAT activities and forming SOD and CAT isoforms in S.salsa seedlings.

  15. Effects of noise exposure level on the relationship between SNPs of SOD1 and the susceptibility to noiseinduced hearing loss (NIHL)%噪声暴露水平对铜锌超氧化物歧化酶基因单核苷酸多态性与噪声性听力损失易感性的影响

    Institute of Scientific and Technical Information of China (English)

    曾文锋; 李旭东; 刘移民; 陈建雄; 苏世标

    2012-01-01

    目的 探讨噪声暴露强度和累积噪声暴露量(cumulative noise exposure,CNE)对铜锌超氧化物歧化酶基因(SOD1)rs2070424,rs10432782单核苷酸多态性(single nucleotide polymorphisms,SNPs)与噪声性听力损失(noise induced hearing loss,NIHL)易感性关系的影响.方法 利用病例对照研究,通过比较同一噪声暴露强度噪声作业人员的左耳3000 Hz频段听阈位移情况,筛选出听阈位移最大的10%个体作为本研究的易感人群组,共201例,听阈位移最小的10%个体作为耐受人群组,共202例,并进行相关的职业卫生调查和问卷调查.噪声暴露强度的检测根据GBZ/T 189.8-2007《工作场所物理因素测量-噪声》,抽取易感人群和耐受人群空腹外周静脉血5 ml用于常规方法抽提基因组DNA,TaqMan探针法化学荧光等位基因鉴别试验检测SNP.结果 在噪声暴露强度85~92 dB(A)组内,rs2070424 AA基因型与GG基因型相比,NIHL的患病风险较低,OR值为0.37(95%CI:0.17~0.80).CNE>82 dB(A)组内,rs2070424 AA基因型是NIHL的保护因素,与GG基因型相比,OR值为0.25(95%CI0.09~0.70).噪声暴露强度85~92 dB(A)组携带rs10432782 GG基因型患NIHL的风险与TT基因型相比OR值为3.17(95%CI:1.16~6.89).GT基因型与TT基因型相比OR值为2.39( 95%CI:1.16~4.97).CNE 75~82 dB(A)组内,携带GG基因型患NIHL的风险与TT基因型相比,OR值为2.35 (95%CI:0.96~5.72),P=0.06,GG基因型可能是NIHL的危险因素.结论 噪声暴露强度和累计噪声暴露量对SOD1rs2070424、rs10432782 SNPs与NIHL.易感性关联存在影响.%Objective To explore the effects of noise exposure level and cumulative noise exposure (CNE) on the relationship between rs2070424 and rs10432782 SNPs in SOD1 and the susceptibility to noiseinduced hearing loss (NIHL).Methods A case-control study was performed for investigating the effects of environmental risk factors on the susceptibility to NIHL in 201sensitive workers and 202

  16. Superoxide dismutase levels and peak expiratory flow in asthmatic children

    Directory of Open Access Journals (Sweden)

    Arie Kurniasih

    2015-09-01

    Full Text Available Background Asthma is a chronic inflammatory process which involve variety of cells such as inflammatory mediators, reactive oxygen species (ROS, and cytokines. The inflammatory process would be exacerbated in the presence of oxidative stress. Superoxide dismutase (SOD is the first important enzyme to protect the respiratory tract against oxidative stress. The decreased of SOD has a correlation with increased of airway obstruction and bronchospasm. Objective To assess for a correlation between superoxide dismutase (SOD levels and peak expiratory flow, as well as to determine the impact of SOD levels for predicting asthma attacks. Methods We conducted a prospective cohort study at Dr. Sardjito Hospital, Yogyakarta, between February and April 2011 involving asthmatic children aged 5-18 years. Subjects’ serum SOD levels and peak expiratory flow were measured at the same time point. We then performed a prospective study following up on the same subjects to find out if they had a recurrent asthma attack within one month of the tests. We also reassessed their peak expiratory flow one month after blood specimens were obtained. Results Thirty-nine patients were enrolled in this study. There was no significant correlation between SOD level and peak expiratory flow [r=0.289; 95%CI -0.025 to 0.47; P=0.074]. However, older age was significantly associated with higher peak expiratory flow (=0.5; 95%CI 3.10 to 11.57; P=0.01. Lower levels of SOD increased the risk of asthma attacks in a month following the initial measurements (RR=5.5; 95%CI 1.6 to 18.9; P=0.009. Conclusion Superoxide dismutase (SOD level is not significantly associated with peak expiratory flow. However, we find a relationship between older age and higher peak expiratory flow and a relationship between lower SOD levels and risk of asthma attacks within one month following the tests.

  17. Superoxide dismutase levels and peak expiratory flow in asthmatic children

    Directory of Open Access Journals (Sweden)

    Arie Kurniasih

    2016-11-01

    Full Text Available involvevariety of cells such as inflammatory mediators, reactive oxygenspecies (ROS, and cytokines. The inflammatory process would beexacerbated in the presence of oxidative stress. Superoxide dismutase(SOD is the first important enzyme to protect the respiratory tractagainst oxidative stress. The decreased of SOD has a correlation withincreased of airway obstruction and bronchospasm.Objective To assess for a correlation between superoxide dismutase(SOD levels and peak expiratory flow, as well as to determinethe impact of SOD levels for predicting asthma attacks.Methods We conducted a prospective cohort study at Dr. SardjitoHospital, Yogyakarta, between February and April 2011 involvingasthmatic children aged 5-18 years. Subjects’ serum SOD levelsand peak expiratory flow were measured at the same time point.We then performed a prospective study following up on the samesubjects to find out if they had a recurrent asthma attack withinone month of the tests. We also reassessed their peak expiratoryflow one month after blood specimens were obtained.Results Thirty-nine patients were enrolled in this study. Therewas no significant correlation between SOD level and peakexpiratory flow [r=0.289; 95%CI -0.025 to 0.47; P=0.074].However, older age was significantly associated with higher peakexpiratory flow (=0.5; 95%CI 3.10 to 11.57; P=0.01. Lowerlevels of SOD increased the risk of asthma attacks in a monthfollowing the initial measurements (RR=5.5; 95%CI 1.6 to 18.9;P=0.009.Conclusion Superoxide dismutase (SOD level is not significantlyassociated with peak expiratory flow. However, we find arelationship between older age and higher peak expiratory flowand a relationship between lower SOD levels and risk of asthmaattacks within one month following the tests. [

  18. Redox properties of a mononuclear copper(II)-superoxide complex.

    Science.gov (United States)

    Tano, Tetsuro; Okubo, Yuri; Kunishita, Atsushi; Kubo, Minoru; Sugimoto, Hideki; Fujieda, Nobutaka; Ogura, Takashi; Itoh, Shinobu

    2013-09-16

    Redox properties of a mononuclear copper(II) superoxide complex, (L)Cu(II)-OO(•), supported by a tridentate ligand (L = 1-(2-phenethyl)-5-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane) have been examined as a model compound of the putative reactive intermediate of peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) (Kunishita et al. J. Am. Chem. Soc. 2009, 131, 2788-2789; Inorg. Chem. 2012, 51, 9465-9480). On the basis of the reactivity toward a series of one-electron reductants, the reduction potential of (L)Cu(II)-OO(•) was estimated to be 0.19 ± 0.07 V vs SCE in acetone at 298 K (cf. Tahsini et al. Chem.-Eur. J. 2012, 18, 1084-1093). In the reaction of TEMPO-H (2,2,6,6-tetramethylpiperidine-N-hydroxide), a simple HAT (hydrogen atom transfer) reaction took place to give the corresponding hydroperoxide complex LCu(II)-OOH, whereas the reaction with phenol derivatives ((X)ArOH) gave the corresponding phenolate adducts, LCu(II)-O(X)Ar, presumably via an acid-base reaction between the superoxide ligand and the phenols. The reaction of (L)Cu(II)-OO(•) with a series of triphenylphosphine derivatives gave the corresponding triphenylphosphine oxides via an electrophilic ionic substitution mechanism with a Hammett ρ value as -4.3, whereas the reaction with thioanisole (sulfide) only gave a copper(I) complex. These reactivities of (L)Cu(II)-OO(•) are different from those of a similar end-on superoxide copper(II) complex supported by a tetradentate TMG3tren ligand (1,1,1-Tris{2-[N(2)-(1,1,3,3-tetramethylguanidino)]ethyl}amine (Maiti et al. Angew. Chem., Int. Ed. 2008, 47, 82-85).

  19. A superoxide dismutase of metacestodes of Taenia taeniaeformis.

    Science.gov (United States)

    Leid, R W; Suquet, C M

    1986-03-01

    Superoxide dismutase was purified from Taenia taeniaeformis metacestodes by sequential ion exchange chromatography on quaternary-amino-ethyl-cellulose, gel filtration chromatography on ACA 44 and ion exchange chromatography on DEAE-cellulose, followed by chromatofocusing on polybuffer exchanger 94. This isolation procedure resulted in the detection of a single protein-staining band on alkaline gels, coincident with enzyme activity. We have, however, detected what appear to be two peaks of enzyme activity within this single protein-staining band. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis using gradient slab gels and analysis under reducing conditions, resulted in the detection of only one protein at an apparent Mr of 16,600, while analysis under non-reducing conditions, gave a single protein of an apparent Mr of 64,000. The isoelectric point of the purified protein is 6.6. Boiling for 3 min completely destroyed the enzyme, whereas incubation for 2 h at 37 degrees C resulted in the loss of 56% of the enzymic activity. Incubation with 10 mM KCN resulted in 83% inhibition of the enzyme. We have detected up to 168 U ml-1 of enzyme activity in the cyst fluid surrounding the parasite in situ. This is the first instance in which any parasite superoxide dismutase has been purified to apparent homogeneity. Parasite-mediated enzymic destruction of superoxide anion can not only protect against oxygen toxicity as a result of normal parasite respiratory processes but also may serve as yet another mechanism used by tissue-dwelling parasites to evade host immunologic attack.

  20. Theoretical determination of the alkali-metal superoxide bond energies

    Science.gov (United States)

    Partridge, Harry; Bauschlicher, Charles W., Jr.; Sodupe, Mariona; Langhoff, Stephen R.

    1992-01-01

    The bond dissociation energies for the alkali-metal superoxides have been computed using extensive Gaussian basis sets and treating electron correlation at the modified coupled-pair functional level. Our computed D0 values are 61.4, 37.2, 40.6, and 38.4 kcal/mol for LiO2, NaO2, KO2, and RbO2, respectively. These values, which are expected to be lower bounds and accurate to 2 kcal/mol, agree well with some of the older flame data, but rule out several recent experimental measurements.

  1. Methylglyoxal as a scavenger for superoxide anion-radical.

    Science.gov (United States)

    Shumaev, K B; Lankin, V Z; Konovalova, G G; Grechnikova, M A; Tikhaze, A K

    2016-07-01

    Methylglyoxal at a concentration of 5 mM caused a significant inhibition of superoxide anion radical (O2 (·-)) comparable to the effect of Tirone. In the process of O2 (·-) generation in the system of egg phosphatidylcholine liposome peroxidation induced by the azo-initiator AIBN, a marked inhibition of chemiluminescence in the presence of 100 mM methylglyoxal was found. At the same time, methylglyoxal did not inhibit free radical peroxidation of low-density lipoprotein particles, which indicates the absence of interaction with methylglyoxal alkoxyl and peroxyl polyenoic lipid radicals. These findings deepen information about the role of methylglyoxal in the regulation of free radical processes.

  2. Differential pH sensitivity of tissue superoxide dismutases

    OpenAIRE

    Patel, Samir P.; Katyare, Surendra S.

    2006-01-01

    Superoxide dismutase (SOD) activities in the human and rat RBCs and rat liver, kidney, brain and heart mitochondria as well as cytosolic fractions were determined by the pyrogallol assay procedure with slight modifications. Measurements were carried out in 0.1 M potassium phosphate buffer pH 8.0 and 9.2 to assess the pH stability of the SODs from various systems. Under these conditions the SODs from different systems including RBCs exhibited differential pH stability i.e. they displayed diffe...

  3. Superoxide anion-induced pain and inflammation depends on TNFα/TNFR1 signaling in mice.

    Science.gov (United States)

    Yamacita-Borin, Fabiane Y; Zarpelon, Ana C; Pinho-Ribeiro, Felipe A; Fattori, Victor; Alves-Filho, Jose C; Cunha, Fernando Q; Cunha, Thiago M; Casagrande, Rubia; Verri, Waldiceu A

    2015-09-25

    Inhibition of tumor necrosis factor-alpha (TNFα) and superoxide anion production reduces inflammation and pain. The present study investigated whether superoxide anion-induced pain depends on TNFα signaling and the role of superoxide anion in TNFα-induced hyperalgesia to clarify the interrelation between these two mediators in the context of pain. Intraplantar injection of a superoxide anion donor (potassium superoxide) induced mechanical hyperalgesia (0.5-5h after injection), neutrophil recruitment (myeloperoxidase activity), and overt pain-like behaviors (paw flinching, paw licking, and abdominal writhings) in wild-type mice. Tumor necrosis factor receptor 1 deficiency (TNFR1-/-) and treatment of wild-type mice with etanercept (a soluble TNFR2 receptor that inhibits TNFα actions) inhibited superoxide anion-induced pain-like behaviors. TNFR1(-/-) mice were also protected from superoxide anion donor-induced oxidative stress, suggesting the role of this pathway in the maintenance of oxidative stress. Finally, we demonstrated that Apocynin (an NADPH oxidase inhibitor) or Tempol (a superoxide dismutase mimetic) treatment inhibited TNFα-induced paw mechanical hyperalgesia and neutrophil recruitment (myeloperoxidase activity). These results demonstrate that TNFα/TNFR1 signaling is important in superoxide anion-triggered pain and that TNFα/TNFR1 signaling amplifies the oxidative stress triggered by superoxide anion, which contributes to sustaining pain and inflammation.

  4. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

    Science.gov (United States)

    Hampton, M B; Kettle, A J; Winterbourn, C C

    1996-09-01

    We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-deficient neutrophils were used. Adding the NADPH oxidase inhibitor diphenyleneiodonium, to prevent superoxide production, decreased the killing rate to 25%, indicating that oxidative killing mechanisms predominate in this system. The rate constant for killing of S. aureus with superoxide dismutase attached was 70% of that for control bacteria linked to inactivated enzyme. Superoxide dismutase had no effect in the presence of diphenyleneiodonium. The rate of killing was decreased to 33% in the presence of azide and to 40% with myeloperoxidase-deficient neutrophils. Superoxide dismutase had no effect in the presence of azide. On the assumption that the oxidative and nonoxidative components of killing can be considered separately, the oxidative rate was decreased by almost half by superoxide dismutase and was about six times lower when myeloperoxidase was inactive. We conclude that myeloperoxidase-dependent processes are strongly favored by human neutrophils as their prime mechanism of oxidative killing of S. aureus and that superoxide makes a direct contribution to killing. Our results also suggest that superoxide acts in conjunction with a myeloperoxidase-dependent pathway.

  5. Dark production of extracellular superoxide by the coral Porites astreoides and representative symbionts

    Directory of Open Access Journals (Sweden)

    Tong Zhang

    2016-11-01

    Full Text Available The reactive oxygen species (ROS superoxide has been implicated in both beneficial and detrimental processes in coral biology, ranging from pathogenic disease resistance to coral bleaching. Despite the critical role of ROS in coral health, there is a distinct lack of ROS measurements and thus an incomplete understanding of underpinning ROS sources and production mechanisms within coral systems. Here, we quantified in situ extracellular superoxide concentrations at the surfaces of aquaria-hosted Porites astreoides during a diel cycle. High concentrations of superoxide (~10’s of nM were present at coral surfaces, and these levels did not change significantly as a function of time of day. These results indicate that the coral holobiont produces extracellular superoxide in the dark, independent of photosynthesis. As a short-lived anion at physiological pH, superoxide has a limited ability to cross intact biological membranes. Further, removing surface mucus layers from the P. astreoides colonies did not impact external superoxide concentrations. We therefore attribute external superoxide derived from the coral holobiont under these conditions to the activity of the coral host epithelium, rather than mucus-derived epibionts or internal sources such as endosymbionts (e.g., Symbiodinium. However, endosymbionts likely contribute to internal ROS levels via extracellular superoxide production. Indeed, common coral symbionts, including multiple strains of Symbiodinium (clades A to D and the bacterium Endozoicomonas montiporae LMG 24815, produced extracellular superoxide in the dark and at low light levels. Further, representative P. astreoides symbionts, Symbiodinium CCMP2456 (clade A and E. montiporae, produced similar concentrations of superoxide alone and in combination with each other, in the dark and low light, and regardless of time of day. Overall, these results indicate that healthy, non-stressed P. astreoides and representative symbionts produce

  6. A lithium-oxygen battery based on lithium superoxide.

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Jun; Lee, Yun Jung; Luo, Xiangyi; Lau, Kah Chun; Wen, Jianguo; Wang, Hsien-Hau; Zhai, Dengyun; Miller, Dean; Jeong, Yo-Sub; Park, Jin-Bum; Curtiss, Larry A.; Amine, Khalil

    2016-01-11

    Although the superoxide of lithium (LiO2) is believed to be a key intermediate in Li-O2 batteries leading to the formation of lithium peroxide, LiO2 has never been observed in its pure state. In this work, we provide evidence that use of a cathode based on a reduced graphene oxide with Ir nanoparticles in a Li-O2 battery results in a LiO2 discharge product formed by single electron transfer without further electron transfer or disproportionation to form Li2O2. High energy X-ray diffraction (HE-XRD) patterns indicates the presence of crystalline LiO2 with no evidence of Li2O2 or Li2O. The HEXRD studies as a function of time also show that LiO2 can be stable in its crystalline form after one week of aging in the presence of electrolyte. The results provide evidence that LiO2 is stable enough that it can be repeatedly charged and discharged with a very low charge potential (~3.2 V) and may open the avenue for a lithium superoxide-based battery.

  7. Superoxide DismutasE in Spring Barley Caryopses

    Directory of Open Access Journals (Sweden)

    Natálie Březinová Belcredi

    2016-01-01

    Full Text Available Superoxide dismutase (SOD activity was determined in caryopses of spring barley grown in field trials in 2004–2006. A total set under study included five malting varieties with hulled grain, three waxy hull-less and hulled varieties (of US origin, seven lines formed by crossing of the above given varieties and four hull-less lines of Czech origin. SOD activity was determined by a modified method using a Ransod diagnostic kit (RANDOX. The method employs xanthine and xanthine oxidase to generate superoxide radicals which react with 2-(4-iodophenyl-3-(4-nitrophenol-5-phenyltetrazolium chloride (INT to form a red formazan dye. Statistically significantly higher activity was measured in the variety Nordus (131 U.g−1 d.m. and line ME1 (128 U.g−1 d.m. compared to the other varieties/lines (66–111 U.g−1 d.m.. The line ME1 had significantly higher SOD activity in grain versus its parental varieties Kompakt (83 U.g−1 d.m. and Krona (78 U.g−1 d.m.. The results of this study proved the availability of varieties/lines with a higher SOD content, the antioxidant effect of SOD can improve quality of beer and food made from barley.

  8. Enzyme superoxide dismutase in grain of barley and malt

    Directory of Open Access Journals (Sweden)

    Natálie Belcrediová

    2006-01-01

    Full Text Available The aim of the work was modification of superoxide dismutase enzyme (SOD, EC 1.15.1.1 activity analysis in barley grain and identical malts with using of the Ransod set. This set from company Randox were used for enzyme determination in blood samples. This method employs xanthine and xanthine oxidase to generate superoxide radicals, which react with tetrazolium chloride to form a red formazan dye. SOD is classified as natural antioxidants and enzyme plays a significant role at detoxication of products of molecular oxygen degradation. The largest rate of SOD occurs in embryo of barley grain. Its presence in barley grain and malt thus inhibits rancidity of grain during storage and undesirable beer flavour. The line Wabet x Washonubet (in grain-104,93 and malt 152,42 U/g dry matter and the variety Annabell (104,65 a 147,21 U/g dry matter had the highest activity of SOD in grain and malt of barley while the lowest activity was measured in the line KM 1910 (73,15 a 88,16 U/g dry matter and variety Tolar (74,34 a 96,44 U/g dry matter.

  9. Mechanism of Action of Sulforaphane as a Superoxide Radical Anion and Hydrogen Peroxide Scavenger by Double Hydrogen Transfer: A Model for Iron Superoxide Dismutase.

    Science.gov (United States)

    Prasad, Ajit Kumar; Mishra, P C

    2015-06-25

    The mechanism of action of sulforaphane as a scavenger of superoxide radical anion (O2(•-)) and hydrogen peroxide (H2O2) was investigated using density functional theory (DFT) in both gas phase and aqueous media. Iron superoxide dismutase (Fe-SOD) involved in scavenging superoxide radical anion from biological media was modeled by a complex consisting of the ferric ion (Fe(3+)) attached to three histidine rings. Reactions related to scavenging of superoxide radical anion by sulforaphane were studied using DFT in the presence and absence of Fe-SOD represented by this model in both gas phase and aqueous media. The scavenging action of sulforaphane toward both superoxide radical anion and hydrogen peroxide was found to involve the unusual mechanism of double hydrogen transfer. It was found that sulforaphane alone, without Fe-SOD, cannot scavenge superoxide radical anion in gas phase or aqueous media efficiently as the corresponding reaction barriers are very high. However, in the presence of Fe-SOD represented by the above-mentioned model, the scavenging reactions become barrierless, and so sulforaphane scavenges superoxide radical anion by converting it to hydrogen peroxide efficiently. Further, sulforaphane was found to scavenge hydrogen peroxide also very efficiently by converting it into water. Thus, the mechanism of action of sulforaphane as an excellent antioxidant has been unravelled.

  10. Superoxide Mediates the Toxicity of Paraquat for Chinese Hamster Ovary Cells

    Science.gov (United States)

    Bagley, Ann C.; Krall, Judith; Lynch, Robert E.

    1986-05-01

    The roles of superoxide and H2O2 in the cytotoxicity of paraquat were assessed in Chinese hamster ovary cells. Neither catalase nor superoxide dismutase inhibited the loss of ability to form colonies when added to the medium. When introduced into the cells, superoxide dismutase but not catalase inhibited the toxicity of paraquat. That superoxide dismutase acted by its known catalytic action is shown by the loss of inhibition when the enzyme was inactivated by H2O2 before being introduced into the cells. The lack of inhibition by catalase, by dimethyl sulfoxide, and by desferoxamine suggests that the toxicity is not mediated by a reaction between H2O2 and superoxide to engender the hydroxyl radical. Exposure of Chinese hamster ovary cells to paraquat may be a suitable means to determine the effects of superoxide anion in cultured cells and the ways in which cells can resist this toxic action.

  11. Superoxide disproportionation driven by zinc complexes with various steric and electrostatic properties.

    Science.gov (United States)

    Wada, Akira; Jitsukawa, Koichiro; Masuda, Hideki

    2013-11-18

    Attractive models: Synthetic Zn(II) complexes were investigated as models of copper-zinc superoxide dismutase. Superoxide underwent a unique disproportionation reaction in the electrostatic sphere of the complexes (see picture; bpy=2,2'-bipyridyl). The effectiveness of the Zn(II) complexes in inducing the disproportionation of superoxide depended on both the Lewis acidity and the coordination geometry of the Zn center.

  12. Mitochondrial superoxide flashes: metabolic biomarkers of skeletal muscle activity and disease

    OpenAIRE

    2011-01-01

    Mitochondrial superoxide flashes (mSOFs) are stochastic events of quantal mitochondrial superoxide generation. Here, we used flexor digitorum brevis muscle fibers from transgenic mice with muscle-specific expression of a novel mitochondrial-targeted superoxide biosensor (mt-cpYFP) to characterize mSOF activity in skeletal muscle at rest, following intense activity, and under pathological conditions. Results demonstrate that mSOF activity in muscle depended on electron transport chain and aden...

  13. Involvement of superoxide and myeloperoxidase in oxygen-dependent killing of Staphylococcus aureus by neutrophils.

    OpenAIRE

    Hampton, M B; A. J. Kettle; Winterbourn, C C

    1996-01-01

    We have used a quantitative assay that measures independent rate constants for phagocytosis and killing of Staphylococcus aureus to investigate the involvement of superoxide and myeloperoxidase in bacterial killing by human neutrophils. To inhibit superoxide-dependent processes, superoxide dismutase was cross-linked to immunoglobulin G and the conjugate was attached to the surface of S. aureus via protein A in its cell wall. Myeloperoxidase was inhibited with azide, and myeloperoxidase-defici...

  14. Permeability transition pore-mediated mitochondrial superoxide flashes regulate cortical neural progenitor differentiation.

    Science.gov (United States)

    Hou, Yan; Mattson, Mark P; Cheng, Aiwu

    2013-01-01

    In the process of neurogenesis, neural progenitor cells (NPCs) cease dividing and differentiate into postmitotic neurons that grow dendrites and an axon, become excitable, and establish synapses with other neurons. Mitochondrial biogenesis and aerobic metabolism provide energy substrates required to support the differentiation, growth and synaptic activity of neurons. Mitochondria may also serve signaling functions and, in this regard, it was recently reported that mitochondria can generate rapid bursts of superoxide (superoxide flashes), the frequency of which changes in response to environmental conditions and signals including oxygen levels and Ca(2+) fluxes. Here we show that the frequency of mitochondrial superoxide flashes increases as embryonic cerebral cortical neurons differentiate from NPCs, and provide evidence that the superoxide flashes serve a signaling function that is critical for the differentiation process. The superoxide flashes are mediated by mitochondrial permeability transition pore (mPTP) opening, and pharmacological inhibition of the mPTP suppresses neuronal differentiation. Moreover, superoxide flashes and neuronal differentiation are inhibited by scavenging of mitochondrial superoxide. Conversely, manipulations that increase superoxide flash frequency accelerate neuronal differentiation. Our findings reveal a regulatory role for mitochondrial superoxide flashes, mediated by mPTP opening, in neuronal differentiation.

  15. Selective superoxide generation within mitochondria by the targeted redox cycler MitoParaquat.

    Science.gov (United States)

    Robb, Ellen L; Gawel, Justyna M; Aksentijević, Dunja; Cochemé, Helena M; Stewart, Tessa S; Shchepinova, Maria M; Qiang, He; Prime, Tracy A; Bright, Thomas P; James, Andrew M; Shattock, Michael J; Senn, Hans M; Hartley, Richard C; Murphy, Michael P

    2015-12-01

    Superoxide is the proximal reactive oxygen species (ROS) produced by the mitochondrial respiratory chain and plays a major role in pathological oxidative stress and redox signaling. While there are tools to detect or decrease mitochondrial superoxide, none can rapidly and specifically increase superoxide production within the mitochondrial matrix. This lack impedes progress, making it challenging to assess accurately the roles of mitochondrial superoxide in cells and in vivo. To address this unmet need, we synthesized and characterized a mitochondria-targeted redox cycler, MitoParaquat (MitoPQ) that comprises a triphenylphosphonium lipophilic cation conjugated to the redox cycler paraquat. MitoPQ accumulates selectively in the mitochondrial matrix driven by the membrane potential. Within the matrix, MitoPQ produces superoxide by redox cycling at the flavin site of complex I, selectively increasing superoxide production within mitochondria. MitoPQ increased mitochondrial superoxide in isolated mitochondria and cells in culture ~a thousand-fold more effectively than untargeted paraquat. MitoPQ was also more toxic than paraquat in the isolated perfused heart and in Drosophila in vivo. MitoPQ enables the selective generation of superoxide within mitochondria and is a useful tool to investigate the many roles of mitochondrial superoxide in pathology and redox signaling in cells and in vivo.

  16. Superoxide Induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism

    Science.gov (United States)

    Al-Khafaji, Ahmed B; Tohme, Samer; Yazdani, Hamza Obaid; Miller, David; Huang, Hai; Tsung, Allan

    2016-01-01

    Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil extracellular traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia, including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and nonparenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination and increased levels of MPO-DNA complexes occurring in a TLR-4–dependent manner. Inhibition of superoxide generation by allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT but not TLR-4 KO mice. To reduce circulating superoxide, we pretreated mice undergoing I/R with allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings. PMID:27453505

  17. Singlet oxygen production in the reaction of superoxide with organic peroxides.

    Science.gov (United States)

    MacManus-Spencer, Laura A; Edhlund, Betsy L; McNeill, Kristopher

    2006-01-20

    [reaction: see text] A selective chemiluminescent probe for singlet oxygen has been employed to detect and quantify singlet oxygen in the reactions of superoxide with organic peroxides. The production of singlet oxygen has been quantified in the reaction of superoxide with benzoyl peroxide (BP). No singlet oxygen was detected in the reactions of superoxide with cumyl peroxide, tert-butyl peroxide, or tert-butyl hydroperoxide. On the basis of these results and on the temperature dependence of the reaction, we proposed a mechanism for singlet oxygen formation in the reaction of superoxide with BP.

  18. State of the art and the dark side of amyotrophic lateral sclerosis

    Institute of Scientific and Technical Information of China (English)

    Antonio; Musarò

    2010-01-01

    Amyotrophic lateral sclerosis(ALS) is a disorder that involves the degeneration of motor neurons,muscle atrophy,and paralysis.In a few familiar forms of ALS,mutations in the superoxide dismutase-1(SOD1) gene have been held responsible for the degeneration of motor neurons.Nevertheless,after the discovery of the SOD1 mutations no consensus has emerged as to which cells,tissues and pathways are primarily implicated in the pathogenic events that lead to ALS.Ubiquitous overexpression of mutant SOD1 in transgenic animals recapitulates the pathological features of ALS.However,the toxicity of mutant SOD1 is not necessarily limited to the central nervous system.Views about ALS pathogenesis are now enriched by the recent discovery of mutations in a pair of DNA/RNA-binding proteins called TDP-43 and FUS/TLS as causes of familial and sporadic forms of ALS.Although the steps that lead to the pathological state are well defined,several fundamental issues are still controversial:are the motor neurons the first direct targets of ALS;and what is the contribution of non-neuronal cells,if any,to the pathogenesis of ALS?The state of the art of ALS pathogenesis and the open questions are discussed in this review.

  19. Intrinsic Membrane Hyperexcitability of Amyotrophic Lateral Sclerosis Patient-Derived Motor Neurons

    Directory of Open Access Journals (Sweden)

    Brian J. Wainger

    2014-04-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a fatal neurodegenerative disease of the motor nervous system. We show using multielectrode array and patch-clamp recordings that hyperexcitability detected by clinical neurophysiological studies of ALS patients is recapitulated in induced pluripotent stem cell-derived motor neurons from ALS patients harboring superoxide dismutase 1 (SOD1, C9orf72, and fused-in-sarcoma mutations. Motor neurons produced from a genetically corrected but otherwise isogenic SOD1+/+ stem cell line do not display the hyperexcitability phenotype. SOD1A4V/+ ALS patient-derived motor neurons have reduced delayed-rectifier potassium current amplitudes relative to control-derived motor neurons, a deficit that may underlie their hyperexcitability. The Kv7 channel activator retigabine both blocks the hyperexcitability and improves motor neuron survival in vitro when tested in SOD1 mutant ALS cases. Therefore, electrophysiological characterization of human stem cell-derived neurons can reveal disease-related mechanisms and identify therapeutic candidates.

  20. Protein Misdirection Inside and Outside Motor Neurons in Amyotrophic Lateral Sclerosis (ALS: A Possible Clue for Therapeutic Strategies

    Directory of Open Access Journals (Sweden)

    Akemi Ido

    2011-10-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a devastating neurodegenerative disease characterized by progressive muscle wasting and weakness with no effective cure. Emerging evidence supports the notion that the abnormal conformations of ALS-linked proteins play a central role in triggering the motor neuron degeneration. In particular, mutant types of superoxide dismutase 1 (SOD1 and TAR DNA binding protein 43kDa (TDP-43 are key molecules involved in the pathogenesis of familial and sporadic ALS, respectively. The commonalities of the two proteins include a propensity to aggregate and acquire detrimental conformations through oligomerization, fragmentation, or post-translational modification that may drive abnormal subcellular localizations. Although SOD1 is a major cytosolic protein, mutated SOD1 has been localized to mitochondria, endoplasmic reticulum, and even the extracellular space. The nuclear exclusion of TDP-43 is a pathological hallmark for ALS, although the pathogenic priority remains elusive. Nevertheless, these abnormal behaviors based on the protein misfolding are believed to induce diverse intracellular and extracellular events that may be tightly linked to non-cell-autonomous motor neuron death. The generation of mutant- or misfolded protein-specific antibodies would help to uncover the distribution and propagation of the ALS-linked proteins, and to design a therapeutic strategy to clear such species. Herein we review the literature regarding the mislocalization of ALS-linked proteins, especially mutant SOD1 and TDP-43 species, and discuss the rationale of molecular targeting strategies including immunotherapy.

  1. Mechanism and kinetics for scavenging superoxide anion by progesterone

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    The chemical reaction of progesterone with superoxide anion in 0.1 mol/L NaHCO3 medium is studied by polarography. Differing from the indirect inhibition of generation by synthesized glucocorticoids in mechanism, the function that progesterone scavenges is ascribed to that directly oxidizes the C == C double bond conjugated with the carbonyl moiety of progesterone molecule to a free radical, and then is reduced to H2O2. The result obtained in this work gives new evidence for biomedical research. The equation of rate constant of the oxidization reaction is de-duced, and the apparent rate constant obtained is 308 L·mol-1·s-1.

  2. Levels of erythrocyte superoxide dismutase activity in Japanese people

    Directory of Open Access Journals (Sweden)

    Ueda,Kazuko

    1978-12-01

    Full Text Available Levels of erythrocyte superoxide dismutase (SOD activitiy in a sample of Japanese people were determined. Blood samples were taken from new-born infants, preschool children, young and old people who had no apparent diseases and also from three anemic patients. Erythrocyte SOD activities in different age groups had a nearly normal distribution. Females had slightly lower activities than males, although the difference was statistically insignificant. The distributions of SOD activities were 12.6 +/- 2.7 (m +/- SD unit/mg Hb in young people and 11.4 +/- 3.0 in old people, indicating that erythrocyte SOD activity falls with aging. Because of low concentration of hemoglobin, SOD activities of old people expressed as unit/ml blood were much lower than in young people. Three anemic patients had slightly lower SOD activity.

  3. Immobilization of superoxide dismutase on Pt-Pd/MWCNTs hybrid modified electrode surface for superoxide anion detection.

    Science.gov (United States)

    Zhu, Xiang; Niu, Xiangheng; Zhao, Hongli; Tang, Jie; Lan, Minbo

    2015-05-15

    Monitoring of reactive oxygen species like superoxide anion (O2(∙-)) turns to be of increasing significance considering their potential damages to organism. In the present work, we fabricated a novel O2(∙-) electrochemical sensor through immobilizing superoxide dismutase (SOD) onto a Pt-Pd/MWCNTs hybrid modified electrode surface. The Pt-Pd/MWCNTs hybrid was synthesized via a facile one-step alcohol-reduction process, and well characterized by transmission electron microscopy, X-ray photoelectron spectroscopy and X-ray diffraction. The immobilization of SOD was accomplished using a simple drop-casting method, and the performance of the assembled enzyme-based sensor for O2(∙-) detection was systematically investigated by several electrochemcial techniques. Thanks to the specific biocatalysis of SOD towards O2(∙-) and the Pt-Pd/MWCNTs - promoted fast electron transfer at the fabricated interface, the developed biosensor exhibits a fast, selective and linear amperometric response upon O2(∙-) in the concentration scope of 40-1550 μM (R(2)=0.9941), with a sensitivity of 0.601 mA cm(-2) mM(-1) and a detection limit of 0.71 μM (S/N=3). In addition, the favorable biocompatibility of this electrode interface endows the prepared biosensor with excellent long-term stability (a sensitivity loss of only 3% over a period of 30 days). It is promising that the proposed sensor will be utilized as an effective tool to quantitatively monitor the dynamic changes of O2(∙-) in biological systems.

  4. Effect of superoxide and superoxide-generating systems on the prooxidant effect of iron in oil emulsion and raw turkey homogenates.

    Science.gov (United States)

    Ahn, D U; Kim, S M

    1998-09-01

    Mechanisms of superoxide.O2--generating systems on the pro-oxidant effect of iron from various sources were studied. Reaction mixtures were prepared with distilled water, oil emulsion, or meat homogenates. Free ionic iron (ferrous and ferric), ferritin and hemoglobin (Hb) were used as iron sources, and KO2 and xanthine oxidase (XOD) systems were used to produce .O2-. Thiobarbituric acid reactive substances (TBARS) values and iron contents of the reaction mixtures were determined. Ferric iron and ferritin, in the presence or absence of superoxide-generating systems, had no catalytic effect on the oxidation of oil emulsion but became pro-oxidants when reducing agent (ascorbate) was present. Ferrous iron and Hb had strong catalytic effects on the oxidation of oil emulsion as shown by TBARS values. Superoxide and H2O2, generated from superoxide-generating systems, oxidized ferrous iron and ascorbate, and lowered the pro-oxidant effect of ferrous iron in oil emulsion. Addition of ferric or ferrous iron increased but Hb did not have any effect on the TBARS values of raw meat homogenates. The reaction mechanisms of superoxide and the superoxide-generating systems on the prooxidant effect of various iron sources indicated that .O2- was a strong oxidizer rather than a reducing agent, and the antioxidant effect of XOD system in oil was caused by the oxidation of ferrous iron to the ferric form by .O2- and/or H2O2.

  5. Mutant TDP-43 deregulates AMPK activation by PP2A in ALS models.

    Directory of Open Access Journals (Sweden)

    Nirma D Perera

    Full Text Available Bioenergetic abnormalities and metabolic dysfunction occur in amyotrophic lateral sclerosis (ALS patients and genetic mouse models. However, whether metabolic dysfunction occurs early in ALS pathophysiology linked to different ALS genes remains unclear. Here, we investigated AMP-activated protein kinase (AMPK activation, which is a key enzyme induced by energy depletion and metabolic stress, in neuronal cells and mouse models expressing mutant superoxide dismutase 1 (SOD1 or TAR DNA binding protein 43 (TDP-43 linked to ALS. AMPK phosphorylation was sharply increased in spinal cords of transgenic SOD1G93A mice at disease onset and accumulated in cytoplasmic granules in motor neurons, but not in presymptomatic mice. AMPK phosphorylation also occurred in peripheral tissues, liver and kidney, in SOD1G93A mice at disease onset, demonstrating that AMPK activation occurs late and is not restricted to motor neurons. Conversely, AMPK activity was drastically diminished in spinal cords and brains of presymptomatic and symptomatic transgenic TDP-43A315T mice and motor neuronal cells expressing different TDP-43 mutants. We show that mutant TDP-43 induction of the AMPK phosphatase, protein phosphatase 2A (PP2A, is associated with AMPK inactivation in these ALS models. Furthermore, PP2A inhibition by okadaic acid reversed AMPK inactivation by mutant TDP-43 in neuronal cells. Our results suggest that mutant SOD1 and TDP-43 exert contrasting effects on AMPK activation which may reflect key differences in energy metabolism and neurodegeneration in spinal cords of SOD1G93A and TDP-43A315T mice. While AMPK activation in motor neurons correlates with progression in mutant SOD1-mediated disease, AMPK inactivation mediated by PP2A is associated with mutant TDP-43-linked ALS.

  6. The French Paradox: Determining the Superoxide-Scavenging Capacity of Red Wine and Other Beverages

    Science.gov (United States)

    Logan, Barry A.; Hammond, Matthew P.; Stormo, Benjamin M.

    2008-01-01

    Plant-derived phenolic compounds such as those found in red wine, tea, and certain fruit juices may protect against cardiovascular disease by detoxifying (scavenging) superoxide and other unstable reactive oxygen species. We present a laboratory exercise that can be used to assess the superoxide-scavenging capacity of beverages. Among the…

  7. Do Superoxide Dismutase (SOD) and Catalase (CAT) protect Cells from DNA Damage Induced by Active Arsenicals?

    Science.gov (United States)

    Superoxide dismutase (SOD) catalyzes the conversion of superoxide to hydrogen peroxide, which can be converted to water and oxygen through the action of catalase. Heterozygous mice of strain B6: 129S7-SodltmlLeb/J were obtained from Jackson Laboratories and bred to produce offspr...

  8. A new formula to calculate activity of superoxide dismutase in indirect assays

    NARCIS (Netherlands)

    Zhang, Chen; Bruins, Marieke E.; Yang, Zhi Qiang; Liu, Shu Tao; Rao, Ping Fan

    2016-01-01

    To calculate superoxide dismutase (SOD) activity rapidly and accurately by indirect SOD assays, a formula based on the ratio of the catalytic speed of SOD to the reaction speed of the indicator with superoxide anion was deduced. The accuracy of this formula was compared with the conventional form

  9. Isolation and characterization of Cu,Zn superoxide dismutase of the shark Prionace glauca.

    Science.gov (United States)

    Galtieri, A; Natoli, G; Lania, A; Calabrese, L

    1986-01-01

    A Cu,Zn superoxide dismutase was purified for the first time from an elasmobranch species (Prionace glauca) and showed the following differences with respect to other animal superoxide dismutases. The enzyme displays a low isoelectric point. The enzyme activity is unusually independent of ionic strength. The isolated enzyme has 30% of its copper in the reduced state.

  10. Comparative analysis of cyanobacterial superoxide dismutases to discriminate canonical forms

    Directory of Open Access Journals (Sweden)

    Prabaharan Dharmar

    2007-11-01

    Full Text Available Abstract Background Superoxide dismutases (SOD are ubiquitous metalloenzymes that catalyze the disproportion of superoxide to peroxide and molecular oxygen through alternate oxidation and reduction of their metal ions. In general, SODs are classified into four forms by their catalytic metals namely; FeSOD, MnSOD, Cu/ZnSOD and NiSOD. In addition, a cambialistic form that uses Fe/Mn in its active site also exists. Cyanobacteria, the oxygen evolving photosynthetic prokaryotes, produce reactive oxygen species that can damage cellular components leading to cell death. Thus, the co-evolution of an antioxidant system was necessary for the survival of photosynthetic organisms with SOD as the initial enzyme evolved to alleviate the toxic effect. Cyanobacteria represent the first oxygenic photoautotrophs and their SOD sequences available in the databases lack clear annotation. Hence, the present study focuses on structure and sequence pattern of subsets of cyanobacterial superoxide dismutases. Result The sequence conservation and structural analysis of Fe (Thermosynechococcus elongatus BP1 and MnSOD (Anabaena sp. PCC7120 reveal the sharing of N and C terminal domains. At the C terminal domain, the metal binding motif in cyanoprokaryotes is DVWEHAYY while it is D-X-[WF]-E-H-[STA]-[FY]-[FY] in other pro- and eukaryotes. The cyanobacterial FeSOD differs from MnSOD at least in three ways viz. (i FeSOD has a metal specific signature F184X3A188Q189.......T280......F/Y303 while, in Mn it is R184X3G188G189......G280......W303, (ii aspartate ligand forms a hydrogen bond from the active site with the outer sphere residue of W243 in Fe where as it is Q262 in MnSOD; and (iii two unique lysine residues at positions 201 and 255 with a photosynthetic role, found only in FeSOD. Further, most of the cyanobacterial Mn metalloforms have a specific transmembrane hydrophobic pocket that distinguishes FeSOD from Mn isoform. Cyanobacterial Cu/ZnSOD has a copper domain and two

  11. Superoxide radicals can act synergistically with hypochlorite to induce damage to proteins

    DEFF Research Database (Denmark)

    Hawkins, Clare Louise; Rees, Martin D; Davies, Michael Jonathan

    2002-01-01

    Activated phagocytes generate both superoxide radicals via a respiratory burst, and HOCl via the concurrent release of the haem enzyme myeloperoxidase. Amine and amide functions on proteins and carbohydrates are major targets for HOCl, generating chloramines (RNHCl) and chloramides (RC(O)NClR'), ......Activated phagocytes generate both superoxide radicals via a respiratory burst, and HOCl via the concurrent release of the haem enzyme myeloperoxidase. Amine and amide functions on proteins and carbohydrates are major targets for HOCl, generating chloramines (RNHCl) and chloramides (RC......(O)NClR'), which can accumulate to high concentrations (>100 microM). Here we show that superoxide radicals catalyse the decomposition of chloramines and chloramides to reactive nitrogen-centred radicals, and increase the extent of protein fragmentation compared to that observed with either superoxide radicals...... or HOCl, alone. This synergistic action may be of significance at sites of inflammation, where both superoxide radicals and chloramines/chloramides are formed simultaneously....

  12. Inhibitory effects of cardols and related compounds on superoxide anion generation by xanthine oxidase.

    Science.gov (United States)

    Masuoka, Noriyoshi; Nihei, Ken-ichi; Maeta, Ayami; Yamagiwa, Yoshiro; Kubo, Isao

    2015-01-01

    5-Pentadecatrienylresorcinol, isolated from cashew nuts and commonly known as cardol (C₁₅:₃), prevented the generation of superoxide radicals catalysed by xanthine oxidase without the inhibition of uric acid formation. The inhibition kinetics did not follow the Michelis-Menten equation, but instead followed the Hill equation. Cardol (C₁₀:₀) also inhibited superoxide anion generation, but resorcinol and cardol (C₅:₀) did not inhibit superoxide anion generation. The related compounds 3,5-dihydroxyphenyl alkanoates and alkyl 2,4-dihydroxybenzoates, had more than a C9 chain, cooperatively inhibited but alkyl 3,5-dihydroxybenzoates, regardless of their alkyl chain length, did not inhibit the superoxide anion generation. These results suggested that specific inhibitors for superoxide anion generation catalysed by xanthine oxidase consisted of an electron-rich resorcinol group and an alkyl chain having longer than C9 chain.

  13. Studies on the Nucleophilicity and Scavenge of Superoxide Ion by Cyclic Voltammetry

    Institute of Scientific and Technical Information of China (English)

    Wei Ying-liang; Dang Xue-ping; Hu Sheng-shui

    2003-01-01

    Superoxide ion was generated by the electrochemical reduction of oxygen at a platinum electrode in dimethylsulphoxide (DMSO). This work was focused on the nucleophilicity and scavenge of electrogenerated-superoxide ion by cyclic voltammetry. The nucleophilic displacement reactions of superoxide ion with ethyl acetate and diethyl adipate were discussed and the reason for remarkable influence of diethyl adipate was elucidated. The scavenging activity of ascorbic acid was evaluated and the result allowed the conclusion that the scavenging ability of ascorbic acid is much lower in DMSO than in aqueous phase. UV-spectrum of electrogenerated superoxide ion in DMSO exhibited a single absorption band with λmax at 275 nm, which certified further that the method of electrogeneration was reliable and superoxide ion was stable in DMSO.

  14. Studies on the Nucleophilicity and Scavenge of Superoxide Ion by Cyclic Voltammetry

    Institute of Scientific and Technical Information of China (English)

    WeiYing-liang; DangXue-ping; HuSheng-shui

    2003-01-01

    Superoxide ion was generated by the electro-chemical reduction of oxygen at a platinum electrode in dimethylsulphoxide (DMSO). This work was focused on the nucleophilicity and scavenge of electrogenemted-superoxide ion by cyclic voltammetry. The nucleophilic displacement reactions of superoxide ion with ethyl acetate and diethyl adipate were discussed and the reason for remarkable influence of diethyl adipate was elucidated. The scavenging activity of ascorbic acid was evaluated and the result allowed the conclusion that the scavenging ability of ascorbic acid is much lower in DMSO than in aqueous phasc UV-spectrum of electrogenerated superoxide ion in DMSO exhibited a single absorption band with λmax at 275 nm, which certified further that the method of electrogeneration was reliable and superoxide ion was stable in DMSO.

  15. Bosentan, a mixed endothelin receptor antagonist, inhibits superoxide anion-induced pain and inflammation in mice.

    Science.gov (United States)

    Serafim, Karla G G; Navarro, Suelen A; Zarpelon, Ana C; Pinho-Ribeiro, Felipe A; Fattori, Victor; Cunha, Thiago M; Alves-Filho, Jose C; Cunha, Fernando Q; Casagrande, Rubia; Verri, Waldiceu A

    2015-11-01

    Bosentan is a mixed endothelin receptor antagonist widely used to treat patients with pulmonary arterial hypertension, and the emerging literature suggests bosentan as a potent anti-inflammatory drug. Superoxide anion is produced in large amounts during inflammation, stimulates cytokine production, and thus contributes to inflammation and pain. However, it remains to be determined whether endothelin contributes to the inflammatory response triggered by the superoxide anion. The present study investigated the effects of bosentan in a mouse model of inflammation and pain induced by potassium superoxide, a superoxide anion donor. Male Swiss mice were treated with bosentan (10-100 mg/kg) by oral gavage, 1 h before potassium superoxide injection, and the inflammatory response was evaluated locally and at spinal cord (L4-L6) levels. Bosentan (100 mg/kg) inhibited superoxide anion-induced mechanical and thermal hyperalgesia, overt pain-like behavior (abdominal writhings, paw flinching, and licking), paw edema, myeloperoxidase activity (neutrophil marker) in the paw skin, and leukocyte recruitment in the peritoneal cavity. Bosentan also inhibited superoxide anion-induced interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α) production, while it enhanced IL-10 production in the paw skin and spinal cord. Bosentan inhibited the reduction of antioxidant capacity (reduced glutathione, ferric reducing antioxidant power, and ABTS radical scavenging ability) induced by the superoxide anion. Finally, we demonstrated that intraplantar injection of potassium superoxide induces the mRNA expression of prepro-endothelin-1 in the paw skin and spinal cord. In conclusion, our results demonstrated that superoxide anion-induced inflammation, pain, cytokine production, and oxidative stress depend on endothelin; therefore, these responses are amenable to bosentan treatment.

  16. The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2.

    Science.gov (United States)

    Maioli, N A; Zarpelon, A C; Mizokami, S S; Calixto-Campos, C; Guazelli, C F S; Hohmann, M S N; Pinho-Ribeiro, F A; Carvalho, T T; Manchope, M F; Ferraz, C R; Casagrande, R; Verri, W A

    2015-04-01

    It is currently accepted that superoxide anion (O2•-) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

  17. Calorimetric Study of Thermal Denaturation of Superoxide Dismutase

    Institute of Scientific and Technical Information of China (English)

    王邦宁; 谈夫

    1994-01-01

    The thermal denaturation of superoxide dismutase (SOD) from bovine erythrocytes was studied at various pH values of different buffers and at various concentrations of solutions of two neutral salts by differential scanning calorimetry. The experiments performed indicate that the PIPES is a buffer non-coordinating with the SOD, and that the binding of the anions studied influences more or less the thermal denaturation of SOD, but the effect on the oxidation form of SOD is more apparent. A new conformer of SOD with lower thermostability was discovered by the experiments performed in different buffers at certain pH values higher than the isoelectric point of SOD, or at higher concentrations of neutral salt solutions. The new conformer may be converted irreversibly into the usual conformer with high thermostability during heating. Based on the thermodynamic parameters obtained in distilled water and by thermodynamic analysis using the Ooi’s model, it is revealed that the large enthalpy △Hdc contributed by

  18. Evaluation of microbiocidal activity of superoxidized water on hospital isolates

    Directory of Open Access Journals (Sweden)

    Aggarwal Ritu

    2010-10-01

    Full Text Available Background: Prevention of nosocomial infections, pre-operative and post-operative complications is directly linked with effective disinfection and decontamination. Microbial decontamination is the most serious challenge to the today′s health care practice despite the abundance of disinfectants and chemicals as there are increasing reports of emergence of resistance to the action of commonly used disinfectants. There is a need to evaluate the efficacy of newer methods of asepsis for better patient management. Aim: This study was designed to evaluate the microbiocidal activity of superoxidized water (SOW on common clinical isolates, ATCC strains, vegetative cells and spores of Bacillus subtilis. Materials and Methods: Bacterial suspensions were treated with SOW and deionized water (control. All the tubes were incubated at 37°C for 0.5, 2.5 and 5.0 min. The number of viable cells was counted. Results: All the clinical isolates and ATCC strains were killed within 0.5 min of exposure to the SOW. Vegetative cells and spores of B. subtilis were killed after 5.0 min. Conclusion: We conclude that SOW is an effective microbiocidal agent for routine hospital use.

  19. Expression of Extracellular Superoxide Dismutase Protein in Diabetes

    Directory of Open Access Journals (Sweden)

    Chul Han Kim

    2013-09-01

    Full Text Available Background Diabetes is characterized by chronic hyperglycemia, which can increase reactiveoxygen species (ROS production by the mitochondrial electron transport chain. The formationof ROS induces oxidative stress and activates oxidative damage-inducing genes in cells. Noresearch has been published on oxidative damage-related extracellular superoxide dismutase(EC-SOD protein levels in human diabetic skin. We investigated the expression of EC-SOD indiabetic skin compared with normal skin tissue in vivo.Methods The expression of EC-SOD protein was evaluated by western blotting in 6 diabeticskin tissue samples and 6 normal skin samples. Immunohistochemical staining was also carriedout to confirm the EC-SOD expression level in the 6 diabetic skin tissue samples.Results The western blotting showed significantly lower EC-SOD protein expression in thediabetic skin tissue than in the normal tissue. Immunohistochemical examination of EC-SODprotein expression supported the western blotting analysis.Conclusions Diabetic skin tissues express a relatively small amount of EC-SOD protein andmay not be protected against oxidative stress. We believe that EC-SOD is related to the alteredmetabolic state in diabetic skin, which elevates ROS production.

  20. Copper, ceruloplasmin, superoxide dismutase and iron parameters in Parkinson's disease.

    Science.gov (United States)

    Tórsdóttir, G; Kristinsson, J; Sveinbjörnsdóttir, S; Snaedal, J; Jóhannesson, T

    1999-11-01

    In a previous study we found copper dyshomeostasis in patients with Alzheimer's disease. In this study, levels of copper in plasma, of ceruloplasmin in serum and ceruloplasmin oxidative activity as well as superoxide dismutase (SOD) activity in erythrocytes were determined in 40 patients with Parkinson's disease and their healthy age- and gender-matched controls. Copper concentrations did not differ significantly in the two groups, whereas both ceruloplasmin concentrations and ceruloplasmin oxidative activity were significantly lower in the patients, also relative to ceruloplasmin mass. SOD activity was not significantly different in the two groups but decreased significantly with the duration of disease. The same was found for ceruloplasmin oxidative activity. Ceruloplasmin oxidative activity and SOD activity did not decrease with age. Levels of serum iron, serum ferritin and total iron binding capacity were determined in about 30 of the patients and an equal number of controls and were not found to differ. Transferrin levels were significantly lower in the patients than in their controls but, conversely, the transferrin saturation was significantly higher in the patients. The results indicate that patients with Alzheimer's disease and Parkinson's disease have defective ceruloplasmin and SOD activities in common and that these defects are not necessarily associated with major disturbances in iron homeostasis.

  1. Superoxide anion production by neutrophils in myelodysplastic syndromes (preleukemia.

    Directory of Open Access Journals (Sweden)

    Takahashi,Isao

    1988-02-01

    Full Text Available Superoxide anion (O2- production by neutrophils from 14 untreated patients with acute nonlymphocytic leukemia (ANLL was significantly less than that of healthy controls (4.93 +/- 1.99 vx 6.20 +/- 1.53 nmol/min/10(6 neutrophils, p less than 0.05. In 10 patients with myelodysplastic syndrome (MDS, however, it was not significantly different from the control level although 6 of the 10 patients had low levels, when individual patients were compared with the lower limit of the control range. An inverse correlation between the O2- production of neutrophils and the percentage of leukemic cells in the marrow existed in ANLL (r = -0.55, p less than 0.01, but not in MDS. Three of 4 MDS patients who died of pneumonia prior to leukemic conversion showed a low level of O2- production. The impaired O2- production by neutrophils from some MDS patients, probably due to the faulty differentiation from leukemic clones, may be one of the causes of enhanced susceptibility to infection.

  2. NADPH Oxidase-Dependent Superoxide Production in Plant Reproductive Tissues.

    Science.gov (United States)

    Jiménez-Quesada, María J; Traverso, José Á; Alché, Juan de Dios

    2016-01-01

    In the life cycle of a flowering plant, the male gametophyte (pollen grain) produced in the anther reaches the stigmatic surface and initiates the pollen-pistil interaction, an important step in plant reproduction, which ultimately leads to the delivery of two sperm cells to the female gametophyte (embryo sac) inside the ovule. The pollen tube undergoes a strictly apical expansion characterized by a high growth rate, whose targeting should be tightly regulated. A continuous exchange of signals therefore takes place between the haploid pollen and diploid tissue of the pistil until fertilization. In compatible interactions, theses processes result in double fertilization to form a zygote (2n) and the triploid endosperm. Among the large number of signaling mechanisms involved, the redox network appears to be particularly important. Respiratory burst oxidase homologs (Rbohs) are superoxide-producing enzymes involved in a broad range of processes in plant physiology. In this study, we review the latest findings on understanding Rboh activity in sexual plant reproduction, with a particular focus on the male gametophyte from the anther development stages to the crowning point of fertilization. Rboh isoforms have been identified in both the male and female gametophyte and have proven to be tightly regulated. Their role at crucial points such as proper growth of pollen tube, self-incompatibility response and eventual fertilization is discussed.

  3. Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction.

    Science.gov (United States)

    English, D; Schell, M; Siakotos, A; Gabig, T G

    1986-07-01

    gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma

  4. Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction

    Energy Technology Data Exchange (ETDEWEB)

    English, D.; Schell, M.; Siakotos, A.; Gabig, T.G.

    1986-07-01

    ..gamma..-hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with /sup 32/P to 4 x 10/sup -4/ M ..gamma..-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, ..gamma..-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10/sup 6/ cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messenger for activation of the NADPH-dependent O/sub 2//sup -/-generating system, neutrophil responses to ..gamma..-hexachlorocyclohexane were compared with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, ..gamma..-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of ..gamma..-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that n intact cells indicated that concurrent degranulation was not required for sustained O/sub 2//sup -/ generation in response to this agent.

  5. Superoxide enhances Ca2+ entry through L-type channels in the renal afferent arteriole.

    Science.gov (United States)

    Vogel, Paul A; Yang, Xi; Moss, Nicholas G; Arendshorst, William J

    2015-08-01

    Reactive oxygen species regulate cardiovascular and renal function in health and disease. Superoxide participates in acute calcium signaling in afferent arterioles and renal vasoconstriction produced by angiotensin II, endothelin, thromboxane, and pressure-induced myogenic tone. Known mechanisms by which superoxide acts include quenching of nitric oxide and increased ADP ribosyl cyclase/ryanodine-mediated calcium mobilization. The effect(s) of superoxide on other calcium signaling pathways in the renal microcirculation is poorly understood. The present experiments examined the acute effect of superoxide generated by paraquat on calcium entry pathways in isolated rat afferent arterioles. The peak increase in cytosolic calcium concentration caused by KCl (40 mmol/L) was 99±14 nmol/L. The response to this membrane depolarization was mediated exclusively by L-type channels because it was abolished by nifedipine but was unaffected by the T-type channel blocker mibefradil. Paraquat increased superoxide production (dihydroethidium fluorescence), tripled the peak response to KCl to 314±68 nmol/L (Psuperoxide and not of hydrogen peroxide. Unaffected by paraquat and superoxide was calcium entry through store-operated calcium channels activated by thapsigargin-induced calcium depletion of sarcoplasmic reticular stores. Also unresponsive to paraquat was ryanodine receptor-mediated calcium-induced calcium release from the sarcoplasmic reticulum. Our results provide new evidence that superoxide enhances calcium entry through L-type channels activated by membrane depolarization in rat cortical afferent arterioles, without affecting calcium entry through store-operated entry or ryanodine receptor-mediated calcium mobilization.

  6. AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function.

    Science.gov (United States)

    Dugan, Laura L; You, Young-Hyun; Ali, Sameh S; Diamond-Stanic, Maggie; Miyamoto, Satoshi; DeCleves, Anne-Emilie; Andreyev, Aleksander; Quach, Tammy; Ly, San; Shekhtman, Grigory; Nguyen, William; Chepetan, Andre; Le, Thuy P; Wang, Lin; Xu, Ming; Paik, Kacie P; Fogo, Agnes; Viollet, Benoit; Murphy, Anne; Brosius, Frank; Naviaux, Robert K; Sharma, Kumar

    2013-11-01

    Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2(+/-)) mice had no evidence of increased renal disease, and Ampka2(-/-) mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity.

  7. Imaging superoxide flash and metabolism-coupled mitochondrial permeability transition in living animals

    Institute of Scientific and Technical Information of China (English)

    Huaqiang Fang; Min Chen; Yi Ding; Wei Shang; Jiejia Xu; Xing Zhang; Wanrui Zhang

    2011-01-01

    The mitochondrion is essential for energy metabolism and production of reactive oxygen species (ROS).In intact cells,respiratory mitochondria exhibit spontaneous "superoxide flashes",the quantal ROS-producing events consequential to transient mitochondrial permeability transition (tMPT).Here we perform the first in vivo imaging of mitochondrial superoxide flashes and tMPT activity in living mice expressing the superoxide biosensor mt-cpYFP,and demonstrate their coupling to whole-body glucose metabolism.Robust tMPT/superoxide flash activity occurred in skeletal muscle and sciatic nerve of anesthetized transgenic mice.In skeletal muscle,imaging tMPT/superoxide flashes revealed labyrinthine three-dimensional networks of mitochondria that operate synchronously.The tMPT/superoxide flash activity surged in response to systemic glucose challenge or insulin stimulation,in an apparently frequency-modulated manner and involving also a shift in the gating mode of tMPT.Thus,in vivo imaging of tMPT-dependent mitochondrial ROS signals and the discovery of the metabolism-tMPT-superoxide flash coupling mark important technological and conceptual advances for the study of mitochondrial function and ROS signaling in health and disease.

  8. How the location of superoxide generation influences the β-cell response to nitric oxide.

    Science.gov (United States)

    Broniowska, Katarzyna A; Oleson, Bryndon J; McGraw, Jennifer; Naatz, Aaron; Mathews, Clayton E; Corbett, John A

    2015-03-20

    Cytokines impair the function and decrease the viability of insulin-producing β-cells by a pathway that requires the expression of inducible nitric oxide synthase (iNOS) and generation of high levels of nitric oxide. In addition to nitric oxide, excessive formation of reactive oxygen species, such as superoxide and hydrogen peroxide, has been shown to cause β-cell damage. Although the reaction of nitric oxide with superoxide results in the formation of peroxynitrite, we have shown that β-cells do not have the capacity to produce this powerful oxidant in response to cytokines. When β-cells are forced to generate peroxynitrite using nitric oxide donors and superoxide-generating redox cycling agents, superoxide scavenges nitric oxide and prevents the inhibitory and destructive actions of nitric oxide on mitochondrial oxidative metabolism and β-cell viability. In this study, we show that the β-cell response to nitric oxide is regulated by the location of superoxide generation. Nitric oxide freely diffuses through cell membranes, and it reacts with superoxide produced within cells and in the extracellular space, generating peroxynitrite. However, only when it is produced within cells does superoxide attenuate nitric oxide-induced mitochondrial dysfunction, gene expression, and toxicity. These findings suggest that the location of radical generation and the site of radical reactions are key determinants in the functional response of β-cells to reactive oxygen species and reactive nitrogen species. Although nitric oxide is freely diffusible, its biological function can be controlled by the local generation of superoxide, such that when this reaction occurs within β-cells, superoxide protects β-cells by scavenging nitric oxide.

  9. Mn(II) oxidation by an ascomycete fungus is linked to superoxide production during asexual reproduction

    Energy Technology Data Exchange (ETDEWEB)

    Hansel, C. M.; Zeiner, C. A.; Santelli, C. M.; Webb, S. M.

    2012-07-16

    Manganese (Mn) oxides are among the most reactive minerals within the environment, where they control the bioavailability of carbon, nutrients, and numerous metals. Although the ability of microorganisms to oxidize Mn(II) to Mn(III/IV) oxides is scattered throughout the bacterial and fungal domains of life, the mechanism and physiological basis for Mn(II) oxidation remains an enigma. Here, we use a combination of compound-specific chemical assays, microspectroscopy, and electron microscopy to show that a common Ascomycete filamentous fungus, Stilbella aciculosa, oxidizes Mn(II) to Mn oxides by producing extracellular superoxide during cell differentiation. The reactive Mn oxide phase birnessite and the reactive oxygen species superoxide and hydrogen peroxide are colocalized at the base of asexual reproductive structures. Mn oxide formation is not observed in the presence of superoxide scavengers (e.g., Cu) and inhibitors of NADPH oxidases (e.g., diphenylene iodonium chloride), enzymes responsible for superoxide production and cell differentiation in fungi. Considering the recent identification of Mn(II) oxidation by NADH oxidase-based superoxide production by a common marine bacterium (Roseobacter sp.), these results introduce a surprising homology between some prokaryotic and eukaryotic organisms in the mechanisms responsible for Mn(II) oxidation, where oxidation appears to be a side reaction of extracellular superoxide production. Finally, given the versatility of superoxide as a redox reactant and the widespread ability of fungi to produce superoxide, this microbial extracellular superoxide production may play a central role in the cycling and bioavailability of metals (e.g., Hg, Fe, Mn) and carbon in natural systems.

  10. Mn(II) oxidation by an ascomycete fungus is linked to superoxide production during asexual reproduction.

    Science.gov (United States)

    Hansel, Colleen M; Zeiner, Carolyn A; Santelli, Cara M; Webb, Samuel M

    2012-07-31

    Manganese (Mn) oxides are among the most reactive minerals within the environment, where they control the bioavailability of carbon, nutrients, and numerous metals. Although the ability of microorganisms to oxidize Mn(II) to Mn(III/IV) oxides is scattered throughout the bacterial and fungal domains of life, the mechanism and physiological basis for Mn(II) oxidation remains an enigma. Here, we use a combination of compound-specific chemical assays, microspectroscopy, and electron microscopy to show that a common Ascomycete filamentous fungus, Stilbella aciculosa, oxidizes Mn(II) to Mn oxides by producing extracellular superoxide during cell differentiation. The reactive Mn oxide phase birnessite and the reactive oxygen species superoxide and hydrogen peroxide are colocalized at the base of asexual reproductive structures. Mn oxide formation is not observed in the presence of superoxide scavengers (e.g., Cu) and inhibitors of NADPH oxidases (e.g., diphenylene iodonium chloride), enzymes responsible for superoxide production and cell differentiation in fungi. Considering the recent identification of Mn(II) oxidation by NADH oxidase-based superoxide production by a common marine bacterium (Roseobacter sp.), these results introduce a surprising homology between some prokaryotic and eukaryotic organisms in the mechanisms responsible for Mn(II) oxidation, where oxidation appears to be a side reaction of extracellular superoxide production. Given the versatility of superoxide as a redox reactant and the widespread ability of fungi to produce superoxide, this microbial extracellular superoxide production may play a central role in the cycling and bioavailability of metals (e.g., Hg, Fe, Mn) and carbon in natural systems.

  11. Oxygen toxicity in Streptococcus mutans: manganese, iron and superoxide dismutase

    Energy Technology Data Exchange (ETDEWEB)

    Martin, M.E.; Strachan, R.C.; Aranha, H.; Evans, S.L.; Salin, M.L.; Welch, B.; Arceneaux, J.E.L.; Byers, B.R.

    1984-07-01

    When cultured anaerobically in a chemically defined medium that was treated with Chelex-100 to lower its trace metal content, Streptococcus mutans OMZ176 had no apparent requirement for manganese or iron. Manganese or iron was necessary for aerobic cultivation in deep static cultures. During continuous aerobic cultivation in a stirred chemostat, iron did not support the growth rate achieved with manganese. Since the dissolved oxygen level in the chemostat cultures was higher than the final level in the static cultures, manganese may be required for growth at elevated levels. In medium supplemented with manganese, cells grown anaerobically contained a low level of superoxide dismutase (SOD) activity; aerobic cultivation increased SOD activity at least threefold. In iron-supplemented medium, cells grown anaerobically also had low SOD activity; aerobic incubation resulted in little increase in SOD activity. Polyacrylamide gel electrophoresis of the cell extracts revealed a major band and a minor band of SOD activity in the cells grown with manganese; however, cells grown with iron contained a single band of SOD activity with an R/sub f/ value similar to that of the major band found in cells grown with manganese. None of the SOD activity bands were abolished by the inclusion of 2 mM hydrogen peroxide in the SOD activity strain. S. mutans may not produce a separate iron-containing SOD but may insert either iron or manganese into an apo-SOD protein. Alternatively, iron may function in another activity (not SOD) that augments the defense against oxygen toxicity at low SOD levels. 28 references, 3 figures, 1 table.

  12. A Manganese Superoxide Dismutase (SOD2)-Mediated Adaptive Response

    Science.gov (United States)

    Grdina, David J.; Murley, Jeffrey S.; Miller, Richard C.; Mauceri, Helena J.; Sutton, Harold G.; Thirman, Michael J.; Li, Jian Jian; Woloschak, Gayle E.; Weichselbaum, Ralph R.

    2013-01-01

    Very low doses of ionizing radiation, 5 to 100 mGy, can induce adaptive responses characterized by elevation in cell survival and reduction in micronuclei formation. Utilizing these end points, RKO human colon carcinoma and transformed mouse embryo fibroblasts (MEF), wild-type or knockout cells missing TNF receptors 1 and 2 (TNFR1−R2−), and C57BL/6 and TNFR1−R2− knockout mice, we demonstrate that intact TNF signaling is required for induction of elevated manganese superoxide dismutase (SOD2) activity (P adaptive responses when cells are challenged at a later time with 2 Gy. In contrast, amifostine’s free thiol form WR1065 can directly activate NF-κB giving rise to elevated SOD2 activity 24 h later and induce an adaptive response in both MEF wild-type and TNF signaling defective TNFR1−R2− cells. Transfection of cells with SOD2 siRNA completely abolishes both the elevation in SOD2 activity and expression of the adaptive responses. These results were confirmed in vivo using a micronucleus assay in splenocytes derived from C57BL/6 and TNFR1−R2− knockout mice that were exposed to 100 mGy or 400 mg/kg amifostine 24 h prior to exposure to a 2 Gy whole-body dose. A dose of 100 mGy also conferred enhanced protection to C57BL/6 mice exposed 24 h later to 100 mg/kg of N-Ethyl-N-nitrosourea (ENU). While very low radiation doses require an intact TNF signaling process to induce a SOD2-mediated adaptive response, amifostine can induce a similar adaptive response in both TNF receptor competent and knockout cells, respectively. PMID:23237540

  13. [The superoxide theory of pathogenesis and therapy of immune disorders].

    Science.gov (United States)

    Lebedev, V V

    2004-01-01

    On the basis of the understanding that there are common development mechanisms for the inflammatory and immune reactions it was established that the activity of the oxidant-antioxidant system (OAS) correlates not only with a severity of the inflammatory reaction but also with a degree of immune disorders. Such disorders were studied in patients with endogenous uveitis and with cancer of the esophagus or uterine cervix, i.e. those nosological forms, which are normally accompanied by OAS decompensation, which comprised a lower activity of primary antioxidants (superoxides of dismutase, catalase, lactoferrin, ceruloplasmin etc.) in patients with pronounced immune disorders. Moreover, a lower content of secondary antioxidants, like vitamin A, ascorbic acid and tocopherol, was registered in the blood of patients with immune disorders. The suppression of the antioxidant system was concomitant with an essentially increased level of lipid peroxidation in all patients. Besides, it was noted that there were intensifying signs of immune disorders primarily observed during irradiation chemotherapy. In this context, a clear-cut correlation was established, in monitoring the body immune status, between degrees of free-radical formation and lipid peroxidation, on the one hand, and an activity of detoxication-system antioxidants, on the other hand,. The OAS correction by direct or indirect-action antioxidants normally improves the clinical course of immune impairments. The indirect-action antioxidants, e.g. synthetic regulatory peptide "Imunofan", induce the increasing activity of primary endogenous antioxidants. An activation of the detoxication antioxidant system, brings about, in such cases, a lower content of inflammation mediators, a recovery of cell-immunity indices and lower parameters of body auto-sensitization. Finally, the antioxidant system in patients with chronic inflammatory or oncological disorders, when recovered, ensures the correction of cell immunity and cuts

  14. Expression of manganese superoxide dismutase in patients with breast cancer

    Directory of Open Access Journals (Sweden)

    Shih-Meng Tsai

    2011-05-01

    Full Text Available Breast cancer has become the second leading cancer among females in Taiwan. Even though the etiology of breast cancer is multifactorial, oxidative stress plays an important role in the carcinogenesis. The purpose of this study was to investigate the expression of manganese superoxide dismutase (MnSOD, one of the major antioxidant enzymes that is involved against oxidative stress, in adjacent cancer-free breast tissues and neoplasm tissues within the same patient. Sixty-five breast cancer patients’ formalin-fixed tissue blocks, including ductal carcinoma in situ (DCIS tissues, invasive ductal carcinoma (IDC tissues, and adjacent cancer-free tissues, were evaluated by immunohistochemical stain. Meanwhile, their demographic and clinical information was also collected. The combined scores of MnSOD-positive cell proportion and MnSOD staining intensity were compared for different tissues within the same patient. The results showed that the mean combined scores of MnSOD expression in adjacent cancer-free tissues (6.33, IDC (5.30, and DCIS (3.78 were significantly different when assessed by repeated-measurement analysis of variance (F=14.17, p<0.001. Additionally, the results revealed that the distribution of strong MnSOD protein expression was 80.0%, 72.3%, and 52.3% in adjacent cancer-free tissues, IDC, and DCIS, respectively. However, there was no statistically significant relationship between the expression of MnSOD and grades of breast cancer or other clinicopathologic variables. We suggest that the expression of MnSOD in neoplasm tissues, independent of the clinicopathologic characters, plays a critical role in breast cancer biology.

  15. Endomorphins 1 and 2 modulate chemotaxis, phagocytosis and superoxide anion production by microglia.

    Science.gov (United States)

    Azuma, Y; Ohura, K; Wang, P L; Shinohara, M

    2001-09-03

    We evaluate the role of endomorphins 1 and 2 on microglial functions. Endomorphins 1 and 2 blocked phagocytosis of Escherichia coli. In addition, both markedly inhibited chemotaxis toward zymosan-activated serum. In contrast, when microglia was preincubated with these endomorphins, followed by incubation with LPS before stimulation with phorbol 12-myristate 13-acetate (PMA) at 200 nM, they potentiated superoxide anion production. Furthermore, when microglia was preincubated with these endomorphins together with PMA at 20 nM, followed by stimulation with PMA at 200 nM, superoxide anion production was potentiated. These results suggest that endomorphins 1 and 2 modulate phagocytosis, chemotaxis and superoxide anion production by microglia.

  16. Production of superoxide in chloroplast thylakoid membranes ESR study with cyclic hydroxylamines of different lipophilicity.

    Science.gov (United States)

    Kozuleva, Marina; Klenina, Irina; Proskuryakov, Ivan; Kirilyuk, Igor; Ivanov, Boris

    2011-04-06

    Accumulation of nitroxide radicals, DCP· or TMT·, under illumination of a thylakoid suspension containing either hydrophilic, DCP-H, or lipophilic, TMT-H, cyclic hydroxylamines that have high rate constants of the reaction with superoxide radicals, was measured using ESR. A slower accumulation of TMT· in contrast with DCP· accumulation was explained by re-reduction of TMT· by the carriers of the photosynthetic electron transport chain within the membrane. Superoxide dismutase suppressed TMT· accumulation to a lesser extent than DCP· accumulation. The data are interpreted as evidencing the production of intramembrane superoxide in thylakoids.

  17. Superoxide dismutase versus ferricytochrome C: determining rate constants for the spin trapping of superoxide by cyclic nitrones.

    Science.gov (United States)

    Weaver, John; Tsai, Pei; Pou, Sovitj; Rosen, Gerald M

    2004-11-26

    Given that spin trapping/electron paramagnetic resonance (EPR) spectroscopy has become the primary technique to identify important biologically generated free radicals, such as superoxide (O(2)(*-)), in vitro and in vivo models, evaluation of the efficiency of specific spin traps to identify this free radical is paramount. Recently, a family of ester-containing nitrones has been prepared, which appears to have distinct advantages for spin trapping O(2)(*-) compared to the well-studied spin traps 5,5-dimethyl-1-pyrroline N-oxide 1 and 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide 2. An important determinant in the selection of a spin trap is the rate constant (k(app)) for its reaction with O(2)(*-), and several different methods have been employed in estimating this k(app). In this paper, the two most frequently used scavengers of O(2)(*-), ferricytochrome c and Cu/Zn-SOD, were evaluated as competitive inhibitors for spin trapping this free radical. Data presented herein demonstrate that SOD is the preferred compound when determining the k(app) for the reaction of O(2)(*-) with spin traps. Using this model, the k(app) for the reaction of nitrone 1, 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide 3, and 5-methoxycarbonyl-5-methyl-1-pyrroline N-oxide 4 with O(2)(*)(-) was estimated to be 24.6 +/- 3.1, 73.0 +/- 12, and 89.4 +/- 1.0 M(-1) s(-1) at pH 7.0, respectively. Several other comparative studies between known spin traps were also undertaken.

  18. Cryo-Trapping the Distorted Octahedral Reaction Intermediate of Manganese Superoxide Dismutase

    Science.gov (United States)

    Borgstahl, Gloria; Snell, Edward H.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportion of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically trap the 6th ligand bound to the active site of manganese superoxide dismutase. Using cryocrystallography and synchrotron radiation, we describe at 1.55A resolution the six-coordinate, distorted octahedral geometry assumed by the active site during catalysis and compare it to the room temperature, five-coordinate trigonal-bipyramidal active site. Gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing off the active site and blocking the escape route of superoxide during dismutation.

  19. Chloramphenicol Mediates Superoxide Production in Photosystem II and Enhances Its Photodamage in Isolated Membrane Particles

    Science.gov (United States)

    Rehman, Ateeq Ur; Kodru, Sandeesha; Vass, Imre

    2016-01-01

    Chloramphenicol (CAP) is an inhibitor of protein synthesis, which is frequently used to decouple photodamage and protein synthesis dependent repair of Photosystem II during the process of photoinhibition. It has been reported earlier that CAP is able to mediate superoxide production by transferring electrons from the acceptor side of Photosystem I to oxygen. Here we investigated the interaction of CAP with Photosystem II electron transport processes by oxygen uptake and variable chlorophyll fluorescence measurements. Our data show that CAP can accept electrons at the acceptor side of Photosystem II, most likely from Pheophytin, and deliver them to molecular oxygen leading to superoxide production. In addition, the presence of CAP enhances photodamage of Photosystem II electron transport in isolated membrane particles, which effect is reversible by superoxide dismutase. It is concluded that CAP acts as electron acceptor in Photosystem II and mediates its superoxide dependent photodamage. This effect has potential implications for the application of CAP in photoinhibitory studies in intact systems. PMID:27092170

  20. Induction of Apoptosis by Superoxide Anion and the Protective Effects of Selenium and Vitamin E

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective The purpose of this study is to investigate the effect of superoxide anion on the apoptosis of cultured fibroblasts and the protective role of selenium and Vitamin E. Methods Cultured fibroblasts (NIH3T3), with or without selenium or vitamin E in the medium, were treated by superoxide anion produced by xanthine/xanthine oxidase reaction system and changes in cell structure and DNA were observed microscopically and electrophoretically. Results Apoptosis was observed when superoxide anion at a concentration of 5 nmol/L or 10 nmol/L had acted on the fibroblasts for 5-10 h. Selenium and Vitamin E in the medium inhibited the apoptosis significantly when their concentrations reached 1.15 mol/L and 2.3 mol/L respectively. Conclusion Selenium and vitamin E have protective effect against the apoptosis induced by superoxide anion. The effect of selenium is more remarkable than that of vitamin E.

  1. Cu(II)-disulfide complexes display simultaneous superoxide dismutase- and catalase-like activities.

    Science.gov (United States)

    Aliaga, Margarita E; Andrade-Acuña, Daniela; López-Alarcón, Camilo; Sandoval-Acuña, Cristián; Speisky, Hernán

    2013-12-01

    Superoxide is a potentially toxic by-product of cellular metabolism. We have addressed here the in vitro ability of complexes formed between copper(II) ions and various biologically-occurring disulfides (RSSR: oxidized glutathione, cystine, homocystine and α-lipoic acid) to react with superoxide. The studied complexes were found to react with superoxide (generated by a xanthine/xanthine oxidase system) at rate constants (kCu(II)-RSSR) close to 10(6)M(-1)s(-1), which are three orders of magnitude lower than that reported for superoxide dismutase (SOD) but comparable to that of several other copper-containing complexes reported as SOD mimetics. The interaction between the tested Cu(II)-RSSR and superoxide, led to the generation and recovery of concentrations of hydrogen peroxide and oxygen that were, respectively, below and above those theoretically-expected from a sole SOD mimetic action. Interestingly, oxygen was generated when the Cu(II)-RSSR complexes were directly incubated with hydrogen peroxide. Taken together, these results reveal that the Cu(II)-RSSR complexes not only have the capacity to dismutate superoxide but also to simultaneously act like catalase mimetic molecules. When added to superoxide-overproducing mitochondria (condition attained by its exposure to diclofenac), three of the tested complexes were able (2-4μM), not only to totally restore, but also to lower below the basal level the mitochondrial production of superoxide. The present study is first in reporting on the potential of Cu(II)-disulfide complexes to act as SOD and catalase like molecules, suggesting a potential for these types of molecules to act as such under physiological and/or oxidative-stress conditions.

  2. A new superoxide-generation inhibitor from rhizome of Anemone raddeana Regel

    Institute of Scientific and Technical Information of China (English)

    Jin Cai Lu; Bei Bei Xu; Song Gao; Hiroyuki Kodam

    2009-01-01

    A new triterpenoid saponins, raddeanoside R19 (1) was isolated from the rhizome of Anemone raddeana Regel. The effects of raddeanoside R19 on superoxide generation in human neutrophil were evaluated. The compound suppressed the superoxide generation induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol 12-myristate 13-acetate (PMA) and araehidonic acid (AA) in a different concentration-dependent manner.

  3. Antioxidative capacity and enzyme activity in Haematococcus pluvialis cells exposed to superoxide free radicals

    Institute of Scientific and Technical Information of China (English)

    刘建国; 张晓丽; 孙延红; 林伟

    2010-01-01

    The antioxidative capacity of astaxanthin and enzyme activity of reactive oxygen eliminating enzymes such as superoxide dismutase (SOD),peroxidase (POD),catalase (CAT) and ascorbate peroxidase (APX) were studied in three cell types of Haematococcus pluvialis exposed to high concentrations of a superoxide anion radical (O2ˉ).The results show that defensive enzymes and astaxanthin-related mechanisms were both active in H.pluvialis during exposure to reactive oxygen species (ROS) such as Oˉ2.Astaxanthin reacte...

  4. Osmotic stress stimulates generation of superoxide anion by spermatozoa in horses.

    Science.gov (United States)

    Burnaugh, L; Ball, B A; Sabeur, K; Thomas, A D; Meyers, S A

    2010-02-01

    The objective of this study was to examine the interplay between osmotic and oxidative stress as well as to determine mechanisms by which osmotic stress increases superoxide generation in spermatozoa of horses. Superoxide production, as measured by dihydroethidium (DHE), increased when spermatozoa of horses were incubated under either hyperosmotic or hyposmotic conditions. This increase in superoxide production was inhibited by the MAP kinase p38 inhibitor, SB203580, and by the superoxide scavenger, tiron. Incubation of spermatozoa under hyperosmotic conditions increased overall protein tyrosine phosphorylation as measured by western blotting techniques; however, a similar increase was not detected when spermatozoa were incubated under hyposmotic conditions. The general protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibitor staurosporine inhibited (P<0.05) tyrosine phosphorylation in samples from cells under hyperosmotic conditions. In addition, the NADPH oxidase inhibitor diphenyleneiodonium (DPI) also inhibited (P<0.05) protein tyrosine phosphorylation in cells under hyperosmotic conditions. In summary, these data indicate that incubation of equine spermatozoa under both hyposmotic and hyperosmotic conditions can increase superoxide anion generation. Under hyperosmotic conditions, this increased generation of superoxide anion was accompanied by increased protein tyrosine phosphorylation.

  5. Macroporous mesh of nanoporous gold in electrochemical monitoring of superoxide release from skeletal muscle cells.

    Science.gov (United States)

    Banan Sadeghian, Ramin; Han, Jiuhui; Ostrovidov, Serge; Salehi, Sahar; Bahraminejad, Behzad; Ahadian, Samad; Chen, Mingwei; Khademhosseini, Ali

    2017-02-15

    Real-time monitoring of metabolically relevant biochemicals released in minuscule amounts is of utmost diagnostic importance. Superoxide anion as a primary member of reactive oxygen species, has physiological and pathological effects that depend on its concentration and release rate. Here we present fabrication and successfully testing of a highly sensitive electrochemical biosensor featuring a three-dimensional macroporous mesh of nanoporous gold tailored to measure the dynamics of extracellular superoxide concentration. Wide and accessible surface of the mesh combined with high porosity of the thin nanoporous gold coating enables capturing the analyte in pico- to nano-molar ranges. The mesh is functionalized with cytochrome-c (cyt-c) and incorporated as a working electrode to measure the release rate of drug-induced superoxides from C2C12 cells through a porous membrane. The device displays a considerably improved superoxide sensitivity of 7.29nAnM(-)(1)cm(-)(2) and a low level of detection of 70pM. Such sensitivity is orders of magnitude higher than any similar enzyme-based electrochemical superoxide sensor and is attributed to the facile diffusion of the analyte through the well-spread nanofeatured gold skin. Superoxide generation rates captured from monolayer myoblast cultures containing about 4×10(4) cells, varied from 1.0 to 9.0nMmin(-)(1) in a quasi-linear fashion as a function of drug concentration. This work provides a platform for the development of highly sensitive molecular electrochemical biosensors.

  6. Elevated peripheral blood mononuclear cell-derived superoxide production in healthy young black men.

    Science.gov (United States)

    Deo, Shekhar H; Holwerda, Seth W; Keller, David M; Fadel, Paul J

    2015-03-01

    Several studies have demonstrated that blacks exhibit elevations in systemic oxidative stress. However, the source(s) and mechanism(s) contributing to the elevation in oxidative stress remain unclear. Given that peripheral blood mononuclear cells (PBMCs) can be a major source of NADPH oxidase-derived superoxide production, we tested the hypothesis that young black men demonstrate greater superoxide production and NADPH oxidase expression in PBMCs compared with whites. PBMCs were freshly isolated from whole blood in young normotensive black (n = 18) and white (n = 16) men. Intracellular superoxide production in PBMCs was measured using dihydroethidium fluorescence, protein expression of NADPH oxidase subunits, gp91(phox) (membranous) and p47(phox) (cytosolic) in PBMCs were assessed using Western blot analysis, and plasma protein carbonyls were measured as a marker of systemic oxidative stress. Black men showed elevated intracellular superoxide production (4.3 ± 0.5 vs. 2.0 ± 0.6 relative fluorescence units; black men vs. white men, P superoxide production or NADPH oxidase subunit protein expression. These findings indicate that black men exhibit greater resting PBMC-derived superoxide production and an upregulation of the NADPH oxidase pathway with a possible contribution to increases in systemic oxidative stress.

  7. Chemiluminescence Imaging of Superoxide Anion Detects Beta-Cell Function and Mass.

    Science.gov (United States)

    Bronsart, Laura L; Stokes, Christian; Contag, Christopher H

    2016-01-01

    Superoxide anion is produced during normal cellular respiration and plays key roles in cellular physiology with its dysregulation being associated with a variety of diseases. Superoxide anion is a short-lived molecule and, therefore, its homeostatic regulation and role in biology and disease requires dynamic quantification with fine temporal resolution. Here we validated coelenterazine as a reporter of intracellular superoxide anion concentration and used it as a dynamic measure both in vitro and in vivo. Chemiluminescence was dependent upon superoxide anion levels, including those produced during cellular respiration, and concentrations varied both kinetically and temporally in response to physiologically relevant fluctuations in glucose levels. In vivo imaging with coelenterazine revealed that beta cells of the pancreas have increased levels of superoxide anion, which acted as a measure of beta-cell function and mass and could predict the susceptibility of mice to diabetes mellitus. Glucose response and regulation are key elements of cellular physiology and organismal biology, and superoxide anion appears to play a fundamental and dynamic role in both of these processes.

  8. Increased salt sensitivity induced by sensory denervation:role of superoxide

    Institute of Scientific and Technical Information of China (English)

    Wei-zhong SONG; Alex F CHEN; Donna H WANG

    2004-01-01

    AIM: To test the hypothesis that production of superoxide in mesenteric resistance arteries is increased and contributes to the development of hypertension induced by sensory denervation plus high salt intake. METHODS:Newborn Wistar rats were given capsaicin 50mg/kg sc on the 1st and 2nd d of life. After weaning, male rats were grouped as follows and treated for 3 weeks with: capsaicin pretreatment plus normal sodium diet (0.5%, CAP-NS),CAP plus high sodium diet (4%, CAP-HS), control plus NS (CON-NS), or CON-HS. Both tail-cuff systolic blood pressure and mean arterial pressure (MAP) were measured in each of the groups. Western blot analysis was used for measurement of manganese superoxide dismutase (MnSOD) and endothelial nitric oxide synthase (eNOS) in the mesenteric resistance arteries. Lucigenin chemiluminescence assay was used for superoxide production in the mesenteric resistance arteries. The Griess method was used for measurement of nitrite/nitrate levels in plasma.RESULTS: Both tail-cuff pressure and MAP were higher in CAP-HS compared with CAP-NS, CON-HS, and CONNS rats (P<0.05). Both MnSOD and eNOS in the mesenteric resistance arteries were increased in CAP-HS compared with CAP-NS, CON-HS, and CON-NS (P<0.05). However, nitrite/nitrate levels in plasma were not different among 4 groups. Acute iv administration of tempol, a membrane-permeable superoxide scavenger, decreased MAP in both CAP-HS and CON-HS when compared with their respective controls. However, the decreases of MAP between these two groups were not different. Chronic treatment with tempol failed to prevent the development of hypertension in CAP-HS rats. Superoxide production in the mesenteric resistance arteries was increased in CAP-HS compared with CAP-NS, CON-HS, and CON-NS (P<0.05). However, chronic treatment with tempol did not prevent the increase of mesenteric superoxide production in CAP-HS rats. CONCLUSIONS:Regardless of increased vascular MnSOD levels, salt sensitive hypertension

  9. Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis.

    Science.gov (United States)

    Cheroni, Cristina; Marino, Marianna; Tortarolo, Massimo; Veglianese, Pietro; De Biasi, Silvia; Fontana, Elena; Zuccarello, Laura Vitellaro; Maynard, Christa J; Dantuma, Nico P; Bendotti, Caterina

    2009-01-01

    In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic Ub(G76V)-GFP /SOD1G93A mice an increase in Ub(G76V)-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of Ub(G76V)-GFP was due to deficient reporter degradation. In some motor neurons the increase of Ub(G76V)-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.

  10. Magnetic Resonance Imaging of Mitochondrial Dysfunction and Metabolic Activity, Accompanied by Overproduction of Superoxide.

    Science.gov (United States)

    Bakalova, Rumiana; Georgieva, Ekaterina; Ivanova, Donika; Zhelev, Zhivko; Aoki, Ichio; Saga, Tsuneo

    2015-12-16

    This study shows that a mitochondria-penetrating nitroxide probe (mito-TEMPO) allows detection of superoxide and visualization of mitochondrial dysfunction in living cells due to the effect of T1 shortening in MRI. Mitochondrial dysfunction was induced by treatment of cells with rotenone and 2-methoxyestradiol (2-ME/Rot). The MRI measurements were performed on 7T MRI. The 2-ME/Rot-treated cells were characterized by overproduction of superoxide, which was confirmed by a conventional dihydroethidium test. In the presence of mito-TEMPO, the intensity of MRI signal in 2-ME/Rot-treated cells was ∼30-40% higher, in comparison with that in untreated cells or culture media. In model (cell-free) systems, we observed that superoxide, but not hydrogen peroxide, increased the intensity of T1-weighted MRI signal of mito-TEMPO. Moreover, the superoxide restores the T1-weighted MRI contrast of mito-TEMPOH, a noncontrast (diamagnetic) analogue of mito-TEMPO. This was also confirmed by using EPR spectroscopy. The results demonstrate that superoxide radical is involved in the enhancement of T1-weighted MRI contrast in living cells, in the absence and presence of mito-TEMPO. This report gives a direction for discovering new opportunities for functional MRI, for detection of metabolic activity, accompanied by overproduction of superoxide, as well as by disturbance of the balance between superoxide and hydrogen peroxide, a very important approach to clarify the fine molecular mechanisms in the regulation of many pathologies. The visualization of mitochondrial activity in real-time can be crucial to clarify the molecular mechanism of the functional MRI in its commonly accepted definition, as a method for detection of neurovascular coupling.

  11. Serine 1179 Phosphorylation of Endothelial Nitric Oxide Synthase Increases Superoxide Generation and Alters Cofactor Regulation.

    Science.gov (United States)

    Peng, Hu; Zhuang, Yugang; Harbeck, Mark C; He, Donghong; Xie, Lishi; Chen, Weiguo

    2015-01-01

    Endothelial nitric oxide synthase (eNOS) is responsible for maintaining systemic blood pressure, vascular remodeling and angiogenesis. In addition to producing NO, eNOS can also generate superoxide (O2-.) in the absence of the cofactor tetrahydrobiopterin (BH4). Previous studies have shown that bovine eNOS serine 1179 (Serine 1177/human) phosphorylation critically modulates NO synthesis. However, the effect of serine 1179 phosphorylation on eNOS superoxide generation is unknown. Here, we used the phosphomimetic form of eNOS (S1179D) to determine the effect of S1179 phosphorylation on superoxide generating activity, and its sensitivity to regulation by BH4, Ca2+, and calmodulin (CAM). S1179D eNOS exhibited significantly increased superoxide generating activity and NADPH consumption compared to wild-type eNOS (WT eNOS). The superoxide generating activities of S1179D eNOS and WT eNOS did not differ significantly in their sensitivity to regulation by either Ca2+ or CaM. The sensitivity of the superoxide generating activity of S1179D eNOS to inhibition by BH4 was significantly reduced compared to WT eNOS. In eNOS-overexpressing 293 cells, BH4 depletion with 10mM DAHP for 48 hours followed by 50ng/ml VEGF for 30 min to phosphorylate eNOS S1179 increased ROS accumulation compared to DAHP-only treated cells. Meanwhile, MTT assay indicated that overexpression of eNOS in HEK293 cells decreased cellular viability compared to control cells at BH4 depletion condition (Psuperoxide generation: S1179 phosphorylation increases superoxide production while decreasing sensitivity to the inhibitory effect of BH4 on this activity.

  12. The Effect of IVIG on Superoxide Generation in Primary Humoral Immunodeficiencies

    Directory of Open Access Journals (Sweden)

    Gulay Sezgin

    2016-01-01

    Full Text Available Primary antibody deficiency (common variable immunodeficiency, Hyper IgM, X-linked agammaglobulinemia and selective Ig A deficiency is a group of heterogeneous diseases characterized by defective antibody production. In primary hypogammaglobulinemias, particularly in patients with common variable immunodeficiency there is an increased generation of reactive oxygen species from monocytes which may be important for both immunopathogenesis and clinical manifestations. The generation of toxic oxygen metabolites may contribute to inflammation and tissue damage associated with phagocytic infiltration, and play role in the pathogenesis of malignancies, autoimmune disorders, acute and chronic pulmonary diseases seen in these patients. In primary immunodeficiencies and functional antibody deficiencies, IVIG act as replacement therapy and several mechanisms of IVIG action have been postulated. In vitro studies with human granulocytes showed stimulation of respiratory burst and promotion of bacterial killing by IVIG. In adult patients with primary humoral immunodeficiency, treated with IVIG showed that IVIG does not affect superoxide generation. We investigated superoxide generation from PMNL in 35 children with hyper IgM syndrome, XLA, CVID and IgA deficiency and 13 healthy children. We also explored the effect of IVIG administration on superoxide generation from granulocytes, white cell count, absolute neutrophil count, absolute lymphocyte count and quantitative CRP levels. There was a substantial increase in superoxide generation from PMNL in patients with XLA, CVID and IgA deficiency. Comparison of the superoxide generation before, 24 hours and one week after IVIG treatment showed no difference. In patients with CVID, quantitative CRP levels before and 24 hours after IVIG revealed significant difference. Other parameters were not changed. It can be concluded that enhanced superoxide generation in patients with XLA, CVID, Ig A deficiency may result from

  13. Species-specific control of external superoxide levels by the coral holobiont during a natural bleaching event

    Science.gov (United States)

    Diaz, Julia M.; Hansel, Colleen M.; Apprill, Amy; Brighi, Caterina; Zhang, Tong; Weber, Laura; McNally, Sean; Xun, Liping

    2016-12-01

    The reactive oxygen species superoxide (O2.-) is both beneficial and detrimental to life. Within corals, superoxide may contribute to pathogen resistance but also bleaching, the loss of essential algal symbionts. Yet, the role of superoxide in coral health and physiology is not completely understood owing to a lack of direct in situ observations. By conducting field measurements of superoxide produced by corals during a bleaching event, we show substantial species-specific variation in external superoxide levels, which reflect the balance of production and degradation processes. Extracellular superoxide concentrations are independent of light, algal symbiont abundance and bleaching status, but depend on coral species and bacterial community composition. Furthermore, coral-derived superoxide concentrations ranged from levels below bulk seawater up to ~120 nM, some of the highest superoxide concentrations observed in marine systems. Overall, these results unveil the ability of corals and/or their microbiomes to regulate superoxide in their immediate surroundings, which suggests species-specific roles of superoxide in coral health and physiology.

  14. [Involvement of carbonate/bicarbonate ions in the superoxide-generating reaction of adrenaline autoxidation].

    Science.gov (United States)

    Sirota, T V

    2015-01-01

    An important role of carbonate/bicarbonate ions has been recognized in the superoxide generating reaction of adrenaline autooxidation in an alkaline buffer (a model of quinoid adrenaline oxidation in the body). It is suggested that these ions are directly involved not only in formation of superoxide anion radical (О(2)(-)) but also other radicals derived from the carbonate/bicarbonate buffer. Using various buffers it was shown that the rate of accumulation of adrenochrome, the end product of adrenaline oxidation, and the rate of О(2)(-)· formation depend on concentration of carbonate/bicarbonate ions in the buffer and that these ions significantly accelerate adrenaline autooxidation thus demonstrating prooxidant properties. The detectable amount of diformazan, the product of nitro blue tetrazolium (NBT) reduction, was significantly higher than the amount of adrenochrome formed; taking into consideration the literature data on О(2)(-)· detection by NBT it is suggested that adrenaline autooxidation is accompanied by one-electron reduction not only of oxygen dissolved in the buffer and responsible for superoxide formation but possible carbon dioxide also dissolved in the buffer as well as carbonate/bicarbonate buffer components leading to formation of corresponding radicals. The plots of the dependence of the inhibition of adrenochrome and diformazan formation on the superoxide dismutase concentration have shown that not only superoxide radicals are formed during adrenaline autooxidation. Since carbonate/bicarbonate ions are known to be universally present in the living nature, their involvement in free radical processes proceeding in the organism is discussed.

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