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Recently, chromogranins were reported to interact specifically with mutant forms of superoxide dismutase that are linked to amyotrophic lateral sclerosis (ALS). This interaction led us to analyze the frequencies of sequence variants of the CHGB gene in ALS patients and matched controls from three different countries. Of particular interest was the finding of the P413L CHGB variant present in 10% of ALS patients (n = 705) as compared to 4.5% in controls (n = 751), conferring a 2.2-fold greater relative risk to develop the disease (P < 0.0001). This effect was mainly contributed by the samples of French origin that yielded a frequency of the P413L variation at 17% in ALS (n = 289) and 5% in controls (n = 448), conferring a 3.3-fold greater risk to develop ALS. Furthermore, the P413L CHGB variant is associated with an earlier age of onset by almost a decade in both sporadic ALS and familial ALS cases. Genetic variation influencing age of onset in ALS had not previously been reported. Expression of fusion CHGB-EGFP constructs in SHSY-5Y cells revealed that the P413L variation can cause defective sorting of CHGB into secretory granules. The finding that CHGB may act as a susceptibility gene and modifier of onset in ALS is consistent with the emerging view that dysfunction of the secretory pathway may contribute to increased vulnerability of motor neurons. association study | modifier gene | motor neuron doi/10.1073/pnas.0902174106

We know a great deal about the cellular response to starvation via AMPK, but less is known about the reaction to nutrient excess. Insulin resistance may be an appropriate response to nutrient excess, but the cellular sensors that link these parameters remain poorly defined. In the present study we provide evidence that mitochondrial superoxide production is a common feature of many different models of insulin resistance in adipocytes, myotubes, and mice. In particular, insulin resistance was rapidly reversible upon exposure to agents that act as mitochondrial uncouplers, ETC inhibitors, or mitochondrial superoxide dismutase (MnSOD) mimetics. Similar effects were observed with overexpression of mitochondrial MnSOD. Furthermore, acute induction of mitochondrial superoxide production using the complex III antagonist antimycin A caused rapid attenuation of insulin action independently of changes in the canonical PI3K/Akt pathway. These results were validated in vivo in that MnSOD transgenic mice were partially protected against HFD induced insulin resistance and MnSOD+/- mice were glucose intolerant on a standard chow diet. These data place mitochondrial superoxide at the nexus between intracellular metabolism and the control of insulin action potentially defining this as a metabolic sensor of energy excess. diabetes | mitochondria | superoxide doi/ 10.1073/pnas.0902380106

Given that the physiology of heine oxygenase-1 (HO-1) encompasses mitochondrial biogenesis, we tested the hypothesis that the HO-1 product, carbon monoxide (CO), activates mitochondrial biogenesis in skeletal muscle and enhances maximal oxygen uptake ([??].sub.2max]) in humans. In l0 healthy subjects, we biopsied the vastus lateralis and performed [??].sub.2max] tests followed by blinded randomization to air or CO breathing (1 h/day at 100 parts/million for 5 days), a contralateral muscle biopsy on day 5, and repeat [??].sub.2max] testing on day 8. Six independent subjects underwent CO breathing and two muscle biopsies without exercise testing. Molecular studies were performed by real-time RT-PCR, Western blot analysis, and immunochemistry. After [??].sub.2max] testing plus CO breathing, significant increases were found in mRNA levels for nuclear respiratory factor-1, peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha], mitochondrial transcription factor-A (Tfam), and DNA polymerase [gamma](Pol[gamma]) with no change in mitochondrial DNA (mtDNA) copy number or [??].sub.2max]. Levels of myosin heavy chain I and nuclear-encoded HO-1, superoxide dismutase-2, citrate synthase, mitofusin-1 and -2, and mitochondrial-encoded cytochrome oxidase subunit-I (COX-I) and ATPase-6 proteins increased significantly. None of these responses were reproduced by [??].sub.2max] testing alone, whereas CO alone increased Tfam and Pol[gamma] mRNA, and COX-I, ATPase-6, mitofusin-2, HO-1, and superoxide dismutase protein. These findings provide evidence linking the HO/CO response involved in mitochondrial biogenesis in rodents to skeletal muscle in humans through a set of responses involving regulation of the mtDNA transcriptosome and mitochondrial fusion proteins autonomously of changes in exercise capacity. heme oxygenase-1; superoxide dismutase-2; peroxisome proliferator-activated receptor-[gamma] coactivator-1[alpha]; nuclear respiratory factor-1; oxygen uptake

We recently demonstrated that (n-3) PUFA trigger the induction of apoptosis in the colon by enhancing phospholipid oxidation and mitochondrial [Ca.sup.2+] accumulation. To further elucidate the mechanisms regulating oxidative stress-induced apoptosis in vivo, a 2 x 2 experiment was designed using both wild type (control) and manganese-dependent superoxide dismutase (SOD2) heterozygous knockout mice ([SOD2.sup.+/-]), which exhibit increased mitochondrial oxidative stress. Mice were fed diets differing only in the type of fat [corn oil or fish oil containing (n-3) PUFA] at 15% by weight for 4 wk. Dietary (n-3) PUFA treatment enhanced (22%) apoptosis in colonic crypts. In addition, SOD2 haploinsufficiency enhanced (20%) apoptosis, which was further increased (36%) by (n-3) PUFA feeding. Dietary lipid source and genotype interactively modulated nitrotyrosine levels (P = 0.027) and inflammation (P = 0.032). These findings demonstrate that the proapoptotic effects of (n-3) PUFA are enhanced in oxidatively stressed [SOD2.sup.+/-] mice. Thus, (n-3) PUFA appear to promote an oxidation-reduction imbalance in the intestine, which may directly or indirectly trigger apoptosis and thereby reduce colon cancer risk.

Superoxide dismutase (SOD) is an enzyme that catalytically removes the superoxide radical (*[O.sub.2.sup.-]) and protects organisms from oxidative damage during normal aging. We demonstrate that not only the cytosolic *[O.sub.2.sup.-] level but also the mitochondrial *[O.sub.2.sup.-] level increases in the deletion mutants of sod-1 gene encoding Cu/Zn SOD in Caenorhabditis elegans (C. elegans). Interestingly, this suggests that the activity of SOD-1. which so far has been thought to act mainly in cytoplasm, helps to control the detoxification of *[O.sub.2.sup.-] also in the mitochondria. We also found functional compensation by other SODs, especially the sod-5 gene, which was induced several fold in the mutants. Therefore. the possibility exists that the compensative expression of sod-5 gene in the sod-I deficit is associated with the insulin/insulin-like growth factor-I (Ins/IGF-1) signaling pathway, which regulates longevity and stress resistance of C. elegans because the sod-5 gene may be a target of the pathway. Key Words: Sod-1--Sod-5--Mitochondrial ROS production--Oxidative stress--Aging.

The structural and dynamical properties of the metal-free form of WT human superoxide dismutase 1 (SOD1) and its familial amyotrophic lateral sclerosis (fALS)-related mutants, T54R and I113T, were characterized both in solution, through NMR, and in the crystal, through X-ray diffraction. We found that all 3 X-ray structures show significant structural disorder in 2 loop regions that are, at variance, well defined in the fully-metalated structures. Interestingly, the apo state crystallizes only at low temperatures, whereas all 3 proteins in the metalated form crystallize at any temperature, suggesting that crystallization selects one of the most stable conformations among the manifold adopted by the apo form in solution. Indeed, NMR experiments show that the protein in solution is highly disordered, sampling a large range of conformations. The large conformational variability of the apo state allows the free reduced cysteine Cys-6 to become highly solvent accessible in solution, whereas it is essentially buried in the metalated state and the crystal structures. Such solvent accessibility, together with that of Cys-111, accounts for the tendency to oligomerization of the metal-free state. The present results suggest that the investigation of the solution state coupled with that of the crystal state can provide major insights into SOD1 pathway toward oligomerization in relation to fALS. amyotrophic lateral sclerosis | NMR | X-ray I mobility | [H.sub.2]O/[D.sub.2]O exchange

The long-term effects of uncoupled mitochondrial respiration by uncoupling protein-2 (UCP2) in mammalian physiology remain controversial. Here we show that increased mitochondrial uncoupling activity of different tissues predicts longer lifespan of rats compared with mice. UCP2 reduces reactive oxygen species (ROS) production and oxidative stress throughout the aging process in different tissues in mice. The absence of UCP2 shortens lifespan in wild-type mice, and the level of UCP2 positively correlates with the postnatal survival of superoxide dismutase-2 mutant animals. Thus UCP2 has a beneficial influence on cell and tissue function leading to increased lifespan. reactive oxygen species; mitochondria; superoxide dismutase-2; longevity; aging; fatty acid oxidation

Misfolding and aggregation of Cu, Zn superoxide dismutase (SOD1) is implicated in neuronal death in amyotrophic lateral sclerosis. Each SOD1 monomer binds to 1 copper and 1 zinc ion and maintains its disulfide bond (Cys-57-Cys-146) in the reducing cytoplasm of cell. Mounting experimental evidence suggests that metal loss and/or disulfide reduction are important for initiating misfolding and aggregation of SOD1. To uncover the role of metals and the disulfide bond in the SOD1 folding, we systemically study the folding thermodynamics and structural dynamics of SOD1 monomer and dimer with and without metal binding and under disulfide-intact or disulfide-reduced environments in computational simulations. We use all-atom discrete molecular dynamics for sampling. Our simulation results provide dynamical evidence to the stabilizing role of metal ions in both dimer and monomer SOD1. The disulfide bond anchors a loop (Glu-49 to Asn-53) that contributes to the dimer interface. The reduction of the disulfide bond in SOD1 with metal ions depleted results in a flexible Glu-49-Asn-53 loop, which, in turn, disrupts dimer formation. Interestingly, the disulfide bond reduction does not affect the thermostability of monomer SOD1 as significantly as the metal ions do. We further study the structural dynamics of metal-free SOD1 monomers, the precursor for aggregation, in simulations and find inhomogeneous local unfolding of [beta]-strands. The strands protected by the metal-binding and electrostatic loops are found to unfold first after metal loss, leading to a partially unfolded [beta]-sheet prone to aggregation. Our simulation study sheds light on the critical role of metals and disulfide bond in SOD1 folding and aggregation. amyotrophic lateral sclerosis | discreet molecular dynamics | protein aggregation | protein stability

Cu, Zn-superoxide dismutase (SOD-1), an enzyme that catalyzes the disproportionation reaction of superoxide to produce oxygen and hydrogen peroxide, thereby protecting cells from oxidative stress, is a homodimer that coordinates one copper and one zinc ion per monomer. [Cu.sup.2+] and [Zn.sup.2+] ions play important roles in enzyme activity and structural stability, respectively. In addition, dimer formation is also essential for fulfilling the function of SOD-1. We here report on the reconstitution and enzyme activities of several metalation states of SOD-1 ([Cu.sub.4-], [Cu.sub.3]Zn-, and [Cu.sub.2][Zn.sub.2]-homodimers). Each metalation state of the reconstituted SOD-1 could be unambiguously differentiated by electrospray ionization mass spectrometry, the metal ions of which had been completely replaced by 99 atom % [sup.63]Cu and [sup.68]Zn stable isotopes. It was found that (1) the [Cu.sub.4]-dimer possessed 84% of the activity of the native enzyme, (2) the Cu-site resisted being coordinated with [Zn.sup.2+] ions while the Zn-site could be bound with [Cu.sup.2+] ions, and (3) the simultaneous addition of the [Cu.sup.2+] and [Zn.sup.2+] ions to generate a fully metalated form produced the multiply metalated SOD-1 ([Cu.sub.4-], [Cu.sub.3]Zn-, and [Cu.sub.2][Zn.sub.2]-dimers), which were clearly distinguishable from one another by the use of the stable isotopes, while the sequential addition of [Zn.sup.2+] followed by the [Cu.sup.2+] ion predominantly produced a [Cu.sub.2][Zn.sub.2]-dimer comparable to the native enzyme.

Although production of reactive oxygen species (ROS) such as superoxide ([O.sub.2.sup..-]) has been implicated in chronic hypoxia-induced pulmonary hypertension (PH) and pulmonary vascular remodeling, the transcription factors and gene targets through which ROS exert their effects have not been completely identified. We used mice overexpressing the extracellular antioxidant enzyme extracellular superoxide dismutase (EC-SOD TG) to test the hypothesis that [O.sub.2.sup..-] generated in the extracellular compartment under hypoxic conditions contributes to PH through the induction of the transcription factor, early growth response-1 (Egr-1), and its downstream gene target, tissue factor (TF). We found that chronic hypoxia decreased lung EC-SOD activity and protein expression in wild-type mice, but that EC-SOD activity remained five to seven times higher in EC-SOD TG mice under hypoxic conditions. EC-SOD overexpression attenuated chronic hypoxic PH, and vascular remodeling, measured by fight ventricular systolic pressures, proliferation of cells in the vessel wall, muscularization of small pulmonary vessels, and collagen deposition. EC-SOD overexpression also prevented the early hypoxia-dependent upregulation of the redox-sensitive transcription factor Egr-1 and the procoagulant protein TF. These data provide the first evidence that EC-SOD activity is disrupted in chronic hypoxia, and increased EC-SOD activity can attenuate chronic hypoxic PH by limiting the hypoxic upregulation of redox-sensitive genes. extracellular superoxide dismutase; redox-sensitive transcription factor; early growth response-1: tissue factor; pulmonary hypertension

Fluid shear stress modulates vascular production of endothelial superoxide anion ([[O.sub.2].sup.-]) and nitric oxide (NO). Whether the characteristics of shear stress influence the spatial variations in mitochondrial manganese superoxide dismutase (Mn-SOD) expression in vasculatures is not well defined. We constructed a three-dimensional computational fluid dynamics model simulating spatial variations in shear stress at the arterial bifurcation. In parallel, explants of arterial bifurcations were sectioned from the human left main coronary bifurcation and right coronary arteries for immunohistolocalization of Mn-SOD expression. We demonstrated that Mn-SOD staining was prominent in the pulsatile shear stress (PSS)-exposed and atheroprotective regions, but it was nearly absent in the oscillatory shear stress (OSS)-exposed regions and lateral wall of arterial bifurcation. In cultured bovine aortic endothelial cells, PSS at mean shear stress ([[tau].sub.ave]) of 23 dyn/[cm.sup.2] upregulated Mn-SOD mRNA expression at a higher level than did OSS at [[tau].sub.ave] = 0.02 dyn/[cm.sup.2] [+ or -] 3.0 dyn x [cm.sup.-2] x [s.sup.-1] and at 1 Hz (PSS by 11.3 [+ or -] 0.4-fold vs. OSS by 5.0 [+ or -] 0.5-fold vs. static condition; P < 0.05, n = 4). By liquid chromatography and tandem mass spectrometry, it was found that PSS decreased the extent of low-density lipoprotein (LDL) nitration, whereas OSS increased nitration (P < 0.05, n = 4). In the presence of LDL, treatment with Mn-SOD small interfering RNA increased intracellular nitrotyrosine level (P < 0.5, n = 4), a fingerprint for nitrotyrosine formation. Our findings indicate that shear stress in the atheroprone versus atheroprotective regions regulates spatial variations in mitochondrial Mn-SOD expression with an implication for modulating LDL nitration. superoxide dismutase; superoxide anion; nitric oxide; nitrotyrosine; low-density lipoprotein

Superoxide dismutase 1 (SOD1) is an abundant copper/zinc enzyme found in the cytoplasm that converts superoxide into hydrogen peroxide and molecular oxygen. Tetrathiomolybdate (ATN-224) has been recently identified as an inhibitor of SOD1 that attenuates FGF-2- and VEGF-mediated phosphorylation of ERK 1/2 in endothelial cells. However, the mechanism for this inhibition was not elucidated. Growth factor (GF) signaling elicits an increase in reactive oxygen species (ROS), which inactivates protein tyrosine phosphatases (PTP) by oxidizing an essential cysteine residue in the active site. ATN-224-mediated inhibition of SOD1 in tumor and endothelial cells prevents the formation of sufficiently high levels of [H.sub.2][O.sub.2], resulting in the protection of PTPs from [H.sub.2][O.sub.2]-mediated oxidation. This, in turn, leads to the inhibition of EGF-, IGF-1-, and FGF-2-mediated phosphorylation of ERK 1/2. Pretreatment with exogenous [H.sub.2][O.sub.2] or with the phosphatase inhibitor vanadate abrogates the inhibition of ERK 1/2 phosphorylation induced by ATN-224 or SOD1 siRNA treatments. Furthermore, ATN-224-mediated SOD1 inhibition causes the down-regulation of the PDGF receptor. SOD1 inhibition also increases the steady-state levels of superoxide, which induces protein oxidation in A431 cells but, surprisingly, does not oxidize phosphatases. Thus, SOD1 inhibition in A431 tumor cells results in both prooxidant effects caused by the increase in the levels of superoxide and antioxidant effects caused by lowering the levels of [H.sub.2][O.sub.2]. These results identify SOD1 as a master regulator of GF signaling and as a therapeutic target for the inhibition of angiogenesis and tumor growth. angiogenesis | cancer | redox | tetrathiomolybdate | ATN-224

Mutations in copper/zinc superoxide dismutase (SOD1) are causative for dominantly inherited amyotrophic lateral sclerosis (ALS). Despite high variability in biochemical properties among the disease-causing mutants, a proportion of both dismutase-active and -inactive mutants are stably bound to spinal cord mitochondria. This mitochondrial proportion floats with mitochondria rather than sedimenting to the much higher density of protein, thus eliminating coincidental cosedimentation of protein aggregates with mitochondria. Half of dismutase-active and [approximately equal to] 90% of dismutase-inactive mutant SOD1 is bound to mitochondrial membranes in an alkali- and salt-resistant manner. Sensitivity to proteolysis and immunoprecipitation with an antibody specific for misfolded SOD1 demonstrate that in all mutant SOD1 models, misfolded SOD1 is deposited onto the cytoplasmic face of the outer mitochondrial membrane, increasing antigenic accessibility of the normally structured electrostatic loop. Misfolded mutant SOD1 binding is both restricted to spinal cord and selective for mitochondrial membranes, implicating exposure to mitochondria of a misfolded mutant SOD1 conformer mediated by a unique, tissueselective composition of cytoplasmic chaperones, components unique to the cytoplasmic face of spinal mitochondria to which misfolded SOD1 binds, or misfolded SOD1 conformers unique to spinal cord that have a selective affinity for mitochondrial membranes. degeneration | motor neuron disease | superoxide dismutase

Advanced glycation end products (AGEs) promote reactive oxygen species (ROS) formation and oxidant stress (OS) in diabetes and aging-related diseases. AGE-induced OS is suppressed by AGER1, an AGE-receptor that counteracts receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR)mediated Shc/Ras signal activation, resulting in decreased OS. Akt, FKHRL1, and antioxidants; e.g., MnSOD, regulate OS. Serine phosphorylation of [p66.sup.shc] also promotes OS. We examined the effects of two defined AGEs [N.sup.[epsilon]]-carboxy-methyl-lysine (CML) and methylglyoxal derivatives (MG) on these cellular pathways and their functional relationship to AGER1 in human embryonic kidney cells (HEK293). Stimulation of HEK293 cells with either AGE compound increased phosphorylation of Akt and FKHRL1 by approximately threefold in a redox-dependent manner. The use of [p66.sup.shc] mutants showed that the AGE-induced effects required Set-36 phosphorylation of [p66.sup.shc]. AGE-induced phosphorylation of FKHRL1 led to a 70% downregulation of MnSOD, an effect partially blocked by a phosphatidylinositol 3-kinase inhibitor (LY-294002) and strongly inhibited by an antioxidant (N-acetylcysteine). These pro-oxidant responses were suppressed in AGER1 overexpressing cells and reappeared when AGER1 expression was reduced by small interfering RNA (siRNA). These studies point to a new pathway for the induction of OS by AGEs involving FKHRL1 inactivation and MnSOD suppression via Ser-36 phosphorylation of [p66.sup.shc] in human kidney cells. This represents a key mechanism by which AGER1 maintains cellular resistance against OS. Thus the decrease of AGER1 noted in aging and diabetes may further enhance OS and reduce innate antioxidant defenses. glycoxidation; aging; diabetes; N-carboxy-methyl-lysine; methylglyoxal; forkhead transcription factors; manganese superoxide dismutase; receptor for advanced glycation end products

Mitochondria are indispensable for bioenergetics and for the regulation of physiological/signaling events in cellular life. Although TNF-[alpha]-induced oxidative stress and mitochondrial dysfunction are evident in several pathophysiological states, the molecular mechanisms coupled with impaired cardiac function and its potential reversal by drugs such as Tempol or apocyanin have not yet been explored. Here, we hypothesize that TNF-[alpha]-induced oxidative stress compromises cardiac function by altering the mitochondrial redox state and the membrane permeability transition pore (MPTP) opening, thereby causing mitochondrial dysfunction. We measured the redox states in the cytosol and mitochondria of the heart to understand the mechanisms related to the MPTP and the antioxidant defense system. Our studies demonstrate that TNF-[alpha]-induced oxidative stress alters redox homeostasis by impairing the MPTP proteins adenine nucleotide translocator and voltage-dependent anion channel, thereby resulting in the pore opening, causing uncontrolled transport of substances to alter mitochondrial pH, and subsequently leading to dysfunction of mitochondria and attenuated cardiac function. Interestingly, we show that the supplementation of Tempol along with TNF-[alpha] restores mitochondrial and cardiac function. tumor necrosis factor-[alpha]; redox state; membrane permeability transition pore protein; cardiac function; cytokines

Byline: I. Hanta (1), S. Kuleci (1), N. Canacankatan (2), A. Kocabas (1) Keywords: Asthma; Oxidative stress; MDA and SOD Abstract: We investigated the oxidant-antioxidant balance and the effect of inhaled corticosteroids on this balance in mild stable asthmatics. Included in the study were 30 mild asthmatic patients (11 male, 19 female, mean age (year) +- SD: 35.1 +- 9.7) and 26 healthy adults (7 male, 19 female, mean age (year) +- SD: 40.8 +- 13.3). In all study groups, the peripheral venous blood samples were taken for plasma malonyldialdehyde (MDA), a parameter of lipid peroxidation caused by the oxidants, and erythrocyte superoxide dismutase (SOD), an antioxidant enzyme. The mean plasma MDA level was lower in the asthmatic group (5.7 +- 1.2 nmol/ml) than in the healthy group (6.3 +- 1.7 nmol/ml) and the mean erythrocyte SOD level was higher in asthmatic group (1086.4 +- 247.4 U/gHb) than in the healthy group (1028.0 +- 230.0 U/gHb). However, there were no significant differences in measurements of both plasma MDA levels and erythrocyte SOD enzyme activities between the groups (respectively, p = 0.1 and p = 0.4). When asthmatic patients were divided into subgroups as "inhaled steroid user" and "no inhaled steroid user", no significant differences were observed in the measurements of either plasma MDA level or erythrocyte SOD enzyme activity between the mentioned subgroups. According to the results of our study, we can say that oxidant-antioxidant balance is not significantly affected in mild asthmatics or measurement of plasma level of MDA and erythrocyte SOD enzyme activity is not sensitive to the oxidant-antioxidant balance in mild asthmatics. Author Affiliation: (1) Department of Chest Diseases, University of Cukurova, School of Medicine, Balcali/Adana, Turkey (2) Department of Biochemistry, University of Cukurova, School of Medicine, Balcali/Adana, Turkey Article History: Registration Date: 01/01/2003 Accepted Date: 23/06/2003

A laboratory experiment that aims to synthesize a manganese complex of complex of ,'-bis(salicylidene)ethane-1,2-diamine ligand (H(sub 2) salen) is described. The superoxide ability of the complex is also investigated. Recommendations on how to expand the experiment for characterization of the metal complex or the ligand are also given.

The semisynthesis and spectroscopic characterization of a series of derivatives with the sequences 243-CACA, 243-CAUA, 243-UACA, and 243-UAUA in the D1 double mutant (C22AC25A) background produced by expressed protein ligation of Sec-containing tetrapeptides to an hCCS-243 truncation is described. The data indicated the involvement of Cys residues in D2 of hCCS formation, stability, and redox potential of the D3 cluster and their significance to the SOD1 thiol/disulfide oxidase activity.d

NMR spectroscopy is used to understand the structure of the SodCII-encoded monomeric Cu, Zn superoxide dismutase from Salmonella enterica. The results provide insight into the functional differences between monomeric and dimeric bacterial Cu, Zn superoxide dismutases and the convergent evolution toward a dimeric structure in prokaryotic and eukaryotic class of enzymes.

Amyotrophic lateral sclerosis (ALS) is a neurological disorder characterized by the selective premature degeneration and death of motor neurons, which control voluntary actions such as breathing and walking. The disease [...]