The major hurdle in understanding Alzheimer's disease (AD) is a lack of knowledge about the etiology and pathogenesis of selective neuron death. In recent years, considerable data have accrued indicating that the brain in AD is under increased oxidative stress and this may have a role in the pathogenesis of neuron degeneration and death in this disorder. The direct evidence supporting increased oxidative stress in AD is: (1) increased brain Fe, Al, and Hg in AD, capable of stimulating free radical generation; (2) increased lipid peroxidation and decreased polyunsaturated fatty acids in the AD brain, and increased 4-hydroxynonenal, an aldehyde product of lipid peroxidation in AD ventricular fluid; (<em>3</em>) increased protein and DNA oxidation in the AD brain; (4) diminished energy metabolism and decreased cytochrome c oxidase in the brain in AD; (5) advanced glycation end products (AGE), malondialdehyde, carbonyls, peroxynitrite, heme oxygenase-1 and <em>SOD</em>-1 in neurofibrillary tangles and AGE, heme oxygenase-1, <em>SOD</em>-1 in senile plaques; and (6) studies showing that amyloid beta peptide is capable of generating free radicals. Supporting indirect evidence comes from a variety of in vitro studies showing that free radicals are capable of mediating neuron degeneration and death. Overall, these studies indicate that free radicals are possibly involved in the pathogenesis of neuron death in AD. Because tissue injury itself can induce reactive oxygen species (ROS) generation, it is not known whether this is a primary or secondary event. Even if free radical generation is secondary to other initiating causes, they are deleterious and part of a cascade of events that can lead to neuron death, suggesting that therapeutic efforts aimed at removal of ROS or prevention of their formation may be beneficial in AD.

The present investigation aimed to use primary liver cell culture obtained from mullet, Liza klunzingeri, to evaluate the toxic effects of benzo[a]pyrene (BaP) and nonylphenol (NP) on the antioxidant defense system. Liver samples taken from 20 L. klunzingeri were digested with 0.1% collagenase IV. The digested cells were then moved to Leibovitz L-15 culture medium and incubated at 25 °C for 2 weeks. 10^-5 mol/l of BaP and 10^-4 mol/l of NP were considered as the half maximal inhibitory concentration (IC50). Cells were then incubated with L-15 medium containing BaP (0[control], 10^-6,2 × 10^-6,<em>3</em> × 10^-6 mol/l) and NP (0[control],10^-5,2 × 10^-5,<em>3</em> × 10^-5 mol/l), and sampling was performed after 6, 12, and 24 h of incubation for measurement of catalase (CAT), superoxide dismutase (<em>SOD</em>), glutathione peroxidase (GPx), lipid peroxidation (LPO), total antioxidant power, and total protein. The lowest concentration of BaP and NP did not have considerable toxic effects on cultivated hepatocytes. The activities of <em>SOD</em>, CAT, GPx, LPO, total antioxidant power, and total protein changed dose-dependently in cells treated with BaP and NP. In conclusion, based on the results, short-term exposure to BaP and NP induced the oxidative stress in cultivated liver cells of L. klunzingeri. The toxicity of both pollutants is mainly because of the induction of the reactive oxygen species (ROS), which lead to cell membrane disruption, damage of cellular metabolism, and interference with cellular macromolecules.

Single-site mutants in the Cu,Zn superoxide dismutase (SOD) gene (SODSOD. The 2.4 A crystal structure of human SOD, along with two other SOD structures, established that all 12 observed FALS mutant sites alter conserved interactions critical to the beta-barrel fold and dimer contact, rather than catalysis. Red cells from heterozygotes had less than 50 percent normal SOD activity, consistent with a structurally defective SOD dimer. Thus, defective SOD is linked to motor neuron death and carries implications for understanding and possible treatment of FALS.

beta-Catenin is a multifunctional protein that mediates Wnt signaling by binding to members of the T cell factor (TCF) family of transcription factors. Here, we report an evolutionarily conserved interaction of beta-catenin with FOXO transcription factors, which are regulated by insulin and oxidative stress signaling. beta-Catenin binds directly to FOXO and enhances FOXO transcriptional activity in mammalian cells. In Caenorhabditis elegans, loss of the beta-catenin BAR-1 reduces the activity of the FOXO ortholog DAF-16 in dauer formation and life span. Association of beta-catenin with FOXO was enhanced in cells exposed to oxidative stress. Furthermore, BAR-1 was required for the oxidative stress-induced expression of the DAF-16 target gene <em>sod</em>-<em>3</em> and for resistance to oxidative damage. These results demonstrate a role for beta-catenin in regulating FOXO function that is particularly important under conditions of oxidative stress.

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of <em>sod</em>-<em>3</em> by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

Autophagy is involved in human diseases and is regulated by reactive oxygen species (ROS) including superoxide (O(2)(*-)) and hydrogen peroxide (H(2)O(2)). However, the relative functions of O(2)(*-) and H(2)O(2) in regulating autophagy are unknown. In this study, autophagy was induced by starvation, mitochondrial electron transport inhibitors, and exogenous H(2)O(2). We found that O(2)(*-) was selectively induced by starvation of glucose, L-glutamine, pyruvate, and serum (GP) whereas starvation of amino acids and serum (AA) induced O(2)(*-) and H(2)O(2). Both types of starvation induced autophagy and autophagy was inhibited by overexpression of <em>SOD</em>2 (manganese superoxide dismutase, Mn-<em>SOD</em>), which reduced O(2)(*-) levels but increased H(2)O(2) levels. Starvation-induced autophagy was also inhibited by the addition of catalase, which reduced both O(2)(*-) and H(2)O(2) levels. Starvation of GP or AA also induced cell death that was increased following treatment with autophagy inhibitors <em>3</em>-methyladenine, and wortamannin. Mitochondrial electron transport chain (mETC) inhibitors in combination with the <em>SOD</em> inhibitor 2-methoxyestradiol (2-ME) increased O(2)(*-) levels, lowered H(2)O(2) levels, and increased autophagy. In contrast to starvation, cell death induced by mETC inhibitors was increased by 2-ME. Finally, adding exogenous H(2)O(2) induced autophagy and increased intracellular O(2)(*-) but failed to increase intracellular H(2)O(2). Taken together, these findings indicate that O(2)(*-) is the major ROS-regulating autophagy.

Longevity is regulated by the daf-2 gene network in Caenorhabditis elegans. Mutations in the daf-2 gene, which encodes a member of the insulin receptor family, confer the life extension (Age) phenotype and the constitutive dauer (a growth-arrested larval form specialized for dispersal) formation phenotype. The Age phenotype is mutually potentiated by two life extension mutations in the daf-2 gene and the clk-1 gene, a homologue of yeast CAT5/COQ7 known to regulate ubiquinone biosynthesis. In this study, we demonstrated that the daf-2 mutation also conferred an oxidative stress resistance (Oxr) phenotype, which was also enhanced by the clk-1 mutation. Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 to the daf-16 gene, a homologue of the HNF-<em>3</em>/forkhead transcription factor. These findings led us to examine whether the insulin-like signaling pathway regulates the gene expression of antioxidant defense enzymes. We found that the mRNA level of the <em>sod</em>-<em>3</em> gene, which encodes Mn-superoxide dismutase (<em>SOD</em>), was much higher in daf-2 mutants than in the wild type. Moreover, the increased <em>sod</em>-<em>3</em> gene expression phenotype is regulated by the insulin-like signaling pathway. Although the clk-1 mutant itself did not display Oxr and the increased <em>sod</em>-<em>3</em> expression phenotypes, the clk-1 mutation enhanced them in the daf-2 mutant, suggesting that clk-1 is involved in longevity in two ways: clk-1 composes the original clk-1 longevity program and the daf-2 longevity program. These observations suggest that the daf-2 gene network controls longevity by regulating the Mn-<em>SOD</em>-associated antioxidant defense system. This system appears to play a role in efficient life maintenance at the dauer stage.

The etiology of the selective neuronal death that occurs in Huntington's disease (HD) is unknown. Several lines of evidence implicate the involvement of energetic defects and oxidative damage in the disease process, including a recent study that demonstrated an interaction between huntingtin protein and the glycolytic enzyme glyceraldehyde-<em>3</em>-phosphate dehydrogenase (GAPDH). Using spectrophotometric assays in postmortem brain tissue, we found evidence of impaired oxidative phosphorylation enzyme activities restricted to the basal ganglia in HD brain, while enzyme activities were unaltered in three regions relatively spared by HD pathology (frontal cortex, parietal cortex, and cerebellum). Citrate synthase-corrected complex II-III activity was markedly reduced in both HD caudate (-29%) and putamen (-67%), and complex IV activity was reduced in HD putamen (-62%). Complex I and GAPDH activities were unaltered in all regions examined. We also measured levels of the oxidative damage product 8-hydroxydeoxyguanosine (OH8dG) in nuclear DNA, and superoxide dismutase (<em>SOD</em>) activity. OH8dG levels were significantly increased in HD caudate. Cytosolic <em>SOD</em> activity was slightly reduced in HD parietal cortex and cerebellum, whereas particulate <em>SOD</em> activity was unaltered in these regions. These results further support a role for metabolic dysfunction and oxidative damage in the pathogenesis of HD.

We aimed to elucidate the possible role of phenotypic alterations and oxidative stress in age-related endothelial dysfunction of coronary arterioles. Arterioles were isolated from the hearts of young adult (Y, 14 weeks) and aged (A, 80 weeks) male Sprague-Dawley rats. For videomicroscopy, pressure-induced tone of Y and A arterioles and their passive diameter did not differ significantly. In A, arterioles L-NAME (a NO synthase blocker)-sensitive flow-induced dilations were significantly impaired (Y: 41+/-8% versus A: <em>3</em>+/-2%), which could be augmented by superoxide dismutase (<em>SOD</em>) or Tiron (but not L-arginine or the TXA(2) receptor antagonist SQ29,548). For lucigenin chemiluminescence, O(2)(.-) generation was significantly greater in A than Y vessels and could be inhibited with <em>SOD</em> and diphenyliodonium. NADH-driven O(2)(.-) generation was also greater in A vessels. Both endothelial and smooth muscle cells of A vessels produced O(2)(.-) (shown with ethidium bromide fluorescence). For Western blotting, expression of eNOS and COX-1 was decreased in A compared with Y arterioles, whereas expressions of COX-2, Cu/Zn-<em>SOD</em>, Mn-<em>SOD</em>, xanthine oxidase, and the NAD(P)H oxidase subunits p47(phox), p67(phox), Mox-1, and p22(phox) did not differ. Aged arterioles showed an increased expression of iNOS, confined to the endothelium. Decreased eNOS mRNA and increased iNOS mRNA expression in A vessels was shown by quantitative RT-PCR. In vivo formation of peroxynitrite was evidenced by Western blotting, and immunohistochemistry showing increased <em>3</em>-nitrotyrosine content in A vessels. Thus, aging induces changes in the phenotype of coronary arterioles that could contribute to the development of oxidative stress, which impairs NO-mediated dilations.

1 The pharmacological effects of endothelium-derived relaxing factor (EDRF), nitric oxide (NO) and prostacyclin on human and rabbit platelets were examined. 2 EDRF is released from porcine aortic endothelial cells, cultured on microcarriers and treated with indomethacin, in sufficient quantities to inhibit platelet aggregation induced by 9,11-dideoxy-9 alpha, 11 alpha-methano epoxy-prostaglandin F2 alpha (U46619) and collagen. <em>3</em> The anti-aggregating activity of EDRF was potentiated by M&B 22948, a selective inhibitor of cyclic GMP phosphodiesterase, and by superoxide dismutase (<em>SOD</em>) and was inhibited by haemoglobin and Fe2+. 4 Both NO and prostacyclin inhibited platelet aggregation. 5 The anti-aggregatory activity of NO, but not that of prostacyclin, was potentiated by M&B 22948 and by <em>SOD</em> and was inhibited by haemoglobin and Fe2+. Thus NO is a potent inhibitor of platelet aggregation whose activity on platelets mimics that of EDRF. 6 It is likely that the inhibitory effect of NO on platelets represents the action of endogenous EDRF and therefore this substance, together with prostacyclin, is a regulator of platelet-vessel wall interactions.

The endothelium is a source of reactive oxygen species in short-term models of hypercholesterolemia and atherosclerosis. We examined a chronic model of atherosclerosis for increased vascular production of superoxide (O2-.) and determined whether endothelial overexpression of superoxide dismutase (<em>SOD</em>) would improve endothelium-dependent relaxation. Superoxide generation was <em>3</em> times higher in isolated aortas from Watanabe heritable hyperlipidemic (WHHL) rabbits (2 to 4 years old) compared with aortas from New Zealand White (NZ) rabbits (4<em>3</em>+/-10 versus 14+/-2 relative light units x min(-1) x mm(-2), n=9, P<0.05). After in vitro transduction with adenovirus containing the gene for CuZn-<em>SOD</em> (AdCMVCuZn-<em>SOD</em>) or extracellular <em>SOD</em> (AdCMVEC-<em>SOD</em>), endothelial O2-. levels in WHHL aortas were significantly reduced. Gene transfer of <em>SOD</em> to WHHL aortas, however, failed to improve the impaired relaxation to acetylcholine or calcium ionophore. By use of the oxidative fluorescent dye hydroethidine, an in situ assay indicated markedly increased generation of O2-. throughout the wall of WHHL aorta, especially within layers of smooth muscle. This finding was confirmed by demonstrating increased O2-. levels in smooth muscle cells cultured from WHHL aorta. We conclude that elevated O2-. levels in atherosclerotic vessels are not confined to the endothelium but occur throughout the vascular wall, including smooth muscle cells. Reduction in endothelial O2-. levels is not sufficient to improve endothelium-dependent relaxation. Generation of reactive oxygen species within the media may contribute to vasomotor dysfunction in atherosclerosis.

BACKGROUND

In the nematode Caenorhabditis elegans the conserved Ins/IGF-1 signaling pathway regulates many biological processes including life span, stress response, dauer diapause and metabolism. Detection of differentially expressed genes may contribute to a better understanding of the mechanism by which the Ins/IGF-1 signaling pathway regulates these processes. Appropriate normalization is an essential prerequisite for obtaining accurate and reproducible quantification of gene expression levels. The aim of this study was to establish a reliable set of reference genes for gene expression analysis in C. elegans.

RESULTS

Real-time quantitative PCR was used to evaluate the expression stability of 12 candidate reference genes (act-1, ama-1, cdc-42, csq-1, eif-<em>3</em>.C, mdh-1, gpd-2, pmp-<em>3</em>, tba-1, Y45F10D.4, rgs-6 and unc-16) in wild-type, three Ins/IGF-1 pathway mutants, dauers and L<em>3</em> stage larvae. After geNorm analysis, cdc-42, pmp-<em>3</em> and Y45F10D.4 showed the most stable expression pattern and were used to normalize 5 <em>sod</em> expression levels. Significant differences in mRNA levels were observed for <em>sod</em>-1 and <em>sod</em>-<em>3</em> in daf-2 relative to wild-type animals, whereas in dauers <em>sod</em>-1, <em>sod</em>-<em>3</em>, <em>sod</em>-4 and <em>sod</em>-5 are differentially expressed relative to third stage larvae.

CONCLUSIONS

Our findings emphasize the importance of accurate normalization using stably expressed reference genes. The methodology used in this study is generally applicable to reliably quantify gene expression levels in the nematode C. elegans using quantitative PCR.

Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as <em>SOD</em> mimics. The same thermodynamic and electrostatic properties that make them potent <em>SOD</em> mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO(<em>3</em>)(*-), peroxyl radical, and less efficiently H(2)O(2). By doing so <em>SOD</em> mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds.

Blood vessels express <em>3</em> isoforms of superoxide dismutase (<em>SOD</em>): cytosolic or copper-zinc <em>SOD</em> (CuZn-<em>SOD</em>), manganese <em>SOD</em> (Mn-<em>SOD</em>) localized in mitochondria, and an extracellular form of CuZn-<em>SOD</em> (EC-<em>SOD</em>). Because there are no selective pharmacological inhibitors of individual <em>SOD</em> isoforms, the functional importance of the different <em>SODs</em> has been difficult to define. Recent molecular approaches, primarily the use of genetically-altered mice and viral-mediated gene transfer, have allowed investigators to begin to define the role of specific <em>SOD</em> isoforms in vascular biology. This review will focus mainly on the role of individual <em>SODs</em> in relation to endothelium under normal conditions and in disease states. This area is important because reactive oxygen species and superoxide anion are thought to play major roles in changes in vascular structure and function in pathophysiology.

BACKGROUND

Complications of ERCP are an important concern. We sought to determine predictors of post-ERCP complications at our institution.

METHODS

GI TRAC is a comprehensive data set of patients who underwent ERCP at our institution from 1994 through 2006. Logistic regression models were used to evaluate 4 categories of complications: (1) overall complications, (2) pancreatitis, (<em>3</em>) bleeding, and (4) severe or fatal complications. Independent predictors of complications were determined with multivariable logistic regression.

RESULTS

A total of 11,497 ERCP procedures were analyzed. There were 462 complications (4.0%), 42 of which were severe (0.<em>3</em>6%) and 7 were fatal (0.06%). Specific complications of pancreatitis (2.6%) and bleeding (0.<em>3</em>%) were identified. Overall complications were statistically more likely among individuals with suspected sphincter of Oddi dysfunction (SOD) (odds ratio [OR] 1.91) and after a biliary sphincterotomy (OR 1.<em>3</em>2). Subjects with a history of acute or chronic pancreatitis (OR 0.78) or who received a temporary small-caliber pancreatic stent (OR 0.69) had fewer complications. Post-ERCP pancreatitis was more likely to occur after a pancreatogram via the major papilla (OR 1.70) or minor papilla (OR 1.54) and among subjects with suspected SOD with stent placement (OR 1.45) or without stent placement (OR 1.84). Individuals undergoing biliary-stent exchange had less-frequent pancreatitis (OR 0.<em>3</em>8). Biliary sphincterotomy was associated with bleeding (OR 4.71). Severe or fatal complications were associated with severe (OR 2.<em>3</em>8) and incapacitating (OR 7.65) systemic disease, obesity (OR 5.18), known or suspected bile-duct stones (OR 4.08), pancreatic manometry (OR <em>3</em>.57), and complex (grade <em>3</em>) procedures (OR 2.86).

CONCLUSIONS

This study characterizes a large series of ERCP procedures from a single institution and outlines the incidence and predictors of complications.

Oxidative stress has been implicated in the pathophysiology of myocardial failure. We tested the hypothesis that oxidative stress can regulate extracellular matrix in cardiac fibroblasts. Neonatal and adult rat cardiac fibroblasts in vitro were exposed to H(2)O(2) (0.05-5 microM) or the superoxide-generating system xanthine (500 microM) plus xanthine oxidase (0.001-0.1 mU/ml) (XXO) for 24 h. In-gel zymography demonstrated that H(2)O(2) and XXO each increased gelatinase activity corresponding to matrix metalloproteinases (MMP) MMP-1<em>3</em>, MMP-2, and MMP-9. H(2)O(2) and XXO decreased collagen synthesis (collagenase-sensitive [(<em>3</em>)H]proline incorporation) without affecting total protein synthesis ([(<em>3</em>)H]leucine incorporation). H(2)O(2) and XXO decreased the expression of procollagen alpha(1)(I), alpha(2)(I), and alpha(1)(III) mRNA but increased the expression of fibronectin mRNA, suggesting a selective transcriptional effect on collagen synthesis. H(2)O(2), but not XXO, also decreased the expression of nonfibrillar procollagen alpha(1)(IV) and alpha(2)(IV) mRNA. To determine the role of endogenous antioxidant systems, cells were treated with the superoxide dismutase (<em>SOD</em>) inhibitor diethyldithiocarbamic acid (DDC, 100 microM) to increase intracellular superoxide or with the glucose-6-phosphate dehydrogenase inhibitor dehydroisoandrosterone <em>3</em>-acetate (DHEA; 10 microM) to increase intracellular H(2)O(2). DDC and DHEA decreased collagen synthesis and increased MMP activity, and both effects were inhibited by an <em>SOD</em>/catalase mimetic. Thus increased oxidative stress activates MMPs and decreases fibrillar collagen synthesis in cardiac fibroblasts. Oxidative stress may play a role in the pathogenesis of myocardial remodeling by regulating the quantity and quality of extracellular matrix.

Superoxide production has been measured in subcellular fractions of <em>SOD</em>-deficient Escherichia coli provided with physiological reductants. Although cytosolic enzyme(s) do generate O2-., the larger portion is produced by autoxidation of components of the respiratory electron-transport chain. At <em>3</em>7 degrees C and with pO2, NADH, and NAD+ levels matching those in vivo, respiring membrane vesicles generate <em>3</em> O2-./10,000 electrons transferred. This corresponds to intracellular O2-. production, in glucose-fed cells, of 5 microM/s. The high <em>SOD</em> content of normal cells restricts O2-. accumulation to 2.10(-10) M, with a moderate gradient from the membrane to the center of the cell. <em>SOD</em>-deficient mutants achieve a much higher steady-state content of O2-.. Rates of superoxide-mediated inactivation of certain enzymes are sufficiently rapid that even 10(-10) M O2-. imposes a significant oxidative stress.

To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-<em>3</em>, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-<em>3</em>, which shows a corresponding decrease in expression in old worms. elt-<em>3</em> regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and <em>sod</em>-<em>3</em>. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-<em>3</em>, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage.

Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm-grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI-1 (N-(<em>3</em>-oxododecanoyl)-L-homoserine lactone) and PAI-2 (N-butyryl-L-homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (<em>SODs</em>), Mn-<em>SOD</em> and Fe-<em>SOD</em>, and the major catalase, KatA. The expression of sodA (encoding Mn-<em>SOD</em>) was particularly dependent on PAI-1, whereas the influence of autoinducers on Fe-<em>SOD</em> and KatA levels was also apparent but not to the degree observed with Mn-<em>SOD</em>. beta-Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI-1/2-deficient double mutant partially restored KatA activity, while the addition of PAI-1 only was sufficient for full restoration of Mn-<em>SOD</em> activity. In biofilm studies, catalase activity in wild-type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild-type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Research into methamphetamine-induced neurotoxicity has experienced a resurgence in recent years. This is due to (1) greater understanding of the mechanisms underlying methamphetamine neurotoxicity, (2) its usefulness as a model for Parkinson's disease and (<em>3</em>) an increased abuse of the substance, especially in the American Mid-West and Japan. It is suggested that the commonly used experimental one-day methamphetamine dosing regimen better models the acute overdose pathologies seen in humans, whereas chronic models are needed to accurately model human long-term abuse. Further, we suggest that these two dosing regimens will result in quite different neurochemical, neuropathological and behavioral outcomes. The relative importance of the dopamine transporter and vesicular monoamine transporter knockout is discussed and insights into oxidative mechanisms are described from observations of nNOS knockout and <em>SOD</em> overexpression. This review not only describes the neuropathologies associated with methamphetamine in rodents, non-human primates and human abusers, but also focuses on the more recent literature associated with reactive oxygen and nitrogen species and their contribution to neuronal death via necrosis and/or apoptosis. The effect of methamphetamine on the mitochondrial membrane potential and electron transport chain and subsequent apoptotic cascades are also emphasized. Finally, we describe potential treatments for methamphetamine abusers with reference to the time after withdrawal. We suggest that potential treatments can be divided into three categories; (1) the prevention of neurotoxicity if recidivism occurs, (2) amelioration of apoptotic cascades that may occur even in the withdrawal period and (<em>3</em>) treatment of the atypical depression associated with withdrawal.

Nonalcoholic fatty liver disease (NAFLD) is one of the most frequent causes of abnormal liver dysfunction, and its prevalence has markedly increased. We previously evaluated the expression of fatty acid metabolism-related genes in NAFLD and reported changes in expression that could contribute to increased fatty acid synthesis. In the present study, we evaluated the expression of additional fatty acid metabolism-related genes in larger groups of NAFLD (n=26) and normal liver (n=10) samples. The target genes for real-time PCR analysis were as follows: acetyl-CoA carboxylase (ACC) 1, ACC2, fatty acid synthase (FAS), sterol regulatory element-binding protein 1c (SREBP-1c), and adipose differentiation-related protein (ADRP) for evaluation of de novo synthesis and uptake of fatty acids; carnitine palmitoyltransferase 1a; (CPT1a), long-chain acyl-CoA dehydrogenase (LCAD), long-chain L-<em>3</em>-hydroxyacylcoenzyme A dehydrogenase alpha (HADHalpha), uncoupling protein 2 (UCP2), straight-chain acyl-CoA oxidase (ACOX), branched-chain acyl-CoA oxidase (BOX), cytochrome P450 2E1 (CYP2E1), CYP4A11, and peroxisome proliferator-activated receptor (PPAR)alpha for oxidation in the mitochondria, peroxisomes and microsomes; superoxide dismutase (<em>SOD</em>), catalase, and glutathione synthetase (GSS) for antioxidant pathways; and diacylglycerol O-acyltransferase 1 (DGAT1), PPARgamma, and hormone-sensitive lipase (HSL) for triglyceride synthesis and catalysis. In NAFLD, although fatty acids accumulated in hepatocytes, their de novo synthesis and uptake were up-regulated in association with increased expression of ACC1, FAS, SREBP-1c, and ADRP. Fatty acid oxidation-related genes, LCAD, HADHalpha, UCP2, ACOX, BOX, CYP2E1, and CYP4A11, were all overexpressed, indicating that oxidation was enhanced in NAFLD, whereas the expression of CTP1a and PPARalpha was decreased. Furthermore, <em>SOD</em> and catalase were also overexpressed, indicating that antioxidant pathways are activated to neutralize reactive oxygen species (ROS), which are overproduced during oxidative processes. The expression of DGAT1 was up-regulated without increased PPARgamma expression, whereas the expression of HSL was decreased. Our data indicated the following regarding NAFLD: i) increased de novo synthesis and uptake of fatty acids lead to further fatty acid accumulation in hepatocytes; ii) mitochondrial fatty acid oxidation is decreased or fully activated; iii) in order to complement the function of mitochondria (beta-oxidation), peroxisomal (beta-oxidation) and microsomal (omega-oxidation) oxidation is up-regulated to decrease fatty acid accumulation; iv) antioxidant pathways including <em>SOD</em> and catalase are enhanced to neutralize ROS overproduced during mitochondrial, peroxisomal, and microsomal oxidation; and v) lipid droplet formation is enhanced due to increased DGAT expression and decreased HSL expression. Further studies will be needed to clarify how fatty acid synthesis is increased by SREBP-1c, which is under the control of insulin and AMP-activated protein kinase.

Familial amyotrophic lateral sclerosis (FALS) is an inherited neurodegenerative disorder of the motor neurons. While 10-15% of cases are caused by mutations in the copper/zinc superoxide-dismutase-1 (<em>SOD</em>-1) gene, the dying-forward hypothesis, in which corticomotoneurons induce anterograde excitotoxic motoneuron degeneration, has been proposed as a potential mechanism. The present study applied novel threshold tracking transcranial magnetic stimulation techniques to investigate the mechanisms underlying neurodegeneration in FALS. Studies were undertaken in 14 asymptomatic and <em>3</em> pre-symptomatic <em>SOD</em>-1 mutation carriers, followed longitudinally for up to <em>3</em>-years. The pre-symptomatic subjects were asymptomatic at the time of their initial study but developed symptoms during the follow-up period. Results were compared to 7 <em>SOD</em>-1 FALS patients, 50 sporadic ALS patients and 55 normal controls. Short-interval intracortical inhibition (SICI) was significantly reduced in <em>SOD</em>-1 FALS (-1.2 +/- 0.6%) and sporadic ALS patients (-0.7 +/- 0.<em>3</em>%) compared to asymptomatic <em>SOD</em>-1 mutation carriers (9.8 +/- 1.5%, P<0.00001) and normal controls (8.5 +/- 1.0%, P<0.00001). SICI reduction was accompanied by increases in intracortical facilitation, motor evoked potential amplitudes and the slope of the magnetic stimulus-response curve. In two pre-symptomatic <em>SOD</em>-1 mutation carriers SICI was completely absent (SICI patient 1, -<em>3</em>.2%; patients 2, -1.<em>3</em>%), while in one subject there was a <em>3</em>2% reduction in SICI prior to symptom onset. These three individuals subsequently developed clinical features of ALS. Simultaneous investigation of central and peripheral excitability has established that cortical hyperexcitability develops in clinically affected <em>SOD</em>-1 FALS patients, similar to that seen in sporadic ALS patients, thereby suggesting that a similar pathophysiological process in evident in both familial and sporadic ALS patients. In addition, the present study has established that cortical hyperexcitability precedes the development of clinical symptoms in pre-symptomatic carriers of the <em>SOD</em>1 mutation, thereby suggesting that cortical hyperexcitability underlies neurodegeneration in FALS.

We review gases that can affect oxidative stress and that themselves may be radicals. We discuss O(2) toxicity, invoking superoxide, hydrogen peroxide, and the hydroxyl radical. We also discuss superoxide dismutase (<em>SOD</em>) and both ground-state, triplet oxygen ((<em>3</em>)O(2)), and the more energetic, reactive singlet oxygen ((1)O(2)). Nitric oxide ((*)NO) is a free radical with cell signaling functions. Besides its role as a vasorelaxant, (*)NO and related species have other functions. Other endogenously produced gases include carbon monoxide (CO), carbon dioxide (CO(2)), and hydrogen sulfide (H(2)S). Like (*)NO, these species impact free radical biochemistry. The coordinated regulation of these species suggests that they all are used in cell signaling. Nitric oxide, nitrogen dioxide, and the carbonate radical (CO(<em>3</em>)(*-)) react selectively at moderate rates with nonradicals, but react fast with a second radical. These reactions establish "cross talk" between reactive oxygen (ROS) and reactive nitrogen species (RNS). Some of these species can react to produce nitrated proteins and nitrolipids. It has been suggested that ozone is formed in vivo. However, the biomarkers that were used to probe for ozone reactions may be formed by non-ozone-dependent reactions. We discuss this fascinating problem in the section on ozone. Very low levels of ROS or RNS may be mitogenic, but very high levels cause an oxidative stress that can result in growth arrest (transient or permanent), apoptosis, or necrosis. Between these extremes, many of the gasses discussed in this review will induce transient adaptive responses in gene expression that enable cells and tissues to survive. Such adaptive mechanisms are thought to be of evolutionary importance.

Peroxynitrite has recently been implicated in the inactivation of many enzymes. However, little has been reported on the structural basis of the inactivation reaction. This study proposes that nitration of a specific tyrosine residue is responsible for inactivation of recombinant human mitochondrial manganese-superoxide dismutase (Mn-<em>SOD</em>) by peroxynitrite. Mass spectroscopic analysis of the peroxynitrite-inactivated Mn-<em>SOD</em> showed an increased molecular mass because of a single nitro group substituted onto a tyrosine residue. Single peptides that had different elution positions between samples from the native and peroxynitrite-inactivated Mn-<em>SOD</em> on reverse-phase high performance liquid chromatography were isolated after successive digestion of the samples by staphylococcal serine protease and lysylendopeptidase and subjected to amino acid sequence and molecular mass analyses. We found that tyrosine <em>3</em>4 of the enzyme was exclusively nitrated to <em>3</em>-nitrotyrosine by peroxynitrite. This residue is located near manganese and in a substrate O-2 gateway in Mn-<em>SOD</em>.