Superoxide Dismutase

Nitro blue tetrazolium has been used to intercept O 2 − generated enzymically or photochemically. The reduction of NBT by O 2 − has been utilized as the basis of assays for superoxide dismutase, which exposes its presence by inhibiting the reduction of NBT. Superoxide ...

Oxygen-free radicals, more generally known as reactive oxygen species (ROS) along with reactive nitrogen species (RNS) are well recognised for playing a dual role as both deleterious and beneficial species. The "two-faced" character of ROS is substantiated by growing body of evidence that ROS within cells act as secondary messengers in intracellular signalling cascades, which induce and maintain the oncogenic phenotype of cancer cells, however, ROS can also induce cellular senescence and apoptosis and can therefore function as anti-tumourigenic species. The cumulative production of ROS/RNS through either endogenous or exogenous insults is termed oxidative stress and is common for many types of cancer cell that are linked with altered redox regulation of cellular signalling pathways. Oxidative stress induces a cellular redox imbalance which has been found to be present in various cancer cells compared with normal cells; the redox imbalance thus may be related to oncogenic stimulation. DNA mutation is a critical step in carcinogenesis and elevated levels of oxidative DNA lesions (8-OH-G) have been noted in various tumours, strongly implicating such damage in the etiology of cancer. It appears that the DNA damage is predominantly linked with the initiation process. This review examines the evidence for involvement of the oxidative stress in the carcinogenesis process. Attention is focused on structural, chemical and biochemical aspects of free radicals, the endogenous and exogenous sources of their generation, the metal (iron, copper, chromium, cobalt, vanadium, cadmium, arsenic, nickel)-mediated formation of free radicals (e.g. Fenton chemistry), the DNA damage (both mitochondrial and nuclear), the damage to lipids and proteins by free radicals, the phenomenon of oxidative stress, cancer and the redox environment of a cell, the mechanisms of carcinogenesis and the role of signalling cascades by ROS; in particular, ROS activation of AP-1 (activator protein) and NF-B (nuclear factor kappa B) signal transduction pathways, which in turn lead to the transcription of genes involved in cell growth regulatory pathways. The role of enzymatic (superoxide dismutase (Cu, Zn-SOD, Mn-SOD), catalase, glutathione peroxidase) and non-enzymatic antioxidants (Vitamin C, Vitamin E, carotenoids, thiol antioxidants (glutathione, thioredoxin and lipoic acid), flavonoids, selenium and others) in the process of carcinogenesis as well as the antioxidant interactions with various regulatory factors, including Ref-1, NF-B, AP-1 are also reviewed.

Lim et al., 2003a, 2003b). miRNAs are encoded in genes 1 Department of Biochemistry and Molecular distinct from the mRNAs whose expression they control. Pharmacology and siRNAs were first identified as the specificity determi-2 Department of Medicine nants of the RNAi pathway, where they act as guides University of Massachusetts Medical School to direct endonucleolytic cleavage of their target RNAs Lazare Research Building (Hamilton and Baulcombe, 1999; Hammond et al., 2000; 364 Plantation Street Zamore et al., 2000; Elbashir et al., 2001a). Prototypical Worcester, Massachusetts 01605 siRNA duplexes are 21 nt double-stranded RNAs that contain 19 base pairs, with 2 nt, 3Ј overhanging ends (Elbashir et al., 2001a; Nykä nen et al., 2001; Tang et al., Summary 2003). Active siRNAs, like miRNAs, contain 5Ј phosphates and 3Ј hydroxyls (Zamore et al., 2000; Boutla et A key step in RNA interference (RNAi) is assembly of al., 2001; Hutvá gner et al., 2001; Nykä nen et al., 2001; the RISC, the protein-siRNA complex that mediates Chiu and Rana, 2002; Mallory et al., 2002). Recent evitarget RNA cleavage. Here, we show that the two dence suggests that siRNAs and miRNAs are function-

Ϫ production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular и O 2 Ϫ production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/ kg per d) produced a similar degree of hypertension, but did not increase vascular и O 2 Ϫ production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of и O 2

Reactive O 2 species (ROS) are produced in both unstressed and stressed cells. Plants have welldeveloped defence systems against ROS, involving both limiting the formation of ROS as well as instituting its removal. Under unstressed conditions, the formation and removal of O 2 are in balance. However, the defence system, when presented with increased ROS formation under stress conditions, can be overwhelmed. Within a cell, the superoxide dismutases (SODs) constitute the first line of defence against ROS. Specialization of function among the SODs may be due to a combination of the influence of subcellular location of the enzyme and upstream sequences in the genomic sequence. The commonality of elements in the upstream sequences of Fe, Mn and CuuZn SODs suggests a relatively recent origin for those regulatory regions. The differences in the upstream regions of the three FeSOD genes suggest differing regulatory control which is borne out in the research literature. The finding that the upstream sequences of Mn and peroxisomal CuuZn SODs have three common elements suggests a common regulatory pathway. The tools are available to dissect further the molecular basis for antioxidant defence responses in plant cells. SODs are clearly among the most important of those defences, when coupled with the necessary downstream events for full detoxification of ROS.

Nitric oxide (NO) mediates several biological actions, including relaxation of blood vessels, cytotoxicity of activated macrophages, and formation of cGMP by activation ofglutamate receptors in cerebellar slices. Nitric oxide synthase (EC 1.14.23.-) immunoreactivit is colocalized with nicotinamide adenine di-nucleotide phosphate diaphorase in neurons that are uniquely resistant to toxic insults. We show that the nitric oxide synthase inhibitors, N0-nitro-L-arginine (EC50 = 20 INM) andNw-nmonomethyl-L-arginine (EC,5 = 170 jtM), prevent neurotoxicity elicited by N-methyl-D-aspartate and related excitatory amino acids. This effect is competitively reversed by L-arginine. Depletion of the culture medium of argnine by argnase or arginine-free growth medium completely attenuates N-methyl-D-aspartate toxicity. Sodium nitroprusside, which spontaneously releases NO, produces dose-dependent cell death that parallels cGMP formation. Hemoglobin, which complexes NO, prevents neurotoxic effects of both N-methyl-D-aspartate and sodium nitroprusside. These data establish that NO mediates the neurotoxicity of glutamate.

To describe the importance of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase working together in human cells against toxic reactive oxygen species, their relationship with several pathophysiologic processes and their possible therapeutic implications. Conclusions: Reactive oxygen species (ROS) are involved in the cell growth, differentiation, progression, and death. Low concentrations of ROS may be beneficial or even indispensable in processes such as intracellular signaling and defense against micro-organisms. Nevertheless, higher amounts of ROS play a role in the aging process as well as in a number of human disease states, including cancer, ischemia, and failures in immunity and endocrine functions. As a safeguard against the accumulation of ROS, several nonenzymatic and enzymatic antioxidant activities exist. Therefore, when oxidative stress arises as a consequence of a pathologic event, a defense system promotes the regulation and expression of these enzymes.

PPARg coactivator 1a (PGC-1a) is a potent stimulator of mitochondrial biogenesis and respiration. Since the mitochondrial electron transport chain is the main producer of reactive oxygen species (ROS) in most cells, we examined the effect of PGC-1a on the metabolism of ROS. PGC-1a is coinduced with several key ROS-detoxifying enzymes upon treatment of cells with an oxidative stressor; studies with RNAi or null cells indicate that PGC-1a is required for the induction of many ROS-detoxifying enzymes, including GPx1 and SOD2. PGC-1a null mice are much more sensitive to the neurodegenerative effects of MPTP and kainic acid, oxidative stressors affecting the substantia nigra and hippocampus, respectively. Increasing PGC-1a levels dramatically protects neural cells in culture from oxidativestressor-mediated death. These studies reveal that PGC-1a is a broad and powerful regulator of ROS metabolism, providing a potential target for the therapeutic manipulation of these important endogenous toxins.

The in vitro production of mammalian embryos suffers from high frequencies of developmental failure due to excessive levels of permanent embryo arrest and apoptosis caused by oxidative stress. The p66Shc stress adaptor protein controls oxidative stress response of somatic cells by regulating intracellular ROS levels through multiple pathways, including mitochondrial ROS generation and the repression of antioxidant gene expression. We have previously demonstrated a strong relationship with elevated p66Shc levels, reduced antioxidant levels and greater intracellular ROS generation with the high incidence of permanent cell cycle arrest of 2-4 cell embryos cultured under high oxygen tensions or after oxidant treatment. The main objective of this study was to establish a functional role for p66Shc in regulating the oxidative stress response during early embryo development. Using RNA interference in bovine zygotes we show that p66Shc knockdown embryos exhibited increased MnSOD levels, reduced intracellular ROS and DNA damage that resulted in a greater propensity for development to the blastocyst stage. P66Shc knockdown embryos were stress resistant exhibiting significantly reduced intracellular ROS levels, DNA damage, permanent 2-4 cell embryo arrest and diminished apoptosis frequencies after oxidant treatment. The results of this study demonstrate that p66Shc controls the oxidative stress response in early mammalian embryos. Small molecule inhibition of p66Shc may be a viable clinical therapy to increase the developmental potential of in vitro produced mammalian embryos.

To determine the role of reactive oxygen species in mammalian longevity, we generated transgenic mice that overexpress human catalase localized to the peroxisome, the nucleus, or mitochondria (MCAT). Median and maximum life spans were maximally increased (averages of 5 months and 5.5 months, respectively) in MCAT animals. Cardiac pathology and cataract development were delayed, oxidative damage was reduced, H 2 O 2 production and H 2 O 2induced aconitase inactivation were attenuated, and the development of mitochondrial deletions was reduced. These results support the free radical theory of aging and reinforce the importance of mitochondria as a source of these radicals.

Nitric oxide, which mediates influences of numerous neurotransmitters and modulators on vascular smooth muscle and leukocytes, can be formed in the brain from arginine by an enzymatic activity that stoichiometrically generates citrulline. We show that glutamate and related amino acids, such as N-methyl-D-aspartate, markedly stimulate arginine-citrulline transformation in cerebellar slices stoichiometrically with enhancement of cGMP levels. N'-monomethyl-L-arginine blocks the augmentation both of citrulline and cGMP with identical potencies. Arginine competitively reverses both effects ofN0-monomethyl-L-arginine with the same potencies. Hemoglobin, which complexes nitric oxide, prevents the stimulation by N-methyl-D-aspartate of cGMP levels, and superoxide dismutase, which elevates nitric oxide levels, increases cGMP formation. These data establish that nitric oxide mediates the stimulation by glutamate of cGMP formation.

The Drosophila melanogaster gene chico encodes an insulin receptor substrate that functions in an insulin/insulin-like growth factor (IGF) signaling pathway. In the nematode Caenorhabditis elegans, insulin/IGF signaling regulates adult longevity. We found that mutation of chico extends fruit fl y median life-span by up to 48% in homozygotes and 36% in heterozygotes. Extension of life-span was not a result of impaired oogenesis in chico females, nor was it consistently correlated with increased stress resistance. The dwarf phenotype of chico homozygotes was also unnecessary for extension of life-span. The role of insulin/IGF signaling in regulating animal aging is therefore evolutionarily conserved.

The effect of growing pea (Pisum sativum L.) plants with CdCl 2 (0-50 mM) on different plant physiological parameters and antioxidative enzymes of leaves was studied in order to know the possible involvement of this metal in the generation of oxidative stress. In roots and leaves of pea plants Cd produced a significant inhibition of growth as well as a reduction in the transpiration and photosynthesis rate, chlorophyll content of leaves, and an alteration in the nutrient status in both roots and leaves. The ultrastructural analysis of leaves from plants grown with 50 mM CdCl 2 , showed cell disturbances characterized by an increase of mesophyll cell size, and a reduction of intercellular spaces, as well as severe disturbances in chloroplast structure. Alterations in the activated oxygen metabolism of pea plants were also detected, as evidenced by an increase in lipid peroxidation and carbonyl-groups content, as well as a decrease in catalase, SOD and, to a lesser extent, guaiacol peroxidase activities. Glutathione reductase activity did not show significant changes as a result of Cd treatment. A strong reduction of chloroplastic and cytosolic Cu,Zn-SODs by Cd was found, and to a lesser extent of Fe-SOD, while Mn-SOD was only affected by the highest Cd concentrations. Catalase isoenzymes responded differentially, the most acidic isoforms being the most sensitive to Cd treatment. Results obtained suggest that growth of pea plants with CdCl 2 can induce a concentration-dependent oxidative stress situation in leaves, characterized by an accumulation of lipid peroxides and oxidized proteins as a result of the inhibition of the antioxidant systems. These results, together with the ultrastructural data, point to a possible induction of leaf senescence by cadmium.

The possible involvement of activated oxygen species in the mechanism of damage by NaCl stress was studied in leaves of four varieties of rice (Oryza sativa L.) exhibiting different sensitivities to NaCl. The 3-week-old rice seedlings were subjected to 0, 6 and 12 dS m −1 salinity ...

Exercise promotes longevity and ameliorates type 2 diabetes mellitus and insulin resistance. However, exercise also increases mitochondrial formation of presumably harmful reactive oxygen species (ROS). Antioxidants are widely used as supplements but whether they affect the health-promoting effects of exercise is unknown. We evaluated the effects of a combination of vitamin C (1000 mg/day) and vitamin E (400 IU/day) on insulin sensitivity as measured by glucose infusion rates (GIR) during a hyperinsulinemic, euglycemic clamp in previously untrained (n ‫؍‬ 19) and pretrained (n ‫؍‬ 20) healthy young men. Before and after a 4 week intervention of physical exercise, GIR was determined, and muscle biopsies for gene expression analyses as well as plasma samples were obtained to compare changes over baseline and potential influences of vitamins on exercise effects. Exercise increased parameters of insulin sensitivity (GIR and plasma adiponectin) only in the absence of antioxidants in both previously untrained (P < 0.001) and pretrained (P < 0.001) individuals. This was paralleled by increased expression of ROS-sensitive transcriptional regulators of insulin sensitivity and ROS defense capacity, peroxisomeproliferator-activated receptor gamma (PPAR␥), and PPAR␥ coactivators PGC1␣ and PGC1␤ only in the absence of antioxidants (P < 0.001 for all). Molecular mediators of endogenous ROS defense (superoxide dismutases 1 and 2; glutathione peroxidase) were also induced by exercise, and this effect too was blocked by antioxidant supplementation. Consistent with the concept of mitohormesis, exercise-induced oxidative stress ameliorates insulin resistance and causes an adaptive response promoting endogenous antioxidant defense capacity. Supplementation with antioxidants may preclude these health-promoting effects of exercise in humans. aging ͉ hormesis ͉ insulin resistance ͉ oxidative stress ͉ reactive oxygen species 1 To whom correspondence should be addressed. E-mail: [email protected]. 2 M.R., K.Z., A.O., and M. Blü her contributed equally to this work. This article contains supporting information online at www.pnas.org/cgi/content/full/ 0903485106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0903485106 PNAS Early Edition ͉ 1 of 6 MEDICAL SCIENCES

02 -, superoxide anion One-electron reduction state, formed in many autoxidation reactions (for example, flavoproteins, redox cycling) H02., perhydroxy radical Protonated form of 02 ', more lipid soluble H202, hydrogen peroxide Two-electron reduction state, formed from 0~. (H02') by dismutation or directly from 02 HO. (OH.), hydroxyl radical Three-electron reduction state, formed by Fenton reaction, metal (iron)-catalyzed Haber-Weiss reaction; highly reactive RO., alkoxy radical Oxygen-centered organic (for example, lipid) radical RO0., peroxy radical Formally formed from organic (for example, lipid) hydroperoxide, ROOH, by hydrogen abstraction ROOH Organic hydroperoxide (for example, lipid-, thymine-OOH) lag 0 2 (also 102) Singlet molecular oxygen, first excited state~ 22 kcal/mol above ground state (triplet) ~02; red (dimol) or infrared (monomol) photoemission RO (also RO*)

The SOD1 mutant mouse is the most widely used model of human amyotrophic lateral sclerosis (ALS). To determine where and when the pathological changes of motor neuron disease begins, we performed a comprehensive spatiotemporal analysis of disease progression in SOD1(G93A) mice. Quantitative pathological analysis was performed in the same mice at multiple ages at neuromuscular junctions (NMJ), ventral roots, and spinal cord. In addition, a patient with sporadic ALS who died unexpectedly was examined at autopsy. Mice became clinically weak at 80 days and died at 131 +/- 5 days. At 47 days, 40% of end-plates were denervated whereas there was no evidence of ventral root or cell body loss. At 80 days, 60% of ventral root axons were lost but there was no loss of motor neurons. Motor neuron loss was well underway by 100 days. Microglial and astrocytic activation around motor neurons was not identified until after the onset of distal axon degeneration. Autopsy of the ALS patient demonstrate...

This study was designed to examine whether endothelin is released from the intima of intact arteries, and whether endothelium-derived nitric oxide regulates its production. Endothelin was detected in the incubating medium of unstimulated pig aortae with, but not in those without endothelium. In preparations with endothelium, thrombin (2-6 U/ml) and the calcium ionophore A23187 (10(-6) M) stimulated the release of the peptide. The basal and thrombin-stimulated production of endothelin were prevented by the protein synthetase inhibitor cycloheximide (10(-6) M). The production of endothelin upon stimulation with thrombin (4 U/ml) was potentiated by L-NG-monomethyl arginine and methylene blue and reduced by superoxide dismutase and 8-bromo cyclic guanosine 5&amp;#39;-monophosphate (GMP), while the basal release of the peptide was unaffected. Thus, (a) endothelin is released from the intimal layer of intact blood vessels, both under basal conditions and after stimulation with thrombin and the calcium ionophore A23187, and (b) endothelium-derived nitric oxide released during stimulation with thrombin inhibits the production of the peptide via a cyclic GMP-dependent pathway.

Oxidative stress is involved in the aetiology of defective embryo development. Reactive oxygen species (ROS) may originate from embryo metabolism and/or embryo surroundings. Embryo metabolism generates ROS via several enzymatic mechanisms. The relative contribution of each source seems different depending on the species, the stage of development, and the culture conditions. Several exogenous factors and culture conditions can enhance the production of ROS by embryos. ROS can alter most types of cellular molecules, and also induce development block and retardation. Multiple mechanisms of embryo protection against ROS exist, and these have complementary actions. External protection, present in follicular and tubal fluids, mainly comprises non-enzymatic antioxidants such as hypotaurine, taurine and ascorbic acid. Internal protection mainly comprises antioxidant enzymes: superoxide dismutase, glutathione peroxidase and gamma-glutamylcysteine synthetase. Transcripts encoding for these enzymes are present in the oocyte, embryo and oviduct. It may be important that these transcripts are stored during oocyte maturation in order to allow the embryo to acquire the aptitude to develop. It is now common to add antioxidant compounds to culture media. Nevertheless, maintaining the pro-oxidant-antioxidant equilibrium in embryos through such supplementation is a complex problem. Further studies are necessary to limit oxidative stress during embryo culture.

The effects of NaCl stress on the activity of antioxidant enzyme such as superoxide dismutase (SOD: EC 1.15.1.1), peroxidase (POD: EC 1.11.1.7), glutathione reductase (GR: EC 1.6.4.2), rate of lipid peroxidation, gas-exchange, chlorophyll content and chlorophyll fluorescence were investigated in two cotton cultivars, Guazuncho and Pora (hybrids between Gossypium hirsutum )/G. arboretum )/G. raimondii ) grown in nutrient solution. Plants were treated with three salt concentrations (50, 100 and 200 mol m (3 NaCl) for 21 days. The SOD activity in Pora increases with the increase in the intensity of NaCl stress, but salt treatment had no significant effect on this enzyme activity in Guazuncho. The POD and GR activities showed similar trends under salt stress, in both cotton cultivars. In Pora, there was an average increase in GR activity of about 53%, but there was no further increase at higher NaCl concentrations. In Guazuncho, no change in GR activity was observed. Net photosynthesis and stomatal conductance decreased in response to salt stress, but Pora showed a smaller reduction in photosynthesis than Guazuncho. The results indicated that stomatal aperture limited leaf photosynthetic capacity in the NaCl-treated plants of both cultivars. However, significant reduction in the leaf chlorophyll contents due to NaCl stress was observed only on Guazuncho. In both cotton cultivars, the photochemical efficiency of PSII was not affected by salt stress. These results suggest that salttolerant cotton varieties may have a better protection against reactive oxygen species (ROS) by increasing the activity of antioxidant enzymes under salt stress. #

When seedlings of two rice (Oryza sativa L. ) cultivars were raised in sand cultures under 500 and 1000 mM Pb(NO 3 ) 2 in the medium, lengths as well as weights of roots and shoots decreased with increase in Pb concentration. Pb-treated seedlings showed elevated levels of lipid peroxides with a concomitant increase in the activities of the enzymes superoxide dismutase (SOD), guaiacol peroxidase, ascorbate peroxidase and glutathione reductase compared to controls. Though Pb was readily absorbed by growing seedlings, its localization was greater in roots than shoots. The level of Pb accumulation in seedlings was far higher than the supplied one. Seedlings grown for 5 Á/20 days in presence of 1000 mM Pb(NO 3 ) 2 showed about 21 Á/177% increase in the level of thiobarbituric acid reacting substances (TBARS) in shoots indicating enhanced lipid peroxidation compared to controls. With increase in the level of Pb treatment in situ peroxidases showed more increase in activity than SOD. Under both controls as well as Pb treatments roots maintained higher activity of these enzymes than shoots. About 87 Á/100% increase in SOD activity, 1.2 Á/5.6 times increase in guaiacol peroxidase activity and 1.2 Á/1.9 times increase in ascorbate peroxidase activity was observed in the roots of seedlings grown for 15 days in presence of 1000 mM Pb in the medium. Under similar treatment conditions about 128 Á/196% increase in glutathione reductase activity was recorded in roots and 69 Á/196% increase in shoots compared to control grown seedlings. Pb treatment resulted in a decline in catalase activity in roots whereas in shoots catalase activity increased in seedlings grown at moderately toxic Pb (500 mM) level whereas a highly toxic Pb (1000 mM) level led to a marked inhibition in enzyme activity. Two catalase isoforms were detected in roots and three in shoots of the seedlings. A highly toxic Pb (1000 mM) level led to decrease in the intensity of two preexisting catalase isoforms in shoots. Results suggest that Pb induces oxidative stress in growing rice plants and that SOD, peroxidases and GR could serve as important components of antioxidative defense mechanism against Pb induced oxidative injury in rice. #

Nitric oxide (NO) and reactive oxygen intermediates (ROIs) play key roles in the activation of disease resistance mechanisms both in animals and plants. In animals NO cooperates with ROIs to kill tumor cells and for macrophage killing of bacteria. Such cytotoxic events occur because unregulated NO levels drive a diffusionlimited reaction with O 2 ؊ to generate peroxynitrite (ONOO ؊ ), a mediator of cellular injury in many biological systems. Here we show that in soybean cells unregulated NO production at the onset of a pathogen-induced hypersensitive response (HR) is not sufficient to activate hypersensitive cell death. The HR is triggered only by balanced production of NO and ROIs. Moreover, hypersensitive cell death is activated after interaction of NO not with O 2 ؊ but with H 2O2 generated from O2 ؊ by superoxide dismutase. Increasing the level of O 2 ؊ reduces NO-mediated toxicity, and ONOO ؊ is not a mediator of hypersensitive cell death. During the HR, superoxide dismutase accelerates O 2 ؊ dismutation to H2O2 to minimize the loss of NO by reaction with O 2 ؊ and to trigger hypersensitive cell death through NO͞H 2O2 cooperation. However, O2 ؊ rather than H 2O2 is the primary ROI signal for pathogen induction of glutathione S-transferase, and the rates of production and dismutation of O 2 ؊ generated during the oxidative burst play a crucial role in the modulation and integration of NO͞H 2O2 signaling in the HR. Thus although plants and animals use a similar repertoire of signals in disease resistance, ROIs and NO are deployed in strikingly different ways to trigger host cell death.

A major impediment in the treatment of neurological diseases is the presence of the blood-brain barrier, which precludes the entry of therapeutic molecules from blood to brain. Here we show that a short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide was synthesized by adding nonamer arginine residues at the carboxy terminus of RVG. This RVG-9R peptide was able to bind and transduce siRNA to neuronal cells in vitro, resulting in efficient gene silencing. After intravenous injection into mice, RVG-9R delivered siRNA to the neuronal cells, resulting in specific gene silencing within the brain. Furthermore, intravenous treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Repeated administration of RVG-9R-bound siRNA did not induce inflammatory cytokines or anti-peptide antibodies. Thus, RVG-9R provides a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the blood-brain barrier.

Melatonin was discovered to be a direct free radical scavenger less than 10 years ago. Besides its ability to directly neutralize a number of free radicals and reactive oxygen and nitrogen species, it stimulates several antioxidative enzymes which increase its efficiency as an antioxidant. In terms of direct free radical scavenging, melatonin interacts with the highly toxic hydroxyl radical with a rate constant equivalent to that of other highly efficient hydroxyl radical scavengers. Additionally, melatonin reportedly neutralizes hydrogen peroxide, singlet oxygen, peroxynitrite anion, nitric oxide and hypochlorous acid. The following antioxidative enzymes are also stimulated by melatonin: superoxide dismutase, glutathione peroxidase and glutathione reductase. Melatonin has been widely used as a protective agent against a wide variety of processes and agents that damage tissues via free radical mechanisms.

Two wheat (Triticum aestivum L.) genotypes viz. Uniculm ‘Gigas’ Line 492 and Kalyansona were used to study the development of oxidative stress and antioxidant capacity in fully expanded flag leaves in response to low nitrogen (N). The genotypes differed in terms of metabolic constituents, green leaf area, membrane integrity and activities of antioxidant enzymes. Uniculm maintained photosynthetic rate and metabolic constituents in spite of large reduction in total flag leaf area. Low N resulted in increased formation of hydrogen peroxide (H2O2) and accumulation of thiobarbituric acid reactive substances
(TBARS) in flag leaves of both the genotypes. However, the activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) were higher in N stressed plants of Uniculm till maturity. In N stressed Kalyansona, activities of SOD and APX declined early as compared to control N plants. The low N induced generation of reactive oxygen species (ROS) was higher in Kalyansona as compared to Uniculm due to lower APX activity and/or the higher SOD/APX or SOD/CAT ratio. Kalyansona was found to be highly sensitive to low N in comparison to Uniculm. The catalase activity did not increase
in low N grown plants of Kalyansona leading to increased hydrogen peroxide in this genotype. The study provides evidence that wheat genotypes respond differentially to N supply.

Manganese superoxide dismutase (SOD2) converts superoxide to oxygen plus hydrogen peroxide and serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in humans has been associated with several chronic diseases, including ovarian cancer and type I diabetes, and SOD2 overexpression appears to suppress malignancy in cultured cells. We have produced a line of SOD2 knockout mice (SOD2m1BCM/SOD2m1BcM) that survive up to 3

Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for ϳ50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining ϳ50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.

The current goal of diabetes therapy is to reduce time-averaged mean levels of glycemia, measured as HbA1c, to prevent diabetic complications. However, HbA1c only explains < 25% of the variation in risk of developing complications. Because HbA1c does not correlate with glycemic variability when adjusted for mean blood glucose, we hypothesized that transient spikes of hyperglycemia may be an HbA1c -independent risk factor for diabetic complications. We show that transient hyperglycemia induces long-lasting activating epigenetic changes in the promoter of the nuclear factor B (NF-B) subunit p65 in aortic endothelial cells both in vitro and in nondiabetic mice, which cause increased p65 gene expression. Both the epigenetic changes and the gene expression changes persist for at least 6 d of subsequent normal glycemia, as do NF-B -induced increases in monocyte chemoattractant protein 1 and vascular cell adhesion molecule 1 expression. Hyperglycemia-induced epigenetic changes and increased p65 expression are prevented by reducing mitochondrial superoxide production or superoxide-induced ␣ -oxoaldehydes. These results highlight the dramatic and long-lasting effects that short-term hyperglycemic spikes can have on vascular cells and suggest that transient spikes of hyperglycemia may be an HbA1c -independent risk factor for diabetic complications.

Oxidative stress is implicated in neuronal apoptosis that occurs in physiological settings and in neurodegenerative disorders. Superoxide anion radical, produced during mitochondrial respiration, is involved in the generation of several potentially damaging reactive oxygen species including peroxynitrite. To examine directly the role of superoxide and peroxynitrite in neuronal apoptosis, we generated neural cell lines and transgenic mice that overexpress human mitochondrial manganese superoxide dismutase (MnSOD). In cultured pheochromocytoma PC6 cells, overexpression of mitochondria-localized MnSOD prevented apoptosis induced by Fe 2ϩ , amyloid ␤-peptide (A␤), and nitric oxide-generating agents. Accumulations of peroxynitrite, nitrated proteins, and the membrane lipid peroxidation product 4-hydroxynonenal (HNE) after exposure to the apoptotic insults were markedly attenuated in cells expressing MnSOD. Glutathione peroxidase activity levels were increased in cells overexpressing MnSOD, suggesting a compensatory response to increased H 2 O 2 levels. The peroxynitrite scavenger uric acid and the antioxidants propyl gallate and glutathione prevented apoptosis induced by each apoptotic insult, suggesting central roles for peroxynitrite and membrane lipid peroxidation in oxidative stress-induced apoptosis. Apoptotic insults decreased mitochondrial transmembrane potential and energy charge in control cells but not in cells overexpressing MnSOD, and cyclosporin A and caspase inhibitors protected cells against apoptosis, demonstrating roles for mitochondrial alterations and caspase activation in the apoptotic process. Membrane lipid peroxidation, protein nitration, and neuronal death after focal cerebral ischemia were significantly reduced in transgenic mice overexpressing human MnSOD. The data suggest that mitochondrial superoxide accumulation and consequent peroxynitrite production and mitochondrial dysfunction play pivotal roles in neuronal apoptosis induced by diverse insults in cell culture and in vivo.

Reactive oxygen (RO) has been identified as an important effector in ageing and lifespan determination 1-3 . The specific cell types, however, in which oxidative damage acts to limit lifespan of the whole organism have not been explicitly identified. The association between mutations in the gene encoding the oxygen radical metabolizing enzyme CuZn superoxide dismutase (SOD1) and loss of motorneurons in the brain and spinal cord that occurs in the life-shortening paralytic disease, Familial Amyotrophic Lateral Sclerosis (FALS; ref. 4), suggests that chronic and unrepaired oxidative damage occurring specifically in motor neurons could be a critical causative factor in ageing. To test this hypothesis, we generated transgenic Drosophila which express human SOD1 specifically in adult motorneurons. We show that overexpression of a single gene, SOD1, in a single cell type, the motorneuron, extends normal lifespan by up to 40% and rescues the lifespan of a short-lived Sod null mutant. Elevated resistance to oxidative stress suggests that the lifespan extension observed in these flies is due to enhanced RO metabolism. These results show that SOD activity in motorneurons is an important factor in ageing and lifespan determination in Drosophila.

The roles of superoxide (O 2 •Ϫ ), peroxynitrite, and carbon dioxide in the oxidative chemistry of nitric oxide ( • NO) are reviewed. The formation of peroxynitrite from • NO and O 2 •Ϫ is controlled by superoxide dismutase (SOD), which can lower the concentration of superoxide ions. The concentration of CO 2 in vivo is high (ca. 1 mM), and the rate constant for reaction of CO 2 with Ϫ OONO is large (pH-independent k ϭ 5.8 ϫ 10 4 M Ϫ1 s Ϫ1 ). Consequently, the rate of reaction of peroxynitrite with CO 2 is so fast that most commonly used scavengers would need to be present at very high, near toxic levels in order to compete with peroxynitrite for CO 2 . Therefore, in the presence of physiological levels of bicarbonate, only a limited number of biotargets react directly with peroxynitrite. These include hemecontaining proteins such as hemoglobin, peroxidases such as myeloperoxidase, seleno-proteins such as glutathione peroxidase, proteins containing zinc-thiolate centers such as the DNA-binding transcription factors, and the synthetic antioxidant ebselen. The mechanism of the reaction of CO 2 with Ϫ OONO produces metastable nitrating, nitrosating, and oxidizing species as intermediates. An analysis of the lifetimes of the possible intermediates and of the catalysis of peroxynitrite decompositions suggests that the reactive intermediates responsible for reactions with a variety of substrates may be the free radicals • NO 2 and CO 3 •Ϫ . Biologically important reactions of these free radicals are, for example, the nitration of tyrosine residues. These nitrations can be pathological, but they also may play a signal transduction role, because nitration of tyrosine can modulate phosphorylation and thus control enzymatic activity. In principle, it might be possible to block the biological effects of peroxynitrite by scavenging the free radicals • NO 2 and CO 3

Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that promote longevity in many organisms. Increased expression of SIRT3 has been linked to an extended life span in humans. Here, we have shown that Sirt3 protects the mouse heart by blocking the cardiac hypertrophic response. Although Sirt3-deficient mice appeared to have normal activity, they showed signs of cardiac hypertrophy and interstitial fibrosis at 8 weeks of age. Application of hypertrophic stimuli to these mice produced a severe cardiac hypertrophic response, whereas Sirt3-expressing Tg mice were protected from similar stimuli. In primary cultures of cardiomyocytes, Sirt3 blocked cardiac hypertrophy by activating the forkhead box O3a-dependent (Foxo3a-dependent), antioxidant-encoding genes manganese superoxide dismutase (MnSOD) and catalase (Cat), thereby decreasing cellular levels of ROS. Reduced ROS levels suppressed Ras activation and downstream signaling through the MAPK/ERK and PI3K/Akt pathways. This resulted in repressed activity of transcription factors, specifically GATA4 and NFAT, and translation factors, specifically eukaryotic initiation factor 4E (elf4E) and S6 ribosomal protein (S6P), which are involved in the development of cardiac hypertrophy. These results demonstrate that SIRT3 is an endogenous negative regulator of cardiac hypertrophy, which protects hearts by suppressing cellular levels of ROS.

Mitochondrial reactive oxygen species (ROS) are mainly produced by the respiratory chain enzymes. The sites for ROS production in mitochondrial respiratory chain are normally ascribed to the activity of Complex I and III. The presence of specific inhibitors modulates reactive oxygen species production in Complex I: inhibitors such as rotenone induce a strong ROS increase, while inhibitors such as stigmatellin prevent it. We have investigated the effect of hydrophilic quinones on Complex I ROS production in presence of different inhibitors. Some short chain quinones are Complex I inhibitors (CoQ2, idebenone and its derivatives), while CoQ1, decylubiquinone (DB) and duroquinone (DQ) are good electron acceptors from Complex I.Our results show that the ability of short chain quinones to induce an oxidative stress depends on the site of interaction with Complex I and on their physical-chemical characteristics. We can conclude that hydrophilic quinones may enhance oxidative stress by interaction with the electron escape sites on Complex I while more hydrophobic quinones can be reduced only at the physiological quinone reducing site without reacting with molecular oxygen.

During normal cellular activities, various processes inside of cells produce reactive oxygen species (ROS). Some of the most common ROS are hydrogen peroxide (H 2 O 2 ), superoxide ion (O 2 À ), and hydroxide radical (OH À ). These compounds, when present in a high enough concentration, can damage cellular proteins and lipids or form DNA adducts that may promote carcinogenic activity. The purpose of antioxidants in a physiological setting is to prevent ROS concentrations from reaching a high-enough level within a cell that damage may occur. Cellular antioxidants may be enzymatic (catalase, glutathione peroxidase, superoxide dismutase) or nonenzymatic (glutathione, thiols, some vitamins and metals, or phytochemicals such as isoflavones, polyphenols, and flavanoids).

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to amplify the heat shock response in cell lines by increasing the binding of heat shock transcription factor-1 to heat shock elements within heat shock gene promoters. Because overexpression of the inducible heat shock protein 70 (Hsp70) was neuroprotective in a culture model of motor neuron disease, this study investigated whether NSAIDs induce Hsp70 and confer cytoprotection in motor neurons of dissociated spinal cord cultures exposed to various stresses. Two NSAIDs, sodium salicylate and niflumic acid, lowered the temperature threshold for induction of Hsp70 in glia but failed to do so in motor neurons. At concentrations that increased Hsp70 in heat shocked glial cells, sodium salicylate failed to delay death of motor neurons exposed to hyperthermia, paraquat-mediated oxidative stress, and glutamate excitotoxicity. Neither sodium salicylate nor the cyclooxygenase-2 inhibitor, niflumic acid, protected motor neurons from the toxicity of mutated Cu/Zn-superoxide dismutase (SOD-1) linked to a familial form of the motor neuron disease, amyotrophic lateral sclerosis. Thus, treatment with 2 types of NSAIDs failed to overcome the high threshold for the activation of heat shock response in motor neurons.