Dismutation Of Superoxide Research Articles

Antioxidant 1 in Hypertension

See related article, pp 476–486 Oxidative stress, defined as an increase in reactive oxygen species, has been implicated in a number of diseases, including hypertension. Superoxide is a particularly detrimental reactive oxygen species in the cardiovascular system because it reacts directly with NO, thereby limiting the amount of NO normally available for processes such as endothelium-dependent relaxation. Superoxide dismutases (SODs) represent a first-line defense against oxidative stress by tempering increases in superoxide via its metabolism (dismutation). Three distinct mammalian isoforms of SOD exist, CuZnSOD (localized mainly to the cytoplasmic space), MnSOD (the predominant isoform in mitochondria), and extracellular SOD (SOD3), a CuZnSOD found exclusively in the extracellular compartment (Figure).1 The fact that there are 3 different gene products that catalyze the same reaction underscores the importance of limiting cellular superoxide. Figure. Expression of the mammalian superoxide dismutase (SOD) isoforms and the importance of copper chaperones. Three distinct mammalian SODs are expressed in specific cellular compartments. CuZnSOD (SOD1) is expressed primarily in the cytoplasmic space; MnSOD (SOD2) is the predominant isoform in mitochondria; and extracellular SOD (EC-SOD; SOD3) is found exclusively in the extracellular space. All 3 isoforms catalyze the dismutation of superoxide to hydrogen peroxide. Both SOD1 and SOD3 require copper as a key cofactor for catalytic activity. The copper chaperone for SOD1 (CCS) delivers copper (Cu2+) specifically to SOD1, whereas antioxidant 1 (Atox1) delivers copper to the transgolgi network, as well as SOD3. The present study by Ozumi et al5 examined the role of Atox1 in limiting hypertension, increases in vascular superoxide, and endothelial dysfunction produced by angiotensin II. The mechanism by which Atox1 limits these effects appears …

Progesterone prevents mitochondrial dysfunction in the spinal cord of wobbler mice

In the Wobbler mouse, a mutation of the Vps54 protein increases oxidative stress in spinal motoneurons, associated to toxic levels of nitric oxide and hyperactivity of nitric oxide synthase (NOS). Progesterone neuroprotection has been reported for several CNS diseases, including the Wobbler mouse neurodegeneration. In the present study, we analyzed progesterone effects on mitochondrial-associated parameters of symptomatic Wobbler mice. The activities of mitochondrial respiratory chain complexes I, II-III and IV and protein levels of mitochondrial and cytosolic NOS were determined in cervical and lumbar cords from control, Wobbler and Wobbler mice receiving a progesterone implant for 18 days. We found a significant reduction of complex I and II-III activities in mitochondria and increased protein levels of mitochondrial, but not cytosolic nNOS, in the cervical cord of Wobbler mice. Progesterone treatment prevented the reduction of complex I in the cervical region and the increased level of mitochondrial nNOS. Wobbler motoneurons also showed accumulation of amyloid precursor protein immunoreactivity and decreased activity and immunostaining of MnSOD. Progesterone treatment avoided these abnormalities. Therefore, administration of progesterone to clinically afflicted Wobblers (i) prevented the abnormal increase of mitochondrial nNOS and normalized respiratory complex I; (ii) decreased amyloid precursor protein accumulation, a sign of axonal degeneration, and (iii) increased superoxide dismutation. Thus, progesterone neuroprotection decreases mitochondriopathy of Wobbler mouse cervical spinal cord.

Effects of arsenite and UVA-1 radiation on calcineurin signaling

Calcineurin is a Ca2+-dependent serine/threonine phosphatase and the target of the immunosuppressive drugs cyclosporin and tacrolimus, which are used in transplant recipients to prevent rejection. Unfortunately, the therapeutic use of this drugs is complicated by a high incidence of skin malignancy, which has set off a number of studies into the role of calcineurin signaling in skin, particularly with respect to cell cycle control and DNA repair. Both UVA1 radiation and arsenic species are known to promote skin cancer development via production of reactive oxygen species. In light of the well-documented sensitivity of calcineurin to oxidative stress, we examined and compared the effects of UVA1 and arsenite on calcineurin signaling.In this paper, we show that physiologically relevant doses of UVA1 radiation and low micromolar concentrations of arsenite strongly inhibit calcineurin phosphatase activity in Jurkat and skin cells and decrease NFAT nuclear translocation in Jurkat cells. The effects on calcineurin signaling could be partly prevented by inhibition of NADPH oxidase in Jurkat cells or increased dismutation of superoxide in Jurkat and skin cells. In addition, both UVA1 and arsenite decreased NF-κB activity, although at lower concentrations, arsenite enhanced NF-κB activity. These data indicate that UVA1 and arsenite affect a signal transduction route of growingly acknowledged importance in skin and that calcineurin may serve as a potential link between ROS exposure and impaired tumor suppression.

Design, synthesis, and characterization of a series of biologically active copper(II) Schiff-base coordination compounds

Four Schiff-base copper(II) complexes, [Cu(L1)(Phen)(H2O)](NO3)2 (1), [Cu(L1)(bipy)(H2O)](ClO4)(H2O)(NO3) (2), [Cu(L1)(Dien)](ClO4)2(H2O) (3), and [Cu(L1)(ImH)(H2O)](ClO4) (4) (L1 = N-[(1)-pyridin-2-ylmethylidene]benzohydrazide, Phen = 1,10-phenanthroline, bipy = 2,2′-bipyridine, Dien = diethylenetriamine, ImH = imidazole), have been synthesized and characterized using various physico-chemical techniques. L1 is coordinated to copper(II) neutral and uninegatively charged tridentate chelating agent via the azomethine nitrogen, pyridine nitrogen, and carbonyl oxygen. The copper(II) complexes are paramagnetic with octahedral stereochemistry. The crystal structures of the complexes reveal the presence of structure consisting of a tridentate N, N, O donor Schiff base, tridentate NNN donor polyamine or N, N donor heterocyclic base and the copper(II) center. In 1, 2, and 4, water is present as a coordinating molecule whereas in 2 and 4 it is also present as lattice water. In discrete monomeric species all complexes show significant hydrogen-bonding interactions. H-bridges are also present in the solid state structure. Complexes 1–4 effectively catalyze the dismutation of superoxide () in alkaline nitro blue tetrazolium assay and IC50 values were determined. The susceptibility of certain strains of bacteria toward the precursors L1 and their complexes were also evaluated.

Abstract 4355: Use of MnSOD tet on transgenic mice as a model for evaluating irradiation protection and mitigation

Abstract Manganese superoxide dismutase (MnSOD) is a mitochondrial enzyme responsible for the dismutation or reduction of superoxide produced by cellular respiration. Absence of MnSOD is lethal two to three days after birth. Following irradiation there is an increase in superoxide production which combined with nitric oxide can result in the formation of the highly reactive radical peroxynitrite. Increased expression of MnSOD in a cell increases survival by the reduction of superoxide produced following irradiation. Decreased MnSOD production has resulted in decreased survival following irradiation. To better understand the role of MnSOD in cellular respiration as well as protecting the cell from irradiation damage, we have developed a MnSOD tet on mouse which has no MnSOD activity. In the absence of doxycycline the homozygous MnSOD tet on mouse does not survive past 2 or 3 days after birth. If doxycycline (2 µg/ml) is added to the water of heterozygous mothers, the MnSOD tet on mice develop normally and are fertile as long as they remain on the doxycycline treated water. Long term bone marrow cultures (LTBMCs) were established from homozygous MnSOD tet on mice, heterozygous tet on mice and control C57BL/6J mice. The cultures from the control mice had more hematopoietic cell activity than the MnSOD tet on cultures. To determine when the MnSOD gene is turned on after addition of doxocycline, bone marrow stromal cell lines were developed from the LTBMCs were incubated in 1 µg doxocycline/ml media for time points up to 24 hr, RNA was extracted and RT-PCR for the MnSOD transgene was performed. By 12 hours there was an increase in MnSOD RNA expression in the MnSOD tet on cells. Removal of the doxycycline resulted in a decrease in MnSOD RNA by 1 hr in the MnSOD tet on cells as well as the heteroxyzous cells. This mouse model will be useful for testing new compounds for radiation protection and mitigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4355. doi:1538-7445.AM2012-4355

Hydrogen peroxide mediates oxidant‐dependent stimulation of arterial smooth muscle L‐type calcium channels

Changes in calcium and redox status influence multiple cellular processes. We tested the hypothesis that the ubiquitous reactive oxygen species hydrogen peroxide mediates oxidant‐dependent stimulation of arterial smooth muscle L‐type calcium channels. Using a combinatorial approach including conventional electrophysiology and total internal reflection fluorescence imaging, we found that application of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L‐type calcium channels. Similarly, oxidant‐dependent stimulation of L‐type calcium channels by the vasoconstrictor angiotensin II was abolished by intracellular application of catalase. Catalase also prevented angiotensin II from increasing localized subplasmalemmal generation of endogenous reactive oxygen species previously associated with colocalized calcium influx through L‐type channels. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with superoxide dismutase had no effect on angiotensin‐dependent stimulation of L‐type calcium channels. We conclude that hydrogen peroxide is necessary for oxidant‐dependent stimulation of rat arterial smooth muscle L‐type calcium channels.

Synthesis, structure and biological activities of mixed ligand copper(II) and nickel(II) complexes of N′-(1E)-[(5-bromo-2-hydroxyphenyl)methylidene]benzoylhydrazone

We have made an effort to synthesize ternary complexes [Cu2(L)2(Him)2]·CH3OH 1, [Cu(L)(bipy)] 2 and [Ni(HL)(PMDT)]BF43 (H2L=N′-(1E)-[(5-bromo-2-hydroxyphenyl)methylidene]benzoylhydrazone; Him=imidazole; bipy=2,2′-bipyridine and PMDT=N,N,N′,N″,N″-pentamethyldiethylenetriamine). Their crystal structures were determined by X-ray diffraction method. Single crystal X-ray of 1 revealed the geometry of phenoxo bridged copper center and the distance between the two Cu(II) ion is 3.245Å. The two copper ions are penta coordinated with distorted square pyramidal geometries. Magnetic measurements of dicopper complex 1 exhibited the presence of antiferromagnetic exchange interaction. The observed trend in the spin Hamiltonian parameters in complexes 1 and 2g||>g⊥>2.0023 and |A⊥|≪|A|||≈163–173×10−4cm−1) indicates dx2-y2 band ground state with tetragonal site symmetry for copper(II). Complex 3 was crystallized in orthorhombic crystal system having six coordination numbers. Superoxide dismutase activities of all these complexes have been revealed to catalyze the dismutation of superoxide (O2-) and IC50 values were evaluated. DNA cleavage properties and antibacterial activity have been measured and discussed.

Ordering of neuronal apoptosis signaling: a superoxide burst precedes mitochondrial cytochrome c release in a growth factor deprivation model

Axonal injury to retinal ganglion cells, a defined central neuron, induces a burst of intracellular superoxide anion that precedes externalization of membrane phosphatidylserine and subsequent apoptotic cell death. Dismutation of superoxide prevents the signal and delays loss of these cells, consistent with superoxide being necessary for transduction of the axotomy signal. However, phosphatidylserine externalization is a relatively late step in apoptosis, and it is possible that the superoxide burst is not an early axotomy signal but rather a result of cytochrome c release from the mitochondrial inner membrane with consequent accumulation of reduced intermediates. Other possibilities are that both superoxide generation and cytochrome c release are induced in parallel by axotomy, or that cytochrome c release potentiates the effect of the superoxide burst. To distinguish these various possibilities, serum-deprived neuronal retinal cells were assayed in vitro for superoxide elevation and release of cytochrome c from mitochondria, and the distribution of these two markers across a large number of cells used to model the temporal ordering of events. Based on this model of factor-dependent cell death, superoxide precedes, and possibly potentiates, cytochrome c release, and thus the former is likely an early signal for certain types of neuronal apoptosis in the central nervous system.

Cytotoxicity of a mitochondriotropic quercetin derivative: Mechanisms

The mitochondriotropic compound 7-O-(4-triphenylphosphoniumbutyl)quercetin iodide (Q-7BTPI) in the μM concentration range caused necrotic death of cultured cells by acting as a prooxidant, with generation of superoxide anion in the mitochondria. Externally added membrane-permeating superoxide dismutase or catalase largely prevented death. Rescue by permeant catalase indicates that the toxicant is H2O2, or reactive species derived from it. Rescue by permeant dismutase suggests the possibility of a chain mechanism of H2O2 production, in which dismutation of superoxide constitutes a termination step. Oxidative stress was due to the presence of free phenolic hydroxyls and to accumulation in mitochondria, since the analogous mitochondriotropic per-O-methylated compound -3,3′,4′,5-tetra-O-methyl,7-O-(4-triphenylphosphoniumbutyl) quercetin iodide (QTM-7BTPI)—or Quercetin itself induced no or little superoxide production and cell death. Q-7BTPI did not cause a significant perturbation of the mitochondrial transmembrane potential or of respiration in cells. On the other hand its presence led to inhibition of glutathione peroxidase, an effect expected to accentuate oxidative stress by interfering with the elimination of H2O2. An exogenous permeable glutathione precursor determined a strong increase of cellular glutathione levels but did not rescue the cells. Death induction was selective for fast-growing C-26 tumoral cells and mouse embryonic fibroblasts (MEFs) while sparing slow-growing MEFs. This suggests a possible use of Q-7BTPI as a chemotherapeutic agent.

Hydrogen peroxide mediates oxidant-dependent stimulation of arterial smooth muscle L-type calcium channels

Changes in calcium and redox homeostasis influence multiple cellular processes. Dysregulation of these signaling modalities is associated with pathology in cardiovascular, neuronal, endocrine, and other physiological systems. Calcium and oxidant signaling mechanisms are frequently inferred to be functionally related. To address and clarify this clinically relevant issue in the vasculature we tested the hypothesis that the ubiquitous reactive oxygen molecule hydrogen peroxide mediates oxidant-dependent stimulation of cerebral arterial smooth muscle L-type calcium channels. Using a combinatorial approach including intact arterial manipulations, electrophysiology, and total internal reflection fluorescence imaging, we found that application of physiological levels of hydrogen peroxide to isolated arterial smooth muscle cells increased localized calcium influx through L-type calcium channels. Similarly, oxidant-dependent stimulation of L-type calcium channels by the vasoconstrictor ANG II was abolished by intracellular application of catalase. Catalase also prevented ANG II from increasing localized subplasmalemmal sites of increased oxidation previously associated with colocalized calcium influx through L-type channels. Furthermore, catalase largely attenuated the contractile response of intact cerebral arterial segments to ANG II. In contrast, enhanced dismutation of superoxide to hydrogen peroxide with SOD had no effect on ANG II-dependent stimulation of L-type calcium channels. From these data we conclude that hydrogen peroxide is important for oxidant-dependent regulation of smooth muscle L-type calcium channels and arterial function. These data also support the emerging concept of hydrogen peroxide as a biologically relevant oxidant second messenger in multiple cell types with a diverse array of physiological functions.

Cupricyclins, Novel Redox-Active Metallopeptides Based on Conotoxins Scaffold

Highly stable natural scaffolds which tolerate multiple amino acid substitutions represent the ideal starting point for the application of rational redesign strategies to develop new catalysts of potential biomedical and biotechnological interest. The knottins family of disulphide-constrained peptides display the desired characteristics, being highly stable and characterized by hypervariability of the inter-cysteine loops. The potential of knottins as scaffolds for the design of novel copper-based biocatalysts has been tested by engineering a metal binding site on two different variants of an ω-conotoxin, a neurotoxic peptide belonging to the knottins family. The binding site has been designed by computational modelling and the redesigned peptides have been synthesized and characterized by optical, fluorescence, electron spin resonance and nuclear magnetic resonance spectroscopy. The novel peptides, named Cupricyclin-1 and -2, bind one Cu2+ ion per molecule with nanomolar affinity. Cupricyclins display redox activity and catalyze the dismutation of superoxide anions with an activity comparable to that of non-peptidic superoxide dismutase mimics. We thus propose knottins as a novel scaffold for the design of catalytically-active mini metalloproteins.

Peritoneal Infection and Inflammation

Peritonitis is a major complication of various disease entities in internal medicine, surgery, and gynecology and, despite treatment advances, remains associated with unacceptably high morbidity and mortality. Causes of peritonitis are manifold including infectious, autoimmune, or chemical processes. A precise knowledge of inflammatory mechanisms in the initiation and establishment of peritoneal inflammation is of pivotal importance for the prevention and treatment of peritonitis. This special issue aims to bring the molecular mechanisms of inflammatory processes in the peritoneal cavity into focus, regardless of the organ system or the specific syndrome they result from. In this special issue of Mediators of Inflammation, we are pleased to present to the reader a series of special features written by designated experts in the field. Local production of proinflammatory mediators at sites of tissue and cell injury within the peritoneal membrane plays a critical role in the propagation of peritoneal inflammation. In addition to infectious causes, various forms of peritoneal damage, induced by surgical trauma and peritoneal dialysis, compromise peritoneal integrity and can ultimately lead to tissue damage, fibrosis, adhesions, or even in the most severe cases the development of encapsulating peritoneal sclerosis. Peritoneal mesothelial cells appear to play a major role in these processes as they are capable of expressing a raft of proinflammatory and immune-modulatory mediators that either directly mediate fibrotic remodelling or attract immune cells to the peritoneal cavity or participate in immune activation. Therapeutic modulation of this sequence of events could provide novel avenues to attenuate peritoneal inflammation and its resulting tissue damage. The present special issue presents novel data on this topic, particularly underscoring the potential impact of the peroxisome proliferator-activated receptor-gamma (PPAR-γ) in regulating peritoneal inflammation and fibrosis. Furthermore, a novel PEGylated Toll-like receptor 7 (TLR7) ligand might be of great value for treatment of mast-cell-mediated neutrophilic inflammation. M. W. J. A. Fieren describes the regulatory role of cytokines, macrophages, and leukocytes during local inflammatory processes in the peritoneal cavity. These processes are of special interest in patients on peritoneal dialysis treatment. A successful peritoneal dialysis treatment requires an intact peritoneal membrane. Treatment can be complicated by infectious or noninfectious sterile peritonitis due to the use of nonphysiologic dialysis solutions. S. Yung and T. M. Chan present an overview of pathophysiological changes to the peritoneal membrane during peritonitis related to peritoneal dialysis. G. Baroni et al. focus on structural changes during peritoneal dialysis-related inflammation. Moreover, experimental data presented in this special issue point to a novel role of the osmosensitive transcription factor NFAT5 in osmolality-induced expression of chemokines in human mesothelial cells. This is of special interest because peritoneal dialysis solutions are usually hypertonic. A rare but fatal complication of peritoneal dialysis treatment is sclerosing peritonitis; in their review article, M. Merkle and M. Wornle overview causes and treatment options, a complication which can also be seen after kidney or liver transplantation. Peritoneal adhesions frequently occur after abdominal surgery. Patients with this complication present with abdominal and pelvic pain or intestinal mechanical obstruction. In this special issue, C. Di Filippo et al. provide evidence for a pivotal role of the ubiquitin-proteasome system in the formation of experimental postsurgical peritoneal adhesions. Furthermore, the authors show a possible therapeutic strategy to prevent adhesion formation by blocking the ubiquitin-proteasome system. W. Kessler et al. present novel data on the particular importance of the vagus nerve for the pathophysiology of peritonitis. According to their data, a functioning vagus nerve has a significant modulating influence on mortality in an animal model of polymicrobial sepsis. In this special issue, T. Kelkka et al. present data in arthritis and peritonitis mice models, where overexpression of SOD3 results in anti-inflammatory effects that are largely independent of NOX2-mediated oxidative bursts. The anti-inflammatory action of extracellular superoxide dismutase (SOD3) is mediated by the dismutation of superoxide into hydrogen peroxide. To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, T. Kelkka et al. used an elegant design, comparing anti-inflammatory effects of overexpression of SOD3 in Ncf1−/− mice with a deficient oxidative burst with that in wild-type mice. The relevance of these findings will need verification in the clinical setting; however, SOD3 is an important redox regulatory enzyme, and polymorphisms in SOD3 gene are associated with COPD, coronary artery disease, myocardial infarction, and acute lung injury. We would like to thank all contributors and reviewers for their support to this special issue and feel sure that readers will learn much about this important topic. Christoph Aufricht Wolfgang Neuhofer Nicholas Topley Markus Wornle

Superoxide Dismutase 3 Limits Collagen-Induced Arthritis in the Absence of Phagocyte Oxidative Burst

Extracellular superoxide dismutase (SOD3), an enzyme mediating dismutation of superoxide into hydrogen peroxide, has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues. In inflamed tissues, superoxide is produced by the phagocytic NOX2 complex, which consists of the catalytic subunit NOX2 and several regulatory subunits (e.g., NCF1). To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, we injected an adenoviral vector overexpressing SOD3 directly into the arthritic paws of Ncf1∗/∗ mice with collagen-induced arthritis. SOD3 reduced arthritis severity in both oxidative burst-deficient Ncf1∗/∗ mice and also in wild-type mice. The NOX2 complex independent anti-inflammatory effect of SOD3 was further characterized in peritonitis, and SOD3 was found to reduce macrophage infiltration independently of NOX2 complex functionality. We conclude that the SOD3-mediated anti-inflammatory effect on arthritis and peritonitis operates independently of NOX2 complex derived oxidative burst.

Superoxide determination using membrane-engineered cells: An example of a novel concept for the construction of cell sensors with customized target recognition properties

Membrane-engineering is a generic methodology for increasing the selectivity of a cell biosensor against a target molecule, by electroinserting target-specific receptor molecules on the cell surface. We have previously reported the construction of an ultra-sensitive superoxide anion (O 2 − ) sensor based on immobilized cells, which have been membrane-engineered with superoxide dismutase (SOD). In the present study, we provide evidence that superoxide dismutation triggered changes to the membrane potential of membrane-engineered fibroblast cells, as confirmed by electrophysiological and fluorescence assays. These changes were associated with changes in [Ca 2+ ] cyt , as revealed by the selective inhibition of intracellular calcium ion traffic. In addition, by conducting selective inhibition assays, we show that electroinserted SOD molecules retained their characteristic catalytic properties. We also investigated the effect of the concentration of electroinserted SOD molecules on the performance of the superoxide assay. Finally, we increased the sensitivity of the sensor by hundredfold to a detection limit of 1 pM O 2 − by changing the concentration of immobilized cells on the performance of the biosensor.

Ca2+-activated K+ channels of small and intermediate conductance control eNOS activation through NAD(P)H oxidase

Ca2+-activated K+ channels (KCa) and NO play a central role in the endothelium-dependent control of vasomotor tone. We evaluated the interaction of KCa with NO production in isolated arterial mesenteric beds of the rat. In phenylephrine-contracted mesenteries, acetylcholine (ACh)-induced vasodilation was reduced by NO synthase (NOS) inhibition with Nω-nitro-l-arginine (L-NA), but in the presence of tetraethylammonium, L-NA did not further affect the response. In KCl-contracted mesenteries, the relaxation elicited by 100nM ACh or 1μM ionomycin was abolished by L-NA, tetraethylammonium, or simultaneous blockade of small-conductance KCa (SKCa) channels with apamin and intermediate-conductance KCa (IKCa) channels with triarylmethane-34 (TRAM-34). Apamin–TRAM-34 treatment also abolished 100nM ACh-activated NO production, which was associated with an increase in superoxide formation. Endothelial cell Ca2+ buffering with BAPTA elicited a similar increment in superoxide. Apamin–TRAM-34 treatment increased endothelial NOS phosphorylation at threonine 495 (P-eNOSThr495). Blockade of NAD(P)H oxidase with apocynin or superoxide dismutation with PEG-SOD prevented the increment in superoxide and changes in P-eNOSThr495 observed during apamin and TRAM-34 application. Our results indicate that blockade of SKCa and IKCa activates NAD(P)H oxidase-dependent superoxide formation, which leads to inhibition of NO release through P-eNOSThr495. These findings disclose a novel mechanism involved in the control of NO production.

Real time qPCR expression analysis of some stress related genes in leaf tissue of Pyrus communis cv. Conférence after infection with Erwinia amylovora

Hydrogen peroxide plays a critical role in the expression of disease resistance in several plant/pathogen interactions. It serves as a substrate for oxidative cross-linking of various plant cell wall components leading to the reinforcement of the cell structure, as a direct toxin against the pathogen and as a signal molecule for the induction of defence-related genes in the adjacent, still healthy tissues. In plant cells, enzymes and redox metabolites act in synergy to carry out the detoxification of hydrogen peroxide and other reactive oxygen species (ROS). Superoxide dismutase (SOD) catalyses the dismutation of superoxide to hydrogen peroxide, catalase (CAT) dismutates hydrogen peroxide to oxygen and water, and ascorbate peroxidase (APX) reduces hydrogen peroxide to water by utilising ascorbate as specific electron donor. These are considered some of the main enzymatic systems for protecting cells against oxidative damage. These enzymes are present in various isozyme forms in several cell compartments and their expression is genetically controlled and regulated both by developmental and environmental stimuli, according to the necessity to remove ROS produced in cells. The aim of this study was to determine the possible role of these antioxidants in the defence mechanism of Pyrus communis cv. Conference leaf tissue after an infection with Erwinia amylovora. Shoots of 2-year-old pear trees cv. Conference were infected with E. amylovora strain SGB 225/12, were mock infected or left untreated. To account for structural changes, not only a difference was made between control, infected and mock-infected leaves, but we also included a distinction between young and old leaves, because it is known that older leaves are less susceptible for fire blight infections. Leaf samples were taken at specific time points after infection and the expression pattern of not necrotic tissue close to the infection site was analysed for their diverse isoforms of SOD, APX and CAT by using real time qPCR. In this study, no striking differences in transcription patterns of these enzymes between control, mock infected and E. amylovora infected leaves were observed. However, a significant difference between the expression levels of some genes in young and old leaves was noticed. These differences could partially explain the different progression rate by which E. amylovora infects, invades and causes necrosis in young and old leaves.

Abstract A175: Role of MnSOD and hydrogen peroxide in rapamycin-induced cytostasis.

Abstract Existing treatments for mantle cell lymphoma (MCL) are non-curative, demonstrating a need for a refined treatment approach. Recent clinical trials have shown promising results with the use of mammalian target of rapamycin (mTOR) inhibitors. While cyclin D1 can be regulated through the mTOR pathway, treatment with mTOR inhibitors often inhibits MCL growth without altering cyclin D1 levels of expression. Thus, the anti-tumor activity of mTOR inhibitors is likely mediated through other key targets. In other malignancies, recent data suggests that increased oxidative stress plays a role in the growth suppressive mechanisms of mTOR inhibitors. Initial studies in our lab indicated that a 48 hour rapamycin treatment of MCL cell lines increased overall levels of reactive oxygen species (ROS). Specifically, we found evidence of increased hydrogen peroxide (H2O2) levels in the cells. One potential source of H2O2 is manganese superoxide dismutase (MnSOD), which is a known tumor suppressor in several cell types. MnSOD catalyzes the dismutation of superoxide to H2O2 in the mitochondria. Thus, it removes one form of ROS while generating another. We hypothesized that the anti-tumor effect of mTOR inhibitors in MCL is mediated by an accumulation of ROS, specifically H2O2, due to an increase in MnSOD protein expression. We investigated the effect of a 48 hour rapamycin treatment on MnSOD in two MCL cell lines and genetic manipulation of MnSOD in Jurkat cells, a T lymphocyte cell line. Rapamycin treatment increased MnSOD in the MCL cell lines as well as Jurkat cells. Overall, sensitivity of cells to rapamycin correlated with high MnSOD levels. Analysis of the results showed that rapamycin treatment increased MnSOD expression in parallel with H2O2. Over-expression of MnSOD in Jurkat cells elevated the level of H2O2 and resulted in a cytotoxic, rather than cytostatic, effect of rapamycin. Treatment with the general ROS scavenger, N-acetyl cysteine, reduced overall ROS, cellular levels of H2O2, and the growth inhibitory effect of rapamycin. These findings indicate that the rapamycin-induced cytostatic effect is characterized by increased levels of MnSOD and H2O2, which is necessary for the full growth inhibitory effect of rapamycin. Rapamycin and its derivatives are generally well accepted as inhibitors of the mTORC1 complex upon initial treatment, but recent studies in other cell lines have shown that long term treatment can have an inhibitory effect on the mTORC2 complex and its downstream targets, such as Akt. Our results found that treatment with rapamycin resulted in a loss of S473 phosphorylation of Akt in the MCL cell lines. Increased levels of MnSOD following treatment of Jurkat cells were found to be due to inhibition of the mTORC2 complex. These results suggest that in MCL cells treated with rapamycin for 48 hours the mTORC2 complex is inhibited. Multiple transcription factors that have been linked to MnSOD regulation are affected by the Akt pathway. By understanding the key signaling molecules and affected pathways in the anti-tumor effects of rapamycin, we may be able to identify additional predictive markers to improve the therapeutic value of mTOR inhibitors, or study drug combinations that will enhance the effect of ROS-induced cytotoxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A175.

Cu,Zn superoxide dismutase: Cloning and analysis of the Taeniasolium gene and Taenia crassiceps cDNA

Cytosolic Cu,Zn superoxide dismutase (Cu,Zn-SOD) catalyzes the dismutation of superoxide (O2-) to oxygen and hydrogen peroxide (H2O2) and plays an important role in the establishment and survival of helminthes in their hosts. In this work, we describe the Taenia solium Cu,Zn-SOD gene (TsCu,Zn-SOD) and a Taenia crassiceps (TcCu,Zn-SOD) cDNA. TsCu,Zn-SOD gene that spans 2.841kb, and has three exons and two introns; the splicing junctions follow the GT-AG rule. Analysis in silico of the gene revealed that the 5′-flanking region has three putative TATA and CCAAT boxes, and transcription factor binding sites for NF1 and AP1. The transcription start site was a C, located at 22 nucleotides upstream of the translation start codon (ATG). Southern blot analysis showed that TcCu,Zn-SOD and TsCu,Zn-SOD genes are encoded by a single copy. The deduced amino acid sequences of TsCu,Zn-SOD gene and TcCu,Zn-SOD cDNA reveal 98.47% of identity, and the characteristic motives, including the catalytic site and β-barrel structure of the Cu,Zn-SOD.Proteomic and immunohistochemical analysis indicated that Cu,Zn-SOD does not have isoforms, is distributed throughout the bladder wall and is concentrated in the tegument of T. solium and T. crassiceps cysticerci. Expression analysis revealed that TcCu,Zn-SOD mRNA and protein expression levels do not change in cysticerci, even upon exposure to O2- (0–3.8nmol/min) and H2O2 (0–2mM), suggesting that this gene is constitutively expressed in these parasites.

Bioinspired Design of a Cu–Zn–Imidazolate Mesoporous Silica Catalyst System for Superoxide Dismutation

An imidazolate-bridged Cu(II)–Zn(II) model compound, Cu(II)-diethylenetriamino-μ-imidazolato-Zn(II)-tris(aminoethyl)amine perchlorate (CZS), was synthesized and encapsulated into various mesoporous silicas (MPSs) to mimic the structure and functionalities of copper zinc superoxide dismutase (CuZnSOD). Two encapsulation methods were studied: covalent bonding and ionic exchange. The encapsulated CZS yielded good thermal stability and enhanced superoxide disproportionation activity in various designed MPS solids mimicking the environment and functionality of native CuZnSOD enzyme. Ionic exchange method generally gave better SOD-like catalytic activities than covalent bonding because the local attraction of SiO– to Cu(II) opens up the active axial position. We employed several spectroscopic techniques: UV–vis, EPR, and EXAFS to characterize the active site-Cu(II) ion of the immobilized CZS and to obtain the structural information of Cu(II) and Zn(II) centers confined in MPS. In the analyses of EPR and EXAFS spectra, we established the geometry of confined copper center changed to a more distorted square planar symmetry than free-form CZS, which can accommodate superoxide anion radical (O2–•) coordinating to copper center in the proper manner. As the result of confinement in MPS, the active site of Cu(II) center gives rise to much increased SOD activities. Inspired by the local environments of guanidinium group in the native CuZnSOD enzyme in facilitating the superoxide dismutation, we further modified the silanol surface of MPS with N-trimethoxysilylpropyl-N,N,N-trimethyl-ammonium chloride (TMAC) preparing MPS-N+ to facilitate the transport of superoxide anion radicals. Optimized activities are associated with fine-tuning of pore size, surface acidity, and positively charged functional group. The spectroscopic studies allow us to establish the structure–reactivity relationship between the nanostructure of MPS and the efficacy of the superoxide dismutation in MPS and to elucidate the mechanism of the SOD-like activity of immobilized model compound in MPS.

Therapeutic Effect of Lecithinized Superoxide Dismutase on Pulmonary Emphysema

No medication exists that clearly improves the mortality of chronic obstructive pulmonary disease (COPD). Oxidative molecules, in particular superoxide anions, play important roles in the COPD-associated abnormal inflammatory response and pulmonary emphysema, which arises because of an imbalance in proteases and antiproteases and increased apoptosis. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide anions. Lecithinized human Cu/Zn- SOD (PC-SOD) has overcome a number of the clinical limitations of SOD, including low tissue affinity and low stability in plasma. In this study, we examine the effect of PC-SOD on elastase-induced pulmonary emphysema, an animal model of COPD. The severity of the pulmonary inflammatory response and emphysema in mice was assessed by various criteria, such as the number of leukocytes in the bronchoalveolar lavage fluid and the enlargement of airspace. Not only intravenous administration but also inhalation of PC-SOD suppressed elastase-induced pulmonary inflammation, emphysema, and dysfunction. Inhalation of PC-SOD suppressed the elastase-induced increase in the pulmonary level of superoxide anions and apoptosis. Inhalation of PC-SOD also suppressed elastase-induced activation of proteases and decreased in the level of antiproteases and expression of proinflammatory cytokines and chemokines. We also found that inhalation of PC-SOD suppressed cigarette smoke-induced pulmonary inflammation. The results suggest that PC-SOD protects against pulmonary emphysema by decreasing the pulmonary level of superoxide anions, resulting in the inhibition of inflammation and apoptosis and amelioration of the protease/antiprotease imbalance. We propose that inhalation of PC-SOD would be therapeutically beneficial for COPD.