Oxidase System Research Articles

P41 - Reactive oxygen species and PI3K/Akt signaling in cancer

Reactive oxygen species (ROS) are chemically reactive molecules containing oxygen and associates with multiple cellular functions such as cell proliferation, differentiation, and apoptosis. In the present study, we showed that Insulin-like growth factor-1(IGF-1) modulates SKOV-3 ovarian cancer cell by regulation of generation of ROS. Akt mediates cellular signaling pathways in association with mammalian target of rapamycin complex (mTOR) and Rac small G protein. Insulin-like growth factor-1 (IGF-1)-induced generation of ROS was completely abolished by phosphatidylinositol 3-kinase (PI3K) (LY294002, 10?µM) or Akt inhibitors (SH-5, 50?µM), whereas inhibition of extracellular-regulated kinase by an ERK inhibitor (PD98059, 10?µM) or inhibition of mammalian target of rapamycin complex 1 (mTORC1) by an mTORC1 inhibitor (Rapamycin, 100?nM) did not affect IGF-1-induced generation of ROS. Inactivation of mTORC2 by silencing Rapamycin-insensitive companion of mTOR (Rictor), abolished IGF-1-induced SKOV-3 cell migration as well as activation of Akt. However, inactivation of mTORC1 by silencing of Raptor had no effect. Silencing of Akt1 but not Akt2 attenuated IGF-1-induced generation of ROS. Expression of PIP3-dependent Rac exchanger1 (P-Rex1), a Rac guanosine exchange factor and a component of the mTOR complex. Silencing of P-Rex1 abolished IGF-1-induced generation of ROS. Finally, inhibition of NADPH oxidase system completely blunted IGF-1-induced generation of ROS, whereas inhibition of xanthine oxiase,cyclooxygenase, and mitochondrial respiratory chain complex was not effective. Given these results, we suggest that IGF-1 induces ROS generation through the PI3K/Akt/ mTOR2/NADPH oxidase signaling axis.

Subkutan siyanür enjeksiyonu ile özkıyım girişimi: Bir olgu sunumu

Cyanide is a powerful poison which cause inhibition of mitochondrial cytochrome oxidase system. Cyanide poisining can present in a wide variety of forms such as inhalation, ingestion or injection. Dermal exposure is rare, very few case reports have been reported about cyanide. In this paper, we present an uncommon case of a suicide attempt with cyanide injection. A 91 year old man with a history of depression admitted to the emergency department after injected cyanide to himself subcutaneously. He was diagnosed with depressive disorder and hospitalized after psychiatric assessment. The psychiatric history of this patient suggests that depression in elderly can give way to serious suicides attempts. Psychiatrists interested in treatment of elderly depressed patients should do careful monitoring about suicide.

Chemiluminescent imaging of transpired ethanol from the palm for evaluation of alcohol metabolism.

A 2-dimensional imaging system was constructed and applied in measurements of gaseous ethanol emissions from the human palm. This imaging system measures gaseous ethanol concentrations as intensities of chemiluminescence by luminol reaction induced by alcohol oxidase and luminol-hydrogen peroxide-horseradish peroxidase system. Conversions of ethanol distributions and concentrations to 2-dimensional chemiluminescence were conducted on an enzyme-immobilized mesh substrate in a dark box, which contained a luminol solution. In order to visualize ethanol emissions from human palm skin, we developed highly sensitive and selective imaging system for transpired gaseous ethanol at sub ppm-levels. Thus, a mixture of a high-purity luminol solution of luminol sodium salt HG solution instead of standard luminol solution and an enhancer of eosin Y solution was adapted to refine the chemiluminescent intensity of the imaging system, and improved the detection limit to 3 ppm gaseous ethanol. The highly sensitive imaging allows us to successfully visualize the emissions dynamics of transdermal gaseous ethanol. The intensity of each site on the palm shows the reflection of ethanol concentrations distributions corresponding to the amount of alcohol metabolized upon consumption. This imaging system is significant and useful for the assessment of ethanol measurement of the palmar skin.

Sensitive electrochemical measurement of hydroxyl radical generation induced by the xanthine–xanthine oxidase system

A sensitive electrochemical measurement system for hydroxyl radical (OH) was developed using enzyme-catalyzed signal amplification. In the presence of 2,6-xylenol as a trapping agent, glucose as a substrate, and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ–GDH) as a catalyst, the amperometric signal of the trapping adduct 2,6-dimethylhydroquinone (DMHQ) produced by the hydroxylation of 2,6-xylenol was able to be amplified and detected sensitively. The limit of detection (signal/noise [S/N]=3) for DMHQ was 1nM. There was no significant interference from urate and other oxidizable compounds in the reaction mixture at the applied potential of 0V versus Ag/AgCl. This method was employed to observe the OH generation induced by the xanthine–xanthine oxidase (XO) system. The reaction rates of the DMHQ production induced from the xanthine–XO system in the presence and absence of various Fe(III) complexes and proteins were compared. Those with a free coordination site on the Fe atom effectively enhanced the OH generation.

Chronic granulomatous disease: why an inflammatory disease?

Chronic granulomatous disease is a primary immunodeficiency caused by mutations in the genes encoding subunits of the phagocytic NADPH oxidase system. Patients can present with severe, recurrent infections and noninfectious conditions. Among the latter, inflammatory manifestations are predominant, especially granulomas and colitis. In this article, we systematically review the possible mechanisms of hyperinflammation in this rare primary immunodeficiency condition and their correlations with clinical aspects.

Chlorovirus PBCV-1 encodes an active copper-zinc superoxide dismutase.

Superoxide dismutases (SODs) are metalloproteins that protect organisms from toxic reactive oxygen species by catalyzing the conversion of superoxide anion to hydrogen peroxide and molecular oxygen. Chlorovirus PBCV-1 encodes a 187-amino-acid protein that resembles a Cu-Zn SOD with all of the conserved amino acid residues for binding copper and zinc (named cvSOD). cvSOD has an internal Met that results in a 165-amino-acid protein (named tcvSOD). Both cvSOD and tcvSOD recombinant proteins inhibited nitroblue tetrazolium reduction of superoxide anion generated in a xanthine-xanthine oxidase system in solution. tcvSOD was chosen for further characterization because it was easier to produce. Recombinant tcvSOD also inhibited a riboflavin photochemical reduction system in a polyacrylamide gel assay, which was blocked by the Cu-Zn SOD inhibitor cyanide but not by azide, which inhibits Fe and Mn SODs. A k(cat)/K(m) value for cvSOD was determined by stop-flow spectrophotometry as 1.28 × 10(8) M(-1) s(-1), suggesting that cvSOD-catalyzed O2 (-) dismutation was not a diffusion controlled encounter. The cvsod gene was expressed as a late gene, and cvSOD activity was detected in purified virions. Superoxide accumulated rapidly during virus infection, and circumstantial evidence indicates that cvSOD aids its decomposition to benefit virus replication. Cu-Zn SOD homologs have been described to occur in 3 other families of large DNA viruses, poxviruses, baculoviruses, and mimiviruses, which group as a clade. Interestingly, cvSOD does not group in the same clade as the other virus SODs but instead groups in an expanded clade that includes Cu-Zn SODs from many cellular organisms. Virus infection often leads to an increase in toxic reactive oxygen species in the host, which can be detrimental to virus replication. Viruses have developed various ways to overcome this barrier. As reported in this article, the chloroviruses often encode and package a functional Cu-Zn superoxide dismutase in the virion that presumably lowers the concentration of reactive oxygen induced early during virus infection.

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

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

Protection of common carp (Cyprinus carpio L.) spermatozoa motility under oxidative stress by antioxidants and seminal plasma.

The protective influence of seminal plasma and the antioxidants catalase (CAT), superoxide dismutase (SOD), and glutathione (GTH) on quality parameters, oxidative stress indices, and antioxidant activity was studied in common carp (Cyprinus carpio) spermatozoa exposed to the xanthine-xanthine oxidase (X-XO) system. Fish spermatozoa were incubated for 5 and 20 min at 4 °C with X-XO concentrations of 1 mM X-0.1 U/mL, 0.6 mM X-0.05 U/mL, 0.3 mM X-0.025 U/mL, and 0.1 mM X-0.0125 U/mL. A dose-dependent reduction in spermatozoa motility and velocity was observed at concentrations of 0.1 mM X-0.0125 U/mL to 1 mM X-0.1 U/mL XO. Increase in spermatozoa motility parameters was recorded following treatment with antioxidants and seminal plasma. The level of the oxidative stress indices lipid peroxidation (LPO) and carbonyl derivatives of proteins (CP) was significantly reduced after addition of CAT, SOD, or GTH along with seminal plasma. Significant differences in SOD, glutathione reductase, and glutathione peroxidase activity were seen in spermatozoa incubated with, compared to that without, seminal plasma at all studied X-XO concentrations. The data demonstrated that CAT, SOD, or GTH in combination with SP can reduce reactive oxygen species stress in fish spermatozoa and improve spermatozoa quality.

Two independent killing mechanisms of Candida albicans by human neutrophils: evidence from innate immunity defects

AbstractInvasive fungal infections, accompanied by high rates of mortality, represent an increasing problem in medicine. Neutrophils are the major effector immune cells in fungal killing. Based on studies with neutrophils from patients with defined genetic defects, we provide evidence that human neutrophils use 2 distinct and independent phagolysosomal mechanisms to kill Candida albicans. The first mechanism for the killing of unopsonized C albicans was found to be dependent on complement receptor 3 (CR3) and the signaling proteins phosphatidylinositol-3-kinase and caspase recruitment domain-containing protein 9 (CARD9), but was independent of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. The second mechanism for the killing of opsonized C albicans was strictly dependent on Fcγ receptors, protein kinase C (PKC), and reactive oxygen species production by the NADPH oxidase system. Each of the 2 pathways of Candida killing required Syk tyrosine kinase activity, but dectin-1 was dispensable for both of them. These data provide an explanation for the variable clinical presentation of fungal infection in patients suffering from different immune defects, including dectin-1 deficiency, CARD9 deficiency, or chronic granulomatous disease.

Antioxidant effect of the active metabolites of tibolone

Certain steroidal compounds have an antioxidant effect in humans. Our aim was to test whether the synthetic steroid tibolone and its metabolites are also able to display such a property. For this, granulocytes from healthy men and women were incubated for two hours with different concentrations (10−7, 10−8, 10−9 M) of either estradiol, tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, Δ4-tibolone, 3α-sulfated-tibolone, 3α-17β-disulfated-tibolone, 3β-sulfated-tibolone or 3β-17β-disulfated-tibolone. Superoxide anion generation of neutrophils was measured by photometry. Results of different steroids were given as percentages of their controls. A more simple superoxide generating system, the xanthine–xanthine oxidase reaction was also tested. We found that granulocyte superoxide production did not differ from the control using 10−9 M of steroids. Using 10−8 M concentration: estradiol (80.9 ± 2.5%); 3β-sulfated-tibolone (83.3 ± 4.7%); 3β-17β-disulfated-tibolone (81.0 ± 4.2%) caused a significant decrease in superoxide production, compared to the control. In addition at 10−7 M, 3β-hydroxytibolone and 3α-sulfated-tibolone also showed antioxidant effects. In the xanthine–xanthine oxidase system estradiol (67.4 ± 1.0%), 3α-sulfated-tibolone (85.8 ± 5.3%), 3α-17β-disulfated-tibolone (71.9 ± 2.5%), 3β-sulfated-tibolone (73.9 ± 5.0%), and 3β-17β-disulfated-tibolone (65.8 ± 3.4%) caused a significant decrease in superoxide production. Conclusively, although tibolone itself did not show significant antioxidant capacity, most of its active metabolites have antioxidant effects.

Colorimetric estimation of human glucose level using γ-Fe2O3 nanoparticles: An easily recoverable effective mimic peroxidase

This article reports simple, green and efficient synthesis of γ-Fe2O3 nanoparticles (NPs) (maghemite) through single-source precursor approach for colorimetric estimation of human glucose level. The γ-Fe2O3 NPs, having cubic morphology with an average particle size of 30nm, exhibited effective peroxidase-like activity through the catalytic oxidation of peroxidase substrate 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H2O2 producing a blue-colored solution. On the basis of this colored-reaction, we have developed a simple, cheap, highly sensitive and selective colorimetric method for estimation of glucose using γ-Fe2O3/TMB/glucose–glucose oxidase (GOx) system in the linear range from 1 to 80μM with detection limit of 0.21μM. The proposed glucose sensor displays faster response, good stability, reproducibility and anti-interference ability. Based on this simple reaction process, human blood and urine glucose level can be monitored conveniently.

Novel Mn-SOD Mimetics Offer Superior Protection Against Oxidative Damages in Hek293 Kidney Cells

A series of ternary Mn (II) complexes have been synthesized employing combination of thiosemicarbazones as primary ligand and bipyridyl/aminoethanethiol as ancillary ligands and structurally characterized. The compounds were examined for their protective effects in Xanthine–Xanthine oxidase-induced oxidative stress in HEK-293 kidney cells using MTT assay, confocal imaging and Western blots. All compounds offer protection against the oxidative stress generated by Xanthine–Xanthine oxidase system, while compounds having aminoethanethiol as ancillary ligands especially exhibit potent effects even at nanomolar concentrations. These compounds seem to operate mechanistically through quenching of ROS and enhancing Mn-SOD expression in mitochondria.

Metabolomic analysis reveals a unique urinary pattern in normozoospermic infertile men.

Normozoospermic infertility has become a common and important health problem worldwide. We designed this metabolomic case-control study to investigate the possible mechanism and urinary biomarkers of normozoospermic infertility. Normozoospermic infertile cases (n = 71) and fertile controls (n = 47) were recruited. A urinary metabolome pattern could discriminate normozoospermic infertile cases from fertile controls. A total of 37 potential biomarkers were identified; these have functionally important roles in energy production, antioxidation, and hormone regulation in spermatogenesis. This gave rise to a combined biomarker pattern of leukotriene E4, 3-hydroxypalmitoylcarnitine, aspartate, xanthosine, and methoxytryptophan pointing to a diagnostic capability (AUC = 0.901, sensitivity = 85.7%, and specificity = 86.8%) in a ROC model; these markers may highlight keynote events of normozoospermic infertility. Stalled medium- and long-chain fatty acid metabolism with improved ketone body metabolism, plus decreased levels of malate and aspartate could result in citrate cycle alterations via a malate-aspartate shuttle in ATP generation in spermatogenesis. Inhibitory alterations in the normal hormone-secreting activity in spermatogenesis were suggested in normozoospermic infertility. Folate deficiency and oxidative stress may jointly impact infertile patients. The disruption of eicosanoid metabolism and xanthine oxidase system, which were tightly associated with energy metabolism and oxidative stress, was also a potential underlying mechanism. In addition, depression might be associated with normozoospermic infertility via neural activity-related metabolites. This study suggests that the urinary metabolome can be used to differentiate normozoospermic infertile men from fertile individuals. Potential metabolic biomarkers derived from these analyses might be used to diagnose what remains a somewhat idiopathic condition and provide functional insights into its pathogenesis.

New chiral 4-substituted 2-cyanoethyl-oxazolines: Synthesis and assessment of some biological activities

This paper describes the synthesis of new enantiomerically pure 2-cyanoethyl-oxazolines in one step starting from a wide range of amino alcohols and 4-ethoxy-4-iminobutanenitrile with high to good yields (73–96%) via an appropriate procedure which can be used for a selective synthesis of mono-oxazolines. A simple operation as well as a practical separation is additional eco-friendly attributes of this method. All the synthesized compounds were identified and characterized with their physicochemical features and their spectral data (1H NMR, 13C NMR and TOFMS ES+). Among the prepared mono-oxazolines, the mono-oxazoline (3a) [3-[(4S)-4-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile] was tested to detect some biological activities. This compound was studied in vitro given the various types of pharmacological properties characterizing these compounds such as antioxidant, antimicrobial and analgesic activities. The antioxidant activity and mechanism of (3a) were identified using various in vitro antioxidant assays including 1,1-diphenyl-2-picryl-hydrazyl (DPPH), and superoxide anion radicals (O2−) scavenging activity. In addition, compared to Quercetin, the tested synthetic product reveals a relatively-strong antiradical activity towards the DPPH (activity percentage of 81.22%) free radicals and significantly decreased the reactive oxygen species such as (O2−) formation evaluated by the non-enzymatic (nitroblue tetrazolium/riboflavine) and the enzymatic (xanthine/xanthine oxidase) systems. Related activity values were, respectively, 66% and 60.30%. The oxazoline (3a) showed a high ability to reduce the O2− generation and proved to be a very potent radical scavenger. On the other hand, the analgesic property of the 3[(4S)-benzyl-4,5-dihydro-1,3-oxazol-2-yl] propanenitrile (3a) was demonstrated. The subcutaneous administration of (3a) produced a significant reduction in the number of abdominal constrictions amounting to 73.81% in the acetic acid writhing test in mice. In addition to these advances, the oxazoline (3a) has been investigated as an antimicrobial agent. Our results showed that this molecule exhibited various levels of antibacterial effect against all the tested bacterial strains.

Metarhizin A suppresses cell proliferation by inhibiting cytochrome c oxidase activity

AimsMetarhizin A was originally isolated from Metarhizium flavoviride as a potent inhibitor of the growth of insect and mammalian cells. In this study, we aimed to understand the molecular targets of metarhizin A involved in its anti-proliferative activity against human cells. Main methodsCell cycle regulators and signaling molecules were examined by immunoblotting using specific antibodies. A mitochondria-enriched fraction was prepared from mouse liver, and mitochondrial activity was monitored using an oxygen electrode. Enzyme activity was measured using purified cytochrome c oxidase and permeabilized cells. Key findingsMetarhizin A inhibits the growth of MCF-7 cells with an IC50 value of ~0.2μM and other cells in a similar manner; a cell cycle-dependent kinase inhibitor, p21, is selectively induced. Significant amounts of reactive oxygen species (ROS) are generated and ERK1/2 is activated in cells treated with metarhizin A. Metarhizin A completely suppresses oxygen consumption by mitochondria, and potently inhibits the activity of cytochrome c oxidase. It induces cell death when MCF-7 cells are cultured under limiting conditions. SignificanceMetarhizin A is a potent inhibitor of cytochrome c oxidase and activates the MAPK pathway through the generation of ROS, which induces growth arrest of cells, and, under some conditions, enhances cell death. The cytochrome c oxidase system is a possible molecular target of metarhizin A.

Effects of lycopene supplementation on oxidative stress markers in adipose tissue of obese rats (LB389)

Background: Obesity has been associated with oxidative stress, and NADPH oxidase system is known as main source of reactive species. Lycopene is a potent antioxidant. However, it is not known the influence of lycopene on NADPH‐oxidase activity in adipose tissue of obese rats. Objective: To analyze the effects of lycopene supplementation on oxidative stress markers, including NADPH‐oxidase activity, in adipose tissue of diet‐induced obese rats. Methods: Male Wistar rats were first fed with hypercaloric diet (HD, n=14) for 6 weeks and afterwards, these rats were randomly assigned to receive HD (n=7) or HD with lycopene (10 mg/Kg BW/d; HD+L, n=7) by gavage for additional 6 weeks. Adipose tissue was collected for evaluation of NADPH‐oxidase activity and other oxidative stress markers, by HPLC system and immunoassay, respectively. Results: In obesity, lycopene treatment for six weeks decreased protein carbonylation (HD: 5.24 ± 0.77 vs HD+L: 3.78 ± 0.50) in visceral adipose tissue, and 4‐HNE in visceral (HD: 0.43 ± 0.04 vs HD+L: 0.36 ± 0.02) and retroperitoneal (HD: 0.40 ± 0.07 vs HD+L: 0,31 ± 0,02) adipose tissue. Absence of difference in NADPH oxidase activity was found between HD and HD+L groups. Conclusion: Lycopene supplementation decreased lipid peroxidation and protein carbonylation and did not interfere in NADPD‐oxidase activity in adipose tissue. These data suggest that lycopene may be an effective strategy in reducing some oxidative stress markers in obesity.Grant Funding Source: Supported by FAPESP #10/06100‐9 and #10/19746‐4

EComment. Application of prostaglandin E1 and pentoxyphylline in clinical mesenteric ischaemia

We read with great interest the original article by San Norberto Garcia and colleagues describing the effects of intra-arterial and intravenous prostaglandin E1 (PGE1) in intestinal ischaemia-reperfusion injury [1]. Using a rat model, the study was designed to assess histopathological score following ileal segment resection, polymorphonuclear leukocytes visualized and myeloperoxidase activity following a well-designed protocol of ischaemia time and reperfusion time with variable route administration of PGE1. The ischaemic insult presents a vicious cycle at the molecular and biochemical level. Vasodilatation occurs with increased microvascular permeability and release of lysosomal hydrolases and ultimately increased proteolysis. This cascade is aggravated by reperfusion, where an already oxygen deprived organ territory is now exposed to oxygen free radicals through oxidation of the hypoxanthine-xanthine oxidase system resulting in the superoxide anion [2]. The main determinants of haemodynamic instability, and subsequent mortality, are the generation of oxygen free radicals and the increased capillary permeability [3]. The spectrum of mesenteric ischaemia encompasses either an acute insult or a chronic phase, and can be caused by thrombosis, embolism, non-occlusive mesenteric ischaemia, and significant venous obstruction. Clearly with acute mesenteric ischaemia, mortality is quite high. As pointed out by the authors, thromboembolic acute occlusion of the superior mesenteric artery is usually unpredictable, so pretreatment is not possible or practical. However, in such a case like the critically ill with non-occlusive mesenteric ischaemia, with early diagnosis and detection, the initiation of continuous intravenous PGE1 may increase survival as noted in a study by Mitsuyoshi and colleagues [4]. The 60-minute ischaemia time and 7 days reperfusion portended the best outcome with regard to Chiu classification of intestinal tissue injury as well as myeloperoxidase activity and polymorphonuclear leukocyte infiltration. Another promising vascular drug that has been evaluated experimentally is pentoxyphylline. It has specific antioxidant properties that directly inhibit the superoxide anion and indirectly blocks xanthine-oxidase enzyme activity. It has been shown to reduce histopathological changes secondary to ischaemia-reperfusion in intestinal experimental models [5]. Again, although in acute mesenteric ischaemia it is impossible to pretreat these patients with PGE1 (or even pentoxyphylline), it may be clinically appropriate to consider such pretreatment in elective revascularization of patients with chronic mesenteric ischaemia or the critically ill in whom high-dose vasoactive agents are necessary for haemodynamic control. Further translational studies are necessary to try delineate whether a functional benefit is clinically realized from a histopathological and anatomic amelioration of the ischaemia-reperfusion injury. Conflict of interest: none declared

Tempol attenuates atherosclerosis associated with metabolic syndrome via decreased vascular inflammation and NADPH-2 oxidase expression

Oxidative stress is an important factor in the generation of vascular injury in atherosclerosis. Chronic administration of fructose in rodents is able to facilitate oxidative damage. In the present study we evaluated the role of Tempol, a superoxide dismutase mimetic, on the effect of high fructose intake in apolipoprotein E-deficient (ApoE-KO) mice. Rodents were fed with fructose overload (FF, 10% w/v) for 8 weeks and treated with Tempol 1 mg/kg/day the latest 4 weeks. Tempol revert the pro-oxidant effects caused by FF, diminished lipid peroxidation and impaired vascular NADPH oxidase system through the downregulation of p47phox expression in the vascular wall. Tempol inhibited the expression of vascular adhesion molecule 1 (VCAM-1) in aorta and reduced the development of atheroma plaques. Our results indicate that tempol attenuates oxidative stress by interfering with the correct assembly of Nox2 oxidase complex in the vascular wall and is able to reduce atherosclerosis. Thus tempol represents a potential therapeutic target for preventing risk factors associated with metabolic syndrome.

Integration of molecular and enzymatic catalysts on graphene for biomimetic generation of antithrombotic species

The integration of multiple synergistic catalytic systems can enable the creation of biocompatible enzymatic mimics for cascading reactions under physiologically relevant conditions. Here we report the design of a graphene-haemin-glucose oxidase conjugate as a tandem catalyst, in which graphene functions as a unique support to integrate molecular catalyst haemin and enzymatic catalyst glucose oxidase for biomimetic generation of antithrombotic species. Monomeric haemin can be conjugated with graphene through π-π interactions to function as an effective catalyst for the oxidation of endogenous L-arginine by hydrogen peroxide. Furthermore, glucose oxidase can be covalently linked onto graphene for local generation of hydrogen peroxide through the oxidation of blood glucose. Thus, the integrated graphene-haemin-glucose oxidase catalysts can readily enable the continuous generation of nitroxyl, an antithrombotic species, from physiologically abundant glucose and L-arginine. Finally, we demonstrate that the conjugates can be embedded within polyurethane to create a long-lasting antithrombotic coating for blood-contacting biomedical devices.

PPARγ and NAD(P)H oxidase system interaction in glycerol-induced acute renal failure: role of gp91phox subunit of NAD(P)H oxidase

Increased NAD(P)H oxidase-dependent free radical generation has been proposed to be a mechanism in glycerol-induced acute renal failure (ARF). Previously, we showed a PPARγ-mediated regulation of free radical generation in ARF. In this study, we examined NAD(P)H oxidase-dependent pathology in ARF and its connection with PPARγ using both Sprague–Dawley rats and gp91phox (+/−) mice. Male gp91phox (+/−) or wild type (+/+) mice were distributed into vehicle and ARF group (50% glycerol; 8 mL/kg bw; i.m.). Animals were placed in metabolic cages for 24 hr and were sacrificed under pentobarbital anesthesia. Urine, plasma and kidneys were processed for biochemical and molecular analysis. Glycerol doubled proteinuria in (+/+) mice (68 ± 4 mg/24 hr) but not in (+/−) mice (43 ± 9 mg/24 hr). This was associated with a markedly reduced creatinine excretion in (+/+) mice (Con: 0.6 ± 0.03 & ARF: 0.37 ± 0.02). Basal plasma and urinary NO was higher in (+/−) mice than the (+/+) type while plasma 8-isoprostane level was lower in (+/−) mice (WT: 165 ± 20; KO: 100 ± 15 pg/mL). Glycerol reduced UNOXV in both (+/+) and (+/−) mice although plasma NO was unchanged. Glycerol also doubled 8-isoprostane in (+/+) (363 ± 22 pg/mL) but not in (+/−) mice (152 ± 20 pg/mL) and this was associated with an increased NAD(P)H oxidase activity in the (+/+) mice. In ARF, PPARγ expression was reduced in (+/+) mice but increased in (+/−) mice. PPARγ activity was also reduced in (+/+) mice but was unchanged in (+/−) mice. We conclude that gp91phox contributes to NAD(P)H oxidase-mediated increased free radical generation in ARF and this may be via reduced PPARγ.