Reactive oxygen species generated by NADPH oxidase are involved in neurodegeneration in the pilocarpine model of temporal lobe epilepsy

Reactive oxygen species (ROS) appear to be involved in several neurodegenerative disorders. We tested the hypothesis that oxidative stress could have a role in the hippocampal neurodegeneration observed in temporal lobe epilepsy induced by pilocarpine. We first determined the spatio-temporal pattern of ROS generation, by means of detection with dihydroethidium oxidation, in the CA1 and CA3 areas and the dentate gyrus of the dorsal hippocampus during status epilepticus induced by pilocarpine. Fluoro-Jade B assays were also performed to detect degenerating neurons. ROS generation was increased in CA1, CA3 and the dentate gyrus after pilocarpine-induced seizures, which was accompanied by marked cell death. Treatment of rats with a NADPH oxidase inhibitor (apocynin) for 7 days prior to induction of status epilepticus was effective in decreasing both ROS production (by an average of 20%) and neurodegeneration (by an average of 61%). These results suggest an involvement of ROS generated by NADPH oxidase in neuronal death in the pilocarpine model of epilepsy. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

Peptide gomesin triggers cell death through L-type channel calcium influx, MAPK/ERK, PKC and PI3K signaling and generation of reactive oxygen species

Gomesin is an antimicrobial peptide isolated from hemocytes of a common Brazilian tarantula spider named Acanthoscurriagomesiana. This peptide exerts antitumor activity in vitro and in vivo by an unknown mechanism. In this study, the cytotoxic mechanism of gomesin in human neuroblastoma SH-SY5Y and rat pheochromocytoma PC12 cells was investigated. Gomesin induced necrotic cell death and was cytotoxic to SH-SY5Y and PC12 cells. The peptide evoked a rapid and transient elevation of intracellular calcium levels in Fluo-4-AM loaded PC12 cells, which was inhibited by nimodipine, an L-type calcium channel blocker. Preincubation with nimodipine also inhibited cell death induced by gomesin in SH-SY5Y and PC12 cells. Gomesin-induced cell death was prevented by the pretreatment with MAPK/ERK, PKC or PI3K inhibitors, but not with PKA inhibitor. In addition, gomesin generated reactive oxygen species (ROS) in SH-SY5Y cells, which were blocked with nimodipine and MAPK/ERK, PKC or PI3K inhibitors. Taken together, these results suggest that gomesin could be a useful anticancer agent, which mechanism of cytotoxicity implicates calcium entry through L-type calcium channels, activation of MAPK/ERK, PKC and PI3K signaling as well as the generation of reactive oxygen species. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

The novel ruthenium-gamma-linolenic complex [Ru(2)(aGLA)(4)Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitro

The present study reports the synthesis of a novel compound with the formula [Ru(2)(aGLA)(4)Cl] according to elemental analyses data, referred to as Ru(2)GLA. The electronic spectra of Ru(2)GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru(2)GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and gamma-linolenic acid (GLA). The properties of Ru(2)GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru(2)GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru(2)GLA enters the cells and ICP-AES elemental analysis found all increase in ruthenium from <0.02 to 425 mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru(2)GLA (22 +/- 5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru(2)GLA (44 +/- 2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18 +/- 1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru(2)GLA exposed cells. The EC(50) for Ru(2)GLA decreased with increasing time of exposure from 285 mu M at 24h, 211 mu M at 48 h to 81 mu M at 72 h. In conclusion, Ru(2)GLA is a novel drug with anti proliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright (C) 2009 John Wiley & Sons, Ltd.

Identification of geodesic chirping Alfven modes and q-factor estimation in hot core tokamak plasmas in ASDEX Upgrade

Alfven eigenmodes (AE) driven by ion cyclotron resonance heating are usually registered by different diagnostic channels in the hot core plasmas of large tokamaks like JET and ASDEX Upgrade. These AE appear very near to the extremum points of Alfven wave continuum, which is modified by the geodesic effect due to poloidal mode coupling. It is shown that the AE spectrum may be explored as the magnetic spectroscopy (like Alfven cascades by Sharapov et al 2001 Phys. Lett. A 289 127) to determine the q-factor minimum and geodesic frequency at the magnetic axis in standard sawtoothed discharges without reversed shear.

Rotation effect on geodesic and zonal flow modes in tokamak plasmas with isothermal magnetic surfaces

The electrostatic geodesic mode oscillations are investigated in rotating large aspect ratio tokamak plasmas with circular isothermal magnetic surfaces. The analysis is carried out within the magnetohydrodynamic model including heat flux to compensate for the non-adiabatic pressure distribution along the magnetic surfaces in plasmas with poloidal rotation. Instead of two standard geodesic modes, three geodesic continua are found. The two higher branches of the geodesic modes have a small frequency up-shift from ordinary geodesic acoustic and sonic modes due to rotation. The lower geodesic continuum is a newzonal flowmode (geodesic Doppler mode) in plasmas with mainly poloidal rotation. Limits to standard geodesic modes are found. Bifurcation of Alfven continuum by geodesic modes at the rational surfaces is also discussed. Due to that, the frequency of combined geodesic continuum extends from the poloidal rotation frequency to the ion-sound band that can have an important role in suppressing plasma turbulence.

Transport control in fusion plasmas by changing electric and magnetic field spatial profiles

For magnetically confined plasmas in tokamaks, we have numerically investigated how Lagrangian chaos at the plasma edge affects the plasma confinement. Initially, we have considered the chaotic motion of particles in an equilibrium electric field with a monotonic radial profile perturbed by drift waves. We have showed that an effective transport barrier may be created at the plasma edge by modifying the electric field radial profile. In the second place, we have obtained escape patterns and magnetic footprints of chaotic magnetic field lines in the region near a tokamak wall with resonant modes due to the action of an ergodic magnetic limiter. For monotonic plasma current density profiles we have obtained distributions of field line connections to the wall and line escape channels with the same spatial pattern as the magnetic footprints on the tokamak walls. (c) 2008 Elsevier B.V. All rights reserved.

High-frequency extensions of magnetorotational instability in astrophysical plasmas

High-frequency extensions of magnetorotational instability driven by the Velikhov effect beyond the standard magnetohydrodynamic (MHD) regime are studied. The existence of the well-known Hall regime and a new electron inertia regime is demonstrated. The electron inertia regime is realized for a lesser plasma magnetization of rotating plasma than that in the Hall regime. It includes the subregime of nonmagnetized electrons. It is shown that, in contrast to the standard MHD regime and the Hall regime, magnetorotational instability in this subregime can be driven only at positive values of dln Omega/dlnr, where Omega is the plasma rotation frequency and r is the radial coordinate. The permittivity of rotating plasma beyond the standard MHD regime, including both the Hall regime and the electron inertia regime, is calculated.

Low-dimensional chaos and wave turbulence in plasmas

We investigated drift-wave turbulence in the plasma edge of a small tokamak by considering solutions of the Hasegawa-Mima equation involving three interacting modes in Fourier space. The resulting low-dimensional dynamics presented periodic as well as chaotic evolution of the Fourier-mode amplitudes, and we performed the control of chaotic behaviour through the application of a fourth resonant wave of small amplitude.

Recurrence quantification analysis of electrostatic fluctuations in fusion plasmas

We have investigated plasma turbulence at the edge of a tokamak plasma using data from electrostatic potential fluctuations measured in the Brazilian tokamak TCABR. Recurrence quantification analysis has been used to provide diagnostics of the deterministic content of the series. We have focused our analysis on the radial dependence of potential fluctuations and their characterization by recurrence-based diagnostics. Our main result is that the deterministic content of the experimental signals is most pronounced at the external part of the plasma column just before the plasma radius. Since the chaoticity of the signals follows the same trend, we have concluded that the electrostatic plasma turbulence at the tokamak plasma edge can be partially explained by means of a deterministic nonlinear system. (C) 2007 Elsevier B.V. All rights reserved.

Anisotropy of thermal stresses in confined dusty plasmas

Anisotropy of thermal stresses in confined dusty plasmas is considered. It is shown that in a multi-component low-temperature plasma containing electrons, ions and dust, the complicated dependence of the ion viscosity on ion temperature gradients leads to a plasma equilibrium state with anisotropic pressure. This pressure anisotropy can be of the order of the ion pressure in some limiting cases, in which the ion Larmor radius or the ion mean free path are of the order of the characteristic length of the plasma nonuniformity. For a sufficiently large dust number density, they contribute to the plasma pressure anisotropy and to its spatial dependence. Currently, it is not yet clear whether this equilibrium state is stable or not. Under these conditions, some convective plasma flows can arise in confinement devices. Therefore, this question needs special consideration.

Mean Motion in Stochastic Plasmas With a Space-Dependent Diffusion Coefficient

Radial transport in the tokamap, which has been proposed as a simple model for the motion in a stochastic plasma, is investigated. A theory for previous numerical findings is presented. The new results are stimulated by the fact that the radial diffusion coefficients is space-dependent. The space-dependence of the transport coefficient has several interesting effects which have not been elucidated so far. Among the new findings are the analytical predictions for the scaling of the mean radial displacement with time and the relation between the Fokker-Planck diffusion coefficient and the diffusion coefficient from the mean square displacement. The applicability to other systems is also discussed. (c) 2009 WILEY-VCH GmbH & Co. KGaA, Weinheim

Tissue-, substrate-, and site-specific characteristics of mitochondrial reactive oxygen species generation

Reactive oxygen species are a by-product of mitochondrial oxidative phosphorylation, derived from a small quantity of superoxide radicals generated during electron transport. We conducted a comprehensive and quantitative study of oxygen consumption, inner membrane potentials, and H(2)O(2) release in mitochondria isolated from rat brain, heart, kidney, liver, and skeletal muscle, using various respiratory substrates (alpha-ketoglutarate, glutamate, succinate, glycerol phosphate, and palmitoyl carnitine). The locations and properties of reactive oxygen species formation were determined using oxidative phosphorylation and the respiratory chain modulators oligomycin, rotenone, myxothiazol, and antimycin A and the Uncoupler CCCP. We found that in mitochondria isolated from most tissues incubated under physiologically relevant conditions, reactive oxygen release accounts for 0.1-0.2% of O(2) consumed. Our findings support an important participation of flavoenzymes and complex III and a substantial role for reverse electron transport to complex I as reactive oxygen species sources. Our results also indicate that succinate is an important substrate for isolated mitochondrial reactive oxygen production in brain, heart, kidney, and skeletal muscle, whereas fatty acids generate significant quantities of oxidants in kidney and liver. Finally, we found that increasing respiratory rates is an effective way to prevent mitochondrial oxidant release under many, but not all, conditions. Altogether, our data uncover and quantify many tissue-, substrate-, and site-specific characteristics of mitochondrial ROS release. (C) 2009 Elsevier Inc. All rights reserved.

1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB`s cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.

Horseradish peroxidase compound I as a tool to investigate reactive protein-cysteine residues: from quantification to kinetics

Proteins containing reactive cysteine residues (protein-Cys) are receiving increased attention as mediators of hydrogen peroxide signaling. These proteins are mainly identified by mining the thiol proteomes of oxidized protein-Cys in cells and tissues. However, it is difficult to determine if oxidation occurs through a direct reaction with hydrogen peroxide or by thiol-disulfide exchange reactions. Kinetic studies with purified proteins provide invaluable information about the reactivity of protein-Cys residues with hydrogen peroxide. Previously, we showed that the characteristic UV-Vis spectrum of horseradish peroxidase compound I, produced from the oxidation of horseradish peroxidase by hydrogen peroxide, is a simple, reliable, and useful tool to determine the second-order rate constant of the reaction of reactive protein-Cys with hydrogen peroxide and peroxynitrite. Here, the method is fully described and extended to quantify reactive protein-Cys residues and micromolar concentrations of hydrogen peroxide. Members of the peroxiredoxin family were selected for the demonstration and validation of this methodology. In particular, we determined the pK(a) of the peroxidatic thiol of rPrx6 (5.2) and the second-order rate constant of its reactions with hydrogen peroxide ((3.4 +/- 0.2) x 10(7) M(-1) s(-1)) and peroxynitrite ((3.7 +/- 0.4) x 10(5) M(-1) s(-1)) at pH 7.4 and 25 degrees C. (C) 2011 Elsevier Inc. All rights reserved.