H(2)O(2)-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca²⁺ overload play key roles in myocardial ischemia-reperfusion (IR) injury but the relationships among ROS, Ca²⁺ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H₂O₂ impairs LV function by causing Ca²⁺ overload by increasing late sodium current (I_Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca²⁺ concentrations (d[Ca²⁺]_i and s[Ca²⁺]_i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H₂O₂ (100 μM, 30 min) increased d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca²⁺]_i and s[Ca²⁺]_i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I_Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca²⁺]_i and LV function, but did not alter effects of H₂O₂. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H₂O₂-induced LV dysfunction, but did not alter [Ca²⁺]_i. Paradoxically, further increases in [Ca²⁺]_i by ATX-II or ouabain, given 10 min after H₂O₂, improved function. The failure of late I_Na inhibitors to prevent H₂O₂-induced LV dysfunction, and the ability of MPG to prevent H₂O₂-induced LV dysfunction independent of changes in [Ca²⁺]_i indicate that impaired contractility is not due to Ca²⁺ overload. The ability of further increases in [Ca²⁺]_i to reverse H₂O₂-induced LV dysfunction suggests that Ca²⁺ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.     

Reactive oxygen species directly modify sodium–calcium exchanger activity in a splice variant-dependent manner

The sodium–calcium exchanger isoform 1 (NCX1) operating in calcium-efflux mode plays an important role in maintaining calcium homeostasis in the heart. Paradoxically, activity of NCX1 in calcium-influx mode contributes to the pathological intracellular calcium overload during cardiac ischemia–reperfusion injury. Reactive oxygen species (ROS) also contribute to myocardial dysfunction in ischemia–reperfusion and are reported to alter NCX1 activity. However, the molecular mechanism(s) by which ROS modifies NCX1 activity have not been elucidated. Therefore, the effects of the ROS, H₂O₂, on recombinant NCX1 splice variants were studied using the patch-clamp technique. H₂O₂ irreversibly increased calcium-influx mode activity in the cardiac NCX1.1 splice variant, without affecting calcium-efflux mode activity. In direct contrast, H₂O₂ inhibited the calcium-influx mode of the vascular NCX1.3 splice variant indicating that these disparate effects of H₂O₂ may be dependent on the exon complement of the alternative splicing region. Using NCX1 splice variants with various exon compositions, the mutually exclusive exons A and B were found to bestow the differential effects of H₂O₂ on NCX1 function. As NCX1 inhibition is a potential therapeutic strategy for ischemia–reperfusion injury, the effects of the NCX1 inhibitor KB-R7943 were examined. KB-R7943 was ~ 7-fold less potent at inhibiting NCX1 activity after H₂O₂ modification. In summary, this study provides insights into the molecular regulation of NCX1 by ROS and indicates that ROS may elicit differential effects in various tissues depending on the exon composition of the splice variant expressed. These results also highlight that the potency of NCX1 inhibitors may be impaired under conditions of oxidative stress.     

p53 and ATF-2 partly mediate the overexpression of COX-2 in H2O2-induced premature senescence of human fibroblasts

Cyclooxygenase-2 and release of prostaglandin E2 are up-regulated in replicative senescence of dermal and prostate fibroblasts and in H₂O₂-induced premature senescence of IMR-90 lung fibroblasts expressing the catalytic subunit of telomerase. Inhibition of cyclooxygenase-2 activity by specific chemical inhibitor or siRNA attenuates the H₂O₂-induced increase of senescence associated β-galactosidase positive cells and attenuates growth arrest. In this work, p38^MAPK activation and increased DNA binding activities of ATF-2 and p53 are shown to mediate cyclooxygenase-2 overexpression in premature senescence.     

Mitochondrial Ca2+ flux and respiratory enzyme activity decline are early events in cardiomyocyte response to H2O2

Oxidative stress is involved in mitochondrial apoptosis, and plays a critical role in ischemic heart disease and cardiac failure. Exposure of cardiomyocytes to H₂O₂ leads to oxidative stress and mitochondrial dysfunction. In this study, we investigated the temporal order of mitochondrial-related events in the neonatal rat cardiomyocyte response to H₂O₂ treatment. At times ranging from 10 to 90 min after H₂O₂ treatment, levels were determined for respiratory complexes I, II, IV and V, and citrate synthase activities, mitochondrial Ca²⁺ flux, intracellular oxidation, mitochondrial membrane potential and apoptotic progression. Complexes II and IV activity levels were significantly reduced within 20 min of H₂O₂ exposure while complexes I and V, and citrate synthase were unaffected. Mitochondrial membrane potential declined after 20 and 60 min of H₂O₂ exposure while intracellular oxidation, declining complex I activity and apoptotic progression were detectable only after 60 min. Measurement of mitochondrial Ca²⁺ ([Ca²⁺]_m) using rhodamine 2 detected an early accumulation of [Ca²⁺]_m occurring between 5 and 10 min. Pretreatment of cardiomyocytes with either ruthenium red or cyclosporin A abrogated the H₂O₂-induced decline in complexes II and IV activities, indicating that [Ca²⁺]_m flux and onset of mitochondrial permeability transition pore opening likely precede the observed early enzymatic decline. Our findings suggest that [Ca²⁺]_m flux represents an early pivotal event in H₂O₂-induced cardiomyocyte damage, preceding and presumably leading to reduced mitochondrial respiratory activity levels followed by accumulation of intracellular oxidation, mitochondrial membrane depolarization and apoptotic progression concomitant with declining complex I activity.     

Salvianolic acid B from Salvia miltiorrhiza inhibits tumor necrosis factor-alpha (TNF-alpha)-induced MMP-2 upregulation in human aortic smooth muscle cells via suppression of NAD(P)H oxidase-derived reactive oxygen species

Activated matrix metalloproteinases (MMPs) in patients with acute coronary syndromes may contribute to plaque destabilization. Tumor necrosis factor-α (TNF-α) enhances NAD (P) H oxidase-dependent reactive oxygen species (ROS) formation and ROS induce MMP-2. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (SalB), derived from a Chinese herb, Salvia miltiorrhiza, on the expression of MMP-2 by TNF-α-treated human aortic smooth muscle cells (HASMCs) were investigated. In this study, salvianolic acid B scavenged H₂O₂ in a dose-dependent manner in test tube. We found that SalB, as well as NADPH oxidase inhibitors, DPI or apocynin, and antioxidant NAC, inhibited TNF-α-induced MMP-2 mRNA, protein expression, and gelatinolytic activity in HASMCs in a concentration-dependent manner. We also observed a dose-dependent decrease in ROS production and NADPH oxidase activity induced by TNF-α in the presence of SalB. SalB also significantly inhibited angiotensin II or H₂O₂-induced MMP-2 mRNA and protein expression and gelatinolytic activity in HASMCs. Our data point out that the importance of NADPH oxidase-dependent ROS generation in the control of SalB inhibition of TNF-α-induced MMP-2 expression and activity.     

Shear stress inhibition of H<Subscript>2</Subscript>O<Subscript>2</Subscript> induced p66<Superscript>Shc</Superscript> phosphorylation by ASK1–JNK inactivation in endothelium

Shear stress protects endothelium from a variety of risk factors for vascular disease. Here, we demonstrate a novel mechanism whereby shear stress inhibited reactive oxygen species (ROS)-triggered signaling cascades in endothelial cells. Stimulation of bovine aortic endothelial cells (BAECs) with H₂O₂ induced a 3.07-fold increase in p66^Shc phosphorylation. This response was fully blocked by pretreatment of cells with specific JNK but not p38 or ERK MAP kinase inhibitor. Further study showed that knocking down of apoptosis signal-regulating kinase 1 (ASK1) by siRNA transfection in cells dramatically inhibited phosphorylation of JNK and p66^Shc elicited by H₂O₂. Pre-perfusion of BAECs cultured in silastic tubes with laminar flow generated by a servo-pump system for 30 min also significantly suppressed H₂O₂-induced phosphorylation of p66^Shc. This was accompanied by quantitatively similar inhibition of ASK1 and JNK phosphorylation and activation. These results suggested that shear stress protects endothelium against oxidant stress by suppression of ASK1–JNK-mediated p66^Shc phosphorylation.     

An Ultrasensitive Electrochemical Immunosensor for HIV p24 Based on Fe3O4@SiO2 Nanomagnetic Probes and Nanogold Colloid-Labeled Enzyme–Antibody Copolymer as Signal Tag

An ultrasensitive portable electrochemical immunosensor for human immunodeficiency virus p24 (HIV p24) antigen detection has been developed, whereby the detection sensitivity was 1000 times higher than that of the ELISA method. Firstly, a novel HRP enzyme–antibody copolymer (EV-p24 Ab2) was synthesized through an EnVision regent (EV, a dextrin amine skeleton anchoring more than 100 molecules of HRP and 15 molecules of anti IgG), then incubated in the secondary antibody of p24. Secondly, the copolymer was immobilized on the gold nanocolloids (AuNPs) to fabricate a novel signal tag (AuNPs/EV-p24 Ab2). Subsequently, a sandwich-type immunoreaction would take place between the capture probe (silicon dioxide-coated magnetic Fe₃O₄ nanoparticles (MNPs) labeled with the primary p24 antibody (MNPs-p24 Ab1)), p24 (different concentrations) and the signal tag [AuNPs/EV-p24 Ab2)] to form the immunocomplex. Finally, the immunocomplex was absorbed on the surface of screen printed carbon electrode (SPCE) by a magnet and immersed in the o-hydroxyl phenol (HQ) and H₂O₂. The large amounts of HRP on the signal tag can catalyze the oxidation of HQ by H₂O₂, which can induce an amplified reductive current. Moreover, the capture probe could improve the accumulation ability of p24 and facilitate its separation from the substrate through the magnet. Under optimal conditions, the proposed immunoassay exhibited good sensitivity to p24 within a certain concentration range from 0.001 to 10.00 ng/mL, with a detection limit of 0.5 pg/mL (S/N = 3). The proposed method can be used for real-time and early detection of HIV-infected people.     

Protection of peroxiredoxin II on oxidative stress-induced cardiomyocyte death and apoptosis

Peroxiredoxin II, a cytosolic isoform of the antioxidant enzyme family, has been implicated in cancer-associated cell death and apoptosis, but its functional role in the heart remains to be elucidated. Interestingly, the expression levels of peroxiredoxin II were decreased in mouse hearts upon ischemia-reperfusion, while they were elevated in two genetically modified hyperdynamic hearts with phospholamban ablation or protein phosphatase 1 inhibitor 1 overexpression. To delineate the functional significance of altered peroxiredoxin II expression, adenoviruses encoding sense or antisense peroxiredoxin II were generated; cardiomyocytes were infected, and then subjected to H₂O₂ treatment to mimic oxidative stress-induced cell death and apoptosis. H₂O₂ stimulation resulted in a significant decrease of endogenous peroxiredoxin II expression, along with reduced cell viability in control cells. However, overexpression of peroxiredoxin II significantly protected from H₂O₂-induced apoptosis and necrosis, while downregulation of this enzyme promoted the detrimental effects of oxidative stress in cardiomyocytes. The beneficial effects of peroxiredoxin II were associated with increased Bcl-2 expression, decreased expression of Bax and attenuated activity of caspases 3, 9 and 12. Furthermore, there were no significant alterations in the expression levels of the other five isoforms of peroxiredoxin, as well as active catalase or glutathione peroxidase-1 after ischemia-reperfusion or H₂O₂ treatment. These findings suggest that peroxiredoxin II may be a unique antioxidant in the cardiac system and may represent a potential target for cardiac protection from oxidative stress-induced injury. Returned for 1. Revision: 14 April 2008 1. Revision received: 25 September 2008     

The Corrosion Behavior of Pure Iron under Solid Na2SO4 Deposit in Wet Oxygen Flow at 500 °C

The corrosion behavior of pure Fe under a Na₂SO₄ deposit in an atmosphere of O₂ + H₂O was investigated at 500 °C by thermo gravimetric, and electrochemical measurements, viz. potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), and surface characterization methods viz. X-ray diffraction (XRD), and scanning electron microscope (SEM)/energy dispersive spectroscopy(EDS). The results showed that a synergistic effect occurred between Na₂SO₄ and O₂ + H₂O, which significantly accelerated the corrosion rate of the pure Fe. Briefly, NaFeO₂ was formed in addition to the customary Fe oxides; at the same time, H₂SO₄ gas was produced by introduction of water vapor. Subsequently, an electrochemical corrosion reaction occurred due to the existence of Na₂SO₄, NaFeO₂, and H₂O. When this coupled to the chemical corrosion reaction, the progress of the chemical corrosion reaction was promoted and eventually resulted in the acceleration of the corrosion of the pure Fe.     

Respiration triggers heme transfer from cytochrome c peroxidase to catalase in yeast mitochondria

In exponentially growing yeast, the heme enzyme, cytochrome c peroxidase (Ccp1) is targeted to the mitochondrial intermembrane space. When the fermentable source (glucose) is depleted, cells switch to respiration and mitochondrial H₂O₂ levels rise. It has long been assumed that CCP activity detoxifies mitochondrial H₂O₂ because of the efficiency of this activity in vitro. However, we find that a large pool of Ccp1 exits the mitochondria of respiring cells. We detect no extramitochondrial CCP activity because Ccp1 crosses the outer mitochondrial membrane as the heme-free protein. In parallel with apoCcp1 export, cells exhibit increased activity of catalase A (Cta1), the mitochondrial and peroxisomal catalase isoform in yeast. This identifies Cta1 as a likely recipient of Ccp1 heme, which is supported by low Cta1 activity in ccp1Δ cells and the accumulation of holoCcp1 in cta1Δ mitochondria. We hypothesized that Ccp1’s heme is labilized by hyperoxidation of the protein during the burst in H₂O₂ production as cells begin to respire. To test this hypothesis, recombinant Ccp1 was hyperoxidized with excess H₂O₂ in vitro, which accelerated heme transfer to apomyoglobin added as a surrogate heme acceptor. Furthermore, the proximal heme Fe ligand, His175, was found to be ∼85% oxidized to oxo-histidine in extramitochondrial Ccp1 isolated from 7-d cells, indicating that heme labilization results from oxidation of this ligand. We conclude that Ccp1 responds to respiration-derived H₂O₂ via a previously unidentified mechanism involving H₂O₂-activated heme transfer to apoCta1. Subsequently, the catalase activity of Cta1, not CCP activity, contributes to mitochondrial H₂O₂ detoxification.Cytochrome c peroxidase (Ccp1) is a monomeric nuclear encoded protein with a 68-residue N-terminal mitochondrial targeting sequence (1). This presequence crosses the inner mitochondrial membrane and is cleaved by matrix proteases (2, 3). Mature heme-loaded Ccp1 is found in the mitochondrial intermembrane space (IMS) in exponentially growing yeast (2, 3) but the point of insertion of its single b-type heme is unknown. Under strict anaerobic conditions, Ccp1 is present in mitochondria as the heme-free form or apoform (4). Once cells are exposed to O₂ and heme biosynthesis is turned on, apoCcp1 converts rapidly to the mature holoenzyme by noncovalently binding heme (5).It is well established that mature Ccp1 functions as an efficient H₂O₂ scavenger in vitro (6). Its catalytic cycle involves the reaction of ferric Ccp1 with H₂O₂ (Eq. 1) to form compound I (CpdI) with a ferryl (Fe^IV) heme and a cationic indole radical localized on Trp191 (W191^+•). CpdI is one-electron reduced by the ferrous heme of cytochrome c (Cyc1) to compound II (CpdII) with ferryl heme (Eq. 2), and electron donation by a second ferrous Cyc1 returns CpdII to the resting Ccp1^III form (Eq. 3):Ccp1^III + H₂O₂ → CpdI(Fe^IV, W191^+?) + H₂O[1]CpdI(Fe^IV, W191^+?) + Cyc1^II → CpdII(Fe^IV) + Cyc1^III[2]CpdII(Fe^IV) + Cyc1^II → Ccp1^III + Cyc1^III + H₂O.[3]Because Ccp1 production is not under O₂/heme control (4, 5), CCP activity is assumed to be the frontline defense in the mitochondria, a major source of reactive oxygen species (ROS) in respiring cells (7). Contrary to the time-honored assumption that Ccp1 catalytically consumes the H₂O₂ produced during aerobic respiration (8), recent studies in our group reveal that the peroxidase behaves more like a mitochondrial H₂O₂ sensor than a catalytic H₂O₂ detoxifier (9–11). Notably, Ccp1 competes with complex IV for reducing equivalents from Cyc1, which shuttles electrons from complex III (ubiquinol cytochrome c reductase) to complex IV (cytochrome c oxidase) in the electron transport chain (12).Because CCP activity in the IMS siphons electrons from energy production, an H₂O₂ sensor role for Ccp1 should be energetically more favorable for the cell. Key evidence for a noncatalytic role for Ccp1 in H₂O₂ removal is that the isogenic strain producing the catalytically inactive Ccp1^W191F protein accumulates less H₂O₂ than wild-type cells (10). In fact, this mutant strain exhibits approximately threefold higher catalase A (Cta1) activity than wild-type cells (10) whereas CCP1 deletion results in a strain (ccp1Δ) with negligible Cta1 activity and high H₂O₂ levels (5). Unlike Cta1, which is the peroxisomal and mitochondrial catalase isoform in yeast (13), the cytosolic catalase Ctt1 (14) exhibits comparable activity in the wild-type, Ccp1^W191F, and ccp1Δ strains (10). Given that both Ccp1 and Cta1 are targeted to mitochondria, we hypothesized that Ccp1 may transfer its heme to apoCta1 in respiring cells.Cta1 is nuclear encoded with embedded mitochondrial and peroxisomal targeting sequences (15). Like Ccp1, each monomer noncovalently binds a b-type heme and mature Cta1 is active as a homotetramer. Synthesis of the Cta1 monomer is under O₂/heme control such that the apoenzyme begins to accumulate only during the logarithmic phase of aerobic growth (16). Hence, its O₂/heme independent production (4, 5) allows apoCcp1 to acquire heme while cells are synthesizing apoCta1. This, combined with our observation that Cta1 activity increases in respiring cells producing Ccp1 or Ccp1^W191F but not in ccp1Δ cells (10), led us to speculate that respiration-derived H₂O₂ triggers heme donation from Ccp1 to apoCta1 within mitochondria.What experimental evidence would support heme donation by Ccp1? It has been demonstrated that mutation of the proximal heme Fe ligand, His175, to a residue with weak or no Fe-coordinating ability produces Ccp1 variants (H175P, H175L, H175R, and H175M) that undergo mitochondrial processing but do not accumulate in isolated yeast mitochondria (17). Presumably, reduced heme affinity allows the Ccp1 variants to unfold and cross the outer mitochondrial membrane (17). Hence, we argued that if wild-type Ccp1 donated its heme, the apoprotein would likewise exit mitochondria. Consequently, we examine here age-dependent Ccp1–green fluorescent protein (Ccp1-GFP) localization in live cells chromosomally expressing Ccp1 C-terminally fused to GFP as well as the distribution of wild-type Ccp1 between subcellular fractions. Because weakening or removal of the proximal Fe ligand on His175 mutation reduces heme affinity (17), His175 oxidation in wild-type Ccp1 should have a similar effect, which we investigate here. We further speculated that in the absence of apoCta1 as an acceptor for its heme, more Ccp1 would remain trapped in the IMS so we compare mitochondrial Ccp1 levels in wild-type and cta1∆ cells. Our combined results support triggering of heme donation from Ccp1 to apoCta1 by respiration-derived H₂O₂. Such H₂O₂-activated heme transfer between proteins has not been reported to date and its implications in H₂O₂ signaling are discussed.     

Distinct roles of p42/p44ERK and p38 MAPK in oxidant-induced AP-1 activation and cardiomyocyte hypertrophy

Cardiac hypertrophy is an adaptive response to a number of heart diseases including myocardial infarction. Although it can be compensatory at first, sustained hypertrophy is often a transition to heart failure. We have found that cardiomyocytes in culture can survive mild doses of H₂O₂ but develop hypertrophy involving activation of p70 S6 kinase 1 (p70S6K1). Here, the role of p42/p44^ERK and p38 MAPK in oxidant-induced hypertrophy is tested. H₂O₂-induced phosphorylation (activation) of p42/p44^ERK and p38 within 10 min of 200 μM H₂O₂ exposure. Although p42/p44^ERK remained highly phosphorylated from 60 to 120 min, the level of p38 phosphorylation reached highest at 60 min and started to decline at 90 min. Inhibiting ERKs with PD98059 attenuated H₂O₂-induced AP-1 activation but did not affect H₂O₂-induced p70S6k1 activation or cardiomyocyte enlargement as measured by increases in cell volume and protein content. In contrast, the p38 inhibitor SB202190 has not inhibitory effect on AP-1 activation but partially prevented H₂O₂ from inducing p70S6K1 activation and cell enlargement. These data suggest that while p42/p44^ERK participates in gene expression associated with hypertrophy, p38 may regulate cell size increase by p70S6K1 activation.     

Direct Electrochemistry and Electrocatalysis of Horseradish Peroxidase Immobilized in a DNA/Chitosan-Fe3O4 Magnetic Nanoparticle Bio-Complex Film

A DNA/chitosan-Fe₃O₄ magnetic nanoparticle bio-complex film was constructed for the immobilization of horseradish peroxidase (HRP) on a glassy carbon electrode. HRP was simply mixed with DNA, chitosan and Fe₃O₄ nanoparticles, and then applied to the electrode surface to form an enzyme-incorporated polyion complex film. Scanning electron microscopy (SEM) was used to study the surface features of DNA/chitosan/Fe₃O₄/HRP layer. The results of electrochemical impedance spectroscopy (EIS) show that Fe₃O₄ and enzyme were successfully immobilized on the electrode surface by the DNA/chitosan bio-polyion complex membrane. Direct electron transfer (DET) and bioelectrocatalysis of HRP in the DNA/chitosan/Fe₃O₄ film were investigated by cyclic voltammetry (CV) and constant potential amperometry. The HRP-immobilized electrode was found to undergo DET and exhibited a fast electron transfer rate constant of 3.7 s⁻¹. The CV results showed that the modified electrode gave rise to well-defined peaks in phosphate buffer, corresponding to the electrochemical redox reaction between HRP(Fe^(III)) and HRP(Fe^(II)). The obtained electrode also displayed an electrocatalytic reduction behavior towards H₂O₂. The resulting DNA/chitosan/Fe₃O₄/HRP/glassy carbon electrode (GCE) shows a high sensitivity (20.8 A·cm⁻²·M⁻¹) toward H₂O₂. A linear response to H₂O₂ measurement was obtained over the range from 2 μM to 100 μM (R² = 0.99) and an amperometric detection limit of 1 μM (S/N = 3). The apparent Michaelis-Menten constant of HRP immobilized on the electrode was 0.28 mM. Furthermore, the electrode exhibits both good operational stability and storage stability.     

Initial Processes of Atomic Layer Deposition of Al2O3 on InGaAs: Interface Formation Mechanisms and Impact on Metal-Insulator-Semiconductor Device Performance

Interface-formation processes in atomic layer deposition (ALD) of Al₂O₃ on InGaAs surfaces were investigated using on-line Auger electron spectroscopy. Al₂O₃ ALD was carried out by repeating a cycle of Al(CH₃)₃ (trimethylaluminum, TMA) adsorption and oxidation by H₂O. The first two ALD cycles increased the Al KLL signal, whereas they did not increase the O KLL signal. Al₂O₃ bulk-film growth started from the third cycle. These observations indicated that the Al₂O₃/InGaAs interface was formed by reduction of the surface oxides with TMA. In order to investigate the effect of surface-oxide reduction on metal-insulator-semiconductor (MIS) properties, capacitors and field-effect transistors (FETs) were fabricated by changing the TMA dosage during the interface formation stage. The frequency dispersion of the capacitance-voltage characteristics was reduced by employing a high TMA dosage. The high TMA dosage, however, induced fixed negative charges at the MIS interface and degraded channel mobility.     

Arsenicals,the Integrated Stress Response,and Epstein–Barr Virus Lytic Gene Expression

Following our observation that clofoctol led to Epstein–Barr virus (EBV) lytic gene expression upon activation of the integrated stress response (ISR), we decided to investigate the impact of As₂O₃ on viral lytic gene expression. As₂O₃ has also been reported to activate the ISR pathway by its activation of the heme-regulated inhibitor (HRI). Our investigations show that As₂O₃ treatment leads to eIF2α phosphorylation, upregulation of ATF4 and TRB3 expression, and an increase of EBV Zta gene expression in lymphoid tumor cell lines as well as in naturally infected epithelial cancer cell lines. However, late lytic gene expression and virion production were blocked after arsenic treatment. In comparison, a small molecule HRI activator also led to increased Zta expression but did not block late lytic gene expression, suggesting that As₂O₃ effects on EBV gene expression are also mediated through other pathways.     

The effects of asthma medications on reactive oxygen species production in human monocytes

Objective: Asthma is a chronic inflammatory airway disease induced by many environmental factors. The inhalation of allergens and pollutants promotes the production of reactive oxygen species (ROS) leading to airway inflammation, hyper-responsiveness, and remodeling in allergic asthma. The effects of asthma medications on ROS production are unclear. The present study investigated the anti-ROS effects of current asthma medications including inhaled corticosteroid (ICS; budesonide and fluticasone), leukotriene receptor antagonist (LTRA; montelukast), long-acting β2 agonists (LABAs; salmeterol and formoterol), and a new extra-LABA (indacaterol). Methods: The human monocyte cell line THP-1 cells were pre-treated with different concentrations of the asthma medications at different time points after hydrogen peroxide (H₂O₂) stimulation. H₂O₂ production was measured with DCFH-DA by flow cytometry. Results: Montelukast, fluticasone, and salmeterol suppressed H₂O₂-induced ROS production. Indacaterol enhanced H₂O₂-induced ROS production. Budesonide and formoterol alone had no anti-ROS effects, but the combination of these two drugs significantly suppressed H₂O₂-induced ROS production. Conclusions: Different asthma medications have different anti-ROS effects on monocytes. The combination therapy with LABA and ICS seemed not to be the only choice for asthma control. Montelukast may also be a good supplemental treatment for the poorly controlled asthma because of its powerful anti-ROS effects. Our findings provide a novel therapeutic view in asthma.     

Regional differences of superoxide dismutase activity enhance the superoxide–induced electrical heterogeneity in rabbit hearts

During myocardial ischemia and the subsequent reperfusion, free radicals are important intermediates of the cellular damage and rhythm disturbances. We examined the effects of superoxide radicals or hydrogen peroxide (H₂O₂) on the action potentials in isolated rabbit Purkinje fibers, atrial muscle and ventricular muscle. Reactive oxygen species (ROS) donors such as adriamycin, xanthine/xanthine oxidase and menadione induced prolongation of APD₉₀ in Purkinje fibers. Menadione (30 µM), the most specific superoxide radical donor, prolonged the action potential duration at 90% repolarization (APD₉₀) by 17% in Purkinje fibers, whereas it shortened the APD by 57% in ventricular muscle, and it did not affect the atrial APD. All these menadione–induced effects were completely blocked by 2,2,6,6–tetramethyl– 1–peperadinyloxy, a superoxide radical scavenger. Superoxide dismutase (SOD) activity was lowest in Purkinje fibers, it was moderate in atrial muscle and highest in ventricular muscle. H₂O₂ shortened the APDs of all three cardiac tissues in a concentration–dependent manner. These results suggest that the different electrical responses to O₂^●– in different cardiac regions may result from the regional differences in the SOD activity, thereby enhancing the regional electrical heterogeneity.Drs. B. H. Choi and K.–Ch. Ha contributed equally to this study.