Effect of sodium nitroprusside on heat resistance of wheat coleoptiles: Dependence on the formation and scavenging of reactive oxygen species

The effect of nitric oxide donor sodium nitroprusside (SNP) on resistance of coleoptiles of 4-day-old etiolated seedlings of wheat (Triticum aestivum L., cv. Elegiya) to damaging heating (10 min at 43°C) and possible dependence of this effect on changes in the activities of enzymes producing and scavenging reactive oxygen species (ROS) were studied. Treatment of coleoptiles with 500 μM SNP considerably boosted generation of superoxide anion radical therein. This effect was substantially suppressed by blocker of calcium channels (lanthanum chloride), calmodulin antagonist (chlorpromazine), and inhibitor of NADPH-oxidase (imidazole) but not by peroxidase inhibitor (salicylhydroxamic acid). NO donor activated antioxidant enzymes (superoxide dismutase, catalase, and soluble peroxidase) and elevated heat resistance of wheat coleoptiles. NO scavenger methylene blue, antioxidant agent ionol, calcium antagonists, and NADPH-oxidase inhibitor imidazole substantially reduced the elevation of heat resistance of wheat coleoptiles induced by NO donor. It was concluded that SNP-induced heat resistance of coleoptiles depended on calcium and ROS, whose production is probably boosted by activation of NADPH-oxidase.     

Chemoattractant-induced cytoplasmic pH changes and cytoskeletal reorganization in human neutrophils. Relationship to the stimulated calcium transients and oxidative burst

The relationships between the chemotactic factor-stimulated mobilization of calcium, activation of the NADPH-oxidase, changes in cytosolic pH, and in the level of polymerized actin in human neutrophils have been examined. The approach taken was to use intracellular calcium chelators, and pharmacologic modulators (both positive and negative) of the NADPH-oxidase to measure the aforementioned responses under conditions where the calcium transients were abrogated and/or the generation of superoxide anions was either inhibited or augmented. The decrease in cytosolic pH induced by chemoattractants was inhibited by the calcium chelator BAPTA and by the diglyceride kinase inhibitor 6-[2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinylethyl ]-7-methyl-5H-thiazolo[3,2-alpha]pyriimidin-5-one (R59022) (this latter compound enhanced the oxidative response of the cells). Furthermore, a specific inhibitor of the NADPH-oxidase (diphenyleneiodonium) had no significant effect on the cytosolic acidification induced by FMLP or leukotriene B4. These results indicate that the initiation of the cytosolic acidification induced by chemotactic factors is a calcium-dependent event that is not directly linked to the activation of the NADPH-oxidase. In contrast, the stimulated polymerization of actin was insensitive to BAPTA, R59022, and diphenyleneiodonium. Thus, neither the calcium transients nor the oxidative burst play a signaling role in the initiation of actin polymerization elicited by chemoattractants. These data indicate that additional investigations are needed to uncover the biochemical basis of the signals initiated in human neutrophils by chemotactic factors that lead to the polymerization of actin and to the cytosolic acidification.     

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O (2) (.-) ) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O (2) (.-) generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)-respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O (2) (.-) in intact and excised roots; ECPOX was an important component of them in both, together with NOX-RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane-redox systems, with different terminal oxidases, activated by different effectors or stresses.     

The elicitor-induced oxidative burst in cultured chickpea cells drives the rapid insolubilization of two cell wall structural proteins

Elicitation of cultured chickpea cells caused rapid insolubilization of two cell wall structural proteins, p190, a putative hydroxyproline-rich glycoprotein and p80, a putative proline-rich protein. This process appeared to result from an H₂O₂-mediated oxidative cross-linking mechanism and was initiated within 5 min and complete within 20 min. Further, elicitation of cells induced a rapid, transient generation of H₂O₂ (oxidative burst), with an onset after 5 min and a maximum H₂O₂-release after 20 min, as measured by a luminol-dependent chemiluminescence assay. Both chemiluminescence and protein insolubilization were suppressed by exogenous application of catalase or diphenylene iodonium, an inhibitor of plasma-membrane NADPH oxidase, respectively. In contrast, exogenous H₂O₂ mimicked the effect of the elicitor, suggesting that the putative oxidative crosslinking of the proteins depends directly on H₂O₂ from the oxidative burst. The peroxidase inhibitor salicylhydroxamic acid blocked both the elicitor- and the exogenous-H₂O₂-stimulated insolubilization, indicating that a peroxidase activity downstream of H₂O₂-supply is required. The protein kinase inhibitor staurosporine blocked the elicitation of the oxidative burst and protein insolubilization. In contrast, the protein phosphatase 2A inhibitor cantharidin accelerated, potentiated and extended the elicited oxidative burst. Cantharidin even stimulated the responses in the absence of the elicitor. The competitive effect of both inhibitors confirms that a coordinated activation of (i) protein kinase(s) and (ii) counteracting protein phosphates(s) is a poised signal transduction step for the induction of an NADPH-oxidase-dependent oxidative burst, which drives the putative peroxidase-catalyzed cross-linking of the cell wall proteins.Abbreviations DPI diphenylene iodonium - Ext-1 extensin-1 - gE1 anti-glycosylated extensin-1 antibodies - HRGP hydroxyp-roline-rich glycoprotein - LDC luminol-dependent chemiluminescence - POD peroxidase - PA polyacrylamide - PRP proline-rich proteins - SHAM salicylhydroxamic acid Financial support by Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie is gratefully acknowledged. We thank Dr. C.J. Lamb (Salk Institute, La Jolla, Calif., USA) and Dr. L.A. Staehelin (University of Colorado, Boulder, Colo., USA) for their kind gifts of antibodies.     

Participation of Nitric Oxide in 24-Epibrassinolide-Induced Heat Resistance of Wheat Coleoptiles: Functional Interactions of Nitric Oxide with Reactive Oxygen Species and Ca Ions

Nitric oxide (NO) effects on heat resistance of wheat (Triticum aestivum L.) coleoptiles induced by 24-epibrassinolide (24-EB) have been investigated. Coleoptiles’ survival after damaging heating (43°С, 10 min) increased when they were treated preliminarily with 5–200 nM of 24-EB. After 24-EB treatment, transient amplification of nitric oxide (NO) and also ROS (superoxide anion-radical (O ₂ ^?? ) and hydrogen peroxide) generation by coleoptiles was noted. Coleoptiles pretreatment with inhibitors of nitrate reductase and an enzyme similar to animal NO-synthase partially removed the increase of NO content caused by the action of 24-EB. Amplification of superoxide anion-radical generation caused by 24-EB was depressed under the influence of imidazole (NADPH-oxidase inhibitor). Calcium antagonists (EGTA and neomycin) largely neutralized the 24-EB-induced increase in generation of both O ₂ ^?? and NO. The increase in NO content in coleoptile tissues caused by 24-EB was almost completely leveled by antioxidants and partly by imidazole. 24-EB-induced enhancement of the superoxide anion-radical generation was partially suppressed by the action of NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) and the inhibitors of nitrate reductase and an enzyme similar to animal NO-synthase. Positive 24-EB effect on the heat resistance of wheat coleoptiles was leveled by PTIO, inhibitors of enzymes that generate NO, antioxidants, an inhibitor of NADPH-oxidase imidazole, and calcium antagonists. A conclusion was made on the role of NO in brassinosteroid signal transduction inducing heat resistance development of coleoptiles and on the functional interaction between NO, ROS, and calcium ions as the signal mediators.     

Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)

The rapid generation of H(2)O(2) by Cd(2+)-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H(2)O(2) has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H(2)O(2) induced by Cd(2+). These data suggest the involvement of an NADPH oxidase-like enzyme leading to H(2)O(2) production through O(2)(*-) dismutation by superoxide dismutase enzymes. To investigate the implication of Ca(2+) channels in a Cd(2+)-induced oxidative burst, different inhibitors of Ca(2+) channels were used. Only La(3+) totally inhibited the generation of H(2)O(2) induced by Cd(2+). However, verapamil and nifedipine, inhibitors of Ca(2+) channels, were not effective. Calmodulin or a Ca(2+)-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H(2)O(2) induced by Cd(2+), suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.     

Calmodulin inhibitors, W-7 and TFP, block the calmodulin-independent activation of NADPH-oxidase by arachidonate in a cell-free system

The calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), or trifluoperazine inhibited not only Fc gamma-receptor mediated cytosolic free Ca2+ increase and O2- generation in macrophages, but also an arachidonate-induced activation of NADPH-oxidase in a cell-free system. Although these results suggested the involvement of Ca2+-calmodulin system, the cell-free activation of NADPH-oxidase occurred in the presence of EGTA and addition of calmodulin had no effect. Furthermore W-7 shifted the optimal concentration of arachidonate required for the activation to a higher level, suggesting that W-7 may block the interaction between arachidonate and NADPH-oxidase system rather than inhibiting a Ca2+-calmodulin system.     

Chemiluminescence response of β-glucan stimulated leukocytes isolated from different tissues and peritoneal cavity of Dicentrarchus labrax

The respiratory burst of leukocytes isolated from sea bass (Dicentrarchus labrax) pronephros, peritoneal cavity (P.C.), spleen and blood, was measured by a chemiluminescence (CL) assay after stimulation with β-glucan. The CL response by P.C. and pronephros leukocytes was significantly higher than that expressed by a similar number of cells separated from spleen and blood. This probably reflects the observation that the proportion of macrophages and neutrophils was highest in the populations of leukocytes from peritoneal cavity and pronephros. Comparative observations showed a higher degree of yeast phagocytosis by leukocytes taken from peritoneal cavity than the pronephros. Moreover phagocytic index evaluated by microscopical observations, indicated that peritoneal macrophages internalised more yeast cells than neutrophils (identified by the peroxidase reaction). Scanning electron microscopy observations were also carried out.Inhibition experiments by a myeloperoxidase inhibitor sodium azide, iodonium-diphenyl-chloride which inhibits NADPH-oxidase, and exogenous superoxide dismutase, which catalyses O−2 dismutation to H₂O₂, supported the correlation between CL and respiratory burst. Treatment with ouabain and DNP suggested that in this response, Ca⁺⁺ pump channels and calmodulin are involved in a metabolic energy-dependent pathway.     

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O₂^•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O₂^•− in a hydrogen peroxide (H₂O₂)-dependent manner. However, this model is incomplete since the source of the initially required H₂O₂ could not be explained. Based on the recently proposed role for H₂O₂-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.     

A monoclonal antibody against peripheral benzodiazepine receptor activities the human neutrophil NADPH-oxidase.

A murine monoclonal antibody, mAb 8523, raised against whole human pro-monocytic U937 cells recognizes an 18 kDa antigen in human neutrophils (PMN), as determined by immunoprecipitation and by immunodetection on Western blots of SDS-PAGE of PMN membrane fractions. That is 18 kDa antigen corresponds to the phagocyte peripheral benzodiazepine receptor (PBZDR) is evidenced by its co-migration with the 18 kDa covalently labeled PBZDR, detected by autoradiography, and their co-modulation upon phorbol-myristate-acetate activation of PMN. Purified mAb 8523 (IgG2b) is able to dose-dependently and specifically stimulate both the basal and the FMLP-induced oxidative burst of intact human PMN, assessed by luminol-amplified chemiluminescence. This property of the first described monoclonal antibody against PBZDR supports the implication of this receptor in NADPH-oxidase activation and consequently in phagocyte-dependent host defense mechanisms.     

Cytosolic and mitochondrial ROS in staurosporine-induced retinal cell apoptosis

In this study, we investigated the involvement of reactive oxygen species (ROS) and calcium in staurosporine (STS)-induced apoptosis in cultured retinal neurons, under conditions of maintained membrane integrity. The antioxidants idebenone (IDB), glutathione-ethylester (GSH/EE), trolox, and Mn(III)tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) significantly reduced STS-induced caspase-3-like activity and intracellular ROS generation. Endogenous sources of ROS production were investigated by testing the effect of the following inhibitors: 7-nitroindazole (7-NI), a specific inhibitor of the neuronal isoform of nitric oxide synthase (nNOS); arachidonyl trifluoromethyl ketone (AACOCF(3)), a phospholipase A(2) (PLA(2)) inhibitor; allopurinol, a xanthine oxidase inhibitor; and the mitochondrial inhibitors rotenone and oligomycin. All these compounds decreased caspase-3-like activity and ROS generation, showing that both mitochondrial and cytosolic sources of ROS are implicated in this mechanism. STS induced a significant increase in intracellular calcium concentration ([Ca(2+)](i)), which was partially prevented in the presence of IDB and GSH/EE, indicating its dependence on ROS generation. These two antioxidants and the inhibitors allopurinol and 7-NI also reduced the number of TdT-mediated dUTP nick-end labeling-positive cells. Thus, endogenous ROS generation and the rise in intracellular calcium are important inter-players in STS-triggered apoptosis. Furthermore, the antioxidants may help to prolong retinal cell survival upon apoptotic cell death.     

Fc epsilon RI signaling of mast cells activates intracellular production of hydrogen peroxide: role in the regulation of calcium signals

Earlier studies, including our own, revealed that activation of mast cells is accompanied by production of reactive oxygen species (ROS) that help to mediate the release of the inflammatory mediators, including histamine and eicosanoids. However, little is known about the mechanisms of ROS production, including the species of oxidants produced. In this study we show that in both the RBL-2H3 mast cell line and bone marrow-derived mast cells, FcepsilonRI cross-linking stimulates intracellular oxidative burst, including hydrogen peroxide (H(2)O(2)) production, as defined with the oxidant-sensitive dyes dichlorofluorescein and scopoletin and the selective scavenger ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one). The oxidative burst was observed immediately after stimulation and was most likely due to an NAD(P)H oxidase. Experiments using selective pharmacological inhibitors demonstrated that activation of tyrosine kinases and phosphatidylinositol-3-kinase is required for induction of the oxidative burst. Blockade of the oxidative burst by diphenyleneiodonium impaired the release of preformed granular mediators, such as histamine and beta-hexosaminidase, and the secretion of newly synthesized leukotriene C(4), whereas selective scavenging H(2)O(2) by ebselen impaired leukotriene C(4) secretion, but not degranulation. Sustained elevation of cytosolic calcium through store-operated calcium entry was totally abolished when ROS production was blocked. In contrast, selective depletion of H(2)O(2) caused a considerable decrease and delay of the calcium response. Finally, tyrosine phosphorylation of phospholipase Cgamma and the linker for activation of T cells, an event required for calcium influx, was suppressed by diphenyleneiodonium and ebselen. These studies demonstrate that activation of the intracellular oxidative burst is an important regulatory mechanism of mast cell responses.     

Lipoxygenase may be involved in cationic liposome-induced macrophage apoptosis

The purpose of this study was to determine the source of reactive oxygen species (ROS) generation and the contribution of ROS to the apoptosis of RAW264.7 cells induced by cationic liposomes. Cationic liposome-induced apoptosis was inhibited by lipoxygenase inhibitors, but not inhibitors of NADPH-oxidase, xanthine oxidase or cyclooxygenase. ROS generation induced by cationic liposomes was also inhibited by the lipoxygenase inhibitor NDGA. Furthermore, lipid peroxidation was observed following liposome treatment, but the apoptosis was not inhibited by the antioxidant alpha-tocopherol. These findings suggested that lipoxygenase is responsible for ROS generation, and ROS but not lipid peroxidation acts as a key mediator in the progress of apoptosis induced by cationic liposomes.     

The lipopolysaccharides of the phytopathogen Xanthomonas campestris pv. campestris induce an oxidative burst reaction in cell cultures of Nicotiana tabacum

The lipopolysaccharides (LPSXcc) of the phytopathogenic bacteria Xanthomonas campestris pv. campestris (X.c.c.) were purified from an exopolysaccharide-deficient mutant strain. The isolated LPSxcc induced an oxidative burst reaction in cell-suspension cultures of the non-host plant tobacco (Nicotiana tabacum L.) SRI. The oxidative burst elicited by LPSXcc differed from that induced by yeast elicitor (YE), a cell wall preparation of baker's yeast. The LPSXcc-induced oxidative burst was characterised by a slow increase in H2O2 production and an extended decline. Both the LPSXcc-and YE-induced oxidative bursts were completely blocked by the NAD(P)H-oxidase inhibitor diphenylene-iodonium. When LPSXcc and YE were applied in combination, a synergistic effect and the establishment of refractory states in the generation of H2O2 were observed. The amount of cytosolic calcium was measured in transgenic tobacco cell cultures carrying the apoaequorin gene by coelenterazine-derived chemiluminescence. Whereas YE induced a calcium peak within 1 min after application, LPSXcc induced a long-term calcium signal without transients. To our knowledge this is the first report on the elicitation of an oxidative burst in plant cell cultures by isolated LPS of a phytopathogenic bacterium.     

Oxidative burst and NO generation as initial response to ischemia in flow-adapted endothelial cells

Shear stress modulates endothelial physiology, yet the effect(s) of flow cessation is poorly understood. The initial metabolic responses of flow-adapted bovine pulmonary artery endothelial cells to the abrupt cessation of flow (simulated ischemia) was evaluated using a perfusion chamber designed for continuous spectroscopy. Plasma membrane potential, production of reactive O2 species (ROS), and intracellular Ca(2+) and nitric oxide (NO) levels were measured with fluorescent probes. Within 15 s after flow cessation, flow-adapted cells, but not cells cultured under static conditions, showed plasma membrane depolarization and an oxidative burst with generation of ROS that was inhibited by diphenyleneiodonium. EGTA-inhibitable elevation of intracellular Ca(2+) and NO were observed at approximately 30 and 60 s after flow cessation, respectively. NO generation was decreased in the presence of inhibitors of NO synthase and calmodulin. Thus flow-adapted endothelial cells sense the altered hemodynamics associated with flow cessation and respond by plasma membrane depolarization, activation of NADPH oxidase, Ca(2+) influx, and activation of Ca(2+)/calmodulin-dependent NO synthase. This signaling response is unrelated to cellular anoxia.     

Oxidative stress induced by P2X7 receptor stimulation in murine macrophages is mediated by c-Src/Pyk2 and ERK1/2

Background

Activation of ATP-gated P2X7 receptors (P2X7R) in macrophages leads to production of reactive oxygen species (ROS) by a mechanism that is partially characterized. Here we used J774 cells to identify the signaling cascade that couples ROS production to receptor stimulation.

Methods

J774 cells and mP2X7-transfected HEK293 cells were stimulated with Bz-ATP in the presence and absence of extracellular calcium. Protein inhibitors were used to evaluate the physiological role of various kinases in ROS production. In addition, phospho-antibodies against ERK1/2 and Pyk2 were used to determine activation of these two kinases.

Results

ROS generation in either J774 or HEK293 cells (expressing P2X7, NOX2, Rac1, p47phox and p67phox) was strictly dependent on calcium entry via P2X7R. Stimulation of P2X7R activated Pyk2 but not calmodulin. Inhibitors of MEK1/2 and c-Src abolished ERK1/2 activation and ROS production but inhibitors of PI3K and p38 MAPK had no effect on ROS generation. PKC inhibitors abolished ERK1/2 activation but barely reduced the amount of ROS produced by Bz-ATP. In agreement, the amount of ROS produced by PMA was about half of that produced by Bz-ATP.

Conclusions

Purinergic stimulation resulted in calcium entry via P2X7R and subsequent activation of the PKC/c-Src/Pyk2/ERK1/2 pathway to produce ROS. This signaling mechanism did not require PI3K, p38 MAPK or calmodulin.

General significance

ROS is generated in order to kill invading pathogens, thus elucidating the mechanism of ROS production in macrophages and other immune cells allow us to understand how our body copes with microbial infections.     

Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.     

Role of hydrogen peroxide in generation of a signal inducing heat tolerance of wheat seedlings

The effects of 1-min-long exposure to 42°C (hardening heating) on heat tolerance and dynamics of ROS (superoxide anion radical and hydrogen peroxide) generation were investigated in the wheat (Triticum aestivum L., cv. Elegiya) seedlings. During the initial 5–30 min after the onset of hyperthermia, ROS generation by roots and shoots was intensified, and superoxide dismutase (SOD) was activated. During the first hour after hardening heating, the seedling tolerance to injurious 10-min-long treatment with high temperature (46°C) decreased but subsequently it gradually rose, reaching maximum in 24 h. Transient accumulation of hydrogen peroxide induced by hardening was suppressed by seedling treatment with H₂O₂ scavenger dimethylthiourea, by inhibitors of NADPH-oxidase (imidazole) and DDC (sodium diethyldithiocarbamate). These compounds considerably reduced favorable effect of hardening on seedling heat tolerance. It was concluded that generation of a signal inducing the development of heat tolerance depended on NADPH-oxidase producing superoxide anion radical and SOD that transforms it into hydrogen peroxide (more stable ROS performing signaling functions).     

Nonstructural 3 protein of hepatitis C virus triggers an oxidative burst in human monocytes via activation of NADPH oxidase

It has been shown that oxidative stress occurs in chronic hepatitis C. Release of reactive oxygen species (ROS) from sequestered phagocytes and activated resident macrophages represents the predominant component of oxidative stress in the liver. However, little is known about the ability of the monocyte to produce ROS in response to protein of hepatitis C virus. In this study, we investigated the ROS production in human monocytes stimulated by several viral proteins of hepatitis C virus. Human monocytes from healthy blood donors were incubated with recombinant viral protein: Core, NS3, NS4, and NS5. ROS production was measured by chemiluminescence. Only NS3 triggered ROS production in human monocytes. Generated ROS were mainly the anion superoxide. NS3 also induced a rapid and transient increase in intracellular calcium concentration measured by a video digital microscopy technique. By using different metabolic inhibitors, we showed that ROS production requires calcium influx, tyrosine kinases, and the stress-activated protein kinase, p38. The study of p47(PHOX) phosphorylation and translocation showed that NADPH oxidase was activated and involved in ROS production induced by NS3. In a second experiment, NS3 inhibited the oxidative burst induced by phorbol 12-myristate 13-acetate. These results indicate that NS3 activates NADPH oxidase and modulates ROS production, which may be involved in the natural history of hepatitis C infection.