The potential physiological implications of polygalacturonic acid-mediated production of superoxide

PGA/OGA/PF represent apoplastic signaling molecules implicated in the control of gene expression and the activity of enzymes involved in defense regulation. However, the underlying mechanisms behind such processes are lacking. Here we unequivocally show using EPR spectroscopy with DEPMPO spin-trap capable of differentiating between ^•OH and ^•O₂⁻ that PGA and PF can produce ^•O₂⁻ by transforming ^•OH. The potential physiological implications of this unique property are discussed. We propose that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H₂O₂ being a downstream secondary messenger.Key words: polygalacturonic acid, pectin, superoxide, hydrogen peroxide, apoplast, redox signalingPGA/OGA/PF represent apoplastic signaling molecules involved in defense regulation.^1–5 For example, they induce de novo enzyme synthesis in the wound-inducible defense reaction⁶ and increase the resistance of plants to pathogens.⁷ However, the underlying mechanisms behind such processes are lacking. Aldington et al.⁸ have postulated that OGAs do not act through a receptor, but rather they owe their activity to some specific physical property. Pertinent to this is the fact that there is a broad range of active OGA structures.⁵ In addition, it has been reported that methylated OGAs are not able to trigger signaling pathways that are activated by OGAs possessing ‘free’ carboxyl groups.^9,10 In contrast to this concept, several research groups have showed that PGA/OGA/PF bind to wall-associated kinases (WAK1 and WAK2).^11–15 However, potential effects of PGA/OGA/PF on the activity of WAK1 or WAK2 have not been observed to date. We propose here that the specific property proposed by Aldington and co-workers,⁸ is in fact the ability of the polymers of galacturonic acid (PGA/OGA/PF) to produce ^•O₂⁻. By taking into account previously proposed mechanisms of reaction of PGA with ^•OH,^16,17 and thermodynamic properties of species potentially involved in the reaction,¹⁸ we hypothesized that PGA could transform the ^•OH radical into ^•O₂⁻. To test our hypothesis we investigated the effects of PGA and pectin on radical production in two different ^•OH-generating systems using EPR spectroscopy with the DEPMPO spin-trap capable of differentiating between ^•OH and ^•O₂⁻.¹⁹The results presented in Figure 1 document the ability of PGA to transform ^•OH to ^•O₂⁻. In addition, our experimental approach showed that pectin shares the ^•O₂⁻-producing ability of its constituent PGA. In the control Fenton system (Fe²⁺ + H₂O₂ → ^•OH + OH⁻ + Fe³⁺) only ^•OH radical was produced (Fig. 1A). However in the presence of PGA or pectin, a significant production of ^•O₂⁻ was detected. Haber-Weiss-like reaction (^•O₂⁻ + H₂O₂ → ^•OH + OH⁻ + O₂) generated ^•OH radical, accompanied by a low level of ^•O₂⁻ (Fig. 1B). The supplementation of PGA or pectin to this system led to sole or pronounced production of ^•O₂⁻, respectively. Under the same experimental settings, no ^•O₂⁻ production was observed for other two major extracellular carbohydrates—cellulose and mannan (Fig. 2).Open in a separate windowFigure 1The ability of PGA and pectin to transform ^•OH radical into ^•O₂⁻. Presented are characteristic EPR spectra of adducts of DEPMPO with the ^•OH radical (/OH) and the ^•O₂⁻ radical (/ooh) in two ^•OH-generating systems: (A) Fenton reaction; (B) Haber-Weiss-like reaction; in the absence (control) or presence of PGA or pectin (15 mgml⁻¹ final concentration). The downward triangle represents the characteristic line of the/OH adduct. The circular symbol represents the characteristic line of the/OOH adduct. The grey lines represent the spectral simulations based on signals of DEPMPO adducts contributing to each spectrum in specific percentages [mean values from four experiments (standard deviations were <5%)].Open in a separate windowFigure 2Characteristic EPR spectra of adducts of DEPMPO with the ^•OH radical (downward triangle) and the ^•O₂⁻ radical (circular symbol) in Haber-Weiss-like ^•OH generating system in the absence (control) or presence of cellulose or mannan (15 mgml⁻¹ final concentration). No ^•O₂⁻ production can be observed in the presence of cellulose or mannan.Presented results illustrate the ability of PGA and pectin to transform ^•OH radical into ^•O₂⁻. Other carbohydrates involved in plant metabolism, such as cellulose and mannan, but also glucose and fructose,²⁰ do not show such properties. In addition, methylated PGA do not produce ^•O₂⁻ in the reaction with ^•OH, but methane,²¹ probably with ^•CH₃ radical as an intermediate.²² This implies that carboxyl groups which are characteristic for PGA play a critical role in the production of ^•O₂⁻. Zegota¹⁶ has proposed that pectin and ^•OH react to produce pectin C(5) radical, which further reacts with molecular oxygen thus forming C(5) peroxyl radical. This radical is unstable, especially at physiological pH values,¹⁷ hence it is further decomposed to carbohydrate fragment(s) and superoxide, via ^•O₂⁻-elimination.^16,17Under in vivo settings, superoxide generated in the apoplast by PGA/OGA/PF can be further dismutated by SOD to H₂O₂, which represents a crucial signaling molecule in plants.^23,24 It is very interesting that signaling properties of H₂O₂ in the plant immune response remarkably overlap with the events initiated by OGA: (1) Similarly to the inverted H₂O₂ gradient across the plant plasma membrane,²⁴ OGA has been reported to activate calcium-dependent protein kinases,²⁵ to provoke membrane depolarization with H⁺ influx and K⁺ efflux,²⁶ and to induce activation of mitogen-activated protein kinases.²⁷ (2) Both, OGA^10,28 and H₂O₂^29–31 provoke an influx of Ca²⁺ from the apoplast into the intracellular compartment. (3) It has been documented that apoplastic generation of ^•O₂⁻ and H₂O₂ follows mechanical stress and the recognition of pathogens,^4,32–34 but also the supplementation of OGAs.^35–37 In addition, it has been reported that the supplementation of OGA to plant cells leads to the increase of apoplastic and total concentration of H₂O₂.^37,38 The enlisted results obtained by others and data presented here imply that PGA/OGA/PF could represent the initiators of redox signaling cascades in stress response, with H₂O₂ being a downstream secondary messenger.Hereby-proposed mechanism of apoplastic production of H₂O₂ by PGA/OGA/PF and SOD, depends on ^•OH radicals. Hence, the central question of our hypothesis is: “Where do apoplastic ^•OH radicals come from, under in vivo conditions?”. Hydrogen peroxide is continually generated in the apoplast by NAD(P) H oxidase/SOD, cell wall peroxidase and other sources during normal metabolism, as reviewed by Neill and co-workers.²⁴ The physiological concentrations of H₂O₂ in plants are not well established,⁴⁰ but it seems that apoplastic and total (fresh weight) concentrations are similar and maintained at around 1 µM.^38–40 In the extracellular compartment, continuous generation of H₂O₂ is balanced by its degradation in ^•OH-generating Fenton reaction which involves redox active metals, such as copper and iron.⁴¹ In principle, ^•OH radicals are further removed by apoplastic ascorbate or cell wall constituents (Fig. 3A).^41–43 However, mechanical wounding (e.g., provoked by cold⁴⁴), degradation of the cell wall by pathogenic enzymes (such as polygalacturonase or pectate lyase⁴⁵) or insect chewing could release PGA/OGA/PF from the cell wall into the apoplast. The presence of PGA/OGA/PF in the apoplast related with these events could drastically change apoplastic redox poise. Positively charged redox active metals readily bind to negatively charged polymers of galacturonic acid.⁴⁶ The close proximity of PGA/OGA/PF to the site of ^•OH production could change the fate of ^•OH. Instead of being scavenged, ^•OH could react with PGA/OGA/PF, which leads to ^•O₂⁻ production and subsequent H₂O₂ re-generation (Fig. 3B). Such ‘recycling’ of H₂O₂ could result in a higher steady-state H₂O₂ concentration in the apoplast and consequent H₂O₂ influx, as H₂O₂ is capable of passing the membrane via passive diffusion and specific aquaporins.⁴⁷ In the stress response, H₂O₂ can traverse the membrane, induce Ca²⁺ influx or diffuse into surrounding healthy tissue to modulate enzyme activity⁴⁸ and initiate gene expression,^23,24,49 crucial for subsequent phases of defense and adaptation. To conclude, PGA/OGA/PF could provide the cell with information about the status of the cell wall affected by stressors, via H₂O₂ signaling.Open in a separate windowFigure 3Schematic presentation of potential effects of PGA/OGA/PF released from stressed wall on apoplastic redox poise and H₂O₂ and Ca²⁺ signaling cascades. (A) Redox processes in apoplast under physiological settings. (B) Redox processes in the apoplast of plant cell exposed to stress. PGA/OGA/PF are released from the cell wall into the apoplast changing the redox poise by transforming ^•OH to ^•O₂⁻ and H₂O₂ (‘H₂O₂ recycling’). This could lead to H₂O₂ accumulation, H₂O₂ influx (via diffusion or peroxiporins) or the activation of Ca²⁺ influx, which leads to the activation of different intracellular responses.     

Reductions in maize root-tip elongation by salt and osmotic stress do not correlate with apoplastic O2*- levels

Background and Aims

Experimental evidence in the literature suggests that O₂^•− produced in the elongation zone of roots and leaves by plasma membrane NADPH oxidase activity is required for growth. This study explores whether growth changes along the root tip induced by hyperosmotic treatments in Zea mays are associated with the distribution of apoplastic O₂^•−.

Methods

Stress treatments were imposed using 150 mm NaCl or 300 mm sorbitol. Root elongation rates and the spatial distribution of growth rates in the root tip were measured. Apoplastic O₂^•− was determined using nitro blue tetrazolium, and H₂O₂ was determined using 2′, 7′-dichlorofluorescin.

Key Results

In non-stressed plants, the distribution of accelerating growth and highest O₂^•− levels coincided along the root tip. Salt and osmotic stress of the same intensity had similar inhibitory effects on root elongation, but O₂^•− levels increased in sorbitol-treated roots and decreased in NaCl-treated roots.

Conclusions

The lack of association between apoplastic O₂^•− levels and root growth inhibition under hyper-osmotic stress leads us to hypothesize that under those conditions the role of apoplastic O₂^•− may be to participate in signalling processes, that convey information on the nature of the substrate that the growing root is exploring.Key words: Root tip growth, Zea mays, salt stress, reactive oxygen species, ROS     

Cigarette smoke-induced kinin B1 receptor promotes NADPH oxidase activity in cultured human alveolar epithelial cells

Pulmonary inflammation is an important pathological feature of tobacco smoke-related lung diseases. Kinin B1 receptor (B1R) is up-regulated in the rat trachea chronically exposed to cigarette-smoke. This study aimed at determining (1) whether exposure to total particulate matter of the cigarette smoke (TPM) can induce B1R in human alveolar epithelial A549 cells, (2) the mechanism of B1R induction, (3) the functionality of de novo synthesized B1R, and (4) the role of B1R in TPM-induced increase of superoxide anion (O₂^●-) level. Results show that A549 cells exposed to 10 μg/ml TPM increased O₂^●- level along with B1R (protein and mRNA) and IL-1β mRNA. In contrast, B2R and TNF-α mRNA were not affected by TPM. The increasing effect of TPM on O₂^●- level was not significantly affected by the B1R antagonist SSR240612. TPM-increased B1R mRNA was prevented by co-treatments with N-acetyl-l-cysteine (potent antioxidant), diphenyleneiodonium (NADPH oxidase inhibitor), IL-1Ra (interleukin-1R antagonist) and SN-50 (specific inhibitor of NF-kB activation) but not by pentoxifylline (TNF-α release inhibitor), indomethacin and niflumic acid (COX-1 and -2 inhibitors). Stimulation of B1R with a selective agonist (des-Arg⁹-BK, 10 μM; 30 min) increased O₂^●-production which was prevented by apocynin and diphenyleneiodonium (NADPH oxidase inhibitors). Data suggest that the increased expression of B1R by TPM in A549 cells is mediated by oxidative stress, IL-1β and NF-kB but not by cyclooxygenases or TNF-α. The amplification of O₂^●- levels via the activation of B1R-NADPH oxidase may exacerbate pulmonary inflammation and contribute to the chronicity of tobacco smoke-related lung diseases.     

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.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.     

Production of Superoxide Anions by Keratinocytes Initiates P. acnes-Induced Inflammation of the Skin

Acne vulgaris is a chronic inflammatory disorder of the sebaceous follicles. Propionibacterium acnes (P. acnes), a gram-positive anareobic bacterium, plays a critical role in the development of these inflammatory lesions. This study aimed at determining whether reactive oxygen species (ROS) are produced by keratinocytes upon P. acnes infection, dissecting the mechanism of this production, and investigating how this phenomenon integrates in the general inflammatory response induced by P. acnes. In our hands, ROS, and especially superoxide anions (O₂ ^•−), were rapidly produced by keratinocytes upon stimulation by P. acnes surface proteins. In P. acnes-stimulated keratinocytes, O₂ ^•− was produced by NAD(P)H oxidase through activation of the scavenger receptor CD36. O₂ ^•− was dismuted by superoxide dismutase to form hydrogen peroxide which was further detoxified into water by the GSH/GPx system. In addition, P. acnes-induced O₂ ^•− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O₂ ^•− with nitric oxide to form peroxynitrites. Finally, retinoic acid derivates, the most efficient anti-acneic drugs, prevent O₂ ^•− production, IL-8 release and keratinocyte apoptosis, suggesting the relevance of this pathway in humans.     

Characterization of oxygen radical formation mechanism at early cardiac ischemia

Myocardial ischemia–reperfusion (I/R) causes severe cardiac damage. Although the primary function of oxymyoglobin (Mb) has been considered to be cellular O₂ storage and supply, previous research has suggested that Mb is a potentially protective element against I/R injury. However, the mechanism of its protective action is still largely unknown. With a real-time fluorescent technique, we observed that at the onset of ischemia, there was a small burst of superoxide (O₂^•–) release, as visualized in an isolated rat heart. Thus, we hypothesize that the formation of O₂^•– correlates to Mb due to a decrease in oxygen tension in the myocardium. Measurement of O₂^•– production in a Langendorff apparatus was performed using surface fluorometry. An increase in fluorescence was observed during the onset of ischemia in hearts perfused with a solution of hydroethidine, a fluorescent dye sensitive to intracellular O₂^•–. The increase of fluorescence in the ischemic heart was abolished by a superoxide dismutase mimic, carbon monoxide, or by Mb-knockout gene technology. Furthermore, we identified that O₂^•– was not generated from the intracellular endothelium but from the myocytes, which are a rich source of Mb. These results suggest that during the onset of ischemia, Mb is responsible for generating O₂^•–. This novel mechanism may shed light on the protective role of Mb in I/R injury.     

Kinin B1 Receptor in Adipocytes Regulates Glucose Tolerance and Predisposition to Obesity

Background

Kinins participate in the pathophysiology of obesity and type 2 diabetes by mechanisms which are not fully understood. Kinin B₁ receptor knockout mice (B₁ ^−/−) are leaner and exhibit improved insulin sensitivity.

Methodology/Principal Findings

Here we show that kinin B₁ receptors in adipocytes play a role in controlling whole body insulin action and glucose homeostasis. Adipocytes isolated from mouse white adipose tissue (WAT) constitutively express kinin B₁ receptors. In these cells, treatment with the B₁ receptor agonist des-Arg⁹-bradykinin improved insulin signaling, GLUT4 translocation, and glucose uptake. Adipocytes from B₁ ^−/− mice showed reduced GLUT4 expression and impaired glucose uptake at both basal and insulin-stimulated states. To investigate the consequences of these phenomena to whole body metabolism, we generated mice where the expression of the kinin B₁ receptor was limited to cells of the adipose tissue (aP2-B₁/B₁ ^−/−). Similarly to B₁ ^−/− mice, aP2-B₁/B₁ ^−/− mice were leaner than wild type controls. However, exclusive expression of the kinin B₁ receptor in adipose tissue completely rescued the improved systemic insulin sensitivity phenotype of B₁ ^−/− mice. Adipose tissue gene expression analysis also revealed that genes involved in insulin signaling were significantly affected by the presence of the kinin B₁ receptor in adipose tissue. In agreement, GLUT4 expression and glucose uptake were increased in fat tissue of aP2-B₁/B₁ ^−/− when compared to B₁ ^−/− mice. When subjected to high fat diet, aP2-B₁/B₁ ^−/− mice gained more weight than B₁ ^−/− littermates, becoming as obese as the wild types.

Conclusions/Significance

Thus, kinin B₁ receptor participates in the modulation of insulin action in adipocytes, contributing to systemic insulin sensitivity and predisposition to obesity.     

Critical role of NADPH oxidase in neuronal oxidative damage and microglia activation following traumatic brain injury

Background

Oxidative stress is known to play an important role in the pathology of traumatic brain injury. Mitochondria are thought to be the major source of the damaging reactive oxygen species (ROS) following TBI. However, recent work has revealed that the membrane, via the enzyme NADPH oxidase can also generate the superoxide radical (O₂ ⁻), and thereby potentially contribute to the oxidative stress following TBI. The current study thus addressed the potential role of NADPH oxidase in TBI.

Methodology/Principal Findings

The results revealed that NADPH oxidase activity in the cerebral cortex and hippocampal CA1 region increases rapidly following controlled cortical impact in male mice, with an early peak at 1 h, followed by a secondary peak from 24–96 h after TBI. In situ localization using oxidized hydroethidine and the neuronal marker, NeuN, revealed that the O₂ ⁻ induction occurred in neurons at 1 h after TBI. Pre- or post-treatment with the NADPH oxidase inhibitor, apocynin markedly inhibited microglial activation and oxidative stress damage. Apocynin also attenuated TBI-induction of the Alzheimer''s disease proteins β-amyloid and amyloid precursor protein. Finally, both pre- and post-treatment of apocynin was also shown to induce significant neuroprotection against TBI. In addition, a NOX2-specific inhibitor, gp91ds-tat was also shown to exert neuroprotection against TBI.

Conclusions/Significance

As a whole, the study demonstrates that NADPH oxidase activity and superoxide production exhibit a biphasic elevation in the hippocampus and cortex following TBI, which contributes significantly to the pathology of TBI via mediation of oxidative stress damage, microglial activation, and AD protein induction in the brain following TBI.     

Visualization of Vascular Ca2+ Signaling Triggered by Paracrine Derived ROS

Oxidative stress has been implicated in a number of pathologic conditions including ischemia/reperfusion damage and sepsis. The concept of oxidative stress refers to the aberrant formation of ROS (reactive oxygen species), which include O₂^•-, H₂O₂, and hydroxyl radicals. Reactive oxygen species influences a multitude of cellular processes including signal transduction, cell proliferation and cell death^1-6. ROS have the potential to damage vascular and organ cells directly, and can initiate secondary chemical reactions and genetic alterations that ultimately result in an amplification of the initial ROS-mediated tissue damage. A key component of the amplification cascade that exacerbates irreversible tissue damage is the recruitment and activation of circulating inflammatory cells. During inflammation, inflammatory cells produce cytokines such as tumor necrosis factor-α (TNFα) and IL-1 that activate endothelial cells (EC) and epithelial cells and further augment the inflammatory response⁷. Vascular endothelial dysfunction is an established feature of acute inflammation. Macrophages contribute to endothelial dysfunction during inflammation by mechanisms that remain unclear. Activation of macrophages results in the extracellular release of O₂^•- and various pro-inflammatory cytokines, which triggers pathologic signaling in adjacent cells⁸. NADPH oxidases are the major and primary source of ROS in most of the cell types. Recently, it is shown by us and others^9,10 that ROS produced by NADPH oxidases induce the mitochondrial ROS production during many pathophysiological conditions. Hence measuring the mitochondrial ROS production is equally important in addition to measuring cytosolic ROS. Macrophages produce ROS by the flavoprotein enzyme NADPH oxidase which plays a primary role in inflammation. Once activated, phagocytic NADPH oxidase produces copious amounts of O₂^•- that are important in the host defense mechanism^11,12. Although paracrine-derived O₂^•- plays an important role in the pathogenesis of vascular diseases, visualization of paracrine ROS-induced intracellular signaling including Ca²⁺ mobilization is still hypothesis. We have developed a model in which activated macrophages are used as a source of O₂^•- to transduce a signal to adjacent endothelial cells. Using this model we demonstrate that macrophage-derived O₂^•- lead to calcium signaling in adjacent endothelial cells.     

Intraphagosomal peroxynitrite as a macrophage-derived cytotoxin against internalized Trypanosoma cruzi: consequences for oxidative killing and role of microbial peroxiredoxins in infectivity

Macrophage-derived radicals generated by the NADPH oxidase complex and inducible nitric-oxide synthase (iNOS) participate in cytotoxic mechanisms against microorganisms. Nitric oxide (^•NO) plays a central role in the control of acute infection by Trypanosoma cruzi, the causative agent of Chagas disease, and we have proposed that much of its action relies on macrophage-derived peroxynitrite (ONOO⁻ + ONOOH) formation, a strong oxidant arising from the reaction of ^•NO with superoxide radical (O₂^˙̄). Herein, we have shown that internalization of T. cruzi trypomastigotes by macrophages triggers the assembly of the NADPH oxidase complex to yield O₂^˙̄ during a 60–90-min period. This does not interfere with IFN-γ-dependent iNOS induction and a sustained ^•NO production (∼24 h). The major mechanism for infection control via reactive species formation occurred when ^•NO and O₂^˙̄ were produced simultaneously, generating intraphagosomal peroxynitrite levels compatible with microbial killing. Moreover, biochemical and ultrastructural analysis confirmed cellular oxidative damage and morphological disruption in internalized parasites. Overexpression of cytosolic tryparedoxin peroxidase in T. cruzi neutralized macrophage-derived peroxynitrite-dependent cytotoxicity to parasites and favored the infection in an animal model. Collectively, the data provide, for the first time, direct support for the action of peroxynitrite as an intraphagosomal cytotoxin against pathogens and the premise that microbial peroxiredoxins facilitate infectivity via decomposition of macrophage-derived peroxynitrite.     

Interplay Among Nitric Oxide and Reactive Oxygen Species: A Complex Network Determining Cell Survival or Death

Programmed cell death (PCD) is an integrated cellular process occurring in plant growth, development, and defense responses to facilitate normal growth and development and better survival against various stresses as a whole. As universal toxic chemicals in plant and animal cells, reactive oxygen or nitrogen species (ROS or RNS), mainly superoxide anion (O₂^−•), hydrogen peroxide (H₂O₂) or nitric oxide (^•NO), have been studied extensively for their roles in PCD induction. Physiological and genetic studies have convincingly shown their essential roles. However, the details and mechanisms by which ROS and ^•NO interplay and induce PCD are not well understood. Our recent study on Cupressus lusitanica culture cell death revealed the elicitor-induced co-accumulation of ROS and ^•NO and interactions between ^•NO and H₂O₂ or O₂-^• in different ways to regulate cell death. ^•NO and H₂O₂ reciprocally enhanced the production of each other whereas ^•NO and O₂^−• showed reciprocal suppression on each other''s production. It was the interaction between ^•NO and O₂-^• but not between ^•NO and H₂O₂ that induced PCD, probably through peroxynitrite (ONOO⁻). In this addendum, some unsolved issues in the study were discussed based on recent studies on the complex network of ROS and ^•NO leading to PCD in animals and plants.Key Words: cell death, nitric oxide, reactive oxygen species, interaction, posttranslational modification     

Insulin Reverses D-Glucose–Increased Nitric Oxide and Reactive Oxygen Species Generation in Human Umbilical Vein Endothelial Cells

Vascular tone is controlled by the L-arginine/nitric oxide (NO) pathway, and NO bioavailability is strongly affected by hyperglycaemia-induced oxidative stress. Insulin leads to high expression and activity of human cationic amino acid transporter 1 (hCAT-1), NO synthesis and vasodilation; thus, a protective role of insulin on high D-glucose–alterations in endothelial function is likely. Vascular reactivity to U46619 (thromboxane A₂ mimetic) and calcitonin gene related peptide (CGRP) was measured in KCl preconstricted human umbilical vein rings (wire myography) incubated in normal (5 mmol/L) or high (25 mmol/L) D-glucose. hCAT-1, endothelial NO synthase (eNOS), 42 and 44 kDa mitogen-activated protein kinases (p42/44^mapk), protein kinase B/Akt (Akt) expression and activity were determined by western blotting and qRT-PCR, tetrahydrobiopterin (BH₄) level was determined by HPLC, and L-arginine transport (0–1000 μmol/L) was measured in response to 5–25 mmol/L D-glucose (0–36 hours) in passage 2 human umbilical vein endothelial cells (HUVECs). Assays were in the absence or presence of insulin and/or apocynin (nicotinamide adenine dinucleotide phosphate-oxidase [NADPH oxidase] inhibitor), tempol or Mn(III)TMPyP (SOD mimetics). High D-glucose increased hCAT-1 expression and activity, which was biphasic (peaks: 6 and 24 hours of incubation). High D-glucose–increased maximal transport velocity was blocked by insulin and correlated with lower hCAT-1 expression and SLC7A1 gene promoter activity. High D-glucose–increased transport parallels higher reactive oxygen species (ROS) and superoxide anion (O₂ ^•–) generation, and increased U46619-contraction and reduced CGRP-dilation of vein rings. Insulin and apocynin attenuate ROS and O₂ ^•– generation, and restored vascular reactivity to U46619 and CGRP. Insulin, but not apocynin or tempol reversed high D-glucose–increased NO synthesis; however, tempol and Mn(III)TMPyP reversed the high D-glucose–reduced BH₄ level. Insulin and tempol blocked the high D-glucose–increased p42/44^mapk phosphorylation. Vascular dysfunction caused by high D-glucose is likely attenuated by insulin through the L-arginine/NO and O₂ ^•–/NADPH oxidase pathways. These findings are of interest for better understanding vascular dysfunction in states of foetal insulin resistance and hyperglycaemia.     

NADPH Oxidase Limits Innate Immune Responses in the Lungs in Mice

Background

Chronic granulomatous disease (CGD), an inherited disorder of the NADPH oxidase in which phagocytes are defective in generating superoxide anion and downstream reactive oxidant intermediates (ROIs), is characterized by recurrent bacterial and fungal infections and by excessive inflammation (e.g., inflammatory bowel disease). The mechanisms by which NADPH oxidase regulates inflammation are not well understood.

Methodology/Principal Findings

We found that NADPH oxidase restrains inflammation by modulating redox-sensitive innate immune pathways. When challenged with either intratracheal zymosan or LPS, NADPH oxidase-deficient p47^phox−/− mice and gp91^phox-deficient mice developed exaggerated and progressive lung inflammation, augmented NF-κB activation, and elevated downstream pro-inflammatory cytokines (TNF-α, IL-17, and G-CSF) compared to wildtype mice. Replacement of functional NADPH oxidase in bone marrow-derived cells restored the normal lung inflammatory response. Studies in vivo and in isolated macrophages demonstrated that in the absence of functional NADPH oxidase, zymosan failed to activate Nrf2, a key redox-sensitive anti-inflammatory regulator. The triterpenoid, CDDO-Im, activated Nrf2 independently of NADPH oxidase and reduced zymosan-induced lung inflammation in CGD mice. Consistent with these findings, zymosan-treated peripheral blood mononuclear cells from X-linked CGD patients showed impaired Nrf2 activity and increased NF-κB activation.

Conclusions/Significance

These studies support a model in which NADPH oxidase-dependent, redox-mediated signaling is critical for termination of lung inflammation and suggest new potential therapeutic targets for CGD.     

Evidence on the Formation of Singlet Oxygen in the Donor Side Photoinhibition of Photosystem II: EPR Spin-Trapping Study

When photosystem II (PSII) is exposed to excess light, singlet oxygen (¹O₂) formed by the interaction of molecular oxygen with triplet chlorophyll. Triplet chlorophyll is formed by the charge recombination of triplet radical pair ³[P680^•+Pheo^•−] in the acceptor-side photoinhibition of PSII. Here, we provide evidence on the formation of ¹O₂ in the donor side photoinhibition of PSII. Light-induced ¹O₂ production in Tris-treated PSII membranes was studied by electron paramagnetic resonance (EPR) spin-trapping spectroscopy, as monitored by TEMPONE EPR signal. Light-induced formation of carbon-centered radicals (R^•) was observed by POBN-R adduct EPR signal. Increased oxidation of organic molecules at high pH enhanced the formation of TEMPONE and POBN-R adduct EPR signals in Tris-treated PSII membranes. Interestingly, the scavenging of R^• by propyl gallate significantly suppressed ¹O₂. Based on our results, it is concluded that ¹O₂ formation correlates with R^• formation on the donor side of PSII due to oxidation of organic molecules (lipids and proteins) by long-lived P680^•+/TyrZ^•. It is proposed here that the Russell mechanism for the recombination of two peroxyl radicals formed by the interaction of R^• with molecular oxygen is a plausible mechanism for ¹O₂ formation in the donor side photoinhibition of PSII.     

A Missing PD-L1/PD-1 Coinhibition Regulates Diabetes Induction by Preproinsulin-Specific CD8 T-Cells in an Epitope-Specific Manner

Coinhibitory PD-1/PD-L1 (B7-H1) interactions provide critical signals for the regulation of autoreactive T-cell responses. We established mouse models, expressing the costimulator molecule B7.1 (CD80) on pancreatic beta cells (RIP-B7.1 tg mice) or are deficient in coinhibitory PD-L1 or PD-1 molecules (PD-L1^−/− and PD-1^−/− mice), to study induction of preproinsulin (ppins)-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD) by DNA-based immunization. RIP-B7.1 tg mice allowed us to identify two CD8 T-cell specificities: pCI/ppins DNA exclusively induced K^b/A_12–21-specific CD8 T-cells and EAD, whereas pCI/ppinsΔA_12–21 DNA (encoding ppins without the COOH-terminal A_12–21 epitope) elicited K^b/B_22–29-specific CD8 T-cells and EAD. Specific expression/processing of mutant ppinsΔA_12–21 (but not ppins) in non-beta cells, targeted by intramuscular DNA-injection, thus facilitated induction of K^b/B_22–29-specific CD8 T-cells. The A_12–21 epitope binds K^b molecules with a very low avidity as compared with B_22–29. Interestingly, immunization of coinhibition-deficient PD-L1^−/− or PD-1^−/− mice with pCI/ppins induced K^b/A_12–21-monospecific CD8 T-cells and EAD but injections with pCI/ppinsΔA_12–21 did neither recruit K^b/B_22–29-specific CD8 T-cells into the pancreatic target tissue nor induce EAD. PpinsΔA_12–21/(K^b/B_22–29)-mediated EAD was efficiently restored in RIP-B7.1⁺/PD-L1^−/− mice, differing from PD-L1^−/− mice only in the tg B7.1 expression in beta cells. Alternatively, an ongoing beta cell destruction and tissue inflammation, initiated by ppins/(K^b/A_12–21)-specific CD8 T-cells in pCI/ppins+pCI/ppinsΔA_12–21 co-immunized PD-L1^−/− mice, facilitated the expansion of ppinsΔA_12–21/(K^b/B_22–29)-specific CD8 T-cells. CD8 T-cells specific for the high-affinity K^b/B_22–29- (but not the low-affinity K^b/A_12–21)-epitope thus require stimulatory ´help from beta cells or inflamed islets to expand in PD-L1-deficient mice. The new PD-1/PD-L1 diabetes models may be valuable tools to study under well controlled experimental conditions distinct hierarchies of autoreactive CD8 T-cell responses, which trigger the initial steps of beta cell destruction or emerge during the pathogenic progression of EAD.     

NO? Binds Human Cystathionine β-Synthase Quickly and Tightly

The hexa-coordinate heme in the H₂S-generating human enzyme cystathionine β-synthase (CBS) acts as a redox-sensitive regulator that impairs CBS activity upon binding of NO^• or CO at the reduced iron. Despite the proposed physiological relevance of this inhibitory mechanism, unlike CO, NO^• was reported to bind at the CBS heme with very low affinity (K_d = 30–281 μm). This discrepancy was herein reconciled by investigating the NO^• reactivity of recombinant human CBS by static and stopped-flow UV-visible absorption spectroscopy. We found that NO^• binds tightly to the ferrous CBS heme, with an apparent K_d ≤0.23 μm. In line with this result, at 25 °C, NO^• binds quickly to CBS (k_on ∼ 8 × 10³ m⁻¹ s⁻¹) and dissociates slowly from the enzyme (k_off ∼ 0.003 s⁻¹). The observed rate constants for NO^• binding were found to be linearly dependent on [NO^•] up to ∼ 800 μm NO^•, and >100-fold higher than those measured for CO, indicating that the reaction is not limited by the slow dissociation of Cys-52 from the heme iron, as reported for CO. For the first time the heme of human CBS is reported to bind NO^• quickly and tightly, providing a mechanistic basis for the in vivo regulation of the enzyme by NO^•. The novel findings reported here shed new light on CBS regulation by NO^• and its possible (patho)physiological relevance, enforcing the growing evidence for an interplay among the gasotransmitters NO^•, CO, and H₂S in cell signaling.     

Lactate Up-Regulates the Expression of Lactate Oxidation Complex-Related Genes in Left Ventricular Cardiac Tissue of Rats

Background

Besides its role as a fuel source in intermediary metabolism, lactate has been considered a signaling molecule modulating lactate-sensitive genes involved in the regulation of skeletal muscle metabolism. Even though the flux of lactate is significantly high in the heart, its role on regulation of cardiac genes regulating lactate oxidation has not been clarified yet. We tested the hypothesis that lactate would increase cardiac levels of reactive oxygen species and up-regulate the expression of genes related to lactate oxidation complex.

Methods/Principal Findings

Isolated hearts from male adult Wistar rats were perfused with control, lactate or acetate (20mM) added Krebs-Henseleit solution during 120 min in modified Langendorff apparatus. Reactive oxygen species (O₂ ^●-/H₂O₂) levels, and NADH and NADPH oxidase activities (in enriched microsomal or plasmatic membranes, respectively) were evaluated by fluorimetry while SOD and catalase activities were evaluated by spectrophotometry. mRNA levels of lactate oxidation complex and energetic enzymes MCT1, MCT4, HK, LDH, PDH, CS, PGC1α and COXIV were quantified by real time RT-PCR. Mitochondrial DNA levels were also evaluated. Hemodynamic parameters were acquired during the experiment. The key findings of this work were that lactate elevated cardiac NADH oxidase activity but not NADPH activity. This response was associated with increased cardiac O₂ ^●-/H₂O₂ levels and up-regulation of MCT1, MCT4, LDH and PGC1α with no changes in HK, PDH, CS, COXIV mRNA levels and mitochondrial DNA levels. Lactate increased NRF-2 nuclear expression and SOD activity probably as counter-regulatory responses to increased O₂ ^●-/H₂O₂.

Conclusions

Our results provide evidence for lactate-induced up-regulation of lactate oxidation complex associated with increased NADH oxidase activity and cardiac O₂ ^●-/H₂O₂ driving to an anti-oxidant response. These results unveil lactate as an important signaling molecule regulating components of the lactate oxidation complex in cardiac muscle.