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     

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.     

A Proposed Mechanism for the Promotion of Prion Conversion Involving a Strictly Conserved Tyrosine Residue in the β2-α2 Loop of PrPC

The transmission of infectious prions into different host species requires compatible prion protein (PrP) primary structures, and even one heterologous residue at a pivotal position can block prion infection. Mapping the key amino acid positions that govern cross-species prion conversion has not yet been possible, although certain residue positions have been identified as restrictive, including residues in the β₂-α₂ loop region of PrP. To further define how β₂-α₂ residues impact conversion, we investigated residue substitutions in PrP^C using an in vitro prion conversion assay. Within the β₂-α₂ loop, a tyrosine residue at position 169 is strictly conserved among mammals, and transgenic mice expressing mouse PrP having the Y169G, S170N, and N174T substitutions resist prion infection. To better understand the structural requirements of specific residues for conversion initiated by mouse prions, we substituted a diverse array of amino acids at position 169 of PrP. We found that the substitution of glycine, leucine, or glutamine at position 169 reduced conversion by ∼75%. In contrast, replacing tyrosine 169 with either of the bulky, aromatic residues, phenylalanine or tryptophan, supported efficient prion conversion. We propose a model based on a requirement for tightly interdigitating complementary amino acid side chains within specific domains of adjacent PrP molecules, known as “steric zippers,” to explain these results. Collectively, these studies suggest that an aromatic residue at position 169 supports efficient prion conversion.     

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.     

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.     

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.     

Ferricytochrome c Directly Oxidizes Aminoacetone to Methylglyoxal,a Catabolite Accumulated in Carbonyl Stress

Age-related diseases are associated with increased production of reactive oxygen and carbonyl species such as methylglyoxal. Aminoacetone, a putative threonine catabolite, is reportedly known to undergo metal-catalyzed oxidation to methylglyoxal, NH₄ ⁺ ion, and H₂O₂ coupled with (i) permeabilization of rat liver mitochondria, and (ii) apoptosis of insulin-producing cells. Oxidation of aminoacetone to methylglyoxal is now shown to be accelerated by ferricytochrome c, a reaction initiated by one-electron reduction of ferricytochrome c by aminoacetone without amino acid modifications. The participation of O₂ ^•− and HO^• radical intermediates is demonstrated by the inhibitory effect of added superoxide dismutase and Electron Paramagnetic Resonance spin-trapping experiments with 5,5′-dimethyl-1-pyrroline-N-oxide. We hypothesize that two consecutive one-electron transfers from aminoacetone (E₀ values = −0.51 and −1.0 V) to ferricytochrome c (E₀ = 0.26 V) may lead to aminoacetone enoyl radical and, subsequently, imine aminoacetone, whose hydrolysis yields methylglyoxal and NH₄ ⁺ ion. In the presence of oxygen, aminoacetone enoyl and O₂ ^•− radicals propagate aminoacetone oxidation to methylglyoxal and H₂O₂. These data endorse the hypothesis that aminoacetone, putatively accumulated in diabetes, may directly reduce ferricyt c yielding methylglyoxal and free radicals, thereby triggering redox imbalance and adverse mitochondrial responses.     

Integrated Analysis of COX-2 and iNOS Derived Inflammatory Mediators in LPS-Stimulated RAW Macrophages Pre-Exposed to Echium plantagineum L. Bee Pollen Extract

Oxidative stress and inflammation play important roles in disease development. This study intended to evaluate the anti-inflammatory and antioxidant potential of Echium plantagineum L. bee pollen to support its claimed health beneficial effects. The hydromethanol extract efficiently scavenged nitric oxide (^•NO) although against superoxide (O₂ ^•−) it behaved as antioxidant at lower concentrations and as pro-oxidant at higher concentrations. The anti-inflammatory potential was evaluated in LPS-stimulated macrophages. The levels of ^•NO and L-citrulline decreased for all extract concentrations tested, while the levels of prostaglandins, their metabolites and isoprostanes, evaluated by UPLC-MS, decreased with low extract concentrations. So, E. plantagineum bee pollen extract can exert anti-inflammatory activity by reducing ^•NO and prostaglandins. The extract is able to scavenge the reactive species ^•NO and O₂ ^•− and reduce markers of oxidative stress in cells at low concentrations.     

Lack of K-Dependent Oxidative Stress in Cotton Roots Following Coronatine-Induced ROS Accumulation

Coronatine [COR] is a novel type of plant growth regulator with similarities in structure and property to jasmonate. The objective of this study was to examine the relationship between increased root vitality induced by 10nM COR and reactive oxygen species scavenging under potassium (K)-replete (2.5mM) and K-deficient (0.05mM) conditions in hydroponic cultured cotton seedlings. K-replete and K-deficient conditions increased root vitality by 2.7- and 3.5-fold, respectively. COR treatment significantly decreased lipid peroxidation in cotton seedlings determined by reduction in MDA levels. These results suggest that COR improves the functioning of both enzymatic and non-enzymatic antioxidant systems. Under K-replete and K-deficient conditions, COR significantly increased the activities of antioxidant enzymes SOD (only for K-repletion), CAT, GPX, and APX comparing; COR also significantly increased DPPH-radical scavenging activity. However, COR led to 1.6- and 1.7-fold increases in superoxide anion (O₂^•-) concentrations, and 5.7- and 2.1-fold increases in hydrogen peroxide (H₂O₂) levels, respectively. Additionally, COR intensified the DAB staining of H₂O₂ and the NBT staining of O₂^•-. Therefore, our results reveal that COR-induced ROS accumulation stimulates the activities of most antioxidant enzymes but does not induce oxidative stress in cotton roots.     

Kinetics of Nitrite Reduction and Peroxynitrite Formation by Ferrous Heme in Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is a pyridoxal phosphate-dependent enzyme that catalyzes the condensation of homocysteine with serine or with cysteine to form cystathionine and either water or hydrogen sulfide, respectively. Human CBS possesses a noncatalytic heme cofactor with cysteine and histidine as ligands, which in its oxidized state is relatively unreactive. Ferric CBS (Fe(III)-CBS) can be reduced by strong chemical and biochemical reductants to Fe(II)-CBS, which can bind carbon monoxide (CO) or nitric oxide (NO^•), leading to inactive enzyme. Alternatively, Fe(II)-CBS can be reoxidized by O₂ to Fe(III)-CBS, forming superoxide radical anion (O₂^˙̄). In this study, we describe the kinetics of nitrite (NO₂⁻) reduction by Fe(II)-CBS to form Fe(II)NO^•-CBS. The second order rate constant for the reaction of Fe(II)-CBS with nitrite was obtained at low dithionite concentrations. Reoxidation of Fe(II)NO^•-CBS by O₂ showed complex kinetic behavior and led to peroxynitrite (ONOO⁻) formation, which was detected using the fluorescent probe, coumarin boronic acid. Thus, in addition to being a potential source of superoxide radical, CBS constitutes a previously unrecognized source of NO^• and peroxynitrite.     

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.     

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.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

A Histidine-Rich Extensin from Zea mays Is an Arabinogalactan Protein

Earlier we isolated a threonine-rich extensin from maize (Zea mays). Here, we report that maize cell suspension cultures yield a new extensin rich in histidine (HHRGP) that also has characteristics of arabinogalactan proteins (AGPs). Thus, chymotryptic peptide maps of anhydrous hydrogen fluoride (HF)-deglycosylated HHRGP showed repetitive motifs related to both extensins and AGPs as follows. HHRGP contains Ala-Hyp₃ and Ala-Hyp₄ repeats that may be related to the classical dicot Ser-Hyp₄ extensin motif by the single T → G (Ser → Ala) base change. Furthermore, HHRGP also contains the repetitive motif Ala-Hyp-Hyp-Hyp-His-Phe-Pro-Ser-Hyp-Hyp related to the Ser-Hyp₄-Ser-Hyp-Ser-Hyp₄ motif of P3-type dicot extensin. However, HHRGP also has AGP characteristics, notably an elevated alanine content, near sequence identity with the known Lolium AGP peptide Ser-Hyp-Hyp-Ala-Pro-Ala-Pro, the putative presence of glucuronoarabinogalactan, and precipitation by Yariv antigen, but β-elimination of arabinogalactan indicates its O-linkage to serine rather than the characteristic O-hydroxyproline link of other AGPs. Although HHRGP might be a “chimera” of two different proteins, i.e. an extensin and an AGP, this is unlikely because one can account for the apparent chimera by the codon relationships of the five common hydroxyproline-rich glycoprotein amino acid residues, Ser, Pro, Thr, Ala (TCx, CCx, ACx, GCx) and histidine (CAT or CAC), which facilitate interconversion of major motifs by single point mutations. Thus, we propose that the extensin family of wall proteins consists of a highly diversified phylogenetic series ranging from basic minimally glycosylated repetitive pro-rich proteins to the highly glycosylated acidic AGPs. To relate this diversity of form and function at the molecular level, we identified putative functional domains hypothetically involved in properties such as reptation, recognition, adhesion, intermolecular cross-linkage, and self-assembly. Not previously noted, peptide palindromes feature prominently in HHRGP: Hyp-Hyp-Ala-Ala-Asn-Ala-Ala-Hyp-Hyp and Hyp-Hyp-Hyp-His-His-His-Hyp-Hyp-Hyp; in P3: Hyp₄-Ser-Hyp-Ser-Hyp₄, and in other extensins. Such palindromes would enhance glycoprotein stereoregularity, thereby possibly promoting quasicrystalline interactions between wall components.     

Kinin B1 Receptor Enhances the Oxidative Stress in a Rat Model of Insulin Resistance: Outcome in Hypertension,Allodynia and Metabolic Complications

Background

Kinin B₁ receptor (B₁R) is induced by the oxidative stress in models of diabetes mellitus. This study aims at determining whether B₁R activation could perpetuate the oxidative stress which leads to diabetic complications.

Methods and Findings

Young Sprague-Dawley rats were fed with 10% D-Glucose or tap water (controls) for 8–12 weeks. A selective B₁R antagonist (SSR240612) was administered acutely (3–30 mg/kg) or daily for a period of 7 days (10 mg/kg) and the impact was measured on systolic blood pressure, allodynia, protein and/or mRNA B₁R expression, aortic superoxide anion (O₂ ^•−) production and expression of superoxide dismutase (MnSOD) and catalase. SSR240612 reduced dose-dependently (3–30 mg/kg) high blood pressure in 12-week glucose-fed rats, but had no effect in controls. Eight-week glucose-fed rats exhibited insulin resistance (HOMA index), hypertension, tactile and cold allodynia and significant increases of plasma levels of glucose and insulin. This was associated with higher aortic levels of O₂ ^•−, NADPH oxidase activity, MnSOD and catalase expression. All these abnormalities including B₁R overexpression (spinal cord, aorta, liver and gastrocnemius muscle) were normalized by the prolonged treatment with SSR240612. The production of O₂ ^•− in the aorta of glucose-fed rats was also measured in the presence and absence of inhibitors (10–100 µM) of NADPH oxidase (apocynin), xanthine oxidase (allopurinol) or nitric oxide synthase (L-NAME) with and without Sar[D-Phe⁸]des-Arg⁹-BK (20 µM; B₁R agonist). Data show that the greater aortic O₂ ^•− production induced by the B₁R agonist was blocked only by apocynin.

Conclusions

Activation of kinin B₁R increased O₂ ^•− through the activation of NADPH oxidase in the vasculature. Prolonged blockade of B₁R restored cardiovascular, sensory and metabolic abnormalities by reducing oxidative stress and B₁R gene expression in this model.     

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.     

Hydrogen peroxide-independent generation of superoxide catalyzed by soybean peroxidase in response to ferrous ion

It is well documented that extracellular alkalization occurs in plants under the challenges by pathogenic microbes. This may eventually induce the pH-dependent extracellular peroxidase-mediated oxidative burst at the site of microbial challenges. By employing the purified proteins of horseradish peroxidase as a model, we have recently proposed a likely role for free Fe²⁺ in reduction of ferric enzyme of plant peroxidases into ferrous intermediate and oxygen-bound form of enzyme known as Compound III which may eventually releases superoxide anion radical (O₂^•−), especially under alkaline condition, possibly contributing to the plant defense mechanism. In the present study, we employed the purified protein of soybean peroxidase (SBP) as an additional model, and examined the changes in the redox status of enzyme accompanying the generation of O₂^•− in response to Fe²⁺ under alkaline condition.     

Pulse radiolysis of aromatic amino acids — State of the art

Summary The pulse radiolysis method as well as the primary processes of water radiolysis and the spectroscopic characteristics of H, OH, HO₂/O₂ ^– and e _aq ^- are briefly presented. Subsequently, kinetic and spectroscopic data of the transients resulting from the resolved multi site attack on aromatic amino acids are discussed. The reactivity of H and e _aq ^- with the same substrates, as well as the effect of oxygen on the major radiolytic processes are reviewed. Finally, the formation of tryptophan radical cation is mentioned shortly. The presented radiation mechanisms are the fundamentals for radiolytic processes occurring in proteins, enzymes and hormones in the living cells.