Participation of nitric oxide reductase in survival of Pseudomonas aeruginosa in LPS-activated macrophages

Nitric oxide (NO) plays a crucial role in the antimicrobial activity of host defense systems. We investigated the function of Pseudomonas aeruginosa NO reductase as a detoxifying enzyme in phagocytes. We found that the growth of the NO reductase-deficient mutant of P. aeruginosa under a microaerobic condition was inhibited by the exogenous NO. Furthermore, the intracellular survival assay within the NO-producing RAW 264.7 macrophages revealed that the wild-type strain survived longer than the NO reductase-deficient mutant. These results suggest that the P. aeruginosa NO reductase may contribute to the intracellular survival by acting as a counter component against the host's defense systems.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Nitric oxide synthase immunoreactivity in the nematode Trichinella britovi. Evidence for nitric oxide production by the parasite

Nitric oxide has been extensively studied as an effector molecule of the host immune response against both protozoa and helminths, but parasites can also produce this molecule, through the action of nitric oxide (NO) synthases or NO synthases-like enzymes. The aim of this study was to verify the possible production of NO by Trichinella britovi L(1) larvae and the enzymes involved in this process. The NO synthase immunoreactivity and putative nitric oxide synthase-activity was analysed using antibodies to mammalian NO synthase III and to nitrotyrosine with immunohistochemistry, gold immunocytochemistry and immunoblot analysis and NADPH-diaphorase histochemistry. Our results show that T. britovi L(1) larvae possess an enzymatic activity capable of producing NO. The localisation of this activity, according to the NADPH-diaphorase histochemistry, is both at the cuticular and the internal level. This localisation is confirmed by nitrotyrosine immunohistochemistry both under optical and electron microscopy. Using the NO synthase III antibody, a similar pattern of labelling was found: in particular, electron microscopy showed a localisation of this immunoreactivity in the cuticle and in the stichocytes, where only the alpha2 granules contained gold particles, mainly concentrated at their periphery. Four polypeptides reacting to the NO synthase III antibody are revealed by Western blotting. Their molecular weight ranged from 38 to 50 kDa. A significant reaction of the anti-nitrotyrosine antibody to polypeptides 95, 60, 48 and 39 kDa from the same sample suggested the presence of different nitrosylated proteins.     

Arsenic-induced oxidative stress in the common bean legume,Phaseolus vulgaris L. seedlings and its amelioration by exogenous nitric oxide

The adverse effects of arsenic (As) toxicity on seedling growth, root and shoot anatomy, chlorophyll and carotenoid contents, root oxidizability (RO), antioxidant enzyme activities, H₂O₂ content, lipid peroxidation and electrolyte leakage (EL%) in common bean (Phaseolus vulgaris L.) were investigated. The role of exogenous nitric oxide (NO) in amelioration of As-induced inhibitory effect was also evaluated using sodium nitroprusside (100 μM SNP) as NO donor and 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (200 μM PTIO) as NO scavenger in different combinations with 50 μM As. As-induced growth inhibition was associated with marked anomalies in anatomical features, reduction in pigment composition, increased RO and severe perturbations in antioxidant enzyme activities. While activity of superoxide dismutase and catalase increased, levels of ascorbate peroxidase, dehydroascorbate reductase and glutathione reductase decreased significantly and guaiacol peroxidase remained normal. The over-accumulation of H₂O₂ content along with high level of lipid peroxidation and electrolyte leakage indicates As-induced oxidative damage in P. vulgaris seedlings with more pronounced effect on the roots than the shoots. Exogenous addition of NO significantly reversed the As-induced oxidative stress, maintaining H₂O₂ in a certain level through balanced alterations of antioxidant enzyme activities. The role of NO in the process of amelioration has ultimately been manifested by significant reduction of membrane damage and improvement of growth performance in plants grown on As + SNP media. Onset of oxidative stress was more severe after addition of PTIO, which confirms the protective role of NO against As-induced oxidative damage in P. vulgaris seedlings.     

Integrated transcriptomic and proteomic analysis of the global response of Wolbachia to doxycycline-induced stress

The bacterium Wolbachia (order Rickettsiales), representing perhaps the most abundant vertically transmitted microbe worldwide, infects arthropods and filarial nematodes. In arthropods, Wolbachia can induce reproductive alterations and interfere with the transmission of several arthropod-borne pathogens. In addition, Wolbachia is an obligate mutualist of the filarial parasites that cause lymphatic filariasis and onchocerciasis in the tropics. Targeting Wolbachia with tetracycline antibiotics leads to sterilisation and ultimately death of adult filariae. However, several weeks of treatment are required, restricting the implementation of this control strategy. To date, the response of Wolbachia to stress has not been investigated, and almost nothing is known about global regulation of gene expression in this organism. We exposed an arthropod Wolbachia strain to doxycycline in vitro, and analysed differential expression by directional RNA-seq and label-free, quantitative proteomics. We found that Wolbachia responded not only by modulating expression of the translation machinery, but also by upregulating nucleotide synthesis and energy metabolism, while downregulating outer membrane proteins. Moreover, Wolbachia increased the expression of a key component of the twin-arginine translocase (tatA) and a phosphate ABC transporter ATPase (PstB); the latter is associated with decreased susceptibility to antimicrobials in free-living bacteria. Finally, the downregulation of 6S RNA during translational inhibition suggests that this small RNA is involved in growth rate control. Despite its highly reduced genome, Wolbachia shows a surprising ability to regulate gene expression during exposure to a potent stressor. Our findings have general relevance for the chemotherapy of obligate intracellular bacteria and the mechanistic basis of persistence in the Rickettsiales.     

Redox and nitric oxide homeostasis are affected in tomato (Solanum lycopersicum) roots under salinity-induced oxidative stress

The nicotinamide adenine dinucleotide phosphate (NADPH) and reduced glutathione (GSH) molecules play important roles in the redox homeostasis of plant cells. Using tomato (Solanum lycopersicum) plants grown with 120 mM NaCl, we studied the redox state of NADPH and GSH as well as ascorbate, nitric oxide (NO) and S-nitrosoglutathione (GSNO) content and the activity of the principal enzymes involved in the metabolism of these molecules in roots. Salinity caused a significant reduction in growth parameters and an increase in oxidative parameters such as lipid peroxidation and protein oxidation. Salinity also led to an overall decrease in the content of these redox molecules and in the enzymatic activities of the main NADPH-generating dehydrogenases, S-nitrosoglutathione reductase and catalase. However, NO content as well as gluthahione reductase and glutathione peroxidase activity increased under salinity stress. These findings indicate that salinity drastically affects redox and NO homeostasis in tomato roots. In our view, these molecules, which show the interaction between ROS and RNS metabolisms, could be excellent parameters for evaluating the physiological conditions of plants under adverse stress conditions.     

Characterizing the effect of nitrosative stress in Saccharomyces cerevisiae

Nitrosative stress has various pathophysiological implications. We here present a detailed characterization on the effect of nitrosative stress in Saccharomyces cerevisiae wild-type (Y190) and its isogenic flavohemoglobin mutant (Δyhb1) strain grown in presence of non fermentable carbon source. On addition of sub-toxic dose of nitrosating agent both the strains showed microbiostatic effect. Cellular respiration was found to be significantly affected in both the strains in presence sodium nitroprusside. Although there was no alteration in mitochondrial permeability potential changes and reactive oxygen species production in both the strains but the cellular redox status is differentially regulated in Δyhb1 strain both in cytosol and in mitochondria indicating cellular glutathione is the major player in absence of flavohemoglobin. We also found important role(s) of various redox active enzymes like glutathione reductase and catalase in protection against nitrosative stress. This is the first report of its kind where the effect of nitrosative stress has been evaluated in S. cerevisiae cytosol as well as in mitochondria under respiratory proficient conditions.     

The effects of anthrax lethal factor on the macrophage proteome: potential activity on nitric oxide synthases

Anthrax lethal factor (LeTx) is a critical virulence factor in toxin-challenged cells, as lethal factor (LF) cleaves mitogen-activated protein kinase kinases (MKKs), inhibiting their activity. The physiological importance of this cleavage for macrophage cytolysis remains unclear, because similar proteolysis has been also observed in LeTx-resistant macrophages. Here, we analyzed in vitro proteomic profiles of Raw264.7 lysates treated with LF. In our experiments, neuronal NO synthase (nNOS) was found to be a fragment, suggesting that LF may act on nNOS cleavage. A similar cleavage of nNOS was shown in LeTx-challenged HEK293 cells expressing nNOS by a transient transfection. However, the cleavage site on nNOS is a unique leader sequence among the NOS family and this LF-mediated cleavage was not observed in iNOS, a major NOS isoform for anti-bactericidal NO production, even though NO level in LeTx-challenged cells was dramatically reduced. Our findings suggest that LF is directly capable of cleaving cellular protein(s) other than MKKs, and that these actions potentiate to promote the cytotoxic mechanisms of anthrax.     

Inactivation of nitric oxide by cytochrome c oxidase under steady-state oxygen conditions

We have developed a respiration chamber that allows intact cells to be studied under controlled oxygen (O₂) conditions. The system measures the concentrations of O₂ and nitric oxide (NO) in the cell suspension, while the redox state of cytochrome c oxidase is continuously monitored optically. Using human embryonic kidney cells transfected with a tetracycline-inducible NO synthase we show that the inactivation of NO by cytochrome c oxidase is dependent on both O₂ concentration and electron turnover of the enzyme. At a high O₂ concentration (70 μM), and while the enzyme is in turnover, NO generated by the NO synthase upon addition of a given concentration of l-arginine is partially inactivated by cytochrome c oxidase and does not affect the redox state of the enzyme or consumption of O₂. At low O₂ (15 μM), when the cytochrome c oxidase is more reduced, inactivation of NO is decreased. In addition, the NO that is not inactivated inhibits the cytochrome c oxidase, further reducing the enzyme and lowering O₂ consumption. At both high and low O₂ concentrations the inactivation of NO is decreased when sodium azide is used to inhibit cytochrome c oxidase and decrease electron turnover.     

Nitric oxide imbalance provokes a nitrosative response in plants under abiotic stress

Nitric oxide (NO), a free radical generated in plant cells, belongs to a family of related molecules designated as reactive nitrogen species (RNS). When an imbalance of RNS takes place for any adverse environmental circumstances, some of these molecules can cause direct or indirect damage at the cellular or molecular level, promoting a phenomenon of nitrosative stress. Thus, this review will emphasize the recent progress in understanding the function of NO and its production under adverse environmental conditions.     

NO对镉胁迫下小麦根系生长发育的生理影响

为了探究外源物一氧化氮（NO）供体硝普钠（sodium nitroprusside,SNP）对Cd²⁺胁迫下小麦根系生长发育和活性氧代谢的影响,以小麦（Triticum aestivum L.）为材料,研究10 mmol/L CdCl₂胁迫下,30 μmol/L硝普钠（含一氧化氮NO）对小麦根系生长发育和活性氧代谢的影响。结果显示,外施SNP后,Cd²⁺胁迫下的小麦根长度、鲜重与干重较单独镉胁迫处理分别上升了48.0%、107.7%和87.3%,根系超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）、抗坏血酸过氧化物酶（APX）的活性分别上升了28.5%、7.4%、19.2%和9.8%,根中超氧自由基（O₂^.-）和过氧化氢（H₂O₂）的含量分别降低了80.5％和47.0％;同时外施SNP,使镉胁迫下小麦根中的可溶性糖含量和脯氨酸含量分别上升了24.7％和22.1%;使根中丙二醛（MDA）含量降低了30.2％;使根系活力上升了15.3%。因此,外源NO在一定程度上可以显著提高小麦根的抗氧化能力,增强小麦的抗逆性,缓解镉对小麦根系的毒害,进而促进小麦幼苗根系的生长发育。     

The proteome of Populus nigra woody root: response to bending

Background and Aims

Morphological and biomechanical alterations occurring in woody roots of many plant species in response to mechanical stresses are well documented; however, little is known about the molecular mechanisms regulating these important alterations. The first forest tree genome to be decoded is that of Populus, thereby providing a tool with which to investigate the mechanisms controlling adaptation of woody roots to changing environments. The aim of this study was to use a proteomic approach to investigate the response of Populus nigra woody taproot to mechanical stress.

Methods

To simulate mechanical perturbations, the taproots of 30 one-year-old seedlings were bent to an angle of 90 ° using a steel net. A spatial and temporal two-dimensional proteome map of the taproot axis was obtained. We compared the events occurring in the above-bending, central bending and below-bending sectors of the taproot.

Key Results

The first poplar woody taproot proteome map is reported here; a total of 207 proteins were identified. Spatial and temporal proteomic analysis revealed that factors involved in plant defence, metabolism, reaction wood formation and lateral root development were differentially expressed in the various sectors of bent vs. control roots, seemingly in relation to the distribution of mechanical forces along the stressed woody taproots. A complex interplay among different signal transduction pathways involving reactive oxygen species appears to modulate these responses.

Conclusions

Poplar woody root uses different temporal and spatial mechanisms to respond to mechanical stress. Long-term bending treatment seem to reinforce the defence machinery, thereby enabling the taproot to better overcome winter and to be ready to resume growth earlier than controls.     

Protective roles of nitric oxide on germination and antioxidant metabolism in wheat seeds under copper stress

Nitric oxide (NO) is a multifunctional gaseous signal in plant. In the present study, we found that pretreatment with NO could significantly improve wheat seeds germination and alleviate oxidative stress against copper toxicity. With the enhancement of copper stress, the germination percentage of wheat seeds decreased gradually. Pretreatment during wheat seed imbibition with sodium nitroprusside (SNP), an NO donor, could greatly reverse the inhibitory effect of the following copper stress to wheat seeds germination. SNP-pretreated seeds also tended to retain higher amylase activities than that of the control without SNP pretreatment. On the other hand, there was no apparent difference in the activities of esterase in wheat seeds pretreated with or without SNP. Further investigations showed that pretreatment with NO donor dramatically stimulated the activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), decreased the activities of lipoxygenases, sustained a lower level of malondialdehyde, and interfered with hydrogen peroxide (H₂O₂) excessive accumulation compared with the control, thereby enhancing the antioxidative capacity in wheat seeds under copper stress. In addition, the seed copper contents were not significant different between those pretreated with SNP and the controls, inferring that protective roles of NO was not responsible for preventing Cu uptake. Kang-Di Hu and Lan-Ying Hu contributed equally to this paper.     

Nitric oxide accumulation is required to protect against iron-mediated oxidative stress in frataxin-deficient Arabidopsis plants

Frataxin is a mitochondrial protein that is conserved throughout evolution. In yeast and mammals, frataxin is essential for cellular iron (Fe) homeostasis and survival during oxidative stress. In plants, frataxin deficiency causes increased reactive oxygen species (ROS) production and high sensitivity to oxidative stress. In this work we show that a knock-down T-DNA frataxin-deficient mutant of Arabidopsis thaliana (atfh-1) contains increased total and organellar Fe levels. Frataxin deficiency leads also to nitric oxide (NO) accumulation in both, atfh-1 roots and frataxin null mutant yeast. Abnormally high NO production might be part of the defence mechanism against Fe-mediated oxidative stress.     

Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells by stem bark of Ulmus pumila L.

This study was designed to isolate and identify a potent inhibitory compound against nitric oxide (NO) production from the stem bark of Ulmus pumila L. Ethyl acetate fraction of hot water extract registered a higher level of total phenolics (756.93 mg GAE/g) and also showed strong DPPH (IC₅₀ at 5.6 μg/mL) and ABTS (TEAC value 0.9703) radical scavenging activities than other fractions. Crude extract and its fractions significantly decreased nitrite accumulation in LPS-stimulated RAW 264.7 cells indicating that they potentially inhibited the NO production in a concentration dependent manner. Based on higher inhibitory activity, the ethyl acetate fraction was subjected to Sephadex LH-20 column chromatography and yielded seven fractions and all these fractions registered appreciable levels of inhibitory activity on NO production. The most effective fraction F1 was further purified and subjected to ¹H, ¹³C-NMR and mass spectrometry analysis and the compound was identified as icariside E₄. The results suggest that the U. pumila extract and the isolated compound icariside E₄ effectively inhibited the NO production and may be useful in preventing inflammatory diseases mediated by excessive production of NO.     

Nitric oxide is involved in light-specific responses of tomato during germination under normal and osmotic stress conditions

Background and Aims Nitric oxide (NO) is involved in the signalling and regulation of plant growth and development and responses to biotic and abiotic stresses. The photoperiod-sensitive mutant 7B-1 in tomato (Solanum lycopersicum) showing abscisic acid (ABA) overproduction and blue light (BL)-specific tolerance to osmotic stress represents a valuable model to study the interaction between light, hormones and stress signalling. The role of NO as a regulator of seed germination and ABA-dependent responses to osmotic stress was explored in wild-type and 7B-1 tomato under white light (WL) and BL. Methods Germination data were obtained from the incubation of seeds on germinating media of different composition. Histochemical analysis of NO production in germinating seeds was performed by fluorescence microscopy using a cell-permeable NO probe, and endogenous ABA was analysed by mass spectrometry. Key Results The NO donor S-nitrosoglutathione stimulated seed germination, whereas the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) had an inhibitory effect. Under WL in both genotypes, PTIO strongly suppressed germination stimulated by fluridone, an ABA inhibitor. The stimulatory effect of the NO donor was also observed under osmotic stress for 7B-1 seeds under WL and BL. Seed germination inhibited by osmotic stress was restored by fluridone under WL, but less so under BL, in both genotypes. This effect of fluridone was further modulated by the NO donor and NO scavenger, but only to a minor extent. Fluorescence microscopy using the cell-permeable NO probe DAF-FM DA (4-amino-5-methylamino-2',7'-difluorofluorescein diacetate) revealed a higher level of NO in stressed 7B-1 compared with wild-type seeds. Conclusions As well as defective BL signalling, the differential NO-dependent responses of the 7B-1 mutant are probably associated with its high endogenous ABA concentration and related impact on hormonal cross-talk in germinating seeds. These data confirm that light-controlled seed germination and stress responses include NO-dependent signalling.     

Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells

Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative-nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P ≤ 0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.     

Mechanism of strong resistance of Helicobacter pylori respiration to nitric oxide

The aim of the present work is to elucidate the mechanism by which the respiration of Helicobacter pylori but not of Escherichia coli shows a strong resistance to nitric oxide (NO). Nitric oxide strongly but reversibly inhibited the oxygen consumption by sonicated membranes from H. pylori and Triton X-100-treated cells. Although the sensitivity of the H. pylori respiration to cyanide was low, it also increased after the treatment with Triton X-100. Kinetic analyses revealed that NO was rapidly degraded by E. coli and the Triton X-100-treated H. pylori, but not by the intact H. pylori. Thus, the low sensitivity to NO might reflect the low affinity of the cytochrome c oxidase for this radical within the membrane/lipid bilayers of H. pylori. Such properties of the oxidase in H. pylori membranes may, at least in part, underlie the mechanism by which this bacterium thrives in NO-enriched gastric juice.     

Differential proteome analysis of a selected bacterial strain isolated from a high background radiation area in response to radium stress

The present study describes the response of a bacterial strain, isolated from a hot spring in an area with the highest levels of natural radiation, under radium ((226)Ra) stress. The bacterium has been characterized as a novel and efficient radium biosorbent and identified as a variant of Serratia marcescens by biochemical tests and molecular recognition. In order to gain insights into key cellular events that allow this strain to survive and undergo (226)Ra adaptation and biosorption, the strain was tested under two experimental conditions of 1000 and 6000 Bq (226)Ra stress. A proteomic approach involving two-dimensional polyacrylamide gel electrophoresis and mass spectrometry was used to identify the differentially expressed proteins under (226)Ra stress. Functional assessment of identified proteins with significantly altered expression levels revealed several mechanisms thought to be involved in (226)Ra adaptation and conferring resistant phenotype to the isolate, including general stress adaptation, anti-oxidative stress, protein and nucleic acid synthesis, energy metabolism, efflux and transport proteins. It suggests that this strain through evolution is particularly well adapted to the high background radiation environment and could represent an alternative source to remove (226)Ra from such areas as well as industrial radionuclide polluted wastewaters.     

Production of nitric oxide by murine macrophages induced by lipophosphoglycan of Leishmania major

Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is the surface glycoconjugate lipophosphoglycan (LPG). In addition, LPG is shown to be useful as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we made attempts to characterize functions of the culture supernatant, and membrane LPG isolated from metacyclic promastigotes of Leishmania major. The purification scheme included anion-exchange chromatography, hydrophobic interaction chromatography and cold methanol precipitation. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by SDS-PAGE and thin layer chromatography. The effect of mLPG and sLPG on nitric oxide (NO) production by murine macrophages cell line (J774.1A) was studied. Both sLPG and mLPG induced NO production in a dose dependent manner but sLPG induced significantly higher amount of NO than mLPG. Our results show that sLPG is able to promote NO production by murine macrophages.