Addition Of Exogenous Nitric Oxide Research Articles

Regulation and microbial response mechanism of nitric oxide to copper-containing swine wastewater treated by Pistia stratiotes

As a signaling molecule, Nitric oxide (NO) has been widely used in abiotic stress mitigation studies.Pistia stratiotes showed a good synergistic removal effect on heavy metals, nitrogen and phosphorus, but the high concentration of copper(Cu) in swine wastewater inhibited the comprehensive removal ability of Pistia stratiotes. At present, it is not clear how the addition of NO regulates the stress resistance mechanism of Pistia stratiotes to copper in swine wastewater, and the microbial response mechanism accompanying this process is not yet clear. Therefore, in the concentration range of 0.31∼4 mg·L−1Cu2+ and NO concentration of 0,0.05 and 0.1 mg L−1, the removal effect of Pistia stratiotes on copper from swine wastewater was studied. The results showed as follows: The treatment of non-available copper in groups M and H increased by 10.67% and 22.31%, respectively, compared with that in group L. The critical point of inhibiting effect of NO on growth rate was 2.03 mg·L−1Cu. By measuring three-dimensional fluorescence spectrum, combined with parallel factor analysis and principal component analysis, it was confirmed that exogenous addition of NO affected the humification degree of dissolved organic matter(DOM) and promoted the chelation of organic matter with copper. With the increase of Cu concentration, the Reyranella and Prosthecobacter with certain copper resistance gradually gained advantages. Redundancy analysis(RDA) showed that Emiticicia had a strong correlation with the removal rates of ammonia nitrogen, total phosphorus and copper in swine wastewater, while hgcI_clade had a strong correlation with the removal rates of total nitrogen. In conclusion, controlling the dosage of NO can effectively improve the tolerance and removal effect of Pistia stratiotes on copper in swine wastewater, which is of great significance for promoting the treatment and resource transformation of swine wastewater.

The involvement of nitric oxide and ethylene on the formation of endodermal barriers in response to Cd in hyperaccumulator Sedum alfredii

Nitric oxide (NO) and ethylene are both important signaling molecules which participate in numerous plant development processes and environmental stress resistance. Here, we investigate whether and how NO interacts with ethylene during the development of endodermal barriers that have major consequences for the apoplastic uptake of cadmium (Cd) in the hyperaccumulator Sedum alfredii. In response to Cd, an increased NO accumulation, while a decrease in ethylene production was observed in the roots of S. alfredii. Exogenous supplementation of NO donor SNP (sodium nitroprusside) decreased the ethylene production in roots, while NO scavenger cPTIO (2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide) had the opposite effect. The exogenous addition of NO affected the ethylene production through regulating the expression of genes related to ethylene synthesis. However, upon exogenous ethylene addition, roots retained their NO accumulation. The abovementioned results suggest that ethylene is downstream of the NO signaling pathway in S. alfredii. Regardless of Cd, addition of SNP promoted the deposition of endodermal barriers via regulating the genes related to Casparian strips deposition and suberization. Correlation analyses indicate that NO positively modifies the formation of endodermal barriers via the NO-ethylene signaling pathway, Cd-induced NO accumulation interferes with the synthesis of ethylene, leading to a deposition of endodermal barriers in S. alfredii.

Insights into the Production and Role of Nitric Oxide in the Antarctic Sea-ice Diatom Fragilariopsis cylindrus.

Nitric oxide (NO) is widely recognized as an important transmitter molecule in biological systems, from animals to plants and microbes. However, the role of NO in marine photosynthetic microbes remains unclear and even less is known about the role of this metabolite in Antarctic sea-ice diatoms. Using a combination of microsensors, microfluidic chambers, and artificial sea-ice tanks, a basic mechanistic insight into NO's dynamics within the Antarctic sea-ice diatom Fragilariopsis cylindrus was obtained. Results suggest that NO production in F.cylindrus is nitrite-dependent via nitrate reductase. NO production was abolished upon exposure to light but could be induced in the light when normal photosynthetic electron flow was disrupted. The addition of exogenous NO to cellular suspensions of F.cylindrus negatively influenced growth, disrupted photosynthesis, and altered non-photochemical dissipation mechanisms. NO production was also observed when cells were exposed to stressful salinity and temperature regimes. These results suggest that during periods of environmental stress, NO could be produced in F.cylindrus as a "stress signa" molecule.

Defective autophagy in vascular smooth muscle cells increases passive stiffness of the mouse aortic vessel wall.

Aging and associated progressive arterial stiffening are both important predictors for the development of cardiovascular diseases. Recent evidence showed that autophagy, a catabolic cellular mechanism responsible for nutrient recycling, plays a major role in the physiology of vascular cells such as endothelial cells and vascular smooth muscle cells (VSMCs). Moreover, several autophagy inducing compounds are effective in treating arterial stiffness. Yet, a direct link between VSMC autophagy and arterial stiffness remains largely unidentified. Therefore, we investigated the effects of a VSMC-specific deletion of the essential autophagy-related gene Atg7 in young mice (3.5months) (Atg7F/F SM22α-Cre+ mice) on the biomechanical properties of the aorta, using an in-house developed Rodent Oscillatory Tension Set-up to study Arterial Compliance (ROTSAC). Aortic segments of Atg7F/F SM22α-Cre+ mice displayed attenuated compliance and higher arterial stiffness, which was more evident at higher distention pressures. Passive aortic wall remodeling, rather than differences in VSMC tone, is responsible for these phenomena, since differences in compliance and stiffness between Atg7+/+ SM22α-Cre+ and Atg7F/F SM22α-Cre+ aortas were more pronounced when VSMCs were completely relaxed by the addition of exogenous nitric oxide. These observations are supported by histological data showing a 13% increase in medial wall thickness and a 14% decrease in elastin along with elevated elastin fragmentation. In addition, expression of the calcium-binding protein S100A4, which is linked to matrix remodeling, was elevated in aortic segments of Atg7F/F SM22α-Cre+ mice. Overall, these findings illustrate that autophagy exerts a crucial role in defining arterial wall compliance.

Nitric oxide-mediated regulation of sub-cellular chromium distribution, ascorbate–glutathione cycle and glutathione biosynthesis in tomato roots under chromium (VI) toxicity

Unprecedented anthropogenic activities have led to contamination of soil and water with toxic metals that present a great threat to crop yields. This situation has compelled researchers to understand metal toxicity responses in order to develop strategies to curtail toxic metal-mediated losses in crop yields. Past decade has witnessed tremendous developments with regard to the role of nitric oxide (NO) in regulating abiotic stresses including toxic metal in crop plants. However, mechanisms related with NO-mediated mitigation of metal toxicity are still less known, and thus investigation in this domain remains underway. Therefore, in this study potential of NO along with its mechanisms of action in mitigating hexavalent chromium [Cr(VI)] toxicity in tomato roots were investigated. Root length and dry weight were declined by Cr(VI) which coincided with increased accumulation of Cr. Major amount of Cr was in the cell wall fraction followed by soluble (including vacuoles) and cell organelles fraction and thus, leading to the cell death in roots. Further, Cr(VI) also declined endogenous NO by inhibiting nitric oxide synthase like activity, and down-regulated ascorbate-glutathione cycle and glutathione biosynthesis, but stimulated oxidative stress markers. In contrast, exogenous addition of NO (as a sodium nitroprusside) reduced toxic effects of Cr(VI) in tomato roots by decreasing Cr accumulation as well as triggering sequestration of Cr into vacuoles and thus collectively protect root from cell death. Moreover, NO also up-regulated ascorbate-glutathione cycle and glutathione biosynthesis, and stimulated phytochelatins, but greatly declined oxidative stress markers. Interestingly, addition of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) further worsened Cr(VI) toxicity, and Cr(VI) toxicity alleviatory effect of NO was partly reversed by the addition of c-PTIO, suggesting that NO has a crucial role in rendering Cr(VI) toxicity tolerance in tomato roots. Collectively, results suggest that NO mitigates Cr(VI) toxicity in tomato roots by reducing Cr and oxidative stress markers accumulation, triggering sequestration of Cr into vacuoles, and up-regulating ascorbate-glutathione cycle and glutathione biosynthesis, and phytochelatins.

Nitric Oxide Enhances Cytotoxicity of Lead by Modulating the Generation of Reactive Oxygen Species and Is Involved in the Regulation of Pb2+ and Ca2+ Fluxes in Tobacco BY-2 Cells.

Lead is a heavy metal known to be toxic to both animals and plants. Nitric oxide (NO) was reported to participate in plant responses to different heavy metal stresses. In this study, we analyzed the function of exogenous and endogenous NO in Pb-induced toxicity in tobacco BY-2 cells, focusing on the role of NO in the generation of reactive oxygen species (ROS) as well as Pb2+ and Ca2+ fluxes using non-invasive micro-test technology (NMT). Pb treatment induced BY-2 cell death and rapid NO and ROS generation, while NO burst occurred earlier than ROS accumulation. The elimination of NO by 2-4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) resulted in a decrease of ROS, and the supplementation of NO by sodium nitroprusside (SNP) caused an increased accumulation of ROS. Furthermore, the addition of exogenous NO stimulated Pb2+ influx, thus promoting Pb uptake in cells and aggravating Pb-induced toxicity in cells, whereas the removal of endogenous NO produced the opposite effect. Moreover, we also found that both exogenous and endogenous NO enhanced Pb-induced Ca2+ effluxes and calcium homeostasis disorder. These results suggest that exogenous and endogenous NO played a critical regulatory role in BY-2 cell death induced by Pb stress by promoting Pb2+ influx and accumulation and disturbing calcium homeostasis.

A Novel Ruthenium-based Molecular Sensor to Detect Endothelial Nitric Oxide

Nitric oxide (NO) is a key regulator of endothelial cell and vascular function. The direct measurement of NO is challenging due to its short half-life, and as such surrogate measurements are typically used to approximate its relative concentrations. Here we demonstrate that ruthenium-based [Ru(bpy)2(dabpy)]2+ is a potent sensor for NO in its irreversible, NO-bound active form, [Ru(bpy)2(T-bpy)]2+. Using spectrophotometry we established the sensor’s ability to detect and measure soluble NO in a concentration-dependent manner in cell-free media. Endothelial cells cultured with acetylcholine or hydrogen peroxide to induce endogenous NO production showed modest increases of 7.3 ± 7.1% and 36.3 ± 25.0% respectively in fluorescence signal from baseline state, while addition of exogenous NO increased their fluorescence by 5.2-fold. The changes in fluorescence signal were proportionate and comparable against conventional NO assays. Rabbit blood samples immediately exposed to [Ru(bpy)2(dabpy)]2+ displayed 8-fold higher mean fluorescence, relative to blood without sensor. Approximately 14% of the observed signal was NO/NO adduct-specific. Optimal readings were obtained when sensor was added to freshly collected blood, remaining stable during subsequent freeze-thaw cycles. Clinical studies are now required to test the utility of [Ru(bpy)2(dabpy)]2+ as a sensor to detect changes in NO from human blood samples in cardiovascular health and disease.

Scavenger Receptor BI Mediates the Uptake and Transcytosis of HDL in Brain Microvascular Endothelial Cells Independent of PDZK1 and Nitric Oxide

The vascular endothelium supplying the brain exhibits very low paracellular and transcellular permeability which protects the brain from fluctuations in ion concentrations and exposure to circulating toxins. The blood‐brain‐barrier (BBB) consists in large part of a continuous monolayer of endothelial cells joined by intercellular tight junctions that effectively occlude the intercellular space, reducing paracellular permeability. At the same time, the impermeability of the blood‐brain barrier is not absolute, allowing the regulated transport of various molecules such as high‐density lipoprotein (HDL). HDL crosses the blood‐brain barrier by transcytosis but technical limitations have made it difficult to elucidate its regulation. Elucidating the mechanisms of HDL transcytosis across the blood‐brain barrier, in particular, may be significant pathologically as its constituent apolipoprotein ApoAI has been demonstrated to confer a protective effect against Alzheimer disease (AD). Using a combination of spinning‐disc confocal and total internal reflection fluorescence microscopy, we examined the internalization and transcytosis of fluorescently labeled HDL by human primary brain microvascular endothelial cell monolayers. Using these approaches, we report that HDL internalization requires dynamin but not clathrin heavy chain and that its internalization and transcytosis are saturable. Internalized HDL partially co‐localized with the scavenger receptor BI (SR‐BI) and knockdown of SR‐BI significantly attenuated HDL internalization. However, we observed that the adaptor protein PDZK1 ‐ which is critical to HDL‐SR‐BI signaling in other tissues ‐ is not required for HDL uptake in these cells. Furthermore, inhibition of endothelial nitric oxide synthase did not prevent HDL transcytosis but increased HDL internalization while the addition of exogenous nitric oxide had no effect on internalization. Together, these data indicate that HDL transcytosis across the blood‐brain barrier involves a non‐canonical signaling pathway downstream of SR‐BI. Our findings may facilitate manipulation of HDL transcytosis to increase delivery of ApoA1 as a potential therapeutic approach for AD and to increase transport of HDL‐like synthetic particles containing therapeutic drug across the blood‐brain barrier to treat neurodegenerative diseases.Support or Funding InformationKaren Fung ‐ Ontario Early Researchers Award from Dr. Gregory Fairn

Abstract 12674: LDL Transcytosis by SR-BI Requires Signaling That is Distinct From Reverse Cholesterol Transport

Introduction: How LDL crosses an intact endothelial layer to initiate atherosclerosis is poorly understood, although it is postulated to involve LDL transcytosis. To date, technical limitations have greatly limited the study of its molecular mechanisms. We have recently devised an assay for quantifying LDL transcytosis by primary human coronary artery endothelial cells and have reported an unexpected role for the scavenger receptor SR-BI. SR-BI is best known as the HDL receptor and mediates reverse cholesterol transport; its downstream signaling involves the adaptor PDZK1 and endothelial nitric oxide synthase (eNOS). However, whether SR-BI internalization and its canonical signaling pathway are required for LDL transcytosis is unknown. Hypothesis: We hypothesized that LDL transcytosis and reverse cholesterol transport require different signaling pathways. Methods: Transcytosis of DiI-labeled LDL by primary human coronary artery endothelial cells was studied by total internal reflection fluorescence microscopy. The role of lipid rafts, PDZK1, eNOS and MAP kinases was studied by siRNA and pharmacological inhibitors. Results: Incubation with LDL induced the internalization of SR-BI, which was enriched in lipid rafts. Knockdown of caveolin-1 significantly attenuated LDL transcytosis. Coronary endothelial cells expressed low levels of PDZK1 and knockdown by siRNA had no effect on transcytosis. Similarly, inhibition of eNOS or addition of exogenous nitric oxide (NO) by NO-donors did not affect LDL transcytosis. Over-expression of G420H-SR-BI, a mutant receptor which does not perform reverse cholesterol transport, actually induced LDL transcytosis while the SR-BI inhibitor BLT-1, which prevents reverse cholesterol transport, had no effect on LDL transcytosis. Finally, inhibition of c-Jun N-terminal kinase (JNK) significantly attenuated LDL transcytosis. Conclusions: SR-BI is internalized during LDL transcytosis but PDZK1 and nitric oxide are not required. Instead, JNK kinase is involved. These data raise the possibility that reverse cholesterol transport and LDL transcytosis by SR-BI are regulated separately. These findings suggest that LDL transcytosis by SR-BI may constitute a novel therapeutic target for atherosclerosis.

P.1.g.019 - New neurotensin (8−13) analogues: hydrolytic stability and pharmacological activity

Canavanine (CAN) is non-proteinogenic aminoacid and a structural analog of arginine (Arg). Naturally, CAN occurs in legumes e.g. jack bean and is considered as a strong allelochemical. As a selective inhibitor of inducible nitric oxide synthase in mammalians, it could act as a modifier of nitric oxide (NO) concentration in plants. Modifications in the content of endogenous reactive nitrogen species (RNS) and reactive oxygen species (ROS) influence root structure and architecture, being also under hormonal control. The aim of the work was to investigate regulation of root growth in tomato (Solanum lycopersicum L. cv. Malinowy Ożarowski) seedling by application of CAN at concentration (10 and 50 μM) leading to 50% or 100% restriction of root elongation. CAN at higher concentration led to slight DNA fragmentation, increased total RNA and protein level. Decline in total respiration rate after CAN supplementation was not associated with enhanced membrane permeability. Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and ONOO− localization; determined mainly in peridermal cells and some border cells. Although, CAN resulted in low RNS production, addition of exogenous NO by usage of NO donors did not reverse its negative effect, nor recovery effect was detected after roots imbibition in Arg. To build up a comprehensive view on mode of action of CAN as root growth inhibitor, it was shown an elevated level of auxin. To summarize, we demonstrated several secondary mode of action of CAN, indicating its toxicity in plants linked to restriction in RNS formation accompanied by simultaneous overaccumulation of ROS.

Toxicity of canavanine in tomato (Solanum lycopersicum L.) roots is due to alterations in RNS, ROS and auxin levels

Canavanine (CAN) is non-proteinogenic aminoacid and a structural analog of arginine (Arg). Naturally, CAN occurs in legumes e.g. jack bean and is considered as a strong allelochemical. As a selective inhibitor of inducible nitric oxide synthase in mammalians, it could act as a modifier of nitric oxide (NO) concentration in plants. Modifications in the content of endogenous reactive nitrogen species (RNS) and reactive oxygen species (ROS) influence root structure and architecture, being also under hormonal control. The aim of the work was to investigate regulation of root growth in tomato (Solanum lycopersicum L. cv. Malinowy Ożarowski) seedling by application of CAN at concentration (10 and 50 μM) leading to 50% or 100% restriction of root elongation. CAN at higher concentration led to slight DNA fragmentation, increased total RNA and protein level. Decline in total respiration rate after CAN supplementation was not associated with enhanced membrane permeability. Malformations in root morphology (shorter and thicker roots, limited number of lateral roots) were accompanied by modification in NO and ONOO− localization; determined mainly in peridermal cells and some border cells. Although, CAN resulted in low RNS production, addition of exogenous NO by usage of NO donors did not reverse its negative effect, nor recovery effect was detected after roots imbibition in Arg. To build up a comprehensive view on mode of action of CAN as root growth inhibitor, it was shown an elevated level of auxin. To summarize, we demonstrated several secondary mode of action of CAN, indicating its toxicity in plants linked to restriction in RNS formation accompanied by simultaneous overaccumulation of ROS.

Nitric oxide inhibition of NaCl secretion in the opercular epithelium of seawater-acclimated killifish, Fundulus heteroclitus.

Nitric oxide (NO) modulates epithelial ion transport pathways in mammals, but this remains largely unexamined in fish. We explored the involvement of NO in controlling NaCl secretion by the opercular epithelium of seawater killifish using an Ussing chamber approach. Pharmacological agents were used to explore the mechanism(s) triggering NO action. A modified Biotin-switch technique was used to investigate S-nitrosation of proteins. Stimulation of endogenous NO production via the nitric oxide synthase (NOS) substrate l-arginine (2.0 mmol l-1), and addition of exogenous NO via the NO donor SNAP (10-6 to 10-4moll-1), decreased the epithelial short-circuit current (Isc). Inhibition of endogenous NO production by the NOS inhibitor l-NAME (10-4moll-1) increased Isc and revealed a tonic control of ion transport by NO in unstimulated opercular epithelia. The NO scavenger PTIO (10-5moll-1) supressed the NO-mediated decrease in Isc, and confirmed that the effect observed was elicited by release of NO. The effect of SNAP on Isc was abolished by inhibitors of the soluble guanylyl cyclase (sGC), ODQ (10-6moll-1) and Methylene Blue (10-4moll-1), revealing NO signalling via the sGC/cGMP pathway. Incubation of opercular epithelium and gill tissues with SNAP (10-4moll-1) led to S-nitrosation of proteins, including Na+/K+-ATPase. Blocking of NOS with l-NAME (10-6moll-1) or scavenging of NO with PTIO during hypotonic shock suggested an involvement of NO in the hypotonic-mediated decrease in Isc Yohimbine (10-4moll-1), an inhibitor of α2-adrenoceptors, did not block NO effects, suggesting that NO is not involved in the α-adrenergic control of NaCl secretion.

Cadmium-induced embryopathy: nitric oxide rescues teratogenic effects of cadmium.

Although Cadmium (Cd) is a well-known heavy metal pollutant and teratogen, the mechanism behind Cd-mediated teratogenicity remains unknown. Previously, we have reported of the protective role of Nitric oxide (NO), a key signaling molecule in the embryonic developmental process, against Thalidomide-induced teratogenicity. The objective of this study was to obtain a mechanistic in-sight of the antiteratogenic potential of NO against Cd-mediated teratogenicity. To achieve this goal, we first studied the effect of Cd on the vasculature of developing embryos and then we investigated whether Cd mediated its effects by interfering with the redox regulation of NO signaling in the early development milieu. We used a chick embryonic model to determine the time and dose-dependent effects of Cd and NO recovery against Cd assault. The effects of Cd and NO recovery were assessed using various angiogenic assays. Redox and NO levels were also measured. Results demonstrated that exposure to Cd at early stage of development caused multiple birth defects in the chick embryos. Exposure to Cd suppressed endogenous NO levels and cGMP signaling, inhibiting angioblast activation and subsequently impairing yolk sac vascular development. Furthermore, Cd-induced superoxide and lipid peroxidation mediated activation of proapoptotic markers p21 and p53 in the developing embryo. Cd also caused the down-regulation of FOXO1, and up-regulation of FOXO3a and Caspase 3-mediated apoptosis. Addition of exogenous NO through a NO donor was able to blunt Cd-mediated effects and restore normal vascular and embryonic development. In conclusion, Cd-mediated teratogenicity occurs as a result of impaired NO-cGMP signaling, increased oxidative stress, and the activation of apoptotic pathways. Subsequent addition of exogenous NO through NO donor negated Cd-mediated effects and protected the developing embryo.

Do β-Cells Generate Peroxynitrite in Response to Cytokine Treatment?

The purpose of this study was to determine the reactive species that is responsible for cytokine-mediated β-cell death. Inhibitors of inducible nitric oxide synthase prevent this death, and addition of exogenous nitric oxide using donors induces β-cell death. The reaction of nitric oxide with superoxide results in the generation of peroxynitrite, and this powerful oxidant has been suggested to be the mediator of β-cell death in response to cytokine treatment. Recently, coumarin-7-boronate has been developed as a probe for the selective detection of peroxynitrite. Using this reagent, we show that addition of the NADPH oxidase activator phorbol 12-myristate 13-acetate to nitric oxide-producing macrophages results in peroxynitrite generation. Using a similar approach, we demonstrate that cytokines fail to stimulate peroxynitrite generation by rat islets and insulinoma cells, either with or without phorbol 12-myristate 13-acetate treatment. When forced to produce superoxide using redox cyclers, this generation is associated with protection from nitric oxide toxicity. These findings indicate that: (i) nitric oxide is the likely mediator of the toxic effects of cytokines, (ii) β-cells do not produce peroxynitrite in response to cytokines, and (iii) when forced to produce superoxide, the scavenging of nitric oxide by superoxide is associated with protection of β-cells from nitric oxide-mediated toxicity.

Effects of exogenous NO supplied with different approaches on cadmium toxicity in lettuce seedlings

The study aimed to test the effects of sodium nitroprusside [SNP, a nitric oxide (NO) donor], supplied with different approaches on cadmium (Cd) toxicity in lettuce seedlings (Lactuca sativa) in a pot experiment. SNP (8.94 mg) was applied into Cd-contaminated soil directly or added into a capsule, a paper bag, starch-coated granules, or foliar application. Cd (50 mg kg− 1) reduced chlorophyll content, caused oxidative stress, increased Cd accumulation in roots and leaves, and inhibited the uptake of calcium (Ca), magnesium (Mg), and iron (Fe). The addition of exogenous NO in Cd-contaminated soil increased chlorophyll content, improved antioxidant enzyme activities, promoted the uptake of Ca, Mg, and Fe, reduced Cd-induced oxidative damages, and inhibited Cd transferred from roots to shoots. Moreover, SNP supplied with different approaches had varied effects on Cd tolerance of lettuce seedlings. The alleviated effect of SNP applied into soil directly was the worst, and the three SNP slow release materials had better alleviation effects on Cd toxicity. Foliar SNP application had the best effects on increasing Cd tolerance in lettuce seedlings.

Nitric oxide alleviates arsenic-induced toxic effects in ridged Luffa seedlings

Hydroponic experiments were conducted to investigate whether exogenous addition of nitric oxide (NO) as sodium nitroprusside (SNP) alleviates arsenic (As) toxicity in Luffa acutangula (L.) Roxb. seedlings. Arsenic (5 and 50 μM) declined growth of Luffa seedlings which was accompanied by significant accumulation of As. SNP (100 μM) protected Luffa seedlings against As toxicity as it declined As accumulation significantly. The photosynthetic pigments and chlorophyll fluorescence parameters such as Fv/Fm, Fv/F0, Fm/F0 and qP were decreased while NPQ was raised by As. However, the toxic effects of As on photosynthesis were significantly ameliorated by SNP. The oxidative stress markers such as superoxide radical, hydrogen peroxide and malondialdehyde (lipid peroxidation) contents were enhanced by As, however, these oxidative indices were diminished significantly in the presence of SNP. As treatment stimulated the activities of SOD and CAT while the activities of APX and GST, and AsA content and AsA/DHA ratio were decreased. Upon SNP addition, along with further rise in SOD and CAT activity, APX and GST activity, and levels of AsA and AsA/DHA ratio were restored considerably. Overall results revealed that significant accumulation of As suppressed growth, photosynthesis, APX and GST activities and decreased AsA content, hence led to the oxidative stress. However, the addition of SNP protected seedlings against As stress by regulating As accumulation, oxidative stress and antioxidant defense system.

Positive Feedback Regulation of Human Inducible Nitric-oxide Synthase Expression by Ras Protein S-Nitrosylation

The production of nitric oxide (NO) by inducible NO synthase (iNOS) regulates many aspects of physiology and pathology. The expression of iNOS needs to be tightly regulated to balance the broad ranging properties of NO. We have investigated the feedback regulation of cytokine-induced iNOS expression by NO in human cells. The pharmacological inhibition of iNOS activity reduced iNOS protein levels in response to cytokine stimulation in a human epithelial cell line (A549 cells) as well as in primary human astrocytes and bronchial epithelial cells. The addition of exogenous NO using a NO donor prevented the reduction in iNOS levels caused by blockade of iNOS activity. Examination of signaling pathways affected by iNOS indicated that NO S-nitrosylated Ras. Transfection of cells with a S-nitrosylation-resistant Ras mutant reduced iNOS protein levels, indicating a role for this Ras modification in the amplification of iNOS levels. Further, the induction of iNOS protein levels correlated with the late activation of the phosphatidylinositol 3-kinase/Akt and mammalian target of rapamycin (mTOR) pathways, and inhibition of these signaling molecules reduced iNOS levels. Altogether, our findings reveal a previously unknown regulatory pathway that amplifies iNOS expression in human cells.

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 (100μM SNP) as NO donor and 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (200μM PTIO) as NO scavenger in different combinations with 50μM 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.

Enhanced nitric oxide production during lead (Pb2 +) exposure recovers protein expression but not presynaptic localization of synaptic proteins in developing hippocampal neurons

We have previously reported that lead (Pb2+) exposure results in both presynaptic and postsynaptic changes in developing neurons as a result of inhibition of the N-methyl-d-aspartate receptor (NMDAR). NMDAR inhibition by Pb2+ during synaptogenesis disrupts downstream trans-synaptic signaling of brain-derived neurotrophic factor (BDNF) and exogenous addition of BDNF can recover the effects of Pb2+ on both presynaptic protein expression and presynaptic vesicular release. NMDAR activity can modulate other trans-synaptic signaling pathways, such as nitric oxide (NO) signaling. Thus, it is possible that other trans-synaptic pathways in addition to BDNF signaling may be disrupted by Pb2+ exposure. The current study investigated whether exogenous addition of NO could recover the presynaptic vesicular proteins lost as a result of Pb2+ exposure during synaptogenesis, namely Synaptophysin (Syn) and Synaptobrevin (Syb). We observed that exogenous addition of NO during Pb2+ exposure results in complete recovery of whole-cell Syn levels and partial recovery of Syn and Syb synaptic targeting in Pb2+-exposed neurons.

A Patatin-Like Protein Protects Toxoplasma gondii from Degradation in a Nitric Oxide-Dependent Manner

Toxoplasma gondii is an obligate intracellular parasite that uses immune cells to disseminate throughout its host. T. gondii can persist and even slowly replicate in activated host macrophages by reducing the antimicrobial effects of molecules such as nitric oxide (NO). A T. gondii patatin-like protein called TgPL1 was previously shown to be important for survival in activated macrophages. Here we show that a T. gondii mutant with a deletion of the TgPL1 gene (ΔTgPL1) is degraded in activated macrophages. This degradation phenotype is abolished by the removal of NO by the use of an inducible NO synthase (iNOS) inhibitor or iNOS-deficient macrophages. The exogenous addition of NO to macrophages results in reduced parasite growth but not the degradation of ΔTgPL1 parasites. These results suggest that NO is necessary but not sufficient for the degradation of ΔTgPL1 parasites in activated macrophages. While some patatin-like proteins have phospholipase A2 (PLA2) activity, recombinant TgPL1 purified from Escherichia coli does not have phospholipase activity. This result was not surprising, as TgPL1 contains a G-to-S change at the predicted catalytic serine residue. An epitope-tagged version of TgPL1 partially colocalized with a dense granule protein in the parasitophorous vacuole space. These results may suggest that TgPL1 moves to the parasitophorous vacuole to protect parasites from nitric oxide by an undetermined mechanism.