Research ReportNitric Oxide Diffusion in Membranes Determined by Fluorescence Quenching
Abstract
Quenching of pyrene derivative fluorescence by nitric oxide was used to evaluate the apparent diffusion coefficients of nitric oxide in artificial and biological membranes. The apparent second-order quenching constants of nitric oxide were obtained from Stern–Volmer plots using methyl- and undecylpyrene derivatives incorporated into liposomes and erythrocyte plasma membranes in order to assess the ability of nitric oxide to interact with molecules located at different positions in the membrane. Diffusion coefficients were estimated from the determined second-order quenching constants and compared to that of oxygen obtained under the same conditions. Oxygen and nitric oxide presented similar diffusional behavior in agreement with their similarity in structures, with the differences observed attributable to the higher lipophilicity of oxygen compared to nitric oxide. In solution, both showed the same quenching efficiency while in liposomes and erythrocyte ghosts oxygen diffusion was twice that of nitric oxide (kO2/kNO= 2). Nitric oxide diffusion coefficients determined at 20°C ranged from 1.3 × 10−5cm2s−1in liposomes to 0.4 × 10−5cm2s−1in surface erythrocyte plasma membranes. Both nitric oxide and oxygen had larger quenching constants for the undecyl derivative compared to the methylpyrene compound incorporated into erythrocyte plasma membranes, indicating an increased solubility of both gases toward the center of the membrane.
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Plasma-generated nitric oxide water: A promising strategy to combat bacterial dormancy (VBNC state) in environmental contaminant Micrococcus luteus
2024, Journal of Hazardous MaterialsThe viable but non-culturable (VBNC) is an inactive state, and certain bacteria can enter under adverse conditions. The VBNC state challenges the environment, food safety, and public health since VBNCs may resuscitate and pose a risk to human health. The aim of this study was to investigate the effect of plasma-generated nitric oxide water (PG-NOW) on airborne contaminant Micrococcus luteus (M. luteus) and examine its potential to induce the VBNC state. The essential conditions for bacteria to enter VBNC state are low metabolic activity and rare or no culturable counts. The results indicated that PG-NOW effectively eliminates M. luteus, and the remaining bacteria are in culturable condition. Moreover, the conventional cultured-based method combined with a propidium iodide monoazide quantitative PCR (PMAxxTM-qPCR) showed no significant VBNC induction and moderate culturable counts. Results from the qPCR revealed that gene levels in PG-NOW treated bacteria related to resuscitation-promoting factors, amino acid biosynthesis, and fatty acid metabolism were notably upregulated. PG-NOW inactivated M. luteus showed negligible VBNC formation and alleviated infection ability in lung cells. This study provides new insights into the potential use of PG-NOW reactive species for the prevention and control of the VBNC state of M. luteus.
Thiols in blood
2022, Redox Chemistry and Biology of ThiolsCirculation through different vascular beds continuously exposes blood components to tissue-derived oxidants. To counteract this continuous challenge, blood counts on a fast and effective set of antioxidant systems. Circulating thiol-containing molecules are key players in blood antioxidant systems. In red blood cells, the peroxiredoxin/thioredoxin/thioredoxin reductase system effectively contributes to the removal of hydrogen peroxide and peroxynitrite. These cells also retain a high concentration of reduced glutathione due to the concerted action of the enzymes dedicated to glutathione biosynthesis and homeostasis. The glutathione/glutathione peroxidase/glutathione reductase system contributes mainly to the removal of lipid hydroperoxides, while glutathione transferase and glutaredoxin participate in detoxification and repairing pathways. On the other hand, plasma is relatively poor in antioxidants—the single reduced cysteine of albumin is the most abundant thiol in plasma and a preferential target of oxidants in this milieu.
Robustness of nitric oxide detoxification to nitrogen starvation in Escherichia coli requires RelA
2021, Free Radical Biology and MedicineReactive nitrogen species and nutrient deprivation are two elements of the immune response used to eliminate pathogens within phagosomes. Concomitantly, pathogenic bacteria have evolved defense systems to cope with phagosomal stressors, which include enzymes that detoxify nitric oxide (•NO) and respond to nutrient scarcity. A deeper understanding of how those defense systems are deployed under adverse conditions that contain key elements of phagosomes will facilitate targeting of those systems for therapeutic purposes. Here we investigated how Escherichia coli detoxifies •NO in the absence of useable nitrogen, because nitrogen availability is limited in phagosomes due to the removal of nitrogenous compounds (e.g., amino acids). We hypothesized that nitrogen starvation would impair •NO detoxification by E. coli because it depresses translation rates and the main E. coli defense enzyme, Hmp, is synthesized in response to •NO. However, we found that E. coli detoxifies •NO at the same rate regardless of whether useable nitrogen was present. We confirmed that the nitrogen in •NO and its autoxidation products could not be used by E. coli under our experimental conditions, and discovered that •NO eliminated differences in carbon and oxygen consumption between nitrogen-replete and nitrogen-starved cultures. Interestingly, E. coli does not consume measurable extracellular nitrogen during •NO stress despite the need to translate defense enzymes. Further, we found that RelA, which responds to uncharged tRNA, was required to observe the robustness of •NO detoxification to nitrogen starvation. These data demonstrate that E. coli is well poised to detoxify •NO in the absence of useable nitrogen and suggest that the stringent response could be a useful target to potentiate the antibacterial activity of •NO.
Metabolic pathways of Chlorella sp. cells induced by exogenous spermidine against nitric oxide damage from coal-fired flue gas
2021, Bioresource TechnologyTo protect microalgae that are used for photosynthetic CO2 fixation against high NO concentrations from coal-fired flue gas, 500 μM exogenous spermidine was added into Chlorella sp. solution resulting in an elevation of biomass yield by 30.5% under 327 ppm NO. Metabolomics, proteomics and enzyme activities were analyzed, revealing three effects of spermidine on Chlorella sp. resistance to NO stress. First, spermidine induced NO fixation in amino acids and their metabolites, mainly in form of 5-oxoproline (1.51-fold), which occurred through intracellular conversion reactions between citrulline and arginine. Accordingly, cellular respiration was strengthened along with a weakened NO inhibition, which enhanced active transport with ATP consumption. Second, spermidine guarded Chlorella sp. against peroxidation damage by improving activity of antioxidant enzymes. Finally, it protected the photosynthetic system of Chlorella sp. by increasing abundance of related enzymes to enhance carbon fixation. Thus exogenous spermidine improved biomass production against NO environment.
Free radical-dependent inhibition of prostaglandin endoperoxide H Synthase-2 by nitro-arachidonic acid
2019, Free Radical Biology and MedicineProstaglandin endoperoxide H synthase (PGHS) is a heme-enzyme responsible for the conversion of arachidonic acid (AA) to prostaglandin H2 (PGH2). PGHS have both oxygenase (COX) and peroxidase (POX) activities and is present in two isoforms (PGHS-1 and -2) expressed in different tissues and cell conditions. It has been reported that PGHS activity is inhibited by the nitrated form of AA, nitro-arachidonic acid (NO2AA), which in turn could be synthesized by PGHS under nitro-oxidative conditions. Specifically, NO2AA inhibits COX in PGHS-1 as well as POX in both PGHS-1 and -2, in a dose and time-dependent manner. NO2AA inhibition involves lowering the binding stability and displacing the heme group from the active site. However, the complete mechanism remains to be understood. This review describes the interactions of PGHS with NO2AA, focusing on mechanisms of inhibition and nitration. In addition, using a novel approach combining EPR-spin trapping and mass spectrometry, we described possible intermediates formed during PGHS-2 catalysis and inhibition. This literature revision as well as the results presented here strongly suggest a free radical-dependent inhibitory mechanism of PGHS-2 by NO2AA. This is of relevance towards understanding the underlying mechanism of inhibition of PGHS by NO2AA and its anti-inflammatory potential.
Detection and quantification of nitric oxide-derived oxidants in biological systems
2019, Journal of Biological ChemistryCitation Excerpt :The partition coefficient in membrane models and human low-density lipoprotein is 4.4–3.4 at 25 °C (14). The diffusion through cell membranes is very rapid (14–17). The permeability coefficients of lipid membranes to NO• range from 18 to 73 cm s−1 (15, 17), similar to that of an equally thick layer of water.
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