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81.
Olof Beck Sören Sandqvist Paul Eriksen Johan Franck Göran Palmskog 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(24):2255-2259
At present drugs of abuse testing using exhaled breath as specimen is only possible for alcohol. However, we recently discovered that using modern liquid chromatography–mass spectrometry technique amphetamine and methamphetamine is detectable in exhaled breath following intake in drug addicts. We therefore undertook to develop a method for determination of methadone in exhaled breath from patients undergoing methadone maintenance treatment. Exhaled breath was collected from 13 patients after intake of the daily methadone dose. The compounds were trapped by filtering the air through a C18 modified silica surface. After elution of any trapped methadone the extract was analysed by a combined liquid chromatography–tandem mass spectrometry method. Recovery of trapped methadone from the filter surface was 96%, no significant matrix effect was observed, and the quantification using methadone-d3 as an internal standard was accurate (<10% bias) and precise (coefficient of variation 1.6–2.0%). Methadone was indisputably identified by means of the mass spectrometry technique in exhaled breath samples from all 13 patients. Identification was based on monitoring two product ions in selected reaction monitoring mode with correct relative ratio (±20%) and correct retention time. Excretion rates ranged from 0.39 to 78 ng/min. No methadone was detected in 10 control subjects. This finding confirms that breath testing is a new possibility for drugs of abuse testing. Collection of exhaled breath specimen is likely to be more convenient and safe as compared to other matrices presently in use. 相似文献
82.
83.
Nitric oxide (NO) plays important roles in plant development, and biotic and abiotic stress responses. In a recent study, we showed that endogenous NO negatively regulates abscisic acid (ABA) signaling in guard cells by inhibiting sucrose nonfermenting 1 (SNF1)-related protein kinase 2.6 (SnRK2.6)/open stomata 1(OST1) through S-nitrosylation. Application of NO breaks seed dormancy and alleviates the inhibitory effect of ABA on seed germination and early seedling growth, but it is unclear how NO functions at the stages of seed germination and early seedling development. Here, we show that like SnRK2.6, SnRK2.2 can be inactivated by S-nitrosoglutathione (GSNO) treatment through S-nitrosylation. SnRK2.2 and the closely related SnRK2.3 are known to play redundant roles in ABA inhibition of seed germination in Arabidopsis. We found that treatment with the NO donor SNP phenocopies the snrk2.2snrk2.3 double mutant in conferring ABA insensitivity at the stages of seed germination and early seedling growth. Our results suggest that NO negatively regulates ABA signaling in germination and early seedling growth through S-nitrosylation of SnRK2.2 and SnRK2.3. 相似文献
84.
Ji Hyung Chung Jiyoung Moon Youn Sue Lee Hye-Kyung Chung Seung-Min Lee Min-Jeong Shin 《Biochemical and biophysical research communications》2014
Arginase may play a major role in the regulation of vascular function in various cardiovascular disorders by impairing nitric oxide (NO) production. In the current study, we investigated whether supplementation of the arginase inhibitor Nω-hydroxy-nor-l-arginine (nor-NOHA) could restore endothelial function in an animal model of diet-induced obesity. Arginase 1 expression was significantly lower in the aorta of C57BL/6J mice fed a high-fat diet (HFD) supplemented with nor-NOHA (40 mg kg-1/day) than in mice fed HFD without nor-NOHA. Arginase inhibition led to considerable increases in eNOS expression and NO levels and significant decreases in the levels of circulating ICAM-1. These findings were further confirmed by the results of siRNA-mediated knockdown of Arg in human umbilical vein endothelial cells. In conclusion, arginase inhibition can help restore dysregulated endothelial function by increasing the eNOS-dependent NO production in the endothelium, indicating that arginase could be a therapeutic target for correcting obesity-induced vascular endothelial dysfunction. 相似文献
85.
Serena Rinaldo Giorgio GiardinaFrancesca Cutruzzolà 《Biochemical and biophysical research communications》2014
The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 heme per monomer (cd1NiR), encoded by the nirS gene. 相似文献
86.
Vasantha Madhuri Kallakunta Andrea StaruchBulent Mutus 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Protein S-nitrosation is an important post-translational modification altering protein function. Interaction of nitric oxide with thiols is an active area of research, and is one of the mechanisms by which NO exerts its biological effects. Biotin switch assay is the method, which has been developed to identify S-nitrosated proteins. The major concern with biotin switch assay includes reducing disulfide which may lead to false positives. We report a modification of the biotin switch assay where sinapinic acid is utilized instead of ascorbate to eliminate potential artifacts in the detection of S-nitrosated proteins.Methods
The denitrosation ability of sinapinic acid was assessed by monitoring either the NO or NO2- released by chemiluminescent NO detection or by the griess assay, respectively. DTNB assay was used to compare disulfide reduction by ascorbate and sinapinic acid. Sinapinic acid and ascorbate were compared in the biotin switch detection of S-nitrosoproteins in RAW 264.7 cells ± S-nitrosocysteine (CysNO) exposure.Results
We show that sinapinic acid has the ability to denitrosate S-nitrosothiols at pH 7.0 and denitrate plus denitrosate at pHs 8 and 8.5. Unlike ascorbate, sinapinic acid degrades S-nitrosothiols, but it does not reduce disulfide bridges.Conclusions
Sinapinic acid denitrosate RSNO and does not reduce disulfides. Thus can readily replace ascorbate in detection of S-nitrosated proteins in biotin switch assay.General significance
The work described is important in view of protein S-nitrosation. In this study we provide an important modification that eliminates artifacts in widely used technique for detecting the S-nitrosoproteome, the biotin switch assay. 相似文献87.
Marek Sanak Anna GieliczKrzysztof Nagraba Marek KaszubaJagoda Kumik Andrew Szczeklik 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(21):1796-1800
Background
Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry.Objective
To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals.Methods
Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor.Results
Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E2 was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D2 products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B4 has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E4. Two main vascular prostanoids: prostacyclin and thromboxane B2 were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF2α isoprostane was increased in smokers.Conclusion
Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment. 相似文献88.
蛋白亚硝基化研究进展及其在植物抗病中的作用 总被引:1,自引:1,他引:0
蛋白亚硝基化(S-nitrosylation)是一种在一氧化氮作用下与蛋白半胱氨酸巯基共价结合,使巯基-SH转化为-SNO的反应。作为一种氧化还原依赖的翻译后调控形式,蛋白亚硝基化对多种蛋白的功能具有调节作用,越来越多的证据表明蛋白亚硝基化在植物抗病中发挥重要的作用。简要介绍了蛋白巯基亚硝基化的特点、检测方法、功能研究以及在植物抗病调节方面的最新进展。 相似文献
89.
Nitric oxide (NO) release from nitric oxide synthases (NOSs) depends on the dissociation of a ferric heme-NO product complex (FeIIINO) that forms immediately after NO is made in the heme pocket. The NOS-like enzyme of Bacillus subtilis (bsNOS) has 10-20 fold slower FeIIINO dissociation rate (kd) and NO association rate (kon) compared to mammalian NOS counterparts. We previously showed that an Ile for Val substitution at the opening of the heme pocket in bsNOS contributes to these differences. The complementary mutation in mouse inducible NOS oxygenase domain (Val346Ile) decreased the NO kon and kd by 8 and 3-fold, respectively, compared to wild-type iNOSoxy, and also slowed the reductive processing of the heme-O2 catalytic intermediate. To investigate how these changes affect steady-state catalytic behaviors, we generated and characterized the V346I mutant of full-length inducible NOS (iNOS). The mutant exhibited a 4-5 fold lower NO synthesis activity, an apparent uncoupled NADPH consumption, and formation of a heme-NO complex during catalysis that was no longer sensitive to solution NO scavenging. We found that these altered catalytic behaviors were not due to changes in the heme reduction rate or in the stability of the enzyme heme-O2 intermediate, but instead were due to the slower NO kon and kd and a slower oxidation rate of the enzyme ferrous heme-NO complex. Computer simulations that utilized the measured kinetic values confirmed this interpretation, and revealed that the V346I iNOS has an enhanced NADPH-dependent NO dioxygenase activity that converts almost 1 NO to nitrate for every NO that the enzyme releases into solution. Together, our results highlight the importance of heme pocket geometry in tuning the NO release versus NO dioxygenase activities of iNOS. 相似文献
90.
Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O2 concentrations and mitochondrial O2 consumption by high-resolution respirometry over a range of O2 concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC50 on [O2] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O2 and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O2-dependent oxidation. It also explains the non-linear dependence of IC50 on O2 concentration, and the hyperbolic increase of c50 as a function of NO concentration. 相似文献