53 research outputs found
Quantification of the 2-Deoxyribonolactone and Nucleoside 5 '-Aldehyde Products of 2-Deoxyribose Oxidation in DNA and Cells by Isotope-Dilution Gas Chromatography Mass Spectrometry: Differential Effects of gamma-Radiation and Fe2+-EDTA
The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC−MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1′-oxidation and the nucleoside 5′-aldehyde of 5′-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC−MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5′-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin γ1I, was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5′-aldehyde per 106 nt per μM in accord with its established minor 1′- and major 5′-oxidation chemistry. Calicheamicin unexpectedly caused 1′-oxidation at a low level of 10 2-deoxyribonolactone per 106 nt per μM in addition to the expected predominance of 5′-oxidation at 560 nucleoside 5′-aldehyde per 106 nt per μM. The two hydroxyl radical-mediated DNA oxidants, γ-radiation and Fe2+−EDTA, produced nucleoside 5′-aldehyde at a frequency of 57 per 106 nt per Gy (G-value 74 nmol/J) and 3.5 per 106 nt per μM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, γ-radiation and Fe2+−EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 106 nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 106 nt per μM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5′-aldehyde of 9.7 and 73 lesions per 106 nt, respectively. γ-Irradiation of the cells caused increases of 0.045 and 0.22 lesions per 106 nt per Gy, respectively, which represents a 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by γ-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between γ-radiation and Fe2+−EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.National Institute of Environmental Health Sciences (ES002109)National Center for Research Resources (U.S.) (RR023783-01)National Center for Research Resources (U.S.) (RR017905-01)National Cancer Institute (U.S.) (CA103146
Dünnschicht-chromatische Trennung einiger nahe verwandter 2-(3′,5′-Dimethyl-4′-arylazo-1′-pyrazolyl)-Benzothiazole
Investigation of the flow establishment of pre-combustion shock trains in a shock tunnel
This work is a fundamental investigation of the behavior of precombustion shock trains in the T4 Stalker Tube at the upper end of the dual-mode combustion regime. Experiments were conducted at a condition that represents flight at Mach 8 at an altitude of 26 km. The test model was a simple axisymmetric duct comprising a short diffuser, an isolator (length/diameter = 4.1), and a constant-area combustor (length/diameter = 15.7). Gaseous hydrogen was used as the fuel and was injected via six portholes equispaced around the perimeter of the duct. Time histories of the surface pressure in the model showed a pressure rise from stable supersonic combustion up to a fuel equivalence ratio of 0.9. At higher fuel levels, a precombustion shock train formed upstream of the point of fuel injection. Between equivalence ratios of 0.9 and 1.1, the shock train established itself and remained stable over a significant portion of the approximately 3 ms test window. Above an equivalence ratio of 1.1, the pressure time histories continued to rise throughout the test window and a stable flow was not established. These results indicate that short-duration facilities like the T4 Stalker Tube can be used for dual-mode combustion studies at fuel levels below a limit that will depend on the particular flow condition and model geometry
Dünnschicht-chromatische Trennung einiger nahe verwandter 2-(3′-Methyl-5′-phenyl-4′-arylazo-1′-pyrazolyl)-Benzothiazol-Derivate
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