Hydrogen-deuterium exchange in gamma-irradiated polycrystalline DL-alanine: a spin-trapping and e.s.r. study

The hydrogen-deuterium exchange reactions in gamma-irradiated DL-alanine in the solid state were investigated by spin-trapping and electron spin resonance (e.s.r) using selectively deuterated DL-alanine. Subsequent to gamma-radiolysis at 30 degrees C, polycrystalline DL-alanine was dissolved in aqueous solutions of 2-methyl-2-nitrosopropane and the extent of H-D exchange of the deamination radicals was followed by e.s.r. After formation of the deamination radicals, four exchange reactions were found to occur between the radicals and the surrounding undamaged molecules. The first reaction, which occurs between the hydrogens of the C-2 carbon of the radicals and those of the methyl groups of the neighbouring molecules, can be followed at room temperature. The three other reactions could be conveniently monitored in gamma-irradiated polycrystalline alanine at 110 degrees C. The first of the other three reactions takes place between the methyl hydrogens of the radicals and the C-2 hydrogens of nearby molecules, while the remaining processes involve exchange between the hydrogen atoms of the amino group and those on the C-2 and C-3 carbon atoms of the deamination radical.     

Hydrated electron-initiated main-chain scission in peptides: an e.s.r. and spin-trapping study.

The reactions of hydrated electrons (eaq-) with 19 tripeptides were investigated. Hydrated electrons were produced by gamma-radiolysis of aqueous peptide solutions containing sufficient sodium formate to remove hydroxyl radicals and hydrogen atoms. t-Butanol was also used to scavenge hydroxyl radicals. The short-lived radicals formed by the reactions of eaq- with the peptides were spin-trapped with t-nitrosobutane to form stable nitroxide radicals and identified by e.s.r. spectroscopy. The tripeptides studied contained two glycine residues. Following the addition of eaq- to tripeptides, C-N bond scission was observed at three sites. Cleavage occurred between the nitrogen of the ammonium group and the alpha-carbon and between the nitrogen of the peptide linkage and the adjoining alpha-carbons. The radicals corresponding to each of these three types of scission were identified. From a comparison of the radical yields of the reaction of eaq- with ala, (ala)2, and poly-DL-ananine with an average degree of polymerization of 1800, it was shown that eaq-, can react with many carbonyl groups of poly-DL-alanine, leading to main-chain scission. Analogous reactions of eaq- with proteins and enzymes may be expected to lead to loss of biological activity.     

Reappraisal of the e.p.r. signals in (post)-ischaemic cardiac tissue.

The present study was designed to measure directly, using e.p.r. spectroscopy, oxygen-derived free radicals in (post)-ischaemic or (post)-anoxic rat hearts. Rat hearts were rapidly freeze-clamped at 77 K under normoxic, anoxic, ischaemic or reperfusion conditions. The samples were measured at three different temperatures (13, 77 and 115 K) and at several microwave power levels, and were compared with isolated rat heart mitochondria. Samples were prepared both by grinding and as tissue cuts. The two preparation techniques gave identical e.p.r. results, which excludes the occurrence of grinding artifacts. No free radical signals linked to reperfusion injury were detected. Several electron transfer centres known in the mitochondrial respiratory chain were measured. The signals previously assigned to post-ischaemic reperfusion injury were found to originate from electron transfer centres of the respiratory chain, predominantly the iron-sulphur cluster S-1 in succinate dehydrogenase. The differences in signal intensity between normoxic, ischaemic and reperfused hearts were found to result from the different redox stages of these centres under the various conditions tested. These findings do not necessarily imply that oxygen-derived free radicals are not formed in cardiac tissue during (post)-ischaemic reperfusion. The constitutive background of paramagnetism from the respiratory chain, however, seriously hampers the direct detection of comparatively low concentrations of free radicals in cardiac tissue. It is therefore expedient to focus future experiments in this field on the use of spin-trapping agents.     

Photochemistry of pyrimidine bases as studied by e.s.r. and spin-trapping

The direct photoexcitation of pyrimidine bases in D2O solutions yields free radicals which could be conveniently identified by spin-trapping with 2-methyl-2-nitrosopropane. Most of the radicals formed were attributed to D-addition to one end of the 5,6 double bond. However, orotic acid and iso-orotic acid yielded N(3) centred free radicals, formed by homolytic cleavage of the N-H bond. No indication could be found for a free radical involvement in the photocleavage of cyclobutane-type pyrimidine dimers.     

Fibroxanthosarcoma of the uterine cervix: cytopathologic and histopathologic manifestations.

A case of fibroxanthosarcoma of the uterine cervix is reported with its cytopathologic manifestations. The cellular features of two cell populations characterized by atypical cells of fibroblastic and histiocytic types, suggests the correct diagnosis. The possibility of uterine sarcoma must be considered in the differential diagnosis of bizarre and unusual cytologic findings.     

The oxidation of some polysaccharides by the hydroxyl radical: an e.s.r. investigation

E.s.r. Experiments employing a flow system in conjunction with the TiIII-H2O2 couple show that dextrans react with the hydroxyl radical (HO.) via indiscriminate attack (except that abstraction of hydrogen atoms from carbons which are both linked by glycosidic bonds and included in the pyranose ring may be inhibited, possibly for steric reasons). Acid- and base-catalysed transformations of first-formed radicals have been demonstrated; the suggestion that such reactions can lead to glycosidic cleavage is supported by viscosity studies which confirm the pH-dependence of radical-initiated degradation. For galacturonan and related compounds, e.s.r. results indicate that reaction with HO. proceeds preferentially via abstraction of the hydrogen on the carbon adjacent to the carboxyl group. The crucial step in the subsequent degradation pathway probably involves a pH-independent rearrangement.     

Comparison of radiolysis products of thymine and thymidine with e.s.r. results.

Radicals determined by e.s.r. spectrometry of irradiated thymine or thymidine and radiolytic products generated under tha ction of gamma rays in aerated aqueous solutions have been compared. This comparison lies mainly in the fact that a radical R gives rapidly the corresponding peroxide ROOH. The authors have isolated and characterized twenty peroxides, i.e., the four isomers cis (-), cis (+), trans (-), trans(+) of 6-hydroperoxy-5-hydroxy-5,6-dihydrothymidine; the four isomers cis (-), cis (+), trans (-), trans (+) of 5-hydroperoxy-6-hydroxy-5,6-dihydrothymidine; 5-hydroperoxy-2-deoxyuridin;cis and trans 6-hydroperoxy-5-hydroxy-5,6-dihydrothymine; cis and trans 5-hydroperoxy-6-hydroxy-5,6-dihydrothymine; 5-hydroperoxymethyl-uracil; 5-hydroperoxy-5,6-dihydrothymine;cis and trans 6-hydroperoxy-5,6-dihydrothymine; 5-hydroperoxy-5-methyl barbituric acid; 5-hydroperoxy-5-methyl hydantoin; trans 5,6-dihydroperoxy-5,6-dihydrothymine. Most of thethymine and thymidine radicals hypothesized or described in the literature were correlated to these peroxides. However, the presence of certain peroxides could not be explained by recognized radicals. Taking advantage of this fact, the existence of new thymine or thymidine radicals so far unknown can be predicted.     

On the e.s.r. determination of radical yields in X-irradiated amino acids.

Studies of the temperature dependence of the areas under the e.s.r. absorption spectra of X-ray-induced free radicals in amino acids at various power levels and of power saturation at different temperatures are reported. They indicate that power saturation is responsible for the anomalous Curie--Weiss behaviour previously reported. The consequences of power saturation to the e.s.r. determination of radical yields are discussed. The effect of impurity e.s.r. signals in amino acids on quantitative e.s.r. determinations is also discussed.     

OH-induced free radicals in purine nucleosides and their homopolymers: e.s.r. and spin-trapping with 2-methyl-2-nitrosopropane

Free radicals produced by X-irradiation of N2O-saturated aqueous solutions of purine nucleosides (2'-deoxyadenosine, adenosine, 2'-deoxyguanosine, 3'-deoxyadenosine, guanosine and inosine) and the corresponding homopolymers (poly A and poly I) have been investigated by the technique of spin-trapping and e.s.r. spectroscopy. 2-Methyl-2-nitrosopropane was used as a spin-trap. For 2'-deoxyadenosine and 2'-deoxyguanosine, the resulting spin-adducts were separated by Bio-Gel P-2 column chromatography and analysed by e.s.r. spectroscopy. For homopolymers, e.s.r. spectra were recorded at 50 degrees C after enzymatic digestion to obtain signals with narrower line width. The e.s.r. signal consisting of only a primary triplet without further splittings, which is consistent with assignment to the trapping of an H-abstraction radical at the C4' position of the sugar moiety, was observed in all cases. For 2'-deoxyguanosine an e.s.r. signal consisting of a secondary triplet was observed. Examinations using other spin-trapping reagents such as PBN, 4-PyOBN and DMPO provided no positive evidence supporting the proposal that this was due to an alpha-nitrogen. The e.s.r. signal consisting of a secondary doublet which further splits into a doublet was observed for 2'-deoxyadenosine, adenosine, 3'-deoxyguanosine, 2'-deoxyguanosine, and inosine, and tentatively associated with a radical centered in the sugar moiety.