Light-Induced Covalent Buffer Adducts to Histidine in a Model Protein.

PURPOSE: Light is known to induce histidine (His) oxidation and His-His crosslinking in proteins. The crosslinking is resulted from the nucleophilic attack of a His to a photooxidized His from another protein. The goal of this work is to understand if covalent buffer adducts on His residues can be generated by light through similar mechanisms in nucleophilic buffers such as Tris and His. METHODS: A model protein (DNase) was buffer exchanged into nucleophilic buffers before light exposure. Photogenerated products were characterized by tryptic peptide mapping with mass spectrometry (MS) analysis. Several buffer adductions on His residues were identified after light exposure. To understand the influencing factors of such reactions, the levels of adducts were measured for six nucleophilic buffers on all His residues in DNase. RESULTS: The levels of adducts were found to correlate with the solvent accessibility of the His residue. The levels of adducts also correlate with the structure of the nucleophile, especially the steric restrictions of the nucleophile. The levels of adducts can be higher than that of other His photoreaction products, including photooxidation and crosslinking. CONCLUSIONS: In nucleophilic buffers, light can induce covalently-linked adducts to His residues.

[Effect of preliminary irradiation on DNA-ase activity and DNA fragmentation degree in the nuclei of Guerin's carcinoma cells].

The effect of preliminary irradiation on DNA-ase activity and DNA fragmentation degree in the nuclei of Guerin's carcinoma was evaluated. It was found that effective growth of malignant cells involved higher concentrations of acid DNA-ase matched by decreased levels of alkaline one which in turn lowered the rates of DNA fragmentation. Low-dose preliminary irradiation stimulated relevant processes.

Photochemistry of naphthalene diimides: EPR study of free radical formation via photoredox process.

Earlier studies have shown that on exposure to UVA, hydroperoxynaphthalene diimide (IA) generates hydroxyl radicals, induces DNA strand scission, and kills cells. Here we employed electron paramagnetic resonance (EPR) and spin trapping to investigate the free radical photochemistry of IA and that of related naphthalene diimides, which are devoid of the hydroperoxyl moiety (N,N'-bis[2-methyl]-1,4,5,8-naphthaldiimide [IB], N,N'-bis[2-thiomethyl-2-methoxyethyl]-1,4,5,8-naphthaldiimide [IC]) and therefore are unable to generate hydroxyl radicals. It is shown that on UV irradiation (>300 nm) in air-free methanol or ethanol solutions all these naphthalene diimides undergo one-electron reduction to corresponding anion radicals, positively identified by EPR. With EPR and a spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO), we found that the photogeneration of the naphthalene diimide radicals is concomitant with the formation of radicals from the solvents, presumably through electron/hydrogen atom abstraction by photoactivated diimides. Irradiation of IA, IB or IC in the presence of oxygen generates superoxide, which was detected as a DMPO adduct. The high photoreactivity of IB and IC supports the notion that hydroperoxide IA can induce oxidative damage via photoprocesses that are independent of *OH generation. These observations could be pertinent to the application of naphthalene diimides as selective photonucleases, PDT anticancer agents or both.

SOS induction by gamma-radiation in Escherichia coli strains defective in repair and/or recombination mechanisms.

Ionizing radiation causes several types of DNA lesions, mainly single- or double-strand breaks and base damage. By means of the chromotest, an assay that allows the level of the SOS response to be monitored via beta-galactosidase enzymatic activity, the roles of several repair (uvrA, recN and oxyR) and recombination (recB, recJ and recO) genes in the response of Escherichia coli to gamma-radiation were studied. The results indicate that all the repair- and recombination-deficient strains were more sensitive to the lethal effects of ionizing radiation. However, the SOS activation pattern was somewhat different. The minimal inducing dose in uvrA and recN mutants was lower than in the wild-type, whereas their SOS response was higher at all doses. Conversely, in the strains lacking an active recB, recJ or recO gene, the doubling dose was almost the same as in the wild-type but the level of induction remained stable over a wide dose range. These findings suggest that neither single- nor double-strand breaks are in themselves direct SOS inducers and that while uvrA, recN and oxyR take part in different repair or protective pathways, apparently recB, recJ and recO participate in damage processing leading to SOS induction, as well as in recombination repair.

Evidence for UV-B-induced DNA degradation in Euglena gracilis mediated by activation of metal-dependent nucleases.

It is demonstrated that in vivo irradiation with artificial UV-B for several hours significantly reduces the amount of large DNA extractable from immobilized Euglena in comparison with non-irradiated controls. This UV-B effect can be eliminated by a drastic reduction of the divalent ion concentration in the extracellular medium, i.e. the substitution of the culture medium by Tris-buffered agarose. Moreover, in vitro degradation of large DNA is demonstrated for crude protein extracts isolated from non-irradiated or UV-B-irradiated Euglena. The nuclease activity is shown for both crude protein extracts and purified nucleases; in both cases, two protein bands possessing nuclease activity are obtained with apparent molecular masses of 26 and 40 kDa and their activity is inhibited by specific nuclease inhibitors, i.e. aurintricarboxylic acid and ATP, applied at a concentration as low as 10(-8) M. Moreover, in vitro, nuclease activity clearly depends on the pH, with an optimum around pH 4.5, and on the ion composition of the extracellular medium. A strong stimulating effect is shown for Ca2+ with an optimum around 10(-4) M; this effect is potentiated by Zn2+ and Mn2+, but strongly counteracted by Mg2+ and the calmodulin inhibitors trifluoperazine and N- (6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W5). These results favour the concept which explains the lethal UV-B effect on Euglena as arising from a change in the general metabolic state of the cell and an activation of a DNA-degrading system, i.e. activation of metal-dependent nucleases (U.K. Tirlapur, D.-P. Häder and R. Scheuerlein, UV-B mediated damage in the photosynthetic flagellate, Euglena gracilis, studied by image analysis, Beitr. Biol. Pflanzen, 67 (1992) 305-317).

[Deoxyribonuclease activity in the blood serum of persons participating in liquidation of the effects of the accident at the Chernobyl nuclear power plant]. / Aktivnost' dezoksiribonukleaz syvorotki krovi lits, uchastvova- iushchikh v likvidatsii posledstvii avarii na Chernobyl'skoi AES.

Deoxyribonuclease activity in blood serum was comparatively analyzed in 90 subjects who had been engaged in liquidation of consequences of the catastrophe at Chernobyl NPS in 1986, and in 55 normal donors. It was found that the mean value of deoxyribonuclease activity in the group of the liquidators was significantly lower as compared to that of donors. A stable decrease of activity of neutral deoxyribonuclease (DNAse I, pH 7.3) was detected in 18 and that of acid deoxyribonuclease (DNAse II) in 9 out of 90 subjects investigated. The anamnesis of most of the patients with lowered deoxyribonuclease activity has revealed transient leukopenia, decreased parameters of T-cellular immunity and phagocytic activity of neutrophils.

[The nuclear desoxyribonuclease (DNAse) spectrum of normal rat thymus cells and after whole-body x-ray irradiation]. / Izuchenie spektra iadernykh dezoksiribonukleaz (DNKaz) kletok timusa krys v norme i posle obshchego rentgenovskogo oblucheniia.

Spectra of thymocyte nuclear DNAases of control and irradiated (4 Gy) rats have been investigated. Using the method of SDS-electrophoresis of nuclear proteins in DNA-polyacrylamide gel (PAAG) the authors managed to discover a number of polypeptides of 35, 32, 17.7, 17.2, and 16.4 kDA molecular mass possessing a DNAase activity. The enzyme of 35 kDA is only active in the presence of Ca2+ and Mg2+ ions. Nucleases of 32, 17.7, 17.2, and 16.4 kDA are active in the presence of Ca2+ ions and inactive in the presence of Mg2+ ions or in the absence of divalent cations. A simultaneous addition of Ca2+ and Mg2+ ions to the incubation medium causes a synergistic effect with respect to the manifestation of these DNAase activities. Nucleases of 32, 17.7, 17.2, and 16.4 kDa only emerge after the preliminary removal of histones by ion exchange chromatography on a column with CM-sephadex C-50. The enzymic activity of 32 kDA protein increases 60 min after irradiation and drops to the control value in 4 h. At the same time, the postirradiation increase in DNAase activity of a low-molecular weight enzyme group remains invariable throughout the entire period of observation (1-4 h). The preinjection of cycloheximide (CHI) prevents the postirradiation degradation of chromatin and, simultaneously, makes the enzymic activity, corresponding to 35 kDA protein, disappear at the electrophoregrams. The experiments with CHI permit to identify the given enzymic fraction as Ca/Mg-dependent endonuclease. This indicates the participation of normally pre-existing Ca/Mg-dependent endonuclease in implementing the process of chromatin enzymic degradation in the irradiated thymocytes.

[Nuclease-aided simulation of the action of radiation on Chinese hamster fibroblasts. The role of double-stranded DNA breaks induced by restrictases in initiating chromosomal disorders]. / Modelirovanie s pomoshch'iu nukleaz deistviia radiatsii na fibroblasty kitaiskogo khomiachka. O roli dvunitevykh razryvov DNK, indutsirovannykh restriktazami, v initsiatsii khromosomnykh narushenii.

Comparison was made between the effectiveness of restrictases inducing double-strand DNA breaks with blunt (Hae III and Eco RV) and cohesive (Hind III and Sal I) ends and that of gamma-radiation on the initiation of chromosome aberrations. The analysis of the spectrum of chromosome aberrations induced in the presence or absence of DNA repair inhibitors, as well as the study of the pattern of cell distribution by the number of DNA breaks per cell showed that the decisive role in the initiation of chromosome mutagenesis is played by the localization of the break in certain sequences of target DNA rather than the type of the break.

[Deoxyribonuclease activity in the tissues of X-ray irradiated animals with alloxan diabetes]. / Aktivnost' dezoksiribonukleaz v tkaniakh pri rentgenovskom obluchenii zhivotnykh s alloksanovym diabetom.

Fractionated X-irradiation (a cumulative dose of 12.9 mC/kg) of animals with alloxan diabetes caused different, with respect to direction and degree, changes in liver nuclease system than those observed in intact animals after similar irradiation. This indicates that insulin is involved in a metabolic response of the organism to irradiation with relatively small doses.

Reversibility of acid and alkaline deoxyribonuclease deficiency in malignant tumor cells.

The nature of DNAse deficiency, which appears to be characteristic for malignant tumor cells, was investigated by the histochemical lead nitrate technique under various experimental conditions. Reappearance of distinct alkaline and acid DNAse activity was observed on the periphery of spontaneously occurring tumor necrosis, at early stages of the in vitro induced tumor necrosis, in necrotic tumor cells after in vivo irradiation and after in vitro treatment with different compounds. A membrane releaser did not reactivate DNAses in viable tumor cells, whereas the homogenate from tumor tissue inhibited DNAses in normal rat liver. These findings indicate that alkaline and acid DNAse deficiency in malignant tumor cells is a reversible phenomenon. This reversal of enzymatic activity has different histochemical and chronological patterns and specific reactivating factors for each DNAse. The masking effect of DNAse activity in malignant tumor cells is probably linked to natural enzyme inhibitors and its reversal to early stages of tumor necrosis.