Differential effects of glutathione depletion and metallothionein induction on the induction of DNA single-strand breaks and cytotoxicity by tert-butyl hydroperoxide in cultured mammalian cells

Induction of DNA single-strand breaks (ssb), their resealing and cytotoxicity by tert-butyl hydroperoxide (t-BuOOH) were investigated in cultured Chinese hamster V79 cells. The effect of the depletion of cellular glutathione (GSH), iron chelation and induction of metallothionein (MT) on these parameters was studied. t-BuOOH in a concentration range of 0.02-0.5 mM induced DNA ssb in a dose-dependent fashion. Strand breakage increased as a function of time up to 1 h. Divalent iron chelator o-phenanthroline suppressed markedly the induction of DNA ssb while the trivalent iron chelator desferrioxamine had no effect. GSH-depletion increased cytotoxicity of t-BuOOH. In contrast, the depletion of GSH did not affect the efficiency of formation of DNA ssb by t-BuOOH and the rate of resealing of the DNA damage. The induction of MT did not influence the efficiency of formation of DNA ssb by t-BuOOH. In summary, while GSH depletion and MT induction affected the formation of DNA ssb and cytotoxicity differently divalent iron was required for both. Therefore, appears likely that DNA breakage and cytotoxicity by t-BuOOH are caused by independent mechanisms.     

Calcium chelator Quin 2 prevents hydrogen-peroxide-induced DNA breakage and cytotoxicity

Exposure of cultured Chinese hamster ovary (CHO) cells to hydrogen peroxide results in the production of extensive DNA breakage which can be prevented by the intracellular calcium chelator Quin 2. This effect occurs at Quin 2 AM concentrations as low as 0.1 microM and is maximal at 1 microM. Addition of the extracellular calcium chelator, EGTA, does not affect the level of DNA breakage generated by H2O2. Quin 2 also significantly reduces cellular toxicity caused by the oxidant. Experiments with spin-trapping techniques demonstrate that Quin 2 does not affect the formation of hydroxyl radicals generated by the action of Fe2+ on H2O2. Quin 2 at high concentrations, similar to those reached within the cell, actually enhanced generation of hydroxyl radical in the absence of other iron chelators under our experimental conditions. These results suggest that H2O2 or H2O2-derived radicals do not directly induce DNA strand breakage in intact mammalian cells; rather, these radicals may disturb intracellular Ca2+ homeostasis which results in secondary reactions ultimately leading to DNA strand breakage. In addition to strand breakage, membrane and protein oxidation probably contribute to the cytotoxic effect of H2O2.     

The dynamics of grape leafhopper Empoasca vitis Göthe populations in southern Switzerland and the implications for habitat management

The life cycle of Empoasca vitis and its most important parasitoid Anagrus atomus in southern Switzerland can be divided into three phases. In phase I the adults of E. vitis move from overwintering sites, i.e. primarily conifers, over deciduous plants into the vineyards. The parasitoid A. atomus on the other hand, appears to overwinter in leafhopper eggs, mainly on roses and blackberries. It subsequently completes one generation in leafhopper eggs primarily on blackberries and hazelnuts, before it attacks E. vitis eggs in the vineyards. For phase II, a population model with time‐varying age structures and stochastic properties was constructed for E. vitis. For this purpose a time‐varying distributed delay model with attrition was constructed. Given the calibrated initial density of overwintering females, the model predicts an unacceptable number of E. vitis for the growing season. However, if egg parasitism of A. atomus and Stethynium triclavatum is introduced into the model as an external variable E. vitis densities are predicted which are economically irrelevant. Inphase III E. vitis adults leave the vineyards for the overwintering sites. The model shows the importance of the parasitoid A. atomus during phase II. A. atomus should be favoured by surrounding the vineyards with host plants carrying leafhoppers’ eggs. Consequently, habitat management measures for E. vitis control could be applied to the surroundings of the vineyards.     

Selective excision of gamma ray damaged thymine from the DNA of cultured mammalian cells.

Three mammalian cell lines (WI-38, SV40-transformed WI-38 and Chinese hamster ovary) were exposed to high doses of 137-Cs gamma rays and their DNA analysed, following various periods of postirradiation incubation, for products of the 5,6-dihydroxy-dihydrothymine type. Within fifteen minutes of incubation at 37 degrees C 70 to 90 percent of these radiation products were removed from acid-precipitable material in all three cell lines. The amount of DNA degradation induced by radiation varied from approximately one percent in WI-38 cells to 15 percent in SV40-transformed WI-38 cells. Comparison of DNA degradation with the amount of thymine radiation product removed indicates that a selective gamma ray-induced excision repair capability exists in mammalian cells. Because of its more rapid kinetics, gamma ray excision repair is probably a distinct process as compared with ultraviolet-induced pyrimidine dimer excision.     

Excision of gamma-ray damaged thymine by E. coli extracts is due to the 5'leads to 3' exonuclease associated with DNA polymerase I

Extracts of $\text{E. coli pol}$Aexl which contains a temperature sensitive 5′→3′ exonuclease function of polymerase I accomplish the selective excision of products of the 5,6-dihydroxy-dihydrothymine type from γ-irradiated DNA and OsO₄-oxidized polyd(A-T) at the permissive temperature (30°) but not at the nonpermissive temperature (42°). The 5′→3′ exonuclease activity of polymerase I, therefore, acts as a repair exonuclease in γ-ray excision repair.     

The stability of deoxycytidine photohydrates in the mononucleotide, oligodeoxynucleotides and DNA

The stability of deoxycytidine photohydrates was determined for deoxycytidylic acid and deoxycytidine residues in oligodeoxynucleotides by optical measurements and in native and denatured DNA by a chemical assay. The half lives at 20° in $\text{10}{}^{\mathrm{?2}}\text{M}$ tris buffer, pH 7.7, were 102 min. for the mononucleotide, 128 min. for dpApGpG, 152 min. for MeOdpTpCpA, 51 min. for denatured $\text{E}\text{.}\text{coli}\text{DNA and 58 min. for native}\text{E}\text{.}\text{coli}$ DNA (at pH 8.1). It is concluded that the stability of deoxycytidine photohydrates is sufficient that they cannot at present be dismissed as lesions of possible biological importance in ultraviolet irradiated cells.     

Characterization of Hg-phytochelatins complexes in vines (Vitis vinifera cv Malbec) as defense mechanism against metal stress

An approach to understand vines (Vitis vinifera) defense mechanism against heavy metal stress by isolation and determination of Hg-phytochelatins (PCs) complexes was performed. PCs are important molecules involved in the control of metal concentration in plants. PCs complex toxic metals through ?SH groups and stores them inside cells vacuole avoiding any toxic effect of free metals in the cytosol. The Hg-PCs identification was achieved by determination of Hg and S as hetero-tagged atoms. A method involving two-dimensional chromatographic analysis coupled to atomic spectrometry and confirmation by tandem mass spectrometry is proposed. An approach involving size exclusion chromatography coupled to inductively coupled plasma mass spectrometry on roots, stems, and leaves extracts describing Hg distribution according to molecular weight and sulfur associations is proposed for the first time. Medium–low molecular weight Hg–S associations of 29–100 kDa were found, suggesting PCs presence. A second approach employing reversed-phase chromatography coupled to atomic fluorescence spectrometry analysis allowed the determination of Hg-PCs complexes within the mentioned fractions. Chromatograms showed Hg-PC₂, Hg-PC₃ and Hg-PC₄ presence only in roots. Hg-PCs presence in roots was confirmed by ESI–MS/MS analysis.     

Structural basis for accommodation of emerging B.1.351 and B.1.1.7 variants by two potent SARS-CoV-2 neutralizing antibodies

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