Proteomic analysis of meiosis and characterization of novel short open reading frames in the fission yeast Schizosaccharomyces pombe.

Meiosis is the process by which haploid gametes are produced from diploid precursor cells. We used stable isotope labeling by amino acids in cell culture (SILAC) to characterize the meiotic proteome in the fission yeast Schizosaccharomyces pombe. We compared relative levels of proteins extracted from cells harvested around meiosis I with those of meiosis II, and proteins from premeiotic S phase with the interval between meiotic divisions, when S phase is absent. Our proteome datasets revealed peptides corresponding to short open reading frames (sORFs) that have been previously identified by ribosome profiling as new translated regions. We verified expression of selected sORFs by Western blotting and analyzed the phenotype of deletion mutants. Our data provide a resource for studying meiosis that may help understand differences between meiosis I and meiosis II and how S phase is suppressed between the two meiotic divisions

Mapping and Analysis of Swi5 and Sfr1 Phosphorylation Sites

The evolutionarily conserved Swi5-Sfr1 complex plays an important role in homologous recombination, a process crucial for the maintenance of genomic integrity. Here, we purified Schizosaccharomyces pombe Swi5-Sfr1 complex from meiotic cells and analyzed it by mass spectrometry. Our analysis revealed new phosphorylation sites on Swi5 and Sfr1. We found that mutations that prevent phosphorylation of Swi5 and Sfr1 do not impair their function but swi5 and sfr1 mutants encoding phosphomimetic aspartate at the identified phosphorylation sites are only partially functional. We conclude that during meiosis, Swi5 associates with Sfr1 and both Swi5 and Sfr1 proteins are phosphorylated. However, the functional relevance of Swi5 and Sfr1 phosphorylation remains to be determined

Extract from Armoracia rusticana and Its Flavonoid Components Protect Human Lymphocytes against Oxidative Damage Induced by Hydrogen Peroxide

DNA damage prevention is an important mechanism involved in cancer prevention by dietary compounds. Armoracia rusticana is cultivated mainly for its roots that are used in the human diet as a pungent spice. The roots represent rich sources of biologically active phytocompounds, which are beneficial for humans. In this study we investigated the modulation of H2O2 genotoxicity using the A. rusticana root aqueous extract (AE) and two flavonoids (kaempferol or quercetin). Human lymphocytes pre-treated with AE, kaempferol and quercetin were challenged with H2O2 and the DNA damage was assessed by the comet assay. At first we assessed a non-genotoxic concentration of AE and flavonoids, respectively. In lymphocytes challenged with H2O2 we proved that the 0.0025 mg·mL−1 concentration of AE protected human DNA. It significantly reduced H2O2-induced oxidative damage (from 78% to 35.75%). Similarly, a non-genotoxic concentration of kaempferol (5 μg·mL−1) significantly diminished oxidative DNA damage (from 83.3% to 19.4%), and the same concentration of quercetin also reduced the genotoxic effect of H2O2 (from 83.3% to 16.2%). We conclude that AE, kaempferol and quercetin probably act as antimutagens. The molecular mechanisms underlying their antimutagenic activity might be explained by their antioxidant properties

Antioxidant activity and antigenotoxicity of seeds and berries extracts from the red, anthocianin-rich Vitis vinifera variety Vinhão and from the white variety Loureiro from the northwestern region of Portugal

Introduction: Vitis vinifera grape berries are rich in polyphenols, mainly in seeds1 and the skin. Red varieties are richer due to the presence of anthocianins but there are also considerable differences also among red grape varieties2. The red variety Vinhão from the Northwest of Portugal is highly concentrated in these compounds3, conferring a great potential for biological activities usually associated to polyphenols. Material & Methods: Ethanolic extracts were prepared from seeds and berries from the red variety Vinhão and the white variety Loureiro. Antioxidant analysis was performed with the reducing power, DPPH and iron chelating assays and genotoxicity/antigenotoxicity were assessed with the DNA topology assay and the comet assay with human lymphocytes. Results: Seed extracts from both varieties displayed strong reducing power and DPPH scavenging activities, while red Vinhão berries displayed higher activities than the white variety (Loureiro). Interestingly, all samples did not chelate iron, suggesting that this property is not involved in antioxidant activity. In addition, all extracts were antigenotoxic in the DNA topology assay and the comet assay with human lymphocytes. Conclusion: As antioxidant and antigenotoxic activities were not coincident in some of the extracts (eg, Loureiro berries), different compounds and mechanisms are probably involved in these bioactivities.This work is funded by: INTERACT project -“Integrative Research in Environment, Agro-Chains and Technology”, no. NORTE-01-0145- FEDER-000017, in its line of research entitled ISAC, co-financed by the European Regional Development Fund (ERDF) through NORTE 2020 (North Regional Operational Program 2014/2020)

Endocrine-independent cytotoxicity of bisphenol A is mediated by increased levels of reactive oxygen species and affects cell cycle progression

Bisphenol A (BPA) is used for the production of plastics and epoxy resins, which are part of packaging materials for food and beverages, and can migrate into food and the environment, thus exposing human beings to its effects. Exposure to BPA has been associated with oxidative stress, cell cycle changes and genotoxicity, and is mediated by its known endocrine-disrupting activity. Possible BPA cytotoxicity without mediation by estrogen receptors has been reported in the previous literature. Here, we show the toxic effects of BPA by live-cell imaging on the fission yeast Schizosaccharomyces pombe, an experimental model lacking estrogen receptors, which were in line with data from flow cytometry on intracellular oxidation (76.4 14.4 % and 19.4 16.1 % of fluorescent cells for BPA treatment and control, respectively; p < 0.05) as well as delay in cell cycle progression (after 90 min of experiment, 48.4 4.30 % and 64.6 5.46 % of cells with a 4C DNA content for BPA treatment and control, respectively; p < 0.05) upon exposure to BPA. These results strongly support the possibility that BPA-induced cell cycle changes can be independent from estrogen receptors and that live-cell imaging is a powerful tool for genotoxic analysis.This work was supported by European Investment Funds by FEDER/COMPETE/POCI - Operational Competitiveness and Internationalization Program, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019. It was also supported by the Slovak grants VEGA 1/0450/18, VEGA 1/0410/18, UK/249/2019, and UK/157/2019.info:eu-repo/semantics/publishedVersio

Extract of Lillium candidum L. Can Modulate the Genotoxicity of the Antibiotic Zeocin

Lilium candidum L. extract (LE) is well known in folk medicine for the treatment of burns, ulcers, inflammations and for healing wounds. This work aims to clarify whether the genotoxic potential of the radiomimetic antibiotic zeocin (Zeo) could be modulated by LE. Our results indicate that LE exerts no cytotoxic, DNA-damaging and clastogenic activity in in Chlamydomonas reinhardtii, Pisum sativum L. and Hordeum vulgare L. test systems over a broad concentration range. Weak but statistically significant clastogenic effects due to the induction of micronuclei and chromosome aberrations have been observed in H. vulgare L. after treatment with 200 and 300 μg/mL LE. To discriminate protective from adverse action of LE different experimental designs have been used. Our results demonstrate that the treatment with mixtures of LE and Zeo causes an increase in the level of DNA damage, micronuclei and “metaphases with chromatid aberrations” (MwA). Clear evidence has been also obtained indicating that pretreatment with LE given 4 h before the treatment with Zeo accelerates the rejoining kinetics of Zeo-induced DNA damage in P. sativum L. and C. reinhardtii, and can decrease clastogenic effect of Zeo measured as frequencies of micronuclei and MwA in H. vulgare L. Here, we show for the first time that LE can modulate the genotoxic effects of zeocin. The molecular mode of action strongly depends on the experimental design and varies from synergistic to protective effect (adaptive response–AR). Our results also revealed that LE-induced AR to zeocin involves up-regulation of DSB rejoining in C. reinhardtii and P. sativum L. cells