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We analyzed the antioxidant properties of Ilex paraguariensis infusion (Ip) popularly known as mate (mä'tā), by using two experimental models: the induction of DNA double-strand breaks (DSB) by hydrogen peroxide (H(2)O(2)) and lethality in Saccharomyces cerevisiae, as well as peroxide and lipoxygenase-induced human low-density lipoprotein (LDL) oxidation. Diploid yeast cells were exposed to different concentrations of H(2)O(2) (5-10 mmol/L) in the absence or presence of Ip infusion (10(-1) v/v) or alpha-tocopherol (10(-2) mol/L). Both mate infusion and alpha-tocopherol significantly decreased the dose dependent DSB number, and the lethality induced by H(2)O(2). Peroxynitrite and lipoxygenase-induced human LDL oxidation are inhibited by Ip extracts in a potent, dose-dependent fashion. Dilutions of 5 x 10(-3) v/v provide 50% +/- 10% inhibition. Finally, Ip extracts are potent direct quenchers of the free radical 1,1-diphenyl-2-picrylhydrazyl. Dilutions of 2 x 10(-2) v/v produced quenching of more than 30%, which was comparable to that obtained with 0.5-1 mmol/L alpha-tocopherol or the quercetin aglycone, respectively. For comparison, total polyphenol content of Ip, green, and black tea (Camelia sinensis) were 6.5 +/- 0.8; 1.8 +/- 0.5; and 1.13 +/- 0.3 mmol of quercetin equivalents per liter, respectively. Their respective free radical quenching activities at dilutions of 1 x 10(-1) v/v were 75% +/- 5%; 35% +/- 5%; and 2% +/- 5%. Ip is thus a rich source of polyphenols and has antioxidant properties comparable to those of green tea which merit further in vivo intervention and cross-sectional studies.

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DNA repair pathways, cell cycle checkpoints, and redox protection systems are essential factors for securing genomic stability. The aim of the present study was to analyze the effect of Ilex paraguariensis (Ip) infusion and one of its polyphenolic components rutin on cellular and molecular damage induced by ionizing radiation. Ip is a beverage drank by most inhabitants of Argentina, Paraguay, Southern Brazil, and Uruguay. The yeast Saccharomyces cerevisiae (SC7Klys 2-3) was used as the eukaryotic model. Exponentially growing cells were exposed to gamma rays (γ) in the presence or absence of Ip or rutin. The concentrations used simulated those found in the habitual infusion. Surviving fractions, mutation frequency, and DNA double-strand breaks (DSB) were determined after treatments. A significant increase in surviving fractions after gamma irradiation was observed following combined exposure to γ+R, or γ+Ip. Upon these concomitant treatments, mutation and DSB frequency decreased significantly. In the mutant strain deficient in MEC1, a significant increase in γ sensitivity and a low effect of rutin on γ-induced chromosomal fragmentation was observed. Results were interpreted in the framework of a model of interaction between radiation-induced free radicals, DNA repair pathways, and checkpoint controls, where the DNA damage that induced activation of MEC1 nodal point of the network could be modulated by Ip components including rutin. Furthermore, ionizing radiation-induced redox cascades can be interrupted by rutin potential and other protectors contained in Ip.

DNA repair, checkpoint pathways and protection mechanisms against different types of perturbations are critical factors for the prevention of genomic instability. The aim of the present work was to analyze the roles of RAD17 and HDF1 gene products during the late stationary phase, in haploid and diploid yeast cells upon gamma irradiation. The checkpoint protein, Rad17, is a component of a PCNA-like complex-the Rad17/Mec3/Ddc1 clamp-acting as a damage sensor; this protein is also involved in double-strand break (DBS) repair in cycling cells. The HDF1 gene product is a key component of the non-homologous end-joining pathway (NHEJ). Diploid and haploid rad17 /rad17 , and hdf1 Saccharomyces cerevisiae mutant strains and corresponding isogenic wild types were used in the present study. Yeast cells were grown in standard liquid nutrient medium, and maintained at 30 • C for 21 days in the stationary phase, without added nutrients. Cell samples were irradiated with 60 Co γ rays at 5 Gy/s, 50 Gy ≤ Dabs ≤ 200 Gy. Thereafter, cells were incubated in PBS (liquid holding: LH, 0 ≤ t ≤ 24 h). DNA chromosomal analysis (by pulsed-field electrophoresis), and surviving fractions were determined as a function of absorbed doses, either immediately after irradiation or after LH. Our results demonstrated that the proteins Rad17, as well as Hdf1, play essential roles in DBS repair and survival after gamma irradiation in the late stationary phase and upon nutrient stress (LH after irradiation). In haploid cells, the main pathway is NHEJ. In the diploid state, the induction of LH recovery requires the function of Rad17. Results are compatible with the action of a network of DBS repair pathways expressed upon different ploidies, and different magnitudes of DNA damage.

Checkpoints are components of signalling pathways involved in genome stability. We analysed the putative dual functions of Rad17 and Chk1 as checkpoints and in DNA repair using mutant strains of Saccharomyces cerevisiae. Logarithmic populations of the diploid checkpoint-deficient mutants, chk1Delta/chk1Delta and rad17Delta/rad17Delta, and an isogenic wild-type strain were exposed to the radiomimetic agent bleomycin (BLM). DNA double-strand breaks (DSBs) determined by pulsed-field electrophoresis, surviving fractions, and proliferation kinetics were measured immediately after treatments or after incubation in nutrient medium in the presence or absence of cycloheximide (CHX). The DSBs induced by BLM were reduced in the wild-type strain as a function of incubation time after treatment, with chromosomal repair inhibited by CHX. rad17Delta/rad17Delta cells exposed to low BLM concentrations showed no DSB repair, low survival, and CHX had no effect. Conversely, rad17Delta/rad17Delta cells exposed to high BLM concentrations showed DSB repair inhibited by CHX. chk1Delta/chk1Delta cells showed DSB repair, and CHX had no effect; these cells displayed the lowest survival following high BLM concentrations. Present results indicate that Rad17 is essential for inducible DSB repair after low BLM-concentrations (low levels of oxidative damage). The observations in the chk1Delta/chk1Delta mutant strain suggest that constitutive nonhomologous end-joining is involved in the repair of BLM-induced DSBs. The differential expression of DNA repair and survival in checkpoint mutants as compared to wild-type cells suggests the presence of a regulatory switch-network that controls and channels DSB repair to alternative pathways, depending on the magnitude of the DNA damage and genetic background.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.

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