Protective effect of Aronia melanocarpa juice against acrylamide-induced cellular toxicity.

Acrylamide (AA) a widely used industrial chemical is also formed during food processing by the Maillard reaction, which makes its exposure to humans almost unavoidable. In this study, we used Schizosaccharomyces pombe as a model organism to investigate AA toxicity (10 or 20 mM concentration) in eukaryotes. In S. pombe, AA delays cell growth causes oxidative stress by enhancement of ROS production and triggers excitement of the antioxidant defence system resulting in the division arrest. Aronia fruit contains a variety of health-promoting substances with considerable antioxidant potential. Therefore, Aronia juice supplementation was tested to evaluate its protective effect against AA-derived perturbations of the organism. Cell treatment with several Aronia juice concentrations ranging from 0 to 2% revealed the best protective effect of 1 or 2% Aronia juice solutions. Both chosen Aronia juice concentrations alleviated AA toxicity through the improvement of the antioxidant cell capacity and metabolic activity by their strong ROS scavenging property. Efficiency of Aronia juice cell protection is dose dependent as the 2% solution led to significantly higher cellular defence compared with 1%. Due to the high similarity of biological processes of S. pombe with higher eukaryotes, the protective effect of Aronia juice against AA toxicity might also apply to higher organisms.

Acrylamide-Derived Ionome, Metabolic, and Cell Cycle Alterations Are Alleviated by Ascorbic Acid in the Fission Yeast.

Acrylamide (AA), is a chemical with multiple industrial applications, however, it can be found in foods that are rich in carbohydrates. Due to its genotoxic and cytotoxic effects, AA has been classified as a potential carcinogen. With the use of spectrophotometry, ICP-OES, fluorescence spectroscopy, and microscopy cell growth, metabolic activity, apoptosis, ROS production, MDA formation, CAT and SOD activity, ionome balance, and chromosome segregation were determined in Schizosaccharomyces pombe. AA caused growth and metabolic activity retardation, enhanced ROS and MDA production, and modulated antioxidant enzyme activity. This led to damage to the cell homeostasis due to ionome balance disruption. Moreover, AA-induced oxidative stress caused alterations in the cell cycle regulation resulting in chromosome segregation errors, as 4.07% of cells displayed sister chromatid non-disjunction during mitosis. Ascorbic acid (AsA, Vitamin C), a strong natural antioxidant, was used to alleviate the negative impact of AA. Cell pre-treatment with AsA significantly improved AA impaired growth, and antioxidant capacity, and supported ionome balance maintenance mainly due to the promotion of calcium uptake. Chromosome missegregation was reduced to 1.79% (44% improvement) by AsA pre-incubation. Results of our multiapproach analyses suggest that AA-induced oxidative stress is the major cause of alteration to cell homeostasis and cell cycle regulation.

Nickel induced cell impairments are negatively regulated by the Tor1 kinase in Schizosaccharomyces pombe.

In our study we investigated the effect of different nickel (NiSO4·6H2O) (Ni) concentrations on cell division, cellular morphology and ionome homeostasis of the eukaryotic model organism Schizosaccharomyces pombe. Target of rapamycin (TOR) protein kinase is one of the key regulators of cell growth under different environmental stresses. We analyzed the effect of Ni on cell strains lacking the Tor1 signaling pathway utilizing light-absorbance spectroscopy, visualization, microscopy and inductively coupled plasma optical emission spectroscopy. Interestingly, our findings revealed that Ni mediated cell growth alterations are noticeably lower in Tor1 deficient cells. Greater size of Tor1 depleted cells reached similar quantitative parameters to wild type cells upon incubation with 400 µM Ni. Differences of ion levels among the two tested yeast strains were detected even before Ni addition. Addition of high concentration (1 mM) of the heavy metal, representing acute contamination, caused considerable changes in the ionome of both strains. Strikingly, Tor1 deficient cells displayed largely reduced Ni content after treatment compared to wild type controls (644.1 ± 49 vs. 2096.8 ± 75 µg/g), suggesting its significant role in Ni trafficking. Together our results predict yet undefined role for the Tor1 signaling in metal uptake and/or metabolism.

Impact of cadmium and nickel on ion homeostasis in the yeast Schizosaccharomyces pombe.

Toxicity of heavy metals to living organisms is a worldwide research topic. Although, much has been discovered about cadmium and nickel impact on biological systems, a lot still remains unclear. We used inductively coupled plasma - optical emission spectroscopy to address the question of the effect of two different heavy metals nickel, and cadmium on intracellular ion balance. Increase or decrease of the content of several essential cations including Ca2+, Na+, K+, Mg2+, Cu2+, Fe3+ in the yeast Schizosaccharomyces pombe was determined. Our results revealed that the cell exposure to high nickel and cadmium concentrations led to significant elevation of Ca2+, Na+, Mg2+, Cu2+, Fe3+ levels in the yeast cell, while the content of K+ decreased. Correlation analyses showing in the presence of nickel and cadmium strong positive correlation among each tested element (Ca2+, Na+, Cu2+, Mg2+ and Fe3+) except for K+, demonstrate the significant impact of heavy metal treatment to ion homeostasis of the cell. Our data indicate that acute nickel and cadmium contamination leads to substantial ionome misbalance in yeast.

Association of HindIII-polymorphism in kappa-casein gene with milk, fat and protein yield in holstein cattle.

The aim of this paper was to evaluate the effect of genetic polymorphism of kappa-casein on milk production in Holstein cattle. Two hundred and ten Holstein cows were used in this study. We established genotype structure of cattle population and calculated allelic frequencies based on PCR-RFLP analyses. The three genotypes: AA (69.52%), AB (27.62%), and BB (2.86%) were detected. Frequency of allele A was 83.33%, and of allele B 16.67%. The Holstein cattle kept in Slovak Republic exhibit a high value of homozygosity (0.7222) and low values of polymorphism information content (0.2392), effective number of alleles (1.3847) and level of possible variability realization (27.91%). The effect of polymorphism of CSN3 gene on average breeding values for milk production traits, such as yield of milk, fat and protein expressed in kilograms, as well as percentage content of fat and protein in milk, has been assessed. In our assessment of the observed traits' variability's dependence on CSN3 gene polymorphism, we detected a statistically significant difference between genotypes only in case of the average breeding value for the percentage of protein in milk.

Genetic polymorphisms of leptin and leptin receptor genes in relation with production and reproduction traits in cattle.

Leptin and leptin receptor genes are considered as production traits markers in dairy or beef cattle. The aim of this study was to verify the associations of polymorphisms in bovine LEP and LEPR genes with production and reproduction traits in Slovak Spotted and Pinzgau cows. Long-life production was evaluated: milk, protein, and fat yield and reproduction traits: age at first calving, calving interval, days open, and insemination interval. In total, 296 blood samples of Slovak Spotted and 85 hair roots samples of Pinzgau cows were analyzed. In order to detect LEP/Sau3AI (BTA 4, inron 2) and LEPR/T945M (BTA 3, exon 20) genotypes PCR-RFLP method was used. In Slovak Spotted and Pinzgau cows allele frequencies were 0.838/0.162 and 0.694/0.306 for A and B LEP variants, and 0.954/0.046 and 0.912/0.088 for C and T LEPR variants, respectively. For testing the associations between SNPs LEP/Sau3AI and LEPR/T945M and evaluated traits, the General Linear Model procedure in SAS Software was used. Statistical analysis showed that SNP LEP/Sau3AI significantly affected milk, protein and fat yield (P<0.05), and age at first calving (P<0.01) in analyzed population of cows. Statistically, SNP LEPR/T945M affected significantly calving interval (P<0.01) only. Results of our study suggest that especially leptin is a candidate gene, which influences mainly milk production traits and might be implemented in breeding strategies to improve the production performance of both analyzed cattle breeds.

Analysis of Slovak Spotted breed for bovine beta casein A1 variant as risk factor for human health.

The goal of work was identification A1 variant of bovine beta casein which involves ischemic heart disease and diabetes mellitus in human. The digestion of A1beta casein can result in the production of bioactive beta casomorphin-7 (BCM-7); this is not the case with A2. This bioactive peptide has been linked to physiological traits that may elicit effects on components of the vascular and immune systems. The material involved 111 Slovak Spotted breed. Bovine genomic DNA was extracted from whole blood by using commercial kit, and used in order to estimate beta-casein genotypes by means of PCR-RFLP method. The PCR products were digested with DdeI restriction enzyme. In the population included in the study were detected all three genotypes, homozygote genotype A1A1 (14 animals), heterozygote genotype A1A2 (37 animals) and homozygote genotype A2A2 (60 animals). In the total population of cattle homozygotes A2A2-0.5405 were the most frequent, while homozygotes A1A1-0.1261 were the least frequent ones. This suggests a superiority of allele A2 (0.7072) which does not produce BCM-7, and thus is safe for human consumption. The expected homozygosity for gene CSN2 is in the population stated a slight increase in homozygosity (0.5858). This caused a slight decrease in the level of possible variability realization (41.80%), which corresponds to the effective number of alleles (1.7071).

Genetic diversity in populations of Slovak Spotted cattle based on single nucleotide polymorphisms analyses.

The aim of this study was to identify SNPs in leptin (LEP), leptin receptor (LEPR) and growth hormone (GH) genes in order to analyze genetic diversity of Slovak Spotted cattle. The total numbers of blood samples were taken from 353 Slovak Spotted cows originating from four farms. Genomic DNA was isolated by phenol-chloroform extraction method and analyzed by PCR-RFLP method. After digestion with restriction, enzymes were detected in whole population of cow's alleles with frequency: LEP/Sau3AI A 0.84 and B 0.16 (±0.0152); LEPR/BseGI C 0.95 and T 0.05 (±0.0089) and GH/AluI L 0.70 and V 0.30 (±0.0188). Based on the observed vs. expected genotypes frequencies populations across loci were in Hardy-Weinberg equilibrium (P\>0.05). Predominant for SNP LEP/Sau3AI was AA genotype (0.70), for SNP LEPR/T945M CC genotype (0.91), and LL genotype (0.48) was most frequent for SNP GH/AluI. The observed heterozygosity of SNPs across populations was also transferred to the low or median polymorphic information content 0.24 (He 0.28), 0.08 (He 0.09) and 0.33 (He 0.47) for LEP, LEPR and GH genes, respectively. Within genetic variability estimating negative values of fixation indexes FIS (-0.09-0.05) and FIT (-0.07-0.03) indicating heterozygote excess were observed. The value of FST indexes (0.018-0.023) shows very low levels of genetic differentiation in allele frequencies of loci among evaluated subpopulations. The low values of genetic distances (0.0018-0.0159) indicated high genetic relatedness among animals in subpopulations caused probably by common ancestry used in breeding program at farms.

Prp4 kinase is required for proper segregation of chromosomes during meiosis in Schizosaccharomyces pombe.

Chromosome segregation during meiosis is a complex process, which leads to production of four haploid gametes from two precursor cells. Reversible phosphorylation of proteins plays a crucial role in this process. The Schizosaccharomyces pombe Prp4 is an essential serine/threonine protein kinase, which belongs to the Clk/Sty family. To study the role of Prp4 in meiosis, we analysed chromosome segregation in a strain carrying conditional analog-sensitive allele of Prp4 protein kinase (prp4-as2). Our data show, that Prp4 protein kinase plays important role in chromosome segregation during meiosis, as revealed by enhanced missegregation of chromosomes in prp4-as2 mutant cells.