Sigma Xi 2014
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Sigma Xi 2014 Sigma Xi 2014 Presentation Transcript

  • + Differential gene expression in Saccharomyces cerevisiae as a result of exposure to (-)-epicatechin, a flavanol antioxidant found in cocoa Gen Selden Pine Crest School
  • + Background  Free radicals, or reactive oxygen species (ROS) are constantly produced from oxygen metabolism in cells  Examples of free radicals: OH, O2 -, H2O2  Oxidative stress – cumulative effect of ROS causing degradation of DNA, lipids, and proteins and damaging the cells  Oxidative stress has been seen to play a role in certain neurodegenerative diseases and cancer  Antioxidants react with free radicals to reduce oxidative stress and improve longevity  Flavanols are a specific type of antioxidant and are thought to be the main antioxidants present in cocoa  Predominant antioxidant properties of cocoa are thought to be mainly a result of (-)-epicatechin
  • + Background  (-)-Epicatechin:  A specific flavanol antioxidant found in cocoa  May have beneficial effects on certain aspects of health including aging and disease  Found in higher concentrations in cocoa than other flavanols such as (+)- catechin and quercetin  Found in higher concentrations in cocoa than in other commonly known sources of antioxidants such as fruits and vegetables  Saccharomyces cerevisiae – Yeast  Simple and rapid reproduction  Easily cultured  Model organism for humans  31% homology between genomes  Humans and yeast share many homologous genes
  • + Background  Longevity genes in yeast  RAS2 - involved in cell proliferation  IDH2 – associated with the oxidative decarboxylation of the citric acid cycle and is necessary for cellular respiration  HST2 – transcriptional silencing, DNA repair, genomic stability, and overall longevity  DNM1 – programmed cell death  Oxidative stress genes in yeast  CTA1 – codes for the enzyme catalase A, which catalyzes the breakdown of H2O2 and other oxidative molecules  GSH1 – codes for GSH, a radical scavenger protein
  • + Purpose  To test the effects of varying concentrations of (-)- epicatechin on the gene expression of the longevity genes RAS2, IDH2, HST2, and DNM1 and the oxidative stress genes CTA1 and GSH1 in Saccharomyces cerevisiae, a model organism for humans
  • + Hypothesis Gene Function Human Homolog % Homology Hypothesis RAS2 Longevity KRAS 54% Upregulated IDH2 Longevity IDH3A 56% Upregulated HST2 Longevity SIRT2 61% Upregulated DNM1 Longevity DNM1L 55% Downregulated CTA1 Oxidative stress CAT 54% Downregulated GSH1 Oxidative stress GCLC 53% Downregulated  RAS2, IDH2, and HST2 are expected to be upregulated because they are involved in cellular processes necessary for longevity  DNM1 is expected to be downregulated because of its involvement in programmed cell death  CTA1 and GSH1 are expected to be downregulated because exposure to the antioxidant molecule (-)-epicatechin may reduce the need for cellular protection against oxidative stress by radical-scavenging proteins
  • + Materials and Methods  Yeast was cultured in YPD media at 25oC while rotating for 13.5 hours and exposed to 6 different concentrations of (-)-epicatechin for 36.5 hours following growth  0 uM, 50 uM, 100 uM, 250 uM, 500 uM, and 750 uM  Total RNA isolated using RiboPure Yeast protocol from Ambion Life Technologies  RNA presence in each sample confirmed using gel electrophoresis  cDNA synthesized from extracted RNA using ProtoScript First Strand cDNA Synthesis Kit  Primers for PCR were designed using www.operon.com  PCR was performed to amplify genes of interest  Gel electrophoresis used to analyze differential gene expression
  • + Confirmation of the presence of total RNA  Presence of total RNA in each sample was confirmed using gel electrophoresis  The 28S and 18S bands confirm RNA – standard bands of total isolated RNA 28S 18S DNA ladder 0 uM 50 uM 100 uM250 uM500 uM750 uM
  • + Gel Electrophoresis of HST2  An increase in expression was seen in the 500 uM and 750 uM samples  Increase in expression signified by more intense bands  Supports hypothesis that HST2 would be upregulated in response to (-)-epicatechin DNA ladder 0 uM 50 uM 100 uM 250 uM 500 uM 750 uM NC DNA ladder
  • + Gel Electrophoresis of GSH1  A decrease in expression was seen in the 500 uM and 750 uM samples  Decrease in expression signified by less intense bands  Supports the hypothesis that exposure to (-)- epicatechin leads to a downregulation of GSH1 DNA ladder 0 uM 50 uM 100 uM250 uM500 uM750 uM NC DNA ladder
  • + Conclusion  HST2 is expressed under normal cellular conditions and seems to be upregulated by relatively high concentrations of (-)- epicatechin  Suggests that (-)-epicatechin improves longevity and upregulates genes necessary for longevity  GSH1 is expressed under normal cellular conditions and expression seems to be downregulated by (-)-epicatechin at relatively high concentrations  Suggests that (-)-epicatechin combats oxidative stress by reducing free radicals, subsequently downregulating oxidative stress genes that are necessary to reduce normal cellular oxidative stress
  • + Future Research  Tests to confirm differential gene expression of the genes that have already been tested (GSH1, HST2)  Test for differential gene expression of remaining genes (RAS1, IDH2, DNM1, CTA1)  Including patterns of expression between the longevity genes and patterns of expression between the oxidative stress genes  Once patterns of expression in the two gene categories have been observed, I will expose yeast to a higher concentration range of (-)-epicatechin (500 uM to 1000 uM)  Because (-)-epicatechin seemed to cause differential gene expression in the samples exposed to the two highest concentrations, 500 uM and 750 uM, in the first trial
  • + Future Research  Certain mitochondrial genes such as COQ3 have also been shown to play a significant role in oxidative stress and the rate of cell aging  Determining differential gene expression of these genes may give a more complete representation of the effects of (-)-epicatechin on longevity and oxidative stress in yeast  Concurrent exposure of yeast to (-)-epicatechin and oxidative stress, in the form of H2O2, may provide a more complete understanding of the effects of (-)-epicatechin on oxidative stress and subsequent regulation of gene expression
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