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The Anti-Toxin Properties of Grape Seed Phenolic Compounds

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The Anti-Toxin Properties of Grape Seed Phenolic Compounds University of Central Florida STARS Electronic Theses and Dissertations, 2004-2019 2014 The Anti-toxin Properties of Grape Seed Phenolic Compounds Patrick Cherubin University of Central Florida Part of the Biotechnology Commons, and the Molecular Biology Commons Find similar works at: https://stars.library.ucf.edu/etd University of Central Florida Libraries http://library.ucf.edu This Masters Thesis (Open Access) is brought to you for free and open access by STARS. It has been accepted for inclusion in Electronic Theses and Dissertations, 2004-2019 by an authorized administrator of STARS. For more information, please contact [email protected]. STARS Citation Cherubin, Patrick, "The Anti-toxin Properties of Grape Seed Phenolic Compounds" (2014). Electronic Theses and Dissertations, 2004-2019. 4827. https://stars.library.ucf.edu/etd/4827 THE ANTI-TOXIN PROPERTIES OF GRAPE SEED PHENOLIC COMPOUNDS by PATRICK WALTER CHERUBIN B.S. University of Central Florida, 2012 A.A. Valencia College, 2010 A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in the Burnett School of Biomedical Sciences in the College of Medicine at the University of Central Florida Orlando, Florida Summer Term 2014 Major Professor: Kenneth Teter © 2014 Patrick Walter Cherubin ii ABSTRACT Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. aeruginosa exotoxin A (ETA), ricin, Shiga toxin, and cholera toxin (CT). To identify individual compounds in grape seed extract that are capable of inhibiting the activities of these AB toxins, we screened twenty common phenolic compounds of grape seed extract for anti-toxin properties. Three compounds inhibited DT, four inhibited ETA, one inhibited ricin, and twelve inhibited CT. Additional studies were performed to determine the mechanism of inhibition against CT. Two compounds inhibited CT binding to the cell surface and even stripped bound CT off the plasma membrane of a target cell. Two other compounds inhibited the enzymatic activity of CT. We have thus identified individual toxin inhibitors from grape seed extract and some of their mechanisms of inhibition against CT. This work will help to formulate a defined mixture of phenolic compounds that could potentially be used as a therapeutic against a broad range of AB toxins. iii ACKNOWLEDGMENTS First and foremost I would like to thank Dr. Ken Teter for allowing me the chance to be part of his team. I was a senior undergraduate when I contacted Dr. Teter about a position in his lab. Dr. Teter invited me to his office, and we discussed about his research. The summer of that same year I applied to an undergraduate research program and I got accepted into the program to work with Dr. Teter on cholera. I have been a research member in Dr. Teter’s lab for over three years now, and it has truly been a rewarding experience. Additionally I would like to thank the rest of my committee members, Dr. Hervé Roy, Dr. Antonis Zervos, and Dr. Otto Phanstiel, for their support and advice throughout the completion of this project. These committee members have “empowered” me throughout the duration of my project. Furthermore I would like to thank Dr. Teter’s current and former staff members for all their support and help. When I started working in Dr. Teter’s lab, I had no knowledge whatsoever on how to conduct any experiments. The staff members were always there to help me. I want to thank Albert, Aimee, Camila, Carly, Chris, David, Helen, Mike, Neyda, Supriyo, and Tuhina for all their help. Each of these lab mates has contributed in a way or another to my training. Lastly, I would like to thank the Biotechnology MS program staff members for all their support and academic advice. Ms. Lisa Vaughn, Mr. Eddie Garcia, and Dr. Saleh Naser have been there to assist me ever since I started the program. Many thanks to each and single one of you! iv TABLE OF CONTENTS LIST OF FIGURES ...................................................................................................................... vii LIST OF TABLES ....................................................................................................................... viii LIST OF ACRONYMS ................................................................................................................. ix CHAPTER 1: INTRODUCTION ................................................................................................... 1 1.1 Corynebacterium diphtheriae and Diphtheria Toxin ................................................................. 1 1.2 Pseudomonas aeruginosa and Exotoxin A ................................................................................ 4 1.3 Ricinus communis and Ricin Toxin ........................................................................................... 6 1.4 Shigella dysentariae and Shiga Toxin ....................................................................................... 8 1.5 Vibrio cholerae and cholera toxin ............................................................................................ 10 1.6 Hypothesis and Aims ............................................................................................................... 13 CHAPTER 2: MATERIALS AND METHODS .......................................................................... 17 2.1 Chemicals and Reagents .......................................................................................................... 17 2.2 Plant Extracts and Phenolic Compounds (with Stock Concentration and Solvent)................. 19 2.3 Buffers...................................................................................................................................... 21 2.4 Toxins ...................................................................................................................................... 21 2.5 Equipments .............................................................................................................................. 22 2.6 Cell Lines ................................................................................................................................. 22 2.7 Data Processing Softwares ....................................................................................................... 22 2.8 Other Materials ........................................................................................................................ 23 2.9 Techniques ............................................................................................................................... 23 CHAPTER 3: RESULTS .............................................................................................................. 33 v 3.1 Grape Seed Extract Inhibits the Activity of DT, ETA and Ricin ............................................ 33 3.2 Phenolic Compounds’ Screens against DT, ETA, Ricin, and ST ............................................ 38 3.3 Inhibition of CT Activity by Phenolic Compounds ................................................................. 41 CHAPTER 4: DISCUSSION ........................................................................................................ 50 LIST OF REFERENCES .............................................................................................................. 54 vi LIST OF FIGURES Figure 1: Molecular mechanism of action of DT. ................................................................................ 3 Figure 2: Molecular mechanism of action of ETA. ............................................................................. 5 Figure 3: Molecular mechanism of action of ricin. .............................................................................. 7 Figure 4: Molecular mechanism of action of ST. ................................................................................ 9 Figure 5: Retrograde trafficking of CT. ............................................................................................. 12 Figure 6: The chemical structures of 8 phenolic compounds. ........................................................... 16 Figure 7: The synthesis of DEA-BAG. .............................................................................................. 27 Figure 8: In vitro DEA-BAG assay. .................................................................................................. 28 Figure 9: Thermolysin assay for CTA1 unfolding. ............................................................................ 30 Figure 10: Vero-d2EGFP to monitor protein synthesis activity. ....................................................... 32 Figure 11: The effect of DT on Vero-d2EGFP signal. ...................................................................... 35 Figure 12: The effect of ETA on Vero-d2EGFP signal. .................................................................... 36 Figure 13: The effect of ricin
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