Jim Delaney, MIT - 1st place oral presentation
Influence of AlkB on 1,N2-ethenoguanine genotoxicity and mutagenicity in E. coli
James C. Delaney1, Ivan D. Kozekov2, Albena Kozekova2, Cintyu Wong3, Catherine L Drennan3, Lawrence J. Marnett2, Carmelo J. Rizzo2, and John M. Essigmann1. (1) Departments of Chemistry and Biological Engineering, Massachusetts Institute of Technology, Room 56-669, 77 Massachusetts Avenue, Cambridge, MA 02139, Fax: 617-253-5445, delaney@mit.edu, (2) Department of Chemistry, Center in Molecular Toxicology and the Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235, (3) Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
A multitude of pathways exist for the formation of DNA damage from exogenous and endogenous reactive agents. One class of damage creates etheno-DNA lesions, whereby an unsaturated two-carbon unit bridges the exocyclic amino group and a ring nitrogen of either the adenine, cytosine, or guanine base. We have previously discovered that etheno-A and etheno-C are substrates for AlkB, which can directly reverse the etheno damage by epoxidation of the double bond, followed by hydrolysis of the epoxide to the diol, and jettisoning of the two carbon bridge as glyoxal. In the present work, we constructed a single-stranded viral genome containing 1,N2-ethenoguanine, which was passaged through AlkB(+) and AlkB(-) E. coli. Assays previously developed in our lab were used to assess the genotoxicity and mutagenicity of the lesion. We find that 1,N2-ethenoguanine gives predominantly -1 deletions, regardless of cell strain; however, G to A and G to T point mutations predominate when SOS-bypass polymerases are induced. While AlkB does diminish the amount of -1 deletions (and the amount of point mutations), the enzyme does not appear to act with vigor on 1,N2-ethenoguanine, as it weakly repaired an oligonucleotide containing the lesion (with respect to etheno-A), as analyzed by ESI-TOF mass spectrometry.
Derrick Seiner, University of Missouri-2nd place oral presentation (tie)
Inactivation of protein tyrosine phosphatase 1B (PTP1B) by the endogenous/dietary aldehyde acrolein
Derrick R. Seiner1, Jason LaButti1, and Kent S. Gates2. (1) Department of Chemistry, University of Missouri-Columbia, 601 South College Avenue, Columbia, MO 65211, drs4zc@mizzou.edu, (2) Departments of Chemistry and Biochemistry, University of Missouri - Columbia, Columbia, MO 65211
PTP1B dephosphorylates the insulin receptor and the insulin receptor substrate, thus serving as the primary negative regulator that "switches off" the insulin signaling pathway. Inhibition of PTP1B activity can potentiate the action of insulin, thus yielding significant biological effects. The alpha,beta-unsaturated aldehyde acrolein occurs naturally in the human diet and is a product of endogenous lipid peroxidation. In the work presented here, we have examined the inactivation of PTP1B by acrolein and have investigated the mechanism underlying phosphatase inactivation by this endogenous aldehyde.
Kyle Brown, Vanderbilt University, 2nd place oral presentation (tie)
Translesion synthesis past the Me-FAPy adduct in oligonucleotides by DNA polymerase
Plamen P. Christov1, Ivan D. Kozekov2, Kyle L. Brown1, Michael P. Stone2, Thomas M. Harris1, and Carmelo J. Rizzo2. (1) Department of Chemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, P.O. Box 1822, Nashville, TN 37235, plamen.p.christov@vanderbilt.edu, (2) Department of Chemistry, Center in Molecular Toxicology and the Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235
Methylating agents react with DNA to give a cationic N7-methyl deoxyguanosine adduct, which can undergo hydrolytic opening of the imidazole ring to form 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (Me-FAPy) 1. We developed a four step synthesis of phosphoroamidite 2 in 24% overall yield, which allowed the preparation of oligodeoxynucleotides containing the Me-FAPy lesion. The in vitro replication of the Me-FAPy lesion was examined with eukaryotic and prokaryotic DNA polymerases and the full-length extension products were sequenced by tandem mass spectrometry. The Me-FAPy adduct was found to be mis-coding however, only the initial insertion of dCTP opposite the adduct could be extended.
Ricardo Vidales, MIT - 1st place grad student poster
Development of an in vitro model for the study of drug-induced bile acid synthesis and transport dysfunction in a 3-D tissue engineered liver microreactor culturing system
J. Ricardo Llamas Vidales1, Ju Liu1, Jung Nyun Kim1, Sharon Karackattu1, Michal Bokayza1, Keith Hoffmaster2, Steve R. Tannenbaum3, and Linda G. Griffith4. (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge, MA 02139, rllamas@mit.edu, (2) Pfizer Research Technology Center, Cambridge, MA 02139, (3) Division of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, (4) Department of Chemical and Biological Engineering, Center for Biomedical Engineering, and Biotechnology Process Engineering Cent, Massachusetts Institute of Technology, Cambridge, MA 02139
Drug-induced bile acid synthesis and transport dysfunction studies in in vitro liver cultures aredifficult to conduct with current widely-used in vitro models due in part to rapid dedifferentiation of primary hepatocytes in culture. A 3D liver microreactor system was developed with the goal of maintaining hepatic functions in long-term primary hepatocyte cultures. Microreactor primary rat hepatocyte culture bile acid synthesis rates have been measured to be closer to physiological and much higher than that of hepatocyte cultures in widely-used in vitro models. Taurocholic acid accumulation data suggests these cultures preserve twice the bile acid transport activity than widely-used in vitro models. Accumulation data also suggests microreactor cultures develop functioning bile canaliculi-like structures, consistent with imaging data. Future work focuses on measuring effects of cholestasis-inducing compounds on bile acid transport and synthesis kinetics, as well as its effect on expression of relevant transporters and enzymes.
Derrick Seiner, University of Missouri- 2nd place grad student poster
Effect of sequence context on rate and yield of interstrand crosslinks generated by abasic sites in duplex DNA
Derrick R. Seiner1, Sanjay Dutta1, and Kent S. Gates2. (1) Department of Chemistry, University of Missouri-Columbia, 601 South College Avenue, Columbia, MO 65211, drs4zc@mizzou.edu, (2) Departments of Chemistry and Biochemistry, University of Missouri - Columbia, Columbia, MO 65211
Abasic sites are one of the most common lesions in cellular DNA. We recently characterized an interstrand crosslink generated by an abasic site in duplex DNA. In the work presented here, we have examined the effects of sequence context on the rate and yield at which a crosslink forms with a guanine residue in the 5'-CAp-3' sequence. In addition we report on the generation of abasic-site-derived interstrand crosslinks with nucleobases other than guanine.
Jennifer Seal, MIT, - 1st place postdoctoral poster
Identification of nitrated proteins from iNOS-induced macrophages using biotin labeling and capture
Jennifer R. Seal, Biological Engineering Division, MIT, 77 Massachusetts Ave, Cambridge, MA 02139, jseal@mit.edu, John S. Wishnok, Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, and Steven R. Tannenbaum, Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139
Tyrosine nitration is an important post-translational modification that is associated with oxidative stress and activation of nitric oxide synthases, leading to increased production of nitric oxide. Described here is a method that combines specific isolation and enrichment of nitrated proteins from complex biological systems. Proteins modified with nitrotyrosine were reduced to their corresponding aminotyrosine followed by biotin coupling. Biotinylated proteins were then enriched by avidin capture followed by 2D gel electrophoresis. Proteins were identified by mass spectrometric methods including confirmation of structure by peptide mass fingerprinting and LC/MS/MS, with data analysis by MASCOT and Spectrum Mill. This method was applied to the analysis of macrophages activated to produce nitric oxide which, through formation of other reactive nitrogen species, leads to tyrosine nitration.
Kun Song, SUNY Stony Brook - 2nd place postdoctoral poster
Computational analysis of the lesion recognition mechanism of formamido-pyrimidine DNA glycosylase
Kun Song1, Carlos De los Santos2, Arthur P. Grollman3, and Carlos L. Simmerling1. (1) Department of Chemistry, Stony Brook University, Stony Brook, NY 11794, kunsong@gmail.com, (2) Department of Pharmacological Sciences, State University of New York, Stony Brook, NY 11794, (3) Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794-8651
8-oxo-guanine (8OG) is the most prevalent form of oxidative DNA damage. In bacteria, 8OG is excised by formamidopyrimidine glycosylase (Fpg) as the initial step in base excision repair. To efficiently excise this lesion, Fpg must discriminate between 8OG and an excess of guanine in duplex DNA. In this poster, we discuss the source of the high degree of selectivity from both structural and energetic basis. The origin of the translocation barrier and the difference between 8OG and normal guanine are investigated. The roles of key residues to the lesion recognition have been analyzed. The comparisons between the enzyme/DNA complexes containing 8OG and normal guanine provide novel insights into the recognition of 8OG by Fpg. Targeted MD simulations are also used to simulate the process of the enzyme/DNA translocation and base eversion. Structural comparison has also been made between Fpg and hOGG1, Fpg's functional homolog in human.
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