EDUCATION
Ph.D., Biochemistry/Microbiology, Division of Biological Science, University of Montana
B.S., Biology, Southeastern Oklahoma State University
PROFESSIONAL EXPERIENCE
UNIVERSITY OF TEXAS- PAN AMERICAN, Edinburg, Texas 2009- present
Assistant Professor
REPLIDYNE, INC., Louisville, Colorado 2000-2008
Senior Scientist II, Project Leader
ENZYCO INC., University of Colorado Health Sciences Center, Denver, Colorado 1998- 2000
Senior Scientist, Group Leader
UNIVERSITY OF NORTH CAROLINA, Chapel Hill, North Carolina 1996-1998
Postdoctoral Fellow
FUNDING
NIH Postdoctoral Fellowship Grant 1 F32 GM19117-01A1, NIH phase I and II SBIR Grant #R44 GM57121
NIH phase II SBIR Grant #GM54482
PUBLICATIONS, PATENTS and PRESENTATIONS
Thirteen patent applications, pending or issued. Two chapters in published books. Seventeen published manuscripts in major peer reviewed journals. Over thirty abstracts and presentations at scientific meetings. Several invited lectures and presentations.
SELECTED PUBLICATIONS
J Antimicro Chemo, (2009), 63(5):954-63
J.Mol.Biol. (1999), 288, 567-577
Antimicrob Agents Chemother,(2009), 53, 86-94
Protein Science, (2000), 9, 1791-1800
PNAS (2008), 105 (52), 20695-20700
J.Biol.Chem., (2000), 275, 20308-20314
J.Biol.Chem.,(2005), 280, 40465-40473
J.Biol.Chem., (2005), 280, 7890-7900
J.Biol.Chem., (2002), 277, 13246-13256
Biochim.Biophys.Acta (2000), 1490, 245-258
J.Biol.Chem., (2002), 277, 13401-13408
Biochim.Biophys.Acta (1999) 1446, 102-114
J.Mol.Biol. (1995) 252, 572-582
Biochemistry, (1998), 37, 1350-1356
Biochemistry, (1997), 36, 7951-7957
Biochim. Biophys. Acta, (1997), 1352, 91-101
RESEARCH
The existing armament of antibiotics for treatment of infectious disease is insufficient for protecting us in long term. The reason for this is the continuing evolutionary selection that results in bacterial resistance to currently used antibiotics. This is becoming more apparent in the hospital setting where resistance to current antibiotics is becoming alarming. This along with the new threats of bacteria as agents of bioterrorism has stimulated interest and funding opportunities for work in this field.
DNA Replication
A multi-subunit enzyme known as the DNA polymerase III holoenzyme carries out replication of bacterial chromosomes. Pol III is a complex composed of holoenzyme core (a2, e, q), the clamp-loading complex (DnaX3, d, d’, c, y) and the sliding processivity clamp (b2). I have identified all the members of the pol III holoenzyme from numerous bacteria, many that are of medical importance and have directed the reconstitution of the replicative holoenzyme polymerase from two Gram+ pathogenic organism and two Gram- pathogenic organisms and shown them to be functional. These systems were developed and used in target based screens to identify possible inhibitors and therefore development of antibacterials. My research plans in DNA replication entail putting together two new replication systems: one from a medically important pathogen and one from an organism that is important as an agent of bioterrorism. The research would be basic in nature and move in a direction that would allow a better understanding of DNA replication in organisms not previously studied. In DNA replication systems there are several components that interact so the scope of the research would be focused on a few important areas to be competitive for available funding.
Protein Synthesis
I developed a plan to put together a purified component protein synthesis system, from a model Gram (-), E. coli, and use this system to look for compounds that could inhibit protein synthesis. For protein synthesis to occur one needs a functional ribosome and several accessory proteins. Ribosomes were prepared and we cloned, expressed and purified recombinant EF-Tu and EF-Ts from E. coli. These are the proteins required to deliver charged tRNA to the ribosome during protein synthesis. E. coli EF-G, required for translocation of tRNAs on the ribosome, was also cloned, expressed and purified. Aminoacyl tRNA synthetases are enzymes required to attach amino acids to their cognate tRNA, thereby charging the tRNA were prepared. Using poly-U mRNA to program the ribosome a homogeneous peptide (poly-Phe) was synthesized. Using a radioactive labeled phenylalanine amino acid the amount of poly-Phe synthesized was monitored and in the presence of possible inhibitors the decrease of poly-Phe synthesis was followed. This provides a powerful tool in a cell-free environment to assay for compounds that have the potential to inhibit protein synthesis in bacteria.
Several focused small molecule compound groups were assayed against this system. We identified a compound series that had moderate inhibitory activity against this E. coli protein synthesis system. Medicinal chemistry collaborators prepared analogs of the compounds that inhibited the system and we carried out the beginnings of some structure-activity relationship (SAR) studies. Assays were designed to monitor the activity of each of the accessory proteins to determine if the activity of an accessory protein was inhibited or if in fact the function of the ribosome was inhibited. We carried out some initial experiments to determine if these compounds could inhibit the growth of Gram (- or +) bacteria in cell cultures. We found that the compounds had activity in particular against some respiratory pathogens including H. influenzae and S. pneumonae.
Functional ribosomes from both Pseudomonas aeruginosa and Bacillus subtilis have been isolated and additional research would be to put together the purified component protein synthesis system from each of these pathogens. These systems could be extended so that they contained all of the aa-RS enzymes and allow synthesis of native proteins from native mRNAs. This would be a novel endeavor and would have the potential to open new areas in cell-free protein synthesis as well as drug discovery.
