David B. Stephens, Ph.

D
Curriculum Vitae
7621 Leawood Blvd. Little Rock, AR 72205-2519 Work phone: 501-661-2801 Home phone: 501-291-4140 Cell phone: 501-772-7621 david.stephens@arkansas.gov

Education
1990 1996 University of Texas, Department of Chemistry and Biochemistry, Austin, TX . - Doctor of Philosophy in Biochemistry under the supervision of Professor Brent L. Iverson . - Title of Dissertation: "Polyclonal Catalytic Antibodies". - 5 years as Research Assistant and 4 years as Teaching Assistant. - Graduate-level coursework in Microbial Genetics and Computational Chemistry. 1983 1989 University of Arkansas, Little Rock, AR. - Bachelor of Science in Chemistry. - Bachelor of Science in Biology. Miscellaneous 1992 - Certified Electronics Technician Training, Austin Community College, Austin, TX 1988 - Electronic Design Technology, NRI, Washington, D.C.

Research Experience
2001-2005 Research as a part-time postdoctoral fellow into the role of Rab small GTPase proteins in vesicular trafficking, using the NIH model organism Dictyostelium discoideum , in the laboratory of Dr. John M. Bush at the University of Arkansas at Little Rock. Investigations into the role of the contractile vacuole as a calcium homeostasis organ, implied by the presence in association with the CV of a proton pump, a calcium-proton antiport, calmodulin, and Rab11 bearing a possible pH-dependent CaM-binding site, for which I was a winner of an NSF Research Opportunity Award fellowship in 2002. Skills Acquired: Dictyostelium cell culture. Transformation by electroporation GST-pulldown Mutagenic PCR. Shuttle vector manipulation. UV confocal microscopy with digital interface. Research grant writing and submission.

1995-1997 Research as a postdoctoral fellow into the solubilization, purification, and reconstitution of the transporter associated with antigen processing (TAP) in the laboratory of Dr. Matthew Androlewicz at the H. Lee Moffitt Cancer Research Center, University of South Florida. TAP is an ATP-hydrolytic peptide pump situated in the endoplasmic reticulum membrane, involved in the peptide charging of the major histocompatibility complex 1. The initial problem was the determination of conditions that would allow for the solubilization of this integral membrane protein without loss of biological activity. Experimental evidence has now been collected which demonstrates peptide binding by TAP solubilized under the proper conditions and reconstituted into proteoliposomes; I determined that an important factor in maintenance of peptide binding function was the replication of the proportions of different phospholipids found in the ER membrane, leading to the publication of the first paper detailing the reconstitution of peptide-binding activity in solubilized TAP. Research was performed on the suitability of immunoaffinity chromatography of solubilized TAP with competition elution utilizing the free TAP epitope, with inconclusive results. Hydrophobicity indices were generated for TAP primary sequences to aid in elucidation of transmembrane domains. Skills Acquired: Mammalian cell culture. Radioiodination of proteins. Liposome and proteoliposome formation. Membrane protein solubilization and reconstitution. Autoradiography. Research grant writing and submission. 1990 1996 Research as a graduate student into production, isolation, and characterization methods for polyclonal catalytic antibodies in the laboratory of Dr. Brent Iverson at the University of Texas at Austin. This research involved the organic synthesis of both transition-state-analog haptens and substrates, injection of and blood collection from laboratory animals, isolation of pure IgG from sera, and a variety of techniques for catalytic and immunoassay. My initial work determined that hapten-affinity chromatography cannot purify catalytic antibodies from whole IgG samples, a stumbling block to earlier attempts; both monoclonal and polyclonal catalytic antibodies failed to elute from a hapten-functionalized column under a wide variety of conditions. Whole IgG fractions were thus used, with preimmunization sera used as a control, and hapten titrations of catalytic reactions were used to estimate catalytic fraction. Studies of both the maturation of the catalytic immune response and variation of response between individuals were performed. Mathematical models predicting the behavior of different distributions of pooled heterogeneous catalysts were constructed. Subsequently, a substrate column for catalytic elution of abzymes was successfully developed, allowing for significant enrichment of the catalytic fraction in the IgG samples. Studies were performed to determine binding kinetics of catalytic fraction for hapten. Skills Acquired: Antibody production NMR spectroscopy. Affinity FPLC. Reverse-phase HPLC. Quantitative UV-Visible spectrophotometry. Enzyme-linked immunosorbent assay. Acrylamide and agarose electrophoresis. DNA ligation. Protein purification. Enzyme kinetics. Phosphoramidite and phosphonium synthetic techniques.

1988 1989 Research as an undergraduate utilizing X-ray crystallography of halo-substituted picolines and nicotinates in the laboratory of Dr. Alcuin F. Gremillion at the University of Arkansas at Little Rock. 1987 1988 Research as an undergraduate involving the characterization of the quaternary structure of deer malate dehydrogenase (EC 1.1.1.40) by starch gel electrophoresis in the laboratory of Dr. Alvan A. Karlin at the University of Arkansas at Little Rock.

Employment Experience
2005-Present Health physicist for the Radiation Control Section, Arkansas Department of Health and Human Services. Responsible for inspection of sources of ionizing radiation, shielding calculations for installations, and dose assessment modeling. Training in hazardous materials and radiological emergency response, environmental sampling, and computer modeling of environmental contamination and biokinetics. 2002 Botany Instructor at the University of Arkansas at Little Rock. 2000-2002 Instructor in Introductory Physical Science at Pulaski Technical College in North Little Rock. 1997-2002 Event Horizon Development (http://www.eventhorizondev.com/html/ehd.htm). Proprietor of scientific and technical consultation business. Expert witness for attorney Hubert Alexander in several cases. Development of web sites. Consultation and tutoring. Research into bioremediation techniques, information retrieval and analysis, and computational chemistry. Independent contractor as Internet resource researcher for Hungry Minds, Inc. (http://www.hungryminds.com/) for a column called Science in the News, StudyWeb (http://www.studyweb.com/) in the areas of Biology and Computational Chemistry, and the Open Directory Project (http://dmoz.org) in the area of Molecular Evolution. 1999 Visiting Assistant Professor of Chemistry, University of Central Arkansas, Conway, Arkansas. 1993-1994 Volunteer high school chemistry teacher, Marywood Children and Family Services, Austin, TX. 1990 Department of Radiochemistry, Arkansas Department of Health, Little Rock, AR Radiochemist working under Dr. George Dilbeck, responsible for the preparation and analysis of various environmental solid and liquid samples for alpha, beta, and gamma radiation counts. Experience in scintillation count, alpha-beta emission count, and gamma spectroscopy. Developed a standard for swabs used to test GC detectors for 63Ni leaks. 1988-1989 Quality Assurance laboratory technician for Tyson Foods of North Little Rock, AR Microbiological assays of product and plant surfaces for Escherichia coli , Listeria monocytogenes , Staphylococcus aureus, and Salmonella , activity titrations of hypochlorite and quaternary ammonium salt antiseptic solutions, free fatty acid titration of cooking oil to determine extent of oxidation, and statistical quality control.

Memberships
- American Chemical Society #2205461 - American Society for Cell Biology #48130 - Health Physics Society #34794 - Mensa.

Publications
Stephens, D.B., and Iverson, B.L.(1993) "Catalytic Polyclonal Antibodies", Biochemical and Biophysical Research Communications 192:1439-1444. Stephens, D.B., Wilmore, B.H., and Iverson, B.L.(1994) "Polyclonal Antibodies and Catalysis", Bioorganic and Medicinal Chemistry 2:1-6. Stephens, D.B., and Androlewicz, M.J.(1997) "Reconstitution of Peptide-binding Activity by TAP in Proteoliposomes", FEBS Letters, 416: 353-358. Stephens, D.B., Thomas, R.E., Stanton, J.F., and Iverson, B.L.(1998) "Polyclonal Antibody Catalytic Variability", Biochemical Journal, 332:127-134. Stephens, D.B., B.N. Mitra, and J.M. Bush (2003) "Analysis and function of Rab GTPases in Dictyostelium discoideum: Key regulators of vesicular membrane transport and multicellular development", Recent Research Developments in Cell Biology, 1:203-236.

Presentations
August, 1993 "Catalytic Polyclonal Antibodies", 206th ACS National Meeting, Chicago, IL. October, 1996 "Progress in the Purification and Reconstitution of Active Human TAP into Proteoliposomes", Suncoast Biomolecular Science Conference, Tampa, FL. December, 2003 "Null Mutation of the Dictyostelium Water Channel Gene AquaporinA (aqpA) Affects Contractile Vacuole Function, Fluid Phase Transport and Phagocytosis", American Society for Cell Biology Annual Meeting, San Francisco, CA

Awards
2002 NSF Research Opportunity Award 2005 Finalist for a CDC Emerging Infectious Diseases grant under the auspices of the Arkansas Department of Health.

Dissertation Abstract
Pointillism, a technique of painting in which small, distinct dots of pure color are applied in patterns to form an image, was developed by Georges Seurat in 1886; the most famous example of this technique may be the painting La Parade de Cirque , a summertime view of a Parisian park. In the perception of the beholder this myriad of distinct pigment points blends to form a smooth concerted view of the scene, a bright happy tableau of picnickers, strollers and boaters. The immune response can be compared to this visual phenomenon; a multitude of responding B-cell clones, each producing a pure antibody molecule with unique primary sequence and binding characteristics for the antigen, are brought to bear against the material stimulating the immune response, resulting in a smooth concerted response to this material. As first noted by Linus Pauling years ago, the immune response develops exquisite binding affinity and selectivity of antibodies for their ground-state antigens, whereas enzymes possess the same binding affinity and selectivity for the transition states of reactions; this observation ultimately gave rise to the concept of producing catalysts using the immune response to develop antibodies against transition state analogs. The advent of catalytic antibodies opened a unique new window for the study of the immune response and biocatalysis. The production of catalytic antibodies, with the use of the immune system as a molecular evolution engine to obtain antibodies possessing binding pockets with high transition state affinity, complements studies such as site-directed mutagenesis of enzymes admirably; the former works from the theoretical direction towards enzyme-like catalysis while the latter is capable of incremental dissection of known biocatalytic sites. The vast majority of catalytic antibody studies have been done with monoclonal antibodies, harvested from immortalized B-cell clones grown in culture. Such monoclonal approaches are preferred for mechanistic studies, but lack perspective as to the range of catalysts obtained and temporal course of response stretching the analogy given above a little further, these have been studies of each point of color in isolation, in a highly artificial environment. Monoclonal studies can also be expensive and time-consuming. Earlier attempts to isolate the full polyclonal catalytic response, in effect to look at the entire panoply of colors in the portrait of the response, had met with failure and had been abandoned. By careful construction of controls and avoidance of hapten affinity chromatography, we were able to characterize a complete immune and catalytic response in mice and rabbits to the phosphonium transition-state analog for the hydrolysis of the triphenylmethyl (trityl) ether protecting group; this is a good choice as there is no known enzyme catalyzing the hydrolysis of this linkage. Our results indicate that hapten affinity chromatography permanently removes and/or denatures the catalytic species present, hampering the earlier studies. Analysis of preimmunization serum for catalytic activity, along with competitive inhibition of immunized serum activity with free hapten molecules, allowed for characterization of the catalytic fraction in the absence of hapten affinity isolation. Initial results indicated an apparent catalytic rate enhancement (k cat/kuncat) of 125 and a Km of 31 M, comparable with monoclonals previously generated against the same moiety. The catalytic response exhibited a later and steeper rise than the immune response, implying onset of catalysis as the immune response matures and antibody-hapten affinity increases. Subsequent experiments showed surprising levels of homogeneity of catalytic response both within and between individuals. Plasmon resonance techniques were used to study binding kinetics, a substrate column was developed to effect catalyst enrichment by activity, and a mathematical treatment of the behavior of pooled catalysts was developed.

Contacts
Dr. Brent L. Iverson University of Texas at Austin Department of Chemistry and Biochemistry 1 University Station A5300 Austin, TX 78712 (512) 471-5053 biverson@mail.utexas.edu Bernard Bevill Radiation Control Section Chief Arkansas Department of Health 4815 W. Markham, Slot 30 Little Rock, AR 72205 (501) 661-2107 brbevill@arkansas.gov Donald Greene Preparedness and Response Arkansas Department of Health 4815 W. Markham, Slot 30 Little Rock, AR 72205 (501) 661-2808 dgreene@arkansas.gov David Snellings Health Systems Licensing and Regulation Arkansas Department of Health 4815 W. Markham, Slot 30 Little Rock, AR 72205 (501) 661-2319 David.Snellings@arkansas.gov