Summer Undergraduate Research Symposium
Thursday, 14 August 2003
McKinly Laboratory
University of Delaware
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Abstracts for Oral Presentations in 061 McKinly Laboratory 1:00 – 3:00 PM

1:00 PM  Steve Brohawn 2:00 PM  Amy van Fossen
1:15 PM  Jennifer Risser 2:15 PM  James Parris
1:30 PM  Christopher McAndrew 2:30 PM  Arthur Suckow
1:45 PM  Alaina Brown 2:45 PM  Amanda Barker

1:00 PM  Role of Metal Ions in Oxidative Protein Folding:
Do flavoprotein sulfhydryl oxidases need metals?

Stephen G. Brohawn, Irena Rudik Miksa, and Colin Thorpe
Department of Chemistry and Biochemistry

Each disulfide bond generated during the oxidative folding of secreted proteins requires removal of 2 electrons.  In higher eukaryotes, sulfhydryl oxidases have been found to catalyze this process with reduction of oxygen to hydrogen peroxide.  Both metal-dependent and flavin-dependent classes of these oxidases have been described.  The first protein sequence of a metal-dependent enzyme, from a copper-containing skin sulfhydryl oxidase, has been recently published and is 51% identical to the sequence for a flavin-dependent sulfhydryl oxidase already studied extensively in our laboratory.  Thus we were concerned that our work on the emerging flavin-dependent sulfhydryl oxidases had missed an important additional copper cofactor.  The present study establishes that the best-studied flavin-dependent oxidase contains no significant copper or other metals, and that copper and zinc are inhibitors of disulfide bond formation.  Studies with zinc as a model divalent metal show that it binds to CxxC centers and drastically perturbs the flow of reducing equivalents in this multi-domain enzyme.  Similarly, copper binds within the active center close to the flavin prosthetic group.  Our metal analyses show that the flavin-dependent oxidase is prone to bind metals and this may have misled earlier workers.  Thus the evidence that the skin sulfhydryl oxidase requires copper (rather than flavin) for enzyme activity should be reexamined. Supported by Beckman Scholars Program and NIH  GM26643.

1:15 P.M. Expression Patterns of Orm1 and Angptl4 During Adipocyte Differentiation

Jennifer Risser, Amanda Peters, John David and David C. Usher
Department of Biological Sciences

Communication between adipocytes and endothelial cells is essential for regulating the amount of free fatty acids allowed to transverse the endothelial layer.  Serial analysis of gene expression (SAGE) used to obtain gene expression profiles of 3T3 L1 adipocytes and preadipocytes suggested two genes potentially responsible for such communication, orosomucoid (Orm1) and angiopoietin-like 4 (angptl4) are expressed.  The concentration of message during differentiation of mesenchymal cells to adipocytes was measured using Real-Time RT-PCR.   Trizol was used to extract RNA from cells harvested daily for two weeks.  An ABI Prism 7000 with SYBR-green detection was used to quantify the relative concentrations of mRNA for the two genes.  After induction of differentiation the 3T3 L1 cells begin to accumulate lipid around day 3 and reach capacity around day 7.  Orm1 expression increased 30-fold and angptl4 expression increased 6-fold greater than the internal marker tata-box binding protein (tbp).   Orm1 and Angptl4 are both upregulated early during differentiation, around day 3.  Orm1 reaches its maximum expression at day 12, whereas angptl4 reaches maximum expression around day 3 and remains static throughout the rest of differentiation.  These results along with previously published data suggest that orm1 and angptl4  secretion by adipocytes may inhibit fatty acid uptake by adipose tissue. Funded by Charles Peter White.

1:30 P.M.   Purification and Characterization of Paraoxonase (PON1)

Chris McAndrew, Ahkil Khanal, and Brian Bahnson
Department of Chemistry and Biochemistry

Human plasma paraoxonase (PON1) has been shown to have arylesterase and paraoxonase activity. This high-density lipoprotein (HDL) associated enzyme exhibits antiatherogenic properties and acts as a detoxifying agent for several chemical warfare agents and insecticides. We show that the reported purification process (Gan et al.) contains a ~68kDa co-purifying contaminant. We have developed a modified procedure using size exclusion chromatography to obtain pure PON1 from human serum. In order to support the current homology model of PON1 developed using the crystal structure of DFPase as a model, a CD spectrum of pure, monodisperse PON1 was measured. Previous attempts were inconclusive due to a high background caused by detergent micelle light scattering. The detergent free form of PON1 has been characterized to exist as monomer, dimer, and higher order soluble aggregates. Thus, detergents are necessary to retain native, monodisperse enzyme. In conjunction with our modified purification procedure, Triton X-100 was exchanged for n-Octyl-beta-D-glucopyranoside (bOG), a detergent with a critical micelle concentration (CMC) of 20mM. Also, bOG was used because of its characteristically low absorbance from 190-250 where CD measurements are made. Buffer concentrations were manipulated to obtain the lowest absorbance in this region without losing enzymatic activity to obtain CD spectra. Although the activity and oligomeric states of PON1 have been characterized in a number of detergents, no data was previously available for its presence in bOG. PON1 was found to have a slightly lower rate of phenylacetate hydrolysis in the presence of this detergent, indicating it may cause slight conformational modifications to the structure of the enzyme. With this information and conclusive CD spectra data, the structure of PON1 and the current homology model can be further characterized. Supported in part by the HHMI Undergraduate Science Education Program.

1:45 P.M. A Thermodynamic Propensity Scale for Type II Polyproline Helices

Alaina M. Brown, and Neal J. Zondlo
Department of Chemistry and Biochemistry

Although type II polyproline (PPII) helices and proline-rich peptides are common structures in globular proteins, little is known about their stability, dynamics, and fundamental energetics. The ability to predict regions of high propensity for PPII helices is crucial to the analysis of PPII-mediated intermolecular and intramolecular interactions.  Sequences of three or more consecutive prolines induce PPII helix formation.  A model peptide (Ac-GPPXPPGY-NH2) was designed with two proline residues on each side of a randomized position, X.  Peptides with each of the twenty amino acids in the randomized position were tested using circular dichroism (CD) spectroscopy to determine the relative stabilization effect on propagation of a PPII helix.  The CD signal of each peptide was compared to the CD signal of the reference peptide (X=P) at 228 nm to determine the relative PPII helix stability.  Beta-branched amino acids (deltadeltaG~ 0.6 kcal/mol), cysteine, serine, and asparagine (deltadeltaG~ 0.3 kcal/mol), were all found to have a significant destabilizing effect on the PPII helix, while all other amino acids were moderately worse than proline (deltadeltaG~ -0.2 kcal/mol).  Helicity was temperature-dependant and pH-dependant for charged residues, but independent of salt and peptide concentration.  This research was funded by a Howard Hughes Medical Institute grant, the American Heart Association, and start-up funds from the University of Delaware for Dr. Zondlo.

2:00  P.M.  Stress Response to GPCR Expression in Yeast

Amy VanFossen and Anne Skaja Robinson
Department of Chemical Engineering

A G-protein coupled receptor (GPCR) is an integral membrane protein that helps to regulate a cell’s response to signals (or molecules).  GPCRs are believed to play a role in heart disease and cancer, so research about these proteins could lead to improved treatment of these conditions. The main goal of our research with GPCRs is to produce large amounts of functional protein, using the host system yeast, so detailed structural information of the proteins can be obtained.  In particular, I have been using the human A2a adenosine receptor (A2a).  A problem faced in previous research with the A2a protein is that there is a slow down in the production of A2a over time.  To understand the mechanism for changes in A2a production, we investigated changes in cell regulation.  The investigation focused on the potential effects of cytosolic stress due to the expression of the foreign protein.  This was tested by using a lac Z reporter gene fused to a heat shock protein promoter in the yeast cells that are expressing A2a.  If the heat shock protein promoter is activated, the lac Z gene will be turned on, which will produce beta-galactosidase in the cell.  A beta-galactosidase assay was performed to determine the relative amounts present in the cells over time during A2a expression.  Preliminary results indicate that a typical stress response pathway is being activated during A2a expression.  This research was supported in part by HHMI undergraduate research funding (ALV) and NSF BES59984312.

2:15 P.M.    JAM-1 Expression during Vasculogenesis and Early Organogenesis

James J. Parris, Patrick B. Kelley, Melinda K. Duncan, and Ulhas P. Naik
Department of Biological Sciences

Cell adhesion molecules of the Ig superfamily play an important role in embryonic development. We have recently shown that JAM-1, a member of this family, is involved in endothelial cell adhesion and migration leading to angiogenesis. We hypothesized that JAM-1 may also play a role in vasculogenesis; however, embryonic expression of JAM-1 is not well characterized. In order to understand the role of JAM-1 in vascular development/function, we studied the expression of JAM-1 during early embryonic development by generating transgenic mice in which a lacZi gene was knocked into the JAM-1 gene. JAM-1 gene expression was determined using histochemical staining for beta-galactosidase in mouse embryos ranging from 9.5 to 12.5 days post coitum (dpc). As expected, JAM-1 is expressed at sites of vasculogenesis, namely, intersomitic vessels, as early as 9.5 dpc. Expression in blood vessels is continuous through 12.5 dpc where staining is clearly visible in both the head and tail of the embryo. JAM-1 is also detected in the otic and olfactory placodes, which form the epithelial linings of the inner ear and nose, and the epithelia of the developing kidneys and lungs, which undergo branching morphogenesis. Thus, JAM-1 may function in the epithelial development of certain tissues. JJP funded by the HHMI Undergraduate Science Education program.

2:30  P.M.    The Ocular Expression and Regulation of Junctional Adhesion Molecule-1

Arthur T. Suckow, Vesselina Cooke, Ulhas P. Naik, William Skarnes,
Bharesh K. Chauhan, Ales Cvekl and Melinda K Duncan
Department of Biological Sciences

Junctional adhesion molecule-1 (JAM-1) is a member of the immunoglobin superfamily involved in the organization of tight junctions and the regulation of leukocyte transmigration.  Recently, a cDNA microarray analysis of transgenic mice overexpressing PAX-6 in lens fiber cells revealed that JAM-1 mRNA expression was 2.5 fold elevated over normal.  This data suggested that JAM-1 gene expression is regulated by PAX-6, a transcription factor essential for normal eye development.  The overexpression of JAM-1 in the PAX-6 transgenic lenses of adult mice was confirmed by RT-PCR.  A LacZ-Neor fusion genetrap was used to disrupt the JAM-1 gene in ES cells to create knockout mice and detect JAM-1 gene activity via B-galactosidase expression.  In the lens, JAM-1 gene activity is detected in the epithelium, cells where high levels of PAX-6 are detected.  Levels decrease during fiber cell differentiation coincident with the downregulation of PAX-6 expression.  In the cornea, the JAM-1 gene is active in the corneal epithelium, a region that requires PAX-6 for normal morphogenesis.  Analysis of JAM-1 null mice revealed a down-regulation of Jam-1 gene expression in the corneal epithelium, suggesting JAM-1 may indirectly regulate its own expression.  Further, histological analysis of the mice demonstrated that the corneal epithelia is thicker than normal and lack a normal squamous layer in the outermost layer of the corneal epithelia; thus, JAM-1 is essential for normal corneal morphogenesis.

2:45  P.M.  Homology Modeling and Analysis of the Human NK2R Ligand Binding Domain

Amanda Barker*, Jason M. Winget+, and Clifford R. Robinson*+
*Department of Chemical Engineering, +Department of Chemistry and Biochemistry, †The Delaware Biotechnology Institute

Approximately 60% of drugs on the market today target members of the G protein-coupled receptor family.  GPCRs are a family of proteins that mediate signaling events in cells throughout the body.  These proteins are characterized by seven hydrophobic transmembrane helices connected by alternating intra- and extracellular loops.  Standard structure determination techniques cannot be easily applied to GPCRs, due to the hydrophobic nature of the transmembrane regions.  Determination of the three-dimensional structure of GPCR ligand recognition sites would result in more effective drug design.  Our goal is to use computational methods to generate models for GPCR ligand binding pockets, and to use protein engineering to produce variants of the receptors corresponding to these models.  Computational three-dimensional representations of two GPCRs, the Neurokinin 1 and 2 receptors, were utilized to generate models of the proteins’ ligand binding pockets.  It was determined that the majority of contact points with the ligand occurred along the extracellular loops of the GPCRs, which is in accordance with previous experimental evidence.  These models will assist in the generation of water-soluble GPCR homologues.  We will use genetic engineering to produce these homologues and purify them for structural and ligand binding studies.  Their low hydrophobicity will make them more tractable targets for structural studies and high-throughput drug screening.

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Created 29 July 2003, Last revised 8 August 2003 by Hal White
Copyright 2003 Harold B. White, Department of Chemistry and Biochemistry, University of Delaware