Abstracts from the Department of Biological Sciences
Undergraduate Summer Research Symposium August 9, 2006

Ordered alphabetically by student's last name

Koemeter-Cox Madden McLaughlin Neal Parker Rocha
 Shankman Stallings,1,2 Tseng Zony,N
Lavender 1  2 Maguire,C Michael Ogbonna Patel,T Romaniello
 Sinanan Sterling 1 2 Vasta
Lebois Maguire,K Mills Oren Pomroy Samant Smith,S Stokes,1,2 Woody
Lewis Manrai Mlynarski Ovadje Reed Scott Snow Swain Yehiri

Cholesterol Depletion in Adipocytes and its Affects on Lipoproteins E and C1
Marysol D. Lavander, Sara Sterling, and David Usher
Department  of Biological Sciences

Apolipoprotein (apo) C1, a 6.6-kD protein, is a protein constituent of triglyceride (TG) -rich chylomicrons, very low density lipoproteins (VLDL) and high density lipoproteins (HDL). The human APOC1 gene is located 5 kb downstream of the human APOE gene on chromosome 19 in the same transcriptional orientation. ApoE mediates the uptake of triglyceride-rich lipoproteins (TRL) by hepatic receptors, where as apoC-I has an inhibitory effect. Both these proteins appear to be involved in cholesterol efflux in adipocytes. In order to find how this process is controlled, the expressions of APOE and APOC1 were tested in 3T3-L1 mouse adipocytes treated with β-cyclodextrin, triiodothyronine (T3) and a combination of β-cyclodextrin and T3. The results showed that β-cyclodextrin decreased expression of the two genes 7-10 fold, T3 decreased expression 2-4-fold and the combination of β-cyclodextrin and T3 inhibited expression 9-14 fold. The results suggest that cholesterol depletion or stimulation of carbohydrate metabolism causes cells to attempt to increase the cholesterol concentration within the cell. This may be due to the formation of lipid droplets which require a boundary of cholesterol, phospholipids and protein. Sponsored by INBRE.

A Role for N-Glycosylation in Differential Growth Control and Drosophila Development:
Phenotypic Analysis of alg10
Evan Lebois and Erica Selva
Department of Biological Sciences

<>Proteins involved in cellular signaling must traverse the secretory pathway where they can undergo a variety of different posttranslational modifications before they can execute their function extracellularly.  In this study we begin to phenotypically characterize Alg10, an enzyme that adds the terminal glucose residue to the growing dolichol-linked oligosaccharide during N-glycosylation prior to its en masse transfer to a nascent polypeptide.  N-glycosylation is often required for correct protein folding and may dictate specificity of extracellular interactions.  Genetic and molecular approaches were developed to characterize the alg10 loss of function phenotype during Drosophila development.  While pleotrophic phenotypes were observed in alg10 mutant embryos, clonal analysis of the alg10 mutation in the developing wing yielded a smaller and rounder adult wing phenotype as compared to wild type.  This implicates a potential link between glycosylation and pathways governing growth control.  Both the Insulin receptor (InR) signaling pathway and the Epidermal growth factor pathway acting through c-myc are known to control growth in Drosophila, roles that are conserved in mammals. To begin to address whether alg10 disrupts InR signaling, I examined the genetic interaction between alg10 and an activated form of InR that produced large disordered eyes.  By removing half the Alg10 activity in this background, an enhanced phenotype was observed with an irregular eye surface, fused ommatidia, and patches of necrotic tissue.  This indicates a genetic interaction between alg10 and the dInR pathway and suggests alg10 influences InR mediated growth control. Supported by HHMI Undergraduate Science Education Grant.


Effects of TGF-β on Prostate Cancer Cell Adhesion to Bone Endothelium under Static and Flow Conditions.
M. Christopher Lewis, II1, Fayth L. Miles2, Jeremy Molligan3, David DeGraff4, Robert A. Sikes5, Carlton R. Cooper6
1Biological Sciences, Lincoln University, 2Cancer Biology Laboratory, Univ. of Delaware, 3Laboratory for Cancer Ontogeny and Therapeutics, Univ. of Delaware

Prostate cancer (PCa) has a proclivity to metastasize to bone. Surgery can eliminate the primary tumor but cannot remove secondary cancer growths, metastases, that have spread throughout the body, e.g. bone for PCa. Once metastasized to bone, these growths cause great pain, inflammation, and increased susceptibility to fractures. Treatment of tumors in bone is very difficult and palliative at best with no curative therapy. Transforming growth factor β1 (TGF-β1) is a peptide growth factor, which through membrane receptors, decreases proliferation and induces differentiation or death in normal cells. TGF-β1 has been found to stop growth of primary, early stage prostate tumors, however, as tumor cells become more aggressive they adapt to the presence of TGF-β1 and respond by increasing expression of extracellular matrix and integrins used for metastasis. The purpose of this study is to understand the role of TGF-β1 in the metastatic process by examining the effects of TGF-β1 treatment on the cellular interaction between PCa cells and bone marrow-derived endothelial cells (BMEC) in vitro. Adhesion assays were used to compare TGF-β1 treated PCa cell adhesion to BMEC versus vehicle treated PCa cell adhesion. Our data shows that C4-2 and C4-2B4, highly metastatic PCa cell lines, have decreased adhesion to BMEC when treated acutely with TGF-β1, while LNCaP cells, which are notably less metastatic, show a marked increase in adhesion. As TGF-β1 is a major cytokine expressed in BMEC, these data support a potential role for this cytokine in the transmigration of PCa cells through BMEC. Funded by IGERT.

Control of Cellular Elasticity by Actin Organization in Osteoblasts
Gregory Madden, Randall L. Duncan, Elizabeth L. Adams, and Jinsong Zhang
Department of Biological Sciences

Osteoblasts respond to mechanical stimuli with increased intracellular signaling and gene expression that ultimately results in bone formation, in vivo.  However, osteoblastic mechanosensitivity is lost upon continued stimulation.  The actin cytoskeleton rapidly polymerizes into stress fibers in response to mechanical loading and we predict that this increase in organization may induce mechanical desensitization.  Parathyroid hormone (PTH) is known to enhance the response of bone to mechanical loads and destabilize the actin cytoskeleton.  We hypothesized that administration of PTH will increase cellular elasticity through disruption of actin.  The cellular elasticity of MC3T3-E1 osteoblasts was quantified using Atomic Force Microscopy [AFM] in ramping mode to establish the stress-strain curve of the cell.  These studies demonstrated that the elasticity of an osteoblast increased two-fold following 50 nM PTH treatment for 30 minutes.  These results suggest that PTH decreases the intracellular tension of osteoblasts by disrupting the actin cytoskeleton.  Continuing studies will identify the effects of shear-induced stress fiber formation on elastic modulus as well determine the changes in elasticity in response to cytoskeleton disrupting agents such as cytochalasin D, latrunculin, and jasplakinolide.  These studies will define the role of cytoskeletal actin organization in response to mechanical loading and provide insight into the cellular mechanisms associated with bone loss in degenerative diseases such as osteoporosis as well as the loss of bone during microgravity experienced in space flight.  Supported by the Howard Hughes Medical Institute and the National Institutes of Health.

Engineering Expression Constructs for the Study of IGFBP-2 Proteolysis
in Metastatic Androgen Independent Prostate Cancer
Christine J. Maguire, David J. DeGraff, and Robert A. Sikes
Department of Biological Sciences

Prostate cancer (PCa) is the most frequently diagnosed non-cutaneous malignancy in American men. As a majority of PCa associated deaths result from the formation of metastatic lesions in bone, there is an intense effort to identify molecular targets to prevent this deadly occurrence. One such therapeutic target is the Insulin-like Growth Factor (IGF) axis. The IGF axis consists of two ligands (IGF-I and IGF-II), two receptors (IGFIR and IGFIIR), and a family of IGF binding proteins (IGFBP 1-6).  While previous studies indicate IGFBP-2 overexpression plays a role in the metastatic behavior of a variety of human cancers, the role of IGFBP-2 in metastatic PCa is unknown.  We are interested in the role of IGFBP-2 in PCa metastasis.  In an effort to mirror clinical PCa in vitro, we utilize the LNCaP human PCa progression model in our IGF studies. This model system consists of the androgen-sensitive (AS) and non-metastatic LNCaP PCa cell line, as well as a series of lineage-related, androgen-insenstive (AI) and metastatic cell lines. Upon 48 hours androgen treatment, LNCaP induces IGFBP-2 proteolysis, while identically treated AI C4-2 has a greatly attenuated ability to do so. We hypothesize that IGFBP-2 proteolytic fragments will decrease the metastatic behavior of C4-2.  The goal of this project is to engineer and express constructs representing the amino acid sequences of IGFBP-2 proteolytic fragments secreted by androgen treated LNCaP.  The use of surrogate assays will then allow for the identification and measurement of any fragment-associated antimetastatic effects on AI and metastatic C4-2. This project was funded by the INBRE program of the Delaware Biotechnical Institute, supported by NIH grant 2 P20 RR016472-06 from the NCRR.

Analysis of the Targeted Gene Repair Transcriptome in Mammalian Systems
Arjun K. Manrai1, Luciana Ferrara2, Hetal Parekh-Olmedo2, and Eric B. Kmiec2
1Department of Physics, Harvard University, 2Department of Biological Sciences, University of Delaware  

Targeted Gene Repair is the process in which a modified single-stranded oligonucleotide directs the exchange of a single mutated DNA base. This correction is site-specific, utilizing the cell’s endogenous repair mechanisms. Upon correction induced by the oligonucleotide, many corrected cells cease active replication, and key proteins of senescence pathways have been implicated through increased activation. We perform the gene repair reaction on DLD-1 cells and carry out microarray analysis using cell-cycle specific oligonucleotide microarrays to profile the transcriptome of gene-repaired cells at 8, 24, and 48 h. After mathematically correcting for background and normalizing the data, we employ biocomputational tools and analyses to construct a regulatory model and explain the response of cells to gene repair. We then use insight gleaned from this experiment to develop and implement a framework for further transcriptional analysis of targeted cells. Specifically, we create and program a model for global expression analysis of targeted cells to be used on data obtained from (Affymetrix) transcriptome-wide arrays, capturing more than 47,000 transcripts. The framework integrates the latest probe-mapping, normalization algorithms, cluster analysis, ontological information, motif finding, and pathway systems-level information into a pipeline for the upcoming analysis. Moreover, we tailor our pipeline to be specifically adapted to future large-scale gene repair microarray experiments using information obtained from the DLD-1 study, by developing computational tools to extract and compile pathway-specific data. This is the first study to obtain and analyze transcriptional profiles of targeted DLD-1 cells and use these analyses for an integrated framework and we hope that, in addition to providing information about the observed regulatory pathways of targeted cells in vitro, this work provides valuable insight for future in vivo gene repair applications. This project was supported by NIH NCRR INBRE grant to Delaware, grant number 2P20RR016472-04, and NIH grant RO1 CA89325.

Seeing Beyond the Human Eye with Modern Microscopy
Eamon McLaughlin1, Kirk Czymmek1,2, Debbie Powell2, Jeanette Miller2, and Liz Adams2
1Department of Biological Sciences and 2Delaware Biotechnology Institute

My project involved the task of creating a way to educate local high school science teachers about the microscopy equipment located in the Bio-Imaging Center at the Delaware Biotechnology Institute (DBI).  The teachers would have a background in the sciences, but no real exposure to the multitude of cutting-edge microscopy equipment located in this facility.  I designed a professional looking PowerPoint presentation displaying the concepts, capabilities, applications and example images of the microscopy equipment to accomplish this goal.  The imaging equipment that was included in my project included a multiphoton/confocal microscope, field emission scanning electron microscope, transmission electron microscope, laser capture microdissection microscope, and atomic force microscope.  For each microscope a brief history and description of the microscope was included in the presentation, followed by images, original animations displaying the basic concepts, and finally example images with real world applications from each microscope.  A chart depicting the range of resolution for each microscope, along with images of objects that would be found at each within the range, was also included to address the often difficult and abstract concept of scale at the microscopic level.  It is expected that this presentation will not only inform the teachers of the capabilities of these technologies, but also inspire a younger generation of scientists to consider the Life Sciences as a career and to use modern microscopy to do cutting edge science.   This project was funded by the National Science Foundation and Delaware EPSCoR.

Vesicular Transport and Excess Residual Cytoplasm in Sperm
Rowan Michael, Minghai Shao, Genevieve Griffiths, and Patricia A. Martion-DeLeon
Department of Biological Sciences

The retention of excess residual cytoplasm (EC) on the sperm flagellum during spermiogenesis is associated with male infertility in a wide variety of mammalian species, including humans. Recent studies have shown that transgenic over-expression of Spam1 and Hyal5, both membrane proteins and reproductive hyaluronidases, resulted in a significant number of sperm with the retention of EC which contained large amounts of these proteins, normally found on the sperm head. Importantly, in EC-containing sperm, the proteins were absent from the head where they play major roles in fertilization. Spam1 mRNA which binds to the microfilament cytoskeleton has been demonstrated to be post-transcriptionally regulated, and it has been shown that disruption of the microfilament cytoskeleton by Cytochalasin-D (CD) increases the testicular mRNA levels. The objective of this study was to test the hypothesis that exposure to CD results in an increased number of sperm with EC due to an upregulation of Spam1 and Hyal5 mRNAs and proteins, whose transport via vesicles is obstructed. To test our hypothesis, sexually mature ICR mice were intra-testicularly injected with CD and the contra-lateral testis injected with the carrier, dimethyl sulphoxide (DMSO). Mice were sacrificed either 24 h, 15 days, or 20 days post-injection and the testes were removed. Testicular Spam1 and Hyal5 mRNA levels were determined 24 h after injection, and the number of caudal and corpus sperm with and without EC were observed for the latter periods. For each animal, significantly (p<0.001) more sperm had EC in the tests compared to the controls treated. Our result reveals a molecular mechanism by which EC sperm are generated. Supported in part by HHMI.

The Gemini virus Coat Protein: Detection of mRNA and Subcellular Localization
Stacey L. Mlynarski, Marie Etarock, R.V. Chowda Reddy, Patricia Nugent, Bertrand Hankoua,
Christian Felton, Marie Paul-Emile, Marie-Teresa Ananfac, Mastingor Desir, and Vincent N. Fondong
Department of Biology, Delaware State University 

The genome of East African cassava mosaic Cameroon virus (EACMCV) have two circular single-stranded DNA (ssDNA) molecules of 2.8 kb and packaged as minichromosomes. The genes of EACMCV are distributed in DNA-A and DNA-B. DNA-A encodes six genes, two sense genes including AV1, which codes for the coat protein (CP) and AV2 protein. The four complementary sense genes include AC1, which codes for the replication associated protein (Rep), AC2 and AC3, which code for the transcription activation protein (TrAP) and the replication enhancer protein (REn), and AC4, which is located within AC1 at the 5’ end, but in a different reading frame (Fig. 1A). DNA-B encodes two genes, BV1 and BC1, which code for the movement protein (MP) and nuclear shuttle protein (NSP), respectively. We studied the ACMV coat protein (CP) detection using Northern blot analysis and subcellular localization using a confocal microscope. Funding from INBRE.

Identification of a Patch of Residues on SV40 T-Antigen Origin Binding Domain Needed for DNA Replication 
Krista Neal, Erin Foster, Daniel T. Simmons
Department of Biological Sciences

Simian Virus 40 (SV40) is used as a good model system to study the highly regulated process of eukaryotic DNA replication. Its genome encodes the multifunctional protein, large T-Antigen (T-Ag), which orchestrates initiation of replication. T-Ag is known to interact with several cellular proteins including Replication Protein A (RPA), Topoisomerase I, and DNA polymerase alpha/primase. RPA also binds to single-stranded viral DNA after the replication origin is melted by T-Antigen. RPA/ssDNA binding interferes with intramolecular hybridization of unwound DNA and facilitates primer formation. Although RPA interacts with T-Ag’s origin binding domain (OBD), the exact binding site is not known. The 3-D structure of T-Ag’s OBD exhibits a face of amino acids that we hypothesize to participate in RPA binding. Single point mutations were generated in this region. In vitro replication assays are being performed with purified mutant T antigens to assess their ability to function in DNA replication. Preliminary results show that three mutants, K174Q, K178E, and H201N, are extremely defective in replication. The three original amino acids form a patch on the surface of the OBD and may constitute a binding site for another protein. Future assays will be performed to characterize the ability of the mutant T-antigens to bind RPA as well as to perform various activities involved in DNA replication. This research is funded by a PHS Grant from the National Cancer Institute.



Alterations in BDNF and TrkB Expression in Response to Neonatal Alcohol Exposure
Ronald C. Ikechi-Ogbonna, C.J. Rocha, A.Y. Klintsova
Behavioral Neuroscience and Psychology

The brain growth spurt occurs during the third trimester of prenatal human development, which corresponds to the first ten postnatal days in rats.  Moderate or high dose of alcohol exposure (AE) during this period of neonatal rat development is an animal model of Fetal Alcohol Spectrum Disorders (FASD).  Physical exercise, such as Wheel Running, in a social environment has been shown to ameliorate certain effects of AE by stimulating the neuronal mechanisms that induce cytogenesis and concurrently brain plasticity.  Brain-Dervied Neurotrophic Factor (BDNF) and its receptor, Tyrosine receptor kinase B (TrkB), contribute to the activation of NMDA receptors found in the postsynaptic membrane. This activation ultimately strengthens synaptic plasticity. We hypothesize that rats exposed to moderate exercise in social environments will show changes in the amount of BDNF and TrkB, in the hippocampal fields CA1 and dentate gyrus.  The presence of BDNF and TrkB is evaluated using densitometry to determine their different levels of expression in animals from the following conditions: Alcohol Exposed Wheel Running (AE-WR), Alcohol Exposed Non-Wheel Running (AE-NWR), Sham Intubated Wheel Running (SI-WR) and Sham Intubated-Non Wheel Running (SI-NWR). We also hypothesize that increased production of BDNF protein or increased expression of TrkB receptor is involved in the mechanism of exercise-promoted brain plasticity. We further hypothesize that AE animals could benefit from exercise-promoted plasticity. Funded in part by a UDRF Grant.

The Search for Genes in Prostate Morphogenesis
Lauretta Ovadje1, Peter S. Nelson2, Cynthia Qian3, Robert Sikes3
1Department of Biology, Lincoln University, PA, 2Department for Human Genomics, Fred Hutchinson Cancer Institute Seattle, WA, <>3Laboratory for Cancer Ontogeny and Therapeutics, University of Delaware, Newark, Delaware

The Urogenital Sinus (UGS) is predetermined to make the prostate gland at embryonic day 15 (E15). The resulting glandular morphogenesis occurs via epithelial budding from both the ventral and dorsal portions of the UGS to give rise to ventral and dorsolateral lobes of the prostate, respectively. The anterior lobe epithelium buds through Wölffian duct mesenchyme. These differences result in different branching patterns and protein expression profiles in the adult tissue of each prostate lobe. We have undertaken a study to determine the regional differences in gene expression of the UGS in order to assemble a Venn diagram of gene expression. Over 8500 UGS genes and 776 prostate genes were assembled into a cDNA microarray. RNA expression profiling was done on microdissected UGS tissue to yield microarray data from UGS mesenchyme (UGM), UGS epithelium (UGE), dorsal half of UGS (dugs) and the ventral half of the UGS (vUGS). Bioinformatics was then utilized to determine which genes are specifically localized to each compartment. Supported by DoD.


Characterization of Post-Translational Modification of the Survival Motor Neuron Protein
Matthew Paoli and Wenlan Wang
A. I. DuPont Hospital for Children

Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by a progressive loss of the spinal motor neurons. The disease is caused by deletion or mutation of survival motor neuron gene (SMN1). The encoded SMN protein is ubiquitously expressed and involved in RNA processing. Yet, reduced levels of SMN in patients selectively result in motor neuron death. This suggests SMN may play a neuronal specific function. The objective of this project is to search for potential post-translational modifications in neurons. We have used a bioinformatics approach to examine the SMN sequence, and 2D Western blotting to analyze a range of cell lines. We found SMN contains two domains and various post-translational modification sites including phosphorylation sites. SMN from fibroblast cells, naïve and NGF-differentiated PC12 cells, and rat spinal cords were post-translationally modified. In addition, the modification pattern varies in the different cells. Interestingly, SMN from differentiated PC12 cell cytoplasm fraction is more abundant and acidic than that in the nucleus. The nature of these post-translational modifications and their potential significance in neurons will be further studied. Supported by Charles Peter White Fellowship.
 
Characterization of the Growth Defect Phenotype of HIP/RPL29 Knock-Out Mice 
Shaila Parker, Daniel Carson, and Catherine Kirn-Safran
Department of Biological Sciences
Our research focus is to understand the function of heparin/heparin sulfate interacting protein, ribosomal protein L29 (HIP/RPL29), particularly in bone development.  Mutant mice that do not express HIP/RPL29 protein were obtained using a gene targeting strategy in which a neomycin phosphotransferase (neo) selection cassette replaces the first three exons of the Hip/Rpl29 gene.  The targeted mutant (tm1) mice displayed global growth defects with an average adult body weight difference of approximately 30% when compared to control littermates. A comprehensive weight study demonstrated that the organ weights were proportional to the reduction in body weight with an exception of the spleen which showed a significant decrease in size when compared to the liver.  We identified similar growth defects in mice carrying another targeted mutation (tm2) that deletes the neo cassette.  Our results indicate that HIP/RPL29 plays a major role during skeletogenesis and suggest an involvement of HIP/RPL29 in hematopoiesis. Funding for this project was provided by the Carson lab and McNair Scholars Program.

Biochemistry of Prox1
Tapan Patel1, Xiaoren Chen1, Li Liao2, Melinda K. Duncan1
1Department of Biological Sciences, 2Department of Computer and Information Sciences

Prox1 is a vertebrate homeobox transcription factor related to Drosophila Prospero and as such plays many important roles during development such as: lymphangiogenesis, hepatocyte migration during liver development, lens fiber cell differentiation, and differentiation of ganglion cells in retinal development. In certain cell types such as the ocular lens, it acts as a true transcription activator by binding to γF-crystallin and βB1 crystallin promoters and activating their expression.  Prox1 can also act as a co-repressor of nuclear hormone receptors SF-1 and LRH1. We hypothesize that these dual functions of Prox1 are achieved through specific protein-protein interactions. A yeast two-hybrid screen of mouse cDNA library led to the discovery of numerous proteins that may potentially interact with Prox1 in vivo. Here we report the interaction of Prox1 with the splicing factor SC35 and the DNA sliding clamp PCNA. Immunofluorescence colocalization data suggest that Prox1-SC35 interaction results in redistribution of intracellular SC35 protein. SC35 is normally clustered in the nucleus giving rise to the famous nuclear speckles; however, in cells expressing Prox1, SC35 is redistributed evenly throughout the nucleoplasm. Furthermore, this interaction leads to the repression of Prox1 mediated transactivation through the PL2 promoter element as determined via CAT assay. Similar repression has been found for Prox1-PCNA interaction as well. To help explain these observations in molecular detail, we propose a docking model of Prox1-PCNA complex whereby the PIP-box of Prox1 binds chemically to the interdomain connecting loop of PCNA. Future in vivo mutagenesis experiments will validate the proposed model. Supported in part by a Beckman and Goldwater Fellowships.

The Role of MeCP2 in  Neuron Development and Bone Homesostasis
Donna Pomroy, Carolyn Schanen, Rose Deeter, and Andrea Ham
A. I. Dupont Hospital for Children.

Rett Syndrome is a severe neurodevelopment disorder affecting young females.  It is characterized by apparently normal early development followed by loss of purposeful use of the hands, slowed brain and head growth, seizures, osteoporosis, loss of speech, and mental retardation.  Mutations in the gene, encoding methly-CpG-binding protein 2 (MeCP2), are the main cause of this syndrome. Although Rett Syndrome is being studied worldwide, it is not known what specific role MeCP2 plays in neuron development and bone homeostasis.  The focus of this project was to optimize staining procedures that would allow successful research in synapse formation and bone development in models of Rett syndrome.  Immunofluorescence was used to differentially stain myocytes, which are used in co-culture with sympathetic neurons to study synapse formation.  Several factors influencing staining outcome were examined to determine their effect on specificity and clarity of the fluorescence signal generated by an antibody to the muscle protein, actinin. In bone, histological techniques, such as safranin O/fast green staining, were used to analyze differences between wild-type mice and mice with a targeted deletion of the Mecp2 gene.  The results of these initial histological studies on bone will lead to further exploration in several areas of bone homeostasis.  Detailed histological studies of these clinically relevant tissues will provide insight into how loss of MeCP2 affects the development and homeostasis of cells that are directly involved in the pathogenesis of this disorder.  This project was funded by INBRE - Supported by NIH grant 2 P20 RR016472-06 from the NCRR and the Schanen/Twiss labortaory at A. I. Dupont Hospital for Children. 

The Effect of the Scale of an Alien Plant Invasion on Native Insect Communities
Erin Reed and Douglas W. Tallamy
Department of Entomology and Wildlife Ecology

Because of their competitive success in unnatural habitats, alien plants are increasingly replacing their native counterparts in both natural and synthetic ecosystems. The goal of this study is to determine the effect of such an invasion on the biomass and diversity of insect herbivores, that being directly related to the productivity of insectivores and animals in higher trophic levels. Data from pilot experiments I conducted suggest the visual and chemical cues of a native host plant may be lost to insect herbivores in areas densely populated with alien plant species. Therefore, areas in which the scale of the alien invasion is minimal may have more insect productivity because natural host plants with which the insects share an evolutionary history are more accessible. To determine insect productivity, Black Cherry trees (Prunus serotina) in an early successional forest surrounded by varying degrees of alien incursion form the centers of circular collecting zones. Native insect herbivores, including galls and leaf miners, are recorded and/or removed from a standard number of leaves on each Black Cherry tree to be later identified and weighed. After a vegetation survey within each collecting zone is conducted, noting both the Diameter at Base Height (DBH) and natural origin of each woody plant, a correlation will be identified between percent alien plant invasion and species richness, insect biomass, diversity (using the Simpson Index), and the occurrence of generalists and specialists. Funding for this research is provided by the National Science Foundation and the University of Delaware Life Sciences Undergraduate Research Scholars Program.

Geographical Distribution of Mpi Allozymes in Platorehestia platensis
Don Romaniello and John McDonald
Department of Biological Sciences
 
There are myriad examples of polymorphisms that are affected by selection, but few of these are completely understood on a physiological and biochemical ground. The role of MPI in amphipods is to process dietary mannose: After mannose has been converted to mannose-6-phosphate by hexokinase using ATP, mannose-6-phosphate is converted my MPI (the enzyme in question) to fructose-6-phosphate, which is fed into the glycolysis pathway to eventually produce more ATP, especially if there is adequate oxygen. It is possible that hypoxic, high-mannose environments may select against less-active MPI genotypes.


Generation of JAM-A overexpressing MDA-MB-231 Cells for Cancer Metastasis and Tumor Growth Study
Ritika Samant, James Parris, Ulhas P. Naik
Department of Biological Sciences

Our lab has identified the transmembrane protein Junctional Adhesion Molecule – A (JAM-A), which is localized at the tight junctions of endothelial and epithelial cells, and is known to be involved in angiogenesis, cell adhesion, and the transmigration of leukocytes out of the bloodstream.  It has also been observed that the highly metastatic breast cancer cell line, MDA-MB-231 (231), expresses low levels of JAM-A which are spread throughout the cell membranes, compared to the lesser metastatic T47D breast cancer cells, in which higher levels of JAM-A are localized to the tight junctions.  We hypothesize that the overexpression of  JAM-A will lessen the overall metastatic ability of 231 cells due to increased cell adhesion and formation of tight junctions.  The aim of this project was to generate human 231 cells overexpressing murine JAM-A protein.  Human 231 cells were transfected either with a pcDNA vector containing murine JAM-A (mJAM-A) cDNA in order to overexpress mJAM-A.  JAM-A overexpression was analyzed by Western blotting and single cell colonies were isolated by limited dilution and by picking isolated colonies.  The transfected cells will be used for subcutaneous injections, to study tumor growth, and intracardiac injections, to study metastasis, on immunocompromised mice.  Funding was provided by HHMI.

Is JAM Making Your Liver Fatty? The Effect of JAM-A on NAFLD
Laura S. Shankman1, Vesselina G. Cooke1, William C. Skarnes2 Ulhas P. Naik1
1Department of Biological Sciences, University of Delaware, Newark, DE, 2The Sanger Institute, Hinxton, Cambridge, CB101, UK

Each year the number of obese United State Citizens increases dramatically. A common precursor to obesity is a disease known as Non-alcoholic Fatty Liver Disease (NAFLD). The progression of NAFLD includes inflammation of the liver caused by large, detrimental fat deposits within the liver cells that ultimately cause cirrhosis of the liver (Foundation 2001). Junction adhesion molecule A (JAM-A), a member of the IgG superfamily, localizes in tight junctions and plays a major role in cell to cell adhesion. JAM-A also participates in leukocyte transmigration through the endothelium via “tunneling” and antigen binding (Bazzoni 2003). We hypothesize that JAM-A assists in the natural progression of NAFLD through neutrophil transmigration and/or regulation of lipid accumulation in the liver as seen in preliminary data. A wild type mouse exposed to a high-fat diet showed an increased lipid accumulation and liver steatosis than a JAM-A null mouse. A systematic experiment, including JAM-A null and wild type mice exposed to either a high-fat or low-fat diet for 25 weeks, was set up to determine JAM-A’s involvement in NAFLD progression. Weekly weights and monthly blood samples are collected to track diet progression. At the end of the trial, the mice will be sacrificed in order to determine the level of NAFLD progression. If the progression of NAFLD increases in the high-fat diet groups, the diet will be deemed effective. JAM-A will have assisted in the development of NAFLD if the level of steatosis increases in the livers of the wild type animals. Research conducted during the past ten weeks was made possible by the INBRE program.

The Influence of IGF-1 and IL-6 on CD44 Expression in the LNCaP Progression Model
Jesse Sinanan1, Lynelle Thorpe, Dave Degraff, Robert A. Sikes, and Carlton Cooper
1Delaware Technical and Community College, Department of Biological Sciences University of Delaware

Prostate Cancer (PCa) is the most frequently diagnosed non-cutaneous cancer in American men. Left untreated, PCa will preferentially metastasize to bone, where treatment is palliative at best. It has been recognized in several studies that selective metastasis of some cancers is, in part, related to the unique cell adhesion molecules (CAM) expressed by the vascular endothelial cells of target organs. CD44, the primary receptor for hyaluronan (HA), is a widely distributed transmembrane glycoprotein and has been identified as an adhesion molecule in/for). In addition to CD44/HA, two factors that are present in the bone microenvironment that influence tumor proliferation and bone matrix alteration are insulin-like growth factor-1 (IGF-1) and interleukin-6 (IL-6) respectively. The influence of these factors, however, has not been investigated in a human PCa progression model, which mirrors its natural clinical progression to increasing androgen-independence and bone metastasis propensity. Therefore, we hypothesize that; IGF-1 and IL-6 influence the expression of CD44, thus modulating the metastatic behavior of advanced PCa. We are therefore investigating the influence of IL-6 and IGF-1 on CD44 expression in the LNCaP human PCa progression model and PC3 subline. The LNCaP model parallels the natural progression of PCa, from the gain of androgen-independence to spontaneous bone involvement, with its LNCaP, C4-2 and C4-2B4 sublines respectively. The expression of CD44 was determined using RT-PCR and qPCR. Funded in part by NIH Bridges grant. numerous cancers. Previous studies provided the first evidence implicating CD44 expression in the adhesion of PCa and breast cancer cells to bone marrow endothelial cells (BMECs

 
Detection of Salmonella in Biosolids
Samantha A. Smith, Diane S. Herson, and Yinan Qi
Department of Biological Sciences and Department of Civil and Environmental Engineering

Biosolids are produced as byproducts of waste water treatment. The presence of Salmonella spp. in this material is of concern because of the illnesses they cause.  In the newly proposed EPA Method 1682, selection of Salmonella spp. occurs on modified semi-solid Rappaport Vassiliadis (MSRV), which contains an antibiotic (novobiocin) and a dye (malachite green) to inhibit non-Salmonella species.  Method 1682 takes several days due to the multiple cultural steps involved.  The standard polymerase chain reaction (PCR) assay is a molecular assay that can be used for the detection of Salmonella spp.  This method takes less time, but the results may be inhibited by the presence of substances contained in biosolids. Our studies first compared the results of biosolids samples in the cultural EPA method 1682 and a PCR assay.  The results indicated that PCR was inhibited in the most concentrated samples.  The use of MSRV as an enrichment step prior to PCR was then tested by removing a sample of cells and media and subjecting the samples to DNA isolation.  The results indicated that PCR was inhibited when using this method.  The effect of vortexing samples in both MSRV and Trypticase Soy Broth (TSB) after the addition of the Instagene matrix was also tested and was found to have no effect on the results of the PCR assay. We then tested several different ways of isolating the cells or DNA from the MSRV and its potential inhibitors.  It was found that heating the sample in a hot water bath for 1-2 minutes to melt the media, followed by centrifugation and removal of the supernatant prior to the addition of Instagene, resulted in the PCR assay agreeing with the results of the cultural method. Funding from: Charles Peter White, Delaware Water Resource Center

Ontogeny of Surfactant Protein B (SP-B) Expression in Avian Lung
Sam Snow and Sabrina M. Brougher
Biology Department, Delaware State University, Dover, DE 19901

Pulmonary surfactant is a mixture of phospholipids and proteins that line the internal surface of the lung of all vertebrates.  It is synthesized and secreted by alveolar type 2 pneumocytes.  In the mammalian system, surfactant functions mainly to reduce surface tension in the airway to maintain inflated alveoli.  Avian lungs, which consist of tubular and rigid parabronchi surrounded by air capillaries for gas exchange, structurally resemble human conducting airways (i.e. bronchioles).  This histological resemblance suggests the idea of looking at avian surfactant in order to better understand the composition and function of surfactant in the upper respiratory tracts of humans.  We are particularly interested in looking at surfactant protein B (SP-B), which is expressed in both avian and human lung.  It is hypothesized that surfactant protein B will be expressed in a developmental and histological pattern similar to that of the human system.  Our aims are to characterize the patterns of surfactant protein expression in embryonic domestic fowl.  Domestic fowl eggs from a local Delaware hatchery will be incubated at 37.5° C and 65% relative humidity.  A subset of 22 embryos will be neocropsied daily, from embryonic day 13 to day 20.  Proteins will be characterized by Western immunoblot, and mRNA expression will be determined by RT-PCR. A better understanding of the physiological properties of avian pulmonary surfactant may lead to relevant applications in biomedical research on human upper respiratory diseases. INBRE -Supported by NIH grant 2 P20 RR016472-06 from the NCRR
    

SPAM1 Expression in Prostate Cancer-a Possible Adhesion Marker for Metastatic Tumor
Erin Stallings, Brittany Stokes, Mehrnoosh Soori, Minghai Shao, Deni Galileo, Patricia A. Martin-Deleon
Department of Biological Sciences

Sperm adhesion molecule 1 (SPAM 1 or PH-20) is encoded by the SPAM 1 gene and is a widely conserved mammalian sperm membrane protein with multifunctional roles in fertilization, and it is best known for its neutral hyaluronidase activity.  The human SPAM 1 gene, which is located on chromosome 7q31, is expressed in a variety of cancers in both reproductive and non-reproductive organs where it is up-regulated with abundant transcript levels. However, its protein has never been studied in any malignant cells.  The objectives of this study are to (1) document the presence of SPAM 1 protein in prostate cancer lines A and C, previously proven to contain SPAM 1 mRNA by RT-PCR; (2) sub-localize the protein in the cells; and (3)  verify its expected hyaluronidase activity.  A 64 kDa SPAM 1 protein was detected by Western analysis and confirmed by immunocytochemistry (ICC), which revealed the localization of the protein in the cytoplasm, but not on the plasma membrane.  Absence of the protein on the plasma membrane was confirmed by Fluorescence Activated Cell Sorting (FACS), which showed no increase in fluorescence in samples treated with SPAM1 antibody compared to pre-immune serum.  Lastly, Hyaluronic Acid Substrate Gel Electrophoresis (HASGE), used to detect hyaluronidase activity of the SPAM 1 protein, revealed the absence of hyaluronidase activity at neutral pH.   Preliminary data suggest that SPAM1 may exert its effect in prostate cancer metastasis via its adhesion properties rather than its enzymatic function, as previously reported. Funded in part by the Department of Defense Grant (DOD)


The Role of ATP Binding Cassette Transporters A1 and G1 in Human Cholesterol Homeostasis<>
Sara E. Sterling, Marysol Lavender and David C. Usher
Department of Biological Sciences

ATP binding cassette transporters, A1 and G1, (ABCA1 and ABCG1) regulate reverse cholesterol transport in mouse adipocyte cells. ABCA1 transports phospholipids and cholesterol to lipid poor High Density Lipoproteins (HDL), while ABCG1 transports cholesterol to large HDL particles.  In order to determine how cholesterol transport might be controlled, the expression of the genes ABCA1 and ABCG1 was tested in β-cyclodextrin, triiodothyronine (T3) and a combination of β-cyclodextrin and triiodothyronine treated 3T3-L1 adipocytes. Results of QPCR assays showed that both cholesterol depletion with β-cyclodextrin and T3 inhibited their expression. These results suggest, as expected that the cholesterol content of the adipocyte influences the expression of genes important to cholesterol efflux. These genes are also suppressed by T3 which is an important regulator of carbohydrate and lipid metabolism; further suggesting that lipogenesis requires increased cholesterol content in these cells. Although it was expected that cholesterol efflux in mouse and human adipocytes would share similarities, differences were found. Using human adipocyte RNA, the expression of ABCA1 and ABCG1 was tested using QPCR. The results indicated that differentiation increased the expression of ABCA1 in human adipocyte cells, as it did in mouse adipocytes, but that ABCG1 did not. In fact ABCG1 was not expressed at all. This in turn suggests that the human ABCA1 transporter may function in mouse differently than in humans or that an ABC transporter other than ABCG1 is involved in cholesterol efflux to large HDL particles.  Sponsored by Howard Hughes Medical Institute		 

SPAM1 Expression in Breast Cancer Cells Is Not Associated with Hyronidase Activity
Brittany Stokes, Erin Stallings, Minghai Shao, Deni Galileo, Monica Mikhail, Patricia A. Martin-DeLeon
Department of Biological Sciences

Sperm adhesion molecule 1 (SPAM 1 or PH-20) is encoded by the SPAM 1 gene and is a broadly conserved mammalian sperm membrane protein with multifunctional roles in fertilization, and it is best known for its neutral hyaluronidase activity. The human SPAM 1 gene, located on chromosome 7q31, is expressed in a variety of cancers in both reproductive and non-reproductive organs where it is up regulated with abundant transcript levels. However, its protein has yet to be studied in any malignant cells. The objectives of this study were to (1) document the presence of SPAM 1 protein in Breast cancer lines T-47D and MDA-MB-231, previously proven to contain SPAM 1 mRNA by RT-PCR; (2) sub-localize the protein in the cells; and (3) verify its expected hyaluronidase activity. Using Western analysis, a 64 kDa SPAM 1 protein was detected and confirmed by immunocytochemistry (ICC), which revealed the localization of the protein in the cytoplasm, but not on the plasma membrane. Absence of the protein on the plasma membrane was confirmed by Fluorescence Activated Cell Sorting (FACS), which showed no increase in fluorescence in samples treated with the SPAM 1 (anti-rabbit donkey) antibody compared to pre-immune serum. Lastly, Hyaluronic Acid Substrate Gel Electrophoresis (HASGE), used to detect hyaluronidase activity of the SPAM 1 protein, revealed the absence of hyaluronidase activity at neutral pH.  Thus, SPAM1 may exert its effect in breast cancer metastasis via its adhesion properties rather than its enzymatic function, as previously reported. Supported by HHMI.

 
Characterization of SMA Fibroblast Cell Lines Using Pyrosequencing
Sarah Swain, Wenlan Wang, Susan Kirwin, and Vicky Funanage
Nemours Biomedical Research, A.I. duPont Hospital for Children, Wilmington, DE

Spinal Muscular Atrophy (SMA) is an autosomal motor neuron disease and is the leading inherited cause of infant and early childhood mortality. SMA is caused by mutation or deletion of the telomeric copy of the gene, SMN1, however all patients retain at least one centromeric copy of the gene, SMN2.  SMN2 produces reduced amounts of full length transcript and SMA results from insufficient levels of SMN protein in motor neurons.  SMA disease severity correlates inversely with SMN2 copy number.  Determining the SMN copy number is crucial for diagnosis and clinical prognosis of SMA. Currently, the most widely used method for characterizing cell lines is restriction digest.  However, we have shown that pyrosequencing is more selective, sensitive, and specific in determining the relative SMN copy number.  In this study we have collected six (6) controls and five (5) SMA type I patient cell lines.  It has been shown by restriction digest that all SMA type I patients were deleted for SMN1.  We will continue to collect cells from the various types of SMA for characterization.  This will enable us to better create a novel assay for drug screening of potential treatments for the disease and to determine the SMN2 copy number that correlates to SMA types I - III.  Future projects will also include optimizing the pyrosequencing method for the addition of an internal control. This research was funded in part by the Howard Hughes Medical Institute Undergraduate Science Education program.

Examination of the Role of JAM-A Homodimerization in Angiogenesis
Wen Allen Tseng and Ulhas P. Naik
Department of Biological Sciences

Junctional adhesion molecule-A (JAM-A) is found at the tight junctions of endothelial and epithelial cells.  It has been demonstrated that JAM-A forms cis-homodimers in vitro and in vivo and that this property is needed for the proper maintenance of selective barriers by epithelial monolayers.  Our lab has shown that signaling through JAM-A plays an essential role in the induction of angiogenesis by basic fibroblast growth factor (bFGF).  This study intends to determine if and how cis-homodimerization of JAM-A is involved in the induction of angiogenesis.  To this end, several mutant JAM-A cDNA constructs were generated via site-directed mutagenesis.  For some constructs, mutations were designed to impair JAM-A homodimerization.  In other constructs, reciprocal mutations were made that were intended to restore cis-homodimerization.  An assay for measuring JAM-A dimerization at the cell surface was developed and will be used to quantitate the extent of homodimerization-impairment for the mutant JAM-A constructs.  The relationship between JAM-A homodimerization and angiogenesis will be studied by examining the pro-angiogenic behavior of human umbilical vein endothelial cells (HUVECs) over-expressing wild-type or mutant JAM-A. This research was supported by the Arnold and Mabel Beckman Foundation and the Barry M. Goldwater Scholarship and Excellence in Education Foundation.

ssDNA Oligonucleotides Arrest Growth of Esophageal Cancer Cells
Casey A. Vasta, Timothy R. Schwartz, and Eric B. Kmiec
Department of Biological Sciences and the Delaware Biotechnology Institute

Gene repair employs single-stranded DNA oligonucleotides (ODNs) to directly alter specific sequences. These ODNs, when transfected into mammalian cells, have been reported to stall the cells in S-phase of the cell cycle. We have shown that a G-rich oligonucleotide, G20, induces apoptosis in human esophageal cancer cells at ≥72 hours. This effect seems to be unique to the G20 sequence; data collected using other sequences such as the A20, T20, and C20 ODNs do not produce the same apoptotic effects. We observe successful transfection by electroporation of the G-rich oligonucleotide in several cell lines, as measured by FACS and confocal microscopy. We examined a variety of oligonucleotide sequences reported to form G-quadruplexes, none of which result in the same effects as the G20 molecule. This demonstrates the uniqueness of the G20 in its initiation of the cellular response.  In our efforts to determine the nature of this novel response, we examined the involvement of chromosomal telomeres; telomeric sequences are G-rich in nature and form quadruplexes. To determine if the G20 ODN is mimicking a cellular telomeric sequence or interfering with proteins that interact with telomeres, we treated cells with a telomerase inhibitor, TMPyP4. While this inhibitor induces a minor G2/M stall, it does not correlate to the extent as the G20.  Funded by INBRE.

Variation in Sperm Hyaluronidase Activity in Men Attending an IVF Clinic
Sarah Woody, Jessica Bruton, Minghai Shao, Patricia A. Martin-Deleon
Department of Biological Sciences
 
Sperm hyaluronidase activity is necessary for degradation of hyaluronan or hyaluronic acid (HA) in the extracellular matrix in several steps in human fertilization. These steps include the penetration of the cumulus oocyte complex, the zona pellucida, and the perivitelline space. To date, the only known sperm hyaluronidase is Sperm Adhesion Molecule 1 (SPAM1 or PH-20), a 64 kDa membrane protein which plays multifunctional roles in mammalian fertilization and which has recently been shown to be a marker of sperm maturation in mice (Chen et al. 2006). SPAM1 is unique among the hyaluronidases, with respect to its neutral hyaluronidase activity. Our lab has recently shown that a second sperm hyaluronidase, HYAL3, is present in human sperm. The objective of this study was to compare the levels of hyaluronidase activity in men using assisted reproductive technologies (ART) at Christiana Hospital, a local facility in Newark, DE. Hyaluronic acid substrate gel electrophoresis (HASGE) revealed varying levels of hyaluronidase activity at neutral pH among the subjects, as reflected by the absence of a band to the presence of bands at various sizes. Using Western blot analysis, attempts were made to correlate the absence or reduction of hyaluronidase activity with the presence of SPAM1 and HYAL3 isoforms.  Further studies are in progress to investigate the presence of acidic hyaluronidase activity in these subjects. Funded by IGERT.

Chondrogenic Marker Expression is Impaired by Inhibition of Heparanase 1 (Hspe 1) by PI-88
Franck A. Yehiri, Anissa J. Brown, Mary C. Farach-Carson
Department of Biological Sciences

Heparanase 1 (Hspe 1) is an endo-β-D-glucuronidase enzyme that catalyzes the hydrolytic cleavage of β-1, 4-glycosidic bond between a D-glucuronidase and D-glucosamine in heparan sulfate (HS).  Although one of the physiological known functions of hspe 1 is to degrade HS and enable the delivery of active heparan sulfate binding growth factors to the extracellular matrix (ECM) of various tissues, the role of heparanase in endochondral ossification is not yet understood.  Based on the importance of heparan sulfate in chondrogenesis, we hypothesized that heparanase would also be involved in growth factor delivery during chondrogenic differentiation; perturbing normal patterns of heparanase expression was expected to impact HS-dependent processes during cartilage differentiation.  In order to investigate the role of hspe 1 in endochondral ossification, we proceeded in inhibiting its expression in ATDC5 cells micromass cultures, with PI-88.  PI-88 is a sulfated oligosaccharide mimic of HS which acts as an inhibitor of hspe 1, thus can prevent the release of heparin binding growth factors from the extracellular matrix (ECM).  ATDC5 cell micromass cultures were differentiated and treated with various concentrations of PI-88 (0.5ng/mL, 1ng/mL, 5ng/mL and 10ng/mL).  To determine glycosaminoglycans (GAG) production during in vitro chondrogenic differentiation of mouse ATDC5 cells, alcian blue staining was employed.  A modest decrease in GAG production was observed in micromass cultures treated with PI-88.  Additional changes in chondrogenic markers such as aggrecan and collagen II will be determined by conventional PCR.

IGF-1  Influences CD44 Expression in  Human Prostate Cancer Cells
Noella M Zony1 and  Lynelle Thorpe2
1 Biological Sciences, Delaware State University, 2Cancer Biology Laboratory, University of Delaware, Biological Sciences.

CD44 is a multifunctional cell surface adhesion molecule that has been implicated in tumor cell invasion and metastasis. Many cancer cell types, including prostate cancer cells, express high levels of CD44. IGF-1 (Insulin-like growth factor 1) is a polypeptide involved in epithelial cell proliferation and survival that has been shown to play a role in prostate cancer progression.  However, the effect of IGF-1 on CD44 expression has not been examined previously in lineage related cell lines of increasing metastatic potential. Therefore we examined the relative expression levels of CD44 in the LNCaP human PCa progression model using Western Blot analysis in order to determine if IGF-1 stimulation affects CD44 expression. Analysis of CD44 protein levels indicated similar levels of CD44 in all cell lines of the LNCaP progression model with higher levels observed in the unrelated PC-3 cell line. Treatment of LNCaP, C4-2, C4-2B4 and PC-3 cell lines with IGF-1, 2ng/ml for 24hours, was done and Western Blot analysis performed to examine CD44 levels. The results indicated a downregulation of CD44 in metastatic C4-2 cells when treated with IGF-1 and an upregulation of CD44 in LNCaP cell lines after treatment with IGF-1. CD44 expression in PC-3 cells was not affected by IGF-1 treatment. These data support previous results that indicate altered regulation of IGF-1 and androgen signaling in the LNCaP progression model and a correlation of high CD44 expression in metastatic prostate cancer.
Biology Abstracts for students with names beginning A-K.
Links: Summer 2006 Undergraduate Research Symposium, Symposium Abstracts from other Colleges and Departments,
Undergraduate Research Summer Enrichment Program,  Unversity of Delaware Undergraduate Research Program, Howard Hughes Undergraduate Program.
Created  1 August 2006. Last up dated 11 September 2006 by Hal White
Copyright 2006, University of Delaware