Abstracts from the Department of Biological Sciences
Undergraduate Summer Research Symposium August 14, 2003

Ordered alphabetically by student's last name

Small Molecule Inhibitors as Cancer Therapeutics in a Prostate Model

Samantha M Allen, W. Nathaniel Brennen, Milton Brown, Robert A. Sikes
Department of Biological Sciences

This project focuses on small molecule inhibitors as cancer therapeutics. VGSC blockers were designed and examined for their ability to inhibit androgen dependent and independent prostate cancer cell growth (PCa) in vitro.  The best lead compound was a hydoxyamide, ICM-I-136. PCa were grown in soft agarose colony forming assays (SACF).  It was found that SACF was inhibited by >95% in all of the prostate cancer cells tested.  Since previous results correlated VGSC expression to metastatic capacity, wound-healing assays were performed to determine the effect of VGSC blockade on cell migration. (Assay in progress).   Thalidomide has been identified as a potent angiogenesis inhibitor.   Derivatives of thalidomide may be more effective then the parent molecule.  Eighteen thalidomide derivatives were tested at 100 µM, in accordance with standard NIH screening practice, in PC3, LNCaP, C4-2 and C4-2B4 cell lines using an in vitro growth assay.  One of these, SC-2-71, a quinazolone, was particularly effective at inhibiting the growth of human endothelial cell cultures and PCa.  We are testing SC-2-71 in a pseudo-in vivo assay on the chriollantric a membrane (CAM) of a growing chicken to test for the inhibition of angiogenesis.   From these initial studies, there is evidence that these thalidomide derivatives may share a common mechanism that inhibits both angiogenesis and prostate cancer cell growth. Funding provided by  Peter White and University of Delaware Research Foundation 

Chloride-dependent Sodium Hydrogen Exchangers in Renal Proximal Tubule Cells

Jennifer Arseneau and Gary Laverty
Department of Biological Sciednces

In renal proximal tubules (PT), sodium/hydrogen exchangers (NHE) on the apical membrane regulate bicarbonate reabsorption and acid/base equilibrium. Parathyroid hormone (PTH) has been known to regulate NHE.  Evidence shows that the NHE may be Cl--dependent in chick proximal tubules (PT) and mammalian tissues.  The present study analyzes this Cl--dependence of NHE by utilizing a pH-sensitive dye 2'7'-bis (carboxyethyl) 5-6-carboxy-fluorescein (BCECF).  A spectrofluorometer with dual-excitation of 505nm and 439nm monitored the intracellular fluorescence.  Opossum kidney (OK) cells in this experiment were used as a control for chick PT.   OK cells were prepulsed with NH4Cl to acidify the cells.  Intracellular pH recovery, due to NHE activity, was monitored in solutions with and without chloride.  A calibration curve using nigercin was used to convert the magnitude of fluorescence into pH.   Preliminary studies show that the recovery rate of OK cells in a solution without chloride is reduced when compared to OK cells in a solution with chloride.  Our initial studies with chick PT cells , however, were unsuccessful and future efforts on chick PT cells will try to optimize loading conditions.  In conclusion, these studies may support previous experiments describing a novel Cl--dependent NHE in other tissues.  This work was supported by HHMI 

Narrowing the Cytological Region of 8J16: 
A Drosophila Ethylmethane Sulfonate Induced Mutation 

Daniella Betts and Erica M. Selva
Department of Biological Sciences

The onset of certain cellular afflictions (such as cancerous growth), stem from improper functioning of one or more of the many steps that may take place between an extracellular signal and an intracellular response, otherwise known as cellular signal transduction. The primary objective of this project is to use Drosophila melanogaster as a model to study the posttranslational events that affect the Hedgehog (Hh) and Wingless (Wg) signal transduction pathways. The Hh and Wg pathways are heavily affected by the extracellular environment and function as important regulatory pathways for organismal development. The goal of this experiment is to locate and clone the gene that carries a mutation classified as 8J16. 8J16 is an ethylmethane sulfonate (EMS) induced mutation that displays Drosophila embryonic phenotypes that are suggestive of irregularity within the Hh and Wg pathways. By primarily using classical genetic techniques such as deficiency and recombination analysis the existing 8J16 region will hopefully be narrowed from the numerous open reading frames (potential genes sites) to a list of seven or fewer candidate gene locales for the mutation. After reducing the existing region, molecular genetic techniques such as In situ-hybridization, and RNA interference analysis will be implemented to identify the gene that is disrupted by the mutation. Currently, the project is in its beginning stages and deficiency complementation analysis is in progress. Funded by UDRF and HHMI.

Hyaluronan Synthase Expression in the LNCaP Progression Model 

Chelsea Bridgewater, Bianca Graves, Freddie Pruitt, Linda Sequeira, 
Robert A. Sikes, and Carlton R. Cooper
Department of Biological Sciences

Hyaluronan (HA), which is involved in normal cell adhesion and migration, may contribute to prostate cancers (PC) metastasis to bone. HA is over-expressed in metastatic PC cell lines derived from bone and acts as an adhesive to Human Bone Marrow Endothelial (HBME) cells. Two isoforms of HA producing enzymes are HAS2 and HAS3. A previous study comparison of PC-3 to LNCaP cells is not entirely complete because the cell lines are unrelated. Therefore, the LNCaP progression model (LNCaP, C4-2, C4-2B4), WiDr (colon cancer which binds poorly to HBME, negative control) and PC-3 (metastatic PC line, positive control) were used to observe the RNA levels of HAS2 and HAS3. It is hypothesized that there will be an increase in HAS2 and HAS3 expression as LNCaP progresses to C4-2B4.  An agarose gel was run to observe the HAS2 and HAS3 in the cell lines. The data show HAS3 expressed in all cell lines, but over-expressed in PC-3, C4-2 and C4-2B4. HAS2 is only present in WiDr, PC-3 and C4-2. We concluded that the HAS3 enzyme contributes to HA production in metastatic PC cells. An adhesion assay will be performed using cells stripped of HA to show its role in PC metastasis. CB supported by the HHMI Undergraduate Science Education Program. 

mRNA Localization in Regenerating Axons

M. Chosseler1, D.L. Willis2, J.-Q. Zheng2, J.L. Twiss2
1IUP Genie Physiologique et Informatique, Poitiers University, France.
2Nemours Biomedical Research, A.I. duPont Hosp for Children, Wilmington, DE, USA.

Our lab previously showed that adult rat DRG neurons have the capacity for intra-axonal protein synthesis and use this mechanism for regenerating injured axons (Zheng et al 2001). Here, we have used cultures of rat DRG neurons, which only extend axons, to obtain a pure preparation of regenerating axons for biochemical and molecular analyses of local protein synthesis. Cultured neurons do not always reproduce the in vivo conditions, thus, we have analyzed mRNA localization in motor axon of adult rats using Ventral spinal roots as a template for RT/PCR. The injured ventral root contained neuronal specific mRNAs. This is consistent with mRNA localization into spinal motor axons in vivo. In other neuronal systems, dendritic RNA localization and translation are regulated by activity. Since nerve injury alters neuronal activity, we asked whether activity may also play a role in nerve regeneration. Supported by the Neours Foundation.

The Roles of tPA and uPA in Pseudoexfoliation Syndrome

Janine E Collinge, Shawn Gallagher, Melinda K Duncan
Department of Biological Sciences

Pseudoexfoliation syndrome (PSX), the primary identifiable cause of open-angle glaucoma, one of the leading causes of blindness.  PSX is characterized by the appearance of lightly colored flakes, consisting of extracellular matrix (ECM) on the lens surface.  Transforming growth factor beta-1 (TGF?-1), a known regulator of ECM metabolism and production, is present at high levels in PSX patients.  High levels of TGF?-1 could cause over-activation of the serine proteases, tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which can break down the ECM.   Preliminary studies showed that there are high levels of tPA associated with PSX material.  This led to the development of the hypothesis that tPA and uPA are overexpressed in PSX lens epithelium and contribute to the production of PSX material.  By RT-PCR, we have demonstrated that tPA and uPA mRNA are expressed by the lens epithelium.  However, total RNA in clinical samples is too small to directly quantitate tPA and uPA expression, so quantitative RT-PCR is necessary for this study.  Currently, standard curves for these genes are being developed, using TBP and GAPDH as internal controls, to determine the ratio of expression levels for tPA and uPA.  Understanding the roles that tPA and uPA may play in PSX may lead to finding a suitable treatments for this disease. This project is funded by a Charles Peter White fellowship.

The Interaction of CRASS-1 and CRASS-1 with Calcium-Integrin-Binding Protein 

William Dunworth, Sairam Palicherla, and Ulhas P. Naik
Department of Biological Sciences

Integrins are cell surface receptors consisting of an alpha and a beta subunit which can heterodimerize to bind extracellular matrix molecules.  One specific integrin, alphaIIbbeta3, is only found in platelets.  Recently, it has been shown that calcium-integrin-binding (CIB) protein binds with the intracellular domain of the alphaIIb subunit to meditate platelet adhesion and spreading.  In order to further understand the function of CIB, our laboratory used the yeast-two hybrid technique, which allowed us to isolate a novel 247 amino acid protein called CIB-regulated-adhesion-spreading-stimulator (CRASS-1).  Preliminary results have indicated that CIB and CRASS-1 interact in a cell-signaling pathway that mediates the adhesion and spreading of cells.  A search through the human genome has identified another protein, CRASS-2 that shares 91% amino-acid identity with CRASS-1.  Producing CRASS-1 and CRASS-2 recombinant proteins in soluble form would allow monoclonal/polyclonal antibody generation as well as binding studies.  To achieve this, CRASS-1 and CRASS-2 cDNA’s were subcloned in pGEX vector to produce GST-fusion proteins.  These constructs were then expressed in E. coli BL-21 strain and induced to produce soluble CRASS-1 and CRASS-2 recombinant proteins which were purified using a Glutathione-Sepharose column.  WD supported by the HHMI Undergraduate Science Education Program. 

Genes Involved in Sperm Function: 
Investigation of Dual Expression of Hyal5 in The Testis and Epididymis

 Mesha Eaton, Hong Zhang, and Patricia A. Martin-Deleon
Department of Biological Sciences

SPAM1, the Sperm Adhesion Molecule 1, is a highly conserved sperm membrane protein with multiple essential roles in mammalian fertilization. This protein belongs to a group of closely linked hyaluronidases residing on mouse chromosome 6A2, and is responsible for the dissolution of the cumulus cells of the oocyte during fertilization. This is necessary for penetration of the sperm. More recently a Spam1 knockout study revealed that fertility was unaffected and suggested that there was a mouse-specific hyaluronidase, Hyal5, with similar functions to Spam1 (Baba et. al., 2002). The goal of this study was to determine if Hyal5 is redundant to Spam1 in regards to its mRNA and protein expression in the epididymis. An mRNA expression analysis was performed using RT-PCR on both the testis and all three regions of the epididymis (caput, corpus, and cauda), which was rendered sperm-free after excessive washing. RT positive and RT negative reactions were performed and the results, although preliminary, suggest the presence of Hyal5 in the cauda. An attempt was made to study the protein expression in the testis with immunohistochemistry, however due to non-specificity of the antibody the results were inconclusive. A new antibody is being generated to continue these studies. ME supported by The Beckman Scholars and McNair Scholars Programs.

The Role of Contactin in the Tangential Migration of Chick Optic Tectum Neurons

Melissa Edgecombe and Deni S. Galileo
Department of Biological Sciences

Contactin is a cell adhesion/recognition molecule and a member of the Immunoglobin Superfamily.  It has been shown to be involved in the formation of neuronal networks in the brain during development, specifically axon growth and fasciculation.  Based on preliminary data from immunostaining of chick optic tecta sections, we have found that contactin is highly expressed in the intermediate axonal layer of the optic tectum during development.  There are two specific aims to the research project.  The first aim is to look for cell autonomous effects when contactin expression is attenuated.  To attenuate contactin, we will infect the chick optic tectum with an anti-sense contactin retroviral vector.  Retroviral vector production through transient transfection has not yielded virus with a sufficiently high titer. To circumvent this problem, we have begun to make and test stable virus producing cell lines.  We hypothesize that tectofugal projection neurons will abnormally extend their axons in a random direction on embryonic day 6.  The second aim of the project is to look for widespread effects of contactin attenuation.  This will be achieved through in vivo electroporation of the anti-sense contactin plasmid into the chick optic tectum.  We hypothesize that the axonal intermediate zone will be visibly disorganized during embryonic days 6-9. 

In Vitro Gene Expression Profiling of Hepatotoxins and DNA Microarray Analysis

Kenneth A. Ewane and Christine M. Glatt1
1duPont Nutrrition and Health

Abstract withheld by request of the authors.
KAE supported by the McNair Program

Mapping Intracellular Trafficking of Ca2+-ATPase Using Glycosylation Mutants 

Rory Flinn and Norman Karin
Department of Biological Sciences

We postulate that the Ca2+-ATPase isoform, SERCA1a, is involved in the biogenesis of the sarcoplasmic reticulum in skeletal muscle. Mapping the intracellular trafficking of SERCA1a would provide insight into how SERCA1a is inserted into the sarcoplasmic reticulum, and how the biogenesis of the sarcoplasmic reticulum occurs. Wild-type SERCA1a does not contain an N-linked glycosylation sequence, and therefore does not have carbohydrates added to it during trafficking. Site-directed mutagenesis was performed to construct a mutant form of SERCA1a containing an N-linked glycosylation site in an area of the cDNA that encodes a luminal loop. Mass spectrometry will reveal which carbohydrates were added to the mutant SERCA1a protein during intracellular trafficking and thus through which organelles the protein traveled. The mutant cDNA will be transfected into the C2C12 cell line (mouse myoblasts derived from skeletal muscle). Lipofectamine 2000 was the best transfection agent of those tested. Different reporter genes were used, but the most practical one was GFP-SERCA1a in the pcDNA3.1 vector. Funding for this project was provided by a Charles Peter White Fellowship. 

Role of Secreted L1/NgCAM in Glioma Invasiveness

Joseph S. Fotos, Alexandra Cretu, Errin Lagow, and Deni S. Galileo
Department of Biological Sciences

Abstract withheld by request of the authors.
Funded by NIH. JSF supported by the HHMI Undergraduate Science Education Program

Biosolids: Rise of the Fecal Coliforms

Sean Gillow, Yinan Qi, Steve Dentel,1 and Diane Herson
Department of Biological Sciences and 1Department of Civil and Environmental Engineering, 

A goal of wastewater treatment (WWT) is to remove pathogens including bacteria, viruses, protozoa, and Helminth ova from domestic sewage.  The resulting residue, called biosolids, can be land applied and used as a soil conditioner or fertilizer.  During the processing of biosolids pathogen monitoring is problematic.  Instead fecal coliforms, which are always present in human waste, serve as the indicator.  At several wastewater treatment plants (WWTP), an increase in fecal coliforms was observed after dewatering.  We have received funding from the Water Environment Research Foundation (WERF) to determine the cause of this increase which may be a result of coliform regrowth or reactivation.  Fecal coliform numbers are determined initially and after the dewatering step in WWT using either A-1 or LTB/EC media.  Previous studies have shown that the use of A-1 may result in false positive results under certain conditions.  Initial studies were done to see if these conditions could be reproduced in a laboratory setting.  Additional studies were done to determine the effect of conditioning and storage of biosolids on fecal coliform numbers.

Induction of Brain Creatine Kinase by Factor AP2 Also Requires Factor NF-Y

Brian Gladnick, Dan Nadel, Yanping Zhang, George R. Molloy
Department of Biological Sciences

Astrocytes (i) provide physical support for neurons in the central nervous system, (ii) supply neurons with nutrients needed for proper functioning, and (iii) take up excess glutamate that is released from neurons.  Most of these processes are highly energy-demanding and require constant regeneration of ATP.  An important enzyme for regenerating ATP in astrocytes and neurons is brain creatine kinase (CKB).  I have investigated how the transcription factors AP2alpha and NF-Y induce transcription of the CKB gene in U87-MG glioblastoma cells.  AP2alpha binds to the GCCAATGGG sequence element located at –50bp in the proximal CKB promoter.  Interestingly, NF-Y binds to the CCAAT element also located at –50bp, which is within the –50bp AP2alpha element.  Transfection experiments have shown that AP2alpha induces transcription of CKB greater than 50-fold.  However, the ability of AP2alpha to induce CKB was dramatically reduced under conditions where factor NF-Y could not function.  This supports the model whereby both AP2alpha and NF-Y become associated with the –50bp GCCAATGGG element and subsequently induce CKB transcription.  Additional transfections in U87-MG showed that simply increasing the levels of factor NF-Y increased CKB transcription by 10-fold.  The results provide new information on how factors AP2alpha and NF-Y function together to regulate CKB mRNA transcription. BG and DN supported by the HHMI Undergraduate Science Education Program.


The Effect of Novel Compounds on the Growth of Human Bone Marrow Endothelial and Human Dermal Microvascular Endothelium Cells: Drug Development for Angiogenesis

Janelle Green, Cliff Poindexter, Alison M. Walls, Robert A. Sikes
Milton L. Brown*, and Carlton R. Cooper
Department of Biological Sciences, University of Delaware, Newark, DE, and 
*Department of Chemistry, University of Virginia-Charlottesville, VA

Angiogenesis has been implicated in the progression and metastasis of prostate cancer.  This process of forming new blood vessels from preexisting vessels occurs as a result of a disruption in the balance of endogenous proangiogenic and antiangiogenic factors.  Complex interactions among cytokines, growth factors, and other molecular pathways can result in a net increase in proangiogenic factors released from tumor cells, endothelial cells, stromal cells, the extracellular matrix, and /or blood cells. Thalidomide is a drug commonly used to inhibit angiogenesis, but this drug has side effects.  In collaboration with a medicinal chemist, we secured five novel compounds derived from thalidomide. We hypothesize that these compounds are as effective as thalidomide and may be useful in the treatment of bone metastasis, a common complication of advanced prostate cancer. Our proposed study is therefore designed to determine the effects of these compounds on the growth of Human Bone Marrow Endothelial (HBME) and Human Dermal Microvascular Endothelial (HDMVE) cells.  HDMVEC served as a control for HBME cells.   A growth assay was performed in the presence or absence of 100uM concentration, the NIH recommended starting dose for any potential drug, of each compound. The data showed that all five compounds significantly inhibited the growth of both HBME and HDMVEC, suggesting these compounds are general angiogenic inhibitors.  Specifically, their effects on HBME cells may indicate their clinical usefulness for treating established bone metastasis. JG supported by the BRIN Program.

Targeted Porcupine Expression to Generate Dominant Adult Wing Phenotypes

Chantal Gwannulla2, Daniella Betts1, Carolyn Lowry1, and Erica M. Selva1
1Department of Biological Sciences, University of Delaware 
and 2Delaware Technical & Community College

The focus of the laboratory is to identify components involved in the posttranslational modification of extracellular signaling molecules to gain a better understanding of how these events modulate signal transduction. Porcupine (Porc) is involved in posttranslational modification of Wingless (Wg) and has been shown to influence Wg signal transduction. Porcupine binds the N-terminal domain of the Wingless and stimulates its posttranslational N-glycosylation by anchoring them at the Endoplasmic Reticulum (ER) membrane. It was previously observed that when porc is misexpressed in the engrailed (en) domain of the developing wing using the UAS-Gal4 expression system, it yielded an adult visible wing phenotype. The goal of my project was to identify wing specific Gal4 lines that when crossed to UAS-porc to misexpress porc yielded a visible adult wing phenotype. Ultimately we will use these results to attempt to develop a porc misexpression fly line that can be used in a modifier F1 screen to identify mutation/gene that interact with porc. Only apteros-Gal4 (ap-Gal4) has some good phenotype. We are still testing more Gal4 lines to try and identify more phenotypes. Funded by the HHMI Program and UDRF. 

Potential Role of Dynamin-Like Protein 1 (DLP1) in Biogenesis of Sarcoplasmic Reticulum

Lawrence David Hall and Norman Karin
Department of Biological Sciences

Dynamins are large GTPases that have been implicated in intracellular membrane trafficking by their ability to constrict and tubulate membranes.  Studies have suggested that mammalian dynamin-like protein, DLP1, has the capacity to tubulate spherical liposomes in vitro, and we postulate that DLP1 performs a similar role in sarcoplasmic reticulum tubule formation.  Initial RT-PCR of total RNA from premyoblasts and 7-day differentiated myotubes (C2C12 cells) showed that DLP1 gene expression was upregulated in differentiated myotubes.  To quantify the increased gene expression, non-competitive semi-quantitative RT-PCR was performed for both undifferentiated and differentiated cells and compared using glyceraldehyde-3-phospate dehydrogenase (GAPDH) as a housekeeping gene to normalize the level of gene expression.  The level of DLP1 protein expression increased approximately two-to three-fold in differentiated myotubes as quantified by Western blotting with a polyclonal antibody.  It has been shown through double immunolabeling of mouse fibroblasts that DLP1 colocalizes with endoplasmic reticulum tubules.  We hope that these studies may implicate DLP1 in the biogenesis of the sarcoplasmic reticulum.  Funding for this project was provided by the Howard Hughes Medical Institute. 

Phenotypic Characterization of 7H24 mutation 
in Drosophila melanogaster

Afeez A. Hazzan¹, Robyn M. Goodman², 
and Erica M. Selva²
 ¹Lincoln University, PA, 
²Department of Biological Sciences

The goal of our laboratory is to understand how posttranslational events influence Wingless (Wg) and Hedgehog (Hh) signaling pathways in Drosophila melanogaster. My research focuses on phenotypic characterization of 7H24, an Ethylmethane sulfonate (EMS) induced recessive lethal mutation that disrupts larval eye development. 7H24 is a maternal effect lethal mutation that has a phenotype consistent with disruption of Hh or Wg signaling. These pathways exist in a feed back loop during embryonic development, but can be separated during imaginal discs development to determine which pathway is disrupted by a mutation. I examined homozygous mutant imaginal discs tissue and homozygous mutants of 7H24 clones in developing heterozygous imaginal discs. FLP/FRT system was used to generate mutant clones. The mutant tissue is distinguished from the wild type by the use of P [heatshock-green fluorescent protein], a visible cell marker that co-segregates with the wild types, therefore labeling the mutant cell by its absence. 7H24 is pupal lethal and it was observed that homozygous 7H24 animals had rough eye, a phenotype caused by an increase or decrease in the number of cells that make up the eyes. In order to examine this phenotype further, I dissected out late instar larval eye discs and examined cell specific markers to determine the structure of the 7H24 developing eye compared to wild type. I found that this mutation most likely disrupts the Hh signaling pathway. AH supported by the HHMI Undergraduate Science Education Program. Research supported by UDRF.

Inhibitory Efficacy of Sodium Channel Blockers as Cancer Therapeutics in a Prostate Model

Rahul Jawa, W. Nathaniel Brennen, Milton L. Brown, Robert A. Sikes
Department of Biological Sciences

We are investigating the utility of novel voltage-gated sodium channel (VGSC) blockers as inhibitors of both androgen dependent and independent prostate cancer (PCa) cells. Our lab has found that these compounds are very effective at inhibiting cell growth in vitro. I have used an ELISA to assess the effect of VGSC blockers on the secretion os a clinically relevant biomarker, prostate specific antigen (PSA). Preliminary data indicate that PSA secretion is decreased in C4-2 cells following treatment with VGSC blockers. Using DNA as a surrogate for cell numbers, I will correlate the PSA secreted per cell to determine the effect of VGSC blockers on PSA secretion. In vivo, the effects of VGSC blockers on tumor growth will be determined using the most promising lead compound, ICM-1-136. Mice were injected with 2 million PC-3 cells per site and have been treated with three different allometrically determined dosages (range 24-240 mg/kg*dy) of ICM-I-136, Phenytoin, and a vehicle control over an 8-week period. Currently, tumor growth is being monitored. My hypothesis is that over the week period, ICM-I-136 will inhibit the tumor growth compared to either Phenytoin or control. These data will allow us to determine the efficacy of ICM-I-136 and to predict the extended survival of murine hosts in the presence of this drug. RJ supported by the HHMI Undergraduate Science Education Program.

Microarray Implicates Several Signal Transduction Pathways 
for Ca2+Inhibition of Adipocyte Differentiation

Erin E. Kenaley, Mary C. Farach-Carson and Kamil A. Akanbi
Department of Biological Sciences

Ca2+ is critical for many important cellular processes and functions.  Ca2+ plays a role in adipocyte metabolism including adipocyte differentiation. An inverse relationship exists between Ca2+ intake and body fat. One study showed that increased Ca2+ intake discourages weight gain and body fat increase and allows for weight loss3. Obese African Americans whose Ca2+ supplementation was increased from 400 to 1000mg/day lost significantly more body fat than those with low Ca2+ intake3.  The objectives of this research were 1) to examine the effects of varying extracellular Ca2+ concentrations on adipocyte differentiation and 2) to identify signal transduction pathways affected by Ca2+ during differentiation. Data showed that 3T3-L1 preadipocytes differentiate readily in low concentrations of extracellular Ca2+. Conversely, 3T3-L1 preadipocytes cultured in higher concentrations of extracellular Ca2+ displayed inhibited differentiation, lowered GPDH activity (a marker of differentiation), and less triglyceride filling. Using microarray, three signal transduction pathways were identified as possible pathways for extracellular Ca2+ inhibition of adipocyte differentiation. Supported in part by the HHMI Undergraduate Science Education Program.


RNA Interference of Topoisomerase I & Its Effect onSV40 DNA Replication

Christine Kosman and Daniel Simmons
Department of Biological Sciences

RNA interference (RNAi) is a technique that uses double stranded RNA to prevent the expression of its corresponding gene. In this project, topo I’s role in cellular replication was studied by the use of RNAi. The critical nature of topo I to cellular growth and function was determined. First, the RNAi oligonucleotide template was designed, which then was transcribed and purified to yield the RNAi of topo I. The RNA was then transfected into monkey cells to study its effect on cellular replication. Several experiments were done to study this effect. First, we compared four different oligonucleotides that target different regions of the topo I mRNA to see which oligo works the best. Second, we compared the effects of different concentrations of RNAi. Third, we varied the length of time the cells were transfected. The results of these experiments determined that RNAi inhibits cell growth and we therefore concluded that topo I is important for cell division. Our next step is to determine the effects of RNAi on the replication of SV40 in monkey cells. This project was funded by the Howard Hughes Medical Institute.

Neuromuscular Junctions in Cerebral Palsy: Structural Organization

Justine Lahaye, Bobbie Boyce, Mary Theroux, MD and Robert Akins, PhD
Nemours Biomedical Research, A. I. duPont Hospital for Children, Wilmington, DE

Cerebral palsy (CP) is a common neurologic disorder in children. It results from damage to the brain occurring before, during, or shortly after birth. A primary deficit in CP is neuromuscular dysfunction, and we have shown that at least some children with CP have abnormally distributed acetylcholine receptors (AChRs) relative to the functional neuromuscular junction (NMJ) as defined by acetylcholine esterase (AChEase) immunoreactivity. In the present study, the relationship between the distribution of AChRs outside the NMJ and the degree of motor-system affect of the CP child was investigated. Biopsies of lower limb muscles (Vastus, Gracilus and Gastrocnemius) were double stained in order to detect AChR and AChEase. Image analysis using a customized software macro was performed, and the Spread of AChR staining outside NMJs was calculated using biopsies of 59 children. Comparisons between ambulatory (n=21) and non-ambulatory (n=38) children indicated that non-ambulatory children had higher median Spread values (p=0.025) and a higher ratio of abnormal (Spread > mean + 2 x S.D. for normal NMJs) to total NMJs than ambulatory children (p=0.023). These results suggest that the level of affect in children with CP is associated with the presence of structurally pathological NMJs. This project was supported by the Nemours Foundation.

The Role of NgCAM in Neuronal Migration in Brain

Peter Lazzopina, Phil Kudish, Deni S. Galileo
Department of Biological Sciences

CRASH syndrome (Corpus callosum hypoplasia, Retardation, Adducted thumbs, Spastic paraplesia, and Hydrocephalus), a congenital birth defect in humans, is responsible for a spectrum of debilitating symptoms caused by the absence or misexpression of the cell adhesion molecule L1.  We believe that comparable abnormalities can be created in developing chick embryo brains by locally modifying expression of the structurally similar and functionally equivalent protein, NgCAM.  NgCAM will be attenuated by use of a 562 base pair antisense sequence that is complementary to the corresponding NgCAM coding sequence.  These sequences will be introduced into the chick brain via the RCASBP(A) replication-competent retroviral vector.  After incorporation into the host genome and expression, they will bind to the host’s NgCAM mRNA and will block its translation.  We hypothesize that this attenuation will cause disruption of normal neuronal development, thereby mimicking the effects of CRASH syndrome.  Effects are expected on migration of a subset of neurons and on axon extension and fasciculation.  In the end, the intention is to create a simpler and more easily manipulated model than the existing mouse models for the study of brain abnormalities in CRASH syndrome.  Supported by HHMI and NIH

Mapping and Cloning of a New Mutation Related to Wingless/Hedgehog Signaling

Carolyn Lowry, Robyn Goodman, Erica M. Selva
Department of Biological Sciences

The focus of our laboratory is to investigate the extracellular interactions that influence signaling pathways between cells.  Correct signaling and signal reception is vital for an organism to correctly develop.  Many diseases in humans have been linked to incorrect cell signaling, cancer being one example.  The information gathered by our research on Drosophila melanogaster can be applied to our knowledge of human cell signaling since many genes and cellular mechanisms are conserved throughout evolution.  Two examples of signaling pathways conserved throughout evolution and that are crucial to vertebrate development are the Wingless (Wg) and Hedgehog (Hh) signaling pathways.  These pathways are perfect as the focus of our study on cell signaling because they are both greatly influenced by the extracellular environment.   7H24, an ethylmethane sulfonate (EMS) mutation, was previously found to be linked to Wg and/or Hh signaling due to its maternal effect embryonic phenotype. The goal of my project is to map 7H24 to its corresponding gene through various genetic mapping techniques, then to determine the consequences of 7H24 on development through genetic and molecular characterization.  By analyzing the effect 7H24 has on Drosophila development, we can gain an understanding of the role the disrupted gene plays in Wg/Hh signaling.  This will help to shed light on the extracellular interactions and regulatory mechanisms required for correct signaling, as well as how disruptions in this signaling can lead to abnormal growth and development. Supported by VWR international and UDRF.

A Novel Protein that Interacts with Calcium and Integrin Binding Protein 
Induces Cellular Adhesion and Spreading

Katherine M. Maddox, Meghna U. Naik, Kristin Eckfeld, Ulhas P. Naik
Department of Biological Sciences

Calcium and Integrin Binding (CIB) protein, which interacts with the integrin alphaIIbbeta3, plays an important role in platelet spreading on immobilized fibrinogen. After screening a human mammary epithelial cDNA library in the yeast two-hybrid system using CIB as bait, a 247 amino acid polypeptide was isolated. This protein was stably expressed in NIH3T3 cells and a 2-3-fold increase in cell surface area was observed using immunofluorescence microscopy. We also observed an increased amount of stress fibers as visualized by phalloidin staining of F-actin. When CIB was overexpressed in these cells we found about a 40% decrease in cell surface area, suggesting that CIB inhibits the ability of this novel protein to induce cell spreading. Therefore, we named this protein CIB Regulated Adhesion and Spreading Stimulator-1 (CRASS-1). Since cell adhesion is a prerequisite for spreading, we asked whether CRASS-1 also effects cell adhesion. In adhesion assays performed with NIH3T3 cells on a fibronectin matrix, approximately a 2-fold increase in cell adhesion was observed in cells expressing CRASS-1 and this adhesion was decreased by 40% when CIB was expressed in these cells. Thus, our studies suggest that together with CIB, CRASS-1 may regulate cell adhesion and migration. KMM was funded by a Charles Peter White summer fellows grant. 

Investigation of the Topologic Form of DNA 
that Serves as a Template for SV40 DNA Replicaton.

David Manna and Daniel Simmons
Department of Biological Sciences

During the process of SV40 DNA replication initiation, four major proteins interact with each other to form an initiation complex that unwinds the origin of replication and begins the process of DNA synthesis.  First, large T antigen, the only viral protein required for DNA replication binds to the origin and forms a double hexamer.  This multifunctional protein must interact with topoisomerase I (topo), DNA pol¦Á/primase (DNA pol), and replication protein A (RPA) in order for DNA replication to initiate.  All three of these proteins have been shown to interact with T antigen in protein binding assays.  First, we determined the effects of each protein on initiating origin unwinding, and second, we investigated the form of circular DNA that serves best as a template in DNA replication.  These experiments were designed to shed light on the initiation of SV40 replication, one of the best known models to study a simplified version of the complicated process of mammalian DNA replication.  Previous data from origin unwinding assays have shown that when increasing amounts of DNA pol are added into in vitro replication conditions containing T and topo, more origin DNA becomes unwound that when DNA pol is absent.  DNA replication assays are now being performed with small circular double stranded DNAs to mimic the conditions used in the DNA unwinding assays.  In this way, we hope to discover the DNA template that serves best in DNA replication. DM supported by the HHMI Undergraduate Science Education Program.

Chondrogenic Differentiation of Murine Embryonic Stem Cells: A Model to Study the Consequences of HIP/RPL29 Gene Targeting.

Elisabeth R. Mari, Richard J. Focht, Daniel D. Carson, Catherine B. Kirn-Safran
Department of Biological Sciences

The heparin/heparan sulfate (Hp/HS) interacting protein (HIP/RPL29) is identical to a ribosomal protein of the large subunit of ribosomes: L29.  It has been previously shown that HIP/RPL29 is expressed throughout growth plate cartilage.  The expression pattern shows that HIP/RPL29 expression is tightly down-regulated during chondrocyte terminal differentiation.  Our hypothesis is that a decrease in HIP/RPL29 expression is associated with a progression towards a late chondrocytic state.  To develop an in vitro system to study the importance of HIP/RPL29 during early chondrogenesis, we analyzed the chondrogenic potential of the totipotent mouse embryonic stem (ES) cell line, AB-1.  This ES cell line was cultured into embryoid bodies (EB) using the “hanging drop” method followed by suspension culture in the presence of bone morphogenetic protein 2 (BMP-2) (2-100 ng/ml).  EBs were plated onto plastic dishes coated or not with a domain of perlecan.  The chondrogenic nature of the plated EBs was determined after 8-9 days of culture on the basis of positive Alcian Blue staining.  Our results show that under these experimental conditions AB-1 ES cells not only differentiate into various morphological structures including cartilage-like aggregates, but also into cardiomyocyte contractile fibers.  Future work will optimize conditions to compare the chondrogenic potential of HIP/RPL29 targeted ES cells versus the parental wild type line.  (This work was supported by NIH grant HD25235 to D.D.C.)  Additional funding was received from the Science and Engineering Scholars Program.

Ammonia Production and Transport Pathway in Response to Metabolic Acidosis in Chick Proximal Tubule Cells. 

Asif Mohammed and Gary Laverty
Department of Biological Sciences

The mode of adaptation to metabolic acidosis was studied in a primary cell culture of chick (Gallus domesticus) proximal tubule (PT) cells.  This was performed under a novel experimental model that allows for polarization of the PT-cells in culture.  Preliminary data indicates that cells under metabolic acidosis exhibit an increased rate of ammonia excretion.  This is accomplished mainly through the catabolism of L-glutamine.   A product of this catabolism is NH4+ which is primarily transported across the apical side of chick PT-cells via a Na+/H+ exchanger. In mammals this exchanger is inhibited by parathyroid hormone (PTH), leading to decreased renal bicarbonate reabsorption. The effect of PTH on chick PT-cells is not well known. Therefore a comparative analysis was done with opossum kidney (OK) cells, an established mammalian cell line.  A three day protocol was used for both cell types with either acidic or normal pH media, with or without PTH.  Samples were taken from the apical and basolateral sides for analysis of ammonia. With the addition of PTH the OK cells did show a net decrease in NH4+ production.  Chick PT-cell responses were not as well defined, which could be due to the use of alternate transporters. AM funded by HHMI 

AP2delta Induces Transcription of Creatine Kinase B 
and Requires PC4 for Maximal Induction 

Daniel Nadel, Brian Gladnick, Yanping Zhang, George R. Molloy
Department of Biological Sciences

Astrocytes maintain synapse function in the brain by taking up the neurotransmitter glutamate and converting it to glutamine.  Both of these reactions are highly energy-demanding and will occur too slowly if ATP is not rapidly regenerated.  In astrocytes and neurons, ATP is rapidly regenerated by the brain creatine kinase (CKB) enzyme.  For brain cells to produce sufficient amounts of CKB enzyme they must regulate transcription of the CKB gene.  Previous transfection experiments have shown that transcription factor AP2alpha can greatly induce transcription of CKB in U87-MG glioblastoma cells, an attractive model cell system for astrocytes.  Additional previous experiments showed that untransfected U87-MG do not contain the AP2alpha protein but rather an AP2 protein resembling AP2delta.  Our transfection experiments have shown that AP2delta can induce transcription of CKB but not as much as AP2alpha.  In addition, we have investigated whether the coactivator protein PC4 cooperates with AP2delta to elicit maximal induction of CKB transcription.  Understanding how AP2delta and PC4 cooperate in regulating CKB transcription will reveal how the levels of the CKB enzyme are maintained to permit proper ATP regeneration in astrocytes and possibly neurons.  DN and BG funded by the Howard Hughes Medical Institute.

Characterization of Calcium Channels in C4-2B4 Prostate Cancer Cells

David A. Nation, Kamil Akanbi, and Mary C. Farach-Carson
Department of Biological Sciences

Prostate cancer cells tend to metastasize to bone, where they proliferate with  the stimulation of local growth factors.  Voltage-sensitive calcium channels (VSCCs) located in the plasma membrane are the primary passageway for extracellular calcium to move into cells and act as a second messenger. Since bone is calcium rich, these channels are potential contributors to the growth  of prostate cancer in bone.  This study aimed to identify the major calcium channel subtypes found in the C4-2B4 prostate cancer cell line in order to find  targets that are not common in normal bone cells. C4-2B4 cells were cultured, total RNA was extracted, and RT-PCR techniques were used to test for expression of the various channel subtypes of the pore-forming alpha1 subunit as well as  the auxiliary beta, alpha2-delta, and gamma subunits. Immunohistochemical staining was then conducted for the L-type alpha1C and alpha1D subunits using specific rabbit derived antibodies to localize these channel subtypes. An Ion  Channel Superarray™ also was conducted using C4-2B4 cells to examine channel expression in response to treatment with 1alpha,25-Dihydroxyvitamin D3, a known growth inhibitor of prostate cancer. Based upon our results, we concluded that  in C4-2B4 cells, the alpha1D subunit seems to be more highly expressed than alpha1C, which is the major channel found in osteoblasts and other bone cells. This study was funded by the Howard Hughes Medical Institute. 

Analysis of Aqueous Humor Proteins in PSX Patients

James O’Leary, Shawn Gallagher, and Melinda Duncan
Department of Biological Sciences

Pseudoexfoliation Syndrome (PSX), a disorder diagnosed by fibrillar extracellular matrix (ECM) deposition in ocular tissue, is a major cause of open-angle glaucoma in addition to ocular surgery complications.  The buildup of ECM observed has led to the hypothesis that PSX is related to the inappropriate breakdown of extracellular matrix and basement membranes in the eye.  Since aqueous humor is the fluid bathing the ocular tissues affected by PSX, SELDI mass spec profiling of aqueous humor proteins was used to identify proteins contributing to PSX pathogenesis.  Initially, the NP20 ProteinArray, a chip whose active spots contain silicon oxide allowing the binding of hydrophilic proteins, was used.  The NP20 protein chip spectrums have contained a number of reproducible mass peaks, though the low accuracy of the specific mass values has necessitated the use of chromatographic surface chips that allow the binding of smaller subsets of proteins.  In parallel, a candidate gene approach has identified the presence of Cathepsin B, a protein associated with the inappropriate breakdown of extracellular matrix in a number of diseases, in both normal and PSX aqueous humor through Western blotting.  Sources of funding include HHMI, Undergraduate research, and PHS.

Creation of Tools to Investigate HIP/RPL29 Function via in vivo Ribozyme Knock-down 
and in vitro Overexpression Approaches

Daniel S. Oristian, Richard J. Focht, Mary C. Farach-Carson, and Catherine B. Kirn-Safran
Department of Biological Sciences

HIP/RPL29 is a heparin/heparan sulfate (Hp/HS) binding protein identical to ribosomal protein L29. Previous studies indicated that HIP/RPL29 is down regulated during the terminal phase of chondrogenesis and that partial reduction of HIP/RPL29 using an in vitro ribozyme knock-down approach accelerates differentiation of C3H/10T1/2 cells into cartilage-like cells. Because cartilage serves as a template for endochondral bone formation, we postulate that a strict regulation of HIP/RPL29 expression is essential for normal bone growth.  Our hypotheses are: 1) HIP/RPL29 down-regulation is essential for chondrocyte terminal differentiation and reciprocally, 2) HIP/RPL29 overexpression prevents terminal chondrocyte differentiation.  In this study, we microinjected the active HIP/RPL29 ribozyme into one-cell stage fertilized embryos and observed subsequent in vitro embryonic development. Future studies will consist of transferring embryos microinjected with HIP/RPL29 ribozyme into pseudopregnant surrogate mothers to study the effect of HIP/RPL29 down-regulation on in vivo bone development.  In parallel we created tools to create stable cell lines overexpressing HIP/RPL29 and cloned the entire sequence encoding for HIP/RPL29 downstream of the strong CMV promoter into an expression vector including a positive neomycin phophotransferase selection cassette to generate stable transfectants using G-418.  Optimal concentrations of G-418 and transfection conditions were established for the chondrocytic cell line ATDC5.  Collectively, these studies will provide valuable tools to study the importance of HIP/RPL29 regulation during in vitro and in vivo chondrogenesis.

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 supported by HHMI Undergraduate Science Education Program.

Cholesterol Efflux and Adipocyte Lipid Homeostasis

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

Adipocytes are known to be important regulators of fatty acid homeostasis.   The Serial Analysis of Gene Expression (SAGE) library of 3T3-L1 adipocytes, constructed in our laboratory, has also implicated them in having an important role in cholesterol homeostasis.  Several genes involved in cholesterol transport, Cav, SREBP-1c, Abca1, and Abcg1, were highly expressed.  To determine relative changes in gene expression from 3T3-L1 fibroblasts to differentiated adipocytes, 3T3-L1 cells were harvested daily during differentiation, and RNA was extracted with trizol.  Relative levels of Cav, SREBP-1c, Abca1, and Abcg1, were determined by real-time RT-PCR, and all showed an increase in level, over TATA box-binding protein, during 3T3-L1 differentiation. AP funded by HHMI Undergraduate Science Education Program

Characterization of Calcium- and Integrin-binding Protein in Lipid Rafts

Scott Pileckas, Meghna U. Naik, and Ulhas P. Naik
Department of Biological Sciences

The complex signal transduction pathways that lead to events such as blood platelet activation and, as a result of this process, hemastasis, involve several key molecules.  One such molecule, calcium- and integrin-binding protein (CIB), a 22-kDa polypeptide, is already known to be greatly involved in the platelet activation pathway.  Thus, it has been hypothesized that CIB, which is highly present in prostate cancer (PC3) cells, might also play a similar signaling role in these cells.  Several membrane-associated signaling molecules are abundant in cholesterol-rich lipid rafts, infolds of the plasma membrane usually characterized by a 22-kDa aptly named protein called caveolin.  In order to study CIB’s relationship with lipid rafts, or caveolae, a detergent extract of the cells was subjected to isopycnography to separate the lipid rafts from total cell extract.  After confirming its general presence in PC3 cells through Western Blot analysis, distribution of caveolin in PC3 cells was determined using the isopycnographically produced fractions correlating to lipid rafts of the cells.  Presence of CIB in the lipid raft fractions was determined through Western Blotting which showed that CIB was not present in these fractions.  Since both CIB and caveolin are not simultaneously localized in the “resting” PC3 cell, it is possible that after stimulation with an agonist, CIB may associate with lipid rafts.  Experiments to confirm this hypothesis are ongoing.  Funded in part by Charles Peter White Scholars Program.

Determining the Functional Role of a Novel CIB Regulated Adhesion 
and Spreading Stimulator (CRASS-2) Protein

Swati Pradhan, Meghna U. Naik, and Ulhas P. Naik
Department of Biological Sciences

Cell-to-cell interactions and cell to extracellular matrix protein interactions play an important role in incurable illnesses like cardiovascular disease and cancer.  We have previously identified a calcium and integrin binding protein (CIB) which is involved in cell migration and cell spreading.  Another protein was identified in our laboratory which is known to interact with CIB.  It was named the CIB Regulated Adhesion and Spreading Stimulator, CRASS-1, because of its involvement in cell spreading.  Human genome search revealed that there exits another protein with 90% similarity to CRASS-1.  We named this protein CRASS-2.  In order to determine the function of CRASS-2, NIH3T3 (mouse fibroblast) cells were transfected with CRASS-2 cDNA.  A cell adhesion assay performed with CRASS-2 or Vector transfected cells indicated that CRASS-2 induced cell adhesion on fibronectin.  Consistent with enhanced adhesion, immunofluorescence experiments exhibited CRASS-2 localization to the stress-fibers of the cell.  Effect of expression of CIB on CRASS-2-induced cell adhesion is ongoing.  By determining the function of CRASS-2 and its regulation by CIB, it will be possible to uncover and establish, more exhaustively, the functional roles and capabilities of both CRASS-2 and CIB.  This will allow us to further expand our knowledge and understanding of cell migration and platelet aggregation that can instigate diseases like cancer, myocardial infarction and stroke. SP funded by the McNair Scholars Program.

Analysis of the Activation of FAK and AKT in Prostate Carcinoma Cells

Freddie L. Pruitt, Kathy Ignatoski, Angelo Evans, Bianca Graves, Ulhas P. Naik
Meghna Naik, Kenneth J. Pienta, Steve Ether, and Carlton R. Cooper
Department of Biological Sciences

It has been well established that androgenic hormones are potent and obligatory survival factors in vivo for secretory epithelial cells of the prostate gland. This dependence becomes apparent in the dramatic wave of apoptosis that is rapidly induced in the prostate in response to the removal of circulating androgen by castration (5).  The major obstacle posed by advanced prostate carcinoma is androgen independence and bone metastasis.  It seems that prostate carcinoma cells growing in the bone develop a characteristic for no longer being dependent on circulating androgen. The adhesion of cancer cells to the bone extra-cellular matrix is mediated through integrins, which activate intracellular signaling pathways that influence the behavior of the cell. We are interested in FAK (focal adhesion kinase); which is believed, when activated, recruits phosphatidylinositol 3-kinase (PI3K) which can directly activate intracellular cell survival pathways. The cell survival function of the PI3K pathway is mediated in part, by Akt-dependent inactivation of proapoptotic proteins (5). Since prostate cancer cells preferentially metastasize to the bone, it is hypothesized that PC-3 cells, when grown on a bone matrix, will produce a higher signal of activated FAK and activated Akt, when compared to PC-3 cells grown on plastic and kidney matrices.  FP supported by the Howard Hughes Medical Institute. 

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.

Do Hyal5 and Spam1 Have Overlapping Protein Expression Patterns?

Stacy Shertok, Hong Zhang, and Patricia A. Martin-Deleon
Department of Biological Sciences

This study was conducted to characterize the expression of a hyaluronidase-like gene member, Hyal5.  Primers were designed from the 3UTR of Hyal5 to ensure that only DNA encoding the mRNA would be amplified.  Immunocytochemistry and immunohistochemistry were performed to determine the cell types in which the protein is expressed.  However, the results for the protein expression are inconclusive.  This is partly due to the fact that a Western Blot revealed the primary antibody used is not specific: it binds to Hyal5, Hyalp1, and Spam1, three closely linked hyaluronidase family members.  A developmental RT-PCR experiment was performed on testis RNA from mice that were the following ages:  27 days, 25 days, 21 days, 18 days, and 12 days.  RT-PCR was also performed on CREM-knockout mice to determine if Hyal-5 has a CRE sequence in its promoter region in order to confirm bioinformatic studies of the gene. This study was supported by grants from the Howard Hughes Medical Institute and National Institutes of Health. 

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 lacks a normal squamous layer; thus, JAM-1 is essential for normal corneal morphogenesis. Supported by the Beckman Scholars Program.

IEC-6 Cell cAMP Response to 1,25-dihydroxyvitamin D3 Treatment

Jane O. Ullah1, Benjamin Rohe2, Susan Safford1, and Mary C. Farach-Carson2
1Lincoln University, 2Department of Biological Sciences, University of Delaware

The active metabolite of vitamin D, 1,25(OH)2D3, acts through nuclear receptor-mediated and plasma membrane initiated mechanisms. In the rat intestinal epithelial cell line, IEC-6, a series of rapid events result from the binding of 1,25(OH) 2D3 to its basolateral receptor including G-protein coupled activation of phospholipase C, increases in intracellular cyclic adenosine monophosphate (cAMP), and transcaltachia (Ca2+ transport). The novel non-isotopic assay that we utilized measures cAMP levels after  activation of adenylate cyclase in response to 1,25(OH)2D3 treatment. This fluorescent cAMP assay (Bridge-It™) is based on the activity of cAMP receptor protein (CRP), a bacterial sequence-specific DNA binding protein. Fluorescence resonance energy transfer (FRET) between the fluorochromes introduced to these DNA molecules detects protein-dependent association of the two DNA fragments. The assay mixture of the Bridge-It™ fluorescence cAMP assay kit contains CRP and the two fluorochrome-labeled DNA half-sites and thus contains all components necessary to generate a fluorescence signal in the presence of cAMP. We used the Bridge-It™ assay to determine if 1,25(OH)2D3 increased cAMP in IEC-6 cells via its membrane receptor. A standard curve was developed over the summer that will allow us to assess cAMP responses in IEC-6 cells that contain varying levels of a candidate 1,25(OH)2D3 membrane receptor. JOU supported by the HHMI Undergraduate Science Education Program.

Expression of Hyalp1, 
a Reproductive Hyaluronidase, 
in the Testis and Extratesticular Pathway 

KaTonna Williams1 and Patricia A. Martin-DeLeon
1Delaware State University 
and Department of Biological Sciences

HYALP1, a member of the hyaluronidase family, is an expressed psuedogene (resulting from two deletions that cause premature termination, Csoka et.al., 1999) located on human chromosome 7.  The mouse homologue, Hyalp1, is a functional gene expressed in the testis along with two other closely linked hyaluronidases, Hyal5 and Spam1 (sperm adhesion molecule 1).  Spam1 is widely conserved and has at least three essential roles in mammalian fertilization. However, a recent study of mice with the null mutation revealed no loss of fertility, suggesting that some of the roles attributed to Spam1 may be shared by its closely linked family members, Hyal5 and Hyalp1.  The aim of this investigation was to characterize the expression of Hyalp1 in the mouse, specifically, to determine if it is present on sperm and in the epididymis, as is the case for Spam1.  To determine its possible presence on sperm, a developmental RT-PCR analysis was performed using RNA from testes of 12, 18, 21, and 25 day-old mice.  Caput and cauda epididymal tissues were similarly analyzed after sperm was removed. RT-PCR showed the presence of Hyalp1 in the adult testis. Attempts at the protein expression by immunohistochemistry gave inconclusive results, partly due to the ineffectiveness of the antibody.  These studies are continuing. KW funded by the BRIN Program. 

Allison Wojcik, Rebekah Parsons, 
and Daniel Simmons
Department of Biological Sciences

Purification of Cellular Factors Involved in Elongation of SV40 DNA Synthesis

Three major proteins are involved in the elongation step of Simian Virus 40 DNA synthesis. PCNA, Replication factor C (RF-C), and Polymerase delta are required for efficient elongation of growing strands.  My initial project was to purify two of these proteins, RF-C and polymerase delta.  RF-C is a complex of five subunits and polymerase delta is comprised of two subunits.  The recombinant baculoviruses expressing these subunits were first tested by immunofluorescence.  The best combinations of virus and insect cells were used for the purification prep.  High 5 insect cells were infected with baculoviruses encoding each individual subunit of the protein.  For RF-C, one of the subunits was 6 His-tagged and had the ability to bind to Ni-agarose beads.  After binding, the Ni-agarose beads were washed and the protein was eluted using imidazole.  Polymerase ? was purified by using two chromatographic columns.  Protein preps were analyzed for concentration and subunit expression by SDS PAGE gels, dot blots, and western blots.  Now that all three proteins have been purified, the next step will be to perform and characterize the elongation reaction.  This project was sponsored by the Howard Hughes Medical Institute. 

The Ability of DeltaEF1 to Regulate BetaB1-crystallin Promoter Activity

Alisha R Yallowitz, Jennifer R Taube, 
and Melinda K Duncan
Department of Biologiocal Sciences

BetaB1-crystallin is a marker for fiber cell differentiation from lens epithelial cells.  Since three potential deltaEF1-consensus binding sites were found in the chicken deltaB1-crystallin promoter, and deltaEF1 represses delta-crystallin expression, we hypothesize that deltaEF1 protein in the lens epithelium represses betaB1-crystallin expression.  In transfected lens epithelial cells, betaB1-crystallin promoter activity increases as its length decreases, which could result from the removal of deltaEF1 binding sites.  Thus, additional truncation constructs of the chicken betaB1-crystallin promoter were created with either zero, one, two, or three deltaEF1 sites. Also, 10 bp substitution mutations were made in each potential deltaEF1 site in the chicken betaB1-crystallin promoter.  These promoter truncations and mutations were ligated upstream of the chloramphenicol acetyl transferase (CAT) reporter gene in a pBasic CAT vector.  This fall, these vectors will be transfected into cultured lens epithelial cells and the effects of the mutations will be determined by measuring CAT activity.  If there is an increase in CAT activity, then the missing or mutated deltaEF1 sites are likely repressing etaB1-crystallin production in lens epithelial cells.  The influence of deltaEF1 on betaB1-crystallin expression will then be tested through co-transfection studies using deltaEF1 expression vectors with wild type and mutant promoters.  Funding sources are: HHMI, UR, and PHS.

Links: Summer 2002 Undergraduate Research Symposium, Symposium Abstracts from other Colleges and Departments,
Undergraduate Research Summer Enrichment ProgramUnversity of Delaware Undergraduate Research Program, Howard Hughes Undergraduate Program.
Created 8 August 2003. Last up dated 11 September 2003 by Hal White
Copyright 2003, University of Delaware