Viability of Helicobacter pylori: Effect of Other Microorganisms Lisa M. Attanasio and Diane S. Herson Department of Biological Sciences Helicobacter pylori is a Gram negative bacterium that is responsible for many gastric illnesses. H. pylori has been declared a Class I carcinogen by the World Health Organization and has infected roughly half of the world’s population. The human stomach is the only known reservoir for the bacteria. The means of infection is currently unknown, however contaminated water is thought to be a mode of transmission. In the aquatic environment other microorganisms may play a role the viability of H. pylori. In this study, the viability of H. pylori was examined in the presence of Escherichia coli, an indicator of water potability, and Pseudomonas aeruginosa, an organism naturally found in ground and surface waters. Preliminary studies indicated an inhibitory effect on H. pylori viability when combined with E. coli in a ratio of 1000:1. A follow-up study involving spent media from E. coli and P. aeruginosa showed that the viability of H. pylori was not affected by any compounds excreted by the two organisms into the water. Future planktonic studies will involve studying the viability of H. pylori after it has been suspended in spent media from a mixed culture. H. pylori is also being studied in an attached state. Biofilms of the microorganism are formed overnight on sterile glass slides. Detection of H. pylori is performed using fluorescent antibodies (FA) that specifically target surface antigens on the bacteria. Future biofilm studies will involve using FA to detect H. pylori in the presence of attached E. coli and P.aeruginosa. INT staining will also be used to determine the viability of attached microorganisms.

 
 

Genomic walking of the turtle (Trachemys scripta elegans)  apolipoprotein C-III gene from the 3’ end Lauren E. Baker, Erin M. Hill, Jennifer A. Rutan, Robin Davis, and David C. Usher Department of Biological Sciences Apolipoprotein C-III (apoC-III), a protein that influences the catabolism of triglyercide-rich lipoproteins, has previously been observed only in mammals. A cDNA sequence was isolated from a turtle, Trachemys scripta elegans, liver expression library, and was shown to be the equivalent of mammalian apoC-III. Like mammalian apoC-III, turtle apoC-III is associated with both high-density lipoproteins and triglyceride-rich lipoproteins and is synthesized mainly by the liver. The human apoC-III gene is located in a linkage group consisting of the apoA-I, apoC-III, and apoA-IV genes. This linkage group can also be seen in turtles. The apoC-III gene is transcribed on the opposite strand of DNA as the apoA-I and apoA-IV gene. In turtles, the apoC-III gene contains four exons and three introns.  The apoC-III gene has been sequenced from the 3’ end to intron II. A 300 bp PstI fragment, a 1093 bp pooled DNA fragment, a 800 bp KpnI fragment and a 255 bp pooled DNA fragment were found. The four fragments were purified, cloned, transformed and sequenced. These sequences span from intron II, exon II, intron I, exon I and ~800 bp into the promoter/enhancer region.

 
 

Establishing a binding order for SV40 T Antigen and cellular proteins DNA polymerase delta, RPA, and Topoisomerase 1 on biotinylated plasmid DNA containing the SV40 origin. Joseph A. Brobst and Daniel T. Simmons, Department of Biological Sciences Order of addition experiments were conducted to determine what influences or effects the presence or absence of one or more of T antigen, Topoisomerase 1, DNA polymerase delta, and Replication Protein A bound to the DNA would have on binding of the remaining proteins when added at different times. A combination of data from multiple order of addition experiments indicated a binding order of T antigen followed by DNA pol, RPA, and Topo 1 respectively.  T antigen signal was consistently strongest, followed by RPA and Topo 1 which were approximately equal and DNA pol which was almost completely absent.  The binding order established as a result of these experiments is somewhat contradictory to previous results obtained in a monopolymerase buffer system, indicating the need for further experimentation.  In addition, a change of the reaction buffer in the experiments to one free of the nucleotide UTP led to a dramatic increase in RPA binding across the board.  An explanation for this phenomenon as a consequence of inhibited DNA replication is speculative at best although it offers interesting insight into previously unobserved behavior of RPA.

 
 

Expression of Calcium Channel Beta-subunit Subclasses  in Ribozyme Transfected ROS 17/2.8 Cells  Dinu Cherian, Mary C. Farach-Carson, Kamil Akanbi Department of Biological Sciences Calcium is an important second messenger used by cells to control many cellular processes. Voltage sensitive calcium channels (VSCCs) are the major pathways through which calcium enters osteoblasts. Calcium channel is composed of various subunits, including alpha 1, beta, alpha 2 delta, and gamma subunits. The alpha 1 is the pore forming subunit and thebeta helps localize it to the membrane. The beta subunit consists of four different subclasses, 1, 2, 3 and 4. The osteoblastic cell line, ROS 17/2.8, expresses the alpha1c subtype of alpha 1 subunit of VSCC. Our laboratory previously used ribozyme technology to knock down (< 10% control) the expression of VSCC alpha1c in ROS 17/2.8 cells. The ribozyme-transfected cells have reduced calcium influx and decreased response to 1,-25(OH)2D3 depolarization. Since the beta subunit is an important prerequisite for channel assembly and for the proper plasma membrane targeting of the VSCCs, we examined the expression patterns of the various subclasses of the beta subunits in ROS 17/2.8 cells (parental), ribozyme-transfected ROS 17/2.8 cells and ribozyme control-transfected ROS 17/2.8 cells using PCR and immunochemistry. We examined whether the loss of alpha1c affected the expression levels of the various subclasses of the beta subunit, or their cellular localization. Our results show that all four beta subunits are present in the ribozyme-transfected knockdown cells, indicating that the biosynthesis of the channel subunits is not coupled. Measurements of the relative abundance of the isoforms are underway. These studies should provide new insights into the biosynthesis and assembly of the VSCC, and its role in regulating calcium permeability in the osteoblast plasma membrane.

 
 

The Roles of tPA and uPA in Pseudoexfoliation Syndrome Janine E. Collinge, Justin DiAngelo, and Melinda K. Duncan Department of Biological Sciences Open-angle glaucoma is one of the leading causes of blindness worldwide. A disease known as pseudoexfoliation syndrome (PSX) is noted as the primary identifiable cause of open-angle glaucoma. PSX is characterized by the appearance of lightly colored flakes on the lens surface. Previous studies showed that these flakes mainly consist of extracellular matrix (ECM) components. Transforming growth factor beta-1 (TGF-B1), a known regulator of ECM metabolism and production, has been found in high levels in PSX patients.  Two important serine proteases associated with ECM metabolism are tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA).  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.  To begin this study, RT-PCR experiments were performed using U-87 and PC-3 cell lines as positive controls. In these experiments it was possible to establish the presence of tPA and uPA in a cataractous non-PSX clinical sample. In order to study this expression on a more quantitative level, internalized controls of the commonly expressed proteins TBP and GAPDH were designed for quantitative RT-PCR.  Currently, all of the genes involved in this study are being cloned and sequenced as a control for quantitative RT-PCR. Understanding the roles that tPA and uPA may play in the development of PSX is one step closer to finding a suitable treatment and perhaps a cure for this disease.

 
 

Age-at-Maturity Estimates for Female Striped Bass in the Delaware Estuary Shanna L. Corbin and Malcolm H. Taylor, Department of Biological Sciences This study was undertaken in collaboration with state biologists working in the Delaware Department of Natural Resources and Environmental Control (DNREC).  The goal was to estimate the age-at-maturity for female striped bass in response to a decline in abundance of striped bass, Morone saxatilis, in the Delaware Estuary.  A sample of migratory striped bass (N=45) was captured from the Delaware Estuary during early spring (March-April) of 2002.  Cross-sections of the ovaries were examined, oocytes were obtained from mechanical teasing of ovarian tissue, and oocyte diameters were measured using graphic imaging software.  Our estimates were compared to age data determined from examination of the scales and ear otoliths by state biologists.  We estimated the percentage of mature ovaries within each age group.  Oocyte diameters ranged variably from 0.07 mm to 0.73 mm.  The criteria used to judge maturity was based on the maximum oocyte diameter per fish.  It was established that fish with a maximum oocyte diameter of 0.40 mm or higher could be considered mature and were capable of spawning in the spring of 2002.  Fish with oocyte measurements smaller than 0.40 mm were classified as immature, with no chance of spawning.  Our data could not conclusively establish the age-at-maturity because the number of fish caught was too small.  It is hoped that an additional catch next spring will improve the precision of our estimates.

 
 

The Role of Cell Adhesion Molecule L1 in Glioma Cell Invasion Alexandra Cretu and Deni S. Galileo, Department of Biological Sciences Cell adhesion molecules play important roles in cell-cell interactions during embryogenesis and tissue repair in the nervous system. One such molecule is L1, a transmembrane adhesion and recognition molecule that belongs to the immunoglobulin supergene family.  L1 mediates processes such as neuronal migration, neurite extension and axon fasciculation. It has been speculated that L1 may facilitate the invasiveness of glioma brain tumors as well. By modifying L1 expression in several human and rat cell lines, we will determine directly the impact of this molecule on invasiveness.  In the chick embryo brain we can produce noninvasive tumors using rat 9L gliosarcoma cells and invasive tumors using rat C6 or human U-87 glioma cells.  We found that rat 9L gliosarcoma cells do not express L1, so the chick homologue NgCAM was stably transfected into them.  Recently, there have been reports suggesting that L1 is proteolytically cleaved (shed) from the surface of some cells, so we are interested if this occurs in glioma cells.  Flow cytometry data suggests that L1 is produced within U-87 and C6 cells, but not expressed on the cell surface.  This is consistent with the concept that L1 is shed from these cells.  Thus, shed L1 might influence the migration of glioma cells.  After confirming expression of L1 in U-87 and C6, L1 will be attenuated and cells will be sorted to negative homogeneity. Invasiveness of the original tumor cells will be compared to cells that are modified for L1 expression for all lines in vivo.  Supported by UDRF.

 
 

The Role of Bcl-2 in Brain Cell Migration and Survival Melanie Evans and Deni S. Galileo, Department of Biological Sciences During vertebrate brain development, neurons migrate along radial glia to their final destinations.  We propose that interactions between extracellular fibronectin and neuronal cell surface integrin receptors facilitate neuronal migration and suppress a normal period of widespread programmed cell death (PCD; apoptosis).   In other systems, similar interactions cause expression of the anti-apoptotic protein Bcl-2.  Bcl-2 is expressed in early chick optic tectum (OT; midbrain), but its role is uncertain.  Indirect immunofluorescence at various embryonic days (E) was used to identify Bcl-2 distribution.  Previously, sensitive fluorescent in situ end labeling was used to identify a wave of PCD in OT on E7.5-E8.  Here, we extended this by performing an independent analysis of apoptosis using Annexin V.   Annexin V binds to phosphatidylserine on the surface of apoptotic cells.  Flow cytometry was used to reveal that significant numbers of E8 OT cells labeled with Annexin V-biotin and, thus, were apoptotic.  Analyses will be performed on E6, before migration takes place, and E9, after mass cell death occurs.  Bcl-2’s role in apoptosis and migration is being tested in vivo by expressing the protein using both a replication-competent, “RCASBP(A) Bcl-2”  and a replication-incompetent retroviral vector.  Multiple embryos have been infected with a replication-competent retroviral vector, and are currently being analyzed for viral expression, ectopic Bcl-2 expression, and morphological abnormalities.  It is hypothesized that the Bcl-2-expressing clones will contain statistically significant greater numbers of neurons and they will be distributed differently, showing that Bcl-2 plays an important role in neuronal migration and survival.

 
 

Creation of tools to generate a conditional allele for HIP/RPL29 Orlando Glasby1,2 and Catherine Kirn-Safran2 1Department of Biological Sciences, Delaware State University, Dover, DE  2Department of Biological Sciences, University of Delaware, Newark, DE HIP is a heparin/heparan sulfate interacting protein identical to ribosomal protein RPL29. It is expressed during both embryogenesis and adult life and is believed to participate in multiple cell processes, such as cell adhesion and protein synthesis.  To study the significance of the HIP/RPL29 protein in vivo, the generation of mice carrying null mutations for the Hip/Rpl29 gene is underway. The HIP/RPL29 expression pattern during early development suggests that early embryonic lethality may result from the total depletion of HIP/RPL29, thereby preventing the study of HIP/RPL29 importance in tissues developing at later stages. For this reason, mice models will be created to examine tissue-specific gene function. A first step will be to generate mouse embryonic stem (ES) cell lines carrying a conditional Hip/Rpl29 allele where essential exons are flanked by two Cre recombinase (loxP) recognition sites. A “three loxP” strategy will be used to target ES cells and will flank, with loxP sites, both the HIP/RPL29 wild type allele and a neomycin phosphotransferase gene expression cassette (neo). A toxin gene, diphtheria toxin A-chain (DT-A) will be used as a selectable negative cassette. The “three loxP” strategy will enable the complete excision of both the floxed Hip/Rpl29 wild type allele and the neo positive selection cassette upon breeding with a tissue-specific Cre recombinase transgenic mouse line. The purpose of this project has been to initiate construction of a targeting vector necessary to generate ES cell lines carrying a Hip/Rpl29 conditional allele. Our goal is to create mouse lines that cannot express HIP/RPL29 in specific tissues and study its consequences on bone formation.

 
 

Genomic Applications of Stabilized Synaptic Complexes Brandy Heckman, Michael Rice, Michael Usher, and Eric Kmiec Department of Biological Sciences and the Delaware Biotechnology Institute The recombination protein Escherichia coli RecA has been shown to catalyze strand exchange between both single and double-stranded DNA. In vitro experiments have shown RecA forms a stable filament when added to single stranded DNA with the use of ATPgS, or other non-hydrolyzable ATP analogs.  This filament, when incubated with a larger double-stranded homologous target, forms a triplex nucleic acid structure known as a displacement loop or D-loop. The addition of a second modified oligonucleotide, complementary to the initial incoming oligonucleotide, results in the formation of a complement stabilized double displacement loop or double D-loop. Upon the removal of RecA protein, evidence has shown D-loops remain stable only in supercoiled DNA targets. Double D-loops, however, have been shown to be much more stable not only in supercoiled DNA, but also in linear and genomic DNA targets. Because of its increased stability, double D-loops help to provide a number of opportunities for diagnostic applications.

 
 

Nucleotide Sequence of the Apolipoprotein A-II Gene  in the turtle, Trachemys scripta elegans Erin Hill, Lauren Baker, Liz Manning, Stacey Karr, Robin Davis,  Robert Hodson, and David Usher, Department of Biological Sciences Apolipoproteins of the red-ear slider turtle Trachemys scripta elegans are being studied to compare the cDNA and gene sequences to other vertebrate species. By comparing sequences from such evolutionarily distant animals, conserved regions should indicate functionally important domains. The gene studied in this project is apolipoprotein A-II (apoA-II). Previously some of this gene’s sequence was determined, including in the 3’ direction, most of Intron 2, Exon 3, Intron 3 and Exon 4. The complete sequence has now been found for Exon 1, Intron 1, Exon 2 and Intron 2 using gene “walking” techniques, yielding a genomic sequence nearly 3 kb long. Comparisons of the genomic DNA of turtles and other animals have not shown much homology along the gene; however, alignments of the cDNA sequences have shown conserved regions, particularly throughout the coding region.

 
 

Apolipoprotein A-IV in Turtles and Humans. Swetha Jarugumilli, Robin Davis, Donna Maslak, and David Usher Department of Biological Sciences Apolipoprotein A-IV (apoA-IV) aids in mammalian in high-density lipoprotein metabolism. It has been found in a linkage group with apoA-I, and apoC-III in humans and mice. This project investigates the presence of the apoA-IV gene in the red ear slider turtle, Pseudymes scripta. The turtle was chosen on the basis of its evolutionary distance from human and other mammalian species.  Finding the apoA-IV in the turtle will allow us to compare it with the mammalian apoA-IV and detect the areas that are conserved to estimate the regions important for the function of apoA-IV. This would play an important role in determining the role of apoA-IV in causing atherosclerosis. To achieve this purpose a genomic library is being constructed using the lambda BlueSTAR vector system. Lambda BlueSTAR is a lambda replacement vector designed for the production of genomic DNA libraries with efficient cloning of DNA fragments 7-20 kbp in size. Currently the first step in the construction of the genomic library, which is the small-scale digestion of the DNA with the Sau 3AI enzyme, has been carried out. The conditions necessary for obtaining the DNA within the best-desired size range have been optimized.

 
 

The Role of NgCAM in Neuronal Migration in Brain Peter Lazzopina, Phil Kudish, and Deni 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 embry brains by modifying expression there 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.  Thus far, PCR has been used to clone the antisense sequence with the proper restriction sites for insertion into the vector.  Additionally, western blotting has been used to examine normal NgCAM expression in the developing chick brain.  Future work will concentrate on cloning the antisense NgCAM sequence into the retrovirus and examination of the resultant effects.  Supported by NIH.

 
 

Endoplasmic Reticulum Arrays Resulting from the Overexpression of SERCA1a in Mouse Ltk-Cells Karla F. Leavens1,2, Suzanne E. Biehn,2, and Norman J. Karin,2 1Dartmouth College and 2Department of Biological Sciences The sarcoplasmic reticulum (SR) is proposed to arise from the endoplasmic reticulum (ER) during skeletal myogenesis. SR/ER Ca2+-ATPase (SERCA) is the main membrane protein found in the SR. Overexpression of the avian fast-twitch isoform, SERCA1a, in mouse Ltk- cells leads to the formation of localized intracellular membrane structures. These “plaques” contain large concentrations of the SERCA1a protein, as seen by immunofluorescent labeling. Previous experiments also showed the presence of other ER proteins, such as BiP and calreticulin. The current project has shown cDNA encoding catalytically-inactive SERCA1a also produces plaques in Ltk- cells, suggesting the effects of the protein are structural, not enzymatic.  An experiment to determine whether the mouse cell’s own SERCA isoform, SERCA2, was present in the plaques was unsuccessful due to a lack of effective antibody. The original mutant SERCA1a cDNA was contained in a plasmid that differed from the plasmid containing wild-type SERCA1a in two respects. There was a single base pair mutation (D351N) which eliminated the ATPase function of the protein. There was also a myc epitope at the end of the SERCA1a cDNA, that was inserted by the lab which designed the plasmid. In order to verify the myc epitope was not altering the function of the mutant SERCA1a cDNA, a new D351N mutant plasmid was made using restriction enzymes to cut out the mutated region, and a ligation reaction to insert the mutant sequence into the digested wild-type plasmid. This new mutant will be transfected into Ltk- cells to see if plaques are formed.

 
 

Perlecan Cell Adhesive Properties Megan Lynam, Anissa Brown, Jeffrey Safran, Daniel Carson,  and Mary C. Farach Carson, Department of Biological Sciences Perlecan (Pln), a large multi-domain heparan sulfate proteoglycan, commonly found in the basement membrane of extracellular cartilage matrix consists of five independently functioning domains (I-V).  With the exception of domain I, the other four domains are known to have sequences similar to various protein families including low density lipoprotein receptor, laminin A, neural cell adhesion molecule (NCAM), the immunoglobulin (IgG) family, and epidermal growth factor (EGF).  Using a bioinformatic approach, 16-18 amino acids long peptides were constructed representing each domain, with sites chosen based on their predicted antigenicity, hydrophilicity, and likelihood of being exteriorly disposed on the mature folded protein. While characterizing the peptides, it was discovered (J, Safran et al, unpublished) the peptide that corresponds to domain IV (TWSKVGGHLRPGIVQSG) displayed a significant level of adhesion activity.  The properties of adhesion were investigted by testing all domain specific peptides in a coated well adhesion assay in comparison to the negative control, BSA, and type I collagen, the positive control. The murine fibroblast cell line, C3H10T1/2, and MG-63, a human osteosarcoma cell line, displayed both cell attachment and spreading when introduced to an adhesive sequence derived from perlecan’s domain IV.  In both C3H10T1/2 and MG-63 cell lines, focal adhesions rapidly formed and assembled the actin cytoskeleton.  The amount of adhesion in both cell lines is dependent on the concentration of perlecan IV peptide present in the coated wells.  Experiments are underway to identify the receptor(s) of perlecan domain IV and to characterize the properties of binding, including the influence of divalent cations.  (Supported by NIH grant DE 13542)

 
 

Deciphering the Interactions Between the Four Major Players  in SV40 Replication Initiation David Manna and Daniel Simmons, Department of Biological Sciences During the process of Simian Virus 40 (SV40) DNA replication initiation there are four major proteins that interact with each other to unwind the origin of replication and form an initiation complex.  First is SV40 Large T antigen (T), the only protein that SV40’s DNA encodes.  This multifunctional protein must interact with Topoisomerase I (Topo I), DNA polymerase alpha/primase (DNA pol), and Replication protein A (RPA) in order for in vitro replication to initiate.  Topo I has been shown to bind to T itself, while the interactions T has with DNA pol and RPA are not from direct binding. The following experiments are designed to test the effects that the four major proteins have on each other while initiating unwinding at the SV40 origin of replication.  These experiments will hopefully shed new light on the initiation of SV40 DNA replication, one of the best know models by which one may study a simplified version of the complicated process of mammalian DNA replication initiation.  Preliminary data shows that when increasing amounts of DNA pol are added into in vitro replication conditions containing T and Topo I, a larger total amount of origin DNA becomes unwound than when T and Topo I alone are acting to unwind the DNA.  New data also show that DNA pol only enhances unwinding to a certain extent, when too much is added, less DNA is unwound and the radioactive signal is disrupted.  This suggests that there is a preferred molar ratio between T, Topo I, and DNA pol when unwinding the origin, and any greater amount of DNA pol will disrupt the initiation complex.

 
 

The Use of Acid Urea to Isolate of Helicobacter pylori from Synthetic Groundwater Joshua C. Miller and Diane S. Herson, Department of Biological Sciences Helicobacter pylori infects approximately two thirds of the world’s population.  It is an etiologic agent for several gastric problems including peptic ulcers and stomach cancer in humans and yet its route of transmission is still unknown.  One possibility is that H. pylori is transmitted by water via a fecal-oral route.  One of the difficulties in the detection and isolation of H. pylori from water is that other bacteria are present in greater numbers and may be inhibitory to H. pylori.  In the acidic environment of the human stomach it has been shown that H. pylori persists by producing urease.  This enzyme converts urea into ammonia, thereby creating a microenvironment with a less acidic pH.  Other organisms do not produce urease and are vulnerable to the low pH of the stomach.  In our studies we attempted to use acid urea treatment of water to reduce the numbers of competing organisms.  In the control experiments presented here, acid urea treatment of water (pH of 3) resulted in an exponential decrease over time of both Escherichia coli and Pseudomonas aeruginosa populations.  The ultimate goal of this study is to develop a method for the isolation of H. pylori from water.  The concentration of acid urea that will inhibit the other bacteria present but allow for H. pylori isolation will be determined.  It is expected that H. pylori will persist for the 6 hours of this study with a lower death rate than either E. coli or P. aeruginosa.

 
 

Effects of HIP/RPL29 Knock-down on In Vitro Chondrocyte Differentiation Stephanie A. Miller, Mary C. Farach-Carson and Catherine B. Kirn-Safran Department of Biological Sciences Heparin/heparan sulfate interacting protein (HIP) is a small, highly basic protein identical to ribosomal protein L29 believed to participate in multiple cell processes, such as cell adhesion, protein synthesis and potentiation of growth factor activity. In situ hybridization and immunohistochemistry showed that HIP/RPL29 is tightly expressed during chondrocyte terminal differentiation. To investigate the role of HIP/RPL29 normal expression during cartilage formation, we designed a ribozyme approach to knock-down HIP/RPL29 expression in a cell culture model for chondrogenesis. The multipotent mouse embryonic skin fibroblast cell line C3H/10T1/2 was stably transfected with an expression vector driving high ubiquitous nuclear expression of a ribozyme flanked by either HIP/RPL29-targeted or control scrambled sequences.  Clones obtained after zeocin selection were further analyzed for integration and expression of the ribozyme constructs using PCR and RT-PCR, respectively.  Semi-quantitative analysis of HIP/RPL29 by Northern and Western Blotting identified at least two clones in which HIP/RPL29 expression levels are perturbed at both mRNA and protein levels.  Preliminary studies showed in one ribozyme-transfected clone that reduced levels of HIP/RPL29 inhibits cell growth and accelerates differentiation of C3H/10T1/2 into cartilage-like cells. These data suggest a role for HIP/RPL29 as a regulator supporting cartilage growth.  Additional clones will be analyzed to demonstrate that reduced expression of HIP/RPL29 is associated with fast progression towards a more differentiated chondrocytic state. Studies on the effect of HIP/RPL29 knock-down will shed light on cartilage formation and maturation of adult cartilage.

 
 

Role of Osteopontin Charge Forms in Renal Stone Formation Ian Musselman, R. Al-Shami, D. Carson, and M.C. Farach-Carson, Department of Biological Sciences Osteopontin (OPN) is a major non-collagenous phosphoprotein located in the bone extracellular matrix. OPN also has  been found in the luminal surfaces of different glandular tissues and in many biological fluids. It is a secreted, highly acidic  protein that binds to hydroxyapatite and Ca2+ in the context of mineralization. Its amino acid sequence contains a conserved Gly- Arg- Gly- Asp- Ser (GRGDS) sequence, which allows it to bind effectively to the integrins. Two charge forms of OPN differing in their extent of phosphorylation have been identified in osteoblasts upon treatment with 1alpha, 25-dihydroxyvitamin D3 . OPN-1 represents the highly phosphorylated protein (pI 4.6) while OPN-2 is the less phosphorylated form (pI 5.1). It is thought that the extent of phosphorylation affects the ability of OPN to regulate crystal formation in solution. A current research focus is to establish a methodology for studying the expression of these OPN forms in human urine that can be applied to a variety of biological fluids. Samples have been collected from both healthy and stone forming elderly individuals of both genders. Using the SELDI ProteinChip technology, a Mass Spectrometry based technique, it is possible to determine a relationship between the levels of each charge form of OPN and the gender or propensity to form renal stones. These data will then be confirmed using Western blot and other conventional analyses. (Supported by NIH grant HD25235 to D. Carson)

 
 

Transcription of the Brain Creatine Kinase Gene in Glioblastoma Cells is Regulated by a Factor Related to Activator Protein 2 (Ap2) Sajid A. Noor, Dianna Willis, Yanping Zhang, and George R. Molloy Department of Biological Sciences Sajid Noor explainiong his poster to Dr. Jeremy Nathans Previous reports from Dr. Molloy’s laboratory have established that forskolin-mediated elevations in cAMP increased transcription of brain creatine kinase (CKB) mRNA in U87 glioblastoma cells despite the absence of a cAMP response element (CRE) in the CKB proximal promoter.  However, the proximal CKB promoter has four AP2 elements and current transient transfection experiments show that transcription of CKB in U87 cells is induced by transcription factor AP2, which is known to be activated by cAMP.  Mutation of the four AP2 elements indicated that the induction of CKB transcription by AP2 is principally mediated through the AP2 element located at –55 bp of the CKB promoter.  One of my experimental goals will be to show that the –55 AP2 element is also important in the forskolin-mediated induction of CKB transcription.  A second goal is to firmly establish the role of AP2 in CKB induction by showing that a dominant-negative form of AP2? can prevent the AP2?-mediated induction of CKB in U87 cells and, importantly, also block the forskolin-mediated increased transcription of CKB mRNA.  A third goal is to determine if induction of CKB transcription involves transcription factor AP2? or, alternatively, a new member of the growing list of AP2-related transcription factors possibly with a glial origin. Since U87 glioblastoma cells are a model cell culture system for astrocytes, the induction of CKB transcription by AP2 may help to determine the mechanism of regulation of CKB expression in glial cells during development and under normal and pathological conditions.

 
 

Effect of Extracellular Calcium on Preadipose Cells (3t3-L1) Barbara Nyatete1,2, Mary C. Farach-Carson2, and Kamil Akanbi2 1Linclon University and 2Department of Biological Sciences, University of Delaware Cells are able to sense and respond to changes in extra cellular Ca+2. Previously this lab showed that elevated extra cellular Ca+2 inhibits 3T3-L1 preadipocytes' differentiation. Since the discovery of calcium sensing receptors on the surface of parathyroid cells, there has been increased interest in extra cellular Ca+2 as a first messenger. The parathyroid cell calcium receptor binds extra cellular Ca+2 and activates phosphilipase- C and adenylyl cyclase through the G-protein isoforms Gq and Gi, respectively. The purpose of these experiments is to decipher the mechanisms way by which elevated extra cellular Ca+2 inhibits the differentiation of 3T3-L1 preadipocytes. Mouse cell line 3T3-L1 was used; 3T3-L1 preadipocytes undergo expansion before terminal differentiation which is characterized by rounding up of the preadipocytes and accumulation of lipid in cells. Treatment of mouse cell line 3T3-L1 cells with variable concentrations of Ca+2 showed fewer lipid filled cells are observed in cells exposed to higher extra cellular Ca+2  concentration as compared to those in lower concentration as revealed by Oil Red O staining. Cells were counted on hemacytometer to see whether their number is affected by increasing extra cellular concentration. The data indicate that increasing the extra cellular Ca+2 affected the cell numbers. There were fewer cells in culture treated with higher Ca+2 concentration than the control. Experiments using inhibitors, pertussis toxin (PTx) a Gi protein inhibitor and U73122, a phospholipase-C (PLC) inhibitor are still on going to examine their effects on the expression of nuclear transcription factors of C/EBP family and PPAR that co-ordinate adipocyte differentiation.

 
 

Expression of Ca2+ Channels in Mouse Pre-Osteoblastic Cells Karyn G. Oberman and Norman J. Karin, Dept. of Biological Sciences Autosomal dominant polycystic kidney disease is a common genetic disorder in humans caused by mutations in one of two genes, PKD1 or PKD2.  The function of the gene products, polycystin-1 and polycystin-2, is not clear; however, polycystin-2, the smaller of the two proteins, is predicted to be a cation channel with high permeability to Ca2+.  Evidence indicates that both gene products are integral proteins of the plasma membrane and that they must form a heterodimer for polycystin-2 to function as a Ca2+ channel.  RT-PCR revealed that both polycystins are expressed in MC3T3-E1 cells, a mouse pre-osteoblastic cell line.  This finding may be related to the fact that autosomal dominant polycystic kidney disease often has skeletal deformities as a side effect.  Although we have found that MC3T3-E1 cells express the mRNA encoding both PKD1 and PKD2, we have been unable to amplify the entire PKD2 sequence in MC3T3-E1 cells.  We therefore hypothesize that MC3T3-E1 cells express an alternate splicing product than the PKD2 expressed in kidney.  Using two sets of primers for different regions of PKD1, we were able to conclude that the PKD1 expressed in MC3T3-E1 cells is authentic.  Because of the ease with which we can amplify PKD1, as well as the middle portion of PKD2, we are confident that the PKD2 expressed in MC3T3-E1 cells is a variant of the previously reported sequence.  The rapid amplification of cDNA ends (RACE) technique is currently being employed in order to obtain the full sequence of PKD2 in osteoblasts.

 
 

Changes in Gene Expression of Co-cultured  Human Bone Marrow  Stromal Cells and Prostate Cancer Cells Roma Patel1,2, Cristiana Savorè2, Mary C. Farach-Carson2, 1Rice University and 2Department of Biological Sciences,  University of Delaware

Prostate cancer has a high tendency to metastasize to bone. Research has shown that the prostate cancer cells in the bone environment gain osteoblast-like phenotype and induce osteoblastic metastases (Fu et al, 2002).  Prostate cancer cells have been shown to preferentially adhere to the endothelium (Cooper et al., 2000) and migrate through it to reach the bone environment. We hypothesized that, before encountering the osteoblasts, the migrating prostate cancer cells are most likely to interact with the bone marrow initiating the cascade of events leading to bone metastasis.  To reproduce the bone marrow extracellular matrix environment in which the cancer cells express their metastatic behavior, we co-cultured immortalized human bone marrow stromal cells with prostate cancer cells of human origin.  We compared the gene expression of the different cells before and after co-culture using membrane-based gene arrays. The expression of a total of 198 genes was analyzed focusing on genes involved in angiogenesis and genes of the TGF? superfamily that influence cellular processes such as adhesion, proliferation, apoptosis and secretion of extracellular matrix.

 
 

Mechanical Loading Effects on VSCC Subunit Expression  and Complex Formation in MC3T3-E1 Cells Erwin C. Puente, Joel J. Bergh, and M.C. Farach-Carson Department of Biological Sciences Bone adapts to mechanical load by altering its mass and geometry.  Osteoblasts, cells that secrete bone matrix, are believed to act as mechanosensors that transduce mechanical stimuli into cellular biochemical signals.  Upon application of mechanical load to osteoblasts, a rapid rise in intracellular Ca2+ occurs.  This elevation is due to an increase in Ca2+ influx through voltage sensitive calcium channels (VSCC) located in the plasma membrane.  VSCC are composed of 4-5 protein subunits, of which the a1 subunit is the site for Ca2+ influx. I hypothesize that addition of mechanical load to cultured osteoblasts will change expression of VSCC a1 subunit mRNA.  Real-Time Polymerase Chain Reaction (PCR) was performed to analyze changes in mRNA expression.  Mechanical load of MC3T3-E1 cells resulted in an increase of a1C mRNA expression but a short-term, temporary decrease in a1H mRNA expression. Presumably, the increase in a1C subunit expression facilitates the increase in Ca2+ permeability. The a1H mRNA expression was not up-regulated and actually decreased after 2 hours of loading.  This difference in mRNA expression between the a1C and a1H subunits may be mediated by cyclooxygenase-2 (COX2). COX2 has been shown to increase during mechanical load and is important in gene regulation.  Future experiments will attempt to determine if the changes in a1 subunit expression due to mechanical load are directly related to COX2.

 
 

Expression Patterns of Apolipoproteins Induced  by 3T3 L1 Adipocyte Differentiation Jennifer Risser, Danielle Skorupa, John David, William Cain and David Usher Department of Biological Sciences Apolipoproteins are nonlipid proteins occurring in plasma lipoproteins, such as high-density lipoprotein (HDL) and low-density lipoprotein (LDL).  Apolipoproteins function in the transport of these lipoproteins.  SAGE (Serial Analysis of Gene Expression) data suggests the expression of six apolipoproteins in 3T3 L1 adipocytes and preadipocytes.  According to the SAGE libraries, apolipoproteins A-I, A-IV, C-I, D, E and M are significantly expressed during adipocyte differentiation.  We are interested in finding the levels of expression of these apolipoproteins in adipocytes as they potentially play a role in the development of atherosclerosis and hyperlipidemia, two conditions that contribute to the onset of coronary artery disease (CAD).  We are using real-time Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) to both verify the expression of the apolipoproteins and to relatively quantify their level of expression during adipocyte differentiation.  Real-time PCR data has confirmed the expression of the six apolipoproteins in both 3T3 L1 adipocytes and preadipocytes.  Upon further investigation, we hope to find the relative levels of expression of these apolipoproteins at specific points during differentiation from fibroblast to mature adipocyte.  We expect that the levels of expression will be greater in adipocytes than preadipocytes, indicating that they are significant to mature adipocyte function.

 
 

Localization of Spam1 Isoforms  on the Immature and Mature Murine Sperm Michelle D. Rulon and Patricia A. DeLeon, Department of Biological Sciences The Sperm Adhesion Molecule 1 (Spam1) is a sperm membrane glycoprotein with three functions in mammalian fertilization, namely: penetration of the cumulus cells, promoting the acrosome reaction, and secondary binding to the zona pellucida.  Previous studies in our lab have shown that in the mouse the protein (67 kDa) is localized on the anterior and posterior regions of the sperm head, produced in the testis and epididymis, and present in different isoforms. One isoform, which has an N-linked glycan that preferentially binds the Lycopersicon esculentum lectin (LEL), is found in caudal sperm and epididymis, but not in testicular Spam1. This lectin was also recently shown by others to stain a 68 kDa mouse protein that binds to the anterior and posterior regions of the sperm head. The similarity of the Spam1 localization and molecular weight to that of the LEL stained protein suggests that the latter is a Spam1 isoform.  We studied the localization of this isoform as a means of determining its possible role in sperm function and maturation.  Sexually mature mice were sacrificed and sperm were collected from the caput (immature) and cauda (mature) epididymis. Immunocytochemical and lectin cytochemical studies were performed.  Slides were analyzed using fluorescence microscopy and computer imaged.  To date, the results indicate that the pattern of distribution of LEL is seen predominantly in caudal sperm, suggesting an association of this protein localization with maturation. Further studies are underway to examine the localization with sperm aging and over- and under-expression of the protein.

 
 

The Use of Real-time RT-PCR to Identify Expression Patterns of Novel Genes During 3T3-L1 Preadipocyte Differentiation. Danielle Skorupa, John David, Jennifer Risser, William Cain, and David Usher Department of Biological Sciences Until recently, adipocytes were viewed as static and inactive storage reserves for fatty acids.  However, current studies done on a variety of diseases such as diabetes, coronary artery disease, atherosclerosis and obesity are all pointing to defects in adipocyte release or uptake of fatty acids as the underlying cause.  Consequently, our laboratory is investigating the differentiation program of the 3T3-L1 preadipocyte cell line by serial analysis of gene expression (SAGE).  Through the recent completion of the 3T3-L1 SAGE library, our laboratory was able to identify several novel genes which were upregulated during the cell line’s differentiation program.  We have found ?-endosulfine, scavenger receptor type B1 (SR-B1), CD36, sterol carrier protein 2 (SCP2) and ATP-binding cassette transporter class A1 (ABC1) to be highly expressed in both preadipocytes and adipocytes.  These genes are all involved in certain aspects of cholesterol metabolism and transport.  The goal of the current study is to validate these findings using real-time RT-PCR to quantitatively analyze mRNA transcripts.  Determining the role of these genes in the preadipocyte differentiation program will provide well-needed information on adipocyte development.

 
 

Ocular Expression of the  JAM Family Members Arthur Suckow and Melinda K. Duncan Department of Biological Sciences

The three members of the junctional adhesion molecule (JAM) family of cell adhesion molecules (CAMs), JAM-1, JAM-2 and JAM-3, are all members of the immunoglobin superfamily.  Recently, our group became interested in this family of CAMs when a cDNA microarray analysis on transgenic mice overexpressing PAX-6 in the lens revealed that JAM-1 mRNA expression was 2.5 fold elevated over normal.  More recently, another group established that heterozygous small eye mice containing mutations for the PAX-6 gene lack functional cell adhesive properties in their corneas.  These data suggested that JAM-1 is present in both the lens and the cornea and that its gene expression is regulated by PAX-6, a transcription factor that is essential for normal development of eye tissue.  In order to confirm the presence of JAM-1, RNA was isolated from both the corneas and lenses of adult mice, as well as from 15-day embryos where the JAM-1 transcript is known to be expressed.  Primers were then designed and the presence of JAM-1, JAM-2 and JAM-3 RNA was confirmed in both the lens and the cornea by RT-PCR analysis.  This analysis also found that JAM-2 RNA was preferentially expressed in the lens and that JAM-3 RNA was preferentially expressed in the cornea.  At the protein level, the presence of the JAM-1 protein in the lens and the cornea was confirmed by both immunohistochemistry and western analysis.  Currently, a developmental expression study of the JAM-1 protein in eye tissue is underway.

 
 

Chariteristics of the Protein Complexes formed at the Human B2 Lamin Origin Terrance Taylor and Daniel Simmons Department of Biological Sciences Our lab is interested in understanding the mechanism of eukaryotic DNA replication. Previous studies have shown that a cell-cycle dependent protein complex forms over a 74bp region of the human origin of replication site fixed to the 3’ end of the B2 Lamin gene. Three proteins were isolated during this analysis (HOXA13, HOXC10 and HOXC13), but by the authors’ admission, none appears to represent a helicase or similar protein (1). The overall goal is to isolate and study the proteins that interact with the B2 Lamin origin sequence and compare them functionally with known helicases such as the SV40 viral protein, T-antigen. Through the ligation of 14 overlapping oligonucleotide primers, the B2 Lamin origin was constructed for use in later experiments. Gel-shifting experiments with the B2 Lamin ori and extracts from a human brain tumor cell line, U-87, have yielded DNA-protein complexes in the presence of a several hundred-fold excess of non-specific competitor. Elution, precipitation, and denaturing of these DNA-protein complexes yield a consistent pattern containing a number of variably sized proteins.

 
 
 

Detecting the Presence of Helicobacter pylori in New Jersey Well Water Samples  Using the Polymerase Chain Reaction – Inhibition Studies Maren Thompson, Pamela Vercellone-Smith, and Diane Herson  Department of Biological Sciences Approximately half of the world’s population is infected by Helicobacter  pylori which causes serious health problems; therefore, it is essential to determine its route of transmission. Evidence has shown a correlation between H. pylori infection and consumption of untreated water, suggesting that water may serve as a route of transmission. Our lab has been collaborating with the New Jersey Department of Environmental Protection to determine if H. pylori is present in New Jersey ground water. 138 well water samples were collected to determine the presence of H. pylori using the Polymerase Chain Reaction (PCR). The majority of the samples (91.3 %) were negative.  The goal of this research was to determine if the low level detection of H. pylori was due to inhibitory substances in the ground water samples.  Environmental samples contain many unknown elements, which can interfere with PCR and prevent amplification of the target gene. To detect inhibition, H. pylori DNA was transferred into each of the water samples.  PCR analysis was then performed to detect the presence of inhibitory materials in the samples.  Our results indicate that less than 10% of the waters tested inhibited PCR. Despite the low level of inhibition, this must be taken into account when using PCR to work with environmental water samples.

 
 

The Effect of Oxidative Stress on Helicobacter pylori Natalie K. Weaver, Brady W. Redmond and Diane S. Herson, Department of Biological Sciences Helicobacter pylori is a common pathogen that colonizes over 50% of the world’s population yet the principal route of transmission is still unresolved.  Although some researchers have proposed a water-borne route of transmission, the environmental conditions under which H. pylori can persist in water have not been fully studied.  This study focuses on the ability of H. pylori, a microaerophilic organism, to persist when incubated under varying levels of oxygen.  Persistence was studied by suspending samples of H. pylori culture in synthetic groundwater and incubating them under varying atmospheric oxygen levels.  H. pylori was able to persist the longest under microaerophilic conditions at 10ºC.  Since H. pylori require a microaerophilic environment, toxic oxygen forms are expected to be particularly harmful to H. pylori.  Real time PCR was used to examine whether the transcription of the superoxide dismutase, catalase, and alkyl hydroperoxidase genes was affected when H. pylori is exposed to atmospheric oxygen levels.

 
 

The Ability of Pax-6 and dEF1 to Regulate  the Production of bB1-Crystallin Alisha Yallowitz, J. R. Taube, and Melinda K. Duncan Department of Biological Sciences The lens is composed of two main cell types: epithelial cells and fiber cells, which develop from elongated epithelial cells.  bB1-crystallin marks the differentiation from lens epithelial to lens fiber cells, and it is possible that repressor transcription factors, such as dEF1 and Pax-6, prevent its expression in lens epithelial cells. 10 bp substitution mutations are being made in the binding sites for dEF1 and Pax-6 along the –432/+30 region of the chicken bB1-crystallin promoter.  The mutations should result in increased expression of the bB1-crystallin promoter in epithelial cells, when compared to the unmodified promoter, because the repressor factors would no longer be able to bind. For one dEF1 site, a –282/+30 construct was made with engineered AccI and XbaI restriction sites.  A PCR temperature gradient was done to determine an annealing temperature that would produce one band of the construct.  After PCR amplification, the construct was ligated into a PCR 2.1-Topo cloning vector and transformed into E. coli.  The plasmid with the insert was then purified and sent for sequencing.  After determining the correct sequence the insert will be ligated into pBasic CAT. A –432/-292 substitution construct will then be made and cloned upstream in the gene, creating a –292/-282 deletion. Additional substitution constructs of dEF1 and Pax-6 sites will be made and transfected into non-differentiated lens cells. By studying the roles that dEF1 and Pax-6 play in bB1-crystallin production it would give us a better understanding of why bB1-crystallin is found only in lens fiber cells.

 
 

Protein Analysis of Helicobacter pylori at Various Temperatures  by 2-D Gel Electrophoresis Erin Zoranski, Shauna Hodge, and Diane Herson Department of Biological Sciences Helicobacter pylori, a Gram negative, microaerophilic bacterium, has been proven to be the cause of many gastric problems such as gastritis. It is also correlated with the formation of peptic ulcers, stomach cancer and B-cell MALT lymphoma. While the route of transmission is still unclear, the fecal-oral and oral-oral routes are possibilities. Water is believed to be one of the potential sources of infection. Previous studies in the Herson lab have shown that H. pylori is able to survive and persist at low temperatures and in simulated ground water. The goal of my research is to determine if proteins are being synthesized or being degraded by H. pylori under various temperatures. Cultures of H. pylori are grown in brucella broth at 37 degrees Celsius, shaking, under microaerophilic conditions and then transferred to 15 degrees Celsius or room temperature under the same conditions. The cells were then washed and lysed with a French pressure cell at 800 psi. Steps were then carried out to concentrate cellular proteins. The sample was then analyzed by 2-D gel electrophoresis and silver stained for development of the gel. The protein profiles created will then be studied to determine whether proteins are degraded or synthesized and proteomics will allow for the identification of these proteins.