Tiffany Crawford, Appalachian State University
Sponsored by: Dr. Ann Erickson
Screening cDNA Libraries for Lysosomal Proenzyme Receptors
In an attempt to isolate the cDNA encoding a newly identified lysosomal proenzyme receptor (LPR), two cDNA libraries, one from mouse embryo tissue and the other from mouse fibroblasts were screened using the yeast two-hybrid system. Two proteins known to bind to the LPR were used "as bait" to identify the cDNA encoding the receptor, procathepsin L, a lysosomal cysteine protease and procathepsin D, a lysosomal aspartic protease. One half of the colonies screened received procat L as bait; the other half received procat D. Yeast were sequentially transformed, first with the DNA binding domain (BD) vector containing the bait and then with the activation domain (AD) vector containing the cDNA library. All transformants colonies were plated in the absence of histidine as the first level of selection to identify protein interactions. Yeast colonies which grew were then assayed for ß-galactosidase activity by blue/white screening as the second level of selection. Two thousand yeast colonies transformed with the mouse embryo cDNA library were screened using the ß-galactosidase assay and all of the yeast colonies transformed with the mouse fibroblast cDNA library were assayed for ß-galactosidase activity. Negative ß-gal activity results were obtained for all colonies screened from both cDNA libraries. Possible explanations for the negative results include: poor yeast cracking efficiency, toxicity of 3-aminotriazole to yeast, neutral pH of the nucleus, failure of the expressed protein to localize to the nucleus, low LPR representation in the cDNA library, incorrect folding of the integral membrane, and weak affinity of proenzymes for LPR.
Anamaria Craice, Cornell College
Sponsored by: Dr. Phil Carl
Optimization of Parameters for Differential Display of cDNA
Differential Display is a method of detecting differences in gene expression between different cell types or cell conditions and can detect genes expressed at very low quantities. Differential display is a favorable method over the traditional technique of subtractive hybridization. Different parameters of the differential display method were investigated in order to optimize the procedure for our purposes. Among the most important of these parameters are the non-radioactive method and the use of 5-nitroindole-substituted primers for amplification of cDNA.
Eric Wagner, Albright College
Sponsored by: Dr. Ken Bott
Defining Genes Essential for Mycoplasma Cell-free Growth and Metabolism
Mycoplasmas are cell wall-less members of a bacterial genus derived from Gram positive ancestors. In addition to lacking cell walls, members of this genus are unique in their physiology and in their use of the genetic code. In nature all Mycoplasmas are parasites of some plants, animals, and insects, but many species can be cultivated in the laboratory if their extremely fastidious nutritional requirements are met. Mycoplasma genitalium, the smallest of all members of this genus has been associated with non-gonococcal urethritis in man. It is a biological curiosity because its genome is the smallest found among all free living species. Its genome is so small that it comes closer to the definitions of the minimal number of genes necessary for survival and for pathogenicity than does any other species. In 1995, its complete genomic sequence was elucidated and predicted by computer analysis to encode 470 proteins. Many of its putative gene products were not recognized by similarity to genes from other species which make up the national sequence database. In fact, there is no experimental evidence relating more than half of its genes to an actual cellular function. The research direction in this laboratory is now attempting to define the genes essential for cell-free growth and metabolism; subsequently we hope to elucidate a subset of genes required for the parasitic metabolism in association with human epithelial cells in culture. Two avenues of experimental approach are (1) obtain mRNA population from axenically growing cell cultures, derive cDNA from that population and clone these derivatives of "functioning genes" into a conventional E. coli host/vector system, and determine the DNA sequence of these clones to compare them with the complete genomic sequence to identify functioning genes and (2) use transposon mediated insertional mutagenesis to randomly inactivate genes thereby establishing a category of "dispensable" functions. Transposon Tn4001, which encodes gentamycin resistance following uptake by electroporation has been the most successful to date. My project involved some participation in each of these aspects of the lab's research, especially the construction of a new plasmid vector from p2062 and pMUT2 to be used in transposon mediated mutagenesis. pMUT2 carries a green fluorescent protein cassette which will make transposon integrations in frame with promoter sequences easier to detect.
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LaKesha A. Tables, St. Ambrose College
Sponsored by: Dr. James A. Raleigh
Immunohistochemical Analysis of Tumor Redox Status
Hypoxia in tumors is a stress that might generate more aggressive tumors. Factors involved include the formation of superoxide anion when oxygen enters previously hypoxic regions. Superoxide anion reacts with nitric oxide -- a normal constituent of many tumors -- to produce peroxynitrite which can be activated to produce a strong oxidizing intermediate that can nitrate tyrosine to produce 3-nitrotyrosine. The reaction is catalyzed by acid and metals and is inhibited by protein sulfhydryl groups that react rapidly with activated peroxynitrite. An important thiol containing protein is metallothionein. We hypothesized that peroxynitrite is activated in hypoxic regions of tumors because these tend to be more acidic than the well-oxygenated regions near blood vessels and that metallothionein, if present, could quench such reactions. The extent of hypoxia in C3H mammary carcinomas was varied by air breathing, carbogen breathing or hydralazine injection. Relative to air breathing, carbogen (95% O2/5% CO2) breathing decreases hypoxia by increasing oxygen supply to tumors. Hydralazine increases hypoxia by dilating blood vessels in normal tissues thereby "stealing" blood supply from tumors. The extent of hypoxia was measured immunohistochemically by means of the hypoxia marker, pimonidazole, and an associated monoclonal antibody (MAb) prepared in our laboratory; 3-nitrotyrosine and metallothionein were identified with commercially available antibodies. Measurement of the different factors was achieved by quantitative image analysis. 3-Nitrotyrosine was undetectable in most tumors. Where it was detected, it was not in the hypoxic regions. Metallothionein was present in the hypoxic regions of tumors. The amount of metallothionein correlated with the extent of hypoxia. We conclude that 3-Nitrotyrosine is not formed in detectable amounts in the hypoxic regions of C3H mammary carcinomas; however, metallothionein is produced in the hypoxic regions of tumors and, although peroxynitrite might be activated in the acidic hypoxic regions, the quenching of peroxynitrite by metallothionein might prevent 3-nitrotyrosine formation.
Nancy J. Gettel, Alma College
Sponsored by: Dr. Ralph S. Quatrano
Characterization of Suppressors of an ARM-Repeat-Containing Protein in Saccharomyces cerevisiae
Asymmetric cell division involves the polarization of spherically symmetric cells which then undergo unequal division perpendicular to the polar axis. The interaction of the actin cytoskeleton with transmembrane proteins plays a key role in asymmetric cell growth. A family of armadillo- repeat-containing proteins is indirectly involved with the binding of the actin cytoskeleton to the transmembrane proteins in multicellular animals. In Saccharomyces cerevisiae, genes encoding two distinct armadillo-repeat-containing proteins have been identified, one of which has been designated ARM1. This gene was disrupted in order to determine if it had any effects on S. cerevisiae growth. Its removal resulted in a cold-sensitive phenotype, causing the Darm1 mutants to be vulnerable to temperatures of 16 C. A high copy suppression screen using a yeast genomic DNA library was performed in order to identify genes that interact with ARM1. Nine different genomic fragments with the ability to suppress the conditional phenotype were found. These suppressors are currently being characterized in order to identify the individual open reading frames (ORFs) responsible for suppression of the phenotype. One ORF which has recently been isolated contains ankyrin-repeats, which may be linked to cytoskeletal function. A second ORF of interest, SCY1, was also identified by others in an unrelated screen to locate high copy suppressors of a GTPase mutant. The relationship of this ORF to ARM1 function is currently being investigated. Tagging of SCY1 with a myc epitope in order to determine its subcellular localization and identify those proteins with which it physically interacts is currently in progress. This should provide information about the function of SCY1 within the cell and its relationship to ARM1. Further characterization of ARM1 and the suppressors will be continued in future experiments.
Mark K. Larson, Concordia College
Sponsored by: Sharon L. Milgram
What are the Protein-Protein Interactions Involved in cAMP-mediated Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator?
The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a chloride channel present at the apical surface of epithelial cells lining the airway, gastrointestinal tract and reproductive tract. CFTR also regulates the conductance of other ion channels located at the apical cell surface. The dual roles of CFTR as an ion channel and a conductance regulator may account for the variety of pathologies seen in cystic fibrosis patients. CFTR is regulated in a cAMP-dependent manner and is phosphorylated in vivo by the cAMP-dependent protein kinase (PKA). CFTR function increases with small changes in intracellular cAMP. Therefore, we hypothesize that a protein known as an A-Kinase Anchoring Protein (AKAP) anchors PKA at the apical surface in proximity to CFTR for efficient regulation. AKAPs bind to the N-terminus of the regulatory subunit (RII) of the PKAII holoenzyme, a binding that localizes PKA to specific sites in cells and is characteristic of the Type II kinase. Stutts et al. demonstrated that the HT31 inhibitor peptide that blocks the interaction between RII and AKAPs decreases CFTR channel activity in patch clamp experiments. This suggests that there is indeed an AKAP that helps target PKAII in the vicinity of CFTR. Using bacterial expression and affinity chromatography, we have generated and purified RIIa and biotinylated it for use in overlay assays of protein extracts prepared from human tracheal cells. In these assays, RIIa binds to AKAPs in protein fractions on nitrocellulose membranes. The biotinylated RIIa probe and HT31 have revealed a number of potential AKAPs in our overlays of human tracheal cells. One protein visualized in the overlay corresponds to a western blot of the AKAP ezrin, and another corresponds to AKAP79. In addition, we have screened human airway epithelial and whole lung bacteriophage l cDNA libraries using the same RIIa probe and isolated a known AKAP (AKAP149) and a novel cDNA that we are characterizing. Furthermore, we intend to use the yeast two-hybrid system to screen an airway epithelial library to identify known and novel RII binding proteins in airway epithelium. In the future, truncated AKAP constructs will be made to identify the RII binding site and membrane targeting domain for novel AKAPs. Mutagenesis of novel and known AKAPs will also be used to determine if the AKAP is involved in CFTR regulation.
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Jody Weinstein, Wells College
Sponsored by: Dr. Nobuyo Maeda
Renal Function in Natriuretic Peptide Receptor A-null Mice
Hypertension is one of the most important risk factors for cardiovascular disease. The symptoms seen in humans with untreated hypertension are 1) compensated cardiac hypertrophy, 2) decompensated ischemic changes in myocardium, and 3) possible death from heart failure or aneurysm. These same hypertrophic heart disease symptoms are seen in gene knockout mice with an absence of the natriuretic peptide receptor A. In these mice, as well as in humans, lethal incidences can be precipitated by stress including changes in diet. The central systems involved in blood pressure regulation are the circulatory system and the kidney. The kidney regulates body fluid volume, which affects circulatory volume, and thus cardiac output. Therefore renal excretory function is essential in regulating blood volume. Volume and altered fluid excretion can function to both initiate hypertension and function to compensate and protect against effects of increased blood pressure. NPRA -/- mice can be used as an animal model of the progression of heart disease associated with hypertension, since these knockout mice are known to have high blood pressure, and high blood pressure is a product of both heart and kidney function. We are interested in assessing whether the kidneys of these NPRA -/- mice, which appear histologically normal, are indeed functioning in an adequate manner. By altering daily dietary sodium intake, we studied the physiological differences between 0 and 2 copy male and female mice regarding renal function. To do so we investigated many variables that could predict kidney function on an NPRA -/- background and compared these to wild type mice.
Stephen Moyer, Goshen College
Sponsored by: Dr. William F. Marzluff & Dr. Robert Duronio
The Role of cyclin E and E2F in Histone Gene Expression
The expression of histone mRNA is strictly coordinated with the replication of DNA. Two important factors controlling DNA replication in the cell cycle are the G1 cyclin cyclin E and the transcription factor E2F. In an attempt to determine the role that both of these factors play in histone mRNA expression, homozygous null cyclin E and E2F mutant Drosophila embryos were stained with a histone 3 mRNA probe. At 10-12 hours after egg deposition at 25 C, the cyclin E mutants did not express H3 mRNA. Therefore, it can be concluded that cyclin E is required for H3 mRNA expression. Additionally, since it is known that E2F is activated normally in cyclin E mutants, the data implies that E2F is not sufficient for H3 mRNA expression. Another aspect of histone expression is the processing of its pre-mRNA. Histone pre-mRNA is unusual in that it does not contain introns or a 3' poly-A tail. Instead the pre-mRNA has a highly conserved stem- loop binding structure at its 3' end. One component necessary for the processing of the pre- mRNA is the stem-loop binding protein (SLBP). SLBPs have been identified in Xenopus, sea urchins, humans, and mice. In this project, band shift assays were performed on the stem-loop binding structure with Drosophila embryo and ovary extracts. Shifts occurred with both extracts, indicating the presence of a site-specific SLBP in Drosophila.
Christie Laming, Hollins College
Sponsored by: Dr. David Brenner
Regulation of Protoporphyrinogen Oxidase Gene Expression By Hemin, DMSO, and Retinoic Acid
Protoporphyrinogen oxidase (PPO) along with the enzyme ferrochelatase (FEC) are terminal enzymes in the heme biosynthetic pathway. Erythroid differentiation of human erythroleukemia (HEL) and K562 erythroleukemia cells was induced using hemin, dimethylsulfoxide (DMSO), and retinoic acid (RA) in DMSO in order to examine the expression of protoporphyrinogen oxidase (PPO). The gene expression analyzed was PPO and FEC. We assayed induced HEL and K562 erythroleukemia cell lines using reverse transcription polymerase chain reactions (RT-PCR). Cells were treated with the appropriate inducer (hemin, DMSO, and RA) and collected at 3 and 7 days. In K562 cells, PPO was induced at day 3 by hemin, RA, and DMSO, and remained constant until day 7. FEC seemed to remain constant in RA and hemin treated cells. In DMSO treated cells, levels decreased at the 3 day time point, then returned to normal by day 7. Thus, it appears that in K562 cells PPO is unregulated during chemically induced erythroid differentiation.
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Tinsley H. Davis, Swarthmore
Sponsored by: Dr. Janne G. Cannon
Comparison of opa Hypervariable Regions in Five Clinical Isolates of Neisseria gonorrhea: Evaluating the Possible Role of Anti-opa Antibodies in Protection from Acute Salpingitis
Pelvic inflammatory disease (PID), also known as acute salpingitis, affects an estimated 1 million women in the United States each year. 20% of these women will develop tubal factor infertility or an increased risk for life-threatening ectopic pregnancies. Sexually transmitted diseases (STDs) are responsible for 60% of PID cases, and 55% of these cases are associated with Neisseria gonorrhea, the causative agent of gonorrhea. N gonorrhea is a gram-negative, obligate human pathogen that infects 2.5 million people per year. There is no vaccine available for gonorrhea due to the antigenic variation exhibited by the surface antigens present on N gonorrhea. Opacity (Opa) proteins are thought to mediate secondary adherence, but their exact role in infection is unproven. There are eleven different opa genes present in the chromosome of the common lab strain FA1090. The opa locus is defined by a combination of two hypervariable regions, HV1 and HV2, separated by a conserved region; each hypervariable region can be characterized as one of six cassettes. These hypervariable regions are thought to be exposed on the cell surface, while the conserved regions may lie in the membrane. A cohort study in Nairobi suggested that antibodies to an increased number of Opa variants reduced a woman's risk of developing acute salpingitis. However, no single anti-Opa antibody was associated with reduced risk of PD. It is important to characterize the Opa proteins found in these strains to determine the epitopes recognized by antiOpa antibodies. Preliminary studies show that the anti-Opa antibodies are directed against the hypervariable regions of the protein. To find similarities among the hypervariable cassettes of opa genes from the five reference strains used in the Nairobi study, I assayed chromosomal DNA from each strain by probing with oligonucleotides derived from each hypervariable cassette in strain FA1090. DNA from four of the five strains hybridized to at least one of the 12 FA1090 HV probes. Three of those four strains bound more than one HV probe. Southern blotting analysis showed that some of the HV probes hybridized to more than one fragment. These results indicate that the hypervariable regions of the clinical isolates from Africa are similar to each other and also have homology to FA1090, a strain isolated in North America. The similarity of HV regions in FA1090 and HV regions in the reference strains is significant because previously it was not known if there was any conservation of HV regions between strains. Sequence analysis of the cloned opa genes is pending, and this information will allow for more detailed analysis of HV regions common to the isolates and FA1090.
Pieter Lagaay, University of California-Santa Cruz
Sponsored by: Dr. Howard Fried
Analysis of Binding of Signal Recognition Particle Protein 19 to Helix 6 of SRP RNA
Signal Recognition Particle (SRP) is a cytoplasmic RNA-protein complex that mediates association of ribosomes with the endoplasmic reticulum in the process of protein secretion. The goal of this project was to define a minimal RNA binding site for one SRP protein, i.e., SRP19, with the eventual aim of determining the 3D structure of SRP19 bound to SRP RNA. SRP19 was expressed in bacteria as a polypeptide containing six N-terminal histidine residues and purified by Ni2+-chelate chromatography. Derivatives of SRP RNA were synthesized by in vitro transcription in the presence of a 32P-labeled ribonucleotide. Different portions of SRP RNA were used as ligands in binding reactions with SRP19 to determine a dissociation constant (Kd). The Kd conveys how much of an affinity SRP19 has with the different SRP RNA substrates, which indicates whether certain nucleotides are crucial for binding between SRP19 and SRP RNA. The degree of binding between SRP RNA and SRP19 was measured by gel mobility shifts. The results showed successful binding of SRP19 to the "S" domain (also called D35) of SRP RNA, with an estimated Kd of 10-8 M. A smaller substrate, helix 6 RNA, did show RNA-protein complex formation but did not demonstrate a gradual increase in binding as seen for D35 RNA. We hypothesized that SRP19 may dimerize at high protein concentration and that dimerization changes SRP19's RNA affinity. To determine if SRP19 self-associates, an experiment was carried out in which equal amounts of SRP19 and an epitope-tagged SRP19 (SRP19-myc) were mixed and subjected to immunoprecipitation with an anti-myc antibody. Only the myc-tagged SRP19 was found in the immunoprecipitate, suggesting that SRP19 may not dimerize. The significance of this project will be in helping to understand how protein molecules bind specifically to their target RNAs.
Kendra Zeiter, Ohio Northern University
Sponsored by: Dr. Mark Peifer
Identification of the Binding Site for p120ctn in Drosophila Cadherin
Proper cell:cell adhesion is critical to the development and maintenance of multicellular organisms. Adherens junctions are a component of the cell adhesion machinery and are essential for organizing cell polarity and integrity of tissues in both vertebrate and invertebrate animals. Adherens junctions are built around transmembrane proteins known as cadherins. The extracellular domain of cadherins interact homophilically with cadherins on neighboring cells. The intercellular domain of cadherin interacts with a number of adapter molecules called catenins. These include Armadillo (the Drosophila homolog of b-catenin) and a-catenin which serve to tether the junction to the actin cytoskeleton. Adherens junctions are not static entities, however. During development and under certain physiological conditions, cells must be able to lose adhesion with one another in order to migrate to other locations within the body. The mechanisms for regulation of adhesion remain a mystery. One likely player is the newly discovered vertebrate catenin, p120ctn. p120ctn becomes tyrosine phosphorylated when cells are treated with certain mitogens. This highly phosphorylated p120ctn has an increased affinity for cadherins and the p120ctn:cadherin is in the noncytoskeletal, and thus non-functional, fraction of the cell. In addition, the level of tyrosine phosphorylation of p120ctn in tumor cells correlates with a less adhesive, more invasive phenotype. In order to understand the role of p120ctn in cancer, we must first understand its normal cellular function. We will approach this problem using the fruitfly Drosophila melanogaster as a model system, as insect adherens junctions are highly homologous to vertebrate adherens junctions at the molecular level. Previously, our lab has cloned a fly p120ctn homolog and demonstrated that it interacts physically with DE-cadherin using the two-hybrid system. I have generated smaller pieces of DE-cadherin and using the two-hybrid system, demonstrated that the p120ctn binding site lies within the first 41 amino acids of the cytoplasmic domain of DEcadherin. These data will allow us to make relevant mutations in p120ctn binding site to test their effects in vivo.
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Leslie E. Sutton, Campbell University
Sponsored by: Dr. Clifford Rinehart
Regulation of Growth Control Genes in Breast and Endometrial Cell Tumors
Endometrial and breast cancers appear to have a close relationship to the aging process. These types of cancers occur more in older than younger people, but the contribution of aging to carcinogenesis is not well understood. One hypothesis considers aging to be a part of the body's genetic plan, postulating that our DNA is programmed for selective gene expression as a function of age. In young adults, certain genes are expressed; however, an older adult will express these genes at different levels. Such an age-related control of gene expression causes old cells to function differently than younger cells. We believe that two main cell types are involved with endometrial and breast cancer. These two cell types, fibroblast and epithelial, are in close proximity to one another. They communicate through biochemical messengers sent from the fibroblasts to epithelial cells. These signals from the fibroblasts direct the functioning of epithelial cells. Using these chemical signals fibroblasts can regulate development and differentiation of epithelial cells. Evidence is accumulating that the regulatory factors secreted by young fibroblasts are different from those secreted by old fibroblasts. This alteration places the epithelial cells of an older individual in a different microenvironment than the epithelial cells of a younger person. We hypothesize that the microenvironment surrounding epithelial cells in an older person loses a portion of the ability to control cellular proliferation, and a large percentage of dysregulated epithelial cells form tumors. Such qualities are in great contrast to the tumor-suppressive chemical environment of epithelial cells in a young person. This relationship between aging, the microenvironment of cells, and cancer points to an age-related change in gene expression as a probable cause of endometrial and breast cancers. Cancer is characterized by unchecked cell growth. Further, the more serious the cancer, the more dysregulated the cellular proliferation. Four genes known to control the growth of epithelial cells were studied in this project. All of the genes in breast cancer and some of the genes in endometrial cancer demonstrated the expected expression pattern; cells in more aggressive types of cancer had less proliferative control than did the cells in milder types of cancer. Future work will study how the biochemicals from fibroblasts effect this alteration in epithelial cell growth control.
Christian Capitini, Drew University
Sponsored by: Dr. Ryszard Kole
Improving Uptake of Antisense Oligonucleotides Using Cationic Lipids Noncovalently Linked to Transferrin
b-Thalassemia is a genetic blood disorder that results from a mutation that causes incorrect splicing pathways, yielding an aberrant b-globin product. Antisense oligonucleotides targeted to the aberrant splice site forces the splicing machinery to select the correct splice site, thus restoring the activity of the damaged gene. The objective of this study was to improve the cellular uptake of these antisense oligonucleotides into mammalian cell lines expressing the b-Thalassemia W52-705 mutation, using cationic lipids noncovalently linked with transferrin, an iron transporter. Since b- Thalassemia affects red blood cells, which have a high demand for iron, their high transferrin receptor density could be utilized to deliver antisense oligonucleotides through receptor-mediated endocytosis. Both HeLa and K562 cell lines were treated with transferrin-lipid complexes containing antisense oligonucleotides for 5 hours. After the transfection, cells were grown for 22 hours and their RNA was isolated for reverse transcription-PCR analysis. The RTPCR products were separated on a 8% polyacrylamide gel and analyzed using autoradiography. Treatment of cells with antisense oligonucleotides restores splicing in a dose-dependent fashion. The addition of transferrin yielded a similar amount of corrected product at a lower concentration of oligonucleotide as compared to using lipid-oligo complexes alone. Transferrin may allow a more cell-specific delivery of oligonucleotides using a ligand-receptor system, and may also facilitate release of oligonucleotide from the endosome after entering the cell.
Julie Hanson, College of St. Scholastica
Sponsored by: Dr. Steven Bachenheimer
Induction of NFkB Binding Activities but not Activation Function Following Infection by Herpes Simplex Virus
Nuclear Factor kappa B (NFkB) is a transcription factor that has attracted attention due to its unusual and rapid regulation, the wide range of genes it controls, its central role in immune and inflammatory and anti-apoptotic responses, and its involvement in several diseases. NFkB is anchored in the cytoplasm by IkB (Inhibitory kappa B). It has been shown that IkB must be phosphorylated proteolyzed for NFkB to be released and translocated to the nucleus. This process can be induced by immunological stimuli, UV irradiation, growth factors, and viral infection. The purpose of this project was to determine if NFkB is translocated to the nucleus following herpes simplex virus (HSV) infection. Classically NFkB forms a heterodimer with a 50 kDa (p50) and a 65 kDa (p65) subunit, although the dimer can consist of a number of other subunits as well. These subunits include: RelB, c-Rel and p52. All subunits contain a Rel homology (RH) domain. This domain is a 300 amino acid multifunctional region that controls dimerization, interaction with IkB, DNA binding, and contains the transactivation domain. The most abundant forms of NFkB are p65/p50, p50/p50, and p65/p52. In order to determine the function of NFkB it is also important to identify what subunits the NFkB is composed of in the nucleus. The p65, Rel B, and c-Rel subunits of NFkB contain a trans-activation domain. Once NFkB translocates to the nucleus and binds DNA it must have the trans-activation domain activated before it can stimulate transcription. Therefore, another aspect of the project was to determine if NFkB in the infected cell nucleus has trans-activation function.
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Julie Harris, Rensselaer Polytechnic Institute
Sponsored by: Dr. Frank Church
Construction of His-tagged Mutants of Heparin Cofactor II
The D-helix of heparin cofactor II (HCII), from amino acids 170-195, contains binding sites, possibly overlapping, for both dermatan sulfate and heparin. One site of relevance is Arg 189, which is thought to be a residue within the D-helix that binds dermatan sulfate. Arginine has a positively charged side chain at physiological pH that is thought to be involved in the formation of the glycosaminoglycan-binding site. By converting this Arg to Lys, Ala, and Glu, all with different side chain characteristics, the effect of the side chain charge on the active site can be assessed. These R189 mutants were created by site-directed mutagenesis and sequenced. Protein activity assays that measure the inhibition of thrombin in the presence and absence of the glycosaminoglycans, heparin and dermatan sulfate, can be used to compare these mutants. One way to purify HCII is by mixing it with heparin sepharose and eluting with a salt gradient. Due to the mutations being made in the heparin binding site, this is an unreliable way to purify Dhelix mutants. By inserting 6 His residues at the carboxy terminus of the HCII amino acid sequence, a different method of purification can be utilized. The 6-His tag will bind to Ni2+-NTA-agarose; by binding the tagged mutant serpin to a Ni2+-NTA-agarose column, it will be isolated without using the D-helix. The first attempt to alter HCII in this way resulted in a mutation of 5 His and a Pro in the tag rather than 6 His. Protein assays showed that this HCII was hyperactive, with a faster rate of inhibition of thrombin both in the presence and absence of glycosaminoglycans. Although its hyperactivity rules it out as a new wild-type HCII, the His5Pro-tagged HCII may be therapeutically useful and was therefore tested for its ability to be purified on a nickel chelate column. The correct 6 His tag has been added separately to both the amino and the carboxy terminus of HCII and their activity will be assayed in future experiments.
Jason Stumpff, Eckerd College
Sponsored by: Dr. Brian Herman
Alteration of Bcl-2 Related Protein Levels in HPV-positive Cervical Carcinoma Cell Lines Following Treatment with Mitomycin C
The Bcl-2 family of proteins have been identified as significant effectors in the regulation of apoptosis. The mechanisms through which these proteins control apoptosis are unknown. It has been suggested that the expression of these apoptotic regulating proteins may be mediated by p53. The E6 protein of high risk human papillomaviruses (HPV), type 16 and 18, has been shown to inactivate p53 function. Our interests were to determine which of the Bcl-2 related proteins are expressed in HPV-positive cervical carcinoma cells and whether their levels are altered following treatment with chemotherapeutic agents. In the current study, expression of Bcl-2, Bax, Bad, and Bak were examined through western blot analysis in HeLa, SiHa, and C33A cells following treatment with the DNA-damaging agent mitomycin C (MMC). Bcl-2, an inhibitor of apoptosis, was expressed in both HPV 18-positive HeLa and HPV-negative, p53-mutated C33A cells with slight induction in both cell lines following MMC treatment. Strong induction of the apoptotic promoting proteins Bax, Bad, and Bak was observed in HeLa cells following MMC treatment. Bax was also induced in HPV 16-positive SiHa cells following MMC treatment. Bad and Bak induction were significant in C33A cells following MMC treatment which suggests a p53- independent pathway for modulation of these proteins. Expression and induction of the Bcl-2 related proteins studied are cell-type specific. This specificity could possibly be explained by HPV status and copy number and by p53 status.
Joy Kendrick, King College
Sponsored by: Dr. Joe D'Ercole
Analysis of Transgenic Mice That Overexpress IGF Binding Protein-5
Insulin-like growth factor-1 (IGF-1) is an anabolic peptide essential for normal fetal and postnatal mammalian growth. It is a major mediator of growth hormone's effects and stimulates cell proliferation and differentiation through interactions with specific cell-surface receptors. IGF-1 is primarily found in the body bound to high affinity IGF binding proteins (IGFBP). These binding proteins can either augment or inhibit IGF-1's interactions with its receptor, thereby either enhancing or reducing IGF-1's effects on the tissue. Among the 6 known IGFBPs, IGFBP-5 has the unique capacity to bind to basal membranes. In order to explore the in vivo actions of IGFBP- 5, we generated transgenic mice that overexpress in the pancreas. In our project we began to critically evaluate pancreatic islet function by performing glucose tolerance tests on IGF-I Tg, IGFBP-5 Tg and normal mice. In addition, we conducted a histological study with IGFBP-5 Tg and normal mice. Our results show that IGFBP-5 Tg mice demonstrate impaired glucose tolerance and islet hyperplasia compared to normal littermate controls.
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