Summer Undergraduate Research Symposium Thursday, 9 August 2001 McKinly Laboratory University of Delaware

Vitamin D receptor (VDR) polymorphisms have been linked with breast and prostate cancers. DRIP130 is one of the 13 components of the Vitamin D Interacting Protein (DRIP) complex, which regulates transcription in cells via interaction with Vitamin D-VDR complexes. The gene encoding DRIP130 maps to ~6q22-q24, which is in a region frequently deleted in malignant melanoma. Colonies of bacteria containing plasmids with a 500 bp portion of DRIP130 and full length DRIP130 have been isolated. A Phage-derived artificial chromosome (PAC), RPCI-5-914N13, containing the DRIP130 gene sequence has also been isolated. This PAC cannot be transfected into mammalian cells because it does not contain a mammalian selection marker. Therefore, experiments to retrofit the PAC insert into the pPAC4 vector are in progress. This procedure will allow selection by blasticidin for future transfection studies to determine its effect on human melanoma metastasis.

Experimental melanoma metastasis can be inhibited by the venom-derived disintegrin eristostatin. Since eristostatin’s interaction with cells usually involves an integrin, and these receptors have been associated with melanoma cell growth and invasion, it is thought that eristostatin’s interaction with melanoma cells involves one or more integrins. Eristostatin conjugated to alkaline phosphatase (ErAP) may be a useful material for studying the interaction of this disintegrin with melanoma cells. The binding of ErAP can be inhibited by unlabeled eristostatin in a manner comparable to previous studies using fluorescent-labeled eristostatin. The purpose of this study was to establish a panel of antibodies to integrin subunits that bind to 1205 LU, WM164, C8161, and MV3 human melanoma cell lysates. The melanoma cell membranes were solublized with 1% Nonidet P-40 in HEPES buffered saline. After centrifugation, the supernatant from the cellular material was assayed for total protein, and 25 m g of protein was separated by SDS-PAGE. The proteins were transferred to nitrocellulose and incubated with an antibody to one of eight integrin subunits. 1205 LU cells were found to strongly express a₃, a₄, a₅, and b₃, and weakly express a_v. WM164 cells possessed the same integrin subunits but showed weaker expression of a₃ and a₅^. C8161 cells also expressed the same integrin subunits as 1205 LU cells, but they appeared to have a lower concentration of a₄. MV3 cells expressed high levels of a₃, a₄, and a₅ and weakly expressed a_v. Unlike the other three cell lines, MV3 cells did not possess the b₃ subunit. The antibodies identified by this method will be used to compete with ErAP in binding to melanoma cells on a dot blot.

Connexin 43 (CX43), a gap junctional protein, is located on chromosome 6 in a region (6q21-q23) frequently deleted in malignant melanomas. Because of this, our hypothesis is that CX43 plays a role in metastasis suppression. RT-PCR revealed that in a panel of human melanoma cell lines, none of the cell lines expressed CX43. To test our hypothesis, CX43 will be transfected into metastatic cell lines. Hygromycin kill curves were performed on each cell line to establish the dose needed for transfection selection. After selection of transfected cells, CX43 expression will be measured. CX43-expressing cell lines will be used in metastasis assays employing nude mice to determine if the cells lose their ability to metastasize (i.e., metastasis suppressed).

Analysis of metastatic C8161 cell lines has shown that regions of chromosome 6 (chr 6) are missing in these cells. A deletion occurring at 6q16.3-q23 has been described. When an intact chr 6 is transferred into these cell lines, metastasis is suppressed indicating that the loss of a gene within the deletion is necessary for metastasis to occur. The specific genes responsible for metastasis suppression and the pathways by which they work are unknown. These genes can be found by comparing the expression of gene(s) and gene products in metastatic cells and non-metastatic cells. To test differential gene expression, various metastatic and non-metastatic cell lines originating from the C8161 parent line were studied for expression of two putative metastasis-suppressor genes. These two genes, Connexin 43 (Cx43) and Vitamin D3 interacting protein (DRIP130), were analyzed using RT-PCR and gel electrophoresis. The total RNA used for these procedures was collected from 15 cell lines originating from C8161. Results indicated the need to analyze multiple gene interactions involved in regulation of metastasis suppression. Microarray experiments will be used to identify additional genes involved in melanoma progression. The pathways in which the genes are involved can then be analyzed to determine possible mechanisms that allow metastases to form. This knowledge will provide a basis for future studies in prevention and/or treatment of malignant melanoma.

Protein expression is an integral part of our laboratory's disintegrin research. Since bacteria are used to produce these proteins, the cell line that can produce the greatest amount of protein is the most desired. Here we compare the expression capabilities of our current E. coli strain, BL21 Gold (G), against that of a newly derived strain, BL21 Codon Plus (CP). The protein produced by both cell lines in this experiment was the 7.5 kDa disintegrin eristostatin. Both strains were transformed with a pGEX-KG plasmid that contained the gene for eristostatin. Cells were lysed and protein was isolated using glutathione resin; the protein was cleaved from the resin using thrombin. HPLC profiles showed that the CP cells expressed a higher concentration of eristostatin than the G cells. This was confirmed by protein yields of 23 µg/L of culture (G) and 116 µg/L of culture (CP). Upon purification of the protein, ADP-induced platelet aggregation was used to determine IC₅₀, because eristostatin will bind to platelets via the integrin receptor "_{IIb$ 3}, and inhibit aggregation. The IC₅₀ values for eristostatin from both strains of cells were comparable to each other and to values previously obtained in our lab. Immunoblots using rabbit anti-eristostatin indicate that the protein was present in the thrombin digests from both cell lines. These results suggest that it would be advantageous to use CP cells for further expression of eristostatin mutations instead of the G cells.

The disintegrin eristostatin, a protein isolated from the venom of the viper Eristocophis macmahoni, has been shown to inhibit experimental melanoma metastasis in a mouse model. The receptor for eristostatin is
unknown, but eristostatin may inhibit melanoma cell metastasis by binding to the integrin alpha4beta1. Expression of alpha4beta1 increases in metastatic melanoma cells, and antibodies to alpha4 have been shown to inhibit melanoma cell metastasis. In this study, we examined the effect of disintegrins on the binding of alpha4 to melanoma cells. We expressed bacterial recombinant eristostatin, echistatin, and seven mutations targeting amino acids in the RGD loop. Human MV3, 1205 LU, and WM164 melanoma cells were incubated with fluorescent-labeled anti-alpha4 and an unlabeled disintegrin and were observed by confocal microscopy. Eristostatin was able to inhibit binding of alpha4beta1 to MV3 and 1205 LU. Truncated eristostatin inhibited binding of anti-alpha4 to WM164 cells when asparagine at position 31 was replaced with a methionine. Echistatin was unable to inhibit binding of anti-alpha4 to the three melanoma cell types. These findings suggest that the RGD motif of eristostatin is required to prevent anti-alpha4 from binding to MV3 cells, and that the asparagine at position 31 is necessary for eristostatin to prevent the binding of anti-a4 to 1205 LU cells. The RGD sequence and the tryptophan at position 30 are critical for eristostatin to prevent the binding of anti-alpha4 to WM164 cells. In a similar study, the effect of disintegrins on the binding of anti-beta1 to MV3, 1205 LU, WM164, and C8161 melanoma cells was examined. Finally, characterization of a number of integrins present on the surface of the melanoma cells was determined using antibodies to alphavbeta3, alpha2, and alpha6.

Relatively little is known about RNA modification reactions and the metabolism of sulfur-containing biomolecules, and s⁴U generation provides a paradigm for both of these critical, ubiquitous biochemical processes. The s⁴U generation in tRNA imparts near-UV sensitivity to tRNA through a photo-induced crosslink with cytidine-13. The formation of s⁴U is catalyzed by two enzymes, IscS and ThiI. ThiI from E. coli appears to have a large N-terminal and a smaller C-terminal domain. N-Terminal domain of ThiI contains a "P loop" motif that is found in a family of enzymes called the PP_i synthatase family. The "P loop" motif catalyzes the adenylation of carbonyl groups for oxygen substitution. This poster presents experiments to determine the roles of the N-terminal and C-terminal domains of ThiI in the activation of O⁴ uridine in the sulfur transfer.

Forensic scientists and art conservators occasionally need to identify glues or other binding media. A reliable identification procedure could be useful to detect fraudulent or highly restored pieces. Animal glues are high molecular weight proteins. Many modern glues are high molecular weight polymers. Information on the composition of glues is not readily available. Samples of different types of glues were decomposed (pyrolyzed) in a Thermal Desoprtion unit (SIS). The volatile products were trapped at the front of a gas chromatographic column, separated and detected by temperature programmed gas chromatography/mass spectroscopy (HP). Thermal desorption (decomposition) conditions (temperature and time) were varied for the glues. Chromatographic conditions (initial temperatures and temperature program) for the separation were also varied. Chromatograms of products from samples of rabbit skin glue, aged hide glue and white glue that were decomposed under similar conditions were reproducibly different.

Water soluble phosphines, such as tris-(2-carboxyethyl)-phosphine (TCEP) and tris-(2-cyanoethyl)-phosphine (TCNP), will reduce disulfide bonds. TCEP is a good substrate of sulfhydryl oxidase; however, the closely related TCNP is not a significant substrate. Kinetic studies using a rapid-reaction stopped-flow spectrophotometer show that TCEP reacts 30-100 times faster than TCNP with a range of model disulfides. These reactions are first-order in both phosphine and disulfide. The reactivity of phosphines presumably depends on the rate-limiting nucleophilic attack of a phosphine on a disulfide. Therefore, the nucleophilicity can be quantified by the pKa value of the phosphorus atom. The pKa of TCEP was determined to be 7.8 by P³¹ NMR – a value much higher than that quoted for TCNP (pKa=1.3). This pKa shift correlated with the increased nucleophilicity of TCEP as expected. These model studies explain, in part, why TCEP is a much better substrate of sulfhydryl oxidase.

In E.coli, the proteins IscS and ThiI work together in the transformation of the uridine at position 8 of tRNA into 4-thiouridine (s⁴U). The s⁴U acts as a near-UV sensor forming a crosslink with cytidine-13 on the same tRNA, thus leaving the tRNA a poor substrate for amino acyl synthetases. Examined eukaryotes contain neither s⁴U nor ThiI homologs, but many have IscS homologs, such as Nfs1p in S. cerevisiae. This project examines the ability of Nfs1p to substitute for IscS as a first step towards generating s⁴U in yeast by expressing thiI. As Nfs1p has been characterized as both mitochondrial and nuclear, the mitochondrial targeting sequence from Nfs1p is being fused to thiI in a yeast expression vector. To conduct in vitro tests, Nfs1p is being expressed in E.coli cells.

The disease RTH occurs due to mutations in the thyroid hormone receptor. Many of these mutations happen at the ligand-binding domain or near regions involved in ligand-dependent gene activation. Proper receptor activity can be recovered by rationally designing ligands that target the receptor and compensate for the mutation.

The development of highly enantioselective reducing processes for conjugated carbonyls into their corresponding alcohols is of great importance in biological related synthesis. The use of chiral ligands in conjunction with reducing agents such as NaBH4 and LiAlH4 have shown to reduced conjugated carbonyls with relatively high control of absolute configuration. The use of a, a, a', a'-Tetraaryl-2, 2-dimethyl-1, 3-dioxolan-4, 5-dimethanol (TADDOL) has been shown to be a highly effective moiety for production of the necessary reducing ligand. In our research, we hope to show that "TADDOL" derivatives 5a-e complexed with LiAlH4 will selectively reduce 1, 1-dimethylethyl 7-oxo-phenylheptanoate 4 into 1, 1-dimethylethyl
(7S)-7-hydroxy-phenylheptanoate 3 in good yield with high enantioselectivity.

Many bacteria modify uridine-8 of tRNA to 4-thiouridine (s⁴U), which serves as a photosensor for near-UV light. In early s⁴U generation assays using cell extracts and tRNA transcript containing [5-³H]uridine ([³H]tRNA), no [5-³H]s⁴U was detected even though [³⁵S]s⁴U was detected in parallel reactions run with [³⁵S]cysteine. What happened to the tritium? Some aminoacyl tRNA synthetases catalyze the wash-out of tritium from [5-³H]uridine at position 8 in tRNA, and their presence in cell extracts could explain the lack of [5-³H]s⁴U. Alternatively, the tritium wash-out might be inherent to s⁴U generation, which would provide mechanistic information. To resolve the source of wash-out, s⁴U generation with [³H]tRNA will be repeated using purified enzymes rather than cell extracts. From preliminary experiments, the [³H]tRNA appears to be radiochemically pure and competent as a substrate. Next, s⁴U generation assays will be performed using the verified [³H]tRNA substrate.

This research focuses on the plant’s natural defense reaction, the hypersensitive response and the involvement that nitric oxide may have in it. The possibility of increasing or accelerating the hypersensitive response could be discovered through the genetic manipulation of the gene or genes responsible for the biochemical pathway, which causes the hypersensitive response. Arabidopsis plants were mutated two ways: through point mutations, and through the insertion of activation tags. These two different screens each required inoculating the plants with Pseudomonas bacteria and an inhibitor of the biochemical pathway responsible for the hypersensitive reaction. Positive plants from both screens are then, crossed with another plant or self-pollinated, brought to seed, and inoculated again. The end goal of both screens is to determine the location of the gene or genes responsible for encoding the nitric oxide synthase enzyme.

The objective of the research is to locate the nitric oxide synthase (NOS) gene in Arapidopsis thaliana. Both ethyl methane sulfanate (EMS) and activation tagged plants were screened. A solution containing Pseudomonas syringae and NOS inhibitor was inoculated into the leaves. After incubation, the plants were then scored for the presence of a hypersensitive response (HR). EMS positives were then crossed to limit the number of mutations present; DNA was extracted, PCR reactions were run, and products were then run on agarose gels to determine linkage groups. This data will lead to the mapping and isolation of the NOS gene.

Macrophages are important modulators of the immune response. We have constructed an expressed sequence tag (EST) library from an avian macrophage cell line (HD11) stimulated with bacterial lipopolysaccharide (LPS). Over 1,200 clones from this library were sequenced and subjected to BLAST analysis. Eleven percent of the clones represent genes with potential immunological functions. Antigen recognition and processing is one essential step in the immune response. One gene that we have identified from the library and are studying in greater detail is the avian homolog to DEC205/gp200mr6. This transmembrane protein is a member of the mannose receptor family. It is also expressed by dendritic and thymic epithelial cells. This protein is believed to be responsible for presenting antigens to T cells. The initial EST clone contained only part of the DEC205 gene. We are using primers designed from the amino acid sequences of the mammalian homologs to DEC205 in RT-PCR to clone the remainder of the avian gene. The avian DEC205 gene is approximately 50% identical to its mammalian counterparts. In order to study the chDEC205 protein, another aspect of this project entails the cloning of a portion of the gene in a prokaryotic expression vector. We will use the protein to elicit the formation of polyclonal antisera in rabbits, which can then be used to study the synthesis and localization of this protein in vivo.

Although many scientists have proposed relationships between the solubility of a protein and electrolyte concentration, the formulations have all been empirical in some way. In order to find a more predictive relationship, an expression for the partial molar Gibbs free energy of a protein () in an aqueous electrolyte solution was derived. The derivation stems from the hypothesis that the excess Gibbs free energy of the protein can be constructed from two terms: one term accounting for the protein-solution interactions by way of the osmotic second virial coefficient and the other term accounting for the change in salt-solution interactions when adding protein. Satisfactory results are seen at dilute protein concentration and low ionic strength of electrolyte. However, calculated values of at the solubility limit for various electrolyte concentrations were not identical as expected. This result could be due to interactions between the solid protein crystals and the electrolyte solution that were neglected in the calculation. Also, the contribution from the protein-solution interaction term was negligible indicating that the behavioral characteristics of the protein in solution are unimportant, or that the osmotic second virial coefficient relation is inappropriate for this model.

Ultrafiltration using a stereospecific binding agent can potentially provide a high throughput, cost-effective means for commercial separation of chiral molecules. The feasibility of using affinity ultrafiltration for chiral separations as been demonstrated in recent studies with selectivities as high as 11 reported for a model system of DL-tryptophan with bovine serum albumin as the stereospecific binding agent. The goal of this study was to investigate the effects of chloride ion and ethanol on the binding of L- and D-tryptophan to bovine serum albumin. Ultrafiltration experiments were performed to determine the concentration of unbound tryptophan at various chloride or ethanol concentrations. The data showed an increase in the free tryptophan concentration as the concentration of chloride or ethanol was increased. Ultrafiltration experiments with a racemic mixture at different chloride concentrations gave a maximum selectivity of 9 for the separation of tryptophan stereoisomers.

The field of biochemical engineering is currently experiencing an explosion of interest and importance in the world of science. In the past, oligomeric protein folding has been understudied due to the lack of technology with which to characterize the intermediates. As our knowledge of protein folding increases, the complexity of the processes which drive these reactions becomes apparent. Such is the case with P22 tailspike, a model protein which has been studied for years. This protein is not disulfide bonded in its final trimeric conformation, and for the first time evidence exists for an intermediate with oxidized sulfhydryl groups not present in the native state. In this project, the 496 and 635 cysteine residues suspected to be involved in disulfide bonding were removed and replaced with serine residues. This double mutant was expressed at 15, 20 and 30 degrees Celsius, and it was found to make trimer. The highest yield occurred at the lowest temperature, where productive folding is most favorable. In light of past folding studies, it can be speculated that either the 613 disulfide bonds to its parallel residue in another chain, or an alternate pathway exists in which disulfide bonding is avoided. In conclusion, the creation of this mutant form presents many interesting questions and more research must be conducted to solve the mystery of the elusive disulfide- bonded intermediates.

Helicobacter pylori is a causative agent for many gastric illnesses. It is thought to be transmitted primarily by the fecal-oral route, and contaminated water may be the source of the organism. Other microorganisms that are present in the aquatic environment may have an inhibitory effect on H. pylori viability and detection. The goal of this study is to examine the interactions between H. pylori and Escherichia coli, a coliform which is used as an indicator of water potability, and Pseudomonas aeruginosa, a microbe that is commonly found in aquatic environments. Initial studies were done using each organism separately prior to studying them in combination. E. coli and P. aeruginosa were incubated in a variety of temperatures (10° , 23° , and 37° C). Both organisms persisted longest at 37° C. Techniques were also developed to detect H. pylori in the presence of E. coli and P. aeruginosa. An acid-urea solution (pH 3) was used to inhibit E. coli, while minimally affecting H. pylori. Below an initial cell concentration of 10⁷ cells/mL, the acid-urea solution limited the cell growth of E. coli.

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, Pseudymes scripta, 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 triglyercide-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. The apoC-III gene is transcribed on the opposite strand of DNA as the apoA-I and apoA-IV genes. The turtle apoC-III gene contains four exons and three
introns. The apoC-III gene has been sequenced from the 3' end to intron 2. Therefore, leaving the promoter/enhancer region, exon 1 and intron 1 to be sequenced. My goal is to sequence that region and then eventually "walk" toward where apoA-IV should be located. A 1.1 kb KpnI fragment, 0.9 and 1.2 kb KpnI fragments and a 1.5 kb SacI fragment were found that should span from intron 2 to the promoter/enhancer region. The 1.1 and 1.5 kb fragments were purified, cloned, transformed and sequenced. However, the sequences were not as expected due to the vector being rearranged. In order to correct this, the 1.1 and 1.5 kb fragments were reamplified, so cloning and transformation could be performed again. In the future, primers will be designed from the promoter/enhancer sequence to allow for "walking" toward the apoA-IV gene to occur.

Contraction of ovaries has been demonstrated in the mummichog, Fundulus heteroclitus and several other teleosts. We have expanded this information by recording spontaneous contractions and the response to acetylcholine in ovaries of the striped killifish, Fundulus majalis; the Atlantic silverside, Menidia menidia; the pumpkinseed, Lepomis gibbosus; and the goldfish, Carassius auratus suspended in a smooth muscle bath containing a modified Ringer's solution. Ovaries of all five species produced similar contractions in response to 10-5 M acetylcholine. Spontaneous contractions in F. majalis, and M. menidia occurred in a series of single peaks with frequencies and amplitudes similar to the pattern previously observed in F. heteroclitus. Contractions observed in C. auratus appeared in single peaks with an increased frequency and amplitude compared to F. heteroclitus. Those seen in L. gibbosus were more variable and had greater amplitude, typically with a series of superimposed higher frequency contractions. These results suggest that ovarian contractions may be a common phenomenon in the Atherinidae and Fundulidae, which share many reproductive characteristics, including the occurrence of semilunar reproductive cycles. The Centrarchidae are nest-building Percoids, which are taxonomically distant from the other species analyzed.

Calcium signals in the cell promote cell proliferation, cell differentiation cell excitation and further signal transduction. The major pathway for calcium to enter the cell is through the plasma membrane voltage sensitive calcium channel (VSCCs). A major subunit of this channel that we studied is the a_1c that serves as the pore forming subunit for calcium entry. We used three sub lines in these experiments: ROS17/2.8, parental line, ribozyme transfected ROS17/2.8, and ribozyme control. A ribozyme that eliminates the a_1c transcript and a control scrambled ribozyme were previously transfected into cultured rat osteosarcoma cells. We examined the effect of ribozyme against the VSCCs on basal growth, and we concluded that the presence of ribozyme in the ROS17/2.8 affects the growth of these cells. We examined the effect of two steroid hormones (17-b -estradiol and 2-methoxyestradiol) on these cell lines. Our results demonstrated that the presence of the active ribozyme does not affect the hormone-stimulated growth of the cells. Lastly we examined the effect of the ribozyme on apoptosis. These experiments are currently underway. We hypothesize that the ribozyme transfected ROS17/2.8 will grow slower than the ROS 17/2.8 parental or control cells, and might be more susceptible to apoptosis. Our results to date indicate that this hypothesis is likely to be correct, demonstrating the essential role of the VSCC in osteosarcoma cell growth.

Cell adhesion molecules play an important role in cell-cell interactions during many critical stages of embryogenesis as well as tissue repair in the nervous system. One such molecule is L1, a transmembrane recognition molecule that belongs to the immunoglobin supergene family and mediates dynamic neurological processes such as neural cell migration, neurite extension and adhesion. This cell adhesion molecule may facilitate in the invasiveness of glioma brain tumors. By modifying the L1 expression directly in several human and rat cell lines, the impact of this molecule on the invasiveness of glioma brain tumors will be better understood. For rat 9L gliosarcoma cell line, which does not express L1, the chick homologue NgCAM was stably transfected into these cells. For rat C6 and human U-87 glioma cells, which heterogeneously express L1, cells will be sorted to positive homogeneity utilizing FACs. Then, L1 will be attenuated by direct gene conversion followed by cell sorting. Invasiveness of the original tumor cells will be compared to the cells that are modified for L1 expression. For in vivo experiments, tumor cells are injected into the early chick embryo brain. After allowing the injected embryos to develop for several days to two weeks, the distance of migration is measured in tissue sections. For future experiments, in vitro assays will also be performed by adding tumor cells to brain cell aggregates and measuring the distance of tumor cell migration intothe aggregate.

DNA polymerase a/primase and Replication Protein A (RPA) are cellular proteins essential to the initiation of SV40 DNA replication. The viral protein T antigen forms a double hexamer on the core origin of SV40 to initiate replication. The specific interactions of DNA polymerase a/primase and RPA with the T-antigen double hexamer during replication initiation have yet to be undetermined. Although other labs have successfully completed purification of these proteins, the process is long and tedious. In an effort to make the proteins faster and cheaper we used the Impact system from NEB to generate intein-tagged proteins. The DNA polymerase subunits; p48pol, p58pol, and p68pol, and the RPA subunits; p70, p32, and p14 were each amplified by PCR. The DNA’s were then cloned into the appropriate PTYB vector from NEB constructs. The constructs were then recloned into the baculovirus transfer vector p1393, which were then co-transfected into insect SF9 cells to generate the recombinant baculoviruses. Recombinant viruses were tested for expression of the protein subunits by immunoflourescence. Expressing viruses were purified by plaque assays and amplified on insect SF9 cells. Currently, DNA polymerase is being purified by binding to chitin beads. The intein tag has a chitin-binding domain on either terminus of the protein. In the presence of chitin beads, the intein undergoes self-cleavage. This releases the protein without any extra residue from the chitin-bound intein tag. The purified protein for both DNA polymerase a/primase and RPA will be tested to determine if and under what conditions they interact with the T-antigen double hexamer. I will use Western blotting to test if the protein binds to the double hexamer, whether it is an ATP-dependent step, and if its binding is dependant on the presence of any other protein. Through this research, the steps of the assembly of the SV40 replication initiation complex on the origin will hopefully be more defined, and the involvement of these cellular proteins will be better understood.

During brain development neurons migrate along radial glia to their final destinations. Throughout migration, a signaling pathway, involving interactions betweens substrates and integrins, facilitates migration and suppresses apoptosis ( a normal period of widespread cell death.) In other systems, integrin substrate interactions are known to cause expression of the anti- apoptotic protein Bcl-2. Bcl-2 is expressed in early chick optic tectum (midbrain), but its specific role in the signaling pathway is uncertain. Immunostaining at various embryonic days (E) shows that Bcl-2 is present in the optic tectum, but the distribution in neurons along radial glia is still unclear. Aside from identifying Bcl-2 in tissue sections, cultured cells on cover slips were also stained and analyzed by confocal and fluorescence microscopy. Further immunostaining along with analysis by flow cytometry will be performed to further analyze Bcl-2, focusing on E6, before migration takes place, and E9, after mass cell death occurs (E7.5-E8). After the tissue has been analyzed for Bcl-2, it’s role in apoptosis and migration will be tested in vivo by expressing the protein using both a replication competent and replication incompetent retroviral vector. Neuronal progenitors will be infected with the Bcl-2 retroviral vector to determine its effect on the number of neurons that survive within a clone throughout the period of early cell death. It is hypothesized that the Bcl-2 expressing clones will contain statistically significant greater numbers of neurons and they will be distributed differently. This will show that Bcl-2 plays an important role in the migration of neurons up radial glia during development.

Neurons migrate along radial glial cell guides during brain development. It has previously been shown that the cell adhesion molecule a 8b 1-integrin and an unidentified substrate play a key role in this interaction. In order to determine exactly which extracellular matrix (ECM) protein acts as the key substrate in this mechanism several procedures were taken. Cryostat tissue sections of the chick optic tectum from embryonic ages day 6-12 (E6-E12) were double-label immunostained for vimentin (a cytoskeleton protein in the radial glia) and one of the suspected ECM molecules: fibronectin, tenascin, osteopontin, laminin, vitronectin. The sections indicated specific staining patterns of fibronectin (Fn) along the radial glia and a less-defined pattern for tenascin. Although the other ECM molecules were present in the sections, positive staining was not apparent. The same immunolocalization was carried out in suspended cells of the optic tectum from various ages and analyzed on the flow cytometer. Quantitative results are still being determined. Future aims include attempting to attenuate fibronectin expression in the developing brain to determine the effect. This will be accomplished by injecting a fibronectin-blocking antibody hybridoma cell line and a retroviral vector in ovo and allowing the brain to develop. It is hypothesized that blocking the proposed substrate (Fn) in the developing OT will cause abnormal neuronal migration and brain growth. In this way, the ECM molecules can be identified as the key a 8b 1-integrin substrate needed for proper interactions of the migrating neuronal cells.

Gene sequences for the apolipoproteins of the red-ear slider turtle Pseudemys scripta are being studied to obtain structure and function data that can be compared to other vertebrate species. The turtle was chosen based on its distant evolutionary separation from human and other mammalian species. The gene studied in this project is apoA-II. Previously some of this gene’s sequence was determined, including in the 3’ direction most of Exon 2, Intron 2, Exon 3, Intron 3, and Exon 4. Currently I am making progress with techniques for advancing toward the 5’ end of the gene using gene "walking" to complete the determination of sequence for Exon 2, Intron 1, and Exon 1.

To attain a better understanding of cancer, it is crucial for researchers to continue identifying and characterizing oncogenes. Although originally identified through platelet aggregation studies, the novel calcium and integrin binding protein CIB became linked to oncogenesis when found to be expressed in breast cancer cell lines. Inquiry into CIB’s role in breast cancer resulted in the isolation of the second novel protein CIBB23.3 (CIB binder, clone 23.3) by a yeast-two-hybrid screening of the mammary epithelial cell library. Preliminary focus formation studies showed the ability of NIH3T3 cells, transfected with a mammalian expression vector (pcDNA 3.1) containing 23.3 to form foci. The purpose of this investigation is to demonstrate or disprove the possible oncogenic function of 23.3 however, the initial experimentations were aimed at confirming the preliminary data. NIH3T3 cells were transfected with the vector containing 23.3, using Kras, a known oncogene, as a positive control and the empty vector as a negative control. The foci were allowed to develop for a duration of fourteen days, after which the results were quantified. Although the data is inconclusive due to difficulties with the controls, foci were present in abundance on plates containing the 23.3 over-expressed cells. Though these results appear promising, modification and repetition of this assay is necessary. Furthermore, to determine if 23.3 is indeed an oncogene, additional assays commonly used for this purpose (i.e. Soft agar colony formation assays, gene expression assays) will be performed.

We are using the 3T3 L1 cell line from mice to characterize preadipocyte gene expression. By constructing a SAGE (Serial Analysis of Gene Expression) library from the 3T3 L1 cell line as well as four libraries using adipocyte cells from two human samples we hope to gain further knowledge of adipocyte gene expression and aid in the determination of what genes lead to the onset of CAD. We began by isolating RNA, which we reverse transcribed to cDNA. Subsequent modifications to the cDNA yielded a 13 bp tag, which can be used to identify a unique transcript. These tags were ligated to form ditags with adapters, which were later amplified. The adapters were then cleaved and the ditags were purified and ligated to form concatemers, which range in size from 200 bp – 2 kb. The 500 – 800 bp region was found to be the most effective for cloning. PCR was used to screen the transformants, of which 40% were found to have inserts larger than 250 bp, equivalent to approximately 10 ditags. We have accumulated more than 100 samples for sequencing, resulting in 3,000 or more tags per library. Our goal is to accumulate at least 30,000 tags per library, which is a sufficient number of tags to identify unique gene transcripts. We hope to analyze both the pre adipocyte and adipocyte 3T3 L1 cells for comparison purposes and to use the information we gather to base our human adipocyte libraries from. Eventually we will be looking at unique protein expression in both types of human adipocyte cell gene sequences to learn more about how gene expression correlates to the different hereditary and acquired coronary artery diseases.

Heparin/Heparan Sulfate (HP/HS)-binding proteins (HIP) have been linked to a variety of cell functions in vitro including growth regulation, modulation of blood coagulation and cell adhesion.  It is also identical at the amino acid level to a previously described murine ribosomal protein.  To create a model in vivo, chimeric male mice carrying a line of cells heterozygous (+/-) for HIP/RPL29 and agouti coat color were mated with C57BL6 females (black coat color).  Possible transmission of the mutation can be identified in offspring by agouti coat color and a positive identification through PCR and Southern Blot analysis.  A single agouti mouse has been produced thus far and data seems to show a negative result.  However, the ambiguity of the results makes a positive ID impossible at this point.  The other approach used, ribozyme technology, lowers the amount of HIP/RPL29 mRNA leaving the nucleus by cleaving its specific transcript.  Transgenic mice carrying this ribozyme within their cells will most likely be a more successful approach than the knockout mice.  Due to the widespread function of HIP/RPL29, it is possible that loosing even one copy of the gene could be lethal.  Therefore a knockout mouse would not be able to survive embryogenesis.  Production of the transgenic mice should be a better investigative tool into the function of HIP/RPL29 in vivo.

Because prostate cancer preferentially metastasizes to bone and exhibits osteoblastic features, it is important to understand the bone microenvironment to know how and why this occurs. Perlecan, an extracellular matrix HSPG most likely has a role in interactions between prostate cancer and bone matrix stromal cells. A ribozyme has been developed to reduce the production of perlecan by cutting the transcript and is used in in vitro studies to verify whether perlecan affects growth and proliferation of prostate cancer cells in bone like conditions. The bone environment in vitro was represented by human bone marrow stromal cells. Proliferation and motility of prostate cancer cells, both normal and ribozyme transfected cells, were analyzed in different conditions. No difference in growth was observed between the parental line and the ribozyme-transfected clones. Wound healing assays did not allow us to establish evidence of reduced motility of the ribozyme-transfected prostate cancer cells. Despite some problematic results, we obtained postive indications regarding the in vitro modeling of the bone environment.

Coronary artery disease is the leading cause of death in the United States (1). Subsequently, an increased knowledge in this field is crucial to developing treatments. The focus of this research is to identify and characterize the lipoproteins of the red-eared slider, Pseudemys scripta, with the expectation of gaining a better understanding of cholesterol and triglyceride transport throughout the body. Apolipoproteins, the proteins found on the surface of lipoproteins, direct the metabolism and the transport of lipids in the body. The turtle was chosen because of its important evolutionary links; many of the apolipoproteins important today evolved around the time birds and reptiles branched off evolutionarily. A 38kD apolipoprotein (P38) was found on a blot of an intermediate-density lipoprotein (IDL)/remnant fraction in a turtle with a highly unusual lipoprotein profile. It is hypothesized that P38 is apoliporptoeinA-IV (apoA-IV). The N-terminal portion of the protein was sequenced and then synthesized (15 amino acids with a C-terminal cysteine). The peptide was conjugated to human low-density lipoprotein (LDL) as well as to ovalbumin. The conjugates were used to stimulate an immune response in mice, and the antiserum was collected. In addition, the spleen lymphocytes from an immunized mouse were fused with a B cell myeloma line (P3) to produce hybridomas secreting monoclonal antibodies. The titers of the immune response from the mouse after a tail IV injection were promising (1:10,000). Although the fusion did not produce a very high number of hybridoma primaries (64 wells with cell growth/ 372 wells plated), the initial screen indicated that nine wells contained hybridomas secreting anti-peptide antibodies. The future plans are to probe phage expression libraries from the turtle intestine and liver to identify P38 in its native form. The protein will then be sequenced and analyzed for apoA-IV homology.

Lysophosphatidic acid (LPA) is a potent calcium signaling agent in osteoblasts, but the endoplasmic reticulum (ER) channel that is activated is unknown. We hypothesize that the channel responsible for Ca²⁺ release from the ER is the type 1 isoform of the ryanodine receptor (RYR1). RT-PCR revealed that mouse osteoblastic cells (MC3T3-E1 cells) express mRNA for RYR1, and the sequenced PCR product had a 99.3% similarity to the previously published sequence of mouse RYR1. Western blot analysis showed RYR1 protein expression in chicken breast, but not in MC3T3-E1 cells, possibly because the expression level in the osteoblasts was too low to be detected. RT-PCR also revealed that MC3T3-E1 cells express mRNA for FKBP12, a binding protein closely associated with RYR1 in skeletal muscle. Antisense experiments are underway to downregulate RYR1 expression and test the function of the RYR1 protein in MC3T3-E1 cells.

SV-40 Large T-Antigen is a multifunctional protein, which is responsible for the transformation of cells upon viral infection. The region of the protein, which this experiment deals with, is the DNA binding domain at the N-Terminal portion of the protein. The DNA binding domain has been elucidated and characterized extensively by the use of deletion mutants. It lies in the region corresponding to residues 131-259. This inquiry utilized N-terminal fragments created in a baculovirus expression system and purified utilizing an intein tag that was cleaved from the protein in the purification process. Once the proteins were purified, they were utilized in gel shift assays to determine the DNA binding characteristics of each individual fragment. It was found that the 1-109 region of the protein actually lowered the DNA binding activity in the gel shift assays of the proteins involved. Proteins missing this region were better suited to DNA binding of both the 112 base pair full origin and 64 base pair minimal origin.

Bone adapts to mechanical stress 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 stress 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 proteins, of which the alpha1 subunit is the site for Ca2+ influx. The purpose of this study is to determine the effects of mechanical loading on VSCC a1 subunit mRNA expression in MC3T3-E1 cells. Changes in mRNA expression in response to chronic mechanical loading are done by comparing RT-PCR results to a standard curve. A stock of alpha1 subunits standards of known concentrations was prepared by cloning each subunit using Taq-polymerase PCR. The amplified products were transformed into E. coli cells, spread on LB-amp plates, positive colonies were grown in an overnight culture, and the plasmids were isolated. Initial attempts to amplify the insert from the plasmid were unsuccessful. Amplification was only possible after the plasmid was linearized with EcoRV. We thus conclude that amplification and hence production of a standard curve can be accomplished by linearizing the vector. The standard curve has yet to be constructed due to problems with the BioRad Real Time PCR machine. Once the standard curve is constructed, the effects of mechanical loading will be further studied.

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 their own admission, none appears to represent a helicase or similar protein [Stanchina et al.(2000) J. Mol. Biol, 299, 667-680]. . The overall goal is to isolate and study the proteins that interact with the B2 Lamin gene sequence and compare them functionally with known helicases such as the SV-40 viral protein, T-antigen. As of this point, procedures to obtain human genomic DNA have been employed and customized PCR conditions using two 21bp oligonucleotide primers have produced several distinct bands of varying length. Separation and purification steps have yielded DNA that in the presence of nuclear extracts from U-87 cells exhibits a distinct DNA-protein binding pattern supporting the presence of and DNA interaction with several different proteins of varying size.

The lens capsule is a thickened basement membrane surrounding the lens. Since the adult capsule is known to contain the HSPG (heparan sulfate proteoglycan) perlecan, which is proposed to be important for lens differentiation, its developmental expression pattern was studied during murine lens development. We hypothesized that a high expression of perlecan would be found in prenatal murine lens development. Eyes were prepared from wildtype embryos of 9, 10, 12, 14, and 16 days post conception (dpcs) as well as newborn (nb), 1 week (wk), and 18 wk wildtype mice. These eyes were sectioned and stained with a polyclonal anti-perlecan antibody, which was detected by immunofluorescence labeling and confocal imagery. Perlecan was found in the earliest basement membranes underlying the lens placode and was maintained in the lens capsule throughout development. However, staining intensity was much higher in embryos than in adults. Since perlecan can work as a structural protein, a direct signaling molecule and an extracellular reservoir for growth factors, we proposed that it plays important roles in lens development.

Helicobacter pylori is a Gram negative, microaerophilic bacterium that has infected approximately half of the world's population. H. pylori has been linked to gastric and peptic ulcer disease and has been classified as a class one carcinogen. These factors make H. pylori a significant health concern. The purpose of this study has been to examine the infectivity of H. pylori after exposure to synthetic groundwater in order to investigate this medium as a possible route of transmission. A major goal of the labs' collaborative work has been to document the correlation between the presence of H. pylori in ground water (especially that found in wells of individual families) and H. pylori infection in the individuals who come in contact with the source. Arguments against the theory suggest that H. pylori does not remain viable in the stressed conditions of water and that such a method of transmission is not feasible. In order to explore further this hypothesis, a mouse model for infection is currently being developed under the guidance of Dr. Paul Fawcett at the A.I. DuPont Hospital for Children. Using balb/c mice, the methods of PCR, serology (Western blots), and culturing of the stomach are being explored as determinants of infection. Once the model is completed, the mice will be orally inoculated with H. pylori after the organism has been exposed to synthetic groundwater for various increments of time. Subsequent evidence of infection would then lend support to the hypothesis that ground water is a route of transmission.

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 determined. The goal of this study is to determine if H. pylori can persist under conditions it may encounter in natural water environments for a period of time long enough for it to spread and cause infection. Free-living and biofilm-associated organisms will be studied. Persistence of free-living organisms was studied by suspending H. pylori in a synthetic buffered moderately hard groundwater followed by incubation under various conditions. Physical and chemical factors considered were temperature and pH. Results have indicated that low water temperatures lead to increased survival of H. pylori and that a wide range of pH levels can be tolerated. Oxygen and nutrient levels will also be studied using this system. Attachment studies were carried out and demonstrated that H. pylori forms biofilms (Figure3). In future studies, biofilm-associated H. pylori will be exposed to the same environmental conditions as free-living organisms.

Two related studies are currently being undertaken to isolate and sequence biologically active proteins in fish. A vitamin D receptor was studied and currently a growth hormone sequence is being studied. A vitamin D membrane binding protein for 1,25-dihydroxyvitamin D3 was previously isolated in chick intestine as well as carp and Atlantic cod enterocytes. The present study tried to expand on that work by finding the analogous binding protein in the fish, Fundulus heteroclitus. Primers that were used to isolate the protein in the chick intestine were used to probe for the protein in the intestine and kidney of F. heteroclitus. The protein could not be found in the fish. The theorized reason that the protein could not be found is that it is not highly conserved, therefore the chick intestine primers would not work on the fish. A second project in this study focused on growth hormone in F. heteroclitus. A partial growth hormone DNA sequence was found for this fish by previous researchers. The present study hopes to expand the partial sequence into a full-length sequence. Currently, primers from the sequenced portion are being used to isolate and sequence the remaining 5' and 3' ends of the sequence from male and female F. heteroclitus pituitaries. The full-length sequence will be used to create recombinant F. heteroclitus growth hormone that will be used in further physiological studies.