Twelve University of Delaware Undergraduate Researchers attended the  EXPERIMENTAL BIOLOGY MEETINGS
in San Diego, April 2 - 6, 2005

The University of Delaware group included four faculty and twelve undergraduates.

Prof. Hal White  Chem & Biochem
Prof. David Usher Biol Sci
Prof. Gary Laverty, Biol. Sci.
Seung Hong, Biol Sci
Agata Bielska, Chem & Biochem
Meghan Bills, Biol Sci.
Meghan Cashman, Chem & Biochem
Glenn Christman
Chem & Biochem
Madaline Gregorits, Biological Sci.
Liang-I Kang
, Biol Sci
Erin Kenaley, Biol Sci
Elisabeth Mari, Biol Sci
Kimberly Miller, Biol Sci
Amanda Peters
Biol Sci
Kristen Reese, Biol. Sci
Mariclair Yandon,
    Del Biotech Inst.

Undergraduate students from the University of Delaware who participated in the American Society for Biochemistry and Molecular Biology's 9th annual Undergraduate Research Poster Competition, San Diego, CA. 4 April 2005. From Back left to front right: Glen Christman,  Liang Kang, Maddy Gregorits, Aggie Bielska, Meghan Cashman, Lis Mari, Kim Miller, Kristen Reese, (front row) Meghan Bills, Erin Kenaley, Marci Yandon, and Mandy Peters.

Alumni Dinner at Cafe Coyote in Old Town San Diego, 2 April 2005. Left to right: Mr. Schmieg, Florence Schmieg, Seung Hong, Liang Kang, Erin Kenaley, Marci's mom, Meghan Bills, Marci Yandon, Kristen Reese, and Maddy Gregorits.
From left to right: Gary Laverty, Mandy Peters, Dan Dries '00, Glenn Christman, Aggie Bielska, Hal White's empty chair, Artie Suchow '04, Allison Olszewski '02, Jen Risser '04, Meghan Cashman, Lis Mari, Justin DiAngelo '02. (Note-Justin apears twice-look in mirror)

Lunch gathering under the McKinly Lab skylight after practice presentations of posters on Wednesday, 9 March.
From left to right: Kim Miller, Glenn Christman, Aggie Bielska, Meghan Cashman, Prof. Hal White, Kristen Reese, Elisabeth Mari, Liang Kang, Maddy Gregorits, Erin Kenaley, Meghan Bills, and Marci Yandon. Mandy Peters not shown.

Our last view of San Diego. Things looks close but it took about an hour by trolley with waiting and transfers to get from the Convention Center (4) three miles to the Comfort Inn (2) right next to Interstate 5  near Old Town (1) and the airport (7). Interstate 5 (6) goes south to Tijuana. Those who walked from the transfer station may have seen the aircraft carrier midway in dock (5). The San Diego Zoo (3) is situated in  Balboa Park.

Delaware students with their posters and abstracts.

Conformational Effects of Protein Phosphorylation

Agata Bielska and Neal J. Zondlo
Department of Chemistry and Biochemistry

Phosphorylation-induced conformational change is an important tool used by cells to regulate metabolic pathways, signal transduction pathways, and transcription.  The objective of this research is to characterize the specific conformational effects of protein phosphorylation using the proteins tau and cJun as a model. Hyperphosphorylation of tau has been shown to cause its dissociation from microtubules and subsequent precipitation into neurofibrillary tangles (NFTs) in Alzheimer’s disease.  In order to study mechanisms by which phosphorylation could lead to NFT formation, various tau-derived peptides were synthesized.  Phosphorylated and non-phosphorylated versions of the peptides were then analyzed using circular dichroism (CD) and NMR spectroscopy.  Significant conformational changes were found in regions which, when hyperphosphorylated, are associated with NFT formation.   These conformational changes were consistent with an increase in type two polyproline helix (PPII) character.
The oncoprotein, cJun, is a transcription factor regulated by phosphorylation.   Peptides derived from cJun were synthesized and analyzed in non-phosphorylated, mono-phosphorylated, and di-phosphorylated forms. Upon phosphorylation, significant conformational changes were observed favoring PPII formation.  The magnitude of these changes increased as the number of phosphorylation sites increased.

Spontaneous Ovarian Contractions in Several Fish Species

Meghan Bills and Malcolm Taylor
Department of Biological Sciences

Spontaneous ovarian contractions have been found in a variety of fish species, ranging from the brackish dwelling mummichog, Fundulus heterclitus, to the common goldfish, Carassius auratus. This study sought to expand what is known about ovarian contraction in fish by examining fish from varied orders and families. In all, eleven species have been studied comprising 6 orders and 8 families of fish. Within each species a minimum of three fish were studied. An ovary from each fish was removed and placed in a muscle bath. It was assessed for contractions using a force-displacement transducer which sends amplified signals to a MacLab recording system. After at least one hour of observation for spontaneous contractions, Acetylcholine (ACh) was added to the muscle bath to test for non-spontaneous contractile ability. All fish responded with the exception of the mudminnow, Umbra pygmaea, a primitive fish related to salmon. Ovarian contraction in fish may be a useful model for study of mammalian smooth muscle function. This research was supported by a Charles Peter White undergraduate research fellowship to MLB.

Evaluation of the Role of T93 and S94 in the Function of Adenylosuccinate Lyase

Meghan Cashman, Mark Segall, & Roberta F. Colman Department of Chemistry. and Biochemistry

Adenylosuccinate lyase (ASL) of Bacillus subtilis is a homotetramer that catalyzes the conversion of adenylosuccinate to AMP and fumarate.  The enzyme has 4 active sites/molecule each of which has amino acids contributed from 3 subunits.  T93 and S94 are conserved in all ASLs and are close to other amino acids previously identified by mutagenesis as being in the active site.  To test their involvement in the enzyme’s function, T93 and S94 were replaced by alanine.  T93A and S94A exhibit circular dichroism spectra superimposable on that of wild-type enzyme, indicating there is no change in secondary structure.  T93A exhibits 0.5% of the Vmax of wild-type ASL with a 10-fold increase in Km for adenylosuccinate.  Intersubunit complementation yields increased activity for T93A when mixed with other inactive ASL mutants with replacements for residues contributed to the active site by different subunits.  The largest increase in activity accompanies the pairing of T93A and K268Q mutants.  H68Q and T93A mutants do not show complementation since both are provided to the active site by the same subunit.  S94A has 65% of the Vmax of wild-type ASL with little change in Km.  pH-Vmax profile measurements reveal a pK2 value for S94A of 7.83 in contrast to 8.32 for the wild-type enzyme.  T93 may orient adenylosuccinate optimally for catalysis; while S94 stabilizes protonated H68/H89, the determinants of pK2.  (Supp. By HHMI & NIH DK60504.)

Characterization of the ThiI-persulfide Intermediate Formed During 4-Thiouridine Biosynthesis

Glenn D. Christman, Chapman M. Wright, Eugene G. Mueller.
Department of Chemistry and Biochemistry

Two enzymes, ThiI and IscS, are involved in the modification of uridine to 4-thiouridine in some prokaryotic tRNAs.  IscS removes a sulfur atom from free cysteine and transfers it to ThiI, and indirect evidence indicates that a persulfide group is formed at Cys-456. A fluorescent derivative of iodoacetamide, I-AEDANS, has been used to trap the putative persulfide group. Both thiol and persulfide groups react with I-AEDANS to displace iodide and form a carbon-sulfur bond.  Reduction is expected to release the labeled persulfide group (which has a disulfide linkage) but not a thiol group (which has a thioester linkage).  In a non-reducing environment, both ThiI and IscS are fluorescently labeled by I-AEDANS. Treatment with reducing agents (either DTT, 2mercaptoethanol, or TCEP) greatly diminished the fluorescence of ThiI, with residual labeling from a small amount of ThiI in the thiol form before the addition of IAEDANS. The tryptic fragment of ThiI containing Cys-456 connected to the fluorescent label was detected using MALDI mass spectroscopy.  The observed mass included one "extra" sulfur, indicating that a persulfide, not a thiol or trisulfide group, was modified by I-AEDANS.  As expected, this fragment was labile to reduction.  This research was supported by the HHMI Undergraduate Science Education Program and the National Institutes of Health (GM59636).
Effects of Hindlimb Suspension on CB. 17 SCIB/beige Mice

Madeline Gregorits, Ronald R. Gomes, Jr. and Robert Sikes
Department of Biological Sciences

The rodent tail suspension model developed in the mid-1970’s is widely used to study the musculoskeletal adaptation to microgravity.  In this model, rodents are suspended by their tails such that the hindlimbs fail to contact the floor.  Under these conditions, the non-weight-bearing hindlimbs rapidly undergo remodeling, characterized by a loss of skeletal muscle and bone mass.  Best characterized using rats; few studies have adapted tail suspension to mice. Thus, the major goals of this study were:  1) Determine if CB. 17 SCID/beige mice can adapt to the stress induced by two and three weeks of tail suspension; 2) Characterize the trabecular bone changes in the unweighted hindlimbs following two and three weeks of tail suspension. To test our hypothesis, five CB. 17 SCID/beige mice were tail-suspended for either two or three weeks. Mice were monitored regularly for changes in body weight, and daily for eating, drinking, and grooming. Blood was collected on day 0, 14, and 21 to be analyzed for serum cortisol, a marker of stress. On day 14 or 21 the animals were sacrificed and the heart, adrenal glands, gastrocnemius, femurs, and tibias harvested.  A significant decrease in body weights of the tail-suspended mice were observed on day 4 of the two and three week suspensions.  Body weights remained ~10% below controls after 14 days of suspension and ~20% below controls after 21 days.  Following 14 days of suspension, gastrocnemius muscle mass and femur dry weights were significantly reduced in tail-suspended mice. Following 21 days of suspension, adrenal gland wet weights and femur whole bone porosity were significantly increased. These findings suggest that CB. 17 SCID/beige mice are capable of remodeling their skeletal and trabecular bone with changes in load-bearing activity; however, increased adrenal gland wet mass and reduced body weights may suggest increased stress levels and poor adaptation to suspension.
The Role of Junctional Adhesion Molecule A (JAM-A) in the Corneal Epithelium

Liang-I Kang, Vesselina Cooke,
 Ulhas P. Naik, and Melinda K. Duncan
Department of Biological Sciences

Junctional Adhesion Molecule A (JAM-A) is a ~38 kDa protein implicated in platelet activation and adhesion, angiogenesis, and the structural integrity of endothelial and epithelial cells. Recently, we showed that JAM-A expression is upregulated in the ocular lens in response to Pax6, a transcription factor important for eye development. The presence of JAM-A, JAM-B, and JAM-C mRNA in the wildtype lens and JAM-A mRNA in the wildtype cornea was confirmed through RT-PCR. Using immunohistochemistry, JAM-A protein was found in the blood vessels of the developing mouse eye as early as 12.5 dpc and in the corneal epithelium by 13.5 dpc. High levels of JAM-A remain in the corneal epithelium throughout adulthood. Mice with an insertion of a promoter-less GEO genetrap construct in the JAM-A gene lack JAM-A staining in the cornea, validating the JAM-A antibody specificity. Both heterozygous and homozygous JAM-A knockout mice express both gene products of the genetrap cassette, placental alkaline phosphatase and -galactosidase, in the corneal epithelium, further confirming this expression pattern. While the eye appears to develop normally in JAM-A knockout mice, older animals have subtle abnormalities in the corneal epithelium. We hypothesize that JAM-A is important to maintain the cornea and future studies will be directed toward determining the function of JAM-A.

Funding: NIH and the Arnold and Mabel Beckman Foundation

Calcium Effects on Ahnak Protein Expression

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

Ahnak is a 700 kDa protein that is expressed in a variety of cells. The precise function of Ahnak remains unknown. Ahnak has been proposed to be involved in actin cytoskeleton rearrangement in some cell types and in calcium homeostasis. Ahnak cellular location is influenced by intracellular calcium levels.  In this study, we assessed the effect of calcium on the expression of Ahnak in 3T3-L1 preadipocytes.  RT-PCR and Western blot analyses revealed that Ahnak is present in the 3T3-L1 preadipocyte cell line. Immunostaining of 3T3-L1 cells with Ahnak specific antibodies showed that Ahnak is localized in the cytoplasm and along the plasma membrane. Immunostaining of proliferating 3T3-L1 cells (in exponential growth phase) cultured in varying levels of extracellular calcium showed that Ahnak protein expression may increase with increasing extracellular calcium levels.  When cytoplasmic calcium levels were raised with thapsigargin or calcium ionophore, Ahnak protein expression increased in the treated cells as compared to control. These data suggest that calcium is an important regulator of Ahnak expression in 3T3-L1 cells.
Effects of Hip/Rpl29 Gene Disruption in Murine Embryonic Stem Cells

Elisabeth Mari, Daniel Carson, and Catherine Kirn-Safran
Department of Biological Sciences

HIP/RPL29 is a multifunctional ribosomal protein with heparan sulfate binding properties that is highly expressed in developing tissues including the inner cell mass of early preimplantation mouse embryos. Recently, we found that HIP/RPL29 transcript and protein are among the most abundant in embryonic stem (ES) cells and are strongly detected in limited amounts of total lysates. Because of HIP/RPL29 abundance in ES cells, we hypothesize that HIP/RPL29 expression is required for basic cell survival and growth.  To evaluate the effect of HIP/RPL29 monoallelic expression, we performed RT-PCR on Hip/Rpl29 +/- ES cell lines using specific primers for HIP/RPL29.  Our results showed that there was no significant change in the steady-state level of both HIP/RPL29 mRNA and protein in +/- versus wild type ES cell lines.  This observation indicates that gene compensation mechanisms are taking place to restore normal levels of expression.  Preliminary transcriptional profiling studies of +/- ES cell lines by RT-PCR suggested that Oct-3/4, a transcription factor known to sustain stem cell renewal and pluripotency, is differentially regulated in +/- ES cell lines when compared to control cell lines.  Future efforts will consist in determining whether concomitant changes in Oct-3/4 protein expression levels are seen in +/- versus +/+ ES cells. In order to investigate the importance of HIP/RPL29 for ES cell growth and survival we are currently performing experiments that will induce the loss of the remaining wild type allele by culturing +/- ES cells carrying the neomycin phosphotransferase gene in the presence of high concentrations of G418. The outcome of this work will determine whether HIP/RPL29 null cell lines are viable.  All the in vitro data generated with Hip/Rpl29 +/- ES cells constitute valuable tools for the disruption of Hip/Rpl29 gene in animals and will help understand HIP/RPL29 function in ES cell metabolism and embryo survival. (This work was supported by Charles Peter White Fellowship [to E. R. M.] and NIH grant HD25235 [to D.D.C.]).

A Role for Hyalp1 in Murine Fertilization

Kimberly A. Miller, Hong Zhang, and Patricia A. Martin-DeLeon
Department of Biological Sciences

The cumulus cells surrounding the unfertilized egg have an extracellular matrix rich in hyaluronic acid and form a formidable barrier to sperm penetration.  The digestion of hyaluronic acid and subsequent penetration of the cumulus cells, required for fertilization, is aided by enzymes dubbed ‘reproductive hyaluronidases,’ of which Spam1 remains the most extensively characterized.  Located on mouse chromosome 6, the gene family also contains Hyalp1, whose encoded protein remains almost fully uncharacterized, in addition to Hyal4, Spam1 (PH-20), and Hyal5.  Recently, it was discovered that sperm from Spam1 knockout mice were able to penetrate the cumulus and successfully fertilize oocytes, indicating that other proteins involved in this process can functionally compensate for a lack of Spam1 (Baba, et al. 2002).  The aim of this study is therefore to investigate the role of Hyalp1 in murine fertilization.  Testicular developmental RT-PCR and immunohistochemistry, and immunocytochemistry on epididymal sperm show colocalization of Hyalp1 on the acrosome cap of mature sperm.  Hyaluronic Acid Substrate Gel Electrophoresis and In vitro fertilization vestment penetration assays demonstrate hyaluronidase activity of Hyalp1 testicular protein and the role of Hyalp1 in the breakdown of hyaluronic acid and the penetration of the cumulus cells surrounding the unfertilized oocyte by mature caput sperm.  Vestment penetration was retarded by the inhibition of Hyalp1 with antiserum.  This implicates a significant role for the murine reproductive hyaluronidase in fertilization.
This work is supported by NIH grant# R01 HD38273 and the Howard Hughs Medical Institute Summer Research Scholar Grant Program.

Influence of Cholesterol on Fatty Acid Synthesis During Adipocyte Differentiation

Amanda Lynn Peters, John David, and David Usher
Department of Biological Sciences

Adipocytes are important regulators of fatty acid homeostasis.  The SAGE library of 3T3-L1 adipocytes, constructed in our laboratory, has implicated cholesterol as having an important role in this process.  The dependence of adipocyte differentiation on cholesterol to form lipid droplets was examined by treating differentiating and control 3T3-L1 cells with -cyclodextrin (CD).  Changes in expression patterns of transcription factors and their target genes needed for triglyceride accumulation and cholesterol efflux were determined.  Expression of SREBP2 and its target genes HMGCR and LDL-R significantly increased soon after CD addition, as did the expression of cholesterol efflux genes ApoE, ABCA1, and ABCG1.  That LXR expression was unaffected by cholesterol depletion, suggests that changes in oxysterol ligand availability leads to a decrease in these genes under treatment.  Decreased expression for other LXR target genes SREBP-1c, FAS, and PPAR occurred much later in the treatment.  Target genes for SREBP-1c and PPAR showed a similar decreased expression in the treatment.  Interestingly, SPOT14, another target gene of SREBP-1c, showed a severe decrease in expression very early during cholesterol depletion, suggesting that SPOT14 is not exclusively under SREBP-1c control.  A decrease in expression of SPOT14, a known G-6-P sensor, implies an attempt to slow fatty acid synthesis when cholesterol is limited.  SPOT14 regulation is currently being investigated.  These results suggest that a complex regulatory network is involved in adipocyte differentiation, which is highly dependent on cholesterol.  Funded by HHMI and SDI.
The Characterization of Hyal3 in Murine Testicular Cells

Kristen L. Reese and Patricia A. Martin-DeLeon,
Department of Biological Sciences

The digestion of hyaluronic acid is required for penetration of the cumulus cells surrounding the egg during fertilization and is implemented by a family of enzymes termed hyaluronidases.  Hyal3, one of the three murine somatic hyaluronidases, has high similarity to the ‘reproductive’ hyaluronidases and is most highly expressed in the testes, but its contribution to the fertilization process and intracellular characteristics are not yet known.  It has recently been determined that sperm from mice lacking a primary reproductive hyaluronidase, Spam1, are able to fertilize oocytes due to a predicted contribution of related hyaluronidases.  However, humans lack a functional reproductive hyaluronidase other than Spam1.  Hyal3 is particularly important to characterize because it has the highest amino acid identity to its human homologue, with similar testicular expression patterns and identity to Spam1.  Therefore, the knowledge obtained in the mouse model can then be applied to humans.  It is the goal of this study to investigate developmental expression of Hyal3 in the testes, its location in sperm, and its role in the dissolution of the extracellular matrix of the cumulus.  Briefly, the methods used are as follows: RT-PCR and Real-Time PCR for quantitative, qualitative, and developmental analyses and Hyaluronic Acid Substrate Gel Electrophoresis to determine hyaluronidase activity.  Thus far, our results demonstrate that Hyal3 is made in germ cells, suggesting its presence in sperm and that the protein demonstrates hyaluronidase activity.  We are currently determining its role in cumulus penetration using antibodies for Hyalp1 and Hyal5 and sperm from Spam1 knockout mice.  This research is funded by the Howard Hughes Medical Institute and NIH grant #R01 HD38273.
This research is funded by the Howard Hughes Medical Institute (KLR) and NIH grant #R01 HD38273 (PAM-D).

Characterization Of Genes Regulated By Infection Of Chick Embryo Fibroblasts (CEFs) With Herpesvirus Of Turkeys (HVT). 

Mariclaire Yandon, Erin Bernberg, Amarin Cogburn,
Robin Morgan
, Joan Burnside
Delaware Biotechnology Institute

HVT is used as a vaccine against Marek’s disease virus (MDV), an alphaherpesvirus that grows in chickens, chick embryos, and chick embryo fibroblast (CEF) cell culture. Infection of CEFs with either MDV or HVT causes the formation of characteristic plaques and relatively few cells in a culture become infected. The low level of infectivity impairs global gene expression studies of host responses to infection, since the infected cells are likely to have a very different gene expression profile than surrounding or uninfected cells. We coupled laser capture microdissection (LCM) with microarray analysis of HVT-infected CEFs to identify regulated genes. To confirm differential expression, a quantitative reverse transcription-polymerase chain reaction was used to compare gene expression in plaques, uninfected cells, as well as cells adjacent to plaques. A large number of regulated genes have no match to any entry in GenBank. Additional sequence information was obtained on several clones, and expression profiles evaluated by dbEST data. This approach adds functional information to previously uncharacterized genes and will improve our understanding of the mechanism of viral disease. Funded by NIH BRIN and USDA NRI Programs.

The trip to the Experimental Biology Meetings in San Diego is being organized by the University of Delaware HHMI Undergraduate Science Education Program with additional support from travel grants from the American Society for Biochemistry and Molecular Biology, the Beckman Scholars Program,  and the University of Delaware's Women Scholars Program. The HHMI Program, the Beckman Scholars Program, Charles Peter White Fellowships, the Biomedical Research Infrastructure Network (BRIN) Program, and the Undergraduate Research Program supported research by the students.

Downtown San Diego from Convention Center

Aggie, Mandy, & Dr. Usher at Convention Center

Convention Center from across the street.

Convention Center from the marina.

Reunion of successive students from Melinda Duncan's lab. Liang Kang '06, Artie Suckow '04, Justin DiAngelo '02, Dan Dries '00.

Blue Hen bus pickup at PHL.

Meghan B., Aggie, and Meghan C. on their way to San Diego.

Liang discovers that the San Diego Trolley Machine doesn't like one dollar bills.

Erin and Marci putting up a poster.

Gary Laverty with his poster and his postdoc advisor.

Seung Hong with her poster.

Don't let that poster get away!

Yes gang, that is the Pacific Ocean behind us.
Meghan Bills and Mandy Peters at the ASBMB Undergraduate Poster Competition

Meghan Cashman, Kim Miller, and Glenn Christman at the ASBMB Undergraduate Poster Competition.

The University of Delaware Delegation at the ASBMB Awards Ceremony.
ASBMB Undergraduate Poster Competition in San Diego 2003
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Created 20 December 2004,  revised 15 April 2005 by Hal White [halwhite at]
Copyright 2005, Harold B. White, Department of Chemistry and Biochemistry, University of Delaware