EXPERIMENTAL BIOLOGY MEETINGS
New Orleans, LA APRIL 18-22, 2009
The University of Delaware group included four faculty and 14 undergraduates.
Hal White Chem & Biochem
Prof. Seung Hong, Biol Sci
Prof. Gary Laverty Biol Sci
Kathleen Cornely, Providence College
|Amber Majid (left)
received a first place award in the American Society for Biochemistry
and Molecular Biology Undergraduate Poster Competition on Sunday, April
19 in New Orleans. Sander Frank (middle) and Stephanie Myrick (right)
received honorable mention awards in the same competition. Since 2001
when University of Delaware students first participated in this
competition, more students from UD have received awards for their
research posters than any other participating school.
of Delaware students who participated in the American Society for
Biochemistry and Molecular Biology Undergraduate Poster Competition on
Saturday, April 18, 2009, in New Orleans, LA. From left to right:
Obinna Mmagu, Laura Sloofman, Sander Frank, Christina
Antonopoulos, Matthew Richards, Meghan Woods, Ryan Wilson, Ritika
Samant, Allison Kasmari, Stephanie Myrick, Tyler Larsen, Kathryn
Teixeira, and Amber Majid. Not shown is Patricia Timothee who competed
in the American Physiological Society's Undergradate Poster Competition.
Christina H. Antonopoulos, Lushanti De Zoysa Ariyananda,
lyase (ASL), a catalyst of
key reactions in purine biosynthesis,
is a homotetramer in which different regions of three subunits
each of four active sites. Human ASL deficiency is an inherited
disease associated with autism and mental retardation. We have
expressed, purified and characterized two disease-associated ASL
and R396H) that are located in different regions of the enzyme. L311 is
central helical region away from the active site, whereas R396 is in
proximity to the entrance to the active site. The Vmax for L311V and
8.6 and ~2.0 μmol/min/mg, respectively, as compared to 11.9 for wild
all three enzymes have comparable affinities for adenylosuccinate.
~25 % less tetramer than wild type while R396H has similar amounts of
as wild type. At 37 °C the specific activities of these enzymes
time and reach a limiting specific activity without significantly
changing the amount
of tetramer. Most ASL deficient patients are compound heterozygotes
L311V/R396H). In vitro generation of hybrid
R396H, only one of which has a His6- tag, followed by their
purification on a
Ni-NTA column and characterization, is in progress in this laboratory
yields insights into the function of ASL in compound heterozygous
by Autism Speaks and HHMI
Undergraduate Science Education grant.)
Recipient of an ASBMB Undergraduate CompetitiveTravel Award.
Sander Frank, Qian Chen, and Robert Sikes
The prostate gland is a significant source of male genitourina<>ry tract morbidity. As developmental processes share several features in common with metastatic cancer, we hypothesize that a better understanding of genes involved in prostate morphogenesis may identify possible targets for therapeutic intervention. The prostate is derived from the urogenital sinus (UGS) which is composed of 4 subdomains: epithelium/mesenchyme (UGE/UGM) and dorsal/ventral halves (UGD/UGV). Each region develops into specific lobes of the prostate with unique properties. This study sought to validate prostate developmental cDNA arrays that showed about 530 UGE/UGM and 35 UGD/UGV differentiated genes. Male UGS were separated either into UGE/UGM or UGD/UGV. Total RNA was extracted and used to synthesize cDNA templates for each subdomain. Quantitative-polymerase chain reaction (Q-PCR) was performed to observe relative mRNA levels and confirmed localization for 10/12 primary and 6/10 secondary compartmental genes. Immunofluorescence (IF) localized 6 genes in different predicted compartments. With this research, we have established the basis for a map of regional gene expression in the murine UGS. This work identifies candidate genes for further study and functional analysis by other developmental laboratories. Funding: NIH DK63919 and P20RR016472. Individual support: UD science and engineering and Stetson awards.
Recipient of an Honorable Mention Award in the ASBMB Undergraduate Poster Competition
metastasis of human prostate cancer cells to bone marrow contributes to
mortality and morbidity. MicroRNAs
(miRNAs) are endogenous ~22nt RNAs that play important regulatory roles
animals and plants by binding to specific messenger RNA (mRNA) targets
either block their translation or trigger their degradation.
evidence indicates that miRNAs are involved in human cancer. Here we
human prostate cancer cell line, LNCaP, and its derivative bone
C4-2B, as a model system to study the roles of miRNAs in prostate
progression. We sequenced more than two million small RNAs from LNCaP
cell lines using a 454 library. Known miRNAs and new small candidate
with expression differences between these two cell lines were selected
analysis. Various bioinformatics tools and gene databases were used to
potential targets for both novel and known microRNAs. Prostate cancer
expression microarray data were used to further narrow the prospective
genes for known microRNAs. Biobase (http://bkl.biobase.de/cgi-bin/bkl/idb/1.0/searchengine/start.cgi)
software was used to place these gene targets in relevant cellular
Ongoing studies seek to explore the potential targets of miRNAs
these pathways. These data provide us with important information about
miRNAs are involved in prostate cancer including identification of new
for modulation of gene expression during progression to bone.
targets and functions of these miRNAs will translate into new means to
prostate cancer metastasis and growth in bone. (Supported
by NCI P01 CA098912 and
Tyler Larsen, Daphne A. Salick, Radhika Nagarkar, and Joel P. Schneider
Department of Chemistry and Biochemistry
Hydrogels are heavily hydrated materials that show considerable promise as artificial extracellular matrices for use in tissue regenerative therapies. The development antibacterial hydrogels has been of great interest to the hydrogel research community as a means to combat the threat of infection during material implantation. We have developed MAX1, a self-assembling b-hairpin peptide hydrogel whose surface exhibits inherent antibacterial activity against several pathogens prevalent in hospital settings. Under physiological conditions, MAX1 self-assembles into a highly crosslinked, mechanically rigid hydrogel whose solvent-exposed fibrils display positive charge, which is thought to be important for antibacterial activity. This study aims to investigate the contributions of a cation-p interaction to the antibacterial activity of a newly designed peptide hydrogel. Cation-p interactions are a common feature of many antibacterial peptides, where they assist in the disruption of bacterial membranes. Thus, a new b-hairpin peptide (RWMAX1) was designed, incorporating a cross-strand Tryptophan/Arginine pair, in the hope of creating a more potent antibacterial hydrogel. The folding and self-assembly properties were assessed using circular dichroism and rheology and the antibacterial activity was investigated against E. coli and S. aureus. Supported by the Beckman Foundation.
skeletal remodeling is a major
focus of attention in translational osteoporosis research. A recent
study including 200 single-nucleotide polymorphisms on human chromosome
revealed CACNA2D2, (one of four α2δ subunits of the voltage sensitive
channel (VSCC) complex), as a novel susceptibility marker for bone
density (BMD) variation. VSCCs are a complex of polypeptides that
osteoblast mechanosensitivity and consist of a pore forming α1 subunit,
intracellular β subunit, a dimer of α2 and δ subunits, and a γ subunit
tissues. This study characterized the structure of the VSCC complex in
osteocyte-like cell line, MLO-Y4, using RT-PCR, Western blot, and
immunostaining. We demonstrated that the T-type Cav3.2 (α1H) subunit is
predominant α1 subunit expressed in osteocytic cells in vitro
cortical bone. Additionally, MLO-Y4
cells express α2δ1, γ7, and β14 subunits. These findings represent a
the expression of VSCC from long lasting (L-type) in osteoblasts to
(Ttype) channels in osteocytes. An association of the α2δ subunit with
channels in osteocytes could stabilize the functional channel and
mechanism for interaction with the extracellular environment. This key
complex may provide a crucial function for mechanosensitive osteocytes
having implications in skeletal remodeling and BMD. (Supported in
part by HHMI
Undergraduate Science Education Grant)
Recipient of First
Place Award in the ASBMB Undergraduate Poster Competition
Recipient of a FASEB MARC Program Poster/Oral Presentation Travel Award.
formulating efficient non-viral DNA delivery vehicles by incorporating
functional layers that can be sequentially cleaved off during the
to expose new features. We have established a simplified model vehicle
consisting of a polycation-condensed DNA core attached via linker
peptides to a
layer of poly(ethylene) glycol (PEG). PEG has been shown to protect
from salt-induced aggregation and immune recognition, but PEG coatings
inhibit DNA delivery at the target cell. This project is focused on
de-PEGylation of the vehicle prior to delivery via the cleavage of
peptides sensitive to matrix metalloproteinase-1 (MMP-1), a
enzyme upregulated by fibroblasts as they migrate through the
matrix. This migration is observed in
tumor stroma and at sites of injury, making MMP-1 secretion a useful
Having established the conditions that stimulate MMP-1 expression by
dermal fibroblasts via Western blotting and immunostaining, we are now
investigating the efficiency of vehicle cleavage using light scattering
combination with salt aggregation assays (Figure 1) to detect PEG layer
release. Further experiments in this area and cell studies are ongoing.
NSF, and UDRF.
Recipient of an
Honorable Mention Award in the ASBMB Undergraduate Poster Competition
Matthew Richards, Benjamin Rohe, and Mary C. Farach-Carson
Perlecan, also called HSPG2, is a heparan sulfate proteoglycan predominantly located in basement membranes and the matrix surrounding endothelial, mesenchymal and stromal cells. The reactive stroma surrounding prostate cancer cell lines produces high levels of the protein which may play a role in delivery of growth and angiogenic factors, aiding survival and growth of metastatic tumors. The overall goal of this project was to study the promoter in order to understand the up-regulation of perlecan in the tumor reactive stroma which occurs via transcriptional increases in perlecan biosynthesis. The sequence for the human HSPG2 promoter was found using public databases and compared to a published human sequence (Iozzo et al., 1997) and a mouse promoter sequence found in online databases. Several transcription factor binding sites of interest were identified for further study including NFkB, CREB, Smad3, Elk-1, c-Jun and TCF/LEF-1. Currently we are working to build a promoter-reporter construct by isolating the promoter region using polymerase chain reaction (PCR) amplification. Our current strategy seeks to optimize the PCR conditions with different sets of primers. The next step will be creating a fluorescence protein reporter construct of the full promoter sequence in order to test the effects of the identified pathways. This research was funded by the HHMI program and NIH/ NCI P01 CA098912.
Recipient of an ASBMB Undergraduate Travel Award.
Effects of Diminished Protein Synthesis on Bone Anabolic ResponseDeletion of a single ribosomal protein, RPL29, increases bone fragility due to diminished protein synthesis and poor tissue quality, suggesting an important link between skeletal tissue growth and efficient protein production. The goal of this study is to determine if reduced capacity to synthesize large volumes of proteins modifies bone anabolic response to mechanical load. To test this idea, tibiae of RPL29-null mice and age-matched wild type (WT) controls were loaded at 20% maximal load, 0.5 Hz for 50 cycles/day, with a five-second rest period inserted between two cycles for two weeks. The cortical bone microstructure and mechanical properties of the non-loaded and loaded tibiae of both RPL29-null and WT mice were analyzed using micro-computed tomography (microCT) and three-point-bending tests, respectively. Results indicate that following loading minor changes occur at the structural and material property levels in both control and null tibiae. Ongoing studies are using dynamic histomorphometry to quantify differences in bone formation capacity between loaded and non-loaded conditions. These studies will establish the importance of high volume protein synthesis for the regulation of bone formation. This work is supported by NIH P20 RR016458-06 and an HHMI Undergraduate Science Education Grant.
to Load in RPL29-deficient Mice
Laura G. Sloofman1, David Chen2, Xiaozhou Zhou2, Christopher Price2, John E. Novotny2,
Liyun Wang2, and Catherine B. Kirn-Safran1
Departments of 1Biological Sciences and 2Mechanical Engineering
Our lab has shown that MDA-MB-231 human breast cancer cells can be isolated from the chick embryo brain after injection into the extra-embryonic vasculature. Others have demonstrated with nude mice that re-injection of these cells results in sublines with enhanced capability to metastasize to the brain. It remained unknown whether this was the result of cells specifically targeting the brain, or increased survival in the brain compared to other organs. We transfected
Recipient of an ASBMB Undergraduate Competitive Travel Award.
Patricia Timothee, Victor Fomin, Kirk Czymmek, and Randall L. Duncan
Department of Biological Sciences
P2X7 receptor activation mediates the load-induced increase in bone formation, in vivo. We find that activation of this receptor produces a rapid contraction of the osteoblast that we hypothesize is modulated by two distinct pathways; RhoA GTPase and PKC. To test this hypothesis, we measured MC3T3-E1 preosteoblast contractions using the Zeiss 5LIVE rapid confocal microscope during activation of the P2X7 receptor in the presence or absence of specific inhibitors of RhoA GTPase and PKC pathways. BzATP, a known agonist of the P2X7 receptor, was added to the cells and changes in cell area following BzATP stimulation were quantified using Differential Interphase Contrast (DIC) microscopy. Addition of 0.5mM BzATP to MC3T3-E1 cells resulted in a 34.14% reduction in cell area. Similar results were seen using a PKC activator, PMA. Non-specific inhibition of PKC with, GF109203X, significantly attenuated the BzATP-induced contraction. Specific inhibition of PKCα, a Ca2+ dependent isoform of PKC, amplified the contraction in response to BzATP, suggesting that PKCα is not responsible for the P2X7 induced contraction. Inhibition of myosin light chain kinase and Rho kinase (ROCK) also failed to block BzATP-induced contractions. These studies suggest that PKC mediates the response of P2X7 receptor activation that may be important in skeletal remodeling. (Supported by INBRE2 P20 RR016472-08 and NIH/NIDDK R01 DK058246)
VI (Prdx6) is an antioxidant enzyme highly expressed in the lungs. Its antioxidant properties are due to its
ability to reduce hydroperoxides found in lung surfactants and thus
toxicity associated with hyperoxia. Prdx6
is a bifunctional protein that contains two distinct
sites. One active site catalyzes a
phospholipase A2 (PLA2) type hydrolysis of phospholipids,
second active site catalyzes the reduction of lipid hydroperoxides
1-cys peroxiredoxins. The structure of
Prdx6 has been solved; however, there is no structural evidence to
its catalytic mechanism. This study is
designed to solve the structure of Prdx6 in complex with MJ33, a PLA2
transition-state inhibitor, in an effort to understand the mechanism
conformational change Prdx6 undergoes during its catalytic cycle. Here we report that Prdx6 has been
successfully purified to homogeneity. Our
crystallization screens have produced protein crystals
diffracted to a resolution of 2.8 Å. This
research is supported by the Chemistry
Recipient of an ASBMB Undergraduate Travel Award.
Hybrid Hydrogels for Use in Vocal Fold Tissue Engineering
Meghan Woods1, Sarah Grieshaber2, and Xinqiao Jia2
Elastin is an extracellular matrix (ECM) protein abundant in vocal folds and other mechanically active tissues. This protein provides these tissues with elastic recoil and strength. Due to difficulties with natural elastin, there is a need to develop scaffolds for vocal fold tissue regeneration that mimics this elasticity and also provides tunability in a range of morphological, biological and mechanical properties. Hybrid polymer-peptide materials are attractive candidates for tissue engineering scaffolds because their synthesis allows for this tunability. In this work, synthetic polymers of poly(ethylene glycol) (PEG) and peptides with the amino acid repeat unit AKAAAKA found in natural elastin were synthesized and cross-linked to form hybrid hydrogels. PEG was functionalized with azide end groups and cross-linked through a copper-catalyzed azide-alkyne cycloaddition reaction with peptides functionalized with multiple alkyne groups. Both linear and 4-arm star PEG polymers with varying molecular weights were used in these trials, and the peptide length and functionality was varied by the number of repeat units. It was found that the swelling ratio and mechanical properties of the hybrid hydrogels can be tuned according to the molecular weight and geometry of each component. This project was funded by HHMI, NIH/NIDCD (1R01DC008965-01), and NSF/DMR (Career: 0643226).
Recipient of an ASBMB Undergraduate Travel Award.
The trip to the Experimental Biology
in New Orleans was organized by the University of Delaware HHMI
Science Education Program with additional support from travel grants
Society for Biochemistry and Molecular Biology, the FASEB MARC Program,
Program, and the Women Scholars Program. The HHMI
Undergradaute Science Education Program, the Arnold
and Mabel Beckman
Scholars Program, The Ronald McNair Program, Charles Peter White
Fund, The Chemistry Alumni
and the Undergraduate Research
supported research by the students.
|The annual dinner
with UD alumni and friends was held at the Bourbon House Resrtaurant on
Bourbon Street on Sunday, April 19. A record number of alumni and
friends joined us this year.
Clockwise from middle front: Allison Kasmari,
Stephanie Myrick, Laura Sloofman, Patricia Timothee, Kathryn Teixeira, Matthew Richards, and Ryan Wilson.
Clockwise from left front: Jens Hemmingsen,
Christina Antonopoulos, Evan Lebois, Obinna Mmagu, Ronald Ogbonna, Meghan Woods, Amber Majid, and Ritika Samant.
Clockwise from left front: Michael Cox, Luis Ralat, Damien Thevenin, Anastasia Fuzaylova, Judith Voet, Don Voet, and Andrew Hollenbach.
Clockwise from left front: Michelle Lazarus, Gary Laverty, Seung Hong, Tyler Larsen, Roberta Colman, Robert Colman, and Sander Frank.
Our bus to PHL.
Christina and Meghan with biochemistry textbook authors, Judy and Don Voet.
A small section of the exhibit floor at EB2009.
Liang Kang looking at Ritika's poster.
Obi being interviewed by Gregory Petsko, president of ASBMB.
Ryan (VP) and Meghan (Pres) receive the outstanding ASBMB-UAN Chapter award for the Northeast US from Ann Aguanno, Regional Director..
In the Ambassador Hotel lobby waiting for the Airport Shuttle.
Christina, Stephanie, and Matt at the Undergraduate Poster Competition.
A night out at Cafe du Monde. (A picture Dr. White didn't take.)
Ryan being interviewed by Gregory Petsko, president of ASBMB.