in Washington, DC, April 28 - May 2, 2007
|Prof. Hal White Chem
Prof. David Usher Biol Sci
Prof. Gary Laverty, Biol. Sci.
Dr. Seung Hong, Biol Sci
Ashley Anttila, Biological Sci
Corrine Decker, Biological Sciences
Vivek Desai, Biological Sciences
Charles Drummer, Biological Sci.
Joseph Feng, Biochemistry
Haag, Biological Sci.
Lindsay Higdon, Biological Sci.
James Kelleher, Biochemistry
Patrick Knerr, Biochemistry
Evan Lebois, Biochemistry
Justin Moses, Biological Sciences
Sarah Swain, Biochemistry
Wen Allen Tseng, Biological Sci
Ready for departure from UD Friday afternoon.
Participants in the ASBMB Undergraduate Poster Competition.
UD students at the Poster Award Ceremony
UD students at the ASBMB Undergraduate Poster Awards Ceremony
Effects of the sodium-hydrogen exchange inhibitor EIPA on chloride secretion in permeabilized monolayers of chick renal proximal tubule cells
Ashley Anttila, Sighvatur S. Arnason1, and Gary Laverty
In voltage-clamped cultured
monolayers of the chick renal
proximal epithelium, parathyroid hormone and the cAMP agonist forskolin
stimulate a positive short circuit current (ISC) that is
to chloride ion (Cl-) substitution and to Cl- channel
blockers, suggesting a stimulated net Cl- secretion.
However, this ISC
response is also blocked by the Na+/H+ exchanger
inhibitor, 5-ethylisopropyl amiloride (EIPA), indicating a possible
between these two transport pathways. To further study these
generated current-voltage curves (I/V curves) on permeabilized
µM amphotericin B to the basolateral side). Control and EIPA (100
pre-treated monolayers were subsequently treated with 1.0 µM
by the Cl- channel blocker NPPB. I/V curves were generated
each addition. Forskolin stimulated ISC (intercept of I/V
19.4 + 3.1 µA/cm2 (n = 7), and EIPA almost
this effect (0.96 + 1.27 µA/cm2, n = 5). On
the other hand,
forskolin decreased the I/V curve slope (conductance) by 19 +
controls, but increased it by 17.18 + 4.4% in EIPA treated
Similarly, NPPB had opposite effects on slope with and without EIPA.
suggest that forskolin may both activate a Cl- secretion
simultaneously inhibit a conductance associated with NHE, and that
transport components may be linked. Supported by NSF IBN 03433478.
Aly Bourreza and John T. Koh
Chemistry and Biochemistry,
Prostate cancer remains the second leading cause of cancer death in men. As prostate tissue is dependent on androgens for growth and homeostasis, anti-androgens, such as flutamide and bicalutamide (Casodex®), used alone or in conjunction with chemical castration have been used in the treatment of prostate cancer for decades. However, as many as 30% -40% of patients treated with anti-androgens become resistant to anti-androgens within five years of treatment. The prevalence of androgen receptor (AR) mutations that caused flutamide resistance led to the adoption of bicalutamide as the drug of choice for prostate cancer treatment six years ago, however new bicalutamide resistant mutants, W741C and W741L, have recently been identified in the clinic. Bicalutamide acts as an agonist with these mutants. It is believed that anti-androgens can be synthesized in such a way that they remain functional antagonists in mutant forms of the androgen receptor.
The novel compounds were synthesized based on the crystal structure of anti-androgen resistant AR mutants. The androgenic and anti-androgenic effects of novel compounds in competition with synthetic androgen R1881 is evaluated using ARE-luciferase reporter gene assays. In addition, anti-androgen resistance can be simulated in vitro by culturing LNCaP cells in the presence of anti-androgens for extended periods of time. Based on structural models we predict that these second generation AR antagonists will be resistant to AR mutations associated with anti-androgen withdrawal syndrome. Funding provided by the Howard Hughes Medical Institute Undergraduate Science Education Program.
The vertebrate lens is composed of epithelial cells and fiber cells, both of which contain crystallins, proteins necessary to maintain the high refractive index of the lens. Mice and humans harboring mutations in bB2-crystallin develop cataracts that have been proposed to be caused by aggregation of mutant βB2-crystallin. However, we have found that mice harboring the Phy mutation of the bB2-crystallin locus develop cataracts associated with profound fibrosis of the adult lens epithelium, expression of abnormally high levels of asmooth muscle actin (aSMA), and the loss of F-actin from lens fiber cells. Although bB2-crystallin expression in the lens initiates at birth, these observations suggest a role for bB2-crystallin in lens cytoskeletal organization. Consequently, we undertook a developmental study of this phenotype. While newborn and 2 week-old crybb2Phy mice exhibited normal lens morphology with no differences in aSMA or F-actin distribution, 4 week-old mutants lost F-actin from the lens fibers and upregulated aSMA expression in the epithelium. The distribution of the intermediate filaments CP49 and filensin was not altered. Upregulation of aSMA in the lens epithelium is associated with epithelial-mesenchymal transitions coupled with the loss of epithelial marker Pax6 and aberrant expression of fiber cell marker cMaf. Remarkably, mutant lens epithelial cells which appear fibrotic maintain Pax6 and do not upregulate cMaf. Overall, these data suggest that bB2-crystallin plays critical roles in the cytoskeletal organization of the lens and suggest that the pathologies seen in bB2-crystallin mutant lenses are not simply due to protein aggregation. Funding provided by grants from the Howard Hughes Medical Institute and the NIH.
Posterior capsular opacification (PCO) is an undesirable wound healing response in which the residual lens cells remaining in the eye after cataract surgery proliferate, migrate into the visual field, and synthesize extracellular matrix molecular similar to those found in scar tissue, damaging the patient’s vision. PCO arises from epithelial mesenchymal transition (EMT) of lens epithelial cells. In other systems, CD44, a receptor for hyaluronan, has been identified to mediate changes in cellular proliferation, migration and cell identity leading to EMT. We hypothesize that the hyaluronan receptor CD44 is involved in EMT of lens cells. CD44 is expressed in the adult lens fiber cells and not in the lens epithelial cells; it is first detected in the newborn lens fiber cells. RT-PCR demonstrated that the “canonical” version of CD44 is the major CD44 splice variant in the lens. In adult mice, CD44 is not expressed in the lens epithelial cells immediately after the cataract surgery, however, its expression highly up-regulates in the lens epithelial cells 1-day following the surgery, therefore CD44 could be an early marker for EMT. α-SMA, an early marker for EMT, is up-regulated in both the WT and CD44 KO lens 2-day after surgery, thus CD44 is not essential for EMT in the lens epithelial cells. Our immediate future work is to study the molecular mechanisms regulating CD44 expression in the lens. Funded by the Beckman Foundation.
Regulation of Lipid Metabiolism in Drosophia
|Until recently it was believed that insulin was the main regulator of lipogenic gene expression. However, it has been shown that nutrients themselves play a role in the regulation of these genes and that many lipogenic genes contain carbohydrate responsive elements (ChRE) within their promoters. These response elements were used to identify ChRE-binding factors that mediate lipogenic gene expression, including carbohydrate responsive element binding protein, ChREBP. We examined the role of the Drosophila homolog of ChREBP, Mio, in the regulation of fatty acid synthesis, triglyceride storage and lipogenic gene expression. Using the Gal 4/ UAS system, we created transgenic flies that expressed a MioRNAi knockdown construct. We assayed triglyceride levels in larvae lacking Mio in their fat bodies and used RT-PCR to examine Fatty Acid Synthase (dFAS) and Acetyl-CoA Carboxylase (dACC) expression. We observed a decrease in larval triglyceride levels in 2 independent transgenic lines. Consistent with a decrease in triglycerides, we also observed a decrease in dFAS and dACC expression. We concluded that Mio is necessary for regulating triglyceride storage as well as lipogenic gene expression. Supported in part by the HHMI EXROP Program.|
Protein Kinase C (PKC)-mediated Actin Disruption Regulates [Ca2+]i Responses to Mechanical Load in Osteoblasts
Joseph Joshua Feng1, Andris Kronbergs1, Victor P Fomin1,
Peter V Usatyuk2, Viswanathan Natarajan2,
and Randall L Duncan1
1Biological Sciences, University of Delaware
2Department of Medicine, University of Chicago
to mechanical load with a rapid and transient increase in
intracellular free Ca concentration ([Ca2+]i) that is essential for
load-induced bone formation. PKC is also rapidly increased during
mechanical stimulation and we postulate that PKC regulates the
[Ca2+]i response through alteration of the actin
cytoskeleton. Using fura-2 loaded MC3T3-E1 preosteoblastic cells, we examined the [Ca2+]i response to hypotonic swelling (HS) during phorbol ester (PMA)-induced PKC activation. PKC activation potentiated the peak [Ca2+]i response to HS by 57% compared to HS alone. A significant increase in the rising slope of the transient was also observed. PKC inhibition with 1 μM GF109203X reduced the peak [Ca2+]i response to 32% below HS alone peak levels. Voltage sensitive calcium or mechanosensitive channel block with nifedipine (10-6M) or gadolinium (5x10-6M),
respectively, abrogated the effects of PKC on the HS-induced [Ca2+]i increase. HS alone increased actin stress fibers in MC3T3- E1 cells. However, PMA (10-6M) disrupted of the HS-induced actin organization. Osteoblasts pretreated with actin cytoskeleton stabilizer, phalloidin, failed to exhibit the PKC potentiation of HSinduced [Ca2+]i. These studies suggest that PKC regulates the [Ca2+]i response to mechanical load in osteoblasts by reorganization of actin and through control of channel activity. Supported by NIH/NIAMS AR43222
through everyday activities are necessary for skeletal development and
function, yet the cellular mechanisms necessary for this response are
unknown. The earliest reaction to
mechanical strain caused by fluid shear in osteoblasts is a rapid
intracellular calcium that occurs through activation of the L-type
sensitive calcium channel (LVSCC). This
calcium influx is necessary to induce bone formation, in vivo.
The actin cytoskeleton of osteoblasts also
responds to mechanical strain with an increase in formation of stress
fibers. Ahnak, a 700kD protein has been
found to interact with both the LVSCC and the f-actin of the
cytoskeleton. We propose that ahnak bridges the
cytoskeleton to the LVSCC and that fluid shear will alter the
this protein in a time dependent manner.
We found that ahnak protein levels rapidly decreased in osteoblasts
within 1 hour of the onset of fluid shear.
Ahnak levels returned within 6 hrs of shear onset, suggesting that
is rapidly degraded and synthesized or is stored in discrete locations
the cell. Cell fragmentation studies to
identify ahnak storage sites within the cell will be correlated with
cytoskeleton disruption and immunofluorescent imaging to determine
ahnak localization during application of shear.
Supported by NIH/NIDDK grant DK058246 and Howard Hughes
Caitlin is pictured
with her father.
During brain development neurons migrate along radial glia to their final destinations. Interactions between radial glial substrates and neuronal integrins facilitate migration. Integrin-substrate interactions have been shown in other systems to induce expression of Bcl-2, a protein that suppresses apoptosis (programmed cell death). Bcl-2 is expressed in the early chick optic tectum (midbrain) where it is hypothesized to promote neuronal survival, but its role has not been demonstrated. Thus, a replication-competent retroviral vector expressing Bcl-2 was injected into the optic tecta in vivo to increase Bcl-2 levels and characterize the effects on tectal architecture formation. We have been characterizing Bcl-2 expression patterns by immunostaining infected and un-infected tectal cryosections. To determine the extent of viral spread we will also immunostain the sections for the viral gag protein. We are also developing an in vitro model that will investigate cell contact mediated survival by using time-lapse microscopy. We have finished constructing a replication-incompetent retroviral vector that encodes Bcl-2 and the marker gene lacZ, which should result in discrete infected cell clones (arrays) with higher numbers of surviving marked neurons. We will infect a group of tecta with this virus and another group with a lacZ-only expressing virus. We will then count and compare the number of cells per clone produced from each virus type. We predict the number of cells per clone with Bcl-2-expressing virus will be greater than control clones. This will implicate Bcl-2 in mediating cell survival during brain development. Supported by the Howard Hughes Medical Institute Undergraduate Program .
Lindsay won honorable mention for her work in the ASBMB Undergraduate Poster Competition. She is pictured with Don Smith, a judge in the competition.
Thyroid hormone responsive Spot 14
(THRSP) is a
transcription factor localized to the liver and adipocytes. In addition to sensing triiodo-L-thyronine
(T3) and glucose levels, previous studies have indicated the expression
14 is regulated by liver X receptor (LXR), a transcription factor
cholesterol. The known target genes of
Spot 14 are specific to lipogenesis, suggesting it as a biochemical
point in the accumulation of lipids. In
adipocytes, however, the regulation and function of Spot 14 have yet to
investigated. In this study, 3T3 L1
mouse adipocytes were depleted of cholesterol and/or supplemented with
agonist or T3 in order to examine the regulation of Spot 14. Gene expression of these treated cells was
measured through quantitative
Department of Chemistry and Biochemistry
Peptides have been designed which undergo intramolecular folding from random coil to β-hairpin conformation, triggered by specific environmental conditions such as temperature, pH and ionic strength. These folded, amphiphilic β-hairpins then intermolecularly self-assemble to form a crosslinked, fibrillar network, converting the bulk material from a free-flowing liquid to a self-supporting, rigid hydrogel. This work reports a new trigger: zinc-induced hydrogelation. A pentadentate ligand with a strong propensity to chelate zinc was synthesized and coupled to the side-chain of diaminopriopionic acid, yielding a zinc-ligating α-amino acid. This residue was subsequently incorporated into a 20 residue β-hairpin peptide composed of two strand regions connected by a four residue type II’ β-turn. Using this approach, a peptide was designed which does not fold and self-assemble until the addition of one or more equivalents of the zinc chloride trigger. Such a responsive system is of particular interest in the development of sensor technology to detect and remediate toxic levels of zinc pollution in water or soil, as well as in the design of microfluidic devices. Funding has been supplied by the Arnold and Mabel Beckman Foundation, the HHMI Undergraduate Biological Sciences Education Program and the National Institute of Health.
of Biological Sciences
this study we begin to phenotypically characterize Alg10, an
enzyme that adds the terminal glucose residue to the growing
oligosaccharide during N-glycosylation prior to its en
masse transfer to a nascent polypeptide. Genetic and
molecular approaches were developed to characterize the alg10
loss of function phenotype during Drosophila development. While pleotrophic phenotypes were observed in alg10
mutant embryos, clonal analysis of the alg10 mutation
in the developing wing yielded a smaller and rounder
adult wing phenotype as compared to wild type. This
implicates a potential link between glycosylation and
governing growth control. Both the
Insulin receptor (InR) signaling pathway and the Epidermal growth
pathway acting through c-myc are known to control growth in Drosophila,
roles that are conserved in
mammals. To begin to address whether alg10
disrupts InR signaling, I examined the genetic interaction between alg10 and an activated form of InR that
produced large disordered eyes. By removing half the Alg10
this background, an enhanced phenotype was observed with an irregular
surface, fused ommatidia, and patches of necrotic tissue. This
a genetic interaction between alg10
and the dInR pathway and suggests alg10 influences
InR mediated growth control. This work
was funded by an HHMI Undergraduate Research Scholarship.
Evan also spoke about his work in the American Society for Biochemistry and Molecular Biology Symposium on "Extracellular Matrix at the Organism Scale."
responds to mechanical stimulation with an increase in bone formation,
in vivo, yet mechanosensitivity is lost upon continued stimulation.
Osteoblasts rapidly increase actin stress fibers in response to
mechanical loading and we predict that this increase
may induce mechanical desensitization. Parathyroid hormone (PTH) enhances the response of bone to mechanical loads and briefly destabilizes the actin cytoskeleton. We postulate that PTH increases cellular elasticity, and hence mechanosensitivity, through disruption of actin stress fibers. The cellular elasticity of MC3T3- E1 osteoblasts was quantified using atomic force microscopy in ramping mode to establish the stress-strain curve of the cell. We found that PTH treatment for 30 min increased the elasticity of an osteoblast two-fold. Confocal microscopy studies demonstrate that
actin stress fiber formation is increased in MC3T3-E1 cells within 15 min of initiation of fluid shear and that addition of 50 nM PTH prevents this increase in actin organization. These results indicate that PTH increases the intracellular elasticity of osteoblasts by
disrupting the actin cytoskeleton that may explain the PTHenhanced response of bone to mechanical loads. Continuing studies will define the elastic modulus of osteoblasts during fluid shear and cytoskeletal disruption and correlate the elastic modulus with cellular responses to mechanical load. (Supported by HHMI and NIH/NIAMS AR043222)
Greg is pictured with his research advisor, Randall Duncan.
Dept of Health, Nutrition, and Exercise Sciences,
University of Delaware
Non-invasive estimates of central blood pressure (CP) using a radial transfer function would be valuable in physiologic studies since central systolic pressure (CSP) better reflects myocardial demand. However, this technique appears to be dependent on the calibration method. The purpose of this study was to determine the effect of different calibration procedures on CP estimates at rest and during cold pressor testing. Fifteen healthy subjects (age 23.5 ± 2 years) underwent blood pressure measurement in the left brachial artery (BP) and simultaneous recording of brachial and radial artery waveforms in the right arm. The right foot was placed in ice water and measurements were repeated 90 seconds later. The radial pressure wave was calibrated using BSP and BDP (Cuff), diastolic and brachial mean arterial pressure (MAP) determined from the brachial wave (BM), or diastolic and MAP calculated as 1/3PP + DP (CM). CP was estimated for each calibration method from the radial wave via a transfer function. Cold pressure testing increased (p<0.05) BSP (109 ± 2 vs. 127 ± 3 mmHg) and BDP (62 ± 2 vs. 77 ± 3 mmHg). Radial SP varied from BSP by + 9 and + 7 mmHg when calibrated using BM and CM respectively. As a result BM-CSP was 6.1 ± 1 and 4.5 ± 1 mmHg greater (p<0.05) than Cuff-CSP and EM-CSP respectively however all 3 methods tracked the cold pressor response similarly. Estimates of CP using a radial transfer function are dependent on the method of calibration. However, the ability to track changes in CP using a transfer function is not dependent on calibration and may provide valuable physiologic information.
of SMA Fibroblast Cell
Spinal Muscular Atrophy (SMA) is an
disease that is caused by mutation or deletion of the survival motor
gene 1 (SMN1). SMN2,
which is 99% identical to SMN1, is present in all
patients but is
unable to compensate for SMN1. A
base change in exon 7 is responsible for
the alternative splicing of SMN2
transcripts. Full-length transcripts are
predominantly produced by SMN1,
whereas SMN2 produces Δ7 mRNA and
little full-length transcript. Thus, SMA
results from insufficient levels of SMN
protein in motor neurons. The severity
of the disease seems to inversely correlate with SMN2
copy number making determination of SMN copy number
crucial for clinical diagnosis and prognosis. Genetic
diagnosis of SMA involves PCR amplification followed by DraI
enzyme digestion analysis. The
disadvantages of this approach include underestimation
of SMN copy number and the lack of a true
quantitative assessment. We are developing
a Pyrosequencing method that allows sensitive and quantitative
determination of SMN copy number using a modified
genotyping assay that includes the cystic fibrosis transmembrane
(CFTR) as an internal standard. SMN
and CFTR are amplified in a multiplex
PCR and then analyzed by Pyrosequencing. SMN copy number can be
determined by comparing the peak height of the SMN
genes to standard peaks in CFTR. This
research was funded in part by the
Howard Hughes Medical Institute Undergraduate Science Education program.
|Angiogenesis is the formation of new blood
vessels from pre-existing ones. It is
in both physiological and pathological processes. Junctional
adhesion molecule-A (JAM-A) is
required for the induction of angiogenesis by basic fibroblast growth
factor. JAM-A is also known to form
homodimers in vitro and in vivo. This study intends
to determine the relationship between JAM-A homodimerization and
angiogenesis. For this purpose,
was performed on JAM-A cDNA constructs to introduce mutations designed
impair homodimerization. Our goal is to
use these constructs to examine the relationship between angiogenesis
homodimerization in a blood vessel endothelial cell model.
In order to confirm that the mutations do, in
fact, impair homodimerization, Chinese hamster ovary (CHO) cells were
with wild-type or mutant JAM-A constructs. The
cells were then treated with a cross-linker,
suberate (BS3), lysed, and subjected to Western blotting for
JAM-A. Putative homodimer bands were seen
twice the molecular weight of monomeric JAM-A. Dimer
bands were detected at similar
intensities for both mutant and wild-type JAM-A, indicating no
homodimerization. A different approach
may be needed to detect any differences. This
research was funded by the Arnold and Mabel Beckman
the Barry M. Goldwater Foundation.
Allen won honorable mention for his work in the ASBMB Undergraduate Poster Competition.
US Capitol Building
Prepared for departure in front of Mckinly Lab
Evan, Joseph, Allen, James, and Justin at the ASBMB poster competition.
Sarah Swain, Dr. Hal White, and James Kelleher.
Alumni Dinner, Clockwise from left:
Dr. Seung Hong, Dr. David Usher, Mrs Usher, Elisabeth Mari (BS '05) and friend,
Greg Madden, and Caitlin Haag.
Alumni Dinner, Clockwise from front left: Dr. Gary Laverty, Ashley Anttila, Linday Higdon, Corrine Decker, Jen Buss (BS '06), Sarah Swain,
Justin Moses, and Aly Bourreza.
Alumni Dinner, Clockwise from left: Charles Drummer, Joseph Feng, Evan Lebois, Allen Tseng, Pat Knerr, James Kelleher,
and Vivek Desai.
Alumni Dinner, Clockwise from front left:
Michael Skinner (BS '96), Eugene Antiopov (BS '02) and guest, Dr. Hal White and daughters Rachel BA '02 and Anna BA '97, and Lilian Pintea.
Dr. White in his role as judge of posters from non-UD students.
Dr. White with Honorable Mention winners Lindsay higdon and Allen Tseng.
Washington DC Convention Center
|Washington DC Convention Center|
Mobile in the Convention Center.
Taking a break from the Scientific Session, UD group see some Washington Sights.
|Washington DC Convention Center||Washington DC Convention Center|
Aly Bourreza with Dr. White.
Sarah, Evan, and Joseph at the Convention Center.
Joseph Feng with his poster.
Justin, Lindsay, Pat, Aly,Sarah, Evan, and Corrine at the Lincoln Memorial.
Justin, Aly, Sarah, and Pat at the Washington Monument.
Ashley answering questions after her symposium talk.
The trip to the Experimental Biology
Meetings in Washington, DC was 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 and the Beckman
Scholars Program. The HHMI Program,
Scholars Program, Charles Peter White Fellowships, and the Undergraduate Research Program
supported research by the students.