Biotechnology
The Biotechnology Laboratory provides a unique technology experience for a large portion of the
Jefferson student body. The program provides both a biotechnology training and research program
designed to transform traditional secondary molecular biology studies into an applications program that
will help students to experience the power of newly discovered research tools associated with
recombinant DNA technologies. The laboratory’s primary mission is to provide a laboratory researchbased program that allows its students to experience topics from bacterial transformation to DNA
mapping. In addition, the program provides students with the experience of new, leading-edge
technologies, such as bioinformatics, western blotting, rtPCR, and DNA sequencing.
Student:
Nathan Allen
Firm:
George Mason University
Mentor:
Dr. Patrick Gillevet
Title:
Determination of Unique
Ecosystems in Gulf of Mexico
Oil Seeps
Background: Below the floor of the Gulf of
Mexico lie vast oil fields. At certain locations,
where the earth between the oil and the ocean is
thin, oil slowly seeps up through the ground and
is discharged into the water. The oil is at the
same temperature as the surrounding water,
which is very cold. This is one of the primary
differences between the oil seeps and the
thermal vents that exist elsewhere on the planet.
Another difference is that the bacteria that form
the base of the food chain are methane digesting
bacteria, as opposed to those at the
hydrothermal vents, which are dependent upon
sulfide compounds. Certain organisms thrive in
this environment, such as giant tube and ice
worms, and many microorganisms. Because of
the large amount of available oil, oil companies
are anxious to drill in the area. However,
environmental groups are concerned that if the
oil platforms disrupt or destroy the area around
the oil seeps, entire ecosystems may be lost.
Mentorship Projects - 2001
Description: This project is designed to
determine the microorganisms that inhabit the
area. Using extractions done by Kate Smith,
DNA from dive four samples was amplified to
determine the presence and abundance of life in
the pushcore. This was done by means of the
Polymerase Chain Reaction, using the primers
27F and 1492R. To ensure accuracy, additional
extractions were completed on the Bush Hill
samples using Bio 101, and similar results were
achieved. Serial dilutions, ranging from 1:10 to
1:10,000, were made with the samples, and
PCRs done with each, to find the ones that
produced optimal results. The best PCR
products, which contained the fluorescent
version of the primer 27F, were then
fingerprinted. Due to the presence of distinct
peaks present in the fingerprint analysis, it was
apparent that many organisms were present in
some abundance. Another PCR was then
performed using non-fluorescent primers, and
these products were ligated, and then
transformed, and placed on plates. Each distinct
colony produced on this agar plate was then
used in a PCR reaction. The PCR, using the
primers M10F and M10R, was successful for
many of the colonies. These products will be
used for sequencing. Once the sequencing is
done, it will be possible to compare the
sequences found with the NIH database to
determine what each of the organisms is, and if
any might be previously unknown life-forms.
Biotechnology
Exhibit
1
Student:
Lisa An
Firm:
Lombardi Cancer Center,
Georgetown University
Medical Center
Mentor:
Dr. Luyuan Li, Assistant
Professor
Title:
Promoter of Vascular Endothelial
Growth Inhibitor
Background: Vascular endothelial growth
inhibitor (VEGI) is a member of the tumor
necrosis family (TNF). It has various functions,
including cell cytotoxicity, immune responses,
and regulation of cell proliferation. VEGI,
member of this family, has about 20-30%
sequence homology to the TNF superfamily and
is expressed mainly in endothelial cells.
Endothelial cells are involved in many different
biological activities including angiogenesis.
During this process, endothelial cells proliferate
and migrate toward the stimulus of
angiogenesis, such as a cancer cell. They
eventually form capillaries; however, this type
of pathological angiogenesis seen in tumors
continues with no check even though new blood
vessels have already formed. Previous research
has shown that VEGI is an endothelial cell
specific negative regulator of angiogenesis.
The gene for VEGI encodes a protein
consisting of 174 amino acids. There are four
isoforms of the protein, including a secreted
form and a form that has characteristics of a
transmembrane protein. It has been shown that
murine colon cancer cells that express the
soluble form of VEGI have significant reduction
of vascularization. The VEGI protein can be
highly valuable in the future of angiogenesisbased cancer therapies.
The promoter of a gene is the site
upstream from the start of the open reading
frame where general transcription factors bind
Mentorship Projects - 2001
to initiate transcription. These general
transcription factors are required to assemble at
the promoter with RNA polymerase II, the
enzyme that transcribes most eukaryotic genes.
This complex binds at the TATA box, which is
a short sequence of DNA that mostly contains
thymine and adenine nucleotides.
Description: This study is aimed at identifying
and locating the promoter of the VEGI gene.
The sequence of about 10 kilobases upstream
from the start codon of the open reading frame
is known. Five possible promoter regions have
been identified through a computer program on
the Berkeley Genome Project website. The
sequence was inputted into the program that
compared it to other known promoters to
generate a list of possible promoter regions.
These five possible promoter regions were
amplified through polymerase chain reaction
(PCR). Then these DNA fragments were
inserted into a vector called pGL3-basic with a
Firefly luciferase reporter gene and transfected
into mammalian cells. Also, the cells were
transfected with an internal control vector and
normalizing vector called pRL-TK, in addition
to the pGL3-basic construct. The pRL-TK
vector contains the Renilla luciferase reporter
gene. This vector will serve as a normalizing
vector to take into account the variability of the
transfection from well to well. During the
luciferase assay, separate readings for the
Renilla and Firefly luciferase reactions were
made.
This study found that the positive
regulator region of the VEGI promoter is found
about 300 base pairs upstream from the start
codon and that the promoter is not endothelial
cell specific. This research has great
significance because once the promoter is
known, further studies may be conducted on
how the gene is regulated.
Biotechnology
Exhibit
2
Student:
Bic Cung
Firm:
Walter Reed Army Institute of
Research
Mentor:
Dr. Malabi Venkatesan
Title:
Construction and Analysis of
ipaB Mutants by Making
Deletions in Two Separate and
Independent Regions of the
Protein
Background: Shigellosis, a severe and highly
infectious disease that kills more than one
million people yearly, is caused by the
enteroinvasive bacteria Shigella. Infection is
transmitted via the oral-fecal route, whereas the
bacteria travel down the gastrointestinal tract
(GIT) and penetrate the epithelial lining of the
large intestine, causing bacillary dysentery.
Clinical manifestations of shigellosis may
include but are not limited to watery diarrhea,
frequent passage of bloody stools with mucus
and acute abdominal pain in combination with
rectal tenemus, fever, tenderness of left colon
upon palpation and presence of faecal
leucocytes.
The ability of Shigella to invade
epithelial cells is encoded by a 220 kb plasmid
termed the “invasion plasmid.” The presence of
Mentorship Projects - 2001
such a plasmid confers on Shigella the ability to
invade and move inter- and intracellularly. A
32 kb region on the invasion plasmid encodes
the major proteins required for invasion. This
invasion-associated region has two operons: a)
the Ipa proteins, which are secreted invasion
plasmid antigens or virulence factors; and b) a
type III secretion system with the mxi/spa
genes, which encode approximately 20 proteins
whose function is to present the effector
molecules (Ipa proteins) on the bacterial
surface.
Description: A short sequence (approximately
15-amino acid in length) in ipaB has been
shown to react to antibody from subjects
challenged with Shigella. To test its function in
IpaB protein using molecular techniques. The
altered IpaB protein, so obtained, is compared
with a normal wild type IpaB protein for
function by using it to complement a Shigella
strain SF620, which contains an IpaB mutation.
SF620 is noninvasive in epithelial cells in tissue
culture. When a normal IpaB protein is
introduced into SF620, using a plasmid clone of
the ipaB gene, invasion is restored in epithelial
cells. Function (or lack thereof) of the deleted
sequence is determined by an invasion assay
done with SF620 complemented with plasmid
clones encoding normal or altered IpaB.
Biotechnology
Exhibit
3
Student:
Bic Cung
Firm:
Walter Reed Army Institute of
Research (WRAIR)
Mentor:
Dr. Malabi Venkatesan
Title:
Analyzing Functions of ipaB
Epitopes in Shigella
Background: Shigellosis, a severe and highly
infectious disease that kills more than one
million people yearly, is caused by the
enteroinvasive bacteria Shigella. Infection is
transmitted via the oral-fecal route. The
bacteria travel down the gastrointestinal tract
(GIT) and penetrate the epithelial cells lining
the distal colon and rectum, causing bacillary
dysentery. Shigella secrets some 15 proteins
that are crucial for invasion (effector molecules)
through the mxi-spa system. The four most
abundant proteins are Ipa (Invasion plasmid
antigen) A, B, C and D. All four Ipa proteins
are crucial to Shigella invasion, and most are
multifunctional. For example, IpaB also plays a
role in forming contact with host cells using
receptors. IpaB has also been implicated as the
factor causing apoptosis in macrophages
because bacteria without IpaB (or mutated IpaB)
have been observed to be unable to escape
phagocytic vesicle.
Mentorship Projects - 2001
Description: The ability of Shigella to invade
epithelial cells is encoded by a 220 kb plasmid
termed the “invasion plasmid”. The presence of
such a plasmid confers on Shigella the ability to
invade and move inter- and intracellularly. A
32 kb region on the invasion plasmid encodes
the major proteins required for invasion. This
invasion-associated region has two operons: a)
the Ipa proteins, which are the secreted invasion
plasmid antigens or virulence factors and b) a
type III secretion system with the mxi/spa
genes, which encode approximately 20 proteins
whose function is to present the effector
molecules (Ipa proteins) on the bacterial
surface.
A short region on ipaB has been shown to
react to antibody from subjects challenged with
Shigella. To correlate the function of those
regions with structure in ipaB, the mutants of
ipaB are created and then transformed into an
ipaB mutant of Shigella (SF620) and tested for
restoration of biological activity. Various
mutants of ipaB are constructed using
Strategene QuickChange Mutagenesis Kit. PCR
is the method by which the mutants are
constructed. Primers are designed with the
desired mutations (ranging from single-base
change to deletion of several amino acids). The
mutants are tested for accuracy by DNA
sequencing. Mutants of ipaB are then
transformed into SF620 and the function of the
mutants are assessed by Western Blot and
invasion Assay.
Biotechnology
Exhibit
4
Student:
Melanie Dispenza
Firm:
National Institutes of Health
Mentor:
Dr. Jonathan Auerbach
Title:
Embryonic Stem Cell-Derived
Dopaminergic Neurons Cocultured with Striatal Explants
Background: Parkinson’s Disease is a
neurodegenerative disease caused by the death
of dopamine-producing (dopaminergic) neurons
located in the substantia nigra of the brain,
resulting in the debilitation of movement and
motor skills. As common treatments are not
efficient enough and only slow the progression
of the disease, new therapies are being
investigated. One possible treatment would be
replacing dying neurons with cells grown in a
lab from embryonic stem (ES) cells. ES cells
can differentiate to form any type of cell found
in the human body, and new research has been
applied to the manipulation of stem cell
differentiation specifically into dopaminergic
(DA) neurons. The most efficient way to do this
remains to be elucidated, as different procedures
and conditions are being proposed and
researched.
This project attempts to grow DA
neurons from ES cells together with striatal
brain tissue, specifically the lateral ganglionic
eminence (LGE). In the midbrain, the striatum
is the target area of DA neurons in the
substantia nigra. The hypothesis is that the
striatal cells from the LGE tissue will help
support the survival and growth of DA neurons
Mentorship Projects - 2001
and possibly help generate them from ES cells.
Different conditions were investigated in an
attempt to reach optimal generation of DA
neurons, which could apply to finding a better
therapy or cure for Parkinson’s Disease.
Description: Stage V ES cells were plated with
fresh embryonic LGE in culture-specific media.
A chicken plasma/thrombin clot was used to
hold the tissue in place, and the cultures were
incubated at 37º C in a rotating drum. Cultures
were removed from incubation at different time
intervals, placed in a fixative, and stained with
fluorescence-conjugated antibodies for
observance of tyrosine hydroxylase-positive
(TH+) neurons with a fluorescent microscope.
The number of TH+ neurons produced in each
culture was used as a measure of survival of DA
neurons. Data was also correlated with
electrophysiological analysis of the synaptic
properties of the DA neurons. If mature DA
neurons can be grown, they must also form
functional synapses with the striatum in order to
be effective therapy for Parkinson’s patients.
(Electrophysiological data will be correlated
with this project in a separate assay).
Results from this project were
cumulative, with one set of data determining the
next set of experimental cultures. Several
different growth conditions were tested with
these co-cultures, using different media and
growth factors such as cyclic adenosine
monophosphate (cAMP), brain-derived
neurotrophic factor (BDNF), and neurotrophin 3
(NT3) in order to reach optimal generation of
DA neurons.
Biotechnology
Exhibit
5
Student:
Megh Duwadi
Firm:
Walter Reed Army Institute of
Research
Mentor:
Drs. Joseph B. Long and
Debra L. Yourick
Title:
Alterations in Gene Expression
in Response to Ischemic Spinal
Cord Injury in Rats
Background: Traumatic injury to the central
nervous system (CNS) is a leading cause of
death in civilian and military populations alike.
The amino acid glutamate (Glu) is the major
excitatory neurotransmitter in the CNS. Upon
insult of ischemic spinal cord (SC) injury, a
model of CNS trauma offering ready
recognition of the degree of neuronal injury by
the obvious measure of hind limb performance,
an overactivation of Glu receptors leads to
substantial increases in extracellular Glu and
intracellular Ca2+. These changes have been
shown to largely mediate neuronal degeneration.
Attenuation of Glu release minimizes further SC
damage; however, it is imperative that this
procedure be performed near the onset of injury
to be successful. Identifying significant changes
in neuronal gene expression may allow the
targeting of specific downstream events caused
by alterations in gene regulation, and
consequently, a greater window for SC injury
treatment. The present study, which examines
the possibility of therapeutic intervention in
downstream changes in gene expression
following CNS trauma in the form of ischemic
SC injury, will perhaps lead to novel therapy in
CNS injury.
Description: Previous studies involving
ischemic SC injury have shown that dramatic
changes in neuronal gene regulation occur both
during and after incidence. By utilizing the
Mentorship Projects - 2001
powerful technique of differential-display
polymerase chain reaction (DD-PCR), these
modifications in expression are being detected.
The identification of gene expression patterns
unique to ischemic SC injury thus allows
neuroprotective therapeutic strategies to be
targeted and performed, reducing the effects of
glutamate mediated- and other types of neuronal
loss, a primary result of SC trauma.
Methodology for the completion of these studies
was investigated, modified, and applied to SC
tissue extracted from SC-injured rats.
Ischemic SC injury was induced in rats
by injection of the endogenous opioid peptide
dynorphin A. Upon insult, dynorphin A
lowered lumbosacral blood flow, elevated
cerebrospinal fluid lactic acid concentrations,
and caused flaccid hindlimb paralysis and
striking neuropathological changes. Anaerobic
metabolism was increased in association with
ischemia.
DD-PCR is a relatively new technique,
which, unlike conventional methods of
measuring modifications in gene regulation,
allows for the comparison of similar tissue types
and the identification and isolation differentially
expressed genes. RNA isolated from dynorphin
A- and saline-injected spinal cord tissue (injured
and noninjured) was reverse transcribed from an
anchor primer containing a poly (dT) region,
which targets the 3’ ends of mRNA. Low
stringency, competitive PCR followed to induce
mismatching – it is this process that
differentiates DD-PCR from related procedures.
By utilizing DD-PCR to analyze genes found in
injured SC tissue, changes in gene expression
levels upon incidence can be readily detected.
Differentially expressed genes appear as bands
present in the track from only one cell type.
Fragments can be excised from the DD gel,
identified using GenBank and BLAST
databases, and used to prepare gene tags for the
study of gene expression level alterations.
Biotechnology
Exhibit
6
Student:
Erwin P. Gianchandani
Firm:
The Institute for Genomic
Research (TIGR)
Mentor:
Dr. William C. Nelson
Title:
Re-Annotation of Helicobacter
pylori and Analysis of
Bioinformatics Techniques
Background: Helicobacter pylori is a
flagellated, motile, curved microaerophilic
Gram-negative rod that accounts for more than
90 percent of all duodenal ulcers and up to 80
percent of all gastric ulcers. The bacterium is
also attributed to mucosal-associated-lymphoidtype (MALT) lymphoma as well as Sudden
Infant Death Syndrome (SIDS) and morning
sickness. In 1997, TIGR successfully
completed the sequencing and annotation of H.
pylori strain 26695. The published genome was
composed of a 1,667,867-basepair circular
chromosome that was predicted to have 1,590
coding regions. However, nearly one-third of
these open reading frames (ORFs) had no
matches to global databases of known genes.
Although many questions about H. pylori’s
pathology were answered with the release of the
26695 genome, discovering the “unknowns”
was still essential to the development of cures
for diseases of the likes of SIDS and MALT.
This project served to re-annotate the entire
sequence of strain 26695 using the enhanced
resources available today in the field of
Bioinformatics. Specifically, the objectives
were as follows: identifying some of the 499
unknown genes from 1997 by matching them
with genes of known type and function;
verifying previous gene assignments, including
updating names and references; and learning the
improvements in bioinformatics techniques by
comparing the new and old data.
Description: The sequence data from 1997
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was analyzed by “GLIMMER,” a computer
program written to recognize coding regions of
microbial DNA. The possible genes that were
found were subsequently fed into computer
algorithms – Blast-Extend-Repraze (BER)
pairwise alignments and Hidden Markov
Models (HMMs) – that matched the ORFs with
genes of known functions. The resulting data
were then combined by “autoBYOB,” which
made preliminary gene assignments by
classifying each gene into one of 113 different
role-specific categories.
In addition to curating the “autoBYOB”
assignments – gene name, type, and role, etc. –
the start sites selected by “GLIMMER” were
checked. It was also important to identify
frameshifts or other disruptions within the
reading frames.
Finally, upon conclusion of the reannotation process, a number of computergenerated outputs became critical in further
understanding H. pylori, including the
following: a “GC”-skew allowed for the
identification of the origin of replication within
the organism; a trinucleotide skew helped
distinguish those genes that may have been
transferred into the genome from other bacteria;
a software program developed since 1997
analyzed rho-independent terminators and assist
in the understanding of operon structures; and
repeat finders located those sequences that occur
more than once within a genome and that may
be ordered into paralogous families.
Data analysis was conducted thereafter
to make a distinction between the 1997
annotation and the 2000 re-annotation. In
addition to investigating new or different gene
classifications, a step-by-step comparison
between the two genomes during the annotation
process – number of genes identified by
“GLIMMER” versus number of genes identified
by 1997 technology, original number of
database “hits” of the two annotations, and the
first classifications by “autoBYOB,” etc. –
validated the hypothesis.
Biotechnology
Exhibit
7
Student:
Brenda Goguen
Firm:
George Mason University, Prince
William Campus
Mentor:
Dr. Patrick M. Gillevet
Title:
Molecular Characterization of
Potential Fish Pathogens in
Waters Where Reported
Pfiesteria piscicida Outbreaks
Have Occurred
Background: Over the past ten years, harmful
algal blooms (HABs) have occurred in Atlantic
slope waters, including in the Chesapeake Bay
region, killing millions of fish and causing
human debilitation. It has been hypothesized
that these events can be attributed to two
endotoxins released by the dinoflagellate
Pfiesteria piscicida. In the protist kingdom, P.
piscicida represents a new family, genus, and
species of dinoflagellate that was first identified
and named the causal agent of fish deaths in
Pamlico Sound, North Carolina in 1988. It has
been proposed that P. piscicida has about 24
varying growth stages, including stellate
amoeboid and flagellated zoospore stages.
Additionally, it is hypothesized that in its
flagellated form, P. piscicida releases
endotoxins which may lead to ulcerated lesions
in fish and to large scale fish kills.
The presence of these endotoxins poses a
concern for ecological and human health and for
commercial and recreational fisheries.
However, growing evidence exists that the fish
kills in the Chesapeake Bay have not been the
result of the proposed toxins excreted by P.
piscicida or Pfiesteria-like organisms. Studies
have reported that the proposed amoeboid forms
of P. piscicida, in fact, do not exist for the
organism. Thus, stellate amoebae may be
mistaken for the reported amoeboid forms of P.
Mentorship Projects - 2001
piscicida. Other causes of the fish lesions in
Virginia waters have been proposed to be the
result of endosymbiotic bacteria associated with
the putative amoeboid stages of P. piscicida.
Description: The purpose of this research was
to molecularly characterize microorganisms
associated with HABs, including potential fish
pathogens in Chesapeake Bay waters where
reported Pfiesteria outbreaks and fish kills have
occurred, to clearly show what the casual agent
is. Bottom soil sediments from five river
systems (Potomac, Patuxent, Chester, Choptank,
and Pocomoke) that discharge into the Bay were
taken prior to this study. In order to molecularly
determine the variety of protists and bacteria in
the rivers, the DNA extracted from the soil
sediments was amplified through polymerase
chain reaction (PCR) with fluorescently labeled
primers specific to protist and bacterial smallsubunit (SSU) regions of the DNA. There is an
abundance of natural length variation in the
SSU rRNA genes in microorganisms which is
directly related to phylogeny. Through the use
of an amplicon length heterogeneity fingerprint
after PCR with fluorescent primers, it was
possible to differentiate between genera and to
determine the microheterogeneity of protists and
bacteria in the samples from the five rivers.
Analysis of the fingerprints found that P.
piscicida was present in small amounts in only
the Pocomoke, Patuxent, and Choptank Rivers,
preliminarily indicating that P. piscicida may
not have had a role in the fish kills.
Additionally, other DNA taken from
experimental fish tanks has been amplified by
PCR and fingerprinted for both the protist and
bacterial regions of the DNA. Analysis of this
data remains to be completed. The fingerprints
showing the bacteria from all samples will be
used to help determine if the fish death is the
result of endosymbiotic bacteria associated with
the putative amoeboid stages of P. piscicida.
Biotechnology
Exhibit
8
Student:
Tina Gupta
Firm:
Walter Reed Army Institute of
Research
Mentor:
Prabhati Ray, Ph.D.
Title:
Protease Response to Alkylation
Damage in Cultured Human Skin
Cells
Background: Nitrogen mustard (HN-2, bis-(2chloroehtylthioethyl)-ether) and sulfur mustard
(HD, bis-(2-chloroethyl) sulfide) are alkylating
agents. HD has been used as a chemical warfare
agent with severe consequences in the past, and
was used more recently in the Iran-Iraq conflict.
Mustard-induced blister formation is an
epidermal event accompanied by a separation of
the dermis from the epidermis due to the
disruption of the connective tissues, possibly by
the action of some protease(s) at the epidermaldermal junction. Recent studies have
demonstrated induction of a tumor-suppressant
nuclear phosphoprotein, p53, in mustard
exposed NHEK. Typically, p53 exists in a
Mentorship Projects - 2001
highly unstable state, with a half-life of 20-30
minutes. The phosphorylation of p53, which
stabilizes the transcription factor, may be
involved in the expression of the mustard
associated protease. Curcumin, a powerful anticancer agent, prevents the phosphorylation of
p53, and other biological molecules.
Description: Protease expression was
identified with Western blotting analysis using a
polyclonal rabbit antibody raised against the HD
induced protease from NHEK purified
previously in this laboratory. The intracellular
calcium chelator, BAPTA-AM reduced HD
induced protease expression. Topical
application of HD on hairless guinea pigs
verified in vivo protease induction. A light
microscopy study demonstrated that mustard
exposed NHEK had remarkable morphological
changes. Treatment of cells with the protease
antisense oligonucleotides blocked
morphological alterations. Western blot
analysis of antisense treated cells is
forthcoming. NHEK incubated in curcumin,
may inhibit protease expression, however results
remain inconclusive.
Biotechnology
Exhibit
9
Student:
Edward Kim
Firm:
Walter Reed Army Institute of
Research
Mentor:
Dr. Rina Das
Title:
Regulation of Fatty Acid Binding
Proteins in Breast and Prostate
Cancer Cells
Background: Epidemiological studies on
cancer of the prostate gland have shown a
positive relationship between the consumption
of dietary fats and development of prostate
cancer. The objective of the research was to
determine the effects of cell growth regulators
such as anti-sense, fatty acids, and cancer
fighting drugs, on the expression of Fatty Acid
Binding Proteins (FAPB’s) and how these
FABP levels in turn regulate the proliferation or
the apoptosis, systematic death, of prostate and
breast cancer cells. FABP’s serve as
transporters of bioactive lipids and whose up or
down regulation may play a role in cancer
growth in prostate and breast cancer cells.
Several studies suggest that FABP’s increase the
solubility of fatty acids in the cell cytoplasm
causing a new diffusion of fatty acids from the
plasma membrane to the intracellular membrane
compartments, making them more easily
available to cause cancer proliferation. The
levels of these FAPB’s can be found by
measuring the mRNA expression levels in
various strains of cancer cells, which are
systematically grown in different mediums and
growing conditions. In preliminary research it
was found that Liver-FAPB and Intestine-FABP
were up-regulated, in increased levels, in cancer
cells, while Adipose-FABP, Epidermal-FAPB,
and Heart-FABP were down-regulated, in
decreased levels, compared to levels in normal
cells. FABP’s could act as potential markers for
detection of prostate or breast cancer. By
adding various cell growth regulators, FABP
levels fluctuated accordingly, which show that
Mentorship Projects - 2001
FABP’s may have some role in prostate and
breast cancer.
Description: Not much is known on how the
levels of FABP’s are regulated in cancer cells.
A comparison was done between normal
prostate cells and cancer cells as a model
system. This study examined the levels of
FABP’s in several prostate normal and cancer
cell lines in order to establish a correlation
between FABP levels and how several growth
regulators affect them. The levels of expression
of mRNA for selected FAPB’s were analyzed
using primers for RT-PCR. These levels could
then be further affected towards or away from
the normal cell levels by the addition of growth
regulators such as cancer fighting drugs, fatty
acids, and anti-sense specific for each FABP.
Cancer cells would be grown in different growth
regulators, such as different fatty acids,
harvested, then analyzed for FABP expression.
These levels would then be compared to a
standardized control culture of cancer cells to
determine the amount of change.
The addition of the cancer fighting drugs
Curcumine, MK 886, and NDGA to the cancer
cells brought the levels of both groups of
FABP’s towards control levels due to the fact
that they are inhibitors of the arachidonic acid
metabolic pathway. Also, by adding anti-sense
of Liver and Breast, which work by blocking the
expression of L-FABP and B-FABP
respectively, the levels of other FABP’s varied
in response to the change in concentration of
one FABP. The addition of fatty acids such as
linoleic and arachidonic acids did increase the
growth of cancer cells, but did not return the
expected results. This shows that the levels of
FABP’s were directly affected by the
proliferation of cancer and normal cells. The
detection of FABP levels in cancer cells and in
the medium in which they grow can provide a
means of identifying the aggressiveness of a
patient’s prostate or breast cancer.
Biotechnology
Exhibit
10
Student:
Jonathan Lasken
Firm:
ARCTECH, Incorporated
Mentor:
Mr. Randy Reed
Title:
The Effect of Humic Acid-Based
Soil Amendments on the
Premature Stages of Plant Radish
Growth
Background: Recently the Department of
Defense has found it necessary to develop a
more environmentally friendly way of disposing
of its surplus propellant. The previous method,
termed open detonation, was the destruction of
the munitions in a pit. Under the Department of
Defense’s “R3 Reduce/Recycle/Reuse” plan
ARCTECH has developed the Actodemil™
technology. The Actodemil™ technology is
used to convert the propellant from missiles,
often TNT or another highly explosive
substance, into a humic acid based fertilizer.
This technology has been proven at Hawthorne
Army Depot, Nevada, and the University of
Nevada Las Vegas has done some preliminary
testing on the fertilizer, declaring it safe for
commercial use. However, little testing has
been done on the effectiveness of this fertilizer
to this point.
Description: The direct resultant of the
Actodemil™ technology is called actosol®-m
(actosol®-mixture). To separate the supernatant
from the humic acid the pH of the actosol®-m is
lowered to below 2 and the actosol®-m is
centrifuged. Sludge forms at the bottom of the
centrifuged container and supernatant forms
above. In this experiment, the supernatant was
Mentorship Projects - 2001
tested as one type of fertilizer and the sludge
was tested as another. The fertilizer made from
the sludge is termed actosol®-x and the one
from the supernatant is termed actosol®-s. Both
of these products were mixed to a forty to one
dilution.
Actosol®-x, actosol®-s, water, and
Professional actosol®, a professionally
manufactured and used fertilizer, were each
applied to five out of twenty pots, all of which
were filled with potting soil. Each pot was
planted with three seeds and their germination
rates and shoot growth were recorded. The
plants were allowed to grow for thirty-five days
at which point, the plants were uprooted from
the ground and both above ground and below
ground biomass were taken. This was done to
see the effects of the various actosols® on the
preliminary plant growth, both in shoot growth
(above ground) and root growth (below ground).
Through this experiment, the various
actosols® were proven to be effective on their
respective plants. Another purpose of this
experiment was to try to infer what is contained
in the supernatant. It is known that the sludge
consists primarily of humic acid, but the
contents of the supernatant were unknown. This
experiment shone some light on what is
contained in the supernatant. There are three
extensions of this experiment. In the first
extension, the same experiment will be
conducted in sandy soil, and in the second
experiment, the actosol®-m will be turned into a
fertilizer of its own and used on an additional
set of pots along with the other four fertilizers
used in the experiment outlined above. The
final extension would be to mix dilutions of the
sludge and supernatant to find the most effective
combination of the two.
Biotechnology
Exhibit
11
Student:
Swan Lee
Firm:
Walter Reed Army Institute of
Research
Mentor:
Dr. Debra Yourick
Title:
Changes in the Blood Brain
Barrier After Inducing Fluid
Percussion Injury and
Hemorrhagic Shock
Background: Traumatic brain injury is one of
the most frequent causes of morbidity and
mortality on the battlefield, and remains the
leading cause of traumatic death in the United
States. The pathophysiological features of brain
injury seen in humans can be recreated through
rodent fluid percussion injury (FPI), which is
considered to be one of the most effective
models of traumatic brain injury. The aspect of
particular interest in this study is the destruction
of the blood brain barrier (BBB). The BBB is
the barrier that exists between the blood and the
cerebrospinal fluid which prevents the passage
of various substances from the bloodstream to
the brain. Traumatic brain injury disrupts the
BBB, which can cause a complete breakdown of
the BBB, or cause the brain to be more
susceptible to leakage of solutes. Since the state
of the BBB influences the effectiveness of
Mentorship Projects - 2001
resuscitation, it is important to characterize the
BBB changes resulting from combined
traumatic brain injury and hemorrhagic
hypotension, to evaluate where loss of BBB
integrity might affect the efficacy of the
resuscitation fluid.
Description: Evans blue dye (EB) was used to
evaluate the blood brain barrier to the
movement of molecules, during or after
combined injury. The dye concentration was
measured and compared in brains from rats that
were uninjured, fluid-percussion injured only,
hemorrhaged only, or injured and hemorrhaged.
Male Sprague-Dawley rats were injured
with a fluid-percussion device, and
subsequently, rats were hemorrhaged to a mean
arterial blood pressure of 40 mmHg over a
period of 15 minutes, and maintained at that
level for 60 minutes. After injection of the
Evans blue dye, rats were resuscitated to 80
mmHg with a lactated Ringer’s solution or
autologous blood for 60 minutes. Brains were
removed, dissected, and EB was extracted in a
50% trichloroacetic acid solution.
Concentration of EB was determined using a
fluorescence plate reader with appropriate
excitation and emission wavelengths for the
dye. The dye content in plasma and brain was
determined, and a ratio calculated for relative
permeability, specifically, percent extravasation.
Biotechnology
Exhibit
12
Student:
Meredith Lowe
Firm:
Food and Drug Administration
Mentor:
Dr. Robin Levis
Title:
The Role of the NS4B
Nonstructural Protein in Dengue
Virus Replication
Background: The goal of this project is to
determine the role of the NS4B nonstructural
protein in dengue virus replication. The dengue
virus is spread among humans by mosquitoes
and is especially prevalent in subtropical and
tropical regions. However, epidemics have
occurred throughout history all over the world,
including the United States. There are an
estimated 100-300 million cases of dengue
infection per year in recent years. The dengue
virus causes 2 different diseases: primary
dengue fever (bone-crushing disease), and
dengue hemorrhagic fever shock syndrome
(DHFSS). There is currently no vaccine or cure
for any of these diseases.
Mentorship Projects - 2001
Description: The purpose of the research was
to determine the effects of mutations in the
NS4B gene on dengue virus replication, viral
release, and protein synthesis. It is hypothesized
that the seven nonstructural proteins in the
dengue genome play a large role in dengue virus
replication, but so far little is known about the
individual proteins. Minimal information about
the NS4B nonstructural protein has been
published, so that protein was the focus of the
experiment.
In this experiment, two cell lines were
transfected with six lethal virus mutants of the
NS4B nonstructural protein and one wild type.
One cell line was a control; the other had been
genetically altered to express NS4B in trans.
Both cell lines were incubated and samples were
extracted every six days for 24 days. Three
assays were performed on the samples: Northern
blot analysis to test virus replication, reversetranscription polymerase chain reaction (RTPCR) to test viral release, and an
immunofluorescence assay (IFA) to detect
NS4B and other viral protein synthesis. This
experiment is one step towards understanding
how the dengue virus replicates, which is crucial
for development of a vaccination.
Biotechnology
Exhibit
13
Student:
Greg Mattingly
Firm:
Department of Pharmacology,
Georgetown University Medical
Center
Mentors:
Dr. Niaz Sahibazada,
Dr. Richard Gillis
Title:
Modulation of Intragastric
Pressure and Fundic Tone in the
Dorsal Motor Nucleus of the
Vagus of the Rat
Background: The native 7 nicotinic
acetylcholine receptor (nAChR) is very
common in both the central and peripheral
nervous systems. They have a high
permeability to calcium, can act presynaptically
to modulate neurotransmitter release, and can
also participate in postsynaptic signalling.
Although the 7 nAChR has been studied and
classified as homomeric when expressed on a
Xenopus oocyte, it has never been characterized
in its native state. Previous studies have linked
the 7 nAChR to an area in the central nervous
system known as the Dorsal Motor Nucleus of
the Vagus (DMV) due to the effects of an 7
antagonist, -bungarotoxin (-BGTX), on the
fundus area of the stomach. -BGTX blocks
fundic tone, indicating that the neurons
projecting to the fundus contain 7 subunits.
adult anesthsized rat regarding the role of 7
nAChR subtype can be confirmed by
electrophysiological studies of single neurons in
the DMV that project to the fundus; (2) to
pharmocologically characterize 7 nAChR on
single DMV neurons; (3) to determine whether
nAChR subtypes other than the 7 subtype are
present on DMV neurons that project to the
fundus.
In order to identify which DMV neurons
project to the fundus, Sprague Dawley rats
(postnatal day 14) were anesthesized with
methoxyflurane, cut open in the stomach area,
and a retrograde DiI tracer was applied to the
fundus. The rats (19-28 days old) were then
decapitated and brainstem slices 250 um thick
were cut using the vibrating microtome. After
being immersed in a physiological solution,
cells bearing the retrograde DiI tracer were
identified visually by flourescence optics.
Using the whole-cell patch clamp method, the
cells were voltage clamped and acetylcholine,
an nAChR agonist, was applied via bath
application. When a viable cell was obtained
(one that responded well to acetylcholine), 7
antagonists such as -BGTX and
methyllycaconitine were pressure injected into
the cell, and the change in current was recorded.
It was found that the currents of the DMV cells
studied were only partially attenuated by BGTX and MLA, indicating that the native 7
receptor is indeed heteromeric. The other
subunit(s) remains to be determined.
Description: The purpose of my project is to
(1) determine whether results obtained from the
Mentorship Projects - 2001
Biotechnology
Exhibit
14
Student:
Vasiliki Michopoulos
Firm:
National Institutes of Health
Mentor:
Dr. Zlatko Trajanoski
Title:
In silico Identification of
Peroxisome ProliferatorActivated Receptor Gamma
Transcriptional Targets
Background: Obesity is an increasing health
problem reaching epidemic proportions in most
western societies. The prevalence of obesity in
much of Europe is 15-20% of the middle-aged
population and is higher in the United States,
especially in minorities such as African or
Mexican Americans where the prevalence in
women is about 40%. Obesity is the main
precursor state of diabetes. Other diseases such
as hypertension, dyslipidaemia, and
cardiovascular diseases are also attributable to
obesity. Recently, a protein member of the
nuclear hormone receptor super-family
designated as PPAR (peroxisome proliferatoractivated receptor gamma) was discovered and
its central role in fat cell differentiation was
identified.
Mentorship Projects - 2001
Description: The specific aim in this study
was to identify additional downstream PPAR
(peroxisome proliferator-activated receptor
gamma) target genes by using in silico methods.
Genes with PPAR responsive elements in their
promoter regions were identified by screening
annotated sequence databases for PPAR binding
motifs.
In order to identify PPAR targets,
database searches were performed and three
computational techniques were used: 1)
Searching for short sequence patterns within the
database entries, 2) Searching for patterns using
a position weight matrix (PWM) from the
TRANSFAC database and programs, 3) Patterns
were searched using a program and a newly
constructed position weight matrix (PWM). Due
to the context-sensitivity and the possibility of
using specialized PWMs, the method of choice
for in silico identification of PPAR targets was
"TargetFinder", leading to a total of 1206
putative targets.
This computational approach enabled the
identification of putative target genes for
PPAR. These target genes can be responsible
for setting the adipogenic program in motion
and hence, enable a more rational design of new
classes of drugs to control obesity and
eventually other diseases.
Biotechnology
Exhibit
15
Student:
Diane Oh
Firm:
Walter Reed Army Institute of
Research
Mentor:
Dr. Rasha Hammamieh
Title:
Expression and Purification of
Fatty Acid Binding Proteins in
E. coli
Background: Intracellular transport of
bioactive lipids is a critical component in the
process by which molecules continuously
stimulate proliferation through interaction with
nuclear receptors. Transport and utilization of
lipids are mediated by an important family of
cytoplasmic proteins, known as fatty acid
binding proteins (FABPs). Several studies
suggest that FABP increase the solubility of
fatty acids in the cell cytoplasm, causing a net
diffusion of fatty acids from the plasma
membrane to the intracellular membrane
compartments.
The members of this multigene family of
FABPs consist of at least seven types whose
amino acid sequences have been obtained from
protein purified from tissues or from cDNA
sequences. The designations for each of the
FABPs have been derived from the tissue from
which it was originally isolated and include
adipocyte (A-FABP), heart or muscle (HFABP), brain (B-FABP), epidermis or psoriasisassociated (E-FABP), liver (L-FABP), intestine
(I-FABP), and myelin or P2 (P2-FABP).
Within this family of FABPs, however,
are two separate classes. In one class is L-FABP
and I-FABP, which were elevated 5-9 fold in
most cancer cells compared to normal primary
cells. In contrast, the other class of FABPs
Mentorship Projects - 2001
including A-FABP, E-FABP, and H-FABP were
severely down-regulated (3-50 fold) in cancer
cells compared to normal cells. This suggests
that there may be a distinct balance between
these two groups of FABP whose up or down
regulation in cells may play a role in prostate
and breast cancer.
Description: In order to discover the effects of
up or down regulating these FABPs in cancer
cells, the FABPs themselves must first be
isolated for use. The purification of FABPs was
done by using the PinPointTM Xa-1 T-Vector to
clone and express the cDNA that codes for
specific FABPs in E. coli. After transformation
of the vector into E. coli, the transformation
cultures were grown and the DNA was isolated
from cells from each culture. In those DNA
samples in which the cDNA insert had been
inserted into the vector successfully, the DNA
was sequenced and checked with existing
sequences in the GeneBank to ensure that the
cDNA had been inserted into the vector in the
correct orientation. IPTG, a protein inducer, was
then added to the E. coli cultures from which the
successful plasmid samples were isolated to
induce the production of the FABP. The
proteins produced were fusion proteins. The
PinPointTM Xa-1 T-Vector carries a segment
encoding a peptide that becomes biotinylated in
E. coli and subsequently functions as a
purification tag. Using an avidin resin, the
protein was specifically removed from an
extract by binding of the covalently linked
biotin on the fusion to the monomeric avidin
linked to the resin. Elution from the resin was
accomplished by incubation with biotin under
nondenaturing conditions. This produced the
purified FABP.
Biotechnology
Exhibit
16
Student:
Rita Portocarrero
Firm:
National Institute for
Neurological Disorders and
Stroke
Mentor:
Dr. Heather Cameron
Title:
Effect of Growth Factors on
Proliferating Neurons in the
Adult Dentate Gyrus
Background: After birth, most neurons in the
central nervous system no longer proliferate,
remaining in the brain until they die. This
characteristic makes it extremely difficult to
reverse damage done to the brain, because the
cells cannot replace themselves. In the 1960s,
scientists discovered that in the dentate gyrus, a
region of the hippocampus, which controls
learning and memory, cells proliferate
throughout adulthood. The direct signals
controlling proliferation are not known, but
several growth factors have been found in the
dentate gyrus, leading scientists to believe that
they may be regulating proliferation.
membrane to trigger a reaction, signaling the
start of mitosis. Many are present in the dentate
gyrus, though how they affect neurogenesis is
unknown. Most likely, the receptors for these
growth factors are present on the dividing cells;
this would suggest that they directly signal cell
proliferation.
Five rat brains were labeled with [3H]
thymidine, a radioactive substance that labels
cells in the S phase of mitosis, and euthanized
by Dr. Heather Cameron. Sections of these
brains were put on slides and
immunohistochemically stained for epidermal
growth factor receptor (EGFR), insulin-like
growth factor receptor (IGFR), and fibroblast
growth factor receptor 1, 2, and 3 (FGFR1,
FGFR2, FGFR3). Then the slides were dipped
in photographic emulsion, and allowed to sit in
the dark for three weeks, so the cells that had
been probed by thymidine would be visibly
labeled under the microscope. Finally, the
slides were analyzed, and the thymidine labeled
cells counted, to determine whether or not the
specific receptors are on the dividing cells. The
results show that all five receptors were present
on some proliferating cell, suggesting that they
directly trigger the start of mitosis in neurons.
Description: Growth factors are ligands that
attach to specific receptors on the cell
Mentorship Projects - 2001
Biotechnology
Exhibit
17
Student:
Andrew Ra
Firm:
Georgetown University,
Department of Neuroscience
Mentor:
Dr. Jean R. Wrathall
Title:
Proliferation of Cells After
Spinal Cord Injury
Background: Spinal cord injury was once
viewed to be incurable. Victims of spinal cord
injury could expect themselves to live in
wheelchairs for the rest of their life, helpless
and without any hope of recovery. However,
recent discoveries in the field of neuroscience
have given those victims of spinal cord injury
hope. Studies indicate that the spinal cord is
able to take steps to heal itself after spinal cord
injury. After spinal cord injury, oligodendrocyte
apoptosis, loss of myelin occur at the injury site
up and down the spinal cord and in various
animal models it is observed that demyelination
occurs most significantly during the first week
after spinal cord injury. Also remyelination
appears to occur at approximately 14 days post
injury and by one month most axons have
become remyelinated, however, not to the level
of myelination observed before spinal cord
injury. Although it is known that remyelination
Mentorship Projects - 2001
occurs, it is unknown what role, if any, cells
such as oligodendrocytes take in the
remyelination process.
Description: Studies have identified an
oligodendrocyte progenitor cell (OPC)
population in the adult central nervous system.
These cells are as abundant as microglia and
there is evidence to show that these progenitor
cells may aid in the remyelination of axons in
the spinal cord. However, this work was done in
vitro and thus it is not known if these cells
actually aid the spinal cord in the healing
process. In the study that we are conducting, we
used bromodeoxyuridine, or BrdU for short, to
show the time course during the first week
following spinal cord injury and the distribution
of cell division in the rat spinal cord at 2
millimeters and 4 millimeters rostral and caudal
to the epicenter. Some of the cells that divide
after spinal cord injury are likely to be
oligodendrocyte precursor cells that will be
involved in remyelinating axons after injury.
Identifying when and where these cells divide is
the first step towards studying how they may be
involved in the healing process. BrdU is thus
incorporated in the experiment because it has
been found to be able to detect the S phase of
cell division.
Biotechnology
Exhibit
18
Student:
Elizabeth Reynolds
Firm:
National Institute of Mental
Health, Clinical Brain Disorders
Branch
Mentor:
Dr. Jeremy M. Crook
Title:
Post Mortem Studies of
Muscarinic Receptor 2 Gene
Expression in Dorsolateral
Prefrontal Cortex of Patients with
Schizophrenia
Background: Schizophrenia is a mental
disorder characterized by auditory
hallucinations, delusional thought and paranoia,
distortion of personality, inappropriate impulse
and emotional response, and difficulty in
learning and memory. Schizophrenia effects
1% of the world’s population. Schizophrenia
has been found in patients from anywhere in
their early teens to well into their seventies.
Most cases of Schizophrenia are diagnosed
between the ages of 15 and 45. Studies have
shown that males and females have equal
chances of being affected by schizophrenia.
The cause of schizophrenia is currently
unknown, however several factors are suspected
to contribute to a persons chances of developing
schizophrenia. While genetics is most likely an
underlying cause of schizophrenia,
environmental factors such as fetal trauma or
infection, season of birth, and viral pandemics
may also be important (Harrison, 1999).
Mentorship Projects - 2001
Description: We have investigated muscarinic
receptor 2 [M2] gene expression differences in
dorsolateral prefrontal cortex [DLPFC] of
patients with schizophrenia or affective
disorders (patients who committed suicide or
had bipolar disorder) using in situ hybridization
histochemistry [ISHH]. This includes: mRNA
density analysis of autoradiograpic images
produced from in situ hybridization and silver
grain analysis of M2 mRNA on tissue on slides.
To identify the localization of M2 receptor
protein we have also performed
immunohistochemistry on tissue from 5 normal
subjects included in ISHH. From film based
studies no significant difference was found
between mRNA levels of patients with
schizophrenia or affective disorder and normal
controls. The lack of a difference in mRNA
levels between schizophrenics and normals
along with findings of Crook et al. 1999, and the
1999 Intel entry of Hilary Glidden who report
reduced [3H]AF-DX 384 binding to receptors in
the DLPFC and caudate-putamen, respectively,
of subjects with schizophrenia, suggests
changes to muscarinic receptor M2 are post
transcriptional. Preliminary results from the
slide-based study show low levels of muscarinic
receptor 2 mRNA associated with pyramidal
neurons in DLPFC. Immunohistochemistry
revealed immunostaining throughout the
neuropil with no staining around dendrites or
cell bodies. This suggests muscarinic receptor
M 2 is presynaptic.
Biotechnology
Exhibit
19
Student:
Won Suh
Firm:
Uniformed Services University
Mentor:
Allen L. Richards, PhD
Title:
Rapid Molecular Diagnosis of
Scrub Typhus Utilizing TaqMan
Assay
Background: Scrub typhus is a bacterial
disease endemic to the Asia-Pacific region,
which has, if left untreated, up to a 50%
mortality rate. Its etiologic agent is Orientia
tsutsugamushi (previously known as Rickettsia
tsutsugamushi), a bacteria transmitted by the
bite of a chigger, a trombiculid mite in its larval
phase. Some symptoms of scrub typhus include
an eschar at the site of the bite, fevers,
headaches, swollen lymph glands, a rash that
spreads to the arms and legs, and at times
pneumonitis. Although this disease has been
around for a long time, it was not much
publicized until U.S. soldiers began to report
infections during World War II and the Vietnam
War. Antibiotic treatments are available
including one of the tetracyclines or
chloramphenicol. However, there have been
recent reports of drug resistant strains of the
bacteria. Working with O. tsutsugamushi is
rather difficult and expensive, because unlike
other types of bacteria, O. tsutsugamushi cannot
be cultured on plates (artificial media), but
Mentorship Projects - 2001
requires living organisms (lab animals or tissue
culture) to grow.
Description: This project focused on the
detection and quantitation of O. tsutsugamushi
in laboratory and animal acquired samples
utilizing a TaqMan assay. The assay takes the
polymerase chain reaction (PCR) technique one
step further. It incorporates a specific
nucleotide probe with a fluorescent dye in order
to provide for an extremely sensitive assay. The
dye and a quencher molecule are bound to each
end of the probe, which attaches specifically to
the targeted region of the O. tsutsugamushi 47
kDa antigen gene being amplified by the Taq
polymerase. In addition to the Taq enzyme’s
ability to make new DNA it also “chews up” the
probe in front of it releasing the fluorescent dye
from close proximity to the quencher molecule,
allowing the fluorescence emitted to be detected
by the AB1 Prism 7700 SDS machine. The
assay has been successful in the detection of
laboratory grown O. tsutsugamushi strains. In
addition, it was found to be effective in
detecting O. tsutsugamushi in mouse blood. In
order to quantify the results of the experiments,
a standard curve was made with dilutions of the
plasmid VR 1012 containing the O.
tsutsugamushi Kato strain 47 kDa antigen gene
produced in the laboratory. Blood from humans
infected with O. tsutsugamushi have not been
tested yet, but this is the next step planned in
evaluating and optimizing this new rapid
molecular diagnostic assay for scrub typhus.
Biotechnology
Exhibit
20
Student:
Grace Wan
Firm:
Georgetown University Medical
Center
Mentor:
Dr. Steven N. Ebert
Title:
The Genetic and Molecular
Mechanisms Underlying Female
Susceptibility to Drug-Induced
Cardiac Arrhythmia
Background: It has been shown that women
are at a far greater risk of developing torsades
de pointes (TdP) in response to drugs such as
antihistamines, antibiotics, antimalarials, and
antiarrhythmics, which act as potassium channel
blockers. TdP is a type of cardiac arrhythmia
occurring in the setting of a lengthened QT
interval, which reflects prolonged cardiac
repolarization and may result in ventricular
fibrillation and cardiac arrest. It is yet unknown
why women are more susceptible to TdP,
although it is suspected that the naturally longer
baseline electrocardiographic rate corrected QT
(QTc) interval may be a factor. The QTc
interval in males begins to shorten at the onset
of puberty when the level of androgens in males
begins to rise and returns to equal that of
women’s at about age 50. This suggests that
one or more of the male hormones may be
responsible for the QTc shortening, and
relatively lower risk of drug-induced TdP in
men. Molecular mechanism studies have shown
at least six chromosomal loci that have been
linked to congenital long QT syndrome (LQTS)
Mentorship Projects - 2001
for drug-induced arrhythmias. One such
mutation is found on chromosome 7 in HERG, a
potassium channel gene that encodes the rapid
component of two major repolarizing potassium
currents, the delayed rectifier (IKr) and the
inward rectifier (IK) current densities.
Preliminary studies have shown that women are
more likely to suffer from mutations on the
HERG gene which alters the IKr density and in
turn results in LQTS. This would explain why
women are more likely to suffer from TdP.
Description: It is logical to assume that male
sex hormones may play a role in the length of
the QT interval and that this effect is regulated
by an effect on HERG. In this study, the effects
that the androgen, dihydrotestosterone (DHT),
has on HERG mRNA and protein were
examined. We determined whether there is an
increase in the concentration of the mRNA and
the HERG protein in castrated male rabbit hearts
that have been treated with either placebo slow
release pellets or DHT slow release pellets. If
concentrations of HERG protein increase in
DHT treated hearts relative to placebo heart, this
will support the hypothesis that androgens
chronically modulate delayed rectifier
potassium current activity through an effect on
HERG gene expression. If concentrations of
HERG mRNA is also increased due to DHT
treatment, then the molecular mechanisms of
androgen action may also affect HERG gene
expression. Experiments were designed to
distinguish between the actions of DHT on
HERG mRNA and on HERG protein.
Biotechnology
Exhibit
21
Student:
Leslie White
Firm:
National Zoo, Molecular
Genetics Laboratory
Mentor:
Dr. Miyoko Chu
Title:
PCR Optimization and Genetic
Comparison of Three Melospiza
georgiana Subspecies Using
Microsatellite DNA
primer. Allele sizes from the three subspecies
were compared with the use of an ABI
automated sequencer and data were analyzed
using the computer program Arlequin.
Comparisons of allele sizes and frequencies help
to determine the degree of genetic divergence
between the Coastal Plains Swamp Sparrow and
the other subspecies and also to estimate levels
of gene flow.
Background: Swamp Sparrows (Melospiza
georgiana) are migratory birds that breed in
North America. The Coastal Plain Swamp
Sparrows (M.g. nigrescens) are morphologically
distinct from the other two subspecies, and they
breed in saltwater marshes, rather than
freshwater marshes. They are also restricted to a
distinct range in the marshlands of Maryland,
Delaware, Pennsylvania, and New Jersey, and
their populations are declining. If the Coastal
Plain Swamp Sparrows are genetically distinct
from other subspecies of Swamp Sparrows,
actions should be taken to protect them from
extinction. Previous projects suggest that any
divergence of the three subspecies must have
occurred recently, as they could not be
distinguished based on mitochondrial DNA.
Studies of microsatellite DNA, or segments of
non-coding repeated sequences, could reveal a
more recent divergence.
Description: In order to compare
microsatellite DNA, the Swamp Sparrow DNA
samples must be amplified through PCR. There
are no primers that have been designed to work
specifically on Swamp Sparrow DNA, so a
major component of the project was optimizing
PCR conditions so that Song Sparrow primers
would amplify Swamp Sparrow DNA. The
factors that were altered most were the amount
of MgCl2 added to the PCR solution and the
annealing temperature.
When optimal PCR conditions were
determined for a primer, Swamp Sparrow DNA
from the three different subspecies was
amplified using fluorescent versions of the
Mentorship Projects - 2001
Biotechnology
Exhibit
22
Mentorship Projects - 2001
Chemical Analysis
Exhibit
23
Mentorship Projects - 2001
Chemical Analysis
Exhibit
24