The University of Toledo Department of Bilogical Sciences

Bowman-Oddy Room 1045 Saturday March 28, 1998 9:15AM- 3:00PM

(click on the presenter name for abstract)

Time	Speaker	Presentation Title	Advisor
9:15		Welcome
9:20	Plenary Session- Dr. John Reeve of Ohio State University
10:00	Coffee Break
10:15	Kevin York	*The isolation and hatching characterization of Ascaris suum* hatching fluid proteins.**	Dr. Patricia Komuniecki
10:30	Bhairavi M. Patel	*Tyrosine-phosphorylated proteins and their developmental regulation in Ascaris suum.*	Dr. Patricia Komuniecki
10:45	Spencer Johnson	Production of a monoclonal antibody against Plakophilin 2.	Dr. Keith Johnson and Dr. Peg Wheelock
11:00	Andrew J. Hill	Creation of a monoclonal antibody.	Dr. Keith Johnson and Dr. Peg Wheelock
11:15	Sujatha Prabhakaran	Characterization of the N-CAD-M phenotype.	Dr. Keith Johnson and Dr. Peg Wheelock
11:30	Michelle DuPlain	Effects of growing conditions of the Cauliflower Mosaic Virus on Arabidopsis thaliana.	Dr. Scott Leisner
11:45	Matt Elkins	Plants aiding drug research: The CaMV/ Turnip plant model system for drug testing.	Dr. Scott Leisner
12:00	Ramsey Chehab	Crystalization of E. coli manganese superoxide dismutase.	Dr. Gloria Borgstahl
12:15	Jennifer L. Kurek	Effects of freezing on soil communities.	Dr. Deb Neher
12:30	Lunch and Award Presentation

Abstract 3rd URS

Kevin M. York
The isolation and characterization of Ascaris suum hatching fluid proteins
Advisor: Dr. Patricia Komuniecki, University of Toledo
Department: Biology

Ascaris suum is a parasitic nematode which, as an adult, infects the small intestines of pigs. Several earlier developmental stages occur within a resistant eggshell. The second-stage larvae hatch from this eggshell, but little is known concerning the biochemical processes involved. The purpose of the present study was to isolate and characterize the proteins in the larval hatching fluid. After artificially hatching the larvae, proteins form the hatching fluid were collected, isolated, and tentatively identified. A. suum is an experimental model for medically important parasites, and information gleaned from these studies could lead to new anti-parasitic drug therapies in humans.

Bhairavi M. Patel
Tyrosine-phosphorylated proteins and their developmental regulation in Ascaris suum.
Advisor: Dr. Patricia Komuniecki, University of Toledo
Department: Biology

Tyrosine phosphorylation is an important process in regulating growth and differentiation. Tyrosine kinase activity is highly regulated during cellular differentiation in many organisms, and the resulting tyrosine-phosphorylated proteins are involved in complex signaling cascades. The parasitic nematode, Ascaris suum, undergoes an aerobic-anaerobic transition during its life cycle. Early embryonic and larval stages are free-living and aerobic, but adult worms reside in the microaerobic small intestine if swine. Signals that regulate this transition have not yet been defined, however it is possible that tyrosine phosphorylation may play a key regulatory role. To determine whether tyrosine phosphorylated proteins are present in A. suum, homogenates have been prepared from adult muscle and intestine and immunoblotted with PY-20, a monoclonal antibody specific for tyrosine-phosphorylated proteins. Studies are currently underway to assess whether any tryosine-phosphorylated proteins are present in these adult tissues. Once positive results are obtained, then homogenates from different developmental stages will be analyzed. Demonstration of such proteins and their developmental regulation would indicate that tyrosine phosphorylation is an important signaling mechanism during the aerobic to anaerobic transition in A. suum.

Spencer Johnson
Production of a monoclonal antibody against Plakophilin 2.
Advisors: Drs. Keith Johnson and Margaret Wheelock, University of Toledo
Department: Biology

Plakophilin 2, a member of the armadillo family of proteins, has been found in the desmosomes of cells and in the karyoplasm of cells where the desmosome is absent. The function of plakophilin 2, although unknown, may be similar in nature to the other arm-protein family members (plakoglobin, p120, b-catenin). One such parallel could be drawn from the wnt-signaling pathway, in which Beta-catenin is involved. Recently, a plasmid containing a fusion protein and a portion of plakophilin was transformed into bacteria. Then an attempt was made to isolate plakophilin 2 fusion protein. Isolation of plakophilin 2 failed because the insertion into the vector occurred out of frame. Once the insert is “in frame” with the fusion protein another attempt will be made to isolate plakophilin 2. Subsequently, this protein will then be injected into mice in order to isolate an antibody specific for plakophilin 2. This monoclonal antibody will then be used to further evaluate plakophilin 2.

Andrew J. Hill
Creation of a monoclonal antibody
Advisors: Drs. Keith Johnson and Margaret Wheelock, University of Toledo
Department: Biology

Desmoglein I, a member of the cadherin family of transmembrane cell-cell adhesion molecules, is found in desmosomes. Desmosomes are cell-surface specialization’s that serve as attachment sites for intermediate filaments. Desmoglein I is found in the upper layers of tissue composed of stratified squamous epithelial cells. This makes desmoglein I a great differentiation marker for the upper layers of the skin. Recently, a fusion protein containing a portion of desmoglein I has been expressed in bacteria. This fusion protein will then be injected into mice in an attempt to isolate a monoclonal antibody against desmoglein I. A monoclonal antibody against desmoglein I will be used in further studies of desmoglein I in the skin.

Sujatha Prabhakaran
Chracterization of the N-CAD-M phenotype.
Advisors: Drs. Keith Johnson and Margaret Wheelock, University of Toledo
Department: Biology

Cadherins are transmembrane, cell-cell adhesion molecules that play an important role in development and tumorigenesis. To examine the cadherin cytoplasm domain, a construct consisting of the cytoplasmic domain N-cadherin with a plasma membrane targeting region added was made (N-ca-M). N-cad-M expressing cells had increased levels of B-catenin, decreased levels of E-cadherin, and decreased cell-cell contact resulting in an altered morphology. To further investigation the N-cad-M phenotype, motility assays were performed to determine if expression on N-cad-M, which alters cell-cell adhesion, also alters the motility of A431 cells. The wound filling assay showed that N-cad-M expressing cells had increased motility. To elucidate the role of b-catenin in the N-cad-M phenotype, A431 cells were transfected to express b-catenin with a stabilizing mutation (b-cat-S37F). The stabilized b-catenin is turned over at a slower rate than endogenous b-catenin. It was hoped that the expression of b-cat-S37F would increase the overall content of b-catenin in the cells, thus mimicking the overexpression of b-catenin in N-cad-M cells and possibly inducing the N-cad-M phenotype. The expression of b-cat-S37F did not appear to raise the overall level of b-catenin and appeared to have no effect on the overall phenotype of the cells. To summarize, this study showed that: 1) expression of b-cat-S37F is not sufficient to cause the N-cad-M phenotype, 2) N-cad-M expression increases the motility of A431 cells.

Michelle A. DuPlain
Effects of growing conditions of cauliflower mosain virus on Arabidopsis thaliana
Advisor: Dr. Scott Leisner, University of Toledo
Department: Biology

Since many plant viruses are similar to animal viruses, they provide good model systems for studying host-pathogen interactions. We are studying the En-2 variety of the small plant Arabidopsis thaliana and its interactions with the Cauliflower Mosaic Virus (CaMV). It has been shown that En-2 plants are resistant to most isolates of CaMV. The NY8153 isolate is able to overcome this resistance but this ability is dependent on the conditions under which the virus is propagated. We are investigating this phenomenon to determine if the virus titer or the other factors are responsible for this conditional resistance-breaking ability. At this point, our data indicates that the virus titer is not responsible for the effects of environment on resistance-breakage by CaMV isolate NY8153. We have also found that the developmental stage of En-2 plants at the time of inoculation influences their degree of susceptibility to NY8153. En-2 plants inoculated at the seven-leaf stage were most resistant to NY8153, while plants of later developmental stages were more susceptible. This data illustrates the effectiveness of the host-pathogen system that we are studying and illustrates the implicit roles of plant and viral factors.

Matthew Elkins
Plants aiding drug research: The CaMV/turnip plant model system for drug testing
Advisor: Dr. Scott Leisner, University of Toledo
Department: Biology

The inhibition of reverse transcriptase is one of the major focuses of drug therapies designed to stop the infection of HIV, the virus that causes AIDS. Through the use of the cauliflower mosaic virus/turnip plant system, the effects of drugs on reverse transcriptase can be studied without any risk of human infection. Reverse transcriptase catalyzes genomic recombination and it is this recombination that accounts for some of HIV’s genetic variability. Turnip plants inoculated with two virus strains containing deletions in the viral genome that prevent either strain from being infectious individually, and thus show symptoms of infection only if reverse transcriptase is active; since this allows for recombination of the two chromosomes to form an intact genome. The inhibition of reverse trancriptase’s recombination properties can also be tested by infecting a turnip plant with one viral strain lacking a non-essential gene and another strain containing a fatal deletion and determining the distribution of the virus in the infected plant with a probe for the non-essential gene. The data thus far collected suggest that the addition of RTI1, a reverse transcriptase inhibiting drug, increases the recombination activity of reverse transcriptase. Further research must be done to test the feasibility if this system for future drug testing.

Ramsey Chehab
Crystalization of E. coli manganese superoxide dismutase
Advisor: Dr. Gloria Borgstahl, University of Toledo
Department: Chemistry

X-ray crystallography has been used to study the structure of superoxide dismutases (SODs), an important class of metalloenyzmes that defend the cell against oxygen-mediated free radical damage. Manganese SODs (MnSODs) prevent oxidative stresses by catalyzing the dismutation of toxic superoxide radicals to oxygen and hydrogen peroxide. Damage inflicted by active superoxide radicals has been implicated in many degenerative processes, including cancer and aging. Thus, the role of these MnSODs is crucial, and in-depth study of their structure is not only vital in understanding their important function, but may lead to new therapeutics for treatment of various degenerative processes. SODs have been studied for the last twenty years and as a result, various SODs have been successfully crystallized and analyzed by several prominent crystallographers. For example, Dr. Martha Ludwig, of the University of Michigan has crystallized MnSOD form Thermus thermophilus, while U.T.’s own Dr. Gloria Borgstahl has crystallized human mitochondrial MnSOD. Current attempts are underway to crystallize MnSOD from Escherichia coli, a bacterium from which MnSOD has never been crystallized. Although E. coli MnSOD is readily available and has been extensively studied biochemically, as yet, the crystal structure of this enzyme has not been solved.

Jennifer Kurek
Effects of freezing on soil communities
Advisor: Dr. Deb Neher, University of Toledo
Department: Biology

Our goal was to test a method for establishing a model system for studying the structure and function of soil communities under natural conditions. Freezing has been used to establish a model soil ecosystem for testing bioindicators by eliminating organisms without changing physical and chemical properties. Intact soil cores collected from Oak Openings MetroPark were assigned randomly to one of five treatments in factorial combination and replicated five times. Cores exposed to freezing (quick or slow at 4 C) followed by 0 or 7 day incubation at constant moisture and temperature were compared to cores not frozen or incubated. For each experimental unit, total numbers of bacteria, fungi, actinomycetes and microarthropods were counted. Microbes were enumerated as colony-forming-units per gram of dry soil and microarthropods were identified to taxonomic order. Freezing, whether slow or quick, did not affect the population numbers of bacteria or actinomycetes, but reduced numbers of microarthropods. Abundance of fungi was reduced more by slow than quick freeze and abundances were greatest in soils treated by quick freeze and one week incubation. Freezing and incubation affected chemical properties including percentage of organic matter, soil moisture, ammonium and nitrate, but not soil pH and electrical conductivity. The results will be used to design future experiments that examine mechanisms between soil biota and ecological processes, such as decomposition and nutrient cycling, to aid in interpretation and calibration of indices for monitoring the health of soils.

3rd URS Photos

Student Presenters

Front row: Suzie Prabhakaran, Ramsey Chelab, Jennifer Kurek, Michelle DuPlain, Bhairavi Patel. Back row: Matt Elkins, Spencer Johnson, Kevin York, Andy Hill.

Symposium Winners

First Place: Suzie Prabhakaran; Plenary Lecturer, Dr. John Reeve of Ohio State University; Runners Up; Ramsey Chelab and Michelle DuPlain.

last updated 29 Dec 2005
please contact brenda.leady@utoledo.edu with comments or concerns

Department of Biological Sciences