The University of Toledo Department of Bilogical Sciences

Bowman-Oddy Labs Room 1045 Saturday March 27, 1999 9:00AM- 3:00PM

(click on the presenter name for abstract)

Time	Speaker	Presentation Title
9:00	Dr. Deborah Neher	Welcome
9:10	Plenary Session- Dr. Deborah E. Goldberg, University of Michigan, Community-Level Consequences of Competition in Plants
10:00	Coffee Break
10:15	Cory D. Dunn	Dexamethasone and EGF affect cell adhesion and growth in A431 cells
10:30	Michelle Ariss	A role for p45 in cell-cell interactions
10:45	Milton E. Pommeranz	Effect of neonatal versus adult forlimb removal on the organization of the dorsal column nuclei in rats
11:00	Eric Kirkendall	c-Jun's role in androgen receptor-mediated transactivartion in the context of chromatin
11:15	Valerie A. Kocsis	The effects of host morphology on virus spread
11:30	Jeffrey A. Connell	Influence of soil temperature on development of Phytophthora root rot on tomato
11:45	Mara Hollaway	Effect of feeding schedule on survival of lark sparrow nestlings
12:00	Pizza lunch
1:00	Jaime Haidet	Cauliflower Mosaic Virus isolate, W260, overcomes resistance in Arabodopsis thaliana ecotype Tsu-0 via passive pathway
1:15	Sushant Khandekar	Identifying genes that interact with mua-3 and mua-6, in C. elegans skeletal muscles
1:30	Mike Ariss	The effect of TGFB1 and BFGF on the growth rate of carpal tunnel syndrome cells
1:45	Katherine Smith	Sequencing of the serotonin receptor gene in Ascaris suum: introduction and methodology
2:00	Peter Davies	Sequencing of the serotonin receptor gene in Ascaris suum: data analysis
2:15	Mark A. Schroeder	The effects of various ligands on cyclic AMP levels in body wall muscle of adult parasitic nematode, Ascaris suum
2:30	Erik Wenninger	Effect of river flow manipulation on wolf spider assemblages at three desert riparian sites
2:45	B.A. Qadri	Intracortical and thalamocortical projections in adult rats that sustained neonatal forelimb removal
3;00	Break
3:15	Awards ceremony

Abstract 4th URS

Cory D. Dunn
Dexamethasone and EGF affect cell adhesion and growth In A431 cells
Advisors: Drs. Jani E. Lewis, Keith R. Johnson and Margaret J. Wheelock, University of Toledo
Department: Biology

Cadherins are the transmembrane components of adherens junctions in epithelial cells. Adherens junctions are linked to the actin cytoskeletons via several cytoplasmic proteins. There is a correlation between loss of cadherin or cytoplasmic proteins and the ability of tumor cells to metastasize. A431 cells are squamous epithelial cells that express E- and P- cadherin and overexpress the epidermal growth factor receptor (EGFr). In contrast to most epithelial cell types, EGF causes cell cycle arrest in A431 cells. Treatment of A431 cells with the steroid dexamethasone (DEX) causes the loss of E- and P- cadherin expression. The morphology, cadherin expression, and growth rate of A431 cells were examined during treatment with DEX, EGF, or both, in order to determine if A431 cells downregulate the cadherins at different rates with or without EGF. Preliminary data indicates that A431 cells express decreased levels of cadherin following treatment with DEX and EGF compared to DEX alone or neither. When A43 1D cells were tested with the same chemicals, it was found that these cells, which were derived from the A431 cell line, are resistant to growth arrest caused by EGF. Finally, an A431 clone, 15BR, that down-regulates cadherin expression was selected for a future differential display experiment to compare gene expression in A431 and A431D cells.

Michelle Ariss
A role for p45 in cell-cell interactions
Advisor: Margaret Wheelock, University of Toledo
Department:Biology

The adherens junction is a crucial complex of proteins that functions in cell-cell adhesion. The extracellular domain of an adherens junction involves cadherin calcium-dependent transmembrane glycoproteins that bind homophilically to induce cell adhesion. The intracellular domain of cadherins bind catenin proteins which function to link cadherins to the actin cytoskeleton. One of the most instrumental proteins involved in cell-cell adhesion is alpha catenin, whose primary function is to associate with beta catenin in order to link cadherins to the actin cytoskeleton. In an attempt to reveal alpha catenin binding partners, Jill Nieset, in the Wheelock/Johnson lab, performed the yeast two hybrid experiment and discovered a positive interaction between the carboxy terminus of alpha catenin and a novel protein which we have named p45. This study explores the role of p45 in cell-cell interactions while also attempting to verify an alphacatenin/p45 interaction in vivo. A mouse monoclonal antibody, 10B10, has been generated against a p45 fusion protein and has been used in western blotting and coimmunoprecipitations to determine the molecular weight of p45 as 45kDa. Using Dounce homogenized cell extract, we have shown p45 to be located primarily in the cytosol. Immunofluorescence of p45 confirmed this localization. Presently we are trying to determine if increasing alpha catenin levels will result in an upregulation of p45. We are utilizing an alpha catenin negative breast carcinoma cell line, MDA MB 468, which we plan to transfect with alpha catenin in an attempt to upregulate p45 expression.

Milton E. Pommeranz
Effect of neonatal versus adult forelimb removal on the organization of the dorsal column nuclei in rats
Advisor: Richard D. Lane, Medical College of Ohio
Department: Biology

While the capacity of somatosensory cortex to reorganize as a consequence of peripheral deafferentation is well established, less is understood about the extent and nature of plasticity that occurs in subcortical areas of the somatosensory neuraxis. In this study, neuroanatomical methods were used to examine the effects of neonatal and adult forelimb removal on the organization of the dorsal column nuclei in adult rats. Volumetric measurements of cytochrome oxidase-stained brainstem sections revealed no significant difference in the size of the cuneate nucleus (CN) ipsilateral to the lesion following neonatal or adult forelimb amputation when compared to normals. Horseradish-peroxidase labeling of sciatic nerve afferents showed evidence of an altered projection pattern of intact hindlimb afferents in the brainstem of rats that sustained neonatal forelimb removal. In normal rates, sciatic nerve afferents from the hindlimb were found to project exclusively to the gracile nucleus. However, following neonatal forelimb amputation, sciatic afferents ipsilateral to the lesion were found to form novel connections within the deafferented CN. No such sprouting of sciatic afferents was observed in the CN of rats that had undergone adult forelimb removal. These results indicate that neonatal forelimb removal results in anatomical expansion of the central projection of hindlimb afferents. The observation that no such expansion of hindlimb related afferents occurs following adult forelimb removal suggests that there might be differences in the nature of the sensory plasticity within the forelimb somatosensory neuraxis following peripheral nerve lesions in adult and developing rats.

Eric Kirkendall
c-Jun's role In androgen receptor-mediated transactivation In the context of chromatin
Advisor: Dr. Lirim Shemshidini, University of Toledo
Department: Biology

Androgens such as testosterone play an essential role in male sexual differentiation. These effects are mediated mainly through its specific receptor, the androgen receptor (AR), a ligand-dependent transcription factor that is a member of the nuclear receptor superfamily. A central DNA-binding domain in the AR recognizes specific DNA sequences in the regulatory regions of androgen responsive genes. The mouse mammary tumor virus (MMTV) promoter provides the best characterized example of transcriptional control by androgens. In addition, the promoters of prostate-specific antigen (PSA) and human kallikrein 2 (hKLK2) are inducible by androgens and are diagnostic markers for prostate cancer. The proto-oncoprotein c-Jun is a component of the transcription factor AP-1 and has been shown to mediate AR-induced transactivation without binding to DNA in a sequence-specific manner. c-Jun's positive effect is dose dependent and both exogenously added and endogenously-induced c-Jun can act on AR. Thus far, c-Jun's role in AR-mediated transcription has been elucidated via transient transfection methods. The importance of this role in vivo, that is, c-Jun activity in turning on androgen-responsive genes in the context of chromatin, has yet to be characterized. My project will examine this possible role by generating stable cell lines by transfecting PC-3 (human prostate cancer) cells with the MMTV-CAT (positive control), PSA-CAT, and hKLK2-CAT reporter genes. Subsequent reporter gene activity analysis should then shed light on c-Jun's role in vivo.

Valerie A. Kocsis
The effects of host morphology on virus spread
Advisor: Dr. Scott Leisner, University of Toledo
Department: Biology

The spread of virus through its host is a critical component of disease. To enhance our knowledge of virus transport, the effects of host morphology on pathogen spread will be examined. In order to test these effects, two initial steps had to be performed. The first was to develop a sensitive technique that will allow four cauliflower mosaic virus (CaMV) isolates to be distinguished in a mixture. To accomplish this, four isolates of CaMV were identified by a polymorphism in gene II using polymerase chain reaction (PCR). The four strains tested were CaN, CM1841, Ca365 and CM4-184. Mixtures of the 4 viruses, examined by PCR, showed that three of these viruses were equally amplified. However, CaN was preferentially amplified in each mixture. This was puzzling because all four viruses possessed essentially the same DNA sequences except for the gene II alterations. Therefore, CaN was eliminated from the experiment. The next step was to establish a method of virus extraction from the host plants. This was accomplished by grinding a small amount of plant tissue in buffer and extracting virus DNA for PCR. These methods will be utilized to study the effects of host morphology on virus distribution in Arabidopsis of differing morphology: one group of plants that was left to grow naturally and another that was apically cut in order to promote branching. Plants exhibiting a high degree of branching were expected to contain a more diverse virus population than those that do not.

Jeffrey A. Connell
Influence of soil temperature on development of Phytophthora root rot on tomato
Advisor: Dr. Deborah Neher, University of Toledo
Department: Biology

Phytophthora is known to cause disease on many major food crops, forest trees, subtropical and tropical fruit trees, berries, nut trees, many ornamental plants, and valuable tropical export crops. Most species of Phytophthora cause root rots, damping off of seedlings, and rots of lower stems. Some attack the foliage, young twigs and the fruit. We inoculated processing tomato seedlings with 11 isolates of P. parasitica to test for pathogenicity and chose the five most virulent isolates for our experiment. Our goal was to compare disease symptoms and plant biomass from tomato plants grown at four different soil temperatures (17.5, 24.5, 27.5, and 33.5 C) and three different inoculum concentrations (0, 2.58, and 32.02 colony forming units (cfu) of viable chlamydospores per g of dry soil). The shoot and root symptom severity were quantified for the proportion of plant organ affected plus biomass of shoots and roots were quantified. The disease severity was greater at 32.02 cfu than at 2.58 cfu, and both were greater than those at 0 cfu. Root symptom severity was greater at 33.5 C than at 22.7 C. Shoot symptom severity was similar across all temperatures. We conclude that inoculum level had more effect on symptom severity than did temperature. The effect of temperature on symptoms was linear with the greatest severity occurring at warmer temperatures. Linear regression showed variation of symptoms explained by biomass was 52.6% for roots and 36.4% for shoots.

Mara Hollaway
Effect of feeding schedule on survival of lark sparrow nestlings
Advisor: Elliot Tramer, University of Toledo
Department: Biology

The purpose of this study was to determine whether or not there was a critical age, in terms of feeding frequency, for the survival of lark sparrow nestlings. Evening (6:00 pm to 9:05 pm) feeding activity was also studied to determine whether a peak feeding period existed during this time. Members of an isolated breeding population of lark sparrows at Oak Openings Metropark in Whitehouse, Ohio were observed form late-May through late-June. Data was collected from nine nests starting on the day of hatching or earliest discovery of hatchlings and ending on the day of fledging or earliest discovery of predation. Each nest was observed for a fifty minute period each evening. Unexpected delays in the observation schedule caused the data sets correlating feeding period within feeding frequency to be incomparable, and therefore inconclusive. Due to unexpected predation, early fledging, and early hatching, the number of samples in the data sets comparing feeding frequency increases with the age of the nestlings. This implies that disturbances within the nest territory are more prone to be detrimental to the young sparrows during the latter stages of the nesting period.

Jaime Haidet
Cauliflower Mosaic Virus isolate, W260, overcomes resistance in Arabidopsis thaIiana ecotype Tsu-O via a passive pathway
Advisor: Dr. Scott Leisner, Department of Biology
Department: Biology

Arabidopsis thaliana ecotype Tsu-0 is resistant to cauliflower mosaic virus (CaMV), isolate CM1841, but susceptible to W260. Tsu-0 resistance could be overcome by W260 in two ways: either the virus suppresses defenses or W260 is not recognized by host defense mechanisms. To determine which of these hypotheses are correct, we first needed to develop a method for distinguishing between different CaMV isolates within the same plant. To do this, CaN, a version of CM1841 in which gene II is replaced with the N gene of beet necrotic yellow vein virus, was used. The N gene provided a sequence polymorphism that could be detected by polymerase chain reaction (PCR). Using primers that amplify a region spanning gene II, a PCR program was developed that amplified both CaN and W260. The fragments generated by PCR are approximately 400 base pairs for CaN and approximately 700 base pairs for W260. Interestingly, CaN was preferentially amplified in comparison to W260. The second set of experiments required that virus be isolated from plants. This was done using a virus extraction protocol and PCR. Finally, to determine the mechanism by which W260 overcomes resistance, Tsu-0 plants were inoculated with the two viruses. When mixtures of CaN and W260 were inoculated to Tsu-0 plants, no symptoms were observed. These preliminary data indicate that the ability of W260 to overcome Tsu-0 resistance is passive, i.e., the virus does not activate the host defense pathway. All of these experiments are still in progress.

Sushant Khandekar
Identifying genes that interact with mua-3 and mua-6, in C. elegans skeletal muscles
Advisor: Dr. John Plenefisch, University of Toledo
Department: Biology

In C. elegans, mua-3 and mua-6 are genes required for maintaining attachments between muscles and exoskeleton. Any mutation in these genes causes complete or incomplete paralysis of the worms. Mutants for the rh195 allele of mua-3 gene have bent heads and are sterile at 25C. Mutants for the rh85 allele of mua-6 gene have extensive muscle detachment, which make it difficult for them to move. Mua-3 encodes a presumed matrix-associated receptor and preliminary experiments suggest that mua-6 may encode a transcription factor. I have taken a genetic approach to identify other genes that interact with mua-3 and mua-6 in regulating muscle attachments. Animals mutant for either rh195 or rh85 were exposed to the chemical mutagen EMS (methane sulfonic acid ethyl ester). I screened almost ten thousand F1 and their progeny in mua-3 and isolated one extragenic suppressor mutation. The results reveal that extragenic suppressors of mua-3 are rare. I am currently genetically mapping this mutation. In mua-6, I screened almost one thousand F1 and their progeny, and identified one suppressor. This mutant is a dominant suppressor, but is itself a homozygous lethal. The suppressor gene is presumably essential for other non-muscle developmental processes. I am currently further characterizing this new mutation.

Mike Ariss
The effect of TGFb1 and bFGF on the growth rate of carpal tunnel syndrome cells
Advisor: Dr. Joana Chakraborty, Medical College of Ohio
Department: Biology

During the last few years, the tissue culture lab at the Medical College of Ohio established 27 cell lines of flexor retinaculum from carpal tunnel syndrome patients. In this project, six cell lines from flexor retinaculum tissues of CTS patients and 2 cell lines from the flexor retinaculum tissues of healthy humans were generated. Once the cells were obtained, they were cultured in a defined medium lacking growth factors for a known period and growth curves were established. The growth curves allowed us to compare the growth kinetics of CTS cells in culture to that of normal flexor retinaculum cells. After conducting the controlled experiment, growth factors were added to the medium. The two growth factors used in this experiment were basic fibroblast growth factor (bFGF) and transforming growth factor (TGFb1). Again, growth curves were established for the CTS and normal cells grown in the presence of growth factors, and these two curves were compared to the original curves produced in the absence of growth factors. After comparing the growth curves, we determined that, in the presence of bFGF and TGFb1, CTS cells had a much higher growth rate than the normal cells.

Katherine Smith
Sequencing the serotonin receptor gene in Ascaris suum: Introduction and methodology
Advisors: Drs. Emilo Duran, Patricia Komuniecki, Richard Komuniecki
Department: Biology

Ascaris lumbricoides infects 1.5 billion people through out the world each year. The worms grow up to 15 to 30 centimeters in length and move with in the hosts gut via the contraction of it's body wall muscle. Ascaris suum a closely related nematode lives in swine’s intestines. Nematodes survival is entirely dependant upon the contraction of the muscular pharynx. Feeding depends upon the continual pumping of the pharyngeal muscle, an ideal target for chemotherapy. Serotonin (5-HT) is an important neurotransmitter involved in pharyngeal muscle contraction in Ascaris. The designing of the current project was to gain a better understanding of the receptors involved in serotonin binding. The specific focus of our study was to obtain the length of the gene for the serotonin receptor. The sequencing began by using many techniques the following primer designing, and using polymerase chain reaction, E.coli transfection, cloning, DNA purification, all involved with gaining the receptor DNA fragments needed before the actual sequencing can begin. Through the knowledge of the receptor sequence a better understanding of the serotonin receptor is achieved, and chemotherapy targeting can begin.

Peter Davies
Sequencing the serotonin receptor gene in Ascaris suum: Data analysis
Advisors: Drs. Emilo Duran, Patricia Komuniecki, Richard Komuniecki
Department: Biology

Parasitic helminths cause significant debilitating effects in humans and livestock throughout the world today. Current treatment for parasitic diseases is inadequate. To address this problem points of attack that are unique to the parasite but which do not affect the host must be identified. Body wall muscle metabolism has been well characterized in the parasitic nematode, Ascaris suum. However the same can not be said for the mechanism of pharyngeal pumping, which is an essential muscular action allowing the nematode to feed. Knowledge of the neurotransmitters involved in regulating this continuous muscular pumping needed to be sought to better understand pharyngeal metabolism. Serotonin (5-HT) is one of the neurotransmitters involved in the modulation of pharyngeal pumping however the mode of action of serotonin with its receptor is not yet fully understood. Sequencing of the gene coding for the serotonin receptor was an important step in this project. It involved collecting nematodes from a slaughterhouse to obtain pharyngeal tissue; obtaining DNA from the tissue and amplifying it up to a suitable quantity for sequencing. The use of techniques such as PCR and E. coli transfection were employed to meet these ends. Each stage of the strategy for obtaining the gene sequence had to be analyzed before moving on to the next stage. Further investigation via sequence entry into Blast Research data base, receptor functional expression, ligand binding assays and looking for conserved amino acid sequences will ultimately reveal enough information to suggest points of chemotheraputic attack against the nematode.

Mark A. Schroeder
The effects of various ligands on cyclic AMP levels in body wall muscle of adult parasitic nematode, Ascaris suum
Advisors: Drs. Wei Chen and Richard Komuniecki, University of Toledo
Department: Biology

The adult porcine nematode, Ascaris suum, resides in the host intestine and has been used as a model system for the development of chemotherapy to control a variety of parasitic nematodes of medical and veterinary importance. Most anthelminthics inhibit processes at the neuromuscular junction. Therefore, the present study was initiated to examine the potential roles of a number of physiologically important ligands on second messenger levels in isolated adult A. suum somatic muscle strips. Specifically, changes in cAMPs levels were measured in the presence of serotonin (5-HT), the neuropeptide, AF2 (khelyrfamide), acetylcholine, the muscarinic cholinergic agonist, carbachol, and octopamine. Preliminary studies indicated that AF2 markedly stimulated cAMP levels, while 5-HT, acetylcholine, carbachol and octopamine had no effect when incubated alone with muscle strips. Surprisingly, 5-HT markedly inhibited the AF2-dependent increase in cAMP levels. These results suggest that muscle AF2 receptors are coupled to adenyl cyclase through Gas, while muscle 5-HT receptors are coupled through Gai/o. The identification of potentially novel pathways of signal transduction in somatic muscle could provide novel targets for chemotherapy. These studies are continuing and are focused on more clearly defining the relationship between AF2, 5-HT, and cAMP levels. The results reported in the present study are at odds with those reported in the literature which suggest that 5-HT increased muscle cAMP levels and stimulated glycogen breakdown. The reason for these differences is unclear, but suggest that the effects of 5-HT on signal transduction and metabolism need to be reexamined more extensively.

Erik Wenninger
Effect of river flow manipulation on wolf spider assemblages at three desert riparian sites
Advisor: William F. Fagan, Arizona State University
Department: Biology

The distribution, abundance, and diversity of wolf spider (Lycosidae) assemblages were investigated via pitfall trapping at three sites near Granite Reef Dam outside Phoenix, Arizona. These three sites featured different moisture and temperature regimes due to the dam, which diverts the Salt River into an urban canal system. Site 1 was a natural riparian area above the dam, along the Salt River; Site 2 was adjacent to a man-made diversion canal; and Site 3 was adjacent to the dry riverbed below the dam. Four lycosid species were found at Site 1, with Pardosa vadosa Barnes 1959 highly dominant. Two species each, though very few total individuals, were found at Sites 2 and 3. Simpson's index of diversity (of lycosids and of all other terrestrial arthropods) was higher for Site 1 than for Sites2-3. Prey availability was comparable among sites, but Site 1 had significantly higher relative soil moisture levels and less extreme substrate and air temperature conditions than did Sites 2 and 3. Spider abundance at each site was independent of prey availability, but instead depended chiefly upon moisture and temperature regimes among sites. The results suggest that wolf spiders experienced a significant effect from disturbance of their habitat by the dam, and that abiotic habitat attributes such as moisture and temperature may be more important for wolf spider abundance than prey availability alone in desert riparian systems.

BA. Qadri
Intracortical and thalamocortical projections in adult rats that sustained neonatal forelimb removal
Advisor: A.S. Stojic, R. D. Lane, W. Rhodes, Medical College of Ohio Department: Biology

In our previous paper, we reported the abnormal expression of hindiimb receptive fields in the stump representation of the primary somatosensory cortex (SI) in rats that sustained neonatal forelimb removal when cortical g-aminobutyric acid (GABA) receptors were pharmacologically blocked. In this study, we attempted to identify the substrate for this functional modification. Three potential substrates were examined: 1) changes in intracortical connections within SI; 2) alterations in the projection pattern of thalamocortical afferents from the ventroposterior lateral (VPL) nucleus to SI; and 3) changes in receptive fields of thalamocortical neurons. We used biotinylated dextran amine and Phaseolus vulgaris leucoagglutinin to examine the intracortical projections associated with the stump and hindlimb representation of the SI. Finally, single-unit recordings from VPL neurons were made to examine the functional organization of this nucleus of neonatally amputated adult rats. Tracer studies demonstrated no significant change in the intracortical connections or VPL projections associated with the stump and hindlimb SI in neonatally amputated rats. Recordings from VPL of neonatally manipulated rats revealed a small, but significant, population of cells (19.0%) within the stump representation that had dual stump and hindlimb receptive fields. Thus, the data suggest that the functional reorganization observed in SI of neonatally amputated rats may reflect functional alterations occurring in its thalamic inputs.

4th URS Photos

Student Presenters

Left to right: Jeffrey Connell, Erik Wenniger, Valerie Kocsis, Eric Kirkendall, Michelle Ariss, Mark Schroeder, Katherine Smith, Michael Ariss, Jaime Haidet, Cory Dunn, Sushant Khandekar.

Symposium Winners

Plenary Lecturer, Dr. Deborah Golberg of the University of Michigan, Runners Up: Jaime Haidet, Cory Dunn and Michael Ariss; First Place Winner: Michelle Ariss.

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

Department of Biological Sciences