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Poster Session Biological; Medical 9:00- 10:00am Kerns Chapel.

Board 1 HIGH DIETARY PROTEIN INTAKE INCREASES SERUM ENZYME ACTIVITY FOLLOWING DAMAGING EXERCISE IN HUMANS. Amy L. Miracle,, PritiRane, (Lonnie M. Lowery, Dept of Nutrition, Rm. 100 Nixson Hall, Kent State University, Kent OH 44242.

Rodent data suggest that high protein intakes can significantly elevate serum creatine kinase (CK) and aspartate transaminase (AST) concentrations following damaging exercise. The purpose of the present study was to determine the effects of protein intake on exercise-induced muscle injury (serum enzyme activity) in humans. The hypothesis was that large protein intakes (>1.8g/kg) increased serum enzymes 24 hours after eccentric exercise compared to moderate or low protein intakes. Twenty-six male volunteers completed either a 40-minute downhill run (n=14) or a 12-set resistance exercise protocol (n=12). The exercise protocols were similar in their induction of tissue damage (enzyme release). Based upon 5-day diet logs, participants were stratified by usual protein intake: "low", <1.4g/kg body mass; "med", 1.4-1.8g/kg; and "high", >1.8g/kg. Protein intakes differed significantly among categories but carbohydrate, fat and energy intake did not (p>0.10). Serum values of CK, AST, alanine transaminase (ALT), and lactate dehydrogenase (LDH) were measured prior to and 24 hours after exercise. Significant differences were found between the high (37.0 [+ or -] 8.8) verses med (23.8 [+ or -] 7.8) (p=0 .0027) and low (22.6 [+ or -] 7.3) (p=0.0034) protein intake groups for ALT. Significance was also found in AST values, with high protein intakes (32.6 [+ or -] 7.5) resulting in larger concentrations compared to med (24.3 [+ or -] 6.5) (p=0.064) and low (25.6 [+ or -] 7.5) (p=0.058) protein intakes. Creatine kinase, similarly, was significantly different between the high (754.4 [+ or -] 651.0) and med (287.3 [+ or -] 120.0) protein intake groups (p=0.032). We conclude that humans respond similarly to rots, in that a high level of protein intake increases enzyme efflux following damaging exercise.

Board 2 EFFECTS OF PHENYLPROPANOLAMINE (PPA) ON BEHAVIOR, PROTEIN LEVELS, AND REPRODUCTIVE HORMONES IN YOUNG FEMALE RATS. Andrea M. Dvorak (Beth B. Pritts Dept of Biology, LeMoyne College, 1419 Salt Springs Rd, Syracuse NY 13214.

Phenylpropanolamine (PPA) is a chemical compound found in a number of weight loss products and over the counter cold medicines. This experiment is designed to test the effects of PPA (in the form of Dexatrim[R], an over the counter diet drug) on young female CD IGS rats. The purpose of this project is to determine if PPA has behavioral effects, as well as having effects similar to those associated with starvation, such as protein degradation leading to muscle wasting. This experiment will use sixteen female rats, divided into a control and experimental groups, both of which will be fed ground rat chow, with the experimental rats receiving Dexitrim[R] mixed into their chow for a total of 30 days. After 20 days of PPA exposure, the rats will swim in a Morris water maze (MWM) once a day for a total of ten trials. There have been reports that those on weight loss products feel more nervous and agitated; therefore, PPA exposure could cause a decreased time to locate the podium. In addition, protein levels in the heart, kidney, liver, and muscle will be determined by a Lowry assay. Because weight loss and a decrease in food intake have been reported to alter menstruation, potential effects of PPA on female reproductive hormone levels will be determined using radioimmunoassay. Reproductive cyclicity will be assessed via vaginal lavage.


Tissue damage and infection initiate a stereotypical sequence of host defense reactions called the acute phase response. The hypothesis of this investigation was that immunological factors like WCC (white cell count), neutrophils (Neut), and interleukin-6 (IL-6) following damaging eccentric exercise are related to biochemical stress markers including total urinary nitrogen (TUN), creatine kinase (CK) and lactate dehydrogenase (LDH)-often described in clinical situations. After obtaining pre-exercise urine and blood measurements, 14 male resistance-trained athletes performed six sets of six repetitions, each at 80% of their one repetition maximum strength (1RM). Blood samples were taken at 2 hr post--exercise and every 24 hr thereafter for five days. They were examined for differential WCC, IL-6, CK and LDH while 24-hour urine samples were assessed for TUN. Analysis of data showed no significant relationships between immune variables and stress markers. Relationships however emerged post-exercise. TUN and Neut were correlated (r=0.67, p=0.009; r=0.75, p=0.002; N=14 at 24 and 48 hr respectively. LDH and IL-6 (r=0.83, p=0.003 and r=0.83, p=0.003 at 2 hr and 24 hr respectively; N=10), CK and IL-6 (r=0.85, p=0.03 and r=0.64, p=0.05 at 2 and 24 hr, respectively), and LDH and WCC were correlated (r=0.67, p=0.010 at 24; N=7). All relationships disappeared by 72 hr. Based upon these findings; we conclude common stress markers following eccentric exercise are related to immune function. Future investigations are needed to determine whether immune stimulation is mechanism behind exercise-induced catabolism

Board 4 A MRI COMPARISON OF THE CAUDATE NUCLEI VOLUMES IN DEPRESSED WOMEN AND NORMAL CONTROLS. Tiffany Frankhauser (Cathy L. Pederson, Wittenberg University, Dept of Biology, PO Box 720, Springfield OH 45501.

Recent evidence suggests that the caudate nucleus may play a role in depression as the destruction of the caudate nucleus leads to changes in behavior and problem, solving. Therefore, it is expected that a significant reduction will be observed in the volume of the caudate nuclei in depressed subjects when compared with nondepressed controls. Fifteen right-handed women ages 20 to 40 were categorized into two groups: six subjects suffering from major depression as indicated by elevations above 85 on the Millon Clinical Multiaxial Inventory, and nine nondepressed controls. There was no signigicant difference between the groups based on age, education, IQ, alcohol intake, or smoking habits (p>0.442, n=15). Using 3D Brainstation on magnetic resonance images, three tracings were taken of the left and right caudate nuclei with a mouse-driven cursor every 2mm in transaxial slices. These values were averaged and the results summed to approximate the total volume of the caudate nuclei. The goal was to determine whether there was a significant difference in the volume of the caudate nuclei in women who were depressed compared to nondepressed controls. Preliminary results of the omnibus test from MANOVA show no significant difference in the left or right caudate nucleus between groups [F(2.12)=1.08, p=0.372, [Eta.sup.2]=0.152].

Board 5 THE EFFECT OF OVER-THE-COUNTER DEHYDROEPIANDROSTERONE (DHEA) ON SERUM SODIUM CONCENTRATION IN MICE. Carrie Mills, Arin Fletcher, Emily Richards, (Donald Troike, don Dept of Biology, Wilmington College, 251 Ludovic St, Wilmington OH 45177.

DHEA, a hormone secreted by the adrenal gland, is believed to enhance a wide range of physiological functions. It first appears in humans in about their seventh year of life and peaks around the age of 25 before gradually declining to very low levels by age 70. This has produced a market for the over-the-counter (OTC) sales of this hormone. A previous study demonstrated that purified DHEA (Sigma Chemical) significantly elevated serum sodium concentrations in male mice. Since OTC-DHEA preparations are derived from plant sources, it was of interest to test the effectiveness of an OTC formulation. In this study 3 groups of 6 male mice were used. Each was administered 0.5 ml of one of three solutions by gavage: vegetable oil alone, vegetable oil containing 1 mg of purified DHEA (Sigma), or vegetable oil containing 1 mg of OTC-DHEA (General Nutrition Corp). Eight hours later tail vein blood was collected from each mouse and the sera separated by centrifugation. Blood samples were also collected prior to gavage. All serum samples were diluted 1/2500 with deionized water and their Na concentrations determined with a Perkins Elmer Atomic Absorption Spectrometer following directions in its procedure manual. Preliminary results at this time indicate that the OTC-DHEA does not elevate serum Na concentrations, unlike the purified product. If this holds true in subsequent experiments, then we question the labeling and effectiveness of OTC-DHEA products.

Board 6 HIPPOCAMPAL VOLUMES AND MEMORY CAPABILITIES IN WOMEN WITH POSTTRAUMATIC STRESS DISORDER SECONDARY TO CHILDHOOD ABUSE. Megan A. Hoffmann, Christina M. Peters, Kelly A. Zander. (Cathy Pederson Wittenberg University, Dept of Biology, PO Box 720, Springfield OH 45501.

Abuse during childhood has been shown to induce Posttraumatic Stress Disorder (PTSD) in some trauma survivors. In this study, right handed women ages 20 to 40 were categorized into three groups: PTSD and abuse group, abuse only group, and normal controls (n=33,11 per group). Dividing the subjects in this way allowed for differentiation between abuse and PTS D effects. Placing subjects into triads, one from each group, compensated for possible variation in age, education, IQ, body mass index, alcohol intake, and nicotine habits (p>.07). After an initial phone questionnaire, subjects answered demographic questions and took the Childhood Trauma Questionnaire, the Trauma Symptoms Inventory, and the Million Multiaxial Clinical Inventory. Subjects were given the Weschler Memory Scales (WMS), were interviewed to determined PTSD status, and had an MRI brain scan taken. Because the hippocampus is the center of learning and memory in the brain, itemized WMS scores were statistically compared using one-way ANOVA testing to assess differences among the three groups. There was no significant difference in the initial omnibus test between groups, covarying for IQ, for WMS age-adjusted subscores of auditory immediate memory, auditory delayed memory, visual immediate memory, visual delayed memory, nor working memory (p=0.445, F(10,50)=1.01, [Eta.sup.2]=0.069). Bilateral hippocampi were traced by a researcher blind to group status. Hippocampal tracings in sagittal slices in which the hippocampi appears is currently in progress.

Board 7 IS GLUCOCORTICOID EXCITOTOXICITY RESPONSIBLE FOR THE DETERIORATION OF THE HIPPOCAMPUS IN POSTTRAUMATIC STRESS DISORDER? Christina M. Peters Kelly A. Zander, (Cathy L. Pederson, Wittenberg University, Dept of Biology, PO Box 720, Springfield OH 45501.

Posttraumatic stress disorder (PTSD) is a mental affliction resulting from exposure to a trauma, such as childhood abuse. The hippocampus contains a high density of glucocorticoid receptors, and it is thought that increased levels of glucocorticoids in PTSD cause excitotoxicity of the hippocampus. If that is the case, other areas of the brain with a high density of glucocorticoid receptors, such as the hypothalamus, should also atrophy in women with PTSD. Twenty-seven right-handed women aged 20-40 who had a history of childhood abuse were divided into three groups: PTSD, abuse only, and normal controls. Each woman received a magnetic resonance image of her brain. A researcher blind to group assignment traced both the right and left hippocampus and hypothalamus. No statistically significant difference was found among the three groups in age, years of education, IQ, body mass index, drinks per year, or pack years of cigarette smoking (p>0.15). AMANOVA omnibus test revealed a significant difference in left and right hippocampal volume (p=0.02, F(2,24)=4.69, [Eta.sup.2]=0.28). While no statistically significant difference was found in the volume of the right hippocampi (p=0.78) between PTSD and normal controls, the left hippocampi were significantly smaller in women with PTSD when compared to normal controls (p=0.02). In contrast, an omnibus test revealed no significant difference between left and right hypothalamic volumes (p=0.64, F(2,24)=0.46, [Eta.sup.2]=0.12) for the three groups. These results indicate that another mechanism may be acting in synergy with glucocorticoid excitotoxicity to cause hippocampal, but not hypothalamic, atrophy.

Board 8 THE TOXICITY OF ALCOHOL ON THE CEREBELLAR VERMIS. Emily L. Kingsley, Megan A. Mehicic, Sarah M. Charles, (Cathy L. Pederson, Wittenberg University, Dept of Biology, PO Box 720, Springfield OH45501-0720.

In examining the adverse effects of ethanol on the neurological function of the brain, our study sought to ascertain the extent of cerebellar vermis deterioration in women who are moderate alcohol drinkers. Damage to this region leads to loss of Purkinje cells which are critical for relaying neuronal messages concerning movement stability. The subjects were eleven right-handed women between the ages of 20 and 36 years who exhibited no significant difference in age, years of education, body mass index, alcohol dependency, pack years of cigarette smoking, drug use, anxiety, depression, and histrionics (p [greater than or equal to] 0.06). The size of the vermis for each subject was determined by averaging the tracing of approximately 25 horizontal slices viewed on magnetic resonance imaging (MRI) using 3D Brainstation[TM]. For each slice in which the vermis was fragmented, the average number of fragments per slice was calculated. The control group included six subjects with an alcohol intake of five drinks or fewer per year(mean=0.54 drinks/year). The experimental group consisted of five women who drank between 120 and 312 drinks per year (mean=200.8 drinks/year). The results of the study indicated no significant difference (p=0.59) in the volume of the cerebellar vermis between the two groups. There was also no statistical variance in the fragmentation of the vermis between the groups (p=0.226). This study demonstrates that minimal damage is seen in moderate alcohol drinkers when compared with nondrinkers.

Board 9 ALLERGENS IN HOUSE DUST MITE EGGS. Ndate Fall, Larry G. Arlian, Marjorie S. Morgan, and R. Jeff. Schumann, Wright State University, Dept of Biological Sciences, Dayton OH 45435.

House dust mites are small arachnids that are prevalent in carpets, sofas, mattresses and bedding in homes in humid climates worldwide. They are the source of multiple potent allergens that trigger allergic reactions in humans and dogs predisposed to allergies. At least 14 groups of allergens have been isolated and characterized; however, there are still others that remain to be characterized. Some of these characterized allergens are associated with digestive enzymes and other internal body proteins, such as tropomyosin. It has been assumed that most of these allergenic proteins are associated with the active life stages of the mites. The purpose of our study was to determine if dust mite eggs are also a source of allergenic proteins. Fresh eggs (more than 1000 and less than 24 hrs old) were collected from confined females, then aqueous extracts were prepared from the eggs. Presence of allergenic products in these extracts was then determined by Western blotting. Blotting was done using serum from dust mite sensitive patients. SDS-PAGE showed that eggs contained multiple soluble proteins with molecular weights between 10 to 230 kDa Antisera from rabbits immunized with whole body mite extract contained antibody directed at some of these egg proteins. When these SDS-PAGE resolve egg proteins were incubated with sera from dust mite sensitive-patients and subjected to autoradiography, several IgE binding proteins were evident. Therefore, house dust mite eggs are a source of allergenic proteins.

Board 10 ASSESSING DIABETES RESISTANCE OF NOD.[Ea.sup.d] TRANSGENIC MICE BY ADOPTIVE TRANSFER OF DIABETOGENIC T-CELL CLONES. Samantha A. Smith, (Matthew S. Hanson, Wittenberg University, Ward Street at N. Wittenberg Ave., PO Box 720, Springfield OH 45501-0720.

Thenon-obese diabetic (NOD) mouse strain serves as an animal model for human Type I diabetes. The most important genetic factors contributing to diabetes susceptibility in humans and NOD mice are the class I and II genes of the major histocompatibility complex (MHC). MHC class I (k and d) and II (I-A and I-E) molecules facilitate the maturation of T lymphocytes that will react against pathogens while also being tolerant of self-molecules. NOD mice express a unique I-[A.sup.g7] molecule, but do not express I-E molecules. Diabetes in NOD mice can be prevented by transgenic introduction of I-E alpha genes (NOD-[Ea.sup.d]), which restores I-E expression. The exact mechanisms by which I-E expression prevents diabetes in NOD mice are unknown. The purpose of our experiments is to determine if diabetes-resistant NOD.[Ea.sup.d] mice are capable of suppressing a highly diabetogenic T cell clone. Five to ten day old NOD.[Ea.sup.d] mice were injected with saline (n=3) or 5-10 * [10.sup.6] diabetogenic T cells (n=9) and followed for diabetes development. Diabetes was diagnosed by assaying for the presence of glucose in the urine. Histological examination of pancreata from recipient mice was used to confirm the destruction of insulin-producing islet beta cells. By 60 days post transfer, 5 of 9 recipients of the T cell clone became overtly diabetic and none of the control saline recipients became diabetic. These results demonstrate that the I-E mediated protective mechanisms, which prevent diabetes in the NOD.[Ea.sup.d] mice, are unable to inhibit the diabetogenicity of a T cell clone when injected during the neonatal period.

Board 11 ACCURACY OF COMBINED DIFFUSE REFLECTANCE AND INTRINSIC FLUORESCENCE IN IDENTIFYING CORONARY ATHEROSCLEROSIS. George O. Angheloiu (1), Joseph T. Arendt, (1) Sweder W. E. van de Poll (1), Markus G. Muelle, (3) Abigail Haka (3), Irene Georgakoudi (3), Barry Kuban (1), Jonathan Myles (1), Maryann Fitzmaurice (4), Michael S. Feld (3), John R. Kramer (1), (1) The Cleveland Clinic Foundation- ND 20, 9500 Euclid Ave., Cleveland OH 44195,(2) Leiden University Medical Center, Postbus 9600, 2300 RC Leiden, The Netherlands, (3) Massachusetts Institute of Technology, 77 Massachusetts Ave., Cambridge MA 02139, (4) Case Western Reserve University, 2085 Adelbert Rd., Cleveland OH 44106.

An intrinsic fluorescence (IF) and diffuse reflectance (DR) based algorithm was designed for coronary atherosclerosis diagnosis of in-vitro tissue. In contrast to fluorescence, IF spectra are free from distortions introduced by tissue scattering and absorption. White light DR and fluorescence emission spectra generated at 11 laser excitation wavelengths ([[lambda].sub.x]) were collected from heart transplant and autopsy cases with an original instrument called FastEEM. IF spectra were extracted by combining DR and fluorescence spectra using a photon migration model. IF spectra were fit to a linear combination of collagen and elastin spectra at [[lambda].sub.x]=342 nm, and of collagen and component C at [[lambda].sub.x]=480 nm. C spectrum was derived from multivariate curve resolution analysis of IF and related to that of ceroid, a lipid oxidation product in atherosclerotic lesions. We calculated the contributions of collagen and elastin to IF at [[lambda].sub.x]=342 and of C at [[lambda].sub.x]=480 nm, and also the contribution of beta-carotene absorption to DR. A coronary atherosclerosis diagnostic algorithm was derived. Specificity, sensitivity and validity were verified by leave-one out cross-validation. Coronary segments (n=110) were studied: 22 normal and intimal fibroplasia and 88 non-calcified and calcified atherosclerotic/atheromatous plaques. An algorithm using percentage of collagen contribution to IF at [[lambda].sub.x]=342 nm, contribution of C to IF at [[lambda].sub.x]=480 nm and that of beta-carotene to DR had sensitivity 95%, specificity 91% and PPV 98%. We demonstrated that fundamental parameters extracted from spectral data can accurately diagnose atherosclerotic lesions using features similar to those used by pathologists.

Board 12 CARDIAC FUNCTION AS MEASURED BY CREATININE CLEARANCES FOLLOWING PERATIVE CORRECTION OF CONGENITAL HEART DEFECTS. Suzanne M. Eggleston, Dr. Nancy Woodley, Ohio Northern University, 214 South Johnson Street, Ada OH 45810 and Dr. A. Marc Harrison, Cleveland Clinic Foundation.

Creatinine is a waste product produced by the body that is normally filtered and completely cleared from the blood by the kidneys. Since creatinine is not reabsorbed or secreted by the kidney, it gives a good indication of the glomerular filtration rate (GFR), which is the rate at which the kidneys filter out waste products. Furthermore, GFR is directly related to heart function because, in order for the kidneys to function properly, they must receive blood from the heart. Creatinine clearance (CrCl) may be used to indicate cardiac function following post-operative correction of congenital heart defects; heart malformations that are present at birth. In this study, post-operative CrCl and thereby cardiac function of neonates with hypoplastic left heart syndrome (HLHS) (n=4) and transposition of the great arteries (TGA) (n=5) was compared to determine if restorative function of the two defects were equivalent. ANOVA conducted at a 95 confidence level were used on data prospectively collected as part of an unrelated study. There was no significant difference between the CrCl of patients with HLHS versus those with TGA. However, the CrCl of HLHS and TGA patients was significantly reduced when compared to the normative value of 39mL/min/([m.sup.2]). This was expected due to the insufficiency of blood flow caused by HLHS and TGA.


In the present study, we examined the effects of an antisense (AS) oligonucleotide (ODN) targeted at monoamine oxidase-B (MAO-B) on the acute release of dopamine following treatment with 3,4-methylenedioxymethamphetamine (MDMA, ecstasy). This was done in order to determine if AS knockdown of MAO-B has an effect on acute dopamine release and/or turnover as assessed by changes in 3,4-dihydroxyphenylacetic acid (DOPAC) levels following treatment with MDMA. Seven Sprague-Dawley rats were surgically implanted with microdialysis cannulae into the caudate putamen; three of these animals were also surgically implanted with an osmotic minipump. The minipump administered the AS ODN to MAO-B at constant rate of 0.5 [micro]L/hr for seven days, yielding a total daily dosage of 600 [rho]mols/day. At the end of the seven-day treatment all of the rats were treated with MDMA (10 mg/kg, sc) and the cerebral dialysate was collected using microdialysis. Samples were collected at 20-minute intervals for a total collection period of 180 minutes. These samples were then analyzed via High Pressure Liquid Chromatography with electrochemical detection. The amounts of dopamine and DOPAC were recorded as a percent of baseline levels. The results showed no change in the levels of acute dopamine in the rats treated with AS ODN to MAO-B as compared with those treated only with the MDMA corresponding with the hypothesis that it does not alter acute dopamine release. A significant (p<0.05) decrease in DOPAC levels was seen in the AS/MDMA treatment group indicating that the AS had, in fact, resulted in a knockdown of MAO-B.


This project seeks to use scanning electron microscopy (SEM) to determine whether damage occurs to Bausch and Lomb PureVision[TM] contact lenses while using commercially available cleaning/disinfecting solutions. PureVision[TM] contact lenses are made from balafilcon A, a silicon hydrogel material containing 36% water. The silicon is made hydrophilic by the Performa[TM] process that provides a build-up resistant surface. PureVision[TM] contact lenses are designed for 30-day extended wear and are currently prescribed for 1 to 7 day continuous wear. Six different types of solutions were used including MiraFlow[R] (CIBA Vision[R]), AOSept[R] (CIBA Vision[R]), UltraCare[R] (Allergan), Opti-Free[R] (Alcon), Opti-Free Express[R] No Rub, and ReNu MultiPlus[R] Multi-Purpose Solution (Bausch & Lomb). Sterile saline was used to rinse the lenses. In a blind study, seven contact lenses were cleaned as directed with each solution. The surface details were observed using SEM with cryogenic preparation. Images were taken at seven-day intervals over a 7-week period. Preliminary results show that the SEM images of those contact lenses viewed at the beginning of the study have little buildup and damage while those viewed after several weeks of the study have more build-up and some pitting associated with both anterior surface and internal composition. With the exception of one solution used, all other solutions showed similar results on the contact lenses. This exception showed considerable build-up, which may have covered the possible damage to the contact lens.

Board 15 STUDIES ON WHITE LEAF SPOT OF WALNUT INCITED BY Microstroma juglandis. David L. Mason, Dept of Biology, Wittenberg University, Springfield OH 45501.

The objective of this study was to investigate the host parasite relations of the white leaf spot disease of Juglans nigra incited by the fungus, Microstroma juglandis. Infections revealed white, powdery, spore-bearing lesions 0.2-1 cm on the lower side of the leaves. The corresponding upper side of the leaf showed the lesions to be slightly bulbous and chlorotic. Histology by means of light and transmission electron (TEM) microscopy clearly revealed the fungal hyphae located between the mesophyll cells which are seen to be hyperplastic and to contain a reduced number of non starch-containing chloroplasts as compared to cells of a non infected leaf. No septal pores or Woronin bodies were detected between the fungal cells, and no direct or haustorial invasion of the host cells by the fungus were ever observed. Somewhat parallel mycelial accumulations beneath stomata were seen extending to form clusters of "basidium-like", spore-bearing, conidiophores that protrude through the opening of stomata. These reproductive structures were viewed approximately five times by means of light, TEM, and scanning electron microscopy (SEM). Spores of the fungus were cultured on various media, including: potato dextrose (PDA), corn meal (CM), and honey peptone (HP). On the three types of media, the spores produced yeast-like blastospores that continued to germinate, forming whitish, yeast-like colonies. No mycelial growth was observed. Fragments of sterilized leaves and ultarfiltrates of homogenized leaf materials added to the various media in conjunction with various temperature variations did not induce the spores to form mycelium. A number of attempts were made to infect leaves from spores produced on the host and from those grown in culture, but no infections occurred.

Board 16 STUDIES ON THE LEAF SPOT OF IRIS INCITED BY Heterosporium iridis. David L. Mason, Dept of Biology, Wittenberg University, Springfield OH 45501.

The objective of this study was to investigate the host parasite relations of the leaf spot of Iris incited by the Deuteromycete, Heterosporium iridis. Leaf infections revealed elongated, reddish-brown, necrotic, lesions surrounded by a yellowish, chlorotic ring of tissue. By means of stereoscopic microscopy, dark, spore-bearing conidiophores were seen extending out of stomata in the necrotic regions. Scanning electron microscopy (SEM) revealed early spore formation and mature 3-5 celled spores covered with spinules on the branching conidiophores. Spores both from nature and from culture transferred to uninfected leaves were seen by SEM to form hyphae that directly penetrate stomata. Some hyphae were also seen forming what appear to be an appressorium. Histology by means of light microscopy revealed that the epidermal and mesophyll cells in the presence of invading fungal hyphae undergo a rapid necrosis. Within this region of dead cells and at their margins only a few segments of hyphae could be detected. Spores of the pathogen were removed from infected leaves and grown in culture on corn meal (CM), potato-dextrose (PDA) and honey peptone (HP) media. Early spore germination, followed in culture by light microscopy, revealed that each cell of the four to five-cell spores produced branching septate mycelium. Within four days following germination, dark, branching, conidiophores bearing elongated, dark, four to five-cells spores could be seen. SEM on cultures clearly revealed the structure of the conidiophores and spinule-covered spores.


The ability to manipulate gene sequences by site-directed mutagenesis is a powerful technique derived from molecular biology. It is possible to create specific amino acid mutations at the DNA level, then characterize these effects at the level of the protein. The use of PCR (polymerase chain reaction) to create these mutations requires the presence of novel restriction enzyme sites to allow for the cloning of these mutant fragments into the wild type gene sequence. The objective of this project is to create a novel EcoRV restriction site in the gene sequence of the lactose permease of Escherichia coli. There are only eleven novel restriction sites currently identified in this gene sequence, with no novel sites in the location of transmembrane domain two, making additional study in this region of the protein difficult. The addition of this new site will allow site directed mutagenesis to be further pursued in the first two transmembrane domains of this protein, opening new questions about the structure and function of this protein. This mutation was created by using downstream flanking restriction sites that incorporate a mutant primer for the novel EcoRV site at the desired location. This PCR fragment was subcloned into a commercially available PCR cloning vector. In a series of several trials at each step, the fragment was restriction digested, isolated and purified using Gene Clean[TM], and then ligated into the plasmid pACYC184. The completion of this project will involve the final cloning of the mutant fragment into the wild type gene in plasmid pQE30-LacY C 148 S. The success of this cloning will be verified by restriction enzyme analysis of the new fragment.

Board 18 PURIFICATION AND CHARACTERIZATION OF STAPHYLOCOCCUS AUREUSSEROTYPE 8 CAPSULAR POLYSACCHARIDE. Elena Bocola-Mavar (1), Diana L. Fagan (1), Jeffrey A. Smiley (2), (1) Youngstown State University, Dept of Biological Sciences and(2) Dept of Chemistry, Youngstown OH 44555.

Infections caused by Staphylococcus aureus remain the number one cause of hospital-acquired infections. It has been shown that the immune response in the host includes forming antibodies against the bacterial capsular polysaccharide (CP). Bacteria containing type 8 CP have been isolated from 44% of the patients with nosocomial infections. The goal of this study is to obtain a pure cell wall carbohydrate of S. aureus expressing type 8 CP. Sodium chloride (2%) supplemented Columbia Broth was used to grow 8 liters of bacteria. After lysing the cells with lysostaphin, and removing nucleic acids with DNAse and RNAse, the CP was separated from other cell components by DEAE Sephacel chromatography. The presence of CP was detected using a Red Tetrazolium test for reducing sugars, as well as the absorbance at 213 nm. Teichoic acid contamination was determined by the microdeterminafion of phosphorus. The ion-exchange column showed the presence of three peaks. Two tested positive for CP, one was eliminated because of teichoic acid contamination. CP was broken down into oligosaccharides with 70% HF. HF solvolysis will allow for spectrometric analysis as a method of identifying the carbohydrate. Purified carbohydrate will be used to develop monoclonal antibodies against type 8 CP.

Board 19 PREPARATION OF TAQ POLYMERASE AND ITS USAGE IN THE PCR DETECTION OF LYME CAUSING PARASITE, Borrelia burgdorferi, IN HARD TICK, Ixodes ricinus. Supriya.S.Pai, (Dr. Marten Edwards, Ohio Wesleyan University, Dept of Zoology, Delaware OH 43015.

Ixodes ricinus ticks are the primary vectors of Lyme Borreliosis in Central Europe, Czech Republic being one of the many affected nations. Clinical manifestations of Lyme Borreliosis include a characteristic rash (erythemia migrans) and flu symptoms. More severe infections may result in facial palsy, cranial nerve lesions, arthritis, mild encephalitis and heart conditions. Borrelia burgdorferi, the causative agent of Lyme Borreliosis, is a gram-negative spirochete that enters the human host during the blood feeding of ticks. The goal of this research project was to study the rate of Borrelia burgdorferi infection amongst the Ixodes ricinus ticks. Borrelia burgdorferi infection was detected in Ixodes ricinus females that were collected in the region of Ceske Budejovice, Czech Republic. Bacterial DNA isolated using the GFX column kit was amplified using the Polymerase Chain Reaction (PCR). DNA was isolated from wild Ixodes ricinus adults and used as a template in PCR reactions that used primers specific for the Borrelia burgdorferi DNA sequences. In order to perform the PCR reactions, recombinant Taq DNA Polymerase was purified from a bacterial expression vector. The activity of the recombinant polymerase was verified using purified samples of Borrelia burgdorferi DNA. This DNA was isolated and purified from cultured Borrelia burgdorferi. Serial dilutions of the DNA sample were used to determine optimum Taq Polymerase activity level. PCR amplified products were loaded onto agarose gels, and 307 bp bands were observed. These bands detected the Borrelia burgdorferi DNA and thus the presence of Borrelia. Out of the 700 ticks analyzed in 35 trials, 245 ticks were found to be infected with Borrelia burgdorferi.

Board 20 CONSTRUCTION AND TESTING OF A PARTICLE INFLOW GUN FOR THE TRANSFORMATION OF PARAMECIUM. Dean Fraga, Erica Keenan, and Whit Schofield, College of Wooster, Biology Dept, Wooster OH 44691.

We have successfully built a particle inflow gun (commonly called a PIG) and used it to transform Paramecium using tungsten and gold particles (1 um in diameter). PIGs represent an economical way to use bioballistics to transform a variety of organisms. We describe how to construct such a gun, its basic principles of operation and provide a summary of our success using both tungsten and gold particles to transform Paramecium with linear and circular pP XV-NEO, a plasmid that can confer paromomycin resistance to Paramecium. Our basic methodology is as previously described in which autogamous cells are collected by centrifugation and resuspended in a simple buffer. The cells are placed in a vacuum chamber beneath a swinge filter containing DNA that has been Ca[Cl.sub.2]-spermidine precipitated onto tungsten or gold particles. A 50 msec blast of pressurized helium gas (80 psi) is used to fire the particles into the Paramecium cells contained in a chamber under vacuum. The cells are removed and after a period of recovery (2-3 days) in culture media, 50 ug/mL paromomycin is added and cells are scored for survival after two days. We found tungsten to be as effective as gold in achieving transformation (0.7 [+ or -] 0.9% vs. 1.6 [+ or -] 1 .7%, respectively, N=3). Circular and linear DNA gave comparable transformation efficiencies (1.64 [+ or -] 1.7% vs 0.53 [+ or -] 0.98%, respectively, N=3). Additional experiments demonstrated that tungsten did not significantly nick the DNA under our conditions when compared to gold treated DNA.

Board 21 CLONING OF GM3 SYNTHASE GENE UNDER THE CONTROL OF DOXYCYCLINERESPONSIVEPROMOTER. Matsha Y. Bratzel, Hany E. Saqr, and Allan J. Yates, Ohio State University, Division of Neuropathology, Room 4166 Graves Hall, 333 W. 10th Ave., Columbus OH 43210.

Gangliosides are a family of sialic acid containing glycolipids that are present in most mammalian cells and highly concentrated in neuronal membranes. Although the cellular functions of gangliosides are not fully understood, studies have shown that changes in ganglioside composition have been correlated with brain tumor grade. GM3 is the simplest ganglioside from which complicated gangliosides are synthesized. It contains two fatty acids, one sialic acid and a disaccharide, and is synthesized by GM3 synthase enzyme. To study the effect of controlled overexpression of GM3 in glioma cells, we cloned GM3 synthase in PREV-TRE vector. This vector has a promoter that is activated only in the presence of tetracycline or tetracycline analogues (e.g. Doxycycline). We digested PREV-TRE with SalI restriction enzyme, then ligated the GM3 synthase gene digested with XhoI to the vector. After ligation, we transformed the cDNA into competent XL 10 E. coli cells and spread the cells onto an ampicillin resistant p late over night at 37U Celsius. We picked all the colonies and began a series of DNA minipreps. DNA from these minipreps were separated using electrophoresis on a 0.7% agarose gel. Potential clones were digested with SspI and XhoI to determine the direction of the inserted gene. Out of eleven clones retrieved, two clones (pMB 101 and pMB 102) contained the GM3 synthase gene in correct orientation. These clones will be used to transfect the GM3 synthase gene into cultured glioma cells. These tranfected cells are essential to study the effects of endogenously synthesized GM3 ongliomabiology.

Board 22 ISOLATION AND CHARACTERIZATION OF FEATHER-DEGRADING AND CELLULOSE-HYDROLYSING STREPTOMYCES. Roshni K. Nuggehalli, Jann M. Ichida, Ohio Wesleyan University, Botany/Microbiology Dept, Delaware OH 43015.

Streptomyces, common soil bacteria that occur on the plumage of about 64% of wild birds, especially bark-probers, are known to degrade feathers. This experiment focused on isolation and characterization of Streptomyces spp. using both selective and differential agar and basal media to determine the most effective techniques. Of 13 strains tested, athermotolerant Streptomyces spp. OWU 1455, isolated from a Downy Woodpecker Dryobates pubescens), was found to degrade feathers in vitro from 28-40 [degrees] C. This strain was most compatible with the keratinase-producing Bacillus licheniformis OWU 1004B when tested by the cross-streak method on Mueller-Hinton agar plates to check for inhibition of B. licheniformis due to antibiotic production by the 13 Streptomyces spp. OWU 1455 and OWU 1004B were used as a dual inoculation for composting feather waste in five 4-L bioreaction vessels, while five control bioreactors were left uninoculated. Many antibiotics are produced as secondary metabolites of Streptomyees species. Pathogenic bacteria isolated from chicken feather waste were inhibited by Streptomyces 1455 as shown by the cross-streak plating method. Of special interest was a multiply antibiotic-resistant strain of Salmonella enterica serovar Enteritidis because antibiotic resistant bacterial infections have been traced to the use of broad-spectrum antibiotics in livestock and poultry feed. Since many Streptomyces spp. are widely used in applied and industrial microbiology, carbon (e.g. several sugars and cellulose) utilization tests were done for additional characterization. Strain 1455 hydrolyzed cellulose especially well with the addition of cellobiose. Because poultry and livestock waste contains large amounts of straw, sawdust and wood chips, thermotolerant bacteria that produce keratinase and cellulase would be of great value in enhancing the composting process.


The lactose permease functions as a symport protein in the inner membrane of Escherichia coli. Working models of the protein transmembrane domains in the membrane are based on hydropathicity plots that identify hydrophobic and hydrophilic regions of the protein and by comparison to other known membrane proteins. We are interested in determining the location of the amino acid boundary in the membrane for one of the periplasmic loops by the use of a membrane impermeable sulfhydryl specific fluorescent probe, Oregon-Green Maleimide. The objective of this project is to create cysteine residue changes in the first periplasmic loop and test the membrane/protein boundary using the fluorescent probe. The three-cysteine mutations were created by PCR based site-directed mutagenesis. The mutant fragments were isolated by restriction enzyme digest, purified by using the Gene Clean[TM] protocol and ligated into an intermediate plasmid vector pACYC 184. The fragments will then be cloned into the wild type gene sequence found in pQE30-LacYC 148S. The sequence will be verified by automated sequencing. The protein will be expressed and purified using Qiagen[TM] nickel columns and tested by fluorescence analysis of SDSPAGE gels. We expect to identify the amino acids located at the membrane boundary of periplasmic loop 1-2 based on the presence or absence of fluorescence in protein preparations.

Board 24 TOTAL BACTERIAL AND COLIFORM LEVELS AT THE OLENTANGY RIVER WETLANDS RESEARCH PARK. Terry D. Hinds. Jr., Judith L. Gardner, Rachel Brown, Hui Suk Jones, and Eugene H. Burns, Jr., Shawnee State University, Dept of Natural Sciences, 940 Second St., Portsmouth OH 45662.

The Olentangy River Wetlands Research Park, Columbus OH, contains one man-made wetland area (wetland 1) that was planted with native plants during construction in 1994 and one (wetland 2) that was not planted. Although much other research has been performed, the bacterial population has not been studied previously. The purpose of this study was to determine total bacterial number in both wetlands and to catalog the effect of water flow through the wetlands on coliform levels. One sample from the river, five samples from five sites within each wetland, and one sample from the swale were taken monthly from October 2000 to August 2001 (except months when the wetlands were frozen). Samples were used for standard plate counts on trypticase soy agar and coliform counts using phenol red lactose broth and eosin methylene blue agar. Samples were subjected to presumptive, confirmed, and completed tests in the multiple tube technique with most probable number analysis. Total bacterial numbers varied from month to month but showed a mean 73.63 % [+ or -] 29.4% decrease in colony forming units (cfu) from the inlet of wetland 1 to its outlet. Wetland 2 increased 50.85% [+ or -] 114.4% in total bacterial numbers in cfu. These differences are not statistically significant (P=0.1336). Coliform concentrations (coliforms/100ml) decreased from 44.4% to 93.9% per month as water traveled from the inlet to the outlet. From seven observations, the mean decrease in coliform concentration (coliforms/100 ml) was 80.04% [+ or -] 17.9% in wetland 1 and 72.12% [+ or -] 17.0% in wetland 2. These differences are not statistically significant (P=0.2801). These data suggest that both wetland areas reduce levels of coliforms as water flows through them, although total bacterial numbers may not decrease.

Board 26 SURVEY OF THE COLEOPTERA AT THE RAVENNA TRAINING AND LOGISTICS SITE. Roger Williams, and Diane Hartzler, Dept of Entomology, OARDC, Ohio State University, Wooster OH 44691.

We are participating in a long-term study of the biological biodiversity at the Ravenna Training and Logistics Site (RTLS). The 2000 growing season was the second year we conducted surveys of the beetles in the RTLS. We utilized experimental traps and attractants from several sources in addition to homemade terrestrial traps, aquatic nets, beating sheets, and hand collecting in various habitats. Counts were made of the numbers caught, and the location of each was recorded. Labels with all pertinent data were placed with specimens and determination to taxon on a separate label. Most determinations were to species by specialists of that group. Abundance ratings were used to give an estimate of population size of species. Several species of beetles were encountered in Ohio for the first time. The combined data from 1999 and 2000 collecting seasons resulted in 682 species, 75 families, and 13,638 specimens represented. In addition to new state records we also have new county records, plus a few rare and unusual specimens which should stimulate interest and prompt further study. This study was administered under the direction of the Division of Natural Areas and Preserves of the Ohio Dept of Natural Resources with the funding supplied by the Ohio Army National Guard.

Board 27 SYNTHETIC PREPARATION OF A POTENT TICK ATTRACTANT FOR TRAP BAITS. William J. Burke, Amanda E. Johnson, Jay A. Yoder, and Peter E. Hanson, Wittenberg University, Dept of Biology and Chemistry, PO Box 720, Springfield OH 45504-0720.

The attractant sex pheromone of ticks consists of a halogenated aromatic ring, 2,6-dichlorophenol. Females release 2,6-DCP during feeding and prompt vigorous attraction responses by males. Our study demonstrates, for the first time, attraction to a mixture of chlorophenols by the American dog tick, Dermacentor variabilis (Say), the principle vector of the agents of Rocky Mountain spotted fever and tularemia. The chlorophenol mixture was prepared by reacting phenol with sulfuryl chloride, S[O.sub.2][Cl.sub.2], in the presence of diisobutylamine. Gas chromatography/mass spectral analysis (GC/MS) indicated that the product mixture obtained by this method contained 2,6-DCP (89.4%),2,4-dichlorophenol(7.6%o),2,4,6-trichlorophenol(2.4%),and 2-chlorophenol (0.6%) (N=4 trials). Interestingly, attraction to this chlorophenol mixture was stronger (ca.20% more pronounced) when compared to the response to the naturally occurring sex pheromone 2,6-DCP (replicates of 15; N=3). This finding is significant in that D. variabilis is not known for its attraction behavior, and few attractants have been identified. We anticipate that this chlorophenol mixture may have practical application as a trap bait for use in tick monitoring and eradication programs.

Board 28 TIMING AND DETECTION OF SEX PHEROMONE PRODUCTION IN TICKS. Jessica L. Pizzuli, and Chris Sanders, Jay A. Yoder, and Peter E. Hanson, Dept of Chemistry and Biology, Wittenberg University, 600 W. Ward St., Springfield OH 45501. Secretion of the attractant sex pheromone by ticks serves to bring members of the mating pair together. The only verified attractant sex pheromone in the American dog tick Dermacentor variablis (Say), vector of Rocky Mountain spotted fever and tularemia in North America, is 2,6-dichlorophenol (2,6-DCP). Females release the pheromone while feeding, prompting nearby males to detach and search for the female emitter. In this study, we used a novel extraction technique (Soxhlet extractor) to isolate 2,6-DCP. Analysis by gas chromatography/mass spectroscopy (GC/MS) was used to compare extracts of nonfed and fed ticks. Eggs, nonfed and fed stages of larvae, nymphs, males, and females were tested (replicates of 100 each, N = 3). 2,6-DCP was detected in fed females as expected. Interestingly, nonfed females and fed and nonfed males and nymphs also contained 2,6-DCP, but larvae and eggs did not. Short-range attraction bioassays using various concentrations of 2,6-DCP and its analogs (2,4-dichlorophenol, 4-chlorophenol, and 2-chlorophenol) showed that males were predictably attracted to 2,6-DCP and that 2,4-dichlorophenol acted as a mimic. Surprisingly, nymphs and adult females also displayed attractiveness toward 2,6-DCP. Because 2,6-DCP is a sex pheromone, only the attraction of males is biologically relevant. We concluded that the activation of receptors for detection of 2,6-DCP and biosynthesis of 2,6-DCP takes place during the nymphal stage of development.


Currently, 15 species of Geolycosa have been described based on a limited number of morphological characteristics. The state of Florida has nine Geolycosa sp., seven living in scrubs and sandhills across the state. The goals of this project are: 1) to estimate the evolutionary relationships among Floridian Geolycosa populations and species and between Floridian Geolycosa and Geolycosa from the rest of the USA and, 2) to examine patterns in the evolution of the two ecotypes of Geolycosa: those that build turrets at the entrance of their burrow and those that don't. We used cytochrome c-oxidase subunit I (COI) DNA sequences and morphological traits in a cladistic analysis. Geolycosa (N = 488) individuals from a total of 63 Florida scrub sites were collected and identified based on morphological characteristics. Total DNAs were extracted, amplified, and sequenced from 74 individuals representing the species G. escambiensis, G. micanopy, G. patellonigra, G.x. xera, G.x. archboldi, G. hubbelli, G. ornatipes, G. wrighti, G. missouriensis, G. rafaelana, G. turricola, and G. pikei. Results to date suggest that: 1) Floridian Geolycosa are not a monophyletic assemblage, 2) G. xera, G. escambiensis, G. hubbelli, G. patellonigra, and G. micanopy are not valid species in a phylogenetic sense, and 3) the two distinct ecotypes of Geolycosa have evolved repeatedly across the state. We also found evidence that the Geolycosa of the entire eastern USA are derived from ancestors in the western Florida Panhandle.


Spider sociality is a rare phenomenon, being observed in fewer than 0.1% of described species. We will study the reproductive behavior, maternal care, and spiderlings social behavior in a small subsocial tarantula from east Africa. The focal species was chosen because it is a member of a genus that has been observed to exhibit prolonged cohabitation of the female and her offspring. In addition, the genus is phylogenetically enigmatic, having been recently moved between different genera and even families. The current hypothesized placement of this genus places it in a poorly defined group of small and widely distributed tarantula species. Ours will be the second description of the sociality of this genus of tarantula, and one of the few studies of the ethology of the social behavior any tarantula. We have secured 32 wild-caught Heterothele villosela from an importer. The spiders were collected in Tanzania. The spiders are being held in 1.0 liter plastic containers with a bark mulch substratum, a piece of bark for a refuge, and a small water dish. The spiders are fed weekly on domestic crickets. The cages are held in a heated room (average temperature 26.6 [degrees] c) with ambient sunlight for photoperiod. We will pair spiders and videotape the matings. When offspring are produced, we will allow some to remain in the containers with their mothers and we will rear some spiderlings solitarily. We will observe and videotape group feeding behavior and other social interactions, and record growth rates.
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Publication:The Ohio Journal of Science
Geographic Code:1USA
Date:Mar 1, 2002
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