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2007 Appalachian Student Research Forum

Abstracts(Names are listed alphabetically by first author in each Division and Category)

Division 1 – UndergraduatesBiomedical Sciences

RELATIONSHIP BETWEEN COLLEGIATE THROWERS’ ABILITY TO PRODUCE MAXIMUM FORCE AND THEIR ABILITY TO POTENTIATE

J.G. Calloway, J.M. Kraska , M.A. Kinser, M.E. Stone and M.W. Ramsey. Department of Kinesiology, Leisure and Sport Sciences, East Tennessee State University, Johnson City, TN 37614

PURPOSE: This investigation examined the relationship between a collegiate thrower’s ability to produce maximum force and post-activation (or post-exercise) potentiation.

METHODS: 5 male and 3 female collegiate throwers (mean + SD; age = 19.8 + 0.9 y; weight = 111.7 + 18.9 kg) participated in a specifically designed 4-week strength and conditioning program. At the end of the 4-week training cycle maximal force and explosive power were measured with and without a potentiation protocol. The ability to produce maximum force was determined by measuring the isometric peak force (IPF). On the first day athletes performed isometric pulls from the power position (mid-thigh) in a custom designed force rack over a force plate (1000 Hz). Along with the isometric pulls the athletes were instructed to perform an overhead ball throw. On day two the athletes performed a 1 RM snatch test and a potentiation protocol consisting of two trials of mid-thigh clean pulls at 60 kg, 100 kg, 120 kg, 140 kg, 160 kg, and 100 kg. Each trial was separated by approximately one to two minutes of rest in order to ensure that full recovery occurred between each trial. All trials were performed on a custom rack placed over a force plate (1000 Hz), two linear position transducers were attached to the ends of the barbell. Individual force time curve analyses were used to determine the peak force, peak velocity, peak power, and peak rate of force development for each trial. Peak rates of force development (RFD) and peak force were determined from the ground reaction forces, while the velocity data were derived from the vertical barbell displacements collected by the linear transducers. Power was then calculated from the force values and the velocity data. A t-Test was used to determine if significant difference existed between pre- and post-potentiation values that were tested. Correlations were calculated using a pearson-r to determine the strength of the relationship between peak isometric force (N), RFD (0-250 ms), and the potentiation of dynamic peak force, peak velocity, and peak power, as well as ball throw (m), and maximum snatch (kg).

RESULTS: Potentiation values for peak force, peak velocity, peak power, and RFD showed significant statistical improvement (p < 0.05). When correlating these potentiation values to IPF the correlations ranged from moderate to strong for peak force (r=0.47), peak velocity (r=0.34), and peak power (r=0.76). Isometric peak force also correlated strongly with ball throw (r=0.76) and maximum snatch (r=0.79).

CONCLUSION: These data indicate that the relationship between the ability to produce force (IPF) and the potentiation ability in athletes are related. Not only will stronger athletes perform


better, but also their performance can be enhanced to an even greater extent, versus a weaker athlete, through potentiation. -------------------------------------------------------------------------------------------------------------------------------

THE RELATIONSHIP BETWEEN POSTMENOPAUSAL HORMONE THERAPY AND THE EXERCISE ELECTROCARDIOGRAM: A RETROSPECTIVE STUDY

Lindsey Perry and Dr. Melessia Webb. Department of Professional Roles and Mental Health Nursing, East Tennessee State University, CON, Johnson City, TN 37614

Postmenopausal Hormone Therapy (PHT) is a hormone regimen consisting of estrogen or estrogen-plus-progestin which is administered to females in order to relieve symptoms of menopause and to prevent osteoporosis. Estrogen has been found to be molecularly similar to the drug digitalis, a positive inotrope. Consequently, PHT may be associated with significant ST-segment changes on the electrocardiogram. A retrospective chart review was conducted with the sample’s inclusion criteria of females, 50 years of age or older who had experienced a stress test followed by a cardiac catheterization in order to investigate a possible relationship between PHT and cardiac-stress testing. The patient’s cardiac catheterization results were used as the reference standard to identify true positive stress test from false positives. The researchers reviewed 128 medical records and collected the following data: demographics, laboratory values, medical history, current list of medications, stress test date and results, cardiac catheterization date and results, and interventions after cardiac catheterization. Results of this study did not identify a higher rate of false-positive results for stress tests in females taking PHT than females who were not taking PHT in the study group. However, females with a lower percentage of coronary artery occlusion of specific arteries, the right coronary artery (RCA), the left anterior descending artery (LAD), and the left circumflex artery (LCx), were associated with a false-positive stress test. In addition, the following medical histories were significant in relation to having a higher rate of false-positive stress tests: (1) no previous myocardial infarction (MI); (2) no invasive cardiac intervention; (3) peripheral vascular disease (PVD); (4) no significant vessel disease; (5) not taking a lipid-lowering agent; (6) not taking an antiplatelet medication; and (7) taking an antiarrythmic medication. It is also noteworthy that 40% (n = 51) of the study population had a false-positive stress test, while only 23% (n = 30) had a true-positive stress test result. Females, who had a higher rate of true-positive stress tests, were those with lower HDL cholesterol levels and a higher percentage of occlusion in the RCA, the LAD, and the LCx. Medical histories associated with a higher percentage of true-positive stress tests were positive history of MI, history of invasive cardiac intervention, having significant vessel disease, females taking a H2-Blocker, females taking an antiplatelet medication, and not taking a Proton Pump Inhibitor. Due to the small number of participants, the effect size was small with a power of 0.169; the researcher recommends that this study be conducted again with a larger sample size in order to increase the power of the study. The results may prove useful in future referral for noninvasive diagnostic testing for heart disease in females.-------------------------------------------------------------------------------------------------------------------------------

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NATURAL VITAMIN E ISOFORMS, GAMMA AND DELTA MODULATE TGFβ 2 IN PC-3 PROSTATE CANCER CELL LINES

Regina Phillips, Chuan Xing Ho, Sarah Whaley, Koyamangalath Krishnan, William Stone and Sharon Campbell. Department of Internal Medicine, East Tennessee State University, COM, Johnson City, TN 37614

In early stages of cancer, TGFβ (primarily TGFβ1) inhibits tumor development, but at later stages of tumor progression, cancer cells evade the growth inhibition by TGFβ. TGFβ is switched from a tumor suppressor to a tumor promoter. Lu et al. has demonstrated that the PC-3 cancer cell line exhibits constitutive expression of NF-ĸB, an anti-apoptotic transcription factor. The constitutive expression of NF-ĸB conveys resistance to Tumor Necrosis Factor alpha (TNF-α), a natural apoptotic stimuli. Transforming Growth Factor β2 (TGFβ2) has been identified as a factor mediating the constitutive activation of NF-ĸB. The Alpha-Tocopherol, Beta Carotene study found that substantially fewer participants taking alpha-tocopherol developed prostate cancer. Helzlsouer et al. furthered these findings by showing that high plasma concentrations of gamma-tocopherol enhanced the effects previously seen with alpha-tocopherol. Further research has shown that vitamin E is able to induce cell death in PC-3 cells with the effect being greatly increased when cells are treated with both TNFα and vitamin E, suggesting a possible mechanism of action for TGFβ induced cell death. In this study, the gamma and delta isoforms of vitamin E were tested for their ability to halt cell proliferation in PC-3 prostate cancer cell lines in comparison with alpha-tocopherol at 20, 40 and 60 µM. By western blotting and ELISA assays, it was found that the gamma and delta isoforms are more potent inhibitors of cell proliferation than alpha-tocopherol. These natural vitamin E isoforms are able to allow PC-3 cells to overcome the resistance to TNFα-mediated cell death. We further demonstrate that these isoforms down regulate the expression of TGFβ2, NF-ĸB, and TGFβ Receptor 1.-------------------------------------------------------------------------------------------------------------------------------

CELL CYCLE REGULATORY PROTEINS IN TASTE CELL TURNOVER IN MICE

Colin Spaulding, Dr. Theresa Harrison and Lorraine Adams. Department of Biology and Department of Anatomy and Cell Biology, East Tennessee State University, COM, Johnson City, TN 37614

The cells in mammalian taste buds are known to regenerate throughout life every 10 – 14 days, but this process of taste receptor cellular turnover, particularly details concerning the regulation of progenitor cell division and differentiation, is still poorly understood. In all cells, cellular division and cell cycle exit prior to differentiation is regulated primarily by cell cycle regulatory proteins (CCRPs) such as cyclin dependent kinases (Cdks), Cdk inhibitors (CKIs), and the cyclin family of proteins. Studies have shown that normal taste buds express the CKI, p27(Kip1) (Hirota et al, 2001). Previous experiments in this laboratory (Adams et al, 2006) demonstrated that animals with null mutations for the p27(Kip1) gene (“knockouts”) have normal taste bud morphology, but an increased rate of cell turnover, and cyclin D2 is the major D cyclin expressed in taste tissues. In the present experiments, we wanted to examine in more detail the expression of p27(Kip1) and cyclin D2 in normal taste buds, and to determine if the absence of p27(Kip1) in mutants results in changes in the expression of D cyclins and/or other CKIs which may act to maintain the generally normal phenotype observed in taste buds of p27(Kip1) knockout mice. We used single and double-label immunohistochemical techniques to evaluate the expression of D cyclin isoforms and proteins of the Cip/Kip family of CKIs in circumvallate

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papillae of adult wild type and p27(Kip1) knockout mice. Light and confocal microscopy techniques were used to analyze the investigated proteins in fluorescent-labeled sections of tissue. Mature taste buds in wild type animals showed between 10 – 15% of their total cells to be labeled for p27(Kip1). Experiments to quantify the occurrence of p27(Kip1) and cyclin D2 labeled cells in the taste buds of wild type mice revealed averages of 5 – 6 cells per taste bud labeled for p27(Kip1) and cyclin D2. Extensive overlap was found between labeled cell groups, such that 55% of p27(Kip1) labeled cells also express cyclin D2, and 78% of cyclin D2 labeled cells were double-labeled for p27(Kip1). When investigating the possibility of upregulation of other CCRPs in the p27 (Kip1) null mutants, the results were negative for other members of the cyclin family (i.e. D1, D3) and, in initial experiments, for other members of the Cip/Kip family of CKIs (p21, p57). Experiments to date indicate that the hypothesis that normal taste bud morphology in knockout animals is maintained due to compensatory increases in expression of other Cip/Kip family CKIs and/or D cyclin proteins is not correct. Other CKIs (i.e. the INK family) may be more important in regulating taste bud cell numbers. Surprisingly, cyclin D2 continues to be expressed by some cells in the taste bud despite the fact that they have apparently withdrawn from the cell cycle. This suggests that, in addition to its role in facilitating transit through the cell cycle, cyclin D2 may play a different role in maturing and/or mature cells within the taste bud.-------------------------------------------------------------------------------------------------------------------------------

FANCONI PROTEINS IN DOUBLE STRANDED DNA REPAIR

Robin Woodard, Ph.D. and Henning Kuich. Department of Natural Sciences, University of Virginia's College at Wise, Wise, VA 24293

Fanconi Anemia is a hereditary form of Aplastic Anemia. Aplastic Anemia is a disorder in which destruction of bone marrow leads to a deficiency of circulating erythrocytes, leukocytes, and platelets. As a result, patients exhibit symptoms such as easy bruising, fatigue, and a decreased ability to fight off infections. In Fanconi Anemia, known mutations lead to a decrease in chromosomal repair in bone marrow cells and their consequent inefficiency in blood cell production. The Fanconi complex necessary to repair these chromosomal breaks is composed of the proteins FancA, FancC, FancG, and FancF. Upon chromosome breakage, FancE accumulates in the nucleus via an interaction with FancC, which in turn leads to the binding of these two proteins to the remainder of the Fanconi Complex. Once the components are assembled, the complex localizes to the chromosome break and initiates a repair pathway. DNA-PK is a kinase known to be vital to repair of double stranded (ds) DNA breaks as well as the joining of ds breaks induced during non-homologous recombination. It is composed of 3 subunits. The subunits Ku-70 and Ku-87 are responsible for binding the DNA and form a dimer upon exposure to ds breaks. This dimer recruits the catalytic subunit (360 kDa), which in turn activates a ds break repair pathway. This study investigated the relationship between the DNA-PK and the Fanconi repair pathway by looking for interactions between proteins of the different pathways in the presence of double stranded breaks. Upon verification of the binding of Ku-70 and Ku-87 to the simulated double stranded breaks, it was shown that FancC in fact does interact with the same ds breaks. This interaction could be attributed to interactions with ds breaks, Ku-70, Ku-87, or any combination of the three. This study was also able to show that FancA does not seem to directly interact with ds breaks or either of the Ku proteins.---------------------------------------------------------------------------------------------------------------------

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Natural Sciences & Mathematics

FERMAT’S LAST THEOREM, A COMBINATORIAL APPROACH

Connie Blalock. Depart of Mathematics, East Tennessee State University, Johnson City, TN 37614. Dr. Debra Knisley, advisor.

Fermat’s Last Theorem is perhaps one of the most famous theorems in all of mathematics. The theorem states that the equation xn + yn = zn does not have positive integer solutions if n > 2. Of course, there are many solutions if n = 2, such as 32 + 42 = 52. The case when n = 2 has a well known geometric interpretation, namely x, y and z are lengths of sides of a right triangle. Fermat claimed to have proof that no solution exists if n > 2, but he simply did not have room in the margin to write the proof down. For more than 300 years, mathematicians tried to prove Fermat’s claim, or find a counter example. Finally, the theorem was proved by Andrew Wiles, a mathematics professor at Princeton, in 1995. His proof, however, was clearly not the proof that Fermat claimed to have since it requires mathematics not yet invented at the time of Fermat.In this work, we approach Fermat’s last theorem using a field of mathematics called combinatorics and techniques that were known at the time of Fermat. In particular we use numerical sequences with alphabets of size x, y, z where each sequence is of length n. In so doing, we demonstrate a combinatorial interpretation rather than a geometric one. This method provides an alternative interpretation of Fermat’s claim.-------------------------------------------------------------------------------------------------------------------------------

CLONING PGT-4 FOR EXPRESSION AND CHARACTERIZATION AS A PUTATIVE GLUCOSYLTRANSFERASE IN CITRUS PARADISI (GRAPEFRUIT)

Epling, L. and McIntosh, C. Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614

Flavonoids are important molecules in plants, including Citrus paradisi. These compounds are of significance because of their probable power as antioxidants and the characteristics they give to plants, including the bitter flavor of grapefruit. Several research projects have identified naringin as being a possible antioxidant, even at nutritionally absorbable levels. Beyond antioxidant capabilities, research has shown correlation with anti-proliferation of cells, inhibition of angiogenesis, inhibition of cellular signaling, and effects on DNA repair enzymes (Gao et. al. 2006). Commercially, it is much harder to sell grapefruit or derived products if there are especially high levels of the bitter compounds, thus further supporting the need for research in this area (McIntosh, Personal Communication).

The bitter flavanone naringin accounts for 40-70% of the dry weight of young grapefruit (Jourdan et. al. 1985). Synthesis of bitter naringin involves a two-step glycosylation of naringenin (McIntosh and Mansell 1990, McIntosh et. al. 1990, Frydman et. al. 2004). In order to be able to control this biosynthetic pathway by the modification of enzymes, clones must be obtained for analysis of structure and function.

This research focuses on characterization of PGT-4, a putative flavonoid glucosyltransferase obtained viaSMART RACE RT-PCR using degenerate primers designed using the PSPG (plant secondary product glucosyltransferase) box sequence. (Strong 2005, Roy Sarkar et. al. 2007). This full length clone contained internal restriction endonuclease recognition sites that prevented it from being inserted in expression vector in the same way as previous PGT clones

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obtained in the McIntosh Lab. Primers were designed to incorporate a Bsa I restriction site with Nco I compatible overhang on the five prime end of the sequence and a Xho I site on the three prime end of the gene sequence for PCR (polymerase chain reaction) modification. These primers were designed to be used in the same PCR reaction, and this modification was performed on PGT-4. The PCR product was intermediately cloned into the PCR-4 TOPO vector. Bsa I and Xho I restriction endonucleases were used to isolate the desired DNA fragment. Cloning is in progress to insert mPGT-4 (modified) in the PCD1 expression vector for transformation into BL21 (DE3) RIL e. coli cells and subsequent induction of protein expression. The expressed proteins will be tested for possible flavonoid glucosyltransferase activity.----------------------------------------------------------------------------------------------------

INTERACTING GALAXIES AND THE EFFECTS ON STAR FORMATION AND GALACTIC STRUCTURE

Sabrina H. Hurlock, Dr. Beverly J. Smith and Dr. Mark Hancock. Department of Physics, Astronomy and Geology, East Tennessee State University, Johnson City, TN 37614

By analyzing images of interacting galaxies, we are able to study the effects that galactic interactions have on the involved galaxies, including new star formation, matter bridges between the galaxies, and changes in overall galactic structure. We obtain the data by making observations, via the internet, from the SARA (Southeastern Association for Research in Astronomy) 0.9 meter telescope at the Kitt Peak National Observatory in Tucson, Arizona. We observe many different interacting galaxies selected from the Arp Atlas in multiple filters, making numerous 10-minute observations in each filter. After making the observations, we analyze the data using the IRAF (Image Reduction and Analysis Facility) program, to remove the bias and dark current, flat-field the data, and co-add the images. The DS9 software is then used to view the final compiled images of each galaxy. At this point, we are able to determine the location and size of new areas of star formation, the size, shape and location of tails and bridges between galaxies, and the transformation of the structure of the galaxies. This research is important in helping us to understand the magnitude of the effects of galactic interaction and what will one day be the fate of our own galaxy.------------------------------------------------------------------------------------------------------------------------------

IN SITU VASCULAR ENDOTHELIAL GROWTH FACTOR LOCALIZATION

Kevin Long. Department of Health Sciences, East Tennessee State University, Johnson City, TN 37614

Vascular Endothelial Growth Factor is the primary molecule that acts upon endothelial cells, triggering the growth of new blood vessels. It is the only known growth factor that acts exclusively on endothelial cells. This factor can be found in the ovaries of all mammals and plays a pivotal role in the estrous cycle. VEGF is known to be more prevalent at different parts of the estrous cycle, but it is not known in which part of the ovaries VEGF production originates.

We have attempted to locate the source of Vascular Endothelial Growth Factor in the ovaries of hamsters and mice. Since we could not locate the growth factor itself, we had to find the mRNA that encodes for VEGF production. The required process for such situ hybridization localization is in situ hybridization. We began this procedure by using template DNA to create an RNA probe through transcription reactions. The probe was made from a vector known as PGEMIII and was cut using EcoRI and SP6 restriction enzymes. Once the probe was made, it was used

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in the staining process of in situ hybridization in order to stain the microscope slides with the ovarian tissue. Because our probe was comprised of RNA, many precautions had to be made in order to keep RNase enzymes out of our solutions and equipment. These enzymes would degrade the probe, rendering it useless for our study.

We attained limited results, but explored several new techniques in discovering the prevalence of VEGF throughout the cycle. We improved our methods through a significant amount of troubleshooting and research. The study will continue and with what we have learned and been able to improve upon, it should lead to extensive findings about VEGF throughout the cycle.-------------------------------------------------------------------------------------------------------------------------------

ASSESSMENT OF PUTATIVE GLUCOSYLTRANSFERASE PGT-2 EXTRACTED FROM CITRUS PARADISI THROUGH CLONING, SEQUENCING AND KINETIC ANALYSIS

Shannon McConnell, Daniel Owens and Cecilia McIntosh. Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614

Flavonoids, a class of secondary metabolites, are a kay focus in plant biochemistry. Flavonoids are polyphenolic compounds consisting of fifteen carbons; two benzene rings attached by a three carbon chain. There is considerable interest in flavonoids with respect to their antioxidant effects and their contribution to human health. Flavonoids are characterized by their molecular structure and research is focused on the common pathway that synthesizes them. With over 5,000 different flavonoid metabolites present in plants, research is focused on the core/common pathway that synthesizes flavonoids. Not much is understood or known about regulation of modification reactions where the synthesis of flavonoid derivatives are concerned, but glyosylation is one of the most common modifications that occur in the synthesis of flavonoids. Glycosylation is a chemical reaction in which glycosyl groups are added to a substrate to produce glycosylated derivatives. The focus of the research was to analyze/evaluate putative glucosyltransferase PGT-2 from grapefruit leaves using techniques such as cloning, sequencing and experiments to test enzyme activity. Over the course of evaluating PGT-2, it was cloned into Topo and PCD1 vectors. The success of PGT-2 being inserted into these vectors was assessed using plasmid isolations along with evidence visualized in DNA gels and verification through sequencing. After confirming that PGT-2 had been inserted in PCD1 it was transformed into BL21 DE3 RIL cells and further evaluated by induction experiments at varying temperatures to evaluate protein production. Current efforts are focused on optimizing induction conditions to increase levels of soluble protein and testing expressed protein for flavonoid GT activity.-------------------------------------------------------------------------------------------------------------------------------

REGULATION OF SABP2: A CRITICAL COMPONENT OF THE PLANT INNATE IMMUNITY

Poonam Sheth, Daniel F. Klessig and Dhirendra Kumar. Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614 and Boyce Thompson Institute, Ithaca, NY 14853

SABP2 has been identified as a receptor for SAR (a form of innate immunity) in plants (Kumar & Klessig, 2003). RNAi mediated silencing of SABP2 makes the plant susceptible to pathogen infection and the plant produces methyl salicylate (MeSA) in its response (Farhad et. al., 2005). MeSA is an ester and is hypothesized to move through the membranes and is also a volatile compound. SABP2 is required in the uninnoculated systemic tissue to convert the inactive

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MeSA into active salicylic acid (SA) by methyl esterase activity. The active SA is critical for basal, local and systemic acquired resistance and therefore it is important to know the regulation of SABP2. A cell suspension culture of Nicotiana tabacum were freshly inoculated by diluting 1:10 in MS media from the cultures that were previously established. The cultures were grown at 20-25º C on a shaker maintained at approximately 130 rpm. After 3-4 days the cells were treated with MeSA, MeJA, nitric oxide donor (GSNO). Cells were collected at various time intervals and used for RNA isolation. Total RNA was isolated using Tri-reagent (Sigma) and 1µg of the RNA was used for cDNA synthesis. Total cDNA was used for amplification of SABP2 and other genes using PCR. PCR products were analyzed by separating on a agarose gel. To confirm the results northern analysis is being carried out using a non radioactive probe (SABP2 cDNA) using the kit from GE Healthcare Life Sciences. For Western analysis polyclonal antibodies (produced in rabbit) against the recombinant SABP2 will be used. All the exciting results of this ongoing experiment will be presented. This study will help us to understand the regulation of a critical component of plant immunity. -------------------------------------------------------------------------------------------------------------------------------

Social & Behavioral Sciences

THE EFFECTS OF NICOTINE ON CONDITIONED PLACE PREFERENCE IN A RODENT MODEL OF PSYCHOSIS

Jennifer Abraham, Yoko E. Ogawa, Elizabeth L. Cooper and Russell W. Brown. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

Increases in sensitivity of the dopamine D2 receptor has been shown to play important roles in behavioral abnormalities in schizophrenia. Past studies from this laboratory have shown that neonatal treatment with the drug quinpirole (a dopamine D2/D3 agonist) results in long-term increases of sensitivity of the D2 receptor that persists throughout the animal’s lifetime. A major health problem within the schizophrenic population is an increased incidence of smoking cigarettes, which contain the psychoactive and addictive ingredient nicotine. This study was designed to analyze the effects of nicotine on conditioned place preference (CPP) in both early postweanling and adolescent rats that were neonatally treated with quinpirole. All rats were admininistered a daily intraperitoneal injection of Quinpirole HCl (dose: 1 mg/kg) for the first three weeks of development (P1-21). Rats were weaned from the female dam at P21. There were two conditioned place preference shuttle boxes used in this study: A two-chamber and a three-chamber apparatus. The two chamber was a black/white shuttle box constructed of plywood that was 30 x 18 x 20 in that either had only a black and white chamber. The three-chamber apparatus was constructed in a similar fashion but had a gray chamber separating the black and white chamber. In experiment 1, beginning on 23 or 31 days of age, animals were conditioned by administering animals nicotine tartarate (0.8 mg/kg free base) in the non-preferred environment (white compartment), whereas saline was given in the black compartment for eight consecutive days, and controls received saline in both compartments. Controls were administered saline was given in both compartments. Results demonstrated that regardless of age, rats neonatally treated with quinpirole demonstrated a significant preference for the black compartment, but nicotine alleviated this preference after eight days of conditioning. Rats neonatally treated with saline and conditioned with nicotine demonstrated a stronger preference for the white compartment. In Experiment 2, rats were conditioned in a similar fashion but in a three chamber apparatus, and a similar result was found: neonatal quinpirole produced a significant preference for the black compartment in both early postweanlings and adolescents that was alleviated by nicotine. These results appear to show that neonatal quinpirole, that results in priming of the D2 receptor, produces a significant increase in stress that is alleviated by nicotine.

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OUTCOMES OF THE APPALACHIAN TEACHING PROJECT

Erika Adams. Department of Family and Consumer Sciences, East Tennessee State University, Johnson City, TN 37614

The Appalachian Teaching Project began in 2000 having in mind bridging the gap between communities and institutions of higher education. The colleges and universities of Appalachia set about to engage with their communities through specific projects designed to stabilize the Appalachian region and contribute to keeping the region’s youth committed to their communities. Developing community projects that will have a continuing presence and a lasting influence, the thirteen participating schools in nine states which have joined the project contribute through community based research projects and service learning which is partially funded by the Appalachian Regional Commission. This poster project was designed to highlight the numerous outcomes of Appalachian Teaching Projects of the 2004-2005 year. Research data was collected through interviews with participating students and faculty, tours of project sites, and attending presentations and conferences. The outcomes of the various projects are as diverse as the Appalachian population they represent.-------------------------------------------------------------------------------------------------------------------

DANGEROUS DRIVING AND FUNDAMENTALISM, SPIRITUALITY, AND RELIGIOSITY

Amber Cline, Chris S. Dula, Robert B. Russell, Kevin Gilmer, Jodie A. Lamb and Julia T. Sutherland. Applied Psychology Laboratory, East Tennessee State University, Johnson City, TN 37614

Risk-taking is a dimension of dangerous driving (Dula & Geller, 2003). Risky driving is a problem because in 2002 alone, over 42,000 people were killed in traffic crashes on U.S. roads (NHTSA, 2003). An interesting set of unpublished pilot data suggested that drivers perceived levels of spirituality, as assessed by a single question, was related to how often people reported wearing safety belts in their vehicles. To explore this concept more in-depth, measures of fundamentalism, spirituality, and religiosity, were given to undergraduate participants along with the Dula Dangerous Driving Index (DDDI, Dula & Ballard, 2003) and a demographic questionnaire assessing a wide variety of traffic safety behaviors and history of traffic crashes. With only a single item of spirituality having previously shown a mild, but significant, positive correlation with reported safety belt use, no specific hypotheses were made with regard to fundamentalism or religiosity as they pertain to risky driving behaviors. It was hypothesized that higher levels of perceived spirituality would be related to lower reported risky driving as measured by the DDDI, and a lower incidence of reported dangerous driving behaviors. A total of 117 undergraduate students participated in an online survey of traffic safety behaviors and personality constructs. Results are discussed and future research directions are proposed in this poster presentation.---------------------------------------------------------------------------------------------------------------------

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SOCIAL DESIRABILITY BIASES AND SELF-REPORT MEASURES OF DANGEROUS DRIVING

Kevin Gilmer, Chris S. Dula, Robert B. Russell, Amber Cline, Jodie A. Lamb and Julia T. Sutherland. Applied Psychology Laboratory, East Tennessee State University. Johnson City, TN 37614

Adolescents and young adults are particularly prone to unintentional injury by motor vehicles crashes. Healthy People 2010 noted that the death rates associated with motor vehicle-traffic injuries are highest in the age group 15 to 24 years (U.S. Department of Health and Human Services, 2001). Thus, the college population is a particularly apropos venue to study traffic safety issues. Measures of driver risk do exist, such as the Dula Dangerous Driving Index (DDDI, Dula & Ballard, 2003), the Propensity for Angry Driving Scale (PADS, DePasquale, Geller, Clarke, & Littleton, 2001), and the Driving Behaviour Inventory (DBI, Gulian, Matthews, Glendon, Davies, & Debney, 1989). A problem all these measures have in common is an obvious item content whereby test takers can readily discern how to answer in a positive manner. This study looks at the construct of Social Desirability, a tendency for people to answer survey items in a manner more positive than is likely the case. Social desirability was hypothesized to be negatively correlated with the DDDI and the PADS. A total of 117 undergraduate students participated in an online survey of traffic safety behaviors and personality constructs. Results are discussed and future research directions are proposed in this poster presentation.-----------------------------------------------------------------------------------------------------------------------------

A CROSS-CULTURAL COMPARISON OF MINIMUM LEGAL DRINKING AGE LAWS AND ACTUAL DRINKING BEHAVIOR

Kristina Hollowell. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

It is no secret that underage drinking exists and is prevalent throughout the United States. Binge drinking is considered a common social problem within our culture, especially among underage drinkers (Miller et al., 2006). The United States’ minimum legal drinking age (MLDA) is 21; one of the highest in the world. This leads one to assume that one of the country’s solutions to prevent drinking is to implement laws forbidding one to consume alcohol until they reach an age approved by the government. However, the law itself does not necessarily guarantee obedience, but rather imposes consequences for use and limits availability of alcohol (Wagenarr, Toomey, & Lenk, 2005). I approached my research with a cross-cultural perspective exploring the relationship between MLDA and alcohol consumption behavior comparing East Tennessee and Amsterdam. The United States and the Netherlands have very different governmental policies concerning alcohol (International Center for Alcohol Policies, 1998). In The US a person is not allowed to purchase or consume alcohol until the age of 21. In contrast, in the Netherlands a person is allowed to purchase beer and wine at the age of 16 and liquor at 18. The participants for my research were all between the ages of 18 and 20 years old; those from Amsterdam could drink legally while those from the United States could not. My measurement consisted of a survey containing 18 questions that asked the participants about their alcohol consumption, consequences of drinking behavior, family alcohol problems, perception of colleagues’ consumption, and their perception of their own consumption. If the United States’ laws are effective in preventing underage drinking, then the U.S. participants should drink less and consequently have fewer negative consequences than the Amsterdam participants who had been able to drink legally anywhere from two to four years at the time they

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filled out the survey. There were 82 participants from Vrije Universiteit and 118 participants from East Tennessee State University. Over 80% of the participants from both universities were considered to be drinkers. At ETSU 44% were binge drinkers while 53.6% at Vrije binge drank. ETSU participants reported a significantly higher rate of negative consequences in their personal relationships than Vrije participants. While only 13.0% of Vrije participants claimed other students’ negatively impacted their life on or around campus, 40.2% of ETSU participants agreed. Students from both universities reported a similar rate of negative effects upon their jobs and school work. Both set of participants perceived their colleagues to drink more than what was actually reported. The results agree and contradict the original hypothesis. Although Vrije participants did report more binge drinking than ETSU participants, the ETSU participants reported more negative consequences of their drinking behavior. Despite all of the attention focused on alcohol consumption, colleges and universities have failed to control binge drinking rates (Glassman, 2002). Weschler and colleagues (2002) showed that approximately 44% of the participants in their study reported binge drinking, a rate almost identical to previous years. Regardless of the legality status adolescents often drink. It is my belief that it is important to teach healthy drinking habits, not abstinence from alcohol. -----------------------------------------------------------------------------------------------------------------------------

METHYLPHENIDATE (RITALIN) EXPOSURE TO ADOLESCENT D2-PRIMED RATS ELIMINATES THE INITIAL HYPOACTIVE RESPONSE TO NICOTINE IN ADULTHOOD

Andrew B. Hughes, A. Brianna Sheppard, Ian D. Longacre and Russell W. Brown. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

Past studies from our laboratory have shown that neonatal quinpirole (dopamine D2/D3 agonist) treatment produces increases in sensitivity of the dopamine D2 receptor, an effect that has some similarities with attention deficit/hyperactivity disorder. A common drug typically used to medicate ADHD is Ritalin (methylphenidate), a drug that has stimulant properties and abuse potential. Additionally, studies have shown that children that are medicated with Ritalin show a significant increased propensity to smoke cigarettes. In this study, we analyzed whether adolescent rats neonatally treated with quinpirole will demonstrate a significant increase in locomotor sensitization when methylphenidate is administered in adolescent in D2-primed and non D2-primed rats. Rats were administered quinpirole, a dopamine D2/D3 agonist from postnatal days (P1-21) and raised to adolescence (P29). Rats were habituated to a locomotor arena, and subsequently administered methylphenidate (Trade name: Ritalin; 5 mg/kg) or saline every other day for three weeks. Additionally a nicotine challenge was performed at postnatal day (P60), in early adulthood, to analyze the effects of nicotine on D2-primed rats exposed to methylphenidate in adolescence. On overall activity counts, results showed that methylphenidate produced rapid sensitization, and D2-primed animals that received methylphenidate began to demonstrate a significant decrease in activity during the third week of testing, suggesting increases in stereotypic behavior and immobility. In fact, a significant increase in immobility was observed during the third week of testing in D2-primed animals administered methylphenidate, supporting this claim. On the nicotine challenge, D2-primed animals exposed to methylphenidate as adolescents demonstrated no hypoactive response to initial nicotine exposure, and in fact demonstrated a significant increase in activity compared to all other groups on the nicotine challenge. These results suggest that D2-priming combined with methylphenidate exposure may lead to elimination of the hypoactive response to initial nicotine, and may be suggestive as to the mechanism of the increased propensity of methylphenidate-treated ADHD patients to smoke cigarettes.-------------------------------------------------------------------------------------------------------------------------------

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THE FUTURE IMPLICATIONS OF INDOOR UV TANNING AT A YOUNG AGE

Leslie King, Preston Visser, Joel Hillhouse and Lana McGrady. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

Indoor UV tanning is a significant health-risk for the development of skin cancer in young adults. Research has shown that it is strongly related to the development of all types of skin cancer including melanoma, the most lethal form of skin cancer. Despite this, the popularity of indoor tanning is strong and growing in young people. The prevalence of indoor tanning is consistently found to be highest among young adult females. This project sought to examine the risks associated with indoor tanning at a young age by comparing groups of individuals who first indoor tanned before and after chosen ages. We surveyed 174 female college students from a regional university (East Tennessee State University) as a part of a larger research project. Using a software program that allows for complete online surveying, subjects were given a survey assessing both their history of indoor UV tanning as well as their intentions to indoor UV tan in the future. In the study we divided the subjects into two groups based on the age of their first indoor UV tanning experience. We then compared the two groups to the amount of times they reported indoor UV tanning in the past twelve months. We found a significant difference between individuals who first indoor UV tanned at age 16 or below compared with those who first indoor UV tanned at ages 17 or above (p=<.01). This suggests that a female is more likely to indoor UV tan at a greater frequency in the future when she is exposed to it at an earlier age. This may have significant implications in potential interventions by encouraging the investigation of the efficacy of targeting younger female populations for pre-behavioral treatment. Importantly, we also found a significant difference between average number of times participants reported indoor UV tanning based on whether or not they first indoor UV tanned before the age of 18 (p=<.01; mean difference of 18.8). Data such as this may be crucial in the debate concerning illegalizing indoor UV tanning for persons under the age of 18 as it demonstrates that females indoor UV tan significantly more in the future if they first indoor UV tan before the age of 18. -------------------------------------------------------------------------------------------------------------------------------

EARLY WOODLAND CERAMIC TYPOLOGY AND CULTURE CHRONOLOGY IN UPPER EAST TENNESSEE

Lucinda M. Langston and Jay D. Franklin. Department of Sociology and Anthropology, East Tennessee State University, Johnson City, TN 37614

Based on research and excavations at Phipps Bend on the Holston River, Robert Lafferty (1978, 1981) developed a model for Early Woodland ceramic typology and chronology for upper East Tennessee. The utility of the model has never been evaluated beyond Phipps Bend, however. In this paper, we examine the Early Woodland ceramic assemblages from several sites in upper East Tennessee in an attempt to place them within the chronological framework developed by Lafferty. Where possible, we use radiocarbon assays as an independent means of assessing the efficacy of the model. -------------------------------------------------------------------------------------------------------------------------------

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AGE AND GENDER DIFFERENCES IN THE VIRTUAL MORRIS WATER MAZE FROM 12-25: A FRESH LOOK AT STRATEGIES

Matthew F. Lazenka and Dr. Russell W. Brown. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

The Morris water maze (MWM) has been utilized as one of the primary behavioral neuroscience tasks in rodents and is an excellent test of spatial memory function (Gerlai, 2001). Two primary versions of the MWM have been utilized: A place version, in which the platform is stationary, and a match-to-place version in which the platform moves to a new location on each day of training. Recently, a virtual version of the MWM has been developed to test human participants on this task (Astur, et al., 1998). In this version of the MWM, an environment is presented on a computer screen that includes a depiction of a three-dimensional extramaze cue environment. Participants must navigate through the environment using arrows on a keyboard to a hidden platform. Past studies have reported significant gender differences on the place version of the vMWM (Driscoll, et al., 2005), but there has not been any reports on the match-to-place version of the vMWM. Similarly, research has also failed to address the differences between adolescents and adults utilizing the match-to-place version, as well as, the effects of gender stereotype threats.

Participants for experiment one were students (12-13 years old) from a public school located on the campus of East Tennessee State University (ETSU). Participants for experiment two were college students (18-25 years) recruited from Psychology and Sociology classes at ETSU. The computer simulation consists of a pool located in the center of a square room with various landmarks surrounding the pool. For experiment one, adolescents were read a script discussing how to perform the task, mentioning that they would have to swim in search of a hidden platform located under an opaque water surface and that the experiment would consist of 20 training trials. In experiment two, the same script was read and another was read revealing that either men or women were superior in the task alluding that this superiority was due to evolutionary significance.

Results from the first experiment showed that adolescent males had reduced latency, heading error and path length. In reference to paths, adolescent males and females did not differ. Both preferred to alternate between a circle and zig-zag strategy; however, their paths are more closely related to those of >60 year olds than college-aged students. This may be a factor paralleling the developing hippocampus with the aging hippocampus.

In experiment two, mentioning male superiority seems to eliminate the gender difference while mentioning female superiority does not entirely. Surprisingly, no significant difference was found within genders. Compared with the strategies previously found with college-aged participants, males differed in their choice of strategy. Males in experiment two circled more than males in that study (Click & Brown, unpublished). This may be influenced by mentioning of a strategy.-------------------------------------------------------------------------------------------------------------------------------

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NICOTINE SENSITIZATION IN ADOLESCENT AND ADULT RATS D2 RECEPTOR PRIMED AS NEONATES: GENDER DIFFERENCES AND NEUROTROPHIC FACTORS

Ian D. Longacre, Marla K. Perna and Russell W. Brown. Department of Psychology, East Tennessee State University, Johnson City, TN 37614 We have demonstrated in past work that neonatal quinpirole (D2/D3 receptor agonist) treatment to rats produces long-term priming of the dopamine (DA) D2 receptor that persists throughout the animal's lifetime. In Experiment 1, the offspring of six male-female Sprague-dawley (SD) rat breeder pairs were administered one daily i.p. injection of quinpirole (1mg/kg) or saline from postnatal days 1-21 (P1-21) and raised to adolescence. After three consecutive days of habituation to the locomotor arena, beginning on P31 all animals were administered one i.p. injection of 0.5 mg/kg free base nicotine tartarate every other day for three weeks. Results showed that neither D2-primed or non D2-primed adolescent female rats demonstrated nicotine-induced hypoactivity early in training, and D2-primed adolescent females showed a significant decrease in activity as compared to females administered nicotine in week 2 and 3 due to increased stereotypic behavior. D2-primed male adolescents demonstrated more rapid sensitization to nicotine than non D2-primed males. In Experiment 2, offspring of seven breeder pairs were given the identical neonatal drug treatment as in Experiment 1 but raised to adulthood (P60). Although initial hypoactivity did not differ across groups, D2-primed adults rats administered nicotine demonstrated significantly more robust sensitization than controls given nicotine during the third week of testing. In both Experiments 1 and 2, one day after testing was complete brain tissue was taken and the nucleus accumbens and frontal cortex were analyzed for brain-derived neurotrophic factor (BDNF). In adolescents, nicotine produced a significant increase in BDNF in the nucleus accumbens that was unaffected by neonatal quinpirole treatment. In adults, Neonatal quinpirole treatment produced a significant decrease in nucleus accumbens BDNF in adult rats that was alleviated by nicotine. These results suggest that sensitization to nicotine is more robust in D2-primed animals, but sensitization is dependent upon both age and sex. Additionally, it does not appear that BDNF plays a significant role in nicotine sensitization in either adolescents or adults, which is contrary to findings with other psychostimulants such as cocaine and amphpetamine. -----------------------------------------------------------------------------------------------------------------------------

GENDER DIFFERENCES IN STIGMA AGAINST TATTOOS

Benjamin A. Martin, Sonia L. Coney and Dr. Chris S. Dula. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

Tattoos have grown in popularity in recent years, mainly brought to the forefront by celebrities and “reality-television” shows involving various tattoo shops. One theory for explaining the proliferation of fleshworks is that with the demise of the mythology of Christianity in the popular imagination (Mercury, 2000). The purpose of the present study was to evaluate gender differences in stigma against tattooing, and two measures of adventurousness; men were expected to have less stigma associated with tattooing and were also expected to be more adventurous. It is notable that previous studies have basically adhered to the quasi-interview method, which may be a limitation to the studies in that it often leads to desirable responding. For this pilot study, 58 undergraduates (9 men and 49 women) participated for modest extra credit. Participants completed a set of surveys that included attitudes regarding tattooing, the Adventurousness Scale (van der Zee & van Oudenhoven, 2000), a version of the EPQV modified to measure adventurousness (Eysenck et al., 1985), and a short demographic

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questionnaire. Men scored higher on the stigma measure than women but this difference was not significant. Significant gender differences were found for scores on both the Adventurousness and EPQV Scales. The impact of a non-significant gender difference in responses to the stigma measure, eludes to an overall social taboo against tattooing. Further research will be aimed at how familiarity with the tattoo process affects overall stigma scores.-------------------------------------------------------------------------------------------------------------------------------

NICOTINE SENSITIZATION IN ADOLESCENT BETA ARRESTIN-2 KNOCKOUT MICE: IMPLICATIONS FOR MECHANISMS OF NICOTINE ADDICTION

Daniel M. Noel, Jennifer A. Correll, A. Brianna Sheppard and Russell W. Brown. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

This study was designed to sensitize adolescent Beta Arrestin-2 knockout (BA-2 KO) mice to the psychostimulant nicotine. Activation of the dopamine D2 receptor has been shown to cause G protein-coupled receptor kinase-dependent receptor phosphorylation, and a robust translocation of Beta-arrestin to the cell membrane and profound receptor internalization by the cell membrane. Thus, it appears that Beta Arrestin-2 plays an important role in cell signaling from the dopamine D2 receptor in response to stimulants. Sensitization to nicotine as measured through locomotor activity in rats has been shown to be an excellent behavioral test for dopaminergic activity and is a clinically relevant test for addiction to drugs. Induction of sensitization has been referred to as the locomotor activity produced by administration of the drug, and expression of sensitization has been referred to as the locomotor activity response after a period of abstinence. Both induction and expression of sensitization was measured in the present study. Finally, nicotine addiction typically begins in adolescence, and research has shown adolescent and adults demonstrated significantly different behavioral responses to nicotine. In this study, 3-4 week old adolescent BA-2 KO and wild type C57/Bl6 mice were habituated to a square locomotor arena for one 30 min session. The following day, animals were administered either nicotine tartarate (s.c, 0.5 mg/kg free base) or saline 10 min before being placed into the locomotor arena for seven (Experiment 1) or 14 consecutive days (Experiment 2). A drug-free abstention period of seven days followed in both experiments, at the end of which animals received a nicotine challenge (0.5 mg/kg free base). In Experiment 1, results showed that BA-2KO mice demonstrated significantly decreased levels of activity as compared to wild type animals during habituation, and also demonstrated an initial hypoactivity to nicotine as compared to wild type controls that received nicotine. In fact, BA-2KO mice treated with nicotine remained in a hypoactive state throughout the first 6 days of sensitization training compared to saline-treated BA2 KO mice as well as wild type controls. However, by seven days, wild type controls receiving nicotine demonstrated equivalent levels of activity to saline controls, and thus did not demonstrate sensitization to nicotine. On the nicotine challenge, however, BA 2 KO mice demonstrated significantly decreased activity levels compared to all other groups, and thus failed to express sensitization to nicotine. In Experiment 2, both BA-2 KO mice and wild type controls demonstrated sensitization to nicotine, although the knockout mice demonstrated significantly lower levels of activity. On the nicotine challenge, BA-2 KO mice did not demonstrate sensitization to nicotine, and in fact demonstrated significantly lower levels of activity. Therefore, it appears that Beta arrestin-2 molecule is necessary for induction of sensitization to nicotine, but does not play a role in expression of nicotine sensitization. These results appear to indicate the importance of the BA-2 protein in locomotor sensitization to nicotine in adolescence.-------------------------------------------------------------------------------------------------------------------------------

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AN INVESTIGATION OF PERFECTIONISM, OPTIMISM, AND SELF-REINFORCEMENT AMONG COLLEGE ATHLETES

Sabrina D. Rohr and Kevin L. Burke. Department of Kinesiology, Leisure, and Sport Sciences, East Tennessee State University, Johnson City, TN 37614

Perfectionism and optimism are common characteristics found in college athletes (Gould & Diefenbach, 2002). However, self-reinforcement is not as commonly studied in sport. Athletes at a southeastern university (N=91) completed the Frost Multidimensional Perfectionism Scale (Frost, Martin, Lahart, & Rosenblate, 1990), Life-Oriented Test Revised (Scheier, Carver, & Bridges, 1994), and Frequency of Self-Reinforcement Questionnaire (Heiby, 1983) during either practice or study hall sessions. Athletes from the following seven sport teams participated: men’s tennis, women’s golf, women’s softball, women’s basketball, men’s baseball, women’s soccer, women’s volleyball, and men’s golf. Results indicated a slightly negative relationship between perfectionism and optimism (r= -.060). With an alpha level of .01 used for all statistical test, the correlation between perfectionism and optimism was found not to be significant, p=.569. Also, the results indicated perfectionism and self-reinforcement had a negative relationship and was found to be significant (r= -.321, p=.002). Conversely, a positive relationship was found between optimism and self-reinforcement with a correlation of r=.306 and found to be significant, p=.003. Although the correlation between perfectionism optimism was negative, the athletes were found to have high levels of perfectionism (M= 106.35) and optimism (M=20.91). Likewise, the athletes were found to have high levels of self-reinforcement (M=20.78). Team sports (M=108.8) indicated higher perfectionism scores than individual sports (M=98.19). However, levels of optimism (team M=20.34, individual M=22.81) and self-reinforcement (team M=20.7, individual M=21.05) were higher in individual sports than team sports. -----------------------------------------------------------------------------------------------------------------------------

ACTIVELY CARING AND PRO-SOCIAL DRIVING

Robert B. Russell, Chris S. Dula, Kevin Gilmer, Amber Cline, Jodie A. Lamb, and Julia T. Sutherland. Applied Psychology Laboratory, East Tennessee State University, Johnson City, TN 37614

According to the U.S. Department of Health and Human Services (USDHHS, 2001), motor vehicle crashes are the leading cause of serious injury in our society. Motor vehicle injuries remain the most costly and fatal of unintentional injuries. The National Safety Council (NSC, 2001) estimated that motor vehicle crashes were the cause of $169 billion in lost wages, medical expenses, and administrative costs. Motor vehicle crashes are the leading cause of death for people ages 1 to 33 (NSC, 2001). While there is a good deal of literature exploring the effect of personality on dangerous driving, it is likely that personality traits also affect positive driving behaviors, or at least attenuate dangerous driving behaviors. Driving behaviors that endanger or have the potential to endanger have been considered as lying on a behavioral spectrum of dangerous driving, including three dimensions: 1) intentional acts of aggression toward others, 2) negative emotions experienced while driving, and 3) risk-taking (Dula & Ballard, 2003; Dula & Geller, 2004). This study looks at the construct of Actively Caring, a tendency for some people to go out of their way, or beyond the call of duty, to be helpful to others. Actively caring was hypothesized to be negatively correlated with all dangerous driving variables. A total of 117 undergraduate students participated in an online survey of traffic safety behaviors and personality constructs. Results are discussed and future research directions are proposed in this poster presentation.---------------------------------------------------------------------------------------------------------------------------

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Division II—Graduate Students 1 - 2 years

Arts & Humanities

SNAPSHOTS OF THE HISPANIC IMMIGRANT COMMUNITY IN APPALACHIA

Vivian Gonzales Gladson. Department of Sociology & Anthropology, East Tennessee State University, Johnson City, TN 37614 The proposed poster presentation focuses on my ethnographic fieldwork with Hispanic immigrant community in Appalachia. Images include brief ethnographic descriptions that illustrate aspects of everyday life and work among Hispanics in Johnson City and nearby communities. My work centers primarily on secondary migration, or the process by which Hispanics have come to Appalachia from other cities in the U.S. The poster presentation includes issues, such as migrant seasonal labor; concerns about documentation and immigration laws; gender and children; social problems within the Hispanic community; and religious belief folklore. Broader themes connecting these issues are processes of acculturation, concepts of "home" and identity, and the continuity of tradition.----------------------------------------------------------------------------------------------------------------------------- Natural Sciences & Mathematics

ISOLATION AND IDENTIFICATION OF THE PUTATIVE GLUCOSYLTRANSFERASE PGT5 FROM CITRUS PARADISI

J.K. Cooke, D. Owens, and C. McIntosh. Department of Biological Sciences and Department of Health Sciences, East Tennessee State University, Johnson City, TN 37614

The isolation of the putative glucosyltransferase clone PGT5 and its resolution from other glucosyltransferases from Citrus paradisi (grapefruit) is described. This clone was obtained by designing two primers based on the sequence of the liminoid glucosyltransferase found in Citrus unshiu. These primers were designed so that PCR products would encompass the signature Plant Secondary Product Glucosyltransferase (PSPG) domain. Forward primer JC2 was taken from an area of the sequence approximately 50 base pairs ahead of the PSPG box. Reverse primer JC4 was taken from an area of the sequence approximately 100 base pairs after the PSPG box toward the 3’ end. A PCR reaction with cDNA from C. paradisi yielded a fragment approximately 500 base pairs in length. This fragment was cloned into TOPO vector and plasmids were isolated from the cells, digested using EcoR1, and sent for sequencing. Analysis of sequences confirmed the presence of the PSPG box and showed >90% homolysis with the liminoid GT in C. unshiu. New primers CU5R and CU6F were designed approximately 75 base pairs from the 5’ and 3’ ends respectively. These primers were used in separate PCR reactions combined with SMARTRACE universal primers in order to "walk out" and obtain additional sequence for this putative GT. These PCR products have been cloned into TOPO vector and isolated, and sequencing results are pending.------------------------------------------------------------------------------------------------------------------------------A COMPUTATIONAL CHEMISTRY STUDY OF SPIN TRAPS

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Tande Jacob Fosso. Department of Chemistry, East Tennessee State University, Johnson City, TN 37614

Many defects in physiological processes are due to free radical damage: reactive oxygen species, nitric oxide and hydroxyl radicals have been implicated in the parthenogenesis of cancer, diabetes mellitus, and rheumatoid arthritis. Also there is growing evidence that aging is due to free radical mediated regulatory processes that result in altered gene expression. The effects and defects of these radicals are studied using biological probes commonly known as spin traps. In this study, theoretical calculations are carried out on the two main types of spin traps (DMPO and PBN) at the density functional theory level (DFT). The energies of the optimized structures of the spin traps and the hydroxyl radical adduct are calculated at the DFT(B3LYP)/6-31G(d), hyperfine calculations in gaseous and aqueous phases are carried out at the DFT (B3LYP)/6-31G(d)/6-311G(2df,p). The dielectric effect on the performance of the spin trap is determined using the polarized continuum model. Calculations show a localization of spin densities in both cases. However, DMPO spin traps are shown to be more stable and more interactive in aqueous environment.

Spin density map of PBN hydroxyl radical adduct

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CHARACTERIZATION OF G.S.INT1, A GROUP IIC INTRON FROM THE THERMOPHILLE GEOBACILLUS STEAROTHERMOPHILUS H. Sun, S.E. Moretz and B.C. Lampson. Department of Health Sciences, East Tennessee State University, Johnson City, TN 37615 and NIH, Rockville, MD Group II introns are small segments of DNA that reside in the chromosome of bacteria or the genome of organelles of primitive eukaryotes. These elements have two very interesting properties. First, they are retrotransposons that can move to a new location in DNA via a reverse transcription mechanism. Second, they form a large ribozyme that mediates self-splicing of the intron from pre-m RNA. Recently, a group II intron type protein with similarity to reverse transcriptase was discovered in the thermophile Geobacillus stearothermophilus strain 10 (Vellore et al., 2004. AEM 70: 7140). The purpose of this work is to clone and characterize the intron that codes for this protein. Because it is found in a thermophile, this intron (and ribozyme) may have novel properties and structural changes that allow it to function at hot temperatures. Numerous copies of the intron protein gene are present in the chromosome of strain 10, but absent from a related ATCC 12980 strain, based on a Southern hybridization

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experiment. Three separate copies of the entire intron plus flanking exon DNA were cloned (and their DNA sequenced) from a HindIII restriction fragment, plasmid library of the strain 10 genome. Because the intron has inserted into different positions in the chromosome the exact junctions of the intron were identified. The intron, designated G.s. Int1, is about (the size varies) 1890 base pairs and codes for a bacterial type C intron ribozyme. Analysis of the nearly completed genome sequence of strain 10 identified at least seven additional copies of the intron, one being a truncated copy. DNA primers, designed to anneal to exon DNA that flanks the intron, were used to detect in vivo splicing of the intron in G. stearothermophilus cells. The intron was cloned into a T7 expression plasmid to allow inducible expression in Escherichia coli. Splicing experiments using these constructions are in progress. Keyword: Geobacillus stearothermophilus; Reverse transcriptase; Group II intron-------------------------------------------------------------------------------------------------------------------------------


TYPICALLY DEVELOPING CHILDREN DISPLAY TWO PRELINGUISTIC ACTS PER MINUTE BEFORE FIRST WORDS

Jessica Brown, Megan Ringley, Tiffany Barber, Chantal Newell and Dr. Kerry Proctor-Williams, PhD, CCC-SLP. Department of Communicative Disorders – Speech-Language Pathology, East Tennessee State University, Johnson City, TN 37614

The goals of this longitudinal study were to define the transitional rates per minute for protoimperatives, protodeclaratives, behavioral regulation, joint attention, social interaction, canonical vocalizations, and total communicative acts at both the prelinguistic and one-word stages of language development. Specifically, we asked: Are the rates of these behaviors the same in both unstructured and uninstructed parent-child free-play and book-reading activities as in those the more structured samples collected with clinicians in Wetherby, Cain, Yonclas, and Walker (1988)? This study examined 29 typically developing children at 7, 10, 13, and 20 months during structured and uninstructed parent-child interactions. Unstructured and uninstructed conditions create a more natural setting for the child and mother, allows for more variability in parent’s responsiveness, and is more consistent with diagnostic procedure in the field of speech language pathology. The following categories statistically significant increased from the prelinguistic to one-word stages and yielded medium effect sizes: total communicative acts, protodeclaratives, and canonical vocalizations. The average rate of total communicative acts used by participants in the prelinguistic stage was 1.11 acts per minute. The average rate of total communicative acts used by participants in their first one word stage was 2.12 acts per minute. This shows that children must use somewhere between 1.11 and 2.12 acts per minute before transitioning into the one-word stage. As well, correlations were conducted to see if rates of total communicative acts and use of canonical vocalizations at early ages predicted later production of words. The sample included a slightly smaller sub-sample of 27 children because two subjects never reached the one word stage. Our results indicated significant relationships between rates of prelinguistic communicative acts and later word production. Our findings generally coincide with those of Wetherby et al. (1988) in that typically developing children display around one communicative act per minute for the prelinguistic stage and that this rate is necessary for transition to the one word stage. Our data predicts that children need to display an overall increase in communicative act functions, with the most critical being

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protodeclaratives, canonical vocalizations, and total communicative acts. Future research should replicate this study using a larger sample size with a more economically and ethnically diverse population to represent more adequately the population as a whole. Future studies need to incorporate longer unstructured activities for mother and child participation in order to elicit a wider range of communicative acts. The results from this study will provide speech- language pathologists with normative guidelines about transitions from the prelinguistic stage to the one-word stage. This information can assist in assessment and therapy of language-impaired children.------------------------------------------------------------------------------------------------------------------------------

AMPHETAMINE SENSITIZATION IN A RODENT MODEL OF PSYCHOSIS

Zackary A. Cope, A. Brianna Sheppard, Ian D. Longacre, Marla K. Perna, Kimberly N. Thompson and Russell W. Brown. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

This study was designed to analyze the effects of amphetamine on locomotor sensitization in a rodent model of psychosis developed in our laboratory. Past studies have shown neonatal quinpirole (dopamine D2/D3 agonist) produces a significant increase in dopamine D2 receptor sensitivity that persists into adulthood, a phenomenon known as D2 receptor priming. An increase in D2 receptor sensitivity is consistent with several behavioral disorders, including schizophrenia. Additionally, a past study from a collaborating laboratory has shown that acute amphetamine (street name: Speed) administration produces a 5-fold increase in dopamine release of D2-primed rats, suggesting that the dopamine system in the brain, which mediates positive reinforcement, is hypersensitive in psychosis. Congruent with this findings, individuals that are diagnosed with schizophrenia demonstrate a 4-5 fold increase in abuse of drugs in the psychostimulant class, of which amphetamine belongs, which suggests that increases in dopamine activity in response to amphetamine may be more reinforcing to schizophrenics than to the normal population. Rats were administered quinpirole (1 mg/kg) or saline from postnatal days 1-11 and raised to adulthood (postnatal day 60). Beginning in adulthood, rats were administered d-amphetamine sulfate (1 mg/kg) or saline every other day for 14 days, resulting in a total of seven exposures to the drug. Approximately 10 min after drug injection, rats were placed in a locomotor arena and overall activity was analyzed using a software program purchased in our laboratory (AnyMaze, Stoelting, Wood Dale, IL). Results showed that D2-primed rats receiving amphetamine demonstrated a significant increase in locomotor activity across all seven days of testing relative to all other groups. This is consistent with past findings demonstrating the D2-primed rats administered amphetamine demonstrated significant increases in dopamine levels, as increases in dopamine levels in the brain typically positive correlate with significant increases in locomotor activity. Controls receiving amphetamine also demonstrated a significant increase in activity over days. Interestingly, D2-primed rats given saline also sensitized to the environment, but this is consistent with past observations in our laboratory and suggests a lack of ability in these animal to habituate to the environment. Seven to fourteen days after locomotor sensitization testing was complete, cerebrospinal fluid samples were taken via microdialysis from the nucleus accumbens core to be analyzed for dopamine levels, and these samples will be assayed in the coming months. -------------------------------------------------------------------------------------------------------------------------------

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EVALUATION OF THE EFFICACY OF AFTER SCHOOL PROGRAMS WITH AT RISK CHILDREN

Heather R. Hyder and Chris S. Dula, PhD. Department of Psychology, East Tennessee State University, Johnson City, TN 37614

The purpose of this study was to evaluate the efficacy of an after school program with children at-risk for poor behavioral outcomes. Participants in the program were referred by school guidance counselors, classroom teachers, the Department of Children’s Services and Juvenile Court Systems. Referrals were largely for lower socioeconomic status children and based on poor academic performance, socially deviant or defiant behavior, and potentially unsafe home environments. Research has suggested that socially deviant behavior, family history of problem behavior, divorce, child abuse and neglect, average or below average academic proficiency, truancy, absenteeism, economic deprivation and higher community crime rates are positively correlated to problem behaviors in children (OJJDP Model Programs Guide). The after school program implemented a standardized program known as Life Skills Training, which focused on improving decision making skills related to socially acceptable behaviors at home and in school, resisting negative peer influences, refusing drugs, and avoiding delinquent behavior. Data were collected by the host community organization and were analyzed by the present authors as independent consultants. With appropriate qualification due to issues with a large number of missing data points and a lack of demographic data, involvement in Life Skills Training produced an increase in knowledge about risky situations and behaviors and an increase in positive attitudes and intentions to resist negative influences and to choose positive behaviors. The evaluation of whether such programs are successful is essential for the effective design and implementation of future programs. Future directions will examine the conditions in which after school programs are most successful and what amount of time in a program produces the most desirable outcome.-----------------------------------------------------------------------------------------------------------------------------

VOICE ONSET TIME AS A CLINICAL INDICATOR OF HYPERFUNCTIONAL VOICE DISORDERS

Brandon Phillips. Department of Communicative Disorders, East Tennessee State University, Johnson City, TN 37614

Voice onset time (VOT) is a measurable time interval that can be used to measure glottal pulsing after the oral release of a stop consonant. VOT is an important measure for distinguishing stop consonant voicing and place of articulation during speech across a variety of languages. This measurement is relatively easy to make and displays the complexity of the coordination between the supralaryngeal and laryngeal mechanisms. Given that VOT reflects the timing differences between supralaryngeal articulation and phonatory gestures, previous studies have examined if VOT can reflect laryngeal dysfnuction. Tyler and Watterson (1991) compared the VOTs of individuals with hyperfunctional voice disorders and individuals with normal laryngeal function, and reported that the individuals with laryngeal hyperfunction displayed longer VOTs for voiced stops than the normal laryngeal function group. However, several extraneous variables were not controlled, including rate of speech, gender of the participants, and vowel context, each of which have been shown to influence VOT values (Ryalls et al., 1997; Whiteside & Irving, 1997; 1998; Robb et al,. 2005). Thus, the purpose of this study was to examine the VOTs of individuals with hyperfunctional voice disorders when compared to the VOTs of individuals with normal voices within English stop consonants while

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controlling for rate, gender, and vowel context. Based on the results of previous research, it was hypothesized that individuals with hyperfunctional disorders would display longer VOTs for voiced stops than individuals without hyperfunctional voice disorders.

Recordings were made of 10 adults between the ages of 18-55 with normal laryngeal function and 10 adults between the ages of 18-55 with hyperfunctional voice disorders. Participants were classified as hyperfunctional or normal by undergoing a diagnostic voice assessment, including auditory-perceptual measures and acoustic measures. Once the participants were divided into two separate groups, they were each instructed to read six short phrases containing English stop consonants in the word-initial position (ex., “Peek at a peacock.”). The stop consonants contained within the phrases were the analyzed for VOT. A mixed analysis of variance was used to determine any between group and/or within group differences.

Results indicated that both the hyperfunctional group and the normal group produced similar VOT’s across all English stop phonemes; thus, no significant differences in VOTs were reported between the groups. The current results differ with previous research; however, the current methodology used was much more strident than previous studies. It is important to mention that laryngeal gestures involved in the contrast between voiced and voiceless stop consonants is only one of many coordinated muscular movements reflected in VOT. Thus, it seems that VOT cannot be used as a reliable measure of laryngeal function by itself.-------------------------------------------------------------------------------------------------------------------------------

NICOTINE-CONDITIONED HYPERACTIVITY IN A RODENT MODEL OF PSYCHOSIS

Brianna Sheppard and Russell W. Brown, Department of Psychology, East Tennessee State University, Johnson City, TN 37614

Nicotine, the addictive psychostimulant drug in cigarettes, has positive reinforcing properties that often become associated with environmental and contextual cues. The schizophrenic population in particular exhibits especially high rates of smoking, which is nearly three times that of the general population. The high incidence of smoking among these patients raises the possibility of an underlying neurochemical mechanism involving schizophrenia and nicotine use in which the midbrain dopaminergic system, also referred to as the mesocorticolimbic pathway, has been implicated. This high incidence of smoking may be further exacerbated by the presence of nicotine-associated cues. Past studies have shown that a nicotine-conditioned context can elicit locomotor hyperactivity in a rodent model of nicotine addiction during a nicotine-free trial. However, the role of context in nicotine addiction has not been examined in an animal model of schizophrenia. The aim of this investigation was to determine the ability of a nicotine-conditioned context to elicit locomotor hyperactivity in a neonatal quinpirole animal model of schizophrenia. Sprague-Dawley rats were treated with either saline or the dopamine D2 receptor agonist quinpirole from postnatal days (P) 1-21 to create long-term D2 receptor supersensitivity that persists throughout the animal's lifetime, a phenomenon known as D2 priming. On P29, the beginning of early adolescence in the rat, animals were habituated to a locomotor arena for three consecutive days. On each day of habituation, all animals were given an injection of saline and placed into a locomotor arena ten minutes later, and each habituation session lasted for ten minutes. This was done to allow animals to adjust to surroundings of the arena. On P32, animals were injected i.p. with either nicotine (0.5 mg/kg free base) or saline and placed in the locomotor arena 10 minutes later. Results showed that D2 primed rats that received nicotine demonstrated a significant increase in activity compared to all other groups, suggesting that the dopamine system may have a hyperactive response to nicotine in D2-primed animals. Interestingly, on the drug-free test, these animals were also

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more active compared to all other groups, suggesting that nicotine may have stronger associative properties in D2-primed as compared to non D2-primed animals.------------------------------------------------------------------------------------------------------------------------------

RELATIONSHIP BETWEEN PARENTAL ATTITUDES TOWARD WEIGHT/WEIGHT CONTORL AND CHILD FEEDING PRACTICE

Liang Wang, Amit Bodhani, Tiejian Wu and Karen Schetzina. Department of Public Health, East Tennessee State University, Johnson City, TN 37614

Childhood obesity is a significant and growing health problem in the US. Secular trend in the prevalence of child obesity in the US suggest that children have become substantially heavier over the last three decades, and as a result their risk for a number of health problems including hypertension and type II diabetes is increasing. Parents are key agents to develop a home environment that fosters healthful eating and physical activity among children and adolescents. Parents can shape their children’s dietary practices, physical activity, sedentary behaviors, and ultimately their weight status in many ways. The purpose of this study was to investigate parental attitudes toward weight/weight control and their associations with child feeding practice/behaviors. Study subjects included 60 mothers or other primary child care givers of children 5-11 years old who were attending a pediatric clinic for health care. Parental attitude toward weight/weight control was assessed using the Dieting Belief Scale (DBS) and child feeding practice was assessed using the Child Feeding Questionnaire (CFQ). Preliminary data analysis indicated a significant association between parental attitudes toward weight/weight control and certain child feeding practice/behaviors.------------------------------------------------------------------------------------------------------------------------------

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Division III—Graduate Students 2+ years

Biomedical Sciences

IDENTIFICATION OF A POSSIBLE FUNCTIONAL CRE-ELEMENT FOUND IN THE PROMOTER OF THE α7 NICOTINIC RECEPTOR SUBUNIT AND ITS ASSOCIATION WITH SCHIZOPHRENIA

Michelle Chandley, Chris Newell, Tracy Wilson, John Beddies and Dr. Barney Miller. Departments of Psychiatry and Anatomy/Cell Biology, East Tennessee State University, COM, Johnson City, TN 37614

Objective: The identification and characterization of a possible Cyclic-AMP response element (CRE) within the promoter region of the α7 subunit gene.

Abstract: Insight regarding the complex etiology of schizophrenia may lie in one of its most well-known symptoms, auditory hallucinations (AH). One abnormality that has been described in schizophrenia, that may contribute to AH, is the lack of auditory gating, i.e. the inability to filter background stimuli and focus on a single auditory. This gating deficit can be demonstrated by attenuation of P50, which is a measurable brainwave evoked by external auditory stimuli. The neural pathway associated with P50 involves the α7 subunit of the acetylcholine nicotinic receptor (α7nAChR), which has high affinity binding to nicotine, and has been implicated in normal auditory gating, suggesting a link between schizophrenia and this receptor. A7nAChR’s involvement is further supported by the high incidence of tobacco use in the diagnosed schizophrenic population, between 85% and 90%, more than triple the normal use. Nicotine up-regulates the expression of α7nAChR, perhaps explaining the seemingly self-medicating behavior. In addition, diminished post-mortem brain and blood levels of the α7 nicotinic receptor have been reported in schizophrenic patients. Transcriptional regulation of the α7nAChR subunit of the nicotinic receptor is poorly understood. Identifying the transcriptional events that lead to expression of α7 in the central nervous system could uncover alterations in these control mechanisms which might establish a bridge between the symptoms (such as AH) and a genetic etiology. A cyclic-AMP response-like element is located in the proximal promoter region of the α7nAChR gene. The purpose of this study was to assess whether this promoter region acts as a functional CRE (cyclic-AMP response element) and to determine if polymorphisms flanking this region, which have been found in schizophrenic patients, have any effect on transcription of the gene. Electrophoretic Mobility Shift Assay (EMSA) was performed using radioactively-labeled oligonucleotides representing the putative CRE region of the α7 promoter and Cyclic-AMP response element binding protein (CREB). Controls included a purchased, concensus CRE-element oligonucleotide, a mutated CRE-element oligonucleotide, and a concensus Activating Protein 2 (AP2) DNA transcription site. Reporter gene constructs were created using the α7nAChR promoter to drive expression of luciferase protein. Chromatin crosslinking/immunoprecipitation assays (CHIP), using antibodies for CREB, were used to isolate DNA bound in vivo to CREB, which were then subjected to PCR using primers specific to the α7 promoter region. Phosphoimaging of the EMSA gels demonstrated a shift in the mobility of the labeled oligonucleotides in the presence of CREB protein. Luciferase reporter gene

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assays using mutated regions of the promoter demonstrated a decrease in gene expression compared to consensus promoter sequences. CHIP assay/PCR analysis of chromatin isolated using CREB antibodies displayed a 400bp amplified band which migrates with the same MW as the band amplified from genomic DNA. The region of DNA in the α7 promoter analyzed in this study could be a previously uncharacterized regulatory promoter element (CRE) in the gene for the α7 subunit of the nicotinic receptor. By characterizing the transcriptional process of α7, new advances in molecular medicine within this pathway may lead to a more effective treatment for schizophrenia. ------------------------------------------------------------------------------------------------------------------------------

A CANNABINOID (WIN 55,212-2) INDUCES APOPTOSIS IN PANCREATIC TUMOR CELLS VIA CB1-DEPENDENT AND CB1-INDEPENDENT MECHANISMS

Theresa Pickle and Douglas Thewke, Department of Biochemistry and Molecular Biology, East Tennessee State University, COM, Johnson City, TN 37614

Pancreatic cancer is the fourth leading cause of cancer related deaths. In 2007, an estimated 37,170 Americans will be diagnosed with cancer of the pancreas and an estimated 33,370 Americans will die of pancreatic cancer. The mean survival rate is ~6 months, and only 4% of patients survive five years. Despite extensive testing, only one chemotherapy agent (gemcitabine) has been found to produce any benefit to these patients, unfortunately, the clinical response rate to gemcitabine is less than 10% with life prolongation being only 6 weeks on average. Cannabinoids, such as 9-tetrahydrocannabinol (THC), the active agent of Cannabis sativa, exhibit anti-tumor properties. Cannabinoids produce their biological effects by engaging specific receptor-mediated signaling pathways. To date, two cannabinoid-specific receptors, designated CB1 and CB2, have been cloned and characterized from mammalian tissue. The current study was conducted to investigate the potential use of cannabinoids as an anti-tumor treatment in pancreatic cancer. Using RT-PCR and immunoblotting we found evidence for expression of CB1 and CB2 in three human pancreatic cancer tumor cell lines (AsPC1, CaPan1, and Mia-PaCa2). Cell viability analysis revealed that treatment of these pancreatic tumor cell lines with Win 55,212-2, a potent CB1/CB2 agonist, results in a significant reduction in cell viability. When AsPC1 cells were subjected to Win 55,212-2 in combination with gemcitabine or 17-AAG, an hsp90 inhibitor, a cumulative decrease in cell viability was observed after 48 hours. Treatment of Mia-PaCa2 and CaPan1 cells with Win 55,212-2 in combination with gemcitabine or 17-AAG did not result in additive growth inhibition. Caspase-3 activity assays revealed that the growth inhibitory effects of Win 55,212-2 involve induction of apoptotic mechanisms. The induction of caspase-3 activity by Win 55,212-2 in Mia-PaCa2 cells was prevented by a CB1 receptor-specific antagonist, AM251, but not by a CB2 receptor-specific antagonist, SR144528, suggesting the mechanism of apoptotic induction in Mia-PaCa2 cells is mediated by CB1. However, AM251 had no effect on the induction of caspase-3 activity in AsPC1 or CaPan1 cells. In conclusion, the results presented here demonstrate that several pancreatic tumor cell lines express cannabinoid receptors and that a cannabinoid, Win 55,212-2, potently inhibits the viability of these pancreatic tumor cells, at least in part, by inducing apoptosis via CB1-dependent and CB1–independent mechanisms. -------------------------------------------------------------------------------------------------------------------------------

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HSPA12B IS INDUCED BY HYPOXIA/REOXYGENATION IN VITRO AND IS PROTECTIVE AGAINST MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN VIVO

Rebecca Steagall, Fang Hua, Chuanfu Li, Nilanjana Maulik and Zhihua Han, Department of Biochemistry and Molecular Biology, East Tennessee State University, COM, Johnson City, TN 37614 and Molecular Angiogenesis and Cardiology Laboratory Department of Surgery, University of Connecticut Medical Center, Farmington, CT 06030

Hypoxia and oxidative stress associated with Ischemia and reperfusion (I/R) represent major mechanisms of tissue injury and organ failure. Angiogenesis is a major protective mechanism against ischemic injury. Recently, we cloned Heat shock protein A12B (HspA12B), the newest member of a newly defined sub-family of proteins related to the Hsp70 family that is predominantly expressed in endothelial cells (ECs) and is required for angiogenesis. Because of its homology to Hsp70 we proposed that it may represent a specialized molecular chaperone that is involved in angiogenesis. In the present study, we found that HspA12B expression was induced by hypoxia and reoxygenation in endothelial cells. We used three models to investigate the hypoxia-induced HspA12B gene expression and its protective effect against I/R-induced myocardial infarction conceivably due to HspA12B stimulated angiogenesis. First, in human coronary artery endothelial cells (HCAECs), knockdown of HspA12B by siRNA resulted in a significant decrease in hypoxia-induced tubular morphogenesis compared to control. Second, in the in vivo C57BL/6 mouse model, we found that angiogenesis was induced by over-expression of HspA12B by adenovirus in a Directed in vivo Angiogenesis Assays (DIVAA). Third, in the in vivo myocardial I/R injury model, the HspA12B adenovirus decreased I/R-induced infarct size in the myocardium. In conclusion, these results suggest that pro-angiogenic HspA12B is induced by hypoxia and may contribute to a cardioprotective effect in myocardial ischemic models. Many studies have shown that pro-angiogenesis treatments protect against I/R. Alternatively, cytoprotective effect could arise from either protection against apoptosis or chaperone activity, both of which have been demonstrated for Hsp70s. We show here that hypoxia induced HspA12B regulated angiogenesis provided protection against I/R injury to the myocardium. We are in the process of elucidating the mechanisms. Ultimately, over-expression of a therapeutic HspA12B transgene using a vector that will provide expression in response to an endogenous pathophysiological stimulus such as hypoxia may protect tissues at risk of ischemia/reperfusion injury.-------------------------------------------------------------------------------------------------------------------------------

HSV-2 CO-INFECTION STIMULATES CHLAMYDIA TRACHOMATIS PERSISTENCE VIA A NOVEL MECHANISM

J. Vanover, S. Deka, J. Sun, J. Kintner, J. Whittimore and R.V. Schoborg, Department of Microbiology, East Tennessee State University, COM, Johnson City, TN 37614 and ViroMed Laboratories/LabCorp, Minnetonka, MN 55343

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Several models of chlamydial persistence have been previously described, including IFN-, IFN-α, IFN-β, and TNF- α exposure and iron, amino acid or glucose deprivation. We hypothesize that HSV-2 co-infection induced chlamydial persistence is not mediated by one of these known inducers of persistence. ELISA analyses demonstrate that accumulation of intracellular ferritin remains similar in co-infected and singly-infected cells, indicating that viral co-infection does not reduce host intracellular iron stores. Supplementation

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with excess amino acids, iron-saturated holotransferrin, or glucose during co-infection also does not restore chlamydial infectivity in co-infected cells, demonstrating that viral-induced persistence is not mediated by depletion of these nutrients. Previously published studies from our laboratory demonstrate that HSV co-infection induces persistence in co-infected HEC-1B cells. Because HEC-1B cells do not produce indoleamine-2,3-deoxygnease (IDO) in response to IFN-γ, these data suggest that viral-induced persistence is not mediated by IFN- . Several studies indicate that the chlamydial trp operon is strongly up-regulated when the organisms are confronted by tryptophan limiting conditions, as occurs during IFN- induced persistence. However, RT-PCR studies indicate that chlamydial trpA mRNA is not up-regulated during co-infection. Additionally, Luminex assays indicate that IFN-, IFN-α, and TNF- α are not released from co-infected HeLa cells. Finally, RT-PCR studies demonstrate that IFN-, IDO, and IFN-β mRNA transcripts are not present in co-infected HeLa cells. Collectively, these data suggest that the synthesis/release of IFN-, IFN-α, IFN-β and TNF- α is not responsible for viral-induced persistence. Co-infection with UV-inactivated, replication incompetent HSV-2 reduces chlamydial infectivity without altering chlamydial genomic DNA accumulation, suggesting that productive HSV replication is not required. Furthermore, co-incubation of fixed, HSV-2 infected inducer cells with viable C. trachomatis infected responder cells suppresses production of infectious chlamydial elementary bodies and stimulates the formation of swollen, aberrantly shaped reticulate bodies. These data: 1) demonstrate that co-infection induced persistence is not mediated by any currently characterized persistence inducer and 2) suggest that HSV attachment to host cell surface receptors can provide the necessary stimulus to alter C. trachomatis development. Thus, we hypothesize that, during HSV-2 attachment, the actions of one or more virion proteins trigger a novel host anti-chlamydial pathway that restricts chlamydial development.-------------------------------------------------------------------------------------------------------------------------------

TRANSCRIPTIONAL REGULATION OF C-REACTIVE PROTEIN EXPRESSION IN HUMAN HEPATOCYTES

Bhavya Voleti, Prem Prakash Singh and Alok Agrawal. Department of Pharmacology, East Tennessee State University, COM, Johnson City, TN 37614

The hepatic synthesis of C-reactive protein (CRP) increases in inflammatory conditions resulting in raised serum CRP levels. The regulation of CRP gene expression in hepatocytes occurs at the transcriptional level. Previously, we reported that a C/EBP-binding site overlapping a NF-ĸB p50-binding site (C/EBP-p50-site) on the CRP promoter was critical for IL-6-induced CRP expression in human hepatoma Hep3B cells. Transcription factors C/EBPβ and p50 occupy the C/EBP-p50-site in the nuclei containing constitutively active or IL-6-activated C/EBPβ. The aim of this study was to identify the transcription factors that occupy the C/EBP-p50-site in the absence of C/EBPβ. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPβ. Such treated nuclei contain only C/EBPς because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPς. EMSA using this extract revealed formation of a C/EBPς-containing complex at the C/EBP-p50-site. This complex also contained RBP-Jĸ, a transcription factor known to interact with the ĸB sites. RBP-Jĸ was required for the formation of C/EBPς-containing complexes. The RBP-Jĸ-dependent C/EBPς-containing complexes were also formed at the C/EBP-p50-site on the CRP promoter in the nuclei of primary human hepatocytes. In transactivation assays, overexpressed C/EBPς abolished both C/EBPβ-induced and (IL-6+IL-1β)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPς occupies the C/EBP-site, an action that requires RBP-Jĸ. Under induced conditions, C/EBPς is replaced by C/EBPβ and p50. We conclude that the switch

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between C/EBPβ and C/EBPς participates in regulating CRP expression. This process utilizes a novel phenomenon, that is, the incorporation of RBP-Jĸ into C/EBPς-complexes solely to support the binding of C/EBPς to the C/EBP-site.------------------------------------------------------------------------------------------------------------------------------

PROTECTIVE MECHANISMS OF NECROSTATIN-1 ON GLUTAMATE-INDUCED TOXICITY IN HT-22 CELLS

Xingshun Xu, Chu C. Chua, Jiming Kong, Richard M. Kostrzewa, Udayasankar Kumaraguru, Ronald C. Hamdy and Balvin H.L. Chua. Department of Pharmacology, East Tennessee State University, COM, Johnson City, TN 37614

Glutamate, a major excitatory neurotransmitter in the central nervous system, plays a critical role in nervous system disorders such as stroke and Parkinson’s disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1, an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that necrostatin-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, necrostatin-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that necrostatin-1 has no antioxidant effects. Interestingly, the protective effect of necrostatin-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that necrostatin-1 significantly blocks the nuclear translocation of AIF (a marker of caspase-independent programmed cell death) and inhibits the integration of BNIP3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that necrostatin-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH-related as well as AIF and BNIP3-related pathways.

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AN ANALYSIS OF LOUDNESS PERCEPTION IN PERSONS WHO EXPERIENCE TINNITUS

Jeffrey A. Py and Marc Fagelson, PhD. Department of Communication Disorders, East Tennessee State University, Johnson City, TN 37614

The purpose of this study was to examine whether persons with normal hearing thresholds and tinnitus performed differently on a test of intensity discrimination when compared to results from persons with normal hearing and without tinnitus. There were two groups of five subjects; Group 1 consisted of normal hearing patients without tinnitus and Group 2 consisted of subjects with normal hearing and subjective complaints of tinnitus. The test stimulus consisted of three trains of five short tone pips. The five tones were presented in one of three patterns; intensity increasing in 0.5 dB steps (producing a cumulative of 2.5 dB increase), intensity decreasing in 0.5 dB steps (producing a cumulative 2.5 dB decrease), or intensity unchanged. Initial tone level was varied randomly around a 50 or 70 dB HL standard in order to minimize onset and offset cues. Verbal judgments were made by test subjects with regard to their perception of a

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stimulus train increase, decrease or no change in stimulus level. Differences between the two groups were compared by 1) proportion of correct responses and 2) proportion of response types (either ascending, descending, or unchanged). There was no significant difference between groups regarding number of correct responses, but there was a significant difference between groups regarding response type. The results indicated a possible processing difference with regard to intensity discrimination in that the patients with tinnitus were either unable to discern, or unwilling to report, a change in signal loudness. ------------------------------------------------------------------------------------------------------------------------------

TRADITIONAL VS COMPUTER-BASED INTERVENTION FOR TREATMENT OF SPEECH SOUND DISORDERS

Ashley Rice, Julie Eanes, Marsha Mallory and A. Lynn Williams. Department of Communicative Disorders, East Tennessee State University, Johnson City, TN 37614

Children with speech sound disorders (SSD) comprise the most common communication disability in preschool and school-aged children (American Speech-Language Hearing Association, 2000). Traditional treatments of SSDs utilize pictured stimuli while working with children at a table. Recent technological advances have resulted in the availability of software programs for treating SSDs using computer-based intervention (CBI). Despite the emergence of software programs, there is little research that has compared the relative effectiveness and benefits of these two treatment formats. Such research would be of value in assisting speech-language pathologists (SLPs) to make decisions regarding the most efficacious and effective treatment strategies for children with SSDs. The purpose of this study was to determine the differences, if any, in treatment outcomes for children receiving a traditional paper format of therapy versus a computer format. The computer software program Sound Contrasts in Phonology (SCIP) was utilized to create materials for both table-top and computer conditions. The SCIP illustrations were printed out for the traditional condition, whereas the computer conditions operated the SCIP illustrations on the computer screen. Participants for this study were two males (3 years, 8 months and 4 years, 7 months) with moderate to profound SSD. A single-subject AB design was used to evaluate the treatment outcomes of each child. Participants were randomly assigned to treatment conditions. Participant 1 received the traditional treatment condition and Participant 2 received the CBI condition. One phonological goal was targeted for each child in the assigned treatment condition. Treatment consisted of five minimal pair contrasts for each targeted phoneme in both treatment conditions. Treatment sessions were held twice weekly for 30 minutes in length per session. A generalization probe of the target sounds in an untrained context was constructed for each participant in order to measure baseline performance as well as learning throughout the course of treatment. The results indicate that the computer condition was an effective service delivery format in achieving improvement for treatment and generalization. In addition, these gains were achieved in less time (5 sessions) as compared to the traditional condition (12 treatment sessions).-------------------------------------------------------------------------------------------------------------------------------

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Division IV—Medical Students

RELATIONSHIP OF MATRIX METALLOPROTEINASES WITH MARKERS OF FETAL LUNG MATURITY

Scott Cook, Tiffany Ford, Gary Randall, John Kalbfleisch and Kevin Breuel. Departments of Obstetrics and Gynecology and Biometry and Medical Computing, East Tennessee State University, COM, Johnson City, TN 37614

Neonatal respiratory distress syndrome (NRDS) remains a major cause of infant morbidity and mortality. The primary cause of neonatal NRDS is a deficiency in the production of surfactant which results in a reduction in surface tension in the alveolar walls and reduced gas exchange by the lungs. Diagnosis of neonates with fetal lung immaturity allows effective intervention which reduces the risk of NRDS. Specialized laboratory tests (L/S Ratio, Optical Density (OD) and PG) used to diagnose lung maturity in amniotic fluid are quite sensitive (95-96%) in identifying infants with immature lungs whom as a result may develop NRDS. However, these tests lack specificity (60-68%) which result in falsely diagnosing infants with immature lungs when they are actually mature. Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are known to regulate extra cellular matrix remodeling and thus are important in the morphogenesis of all the fetal organs. The objective of this study was to determine whether amniotic fluid levels of various MMPs are correlated with L/S ratio, OD or levels of PG and thus might be used to assess fetal lung maturity (FLM). Seventy-eight, amniotic fluid samples, previously collected for assessment of FLM, were removed from storage (-80°C) and processed for analysis of MMP (1, 2, 3, 7, 8 and 12) levels. Levels of various MMPs were quantified with a Fluorokine MAP multiplex assay kit run on the Luminex 100 instrument. L/S and OD measures were related to MMPs with single and multiple regression analysis. Additionally, mean MMPs were compared for presence (Mature) and absence (Immature) of PG banding subgroups. P = 0.05 or smaller was used for statistical significance. The L/S and OD were always positively correlated with MMP3 levels (P<0.01) irrespective of whether the other MMPs are included in the regression analysis. Mean levels of MMP3 (270±40 pg/ml) and MMP7 (19,447±3565 pg/ml) in amniotic fluid samples with PG present were higher (P<0.05) when compared to samples without PG (142±32 and 6,031±1659, respectively). In conclusion, MMP3 and MMP7 may be candidates for further study in an attempt to identify a predictor of FLM that is both sensitive and specific.------------------------------------------------------------------------------------------------------------------------------

BINDING OF C-REACTIVE PROTEIN TO IMMOBILE IMMUNE COMPLEXES

Christopher L. Cropsey, Madathilparambil V. Surech, Sanjay K. Singh and Alok Agrawal. Department of Pharmacology, East Tennessee State University, COM, Johnson City, TN 37614

C-reactive protein (CRP) participates in the first line of defense against pathogens. The exact mechanism through which CRP functions in vivo has not been defined yet. In vitro, CRP has been shown to bind to Fcγ receptor IIa (also called as CD32). CD32 is present on the cells of

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the immune system and is the receptor for IgG-containing immune complexes. In this study, we explored CRP-CD32 interaction in an ELISA-based solid-phase binding assay using recombinant CD32-coated plates. CRP bound to CD32 in a dose-dependent manner. Surprisingly, when CD32 was blocked with anti-CD32 IgG antibodies, the binding of CRP to CD32 was not inhibited, indicating a possible interaction between CRP and Cd32-anti-CD32 complexes. Accordingly, when normal huma IgG-coated plates were used in the assay, we observed a dose-dependent binding of CRP to igG. The inability of the binding of a mutant form of CRP to IgG further showed the specificity of CRP-IgG interaction. CRP also bound to immune complexes prepared from BSA and Anti-BSA antibodies, indicating that the presence of the antigen did not affect the binding of CRP to IgG. CRP did not bind to immune complexes in the fluid-phase. The capability of CRP to interact with immobile immune complexes suggests that CRP may also bind to immune complex-occupied CD32-bearing cells and modulate intracellular signaling an subsequent inflammatory responses triggered by the binding of immune complexes to CD32. Our findings have implications for the functions of CRP in the antibody-mediated clearance of pathogens and in immune complex-mediated pathologic conditions.------------------------------------------------------------------------------------------------------------------------------

INHIBITORY EFFECTS OF BAICALEIN ON PRODUCTION OF SELECTED INFLAMMATORY CYTOKINES BY IL-1Β-ACTIVATED HUMAN MAST CELLS

Justin R. Sigmon, Chia-Jung Hsieh, Kenton Hall, Guha Krishnaswamy, Tuanzhu Ha, Chuanfu Li and David S. Chi. Departments of Internal Medicine and Surgery, East Tennessee State University, COM, Johnson City, TN 37604

Human mast cells play an integral role in the inflammatory response by mediating the production of inflammatory cytokines. Baicalein (BAI), a compound isolated from the traditional Chinese medicinal herb Scutellaria baicalensis Georgi, has been shown to have anti-inflammatory effects. We examined the effects of BAI on the production of inflammatory cytokines, IL-6, IL-8, and MCP-1 from IL-1β-activated human mast cells (HMC-1). Two mL of HMC-1 at 1 x 106 cells/mL were cultured with BAI (at predetermined non-toxic concentrations) in the presence or absence of IL-1β (10 ng/mL) for 24 hrs. The supernatants were harvested and assayed for cytokines by ELISA. BAI alone did not induce cytokine production from HMC-1. However, BAI (15 and 30 µM) significantly decreased production of IL-6 and MCP-1 (p<0.0004) from IL-1β-activated HMC-1. BAI at all tested concentrations (1.8 to 30 µM) significantly decreased the production of IL-8 (all p<0.009) from IL-1β-activated HMC-1 in a dose dependent manner. The results show that BAI had inhibitory effects on the production of selected inflammatory cytokines, most notably IL-8, and may be useful in the development of novel anti-inflammatory therapies. (Supported by the Chair of Excellence in Medicine, the Ruth R. Harris Endowment, and RDC of ETSU.)------------------------------------------------------------------------------------------------------------------------------

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Division V—Residents/Post-Doctoral

FellowsREVIEW OF LITERATURE ON PROTEINURIA IN PREECLAMPSIA

Rachna Bharti, MD. Department of Family Medicine, East Tennessee State University, COM, Johnson City, TN 37614

BACKGROUND INFORMATION: The research comparing 24 h urine protein to urine protein/creatinine ratio in women with suspected preeclampsia has been ongoing since 1990 (most recent study published in December 2006), however many physicians are not comfortable implementing the use of spot urine protein/creatinine ratio for diagnosing preeclampsia.

OBJECTIVES OF THE STUDY: To review the current literature comparing 24 h urine protein and urine protein/creatinine ratio methods and draw conclusions about the significance and validity of the findings of the research. To identify the most appropriate method for diagnosing preeclampsia.

METHODOLOGY: Over 20 articles were retrieved via Medline regarding the methods for diagnosing proteinuria, more than half of which focused specifically on pregnant women. The outcomes of the studies in the articles were then reviewed for statistical significance. Studies were identified as either supportive or non-supportive of the protein/creatinine ratio for diagnosis of preeclampsia as compared to 24 h urine protein collection.

RESULTS: Nearly all of the research articles reviewed support the use of urine protein/creatinine ratio as an alternative to 24 h urine protein for diagnosis of preeclampsia. Only one study done in 2003 found that the protein/creatinine ratio should not be used an alternative to the 24 h urine protein. Although this study found a significant relationship between the two methods (p<.0001), the correlation coefficient was cited as being too low (r2=0.41). However, another study in the same year showed a very strong correlation between the two methods (r2=.929). A majority of the studies agreed that the protein/creatinine ratio method was simpler, faster, and could be a more practical alternative for assessment of proteinuria.

CONCLUSION: The research supports that the urine protein/creatinine ratio is nearly equivalent to the 24 h urine protein analysis. The urine protein/creatinine ratio may be used as reliable method for diagnosing preeclampsia in pregnant women. -------------------------------------------------------------------------------------------------------------------------------

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SURGICAL SIMULATION OF COLD KNIFE CONIZATION AND SUCTION DILATION AND CURETTAGE PROCEDURES USING A PATIENT SIMULATOR AND PAPAYA MODEL Foulk, Brooke E., Eason, Martin J. and Olsen, Martin E. Department of Obstetrics and Gynecology, East Tennessee State University, COM, Johnson City, TN 37614 Objective: Our purpose was to equip residents in performing gynecologic surgical procedures using a patient simulator and papaya model. Some training institutions have used papayas, cantaloupes or other fruits as uterine models for practice of various surgical procedures. We proposed to further the realism of this design and take advantage of this learning tool in combination with our institution’s innovative patient simulator lab to better represent actual gynecologic patients and surgical procedures. Background: Similar to Paul and Nobel’s 2005 description,1 we planned to teach and practice suction dilation and curettage with the papaya model. However, we incorporated our NOELLE™ Birthing Simulator for a more realistic experience. Also, we added to this the simulation of a cold knife conization on the model prior to the suction dilation and curettage.

Methods: Using the papaya as a uterine model, the resident performed two surgeries. The papaya is placed into the NOELLE™ Birthing Simulator, with its stalk representing the cervix inside the vaginal vault. 1. A cold knife conization was performed by the learner using standard surgical instrumentation, with removal of a small, cone-shaped section from the simulated cervix. 2. Next, a suction dilation and curettage was undertaken inside the papaya, which represents the intrauterine cavity. The fruit’s internal contents and seeds, which have a consistency similar to products of conception, were removed. Injection of red dye prior to the procedure allows this material to demonstrate an appearance similar to products of conception. Results: Resident participants of all training levels have undertaken this simulation. Residents found the educational experience to be valuable and an effective teaching tool which prepared them for and realistically simulated actual surgeries on human patients. Conclusions: This papaya model enables the learning curve to rapidly increase in a controlled setting, with unlimited “patients” and time. This allows interns and even students to act as primary surgeon in a practical experience not typically provided at their levels of training. It is well-suited for increasing the learner’s familiarization with the techniques and instrumentation of the simulated procedures. Discussion: We agree with Paul and Nobel’s suggestion “that simulation is an effective first step in teaching uterine aspiration procedures.” The simulation provides an accurate surgical experience with the potential for competency evaluation and pre- and post-procedure testing and safety analysis. The risks involved with live patients and the costs associated with a real-time operating room, staff, anesthesia, and instruments are avoided.

1. Paul, M. and Nobel, K. Papaya: A Simulation Model for Training in Uterine Aspiration, Fam Med 2005; 37 (4):242-4.----------------------------------------------------------------------------------------------------------------------------

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ALVEOLAR HEMORRHAGE: A RARE, BUT UNDERDIAGNOSED COMPLICATION OF TREATMENT WITH GLYCOPROTEIN IIB/IIIA INHIBITORS

Said B. Iskandar, MD, Ehab S. Kasasbeh, MD and Philip D Henry, MD, FACC. Department of Internal Medicine, Division of Cardiovascular Disease, East Tennessee State University, COM and Veterans Affairs Medical Center, Mountain Home, TN, USA

Objective: Alveolar hemorrhage (AH) is a very rare complication of treatment with GP IIb/IIIa inhibitors. Hemoptysis, a constant sign, lacks in specificity, and may occur in confounding syndromes such as pulmonary edema, pulmonary infarction and pneumonia. Here, we performed a large scale retrospective analysis to define the incidence and risk factors of AH in the setting of GP IIb/IIIa inhibitors therapy.

Methods: We reviewed for the period extending from August 1998 to January 2005 consecutive histories of acute myocardial infarction patients receiving coronary arteriography and treatment with either eptifibatide or abciximab. Concomitantly admitted acute myocardial infarction patients not treated with GP IIb/IIIa inhibitors were reviewed and served as a control group. Potential co-variates including pulmonary disease, pulmonary hypertension, smoking, and use of other anticoagulant or anti-platelet agents were evaluated.

Results: Six of 1,810 patients (0.33%) receiving eptifibatide and five of 3,648 patients (0.14%) receiving abciximab exhibited typical symptoms and signs of AH. Contrarily, only one of 4,136 patients (0.025%) receiving no GP IIb/IIIa inhibitors presented with similar symptoms and signs. Statistically significant differences were found between control patients and patients receiving eptifibatide alone (P=0.004). There was also a significant difference between untreated patients and those receiving eptifibatide and abciximab (P=0.017). No differences were found between eptifibatide and abciximab-treated patients (P=0.19) or between abciximab and untreated controls patients (P=0.105).

Conclusions: AH is a very rare complication of treatment with GP IIb/IIIa inhibitors. Its incidence ranged from 0.14% in patients treated with abciximab to 0.33 % in those receiving eptifibatide. Compared to control group, patient treated with GP IIb/IIIa inhibitors had a statistically increased risk for AH.

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FATAL IATROGENIC HYPERPHOSPHATEMIA: A GROWING DISASTER

Dima Nassour, Kosseifi Semaan, Byrd Ryland and Roy Thomas, IV. Department of Internal Medicine, East Tennessee State University, COM, Johnson City, TN 37614

Fleet Phospho Soda is a widely used colorectal laxative, for which the safety profile has been clearly questioned. Its inadvertent use, however, may be associated with a plethora of adverse effects, some of which may be life-threatening. We are reporting an elderly patient who was given sodium phosphate enemas, which resulted in marked hyperphosphatemia and hypocalcemia followed by his demise.

Case presentation: A 77 year old man who was admitted to the hospital for an acute diffuse abdominal distention, nausea, non-bloody vomitus. His past medical history include coronary artery disease, remote history of cerebrovascular accident, congestive heart failure,

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hypertension, vascular dementia, chronic renal disease with last previous creatinine levels being within normal limits, non-insulin dependent diabetes mellitus. His past surgical history was significant for remote open cholecystectomy, transurethral retrograde prostatectomy. Medications included Aspirin/Dipyridamole, Furosemide, Glyburide, Metoprolol succinate, Omeprazole, Tamulosin and Nitroglycerine tansdermal patch. On initial presentation, patient blood pressure was 139/87 mm of Hg, heart rate of 78 per min, respiratory rate 22/min, and temperature 97.5 °F. On physical exam, patient was awake, demented. Head and neck examination revealed dry oral mucosa otherwise within normal. Cardiovascular and pulmonary exam was also within normal. His abdomen was distended, with sluggish bowel sounds and diffuse tenderness on palpation. Rectal exam was normal with the absence of stools in rectal vault. Admission laboratory data were consistent with acute pancreatitis, creatinine 1.8 (Normal 0.8-1.5 mg/dL), cholestatic liver disease with total bilirubin level 2.7 (Normal 0.2-1 mg/dL). Patient was kept NPO and gastric decompression was performed. Flat and upright abdominal radiograph revealed ileus with possibility of partial small bowel obstruction. Subsequently, he was given a fleet phosphate enema every 4 hours for a total of 6 units. CT- scan of the abdomen and pelvis without intravenous contrast showed evidence of acute interstitial pancreatitis. Over 24 hour period, patient's condition deteriorated, to become hypotensive and in respiratory distress. Patient was transferred to the medical intensive care unit, and initial resuscitative measures were initiated. Shortly after that, he coded and died. Pre-mortem blood work up results came back post-mortem, revealing a calcium level of 4.8 (Normal 8.4-10.2 mg/dL) that was previously normal on admission and a phosphorus level of 16.0 (2.7-4.5 mg/dL)with normal amylase and lipase levels. We assume that our patient died from iatrogenic complications of hyperphosphatemia, hypocalcemia leading to cardiopulmonary arrest and demise.

Conclusion: Since fleet phosphate enemas are widely used for various indications, physicians from all specialties need to be alerted regarding their injudicious use that can lead to an increase in morbidity and mortality. A discussion about the etiology, pathogenesis, clinical presentation, diagnosis and treatment of hyperphosphatemia will also be provided. -------------------------------------------------------------------------------------------------------------------------------

SPONTANEOUS SPLENIC RUPTURE ASSOCIATED WITH STREPTOCOCCAL VIRIDANS ENDOCARDITIS

Dima Nassour, Imran Samnani and Dafer Haddadin. Department of Internal Medicine, East Tennessee State University, COM, Johnson City, TN 37614 and Medical Service, James H. Quillen VAMC, Johnson City, TN

Splenic infarction in infective endocarditis (IE) is not uncommon, though abscess formation is less common. Mycotic aneurysm (MA) can be the end result of arterial wall weakening caused by septic emboli. MA leading to spontaneous splenic rupture is a rare complication of splenic infarction due to embolism. Streptococcus viridians IE complicated by splenic rupture and cerebral embolization in the same patient is extremely rare. We report a case of spontaneous splenic rupture and ischemic stroke associated with Streptococcus viridans endocarditis along with a literature review.

Case presentation: A 46-year-old Caucasian male patient presented with unsteady gait, headaches, multiple falls, fever, non-specific abdominal pain, weight loss and loss of appetite of three week duration. Three months prior to this presentation, patient had dental cleaning. His past medical history was significant for sexually transmitted diseases in the past, negative HIV

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test done 6 months before presentation, smoking disorder and remote history of intravenous drug use along with marijuana and cocaine abuse. Admission vital signs were stable besides temperature of 101 F. Significant findings on physical examination revealed a soft systolicejection murmur best heard at the left sternal border, grade III/VI. Significant neurological examination revealed gait disturbance, decreased motor power in right arm and numbness in right leg. Rest of physical examination was within normal limits. Initial laboratory data revealed white cell count of 5200, 92% segments, platelet counts of 197,000, hemoglobin 10.1 g/dl, creatinine 1.0 mg/dl, elevated sedimentation rate (64 millimeters in the first hour) and C-Reactive protein (29 mg/L), albumin 2.3g/dl. Serology tests for syphilis, hepatitis A, B, C viruses and HIV were negative. His blood cultures were positive for Streptococcus viridans. Brain magnetic resonance imaging including angiogram showed occlusion of the right insular branch of the MCA along with severe atherosclerotic narrowing of the distal first portion of the right middle cerebral artery with right insular gyrus and corona radiata white matter ischemia. Additional ischemia involved the medial superior left cerebellum. CT abdomen showed small bilateral pleural effusions and a 3.0 cm lesion in the superior margin of the spleen consistent with infarct. Trans-esophageal echocardiogram showed a large 1.7 cm aortic valve (AV) vegetation with significant AV prolapse and severe aortic regurgitation. A diagnosis of AV endocarditis with embolisation phenomenon to brain, spleen was made. Therapy with ceftriaxone and gentamicin was started. While patient was in the process of work-up prior to AV replacement surgery, he started complaining of left pleuritic chest pain associated with left sided abdominal pain and fever of 100 F. Repeated laboratory data showed worsening leukocytosis up to 30,000, creatinine up to 3.3 mg/dl and drop in Hgb of 5 g/dL. Repeat CT abdomen showed new splenic hemorrhage with hematoma formation and hemoperitoneum. He received supportive care with blood products. Angiogram of abdominal vessels showed bleeding aneurysm in the splenic artery. Patient underwent splenectomy with operative findings of severely lacerated ruptured spleen with splenic artery aneurysm. Histopathology confirmed the mycotic aneurysm in the splenic artery. He was discharged after a prolonged hospital course. IE, not only, affects the heart but also other organs through other mechanisms, including embolisation and immunological phenomena. Recognizing IE and its complications, with its salient features, is very important when treating such patients for better outcome.-------------------------------------------------------------------------------------------------------------------------------

IDENTIFICATION AND CHARACTERIZATION OF PUTATIVE FLAVONOID GLUCOSYLTRANSFERASES FROM CITRUS PARADISI

Daniel K. Owens and Cecilia A. McIntosh. Department of Biological Sciences and School of Graduate Studies, East Tennessee State University, Johnson City, TN, 37614

Flavonoids are a diverse group of natural products that are ubiquitous in higher plants with over 6000 naturally occurring compounds having been identified. These compounds are associated with a number of essential physiological roles in planta and have actions that are agriculturally and pharmacologically important. The addition of sugars is a predominant modification reaction in flavonoid biosynthesis, particularly in grapefruit where up to 70% of the dry weight of very young fruit consists of flavanone glycosides. Glycosylation has a number of effects upon the solubility, stability, and subsequent availability of metabolic products. The glycosylation of flavonoids also has an influence upon the quality of foods and food products. In particular, flavonoid glycosides convey taste characteristics in citrus making flavonoid glucosyltransferases interesting targets for biotechnology applications in these species. To investigate glycosylation of flavonoids in Citrus paradisi, putative glucosyltransferase clones are being isolated from young leaf cDNA employing BD SMART™ RACE reactions with degenerate primers designed against the plant secondary product glucosyltransferase (PSPG) box signature motif. Complete

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gene sequences are identified by primer walking, cloned into the Novagen pET® system for recombinant protein expression, and purified by metal affinity chromatography. Ultimately, the recombinant enzymes will be tested for substrate usage and thoroughly characterized by biochemical assay. While new putative glucosyltransferases continue to be sought, PGT3 has been established in the pET® system and the production of soluble recombinant protein verified. Efforts to further develop metal affinity chromatography and begin biochemical assays are currently underway. -------------------------------------------------------------------------------------------------------------------------------

PREVALENCE OF PREMENSTRUAL DYSPHORIC DISORDER (PMDD):UNITED STATE NATIONAL COMORBIDITY SURVEY REPLICATION (NCS-R)

Avani Prabhakar MBBS MPH, Merry N. Miller, MD and Deepak Prabhakar MD MPH. Department of Internal Medicine and Department of Psychiatry and Behavioral Sciences, East Tennessee State University, COM, Johnson City, TN 37614 and Department of Epidemiology, University of North Texas Health Science Center,Fort Worth, TX 76107

Although premenstrual worsening of mood disorder has been reported in medical literature since the days of Hippocrates, it was not until 1931 when Robert Frank coined the term premenstrual tension syndrome. In October 1998 medical community accepted a term called Premenstrual Dysphoric Disorder (PMDD) as a distinct clinical entity. Women who have major depressive disorder (MDD) through out the month are by definition excluded from the diagnosis of PMDD or PMS. The concept of Premenstrual Exacerbation of MDD (PMED) has not been recognized in DSM-IV, but has been reported in literature informally. DSM-IV Task Force on Nomenclature and Statistics decided to include PMDD as an example of a mood disorder not otherwise specified and put the criteria in the appendix. Objective of this study is to investigate the spectrum of premenstrual depressive syndrome that occur across the women’s reproductive life span, estimate the prevalence of PMDD, Dysphoric PMS, and PMED in the United States Population using National Comorbidity Survey: Replication 2001-2003 (NCS-R) Data. We used the NCS-R data to assess the prevalence rates, NCS-R was a fairly comprehensive survey and its data is representative of the US trends. Survey investigators used the World Health Organization (WHO) Composite International Diagnostic Interview (CIDI) questionnaire, a fully structured instrument designed for use by trained interviewers, where diagnosis is based on DSM-IV criteria. The prevalence of self-reported PMDD among US household-residing adult females in their reproductive age group was estimated to be 1.9%, when a previous episode of MDD was included in the analysis the prevalence rate rose to 2.8%, 0.9% of women report PMED. The prevalence of dysphoric PMS is estimated to be 10.8%. Amongst Caucasians prevalence of PMDD, dysphoric PMS, PMED, was, 2.2%, 11.5%, 0.9% respectively, for African Americans prevalence of PMDD, dysphoric PMS, PMED, was,0.6%, 7.9%, 0.8% respectively and for other race categories the prevalence of PMDD, dysphoric PMS, PMED, was, 1%, 8.5%, 1.1% respectively. Approximately 25% of adult females suffering from PMDD and 25% of those suffering from dysphoric PMS received professional treatment at least once in the previous 12 months at the time of the interview, while only 11% of adult females suffering from PMED received professional treatment at least once in the last 12 months at the time of the interview. If we extrapolate this data on the US population according to the 2000 census, there are roughly 150 million adult females in their reproductive age in the US suffering from PMDD and around 750 million adult females in their reproductive age suffer from dysphoric PMS. Strict definition of PMDD underestimates the prevalence. Our criterions were in sync with the DSM-IV PMDD definition, but have their own limitation due to the methodology of data collection. The data has been collected retrospectively, cross-sectionally, and thus prospective measurement of

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premenstrual symptoms in two consecutive months is not achieved in this data. Our analysis demonstrates that spectrum of premenstrual depressive syndrome is widely prevalent in women of reproductive age.

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RISK OF BREAST AND OTHER CANCERS DUE TO HYPERPROLACTINEMIA CAUSED BY ANTIPSYCHOTICS (NEUROLEPTICS) OR OTHER MEDICATIONS: A LITERATURE REVIEW Umesh Vyas, MD. Department of Psychiatry, East Tennessee State University, COM, Johnson City, TN 37614 BACKGROUND: Breast cancer is the most common cancer in females, and is the second most common cause of death. There are several factors which increase a woman's risk for the development of breast cancer. Some reports suggest that neuroleptics and other dopamine antagonists increase the risk of breast cancer due to hyperprolactinemia. There are other reports which suggest that they may decrease the risk of cancer especially in the rectum, colon and prostate. Additionally, there is evidence that patients with Parkinson's disease have lower rates of breast cancer and other types of malignancies. OBJECTIVE: A literature review was performed to extract evidence and to evaluate risk or benefit from hyperprolactinemia caused by these medications. METHOD: Pubmed.gov and other online resources were searched by using pre-determined key words. RESULTS: Most studies report no increased risk of breast cancer associated with use of these medications. Only one study reported a positive correlation between neuroleptic induced hyperprolactinemia and an increased risk of breast cancer. Other studies report inconclusive data. CONCLUSION: At this time we do not have definitive data suggesting increased risk of breast cancer secondary to hyperprolactinemia caused by antipsychotics. Thus, further studies are desirable. Author will discuss this literature review in detail in poster presentation.------------------------------------------------------------------------------------------------------------------------------

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Division VI—Case History

SPONTANEOUS RUPTURE OF SPLEEN: DIAGNOSTIC DILEMMA, A CASE REPORT

Rajesh Shivaji Kadam, MD, Hetal K. Brahmbhatt, MD and Avani Prabhakar, MBBS MPH. Department of Internal Medicine and Department of Psychiatry and Behavioral Sciences, East Tennessee State University, COM, Johnson City, TN 37614

Spontaneous rupture of the spleen has been defined as rupture of the spleen in the absence of either trauma or any splenic pathology that has been reported after apparently minor insults such as coughing and vomiting. To the date there are only two cases of spontaneous rupture of spleen that have been reported in the literature and cough was supposed to be the underlying culprit. The authors report a case of suspected spontaneous rupture of spleen secondary to cough in a 54 year old Caucasian female, who recovered well after appropriate diagnosis, and timely splenectomy. A fifty four year old Caucasian female was brought in by emergency medical services. Initial encounter revealed an intubated and unresponsive female who was in shock. Abdomen was mildly distended with hypoactive bowel sounds, and no mass or organomegaly was appreciated. Lab data was remarkable for hemoglobin of 8.9 gram per deciliter, hematocrit of 27.6. CT scan of head done at the time of admission showed no acute abnormality, and chest x ray was unremarkable. CT scan of Abdomen showed large hemoperitoneum secondary to a ruptured spleen. Patient was emergently taken to Operation Theater just as CT scan was completed, there she underwent emergent abdominal exploratory surgery where a ruptured spleen with a large subcapsular hematoma was found, and prompt splenectomy was performed. Patient survived the surgery and had a slow post operative recovery. Culture results from blood, sputum, and urine were all negative, and further serum tests provided no evidence of acute viral infection but still Epstein Barr virus (EBV) serology report was pending, which later on turned in positive, patient had no history of any viral infection or malignancy. Splenic histopathology was suggestive of some degree of reactive hyperplasia although the significance of this finding was uncertain. There are 5 criteria identified for the diagnosis of spontaneous splenic rupture that, there should be no history of trauma to the spleen, no evidence of disease in organs other than the spleen that are known to affect the spleen, no perisplenic adhesions or scarring of the spleen, spleen should be normal on both gross inspection and histologic examinations and convalescent, acute phase sera should not show any significant rise in viral antibody titers suggestive of recent infection with viral types associated with splenic involvement. This case meets the first 4 criterion and the EBV serology is at best 91% specific therefore the authors believe that a spontaneous rupture of spleen is still a possibility until we completely rule out EBV infection. If truly spontaneous this will be the third case of spontaneous splenic rupture secondary to coughing. The clinician must be alert to the possibility of occult splenic rupture because the consequences of delayed diagnosis and treatment of this condition are grave and potentially fatal. Fortunately for our patient who recovered very well, splenic rupture was diagnosed promptly and she was taken to surgery for a successful splenectomy.-------------------------------------------------------------------------------------------------------------------------------

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A CASE OF CEFTRIAXONE INDUCED NEUTROPENIA

Avani Prabhakar, MBBS MPH and Hetal K. Brahmbhatt, MD. Department of Internal Medicine and Department of Psychiatry and Behavioral Sciences, East Tennessee State University, COM, Johnson City, TN 37614

Neutropenia has been reported in the literature as one of the rare but serious complication of ceftriaxone therapy. Nine cases to the knowledge of these authors have been reported to date, two of these cases have been reported secondary to prolonged ceftriaxone therapy, both of which resulted in secondary infection due to neutropenia. We report reversible neutropenia and transient elevation of liver transaminases; rare complications from ceftriaxone therapy that developed in a patient who presented to emergency room with altered mental status and received 2 grams of intravenous ceftriaxone. With in twelve hours of ceftriaxone administration, patient developed severe neutropenia with absolute neutrophil count of 100cells/deciliter. The patient subsequently required hospitalization for observation under neutropenic precautions, the neutropenia and elevated transaminases resolved after conservative treatment and cessation of therapy. A case report is prepared of this unusual clinical scenario and the authors recommend that the clinicians must be vigilant to the possibility of this rare but serious side effect of this commonly used antibiotic because the consequences of delayed recognition and management are grave and potentially fatal. Fortunately the patient in this case scenario recovered very well, ceftriaxone induced neutropenia was identified promptly and she was monitored under neutropenic precautions, moreover neutropenia was transient and leukocyte count returned to baseline with in 24 hours. In general neutropenia may develop in 15% of patients receiving high dose of beta-lactam antimicrobials a group of antibiotics to which ceftriaxone belongs. In particular neutropenia develops in patients treated for ten days or more, but only 4.2% develop infection secondary to it and neutropenia completely resolves after few days of drug withdrawal in most of the cases. There are a couple of mechanism suggested in the literature for beta-lactam induced neutropenia, the first one is direct toxic effect of beta-lactam antibiotics on granulocyte progenitor cells and the second one is presence of leucoagglutinins complement fixing antibodies and antigranulocyte antibodies. The consequences of severe neutropenia include life threatening bacterial infections, thus it is mandatory to monitor blood counts serially in patients who receive ceftriaxone in a high daily dose, a total high dose or are treated for a long duration. -------------------------------------------------------------------------------------------------------------------------------

ENTEROSCOPIC RETRIEVAL: A POSSIBLE TREATMENT MODALITY FOR DISPLACED ESOPHAGEAL STENT

Raja S. Vadlamudi, MD, MPH and Mark F. Young, MD. Department of Internal Medicine, East Tennessee State University, COM, Johnson City, TN 37614

Esophageal Stent migration is an important complication (6% to 18%). Stent migrations are usually treated with conservative measures. We present a case of stent migration where the stent is removed transorally using enteroscope.

A 48 year old male smoker with intermittent dysphagia to solids and GERD underwent EGD with biopsy that showed squamous cell carcinoma of the distal esophagus. Exploratory laparotomy showed metastatic involvement of the celiac lymph nodes. He underwent chemo and radiation therapies without any complications. Later esophagectomy with splenectomy was done and he developed an esophageal anastomotic leak. An esophageal deficit of 1.5 cm was identified on EGD and was reduced using surgical clips. A silicone coated Boston Scientific Polyflex®

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esophageal stent was also placed at the anastomotic site. Few days before the proposed stent removal, he developed abdominal discomfort. The abdominal x-rays showed possible distal migration of the stent in to the left upper quadrant. He underwent enteroscopy and the stent, initially found in the jejunum, was pulled into third part of the duodenum. For lack of facility to use forceps with enteroscope, a pediatric colonoscope was used to remove the stent transorally using rat toothed forceps without any complications (Figure 1 & 2). Neither small bowel defects nor distal esophageal deficits were noted on repeat enteroscopy.

This case suggests the use of enteroscopy with rat tooth forceps for retrieval of migrated esophageal stents depending on the site of displacement and thus contradicting the use of only conservative measures for stent migration.Figures:

Figure 1: Third Part of the Duodenum with the Esophageal Stent

Figure 2: Stent Attached to Rat Toothed Forceps (Solid Black Arrow)

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Post-Baccalaureate Oral Presentations

B. CARROLL REECE OF BUTLER, TENNESSEE: HIS EDUCATION AND FORMATIVE YEARS

Margaret Carr, MALS. East Tennessee State University, Johnson City, TN 37614

One aspect of this project is to consider the educational history of the Butler community in Johnson County, Tennessee, and the effects of that experience on one individual – B. Carroll Reece. Reece became the Congressional Representative from Tennessee’s first district in 1921, a post he held for nearly 40 years. Reece, born in 1889 in Butler, graduated from Watauga Academy in 1911 and also attended Carson-Newman College and New York University. His career included service in the U.S. Army during World War I, and he served as a teacher, principal, and professor in schools such as elementary schools in Carter County and at New York University. The educational history of the Butler Community in Johnson County, Tennessee – particularly the Watauga Academy – and the academic career of Carroll Reece serve as an illustration of the development of education in the region. In much of Northeast Tennessee, public education was poorly funded and standards of instruction were not well established. Private academies filled some of this void, as did the Baptist-run Watauga Academy, which was the last incarnation of two earlier private academies. Information was collected from the Reece papers housed in the Archives of Appalachia at ETSU, from the Butler Museum in Butler, and from media from the time period covered (Elizabethton Star and other periodicals); this information was integrated into data compiled from published materials regarding the history of education in the region and the Watauga Academy, in particular. The research sheds new light on the education and career of Carroll Reece and examines the influences of his community and educational institutions. ---------------------------------------------------------------------------------------------------------------------

CHARACTERIZATION OF THE PATHWAY LEADING TO THE SYNTHESIS OF SALICYLIC ACID IN PLANTS UNDER ATTACK BY PATHOGEN

Alexander Eddo and Dhirendra Kumar. Department of Biological Science, East Tennessee State University, Johnson City, TN 37614

Salicylic acid (SA) a plant hormone mediates plant defense against pathogens, accumulating in infected and uninfected distal leaves in response to the attack. Two pathways have been implicated in SA production and one of them the Phenylalanine ammonia-lyase (PAL) pathway has now been suggested to play only a minor role in plant defense. The other pathway leading to SA synthesis is through isochorismate synthase (ICS) (an enzyme localized in plant plastids). Both PAL and ICS pathway synthesize SA from chorismate - the end product of the Shikimate pathway. In plants and bacteria this pathway is responsible for biosynthesis of aromatic amino acids. Plants which either fail to accumulate SA or do not produce SA are unable to fight the pathogen and are susceptible to pathogen infection (nahG transgenic plant and the SID2, Arabidopsis mutant plant respectively). However mutant plants which accumulate high levels of SA are shown to be more resistant and exogenous application of SA induces the production of pathogenesis related (PR) defense proteins and enhanced resistance. Some of the plants have

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Resistance genes encoding for R-proteins. These R proteins can in some cases recognize the avirulence (avr) proteins introduced by pathogen into the plant host cell leading to resistance

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response. Absence of corresponding ‘R’ protein against the avr protein allows the pathogen to manipulate the host defenses as it wants. In case of tobacco resistance protein (N) which recognizes the avr from tobacco mosaic virus (TMV), the interaction results in ~50 fold increase in SA levels. Signaling pathway following this interaction leading to SA biosynthesis is not fully understood. In order to characterize this biochemical pathway we have done RT-PCR based expression profiling of the known genes of this pathway. This was done to determine the component which receives the initial signal of the positive interaction of the R and avr protein. Resistant and susceptible tobacco (model plant) plants were infected with TMV and were used in this study. Results of this work will indicate which enzymes are involved in the signaling to make SA when resistance is mounted against infection. Better understanding of the plant’s natural defense mechanism will help us to sensitize it under normal conditions so that a faster response can be mounted under actual pathogen attack leading to better resistance. ---------------------------------------------------------------------------------------------------------------------

YEAST TWO-HYBRID ANALYSIS OF PROTEIN-PROTEIN INTERACTIONS INVOLVING A MICROSPORIDIA ADAM (A DISINTEGRIN AND METALLOPROTEASE) PROTEIN

Carrie E. Jolly and J. Russell Hayman. Department of Microbiology, East Tennessee State University, COM, Johnson City TN, 37614

Microsporidia are spore-forming, obligate intracellular fungal-like protists typically associated with opportunistic infections in immunocompromised individuals. Our previous research has demonstrated a direct relationship between adherence of microsporidia spores to the surface of host cells and infectivity in vitro. As for many other pathogenic organisms, adherence of microsporidia to the host cell surface may be a prerequisite to infection. In an effort to better understand adherence, we have turned our attention to determining what microsporidia proteins may be involved in this process. Through examination of the Encephalitozoon cuniculi genome database, we identified a gene encoding a protein with sequence homology to members of the ADAM (a disintegrin and metalloprotease) family of type I transmembrane glycoproteins. ADAM proteins are involved in a variety of biological processes including cell adhesion, proteolysis, cell fusion, and signaling. Through the use of immunoelectron transmission microscopy, we have determined that the microsporidial ADAM protein or MADAM is localized to at least three distinct regions of Encephalitozoon intestinalis spores. These areas include the surface exposed exospore, the plasma membrane, and the polar sac-anchoring disc complex (a bell-shaped structure at the spore apex involved in the infection process). Immunofluorescence assays show that MADAM is also located on the extruded polar tube, which is the organelle used to infect the host cell. Since ADAM proteins are known to be involved in cell adhesion and proteolysis, we employed a yeast two-hybrid system to identify potential proteins capable of interacting with MADAM. Of the cDNA clones analyzed thus far, several contain an insert that encodes for polar tube protein 3 (PTP3). Additional yeast two-hybrid analyses demonstrate that the interaction between MADAM and PTP3 passes the most stringent selection process, whereas, other polar tube proteins (PTP1 and PTP2) do not. Current studies are underway to confirm that PTP3 interacts with MADAM by co-immunoprecipitation assays. Further characterization of the interactions that occur between MADAM and PTP3 may lead to a better understanding of the events that occur during microsporidia host cell infection. ---------------------------------------------------------------------------------------------------------------------

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THE HOST ADHERENS JUNCTION MOLECULE NECTIN-1 IS DOWN-REGULATED IN CHLAMYDIA TRACHOMATIS-INFECTED GENITAL EPITHELIAL CELLS

Jingru Sun, Jennifer Kintner and Robert V. Schoborg. Department of Microbiology, East Tennessee State University, COM, Johnson City, TN 37614

Although Chlamydia trachomatis (serovars D-K) is the most commonly reported bacterial sexually transmitted disease agent in the US, it is still not clear how chlamydial infection causes disease. Nectin-1, a member of the immunoglobulin superfamily, is a Ca 2+-independent homophilic and heterophilic cell adhesion protein that has been implicated in the organization of E-cadherin-based adherens junctions (AJs) and claudin-based tight junctions (TJs) in epithelial cells. Nectin-1 also plays key roles in regulating intracellular signaling molecules. For example, activation of Cdc42 and Rac through c-Src by nectin-1 regulates cell-cell adhesion, gene expression, and cell polarization. Using Western blot analyses, our laboratory has demonstrated that accumulation of host cellular nectin-1 is significantly decreased in C. trachomatis serovar E infected non-polarized HeLa cells. Nectin-1 was decreased by as much as 85% by 48 hours post infection (hpi); this decrease was sustained until 72 hpi. Similar results were also obtained using polarized HeLa cell cultures. Indirect immunofluorescence assay further supported the fact that accumulation of nectin-1 is down-regulated by C. trachomatis infection. Down-regulation of nectin-1 in C. trachomatis-infected cells is prevented by chloramphenicol exposure, demonstrating that C. trachomatis protein synthesis and/or replication is required for this effect. To determine whether persistent chlamydial infection also reduces nectin-1 accumulation, C. trachomatis infected HeLa cells were exposed to penicillin G and harvested at 48 hpi. Nectin-1 levels decreased in the presence of penicillin G, indicating that nectin-1 accumulation can indeed altered by persistent chlamydiae. Interestingly, N-cadherin-dependent cell-cell junctions can be disrupted by C. trachomatis infection, as reported by Ramsey KH et al. 2002. Taken together, these data suggest that C. trachomatis may disrupt adherens junctions, at least in part, by diminishing nectin-1 expression. Because other investigators have observed release of chlamydia-infected epithelial cells in vitro (from monolayers) and in vivo (from tissues), we speculate that chlamydia modulated down-regulation of adhesion molecule and subsequent disruption of host cell adherence may be involved in chlamydial dissemination or pathogenesis. ---------------------------------------------------------------------------------------------------------------------

TRANSCRIPTIONAL REGULATION OF C-REACTIVE PROTEIN EXPRESSION IN HUMAN HEPATOCYTES

Bhavya Voleti, Prem Prakash Singh and Alok Agrawal. Department of Pharmacology, East Tennessee State University, COM, Johnson City, TN 37614

The hepatic synthesis of C-reactive protein (CRP) increases in inflammatory conditions resulting in raised serum CRP levels. The regulation of CRP gene expression in hepatocytes occurs at the transcriptional level. Previously, we reported that a C/EBP-binding site overlapping a NF-ĸB p50-binding site (C/EBP-p50-site) on the CRP promoter was critical for IL-6-induced CRP expression in human hepatoma Hep3B cells. Transcription factors C/EBPβ and p50 occupy the C/EBP-p50-site in the nuclei containing constitutively active or IL-6-activated C/EBPβ. The aim of this study was to identify the transcription factors that occupy the C/EBP-p50-site in the absence of C/EBPβ. Accordingly, we treated Hep3B nuclear extract with a C/EBP-binding consensus oligonucleotide to generate an extract lacking active C/EBPβ. Such treated nuclei contain only C/EBPς because the C/EBP-binding consensus oligonucleotide binds to all C/EBP family proteins except C/EBPς. EMSA using this extract revealed formation of a C/EBPς-

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containing complex at the C/EBP-p50-site. This complex also contained RBP-Jĸ, a transcription factor known to interact with the ĸB sites. RBP-Jĸ was required for the formation of C/EBPς-containing complexes. The RBP-Jĸ-dependent C/EBPς-containing complexes were also formed at the C/EBP-p50-site on the CRP promoter in the nuclei of primary human hepatocytes. In transactivation assays, overexpressed C/EBPς abolished both C/EBPς-induced and (IL-6+IL-1β)-induced CRP promoter-driven luciferase expression. These results indicate that under basal conditions, C/EBPς occupies the C/EBP-site, an action that requires RBP-Jĸ. Under induced conditions, C/EBPς is replaced by C/EBPβ and p50. We conclude that the switch between C/EBPβ and C/EBPς participates in regulating CRP expression. This process utilizes a novel phenomenon, that is, the incorporation of RBP-Jĸ into C/EBPς-complexes solely to support the binding of C/EBPς to the C/EBP-site.---------------------------------------------------------------------------------------------------------------------

INVOLVEMENT OF TNF RECEPTOR APOPTOSIS INDUCING LIGAND (TRAIL) IN NK CELL MEDIATED ANTI-HSV ADAPTIVE IMMUNE RESPONSE

Stacie N. Woolard, Subhadra Nandakumar, Yagita H and Uday Kumaraguru. Department of Microbiology, East Tennessee State University, COM, Johnson City, TN-37614 and Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

Linking the innate and acquired immune system has been a major topic of research leading to the examination of certain cells that could facilitate this interaction. Two such cells are the Dendritic cell (DC), the professional APC, and the Natural Killer (NK) cell, which are recruited and activated in a viral response. The cross-talk of DCs and NKs has been thoroughly examined yet the precise role of NK cells in this process has yet to be demonstrated. The DCs from infected site migrate to the draining lymphoid organ, but transfer the cargo to the lymphoid resident CD8α+ DC through an undefined process. The CD8α+ DC then process and cross-present the viral epitope to the T cells. We hypothesized that the NK cells may play a predominant role in this process. Using PK136 Tg mice (NK cell less mouse) and wild type C57BL/6 mice we show that the adaptive immune response to HSV was impaired. To further test if NK cell contributed to this impairment, we looked at NK cell interaction with DC. Our results suggest the interaction to be reciprocal in that the antigen bearing migratory immature DC stimulated the NK cells (reduced MHC-I and IL-12). The activated NK cells killed the immature DC (iDC) resulting in an inflammatory milieu. This in turn stimulates the lymphoid resident CD8α+ DC to acquire the apoptotic DCs, process and cross-present the viral epitope to T cells. In addition, the NK cell produced IL-2 and IL-15 act as T cell growth factor. Our studies on the mechanism suggested that the NK cell used the TNF receptor apoptosis inducing ligand (TRAIL) to deliver the death signal to the iDC. This was further confirmed in vivo by blocking the pathway using anti-TRAIL Mab. Hence, we may conclude that HSV infection prevents maturation of DCs, but upon arrival at the lymphoid organ stimulate the NK cells, they reciprocate by killing them through TRAIL. The resulting inflammatory milieu then aids the lymphoid resident DC to take up the antigen bearing dead DC, process and cross-presents the viral epitope resulting in optimized immune activation.Acknowledgements: Department of Microbiology, ETSU - Start up Funds and Dr. Wyrick.---------------------------------------------------------------------------------------------------------------------

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Resident/Post-Doc Oral Presentations

WEGENER’S GRANULOMATOSIS AND PURULENT PERICARDITIS: THE FIRST CASE REPORT

Admad Halawa, MD, Bobby Keith, MD and Ryland P. Byrd, Jr. MD. Department of Internal Medicine, East Tennessee State University, COM, Johnson City, TN 37614

Wegener's granulomatosis is a systemic vasculitis presents with variable multi-organ involvement. Different cardiac manifestations, like myocarditis or valvulitis, have been described before as part of wegener's pathogenesis, but the most common cardiac features were pericarditis and coronary arteritis. Fibrinous and constrictive pericarditis are well identified in Wegener's patients who suffered cardiac impairment. To the best of our knowledge, we present the first case report of purulent pericarditis during an acute Wegener's exacerbation. Our patient is a young female presented with a typical vasculitis picture including severe pulmonary nodular infiltrates, arthritis, and neuritis. She also developed a massive pericardial effusion. Pus was evacuated from the pericardium during pericardectomy and it grew no organisms. Immunosuppressive therapy dramatically improved the systemic inflammation and resolved the pulmonary and cardiac defect. ------------------------------------------------------------------------------------------------------------------------------

OLDER GALAXY PAIR HAS SURPRISINGLY YOUTHFUL GLOW

Mark Hancock et al. Department of Physics, Astronomy, and Geology, East Tennessee State University, Johnson City, TN 37614

A pair of interacting galaxies might be experiencing the galactic equivalent of a mid-life crisis. For some reason, the pair, called Arp 82, didn't make their stars early on as is typical of most galaxies. Instead, they got a second wind later in life (about 2 billion years ago) and started pumping out waves of new stars as if they were young again. Our new observations are from NASA's Galaxy Evolution Explorer, NASA's Spitzer Space Telescope and the Southeastern Association for Research in Astronomy Observatory at Kitt Peak, Arizona. Arp 82 is an interacting pair of galaxies with a strong bridge and a long tail. NGC 2535 is the big galaxy and NGC 2536 is its smaller companion. The disk of the main galaxy looks like an eye, with a bright ‘pupil’ in the center and oval-shaped ‘eyelids.’ Dramatic ‘beads on a strin’ features are visible as chains of evenly spaced star-formation complexes along the eyelids. These are presumably the result of large-scale gaseous shocks from a grazing encounter. The colors of this galaxy indicate that the observed stars are young to intermediate in age, around 2 million to 2 billion years old, much less than the age of the universe (13.7 billion years). The pair first burst with new star formation about 2 billion years ago after swinging by each other. A second close passage more recently resulted in yet another batch of star formation. The puzzle is: why didn't Arp 82 form many stars earlier, like most galaxies of that mass range? Scientifically, it is an oddball and provides a relatively nearby lab for studying the age of intermediate-mass galaxies. In more popular terms, think of this as an example of arrested development. For some reason, it took a kick-in-the-pants to get the stars forming recently, whereas most other galaxies of that mass range formed their stars much earlier (between 4 and 8 billion years ago).---------------------------------------------------------------------------------------------------------------------

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EFFECTS OF PATHOGEN ASSOCIATED MOLECULAR PATTERNS (PAMPS) ON ANTI-VIRAL IMMUNE MEMORY

Subhadra Nandakumar and Uday Kumaraguru. Department of Microbiology, East Tennessee State University, COM, Johnson City, TN 37614

Our immune memory needs to be refreshed from time to time. Immune system of individuals living in a sanitized society will eventually atrophy and go into immune-amnesia. We know that some vaccines wear off after a few decades, but upon re-exposure the response is quicker and better and there is an upward shift in memory frequency. This new set-point is likely higher than the primary set-point and varies with individuals based on the exposure pattern to other microbes or its components. Our investigations were designed to test such differences of non-specific stimulation on anti-viral adaptive immune response by bacteria or its components in lowering the threshold of activation. We exposed young mice to various TLR ligands in the first month after birth and analyzed for their ability to resist and survive challenge with virus at the adult phase. Secondly, pre-immune animals were exposed to TLR ligands in the absence of cognate antigen to determine the impact on immune memory response. The mice that were in the simulated dirty environment were better able to resist lethal challenge and needed 10 times more virus to show symptoms of CNS infection with HSV. This was attributed to the up-regulation of certain key co-stimulatory molecules (such as 41BB, OX40, CD40) on antigen presenting cells (APC), modulation of cytokines, cytokine receptors and anti-apoptotic molecules on APCs and T cells that are known to be critically involved in memory expansion and maintenance. In addition, pre-immune mice given TLR ligands in the absence of cognate antigen showed increase in frequency and quality. The frequency was measured in vitro by HSV specific tetramer and in vivo by BrdU incorporation and CFSE dilution. The quality was assessed by measuring intracellular expression of cytokines (IFNγ and TNFα) and release of perforin and granzymes by assessing lysosome-associated membrane proteins (CD107a and b). The timing, dose and combination of TLR ligands exposure seem to have an impact on the positive outcome. These studies provide insights into the role of hyper-sanitized environment versus PAMP mediated signaling in adaptive immune response and long term memory.

Acknowledgements: ETSU-RDC major grant support, Start up Funds, Department of Microbiology and Dr. Wyrick.--------------------------------------------------------------------------------------------------------------------

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Poster – Non-CompetitiveBiomedical Sciences

CHARACTERIZATION OF BINDING AND TRANSPORT AND INITIAL IDENTIFICATION OF THE GENES INVOLVED IN THE BIOSYNTHESIS AND TRANSPORT OF SCHIZOKINEN, A DIHYDROXAMATE SIDEROPHORE PRODUCED BY RHIZOBIUM LEGUMINOSARUM IARI 917

D. Hammond, E. Storey, B.C. Lampson and R.N. Chakraborty. Department of Health Sciences, East Tennessee State University, Johnson City, TN 37614

Siderophores are small organic molecules produced by many microorganisms under iron deficient conditions. They scavenge ferric ions and transport them across the cell membranes into the cell via a multicomponent transport system. With a very few exceptions iron is a vital element for the survival of most living cells. This is because iron serves as a cofactor for the components of electron transport chain and a variety of oxidoreductases. Iron is present in abundance in earth’s crust; however, under physiological pH and aerobic conditions it is not available to the microorganism as it forms an insoluble ferric oxyhydroxide polymer. Siderophore mediated iron transport is one of the ways in which microorganisms acquire iron to overcome the shortage of iron. Rhizobia form a symbiotic relationship with its host plant and fix atmospheric nitrogen, making it available to the plant in the form of ammonia. Siderophores have been described in a variety of Rhizobium species and are thought to play an important role in the nodulation process. We have already characterized the siderophore produced by Rhizobium leguminosarum IARI 917 to be ‘schizokinen’ and have also tried to identify the genes involved in its biosynthesis. Random mutations of strain IARI 917 were done using mini Tn5 mutagenesis. Siderophore overproducers and non-producers were detected on CAS (chrom azurol S) plates. DNA sequence analysis of several non-producer mutants carrying the transposon revealed a gene similar to alpha keto acid reductase enzyme, which is already reported to be involved in siderophore biosynthesis besides amino acid biosynthesis. The sequence analysis of other mutants have shown similarities to amino transferases (may be involved in biosynthesis of schizokinen as it does contain amino groups). It is important to characterize siderophore mediated iron transport systems in Rhizobia for their agricultural significance as well as to understand in general the mechanism of iron transport in gram-negative organisms. ----------------------------------------------------------------------------------------------------------------------------

Natural Sciences & Mathematics

CUTICLE MICROMORPHOLOGY OF TSUGA

Graham Cooke and Yusheng Liu. Department of Biology, East Tennessee State University, Johnson City, TN 37614

Cuticle micromorphology from leaves of all 8 species of the genus Tsuga, and one cultivated variant are being studied by both light microscopy and scanning electron microscopy for the first time. Specimens collected from the ETSU Arboretum exhibit characteristic differences between

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species at a micromorphological level. Features of the axial and abaxial cuticle are in the process of being described for all 8 species and one species cultivar. When completed we expect to use the description of 28 separate characters, 7 from the outer cuticle and 21 from the inner cuticle. These characters should provide additional characterization of the species within the genus, and will provide additional detail to aid in the reconstruction of the phylogeny of Tsuga. This will also provide a basis for the identification of fossilized cuticle. In addition, the stomatal density will also be quantified in an attempt to reconstruct characteristics paleoclimates, and will be applied in modeling of future earth climates.-----------------------------------------------------------------------------------------------------------------------------

ROLE OF SALICYLIC ACID METHYL TRANSFERASE IN PLANT DISEASE RESISTANCE SIGNALING

Tazley Hotz, Poonam Sheth and Dhirendra Kumar. Department of Biological Sciences, East Tennessee State University, Johnson City, TN 37614

Salicylic acid (SA) has been shown to play an important role in plant defense response against microbial pathogens. SA is synthesized in the plant chloroplasts from chorismate, the end product of the shikimate pathway, through two biochemical pathways: Isochorismate synthase (ICS) and phenylpropanoid (PAL). SA produced is lipid immobile and cannot pass through the chloroplast membrane to enter into the cytoplasm to mediate the disease resistance signaling. It has been suggested that SA synthesized in the chloroplast is converted into lipid mobile methyl salicylate (MeSA) by an enzyme, salicylic acid methyl transferase (SAMT). To investigate the role played by SAMT in SA-mediated disease resistance signaling, we examined the expression of SAMT in resistant tobacco’s response to tobacco mosaic virus (TMV). To compare SAMT expression, we also studied tobacco plants susceptible to TMV expression and a transgenic tobacco plant (NahG) which does not accumulate SA. Expression studies were performed using semi-quantitative RT-PCR. To investigate the role of SAMT in plant immunity, we are in the process of making transgenic tobacco by either over expressing SAMT or by knocking out its expression using RNAi. These transgenic lines will be infected with viral bacterial and fungal pathogens to determine the role of SAMT in plant defense. Understanding the biochemical pathways involved in plant defense response against pathogens will help to develop crop plants showing better resistance to ever-evolving pathogens. -------------------------------------------------------------------------------------------------------------------------------

Social and Behavioral Sciences

VOCAL FUNCTION EXERCISES (VFE) VERSUS RESONANT VOICE THERAPY (RVT) FOR THE TREATMENT FOR HYPERFUNCTIONAL VOICE DISORDERS

Shelley Bell, Brittany Kidd, Cora Leemkuil, Annie Smith and Christopher McCrea. Department of Communicative Disorders, East Tennessee State University, Johnson City, TN 37614

This case study used patient-based treatment outcome measures combined with acoustic analysis to compare vocal function exercises (VFE) and resonant voice therapy (RVT) for the treatment of an individual with mild-moderate hyperfunctional voice disorder. The participant was a 44 year old, first language general American English speaker, with no history of previous speech-language impairment. The participant underwent six weeks of RVT, followed by a six-week withdrawal, and six weeks of VFE. Auditory-perceptual and laryngeal imaging data were

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collected before and after each six-week treatment phase. In addition, acoustic data were collected during each treatment session. Laryngeal results indicated that the participant displayed some noticeable changes in vocal fold function across both treatment protocols. Acoustic results indicated that the participant’s loudness was consistent during pre-treatment, mid-treatment, and post-treatment measures and that both treatments were effective in improving the cycle-to-cycle vibration patterns of the participant’s vocal folds. No noticeable differences between the two treatments were noted; however, according to patient ratings, it seemed that the RVT was preferred over VHI. Although several questions remain unanswered, the results of this case study represent evidence to support the clinical utility of RVT and VFE within hyperfunctional voice disordered patients.

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WEBSITE USABILITY

Kevin Bowes. Department of Computer and Information Science, East Tennessee State University, Johnson City, TN 37614

The usability of a website has many benefits, greater respect for the company and designer, a wider user base, a greater range of supported user devices (Browsers, PDAs, Cell phones), and easy access to information. The information available on East Tennessee State University’s College of Business and Technology’s Career Services’ Job listings website was difficult to access and maintaining the site was just as difficult. Building a new website, focusing on the users, to encompass the job listings and other facets of the Career Services department (such as statistics gathering) was the best method of solving this problem. So, a new website was built to facilitate these needs. This website is a good starting point toward great user experience for the Career Services Department and these methods can be used for any data driven website.-----------------------------------------------------------------------------------------------------------------------------

TEMPORALIZATION OF AN EXISTING ACCREDITATION DATABASE

Matthew Campbell. Department of Computer and Information Sciences, East Tennessee State University, Johnson City, TN 37614

Storage of accreditation data in a database is an easy and quick solution provided the criteria for accreditation are unchanging. An accreditation database should be able to store data about current accreditation criteria and data, as well as previous versions of criteria and data. In order to do this, the database must be temporalized. Research on Temporal Relational Databases has progressed greatly since Gadia and Yeung’s generalized model was introduced in the 1980’s. Temporal databases allow for previous versions of data to be placed in the same table as the current data and to be accessed very easily. For the accreditation database, a valid time approach was used for tables requiring temporalization. Valid time is the time at which the data was the current version. All tables in the database were analyzed to see if temporalization was necessary, and the tables that store the goals for the university, college, and department, as well as the table that stores the requirements for accreditation by the accreditation organization, ABET, were the only ones that required temporalization.

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