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Appalachian Student Research Forum

Office of Research and Sponsored Programs

Division I

Abstracts Submitted:Division I - Undergraduate students - Biomedical Sciences


THE MECHANISMS OF IL-1BETA AND EPINEPHRINE INDUCED INFLAMMATION IN MAST CELLS AND ITS ROLE IN ATHEROSCLEROSIS

S. Matthew Fitzgerald 1, Guha Krishnaswamy 1, Steven A. Lee 1, Shau-Ku Huang 2, David S. Chi 1. 1East Tennessee State University Dept. of Internal Medicine Johnson City, TN 37614. 2The Johns Hopkins Asthma and Allergy Center, Baltimore MD 21224.

Atherosclerosis is the most common type of arteriosclerosis and is characterized by lipid deposition in the intimal layers of large arteries which causes them to become hard and inelastic. These lipid deposits are called plaques and may dislogde and obstruct blood flow to the heart. Data over the past few years suggests that lipid gorged macrophages called foam cells that reside in the shoulders of these plaques may play a pivotal role in dislodging them. Postmortem pathology results from the area around ruptured plaques also show increased presence of mast cells, a highly potent, aggressive inflammatory cell. A myriad of proinflammatory cytokines like interleukin (IL)-1beta, IL-6, IL-8, and IL-13 found in these plaques suggest an important role for inflammation. We aimed to investigate the inflammatory role of mast cells in atherosclerosis and the mechanisms by which proinflammatory cytokines are signaled in these cells. Our previous study with human mast cells (HMC-1) has shown that epinephrine, a catecholamine released during stress responses, synergizes with IL-1beta to produce IL-6, IL-8, and IL-13 levels above either stimulus alone. IL-6, IL-8, and IL-13 are highly inflammatory cytokines that may play a role in plaque rupture. The increase of IL-6, IL-8, and IL-13 as seen with ELISA was paralleled by RT-PCR. Results also showed a major role for p38 MAPK and NF-kappaB in signaling these cytokines. Flow cytometry showed the expression of both beta1 and beta2 adrenoceptors on mast cells. Propranolol but not the beta1 adrenoceptor specific drug atenolol blocked epinephrine enhancement of IL-6, IL-8, and IL-13 production suggesting that the beta2 receptor is the major pathway used by epinephrine in these cells. The immunosuppressive drugs prednisone, dexamethasone, and cyclosporine decreased IL-6, IL-8, and IL-13 production. These data provide a possible and important role for mast cells in atherosclerosis. The data also show that a stress hormone, epinephrine, with IL-1beta, an acute phasecytokine, can induce IL-6, IL-8, and IL-13 production above epinephrine or IL-1beta alone. The mechanisms into this signaling give novel insights into specific drug targets for atherosclerosis. (Supported by NIH grant HL63070, the Ruth R. Harris Endowment , and RDC of ETSU.)


THE PURIFICATION AND CHARACTERIZATION OF RECOMBINANT HUMAN MAST CELL CHYMASE

Jessica R. Vencill and David A. Johnson, Department of Biochemistry and Molecular Biology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614

Recombinant human chymase (rhChymase) is a chymotrypsin-like serine protease that is found in skin and heart mast cells. Mast cells are primarily found in the connective tissues and tend to be concentrated around the blood vessels. When mast cells are activated by various agents including the binding of antigens to cell surface IgE they degranulate, releasing their cytoplasmic granules, which contain chymase, tryptase (trypsin-like serine protease), heparin and histamine. The Pichia pastoris yeast expression system had been used to express rhChyamse as an active enzyme into culture media. The object of this project was to purify the rhChymase and to characterize it. Stability studies were performed to choose buffers for maintaining maximum activity. The stability experiments showed that chymase was more stable in high ammonium sulfate concentrations and at low pH. Also, octyl glucoside, a detergent, was used to aid in the stability of the protease. Consequently, it was found that the combination of pH 6.0 buffer, octyl glucoside, and high salt maintained optimal activity. The synthetic peptide substrate used to measure activity was Succinyl-Ala-Ala-Pro-Phe-p-Nitroanilide, which produces a yellow color when cleaved by chymase. Protein concentration was based on absorbance at 280 nm and protein purity was assessed by SDS-PAGE. Hydrophobic interaction chromatography on a butyl column, followed by affinity chromatography on a heparin column resulted in two peaks of activity. Peak B was highly purified and accounted for the majority of the enzyme. The purified rhChymase produced a band of 33 kDa on SDS-PAGE and was found to be 96% active via titration with Eglin C, a leech derived inhibitor that forms a tight 1:1 complex. Deglycosylation experiments with PNGaseF proved that the rhChymase product contained Asn-linked carbohydrate and that the core protein was of the expected size. The yield of active rhChymase was 22%. Supported by NIH grant R15 AI45549 to DAJ and an East Tennessee State University Student-Faculty Collaborative Grant through undergraduate honor program to JRV and DAJ.


THE EFFECTS OF INSULIN AND METFORMIN ON THE EMOTIONAL WELL BEING OF WOMEN WITH POLYCYSTIC OVARY SYNDROME

Jessica R. Lott and Dr. Judy G. McCook, College of Nursing, East Tennessee State University, Johnson City, Tennessee, 37614

Polycystic Ovary Syndrome (PCOS), the most common endocrine disorder in women, has significant emotional and physical health concerns. Studies regarding emotional distress, which have been largely overlooked until recently, report significant negative mood affects in women with PCOS. This study investigated the effects of insulin levels and medication therapy (metformin (Glucophage)) on the emotional well being of women with PCOS. This investigation is a further analysis of the original study: McCook, J.G. (2002). "The Influence of Hyperandrogenism, Obesity, and Infertility on the Psychosocial Health and Well Being of Women with Polycystic Ovary Syndrome." A cross-sectional, correlational design was utilized to study a convenience sample of 128 women with PCOS. The majority of subjects were residents of Southern Appalachia, married, Caucasian, with a mean age of 30 years. Lab work, including insulin levels, was drawn during the same office visit in which the questionnaires were completed. Metformin use was self-reported and emotional well being was determined through use of three self-report psychometric instruments: the Derogatis Brief Symptoms Inventory (BSI), Derogatis Affects Balance Scale (DABS), and the PCOS Health-Related Quality of Life Questionnaire (PCOSQ) (Cronin et al., 1998). Women with elevated fasting insulin levels (suggestive of insulin-resistance) reported higher phobic anxiety symptoms on the BSI. Women on metformin therapy had fasting insulin levels which positively correlated ( p=.01) with their reported DABS scores for anxiety, depression, guilt, hostility, negative affective total scores, and affects balance index scores. Lower body weight concerns were reported in those women undergoing metformin therapy. Monitoring insulin levels is indicated on all women with PCOS. However, these study findings suggest PCOS women with elevated fasting insulin levels had more phobic anxiety symptoms than those with normal insulin levels. Women on metformin therapy with elevated insulin levels reported higher levels of negative affects including depression, guilt, and hostility than those with normal insulin levels. Women on metformin therapy reported lower body weight concerns, thus improving their health-related quality of life score, an indirect measurement of emotional well being. A comprehensive approach is required to treat both the physical and emotional aspects of caring for women with PCOS. Early diagnosis and treatment are key to managing morbidity issues which include cardiovascular disease, diabetes, and depression. Psychosocial surveillance includes screening, treatment and referral of women with PCOS who are experiencing emotional distress. Findings here suggest women with elevated insulin levels are at increased risk for negative affects. A plan of care for women with PCOS includes lifestyle modifications, such as diet and exercise, since both influence insulin levels.


ALTERNATIVE MEDICINE EDUCATION IN FNP SCHOOLS WITHIN THE SOUTHEASTERN UNITED STATES.

Amanda D. Delph, East Tennessee State University, College of Nursing, Johnson City, TN, 37614

Alternative Medicine (AM) is an integral part of the health care regimen for many Americans. However, it is reported that the majority of the AM modalities used by patients are never disclosed to their physician (Lubkin & Larsen, 1998). This deficit of communication can result in serious and, even, life-threatening consequences. Furthermore, it is chiefly considered to be a direct result of lack of staff training (Kreitzer, Mitten, Harris, Shandeling, 2002). This original study was designed to assess the quantity of AM education currently taught in FNP programs within the southeast, any future plans to include AM education into the curriculum, and faculty and school certification in relation to AM. The study utilized a survey designed by the principle investigator to gather descriptive statistics via the internet. Fifty-one FNP faculty members, from various nursing schools in seven southeastern states, were selected to receive the survey. Thirteen FNP faculty responded; a 25 percent response rate. Results indicated that less than one-third percent (n=4) of the FNP programs surveyed offered specific AM courses, and even fewer schools required their students to attend the course(s). A higher percentage of schools indicated they incorporated minimal to extensive amounts of AM content into already established classes. However, herbal remedies and therapeutic touch were they only modalities considered to be extensively covered in the curriculum. Fifteen percent (n=2) of the respondents, that stated their school did not currently include AM education within the curriculum, declared there had plans to incorporate it in the future, and another 23 percent (n=3) of schools, that were currently teaching AM, planned to increase the amount of content. The vast majority of respondents stated that there school did not belong to any AM organization or they were not aware of such a relationship. However, approximately one-third (n=4) of participants indicated that one or more FNP faculty member(s) was certified in an alternative therapy. Overall, the results support the idea that the health care community, as a whole, does not place much merit in AM. This lack of trust leads to lack of interest and study; which, in turn, yields knowledge deficits. Limitations of this study include a small sample size and low response rate. Both limitations should be addressed to increase the generalization of the results.


Healthy Diets and Cancer Prevention: Fruits, Vegetables, Chocolate, Wine and Tea

Joseph Falcone, Jennifer Dean, and Dr. Phillip Musich, Department of Biochemistry & Molecular Biology, J. H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614

A healthy diet reduces ones chances of getting certain cancers. One may not think of dark chocolate and wine as part of this diet. How might components of these products help in the prevention of skin and other types of cancers? Oxidation can cause DNA to become nicked (broken) and/or may cause base damage that can lead to mutations in the genetic material (a pro-mutagenic effect). DNA also can be damaged by exposure to ultraviolet (UV) light. UV light initiates a cross-linking between two adjacent thymine bases in DNA. If these thymine dimers are not repaired mutations may result that can be fatal to the cell or lead to cancer. The purpose of this study was to measure the ability of flavonoids to protect DNA against oxidant- and UV-induced mutations. Flavonoids are found in plants, especially in fruits and vegetables, but also in chocolate, wine and tea. They are reported to have anti-oxidant and anti-inflammatory activities, and are capable of scavenging the very damaging hydroxyl radicals. Flavonoids include compounds that give plants their bright colors (red and blue anthocyanins) and also act as UV filters by blocking the UV radiation from damaging the plants. Flavone was the main flavonoid used in this study, which employed two different assay s systems. The first assay was a DNA oxidation-induced mutation, and the second monitored cellular UV-induced mutations. In the first assay the 2.8 kb pTZ19U plasmid was exposed to hydroxyl radicals generated by Fenton chemistry. The treated plasmid was transformed into E. coli cells and the integrity of the plasmid tested by measuring cell viability on ampicillin-containing plates. If the plasmid was oxidatively damaged in its ampicillin resistance and/or origin of replication genes, the transformed cells would not be viable on the ampicillin plates. The data obtained showed that flavonoids added before DNA treatment afforded plasmid protection against hydroxyl radical damage. In the cellular UV-damage assay flavonoids were added to E. coli cells containing the plasmid pAW9. This 2.5 kb plasmid consists mostly of the origin of replication and a tetracycline resistance gene. If damaged in either of these regions the cells will not be viable on tetracycline plates. The cells were exposed to UV light and then plated to measure plasmid and cellular functionality. This assay reveled that flavonoids also afforded protection against UV-induced damage. Together these data demonstrated that flavonoids afforded protection against both oxidative and UV-induced DNA and cellular damage. These data further validate the consumption of fruits, vegetables and products such as dark chocolate and wine as means to improve ones chances in prevention of cancer. These data also suggest that the incorporation of flavonoids into sunscreens may help protect against skin cancer, similar to one of their natural functions in plants. (Support by the Howard Hughes Medical Institute, the McNair Research Program, and an East Tennessee State University Student-Faculty Collaborative Grant through the Undergraduate Honors Program).


Analysis of Rat Testicular Tissue Subjected to Simulated Microgravity

Amy Beck and Allan Forsman, Department of Health Sciences, East Tennessee State University, College of Public and Allied Health, Johnson City, TN 37614

Space flight has been shown to have many adverse effects on various systems throughout the body. Because the opportunity to place research animals on board a space shuttle or the international space station is rare and infrequent various techniques have been designed to simulate the effects of microgravity, while being conducted in Earth based laboratories. A commonly used technique is known as antiorthostatic suspension, also often referred to as hind limb suspension. This technique raises the hind portion of the research animal so that its hind limbs are non-weight bearing. This places the animal in roughly a 30 head down tilt position. This results in cephalad fluid shifts similar to those seen in actual space flight. This technique has also been shown to mimic other physiological parameters that are affected during space flight. This study examined testicular tissue from rats subjected to a 7 day antiorthostatic suspension. Because this tissue was acquired through a tissue sharing program with BioServe Space Technologies some of the experimental animals were also injected with Interleukin 1 receptor antagonist (IL-1 ra) which was hoped to ameliorate some of the effects of antiorthostatic suspension. The tissue was embedded in paraffin, sectioned, and stained using standard H & E staining. The tissue was then qualitatively ranked according to the health of the seminiferous tubules. Our findings indicate that antiorthostatic suspension had adverse effects on the tissue that comprises the walls of the seminiferous tubules. These tubule walls consist of primarily Sertoli cells which form the blood-testis barrier. There was a significant difference between suspended animals and cage control animals. Treatment of animals with IL-1 ra had no effect, either positive or negative, on testicular tissue in both cage control and suspended animals. These findings indicate that space flight may have harmful effects on the seminiferous tubules, more specifically the blood-testis barrier, of astronauts. These effects could lead to an inability to produce viable sperm or at least produce adequate numbers of viable sperm. This would greatly impair the ability to reproduce during long term space flight. This would be problematic when colonizing space stations or other planets/moons that have gravity less than that of Earth. This study directly addresses NASAs goal of investigating an organisms ability to reproduce in microgravity conditions.


NOVEL FOCAL ADHESION KINASE EXPRESSION PATTERNS IN THE HUMAN RETINA

Sarah E. Collier and Michelle M. Duffourc, Ph.D. Department of Pharmacology, Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614

Focal adhesion kinase (FAK) is a protein tyrosine kinase important for cell motility, cell survival (resistance to apoptosis) and axon path-finding during central nervous system development. FAK is a structurally unique tyrosine kinase with a central catalytic domain flanked by large N- and C-terminal domains. The C-terminal domain of FAK contains a focal adhesion targeting domain and provides docking sites for several cytoskeletal and signaling molecules. The functional role of the N-terminal domain is not well understood but it contains a structural domain (4.1/JEF) which mediates interactions with plasma membrane proteins. FAKs N-terminus interacts with beta-integrin cytoplasmic tails and growth factor receptors, suggesting that it is important for coordinating input signals from a variety of sources. The object of this study was to characterize FAK expression patterns in the human retina. Using RT-PCR and Western blot analysis, we identified novel splice variants of FAK with altered expression patterns between normal human retina and tissue culture model of retinoblastoma (Y79 cells). The PCR amplimers were TA-cloned into pCR II (Invitrogen), characterized and sequenced in their entirety. The predicted protein structure of these one of these variants, FAK1-2, indicates that it has several amino acid residues inserted throughout the catalytic domain, suggesting that this variant may display altered kinetic parameters compared to normal FAK. In addition, this variant does not contain the exon immediately upstream of the initiator codon, which could lead to changes in message stability or regulation of its expression. Examination of a second FAK variant, FAK2-2, demonstrates that it lacks an exon immediately downstream of the initiator codon. The splicing out of this exon results in a frame shift in the message. This change results in actually two predicted proteins a small peptide corresponding to the amino terminal region of FAK, and a 101 kD protein encoding the C-terminal end of FAK translated from a different reading frame. While the finding that alternative splicing can be used to generate proteins with alternative amino termini is not new, the use of alternative splicing coupled with a frameshift to generate an amino terminal deletion mutant has never been previously described. This observation is especially important as it is the N-terminal domain which mediates FAK interactions with membrane receptors. Hence, with a truncated N-termini, the variant will be unable to interact with the membrane receptors known to stimulate FAK activity and these variants are predicted to demonstrate altered signaling pathways. Western analysis with antibodies against the N- and C-termini of FAK indicate the presence of multiple FAK-immunoreactive peptides in Y79 cells. Studies are currently underway to assess the functional importance of these variants.


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