Section V: Biomedical Sciences Bailey Science Center, room 3017 Seyed H. Hosseini, presiding.
9:15 EFFECTS OF LIGHT EXPOSURE AND VITAMIN D ON THE DEVELOPMENT OF MYELIN BASIC PROTEIN IN THE MYELIN SHEATH OF NEURONAL AXONS IN JUVENILE ZEBRAFISH **, Callie Holloway * and Linda G. Jones, Young Harris College, Young Harris, GA 30582. Higher latitudes and one's relative exposure to sunlight have been correlated with the incidence of multiple sclerosis (MS). This degenerative autoimmune disorder targets the myelin sheath of nerves, an important component of which is myelin basic protein (MBP). In this study we assessed the levels of MBP by Western blotting using a rabbit polyclonal antibody against MBP and a chromagenic secondary antibody. Our preliminary experiments have confirmed our previous findings that zebrafish (Danio rerio) embryos raised in the dark for seven days post fertilization (dpf) express more MBP than do those raised with exposure to a full-spectrum light source. In that exposure to sunlight is associated with the production of vitamin D, we also investigated whether treatment with vitamin D would alter the levels of MBP found in zebrafish embryos in the two different light exposure groups. Our results to date suggest that exposure to vitamin D enhances the level of MBP found in embryos exposed to light but reduces the level in embryos raised in the dark. We are in the process of confirming these findings and determining a dose-response of this effect of vitamin D. Funding for this project was from the Young Harris College Undergraduate Research Initiative.
9:30 ALTERATION OF GLUT1 AND GLUT4 IN L6 MUSCLE CELLS IN RESPONSE TO GLUCOSE LEVELS AND INSULIN **, Cameron Medina * and Linda G. Jones, Young Harris College, Young Harris, GA 30582. The uptake of glucose into skeletal muscle cells is mediated by the glucose transporters, GLUT1 and GLUT4, the latter being subject to insulin regulation. In that obesity and diabetes are associated with changes in the levels and/or cellular location of the transporters, we are investigating whether the levels of available glucose (1000 mg/L or 4500 mg/L) with or without insulin (3-300 nM) will alter the levels of the transporters in cultured L6 muscle cells. L6 cells are being cultured under standard conditions and subcultured as needed to avoid confluency. Cell lysates from L6 cells incubated under experimental conditions for 48 hours will be subjected to SDS-PAGE and Western blotting in order to determine whether amounts of the transporters are altered. In order to determine whether insulin treatments and/or levels of glucose promote a differential translocation of the transporters, immunocytochemistry (ICC) will be performed on cells fixed in the culture dishes following the same experimental design. A fluorescent secondary antibody will be used to visualize cellular location of GLUT1 and GLUT4. Funding for this project was from the Young Harris College Undergraduate Research Initiative.
9:45 CHEMOTAXIS OF NEUTROPHILS IN A TAIL-FIN WOUND MODEL IN JUVENILE ZEBRAF1SH **, Emalyn Cork * (1), Kacey Mille * (1), Chris Heard * (2) and Linda G. Jones (1), (1) Young Harris College, Young Harris, GA 30582 and 'Georgia College & State University, Milledgeville, GA 31061. The chemotaxis of neutrophils to damaged tissue is an important inflammatory process. Hydrogen peroxide ([H.sub.2][O.sub.2]), known for its oxidative damage at higher concentrations, is thought, at low concentrations, to provide the chemotactic signal to attract neutrophils to the wound site. More specifically, endogenously produced hydrogen peroxide has been implicated in the mediation of the chemotaxis of neutrophils to a tail-fin wound in zebrafish. a widespread model for studying the regenerative and inflammatory responses to wounds. In this study, we examined the chemotactic response to H202, administered exogenously while wounding, to determine whether such treatment enhanced neutrophil migration to the wound site. Four day post fertilization (pdf) zebrafish were wounded using a 26 g needle in the presence or absence of 0.3% [H.sub.2][O.sub.2]. At selected time points, fish were anesthetized, fixed and stained using Sudan Black. Neutrophils at the wound site were then counted under a light microscope. Our results to date suggest that 0.3% [H.sub.2][O.sub.2], promotes a significant increase in the number of neutrophils that migrate to the wound site compared to wounding alone. We plan to confirm these data and to determine whether the addition of the antioxidants glutathione or catalase inhibit the enhanced migration of neutrophils to the wound site. Funding for this project was from the Young Harris College Undergraduate Research Initiative.
10:00 Section Business Meeting
PLANT BASED AS ANTIFUNGAL AGENT AGAINST ASPERGILLUS SPECIES IN GEORGIA PEANUTS **, Reesheda Gilbert *, Department of Biology and Physics, Kennesaw State University, Kennesaw, GA 30144 and College of Health Sciences, University of Northampton, UK. Aspergillus species has shown to be a cumbersome invader of storage grains, peanuts, and nut trees. This commonly encountered fungus contaminates food, plants, and feed for commercially raised animals. Specifically A. flavus and A. parasiticus are two species of Aspergillus that naturally produce aflatoxins that infect peanuts and can be carcinogenic. In the United States, a total 20ppb is the maximum permitted level for human consumption. Research has shown that aflatoxin exposure of (>6000mg) leads to acute toxicity while prolonged exposure to minute doses were carcinogenic). A. flaws is the most common strain of Aspergillus that causes crop contamination and manifesting as a common threat to peanut industries worldwide. Safe and ecological friendly methods for controlling A. flavus with antimicrobial compounds such as essential oils are being explored to substitute chemically based fungicides. Although genetically modified organisms are being persuaded as another method of control, they are not cost-effective. Essential oils derived from aromatic plants such as cinnamon and clove have clinically displayed antifungal characteristics. Our study tested the antifungal effects of cinnamon and clove oil vapors separately. A. flavus spores were exposed to different concentrations at 24. 72, and 96 h and incubated for seven days. Exposure time correlates with both the growth of A. flavus and zone of inhibition. Further studies should focus on active ingredients of vapors to show their potential as biological control agents. Acknowledgements for this study are presented to the following: funding provided by NSF and LSAMP Peach Grants, Department of Biology and Physics at Kennesaw State University, Dr. K. Gkatzionis. Northampton, UK. Army Lester and Premila Achar. Kennesaw State University, and Dr. Seenivasa, University of Mysore India.
NLRP 3 INFLAMMASOME ASSEMBLY IS REQUIRED FOR CASPASE ACTIVATION DURING CHLAMYDIA INFECTION **, Danielle N. McKeithen * (1), (2), Yusuf Omosun (1), E. Caroline Kibakaya * (1), Francis O. Eko (1), Joseph U. lgietseme (1), (3), Godwin A. Ananaba (2) and Qing He (1). (1) Department Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, Atlanta, GA 30310. (1) Department of Biology. Clark Atlanta University, Atlanta, GA 30314 and (3) Centers for Disease Control & Prevention (CDC), Atlanta, GA 30333. Chlamydia. which induces inflammation and infertility in women, is a major infectious bacterial agent that causes sexually transmitted disease (STD) worldwide. The purpose of this study is to determine the role of the inflamma-some in bone marrow dendritic cells (BMDCs) of Interleukin 10 knock out (IL-10KO) mice Chlamydia infection. BMDCs were harvested from wild type (WT) and IL-10KO mice and pulsed with C. muridarum. Our results showed higher inflammasome, pro-caspase-1, and, pro-IL -1[beta] expression in IL-10KO DCs, but no difference in the inflam-masome assembly at early infection time point. After 2hr infection there was reduced co-localization of NALP3, ASC, and, caspase 1 in IL-10KO DCs. The NLRP3 inflamma-some sub-aggregates are highly expressed in IL-10KO mice, but at the macro-molecular level appear to form incomplete aggregates leading to a reduction in activated NLRP3 inflammasomes. While in WT there appears to be robust activation of most inflammasomes, thus leading to increased apoptosis. This research is supported by Title II Grant#22210J and NIH#1SC1AII03041-01A1.
AN ASSESSMENT OF THE TOXICITY OF NOVEL ANALOGUES OF PERSIN **, Heather D. Perry *, David N. Aban *. Thomas D. Crute and Richard D. Griner, Georgia Regents University, Augusta, GA 30904. Persin is a compound produced in avocado leaves that has been shown previously to be cytotoxic to mammary epithelium and to induce apoptosis in several breast cancer cell lines. However, at slightly higher doses administered in vivo, persin demonstrated cardiotoxicity. Thus, the search for safer analogues has been ongoing. Specifically, our lab has synthesized analogues of persin in an effort to identify other compounds that share persin's ability to induce apoptosis in breast cancer cells. Two analogues, a secondary alcohol synthesized from oleic acid (CG1) and its derived acetate (CG2) have been tested against the human breast cancer cell line MCF-7. In initial screenings, as little as 30uM of CG1 reduced cell viability to only 3% of control, while as much as 1mM CG2 had no impact on cell viability (96% of control). It will now be important to determine if these compounds are acutely toxic or if they are inducing apoptosis. Thus, assays for specific markers of apoptosis will be conducted. If apoptosis is detected, additional studies will examine the ability of these compounds to stabilize/bundle microtubules and to elicit an increase in cellular ceramide levels as have been shown for persin. Ultimately a link between the three events of increased ceramide levels, bundling of microtubules, and initiation of apoptosis will be sought. Synthesis of additional analogues is planned, and these will be screened and tested in a similar manner.
ANTIMICROBIAL PROPERTIES OF EGG WHITE ON THE BACTERIA AEROMONAS HYDROPH1LA. STAPHYLOCOCCUS AUREUS, AND ESCHERICH1A COLI **, Natalie Mllem *. Joshua Butler *, Stephen Gitau *. Army Lester and Donald McGarey, Kennesaw State University. Kennesaw, GA 30144. Background: The antimicrobial properties of egg white have long been documented: however the mechanism of this natural process is not well understood. The objective of this study was to determine the lethal point of egg white on various concentrations of the bacteria Aeromonas hydrophila. Escherichia coli and Staphylococcus aureus. Methods: One milliliter of egg white from three-day-old fertilized eggs was aseptically transferred to sterile test tubes. Aeromonas hydrophila, Staphylococcus aureus and Escherichia coli at cell counts ranging from 1 x[10.sup.3] to 1 x [10.sup.6] colony forming units (CFUs) were inoculated into the egg white, and incubated and cultured over a three-day period. In a separate experiment, 1 x [10.sup.3] to 1 x [10.sup.6] CFUs of these bacteria were inoculated 1cm from the developing embryo in a shell-less environment and tracked for three days for embryo survival and bacterial persistence. Results: In egg white inoculated with 1 x [10.sup.3] to 1 x 10 cells (1 CFU/microliter up to 100 CFUs/microliter egg), no bacteria were recovered after 24 hours. At 5 x [10.sup.5] CFUs (500 CFUs/microliter egg white) a severe drop in population within 24-48 hours, then cell repopulated in 50% of the tubes. At 1 x [10.sup.6] cell (1000 cells/1 microliter of egg white) the antimicrobial action of egg white was overwhelmed with no reduction in bacterial CFUs. In the shell-less egg experiment, 1 x [10.sup.3] CFUs of each bacterium was lethal to the developing embryo. Discussion: Results clearly show that egg white has antimicrobial properties that destroy pathogen at concentrations of 100 CFUs/microliters or less, but appears to reach a lethal concentration (50%) at 500 CFUs/microliters. Inoculation of 1x10 CFUs 1 cm away from the embryo is lethal, most likely as the inoculation is on the yolk and avoids the antimicrobial action of egg white.
RNA INTERFERENCE (RNAI) STRATEGIES TO REDUCE FITNESS OF INSECT PESTS **, Neha Reddy * (1) and Wayne B. Hunter (2). (1) Lincoln Park Academy, Ft. Pierce. FL 34950 and (2) United States Depart. Agriculture--ARS, Ft. Pierce, FL 34945. RNA interference (RNAi) is a naturally occurring cellular process that results in the down regulation of targeted messenger RNA transcripts. RNAi was utilized to reduce the fitness of an insect pest, the Asian citrus psyllid. (Diaphorina citri), which transmits a deadly disease of citrus trees. Citrus greening disease (Huanglongbing) has resulted in the loss of over $3 billion and over 6,000 jobs since 2006. We designed a double stranded RNA (dsRNA) treatment which was administered to psyllids using a cut plant feeding assay. Each of the three trials was comprised of four cages of plant shoots which absorbed the dsRNA, and two cages of plant shoots in water only. Each cage consisted of 12 adult psyllids. Observations of mortality were taken daily for 11 days. Prior to insect exposure, the set up of plant shoots in cages were screened for three days for any signs of wilting or contamination. Only healthy looking plant cuttings were used in the experiment. Initial trials showed that the dsRNA treatment induced significant insect mortality over controls. RNAi approaches are highly specific due to their design based on insect sequences, thus demonstrating tremendous potential as a means of pest management. Studies are still in progress.
STUDY OF THE GENE REGULATORY NETWORK FOR SEA URCHIN PIGMENT CELLS DEVELOPMENT **, Antonio C. Ortiz * and Cristina Calestani, Valdosta State University, Valdosta, GA 31698. Embryogenesis is a remarkably dynamic and precisely regulated process, and the exact genetic programming required has not been entirely uncovered. We utilize Strongylocentrotus purpuratus to study the process of cell differentiation during embryogenesis. Our objective is to gain further understanding of the gene transcriptional regulation of differentiation genes, using as a model the development of larval pigment cells, which are part of the larval immune system. We study cis-regulatory elements, more specifically those controlling the expression of the differentiation gene polyketide synthase (pks) in pigment cells. Comparative genomics was performed utilizing the software FamilyRelationsII, MEME, and SANN in order to predict cisregulatory modules and putative DNA-binding sites in the -3kb region of the pks promoter. The SANN and MEME analyses were done utilizing five co-expressed pigment cell genes: flavin-monoxidase 1, 2, 3, sulfotransferase, and pks. Furthermore, we compared them in two additional sea urchin species: Allocentrotus purpuratus and Strongilocentrotus fransiscanus. From these analyses we will select putative sites to test via site-directed mutagenesis within the -3kb promoter. We then utilized the Yeast-One-Hybrid system to identify putative transcription factors that bind to the pks promoter. As a result of this screen Otx and Eve have been identified and will be confirmed by site-directed mutagenesis and gene knock-down approaches.
PERFLUOROOCIANOIC ACID (PFOA) DECREASES STEROIDOGENESIS IN MOUSE LEYDIG TUMOR CELLS **, S. Tadros * and J.D. Cannon, Augusta State University. Augusta, GA 30904. Perfluorooctanoic acid (PFOA) is a synthetic long-chain perfluorinated chemical that has been widely used in consumer and industrial products, such as pesticides, non-stick cookware, and weather- and fire-resistant clothing. PFOA has been identified as an environmental contaminant and recently has been suggested to be an endocrine disruptor. The goal of the present study is to examine the effect of PFOA on steroid production in mouse Leydig Tumor cells (mLTC-1). Cells were treated with concentrations of PFOA ranging from 100nM to 100[micro]M for 24h before being stimulated for 4h with human chorionic gonadotropin (hCG). Media was collected and progesterone measured using an ELISA (ALPCO). PFOA concentration of 100pM reduced hCG-stimulated progesterone synthesis by over 70% compared to hCG-stimulated controls. Twenty-four hour treatment of cells with the above concentrations showed no significant decrease in cell viability, as measured by the CellTiter-Blue[R] Cell Viability Assay (Promega), suggesting that the decline in steroidogenesis was not due to reduced cell numbers. It is expected that repeating the above experiments will result in the same findings, confirming that PFOA does in fact decrease progesterone production in mLTC-1 cells. Because this compound is environmentally persistent. further studies should be done to examine its effect on human health, including reproductive health, and to understand its mechanism of action.
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|Title Annotation:||SATURDAY PAPER PRESENTATIONS|
|Publication:||Georgia Journal of Science|
|Article Type:||Conference notes|
|Date:||Mar 22, 2013|
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