Cellular, molecular and developmental biology.

Chair: Lauren Brandon, Mississippi University for Women

Vice-chair: Bernadette Connors, Millsaps College

THURSDAY MORNING

Oak

9:00 Opening Remarks

O2.01

9:10 ACTIVITY ASSAY OF A COTTON FIBER RING-TYPE UBIQUITIN LIGASE

Meng-Hsuan Ho, Din-Pow Ma

Mississippi State University 

The ubiquitin-proteasome proteolysis pathway, responsible for the degradation and short-lived proteins, regulates a wide variety of cellular processes in eukaryotes. The ubiquination of target proteins for degradation requires sequential action by three enzymes: an ubiquitin-activating enzymes (E1), an ubiquitin conjugating enzyme (E2), and an ubiquitin ligase (E3). There are two main classes of E3 ligases, the HECT domain and the RING-type ligases. In plants, the RING-type ubiquitin E3s are encoded by a very large gene family. It has been reported that there are 469 RING-type protein genes in the Arabidopsis genome, suggesting that plant RING-type ubiquitin E3s have many specific target substrates. Using a cotton E2 (GhUBC 1) as bait in a bacterial two-hybrid system, we had previously cloned two unique fiber cDNAs, GhRIN1 and GhRIN2, encoding RING-type ubiquitin ligases. The GhRIN1 protein derived from the full-length GhRIN1 cDNA consists of 338 aa and has the highest homologies with E3 homologs from Arabidopsis (AAN18152 encoded by AT3g19950) and Rice (Oryza sativa) (BAD67937) by BLast searches. GhRIN1 contains eight His and Cys residues (C3H2C3) in the RING domain and is classified as the RING-H2 protein. To understand the function of the GhRIN1 protein, GhRIN1 was tagged with GST (glutathione-S-transferase) and over-expressed in E. coli. The over-expressed GhRIN1 protein had the E3 ligase activity and was capable of catalyzing the formation of polyubiquitin chains via in vitro auto-ubiquitination.

O2.02

9:30 IgD EXPRESSION IN CHANNEL CATFISH, ICTALURUS PUNCTATUS

Shenika Kelly (1), Eva Bengten (2), Eva-Stina Edholm (2), Melanie Wilson (2)

(1) Alcorn State University, (2) University of Mississippi Medical Center 

The overall goal of this project is to test the hypothesis that channel catfish (Ictalurus punctatus) secreted immunoglobulin delta (IgDsec) is bound to the surface of a granular cell population by an activation inducible IgD binding receptor. The catfish IgD homolog is a chimeric molecule consisting of a rearranged variable heavy (VH), diversity (D) and joining (JH) segment spliced to the first IgM constant domain, followed by seven IgD constant domains and either a transmembrane domain or a secreted tail. Cell separation studies of peripheral blood leukocytes (PBL) using anti-IgD and anti-IgM monoclonal antibodies revealed that IgD is found on surfaces of three distinct cell types; IgM+/IgD+ or IgM-/IgD+ B cells and IgM-/IgD+ granular cells, of unknown origin that don't express any Ig message, but are armed with exogenously produced IgD via a putative IgD-binding receptor. Induction of IgD staining on granulocytes was observed in response to over-night incubation with the mitogen concavlin A. Finally, recombinant (r) IgD proteins encompassing different IgD domains were generated in a FLAG epitope tag expression vector. The resulting proteins were purified from total cell lysates using an anti-FLAG column and verified by Western blot. Future experiments include co-culture of concavlin A stimulated cells with rIgD proteins to map the part of IgD that binds to the cells and co-immunoprecipitations will be done to detect the putative IgD receptor.

O2.03

9:50 PARTIALLY PURIFIED FRACTIONS OF LEAF EXTRACTS OF OCIMUM GRATISSIMUM EXHIBIT HIGHER ANTI-PROLIFERATION ACTIVITY IN PROSTATE ADENOCARCINOMA CELLS

Stephen Ekunwe (1), Melvanique Thomas (1), Jelani Zarif (1), Lauraetta Lockridge (1), Yong Chen (2), Xiaopu Zhang (2), Hengshan Wang (2)

(1) Jackson State University, (2) Guangxi Normal University, China

Prostate Cancer is the third leading cause of cancer related deaths of men 50 years and older in the U.S. It affects African Americans disproportionately. There is currently no effective treatment for prostate cancer. Radical prostatectomy or brachytherapy, with the undesirable side effects of erectile dysfunction (ED) and urinary incontinence (UI), are the current treatments for the disease. Lack of effective treatment drives the effort to discover and develop new, affordable, highly efficacious prostate cancer drugs with few or no serious adverse side effects. Previous in-vitro studies have shown that the aqueous extracts of the edible medicinal herb Ocimum gratissimum (Og), inhibit the proliferation of prostate adenocarcinoma (PC-3) cells. This leads us to believe that Og may harbor novel cancer-fighting compounds that need to be isolated. In this study, Og powder was sequentially extracted with different organic solvents to obtain various fractions. We hypothesized that the anti-proliferation activity of these fractions will be significantly greater than that of the aqueous extract. This hypothesis was tested by treating PC-3 cells with 1.61mg/ml of each fraction. Compared to treatment with 2.0mg/ml of aqueous extract, the fractions showed greater anti-proliferation activity against PC-3 cells. Fraction P2, the most active fraction, and fraction P3-2 showed a 5000-fold and a 537-fold higher anti-proliferation activity than the aqueous extracts respectively. These findings suggest that the organic solvent fractions of Og may contain more of its bioactive component(s).

O2.04

10:10 Biofilm Formation is Regulated by Msa in Staphylococcus aureus

Antony Schwartz, Karthik Sambanthamoorthy. Mohamed Elasri

The University of Southern Mississippi

Staphylococcus aureus is a Gram-positive bacterium that causes a wide array of community-acquired and nosocomial infections. Infections caused by S. aureus are progressively more difficult to treat owing to the increasing prevalence of antibiotic resistant strains. In addition, S. aureus forms biofilms. Generally, biofilm bacteria show much greater resistance to antibiotics due to changes in gene expression or physiology associated with surface-attached existence. The global regulator, sarA, regulates biofilm formation under both in vitro and in vivo conditions. Recently, we have elucidated the function of a novel locus, msa (modulator of SarA), that is required for the full expression of sarA. In this study, we show that the msa mutant exhibits a significant decrease in biofilm formation under static and flow conditions. Further analysis of the mutant biofilm using GFP and confocal microscopy revealed differences in morphology and thickness when compared to the wild type. Expression studies on biofilm-related genes showed that mutation of msa decreased the expression of arcA, arlS, atl, clfA, and icaR. Collectively, these results indicate that Msa plays a critical role in biofilm formation.

10:30 Break

O2.05

11:00 MSA EXPRESSION WITHIN THE STAPHYLOCCOCUS AUREUS BIOFILM

Victoria Jones (1), Antony Schwartz (2), Mohamed Elasri (2)

(1) Mississippi University For Women, (2) University of Southern Mississippi

Staphylococcus aureus is a gram positive bacterium that causes a myriad of diseases that range from superficial to deadly infections. These infections are caused by variety of virulence factors that are coordinately controlled by global regulators. One such regulator is the staphylococcal accessory regulator (sarA). SarA regulates over 100 genes that are involved in pathogenesis and the general maintenance of the cell. Also, SarA regulates biofilm formation. A recent study demonstrated that Msa (Modulator of sarA) modulates the expression of sarA and other virulence factors. The aim of this study is to construct a Green Fluorescence Protein (GFP) reporter system whose expression is regulated by the msa promoter. This reporter system will be used to visualize the spatial expression of Msa within a staphylococcal biofilm formed in a flow-cell apparatus.

O2.06

11:20 I1-1[beta]-MEDIATED UP-REGULATION OF CXCL12 WITHIN ASTROCYTES DURING EAE 

Hong Loan Nguyen (1), Robyn Klein (2), Erin McCandless (2), Denise Dorsey (2), Bo Zhang (2), Leroy Johnson

(1) Alcorn State University, (2) Washington University in St. Louis School of Medicine

Multiple Sclerosis (MS) is an autoimmune disease of the central nervous system. Experimental autoimmune encephalomyelitis (EAE), a mouse model for MS, is induced by immunization with myelin proteins or adoptive transfer of myelin-specific CD4+ T cells. Recent research using the EAE model indicates that the chemokine CXCL12, is increased in the CNS during EAE and acts to limit inflammation at the blood-brain barrier (BBB). CXCL12 is normally found at endothelial cell basolateral surfaces and serves to localize infiltrating immune cells to the perivascular space, preventing CNS entry. During CNS autoimmune diseases, the pattern of CXCL12 is altered such that basolateral polarity is lost. This altered CXCL12 expression is associated with loss of immune cell localization to the perivascular space in increased parenchymal migration. Preliminary work has determined that the cytokine IL-1[beta], which is also increased in the CNS during MS and EAE, is capable of redistributing CXCL12 expression at the BBB. We identified astrocytes as the source of redistributed CXCL12 at the BBB in vivo in mice with EAE or in those treated with IL-1[beta]. Primary cultures of astrocytes treated with IL-1[beta] displayed a dose-dependent up-regulation of CXCL12 mRNA. Primary cultures of oligodendrocyte precursor cells (OPCs), the myelinating glial cells of the CNS, increased their expression of myelin oligodendrocyte glycoprotein (MOG) mRNA, suggesting that CXCL12 promotes OPC maturation. Thus IL-1[beta] mediated increase in CXCL12 expression within astrocytes during CNS autoimmune diseases has consequences for both BBB integrity and remyelination.

THURSDAY AFTERNOON

Oak

O2.07

1:30 SULFUR METABOLISM IN THE DIMORPHIC FUNGUS HISTOPLASMA CAPSULATUM

Melissa Adams, Brooke Wheeler, Glen Shearer

The University of Southern Mississipi

Histoplasma capsulatum is a dimorphic fungus that causes histoplasmosis, a common respiratory disease in man. The organism grows in soil in a differentiated, multicellular MOLD form and de-differentiates to a simple budding YEAST in the lungs. This M-Y shift is required for disease. Sulfhydryl groups (particularly cysteine) are required for the M-Y transition. Cysteine dioxygenase (CDO1) produces cysteine sulfinic acid (CSA) from cystiene. CSA is a key intermediate in cysteine metabolism. CDO1 is thought to regulate the intracellular level of free cysteine or provide a product necessary for the transition to the yeast state. The role of cysteine in dimorphism is unclear, but it may act via modulation of intracellular redox. Other pathways involved in cysteine metabolism, and presumably redox control, include gamma-glutamyl cysteine ligase (GSH1) and Glutathione synthetase (GSH2) which result in cysteine being converted to glutathione. The expression and enzyme activity of these three genes are being studied. CDO1, which has been previously isolated in our lab, appears to be expressed in both M and Y phases of the organism while GSH1 and GSH2 appear to be expressed only in the yeast phase. Transcript levels are being measured by northern blotting and real Time PCR. Experiments are underway to determine if enzyme levels correlate with transcript levels.

O2.08

1:50 DIFFERENTIAL EXPRESSION OF THE M46 GENE IN THE MOLD AND YEAST FORMS OF THE PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM

Davida Crossley, Rupesh Patel, Glen Shearer

The University of Southern Mississipi

Histoplasma capsulatum, is the etiologic agent for the respiratory disease histoplasmosis. This dimorphic fungus grows as a multicellular mold in the soil and converts to a budding yeast growth form in the lungs. This mold-to-yeast conversion, which is a requirement for pathogenesis, can also be accomplished in the lab my switching incubation temperature from 25 C to 37 C. To understand the molecular basis of dimorphism we have isolated numerous mold-specific and yeast-specific genes. The subject of this study, the M46 gene, was originally isolated as a mold-specific gene in strain G186AS. Recent data, however, have shown that M46 is expressed in the mold morphotype of strains G186AS and Downs, but is transcriptionally silent in G184AS and G217B strains. The complete ORF is present in all strains and the putative promoter sequence appears to be intact. The reason for lack of transcription in the latter strains could be due to a cis- or trans-acting problem. To address this question, we have constructed M46 promoter- fusions with a GFP (green fluorescent protein) reporter to analyze promoter function. Fluorescent microscopy data has shown that M46 promoters from all four strains were functional. This analysis allows us to conjecture that M46 is silent in strains G184AS and G217B due to a missing trans regulating factor(s). Future studies will attempt to identify this missing trans-acting factor(s) and construction of an M46 knockout to investigate the function of M46.

O2.09

2:10 MAPPING CANDIDATE GENES INVOLVED IN TOMATO FRUIT CUTICLE BIOSYNTHESIS 

Vivian Smith (1), Leroy Johnson (1), Keith McGee (1)

(1) Alcorn State University, 2Cornell University

Most of the candidate genes used for mapping were found in Arabidopsis and few in other plants. Using the tomato ESTs databank, homologues gene sequences were found in the tomato genome. DNA sequences of these genes were amplified and sequenced from the cultivated tomato (M82) and the wild species S. pennelli genomic DNA. The polymorphism between the sequences of the two lines was used to design CAPS (Cleaved Amplified Polymorphic Sequences) genetic markers that were mapped to the Tomato-EXPEN 200 map (http:/www.sgn.cornell.edu/cview/). There were twelve genes that were found in Arabidopsis: LACS, CER5, CYPA8, FDH, CER6, CYPA2, BDG, CER5, CYPA6, ECR, FAD2 and FATB. These Arabidopsis genes are also fruit specific genes, which is why a homologous comparison was found in tomato fruits. LACS was found on chromosome 1, CER5 was found on chromosome 3, CYPA8 was found on chromosome 4, FDH was found on chromosome 5, CER6 was found on chromosome 6, CYPA2 was found on chromosome 7, and BDG was found on chromosome 10. The other genes were not mapped on the tomato chromosome because of problems with DNA sequencing. Only one of the candidate genes was found to show some potential to be the gene causing cuticle mutation, and that gene was LACS. The other genes did not prove to be the genes causing the mutation in the phenotype of the CM (cuticle mutation) lines of tomatoes.

O2.10

2:30 ELUCIDATION OF THE TRANSMEMBRANE TOPOLOGY OF MSA IN STAPHYLOCOCCUS AUREUS.

Hope Ferguson, Vijayaraj Nagarajan, Mohamed Elasri

The University of Southern Mississippi

Staphylococcus aureus is an opportunistic pathogen that inhabits the skin and mucosal membranes of humans. Treatment of S. aureus infections is increasingly difficult due to antibiotic resistance. S. aureus causes a wide variety of infections using a large number of virulence factors. Many virulence factors contributing to the pathogenicity of S. aureus are regulated by global regulators such as SarA. One of the promising approaches to combating antibiotic resistance is to target these global regulators. We have recently identified a protein that is essential for SarA expression. Bioinformatics analysis of Msa shows that it is a putative membrane protein. The predicted topology of the Msa is IN-OUT with three putative transmembrane segments (from amino acid positions 27-47, 54-75, 108-125). The N-terminus is predicted to be in the cytoplasmic side of the membrane while the C-terminus is predicted to be outside the membrane. Secondary structure prediction results indicate the presence of four distinct helical regions. One helical region corresponds to the cytoplasmic helix while the other three correspond to the integral membrane helices. The goal of this study is to determine the transmembrane topology of Msa using GFP (green fluorescent protein) and phoA (alkaline phosphatase) fusions. Analysis of the Msa sequence with eight signal peptide prediction programs showed a consensus that Msa has a cleavable N-terminal signal peptide that mediates its targeting into the membrane. We will also use these fusions to determine if Msa utilizes a signal peptide for translocation into the membrane.

6:00 Dodgen Reception and Divisional Poster Sessions Please set up between 4:00p and 4:30p Location: Grand Ballroom

P2.01

SIGNAL INDUCED CHANGES IN THE SUBCELLULAR LOCATIONS OF IRAK-1--INTEGRATES LPS AND TNF R1 SIGNALING

Tamica Collins (1), Angela Lockett (2), Joseph A. Cameron (1), Maureen Harrington (1)

(1) Jackson State University, (2) Indianapolis University/Purdue University

The innate immune system is present in nearly all multicellular organisms and is regulated in part by pathogen associated molecular pattern receptors. The interleukin-1 receptor associated kinase-1 (IRAK-1) is involved in the activation of the transcription factor NF-kB in response to bacterial derived lipopolysaccharide (LPS) and the pro-inflammatory cytokines, TNF[alpha] and IL-1. Our research is focused on how the subcellular location of IRAK-1 is regulated by different stimuli. Mouse embryonic fibroblasts (MEFS) treated with LPS for 1 hour were harvested, separated into cytosolic and membrane fractions and subjected to SDS-polyacrylamide gel electrophoresis. Western blots were prepared, and probed for IRAK-1. Under steady-state conditions the IRAK-1 protein is located in the plasma membrane, cytosol and in a pelleted fraction which contains non-detergent soluble proteins. In response to LPS, IRAK-1 decreases in the cytosol and the amount of post-translationally modified IRAK-1 increases in the plasma membrane and the pellet. Next we examined whether cholesterol is required for the LPS induced modification of IRAK-1 in the pellet or plasma membrane. In cells, in which the cholesterol was depleted from the plasma membrane, the LPS induced modification of IRAK-1 is reduced. LPS also induces binding of modified IRAK-1 to the type I TNF[alpha] receptor. If MEFs are pretreated with 0.4% ethanol, which disrupts lipid rafts, IRAK-1 binding to the type I TNF receptor is enahanced. (Supported in part by NIGMS-NIH Grant No. R25GM67592)

P2.02

Unique regulation of Fe-superoxide dismutase (Fe-SOD) by Ferric Uptake Regulator in Azotobacter vinelandii.

Young-Man Kwon, Yean-Sung Jung

Mississippi State University

Background:In Escherichia coli and Pseudomonas aeruginosa, Fur protein positively regulates the expression of sodB encoding Fe-SOD through small RNAs, RyhB or PrrFs. Azotobacter vinelandii also possesses fur and prrf genes encoding Fur and PrrF, respectively. Method:To study the effect of Fur on FeSOD in A. vinelandii, fur gene was inactivated by insertion of kanamycin resistance cassette. Ability of the mutant to grow aerobically was measured in both fixed-nitrogen and nitrogen-fixing conditions. On-gel activity assay and immunoblot analysis were used to estimate Fe-SOD activity and polypeptide levels, respectively, in

cell extracts. Atomoc absorption was used to measure iron levels in wild type and mutant cells. Results: The mutant failed to grow diazotropically at normal growth condition (200 rpm and 30 [degrees]C), but started to grow slowly when the aeration was decreased, suggesting that oxidative stress is responsible for growth deficiency. Indeed, the mutant showed declined Fe-SOD activity, but similar levels of its polypeptide, suggesting that a defect in the process of Fe insertion into apoSOD contributes to the decreased activity. The mutant accumulated more irons than wild type. Thus, iron shortage is not the factor causing apoSOD production. Conclusion: the results showing that the mutant has wild-type levels of Fe-SOD mainly as an apoform suggest a novel mechanism of Fur on the modulation of Fe-SOD activity in A. vinelandii.

P2.03

CAMPTOTHECIN MODIFIES LIPOPOLYSACCHARIDE/PHORBOL-12-MYRISTATE-13-ACETATE INDUCTION OF INTERLEUKIN-1 RECEPTOR ANTAGONIST IN HUMAN FIBROBLASTS.

Stephen LeBlanc, John Jenkins

University of Mississippi Medical Center

Interleukin-1 receptor antagonist (IL-1Ra) is a member of the IL-1 family of cytokines. IL-1Ra has anti-inflammatory properties mediated via competitive IL-1 receptor binding, inhibiting IL-1alpha/beta signaling. Lipopolysaccharide (LPS) and phorbol-12-myristate-13-acetate (PMA) have a synergistic effect on production of an intracellular form of IL-1Ra in fibroblasts. Camptothecin (CPT) is an anti-tumor drug that binds and inhibits topoisomerase I, inducing apoptosis at high doses. We examined the effects of CPT on LPS/PMA-induced IL-1Ra in human fibroblasts. We cultured CRL-2522 (normal foreskin fibroblast), CRL-1475 (normal neonatal skin fibroblast), and synovial fibroblasts from a patient with rheumatoid arthritis with and without LPS/PMA with different concentrations of CPT. We measured IL-1Ra mRNA and protein expression by qualitative RT-PCR and sandwich ELISA respectively. High concentrations (10-4-10-5 M) of CPT inhibit the effects of LPS/PMA on IL-1Ra gene expression. LPS/PMA-induced IL-1Ra protein expression was also inhibited by high doses of CPT. Cell death and apoptosis, measured by staining techniques, did not account for this CPT-induced inhibition of IL-1Ra protein. Conversely, in low concentrations (10-6-10-7 M), CPT augments IL-1Ra gene expression. CPT elicits a biphasic dose-effect response on LPS/PMA-induced IL-1Ra in fibroblasts. This complex regulation of intracellular IL-1Ra suggests an alternate function other than binding cell surface IL-1 receptors.

P2.04

HETEROLOGOUS EXPRESSION OF HUMAN PHOSPHOLIPASE D IN BUDDING YEAST

Ilma Patel, Graham Howard, Bernadette Connors

Millsaps College

Phospholipase D (PLD) is a ubiquitous enzyme responsible for the catalysis of phospholipids in many organisms, ranging from bacteria, yeasts, plants, and mammals. Defects in PLD activity are associated with nonalcoholic fatty liver disease and also growth of effector T-lymphocytes in humans. PLD catalyzes the hydrolysis of phosphatidylcholine (PC) to form phosphatidic acid (PA) and choline. PA is further hydrolyzed by PA phosphohydrolase to form diacylglycerol (DAG), both of which are second messengers in activities of many protein kinases. Additionally, PLD is capable of performing transphosphatidylation reactions in which PA is transferred from PC to a primary alcohol to form phosphatidylethanol. These activities are induced by numerous agents, such as ethanol, galactose or glucose, and potassium acetate. In Saccharomyces cerevisae cells lacking PLDSpo14 activity show growth defects on nonfermentable carbon sources, and homozygous diploids are unable to sporulate. In an attempt to understand whether human PLD (hPLD) can function similarly to the yeast PLDSpo14 we cloned hPLD isoform 1 (hPLD1) downstream of the galactose-inducible gal1 promoter, and transformed both haploid and diploid strains of S. cerevisae deleted for PLD1Spo14 with the recombinant plasmid. To assay for functional replacement of PLD activity, we are currently examining growth of the strain expressing hPLD1 on glycerol and ethanol, and determining sporulation efficiencies. Determining the similarities between the yeast PLDSpo 14 and hPLD can assist us in further understanding the reactions catalyzed by PLD.

(Supported by NIH Grant Number RR 016476 from the MFGN INBRE Program of the National Center for Research Resource).

P2.05

THE ROLE OF DIA2 AND CDC20 IN UV-ACTIVATED REPAIR PATHWAYS

Bernadette Connors, Leah Strickland, Graham Howard

Millsaps College

DNA replication requires the regulation and stabilization of replication forks to ensure that the genome is replicated only once per cell cycle. One means to regulate this process is through protein degradation, accomplished in part by the Anaphase Promoting Complex (APC) and the Skp1-Cdc53/Cullin-F-box (SCF). Dia2 is a putative SCF protein that is a member of a complex involved in protein degradation. The primary focus of the research was to determine whether a relationship between Dia2 and the APC exists, and the nature of this interaction. To examine this interaction, the ability of strains carrying the temperature sensitive cdc20-1 allele and [DELTA]dia2 to survive when grown on various drug-containing media were determined. The capacities of the [DELTA]dia2 and [DELTA]dia2/cdc20-1 mutant strains to evoke the RNR3-lacZ damage repair pathway were also examined. Strains carrying the [DELTA]dia2/cdc20-1 and cdc20-l mutations were found to have increased survival rates upon exposure to UV radiation (50-200 J/m2) relative to unirradiated samples. However, the Adia2 and Adia2/cdc20-1 strains were diminished in their capacity to evoke the RNR3-lacZ damage repair system following exposure to UV radiation, while cdc20-1 strains exhibited ability comparable to wild type cells. Our characterization of these mutant strains suggests that both Cdc20 and Dia2 have roles in the cell's response to UV radiation, although we anticipate that they act along different pathways. (Supported by NIH Grant Number RR 016476 from the MFGN INBRE Program).

P2.06

PRODUCTION AND CHARACTERIZATION OF MONOCLONAL ANTIBODIES TO ENDOTHELIAL MONOCYTE ACTIVATING POLYPEPTIDE II (EMAP II)

Lillian Brady (2), Matthias Clauss (1)

(1) Indiana University School of Medicine, (2) Alcorn State University

EMAP II is an antiangiogenic factor and thus maybe of relevance for diseases. The aim of this project was to produce large quantities of antibody as well as characterize and sub-clone a rat anti-mouse EMAP II monoclonal antibody clone, the monoclonal antibody 7/1. First, by cultivating the 7/1 clone, we tested whether the concentration of cells in growth medium affected the growth of cells after thawing. Next, we tested the ability of the 7/1 F5 clone to recognize the EMAP II antigen. Although the monoclonal antibody was reacting with EMAP II very well at higher concentrations, at lower concentrations the polyclonal antiserum turned out to be more sensitive. In order to exclude the possibility that 7/1 was not a real monoclonal line, we decided to sub clone, placing, theoretically, one cell/96-well plate. We compared the supernatants of the 7/1 F5 clone and 7/1 E10 clone with the parental 7/1 clone. The 7/1 E10 clone showed lower OD in comparison to 7/1 and 7/1 F5 at high dose of EMAP II. With the low dose of EMAP II all three clones resulted in similar OD values. It was concluded that the 7/1 hybridoma clone was established as a producer of the desired antibody against EMAP II. After testing the dose dependence of EMAP II in an ELISA, we found that at lower concentrations of EMAP II, the 7/1 was less sensitive than the polyclonal antibody.

P2.07

Affinity Of Ramelteon For Competition To 2-[125I]Iodomelatonin Binding To The Super High And High Affinity States Of The hMT1 And hMT2 Melatonin Receptors Expressed In Mammalian Cells

Desmond Henderson (2), Yomayra Guzman (1), Margarita Dubocovich (1), Rajendram Rajnarayanan (2)

(1) Northwestern University, (2) Tougaloo College

Melatonin (5-methoxy-N-acetyltryptamine), a hormone released at night activates two G-protein coupled receptors, the MT1 and MT2. The melatonin receptor agonist ramelteon ((S)-N-[2-(1, 6, 7, 8-tetrahydro-2H-indeno-[5,4-b] furan-8-yl) ethyl] propionamide) promotes sleep and modulates circadian rhythms (Curr Opinion Inv Drugs 1:114, 2005). The goal of this project was to determine the affinity of ramelteon for competition to 2-[125I]iodomelatonin binding to the super high and high affinity states of the hMT1 and hMT2 melatonin receptors stably expressed in Chinese Hamster Ovary (CHO) (Mol Pharmacol 50:166, 1996). 2-[125I]Iodomelatonin (90 pM) binding was determined in membranes from CHO-hMT1 and CHO-hMT2 in 50 mM Tris-HC1/10 mM MgC12 for 1hr at 25[degrees]C, followed by rapid filtration, and counting in a [gamma] counter. Melatonin and ramelteon (0.3pM - 100nM) competition for 2-[125I]iodomelatonin binding to the hMT1 and hMT2 receptors were biphasic. The affinity constants (IC50, n=3-5) for ramelteon to the super high and high affinity states were: 5.1 + 2pM and 0.5 + 0.03nM for the hMT1, and 0.44 + 0.1pM and 1.2 + 0.2nM for the hMT2, respectively. This model will be used to assess changes in receptor affinity to the super high and high affinity states of the receptors following exposure of CHO-hMT1 and CHO-hMT2 cells to ramelteon (0.3 and 30 nM). Results from these experiments will be presented and discussed.

P2.08

MOLECULAR AND CELLULAR EFFECTS OF LEAD ON HUMAN EPITHELIAL CELLS (HACAT)

Towanta Reese, Kenneth Ndebele, Barbara Graham-Evans

Jackson State University

Lead is a highly toxic metal that has been around for many years. It is omnipresent in the environment and the quantity of exposure is commonly found in all mankind. The major environmental sources of exposure to lead include leaded paint, auto emissions, and drinking water. Despite the dramatic reduction in sources of lead exposure, lead poisoning remains a reality for a high number of people in this country. In this study, we investigated the cellular and molecular effects of inorganic lead on human epithelial keratinocytes (HaCaT cells). This research was designed to evaluate the dose-response relationship by determining cell proliferation using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay for cell viability. Data obtained from this assay has shown the LD50 (the lethal dose needed to kill half (50%) of a population) of HaCaT cells exposed to lead for 24 hours to be 60ppm[+ or -].04 while at 48 hours, the LD50 was 72ppm[+ or -].05. These results indicated that lead induces cytotoxicity in a dose and time dependent manner. Based on these findings, we will use lower concentrations of lead to determine the apoptotic rate and cell damage when exposed to HaCaT cells.

P2.09

EFFECTS IN SLEEP DEPRIVED RATS OF AN ANTIDEPRESSANT AND A SLEEP AID, ALONE OR IN COMBINATION ON SLEEP PATTERNS

Theresa Raymond (2), Jorge Lopez (1), Laura Godfrey (1), Channing McLaurin (1), Howard Roffwarg (1), James Shaffery (1)

(1) University of Mississippi Medical Center, (2) Murrah High School 

Stress and sleep deprivation, both of which are associated with depression, have been demonstrated to independently reduce sleep. Fluoxetine (FLX) is a selective serotonin reuptake inhibitor (SSRI) antidepressant that we use, alone, and, in combination with a sleep aid (SA), which regularizes sleep, to examine their independent and combined effects on sleep patterns in sleep deprived (SD) rats. This study will examine whether combined FLX and SA treatment have a positive and, perhaps, synergistic affect on sleep patterns. The sleep deprivation caused by cage-shaking at sleep-onsets has proven previously to be successful. At sleep-onsets, animals are allowed to sleep for 10-240 seconds before being awakened. SA-treated animals are expected to lose less sleep and return to sleep more quickly. The animals not being pretreated with FLX will be receiving saline on the same schedule as those animals being pretreated. Throughout the three days of SD, either a SA or an equal volume of saline will be injected to all animals. An important control in this study is that all animals are shaken the same number of times per 24-hrs in the sleep-deprivation condition. Our prediction is that the SA, alone or in combination with FLX, will promote normal sleep in SD rats.

2.10

THE MOLECULAR MECHANISMS FOR THE EXPRESSION, SECRETION AND UNIPOLAR TARGETING OF ICSA IN SHIGELLA FLEXNERI

Triet La, Roberts Brandy, Davis Leanna, Lauren Brandon

Mississippi University for Women

The Gram negative bacterium Shigella flexneri causes shigellosis, a form of dysentery, leading to 1.1 million deaths world wide per annum. It expresses a virulence protein, IcsA that is responsible for the motility of the bacterium from one infected cell to another. An understanding of the molecular mechanisms for the expression and surface localization of IcsA will help us to further understand the mechanism of Shigella mediated pathogenesis. A number of complete knockout mutations have been generated by transposon mutagenesis in Shigella flexneri strain JS11.0 which is isogenic to wild type Shigella only the icsA::phoA fusion gene has replaced wild type icsA. The PhoA component of this fusion construct shows whether IcsA has been secreted across the cytoplasmic membrane to the periplasm of Shigella since the PhoA is only active in the periplasm. Transposition events that disrupt genes that are responsible for either the targeting of the IcsA::PhoA fusion protein at the inner face of the cytoplasmic membrane, the secretion of this protein across the cytoplasmic membrane or the expression of this protein are the focus of our research. We have currently identified 52 mutations and have mapped 19 of these mutations by complementation analysis. We are currently using complementation and/or recombination, single primer per and cloning to identify the remainder of these mutations. Western blot analysis has been employed to distinguish between targeting/secretion deficient mutants and expression deficient mutants.

P2.11

THE MOLECULAR AND CELLULAR MECHANISMS OF MERCURY ON HUMAN JURKAT T-CELLS

Anthony Powell, Barbara Graham-Evans, Kenneth Ndebele

Jackson State University

Mercury is widespread and is a highly toxic pollutant. There are three differnet forms of mercury and they include: elemental, inorganic, and organic. It is widely known that exposure to mercury can occur through inhalation, ingestion or dermal absorption. In the human body, mercury accumulates in the kidney, liver, blood, and brain. The immune system is affected by mercury, but long term effects are not known. We hypothesize that mercury will cause adverse effects on immune cells but at a higher concentration that necessary for other cells due to immune cells ability to fight off infection and other foreign agents. In this study, we will use human Jurkat T-cells (clone E6-1) as a model to evaluate the cytotoxicity of mercury based on the MTT assay (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). Data obtained from this assay will show the LD10 (the lethal dose of mercury needed to kill 10% of the t-cells) when exposed to mercury. Our results show that mercury when exposed to T-cells for 24 hours had an LD10 of 120ppm[+ or -]0.004 while at 48hours, the LD10 was 91ppm[+ or -]6.0. These results indicate that mercury induces cytotoxicity in a dose and time dependent manner. Based on these findings, we will use the LD 10 to determine the rate of apoptosis and DNA damage of Jurkat T-cells when exposed to inorganic mercury.

P2.12

SERUM LACTATE DEHYDROGENASE [LDH] AND BILIRUBIN [DIRECT] AS BIOMARKERS OF ARSENIC TRIOXIDE INDUCED HEPATOTOXICITY IN SPRAGUE-DAWLEY RATS.

Anita Patlolla, Donee' McAllister, Paul Tchounwou

Jackson State University

Arsenic trioxide (As203) is a known human carcinogen; it exerts its toxic effect through impairment of cellular respiration. Although the evidence of carcinogenicity of arsenic seems strong, the mechanism by which it produces tumors is not completely understood. The aim of the study was to determine the effect of As203 on the activities of specific enzymes lactate dehydrogenase [LDH] and bilirubin [direct], which may be useful as biomarkers of hepatotoxicity. Four groups of six male rats each weighing 60 + 2 g were used in this study. As203 was intraperitoneally administered to the rats at the doses of 5, 10, 15, 20mg/kg body weight (B W), one dose per 24 hour given for five days. A control group of 6 animals was injected with distilled water. Following anesthetization blood was collected using heparinised syringes. Enzyme activities were determined using colorimetric assay kits. The absorbance was read at wavelengths of 340 nm [LDH] and 546 nm [bilirubin] using visible spectrophotometer. As203 exposure significantly elevated the level of bilirubin (direct) in the treated groups [44 - 81 [micro]mol/L] compared to control [23[micro]mol/L]. There was no significant effect in the serum LDH activity [70.3 + 9.23 - 79.0 + 3.4 U/L], however, the 20 mg/kg dose of As203 exhibited the most activity [90.6 + 8.5 U/L] compared to control [47.6 + 2.31]. These findings indicate that, of the two liver enzymes examined, serum bilirubin (direct) may be a better biological marker in examining arsenic-induced hepatotoxicity.

P2.13

THE HYPERTHERMOPHILIC MICROBIOLOGICAL POPULATIONS OF SUBTERRANEAN HYDROCARBON DEPOSITS

Julianna Isaac, Magan Green, Lewis Brown

Mississippi State Univeristy

In order in increase oil production from nearly depleted oil fields, microorganisms are being used. The microbes in the oil strata are in the form of ultramicobacteria (UMB) and cannot be seen using the light microscope. At the high temperatures of some oil strata the microorganisms are even smaller. In order to detect their presence, a differential staining technique to stain DNA has been employed coupled with examination using the electron microscope. It has been shown in this project that the UMB's can be revived and will proliferate when appropriate nutrients are supplied. Specially designed culture devices are being used to incubate microorganisms at temperatures above 100[degrees]C. Proof of growth of the microbes has been confirmed by the production of carbon dioxide from the oil.

P2.14

INHIBITION POTENCY OF ORGANOPHOSPHATES ON HUMAN CARBOXYLESTERASE 1 PROTEIN Katye Herring, Abdolsamad Borazjani, Allen Crow, Matt Ross Mississippi State University

Hydrolysis of cholesteryl esters in macrophages is the rate-limiting step in the biochemical pathway that removes excess free cholesterol from these scavenger cells. The enzyme that catalyzes the hydrolysis of cholesteryl esters in macrophages is a carboxylesterase (CE) protein. We hypothesize that oxidized metabolites of organophosphate insecticides can inhibit the activity of human CE (human carboxylesterase 1, hCE1), which may inappropriately reduce macrophage reverse cholesterol transport. As an initial step we have examined the inhibitory effect of three organophosphates (chlorpyrifos oxon, CPO; paraoxon, PO; methyl paraoxon, MPO) on the hydrolytic activity of pure recombinant hCE1 protein. Pure hCE1 protein was incubated with increasing concentrations of oxons (0.001nM-10?M) in a 96-well plate for 10 min at room temperature followed by addition of an ester substrate (p-nitrophenyl valerate, pNPV). The rate of cleavage of pNPV by hCE1 was determined by the increase in absorbance at 405 nm using a plate reader. The following results were obtained. When pure hCE1 protein was treated with increasing amounts of CPO, PO, and MPO, potent inhibition of hydrolytic activity was observed in the nM range. The 50% inhibitory concentrations (IC50) for PO, CPO, and MPO were 0.22[+ or -]0.04 nM, 0.48[+ or -]0.28 nM, and 0.91[+ or -]0.29 nM, respectively. These results demonstrate that strikingly low concentrations of organophosphates can lead to inhibition of hCE1 activity and such interactions may elicit adverse effects on cholesterol metabolism in cellular and organismal contexts. [Supported by NIH R15ES015348]

P2.15

SCREENING FOR SYNTHETIC LETHAL MUTANTS OF LEXA AND DINB DELETION MUTATIONS IN ESCHERICHIA COLI.

Leonard Addae (1), Susan Cohen (1), Graham Walker (1), Leroy Johnson (1)

(1) Alcorn State University, (2) MIT 

All organisms have evolved a variety of mechanisms to ensure continuous DNA replication since it contains all the genetic information required for growth and proper functioning. Escherichia coli possess a set of SOS regulatory genes which are induced when replication is blocked by DNA damage. In an SOS induced cell, LexA, a transcriptional repressor is deactivated leading to the transcription of ~40 genes. DinB, a member of the Y family DNA polymerase (Pol IV), has been identified to be induced by the SOS response following DNA damage and allows the polymerization of DNA past lesions. This study is to identify mutants that show a synthetic lethality with deletion mutations of lexA and dinB. We applied the screening method for synthetic lethality in E.coli to select for transposon mutants that are lethal in combination with dinB or lexA delete mutations. Mutants were selected that require the gene of interest for survival. We identified 85 potential mutants of lexA delete mutation, and one mutant passed all the validation tests. More screening needs to be done in order to identify a dinB delete mutant.

P2.16

THE AQUAPORIN GENE FAMILY: EXPRESSION IN NORMAL AND NEOPLASTIC TISSUES

Christopher Nevels (1), Hari Cohly (1), Rajendram Rajnarayanan (2), Cynthia Jeffries (1), Raphael Isokpehi (1)

(1) Jackson State University, (2) Tougaloo College

The maintenance of water balance in cells is critical to the survival of all living organisms. Aquaporin water channels facilitate tumor cell migration and metastasis. Thus inhibition of aquaporins may be useful in reducing the metastatic potential of tumors. Our objective was to determine the expression of the 13 human aquaporins (AQP0 to AQP12) in normal and neoplastic tissues by analyzing Serial Analysis of Gene Expression (SAGE) Tag counts. SAGE data represent absolute expression of genes. We have developed a computational pipeline to construct a dataset of expression levels (tags per 200,000) of the human aquaporin gene family from Digital Northern results of 266 SAGE libraries (188 neoplastic and 78 normal tissues) that express two housekeeping genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Actin B (ACTB). The dataset was visualized using MultiExperiment Viewer (MeV) and converted into binary-encoded patterns representing evidence of gene expression. Our analysis revealed that AQP0, AQP1 and AQP3 were expressed in over 100 tissues. AQP1 and AQP4 were highly expressed in brain neoplastic tissues. AQP8 was expressed in only 5 tissues. We compared the expression levels of AQP1 and AQP4 in 20 SAGE Libraries developed from graded astrocytic tumor samples and found at least 5-fold over-expression of AQP4 in 7 samples. In conclusion, we have provided patterns of gene expression for the human aquaporin family in normal and neoplastic tissues; and provided prioritized datasets for further analysis. Acknowledgements: RCMI-CEH NIH G12RR13459-09, Mississippi NSF-EPSCoR (EPS-0556308), Mississippi Functional Genomics Network.

P2.17

AUTOMATIC EXTRACTION OF INFLUENZA VIRUS PATHOGENICITY FROM BIOMEDICAL LITERATURE

Antoneicka Harris (1), Raphael Isokpehi (1), Hari Cohly (1), Nyasha Chambwe (1), Rajendram Rajnarayanan (2)

(1) Jackson State University, (2) Tougaloo College

The ability to cause disease by avian influenza viruses is categorized as low or high pathogenicity. Both types of viruses cause influenza outbreaks in poultry worldwide. Furthermore, High-pathogenicity avian influenza (HPAI) viruses have emerged from low-pathogenicity avian influenza (LPAI) viruses. The antigenic nomenclature of Influenza A viruses includes the combination of two groups of proteins: the Hemaglutinnin (H) and Neuraminidase (N). There are 16 H types (H1-H16) and 9 N types (N1-N9) resulting in a total of 144 possible symbols. The highly pathogenic H5N1 subtype is responsible for widespread epidemics in commercial poultry. As of November 2007, 204 human deaths were due to H5N1 infection. We are interested in identifying novel inferences on the pathogenicity of influenza viruses from biomedical literature. In this study, we compiled a corpus of PubMed abstracts in which at least one nomenclature symbol was mentioned and then generated a binary matrix to determine co-occurring symbols. Our computational pipeline extracted 5,770 abstracts. Sixty-eight symbols were not mentioned in the abstracts. The 14 symbols pairing exclusively with H5N1 in 174 abstracts were H1N1, H2N2, H3N2, H3N8, H5N2, H5N3, H5N8, H5N9, H6N1, H7N1, H7N7, H9N1, H9N2, and H10N7. We are currently annotating these groups of abstracts with semantic categories and relations that will facilitate reasoning on pathogenicity of influenza viruses. Acknowledgements: Mississippi NSF EPSCoR EPS-0556308); MARC-U*STAR (5-T34-GM007672-28); and the RCMI-CEH (NIH- G12RR13459-09).

P2.18

DOES THE GENETIC POLYMORPHISM INVOLVING A VARIABLE NUMBER OF TANDEM REPEATS IN GLYCOPROTEIN IB DIRECTLY INFLUENCE PLATELET ACTIVATION BY VON WILLEBRAND FACTOR?

Stacy Brown (1), John Kermode (1)

(1) Alcorn State University, (2) University of Mississippi Medical Center

Von Willebrand factor (VWF) is a blood glycoprotein involved in thrombosis and hemostasis. Glycoprotein Ib (GpIb) is a portion of a blood platelet receptor for von Willebrand factor; interaction of VWF with this receptor triggers platelet activation. Within the gene for GpIb there is a genetic polymorphism that involves a variable number of tandem repeats (VNTR) of a 39 base pair sequence (nucleotidesl243-1281) encoding for 13 amino acid in the extracellular portion of the Gplb. This sequence may occur once (VNTR-D allele), twice (VNTR-C), or three times (VNTR-B). A series of healthy volunteers were genotyped for the VNTR polymorphism and assessed for platelet responsiveness in vitro. The VNTR genetic polymorphism was detected by agarose gel electrophoresis of a PCR product covering the repeat region. Platelet activation was detected through [14C]serotonin release and assessment of the calcium signaling that occurs when VWF binds to the Gplb receptor. Data from our pilot study suggests that the degree of platelet activation increases in direct proportion with the number of tandem repeats in the GpIb gene. However, our sample was too small to yield a conclusive result. There was not enough consistency in the data to confirm the hypothesis. A more extensive study with a greater number of participants is required to make a definitive assessment of the influence of this polymorphism on platelet activation.

P2.19

CELLULAR AND MOLECULAR TARGETS OF MERCURY TOXICITY IN HUMAN RENAL PROXIMAL (HK-2) CELLS

Shaneka Simmons, Dwayne Sutton, Raphael Isokpehi

Jackson State University

Mercury is amongst the most environmentally abundant nephrotoxicants known to accumulate in the kidneys, leading to cellular injury and alterations in renal gene expression. However, the mechanisms underlying these events remain largely unknown. This study was designed to identify potential cellular and molecular targets of mercury toxicity in human proximal tubule (HK-2) cells. A combination of cellular assays and bioinformatics approaches were used to evaluate cytotoxicity of HK-2 cells exposed to mercury and to understand mechanistic pathways associated with genes that interact with mercury. Following 24 hour exposure of HK-2 cells to mercury, an MTT assay was performed to determine cytotoxicity. Results of these studies demonstrated that mercury exposure reduced cell viability in a dose-dependent manner. A lipid peroxidation assay was performed to evaluate the role of oxidative stress (as MDA generation) in mercury induced cytotoxicity to HK-2 cells, revealing selective perioxidative damage at 4 [micro]g/ml. For the bioinformatics study, we retrieved 20 human genes from the Comparative Toxicogenomics Database that are curated from literature to interact with mercury. We then extracted Gene Ontology (GO) annotations assigned to the genes from the PubGene Database and constructed a citation co-occurrence gene network for each gene. Based on statistically significant values, the data extracted was used to identify potential target pathways of cellular and molecular toxicity for mercury.

Acknowledgements: Mississippi NSF-EPSCoR "Innovations through Computational Sciences" Award (EPS-0556308); CMCM (W912HZ-04-2-0002); and RCMI Center for Environmental Health (NIH-NCRR G12RR13459-09)

P2.20

Ductal Carcinoma Cell Growth is Vitiated by Vernonia Amygdalina Extracts

Lecia Gresham, Ernest Izevbigie Jackson State University

Breast cancer is the most commonly diagnosed cancer and second leading cause of cancer-related deaths in women. Although, the incidence of breast cancer is highest in White Women (WW), African American women (AAW) have higher mortality rate than other racial groups in the U.S. One of the reported reasons for this disparity is that AAW are more likely to be diagnosed with estrogen receptor negative (ER-) breast cancer (a more aggressive breast cancer, with less treatment options). The human ductal carcinoma cells (BT549) are reported to express little or no (ER-[alpha]), and thus represent a suitable model to study estrogen-independent cell growth. Therefore, the objective of this study was to assess the growth inhibitory activity of Vernonia amygdalina (VA) in these cells. The cells were propagated in tissue culture plates with RPMI-1640 supplemented with 10% FBS and 1% penicillin-streptomycin at 37C in a 95% air/5% CO2 humidified incubator. Mitosis was determined by DNA synthesis assays and confirmed cell counts using a hemacytometer. Exposure of BT549 to increasing concentrations of aqueous VA abrogated cell growth in a concentration-dependent fashion: VA at concentrations of 10, 100, and 1000 [micro]g/ml inhibited BT549 cell viability by 20%, 25% (p<0.05), and 50% (p<0.05) respectively compared to the controls. Increasing concentrations of Taxol alone and in combination with VA were ineffective on DNA synthesis. These data suggest that these cells are insensitive to Taxol and ER-breast cancer patients may benefit from VA consumption.

FRIDAY MORNING

Oak

O2.11

9:00 IDENTIFYING MAMMALIAN PICOT (PROTEIN INTERACTING COUSIN OF THIOREDOXIN)

INTERACTING PROTEINS

Bolanle Bukoye (1), Ning-Hui Cheng (2)

(1) Mississippi University for Women, (2) Baylor College of Medicine 

Glutaredoxin is a member of the thioredoxin fold family that is involved in cellular redox homeostasis in bacteria, yeast, and mammals. It is also found in plants although its function in plants is still unclear. Glutaredoxins have been found to contain a newly identified Protein Interacting Cousin of Thioredoxin (PICOT) domain. Although, determining the function of PICOT has been challenging, the purpose of this research was to dissect the PICOT-mediated signaling involved in cell growth, proliferation, organ development, growth hormone mediated growth, and nutrient metabolism. Using yeast bait to screen a mouse cDNA library, 125 PICOT interacting proteins were identified. We isolated plasmid DNA from yeast strains harboring both bait and prey plasmid DNA and investigated four colonies in an ongoing process to determine the identify of the PICOT-domain proteins through amplification, re-transformation into a yeast two-hybrid system for confirmation, sequencing, and homology searches

O2.12

9:20 THE ROLES OF CDC20 AND RAD4 IN DOUBLE STRAND BREAK REPAIR

Bernadette Connors, Lauren Rochelle, Asela Roberts, Graham Howard, Leah Strickland

Millsaps College

Faulty DNA replication and repair can lead to severe human diseases such as xeroderma pigmentosum (XP), a disorder characterized by extreme photosensitivity and a greater than 1000-fold increased risk of cutaneous and ocular neoplasms. Our studies have suggested a link between Cdc20, a known regulator of mitotic exit, and nucleotide excision repair (NER). Our initial experiments demonstrated that in Saccharomyces cerevisae NER defective mutants containing both a deletion of RAD4 and the cdc20-1 allele are more UV sensitive than either single mutant alone. Reintroduction of RAD4 or CDC20 under the control of a galactose inducible promoter gal1 into [DELTA]rad4 and [DELTA]rad4/cdc20-1 strains returned moderate UV sensitivity to both. Double-strand break repair and the ability to evoke the RNR3 repair pathway were also significantly diminished in strains carrying [DELTA]rad4/cdc20-1 compared with those harboring [DELTA]rad4 or cdc20-1 alone. Microscopic analyses of RAD4-GFP and RAD4-GFP/cdc20-1 strains showed a significant decrease in RAD4 fluorescence upon UV irradiation when cdc20-l was present. These data strongly support an interaction between CDC20 and RAD4. Two homologues of RAD4, RAD33 and RAD34, will also be tested in this manner to uncover any functional overlap between Rad4 and these two proteins. The ultimate goal of this research is to come to a better understanding of mechanisms related to defects in nucleotide excision repair, defects that have direct implications on development of certain types of cancer in higher eukaryotes.

O2.13

9:40 A CLPP MEDIATED REGULATORY NETWORK IN STAPHYLOCOCCUS AUREUS REVEALED BY SAMMD

Vijayaraj Nagarajan, Mohamed Elasri

Department of Biological Sciences, The University of Southern Mississippi

SAMMD is a meta-database for Staphylococcus aureus microarray data (SAMMD: http://bioinformatics.org/sammd/) that we recently developed. SAMMD contains easily retrievable data from 85 different published microarray experiments done so far in S. aureus, with more than 12,000 records for individual transcriptomic status of S. aureus genes. We mined SAMMD for the expression status of 99 known and hypothetical transcriptional regulators. Our analysis showed that stringent response, murF, clpP, sarA and cold shock as the top five conditions controlling 35,22,19,18,17 regulators respectively. Heat shock upregulates 11 regulators while none are downregulated. In contrast cold shock downregulates 14 regulators while upregulating only three. The response regulator saeR is the most affected regulator, appearing in at least 17 different experimental conditions. Nine regulators including cspC and lacR do not seem to be affected at all in any of the 80 different experiments. Several transcriptional regulators seem to be regulated in only one direction (either upregulated or downregulated), for example a fur homolog (SA1329) is upregulated at least in nine different conditions, while it is not down regulated in any of the known conditions. We show that about half of the clpP regulated genes are actually regulated through sigB, sarA, agrA and mgrA. clpP thus acts as a major hub for proteolysis mediated regulatory network in S. aureus, mediating the signal and the response at least during the cold shock induced stress.

O2.14

10:00 Fast Identification of Clustered Regularly Interspaced Palindromic Repeats

Charles Bland

Mississippi Valley State University 

Clustered Regularly Interspaced Palindromic Repeats (CRISPRs) are a novel type of direct repeat found in a wide range of bacteria and archaea. CRISPRs are beginning to attract attention because of their proposed mechanism; that is, defending their hosts against invading extrachromosomal elements such as viruses. Existing repeat detection tools do a poor job of identifying CRISPRs due to the presence of unique spacer sequences separating the repeats. In this study, a new tool, CRT, is introduced that rapidly and accurately identifies CRISPRs in large DNA strings, such as genomes and metagenomes. CRT's approach to detecting repetitive sequences is straightforward. It uses a simple sequential scan of a DNA sequence and detects repeats directly without any major conversion or preprocessing of the input. This leads to a program that is easy to describe and understand; yet it is very accurate, fast and memory efficient, being O(n) in space and O(nm/l) in time. CRT was compared to CRISPR detection tools, Patscan and Pilercr. In terms of correctness, CRT was shown to be very reliable, demonstrating significant improvements over Patscan for measures precision, recall and quality. When compared to Pilercr, CRT showed improved performance for recall and quality. In terms of speed, CRT proved to be a huge improvement over Patscan. Both CRT and Pilercr were comparable in speed, however CRT was faster for genomes containing large numbers of repeats.

O2.15

10:20 THE ROLE OF THE SSA1 GENE IN THE SPONTANEOUS FORMATION OF THE [URE3] PRION OF SACCHAROMYCES CEREVISIAE

Katie Brinkman, Ross E. Whitwam

Mississippi University for Women

Ssal is a chaperone protein required for propagation of stably-established [URE3] prions in Saccharomyces cerevisiae. We have knocked out the Ssal gene from a prion-free strain of S. cerevisiae. These yeast are still able to spontaneously convert to the prion state, suggesting that this gene is not required to establish the prion state, just to maintain it. To investigate the possibility that Ssal, while not strictly required to establish the prion state, may aid in spontaneous prion formation, we are investigating whether the rates of spontaneous prion formation are altered in prion-free yeast that lack Ssa1. We are also interested in the rates of prion elimination in ssa1 strains of yeast that have spontaneously converted to the [URE3] state. This information should tell us something about Ssa1's role in prion propagation.

O2.16

10:40 SPONTANEOUS FORMATION OF THE [URE3] PRION IN SACCHAROMYCES CEREVISIAE IS NOT A STRICTLY RANDOM PROCESS

Mary Oyeleye, Michael Lee, Ross E. Whitwam

Mississippi University for Women

Prions are misfolded and infectious forms of cellular proteins. The [URE3] prion of baker's yeast, Saccharomyces cerevisiae, is the prion form of the Ure2 protein. At a very low rate, the properly-folded, functional form of the Ure2 protein can spontaneously misfold into the prion form. Very little is known about the mechanism of this spontaneous misfolding and about whether misfolded Ure2 protein alone is sufficient to establish the prion state or whether additional cellular factors are required. We have followed the rates of spontaneous prion formation in prion-free cultures of yeast. The rates of spontaneous [URE3] formation in prion-free yeast were highly variable throughout the growth curve of the yeast. Certain time points in the growth curve were consistently associated with the lowest rates of spontaneous prion formation, while other time points were consistently associated with the highest rates. Investigations of parallel cultures of prion-free yeast also suggest that there are constraints on when spontaneous prion formation can occur. Together these results suggest that other cellular factors whose levels vary during culture growth are necessary to establish the stable [URE3] state and prion formation is not a strictly random process governed only by the chance misfolding of Ure2 protein.

11:00 Closing remarks, divisional meeting, and awards for poster and platform presentations.