Cellular, Molecular and Developmental Biology.

Chair: Mary L. Haasch, University of Mississippi

Vice-chair: Stephen I.N. Ekunwe, Jackson State University

THURSDAY MORNING

Cedar Room

8:00 THIOESTERASE RIBOZYMES

Danning Huang* and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406

The theory of RNA World tries to describe an intermediate period between living system and non-living system. In a hypothesized RNA world, all essential biochemical reactions would have to be catalyzed by RNA enzymes, or socalled ribozymes. Many ribozymes with different activities have been isolated during the past decade. In our work, we are trying to link different ribozyme activities to construct metabolic pathways--RNA-based metabolic pathways. A series of ribozyme activities involving coenzyme A (CoA) have been demonstrated in our laboratory. The current work expands such CoA-centered pathway by generating new ribozymes that can catalyze the hydrolysis of thioesters of CoA. Many different RNA sequences have been isolated that show thioesterase activities. These newly isolated ribozymes increase ribozyme repertoire, and demonstrate the plausibility of complex metabolic systems in the RNA world.

8:20 QUANTITATIVE ANALYSIS OF THE INTERACTION BETWEEN A CYCLIC AMPHIPATHIC PEPTIDE WITH LIPID MEMBRANES

Kelley Counts (1*), Peter Butko (1), Maria Ngu-Schwemlein (2), and Oluyemisi Adeyemi (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) University of South Alabama, Mobile, AL 36688

In this work we studied the interaction of a synthetic amphipathic cyclic octapeptide Octa-1 with model lipid membranes. The hypothesis of this study is that the positively-charged Octa-1 will interact with membranes and that this interaction will be more significant with the membranes containing negatively-charged lipids. The hypothesis was tested using small unilamellar vesicles (SUV) made of egg 1,2-diacylsn-glycero-3-phosphocholine and L-[alpha]-phosphatidylglycerol in ratios of 10:0, 9:1, and 3:1. Octa-1 contains a single tryptophan residue, which was employed to determine the strength of the interaction, location and orientation of the membrane-bound peptide by fluorescence spectroscopy. Quantitative analysis of the data yielded values of the association constants for the peptide-lipid interaction. It was found that binding to the 10:0 and 9:1 SUV is well described by a single association constant, whereas binding to the 3:1 SUV requires two association constants. The second binding constant can be due to hydrophobic interaction between the nonpolar part of the peptide and the core of the lipid bilayer or due to aggregation of the peptide on the membrane. There is a strong correlation between the values of the association constant and the mole fraction of the negatively charged lipid. Results confirm that the electrostatics play a dominant role in the peptide-lipid interaction and provide a quantitative insight into the binding.

8:40 INTERACTION OF THE BT TOXIN Cyt1A WITH A LIPID MONOLAYER

Shalawn Clark (1*), Marianne Pusztai-Carey (2), and Peter Butko (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) Case Western Reserve University, Cleveland, OH 44106

Cyt1A is a protein produced by Bacillus thuringiensis var. israelensis. In vitro, it exhibits cytolytic activity against a range of insect and mammalian cells; in vivo it acts specifically on cells of Diptera larvae. Previously we put forward a hypothesis that, interacting with phospholipids, Cyt1A may act as a detergent rather than a pore former. We used monolayers of 1,2-diacyl-sn-glycero-3-phosphocholine isolated from egg(PC) at air/water interface to monitor toxin-lipid interaction. Specifically we examined by Langmuir trough if Cyt1A inserts into the lipid monolayer, insertion is required for pore formation. As a positive control we used alpha-hemolysin, a toxin that, upon lowering the pH, is known to insert into the membrane and form pores. When alpha-hemolysin was placed in the subphase of the PC monolayer a 115% increase in the surface pressure was observed upon lowering the pH, indicating the toxin inserted into the monolayer. A similar experiment was performed with Cyt1A injected into the subphase at the same toxin/lipid molar ratio and the same initial surface pressure of the lipid monolayer. An approximately 50% increase in the surface pressure was observed, with slower kinetics. No increase was observed with the negative control--cytochrome c. The slight increase in surface pressure from Cyt1A may be due to penetration between the lipid polar headgroups and not by insertion into the lipid monolayer.

9:00 UNKNOWN CALCIUM STORES IN MURINE MESENTERIC ARTERIOLE SMOOTH MUSCLE CELLS

Tamara Williams* and Sean M. Wilson, Mississippi University for Women, Columbus, MS 39701 and University of Mississippi, University, MS 38677

Calcium plays many important roles in cellular and bodily health including working as an intracellular signal for muscle contraction, secretion, and cell proliferation. Though much is known about calcium's role in the cell, much has yet to be learned. Antidotal evidence suggests that there are unstudied [Ca.sup.2+] stores in mouse mesenteric arteriole smooth muscle cells. Data was collected and analyzed to determine the percentage of cytosolic calcium that is released from intracellular stores and how the store's [Ca.sup.2+] release changes with aging. Cytosolic calcium was measured using techniques developed by Grynkiewicz et al. 1985. Isolated smooth muscle cells were loaded with Fura-2 AM, a fluorescent dye that binds to diatomic ions and has a high affinity for [Ca.sup.2+]. A xenon arc lamp was used to illuminate the dye at wavelengths of 340 and 380 nm. Upon excitation, the cells emitted light that was collected by a CCD camera, which is part of a fluorescent imaging system mounted to an inverted microscope. The appropriate equations were then used to determine the amount of [Ca.sup.2+] stored in various organelles of the cell. We found that [Ca.sup.2+] from these potentially unstudied sources were present in the cell, and in older cells, the [Ca.sup.2+] content was significantly greater than in younger cells.

9:20 Break

9:40 INHIBITION OF THE PALMITOYLATION OF THE RAS ONCOGENE IN YEAST CELLS

Tonya Dement (1*), Cheryl Budde (2), and Robert J. Deschenes (2), (1) Mississippi University for Women, Columbus, MS 39701 and (2) University of Iowa, Iowa City, IA 52242

Saccharomyces cerevisiae is an excellent model organism for the study of the activation of the Ras oncoprotein. Yeast cells activate Ras through farnesylation, proteolysis, methylation, and palmitoylation in the same manner as multicellular organisms. Palmitoylation is the final step in the Ras processing pathway before activated Ras is transported to the cell membrane where it signals tumor growth. To test the hypothesis that inhibiting Ras palmitoylation would inhibit Ras signaling, we constructed heat-sensitive and galactose-sensitive Ras yeast in which heat shock and galactose, respectively, induce Ras-mediated cell death. We then tested the ability of 2-bromopalmitate to inhibit palmitoylation of the Ras oncoprotein in these strains. Inhibition of Ras palmitoylation should disrupt the activation of Ras in the yeast which in turn should reduce the ability of activated Ras to confer heat-sensitivity and galactose-sensitivity in these strains. We have not as of yet found appropriate conditions that allow heat shock to discriminate between cells expression wild type and activated Ras proteins. However, we have seen signs of 2-bromopalmitate inhibition of Ras-mediated cell death in the galactose-sensitive Ras cells, but we are still in the process of working out the optimal conditions for maximizing inhibition.

10:00 AMYLOID [beta] EXPRESSION REDUCES THE ACTIVITY OF cAMP RESPONSE ELEMENTBINDING PROTEIN (CREB) IN HIPPOCAMPUS OF TRANSGENIC MICE MODELS OF ALZHEIMER'S DISEASE

Yanan Xu*, Evan Comeaux, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406

Amyloid [beta]-peptide (A[beta]), the aberrant product of A[beta] precursor protein (APP), represents the hallmark of Alzheimer's disease (AD). Its continuous deposition and accumulation are thought to cause degeneration of neuronal cells and subsequent cognitive impairment. Transgenic animal model over-expressing A[beta] exhibits learning and memory impairments. A molecule implicated in neuronal plasticity and long-term memory is the cAMP responsible element binding protein (CREB). In this study, we investigated the possibility of whether the activity of CREB is affected in different brain regions using the double (APP/PS1) transgenic mice model of AD and the effect of EGb 761 treatment, a standardized Ginkgo biloba extract known to improve age-dependent dementia. We found that the activity of CREB (phosphorlated) is dramatically reduced in the hippocampus of the double transgenic mice (APP/PS1) compared to the wild type controls. The phosphorylation of CREB is also decreased in the cortex of the double transgenic mice, although not as remarkable as that in the hippocampus. No changes of phosphorylated CREB were observed in the cerebellum of the mice. The expression level of total CREB level remains the same in all brain regions tested. And besides the Ab oligmers are attenuated in hippocampus, cortex and cerebellum of the brain after the treatment of EGb 761. These observations suggest that brain-region-specific toxicity of A[beta] may be accompanied by its interferences with activity of CREB and EGb 761 have effects on the ameliorating the amyloid toxicity.

10:20 EGb 761, AN EXTRACT OF GINKGO BILOBA LEAVES, ALLEVIATE TOXICITY INDUCED BY AMYLOID-BETA EXPRESSION IN THE TRANSGENIC C. ELEGANS MODEL

Yanjue Wu*, Zhixin Wu, Astrid Gutierrez, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406

Alzheimer's disease (AD) is associated with pathological features such as amyloid-[beta] (A[beta]) deposits, neurofibrillary tangles, plaques and degeneration of neurons in the brain. Although the pathogenesis of AD is not fully understood, A[beta] aggregation has been postulated to link oxidative stress and neurodegeneration in AD. This hypothesis remains controversial because the relationship between A[beta] aggregation, oxidative stress indicators and cell toxicity has not been defined in vivo. We have previously reported that the standardized Ginkgo biloba extract Egb 761 inhibited A[beta] aggregation in solution and in an A[beta]-expressing neuroblastoma cell line. In this study, we use an inducible A[beta]-expressing transgenic Caenorhabditis elegans strain (CL4176) to correlate A[beta] expression with its toxicity and with the levels of reactive oxygen species (ROS) in the organism. Our results demonstrated that the A[beta] expression-induced paralysis was delayed in the C. elegans fed with EGb 761 (100 mg/ml). We also found A[beta] deposits in pharyngeal region of another strain CL2006, which constitutively expresses high levels of human A[beta], were significantly decreased in EGb 761 treated C. elegans compared with those untreated controls. A[beta] oligomers and dimers were the aggregate species inhibited by EGb 761 in both strains. The inhibitory activity of EGb 761 against A[beta] deposition and paralysis correlates with its ability to attenuate hydrogen peroxide in the transgenic C. elegans. These findings together with our previous observation suggest that oligomerization of A[beta] is crucial for A[beta] toxicity, and the herbal extract EGb 761 has a clear therapeutic potential for prevention and treatment of AD, at least in part, via its anti-oxidative and anti-oligomerization activities.

10:40 MODULATION OF OXIDATIVE FREE RADICALS AND LOCOMOTIVE MOVEMENT BY EGCG AND RESVERATROL IN TRANSGENIC C. ELEGANS MODEL

Marishka K. Brown (1*), Julie Smith (2), and Yuan Luo (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) Louisiana State University, Baton Rouge, LA70803

Experimental data implicate the beneficial effects of antioxidant therapies to negate the detrimental consequences of elevated free radical levels, a possible contributing factor in both the aging process and the development of neurodegenerative pathologies such as Alzheimer's disease (AD). Previous data have shown the neuroprotective effects of the green tea constituent epigallocatechin gallate (EGCG) and the red wine constituent resveratrol on rat hippocampal neurons against amyloid-[beta] (A[beta]) induced neurotoxicity. In this study, EGCG and resveratrol were applied in an in vivo model of AD using a transgenic strain of Caenorhabditis elegans nematodes with constitutive expression of human A[beta][.sub.1-42]. Both EGCG and resveratrol are shown to significantly attenuate elevated levels of free radicals. Combined treatment with EGCG and resveratrol did not show any additive effect. The degree of attenuation by EGCG is much more profound than that of Ginkgo biloba extract previously demonstrated. The anti-oxidative effects of EGCG correlate with an enhancement of locomotive behavior. These results suggest that the antioxidant effect of EGCG and resveratrol against A[beta] toxicity may have important therapeutic value in slowing down the progress, or even preventing AD.

11:00 AN IN VITRO APPROACH TO CHARACTERIZE STATE-SELECTIVE JANUS KINASE 2 INHIBITORS

Kanakadurga Kundrapu*, Kiranam Chatti, and Roy J. Duhe, University of Mississippi Medical Center, Jackson, MS 39216

Janus kinases are cytoplasmic protein tyrosine kinases with crucial physiological roles. The binding of various class II cytokines to their receptors initiates JAK activation, which in turn causes proliferation or differentiation in cells of various lineage. Uncontrolled JAK2 activity may contribute to the progression of certain cancers. Considerable interest exists in developing JAK-targeted inhibitors. We are currently using an in vitro approach to identify and characterize selective inhibitors of rat JAK2 activity. Several approaches to develop a quantitative biochemical assay for rJAK2 activity have been under way in our laboratory. One of our novel findings has been that rJAK2 exists in at least two distinct states of activity. The phosphorylation of tyrosines within the activation loop of rJAK2 appears to increase its autocatalytic efficiency with respect to ATP, with highest activity when its activation loop is phosphorylated and lower activity when the activation loop is unphosphorylated. We are determining whether this implied difference in ATP binding affinities between the two states of rJAK2 also extends to a difference in affinities for the acceptor substrates, such as the STATs. Based on our demonstration of the differential behavior of these two states towards ATP, we propose that a JAK2 inhibitor(s) could selectively distinguish between these two activity states. State-selective inhibitors of JAK tyrosine kinase activity would represent a novel class of potential therapeutic agents.

THURSDAY AFTERNOON

Cedar Room

1:40 CHARACTERIZATION OF NOVEL VIRULENCE FACTORS IN STAPHYLOCOCCUS AUREUS

Karthik Sambanthamoorthy* and Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS 39406

Staphylococcus aureus is an extraordinarily versatile pathogen causing a wide variety of infections ranging from superficial infections (skin abscess, wound infections), to toxemic syndromes (food poisoning) to life threatening conditions (osteomyletis, endocarditis). S. aureus has developed resistance to an array of antibiotics like methicillin and vancomycin, forcing the need for development of new drugs to combat staphylococcal infections. S. aureus expresses a wide variety of virulence factors such as cell bound proteins (e.g., adhesins) or exoproteins (toxins). The cell bound proteins are expressed early to establish infection and repressed after colonization whereas the exoproteins are repressed early and expressed late during the infection. The coordination of the expression of virulence factors is under the control of two global regulatory systems, the accessory gene regulator (agr) and the staphylococcal accessory regulator (sarA). The agr system is a quorum sensing system in which the S. aureus cells communicate with each other to coordinate expression of virulence factors. sarA regulates the virulence factors via two pathways, an agr-dependent pathway in which sarA activates agr at the transition between the exponential and post exponential growth phase and an agr-independent pathway in which sarA regulates virulence factors directly. There is evidence for the existence of important upstream and downstream factors that modulate sarA function. Our goal is to identify and characterize these new factors which represent potential new therapeutic targets for the prevention and treatment of Staphylococcal infections.

2:00 THE ROLE OF NOVEL QUORUM SENSING IN THE REGULATION OF STAPHYLOCOCCAL VIRULENCE FACTORS.

Cassie R. Crenshaw* and Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS 39406

Staphylococcus aureus is an opportunistic pathogen responsible for a myriad of infections including endocarditis, osteomyelitis, toxic shock syndrome, and food borne illness. S. aureus produces numerous virulence factors. These virulence factors can be grouped into two categories: cell bound proteins (adhesions) and secreted proteins (exotoxins). Cell bound proteins are expressed in the early stages of infection when cell density is low. Secreted proteins are expressed in the late stages of infection when cell density is high. Expression of many virulence factors is coordinated by the agr quorum sensing system. Recently a new quorum sensing system, luxS, has been discovered. Our goal is to determine the role of luxS in the regulation of staphylococcal virulence factors. To do this, we have mutated the luxS gene. We will analyze expression of agr regulated virulence factors in the mutant and compare with the wild type.

2:20 REGULATION OF VIRULENCE FACTORS IN STAPHYLOCOCCUS AUREUS

Antony Schwartz* and Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS 39406

Staphylococcus aureus is a gram-positive bacterium that causes a large number of community-acquired and nosocomial infections. Quorum sensing system allows S. aureus to regulate expression of virulence factors according to cell density. In this study, we are evaluating the role of luxS, a novel quorum sensing system in the regulation of virulence factors. Also, we are investigating possible crosstalk between two known global regulators, the accessory gene regulator (agr) and the staphylococcal accessory regulator (sarA) and the luxS system. luxS expression will be studied in the sarA[.sup.-], agr[.sup.-], and the sarA[.sup.-]/agr[.sup.-] strains. Likewise, sarA[.sup.-] and agr[.sup.-] expression will be studied in the luxS[.sup.-] system. The ultimate goal of this study is to elucidate the intricate virulence regulatory systems in S. aureus, providing for novel therapeutic target areas.

FRIDAY MORNING

Cedar Room

8:00 Divisional Poster Session

ANALYSIS OF HYPOXIA INDUCIBLE FACTOR IN GRASS SHRIMP BY DNA MICROARRAY

Donald Sittman (1*), Tiandao Li (2), and Marius Brouwer (2), (1) University of Mississippi Medical Center, Jackson, MS 39216 and (2) University of Southern Mississippi, Gulf Coast Research Laboratory, Ocean Springs, MS 39564

A pilot study was initiated to identify the hypoxia inducible factor from the grass shrimp, Palaemonetes pugio by DNA microarray technology to determine if the changes in the expression levels can serve as indicator of dissolved oxygen stress in Gulf of Mexico. Eighty clones from grass shrimp were chosen for PCR amplification and digestion. Purified cDNA and control clones were spotted in duplicate arrays on UltraGAPS coated slides using the VersArray ChipWriter. Total RNA was extracted from normoxic shrimp and hypoxic shrimp (Day 3, Day 7, and Day 14). The RNA was labeled with Amino-Allyl-dUTP. Slides were hybridized with the RNA from normoxic and hypoxic shrimps. Hybridized arrays were scanned with the VersArray ChipReader using TIGR Spotfinder for initial spot finding. Raw data was imported into R and analyzed for normalization. Different microarray tools were involved for clustering, visualization, classification, statistical analysis and biological theme discovery. DNA microarray technology is a powerful technology that will substantially increase the speed of molecular biological research, and hypoxia inducible factor will be used as bioindicators of hypoxia-related stress in marine environment.

IMMUNOHISTOCHEMICAL ANALYSIS OF PLACENTAL CYTOKINE RECEPTORS IN A REDUCED UTERINE PERFUSION PRESSURE (RUPP) MODEL OF PREECLAMPSIA IN SPRAGUE-DAWLEY RATS

Tribetta Spires*, Kedra Wallace, Jennifer Craft, Robin Rockhold, and William Bennett, University of Mississippi Medical Center, Jackson MS 39216

Preeclampsia is a hypertensive disease that affects approximately 5-7% of all first pregnancies, and is one of the leading causes of maternal and neonatal morbidity and mortality worldwide. Although the cause of preeclampsia is still unknown, it is hypothesized that a lack of blood flow to the placenta causes an increase in the release of placental cytokines such as interleukin-1 beta (IL-1[beta]), interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor (TNF-[alpha]). Inflammatory cytokines are then though to mediate endothelial dysfunction in the blood vessels of the kidney, reflected by an increased production of vasoconstrictive factors. This mechanism may explain the etiology of preeclampsia in human pregnancy. Placental tissues were obtained from control pregnant rats and those undergoing experimentally reduced uterine perfusion (RUPP). Immunohistochemical analysis were performed by standard ABC staining techniques utilizing five micrograms of primary antibody specific for the placental cytokine of interest. Slides were examined for immunohistochemical staining by light microscopy (40X). Increased staining for the IL-6 and IL-10 receptors was observed in placentas from animals undergoing the RUPP procedure when compared to controls. This increased expression of placental cytokine receptors could contribute to the increased cytokine protein production and subsequent endothelial dysfunction that occurs in women with preeclampsia

CLONING AND ANALYSIS OF THE CYSTEINE DIOXYGENASE (CDO1) GENE FROM THE DIMORPHIC PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM.

Glen Shearer* and Sarah Hasler, University of Southern Mississippi, Hattiesburg, MS 39406

The dimorphic fungus Histoplasma capsulatum is the etiologic agent of histoplasmosis, a disease which afflicts an estimated 500,000 Americans each year. Histoplasma grows in soil in a differentiated form as a multicellular, filamentous mold. After inhalation of spores or mold fragments, the organism de-differentiates into a single-celled budding yeast. This mold-to-yeast conversion is an absolute requirement for pathogenesis. Sulfhydryl metabolism is known to be involved in this dimorphic process. The yeast morphotype, but not the mold morphotype, has cysteine dioxygenase activity, which converts cysteine into cysteine sulfinic acid. Toward our goal of understanding the molecular basis of this dimorphism, we have isolated the cysteine dioxygenase gene (CDO1) from Histoplasma capsulatum strain G186AS. The Histoplasma CDO1 coding sequence shows strong similarity to a hypothetical CDO protein from the fungus Neurospora crassa and weak, but significant, similarity to human and rat CDO genes. Preliminary analyses indicate the gene is differentially regulated in yeast and mold cells. Studies are underway to quantitate CDO1 mRNA levels in yeast and mold morphotypes of several strains of Histoplasma to better elucidate the role of this gene in dimorphism.

CHARACTERIZATION OF THE MOLD-SPECIFIC M46 GENE IN THE DIMORPHIC PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM.

Glen Shearer* and Davida Crossley, University of Southern Mississippi, Hattiesburg, MS 39406

Histoplasma capsulatum, a dimorphic fungus, is the infectious agent responsible for the respiratory disease histoplasmosis. The fungus, grows as a multicellular mold in the soil. Once the soil is disturbed, spores are released and inhaled into the lungs. In the lungs, the fungus converts to the unicellular yeast morphotype. This mold-to-yeast conversion, which is a requirement for pathogenesis, can also be accomplished in the lab by switching incubating temperature from 25 [degrees]C to 37 [degrees]C. To understand the molecular basis of dimorphism we have isolated several mold-specific and yeast-specific genes. The subject of this study, the M46 gene, is mold-specific. Recent data have shown that M46 is expressed in the mold morphotype of G186AS and Downs strains, but is transcriptionally silent in G184AS and G217B strains. To determine the reason for this lack of transcription in the latter strains we have compared the genomic sequence for the M46 locus in all four strains. Preliminary analysis indicates that the non-expressing strains have a large sequence rearrangement at the M46 locus. Work is ongoing to determine the extent of this rearrangement and the effect of this mutation on M46 promoter function.

BACTERIAL ACQUISITION OF ANTIMICROBIAL RESISTANCE IN FRESHWATER WETLAND ENVIRONMENTS

Andrekeus Lee (1*), L. Halda-Alija (1), L. Silas (1), S. Moktan (2), S. Khan (2), and M. Jacob (2), (1) University of Mississippi, University, MS 38677 and (2) National Center for Natural Products Research, University of Mississippi, University, MS 38677

The aim of this study was to select Enterobacteriaceae, potential human and animal pathogens from pristine wetlands, and assess virulence factors that allow microorganisms to cause an infection. Assessed virulence factors include antibiotic resistance, presence of plasmids and capsules. Enteric bacteria were analyzed using classical microbiological tests, AP120E, 16S RNA sequencing, and their susceptibility to a panel of antibiotics assessed using NCCLS protocols. Z prime statistical analysis method was employed to determine the assay/screen quality. The 16S rRNA sequences and antibiotic resistance genes were analyzed using the algorithms BLAST (National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov]) and ClustalX. K. pneumoniae, Enterobacter cloacae, and E. asburiae, known human pathogens, were identified. K. pneumoniae 16S rRNA gene sequence showed the significant hit (E < 0.001) with the unculturable bacteria obtained from feces of elderly individuals (accession no. AB099804) when sequences from Genbank database were used. The rate of antibiotic resistance was high for ampicillin and cephalosporins for all strains tested. Capsules were present in all enteric strains tested but presence of plasmids was not determined. We are currently assessing a genetic linkage between antibiotic resistant and virulent genes. The antibiotic resistance of assessed strains and presence of capsules (protect microorganisms from phagocytosis) suggest that tested environmental bacteria present potential pathogens. It is not clear yet if there is a genetic linkage of resistance and virulence. However, plasmids were not detected in investigated bacteria. These results suggest that there is a little chance for the transmission of virulent factors to other environmental bacteria.

ENVIRONMENTAL INDUCTION OF CYP1A-, CYP2M1-, AND CYP2K1-LIKE PROTEINS IN TROPICAL FISH SPECIES BY PRODUCED FORMATION WATER ON THE NORTHWEST SHELF OF AUSTRALIA

Shiqian Zhu (1*), Susan CodiKing (2), and Mary L. Haasch (1), (1) University of Mississippi, University, MS 38677 and (2) Australian Institute of Marine Science, Townsville, Qld 4810, Australia

Normal operation of oil well platforms results in the discharge of "produced formation water" (PFW). The expression of CYP1A, CYP2M1- and 2K1-like proteins was examined in Gold-Spotted Trevally (Carangoides fulvoguttatus) and Bar-Cheeked Coral Trout (Plectropomus maculatus) as possible biomarkers of PFW impact. The results of this pilot study indicated PFW contamination near the Harriet A platform may contribute to induction of CYP1A- and 2M1-like proteins in Trevally, while other contaminants may induce a CYP2K1-like protein. In a 2003 caged fish study, Stripey seaperch (Lutjanus carponotatus) were caught at a clean site, then distributed to three caging sites: A (near field), B (far field) and C (reference site). Fish were sampled at time (T) zero, T = 3 and T = 10 days. Significant increases of CYP1A, one CYP2K1- and two CYP2M1-like proteins were noted at Site A at T = 10. For the other CYP2K1-like protein, a significant increase was observed at site Aonly at T = 3, but not at T = 10. Prevailing winds switched during sampling, moving the surface water due west, therefore exposing the fish to different components of PFW that may possibly induce this CYP2K1-like protein. These results indicate that CYP1A protein is sensitive to PFW exposure and may act as a good biomarker. Importantly, statistically significant environmental induction of both CYP2M1- and CYP2K1-like proteins in tropical fish due to PFW exposure has not previously been described and represents possible new biomarkers (other than CYP1A) of PFW fraction-specific contamination.

ENHANCING TOXICITY OF TOPOISOMERASE II INHIBITORS USING A c-MYC INHIBITING POLYPEPTIDE

Gene Bidwell III* and Drazen Raucher, University of Mississippi Medical Center, Jackson, MS 39216

Topoisomerase II inhibitors are widely used in cancer chemotherapy. However, their use is limited by severe adverse effects to normal tissues, including cardiotoxicity. One approach to reduce the cytotoxicity in normal tissues may be to sensitize cancer cells to the toxicity of these agents, allowing them to be administered in a lower and safer dose. Here, we report that pretreatment of cells with a polypeptide which inhibits c-Myc transcriptional function causes cells to be more susceptible to the topoisomerase II inhibitors doxorubicin and etoposide. Inhibition of c-Myc and Max dimerization by this polypeptide leads to as much as a two-fold reduction in the doxorubicin and etoposide IC50 in three different cell lines tested. Furthermore, the c-Myc inhibitor enhances doxorubicin or etoposide induced accumulation in G2 or M phase of the cell cycle. We have shown that this effect is not due to enhanced drug accumulation or inhibited drug efflux. Rather, it is likely due to the transcriptional consequences of c-Myc inhibition, specifically reduction in the levels of the polyamine synthesizing enzyme ornithine decarboxylase. Polypeptides which inhibit c-Myc transcriptional function may prove to be a useful tool in combination therapy with topoisomerase II inhibiting drugs.

TNF-ALPHA INDUCED ENDOTHELIN PRODUCTION BY ENDOTHLIAL CELLS; INFLUENCE OF ESTROGEN AND PROGESTERONE

Babbette LaMarca*, Lindsay Roberts, Jennifer Craft Bain, and Joey P. Granger, University of Mississippi Medical Center, Jackson, MS 39216

We previously reported that chronic infusion of TNF-[alpha], at concentrations mimicking plasma levels in preeclamptic women, increased blood pressure and decreased renal function in pregnant rats. In sharp contrast, TNF-[alpha] had no effect in virgin rats. These data suggest that the hormonal environment of pregnancy may influence the vascular actions of TNF-[alpha]. To test this hypothesis, we examined the effects of TNF-[alpha] on endothelin production by endothelial cells in the presence of estrogen (E) and/or progesterone (P). HUVECs were exposed to varying concentrations of TNF-[alpha] with or without hormone for eight hours. The cell culture supernatant was examined using elisas specific for endothelin (ET-1) and soluble intercellular adhesion molecule (sICAM). We found that TNF-[alpha] caused a dose-dependent increase in production of ET-1. In the presence of E or P, TNF-[alpha] increased ET-1 production, however, not significantly greater than what is achieved with TNF-[alpha] alone. In contrast, the combination of E and P significantly enhanced expression and secretion of sICAM by TNF-[alpha]. We conclude that the enhanced blood pressure response to TNF-[alpha] during pregnancy is not likely due to enhanced TNF-[alpha] induced production of ET-1 by sex steroids. However, estrogen and progesterone may play a role by increasing TNF-[alpha] stimulated expression of adhesion molecules, leukocyte activation, and superoxide production.

EFFECT OF EARLY INHIBITION OF THE RENIN ANGIOTENSIN SYSTEM ON THE DEVELOPMENT OF HYPERTENSION IN A MODEL OF LOW BIRTH WEIGHT INDUCED BY REDUCED UTERINE PERFUSION IN THE PREGNANT RAT

Barbara T. Alexander (1*), Antionette Dawson (2), Norma Ojeda (2), W. Russell Johnson (2), and Andrew E. Hendon (2), (1) Murrah High School, Jackson, MS 39202 and (2) University of Mississippi Medical Center, Jackson, MS 39216

Low birth weight (LBW) is a suggested risk factor for development of hypertension. We previously reported that reduced uterine perfusion initiated at day 14 of gestation in the pregnant rat results in LBW offspring that develop hypertension. The purpose of this study was to determine if early, short term blockade of the renin angiotensin system (RAS) would prevent the development of hypertension in this model of LBW. Weight at birth was significantly reduced in offspring from pregnant rats with reduced uterine perfusion as compared to offspring from control pregnant rats (5.9 [+ or -] 0.2 vs. 6.6 [+ or -] 0.2 grams, P < 0.05; growth restricted vs. control, respectively). At two weeks of age, animals were randomly assigned to either an untreated or treated group (angiotensin converting enzyme inhibitor, enalapril, 10 mg/kg/day, administered by gavage for two weeks). Mean arterial pressure (MAP) was measured at four, eight and twelve weeks of age in conscious chronically instrumented animals. In the untreated group, MAP was elevated in growth restricted offspring relative to control offspring at 4, 8, and 12 weeks of age. Early blockade of the RAS did not decrease blood pressure in control offspring at 4, 8 or 12 weeks of age. Early blockade of the RAS initially attenuated hypertension in growth restricted offspring at 4 weeks of age, a decrease of 10 mm Hg, however this attenuation did not persist at 8 or 12 weeks of age. These results suggest that the RAS may play a role in the development of hypertension in this model of LBW induced by reduced uterine perfusion.

TESTING THE BIOCOMPATIBILITY OF SC3 HYDROPHOBIN-COATED PARMAX

Harriet Crockett (1*), Yanan Xu (2), and Yuan Luo (2), (1) Alcorn State University, Alcorn State, MS 39096 and (2) University of Southern Mississippi, Hattiesburg, MS 39406

Evaluations of a Class I fungal protein was done to test the biocompatibility of a high performance polymer. Sc3 hydrophobins are Class I fungal self-assembling proteins that when coated on a polymer makes them more adhesive to hydrophobic and hydrophilic counterparts. Sc3 hydrophobincoated Parmax is the high performance polymer that was utilized in this project. Cellular adhesion and quantitation tests using rat Neuroblastoma and Pheochromacytoma cells were performed to access the biocompatibility of the Sc3 coated Parmax. Parmax is derived from chlorobenzene and is based on a string of substituted and unsubstituted phenylene rings that produce a rigid structure. This polymer can be made into small and transparent films that make them easy for biocompatible testing. My results show that, Sc3 hydrophobins did not serve as satisfactory adhesion substrates for the Neuroblastoma cell line. However, the pheochromacytoma cells continued to grow on a regularly basis. Quantitation of the cells was not successful due to limited samples and time. Cell numbers were relatively low or too low to count. Further biocompatible testing including a viability test should be conducted to ensure the affects of Sc3 hydrophobin-coated Parmax.

HOW ODORS OF GROUP-HOUSED PREGNANT FEMALES AFFECT THE OLFACTORY DEVELOPMENT OF ISOLATED PUPS

Alda Shepherd (1*), Hunter Honeycutt (2), and Jeff Alberts (2), (1) Alcorn State University, Alcorn State, MS 39096 and (2) Indiana University, Bloomington, IN 47405

Little is known about how social influences can affect mice prenatally. It has been found that pups born to pregnant females placed in late social situations during pregnancy have accelerated olfactory development in comparison to pups born to mothers who were isolated. Pups of social situations can distinguish scents of their home bedding from clean bedding on postpartum Day 3 or 4; however pups of isolated mothers do not distinguish scents of their home bedding until postpartum Day 5 or 6. Bedding of group-housed females added to bedding of isolated mothers also produces a similar acceleration. This study was designed to decipher if actual contact with the mice or their bedding creates the acceleration or if exposure to only the odor of group-housed mice can produce the same affect. It was found that the exposure of these airborne odors seems to have no affect on the pups of the isolated mother. Tactile contact or exposure to soiled bedding appears to be the source of acceleration.

THE INHIBITORY EFFECTS OF PB ON NMDA RECEPTORS, SYNAPSIN AND CA-MK II IN PRIMARY CULTURED NEURONAL CELLS

LaTonya Turner*, Shang-Zhi Xu, and Bettaiya Rajanna, Alcorn State University, Alcorn State, MS 39096

The objective of present study is to examine dose-dependent and time-course effects of Pb on NMDAR (NR1C1, NR2A, and NR2B), synapsin and calcium/calmodulin kinase II (CaM KII) in primary cultured neuronal cells. Neuronal cells were isolated from the brain of the fetus of Sprague Dawley pregnant rats at 18-20th gestation and planted in 6 well plates (N = 4). Cells were exposed to Pb at [10.sup.-10]-[10.sup.-7] M for 24 h and then harvested. Later on, [10.sup.-7] M of Pb was incubated with cells for 30 min, 8 h, 14 h, 20 h, 36 h, and 48 h. Antigens of NMDAR, synapsin and CaM KII were first enriched with protein a agarose by immunoprecipitation, and later detected by western blotting with specific antibodies. Meanwhile, total RNA was extracted from harvested cells with Trizol. RT-PCR was employed to detect mRNA expressions of NMDAR with specific primers. The results showed that Pb significantly reduced mRNA expressions of these NMDAR subunits. Protein levels were slightly reduced, but reduction was significant at [10.sup.-8] and [10.sup.-7] M of Pb in comparison with actin control. From exposing for 20 h, Pb gradually reduced protein levels all NMDAR until 48 h. In addition, Pb at [10.sup.-8] and [10.sup.-7] M significantly decreased protein levels of synapsin and CaM KII. Our study suggests that Pb inhibits not only NMDAR subtypes but also synapsin and CaM KII directly. (Supported by NIH/NIGMS/MBRS-SCORE#GM55356.)

SPERM MOVEMENT IN MICROFLUIDIC DEVICES

Brittney Green (1*), Dragos Amerie (2), Sachiko Koyama (2), Milos Novotony (2), and Stephen C. Jacobson (2), (1) Alcorn State University, Alcorn State, MS 39096 and (2) Indiana University, Bloomington, IN 47405

Spermatazoa have been known to exhibit chemotactic behavior in the presence of follicular and oviductal fluids, the oocyte microenvironment, and some hormones. Despite these findings, the components of the materials that are responsible the the chemotactic behavior of sperm are not known. Microfluidics, a relatively new science that examines the behavior of fluids at a microscopic level, can provide a steady state environment in which sperm movement and chemotaxis can be studied in real time. An important aspect in studying the chemotaxis of sperm using complex microfluidic devices, or chips, is to understand how sperm move in the apparatus. The main goal of this study is to observe the motion of spermatozoa so that we will be able to carry out a more efficient chemotaxis assay using microfluidic devices. The observations of sperm movement will be used to find a microfluidic device that has microchannels large enough to allow sperm to swim closer to their natural swim pattern, but small enough to produce a complex gradient that will allow accurate control of the concentration of possible chemoattractants.

USING RNA INTERFERENCE FOR UNDERSTANDING THE FUNCTION OF SMN PROTEIN

Robert Walker (1*), Natalia Singh (2), and Elliot Androphy (2), (1) Alcorn State University, Alcorn State, MS 39096 and (2) University of Massachusetts Medical School, North Worcester, MA 01655

Spinal Muscular Atrophy (SMA) is a common autosomal recessive disorder which causes the loss of motor neuron function in the spinal cord. One in 6,000 babies are born every year with SMA, and one in 40 people carry the mutation that causes it. Children born of parents that are both carriers have a 25% chance that they will develop SMA. SMA is usually divided into three different types. Type 1 is the most severe with the onset occurring in early childhood, and type 3 is the least severe, with onset in early adulthood. The most common cause of SMA is deletion of the SMN gene, which expresses the survival motor neuron (SMN) protein. A second nearly identical copy of the SMN gene called SMN2 expresses very low levels of active SMN protein and is unable to fully substitute for the missing SMN protein. The SMN protein is ubiquitously expressed and its function is essential in all organisms studied, including mouse, Caenorhabditis elegans and Schizosaccharomyces pombe. Motor neurons are particularly sensitive to SMN reduction compared with other cells. The function of SMN1 in motor neurons is still unknown. To understand the function of SMN we used a technique called RNA interference (RNAi), which leads to reduced levels of a specific protein. The RNA interference pathway is shown in Figure 1. One of the key role players in RNAi is small interfering or siRNA. There are several ways to generate siRNA. For our long term studies we used a tetracycline-inducible siRNA-expression vector with a luciferase reporter system to quickly check whether the selected siRNA was effective. Although the presence of siRNA decreased luciferase activity four fold, meaning our siRNA was working, we would need to try additional siRNAs to improve the silencing effect.

TOXICITY EVALUATION OF ACRYLAMIDE ON THE EARLY LIFE STAGES OF THE ZEBRAFISH EMBRYOS (DANIO RERIO)

Hattie Spencer* and Joseph Wahome, Mississippi Valley State University, Itta Bena, MS 38941

Abstract: Embryos of the Zebrafish (Danio rerio) were exposed to serial concentrations of acrylamide (0, 100, 300, and 500 mg/L) to investigate its sub-chronic effects on embryonic development. Eggs/embryos less than 24 hrs old were exposed under static non-renewal conditions until hatching. The toxic endpoints evaluated included: egg/embryo viability, hatchability, and morphological/developmental abnormalities. The acute toxicity test resulted in a 48 h-L[C.sub.50] of 585 mg/L for egg viability. Exposure of eggs to sub-chronic concentrations significantly reduced hatchability and larval survival, in a concentration dependent manner. At 500 mg/L, the highest test concentration, the survival of embryos was greatly reduced within 24hrs of exposure. The lower test concentration, 100 mg/L, produced a significant number of developmental effects to the Zebrafish, including dorsal tail flexure, severe pericardial edema, facial and cranial defects and increase heartrate (150 bpm). Premature hatching and developmental arrest was observed in all concentrations (except control). The severity of these abnormalities was concentration-dependent. It can be concluded from these results that acrylamide is teratogenic and also indicates the ecological significance of sublethal toxicity testing using the zebrafish embryos.

EFFECT OF pH ON INTERLEUKIN-4 ANTIGEN-ANTIBODY INTERACTIONS AS ASSESSED BY ENZYME-LINKED IMMUNOASSAYS

Marie Winston*, Robert C. Sizemore, and Marta Piva, Alcorn State University, Alcorn State, MS 39096

Certain opioid peptides and their metabolic derivatives affect the production of interleukin-4 (IL-4) by immune cells. IL-4 drives the immune response against allergens and certain parasites. Usually the amount of IL-4 present in cell supernatants is very low and requires the use of undiluted samples in the enzyme-linked immunoassay (ELISA). For this reason, we sought to determine if: (1) increases in incubation time and ConA concentration alter the pH of the cell supernatants and (2) changes in the pH of the cell supernatants have a significant effect on the interaction between IL-4 and the ELISA capture antibody. ELISA calibration curves consisted of different concentrations of recombinant mouse IL-4 in serum-free medium from pH 5.0 to 7.5. Splenocytes were incubated for various periods in the presence of different amounts of ConA. At the end of the incubation period, cell supernatants were collected and their final pH and IL-4 concentration measured. Longer incubation times and higher ConA concentrations caused a progressive acidification of the cell supernatants. The slopes of the calibration curves (dependent variable-Y) were plotted against the pH of cell supernatants (independent variable-x), resulting in a bell-shaped function that reached a maximum at pH 6.2. The limit of detection for the assay was 1 pg/mL for 5.7 < pH < 7.2. Since IL-4 ELISA results are considerably affected by the pH of the cell supernatants in addition to the cytokine concentration itself, we conclude that accurate comparisons can be only made using samples and calibration curves of similar pH.

ALPHA7 NICOTINIC RECEPTORS: ROLE IN ETHANOLINDUCED NEUROTOXICITY?

Leon Thomas* and Christopher M. deFiebre, Alcorn State University, Alcorn State, MS 39096 and University of North Texas Health Science Center, Fort Worth, TX 76106

Alpha7 nicotinic acetylcholine receptors (nAChRs) have been suggested to be involved in modulating neuronal viability because agents that act at alpha7 receptors are protective against a variety of neurotoxins, including ethanol. Preliminary data in primary neuronal cultures derived from [alpha]7 nAChR null mutant mice have suggested that the absence of [alpha]7 nAChRs increases susceptibility to ethanol-induced neurotoxicity without affecting baseline neuronal viability. Here we attempted to assess whether the results obtained in cell culture (in vitro) were predictive of what would be seen in whole animals (in vivo). Using a whole animal model of binge-drinking induced neurotoxicity, we proposed to test the hypothesis that subchronic treatment with high doses of ethanol would result in greater deficits in learning ability in alpha7 null mutant mice than in wild type mice. This hypothesis is based on the assumption that null mutant mice would show greater ethanol-induced neurotoxicity and that this would result in greater deficits in learning. Mice were to be treated for 4 days, 3 times daily. The starting dose was to be 8 g/kg with subsequent doses determined by scoring animals on level of intoxication. Animals were then to be withdrawn from ethanol for 3 days prior to testing for learning behavior in a T-maze and in a passive avoidance paradigm. Unexpectedly, most of the null mutant and heterozygous mice died following a single administration of ethanol rendering continuation of the study not feasible. These data, while unexpected, support the hypothesis that null mutation of the alpha7 nAChR gene results in a greater susceptibility to the toxic effects of ethanol. Whether this increased susceptibility is to neurotoxic or other toxic effects remains to be determined. Both in vitro and in vivo studies suggest that alpha7 nAChRs may be a useful site for drug development for agents to protect against ethanol-induced toxicity in alcohol overdose.

ASSESSING SUBNANOMOLAR CONCENTRATIONS OF ENDOGENOUS OPIOID PEPTIDES BY NON-RADIOACTIVE METHODS

Chavez Carter*, Robert C. Sizemore, and Marta Piva, Alcorn State University, Alcorn State, MS 39096

Methionine-enkephalin (ME) is an opioid pentapeptide (YGGFM) that affects the immune response. Mitogens such as Concanavalin A (ConA) stimulate the production of preproenkephalin. Very low concentrations of endogenous ME accumulate in cell supernatants. We have developed a non-radioactive procedure consisting of solid-phase extraction followed by enzyme-linked immunoassay (ELISA) to assess accurately ME concentrations in splenocyte-conditioned culture media. We set up experiments to ascertain the effect of concentrated, solid-phase extracted culture medium on ME ELISA and ME recovery after the solid-phase extraction step. Phenol red-free culture medium (AXL) was solid-phase. The solid residue was dissolved in phosphate saline buffer. The second experiment was performed by adding ME to AXL to a final concentration of [10.sup.-9] M. ME ELISA was linear between [10.sup.-9] and [10.sup.-6] M, had a limit of detection of 1 X [10.sup.-8] M and a coefficient of variation 6% or less. The ELISA calibration curves for SP-AXL and AXL were parallel; however, their intercepts differed considerably. Therefore, SP-AXL was included as control (absorbance value = 100%) in subsequent immunoassays. At [10.sup.-9] M, 65% of the added amount ME was recovered after solid-phase extraction and freeze-drying. The concentrated cell-conditioned medium contained 2 X [10.sup.-8] M of ME, a value that fell within the calibration curve linear range. In conclusion, a sensitive and specific methodology was developed to measure subnanomolar concentrations of endogenous ME in cell supernatants. This assay can be performed with no extensively purified samples and has the advantage of avoiding the use of radioactive materials.

CHARACTERIZATION OF ALCOHOL METABOLIZING ENZYMES IN JAPANESE MEDAKA

Asok K. Dasmahapatra*, Xueqing Wang, Erin Williams, and Mary L. Haasch, University of Mississippi, University, MS 38677

Cardiovascular defects in developmental ethanol exposure have been identified in both human and animal models and used as a phenotypic feature of fetal alcohol syndrome (FAS). The molecular mechanism of these developmental abnormalities has not yet been fully characterized. We used the Japanese medaka developmental model to evaluate cardiovascular defects produced by acute ethanol exposure. Viable medaka eggs within 1h of fertilization were exposed to ethanol (0-400 mM) in hatching solution for 48 h. cardiovascular development was observed from 1-7 dpf. The embryos exposed to low ethanol concentrations (0-100mM) exhibited active blood circulation at 2 dpf but active circulation was significantly delayed at higher ethanol concentrations (200-400mM). Moreover, embryos exposed to these higher concentrations (200-400mM) developed tube heart and blood clots in the Blood Island and in the circulatory vessels. We hypothesized that the cardiovascular abnormalities seen in developing Japanese medaka were due to one or more metabolites of ethanol metabolism. To examine this possibility, we have begun to characterize the genes for alcohol metabolizing enzymes in medaka. By applying PCR-based technologies, we previously cloned and characterized two alcohol dehydrogenase (ADH) enzyme cDNAs from the liver tissue of medaka. Additional work has yielded partial clones of two subtypes of aldehyde dehydrogenase cDNA (ALDHa and ALDHb) in adult medaka liver. ALDHa mRNA showed tissue specific expression, however ALDHb was ubiquitously expressed (brain, eye, GI, gill, heart, kidney, liver, muscle, skin, spleen and ovary). Supported in part by ETRP, Department of Pharmacology and ORSP of the University of Mississippi.

REGIO-SPECIFIC ([omega] TO [omega]-6) FATTY ACID (LAURIC, MYRISTIC, AND PALMITIC) HYDROXYLATION IN HUMAN POOLED LIVER AND RECOMBINANT MICROSOMES

Mary L. Haasch* and James C. Allgood, University of Mississippi, Hattiesburg, MS 38677

Most studies of lauric acid hydroxylation have focused on only two hydroxylation products, [omega], and [omega]-1. Modifications of an HPLC method (Lemaire et al., 1992) to include GC-MS (Buhler et al., 1997) have allowed more sub-terminal hydroxylation products including [omega]-2 to [omega]-6 to be quantified. Further modifications now allow the measurement of not only lauric acid hydroxylation but also the hydroxylation of myristic and palmitic acids (Holmes et al., 2004). We have used a modification of the method to allow measurement of not only of [omega] and [omega]-1 but also [omega]-2 to [omega]-6 sub-terminal hydroxylation products. Previously we have shown regio-specific lauric acid hydroxylation in human pooled liver and recombinant microsomes. Preliminary evidence indicated significant lauric acid subterminal hydroxylation, specifically [omega]-2, [omega]-5, and [omega]-6 by both human liver male and female pooled microsomes, with only female microsomes catalyzing [omega]-1 while products of [omega]-3, [omega]-5, and [omega]-6 were produced by human recombinant CYP4A11. Interestingly, CYP2B6 produced significant lauric acid subterminal hydroxylation at the [omega]-1, [omega]-2, and [omega]-3 positions with high [omega]-6 activity. Additionally the source of the recombinant enzyme and the source of the cytochrome P450 reductase influenced the lauric acid hydroxylation pattern by CYP2E1. The sub-terminal hydroxylation of myristic and palmitic acids has not previously been reported. There is potential for subterminal hydroxylation products to act as second messengers in cellular signal transduction or to interfere with steroid biotransformation. These hypotheses will be investigated in future research.

INHIBITION OF HUMAN CYTOCHROME CYP1 ENZYMES BY FLAVONOIDS OF ST. JOHN'S WORT

Amit Chaudhary* and Kristine L. Willett, University of Mississippi, University, MS 38677

As with many available herbal preparations, components of St. John's wort are not completely characterized with respect to their interaction with drug metabolizing enzymes. Four flavonoids present in St. John's wort and apigenin were studied for their ability to inhibit TCDD-induced EROD activity in 22Rv1 human prostate cancer cells. Quercetin (IC50: 4.1 [micro]M), kaempferol (3.7 [micro]M), myricetin (3.0 [micro]M) and apigenin (3.1 [micro]M) caused significant inhibition of TCDD-induced EROD activity whereas amentoflavone did not cause inhibition. CYP1B1 is involved in metabolizing both polycyclic aromatic hydrocarbons and estradiol to potentially carcinogenic intermediates, and it is also over-expressed in human cancer cells. In order to identify flavonoids that specifically inhibited CYP1B1, eight flavonoids in St. John's wort and apigenin were screened for their selective inhibition of recombinant CYP1B1 versus CYP1A1. IC50s for CYP1B1 inhibition ranged from 0.22 [micro]M for apigeninto 18.34 [micro]M for hyperoside. CYP1A1 IC50s ranged from 0.91 [micro]M for hyperoside to 63.7 [micro]M for quercetin. Seven flavonoids, quercetin (34-fold), apigenin, myricetin, kaempferol, amentoflavone, quercetrin, and isoquercetin, were more selective for CYP1B1 EROD inhibition compared to CYP1A1. Rutin did not inhibit in either system whereas hyperoside significantly inhibited CYP1A1 compared to CYP1B1. Apigenin, quercetin, and amentoflavone were competitive inhibitors of CYP1B1 with Kis of 0.02, 0.08, and 0.51 [micro]M, respectively. By distinguishing relative roles of cytochrome P450s (specifically CYP1B1 vs. CYP1A1) in different human cancer cell lines, methodologies can be developed to provide better diagnostics and possibly new therapies against cancer formation.

THE snxA1 AND nimA5 MUTATIONS OF ASPERGILLUS NIDULANS INTERACT TO AFFECT MITOTIC SPINDLE STRUCTURE AND THE DNA SYNTHESIS CHECKPOINT

Ryan Day*, Brandon Fontenelle, Shruti Chandna, and Sarah Lea McGuire, Millsaps College, Jackson, MS 39210

The snxA gene interacts with NIM[X.sup.cdc2] to affect mitosis, and its mutation causes abnormal nuclear morphology at 17[degrees]C, while the nimA gene affects the nuclear import of NIM[X.sup.cdc2] and when mutated blocks mitotic entry at 42[degrees]C. To characterize the effects of the snxA1 mutation on microtubule and nuclear structure, strains expressing a GFP-tubA ([alpha]-tubulin) gene were generated with either the snxA1 or nimA5 mutations alone or with a snxA1/nimA5 double mutant. At 17[degrees]C, snxA1/GFP-tubA cells had severe nuclear defects, thickened hyphae, abnormal spindle structures, and abnormal interphase microtubule arrays. Mitotic spindles were highly variable in length. Some spindles had no nuclei attached to them, while yet others were bifurcated or trifurcated and had fragmented, variably condensed nuclei along their lengths. Similar abnormal nuclei and spindle structures were observed when snxA1/nimA5 strains were germinated at 32[degrees]C and upshifted to 42[degrees]C for 3 hours. Additionally, snxA1 was shown to suppress a checkpoint defect observed in nimA5 cells, as the snxA1/nimA5 double mutants were able to grow in the presence of 20 mM hydroxyurea, where nimA5 cells were sensitive at 10 mM hydroxyurea. Cloning and further characterization of the snxA gene will provide clues to the interactions of NIMXCDC2, NIMA, SNXA, and the regulation of mitotic spindle formation. Supported by NIHGM55885-03 and the Mississippi Functional Genomics Network.

CELLULAR UPTAKE OF CELL PENETRATING PEPTIDES FUSED TO ELASTIN-LIKE POLYPEPTIDES: EFFICIENCY AND MECHANISM

Iqbal Massodi*, Gene Bidwell III, and Drazen Raucher, University of Mississippi Medical Center, Jackson, MS 39216

Translocation through the plasma membrane is a major limiting step for the cellular delivery of macromolecules. Several cell-penetrating peptides (CPP) have been demonstrated to allow efficient internalization of various molecular "cargo" to targets within the cytoplasm and nucleus of eukaryotic cells. It has been shown that their delivery efficiency and cellular uptake is dependent on cargo. However, the mechanism used by CPP for cell entry is largely unknown. We evaluated efficiency and mechanism of cellular uptake of penetratin, Tat (48-60) and the membrane translocating sequence from (MTS) fused to elastin-like polypeptides (CPP-ELP). Elastin-like polypeptides (ELPs) are biopolymers composed of the pentapeptide repeats Val-Pro-Gly-Xaa-Gly (MW 51kD). They have several attractive features making ELPs useful as polymeric carriers for the delivery of therapeutics. Therefore, improving efficiency by fusing ELPs to CPP and understanding the mechanism of their cellularinternalization could contribute to development of new therapeutic approaches. Flow cytometry of fluorescein labeled CPP-ELPs showed ten fold enhancement in cellular association of Pen-ELP, but no significant increase in cellular association of Tat-ELP or MTS-ELP was seen in HeLa cells as compared to ELP without CPP. Our trypan blue quenching assay showed that 70% of CPP-ELPs associated with cells were initially only attached to the cell surface, but they were internalized completely 24 hours later. That CPP-ELPs are initially only attached to the cell surface was confirmed with confocal fluorescence microscopy. Cells observed immediately after incubation with rhodamine conjugate of the CPP-ELPs showed preferential plasma membrane localization. The internalization of all CPP-ELPs was impaired dramatically at 4[degrees]C, indicating that CPP-ELPs are internalized by the endocytotic pathway.

USING ABSORBANCE DIFFERENCE SPECTROSCOPY TO INVESTIGATE THE INTERACTIONS OF SELECTED INHIBITORS WITH THE BREAST CANCER MOLECULAR TARGET CYTOCHROME P450 AROMATASE

Jonathan Priester* and Stanley V. Smith, Murrah High School, Jackson, MS 39202 and University of Mississippi Medical Center, Jackson, MS 39216

Research into the initiation and progression of breast cancer has led to many breakthroughs in fighting this dreaded illness. The identification of several molecular targets has resulted from this research. One of the molecular targets is cytochrome P450 aromatase. Inhibition of cytochrome P450 aromatase activity results in decreased production of estrogens and is the basis of the efficacy of aromatase inhibitors towards breast cancer. Cytochrome P450 aromatase is a member of the cytochrome P450 superfamily of enzymes. These enzymes are involved in a number of important cellular processes such as xenobiotic metabolism and the synthesis and metabolism of endogenous compounds. A common feature of the cytochrome P450s is the presence of a heme moiety liganded to a cysteinylthiolate at the enzyme active site. This allows the cytochrome P450s to bind molecular oxygen and modulate its reactivity through a series of controlled electron transfers and typically results in the insertion of one atom of molecular oxygen into the substrate. Cytochrome P450 aromatase utilizes these features to convert androgens into estrogens via a complex three-step mechanism. The goal of our investigation was to characterize the binding of aromatase inhibitors to cytochrome P450 aromatase. We estimated the binding affinities by measuring the changes in the spin-state of the heme-iron moiety at the aromatase active site using absorbance difference spectroscopy. The results indicate that the second-generation steroidal inactivator formestane binds tightly to aromatase but not as tightly as a natural substrate (androstenedione). These results will be incorporated into a molecular model for aromatase which will be used to suggest new approaches for inhibitor design. Supported by American Cancer Society #IRG-98-275-04 and the Base Pair Program.

Divisional talks begin

10:00 CHARACTERIZATION OF THE STRUCTURAL AND FUNCTIONAL IDENTITY OF THE GROELI CHAPERONE IN MYCOBACTERIUM SMEGMATIS

Amrita Balachandran (1*), Anil Ojha (2), and Graham Hatfull (2), (1) Mississippi University for Women, Columbus, MS 39701 and (2) University of Pittsburgh, Pittsburgh, PA 15260

The Mycobacteria have two GroEL chaperones, namely, GroEL1 and GroEL2. In Mycobacterium smegmatis specifically, it has previously been observed that although the GroEL1 and GroEL2 proteins show 60% homology in their amino acid sequences, GroEL1 is not essential for the survival of the organism. GroEL2, on the other hand, is essential. Moreover, groEL1 knock-out mutants show a phenotypic defect in biofilm maturation. These observations suggest possible functional differences between GroEL1 and GroEL2. Our project was aimed toward identifying the amino acid residues of M. smegmatis GroEL1 that characterize its distinguishing identity and function in relation to GroEL2. The experimental design involved the construction of groEL1 and groEL2 mutants, and the testing of their ability and extent of biofilm formation in comparison to the wild type M. smegmatis mc2155 strain. The results show that a chimeric construct constituting a 174 bp C-terminal groEL1 fragment fused to a 1425 bp N-terminal groEL2 fragment does not complement the defective biofilm phenotype in groEL1 knock-out M. smegmatis strains. Ongoing stages of the experimental process could possibly confirm the identification of random mutations in groEL1 that impede efficient biofilm formation, and analyze biofilms formed by two additional chimeric constructs. Further understanding of the distinct amino acid identity and structure of M. smegmatis GroEL1 could perhaps further the possibility of targeting the pathogenecity of M. tuberculosis.

10:20 RNA 5' END LABELING BY FLUOROPHOREAMP-INITIATED TRANSCRIPTION AND APPLICATIONS IN RNA STRUCTURE-FUNCTION INVESTIGATION

Na Li (1*), Faqing Huang (1), and C.J. Yu (2), (1) University of Southern Mississippi, Hattiesburg MS 39406 and (2) AdeGenix, Inc., Monrovia, CA 91016

Specific labeling at the either 3' or 5' end of RNA is essential for a variety of applications in RNA structure-function investigation. Conventionally, 5' end labeling is usually achieved through PNK-catalyzed phosphorylation of RNA by [[gamma]-[P.sup.32]]-ATP. The procedure requires the use of a high-energy radioisotope and multi-steps of labeling and purification. Furthermore, it may be difficult to label some RNA molecules if they form 5'-recessed duplex structure. In addition to the hazardous nature of [P.sup.32], the relatively short half-life of [P.sup.32] means frequent replacement of "old" radioactive ATP with fresh one. We have developed a simple method for efficient 5' end labeling of RNA via a single-step transcription. We demonstrate here the utility of 5' cyanine dye-labeled RNA for mapping the reaction sites of newly isolated ribozymes. These ribozymes possess 5' duplex structures with 3' overhangs. To define the reaction sites of ribozymes, 5' cyanine dye-labeled RNAs were partially hydrolyzed by lead-induced phosphodiester bond cleavage and fractionated by denaturing PAGE. Fluorescence signals were directly detected by a regular phosphorimager (Bio-Rad). The RNA ladder patterns revealed well-defined reaction sites (the location of reactive nucleotides) within ribozymes. 5' Fluorophore-labeled RNA prepared by in vitro transcription may find broad applications in various fields of biochemistry, biophysics, molecular biology, and biomedicine.

10:40 EVALUATION OF CPG METHYLATION OF SOCS GENES AS BIOMARKERS FOR JAK-2-INHIBITOR SENSITIVITY IN HUMAN BREAST CANCER CELL LINES

Ashley Jenkins (1*), Roy J. Duhe (2), and John K. Smith (2), (1) Alcorn State University, Alcorn State, MS 39096 and (2) University of Mississippi Medical Center, Jackson, MS 39216

The regulation of the prolactin/JAK/STAT/SOCS pathway is essential in many biological processes, such as mammary gland development. Our lab is working to develop drugs that target abnormally active forms of Janus Kinases (JAK). We are also trying to find molecular markers that can identify cancer patients who will benefit from these drugs. It was recently discovered that SOCS-1 and SOCS-3 (Suppressors Of Cytokine Signaling) are silenced in several cancers, but not in normal tissue. The SOCS silencing occurs due to the hypermethylation of "CpG islands" in the socs gene. This removes an important negative feedback mechanism that normally inactivates the JAKs. Methylation silencing of the socs gene was found in 20-60% of the tumors examined, which included patient-derived hepatocellular carcinomas, pancreatic ductal neoplasms, multiple myelomas, acute myeloid leukemia, hepatoblastomas, and human lung cancers. Because the regulation of the prolactin signaling pathway is so important in normal breast development, we suspect that the loss of control of this pathway via socs methylational silencing might lead to breast cancer. Methylation of CpG islands can be measured by methylation-specific PCR, which distinguishes between unmethylated and methylated DNA in a given region. Ultimately, the methylation status of socs-1, socs-2, and/or socs-3 may be used to identify circumstances in which JAK2 inhibitors can be used to treat patients with breast cancer.

11:00 USE OF SITE-DIRECTED MUTAGENESIS TO INVESTIGATE THE MECHANISM OF REDOX REGULATION OF JANUS KINASES

Naila Mamoon*, Kiranam Chatti, Sheeyong Lee, and Roy J. Duhe, University of Mississippi Medical Center, Jackson, MS 39216

Many cytokines, growth factors and hormones signal through the Janus protein tyrosine kinases (JAKs), dysregulation of which has been implicated in a number of cancers. The catalytic activity of JAKs is modulated by their redox states; activity is abolished by oxidation and restored upon reduction. Pretreatment of JAK2 with NEM, an agent that selectively alkylates thiols, also abolished the autokinase activity of JAK2, providing additional evidence that cysteine thiols are essential for the maintenance of activity. The reductive stimulation of JAK activity may underlie cytokine-independent proliferation of leukemic T-cells overexpressing thioredoxin (Trx), a physiological reductant. Trx restored autokinase activity of oxidatively inhibited JAK2 in vitro, while redox-inactive Trx did not. We have evaluated the possibility that specific cysteine residues within JAKs are critical for the redox sensitivity of the enzyme. Using site-directed mutagenesis, nine cysteine residues (675, 722, 748, 787, 866, 917, 961, 1094, and 1105), within JAK2 were individually and progressively mutated to serines and the effects of these mutations were tested in autophosphorylation and transphosphorylation assays. All nine of the cysteine to serine point mutants retained redox reversibility and demonstrated some level of catalytic activity. When all nine cysteines were converted to serines, the enzyme lost its ability to autophosphorylate under in vitro assay conditions. Under cellular assay conditions, enzyme activity was markedly reduced but not completely obliterated. These results suggest that oxidation of specific cysteine thiols modulates the catalytic activity of JAKs, a feature that may be exploited in JAK-targeted cancer therapy.

FRIDAY AFTERNOON

Cedar Room

12:00-3:00 PLANTING THE SEEDS OF A BIOSCIENCE INDUSTRY IN MISSISSIPPI

Revolutionary advancements in biological research have spawned new business opportunities. In most regions of the United States, collaborations between innovative scientists and technologists have resulted in entrepreneurial start-up companies manufacturing novel products in pharmaceutical, medical, agricultural, chemical, and environmental industry sectors. Bioscience and biotechnology industrial development is at an early stage in Mississippi, and the membership of the Mississippi Academy of Sciences should be active participants in this process. This symposium will bring together bioscience industry experts with a broad range of perspectives, from the scientific innovators who bring novel ideas to the business community to the venture capitalists who provide start-up funding for bioscience business proposals.

Dr. Roy J. Duhe, Associate Professor of Pharmacology and Toxicology at the UMMC and MAS Director (2003-2006): "Promises and pitfalls of biotechnology and bioscience industries in Mississippi";

Dr. Garth Powis, Director of Basic Research at the Arizona Cancer Center and co-founder of ProlX Pharmaceuticals: "Academic entrepreneurship";

Dr. Lyn Stabler, Vice President of Policy and Analysis at the Mississippi Technology Alliance: "Policy initiatives for bioscience development in Mississippi";

Dr. Larry Walker, Director of the National Center for Natural Products Research at the University of Mississippi: "Spin-off products from academic research in Mississippi";

Mr. Frank Montgomery, Managing Partner of Moss Forest Venture: "The investment community's perspective";

Dr. Greg Perkins, President, BioDerm Sciences, Inc.: "How to attract biopharmaceutical industry to Mississippi."

Divisional talks resume

3:00 ASSEMBLING REPORTER CONSTRUCTS TO INVESTIGATE INTERFERON-MEDIATED REGULATION OF THE ONCOGENIC HERPESVIRUS MAREK'S DISEASE VIRUS

Joy Wall (1*), Ross Whitwam (1), and Shane Burgess (2), (1) Mississippi University for Women, Columbus, MS 39701 and (2) Mississippi State University, Starkville, MS 39762

Marek's Disease Virus (MDV) is an [alpha]-herpesvirus that causes lymphomas in chickens. MDV is an economic concern to the poultry industry and a model for human lymphomagenesis. Phosphoprotein 38 (pp38), a MDV gene product, is thought to be involved in the latent/lytic transition of MDV and may exploit interferon for use in its regulation. The pp38 promoter region has two regions which the TransFac database identified as Interferon Response Element (IRE) binding sites. We wish to investigate whether IREs regulate pp38 expression. Reporter constructs are being assembled containing PCR-amplified regions of the pp38 promoter in front of a green fluorescent protein (GFP) reporter gene. The amplicons from the pp38 promoter were chosen to yield no IREs, one IRE, or two IREs. All were successfully amplified. The plasmid pd2EGFP-N1 contains a GFP gene behind the cytomegalovirus promoter. This promoter was digested out of the plasmid and ligations are under way to insert the three pp38 amplicons in front of the GFP gene. Once reporter constructs with each amplicon, in both possible orientations have been successfully assembled they will be transformed into chicken embryo fibroblast (CEF) cultures. How GFP expression in the CEF cultures responds to varying recombinant interferon concentrations will be tested to offer insight into the role of IREs as pp38 regulatory elements.

3:20 DYNAMICS OF NUCLEOLAR PHOSPHOPROTEIN B23 AND ITS MUTANTS IN HeLa CELLS

Sandeep S. Negi* and Mark Olson, University of Mississippi Medical Center, Jackson, MS 39216

B23/NPM is an abundant multifunctional nucleolar phosphoprotein involved in ribosome biogenesis. Activities of protein B23 include nucleic acid binding, ribonuclease and molecular chaperone activity. It is phosphorylated during interphase by casein kinase 2 (CK2) (Ser125) and by cyclin dependant kinase 1 (cdk1) (Thr199, 219, 234, and 237) during mitosis. We studied the effect of these phosphorylations on the dynamics of B23 in the nucleolus. Fluorescence recovery after photobleaching (FRAP) studies showed that the mutant (Ser125 to Ala) recovered slower than the wild type, suggesting that phosphorylation reduces affinity for other nucleolar components. Further studies on the phosphorylation mimicking mutant (Ser125 to Glu) showed no difference in the mobility. This may indicate that B23 mostly remains phosphorylated during interphase. Indirect-immunofluroscence studies done with an antibody against phosphorylated Thr199 revealed that B23 is phosphorylated at this site at the start of the mitosis and is dephosphorylated during early anaphase. This site had little or no signal in interphase cells. We hypothesize that these cdk1 phosphorylations affect the nucleic acid binding activity of B23. A Thr to Glu mutant of the cdk1 sites was studied in the interphase cells. This mutant and other members of the nucleoplasmin family, which lack the C-terminal nucleic acid binding region, B23.2 and NPM3, showed a greater mobility/faster recovery than the B23.1, indicating the importance of this segment.

3:40 Divisional Business Meeting