Cellular, molecular and developmental biology.

Chair: Barbara Graham-Evans, Jackson State University 

Vice-chair: Lauren Brandon, Mississippi University for Women

THURSDAY MORNING

Bost Auditorium South

8:00 BIOLOGY OF PREIMPLANTATION PORCINE EMBRYOS IN VITRO

Hongfeng Wang*, Song-yi Jung, Nelida Rodriguez-Osorio, Kristen Faith Garrison, Kristi Wolgemuth, Laura Samantha Greer, Jean Magloire Nguekam Feugang, and Erdogan Memili, Mississippi State University, Mississippi State, MS 39762

Early mammalian development involves fascinating molecular and cellular programming events as the transition from maternal to embryonic control of development occurs during preimplantation embryogenesis. We aimed at determining transcriptional and translational controls of development in porcine preimplantation embryos using specific inhibitors of mRNA (alpha-amanitin) and protein (cycloheximide) synthesis. Following in vitro maturation and fertilization, presumptive zygotes were cultured in PZM-3 medium, at 39[degrees]C under 5% CO2 in a humidified environment. Starting at 24 hours or 84 hours post-insemination (hpi) embryos were transferred to culture drops containing 25 [micro]g/ml alpha-amanitin or 2 [micro]g/ml cycloheximide for a 60-hour exposure. The effects of inhibitors on cleavage (48 hpi) and blastocyst (144 hpi) rates were recorded. Data were analyzed by SAS 9.1, and p>0.05 were considered significant. None of the two inhibitors allowed blastocyst formation, irrespective of the period of exposure (P>0.05), while 16.5% of the cleaved embryos in the control group developed until the blastocyst stage. The early exposure (24hpi) to cycloheximide significantly reduced the cleavage rate compared to the control (20% vs. 36%; P<0.01). Cleavage rate in the alpha-amanitin treated-group was similar to the control one (p>0.05). We concluded that the novo-synthesis of mRNA transcripts is not necessary at the early stages of porcine embryo development, but is critical for later stages (after 84 hpi) and, the neo-synthesis of protein is a crucial event occurring throughout the porcine embryo development. Further investigations are needed to identify the relevant genes whose products are involved in cleavage and blastocyst formation.

8:15 A COMPARATIVE ANALYSIS OF THE EARLY EMBRYONIC RESPONSES OF THE GIANT DANIO (DANIO AEQUIPINNATUS) AND ZEBRAFISH (DANIO RERIO) TO RETINOIC ACID

Corretha Ingram*, Stephen D'Surney, and Michael Smith, University of Mississippi, University, MS 38677

Retinoic acid is one of the principal biologically active forms of Vitamin A. Even though retinoic acid is essential for development, excess amounts can cause abnormalities. In this experiment, the effects of retinoic acid on the early embryonic development of the zebrafish (Danio rerio) and giant danio (Danio aequipinnatus) were studied. These two fish are members of the Cyprinidae family. Various concentrations of retinoic acid were exposed to the embryos to monitor development. The concentrations were control, 700ng/L, 725ng/L, 750 ng/L, 775ng/L, and 800 ng/L. This experiment focuses to determine the effects of retinoic acid on early embryonic development in fish and compare these effects between the related species.

8:30 DELETION OF p38[alpha]a MAP KINASE IN MOUSE EMBRYONIC STEM CELLS RESULTS IN UP-REGULATION OF SMOOTH MUSCLE CELL MARKERS DURING DIFFERENTIATION

Jianming Ye*, Suja Rajan, Faqing Huang, and Yanlin Guo, University of Southern Mississippi, Hattiesburg, MS 39406

p38 mitogen-activated protein (MAP) kinase [alpha]a (p38[alpha]a) is a broadly expressed signaling molecule that regulates various cellular processes. Most studies of p38[alpha]a have been focusing on adult somatic cells, but little is known about its function in stem cells. Our previous study has shown that mouse embryonic stem (ES) cells isolated from embryos of p38[alpha]a knockout mice (p38[alpha]a-/- ES cells) have altered cell proliferation and adhesion properties from wild type cells (p38[alpha]a+/+). The objective of this study was to determine the role of p38[alpha]a in mouse ES cell differentiation. Using protocols that allow ES cells to differentiate into different types of cells, we investigated the role of p38[alpha]a in smooth muscle cell (SMC) differentiation from wild type ES cells (p38[alpha]a+/+) and p38[alpha]a deficient (p38[alpha]a+/- and p38[alpha]a-/-) ES cells. Although p38[alpha]a-/-ES cells were able to differentiate to SMC, deletion of p38[alpha]a resulted in an increase of expression of SMC markers, [alpha]a-actin (SMA) and SM22[alpha]a. However, SMC differentiated from p38[alpha]a-/-ES cells displayed disorganized SMA filament structures. Our results suggested that p38[alpha]a controls the organization of SMA and negatively regulates SMC marker expression. This is in contrast to adult SMC differentiation where in most cases the activation of p38[alpha]a is required for the expression of SMC markers, indicating that p38[alpha]a plays different roles in adult SMC and embryonic SMC differentiation. This work is supported by grants from the National Institutes of Heath R15HL081126 and R21HL08273 (YLG).

8:45 CHARACTERIZATION OF CANDIDATE TUMOR SUPPRESSORS RIN AND CCK IN NEUROBLASTOMA CELLS

Tamica Collins (1*), William Weiss (2), and Chris Hackett (2), (1) Jackson State University, Jackson, MS 39217 and (2) University of California San Francisco, San Francisco, CA 94143

Neuroblastoma is a malignant tumor of neural crest origin and is the third most common cancer in children. Overexpression of MYCN in neural crest tissue causes neuroblastoma in mice. RIN and CCK were identified as underexpressed in a genetic screen in mouse neuroblastoma. Our research focused on the overexpression of RIN and CCK, possible tumor suppressors, in neuroblastoma cells. We hypothesized that if RIN and CCK are possible tumor suppressors that they will stop neuroblastoma cells from surviving and proliferating. We co-expressed RIN and CCK with Green Flourescent Protein (GFP) and in two neuroblastoma cell lines, Kelly and SKNBE2. Kelly expresses high levels of MYCN and SKNBE2's express low levels of MYCN. We transiently transfected a packaging cell line (Phx A) and produced retrovirus containing the possible tumor suppressors. We infected the neuroblastoma cells with retrovirus and observed no GFP. We found that the retroviral vectors are getting into the packaging cell lines and GFP is being expressed. We also tested to see if retrovirus was being produced by infecting a kidney cell line (293T) which has a high infection rate. GFP was not expressed which may also indicate that there is no retrovirus present in neuroblastoma cells. We tested a new cell line and obtained negative results. Based on these results we will clone an inducible RIN construct using a topo cloning kit and a pT -Rex-DEST Gateway vector allowing high-Ievel tetracycline-regulated expression of the gene of interest (RIN) in neuroblastoma cells.

9:00 OVEREXPRESSION AND PURIFICATION OF NEURAL CADHERIN

Bolanle Bukoye* and Susan D. Pedigo, Mississippi University for Women, Columbus, MS 39701 and University of Mississippi, University, MS 38677

Cadherins are acidic proteins involved in calcium dependent cell adhesion. Cell adhesion molecules are required to instruct particular cells to remain at one particular site to encourage association with neighboring cells or to prevent these associations and migrate directionally. Cadherins communicate adhesion with neighboring cell to actin cytoskeleton through catenin and other proteins. This adhesion process is homophilic, in which cadherins associate specifically with identical cadherins on neighboring cell surfaces. Cadherins are a large transmembrane protein difficult to study as a whole unit. As a result of this, we have simplified them into minimum functional units that are possible to study. We study a construct of Neural Cadherin with Domains 1 and 2 (NCAD12). Domains are 7 strand barrel structures capable of independent folding.-NCAD12 is capable of calcium binding and assembly into dimeric structures. Calcium binding occurs at the interface between each domain. Each domain is made up of approximately 110 amino acid residues. The goal of the current research is to characterize the calcium binding properties of NCAD12. This protein contains two tryptophan residues, one in Domain 1 and one in Domain 2. This presentation reports studies of the change in CD signal as a function of calcium added to the cuvette. These are the first studies of this sort ever performed on NCAD12.

9:15 Break

9:30 ROLE OF COMPLEX FORMATION OF ANGIOTENSIN II RECEPTOR AT1 WITH INSULIN RECEPTOR BETA-SUBUNIT IN THE DEVELOPMENT OF INSULIN RESISTANCE

Ravindra Kolhe*, Nara Gavini, and Lakshmi Pulakat, Mississippi State University, Mississippi State, MS 39762

Angiotensin II (Ang II) hormone has been implicated in the pathogenesis of insulin resistance and inhibitors of Ang II receptor AT1 are shown to improve insulin sensitivity. Here we show that in the skeletal muscle tissue of SHR rats, Insulin Receptor (IR) beta-subunit forms a complex with the AT1 receptor and co-immunoprecipitates with IR-beta. Such strong AT1-IR association was not observed in normo-tensive rat tissue. To our knowledge this is the first report that shows AT1 can associate with IR-beta in mammalian tissue and that such association might play a role in the regulation of signaling by IR-beta. We further demonstrate that a 2-hour pre-incubation with Ang II (at concentrations 50pM to 1fYM) significantly inhibits 1251-insulin binding to IR in human cell line MCF-7. This effect was not seen when Ang II exposure was performed for shorter periods. The two-hour exposure to Ang II also led to the formation of a protein complex containing AT1 and IR-beta, similar to that seen in skeletal muscle tissue of SHR rats. Both AT1-IR association and differential tyrosine phosphorylation of IR-beta and associated proteins were inhibited when the cells were pretreated with the AT1 antagonist losartan. These observations suggest that continuous presence of Ang II would result in sequestering IR in the AT1-IR complex and prevent IR from binding insulin. It also coincided with differential tyrosine-phosphorylation of IR beta-subunit and associated proteins, than that generated when IR was activated by insulin. Therefore, we infer that conformational alterations in IR caused by AT1-IR-beta association underlie the development of Ang II-induced insulin resistance. Based on these data we propose a model for AT1-mediated insulin resistance that involves receptor level interaction between the AT1 and the IR.

9:45 MULTIPLE CYSTEINE RESIDUES ARE CRITICAL TO JAK2-MEDIATED CATALYSIS

John K. Smith (1), Naila M. Mamoon (1), Kiranam Chatti (2), Sheeyong Lee (1), Kanakadurga Kundrapu (1), and Roy Duhe (1*), (1) University of Mississippi Medical Center, Jackson, Mississippi 39216 and (2) State University of New York, Stony Brook, New York 11794

The redox regulation of JAK2 is poorly understood, and there are contradictory reports as to whether the enzyme's activity is inhibited or stimulated by oxidizing conditions in the cell. Here we demonstrate that multiple cysteine residues within the JAK2 catalytic domain are crucial for enzymatic activity. The enzyme is catalytically inactive when oxidized; activity can be restored via reduction to the thiol state. A series of recombinant variants of JAK2 were overproduced using the baculoviral expression vector system. A truncated variant of JAK2, GST/(N[delta]d661)rJAK2, provided evidence that the amino-terminal autoinhibitory domain was not essential for direct redox regulation and that only nine potentially reactive cysteine residues were involved. The effect of individually and combinatorially altering these nine cysteines was examined via cysteine-to-serine mutagenesis. This identified four cysteine residues in the catalytic domain (Cys866, Cys917, Cys1094 and Cys1105) that cooperatively maintain JAK2's catalytic competency. Our data are consistent with a direct mechanism for redox regulation of JAK2 via oxidation and reduction of critical cysteine residues. Seemingly contradictory reports of oxidative stimulation of JAK2 activity might be best explained through indirect mechanisms involving either the oxidative inhibition of JAK2-interacting biomolecules, such as phosphotyrosine phosphatases, or the oxidative activation of signal transduction pathways which intersect with JAK2.

10:00 REGULATION OF MATRIX METALLOPROTEINASES BY P38 MAP KINASE DURING VESSEL FORMATION IN A THREE-DIMENSIONAL CELL CULTURE SYSTEM

Suja Rajan*, Jianming Ye, Faqing Huang, and Yanlin Guo, University of Southern Mississippi, Hattiesburg, MS 39406

In a conventional culture dish (two-dimensional plastic surface), the major activity of endothelial cells is proliferation with limited morphological change. When cultured in a 3-dimensional (3D) collagen matrix, endothelial cells undergo a series of morphological changes starting with the development of intracellular vacuoles, followed by cell elongation, adjacent cells then coalesce to form tube-like structures. This process mimics the steps of vessel formation in angiogenesis. Using this cell culture as an in vitro angiogenesis model, our previous study has shown that p38 mitogen activated protein kinase (p38) plays multiple roles in controlling cell proliferation, viability and morphogenesis during tube formation. In this study, we investigated the role of p38 in the regulation of matrix metalloproteinases (MMPs). MMPs are secreted enzymes that hydrolyze extracellular matrix (ECM) proteins. Controlled degradation of ECM is critical for cell migration and invasion during blood vessel formation and tissue remodeling. Our results indicated that inhibition of p38 by a pharmaceutical inhibitor SB203580 significantly increased the enzymatic activity of MMP-1 and MMP-9 as determined by zymography analysis and the expression of MMP-1 and MMP-9 as determined by real-time quantitative PCR analysis. These results indicate that p38 plays an important role in the regulation of cell invasion and migration in the 3D cell culture. We are currently using RNA interference method to investigate specific roles of p38[alpha]a and p38[beta], two major p38 isoforms, in the regulation of the expression of MMP-1 and MMP-9. This work is supported by grants from the National Institutes of Heath R15HL081126 and R21HL08273 (YLG).

10:15 CHARACTERIZATION AND FUNCTIONAL ANALYSIS OF A COTTON FIBER GENE ENCODING A RING-TYPE UBIQUITIN LIGASE

Meng-Hsuan Ho* and Din-Pow Ma, Mississippi State University, Mississippi State, MS 39762

The ubiquitin-proteasome proteolysis pathway, responsible for the degradation of abnormal and short-lived proteins, regulates a wide variety of cellular processes in eukaryotes. The ubiqination of target proteins for degradation requires sequential action by three enzymes: an ubiquitin-activating enzyme (E 1), an ubiquitin conjugating enzyme (E2), and an ubiquitin ligase (E3). In plants, the RING type ubiquitin E3s are classified into two groups, single subunits and multiple subunit E3s. The multiple SCF E3s contain four subunits: Skp1, cullin1, Rbx1, and an F-box protein. It has been reported that there are 469 RING type protein genes and about 700 F-box protein genes in the Arabidopsis genome. The presence of a large number of RING type and F-box proteins in Arabidopsis suggest that plant ubiquitin E3s have many specific target substrates. Using the cotton E2 (GhUBC1) as bait in a bacterial two-hybrid system, we have cloned two unique fiber cDNAs, GhRINI and GhRIN2, encoding RING type ubiquitin ligases. The GhRIN1 protein derived from the full-length GhRIN1 cDNA contains 338 aa and has the highest homologies with E3 homologs from Arabidopsis (AAN18152 encoded by AT3g19950) and rice (Oryza sativa) (BAD67937) by Blast searches. The real time RT-PCR data reveals that GhRIN1 is highly expressed in fiber in a developmental manner. Our experimental results suggest that protein ubiquitination occurs in fiber cells and the ubiquitin-proteosome pathway may control fiber development and differentiation.

10:30 Break

10:45 CDC20 LINKS NUCLEOTIDE EXCISION REPAIR TO CELL CYCLE PROGRESSION IN SACCHAROMYCES CEREVISIAE

Lauren Rochelle, Mohamed Hajj, Kate Goodwin, and Bernadette Connors*, Millsaps College, Jackson, MS 39210

Considering the importance and universality of our genetic material, its accurate replication and transmission are vital to the survival of cells and the more complex organisms they compose. Faulty DNA replication and repair can lead to severe human diseases such as xeroderma pigmentosum, a disorder characterized by extreme photosensitivity and a greater than 1000-fold increased risk of cutaneous and ocular neoplasms. This study intends to identify the link between cell cycle progression and nucleotide excision repair (NER). NER mechanisms halt the cell cycle upon detection of single and double stranded DNA breaks caused by exposure to UV radiation. Our studies have suggested a link between cdc20p, a known regulator of mitotic exit, and NER. Drug toxicity assays in Saccharomyces cerevisae revealed that selected NER-defective mutants with diminished cdc20p activity are more UV sensitive than either single mutant alone. Overexpression of cdc20p rescued the UV sensitivity of selected mutants, namely, rad2- and rad4-defective strains. Microscopic examination of rad2-GFP revealed that its expression is elevated in G1/S phases, while end-joining experiments demonstrated a novel role of cdc20p in the cell's ability to repair damaged DNA in rad4-defective mutants. Future work will involve a dissection of the physical relationship between NER proteins and cdc20p, as well as other proteins that regulate mitotic exit. 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.

11:00 THE S. CEREVISIAE KIN3 GENE INTERACTS WITH A DNA DAMAGE REPAIR GENE

Terrel Sugar*, Yulon Stewart, and Sarah Lea McGuire, Millsaps College, Jackson, MS 39210

The Saccharomyces cerevisiae KIN3 gene encodes a nonessential protein kinase whose function is not well understood in budding yeast. It is a member of a heterogeneous family of protein kinases, the NIMA family, most of which are involved in mitotic progression. To understand the functions of this protein kinase in budding yeast, we have generated a KIN3 deletion strain and characterized the effects of this deletion on the cells. Deletion cells are larger, with nearly twice the cell volume compared to non-deleted cells. We are performing FACS analysis to determine if these cells are delayed during cell cycle progression, which could lead to larger cell size. The deletion strain exhibits a resistance to the microtubule poison benomyl, and sensitivity to the microtubule poison nocodazole, suggesting that KIN3 may be involved in mitotic spindle function. In addition, we have completed synthetic genetic analysis and identified several genes that interact with KIN3. Most notably, a strain with a deletion in MMS22, a DNA repair gene, exhibits a synthetic defect in combination with a KIN3 deletion. Deletion of KIN3 abrogates the MMS sensitivity of an MMS22 deletion, suggesting that KIN3 may be involved in DNA damage repair or in the DNA damage checkpoint pathway.

11:15 DNA MICROARRAY BASED TRANSCRIPTIONAL PROFILE OF MSA MUTANT OF S.AUREUS

Vijayaraj Nagarajan* and Mohamed O.Elasri, University of Southern Mississippi, Hattiesburg, MS 39406

Staphylococcus aureus is an important pathogen that causes severe infections in humans. S. aureus produces a large number of virulence factors and possesses complex regulatory networks. Previously we identified a novel gene, msa that is involved in regulating several virulence factors. We also showed that msa modulates the activity of the global regulator sarA. In this report, we studied the effect of msa mutation at global level, by analyzing the DNA microarray based transcriptional profile of the msa mutant. Results showed that msa strongly regulates genes related to transport/binding and cell envelope. Several virulence factors including global regulators were also affected by msa. These results support our hypothesis that msa mediates interaction of S.aureus with its environment.

11:30 MULTIDIMENSIONAL PROTEIN IDENTIFICATION TECHNOLOGY-BASED PROTEOGENOMIC MAPPING OF CHANNEL CATFISH VIRUS GENOME

Dusan Kunec*, Bindu Nanduri, and Shane C. Burgess, Institute for Digital Biology, Mississippi State University, Mississippi State, MS 39762

Viruses have overlapping and spliced ORFs, small ORFs and often use alternate translational start sites meaning that accurate annotation of viral genomes using computational gene prediction not possible. Proteogenomic mapping, an independent and complementary experimental measure of ORF expression, can be used for improving genome annotation. Channel catfish virus (CCV) is a simple comparative model for understanding herpesvirus biology and evolutionary relationships within the family Herpesviridae. The canonical CCV genome sequence currently has 76 predicted ORFs. Here we have developed a novel statistical method for assigning probabilities to peptide identifications by mass spectrometry and produced a novel proteogenomic map of the canonical CCV genome sequence. This statistical method allows users to explore proteomics data at user-defined levels of confidence. Traditionally trypsin is used as a protease for proteomics. But many proteins when digested using trypsin do not produce peptides within the size limitations to be identified by mass spectrometry or to be useful as unique identifiers. Also tryptic peptides cannot overlap. To generate much greater proteome coverage, to yield overlapping peptides and to further improve confidence we used partial proteinase K digestion. At P<0.05 we identified 27/76 previously predicted ORFs using trypsin and 55/76 with proteinase K (25 were common to both enzymes). Furthermore, we identified 258 unpredicted peptides, which in turn identified 78 novel ORFs. Our data more than doubles the annotated protein coding capacity of the CCV genome.

11:45 KNOCKING OUT CHAPERONE PROTEIN GENES IN [URE3] STRAINS OF SACCHAROMYCES CEREVISIAE

Katie Brinkman* and Ross E Whitwam, Mississippi University for Women, Columbus, MS 39701

The [URE3] prion of baker's yeast, Saccharomyces cerevisiae, is a misfolded protein capable of misfolding properly-folded versions of the same protein in the same cell. Continued propagation of yeast prions requires the presence of several chaperone proteins in yeast cells, including the Hsp104 protein and the Ssal protein. We wish to investigate whether these chaperone proteins are also required for the establishment of the [URE3] state, as opposed to its propagation. We are knocking out the HSP104 and SSA1 genes in prion-free strains of yeast. Gene knock is being accomplished by targeting the nourseothricin acetyl transferase (NAT) gene to the loci of interest and selecting for nourseothricin resistance in transformants. The knock-out strains will be used to investigate how the absence of those genes affects rates of spontaneous [URE3] formation in yeast.

THURSDAY AFTERNOON

Bost Auditorium South

1:30 RATES OF SPONTANEOUS FORMATION OF THE [URE3] PRION IN CULTURES OF SACCHAROMYCES CEREVISIAE

Michael Lee* and Ross E Whitwam, Mississippi University for Women, Columbus, MS 39701

The [URE3] prion of Saccharomyces cerevisiae is a misfolded form of the Ure2 protein that forms amyloid fibers and can be transmitted to mating partners and offspring. It serves as a useful model for mammalian prions and amyloidoses. We investigated how rates of spontaneous [URE3] formation in prion-free yeast varied during the growth of yeast cultures. Both prion-free yeast and isogenic [URE3] strains showed essentially identical growth curves in YPD medium, suggesting that the prion is not deleterious to growth under high-nutrient conditions. 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. These results suggest that other cellular factors whose levels vary during culture growth are necessary to establish the stable [URE3] state.

1:45 MODELLING OF CD[30.sup.HIGH] LYMPHOMAS USING MAREK'S DISEASE IN CHICKEN AS A NATURAL ANIMAL MODEL

Shyamesh Kumar*, Joram Buza, Bindu Nanduri, Shane C. Burgess, and Shyamesh Kumar*, Institute for Digital Biology, Mississippi State University, Mississippi State, MS 39762

Lymphomas are the 6th leading cause of human death due to cancer in the USA. A diverse range of Hodgkin's and many Non-Hodgkin's lymphoma over-express the CD30 antigen. The CD[30.sup.+] cells are neoplastic transformed cells and control lymphoma formation. Marek's disease(MD) in chicken is the only naturally occurring model for CD[30.sup.+] lymphomas. All chickens are susceptible to MDV infection but MD develops only in susceptible genotypes. MD progression can be divided into cytolytic, latent, and tumor phases. The neoplastic-transformed cells (CD[4.sup.+]CD[30.sup.+]) develop during the cytolytic phase in both the susceptible and resistant genotypes but only in MD-susceptible genotypes do gross lymphomas develop. During the cytolytic and latent phases of MDV infection in both genotypes, most lymphoma-infiltrating cells are CD[4.sup.+] T cells. However, after this stage (21dpi) lymphomas increase in size only in susceptible genotypes and most infiltrating cells are CD[4.sup.+] T cells. Conversely, in resistant chicken genotypes lymphomas decrease in size and most infiltrating cells are CD[8.sup.+] T cells. We hypothesize that the proteome of MDV-transformed cells is altered resulting in aberrant signaling pathways. We also hypothesize that differences exist between the resistant and susceptible genotypes in 1) the profile of cytokine within lymphoma that promote either CD[8.sup.+] T-cell mediated immunity or proliferation of CD[4.sup.+] T and 2) peptides presented to cytotoxic T-lymphocytes (CTL) such that only resistant genotypes load lymphoma-protective epitopes that activate CTL killing of lymphomas. The CD[30.sup.+] and CD[30.sup.-] cells from gross lymphomas in susceptible genotypes will be isolated by magnetic cell sorting, fractionated by differential detergent fractionation (DDF) and proteins identified by 2D LC-MS/MS. Protein functional analysis will be performed using gene ontology (GO) and pathway analysis softwares. Tissue sections from microscopic lesions at 21 dpi will be sectioned by laser capture micro-dissection (LCM) and the mRNA will be assayed for Th1 and Th2 using RT-PCR to obtain information on possible role of cytokines in lymphoma progression or regression. MHC-I and the associated peptides will be co-immunoprecipitated and the peptides identified by 2D LC-MS/MS to facilitate comparison of peptides presented to CTL by susceptible and resistant chicken genotypes.

2:00 DELETION AND EPITOPE-TAGGING OF CELL CYCLE GENES USING UNCLONED PCR FUSION PRODUCTS AND HOMOLOGOUS RECOMBINATION IN ASPERGILLUS NIDULANS

Marlena Mattingly (1*), Megan Jackson (2), David Tuck (3), and P.M. Mirabito (4), (1) Belhaven College, Jackson, MS 39202, (2) Berea College, Berea, KY 40404, Western Kentucky University, Bowling Green, KY 42101, and (4) University of Kentucky, Lexington, Kentucky 40506

Three standard approaches to investigate gene function are 1) inactivate the gene (e.g. gene knockouts) and determine the effect on the organism; 2) determine the cellular and subcellular location of the protein; and 3) determine with which other proteins the protein of interest physically interacts. The key to all three approaches is the ability to replace the endogenous gene with altered forms created in vitro (gene replacement). Although gene replacements are the "industry standard", they have been laborious and time consuming in all eukaryotes except budding yeast (Saccharomyces cerevisiae). Recent technological advances have made gene replacements in several fungi as facile as with yeast. Here we apply this technology to initiate the investigation of six Aspergillus nidulans genes implicated in cell cycle regulation. Five are hypothesized to function with the Anaphase Promoting Complex/Cyclosome (aka APC/C), which is an ubiquitin ligase that regulates multiple cell cycle events. Two, afrA and afrB, are implicated as cell cycle-stage-specific activators of the APC/C. Three, ubc3, ubc4, and ubc11, are implicated as ubiquitin conjugating enzymes required for APC/C function. The last, sv9, was originally identified as required for nuclear division but has since been implicated in lipid metabolism. We report the successful deletion (knock-out) of all six genes, three of which are essential (afrA, ubc4, and sv9). We also report the isolation of A. nidulans strains that probably contain epitope-tagged versions of the genes for use in future cytological localization and protein-interaction studies.

2:15 MSU'S INSTITUTE FOR DIGITAL BIOLOGY: A MISSISSIPPI RESOURCE FOR POST GENOMIC BIOLOGY

Shane C. Burgess* and Susan M. Bridges, Institute for Digital Biology, Mississippi State University, Mississippi State, MS 39762

The MSU Institute for Digital Biology (IDB) is a new multidisciplinary institute that combines MSU's strengths in post-genomic life sciences and computation. Supported from the "grassroots" we leverage our campus-wide expertise in multidisciplinary interdependent ways. We would like to expand our collaborations statewide to help to promote Mississippi's economic growth. The IDB aims to move the traditional academic triad of research, teaching and service to discovery, learning, and engagement. Discovery work at the IDB aims to improve the human condition by improving health, food safety, food production and quality, bio-fuels, and bio-security. Discovery work is funded by grants from the National Science Foundation, the National Institutes for Health, the United States Department of Agriculture, the Office of Naval Research, and the Department of Energy. We will discuss some examples of discovery work and our also our learning community.

2:30 A JACKPOT TEST OF THE SPONTANEOUS FORMATION OF THE [URE3] PRION IN SACCHAROMYCES CEREVISIAE

Mary Oyeleye* and Ross E Whitwam, Mississippi University for Women, Columbus, MS 39701

Prions are misfolded and infectious forms of cellular proteins. They are capable of propagating themselves by inducing properly-folded forms of the same protein to misfold. 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 performed a variation of Luria and Delbruk's jackpot test on the rates of spontaneous [URE3] formation in prion-free yeast. When multiple samples from the same culture were plated on a medium that selects for [URE3]-containing cells, the mean number of [URE3] cells per sample was ten-folded lower than the variance, suggesting divergence from a Poisson distribution. However, when samples from multiple individual cultures were plated on the selective medium, the variance in the number of [URE3] cells per sample was an order-of-magnitude higher than the variance seen in the samples from the individual culture. We believe this supports the hypothesis that, while misfolding of the Ure2 protein is a random event, establishment of the self-propagating [URE3] state is dependent on other cellular factors which are not consistently present.

2:45 Break

3:00 ROLE OF MSA IN REGULATING BIOFILM FORMATION IN STAPHYLOCOCCUS AUREUS

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

Staphylococcus aureus is a 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 (osteomyelitis, endocarditis). The ability of this pathogen to form biofilms plays an important role in the establishment of infection. S. aureus expresses a wide variety of virulence factors that can be classified as either cell bound proteins (e.g., adhesins) or exoproteins (toxins). 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 that regulates expression of several virulence factors according to cell density. sarA is a second global regulator that controls the expression of virulence factors in two pathways, an agr-dependent pathway and an agr-independent pathway. We have identified and characterized a new gene, msa, that modulates the expression of SarA and several virulence factors. We have also discovered that msa plays a role in biofilm formation. Mutation of msa results in delayed biofilm formation in vitro and altered the expression of several biofilm forming genes.

3:15 SITE SPECIFICITIES OF HENNEGUYA EXILIS IN CHANNEL CATFISH (ICTALURUS PUNCTATUS)

Angela P. Brandon (1*), Linda M. Pote (1), and Andrew Goodwin (2), (1) Mississippi State University, Mississippi State, MS 39762 and (2) University of Arkansas, Pine Bluff, AR 71601

Myxozoans are common metazoan parasites of marine and freshwater fish. Previous research identified myxozoans based on their morphological characteristics and site preference within the fish host. Recently with the advent of molecular techniques such as PCR and genomic sequencing, traditional identification based on morphology and location in the host has been shown to be inconsistent. Tumor-like growths containing Henneguya sp. myxospores were observed on the caudal fins and kidneys of naturally infected channel catfish (Ictalurus punctatus). Six samples of caudal fin lesions and two kidney lesions were isolated from fish collected in Mississippi and Arkansas. Each of these samples contained myxospores morphologically resembling Henneguya exilis. DNA was isolated from these spores and the 18s rDNA was amplified with generic Henneguya spp. primers and sequenced. The results indicated that these spores were H. exilis. Currently, H. exilis has not been reported in tissues other than the interlamellar troughs of the gills of channel catfish, making the discovery of these spores in the caudal fin and kidney a new site preference for this organism in catfish. This confirms that H. exilis can infect organs other than the gills in channel catfish.

3:30 EXPRESSION ANALYSIS OF THE MOLD-SPECIFIC M46 GENE IN FIVE STRAINS OF THE PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM

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

Histoplasma capsulatum (Hc) is the etiologic agent for the respiratory disease histoplasmosis which afflicts an estimated 500,000 Americans each year. This dimorphic fungus grows in the soil as a multicellular mold. Once the soil is disturbed, spores or mold fragments are aerosolized and are inhaled into the lungs where the organism shifts to a unicellular yeast growth form. Without this mold-to-yeast conversion disease cannot occur. To understand the molecular basis of dimorphism, we have isolated several mold-specific and yeast-specific genes. The subject of this study is the mold-specific M46 gene. Northern blot analysis has shown that M46 is expressed in two strains of Hc (G186AS and Downs strains), but is transcriptionally silent in two other strains (G184AS and G217B strains). Recent analysis of WU24, which like the Downs strain is in the NAm1 class of H. capsulatum, has shown that M46 is expressed only in the mold morphotype. Sequence analysis has shown that the complete M46 gene is present in all of these strains. Therefore we have constructed M46 promoter-GFP fusions to determine if the promoters from the two non-expressing strains are nonfunctional or if these two strains are defective in some trans-regulating factor(s). Fluorescent microscopy data has shown that M46 promoters from all four strains were functional. This analysis allows us to hypothesize that M46 is not transcriptionally silent in strains G184AS and G217B due to a non-functional promoter but due to a missing trans regulating factor(s). Future work will focus on identifying any missing trans regulating factors that may be causing transcriptional silence in the latter strains.

3:45 THE CO-EXPRESSION OF AMPKA, AMPKB, AND AMPK[GAMMA] FROM TRYPANOSOMA BRUCEI

Jessica Lewis (1*), James C. Morris (2), and Meredith T. Morris (2), (1) Mississippi University for Women and (2) Clemson University, Clemson, SC 29634

Trypansoma brucei is a protozoan parasite with a complex life cycle which involves the tsetse fly host and various mammalian hosts. In the mammalian blood stream, the parasites express variable surface glycoproteins. The coordinated expression of these surface molecules is essential to the survival of the parasite. In T. brucei, adenine monophosphate-activated protein kinase (AMPK) plays a role in the regulation of surface molecule expression. AMPK consists of three subunits: AMPK[alpha], AMPK[beta], and AMPK[gamma]. We have identified candidate open reading frames (ORFs) that might encode the three AMPK subunits. However, we need to demonstrate that these open reading frames express gene products that function as the heterotrimeric AMPK complex. We have cloned the putative AMPK[gamma] and AMPK[beta] ORFs into a pQE30 expression vector. When the expression construct was transformed into E. coli, both ORFs were expressed, but the protein products were insoluble. Work is continuing to clone the putative AMPK[alpha] ORF into the expression vector with the putative AMPK[gamma] and AMPK[beta] ORFs and to express and purify the protein complex in E. coli in its functional heterotrimeric form.

FRIDAY MORNING

Bost Auditorium South

8:30 THE ROLE OF CSOS2 PROTEIN IN THE CARBOXYSOME OF HALOTHIOBACILLUS NEAPOLITANUS

Zhicheng Dou*, Jessup M. Shively, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi. Hattiesburg, MS, 39406

Carboxysomes are biological nanoreactors in some photo- and chemoautotrophic prokaryotes which can transform inorganic C[O.sub.2] into metabolizable sugars. CsoS2 protein is one of the carboxysome shell proteins and it exists in two physically distinct forms of approximately 85 and 130 kDa, respectively. It is our goal to understand what is the difference between these two protein products CsoS2A and CsoS2B and their roles in the self-assembly processes of carboxysomes in vivo to ultimately utilize that knowledge for the development of biopolymer based materials. To obtain the requisite large amounts of CsoS2, the csoS2 gene was inserted into the pPROEX and IMPACT prokaryotic protein expression vectors and recombinant CsoS2 protein was produced. In both expression systems, CsoS2A and CsoS2B were expressed. However, only CsoS2B can be purified in the IMPACT system because of its C-terminal tag, while CsoS2A protein doesn't have the C-terminal tag by the immunoblotting analysis. The polyclonal antibody against CsoS2B protein can recognize these two polypeptides, which indicate these two variants have the similar epitopes. So, through these experimental results, we conclude that CsoS2B protein encodes the full length CsoS2 protein and CsoS2A may be a truncated form of CsoS2 protein. The mechanism of CsoS2 protein expression pattern is being investigated. Simultaneously, various strategies are being explored to increase solubility and concentration of purified recombinant CsoS2 protein in preparation for X-ray crystallography studies.

8:45 REGULATION OF ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS

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

Infections caused by hospital and community-acquired Methicillin-Resistant Staphylococcus aureus (MRSA) have been on a steady rise. The mec operon encodes for Penicillin Binding Protein 2a (PBP2a) that has decreased binding affinity for [beta]-lactam antibiotics. Results of a recent study from our group suggest that a novel regulatory protein Msa (Modulator of SarA) may be associated with methicillin resistance in S. aureus. In this study, we hypothesize that Msa regulates the expression of the mec operon. MIC of methicillin was reduced by several fold in the msa mutant compared to the wild type. Further analysis using quantitative real-time PCR (qRT-PCR) showed undetectable levels of mecA expression in the mutant. Protein levels in the wild type, mutant and complement strains were evaluated using custom-made anti-PBP2a polyclonal antibody. Results of this study suggest that Msa is involved in antibiotic resistance in S. aureus.

9:00 SITE DIRECTED MUTAGENSIS OF THE csoS3 GENE OF HALOTHIOBACILLUS NEAPOLITANUS

Sara Johnson *, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406

Carboxysomes are proteinaceous microcompartments that play an integral part in the metabolism of cyanobacteria and many chemolithotrophic bacteria. These polyhedral inclusions are filled with the central C[O.sub.2] fixing enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and enhance the catalytic ability of RuBisCO by contributing to a C[O.sub.2] concentrating mechanism (CCM). The CCM acts to increase the concentration of C[O.sub.2] within the carboxysome, driving C[O.sub.2] fixation forward by mass action. A carbonic anhydrase (CA) believed to create C[O.sub.2], the substrate of RuBisCO, from abundant cytosolic HC[O.sub.[3.sup.-]], has been found within the shell of carboxysomes in the chemolithoautotrophic sulfur bacterium Halothiobacillus neapolitanus. Using x-ray crystallography, the carboxysomal CA (CsosCA) was determined to be a member of a new subclass of [beta]-CA, containing one biologically relevant zinc-binding active site. A mechanism has been proposed involving three amino acid residues that serve as zinc ligands and are conserved in all [beta]-CAs. To determine the importance of the conserved residues, site-directed mutagenesis of one of the zinc-binding residues (Cys 253) was attempted using PCR techniques. Stop-flow analysis of the recombinant protein will be used to determine the effect the mutation has on the enzymatic activity of the protein. The activity will then be compared to that of wild-type CsosCA to determine the importance of the mutated residue in the process of C[O.sub.2] hydration and HC[O.sub.3]- dehydration.

9:15 FLUORESCENCE STUDY OF THE INTERACTION BETWEEN THE TAT-PTD PEPTIDE AND THE LIPID BILAYER

Venkataswarup Tiriveedhi* and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406

The mechanism of internalization of cationic peptides into the cells is unknown. We used fluorescence spectroscopy to study the interaction between Protein Transduction Domain of the HIV-1 TAT protein (TAT-PTD; residues 47-60 of TAT, fluorescently labeled with tryptophan) and the lipid bilayer in the form of small unilamellar lipid vesicles [SUV]. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity and increase in anisotropy upon the interaction with SUV, which was proportional with the negative charge density in the membrane. Kinetic analysis of the interaction showed two apparent dissociation constants. Kd1 was independent of the negative charge density and accounted for 24% of the interaction, whereas Kd2, contributing 76% to the interaction, decreased linearly with the density of negative charge in the membrane, suggesting an electrostatic nature of the latter interaction. The former could not be inhibited by high salt, suggesting its van der Waals or hydrophobic nature. TAT-PTD did not dissipate membrane potential (165 mV, negative inside), nor did it affect fluorescence anisotropy of the membrane fluorescence probes TMA-DPH or DPH, indicating preserved membrane integrity upon TAT-PTD binding. When the pyrene-labeled phosphatidyl glycerol was included in the membrane, TAT-PTD induced pyrene excimer formation, but only at the temperature above the melting point of the lipid. Thus, the two prerequisites for a strong binding of TAT-PTD to the lipid membrane are the presence of the negative charge and the liquid crystalline phase of the lipid bilayer.

9:30 CONSTRUCTION AND MODIFICATION OF A SINGLE-CHAIN FV ANTIBODY BIOSENSOR FOR THE DETECTION OF SELECTED BACTERIA

Scott Walper*, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406

The specificity of antibody molecules for their target antigens can be a utilized to generate a biosensor for the detection of ubiquitous pseudomonas species. Given the difficulties associated with expression of full-length IgG molecules in both bacterial and plant cultures, a single-chain antibody fragment (scFv) will be constructed and transformed into a bacterial culture to allow for high level expression and rapid assessment of gene mutagenesis. Once isolated, multiple scFv will be combined into a single gene construct to allow for either improved sensitivity of detection, or to add specificities for other antigens giving the biosensor multi-functionality of detection.

9:45 CHARACTERIZATION OF THE SHELL PROTEINS OF MICROCOMPARTMENTS INVOLVED IN ETHANOLAMINE CATABOLISM IN SALMONELLA ENTERICA

Balaraj B. Menon*, Andrea G. Marshall, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406

Bacterial microcompartments and related polyhedral organelles that are assembled in response to varying environmental cues have been observed and characterized in several chemolithoautotrophic systems. Salmonella enterica serovar Typhimurium, the causative agent of a form of human gastroenteritis, synthesizes one such bacterial microcompartment that aids in the catabolism of ethanolamine, a substrate found in the human gut. The ethanolamine utilization (eut) operon, which is induced under aerobic growth conditions in the presence of ethanolamine and cyanocobalamin, is comprised of seventeen genes, five of which encode the shell proteins EutK, -L, -M, -N, and -S. All of these proteins bear significant homology to known carboxysomal shell proteins of autotrophic bacteria. Aside from limited genetic studies aimed at dissecting the biochemical role of these microcompartments in ethanolamine catabolism, very few studies pertaining to the structural understanding of microcompartment assembly have been attempted. The primary focus of this study is thus targeted at characterizing the architecture of microcompartments involved in ethanolamine catabolism. Crystallographic analyses, protein-protein interaction studies, and reconstitution experiments using recombinant shell proteins are underway. To this end, His6-tagged recombinant versions of all putative Eut shell proteins have been purified.

10:00 CLONING AND EXPRESSION OF THE EUTN GENE PRODUCT FROM SALMONELLA ENTERICA

Andrea G. Marshall*, Balaraj B. Menon, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406

Salmonella enterica serovar Typhimurium is a pathogen that resides in the gut of both wild and domestic animals. Ingestion of contaminated meats such as beef and poultry is the most common route for S. enterica infection in humans. Under aerobic conditions S. enterica can utilize ethanolamine, a chemical found in the human gut, as a sole source of carbon, nitrogen, and energy. When grown on ethanolamine, the bacterium forms polyhedral organelles that are similar in appearance to the carboxysomes found in certain photosynthetic and chemolithautotrophic bacteria. The 17 gene ethanolamine utilization (eut) operon of S. enterica contains five genes that are believed to code for the shell proteins of the polyhedral organelle, based upon homology of their primary sequences with those of known carboxysomal shell proteins. Although genetic-studies have been performed to elucidate the role of these bacterial microcompartments in the catabolism of ethanolamine, few studies have addressed the structure of the proteins that compose the organelle. The gene of one of these proteins, eutN, was amplified by polymerase chain reaction (PCR) and purified using gel electrophoresis. The gene was then ligated into the pProEX HTb expression vector and the construct used to transform competent Escherichia coli cells. The insert was confirmed by DNA sequencing. Conditions for protein expression were optimized for high yield of recombinant EutN protein. Future studies will involve crystallographic analyses, protein interaction studies, and antibody development.

10:15 Divisional Business Meeting

FRIDAY AFTERNOON

Bost Auditorium North

3:00 Divisional Poster Session

FLUORESCENCE RESONANCE ENERGY TRANSFER STUDIES ON THE INTERACTION BETWEEN PAMAM DENDRIMER AND THE LIPID MEMBRANE

Venkataswarup Tiriveedhi (1), Shehzeen Shams (1*), Kelly M Kitchens (2), Hamid Ghandehari (2), and Peter Butko (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) University of Maryland, Baltimore, MD 21201

Dendrimers, highly branched complex macromolecules, have been of particular interest in the biomedical field for their extensive use in the intracellular drug delivery. The exact mechanism of penetration of these highly positively charged macromolecules through the hydrophobic core of the lipid bilayer is not known. However, various yet to be proven theories have been put forward like inverted micelle formation or adsorptive endocytosis. We studied the interaction of the fluorescein-labeled PAMAM (polyamidoamine) dendrimers (G1, first generation, Mr = 1430 with eight positive charges on the surface) with negatively-charged small unilamellar lipid vesicles (SUV) composed of egg phosphatidylcholine/phosphatidylglycerol/lissamine-rhodamine-phosphatidylethanolamine (73:25:2). When the fluorescent dendrimer was mixed with the rhodamine labeled SUV, fluorescence resonance energy transfer was observed between the fluorescein donor and the rhodamin acceptor with efficiency of approximately 60%. These data were useed to calculate the average distance between the two fluorophores in our system, which was found to be 4.8 [+ or -] 0.3 nm. These results are consistent with the hypothesis that the dendrimer attaches to the surface of the lipid vesicle and does not significantly penetrate through the hydrophobic core of the lipid bilayer

EFFECTS OF VALPROATE AND ALCOHOL ON CADHERIN EXPRESSION

Brittney Tate*, and Ujjwal K. Rout, University of Mississippi Medical Center, Jackson, MS 39216

Exposure to valproate or alcohol during pregnancy results in neurobehavioral problems including autism in the offspring. Although it is apparent that these teratogens disturb the normal development of the brain, the mechanism of this phenomenon is not completely known. Adhesion molecules play critical role in the development of the brain. Therefore, it is possible that these teratogens may alter the expression and function of adhesion molecules in the neuronal cells causing their abnormal proliferation, differentiation and migration in the developing brain. In the present investigation, neuron-like PC12 cells were used as an in vitro model to examine the effects of valproate and alcohol on the expression levels of adhesion molecule N-Cadherin that regulate cell-cell interactions. Both high and low concentrations of valproate and alcohol were used for the study. Concentrations of Nerve Growth Factor that cause differentiation of PC12 cells into neuronal phenotype were also varied. Cell lysate from untreated and treated cells were subjected to protein concentration determination and Western Blotting analysis to examine the expression levels of N-Cadherins using Alpha Imager software. Results show that both valproate and alcohol changes expression levels of N-Cadherin in PC12 cells. Presence of NGF also influenced valproate and alcohol induced changes in the N-Cadherin expression. Our data indicate that exposure to valproate and alcohol during pregnancy may alter brain development by changing cell-cell interactions mediated by N-cadherins. Supported by a grant from NIH (NIH/NCRR P20 RR017701).

THE CsoS1 PROTEINS AND OrfA/B: MAJOR AND MINOR COMPONENTS OF THE CARBOXYSOME SHELL.

Fei Cai (1*), Sabine Heinhorst (1), Jessup M. Shively (2), and Gordon C. Cannon (1), (1) University of Southern Mississippi, MS 39406 and (2) Clemson University, Clemson, SC 29634

Carboxysomes belong to a family of microcompartments or biological nanoreactors that are found in many autotrophic bacteria and serve to facilitate bacterial C[O.sub.2] fixation. The polyhedral particles consist of a proteinaceous shell and are filled with the enzyme that catalyzes the C[O.sub.2] fixation reaction. To understand the molecular mechanism by which carboxysomes enhance RubisCO catalysis, efforts are underway to determine the three-dimensional structure of carboxysomes in the model bacterium Halothiobacillus neapolitanus. All eight polypeptide components of the carboxysome have been expressed successfully in Escherichia coli and were purified via affinity chromatography. Crystal structures have been obtained for the major shell components CsoS1A, CsoS1B and CsoS1C, which share over 90% amino acid sequence identity. Crystal packaging and biochemical characteristics suggest that these proteins form hexameric units that may well represent protomers in the carboxysome assembly pathway. Two-dimensional gel electrophoresis combined with immunoblotting indicated that the proteins OrfA and OrfB, although not abundant carboxysome components, are present in the shell. X-ray diffraction studies of OrfA and OrfB are currently underway to determine potential higher order (multimeric) structures formed by these polypeptides that might provide clues about their location and function in the carboxysome shell.

CHARACTERIZATION OF PHOSPHORYLATED PEPTIDES USING NANOSPRAY IONIZATION MASS SPECTROSCOPY

Jason Adams* and Bettye Sue Hennington, Tougaloo College, Jackson, MS 39174

Tandem Mass Spectroscopy (MS/MS) is used to obtain the amino acid sequence of proteins. a-casein was selected as a standard and was predicted to have several sites of phosphorylation. It was expected that a minimum of two phosphorylated peptides would be identified along with the exact sites of phosphorylated amino acids. An in-solution trypsin protocol was used and the resulting a-casein peptides were loaded into an Fe++-activated Immobilized Metal Affinity Column (IMAC) that was used to capture the phosphorylated peptides. The peptides were eluted onto a C-18 column, analyzed with MS and MS/MS and analyzed Bioworks. One quadruply-phosphorylated peptide of a-casein was detected. The spectra also showed that the peptide lost up to four phosphates when it was exposed to Collision Induced Dissociation during MS/MS. Our a-casein findings matched the information that has been previously reported by other mass spectrometer labs. Once it was determined that we could capture and detect phosphorylated peptides and determine the phosphorylation sites, Rap 1, a yeast transcription factor, was isolated from Saccharomyces cereviseae using 1-D electrophoresis and Western blot techniques. After trypsinization, Rap 1 peptides were loaded onto the C-18 column and eluted. No phosphorylated peptides were detected. Bioworks confirmed the presence of a yeast transcription factor, but did not comfirm that it was Rap 1.

CONSTRUCTION OF A MINIMAL HETEROLOGOUS URA5-COMPLEMENTING SHUTTLE VECTOR FOR USE IN THE DIMORPHIC PATHOGENIC FUNGUS, HISTOPLASMA CAPSULATUM

Madhavi Patel* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406

Approximately 40 million Americans have contracted the respiratory mycotic disease, histoplasmosis. This disease is caused by an infection with the fungus Histoplasma capsulatum (He). This fungus is a dimorphic parasitic species that grows naturally in damp soil in a multicellular saprophytic mycelial (mold) form at 25[degrees]C. The species is capable of undergoing a morphological shift from the environmental multicellular mold form to a unicellular pathogenic yeast in the infected host (or in the lab at 37C). Because the mold-to-yeast shift is an absolute requirement for disease, an understanding of the molecular genetics of this dimorphism is vital to control this important disease. A critical roadblock for current molecular biology research is the lack of a small, facile shuttle vector to study dimorphism associated genes. Therefore, we have constructed a vector with the smallest functional URA5 marker (AnURA5) from the fungus Aspergillus nidulans. The AnURA5 gene was first resected to identify the smallest promoter that maintained full expression in Histoplasma capsulatum. These experiments demonstrated that a 1.4 kb AnURA5 marker was the smallest fragment maintaining high transformation efficiency. This heterologous marker was cloned into a Hc telomere vector for future use in genetic knockout experiments. Interestingly, the resection experiments showed that removing approximately 200 bp of 5' promoter sequence abolished promoter function while removal of 400 bp or more restored function. Work is ongoing to confirm this observation in other strains of Histoplasma and to determine the cause of this loss of function.

LIPID SPECIFICITY OF THE BT TOXIN/MEMBRANE INTERACTIONS

Kerrick Nevels (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

Cyt1 A is a cytolytic toxin produced by the spore-forming bacterium Bacillus thuringiensis var. israelensis and is used in insecticide preparations. Understanding the molecular details of how the toxin changes conformation in the presence of lipid membrane is important for elucidating the toxin's mode of action. Previous binding studies were performed using membranes made of chemically undefined lipid egg PC. Here we studied lipid specificity of Cyt1 A binding regarding saturation of fatty acy1 chain and chemical nature of the lipid head-group. Fluorescence of tryptophan was used as a measure of binding. Results show that Cyt1 A binds to the membranes made of the saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) to a similar extent as to those made of the egg PC lipid mixture. This suggests that Cyt1 A has no preference for binding to vesicles composed of either saturated or unsaturated lipids. Results from the experiments designed to study effects of lipid fluidity on Cyt1 A binding will also be presented.

EVALUTION OF ACTIVITY OF CRUDE EXTRACT OF OCIMUM GRATISSIMUM LEAVES ON HUMAN HEPATOCELLULAR CARCINOMA (HEPG2) CELLS, IN VITRO

Derrick White*, and Stephen Ekunwe, Jackson, MS 39206 Jackson State University, Jackson, MS 39217

Cancer is the second leading cause of death in humans. Statistics show that in 2006, of the estimated 1,399,790 new cases, 564,830 will die. Traditional treatments for cancer have shown some significant results. However, the incidence of cancers is still rising. In this study, the effects of crude extract from leaves of the edible plant Ocimum gratissimum (Og) on human hepatocellular carcinoma (HepG2) cells were investigated. It was hypothesized that crude extract of Og will inhibit growth of, be cytotoxicity to, and cause DNA fragmentation in HepG2 cells. To test this hypothesis, HepG2 cells were cultured at 37[degrees]C in a humidified 5% C[O.sub.2] incubator in complete growth medium. The cells were allowed to grow until they became 60-65% confluent. They were serum starved for 24hrs, and then treated with Og extract at the following concentrations: 0.25, 0.50, 1.0, 2.0, 4.0, 8.0, 12.0, and 16.0mg/ml. Cell growth was assessed using thymidine incorporation assay. It was found that the Og extract inhibited the growth of HepG2 cells in a dose dependent manner. Trypan blue dye exclusion assay was used to test the cytotoxicity of the extract. Results of cytotoxicity evaluation show that HepG2 cells survive low concentrations of Og extract. DNA fragmentation test show that Og extract causes DNA fragmentation in HepG2 cells at all concentrations. These results show that crude extract of Og causes DNA fragmentation and inhibits the growth of HepG2 cells. These results warrant more detailed investigation of Og and its potential cancer fighting properties.

CYP2B 1/2 MESSENGER RNA WITHIN THE LIVER

Mary Beth Dail*, Shane C. Burgess, E.Meek, J.Wagner, and J.E.Chambers, Mississippi State University, Mississippi State, MS 39762

Research suggests that CYP2B 1/2 enzyme activity is differentially distributed within the liver and that this distribution is altered by various chemicals. This study examined CYP2B 1/2 mRNA levels in different parts of the liver's functional unit. Rats were treated with either i.p. phenobarbital in saline at 80mg/kg/day for 5 days or dieldrin in corn oil p.o. at 2.5 or 5.0 mg/kg/day for 13 days. LCM and duplex QRT-PCR were used to measure the CYP2B 1/2 mRNA produced in bands of liver cells isolated from 3 locations along the path of blood flow. CYP2B 1/2 mRNA in whole liver was also determined. CYP2B 1/2 enzyme activity was measured through 16sshydroxy-testosterone formation. General CYP activity was determined by desulfuration assays. Whole liver samples exhibited significant increases in CYP2B 1/2 mRNA: 5.7-fold, phenobarbital; 29-fold, dieldrin at 5.0 mg/kg; and 38,166-fold, dieldrin at 2.5 mg/kg. A similar inverse dose response was seen for CYP2B 1/2 enzyme activity, but not for general CYP enzyme activity. All LCM band samples showed significant increases in CYP2B 1/2 mRNA. Dieldrin at 2.5 mg/kg caused increases as blood flowed through the acinus: periportal, 320-fold; midzonal, 538-fold; and centrilobular, 1,418-fold. A different pattern was observed in phenobarbital treated rats: periportal, 1,031-fold; midzonal, 4,973-fold; and centrilobular, 1,136-fold. The differences indicate the variable nature of CYP2B 1/2 response following chemical exposure and add to the fund of knowledge required for a future model of liver function. Supported by NIH P20RR017661

CYTOTOXICITY STUDIES USING NITOGEN ONIUM SALTS

Emily Stewart*, Nancy Salloum, Wolfgang Kramer, Millsaps College, Jackson, MS 39210

The cytotoxicity of nitrogen onium salts was tested using Saccharomyces cerevisiae as a system. Drug uptake as well as retention studies were performed.

OPTIMIZATION OF ORGANIC WASTE BASED MEDIA SUSTAINABLE TO BIOHYDROGEN PRODUCTION

Hanqing Dong, Anberitha Matthews, Michael Cunningham*, Nara Gavini, and Lakshmi Pulakat, Mississippi State University, Mississippi State, MS 39762

Biohydrogen production is a promising novel energy source whose developments remain hindered due to the physiological and biochemical restrictions from the hydrogen-yielding microorganism. Studies on the soil bacterium Azotobacter vinelandii indicate that it yields hydrogen as a by-product of nitrogen fixation; and its characteristics such as high respiratory rates, the ability of expressing anaerobic proteins, and lower growth requirements project the possibility of bridging the diazotroph and hydrogenase. We explored its capability of utilizing carbon compounds from various industrial wastes. The growth of the Azotobacter wild type strain was monitored in Burk's standard nitrogen media ([BN.sup.+]), Burk's nitrogen-free media (B[N.sup.-]) and modified B[N.sup.+]/B[N.sup.-] media in which sugar is substituted by different waste extracts respectively. When nitrogen was present, the strain grew best in the standard media; however, in absence of nitrogen source, the yam-peel based media appeared to provide the optimal environment for the growth. In addition we also expressed the Fe-nitrogenase alone by growing a mutated Azotobacter strain DJ54 in which Mo-dependant nitrogenase is not functional and in this strain the Fe-nitrogenase was induced by high iron concentration in the aforementioned biowaste-based media. Azotobacter DJ54 exhibited highest growth rate. These observations suggest that the industrial residual media are sufficient for Azotobacter nitrogenase activity and probably biohydrogen production.

SEQUENCE AND EXPRESSION ANALYSIS OF THE AAT1 AND SOX1 GENES FROM THE CYSTEINE PATHWAY OF THE DIMORPHIC PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM

Yuan-Chi Lin* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406

Histoplasma capsulatum (Hc) is a dimorphic fungus which can be found in the soil in a multicellular mold form. When conidia or mold cells are inhaled, they will undergo a morphology shift and grow as unicellular yeast in the lungs of the infected individual. This mold-to-yeast shift is required for disease to occur. Sulfur metabolism, in particular cyst(e)ine, is known to play a critical role in this dimorphic process. The focus of this report involves two genes in the sulfur metabolic pathway: aspartate aminotransferase (AAT1) and sulfite oxidase (SOX1). We have isolated the Hc AAT1 and SOX1 genes and used northern blots and real-time PCR to measure the expression in the yeast and mold morphotypes of Hc. Transcripts from both genes are low abundance and the AAT1 transcript appears to be yeast specific. Currently work is underway to knockout both genes to examine their role in yeast-mold dimorphism in this important pathogen.

THE ROLE OF THE SIDE DOOR OF CARBOXYLESTERASES IN THE HYDROLYSIS OF ESTERS

Matt K. Ross*, Abdolsamad Borazjani, Tim M. Streit*, Mississippi State University, Mississippi State, 39762

Carboxylesterase (CE) enzymes catalyze the hydrolysis of esterified chemicals, including pesticides, drugs, and lipids. We have been investigating the substrate specificity of mammalian CEs toward pyrethroid insecticides. However, how CEs recognize pyrethroids and catalyze their hydrolysis is incompletely characterized. To fill this gap, we have commenced studies using a model CE derived from Bacillus subtillis (termed pnbCE), which is highly conserved both in sequence and structure with mammalian CEs. pnbCE variants were constructed with site-specific amino acid modifications at a location involved in substrate recognition and catalysis. Here, we show that amino acid substitutions in the 'side-door' domain of pnbCE are important for the hydrolysis of the pyrethroid trans-permethrin and some model ester compounds. Our results demonstrate that six site-specific mutants at position 362 (L362R, L362K, L362D, L362E, L362A, and L362V) exhibited markedly decreased [V.sub.max] values (3-7-fold) compared to wild-type CE when rates of hydrolysis of trans-permethrin were examined. However, changes in [K.sub.m] values were not markedly different for the mutant CEs. Viscosity variation studies using both wild type and L362R mutant enzyme demonstrated that rates of substrate association ([k.sub.1]) and covalent acylation of pnbCE ([k.sub.2]) by the ester substrate were significantly lower for the L362R enzyme. These data demonstrate the impact of a single amino acid substitution, in a domain adjacent to the active site, on enzyme function. [Supported by NIH P20RR017661]

CHRONIC DEVELOPMENTAL EXPOSURE TO ORGANOPHOSPHATES ELEVATES DOPAMINE LEVELS AND ALTERS NICOTINIC ACETYLCHOLINE SUBUNIT RNA EXPRESSION

Timothy W. Brown*, and Jeffrey B Eells, Mississippi State University, Mississippi State, MS 39762

Organophosphates (OP) inhibit acetylcholinesterase, the enzyme responsible for the inactivation of synaptic acetylcholine, which results in elevated acetylcholine neurotransmission. Acetylcholine neurotransmission is widespread throughout the brain and can affect multiple neurotransmitter systems including the mesencephalic dopamine system. The current experiment investigated how chronic, developmental exposure to the organophospates chlorpyrifos (CPS) or methyl parathion (MPT) affects dopamine levels and dopamine neuron gene expression. Newborn rats were treated with corn oil vehicle, CPS or MPT via gastric lavage daily from postnatal day (PND) 1-21. Rats were sacrificed at PND 21 and 50 and tissue collected and frozen. Dopamine levels were measured in the striatum using high performance liquid chromatography with electrochemical detection and gene expression in the substantia nigra was measured with quantitative real-time PCR. In the striatum, CPS and MPT treatment resulted in a trend toward elevated levels of dopamine, and DOPAC, with a statistically significant elevation in HVA levels. At PND 50, dopamine levels were reduced after CPS but not MPT. Additionally, dopamine turnover was significantly reduced after CPS due to reduced dopamine and a slight increase in DOPAC. Both CPS and MPT treatment caused a significant alteration in the ratio of [alpha]a7 to [alpha]a6 nicotinic acetylcholine receptor subunit expression in the substantia nigra at PND 21. At 50 days, the expression of [alpha]a7 to [alpha]a6 nicotinic acetylcholine receptor subunit was altered only after MPS treatment. These data demonstrate that chronic, developmental inhibition of acetylcholinesterase can have a significant and persistent effect on dopamine neuron function.

EMBRYONIC GENOME ACTIVATION IN BOVINE

Muge Misirlioglu (1), Grier Page (2), Hakan Sagirkaya (1), Abdullah Kaya (3), John J. Parrish (3), Neal L. First (1), and Erdogan Memili (1*), (1) Mississippi State University, Mississippi State, MS 39762, (2) University of Alabama, Birmingham, AL 35294 and (3) University of Wisconsin, Madison, WI 53706

Global activation of embryonic genome is one of the most critical events of early mammalian development. Maternal proteins and RNAs in oocyte support early embryonic development while a number of zygotic and embryonic genes are expressed at a stage specific manner leading to embryonic genome activation (EGA). However, the identities of embryonic genes expressed and the mechanism(s) of EGA are not well understood in the bovine. The aim of this study was to analyze gene expression at two key stages of bovine development, matured oocytes (MII) and 8-cell stage embryos that constitute rich maternal supply of materials for development and a stage during which EGA takes place respectively. Global transcrip-tome of MII oocytes, 8-cell embryos and 8-cell embryos treated with transcription inhibitor alpha-amanitin using bovine DNA microarrays (Affymetrix, Inc.) that contains probe sets representing over 23, 000 transcripts including assemblies from approximately 19, 000 UniGene Clusters. Total of 3, 972 genes showed significant expression (<2 fold, P<0.01). Key genes in regulation of transcription, chromatin structure cell adhesion and signal transduction were up-regulated at the 8-cell stage (as compared to alpha-amanitin and MII oocyte controls) while genes in DNA methylation and metabolism were up-regulated in the MII oocytes. These changes in gene expression related transcriptional machinery, chromatin structure and the other cellular functions occurring during several cleavage stages are expected result in a unique chromatin structure capable of maintaining totipotency during embryogenesis and leading to differentiation during the postimplantation development.

EFFECTS OF LEAF EXTRACT OF NIGERIAN BASIL, OCIMUM GRATISSIMUM, ON THE GROWTH OF HUMAN PROSTATE CANCER CELLS (PC-3) IN VITRO

Jelani Zarif* and Stephen Ekunwe, Jackson State University Jackson, MS 39217

Prostate Cancer is the third leading cause of cancer death of men in the U.S and it affects African-American men disproportionately. Various approaches have been taken to prevent this disease. One of the main approaches is chemotherapy which usually has harsh side effects. Therefore new approaches that have few adverse side effects are highly sought after. It is believed that edible herbs would provide such desirable treatment. A potential candidate is extracts from the leaves of Nigerian basil, Ocimum gratissimum (Og). These extracts, which are consumed as food in teas and soups, have been used in folk medicine to treat headaches, itching, hemorrhoids and pinkeye. Therefore, it is hypothesized that Og extract will inhibit growth of and will be cytotoxic to PC-3 cells, in vitro. To test this hypothesis, heated and unheated Og extracts were used to treat serum-starved sub-confluent PC-3 cells for 18 hours at the following concentrations: .25, .50, 1.0, 2.0, 4.0, 8.0, 12.0, and 16.0 mg/ml. The cells were then labeled with [[.sup.3.H]]-thymidine for 4-6 hours and counted in a Scintillation Analyzer. Trypan blue dye exclusion assay was used to measure cell viability after treatment with the Og extract. Results showed that Og extract is thermostable and it inhibited growth of PC-3 cells in a dose dependent manner. Trypan blue dye exclusion assay show that Og extract was cytotoxic to PC-3 cells especially at higher concentrations: 8.0, 12.0 and 16.0 mg/ml. These results suggest that Og extract may possess potential cancer-fighting agents.

PHOTOCHEMICAL & REDOX CONTROL OF HYDROGEN GAS PRODUCTION IN CYANOBACTERIA

Daya Cohly (1*), Zandra Cole (1), Raphael Isokpehi (1), Rajendram V Rajnarayanan (2) Hari H P Cohly (1), (1) Jackson State University, Jackson, MS 39217, and (2) Tougaloo College, Jackson, MS 39174

Cyanobacteria exists in vegetative form during the daylight while it has heterocytsts during the night time. Under the influence of limiting conditions of nitrogen and/or blue light the equilibrium shifts towards the right while the equilibrium shifts towards the left in the presence of oxygen and/or red light. Water and carbon dioxide (C[O.sub.2]) is transported in the interior of the cyanobacteria by aquaporins (AQP). In the daytime the transport of C[O.sub.2] via AQP is facilitated by AQP but is inhibitory for hydrogen gas production. This C[O.sub.2] is converted to HC[O.sup.3-] in the interior which is eventually converted to carbohydrates. During the night these carbohydrates are converted enzymatically to [H.sub.2] gas. It could be hypothesized that there could be a direct relationship between the number of AQPs present and the amount of hydrogen gas produced. The mercury inhibition of AQPZ in the daylight is more pronounced where it affects the transport of water and CO2. This results in lesser concentration of carbohydrates available for enzymatic conversion in to [H.sub.2] gas during the night. Shifting of the photochemical redox equibrium reaction to the right hand side would lead to maximal production of [H.sub.2] gas. Further investigations on the combination of [N.sub.2], blue light and AQPZ modulators would provide more insights in to molecular and photochemical control of [H.sub.2] gas production in cyanobacteria. Acknowledgements: Mississippi NSF-EPSCoR "Innovations through Computational Sciences" Award (EPS-0556308); and the Research Centers in Minority Institutions (RCMI)--Center for Environmental Health (NIH-NCRR G12RR13459-09)

KAPPA-OPIOID RECEPTOR (KOR) INVOLVEMENT IN HYPOTHALAMIC ACTIVATION OF HYPOTHALAMO-PITUITARY-ADRENAL (HPA) AXIS: ROLE FOR BUTORPHANOL?

Deandria Magee (1*), George Howell (2), Melissa Griggs (2), Rob Rockhold (2), (1) Tougaloo College, Tougaloo, MS 39174 and (2) University of Mississippi Medical Center, Jackson, MS 39216

Butorphanol is a narcotic analgesic with agonist efficacy at both KOR- and mu-opioid receptors. Acute administration of butorphanol stimulates the HPA axis, beginning with neuronal activation in the paraventricular nucleus of the hypothalamus (PVN), as indicated by an increase in c-Fos immunoreactivity. This neuronal activation is hypothesized to result from stimulation of the KOR by butorphanol. Male Sprague-Dawley rats received intracerebroventricular (i.c.v.) pretreatment with the selective KOR antagonist, nor-binaltorphimine (nor-BNI; 20 [micro]g/5 [micro]l), or saline 60 minutes prior to intravenous administration of butorphanol (10.0 mg/kg). Blood was withdrawn 30 minutes thereafter to assess plasma corticosterone concentrations as a marker of HPA axis activity. PVN neuronal activity was determined by c-Fos immunohistochemistry Pretreatment with nor-BNI, followed by saline treatment, slightly increased c-Fos immunoreactivity. Saline pretreatment, followed by butorphanol, increased both c-Fos immunoreactivity and plasma corticosterone levels. However, pretreatment with nor-BNI blocked neither butorphanol-induced PVN c-Fos activation nor increases in corticosterone. While these data do not support KOR actions of butorphanol, a companion report will document that higher (35 [micro]g) doses of nor-BNI effectively reduce the stimulatory effect of butorphanol on neuronal activity. (DM was supported by a fellowship from the Mississippi Functional Genomics Network)

ENGINEERING OF A NOVEL INHBITOR OF BIOFILM-ENCAPSULATED PATHOGEN

Madhavi p, Gavini*, Mississippi School for Mathematics and Science, Columbus, MS 39701 and Mississippi State University, Mississippi State, MS 39762

Pseudomonas infection is a leading cause of death among patients with Ams, Cystic Fibrosis, cancer and burn victims. These opportunistic pathogens are multidrug resistant. They produce biofilms that protect bacteria from phagocytosis and antibiotics. Therefore, treating Pseudomonas infections requires biofilm inhibitors. The aim of this research is to engineer a method to inhibit growth ofbiofilm-forming pathogens. Since non-biofilm forming Pseudomonas is abundant in environment, the hypothesis is that herbal products would contain biofilm-inhibiting compounds. An abiotic technique was developed to isolate a biofilm-forming Pseudomonas from the non-biofilm forming lab-stock of Pseudomonas aeruginosa P AO I. The resulting clonal isolate designated Pseudomonas aeruginosa MG1 could produce biofilm. Effects often herbal extracts/compounds on the growth of PA01 and MG1 were tested by spot testing and growth curve analysis. Terminalia chebula (Tc) extract was the most potent inhibitor of both Pseudomonas strains and was able to inhibit their growth even after over 100 fold dilution. Microarray analysis of gene expression and Real-time Polymerase Chain Reaction amplification showed that Tc can reduce or inhibit expression of genes needed for establishing biofilm and infection. The anti-microbial compound in Tc is water soluble, functional after autoclaving and at pH 7.0, and has a size <0.1 micron. It is ideal for developing anti-microbial sprays and inhalers to treat Pseudomonas infections. Author acknowledges LSBI at MSU for the generous gift of microarrays and facility.

RELATIONSHIP BETWEEN THE NUMBER OF AQUAPORINS AND HYDROGEN GAS PRODUCTION IN CYANOBACTERIA

Zandra Cole (1*), Daya Cohly (1), Raphael Isokpehi (1), Rajendram V Rajnarayanan (2) Hari H P Cohly (1), (1) Jackson State University, Jackson, MS 39217 and (2) Tougaloo College, Jackson, MS 39174

Water and carbohydrate are the primary sources for hydrogen production in cyanobacteria. Water and carbon dioxide (C[O.sub.2]) is transported in the interior of the cyanobacteria by aquaporins (AQP). In the daytime the transport of C[O.sub.2] via AQP is facilitated by AQP but is inhibitory for hydrogen gas production. This C[O.sub.2] is converted to HC[O.sub.3-] in the interior which is eventually converted to carbohydrates. During the night these carbohydrates are converted enzymatically to [H.sub.2] gas. It could be hypothesized that there could be a direct relationship between the number of AQPs present and the amount of hydrogen gas produced. Hydrogen production occurs within at least 14 Cyanobacteria genera, under a vast range of culture conditions. One possibility is that there is efficient transport of C[O.sub.2] via a single AQP and the other is that the cumulative transport of C[O.sub.2] via multiple AQPs leading to increased [H.sub.2] production. To this end we explored 20 annotated genomes of cyanobacteria comprising of about 62,262 genes. Gleobacter PCC 7421 with 3 known AQPs shows increased [H.sub.2] gas production in comparison to Synechocystis sp. PCC 6803 which has only one AQP. There is a clear correlation of the number of AQPs and [H.sub.2] gas production in the AQPs examined in the study. We are currently examining the number of AQPs present in the unannotated bacteria Anabaena variabilis which produces 10-fold more hydrogen than Gleobacter. Acknowledgement: Mississippi NSF-EPSCoR "Innovations through Computational Sciences" Award (EPS-0556308); and the Research Centers in Minority Institutions (RCMI)--Center for Environmental Health (NIH-NCRR G12RR13459-09)

DEVELOPMENT OF PRIMARY CULTURE OF ENTEROCYTES TO STUDY ADAPTIVE REGULATION OF INTESTINAL NUTRIENT ABSORPTION

Yasuhiro Kimura*, Dwaine A. Braasch, and Randal K. Budding-ton, Mississippi State University, Mississippi State, MS 39762

Small intestine tissues exposed to adenosine or AMP rapidly (<10 min) and reversibly increase (~2-fold) glucose uptake. Since signaling networks, and mechanisms of adaptation are difficult to define using intact tissues, we sought a cultured cell model. Although the colon adenocarcinoma cell line, Caco-2, is commonly used as a model for enterocytes, glucose accumulation decreases by 70% and 15% after exposure to adenosine and AMP, respectively. Although IEC-6 cells, a spontaneously immortalized cell line (derived from crypt cells of rat ileum) respond to adenosine and AMP through increases in glucose uptake of 307% and 322% respectively, the response requires plating on Matrigel (a cell binding enhancer) and exposure to 100 nM dexamethasone for 72 h to induce differentiation. Cells of a different tissue origin and degree of differentiation require long-term sterile culture. The use of undifferentiated cells further requires the induction of differentiation. We evaluated mix population primary cultures of enterocytes liberated from BALB/c mouse intestine. Isolated enterocytes were cultured on membrane inserts (PET; 0.4 [micro]m pore size) for 8 h. Subsequent exposure to adenosine and AMP increased glucose uptake by 6- and 9-fold. Therefore, primary cultures of enterocytes can be used to identify molecules that regulate transport functions, and define the associated signaling networks and mechanisms, without depending on long-term, rigorous culture conditions. Moreover, the use of freshly harvested enterocytes provides opportunities to obtain data that are physiologically relevant.

EFFECT OF HETEROTROPHY ON OXIDATIVE STRESS IN THE HERMATYPIC CORAL CAULASTREA ECHINULATA

Bridget Napier*, Patricia M. Biesiot, and Shiao Y. Wang, University of Southern Mississippi, Hattiesburg, MS 39406

Coral nutrition involves host heterotrophy and symbiont autotrophy. Previous studies in other labs have found that: 1. heterotrophy by corals lead to increased productivity by zooxanthellae; 2. both photosynthetic and respiring cells produce reactive oxygen species (ROS) that, if left unquenched, damage cell components and can cause cell death; 3. antioxidant enzymes (AE) function to quench ROS; and 4. AE capacity increases in response to damage by ROS. Our study examined the putative relationship between heterotrophy and subsequent oxidative stress that may arise during periods of increased photosynthesis. Caulastrea echinulata colonies were fragmented and individual polyps were assigned to one of two groups (fed and unfed). Fed polyps were given grass shrimp the morning of the test whereas unfed polyps were fasted. Both groups were exposed to sunlight for 8 h. Activities of two AE [superoxide dismutase (SOD) and catalase (CAT)] were measured at 0, 4, 6, and 8 h. Activities of both SOD and CAT were higher in fed than unfed polyps; however, only SOD activity of fed polyps was statistically significant. Highest SOD activity occurred at 4 h and declined thereafter. Highest CAT activity occurred at 6 h but it was not significant. The lag in peak CAT activity may be due to coupled activity of the enzymes; CAT is known to remove toxic hydrogen peroxide produced by SOD.

ALTERED DOPAMINE LEVELS IN THE NUCLEUS ACCUMBENS OF ISOLATED AND GROUP REARED NURR1-NULL HETEROZYGOUS MICE FOLLOWING ADMINISTRATION OF AMPHETAMINE BY MICRODIALYSIS

Talisha M Moore*, Timothy W Brown, and Jeffrey B Eells, Mississippi State University, Mississippi State, MS 39762

One hypothesis of schizophrenia is that altered dopaminergic transmission attributes to the etiology. Transgenic technology disturbing the Nurr1 gene demonstrated that it is essential for dopamine (DA) neuron development. Nurr1--null heterozygous mice (+/-) possess decreased tissue dopamine levels in the nucleus accumbens (NuAcc) and, when raised in isolation, have disrupted sensorimotor gating, a behavior typical of schizophrenia. To determine the effect of isolation on DA release, male Nurr1 (+/-) and (+/+) mice were randomly reared in groups or isolation at 18-21 days. At 4-6 months microdialysis was conducted within the shell of the NuAcc using amphetamine and CNS. All samples were measured for stimulated DA, DOPAC, and HVA using HPLC with electrochemical detection. At basal levels, the +/- genotype significantly affected DA release in group raised mice. At the 10 & muM level, there were no significant differences observed across groups. At the 100 & muM level, (+/-) genotype did not affect DA release, however when combined with isolation, +/- IS mice possessed the greatest level of amphetamine stimulated DA release. In terms of DOPAC, (+/-) mice possessed no significant differences under basal conditions, displayed inconsistent trends at the 10 & muM level, and when combined with isolation, displayed elevated DOPAC levels at the 100 & muM level. No differences in HVA were observed across groups. These data suggest that isolation differentially affected DA release in the shell of the NuAcc of +/- mice which may underlie the disruptions in sensorimotor gating in these mice and may be relevant to disruptions in sensorimotor gating observed in schizophrenia.

HEPATIC CYTOCHROME P450 ACTIVITY IN A ZEBRAFISH ALCOHOLIC LIVER DISEASE MODEL

Keila Brown*, Stanley Smith, Veronica Levison, Christine A. Purser, Tina G. Smith, and Rodney C. Baker, Tougaloo College, Tougaloo, MS 39174

Alcoholic liver disease results in high morbidity and mortality rates in affected individuals. It is a complex disease resulting in a number of cellular consequences that can lead to tissue damage and liver dysfunction. The hepatic cytochrome P450s have been implicated in the progression of alcoholic liver disease via the generation of reactive oxygen species and other reactive intermediates. Using a zebrafish (Danio rerio) experimental model for alcoholic liver disease, we investigated the role of hepatic cytochrome P450s. Zebrafish were treated for 5-7 days with 50 mM ethanol in the habitat water and compared to zebrafish not exposed to ethanol. The microsomal fractions of the zebrafish livers were isolated after cellular disruption and differential centrifugation. The microsomes were then assayed for cytochrome P450 activity. We chose testosterone as the test substrate. The site of testosterone hydroxylation suggests which cytochrome P450 isoform is responsible. We assayed product formation using HPLC and gas chromatography/mass spectrometry (GC/MS). Surprisingly, HPLC analysis indicated no apparent hydroxylation of testosterone. However, we observed a consistent decrease in the parent testosterone peak that was dependent on the presence of zebrafish microsomes. This occurred in both the control and ethanol-treated samples. We analyzed the reaction products in more detail via GC/MS. From the GC/MS results, the disappearance of testosterone correlated with the appearance of products that were identified in the mass spectral reference library as stanolone and dihydroandrosterone. Further experiments are underway to characterize this activity and identify the cytochrome P450 isoform involved.

OPTIMIZATION OF GENE EXPRESS PROFILING (GEXP) MULTIPLEX QUANTITATIVE PCR TO MEASURE GENE EXPRESSION IN MESENCEPHALIC DOPAMINE NEURONS

Jeffrey B. Eells*, Timothy W. Brown, and Banu Elibol-Flemming, Mississippi State University, Mississippi State, MS 39762

Mesencephalic dopamine neurons are involved in neurological processes such as motivation, cognition and control of movement. As such, alterations in dopamine neuron function have been implicated several diseases and disorders including schizophrenia, Parkinson's disease, attention deficit hyperactivity disorder and drug addiction. Gene eXpress Profiling (GeXP) Multiplex RT-PCR is a recently introduced technology that has the capability to quantitatively measure the expression of up to 30 genes in one PCR reaction. This assay produces multiple fluorescently labeled PCR products of the genes of interest that differ in length by 5-6 base pairs which can be analyzed by the Beckman CEQ 8000 capillary electrophoresis. The goal of the current project was to develop a multiplex PCR reaction to measure dopamine neuron gene expression in order to ultimately assess how dopamine neuron gene expression is affected by pathological conditions. The multiplex reaction consists of primers for 1) extracellular receptors expressed on dopamine neurons, 2) dopamine neuron transcription factors, 3) dopamine neurotransmission genes and 4) internal control genes ([beta]-actin and GAPDH). Singlet reactions produced fragments of the appropriate size in 26 of the 28 primer pairs tested with no undersigned fragments. In the multiplex of mouse whole brain, 18 fragments were identified. These preliminary results demonstrate that multiplex PCR reactions can be developed as a high through-put method to quantify gene expression of a focused subset of genes. Future studies will continue to optimize the multiplex reaction as well as further test the quantitative properties using known concentrations of gene targets.

MOLECUALR ANALYSIS OF INTERACTION OF INSULIN RECEPTOR BETA-SUBUNIT WITH ANGIOTENSIN II RECEPTOR AT2

Maya Ramdas*, Ravindra Kolhe, Nara Gavini, and Lakshmi Pulakat, Mississippi State University, Mississippi State, MS 39762

Angiotensin II (Ang II) is a multifunctional hormone implicated in the regulation of blood pressure and in the pathogenesis of hypertension, insulin resistance and metabolic syndrome. We have shown previously that the Ang II receptor AT2 can function in a ligand-independent manner and that the AT2 can interact with the cytoplasmic ATP-binding region of receptor tyrosine kinase (RTK) ErbB3. Such interaction could be significant in the regulation of growth promoting signaling by RTKs. To further investigate regulation of receptor tyrosine kinase signaling by the AT2, we analyzed whether the AT2 interacts with the Insulin receptor IR. Here we show that a truncated AT2 receptor (amino acids 226-363) interacts strongly with the IR [beta] subunit in yeast two-hybrid (Y2H) assay. We also show that a chimeric AT2-AT1 receptor (third intracellular loop of the AT2 is replaced with that of the AT1) could also interact strongly with the IR [beta] subunit in Y2H assay. The Ang II receptor AT1 also showed week interaction with the IR-beta. However, a truncated AT2 in which the C-terminal cytoplasmic region was deleted did not interact. These results suggested that the C-terminal cytoplasmic domain of AT2 is essential for this interaction. In addition, our studies using Chinese Hamster Ovary (CHO) cells showed that transient expression of the AT2 could result in AT2-IR-beta complex formation and lack of phosphorylation of IR-beta. Based on these data, we propose a model that involves receptor level interaction between the AT2 and IR in response to transient expression of AT2.

INTERACTIONS OF PAMAM DENDRIMERS WITH MODEL LIPID MEMBRANES

Venkataswarup Tiriveedhi (1*), Kelly M Kitchens (2), Hamid Ghandehari (2), and Peter Butko (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) University of Maryland, Baltimore, MD 21201

Dendrimers and other cationic macromolecules in general are considered potential vehicles for intracellular drug delivery. We used fluorescence spectroscopy to study the interaction of cationic PAMAM (polyamidoamine) dendrimers with model lipid bilayers. When the dendrimers interacted with small unilamellar vesicles (SUV) loaded with the self-quenching fluorophore calcein they did not induce release of calcein neither with neutral SUV (made of egg phosphatidylcholine) nor negatively charged SUV (with 25% egg phosphatidylglycerol). The G1 (generation 1, Mr = 1430) dendrimer was labeled with fluorescein and the G4 dendrimer (Mr = 14215) with a similar fluorophore Oregon green, and their membrane interactions were followed by changes in fluorescence anisotropy during titrations with lipid. The saturating value of anisotropy changed linearly with the density of negative charge in the membrane. The addition of high salt caused redissociation of the dendrimers from SUV. These two observations prove the electrostatic nature of the interaction. Analysis of the binding data yielded values of the dissociation constants Kd. For G1 dendrimer, Kd = 30 [+ or -] 16 mM with neutral SUV and Kd = 11 [+ or -] 3 mM with negatively charged SUV. For G4, which carries much higher charge on its surface, exhibited lower dissociation constants, Kd = 16 [+ or -] 7 mM with neutral SUV and 5 [+ or -] 1 mM with negatively charged SUV. We conclude that the interaction of PAMAM dendrimers with lipid membrane is electrostatic and the dendrimer binding does not significantly disrupt the bilayer integrity.

IMMOBILIZATION OF ANTIBODY ON A QUARTZ CRYSTAL FOR DETECTION OF PSEUDOMONAS

Amy Denson*, Scott Walper, Sabine Heinhorst, Gordon C. Cannon, and Jeffrey A. Evans, University of Southern Mississippi, Hattiesburg, MS 39406

Quartz crystal microbalance (QCM) biosensors have been developed for the detection of various bacteria and viruses. The QCM system measures resonant frequency decreases proportional to the mass addition deposited on the crystal surface. Current techniques employed in the development of biosensors include attachment of antibodies via covalent coupling to an amine-functionalized polymer. To couple antibodies specific for Pseudomonas species to the crystal surface, polyethyleneimine (PEI) was used followed by glutaraldehyde treatment. After verifying immobilization of the antibody to the crystal surface, experiments were conducted to determine the optimal concentration of antibody needed to coat the crystal surface for detection of bacteria.

CHARACTERIZATION OF OVEREXPRESSION OF KIN3 IN S. CEREVISIAE

Andrew Gilder*, Yulon Stewart, and Sarah Lea McGuire, Millsaps College, Jackson, MS 39210

The KIN3 gene of Saccharomyces cerevisiae encodes a poorly understood protein kinase, the deletion and overexpression of which have been previously reported to have no effects on cells. Because we have recently found that deletion of KIN3 leads to a number of previously uncharacterized subtle cellular effects, we have undertaken a study of the effects of overexpression of KIN3 on cells. To do this, we amplified the KIN3 gene from strain Y2454, cloned the amplification product into a Topo-TA vector, and then directionally cloned into the pYES vector, putting KIN3 under control of the GAL1 promoter. This vector was then transformed into Y2454 cells and growth tested under inducing and repressing conditions. No differences in plate growth rates were observed under any conditions, compared to the control strain. We are currently performing FACS analysis and microscopy to determine if more subtle effects on the cells occur due to overexpression. We also plan to transform our KIN3 deletion strain with the overexpression vector to determine if overexpression leads to reversion of the deletion phenotype.

CULTURE MAKES A DIFFERENCE IN DEVELOPMENT AND MOLECULAR BIOLOGY OF PORCINE PREIMPLANTATION EMBRYOS IN VITRO

Song-yi Jung*, Hongfeng Wang, Nelida Rodriguez-Osorio, Kriston Faith Garrison, Kristi Wolgemuth, Laura Samantha Greer, Jean Magloire Nguekam Feugang, and Erdogan Memili, Mississippi State University, Mississippi State, MS 39762

In vitro production of pig embryos is important both for basic biomedical research and for animal biotechnology. However, their efficiency and quality are sub par. To determine developmental biology of porcine embryos cultured in two semi-defined media, presumptive zygotes were produced in vitro. Using a 2 x 2 factorial treatment arrangement the effect of the modified culture medium (NCSU-23 + 0.4% BSA or PZM-3 + 0.3% BSA) and additives (10ng/mL Leptin, 10ng/mL EGF, 10-9M Melatonin, or the combination of all three additives) was analyzed. The rates of cleavage and blastocyst development were evaluated at 48 hours post insemination (hpi) and 144 hpi, respectively. The PZM-3 provided better development at all stages as compared to the NCSU-23 medium (cleavage rates: 41% vs. 38%; blastocyst rates: 16% vs. 10% respectively; P<0.05). In the PZM-3 medium, the cleavage rates seem to increase in the EGF and combination groups compared to the control group (49% and 48% vs. 41%; p=0.07). At the blastocyst stage however, the EGF showed the lowest result (9%), while there were no statistical differences between the other groups (p>0.05). In the NCSU-23 medium, only the leptin and melatonin provided a higher number of cleaved embryos as compared to the control (42% for leptin and melatonin vs. 38% for the control). Blastocyst development was similar for the all the groups. We concluded that PZM-3 medium provided better development rates to blastocysts than NCSU-23.

IDENTIFICATION AND INITIAL CHARACTERLZATION OF SINGLE NUCLEOTIDE POLYMORPHISMS OF MAIZE GLOSSY 15

Biing-Ru Wu (1*), Thomas D. Brooks (2), Dawn S. Luthe (3), Jeff R. Wilkinson (1), and W. Paul Williams (2), Mississippi State University, Mississippi State, MS 39762, (2) USDA-ARS Corn Host Plant Resistance Research Unit, Mississippi State, MS, 39762, and (3) Pennsylvania State University, PA, 16802

Persistence of juvenile leaves in maize increase predation by fall army worm. Transition of maize shoots from the juvenile to the adult stage is affected by a variety of leaf epidermal cell traits, including: leaf hairs, cell wall characteristics, and presence of epicuticular waxes such as those regulated by the maize putative transcription factor of Glossy 15 (g115). In maize the first two juvenile leaves are maintained by the kernel, whereas juvenile leaf status in the subsequent leaves is maintained by the expression of Glossy 15 (g115). Thus, different rates of glossy 15 gene expression results in varying transitional periods. Glossy15 has significant sequence similarity to the Arabidopsis regulatory gene APETALA2. This factor regulates floral organ identity and ovule development in Arabidopsis. Studies of APETAL2 have shown that single nucleotide polymorphism (SNP) of microRNA (miR 172) target site controls the expression rate in Arabidopsis. However, sequence comparisons of four maize inbred lines (MP313E, MP608, TX601, B73) revealed no nucleotide differences in the microRNA target site. However, from comparisons of g115 and apetala2 promoters, AP1, and AP2 domains two SNPs were identified in the AP2 domain-2. These SNPs may indicate regulation occurs within the coding region instead of the microRNA site for maize. To ascertain the relationship AP2 may have to gll5 regulation site directed mutagenesis will be performed and the resulting changes in expression rates will be measured.

GENERATION OF GENE SPECIFIC MARKERS ASSOCIATED WITH RESISTANCE TO ASPERGILLUS FLAVUS AND ALFATOXIN FOR USE IN MARKER ASSISTED SELECTION OF HYBRID ZEA MAYS LINES

Erik Mylroie (1*), Rowena Y. Kelley (1), W. Paul Williams (2), Thomas D. Brooks (2), Jeff R. Wilkinson (1), (1) Mississippi State University, Mississippi State, MS 39762 and (2) USDA-ARS Corn Host Plant Resistance Research Unit, Mississippi State, MS 397562

Apergillus flavus is a fungal pathogen that infects tree nuts, maize, cotton, and peanuts. After infection, A. flavus often produces aflatoxins, which are mutagenic, teratogenic, and immunosuppressive compounds known to cause hepatocellular carcinoma. To limit exposure to this naturally occurring carcinogen, the FDA has placed action levels of 20 ppb for human consumption. Maize losses due to aflatoxin contaminated products is estimated to be half a billion dollars annually. Since an economically feasible method does not exist to eliminate or remove aflatoxin from contaminated corn, breeding programs aimed at increasing corna[euro][TM]s resistance to A. flavus are a key component in efforts to reduce aflatoxin contamination. The USDA has released maize lines that show resistance; however, introgression of resistance into production lines has been limited due to a lack of resistance specific markers. Microarray analysis of one a[euro]ceresistanta[euro][dot] line (Mp313E) identified 234 genes that are differentially expressed when compared to the susceptible line (Va35) during A. flavus infection. Of these 234 genes, we have mapped 64 to the 10 maize chromosomes. Out of the 64 genes, 25 map directly to the Quantitative Trait Loci (QTL) on chromosomes 2, 3, and 4, which account for approximately 48% of resistance in Mp313E. Using loci specific primers, these 25 genes have been PCR amplified and are being sequenced to identify single nucleotide polymorphisms (SNP). Gene specific primers associated with resistance will be designed using SNPa[euro][TM]s for subsequent use in marker assisted selection (MAS) of hybrid maize lines.

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF A PREDICTED DROSOPHILA GLUTAMINYL CYCLASE 

Amanda Parker* and Robert C. Bateman, Jr., University of Southern Mississippi, Hattiesburg, MS 39406

Glutaminyl cyclase (QC) is responsible for the N-terminal conversion of glutamate to pyroglutamate. Though the N-terminal pyroglutamate is found in many different peptides in virtually all species, including Drosophila, QC activity has not been observed in Drosophila. Sequence homology analyses revealed protein sequences in Drosophila with high sequence homology to human QC. To evaluate the enzymatic activity of the predicted Drosophila QC sequence a bacterial fusion protein was designed which contained the predicted amino acid sequence for Drosophila QC along with a purification tag. Expression of the fusion protein and subsequent purification revealed a protein of approximately 44kD. The 44kD protein was evaluated using two previously published QC activity assays. Both assays revealed QC activity in the fusion protein, suggesting the Drosophila protein is a QC. The Drosophila QC revealed similar activity as the human QC and was also inhibited with published QC inhibitors. Further analyses will be completed to compare the structural properties of the Drosophila QC to the human QC.

EFFECT OF MELATONIN ON DEVELOPMENT OF PORCINE PREIMPLANTATION EMBRYOS IN VITRO

Nelida Rodriguez-Osorio (1*), In Ju Kim (1), Song-yi Jung (1), Abdullah Kaya (2), and Erdogan Memili (1), (1) Mississippi State University, Mississippi State, MS 39762 and (2) University of Wisconsin, Madison, WI 53706

Melatonin, a reactive oxygen species scavenger, prevents apoptosis and has been used for improving embryo in vitro development. This study aimed at determining the effects of melatonin on porcine embryo cleavage, blastocyst rates, and blastocyst cell number. Porcine abattoir-derived oocytes were subjected to standard in vitro maturation and fertilization procedures. Fertilized oocytes cultured in PZM-3 medium supplemented with Melatonin at concentrations of [10.sup.-3], [10.sup.-6], [10.sup.-9] and [10.sup.-12] M, 5 hours after insemination (hpi), and cultured in a humidified incubator with 5% C[O.sub.2] at 39[degrees]C. Cleavage rates were assessed 48 hpi, and blastocyst rates determined at day 6 post fertilization. The number of cells per embryo was assessed after DAPI epifluorescent staining. Experiments were repeated 8 times and the results were analyzed using t-test. At [10.sup.-9] M concentration, Melatonin had a positive effect on cleavage rates with 45%[+ or -]1.94 (mean[+ or -]SEM) against 39%[+ or -]2.01 in the control group (p<0.05). Conversely, at [10.sup.-3] M, melatonin had a negative effect on cleavage rate with 32[+ or -]5.59%. Blastocyst rates were also higher for the Melatonin [10.sup.-9] M group with 43%[+ or -]0.79 and lower, in the group supplemented with [10.sup.-3] M melatonin 19%[+ or -]0.71 (p<0.05). There were no differences in embryo development from the groups with Melatonin at concentrations [10.sup.-12] M and [10.sup.-6] M. Blastocyst cell numbers were significantly higher in the Melatonin [10.sup.-9] M group, with 65[+ or -]3.22 (cell number[+ or -]SEM), compared to 50[+ or -]2.07 for the control group (p<0.01). Our data indicate that melatonin ([10.sup.-9] M) supports development of porcine preimplantation embryos and increases total number of cells in the blastocysts.

DEVELOPMENT OF A ZEOCIN RESISTANCE CASSETTE FOR MOLECULAR GENETIC RESEARCH IN THE PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM

Brooke Wheeler* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406

Histoplasma capsulatum is a dimorphic fungus which can exist in mold form at 25[degrees]C and morph into a yeast form at 37[degrees]C. The conversion to the yeast form is required for pathogenesis. Histoplasma capsulatum is the causative agent for the disease histoplasmosis. This disease causes an estimated 500,000 infections each year in the United States alone. The fungus is endemic to the Mississippi and Ohio River valleys; however, it can be found throughout the world. An impediment for understanding the molecular genetics of the mold-to-yeast dimorphism is the lack of dominant selectable genetic markers. Therefore, we undertake this study to develop an efficient Zeocin-resistance cassette. A heterologous promoter from Aspergillus nidulans transcription elongation factor I gene, a Zeocin resistance gene isolated from Stretoalloteichus hindustan-us, and a TrpC terminator from Aspergillus nidulans were fused via PCR. The resulting fragment was then placed in a telomeric vector. In one construct a second selectable marker, AnURA5 was included. The vector was then electroporated into H. capsulatum to determine the effectiveness. If this construct is effective, a new dominant selectable marker will be available for molecular genetic research. The heterologous promoter will be resected to determine the minimal functional sequence.

ELUCIDATION OF THE SULFUR METABOLIC PATHWAY IN THE DIMORPHIC PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM: ISOLATION AND CHARACTERIZATION OF THE CDO1, GSH1 AND GSH2 GENES

Melissa Adams* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406

The dimorphic fungus, Histoplasma capsulatum is the causative agent of histoplasmosis, the most common systemic fungal disease of man. Histoplasma grows in certain soils as a multicellular saprophytic mold. In the lungs of an infected host, a shift to the unicellular parasitic yeast occurs. The de-differentiation from the multicellular mold to the unicellular yeast morphotype is required for the progression of the disease. Sulfhydryl groups (particularly cysteine) are necessary for the mold to yeast transition. Several pathways are involved in cysteine metabolism in Histoplasma. In order to better understand the molecular basis of the dimorphism, the different pathways are being elucidated. The cysteine dioxygenase (CDO1) gene from Histoplasma capsulatum has been isolated in our lab. Reports in the literature indicate that CDO1 enzyme activity is only found in the yeast morphotype. Our data however show that the CDO1 transcript is abundant in both the mold and yeast morphotypes. Either the reports in the literature that CDO1 enzyme activity is yeast specific are incorrect or some level of post-transcriptional control is involved. To study this question, we are conducting enzyme assays and correlating this data to transcript levels measured by northern blotting and real time PCR. In addition, we are beginning studies to examine two enzymes involved in glutathione synthesis (gamma-glutamyl cysteine synthetase, GSH1 and glutathione synthetase, GSH2).

CHARACTERIZATION OF LATE EMBRYONIC B-CELL STAGES IN CHICKEN

Balazs Felfoldi*, Amanda M. Cooksey, and G. Todd Pharr, Mississippi State University, Mississippi State, MS 39762

The bursa of Fabricius serves as a primary lymphoid organ for the development of a diverse repertoire of B-cells. B-cell development is initiated between embryonic days (ED) 8-14 when the bursa anlage is populated by a single wave of committed stem cells expressing the sialyl Lewis X carbohydrate (SLEX). Between ED 15-17, there is a phenotypic transition in cell surface glycosylation from SLEX to high levels of a related carbohydrate structure termed Lewis X (LEX). The switch in surface glycosylation correlates with the onset of repertoire development by immunoglobulin-gene conversion. A thorough proteomic analysis of the two B-cell stages is needed to understand their perspective physiology and to identify the genes controlling the progression from one developmental stage to the next. The objective was to identify proteins expressed by developing B-cells before and after the SLEX to LEX transition. Using mass spectrometry, we identified 612 proteins in ED 15 B-cells and 823 in ED18 B-cells. The differentially expressed proteins were cell-adhesion apoptosis-associated and signal transduction molecules. The ED 15 B-cells express proteins that show a stem cell-like phenotype with anti-apoptotic and cell-adhesion molecules associated with homing to the bursa. The ED18 B-cells express pro-apoptotic proteins, cell adhesion molecules with both homo- and heterophilic binding activity and various immune response receptors. Future studies will evaluate mRNA levels and will visualize the expression patterns of genes on a histological level.

IDENTIFICATION OF DOCKING SITE MUTATIONS ON THE AMINO TERMINUS OF NIFM, WHICH ACTS AS A REGULATORY REGION FOR THE PEPTIDYL PROLYL CIS/ TRANS ISOMERASES ACTIVITY OF NIFM

Vandana Chaturvedi*, Lakshmi Pulakat, Nara Gavini, Mississippi State University, Mississsippi State, MS 39762

The Parvulins are ubiquitous and a highly conserved family of Peptidyl prolyl cis/trans isomerases (PPIases). The ESS1 in Saccharomyces cerevisiae was the first parvulin to be found in eukaryotes. Mutations in the ESS1 protein rendered the yeast strains temperature sensitive. The ESS1 temperature sensitive strains (ESS[1.sup.TS]) grew like the wild type at lower temperature but were unable to grow at restrictive temperatures. A homology search showed that the carboxy terminus of NifM from Azotobacter vinelandii protein, which is 292 amino acid long, shows a high structural homology with the PPIase domain from the eukaryotes. The common motifs and putative catalytic residues were also conserved. The 876-basepair-nif m when cloned into pYES2.1 TOPO vector downstream of galactose promoter and then transformed into ESS[1.sup.TS] strain H164R. was unable to rescue the ESS[1.sup.TS] mutants, the truncated NifM containing only the C terminal PPIase domain could do so. However when nifH, the substrate for nifm, is present along with entire nifm, the ESS[1.sup.TS] mutants could grow at restrictive temperature. This elucidates that the substrate nifH is binding at amino terminus of the NifM and thereby opening and exposing the carboxy terminus catalytic domain for the isomerisation activity. The above results shows that the amino terminus of NifM is conformationally blocking the catalytic activity of its carboxy terminus. A mutant of amino terminus of NifM, with mutations located at position 33, 146 and 155 amino acids showed loss of activity with the PPIase domain. This was defined as docking site mutant.

EVALUATION OF FACTORS AFFECTING FUNCTIONAL ANNOTATION QUALITY IN EUKARYOTIC GENOMES

Teresia Buza*, Nan Wang, Susan M. Bridges, Shane C. Burgess, and Fiona McCarthy, Mississippi State University, Mississippi State, MS 39762

High-throughput technologies, such as DNA microarray-based gene expression profiling and proteomics provide valuable information on the genes and gene products. The functional interpretation of microarray and proteomics data is a time-consuming and poses major challenge to many researchers. The Gene Ontology (GO), a controlled vocabulary for describing the biological function of a gene product, is the de facto standard for functional annotation in any organism. A major problem with GO is that researchers do not know the quality of GO entries that they are using to link genes with functions. Additionally, investigators may not be aware of any changes in GO usage. We developed a stable method that take into consideration the GO breadth, depth and quality and we combine these aspects to determine the overall GO annotation quality (GAQ) for a species. We used three different gene datasets to determine the GAQ scores in each organism. The results revealed that GAQ is species-specific and is positively correlated to the quality and depth of GO terms. GAQ represent a powerful stable new approach for showing how good GO is at the time of functional modeling of genes and gene products. Improved GAQ will lead to improved modeling of microarray and proteomics data. Unlike other GO tools, GAQ can be applied in any species.

FOLDING PATHWAY OF THE FE-PROTEIN: EFFECT OF AMINO ACID REPLACEMENTS ON NIFM INDEPENDENT FE-PROTEIN CONFORMATION

Girish V Jamnekar*, Lakshmi Pulakat and Nara Gavini, Mississippi State University, Mississippi State, MS 39762

Nitrogenase is composed of two distinct components, the Fe-protein and the MoFe-protein. The Fe protein is an electron donor for the reduction of Nitrogen in MoFe-protein. The Fe-protein requires several accessory genes of the nif-gene cluster to form sn functional unit of Nitrogenase. The NifM is an accessory gene, which is a PPIase, is required for the folding of the Fe-protein. The PPIase alter the protein conformation through isomerization of peptidyl-prolyl bonds of N-terminal of the proline residue. The NifM protein is inactive in Azotobacter vinelandii strain (AV98) due to insertion of ka[n.sup.r] cassette in the nifM and exhibits a Ni[f.sup.-] phenotype. Therefore, the NifH is unable to undergo cis/trans isomerization leading to improper folding. Using chemical mutagenesis, the nifH mutants were generated and. In the present study, the nifH mutant, pBG1161 having mutations leading to change in amino acids in the region 210-230aa. It was studied in Azotobacter vinelandii strain (AV98). Growth curve analysis showed that the pBG1161 with mutant nifH was able to change phenotype of A. vinelandii (Av98) to Ni[f.sup.+]. To understand NifM independence of the mutant Fe-protein, we studied the interactions between small regions NifH regions using Bacteriomatch Two Hybrid System. The Fe-protein was dissected into three distinct smaller regions, 30-73aa (F1), 205-240aa (F2) and 240-289aa (F3). These analyses, combined with molecular modeling analysis showed that the region spanning amino acids 210 to 230 may be involved in the active conformation of the Fe-protein of nitrogenase in the absence of NifM.

ASSEMBLY OF THE LANTIBIOTIC MUTACIN 1140 IN MEMBRANES

James L. Smith (1*), Shawanda Wilson-Stanford (1*), Eefjan Breukink (2), Hester E. Hasper (2), and Jan Novak (3), (1) Mississippi State University, Mississippi State, MS 39762, (2) Utrecht University, The Netherlands, and (3) University of Alabama, Birmingham, AL 35294

Multi-drug resistance has lead to a great demand for new antibiotic development. Mutacin 1140 and nisin A, are posttranslationally modified peptide antibiotics belonging to a family of antibiotics called type A lantibiotics (lanthionine-containing antibiotics). There is a high degree of structural and sequence similarities in the N-terminal rings A and B of both nisin and mutacin 1140, which is known to be a lipid II binding domain. Nisin function by binding to lipid II and forming transmembrane pores causing the efflux of ions, ATP, and other essential cellular components. More recently, another antimicrobial mechanism of activity has been reported, where mutacin 1140 and nisin abduct lipid II from the site of new cell wall synthesis. Further experimentation using fluorescence techniques reveals that mutacin 1140 has a higher affinity for lipid II than nisin and that the lipid II-mutacin 1140 assembly differs from the assembly of lipid II-nisin complexes. These differences may be attributed to the fact that nisin is a pore former and mutacin does not form pores in membrane system used in our experimental systems or in vivo against Streptococcus rattus, a bacterium known to be sensitive to mutacin 1140. Furthermore, we show that mutacin 1140 has a strong propensity for complex formation by electron microscopy.

AN ADDITIONAL BIOCHEMICAL MECHANISM FOR PERTUSSIS TOXIN-MEDIATED NEURONAL DAMAGE

Mary Hetrick (1*), Dieter Knowle (2), Nitin Warier (2), Nara Gavini (1), and Lakshmi Pulakat (1), (1) Mississippi State University, Mississippi State, MS 39762 and (2) Bowling Green State University, Bowling Green, OH 43403

Infants are vulnerable victims of whooping cough and neurological damage is often an after-effect among infected infants. The known biochemical mechanism for S1-mediated pathogenesis is ADP-ribosylation of Gi protein and disruption to the signaling of cognate receptor. Signaling by the Angiotensin II receptor AT2 is inhibited by Ptx, although whether the AT2 receptor activates Gi & alpha is unclear. High-level expression of AT2 is exhibited in infant brain implicating a role for this molecule in neuronal development. We have shown through yeast two-hybrid analysis that the S1 subunit can directly interact with the 3rd ICL and cytoplasmic tail of AT2. To determine whether S1 interacted with AT2 in mammalian cells, we partially purified a His-tagged S1 subunit and tested whether it could interact with AT2 expressed in Chinese Hamster Ovary (CHO) cells. Our studies showed that AT2 could co-immunoprecipitate with S1, however mutations in the 3rd ICL or C-terminus of AT2 inhibited this interaction in mammalian cells. Based on these studies, we contend that S1 inhibits AT2-mediated signaling via sterical inhibition. The significance of this observation is that S1 uses sterical hindrance of AT2 via direct interaction to cause pathological effects. This raises the question of whether the current acellular vaccines, which involve mutations in the catalytic region of the S1 subunit, are safe or possess the capability to interfere with AT2-mediated signaling.

QUANTITATIVE SERUM PROTEOMICS OF A TRYPTOPHAN DEPLETED DIET USING [.sup.18.O] LABELING AND A RUBISCO INTERNAL STANDARD

Amanda M. Cooksey (1*), Marek D. Koter (2), Alejandro Corzo (1), Timothy S. Cummings (1), Michael T. Kidd (1), and Shane C. Burgess (1), (1) Mississippi State University, Mississippi State, MS 39762 and (2) University of California, Irvine, CA 92697

Tryptophan plays an important role in vertebrate metabolism as not only a building block of proteins, but also as a precursor of serotonin, melatonin, niacin and kynurenines, which influence immune tolerance. Here we use a quantitative proteomic analysis of serum to model tryptophan deficiency. We applied bidirectional [.sup.16/18.O] labeling to serum proteins from chickens fed on tryptophan depleted and adequate diets, using the plant protein Rubisco as an internal standard. The proteins were trypsin digested and processed by 2-dimensional liquid chromatography electrospray ionization tandem mass spectrometry (2D-LC ESI [MS.sup.2]). The resulting mass spectra were analyzed using the SEQUEST algorithm and the ProteinMapper program to identify up- and down-regulated proteins. We identified 4161 proteins labeled bidirectionally, of which 46 were up- and 90 were down-regulated. Using Ingenuity Pathways Analysis (IPA) software, we found tryptophan deficiency influences not only protein synthesis, energy production and immune response, but also cell/tissue morphology, cell cycle progression and cancer. From these results, we are able to conclude that tryptophan plays a role in a number of important physiological processes, further elucidating the role of tryptophan and emphasizing the importance of this essential amino acid.

SITE DIRECTED MUTAGENESIS OF THE CARBOXYSOME CARBONIC ANHYDRASE OF HALOTHIOBACILLUS NEAPOLITANUS

Charles Murin*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg MS, 39406

Carboxysomes are proteinaceous organelles that sequester ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), the primary carbon fixing enzyme of autotrophic bacteria. These microcompartments are thought to participate in a carbon concentrating mechanism (CCM) through the action of a carbonic anhydrase (CA), which is associated with the carboxysome protein shell. The CA catalyzes the reversible dehydration of HCO3- and is believed to enhance the catalytic efficiency of RubisCO within the carboxysome by converting bicarbonate to C[O.sub.2] for fixation. Insertion mutagenesis of the CA gene (csoSCA) of H. neapolitanus with a kanamycin resistance cassette and subsequent complementation with a wild type allele has revealed that the carboxysomal CA is vital for growth of the bacteria at ambient CO2 levels. The structure of the carboxysomal CA (CsoSCA) was elucidated by x-ray crystallography and revealed an active site with a zinc ion that is coordinated by Cys173, His242, and Cys253 residues. In addition, Asp406 and Arg457 are believed to be crucial residues of the active site. Site-directed replacement of Asp406 with Glu was performed and the resultant mutant CA was expressed as recombinant protein in Escherichia coli to assess the role of Asp406 in enzymatic catalysis of CA by a stopped-flow spectrophotometric assay. Combined with results from the analysis of other mutant versions of the carboxysomal CA, these results will be used to elucidate the enzyme's catalytic mechanism.

IDENTIFICATION OF WOOD DECAY FUNGI BY FUNGAL AND BASIDIOMYCETE INTERNAL TRANSCRIBED SPACER PRIMERS

M.L. Prewitt (1*), S. V. Diehl (1), T.C. McElroy (2), and W.J. Diehl (1), (1) Mississippi State University, Mississippi State, MS 39762 and (2) Kennesaw State University, Kennesaw, GA 30144

Wood decay is desirable when it occurs in carbon recycling and a nuisance when it occurs in our homes causing major repair expenses. Traditional methods to protect wood from decay have focused on the use of chemical preservatives as broad spectrum pesticides without knowing the identity of the specific organism or organisms responsible for the decay. Basidiomycete fungi are the principle decomposers of wood and in order to better control and understand wood decomposition, a method to rapidly identify decay fungi is needed. The objective of this study was to compare Restriction Length Polymorphism Length Polymorphisms (RFLP) and sequence data from the internal transcribed spacer regions amplified with general fungal and basidiomycete fungal primers in identifying isolates of selected wood decay fungi. DNA was extracted from pure fungal cultures, amplified with either general fungal or basidiomycete primers, sequenced or digested separately by four enzymes: Hinfl, HaeIII, Alu and Taq. Results from this study indicated that isolates of Phanerochaete clustered separately from isolates of Trametes in both RFLP data and sequence data. Isolates of T. hirsuta and T. versicolor showed separate clustering patterns. However isolates within Gloephyllum did not show a distinct clustering pattern. RFLP produces similar clustering patterns as sequence data in distinguishing isolates of Phanerochate chyrsosporium, Trametes versicolor and Trametes hirstua.

BOVINE GERMINAL VESICLE OOCYTE : A SYSTEMS APPROACH

Divyaswetha Peddinti*, Bindu Nanduri, Erdogan Memili, and Shane C. Burgess, Institute for Digital Biology, Mississippi State University, Mississippi State, MS 39762

The bovine oocyte is a model for studying reproduction, fertility and reproductive diseases in humans. Oocyte undergoes many transitions in its life span that must evoke a dynamic network of gene expressions. The recent completion of the bovine genome sequence provides the opportunity to study oocyte development at systems level by applying functional genomics techniques. Describing the proteome of the oocyte will allow us to model protein interaction networks and pathways involved in its maturation. The objective of this study is to define the proteome of GV stage oocyte using high-resolution differential detergent fractionation (DDF) proteomics and apply systems approaches to model its function. Follicles of 2-8 mm, containing oocytes were collected and proteins extracted using differential detergent fractionation (DDF). Proteomic analysis was done using duplicate samples and 1950 proteins were identified using SEQUEST. Pathways involved in oocyte maturation and development and its phenotype were identified using Ingenuity pathway analysis (IPA). Since bovine proteins are not accepted by IPA, by BLAST searches we identified human orthologs of our bovine proteins for IPA analysis. Of the 48 networks found, majority of oocyte proteins were identified in a network involved in cellular growth and proliferation. To bring about this prolifera-tive function in oocyte, signaling pathways are necessary. We analyzed the various signaling pathways that the identified oocyte proteins represent. We identified 18 signaling pathways at IPA significance threshold. EGF signaling, PPAR signaling, IGF-1 signaling, VEGF signaling, and PDGF signaling is necessary for oocyte maturation and we identified all these signaling pathways in our DDF oocyte proteome. However, the most significant signaling pathway identified was cell cycle: G2/M DNA damage checkpoint regulation. This pathway could help maintain the integrity of oocyte genome during different stages of development. Our oocyte proteome systems analysis confirmed the expression of a number of proteins that can describe both oocyte phenotype and maturation and development. The better we understand the interaction networks involved in oogenesis, the more rational our approach will be to modulate bovine fertility.

REGULATION OF DBF4-DEPENDENT KINASE IN SACCHAROMYCES CEREVISIAE

Asela Roberts, Marilyn Burke, and Bernadette Connors*, Millsaps College, Jackson, MS 39210

In Saccharomyces cerevisiae, dbf4p initiates DNA synthesis by activating and escorting cdc7p to origins of replication, after which the protein is proteolyzed. Dbf4p levels peak at the onset of S phase and are maintained until late mitosis, whereas cdc7p levels are maintained at a constant level through the cell cycle. It is hypothesized that regulation of DDK is accomplished in part through the proteolysis of dbf4p, as well as by changes in its association with cdc7p. My research aims to investigate the regulated proteolysis of dbf4p, and examine the genetic interactions between DDK and spindle checkpoint genes that control exit from mitosis. In order to elucidate dynamic biochemical interactions, an HA-tagged version of dbf4p was used in coimmunoprecipitation experiments with cells blocked in early S phase as well as late mitosis. Furthermore, both deletion and mutational analysis will be used to identify sites of direct interaction between dbf4p and its interacting partner(s). With the goal of revealing genetic interactions between dbf4 or cdc7 and genes known to regulate the metaphase to anaphase transition, crosses between either dbf4-1 or cdc7-7 and selected strains deleted for nonessential spindle checkpoint genes were performed. Tetrad analysis has shown novel interactions between both dbf4 or cdc7 and many of these genes. Since disruption of the orderly progression of cell cycle events often leads to unrestrained cell growth and predisposition to cancer, research into the molecular mechanisms that regulate these processes is consequently of great medical and scientific interest. Characterization of DDK regulation will help to elucidate the controls involved in this orderly progression of the cell cycle among eukaryotic organisms.

ERYTHROMYCIN BLOCKS NON-SELECTIVE CATION CHANNELS IN THE AIRWAY MUCUS GLAND CELLS

Huiling Liu, Bela Kanyicska, and Jerry Farley*, University of Mississippi Medical Center, Jackson, Mississippi 39216

Erythromycin (EM), a macrolide antibiotic, has been known to directly inhibit airway mucus secretion that is independent of its anti-bacterial and anti-inflammatory effects. In this study, we examined the mechanisms of erythromycin actions. In Ussing chamber studies, EM, applied serosally but not apically, blocked ACh or thapsigargin-induced short-circuit current in the plateau phase, and non-selective cation channel blockers such as 2-APB and SKF96365, had similar effects to EM. Under whole-cell patch clamp, ACh or thapsigargin induced non-selective cation current that was blocked by EM, 2-APB, SKF96365, and [Gd.sup.+3]. Real-time RT-PCR detected mRNA expressions for TRPC-1, 3, 4 and 6 in the freshly isolated mucus gland cells. Taken together, these data demonstrate that EM inhibits ACh- or thapsigargin-induced capacitive [Ca.sup.2+] entry by blocking [Ca.sup.2+] influx via non-selective cation channels. These data suggest that EM inhibits mucus secretion by blocking [Ca.sup.2+] entry required for mucus release. This study was supported by a grant from American Heart Association to J.M.F.

COMPARATIVE STUDIES OF VERNONIA AMYGDALINA AND TAMOXIFEN IN MCF- 7 TUMORAL CELLS

Jetaime C. Ross*, Michael Opata, and Ernest. B. Izevbigie, Jackson State University, Jackson, MS 39217

Breast cancer claims the lives of approximately 40,000 women annually in the U.S. One in every eight women will be diagnosed with the disease in her life time. Botanicals may be combined with conventional cancer drugs to optimize treatment outcome. Evidence suggests that a Nigerian edible leaf extracts [V. amygdalina (VA)] may be anti-tumorigenic. The effects of co-treatment of cells with VA and conventional breast cancer drug(s) are unknown. The objective of the study was to assess the effects of Tamoxifen (TAM), VA, and the combination of TAM and VA on cell number and DNA synthesis in MCF-7 cells. Cell number was determined using a hemacytometer and DNA synthesis was determined by [[.sup.3.H]]thymidine incorporation assays. TAM treatment inhibited cell growth in a concentration-dependent fashion (0 TAM, 0%; 1 [micro]M TAM, 25 [+ or -] 10%; 10 [micro]M, 51.3 [+ or -] 0%; and 100 [micro]M, 98 [+ or -]0.4% (P <0.05) cell growth inhibition). Likewise, VA inhibitory action was also concentration-dependent [0 VA, 0%; 50 [micro]g/ml, 25.8 [+ or -]9% (P <0.05); 150 [micro]g/ml, 42.0 [+ or -] 24.7%; and 450 [micro]g/m/, 51.7[+ or -] 14.9% (P <0.01] cell growth inhibition). CONCLUSION: TAM [IC.sub.50] was reduced by approximately ten-fold in the presence of VA (1[micro]g/ml [+ or -] 0.08 with VA vs. 10 [micro]g/ml [+ or -] 1.2 without VA). These data suggest that VA, upon further assessment through clinical studies, may reduce the therapeutic dosage of TAM and its unwanted side effects, and improve the quality of life for cancer patients.

ROLE OF NIFH GLU112 IN BINDING TO NIFK OF NITROGENASE COMPLEX

Hanqing Dong, Anberitha Matthews*, Lakshmi Pulakat, and Nara Gavini, Mississippi State University, Mississippi State, MS 39762

In Azotobacter vinelandii, biological nitrogen fixation is catalyzed by nitrogenase complex, which is composed of the nitrogenase dehydrogenase (NifH, 289aa) and nitrogenase (NifDK, 492aa and 523aa respectively). The proper interaction between NifH and NifK is essential for the enzyme conformation as well as the orientation of electron flow. It has been reported that one of the important factors facilitating the interprotein communication is the carbodiimide crosslink formed by Glul 12 of NifH and Lys400 of NifK. To determine how the interaction is influenced by the characteristics of the amino acids available at position 112 of NifH, we introduced site mutations by site-directed mutagenesis to the codon encoding for Glu112 thus generating proteins carrying various residues instead of glutamic acid. The resulting mutants include Glu112Asn and Glul 12Lys, both of which are differently charged compared to the residue found in the wild type strain. Growth analyses indicated that both mutant strains are capable of propagation under nitrogen-deficit conditions although the growth rate is approximately 15% lower than that of wild type strain. Superimposed crystal structures of the mutant proteins also highlight the possibility that both mutated residues can be appropriately accommodated. Therefore the charge carried by the amino acid at position 112 of NifH plays a minor role in the interaction whereas; a more important factor is the length of the side chains.

CONSTRUING THE ROLES OF ETHYLENE AND JASMONIC ACID IN THE EXPRESSION OF THE MAIZE HERBIVORE DEFENSE PROTEIN C MIR1

Arunkanth Ankala (1*), Dawn S. Luthe (2), Paul Williams (3), and Jeff R. Wilkinson (1), (1) Mississippi State University, Mississippi State, MS 39762, (2) Pennsylvania State University, University Park, PA, 16802, and (3) USDA-ARS Corn Host Plant Resistance Research Unit, Mississippi State, MS 39762

Maize genotypes like MP708 that are resistant to insect feeding employ a novel herbivore defense cysteine proteinase (Mir1), which efficiently retards the growth of Spodoptera frugiperda (fall army worm). The regulatory signals in maize responsible for inducing defense mechanism against insect feeding are orchestrated by two major plant hormones, Ethelyne and Jasmonic acid. To investigate the roles of Ethelyne and Jasmonic acid in the signal transduction pathway of Mir1 expression, the resistant genotype (MP708) was treated with exogenous ethylene and total protein was isolated. Western blot analysis of the treated samples showed increases in the Mir1 protein levels when compared to untreated samples. This increase in Mir1 indicates that ethylene alone is sufficient to induce expression. Subsequent western blots of Ibuprofen and Antipyrine, two Jasmonic acid inhibitors, treated plants revealed a decrease in Mir1 when compared to control plants. However, treatments with exogenous Jasmonic acid alone result in no increase in mir1 expression. It is clear that Jasmonic acid is involved in regulation of Mir1 expression, though the exact mechanisms are not understood. To investigate the role of Jasmonic acid and how interactions with Ethelyne effect Mir1 expression, MP708 lines will be treated with exogenous ethylene, ethylene inhibitors, exogenous Jasmonic acid and Jasmonate inhibitors alone and in combination. Expression of Mir1 will be evaluated and a model of hormonal regulation of the insect resistant protein will be constructed.

THE ROLE OF DETOXICATION ENZYMES IN THE AGE-RELATED TOXICITY DIFFERENCES OF ORGANOPHOSPHATE INSECTICIDES

Valerie C. Beasley, Edward Meek, Howard W. Chambers, Janice E. Chambers, Russell L. Carr, Mississippi State University, Mississippi State, MS 39759

Organophosphate (OP) insecticides are more toxic to juvenile animals than they are to adults. It is thought that the reduced vulnerability of adults is mediated primarily by the higher levels of detoxication enzymes such as the carboxylesterases and non-target cholinesterases. It is thought that if these enzymes were not present in the adult, the toxicity level of an OP would be similar in adults and juveniles. To test this, adult rats and juvenile rats at postnatal day 1 (PND1) and PND12 were treated with a similar dosage of paraoxon (P=O) and brain cholinesterase (ChE) inhibition was determined. Additional adult animals were then pre-treated with specific inhibitors of the carboxylesterases and non-target cholinesterases prior to exposure to P=O and brain ChE inhibition was compared to the inhibition obtained without the pre-treatment. A dosage of, 0.25 mg/kg resulted in 18%, 90%, and 96% inhibition in adults, PND12, and PND1 animals, respectively. However, when detoxication enzyme activity was eliminated in adults prior to P=O treatment, the level of ChE inhibition increased to 86% which is comparable to that observed in the juvenile animals. These data suggest that the greater vulnerability of juvenile animals to OP insecticides is due to, for the most part, the lack of protective esterases which are present in the adult. (Supported by NIH R01 ES011287).

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

Brandy N. Roberts, Catrina Prather, Amanda Williams, Jessica R. Walker, Ashley Garriga, and Lauren Brandon*, Mississippi University for Women, Columbus, MS 39701

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 will continue to use complementation or recombination to map the remainder of these mutations.

FUNCTIONAL ANALYSIS OF A ModC HOMOLOG IN THE Azotobacter vinelandii nif-GENE CLUSTER

Sangeetha Shivaji*, K. Raja, Lakshmi Pulakat, and Nara Gavini, Mississippi State University, Mississippi State, MS 39762

The nif genes of the nif regulon are required for production of MoFe-protein in Azotobactor vinelandii. This regulon also contains several other potential genes (ORFs). Amongst these, ORF10 is of particular importance because its gene product is similar to the molybdate transporter protein ModC. ModE is a molybdate-dependent regulator of several operons including the molybdate transport system of modABC. Analysis of the ORF10 promoter revealed potential binding sites for RpoN, NifA, and ModE, indicating regulation based on nitrogen availability and intracellular molybdate concentrations, respectively. To further investigate ORF10, we constructed the mutant-ORF10 A. vinelandii strain MH5200. This was accomplished by cloning the 1.66kb sequence of ORF10 and flanking regions into a plasmid and subsequently interrupting ORF 10 with Ka[n.sup.R] gene via Pst1-digestion. The resultant plasmid was introduced into wild-type A. vinelandii. Transformants exhibiting kanamycin-resistance, which indicated genomic incorporation of Ka[n.sup.R]-interrupted ORF 10 since plasmid replication cannot occur in A. vinelandii, were selected. Comparisons of mutant growth with wild-type, in molybdate-limited conditions, showed that the ORF10 mutation can be detrimental to diazotrophic growth and implied that ORF 10 plays a role in the maturation and assembly of FeMo-cofactor.

THE ROLE OF NIF-SPECIFIC CLPX IN AZOTOBACTER VINELANDII

Hanqing Dong*, Preeti Patil, Lakshmi Pulakat, and Nara Gavini, Mississippi State University, Mississippi State, MS 39762

Nitrogenase, which catalyzes the biological reduction of atmospheric nitrogen, consists of the Fe and the MoFe protein encoded by nifH and nifDK genes respectively. The Orf9 of Azotobacter vinelandii exhibits about 53% identity and 75% similarity with ClpX. ClpX is an ATPase involved in substrate recognition. It interacts with C1pP to form ClpXP, which is an ATP dependent protease complex found in many prokaryotes and eukaryotes. In order to decipher the functional role of Orf9 in the biogenesis of nitrogenase, we have investigated its interaction with proteins encoded by nif-structural genes, the nifHDK, by utilizing BacterioMatch[TM] Two Hybrid system. The DNA corresponding to orf9 was PCR amplified and cloned in pBT and nifH, nifD, and nifK were cloned in pTRG. The interactions of Orf9 with Nif proteins were detected by analyzing the expression of the reporter genes, the lacZ and bla. The Orf9 showed positive interaction with NifK, whereas no detectable interaction was observed between NifD and NifH. Amino acid sequence comparisons revealed that carboxyl terminus domain of NifK shares about 36% homology with the SsrA tag, the ClpX recognition sequence. A deletion in carboxyl terminus domain of NifK abolished its interaction with Orf9. Furthermore, based on an analysis of interactions of ClpX with the three Nif proteins, we proposed that the Orf9 is a nif specific ClpX and its putative functions include a role in MoFe-protein assembly. The growth analysis also indicated that Orf9 plays an important role in nitrogenase activity during pH stress conditions.