Cellular, molecular and developmental biology.
Vice-chair: Lidija Halda-Alija, University of Mississippi
Exhibit Hall A2
8:30 ETHANOL TOXICITY IN JAPANESE MEDAKA DEVELOPMENT
Xueqing Wang (1), Asok Dasmahapatra (1), and John Boyle (2*), (1) University of Mississippi, University, MS 38677 and (2) Mississippi State University, Mississippi State, MS 39762
Ethanol is well-known as a developmental toxicant; however, the molecular mechanism(s) of this toxicity remains unclear. We have used Japanese medaka (Oryzias latipes) development as a model system to determine associations between ethanol metabolism and ethanol-mediated developmental toxicity. Like all other vertebrates, alcohol dehydrogenase (ADH) initiates alcohol metabolism in medaka. Previously, we cloned two full-length cDNAs encoding two ADH isozymes from the liver tissue of adult medaka. Phylogenetic analysis and computer modeling of the deduced amino acid sequence data identified one of them as an orthologue of mammalian Adh5 and the other as an orthologue of zebrafish Adh8. To study the effect of ethanol on regulation of ADH5 and ADH8 protein and mRNA expression during embryonic development of medaka, fertilized eggs were exposed to waterborne ethanol concentrations of 100 and 400 mM during the first two days of development and sacrificed in ovo at 2, 4, and 6 days post fertilization (dpf). Expression of Adh5 and Adh8 mRNAs were separately determined by semi-quantitative and quantitative real-time RT-PCR. The results indicate that both Adh5 and Adh8 mRNA expression were unaltered in embryos treated with ethanol. The results indicate that ethanol may modulate embryonic development in medaka in a different pathway without affecting ADH locus.
8:45 ANTIBODY TITERS IN FOUR DIFFERENT POPULATIONS OF ATLANTIC BOTTLENOSE DOLPHINS' (TURSIOPS TRUNCATUS) SERUM SAMPLES TO ERYSIPELOTHRIX RHUSIOPATHIAE
Amaziah Coleman* and Rhonda Patterson, University of Southern Mississippi, Hattiesburg, MS 39406 and Western Kentucky University, Bowling Green, KY 42101
Erysipelothrix rhusiopathiae isolated from a septicemic Pacific bottlenose dolphin (Tursiops gilli) provided by the Navy Marine Mammal program was used to perform an enzyme linked immunosorbent assay (ELISA). The ELISA was developed using Frasch (Frasch and Chapman, 1972) extracted antigen of the isolate as the ELISA capture antigen. An indicator system was developed consisting of biotin labeled rabbit anti-Tursiops truncatus IgG, alkaline phosphatase labeled avidin, and paranitrophenyl phosphate as the chromogenic substrate. The ELISA was used to evaluate anti-E. rhusiopathiae titers in a series of serum samples from four populations of Atlantic bottlenose dolphins (Tursiops truncatus). The four dolphin populations indicated consisted of free ranging dolphins from South Carolina living among heavy human traffic, free ranging dolphins from Florida living among low human traffic, captive dolphins from California living among heavy human traffic, and captive dolphins from the Bahamas living among low human traffic. Results indicated that, with the exception of one population, the majority of individuals in each population expressed a titer of 1/1,000. Results further showed that wild dolphins and dolphins living among heavy human traffic had the greatest number of individual dolphins expressing higher titers, while captive dolphins and dolphins living among low human traffic had the greatest number of individual dolphins expressing lower titers.
9:00 THE IMPORTANCE OF C-TERMINUS AXIN
Cherry Lockett (1*), Jie Zheng (2), and Youming Shao (2), (1) Alcorn State University, Lorman, MS 39096 and (2) St. Jude Children's Research Hospital, Memphis, TN 38105
Axin serves as a scaffold protein that binds directly to many proteins involved in the Wnt signaling pathway. It promotes the phosphorylation of a catenin by driving the formation of a complex with APC and GSK3. As such, axin is considered a negative effector of the pathway and functions as a tumor suppressor. However, when the axin-APC-GSK3 mediated degradation of a catenin is inhibited by Wnt signals that activate dishevelled (Dv1) or mutation of components of the Wnt signaling pathway, the concentration of a catenin in the cell increases, leading to an increased cell proliferation and possibly cancer. Axin contains a Dix domain at its C-terminus, and dishevelled protein contains a Dix domain at its N-terminus. The Dix domains of axin and dishevelled can form a heterodimer that leads to a decrease in phosphorylation of a catenin (Jin 2005). Therefore, the aim of the study is to produce the C-terminus of the axin protein to determine its structure for further observations of the interaction of the Dix domains of axin and dishevelled. A sample that had enough protein for nuclear magnetic resonance, NMR, studies was produced; however, it was not fully refolded and contained impurities. Therefore, it could not be used for the determination of the axin structure. Once the axin NMR structure is determined, it could be used to design a small molecule that will bind and inhibit axin C-terminus Dix domain from forming a heterodimer with dishevelled N-terminus Dix domain. This will hopefully aid in the development of drugs for cancer therapies.
9:15 FLUORESCENCE STUDIES ON THE INTERACTIONS OF TAT-PTD WITH LIPID BILAYER IN VITRO
Venkataswarup Tiriveedhi* and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406
Many researchers have shown the transfer of the positively charged protein-transduction domains [PTDs] through the living cells in a rapid and an apparently energy-independent manner by as yet unknown mechanism. Several authors have hypothesized an inverted micelle formation and/or adsorptive endocytosis as possible but not yet proven models to explain the transfer through the lipid bilayer. We used fluorescence spectroscopy to study the interaction between PTD of the HIV-1 TAT protein (TAT-PTD; residues 47-60 of TAT, fluorescently labeled with tryptophan) and the lipid bilayer in the presence of various fluorescence membrane probes. The TAT-PTD tryptophan exhibited a decrease in fluorescence intensity upon interaction of TAT-PTD with negatively charged lipid bilayers. The decrease was proportional with the amount of negative charge in the membrane. When TAT-PTD interacted with small unilamellar vesicles [SUVs] labeled with 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene [TMA-DPH], fluorescence resonance energy transfer [FRET] from tryptophan to TMA-DPH with an approximate efficiency of 10% was observed. No energy transfer was detected when the SUVs were labeled with 1,6-dipheny1-1,3,5-hexatriene (DPH), which partitions to the core of the lipid bilayer. No change in fluorescence anisotropy of either TMA-DPH or DPH was observed upon the interaction with TAT-PTD, indicating no significant disruption or perturbation of the lipid bilayer by the peptide. TAT-PTD did not cause dissipation of membrane potential (165 mV, negative inside) in negatively charged large unilamellar vesicles [LUVs]. The data suggest that TAT-PTD associates electrostatically with the bilayer surface and does not significantly perturb, and penetrate into, the bilayer core.
9:30 DETERMINATION OF INTRAMOLECULAR DISTANCES IN THE BACILLUS THURINGIENSIS TOXIN Cyt1A BY FRET AND MOLECULAR DYNAMICS SIMULATION
Kerrick Nevels (1*), Xiaochuan Li (1), Dexuan Xie (2), and Peter Butko (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) University of Wisconsin, Milwaukee, WI 53211
Cyt1A is an insecticidal cytolytic toxin produced by the spore-forming bacterium Bacillus thuringiensis var. israelensis. Knowledge of molecular details of the toxin's conformation changes in the presence of lipid membranes is important for elucidating the toxin's mode of action. Cyt1A contains a single cysteine residue. Labeling that residue with the fluorescence probe 5-((((2-iodoacety1)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) allowed for precise determination of the distance between the cysteine and a pair of two neighboring tryptophans by fluorescence resonance energy transfer (FRET). The distance was found to be 2.5 +/- 0.3 nm in Cyt1A in water. The same distance was also determined in silico--in the molecular dynamics model of Cyt1A. The agreement between the two numbers gives credence to our computer model of Cyt1A and at the same time confirms the predicted structure of Cyt1A in water. The magnitudes of FRET and the 1,5-IAEDANS fluorescence varied with increasing concentration of lipid (small unilamellar vesicles of egg phosphatidylcholine). The response was nonmonotonous: an initial increase was followed by a decrease at higher lipid concentrations. Interpretation of these results will be aided by quenching studies and measurements of FRET between 1,5-IAEDANS and membrane probes. Hypothetical lipid-induced conformation changes that are consistent with the experimental and simulation data will be presented.
9:45 CONSTRUCTION OF GENES THAT EXPRESS HISTONE-H1.S-PEPTIDE FUSION PROTEINS
Jason Adams (1*), Sonja Clemmons (2), and Susan E. Wellman (2), (1) Tougaloo College, Tougaloo, MS 39174 and (2) University of Mississippi Medical Center, Jackson, MS 39216
Fusion proteins are proteins created through genetic engineering from two or more proteins. The fusion proteins that we constructed were made from DNA sequences of histone-H1, which codes for the H1 protein, and the S-tag, which codes for the S-peptide. Our overall goal is to understand how H1 histones bind to DNA and how they condense chromatin. H1 proteins are linker proteins involved in the packaging of eukaryotic chromatin. The S-peptide consists of 15 amino acids. To determine if a fusion protein was successfully constructed, the FRET Works S-Tag Assay was performed on the non-purified H1 protein extract. When adding S-protein to the S-peptide, ribonuclease becomes active. The ribonuclease then cuts up the added substrate of poly C RNA. The released ribonucleotides absorb ultraviolet light and causes an increase in absorption which confirmed the presence of the fusion S-tag-H1 histone. This project was supported by the Mississippi Functional Genomic Network.
10:15 LOCALIZATION OF KIN3 AND CHARACTERIZATION OF THE EFFECTS OF KIN3 DELETION IN SACCHAROMYCES CEREVISIAE
John Gibson, Michael P. Jackson, and Sarah Lea McGuire*, Millsaps College, Jackson, MS 39210
The Kin3 gene of Saccharomyces cerevisiae encodes a non-essential serine-threonine protein kinase which has not been extensively studied. Kin3 is the homolog of the Aspergillus nidulans nimA gene, which is essential for mitotic entry downstream of the Cdc2 master mitotic regulator. It has been shown to localize to both chromatin and spindle pole bodies in A. nidulans and the fission yeast, Schizzosaccharomyces pombe. As part of efforts to characterize the Kin3 gene and its effects on cellular processes, we are characterizing the phenotype of a Kin3 deletion strain. Studies to date indicate that growth kinetics of the Kin3 deletion strain are similar to those of wild-type cells; both strains reach log phase growth and stationary phase growth with similar kinetics. However, the average number of cells at both log phase and stationary phase for Kin3 deletion cells is 50% less than that of wild-type cells. This suggests that the Kin3 deletion cells saturate at a lower cell density. In addition, the budding index of cells at log phase averages 62% budded in Kin3 deletion cells, compared to 52% budded in wild-type cells. This suggests that a higher percentage of Kin3 deletion cells have committed to completing cell division than wild type cells; they thus traverse the cell cycle with different kinetics. We are in the process of characterizing the effects of various drugs on the cell cycle of Kin3 deletion cells compared to wild-type cells. In addition, we are generating a YFP::kin3 strain for localization of Kin3 in Saccharomyces cerevisiae. This project was supported by NIH Grant Number RR 016476 from the MFGN INBRE Program of the National Center for Research Resources.
10:30 GENOME-WIDE INTERACTIONS AND EFFECTS OF THE [URE3] PRION OF SACCHAROMYCES CEREVISIAE
Sidney Taylor (1*), Piro Cipi (2), Amy Newton (1), and Ross Whitwam (1), (1) Mississippi University for Women, Columbus, MS 39701 and (2) University of Southern Mississippi, Hattiesburg, 39406
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 are investigating two aspects of [URE3] behavior. First, we are using microarray analysis to compare the gene expression profile of prion-containing [URE3] strains to prion-free [ure-o] strains with the goal of identifying differentially-regulated genes which might cause detectable phenotype changes in the two states. Currently, the only screen for the presence of the [URE3] prion is involves lack of growth when the prion is absent. Second, we are performing a screen of a gene-deletion library of S. cerevisiae looking for individual gene deletions which either cure the [URE3] state or facilitate amyloid formation with the goal of identifying genes which either help or hinder [URE3]'s ability to misfold into amyloids. Progress towards these goals will be discussed.
10:45 EXPRESSION ANALYSIS OF THE MOLD-SPE-CIFIC M46 GENE IN THE DIMORPHIC PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM
Davida Crossley* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406
Histoplasma capsulatum, is the causative agent for the respiratory disease histoplasmosis which afflicts an estimated 500,000 Americans each year. The dimorphic fungus grows as a multicellular mold in the soil. Once the soil is disturbed, spores are dispersed and inhaled into the lungs. In the lungs the fungus converts to the unicellular yeast morphotype. This mold-to-yeast conversion, which is a requirement for pathogenesis, can also be accomplished in the lab by switching incubation temperature from 25C to 37C. 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. Recent data have shown that M46 is expressed in the mold morphotype of G186AS and Downs strains, but is transcriptionally silent in G184AS and G217B strains. The reason for this lack of transcription in the latter strains could be due to a promoter lesion, or a missing trans-regulating factor. Currently we are investigating the M46 promoter function for all four strains by fusing the M46 promoter to a LacZ reporter. These promoter-reporter fusions are being tested for expression in both a M46 silent strain (G184AS) and a M46 expressing strain (G186AS). This data will allow us to determine if G184AS and G217B have non-functional promoters or missing transacting factors. Based on this analysis, future work will focus on identification of the promoter lesion or isolation of the missing trans-acting factor.
11:00 SIZE IDENTIFICATION OF AMINOACYL-TRNA SYNTHETASES FROM PLANTS
Timothy A. Pursley, Jason Littleton, Joshua K. Gardner, and Jeffrey Evans*, University of Southern Mississippi, Hattiesburg, MS 39406
The existence of a complex of aminoacyl-tRNA synthetases in plants is still in question. We investigated the possibility a plant synthetase complex by examining the size distribution of the enzymes for leucine and proline. These enzymes in animals are known to be in a large molecular weight complex. One source of plant enzymes we used was wheat germ. We extracted the wheat germ with cold buffer, centrifuged the extract to produce a post-organelle extract, and applied this to a Sephacryl S300 gel column of an HPLC system. The column fractions were assayed for leucyl-tRNA synthetase (LRS) activity. The LRS was found exclusively in the void volume of the column, consistent with its existence in a large molecular weight complex.
11:15 CHARACTERIZATION OF THE GENES INVOLVED IN THE PATHOGENESIS OF STAPHYLOCOCCUS AUREUS
Latrecia Lewis* and Mohamed O. Elasri, The Unviersity of Southern Mississippi, Hattiesburg, MS 39406
Staphylococcus aureus is a very versatile pathogen causing a variety of infections ranging from superficial infections (skin lesions) to toxemic syndromes (food poisoning) to life threatening conditions (osteomyletis, septicemia). S. aureus has developed resistance to several antibiotics like methicillin and vancomycin, prompting development of new drugs to treat staphylococcal infections. Staphlococccal infections are caused by a wide array of virulence factors. The virulence factors are either surface proteins or secreted proteins. The surface proteins are expressed early and repressed late during infection; the secreted proteins are repressed early and expressed late during the infection. The regulation of virulence factors is under the control of two global regulatory systems, the accessory gene regulator (agr) and the staphylococcal accessory regulator (sarA). In this study we aim to identify and characterize accessory genes that modulate sarA functions. We have used random mutagenesis to identify these genes.
11:30 REGULATION OF STAPHYLOCOCCUS ACCESSORY REGULATOR (SarA) 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 (osteomyletis, endocarditis). S. aureus has developed resistance to an array of antibiotics like methicillin and vancomycin, forcing the need for development of new drugs to combat staphylococcal infections. S. aureus expresses a wide variety of virulence factors such as cell bound proteins (e.g., adhesins) or exoproteins (toxins). The cell bound proteins are expressed early to establish infection and repressed after colonization whereas the exoproteins are repressed early and expressed late during the infection. The coordination of the expression of virulence factors is under the control of two global regulatory systems, the accessory gene regulator (agr) and the staphylococcal accessory regulator (sarA). The agr system is a quorum sensing system in which the S. aureus cells communicate with each other to coordinate expression of virulence factors. sarA regulates the virulence factors via two pathways, an agr-dependent pathway in which sarA activates agr at the transition between the exponential and post exponential growth phase and an agr-independent pathway in which sarA regulates virulence factors directly. There is evidence for the existence of important upstream and downstream factors that modulate sarA function. We have identified and characterized a new gene which activates sarA transcription and regulates several virulence factors.
11:45 REGULATION OF VIRULENCE BY QUORUM SENSING IN STAPHYLOCOCCUS AUREUS
Antony Schwartz* and Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS 39406
Staphylococcus aureus is a Gram-positive bacterium that causes a wide variety of community-acquired and nosocomial infections. Infections caused by S. aureus are increasingly difficult to treat owing to the increasing prevalence of antibiotic-resistant strains. Quorum sensing system, among other mechanisms, allows S. aureus to regulate expression of virulence factors according to cell density. The role of luxS, a novel quorum sensing system, has not been evaluated in S. aureus. In this study, we hypothesized that the luxS system regulates the expression of virulence factors and that luxS interacts with the accessory gene regulator (agr). To test this hypothesis, a luxS knock-out was generated via site-directed integration. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was used to compare luxS expression in the wild type and the luxS mutant. Expression of several virulence factors was also measured in the luxS mutant.
Exhibit Hall B
6:00 Divisional Poster Session
INHIBITION OF BREAST CANCER CELL GROWTH BY A NEW CLASS OF RING-SUBSTITUTED DIMS
Tamica Collins (1*), Kathy Vanderlaag (2), and Stephen Safe (1), (1) Jackson State University, Jackson, MS 39217 and (2) Texas A & M University, College Station, TX 77840
Breast cancer is one of the leading causes of death in women and the mortality rate has not changed for the past five decades. Over the past few decades scientists have developed breast cancer chemotherapeutics that target a diverse range of cellular pathways. A new class of compounds called ring-substituted DIMs(Br and Cl series), was tested for their breast cancer cell growth inhibition potential. A 6-day growth inhibition study was carried out in MCF-7 and MDA-MB-231 human breast cancer cell lines. 4,4'-dibromoDIM was the most potent in the MCF-7 cell line. The other ring-substituted DIMs tested all had similar levels of potency in MCF-7 cells. In the MDA-MB-231 cell line all of the ring-substituted DIMs exhibited a similar potency in terms of breast cancer cell growth inhibition. To determine how the ring-substituted DIMs are inhibiting breast cancer cell growth, various mechanisms of growth inhibition are currently being explored.
LIGAND BINDING PROPERTIES OF THE BREAST CANCER MOLECULAR TARGET AROMATASE
Jonathan Priester* and Stanley V. Smith, Murrah High School, Jackson, MS 39202 and University of Mississippi Medical Center, Jackson, MS 39216
The goals of our investigations are to better understand the features that govern the binding interactions of selected substrates and inhibitors to cytochrome P450 aromatase. We previously characterized the binding of formestane to aromatase and compared it to that of the natural substrate androstenedione. In our current study, we characterized the binding features of the aromatase inhibitor exemestane. The [K.sub.s] of exemestane was 112 nM. Compared to the [K.sub.s] of formestane (233 nM), exemestane exhibited higher binding affinity. However, both exemestane and formestane had lower affinities than the natural substrate androstenedione ([K.sub.s] = 63 nM). The mechanisms of binding by exemestane and formestane were also similar. In addition, we investigated the effects binding of the inhibitors had on the spectroscopic features of the aromatase heme environment. We allowed aromatase to proceed through the catalytic cycle in the presence of substrate and/or inhibitors. We then reduced the samples, added the probe ligand (CO), and performed wavelength scans to identify spectral features. The ferrous carbonmonoxy peak at 450 nm, which is indicative of functional cytochrome P450, disappeared in all cases. The 420 nm peak indicating locally misfolded aromatase was present in all cases. It was most pronounced upon exemestane binding to aromatase. This suggests the possibility of differential stabilization of aromatase by exemestane relative to the substrate testosterone and the inhibitor formestane. Further investigations are underway to better characterize the spectroscopically-observable species and their interconversions. (Supported by the Base Pair Program and American Cancer Society #IRG-98-275-04)
OPTIMIZATION OF BACTERIAL GROWTH CONDITIONS AND ISOLATION TECHNIQUES FOR RECOMBINANT CARBOXYSOMES PRODUCED IN ESCHERICHIA COLI
Sara Johnson (1*), Rachel Boone (2), Joseph Sapp (1), Gordon C. Cannon (1), and Sabine Heinhorst (2), (1) University of Southern Mississippi, Hattiesburg, MS 39406 and (2) Texas State University, San Marcos, TX, 78666
Carboxysomes, proteinaceous microcompartments that enhance the catalytic ability of ribulose-1,5-bisphosphate carboxylase/oxygenase to fix CO2, play an integral part in the metabolism of cyanobacteria and many chemolithoautotrophic bacteria. While investigation into the structure and function of these inclusions has ensued for more than 20 years, research has been hindered by the slow growth rate and complex culture conditions of the carboxysome producing bacteria. Attempts at expression of the carboxysome operon from Halothiobacillus neapolitanus in Escherichia coli have been made to ease the research process. To date, proper assembly of carboxysomes in the host bacterium has not been shown; however, transformation with the pGroESL plasmid and pTnlPET vector has produced evidence that the transformants are able to form carboxysome shell plates. To optimize carboxysome expression, manipulation of the growth conditions of the transformed E. coli was implemented. Various lysis conditions were employed to determine the most effective procedure for isolation of assembled carboxysomes.
ISOLATION AND CHARACTERIZATION OF ASPARTATE AMINOTRANSFERASE (AAT1) AND SULFITE OXIDASE (SOX1) GENES FROM THE PATHOGENIC FUNGUS HISTOPLASMA CAPSULATUM
Yuan-Chi Lin* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406
Histoplasma capsulatum is a pathogenic dimorphic fungus which is the etiologic agent of the common respiratory disease histoplasmosis. H. capsulatum grows in a differentiated multicellular mold form in the soil and converts to an undifferentiated single cell yeast form in the lungs of the infected individual. Environmental stimuli such as temperature of incubation and nutritional factors are important for the transition between mold and yeast forms. Sulfur metabolism in particular plays a critical role in this dimorphic process. The yeast morphotype but not the mold, for example, has a cysteine dioxygenase activity and is a cysteine auxotroph. To begin studies on the role of sulfur metabolism in dimorphism, we are isolating genes important in the central sulfur metabolic pathway. The focus of this report involves the genes for aspartate aminotransferase (AAT1) and sulfite oxidase (SOX1). Both genes were isolated by 5' RACE (Rapid Amplification of cDNA Ends) PCR and 3' RACE PCR. Currently real-time PCR and northern blot studies are underway to determine the expression of both genes in the mold and yeast morphotypes. Future work will include attempts to knockout these genes to evaluate the effects of loss of function on dimorphism.
GENETIC INTERACTIONS OF THE SACCHAROMYCES CEREVISIAE KIN3 GENE
Michael P. Jackson and Sarah Lea McGuire*, Millsaps College, Jackson, MS 39210
Appropriate control of the eukaryotic cell cycle is essential for cell survival. The main goal of our laboratory is to gain a better understanding of the molecular mechanisms involved in controlling the G2-M transition. Our recent studies have focused on characterization of the Kin3 gene of Saccharomyces cerevisiae. Kin3 encodes a serine-threonine protein kinase that is the homolog of the A. nidulans nimA kinase; Kin3 is a non-essential gene in S. cerevisiae that has not been examined extensively. Because nimA appears to affect a variety of processes in A. nidulans, we are interested in identifying the various cellular processes with which Kin3 is involved in S. cerevisiae. To accomplish this, we are using synthetic genetic analysis in S. cerevisiae to identify the non-essential genes with which Kin3 interacts. A Kin3 deletion strain was created and used as a query strain to determine synthetic lethal interactions with known non-essential genes, using an ordered library of strains carrying non-essential deletions and a robotic pinning device. Putative synthetic lethal interactions identified in this mass screening are currently being tested by performing individual genetic crosses with the Kin3 deletion strain to confirm the synthetic lethal interactions. Thus far, the mass screening data indicate numerous cellular processes that interact with Kin3, with identified interacting genes including those involved in mitosis, cell bud growth, actin filament organization, ubiquitin-mediated proteolysis, and DNA repair. These experiments will aid in the elucidation of the cellular and molecular mechanisms affected by Kin3. This project was supported by NIH Grant Number RR 016476 from the MFGN INBRE Program of the National Center for Research Resources.
HYPERTENSION IN MALE GROWTH RESTRICTED OFFSPRING IS ABOLISHED BY CASTRATION SUGGESTING A ROLE FOR TESTOSTERONE IN THE FETAL PROGRAMMING OF HYPERTENSION
Norma Ojeda (1*), Daniela Grigore (2), Elliott B. Robertson (2), and Barbara T. Alexander (2), (1) University of Mississippi, University, MS 38677 and (2) University of Mississippi Medical Center, Jackson, MS 39216
The fetal environment is now considered a contributing factor in the etiology of cardiovascular disease and hypertension. Our laboratory has characterized a model of intrauterine growth restriction (IUGR) induced by placental insufficiency whereby growth restricted offspring develop hypertension between 4 and 6 weeks of age. After puberty, however, male growth restricted offspring remain hypertensive, whereas hypertension in female growth restricted offspring is abolished. Thus, the purpose of this study was to determinate whether testosterone plays a role in mediating sex differences in this model of fetal programming. Mean arterial pressure (MAP) was measured in conscious, chronically instrumented male control and male IUGR offspring from 6 to 12 weeks of age followed by collection of plasma for determination of testosterone (T). MAP (12 weeks: 139[+ or -]2 vs.129[+ or -]2 mmHg) and T (12 weeks: 399[+ or -]75 vs.166[+ or -]30 ng/ml) were significantly increased in male IUGR offspring as compared to male control offspring, respectively. Gonadectomy (CTX) at 10 weeks of age abolished hypertension in male IUGR offspring (MAP measured by telemetry from 13 weeks: 137[+ or -]1 vs. 117[+ or -]1 to 16 weeks: 144[+ or -]1 vs. 123[+ or -]2 mmHg, CTX vs. Sham, respectively). Thus, these results suggest that testosterone is associated with sex differences in the fetal programming of hypertension in this model of IUGR.
REGULATED PROTEOLYSIS OF MITOTIC PROTEINS IN SACCHAROMYCES CEREVISIAE
Lindsey Gaskin, Mohamed Hajj, and Bernadette Connors*, Millsaps College, Jackson, MS 39210
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. In the budding yeast Saccharomyces cerevisiae, Dbf4p initiates DNA synthesis by activating and escorting Cdc7p to origins of replication. Dbf4p is subsequently targeted for destruction by the Anaphase Promoting Complex (APC/C) through D-box motifs, thus preventing reinitiation of DNA synthesis. Many known substrates containing D-box motifs are targeted by the APC/C through one of two necessary mediators, Cdc20p/Fizzy and Cdh1p. We are taking both a genetic and biochemical approach to understand the regulated proteolysis of Dbf4p and selected mitotic proteins in Saccharomyces cerevisiae. Confocal microscopy is being used with strains that harbor both YFP::dbf4 and GFP::cdc20 or GFP::cdh1 to establish localization of these proteins through the cell cycle. Two-hybrid assays are also underway to determine if physical interactions between Dbf4p and either Cdc20p or Cdh1p exist. Both deletion and mutational analyses will then be used to identify sites of direct interaction between Dbf4p and the mediator, and TAP-tagged versions of Dbf4p will be used to coimmunoprecipitate the complex in order to ascertain this interaction at the biochemical level. Additionally, synthetic genome analysis is being performed using a temperature sensitive allele of cdc20 in order to dissect genetic interactions. Characterization of the proteolysis of Dbf4p will help to elucidate the controls involved on the orderly progression of the cell cycle among eukaryotic organisms.
MOLECULAR MECHANISMS FOR THE EXPRESSION, SECRETION AND UNIPOLAR LOCALIZATION OF ICSA IN SHIGELLA FLEXNERI
K. M. Ellis, B. N. Roberts, L. D. Brandon, Mississippi University for Women, Columbus, MS 39701
IcsA is an outer membrane protein that is essential for the directed intracellular movement and intercellular dissemination of Shigella in colonic epithelial cells. IcsA is targeted to the old pole of the bacilli where the bacteria recruit actin filaments for directed movement within and between colonic epithelial cells. IcsA must be expressed at the old pole for the organism to spread effectively. The mechanism for IcsA expression and targeting to the old pole of Shigella is not completely understood. Shigella leads to an estimated 1.1 million deaths per year and there are currently no reliable vaccines against shigellosis. By understanding the molecular mechanisms responsible for the expression and surface localization of IcsA, this enables one to further understand the mechanism of pathogenesis in Shigella spp. and may lead to a more effective vaccine against shigellosis. The molecular mechanisms involved in the expression and polar targeting of IcsA will be defined through the development of a genetic screen using reporter constructs in a variety of unknown backgrounds or in a mutant library. Mutant libraries were generated using ethylmethanesulphonate (EMS), 2-aminopurine, and ultraviolet light. The purpose of the genetic screen is to identify mutants that either prevent IcsA polar localization or prevent IcsA from being expressed.
ACTIVATION OF THE RENIN ANGIOTENSIN SYSTEM FOLLOWS DEVELOPMENT OF HYPERTENSION IN A MODEL OF INTRAUTERINE GROWTH RESTRICTION INDUCED BY PLACENTAL INSUFFICIENCY IN THE RAT
Daniela Grigore*, Norma Ojeda, Elliott B. Robertson, and Barbara T. Alexander, University of Mississippi Medical Center, Jackson, MS 39216
Reduced uterine perfusion initiated in late gestation in the rat results in intrauterine growth restriction (IUGR) and development of hypertension between 4 to 6 weeks of age in male growth restricted offspring. A role for renin angiotensin system (RAS) involvement in mediating IUGR-induced hypertension is suggested as RAS blockade abolishes hypertension at 12 week of age in adult male growth restricted offspring. The purpose of this study was to examine renal expression of RAS components in male growth restricted offspring. At 6 weeks of age no difference in renal mRNA expression of angiotensinogen (Aogen) or renin as determined by real time PCR was observed in either the cortex or medulla of growth restricted offspring compared to control offspring. At 12 weeks of age renal mRNA expression of angiotensinogen (Aogen) was increased 4-fold in the cortex and 2-fold in the medulla of growth restricted offspring compared to control offspring. Renal mRNA expression of renin was also significantly elevated at 12 weeks of age in both the cortex, a greater than 2.5 fold increase, and medulla, a greater than 2-fold increase, in growth restricted offspring relative to control. Thus, activation of the renal RAS is present after development of hypertension in this model of fetal programming.
EXPRESSION RESULT FROM FIVE ENDOMETRIAL TUMOR/CONSTITUTIVE TISSUE PANELS
Keila Brown (1*), Margot Kaelbling (2), Warren May (2), and Charles Streckfus (2), (1) Tougaloo College, Tougaloo, MS 39174 and (2) University of Mississippi Medical Center, Jackson, MS 39216
Our aim was to assess if microarray analysis of endometrial tumors can detect altered gene expression associated with this tumor type. We hypothesized that the tumors will show altered gene expression of some genes when compared to uninvolved tissue of the same patient. A connection between altered gene expression and tumorigenesis may facilitate diagnosis, successful therapeutic involvement, and/or lead to the development of effective pharmacogenic agents. We analyzed five endometrial tumor/constitutive tissue panels. Total RNA was isolated from tumor and uninvolved tissue, reverse-transcribed into cDNA, and labeled with fluorescent dyes, either directly with Cyanine 3- or Cyanine 5-conjugated nucleotides or indirectly with Alexa 488 or Alexa 594 using a two-step method. The tumor and uninvolved samples were labeled with different dyes and then simultaneously hybridized to a microarray. We used "Human SS-H19k7" microarrays from the University Health network which contain 19,008 human Expressed Sequence Tags of 100-150 base pairs. After pre-processing the data, expression ratios were calculated and genes ordered using a Student's t-test and an analysis similar to Statistical Analysis for Microarrays. A nearly equal number of genes were down-regulated (50) as were u p-regulated (57). Many of the genes are known to be involved in cell cycle regulation; some are cancer-related genes. We used one-fifth less RNA for each Alexa-labeled sample than for each Cyanine-labeled sample. This research was supported by the Mississippi Functional Genomic Network.
CONSTRUCTION OF A GFP REPORTER VECTOR FOR THE INSERTION INTO Xho1 SITE OF HISTOPLASMA CAPSULATUM TELOMERE VECTOR pRPUT1
Evangeline Deer (1*), Glenn Shearer (2), and Davida Crossley (2), (1) Tougaloo College, Tougaloo, MS 39174 and (2) University of Southern Mississippi, Hattiesburg, MS 39406
Pathogenic fungi such as Histoplasma capsulatum can grow in two different forms: yeastlike or mycelial form based on its environment. This reversible process, known as dimorphism, infects humans and other animals located in temperate, subtropical, and tropical zones in the United States and in some areas of the South and Central America. In order to construct a green fluorescent protein (GFP) reporter vector to be used in the pathogenic fungus Histoplasma capsulatum, a DNA fragment containing the glyceraldehyde phosphate dehydrogenase (gpd) promoter, from the fungus Aspergillus, was fused to GFP, and later inserted into a Histoplasma capsulatum telomere vector (pRPUT1). The gpd-GFP fragment was isolated by agarose gel electrophoresis, and then purified by ZymoClean. Several experiments were conducted to produce clones with the desired insertion isolated, but none prevailed. Continuous work is currently in progress to construct a reporter for this and other fungal pathogens. This project was supported by the Mississippi Functional Genomic Network.
MECHANICAL STRAIN DIFFERENTIALLY ACTIVATES RHO FAMILY GTPASES IN FETAL LUNG TYPE II EPITHELIAL CELLS
Samuel Haile (1*) and Juan Sanchez-Esteban (2), (1) Jackson State University, Jackson, MS 39217 and (2) Brown University Medical School, Providence, RI 02912
Previous research has shown that mechanical forces play a key role in fetal lung development. The purpose of this study is to determine whether mechanical strain activates the Rho family GTPases in fetal lung type II cells. We hypothesized that mechanical stretch would activate Rho to induce stress fibers formation in fetal lung type II cells. During intrauterine life, the fetus makes episodic breathing movements that generate ~5% changes of alveolar surface area. The cytoskeleton consists of a complex network of protein filaments that extends throughout the cytoplasm. Mechanical forces applied to the cytoskeleton via integrin receptors induce reorganization of the actin filaments, and trigger a variety of downstream signaling pathways. This regulation of actin polymerization is orchestrated by the Rho family GTPases (Cdc42, Rac, and Rho). Fetal rat lungs were obtained from timed--pregnant Sprague--Dawley rats at E19 of gestation and cultured overnight on silastic membranes pre-coated with laminin. Cells stretched at 5% for 60 cycles/minute for 1, 5, 15, and 30 minutes to stimulate mechanical forces during fetal lung development. Cells were then assessed for Rho family GTPase activation. After protein isolation, samples were incubated with PAK-PBD beads to pull down the activated Rho GTPase protein. Protein samples were separated by one-dimensional 16.5% SDS-PAGE and transferred to nitrocellulose membranes. To detect GTPases, blots were incubated with anti-GTPase monoclonal antibody and detected by enhanced chemiluminescence. As hypothesized, Rho was differentially activated by mechanical stretch. Future experiments will include studying downstream activators of Rho, such as ROCK and myosin light chain (MLC) using an in vitro kinase assay and western blot respectively.
MEASURING THE CELLULAR ADENYLATE ENERGY CHARGE DURING THE DEREPRESSION OF THE PYRIMIDINE PATHWAY
Jayson Shaifer (1*), Mauricio Rodriguez (2), Melinda Wales (2), and Jim Wild (2), (1) Jackson State University, Jackson, MS 39217 and (2) Texas A & M University, College Station, TX 77840
Pyrimidine metabolism is characterized by a number of synthetic and salvage reutilization reactions. The pyrimidine pathway is repressed in the presence of uracil and derepressed in its absence. An important measure of metabolic transition inside the cell is that of the adenylate energy charge. The energy charge is calculated from the three adenylates ATP, ADP and AMP intracellular concentrations. The goal of this research is to measure the cell's energy charge during the derepression of the pyrimidine pathway at different time intervals. HPLC analyses of cellular extracts were taken during different time intervals following derepression to quantify the energy charge that reflects the relative number of high-energy charge phosphate bonds in the adenylate pool. The energy charge of a cell can vary mathematically from 0 (all AMP) to 1 (all ATP). The healthy cell has an energy charge greater than 0.8 and the cell is non-functional when it reaches a value of approximately 0.5. The metabolic nucleotide pools were analyzed by HPLC. The concentrations of AMP and ADP decreased following the removal of uracil and the derepression of the pyrimindine path way. ATP increased after the removal of uracil. These results are indication that during the derepression of the pyrimindine pathway in bacteria that the AEC increases. This research was supported by NIMH-COR grant MH-16926.
ISOLATION AND EXPRESSION OF RECOMBINANT FUSION PROTEIN OF LECTIN LIKE TRANSCRIPT-1(LLT1)
JeT'aime Ross (1*), Porunelloor Mathew (2), Stephen Mathew (2), and Nowland Bambard (2), (1) Jackson State University, Jackson, MS 39217 and (2) University of North Texas, Fort Worth, TX 761071
Natural killer cell functions are regulated by signals through activating and inhibitory receptors. One of these receptors is lectin-like transcript (LLT1), which is a member of the novel lectin superfamily. This research focuses on the use of a purified plasmid DNA pSec-LLT1 to transfect into mammalina cells to produce the fusion proteins. The recombinant fusion proteins containing the extracellular region of LLT1 were then used to screen various cell lines for expression of the ligand for LLT1. Purified plasimid DNA of pSec-LLT1 was isolated and purified by double cesium chloride DNA maxiprep. The DNA was digested with restriction enzymes BamHI and XhoI to check the LLT1 insert. The purified DNA was then transiently transfected into B16F10 cells and the supernatants containing the fusion proteins were concentrated and run on a SDS-PAGE gel and confirmed by western blot. The fusion protein was used to check the expression of the ligand on various mammalian cell lines. Preliminary flow cytometric analysis with some of the mammalian cell lines showed biding with the fusion protein. The recombinant fusion protein would enable us to in vestigate natural ligand of LLT1.
POSTNATAL DEVELOPMENT, BEHAVIOR, AND MOTOR FUNCTION IN GROWTH RESTRICTED OFFSPRING
Antoinettte Dawson (1*), Norma Ojeda (2), Daniela Grigore (2), Elliott B. Robertson (2), and Barbara T. Alexander (2), (1) Murrah High School, Jackson, MS 39202 and (2) University of Mississippi Medical Center, Jackson, MS 39216
Reduced uterine perfusion initiated in late gestation in the rat results in intrauterine growth restriction (IUGR) and hypertension in growth restricted offspring. The purpose of this study was to examine the effects of IUGR on postnatal development, behavior, and motor function in growth restricted offspring. Two groups of animals were tested; normal birth weight (control) offspring from the control pregnant rats and growth restricted offspring from pregnant rats who underwent reduced uterine perfusion in late gestation. After delivery, evaluation for developmental milestone was performed in pups from 1 to 21 days of age. Beginning at day one, pups were weighed daily and assessed for neonatal milestones. In the pre-weaning stage, control offspring mastered open field assessment and negative geotaxis earlier than growth restricted offspring. However, growth restricted offspring mastered rooting earlier than control offspring. In another group of animals, open-field and rotarod tests were assessed at four and six weeks of age. Neurobehavioral evaluation of growth restricted offspring revealed no alterations in motor function (rotarod) and activity level (open-field). These results suggest that reduced uterine perfusion results in IUGR that is not associated with motor deficiency.
FFECT OF [mu]-OPIOID RECEPTOR ANTAGONIST, [beta]-FUNALTREXAMINE ON METHAMPHETAMINE-IN-DUCED STREOTYPED BEHAVIORS IN MICE
Simira Carothers* and Xine Shen, Tougaloo College, Tougaloo, MS 39174 and University of Mississippi Medical Center, Jackson, MS 39216
This study is to determine the role of [mu]-opioid subtype in methamphetamine-induced stereotyped behaviors by using a [mu]-opoid receptor antagonist, a-funaltrexamine. We hypothesized that [beta]-funaltrexamine reduces methamphetamine-mediated behavioral responses through a dis-inhibitory mechanism to alter dopaminergic activity. It is well known that repeated use of methamphetamine at high dose can produce stereotyped behaviors. Stereotypic behaviors in mice include constant sniffing and grooming. Mice were injected with methamphetamine (10 mg/kg, i.p.) for 7 continuous days. On day 10, mice received an i.c.v. injection of [beta]-funaltrexamine ([micro]g) or saline (control) and were challenged with an injection of methamphetamine (10 mg/kg) 24 hours after [beta]-funaltrexamine injection. No significant effect of a single i.c.v. injection of [beta]-funaltrexamine on methamphetamine-induced stereotyped behaviors was detected in the limited experiment animals. The results need to be verified by increased animal numbers. This project was supported by the Mississippi Functional Genomic Network.
IDENTIFICATION OF SNP'S ON LDH-B GENE IN BOVINE
Deanna Baker*, and Rachel Beecham, Mississippi Valley State University, Itta Bena, MS 38941
Lactate dehydrogenase (LDH) converts lactate to pyruvate in the reverse direction and pyruvate to lactate in the forward direction. LDH is used in the body through to process of glycolysis. This process continues do to LDH turning NAD back into NADH through the reaction equation: Pyruvate + NADH [right arrow] LDH [right arrow] lactate + NAD. Two genes control the production of LDH and have been named LDH-A and LDH-B. The experiment started out with 20 samples at 20 ng/[micro]L of: pure Angus, pure Brahman, Angus/Brahman crossbreed, and Brahman/Angus crossbreed. The 80 samples came from an agriculture research farm in Booneville, AR. The samples were amplified using PCR (polymerase chain reaction) procedure. After this, the PCR products are purified through a 1% agarose gel and electrophoresis (for 1 hour/100 volts). The samples are then extracted from the gel using the Qiagen MiniElute Kit for PCR products. The samples were analyzed and 5 SNPs were found on the LDH-B gene in cattle. Out of those 5 SNPs there were 2 that changed the amino acid chain. These SNPs could be used to further research on the association between them and LDH activities in cattle.
CHARACTERIZATION OF THE SNXB1 MUTATION OF ASPERGILLUS NIDULANS
Michael Yablick and Sarah Lea McGuire*, Millsaps College, Jackson, MS 39110
The snxB1 mutation has been isolated as an extragenic suppressor of the nimX2 mutation of the filamentous fungus Aspergillus nidulans. It affects the cell cycle and causes compact, hyper-septating colonies when grown on rich media. To better understand its role in the cell cycle and identify what other molecules SNXB might interact with, a series of characterization assays and cloning attempts with the AMA-Not1 genomic library have been undertaken. A time-course microscopy assay measured relationships between germination, branching, and septation. It provided a numerical characterization of snxB1, and data suggest that colonies are not hyper-branching, and that septation may be uncoupled from nuclear division. A plate-growth assay in the presence of the DNA damaging agents hydroxyurea and camptothecin investigated defects in cell cycle checkpoints and possible interactions of SNXB with [ANKA.sup.WEE1] and [NIMT.sup.CDC25]. Data show camptothecin sensitivity of the snxB1/nimT23 mutant, which suggests an interaction with NIMT that may mediate the damage response between the ATR pathway and tyrosine phosphorylation of [NIMX.sup.CDC2]. Unsuccessful cloning attempts of snxB have provided insight into a more efficient transformation protocol that will likely work in the future. Understanding snxB may allow us to better understand similar cell cycle controls in humans, and provide insight into mechanisms that cause cancer.
GROWTH INHIBITION IN JAPANESE MEDAKA (ORYZIAS LATIPES) FISH EXPOSED TO TETRACHLOROETHYLENE
Hattie Spencer (1*), Wedad R. Hussein (2), and Paul B. Tchounwou (2), (1) Mississippi Valley State University, Itta Bena, MS 38941 and (2) Jackson State University, Jackson, MS 39217
A recent study in our laboratory has demonstrated that tetrachloroethylene (TCE) is acutely toxic to Japanese medaka (Oryzias latipes) larvae with a 96 hr-LC50 of 18 (17-19) mg/mL (Spencer et al, 2002). In the present study we hypothesize that TCE exposure induces a developmental effect in Japanese medaka. Growth and age specific sensitivity of Japanese medaka larvae were studied with four age groups (7, 14, 21 and 28 days old) to determine tetrachloroethylene effects on these parameters. The medaka larvae were exposed for 96 hours in a single concentration (10 mg/mL) of TCE. The toxic endpoints evaluated were larvae weight, length, water content and protein concentration. The study revealed that exposure of medaka larvae to this sub-acute concentration of TCE significantly reduced length and weight in the treated group. The difference in growth between control and treated groups was more obvious in age versus length, than in age versus weight. The dry weight-fresh weight ratio (dw/fw) was shown to be higher in the control group. Water content in TCE-treated medaka was higher than in the control group, and younger fry had more water content than older ones. A higher protein concentration was also observed in TCE-treated medaka compared to the control group. These results indicate that TCE has a profound effect on the growth and development of Japanese medaka larvae.
Meeting Room 1
8:30 PLATELET POLYMORPHISMS IN RELATION TO VON WILLEBRAND FACTOR SIGNALS
Shawna Clark (1*), Mohan S. Chitta (2), Qi Zheng (2), and John C. Kermode (2), (1) Tougaloo College, Tougaloo, MS 39174 and (2) University of Mississippi Medical Center, Jackson, MS 39216
Cardiovascular disease is the most frequent cause of death in the United States. Prior studies have identified several risk factors for this disease, including natural genetic variations (polymorphisms) of proteins involved in coagulation, thrombosis and lipid metabolism. This study focused on polymorphisms in platelet glycoprotein GpIb, a receptor for von Willebrand factor (VWF). There is tentative evidence that two polymorphisms in platelet GpIb may influence the risk of an individual developing cardiovascular disease. One causes a change from threonine (Thr) to methionine (Met) at amino acid residue 145 in the GpIb [alpha]a-chain. The other affects the number of tandem repeats of a 13-residue sequence in the extracellular portion of GpIb. The goal of this study was to develop a reliable method to assay the Thr/Met polymorphism and conduct a preliminary assessment of its effect on signaling in VWF in the platelet. Blood was drawn from healthy volunteers. Platelets were isolated and RNA was extracted from them. The RNA was converted into cDNA by reverse transcription. By using PCR, different polymorphic variants of GPIB a together with GPIX were cloned. Now we are currently working on expressing these variants in mammalian cells to study how they affect the signaling of VWF. [Supported by the Mississippi Functional Genomics Network, National Science Foundations, and the American Heart Association (Southeast Affiliate)]
8:45 NEOPLASTIC TRANSFORMATION OF THE CD[30.sup.HIGH] LYMPHOMAS USING MDV-TRANSFORMED LYMPHOMAS IN CHICKENS AS A MODEL
Jeremy Whitten (1*), Jorma Buza (2), and Shane Burgess (2), (1) Mississippi University for Women, Columbus, MS 39701 and (2) Mississippi State University, Mississippi State, MS 39762
We are conducting proteomic studies to investigate genes critical to the neoplastic transformation of the CD[30.sup.high] lymphomas using Marek's disease virus (MDV) transformed lymphomas in chicken as a model. We investigated the clonality of CD[30.sup.high] MD lymphoma cells based on RNA expression for T-cell receptors and immunofluorescence of lymphoma sections. Twenty five chicks were infected with MDV strain GA at a dose of 500 pfu through the intraperitoneal route. Twelve chicks were kept as uninfected control and housed separately. Infection was confirmed two weeks later by amplification of MDV DNA fragment in peripheral blood mononuclear cells using polymerase chain reaction. The infected chicks started showing clinical signs of MD starting 4 weeks after infection. Lymphomas were found in kidney, spleen, heart, liver, schiatic nerve, ovary and testis, from which samples were collected, snap frozen in liquid nitrogen and later transferred to -80C to be used in immnofluorescence studies. Samples for studies on RNA expression were collected from 10 non-infected control chicks and 10 infected chicks (from lymphomatous and non-lymphomatous organs). The organs sampled included heart, proventriculus, liver, spleen, kidney, lung, schiatic nerve, brachial nerve, ovary and testis. Further work on these samples will be done at later date.
9:00 DEVELOPMENT OF ELASTIN-LIKE POLYPEPTIDE AS A MACROMOLECULAR CARRIER FOR A CYTOTOXIC PEPTIDE
Preeti Kumar* and Drazen Raucher, Mississippi College, Clinton, MS 39058 and University Medical Center, Jackson, MS 39216
Chemotherapy, the 21st century's most popular treatment for cancer, exhibits nonspecific, off-target toxicity. Chemotherapy tends to kill not only the cancer cells, but other healthy cells in the body also. Therefore, there is a need for specific targeting. The tumor vasculature is more permeable to diffusion of large molecules from the bloodstream than normal capillary beds, and lymphatic drainage is limited. This phenomenon is called the enhanced permeability and retention effect, and leads to the accumulation of macromolecular drugs at the tumor site. This study uses a macromolecule called PEN-ELP-(KLAKLAK)2, which is a thermally responsive polypeptide capable of inducing apoptosis in target cells. The root of this macromolecule is Elastin-like Polypeptide (ELP), which forms aggregates when heated above body temperature. The hypothesis is that systemically circulating ELP will aggregate and accumulate at the tumor site where local hyperthermia will be applied. Attached to ELP in this study is penetratin (Pen), a 16 amino acid peptide known to facilitate transport of the macromolecule across cell membranes, and a pro-apoptotic peptide (KLAKLAK)2, known to induce mitochondrial swelling, cytochrome c release, and apoptosis. PEN-ELP-(KLAKLAK)2 was designed and genetically engineered in the lab. The protein was expressed in E. Coli and then purified using inverse transition cycling. Toxicity of Pen-ELP-(KLAKLAK)2 was demonstrated in melanoma (SK-MEL-2 and Malme-3M), ovarian (SK-OV-3), breast (MCF-7), and cervical (Hela) cancer cell lines. Each of the cell lines responded to a 72 h exposure to the polypeptide with a concentration-dependent inhibition. Growth inhibition was also demonstrated in MCF-7 cells by a cell growth curve. The invitro toxicity of Pen-ELP-(KLAKLAK)2 demonstrated here shows promise for this molecule as a future thermally-targeted therapy.
9:15 GENERATION OF CARDIOMYOCYTES STABLY OVEREXPRESSING THE LEPTIN RECEPTOR
Roshni Trehan* and David Stec, Tougaloo College, Tougaloo, MS 39174 and University of MIssissippi Medical Center, Jackson, MS 39216
There are several genes that have been linked to obesity. Among them is the (ob) obese gene. This gene encodes the leptin hormone. The objective of this research is to create cardiomyocytes which overexpress the leptin receptor to determine if leptin can protect cardiomyocytes from damage caused by exposure to excess fatty acids. Initial methods used were RNA isolation from a cardiomyocyte cell line (H9C2) stably overexpressing the long form of the rat leptin receptor (ObRb) ... Increased expression of the ObRb mRNA was determined in 3 different stable cell lines from each method by real-time PCR using primers specific to long form of the rat leptin receptor. The Fold Induction results for clone 1, 2, and 3 were as follows respectively: 2.35, 4.59, 4.59. Based on the results from the RT-PCR, on the basis of the threshold cycles, the cardiomyocytes transfected with cytomegalovirus promoter and had higher levels of leptin are the 285/264 cells, clones 1, 2 and 3. Program Supporter: Jackson Heart Study
9:30 IMPLEMENTATION OF A CHEMICALLY INDUCIBLE RNAI CONSTRUCT FOR POSTTRANSCRIPTIONAL SILENCING IN ARABIDOPSIS THALIAN
Scott Walper*, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406
To avoid the potential of a lethal effect from constitutive expression of an RNAi construct in Arabidopsis thaliana, a chemically inducible vector was utilized to study the effects of a gene knockout for the plastid nucleoid protein DCP68. This construct contains regulatory regions upstream of a constitutive promoter that inhibits expression of the RNAi construct in the absence of B-estradiol. Once induced, the expressed construct forms a double stranded RNA molecule homologous to exon 2 of the DCP68 gene. The plant immune response to this dsRNA molecule results in the destruction of the mRNA for the DCP68 protein, limiting or eliminating synthesis of the protein in vivo. Phenotypic effects of this knockout in transformants will be compared to characteristics of non-induced wild-type plants.
9:45 USING THE VARIABLE REGIONS OF THE RDNA CLUSTER OF HISTOPLASMA CAPSULATUM FOR AN ANTISENSE APPROACH TO GENE SILENCING
Melissa Adams* and Glen Shearer, University of Southern Mississippi, Hattiesburg, MS 39406
The dimorphic fungus Histoplasma capsulatum is the causative agent of histoplasmosis. The saprophytic multicellular mold form is found in the soil at 25C. When spores are inhaled a shift occurs and the mold becomes a unicellular yeast in the lungs of the infected individual. The yeast form is the "pathogenic form" and thus the mold-to-yeast shift is critical for pathogenesis. Determining which genes play a role in the dimorphic shift and what role the genes play is vital to our understanding of the molecular basis of this developmental shift and may yield data useful to develop new therapeutic modalities. Current methods to create genetic knockouts to study the role of particular genes is difficult and time-consuming in this organism. The use of an antisense ribosome, as reported by Sweeny and Yao in Tetrahymena (Proc Natl Acad Sci U S A. 1996. 93:8518-23), can be an alternative to these labor intensive gene knockouts. For most organisms, the antisense ribosome would not be an option because of the high copy numbers of rDNA. Preliminary data indicate that Histoplasma has a low or single copy number. Antisense ribosomes contain a small fragment of antisense target gene cDNA inserted into a non-essential region of the large subunit rDNA. Hybridization of this sequence with mRNA during translation results in post transcriptional gene silencing. The rDNA cluster of Histoplasma has been isolated and compared with other organisms to identify these nonessential (variable) regions. The D2 (divergent region #2), a likely candidate for replacement with antisense cDNA, has now been isolated and experiments are currently underway to determine the validity of such an antisense ribosome approach in H. capsulatum.
10:15 THE STRUCTURE AND FUNCTION OF CsoS2 PROTEIN IN THE CARBOXYSOME
Zhicheng Dou*, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406
Carboxysomes play a very important role in the assimilation of C[O.sub.2] by some photo- and chemoautotrophic prokaryotes. One of the proteins that comprise the carboxysome shell is CsoS2, which is expressed in two different forms (CsoS2A and CsoS2B) of approximately 85 and 130 kDa, respectively. Understanding the molecular differences between these two CsoS2 variants and determining the protein![.sup.-]s three-dimensional structure can help to elucidate the function of CsoS2 in the carboxysome shell and the role the protein may play in carboxysome assembly. To obtain large amounts of CsoS2 protein, the csoS2 gene was inserted into several different prokaryotic expression vectors and recombinant CsoS2 protein was produced. In the pPROEX system, CsoS2 protein was expressed as two polypeptides that correspond to CsoS2A and CsoS2B. Only the CsoS2B form was expressed in the IMPACT system. Various strategies are being explored to address solubility and concentration of purified recombinant CsoS2 protein in preparation for X-ray crystallography studies.
10:30 EFFECT OF A MUTATION IN THE GENE ENCODING CARBONIC ANHYDRASE ON CO2 FIXATION IN HALOTHIOBACILLUS NEAPOLITANUS CARBOXYSOMES
Sandipan Dawn*, C. Daniel Murin, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406
In chemolithoautotrophic bacteria like Halothiobacillus neapolitanus, C[O.sub.2] fixation is catalyzed by ribulose-1,5-bisphos-phate carboxylase/oxygenase (RuBisCO), which is packaged into polyhedral proteinaceous microcompartments called carboxysomes. The only carbon substrate for RuBisCO is C[O.sub.2], so cytosolic HC[O.sub.3.sup.-] must be converted to C[O.sub.2] before the enzyme can utilize it as a substrate. A carbonic anhydrase (CA) in the carboxysome shell, the CsoS3 protein, ensures this rapid conversion between inorganic carbon species and is believed to provide RuBisCO with a substrate concentration that supports optimal activity. To test the premise that carboxysomal CA has an enhancing effect on RuBisCO activity, carboxysomes from a csoS3 mutant, which produces inactive CA due to an insertion of a kanamycin resistance cassette within its coding sequence, were assayed for RuBisCO activity. Preliminary evidence suggests that in the mutant, which requires elevated C[O.sub.2] levels for growth, carboxysomal C[O.sub.2] fixation is only one third as efficient as it is in wild type carboxysomes. Taken together with the ability to complement the mutant phenotype with a wild type copy of the csoS3 gene, these results strongly suggest that the carboxysomal CA plays a crucial role in the catalytic enhancement of RuBisCO.
10:45 QUANTITATIVE EVALUATION OF CARBOXY-SOME GENE EXPRESSION IN HALOTHIOBACILL-US NEAPOLITANUS AT TRANSCRIPTIONAL LEVEL
Fei Cai*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406
Carboxysomes are polyhedral bodies found in cyanobacteria and in chemoautotrophic bacteria. This microcompartment is surrounded by a monolayer protein shell, with RuBisCO (D-ribulose-1, 5-bisphosphate carboxylase/oxygenase) sequestered inside. The carboxysome genes of H. neapolitanus are arranged in a putative operon. However, stoichiometric analysis showed large differences between individual peptide components in purified carboxysomes, suggesting that the expression of individual genes in the operon must be differently regulated. To understand the molecular mechanism of carboxysome gene regulation, quantitative transcript analysis by real-time RT PCR is underway. The mRNA copy number differs from one to one by one to two orders of magnitude before individual genes suggesting that carboxysome gene expression is regulated at the transcriptional level.
11:00 THE MOLECULAR MECHANISMS FOR THE EXPRESSION, SECRETION, AND UNIPOLAR LOCALIZATION OF ICSA IN SHIGELLA FLEXNERI
B. N. Roberts, K. M. Ellis, L. D. 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 responsible for the motility of the bacterium within the infected cell and from one cell to another. By understanding the molecular mechanisms responsible for the expression and surface localization of IcsA, this enables us to further understand the mechanism of pathogenesis in Shigella spp. Mutant libraries were generated using ethylmethanesulphonate (EMS), 2-aminopurine, and ultraviolet light in a mutagenic screen to study the affects of these mutations on the expression, secretion, and unipolar localization of IcsA. Acknowledgments: MFGN-INBRE NIH grant.
11:15 THE EFFECTS OF SEVERAL SELECTED BREAST CANCER DRUGS ON AROMATASE ACTIVITY
Heather J. Hilderbrand (1*), Tina G. Smith (2), and Stanley V. Smith (2), (1) Delta State University, Cleveland, MS 38733, and (2) University of Mississippi Medical Center, Jackson, MS 39216
Cytochrome P450 aromatase is involved in converting androgens to estrogens. It is a member of the cytochrome P450 superfamily of hemeprotein enzymes. Aromatase is a molecular target for breast cancer therapy in scenarios where estrogen signaling is driving the cancer cell proliferation. By inhibiting aromatase activity from peripheral tissues, estrogen levels and ultimately cell proliferation are greatly decreased. The goals of our studies were to develop a protocol for an HPLC-based assay of aromatase activity and to use this protocol to investigate effects of selected breast cancer drugs on aromatase activity. Assay mixtures contained aromatase, cytochrome P450 NADPH reductase, a NADPH-regenerating system, testosterone (substrate), deoxycortisol (internal standard), and appropriate buffers. We assayed at 37[degrees]C for 30 minutes, extracted the mixtures with acetonitrile, and performed HPLC. We used a C18 reverse-phase column to resolve products and identified them by absorbance detection in conjunction with comparison to standards. In agreement with previous binding studies, both anastrozole and letrozole inhibited activity at micromolar concentrations with letrozole being the more potent inhibitor. The steroidal inactivators exemestane and formestane inhibited aromatase activity at all concentrations tested. The early generation inhibitor aminoglutethemide demonstrated stereospecific inhibition with the R-isomer being more potent than the S-isomer. The results provide confirmation of the ligand binding results obtained in our laboratory. (Supported by the Mississippi Functional Genomics Network REO program and the American Cancer Society #IRG-98-275-04)
11:30 INVESTIGATION OF THERMAL DENATURATION AS A TOOL FOR STUDYING H[1.sub.E]-DNA BINDING
Channing K. Sly (1,2*) and Susan E. Wellman (1), (1) University of Mississippi Medical Center, Jackson, Mississippi 39216 and (2) Murrah High School, Jackson, Mississippi 39202
Histone H[1.sub.E] is involved in the folding and compaction of DNA in cells. This histone is the most abundant type in mammalian cells. Previous studies of binding using H[1.sub.E] yielded inconsistent results. We investigated the possibility that the inconsistent results were due to atechnical problem. The method that was used was thermal denaturation of DNA. In this method, solutions of DNA containing various concentrations of H[1.sub.E] are monitored in a UV spectrophotometer as the solutions are heated. DNA denatures into single strands when it is heated, and the denaturation is accompanied by an increase in UV absorbance. Proteins that bind to DNA will inhibit the denaturation and therefore the thermal denaturation curve will be changed in the presence of DNA-binding proteins. We conducted experiments to determine the effects of 1) equilibrating solutions for longer periods of time before beginning experiments and 2) equilibrating longer at each temperature during thermal denaturation. Our results indicated that there was no difference in data gathered when solutions were heated at one degree per minute or one degree per five minutes. We observed that the thermal denaturation curves were different for solutions incubated for less than about 24 hours. After one day, no changes were seen in the thermal denaturation curves. (Support was provided through BasePair.)
11:45 ELUCIDATING THE MECHANISTIC BASIS FOR REDOX-REGULATION OF JANUS KINASE 2
John K. Smith (1), Naila M. Mamoon (1), Sheeyong Lee (1), Kiranam Chatti (2), and Roy Duhe (1*), (1) University of Mississippi Medical Center, Jackson, MS 39316 and (2) State University of New York at Stony Brook, Stony Brook, New York 11794
Our laboratory has shown that the catalytic activity of the protein-tyrosine kinase JAK2 is reversibly regulated by oxidizing and reducing reagents. Upon oxidation, the enzyme is no longer catalytically active, and catalytic activity is restored upon reduction of the enzyme. However, there are other reports suggesting that JAK2's activity is stimulated by intracellular oxidation, although it is unclear whether this phenomenon occurs through direct or indirect mechanisms. We propose that the oxidative inhibition of JAK2 occurs directly via thiol-disulfide interconversions of critical cysteine residues. This hypothesis has been tested by site-directed mutagenesis of JAK2. Eighteen of the twenty-seven cysteines were eliminated from consideration through deletion mutagenesis experiments which demonstrated that those cysteines were superfluous for direct redox regulation. The remaining nine cysteine residues were individually and combinatorially converted to serine residues, which identified four cysteines near the catalytic pocket that cooperatively maintain JAK2's catalytic activity. The essential role of these residues was corroborated by alanine substitution and additional biochemical characterizations.
12:00 Divisional Meeting
|Printer friendly Cite/link Email Feedback|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Jan 1, 2006|
|Previous Article:||Agriculture and plant science.|
|Next Article:||Chemistry and chemical engineering.|