Cellular, Molecular and Developmental Biology.
Vice-chair: Mary L. Haasch, University of Mississippi
Section I The DNA World
9:30 CONSTRUCTS FOR INVESTIGATING THE EFFECT OF INTERFERON RESPONSE ELEMENTS ON THE EXPRESSION OF MAREK'S DISEASE VIRUS PP38 GENE
Ann-Marie Ziegeler (1)*, Ross Whitwam (1), and Shane Burgess (2), (1) Mississippi University for Women, Columbus, MS 39701, and (2) Mississippi State University, Starkville, MS 39762
Marek's disease (MD) virus (MDV) is a lymphomagenic herpesvirus of chickens. MD is a biomedical model for human lymphomagenesis. A 38 kiloDalton MDV gene product, phosphoprotein 38 (pp38), is critical to MDV's life cycle and is unique amongst herpesviruses. Pp38 is postulated to have evolved in response to chicken interferons. TransFac database searches reveal the pp38 promoter has two interferon response element (IRE) consensus binding sites. To determine whether these IRE sites are functional, reporter constructs are being created. The expression vector used is pd2EGFP-N1, which encodes green florescent protein (GFP) 3' of the multiple cloning site (MCS). The constituive cytomegalovirus (CMV) promoter is 5' to the MCS. The promoter was excised from pd2EGFP-N1. Three regions of the pp38 promoter were amplified by PCR. The first is immediately proximal to the transcription start site and includes no IRE sites. The second extends 5' to include one IRE site. The third extends further 5' to include both IRE sites. Optimizing primer design for the PCRs was not trivial. Desired promoter fragments were amplified and all three amplicons will be inserted 5' of the GFP gene replacing the CMV promoter. Each construct will be transfected into chicken embryo fibroblasts to determine the effects of the IRE sites on pp38 expression in differing concentrations of interferon.
9:45 EXAMINATION OF CARBOXYSOME GENE STRUCTURE IN THIOMICROSPIRA CRUNOGENA
Kristi Budzinski*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406
Most chemoautotrophic bacteria and all cyanobacteria contain carboxysomes, polyhedral inclusion bodies that enhance the fixation of carbon dioxide by ribulose-1,5-bisphosphate carboxylase/oxygenase. The carboxysome genes of most chemoautotrophic bacteria are arranged in a putative operon (cso), whereas the carboxysome genes of cyanobacteria, ccmKLMN, are only loosely clustered. The arrangement of carboxysome genes in Thiomicrospira crunogena as examined in this study. T. crunogena is an obligate chemolithoautotroph found in deep sea hydrothermal vents in the East Pacific Rise. Electron microscopy of T. crunogena revealed that the bacteria contain cso-type carboxysomes, like other chemoautotrophs. However, the carboxysome polypeptide composition of T. crunogena differs from that of other chemoautotrophic bacteria such as Halothiobacillus neapolitanus. Southern blotting will be performed to determine the gene cluster organization.
Section II The RNA World
10:00 RIBOZYME-CATALYZED AMINOACYLATION FROM ACETYL COA
Na Li* and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406
According to the RNA world hypothesis, the evolution of coded protein synthesis was a critical step in the transition from the RNA world to our contemporary biological systems. Evolution in the RNA world would have resulted in ribozyme-catalyzed acyl-transfer reactions, from which coded protein synthesis would have then been evolved. Therefore, an RNA-only-based protein synthesis system would require a set of RNA molecules capable of synthesizing aminoacyl-RNAs, which are produced by the protein aminoacyl-tRNA synthetases in extant biology. We report a new class of highly efficient ribozymes generated by directed in vitro evolution that can catalyze the aminoacylation from aminoacetyl CoA. Thioesters of coenzyme A (CoA) play a prominent role in living cells by serving as essential intermediates in numerous metabolic processes. With its "high-energy" thioester bond yet sufficient chemical stability, a thioester is an excellent intermediate for acyl group transfer reactions. In contrast to tRNA aminoacylation from aminoacyl adenylates, thioester-activated amino acids are used in current work as the substrates to mimic the tRNA charging catalyzed by protein aminoacyl-tRNA synthetases in modern biological systems. Combined with our previously isolated ribozymes that make CoA and its thioesters, the isolation of this new class of ribozymes lends further support to the hypothesis that thioesters of coenzyme A could have played a key role in biological evolution and provides clues to the complexities of the RNA world.
10:15 SOLID-PHASE SYNTHESIS OF DOUBLE FLUORESCENT-LABELED RNA BY cDNA-SPLINTED LIGATION
Guocan Wang* and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406
Fluorescence resonance energy transfer (FRET) is a spectroscopic method that provides distance information on macromolecules in solution in the range 20-100 [Angstrom]. It is therefore a suitable probe to determine distances in RNA molecules and analyze their global structure. Fluorescence resonance energy transfer has been applied successfully in the structural investigation, real-time kinetics of conformational changes, monitoring ion binding, and thermodynamic stability analysis of different conformers. Site-specifically labeling RNA with fluorophores is critical for FRET and current methods for synthesis of fluorescent RNA depends heavily on solid-phase chemical synthesis, which is limited by coupling efficiency of solid-phase synthesis and availability of stable phosphoramidites for solid-phase synthesis. We describe a simple method to prepare fluorescent RNA for FRET on solid-phase matrix, thiopropyl 6B beads. A large RNA molecule is divided into three parts, which are transcribed individually using adenosine-derivatives as initiators. The 5' end piece was initiated with dephos-coenzyme A, which can be immobilized on thiopropyl 6B by a disulfide bond. The middle RNA piece is initiated with N6-HDA-AMP, and can be ligated to the first RNA piece by a cDNA-bridged ligation method. Reaction of the N6 amino group of the labeling site with fluorescein succinimidyl ester produces site-specific labeling. In the same way, the 3' end RNA piece can be ligated to first two RNA pieces. Reaction with the second fluorophore, cyanine 3, complete the process of dual fluorophore-labeling. This solid-phase based RNA labeling procedure should enable simple and efficient preparation of fluorescent RNA for FRET study.
10:45 COENZYME SYNTHESIS AND UTILIZATION BY RNA AND THE RELATIONSHIP BETWEEN RIBOZYME SIZE AND CATALYTIC ACTIVITY
Tricia Coleman*, Guocan Wang, Na Li, and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406
A novel adenosine-initiated transcription system based on the T7 class II promoter [phi] 2.5 has been developed to explore the ability of RNA to utilize adenosine derivatives in RNA catalysis. First, we have isolated ribozymes that can catalyze the formation of CoA, NAD and FAD from their respective precursors (phosphopantetheine, NMN, and FMN), suggesting a plausible mechanism of coenzyme synthesis and utilization in the RNA world. Since these coenzymes all contain an adenosine moiety, we have exploited our adenosine-initiated transcription method to incorporate CoA, NAD and FAD onto the 5'-ends of RNA transcripts to explore a broadened functional capacity of RNA. The CoA-initiated transcription method has been used to isolate a series of thioester synthetase ribozymes. This work is currently being extended to include the isolation of ribozymes capable of peptide synthesis from acyl CoA thioester intermediates. All of the ribozymes described above are linked by an overall scheme, where the multiple chemical steps required to synthesize peptide bonds from thioester intermediates would constitute a synthetic pathway catalyzed by a series of individual metabolic ribozymes in an entirely RNA-based metabolic pathway. Both the coenzyme synthetase ribozymes and the thioester synthetase ribozymes were isolated from a size heterogeneous RNA pool containing RNA with random regions of 30, 60, 100, and 140 nucleotides. In addition, we have isolated several ester synthesizing ribozymes from the same heterogeneous pools. Comprehensive kinetic analyses have been conducted on these ribozymes and there appears to be an optimal catalytic size around 60 nucleotides. Our work may provide significant insight into the RNA world complexity and the origin and evolution of coenzymes.
11:00 ISOLATION OF NOVEL RIBOZYMES WITH THIOESTERASE ACTIVITIES
Danning Huang* and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406
In a hypothesized RNA world, all essential biochemical reactions would have to be catalyzed by RNA enzymes, or ribozymes. First discovered in living systems, ribozymes can also be isolated from artificial random RNA libraries, thanks to the development of powerful in vitro selection techniques. Many ribozymes with a variety of activities have been isolated during the past decade. In our work, we are trying to link different ribozymic activities to form metabolic pathways--RNA-based metabolic pathways. A series of ribozyme activities involving coenzyme A (CoA) have been demonstrated in our laboratory. The current work expands such CoA-centered pathway by generating new ribozymes that can catalyze the hydrolysis of thioesters of CoA.
11:15 POST-TRANSCRIPTIONAL SILENCING OF ARABIDOPSIS THALIANA FERREDOXIN:SULFITE-REDUCTASE BY RNA INTERFERENCE
Scott Walper*, Steven Adamson, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406
The product of the ferredoxin:sulfite reductase (SiR) gene is a bifunctional protein that functions as a DNA binding protein in chloroplast nucleoids and plays an integral part in the plant's sulfur assimilation pathway. In an attempt to better characterize the function of this protein in Arabidopsis thaliana, a binary vector was constructed that contains a portion of the gene sequence in both its sense and antisense orientation. An Agrobacterium tumefaciens bacterial host containing the plasmid construct can then be used to insert the desired sequence into the plant genome. When integrated into the plant genome, this construct silences the SiR gene without direct mutation of the gene itself. The sense/antisense combination forms a double-stranded RNA molecule due to the complementary nature of their sequences when transcribed. This structure stimulates a cellular response that cleaves the double-stranded RNA molecule into small fragments, which are then free to bind to homologous RNA sequences, furthering the cleavage of double-stranded RNA molecules. As these fragments bind the SiR transcript, the mRNA is also cleaved into small non-functional segments. Those plants that successfully integrate the DNA sequence will be used in further studies to characterize the effects of a functional sulfite reductase knockout in planta.
11:30 INCORPORATION AND INTRACELLULAR MAPPING OF HIS-TAGGED p43/EMAP II WITHIN THE MULTIENZME AMINOACYL-tRNA SYNTHETASE COMPLEX
Angela A. Reiken (1)*, Cindy L. Wolfe (2), and Mona T. Norcum (1), (1) University of Mississippi Medical Center, Jackson, MS 39216, and (2) Tougaloo College, Tougaloo, MS 39174
Amino acids are covalently attached to their cognate tRNAs through the enzymatic action of aminoacyl-tRNA synthetases. In multicellular eukaryotes, nine of these enzymes form a distinct high molecular mass complex. Three auxiliary proteins (p43, p38, and p18) are also components of the multisynthetase complex. Previous studies have identified p43 as a precursor form of endothelial-monocyte activating protein II (EMAP II). This is an inflammatory cytokine involved in apoptosis. Primarily localized near the center of the multisynthetase complex, little to no p43 exists as a free polypeptide. In this study, human embryonal kidney cells were transfected with a plasmid expressing p43 tagged with his-6 and an additional epitope (V5). Immunoblot analysis showed that multisynthetase complex isolated from transfected cells contained his-tagged p43/EMAP II. This is the first demonstration that plasmid expressed tagged p43 can be incorporated into this particle. Immunofluorescence utilizing anti-p43 or anti-V5 antibodies showed that the complex is found in both the cytoplasm and nucleus of transfected cells. This is consistent with data from previous immunocytochemical studies. This work was supported by NSF grants MCB-0090539 and MCB-0215940.
11:45 PURIFICATION AND INITIAL ELECTRON MICROSCOPY OF VALYL-tRNA SYNTHETASE-ELONGATION FACTOR 1H COMPLEX
Shoulei Jiang*, J. Anthony Warrington, and Mona T. Norcum, University of Mississippi Medical Center, Jackson, MS 39216
In mammalian cells, valyl-tRNA synthetase (ValRS, 140 kDa) forms a stable ca. 700 kDa heterotypic complex with elongation factor EF-1H (ValRS-EF1H complex). EF1H is the "heavy form" of the translation elongation factor 1. It is a pentameric complex that contains four subunits a, b, g, and d in molar ratio 2:1:1:1. This complex is unusual because it catalyzes sequential reactions in protein biosynthesis. Specifically, valyl-tRNA is synthesized, as well as transferred to the ribosome. The valRS-EF1H complex is highly susceptible to proteolysis and to date little structural information has been determined. We have successfully purified ValRS-EF1H complex from K562 human erythroleukemia cells to near homogeneity. The isolation method uses polyethylene glycol fractionation, S and Q ion-exchange chromatography. Valyl-tRNA synthetase activity, SDS-PAGE silver staining gel patterns and immunoblot analysis verified the composition of the complex. Gel-filtration HPLC confirmed the 700 kDa mass. Initial negatively stained electron micrographs show a distinct particle. This indicates that the preparation is suitable for calculation of its three-dimensional structure. This work was supported by NSF grants MCB-0090539 and MCB-0215940.
Section III The Protein World
1:00 GELATIN AS A SUBSTRATE FOR PROTEASE DETECTION ON A QUARTZ CRYSTAL MICRO-BALANCE
James Daryl Pollard (1), Clifton C. Watkins (2), Newton C. Fawcett (1), and Jeffrey Evans (1)*, (1) University of Southern Mississippi, Hattiesburg, MS 39406, and (2) Mississippi Valley State University, Ita Bena, MS
Quartz crystal microbalance (QCM) technology allows for the measurement of small mass changes on its surface by relating these mass changes to changes in frequency of an oscillating quartz crystal. The QCM is mass sensitive in the nanogram range with the frequency of the crystal decreasing as mass is attached. Consequently, the QCM is able to quantify the interactions between biomolecules such as proteases or enzymes that catalyze important physiological reactions. Proteases have important roles in industry, food processing, medical treatment, but ironically can be weaponized and used in biological and chemical warfare. Consequently, safe, accurate, and efficient protease assays are needed to determine the activity of various proteases. The QCM offers the possibility of protease activity detection by relating a mass change on its surface caused by protease cleavage to a commensurate frequency change. Two methods were performed in developing this assay. In this first method, gelatin immobilized by protein absorption to the crystal surface had a certain mass and frequency. The protease trypsin was introduced to the crystal surface and cleaved or released gelatin from the crystal surface and caused a frequency increase. In the second method, trypsin and gelatin were incubated together and then applied to the crystal surface. This produced a larger frequency decrease than a control gelatin with no trypsin. It was concluded that the QCM is able to detect protease cleavage using both methods.
1:15 AGGREGATION OR SELF-ASSEMBLY OF THE CYT1A ENDOTOXIN FROM BACILLUS THURINGIENSIS VAR. ISRAELENSIS
Slobodanka D. Manceva (1)*, Marianne Pusztai-Carey (2), and Peter Butko (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406, and (2) Case Western Reserve University
The delta-endotoxin Cyt1A from Bacillus thuringiensis var. israelensis is used in commercial preparations of environmentally safe insecticides. Current hypothesis about its mode of action is that Cyt1A self-assembles into well-defined cation-selective channels in cell membranes, which result into a colloid-osmotic lysis of the cells. Recently a new hypothesis has been put forward suggesting that Cyt1A rather aggregates on the membrane surface and acts in a detergent-like manner. We used sodium dodecyl sulfate polyacrylamide gel electro-phoresis (SDS PAGE) and time-resolved fluorescence of Tryptophan (Trp) to distinguish between Cyt1A's self-assembly (pore-forming model) and aggregation (detergent-like model). SDS PAGE revealed that in the presence of lipid Cyt1A forms a broad range of molecular-weight protein aggregates, some of which are so large that they do not enter the gel. If Cyt1A acts as a pore former then it should self-assemble into well-defined pores consisting of no more than six protein molecules. Trp fluorescence life-time measurements reveal that Cyt1A, in the absence of lipid, has a lifetime of 4.46 ns. Cyt1A binding to the lipid results in a 50% decrease in the quantum yield of Trp fluorescence, but the lifetime does not change, which indicates static quenching of Trp fluorescence, presumably due to the toxin aggregates in the membrane. Our data suggest that Cyt1A aggregates, rather than self-assembles, on the membrane surface.
1:30 INTERACTION OF A CYCLIC AMPHIPATHIC PEPTIDE WITH LIPID MEMBRANES
Kelley Counts (1)*, Oluyemisi Adeyemi (1), Maria Ngu-Schwemlein (2), and Peter Butko (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406, and (2) University of Southern Alabama, Mobile, AL
Cyclic peptides serve a multitude of functions in cell biology, such as cellular adherents, inhibitors, regulators, and antibiotics. In this work we studied the interaction of a synthetic amphipathic cyclic octapeptide Octa-1 with model lipid membranes. The hypothesis of this study is that the positively-charged Octa-1 will interact with membranes and that this interaction will be more significant with the membranes containing negatively-charged lipids. The hypothesis was tested using small unilamellar vesicles (SUV) made of egg 1,2-diacyl-sn-glycero-3-phosphocholine (PC, a neutral lipid) and L-a-phosphatidylglycerol (PG, negatively-charged) in ratios of 10:0, 9:1, and 3:1. Octa-1 contains a single tryptophan residue, which was employed to determine the strength of the interaction and the location and orientation of the membrane-bound peptide by fluorescence spectroscopy. A calcein-release assay was used to test the lytic activity of the peptide. It was established that Octa-1 interacts with both the neutral and negatively-charged membranes, but the interaction is stronger with the charged SUV. The peptide is located deeper in the lipid bilayer when the membrane is negatively charged. The lytic activity of the peptide is detected only at high concentrations (micromolar) and it correlates with the negative charge on the membrane.
1:45 CHANGES IN EXPRESSION LEVELS OF FERREDOXIN:SULFITE REDUCTASE WITH CELL AGE IN A SOYBEAN SUSPENSION CULTURE
Jeffrey Broussard (1)*, Steven Adamson (2), Gordon C. Cannon (2), and Sabine Heinhorst (2), (1) Centenary College, Shreveport, LA 71134, and (2) University of Southern Mississippi, Hattiesburg, MS 39406
Chloroplasts of plant cells do not contain a nucleus. Instead, the multiple copies of the organellar genome are compacted into complexes known as nucleoids by a set of proteins. Previous work in our lab had characterized an abundant nucleoid protein (DCP68) from plastids of cultured soybean cells that is able to compact DNA in vitro. This protein was later identified as ferredoxin:sulfite reductase (SiR), an enzyme of the sulfur assimilation pathway. In light of the bifunctionality of this protein, it is important to determine which regulatory strategies plants employ to modulate abundance and biological function of DCP68/SiR. It is well documented that the protein composition of plastid nucleoids changes during plant development and maturation, and that these nucleoid remodeling events are accompanied by changes in plastid transcription and DNA replication activity. Since DCP68/SiR inhibits DNA replication in vitro, it was of interest to determine whether the protein's abundance in cultured soybean cells undergoes changes as the cells age. Soluble protein extracts were prepared from cells harvested at different times post transfer into fresh medium. The proteins were separated by SDS-PAGE, electroblotted onto nitrocellulose and the abundance of DCP68/SiR probed with antiserum against recombinant SiR from Arabidopsis thaliana. Our results revealed a decrease in abundance of the 68 kDa DCP68/SiR polypeptide with increasing cell age, and a concomitant increase in the abundance of a 63 kDa band species that is likely to be a proteolytic processing product of the 68 kDa protein.
2:15 EFFECT OF SULFUR METABOLITES ON THE EXPRESSION OF THE PLASTID NUCLEOID PROTEIN FERREDOXIN:SULFITE REDUCTASE IN CULTURED SOYBEAN CELLS
Brittney Hemba*, Steven Adamson, Gordon C. Cannon, and Sabine Heinhorst, University of Southern Mississippi, Hattiesburg, MS 39406
Ferredoxin:sulfite reductase (SiR) in higher plants is a bifunctional protein serving as an abundant DNA-binding protein of plastid nucleoids and as a member of the sulfur assimilation pathway. Because of the protein's dual role in the organelle, the regulatory mechanisms that govern its expression in the plant cell are of particular interest. The abundance of SiR in the plastid is likely to affect its association with the nucleoid and could have consequences for structure and function of the DNA/protein complex, since SiR was shown to compact DNA and inhibit DNA replication in vitro. As a step towards understanding the regulation of SiR expression, the role of the key sulfur metabolites O-acetyl-L-serine (OAS), L-cysteine, and glutathione in controlling SiR expression was examined. L-cysteine and glutathione down-regulate the expression of several sulfur assimilatory enzymes, likely through a feedback control mechanism, whereas the sulfur biosynthesis pathway activator, OAS, upregulates the expression of these enzymes.
2:30 REGULATION OF FERREDOXIN:SULFITE REDUCTASE DURING DEVELOPMENT OF ARABIDOPSIS THALIANA
Steven Adamson*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406
Sulfite reductase (SiR) [EC 184.108.40.206] from higher plants catalyzes the six-electron reduction of sulfite to produce sulfide. Siroheme and an iron-sulfur cluster aid in catalysis, and in photosynthetic tissues, ferredoxin serves as the electron donor. Our research group recently discovered SiR is an abundant DNA-binding protein present in plastid nucleoids of higher plants. Our continued research is focusing upon the regulation of this bifunctional plastid protein. To further explore cellular strategies that coordinate which role SiR plays in response to internal and external developmental cues, the abundance of SiR protein its transcript levels in leaf and non-photosynthetic root tissues were determined. SiR protein levels were found to be low in young root tissue and to accumulate as the root matures. A relatively small difference in SiR levels, on the other hand, was observed between leaf tissue obtained from young and mature Arabidopsis plants. SiR is a phosphoprotein, and a strong increase in its DNA-binding affinity upon dephosphorylation in vitro had been observed previously. Since posttranslational modification of nucleoid proteins might be an in vivo regulatory strategy to modulate structure and function of the plastid protein/DNA complexes, the isoelectric point of SIR is currently being examined as a first step towards assessing potential changes in the phosphorylation state of SiR during development.
2:45 THE CARBOXYSOME [epsilon]-CARBONIC ANHYDRASE: FACILITATING C[O.sub.2] FIXATION
Eric B. Williams*, Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406
Many autotrophic bacteria contain an energy-dependent carbon concentrating mechanism (ccm) that functions to increase the intracellular concentration of dissolved inorganic carbon ([C.sub.i]). One component of the ccm is the carboxysome, which functions to convert [C.sub.i] into reduced carbon metabolites. Carboxysomes are found in many autotrophic organisms and contain the C[O.sub.2] fixing enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) bounded by a 3-4 nm protein shell. It is thought that the sequestration of RuBisCO into carboxysomes enhances the catalytic efficiency of C[O.sub.2] fixation. Recently, the carboxysome shell (CsoS) protein CsoS3 was shown to have carbonic anhydrase (CA) activity. Carbonic anhydrase catalyzes the reversible dehydration of bicarbonate to C[O.sub.2], which is the only substrate for the RuBisCO catalyzed carboxylation reaction. Because the majority of the [C.sub.i] in the cytosol exists as bicarbonate, a rapid conversion from bicarbonate on the exterior of the carboxysome to C[O.sub.2] in the interior is needed to saturate the RuBisCO catalytic site. It may be possible that CsoS3 facilitates C[O.sub.2] fixation by directionally converting bicarbonate to C[O.sub.2] towards the interior of the carboxysome and that carboxysomes are resistant to C[O.sub.2] loss by diffusion. To test these possibilities, different states of carboxysomes were analyzed by stopped flow spectroscopy and investigations of CsoS3 catalysis were performed to determine the favored direction of the enzymatic interconversion of bicarbonate and C[O.sub.2].
3:00 OPTICAL DIFFERENCE SPECTROSCOPY TO MEASURE BINDING OF WARFARIN ENANTIOMERS TO CYTOCHROME P450 3A4
Juliette Sandifer*, Gary Reid Bishop, and Stanley V. Smith, Tougaloo College, Tougaloo, MS 39174; Mississippi College, Clinton, MS 39058; and University of Mississippi Medical Center, Jackson, MS 39216
Cytochrome P450s are a superfamily of hemeproteins found throughout the plant and animal kingdoms. Cytochrome P450s catalyze a number of reactions on a broad array of structurally distinct compounds. They are involved in many cellular processes but are particularly relevant to drug metabolism. Human cytochrome P450s 1A2, 2C9, 2C19, 2D6, and 3A4 are the major isoforms involved in drug metabolism and contribute to metabolism of the majority of the drugs used in clinical settings. Discovering the features at the molecular level governing substrate recognition and binding is the key to better understanding the structure/function relationships in the P450 superfamily. In this study we sought to characterize the binding of warfarin enantiomers to cytochrome P450 3A4. Warfarin is extensively metabolized to inactive metabolites by the cytochrome P450s. Using optical difference spectroscopy, we determined spectral binding constants (Ks) for racemic (R/S), R-, and S-warfarin binding to cytochrome P450 3A4. The estimated Ks values are in the [micro]M range for the pure enantiomers and the R/S mixture. The Ks for S-warfarin indicated that its binding affinity for P450 3A4 was 2-3 fold higher than that of R-warfarin. We have access to detailed structural information on R- and S-warfarin. The results obtained in this study are a prelude to more detailed studies aimed at measuring the effects warfarin enantiomers have on the conformational dynamics of cytochrome P450 3A4 and to eventually creating structural models for cytochrome P450 3A4 based on the experimental data.
3:15 Divisional Poster Session
IMPROVING TRANSFORMATION EFFICIENCY IN HISTOPLASMA CAPSULATUMBY MODULATING ELECTROPORATION TIME CONSTANTS OR PULSE SHAPES
Selena Young (1)*, Glenmore Shearer, Jr. (2), and Rebecca Phelps (2), (1) Alcorn State University, Alcorn State, MS 39096, and (2) University of Southern Mississippi, Hattiesburg, MS 39406
Histoplasma capsulatum is the etiological agent of histoplasmosis, a common respiratory mycosis of humans. This dimorphic fungal pathogen grows at 25 [degrees]C in the soil as a differentiated multicellular mold and at 37 [degrees]C in the lungs of the host as an undifferentiated single-cell yeast. Its occurrence is worldwide and it affects approximately 500,000 people every year. The identification of the dimorphism genes can be important in the treatment of the disease. Electroporation is an important tool that was used to determine the genes responsible for dimorphism. Several different time constants were tested in order to determine the optimal transformation efficiency. Time constants of 8 ms to 20 ms were used. The results indicate that the highest time constant, 20 ms, yielded the best transformation efficiency. Because the efficiency was still increasing at 20 ms, it was not certain that the optimal time constant was found. More experiments at even higher time constants are necessary in order to determine optimal time constant efficiency.
AUTOMATED DNA EXTRACTION
Bianca Clark (1)*, Stephanie Warren (2), and Don Sittman (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) University of Mississippi Medical Center, Jackson, MS 39216
Automated robotics liquid-handling systems can aid researchers in many high-throughput laboratory procedures. Through the use of additional, assorted labware these machines can perform DNA extraction, RNA extraction, PCR purification, automated gel loading, etc. Each of these procedures can be performed in a relatively short amount of time producing high quality results; to perform these procedures, programs or templates must be written for the instruments. Some programs are available from the companies that make the robots or that make nucleic acid purification kits. The objective of this study was to successfully carry out DNA extraction using a Multi-PROBE II automated liquid-handling system. To accomplish this, DNA extraction kits (some of which included templates) were obtained from Promega, Millipore, and Qiagen. Each of these kits provided the solutions needed to carry out DNA extraction, and the templates provided the parameters to carry out the necessary actions (vacuuming of plates, mixing of solutions, pipetting, etc.). As designed these kits provide high quality DNA. Templates were modified or created for each kit. In conclusion, only the Qiagen kit with an extensively modified template was compatible with the Muliprobe II.
CONSTRUCTION OF PLANT BACTERIAL ARTIFICIAL CHROMOSOME (BAC) LIBRARIES
Anna Nelson (1)*, Breonica Carter (1), and Daniel Peterson (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) Mississippi State University, Mississippi State, MS 39762
The construction and characterization of a bacterial artificial chromosome (BAC) library for the plant species Gossypium arboreim was carried out through intensive chemical processes. A protocol was first developed to isolate high molecular weight nuclear DNA for the plant species. DNA analysis was performed to examine if the lengths of the DNA were sufficient. This was followed by an isolation and preparation of the BAC vector, which includes a restriction digest of the vector to produce cohesive termini (sticky ends) and dephosphorylation. A bacterial culture was plated and a Miniprep and Not1 digest was run to isolate plasmids from the bacterial cultures. A pulse-field electrophoresis gel was run to test for "true-positive." Test restrictions digest; first and second size selections, isolation of size-selected DNA from agarose and a ligation, test transformation, and Not1 digest were followed to obtain DNA between lengths of 100 kb to 350 kb. It also provides information on the success of the ligation and transformation using plant DNA as the source of inserts. After the acquired insert DNA was attained a mass transformation was done. This was by the spreading of agar on a plate and creating a colony of bacteria where each tray should contain between 2000-3000 colonies, and a vast majority of the clones should be white. When this was successfully done a robot, The Genetix Qbot, picked the white clones and placed them into freezing media in microtiter plates to create an ordered BAC library.
BINDING OF TRYPTOPHAN CONTAINING HISTONE H1[degrees]
Sonya Brown (1)*, Yuguang Song (2), and Susan Wellman (2), (1) Millsaps College, Jackson, MS 39210, and (2) University of Mississippi Medical Center, Jackson, MS 39216
H1[degrees] proteins are linker histones involved in the packaging of eukaryotic chromatin. To investigate the binding of these histones to double-stranded DNA, we created a mutant histone, H1[degrees]-W, which exhibited fluorescence. (Wild type H1[degrees] proteins are not fluorescent.) The QuikChangeTM Site-Directed Mutagenesis kit was used to replace the amino acid phenylalanine with the fluorescent amino acid tryptophan. Using an excitation wavelength of 280 nm and emission interval of 350-430 nm, we determined that significant fluorescence of the H1[degrees]-W protein was not detectable for concentrations of less than approximately 2 [micro]M. To investigate differences in binding site size on DNA and the binding affinity, we titrated both the H1[degrees] and H1[degrees]-W histones with varying concentrations of T4 Cyt DNA. Thermal denaturation curves were simulated. Analysis of this data suggests that the binding site size for the H1[degrees] protein is n = 8, where n is the number of base pairs bound by one histone, and that the affinity constant, K, is 1 X [10.sup.8] [M.sup.-1]. The values for the affinity and binding site size (respectively K = 9 X [10.sup.7] [M.sup.-1] and n = 7) for H1[degrees]-W are slightly smaller than those of H1[degrees]. However, data obtained from the study of a previously constructed H1[degrees]-W mutant suggested that the affinity is 7 X [10.sup.8] [M.sup.-1] and binding site size is 1[degrees]. Because of the variability of the H1[degrees]-W data, we are unsure of how much error is introduced into the data because of the experimental method.
SCREENING FOR CELL DEDIFFERENTIATION MUTANTS IN ARABIDOPSIS
June Sun*, Wai-Foong Hong, and Zhaohua Peng, Mississippi State University, Mississippi State, MS 39762
Most plant cells are totipotent, meaning that the differentiated cells are capable of regenerating new plants. This remarkable regeneration ability has been shown to depend on the function of cell dedifferentiation. By definition, cell dedifferentiation is a reverse process in which the fully specialized cells return to their primitive cell forms-the stem cell. The dedifferentiated cells are capable of reassigning their functions and destinations. Recently, it has been shown that cell dedifferentiation is essential for tissue regeneration (wounding repair) and cloning, and tightly associated with carcinogenesis in mammals, including human beings. In order to understand the mechanisms of cell dedifferentiation, Arabidopsis was used as a model plant for isolation of the related genes. In our mutant screening system, different levels of chemical induction were tested. A range of mutants were isolated, monitored, and recorded. Currently, we are analyzing the cell dedifferentiation related genes that have been identified from the mutants. Spatial and temporal expressions of these genes are going to be conducted in the future experiments. This work is significant because it may contribute to asexual propagation in plants and tissue/organ regeneration and cancer prevention in medical sciences.
SUB-PROTEOME ANALYSIS OF CHROMATIN AND ITS ASSOCIATED PROTEINS IN ARABIDOPSIS THALIANA
Lifeng Zhang*, Brahma Nanda Chitteti, and Zhaohua Peng, Mississippi State University, Mississippi State, MS 39762
Chromatin and its associated proteins play an essential role in cell differentiation and organ formation. Identification of proteins and their functions from different subcellular compartments is a branch of Proteomics. To identify protein expression profile of different tissues in Arabidopsis thaliana, we have developed a high efficient chromatin isolation method using differential centrifugation and sucrose gradients. The expression patterns of pure chromatin subproteome from different tissues have been showed on 2-D gels. Some proteins have been identified by MALDI-TOF and LC/MS/MS mass spectrometry analyses. Rest of chromatin and its associated proteins are going to be identified in the near future.
MEASURING SURFACE PRESSURE OF LIPIDS AT THE AIR/WATER INTERFACE
Shalawn Clark* and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406
Langmuir trough is an instrument that measures surface pressure monolayers of amphiphilic molecules at the air/water interface. The aim of our initial experiments was to test our setup with known lipid systems. We used monolayers of 1,2-diacyl-sn-glycero-3-phosphocholine isolated from egg (PC) and the synthetic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). At low surface pressures the lipid is in the two-dimensional gaseous state (G). Upon compression it will undergo a phase transition to the liquid-expanded state (L1). When the L1 state is subject to further compression a transition to the liquid-condensed state (L2) occurs. When the L2 state is further compressed the lipid will reach the solid state (S). Once the monolayer has reached the S state it cannot be further compressed and it changes into a three-dimensional structure, presumably a multilamellar form, which is marked by a rapid decrease in surface pressure. The isotherms constructed from our data demonstrate the existence of the known states and phase transitions. The present results represent control data for the future project, in which we will investigate protein-lipid interactions of cytolytic peptides and toxins.
PURIFICATION OF THE GLOBULAR DOMAIN OF HISTONE H1[degrees]
Kelly Woods (1)*, Eric George (2), and David T. Brown (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) University of Mississippi Medical Center, Jackson, MS 39216
Nucleosomes, the fundamental unit of eukaryotic chromatin, are composed primarily of DNA and histones. Histones are the most abundant proteins associated with eukaryotic DNA. One class of histones, known as the H1 or linker histones, bind to DNA to stabilize two full turns of DNA around the nucleosome core. Histone H1 facilitates the folding and stabilization of the 30 nm chromatin fiber and is believed to function as a non-specific repressor of transcription. Histone H1 has a tripartite structure consisting of a central globular domain and extended highly basic amino- and carboxy-terminal tails. The central globular domain is believed to bind to the nucleosome near the DNA entry and exit points. A long term goal of our laboratories is to understand this interaction in greater detail. Towards this end we have cloned the gene for a histone H1 variant called H1[degrees] and constructed a number of mutants in which the amino acid coding capacity has been changed by in vitro mutagenesis. These mutants will be analyzed by a number of in vivo and in vitro techniques. The major goal of this study was to develop methods for the production of large amounts of these proteins and for purification of the proteins to a sufficient level for analysis by circular dichroism and X-ray crystallography. Circular dichroism will determine the amount of alpha helical content of a protein and allow a preliminary determination of whether the secondary structure of our mutants is disrupted. Ultimately, X-ray crystallography will allow the three-dimensional structure of these proteins to be determined.
REGIO-SPECIFIC ([omega] TO [omega]-6) LAURIC ACID HYDROXYLATION IN HUMAN RECOMBINANT AND PEROXISOME PROLIFERATOR-TREATED JUVENILE CATFISH MICROSOMES
Mary L. Haasch*, Annette W. Ford, and Jimmy C. Allgood, University of Mississippi, University, MS 38677
Most studies of lauric acid hydroxylation have focused on only two hydroxylation products, [omega] and [omega]-1. Modifications of an HPLC method (Lemaire et al., 1992) to include GC-MS (Buhler et al., 1997) have allowed more sub-terminal hydroxylation products including [omega]-2 to [omega]-6 to be quantified. Previously we have shown that in male rat liver microsomes, [omega] to [omega]-2 hydroxylation products of lauric acid, significantly increase in response to peroxisome proliferating agents (PPAs). Interestingly, adult catfish exposed by i.p. injection, to the PPAs, clofibrate (CLO) or the more potent ciprofibrate (CPR), did not exhibit any changes in hepatic microsomal lauric acid hydroxylase (LA-OHase) activities, although kidney microsomal [omega] and [omega]-6 products were increased significantly. In rainbow trout, [omega]-1 lauric acid hydroxylation is catalyzed by CYP2K1 and [omega]6 by CYP2M1 (Buhler and Wang-Buhler, 1998). Induction of CYP2K1/2M1-like immunoreactive proteins in catfish by 48 hr i.p. injection of CLO and CPR has been shown (Haasch, 1996). Waterborne, 48 hr exposure of juvenile channel catfish (Ictalurus punctatus; size range 7.24 g to 34.32 g) to 1 ppm CLO (4.12 nM) or 2,4--dichlorophenoxyacetic acid (2,4-D; 4.52 nM) produced significant increases in both [omega]-1 and [omega]-5 hydroxylation products. Unexpectedly, [omega]-1 activity was absent in juvenile control and vehicle (DMSO) treated fish although we have previously shown that adults have relatively high [omega]-1 activity (Haasch, 1998). Regio-specific lauric acid hydroxylation was also investigated in human recombinant microsomes for isozyme control, CYP4A11 and CYP2E1. There is potential for sub-terminal hydroxylation products to act as second messengers in cellular signal transduction or to interfere with steroid biotransformation. These hypotheses will be investigated in future research. (Work supported by ES07929)
CHARACTERIZATION OF THE INTERACTION BETWEEN PROTEINS B23 AND NPM3
Nian Huang* and Mark Olson, University of Mississippi Medical Center, Jackson, MS 39216
B23/NPM is a predominant nucleolar protein, which has multiple activities, including nucleic acids binding, ribonuclease, interacting with the HIV-1 Rev protein and working as a molecular chaperone. In an effort to find the interacting partners of protein B23 a yeast two hybrid screen was performed. Several potential interacting partners were identified, including two ribosomal proteins. Multiple positive clones were found to contain a cDNA for protein NPM3, which belongs to the nucleoplasmin family of which protein B23 is also a member. B23 and some of its deletion mutants were able to be co-immunoprecipitated by FLAG tagged NPM3 in vivo. Using indirect immuno-fluorescence microscopy B23 and NPM3 co-localized in the nucleolus and both of their locations were dependent on pre-rRNA transcription. These two proteins were analyzed in whole cell extracts subjected to sucrose gradient ultracentrifugation. Unlike B23, of which most sedimented at 10S, NPM3 sedimented both near the top of the gradient and at 90S. This suggests although both of the proteins are involved with ribosome assembly, they may have different roles that depend on the components with which they associate.
8:30 Mississippi Functional Genomics Network (MFGN) Session
9:00 Divisional Poster Session
CELLULAR MECHANISMS REGULATING NOREPINEPHRINE TRANSPORTER ACTIVITY
P.G. Mandela* and G.A. Ordway, University of Mississippi Medical Center, Jackson, MS 39216
Neurotransmission at noradrenergic terminals is terminated by reuptake of norepinephrine (NE) back into cytoplasm via the norepinephrine transporter (NET). Given its pivotal role in neurotransmission, regulation of NET function has important implications for brain function in health and disease. This study examined the potential involvement of NE synthesis, storage and release in regulating NET function. Alpha-methyltyrosine (AMT) is a tyrosine hydroxylase inhibitor that abolishes NE synthesis. Treatment of NET-expressing cells (SK-N-SH and PC12 cells) with 5 to 100 [micro]M AMT did not affect NET function. Reserpine blocks vesicular storage of NE by blocking the vesicular monoamine transporter (VMAT) that is located on secretory vesicles. Brief exposure to reserpine reduced reuptake of NE by the NET in both PC12 and SK-N-SH cells. In PC12 cells, reserpine decreased uptake via the NET at concentrations as low as 10 nM, while reserpine's actions on NET activity were seen only at concentrations 5 [micro]M and above in SK-N-SH cells. In SK-N-SH cells, reserpine's effect on NE uptake at the NET was rapid, reaching a plateau within 5 min. Reserpine's effect on NE uptake was independent of protein kinase C and CAM kinase II mechanisms. Reserpine did not decrease the affinity (Km) of the NET for NE, but decreased the maximum velocity of the transporter (Vmax). Support Contributed By: MH 58211 and MH/AG 02031
GCR2, A TRANSCRIPTIONAL ACTIVATOR OF GLYCOLYTIC GENES, ALSO MEDIATES CELL CYCLE PROGRESSION
Je'Tua Eadon (1)*, Balaraj Menon (2), and George M. Santangelo (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) University of Southern Mississippi, Hattiesburg, MS 39406
Saccharomyces cerevisiae is a model organism of choice for studying various metabolic and cellular processes. GCR2, along with GCR1 and RAP1, is involved in the transcriptional activation of glycolytic genes. CLN1, CLN2 and CLN3 represent a family of cyclins, which are needed for transition from the [G.sub.1] to S phase of the cell cycle. We constructed different GCR2 and CLN deletion mutants (gcr2[DELTA], gcr2[DELTA] cln1[DELTA] cln2[DELTA], and gcr2[DELTA] cln3[DELTA]) and tested for their growth phenotypes at the permissive (23 [degrees]C) and restrictive temperatures (16 [degrees]C and 37 [degrees]C). The gcr2[DELTA] cln1[DELTA] cln2[DELTA] and gcr2[DELTA] cln3[DELTA] mutants were found to be both temperature and cold sensitive. Moreover, the gcr2[DELTA] cln1[DELTA] cln2[DELTA] and gcr2[DELTA] cln3[DELTA] mutants showed a different arrest phenotype after shifting to 37 [degrees]C in liquid medium. The gcr2[DELTA] cln1[DELTA] cln2[DELTA] mutants arrested as elongated or filamentous cells, while the gcr2[DELTA] cln3[DELTA] mutants arrested predominantly as unbudded cells, indicative of a [G.sub.1] phase arrest. The gcr2[DELTA] mutant by itself showed no change in phenotype or temperature sensitivity. Although further experiments need to be performed to elucidate the exact role of GCR2 in growth and cell cycle progression, preliminary studies suggest that a defect in glycolysis may seem to misregulate cell cycle progression.
A THERMALLY TARGETED POLYPEPTIDE INHIBITOR OF C-MYC TRANSCRIPTIONAL ACTIVATION
Adrienne Rembert (1)*, Gene L. Bidwell III (2), Melissa A. Skertich (2), and Drazen Racher (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) University of Mississippi Medical Center, Jackson, MS 39216
C-Myc is an oncogene that causes cells to divide at an uncontrollable rate when it interacts with Max. This c-Myc-Max interaction activates target genes that allow the cell to continue to divide. The current initial study explores the effect of cisplatin, an antimitotic drug, and PenELP (Penetratin elastin-like polypeptide) on T24 cell growth at different temperatures. ELP is a polymer that can be thermally targeted to tumor cells and penetratin is a substance that mediates uptake of the ELP by cells. In a human system, PenELP binds the cisplatin and carries the drug to the laser-heated rapidly growing cells. PenELP aggregates in the tumor cells to deliver an increased concentration of the drug. T24 cells, a bladder cancer cell line, were treated with cisplatin alone, PenELP alone and combined cisplatin/PenELP. The cells were then heated to different temperatures and changes in cell growth were recorded. The results indicate that 4-day cisplatin treatment was more effective at killing tumor cells than 4-day PenELP teatment. The 4-day cisplatin treatment followed by a 2-day PenELP treatment was most effective with more cells being killed at 42 [degrees]C. Further studies will be done to find the optimal temperature for thermal targeting of the cisplatin/PenELP combination treatment.
STRATEGIES FOR FUNCTIONAL GENOMICS IN SYNECHOCOCCUS ELONGATUS PCC 7942
Kimberly Baker (1)*, Carolyn Holtman (2), and Susan S. Goldman (1), (1) Jackson State University, Jackson, MS 39217, and (2) Texas A & M University, College Station, TX
The simplicity of cyanobacteria allows for its functionality. They are the simplest organisms known to have circadian rhythm functions. Circadian rhythms are daily cycles responsible for gene expression in response to changes in the day/night cycle. The objective of this project is to mutagenize and identify every gene that has an altered circadian rhythm function. The focus of this paper is on the strategies used to undergo the subcloning and mutagenesis of genomic DNA and introduction of mutants into the host strain of cyanobacteria, Synechococcus elongatus PCC 7942. The strategies include subcloning genomic DNA from cosmids to plasmids, mutagenesis of that DNA and placing mutant DNA in the S. elongatus genome. As a result of these processes a broader and more analytical perspective of molecular biological processes and gene manipulation has been obtained.
FLUORESCENT RECOVERY AFTER PHOTOBLEACHING (FRAP) STUDIES OF NUCLEOLAR PHOSPHOPROTEIN B23 AND ITS MUTANTS IN HeLa CELLS
Sandeep S. Negi* and Mark Olson, University of Mississippi Medical Center, Jackson, MS 39216
B23 is an abundant multifunctional nucleolar phosphoprotein involved in ribosome biogenesis. Different activities associated with protein B23 include nucleic acid binding, ribonuclease and molecular chaperone activity. It is phosphorylated during interphase by protein kinase casein kinase 2 (CK2) and by cyclin dependant kinase 1 (cdk1) during mitosis. Previous studies have shown that many nucleolar components involved in ribosome biogenesis exchange rapidly between nucleolus and nucleoplasm. We studied the effect of CK2 phosphorylation on the dynamics of B23 in the nucleolus. We made a green fluorescent protein (GFP) tagged mutant of the wild type protein by changing the ser (125) to ala (125) by overlap extension. This mutant cannot be phosphorylated by CK2 and localize to the nucleolus as confirmed by fluorescent microscopy. Fluorescent recovery after photo bleaching (FRAP) studies show that the wild type protein and the mutant have different dynamics in the living cell. The recovery of this mutant is slower than the wild type. This implies that the mutant exchange more slowly with nucleoplasmic pool of free GFP tagged mutant protein than the wild type protein. Further studies on phosphorylation mimicking mutants (by changing ser to glu) are being conducted. These studies will give us an insight into the role of phosphorylation in regulation of B23 dynamics in the nucleolus.
ENVIRONMENTAL INDUCTION OF CYP2M1-LIKE PROTEINS IN TWO TROPICAL FISH SPECIES BY PRODUCED FORMATION WATER ON THE NORTHWEST SHELF OF AUSTRALIA
Shiqian Zhu (1)*, Susan Codi (2), and Mary L. Haasch (1), (1) University of Mississippi, University, MS 38677, and (2) Australian Institute of Marine Science, Townsville, Qld, Australia
There is a paucity of information available regarding hydrocarbon exposure of tropical fish species inhabiting the waters near oil and gas platforms on the Northwest Shelf of Australia. In 1998, a pilot study was funded by Apache Energy Pty Ltd to evaluate the use of sub-lethal stress indicators in fish to assess exposure to produced formation water (PFW). Tropical fish, the Gold-Spotted Trevally (Carangoides fulvoguttatus) and the Bar Cheeked Coral Trout (Plectropomus maculatus), were obtained from two sites impacted by PFW discharge, Harriet A (near field) and Harriet C (far field) and a non-impacted reference site, the Montebello Islands. The purpose of this study was to examine the expression of the cytochrome P450 isozyme, CYP2M1. Microsomal fractions from Trevally had two immunodetectable CYP2M1-like proteins, while Coral Trout had only one immunodetectable CYP2M1-like protein. The significant increases of CYP2M1-like proteins in Trevally were observed at both PFW impacted sites compared to the reference site. These results indicate that different tropical fish species may have different expression patterns of immunodetectable CYP2M1-like proteins, and that PFW contamination near the oil well platforms may possibly induce CYP2M1-like proteins; male fish might be more sensitive than female fish. A previous study had shown increases in CYP2K1-like proteins. Increases in CYP2M1-like proteins in fish have not previously been described as a consequence of environmental petroleum hydrocarbon exposure.
ADH3 PROTEIN EXPRESSION AND DEVELOPMENTAL EFFECTS OF ETHANOL TOXICITY IN ZEBRA FISH (DANIO RERIO)
Erika Brown (1)*, Keri Van Derel (2), and Michael J. Carvan III (2), (1) Jackson State University, Jackson, MS 39217, and (2) Great Lakes WATER Institute, Milwaukee, WI 53204
Fetal Alcohol Syndrome (FAS) is a pattern of birth defects, such as physical and mental retardation, craniofacial malformations and joint abnormalities that occur during the development as a result of alcohol consumption during pregnancy. Zebra fish are used as a model system because they are easily bred and when exposed to ethanol they suffer from the same physical deformities and anomalies as humans do. In zebrafish, alcohol dehydrogenase (ADH3) is presumably responsible for metabolizing alcohol. In a study conducted by Dasmahapatra et al (2001), the EK and AB strains of the zebra fish were found to be more resistant to the ethanol when compared to the more sensitive TU strain. Using the AB and TU strains, it was found that the TU strain has higher levels of ADH3A mRNA than the AB strain, but it was expected to see more ADH3A mRNA in the AB strain since it is resistant to ethanol. The purpose of the present study was to investigate whether there would be a correlation between increased ADH3A mRNA and ADH3A protein levels. The cell lysate of the cloned ADH3A protein was used to check the antibody and showed that the antibody was recognizing the protein. The results from the western blots were not optimal. From this study, we concluded that in future experiments the assays would have to be repeated with serial dilutions of the antibody and protein to optimize the conditions. The experiment should repeated with the various treatment groups. This research was sponsored by NIMH-COR grant MH16926.
THE EFFECT OF COENZYME Q10 ON COGNITION IN AGING MICE
Harriet Crockett (1)*, Michael Forster (2), Scott Coleman (2), Nathalie Sumien (2), and Margaret Rutledge (2), (1) Alcorn State University, Alcorn State, MS 39096, and (2) University of North Texas Health Science Center, Fort Worth, TX
Oxidative stress involves increase in free-radical formation and a decrease in the levels of antioxidants in the mitochondria of the cell. Coenzyme Q10 is an antioxidant that prevents oxygen free-radical formation in the cell. The memory of coenzyme Q10 supplemented C57BL/6 mice were tested. The goal of this project was to determine if a dietary supplement of coenzyme Q10 could lessen the rate of oxidative stress occurring in the cell and improve impaired memory performance. Twenty-one-month-old mice were placed on a diet supplemented with coenzyme Q10 for 12 weeks. Four-month-old (young) and twenty-one-month-old mice (old) were used as non-supplemented controls. Assessments of memory were made using retention of habituation in the old and young mice in a LMA. The mice were given two LMA tests and horizontal activity was measured. The first LMA test was done 6 weeks after the diet began, and the second test was done after 12 weeks on the diet. Each test was done in four sessions that were 4-minutes in length. Trend suggested that young controls showed better retention than old controls. However, in this experiment, statistical analyses showed no effect, which probably accounted for the unequal number of young controls as compared to the number of old controls. Evidence obtained from mice that were supplemented with low concentrations of coenzyme Q10, suggested that the dietary supplement did improve retention of habituation. Mice given high concentrations of coenzyme Q10 showed either no effect or a detrimental effect to the memory of the mice.
POPULATIONSTRUCTURE OF TRIBOLIUM CAST ANEUM
Robert Walker (1)*, Mike Wade (2), and Jeffery Demuth (2), (1) Alcorn State University, Alcorn State, MS 39096, and (2) Indiana University, Bloomington, IN 47401
The purpose of this research study is to determine whether or not there is a correlation between genetic and geographical distance. The number of genetic differences that distinguish two populations is their "genetic distance." When the populations do not exchange genes, genetic distance between two populations tends to increase because of the combined forces of mutation, random genetic shifts, and natural selection. To investigate whether genetic and geographic distance are correlated in the flour beetle, we used a technique referred to as Amplified Fragment Length Polymorphism (AFLP). AFLP fingerprints can be used to distinguish even very closely related organisms, including near isogenic lines. Most importantly, AFLPs have been shown to be reproducible and reliable. Numerous individuals from ten populations were fingerprinted using multiple primer pairs. After analyzing results from a single primer pair, these results suggest that there is no direct correlation between genetic distance and geographic distance.
ENHANCED LEVELS OF ANTIOXIDANT ENZYMES AND LIPID PEROXIDATION PRODUCTS IN LIVER AND KIDNEY OF RATS EXPOSED TO LEAD
Levenia Baker (1)*, Yallapragada Prabhakara Rao (2), Bettiaya Rajanna (1), Christopher D. Bennett (1), Jon J. Brice (1), Samuel L. White (1), and Kiran Kumar (2), (1) Alcorn State University Alcorn State, MS 39096, and (2) Andhra University, Visakhaptanam, India
Lead is an environmental pollutant that affects several enzyme systems in liver and kidney. Liver and kidney are vital organs of the body, they show important detoxifying mechanisms to metal toxicity. In general, both tissues are provided with quantities of antioxidant enzymes namely catalase, superoxide dismutase and glutathione-S-transferase compared to other organs. The objective of the current study is to determine the effect of lead on antioxidant enzymes as well as lipid peroxidation products in liver and kidney. Lead acetate (500 ppm) was administered to rats (Wistar strain, male) through drinking water. Controls were given a similar dose of sodium acetate in water. The animals were sacrificed at intervals of 1, 4, and 8 weeks by cervical dislocation, liver and kidney tissues were isolated. The tissues were washed with ice cold normal saline and processed for the estimation of the antioxidant enzymes named above and lipid peroxidation products. The results indicated a gradual and significant increase in all the antioxidant enzymes (P<0.05) and lipid peroxidation products in both tissues as compared to their respective controls and this increase was found to be time-dependent. The data also indicated that these effects were more in liver than kidney. The results suggest that lead exerts oxidative stress on both the tissues but liver had a higher response rate of lead induced formation of reactive oxygen species than kidney. (Supported by NIH/FIC/MIRT #T37 TW00132 and NIH/NIGMS/MARC GM #08739)
INHIBITORY EFFECT OF LEAD ON IKK-ALPHA-NF-KAPPA B-NNOS IN RAT BRAIN
Chun-juan Shan*, Shang-Zhi Xu, and Bettaiya Rajanna, Alcorn State University, Alcorn State, MS 39096
Nitric oxide (NO) retrograde plays an important role in regulating synaptic vesicle endocytosis and recycling. Neuronal nitric oxide synthase (nNOS) is mainly responsible for its production in the brain. Transcription factor NF-kappa B is involved in nNOS regulation and it is activated by IkB kinase a (IKK-alpha) in response to stimulation. Although it is reported that nNOS was inhibited by lead exposure, our hypothesis is whether NF-kappa B and IKK-alpha are affected by lead exposure in development rat brain as well? SD rats were used to test our hypothesis. As female rats have gestated, rats started to drink 0.1% lead acetate until day 21. The pups were weaned and back to drink distilled water (DDW). Brain stem (BS), cerebellum (CB), hippocampus (HC), and frontal cortex (FC) of rat brain on postnatal day (PND) 1, 5, 7, 10, 51, 30, and 45 were collected. Our results demonstrated that on PND1 not only nNOS but also NF-kappa B and IKK-alpha were decreased by lead in the above regions particularly in HC and FC; Inhibition had been strengthened on PND 5 and continue until PND 10. However, on PND 45 nNOS, NF-kappa B and IKK-alpha were recovered in all the parts of rat brain. It was suggested that nNOS, NF-kappa B and IKK-alpha were inhibited by lead treatment in earlier phase, but restored after the influence of lead was declined.
PKC ACTIVITY AND PROTEIN EXPRESSION WERE BOTH INHIBITED BY LEAD EXPOSURE IN THE BRAIN OF RATS
Latoya Bullock*, Kimberly Cornelius, Shang-Zhi Xu, and Bettaiya Rajanna, Alcorn State University, Alcorn State, MS 39096
There are at least 11 distinct isoforms of Protein kinase C (PKC). The differences among the PKC isoforms are attributed to the calcium or diacylglycerol-dependence activation of each. Most PKC isoforms are involved in the regulation of neurotransmitters, neuron growth, and anti-apoptosis. In the present study, when female rats were gestated, rats started to drink 0.1% lead acetate until 21st day of pups. The pups were weaned. Then they started back drinking distilled water (DDW). The brain stem (BS), cerebellum (CB), hippocampus (HC), and frontal cortex (FC) of the rat brain on postnatal day (PND) 1, 5, 7, 10, 15, 30, and 45 were collected. The PKC activities of total, calcium-independent, and calcium-dependent were determined by transferring [gamma]-[P.sup.32]-ATP to histone. Immunoprecipitation and western blotting were used to assay protein expressions of PKC isoforms. Our results indicated that on PND 1, 5, and 10 total PKC activity was significantly inhibited by lead; most of them were calcium-dependent. On PND 1, PKC-[gamma] both in cytosolic and membrane fraction were decreased in HC and FC by lead, but other PKC isoforms did not occur; On PND 10, all of the above PKC isoforms were inhibited in the BS, CB, HC, and FC membrane fraction by lead in comparison with control group. On PND 45, the above PKC isoforms in lead treated group were restored except [epsilon]. It suggests that not only is PKC activity dramatically inhibited by lead exposure in the developing brain of rats but protein expression is also.
A QUICK AND RAPID TEST FOR POLYAMINES IN URINE
Jana Causey*, Amber Mcilwain, and Robert C. Bateman, Jr., University of Southern Mississippi, Hattiesburg, MS 39406
This project is aimed at finding better methods for determining the effectiveness of cancer treatments. In patients with successful curative surgical treatment, all preoperatively elevated urinary polyamine concentrations markedly decreased and returned to normal, whereas they were elevated and increased further in patients with proven relapse of the tumor and/or metastasis in different organs. We have developed rapid dipstick tests that will detect the presence of polyamines in urine. The stick will turn different shades of purple that can be matched to a standard shade to determine the concentration of polyamines present in the sample. Unfortunately, putrescine is often acetylated and interferes with the validity of the dipsticks. We have obtained a plasmid that contains the deacetylase. We present here the expression of the bacterial deacetylase and incorporation into the urine diamine assay.
INCREASED TOXICITY OF HALOGINATED HYDRO-CARBONS WITH THE DEGREE OF HALOGENATION: A CYTOGENETIC STUDY IN MICE BONE MARROW CELLS
Babu P. Patlolla (1)*, Anita K. Patlolla (2), and B.S. Sekhon (2), (1) Alcorn State University, Alcorn State, MS 39096, and (2) Jackson State University, Jackson, MS 39217
Halogenated aliphatic hydrocarbons have long been regarded as a pharmacological and toxicological entity. They are widely used in industry as solvents, degreasing agents, plastisizers and chemical intermediates in the manufacturing of other chemicals. Major halogenated compounds are the chlorinated hydrocarbons. In this study three chlorinated hydrocarbons were tested for their cytogenetic effects in mice bone marrow cells. Five different concentrations of 1,1-dichloroethane (500, 400, 300, 200, and 100 mg/Kg body weight), 1,1,1-trichloroethane (200, 150, 100, 50, and 10 mg/Kg body weight), and 1,1,2,2-tetrachloroethane (50, 40, 30, 20, and 10 mg/Kg body weight) were used to see the effect on chromosomal aberrations, mitotic index and micronuclei. Results indicated that the toxicity was highest in 1,1,2,2-tetrachloro-ethane and lowest in 1,1-dichloroethane. The increased effect could be due to the increasing degree of halogenation.
Section IV Cellular Models
9:30 THE RAP1/GCR1/GCR2 TRANSCRIPTIONAL ACTIVATION COMPLEX ASSOCIATES WITH THE NUCLEAR ENVELOPE
Balaraj Menon (1)*, Kristine A. Willis (2), Satish Pasula (1), Nayan Sarma (1), Kellie E. Barbara (1), Rebecca Phelps (1), Brenda Andrews (2), and George M. Santangelo (1), (1) University of Southern Mississippi, Hattiesburg, MS 39406, and (2) University of Toronto, Toronto, ON M5S1A8
The budding yeast Saccharomyces cerevisiae is an excellent model organism with which to study various eukaryotic cellular processes. The transcriptional activator Gcr1 in S. cerevisiae enhances expression of ribosomal protein genes, glycolytic genes, and all three G1 cyclin genes (CLN1, CLN2, and CLN3). Deletion of GCR1 causes delayed passage through Start in the cell cycle due to loss of the response to glucose. Mass spectrometry of partially purified Gcr1 detected copurification of both Kap123 (a karyopherin that associates with nuclear pores and is involved in protein import/export) and the replication factor Mcm2; we have also successfully immuno-precipitated complexes that contain both Gcr1 and Kap123. Since Gcr1 (785 residues total) contains transmembrane domains (residues 64-92 and 191-219) and localizes to the yeast nucleus, we investigated the possibility that it functions at the nuclear periphery. We found that Gcr1 was indeed present in purified yeast nuclear envelope fractions. Further, co localization and Fluorescence Recovery after Photobleaching (FRAP) studies suggest that Gcr1 and associated factors (Rap1 and Gcr2) are components of the same relatively immobile nuclear structure.
9:45 THE USE OF AG490, A PROTEIN TYROSINE KINASE INHIBITOR, TO GENERATE NON-TYROSINE-PHOSPHORYLATED RAT JANUS KINASE 2 MUTANTS IN SF21 INSECT CELLS
Kimberly Cornelius (1)*, Kiranam Chatti (2), and Roy J. Duhe (2), (1) Alcorn State University, Alcorn State, MS 39096, and (2) University of Mississippi Medical Center, Jackson, MS 39216
Janus kinases (JAKs) are intracellular protein tyrosine kinases, which regulate proliferation, differentiation and apoptosis through their role in cytokine and endocrine hormone signal transduction. Phosphorylation of tyrosines within the activation loop of JAK2 alters the efficiency of catalysis with respect to ATP. We intend to develop JAK2-targeted anticancer drugs by exploiting the differential catalytic properties of phosphorylated vs. non-phosphorylated JAK2. When rat JAK2 mutants are overexpressed in Sf21 insect cells, they are purified as a heterogeneous mixture of tyrosine-phosphorylated and non-phosphorylated enzymes. Our objective was to maximize production of non-phosphorylated JAK2 proteins by treating infected Sf21 cells with AG490 during the production of these proteins. Recombinant baculoviruses expressing glutathione-S-transferase-tagged forms of rat JAK2 were used to infect Sf21 insect cells, which were then treated with AG490. Cells were harvested after 48 hours, aliquots of cell lysates were boiled with sample buffer and resolved by SDS-PAGE, transferred to PVDF membrane, probed with polyclonal anti-JAK2 and monoclonal anti-phosphotyrosine antisera. Our results showed that phosphorylated JAK2 was in each sample except for 200 [micro]M AG490 samples. We concluded that treating Sf21 cells with low concentrations of AG490 did not significantly alter phosphorylation status of JAK2, and treatment of Sf21 cells with concentrations of 100 [micro]M or greater impaired production of JAK2.
10:00 THIOREDOXIN-MEDIATED ENHANCEMENT OF JANUS KINASE ACTIVITY
Naila Mamoon*, Sheeyong Lee, and Roy J. Duhe, University of Mississippi Medical Center, Jackson, MS 39216
Thioredoxin (Trx), a dithiol oxidoreductase has been reported to promote cell growth by enhancing the mitogenic effect of cytokines. Overexpression of Trx occurs in certain leukemias wherein cells proliferate without cytokine stimulus, a phenomenon attributed to constitutive JAK3 activation. The catalytic activities of JAK2 and JAK3 are modulated by their redox states; enzymatic activity is abolished by oxidation and restored upon reduction by dithiol reducing agents. We hypothesize that the observed constitutive activation of JAK3 occurs due to high intracellular levels of Trx, a biological reductant, which maintains JAK3 in its maximally active state, leading to unabated proliferation of T-cells in the absence of mitogenic stimulation by IL-2. Using recombinant Trx and GST-rJAK2 in an in vitro assay we demonstrate that Trx restores autokinase activity of oxidatively-inhibited JAK2 while a redox-inactive mutant form of Trx does not. Experiments are underway to demonstrate a similar effect of Trx on endogenous JAK2 and JAK3 and to identify the cysteines that confer redox sensitivity to these enzymes. Using IL-2/JAK3 and IL-3/JAK2 signal transduction models, we are currently investigating the effects of high levels of Trx on cellular proliferation. Preliminary data show that exogenously added Trx enhances the mitogenic effect of cytokines. Pretreatment with purified Trx countered the growth-inhibitory effect of DNCB, a thioredoxin reductase inhibitor. The overall goal of this project is to validate a novel target for redox-based chemotherapy.
10:15 AN IN VITRO APPROACH TO CHARACTERIZE STATE-SELECTIVE JANUS KINASE 2 INHIBITORS
Kanakadurga Kundrapu*, Kiranam Chatti, and Roy J. Duhe, University of Mississippi Medical Center, Jackson, MS 39216
Janus kinases are cytoplasmic protein tyrosine kinases with crucial physiological roles. The binding of various class II cytokines to their receptors initiates JAK activation, which in turn causes proliferation or differentiation in cells of various lineage. Uncontrolled JAK2 activity may contribute to the progression of certain cancers. Considerable interest exists in developing JAK-targeted inhibitors. We are currently using an in vitro approach to identify and characterize selective inhibitors of rat JAK2 activity. Several approaches to develop a quantitative biochemical assay for rJAK2 activity have been under way in our laboratory. One of our novel findings has been that rJAK2 exists in at least two distinct states of activity. The phosphorylation of tyrosines within the activation loop of rJAK2 appears to increase its autocatalytic efficiency with respect to ATP, with highest activity when its activation loop is phosphorylated and lower activity when the activation loop is unphosphorylated. We are determining whether this implied difference in ATP binding affinities between the two states of rJAK2 also extends to a difference in affinities for the acceptor substrates, such as the STATs. Based on our demonstration of the differential behavior of these two states towards ATP, we propose that a JAK2 inhibitor(s) could selectively distinguish between these two activity states. State-selective inhibitors of JAK tyrosine kinase activity would represent a novel class of potential therapeutic agents.
10:45 IDENTIFICATION OF CYSTEINE RESIDUES RESPONSIBLE FOR REDOX SENSITIVITY OF JANUS KINASE 2 VIA SITE-DIRECTED MUTAGENESIS
Jay Craddock (1)*, Kiranam Chatti (2), and Roy J. Duhe (2), (1) Millsaps College, Jackson, MS 39210, and (2) University of Mississippi Medical Center, Jackson, MS 39216
Janus protein tyrosine kinases are crucial transducers of signals originating when cytokines bind to their receptors. We have shown that the activity of JAK2 is sensitive to the redox status of the enzyme. It is fully active when reduced mildly and completely catalytically inactive with mild oxidation. This reversible redox sensitivity suggests the interconversion of disulfide/dithiol bonds between cysteine residues within (or near) the catalytic site of JAK2. There are nine cysteine residues within the C-terminal domains of rat JAK2 that may participate in the dithiol-disulfide exchange and thus contribute to the redox sensitivity. Using a recombinant form of JAK2 subcloned into a baculoviral transfer vector (pAcGHLT-A:(Ndelta661)rJAK2), we are creating a progressive series of site-directed mutant enzymes, with each successive mutant harboring an additional substitution of serine for each of the nine cysteine residues. Each mutation is being verified by DNA sequencing. The mutant transfer vectors will be used to create recombinant baculoviruses to produce JAK2 variants in infected insect cells. Preliminary work using nine single cysteine-to-serine mutants showed no significant loss of redox sensitivity in any single mutant. Our hypothesis is that the enzyme will lose redox sensitivity after all critical cysteines are converted to serines. The identification of the critical cysteine(s) may provide a useful structural determinant for the design of novel JAK2-targeted drug candidates.
11:00 THE LOCALIZATION OF HOST AND VIRAL DNA DEPENDENT RNA POLYMERASE II IN IRIDOVIRUS INFECTED CELLS
Jinghe Mao (1)*, Dexter Whitley (1), Locke Bryant (2), and V.G. Chinchar (2), (1) Tougaloo College, Tougaloo, MS 39174, and (2) University of Mississippi Medical Center, Jackson, MS 39216
Frog Virus 3 is a member of the family Iridoviridae. The cascade of viral gene expression is divided into two stages, early and late, that take places within the nucleus and cytoplasm, respectively. It has been hypothesized that both host and viral DNA dependent RNA polymerases II (Pol II) are involved in this process. Host Pol II is utilized for the transcription of immediate early and early genes, and viral transcriptional machinery (especially viral Pol II) is responsible for late gene expression. The objectives of this study are to localize the host and viral Pol II in mock and virus infected cells and to determine whether viral infection leads to a re-distribution and modification of host Pol II. An indirect immunofluorescent assay, using the 8WG 16 monoclonal antibody (specific to largest subunit of cellular Pol II), was done for visualization. In addition, western blot analysis was performed to detect for possible phosphorylation of Pol II. Preliminary data showed that host Pol II are mainly present within the nucleus of mock-infected cells, following infection, they migrated into cytoplasm and subjected to some modification. The results of this project will help us understand the mechanisms of transcriptional regulation in iridovirus-infected cells.
11:15 CHARACTERIZATION OF THE ASPERGILLUS NIDULANS snxB1 MUTANT PHENOTYPE
Ryan Day* and Sarah Lea McGuire, Millsaps College, Jackson, MS 39210
The filamentous fungus Aspergillus nidulans grows by germ tube extension from a uninucleate spore. As the germ tube extends, the nuclei undergo synchronous mitosis and daughter nuclei migrate into the germ tube. After the third mitotic division, the cell begins to lay down incomplete septa, separating the hypha into numerous multinucleate compartments. While the general mechanisms that control mitosis are understood, the molecular mechanisms involved in coordination of septation (the equivalent of cytokinesis) and nuclear division remains a mystery. Our laboratory has recently identified snxB1, a mutation that suppresses the heat sensitivity of the nimX2cdc2 mutation and causes increased septation in the hyphae. This increased septation leads to smaller cell size and compact colony growth. We have begun experiments aimed at further characterizing the effects of the snxB1 mutation and at cloning the snxB gene by complementation of the mutant phenotype. Our preliminary results suggest that snxB1 causes loss of mitotic synchronization in cellular compartments following sublethal DNA damage and allows septation in the presence of hydroxyurea, but that the timing of mitosis in these mutants is similar to wild type. The mutant phenotype is osmotic remedial, which obfuscates cloning by complementation and selection. We are attempting to clone the gene by complementation and screening of transformants. Molecular characterization of this gene will allow a better understanding of the relationship between cytokinesis and nuclear division in this organism.
11:30 ANALYSIS OF PROTEINS ASSOCIATED WITH PP38 IN CELLS INFECTED WITH MAREK'S DISEASE VIRUS
Angela Pippin (1)*, Ross Whitwam (1), and Shane Burgess (2), (1) Mississippi University for Women, Columbus, MS 39701, and (2) Mississippi State University, Starkville, MS 39762
Marek's disease (MD) virus (MDV) is a naturally-occurring lymphomagenic a-herpesvirus of domestic fowl. MDV is also a model for human lymphomagenesis and for virus evolution towards hypervirulence. A 38 kiloDalton phosphoprotein (PP38), encoded by MDV, is expressed by MDV lytically-infected cells. PP38 is unique to MDV and, although not oncogenic, is critical to the pathogenesis of MD lymphoma. PP38's function remains enigmatic. Our goal was to identify proteins that interact with pp38 so as to infer the function of PP38 using proteomics--a so-called "guilt by association" approach. Associated proteins will be co-immunoprecipitated with PP38 from MDV-infected cells (using a monoclonal antibody). Protein identification will use liquid chromatography electrospray ionization tandem mass spectrometry. A source of MDV-infected cells was needed. Chicken embryo fibroblast (CEF) cells provide a source of cells for MDV infection. We established viable primary and secondary in vitro chicken embryo fibroblast cultures. These cells were stored at -80 [degrees]C and resurrected when needed. Primary and resurrected CEF cultures were successfully infected with MDV in vitro. Plaque assays confirm infection and provided infection titers. Our results will allow work to begin towards immunoprecipitation of PP38 and its cellular partners. In parallel, and in preparation for the proteomics analysis, a number of proteomics experiments were done using model tissues. These proteomics experiments will be briefly described.
Section V Organism Models
1:30 REGULATION OF VIRULENCE FACTORS BY QUORUM SENSING IN STAPHYLOCOCCUS AUREUS
Davida Crossley* and Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS 39406
Staphylococcus aureus is capable of causing a wide range of infections such as food poisoning, toxic shock syndrome, endocarditis, and osteomyelitis. Due to the increasing antibiotic resistance, it has become extremely important to find new targets to combat staphylococcal infections. One possible target is quorum sensing which allows S. aureus to regulate gene expression according to cell density. S. aureus uses a well characterized quorum sensing system, agr, to control the expression of several virulence factors. During growth, a signal molecule will accumulate in the medium as the number of cells increases. When cell density is low, surface proteins are expressed and when cell density is high, secreted proteins are expressed. Recently, a novel quorum sensing system (luxS) has been discovered in the marine bacterium Vibrio harveyi, where it regulates bioluminescence. LuxS has been discovered in a wide variety of bacteria. In E. coli, Streptococcus pyogenes, and Clostridium perfringens, luxS regulates virulence factors. In this study we will investigate the role of luxS in the regulation of virulence factors in S. aureus. We will examine the effect of luxS mutation on the expression of agr, sarA, and other virulence factors. We will also investigate the possible interaction between luxS, sarA, and agr system by examining the effect of agr, and sarA mutations on the expression of luxS.
1:45 REGULATION OF VIRULENCE FACTORS IN STAPHYLOCOCCUS AUREUS
Karthik Sambanthamoorthy* and Mohamed O. Elasri, University of Southern Mississippi, Hattiesburg, MS 39406
Staphylococcus aureus is an extraordinarily versatile pathogen causing a plethora of infections ranging from superficial infections (skin abscess, wound infections), to toxemic syndromes (food poisoning), to life-threatening diseases (osteomyletis, endocarditis). S. aureus has developed resistance to a wide range 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. The virulence factors are either cell bound proteins (e.g., adhesins) or secreted proteins (toxins). The cell bound proteins are expressed early and repressed late during infection. On the other hand, 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). 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 factors that modulate sarA function. Our goal is to identify and characterize these new factors which represent potential new therapeutic targets for the prevention and treatment of staphylococcal infections.
2:00 ANTIOBIOTIC RESISTANT COLIFORM BACTERIA IN FECES OF FREE-RANGE AND COMMERCIALLY RAISED CHICKEN
Daniel Murin* and Sabine Heinhorst, Oak Grove High School, Hattiesburg, MS 39402, and University of Southern Mississippi, Hattiesburg, MS 39406
Antibiotic resistant bacteria are a problem that has become more apparent in recent years. While the origins of antibiotic resistant bacteria are unknown, there is strong evidence for a connection between the use of antibiotics in agricultural feeds and the emergence of coliform bacteria that are resistant to those antibiotics. In this study, 276 bacterial clones from chicken raised in commercial henhouses and 190 isolates from free-range chicken were screened for their resistance to ampicillin, tetracycline, and erythromycin. Plasmid DNA was subsequently extracted from 56 clones and examined by gel electrophoresis. Selected plasmids were used to transform a lab strain of Escherichia coli, and transformants were selected on media containing antibiotics. Plasmid DNA recovered from individual transformants was digested with restriction enzymes to compare the resulting DNA fragment patterns with those obtained from the plasmid DNA that was used to transform E. coli. Results from this study showed that antibiotic resistance was not limited to coliform bacteria from commercially raised chicken and was widespread among free-range animals. Plasmid DNA obtained from resistant bacterial isolates was capable of transferring the ampicillin resistance trait to E. coli.
2:15 CHEMICAL MODULATION OF AGE-DEPENDENT TISSUE DEGENERATION
Astrid Gutierrez-Zepeda*, Zhixin Wu, Kenneth J. Curry, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406
Caenorhabditis elegans is used to study aging and many human diseases due to its short life span and the existing genomic resemblance to humans. We have previously demonstrated that the Ginkgo biloba extract, EGb 761, extends life span of C. elegans. In this study, C. elegans were used to determine biochemical processes that regulate the onset of muscle degeneration due to aging, using chemical manipulation, behavioral assay, fluorescence microscopy, and electron microscopy (EM) techniques. An average of thirty C. elegans in each group were treated with vehicle EGb 761, nicotinamide (a chemical known to inhibit SIR-2 gene), or in combination. The motility behavior was scored based on the nematode's movement. Three different movements were set as standards: A movement (body movement without stimulation), B movement (body movement only with stimulation), and C movement (head movement only with stimulation). As the C. elegans grew older, the A movement declined and the C movement became prevalent. It was observed that nicotinamide caused an earlier onset of C movement in the nematodes, compared to the control and other treatments. Nicotinamide also hastened muscle degeneration, which was detected by fluorescence microscopy. Ultrastructure changes observed under EM showed a delay in the age-dependent muscle degeneration by EGb 761 treatment. These results suggest that muscle degeneration due to aging can be modulated by the use of certain drugs, probably via regulation of the longevity assurance genes.
2:30 EGb 761, AN EXTRACT OF GINKGO BILOBA LEAVES, ALLEVIATE TOXICITY INDUCED BY A[beta] IN TRANSGENIC C. ELEGANS MODEL OF ALZHEIMER'S DISEASE
Yanjue Wu*, Zhixin Wu, Astrid Gutierrez-Zepeda, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406
Alzheimer's disease (AD), the most common cause of decline in cognitive performance in elderly people, is associated with pathological features such as [beta]-amyloid (A[beta]) deposits, neurotic plaques, neurofibrillary tangles, and degeneration of synapses, neurons in the brain. Although the pathogenesis of AD is not fully understood, recent studies indicates that the A[beta] deposits and the local reactions of different cells play major roles in the development of the disease. We used an inducible A[beta]-expressing transgenic Caenorhabditis elegans strain CL4175 to correlate A[beta] deposits with its toxicity and with production of reactive oxygen species (ROS). Our results demonstrated that the A[beta] expression-induced paralysis was delayed in the C. elegans fed with EGb 761, an extract from the Ginkgo biloba leaves known to have neuroprotective effects. We also found A[beta] deposits in pharyngeal region of another strain CL2006, which was engineered to constitutively express high levels of the human beta amyloid peptide, were significantly decreased in EGb761 treated C. elegans than those untreated controls. Using the immunoblotting, we show A[beta] oligmer dispersed in CL2006 after EGb 761 treatment. Our results indicated A[beta] toxicity in vivo could be offset by EGb 761, at least in part, via anti-oxidative and anti-oligomerization activities.
2:45 ATTENUATION OF ELEVATED OXIDATIVE FREE RADICALS IN ALZHEIMER'S DISEASE C. ELEGANS MODEL BY GINKGO BILOBA EXTRACT
Julie Smith* and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406
Research indicates that cellular insults resulting from free radicals may be a major contributor to the neurotoxicity and pathology of Alzheimer's Disease (AD). According to the Amyloid [beta] A[beta]-induced oxidative stress theory of AD, internal expression of A[beta] would cause a rise in the levels of reactive oxygen species (ROS). To determine whether ROS levels are increased by endogenous A[beta] expression, we employed an AD-associated transgenic Caenorhabditis elegans model constitutively expressing human AD. Significantly higher ROS levels were observed in AD-associated models compared to wild type counterparts. The elevated levels were attenuated by pretreatment with EGb761, a standardized Ginkgo biloba extract with potent antioxidant properties used extensively in clinical trials. Additionally, of the individual extract constituents tested, the most dramatic attenuation of ROS levels was achieved by pretreatment with the flavonoids, kaempferol, and quercetin. Validity of this assay is supported by using a superoxide dismutase (SOD)-deficient C. elegans strain displaying an oxidation-sensitive phenotype, showing a dramatic increase in ROS levels compared with controls. Furthermore, an age-dependent increase in [H.sub.2][O.sub.2]-related ROS was observed in the wild type C. elegans, which was accelerated in the AD-associated C. elegans mutants. These results support the hypothesis of the involvement of A[beta] and ROS in association with AD, suggesting that in vivo modulation of intracellular oxygen free radical levels is one mechanism through which EGb761 provides neuroprotection.
3:00 Divisional Business Meeting
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|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Jan 1, 2004|
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