Cellular, molecular and developmental biology.
Vicechair: Ross Whitwam, Mississippi University for Women
8:30 Special Joint Session with the Science Education Division
Introduction; Howard Walters and Roy J. Duhe, J.L. Scott Marine Education Center and Aquarium, Biloxi, MS 39530, and University of Mississippi Medical Center, Jackson, MS 39216
8:45 STRUCTURE VISUALIZATION IN BIOCHEMISTRY EDUCATION: SEEING IS BELIEVING
Robert Bateman, University of Southern Mississippi, Hattiesburg, MS 39406
With the advent of powerful desktop and laptop personal computers has come a wealth of computational tools that can aid in the understanding of chemical and biochemical concepts. This talk will survey several of the free structure visualization software packages that are currently used in biochemistry instruction. The emphasis will be on the most effective uses of these tools both inside and outside the lecture classroom.
9:30 Introduction; Roy J. Duhe
Session 1: "Molecular Insights from Biophysical Techniques"
9:45 DETECTION OF PROTEASE ACTIVITY WITH A BIOSENSOR
Daryl [Pollard.sup.1], Patina [Thompson.sup.2], Amy [Denson.sup.1], Steven [Adamson.sup.1], Newton [Fawcett.sup.1], and Jeffrey [Evans.sup.1*], [University.sup.1] of Southern Mississippi, Hattiesburg, MS 39406, and [Alcorn.sup.2] State University, Lorman, MS 39096
The quartz crystal microbalance (QCM) is a biosensor that can measure mass changes accompanying biochemical processes. A decrease in mass occurring on the biosensor's quartz surface is sensed as an increase in vibrational frequency of the crystal. Approximately a one Hz increase in frequency represents a 1 nanogram mass decrease on the crystal. We examined whether the QCM biosensor could detect proteases by detecting the change in mass of protein that accompanies proteolytic cleavage. In testing for protease activity we first attached the protease substrate casein to the surface of the biosensor. When the casein coated biosensor was incubated with the protease trypsin, a mass change occurred as casein was released from the biosensor. This mass change representing protease activity was detected by the biosensor.
10:00 INTERACTION OF AN HIV PEPTIDE WITH MODEL MEMBRANES
Yuko [Tsutsui.sup.*], Slobodanka D. Manceva, and Peter Butko, University of Southern Mississippi, Hattiesburg, MS 39406
Trans-activator protein (TAT) is a protein produced by HIV. This peptide is thought to have a translocation activity and can carry fused molecules into the cytosol of the cell by forming an inverted micelle. Our interest was to test the hypothesis if this peptide can translocate itself through phospholipid membranes. The peptide used in this study has tryptophan (Trp), an aromatic amino acid, as the end of the peptide chain so we were able do studies of fluorescent peptide/lipid interactions. In this study we used large unilamellar vesicles (LUVs) as model membranes. Studies done with neutral (phosphatidyl choline-PC) and positively charged (10% phosphatidyl glycerol PG/PC) LUVs suggest that the peptide lipid interactions are due to the electrostatic forces between negatively charged peptide and positively charged LUVs. Experiments done on release of florescence dye entrapped in LUV show that a high concentration of the peptide was necessary in order for 50% dye to be released, implying that the peptide does not destroy LUVs by making a pore. By using quenching of the Trp fluorescence with acrylamide we obtained a quenching constant, which was the same as the one acquired for free Trp in a solution. These two studies show that the peptide does not insert in the lipid membrane, and it rather destroys them through a different mode of action.
10:15 MODE OF ACTION OF BACILLUS THURINGIENSIS d-ENDOTOXIN CYTlA: DETERGENT OR PORE FORMER?
Slobodanka D. [Manceva.sup.*] (1), Marianne Pusztai-Carey (2), Paul Russo (3), and Peter Butko (1), (1.) University of Southern Mississippi, Hattiesburg, MS 39406; (2.)Case Western Reserve University, Cleveland, OH; and (3.) Louisiana State University, Baton Rouge, LA
Cyt1A is a d-endotoxin from Bacillus thuringiensis var. israelensis, which is highly toxic to the Diptera larvae. It is used as an environmentally safe insecticide, although its cytolytic mechanism is a subject of controversy. It is believed that it makes pores or cation -selective channels in lipid membranes. However, a mass of evidence collected in the last few years does not support that hypothesis. We propose that this protein commences cell death by destroying the cell membrane in a detergent-like manner, rather than by inserting into the membrane and forming a pore. In this work we tested our hypothesis by employing fluorescence photobleaching recovery (FPR), dynamic light scattering (DLS) and electron microscopy (EM) to study Cyt1A-induced changes in size distributions of large unilamellar vesicles (LUV). Upon addition of the toxin to the LUV with a diameter of 100 nm, a new population of lipid particles with a diameter of 54 nm appeared. Since no change in the vesicle size is expected when a pore is f ormed in its membrane, our results give support to the detergent-like model of Cyt1A's action
10:30 INHIBITION OF RGS PROTEINS
Leighton [Janes.sup.*] (1), R.R. Neubig (2), and N.E. Outlaw (1), (1.) Delta State University, Cleveland, MS 38733, and (2.) University of Michigan Medical School, Ann Arbor, MI 48103
Inhibition of RGS interactions can lead to assessing RGS function and specificity in cells and provide a basis for understanding mechanisms of therapeutic drugs that target RGS interactions. RGS inhibitor proteins include YTI3 (lactam), YJl4 (lactam), and YJ16 (BU)-3. Of the peptides tested, YJ 13 is the most potent inhibitor of RGS stimulation and is the best fit for the Alpha subunit binding pocket. The inhibitor peptides are more specific to RGS4 than RGS8. YJ13 may be modified to better inhibit RGS stimulation with more specificity.
Session 2: "The Evolution of Biological Catalysis"
11:00 RNA STRUCTURE PROBING AND ANALYSIS
Rishi [Agarwal.sup.*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406
The "RNA World" hypothesis describes a preprotein world where RNA was able to both carry genetic information (later evolved into DNA) and act as biocatalysts (most of them replaced by proteins). To support the RNA world hypothesis, numerous artificial RNA enzymes (ribozymes) have been isolated by the powerful combinatorial technique called SELEX. Structural and functional analysis of RNA can provide mechanistic basis for RNA catalysis. A self-capping ribozyme named Iso6, an RNA previously isolated in vitro, was employed to investigate the various structural changes that may occur upon binding with divalent metal ions, primarily [Ca.sup.2+] Structural probing of Iso6, i.e., using a variety of chemical and enzymatic probes, was used to search for possible sites of protection, binding sites or conformational changes. Initial experiments were conducted by searching for optimal chemical and enzyme concentrations by which Iso6 was either cut randomly or specifically. The specific cuts of RNA could provide structura l information of Iso6, while random digestion of Iso6 yielded markers to map nucleotide locations. Reagents used included chemicals [Pb.sup.2+], DMS, and DEPC and enzymes RNase T1 and nuclease SI. Different sizes of RNA fragments were fractionated by denaturing polyacrylamide gel electrophoresis, and quantitation of individual RNA bands was achieved by phosphorimaging. Many probing experiments under different conditions were conducted. The results demonstrated applicability of our RNA probing methods to the investigation of RNA structure and mechanism in general. However, we have yet to locate specific structural and conformational changes upon metal and substrate binding. Further experiments will be necessary to reveal the structural information from Iso6 RNA.
11:15 IN VITRO EVOLUTION OF A THIOESTER SYNTHASE RIBOZYME
Tricia M. [Coleman.sup.*] and Faqing Huang, University of Southern Mississippi, Hattiesburg, MS 39406
Thioesters are important intermediates in the metabolism of modern organisms. Coenzyme A (CoA), a common coenzyme, participates in various biochemical pathways, functioning through the formation of thioesters via its sulfhydryl group. Our lab has previously isolated RNA sequences capable of catalyzing the synthesis of CoA, NAD, and FAD from their respective precursors, thereby supporting the availability of these coenzymes in an 'RNA world, In order to demonstrate the utility of these coenzymes as well as the plausibility of thioester synthesis in an RNA world, an iterative in vitro evolution procedure was used to identify a series of catalytic RNAs (ribozymes) with thio ester synthase activity. Active sequences were recovered from a large heterogeneous pool of3O-, 60-, 100-, and 140nucleotide random-sequence, coenzyme A-linked RNA molecules. Biotinyl adenylate was chemically synthesized and used as a reactant for the thioesterification reaction, the end result of being the formation of a covalent C-S bond be tween the carboxyl of biotin-AMP and the sulfhydryl of CoA. Active ribozymes from all four different size pools were isolated and product formation has been confirmed by streptavidin gel mobility shift assays, HPLC analysis and mass spectroscopy. Ribozymes were selected with a series of divalent metal cofactors as well as imidazole. Which of these metals is required for catalysis is being determined and kinetic parameters are being investigated.
11:30 SCREENING OF ARABIDOPSIS THALIANA INSERTION MUTANTS FOR A KNOCKOUT IN THE SULFITE REDUCTASE GENE
Sudha [Sankaran.sup.*], Gordon C. Cannon, and Sabine Heinhorst, The University of Southern Mississippi, Hattiesburg, MS 39406
Chloroplasts are semi-autonomous organelles much like the mitochondria, having their own genome. Their DNA is compacted into nucleoids by specific DNA binding proteins, and structure and morphology of these nucleoids are vital to the different roles these complexes play in the replication and transcription of the chloroplast genome. One protein has been characterized that is associated with soybean (Glycine max) chloroplast nucleoids: the DNA compacting protein DCP68, which was also identified as a sulfite reductase. The amino acid sequence deduced for this protein has approximately 90% homology with sulfite reductase from Arabidopsis thaliana and other plants. We are currently searching for an A. thaliana line that has a knockout insertion in its sulfite reductase gene to study the biological effects of this mutation on nucleoid structure and function. First-round screening of A. thaliana mutant pools with gene-specific primers has yielded six potential knockout candidates. Current sequencing efforts are gea red towards elucidating the location of the insertions and assessing their usefulness for the planned study.
11:45 THE RATE ENHANCEMENT EFFECTS BY ENCAPSULATION OF RIBULOSE-l,5-BISPHOSPHATE CARBOXYLASE WITHIN A CARBOXYSOME
Eric B. [Williams.sup.*], Sabine Heinhorst, and Gordon C. Cannon, University of Southern Mississippi, Hattiesburg, MS 39406
The encapsulation of ribulose-1,5-bisphosphate carboxylase (RuBisCO) within a carboxysome has been shown to increase the enzyme's catalytic capabilities. One hypothesis states that the putative enhancement of the RuBisCO activity within the carboxysome results from the enzyme's co-localization with carbonic anhydrase (CA), which provides saturating levels of [CO.sub.2] for the carboxylation reaction. To test this hypothesis, various concentrations of a potent CA inhibitor were used to inhibit the endogenous CA that is thought to be located within the carboxysome. In addition, exogenous CA was added to carboxylation reactions to determine if there was any enhancement of the rate of carboxysomal [CO.sub.2] fixation. Both approaches showed no significant differences in RuBisCO activity. The possibility that the carboxysome shell is differentially permeable for [CO.sub.2] and its competitor for the active site of RuBisCO, [O.sub.2], was examined. Assays were performed to distinguish the differences in RuBisCO cat alytic activity under controlled atmospheric conditions of 100% and 0% [O.sub.2] to test whether selective exclusion of oxygen plays a significant role in the increase of the catalytic capabilities of RuBisCO by the carboxysome shell.
2:00 Session 3: "Poster Party"
PHYSICAL AND CHEMICAL PROPERTIES OF LIDOCAINE-INCORPORATED HYDROPHILIC FILMS
PRODUCED VIA HOT-MELT EXTRUSION
Lakeshia [Moore.sup.*] and Michael Repka, University of Mississippi, University, MS 38677
Hot-melt extrusion is one of the most widely used techniques in the industry of plastics. Today, more than one-half of all plastic products are produced by this method. Hot-melt extrusion technology is also a viable process to produce thin, flexible, and stable hydrophilic and hydrophobic films. For pharmaceutical applications hot-melt extrusion offers many advantages over the traditional ways of producing films. Shorter and more efficient processing times to the final product, environmental advantages due to the elimination of organic solvents, less labor and equipment demands, favorable cost, and less time of the drug subjected to heat are only a few of the many rewards. Hot-melt extrusion equipment consists of an extruder, a hopper, melting zones, dies, collection equipment, and monitoring tools. In this particular project hot-melt extrusion is utilized to prepare hydroxypropyl cellulose (HPC) and hydroxypropyl methylcellulose (HPMC) films containing lidocaine as a model drug.
THE EFFECT OF EAT-16 AND GPB2 ON THE COMPLEX OF M2 AND GAO-l ON SF9 CELLS
Johnathan [Collins.sup.*] (1), T.K. Harden (2), and H.E. Outlaw (1), (1.) Delta State University, Cleveland, MS 38733, and (2.) University of North Carolina Medical School, Chapel Hill, NC 27599
The goal of this project was to determine if Caenorhabditis elegans GPB2-EAT16 acts as a bg in hormone signaling. Complex formation of EAT 16-GPB2 was demonstrated and expression characteristics for the M2 muscarinic receptor in SF9 cell membranes were optimized.
MOLECULAR CLONING OF HOXA1 FROM RAT FETAL BRAIN TISSUE
Katrina S. [Parker.sup.*] (1), Ibrahim O. Farah (1), and R. Thomas Zoeller (2), (1.) Jackson State University, Jackson, MS 39217, and (2.) University of Massachusetts, Amherst, MA 01003
The vertebrate hindbrain is subdivided into a series of compartments called rhombomeres. During development of the hindbrain certain genes are expressed to differentiate the compartments. One gene in particular, Hoxa1, is expressed in the hindbrain exclusively during the period of neural tube formation determining segmentation and identity in the developing hindbrain and associated structures. It is expressed in the embryo, in the adult intestine and in tumors, specifically in carcinoma cells. According to recent studies, knockout of Hoxa1 will cause segmentation defects leading to the partial deletion of rhombomeres. For example, in the rat, the misexpression of Hoxa1 leads to ectopic differentiation of vestibuloacoustic (VII) motor neuron in rhombomere 2. Our objective was to efficiently clone Hoxa1 using RNA from rat fetal brain tissue. Rt-PCR results revealed the amplicon of Hoxa1 was obtained using random hexamer, oligo-dT, and the reverse primer of Hoxa1 with a denature temperature of 550C. Our amplicon was exactly 750 bp, the size needed for transformation. PCR results revealed an amplicon at 1200 bp with a denature temperature of 600C using only the random hexamer RNA. After excising the amplicon and transforming, the cells were placed in a midiprep for growth. We concluded that our cDNA successfully transformed. Our ability to clone this gene will enable us to determine its role in cancer and the consequent opening of new frontiers in understanding molecular mechanisms in cancer development, progression and therapy.
EXPRESSION AND PURIFICATION OF ACTIVATION DOMAINS FROM A EUKARYOTIC TRANSCRIPTION FACTOR
Sonya D. [Barner.sup.*] and Gary R. Kunkel, Jackson State University, Jackson, MS 39217, and Texas A&M University, College Station, TX 77843
The most characteristic requirement of gene control in multicellular organisms is the execution of precise developmental decisions so that the right gene is activated in the right cell at the right time during the development of many cell types that all form a multicellular organism. Often this control is at the level of transcription of genes. The main goal of the project is to analyze a protein that controls transcription of some genes. This protein called SPH Binding factor (SBF), contains two different regions that are necessary to stimulate transcription of different types of genes. We cloned one of these regions into a plasmid in order to express it in Escherichia coli. To clone specific DNA fragments in a plasmid vector, the fragment was produced and then inserted into the vector DNA. Restriction enzymes and DNA ligases were used to produce these recombinant DNA molecules. Once the clone of cells bearing the desired segment of DNA was isolated, a large amount of this protein fragment of SBF was purifie d so that further study could take place.
7-KETOCHOLESTEROL INHIBITS IL-IRA IN HUMAN MONOCYTES
John C. [Harris.sup.*] (1), John K. Jenkins (2), Kenneth Ndebele (1) and Ibrahim O. Farah (1), (1.) Jackson State University, Jackson, MS 39204, and (2.)University of Mississippi Medical Center, Jasckson, MS 39216-4505
Interleukin-1 (IL-1) is a pro-inflammatory cytokine that has been shown to be involved in early atherogenesis by activating endothelial cells and monocytes. Its biologic effects are regulated at multiple levels including the coordinate expression of a novel receptor antagonist, IL-1Ra, which binds IL-1 receptors without transducing a signal. IL-1Ra exists in cell associated and secreted forms and is induced in monocytes by bacterial lipopolysaccharide (LPS) and phorbol-13-myristate acetate (PMA). Proatherogenic oxysterols are known to induce IL-1 in monocytes. The intracellular isoform inhibits IL-1-induced transcription through a non-receptor mediated mechanism. We investigated the effect of pro-atherogenic mediators on monocyte IL-1Ra production. Hypothesis: icIL-1Ra is down-regulated by oxysterols (7-ketocholesterol, KS) which facilitate the atherogenic process. Methods: Human monocytic cell lines (THP-1, U937) were plated at 1 M/m1 in RPMI with 10% FBS in twenty-four well plates, and stimulated for 48 hrs with PMA and/or LPS with and without KS. A sandwich ELISA employing commercially available antibodies was used to measure supernatant and cell-associated IL-1Ra. Results: As expected in U937s, PMA and/or LPS induced high levels of IL-1Ra. KS (40 [micro]g/m1) inhibited IL- 1Ra production, 87%, 43%, and 83% in PMA, LPS, or PMA/LPS-stimulated cells, respectively. In THP-1 cells, KS also inhibited PMA and LPS induced IL-1Ra. Inhibition of secreted IL-1Ra was primarily seen in U937 cells. Both forms were inhibited in THP-is. The atherogenic effects of oxysterols include inhibition IL-1Ra production in monocytes, inducing an imbalance of IL-1Ra:IL-1 atherogenesis.
TOLERATED XENOGRAPHS USING VIRUS STEALTH TECHNOLOGY
Lawson [Safley.sup.*] (1), H.E. Outlaw (1), and Mark [Crew.sup.2], (1.) Delta State University, Cleveland, MS 38733, and (2.) University of Arkansas Medical School, Little Rock, AR 73203
The objective is to examine the structure and function of MHC Class I transmembrane and cytoplasmic domains. Reduction of pig MHC class I proteins increases human NK cell-mediated lysis and decreases human T cell-mediated lysis. It was further noted that ICP47 is able to inhibit MHC cell-surface expression.
DETERMINATION OF GLUTAMATE DEGRADATION BY YERSINIA PESTIS
Shaneka [Simmons.sup.*] and Robert Brubaker, Jackson State University, Jackson, MS 39217, and Michigan State University, East Lansing, MI 48824
Essential virulence genes such as V antigen and Yersinia Outer Membrane Proteins (Yops) are carried on an ~70-kb low-calcium-response (Lcr) plasmid. These virulence genes play a role in the blockage of inflammation and the expression of cytotoxins within host cells. Duplication of the amount of [Ca.sup.2+] (2.5 mM) and [Mg.sup.2+] (1.5 mM) present in blood permits vegetative growth of Y. pestis in artificial media with repression of V antigen and other virulence factors encoded on the Lcr plasmid. Bacteriostatis and production of virulence factors occur in intracellular fluid that lacks [Ca.sup.2+] and contains ~20 mM [Mg.sup.2+]. Upon loss of the Lcr plasmid, Y. pestis acquires the ability to grow at 37[degrees]C without added [Ca.sup.2+] and does not produce Yops or V antigen. To address the role that the low-calcium-response mechanism plays in host cell death, several experiments were conducted. The Sigma Plot computer program was used to analyze and assign the best linear fit to graphs pertaining to Lcr n utritional requirements. Maximum optical densities were achieved through the replacement of [Na.sup.+] with [K.sup.+] or [Li.sup.+]. Combination of [Na.sup.+] and L-glutamate resulted in toxicity of Lcr cells due to evident loss of aspartase. These results suggest that in the absence of [Ca.sup.2+] and presence of [Mg.sup.2+], [Na.sup.+], and L-glutamate Y. pestis undergoes abrupt bacteriostatis. Since Y. pestis lacks aspartase activity in this environment, the bacteria converted L-glutamate into aspartic acid during the time of bacterial restriction. In an effort to rid itself of asparate, the bacteria may release excess aspartic acid into host cells. Since excess aspartate is harmful to the host, it is possible that the host converts aspartic acid back into glutamic acid, causing constant conversions of these amino acids between bacteria and victim, at the victim's expense. Therefore, cell death could possibly be associated with anti-inflammation, cytotoxicity, and the constant effort of the host to convert aspartate back into glutamate.
IN VITRO CYTOTOXICITY OF HUMAN LIVER CARCINOMA CELL LINE (HEPG2) EXPOSED TO CADMIUM CHLORIDE
Shaneka [Simmons.sup.*] and Barbara A. Wilson, Jackson State University, Jackson, MS 39217
Cadmium, a naturally occurring heavy metal, is widely used in industry and consumer products. It is expelled into air, water, or soil from the burning of fossil fuels and spills at toxic or hazardous waste disposal sites. Cadmium chloride has been classified a human carcinogen. Ingested cadmium accumulates in the kidney. Elevated levels of respiratory, prostate, and other cancers have been shown in persons occupationally exposed to cadmium. The goals of this project are: (1) to determine the acute toxicity cadmium chloride ([CdCI.sub.2]) using the trypan blue dye exclusion and lactate dehydrogenase (LDH) assays; and (2) to determine the cellular response mechanism of [CdCl.sub.2] in human hepatic carcinoma cell (HepG2) lines; and (3) to assess the genotoxic effects of [CdCl.sub.2] in HepG2 cells. Based on data collected for determining percent (%) cell viability at the 24-, 48-, and 72-hour exposure, the experimental LC50 value for cadmium chloride appears to range between 1.5-2.5 ppm. These LC5O values repre sent the concentrations of chemicals required to produce 50% cell death. Western blot analysis was used to evaluate p53, HSP70, cfos and Bcl-2 cellular protein expression. The findings provided in this study indicate a possible mechanism for [CdCl.sub.2] cytotoxicity effects in HepG2 cells. This work is supported by NSF-STARGE.
ACUTE CYTOTOXICITY AND CELL MEDIATED RESPONSE IN HUMAN LIVER CARCINOMA CELL LINE (HEPG2) FOLLOWING EXPOSURE TO 2,4,6- TRINITROTOLUENE
Joyce Y. [Belcher.sup.*] and Barbara A. Wilson, Jackson State University, Jackson, MS 39217
2,4,6-trinitroluene is an explosive used in military shells, bombs and grenades. It enters the environment through liquid and solid wastes that result from manufacturing, processing, or recycling of the compound in military arsenals. The goals of this project are: (1) to determine the acute toxicity of 2,4,6-trinitrotoluene (TNT) using the trypan blue dye exclusion and lactate dehydrogenase (LDH) assays; (2) to determine the cellular response mechanism of TNT in human hepatic carcinoma cell (HepG2) lines; and (3) to assess the genotoxic effects of TNT in HepG2 cells. Based on data collected for determining percent (%) cell viability at the 24-, 48-, and 72-hour exposure, the experimental LC50 value for TNT appears to range within 6 and 7 ppm during the 48 and 72 hours of exposure. These LC50 values represent the concentrations of chemicals required to produce 50% cell death. Western blot analysis was used to evaluate NFkB gene expression. The tumor suppressor gene product p53, which is a critical mediator of the cellular response to DNA damage, was also evaluated in HepG2 cells treated with TNT. Findings provided by this work may indicate a possible cellular pathway by which TNT causes cytotoxicity in HepG2 cells. This research is supported by the Office of Naval Research for Interns in Biomolecular Sciences.
CELL SURVIVAL AND CELLULAR RESPONSE MECHANISMS OF HUMAN LIVER CARCINOMA CELL LINES (HEPG2) TREATED WITH ARSENIC TRIOXIDE
Howard [Loving.sup.*] and Barbara A. Wilson, Jackson State University, Jackson, MS 39217
People working or living near industries and facilities that manufacture various products may be exposed to higher than background levels of hazardous substances. Arsenic, a naturally occurring metal, is released into the air when contaminated materials are burned. It enters the body through inhalation, ingestion, and direct contact. The maximum contaminant level for arsenic exposure is 50 [micro]g/L in humans. Health effects associated with arsenic exposure include diabetes, cardiovascular disease, hearing loss, and neurological and neurobehavioral effects. The maximum contaminant level for arsenic exposure is 50 [micro]g/L. The goals of this project are: (1) to determine the toxicity of arsenic trioxide using the lactate dehydrogenase (LDH) assay; (2) to determine the cellular response mechanism of arsenic in human hepatic carcinoma cell line (HepG2); and (3) to assess the genotoxic effects of arsenic in HepG2 cells. To conduct this experiment, HepG2 cells were seeded at 1x10 (6) cells/ml and exposed to the chemicals for 48 hours. LDH analysis was used to determine the lethal concentration (LC50). Total protein concentration was determined using the Bradford Assay. Western blot analysis was used to evaluate p53 cellular protein expression. And, apoptosis was assessed by DNA fragmentation. Results indicated that the LC5O value for arsenic trioxide was shown to be 11-12.5 ppm. The tumor suppressor gene product p53, which is a critical mediator of the cellular response to DNA damage, was expressed after treatment of HepG2 cells with arsenic trioxide. This work is supported by NSF-STARGE and NIH-RCMI.
INDUCTION PROFILE AND CELLULAR RESPONSE MECHANISMS OF HUMAN LIVER CARCINOMA CELL (HEPG2) LINE TREATED WITH ARSENIC TRIOXIDE, CADMIUM CHLORIDE, AND 2,4,6-TRINITROTOLUENE
Barbara A. [Wilson.sup.*] and Cedrick Whitaker, Jackson State University, Jackson, MS 39217
Metals are an important and emerging class of carcinogens as are residues from military arsenals. At least three metals, arsenic trioxide and cadmium chloride have been shown to be very potent carcinogens in laboratory animals. All three of these compounds have been classified as human carcinogens. The goals of this project are to: (1) to determine the acute toxicity of arsenic trioxide ([As.sub.2][O.sub.3]), cadmium chloride ([CdCl.sub.2]) and 2,4,6-trinitrotoluene (TNT) using the trypan blue dye exclusion and lactate dehydrogenase (LDH) assays, and (2) to determine the cellular response mechanism of [As.sub.2] [O.sub.3], [CdCl.sub.2], and TNT in human hepatic carcinoma cell line (HepG2). Based on data collected for determining percent (%) cell viability the experimental [LC.sub.50] value for [As.sub.2][O.sub.3] appears to range between 6 and 12 ppm after 48 hours exposure. For TNT the [LC.sub.50] value appears to range within 6 and 7 ppm between 48 and 72 hours of chemical exposure, and for cadmium chloride the [LC.sub.50] value appears to range between 1.5 to 2 ppm. To assess the acute cytotoxicity and cellular response mechanism of [As.sub.2][O.sub.3], [CdCl.sub.2], and TNT, human liver carcinoma cells (HepG2) were exposed to each compound at various time intervals. Total protein concentration was determined using the Bradford Assay. Western blot analysis was used to evaluate cellular protein expression. Programmed death was assessed by DNA fragmentation. The tumor suppressor gene product p53 induction profile was also determined. This work is supported by NSF-STARGE and the Office of Naval Research for Interns in Biomolecular Sciences.
EFFECT OF THREE CHLORINATED HYDROCARBONS ON DNA DAMAGE IN MICE LIVER CELLS
Babu P. [Patlolla.sup.*] (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
The decay of the environment and the resultant effect on the human health grows daily. Pesticides and several cleaning products are used in much higher quantities than are actually needed. Halogenated hydrocarbons have long been regarded as pharmacological and toxicological entities and are widely used in industry as solvents, degreasing agents, plasticizers and chemical intermediates in the manufacturing of other chemicals (Tafazoli and Kirsch-Volders, 1996). Rodent animal bioassays are considered to be valuable tools for investigating toxicological effects of environmental pollutants. In the present study five different concentrations of 1,1-dichloroethane; 1,1,ltrichloroethane and 1,1,2,2-tetrachloroethane were used to test the DNA damage in mice liver cells. Ethanol was used as the solvent control. The procedures of Mertens and Hammersmith (1995) for DNA extraction and gel electrophoresis were followed. All three chlorinated hydrocarbons showed an increased migration of DNA in electrophoresis when compare d with the control.
lNHIBITION OF HEPATIC CYTOCHROME P450 BY CHLORPYRIFOS AND METHYL PARATHION IN NEONATAL RATS
Patrick B. [Kyle.sup.*], K.E. Schneider, R.C. Baker, and R.E. Kramer, University of Mississippi Medical Center, Jackson, MS 39216
The adult pattern of hepatic cytochrome P450 enzymes is imprinted neonatally. Organophosphorus compounds, through induction and oxidative desulfuration-dependent inactivation of P450 with subsequent changes in endogenous hormone metabolism, might cause changes in the levels and pattern of cytochrome P450 in neonates which could persist into adulthood. Studies were performed to examine the effects of chlorpyrifos (2 mg/kg) and methyl parathion (5 mg/kg) on hepatic P450s of neonatal rats. Pups of each sex were treated dermally once daily from postnatal day 3 to postnatal day 15. The rate (*) of NADPH-dependent conversion of methyl parathion to methyl paraoxon ((*.) nmol/min/mg prot) in microsomes from livers of control pups decreased over 20 minutes from 0.91[+ or -]0.07 to 0.34[+ or -]0.22; an effect consistent with desulfuration-dependent inactivation of P450. Neither rate was affected by methyl parathion treatment (0.74[+ or -]0.06, 0.46[+ or -]0.16). In contrast, chlorpyrifos treatment inhibited the initial rate by ~45% (0.51[+ or -]0.04) and the final rate by ~90% (0.05[+ or -]0.03); the latter effect suggesting that chlorpyrifos is a more efficient suicide substrate than is methyl parathion. In contrast, both chlorpyrifos (0.14[+ or -]0.01 versus 0.09[+ or -]0.01 nmol/mg prot) and methyl parathion (0.12[+ or -]0.01) increased total hepatic microsomal cytochrome P450 content. These data indicate that the effects of organophosphorus compounds on hepatic P450s reflects a combination of inhibition and induction. The mechanisms and specific isozymes involved remain to be identified. (Grant R06/CCR419466 from the CDC).
A DISCUSSION OF GLUTAMINYL CYCLASE IN THE RAT BRAIN
Kimberly Cornelius, Alcorn State University, Alcorn State, MS 39069
Glutaminyl cyclase also known as QC, is an enzyme that catalyzes the cyclization of N-terminal glutamine of peptides into pyroglutamic acid and releases the amide nitrogen in the form of ammonia. It has been identified in plants, bacteria, bovine and porcine pituitary, and secretory vesicles from brain, adrenal, medulla, and Blymphocytes. Protein purification, antibody purification, gel electrophoresis, western blotting, immunofluorescent staining, and light microscopy, were utilized for this research. The light microscopy work was an attempt to locate QC in the sections of a rat. The sections were probed with a primary antibody that recognizes QC as well as a secondary antibody to fluoresce the site of the recognized protein. The Western Blots of the brain tissue show that QC is present in the brain. While the microscopy work shows mixed results. To get a better understanding of what is going on, a more comprehensive microscopy study must be performed as well as different blotting techniques. QC is still a m ystery, while peptidyl-glycine alpha-amidating monooxygenase (PAM), a similar protein has been well characterized.
Session 4: "Molecular Toxicology"
8:30 THE EFFECT OF HUMIC ACID ON MICROBIAL ECOTOXICITY OF 1-AMINOPYRENE DURING SOLAR PHOTOLYSIS PROCESS
Ana L. Balarezo, Veleka N. Jones, and Huey-Min [Hwang.sup.*], Jackson State University, Jackson, MS 39217
1-Aminopyrene (1-AP), a polycyclic aromatic hydrocarbon (PAH) compound, is a major metabolite during biotransformation of 1-nitropyrene by microflora in natural environment and in the guts of animals and humans. Under UV-A irradiation, 1-AP has been shown to cause light-induced DNA single stranded cleavage. The humic substances in aquatic ecosystems can influence the bioavailability, toxicity and fate of organic xenobiotics. Therefore, photochemical fate and effect of PAHs in natural aquatic environments may differ significantly across sites. The objectives of this study are to assess the effect of HA on microbial ecotoxicity of 1-AP during photolysis process. Microbial ecotoxicity of 1-aminopyrene (1-AP) during different time courses in the presence and absence of humic acids (HA) was measured with spread plate counting and microbial mineralization [of.sup.14] C-D-glucose. At 1-AP concentration of 10 [micro]M, microbial heterotrophic activity was significantly inhibited in the presence of HA (20-60 ppm) in l ight and in darkness. Exposure to HA in light and darkness, however, does not inhibit bacterial viability at the HA concentration range administered. Therefore, inhibition on microbial activity in darkness could have been caused by the decrease of glucose availability, instead of toxicity of HA. Funding support: (1) NIH-RCMI 1G12RR12459-0l and NIH-MBRS S06GM08047 (to JSU); and (2) U.S. Department of the Army #DAAD 19-01-1-0733 to JSU.
8:45 LIGHT-INDUCED DNA DAMAGE BY COSMETICS AND SKIN CARE PRODUCTS
Karishma A. [Parekh.sup.*], Yuguo Jiao, and Hongtao Yu, Jackson State University, Jackson, MS 39217
Skin care products and cosmetics are used on a regular basis for appearance, conditioning, and protection. It is known that skin care products are capable of causing mild allergic reactions but previous studies are inconclusive on the effects of these chemicals. These products are usually a complex mixture of chemicals with distinct properties. Especially, since the chemicals in the skin are subject to light irradiation, the combination effect of light and these chemicals needs to be studied. In this research, we have identified ten chemicals found in cosmetics and skin care products that may cause damage to DNA in combination with repeated exposure to UVA radiation. The chemicals used in these experiments are: methylparaben, 2-hydroxy-4-methoxy-benzophenone, 2-phenylbenzimidazole, 4-methoxy-cinnamic acid, azulene, vitamin E, vitamin E acetate, 4-chloro-m-phenylenediamine, 2,4-toluenediamine, and 4-amino-2-nitrophenol. Upon UVA irradiation, 2-phenylbenzimidazole, azulene, and 4-chloro-m-phenylenediamine cause DNA single strand cleavage. The other chemicals show little variance from the control and are therefore considered not causing DNA damage. The UVA-induced cleavage of supercoiled plasmid DNA is dependent upon concentration of these chemicals and UVA dosage. A longer irradiation time or higher concentration induces more DNA cleavage. Since the combination of these chemicals and UVA light induce DNA cleavage, they can be more toxic and carcinogenic to humans when in combination with UVA light. This research is in part supported by the [NASASHARP.sup.+] Program and the US Army Research Office DAAD 19-01-10733 to JSU.
9:00 SELECTIVE CYTOTOXICITY OF ARSENIC AND CADMIUM ON HEPG2 AND RAT PRIMARY LIVER CELLS: DIFFERENTIAL RESPONSE TO PIFITHRIN
Rowshan [Begum.sup.*] and Ibrahim O. Farah, Jackson State University, Jackson, MS 39217
Pifithrin or PFT-a is a reversible inhibitor of p53 function. In response to genotoxic agents p53 instructs the cells to perform DNA repair or to commit suicide. Our objective was to study the degree of differential protection PFT-a offers to primary rat liver cells and HepG2 cells to toxic effects of arsenic and cadmium. Following growth to 90% confluency under standard conditions, cells were exposed to toxic agents simultaneously with PFT-a (10 ppm). Surviving cells were detected following exposure to fluorescein diacetate (FDA) and ascent fluorospectroscopy. Percent survival was calculated using regression analysis. Mean LC-50 of HepG2 cells following exposure to arsenic was 13.7 [+ or -] 1.0 ppm with PFT-a and 13.4 [+ or -] 0.6 ppm without it. For rat primary liver cells, LC-50 was found to be 670 [+ or -] 2.5 ppm with and 573.15 [+ or -] 1.9 ppm without PFT-a. With cadmium, LC-50 for HepG2 cells was found to be 6.95 [+ or -] 2.5 ppm with PFT-a and 7.35 [+ or -] 1.9 ppm without it. In rat primary liver ce lls, LC-50 was found to be 57.72 [+ or -] 0.8 ppm and 58.1 [+ or -] 5.5 ppm, respectively with and without PFT-a. Results at this level for rat primary liver cells and arsenic were significant. Inherent response of the two cell lines to the toxic agents were also apparent. Use of Pifithrin as a protective agent for normal cells from the side affects of cancer chemotherapy is thus suggestive.
9:15 THE SNXA1 MUTATION OF ASPERGILLUS NIDULANS AFFECTS THE DNA DAMAGE CHECKPOINT
James S. Goode, Ann C. Long, and Sarah Lea [McGuire.sup.*], Millsaps College, Jackson, MS 39210
The DNA damage checkpoint functions to protect cells from mutation by halting progression into mitosis in the presence of damaged DNA. One mechanism by which this checkpoint functions is to inhibit the activity of the universal mitotic regulator, p34cdc2. Using the filamentous fungus Aspergillus nidulans as a model system, we have generated a series of mutations which affect the activity of the A. nidulans p34cdc2 homolog, NIMXCDC2. One of these mutations, snxA 1, abrogates the DNA damage checkpoint such that cells enter mitosis in the presence of what would normally be a sublethal amount of DNA damage. We have undertaken genetic, cytologic, and biochemical analyses of the effects of the snxA 1 mutation on the DNA damage checkpoint. Double mutant analyses indicate that snxA 1 functions in the same DNA damage checkpoint pathway as the UVSBRAD3 protein, a protein kinase similar to the human protein that leads to ataxia telangiectasia when mutated. Cytologic analyses suggest that, like a mutation in UVSBRAD3, mu tation of snxA l leads to re-replication of DNA in the absence of mitosis. Biochemical analyses indicate that SNXA does not function by affecting the activity of NIMXCDC2, which is a major component of the DNA damage checkpoint pathway. These data suggest that either SNXA functions downstream of NIMIX activity or it functions to localize NIMX or regulators of NIMX in response to DNA damage.
9:30 DIFFERENT PATHWAYS USED BY GINKGO BILOBA EXTRACT AND VITAMIN E PROVIDE NEUROPROTECTION OF PCl2 CELLS FROM APOPTOSIS DUE TO MULTIPLE CELLULAR INSULTS
Julie V. [Smith.sup.*], Adam J. Burdick, and Yuan Luo, University of Southern Mississippi, Hattiesburg, MS 39406
Standard Ginkgo biloba extract (EGb 761) has neuroprotective effects. The cellular mechanism remains unclear. Oxidative stress has been implicated in nerve cell death occurring in a variety of neurological disorders. Determination of neuroprotective mechanisms by antioxidants is of importance for understanding the molecular basis of neurodegeneration. PC12 cells undergoing apoptosis are a well-accepted model for studying the potential mechanisms of protection. Here we have determined in PC12 cells the effects of pre-treatment of EGb 761, its components Bilobalide B (BB), Ginkolide B (GB) and the antioxidant Vitamin E on cell survival following multiple forms of apoptosis induction: serum-deprivation, Staurosporine treatment, and treatment with Amyloid [beta] protein. Determinative assays include: TUNEL assay for detection of DNA fragmentation, Mitosensor assay for fluorescent staining of mitochondrial membranes, and Caspase 3 activity assay. Our results show that pre-treatment with EGb 761 or Vitamin E preven ted serum deprivation-, Staurosporine-, and Amyloid [beta]-induced apoptosis. EGb 761, but not Vitamin E, inhibited Staurosporine-induced activation of caspase 3. BB and GB show more significant inhibition than EGb 761. These results suggest that different cellular mechanisms may underlie multiple neuroprotective effects of EGb 761. Acknowledgement: This work is supported by NIH.
Session 5: "Cytokine Signaling"
10:00 TRANSCRIPTIONAL REGULATION OF INTRACELLULAR INTERLEUKlN-1 RECEPTOR ANTAGONIST
John K. Jenkins, University of Mississippi Medical Center, Jackson, MS 39216
Interleukin-1 (IL-1) is a potent pro-inflammatory cytokine. Its biologic effects are regulated at many levels including the presence of different isoforms of a novel and specific receptor antagonist, interleukin-1 receptor antagonist (IL-1Ra). One secreted and several intracellular isoforms of IL-1Ra (sIL-1Ra and icIL-1Ra) exist. sIL-1Ra binds IL-1R and inhibits IL-1 binding. It has been hypothesized that inadequate amounts of IL-1Ra to counter the effects of IL-1 occur in several inflammatory diseases. IcIL-1Ra, interestingly, though not secreted, does bind IL-1R. Its biologic function is unclear, but it is thought to inhibit IL-1 mediated events downstream of IL- 1R resulting in reduced IL-1-induced gene expression. The two isoforms of IL-1Ra are differentially and coordinately expressed from a single gene. We have examined the mechanisms of the transcription regulation of the icIL-1Ra gene to better understand its role in IL-1 homeostasis. icIL-1Ra is constitutively expressed in most epithelial cells (whic h do not express sIL-1Ra) at high levels. icIL-1Ra is a very late gene product of endotoxin-stimulated monocytes, which typically express sIL-1Ra early in response to this stimuli. We have cloned the 4.5 kb of 5' sequence and created deletional mutants. Transient transfections with promoter deletions indicated that the constitutive expression in epithelial cells and inducible expression in monocytes mapped to different promoter regions. Electrophoretic mobility shift assays of potential transcription factor binding sites in the proximal icIL- 1Ra promoter indicated different transcription factors are involved in the epithelial cell expression and "late"-induced monocyte expression. Furthermore, different epithelial cells use similar transcription mechanisms for icIL-1Ra expression.
10:15 INTRACELLULAR INTERLEUKIN-1 RECEPTOR ANTAGONIST IS DEFICIENT IN RHEUMATOID ARTHRITIS FIBROBLASTS
John K. Jenkins, University of Mississippi Medical Center, Jackson, MS 39216
Interleukin-1 (IL-1) is a potent pleiotropic pro-inflammatory cytokine. Its biologic effects are regulated by a novel and specific receptor antagonist, interleukin-1 receptor antagonist (IL-1Ra). A secreted isoform of IL-1Ra (sIL-1Ra) binds IL-1R, inhibits IL-1 binding, and was recently approved to treat rheumatoid arthritis (RA). An intracellular isoform (icIL-1Ra) is not secreted and does bind IL-1R, inhibiting IL-1-induced gene transcription downstream of IL-1R. We hypothesized that the inflammatory cytokine milieu in the RA joint differentially suppresses icIL-1Ra compared to noninflammatory osteoarthritic (OA) joints. We cultured human fibroblasts from OA and RA joints and stimulated them with phorbol-13-myristate acetate (PMA), bacterial lipopolysaccharide (LPS) and inflammatory cytokines known to be present in the RA joint. IL-1Ra protein was measured by sandwich ELISA and Western blot. mRNA was detected using a differential RT-PCR method. icIL-1Ra is the primary isoform expressed in all fibroblast typ es by LPS, PMA, IL-1 and TNF. FGF-1,2, GM-CSF and IL-6 do not induce IL-1Ra. Using signaling inhibitors, maximal expression of fibroblast icIL-1Ra required PMA plus one additional signal. OA fibroblasts responded to LPS, TNF and IL-1 with relatively more icIL-1Ra expression than RA fibroblasts. mRNA analysis confirmed these results. We have identified some of the mechanisms by which the inflammatory milieu may induce icIL-1Ra in synovial fibroblasts. RA fibroblasts do not produce as much icIL-1Ra as OA cells suggesting an intrinsic defect in these transformed cells.
10:30 THE ROLE OF GATA-3 IN INTERLEUK1N-13 (IL-13) TRANSCRIPTIONAL ACTIVATION
Kendria [Holt.sup.*] (1), Sheree Watson (1), Pamela G. Banks (1), and Steve Georas (2), (1.) Jackson State University, Jackson, MS 39217, and (2.) Johns Hopkins University, Baltimore, MD
Asthma is drastically increasing in prevalence, morbidity, and mortality in many developed countries. There is a need to better understand this disease on the molecular level. T lymphocytes play critical role in asthma pathology. The pathophysiological manifestations of asthma are directly related to cytokines produced by Th2 cells. Interleukin-13 (IL- 13) is important in asthma because of its effects on mucus production and airway hyperresponsiveness. Previous research suggests that GATA-3, a Th2 specific transcription factor, directly activates IL-13 transcription. The objective of this study was to establish whether GATA-13 binds to the GATA site of the IL-13 promoter by mutation of the GATA sequence. The IL-13 312 luc construct was synthesized using PCR. The IL-13 promoter GATA site was mutated by site-directed mutagenesis. Jurkat T cells were then transfected with reporter plasmids. Cells were stimulated with calcium ionophore and PMA followed by cell lysis and analysis of reporter gene expression by lum inometry. Results indicate that IL-i3 promoter activity is highly inducible by calcium and PKC signals in Jurkat cells. Mutation of the GATA-3 binding site inhibits IL-13 promoter activity. Enhancement of cg mutant by overexpressed GATA-3 suggests that GATA-3 can activate IL-13 independent of DNA binding.
10:45 THIOREDOXIN-ENHANCED JAK ACTIVITY: A TARGET FOR REDOX-BASED CHEMOTHERAPY?
Roy J. [Duhe.sup.*], Sheeyong Lee, Naila Mamoon, John K. Smith, Kiranam Chatti, Kanadadurga Kundrapu, and Amy Marks, University of Mississippi Medical Center, Jackson, MS 39216
The over-expression of thioredoxin, a redox protein, has been reported in a number of leukemias and carcinomas, including HTLV-1-transformed Adult T cell Leukemia/Lymphoma (ATLL) cells. The hallmark of ATLL pathogenesis is the development of IL-2-independent T cell proliferation, which appears to result from the gradual development of constitutive JAK3 activation. While these observations may at first seem unrelated, they are hypothetically linked by the fact that the catalytic activities of Janus kinases (JAKs) are modulated by their redox states. Both JAK2 and JAK3 are maximally active in a reduced state and inactive in an oxidized state; these states are biochemically reversible. Since Janus kinases are essential components in mitogenic signal transduction, the thioredoxin-mediated reduction of Janus kinases is expected to maximize the mitogenic signal to the cell, conceivably resulting in IL-2-independence and neoplastic transformation. Our hypothesis is that cellular over-expression of thioredoxin enhanc es the enzymatic activity of JAK via its ability to maintain JAK in a reduced state, and that this biochemical mechanism can be targeted for cancer chemotherapy. Both in vitro and cellular experiments are designed to test this hypothesis. If this hypothesis is correct, it will allow us to rationally evaluate disulfide- and diselenide-based compounds as a novel class of cancer chemotherapeutic drugs.
11:15 Divisional Business Meeting and Student Awards Presentation
|Printer friendly Cite/link Email Feedback|
|Title Annotation:||various articles|
|Publication:||Journal of the Mississippi Academy of Sciences|
|Date:||Jan 1, 2002|
|Previous Article:||Agriculture and plant science.|
|Next Article:||Chemistry and chemical engineering.|