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Bacteria are one of the oldest life forms on earth. They have a wide range of different shapes, and they are found in almost every possible habitat on the planet. Many bacteria are harmless, some are beneficial, and some are pathogenic and cause diseases such as tuberculosis, cholera, and syphilis. Staphylococcus aureus (S. aureus) is the most common species that causes staph infections and a range of illnesses. The emergence of drug-resistant pathogenic bacteria has created a true health crisis and a serious problem for hospitals where many patients contract a staphylococcal infection. There is a need for sensitive methods to detect the presence of trace amounts of bacteria to ensure sterility and for convenient kits to distinguish living and dead cells. We have developed a new type of fluorescent triazaboropyridinium (HPY) dyes for biological and biomedical applications. The goal of this project is to investigate the interactions of a series of HPY dyes with live and dead bacteria cells, to determine the effects of different dye structures on cell staining, and to identify possible dyes for use in detection and to determine cell viability.
We will investigate bacteria stained with HPY dyes that are neutral, charged, or capable of reacting with intracellular proteins using fluorescent microscopy. These results will be compared with existing commercial dyes that are known to stain bacteria.
VITAMIN C IN INVERTEBRATES: IS IT THERE AND HOW IS IT MADEMaria Pedraza, Alexander Patananan, Lauren Budenholzer, Steven Clarke.
University of California, Los Angeles, Los Angeles, CA.
Vitamin C is an important antioxidant that neutralizes free radicals in cells, participates in a variety of enzymatic reactions, and helps protect against human diseases. There are three main vitamers of vitamin C including dehydroascorbate, D-isoascorbate, and L-ascorbate. The importance of L-ascorbate, the most biologically active vitamer, is well established in vertebrates, yet its biosynthesis is poorly characterized in invertebrates.
Caenorhabditis elegans, a commonly used invertebrate model organism, requires ascorbate for several of its enzymes in its vertebrate homologs. Therefore, we are investigating the levels of ascorbate or similar species, such as erythroascorbate and arabinoascorbate, in C. elegans, and how they are biosynthesized. To identify these compounds, we optimized several methods using normal and reverse-phase, high-performance liquid chromatography (HPLC) and gas-chromatography mass spectrometry (GC-MS). We will then explore the biochemical pathways for the biosynthesis of these molecules. Finding a novel pathway in C. elegans will broaden our understanding of the synthesis of vitamin C and its importance to organisms. Most importantly, our research can provide insight into putative human homologs that may affect health.
DEVELOPMENT AND VALIDATION OF A HIGH THROUGHPUT SCREENING ASSAY FOR THE DETERMINATION
OF CIDAL ANTIMALARIALSYandira Salinas, Armand Guiguemde.
St. Jude Children’s Research Hospital, Memphis, TN.
Malaria kills 1 million people per year, making it one of the most deadly infectious diseases. Despite the great worldhealth impact of malaria, only a limited number of therapeutic agents are available. The malaria parasite’s ability to quickly develop resistance to treatments poses a threat to world health. Malaria drugs are either cidal or cytostatic.
Cidal compounds act faster and are preferred in the clinic because the development of resistance to cidal compounds is lower. In addition, rapid clearance of the parasite reduces transmission. Using a high throughput screening approach and a 3D7 luciferase transfected strain, we developed a novel assay that would allow us to discriminate between cidal and cytostatic compounds early in the drug development/discovery process. This assay allows the determination of the speed of action of a compound in a 12-hour experiment. Previous methods measure parasite out-growth in a 72-hour experiment. As a pilot study, a small library of 32 antimalarials containing previously reported cidal and cytostatic compounds was screened. This assay was able to accurately discriminate between cidal and cytostatic compounds. Herein, we report the development and validation of this high-throughput screening assay for the determination of fast acting antimalarials.
Forkhead box M1 (FoxM1) transcription factors are essential for G1-S and G2-M cell cycle progression. FoxM1 is over expressed in many cancer types. FoxM1 is normally held inactive by a possible physical interaction between the N-terminal domain (NTD) and the C-transactivation domain (TAD). In order to test for a possible physical interaction between NTD and TAD, each individual domain will be purified. To test for direct interactions between NTD and TAD, GST-tagged TAD will be incubated with pure NTD in a pull-down experiment. This experiment will provide qualitative information about the interaction between the domains. To generate quantitative data, isothermal titration calorimetry will be employed.
DETERMINING THE ROLE(S) OF PRIME-SIDE RESIDUES IN MACROMOLECULAR INHIBITION OF TRYPSINFOLD SERINE PROTEASESCommodore St. Germain, Teaster Baird, Jr.
San Francisco State University, San Francisco, CA.
Trypsin-fold serine proteases are among the most abundant of all proteases. By identifying residues that are important for enzyme inhibitor interaction in trypsin, we may advance the development of protease-based therapies in general. Lysine-60 (K60) is a highly conserved prime-side residue that may play many roles in trypsin. In cocrystals, K60 hydrogen bonds to tyrosine-39 (Y39) and is positioned to restrict conformational mobility of phenylalanine-41 (F41), possibly limiting the observed hydrogen bond interactions between those residues and macromolecular inhibitors. Substitution of K60 with other amino acid residues may disrupt these interactions and provide insight into their significance with respect to inhibitor binding. To test this hypothesis, we created trypsin variants K60G, K60A, K60V, K60I, and K60R and characterized them with respect to their activities and sensitivities to the macromolecular inhibitors soybean trypsin inhibitor (SBTI) and bovine pancreatic trypsin inhibitor (BPTI). Our initial results show, compared to wild-type trypsin, K60A and K60V are catalytically indistinguishable, more resistant to autolysis, and more sensitive to inhibition by SBTI and BPTI. Further experiments with ecotin and other trypsin variants can provide additional insight on specific interactions. (This work was supported by NSF CAREER Award MCB-0643988-02 and NIH MARC T34- GM008574.) FRI-106
HTLV-1 PROMOTER REGION NUCLEOSOME BINDING OF THE TRANSCRIPTIONAL COACTIVATOR P300 VIA
ITS BROMODOMAIN AND PHD FINGER DOMAINJulio Flores Servin, Whitney Luebben, Jennifer Nyborg.
Colorado State University, Fort Collins, CO.
Transcription from the integrated human T cell leukemia virus type-1 (HTLV-1) has been shown to require histone acetylation within promoter region nucleosomes. These acetylation events are carried out by the recruitment of p300 to the viral promoter region via interactions with the cellular transcription factor pCREB and the viral encoded oncoprotein Tax. The protein p300 is a large transcriptional coactivator protein with a histone acetyl-transferase (HAT) domain and multiple other domains, including a bromodomain and PHD finger domain. Research has shown that acetylation of histone H3-K14 is crucial for nucleosome disassembly and transcription activation, although the exact mechanism of nucleosome disassembly is not completely understood. Additionally, work on non-HTLV-1 nucleosomes has suggested that the bromodomain and adjacent PHD finger domain of p300 are involved in the binding of p300 to acetylated K residues on histone tails, resulting in an increased binding affinity of p300 to the nucleosome.
Bromodomain and PHD finger domain binding have yet to be studied on the promoter region nucleosomes of HTLVSince p300 and histone H3-K14 acetylation are crucial components of efficient HTLV-1 transcription, we have tested the hypothesis that bromodomain binding to the HTLV-1 promoter region nucleosomes requires the presence of acetylated lysine residues and specifically histone H3-K14Ac. Using an immobilized HTLV-1 promoter template assembled into chromatin with various histone acetylation modifications, we observed binding of the bromodomain to the HTLV-1 promoter region. Our results suggest that bromodomain binding to the HTLV-1 promoter requires the presence of acetylated histone lysine residues but not necessarily just histone H3-K14Ac.
19 UNDERGRADUATE POSTER ABSTRACTS
THE EFFECTS OF OSMOTIC STRESS ON NUCLEAR PHYSICAL PROPERTIES IN ARABIDOPSIS THALIANASam Yang, Amy Rowat.
University of California, Los Angeles, Los Angeles, CA.
Chromatin density is critical for regulating gene expression, but what sets the volume of the cell nucleus remains poorly understood. In plants, nuclear volume increases linearly with DNA content. However, in metazoan nuclei, extracellular osmolarity can decrease nuclear volume and alter chromatin condensation. Despite the changes in extracellular osmolarity to which plants are subjected, the effects of osmotic stress on their nuclear physical properties remain unexplored. The aim of the present study is to determine whether osmotic stress in A. thaliana alters chromatin density in vivo. We hypothesize that changes in chromatin density occur under osmotic stress and may thereby trigger changes in gene expression. To investigate the effects of hyperosmotic stress on chromatin density and nuclear volume, root tissue from A. thaliana is incubated in controlled mannitol solutions of varied concentration.
Thereafter, laser scanning confocal microscopy and a DNA intercalating dye with emission intensity linear to DNA content is used to concurrently measure nuclear volume and DNA content. Subsequent image analysis reveals the relationship between osmotic stress and chromatin density. A further understanding of the mechanisms by which osmotic stress initiates gene expression changes may provide additional insight to the challenge of producing drought-tolerant plants.
CONSTRUCTION AND ASSEMBLY OF PLANT EUKARYOTIC INITIATION FACTOR 3Valeria Arauz, Karen Browning.
University of Texas at Austin, Austin, TX.
The purpose of this research is to produce the plant eukaryotic initiation factor 3 (eIF3) by expressing its individual subunits in bacteria and allowing complex formation in vitro. In producing a functional plant eIF3, we would be able to study its structure and function in translation initiation. The reconstruction of the 800 kDa human eIF3 has been recently achieved, and the complex was assembled in a stepwise manner where the dimer of subunits eIF3a and eIF3c was utilized as the fundamental framework with a subsequent stable octamer formation consisting of subunits a, c, e, k, l, m, f, and h. Based on the success of human eIF3 complex formation in vitro, the lab has cloned the plant eIF3 core octamer subunits into 3 expression vectors: ELMK-pACYC, AFHC-pRSF, and k-pET (k with an N-terminal His tag). The plan is to first determine optimal conditions for soluble expression of each expression cassette in bacteria and then mix soluble fractions obtained from each bacterial preparation in hopes of complex formation.
Octamer formation would be determined using affinity purification of eIF3k with the N-terminal His tag. Upon successful octamer formation, we will express the rest of the plant eIF3 subunits in attempts to form the complete 13 subunit eIF3 complex in vitro. The reconstruction of eIF3 in vitro would allow us to further study the biochemical functions of this important complex in translation initiation.
MIG-10 GENE RESCUE IN CAENORHABDITIS ELEGANSValentina Chee, Barbara Lyons.
New Mexico State University, Las Cruces, NM.
The long-term goal of our research is the clarification of Grb7 protein function in cell migration with respect to the establishment of secondary tumors in cancer. Study of the homologous protein Mig-10 in Caenorhabditis elegans (C.
elegans) provides a suitable whole organism model for exploration of Grb7 function. Both wild-type and mutated (with a truncated Mig-10 gene) C. elegans strains are utilized in these studies. C. elegans strains missing expression of the Mig-10 gene suffer several phenotypes with varying degrees of penetrance. These phenotypes include foreshortened excretory canals, compromised egg-laying ability, withered tails, and uncoordinated movement. The phenotypes result from deregulated axonal outgrowth of neurons responsible for development of the affected bodily regions.
We hypothesize that reintroduction of the Mig-10 gene (using gene-gun mediated plasmid transfer) into the Mig-10 deficient C. elegans strains will result in subsequent normal (or near-normal) progeny. Thus, we hope to develop a working Mig-10 gene rescue C. elegans system. With this system in hand, the future goal is to use the same technology to introduce a Mig-10/Grb7 chimeric gene. If expression of this chimeric gene attains the same rescue of the Mig-10-deficient phenotype, the implication could be that the Grb7 protein has similar cell migration function to the Mig-10 protein. Such a result may provide valuable insight into the role of Grb7 in cell migration signaling.
Many deadly neurodegenerative diseases arise from protein misfolding and aggregation. With its unmatched atomic resolution, NMR spectroscopy is ideally suited to provide structural and dynamic insights to develop rational cures.