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A recently discovered MCP is involved in propanediol degradation via a glycyl radical enzyme. This MCP is found in enteropathogenic bacteria, and has been named the Grp microcompartment. Grp is found in at least 23 different bacterial species, some of which contain a particularly divergent BMC shell protein homolog. We aim to characterize this unique Grp shell protein to gain understanding of its biological function. Using X-ray crystallography, we have determined the structure at 2.7 Å resolution. Additionally, we used site-directed mutagenesis and electronic absorption spectroscopy to show that this shell protein binds an iron sulfur cluster at its central pore. Our work has provided the first shell protein structure from the Grp microcompartment, and has revealed a new type of BMC metalloprotein. Our findings suggest the potential importance of this Grp shell protein in electron or cofactor transport across the shell.
THE EFFECT OF MAGNESIUM ION ON THE IN VITRO HOMOLOGOUS RECOMBINATION MEDIATED BY A
HYPERACTIVE E.COLI RECA MUTANTJoylyn King1, Shelley Lusetti2.
New Mexico State University, Shiprock, NM, 2New Mexico State University, Las Cruces, NM.
1 The bacterial RecA protein (352 amino acids) is the main recombinase that contributes an important role in nonmutagenic, recombinational DNA repair. The DNA strand exchange assay is commonly used to test the RecA homologous recombination activity in vitro. RecA protein bound to DNA hydrolyzes ATP. One molecule of magnesium ion interacts with one molecule of ATP. However, an additional magnesium ion is needed for an efficient E. coli RecAcatalyzed DNA strand exchange reaction. Previous experiments suggest that magnesium ion directly interacts with the RecA protein, likely altering the conformation of the protein’s negatively charged C terminus. This is thought to constitute a molecular switch that auto-regulates RecA activity. Several RecA C-terminal deletion mutants are hyperactive relative to wild type. Our lab and others have also identified a RecA mutant altered by point mutation at position 217 that exhibits similar hyperactivity to the C-terminal deletion mutants. In the current project, we have combined one RecA C-terminal deletion mutant with this point mutation at position 217 to understand whether the mechanisms of hyperactivity for the two mutants are independent. This project seeks to provide mechanistic evidence
DISSECTING INTERACTIONS BETWEEN BAM A AND BAM D VIA BINDING AND FLUORESCENCE STUDIESStephanie Castillo1, Marcelo Sousa2.
University of Central Florida, Orlando, FL, 2University of Colorado Boulder, Boulder, CO.
1 The Gram-negative outer membrane contains integral β-barrel proteins that create pores in the membrane to allow nutrients and solutes into the cell, and waste products out. The mechanism used by the cell to integrate the β-barrel outer membrane proteins (OMPs) into the membrane surface is the β-barrel assembly machine (BAM) complex, a five-member protein complex located at the outer membrane that folds and inserts the OMPs into the outer membrane bilayer. BamA, an integral membrane protein, acts as the core of the BAM complex and interacts with the associated lipoproteins BamB/C/D/E via its periplasmic POTRA domains. BamD plays a critical role in the BamA-mediated OMP folding pathway; we anticipate that the interaction between BamA and BamD will reveal details of their functions in the BAM complex. The structure of BamD is identified as a series of TPR domains that act as a structural scaffold and interacts with the POTRA domains of BamA, but the orientation of BamD relative to BamA is not identified. If we are able to bind and pull down purified BamD to BamA, we can determine the exact orientation of this interaction through fluorescence studies and structural biochemistry. Fluorescent thiol-reactive probes will be attached to BamA and to BamD. Labeled BamA to BamD will then be allowed to bind, and fluorescence spectroscopy will be used to measure various distances between the probes and determine their relative orientation. This will provide more structural clues on how these proteins are interacting in the BAM complex.
REGULATION OF HDL CHOLESTEROL METABOLISM AND THE IMPACT OF LONG-TERM FEEDING OF
DIETARY JOJOBA OIL AND CHOLESTEROL ON THE DEVELOPMENT OF ATHEROSCLEROSIS IN NEW
ZEALAND WHITE RABBITSAnahis Rincon, Raymond Garcia.
California State University, Los Angeles, Los Angeles, CA.
Dietary jojoba seed oil has been shown to maintain high-density lipoprotein (HDL) concentrations at an elevated level in hypercholesterolemic New Zealand White (NZW) rabbits, suggesting that jojoba oil regulates HDL metabolism and has a protective effect against atherosclerosis, one of the leading causes of death worldwide. We hypothesize that jojoba oil decreases the development of atherosclerosis in NZW rabbits. In this study, NZW rabbits will be fed either a normal chow (N), 3% jojoba seed oil (J), 1% cholesterol (C), or 1% cholesterol + 3% jojoba seed oil (CJ) for 13 weeks. Throughout the study, serum cholesterol concentrations will be monitored via enzymatic assays. Rabbits will be euthanized at 7, 9, 11, and 13 weeks and aortas will be extracted and assessed for atherosclerosis. N- and J-fed rabbits are expected to show no statistical change in their serum lipoprotein concentrations with no development of atherosclerotic lesions. C-fed rabbits are expected to have decreased HDL concentrations with significant atherosclerotic lesion development, while CJ-fed rabbits are expected to have a higher HDL concentration and a lower development of atherosclerotic lesions than those fed the C diet. Validation of the proposed hypothesis will provide a better understanding of how dietary jojoba seed oil, in the presence of dietary cholesterol, regulates HDL metabolism and the development of atherosclerosis. (CSU-LSAMP is supported by the National Science Foundation under Grant # HRD-0802628 and the CSU Office of the Chancellor.) SAT-110
CHARACTERIZATION OF SECRETED EFFECTOR PROTEINS FROM CHLAMYDOPHILA PNEUMONIAE
St.Mary’s University, San Antonio, TX.
Chlamydiae are obligate, intracellular Gram-negative bacteria known to cause various health problems in humans.
Chlamydophila pneumoniae is responsible for an infection of the upper respiratory tract that also causes atypical pneumonia, with evidence linking it to the development of atherosclerosis and coronary artery disease. The infectious cycle of chlamydiae is a unique biphasic life cycle in which small extracellular elementary bodies (EBs) attach themselves to the epithelium of susceptible host cells. These metabolically inactive bacterial forms, upon entry into the cell, reside exclusively in a vesicle termed inclusion. Inside the inclusion, EBs differentiate into larger, non-infectious, metabolically active reticulate bodies (RBs) that divide using binary fission. Within 48 hours, RBs transform back to
31 UNDERGRADUATE POSTER ABSTRACTS
EBs that are released from the inclusion to infect other cells. Chlamydiae interact with the host cells and manipulate their cell signaling and immune response by secreting effector proteins. Among these proteins secreted during Chlamydophila pneumoniae infection are products of a cluster of six homologous genes encoding the effector proteins cpn0794 through cpn0799. Although it has been observed that cpn0796 and cpn0797 are secreted into the host cell and localized in the cytoplasm, the role of these proteins in infection is yet to be determined. We are interested in studying the structures and roles of cpn0794 through cpn0799 during infection using biophysical techniques including X-ray crystallography and analytical ultracentrifugation.
USAGE OF YEAST BIOASSAYS IN TESTING AGAINST ESTROGEN-DISRUPTIVE COMPOUNDS IN
ENVIRONMENTAL SAMPLESReina Hernandez, Jeffrey Sivils, Marc Cox.
University of Texas at El Paso, El Paso, TX.
Endocrine-disrupting chemicals (EDCs) are chemicals that interfere with the endocrine system in humans and animals. These nonsteroidial substances have the ability to bind to the human estrogen receptor and have been found to mimic the activity of estradial (Estrogen 17 ß-Estradial), the natural form of estrogen. They are widely distributed within our environment; are associated with certain cancers, reproductive disorders, and developmental disorders;
and have been previously detected in agriculturally intense watersheds. This study aims to use yeast bioassays to test for estrogenic activity in water and sediment sample extracts from experiments designed to measure 17 ß-estradiol and estrogen degradation in water and sediment. Synthetic complete media (SC-UW) and DSY-219 yeast strains were used to test 24 water samples using receptor-mediated ß-galactosidase reporter assays. Throughout a 3-month period, results were examined from a number of 4-hour yeast experiments, and samples displayed significant estrogenic activity. These results will be correlated with additional data from chemical analysis in the future. These studies will ultimately aid in the understanding of the environmental fate, transport and degradation of these specific endocrine-disrupting chemicals.
THE EFFECTS OF THE CITRUS FLAVONOID NOBILETIN ON HEPATOSTEATOSIS IN HEPG2 CELLSMaria Maureene Batangan, Joan Kuh.
Chaminade University of Honolulu, Honolulu, HI.
Nonalcoholic steatohepatitis (NASH), also known as nonalcoholic fatty liver disease, is defined as inflammation of the liver due to abnormal lipid levels. It is believed to be the first stage of liver damage leading to liver fibrosis and cirrhosis. Fatty liver disease occurs most often in overweight, pre-diabetic and diabetic individuals. NASH affects 2% to 5% of Americans, with an additional 10% to 20% of Americans who may have elevated levels of lipids in their liver, but no inflammation. This study examined the effects of nobiletin, a polymethoxylated flavonoid from citrus peel, on inflammation induced in a human liver cell line HepG2. Inflammation was induced by exposing the cells to physiological levels of palmitic acid (PA) and insulin found in individuals who are obese and/or pre-diabetic, with and without nobiletin. Oil red O (ORO) staining was conducted to qualitatively and quantitatively analyze the amount of lipids. Inflammation was quantified using an ELISA to detect IL-8, an inflammation biomarker, secreted into the media.
Preliminary results show that cells treated with PA in the presence of nobiletin secrete lower levels of IL-8 than cells treated with PA alone. However, in the presence of both PA and insulin, the nobiletin does not reduce the levels of IL-8 secreted. This suggests insulin interferes with the effect of nobiletin on inflammation. We will present additional data on lipid levels as assessed by ORO staining as well as results of further investigation on the effects of nobiletin on the cells treated with insulin and PA.
The three-spine stickleback (Gasterosteus aculeatus) is a euryhaline fish species that inhabits a wide variety of environments ranging from freshwater to seawater with some freshwater populations being landlocked. Gill Na+/ K+-ATPase plays an important role in the animal’s ability to regulate whole body ion and water levels allowing these fish to migrate between environments with varying salinities. Previous studies have shown that multiple isoforms of the Na+/K+-ATPase alpha subunit are expressed in fish gills, and their levels change during acclimation to changing salinity. The beta subunit of the Na+/K+-ATPase is required for normal function of the alpha subunit, but very little is known about the expression patterns of this protein during salinity acclimation of fish. The genome of G.aculeatus has been sequenced and two distinct Na+/K+-ATPase beta isoforms have been identified. This study designed real time PCR primers for each beta subunit and determined their relative expression in gills of wild G. aculeatus collected from either freshwater or seawater and following acclimation to either freshwater or seawater for 14 days. Overall enzyme activity of the Na+/K+-ATPase increased during acclimation to both increased and decreased salinity. These results will help us better understand the role of the beta subunit in Na+/K+-ATPase physiology and its importance during salinity acclimation of fishes.
EFFECTS OF THE DIAMETER DISTRIBUTION OF AXONS ON ACTION POTENTIAL PROPAGATIONVELOCITIES Karla Terrazas, Fidel Santamaria.
University of Texas at San Antonio, San Antonio, TX.