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«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»

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Despite extensive research, multiple sclerosis (MS) remains a disease that lacks a definitive diagnostic test. Even after a diagnosis of MS has been established, currently it cannot be reliably predicted whether and when disease relapses will occur, thus patients may undergo years of unnecessary treatments. Additionally, because the current treatments for MS can produce dramatically different outcomes in individual patients, there is a critical need to develop biomarkers for treatment efficacy and resistance. We have recently developed a novel quantitative microwave & magnetic (M2) proteomics method to quantitatively measure changes in proteome expression during experimental autoimmune encephalomyelitis (EAE), the standard murine model of MS. Our data revealed significant consistent changes in the proteome over the disease course. Changes in the expression of protein isoforms correlated either with time (days after EAE induction) or disease severity (clinical score). Interestingly, we have identified unique changes in the proteome prior to the onset of clinical symptoms. Those preonset changes may allow us to predict the subsequent severity of the disease. Last, we have identified that glucocorticoid-drugs (GCs) have an effect on these changes with a strong statistical correlation to EAE severity and more importantly, these changes closely mirror disease progression independent of any cytokine response to the GCs. Thus, homologous human biomarkers may be useful to accurately and reliably predict disease initiation, clinical severity, and GC drug efficacy. Importantly, these biomarkers illuminate some of the key mechanisms that contribute to the disease and may be useful to develop new therapeutic targets for multiple sclerosis.

Room 210B

THE PHOSPHOGLYCERATE GENE FAMILY – AN EXAMPLE OF EVOLUTION BY GENE DUPLICATION

Pedro Trejo Jr., John R. McCarrey.

University of Texas at San Antonio, San Antonio, TX.

Susumo Ohno proposed the principle of evolution by gene duplication, by which one duplicate copy maintains the original gene function while the other diverges and undertakes new gene function(s) by acquiring key sequence changes. We postulate that the mammalian phosphoglycerate kinase (Pgk) gene family exemplifies Ohno’s principle. We used a bioinformatics approach to mine mammalian genome databases to identify such key sequence changes in the 5’ regulatory region, responsible for transcriptional regulation; the 3-UTR region, responsible for post-transcriptional regulation; and the coding sequence, responsible for protein functions. Among 11 eutherian species examined, we found that Pgk1 promoters have conserved a CpG-island, a pair of GC-boxes, a pair of CAAT-boxes, and an NF-1 sequence, whereas Pgk2 promoters have lost the CpG-island, retained a single GC-box and a single CAAT-box, and gained an enhancer sequence (E3/E4) with homology to an NF-1 element. We also found that Pgk1 3’-UTRs have retained several conserved elements, but that the Pgk2 3’-UTRs have evolved two unique polypyrimidine-tract binding-protein, 2 elements not present in the Pgk1 3’-UTRs. We have not yet identified sequences in the coding region related to sperm-specific localization. We are currently examining Pgk genes from metatherian (opossum and tammar) and prototherian (platypus) species to discover more extensive evolutionary history of the divergence of the mammalian Pgk promoters and 3’-UTRs. Our results to date support the idea that the mammalian Pgk gene family is an example consistent with Ohno’s theory. (Partially funded by NIGMS MBRS-RISE GM060655.) Room 211

DETERMINING MITOTIC INHERITANCE OF THE ENDOPLASMIC RETICULUM

Anthony Eritano, Blake Riggs.

San Francisco State University, San Francisco, CA.

A well studied aspect of cell biology is mitosis, specifically DNA segregation. Despite decades of study that have gone into examining DNA segregation, little is known about the segregation of the cytoplasm and its components. Golgi and mitochondria have been the focus of the cytoplasmic inheritance research; mitotic partitioning and inheritance of the endoplasmic reticulum (ER) are poorly understood. Recent studies from our laboratory demonstrated a centrosomal localization of ER during mitosis, which suggests a possible mechanism of inheritance. We hypothesize that the ER follows a set pattern of inheritance dictated by the selective passage of newly created ER from an original, unaltered (template) ER. Transgenic flies will be created expressing a photo-convertible GFP tagged to the ER resident protein,

332 ORAL ABSTRACTS

PDI (PDI-EOS). We will photo convert ER membrane patches in actively dividing cells in the developing Drosophila embryo and track patterns of movement and inheritance. To identify potential genes that act as a template for ER inheritance, we will perform a small-scale RNAi screen in Drosophila S2 cells that express PDI-GFP and RFP tagged histones. Genes were selected that have been classified as ER stress proteins to disrupt proper function, motor proteins to disrupt movement, and ER structure and morphology proteins to disrupt ER topology. These targets will be examined for proper ER movement in embryos. These selected targets will allow for the identification of templates for ER partitioning and will help identify proteins that are vital for ER segregation during mitosis and lead to a mechanism of inheritance.





Room 211

CHARACTERIZATION OF THE CELL MORPHOLOGY DIFFERENCES BETWEEN PREDIAPAUSE AND

DIAPAUSE EMBRYOS OF THE KILLIFISH AUSTROFUNDULUS LIMNAEUS

Lee Toni, Pamela Padilla.

University of North Texas, Denton, TX.

Austrofundulus limnaeus is a freshwater, annual killifish from Venezuela that produces embryos capable of entering three distinct stages of diapause. Diapause II (DII) is a state of developmental arrest that can last over 100 days and confers extreme anoxia tolerance. Diapause I and III are inducible while II is obligate. DII occurs in ideal conditions (25 oC) whereas incubation at 30 oC induces a bypass of diapause II. DII provides an opportunity to study an arrest of development in a vertebrate animal without exposing the organism to compounding stresses. Currently, little is known about the molecular mechanisms that induce and maintain this developmentally arrested state. We hypothesize that downregulation of gene expression is central to inducing DII. Our aim is to use assays that are principally based on immunohistochemistry to characterize, at the cellular level, differences in cell morphology between pre-DII and DII embryos. We are looking at changes in chromatin modifications, which are known to be involved in the repression or activation of developmental genes, as well as changes in mitotic cell division. Preliminary results show a change in the signal localization of H3K27, which is associated with gene silencing, as the animal transitions between stages of pre-DII and DII. Analysis of H3K4, which is associated with active transcription, shows little change across diapause states although this may represent an energy homeostatic strategy. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.

Room 210B

POST-TRANSLATIONAL REGULATION OF ENOS VIA ESTROGEN RECEPTORS-ALPHA/BETA AND G

PROTEIN-COUPLED ESTROGEN RECEPTOR IN UTERINE ARTERY ENDOTHELIAL CELLS

Mayra Pastore, Saira Talwar, Rosalina Villalon-Landeros, Ronald Magness.

University of Wisconsin-Madison, Madison, WI.

Endothelial nitric oxide production is partly responsible for maintenance of vasodilatation during physiologic states of high estrogen levels such as pregnancy. Estrogen receptors (ER-α/-β) are nuclear transcription factors; however, they induce vasodilatory responses via activation of endothelial nitric oxide synthase (eNOS). Recently, the G proteincoupled estrogen receptor (GPER) has been implicated in estrogenic regulation of the vasculature. It is unknown if eNOS regulation is dependent on ER-α/-β or GPER. We hypothesize that ER-α/-β and GPER localize to the plasma membrane of uterine artery endothelial cells (UAEC) and change eNOS phosphorylation patterns indicative of activation. Vehicle or estradiol-17β treated UAECs were fixed and immunogold-labeled for visualization of caveolae, ER-α, ER-β, and GPER using transmission electron microscopy. UAECs were treated with vehicle or increasing concentrations of estradiol-17β, ER-α (PPT), or GPER (G1) agonists. GPER expression and changes in eNOS stimulatory phosphorylation sites Ser1177 and Ser635 vs. inhibitory site Thr495 were evaluated via western blot.

Immunogold labeling revealed that ER-α/-β localize to the plasma member, the cytosol, and the nucleus. GPER was also detected. Increasing estradiol-17β concentrations augmented eNOS phosphorylation at Ser1177 and Ser635 with decreased phosphorylation at Thr495. PPT (0.1 nM and 100 nM) only increased the eNOS phosphorylation at Ser635. G1 (100 nM and 1 μM) increased eNOS phosphorylation at Ser1177, not Ser635, but decreased Thr495 Graduate Oral phosphorylation at all doses. In conclusion, ER-α/-β localizes to the plasma membrane, cytosol, and nucleus;

estradiol-17β mediates changes in eNOS multisite phosphorylation; ER-α mainly mediates an increase in stimulatory phosphorylation of eNOS; and eNOS multisite phosphorylation also occurs via a GPER and ER-α/β-independent mechanism.

333 ORAL ABSTRACTS

Room 211

A GENETIC SUPPRESSOR OF PEROXISOME DEFECTS REVEALS COMPENSATORY MUTATIONS IN

INTERACTING PEROXINS IN ARABIDOPSIS

Kim Gonzalez1, Sarah Ratzel1, Jeanne Rasbery2, Bethany Zolman3, Bonnie Bartel1.

Rice University, Houston, TX, 2University of Kentucky, Lexington, KY, 3University of Missouri-St. Louis, St. Louis, MO.

1 Peroxisomes are eukaryotic organelles necessary for development in plants and humans because they are home to essential metabolic functions such as fatty acid boxidation. Enzymes enter peroxisomes with the assistance of peroxins (PEX proteins), but the mechanistic details of peroxin functions are incompletely understood. In humans, defects in peroxins can cause peroxisomal biogenesis disorders, inherited disorders often conferring mental retardation and death during infancy. A subset of peroxisomal biogenesis disorders, Zellweger syndrome, is often caused by mutations in PEX1 and PEX6, genes encoding interacting ATPases essential for peroxisome biogenesis.

PEX1 and PEX6 assist in recycling PEX5, a receptor for proteins targeted to the peroxisomal matrix. In plants, pex6 mutants display defects in development, peroxisome metabolism, PEX5 recycling, and peroxisomal matrix protein import. Suppressor screens often reveal interacting proteins and reveal dependent or related cellular processes.

We have isolated and characterized mutants that ameliorate the physiological defects of the loss-of-function pex6-1 mutation in Arabidopsis thaliana. Intriguingly, one suppressor carries a mutation in PEX1. The pex1-1 allele partially suppresses the metabolic and physiological defects of pex6-1, but does not seem to restore PEX5 recycling to wildtype levels. Our ongoing experiments are examining the allele- and peroxin-specificity of the pex1-1 suppression.

Completion of this research will reveal strategies to compensate for defects in peroxins and possibly rescue defects in other organisms with peroxisomal biogenesis disorders. (This research is supported by the NSF and the Welch Foundation.)

ECOLOGY

Room 209

PREDICTING SMALL SCALE DISTURBANCE FROM LARGE SCALE ENVIRONMENTAL FORCES

Dan Orr, Corey Garza.

California State University, Monterey Bay, Seaside, CA.

Understanding and predicting small-scale disturbance by large scale environmental forces has important implications for natural resource management and understanding potential consequences resulting from climate change. This is particularly important when considering disturbance to the foundation species within a community such as coral reefs or large trees in a forest. The California mussel Mytilus californianus is a sessile foundation species that forms massive aggregations along rocky shores in the north-eastern Pacific. In a multilayer mussel bed, each subsequent layer is more weakly attached than the next. The bottom layers can become a sediment trap leading to mortality and weakening the entire structure. This counters the traditional understanding that disturbance is generated solely by external forces distributed randomly across the mussel bed and suggests disturbance across mussel beds may be predictable. We hypothesized that small-scale mussel bed disturbance by large-scale environmental forces can be predicted. Here we began by linking large-scale environmental forces to small-scale sites. We measured maximum wave velocity across mussel beds at 3 sites. We calculated the mean maximum wave velocity (MMWV) experienced at each site for each 24-hour sampling period. Using GIS and remote sensing technologies, we fit a model to the observed MMWV. This was the first step in the empirical testing of our hypothesis. Understanding and predicting small-scale disturbance from large scale environmental forces would have implications on other communities that depend on foundation species. This has important implications regarding climate change and natural resource and ecosystem management.

Room 209

THE VALUE OF HABITAT DIVERSITY IN MARINE RESERVES: SPINY LOBSTER AND SHEEPHEAD USE OF

THE INTERTIDAL ZONE AT THE SANTA CATALINA ISLAND MARINE PROTECTED AREA

Sean Windell, Corey Garza.

California State University, Monterey Bay, Seaside, CA.

Marine protected areas (MPAs) are a relatively new approach to managing exploited marine species. However, in order for MPAs to be effective, a diversity of habitats that incorporate all necessary ecosystem services for targeted

334 ORAL ABSTRACTS



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