«Strengthening the Nation through Diversity, Innovation & Leadership in STEM San Antonio,Texas · October 3-6, 2013 Get Connected! Connect with the ...»
MUC16/CA125 is a high molecular weight, heavily glycosylated transmembrane mucin primarily found at the apical surface of normal epithelia. Mucins protect and lubricate against pathogenic infections. Membrane mucins are aberrantly over-expressed in many cancers including those of the uterus, ovary, and pancreas. Expression of MUC1, a well characterized mucin, is highly stimulated by the proinflammatory cytokines, tumor necrosis factor α (TNFα), and interferon γ (IFNγ), as well as by progesterone in various cellular contexts. High levels of all 3 factors are detected in uterine tissues at the same time that MUC1 is highly expressed. Also, we previously demonstrated that rosiglitazone inhibits MUC1 expression in a variety of contexts. We hypothesized that these same factors also might stimulate MUC16 expression. In this study, we describe that MUC16 mRNA and protein expression is stimulated 2 to 3-fold by TNFα (2.5 ng/ml) alone and 3 to 4-fold by IFNγ (20 IU/ml) alone in IHEEC cells, a telomerase immortalized human endometrial epithelial cell line. Interestingly, combined treatment with both cytokines resulted in a large (20 to 60-fold), synergistic stimulation of MUC16 mRNA and protein expression. Cytokine stimulation of MUC16 expression also was observed in several other cell lines indicating that this may be a general response. We currently are examining rosiglitazone responses and how the cytokine response is mediated at the level of the MUC16 promoter.
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DEVELOPMENT OF AVIAN INNER EAR HAIR CELL CHIMERASGiovanni Diaz1, Kazuo Oshima2, Stefan Heller2.
San Francisco State University, San Francisco, CA, 2Stanford University School of Medicine, Palo Alto, CA.
1 One in five Americans have hearing loss. Hearing loss results from loss of inner ear sensory hair cells due to loud noises, aging, toxic chemicals, or genetic predisposition. In mammals, lost hair cells cannot be regenerated naturally.
The Heller Laboratory has recently developed in vitro guidance methods for pluripotent mouse and human stem cells to generate otic progenitor cells. The differentiation of these progenitors into mature sensory hair cells has been promoted by coculture with non-sensory cells isolated from the developing chicken inner ear. Our goal is to increase the efficacy of otic cell differentiation and maturation by developing in ovo transplantation and in vitro cell suspension assays of mouse and human progenitor cells into the chicken embryo otocyst. We hypothesize that the developing chicken inner ear provides a superior niche for differentiation into sensory hair cells. We are working in parallel on both an efficient in ovo and in vitro avian-mammalian chimera model that would utilize the developing chicken inner ear as a micro niche for differentiation into hair cells and potential identification of factors involved in regeneration.
Our objective is to optimize conditions for both Chimera in ovo transplantation and in vitro cell suspension model systems. Our studies will identify optimal conditions for differentiation of both models including the number of cells per suspension, medium, dissociation enzymes, culture duration, and ratio of mouse/human to chicken cells. The proposed study will provide an efficient platform for investigating the potential use of stem cell transplantation therapy to restore hearing.
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ELUCIDATING THE ROLE OF WNT SIGNALING IN XENOPUS NEURAL CREST INDUCTIONStefanie Monica, Richard Harland.
University of California, Berkeley, Berkeley, CA.
The neural crest (NC) is a vertebrate-specific population of multipotent cells, often referred to as the fourth germ layer because of their unique ability to migrate all over the body and form many different types of tissue. Precise control of BMP and Wnt signaling in both space and time is necessary for proper NC induction. While the distinct roles of Wnt and BMP signaling are well studied, the precise dose-response relationship between Wnt signaling and NC induction has not been elucidated. In this work, we aim to better understand the role of canonical Wnt signaling in NC formation. We used a small molecule activator of Wnt signaling (6-bromoindirubin-3’-oxime) and a small molecule inhibitor of BMP signaling (LDN193189) to systematically alter signaling in Xenopus laevis embryos. Using neuralized animal cap explants of Xenopus laevis embryos, we tested how different levels of Wnt signaling affect NC induction.
In situ hybridization and RT-PCR were employed to quantify the level of gene expression of NC markers including snail2, foxd3, Ap-2, and SoxE family members Sox8, Sox9, and Sox10. Preliminary results confirm that the LDN drug induces neural tissue in animal caps, as evidenced by an increase in neural markers Otx2 and Sox2. Additionally, treatment with both LDN and BIO induces NC tissue as demonstrated by an increase in NC markers snail2 and foxd3.
With this work, by regulating the degree of Wnt activation, we aim to describe the molecular mechanisms underlying the reiterative role of Wnt signaling in neural crest induction and maturation.
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DISCOVERY OF A MACROPHAGE-BIGH3-APOPTOTIC AXIS IN DIABETIC NEPHROPATHYRobert Moritz, Richard LeBaron.
University of Texas, San Antonio, San Antonio, TX.
Graduate Poster Diabetic nephropathy (DN) is the most prevalent cause of end-stage renal disease. DN is manifested, in part, by extracellular matrix (ECM) accumulation in the kidney and renal cell injury. To further clarify the nature of ECM in diabetic renal damage, we are investigating the TGFβ-induced proapoptotic ECM protein transforming growth factor beta-induced gene human clone 3 (BIGH3). Our study has uncovered a mechanistic link between macrophages, the synthesis and secretion of BIGH3, and the induction of apoptosis in kidney cells. Our histological examinations of diabetic mouse kidney cortex revealed an increase in infiltrating macrophages and BIGH3 protein in the interstitial matrix and basal lamina when compared to control cortex. Macrophages cultured under diabetic conditions enriched their medium with TGFβ1. This “diabetic” macrophage-conditioned medium (dMCM) promoted BIGH3 expression and apoptosis in renal proximal tubule cells (RPTEC). Exposing these cells to recombinant BIGH3 also induced apoptosis.
Importantly, an inhibitor of TGFβ1 receptor signaling decreased dMCM-induced RPTEC apoptosis, as did anti-BIGH3 antibody. These results provide evidence for a macrophage-BIGH3-apoptotic axis in progression of DN. Our previous
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studies have implicated integrins in BIGH3-induced apoptosis. Uncovering the integrin type involved in our proposed mechanistic axis is the next objective of our research on understanding BIGH3 actions in DN. Collectively our findings are expected to lead to novel agents and targets for clinical intervention in diabetic nephropathy.
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ROLE OF INTERPHOTORECEPTOR RETINOID-BINDING PROTEIN ON THE TRANSFER OF ATROL AND
11CROL IN THE CONE VISUAL CYCLEAndrew Mendiola1, Brandi Betts-Obregon1, Joshua Mimum1, Federico Gonzalez-Fernandez2, Andrew Tsin1.
University of Texas at San Antonio, San Antonio, TX, 2University of Buffalo, Buffalo, NY.
1 There is a growing body of experimental evidence implicating interphotoreceptor retinoid-binding protein (IRBP) as a key protein involved in transferring all-trans retinol (atROL) and 11-cis-retinol (11cROL) in the novel cone-specific visual cycle. It is well known that in the classical visual cycle, retinoids are transferred between the retinal pigment epithelium (RPE) and the photoreceptor cells; however, in the cone cycle, retinoids are transferred between Müller glial cells and cone photoreceptors. Recently we have shown that IRBP binds to and protects atROL and 11cROL in a light-dependent manner in situ. We now hypothesize that IRBP not only functions to protect retinoids from degradation, but also is responsible for the transfer of retinoids between Müller cells and cone photoreceptors.
To test this, primary Müller cells will be explanted from freshly isolated chicken retinas and grown to confluence (approx. 14 days). Cells will then be treated with 2 µM of either 11cROL or atROL along with 0, 1.0, 10, or 100 µM IRBP and incubated for 24 hrs at 37 C + 5% CO2 under photopic conditions. Following the 24 hr incubation, cells will be harvested and homogenates prepared. Retinoids will be extracted and then identified and quantified by HPLC.
Results from these experiments will provide evidence supporting IRBP’s role in vitamin A regeneration in the cone visual cycle, a molecular mechanism that remains largely unknown.
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KINETOCHORE COMPONENT LOCALIZATION AND FUNCTION DURING SPERMATOGENESIS MAY DEPEND
ON SPERM-SPECIFIC PP1 PHOSPHATASESJoseph Beyene, Diana Chu.
San Francisco State University, San Francisco, CA.
Male infertility affects millions of couples within the US. Male fertility and sperm function depend upon proper chromosome segregation during meiosis. However, little is known about the molecular components required for chromosome segregation during sperm meiosis. Our lab examines the role that PP1 phosphatases, GSP-3/4, have in C. elegans sperm meiosis. GSP-3/4 are sperm-associated protein phosphatases that are 98% identical. While gsp-3/4 mutants are infertile and gsp-3/4 mutant sperm chromosomes fail to segregate properly during meiosis, the specific roles GSP-3/4 have in chromosome segregation remains unknown. During meiosis, microtubules attach to kinetochores to pull chromosomes apart. We have found that GSP-3/4 colocalize with kinetochore components. Thus, we hypothesize that GSP-3/4 are required for the correct localization and function of kinetochore components during spermatogenesis. Consistent with this, we have found that a kinetochore component called HCP-2 mislocalizes in gsp-3/4 male mutants, suggesting a dependency upon GSP-3/4 for kinetochore localization. We are using cytology to visualize if GSP-3/4 regulate additional kinetochore components. These results will elucidate the extent to which GSP-3/4 regulate kinetochore localization during spermatogenesis. Furthermore, we aim to visualize kinetochore and microtubule interactions in gsp-3/4 male mutants. This will allow us to observe how changes in kinetochore components influence microtubule attachment to sperm chromosomes. In gsp-3/4 mutants, we anticipate that there will be aberrant kinetochore localization along with abnormal attachment of microtubules to chromosomes. Our work will demonstrate sperm-specific aspects of kinetochore components and their dependency on GSP-3/4 for proper localization.
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A GENOMICS AND PROTEOMICS APPROACH TO STUDY PLANT DEFENSE RESPONSE AGAINST INSECTHERVIBORE Maria Lockwood, Jurgen Engelberth.
University of Texas at San Antonio, San Antonio, TX.
Plants receiving green leafy volatiles (GLV) prime their defenses resulting in a stronger and faster response when under actual attack. This has been tested to date on a variety of plant species. In a metabolic analysis of different plant species, we found that free fatty acids accumulate rapidly upon treatment with GLV, and that this increase in free
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fatty acids enhanced the response to subsequent insect elicitor (IE) treatment. Microarray analysis of transcriptional networks activated in corn in response to GLV or IE in corn (Zea mays) identified specific transcriptional networks.
However, in both studies, the temporal information was limited and did not allow for a more transcriptional analysis.
Therefore, we plan to expand these first studies by analyzing a longer time frame. Since no such study has ever been performed with dicot plant, we plan to perform a similar analysis with Arabidopsis. The goal of this activity is to identify the transcriptional networks activated by GLV in different plant species. We will identify similarities and differences in response to these volatile signals. We will characterize the consequences of this signaling by monitoring changes in the proteome. Additionally, we will analyze transcriptional networks in response to IE in 2 plant species and compare those with the respective proteome. The outcome of this study will help to establish GLV as volatile signaling compounds and their acceptance as a major plant hormone. (Partially funded by NSF LSAMP-BD 1249284.) Ballroom C - 137
WHOLE GENOME TRANSCRIPT PROFILING ANALYSIS OF TDRD7 NULL MOUSE MUTANT LENSCarrie Barnum, Salil Lachke.
University of Delaware, Newark, DE.