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TRANSCRIPTOME-TO-REACTOME™ BIOSIMULATION: BASAL FOREBRAIN CHOLINERGIC NEURON
NEUROTROPHIN SIGNALINGJonathan Morris, Clyde Phelix, George Perry.
The University of Texas at San Antonio, San Antonio, TX.
Signaling of cholinergic basal forebrain (CBF) neurons is critical for survival and plasticity. Human CBF neurons revealed a shift in neurotrophin receptors toward cell death in Alzheimer’s disease (AD). In this study, transcriptomics from mouse basal forebrain cholinergic neurons (BFCNs; NCBI GEO GSE13379) were used to derive parameters for deterministic kinetic model of nerve growth factor (NGF) signaling. Transcriptome-To-Reactome(TTR)™ biosimulation was performed using COPASI; 11 compartments, 435 species, and 263 reactions. Two hundred forty-five genes were used to determine initial values of species and kinetic values of reactions. BFCN model biosimulations were run with 2 doses of NGF, 500 µM and 10 mM, as a bolus, for 10 and 240 seconds. This tested p75NTR and TrkA receptor mechanisms. Another test used 25 µM brain derived neurotrophic factor and 10 µM NGF continuously for 60 min, evaluating stimulation of p75NTR,TrkA, and TrkB. From human results, downregulation of TrkA (50%) and TrkB (60%), corresponding-parameters in TTR™ biosimulation were decreased. Baseline results were validated on neuronal calcium levels mediated via phospholipase C-γ and inositol-3-phosphate at both bolus doses of NGF alone.
With corresponding parameters decreased in TTR™ biosimulation, reaction flux for c-RAF1 phosphorylation of MEK1 was delayed to peak value by 1.5 min from exposure, but peak value was increased 5 times. Moreover, a right shift of flux over time was observed with B-RAF phosphorylation of MEK1. These results show transient responses to neurotrophins accentuated, whereas sustained MEK activating pathway is blunted in AD, and may represent potential mechanisms for failure of prosurvival pathways in Alzheimer’s disease.
COMPARING MICROARRAY AND QPCR RESULTS TO VALIDATE AGE-RELATED CHANGES IN GENEEXPRESSION Peggy Hogan Hatfield1, Tony Parenti2, Michael Halbisen2, Amy Ralston2.
Gavilan College, Gilroy, CA, 2University of California, Santa Cruz, Santa Cruz, CA.
1 The effects of aging contribute to a multitude of negative variables within organisms. If it were possible to slow, stop, or reverse some portion of the damage that the aging process causes, the benefits to health would be immense.
Pluripotent stem cells have the ability to become any cell type in the adult body and may one day be used to fix cells that have been damaged by aging. There is great interest in using induced pluripotent stem cells (iPSCs) for cellular therapy. However, since these cells are reprogrammed from differentiated cells, it is not clear whether cells from aged individuals can produce iPSCs of the same quality as cells from younger individuals. Our lab has a microarray dataset ( 20,000 genes) that indicates there are gene expression differences between differentiated cells and iPSCs generated from young and old mice. Quantitative PCR (qPCR) will be used to validate these results for a smaller subset of genes related to insulin signaling factors since insulin signaling has been known to affect aging processes.
We expect that the qPCR results will match the microarray analysis, thus potentially providing a more efficient and inexpensive way of comparing specific gene expression profiles. These results will be useful for future experiments designed to improve iPSC quality from aged individuals.
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Biological Sciences SAT-493
BIOCHEMICAL CHARACTERIZATION OF THE RBFOX PROTEINSAdrian Hernandez, Niroshika Keppetipola, Douglas Black.
University of California, Los Angeles, Los Angeles, CA.
The Rbfox proteins are a family of homologous splicing regulators composed of Rbfox 1, Rbfox 2 and Rbfox 3 proteins, which play important roles in alternative splicing processes. The Rbfox proteins contain a highly conserved RNA recognition motif which binds specifically to the sequence element UGCAUG. The Rbfox proteins act as splicing activators when bound downstream and splicing inhibitors when bound upstream of regulated exons. To characterize the upstream and downstream Rbfox complexes, recombinant Rbfox and Rbfox RRM clones were expressed in E. coli, purified, and assayed in vitro for binding to RNA containing a UGCAUG element. A mutant RNA sequence with the UGCAUG mutated to UCAGUC was also assayed. The mutant did not show any RNA-protein complex with Rbfox 3. These results confirmed that the UCGAUG hexanucleotide is critical for mediating the interaction between the Rbfox 3 protein and the RNA. Based on RbFox 2 RRM and Rbfox 3 RRM protein binding assays, it appears there must be interactions, other than the Rbfox RRM-RNA interactions, required for the Rbfox proteins to bind to the UGCAUG sequence located in the downstream control sequence of the csrc alternative N1 exon. This knowledge, coupled with previous hypotheses that the N-and C-terminus of the Rbfox proteins are involved in protein-protein interactions, underscore the importance of determining the types of interactions between Rbfox proteins and splicing associated with neurodegenerative and muscular dystrophy disorders. This may ultimately lead to insights as to how these disorders progress.
EVALUATION OF ENDOTHELIAL PROGENITOR CELL POPULATIONS IN HUMAN OMENTUM AND
SUBCUTANEOUS TISSUEBruktawit Goshu, Samuel Lake, Daniel Peterson.
Rosalind Franklin University of Medicine and Science, Chicago, IL.
Angiogenesis is thought to be one of the ways the omentum responds to injury in situ and why omental flap transfer is so helpful in wound healing. Studies have shown that endothelial progenitor cells (EPCs) induce and facilitate angiogenesis. Therefore finding EPCs in the omentum would provide evidence that the omentum contributes to wound healing through promotion of angiogenesis. We hypothesize there will be more EPCs in the omentum than in subcutaneous fat because the omentum is highly vascularized. We will test for the presence of EPCs in these two tissue types and in control hMSCs using the antibodies CD34, CD309, and CD133 and the corresponding isotypes.
Each cell type will be plated, counted, and given a mixture of the antibodies or isotypes and stained with DAPI. Flow cytometry will be run on each sample. Comparison of the flow cytometric antibody data versus isotope data will differentiate between specific and nonspecific binding. The flow cytometry results will be analyzed for EPC population frequency in the two tissues and in control hMSCs. If our hypothesis is true, we will conduct in vitro angiogenesis assays to see if tubules form. If formation of tubules is detected, we would isolate EPCs from human omentum and evaluate their ability to contribute to wound healing in a mouse model of impaired wound healing. Demonstrating that human omental EPCs promote wound healing could lead to new stem cell therapies for wound healing in humans.
TOWARD AN UNDERSTANDING OF HERPES VIRUS RRM2 AND HOST CELL METABOLISMTaylor Brown, William Sullivan, Heather Christofk.
University of California, Los Angeles, Los Angeles, CA.
Ribonucleotide reductase small subunit (RRM2) is a component of the enzyme that catalyzes the reduction of ribonucleotides to deoxyribonucleotides, which is essential for DNA replication. It is ubiquitously expressed in human cells and has a viral homolog conserved throughout the herpes virus family, suggesting an important role in viral replication. Human RRM2 is known to be regulated by phosphorylation at serine residue 377, and notably there is conservation of an aspartic acid in the viral protein at a position adjacent to the serine residue. This observation has led to the hypothesis that the negatively charged aspartic acid lies in the same pocket as the serine residue and mimics the human phosphorylation event, resulting in constitutively heightened activity of viral RRM2 that promotes viral replication. To test this hypothesis, nontransformed human breast epithelial cells were engineered to stably overexpress RRM2 or mutant versions of the protein, corresponding to amino acid substitutions S376D and S377D.
Expression of the S377D mutant was not tolerated by the cells, causing rapid apoptosis during selection. Only low levels of RRM2 expression were detected via immunoprecipitation of cells selected for expression of the wild-type and
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S376D constructs. These results suggest cells cannot tolerate constitutively elevated levels of RRM2. To overcome the observed repression of constitutive RRM2 expression, the wild-type, S376D, and S377D RRM2 constructs will be subcloned into a doxycycline-inducible vector. Using this system, the cellular effects of elevated wild-type and mutant RRM2 expression will be investigated and compared via proliferation assays, cell cycle analysis, and metabolic profiling.
SGTΑ: AN IDENTIFIED INDEPENDENT REGULATOR OF STEROID HORMONE RECEPTOR FUNCTIONOmar Gutierrez, Marc Cox.
University of Texas at El Paso, El Paso, TX.
Steroid hormone receptors (SHR) are responsible for maintaining a reproductive, developmental, and physiological balance in eukaryotes. The receptors glucocorticoid (GR), progesterone (PR), mineralococorticoid (MR), estrogen (ER), and androgen (AR) belong to the nuclear receptor superfamily. Essentially, SHRs need the Hsp70-Hsp90 assembly system for receptor maturation to translocate to the nucleus and become functionally active. A variety of proteins regulate receptor maturation. A recently identified protein called SGTα (human small glutamine rich TPR-containing protein alpha) has been described as a regulator of AR’s activity. Prior experiments in our lab have demonstrated that SGTα binds to both Hsp70 and Hsp90 to negatively regulate AR’s, GR’s, and PR’s activity in both yeast and mammalian cells. FKBP52, a specific positive AR and GR regulator, shares the same specificity for receptor activity (MEEVD motif). However, SGTα-mediated downregulation of AR’s activity does not appear to be the result of simple competition with FKBP52 for binding Hsp90 because the observed effect in yeast is independent of the presence of FKBP52. In addition, another TPR protein called Cpr7 that positively regulates AR is present in yeast.
Thus, we assessed the ability of SGTα to regulate AR’s activity in yeast-based assays in the presence and absence of Cpr7. We observed that SGTα can abrogate AR’s activity independent of a competition for the MEEVD motif on Hsp90. These observations suggest there may be another role for SGTα at a transcriptional level independent of its TPR region. Future studies aim to identify how the structural function SGTα regulates AR’s activity.
EFFECTS OF ACID AND NONACID TREATMENT OF AG@BATIO3 NANOPARTICLE TOXICITY TO RHESUS
MONKEY ENDOTHELIAL CELLS IN CULTUREIsidro Obregon, Brandi Obregon, Brian Yust, Dhiraj Sardar, Andrew Tsin.
University of Texas at San Antonio, San Antonio, TX.
Nanoparticles are presently being studied for optical and biomedical applications such as medical imaging and drug delivery. Nanoparticles impact the cellular environment because of their size, shape, and composition. How these factors affect cell viability is not fully understood. The purpose of this study is to test the cellular toxicity of silver-coated (Ag@) acid (AT) and nonacid (NA)-treated BaTiO3 nanoparticles on rhesus monkey retinal endothelial cells (RhREC’s) in culture. The addition of silver to the nanoparticles increases their nonlinear optical properties significantly, making the Ag@BaTiO3 nanoparticles good candidates for nonlinear microscopy contrast agents.
We hypothesize that acid treating the Ag@BaTiO3 nanoparticles will cause a decrease in cell viability at higher concentrations when compared to NA-Ag@BaTiO3 nanoparticles. RhREC’s were treated with Ag@AT-BaTiO3 and Ag@NA-BaTiO3 at concentrations of 0, 1.0, 10.0, and 100 µg/ml for 24 h at 37 °C + 5% CO2. After the 24 h incubation with the respective nanoparticles, cell viability was determined using the trypan-blue exclusion method. Cells treated with 0, 1.0, and 10.0 µg/ml of Ag@AT-BaTiO3 showed no effect on cell viability. Treatment with 100 µg/ml of Ag@ATBaTiO3 showed a decrease of 10% in viable cells. Comparatively, cells treated with 0, 1.0, and 10 µg/ml of Ag@NABaTiO3 showed no effect on cell viability while the 100 µg/ml treatment resulted in a decrease of 51% in viable cells.
These results show that Ag@BaTiO3 nanoparticles affect cell viability when delivered at a high concentration and that acid treatment protects RhREC from nanoparticle-induced cell toxicity.