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POSTER #45:
Wnt/Frizzled Expression Signature in a Mouse Model of Inflammatory Arthritis
Bryan Dieffenbach
Dr. Mary Corr
The development of rheumatoid arthritis in humans is dependent on the production of autoantibodies. However the joint destruction seen is also mediated by the lining cells of the joint. These cells proliferate in rheumatoid arthritis, produce inflammatory cytokines and matrix metalloproteinases. Theses fibroblasts direct invade the bone and produce erosions. The joint cartilage is also destroyed. In recent years the wnt signaling pathway has bee described as not only playing an active role in the bony changes seen in inflammatory arthritis, but possibly contributing to the aberrantly activated phenotype of the synovial lining cells. Here we examined the expression signature of selected family members of the wnt/frizzled family and specific target genes. Wild type mice were injected with arthritogenic serum and sacrificed at different times at the onset peak and resolution phases of the arthritis. The mRNA expression of wnt and frizzled family members increased dramatically at the onset, but rapidly declined. The target genes however remained elevated throughout the course suggesting that the effector proteins had a lasting effect in this model.
POSTER #46:
Bim protein regulation in cell apoptosis
Angela Ho
Dr. Paul Insel
G-protein-coupled receptors (GPCRs) and their regulation of G proteins are major mechanisms in signal transduction. Among the most widely studied GPCR systems are â-adrenergic receptors. Epinephrine and norepinephrine are the endogenous ligands that bind â-adrenergic receptors and activate the associated trimeric (áâã) G protein. After the Gá subunit is activated by a GPCR, it exchanges GDP for GTP and then activates effectors, such as adenylyl cyclase; Gâã subunits also can activate effectors.. Adenylyl cyclase converts ATP to cyclic AMP (cAMP), a 2nd messenger that regulates various cellular signaling and responses, such as glycogen usage and cellular growth, differentiation and death (apoptosis) responses.. Many human cancers are associated with alteration in such growth and death responses.Previous studies have shown that Bim, a Bcl-2 protein family member, is a pro-apoptotic protein regulated by the cAMP pathway. We hypothesize that Bim is sufficient and necessary to trigger cell apoptosis. To test this hypothesis, double transgenic cell lines are being made to conditionally express Bim. The data thus far show that up-regulation of Bim is sufficient to induce apoptosis. Bim short hairpin RNAs are being produced to test if Bim is necessary for apoptosis. This part of the research is still in progress.
POSTER #47:
Exploring plant immune signaling through the Arabidopsis and Pseudomonas syringae interaction
Amanda Mason
Dr. Steven Briggs
The Arabidopsis-Pseudomonas syringae pv tomato (Pto) interaction is an intensely studied model system for plant immunity. In order to characterize different aspects of plant immunity we first explored the ability of different microbe associated molecular patterns, MAMPS to induce certain defense marker genes. Additionally we explored specific single bacterial avirulence effectors for their ability to suppress MAMP induced defense signaling in R-gene-deficient plants (Chapter 1). Flagellin (flg22), cellulase, and salicylic acid induced the chosen marker genes in different ways, but plants seemed to respond to a lesser extent to the E.coli compound Kdo2-lipid A, which induces defense in mammalian cells. The Pseudomonas effectors avrRpm1, avrRpt2, avrRps4, avrB, and avrPphB did not clearly suppress any of the marker-gene inductions. Moreover, some induction of markers was observed, suggesting recognition of the effectors by unknown (R-) proteins. We also explored the effects that light and temperature have on the hypersensitive response (HR) during the resistance (R)-gene defense response induced by expression of a single bacterial effector (Chapter 2). As expected deprivation of light delayed the HR. Surprisingly, and in contrast to other studies, HR was functional at high temperatures, and instead HR was delayed at low temperature (17°C). Since the function of many proteins in immune signaling is unknown, as well as potential protein-protein interactions, we explored the interactome of the protein NDR1. We identified 286 potential NDR1 interacting proteins with potential roles in plant immunity. Currently, candidate proteins are being tested for their role in defense.
POSTER #48:
A Screen for NS1 Dominant Negative Peptides that Function as a Treatment for Influenza A Infection
Alex Sarkisian
Dr. Ethan Bier
Major pandemics of Influenza A have caused the deaths of several million people in the past century alone. Discovering treatments to combat influenza A infection could save many lives in the event of an outbreak. We are attempting to generate new ways of treating influenza by creating dominant negative (DN) inhibitors to the influenza non-structural protein, NS1, which has an important role in suppressing the host immune response and is critical for viral replication. We have generated a stock of Drosophila melanogaster carrying a genomic insertion of a NS1 transgene flanked by an N-terminal HA tag and a C-terminal Myc tag. Expression of NS1 conditionally in the wing using the yeast derived UAS/Gal4 system causes a visible wing phenotype including expansion between wing veins L3 and L4 and partial loss of L4. We have conducted a screen in which the mutagen delta 2-3 transposase was used to create deletions and truncations directed to the NS1 transgene in flies. From this screen, we have isolated two putative NS1 DN alleles that produce truncated NS1 proteins and can suppress the wing phenotype caused by the expression of the wild-type copy. Furthermore, these alleles produce no phenotype when expressed on their own indicating a low probability of serious off target effects if used as treatments in humans. We are now in the process of characterizing the precise molecular changes in these DN peptides and will then test their efficacy in suppressing influenza infection in mammalian tissue culture cells.
POSTER #49:
Membrane Porters of ATP-binding Cassette (ABC) Transport Systems are Polyphyletic
Bin Wang
Dr. Milton H. Saier
The ATP-binding cassette (ABC) superfamily consists of both importers and exporters. These transporters have, by tradition, been classified according to the ATP hydrolyzing constituents which are monophyletic. The evolutionary origins of the transmembrane porter proteins/domains are not known. We here demonstrate that ABC exporters are polyphyletic, having arisen at least three times independently. ABC-1 porters arose by intragenic triplication of a primordial two transmembrane segment (TMS)-encoding genetic element, yielding 6 TMS proteins. ABC-2 porters arose by intragenic duplication of a dissimilar 3 TMS-encoding genetic element, yielding a different family of nonhomologous 6 TMS proteins. ABC-3 porters arose by duplication of a primordial 4 TMSencoding genetic element, yielding either 8 or 10 TMS proteins. We assign 48 of the 50 currently recognized families of ABC exporters to the three evolutionarily distinct ABC types.
POSTER #50:
Protein kinase A (PKA) and Exchange protein activated by cAMP-1 (Epac-1) are pro- and anti-apoptotic mediators, respectively, in chronic lymphocytic leukemia
Eric Apaydin
Dr. Paul Insel
Chronic lymphocytic leukemia (CLL), the most common adult leukemia in the Western world, is associated with the accumulation of B-lymphocytes due to decreased apoptosis. The second messenger 3’5’-cyclic adenosine monophosphate (cAMP) promotes apoptosis of peripheral blood mononuclear cells (PBMC) isolated from patients with CLL but the mechanisms involved in this response have not been defined. The actions of cAMP are mediated by protein kinase A (PKA) and Exchange protein activated by cAMP-1 (Epac-1). Using real-time PCR, immunoblotting and an activity assay, we find that compared to PBMC from healthy subjects, CLL-cells have elevated Epac-1 mRNA transcript, protein and activity. Furthermore, use of FACS (to assess annexin 5 staining, a marker for apoptosis) reveals that 48 hr treatment with an Epac-selective analog (8-pCPT-2Me-cAMP (8Me), 50mM) inhibits apoptosis of CLL cells but that a PKA-selective analog (N6- Phenyladenosine-cAMP (N6), 50mM) induces apoptosis. Inhibition of Rap-1, the downstream mediator of Epac-1, with GGTI-298 (1nM-10mM, 48h) increases apoptosis of CLL-cells in a concentration dependent manner. These results reveal that CLL is associated with increased Epac-1 expression and that unlike PKA activation, Epac-1 activation (in a Rap-1 dependent manner) enhances the survival of the malignant B cells. This anti-apoptotic action of Epac opposes the pro-apoptotic effects of PKA. We conclude that approaches that decrease the expression and function of Epac-1 or downstream mediators of Epac may be beneficial for the treatment of CLL by increasing the pro-apoptotic effects of cAMP via PKA.
POSTER #51:
The acyl-CoA binding protein, Rppa09976, is required for the selective degradation of peroxisomes in Pichia pastoris
Katharine Ozeki
Dr. Suresh Subramani
It is imperative for organisms to find a way to recycle and degrade cytoplasmic constituents and eliminate unnecessary organelles. Autophagy is a tightly-regulated, non-selective degradation process that plays an integral role in cellular homeostasis by sequestering ubiquitous proteins or organelles, and delivers such cargo to the vacuole so that they may be degraded and their constituents recycled. For instance, peroxisomes are single membrane-bound organelles required for certain metabolic pathways including both alpha- and beta-oxidation of fatty acids, decomposition of harmful hydrogen peroxide molecules, the production of penicillin in certain fungi and photorespiration in plants. When peroxisomes become redundant, they are selectively delivered to the vacuole in an autophagy-related pathway, termed pexophagy. While most proteins required for autophagy are also required for pexophagy, only two proteins, Atg26 and Atg30, are known to be solely required for pexophagy, but not autophagy-related pathways. Here, we identified a novel peroxisomal membrane-associated acyl-CoA binding protein (ACBP) domain-containing protein, Rppa09976, also required for the selective degradation of peroxisomes, but dispensable for other autophagy-related pathways. Unlike most peroxisomal membrane proteins, Rppa09976 is a type II PMP, trafficking from the ER to peroxisomes, and degrades independent of pexophagy, autophagy and vacuolar proteolysis. Furthermore, we have identified a putative mammalian homolog, the murine peroxisomal MmAcbd5 protein, which, like Rppa09976, contains an N-terminal acyl-CoA binding protein (ACBP) domain. This discovery has led to a deeper understanding of the mechanism underlying the selective sequestration and degradation of peroxisomes, a process that may apply to higher organisms.
POSTER #52:
Characterization of UNUSUAL LATERAL ORGANS (ULO), a miRNA Regulated F-Box
Peter Smith
Dr. Jeff Long
The high quality annotation of the Arabidopsis thaliana genome allows biologists to predict the target transcripts of known microRNAs (miRNAs). The transcript of Unusual Lateral Organs (ULO) is a predicted target of the miRNA394 family. ULO is predicted to contain an F-BOX domain and a Kelch protein-protein interaction domain by amino acid sequence similarity. MiRNA resistant versions of ULO result in polarity defects in all lateral organs derived from the shoot apical meristem. These defects are similar to what is seen in loss-of-function alleles of the class III HD-ZIP genes, indicating ULO may negatively regulate this gene family. Genetic interactions between HD-ZIP III mutants and miRNA resistant alleles of ULO support this finding. Our current work aims to determine if the negative regulation of HD-ZIP III genes by ULO is direct and to better understand the roles ULO plays during lateral organ development.
POSTER #53:
Cloning and Expression of Protein Tyrosine Kinases in the Medicinal Leech
Bailey Zhao
Dr. Eduardo Macagno
Some non-receptor protein tyrosine kinases (PTKs) are thought to play an important role in axonogenesis. I have partially cloned and sequenced two such PTKs found in Hirudo medicinalis: focal adhesion kinase (FAK) and Abelson kinase (Abl). Comparative analysis of the protein sequences of these two kinases reveal that they are closer in homology to vertebrate species than to other invertebrates, such as insects. Whole-mount in situ hybridization (WISH) showed that leech FAK mRNA is strongly expressed in the ganglia of the central nervous system and nephridia, as well as potential expression in the bilateral Comb cells. Leech Abl mRNA is strongly expressed in the ganglia and nephridia across all trials, while some WISH trials show expression in the bilateral heart tubes, Comb cells, as well as sensilla clusters. Attempts to knock down expression of Abl have thus far been inconclusive.