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  • Undergraduate Poster Abstracts
  • Cell/Molecular Biology

    THU-G13 EVALUATION OF NOVEL IMMUNOCYTOKINES THAT PREFERENTIALLY TARGET HIGH OR INTERMEDIATE AFFINITY IL-2 RECEPTORS

    • Zulmarie Perez Horta ;
    • Paul Sondel ;

    THU-G13

    EVALUATION OF NOVEL IMMUNOCYTOKINES THAT PREFERENTIALLY TARGET HIGH OR INTERMEDIATE AFFINITY IL-2 RECEPTORS

    Zulmarie Perez Horta, Paul Sondel.

    University of Wisconsin-Madison, Madison, WI.

    The hu14.18-IL-2 immunocytokine (IC) is a fusion protein combining human IL-2 to the heavy chains of an anti-GD2 monoclonal antibody (mAb). Clinical trials have shown that IC treatment can lead to complete responses; however, therapy is limited by IL-2-induced toxicity. Evidence suggests that the anti-tumor efficacy of IL-2 is achieved through activation of immune cells expressing high-affinity IL-2 receptors (αβγ IL-2Rs) while IL-2-induced toxicity is related to over stimulation of immune cells expressing intermediate affinity IL-2 receptors (βγ IL-2Rs). To study the effects of IL-2R selectivity in efficacy and toxicity, we have tested IC35 and ICSK, a novel generation of ICs with varying levels of affinity for βγ IL-2Rs. We evaluated the ability of IC35 and ICSK to induce proliferation of mouse and human cells expressing αβγ- or βγ-IL-2Rs and found that both ICs maintained activation of both human and mouse αβγ-IL-2Rs. In contrast, IC35 had a 50-fold reduction or complete loss of its ability to stimulate proliferation of human and mouse cells expressing βγ-IL-2Rs, respectively. Notably, ICSK stimulated proliferation of human cells expressing βγ-IL-2Rs comparable to IC, while it had 10-fold increase in its ability to stimulate mouse cells expressing βγ-IL-2Rs. Our data shows that ICSK can be used in mouse models to study the effects of over-stimulation of βγ-IL-2Rs and its relation to IL-2-induced toxicity. In addition, IC35’s reduced ability to stimulate human βγ-IL-2Rs suggests it is a potential candidate for clinical trials where we expect to see good anti-tumor effects without dose-limiting IL-2 toxicity.

    THU-G4 CHARACTERIZING POST-TRANSLATIONAL MODIFICATIONS AS THE KILLIFISH AUSTROFUNDULUS LIMNAEUS ENTERS A STATE OF SUSPENDED ANIMATION

    • Lee Toni ;
    • Pamela Padilla ;

    THU-G4

    CHARACTERIZING POST-TRANSLATIONAL MODIFICATIONS AS THE KILLIFISH AUSTROFUNDULUS LIMNAEUS ENTERS A STATE OF SUSPENDED ANIMATION

    Lee Toni, Pamela Padilla.

    University of North Texas, Denton, TX.

    Austrofundulus limnaeus is a species of annual killifish which inhabits ephemeral ponds in South America. The species is able to survive harsh desiccating ponds across seasons due to their ability to produce robust embryos. The embryos of this species are capable of entering a developmental arrest, termed diapause, which precedes the onset of drought. While in this state, embryos exhibit the greatest tolerance to anoxia of any characterized vertebrate at 25 °C. Currently, little is known about the molecular mechanisms which induce and maintain this developmentally arrested state. We hypothesize that downregulation of gene expression is central to inducing diapause. To test our hypothesis, we have chosen to examine chromatin modifications which are well characterized as modulators of gene expression in other systems. Although the repertoire for molecular assays in this system is growing, there are few relative to model organisms readily available. As such, the techniques utilized in this work had to be adapted and developed from other systems. Preliminary results utilizing adapted immunobased assays show significant changes in the global amount of H3K27me1-3 and H3K4me1-3 as the embryos progress from early embryogenesis through the exit of diapause. This will lay the foundation for future experiments which may identify exactly which genes are poised to expression or silencing. This research is leading to a greater molecular understanding of the remarkable phenomenon of obligate developmental arrest in a vertebrate embryo.

    THU-G14 POLYSACCHAROPEPTIDE INHIBITS VIRAL REPLICATION IN HIV-INFECTED PERIPHERAL-BLOOD MONONUCLEAR CELLS

    • Madeline Rodriguez ;
    • Nawal Boukli ;

    THU-G14

    POLYSACCHAROPEPTIDE INHIBITS VIRAL REPLICATION IN HIV-INFECTED PERIPHERAL-BLOOD MONONUCLEAR CELLS

    Madeline Rodriguez, Nawal Boukli.

    Universidad Central del Caribe, Bayamon, PR.

    Human immunodeficiency virus (HIV) is one of the most studied and challenging diseases, which is characterized by high and rapid replication. Currently used regimens of highly active antiretroviral therapy (HAART) are generally expensive and relatively toxic. There is a major need to identify a compound that can prevent and/or alleviate the adverse effects occurring post-HIV-1 infection. Polysaccharopeptide (PSP) obtained from different edible mushrooms, especially Coriolus versicolor, has demonstrated to be an immune system builder that may have anti-HIV-1 and anti-inflammatory properties. In vitro cytotoxicity and dose assays were applied to evaluate PSP anti-HIV-1 activity on peripheral blood mononuclear cells (PBMC) post HIV-1 infection. The optimal dose effective in reducing HIV replication and maintaining PBMC alive in culture was 200 ug/ml PSP. HIV-1 viral replication inhibition was determined by p24 ELISA assay; HIV replication was inhibited 48.9% after one administration of 200 ug/ml PSP during 3 days of treatment and 72.6% after 2 administrations of PSP during 6 days of treatment. PSP increasing expression of toll-like receptor 4 (TLR4) in HIV-infected PBMC suggest an important role in the innate immune response. Moreover, TLR4 increased expression of RANTES and IL-8, which are key chemokines that have been shown to diminish viral replication. Taken together, data from this study show the anti-HIV-1 activity of PSP immune response. Mechanistic studies are in progress to understand the cellular and molecular mechanisms behind PSP HIV-1 inhibition. Findings from these studies may lead to novel therapeutics, potentially preventing further spread of HIV-1 infection and disease progression.

    FRI-G4 UNDERSTANDING HOW C. ELEGANS’ H3.3 HISTONE VARIANT AFFECTS THE STABILITY AND ASSOCIATION OF THE NUCLEOSOME CORE PARTICLE

    • Dagimhiwat Legesse ;
    • Andrea Colmen ;
    • Issa Esaid ;
    • Raymond Esquerra ;

    FRI-G4

    UNDERSTANDING HOW C. ELEGANS’ H3.3 HISTONE VARIANT AFFECTS THE STABILITY AND ASSOCIATION OF THE NUCLEOSOME CORE PARTICLE

    Dagimhiwat Legesse, Andrea Colmen, Issa Esaid, Raymond Esquerra.

    San Francisco State University, San Francisco, CA.

    DNA is compacted into structural units by histone proteins. The DNA-protein complex, the nucleosome core particle (NCP), is composed of 2 H2A-H2B dimers that associate along both sides of the (H3-H4)2 tetramer. Naturally, there are several variants of each histone protein, and these variants are posited to play important roles in the dynamic regulation of transcriptional activation or deactivation by affecting the stability of nucleosome. Changes in accessibility due to incorporation of variants into the NCP allow for fine control of the gene expression during essential biological benchmarks such as cell differentiation, development, and repair. The (H3-H4)2 tetramer plays a central role in the assembly, disassembly, and positioning of nucleosomes; little is known about how the histone H3.3 variant affects the stability of the tetramer. Although H3.3 only differs from canonical H3 by 5 amino acids, it has a large effect on the dynamic accessibility of the NCP. The purpose of this study is to interrogate how the histone variant (H3.3-H4)2 tetramer differs in folding and assembly compared to the canonical (H3-H4)2 tetramer. Our fundamental hypothesis is (H3.3-H4)2 tetramer will be significantly less stable than the canonical tetramer due to losses of (H3-H4) dimer ionic interactions. Specifically, we will assess differences in stability by using a combination of circular dichroism and fluorescence using chemical denaturation as a function of ionic strength. In addition, understanding how ionic strength affects the dissociation of the (H3-H4)2 tetramer will help in understanding the physical factors that govern the dynamic assembly and disassembly of the NCP.

    FRI-G14 PHOSPHOREGULATION OF SPERM MEIOTIC CHROMOSOME SEGREGATION

    • Luis Quintanilla ;
    • Vanessa Cota ;
    • Thais Godoy-Cintra ;
    • Diana S. Chu ;

    FRI-G14

    PHOSPHOREGULATION OF SPERM MEIOTIC CHROMOSOME SEGREGATION

    Luis Quintanilla, Vanessa Cota, Thais Godoy-Cintra, Diana S. Chu.

    San Francisco State University, San Francisco, CA.

    Phosphoregulation, a significant process that regulates key cellular activities, relies on a fine balance between kinases and phosphatases. Serine/threonine kinases and phosphatases are responsible for mediating key chromosome segregation events: the interaction with microtubules and kinetochores which help pull chromosomes apart, and the removal of cohesion between chromosomes before they separate. A group of key meiotic serine/threonine PP1 phosphatases found only in C. elegans sperm, called GSP-3 and GSP-4 (GSP-3/4), are required for proper chromosome segregation specifically in male meiosis. Chromosome segregation fails when GSP-3/4 are absent in sperm and result in infertility. Although GSP-3/4 are essential for proper sperm meiotic chromosome segregation, their phosphoregulatory relationship at the kinetochore and cohesion remains largely unknown. Thus, we hypothesize that GSP-3/4 are dually responsible for regulating kinetochore and cohesion regions by opposing kinase activities at these sites. Here, we show that GSP-3/4 are essential for proper localization of BUB-1, a kinase located at the outer kinetochore during sperm meiosis. Furthermore, we show that GSP-3/4 are not required for proper localization of AIR-2, a microtubule and cohesion destabilizing kinase, during sperm formation, suggesting that removal of cohesion should be unimpaired, yet sister chromatids fail to separate during meiosis II. Lastly, preliminary results suggest that PLK-1, a kinase that acts at the kinetochore and cohesion regions as sperm develop, is not affected by GSP-3/4, suggesting that GSP-3/4 activity is restricted around kinetochore and cohesion during sperm meiosis. Together, these results elucidate the phosphoregulatory relationships at the kinetochore and cohesion during the male meiotic event.

    FRI-G5 BIOCHEMICAL DISSECTION OF THE MLKL-MEDIATED EXECUTION OF NECROPTOSIS

    • Eduardo Reynoso Moreno ;
    • Zhigao Wang ;

    FRI-G5

    BIOCHEMICAL DISSECTION OF THE MLKL-MEDIATED EXECUTION OF NECROPTOSIS

    Eduardo Reynoso Moreno, Zhigao Wang.

    The University of Texas Southwestern Medical Center, Dallas, TX.

    Necroptosis is a tightly regulated cell death program that has garnered considerable attention in recent years for its implication in several human diseases associated with infection, inflammation, ischemic injury, and neurodegeneration. However, despite its clinical relevance, the mechanistic details of the necroptotic pathway remain poorly understood. Several clues about this process have been revealed through investigation of the TNFα-induced cell death pathway. These studies have led to the identification of key regulators of necroptosis including RIP1, RIP3, and MLKL which form the core of the death-inducing complex known as the necrosome. Recently, accumulating evidence has pointed to MLKL as the main executioner of necroptosis. Upon phosphorylation by RIP3, MLKL forms large oligomeric complexes that translocate from the cytosol to membrane-rich cellular compartments where it interacts with various types of lipids. These observations indicate that MLKL targets membranous structures, providing a potential mechanism as to how necrotic cells lose the integrity of their membranes, a classic hallmark of necrosis. Interestingly, our preliminary data indicate that lysosomal membranes are ruptured during necroptosis. Therefore, we hypothesized that lysosome rupture and subsequent release of damaging, non-discriminant hydrolytic enzymes is a critical step of MLKL-mediated execution. To investigate this process, we have been developing an in vitro biochemical assay to study the membrane-disrupting function of MLKL. The development of this assay will help elucidate the executioner function of MLKL as well as help identify downstream mediators of necroptosis that can potentially serve as therapeutic targets for treating necrosis-associated diseases.

    FRI-G13 THE CONTRIBUTION OF MITOCHONDRIAL CALCIUM-SIGNALING PROTEINS STEP1 AND ENKURIN IN THE LYTIC CYCLE OF TOXOPLASMA GONDII

    • Elizabeth Gray ;
    • Marc-Jan Gubbels ;

    FRI-G13

    THE CONTRIBUTION OF MITOCHONDRIAL CALCIUM-SIGNALING PROTEINS STEP1 AND ENKURIN IN THE LYTIC CYCLE OF TOXOPLASMA GONDII

    Elizabeth Gray, Marc-Jan Gubbels.

    Boston College, Chestnut Hill, MA.

    Toxoplasma gondii is a ubiquitous intracellular parasite that can cause the life-threatening disease toxoplasmosis. Critical for the pathogenic stage of the parasite is the lytic cycle. Calcium signaling is critical to each step of the lytic cycle, yet the specifics behind many of these calcium signaling pathways are still being explored. Of particular note, there is a dearth in knowledge of the role the parasite mitochondrion, a known source of calcium, plays in the lytic cycle. Using the Toxoplasma genome database, we identified 2 proteins with hallmarks of calcium signaling proteins, STEP1 and Enkurin, that we hypothesize contribute to the regulation of mitochondrial calcium. STEP1 is a constitutive, highly expressed protein with 3 genuine calcium-binding EF-hand domains found only in apicomplexans. Another protein of interest, Enkurin, is found broadly in eukaryotes and is known in mammals to interact with the calcium-sensing protein calmodulin and the transient receptor potential canonical (TRPC) cation channel. Each protein was fused to a C-terminal fluorescent tag and found to localize to the mitochondrion in live parasites. Through functional dissection and binding partner identification of these 2 proteins, we seek to understand the connection between the mitochondrion and calcium signaling during the lytic cycle. This understanding of the lytic cycle will contribute in the future to improved outcomes for those afflicted with toxoplasmosis.

    FRI-G15 STIFF SKIN SYNDROME: DETERMINING THE RELATIONSHIP BETWEEN FIBRILLIN-1 AND RGD-BINDING INTEGRINS

    • Joselyn Del Cid ;
    • Dean Sheppard ;

    FRI-G15

    STIFF SKIN SYNDROME: DETERMINING THE RELATIONSHIP BETWEEN FIBRILLIN-1 AND RGD-BINDING INTEGRINS

    Joselyn Del Cid, Dean Sheppard.

    University of California, San Francisco, San Francisco, CA

    Systemic sclerosis (SSc) is an autoimmune disease in which healthy adults develop fibrosis of the skin and internal organs. Due to the difficulty of studying SSc, research has focused on studying the more genetically tractable form of scleroderma, stiff skin syndrome (SSS). SSS is a rare but autosomal dominant form of scleroderma that was initially described in 1971. The fibrosis phenotype has been mapped to specific mutations in the sole Arg-Gly-Asp (RGD) sequence-containing domain of fibrillin-1. Fibrillin-1 is a 350 kda extracellular glycoprotein that is the major structural component of microfibrils and helps initiate elastic fiber assembly. Fibrillin-1-mediated adhesion of fibroblasts is regulated by integrin binding to the RGD motif in the fourth TB domain. Fibrillin-1 binding to integrins has been demonstrated for αvβ3, α5β1, and αvβ6 integrins, but these findings were shown to be cell-type specific. Our data shows that most RGD-binding integrins mediate adhesion to fibrillin-1 at high coating concentrations. Adhesion to low concentrations appears to be mediated primarily by αvβ3 and αvβ1. Furthermore, adhesion to the human Trp to Cys mutated fibrillin-1 fragment (WC) is abrogated for cell lines expressing only α5β1 and αvβ5. In contrast, αvβ6 and αvβ3 bind strongly to the WC mutant, suggesting that the structural changes imposed by the mutation are not recognized by all RGD-binding integrins. The goal of this work is to determine the relationship between fibrillin-1 and RGD-binding integrins. Understanding how the mutation changes the dynamics of integrin binding could highlight a mechanism for the development of therapeutics in SSc.

    THU-G15 CHARACTERIZATION OF TRANSCRIPTIONAL CONTROL OF DERG, A HOMOLOG OF A CAUSATIVE GENE FOR LONG QT SYNDROME

    • Erick Palacios ;
    • Enrique Massa ;
    • James Bolton ;
    • Rudolf Bohm ;

    THU-G15

    CHARACTERIZATION OF TRANSCRIPTIONAL CONTROL OF DERG, A HOMOLOG OF A CAUSATIVE GENE FOR LONG QT SYNDROME

    Erick Palacios, Enrique Massa, James Bolton, Rudolf Bohm.

    Texas A&M University-Kingsville, Kingsville, TX

    Neuronal and cardiac cell activity are regulated by various types of voltage-gated ion channels that conduct sodium, calcium, chloride, or potassium ions. The hERG channel is a voltage-gated K+ channel crucially involved in neuronal and cardiac repolarization; thus, mutation in this ion channel could render cells hyperexcitable by altering refractory time and shifting resting potential. We are investigating the Drosophila melanogaster ortholog of this mutant channel-mutant seizure, by measuring and quantifying seizure-like behaviors and characterizing the transcriptional regulation of these channels. In these studies, we constructed serially deleted regions of the promoters for DERG that should drive expression of the DERG mRNA and subsequent rescue of the seizure mutant phenotype. The transgenic flies, bearing the different serial deletions, will be evaluated for rescue of the DERG mutant phenotype, which in turn will aid in localization of the regulatory elements necessary for the expression of DERG in the nervous system.