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In acute myeloid leukemia (AML), abnormal myeloid precursors accumulate in the bone marrow and blood. Most AML patients die within 1 year of diagnosis. Unfortunately, current therapies do not effectively kill AML cells in the bone marrow, thus new therapies are needed. Our lab recently found that signaling by SDF-1/CXCR4, a chemokine abundant in the bone marrow and its chemokine receptor expressed on most AML cells, causes apoptosis of AML cells via a mechanism that is normally inhibited in the bone marrow by nearby osteoblasts. We hypothesized that, for AML cells expressing high levels of CXCR4, the C-terminus of CXCR4 mediates SDF-1–dependent apoptosis via either G proteins or non-G protein mechanisms. Using the human AML cell line KG1a in vitro and molecular genetic approaches, we found that both the CXCR4 C-terminus and PKC activity were necessary for the SDF-1/CXCR4dependent apoptosis of AML cells. In contrast, Gαi, Src, PLC activation, and β-arrestin1 are not required. Ongoing research aims to identify other molecules required for this pathway. Elucidating the mechanisms of SDF-1/CXCR4 apoptosis could permit the design of more effective therapies that treat AML by enhancing the death of AML cells in the bone marrow via endogenous SDF-1/CXCR4.
ROLES OF MACROPHAGES AND BIGH3 IN RETINAL MICROVASCULAR CELLSFatemeh Reza Poor, Richard LeBaron.
University of Texas at San Antonio, San Antonio, TX.
Diabetic retinopathy (DR) is the leading cause of blindness in the United States. An early hallmark of DR is the loss of pericytes, which help to maintain the integrity of the blood vessels. The underlying cause for pericyte dropout is not known, although it can lead to microaneurysms, hypoxia, and damaging angiogenesis. We propose a novel pathway in which the secretion of TGFβ-Induced Gene Human Clone 3 (BIGH3), a pro-apoptotic protein, is upregulated by macrophage-derived TGFβ in the retina of diabetic patients. Immunohistochemistry (IHC) was performed to compare levels of BIGH3 and macrophage infiltration in the eyes of healthy and diabetic mice. We studied BIGH3 expression and protein upregulation by rhesus retinal endothelial cells (RhREC) and human retinal pericytes (RPC) in response to exogenous TGFβ and medium conditioned by macrophages that were normal, called macrophage-conditioned medium (MCM), or macrophages precultured in high glucose and high LDL diabetic conditions, called diabetic macrophage-conditioned medium (dMCM). IHC showed increased macrophage infiltration and BIGH3 localization in eyes from diabetic mice as compared with the normal mice. BIGH3 expression was increased in RhRECs and RPCs in response to dMCM, and TGFβ increased BIGH3 expression. Polyclonal BIGH3 antibodies blocked apoptosis. We found that TGFβ secreted by diabetic macrophages upregulated BIGH3 expression and promoted BIGH3-dependent apoptosis in RPCs and RhRECs. This mechanism may contribute to pericyte loss and microvascular damage seen in the initial stages of diabetic retinopathy and could lead to better understanding of this disease.
IFN-ϒ IS PROTECTIVE IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS BY LIMITING LIPID
PEROXIDATION DAMAGE TO MYELIN SHEATHSRachel Robinson1, Rebecca Sosa2, Thomas Forsthuber2.
Texas Lutheran University, Seguin, TX, 2University of Texas at San Antonio, San Antonio, TX.
1 Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS), affecting over 400,000 individuals in the U.S. MS is characterized by demyelination and inflammation of the CNS. Proinflammatory cytokines are believed to promote disease in MS and its animal model experimental autoimmune encephalomyelitis (EAE), whereas the role of IFN-γ is less clear. Higher levels of IFN-γ are found in the CNS of MS patients as compared with healthy individuals, yet mice genetically deficient in IFN-γ or its receptor (IFN-γ-/- or IFN-γR-/-) develop more severe EAE than wild type (Wt) mice, arguing for a protective role of this cytokine. We previously found that increased EAE severity in IFN-γ-/- and IFN-γR-/- mice corresponded to an increase in myelin debris in CNS lesions.
Since it is believed that peroxidation of myelin lipoproteins may contribute to disease severity in MS, we hypothesize that IFN-γ may play a protective role in EAE/MS by limiting lipid peroxidation of myelin debris. We will test this hypothesis by inducing EAE in IFN-γR-/- and Wt mice and determining whether treatment with inhibitors of lipid peroxidation ameliorates clinical disease. Treatment effects on lipid peroxidation of myelin debris, demyelination, and inflammation will be corroborated in CNS tissue by confocal microscopy, flow cytometry, and thiobarbituric acid reactive substances (TBARS) assay. We anticipate that treatment with lipid peroxidation inhibitors will ameliorate EAE in IFN-γR-/- to Wt levels and decrease lipid peroxidation of CNS tissue. If correct, inhibitors of lipid peroxidation could open up new avenues for the treatment of multiple sclerosis.
USING PHAGE DISPLAY TO PRODUCE ANTIBODIES AGAINST BOVINE SERUM ALBUMINRobin Kaai, Thomas Premeaux, Matthew Tuthill.
Kapi`olani Community College, Honolulu, HI.
Current methods for monoclonal antibody production utilize hybridoma technology. This requires the repeated immunization of mice followed by the harvest and fusion of spleen cells with an immortalized cell line in order to produce and select for antigen-specific antibody-producing clones. Because of the cost, time, and expertise required for traditional monoclonal production, we are now investigating the efficacy of using phage display to generate novel antibodies. The aim of this study is to develop proficiency in phage display and allow for a supplemental approach to produce antibodies at our campus Monoclonal Antibody Training and Service Center. Phage display utilizes a library of modified bacteriophages that display single-chain variable fragments (ScFvs) of diverse specificities. Through repeated antigen-targeted biopanning and bacterial transformation, target phages with the desired specificity can then be isolated and amplified. Thus far, we have been successful in replicating the phagemid library and have performed various infection assays to validate the phage library as well as the background genotypes of the vectors involved.
Initial studies will target bovine serum albumin (BSA), a serum protein derived from cow blood which functions in vivo as a carrier protein for steroids, fatty acids, and thyroid hormones. Once successful phages have been isolated, amplified, and banked, this technology will allow for epitopic tagging of antigens, immunoprecipitation, antigen isolation and characterization, and recombinant modifications of antibody variable regions. Moreover, this platform will allow us to increase antibody output, expand to more topical targets, involve more students in antibody production, and incorporate components of the process into select lab courses.
DEVELOPMENTAL ROLE OF THE TRANSCRIPTION FACTOR ZBTB24 IN DANIO RERIOGladys Rimkus, Seth Frietze.
Northern New Mexico College, Espanola, NM.
Transcription factors are proteins that are responsible for the initiation and regulation of transcribing gene sequences from DNA into mRNA by binding to a sequence-specific DNA region. Mutations in humans of the transcription factor ZBTB24 are associated with facial-cranial anomalies, immunodeficiency, and other abnormalities. Because this transcription factor is conserved in Danio rerio (zebrafish), we hypothesize that a knockdown of transcription factor ZBTB24 in zebrafish embryos will result in defective developmental characteristics resulting in phenotypic abnormalities. Accordingly, by injecting embryos in the 1, 2, or 4-cell stage of development with the ZBTB24-blocking morpholinos, we expect it will negatively affect the development of the zebrafish. Observing the growing Danio rerio until maturity, phenotypic characteristics will be recorded and compared to wild-type danios. We will also use a yeast
REDUCTION OF FIBROSIS IN TYPE 2 DIABETES BY (-)-EPICATECHINLila Peltekian, Katrina Yamazaki.
California State University, Los Angeles, Los Angeles, CA.
Fibrosis, the buildup of connective tissue (mainly collagen), decreases the contractility and elasticity of the heart which can lead to heart failure. In this study, we looked at the effects of (-)-epicatechin (EPI) on reducing the progression of type 2 diabetes and fibrosis in vivo. Type 2 diabetes was induced by feeding rats a high energy diet (HED) consisting of 10% lard and 20% glucose. After 4 weeks, a low dose streptozotocin (30 mg/kg) was given IP to cause partial dysfunction of pancreatic β-cells and suppress insulin secretion. Control animals were maintained on normal chow and received an IP injection of vehicle (water). Animals were also treated with EPI (1 mg/kg/day) or water by oral gavage. Results demonstrated that diabetic animals had significant weight gain (~44%) and increased blood glucose levels (519 mg/dL) compared to control (37% and 185 mg/dL, respectively). EPI significantly reduced changes in body weight (~33%) and blood glucose levels (351.2 mg/dL) compared to diabetic animals. Histological analysis elevated collagen levels in diabetic hearts compared to control and EPI-treated animals. In conclusion, our results demonstrate the ability of EPI to reduce body weight, plasma glucose levels, and fibrosis in type 2 diabetes. (This study was supported and funded by CSU-LSAMP which is supported by the National Science Foundation under Grant # HRDand the CSU Office of the Chancellor.) SAT-499
MOLECULAR CLONING OF CHONDROITINASE ABC FROM PROTEUS VULGARIS FOR USE IN
GLYCOSAMINOGLYCAN RESEARCHOrlando Antelope, April Joice, Caitlin Mencio, Kuberan Balagurunathan.
University of Utah, Salt Lake City, UT.
Many biological processes involve the use of glycosaminoglycans (GAGs), which are composed of alternating hexosamine and uronic acid residues. Chondroitin sulfate (CS), belonging to a subset of GAGs called galactosaminoglycans (GalAGs), is known to inhibit nerve regeneration following an acute injury to the central nervous system (CNS). The enzyme chondroitinase ABC (ChABC) depolymerizes CS into tetrasaccharides and disaccharides.
The removal of CS polysaccharides at the sight of CNS injury promotes the recovery of the adjacent neurons. This project focuses on the cloning of ChABC, which is used in the lab for further research into nerve regeneration. Using Proteus vulgaris genomic DNA as a template, a forward primer was designed to include an XhoI restriction site, and the reverse primer was designed to include a BamHI restriction site. The ChABC DNA was amplified and cloned into the pET-19b vector. Efforts are currently under way to express, purify, characterize, and utilize this current enzyme.
This technique, along with other variations, will then be used to efficiently produce other pure GAG enzymes. Results will be accessible in this presentation.
POLY (N-ISOPROPYL ACRYLAMIDE)-COATED SURFACES: INVESTIGATION OF CYTOTOXICITY OF CPNIPAMLyndsay Stapleton, Heather Canavan.
Center for Biomedical Engineering, University of New Mexico, Albuquerque, NM.
Poly (N-isopropyl acrylamide) (pNIPAM) is a thermoresponsive polymer that undergoes a phase change at a physiologically relevant temperature range, which leads to cell release. Above its lower critical solution temperature (LCST approximately 32 °C), pNIPAM is relatively hydrophobic and mammalian cells can be cultured on pNIPAMgrafted surfaces. When the temperature is lowered below the LCST, the polymer is hydrophilic and becomes hydrated. In this state, its chains become more extended and cells detach as intact cell sheets. These cell sheets can then be used to engineer tissues. Before the detached cell sheets can be used on humans, the cytotoxicity of the surfaces must be assessed. In recent studies, cytotoxicity testing showed an abnormal increase in the cytotoxicity of the polymer cpNIPAM. In this work, we investigate the reasons behind this anomaly. The cpNIPAM-coated surfaces
53 UNDERGRADUATE POSTER ABSTRACTS
were evaluated for their thermoresponse and surface chemistry using standard surface science techniques (e.g., goniometry, X-ray photoelectron spectroscopy). The relative biocompatibility of the substrates was evaluated using bovine aortic endothelial cells and MTS, live/dead, plating efficiency. We find that the diminished cell viability of BAECs exposed to cpNIPAM substrates is due to a combination of factors, including the inclusion of short chain length polymers, the presence of unreacted catalyst, and other factors. This work will provide valuable insights into the cytotoxicity of cpNIPAM-coated surfaces, and therefore, into the applicability for human subjects of cells grown on this surface.
MEASUREMENT OF RELATIVE TELOMERE REPEATS IN HUMAN CANCER CELL LINES USING A
MONOCHROME MULTIPLEX QUANTITATIVE PCR ASSAYAllison Gomez1, Daniel Edelman2, David Petersen2, Paul Meltzer2.
National Cancer Institute, Los Angeles, CA, 2National Cancer Institute, Bethesda, MD.