Session two genetics cellular and molecular biology I.
EFFECT OF A FUNCTIONAL PBI-F2 PROTEIN ON VIRAL RNA EXPRESSIONS OF THE 2009 PANDEMIC HINI VIRUS. Mary J. Dickey *, Christian Brothers University, Memphis. Tennessee (MD), and St. Jude Children's Research Hospital, Memphis, Tennessee (HM, RW), The PB1-F2 protein has previously been attributed to increased pathogenicity of influenza A viruses. The currently circulating 2009 pandemic H1N1 strains have 3 stop codons in the open reading frame of its PB1-F2 protein rendering it nonfunctional. Our goal was to investigate the role of a functional PB1-F2 protein in viral RNA expression levels of the 2009 pandemic HlNl virus. RNA samples isolated from cells infected with recombinant viruses generated from two backbones representative of the seasonal influenza virus and the 2009 pandemic H1N1 virus were analyzed by primer extension assays to detect the amount of viral RNAs. Our findings show that cells infected with a functional PB1-F2 resulted in increased viral RNA expression levels. It can therefore be inferred that obtainment of a functional PB1-F2 protein from another influenza A virus would result in increased pathogenicity of the currently circulating 2009 pandemic H1N1 virus.
DETERMINATION OF THERAPEUTIC TRANSGENE DOSE. Rachel E. Haag *, Jessica Hines-Beard, Anand Patel, Siddarth Desai, and Tonia Rex, Christian Brothers University, Memphis, Tennessee, and The University of Tennessee Health Science Center Hamilton Eye Institute. Memphis, Tennessee. Erythropoietin (EPO), a transgene, can be used in gene therapy, but levels of expression need to be controlled in order to deliver therapeutic levels. We tested an inducible promoter (tet) and assessed levels of transgene expression (enhanced green fluorescent protein, EGFP, nuorescence) and protection in the retinal degeneration slow (rds) mouse after treatment with different amounts of inducer (doxyeycline). Transgenic rds mice were injected subretinally with a recombinant adeno-associated virus serotype 2/1 containing tet driving expression of eGFP and Epo (rAAV2/1.tet.eGFP.Epo) and then treated with different doses of doxyeycline water. EGFP fluorescence was measured at PD30, and 60, and the outer nuclear layer thickness was measured. EGFP fluorescence intensity increased with increasing concentration of doxycycline and fluorescence increased with duration of treatment. Transgene production of EPO was controlled in a dose dependent manner and a therapeutic level was identified.
ANALYSIS OF TRANSIENT RECEPTOR POTENTIAL VA-NTLLOID 1 RECEPTOR EXPRESSION IN Y-79 RETINOBLASTOMA CELLS. Dominique Garcia Robles *, Luiz R. G. Britto, and Mauro Leonelli, Christian Brothers University, Memphis. Tennessee. and Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo, Brazil. The study aimed to evaluate the receptor expression of TRPV1 on human retinoblastoma cell line Y-79, as well as identifying the presence of the synthesizing enzyme of two nitric oxide synthase (NOS) isoforms. Immunocytochemistry of Y-79 cells revealed TRPV1 expression and real-time PCR further supported these findings with the presence of RNA expression for TRPV1. Previously extracted rat retina samples were paired with Y-79 cells and immunocytochemistry techniques used to determine the presence of NOS enzyme colocalization with TRPV1. Immunocytochemistry indicated the presence of one NOS isoform enzyme in both the retina and Y-79 cells. Some of the NOS positive cells also were positive for TRPV1. Activation of TRPV1 leads to an influx of calcium and .sodium ions, while the endothelial NOS (eNOS) and neuronal NOS (nNOS) pathways are activated in the presence of calcium ions.
USING GLAUCOMA-RELATED PHENOTYPES OF MICE TO IDENTIFY GENES THAT CONTROL THE SEVERITY OF GLAUCOMA. Natalie E. Hurt * and Monica Jablonski, Christian Brothers University. Memphis. Tennessee, and The University of Tennessee Health Science Center Hamilton Eye Institute, Memphis. Tennessee. Glaucoma is a set of eye diseases that damages the optic nerve, resulting in vision loss. BXD inbred mice develop glaucoma similar to pigment dispersion glaucoma in humans. The mice develop varying degrees of nerve cell damage characteristic of glaucoma. The mice have different phenotypes of severity compared to other mice the same age. The severities can be used to locate genes responsible for the phenotypes. The optic nerves of the mice were removed, processed, and prepared for slides. The slides were scanned into the computer and categorized into five phenotypes of increasing severity of optic nerve damage. Preliminary results showed that five different phenotypes of severity existed in the mice as expected. With these phenotypes, the chromosomal location of the genes responsible for severity of glaucoma can be found with ongoing research and matched with a corresponding gene in humans in order lo provide a better understanding of glaucoma.
ANALYSIS OF THE FREQUENCY OF CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) GENE MUTATIONS IN THE AFRICAN-AMERICAN POPULATION IN MEMPHIS. Matthew G. Charles * and Anjaparavanda P. Naren, Christian Brothers University. Memphis. Tennessee, and The University of Memphis Health Science Center, Memphis, Tennessee. Mutations in the Cystic Fibrosis transmembrane conductance regulator (CFTR) gene cause the genetic disease, Cystic Fibrosis (CF). CF is a lethal autosomal recessive disease most common among Caucasians, yet its increasing incidence among other ethnic groups is significant. The goal of this project was lo analyze the CFTR mutation distribution in African American CF-patients in the Memphis area and to characterize the mutant proteins at the molecular level. The study showed that among the CF patients in the Memphis area, 71% of African Americans have the deltaF508 mutation in allele 1, and uniquely, none of the patients have deltaF508 mutation in allele 2 (n = 21), demonstrating a very unique CFTR mutation distribution. Among the Caucasian CF patients, 86% have deltaF508 in allele 1 and 60% have deltaF508 in allele 2 (n = 108). This study will contribute to the data on CFTR mutation frequency among African Americans in the U.S.
THAP1 SEQUENCE VARIANTS IN DYSTONIA PATIENTS IN POLISH COHORT. Carrie A. Le *, Jiangfeng Xiao, and Mark S. Ledoux. Christian Brothers University, Memphis, Tennessee (CL), and The University of Tennessee Health Science Center, Memphis, Tennessee (JX, ML). Sequence variants in the THAP1 gene have been found to be associated with DYT6 dystonia. THAP1 encodes a transcription factor that contains a conserved zinc-dependent DNA-binding domain which when mutated causes transcriptional dysregulation. In this study, 284 dystonia patients of Caucasian European descent were genotyped for sequence variants in all three exons and its intron boundary of the THAP1 gene using both the high-resolution melting and direct sequencing protocols. A c.238A>G variant in exon 2 were found in a 50-year-old male patient with cervical dystonia whose symptom started 5yearsago. Substitutions of e.71+126T>C in intron 1 of THAP1 were found in 25 dystonia subjects, while another 13 subjects had a sequence variant located in the 5'-untranslatcd region of the c.-237_236GA>TT. We revealed that only the c.238A>G variant may contribute to the cause of dystonia.
CYTOCHROME P450 1B1 GENE DISRUPTION PROTECTS AGAINST RENAL DYSHOMEOSTASIS AND DAMAGE ASSOCIATED WITH ANGIOTENSIN II INFUSION IN MICE. Larry J. Anderson *, Brett L. Jennings, Jason Porter, Anne M. Estes, and Kafait U. Mailk, Christian Brothers University, Memphis, Tennessee (LA), and The University of Tennessee Health Science Center. Memphis, Tennessee (BJ, JP, AE, KM). This study investigated the contribution of cytochrome P4501B1 (CYP1B1) to angiotensin II (Ang-II)-induced hypertension and associated renal pathophysiology. Infusion of Ang-II (1 mg/kg/day) for 14 d increased blood pressure in Cyp[1b1.sup.+/+] mice, which was reduced in Cyp[1b1.sup.-/-] mice. Ang-II-induced hypertension was associated with increased renal CYP1B1 and NADPH oxidase activity. Ang-II infusion increased water consumption and urine volume in Cyp[1b1.sup.+/+], but not Cyp[1b1.sup.-/-] mice. Urine analysis revealed alterations in osmolality, sodium and potassium excretion, proteinuria, and albuminuria, indicating renal dysfunction in Ang-II-infused Cyp[1b1.sup.+/+], but not Cyp[1b1.sup.-/-] mice. Immunohistological analysis revealed interstitial fibrosis and vascular hypertrophy, as demonstrated by increased [alpha]-smooth muscle actin staining in Ang-II-infused Cyp[1b1.sup.+/+], but not Cyp[1b1.sup.-/-] mice. Cyp[1b1.sup.+/+] mice infused with Ang-II displayed decreased renal blood Mow, increased renal vascular reactivity and resistance, and endothelial dysfunction that was absent in Cyp[1b1.sup.-/-] mice. These data suggest that renal dyshomeostasis associated with Ang II-induced hypertension is dependent on CYP1B1 activity.
DR. JAMES FELLS, MODERATOR
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|Title Annotation:||ORAL PRESENTATIONS; glaucoma; avian viruses in Western Africa; renal dyshomeostasis|
|Publication:||Journal of the Tennessee Academy of Science|
|Date:||Sep 1, 2011|
|Previous Article:||Session one animal behavior and ecology.|
|Next Article:||Session three A clinically related investigations.|