DOCSLIB.ORG

RNA Isolation and Quantitative Analyses of Gabra3 Editing/Expression

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
RNA Isolation and Quantitative Analyses of Gabra3 Editing/Expression SUPPLEMENTAL METHODS RNA isolation and quantitative analyses of Gabra3 editing/expression RNA was isolated and purified from mouse brain using Versagene RNA tissue and DNase kits (Gentra Systems, Minneapolis, MN) according to the manufacturer’s instructions. Unless otherwise specified, all brain tissues were isolated from 129S6 (12- 14 week) or FVB/J (12 week) male mice. Adult rat whole brain RNA (Sprague-Dawley) was isolated as previously described (Burns et al., 1997) and human whole brain RNA was purchased from BioChain Institute, Inc. (Hayward, CA). Complementary DNA was generated from 2 µg of total RNA using random priming with AMV reverse transcriptase (Promega, Madison, WI). PCR amplification was performed with specific sense and antisense primers designed from GenBank sequences (Gabra1, NM_010250; Gabra2, NM_008066; Gabra3, NM_008067; Gabra4, NM_010251; Gabra5, NM_176942; Gabra6, NM_008068) to generate amplicons corresponding to the entire region of the predicted I/M site duplex within Gabra3 transcripts or the corresponding region for other GABAA subunit isoforms (Supplementary Table 1). Bulk sequence analysis of PCR products was performed with an internal primer using BigDye Terminator chemistry and an ABI 3730xl analyzer (Applied Biosystems). To quantify the extent of editing in mouse brain RNA, RT-PCR products were subcloned into pBKSII+ (Stratagene, La Jolla, CA) and DNA was extracted by heat lysis for use as the template for a custom-designed TaqMan SNP genotyping assay (Supplementary Table 1; Applied Biosystems). Post-run analysis was performed with an ABI Prism 7900HT system (SDS v2.3; Applied Biosystems). For quantitative analysis of Gabra3 mRNA expression, total RNA from whole mouse brain or dissected brain regions was reverse transcribed with random primers and the generated cDNAs were used as templates in a TaqMan MGB gene expression assay for Gabra3 (Mm00433440_m1) with 18S rRNA as an endogenous control (product no. 4319413E) from Applied Biosystems. Real-time PCR and subsequent analyses were performed using an ABI prism 7900HT (SDS v2.3; Applied Biosystems), and all reactions were run in triplicate and quantified as described previously (Feng et al., 2006). Minigene construction and analysis A wild-type Gabra3 minigene (543 bp), extending from a region in exon 6 through exon 9, was constructed by subcloning a non-edited Gabra3 amplicon generated from E15 mouse brain RNA into the pEGFP-C1 expression plasmid (Invitrogen, Carlsbad, CA). Mutagenesis was performed by PCR with Pfu Ultra (Stratagene, La Jolla, CA) in the presence of 5% DMSO. Template DNA was digested with DpnI (New England Biolabs, Ipswich, MA) and the PCR product was transformed into DH5α- competent cells; all constructs were verified by DNA sequence analysis. 60-mm plates of HeLa cells, cultured in AMEM with 10% (v/v) FBS, were cotransfected using FuGENE 6 (Roche, Indianapolis, IN) with 0.5 µg of a Gabra3 minigene along with 4µg of either an empty pRC-CMV expression vector (Promega, Madison, WI) or pRC-CMV containing the full-length cDNA encoding rat ADAR1 (p150) or rat ADAR2b, each with a FLAG epitope tag on the 5’-end. Cells were harvested 36 hours after transfection, RNA was isolated and purified using Versagene RNA cell and DNase kits (Gentra Systems) and whole cell extracts were prepared from a parallel sample for Western blotting analysis, as described previously (Schreiber et al., 1989). Western blotting analysis to quantify ADAR1/ADAR2 expression was performed using an anti-FLAG M2 monoclonal antibody (1:1000 v/v, Sigma-Aldrich, F3165) and an anti-β-tubulin polyclonal antibody (1:1000 v/v; Santa Cruz Biotechnology, sc-9104), detected with an Alexa Fluor 680-conjugated anti-rabbit (Molecular Probes, A21109) or IRDye800-conjugated anti-mouse secondary antibody (Rockland, 610-732-124), respectively. Fluorescence was quantified using an Odyssey infrared imaging system (LI- COR Biotechnology). Analysis of editing was quantified by bulk sequence analysis of RT-PCR amplicons, generated as described above, but using 0.5 mg of RNA template and a transcript-specific RT primer containing a unique tail that was used to prime the PCR reaction (Supplementary Table 2), as described previously (Rueter et al., 1995). Quantification was performed directly from electropherogram traces using a method adapted from Kwok et al. (Kwok et al., 1994) demonstrating that relative nucleotide peak heights are maintained from trace-to-trace when comparing the same region of a DNA sequence. The analysis was performed by dividing the height of the A peak at the editing site (I/M site) in a minigene sequence by an average of the heights of the three preceding adenosine peaks to provide a height normalized to the signal of that specific trace. A parallel analysis was performed on a control PCR amplicon generated from a non-edited Gabra3 plasmid. The percentage of non-edited RNA in the population was calculated by dividing the normalized A peak height from a transfected sample by the normalized height from the non-edited plasmid control sample. Expression of recombinant GABAA receptors Complementary DNAs encoding the human GABAA receptor α3(I), α3(M) and γ2L subunits were individually subcloned into the mammalian expression vector pcDNA3.1 (Invitrogen, Carlsbad, CA); cDNA encoding the human GABAA receptor β3 subunit was subcloned into pCMV6-XL5 (Origene, Rockville, MD). HEK293T cells were transfected using a calcium phosphate precipitation technique, as previously described (Lagrange et al., 2007). Cells were transfected with a total of 12 µg GABAA receptor subunit, 4 µg of each subunit plasmid (ratio 1:1:1) as well as 2 µg of pHook-1 (Invitrogen, Carlsbad, CA) so that immunomagnetic bead selection could be performed on day two. Following selection, the cells were plated on 35 mm dishes, and recordings were made on day three, approximately 18-36 h after selection. For editing analysis, RNA was isolated from HEK293T cells 2 days post- transfection using TRI Reagent (Molecular Research Center) and RT-PCR and bulk sequence analysis was performed as described for Gabra3 minigene analysis. Electrophysiological recording and drug application Whole cell voltage-clamp recordings were performed on transiently transfected HEK293T cells. Cells were bathed in an external solution consisting of (in mM): NaCl 142, CaCl2 1, KCl 8 MgCl2 6, Glucose 10, HEPES 10, (pH 7.4, ≈320 - 340 mOsm) throughout the duration of the experiment. All recordings were done at room temperature. Glass micropipettes were formed from thin-walled borosilicate glass with a filament (World Precision Instruments, Sarasota, FL) with a P2000 laser electrode puller (Sutter Instruments, San Rafael, CA) and fire polished with a microforge (Narishige, East Meadow, NY). Microelectrodes used for lifted cell recording had resistances of 0.7- 1.6 MΩ when filled with an internal solution consisting of (in mM): KCl 153, MgCl2 1, 2+ HEPES 10, EGTA 5, Mg -ATP 2 (pH 7.3, ≈ 300 - 310 mOsm) to produce an ECl of approximately 0 mV. Membrane voltages were clamped at –20 mV using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA) amplifier. GABA was applied to lifted cells with a fast drug application device in which a Warner SF-77B Perfusion Fast-Step (Warner Instrument Corporation, Hamden, CT) motor was used to rapidly translate a multibarrel square glass pipette (Friedrich and Dimmock, Millville, NJ), resulting in open-tip solution exchange times consistently ≤ 800 µs and solution exchange time around lifted cells of ≈ 1 ms(Lagrange et al., 2007). To minimize the error induced by uncompensated series resistance error, all currents larger than 6 nA were excluded from kinetic analysis. Considering the low access resistance (generally 2-3 MΩ) and small cells (membrane capacitance usually 8-15 pF), this cutoff amplitude was expected to keep the membrane potential error difference between the groups to a minimum (≤ 2-3 mV). We found no correlation between current amplitude and any of the reported kinetic parameters. To confirm these results, all kinetic data were independently analyzed using linear regression to assess the interaction between isoform, current amplitude, and kinetic parameters. All of these fits showed the rise time and deactivation rates to be dependent on the isoform. Finally, an independent statistical analysis of electrophysiological data was done by pairing each current from non-edited receptor with an amplitude-matched (± 15%) partner in the edited receptor group, followed by a Wilcoxon matched pairs test (data not shown). For generation of concentration-response relationships, peak GABAA receptor currents evoked by randomly sequenced concentrations of GABA were recorded with at least 45 s of wash between each application. This time was empirically determined to be sufficient for complete recovery from desensitization (data not shown). The responses were normalized to the current elicited by 1 mM GABA after a prolonged wash in external solution during each sweep. Data were excluded if there was a greater than 15% rundown of the maximal response between sweeps. Data analysis Currents were low-pass filtered at 2 kHz, digitized at 5-10 kHz, and analyzed using the pClamp 9 software suite. For those cells with very small (<50 pA) currents, rise time, desensitization, and deactivation were not determined. Current amplitudes and 10- 90% rise times were measured using the Axon Instruments Clampfit 9 software package. The activation rate was calculated as the inverse of the 10-90% rise time. The desensitization and deactivation time courses of GABAA receptor currents were fit using the Levenberg-Marquardt least squares method with up to six component exponential (-t/τn) functions of the form: Σ ane + C where t is time, n is the best number of exponential components, an is the relative ampl0itude of the nth component, τn is the time constant of the nth component, and C is the residual current at the end of the GABA application. Additional components were accepted only if they significantly improved the fit, as determined by an F-test automatically performed by the analysis software on the sum of squared residuals.
Load more

Recommended publications
  • Cognition and Steroidogenesis in the Rhesus Macaque
    Cognition and Steroidogenesis in the Rhesus Macaque Krystina G Sorwell A DISSERTATION Presented to the Department of Behavioral Neuroscience and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy November 2013 School of Medicine Oregon Health & Science University CERTIFICATE OF APPROVAL This is to certify that the PhD dissertation of Krystina Gerette Sorwell has been approved Henryk Urbanski Mentor/Advisor Steven Kohama Member Kathleen Grant Member Cynthia Bethea Member Deb Finn Member 1 For Lily 2 TABLE OF CONTENTS Acknowledgments ......................................................................................................................................................... 4 List of Figures and Tables ............................................................................................................................................. 7 List of Abbreviations ................................................................................................................................................... 10 Abstract........................................................................................................................................................................ 13 Introduction ................................................................................................................................................................. 15 Part A: Central steroidogenesis and cognition ............................................................................................................
    [Show full text]
  • Editing Modifies the GABAA Receptor Subunit A3
    Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press REPORT Editing modifies the GABAA receptor subunit a3 JOHAN OHLSON,1 JAKOB SKOU PEDERSEN,2 DAVID HAUSSLER,2,3 and MARIE O¨ HMAN1 1Department of Molecular Biology and Functional Genomics, Stockholm University, SE-106 91 Stockholm, Sweden 2Center for Biomolecular Science and Engineering, University of California Santa Cruz, Santa Cruz, California 95064, USA 3Howard Hughes Medical Institute, University of California at Santa Cruz, Santa Cruz, California 95064, USA ABSTRACT Adenosine to inosine (A-to-I) pre-mRNA editing by the ADAR enzyme family has the potential to increase the variety of the proteome. This editing by adenosine deamination is essential in mammals for a functional brain. To detect novel substrates for A-to-I editing we have used an experimental method to find selectively edited sites and combined it with bioinformatic techniques that find stem–loop structures suitable for editing. We present here the first verified editing candidate detected by this screening procedure. We show that Gabra-3, which codes for the a3 subunit of the GABAA receptor, is a substrate for editing by both ADAR1 and ADAR2. Editing of the Gabra-3 mRNA recodes an isoleucine to a methionine. The extent of editing is low at birth but increases with age, reaching close to 100% in the adult brain. We therefore propose that editing of the Gabra-3 mRNA is important for normal brain development. Keywords: RNA editing; adenosine deaminase; ADAR; GABA receptor; Gabra-3 INTRODUCTION At this site a CAG, coding for glutamine, is modified to CIG, which is read as an arginine codon (CGG).
    [Show full text]
  • Rare GABRA3 Variants Are Associated with Epileptic Seizures, Encephalopathy and Dysmorphic Features
    doi:10.1093/brain/awx236 BRAIN 2017: 140; 2879–2894 | 2879 Rare GABRA3 variants are associated with epileptic seizures, encephalopathy and dysmorphic features Cristina Elena Niturad,1,* Dorit Lev,2,3,4,* Vera M. Kalscheuer,5,6,* Agnieszka Charzewska,7 Julian Schubert,1,8 Tally Lerman-Sagie,3,4,9 Hester Y. Kroes,10 Renske Oegema,10 Monica Traverso,11 Nicola Specchio,12 Maria Lassota,13 Jamel Chelly,14 Odeya Bennett-Back,15 Nirit Carmi,3,4,10 Tal Koffler-Brill,16 Michele Iacomino,11 Marina Trivisano,12 Giuseppe Capovilla,17 Pasquale Striano,18 Magdalena Nawara,7 Sylwia Rzon´ca,7 Ute Fischer,5,6 Melanie Bienek,5 Corinna Jensen,5,z Hao Hu,5,§ Holger Thiele,19 Janine Altmu¨ller,19,20 Roland Krause,8 Patrick May,8 Felicitas Becker,1 EuroEPINOMICS Consortium, Rudi Balling,8 Saskia Biskup,21 Stefan A. Haas,22 Peter Nu¨rnberg,19 Koen L. I. van Gassen,10 Holger Lerche,1 Federico Zara,11,* Snezana Maljevic1,*,f and Esther Leshinsky-Silver2,3,16,*,† *These authors contributed equally to this work. †Deceased. Genetic epilepsies are caused by mutations in a range of different genes, many of them encoding ion channels, receptors or transporters. While the number of detected variants and genes increased dramatically in the recent years, pleiotropic effects have also been recognized, revealing that clinical syndromes with various degrees of severity arise from a single gene, a single mutation, or from different mutations showing similar functional defects. Accordingly, several genes coding for GABAA receptor subunits have been linked to a spectrum of benign to severe epileptic disorders and it was shown that a loss of function presents the major correlated pathomechanism.
    [Show full text]
  • Molecular Mechanisms Driving Prostate Cancer Neuroendocrine Differentiation
    Molecular mechanisms driving prostate cancer neuroendocrine differentiation Submitted by Joseph Edward Sutton Supervisory team: Dr Amy Poole (DoS) Dr Jennifer Fraser Dr Gary Hutchison A thesis submitted in partial fulfilment of the requirements of Edinburgh Napier University, for the award of Doctor of Philosophy. October 2019 School of Applied Sciences Edinburgh Napier University Edinburgh Declaration It is hereby declared that this thesis is the result of the author’s original research. It has been composed by the author and has not been previously submitted for examination which has led to the award of a degree. Signed: II Dedication This thesis is dedicated to my grandfather William ‘Harry’ Russell, who died of stomach cancer in 2014. Thank you for always encouraging me to achieve my ambitions, believing in me and for retaining your incredible positivity and sense of humour, even at the very end of your life. III Acknowledgements First of all, I would like to acknowledge my parents, who dedicated so much effort and energy into helping me to achieve my lifelong ambition of becoming a scientist. From taking me to the Natural History and Science Museums in London as a child, to tolerating my obsession with Jurassic Park and continuing to support me in both of your unique yet equally important ways, thank you. I would also like to thank my PhD supervisors Dr Amy Poole and Dr Jenny Fraser, not only for their excellent scientific guidance but also for their great banter and encouragement along the way. Thank you for seeing some potential in me, taking a chance on me and for helping me to continue my scientific journey.
    [Show full text]
  • Gabaergic Signaling Linked to Autophagy Enhances Host Protection Against Intracellular Bacterial Infections
    ARTICLE DOI: 10.1038/s41467-018-06487-5 OPEN GABAergic signaling linked to autophagy enhances host protection against intracellular bacterial infections Jin Kyung Kim1,2,3, Yi Sak Kim1,2,3, Hye-Mi Lee1,3, Hyo Sun Jin4, Chiranjivi Neupane 2,5, Sup Kim1,2,3, Sang-Hee Lee6, Jung-Joon Min7, Miwa Sasai8, Jae-Ho Jeong 9,10, Seong-Kyu Choe11, Jin-Man Kim12, Masahiro Yamamoto8, Hyon E. Choy 9,10, Jin Bong Park 2,5 & Eun-Kyeong Jo1,2,3 1234567890():,; Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain; however, the roles of GABA in antimicrobial host defenses are largely unknown. Here we demonstrate that GABAergic activation enhances antimicrobial responses against intracel- lular bacterial infection. Intracellular bacterial infection decreases GABA levels in vitro in macrophages and in vivo in sera. Treatment of macrophages with GABA or GABAergic drugs promotes autophagy activation, enhances phagosomal maturation and antimicrobial responses against mycobacterial infection. In macrophages, the GABAergic defense is mediated via macrophage type A GABA receptor (GABAAR), intracellular calcium release, and the GABA type A receptor-associated protein-like 1 (GABARAPL1; an Atg8 homolog). Finally, GABAergic inhibition increases bacterial loads in mice and zebrafish in vivo, sug- gesting that the GABAergic defense plays an essential function in metazoan host defenses. Our study identified a previously unappreciated role for GABAergic signaling in linking antibacterial autophagy to enhance host innate defense against intracellular bacterial infection. 1 Department of Microbiology, Chungnam National University School of Medicine, Daejeon 35015, Korea. 2 Department of Medical Science, Chungnam National University School of Medicine, Daejeon 35015, Korea.
    [Show full text]
  • Cross-Tissue Analysis of Gene and Protein Expression in Normal and Cancer Tissues Received: 20 January 2016 Idit Kosti, Nishant Jain, Dvir Aran, Atul J
    www.nature.com/scientificreports OPEN Cross-tissue Analysis of Gene and Protein Expression in Normal and Cancer Tissues Received: 20 January 2016 Idit Kosti, Nishant Jain, Dvir Aran, Atul J. Butte & Marina Sirota Accepted: 30 March 2016 The central dogma of molecular biology describes the translation of genetic information from mRNA Published: 04 May 2016 to protein, but does not specify the quantitation or timing of this process across the genome. We have analyzed protein and gene expression in a diverse set of human tissues. To study concordance and discordance of gene and protein expression, we integrated mass spectrometry data from the Human Proteome Map project and RNA-Seq measurements from the Genotype-Tissue Expression project. We analyzed 16,561 genes and the corresponding proteins in 14 tissue types across nearly 200 samples. A comprehensive tissue- and gene-specific analysis revealed that across the 14 tissues, correlation between mRNA and protein expression was positive and ranged from 0.36 to 0.5. We also identified 1,012 genes whose RNA and protein expression was correlated across all the tissues and examined genes and proteins that were concordantly and discordantly expressed for each tissue of interest. We extended our analysis to look for genes and proteins that were differentially correlated in cancer compared to normal tissues, showing higher levels of correlation in normal tissues. Finally, we explored the implications of these findings in the context of biomarker and drug target discovery. In recent years, techniques used to conduct tissue-wide analysis of gene expression, such as microarrays and RNA sequencing technologies (RNA-Seq), have become widely used1,2.
    [Show full text]
  • Characterisation of GABAA Receptors and Cation-Chloride Cotransporters in the Uterus and Their Role in Pre-Term Labour
    Characterisation of GABAA receptors and cation-chloride cotransporters in the uterus and their role in pre-term labour Melissa Linda Sutherland December 2017 Supervisors: Dr. Amy V. Poole, Dr. Jennifer A. Fraser, Dr. Claire Garden. A thesis submitted in partial fulfilment of the requirements of Edinburgh Napier University, for the award of Master by Research Declaration It is hereby declared that this thesis is the result of the author’s original research. It has been composed by the author and has not been previously submitted for examination, which has led to the award of a degree or professional qualification. Signed: Date: Contents page Abbreviations .............................................................................................. 1 Acknowledgements ................................................................................... 3 Abstract ......................................................................................................... 4 CHAPTER 1. Introduction ......................................................................... 5 1.1-aminobutyric acid (GABA) .............................................................. 5 1.2 GABA receptor structure and function .......................................... 5 Figure 1.1 Schematic diagram of the GABAA subunit and receptor ......................................................................................................... 6 1.3 GABAARs role in development central nervous system ..........................................................................................................
    [Show full text]
  • Ion Channels
    UC Davis UC Davis Previously Published Works Title THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Ion channels. Permalink https://escholarship.org/uc/item/1442g5hg Journal British journal of pharmacology, 176 Suppl 1(S1) ISSN 0007-1188 Authors Alexander, Stephen PH Mathie, Alistair Peters, John A et al. Publication Date 2019-12-01 DOI 10.1111/bph.14749 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2019/20: Ion channels. British Journal of Pharmacology (2019) 176, S142–S228 THE CONCISE GUIDE TO PHARMACOLOGY 2019/20: Ion channels Stephen PH Alexander1 , Alistair Mathie2 ,JohnAPeters3 , Emma L Veale2 , Jörg Striessnig4 , Eamonn Kelly5, Jane F Armstrong6 , Elena Faccenda6 ,SimonDHarding6 ,AdamJPawson6 , Joanna L Sharman6 , Christopher Southan6 , Jamie A Davies6 and CGTP Collaborators 1School of Life Sciences, University of Nottingham Medical School, Nottingham, NG7 2UH, UK 2Medway School of Pharmacy, The Universities of Greenwich and Kent at Medway, Anson Building, Central Avenue, Chatham Maritime, Chatham, Kent, ME4 4TB, UK 3Neuroscience Division, Medical Education Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK 4Pharmacology and Toxicology, Institute of Pharmacy, University of Innsbruck, A-6020 Innsbruck, Austria 5School of Physiology, Pharmacology and Neuroscience, University of Bristol, Bristol, BS8 1TD, UK 6Centre for Discovery Brain Science, University of Edinburgh, Edinburgh, EH8 9XD, UK Abstract The Concise Guide to PHARMACOLOGY 2019/20 is the fourth in this series of biennial publications. The Concise Guide provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties.
    [Show full text]
  • Reductions in Midbrain Gabaergic and Dopamine Neuron Markers Are Linked in Schizophrenia Tertia D
    Purves‑Tyson et al. Mol Brain (2021) 14:96 https://doi.org/10.1186/s13041‑021‑00805‑7 RESEARCH Open Access Reductions in midbrain GABAergic and dopamine neuron markers are linked in schizophrenia Tertia D. Purves‑Tyson1,2*† , Amelia M. Brown1†, Christin Weissleder1, Debora A. Rothmond1 and Cynthia Shannon Weickert1,2,3* Abstract Reductions in the GABAergic neurotransmitter system exist across multiple brain regions in schizophrenia and encompass both pre‑ and postsynaptic components. While reduced midbrain GABAergic inhibitory neurotransmis‑ sion may contribute to the hyperdopaminergia thought to underpin psychosis in schizophrenia, molecular changes consistent with this have not been reported. We hypothesised that reduced GABA‑related molecular markers would be found in the midbrain of people with schizophrenia and that these would correlate with dopaminergic molecular changes. We hypothesised that downregulation of inhibitory neuron markers would be exacerbated in schizophre‑ nia cases with high levels of neuroinfammation. Eight GABAergic‑related transcripts were measured with quantita‑ tive PCR, and glutamate decarboxylase (GAD) 65/67 and GABAA alpha 3 (α3) (GABRA3) protein were measured with immunoblotting, in post‑mortem midbrain (28/28 and 28/26 control/schizophrenia cases for mRNA and protein, respectively), and analysed by both diagnosis and infammatory subgroups (as previously defned by higher levels of four pro‑infammatory cytokine transcripts). We found reductions (21 – 44%) in mRNA encoding both presynaptic and postsynaptic proteins, vesicular GABA transporter (VGAT ), GAD1, and parvalbumin (PV) mRNAs and four alpha subunits (α1, α2, α3, α5) of the GABAA receptor in people with schizophrenia compared to controls (p < 0.05). Gene expres‑ sion of somatostatin (SST) was unchanged (p 0.485).
    [Show full text]
  • Distinct Diagnostic and Prognostic Values of Γ‑Aminobutyric Acid Type a Receptor Family Genes in Patients with Colon Adenocarcinoma
    ONCOLOGY LETTERS 20: 275-291, 2020 Distinct diagnostic and prognostic values of γ‑aminobutyric acid type A receptor family genes in patients with colon adenocarcinoma LING YAN1, YI‑ZHEN GONG1, MENG‑NAN SHAO2, GUO‑TIAN RUAN1, HAI‑LUN XIE1, XI‑WEN LIAO3, XIANG‑KUN WANG3, QUAN‑FA HAN3, XIN ZHOU3, LI‑CHENG ZHU4, FENG GAO1 and JIA‑LIANG GAN1 1Department of Colorectal and Anal Surgery, The First Affiliated Hospital of Guangxi Medical University; 2Life Sciences Institute, Guangxi Medical University; 3Department of Hepatobiliary Surgery, The First Affiliated Hospital of Guangxi Medical University; 4Department of Immunology, School of Preclinical Medicine, Guangxi Medical University, Nanning, Guangxi Zhuang Autonomous Region 530021, P.R. China Received July 11, 2019; Accepted February 7, 2020 DOI: 10.3892/ol.2020.11573 Abstract. In the present study, the significance of GABAA of cell matrix adhesion, integrin binding, angiogenesis, endo- genes in colon adenocarcinoma (COAD) were investigated thelial growth factor and endothelial migration regulation in from the view of diagnosis and prognosis. All data were patients with COAD with GABRD overexpression. GABRB1, achieved from The Cancer Genome Atlas. Overall survival GABRD, GABRP and GABRQ were associated with the was analyzed by the Kaplan‑Meier analyses and Cox prognostic factors of COAD. The expression levels of regression model and the hazard ratios and 95% confidence GABRA2, GABRA3, GABRB2, GABRB3, GABRG2, GABRD interval were calculated for computation. The Database for and GABRE may allow differentiation between tumor tissues Annotation, Visualization and Integrated Discovery, and the and adjacent normal tissues. Biological Networks Gene Ontology (BiNGO) softwares were applied to assess the biological processes and Kyoto Introduction Encyclopedia of Genes and Genomes (KEGG) was used for pathway analysis to predict the biological function of GABAA Colorectal cancer (CRC) is a type of malignant tumor origi- genes.
    [Show full text]
  • Ligand-Gated Ion Channels
    S.P.H. Alexander et al. The Concise Guide to PHARMACOLOGY 2015/16: Ligand-gated ion channels. British Journal of Pharmacology (2015) 172, 5870–5903 THE CONCISE GUIDE TO PHARMACOLOGY 2015/16: Ligand-gated ion channels Stephen PH Alexander1, John A Peters2, Eamonn Kelly3, Neil Marrion3, Helen E Benson4, Elena Faccenda4, Adam J Pawson4, Joanna L Sharman4, Christopher Southan4, Jamie A Davies4 and CGTP Collaborators L 1 School of Biomedical Sciences, University of Nottingham Medical School, Nottingham, NG7 2UH, UK, N 2Neuroscience Division, Medical Education Institute, Ninewells Hospital and Medical School, University of Dundee, Dundee, DD1 9SY, UK, 3School of Physiology and Pharmacology, University of Bristol, Bristol, BS8 1TD, UK, 4Centre for Integrative Physiology, University of Edinburgh, Edinburgh, EH8 9XD, UK Abstract The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/ doi/10.1111/bph.13350/full. Ligand-gated ion channels are one of the eight major pharmacological targets into which the Guide is divided, with the others being: ligand-gated ion channels, voltage- gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets.
    [Show full text]
  • Expression of the Eight GABAA Receptor Α Subunits in The
    bioRxiv preprint doi: https://doi.org/10.1101/244590; this version posted January 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 1 2 3 4 Expression of the Eight GABAA Receptor α Subunits in the 5 Developing Zebrafish Central Nervous System 6 7 8 Bryan Monesson-Olson1, 2, Jon J. McClain2, Abigail E. Case2, Hanna E. Dorman2, Daniel R. 9 Turkewitz3, Aaron B. Steiner3 and Gerald B. Downes1, 2* 10 11 12 13 1Neuroscience and Behavior Graduate Program 14 2Biology Department, University of Massachusetts, Amherst, MA 01003, USA 15 3Department of Biology and Health Sciences, Pace University, Pleasantville, NY 10570, USA 16 17 18 *Corresponding author: 19 Gerald B. Downes 20 Email: [email protected] bioRxiv preprint doi: https://doi.org/10.1101/244590; this version posted January 9, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. 21 ABSTRACT 22 GABA is a robust regulator of both developing and mature neural networks. It exerts 23 many of its effects through GABAA receptors, which are heteropentamers assembled from a 24 large array of subunits encoded by distinct genes. In mammals, there are 19 different GABAA 25 subunit types, which are divided into the α, β, γ, δ, ε, π, θ and ρ subfamilies.
    [Show full text]