And -Globin in Mesencephalic Dopaminergic Neurons and Glial Cells
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A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated. -
Biochemical and Cellular Studies of Vertebrate Globins
Biochemical and Cellular Studies of Vertebrate Globins By Shun Wilford Tse Thesis submitted for the degree of Doctor of Philosophy School of Biological Sciences University of East Anglia September 2015 © This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that no quotations from the thesis, nor any information derived there-from may be published without the author's prior, written consent. Abstract Human cytoglobin is a small heme-containing protein in the globin superfamily with a wide range of tissue and organ distribution. Although several cellular functions have been proposed for cytoglobin, the exact physiological function is still not fully defined. Recently, cytoglobin has been implicated to have a regulatory role in cancer cells to control cell proliferation and migration depending on cellular oxygen level. In order to gain a better understanding of a structure-to-function relationship of cytoglobin as a heme-protein and to evaluate its possible physiological function(s) in cancer cells, a combination of techniques, including protein engineering and advanced spectroscopies, was deployed. In this study, recombinant human cytoglobin purified from E.coli was purified as a monomeric protein, but displayed a dimeric property in solution. An intra-molecular disulphide bond is formed within the protein which has a redox potential at ca -280 mV. Advanced spectroscopic studies confirmed a low-spin bis-histidyl heme in cytoglobin in both ferric and ferrous state regardless of the state of the disulphide bond. Furthermore, nitrite reductase activitiy in globins was investigated in detail using myoglobin as a model to explore the biochemical basis of the distal histidine residue in determining activity. -
Supporting Information
Supporting Information Biagioli et al. 10.1073/pnas.0813216106 SI Text and the procedure was repeated twice. Single cells were then RNA Isolation, Reverse Transcription, qPCR, Cloning, and Sequencing. resuspended in panning buffer and incubated on lectin-coated RNA was extracted from cell lines and blood by using TRIzol panning plates for 15 min at room temperature. Nonadherent reagent (Invitrogen) and following vendor instructions. cells were transferred to the next panning plate (four pannings, RNA was extracted from LCM- or FACS-purified cells with an 15 min each). Then, nonadherent cells were collected, centri- Absolutely RNA Nanoprep Kit (Stratagene). Single-strand fuged, and resuspended in serum-free neuronal medium or PBS. cDNA was obtained from purified RNA by using the iSCRIPT A similar procedure was also followed for the dissociation of cDNA Synhesis Kit (Bio-Rad) according to the manufacturer’s cortical and hippocampal astrocytes and oligodendrocytes. instructions. Quantitative RT-PCR was performed by using A cell strainer with 70-m nylon mesh was used to obtain a SYBER-Green PCR Master Mix and an iQ5 Real-Time PCR single-cell suspension (BD Falcon) before sorting. 7-AAD Detection System (Bio-Rad). (Beckman–Coulter) was added to the cell suspension to exclude Quantitative RT-PCR was performed with an iCycler IQ dead cells. A high-speed cell sorter (MoFlo) was used to sort (Bio-Rad); -actin was used as an endogenous control to nor- subpopulation of cells expressing GFP. Sorting parameters used malize the expression level of target genes. Primers were chosen for the three different populations are visualized in Fig. -
Adult, Embryonic and Fetal Hemoglobin Are Expressed in Human Glioblastoma Cells
514 INTERNATIONAL JOURNAL OF ONCOLOGY 44: 514-520, 2014 Adult, embryonic and fetal hemoglobin are expressed in human glioblastoma cells MARWAN EMARA1,2, A. ROBERT TURNER1 and JOAN ALLALUNIS-TURNER1 1Department of Oncology, University of Alberta and Alberta Health Services, Cross Cancer Institute, Edmonton, AB T6G 1Z2, Canada; 2Center for Aging and Associated Diseases, Zewail City of Science and Technology, Cairo, Egypt Received September 7, 2013; Accepted October 7, 2013 DOI: 10.3892/ijo.2013.2186 Abstract. Hemoglobin is a hemoprotein, produced mainly in Introduction erythrocytes circulating in the blood. However, non-erythroid hemoglobins have been previously reported in other cell Globins are hemo-containing proteins, have the ability to types including human and rodent neurons of embryonic bind gaseous ligands [oxygen (O2), nitric oxide (NO) and and adult brain, but not astrocytes and oligodendrocytes. carbon monoxide (CO)] reversibly. They have been described Human glioblastoma multiforme (GBM) is the most aggres- in prokaryotes, fungi, plants and animals with an enormous sive tumor among gliomas. However, despite extensive basic diversity of structure and function (1). To date, hemoglobin, and clinical research studies on GBM cells, little is known myoglobin, neuroglobin (Ngb) and cytoglobin (Cygb) repre- about glial defence mechanisms that allow these cells to sent the vertebrate globin family with distinct function and survive and resist various types of treatment. We have tissue distributions (2). During ontogeny, developing erythro- shown previously that the newest members of vertebrate blasts sequentially express embryonic {[Gower 1 (ζ2ε2), globin family, neuroglobin (Ngb) and cytoglobin (Cygb), are Gower 2 (α2ε2), and Portland 1 (ζ2γ2)] to fetal [Hb F(α2γ2)] expressed in human GBM cells. -
Technical Note, Appendix: an Analysis of Blood Processing Methods to Prepare Samples for Genechip® Expression Profiling (Pdf, 1
Appendix 1: Signature genes for different blood cell types. Blood Cell Type Source Probe Set Description Symbol Blood Cell Type Source Probe Set Description Symbol Fraction ID Fraction ID Mono- Lympho- GSK 203547_at CD4 antigen (p55) CD4 Whitney et al. 209813_x_at T cell receptor TRG nuclear cytes gamma locus cells Whitney et al. 209995_s_at T-cell leukemia/ TCL1A Whitney et al. 203104_at colony stimulating CSF1R lymphoma 1A factor 1 receptor, Whitney et al. 210164_at granzyme B GZMB formerly McDonough (granzyme 2, feline sarcoma viral cytotoxic T-lymphocyte- (v-fms) oncogene associated serine homolog esterase 1) Whitney et al. 203290_at major histocompatibility HLA-DQA1 Whitney et al. 210321_at similar to granzyme B CTLA1 complex, class II, (granzyme 2, cytotoxic DQ alpha 1 T-lymphocyte-associated Whitney et al. 203413_at NEL-like 2 (chicken) NELL2 serine esterase 1) Whitney et al. 203828_s_at natural killer cell NK4 (H. sapiens) transcript 4 Whitney et al. 212827_at immunoglobulin heavy IGHM Whitney et al. 203932_at major histocompatibility HLA-DMB constant mu complex, class II, Whitney et al. 212998_x_at major histocompatibility HLA-DQB1 DM beta complex, class II, Whitney et al. 204655_at chemokine (C-C motif) CCL5 DQ beta 1 ligand 5 Whitney et al. 212999_x_at major histocompatibility HLA-DQB Whitney et al. 204661_at CDW52 antigen CDW52 complex, class II, (CAMPATH-1 antigen) DQ beta 1 Whitney et al. 205049_s_at CD79A antigen CD79A Whitney et al. 213193_x_at T cell receptor beta locus TRB (immunoglobulin- Whitney et al. 213425_at Homo sapiens cDNA associated alpha) FLJ11441 fis, clone Whitney et al. 205291_at interleukin 2 receptor, IL2RB HEMBA1001323, beta mRNA sequence Whitney et al. -
Supplementary Table S1. Correlation Between the Mutant P53-Interacting Partners and PTTG3P, PTTG1 and PTTG2, Based on Data from Starbase V3.0 Database
Supplementary Table S1. Correlation between the mutant p53-interacting partners and PTTG3P, PTTG1 and PTTG2, based on data from StarBase v3.0 database. PTTG3P PTTG1 PTTG2 Gene ID Coefficient-R p-value Coefficient-R p-value Coefficient-R p-value NF-YA ENSG00000001167 −0.077 8.59e-2 −0.210 2.09e-6 −0.122 6.23e-3 NF-YB ENSG00000120837 0.176 7.12e-5 0.227 2.82e-7 0.094 3.59e-2 NF-YC ENSG00000066136 0.124 5.45e-3 0.124 5.40e-3 0.051 2.51e-1 Sp1 ENSG00000185591 −0.014 7.50e-1 −0.201 5.82e-6 −0.072 1.07e-1 Ets-1 ENSG00000134954 −0.096 3.14e-2 −0.257 4.83e-9 0.034 4.46e-1 VDR ENSG00000111424 −0.091 4.10e-2 −0.216 1.03e-6 0.014 7.48e-1 SREBP-2 ENSG00000198911 −0.064 1.53e-1 −0.147 9.27e-4 −0.073 1.01e-1 TopBP1 ENSG00000163781 0.067 1.36e-1 0.051 2.57e-1 −0.020 6.57e-1 Pin1 ENSG00000127445 0.250 1.40e-8 0.571 9.56e-45 0.187 2.52e-5 MRE11 ENSG00000020922 0.063 1.56e-1 −0.007 8.81e-1 −0.024 5.93e-1 PML ENSG00000140464 0.072 1.05e-1 0.217 9.36e-7 0.166 1.85e-4 p63 ENSG00000073282 −0.120 7.04e-3 −0.283 1.08e-10 −0.198 7.71e-6 p73 ENSG00000078900 0.104 2.03e-2 0.258 4.67e-9 0.097 3.02e-2 Supplementary Table S2. -
Recombinant Human Hemoglobin Subunit Theta-1/HBQ1 (N-6His)
9853 Pacific Heights Blvd. Suite D. San Diego, CA 92121, USA Tel: 858-263-4982 Email: [email protected] 32-8592: Recombinant Human Hemoglobin Subunit theta-1/HBQ1 (N-6His) Gene : HBQ1 Gene ID : 3049 Uniprot ID : P09105 Description Source: E.coli. MW :17.7kD. Recombinant Human Hemoglobin subunit theta-1 is produced by our E.coli expression system and the target gene encoding Met1-Arg142 is expressed with a 6His tag at the N-terminus. Hemoglobin subunit theta-1 is a protein that in humans is encoded by the HBQ1 gene. Theta-globin mRNA is originally found in human fetal erythroid tissue but not in adult erythroid or other nonerythroid tissue. Theta-1 is a member of the human alpha-globin gene cluster that includes five functional genes and two pseudogenes. Research supports a transcriptionally active role for the gene and a functional role for the peptide in specific cells, possibly those of early erythroid tissue. Hemoglobin has a quaternary structure characteristically composed of many multi- subunit globular proteins. Most of the amino acids in hemoglobin form alpha helices, connected by short non-helical segments. Hydrogen bonds stabilize the helical sections inside this protein, causing attractions within the molecule, folding each polypeptide chain into a specific shape. Hemoglobin's quaternary structure comes from its four subunits in roughly a tetrahedral arrangement. Product Info Amount : 10 µg / 50 µg Content : Lyophilized from a 0.2 µm filtered solution of 20mM PB, 150mM NaCl, pH 7.0. Lyophilized protein should be stored at -20°C, though stable at room temperature for 3 weeks. -
Neuroglobin and Cytoglobin Fresh Blood for the Vertebrate Globin Family
EMBO reports Neuroglobin and cytoglobin Fresh blood for the vertebrate globin family Alessandra Pesce, Martino Bolognesi+, Alessio Bocedi1, Paolo Ascenzi1, Sylvia Dewilde2, Luc Moens2, Thomas Hankeln3 & Thorsten Burmester4 Department of Physics–INFM and Center for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, I-16146 Genova, 1Department of Biology, University ‘Roma Tre’, Viale Guglielmo Marconi 446, I-00146 Roma, Italy, 2Department of Biochemistry, University of Antwerp, Universiteitsplein 1, B-2610 Antwerp, Belgium, 3Institute of Molecular Genetics, Johannes Gutenberg University of Mainz, Becherweg 32, D-55099 Mainz and 4Institute of Zoology, Johannes Gutenberg University of Mainz, Müllerweg 6, D-55099 Mainz, Germany Received August 7, 2002; revised October 8, 2002; accepted October 18, 2002 Neuroglobin and cytoglobin are two recently discovered (Wittenberg, 1970, 1992; Antonini and Brunori, 1971; Perutz, members of the vertebrate globin family. Both are intracellular 1979, 1990; Dickerson and Geis, 1983; Bunn and Forget, 1986; proteins endowed with hexacoordinated heme-Fe atoms, in Brunori, 1999; Weber and Vinogradov, 2001; Merx et al., 2002), their ferrous and ferric forms, and display O2 affinities comparable although they can also carry out enzymatic functions (Minning with that of myoglobin. Neuroglobin, which is predominantly et al., 1999; Ascenzi et al., 2001). expressed in nerve cells, is thought to protect neurons from Four types of globin, differing in structure, tissue distribution hypoxic–ischemic injury. It is of ancient evolutionary origin, and likely in function, have been discovered in man and other and is homologous to nerve globins of invertebrates. vertebrates: hemoglobin, myoglobin, neuroglobin and cyto- Cytoglobin is expressed in many different tissues, although at globin. -
Supplementary Methods
Supplementary methods Human lung tissues and tissue microarray (TMA) All human tissues were obtained from the Lung Cancer Specialized Program of Research Excellence (SPORE) Tissue Bank at the M.D. Anderson Cancer Center (Houston, TX). A collection of 26 lung adenocarcinomas and 24 non-tumoral paired tissues were snap-frozen and preserved in liquid nitrogen for total RNA extraction. For each tissue sample, the percentage of malignant tissue was calculated and the cellular composition of specimens was determined by histological examination (I.I.W.) following Hematoxylin-Eosin (H&E) staining. All malignant samples retained contained more than 50% tumor cells. Specimens resected from NSCLC stages I-IV patients who had no prior chemotherapy or radiotherapy were used for TMA analysis by immunohistochemistry. Patients who had smoked at least 100 cigarettes in their lifetime were defined as smokers. Samples were fixed in formalin, embedded in paraffin, stained with H&E, and reviewed by an experienced pathologist (I.I.W.). The 413 tissue specimens collected from 283 patients included 62 normal bronchial epithelia, 61 bronchial hyperplasias (Hyp), 15 squamous metaplasias (SqM), 9 squamous dysplasias (Dys), 26 carcinomas in situ (CIS), as well as 98 squamous cell carcinomas (SCC) and 141 adenocarcinomas. Normal bronchial epithelia, hyperplasia, squamous metaplasia, dysplasia, CIS, and SCC were considered to represent different steps in the development of SCCs. All tumors and lesions were classified according to the World Health Organization (WHO) 2004 criteria. The TMAs were prepared with a manual tissue arrayer (Advanced Tissue Arrayer ATA100, Chemicon International, Temecula, CA) using 1-mm-diameter cores in triplicate for tumors and 1.5 to 2-mm cores for normal epithelial and premalignant lesions. -
A Gene-Environment Study of Cytoglobin in the Human and Rat Hippocampus
A Gene-Environment Study of Cytoglobin in the Human and Rat Hippocampus Christian Ansgar Hundahl1,3, Betina Elfving2, Heidi Kaastrup Mu¨ ller2, Anders Hay-Schmidt3, Gregers Wegener2,4* 1 Centre of Excellence for Translational Medicine, University of Tartu, Tartu, Estonia, 2 Translational Neuropsychiatry Unit, Department of Clinical Medicine, Aarhus University, Aarhus, Denmark, 3 Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark, 4 Unit for Drug Research and Development, School of Pharmacy (Pharmacology), North-West University, Potchefstroom, South Africa Abstract Background: Cytoglobin (Cygb) was discovered a decade ago as the fourth vertebrate heme-globin. The function of Cygb is still unknown, but accumulating evidence from in vitro studies point to a putative role in scavenging of reactive oxygen species and nitric oxide metabolism and in vivo studies have shown Cygb to be up regulated by hypoxic stress. This study addresses three main questions related to Cygb expression in the hippocampus: 1) Is the rat hippocampus a valid neuroanatomical model for the human hippocampus; 2) What is the degree of co-expression of Cygb and neuronal nitric oxide synthase (nNOS) in the rat hippocampus; 3) The effect of chronic restraint stress (CRS) on Cygb and nNOS expression. Methods: Immunohistochemistry was used to compare Cygb expression in the human and rat hippocampi as well as Cygb and nNOS co-expression in the rat hippocampus. Transcription and translation of Cygb and nNOS were investigated using quantitative real-time polymerase chain reaction (real-time qPCR) and Western blotting on hippocampi from Flinders (FSL/ FRL) rats exposed to CRS. Principal Findings: Cygb expression pattern in the human and rat hippocampus was found to be similar. -
Simultaneous Detection of Target Cnvs and Snvs of Thalassemia by Multiplex PCR and Next‑Generation Sequencing
MOLECULAR MEDICINE REPORTS 19: 2837-2848, 2019 Simultaneous detection of target CNVs and SNVs of thalassemia by multiplex PCR and next‑generation sequencing DONG-MEI FAN1*, XU YANG2*, LI-MIN HUANG1, GUO-JUN OUYANG3, XUE-XI YANG1 and MING LI1 1Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, Guangdong 510515; 2Clinical Innovation and Research Center, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong 518110; 3Guangzhou Darui Biotechnology Co., Ltd., Guangzhou, Guangdong 510663, P.R. China Received May 16, 2018; Accepted December 3, 2018 DOI: 10.3892/mmr.2019.9896 Abstract. Thalassemia is caused by complex mechanisms, Introduction including copy number variants (CNVs) and single nucleotide variants (SNVs). The CNV types of α-thalassemia are typi- Thalassemia is caused by copy number variants (CNVs) and cally detected by gap-polymerase chain reaction (PCR). The single nucleotide variants (SNVs) in the α-globin (HBA) or SNV types are detected by Sanger sequencing. In the present β-globin (HBB) genes that result in the absence or lack of α- or study, a novel method was developed that simultaneously β-globin chains, and ultimately hemolytic anemia. It is esti- detects CNVs and SNVs by multiplex PCR and next-genera- mated that ~7% of the world population carries the gene for the tion sequencing (NGS). To detect CNVs, 33 normal samples disease (1), and the birth rate of children with hemoglobin (Hb) were used as a cluster of control values to build a baseline, disorders is ≥2.4% per year (2). Thalassemia occurs most in the and the A, B, C, and D ratios were developed to evaluate-SEA, Mediterranean region, East South Asia, and the subcontinents -α4.2, -α3.7, and compound or homozygous CNVs, respectively. -
Light and Electron Microscopy Characteristics of the Muscle Of
Original article J Clin Pathol: first published as 10.1136/jcp.2007.051060 on 1 October 2007. Downloaded from Light and electron microscopy characteristics of the muscle of patients with SURF1 gene mutations associated with Leigh disease M Pronicki,1 E Matyja,2 D Piekutowska-Abramczuk,3 T Szyman´ska-De˛bin´ska,1 A Karkucin´ska-Wie˛ckowska,1 E Karczmarewicz,4 W Grajkowska,1 T Kmiec´,5 E Popowska,3 J Sykut-Cegielska6 1 Department of Pathology, ABSTRACT complex IV (cytochrome c oxidase (COX)) has The Children’s Memorial Health Aims: Leigh syndrome (LS) is characterised by almost been confirmed in humans.34 The SURF1 muta- Institute, Warsaw, Poland; identical brain changes despite considerable causal 2 Department of Metabolic tions were found invariably and almost exclusively Diseases, Endocrinology and heterogeneity. SURF1 gene mutations are among the in patients with generalised COX-deficit coexistent Diabetology, The Children’s most frequent causes of LS. Although deficiency of with LS features. Nearly 60 such patients and over Memorial Health Institute, cytochrome c oxidase (COX) is a typical feature of the 3 35 different mutations have been reported so far in Warsaw, Poland; Department muscle in SURF1-deficient LS, other abnormalities have the literature.5–32 SURF1 encodes for one of several of Medical Genetics, The Children’s Memorial Health been rarely described. The aim of the present work is to proteins involved in proper assembly of the active Institute, Warsaw, Poland; assess the skeletal muscle morphology coexisting with COX complex. 4 Department of Biochemistry SURF1 mutations from our own research and in the Despite numerous detailed reports in muscle and Experimental Medicine, literature.