DOCSLIB.ORG

Epigenetic Changes in the Hypothalamus of Offspring Following Maternal Undernutrition

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Epigenetic Changes in the Hypothalamus of Offspring Following Maternal Undernutrition Epigenetic changes in the hypothalamus of offspring following maternal undernutrition A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences Ghazala Begum September 2013 Contents Page No. List of Figures 5 List of Tables 8 Abstract 9 Declaration 10 Copyright Statement 10 Abbreviations 11 Nomenclature 13 Acknowledgements 14 Chapter 1 15 Introduction 15 1.1 The implications of maternal programming 16 1.1.1 Maternal undernutrition induces programming 17 1.1.2 Maternal overnutrition induces programming 18 1.1.3 Stress-induced maternal programming 19 1.1.4 Twinning as a programming paradigm 20 1.2 Programming of the hypothalamic energy regulating pathway 21 1.2.1 Hypothalamic energy regulating pathway 21 1.2.2 Role of glucocorticoids in the hypothalamic energy regulating pathway 26 1.2.3 Effects of maternal undernutrition on the offspring’s energy regulating 27 athway pathway 1.3 Programming of the HPA axis 31 1.3.1 Key components of the HPA axis 31 1.3.2 Effects of maternal undernutrition on the offspring’s HPA axis 32 1.4 The potential involvement of epigenetics in programming 34 1.4.1 DNA methylation 35 1.4.2 Histone Modifications 37 1.4.3 The GR gene as a potential target for epigenetic modification 38 1.4.4 Epigenetic alterations of fetal GR following maternal programming 39 1.4.5 POMC as a potential target of epigenetic modification 40 1.4.6 Epigenetic alterations of fetal POMC following maternal nutritional 40 dsjdsddsdinsults 1.5 Overview 42 1.5.1 Maternal undernutrition sheep model 42 1.5.2 Aims 44 1.6 Alternative format 47 Chapter 2 49 Methods 49 2.1 Animal Management 50 2.2 DNA, RNA and protein purification from brain tissues 51 2.3 Whole blood DNA and protein purification 52 2.4 Bioinformatic analysis 53 2.5 DNA methylation analysis 53 2.6 mRNA expression analysis 55 2.6.1 mRNA expression analysis during the fetal study 55 2 2.6.2 mRNA expression analysis during the twin study 55 2.6.3 mRNA expression analysis during the adult study 56 2.7 Chromatin immunoprecipitation 57 2.7.1 Chromatin immunoprecipication for fetal tissues 57 2.7.2 Chromatin immunoprecipitation for adult brain tissues 58 2.8 Western blots 59 2.9 POMC ELISA 60 2.10 DNA methyltransferase activity/inhibition assay 61 2.11 Buffers 61 2.12 Statistical analysis 62 Chapter 3: Publication I 63 Epigenetic changes in the hypothalamic pro-opiomelanocrotin and glucocorticoid ssreceptor genes in the ovine fetus after periconceptional undernutrition Chapter 4: Publication II 64 Epigenetic changes in fetal hypothalamic energy regulating pathways are dlassociated with maternal undernutrition and twinning 4.1 Supplemental data 65 Chapter 5: Publication III 67 Maternal undernutrition programs tissue-specific epigenetic changes in the bglucocorticoid receptor in adult offspring Chapter 6 68 Maternal undernutrition induces tissue specific changes in POMC in adult 68 gioffspring 6.1 Introduction 69 6.1.1 Aims 70 6.2 Materials and methods 71 6.2.1 Animal management 71 6.2.2 Isolation of DNA and RNA and protein from tissue and blood 71 6.2.3 DNA methylation enrichment 71 6.2.4 Chromatin immunoprecipitation of H3K9AC and H3K27me3 71 6.2.5 qRT-PCR analysis 72 6.2.6 Western blot 72 6.2.7 Statistical analysis 72 6.3 Epigenetic changes in hypothalamic POMC 73 6.4 Comparable levels of hypothalamic POMC protein 75 6.5 Epigenetic changes in the POMC promoter amplicon in the pituitary of 76 fddffdpericonceptionally undernourished adult offspring 6.6 POMC mRNA and protein levels in the pituitary of adult offspring 77 6.7 POMC status in the peripheral leukocytes of maternally undernourished adult 78 vcvccoffspring 6.8 Summary 79 Chapter 7 81 Discussion 7.1 The impact of the length and timing of maternal undernutrition on the 83 3 hghghoffspring in sheep 7.2 Epigenetic changes in hypothalamic GR as a consequence of maternal 84 ghghgprogramming persisting from fetal life to adulthood 7.3 Epigenetic changes in hypothalamic POMC as a consequence of maternal 87 ghghhprogramming 7.4 Tissue specific changes in GR and POMC 90 7.5 NPY expression in the hypothalami of maternally underfed offspring 92 7.6 Sex-specific outcomes in the maternally undernourished offspring 93 7.7 Twinning as an intrauterine programming paradigm 94 7.8 Phenotypic outcome in the adult offspring subject to periconceptional 95 cvcvcundernourishment 7.9 Mechanisms of action underlying epigenetic changes in the offspring as a 97 cvcvvconsequence of maternal programming 7.10 Conclusion 98 7.11 Future work 99 7.11.1 Analysis of the role of exon 1 of the GR gene in the hypothalamus 99 7.11.2 POMC hypothalamic specific enhancer region 102 7.11.3 Candidate gene approach verses genome wide analysis 103 7.11.4 Potential models of maternal nutritional programming 104 Chapter 8 107 References Chapter 9 128 Appendix Final word count is 52,545. 4 List of Figures Page No. Page No. in thesis in paper Chapter 1: Figure 1.01. Programming paradigms 17 Figure 1.02. Tissue specific POMC processing 22 Figure 1.03. Hypothalamic appetite regulatory pathway 25 Figure 1.04. The HPA axis 31 Figure 1.05. DNA methylation 37 Figure 1.06. Histone modifications 38 Figure 1.07. The GR exon 1 promoter region 39 Figure 1.08: Maternal programming sheep model 43 Chapter 2: Figure 2.01. RNA integrity gel 52 Figure 2.02. RNA integrity of whole and ventral hypothalamic 52 samples Chapter 3: FIG. 1. POMC and GR gene region screening to identify highly 3656 conserved, CpG-rich regions. FIG. 2. HPA axis activity in fetal sheep from control ewes or 3657 ewes subjected to periconceptional undernutrition (underfed from 60 d before conception to 30 d after conception). FIG. 3. Epigenetic changes associated with the POMC gene in 3658 the fetal hypothalamus. Fetal hypothalamic tissue samples were obtained from normal and underfed maternal sheep (underfed from 60 d before to 30 d after conception). FIG. 4. Expression of the POMC gene in the fetal hypothalamus. 3658 Fetal hypothalamic tissue samples were obtained from control and underfed (from 60 d before conception to 30 d after conception). FIG. 5. Presence of H3K9Ac associated with the GR gene 3659 promoter in the fetal hypothalamus 5 Page No. Page No. in thesis in paper FIG. 6. Methylation of GR gene promoter region in the fetal 3659 hypothalamus. Hypothalamic tissue samples were obtained from control and underfed (from 60 d before conception to 30 d after conception) fetal sheep. FIG. 7. Expression of GR gene in the fetal hypothalamus. Fetal 3660 hypothalamic tissue samples were obtained from control and underfed maternal sheep (from 60 d before conception to 30 d after conception); 2 μg of total RNA were used to quantify expression levels of GR. FIG. 8.The effect of different periods of periconceptional 3661 undernutrition on hypothalamic GR, POMC, and NPY methylation and gene expression. The −60 to +30 group (UN −60 to +30) was underfed from 60 d before conception to 30 d after conception. The −60 to 0 group (UN −60 to 0) were fed the same diet as the −60 to +30 group but were allowed to feed ad libitum from conception. The −2 to +30 group (UN −2 to +30) were fed the same diet for 30 d after conception. Fetal ventral hypothalamic tissue samples, enriched for the ARC, were obtained from all sample groups at d 131 of gestation Chapter 4: Figure 1.Summary of ENCODE data from different human cell 1696 lines. Data analysis from the UCSC genome browser depicts cell line-specific changes in chromatin over the POMC promoter marker region. Figure 2.Histone modifications of the POMC promoter in 1698 response to twinning and periconceptional maternal undernutrition. Figure 3.DNA methylation and expression levels of fetal 1699 hypothalamic neuropeptides Figure 4.Changes in the histone patterns of the GR promoter as a 1700 result of twinning and maternal periconceptional undernutrition. Figure 5.Effects of twinning and periconceptional maternal 1700 undernutrition on fetal hypothalamic GR promoter methylation and GR mRNA expression levels. Figure 6.Fetal HPA axis dynamics following twinning and 1701 maternal periconceptional undernutrition. 4.1 Supplemental data: Fig. S1. Summary of ENCODE data 65 6 Page No. Page No. in thesis in paper Fig. S2. NPY mRNA expression levels determined using qRT- 66 PCR. Groups were compared to the control via the one-way ANOVA with Tukey HSD Post Hoc test. *p<0.05, **p<0.01, ***p<0.005. Chapter 5: Figure 1. Periconceptional undernutrition is associated with 4 altered glucocorticoid receptor (GR) epigenetic status in the ventral hypothalamus of adult offspring. Figure 2. Altered glucocorticoid receptor (GR) expression levels 5 in the ventral hypothalamus of adult offspring following periconceptional undernutrition. Figure 3. Ventral hypothalamic neuropeptide mRNA expression. 6 Figure 4. Altered epigenetic and expression status of 6 hippocampal glucocorticoid receptor (GR) epigenetic and expression status in offspring from undernourished mothers. Figure 5. No change in glucocorticoid receptor (GR) promoter 7 methylation or protein expression in leukocytes. Figure 6. Increased fat:lean mass ratio in adult males whose 8 mothers were periconceptionally undernourished Chapter 6: Figure 6.01. Altered epigenetic status of the POMC gene in the 74 ventral hypothalamus of maternally undernourished adult offspring. Figure 6.02. Similar levels of hypothalamic POMC protein in 75 adult animals following periconceptional undernutrition. Figure 6.03. Gender specific epigenetic changes in the POMC 76 promoter region in the pituitary in maternally undernourished adult offspring. Figure 6.04. Comparable POMC mRNA and protein levels in the 77 pituitary.
Load more

Recommended publications
  • Memoranaurns by the Participants in Signes Par Les Partici- I the Meeting
    Memoranda are state- Les Memorandums ments concerning the exposent les conclu- /e , conclusions or recom- sions et recomman- M e mmooranrantedaa mendations of certain dations de certaines / a t w / /WHO scientific meet- /reunions scientifiques ings; they are signed de /'OMS; ils sont Memoranaurns by the participants in signes par les partici- I the meeting. pants a ces reunions. Bulletin ofthe World Health Organization, 62 (2): 217-227 (1984) © World Health Organization 1984 Immunodiagnosis simplified: Memorandum from a WHO Meeting* Technologies suitable for the development ofsimplified immunodiagnostic tests were reviewed by a Working Group of the WHO Advisory Committee on Medical Research in Geneva in June 1983. They included agglutination tests and use ofartificialparticles coated with immunoglobulins, direct visual detection of antigen-antibody reactions, enzyme- immunoassays, and immunofluorescence and fluoroimmunoassays. The use of mono- clonal antibodies,in immunodiagnosis and of DNA /RNA probes to identify viruses was also discussed in detail. The needfor applicability of these tests at three levels, i.e., field conditions (or primary health care level), local laboratories, and central laboratories, was discussed and their use at thefield level was emphasized. Classical serological techniques have been used for All these tests can be carried out in laboratories that a long time for diagnostic purposes, e.g., for con- are equipped with basic instruments as well as special- firmation of clinical diagnoses, epidemiological ized apparatus (e.g., gamma counters for RIA, ultra- studies, testing of blood donors, etc. Some of these violet microscopes for IMF, etc.), which are usually techniques have been standardized to a high degree of available only in the larger, central laboratories.
    [Show full text]
  • Radioimmunoassay in Developing Countries: General Principles
    XA9847613 Chapter 16 RADIOIMMUNOASSAY IN DEVELOPING COUNTRIES (General principles) R.D. Piyasena Radioimmunoassay (RIA) is probably the most commonly performed nuclear medicine technique. It is an in vitro procedure, where no radioactivity is administered to the patient. But this alone is not the reason for its widespread use. It provides the basis for extremely sensitive and specific diagnostic tests, and its use in present day medicine has brought a virtual information explosion in terms of understanding the pathophysiology of many diseases. The fact that the technology involved is within the technical and economic capabilities of the developing world is evident from the increasing demand for its introduction or expansion of existing services. RIA facilities need not be restricted to urban hospitals, as in the case of in vivo nuclear medicine techniques, but may be extended to smaller district hospitals and other laboratories in peripheral areas. It is also possible to send blood samples to a central laboratory so that a single centre can serve a wide geographical area. There are many laboratories in the industrialized world that receive a major proportion of samples for assay by mail. In recent years, substantial RIA services have been established in many of the developing countries in Asia and Latin America. The International Atomic Energy Agency (IAEA) and World Health Organisations (WHO) have made vital contributions to these activities and have played a catalytic role in assisting member states to achieve realistic goals. In the past five years, more than 250 individual RIA laboratories in developing member states have been beneficiaries of IAEA projects.
    [Show full text]
  • Frequently Asked Questions
    Frequently Asked Questions 03-DFAQ-02a version 1 Contents 1. COMMERCIAL INFORMATION .................................................................................................... 4 1.1. How can I place an order? ...........................................................................................................4 1.2. How can I obtain a price? ............................................................................................................4 1.3. Does DIAsource provide the kit components separately? ...........................................................4 1.4. How can I address a complaint? ..................................................................................................4 2. REGULATORY INFORMATION ..................................................................................................... 4 2.1. Do the DIAsource kits contain dangerous substances? ...............................................................4 2.2. Are the DIAsource assays CE mark and/or FDA approved? .........................................................4 2.3. May the DIAsource assays be sold in Canada? ............................................................................5 3. TECHNICAL INFORMATION ......................................................................................................... 5 3.1. How can I receive a technical information? ................................................................................5 3.2. What is the principle of an immunoassay (ELISA – RIA – IRMA)? ................................................5
    [Show full text]
  • Design, Visualize and Detect
    Thermo Scientific Pierce Antibody Immunostaining Guide Design, visualize and detect Immunostaining Primary Antibodies • Secondary Antibodies • Staining Dyes • Kits Thermo Scientific Table of Contents Pierce Antibody Immunostaining Guide Page Introduction 1-3 The Cell 4-5 Mammalian Cell Type Choices 6-8 Immunohistochemistry 9-12 Immunofluorescence 13-22 Secondary Antibodies 23-27 Primary Antibodies by Cellular Structures 28-31 by Research Areas 32-43 by Cell Signaling 44-59 by Biological Processes 60-76 Left: Detection of mouse anti-α-tubulin in an A549 cell in Telophase with Thermo Scientific DyLight Dye 550-GAM. Chromosomes (orange) at the poles become Introduction diffuse, while nuclei (blue) divide into two future cells. Immunofluorescence (IF) and immunohistochemistry (IHC) are two methods commonly used to detect proteins in a cellular context. Immunofluorescent detection of proteins can be performed on both fixed cells in culture and on paraffin or frozen tissue sections. The advantages of using IF to detect cellular proteins includes the ability to visualize the subcellular location of protein(s) of interest, assess both protein expression and post-translational modifications, and design multiplex experiments. When IF detection is extended to tissues sections (IHC), a higher level of resolution is achieved because researchers are analyzing target protein(s) in a near physiological state, making it ideal for assessing normal and disease tissues. To order, call 800-874-3723 or 815-968-0747. Outside the United States, contact your local branch office or distributor. 1 Need Antibodies? Build a Better Antibody Introduction We have over 30,000 antibodies in 42 research areas. Use our custom services to produce antibodies you can trust.
    [Show full text]
  • A New Human Chromogranin a (Cga) Immunoradiometric Assay Involving Monoclonal Antibodies Raised Against the Unprocessed Central Domain (145–245)
    British Journal of Cancer (1999) 79(1), 65–71 © 1999 Cancer Research Campaign A new human chromogranin A (CgA) immunoradiometric assay involving monoclonal antibodies raised against the unprocessed central domain (145–245) F Degorce1, Y Goumon2, L Jacquemart1, C Vidaud1, L Bellanger1, D Pons-Anicet1, P Seguin1, MH Metz-Boutigue2 and D Aunis2 1CIS biointernational, Division In Vitro Technologies, BP175, 30203 Bagnols-sur-Cèze, France; 2Institut National de la Santé et de la Recherche Médicale, INSERM U-338, Biologie de la Communication Cellulaire, 67084 Strasbourg, France Summary Chromogranin A (CgA), a major protein of chromaffin granules, has been described as a potential marker for neuroendocrine tumours. Because of an extensive proteolysis which leads to a large heterogeneity of circulating fragments, its presence in blood has been assessed in most cases either by competitive immunoassays or with polyclonal antibodies. In the present study, 24 monoclonal antibodies were raised against native or recombinant human CgA. Their mapping with proteolytic peptides showed that they defined eight distinct epitopic groups which spanned two-thirds of the C-terminal part of human CgA. All monoclonal antibodies were tested by pair and compared with a reference radioimmunoassay (RIA) involving CGS06, one of the monoclonal antibodies against the 198–245 sequence. It appears that CgA C-terminal end seems to be highly affected by proteolysis and the association of C-terminal and median-part monoclonal antibodies is inadequate for total CgA assessment. Our new immunoradiometric assay involves two monoclonal antibodies, whose contiguous epitopes lie within the median 145–245 sequence. This assay allows a sensitive detection of total human CgA and correlates well with RIA because dibasic cleavage sites present in the central domain do not seem to be affected by degradation.
    [Show full text]
  • Diagnosis of Brucellosis by Serology Klaus Nielsen* Animal Diseases Research Institute, 3851 Fallow®Eld Road, Nepean, Ont., Canada K2H 8P9
    Veterinary Microbiology 90 (2002) 447±459 Diagnosis of brucellosis by serology Klaus Nielsen* Animal Diseases Research Institute, 3851 Fallow®eld Road, Nepean, Ont., Canada K2H 8P9 Abstract Serological diagnosis of brucellosis began more than 100 years ago with a simple agglutination test. It was realized that this type of test was susceptible to false positive reactions resulting from,for instance,exposure to cross reacting microorganisms. It was also realized that this test format was inexpensive,simple and could be rapid,although results were subjectively scored. Therefore,a number of modi®cations were developed along with other types of tests. This served two purposes: one was to establish a rapid screening test with high sensitivity and perhaps less speci®city and a con®rmatory test,usually more complicated but also more speci®c,to be used on sera that reacted positively in screening tests. This led to another problem: if a panel of tests were performed and they did not all agree,which interpretation was correct? This problem was further compounded by the extensive use of a vaccine which gave rise to an antibody response similar to that resulting from ®eld infection. This led to the development of an assay that could distinguish vaccinal antibody,starting with precipitin tests. These tests did not perform well,giving rise to the development of primary binding assays. These assays,including the competitive enzyme immunoassay and the ¯uorescence polarization assay are at the apex of current development,providing high sensitivity and speci®city as well as speed and mobility in the case of the ¯uorescence polarization assay.
    [Show full text]
  • Detailed Follow-Up of Hepatitis B Surface Antigen Positive Blood Donors
    Open Research Online The Open University’s repository of research publications and other research outputs Detailed follow-up of hepatitis B surface antigen positive blood donors. Thesis How to cite: Brown, Susan (1992). Detailed follow-up of hepatitis B surface antigen positive blood donors. The Open University. For guidance on citations see FAQs. c 1992 The Author https://creativecommons.org/licenses/by-nc-nd/4.0/ Version: Version of Record Link(s) to article on publisher’s website: http://dx.doi.org/doi:10.21954/ou.ro.00010184 Copyright and Moral Rights for the articles on this site are retained by the individual authors and/or other copyright owners. For more information on Open Research Online’s data policy on reuse of materials please consult the policies page. oro.open.ac.uk 1 Faculty of Biology. Dissertation for the Degree of Bachelor of Philosophy, Susan Brown North London Blood Transfusion Centre Colindale. Detailed follow-up of hepatitis B surface antigen positive blood donors. fMav 1992. ProQ uest Number: 27919423 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent on the quality of the copy submitted. in the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 27919423 Published by ProQuest LLC (2020). Copyright of the Dissertation is held by the Author. Ail Rights Reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC.
    [Show full text]
  • A Two-Site Immunoradiometric Assay for Serum Calcitonin Using Monoclonal Anti-Peptide Antibodies
    Henry Ford Hospital Medical Journal Volume 35 Number 2 Second International Workshop on Article 15 MEN-2 6-1987 A Two-Site Immunoradiometric Assay for Serum Calcitonin Using Monoclonal Anti-Peptide Antibodies Philippe Motte Malika Ait-Abdellah Pascal Vauzelle Paule Gardet Claude Bohuon See next page for additional authors Follow this and additional works at: https://scholarlycommons.henryford.com/hfhmedjournal Part of the Life Sciences Commons, Medical Specialties Commons, and the Public Health Commons Recommended Citation Motte, Philippe; Ait-Abdellah, Malika; Vauzelle, Pascal; Gardet, Paule; Bohuon, Claude; and Bellet, Dominique (1987) "A Two-Site Immunoradiometric Assay for Serum Calcitonin Using Monoclonal Anti- Peptide Antibodies," Henry Ford Hospital Medical Journal : Vol. 35 : No. 2 , 129-132. Available at: https://scholarlycommons.henryford.com/hfhmedjournal/vol35/iss2/15 This Article is brought to you for free and open access by Henry Ford Health System Scholarly Commons. It has been accepted for inclusion in Henry Ford Hospital Medical Journal by an authorized editor of Henry Ford Health System Scholarly Commons. A Two-Site Immunoradiometric Assay for Serum Calcitonin Using Monoclonal Anti-Peptide Antibodies Authors Philippe Motte, Malika Ait-Abdellah, Pascal Vauzelle, Paule Gardet, Claude Bohuon, and Dominique Bellet This article is available in Henry Ford Hospital Medical Journal: https://scholarlycommons.henryford.com/ hfhmedjournal/vol35/iss2/15 A TVo-Site Immunoradiometric Assay for Serum Calcitonin Using Monoclonal Anti-Peptide Antibodies Philippe Motte,* Malika Ait-Abdellah, Pascal Vauzelle, Paule Gardet, Claude Bohuon, and Dominique Beliet We have produced a library of monoclonal antibodies of various affinities by immunizing mice with synthetic calcitonin (CT) 1-32. These monoclonal antibodies defined two antigenic determinants on the molecule ofCT.
    [Show full text]
  • LECTIN-ENZYME BINDING ASSAYS Development of the Technique and Applications in Biochemistry and Medicine
    LECTIN-ENZYME BINDING ASSAYS Development of the technique and applications in biochemistry and medicine LECTINE-ENZYM BINDINGS ANALYSES Ontwikkeling van de techniek en enige toepassingen in de biochemie en de geneeskunde PROEFSCHRIFT Ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam op gezag van de Rector Magnificus Prof. Dr. A.H.G. Rinnooy Kan en volgens besluit van het College van Dekanen. De openbare verdediging zal plaatsvinden op woensdag 18 januari 1989 om 15.45 uur door Jan Maurits Pekelharing geboren te Eindhoven 1989 DRUKKERU J.H. PASMANS B.V., 's-GRAVENHAGE PROMOTIECOMMISSIE Promotor: Prof. Dr. B. Leijnse Overige leden: Prof. Dr. E.H. Cooper, MD (Leeds) Prof. Dr. H.G. van Eijk Prof. Dr. J.BJ. Soons (Utrecht) Aan moeder Saar, voor het creatieve Aan vader Johan, voor het nuchtere CIP-DATA KONINKLIJKE BIBLIOTHEEK, DEN HAAG Pekelha~ing, Jan Maurits Lectin-enzyme binding assays : development of the technique and applications in biochemistry and medicine I Jan Maurits Pekelharing. - CS.l. : s.n.J C's-Gravenhage Pasmans). -Ill. Thesis Rotterdam. -With index, ref. -With summary in Dutch ISBN 90-9002656-8 SISO 546 UDC 547.96(043.3) Subject heading: lectin-enzyme binding assays. 5 CONTENTS Chapter 1: Lectins and the investigation of plasma protein glycosylation 1.1. Introduction 11 1.2. Lectins 11 1.2.1. History 11 1.2.2. Definitions 12 1.2.3. Occurrence 13 1.2.4. Specificity 13 1.2.5. Lectin methods 14 1.3. Protein glycosylation 15 1.3.1. Roles of glycosylation 17 1.3.2. Changes in plasma glycoprotein glycosylation 20 1.4.
    [Show full text]
  • Clinical Applications of Direct Antiglobulin Test
    Blood, Heart and Circulation Review Article ISSN: 2515-091X Clinical applications of direct antiglobulin test Jeong-Shi Lin1,2,3* 1Division of Transfusion Medicine, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 2Division of Hematology, Department of Medicine, Taipei Veterans General Hospital, Taipei, Taiwan 3National Yang-Ming University School of Medicine, Taipei, Taiwan Abstract The direct antiglobulin test (DAT) is used to detect immunoglobulin and/or complement on the surface of red blood cells (RBCs). The DAT is valuable in the investigation of autoimmune hemolytic anemia, drug-induced immune hemolysis, hemolytic disease of newborn, hemolytic transfusion reactions, and passenger lymphocyte syndrome. There are several limitations of DAT, such as sensitivity, false positive and false negative. The patient's clinical history, diagnoses, and other laboratory test results should also take into consideration for DAT interpretation. Introduction Gel microcolumn DATs were more sensitive than tube agglutination and affinity microcolumn DATs [12]. Gel microcolumn DAT is a The direct antiglobulin test (DAT), also known as ' direct Coombs’ better alternative to conventional tube DAT for detecting red cell test', was found more widespread notoriety after been described in 1945 bound antibodies in various clinical conditions [13]. The sensitive gel by Cambridge immunologist Robin Coombs. The DAT is used to detect technology has enabled the hematologist not only to diagnose some immunoglobulin, complement, or both on the surface of red blood AIHA patients with negative DAT, but also to characterize red cell cells (RBCs). The indirect antiglobulin test (IAT) is used to detect red bound autoantibodies with regard to their class, subclass and titer in a cell antibodies in patient serum.
    [Show full text]
  • Serology of Paracoccidioidomycosis
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Repositório Institucional UNIFESP Mycopathologia (2008) 165:289–302 DOI 10.1007/s11046-007-9060-5 Serology of paracoccidioidomycosis Zoilo Pires de Camargo Received: 10 July 2007 / Accepted: 30 August 2007 Ó Springer Science+Business Media B.V. 2007 Abstract This review provides the background for Keywords Antigens Á Mycosis Á understanding the role of a battery of diagnostic Paracoccidioidomycosis Á Paracoccidioides methods in paracoccidioidomycosis (PCM). This brasiliensis Á Serology systemic mycosis is a disease endemic in many regions of Latin America, with sporadic cases also occurring throughout the world (mycosis of importa- Introduction tion). Although excellent laboratory methods for diagnosis are available, there are deficiencies that The fungus Paracoccidioides brasiliensis is must be met by continued research. Understanding ensconced in the nature in Latin America, but its the uses and limitations of a battery of laboratory exact niche is not known. The expanding human and methods is essential to diagnose PCM. Clinicians and non-human populations of endemic areas provide a laboratory directors must be familiar with the uses continuing supply of individuals who are susceptible and limitations of a battery of serologic and myco- to infection with P. brasiliensis. Paracoccidioidomy- logical tests to accurately diagnose of PCM. cosis (PCM) is caused by the inhalation of conidia Antibody and antigen detections are valuable found in nature and under adverse nutritional condi- adjuncts to histopathology and culture. More tions, the fungus sporulates forming conidia which recently, the gp43 and gp70 antigen detection assay are less than 5 lm in diameter and can easily reach have improved the methodology of diagnosis of this the alveoli when inhaled, and a lung-lymph node mycosis, which improves reproducibility and facili- primary complex develops.
    [Show full text]
  • Cross-Talk Between Gonadotropin- Releasing
    CROSS-TALK BETWEEN GONADOTROPIN- RELEASING HORMONES AND PROGESTERONE RECEPTOR IN NEUROENDOCRINE CELLS by BEUM-SOO AN D.V.M., Chung-buk National University, 2000 M.Sc, Chung-buk National University, 2002 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Reproductive & Developmental Sciences) THE UNIVERSITY OF BRITISH COLUMBIA March 2007 © Beum-soo AN, 2007 ABSTRACT Hypothalamic gonadotropin-releasing hormone (GnRH) is a decapeptide that plays a pivotal role in mammalian reproduction. It is hypothesized that progesterone (P4) may regulate GnRH I, GnRH II (a second form of GnRH) and GnRH I receptor (GnRH I R) at the transcriptional level. Alternatively, GnRHs may stimulate transactivation of the progesterone receptor (PR), thereby, modulating gonadotropin subunit gene expression. Treatment of human neuronal cells with P4 suppressed GnRH I R promoter activity. This P4-stimulated inhibition was enhanced when PR A was over-expressed. With respect to the two GnRHs, P4 increased GnRH I mRNA levels, but did not significantly affect GnRH II gene expression. Regulation of gonadotropin production involves interplay between steroids and neuro• peptides, thus we have examined the effects of GnRHs on PR activation in pituitary cells. Treatment with GnRHs increased a progesterone response element (PRE)-luciferase reporter gene activity. PR was phosphorylated at Ser294 and translocated into nucleus after GnRH treatment in the absence of P4. Interactions between the PR and several coactivators were examined, and treatment with GnRHs specifically induced PR: Steroid Receptor Coactivator-3 (SRC-3) interaction. In chromatin immunoprecipitation assays, recruitment of PR and SRC-3 to the PRE reporter gene was also increased by GnRHs.
    [Show full text]