Histochemistry of Single Molecules: Methods and Protocols by Carlo Pellicciari and Marco Biggiogera


7759a6668166b0d-261x361.jpg Author Carlo Pellicciari and Marco Biggiogera
Isbn 9781493967872
File size 16.20MB
Year 2017
Pages 341
Language English
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Category chemistry



 

Methods in Molecular Biology Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 Histochemistry of Single Molecules Methods and Protocols Edited by Carlo Pellicciari and Marco Biggiogera Department of Biology and Biotechnology, University of Pavia, Pavia, Italy Editors Carlo Pellicciari Department of Biology and Biotechnology University of Pavia Pavia, Italy Marco Biggiogera Department of Biology and Biotechnology University of Pavia Pavia, Italy ISSN 1064-3745     ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-6787-2    ISBN 978-1-4939-6788-9 (eBook) DOI 10.1007/978-1-4939-6788-9 Library of Congress Control Number: 2016962655 © Springer Science+Business Media LLC 2017 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Cover Illustration: Double staining of claudin-5 (red fluorescence) and beta-actin (green) in the glomerulus of mouse kidney in a formalin-fixed paraffin-embedded tissue. Beta-actin and claudin-5 colocalize along the capillary walls showing yellow to orange color, which might correspond to pedicels of podocytes. Beta-actin is also demonstrated in the mesangial cells. Nuclei were stained with the DNA-specific dye DAPI (blue fluorescence). (Courtesy of Shuji Yamashita and Osamu Katsumata.) Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A. Preface The story of histochemistry goes a long way back and, in the last 150 years, has evolved in parallel with the operating instruments, from bright field microscopy to super-resolution microscopy. In this evolution resides the uniqueness of this science. Compared to the biochemical and molecular methods, the distinctive feature of histochemical techniques is the ability to localize chemical species in the very place (in a tissue, or a cell or an organelle) where they exist, are produced, or operate in vivo; this makes histochemistry an irreplaceable tool in basic and applied biomedical research. Actually, over the last fifteen years, about 400,000 “histochemical” articles have been published in qualified international journals (according to the Scopus database). This demonstrates the impact of histochemistry on a wide variety of research subjects (from cell and tissue biology to anatomy and pathology, from zoology to botany, from ecology to nanotechnology), where histochemistry is essentially used for localizing (and often quantifying) in situ single molecules or molecular complexes to relate structural organization and function. This book aims at providing an (certainly nonexhaustive) overview of histochemical techniques, through a series of lab-tested protocols for the detection of specific molecules or metabolic processes, both at light and electron microscopy. More in detail, the book is divided into six parts covering a variety of chemical targets. The first part is on vital histochemistry, including overviews on single-cell histochemistry and autofluorescence. In these chapters, the detection of enzymatic activities is shown through the protocols for detection of peptidases, and a series of enzyme-histochemical methods are described for investigating functional histology in different invertebrate taxa. Lectin histochemistry is represented by an overview followed by the use of lectins to detect glycosylation-specific cell types, cancer cells, or apoptotic cells. Histochemistry of proteins used to be the hardest part of the histochemical course for a student in the 1970s, with a huge number of reactions for different chemical groups; nowadays antibodies have the stage, and in the third part, they are used to detect proteins marking neuronal differentiation, the myogenic progenitors, or autophagy. An essential prerequisite for all these techniques is antigen preservation and detection, which is often made problematic by sample fixation and embedding; a chapter is therefore devoted to the most suitable protocols for retrieving antigens in formalin-fixed paraffin-embedded specimens and frozen sections. The fourth part on lipid histochemistry offers basics in fixation and tissue processing, staining for myelin, lipids in yeast, and single lipid droplets. Nuclear histochemistry, after an overview of DNA fluorochromes, contains protocols for staining and labeling DNA and RNA at electron microscopy as well as for single- and double-strand breaks detection. The last part is on plant histochemistry and deals with nuclear proteins, plant secretory structures, and acetogenins. v vi Preface From this short list of contents, covering molecules, tissues, and species very far from each other, a simple conclusion may be drawn: histochemistry can be, and actually is, applied in all these cases and with successful results. With their specificity and resolution, histochemical and cytochemical methods are more than alive today and can effectively help scientists in very different research fields to elucidate biological issues through a unique approach to molecular biology in situ. Pavia, Italy  Carlo Pellicciari Marco Biggiogera Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi Part I  Vital Histochemistry   1 Single Cell Cytochemistry Illustrated by the Demonstration of Glucose-6-Phosphate Dehydrogenase Deficiency in Erythrocytes . . . . . . . . . Anna L. Peters and Cornelis J.F. van Noorden   2 Autofluorescence Spectroscopy for Monitoring Metabolism in Animal Cells and Tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Anna C. Croce and Giovanni Bottiroli   3 Enzyme-Histochemistry Technique for Visualizing the Dipeptidyl-Peptidase IV (DPP-IV) Activity in the Liver Biliary Tree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vittorio Bertone, Eleonora Tarantola, and Isabel Freitas   4 Histochemical Demonstration of Tripeptidyl Aminopeptidase I . . . . . . . . . . . . Mashenka B. Dimitrova, Dimitrinka Y. Atanasova, and Nikolai E. Lazarov   5 Enzyme Histochemistry for Functional Histology in Invertebrates . . . . . . . . . . Francesca Cima 3 15 45 55 69 Part II Lectin Histochemistry   6 Lectin Histochemistry: Historical Perspectives, State of the Art, and the Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Susan A. Brooks   7 Isolation of Viable Glycosylation-Specific Cell Populations for Further In Vitro or In Vivo Analysis Using Lectin-Coated Magnetic Beads . . . . . . . . . . Ellie-May Beaman, David R.F. Carter, and Susan A. Brooks   8 Lectin Histochemistry for Metastasizing and Non-­metastasizing Cancer Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gerrit Wolters-Eisfeld and Udo Schumacher   9 The Use of Lectin Histochemistry for Detecting Apoptotic Cells in the Seminiferous Epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vicente Seco-Rovira, Ester Beltrán-Frutos, Jesús Hernández-Martínez, Concepción Ferrer, and Luis Miguel Pastor 93 109 121 133 Part III  Protein Histochemistry 10 Heat-Induced Antigen Retrieval in Immunohistochemistry: Mechanisms and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Shuji Yamashita and Osamu Katsumata vii viii Contents 11 Detecting Neuronal Differentiation Markers in Newborn Cells of the Adult Brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 Sara Bonzano and Silvia De Marchis 12 Characterizing Satellite Cells and Myogenic Progenitors During Skeletal Muscle Regeneration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179 Nicolas A. Dumont and Michael A. Rudnicki 13 Immunohistochemical Detection of the Autophagy Markers LC3 and p62/SQSTM1 in Formalin-Fixed and Paraffin-­Embedded Tissue . . . . . . . 189 Sabina Berezowska and José A. Galván Part IV Lipid Histochemistry 14 Tissue Fixation and Processing for the Histological Identification of Lipids . . . Víctor Carriel, Fernando Campos, José Aneiros-Fernández, and John A. Kiernan 15 Staining Methods for Normal and Regenerative Myelin in the Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Víctor Carriel, Antonio Campos, Miguel Alaminos, Stefania Raimondo, and Stefano Geuna 16 Nile Red Staining of Neutral Lipids in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . Kerry Ann Rostron and Clare Louise Lawrence 17 Staining of Lipid Droplets with Monodansylpentane . . . . . . . . . . . . . . . . . . . . Bo-Hua Chen, Huei-Jiun Yang, He-Yen Chou, Guang-Chao Chen, and Wei Yuan Yang 197 207 219 231 Part V Nuclear Histochemistry 18 Fluorochromes for DNA Staining and Quantitation . . . . . . . . . . . . . . . . . . . . . Giuliano Mazzini and Marco Danova 19 Osmium Ammine for Staining DNA in Electron Microscopy . . . . . . . . . . . . . . Irene Masiello and Marco Biggiogera 20 DNA Labeling at Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nicolas Thelen and Marc Thiry 21 Visualizing RNA at Electron Microscopy by Terbium Citrate . . . . . . . . . . . . . . Marco Biggiogera and Irene Masiello 22 Two-Tailed Comet Assay (2T-Comet): Simultaneous Detection of DNA Single and Double Strand Breaks . . . . . . . . . . . . . . . . . . . . . . . . . . . . Elva I. Cortés-Gutiérrez, José Luis Fernández, Martha I. Dávila-­Rodríguez, Carmen López-Fernández, and Jaime Gosálvez 239 261 269 277 285 Part VI  Plant Histochemistry 23 Detection of Endogenous Nuclear Proteins in Plant Cells: Localizing Nuclear Matrix Constituent Proteins (NMCPs), the Plant Analogs of Lamins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297 Malgorzata Ciska and Susana Moreno Díaz de la Espina Contents ix 24 Histochemical Analysis of Plant Secretory Structures . . . . . . . . . . . . . . . . . . . . 313 Diego Demarco 25 A Histochemical Technique for the Detection of Annonaceous Acetogenins . . . . 331 Guillermo Laguna-Hernández, Alicia Enriqueta Brechú-Franco, Iván De la Cruz-Chacón, and Alma Rosa González-Esquinca Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339 Contributors Miguel Alaminos  •  Tissue Engineering Group, Department of Histology, Faculty of Medicine, University of Granada, Granada, Spain; Instituto de Investigación Biosanitaria Ibs, Granada, Spain José Aneiros-Fernández  •  Pathology Unit, Hospital Universitario Virgen de las Nieves, Granada, Spain Dimitrinka Y. Atanasova  •  Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria; Department of Anatomy, Faculty of Medicine, Trakia University, Stara Zagora, Bulgaria Ellie-May Beaman  •  Department of Biological & Medical Sciences, Oxford Brookes University, Headington, Oxford, UK Ester Beltrán-Frutos  •  Department of Cell Biology and Histology, School of Medicine, IMIB-Arrixaca, University of Murcia, Espinardo, Murcia, Spain Sabina Berezowska  •  Institute of Pathology, University of Bern, Bern, Switzerland Vittorio Bertone  •  Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy Marco Biggiogera  •  Department of Biology and Biotechnology, Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy Sara Bonzano  •  Department of Life Sciences and Systems Biology (DBIOS), University of Turin, Turin, Italy; Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano, Turin, Italy Giovanni Bottiroli  •  Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy Alicia Enriqueta Brechú-Franco  •  Facultad de Ciencias, Universidad Nacional Autónoma de México, Colonia Universidad Nacional Autónoma de México, Coyoacán, Ciudad de México, Mexico Susan A. Brooks  •  Department of Biological & Medical Sciences, Oxford Brookes University, Headington, Oxford, UK Antonio Campos  •  Tissue Engineering Group, Department of Histology, Faculty of Medicine, University of Granada, Granada, Spain; Instituto de Investigación Biosanitaria Ibs, Granada, Spain Fernando Campos  •  Tissue Engineering Group, Department of Histology, Faculty of Medicine, University of Granada, Granada, Spain Víctor Carriel  •  Tissue Engineering Group, Department of Histology, Faculty of Medicine, University of Granada, Granada, Spain; Instituto de Investigación Biosanitaria Ibs, Granada, Spain David R.F. Carter  •  Department of Biological & Medical Sciences, Oxford Brookes University, Headington, Oxford, UK Bo-Hua Chen  •  Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Taipei, Taiwan; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan xi xii Contributors Guang-Chao Chen  •  Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Taipei, Taiwan; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan He-Yen Chou  •  Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Francesca Cima  •  Department of Biology, University of Padova, Padova, Italy Malgorzata Ciska  •  Department of Cell and Molecular Biology, Biological Research Center, CSIC, Madrid, Spain Elva I. Cortés-Gutiérrez  •  Department of Genetics, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, Mexico Anna C. Croce  •  Institute of Molecular Genetics (IGM), CNR, Pavia, Italy Iván De la Cruz-Chacón  •  Instituto de Ciencias Biológicas, Universidad de Ciencias y Artes de Chiapas, Tuxtla Gutiérrez, Chiapas, Mexico Marco Danova  •  Department of Medicine, Azienda Socio-Sanitaria Territoriale, Pavia, Italy Martha I. Dávila-Rodríguez  •  Department of Genetics, Centro de Investigación Biomédica del Noreste, Instituto Mexicano del Seguro Social, Monterrey, Mexico Diego Demarco  •  Laboratório de Anatomia Vegetal, Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Sao Paulo, Brazil Mashenka B. Dimitrova  •  Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria Nicolas A. Dumont  •  Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Faculty of Medicine, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada; Sainte-Justine Hospital Research Center and Department of Rehabilitation, Faculty of Medicine, University of Montreal, Montreal, QC, Canada Susana Moreno Díaz de la Espina  •  Department of Cell and Molecular Biology, Biological Research Center, CSIC, Madrid, Spain José Luis Fernández  •  Genetics Unit, INIBIC, Complejo Hospitalario Universitario A Coruña and Laboratory de Genética Molecular y Radiobiología, Centro Oncológico de Galicia, La Coruña, Spain Concepción Ferrer  •  Department of Cell Biology and Histology, School of Medicine, IMIB-Arrixaca, University of Murcia, Espinardo, Murcia, Spain Isabel Freitas  •  Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy José A. Galván  •  Institute of Pathology, University of Bern, Bern, Switzerland Stefano Geuna  •  Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Torino, Italy Alma Rosa González-Esquinca  •  Instituto de Ciencias Biológicas, Universidad de Ciencias y Artes de Chiapas, Tuxtla Gutiérrez, Chiapas, Mexico Jaime Gosálvez  •  Unit of Genetics, Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain Jesús Hernández-Martínez  •  Department of Cell Biology and Histology, School of Medicine, IMIB-Arrixaca, University of Murcia, Espinardo, Murcia, Spain Osamu Katsumata  •  Department of Anatomy, Kitasato University School of Medicine, Kanagawa, Japan Contributors xiii John A. Kiernan  •  Department of Anatomy and Cell Biology, University of Western Ontario, London, ON, Canada Guillermo Laguna-Hernández  •  Facultad de Ciencias, Universidad Nacional Autónoma de México, Colonia Universidad Nacional Autónoma de México, Coyoacán, Ciudad de México, Mexico Clare Louise Lawrence  •  School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, Lancashire, UK Nikolai E. Lazarov  •  Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria; Department of Anatomy and Histology, Medical University of Sofia, Sofia, Bulgaria Carmen López-Fernández  •  Unit of Genetics, Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain Silvia De Marchis  •  Department of Life Sciences and Systems Biology (DBIOS), University of Turin, Turin, Italy; Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano, Turin, Italy Irene Masiello  •  Department of Biology and Biotechnology, Laboratory of Cell Biology and Neurobiology, University of Pavia, Pavia, Italy Giuliano Mazzini  •  Institute of Molecular Genetics, CNR, Pavia, Italy; Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy Cornelis J.F. van Noorden  •  Department of Cell Biology and Histology, Academic Medical Centre, Amsterdam, The Netherlands Luis Miguel Pastor  •  Department of Cell Biology and Histology, School of Medicine, IMIB-Arrixaca, University of Murcia, Espinardo, Murcia, Spain Anna L. Peters  •  Department of Intensive Care, Academic Medical Centre, Amsterdam, The Netherlands Stefania Raimondo  •  Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Turin, Italy Kerry Ann Rostron  •  School of Pharmacy and Biomedical Sciences, University of Central Lancashire, Preston, Lancashire, UK; School of Biological Sciences, University of Reading, Reading, Berkshire, UK Michael A. Rudnicki  •  Centre for Stem Cell Research, Ottawa Hospital Research Institute, Ottawa, ON, Canada; Faculty of Medicine, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, Canada Udo Schumacher  •  Department of Anatomy and Experimental Morphology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany Vicente Seco-Rovira  •  Department of Cell Biology and Histology, School of Medicine, IMIB-Arrixaca, University of Murcia, Espinardo, Murcia, Spain Eleonora Tarantola  •  Department of Biology and Biotechnology “Lazzaro Spallanzani”, University of Pavia, Pavia, Italy Nicolas Thelen  •  Giga Neurosciences, Department of Life Sciences, Laboratory of Cell Biology, University of Liège, Liège, Belgium Marc Thiry  •  Giga Neurosciences, Department of Life Sciences, Laboratory of Cell Biology, University of Liège, Liège, Belgium Gerrit Wolters-Eisfeld  •  Medical Glycobiology Group, Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany xiv Contributors Shuji Yamashita  •  Department of Pathology, School of Medicine, Keio University, Tokyo, Japan Huei-Jiun Yang  •  Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan Wei Yuan Yang  •  Chemical Biology and Molecular Biophysics Program, Taiwan International Graduate Program, Taipei, Taiwan; Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan; Institute of Biochemical Sciences, College of Life Sciences, National Taiwan University, Taipei, Taiwan Part I Vital Histochemistry Chapter 1 Single Cell Cytochemistry Illustrated by the Demonstration of Glucose-6-Phosphate Dehydrogenase Deficiency in Erythrocytes Anna L. Peters and Cornelis J.F. van Noorden Abstract Cytochemistry is the discipline that is applied to visualize specific molecules in individual cells and has become an essential tool in life sciences. Immunocytochemistry was developed in the sixties of last century and is the most frequently used cytochemical application. However, metabolic mapping is the oldest cytochemical approach to localize activity of specific enzymes, but in the last decades of the previous century and the first decade of the present century it almost became obsolete. The popularity of this approach revived in the past few years. Metabolism gained interest as player in chronic and complex diseases such as cancer, diabetes, neurodegenerative diseases, and vascular diseases and both enzyme cytochemistry and metabolic mapping have become important tools in life sciences. In this chapter, we present glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most prevalent enzyme deficiency worldwide, to illustrate recent developments in enzyme cytochemistry or metabolic mapping. The first assays which were developed quantified enzyme activity but were unreliable for single cell evaluation. The field has expanded with the development of cytochemical single cell assays and DNA testing. Still, all assays—from the earliest developed tests up to the most recently developed tests—have their place in investigations on G6PD activity. Recently, nanoscopy has become available for light and fluorescence microscopy at the nanoscale. For nanoscopy, cytochemistry is an essential tool to visualize intracellular molecular processes. The ultimate goal in the coming years will be nanoscopy of living cells so that the molecular dynamics can be studied. Cytochemistry will undoubtedly play a critical role in these developments. Key words Cytochemistry, History, Metabolic mapping, Glucose-6-phosphate dehydrogenase deficiency 1 Introduction Cytochemistry is the discipline that is applied to visualize specific molecules in individual cells and has become an essential tool in life sciences. Most users of cytochemical techniques do not realize nowadays that they are performing cytochemistry when they are labeling specific cellular proteins for flow cytometry and cell sorting, because this has become routine. Flow cytometry and cell Carlo Pellicciari and Marco Biggiogera (eds.), Histochemistry of Single Molecules: Methods and Protocols, Methods in Molecular Biology, vol. 1560, DOI 10.1007/978-1-4939-6788-9_1, © Springer Science+Business Media LLC 2017 3 4 Anna L. Peters and Cornelis J.F. van Noorden sorting are undoubtedly the most frequently-used applications of cytochemistry these days. Besides, cytochemistry has been an essential tool in electron microscopical analysis of specific molecules in cells to understand functioning of subcellular compartments. Here, electron dense markers have to be used instead of fluorescence markers. Recently, nanoscopy has become available for light and fluorescence microscopy at the nanoscale using approaches such as STED, SIM, PALM, and STORM [1–3]. These techniques do not obey the law of Abbe with respect to the limit of spatial resolution when using light to obtain images because of the wavelength character of light. For nanoscopy, cytochemistry is essential to visualize intracellular molecular processes. The ultimate goal in the coming years will be nanoscopy of living cells so that intracellular molecular dynamics can be studied. Text books will then be rewritten in a similar way as in the past decades on the basis of living cell imaging in the micrometer range using cytochemistry and confocal microscopy. Cytochemistry as it is applied these days is almost exclusively based on immunocytochemistry to visualize specific proteins. It should be realized that the presence of a protein in (sub)cellular compartments does not automatically mean that this protein exerts the activity the researcher thinks that he/she investigates. It becomes more and more clear that epigenetic and posttranslational modifications of a protein, its microenvironment and/or its moonlighting (i.e., exerting different functions) are essential aspects of cellular proteins. Therefore, tools to visualize activity of proteins in situ are urgently needed to increase our understanding of cellular functions in health and disease. One such cytochemical approach is the visualization of the activity of a specific type of protein, enzymes, using enzyme cytochemistry or metabolic mapping. In fact, it is the oldest cytochemical approach of single molecule cytochemistry since Gomori published his cytochemical study on phosphatase activity in 1939 [4]. Immunochemistry was developed in the 1960s of last century and in situ hybridization for the visualization of specific mRNAs as measure of expression of a specific gene even later. Nevertheless, enzyme cytochemistry or metabolic mapping never grew into a position in cytochemistry that it deserves. In fact, in the last decades of the previous century and the first decade of the present century it almost became obsolete. However, since metabolism has gained novel interest as players in chronic and complex diseases such as cancer, diabetes, neurodegenerative diseases, and vascular diseases, enzyme cytochemistry or metabolic mapping has revived tremendously [5, 6]. In this chapter, we present the cytochemical visualization of glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most prevalent enzyme deficiency worldwide, to illustrate recent developments in enzyme cytochemistry or metabolic mapping. G6PD is the key enzyme in the oxidative pentose phosphate pathway (PPP). History of Single Cell Cytochemistry 5 Fig. 1 The NADPH-producing steps of the oxidative pentose phosphate pathway. 6PG 6-phosphogluconate, 6PGD 6-phosphogluconate dehydrogenase, 6PGL 6-phosphogluconolactone, G6P glucose-6-phosphate, G6PD glucose-6-­ phosphate dehydrogenase, GL gluconolactonase, R5P ribulose-5-phosphate This pathway is one of the major pathways in which nicotinamide adenine dinucleotide phosphate (NADP+) is converted into its reduced form NADPH, which is essential for the protection against reactive oxygen species (ROS) in cells and in particular erythrocytes (Fig. 1) [7, 8]. G6PD deficiency can lead to acute hemolytic anemia (AHA), chronic non-spherocytic hemolysis and hyperbilirubinemia resulting in neonatal kernicterus [9]. G6PD deficiency is the most common enzyme deficiency and worldwide an estimated 300–400 million people carry at least one deficient G6PD gene [10, 11]. The deficiency is mainly found in Africa, Asia, and Mediterranean Europe, areas where malaria is endemic or has been endemic [10, 12]. In these areas, G6PD deficiency has been found to protect against severe malaria infection [13]. 2  Early History of G6PD Deficiency Accounts of G6PD deficiency can be traced back to Pythagoras. The ancient Greek philosopher and mathematician reportedly forbade his followers to eat fava beans (Vica faba), possibly because of their potential to induce favism [14, 15]. Favism is a pathological disorder in which a G6PD-deficient individual develops AHA after ingestion of fava beans. Reports of favism date back centuries and investigations into its pathogenesis intensified in the first half of the twentieth century. In the early decades, several reports of favism were published which coincided with cases of AHA after ingestion of 6-methoxy-8-aminoquinoline drugs for the treatment of malaria 6 Anna L. Peters and Cornelis J.F. van Noorden [16]. Detailed research was performed on the metabolism of the erythrocyte, and in 1954 induction of AHA by primaquine (a similar compound as 6-methoxy-8-aminoquinoline) was attributed to G6PD deficiency [17]. Some years later, the similarities between AHA induced by primaquine and by favism became apparent and the link between favism and G6PD deficiency was established [18, 19]. It was observed that G6PD deficiency did not transfer from father to son and that all males who carry the gene show full expression. It thus was concluded that G6PD deficiency is transferred X-chromosomally. Males can be hemizygously deficient, while females can be homozygously deficient or heterozygously deficient. Heterozygously-deficient women have a mixed population of G6PD-sufficient and G6PD-deficient erythrocytes, owing to random inactivation of one of the two X-chromosomes. This process is known as lyonization [20]. 3  Enzymology G6PD is the rate-limiting enzyme of the PPP. The enzyme is a monomer of 515 amino acids and has a molecular mass of over 59 kDa [21]. Over 300 variants have been identified based on enzyme kinetics and physicochemical characteristics [22]. G6PD is activated by formation of a dimer or tetramer that contains tightly bound NADP+ [23]. Active G6PD initiates the PPP in which NADP+ is converted into NADPH, and G6P is converted into a pentose sugar, ribulose-5-phosphate, precursor of DNA, RNA, and ATP. NADPH is the most important reducing agent in the cytoplasm (Fig. 1) [7, 8]. During aging of the erythrocyte, the quantity of active G6PD decreases. The cells are not able to synthesize new proteins as mature erythrocytes do not have a nucleus, mitochondria, or ribosomes [24]. This results in increased susceptibility to oxidative stress in senescent erythrocytes. This natural process occurs more rapidly in G6PD deficiency. The deficiency causes increased susceptibility of erythrocytes to H2O2 and other ROS that can lead to AHA. Still, in daily life, G6PD deficiency is usually clinically silent because only 1–2 % of the total NADPH production capacity is used in healthy erythrocytes, even in episodes of hemolytic stress [25]. However, numerous drugs and chemicals, such as primaquine and dapsone, ingestion of fava beans, and stress (for example, infection) can induce AHA in G6PD-deficient individuals [12]. The severity of G6PD deficiency is usually measured in four classes in which class I is the most severe form of deficiency and class IV has near to normal function (Table 1) [25, 26]. Mutations in the region of the enzyme where NADP+ or G6P bind cause severe loss of G6PD activity and are associated with class I ­deficiencies. These deficiencies are rare but can induce transfusion History of Single Cell Cytochemistry 7 Table 1 Classes of G6PD deficiency Percentage of normal G6PD function, % WHO class Severity Class I Severe mutations with chronic non-spherocytic hemolytic anemia ≤10 Class II Intermediate 10 Class III Mild 10–60 Class IV Asymptomatic 60–100 Class V Increased function >100 The WHO designates four classes of G6PD deficiency [25]. Sometimes a fifth class of mutations causing increased G6PD activity is included [26] dependency as patients in this class usually suffer from chronic non-spherocytic hemolytic anemia. The other classes of deficiency are usually asymptomatic unless patients are in contact with agents that induce hemolytic stress. 4  Investigations on G6PD Activity In 1967, the World Health Organization (WHO) published recommendations on the standardisation of investigations of G6PD deficiency [27]. At this time, several G6PD-enzyme variants had been detected and in this bulletin it was recommended that all investigations should include spectrophotometric estimations of erythrocyte G6PD activity, electrophoretic enzyme migration, the Michaelis constant for G6P, thermal stability and the relative rate of utilization of G6P. The gold standard for detection of G6PD deficiency was spectrophotometry. Several tests had already been developed on the basis of dye decolorization methods (brilliant cresyl blue), methemoglobin reduction and reoxygenation, spot testing with tetrazolium salts, and fluorescent spot tests. However, spectrophotometry was the only assay in which G6PD activity could be quantified, thus it became the golden standard. 5  Diagnosis of G6PD Deficiency Diagnosing G6PD deficiency in hemizygous males is (usually) straight forward. In hemizygous males, all erythrocytes express the deficiency and both qualitative and quantitative assays can detect this type of deficiency. The same holds for homozygously deficient 8 Anna L. Peters and Cornelis J.F. van Noorden Fig. 2 Erythrocytes after cytochemical staining of glucose-6-phosphate dehydrogenase (G6PD) activity showing stained G6PD-containing erythrocytes (arrows) and unstained G6PD-deficient erythrocytes (arrowheads). The figure illustrates heterozygous deficiency with a mixed population of G6PD-containing erythrocytes and G6PD-deficient erythrocytes. Bar: 40 μm females. However, detection of heterozygously deficient females can be challenging as heterozygously deficient women have a mixed population of G6PD-sufficient and G6PD-deficient erythrocytes, owing to random inactivation of one of the two X chromosomes (Fig. 2) [20]. Diagnosing G6PD deficiency in these women can be difficult as many assays are based on quantitative methods that can miss females with favourable lyonization [28, 29]. Analysis of patients who recently suffered from AHA can also result in false negative test results, because young erythrocytes express normal G6PD activity and can thus mask G6PD deficiency. However, repeating of the test after a few weeks circumvents this problem. 6  Early Quantitative Assay: Spectrophotometry Spectrophotometry is a technique in which the light reflecting and/or light-absorbing potential of a molecule is quantified. To detect G6PD deficiency, the light transmittance of a solution with a hemolysate of blood is measured. The assay is based on the difference in absorbance of NADPH and NADP+ when excited with light of a wavelength of 340 nm. The assay is more reliable than most qualitative assays for detection of heterozygous females. However, it still can miss heterozygously deficient females with favorable lyonization. An alternative spectrophotometric method used for the detection of G6PD deficiency is based on inhibition by chromate of glutathione reductase (GSSG-R) activity in normal

Author Carlo Pellicciari and Marco Biggiogera Isbn 9781493967872 File size 16.20MB Year 2017 Pages 341 Language English File format PDF Category Chemistry Book Description: FacebookTwitterGoogle+TumblrDiggMySpaceShare This detailed volume explores numerous histochemical techniques through a series of lab-tested protocols for the detection of specific molecules or metabolic processes, both at light and electron microscopy. More in detail, the book is divided into six sections covering a variety of chemical targets. It begins with a section on vital histochemistry and continues with chapters on histochemistry as it relates to lectins, proteins, lipids, DNA and RNA, as well as plants. The volume also contains four overview chapters on vital histochemistry, lectin histochemistry, and DNA fluorochromes. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, Histochemistry of Single Molecules: Methods and Protocols aims to effectively help scientists in very different research fields to elucidate biological issues though a unique approach to molecular biology in situ.     Download (16.20MB) Unconventional Protein Secretion: Methods and Protocols Auxins and Cytokinins in Plant Biology Chemical Neurobiology: Methods And Protocols Chromatin Immunoprecipitation Assays: Methods and Protocols Dna Replication: Methods And Protocols, 2nd Edition Load more posts

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