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Functional Glycomics
1st Edition, Volume 417 - November 21, 2006
Editor: Minoru Fukuda
Language: English
Hardback ISBN:9780121828226
9 7 8 - 0 - 1 2 - 1 8 2 8 2 2 - 6
eBook ISBN:9780080469003
9 7 8 - 0 - 0 8 - 0 4 6 9 0 0 - 3
In the past decade, there has been an explosion of progress in understanding the roles of carbohydrates in biological systems. This explosive progress was made with the efforts in…Read more
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In the past decade, there has been an explosion of progress in understanding the roles of carbohydrates in biological systems. This explosive progress was made with the efforts in determining the roles of carbohydrates in immunology, neurobiology and many other disciplines, examining each unique system and employing new technology. This volume represents the second of three in the Methods in Enzymology series, including Glycobiology (vol. 415) and Glycomics (vol. 416), dedicated to disseminating information on methods in determining the biological roles of carbohydrates. These books are designed to provide an introduction of new methods to a large variety of readers who would like to participate in and contribute to the advancement of glycobiology. The methods covered include structural analysis of carbohydrates, biological and chemical synthesis of carbohydrates, expression and determination of ligands for carbohydrate-binding proteins, gene expression profiling including micro array, and generation of gene knockout mice and their phenotype analyses.
Graduate students, researchers in academics and industry studying biochemistry and glycobiology.
Contributors to Volume 417
Publisher Summary
Preface
Publisher Summary
Contents of Previous Volumes
Section I. N-Glycan Function Revealed by Gene Inactivation
Cytokine Sensitivity and N-Glycan Processing Mutations
Abstract
Introduction
Materials
Methods
Notes
Acknowledgments
Phenotype Changes of Fut8 Knockout Mouse: Core Fucosylation Is Crucial for the Function of Growth Factor Receptor(s)
Abstract
Overview
Materials and Methods
Section II. Neural Cell Function
Cellular and Molecular Analysis of Neural Development of Glycosyltransferase Gene Knockout Mice
Abstract
Overview
Acknowledgment
Roles of Glycolipids in the Development and Maintenance of Nervous Tissues
Abstract
Overview
Genetic Approaches
Fundamental Behavior Analysis
Surgical Approaches for Repair Potential
Morphological Approaches
Analysis of Neural Cell Functions in Gene Knockout Mice: Electrophysiological Investigation of Synaptic Plasticity in Acute Hippocampal Slices
Abstract
Overview
Acknowledgments
Analysis of Neural Cell Function in Gene Knockout Mice: Behavior
Abstract
Introduction
Materials
Methods
Acknowledgments
Electrophysiological Analysis of Interactions Between Carbohydrates and Transmitter Receptors Reconstituted in Lipid Bilayers
Abstract
Overview
Acknowledgment
Section III. Notch Signaling and Muscular Dystrophy
Methods for Analysis of O-Linked Modifications on Epidermal Growth Factor-Like and Thrombospondin Type 1 Repeats
Abstract
Overview
Analysis of O-Fucose and O-Glucose Glycan Structure by Metabolic Radiolabeling Coupled with Alkali-Induced β-Elimination
Glycosylation Site Mapping by Mass Spectrometry
Acknowledgments
Roles of O-Fucosyltransferase 1 and O-Linked Fucose in Notch Receptor Function
Abstract
Overview
Enzyme Assay for O-Fucosyltransferase 1 Activity
Downregulation of Ofut1 by Use of RNAi Techniques
Quantification of the O-Fucose Level
Cell Surface Notch Staining
Notch Secretion Assay
Western Blotting for Notch
Notch-Ligand Binding Assay
Notch–OFUT1 Binding Assay
Acknowledgments
Roles of O-Fucose Glycans in Notch Signaling Revealed by Mutant Mice
Abstract
Overview
Glycosylation of Notch Receptors and Notch Ligands
Pofut1 Is an Essential Component of the Canonical Notch Signaling Pathway
Fringe Is a Modulator of Notch Signaling
β4GalT-1 Is a Novel Regulator of Notch Signaling
Mutations in Other Glycosyltransferase Genes
Acknowledgments
Defect in Glycosylation that Causes Muscular Dystrophy
Abstract
Overview
Purification of α-DG
Method for Glycoprotein Enrichment and Western Blot Analysis (Laminin Overlay Assay)
Method for Laminin Overlay Assay
Solid-Phase Binding Assay
Assay for POMT and POMGnT1 Activities
Preparation of Membrane Fraction for Enzyme Sources from Brain
Preparation of GST-α-DG
POMT Assay
Expression and Enzymatic Activity of POMT1 Mutants
POMGnT1 Assay
POMGnT1 Assay
Expression and Enzymatic Activity of POMGnT1 Mutants
Section IV. Glycolipid Function
Identification and Analysis of Novel Glycolipids in Vertebrate Brains by HPLC/Mass Spectrometry
Abstract
Overview
Isolation of PtdGlc from Developing Brains
Extraction of Lipids
Purification of PtdGlc
Identification of PtdGlc by Normal-phase HPLC/Electrospray Ionization Ion-trap Mass Spectrometry
Modulation of Growth Factor Signaling by Gangliosides: Positive Or Negative?
Abstract
Overview
Methods
Results and Conclusion
Acknowledgments
Activation of Natural Killer T Cells by Glycolipids
Abstract
Overview
Vα14i NKT Cell Isolation
In Vitro Antigen Presentation Assay
In Vivo iNKT Cell Activation Assay
Biochemical Assays
Section V. Glycolipid Interaction
Determination of Glycolipid–Protein Interaction Specificity
Abstract
Overview
Materials
Methods
Notes
Analysis of Carbohydrate–Carbohydrate Interactions Using Gold Glyconanoparticles and Oligosaccharide Self-Assembling Monolayers
Abstract
Overview
Marine Sponges
Synthesis of Spacer-Containing Oligosaccharides
Synthesis of Thiol-Spacer–Containing Oligosaccharides
Synthesis of Gold Nanoparticles
Analysis of Gold Glyconanoparticles
Aggregation Experiments Using TEM
Atomic Force Microscopy
Section VI. Galectin Function
Galectin Interactions with Extracellular Matrix and Effects on Cellular Function
Abstract
Overview
Chemicals and Reagents
Preparation of ECM
T Cell–ECM Adhesion Assays
Cell Migration through ECM
Assessing Cell Death on Matrigel
Conclusions
Acknowledgments
Galectins in Apoptosis
Abstract
Overview
Regulation of Apoptosis by Extracellular Galectins
Regulation of Apoptosis Demonstrated by Use of the Gene Transfection Approach
Preparation of Recombinant Galectin-3
Overexpression of Galectin-3 in Eukaryotic Cells
Acknowledgments
On the Role of Galectins in Signal Transduction
Abstract
Overview
The Role of Extracellular Galectins in Signal Transduction
The Role of Intracellular Galectins in Signal Transduction
Construction of Expression Vectors
Binding Assays for Extracellular Galectins
Analyses of Phosphorylated Proteins
Kinase Assays
Section VII. Newly Developed Field
Helicobacter pylori Adhesion to Carbohydrates
Abstract
Overview
Characterization of Bacterial Adherence by In Vitro Binding to Host Tissue
Glycoprotein Array for Screening and Identification of Adhesin Binding Properties
Analyses of Binding Capacity Based on of RIA and Scatchard Affinity Assays a Nonradioactive Alternative Based on Fluorescent Glycoconjugates
A Nonradioactive Alternative to Test for H. pylori Binding Properties in Solution: Fluorochrome-labeled Glycoconjugates
H. Pylori Binding Assay Using Fluorescent Glycoconjugates
Affinity Analysis by RIA According to Scatchard
ELISA Analysis of H. pylori Binding to Immobilized Lewis b Glycoconjugate
Receptor Activity–directed Affinity Tagging (Retagging) Technique for Adhesin Protein Identification
Phylogenetic Methods for Detecting Adaptive Change
Knock-out of Adhesin Gene: General Strategy for Genetic Constructions by PCR without Recombinant DNA Cloning
Identification of Bacterial Clones with Rare and/or Induced Receptor Binding Properties by Use of Glycoprobes
Characterization of Spatial Expression of H. pylori Virulence Products in Gastric Mucosa by In Situ Hybridization
Application of Laser Tweezers Technique for Analyzes of Binding Strength
Analyses of Nonopsonic Bacterial–Cell Interactions by Chemiluminescence Measurements of Released Oxidative Burst Metabolites
Acknowledgments
Caenorhabditis elegans Carbohydrates in Bacterial Toxin Resistance
Abstract
Overview
Procedure for EMS Mutagenesis
Isolation of Glycosphingolipids from C. elegans by Svennerholm Partitioning
Analysis of Glycolipids by Thin Layer Chromatography
Isolation of Specific Glycosphingolipid Components
N-Glycans Are Involved in the Response of Caenorhabditis elegans to Bacterial Pathogens
Abstract
Overview
The Biology of Caenorhabditis elegans
The Structure and Biosynthesis of GlcNAcβ1-N-Asn Glycans (N-Glycans) in C. elegans
The Function of N-Glycans in C. elegans
Experimental
Results
Effects of Pseudomonas aeruginosa Strain 14 on PGS Medium on the Survival of C. elegans
Production of gly-13 and gly-14 Phenotypes by RNA-mediated Interference (RNAi)
Rescue of the Mutant gly-13 Phenotype by a Wild-type gly-13 Extrachromosomal Array
Discussion
Acknowledgments
Detection of Cytoplasmic Glycosylation Associated with Hydroxyproline
Abstract
Overview
In Vivo Detection of HyPro-dependent Glycosylation by Mr-Shift Analysis