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Contains 51 papers presenting readily reproducible preparative and analytical methods for the detailed analysis of proteoglycans, highly complex macromolecules found in mammalian and lower organisms. Suitable for both novice and experienced researchers, the protocols guide readers through the process of isolating intact proteoglycans from tissues and cultured cells, establishing the composition of their carbohydrate moieties, and generating strategies for prokaryotic and eukaryotic expression. Also presented are detailed techniques for the suppression of specific proteoglycan genes, for the detection of mutant cells and their degradation products, and for studying specific interactions between proteoglycans and extracellular matrix proteins and with growth factors and their receptors. Each section includes a general introduction, a detailed materials section, and a step-by-step methods section, accompanied by extensive notation including troubleshooting tips and practical considerations. Written by and of likely interest to molecular biologists, cell biologists, pathologists, biochemists, geneticists, oncologists, hematologists, rheumatologists, infectious disease specialists, and pediatricians.Book News, Inc.

Book Info

Thomas Jefferson Univ., Philadelphia, PA. Offers analytical and preparative methodologies for the analysis of glycosaminoglycans and details experimental strategies for inhibiting proteoglycan gene expression. For molecular and cellular biologists, biochemists, pathologists, and geneticists.

Preface
Contributors
1 Isolation of Proteoglycans from Cell Cultures and Tissues 3
2 Isolation of Proteoglycans from Tendon 9
3 Purification of Proteoglycans from Mineralized Tissues 19
4 Purification of Perlecan from Endothelial Cells 27
5 Isolation and Characterization of Nervous Tissue Proteoglycans 35
6 Analysis of Proteoglycans and Glycosaminoglycans from Drosophila 41
7 Cartilage and Smooth Muscle Cell Proteoglycans Detected by Affinity Blotting Using Biotinylated Hyaluronan 53
8 Preparation of Proteoglycans for N-Terminal and Internal Amino Acid Sequence Analysis 67
9 High-Specific-Activity [superscript 35]S-Labeled Heparan Sulfate Prepared from Cultured Cells 79
10 Selective Detection of Sulfotransferase Isoforms by the Ligand Affinity-Conversion Approach 91
11 Particulate and Soluble Glycosaminoglycan-Synthesizing Enzymes: Preparation Assay and Use 103
12 Disaccharide Composition of Hyaluronan and Chondroitin/Dermatan Sulfate: Analysis with Fluorophore-Assisted Carbohydrate Electrophoresis 117
13 Integral Glycan Sequencing of Heparan Sulfate and Heparin Saccharides 129
14 Analytical and Preparative Strong Anion-Exchange HPLC of Heparan Sulfate and Heparin Saccharides 141
15 Proteoglycans Analyzed by Composite Gel Electrophoresis and Immunoblotting 149
16 Quantitation of Proteoglycans in Biological Fluids Using Alcian Blue 159
17 Cellulose Acetate Electrophoresis of Glycosaminoglycans 175
18 Disaccharide Composition in Glycosaminoglycans/Proteoglycans Analyzed by Capillary Zone Electrophoresis 181
19 Intact and Oligomeric Glycosaminoglycans Analyzed with Capillary Electrophoresis 193
20 Recombinant Expression of Proteoglycans in Mammalian Cells: Utility and Advantages of the Vaccinia Virus/T7 Bacteriophage Hybrid Expression System 201
21 Purification of Recombinant Human Decorin and Its Subdomains 221
22 Prokaryotic Expression of Proteoglycans 231
23 Proteoglycan Gene Targeting in Somatic Cells 239
24 Constitutive and Inducible Antisense Expression 251
25 Cell-Mediated Transfer of Proteoglycan Genes 261
26 Morphological Evaluation of Proteoglycans in Cells and Tissues 271
27 Expression and Characterization of Engineered Proteoglycans 291
28 Intracellular Localization of Engineered Proteoglycans 301
29 Selection of Glycosaminoglycan-Deficient Mutants 309
30 Xyloside Priming of Glycosaminoglycan Biosynthesis and Inhibition of Proteoglycan Assembly 317
31 Inhibition of Heparan Sulfate Synthesis by Chlorate 325
32 Detection of Proteoglycan Core Proteins with Glycosaminoglycan Lyases and Antibodies 329
33 Proteoglycan Core Proteins and Catabolic Fragments Present in Tissues and Fluids 335
34 Degradation of Heparan Sulfate by Nitrous Acid 347
35 Degradation of Heparan Sulfate with Heparin Lyases 353
36 Degradation of Chondroitin Sulfate and Dermatan Sulfate with Chondroitin Lyases 363
37 In Vitro Assays for Hyaluronan Synthase 373
38 Hyaluronidase Activity and Hyaluronidase Inhibitors: Assay Using a Microtiter-Based System 383
39 Detecting Hyaluronidase and Hyaluronidase Inhibitors: Hyaluronan-Substrate Gel and -Inverse Substrate Gel Techniques 391
40 Affinity Coelectrophoresis of Proteoglycan - Protein Complexes 401
41 Binding Constant Measurements for Inhibitors of Growth Factor Binding to Heparan Sulfate Proteoglycans 415
42 Interaction of Proteoglycans with Receptor Tyrosine Kinases 427
43 Measuring Single-Cell Cytosolic Ca[superscript 2+] Concentration in Response to Proteoglycans 435
44 Serum Amyloid A Peptide Interactions with Glycosaminoglycans: Evaluation by Affinity Chromatography 449
45 Interactions of Lipoproteins with Proteoglycans 457
46 Hyaluronan and Hyaluronan-Binding Proteins: Probes for Specific Detection 479
47 Novel Confocal-FRAP Analysis of Carbohydrate - Protein Interactions Within the Extracellular Matrix 487
48 Regulatory Roles of Syndecans in Cell Adhesion and Invasion 495
49 Optical Biosensor Techniques to Analyze Protein-Polysaccharide Interactions 505
50 Phage Display Technology to Obtain Antiheparan Sulfate Antibodies 519
51 Tissue-Specific Binding by FGF and FGF Receptors to Endogenous Heparan Sulfates 535
Index 547