Biochemistry Books

Book SynopsisFungi have an integral role to play in the development of the biotechnology and biomedical sectors. The fields of chemical engineering, Agri-food,Biochemical, pharmaceuticals, diagnostics and medical device development allemploy fungal products, with fungal biomolecules currently used in a wide range of applications, ranging from drug development to food technology and agricultural biotechnology. Understanding the biology of different fungi in diverse ecosystems, as well as their biotropic interactions with other microorganisms, animals and plants, is essential to underpin effective and innovative technological developments.Fungal Biomolecules is a keystone reference, integrating branches of fungal product research into a comprehensive volume of interdisciplinary research. As such, it:reflects state-of-the-art research and current emerging issues in fungal biology and biotechnology reviews the methods and experimental work used to invTable of ContentsContributors ix Foreword xiii Preface xvii Section 1 Fungi as cell factories 1 Chapter 1 Fungal biofilms: An overview 3 Virginia Medeiros de Siqueira Chapter 2 Fungal biomolecules for the food industry 11 Quang D. Nguyen, Erika Bujna, Gabriella Styevko, Judit M. Rezessy-Szabo and Agoston Hoschke Chapter 3 Fungal biocatalysts in the textile industry: Whole?]cell systems in real textile wastewater treatment 39 Federica Spina, Valeria Tigini, Valeria Prigione and Giovanna Cristina Varese Chapter 4 Discovery of fungal enzymes and pathways 51 Aleksandra Mitrovic and Anton Glieder Chapter 5 Fungal laccase in the textile industry 63 Susana Rodriguez?]Couto Section 2 Production of recombinant peptides 73 Chapter 6 Lignocellulose?]degrading enzymes: An overview of the global market 75 Paula M. D. Jaramillo, Helder A. R. Gomes, Antonielle V. Monclaro, Caio O. G. Silva and Edivaldo X. F. Filho Chapter 7 Recent advancements in the role of volatile organic compounds from fungi 87 Lourdes Macias?]Rodriguez, Hexon Angel Contreras?]Cornejo, Jesus Salvador Lopez?]Bucio and Jose Lopez?]Bucio Chapter 8 Peptaibiotics and peptaibols from fungi 101 Susanne Zeilinger Section 3 Fungal secondary metabolites and synthesis 115 Chapter 9 Biosynthesis of silver nanoparticles by fungi 117 Ana Olivia de Souza and Alexandre Gomes Rodrigues Chapter 10 Fungal biomolecules as modulators of growth and pathogenesis 137 Rajesh N. Patkar and Naweed I. Naqvi Chapter 11 Fungi as a source of antitumour agents 145 Shin Yee Fung and Nget Hong Tan Chapter 12 Fungal mycotoxins: an overview 153 Svetlana V. Malysheva, Jose Diana Di Mavungu and Sarah De Saeger Chapter 13 Fungal pigments: An overview 173 Marcela C. Pagano and Partha P. Dhar Chapter 14 An overview regarding bioherbicide and their production methods by fermentation 183 Rodrigo Klaic, Raquel C. Kuhn, Edson L. Foletto, Valeria Dal Pra, Rodrigo J. S. Jacques, Jerson V. C. Guedes, Helen Treichel, Altemir J. Mossi, Debora Oliveira, J. Vladimir Oliveira, Sergio L. Jahn and Marcio A. Mazutti Section 4 Regulation of fungal secondary metabolism 201 Chapter 15 The role of nutrients in fungal development and pathogenesis 203 YiZhen Deng, Fan Yang and Naweed I. Naqvi Chapter 16 Role of nitrogen sources in regulation of fungal secondary metabolism 213 Bidisha Sharma and Dhruva Kumar Jha Chapter 17 Regulatory genes in fungal secondary metabolism 225 Jaswinder Kaur and Catherine Collins Chapter 18 Fungal metabolic diversity 239 Andrei Stecca Steindorff, Gabriela F. Persinoti, Valdirene Neves Monteiro and Roberto Nascimento Silva Chapter 19 Secreted biomolecules in fungal plant pathogenesis 263 Neil Andrew Brown and Kim E. Hammond?]Kosack Section 5 Developments and advancements 311 Chapter 20 Fungal molecular taxonomy: an overview 313 Marcela C. Pagano and Luiz H. Rosa Chapter 21 Fungal biomolecules in wines and beer industries 323 Carlos Roberto Felix and Eliane Ferreira Noronha Chapter 22 Biosorption and bioaccumulation of chromium VI by different fungal species 333 Dhara Shukla and Padma S. Vankar Chapter 23 Fungal biomolecules in plant growth promotion 345 Hexon Angel Contreras?]Cornejo, Lourdes Macias?]Rodriguez and Jose Lopez?]Bucio Chapter 24 Fungal biomolecules and their implications 363 Andleeb Zehra, Manish Kumar Dubey, Arti Tiwari, Mukesh Meena, Punam Kumari, Vivek Kumar Singh, Vijai Kumar Gupta and R. S. Upadhyay Index 377

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Book SynopsisA key resource for toxicologists across a broad spectrum of fields, this book offers a comprehensive analysis of molecular modelling approaches and strategies applied to risk assessment for pharmaceutical and environmental chemicals. Provides a perspective of what is currently achievable with computational toxicology and a view to future developments Helps readers overcome questions of data sources, curation, treatment, and how to model / interpret critical endpoints that support 21st century hazard assessment Assembles cutting-edge concepts and leading authors into a unique and powerful single-source reference Includes in-depth looks at QSAR models, physicochemical drug properties, structure-based drug targeting, chemical mixture assessments, and environmental modeling Features coverage about consumer product safety assessment and chemical defense along with chapters on open source toxicology and big data Table of ContentsList of Contributors xvii Preface xxi Acknowledgments xxiii Part I Computational Methods 1 1 AccessibleMachine Learning Approaches for Toxicology 3Sean Ekins, Alex M. Clark, Alexander L. Perryman, Joel S. Freundlich, Alexandru Korotcov, and Valery Tkachenko 1.1 Introduction 3 1.2 Bayesian Models 5 1.2.1 CDD Models 7 1.3 Deep LearningModels 13 1.4 Comparison of Different Machine LearningMethods 16 1.4.1 Classic Machine LearningMethods 17 1.4.1.1 Bernoulli Naive Bayes 17 1.4.1.2 Linear Logistic Regression with Regularization 18 1.4.1.3 AdaBoost Decision Tree 18 1.4.1.4 Random Forest 18 1.4.1.5 Support Vector Machine 19 1.4.2 Deep Neural Networks 19 1.4.3 Comparing Models 20 1.5 FutureWork 21 Acknowledgments 21 References 21 2 Quantum Mechanics Approaches in Computational Toxicology 31Jakub Kostal 2.1 Translating Computational Chemistry to Predictive Toxicology 31 2.2 Levels of Theory in Quantum Mechanical Calculations 33 2.3 Representing Molecular Orbitals 38 2.4 Hybrid Quantum and Molecular Mechanical Calculations 39 2.5 Representing System Dynamics 40 2.6 Developing QM Descriptors 42 2.6.1 Global Electronic Parameters 42 2.6.1.1 Electrostatic Potential, Dipole, and Polarizability 43 2.6.1.2 Global Electronic Parameters Derived from Frontier Molecular Orbitals (FMOs) 45 2.6.2 Local (Atom-Based) Electronic Parameters 47 2.6.2.1 Parameters Derived from Frontier Molecular Orbitals (FMOs) 48 2.6.2.2 Partial Atomic Charges 51 2.6.2.3 Hydrogen-Bonding Interactions 51 2.6.2.4 Bond Enthalpies 53 2.6.3 Modeling Chemical Reactions 53 2.6.4 QM/MM Calculations of Covalent Host-Guest Interactions 56 2.6.5 Medium Effects and Hydration Models 59 2.7 Rational Design of Safer Chemicals 61 References 64 Part II Applying Computers to Toxicology Assessment: Pharmaceutical, Industrial and Clinical 69 3 Computational Approaches for Predicting hERG Activity 71Vinicius M. Alves, Rodolpho C. Braga, and Carolina Horta Andrade 3.1 Introduction 71 3.2 Computational Approaches 73 3.3 Ligand-Based Approaches 73 3.4 Structure-Based Approaches 77 3.5 Applications to Predict hERG Blockage 77 3.5.1 Pred-hERGWeb App 79 3.6 Other Computational Approaches Related to hERG Liability 82 3.7 Final Remarks 83 References 83 4 Computational Toxicology for Traditional Chinese Medicine 93Ni Ai and Xiaohui Fan 4.1 Background, Current Status, and Challenges 93 4.2 Case Study: Large-Scale Prediction on Involvement of Organic Anion Transporter 1 in Traditional Chinese Medicine-Drug Interactions 99 4.2.1 Introduction to OAT1 and TCM 99 4.2.2 Construction of TCM Compound Database 101 4.2.3 OAT1 Inhibitor Pharmacophore Development 101 4.2.4 External Test Set Evaluation 102 4.2.5 Database Searching 102 4.2.6 Results: OAT1 Inhibitor Pharmacophore 103 4.2.7 Results: OAT1 Inhibitor Pharmacophore Evaluation 104 4.2.8 Results: TCM Compound Database Searching Using OAT1 Inhibitor Pharmacophore 104 4.2.9 Discussion 110 4.3 Conclusion 114 Acknowledgment 114 References 114 5 PharmacophoreModels for Toxicology Prediction 121Daniela Schuster 5.1 Introduction 121 5.2 Antitarget Screening 125 5.3 Prediction of Liver Toxicity 125 5.4 Prediction of Cardiovascular Toxicity 127 5.5 Prediction of Central Nervous System (CNS) Toxicity 128 5.6 Prediction of Endocrine Disruption 130 5.7 Prediction of ADME 135 5.8 General Remarks on the Limits and Future Perspectives for Employing Pharmacophore Models in Toxicological Studies 136 References 137 6 Transporters in Hepatotoxicity 145Eleni Kotsampasakou, Sankalp Jain, Daniela Digles, and Gerhard F. Ecker 6.1 Introduction 145 6.2 Basolateral Transporters 146 6.3 Canalicular Transporters 148 6.4 Data Sources for Transporters in Hepatotoxicity 148 6.5 In Silico Transporters Models 150 6.6 Ligand-Based Approaches 150 6.7 OATP1B1 and OATP1B3 150 6.8 NTCP 154 6.9 OCT1 154 6.10 OCT2 154 6.11 MRP1, MRP3, and MRP4 155 6.12 BSEP 155 6.13 MRP2 156 6.14 MDR1/P-gp 156 6.15 MDR3 157 6.16 BCRP 157 6.17 MATE1 158 6.18 ASBT 159 6.19 Structure-Based Approaches 159 6.20 Complex Models Incorporating Transporter Information 160 6.21 In Vitro Models 160 6.22 Multiscale Models 161 6.23 Outlook 162 Acknowledgments 164 References 164 7 Cheminformatics in a Clinical Setting 175Matthew D. Krasowski and Sean Ekins 7.1 Introduction 175 7.2 Similarity Analysis Applied to Drug of Abuse/Toxicology Immunoassays 177 7.3 Similarity Analysis Applied toTherapeutic Drug Monitoring Immunoassays 187 7.4 Similarity Analysis Applied to Steroid Hormone Immunoassays 191 7.5 Cheminformatics Applied to "Designer Drugs" 195 7.6 Relevance to Antibody-Ligand Interactions 202 7.7 Conclusions and Future Directions 203 Acknowledgment 204 References 204 Part III Applying Computers to Toxicology Assessment: Environmental and Regulatory Perspectives 211 8 Computational Tools for ADMET Profiling 213Denis Fourches, Antony J.Williams, Grace Patlewicz, Imran Shah, Chris Grulke, JohnWambaugh, Ann Richard, and Alexander Tropsha 8.1 Introduction 213 8.2 Cheminformatics Approaches for ADMET Profiling 214 8.2.1 Chemical Data Curation Prior to ADMET Modeling 215 8.2.2 QSAR Modelability Index 217 8.2.3 Predictive QSAR Model DevelopmentWorkflow 218 8.2.4 Hybrid QSAR Modeling 220 8.2.4.1 Simple Consensus 223 8.2.4.2 Mixed Chemical and Biological Features 223 8.2.4.3 Two-Step HierarchicalWorkflow 224 8.2.5 Chemical Biological Read-Across 226 8.2.6 Public Chemotype Approach to Data-Mining 229 8.3 Unsolved Challenges in Structure Based Profiling 230 8.3.1 Biological Data Curation 231 8.3.2 Identification and Treatment of Activity and Toxicity Cliffs 233 8.3.3 In Vitro to In Vivo Continuum in the Context of AOP 233 8.4 Perspectives 234 8.4.1 Profilers on the Go with Mobile Devices 235 8.4.2 Structure–Exposure–Activity Relationships 236 8.5 Conclusions 237 Acknowledgments 237 Disclaimer 237 References 238 9 Computational Toxicology and Reach 245Emilio Enfenati, Anna Lombardo, and Alessandra Roncaglioni 9.1 A Theoretical and Historical Introduction to the Evolution Toward Predictive Models 245 9.2 Reach and the Other Legislations 247 9.3 Annex XI of Reach for QSARModels 248 9.3.1 The First Condition of Annex XI and QMRF 249 9.3.2 The Second Condition and the Applicability Domain 251 9.3.3 TheThird Condition of Annex XI, and the Use of the QSAR Models 252 9.3.4 Adequate and Reliable Documentation of the Applied Method 254 9.4 The ECHA Guidelines and the Use of QSAR Models within ECHA 255 9.4.1 Example of Bioconcentration Factor (BCF) 255 9.4.2 Example of Mutagenicity (Reverse-Mutation Assay) Prediction 260 9.5 Conclusions 266 References 266 10 Computational Approaches to Predicting Dermal Absorption of Complex Topical Mixtures 269Jim E. Riviere and Jason Chittenden 10.1 Introduction 269 10.2 Principles of Dermal Absorption 270 10.3 Dermal Mixtures 274 10.4 Model Systems 275 10.5 Local Skin Versus Systemic Endpoints 277 10.6 QSAR Approaches to Model Dermal Absorption 278 10.7 PharmacokineticModels 281 10.8 Conclusions 284 References 285 Part IV New Technologies for Toxicology, Future Perspectives 291 11 Big Data in Computational Toxicology: Challenges and Opportunities 293Linlin Zhao and Hao Zhu 11.1 Big Data Scenario of Computational Toxicology 293 11.2 Fast-Growing Chemical Toxicity Data 295 11.3 The Use of Big Data Approaches in Modern Computational Toxicology 299 11.3.1 Profiling the Toxicants with Massive Biological Data 299 11.3.2 Read-Across Study to Fill Data Gap 301 11.3.3 Unstructured Data Curation 302 11.4 Challenges of Big Data Research in Computational Toxicology and Relevant Forecasts 303 References 304 12 HLA-Mediated Adverse Drug Reactions: Challenges and Opportunities for Predictive Molecular Modeling 313George van Den Driessche and Denis Fourches 12.1 Introduction 313 12.2 Human Leukocyte Antigens 314 12.2.1 HLA Proteins 314 12.2.2 ADR–HLA Associations 316 12.2.3 HLA-Drug-Peptide Proposed T-Cell Signaling Mechanisms 321 12.3 Structure-Based Molecular Docking to Study HLA-Mediated ADRs 322 12.3.1 Structure-Based Docking 324 12.3.2 Case Study: Abacavir with B*57:01 326 12.3.3 Limitations 332 12.4 Perspectives 334 References 335 13 Open Science Data Repository for Toxicology 341Valery Tkachenko, Richard Zakharov, and Sean Ekins 13.1 Introduction 341 13.2 Open Science Data Repository 342 13.3 Benefits of OSDR 344 13.3.1 Chemically and Semantically Enabled Scientific Data Repository 344 13.3.2 Chemical Validation and Standardization Platform 346 13.3.3 Format Adapters 347 13.3.4 Open Platform for Data Acquisition, Curation, and Dissemination 350 13.3.5 Dataledger 350 13.4 Technical Details 351 13.5 FutureWork 353 13.5.1 Implementation of Ontology-Based Properties 356 13.5.2 Implementation of an Advanced Search System 357 13.5.3 Implementation of a Scientist Profile, Advanced Security, Data Sharing Capabilities and Notifications Framework 357 References 358 14 Developing Next Generation Tools for Computational Toxicology 363Alex M. Clark, Kimberley M. Zorn, Mary A. Lingerfelt, and Sean Ekins 14.1 Introduction 363 14.2 Developing Apps for Chemistry 364 14.3 Green Chemistry 364 14.3.1 Green Solvents and Lab Solvents 367 14.3.2 Green Lab Notebook 370 14.4 Polypharma and Assay Central 374 14.4.1 Future Efforts with Assay Central 380 14.5 Conclusion 382 Acknowledgments 383 References 383 Index 389

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Book SynopsisA guide to state-of-the-art molecular tools for monitoring and managing the toxigenicity of cyanobacteria Runaway eutrophication and climate change has made the monitoring and management of toxigenic organisms in the world s bodies of water more urgent than ever.Table of ContentsList of Contributors xix About the Editors xxiii About the Book xxvii Preface xxix Acknowledgments xxxi 1 Introduction 1Rainer Kurmayer, Kaarina Sivonen, and Nico Salmaso 1.1 A Brief Historical Overview 1 1.2 The Genetic Basis of Toxin Production 2 1.2.1 Microcystin and Nodularin 2 1.2.2 Cylindrospermopsin 5 1.2.3 Saxitoxin 6 1.2.4 Anatoxin 8 1.3 Application of Molecular Tools 8 1.4 Laboratory Safety Issues 13 1.5 References 14 2 Sampling and Metadata 19Rainer Kurmayer, Guntram Christiansen, Konstantinos Kormas, Wim Vyverman, Elie Verleyen, Vitor Ramos, Vitor Vasconcelos, and Nico Salmaso 2.1 Introduction 19 2.2 Handling of Samples 20 2.3 Sample Contamination 21 2.4 Sampling 21 2.4.1 Quantitative Depth-Integrated and Discrete Sampling 21 2.4.2 Qualitative Plankton Net Sampling 22 2.4.3 Surface (Scum Material) Sampling 22 2.4.4 Benthic (Terrestrial) Cyanobacteria Sampling 22 2.4.5 Food Supplement Sampling 22 2.4.6 Isolation of Single Colonies/Filaments 22 2.5 Subsampling Food Supplement Samples 23 2.6 Sampling of Nucleic Acids 23 2.7 General Conclusions 24 2.8 References 24 SOP 2.1 Sampling and Filtration (DNA) 26Rainer Kurmayer and Konstantinos Kormas SOP 2.1.1 Introduction 26 SOP 2.1.2 Experimental 26 SOP 2.1.3 Procedure 27 SOP 2.1.4 Notes 28 SOP 2.1.5 References 29 SOP 2.2 Sampling of Benthic Cyanobacteria 29Wim Vyverman and Elie Verleyen SOP 2.2.1 Introduction 29 SOP 2.2.2 Experimental 30 SOP 2.2.3 Procedure 30 SOP 2.2.4 Notes 31 SOP 2.2.5 References 31 SOP 2.3 Isolation of Single Cyanobacteria Colonies/Filaments 32 Rainer Kurmayer SOP 2.3.1 Introduction 32 SOP 2.3.2 Experimental 32 SOP 2.3.3 Procedure 33 SOP 2.3.4 Notes 33 SOP 2.3.5 References 33 SOP 2.4 Sampling Food Supplements 34Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos SOP 2.4.1 Introduction 34 SOP 2.4.2 Experimental 35 SOP 2.4.3 Procedure (Fig. 8.3) 35 SOP 2.4.4 Notes 36 SOP 2.4.5 References 36 SOP 2.5 Sampling and Filtration (RNA) 37Rainer Kurmayer and Guntram Christiansen SOP 2.5.1 Introduction 37 SOP 2.5.2 Experimental 37 SOP 2.5.3 Procedure 38 SOP 2.5.4 Notes 38 SOP 2.5.5 References 38 SOP 2.6 Sampling of Abiotic and Biotic Data and Recording Metadata 39Elie Verleyen, Maxime Sweetlove, Dagmar Obbels, and Wim Vyverman SOP 2.6.1 Introduction 39 SOP 2.6.2 Experimental 39 SOP 2.6.3 Type of Metadata and Additional Biotic and Abiotic Data 40 SOP 2.6.4 Notes 41 SOP 2.6.5 References 42 3 Isolation, Purification, and Cultivation of Toxigenic Cyanobacteria 43Sigrid Haande, Iwona Jasser, Muriel Gugger, Camilla H.C. Hagman, Annick Wilmotte, and Andreas Ballot 3.1 Introduction 43 3.2 Methodical Principles for Cyanobacterial Isolation, Purification, and Cultivation 44 3.2.1 Sampling, Identification, and Treatments Prior to the Isolation of Cyanobacteria 44 3.2.2 Traditional Techniques for the Isolation and Purification of Cyanobacteria 45 3.2.3 Culture Media Preparation 47 3.2.4 Cultivation Conditions 48 3.3 General Conclusions 49 3.4 References 49 SOP 3.1 Isolation, Purification, and Clonal Isolate Testing 51Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot SOP 3.1.1 Introduction 51 SOP 3.1.2 Experimental 51 SOP 3.1.3 Procedure 52 SOP 3.1.4 Notes 54 SOP 3.1.5 References 54 SOP 3.2 Isolation of Picocyanobacterial Cells by Flow Cytometer (FCM) Sorting 55Ewa Koz³owska and Iwona Jasser SOP 3.2.1 Introduction 55 SOP 3.2.2 Experimental 56 SOP 3.2.3 Procedure 56 SOP 3.2.4 Notes 58 SOP 3.2.5 References 59 SOP 3.3 Axenization 60Muriel Gugger SOP 3.3.1 Introduction 60 SOP 3.3.2 Experimental 60 SOP 3.3.3 Procedure 61 SOP 3.3.4 Notes 63 SOP 3.3.5 References 63 SOP 3.4 Culture Media (Solid and Liquid) 64Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot SOP 3.4.1 Introduction 64 SOP 3.4.2 Experimental 64 SOP 3.4.3 Procedure 65 SOP 3.4.4 Notes 68 SOP 3.4.5 References 68 SOP 3.5 Strain Maintenance (Living Cultures) 69Sigrid Haande, Camilla H.C. Hagman, and Andreas Ballot SOP 3.5.1 Introduction 69 SOP 3.5.2 Experimental 69 SOP 3.5.3 Procedure 70 SOP 3.5.4 Notes 72 SOP 3.5.5 References 73 SOP 3.6 Cryopreservation and Recovery 73Muriel Gugger SOP 3.6.1 Introduction 73 SOP 3.6.2 Experimental 74 SOP 3.6.3 Procedure 75 SOP 3.6.4 Notes 78 SOP 3.6.5 References 78 4 Taxonomic Identification of Cyanobacteria by a Polyphasic Approach 79Annick Wilmotte, H. Dail Laughinghouse IV, Camilla Capelli, Rosmarie Rippka, and Nico Salmaso 4.1 Introduction 79 4.2 Nomenclature and Classification of Cyanobacteria 82 4.3 Microscopy 84 4.3.1 Light Microscopy 84 4.3.2 Autofluorescence Microscopy 86 4.4 Molecular Markers: Single Loci 87 4.5 Molecular Markers: Multiple Loci 94 4.5.1 Multilocus Sequence Typing (MLST) and Multilocus Sequence Analysis (MLSA) 94 4.5.2 Genome-Based Extension of MLST and MLSA 96 4.6 Molecular Typing Methods Based on Gel Electrophoresis 96 4.7 Denaturing Gradient Gel Electrophoresis (DGGE) 97 4.8 Taxonomic and Molecular Databases 97 4.9 The Polyphasic Approach 98 4.10 Final Considerations 105 4.11 References 106 SOP 4.1 Taxonomic Identification by Light Microscopy 120Nico Salmaso, Rosmarie Rippka, and Annick Wilmotte SOP 4.1.1 Introduction 120 SOP 4.1.2 Experimental 121 SOP 4.1.3 References 124 SOP 4.2 Polyphasic Approach on Cyanobacterial Strains 125Nico Salmaso, Camilla Capelli, Rosmarie Rippka, and Annick Wilmotte SOP 4.2.1 Introduction 125 SOP 4.2.2 Experimental 126 SOP 4.2.3 References 131 5 Nucleic Acid Extraction 135Elke Dittmann, Anne Rantala-Ylinen, Vitor Ramos, Vitor Vasconcelos, Guntram Christiansen, and Rainer Kurmayer 5.1 Introduction 135 5.2 Specific Extraction Procedures and Storage 137 5.2.1 DNA Extraction from Laboratory Strains 137 5.2.2 DNA Extraction from Field Samples 137 5.2.3 DNA Extraction from Food Supplements 137 5.2.4 RNA Extraction from Laboratory Strains 138 5.2.5 RNA Extraction from Field Samples 138 5.2.6 Single Colony and Filament Analysis 138 5.2.7 Whole Genome Amplification 139 5.2.8 Nucleic Acid Storage 139 5.3 References 139 SOP 5.1 Standard DNA Isolation Technique for Cyanobacteria 140Elke Dittmann SOP 5.1.1 Introduction 140 SOP 5.1.2 Experimental 140 SOP 5.1.3 Procedure 141 SOP 5.1.4 Notes 141 SOP 5.1.5 References 142 SOP 5.2 DNA Isolation Protocol for Cyanobacteria with Extensive Mucilage 143Guntram Christiansen, Elisabeth Entfellner, and Rainer Kurmayer SOP 5.2.1 Introduction 143 SOP 5.2.2 Experimental 143 SOP 5.2.3 Procedure 144 SOP 5.2.4 Notes 145 SOP 5.2.5 References 145 SOP 5.3 Quantitative DNA Isolation from Filters 145Rainer Kurmayer SOP 5.3.1 Introduction 146 SOP 5.3.2 Experimental 146 SOP 5.3.3 Procedure 147 SOP 5.3.4 Notes 148 SOP 5.3.5 References 148 SOP 5.4 Genomic DNA Extraction from Single Filaments/Colonies for Multiple PCR Analyses 149Guntram Christiansen, Chen Qin, and Rainer Kurmayer SOP 5.4.1 Introduction 149 SOP 5.4.2 Experimental 149 SOP 5.4.3 Procedure 150 SOP 5.4.4 Notes 151 SOP 5.4.5 References 151 SOP 5.5 Whole Genome Amplification Using Bacteriophage Phi29 DNA Polymerase 151Guntram Christiansen and Rainer Kurmayer SOP 5.5.1 Introduction 151 SOP 5.5.2 Experimental 152 SOP 5.5.3 Procedure 152 SOP 5.5.4 Notes 152 SOP 5.5.5 Reference 153 SOP 5.6 DNA Extraction from Food Supplements 153Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos SOP 5.6.1 Introduction 153 SOP 5.6.2 Experimental 153 SOP 5.6.3 Procedure 154 SOP 5.6.4 Notes 155 SOP 5.6.5 References 156 SOP 5.7 RNA Extraction from Cyanobacteria 156Guntram Christiansen and Rainer Kurmayer SOP 5.7.1 Introduction 156 SOP 5.7.2 Experimental 156 SOP 5.7.3 Procedure 158 SOP 5.7.4 Notes 158 SOP 5.7.5 References 159 SOP 5.8 cDNA Synthesis 159Guntram Christiansen and Rainer Kurmayer SOP 5.8.1 Introduction 159 SOP 5.8.2 Experimental 159 SOP 5.8.3 Procedure 160 SOP 5.8.4 Notes 161 SOP 5.8.5 References 161 6 Conventional PCR 163Elke Dittmann, Anne Rantala-Ylinen, Kaarina Sivonen, Ilona Ga²ga³a, Joanna Mankiewicz-Boczek, Samuel Cirés, Andreas Ballot, Guntram Christiansen, Rainer Kurmayer, Vitor Ramos, Vitor Vasconcelos, and Martin Saker 6.1 Introduction 163 6.2 Principle of PCR and Available Enzymes 164 6.2.1 Primer Development 165 6.2.2 Setup of PCR Conditions for DNA and Single Colony Analysis 168 6.2.3 Gel Electrophoresis and Documentation 168 6.2.4 Troubleshooting of PCR Results 168 6.2.5 PCR Product Downstream Processing (RFLP, Cloning, Sequencing) 169 6.3 Special Notes 170 6.4 References 170 SOP 6.1 PCR Detection of Microcystin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 172Elke Dittmann SOP 6.1.1 Introduction 172 SOP 6.1.2 Experimental 172 SOP 6.1.3 Procedure 173 SOP 6.1.4 Notes 174 SOP 6.1.5 Reference 174 SOP 6.2 PCR Detection of Microcystin and Nodularin Biosynthesis Genes in the Cyanobacterial Orders Oscillatoriales, Chroococcales, Stigonematales, and Nostocales 175Elke Dittmann, Joanna Mankiewicz-Boczek, and Ilona Ga²ga³a SOP 6.2.1 Introduction 175 SOP 6.2.2 Experimental 175 SOP 6.2.3 Procedure 177 SOP 6.2.4 Notes 177 SOP 6.2.5 References 178 SOP 6.3 Genus-Specific PCR Detection of Microcystin Biosynthesis Genes in Anabaena/Nodularia and Microcystis and Planktothrix, Respectively 179Anne Rantala-Ylinen and Kaarina Sivonen SOP 6.3.1 Introduction 179 SOP 6.3.2 Experimental 179 SOP 6.3.3 Procedure 181 SOP 6.3.4 Notes 181 SOP 6.3.5 References 181 SOP 6.4 PCR Detection of Anatoxin Biosynthesis Genes Combined with RFLP Differentiation of the Producing Genus 182Anne Rantala-Ylinen and Kaarina Sivonen SOP 6.4.1 Introduction 182 SOP 6.4.2 Experimental 182 SOP 6.4.3 Procedure 183 SOP 6.4.4 Notes 184 SOP 6.4.5 Reference 184 SOP 6.5 PCR Detection of the Saxitoxin Biosynthesis Genes, sxtA, sxtX, sxtH, sxtG, and sxtI 185Andreas Ballot and Samuel Cirés SOP 6.5.1 Introduction 185 SOP 6.5.2 Experimental 187 SOP 6.5.3 Procedure 187 SOP 6.5.4 Notes 188 SOP 6.5.5 References 189 SOP 6.6 PCR Detection of the Cylindrospermopsin Biosynthesis Gene cyrJ 189Samuel Cirés and Andreas Ballot SOP 6.6.1 Introduction 189 SOP 6.6.2 Experimental 190 SOP 6.6.3 Procedure 191 SOP 6.6.4 Notes 191 SOP 6.6.5 References 192 SOP 6.7 PCR from Single Filament of Toxigenic Planktothrix 193Qin Chen, Guntram Christiansen, and Rainer Kurmayer SOP 6.7.1 Introduction 193 SOP 6.7.2 Experimental 193 SOP 6.7.3 Procedure 194 SOP 6.7.4 Notes 195 SOP 6.7.5 References 195 SOP 6.8 Analysis of Microcystin Biosynthesis Gene Subpopulation Variability in Planktothrix 196Rainer Kurmayer SOP 6.8.1 Introduction 196 SOP 6.8.2 Experimental 196 SOP 6.8.3 Procedure 197 SOP 6.8.4 Notes 197 SOP 6.8.5 References 198 SOP 6.9 PCR Detection of Microcystin Biosynthesis Genes from Food Supplements 199Vitor Ramos, Cristiana Moreira, and Vitor Vasconcelos SOP 6.9.1 Introduction 199 SOP 6.9.2 Experimental 199 SOP 6.9.3 Procedure 201 SOP 6.9.4 Notes 202 SOP 6.9.5 References 203 7 Quantitative PCR 205Anne Rantala-Ylinen, Henna Savela, Kaarina Sivonen, and Rainer Kurmayer 7.1 Introduction 205 7.2 Primer/Probe Design 206 7.3 Optimization 208 7.4 Absolute Quantification 208 7.5 Relative Quantification 209 7.6 Calibration of qPCR Results 209 7.7 General Conclusions 210 7.8 References 210 SOP 7.1 Optimization of qPCR Assays 211Rainer Kurmayer SOP 7.1.1 Introduction 211 SOP 7.1.2 Experimental 211 SOP 7.1.3 Procedure 212 SOP 7.1.4 Notes 213 SOP 7.1.5 References 213 SOP 7.2 Calibration of qPCR Results 214Rainer Kurmayer SOP 7.2.1 Introduction 214 SOP 7.2.2 Experimental 214 SOP 7.2.3 Procedure 215 SOP 7.2.4 Notes 217 SOP 7.2.5 References 217 SOP 7.3 Quantification of Potentially Microcystin/Nodularin-Producing Anabaena, Microcystis, Planktothrix, and Nodularia 218Anne Rantala-Ylinen, Kaarina Sivonen, and Rainer Kurmayer SOP 7.3.1 Introduction 218 SOP 7.3.2 Experimental 219 SOP 7.3.3 Procedure 219 SOP 7.3.4 Notes 221 SOP 7.3.5 References 221 SOP 7.4 Relative Quantification of Microcystis or Planktothrix mcy Genotypes Using qPCR 222Rainer Kurmayer SOP 7.4.1 Introduction 222 SOP 7.4.2 Experimental 222 SOP 7.4.3 Procedure 224 SOP 7.4.4 Notes 225 SOP 7.4.5 References 225 SOP 7.5 Quantification of Transcript Amounts of mcy Genes in Planktothrix 226Guntram Christiansen and Rainer Kurmayer SOP 7.5.1 Introduction 226 SOP 7.5.2 Experimental 226 SOP 7.5.3 Procedure 227 SOP 7.5.4 Notes 228 SOP 7.5.5 References 228 SOP 7.6 Quantification of Potentially Cylindrospermopsin-Producing Chrysosporum ovalisporum 229Rehab El-Shehawy and Antonio Quesada SOP 7.6.1 Introduction 229 SOP 7.6.2 Experimental 229 SOP 7.6.3 Procedure 230 SOP 7.6.4 Notes 231 SOP 7.6.5 References 231 SOP 7.7 qPCR Detection of the Paralytic Shellfish Toxin Biosynthesis Gene sxtB 231Henna Savela SOP 7.7.1 Introduction 231 SOP 7.7.2 Experimental 232 SOP 7.7.3 Procedure 233 SOP 7.7.4 Notes 234 SOP 7.7.5 References 234 SOP 7.8 Application of the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) Guidelines to Quantitative Analysis of Toxic Cyanobacteria 234Henna Savela SOP 7.8.1 Introduction 234 SOP 7.8.2 Sampling 235 SOP 7.8.3 Sample Preparation and DNA Extraction 235 SOP 7.8.4 Target Information and Oligonucleotide Design 235 SOP 7.8.5 qPCR Protocol 238 SOP 7.8.6 qPCR Validation 239 SOP 7.8.7 Data Analysis 239 SOP 7.8.8 Reference 239 8 DNA (Diagnostic) and cDNA Microarray 241Anne Rantala-Ylinen, Kaarina Sivonen, and Annick Wilmotte 8.1 DNA (Diagnostic) Microarray 241 8.1.1 Introduction 241 8.1.2 Methodological Principles 242 8.1.3 General Conclusions 243 8.1.4 References 243 8.2 cDNA Microarray for Cyanobacteria 244Hans C.P. Matthijs and J. Merijn Schuurmans 8.2.1 Introduction 244 8.2.2 Principles of Microarray Use 244 8.2.3 Considerations for Experimental Design 245 8.2.4 Microarray: Practical Approach 246 8.2.5 Microarray: Data Analysis 246 8.2.6 References 246 SOP 8.1 DNA-Chip Detection of Potential Microcystin and Nodularin Producing Cyanobacteria in Environmental Water Samples 248Anne Rantala-Ylinen and Kaarina Sivonen SOP 8.1.1 Introduction 248 SOP 8.1.2 Experimental 249 SOP 8.1.3 Procedure 250 SOP 8.1.4 Notes 253 SOP 8.1.5 References 253 SOP 8.2 cDNA Microarrays for Cyanobacteria 254J. Merijn Schuurmans and Hans C.P. Matthijs SOP 8.2.1 Introduction 254 SOP 8.2.2 Experimental 254 SOP 8.2.3 Procedure 256 SOP 8.2.4 Notes 259 SOP 8.2.5 Reference 261 9 Analysis of Toxigenic Cyanobacterial Communities through Denaturing Gradient Gel Electrophoresis 263Iwona Jasser, Aleksandra Bukowska, Jean-Francois Humbert, Kaisa Haukka, and David P. Fewer 9.1 Introduction 263 9.2 Main Applications of the Method 264 9.3 Possible Applications 264 9.4 DGGE Procedure 265 9.5 General Conclusions Including Pros and Cons of the Method 267 9.6 Optimization of the Method and Troubleshooting 267 9.7 References 268 SOP 9.1 DGGE-mcyA Conditions 270Aleksandra Bukowska and Iwona Jasser SOP 9.1.1 Introduction 270 SOP 9.1.2 Experimental 270 SOP 9.1.3 Procedure 272 SOP 9.1.4 Notes 275 SOP 9.1.5 References 275 10 Monitoring of Toxigenic Cyanobacteria Using Next-Generation Sequencing Techniques 277Li Deng, Maxime Sweetlove, Stephan Blank, Dagmar Obbels, Elie Verleyen, Wim Vyverman, and Rainer Kurmayer 10.1 Introduction 277 10.2 Specific Procedures 279 10.2.1 16S rRNA Gene Amplicon Library Preparation 279 10.2.2 Amplicon Purification, Quantification and Pooling 280 10.2.3 Sequencing 280 10.2.4 Bioinformatic Exploration of Sequencing Results 281 10.2.5 General Conclusions Including Pros and Cons of the Method 281 10.2.6 References 281 10.3 Bioinformatic Processing of Amplicon Sequencing Datasets 283Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman 10.3.1 Introduction 283 10.3.2 Sequencing Platforms 283 10.3.3 Data Formats 284 10.3.4 Error Associated with NGS Data 285 10.3.5 OTU Delineation: Choosing a Similarity Threshold 286 10.3.6 Conclusions 286 10.4 References 286 SOP 10.1 Standard Technique to Generating 16S rRNA PCR Amplicons for NGS 288Li Deng, Stephan Blank, Guntram Christiansen, and Rainer Kurmayer SOP 10.1.1 Introduction 288 SOP 10.1.2 Experimental 288 SOP 10.1.3 Procedure 289 SOP 10.1.4 Notes 290 SOP 10.1.5 References 290 SOP 10.2 Bioinformatics Analysis for NGS Amplicon Sequencing 291Maxime Sweetlove, Dagmar Obbels, Elie Verleyen, Igor S. Pessi, Annick Wilmotte, and Wim Vyverman SOP 10.2.1 Introduction 291 SOP 10.2.2 Experimental 291 SOP 10.2.3 Practical Tips and Alternatives for Quality Filtering 298 SOP 10.2.4 References 298 11 Application of Molecular Tools in Monitoring Cyanobacteria and Their Potential Toxin Production 301Vitor Ramos, Cristiana Moreira, Joanna Mankiewicz-Boczek, and Vitor Vasconcelos 11.1 Introduction 301 11.2 Possible Applications 303 11.3 Checklist of Publications, Applications and Lessons from Practice 315 11.3.1 Molecular-Based Studies on (Toxic) Cyanobacteria: Overview of Methods Being Used, and Generic Findings and Concerns 315 11.3.2 The Need for Complementary Approaches 316 11.3.3 Interpreting Results 316 11.3.4 Choice of Molecular Tools for Toxigenicity Assessment 317 11.3.5 Common and Possible Applications of Molecular Tools 318 11.4 General Conclusions 321 11.5 Acknowledgments 324 11.6 References 324 Appendix: Supplementary Tables 335 Cyanobacterial Species Cited in the Book 376 Glossary 379 Index 393

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Book SynopsisTable of Contents1 The Chemical Foundations of Biochemistry 1 Chemistry in Context 1 1.1 General Chemical Principles 2 1.1.1 The basic principles of thermodynamics describe all systems 2 1.1.2 Equilibrium describes a specific thermodynamic state 7 1.1.3 Kinetics describes the rate of chemical reactions 8 1.1.4 Thermodynamics, equilibrium, and kinetics are used together to describe biochemical reactions and systems 9 1.2 Fundamental Concepts of Organic Chemistry 11 1.2.1 Chemical properties and reactions can be sorted by functional groups 11 1.2.2 The solubility and polarity of a molecule can be determined from its structure 12 1.2.3 Reaction mechanisms attempt to explain how a reaction occurs 13 1.2.4 Many biochemical molecules are polymers 14 1.3 The Chemistry of Water 15 1.3.1 The structure of water provides clues about its properties 16 1.3.2 Hydrogen bonding among water molecules is one of the most important weak forces in biochemistry 17 1.3.3 Water can ionize to acids and bases in biochemical systems 18 2 Nucleic Acids 26 Nucleic Acids in Context 26 2.1 Nucleic Acids Have Distinct Structures 27 2.1.1 Nucleic acids can be understood at the chemical level 28 2.1.2 The complex shapes of nucleic acids are the result of numerous weak forces 30 2.2 Nucleic Acids Have Many Cellular Functions 37 2.2.1 Replication is the process by which cells copy DNA 37 2.2.2 Transcription is the copying of DNA into an RNA message 40 2.2.3 Translation is the synthesis of proteins by ribosomes using an mRNA code 41 2.2.4 Regulation of replication, transcription, and translation is critical to an organism’s survival and propagation 42 2.2.5 Viruses and retroviruses use the cell’s own machinery to reproduce 43 2.3 The Manipulation of Nucleic Acids Has Transformed Biochemistry 45 2.3.1 DNA can be easily manipulated and analyzed in vitro 45 2.3.2 DNA can be used to drive protein expression 53 2.3.3 Techniques can be used to silence genes in organisms 59 3 Proteins I: An Introduction to Protein Structure and Function 67 Proteins in Context 67 3.1 Amino Acid Chemistry 69 3.1.1 The structure of amino acids dictates their chemical properties 69 3.1.2 The side chains of amino acids impart unique properties 70 3.1.3 Amino acids have various roles in biochemistry 72 3.2 Proteins Are Polymers of Amino Acids 74 3.2.1 Both peptides and proteins are polymers of amino acids 74 3.2.2 The peptide bond has special characteristics 76 3.2.3 Many proteins require inorganic ions or small organic molecules to function 76 3.2.4 Amino acids can be modified within proteins 77 3.3 Proteins Are Molecules of Defined Shape and Structure 79 3.3.1 The primary structure of a protein is its amino acid sequence 79 3.3.2 The secondary structure of a protein is comprised of a few conserved structures 81 3.3.3 The tertiary structure of a protein is the organization of secondary structures into conserved motifs 84 3.3.4 The quaternary structure of a protein describes how individual subunits interact 88 3.4 Examples of Protein Structures and Functions 89 3.4.1 Aquaporin, a transmembrane pore 90 3.4.2 Chymotrypsin, an enzyme 90 3.4.3 Collagen, a structural protein 91 3.4.4 Hemoglobin, a transport protein 92 3.4.5 Immunoglobulins, binding proteins 92 3.4.6 Insulin, a signaling protein 93 3.4.7 Myosin, a molecular motor 94 4 Proteins II: Enzymes 101 Enzymes in Context 101 4.1 Regarding Enzymes 103 4.1.1 Enzymes are protein catalysts 103 4.1.2 Enzymatically catalyzed reactions can be categorized 105 4.1.3 How do enzymes work? 106 4.2 Enzymes Increase Reaction Rate 108 4.2.1 A review of chemical rates 108 4.2.2 The Michaelis-Menten equation relates enzymatic rates to measurable parameters 109 4.2.3 The Michaelis constant has several meanings 111 4.2.4 Kinetic data can be graphically analyzed 112 4.2.5 kcat/KM is a measure of catalytic efficiency 112 4.2.6 Some enzymes approach catalytic perfection 112 4.2.7 Enzymatic reactions may be inhibited through one of several different mechanisms 113 4.2.8 Many reactions have more than one substrate 116 4.3 The Mechanism of an Enzyme Can Be Deduced from Structural, Kinetic, and Spectral Data 117 4.3.1 Enzymatically catalyzed reactions have common properties 118 4.3.2 Examining examples of enzymatic reaction mechanisms illustrates underpinning principles 119 4.3.3 Mechanisms are elucidated using a combination of experimental techniques 125 4.4 Examples of Enzyme Regulation 127 4.4.1 Covalent modifications are a common means of enzyme regulation 127 4.4.2 Allosteric regulators bind at sites other than the active site 129 5 Membranes and an Introduction to Signal Transduction 138 Biochemistry in Context 138 5.1 Membrane Structure and Function 139 5.1.1 The chemical properties of the membrane components dictate the characteristics of the membrane 140 5.1.2 Other aspects of membrane structure 145 5.1.3 Membrane fusion and membrane budding 146 5.2 Signal Transduction 149 5.2.1 General principles underlie signal transduction 149 5.2.2 The protein kinase A (PKA) signaling pathway is activated by cyclic AMP 150 5.2.3 Insulin is an important metabolic regulator and growth factor 153 5.2.4 The AMP kinase (AMPK) signaling pathway coordinates metabolic pathways in the cell and in the body 154 6 Carbohydrates I: Mono- and Disaccharides, Glycolysis, Gluconeogenesis, and the Fates of Pyruvate 161 Carbohydrates in Context 161 6.1 Properties, Nomenclature, and Biological Functions of Monosaccharides 163 6.1.1 Monosaccharides are the simplest carbohydrates 163 6.1.2 Monosaccharides form hemiacetals and hemiketals 165 6.1.3 Monosaccharides form heterocyclic structures 165 6.1.4 Monosaccharides can be chemically modified 167 6.1.5 Carbohydrates can be classified as reducing sugars or nonreducing sugars 168 6.2 Properties, Nomenclature, and Biological Functions of Complex Carbohydrates 170 6.2.1 Common disaccharides include lactose, sucrose, and maltose 170 6.2.2 Trisaccharides and oligosaccharides contain three or more monosaccharide units bound by glycosidic linkages 173 6.2.3 Common polysaccharides function to store energy or provide structure 174 6.3 Glycolysis and an Introduction to Metabolic Pathways 177 6.3.1 Metabolic pathways describe how molecules are built up or broken down 178 6.3.2 Glycolysis is the process by which glucose is broken into pyruvate 180 6.4 Gluconeogenesis 193 6.4.1 Gluconeogenesis differs from glycolysis at four reactions 193 6.4.2 The regulation of gluconeogenesis takes place at several different levels 195 6.5 The Fates of Pyruvate 196 6.5.1 Pyruvate can be decarboxylated to acetyl-CoA by pyruvate dehydrogenase 197 6.5.2 Pyruvate can be converted to lactate by lactate dehydrogenase 200 6.5.3 Pyruvate can be transaminated to alanine 201 6.5.4 Pyruvate can be carboxylated to oxaloacetate by pyruvate carboxylase 201 6.5.5 Microbes can decarboxylate pyruvate into acetaldehyde 201 7 The Common Catabolic Pathway: Citric Acid Cycle, the Electron Transport Chain, and ATP Biosynthesis 209 Electron Transport in Context 209 7.1 The Citric Acid Cycle 211 7.1.1 There are eight reactions in the citric acid cycle 212 7.1.2 The citric acid cycle is regulated at multiple places and by several different mechanisms 218 7.1.3 Anaplerotic reactions of the citric acid cycle replenish intermediates 219 7.2 The Electron Transport Chain 223 7.2.1 Electron transport occurs through a series of redox active centers from higher to lower potential energy 224 7.2.2 Complex I (NADH dehydrogenase) transfers electrons from NADH to ubiquinone via a series of iron-sulfur centers 229 7.2.3 Complex II is the citric acid cycle enzyme succinate dehydrogenase 231 7.2.4 Complex III is ubiquinone/cytochrome c reductase 233 7.2.5 Cytochrome c is a soluble electron carrier 235 7.2.6 In complex IV, oxygen is the terminal electron carrier 235 7.2.7 The entire complex working as one: the respirasome 238 7.3 ATP Biosynthesis 241 7.3.1 The structure of ATP synthase underlies its function 241 7.3.2 ATP synthase acts as a molecular machine driving the assembly of ATP molecules 243 7.3.3 Other ATPases serve as ion pumps 245 7.3.4 Inhibitors of the ATPases can be powerful drugs or poisons 246 8 Carbohydrates II: Glycogen Metabolism, the Pentose Phosphate Pathway, Glycoconjugates, and Extracellular Matrices 253 Polysaccharides in Context 253 8.1 Glycogen Metabolism 255 8.1.1 Glycogenesis is glycogen biosynthesis 256 8.1.2 Glycogenolysis is glycogen breakdown 257 8.1.3 The regulation of glycogenesis and glycogenolysis 259 8.2 The Pentose Phosphate Pathway 264 8.2.1 The oxidative phase of the pentose phosphate pathway produces NADPH and ribulose-5- phosphate 264 8.2.2 The nonoxidative phase of the pentose phosphate pathway results in rearrangement of monosaccharides 266 8.2.3 Regulation of the pentose phosphate pathway 267 8.2.4 The pentose phosphate pathway in health and disease 268 8.2.5 Xylulose-5-phosphate is a master regulator of carbohydrate and lipid metabolism 271 8.3 Carbohydrates in Glycoconjugates 273 8.3.1 Glycoproteins 273 8.3.2 Glycolipids 276 8.3.3 Proteoglycans and non-proteoglycan polysaccharides 279 8.3.4 Peptidoglycans 282 8.4 Extracellular Matrices and Biofilms 284 8.4.1 Eukaryotic extracellular matrix proteins 284 8.4.2 Biofilms are composed of microbes living in a secreted matrix 290 9 Lipids I: Fatty Acids, Steroids, and Eicosanoids; Beta-Oxidation and Fatty Acid Biosynthesis 300 Lipids in Context 300 9.1 Properties, Nomenclature, and Biological Functions of Lipid Molecules 302 9.1.1 Fatty acids are a common building block of many lipids 302 9.1.2 Neutral lipids are storage forms of fatty acids or cholesterol 305 9.1.3 Phospholipids are important in membrane formation 306 9.1.4 All steroids and bile salts are derived from cholesterol 309 9.1.5 Eicosanoids are potent signaling molecules derived from 20-carbon polyunsaturated fatty acids 310 9.2 Fatty Acid Catabolism 311 9.2.1 Fatty acids must be transported into the mitochondrial matrix before catabolism can proceed 312 9.2.2 Fatty acids are oxidized to acetyl-CoA by β-oxidation 312 9.2.3 Beta-oxidation is regulated at two different levels 316 9.3 Fatty Acid Biosynthesis 317 9.3.1 Two major enzyme complexes are involved in fatty acid biosynthesis 318 9.3.2 Elongases and desaturases in the endoplasmic reticulum increase fatty acid diversity 326 9.3.3 The formation of malonyl-CoA by acetyl-CoA carboxylase is the regulated and rate-determining step of fatty acid biosynthesis 327 9.4 Ketone Body Metabolism 329 9.4.1 Ketone bodies are made from acetyl-CoA 330 9.4.2 Ketone bodies can be thought of as a water-soluble fuel source used in the absence of carbohydrates 330 9.5 Steroid Metabolism 332 9.5.1 Cholesterol is synthesized in the liver through the addition of energetically activated isoprene units 332 9.5.2 Steroid hormones are derived from cholesterol 336 9.5.3 Bile salts are steroid detergents used in the digestion of fats 337 9.6 Eicosanoid and Endocannabinoid Metabolism 339 9.6.1 Eicosanoids are classified by the enzymes involved in their synthesis 339 9.6.2 Endocannabinoids such as anandamide are also arachidonate derivatives 341 10 Lipids II: Metabolism and Transport of Complex Lipids 351 Complex Lipids in Context 351 10.1 Phospholipid Metabolism 353 10.1.1 Glycerophospholipids are derived from phosphatidate or diacylglycerol using activated carriers 354 10.1.2 Sphingolipids are synthesized from ceramide 355 10.1.3 Phospholipases and sphingolipases cleave at specific sites 358 10.2 Digestion of Triacylglycerols 361 10.2.1 Triacylglycerol digestion begins in the gastrointestinal tract 361 10.2.2 Dietary lipids are absorbed in the small intestine and pass into lymph before entering the circulation 363 10.2.3 Several molecules affect neutral lipid digestion 365 10.3 Transport of Lipids in the Circulation 367 10.3.1 Lipoproteins have a defined structure and composition, and transport lipids in the circulation 367 10.3.2 The trafficking of lipoproteins in the blood can be separated conceptually into three different paths 369 10.3.3 Brain lipids are transported on apo E-coated discs 372 10.3.4 Fatty acids and hydrophobic hormones are transported by binding to carrier proteins 374 10.4 Entry of Lipids into the Cell 377 10.4.1 Fatty acids can enter the cell via diffusion or by protein-mediated transport 377 10.4.2 Lipoprotein particles and many other complexes enter the cell via receptor-mediated endocytosis 377 10.5 Neutral Lipid Biosynthesis 380 10.5.1 Triacylglycerols are synthesized by different pathways depending on the tissue 380 10.5.2 Triacylglycerol metabolism and phosphatidate metabolism are enzymatically linked 382 10.6 Lipid Storage Droplets, Fat Storage, and Mobilization 384 10.6.1 Bulk neutral lipids in the cell are stored in a specific organelle, the lipid storage droplet 384 10.6.2 Specific phosphorylation of lipases and lipid droplet proteins regulate lipolysis (triacylglycerol breakdown) 384 10.6.3 Triacylglycerol metabolism is regulated at several levels 387 10.7 Lipid Rafts as a Biochemical Entity 388 10.7.1 Lipid rafts are loosely associated groups of sphingolipids and cholesterol found in the plasma membrane 388 10.7.2 Lipid rafts have been broadly grouped into two categories: caveolae and non-caveolar rafts 388 11 Amino Acid and Amine Metabolism 397 Amine Metabolism in Context 397 11.1 Digestion of Proteins 399 11.1.1 Protein digestion begins in the stomach 399 11.1.2 Protein digestion continues in the small intestine, aided by proteases 401 11.1.3 Amino acids are absorbed in the small intestine 401 11.1.4 Amino acids serve many biological roles in the organism 403 11.2 Transamination and Oxidative Deamination 404 11.2.1 Ammonia can be removed from an amino acid in two different ways 405 11.2.2 The glucose–alanine shuttle moves nitrogen to the liver and delivers glucose to tissues that need it 405 11.2.3 Glutamine is also important in nitrogen transport 406 11.3 The Urea Cycle 409 11.3.1 Ammonia detoxification begins with the synthesis of carbamoyl phosphate 409 11.3.2 The urea cycle synthesizes urea and other metabolic intermediates 409 11.3.3 Nitrogen metabolism is regulated at different levels 412 11.3.4 Mechanisms for elimination of nitrogenous wastes differ between mammals and non-mammals 412 11.3.5 Some mammals have adapted to high- or low-protein diets 413 11.4 Pathways of Amino Acid Carbon Skeleton Scavenging 414 11.4.1 Three-carbon skeletons produce pyruvate 416 11.4.2 Four-carbon skeletons produce oxaloacetate 417 11.4.3 Five-carbon skeletons produce α-ketoglutarate 418 11.4.4 Methionine, valine, and isoleucine produce succinyl-CoA 418 11.4.5 Other amino acids produce acetyl-CoA, acetoacetate, or fumarate 418 11.5 The Detoxification of Other Amines and Xenobiotics 423 11.5.1 Phase I metabolism makes molecules more hydrophilic through oxidative modification 424 11.5.2 Phase II metabolism couples molecules to bulky hydrophilic groups 427 11.6 The Biochemistry of Renal Function 429 11.6.1 Molecules smaller than proteins are filtered out of the blood by the glomerulus 430 11.6.2 Water, glucose, and electrolytes are reabsorbed in the proximal convoluted tubule, loop of Henle, and distal convoluted tubule 431 12 Regulation and Integration of Metabolism 440 Metabolism in Context 440 12.1 A Review of the Pathways and Crossroads of Metabolism 442 12.1.1 The pathways of metabolism are interconnected 442 12.1.2 Several metabolites are at the intersection of multiple pathways 443 12.2 Organ Specialization and Metabolic States 446 12.2.1 Different organs play distinct roles in metabolism 447 12.2.2 The organism shifts between different metabolic states depending on access to food 450 12.3 Communication between Organs 455 12.3.1 Organs communicate using hormones 455 12.3.2 Hormonal signals to the brain regulate appetite and metabolism 458 12.3.3 Transcription factors and histone acetylases and deacetylases regulate metabolism in the longer term 460 12.4 Metabolic Disease 463 12.4.1 Diseases of excess: obesity, diabetes, and metabolic syndrome 463 12.4.2 Diseases of absence: starvation, cachexia, and cancer 467 12.4.3 Diseases of indulgence: alcohol overconsumption 468 12.4.4 Other metabolic states 469 Techniques 477 solutions 493 Glossary 509 Index I-1

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Book SynopsisWith BPA in baby bottles, mercury in fish, and lead in computer monitors, the world has become a toxic place. But as Emily Monosson demonstrates in her groundbreaking new book, it has always been toxic. When oxygen first developed in Earth's atmosphere, it threatened the very existence of life: now we literally can't live without it. According to Monosson, examining how life adapted to such early threats can teach us a great deal about today's (and tomorrow's) most dangerous contaminants. While the study of evolution has advanced many other sciences, from conservation biology to medicine, the field of toxicology has yet to embrace this critical approach. In "Evolution in a Toxic World", Monosson seeks to change that. She traces the development of life's defence systems - the mechanisms that transform, excrete, and stow away potentially harmful chemicals - from over three billion years ago to today. Beginning with our earliest ancestors' response to ultraviolet radiation, Monosson explores the evolution of chemical defences such as antioxidants, metal binding proteins, detoxification, and cell death. As we alter the world's chemistry, these defences often become overwhelmed faster than our bodies can adapt. But studying how our complex internal defence network currently operates, and how it came to be that way, may allow us to predict how it will react to novel and existing chemicals. This understanding could not only lead to better management and preventative measures, but possibly treatment of current diseases. Development of that knowledge starts with this pioneering book.

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Book SynopsisSince the publication of the bestselling second edition of John Walker’s widely acclaimed Protein Protocols Handbook, there have been continual methodological developments in the field of protein chemistry. This greatly enhanced third edition introduces 57 critically important new chapters, as well as significantly updating the previous edition's tried-and-true methods. Although the timely new chapters are spread throughout all of the book, the vital section on post-translational modifications has been expanded most to reflect the increasing importance of these modifications in the understanding of protein function. Each readily reproducible method follows the highly praised format of the Methods in Molecular Biology™ series, offering a concise summary of its basic theory, a complete materials list, a step-by-step protocol for its successful execution, and extensive notes on avoiding pitfalls, or on modifying the method to function within your own experimental circumstances. The expert authors of each chapter have demonstrated a hands-on mastery of the methods described, fine-tuned here for optimal productivity. Comprehensive, cutting-edge, and highly practical, The Protein Protocols Handbook, Third Edition is today's indispensable benchtop manual and guide, not only for all those new to the protein chemistry laboratory, but also for those established workers seeking to broaden their armamentarium of techniques in the urgent search for rapid and robust resultsTable of ContentsPart I : QUANTITATION OF PROTEINS 1. Protein Determination by UV Absorption A. Aitken and M.P. Learmonth 2. The Lowry Method for Protein Quantitation J. H. Waterborg 3 The Bicinchoninic Acid (BCA) Assay for Protein Quantitation J.M. Walker 4 The Bradford Method for Protein Quantitation N.Kruger 5. Ultrafast Protein Determinations Using Microwave Enhancement R. Akins and R. S. Tuan 6. The Nitric Acid Methods for Protein Estimation in Biological Samples S.A. Boerner, Y.K. Lee, S.H. Kaufmann and K. Bible 7. Quantitation of Tryptophan in Proteins A. Aitken and M.P. Learmonth 8. Kinetic Silver Staining of Proteins D. D. Root and K. Wang 9. Quantitation of cellular proteins by flow cytometry T. D. Friedrich , F.A. Ray, R.L. Smith and J. M. Lehman 10. Quantitation of cellular proteins by laser scanning cytometry T.D. Friedrich, R.L.Smith and J.M. Lehman Part II : ELECTROPHORESIS OF PROTEINS AND PEPTIDES AND DETECTION IN GELS 11 Protein Solubility in 2D Electrophoresis: Basic principles and issues T.Rabilloud 12 Mouse and Human Tissues Sample Preparation for 2-D Electrophoresis C. Zabel and J. Klose 13 Plant Protein sample preparation for 2DE S. Carpentier , R.Swennen and B. Panis 14 Preparation of bacterial samples for 2-D PAGE. B. Vandahl, G. Christiansen and S. Birkelund 15 Preparation of bodily fluids for 2-D PAGE Sullivan, H.Brzeski, J. Ganesalingam, and M. Mayr 16 Immunoaffinity Depletion of high abundance plasma and serum proteins L.A. Echan and D. Speicher 17 Preparation of Yeast samples for 2D PAGE J. Norbeck 18 Membrane Protein Preparation Using Aqueous Polymer Two Phase Systems J. Schindler and H-G Nothwang 19 Subcellular fractionation of small sample amounts H.G Nothwang, I. Guillemin and J. Schindler 20. Nondenaturing Polyacrylamide Gel Electrophoresis of Proteins J.M. Walker 21 SDS Polyacrylamide Gel Electrophoresis of Proteins J.M. Walker 22 Gradient SDS Polyacrylamide Gel Electrophoresis of Proteins J.M. Walker 23. SDS-Polyacrylamide Gel Electrophoresis of Peptides R. C. Judd 24 Blue native gel electrophoresis (BN-PAGE) Elke A. Dian, J. Rassow and Christian Motz 25 Separation of proteins by gel electrophoresis in the Tris-Taurine-HCl system S.Luche, M. Chevallet C. Lelong and T. Rabilloud 26 Cetyltrimethylammonium Bromide Discontinuous Gel Electrophoresis of Proteins: M -Based Separation of Proteins with Retained Native Activity R.E. Akins and R. S. Tuan 27 Acetic Acid-Urea Polyacrylamide Gel Electrophoresis of Basic Proteins J. H. Waterborg 28 Acid-Urea-Triton Polyacrylamide Gel Electrophoresis of Histones J. H. Waterborg 29 Isoelectric Focusing of Proteins in Ultra-Thin Polyacrylamide Gels J. M. Walker 30 Serial immobilized pH gradient isoelectric focusing over pH 4-9 S.Poznanovic, W. Wozny, H. Zengerling, G.P. Schwall and M. A. Cahill 31. Radiolabelling of Eukaryotic Cells and Subsequent Preparation for 2-D < N. Bizios 32 Two-Dimensional PAGE Using Carrier Ampholyte pH Gradients in the First Dimension P.Gravel 33 Vertical agarose electrophoresis and electroblotting of high molecular weight proteins M. Greaser and C.M. Warren 34 2D PAGE of high molecular weight proteins M. Oh-Ishi and T.Maeda 35 Casting immobilised pH gradients E. Gianazza 36 Nonequilibrium pH Gel Electrophoresis J. Carroll 37 Microchip capillary electrophoresis B.A. Fogarty, N.A.Lacher and S.M. Lunte 38 Protein Separations in Microfluidic Chips A. Chow and B.Fathollahi 39 Difference gel electrophoresis (DIGE) D. Friedman and K.S. Lilley 40 Comparing 2-D Electrophoresis Gels Across Internet Databases P. Lemkin, G.C.Thornwall, and J.A. Evans 41 Quantification of Radiolabeled Proteins in Polyacrylamide Gels 25.4.07 W.R. Springer 42 Differential ProteoTope radioactive quantification of protein abundance ratios W.Wozny, G.P.Schwall, C.S. Sastri, S.Poznanovic, W. Stegmann, Hunzinger, K. Groebe and M.A. Cahill 43 Quantitation of Proteins on Polyacrylamide Gels J. Smith 44 Using SDS-PAGE and scanning laser densitometry to measure proteins Aaron P. Miles and A. Saul 45 Rapid and Sensitive Staining of Unfixed Proteins in Polyacrylamide Gels with Nile Red J-R. Daban, S. B.A. Bermudez amd F.J. Alba 46 Zinc reverse staining technique C. Fernandez-Patron 47 Protein Staining with Calconcarboxylic Acid in Polyacrylamide Gels Wei-Tao Cong, Sun-Young Hwang, Li-Tai Jin and Jung-Kap Choi 48 Detection of Proteins in Polyacrylamide Gels by Silver Staining M. J. Dunn 49 Background-free Protein Detection on Polyacrylamide Gels and on Electroblots Using Transition Metal Chelate Stains W.F. Patton 50 Detection of Proteins in Polyacrylamide Gels by Fluorescent Staining M. J. Dunn 51 Detection of Glycoproteins in Gels and Blots Nicolle Packer, Malcolm S Ball, Peter L Devine, and Wayne F Patton 52 Staining of Glycoproteins/Proteoglycans on SDS-Gels H.J. Møller and J.H. Poulsen 53. Detection of Proteins and Sialoglycoproteins in Polyacrylamide Gels Using Eosin X stain F. Lin & G. Wise 54 Pro-Q Diamond phosphoprotein staining G.K. Agrawal and J.J. Thelen 55. Electroelution of Proteins from Polyacrylamide Gels P. Jenö and Martin Horst 56 Autoradiography and Fluorography of Acrylamide Gels A. Circolo and S. Gulati 57 Proteolytic Activity Detection by Two-Dimensional Zymography J.Wilkesman Part III : BLOTTING AND DETECTION METHODS 58. Protein Blotting by Electroblotting M. Page and R.Thorpe 59. Protein Blotting by the Semi-dry Method P. Gravel 60. Protein Blotting by the Capillary Method J.M. Walker 61. Western Blotting of basic proteins electrophoretically resolved on acid-urea-Triton-polyacrylamide gels G.P. Delcuve and J. R. Davie 62 Immunoblotting of 2-DE Separated Proteins Magi and L Bianchi 63 High efficiency blotting of high-molecular weight proteins M. Greaser and D.R. Swartz 64. Alkaline Phosphatase Labeling of IgG Antibody G. B.Wisdom 65. b-Galactosidase Labeling of IgG Antibody G. B. Wisdom 66. Horseradish Peroxidase Labeling of IgG Antibody G. B. Wisdom 67. Digoxigenin (DIG) Labelling of IgG Antibody G. B. Wisdom 68. Conjugation of fluorochromes to antibodies S-Y. Mao 69. Coupling of Antibodies with Biotin R.P. Haugland and Wendy W. You 70. Preparation of Avidin Conjugates R.P. Haugland and M.K. Bhalgat 71. MDPF Staining of Proteins on Western Blots F. Alba and J.R. Daban 72. Copper Iodided Staining of Proteins and its silver enhancement D. D. Root and K. Wang 73. Detection of Proteins on Blots using Direct Blue 71 Wei-Tao Cong, Sun-Young Hwang, Li-Tai Jin and Jung-Kap Choi 74 Detection of proteins on Western blots using colorimetric and radiometric vusialization of Secondary Ligands N. J. Kruger 75. Identification of Glycoproteins on Nitrocellulose Membranes Using Lectin Blotting P. Gravel 76. A Sensitive Method to Quantitatively Detect Total Protein on Membranes after Electrophoretic Transfer Using Avidin- or Streptavidin-Biotin W.J. LaRochelle 77. Detection and Quantification of Proteins on Immunoblots using Enhanced Chemiluminescence J. Young 78 Reutilization of Western Blots After Chemiluminescent Detection or Autoradiography S. Kaufmann 79 The use of quantum dot luminescent probes for Western blot analysis S. Makrides, C. Gasbarro and J.M.Bello 80 The use of infrared fluorescent dyes in quantitative immunoblotting C-H Yang, C. Kasbek, and H. A. Fisk 81 The use of infrared fluorescent dyes in immunofluorescence microscopy C. Kasbek, C-H Yang and H. A. Fisk Part IV : CHEMICAL MODIFICATION OF PROTEINS AND PEPTIDE PRODUCTION, PURIFICATION AND CHARACTERISATION 82 Carboxymethylation of Cysteine Using Iodoacetamide/Iodoacetic Acid A. Aitken and Michele Learmonth 83 Performic Acid Oxidation A. Aitken and Michele Learmonth 84 Succinylation of Protein A. Aitken and Michele Learmonth 85 Pyridylethylation of Cysteine Residues M. Ward 86. Side-Chain Selective Chemical Modifications of Proteins D. S. Tawfik 87 Nitration of Tyrosines D. S. Tawfik 88 Ethoxyformylation of Histidine D. S. Tawfik 89 Modification of Arginine Side Chains with p-Hydroxyphenylglyoxal D. S. Tawfik 90 Amidination of Carboxyl Groups D. S. Tawfik 91 Amidination of Lysine Side Chains D.S. Tawfik 92 Modification of tryptophan with 2-Hydroxy-5-Nitrogenzylbromide D. S. Tawfik 93. Modification of Sulhydryl Groups with DTNB D. S. Tawfik 94 Chemical Cleavage of Proteins at Methionyl-X Peptide Bonds B.J. Smith 95 Chemical Cleavage of Proteins at Tryptophanyl-X Peptide Bonds B. J. Smith 96 Chemical Cleavage of Proteins at Aspartyl-X Peptide Bonds B. J.Smith 97 Chemical Cleavage of Proteins at Cysteinyl-X Peptide Bonds B. J. Smith 98 Chemical Cleavage of Proteins at Asparaginyl-Glycyl Peptide Bonds B.J. Smith 99 Enzymatic Digestion of Proteins in Solution and in SDS Polyacrylamide Gels K.L. Stone, E.E.Gulcicek and K.R. Williams 100 On-PVDF protein digestions for N-terminal sequencing and peptide mass fingerprinting Victoria Pham, William Henzel & Jennie R Lill 101 Enzymatic Digestion of Proteins on PVDF membranes J. Fernandez and S. Mische 102 Reverse Phase HPLC Separation of Enzymatic Digests of Proteins K.L. Stone and K.R. Williams 103 Peptide Mapping by Two-Dimensional Thin-Layer Electrophoresis-Thin-Layer Chromatography R. C. Judd 104 Peptide Mapping by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis R. C. Judd 105 Peptide Mapping by High-Performance Liquid Chromatography P. Højrup 106 Production of Protein Hydrolysates Using Enzymes J. M. Walker and P. J. Sweeney 107 Amino Acid Analysis by Precolumn Derivatization with 1- Fluoro-2,4-Dinitrophenyl-5-L-Alanine Amide (Marfey’s Reagent) S. Kochhar and P. Christen 108 Amino acid analysis in protein hydrolysates using anion exchange chromatography and IPAD detection P. Jandik, Jun Cheng and N. Avdalovic 109 Validation of amino acid analysis A. Reason 110 Molecular Weight Estimation for Native Proteins Using High-Performance Size Exclusion Chromatography G. B. Irvine 111 Detection of Disulfide-Linked Peptides by HPLC A. Aitken and M. Learmonth 112 Detection of Disulfide-Linked Peptides by Mass Spectrometry A.Aitken and M. Learmonth 113 Diagonal Electrophoresis for Detecting Disulfide Bridges A. Aitken and M. Learmonth 114. Estimation of Disulfide Bonds Using Ellman’s Reagent A. Aitken and M. Learmonth 115 Quantitation of Cysteine Residues and Disulfide Bonds by Electrophoresis A. Aitken and M. Learmonth 116 N-Terminal sequencing of the N-terminally Modified Proteins R. Kamp and Hisashi Hirano 117 De-blocking of Proteins containing N-Terminal Pyroglutamic Acidc J. Mozdzanowski 118 Detection and characterization of protein mutations by mass spectrometry Y. Wada 119 Peptide Sequencing by Nanoelectrospray Tandem Mass Spectrometry O. Nørregaard Jensen and M. Wilm 120 Protein identification by Peptide mass fingerprinting using MALDI-TOFF Mass spectrometry J. Webster and D. Oxley 121 Protein ladder sequencing Rong Wang and Brian T. Chait 122. Sequence Analysis with WinGene/WinPep L. Hennig 123 HPLC and MS of integral membrane proteins J. Whitelegge 124 Enrichment of Serum Peptides and Analysis by MALDI-TOF Mass Spectrometry H.W. Ressom, R.S.Varghese and R. Goldman 125 Computational Methods for the Analysis of MALDI-TOF Spectra to Discover Peptide Serum Biomarkers Y. An, H.W. Ressom and R. Goldman Part V : POST-TRANSLATIONAL MODIFICATIONS 126 Simple tools for complex N-glycan analysis A-C. Fitchette, M. Benchabane, T. Paccalet, L. Faye and V. Gomord 127 A Lectin-Binding Assay for the Rapid Characterization of the Glycosylation of Purified Gycoproteins M.T. Goodarzi, A. Fotinopoulou and G. A. Turner 128 Chemical Methods of Analysis of Glycoproteins E.F. Hounsell, M.J. Davies and K.D. Smith 129 Monosaccharide Analysis by HPAEC E.F. Hounsell, M.J. Davies and K.D. Smith 130 Monosaccharide Analysis by GC E.F. Hounsell, M.J. Davies and K.D. Smith 131 Determination of monosaccharide linkage and Substitution Patterns by GC.MS Methylation Analysis E.F. Hounsell, M.J. Davies and K.D. Smith 132 Sialic Acid Analysis by HPAEC-PAD E.F. Hounsell, M.J. Davies and K.D. Smith 133 Chemical Release of O-Linked Oligosaccharide Chains E.F. Hounsell, M.J. Davies and K.D. Smith 134 O-Linked Oligosaccharide Profiling by HPLC E.F. Hounsell, M.J. Davies and K.D. Smith 135 O-Linked Oligosaccharide Profiling by HPAEC-PAD E.F. Hounsell, M.J. Davies and K.D. Smith 136 Release of N-Linked Oligosaccharide Chains by Hydrazinolysis T.Mizuochi and E.F. Hounsell 137 Enzymatic Release of O- and N-Linked Oligosaccharide Chains E.F. Hounsell, M.J. Davies and K.D. Smith 138 N-Linked Oligosaccharide Profiling by HPLC on Porous Graphitized Carbon (PGC) E.F. Hounsell, M.J. Davies and K.D. Smith 139 N-linked Oligosaccharide Profiling by HPAEC-PAD E.F. Hounsell, M.J. Davies and K.D. Smith 140 HPAE-PAD analysis of monosaccharides released by exoglycosidase digestion Weitzhandler, M., J. Rohrer, J,R Thayer and N. Avdalovic 141 Microassay Analyses of Protein Glycosylation N.K.C. Wong, N. Kanu, N. Thandrayen, G,J.Rademaker, C.I. Baldwin, D.V. Renouf and E.F. Hounsell 142 Polyacrylamide gel electrophoresis of fluorophore-labeled carbohydrates from glycoproteins B. Brandley, J. Klock and C. Starr 143 HPLC Analysis of fluorescently labelled glycans A. Merry and S. Astrautsova 144 Glycoprofiling purified glycoproteins using Surface Plasmon Resonance A. Fotinopoulou and G.A. Turner 145 Sequencing Heparan Sulphate Saccharides J. E. Turnbull 146 Analysis of glycoprotein heterogeneity by Capillary Electrophoresis and Mass Spectrometry A. Hooker and D.C. James 147 Affinity chromatography of oligosaccharides and glycopeptides with immobilized lectins K. Yamamoto 148 In-Gel Enzymatic Release of N-glycans D. Harvey 149 Analysis of N-Linked glycans by mass spectrometry D. Harvey 150 MS Analysis of Protein glycosylation N.Takemori, N. Komori and H. Matsumoto 151 Mapping protein N-glycosylation by COFRADIC B. Ghesquiere, J. Vandekerckhove, and K. Gevaert 152 Mass spectrometric analysis of O-linked glycans released directly from Glycoproteins in gels using ß-elimination A.M. Taylor and J. Thomas-Oates 153 Glycopeptide analysis using LC/MS and LC/MSn S.Itoh, D. Takakura, N. Kawasaki and T. Yamaguchi 154 Identification of Vitamin K-Dependent Proteins Using a Gla-specific Monoclonal Antibody K. Hansson 155 The identification of protein S-Nitrocysteine T.M Greco, S.L.Stamer, D.C. Liebler and H. Ischiropoulos 156 Detection of nitrotyrosine-containing proteins Xianquan Zhan and D. Desiderio 157 Mass spectrometric determination of protein Ubiquitination C. E. Parker, M.R.E. Warren, V. Mocanu, S.F. Greer and C.H. Borchers 158 Detection of Sumoylated Proteins Ok-Kyong Park-Sarge and K. Sarge 159 Efficient enrichment of intact phosphorylated proteins by modified immobilized metal-affinity chromatography A. Dubrovska 160 Analyzing Protein Phosphorylation J. Colyer 161 Mass spectrometric analysis of protein phosphorylation S. Gander, A. Cremonesi, J. Chicher, S. Moes and P. Jenö 162 Protein microarrays for phosphorylation studies B. Kersten and T. Feilner 163. Two Dimensional Phosphopeptide Mapping H. Nagahara, R.R. Latek, S.A. Ezhevsky and S.F. Dowdy 164 Identification of Proteins Modified by Protein (D-Aspartyl/L-Isoaspartyl) Carboxyl Methyltransferase D. Weber and P.N. McFadden 165 Analysis of Tyrosine-o- sulfation Jens R. Bundgaard, J.W. Sen, A.H. Johnsen and J.F. Rehfeld 166 Analysis of Protein Palmitoylation by metabolic radiolabeling methods K.H. Pedone, L.S. Bernstein, Maurine E. Linder and J.R. Hepler 167 Incorporation of radiolabeled prenyl alcohols and their analogs into mammalian cell proteins: a useful tool for studying protein prenylation A. Corsini, C.C. Farnsworth, P. McGeady, M.H. Gelb and J.A. Glomset 168 Labelling and analysis of isoprenylated proteins in cells D.A. Andres, D.C. Crick, H.P.Spielmann and C. J. Waechter Part VII : ANTIBODY TECHNIQUES 169 Antibody Production R. Burns 170 Production of Antibodies Using Proteins in Gel Bands S.A. Amero, T.C. James and S.C.R. Elgin 171 Raising Highly Specific Polyclonal Antibodies Using Biocompatible Support Bound Antigens M. Diano and A. Le Bivic 172 Production of antisera using peptide conjugates T. E. Adrian 173 Small-molecule protein conjugation procedures S. Thompson 174 The Chloramine T Method for Radiolabeling Protein G.S. Bailey 175 The Lactoperoxidase Method for Radiolabeling Protein G.S. Bailey 176 The Bolton and Hunter Method for Radiolabeling Protein G.S. Bailey 177 Preparation of 125I-labelled peptides and proteins with high specific activity using iodogen M. Conlon 178 Purification and Assessment of Quality of Radioiodinated Protein G.S. Bailey 179 Purification of IgG by Precipitation with Sodium Sulfate or Ammoniun Sulfate M. Page and R. Thorpe 180 Purification of IgG Using Caprylic Acid M. Page and R. Thorpe 181 Purification of IgG Using DEAE-Sepharose Chromatography M. Page and R. Thorpe 182 Purification of IgG Using Ion-Exchange HPLC M. Page and R. Thorpe 183 Purification of IgG by Precipitation with Polyethylene Glycol (PEG) M. Page and R. Thorpe 184 Purification of IgG Using Protein A or Protein G M. Page and R. Thorpe 185 Purification of IgG Using Size Exclusion High Performance Liquid Chromatography (SE-HPLC) C. Dolman and R. Thorpe 186 Purification of IgG Using Affinity Chromatography on Antigen-Ligand Columns M. Page and R. Thorpe 187 Purification of IgG Using Thiophilic Chromatography M. Page and R. Thorpe 188 Analysis of IgG Fractions by Electrophoresis M. Page and R. Thorpe 189 Purification of Immunoglobulin Y (IgY) from Chicken Eggs C.R. Bird and R. Thorpe 190 Affinity Purification of Immunoglobulins Using PAM (Protein A Mimetic) G. Fassina G. Palombo, A. Verdoliva and M. Ruvo 191 Detection of Serological Cross Reactions by Western Cross Blotting P. Hemmerl, A. Hartl, J. Freund and J.Thalhamer 192 Enzymatic Digestion of Monoclonal Antibodies S. Andrew 193 How to Make Bispecific Antibodies R. French 194 Antigen Measurements Using ELISA W. Jordan 195 Enhanced chemiluminescence immunoassay R. Stott 196 Immunoprecipitation and Blotting S. Thompson 197 Determination of Epitope by Mass Spectrometry C.Hager-Braun and K. B. Tomer 198 Immunogen Preparation and Immunization Procedures for Rats and Mice M. Page and R. Thorpe 199 Making Hybridomas R. Burns 200 Growing hybridomas G. Entrican, C. Jepson and D. Deane 201 Mouse Hybridomas as an Entryway to Monoclonal Antibody Design and Production E. Mechetner 202 Pitfalls E. Mechetner 203 Recombinant antibody expression and purification A. Knappik and R. Brundiers 204 Screening Hybridoma Culture Supernatants Using Solid-Phase Radiobinding Assay M. Page and R. Thorpe 205 Screening Hybridoma Culture Supernatants Using ELISA M. Page and R. Thorpe 206 Growth and Purification of Murine Monoclonal Antibodies M. Page and R. Thorpe 207 Affinity purification techniques for monoclonal antibodies A. Schwarz 208 A rapid method for generating large numbers of high-affinity monoclonal antibodies from a single mouse N.T. Man and G.E. Morris

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