Elsevier S & T

Oxygen Biology and Hypoxia

Sies, Helmut

In stock
No reviews
74.000 KD inc. VAT
License
Table of contents
  • Contentsv
  • Contributorsxiii
  • Prefacexxi
  • Volumes in Seriesxxiii
  • Section I: Hypoxia-Inducible Factor1
  • Chapter 1: Hypoxia-Inducible Factors Per/ARNT/Sim Domains: Structure and Function3
  • 1. Introduction4
  • 2. Delineation of the HIF PAS Domains5
  • 3. Expression and Characterization of HIF PAS Domains7
  • 3.1. Recombinant protein expression7
  • 3.2. Limited proteolysis8
  • 3.3. Solution NMR spectroscopy10
  • 4. Assessing PAS Domain Protein-Protein Interactions11
  • 4.1. NMR spectroscopy11
  • 4.2. Reporter genes of HIF function13
  • 4.3. Preparation of nuclear lysates15
  • 4.4. Coimmunoprecipitation of HIF-alpha and HIF-beta15
  • 4.5. Electrophoretic mobility shift assay17
  • 5. Discussion18
  • Acknowledgments20
  • References21
  • Chapter 2: Hypoxia-Inducible Factor Prolyl-Hydroxylase: Purification and Assays of PHD225
  • 1. Introduction26
  • 2. Preparation of Purified PHD2 from a Bacterial Source28
  • 3. Assaying of PHD2 Activity29
  • 4. Indirect Measurements of PHD2 Activity29
  • 4.1. 1-[14C]-CO2 capture assay30
  • 4.2. Fluorescence derivatization of 2OG32
  • 4.3. Oxygen consumption assay33
  • 4.4. 1-[14C]succinate quantification35
  • 5. Direct Measurements of PHD2 Hydroxylation Activity35
  • 5.1. LC/MS identification of hydroxylated HIF-1alpha 556 to 57435
  • 5.2. pVHL capture assay36
  • 6. Binding Assays37
  • 7. Comparison of Assay Formats38
  • References25
  • Chapter 3: Determination and Modulation of Prolyl-4-Hydroxylase Domain Oxygen Sensor Activity43
  • 1. Introduction44
  • 2. Production of Functionally Active PHDs48
  • 3. Determination of PHD Activity by VHL Binding to Peptides Derived from the HIF-1alpha ODD Domain48
  • 4. Determination of Prolyl-4-Hydroxylation by Oxidative Decarboxylation of 2-Oxoglutarate51
  • 5. Crude Tissue Extracts are not a Suitable Source of PHD Activity for the 2-Oxoglutarate Conversion53
  • 6. Thin Layer Chromatography to Assess the Purity of [5-14C] 2-Oxoglutarate53
  • 7. Application of the 2-Oxoglutarate Conversion Assay to Protein Targets55
  • 8. Conclusions55
  • Acknowledgments25
  • References57
  • Chapter 4: Characterization of Ankyrin Repeat-Containing Proteins as Substrates of the Asparaginyl H61
  • 1. Introduction62
  • 2. Experimental Techniques65
  • 2.1. Production of FIH-165
  • 2.2. Protein quantitation68
  • 2.3. Production of substrates69
  • 2.4. CO2 capture assays71
  • 2.5. Interaction assays76
  • 2.6. In vitro pull-down assay76
  • 2.7. Co-immunoprecipitation assay79
  • 3. Discussion/Conclusion82
  • Acknowledgments25
  • References83
  • Chapter 5: Transgenic Models to Understand Hypoxia-Inducible Factor Function87
  • 1. Introduction88
  • 2. Hypoxia Response Pathway Genes and Development90
  • 2.1. HIF-alpha subunits90
  • 2.2. HIF-beta/ARNT subunits91
  • 2.3. pVHL92
  • 2.4. PHDs92
  • 2.5. VEGF93
  • 3. HIF in Physiology93
  • 3.1. HIF-1alpha heterozygotes93
  • 3.2. Conditional knockouts using the loxP/cre recombinase system94
  • 3.3. Liver94
  • 3.4. Myeloid lineage94
  • 3.5. Lymphocytes95
  • 3.6. Hematopoiesis95
  • 3.7. Skeletal muscle95
  • 3.8. Heart and cardiovascular system96
  • 3.9. Mammary gland96
  • 3.10. Colon96
  • 3.11. Chondrocytes96
  • 3.12. Skin97
  • 3.13. Motor neurons and brain97
  • 4. HIF Function in Tumor Biology98
  • 4.1. First insights-xenografts98
  • 4.2. Astrocytoma99
  • 4.3. Mammary carcinogenesis99
  • 4.4. pVHL and VHL disease99
  • 5. Summary100
  • References25
  • Chapter 6: The Silencing Approach of the Hypoxia-Signaling Pathway107
  • 1. A Brief History of RNAi108
  • 2. The Hypoxia-Signaling Pathway109
  • 3. HIF-alpha Stability110
  • 3.1. The family of the HIF prolyl-hydroxylases110
  • 3.2. Acetylation/deacetylation (ARD1/HDAC1)114
  • 4. HIF Activity114
  • 4.1. The factor-inhibiting HIF114
  • 5. HIF-1/HIF-2 Target Gene Specificity116
  • 6. RNAi as a New Potential Therapeutic Strategy116
  • Acknowledgments118
  • References118
  • Chapter 7: Cellular and Developmental Adaptations to Hypoxia: A Drosophila Perspective123
  • 1. Introduction124
  • 2. Drosophila melanogaster as a Model System to Study Physiological Responses to Hypoxia124
  • 3. Experimental Advantages of the Model System125
  • 4. The Drosophila Respiratory System126
  • 5. Occurrence of a Drosophila System Homologous to Mammalian HIF128
  • 6. Regulation of Sima by Oxygen Levels131
  • 7. Role of Sima and Fatiga in Drosophila Development132
  • 8. Hypoxia-Inducible Genes and the Adaptation of Drosophila to Oxygen Starvation134
  • 9. Regulation of Sima by the PI3K and TOR Pathways134
  • 10. Role of the HIF System in Growth Control and Cell Size Determination136
  • 11. Concluding Remarks138
  • Acknowledgments139
  • References139
  • Section II: Erythropoietin145
  • Chapter 8: Constitutively Overexpressed Erythropoietin Reduces Infarct Size in a Mouse Model of Perm147
  • 1. Introduction148
  • 2. Material and Methods149
  • 2.1. Animals and surgery149
  • 2.2. Determination of infarct size and immunohistochemical analysis150
  • 3. Results151
  • 3.1. EPO plasma levels and EPO-R expression in the myocardium151
  • 3.2. Cardiac protection by EPO153
  • 3.3. Infarct size correlates with WT1 staining153
  • 4. Discussion153
  • Acknowledgments
  • References
  • Chapter 9: Use of Gene-Manipulated Mice in the Study of Erythropoietin Gene Expression157
  • 1. Introduction158
  • 2. Materials161
  • 2.1. Genetic manipulation of mouse lines161
  • 2.2. Hypoxic chamber161
  • 2.3. BAC clones162
  • 2.4. Plasmids and bacterial strains for recombination of BAC clones162
  • 2.5. Antibodies and immunohistochemistry162
  • 3. Methods and Results163
  • 3.1. Real-time and noninvasive monitoring of EPO activity in vivo163
  • 3.2. Analysis of erythropoietic and non-erythropoietic function of EPO-EPOR pathway in vivo165
  • 3.3. Transgenic mouse-expressing GFP under the control of Epo gene regulatory region166
  • 3.4. Regulatory region sufficient for in vivo Epo gene expression168
  • 3.5. Identification of the REP cell169
  • 3.6. Essential cis-elements for cell type-specific and inducible Epo gene expression in vivo171
  • 4. Conclusion173
  • Acknowledgments
  • References
  • Chapter 10: Control of Erythropoietin Gene Expression and its Use in Medicine179
  • 1. Introduction180
  • 2. Native EPO Gene Expression and its Pharmacologic Stimulation
  • 3. EPO Gene Transfer
  • 4. Recombinant EPOS
  • 5. Conclusions190
  • References
  • Chapter 11: Role of Hypoxia-Inducible Factor-2alpha in Endothelial Development and Hematopoiesis199
  • 1. Introduction200
  • 2. Vasculogenesis/Angiogenesis and HIFS200
  • 2.1. HIF-2a null mice201
  • 2.2. HIF-2alpha knockdown mice201
  • 2.3. HIF-1alpha null mice203
  • 2.4. HIF-1alpha null EC204
  • 3. HIF-1beta/ARNT Null Mice204
  • 4. Neovascularization and HIFS205
  • 4.1. HIF-2alpha knockdown mice205
  • 4.2. HIF-1alpha null EC210
  • 5. Hematopoiesis and HIFS211
  • 5.1. HIF-2alpha null mice211
  • 5.2. HIF-2alpha knockdown mice212
  • 5.3. HIF-1a null mice213
  • 5.4. HIF-1beta/ARNT null mice213
  • 6. Conclusion214
  • References
  • Section III: Hypoxia and Adaptation219
  • Chapter 12: Organ Protection by Hypoxia and Hypoxia-Inducible Factors221
  • 1. Introduction222
  • 2. From Ischemic to Hypoxic Preconditioning
  • 3. Hypoxia-Inducible Transcription Factors
  • 4. Strategies to Activate HIF and HIF Target Genes
  • 4.1. Hypoxic hypoxia and carbon monoxide
  • 4.2. Inhibition of HIF prolyl hydroxylases229
  • 4.3. Additional strategies to activate the HIF pathway230
  • 5. Hypoxic Preconditioning and HIF231
  • 5.1. Protective role of HIF target genes231
  • 5.2. HIF activation and organ protection232
  • 6. HIF in Chronic Hypoxic/Ischemic Diseases
  • 7. Conclusions and Perspectives
  • References
  • Chapter 13: Hypoxia and Regulation of Messenger RNA Translation247
  • 1. Introduction248
  • 2. Changes in Global mRNA Translation During Hypoxia
  • 2.1. Kinetics and oxygen dependency250
  • 2.2. Influence of genetic background250
  • 3. Molecular Mechanisms that Regulate mRNA Translation During Hypoxia
  • 3.1. Translational regulation during hypoxia by eIF2alpha phosphorylation252
  • 3.2. Translational regulation during hypoxia by eIF4F complex availability254
  • 3.3. Translational regulation by eEF2 phosphorylation255
  • 4. Methods Employed to Study mRNA Translation During Hypoxia256
  • 4.1. Protein synthesis256
  • 4.2. The polysome assay256
  • 4.3. Enzymatic activity of reporter constructs267
  • 5. Protocols268
  • 5.1. 35S methionine labeling268
  • 5.2. Polysome fractionation269
  • References
  • Chapter 14: Hypoxia and the Unfolded Protein Response275
  • 1. Introduction276
  • 1.1. Mechanisms of cellular adaptation to low oxygen environment276
  • 1.2. Causes of ER stress and UPR277
  • 1.3. The PERK-eIF2alpha-ATF4 arm of the UPR278
  • 1.4. The IRE1-XBP1 arm of the UPR279
  • 1.5. The ATF6 pathway280
  • 1.6. Consequences of aberrant UPR induction for tumor formation281
  • 2. Methods Employed in Detecting Hypoxic Induction of ER Stress282
  • 2.1. Events proximal to ER stress282
  • 2.2. Events distal to ER stress286
  • Acknowledgments
  • References
  • Section IV: Hypoxia and Tumor Biology295
  • Chapter 15: Tumor Hypoxia in Cancer Therapy297
  • 1. Hypoxia in Human Tumors298
  • 2. The Dynamic Nature of Hypoxia in Tumors300
  • 3. Consequences of Tumor Hypoxia for Cancer Treatment300
  • 4. Size of the Oxygen Effect with Radiation302
  • 5. The Influence of Tumor Hypoxia on Cancer Treatment by Radiotherapy303
  • 6. Influence of Tumor Hypoxia on Response to Chemotherapy307
  • 7. Exploiting Hypoxia in Cancer Treatment308
  • 7.1. Hypoxic cytotoxins308
  • 7.2. Hypoxia-selective gene therapy311
  • 7.3. Targeting HIF-1312
  • 7.4. Exploiting tumor necrosis with obligate anaerobes314
  • References
  • Chapter 16: HIF Gene Expression in Cancer Therapy323
  • 1. Introduction324
  • 2. Experimental Procedures326
  • 2.1. Induction by hypoxia or chemical mimetics326
  • 2.2. Genetic mutations327
  • 2.3. HIF hydroxylation and other posttranslational modifications328
  • 2.4. HIF inhibitors329
  • 2.5. RNA interference331
  • 2.6. HIF knockout mice332
  • 2.7. Somatic cell HIF knockout332
  • 2.8. HIF activity (reporter assays)333
  • 2.9. HIF responsive elements333
  • 2.10. RNA analysis333
  • 2.11. Quantitative RT-PCR334
  • 2.12. Western blot analysis334
  • 2.13. Chromatin immunoprecipitation334
  • 2.14. HEEBO microarray analysis335
  • 3. Conclusions337
  • Acknowledgments
  • References
  • Chapter 17: Analysis of Hypoxia-Inducible Factor 1alpha Expression and its Effects on Invasion and M347
  • 1. Introduction347
  • 2. Protocol 1: HIF-1alpha Immunohistochemistry349
  • 3. Protocol 2: Invasion Assay350
  • 4. Protocol 3: Transepithelial Resistance Measurement of Cell-Cell Adhesion351
  • 5. Protocol 4: Analysis of MRNA Expression by QRT-PCR351
  • References
  • Chapter 18: Macrophage Migration Inhibitory Factor Manipulation and Evaluation in Tumoral Hypoxic Ad355
  • 1. Introduction356
  • 2. Modulation of MIF Levels by Targeted shRNAs and Assessment of Knockdown Efficiency357
  • 2.1. MIF-specific shRNA transfection357
  • 2.2. Assessment of MIF knockdown and associated phenotypes by RT-PCR359
  • 2.3. Assessment of MIF knockdown and associated loss of HIF-alpha stability by Western blotting360
  • 2.4. Enzymatic analyses361
  • 3. Analysis of MIF-Dependent CSN5 and COP9 Signalosome Function362
  • 3.1. CSN5 co-immunoprecipitations363
  • 3.2. CSN-dependent deneddylation363
  • 3.3. Determination of CSN-associated versus-disassociated CSN5364
  • 4. Determination of Tumor-Associated MIF Expression and MIF Polymorphic Disparity364
  • 4.1. Immunohistochemistry of MIF and CSN5 tumor expression levels and correlation to hypoxic adaptat365
  • 4.2. MIF plasma analysis and genomic DNA extraction365
  • 4.3. Genotyping of MIF -173 G/C and MIF5-8 CATT repeats from human samples366
  • 5. Conclusions367
  • References367
  • Chapter 19: The von Hippel-Lindau Tumor Suppressor Protein: An Update371
  • 1. Introduction372
  • 2. Regulation of Epithelial Differentiation by pVHL373
  • 2.1. E-cadherin373
  • 2.2. The primary cilium373
  • 3. Crosstalk between c-Met and VHL374
  • 4. Regulation of Neuronal Apoptosis by pVHL375
  • 5. Possible Links Between p53 and pVHL376
  • 6. Regulation of pVHL by Phosphorylation376
  • 7. Polyubiquitylation of pVHL377
  • 8. Mouse Models for Studying pVHL Function377
  • References
  • Chapter 20: Hypoxia-Inducible Factor 1 Inhibitors385
  • 1. Introduction386
  • 2. Cell-Based High Throughput Screens387
  • 2.1. Cell-based HTS protocol389
  • 2.2. Validation of active "hits" from HTS390
  • 2.3. Small molecule inhibitors of HIF-1 identified in cell-based assays392
  • 3. Cell-Free Assays393
  • 3.1. Inhibition of HIF-1 DNA binding393
  • 3.2. Inhibition of protein-protein interaction395
  • 3.3. Inhibition of HIF-1 transcriptional activity397
  • 4. Bioassay-Directed Isolation of Natural Product HIF-1 Inhibitors398
  • 5. Conclusions399
  • Acknowledgments
  • References400
  • Section V: Hypoxia and Inflammatory Mediators403
  • Chapter 21: Regulation of Hypoxia-Inducible Factors During Inflammation405
  • 1. Introduction406
  • 2. Regulation of HIF at the Transcriptional Level408
  • 3. Regulation of HIF at the Translational Level410
  • 4. Regulation of HIF-1alpha at the Posttranslational Level411
  • 5. Regulation of HIF-1 Activity413
  • 6. Perspectives414
  • 7. Conclusions414
  • Acknowledments
  • References
  • Chapter 22: Superoxide and Derived Reactive Oxygen Species in the Regulation of Hypoxia-Inducible Fa421
  • 1. Introduction422
  • 2. Reactive Oxygen Species Act as Signaling Molecules423
  • 3. HIFs are Sensitive to Oxygen424
  • 4. Reactive Oxygen Species Modulate HIF425
  • 5. How are HIFs Regulated by Reactive Oxygen Species?427
  • 5.1. Regulation of HIF-alpha synthesis by reactive oxygen species?427
  • 5.2. Direct regulation of HIF-a by reactive oxygen species428
  • 5.3. Regulation of HIF by reactive oxygen species via interference with a regulatory signaling pathw428
  • 6. Summary431
  • 7. Methods431
  • 8. The Cytochrome C Reduction Assay for Detection of Extracellular Reactive Oxygen Species431
  • 9. Chemiluminescence Assay for Detection of Extracellular Reactive Oxygen Species432
  • 10. Measuring Intracellular Production of Reactive Oxygen Species using Fluorescent Dyes433
  • 10.1. DCF fluorescence434
  • 10.2. Hydroethidine fluorescence434
  • 10.3. Dihydrorhodamine fluorescence435
  • 10.4. ROS measurements in tissues436
  • 11. Detection of Reactive Oxygen Species by Electron Paramagnetic Resonance436
  • Acknowledgments
  • References438
  • Chapter 23: Genetics of Mitochondrial Electron Transport Chain in Regulating Oxygen Sensing447
  • 1. Introduction448
  • 2. Detecting HIF-1alpha Protein Levels449
  • 2.1. Background449
  • 2.2. Materials450
  • 2.3. Equipment451
  • 2.4. Methods451
  • 3. Detecting Intracellular ROS Levels452
  • 3.1. Background452
  • 3.2. Materials453
  • 3.3. Equipment453
  • 3.4. Methods453
  • 4. Method 1: Examining Hypoxic Stabilization of HIF-1alpha Protein in Cells Containing RNAI against454
  • 4.1. Background454
  • 4.2. Materials454
  • 4.3. Equipment455
  • 4.4. Methods455
  • 5. Method 2: Examining the Role of ROS Generated from Mitochondrial Electron Transport in Hypoxic St458
  • 5.1. Background458
  • 5.2. Materials458
  • 5.3. Methods458
  • 6. Concluding Remarks459
  • Acknowledgments
  • References
  • Chapter 24: Hypoxia-Inducible Factor-1alpha Under the Control of Nitric Oxide463
  • 1. HIF-1 and Oxygen Sensing464
  • 2. Nitric Oxide: A Multifunctional Messenger465
  • 3. Accumulation of HIF-1alpha and Activation of HIF-1 by NO467
  • 4. Superoxide Stabilizes HIF-1alpha but Antagonizes NO Actions470
  • 5. Hypoxic Signal Transmission is Antagonized by NO472
  • 6. Summary and Conclusions473
  • Acknowledgments
  • References475
  • Chapter 25: Hypoxic Regulation of NF-kappaB Signaling479
  • 1. Background480
  • 2. Treatment Protocols for Cellular Hypoxia Studies481
  • 2.1. Exposure of cells to ambient atmospheric hypoxia482
  • 2.2. Inhibition of hydroxylases in cultured cells482
  • 2.3. Manipulation of NF-kappaB signaling in cultured cells483
  • 3. Measurement of NF-kappaB Activity in Cultured Cells484
  • 3.1. NF-kappaB-luciferase promoter-reporter assay484
  • 3.2. NF-kappaB DNA-binding assay485
  • 3.3. Immunoblotting analysis488
  • 3.4. Peptide pull-down assay490
  • 4. Summary/Conclusions491
  • References
  • Author Index493
  • Subject Index535
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739704
  • ISBN-13 9780080554860
  • Author Sies, Helmut
  • Category Science
  • Subject Molecular Biology

Do you have questions about this book?

Ask an expert!

Description eBook delivery & access Reviews
For over fifty years the Methods in Enzymology series has been the critically acclaimed laboratory standard and one of the most respected publications in the field of biochemistry. The highly relevant material makes it an essential publication for researchers in all fields of life and related sciences. This volume features articles on the topic of oxygen biology and hypoxia.