Amyloid, prions, and other protein aggregates, Part B

000 fm.pdf......Page 2
001 preface.pdf......Page 3
002 toc.pdf......Page 6
003 contributors.pdf......Page 9
Introduction......Page 13
The Birth of PMCA......Page 15
The Molecular Mechanism of Species Barrier and Prion Strains Phenomena......Page 16
Screening for Inhibitors of Prion Propagation......Page 17
Extension to Other Protein Misfolding Diseases......Page 18
Sonicator......Page 19
Preparation of Prion-Infected Samples from CNS......Page 20
Preparation of Prion-Infected Samples from Peripheral Sources......Page 21
Other Sources of PrPSc such as CSF, Saliva, Milk, Urine, and Feces......Page 22
Normal Brain Homogenate......Page 23
Lipid Rafts......Page 24
Automated PMCA Procedure......Page 25
Serial Automatic PMCA (saPMCA) Procedure......Page 27
Detection of Amplified Product......Page 28
References......Page 29
Fractionation of Prion Protein Aggregates by Asymmetrical Flow Field-Flow Fractionation......Page 32
Field-Flow Fractionation......Page 33
Separation of PrP-res Aggregates by Asymmetrical FlFFF......Page 36
References......Page 42
Analysis of Amyloid Aggregates Using Agarose Gel Electrophoresis......Page 45
Yeast Prions [PSI+] and [PIN+]......Page 46
Protein Electrophoresis in Agarose......Page 48
Transfer of Proteins from Agarose Gels and Immunostaining......Page 49
Analysis of [PSI+] and [PIN+] Prion Subparticles from Yeast Lysates by Electrophoresis in Agarose......Page 52
Analysis of Sup35NM Amyloid Fibers......Page 53
References......Page 57
Characterization of Systemic Amyloid Deposits by Mass Spectrometry......Page 61
Introduction......Page 62
Tissue Specimens......Page 63
Protein/Peptide Preparation......Page 64
Tandem Mass Spectrometry (MS/MS)......Page 65
MS/MS Analyses of Amyloid Deposits......Page 66
Comments......Page 73
References......Page 74
Proteomics of Polyglutamine Aggregates......Page 76
Preparing Neuro 2A Cells Carrying Ecdysone-Inducible tNhtt-150Q-EGFP or tNhtt-150Q-EGFP-NLS Gene......Page 77
EGFP-Fused Cytoplasmic PolyQ Aggregates......Page 79
Nuclear Aggregates......Page 81
SDS-PAGE of the Purified Aggregates......Page 82
Procedure......Page 84
Condition of HPLC......Page 85
Data Analysis......Page 86
References......Page 89
Introduction......Page 90
Technical Comment......Page 93
Protein Extraction from LCM Caps......Page 94
Preparation of Abeta Plaque Proteins for Gel LC-MS/MS......Page 96
Abeta Plaque Protein Identification......Page 98
Abeta Plaque Protein Quantification......Page 100
Technical Comment......Page 102
Conclusion......Page 103
References......Page 104
Introduction......Page 107
MALDI Matrix Deposition......Page 108
Fixation, Staining......Page 109
MALDI Mass Spectrometric Imaging......Page 111
MSI Data Analysis......Page 112
Clustering......Page 114
Identification......Page 116
Conclusions......Page 117
References......Page 118
Imaging Polyglutamine Deposits in Brain Tissue......Page 120
Polyglutamine Aggregate Formation......Page 121
Polyglutamine Aggregation Reactions In Vitro and In Situ......Page 122
Introduction......Page 123
Human and Animal Tissues and Tissue Preparation......Page 124
Peptide Design, Synthesis, and Purification......Page 125
Solutions......Page 127
Protocol for Polyglutamine Recruitment and Elongation......Page 128
Immunohistochemical Detection of Polyglutamine......Page 129
Solutions......Page 131
Protocol for Detection of Polyglutamine......Page 132
Acknowledgments......Page 134
References......Page 135
Introduction......Page 137
Synthesis of X-34......Page 138
X-34 Histostaining Protocol......Page 140
X-34 Is a Marker of the Full Spectrum of Amyloid Pathology in AD Brains......Page 142
Hippocampus......Page 143
Caudate Nucleus and Cerebellum......Page 145
Dystrophic Neurites and Neuropil Threads......Page 146
Comparative Analysis: X-34, Thioflavin S (ThioS) and Abeta Immunohistochemistry (IHC)......Page 147
Concentration-Dependent Detection of Selective Amyloid Pathology with X-34 and X-34-Dimethoxy Fluorescent Histostaining......Page 148
Combining X-34 Histostaining with IHC......Page 150
X-34 Histostaining of Peripheral Amyloidosis......Page 152
Discussion......Page 153
References......Page 156
Visualizing Pathology Deposits in the Living Brain of Patients with Alzheimer\'s Disease......Page 159
Introduction......Page 160
Noninvasive In Vivo Visualization of Neuropathological Deposits......Page 162
Characterization of [F-18]FDDNP Binding to Abeta Fibrils and SPs......Page 164
Preparation of [F-18]FDDNP......Page 166
PET Scanning Protocol......Page 167
Logan Plot Graphical Analysis of Kinetic PET Data......Page 168
Example of Logan Plot Graphical Analysis of [F-18]FDDNP PET Data......Page 169
Acknowledgments......Page 171
References......Page 172
Introduction......Page 176
Microimaging Apparatus......Page 177
Preparation of Radioiodinated Proteins......Page 179
Procedure for the Chloramine T Radioiodination of Proteins......Page 180
Immunoreactivity Protocol for Radiolabeled 11-1F4 mAb......Page 182
Radiolabeling using the Succinimidyl-iodobenzoate (SIB) Method......Page 183
AL Amyloidoma......Page 184
MicroSPECT and CT Image Acquisition......Page 186
Image Coregistration......Page 187
SPECT Image Reconstruction......Page 188
Image Visualization and Analysis......Page 189
Biodistribution......Page 190
Autoradiography......Page 191
Summary......Page 193
References......Page 194
Introduction......Page 198
Purification Protocol......Page 202
Preparation of Different Conformations of Sup-NM Amyloid......Page 203
Preparation of Prions from an In Vivo Source......Page 204
Protein Transformation into Yeast......Page 206
Determination of Prion Conversion Efficiency and Prion Strain Phenotypes......Page 208
Application to the Study of Other Prion or non-Mendelian Elements......Page 210
References......Page 211
Introduction......Page 214
Modeling Amyloid Toxicity in Yeast......Page 215
Generating a Yeast Model of Amyloid Toxicity......Page 218
Obtaining Gene Mutations in Yeast......Page 220
Overexpression Screens......Page 222
Pooling the YKO Library Strains......Page 225
High-Efficiency Yeast Transformation......Page 226
Amplification and Sequencing of Molecular Barcode......Page 227
One Step Buffer (prepare fresh)......Page 228
Spotting Assays......Page 229
Systematic Approaches to Genetic Screening......Page 230
Conclusion......Page 231
References......Page 232
Introduction......Page 236
Cell Lines Chronically Infected with TSEs......Page 237
N2a Cell-Based High-Throughput PrP-res Inhibition Assay......Page 238
Dot-Blot Procedure and Immunodetection of PrP-res on Membranes......Page 240
Inhibitors Found with This Assay......Page 241
The Use of Transgenic Mice for In Vivo Anti-Scrapie Testing......Page 242
Conclusion......Page 244
References......Page 245
A Drosophila Model of Alzheimer\'s Disease......Page 248
What Is it About Alzheimer\'s Disease That We Want To Model?......Page 249
Simple Models Based on Secreted Abeta Peptides......Page 250
Modeling the Generation of Abeta from Human Amyloid Precursor Protein......Page 251
Histological Assessment of a Drosophila Model of AD......Page 255
Climbing and Other Locomotor Assays......Page 256
Rough Eye Phenotype Assessment and the Pseudopupil Assay......Page 258
Pavlovian Olfactory Learning Assays......Page 259
Genetic Screens......Page 260
Chemical Mutagenesis Screens......Page 261
P-Element-Based Screens......Page 262
Drug Screens......Page 263
References......Page 266
Introduction......Page 270
Invertebrate Models of Neurodegenerative Disease......Page 271
C. elegans Models of Polyglutamine Disease......Page 272
Visualization of Protein in a Live, Multicellular Organism......Page 274
Plasmid Construction......Page 275
High-Resolution Imaging in C. elegans......Page 276
Fluorescence Recovery After Photobleaching......Page 277
FRAP Data Analysis......Page 279
FRAP Analysis of Polyglutamine Proteins in a Multicellular Organism......Page 280
Fluorescence Resonance Energy Transfer (FRET)......Page 282
FRET Reveals Intermolecular Interactions of Polyglutamine Proteins in a Multicellular Organism......Page 283
Aging Influences the Threshold for Polyglutamine Aggregation and Toxicity......Page 284
Genome-Wide RNAi Screening Defines Novel Regulators of Polyglutamine Aggregation and Toxicity......Page 286
Genome-wide RNAi Screening in C. elegans......Page 288
Using C. elegans to Identify Modifiers of Polyglutamine Pathogenesis......Page 290
References......Page 292
Introduction......Page 297
Equilibrium......Page 298
Clusters......Page 301
Linear Structures......Page 302
Three-Dimensional Clusters......Page 304
Internal Changes......Page 306
The Concentration of Nuclei......Page 307
The Outcome......Page 308
Other Approaches......Page 309
Heterogeneous Nucleation......Page 310
References......Page 311
Introduction......Page 312
Protein Threading Using PROSPECT......Page 314
Threading Energy Functions......Page 315
Protein Threading and Model Construction......Page 316
Model Quality Evaluation......Page 317
Molecular Dynamics Simulations......Page 319
Simulation Result Analysis......Page 321
Discussion......Page 322
References......Page 323
Ab initio Discrete Molecular Dynamics Approach to Protein Folding and Aggregation......Page 327
Introduction......Page 328
Applications to Abeta Folding and Aggregation......Page 329
In Vitro Findings......Page 330
Four-Bead Model with Hydrogen Bonding: Planar beta-sheet Assemblies and the Role of Glycines......Page 331
Four-Bead Model with Amino Acid-Specific Hydropathic Interactions: Abeta40 versus Abeta42 Oligomer Formation......Page 332
United-Atom Model: Abeta(21-30) Folding Initiated by a Hydrophobic Packing between V24 and K28......Page 333
Structural Hypotheses Derived from the DMD Studies......Page 335
Discrete Molecular Dynamics Method......Page 337
Backbone Hydrogen Bond......Page 338
Amino Acid-Specific Interactions Caused by Hydropathy......Page 339
United-Atom Model Implementation......Page 341
Amino Acid-Specific Interactions Caused by Hydropathy......Page 343
Amino Acid-Specific Interactions Caused by Charge......Page 344
Limitations of the DMD Approach......Page 345
Conclusion......Page 347
References......Page 348
Introduction......Page 352
Protein Models......Page 355
CHARMM......Page 356
Simulation Results on Fibril Structure Using All-Atom MD......Page 360
Intermediate-Resolution Protein Model with Discontinuous Molecular Dynamics......Page 362
Discontinuous Molecular Dynamics......Page 363
Intermediate Resolution Protein Models-PRIME......Page 367
Simulation Results on Fibril Formation and Structure using DMD......Page 370
Conclusion......Page 375
References......Page 376
subject index.pdf......Page 380
zauthor index.pdf......Page 389
zMIE index.pdf......Page 417