Animal Cell Technology: Basic & Applied Aspects (eBook)

Preface 6
Contents 9
Non-fucosylated Therapeutic Antibodies: The Next Generation of Therapeutic Antibodies 16
1 Introduction 17
2 Current Feature of Therapeutic Antibodies 18
3 Human Serum IgG Inhibits Therapeutic Antibody-Induced ADCC 19
4 Fucosylated Therapeutic Antibodies Spoil the Non-Fucosylated Antibody-Induced ADCC 20
5 Manufacturing of Non-Fucosylated Therapeutic Antibodies 20
References 21
Selective Expansion of Genetically Modified T Cells Using a Chimeric IL-2 Receptor for Cancer Therapy 25
1 Introduction 25
2 Materials and Methods 27
2.1 Vector Construction 27
2.2 Cell Culture 27
2.3 Vector Transfection and Selection of the Transfectants 27
2.4 Cell Proliferation Assay 28
2.5 Western Blotting 28
3 Results and Discussion 28
3.1 Selective Expansion of Genetically Modified Cells 28
3.2 Chimeric IL-2R Transduces a Proliferative Signal in Response to HEL or Fluorescein-Conjugated BSA (BSA-FL) 29
3.3 Signal Transduction of the Chimeric IL-2Rs 31
References 32
Effects of Serum and Growth Factors on HEK 293 Proliferation and Adenovirus Productivity 33
1 Introduction 33
2 Results and Discussion 34
2.1 Effect of Serum Concentration on Cell Number and Viability in Basal and Serum Free Media 34
2.2 Effect of Serum Concentrations on Virus Productivity 35
2.3 The Effect of Insulin and Insulin-Like Growth Factors on Propagation 35
2.4 The Effect of Insulin and Insulin-Like Growth Factors on Virus Productivity 35
3 Conclusions 37
References 37
Simple and Efficient Establishment of Recombinant Protein Hyper-Producing Cells by Using RAS-Amplified CHO Cell Line 38
1 Introduction 38
2 Materials and Methods 39
2.1 Cells and Cell Culture 39
2.2 Establishment of Recombinant CHO Cells 39
2.3 Determination of the Concentration of Secreted AE6F4 Antibody 40
3 Results and Discussion 40
3.1 Evaluation for Availability of the CHO-hp1 Cell at the Single Cell Cloning 40
3.2 Evaluation of Marker Protein for the Isolation of Single Cell Cloning 41
3.3 Evaluation for Productivity of AE6F4 in Hyper-Producing Cell Line 41
References 42
Effects of Sericin on Promoting Proliferation and Inhibiting Apoptosis of Mammalian Cells 43
1 Introduction 43
2 Materials and Methods 44
2.1 Preparation of Sericin 44
2.2 Cell Line and Culture Conditions 44
2.3 Cell Proliferation Assay 44
2.4 Cell Death Induced by Heat Stress 44
2.5 Caspase Activity 44
2.6 Transcriptome Analysis 45
3 Results and Discussion 45
3.1 The Effect of Sericin on Cell Proliferation and Survival 45
3.1.1 The Effect of Sericin on Proliferation During Growth Factor Starvation 45
3.1.2 Effect of Sericin on Heat Stress-Induced Cell Death 46
3.2 Mechanism of Action of Sericin 46
3.2.1 Effect of Sericin on Caspase Activity 46
3.2.2 Detection of Genes Involved in the Mode of Action of Sericin 47
References 48
Molecular Biological Analysis of Mitogenic and Anti-Apoptotic Mechanisms of Sericin 49
1 Introduction 49
2 Materials and Methods 50
2.1 Cell Line and Culture Conditions 50
2.2 DE Analysis 50
2.2.1 First Dimension Isoelectric Focusing (IEF) 50
2.2.2 Second Dimension SDS-PAGE 50
3 Results and Discussion 51
3.1 Examination for Determining Optimal Concentration of Sericin 51
3.2 Proteome Analysis Using 2-DE 51
References 52
Novel Serum-Free Cryopreservation of Mammalian Cells Using Sericin 53
1 Introduction 53
2 Materials and Methods 54
2.1 Cell Lines and Culture Conditions 54
2.2 Determination of Cell Number and Viability 54
2.3 Cryopreservative Solutions 54
2.4 Cryopreservation 55
2.5 Thawing 55
2.6 Evaluation of Cellular Functions 55
3 Results 55
3.1 Effect of Sericin Solution on Cryopreservation of Insulinoma Cells 55
3.2 Effect of Sericin Solution on Cryopreservation of Hybridoma Cells 56
4 Conclusion 57
References 57
The Effect of Interleukin-6 and Leukemia Inhibitory Factor on Hybridoma Cells 58
1 Introduction 58
2 Materials and Methods 59
2.1 Cell Lines and Culture Conditions 59
2.2 Cell Proliferation Assay 59
2.3 DNA Ladder Assay 59
3 Results 60
References 62
In-Situ Observation of a Cell Growth Using Surface Infrared Spectroscopy 63
1 Introduction 63
2 Experiment 64
3 Result and Discussion 65
4 Conclusion 67
References 68
Scale-Down Perfusion Process for Recombinant Protein Expression 69
1 Introduction 70
2 Material and Methods 70
3 Results 71
3.1 Perfusion Bioreactor Compared to Push Up and Simulation of Perfusion Scale-Down Models 71
3.2 Lipids Study: Batch Mode and Simulation of Perfusion 73
4 Discussion and Conclusions 73
References 75
Computer-Based Matrix to Evaluate Optimal Medium Delivery Format for Biopharmaceutical Production 76
1 Introduction 77
2 Economic Modeling 78
3 Results 79
4 Discussion and Conclusions 80
4.1 Comparison of Liquid vs. Dry Media Formats 80
4.2 Comparison of Powdered vs. AGT Media Formats 81
4.3 Conclusion 82
References 82
A Serum Substitute for Fed-Batch Culture of Hybridoma Cells 83
1 Introduction 83
2 Strategies for Serum-Free Fed-Batch Culture 84
3 Materials and Methods 85
3.1 Cell Line and Culture Medium 85
3.2 Culture Conditions 85
3.3 Analytical Methods 86
4 Results and Discussion 86
5 Conclusion 88
References 88
Effects of Sugar Chain Precursors on Recombinant Protein Production in BHK Cells 89
1 Introduction 89
2 Materials and Methods 90
2.1 Cell Line and Culture Conditions 90
2.2 Proliferation Assay 90
2.3 Determination of EPO Concentrations 91
2.4 Two-Dimensional Electrophoresis 91
3 Results and Discussion 91
3.1 Effects of ManNAc on BHK Cell Proliferation 91
3.2 EPO Productivity 92
3.3 Two-Dimensional Electrophoresis of EPO Samples 92
References 94
Promoting Non-Hematopoietic Cell Proliferation by Chimeric Receptors 95
1 Introduction 95
2 Materials and Methods 96
2.1 Vector Construction 96
2.2 Cell Culture 96
2.3 AMEGA Selection 97
2.4 Flow Cytometry 97
3 Result 98
3.1 AMEGA on Ba/F3 98
3.2 Growth Assay of Ba/S-EGFR and Ba/S-fms 99
3.3 AMEGA on NIH/3T3 99
3.4 Growth Assay of NIH/S-fms 100
4 Discussion 101
References 101
Efficient Acquisition of Antigen-Specific Human Monoclonal Antibody by Using Peripheral Blood Mononuclear Cells Immunized In V 102
1 Introduction 103
2 Methods 103
2.1 In Vitro Immunization 103
2.2 Construction of Phage Antibody Library 103
2.3 Detection and Production of ME-Specific Antibody 103
3 Results and Discussion 104
References 105
Immunomodulatory Effects of Orally Administered Bifidobacterium Components on Intestinal Lymphoid Tissues 106
1 Introduction 106
2 Materials and Methods 107
2.1 Mice 107
2.2 Bacteria and Preparation of Bifidobacterium Immunomodulator (BIM) Derived from Sonicated B. Pseudocatenulatum 7041 107
2.3 Culture of Immune Tissue Cells and Cytokine and IgA Determination 107
2.4 Fluorescent Staining of Bp 108
2.5 Bacterial Localization in PP Frozen Sections 108
3 Results 108
3.1 Culture Supernatant IgA and Cytokines from PP, CF and MLN Cells Derived from Experimental Mice After Oral Administration 108
3.2 The Bacterial Localization in PP After Single-Shot Oral Administration of Bp 109
4 Discussion 109
References 110
Murine Intestinal Bacteria Modulate Antigen-Specific Cytokine Production by Intestinal Immune Cells Derived from Germ-Free T 111
1 Introduction 112
2 Materials and Methods 112
2.1 Preparation of Intestinal Bacteria 112
2.2 Mice 113
2.3 Preparation of CD4+ Cells and Thy1.2 Cells from Mesenteric Lymph Nodes (MLN) 113
2.4 Cell Culture 113
2.5 Measurements of Cytokine Production 113
3 Results and Discussion 113
References 115
Spleen Cells Derived from Male Non-Obese Diabetic Mice are Capable of Suppressing the Autoantigen-Specific Production of I 116
1 Introduction 116
2 Materials and Methods 117
3 Results and Discussion 117
References 120
Highly Efficient Antibody Production by Improving Cell Survival Using Sericin 121
1 Introduction 121
2 Materials and Methods 122
2.1 Cell Line and Culture Conditions 122
2.2 Determination of Antibody Concentration 122
3 Results and Discussion 122
References 126
Generation of Human Monoclonal Antibody Specific for Propionibacterium Acnes by In Vitro Immunization 127
1 Introduction 128
2 Methods 128
2.1 Cells and Bacterial Culture 128
2.2 In Vitro Immunization 128
2.3 ELISA and ELISPOT 128
2.4 Generation of Single-Chain Fv Antibody 129
2.5 Selecton of Antigen-Specific Phage Antibody by Solid-Phase Panning 129
3 Results and Discussion 131
References 131
Construction of Multi-layered Cell Sheet Using Magnetite Nanoparticles and Magnetic Force 132
1 Introduction 133
2 Materials and Method 133
2.1 Preparation of MCLs 133
2.2 Cell Culture 133
2.3 Cell Sheets Construction 134
2.4 Transplantation of MSC Sheet 134
2.5 Electrical Conduction Within CM Sheet 134
2.6 Construction of NHDF Sheets Including HUVECs 135
3 Results and Discussion 135
3.1 MSC Sheets 135
3.2 CM Sheets 136
3.3 Fibroblast Sheet Involving Capillaries 137
References 138
Anti-histone H1 Autoantibody: An Inducible Immunosuppressive Factor in Liver Transplantation 139
1 Introduction 140
2 Materials and Methods 140
2.1 Animals, OLT, and Post-OLT Serum 140
2.2 Evaluation of Immunosuppressive Activity 140
2.3 SDS-PAGE and Western Blot Analyses 141
2.4 Purification and Structural Analysis of Autoantigens Recognized by Post-OLT Serum IgG 141
2.5 Generation of MLR-Neutralizing Anti-histone H1 mAb 142
2.6 Serum-Free Culture of Hybridoma Cells and Purification of Anti-histone H1 mAb 16G9 142
2.7 Flow Cytometry 142
3 Results and Discussion 142
3.1 Anti-histone H1 Autoantibody Identified as a Major Immunosuppressive Factor in Post-OLT Serum 142
3.2 Immunosuppressive Activity of Anti-histone H1 Antibody In Vitro and In Vivo 143
3.3 Generation of MLR-Neutralizing Anti-histone H1 mAb, 16G9 143
3.4 16G9 mAb Specifically Reacts with Murine Leukocytes 144
Anti-histone H1 Autoantibody Directly Acts on T Cells to Exert Its Immunosuppressive Activity 146
1 Introduction 147
2 Materials and Methods 147
2.1 Animals and Anti-histone H1 mAb, 16G9 147
2.2 Murine Allogeneic MLR 147
2.3 T Cell Proliferation Assay 148
2.4 Flow Cytometry 148
2.5 Statistical Analysis 149
3 Results and Discussion 149
3.1 Anti-histone H1 mAb 16G9 Suppresses T Cell Activation upon Stimulation with Allogeneic DCs 149
3.2 16G9 mAb Suppresses T Cell Activation and IL-2 Production in Response to TCR Ligation 149
3.3 Specific Binding of Anti-histone H1 mAb 16G9 to Purified T Cells 150
References 151
The Effect of Culture Conditions on Liver Function and Proliferation of Hepatic Cells for Bio-Artificial Liver 152
1 Introduction 152
2 Materials and Methods 153
2.1 Cell Lines and Culture Conditions 153
2.2 Seeding and Medium Change 153
2.3 Morphology 153
2.4 Cell Proliferation 154
2.5 Albumin Productivity 154
3 Results and Discussion 154
3.1 Morphology 154
3.2 Cell Proliferation 154
3.3 Albumin Productivity 156
References 156
The Effect of Scaffold on the Morphology and Insulin Secretion of Islet Cells 157
1 Introduction 157
2 Materials and Methods 158
2.1 Islet Cell Preparation 158
2.2 Islet Cell Culture 158
2.3 ECM Proteins 158
2.4 Morphological Assay 158
2.5 Insulin Secretion in Response to Glucose Level 159
3 Results and Discussion 159
3.1 Effect of ECM on Morphology 159
3.2 Effect of ECM on Insulin Release in Response to Glucose Level 159
References 162
Sterilization of Chicken Primordial Germ Cells 163
1 Introduction 163
2 Materials and Methods 164
2.1 Sterilization by UV 164
2.2 Sterilization by g Ray Using 60Co 164
2.3 Sterilization by Removal of Cells from the Center of Area Pellucida 165
2.4 Culture of Manipulated Embryos and Count of PGCs 165
3 Results and Discussion 166
3.1 Viability of Embryos 166
3.2 Reduction of the Number of Circulating PGCs in Sterilized Eggs 166
References 167
Protein Expression by Human Intestinal Epithelial Cells in Response to Wastewater Constituents 169
1 Introduction 169
2 Materials and Methods 170
2.1 Cell Culture 170
2.2 Cell Treatment Conditions 170
2.3 Proteomics 171
2.4 Mass Spectrometry 171
3 Results and Discussion 172
References 174
In Vitro Cytotoxic Effects of Tin Compounds on Normal Human Astrocytes 175
1 Introduction 175
2 Materials and Methods 176
2.1 Material Preparation 176
2.2 Astrocyte Cell Culture 176
2.3 MTT Assay for Cell Proliferation 177
2.4 Scrape-Loading and Dye Transfer (SLDT) Assay 177
2.5 Expression of Gap Junctional and Neural Cell Marker Genes 177
2.6 Statistical Analysis 178
3 Results and Discussion 178
4 Conclusion 179
References 180
Effects of Tin Compounds on Human Chondrogenic Activity In Vitro 181
1 Introduction 181
2 Materials and Methods 182
2.1 Medium and Materials Used for Cell Culture 182
2.2 Cells and Culture Methods 182
2.3 Cell Proliferation Study 182
2.4 Cell Differentiation Assay 183
2.5 Real-Time Polymerase Chain Reaction (PCR) 183
2.6 Statistical Analysis 183
3 Results 184
3.1 Cell Proliferation 184
3.2 Cell Differentiation 184
4 Discussion 184
References 185
Construction of a Fluorescein-Responsive Chimeric Receptor with Strict Ligand Dependency and Analysis of the Role of Erythropo 187
1 Introduction 188
2 Methods and Results 188
3 Discussion 191
References 193
Nuclear Structures Regulate Liver-Specific Expression of the Tryptophan Oxygenase Gene 194
1 Introduction 194
2 Materials and Methods 195
2.1 Isolation and Culture of Rat Hepatocytes 195
2.2 Nuclear Matrix Isolation 195
2.3 Chromatin Immunoprecipitation (ChIP) Assay 196
3 Results 196
3.1 A Potential MAR Was Detected Upstream of the TO Gene 196
3.2 Upstream of the TO Gene Is Specifically Associated with Nuclear Matrices In Vivo 197
3.3 GR Is Associated with the Region Near the MAR of the TO Gene In Vivo 197
4 Discussion 199
References 199
CCAAT/Enhancer-Binding Protein Beta Controls Differentiation-Specific Expression of Chromatin Remodeling Factor BRM 201
1 Introduction 201
2 Materials and Methods 202
2.1 Cell Culture 202
2.2 Plasmids 203
2.3 Luciferase Reporter Gene Assay 203
3 Results 203
3.1 Identification of Promoter Regions of the brg1 and brm Gene 203
3.2 Analyses of Promoter Proximal Regions of mbrm and mbrg-1 Genes by Luciferase Reporter Assay 204
4 Discussion 206
References 206
Involvement of 67 kDa Laminin Receptor on Cellular Uptake of Green Tea Polyphenol Epigallocatechin-3-O-Gallate in Caco-2 Cel 208
1 Introduction 208
2 Materials and Methods 209
2.1 Reagents 209
2.2 Cell Culture 209
2.3 Cell Construction 209
2.4 Western Blot Analysis 209
2.5 Flow Cytometric Analysis 210
2.6 Surface Plasmon Resonance Biosensor Assay 210
2.7 Growth Inhibitory Activity Assay 210
2.8 Cellular Uptake Analysis 210
2.9 Statistical Analysis 210
3 Results and Discussion 211
References 212
Inositol Derivatives Stimulate Glucose Transport in Muscle Cells 213
1 Introduction 213
2 Materials and Methods 214
2.1 Materials 214
2.2 Cell Culture 214
2.3 Glucose Uptake Assay 214
2.4 Ex Vivo Assay 215
2.5 Western Blot Analysis 215
3 Results 215
3.1 Inositol Derivatives Stimulate Glucose Uptake in L6 Myotubes 215
3.2 Inositol Derivatives Stimulate Translocation of GLUT4 to the Plasma Membrane Ex Vivo 216
4 Discussion 217
References 218
Hair Growth Regulation by an Aromatic Plant Extract 219
1 Introduction 219
2 Materials and Methods 220
2.1 Cells and Cell Culture 220
2.2 Sample Extract 220
2.3 MTT Assay 221
2.4 Cell Cycle Assay 221
2.5 Hair Growth Promotion Assay In Vivo 221
3 Results and Discussion 222
3.1 Effect of Tunisian Aromatic Plant Extract on Human Dermal Papilla Cell Growth 222
3.2 Effect of Tunisian Aromatic Plant Extract on HFDPCs Cell Cycle 222
3.3 Hair Growth Promotion Activity of Tunisian Aromatic Plant Extract 223
References 224
Screening of Various Tunisian Olive Oils for Their Inhibitory Effect on Beta-Hexosaminidase Release by Basophilic Cells 226
1 Introduction 226
2 Materials and Methods 227
2.1 Reagents 227
2.2 Preparation of Olive Oil Sample 227
2.3 Cell and Cell Culture 228
2.4 b-Hexosaminidase Assay at Different Stages 228
2.5 Histamine Release Assay 229
2.6 Statistical Analysis 229
3 Results 229
3.1 Effect of Various Olive Oils on the b-Hexosaminidase Release at the Antigen-Antibody Binding Stage 229
3.2 Effect of Various Olive Oils on the b-Hexosaminidase Release at the Antibody-Receptor Binding Stage 230
3.3 Inhibitory Effect of Olive Oil on Histamine Release from KU812 Cells 231
4 Discussion 231
References 232
Leaf Extracts from Tunisian Olive Cultivars Induce Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells 234
1 Introduction 235
2 Materials and Methods 235
2.1 Cell Line and Cell Maintenance 235
2.2 Sampling 235
2.3 Cell Proliferation Assay 235
2.4 Cell Differentiation Assays 236
2.5 Neutral Red Assay 236
3 Results and Discussion 236
3.1 Growth Inhibition of HL60 Cells by Olive Leaf Extracts 236
3.2 Morphological Examination 237
3.3 Induction of Differentiation by Olive Leaf Extracts 237
3.4 Effect of Oleuropein and Luteolin on HL60 Cell Viability 239
References 239
In Vitro Observation of the Effect of Intestinal Bacteria on IgA Production by Immunocytes in the Large Intestine: Comparison 241
1 Introduction 242
2 Materials and Methods 242
2.1 Mice 242
2.2 Cell Preparation 242
2.3 Flow Cytometric Analysis 243
2.4 Enzyme-Linked Immunospots 243
2.5 Detection of IL-6 Products 243
2.6 Isolation of B220+ Cells and IgA-Plasma Cells 243
2.7 Measurement of Total IgA 244
3 Results 244
3.1 The Frequency of IgA-Plasma Cells and IgA-Secreting Cells in L-LP 244
3.2 L-LP Lymphocytes’ IL-6 Production 245
3.3 IgA Production by IgA-Plasma Cells and B220+ Cells Isolated from L-LP 245
4 Discussion 246
References 246
Differentiation of Human Leukemia Cell Line HL-60 by a Polyacetylenic Compound from Hedera Rhombea 247
1 Introduction 247
2 Materials and Methods 248
2.1 Cell Culture 248
2.2 Materials 248
2.3 MTT Assay 249
2.4 Specific and Nonspecific Esterase Double Staining 249
2.5 Time-Dependent Change in Cell Cycle Kinetics 250
3 Results and Discussion 250
3.1 Time-Dependent Change in HL-60 Cell Viability 250
3.2 Specific and Nonspecific Esterase Double Staining 251
3.3 Time-Dependent Change in Cell Cycle Kinetics 251
References 252
Effect of Tunisian Plant Extract on Melanogenesis 253
1 Introduction 253
2 Materials and Methods 254
2.1 Cell Culture 254
2.2 Measurement of Melanin Content and Microscopic Examination 254
2.3 Cell Viability 255
2.4 Western Blot Analysis 255
3 Results and Discussion 255
3.1 Tunisian Aromatic Plant Does Not Induce Cell Morphological Change 255
3.2 Tunisian Aromatic Plant Promotes Melanin Synthesis by B16 Cells 256
3.3 Effects of Tunisian Aromatic Plant on B16 Cell Viability 256
3.4 Tunisian Aromatic Plant Does Not Influence the Signaling Pathway Related to Melanogenesis 256
3.5 Tunisian Aromatic Plant Does Not Affect Tyrosinase Protein Expression in B16 Cells 257
References 257
“Nordenau Phenomenon” – Application of Natural Reduced Water to Therapy 259
1 Introduction 260
2 Material and Method 260
3 Results and Discussion 261
4 Conclusion 264
References 264
Anti-melanogenic Activity of Ergosterol Peroxide from Ganoderma lucidum on a Mouse Melanoma Cell Line 266
1 Introduction 266
2 Materials and Methods 267
2.1 Isolation of Active Compounds from Ganoderma lucidum 267
2.2 Effect of Triterpenes on Melanogenesis and Cell Growth 267
2.3 Fluorescence Microscopy 267
3 Results and Discussion 268
3.1 Anti-Melanogenic Compound from Ganoderma lucidum 268
3.2 Effect on the Level of TRP-1 in B16 10F7 Cells 269
References 270
Differentiation-Inducing Activities by Lupane Triterpenes from Lactuca indica on a Mouse Melanoma Cell Line 271
1 Introduction 271
2 Materials and Methods 272
2.1 Isolation of Active Compounds from Lactuca indica 272
2.2 Effect of Triterpenes on Melanogenesis and Cell Growth 272
2.3 Western Blotting 273
2.4 Fluorescence Microscopy 273
3 Results and Discussion 273
3.1 Active Compounds 273
3.2 Signaling Mechanisms 273
References 276
Immunoglobulin Production Stimulating Effect of Soy-Derived Proteins 278
1 Introduction 279
2 Materials and Methods 279
2.1 Reagents and Cells Culture 279
2.2 Enzyme-Linked Immunosorbent Assay (ELISA) 279
2.3 Neutralization of Interleukin-6 280
2.4 Reverse Transcription–Polymerase Chain Reaction (RT-PCR) 280
2.5 Anion-Exchange Chromatography 281
2.6 Sodium Dodecyl Sulfate–Poly Acrylamide Gel Electrophoresis 281
3 Results and Discussions 281
References 283
Immunostimulation Effect of the Jellyfish Collagen 284
1 Introduction 284
2 Materials and Methods 285
2.1 Preparation of Jellyfish Extract 285
2.2 Cells and Cell Culture 285
2.3 Assay of the Immunostimulating Activity 286
3 Results and Discussion 286
3.1 The Effect of Jellyfish Extract on IgM Production of HB4C5 Cells 286
3.2 Effect of Jellyfish Extract on Ig and Cytokine Production of hPBL 287
3.3 The Active Substance in Jellyfish Extract 287
3.4 Effects of Jellyfish Collagen on mRNA Expression Level for Ig and Cytokines 288
3.5 Effects of Jellyfish Collagen on Post-transcription Process 288
3.5.1 The Stimulation Effect of Collagen on Transcription-Suppressed HB4C5 Cells 288
3.5.2 The Stimulation Effect of Collagen on Translation-Suppressed HB4C5 Cells 289
3.5.3 The Stimulation Effect of Collagen on HB4C5 Cells Suppressed the Post-translation Activity 290
References 290
Mycotoxin Nivalenol Induces Apoptosis and Intracellular Calcium Ion-Dependent Interleukin-8 Secretion but Does Not Exert Mutag 291
1 Introduction 291
2 Materials and Methods 292
2.1 Chemicals and Cells 292
2.2 Examination of DNA Fragmentation 292
2.3 Determination of IL-8 Levels 292
2.4 Evaluation of Mutagenicity (umu Test) 293
2.5 Statistics 293
3 Results and Discussion 293
References 295
Development of an In Vitro System for Screening the Ligands of a Membrane Glycoprotein CD36 297
1 Introduction 298
2 Materials and Methods 298
2.1 Materials 298
2.2 Cell Culture and Establishment of Stable Transformant 299
2.3 Western Blotting Analysis 299
2.4 Immunofluorescence 299
2.5 Binding 300
2.6 MAP Kinase Activation Assay 300
3 Results and Discussion 300
3.1 Establishment of Stable Transformants Expressing hCD36 or mCD36 300
3.2 Binding of OxLDL to the CD36-Expressing Transformants 301
3.3 Binding of LDL to the CD36-Expressing Transformants 301
3.4 MAP Kinase Activation 302
References 304
MTT Reduction by Flavonoids in the Absence of Cells: Influence of Medium Type and Serum 306
1 Introduction 306
2 Materials and Methods 307
2.1 Materials 307
2.2 Preparation of Flavonoids 308
2.3 MTT Assay 308
3 Results 308
4 Discussion 310
References 312
Characterization of Highly Reactive Sequences for Transglutaminase 2 and Factor XIIIa 314
1 Introduction 314
2 Materials and Methods 315
2.1 Screening Using Phage-Displayed Random Peptide Library (Fig. 1) 315
2.2 Production of Peptide-GST Fusion Proteins 316
2.3 Assessments of Substrate Preference in TGase Reaction 317
2.4 Assessments of Synthetic Peptide as TGase Substrate 317
3 Results and Discussion 317
3.1 Screening of the Preferred Substrate Sequence Using a Phage-Displayed Peptide Library 317
3.2 Evaluation of Identified Peptide Sequence as GST Fusion Protein and Labeled Primary Amine 318
3.3 The Peptide Sequence Exhibited the Specific Inhibition Activity for Cross-Linking Reaction 319
References 320
Some Characteristics of UNC-51 Phosphorylations of Both Actins and Tubulins 321
1 Introduction 321
2 Materials and Methods 322
2.1 Materials and Chemicals 322
2.2 Plasmid Constructs 322
2.3 Cell-Transfection, Purification of FLAG-UNC-51 323
2.4 In Vitro Kinase Assay 323
3 Results 323
3.1 Time Courses of UNC-51 Phosphorylations of a/b Tubulins and Actins 323
3.2 Temperature Effects on UNC-51 Phosphorylations of a/b Tubulins and Actins 323
3.3 pH Effects on UNC-51 Phosphorylations of a/b Tubulins and Actins 324
3.4 Mg2+Promotes UNC-51 Kinase Activity 325
4 Discussion 325
References 326
Protein Phosphotase 1a Reverses UNC-51 Phosphorylations of Both Actins and Tubulins and a New Model of UNC-51-Inducing Axo 328
1 Introduction 328
2 Materials and Methods 329
2.1 In Vitro Dephosphorylation of UNC-51 Phosphorylations of Tubulins and Actins by PP1a 329
3 Results and Discussion 329
3.1 PP1a Can Dephosphorylate UNC-51 Phosphorylations of Actins and Tubulins 329
3.2 A New Model of UNC-51 Mediated Cytoskeletal Dynamics 331
References 331
Overexpression of Conserved Kinase UNC-51 Inhibits the Transferrin’s Endocytosis into the Mammalian Cells 333
1 Introduction 333
2 Materials and Methods 334
2.1 Plasmid Constructs 334
2.2 Cell Transfection and Confocal Immunofluorescence Microscopy 334
3 Results and Conclusion 334
References 335