Shirahata / Ikura / Nagao Animal Cell Technology: Basic & Applied Aspects

Proceedings of the 19th Annual Meeting of the Japanese Association for Animal Cell Technology (JAACT), Kyoto, Japan, September 25-28, 2006
2009
ISBN: 978-1-4020-9646-4
Verlag: Springer Netherlands
Format: PDF
Kopierschutz: 1 - PDF Watermark

E-Book, Englisch, Band 15, 357 Seiten

Reihe: Animal Cell Technology: Basic & Applied Aspects

Animal cell technology is a growing discipline of cell biology which aims not only to understand structures, functions and behaviors of differentiated animal cells, but also to ascertain their abilities to be used for industrial and medical purposes. The goal of animal cell technology includes the clonal expansion of differentiated cells, the optimization of their culture conditions, modulation of their ability to produce proteins of medical and pharmaceutical importantance, and the application of animal cells to gene therapy, artificial organs and the production of functional foods. This volume gives the readers a complete review of the present state-of-the-art and will be useful for those working in either academic environments or in the biotechnology and pharmaceutical sectors, particularly cell biologists, biochemists, molecular biologists, immunologists, biochemical engineers and all other disciplines related to animal cell culture.

Autoren/Hrsg.

Shirahata, Sanetaka

Ikura, Koji

Nagao, Masaya

Ichikawa, Akira

Teruya, Kiichiro

Inhaltsverzeichnis

1;Preface;6
2;Contents;9
3;Non-fucosylated Therapeutic Antibodies: The Next Generation of Therapeutic Antibodies;16
3.1;1 Introduction;17
3.2;2 Current Feature of Therapeutic Antibodies;18
3.3;3 Human Serum IgG Inhibits Therapeutic Antibody-Induced ADCC;19
3.4;4 Fucosylated Therapeutic Antibodies Spoil the Non-Fucosylated Antibody-Induced ADCC;20
3.5;5 Manufacturing of Non-Fucosylated Therapeutic Antibodies;20
3.6;References;21
4;Selective Expansion of Genetically Modified T Cells Using a Chimeric IL-2 Receptor for Cancer Therapy;25
4.1;1 Introduction;25
4.2;2 Materials and Methods;27
4.2.1;2.1 Vector Construction;27
4.2.2;2.2 Cell Culture;27
4.2.3;2.3 Vector Transfection and Selection of the Transfectants;27
4.2.4;2.4 Cell Proliferation Assay;28
4.2.5;2.5 Western Blotting;28
4.3;3 Results and Discussion;28
4.3.1;3.1 Selective Expansion of Genetically Modified Cells;28
4.3.2;3.2 Chimeric IL-2R Transduces a Proliferative Signal in Response to HEL or Fluorescein-Conjugated BSA (BSA-FL);29
4.3.3;3.3 Signal Transduction of the Chimeric IL-2Rs;31
4.4;References;32
5;Effects of Serum and Growth Factors on HEK 293 Proliferation and Adenovirus Productivity;33
5.1;1 Introduction;33
5.2;2 Results and Discussion;34
5.2.1;2.1 Effect of Serum Concentration on Cell Number and Viability in Basal and Serum Free Media;34
5.2.2;2.2 Effect of Serum Concentrations on Virus Productivity;35
5.2.3;2.3 The Effect of Insulin and Insulin-Like Growth Factors on Propagation;35
5.2.4;2.4 The Effect of Insulin and Insulin-Like Growth Factors on Virus Productivity;35
5.3;3 Conclusions;37
5.4;References;37
6;Simple and Efficient Establishment of Recombinant Protein Hyper-Producing Cells by Using RAS-Amplified CHO Cell Line;38
6.1;1 Introduction;38
6.2;2 Materials and Methods;39
6.2.1;2.1 Cells and Cell Culture;39
6.2.2;2.2 Establishment of Recombinant CHO Cells;39
6.2.3;2.3 Determination of the Concentration of Secreted AE6F4 Antibody;40
6.3;3 Results and Discussion;40
6.3.1;3.1 Evaluation for Availability of the CHO-hp1 Cell at the Single Cell Cloning;40
6.3.2;3.2 Evaluation of Marker Protein for the Isolation of Single Cell Cloning;41
6.3.3;3.3 Evaluation for Productivity of AE6F4 in Hyper-Producing Cell Line;41
6.4;References;42
7;Effects of Sericin on Promoting Proliferation and Inhibiting Apoptosis of Mammalian Cells;43
7.1;1 Introduction;43
7.2;2 Materials and Methods;44
7.2.1;2.1 Preparation of Sericin;44
7.2.2;2.2 Cell Line and Culture Conditions;44
7.2.3;2.3 Cell Proliferation Assay;44
7.2.4;2.4 Cell Death Induced by Heat Stress;44
7.2.5;2.5 Caspase Activity;44
7.2.6;2.6 Transcriptome Analysis;45
7.3;3 Results and Discussion;45
7.3.1;3.1 The Effect of Sericin on Cell Proliferation and Survival;45
7.3.1.1;3.1.1 The Effect of Sericin on Proliferation During Growth Factor Starvation;45
7.3.1.2;3.1.2 Effect of Sericin on Heat Stress-Induced Cell Death;46
7.3.2;3.2 Mechanism of Action of Sericin;46
7.3.2.1;3.2.1 Effect of Sericin on Caspase Activity;46
7.3.2.2;3.2.2 Detection of Genes Involved in the Mode of Action of Sericin;47
7.4;References;48
8;Molecular Biological Analysis of Mitogenic and Anti-Apoptotic Mechanisms of Sericin;49
8.1;1 Introduction;49
8.2;2 Materials and Methods;50
8.2.1;2.1 Cell Line and Culture Conditions;50
8.2.2;2.2 DE Analysis;50
8.2.2.1;2.2.1 First Dimension Isoelectric Focusing (IEF);50
8.2.2.2;2.2.2 Second Dimension SDS-PAGE;50
8.3;3 Results and Discussion;51
8.3.1;3.1 Examination for Determining Optimal Concentration of Sericin;51
8.3.2;3.2 Proteome Analysis Using 2-DE;51
8.4;References;52
9;Novel Serum-Free Cryopreservation of Mammalian Cells Using Sericin;53
9.1;1 Introduction;53
9.2;2 Materials and Methods;54
9.2.1;2.1 Cell Lines and Culture Conditions;54
9.2.2;2.2 Determination of Cell Number and Viability;54
9.2.3;2.3 Cryopreservative Solutions;54
9.2.4;2.4 Cryopreservation;55
9.2.5;2.5 Thawing;55
9.2.6;2.6 Evaluation of Cellular Functions;55
9.3;3 Results;55
9.3.1;3.1 Effect of Sericin Solution on Cryopreservation of Insulinoma Cells;55
9.3.2;3.2 Effect of Sericin Solution on Cryopreservation of Hybridoma Cells;56
9.4;4 Conclusion;57
9.5;References;57
10;The Effect of Interleukin-6 and Leukemia Inhibitory Factor on Hybridoma Cells;58
10.1;1 Introduction;58
10.2;2 Materials and Methods;59
10.2.1;2.1 Cell Lines and Culture Conditions;59
10.2.2;2.2 Cell Proliferation Assay;59
10.2.3;2.3 DNA Ladder Assay;59
10.3;3 Results;60
10.4;References;62
11;In-Situ Observation of a Cell Growth Using Surface Infrared Spectroscopy;63
11.1;1 Introduction;63
11.2;2 Experiment;64
11.3;3 Result and Discussion;65
11.4;4 Conclusion;67
11.5;References;68
12;Scale-Down Perfusion Process for Recombinant Protein Expression;69
12.1;1 Introduction;70
12.2;2 Material and Methods;70
12.3;3 Results;71
12.3.1;3.1 Perfusion Bioreactor Compared to Push Up and Simulation of Perfusion Scale-Down Models;71
12.3.2;3.2 Lipids Study: Batch Mode and Simulation of Perfusion;73
12.4;4 Discussion and Conclusions;73
12.5;References;75
13;Computer-Based Matrix to Evaluate Optimal Medium Delivery Format for Biopharmaceutical Production;76
13.1;1 Introduction;77
13.2;2 Economic Modeling;78
13.3;3 Results;79
13.4;4 Discussion and Conclusions;80
13.4.1;4.1 Comparison of Liquid vs. Dry Media Formats;80
13.4.2;4.2 Comparison of Powdered vs. AGT Media Formats;81
13.4.3;4.3 Conclusion;82
13.5;References;82
14;A Serum Substitute for Fed-Batch Culture of Hybridoma Cells;83
14.1;1 Introduction;83
14.2;2 Strategies for Serum-Free Fed-Batch Culture;84
14.3;3 Materials and Methods;85
14.3.1;3.1 Cell Line and Culture Medium;85
14.3.2;3.2 Culture Conditions;85
14.3.3;3.3 Analytical Methods;86
14.4;4 Results and Discussion;86
14.5;5 Conclusion;88
14.6;References;88
15;Effects of Sugar Chain Precursors on Recombinant Protein Production in BHK Cells;89
15.1;1 Introduction;89
15.2;2 Materials and Methods;90
15.2.1;2.1 Cell Line and Culture Conditions;90
15.2.2;2.2 Proliferation Assay;90
15.2.3;2.3 Determination of EPO Concentrations;91
15.2.4;2.4 Two-Dimensional Electrophoresis;91
15.3;3 Results and Discussion;91
15.3.1;3.1 Effects of ManNAc on BHK Cell Proliferation;91
15.3.2;3.2 EPO Productivity;92
15.3.3;3.3 Two-Dimensional Electrophoresis of EPO Samples;92
15.4;References;94
16;Promoting Non-Hematopoietic Cell Proliferation by Chimeric Receptors;95
16.1;1 Introduction;95
16.2;2 Materials and Methods;96
16.2.1;2.1 Vector Construction;96
16.2.2;2.2 Cell Culture;96
16.2.3;2.3 AMEGA Selection;97
16.2.4;2.4 Flow Cytometry;97
16.3;3 Result;98
16.3.1;3.1 AMEGA on Ba/F3;98
16.3.2;3.2 Growth Assay of Ba/S-EGFR and Ba/S-fms;99
16.3.3;3.3 AMEGA on NIH/3T3;99
16.3.4;3.4 Growth Assay of NIH/S-fms;100
16.4;4 Discussion;101
16.5;References;101
17;Efficient Acquisition of Antigen-Specific Human Monoclonal Antibody by Using Peripheral Blood Mononuclear Cells Immunized In V;102
17.1;1 Introduction;103
17.1.1;2 Methods;103
17.1.2;2.1 In Vitro Immunization;103
17.1.3;2.2 Construction of Phage Antibody Library;103
17.1.4;2.3 Detection and Production of ME-Specific Antibody;103
17.2;3 Results and Discussion;104
17.3;References;105
18;Immunomodulatory Effects of Orally Administered Bifidobacterium Components on Intestinal Lymphoid Tissues;106
18.1;1 Introduction;106
18.2;2 Materials and Methods;107
18.2.1;2.1 Mice;107
18.2.2;2.2 Bacteria and Preparation of Bifidobacterium Immunomodulator (BIM) Derived from Sonicated B. Pseudocatenulatum 7041;107
18.2.3;2.3 Culture of Immune Tissue Cells and Cytokine and IgA Determination;107
18.2.4;2.4 Fluorescent Staining of Bp;108
18.2.5;2.5 Bacterial Localization in PP Frozen Sections;108
18.3;3 Results;108
18.3.1;3.1 Culture Supernatant IgA and Cytokines from PP, CF and MLN Cells Derived from Experimental Mice After Oral Administration;108
18.3.2;3.2 The Bacterial Localization in PP After Single-Shot Oral Administration of Bp;109
18.4;4 Discussion;109
18.5;References;110
19;Murine Intestinal Bacteria Modulate Antigen-Specific Cytokine Production by Intestinal Immune Cells Derived from Germ-Free T;111
19.1;1 Introduction;112
19.2;2 Materials and Methods;112
19.2.1;2.1 Preparation of Intestinal Bacteria;112
19.2.2;2.2 Mice;113
19.2.3;2.3 Preparation of CD4+ Cells and Thy1.2 Cells from Mesenteric Lymph Nodes (MLN);113
19.2.4;2.4 Cell Culture;113
19.2.5;2.5 Measurements of Cytokine Production;113
19.3;3 Results and Discussion;113
19.4;References;115
20;Spleen Cells Derived from Male Non-Obese Diabetic Mice are Capable of Suppressing the Autoantigen-Specific Production of I;116
20.1;1 Introduction;116
20.2;2 Materials and Methods;117
20.3;3 Results and Discussion;117
20.4;References;120
21;Highly Efficient Antibody Production by Improving Cell Survival Using Sericin;121
21.1;1 Introduction;121
21.2;2 Materials and Methods;122
21.2.1;2.1 Cell Line and Culture Conditions;122
21.2.2;2.2 Determination of Antibody Concentration;122
21.3;3 Results and Discussion;122
21.4;References;126
22;Generation of Human Monoclonal Antibody Specific for Propionibacterium Acnes by In Vitro Immunization;127
22.1;1 Introduction;128
22.2;2 Methods;128
22.2.1;2.1 Cells and Bacterial Culture;128
22.2.2;2.2 In Vitro Immunization;128
22.2.3;2.3 ELISA and ELISPOT;128
22.2.4;2.4 Generation of Single-Chain Fv Antibody;129
22.2.5;2.5 Selecton of Antigen-Specific Phage Antibody by Solid-Phase Panning;129
22.3;3 Results and Discussion;131
22.4;References;131
23;Construction of Multi-layered Cell Sheet Using Magnetite Nanoparticles and Magnetic Force;132
23.1;1 Introduction;133
23.2;2 Materials and Method;133
23.2.1;2.1 Preparation of MCLs;133
23.2.2;2.2 Cell Culture;133
23.2.3;2.3 Cell Sheets Construction;134
23.2.4;2.4 Transplantation of MSC Sheet;134
23.2.5;2.5 Electrical Conduction Within CM Sheet;134
23.2.6;2.6 Construction of NHDF Sheets Including HUVECs;135
23.3;3 Results and Discussion;135
23.3.1;3.1 MSC Sheets;135
23.3.2;3.2 CM Sheets;136
23.3.3;3.3 Fibroblast Sheet Involving Capillaries;137
23.4;References;138
24;Anti-histone H1 Autoantibody: An Inducible Immunosuppressive Factor in Liver Transplantation;139
24.1;1 Introduction;140
24.2;2 Materials and Methods;140
24.2.1;2.1 Animals, OLT, and Post-OLT Serum;140
24.2.2;2.2 Evaluation of Immunosuppressive Activity;140
24.2.3;2.3 SDS-PAGE and Western Blot Analyses;141
24.2.4;2.4 Purification and Structural Analysis of Autoantigens Recognized by Post-OLT Serum IgG;141
24.2.5;2.5 Generation of MLR-Neutralizing Anti-histone H1 mAb;142
24.2.6;2.6 Serum-Free Culture of Hybridoma Cells and Purification of Anti-histone H1 mAb 16G9;142
24.2.7;2.7 Flow Cytometry;142
24.3;3 Results and Discussion;142
24.3.1;3.1 Anti-histone H1 Autoantibody Identified as a Major Immunosuppressive Factor in Post-OLT Serum;142
24.3.2;3.2 Immunosuppressive Activity of Anti-histone H1 Antibody In Vitro and In Vivo;143
24.3.3;3.3 Generation of MLR-Neutralizing Anti-histone H1 mAb, 16G9;143
24.3.4;3.4 16G9 mAb Specifically Reacts with Murine Leukocytes;144
25;Anti-histone H1 Autoantibody Directly Acts on T Cells to Exert Its Immunosuppressive Activity;146
25.1;1 Introduction;147
25.2;2 Materials and Methods;147
25.2.1;2.1 Animals and Anti-histone H1 mAb, 16G9;147
25.2.2;2.2 Murine Allogeneic MLR;147
25.2.3;2.3 T Cell Proliferation Assay;148
25.2.4;2.4 Flow Cytometry;148
25.2.5;2.5 Statistical Analysis;149
25.3;3 Results and Discussion;149
25.3.1;3.1 Anti-histone H1 mAb 16G9 Suppresses T Cell Activation upon Stimulation with Allogeneic DCs;149
25.3.2;3.2 16G9 mAb Suppresses T Cell Activation and IL-2 Production in Response to TCR Ligation;149
25.3.3;3.3 Specific Binding of Anti-histone H1 mAb 16G9 to Purified T Cells;150
25.4;References;151
26;The Effect of Culture Conditions on Liver Function and Proliferation of Hepatic Cells for Bio-Artificial Liver;152
26.1;1 Introduction;152
26.2;2 Materials and Methods;153
26.2.1;2.1 Cell Lines and Culture Conditions;153
26.2.2;2.2 Seeding and Medium Change;153
26.2.3;2.3 Morphology;153
26.2.4;2.4 Cell Proliferation;154
26.2.5;2.5 Albumin Productivity;154
26.3;3 Results and Discussion;154
26.3.1;3.1 Morphology;154
26.3.2;3.2 Cell Proliferation;154
26.3.3;3.3 Albumin Productivity;156
26.4;References;156
27;The Effect of Scaffold on the Morphology and Insulin Secretion of Islet Cells;157
27.1;1 Introduction;157
27.2;2 Materials and Methods;158
27.2.1;2.1 Islet Cell Preparation;158
27.2.2;2.2 Islet Cell Culture;158
27.2.3;2.3 ECM Proteins;158
27.2.4;2.4 Morphological Assay;158
27.2.5;2.5 Insulin Secretion in Response to Glucose Level;159
27.3;3 Results and Discussion;159
27.3.1;3.1 Effect of ECM on Morphology;159
27.3.2;3.2 Effect of ECM on Insulin Release in Response to Glucose Level;159
27.4;References;162
28;Sterilization of Chicken Primordial Germ Cells;163
28.1;1 Introduction;163
28.2;2 Materials and Methods;164
28.2.1;2.1 Sterilization by UV;164
28.2.2;2.2 Sterilization by g Ray Using 60Co;164
28.2.3;2.3 Sterilization by Removal of Cells from the Center of Area Pellucida;165
28.2.4;2.4 Culture of Manipulated Embryos and Count of PGCs;165
28.3;3 Results and Discussion;166
28.3.1;3.1 Viability of Embryos;166
28.3.2;3.2 Reduction of the Number of Circulating PGCs in Sterilized Eggs;166
28.4;References;167
29;Protein Expression by Human Intestinal Epithelial Cells in Response to Wastewater Constituents;169
29.1;1 Introduction;169
29.2;2 Materials and Methods;170
29.2.1;2.1 Cell Culture;170
29.2.2;2.2 Cell Treatment Conditions;170
29.2.3;2.3 Proteomics;171
29.2.4;2.4 Mass Spectrometry;171
29.3;3 Results and Discussion;172
29.4;References;174
30;In Vitro Cytotoxic Effects of Tin Compounds on Normal Human Astrocytes;175
30.1;1 Introduction;175
30.2;2 Materials and Methods;176
30.2.1;2.1 Material Preparation;176
30.2.2;2.2 Astrocyte Cell Culture;176
30.2.3;2.3 MTT Assay for Cell Proliferation;177
30.2.4;2.4 Scrape-Loading and Dye Transfer (SLDT) Assay;177
30.2.5;2.5 Expression of Gap Junctional and Neural Cell Marker Genes;177
30.2.6;2.6 Statistical Analysis;178
30.3;3 Results and Discussion;178
30.4;4 Conclusion;179
30.5;References;180
31;Effects of Tin Compounds on Human Chondrogenic Activity In Vitro;181
31.1;1 Introduction;181
31.2;2 Materials and Methods;182
31.2.1;2.1 Medium and Materials Used for Cell Culture;182
31.2.2;2.2 Cells and Culture Methods;182
31.2.3;2.3 Cell Proliferation Study;182
31.2.4;2.4 Cell Differentiation Assay;183
31.2.5;2.5 Real-Time Polymerase Chain Reaction (PCR);183
31.2.6;2.6 Statistical Analysis;183
31.3;3 Results;184
31.3.1;3.1 Cell Proliferation;184
31.3.2;3.2 Cell Differentiation;184
31.4;4 Discussion;184
31.5;References;185
32;Construction of a Fluorescein-Responsive Chimeric Receptor with Strict Ligand Dependency and Analysis of the Role of Erythropo;187
32.1;1 Introduction;188
32.2;2 Methods and Results;188
32.3;3 Discussion;191
32.4;References;193
33;Nuclear Structures Regulate Liver-Specific Expression of the Tryptophan Oxygenase Gene;194
33.1;1 Introduction;194
33.2;2 Materials and Methods;195
33.2.1;2.1 Isolation and Culture of Rat Hepatocytes;195
33.2.2;2.2 Nuclear Matrix Isolation;195
33.2.3;2.3 Chromatin Immunoprecipitation (ChIP) Assay;196
33.3;3 Results;196
33.3.1;3.1 A Potential MAR Was Detected Upstream of the TO Gene;196
33.3.2;3.2 Upstream of the TO Gene Is Specifically Associated with Nuclear Matrices In Vivo;197
33.3.3;3.3 GR Is Associated with the Region Near the MAR of the TO Gene In Vivo;197
33.4;4 Discussion;199
33.5;References;199
34;CCAAT/Enhancer-Binding Protein Beta Controls Differentiation-Specific Expression of Chromatin Remodeling Factor BRM;201
34.1;1 Introduction;201
34.2;2 Materials and Methods;202
34.2.1;2.1 Cell Culture;202
34.2.2;2.2 Plasmids;203
34.2.3;2.3 Luciferase Reporter Gene Assay;203
34.3;3 Results;203
34.3.1;3.1 Identification of Promoter Regions of the brg1 and brm Gene;203
34.3.2;3.2 Analyses of Promoter Proximal Regions of mbrm and mbrg-1 Genes by Luciferase Reporter Assay;204
34.4;4 Discussion;206
34.5;References;206
35;Involvement of 67 kDa Laminin Receptor on Cellular Uptake of Green Tea Polyphenol Epigallocatechin-3-O-Gallate in Caco-2 Cel;208
35.1;1 Introduction;208
35.2;2 Materials and Methods;209
35.2.1;2.1 Reagents;209
35.2.2;2.2 Cell Culture;209
35.2.3;2.3 Cell Construction;209
35.2.4;2.4 Western Blot Analysis;209
35.2.5;2.5 Flow Cytometric Analysis;210
35.2.6;2.6 Surface Plasmon Resonance Biosensor Assay;210
35.2.7;2.7 Growth Inhibitory Activity Assay;210
35.2.8;2.8 Cellular Uptake Analysis;210
35.2.9;2.9 Statistical Analysis;210
35.3;3 Results and Discussion;211
35.4;References;212
36;Inositol Derivatives Stimulate Glucose Transport in Muscle Cells;213
36.1;1 Introduction;213
36.2;2 Materials and Methods;214
36.2.1;2.1 Materials;214
36.2.2;2.2 Cell Culture;214
36.2.3;2.3 Glucose Uptake Assay;214
36.2.4;2.4 Ex Vivo Assay;215
36.2.5;2.5 Western Blot Analysis;215
36.3;3 Results;215
36.3.1;3.1 Inositol Derivatives Stimulate Glucose Uptake in L6 Myotubes;215
36.3.2;3.2 Inositol Derivatives Stimulate Translocation of GLUT4 to the Plasma Membrane Ex Vivo;216
36.4;4 Discussion;217
36.5;References;218
37;Hair Growth Regulation by an Aromatic Plant Extract;219
37.1;1 Introduction;219
37.2;2 Materials and Methods;220
37.2.1;2.1 Cells and Cell Culture;220
37.2.2;2.2 Sample Extract;220
37.2.3;2.3 MTT Assay;221
37.2.4;2.4 Cell Cycle Assay;221
37.2.5;2.5 Hair Growth Promotion Assay In Vivo;221
37.3;3 Results and Discussion;222
37.3.1;3.1 Effect of Tunisian Aromatic Plant Extract on Human Dermal Papilla Cell Growth;222
37.3.2;3.2 Effect of Tunisian Aromatic Plant Extract on HFDPCs Cell Cycle;222
37.3.3;3.3 Hair Growth Promotion Activity of Tunisian Aromatic Plant Extract;223
37.4;References;224
38;Screening of Various Tunisian Olive Oils for Their Inhibitory Effect on Beta-Hexosaminidase Release by Basophilic Cells;226
38.1;1 Introduction;226
38.2;2 Materials and Methods;227
38.2.1;2.1 Reagents;227
38.2.2;2.2 Preparation of Olive Oil Sample;227
38.2.3;2.3 Cell and Cell Culture;228
38.2.4;2.4 b-Hexosaminidase Assay at Different Stages;228
38.2.5;2.5 Histamine Release Assay;229
38.2.6;2.6 Statistical Analysis;229
38.3;3 Results;229
38.3.1;3.1 Effect of Various Olive Oils on the b-Hexosaminidase Release at the Antigen-Antibody Binding Stage;229
38.3.2;3.2 Effect of Various Olive Oils on the b-Hexosaminidase Release at the Antibody-Receptor Binding Stage;230
38.3.3;3.3 Inhibitory Effect of Olive Oil on Histamine Release from KU812 Cells;231
38.4;4 Discussion;231
38.5;References;232
39;Leaf Extracts from Tunisian Olive Cultivars Induce Growth Inhibition and Differentiation of Human Leukemia HL-60 Cells;234
39.1;1 Introduction;235
39.2;2 Materials and Methods;235
39.2.1;2.1 Cell Line and Cell Maintenance;235
39.2.2;2.2 Sampling;235
39.2.3;2.3 Cell Proliferation Assay;235
39.2.4;2.4 Cell Differentiation Assays;236
39.2.5;2.5 Neutral Red Assay;236
39.3;3 Results and Discussion;236
39.3.1;3.1 Growth Inhibition of HL60 Cells by Olive Leaf Extracts;236
39.3.2;3.2 Morphological Examination;237
39.3.3;3.3 Induction of Differentiation by Olive Leaf Extracts;237
39.3.4;3.4 Effect of Oleuropein and Luteolin on HL60 Cell Viability;239
39.4;References;239
40;In Vitro Observation of the Effect of Intestinal Bacteria on IgA Production by Immunocytes in the Large Intestine: Comparison ;241
40.1;1 Introduction;242
40.2;2 Materials and Methods;242
40.2.1;2.1 Mice;242
40.2.2;2.2 Cell Preparation;242
40.2.3;2.3 Flow Cytometric Analysis;243
40.2.4;2.4 Enzyme-Linked Immunospots;243
40.2.5;2.5 Detection of IL-6 Products;243
40.2.6;2.6 Isolation of B220+ Cells and IgA-Plasma Cells;243
40.2.7;2.7 Measurement of Total IgA;244
40.3;3 Results;244
40.3.1;3.1 The Frequency of IgA-Plasma Cells and IgA-Secreting Cells in L-LP;244
40.3.2;3.2 L-LP Lymphocytes’ IL-6 Production;245
40.3.3;3.3 IgA Production by IgA-Plasma Cells and B220+ Cells Isolated from L-LP;245
40.4;4 Discussion;246
40.5;References;246
41;Differentiation of Human Leukemia Cell Line HL-60 by a Polyacetylenic Compound from Hedera Rhombea;247
41.1;1 Introduction;247
41.2;2 Materials and Methods;248
41.2.1;2.1 Cell Culture;248
41.2.2;2.2 Materials;248
41.2.3;2.3 MTT Assay;249
41.2.4;2.4 Specific and Nonspecific Esterase Double Staining;249
41.2.5;2.5 Time-Dependent Change in Cell Cycle Kinetics;250
41.3;3 Results and Discussion;250
41.3.1;3.1 Time-Dependent Change in HL-60 Cell Viability;250
41.3.2;3.2 Specific and Nonspecific Esterase Double Staining;251
41.3.3;3.3 Time-Dependent Change in Cell Cycle Kinetics;251
41.4;References;252
42;Effect of Tunisian Plant Extract on Melanogenesis;253
42.1;1 Introduction;253
42.2;2 Materials and Methods;254
42.2.1;2.1 Cell Culture;254
42.2.2;2.2 Measurement of Melanin Content and Microscopic Examination;254
42.2.3;2.3 Cell Viability;255
42.2.4;2.4 Western Blot Analysis;255
42.3;3 Results and Discussion;255
42.3.1;3.1 Tunisian Aromatic Plant Does Not Induce Cell Morphological Change;255
42.3.2;3.2 Tunisian Aromatic Plant Promotes Melanin Synthesis by B16 Cells;256
42.3.3;3.3 Effects of Tunisian Aromatic Plant on B16 Cell Viability;256
42.3.4;3.4 Tunisian Aromatic Plant Does Not Influence the Signaling Pathway Related to Melanogenesis;256
42.3.5;3.5 Tunisian Aromatic Plant Does Not Affect Tyrosinase Protein Expression in B16 Cells;257
42.4;References;257
43;“Nordenau Phenomenon” – Application of Natural Reduced Water to Therapy;259
43.1;1 Introduction;260
43.2;2 Material and Method;260
43.3;3 Results and Discussion;261
43.4;4 Conclusion;264
43.5;References;264
44;Anti-melanogenic Activity of Ergosterol Peroxide from Ganoderma lucidum on a Mouse Melanoma Cell Line;266
44.1;1 Introduction;266
44.2;2 Materials and Methods;267
44.2.1;2.1 Isolation of Active Compounds from Ganoderma lucidum;267
44.2.2;2.2 Effect of Triterpenes on Melanogenesis and Cell Growth;267
44.2.3;2.3 Fluorescence Microscopy;267
44.3;3 Results and Discussion;268
44.3.1;3.1 Anti-Melanogenic Compound from Ganoderma lucidum;268
44.3.2;3.2 Effect on the Level of TRP-1 in B16 10F7 Cells;269
44.4;References;270
45;Differentiation-Inducing Activities by Lupane Triterpenes from Lactuca indica on a Mouse Melanoma Cell Line;271
45.1;1 Introduction;271
45.2;2 Materials and Methods;272
45.2.1;2.1 Isolation of Active Compounds from Lactuca indica;272
45.2.2;2.2 Effect of Triterpenes on Melanogenesis and Cell Growth;272
45.2.3;2.3 Western Blotting;273
45.2.4;2.4 Fluorescence Microscopy;273
45.3;3 Results and Discussion;273
45.3.1;3.1 Active Compounds;273
45.3.2;3.2 Signaling Mechanisms;273
45.4;References;276
46;Immunoglobulin Production Stimulating Effect of Soy-Derived Proteins;278
46.1;1 Introduction;279
46.2;2 Materials and Methods;279
46.2.1;2.1 Reagents and Cells Culture;279
46.2.2;2.2 Enzyme-Linked Immunosorbent Assay (ELISA);279
46.2.3;2.3 Neutralization of Interleukin-6;280
46.2.4;2.4 Reverse Transcription–Polymerase Chain Reaction (RT-PCR);280
46.2.5;2.5 Anion-Exchange Chromatography;281
46.2.6;2.6 Sodium Dodecyl Sulfate–Poly Acrylamide Gel Electrophoresis;281
46.3;3 Results and Discussions;281
46.4;References;283
47;Immunostimulation Effect of the Jellyfish Collagen;284
47.1;1 Introduction;284
47.2;2 Materials and Methods;285
47.2.1;2.1 Preparation of Jellyfish Extract;285
47.2.2;2.2 Cells and Cell Culture;285
47.2.3;2.3 Assay of the Immunostimulating Activity;286
47.3;3 Results and Discussion;286
47.3.1;3.1 The Effect of Jellyfish Extract on IgM Production of HB4C5 Cells;286
47.3.2;3.2 Effect of Jellyfish Extract on Ig and Cytokine Production of hPBL;287
47.3.3;3.3 The Active Substance in Jellyfish Extract;287
47.3.4;3.4 Effects of Jellyfish Collagen on mRNA Expression Level for Ig and Cytokines;288
47.3.5;3.5 Effects of Jellyfish Collagen on Post-transcription Process;288
47.3.5.1;3.5.1 The Stimulation Effect of Collagen on Transcription-Suppressed HB4C5 Cells;288
47.3.5.2;3.5.2 The Stimulation Effect of Collagen on Translation-Suppressed HB4C5 Cells;289
47.3.5.3;3.5.3 The Stimulation Effect of Collagen on HB4C5 Cells Suppressed the Post-translation Activity;290
47.4;References;290
48;Mycotoxin Nivalenol Induces Apoptosis and Intracellular Calcium Ion-Dependent Interleukin-8 Secretion but Does Not Exert Mutag;291
48.1;1 Introduction;291
48.2;2 Materials and Methods;292
48.2.1;2.1 Chemicals and Cells;292
48.2.2;2.2 Examination of DNA Fragmentation;292
48.2.3;2.3 Determination of IL-8 Levels;292
48.2.4;2.4 Evaluation of Mutagenicity (umu Test);293
48.2.5;2.5 Statistics;293
48.3;3 Results and Discussion;293
48.4;References;295
49;Development of an In Vitro System for Screening the Ligands of a Membrane Glycoprotein CD36;297
49.1;1 Introduction;298
49.2;2 Materials and Methods;298
49.2.1;2.1 Materials;298
49.2.2;2.2 Cell Culture and Establishment of Stable Transformant;299
49.2.3;2.3 Western Blotting Analysis;299
49.2.4;2.4 Immunofluorescence;299
49.2.5;2.5 Binding;300
49.2.6;2.6 MAP Kinase Activation Assay;300
49.3;3 Results and Discussion;300
49.3.1;3.1 Establishment of Stable Transformants Expressing hCD36 or mCD36;300
49.3.2;3.2 Binding of OxLDL to the CD36-Expressing Transformants;301
49.3.3;3.3 Binding of LDL to the CD36-Expressing Transformants;301
49.3.4;3.4 MAP Kinase Activation;302
49.4;References;304
50;MTT Reduction by Flavonoids in the Absence of Cells: Influence of Medium Type and Serum;306
50.1;1 Introduction;306
50.2;2 Materials and Methods;307
50.2.1;2.1 Materials;307
50.2.2;2.2 Preparation of Flavonoids;308
50.2.3;2.3 MTT Assay;308
50.3;3 Results;308
50.4;4 Discussion;310
50.5;References;312
51;Characterization of Highly Reactive Sequences for Transglutaminase 2 and Factor XIIIa;314
51.1;1 Introduction;314
51.2;2 Materials and Methods;315
51.2.1;2.1 Screening Using Phage-Displayed Random Peptide Library (Fig. 1);315
51.2.2;2.2 Production of Peptide-GST Fusion Proteins;316
51.2.3;2.3 Assessments of Substrate Preference in TGase Reaction;317
51.2.4;2.4 Assessments of Synthetic Peptide as TGase Substrate;317
51.3;3 Results and Discussion;317
51.3.1;3.1 Screening of the Preferred Substrate Sequence Using a Phage-Displayed Peptide Library;317
51.3.2;3.2 Evaluation of Identified Peptide Sequence as GST Fusion Protein and Labeled Primary Amine;318
51.3.3;3.3 The Peptide Sequence Exhibited the Specific Inhibition Activity for Cross-Linking Reaction;319
51.4;References;320
52;Some Characteristics of UNC-51 Phosphorylations of Both Actins and Tubulins;321
52.1;1 Introduction;321
52.2;2 Materials and Methods;322
52.2.1;2.1 Materials and Chemicals;322
52.2.2;2.2 Plasmid Constructs;322
52.2.3;2.3 Cell-Transfection, Purification of FLAG-UNC-51;323
52.2.4;2.4 In Vitro Kinase Assay;323
52.3;3 Results;323
52.3.1;3.1 Time Courses of UNC-51 Phosphorylations of a/b Tubulins and Actins;323
52.3.2;3.2 Temperature Effects on UNC-51 Phosphorylations of a/b Tubulins and Actins;323
52.3.3;3.3 pH Effects on UNC-51 Phosphorylations of a/b Tubulins and Actins;324
52.3.4;3.4 Mg2+Promotes UNC-51 Kinase Activity;325
52.4;4 Discussion;325
52.5;References;326
53;Protein Phosphotase 1a Reverses UNC-51 Phosphorylations of Both Actins and Tubulins and a New Model of UNC-51-Inducing Axo;328
53.1;1 Introduction;328
53.2;2 Materials and Methods;329
53.2.1;2.1 In Vitro Dephosphorylation of UNC-51 Phosphorylations of Tubulins and Actins by PP1a;329
53.3;3 Results and Discussion;329
53.3.1;3.1 PP1a Can Dephosphorylate UNC-51 Phosphorylations of Actins and Tubulins;329
53.3.2;3.2 A New Model of UNC-51 Mediated Cytoskeletal Dynamics;331
53.4;References;331
54;Overexpression of Conserved Kinase UNC-51 Inhibits the Transferrin’s Endocytosis into the Mammalian Cells;333
54.1;1 Introduction;333
54.2;2 Materials and Methods;334
54.2.1;2.1 Plasmid Constructs;334
54.2.2;2.2 Cell Transfection and Confocal Immunofluorescence Microscopy;334
54.3;3 Results and Conclusion;334
54.4;References;335