Cheung, Hang Kei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 138-149). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.vi / List of Abbreviations --- p.x / List of Figures --- p.xi / List of Tables --- p.xiii / Chapter Chapter 1: --- Introduction of Lectins --- p.1 / Chapter 1.1 --- General Introduction --- p.1 / Chapter 1.1.1 --- Definition and History of Lectins --- p.1 / Chapter 1.1.2 --- More than Just Carbohydrate Binding --- p.2 / Chapter 1.1.3 --- Classification of Lectins --- p.3 / Chapter 1.2 --- Plant Lectins --- p.4 / Chapter 1.2.1 --- History of Plant Lectins --- p.4 / Chapter 1.2.2 --- Occurrence of Plant Lectins --- p.5 / Chapter 1.3 --- Physiological Roles of Plant Lectins --- p.6 / Chapter 1.3.1 --- Lectins as Storage Proteins --- p.6 / Chapter 1.3.2 --- Lectins as Defense Proteins --- p.7 / Chapter 1.3.3 --- Lectins as mediator in symbiosis with bacteria --- p.8 / Chapter 1.4 --- Biological Activities of Plant Lectins --- p.9 / Chapter 1.4.1 --- Immunomodulatory Activity --- p.9 / Chapter 1.4.2 --- Lectins and Cancer --- p.10 / Chapter 1.4.3 --- A ntiviral A ctivity --- p.12 / Chapter 1.5 --- Lectins in Glycomic Study --- p.14 / Chapter 1.5.1 --- Background --- p.14 / Chapter 1.5.2 --- Glyco-catch method --- p.15 / Chapter 1.5.3 --- Lectin Blot Analysis --- p.16 / Chapter 1.6 --- Aim of current study --- p.17 / Chapter Chapter 2: --- Purification and Characterization of a Lectin from Musa acuminata --- p.19 / Chapter 2.1 --- Introduction --- p.19 / Chapter 2.2 --- Materials and Methods --- p.20 / Chapter 2.2.1 --- Purification Scheme --- p.20 / Chapter 2.2.2 --- Assay of Hemagglutinating A ctivity --- p.21 / Chapter 2.2.3 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis --- p.22 / Chapter 2.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.22 / Chapter 2.2.5 --- Protein Concentration Determination --- p.22 / Chapter 2.2.6 --- N-terminal amino acid sequence analysis --- p.22 / Chapter 2.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.23 / Chapter 2.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.23 / Chapter 2.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.24 / Chapter 2.2.10 --- Assay of Nitric Oxide Production by Murine Peritoneal Macrophages --- p.25 / Chapter 2.2.11 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.25 / Chapter 2.2.12 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.26 / Chapter 2.2.13 --- RNA Extraction --- p.27 / Chapter 2.2.14 --- Reverse Transcription: First Strand cDNA Synthesis --- p.28 / Chapter 2.2.15 --- Polymerasae Chain Reaction (PCR) --- p.28 / Chapter 2.3 --- Results --- p.32 / Chapter 2.4 --- Discussion --- p.46 / Chapter Chapter 3: --- Purification and Characterization of a Lectin from Gymnocladus chinensis Baill. --- p.49 / Chapter 3.1 --- Introduction --- p.49 / Chapter 3.2 --- Material and Methods --- p.50 / Chapter 3.2.1 --- Purification Scheme --- p.50 / Chapter 3.2.2 --- Assay of Hemaggl utinating Activity --- p.51 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.51 / Chapter 3.2.4 --- Molecular Mass Determination by FPLC Gel Filtration --- p.51 / Chapter 3.2.5 --- Protein Concentration Determination --- p.51 / Chapter 3.2.6 --- N-terminal amino acid sequence analysis --- p.52 / Chapter 3.2.7 --- Inhibition of Lectin-induced Hemagglutination by Carbohydrates --- p.52 / Chapter 3.2.8 --- Effect of Temperature and pH on Lectin-induced Hemagglutination --- p.52 / Chapter 3.2.9 --- Assay of Mitogenic Activity on Murine Splenocytes --- p.52 / Chapter 3.2.10 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.52 / Chapter 3.2.11 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.53 / Chapter 3.2.12 --- Assay of Anti-fungal Activity --- p.53 / Chapter 3.3 --- Results --- p.56 / Chapter 3.4 --- Discussion --- p.67 / Chapter Chapter 4: --- Introduction to Protease Inhibitors --- p.70 / Chapter 4.1 --- General Introduction --- p.70 / Chapter 4.2 --- Serine Protease Inhibitors --- p.71 / Chapter 4.2.1 --- Kunitz Type Serine Protease Inhibitors --- p.73 / Chapter 4.2.2 --- Bowman-Birk Type Serine Protease Inhibitors (BBI) --- p.74 / Chapter 4.2.3 --- Squash Type Serine Protease Inhibitors --- p.75 / Chapter 4.3 --- Roles of Pis in Plants --- p.76 / Chapter 4.3.1 --- Pis as a defense protein --- p.76 / Chapter 4.3.2 --- Pis in seed germination --- p.78 / Chapter 4.4 --- Applications of Protease Inhibitors --- p.79 / Chapter 4.4.1 --- Pis in Cancer Prevention --- p.79 / Chapter 4.4.2 --- Pis in Crop Protection --- p.81 / Chapter 4.5 --- Aim of Current Study --- p.83 / Chapter Chapter 5: --- Isolation and Characterization of a Trypsin Inhibitor from the seeds of Lens culinaris --- p.84 / Chapter 5.1 --- Introduction --- p.84 / Chapter 5.2 --- Materials and Methods --- p.86 / Chapter 5.2.1 --- Purification Scheme --- p.86 / Chapter 5.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.87 / Chapter 5.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.88 / Chapter 5.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.88 / Chapter 5.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.88 / Chapter 5.2.6 --- Protein Concentration Determination --- p.89 / Chapter 5.2.7 --- N-terminal amino acid sequence analysis --- p.89 / Chapter 5.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.89 / Chapter 5.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.90 / Chapter 5.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.90 / Chapter 5.2.11 --- Assay of Anti-fungal Activity --- p.90 / Chapter 5.3 --- Results --- p.93 / Chapter 5.4 --- Discussion --- p.103 / Chapter Chapter 6: --- Isolation and Characterization of trypsin inhibitors trom the seeds of Vigna mungo (L.) Hepper --- p.106 / Chapter 6.1 --- Introduction --- p.106 / Chapter 6.2 --- Materials and Methods --- p.107 / Chapter 6.2.1 --- Purification Scheme --- p.107 / Chapter 6.2.2 --- Assay of Trypsin-Inhibitory Activity --- p.109 / Chapter 6.2.3 --- Assay of Chymotrypsin-Inhibitory Activity --- p.109 / Chapter 6.2.4 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis --- p.109 / Chapter 6.2.5 --- Molecular Mass Determination by FPLC Gel Filtration --- p.109 / Chapter 6.2.6 --- Protein Concentration Determination --- p.109 / Chapter 6.2.7 --- N-terminal amino acid sequence analysis --- p.110 / Chapter 6.2.8 --- Effect of DTT on the inhibitory activity of trypsin inhibitor --- p.110 / Chapter 6.2.9 --- Assay of Antiproliferative Activity on Tumor Cell Lines --- p.110 / Chapter 6.2.10 --- Assay of HIV-1 Reverse Transcriptase Inhibitory Activity --- p.110 / Chapter 6.2.11 --- Assay of Anti-fungal Activity --- p.110 / Chapter 6.3 --- Results --- p.113 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7: --- General Discussion --- p.135 / References --- p.138

Ho, Sai Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 144-164). / Abstracts in English and Chinese. / TABLE OF CONTENTS --- p.ii / ABSTRACT --- p.xii / 撮要 --- p.xv / LIST OF ABBREIVIATIONS --- p.xvi / LIST OF TABLES --- p.xvii / LIST OF FIGURES --- p.xix / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Overview of lectins --- p.1 / Chapter 1.1.1 --- History of lectins --- p.1 / Chapter 1.1.2 --- Definitions of lectins --- p.2 / Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2 / Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4 / Chapter 1.1.5 --- Structure of plant lectins --- p.4 / Chapter 1.1.6 --- Biological function of plant lectins --- p.5 / Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5 / Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6 / Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7 / Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7 / Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7 / Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9 / Chapter 1.1.7 --- Background of legume lectins --- p.11 / Chapter 1.1.7.1 --- Structure of legume lectins --- p.11 / Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12 / Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14 / Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15 / Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17 / Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19 / Chapter 1.2.3.1 --- Medical application --- p.19 / Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22 / Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25 / Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27 / Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28 / Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31 / Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33 / Chapter 1.3.2.1 --- Classification of chitinase --- p.34 / Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34 / Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35 / Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36 / Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38 / Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40 / Chapter 1.4 --- Rationale of the present study --- p.42 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.43 / Chapter 2.2 --- Preparation of crude extract --- p.44 / Chapter 2.2.1 --- Hokkaido large black soybean --- p.44 / Chapter 2.2.2 --- Emperor banana --- p.45 / Chapter 2.3 --- Purification --- p.45 / Chapter 2.4 --- Chromatography --- p.46 / Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46 / Chapter 2.4.2 --- Affi-gel Blue gel --- p.47 / Chapter 2.4.3 --- SP-Sepharse --- p.48 / Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49 / Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50 / Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50 / Chapter 2.6 --- Protein concentration determination --- p.54 / Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54 / Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56 / Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56 / Chapter 2.10 --- Thermal stability determination assays --- p.57 / Chapter 2.10.1 --- Stability at various temperatures --- p.57 / Chapter 2.10.2 --- Stability at 100°C --- p.57 / Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58 / Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58 / Chapter 2.13 --- Assay of antifungal activity --- p.58 / Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60 / Chapter 2.15 --- Assay of antibacterial activity --- p.61 / Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61 / Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62 / Chapter 2.18 --- Assay of mitogenic activity --- p.63 / Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective Sources / Chapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybean / Chapter 3.1.1 --- Introduction --- p.65 / Chapter 3.1.2 --- Results --- p.66 / Chapter 3.1.3 --- Purification --- p.68 / Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69 / Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70 / Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71 / Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72 / Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73 / Chapter 3.1.4 --- Characterization of Lectin --- p.74 / Chapter 3.1.4.1 --- Molecular mass determination --- p.74 / Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76 / Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77 / Chapter 3.1.4.4 --- Thermal stability --- p.78 / Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80 / Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81 / Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82 / Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83 / Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84 / Chapter 3.1.5 --- Discussion --- p.86 / Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybean / Chapter 3.2.1 --- Introduction --- p.93 / Chapter 3.2.2 --- Results --- p.94 / Chapter 3.2.3 --- Purification --- p.95 / Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96 / Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98 / Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99 / Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100 / Chapter 3.2.4.1 --- Molecular mass determination --- p.100 / Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102 / Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103 / Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104 / Chapter 3.2.4.5 --- pH and thermal stability --- p.105 / Chapter 3.2.5 --- Discussion --- p.106 / Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor Banana / Chapter 3.3.1 --- Introduction --- p.108 / Chapter 3.3.2 --- Results --- p.109 / Chapter 3.3.3 --- Purification --- p.111 / Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112 / Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113 / Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114 / Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114 / Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115 / Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118 / Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121 / Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121 / Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122 / Chapter 3.3.4.3 --- Thermal stability --- p.124 / Chapter 3.3.4.4 --- pH stability --- p.125 / Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125 / Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126 / Chapter 3.3.4.7 --- Discussion --- p.127 / Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131 / Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131 / Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131 / Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132 / Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133 / Chapter 3.3.5.3 --- Discussion --- p.136 / Chapter Chapter 4 --- general discussion --- p.138 / References --- p.144