Lo Sze Chung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 100-113). / Acknowledgements --- p.i / Abstract --- p.ii / Table of Contents --- p.iv / List of Figures and Tables --- p.vii / Abbreviations --- p.xii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Edible mushrooms --- p.1 / Chapter 1.1.1 --- Pleurotus sajor-caju --- p.1 / Chapter 1.1.2 --- Flammulina velutipes --- p.2 / Chapter 1.2 --- Lignocellulose and phenolic monomers --- p.4 / Chapter 1.2.1 --- Sources of phenolic monomers --- p.4 / Chapter 1.2.2 --- Toxicity of phenolic monomers --- p.10 / Chapter 1.3 --- Fungal laccases --- p.13 / Chapter 1.3.1 --- Occurrence --- p.13 / Chapter 1.3.2 --- Laccase reaction --- p.14 / Chapter 1.3.3 --- Physiological functions --- p.18 / Morphogenesis --- p.18 / Pathogenicity --- p.19 / Lignin degradation --- p.20 / Chapter 1.4 --- Purpose of study --- p.22 / Chapter 2. --- Materials and Methods --- p.24 / Chapter 2.1 --- General --- p.24 / Chapter 2.1.1 --- Organisms --- p.24 / Chapter 2.1.2 --- Culture medium --- p.24 / Chapter 2.1.3 --- Addition of phenolic compounds --- p.24 / Chapter 2.2 --- Effect of phenolic monomers on the growth of mushroom mycelium on agar plates --- p.25 / Chapter 2.3 --- Effect of phenolic monomers on the production of fungal biomass in liquid culture --- p.25 / Chapter 2.4 --- Effect of phenolic monomers on the extracellular laccase produced by P. sajor-caju and F. velutipes --- p.26 / Chapter 2.5 --- Assay of laccase activity --- p.26 / Chapter 2.6 --- Electrophoresis patterns of laccase proteins --- p.27 / Chapter 2.6.1 --- Non-denaturing polyacrylamide gel electrophoresis --- p.27 / Chapter 2.6.2 --- Localization of laccase activity --- p.27 / Chapter 2.7 --- Purification of extracellular laccases from P. sajor-caju --- p.28 / Chapter 2.7.1 --- Inoculum preparation --- p.28 / Chapter 2.7.2 --- Culture conditions --- p.28 / Chapter 2.7.3 --- Concentration of culture supernatant --- p.29 / Chapter 2.7.4 --- Ammonium sulphate fractionation --- p.29 / Chapter 2.7.5 --- Anion exchange chromatography --- p.29 / Chapter 2.7.6 --- Preparative polyacrylamide gel electrophoresis --- p.30 / Chapter 2.7.7 --- Protein detection and quantification --- p.30 / Chapter 2.8 --- Characterization of Laccase Protein --- p.31 / Chapter 2.8.1 --- "Effect of pH, temperature and substrate concentration" --- p.31 / Chapter 2.8.2 --- Effect of inhibitors --- p.32 / Chapter 2.8.3 --- Determination of isoelectric point --- p.32 / Chapter 2.8.4 --- Determination of molecular weight --- p.33 / Chapter 3. --- Results --- p.34 / Chapter 3.1 --- Effect of phenolic monomers on the growth of P. sajor-caju and F. velutipes --- p.34 / Chapter 3.1.1 --- P. sajor-caju --- p.34 / Chapter 3.1.2 --- F. velutipes --- p.38 / Chapter 3.2 --- Effect of phenolic monomers on laccase production by P. sajor-caju and F. velutipes --- p.41 / Chapter 3.2.1 --- P. sajor-caju --- p.45 / Chapter 3.2.2 --- F. velutipes --- p.49 / Chapter 3.3 --- Electrophoretic patterns of extracellular laccase --- p.53 / Chapter 3.3.1 --- P. sajor-caju --- p.53 / Chapter 3.3.2 --- F. velutipes --- p.56 / Chapter 3.4 --- Purification of laccase protein from P. sajor-caju --- p.58 / Chapter 3.4.1 --- Separation of laccase proteins --- p.58 / Chapter 3.4.2 --- Purification of laccase IV --- p.59 / Chapter 3.5 --- Characterization of laccase IV from P. sajor-caju --- p.64 / Chapter 3.5.1 --- Optimum temperature and thermostability --- p.64 / Chapter 3.5.2 --- Optimum pH and pH stability --- p.67 / Chapter 3.5.3 --- Substrate concentration --- p.70 / Chapter 3.5.4 --- Effect of inhibitors --- p.74 / Chapter 3.5.5 --- Isoelectric point --- p.74 / Chapter 3.5.6 --- Molecular weight --- p.74 / Chapter 4. --- Discussion --- p.78 / Chapter 4.1 --- Phenolic monomers and the growth of P. sajor-caju and F. velutipes --- p.78 / Chapter 4.2 --- Phenolic monomers and laccase production by P. sajor- caju and F. velutipes --- p.81 / Chapter 4.3 --- Electrophoretic patterns of laccase proteins --- p.83 / Chapter 4.4 --- Physiological functions of laccase --- p.85 / Chapter 4.5 --- Purification of selected laccase protein from P. sajor-caju --- p.88 / Chapter 4.6 --- Properties of laccase IV from P. sajor-caju --- p.89 / Chapter 4.6.1 --- Optimum temperature and thermostability --- p.89 / Chapter 4.6.2 --- Optimum pH and pH stability --- p.90 / Chapter 4.6.3 --- Effect of inhibitors --- p.92 / Chapter 4.6.4 --- Km --- p.93 / Chapter 4.6.5 --- Isoelectric point --- p.94 / Chapter 4.6.6 --- Molecular weight --- p.94 / Chapter 4.7 --- Future works --- p.96 / Chapter 5. --- Conclusion --- p.98 / Chapter 6. --- References --- p.100

by Cheung Pui Yi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 124-135). / Abstract --- p.ii / Acknowledgements --- p.iv / List of Tables --- p.viii / List of Figures --- p.ix / List of Abbreviations --- p.xiii / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- General Background --- p.1 / Chapter 1.2 --- Occurrence and Structure of Cellulose --- p.1 / Chapter 1.3 --- Occurrence and Structure of Hemicelluloses --- p.4 / Chapter 1.4 --- Biodegradation of Cellulose and Hemicelluloses --- p.4 / Chapter 1.4.1 --- Cellulolytic and Hemicellulolytic Microorganisms --- p.4 / Chapter 1.4.2 --- Enzymes Involved in Cellulose Degradation --- p.10 / Chapter 1.4.2.1 --- "Endo-1,4-β-glucanases" --- p.12 / Chapter 1.4.2.2 --- "Exo-1,4-β-glucanases" --- p.14 / Chapter 1.4.2.3 --- β-Glucosidases --- p.16 / Chapter 1.4.2.4 --- Oxidative Enzymes --- p.18 / Chapter 1.4.3 --- Synergistic Action between Cellulolytic Enzymes --- p.19 / Chapter 1.4.4 --- Enzymes Involved in Hemicellulose Degradation --- p.21 / Chapter 1.4.4.1 --- "Endo-1,4-β-xylanases" --- p.22 / Chapter 1.4.4.2 --- β-Xylosidases --- p.24 / Chapter 1.4.4.3 --- Other Xylanolytic Enzymes --- p.24 / Chapter 1.4.5 --- Synergistic Action between Hemicellulolytic Enzymes --- p.25 / Chapter 1.5 --- Flammulina velutipes --- p.26 / Chapter 1.6 --- Aims of the Present Investigation --- p.27 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Organism --- p.28 / Chapter 2.2 --- Culture Medium --- p.28 / Chapter 2.3 --- Determination of the Optimal Growth pH of Flammulina velutipes --- p.29 / Chapter 2.4 --- "Preparation of Inoculum, Cultivation and Harvest of Fungal Cultures" --- p.30 / Chapter 2.5 --- Enzyme Assays --- p.30 / Chapter 2.5.1 --- "Exo-1,4-β-glucanase" --- p.30 / Chapter 2.5.2 --- "Endo-1,4-β-glucanase" --- p.31 / Chapter 2.5.3 --- "Endo-1,4-β-xylanase" --- p.34 / Chapter 2.5.4 --- Extracellular β-Glucosidase --- p.36 / Chapter 2.5.5 --- Cell-Associated β-Glucosidase --- p.38 / Chapter 2.5.6 --- Extracellular β-Xylosidase --- p.38 / Chapter 2.5.7 --- Cell-Associated β-Xylosidase --- p.38 / Chapter 2.6 --- Determination of Optimal Temperatures for Cellulolytic and Xylanolytic Enzymes --- p.39 / Chapter 2.7 --- Determination of the Optimal pH for Enzyme Reaction --- p.39 / Chapter 2.8 --- Protein Determination --- p.39 / Chapter 2.9 --- Determination of Enzyme Induction Patterns --- p.42 / Chapter 2.10 --- Elucidation of Cellulase Production Patterns in F. velutipes --- p.43 / Chapter 2.10.1 --- Native Polyacrylamide Gel Electrophoresis --- p.43 / Chapter 2.10.2 --- Activity Staining for Endoglucanases --- p.43 / Chapter 2.10.3 --- Activity Staining for β-Glucosidases --- p.44 / Chapter 2.10.4 --- Protein Staining --- p.44 / Chapter 2.10.5 --- Preparative Polyacrylamide Gel Electrophoresis --- p.44 / Chapter 2.10.6 --- Separation of Proteins and Partial Purification of Different Cellulase Species after Preparative Polyacrylamide Gel Electrophoresis --- p.45 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Determination of the Optimal pH for Fungal Growth --- p.46 / Chapter 3.2 --- Determination of the Optimal Temperature for Cellulolytic and Xylanolytic Enzyme Activity --- p.48 / Chapter 3.3 --- Determination of the Optimal pH for Enzyme Reaction --- p.64 / Chapter 3.4 --- Time Course Experiments on the Production of Cellulolytic and Hemicellulolytic Enzymes --- p.72 / Chapter 3.4.1 --- Production of Cellulolytic Enzymes --- p.72 / Chapter 3.4.2 --- Production of Hemicellulolytic Enzymes --- p.77 / Chapter 3.5 --- Determination of Enzyme Induction Patterns --- p.82 / Chapter 3.5.1 --- Induction of Exoglucanase Production --- p.82 / Chapter 3.5.2 --- Induction of Endoglucanase Production --- p.84 / Chapter 3.5.3 --- Induction of Extracellular β-Glucosidase Production --- p.86 / Chapter 3.5.4 --- Induction of β-Xylanase Production --- p.88 / Chapter 3.5.5 --- Induction of Extracellular β-Xylosidase Production --- p.90 / Chapter 3.5.6 --- Changes in Extracellular Protein Levels in DMS Media Supplemented with Different Substrates --- p.92 / Chapter 3.5.7 --- Changes in Reducing Sugar Levels in DMS Media Supplemented with Different Substrates --- p.94 / Chapter 3.6 --- Partial Purification of Different Cellulases Species Produced by Flammulina velutipes --- p.96 / Chapter 3.6.1 --- Native Polyacrylamide Gel Electrophoresis --- p.96 / Chapter 3.6.2 --- Activity Staining for Endoglucanases --- p.96 / Chapter 3.6.3 --- Activity Staining for β-Glucosidases --- p.96 / Chapter 3.6.4 --- Assay of Cellulolytic Enzymes after Preparative Polyacrylamide Gel Electrophoresis --- p.101 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Optimal Conditions for Cellulolytic and Hemicellulolytic Enzymes of F. velutipes --- p.105 / Chapter 4.1.1 --- Optimal Temperature for Enzymic Reaction --- p.105 / Chapter 4.1.2 --- Optimal pH for Enzymic Reaction --- p.106 / Chapter 4.2 --- Production of Cellulolytic and Hemicellulolytic Enzymes --- p.109 / Chapter 4.2.1 --- Production of Cellulolytic Enzymes --- p.109 / Chapter 4.2.2 --- Production of Hemicellulolytic Enzymes --- p.110 / Chapter 4.3 --- Enzyme Induction Patterns --- p.111 / Chapter 4.4 --- Partial Purification of Different Cellulase Species Produced by Flammulina velutipes --- p.116 / Chapter 4.5 --- Conclusion --- p.121 / Chapter 4.6 --- Further Studies --- p.123 / List of References --- p.124

Leung Yiu Kwong, Michael. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 155-161). / Acknowledgements --- p.i / Abbreviations --- p.ii / Aim and scope of this dissertation --- p.v / Abstract --- p.vi / Table of contents --- p.viii / Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tumor Biology --- p.3 / Chapter 1.3 --- The Defence Mechanisms --- p.4 / Chapter 1.3.1 --- Non-specific defence mechanisms --- p.5 / Chapter 1.3.2 --- Specific defence mechanisms --- p.6 / Chapter 1.4 --- Effector Mechanisms in Anti-tumor Immunity --- p.7 / Chapter 1.4.1 --- B-cell --- p.8 / Chapter 1.4.2 --- "Natural killer (NK) cells (Non-T, Non-B)" --- p.8 / Chapter 1.4.3 --- Macrophages --- p.9 / Chapter 1.4.4 --- Cytolytic T-lymphocytes (CTLs) --- p.10 / Chapter 1.5 --- Cancer Treatment --- p.10 / Chapter 1.5.1 --- Surgery --- p.10 / Chapter 1.5.2 --- Radiotherapy --- p.12 / Chapter 1.5.3 --- Drug therapy --- p.12 / Chapter 1.5.4 --- Gene therapy --- p.13 / Chapter 1.5.5 --- lmmunotherapy --- p.13 / Chapter 1.6 --- Non-cytotoxic Antitumor Polysaccharides of Fungi --- p.14 / Chapter 1.6.1 --- Yeast polysaccharides --- p.14 / Chapter 1.6.2 --- Lichen polysaccharides --- p.15 / Chapter 1.6.3 --- Fungal polysaccharides --- p.18 / Chapter 1.7 --- Fungi and their Polysaccharides --- p.20 / Chapter 1.7.1 --- Reserve carbohydrates --- p.20 / Chapter 1.7.2 --- Structural polysaccharides --- p.21 / Chapter 1.8 --- The Architecture of the Fungal Cell Wall --- p.22 / Materials and Methods --- p.26 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Animals --- p.27 / Chapter 2.1.2 --- Mushrooms --- p.27 / Chapter 2.1.3 --- "Buffers, culture media and chemicals" --- p.27 / Chapter 2.1.4 --- Cell lines --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Screening of β-(l→3)-D-glucan --- p.35 / Chapter 2.2.2 --- Extraction and Fractionation of Flammulina velutipes --- p.35 / Chapter 2.2.3 --- Characterisation of Flammulina velutipes --- p.38 / Chapter 2.2.3.1 --- The determination of carbohydrate content of F.V fractions --- p.38 / Chapter 2.2.3.2 --- The determination of protein content of F.V. fractions --- p.39 / Chapter 2.2.3.3 --- The determination of uronic acid content of F.V fractions --- p.39 / Chapter 2.2.3.4 --- The determination of component sugar units of F.V fractions --- p.39 / Chapter 2.2.3.5 --- Periodate uptake of F.V. fractions --- p.40 / Chapter 2.2.3.6 --- Limulus amebocyte lysate (LAL) coagulation assay --- p.40 / Chapter 2.2.3.7 --- The digestion of F.V. fractions with laminarinase --- p.41 / Chapter 2.2.3.8 --- The Secondary and tertiary structure determination of FH and SFA1 --- p.42 / Chapter 2.2.3.9 --- Molecular weight estimation of FH and SFA1 --- p.43 / Chapter 2.2.3.10 --- Vascular dilation and hemorrhage (VDH) activity of F.V. fractions / Chapter 2.2.4 --- Isolation and preparation of cells --- p.43 / Chapter 2.2.4.1 --- Bone marrow cell --- p.43 / Chapter 2.2.4.2 --- Peritoneal exudate cell (PEC) --- p.44 / Chapter 2.2.4.3 --- Splenocytes --- p.44 / Chapter 2.2.4.4 --- Depleting mouse T-cells by anti-mouse T-cell antigen antibody plus complement treatment --- p.45 / Chapter 2.2.4.5 --- Depleting mouse B-cells by Cedarlane column kit --- p.45 / Chapter 2.2.5 --- Assays for the cytotoxicity of Flammulina velutipes --- p.45 / Chapter 2.2.5.1 --- Brine shrimp assay --- p.45 / Chapter 2.2.5.2 --- In vitro cytotoxicity of FH and SFA1 on bone marrow cells of female BALB/c mice --- p.46 / Chapter 2.2.5.3 --- In vivo cytotoxicity of FH and SFA1 on female BALB/c mice --- p.47 / Chapter 2.2.6 --- "Assays for the immunomodulatory activities of Flamm""lina velutipes" --- p.47 / Chapter 2.2.6.1 --- In vitro mitogenic activities of FH and SFA1 on murine lymphocytes --- p.47 / Chapter 2.2.6.2 --- In vitro mitogenic activities of FH and SFA1 with PMB on murine lymphocytes --- p.48 / Chapter 2.2.6.3 --- In vitro mitogenic activities of FH and on T-cell depleted murine lymphocytes --- p.48 / Chapter 2.2.6.4 --- In vitro mitogenic activities of FH on B-cell depleted murine lymphocytes --- p.49 / Chapter 2.2.6.5 --- In vitro co-mitogenic activitiy of FH and SFA1 on murine lymphocytes --- p.49 / Chapter 2.2.6.6 --- In vitro mitogenic activities of FH and SFA1 on murine bone marrow cells --- p.50 / Chapter 2.2.6.7 --- In vivo mitogenic activities of FH and SFA1 on murine lymphocytes --- p.50 / Chapter 2.2.6.8 --- Effect of FH and SFA1 on the enhancement of first antibody production of SRBC immunised mice --- p.51 / Chapter 2.2.6.9 --- Effect of FM and SFA1 on the in vitro phagocytic activity of murine macrophage --- p.51 / Chapter 2.2.6.10 --- Effect of FM and SFA1 on the in vivo phagocytic activity of murine macrophage --- p.51 / Chapter 2.2.6.11 --- In vivo migration of macrophage in FH- and SFAl-treated mice --- p.53 / Chapter 2.2.6.12 --- Effect of FH and SFA1 on the enhancement of murine PEC cytostatic activity --- p.53 / Chapter 2.2.6.13 --- Effect of FH and SFA1 on the Fc receptor expression of peritoneal exudate cells --- p.54 / Chapter 2.2.6.14 --- Effect of FH and SFA1 on murine serum cytokine level --- p.55 / Chapter 2.2.6.15 --- Effect of FH and SFA1 on murine serum TNF level --- p.55 / Chapter 2.2.6.16 --- Effect of FH and SFA1 on the augmentation of SRBC lysing ability of murine serum --- p.56 / Chapter 2.2.7 --- Assays for the anti-tumor activities of Flammulina velutipes --- p.57 / Chapter 2.2.7.1 --- In vitro anti-tumor activity of FH and SFA1 --- p.57 / Chapter 2.2.7.2 --- Effect of FH and SFA1 on the growth of murine transplantable tumor invivo --- p.58 / Chapter 2.2.8 --- Statistical analysis --- p.59 / "Screening, Purification, Fractionation and Characterisation of β-(l→3)-D-glucan(s) from Flammulina velutipes" --- p.60 / Introduction --- p.61 / Results --- p.62 / Chapter 3.1 --- Screening of β-(l→3)-D-Glucan --- p.62 / Chapter 3.2 --- Extraction and Fractionation of Flammulina velutipes --- p.62 / Chapter 3.3 --- The Determination of Carbohydrate Content of F.V. Fractions --- p.65 / Chapter 3.4 --- The Determination of Protein Content of F.V. Fractions --- p.65 / Chapter 3.5 --- The Determination of Uronic Acid Content of F.V. Fractions --- p.69 / Chapter 3.6 --- The Determination of Component Sugar Units of F.V. Fractions --- p.69 / Chapter 3.7 --- Periodate Uptake of F.V. Fractions --- p.69 / Chapter 3.8 --- Limulus Amebocyte Lysate (LAL) Coagulation Assay --- p.73 / Chapter 3.9 --- The Digestion of F.V. Fractions with Laminarinase --- p.73 / Chapter 3.10 --- The Secondary and tertiary Structure Determination of FH and SFA1 --- p.80 / Chapter 3.11 --- Molecular Weight Estimation of FH and SFA1 --- p.82 / Chapter 3.12 --- "Vascular Dilation and Hemorrhage (VDH) Activity of FH, SFA1 and lFA1" --- p.82 / Discussion --- p.90 / The Toxicity of Flammulina velutipes --- p.96 / Introduction --- p.97 / Results --- p.97 / Chapter 4.1 --- Lack of Cytotoxicity of Flammulina velutipes to Brine Shrimp --- p.97 / Chapter 4.2 --- Lack of Cytotoxicity of Flammulina velutipes to Murine Bone Marrow Cells --- p.99 / Chapter 4.3 --- Lack of Cytotoxicity of Flammulina velutipes to Mouse --- p.99 / Discussion --- p.102 / The Immunomodulatory Activities of Flammulina velutipes --- p.103 / Introduction --- p.104 / Results --- p.105 / Chapter 5.1 --- Effect of Flammulina velutipes on Murine Lymphocytes --- p.105 / Chapter 5.2 --- Effect of Flammulina velutipes on Murine Macrophage --- p.115 / Chapter 5.3 --- Effect of Flammulina velutipes on Murine Serum Cytokine and Complement Level --- p.125 / Discussion --- p.133 / The Anti-tumor Activities of Flammulina velutipes --- p.136 / Introduction --- p.137 / Results --- p.137 / Chapter 6.1 --- In Vitro Anti-Tumor Activity of FH and SFA1 --- p.137 / Chapter 6.2 --- Effect of FH and SFAI on the Growth of Murine TransplantableTumors --- p.138 / Discussion --- p.145 / General Discussion --- p.146 / General Discussion and Future Perspectives --- p.147 / References --- p.154