by Chuen Ka Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 134-166). / Abstracts in English and Chinese. / ACKNOWLDEGEMENT --- p.ii / PUBLICATIONS --- p.iii-iv / ABBREVIATIONS --- p.v-viii / INDEX FOR FIGURES --- p.ix xii / INDEX FOR TABLES --- p.xiii / ABSTRACT (Chinese and English) --- p.xiv-xvi / TABLE OF CONTENT --- p.xvii / Chapter 1. --- INTRODUCTION --- p.1-37 / Chapter 2. --- OBJECTIVES --- p.38-40 / Chapter 3. --- METHODS AND MATERIALS --- p.41 -70 / Chapter 4. --- RESULTS AND DISCUSSION --- p.71-130 / Chapter 5. --- GENERAL DISCUSSION AND CONCLUSION --- p.131-133 / BIBLIOGRAPHY --- p.134-166

Lam Sze Kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves 172-200). / Abstracts in English and Chinese. / Acknowledgements --- p.ii / Table of contents --- p.ii / Abstract --- p.xii / 撮要 --- p.xv / List of Abbreviations --- p.xvi / List of Tables --- p.xvii / List of Figures --- p.xix / TABLE OF CONTENTS / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1. --- Overview of Chitinases --- p.3 / Chapter 1.1.1. --- Classification of Chitinases --- p.7 / Chapter 1.1.1.1. --- Family 19 Chitinases --- p.7 / Chapter 1.1.1.1.1. --- Class I Chitinases --- p.9 / Chapter 1.1.1.1.2. --- Class II Chitinases --- p.10 / Chapter 1.1.1.1.3. --- Class IV Chitinases --- p.10 / Chapter 1.1.1.1.4. --- Class V Chitinases --- p.11 / Chapter 1.1.1.1.5. --- Class VI Chitinases --- p.11 / Chapter 1.1.1.2. --- Family 18 Chitinases --- p.12 / Chapter 1.1.1.2.1. --- PR-8/Class III Chitinases --- p.12 / Chapter 1.1.1.2.2. --- PR-11 Chitinases --- p.15 / Chapter 1.1.1.3. --- The PR-4 Family --- p.16 / Chapter 1.1.2. --- Catalytic Mechanism of Chitinases --- p.19 / Chapter 1.1.2.1. --- Catalytic Mechanism of Family 18 Chitinases --- p.20 / Chapter 1.1.2.2. --- Catalytic Mechanism of Family 19 Chitinases --- p.21 / Chapter 1.1.3. --- Biological Properties of Chitinases --- p.22 / Chapter 1.1.3.1. --- Antifungal Activity of Chitinases in vitro --- p.22 / Chapter 1.1.3.2. --- Antifungal Activity of Chitinases in vivo --- p.23 / Chapter 1.1.3.3. --- Other Functions --- p.23 / Chapter 1.2. --- Overview of Ribonucleases --- p.25 / Chapter 1.2.1. --- Classification of Ribonucleases --- p.26 / Chapter 1.2.1.1. --- RNase T1 Family --- p.26 / Chapter 1.2.1.1.1. --- Action Mechanism of RNase T1 Family --- p.32 / Chapter 1.2.1.2. --- RNase T2 Family --- p.34 / Chapter 1.2.1.2.1. --- Action Mechanism of RNase T2 Family --- p.36 / Chapter 1.2.2. --- Biological Activities of Plant Ribonucleases --- p.38 / Chapter 1.2.2.1. --- Phosphate Remobilization --- p.38 / Chapter 1.2.2.2. --- Senescence --- p.39 / Chapter 1.2.2.3. --- Programmed Cell Death --- p.40 / Chapter 1.2.2.4. --- Plant Defense --- p.41 / Chapter 1.2.2.5. --- RNA Processing and Decay --- p.43 / Chapter 1.2.2.6. --- Antitumor Activities --- p.43 / Chapter 1.3. --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.45 / Chapter 1.3.1. --- General properties of RIPs --- p.46 / Chapter 1.3.1.1. --- Classification of RIPs --- p.46 / Chapter 1.3.2. --- Activities of Ribosome-inactivating Proteins --- p.52 / Chapter 1.3.2.1. --- RNA N-glycosidase activity --- p.52 / Chapter 1.3.2.2. --- Protein synthesis inhibitory activity --- p.58 / Chapter 1.3.2.3. --- Abortifacient activity --- p.59 / Chapter 1.3.2.4. --- Immunosuppressive activity --- p.60 / Chapter 1.3.2.5. --- Antiviral activity --- p.61 / Chapter 1.3.3. --- Roles of RIPs in plants --- p.63 / Chapter 1.3.3.1. --- Defensive role of RIPs in plants --- p.63 / Chapter 1.3.3.2. --- Role of RIPs in stress adaptation in plants --- p.66 / Chapter 1.3.4. --- Possible application of RIPs --- p.67 / Chapter 1.3.4.1. --- Use of RIPs in therapies --- p.67 / Chapter 1.3.4.1.1. --- Antiviral agents --- p.67 / Chapter 1.3.4.1.2. --- Immunotoxins --- p.68 / Chapter 1.3.4.1.3. --- Anti-HIV drugs --- p.69 / Chapter 1.3.4.2. --- Use of RIPs in agriculture --- p.71 / Chapter 1.4. --- Overview of the PR-5 Family: Thaumatin-Like Proteins (TLPs) --- p.72 / Chapter 1.4.1. --- Occurrence of Thaumatin-Like Proteins --- p.76 / Chapter 1.4.2. --- Biological properties of TLPs --- p.77 / Chapter 1.4.2.1. --- Antifungal Activity --- p.77 / Chapter 1.4.2.2. --- TLPs as Anti-Freeze Protein --- p.78 / Chapter 1.4.3. --- Biotechnological Application ´ؤ Transgenic Plants --- p.79 / Chapter Chapter 2 --- Materials and Methods --- p.81 / Chapter 2.1. --- Materials --- p.81 / Chapter 2.2. --- Preparation of Crude Extract --- p.82 / Chapter 2.3. --- Purification --- p.83 / Chapter 2.4. --- Chromatography --- p.84 / Chapter 2.4.1. --- CM-Cellulose Chromatography --- p.84 / Chapter 2.4.2. --- Mono S® HR 5/5 and Mono Q® HR 5/5 --- p.85 / Chapter 2.4.3. --- Affi-gel Blue gel --- p.86 / Chapter 2.4.4. --- Superdex75 --- p.87 / Chapter 2.5. --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.88 / Chapter 2.6. --- Protein Concentration Determination --- p.89 / Chapter 2.7. --- Preparation of Rabbit Reticulocyte Lysate --- p.90 / Chapter 2.8. --- Determination of N-terminal Amino Acid Sequence --- p.91 / Chapter 2.9. --- Biological Activity Assays --- p.92 / Chapter 2.9.1. --- Assay for Antifungal Activity --- p.92 / Chapter 2.9.2. --- Assay for Cell-Free Translation Inhibitory Activity --- p.93 / Chapter 2.9.3. --- Assay of Cytotoxic Activity on Cancer Cell Lines --- p.94 / Chapter 2.9.4. --- Assay for HIV-1 Reverse Transcriptase (RT) Inhibitory Activity --- p.95 / Chapter 2.9.5. --- Assay of Mitogenic Activity --- p.97 / Chapter 2.9.6. --- Assay for N-Glycosidase Activity --- p.98 / Chapter 2.9.6.1. --- RNA Extraction --- p.98 / Chapter 2.9.6.2. --- Aniline Treatment --- p.99 / Chapter 2.9.6.3. --- Formaldehyde Gel Electrophoresis --- p.99 / Chapter 2.9.7. --- Assay of Ribonuclease Activity --- p.100 / Chapter 2.9.7.1. --- Assay for Yeast tRNA --- p.100 / Chapter 2.9.7.2. --- Activity toward Polyhomoribonucleotides --- p.100 / Chapter Chapter 3 --- Purification and Characterization of Pathogenesis-Related Proteins from their Respective Sources --- p.101 / Chapter 3.1. --- Purification and Characterization of Chitinase and Ribonuclease from the Roots of Panax notoginseng --- p.102 / Chapter 3.1.1. --- Introduction --- p.102 / Chapter 3.1.2. --- Results --- p.104 / Chapter 3.1.3. --- Purification --- p.107 / Chapter 3.1.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.108 / Chapter 3.1.3.2. --- Affinity Chromatography on Affi-gel Blue gel --- p.111 / Chapter 3.1.3.3. --- Cation-Exchange Chromatography on Mono S Column --- p.114 / Chapter 3.1.3.4. --- Gel Filtration on Superdex 75 Column --- p.115 / Chapter 3.1.4. --- Characterization of Chitinase --- p.117 / Chapter 3.1.4.1. --- N-terminal Amino Acid Sequence --- p.117 / Chapter 3.1.4.2. --- Assay for Antifungal Activity --- p.118 / Chapter 3.1.4.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.120 / Chapter 3.1.4.4. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.120 / Chapter 3.1.5. --- Characterization of Ribonuclease --- p.121 / Chapter 3.1.5.1. --- N-terminal Amino Acid Sequence --- p.121 / Chapter 3.1.5.2. --- Assay for Ribonuclease Activity --- p.122 / Chapter 3.1.5.3. --- Assay for Cell-Free Translation-inhibitory Activity --- p.125 / Chapter 3.1.5.4. --- Assay for Antifungal Activity --- p.125 / Chapter 3.1.5.5. --- Assay for Antiproliferative Activity --- p.126 / Chapter 3.1.6. --- Discussion --- p.127 / Chapter 3.2. --- Purification and Characterization of Ribosome-Inactivating Protein and Antifungal Protein from the mushroom Lyophyllum shimeji --- p.131 / Chapter 3.2.1. --- Introduction --- p.131 / Chapter 3.2.2. --- Results --- p.132 / Chapter 3.2.3. --- Purification --- p.134 / Chapter 3.2.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.135 / Chapter 3.2.3.2. --- Affinity Chromatography on Affi-gel Blue Gel --- p.137 / Chapter 3.2.3.3. --- Cation-Exchange Chromatography on Mono S --- p.140 / Chapter 3.2.4. --- Characterization of Ribosome-Inactivating Protein and Antifungal Protein from Lyophyllum shimeji --- p.142 / Chapter 3.2.4.1. --- N-terminal Amino Acid Sequence --- p.142 / Chapter 3.2.4.2. --- Assay for Antifungal Activity --- p.144 / Chapter 3.2.4.3. --- Assay for N-glycosidase Activity --- p.147 / Chapter 3.2.4.4. --- Assay for Mitogenic Activity --- p.147 / Chapter 3.2.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.148 / Chapter 3.2.5. --- Discussion --- p.150 / Chapter 3.3. --- Purification and Characterization of Ribosome-inactivating Protein from the Hypsizigus marmoreus --- p.153 / Chapter 3.3.1. --- Introduction --- p.153 / Chapter 3.3.2. --- Result --- p.154 / Chapter 3.3.3. --- Purification --- p.155 / Chapter 3.3.3.1. --- Cation-Exchange Chromatography on CM-Cellulose --- p.156 / Chapter 3.3.3.2. --- Affinity-Chromatography on Affi-gel Blue Gel --- p.158 / Chapter 3.3.3.3. --- Anion-Exchange Chromatography on Mono Q Column --- p.160 / Chapter 3.3.4. --- Characterization of Ribosome-inactivating Protein from Hypsizigus marmoreus --- p.162 / Chapter 3.3.4.1. --- N-terminal Amino Acid Sequence --- p.162 / Chapter 3.3.4.2. --- Assay for Cell-Free Translation-Inhibiting Activity --- p.163 / Chapter 3.3.4.3. --- Assay for Antifungal Activity --- p.164 / Chapter 3.3.4.4. --- Assay for N-glycosidase Activity --- p.166 / Chapter 3.3.4.5. --- Assay for HIV-1 Reverse Transcriptase Inhibitory Activity --- p.166 / Chapter 3.3.4.6. --- Assay for mitogenic Activity --- p.167 / Chapter 3.3.4.7. --- Assay for Antiproliferative Activity --- p.167 / Chapter 3.3.5. --- Discussion --- p.159 / Chapter Chapter 4 --- General Discussion --- p.170 / References --- p.172

Objetivos: Comparar os efeitos dos tratamentos com montelucaste e dexametasona no recrutamento eosinofílico e na avaliação de células positivas para eotaxina, RANTES, fibronectina, IGF-I e NF-B tanto no parênquima pulmonar distal, quanto nas vias aéreas de cobaias com inflamação alérgica crônica. Métodos: As cobaias receberam inalação com ovoalbumina (grupo OVA- 2 vezes semanais, durante 4 semanas, totalizando 7 inalações). Após a quarta inalação, as cobaias foram tratadas com montelucaste (grupo OVA-M: 10mg/Kg/VO/dia) ou dexametasona ( grupo OVA-D: 5mg/Kg/IP/dia). Após 72 horas da sétima inalação, as cobaias foram anestesiadas e os pulmões foram removidos e submetidos a avaliação histopatológica. Resultados: Os tratamentos com montelucaste e dexametasona reduziram o número de eosinófilos tanto no parênquima pulmonar distal quanto nas vias aéreas, quando comparados ao grupo OVA (p<0.05). No parênquima pulmonary distal, ambos os tratamentos foram efetivos na redução de células positivas para RANTES, NF-B e fibronectina, quando comparados ao grupo OVA (p<0.001). O tratamento com montelucaste mostrou melhor eficácia na redução de células positivas para eotaxina, quando comparado ao tratamento com dexametasona (p<0.001), por outro lado, o tratamento com dexametasona mostrou-se mais significativo na redução de células positivas para IGF-I, quando comparado ao tratamento com montelucaste (p<0.001). Nas vias aéreas, ambos os tratamentos foram efetivos na redução de células positivas para IGF-I, RANTES e fibronectina, quando comparados ao grupo OVA (p<0.05). O tratamento com dexametasona foi mais efetivo na redução de células positivas para eotaxina e NF-B, quando comparado ao tratamento com montelucaste (p<0.05). Conclusões: Neste modelo animal, ambos os tratamentos foram efetivos no controle da resposta inflamatória, tanto no parênquima pulmonar distal, quanto nas vias aéreas / Aims: Compare the effects of montelukast or dexamethasone treatments on eosinophilic recruitment, eotaxin, RANTES, fibronectin, IGF-I and NF-B positive cells of distal lung parenchyma and also in airway walls of guinea pigs (GP) with chronic allergic inflammation. Methods: GP were inhaled with ovalbumin (OVA group-2x/week/4weeks). After 4th inhalation, GP were treated with montelukast (M group: 10mg/Kg/PO/day) or dexamethasone (D group: 5mg/Kg/IP/day). After 72 hrs of 7th inhalation, GP were anesthetised, lungs were removed and submitted to histopathological evaluation. Results: Montelukast and dexamethasone treatments reduced the number of eosinophils both in airway wall as well as in distal lung parenchyma compared to OVA group (p<0.05). On distal parenchyma both montelukast and dexamethasone were effective in reducing RANTES, NF-B and fibronectin positive cells compared to OVA group (p<0.001). Montelukast was more effective in reducing the eotaxin positive cells on distal parenchyma compared to dexamethasone treatment (p<0.001), while there was a more expressive reduction of IGF-I positive cells in OVA-D group (p<0.001). On airway walls, both montelukast and dexamethasone were effective in reducing IGF-I, RANTES and fibronectin positive cells compared to OVA group (p<0.05). Dexamethasone was more effective reducing the number of eotaxin and NF-kB positive cells than Montelukast (p<0.05). Conclusions: In this animal model, both treatments were effective in modulating the eosinophilic response in distal lung parenchyma and in airway wall, contributing to a better control of the inflammatory response in distal lung parenchyma as well as in airway walls. Dexamethasone treatment induced a greater reduction of NF-B expression in airway walls which suggests one of the mechanisms that explains the higher efficacy of this therapeutic approach

Introdução: Os pacientes com bronquiolite obliterante (BO) costumam apresentar acometimento importante da função pulmonar que resulta em hipoxemia crônica e limitação da atividade física. Não há terapêutica de grande eficácia na BO, a resposta aos broncodilatadores costuma ser pobre, entretanto, não se conhece a resposta broncodilatadora a um agente anticolinérgico de longa ação como o brometo de tiotrópio (BT). Já se demonstrou eficácia e segurança do BT em adultos com doença pulmonar obstrutiva crônica (DPOC) com resposta broncodilatadora significativa e sustentada em dose única diária. Objetivo: Avaliar se existe melhora do grau de obstrução brônquica e do aprisionamento aéreo, através de medidas funcionais, após o uso de dose única de brometo de tiotrópio por via inalatória comparado a placebo em crianças e adolescentes com BO. Métodos: Ensaio clínico prospectivo, duplo cego, randomizado, placebocontrolado e cruzado em pacientes com BO estáveis na faixa etária de 6 a 16 anos. Espirometrias e pletismografias foram realizadas antes e aos 30, 60, 120, 180 minutos e 24 horas após a inalação de 18 mcg de tiotrópio ou placebo. Após 7-14 dias, os medicamentos foram invertidos e os procedimentos repetidos. As mudanças nos parâmetros de função pulmonar em cada momento foram comparadas com o basal através da análise de variância (ANOVA) e pós-teste de Tukey e as diferenças entre todos os momentos versus o basal nos grupos tiotrópio versus placebo foram comparados usando o teste de Friedman. Resultados: Trinta pacientes participaram do estudo (23 do sexo masculino, 7 do feminino; idade 10,9±2,8a ), com valores basais de função pulmonar (% do previsto) de CVF, VEF1, VEF1/CVF, FEF25-75%, CI, CPT, VR, VR/CPT, resistência das vias aéreas (raw) e condutância (sGaw) de 75±15, 48±14, 59±11, 22±11, 64±23, 120±19, 281±101, 49±13, 250±65 e 23±9, respectivamente. Diferenças estatisticamente significantes foram observadas após inalação do tiotrópio nos seguintes parâmetros comparados ao tempo basal: CVF em 60/120/180 min/24h, VEF1 em 30/60/120/180min, VEF1/CVF em 60/120/180min, FEF25- 75% em 60/120/180min, VR em 30/60/120/180min, CPT em 30/120/180min, VR/CPT em 30/60/120/180min, raw em 30/60/120/180min/24h e sGaw em 30/60/120/180min/24h. Na fase placebo não houve diferença estatisticamente significante em nenhum parâmetro funcional em nenhum momento após a administração. As diferenças entre as medidas funcionais comparando o grupo tiotrópio versus o grupo placebo foram estatisticamente significantes. Conclusões: O brometo de tiotrópio, após dose única, diminuiu agudamente o grau de obstrução e de aprisionamento aéreo por até 24 horas em crianças com BO. Estudos de longo prazo são necessários para se avaliar o papel do BT na terapêutica desses pacientes / Introduction: Patients with bronchiolitis obliterans (BO) usually have severe airflow obstruction that results in chronic hypoxemia and limitation of physical activity. There is no efficient therapy for BO, bronchodilator response is usually poor, however, the bronchodilator response to a long action anticholinergic agent such as tiotropium bromide (TB) is not known. Efficacy and safety of TB with one daily administration has already been shown in chronic obstructive pulmonary disease (COPD) with significant and sustained bronchodilator response. Objective: Verify through functional measurements whether the level of bronchial obstruction and air trapping was improved by the administration of a single dose of TB by inhalation when compared to placebo in children and adolescents with BO. Methods: A randomized, double blind, placebo-controlled, crossover, prospective study in stable BO patients, 6 to 16 years of age. Spirometry and plethysmography were performed before and at 30, 60, 120 and 180 minutes and 24 hours after inhalation of 18 mcg of tiotropium or a placebo. After 7-14 days, the drugs were inverted, and the procedures were repeated. The changes in lung function parameters at each time point were compared to the baseline by analysis of variance (ANOVA) and Tukey\'s post-test and the differences in all time points assessment versus baseline in tiotropium versus placebo groups were compared using the Friedman test. Results: Thirty patients were enrolled in the study (23 male, 7 female; age 10.9±2.8 y) with baseline lung function values (% predicted) of FVC, FEV1, FEV1/FVC, FEF25-75%, IC, TLC, RV, RV/TLC, airway resistance (raw) and conductance (sGaw) of 75±15, 48±14, 59±11, 22±11, 64±23, 120±19, 281±101, 49±13, 250±65 and 23±9, respectively. Statistically significant differences were observed after tiotropium inhalation in the following parameters compared to baseline: FVC at 60/120/180min/24h, FEV1 at 30/60/120/180min, FEV1/FVC at 60/120/180min, FEF25-75% at 60/120/180min, RV at 30/60/120/180min, TLC at 30/120/180min, RV/TLC at 30/60/120/180min, raw at 30/60/120/180min/24h and sGaw at 30/60/120/180min/24h. For the placebo group, no significant differences were observed in any lung function parameters at any time. The differences between the functional measurements comparing the tiotropium versus placebo groups were statistically significant. Conclusions: Tiotropium bromide, after a single dose, acutely decreased airway obstruction and air trapping for up to 24 hours in children with BO. Long-term studies are necessary to evaluate the role of BT in the management of these patients

植物合成多種發病機理相關蛋白以對抗病原體的侵襲。植物發病機理相關蛋白包括：核糖核酸酶；抗真菌蛋白；凝集素；胰蛋白酶抑制因子等。這些發病機理相關蛋白具有抗病毒，抗細菌，抗真菌，免疫調節及抗腫瘤等活性。從六種植物中提純了七個發病機理相關蛋白，包括三個凝集素，一個核糖核酸酶，兩個種抗真菌蛋白及一個胰蛋白酶抑制因子。 / 從西洋參須中提純了新的核糖核酸酶。核糖核酸酶分子量為26kDa，具有特异N末端氨基酸序列。此核糖核酸酶在 pH5 及 60℃ 條件下活性最高。它能抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 從粉色菜豆及日本大花豆中提純了兩種凝集素。它們由兩個分子量為32kDa的亞基構成雙倍體。他們的活性穩定于0-60℃及3-12 pH。粉色菜豆凝集素的特异性糖基為木糖，日本大花豆凝集素的特异性糖基為半乳糖。從太子參中提純的凝集素分子量為33kDa，其活性穩定于0-60℃及2-5 pH。 這三種凝集素都具有抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。 / 提純的胰蛋白酶抑制因子分子量為21kDa。具有高耐熱及耐酸鹼性并表現出抑制腫瘤細胞分裂及抑制人類後天免疫力缺乏症候群病毒逆轉錄酶活性。從豇豆中提純的抗真菌肽分子量為5447Da，具有類防御素N末端氨基酸序列。 / Plants produce a diversity of proteins with antipathogenic activities. These proteins comprise among others, (i) ribonucleases, (ii) antifungal proteins, (iii) lectins and (iv) trypsin inhibitor with antiviral, antifungal and anticancer activities. The aim of this project was to isolate antipathogenic plant proteins including a ribonuclease from American ginseng branch roots, a trypsin inhibitor from rambutan seeds, defensin-like antifungal peptides from borlotti beans and king pole beans, and lectins from borlotti beans, Japanese large pinto beans and Pseudostellaria heterophylla. / The isolated 26-kDa ginseng branch root ribonuclease was monomeric with a novel N-terminal amino acid sequence. It exhibited maximal robonucleolytic activity toward yeast tRNA at pH 5 and 60℃. It inhibited proliferation of MCF7 human breast cancer cells and HepG2 human hepatoma cells. It also inhibited the activity of HIV-1 reverse transcriptase. / Both borlotti bean lectin and Japanese large pinto bean lectin were dimeric with a subunit molecular mass of 32-kDa. They were stable from 0℃ to 60℃ and from pH 3 to pH 12. Borlotti bean lectin was xylose-specific whereas Japanse large pinto bean lectin was galactose-specific. The 33-kDa Pseudostellaria heterophylla lectin could not be inhibited by the simple sugars tested. It was stable from 0℃ to 60℃ and from pH 2 to 5. All three isolated lectins suppressed proliferation of MCF7 and HepG2 cells and inhibited HIV-1 reverse transcriptase. / The isolated 21-kDa rambutan trypsin inhibitor has relatively high pH stability and thermostability, and exhibited HIV-1 reverse transcriptase inhibitory activity and antiproliferative activity toward a variety of tumor cells. The isolated 5447-Da king pole bean defensin-like peptide inhibited fungal growth. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhao, Yuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 202-222). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Declaration --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of Tables --- p.x / List of Figures --- p.xii / Chapter Chapter 1 --- Overview of Plant Defense-related Protein --- p.1 / Chapter 1.1 --- Overview of Lectins and hemagglutinins --- p.4 / Chapter 1.1.1 --- History and definition of lectins and hemagglutinins --- p.4 / Chapter 1.1.2 --- Occurrence and distribution of plant lectins --- p.6 / Chapter 1.1.3 --- Classification of lectins --- p.7 / Chapter 1.1.3.1 --- Classification of lectins on the basis of overall structure of lectin subunits --- p.7 / Chapter 1.1.3.2 --- Classification of lectins based on binding specificty to carbohydrates --- p.11 / Chapter 1.1.3.3 --- Classification of lectins according to families --- p.12 / Chapter 1.1.3.3.1 --- Legume lectins --- p.12 / Chapter 1.1.3.3.2 --- Monocot mannose-binding lectins --- p.13 / Chapter 1.1.3.3.3 --- Other lectins --- p.14 / Chapter 1.1.4 --- Defensive role of plant lectins --- p.15 / Chapter 1.1.5 --- Applications of plant lectins --- p.18 / Chapter 1.1.5.1 --- The antibacterial activity --- p.18 / Chapter 1.1.5.2 --- Anti-insect activity --- p.19 / Chapter 1.1.5.3 --- Antifungal activity --- p.21 / Chapter 1.1.5.4 --- The antiviral activity --- p.22 / Chapter 1.1.5.5 --- Lectin affinity chromatography --- p.23 / Chapter 1.1.5.6 --- Lectin microarray --- p.23 / Chapter 1.2 --- Overview of Ribonucleases --- p.26 / Chapter 1.2.1 --- History and definition of Ribonucleases --- p.26 / Chapter 1.2.2 --- Classification of Ribonucleases --- p.27 / Chapter 1.2.2.1 --- T1 Ribonucleases family --- p.27 / Chapter 1.2.2.2 --- RNase T2 family --- p.28 / Chapter 1.2.3 --- Biological activities of plant ribonucleases --- p.28 / Chapter 1.2.3.1 --- Phosphate remobilization --- p.28 / Chapter 1.2.3.2 --- Senescence --- p.29 / Chapter 1.2.3.3 --- Programmed cell death --- p.30 / Chapter 1.2.3.4 --- Plant defense --- p.31 / Chapter 1.2.3.5 --- RNA processing and decay --- p.32 / Chapter 1.2.3.6 --- Antitumor activities --- p.33 / Chapter 1.3 --- Other plant pathogen-related proteins --- p.34 / Chapter 1.3.1 --- Overview of chitinase --- p.34 / Chapter 1.3.1.1 --- Classification of chitinases --- p.35 / Chapter 1.3.1.2 --- Biological properties of chitinases --- p.38 / Chapter 1.3.2 --- Overview of plant ribosome-inactivating proteins (RIPs) --- p.41 / Chapter 1.3.2.1 --- Classification of RIPs --- p.42 / Chapter 1.3.2.2 --- Roles of RIPs in plants --- p.44 / Chapter 1.3.2.3 --- Possible application of RIPs --- p.46 / Chapter 1.3.3 --- Overview of thaumatin-like proteins (TLPs) --- p.50 / Chapter 1.3.3.1 --- Occurrence of TLPs --- p.51 / Chapter 1.3.3.2 --- Biological properties of TLPs --- p.52 / Chapter 1.4 --- Aim of this study --- p.54 / Chapter Chapter 2 --- Isolation of a lectin and an antifungal protein from Phaseolus vulgaris cv. Borlotti beans / Chapter 2.1 --- Introduction --- p.55 / Chapter 2.2 --- Materials and Methods --- p.55 / Chapter 2.3 --- Results --- p.64 / Chapter 2.4 --- Discussion --- p.79 / Chapter Chapter 3 --- Isolation of a lectin from Pinto beans (Phaseolus vulgaris pinto bean) / Chapter 3.1 --- Introduction --- p.82 / Chapter 3.2 --- Materials and Methods --- p.83 / Chapter 3.3 --- Results --- p.87 / Chapter 3.4 --- Discussion --- p.103 / Chapter Chapter 4 --- Isolation of a lectin from Pseudostellaria hetorophylla roots / Chapter 4.1 --- Introduction --- p.105 / Chapter 4.2 --- Materials and Methods --- p.107 / Chapter 4.3 --- Results --- p.110 / Chapter 4.4 --- Discussion --- p.122 / Chapter Chapter 5 --- Isolation of a ribonuclease from branch roots of American ginseng (Panax quinquefolium) / Chapter 5.1 --- Introduction --- p.124 / Chapter 5.2 --- Materials and Methods --- p.126 / Chapter 5.3 --- Results --- p.129 / Chapter 5.4 --- Discussion --- p.142 / Chapter Chapter 6 --- Isolation of a trypsin inhibitor in rambutan (Nephelium lappaceum L) seeds / Chapter 6.1 --- Introduction --- p.144 / Chapter 6.2 --- Materials and Methods --- p.147 / Chapter 6.3 --- Results --- p.152 / Chapter 6.4 --- Discussion --- p.163 / Chapter Chapter 7 --- Isoation of a defensin-like antifungal peptide from Phaseolus vulgaris cv. 'King Pole Bean' / Chapter 7.1 --- Introduction --- p.168 / Chapter 7.2 --- Materials and Methods --- p.170 / Chapter 7.3 --- Results --- p.173 / Chapter 7.4 --- Discussion --- p.181 / Chapter Chapter 8 --- Overall discussion --- p.183 / References --- p.186

Fatal and non-fatal opioid overdoses resulting from opioid induced respiratory depression are a significant problem throughout the world. Whilst the opioid receptor antagonist, naloxone hydrochloride, can effectively reverse opioid overdoses, its use is limited because of the adverse effects it produces. These include severe withdrawal and the reversal of analgesia produced by opioid receptor agonists. In this project, the peripherally acting opioid receptor antagonist, naloxone methiodide, was investigated for its potential to reverse opioid induced respiratory depression without altering centrally mediated effects, such as withdrawal. In the publications presented in this thesis, naloxone hydrochloride and naloxone methiodide were shown to effectively reverse the decreases in respiratory rate produced by the administration of morphine, methadone and heroin in mice. Naloxone hydrochloride and naloxone methiodide also reversed the analgesia produced by these opioid receptor agonist treatments, but only naloxone hydrochloride induced significant withdrawal. The doses of naloxone methiodide required to produce the effects described above were higher than the naloxone hydrochloride doses required. Radioligand binding techniques indicated that this was due to a difference in the affinity of naloxone hydrochloride and naloxone methiodide for µ, δ and κ opioid receptor binding sites. Radioligand binding techniques were also used to confirm that naloxone methiodide, or its metabolites, could not readily cross the blood brain barrier. Therefore, the effects of naloxone methiodide appear to be mediated outside the central nervous system. The final publication aimed to extend our knowledge of opioid induced respiratory depression by utilising new radiotelemetry technology to test the efficacy of naloxone methiodide in rats subjected to a chronic opioid administration regime. This experiment showed that circadian rhythm plays a role in the development of tolerance to the cardiorespiratory effects of continuous and chronic methadone administration, and that naloxone hydrochloride and naloxone methiodide treatment can increase respiratory rate and heart rate after this methadone administration. Therefore, naloxone methiodide can effectively antagonise the peripheral effects produced by opioid receptor agonists. Peripherally acting opioid receptor antagonists should be developed in the future to prevent or treat the adverse effects of opioid receptor agonists. / Thesis (Ph.D.)--Department of Clinical and Experimental Pharmacology, 2004.

Graduation date: 2006 / We hypothesized that calpain activity is elevated in response to muscle damage. To test this hypothesis, we examined the degradation of α-fodrin into its 150 and 145 kDa fragments following either 20 eccentric or isometric contractions. In addition, experiments were performed in the presence or absence of E-64-d, a calpain inhibitor. Both EDL and SOL muscles displayed significant differences (p<0.003 and p<0.002 respectively) between the raw and normalized 150 and 145 kDa α-fodrin fragments of the DMSO + E-64-d compared to the other bath treatments. Based on our model of exercise-induced muscle damage, we expected to see greater levels of 150 and 145 kDa α-fodrin fragments in those muscles that performed the eccentric protocol. However, there was no evidence that eccentric muscle damage increased the levels of 150 and 145 kDa α-fodrin fragments over the levels observed in the isometric trials. These findings suggest that the magnitude of damage was insufficient to activate calpains.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 85-105).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains xii, 263 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 233-242).

Tumour cell dissemination is a major issue with the treatment of cancer, thus new therapeutic strategies which can control this process are needed. Antagonism of integrins highly expressed in tumours is one potential strategy. The integrins are transmembrane glycoprotein adhesive receptors. Two of the integrins, αVβ3 and αIIbβ3, are highly expressed in a number of tumours and induce bi-directional signalling through their interaction with extracellular matrix proteins, and growth factor receptors. Through this signalling they play an important role in a number of cellular processes that are involved in tumour dissemination such as tumour growth, migration, invasion, metastasis and angiogenesis. Dual αIIbβ3 and αVβ3 integrin antagonism will have a direct effect on β3-expressing tumour cells that leads to the inhibition of cell migration and dissemination. Furthermore, through targeting tumour cell interaction with endothelial cells and platelets, this will also lead to inhibition of angiogenesis and metastasis. The aim of this project was to characterise the expression of αVβ3 and αIIbβ3 integrin in a panel of tumour cell lines and in human tumour xenograft samples, and to develop and utilise cell-based models to investigate potential novel β3 antagonists. The expression of αV and β3 subunits was detected in xenograft tissue using immunoblotting techniques. A panel of cell lines of different tumour types including melanoma, prostate, breast, colon and non small cell lung carcinoma was then characterised for αVβ3 and αIIbβ3 integrin expression using immunoblotting and immunocytochemistry. Melanoma cell lines demonstrated the strongest αVβ3 expression. No αIIbβ3 integrin expression was seen in any of the cell lines evaluated. A selection of cell lines with varying αVβ3 expression were then used to develop a functional test for cell migration, the scratch wound healing assay. Migration of tumour cells that expressed αVβ3 integrin was inhibited by the known β3 antagonists, cRGDfV peptide and LM609 antibody. A panel of 12 potential novel β3 integrin antagonists was screened for cytotoxicity and activity in the validated scratch assay. ICT9055 was the most effective antagonist in inhibition of M14 cell migration as determined by the scratch assay, with an IC₅₀ of < 0.1 μM. Therefore the work presented in this thesis has established models and tools for evaluating potential novel β3 integrin antagonists, and identified a promising molecule to progress for further preclinical evaluation.