by Yung Mei Hing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 130-135. / ACKNOWLEDEMENTS --- p.i / ABSTRACT --- p.ii / CONTENTS --- p.iii / ABBREVIATIONS --- p.viii / Chapter CHAPTER ONE --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Chemistry and Structure of Trichosanthin --- p.1 / Chapter 1.1.1 --- Chemistry --- p.1 / Chapter 1.1.2 --- Primary Structure --- p.2 / Chapter 1.1.3 --- Three-dimensional Structure --- p.4 / Chapter 1.2 --- Biological Activities of Trichosanthin --- p.6 / Chapter 1.2.1 --- Abortifacient Properties --- p.6 / Chapter 1.2.1A --- Termination of Mid-term Gestation --- p.6 / Chapter 1.2.1B --- Inhibition of Early Pregnancy --- p.12 / Chapter 1.2.2 --- Immunological Properties --- p.13 / Chapter 1.2.2A --- Antigenicity and Allergenicity --- p.13 / Chapter 1.2.2B --- Immunosuppressive Effects --- p.14 / Chapter 1.2.3 --- Anti-tumour Activity --- p.15 / Chapter 1.2.4 --- Ribosome-inactivating Activity --- p.16 / Chapter 1.2.5 --- Human Immunodeficiency Virus (HIV) Inhibitory Activity --- p.20 / Chapter 1.3 --- Objectives and Strategy of Cloning the cDNA of Trichosanthin --- p.21 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.24 / Chapter 2.1 --- General Techniques --- p.24 / Chapter 2.1.1 --- Extraction with Phenol --- p.24 / Chapter 2.1.2 --- Ethanol Precipitation --- p.24 / Chapter 2.1.3 --- Spectrophotometry --- p.25 / Chapter 2.1.4 --- Restriction Digestion of DNA --- p.25 / Chapter 2.1.5 --- End-labelling DNA with Recessed 3'-ends --- p.25 / Chapter 2.1.6 --- Agarose Gel Electrophoresis of DNA --- p.26 / Chapter 2.1.7 --- Elution of DNA from Agarose Gel --- p.26 / Chapter 2.1.8 --- Minipreparation of Bacteriophage A DNA from Plate Lysates --- p.27 / Chapter 2.1.9 --- Minipreparation of Plasmid DNA --- p.28 / Chapter 2.1.10 --- Large-scale Preparation of Plasmid DNA --- p.29 / Chapter 2.1.10A --- By Equilibrium Centrifugation in CsCl-Ethidium Bromide Gradient --- p.29 / Chapter 2.1.10B --- By Using QIAGEN-pack 100 Cartridge --- p.31 / Chapter 2.1.11 --- Preparation and Transformation of Competent Escherichia coli --- p.32 / Chapter 2.1.12 --- Preparation of Nucleic Acid Probes --- p.34 / Chapter 2.1.12A --- By Nick Translation --- p.34 / Chapter 2.1.12B --- By Random-primed Labelling --- p.34 / Chapter 2.1.13 --- Sephadex G-50 Spun-column Chromatography --- p.35 / Chapter 2.1.14 --- Monitoring the Progress of Probe Labelling Reactions --- p.37 / Chapter 2.1.15 --- Liquid Scintillation Counting --- p.37 / Chapter 2.1.16 --- Southern and Northern Hybridizations --- p.38 / Chapter 2.1.17 --- Autoradiography --- p.41 / Chapter 2.2 --- Techniques for Constructing a Complementary DNA (cDNA) Library --- p.42 / Chapter 2.2.1 --- Extraction and Purification of Plant RNA --- p.42 / Chapter 2.2.2 --- Electrophoresis of RNA in Agarose Gel Containing Formaldehyde --- p.44 / Chapter 2.2.3 --- Ohgo(dT)-cellulose Column Chromatography --- p.46 / Chapter 2.2.4 --- In Vitro Translation in Rabbit Reticulocyte Lysate --- p.47 / Chapter 2.2.5 --- SDS-polyacrylamide Gel Electrophoresis (SDS-PAGE) of In Vitro Translation Products --- p.48 / Chapter 2.2.6 --- cDNA Synthesis --- p.50 / Chapter 2.2.6A --- First Strand Synthesis --- p.50 / Chapter 2.2.6B --- Second Strand Synthesis --- p.51 / Chapter 2.2.6C --- Purification of the Double-stranded cDNA (ds cDNA) --- p.51 / Chapter 2.2.7 --- Methylation of the cDNA with EcoRI Methylase --- p.52 / Chapter 2.2.8 --- Addition of EcoRl Cohesive Termini onto the cDNA --- p.52 / Chapter 2.2.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA --- p.53 / Chapter 2.2.10 --- Ligation of the cDNA with EcoRI-digested λgt10 --- p.55 / Chapter 2.2.11 --- In Vitro Packaging --- p.56 / Chapter 2.2.12 --- Titration of the λgt10 Library V --- p.56 / Chapter 2.3 --- Screening the cDNA Library in λgt10 with a Nucleic Acid Probe --- p.57 / Chapter 2.4 --- Subcloning DNA Fragments in pUC18 --- p.59 / Chapter 2.4.1 --- Dephosphorylation of Linearized pUC18 with Protruding 5' Termini --- p.59 / Chapter 2.4.2 --- Ligation of Foreign DNA with Linearized pUC18 with Cohesive Termini --- p.60 / Chapter 2.5 --- DNA Sequencing on Double-stranded Templates --- p.60 / Chapter 2.5.1 --- DNA Sequencing Reaction --- p.61 / Chapter 2.5.1A --- Alkaline Denaturation of Double-stranded Plasmid Template --- p.61 / Chapter 2.5.1B --- Labelling Reaction and Termination Reactions --- p.61 / Chapter 2.5.2 --- DNA Sequencing Electrophoresis --- p.62 / Chapter CHAPTER THREE --- CONSTRUCTION AND CLONAL SCREENING OF cDNA LIBRARY FROM ROOT TUBERS OF TRICHOSANTHES KIRILOWII MAXIMOWICZ --- p.65 / Chapter 3.1 --- Introduction --- p.65 / Chapter 3.2 --- Isolation of Total RNA from Root Tubers --- p.69 / Chapter 3.3 --- Purification of Poly(A)+ RNA from Total RNA --- p.70 / Chapter 3.4 --- Northern Blot Analysis of the Poly(A)+ RNA --- p.72 / Chapter 3.5 --- In Vitro Translation of the Poly(A)+ RNA --- p.75 / Chapter 3.6 --- cDNA Synthesis --- p.78 / Chapter 3.7 --- Southern Blot Analysis of the cDNA --- p.81 / Chapter 3.8 --- Addition of EcoRI Cohesive Termini to the cDNA --- p.81 / Chapter 3.9 --- Removal of Excess EcoRI Linkers and Size Fractionation of the cDNA vi --- p.84 / Chapter 3.10 --- Ligation of the Size Fractionated cDNA with EcoRI-digested λgt10 and In Vito Packaging of the A Hybrids --- p.85 / Chapter 3.11 --- Analysis of cDNA Inserts --- p.85 / Chapter 3.12 --- Clonal Screening of the cDNA Library --- p.88 / Chapter 3.13 --- Characterization of Positive Clones --- p.88 / Chapter 3.14 --- Subcloning Positive Inserts into pUC18 --- p.92 / Chapter 3.15 --- Discussion --- p.93 / Chapter CHAPTER FOUR --- DNA SEQUENCING OF POSITIVE CLONES --- p.99 / Chapter 4.1 --- Introduction --- p.99 / Chapter 4.2 --- Sequencing Strategies --- p.104 / Chapter 4.2.1 --- pTCS48210 Sequencing --- p.m / Chapter 4.2.2 --- pTCS5021 Sequencing --- p.107 / Chapter 4.3 --- Results and Discussion --- p.108 / Chapter 4.3.1 --- Nucleotide Sequence and Deduced Amino Acid Sequence --- p.108 / Chapter 4.3.2 --- Secondary Structure Predicted from the Amino Acid Sequence Encoded by the Trichosanthin cDNA --- p.116 / APPENDIX A --- p.124 / APPENDIX B --- p.126 / APPENDIX C --- p.129 / REFERENCES --- p.130

"July 2004." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (p. 166-177) / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Previous research showed that the C-terminal tail of TCS can be deleted to generate a mini-TCS (C7-TCS) with antigenicity. The second topic of my study is to resolve the role of the C-terminal of TCS. Structure of C7-TCS showed that deletion of the C-terminal tail destabilizes the protein structure and makes Trp192 more solvent exposed. The relationship between the C-terminal tail and Trp192 was determined by mutating Trp192 to Phe in wild-type TCS and C7-TCS, generating W192F-TCS and W192F-C7-TCS. The crystal structure of C7-TCS, [W192F]-TCS and [W192F]-C7-TCS were determined and compared. Trp192 was identified as an important residue in stabilizing the conformation of TCS. Besides, the accumulative effect of Trp192 and the C-terminal tail is significant on the ribosome-inactivating activity. By comparing the structures, it was found that, the hydrogen bond formed by amino acids 240 and 35 seems to be essential for the structure and amino acid 240 should be a critical residue for the connection of the N-terminal and C-terminal domains in trichosanthin. / Ribosome-inactivating activity is the most important activity of TCS and RIPs. Therefore, the third topic of my study is to find the important of interaction between TCS and ribosomal proteins. Two ribosomal proteins, P0 and P1, have been identified previously to interact with TCS. By yeast two-hybrid screening, three cut of ten charge residues in TCS were identified to be the interaction sites between TCS and ribosomal protein P0. The interaction region was located on the surface of TCS near the entrance to the active pocket. The interaction with P0 was shown to be carried out by electrostatic interaction between the positively charge residues of TCS. However, the mutation of all the concerned residues in TCS gave only a mild reduction in inhibiting the protein synthesis of an in vitro reticulocyte translation system, showing that the interaction between TCS and P0 only plays a minor role in the ribosomal inactivating activity of TCS. / The first topic of my research is to find the role of Glu-85. The structure of [E85Q]-TCS and AMP complex was obtained. It is deduced that there are two sites for substrate binding in TCS, one is for recognition and another ion hydrolysis. The structure also indicated that protonation of substrate adenine is carried out by a water molecule in the active pocket of TCS during its N-glycosidase action. / Trichosanthin (TCS) is a Chinese medicinal protein isolated froth the root tuber of Trichosanthes kirilowi Maximowicz. It is a 27kDa protein with multiple pharmacological properties, including abortifacient, anti-tumor and anti-human immunodeficiency virus (HIV). It is believed that the pharmacological properties of TCS are related to ribosome-inactivation, by breaking, the specific glycosidic bond of adenine 4324 from the 28S rRNA. / Too Hiu Mei. / "February 2006." / Advisers: Pang-Chui Shaw; Kam-Bo Wong. / Source: Dissertation Abstracts International, Volume: 67-11, Section: B, page: 6213. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 164-175). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Tang Sze-Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 104-118). / Abstracts in English and Chinese. / Abstract --- p.i / Abstract (Chinese) --- p.iii / Acknownledgement --- p.iv / List of Abbreviations --- p.v / List of Tables --- p.vii / List of Figures --- p.viii / Table of Contents --- p.xi / Chapter Chapter 1 - --- Introduction / Chapter 1.1 --- Trichosanthes spp --- p.1 / Chapter 1.1.1 --- Use of Trichosanthes --- p.2 / Chapter 1.1.2 --- Trichosanthin --- p.2 / Chapter 1.1.3 --- Karasurin --- p.5 / Chapter 1.1.4 --- Ribosome Inactivating Proteins --- p.6 / Chapter 1.1.5 --- Immunosuppresion --- p.7 / Chapter 1.1.6 --- Anti-Cancer Activity --- p.8 / Chapter 1.1.7 --- Miscellaneous Uses --- p.8 / Chapter 1.2 --- Cancer --- p.9 / Chapter 1.2.1 --- Oncogenes --- p.10 / Chapter 1.2.2 --- Tumor-Suppressor Genes --- p.11 / Chapter 1.2.3 --- Stability Genes --- p.12 / Chapter 1.2.4 --- Types of Cancer --- p.13 / Chapter 1.2.5 --- Cancer Therapy --- p.13 / Chapter 1.2.6 --- Apoptosis --- p.14 / Chapter 1.3 --- Chronic Myelogenous Leukemia (CML) --- p.17 / Chapter 1.3.1 --- Philadelphia Chromosome and BCR-ABL gene --- p.18 / Chapter 1.3.2 --- Treatment of CML --- p.20 / Chapter 1.4 --- Dendritic Differentiation of LC976 on K-562 --- p.20 / Chapter 1.4.1 --- Dendritic Cells --- p.21 / Chapter 1.4.2 --- Cancer Vaccine Development of Leukemia --- p.22 / Chapter 1.4.3 --- Dendritic differentiation of K-562 cells --- p.23 / Chapter 1.5 --- Perspective of the Project --- p.23 / Chapter Chapter 2 - --- Materials and Methods / Chapter 2.1 --- Materials / Chapter 2.1.1 --- Chemicals and Reagents --- p.25 / Chapter 2.1.2 --- Bioassay Kits --- p.26 / Chapter 2.1.3 --- Human Cell Lines --- p.26 / Chapter 2.1.4 --- Lab Wares and Equipments --- p.28 / Chapter 2.2 --- Extraction of LC9 --- p.76 / Chapter 2.2.1 --- Chemical Properties of the Lead Compound --- p.28 / Chapter 2.2.2 --- Crude Extraction of Trichosanthes sp --- p.29 / Chapter 2.2.3 --- Purification by Reversed-Phase Column --- p.29 / Chapter 2.2.4 --- Lyophilization and Preparation of LC976 --- p.31 / Chapter 2.3 --- Anti-Proliferation Effect of LC976 on Human Cell Lines / Chapter 2.3.1 --- Maintenance of Cell Lines --- p.32 / Chapter 2.3.2 --- MTT Assay --- p.32 / Chapter 2.3.3 --- BrdU Cell Proliferation ELISA --- p.34 / Chapter 2.4 --- Apoptosis Induction on K-5 --- p.62 / Chapter 2.4.1 --- PI Staining --- p.35 / Chapter 2.4.2 --- Annexin V-FITC FACS Analysis --- p.36 / Chapter 2.4.3 --- Caspase Activation --- p.37 / Chapter 2.5 --- Effect on Normal Human Lymphocytes / Chapter 2.5.1 --- Preparation of Human Normal Lymphocytes --- p.38 / Chapter 2.5.2 --- MTT Cell Viability Assay --- p.38 / Chapter 2.5.3 --- PI Staining --- p.39 / Chapter 2.5.4 --- Annexin V-FITC FACS Analysis --- p.39 / Chapter Chapter 3 - --- Results / Chapter 3.1 --- Extraction of LC976 --- p.40 / Chapter 3.2 --- LC976 Inhibited Proliferation of Human Cell Lines / Chapter 3.2.1 --- MTT Assay --- p.41 / Chapter 3.2.2 --- BrdU Cell Proliferation ELISA --- p.52 / Chapter 3.3 --- LC976 Induced Apoptosis in K-562 Cells / Chapter 3.3.1 --- PI Staining --- p.63 / Chapter 3.3.2 --- Annexin V-FITC FACS Analysis --- p.70 / Chapter 3.3.3 --- Caspase Activation --- p.73 / Chapter 3.4 --- Effect on Normal Human Lymphocytes / Chapter 3.4.1 --- MTT Cell Viability Assay --- p.76 / Chapter 3.4.2 --- PI Staining --- p.78 / Chapter 3.4.3 --- Annexin V-FITC FACS Analysis --- p.82 / Chapter Chapter 4 - --- Discussion / Chapter 4.1 --- Extraction of LC976 --- p.85 / Chapter 4.2 --- LC976 Inhibited Proliferation of Human Cell Lines / Chapter 4.2.1 --- MTT Assay --- p.86 / Chapter 4.2.2 --- BrdU Cell Proliferation ELISA --- p.88 / Chapter 4.3 --- LC976 induced Apoptosis in K-562 Cells / Chapter 4.3.1 --- PI Staining --- p.90 / Chapter 4.3.2 --- Annexin V-FITC Analysis --- p.95 / Chapter 4.3.3 --- Caspase Activation --- p.96 / Chapter 4.4 --- Effect on Normal Human Lymphocytes / Chapter 4.4.1 --- MTT Cell Viability Assay --- p.98 / Chapter 4.4.2 --- PI Staining --- p.99 / Chapter 4.4.3 --- Annexin V-FITC FACS Analysis --- p.100 / Chapter 4.5 --- Conclusion --- p.103 / Reference --- p.104

Background. Traditional plant use was studied in Lao PDR. Research focused on medicinal plant use by the Brou, Saek and Kry ethnic groups, traditional plant repellents against parasitic arthropods and leeches, and the phylogeny and biogeography of the medicinally-important snake gourd genus (Trichosanthes, Cucurbitaceae).  Methods. The ethnobiology research used a combination of structured interviews, village surveys, botanical collecting, hydro-distillation, GC-MS analysis, literature studies, and laboratory experiments. The plant systematics research used a combination of morphological studies, molecular biology laboratory work, and phylogenetic, dating and biogeographical analysis.  Results. Informants reported the use of close to 100 species to repel arthropods and leeches, many of which have constituents with documented efficacy.  Brou, Saek and Kry informants use over 75 plant species for women’s healthcare, mainly during the postpartum period for steam sauna, steam bath, hotbed, mother roasting, medicinal decoctions and infusions, and postpartum diet.  A molecular phylogeny of Trichosanthes and Gymnopetalum using a broad sampling of ~60% of their species and 4756 nucleotides of nuclear and plastid DNA shows that Gymnopetalum is nested within Trichosanthes. Fossil-calibrated Bayesian molecular dating of the Trichosanthes phylogeny reveals an early Oligocene origin of the genus, and many of the extant sections originating and diversifying during the Miocene. Biogeographical analysis shows a likely East or South Asian origin of Trichosanthes, with lineages diversifying and spreading throughout Australasia from the early Pliocene to the Pleistocene.  Discussion. Traditional plant use in Lao PDR is common and widespread. The presence among the repellent species of economical alternatives to costly synthetic repellents is tenable, and the subject of ongoing studies.  Postpartum traditions and medicinal plant use are essential parts of childbirth and postpartum recovery in these ethnic groups, and many other groups in Lao PDR. Efforts to improve maternal healthcare and reduce maternal and infant mortality need to integrate these traditions with modern notions of healthcare to achieve wider adoption. Documenting all possible uses of commonly used medicinal plant species shows that similarity in use between these ethnic groups is relatively low considering that they share, and have shared for many generations, the same environment and resources. A lack of effective cures leads to a process of continuous innovation, where effective cures are shared between cultures, but remedies of only cultural importance, or those under evaluation are culture-specific.  The Trichosanthes phylogeny implies the merging of Gymnopetalum into Trichosanthes, and this is done using available names or new combinations. A synopsis of Trichosanthes, the new combinations, and a revision of the species in Australia, are made and presented.  Conclusions. Traditional plant use is widespread in Lao PDR, and of significance to many people as a source of primary healthcare and inexpensive repellents. The important medicinal plant genus Trichosanthes includes Gymnopetalum, and has a complex biogeographic history with multiple colonization events of Australasia.