by Wei-Zai Shen. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 170-193) / 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.

"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.

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Huang Hai. / "May 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. (158-183). / 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.

Chu Lai On. / Thesis submitted in: July 2004. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 152-172). / Abstracts in English and Chinese. / Acknowledgements --- p.2 / Abstract --- p.3 / Table of Content --- p.7 / Abbreviations --- p.12 / Naming system for mutant proteins --- p.13 / Abbreviations for amino acid --- p.14 / Chapter Chapter 1 --- Introduction --- p.15 / Chapter 1.1 --- Structure-function relationship of trichosanthin --- p.18 / Chapter 1.2 --- Properties of acidic ribosomal P-proteins --- p.21 / Chapter 1.3 --- Interaction among P-proteins and trichosanthin --- p.25 / Chapter 1.4 --- Properties of eukaryotic elongation factor 2 and interaction with P-proteins --- p.26 / Chapter 1.5 --- "Objectives and strategy of studying the interaction among trichosanthin, P-proteins and eukaryotic elongation 2" --- p.30 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- General techniques --- p.33 / Chapter 2.1.1 --- Preparation and transformation of Escherichia coli competent cells --- p.33 / Chapter 2.1.2 --- Minipreparation of plasmid DNA using Wizard Plus SV Minipreps DNA purification kit from Promega --- p.34 / Chapter 2.1.3 --- Agarose gel electrophoresis of DNA --- p.36 / Chapter 2.1.4 --- Purification of DNA from agarose gel using Wizard SV Gel and PCR Clean-Up System from Promega --- p.36 / Chapter 2.1.5 --- Polymerase Chain Reaction (PCR) --- p.37 / Chapter 2.1.5.1 --- Basic Protocol --- p.37 / Chapter 2.1.5.2 --- Generation of P2 truncation mutants --- p.38 / Chapter 2.1.5.3 --- Generation of TCS mutants --- p.39 / Chapter 2.1.6 --- Restriction digestion of DNA --- p.41 / Chapter 2.1.7 --- Ligation of DNA fragments --- p.41 / Chapter 2.1.8 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.42 / Chapter 2.1.9 --- Staining of protein in polyacrylamide gel --- p.45 / Chapter 2.2 --- Expression and purification of recombinant proteins --- p.46 / Chapter 2.2.1 --- "Bacterial culture, harvesting and lysis" --- p.46 / Chapter 2.2.2 --- Purification of recombinant TCS and mutants --- p.47 / Chapter 2.2.3 --- Purification of acidic ribosomal protein P2 and mutants --- p.48 / Chapter 2.2.4 --- Purification of MBP-fusion proteins --- p.50 / Chapter 2.3 --- Purification of eEF2 from rat livers --- p.51 / Chapter 2.4 --- In vitro binding assay by NHS-activated Sepharose resin --- p.53 / Chapter 2.4.1 --- Coupling of protein sample to NHS-activated Sepharose resin --- p.53 / Chapter 2.4.2 --- In vitro binding of protein sample to coupled NHS-activated resin --- p.54 / Chapter 2.5 --- Ribosome-inactivated activity assay using rabbit reticulocyte lysate in vitro translation system --- p.55 / Chapter 2.6 --- Circular dichroism (CD)spectrometry --- p.57 / Chapter 2.7 --- Isothermal titration calorimetry (ITC) experiment --- p.57 / Chapter 2.8 --- Surface plasmon resonance (SPR) experiment --- p.58 / Chapter 2.8.1 --- Immobilization of P2 onto aminosilane cuvette --- p.58 / Chapter 2.8.2 --- Interaction between eEF2 and immobilized P2 --- p.60 / Chapter 2.9 --- Preparation of Anti-P antibody --- p.61 / Chapter 2.10 --- Western blotting of protein --- p.62 / Chapter 2.11 --- Reagents and buffer --- p.64 / Chapter 2.11.1 --- Reagents for competent cell preparation --- p.64 / Chapter 2.11.2 --- Nucleic acids electrophoresis buffer --- p.65 / Chapter 2.11.3 --- Media for bacterial culture --- p.66 / Chapter 2.11.4 --- Buffers for TCS purification --- p.67 / Chapter 2.11.5 --- Buffers for eEF2 purification --- p.68 / Chapter 2.11.6 --- Reagents for SDS-PAGE --- p.68 / Chapter 2.11.7 --- Reagents and buffers for Western blot --- p.70 / Chapter 2.11.8 --- Reagents and buffers for coupling sample proteins to NHS-activated Sepharose resin --- p.72 / Chapter 2.11.9 --- Reagents and buffers for in vitro binding assay --- p.72 / Chapter 2.11.10 --- Reagents and Buffers for surface plasmon resonance --- p.72 / Chapter 2.12 --- Sequences of primers --- p.73 / Chapter Chapter 3 --- Interaction between TCS and P2 --- p.80 / Chapter 3.1 --- Introduction --- p.80 / Chapter 3.2 --- Interaction between TCS and P-proteins in rat liver lysate --- p.83 / Chapter 3.3 --- Construction of TCS mutants --- p.85 / Chapter 3.4 --- Expression and purification of TCS mutants --- p.87 / Chapter 3.5 --- Biological assay of TCS mutants --- p.91 / Chapter 3.6 --- Physical interaction of TCS mutants and P2 by surface plasmon resonance (SPR) --- p.94 / Chapter 3.7 --- Discussion --- p.100 / Chapter Chapter 4 --- Mapping the region of P2 that binds TCS and eEF2 --- p.104 / Chapter 4.1 --- Introduction --- p.104 / Chapter 4.2 --- Construction of P2 truncation mutants --- p.106 / Chapter 4.3 --- Expression and purification of P2 truncation mutants --- p.107 / Chapter 4.4 --- Mapping the region of P2 that binds TCS --- p.111 / Chapter 4.4.1 --- Interaction between TCS and P2 mutants by in vitro binding assay --- p.111 / Chapter 4.4.2 --- Interaction study of TCS and P2 mutant by isothermal titration calorimetry (ITC) --- p.116 / Chapter 4.5 --- Mapping the region of P2 that binds eEF2 --- p.120 / Chapter 4.5.1 --- Purification of eEF2 from rat liver --- p.120 / Chapter 4.5.2 --- Physical interaction of P2 and eEF2 by surface plasmon resonance (SPR) --- p.126 / Chapter 4.5.3 --- Interaction between eEF2 and P2 mutants by in vitro binding assay --- p.128 / Chapter 4.6 --- Mapping the C-terminal region of P2 by MBP-fusion proteins --- p.130 / Chapter 4.6.1 --- Construction and purification of MBP-fusion proteins --- p.131 / Chapter 4.6.2 --- "Interaction among eEF2, TCS and MBP-fusion proteins by in vitro binding assay" --- p.133 / Chapter 4.7 --- Discussion --- p.137 / Chapter Chapter 5 --- Effect of C-17 peptide on TCS biological activity --- p.143 / Chapter 5.1 --- Introduction --- p.143 / Chapter 5.2 --- Ribosome-inactivating activity of TCS with C-17 peptide --- p.145 / Chapter 5.3 --- Discussion --- p.147 / Chapter Chapter 6 --- Conclusion and suggestions for future study --- p.149 / References --- p.152 / Appendix --- p.173

Yau, Kwok Hei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 64-71). / Abstracts in English and Chinese. / Chapter Chapter 1: --- Introduction --- p.1 / Trichosanthin --- p.2 / Apoptosis --- p.10 / Herpes Simplex Virus --- p.16 / Conclusion --- p.28 / Chapter Chapter 2: --- Materials and Methods --- p.29 / Cell lines and virus --- p.30 / Infectivity assay --- p.30 / Treatment of cells and virus infection --- p.32 / MTT assay for cytotoxicity --- p.34 / Preparation of cell lysate --- p.35 / Bradford assay for protein concentration --- p.36 / Western blot analysis --- p.37 / ELISA for quantification of HSV-1 antigen --- p.38 / Statistical analyses --- p.39 / Chapter Chapter 3: --- Results --- p.40 / Cytotoxicity and anti-herpetic activity of TCS and CHX --- p.41 / Selective cytotoxicity of TCS toward HSV-1 infected cells --- p.44 / Selective cytotoxicity is implicated in the antiviral activity of TCS --- p.50 / The effect of selective cytotoxicity on TI value --- p.53 / Chapter Chapter 4: --- Discussion --- p.55 / References --- p.64

Sha Ou. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves 167-189). / Abstracts in English and Chinese. / abstract --- p.i / 中文摘要 --- p.iv / acknowledgements --- p.vii / Chapter chapter 1. --- introduction --- p.1 / Chapter 1.1 --- Overview --- p.1 / Chapter 1.2 --- Ribosome-inactivating proteins (RIPs) --- p.1 / Chapter 1.2.1 --- Classification --- p.2 / Chapter 1.2.2 --- Structure --- p.3 / Chapter 1.2.3 --- Enzymatic activities --- p.3 / Chapter 1.3 --- Type II RIPs --- p.5 / Chapter 1.3.1 --- Ricin --- p.5 / Chapter 1.3.2 --- Ricinus communis agglutinin (RCA) --- p.6 / Chapter 1.3.3 --- Intracellular mechanism --- p.7 / Chapter 1.3.4 --- Application of RIPs in neuroscience research: suicide axonal transport --- p.10 / Chapter 1.4 --- Type I RIPs --- p.12 / Chapter 1.4.1 --- Trichosanthin (TCS) --- p.12 / Chapter 1.4.2 --- Ricin A chain (RTA) --- p.15 / Chapter 1.4.3 --- Medical applications: immunolesioning and immunotherapy --- p.16 / Chapter 1.5 --- The types of Cell death --- p.17 / Chapter 1.5.1 --- Necrosis --- p.18 / Chapter 1.5.2 --- Apoptosis --- p.18 / Chapter 1.6 --- Inflammations --- p.21 / Chapter 1.6.1 --- Acute inflammation --- p.21 / Chapter 1.6.2 --- Chronic inflammation --- p.22 / Chapter 1.6.3 --- Retinitis --- p.22 / Chapter 1.7 --- Eye model for neurotoxicity studies in CNS --- p.23 / Chapter 1.8 --- Objective of present study --- p.24 / Chapter CHAPTER 2. --- MATERIALS AND METHODS --- p.25 / Chapter 2.1 --- Plan of this chapter --- p.25 / Chapter 2.2 --- Toxins and methods used --- p.25 / Chapter 2.3 --- Animals --- p.26 / Chapter 2.4 --- Preparation of toxin solutions --- p.27 / Chapter 2.4.1 --- RIP solutions --- p.27 / Chapter 2.4.2 --- Labeling type I RIPs with fluorescence --- p.27 / Chapter 2.4.3 --- Control solutions --- p.29 / Chapter 2.5 --- Administrations of solutions --- p.30 / Chapter 2.5.1 --- Basic procedures of vitreous chamber injection --- p.30 / Chapter 2.5.2. --- Injection of trichosanthin (TCS) --- p.31 / Chapter 2.5.3 --- Injection of ricin A chain (RTA) --- p.31 / Chapter 2.5.4 --- Injection of ricinus communis agglutinin (RCA) --- p.32 / Chapter 2.5.5 --- Administration of FITC-TCS --- p.33 / Chapter 2.5.6 --- Administration of FITC-RTA --- p.33 / Chapter 2.6 --- Retinal tissue processing --- p.33 / Chapter 2.6.1 --- Paraffin method --- p.34 / Chapter 2.6.2 --- Cryostatic method --- p.35 / Chapter 2.6.3 --- Electron microscopic method --- p.35 / Chapter 2.7 --- General effects of RIPs on rat retinas --- p.36 / Chapter 2.7.1 --- Hematoxylin-and-eosin staining --- p.36 / Chapter 2.7.2 --- Retinal thickness --- p.37 / Chapter 2.7.3 --- Pathological changes --- p.38 / Chapter 2.7.4 --- Dosage study on TCS --- p.39 / Chapter 2.7.5 --- Statistics --- p.40 / Chapter 2.8 --- Mechanisms of cell death --- p.40 / Chapter 2.8.1 --- Terminal dUTP nick-end labeling (TUNEL) --- p.40 / Chapter 2.8.2 --- Immunohistochemistry for caspase-3 --- p.42 / Chapter 2.8.3 --- Double staining of cleaved caspase-3 and TUNEL --- p.42 / Chapter 2.8.4 --- Electronic microscope observation --- p.43 / Chapter 2.9 --- Entry of type I RIPs into cells --- p.43 / Chapter 2.9.1 --- Propidium iodide staining --- p.43 / Chapter 2.9.2 --- Immunohistochemical localization of Muller cells --- p.44 / Chapter 2.9.3 --- Double staining of Muller cells and TUNEL --- p.44 / Chapter 2.9.4 --- Confocal microscope --- p.44 / Chapter 2.10 --- Reactions of glial cells --- p.45 / Chapter CHAPTER 3. --- RESULTS --- p.47 / Chapter 3.1 --- Preparation of fluorescein-type I RIP conjugates --- p.47 / Chapter 3.1.1 --- Conjugate of FITC-TCS --- p.47 / Chapter 3.1.2 --- Conjugate of FITC-RTA --- p.47 / Chapter 3.2 --- Effects of TCS on retina --- p.47 / Chapter 3.2.1 --- Retina cell count - a dose-dependence study --- p.48 / Chapter 3.2.2 --- Retinal thickness measurement - a time-course study --- p.49 / Chapter 3.2.3 --- Pathological changes --- p.50 / Chapter 3.3 --- Effects of RTA on retina --- p.51 / Chapter 3.3.1 --- Retinal thickness measurement - a time-course study --- p.51 / Chapter 3.3.2 --- Pathological changes --- p.53 / Chapter 3.4 --- Effects of RCA on retina --- p.54 / Chapter 3.4.1 --- Retinal thickness measurement --- p.54 / Chapter 3.4.2 --- Pathological changes --- p.55 / Chapter 3.5 --- Summary of results: general effects of RIPs --- p.56 / Chapter 3.6 --- Cell death - TUNEL method --- p.56 / Chapter 3.6.1 --- TCS experiment --- p.57 / Chapter 3.6.2 --- RTA experiment --- p.58 / Chapter 3.6.3 --- RCA experiment --- p.58 / Chapter 3.7 --- Cell death 一 cleaved caspase-3 immunohistochemistry --- p.58 / Chapter 3.7.1 --- TCS experiment --- p.59 / Chapter 3.7.2 --- RTA experiment --- p.59 / Chapter 3.8 --- EM observation --- p.59 / Chapter 3.8.1 --- TCS experiment --- p.59 / Chapter 3.8.2 --- RTA experiment --- p.60 / Chapter 3.9 --- Summary of results: mode of cell death --- p.60 / Chapter 3.10 --- Localisation of type I RIPs --- p.61 / Chapter 3.10.1 --- FITC-TCS --- p.62 / Chapter 3.10.2 --- FITC-TCS and Muller cell double staining --- p.63 / Chapter 3.10.3 --- Muller cell and TUNEL double staining --- p.64 / Chapter 3.10.4 --- FITC-RTA --- p.64 / Chapter 3.10.5 --- Summary of results: route of intoxication --- p.65 / Chapter 3.11 --- Glial cell reactions after RIP treatment --- p.65 / Chapter 3.11.1 --- TCS experiment --- p.65 / Chapter 3.11.2 --- RTA experiment --- p.66 / Chapter 3.11.3 --- RCA experiment --- p.67 / Chapter 3.11.4 --- Summary of results: glial reactions --- p.67 / Chapter CHAPTER 4. --- DISCUSSION --- p.69 / Chapter 4.1 --- General effects of RIPs on rat retinas --- p.69 / Chapter 4.1.1 --- Effects of trichosanthin (TCS) --- p.69 / Chapter 4.1.2 --- Effects of ricin A chain (RTA) --- p.71 / Chapter 4.1.3 --- Effects of ricinus communis agglutinin (RCA) --- p.73 / Chapter 4.2 --- The mechanisms of cell death --- p.74 / Chapter 4.2.1 --- Cell death caused by TCS --- p.75 / Chapter 4.2.2 --- Caspase-3 and the retina of RCS rat --- p.77 / Chapter 4.2.3 --- Cell death caused by RTA --- p.78 / Chapter 4.2.4 --- Cell death caused by RCA --- p.80 / Chapter 4.2.5 --- Mechanism of RTA - induced necrosis --- p.81 / Chapter 4.3 --- The mechanisms of type I RIPs entering cells --- p.82 / Chapter 4.3.1 --- Transport of TCS in retinal cells --- p.82 / Chapter 4.3.2 --- The uptake of Pure FITC by rat retina --- p.85 / Chapter 4.4 --- Reactions of glial cells --- p.85 / Chapter 4.4.1 --- Glial cell reactions in TCS experiment --- p.86 / Chapter 4.4.2 --- Glial cell reactions in RTA and RCA experiments --- p.87 / Chapter 4.5 --- Possible applications of RIPs on retinal studies --- p.88 / Chapter 4.5.1 --- Potential applications of TCS --- p.88 / Chapter 4.5.2 --- Possible uses of RTA and RCA --- p.90 / Chapter CHAPTER 5. --- CONCLUSIONS --- p.91 / "FIGURES, TABLES, GRAPHS, AND LEGENDS" --- p.93 / APPENDICES --- p.154 / Appendix A Source of materials --- p.154 / Appendix B Dosages for vitreous chamber injection --- p.156 / Appendix C Protocol of conjugate fluorescein to proteins --- p.157 / Appendix D Electronic Microscope methods --- p.160 / Appendix E Histological methods --- p.162 / Appendix F Protocols of TUNEL --- p.163 / Appendix G Protocols of Immunohistochemistry staining --- p.165 / REFERENCES --- p.167

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.