Lu Shanxiang. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 122-135). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Figures --- p.xiii / List of Tables --- p.xv / List of Abbreviations --- p.xvi / Chapter Chapter 1. --- General Introduction --- p.1 / Chapter 1.1 --- Lysine-rich protein from winged bean --- p.2 / Chapter 1.1.1 --- Discovery --- p.2 / Chapter 1.1.2 --- Applications in enhancing nutritional values --- p.2 / Chapter 1.2 --- Plant secretory pathway --- p.4 / Chapter 1.2.1 --- Overview of plant secretory pathway --- p.4 / Chapter 1.2.2 --- Three models on protein transportation from ER to Golgi --- p.6 / Chapter 1.2.3 --- Brefeldin A: inhibitor of secretion --- p.9 / Chapter 1.2.4 --- Markers for different organelles --- p.10 / Chapter 1.3 --- Tobacco bright yellow 2 (BY-2) cell system --- p.11 / Chapter 1.3.1 --- Origin of BY-2 cell line --- p.12 / Chapter 1.3.2 --- Characteristics of BY-2 cell line --- p.12 / Chapter 1.4 --- Use of fluorescent proteins as reporters --- p.13 / Chapter 1.4.1 --- GFP and its derivatives --- p.13 / Chapter 1.4.2 --- Reporter system --- p.15 / Chapter 1.4.3 --- Applications of GFP and its derivatives in plants --- p.16 / Chapter 1.5 --- Temperature effects on plants --- p.17 / Chapter 1.6 --- Project objectives --- p.18 / Chapter Chapter 2 --- Subcellular localization of LRP in winged bean (Psophocarpus tetragonolobus)seeds --- p.20 / Chapter 2.1 --- Introduction --- p.21 / Chapter 2.2 --- Materials and Methods --- p.21 / Chapter 2.2.1 --- Chemicals --- p.21 / Chapter 2.2.2 --- Plant materials --- p.21 / Chapter 2.2.3 --- Antibodies --- p.22 / Chapter 2.2.4 --- Western blot --- p.23 / Chapter 2.2.4.1 --- Protein extraction --- p.23 / Chapter 2.2.4.2 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.23 / Chapter 2.2.4.3 --- Immunodetection --- p.24 / Chapter 2.2.5 --- Confocal Immunofluorescence --- p.25 / Chapter 2.2.5.1 --- Preparation of samples for immuno-labeling --- p.25 / Chapter 2.2.5.2 --- Immuno-labeling --- p.26 / Chapter 2.2.5.3 --- Collection and analysis of confocal fluorescent images --- p.27 / Chapter 2.2.6 --- Immuno transmission electron microscope (TEM) study --- p.28 / Chapter 2.2.6.1 --- Preparation of samples --- p.28 / Chapter 2.2.6.2 --- Immuno-labeling --- p.29 / Chapter 2.3 --- Results --- p.31 / Chapter 2.3.1 --- Anti-alpha-TIP and anti-LRP antibodies have good specificity in winged bean seeds --- p.31 / Chapter 2.3.2 --- Anti-a-TIP antibodies could label the PSVs of winged bean seeds specifically --- p.31 / Chapter 2.3.3 --- LRP was localized outside of PSVs --- p.33 / Chapter 2.3.4 --- Immuno-TEM localization of LRP --- p.35 / Chapter 2.4 --- Conclusion and discussion --- p.40 / Chapter Chapter 3 --- Generation of Transgenic Tobacco BY-2 Cell Lines Expressing YFP and LRP Fusions --- p.41 / Chapter 3.1 --- Introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.42 / Chapter 3.2.1 --- Primers --- p.42 / Chapter 3.2.2 --- Plant materials --- p.43 / Chapter 3.2.3 --- Bacterial strains --- p.44 / Chapter 3.2.4 --- Construction of fusion constructs --- p.44 / Chapter 3.2.4.1 --- Four fusion constructs of LRP and YFP --- p.44 / Chapter 3.2.4.2 --- His-tag-YFP fusion construct --- p.45 / Chapter 3.2.4.3 --- Cloning of the fusion protein genes into Agrobacterium binary vector pBI121 --- p.45 / Chapter 3.2.5 --- Confirmation of the fusion constructs --- p.53 / Chapter 3.2.6 --- Transformation of Agrobacterium by electroporation --- p.53 / Chapter 3.2.7 --- "Transformation, selection and suspension of tobacco BY-2 cells" --- p.54 / Chapter 3.2.8 --- "Transformation, screening and induction of E. coli BL21-DE3 for expression of His-tagged YFP" --- p.55 / Chapter 3.2.9 --- Protein extraction --- p.55 / Chapter 3.2.9.1 --- Protein fractionation from BY-2 cells --- p.55 / Chapter 3.2.9.2 --- protein extraction from E. coli of BL21-DE3 --- p.56 / Chapter 3.2.10 --- Immunolabeling of suspension cultured cells --- p.56 / Chapter 3.2.11 --- Raising anti-GFP antibodies --- p.57 / Chapter 3.2.12 --- Dot blot analysis --- p.58 / Chapter 3.2.13 --- Affinity purification of proteins and antibodies --- p.59 / Chapter 3.2.13.1 --- Metal affinity resin column for protein purification --- p.59 / Chapter 3.2.13.2 --- Cyanogens bromide (CNBr) activated sepharose column for antibody purification --- p.60 / Chapter 3.2.14 --- SDS-PAGE and western blot analysis --- p.61 / Chapter 3.2.15 --- Antibodies --- p.61 / Chapter 3.3 --- Results --- p.62 / Chapter 3.3.1 --- Two transgenic BY-2 cell lines showed different fluorescent signal patterns --- p.62 / Chapter 3.3.2 --- Two cell lines showed different fluorescent signal stability --- p.63 / Chapter 3.3.3 --- The two fusion proteins were localized in different places in the BY-2 cells --- p.67 / Chapter 3.3.4 --- """Green"" E. coli expressed the recombinant YFP" --- p.69 / Chapter 3.3.5 --- Expressed recombinant YFP could not be affinity purified --- p.69 / Chapter 3.3.6 --- Raised polyclonal anti-GFP antibodies showed good specificity --- p.69 / Chapter 3.4 --- Conclusion and discussion --- p.75 / Chapter Chapter 4 --- Characterization of SpYFP-LRP Fusion in Transgenic BY-2 cells --- p.76 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Materials and Methods --- p.77 / Chapter 4.2.1 --- Plant materials --- p.77 / Chapter 4.2.2 --- BFA and heat treatment --- p.77 / Chapter 4.2.3 --- Confocal immunolabeling --- p.78 / Chapter 4.2.4 --- Conventional TEM study --- p.78 / Chapter 4.2.5 --- Immuno TEM using Lowicryl resin and LR White resin --- p.80 / Chapter 4.3 --- Results --- p.82 / Chapter 4.3.1 --- "BFA induced the SpYFP-LRP-marked organelle to form ""BFA-induced"" compartments" --- p.82 / Chapter 4.3.2 --- Partial recovery from BFA treatment --- p.84 / Chapter 4.3.3 --- SpYFP-LRP was localized in BFA-induced compartments --- p.87 / Chapter 4.3.4 --- BFA treatment induced the formation of various compartmentsin SpYFP-LRP cells --- p.90 / Chapter 4.3.5 --- BFA-induced structures contain SpYFP-LRP --- p.99 / Chapter 4.3.6 --- Elevated temperature affected the signal pattern but not the localization of SpYFP-LRP in transgenic BY-2 cells --- p.100 / Chapter 4.3.7 --- Elevated temperature treatment induced the SPYFP-LRP cells to form new vesicular compartments --- p.105 / Chapter 4.4 --- Conclusions and Discussion --- p.112 / Chapter 4.4.1 --- BFA treatment --- p.112 / Chapter 4.4.2 --- Heat treatment --- p.114 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.116 / Chapter 5.1 --- Summary --- p.117 / Chapter 5.2 --- Future perspectives --- p.119 / Reference --- p.122

Antifungal proteins belong to a group of plant defense proteins, present in different plant tissues ranging from leaves, stems, roots to seeds. Upon fungal invasions, these proteins accumulate at the infection site to inhibit the growth of pathogens. Some antifungal proteins were found to have antifreeze and antiviral characteristics as well. Antifungal proteins are structurally diverse. Thaumatin-like proteins, chitinases, ribosome inactivating proteins, defensins, cyclophilin-like proteins and miraculin-like proteins are members of the family of antifungal proteins. There are antifungal proteins with other structures such as cysteine protease inhibitors and peptides. Apart from plant defense, antifungal proteins have been reported to affect the growth of murine immune cells including splenocytes, macrophages and bone marrow cells, as well as leukemia cells. / In my study, five antifungal proteins were isolated from plant storage tissues that were bulbs and seeds abundant in proteins, comprising a chitinase-like antifungal proteins from black turtle beans (Phaseolus vulgaris cv. black turtle), an 8-kDa antifungal peptide from green chickpea ( Cicer arietinum cv green chickpea), two thaumatin-like proteins from two kinds of chestnut (Castanopsis chinensis and Castanea mollissima) and a 7.1-kDa antifungal peptide from the Chinese daffodil (Narcissus tazetta var. chinensis). In the majority of cases, the antifungal proteins were unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel, CM-cellulose and Mono S. They showed different antifungal potencies on various phytopathogenic fungi examined including Botrytis cinerea, Fusarium oxysporum, Mycosphaerella arachidicola and Physalospora piricola. Only the daffodil and Castanopsis antifungal proteins were active on all four fungal species. The remaining three antifungal proteins inhibit 2-3 of the 4 fungi. / The purified proteins were characterized for various biological activities such as the inhibitory activity on HIV-1 reverse transcriptase and cell-free translation in a rabbit reticulocyte system. All proteins except those from daffodil bulbs and black turtle beans exhibited HIV-1 reverse transcriptase inhibitory activity. The activity of purified proteins on the proliferation of normal and tumor cells in vitro, including murine splenocytes and leukemia L1210 cells, was also studied. Daffodil antifungal protein was the only protein found to have a stimulatory activity on mouse splenocytes and an inhibitory activity on leukemia cells. (Abstract shortened by UMI.) / Chu Kin-tak. / "June 2005." / Adviser: T. B. Ng. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3574. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 175-183). / 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. / Abstract in English and Chinese. / School code: 1307.

by Lam Suet Ling. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 109-121). / Acknowledgements --- p.i / Table of contents --- p.ii / Abstract --- p.vii / List of Abbreviations --- p.ix / List of Tables --- p.x / List of Figures --- p.xi / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Ribosome-inactivating proteins (RIPs) --- p.3 / Chapter 1.1.1 --- Types of RIPs --- p.4 / Chapter 1.1.1.1 --- Type I RIPs --- p.5 / Chapter 1.1.1.2 --- Type II RIPs --- p.7 / Chapter 1.1.2 --- Physicochemical properties --- p.7 / Chapter 1.1.3 --- N-glycosidase activity of RIPs --- p.8 / Chapter 1.1.3.1 --- Specificity of N-glycosidase activity --- p.10 / Chapter 1.1.3.2 --- Inhibition of protein synthesis --- p.11 / Chapter 1.1.4 --- Other enzymatic and biological activities of RIPs --- p.11 / Chapter 1.1.4.1 --- Enzymatic activities --- p.11 / Chapter 1.1.4.2 --- Multiple depurination --- p.13 / Chapter 1.1.4.3 --- RNase activity --- p.14 / Chapter 1.1.4.4 --- DNase activity --- p.15 / Chapter 1.1.4.5 --- Biological activities --- p.16 / Chapter 1.1.5 --- Storage of RIPs in plant cells --- p.17 / Chapter 1.1.5.1 --- RIPs targeted to subcellular compartments --- p.18 / Chapter 1.1.5.2 --- Cytoplasmic RIPs --- p.20 / Chapter 1.1.6 --- Physiological roles of RIPs --- p.22 / Chapter 1.1.6.1 --- Defensive role in plants --- p.22 / Chapter 1.1.6.2 --- Metabolic role of RIPs --- p.26 / Chapter 1.1.6.3 --- RIPs as storage proteins --- p.26 / Chapter 1.1.7 --- Application of RIPs --- p.27 / Chapter 1.1.7.1 --- Therapeutic applications --- p.27 / Chapter 1.1.7.2 --- Possible use of RIPs in agriculture --- p.30 / Chapter 1.2 --- Objectives of the present study --- p.31 / Chapter 1.2.1 --- Rationale of the study --- p.31 / Chapter 1.2.2 --- Outline of the thesis --- p.32 / Chapter Chapter 2 --- Screening of hitherto unexplored plant species for RIPs --- p.33 / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.2 --- Materials and methods / Chapter 2.2.1 --- Materials --- p.36 / Chapter 2.2.2 --- Preparation of crude powder --- p.36 / Chapter 2.2.3 --- Protein determination --- p.38 / Chapter 2.2.4 --- Preparation of rabbit reticulocyte lysate --- p.38 / Chapter 2.2.5 --- Protein synthesis inhibition assay --- p.39 / Chapter 2.3 --- Results / Chapter 2.3.1 --- Preparation of crude powder --- p.41 / Chapter 2.3.2 --- Protein synthesis inhibition assay --- p.41 / Chapter 2.4 --- Discussion --- p.43 / Chapter Chapter 3 --- Purification of RIP from garden pea (Pisum sativum) --- p.45 / Chapter 3.1 --- Introduction --- p.46 / Chapter 3.2 --- Materials and methods / Chapter 3.2.1 --- Materials --- p.50 / Chapter 3.2.2 --- Purification of RIP from garden pea --- p.52 / Chapter 3.2.3 --- Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.54 / Chapter 3.2.4 --- Precautions for working with RNA --- p.56 / Chapter 3.2.5 --- N-glycosidase assay --- p.57 / Chapter 3.2.6 --- Quantitation of RNA --- p.60 / Chapter 3.3 --- Results / Chapter 3.3.1 --- Quantitation of RNA --- p.61 / Chapter 3.3.2 --- Affinity chromatography on Affi-gel Blue gel --- p.61 / Chapter 3.3.3 --- Iminodiacetic acid-agarose chromatography --- p.64 / Chapter 3.3.4 --- Cation exchange chromatography on Resource-S --- p.66 / Chapter 3.3.5 --- Gel filtration on Superose 12 HR 10/30 --- p.69 / Chapter 3.3.6 --- "Assessment of purity, yield and activity" --- p.72 / Chapter 3.4 --- Discussion --- p.74 / Chapter Chapter 4 --- Physicochemical and biological properties of garden pea RIP --- p.77 / Chapter 4.1 --- Introduction --- p.79 / Chapter 4.2 --- Materials and methods / Chapter 4.2.1 --- Materials --- p.81 / Chapter 4.2.2 --- Molecular weight determination --- p.82 / Chapter 4.2.3 --- Subunit composition --- p.82 / Chapter 4.2.4 --- Isoelectric focusing (IEF) --- p.83 / Chapter 4.2.5 --- Detection of glycoproteins --- p.84 / Chapter 4.2.6 --- N-terminal amino acid sequence --- p.84 / Chapter 4.2.7 --- Inhibition of cell-free protein synthesis --- p.86 / Chapter 4.2.8 --- N-glycosidase activity --- p.86 / Chapter 4.2.9 --- Deoxyribonuclease activity --- p.87 / Chapter 4.2.10 --- Activity towards tRNA --- p.88 / Chapter 4.3 --- Results / Chapter 4.3.1 --- Molecular weight determination --- p.89 / Chapter 4.3.2 --- Subunit composition --- p.91 / Chapter 4.3.3 --- Isoelectric focusing (IEF) --- p.92 / Chapter 4.3.4 --- Detection of glycoproteins --- p.94 / Chapter 4.3.5 --- N-terminal amino acid sequence --- p.96 / Chapter 4.3.6 --- Inhibition of cell-free protein synthesis --- p.97 / Chapter 4.3.7 --- N-glycosidase activity --- p.99 / Chapter 4.3.8 --- Deoxyribonuclease activity --- p.101 / Chapter 4.3.9 --- Activity towards tRNA --- p.102 / Chapter 4.4 --- Discussion --- p.103 / Chapter Chapter 5 --- General discussion and conclusion --- p.106 / References --- p.109

Thesis (M.S.)--Oregon State University, 2008. / Printout. Includes bibliographical references (leaves 100-106). Also available on the World Wide Web.

No description available.

Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. It is reported here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18-20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these "nodule-specific" host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a "nodule-specific" antiserum with in vitro translation products of root nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7-11% of the total ('35)S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000-20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for by the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. / Apart from the low molecular weight nodulins, a 35,000 MW polypeptide present in the nodule cytoplasm was also identified as "nodule-specific". This protein, referred to as nodulin-35, represents about 4% of the total cytoplasmic protein in root nodules, and its appearance is not affected by mutations in several nodulating strains of Rhizobium. Nodulin-35 was not detected in uninfected soybean, bacteroids or free-living Rhizobium, and it appears to be synthesized by the plant during the formation of root nodules. / Whereas the transformation of free-living Rhizobium into bacteroids is accompanied by substantial changes within the population of cytoplasmic proteins, the majority of plant polypeptides from nodules are also present in uninfected (non-nodulated) roots. Hence, to further identify and isolate the "nodule-specific" proteins, it was essential to develop several immunological procedures, including a preparative adsorption of antibodies with antigens, the multiple immunoreplica technique, and isolation of a single-copy mRNA by immunoprecipitation of the nascent peptide-polysome complex, which are described in this thesis. / In addition, two polypeptides of bacterial origin were found to be cross-reactive with the "nodule-specific" antiserum, suggesting that they are secreted into the host cell cytoplasm during symbiotic nitrogen fixation.

Graduation date: 1992

Thesis (M. Ag. Sci.)--University of Adelaide, 1988. / Typescript. Includes bibliographical references (leaves 51-61).

Thesis (Ph.D.) -- University of Adelaide, School of Agriculture & Wine, Faculty of Sciences, 2005. / "November, 2004" Bibliography: leaves 209-226.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains ix, 65 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 58-62).