published_or_final_version / abstract / Chemistry / Master / Master of Philosophy

Histidine-rich glycoprotein (HRG) is a plasma protein that regulates key cardiovascular processes such as coagulation, angiogenesis and immune response. The protein consists of six distinct functional domains: two N-terminal domains (N1 and N2), two proline-rich regions (PRR1 and PRR2), a central histidine-rich region (HRR) and a C-terminal domain. The HRR binds Zn²⁺, which alters the affinity of HRG towards various ligands including the anticoagulant, heparin. A key aim of this study was to structurally characterise HRG. The 1.93 Å crystal structure of the HRG N2 domain presented here represents the first crystallographic snapshot of the molecule. The N2 domain is cystatin-like and N-glycosylated at Asn184. An S-glutathionyl adduct was observed at Cys185, providing in vivo evidence that release of an anti-angiogenic HRR/PRR fragment is controlled in part by a redox mechanism, representing a novel further role for GSH in regulation of angiogenesis. Since Zn²⁺ regulates some of the functions of HRG, the dynamics of Zn²⁺ in plasma were investigated using a combination of ITC, ELISA and thrombin assay systems. Zn²⁺ is normally associated with albumin in circulation, but its ability to bind Zn²⁺ is allosterically inhibited upon fatty acids binding to albumin. Elevated plasma fatty acid levels are associated with some disease states. It is proposed that this may alter the proportion of Zn²⁺ bound to HRG, which could in turn activate thrombin to promote coagulation. These studies provide evidence to suggest that Zn²⁺-dependent activation of HRG (following fatty acid binding to albumin) may play a role in the development of haemostatic complications in susceptible individuals. Finally, the Zn²⁺ binding ability of albumin was probed in order to locate unidentified sites using recombinant albumin mutants. H9A, H67A, E252A, D256A and H288A mutants all exhibited diminished Zn²⁺ binding ability, indicating that these residues are involved directly or indirectly in Zn²⁺ binding.

Yam Hin-Fai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1990. / Bibliography: leaves 138-157. / ABSTRACT --- p.2 / ACKNOWLEDGEMENT --- p.5 / CONTENT --- p.6 / Chapter I. --- INTRODUCTION --- p.8 / Chapter II. --- LITERATURE REVIEWS / Chapter 1. --- Esophagus and Esophageal Carcinoma --- p.11 / Chapter 2. --- Characterization of Cell Line --- p.23 / Chapter 3. --- Membrane Surface --- p.26 / Chapter 4. --- Differentiation and Cancer --- p.36 / Chapter 5. --- Calcium Ion --- p.42 / Chapter III. --- MATERIALS AND METHODS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.48 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.57 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.62 / Chapter 4. --- Characterizations of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.65 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.71 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.73 / Chapter IV. --- RESULTS / Chapter 1. --- Characterizations of EC/CUHK2 Cell Line --- p.74 / Chapter 2. --- SQM1 Localization on EC/CUHK2 Cells --- p.81 / Chapter 3. --- SQM1 Localization on Other Cells and Cell Lines --- p.83 / Chapter 4. --- Characterization of EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.87 / Chapter 5. --- SQM1 Localization on EC/CUHK2 Cells in Different Extracellular Calcium Ion Concentrations --- p.96 / Chapter 6. --- SQM1 Localization on EC/CUHK2 Cells with Changes of Extracellular Calcium Ion Concentrations --- p.105 / Chapter V. --- DISCUSSIONS / Chapter 1. --- Characterizations of Carcinoma Cell Line --- p.107 / Chapter 2. --- SQM1 Distribution on Esophageal Cancer Cells --- p.118 / Chapter 3. --- SQM1 Distribution on Other Cells --- p.122 / Chapter 4. --- Calcium-Induced Differentiation of Esophageal Carcinoma Cells --- p.125 / Chapter 5. --- SQM1 Distribution on Calcium-Induced Esophageal Carcinoma Cells 6 --- p.132 / Chapter VI. --- CONCLUSION --- p.136 / Chapter VII. --- REFERENCES --- p.138 / Chapter VIII. --- ILLUSTRATIONS --- p.158

Thesis (Masters Diploma (Medical Technology) -- Cape Technikon, Cape Town, 1992 / Gastric cancer, a fatal malignancy, is endemic in the Coloured population of the Western Cape region of South Africa. Diagnosis is based mainly on histologic investigation with patients of either sex being mainly between 40-60 years of age. The extent to which genetic and environmental influences play a role in the aetiology of the disease is unknown. This study is an attempt to biochemically characterise gastric mucins or mucus glycoproteins, (the main gel forming components of crude mucus scrapings off the mucosal surface), in carcinoma of the stomach (HCA), as compared to those in ulcer disease (HGU), post mortem specimens (PM) and samples obtained from organ transplant donor stomachs (HD). The aim of this study is the development of a diagnostic marker within mucus secretions for the detection of pre-malignant disease amongst the high risk population of the Western Cape region of South Africa. Mucins were extracted from crude mucus gel scrapings according to a carefully designed technique in which proteolytic inhibitors were used to minimise the possibility of endogenous proteolysis in the laboratory through possible contamination. Two density gradient ultra-centrifugation steps for 48 hours each at 105,000g in caesium chloride, a well established standard isolation procedure for mucins, gave a yield of pure mucins which fractionated at a density of approximately 1.41gjml in all groups. These mucins, from the HO, PH, HGU and HCA groups eluted mainly in the included volume of a Sepharose 2B column as broad, polydisperse peaks, suggesting that they were degraded and comprised mainly lower molecular weight PAS positive material in relation to large polymeric gel forming mucin.

The aim of the present study was to assess whether the changes in the epithelial glycoproteins seen in the mucosa adjacent to tumors are specific premalignant markers or secondary reactive phenomena. A secondary objective was to assess whether ulcerative colitis and Crohn's Disease could be distinguished from one another histochemically. The carbohydrate prosthetic groups from colonic epithelial glycoproteins were characterized histologically and histochemically from 17 cases of ulcerative colitis, 21 cases of Crohn's Disease and 19 cases of diverticular disease. Two histochemical parameters - the relative proportion of sulpho- and sialomucin and the side-chain substitution pattern of O-acetylated sialic acid - were assessed using a battery of seven histochemical techniques. Serial sections from each specimen were also evaluated morphologically, using hematoxylin and eosin. In addition, the patterns of O-acetylated side-chain sialic acid from the three inflammatory bowel diseases were compared to data previously acquired from the mucosa adjacent to colonic tumors. Results indicate that neither focal changes nor the predominance of sialomucins are specific to the mucosa adjacent to tumors. As well, changes in one histochemical parameter were independent of changes in the other parameter. No histochemical class of epithelial glycoproteins was specific to any of the inflammatory bowel diseases and, therefore, it was not possible to distinguish between ulcerative colitis and Crohn's Disease on the basis of the histochemical techniques used in the present study. It was also noted that the histochemical changes in ulcerative colitis, Crohn's Disease and diverticular specimens were not related to the degree of inflammation. Finally, as a group, Crohn's Disease specimens showed a loss of sulphomucin-sialomucin gradient along the length of the crypts. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Graduation date: 1993

[Truncated abstract] Human complement receptor 2 (CR2/CD21) is a cell surface glycoprotein detected on specific cells involved in immunity, which binds complement C3 cleavage fragments, cellular ligands IFN-? and CD23 as well as the EBV coat protein, gp350/220. During the early stages of B-cell development CR2/CD21 is silenced. Expression is initiated on immature B-cells escaping negative selection. During peripheral maturation CR2/CD21 is up-regulated with B-cell sub-populations showing distinctive surface levels (comparatively low, intermediate or high). CR2/CD21 is silenced upon terminal plasmacytic differentiation. Appropriate timing and expression level of CR2/CD21 is important for the development of a healthy B-cell repertoire. Previous studies have identified sequences within the proximal promoter and first intron of CR2/CD21 that cooperate within native chromatin to control cell-specific silencing. Further, analysis of cultured human cells has revealed chromatin structural variation causing DNase I hypersensitivity at these regulatory sites in a CR2/CD21-expressing mature B-cell line (Raji) which are absent in a non-lymphoid cell type (K562). The primary focus of the present study involved characterising chromatin structural variation over previously recognized DNase I hypersensitive regions at the CR2/CD21 locus in human cells to understand how chromatin structure might regulate developmental expression of CR2/CD21. ... These studies provide evidence that notch signaling influences CR2/CD21 expression in human cell lines. First, in vivo binding of CBF1 to CR2/CD21 sequences in the proximal promoter and CRS implies that CR2/CD21 is a direct target of notch activation. Second, the effect of exogenous notch signalling molecules on CR2/CD21 proximal promoter activity was modulated by factors binding tandem E-boxes near the transcriptional start site suggesting that the notch pathway may also influence CR2/CD21 expression via control of HLH molecules. Third, initiation of CR2/CD21 expression was observed in a nonexpressing pre-B cell line (Reh) by co-culture with stromal cells expressing a notch ligand (OP9-DL) but not control stroma (OP9-GFP). Together, these findings support a role for notch regulation of B-cell maturation and invite speculation that initiation of CR2/CD21 expression following negative selection of immature B-cells involves crosstalk between HLH transcriptional regulators and the notch pathway. Furthermore, the Reh/OP9-DL co-culture system may provide a model to directly study the relationship between cell signalling molecules, transcription factor regulation, chromatin structural variation and differentiation of B-cells.