No description available.

No description available.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No description available.

The investigations presented in this thesis form part of a systematic international effort to establish the structures of the capsules produced by the bacterial genera, Escherichia coli and Klebsiella (family enterobacteriaceae). These bacteria are of medical interest as they are opportunistic pathogens and are frequently responsible for serious infections in animals and man. Invasive strains are invariably surrounded by a structurally complex polysaccharide capsule which contributes to the organism's ability to attenuate non-specific host defence mechanisms or, in some instances, to completely prevent an immune response. A knowledge of the chemical composition and structure of the capsule is, therefore, of great value as it provides insight into the mechanisms involved in this process. The E. coli, in particular, have generated considerable interest as their capsules are more structurally diverse and cross-reactivity with other, more pathogenic bacteria has also been demonstrated. Accordingly, the structures of three previously unstudied E. coli K-antigens viz. those produced by serotypes 020:K83:H26, 020:K84:H26, and 09:K48:H9 have been established by chemical and spectroscopic means and are presented in this thesis. In addition, a reinvestigation of the structure of the capsule produced by Klebsiella K15 using a novel enzymatic approach was also undertaken and a revised structure is proposed . The E. coli K48 polysaccharide is of special interest as it was found to contain a new diacetamido trideoxy hexose hitherto unrecorded. A synthesis for this saccharide is also presented. Finally, the application of lithium dissolved in ethylenediamine for the degradation of amino sugar-containing polysaccharides was also investigated using the capsular polysaccharides produced by E. coli serotypes K38 and K84 as model compounds.

Previous studies had demonstrated the presence of the p70 (CAMAL) molecule in human myeloid leukemia cells and the promyelocytic leukemia cell line HL60, but not in equivalent preparations of normal cells (Malcolm et al., 1982, 1984; Shipman et al., 1983; Logan et al., 1984). Subsequent studies demonstrated that the p70 (CAMAL) protein was detectable and expressed in human myeloid leukemia cells and the leukemic cell lines HL60, KG1, K562 and U937. The association of p70 (CAMAL) expression with human myeloid leukemia cells prompted its consideration as a candidate leukemia-associated antigen. The demonstration, following CAMAL purification and peptide sequencing, that two tryptic peptides (tp27, tp31) displayed significant homology to sequences present in human serum albumin (HSA) and human alpha-1-fetoprotein (AFP), while one tryptic peptide (tp20) displayed unique peptide sequence, suggested that CAMAL might represent a protein that was structurally and functionally related to the albumins. Consequently, a comparative biochemical analysis of CAMAL and HSA was initiated. The results of the comparative studies demonstrated that although CAMAL and HSA shared conformational antigenic determinants, both proteins were also shown to be distinct molecules by a number of other criteria. The possibility that the CAMAL preparation, used for protein sequencing and comparative studies, was contaminated with HSA was thought likely, in view of the HSA/AFP-related peptide sequences from the CAMAL tryptic peptide sequence analysis. However, other results, particularly the antibody reactivity and ligand binding studies, showed that the CAMAL preparation was not contaminated with HSA. The unique CAMAL tryptic peptide (tp20) sequence supported further the contention that CAMAL was a distinct protein with regions homologous to HSA and AFP. Further analysis of the CAMAL molecule, through extensive protein sequencing, will be, in all likelihood, the only means by which to establish the degree of relatedness between CAMAL, HSA and AFP. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

MALA-2 (Murine Lymphocyte Activated Antigen-2) is a murine cell surface antigen that is detected at high concentration on activated, proliferating lymphocytes, but only weakly on resting lymphocytes. It is thought to play an important role in lymphocyte activation since the rat monoclonal antibody YN1/1.7.4 which recognizes MALA-2 is capable of inhibiting the mixed lymphocyte reaction. Considering the central role of lymphocyte activation to the generation and maintenance of the immune response, I undertook the purification and biochemical characterization of MALA-2. In these studies, MALA-2 was isolated and purified to homogeneity using immobilized YN1/1.7.4 monoclonal antibody and sodium docecylsulphate-polyacrylamide gel electrophoresis. Biochemical characterization studies revealed that MALA-2 is a Mr 95-100 kD glycoprotein containing a protein backbone of approximately 66 kD, and N-linked carbohydrate chains amounting to a Mr of approximately 35 kD. Two dimensional gel electrophoresis suggested that MALA-2 has an isoelectric point of 4.9. Although it was previously suspected that MALA-2 might be associated with the transferrin receptor on the cell surface, this was shown not to be the case on NS-1 cells. Additionally, ³²P-orthophosphate labelling of MALA-2 on NS-1 or MBL-2 cells could not be detected. Finally, the partial amino acid sequence of MALA-2 was determined by sequencing trypsin-generated peptides from purified MALA-2. Computer-assisted homology comparisons of the MALA-2 partial amino acid sequences with other known sequences showed that MALA-2 shared its most consistent homology with a class of proteins known as the immunoglobulin superfamily. Subsequent to this study, the partial amino acid sequences obtained within this study were used to construct oligonucleotide probes. These probes were used for the screening of cDNA libraries, facilitating the successful cloning of the MALA-2 gene. This, in turn, resulted in the identification of MALA-2 as the murine counterpart of the human ICAM-1 molecule, a protein known to play a significant role in intercellular adhesion and lymphocyte activation within the immune system. Significantly, results obtained from the biochemical characterization of MALA-2 carried out in this thesis have been confirmed by the subsequent nucleotide sequence data from the cloning of MALA-2. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

published_or_final_version / Pathology / Master / Master of Philosophy

No description available.