Proefschrift - Utrecht.

Thesis (S.M.)--University of Chicago, Department of Microbiology. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (S.M.)--University of Chicago, Department of Microbiology. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1949. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Lectins are proteins or glycoproteins of non-immune origin, capable of specific interaction with, and reversible binding to carbohydrate moieties of complex glycoconjugates. Lectins are ubiquitous in nature and are present in all living organisms. In nature lectins are implicated in cell recognition and adhesion processes, and have been described as "second generation" mucoadhesives. The aim of this project was to identify lectins that could be incorporated into dosage forms to allow their retention within the oral cavity. This would allow prolonged localised drug delivery. Initial screening studies on human buccal cells, usmg the avidin-biotincomplex/ diaminobenzidine method suggested that all the lectins tested appeared to bind to varying degrees, as visualised by a diaminobenzidine (DAB) precipitate to the oral epithelial surface. The stain intensities were analysed semi-qualitatively with E micro densitometer GN5 (Barr and Stroud). A substantial reduction in lectin binding was observed after exposing buccal cells to a series of lectin solutions pre-treated with secretor and non-secretor saliva. However when bound to the buccal cells, there was little displacement of lectins on exposure to either saliva types. Kinetic binding studies revealed the presence of the lectins from Pisum sativum and Arachis hypogaea, after only 20 s contact. There were some differences in lectin binding evident between rat oral tissue and human buccal cells, but those that bound avidly to both were selected for furthe studies. In order to allow a quantitative assessment of binding, the lectins from Canavali ensiformis, Arachis hypogaea and Triticum vulgaris were labelled with Technetium-99n (Tc-99m) using a cyclic diethylenetriamine pentaacetic acid conjugation technique. In this preliminary study it was found that 1.18 fmoles of the Canavalia ensiformis lectir 3.27 fmoles of the Arachis hypogaea lectin and 11.11 fmoles of the Triticum vulgari lectin bound per buccal cell. This was reduced when Tc-99m-labelled Canavali ensiformis lectin was inhibited by the presence of 40% w/v glucose solution. It was estimated that a therapeutic dose of a potential dosage form could be conjugated to the Triticum vulgaris lectin within the oral cavity, thus demonstrating the feasibility of using this, and possibly other lectins in a drug delivery strategy. Preliminary in vivo studies in the conscious rat model suggested that the Tc-99m-labelled Canavalia ensiformis an Triticum vulgaris lectins were present within the oral cavity, 1 hand 2 h respectively after application, with about 25% of the Canavalia ensiformis lectin being lost into the stomach through swallowing. Transmission electron microscopy studies were initiated to determine the exact sites of lectins binding to the human buccal cell. Fresh, unfixed human buccal cells were incubated in a solution containing 20 nm gold-labelled Canavalia ensiformis lectin. was found that after processing, the lectin bound to the external surface of the buccal cells, or to external mucin. No binding was observed when pre-fixed human buccal cells were used, indicating that fixation affects the glucose/ mannose-containing binding site Canavalia ensiformis lectin binding to the buccal cells was partially inhibited by the presence of glucose, the hapten sugar. It was concluded that lectins will bind to the rat oral epithelial and human buccal cell surfaces to varying degrees, and will persist, even in the presence of saliva. In particular the lectin from Triticum vulgaris was shown to have good stability in the conscious rat model for up to 2 h, and shows great promise for use in a potential lectin-containing drug delivery system.

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Mycobacterium tuberculosis (Mtb) utilizes different metabolic pathways for its survival during infection. Enzymes of these pathways are often targets for antibiotic development. Genetic studies indicate the importance of the serine and biotin biosynthetic pathways for Mtb survival. In this study, enzymes from these pathways were characterized using X-ray crystallographic and biochemical studies. D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step of phosphorylated serine biosynthesis. In comparison to other forms of PGDH, the Mtb enzyme has an insertion near its C-terminus. This insertion results in two different conformations of the subunits in the tetramer, leading to two different environments for cofactor binding. This intervening domain might provide a second binding site for hydroxypyruvic acid phosphate (HPAP) that is responsible for substrate inhibition. Analysis of the HPAP-bound Mtb PGDH active site reveals the residues (Arg52, Arg131, and Arg233) involved in substrate interaction and provides insights into a possible enzyme mechanism. Mtb PGDH is feedback inhibited by the end product Lserine. Examination of the serine-bound PGDH structure elucidates the key players (Tyr461, Asp463, and Asn481) involved in this allosteric inhibition, as well as the resultant conformational changes at the regulatory domain interface. Preliminary biochemical studies of the first enzyme in Mtb biotin biosynthesis, 7- keto-8-aminopelargonic acid (KAPA) synthase show that it exists as a dimer in solution and has higher substrate affinity than the E. coli enzyme. The second enzyme, 7, 8-diaminopelargonic acid synthase (DAPAS) uses Sadenosyl methionine and KAPA as substrates in a bi-bi ping-pong mechanism. A comparison of the substrate analog sinefungin-bound Mtb DAPAS structure with a KAPA-bound DAPAS model provides a basis for the dual-substrate recognition. Tyr25 is a key player in the substrate specificity in DAPAS; this was confirmed by mutation studies. In certain Bacillus species, a Phe replaces this Tyr. The KAPA-bound B. subtilis DAPAS structure shows an alteration in the KAPA binding mode. Substrate and product bound structures of the third enzyme, dethiobiotin synthetase (DTBS) in Mtb reveal the important residues involved in its catalysis and provide framework for a possible enzyme mechanism. Comparison to the DTBS structures from E. coli and H. pylori reveals differences in local conformations.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 113-126).

Bibliography: leaves 218-227. / 227 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / A series of potential receptors for biotin derivatives based on the 2,6-pyridinedicarboxamide motifs were investigated. / Thesis (Ph.D.)--University of Adelaide, Dept. of Chemistry, 2000?