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published_or_final_version / abstract / Botany / Master / Master of Philosophy

Condensed tannins (CT) are polyphenolic compounds that have demonstrated biological activities in ruminants including suppression of enteric methane (CH4) production, protein binding and suppression of gastrointestinal nematode (GIN) infections. Some forage CT have been reported to be biologically active, whereas others have demonstrated no biological activity at all. While the chemical structure of CT has been postulated to be a key contributing factor affecting biological activity, the specific factors that determine whether or not CT from a specific forage have bioactive properties remain unknown. Results from previous studies have shown that as molecular weight of CT increases, CT biological activity also increases. Others have reported no effect of CT molecular weight on biological activity. The relationship between molecular weight of CT and CT biological activity remains inconclusive. The effect of molecular weight of CT from a variety of warm-season perennial legumes commonly consumed by ruminants on biological activity has not been adequately explored. The objectives of this study were to determine if molecular weight of CT from warm-season perennial legumes could predict the biological activity of CT relative to suppression of enteric CH4 production, protein-binding ability (PB) and anthelmintic activity, and to compare the biological activity of CT from native warm-season perennial legumes to that of the introduced species Lespedeza cuneata, a plant that has gained attention in recent years due its anthelmintic properties. All or a combination of the following warm-season perennial legume species were evaluated for in vitro gas production, protein-precipitable phenolics (PPP) and PB, and percent larval migration inhibition (LMI). Eight North American native warm-season perennial legumes: Leucaena retusa Benth. (littleleaf leadtree), Desmanthus illinoensis (Michx.) MacMill. Ex B.L. Rob. & Fernald (Illinois bundleflower), Lespedeza stuevei Nutt. (tall lespedeza), Mimosa strigillosa Torr. & A. Gray (powderpuff), Neptunia lutea (Leavenworth) Benth. (yellow puff), two ecotypes of Acacia angustissima var. hirta (Nutt.) B.L. Rob (prairie acacia), Desmodium paniculatum (L.) DC. var. paniculatum (panicledleaf ticktrefoil), and two introduced legumes: Arachis glabrata Benth. (rhizoma peanut) and Lespedeza cuneata (Dum. Cours.) G. Don (sericea lespedeza) were included. In vitro CH4 production regressed on CT MW resulted in a R2 of 0.0009 (P = 0.80). There was no correlation between PPP or PB and MW of CT (R^2 0.11; P = 0.17 and R^2 0.02; P = 0.54, respectively). There was a weak correlation between CT MW and percent LMI (R^2 0.34; P = 0.05). The results of our study strongly suggested that CT MW does not explain the biological activities of enteric methane suppression or protein-binding ability. Condensed tannin MW may be involved in anthelmintic activity of CT from the forage legumes surveyed. North American native legumes containing biologically active CT, as compared to introduced species, were identified as having promise for use in ruminant diets.

In eukaryotes, the endoplasmic reticulum is the site where folding of secretory proteins and the assembly of multimeric cell surface receptors take place. These processes are mediated by molecule chaperones that include the ER membrane bound chaperone calnexin and the sequence related calreticulin. Using a PCR strategy, a homologue for the mammalian calnexin/calreticulin family, CNE1, was isolated in S. cerevisiae. The CNE1 gene product, Cne!p, is an integral membrane glycoprotein of the ER. Disruption of the CNE1 gene did not lead to inviable cells or to gross effects on the levels of secreted wild type proteins. However, in CNE1 disrupted cells, there was an increase in the cell-surface expression of a normally intracellularly retained temperature sensitive mutant of the $ alpha$-pheromone receptor, Ste2-3p. In addition, an increase in the secretion of heterologously expressed mammalian $ alpha sb1$-antitrypsin was also observed in CNE1 disrupted cells. In order to study calnexin function in another genetically manipulable organism, a Schizosaccharomyces pombe calnexin homologue was sought. Using a similar PCR strategy, a S. pombe calnexin homologue, $cnx1 sp+$, was identified. The $cnx1 sp+$ gene product, Cnx1p, was shown to be a calcium binding type I integral membrane glycoprotein. Unlike the sequence related S. cerevisiae CNE1 gene, the $cnx1 sp+$ gene was essential for cell viability. Full length Cnx1p was able to complement the $cnx1 sp+$ gene disruption but full length mammalian calnexin could not. The ER lumenal domain of Cnx1p, which was secreted from cells, was capable of complementing the $cnx1::ura4 sp+$ lethal phenotype. Both wild type PI M1 (Val 213) $ alpha sb1$-antitrypsin and the ER retained PI Z variant were expressed in S. pombe cells. As in mammalian cells, wild type $ alpha sb1$-antitrypsin was normally secreted whereas the PI Z variant was retained intracellularly. Rescue of the secretion defective phenotype of the PI Z variant occurred in

The molecular biology of human genetic disorders is under intensive investigation at present. In those cases where the disorder is clearly defined in terms of altered gene structure, possibilities may exist for the correction of the disorder by insertion of normal genes through the process of DNA transfection. A possible method for the transfer of genetic material is by attempting to attach DNA to a protein which has specific receptors on cells and which undergoes receptor-mediated endocytosis. By this means one might be able to get DNA into cells. This thesis deals with experimental work on the chemical modification of human serum transferrin by means of water-soluble carbodiimides. The resulting N-acylurea transferrins bind DNA in a reversible manner. Characteristics and properties of the binding interactions are dealt with in detail. N-acylurea derivatives of transferrin were prepared with the water-soluble carbodiimides, N-ethyl-N' -(3-dimethylaminopropyl) carbodiimide and N-ethyl-N' -(3-trimethylpropylammonium) carbodiimide iodide. Reactions were carried out under mild conditions at room temperature for 48-72 hours. [³ H] N-ethyl-N' -(3-trimethylpropylammonium)carbodiimide iodide was used for the determination of covalently attached N-acylurea groups in the protein. Changes in charge properties were determined by agarose gel electrophoresis. Carbodiimide modification of proteins is thought to occur at side chain carboxyl groups of glutamic and aspartic acid residues. This was confirmed by the use of Staphylococcus aureus V8 protease, which cleaves peptide bonds at the carboxyl side of glutamic and aspartic acid residues, but not in the case of substituted side chain carboxyl groups. Through the use of puromycin as a nucleophile it has been shown that other functional groups were not activated upon reaction of transferrin with carbodiimide. The carbodiimide-modified proteins bind various types of DNA and RNA in a reversible manner. Low concentrations of N-acylurea transferrin retarded the migration of pBR322 DNA, M 13 mp 8 single-stranded DNA and Pst 1 restricted lambda DNA on agarose gel electrophoresis, while at higher concentrations the DNA was unable to enter the gel. Nitrocellulose filter binding assays showed that binding of DNA to Nacylurea transferrins was rapid, dependent on concentration of the modified transferrin and sensitive to ionic conditions. Binding was found to occur mainly through electrostatic interactions between phosphate groups of DNA and N-acylurea groups. These conclusions were based on experiments which showed that protein-DNA complexes were dissociated by increasing salt concentrations and by heparin. Non-electrostatic interactions such as hydrophobic interactions and hydrogen bonding are also involved in binding, since half dissociation of complexes, induced by chaotropic salts, KSCN and NaC10₄occurs at lower concentrations of salt than in the case of NaCl. Also RNA polynucleotides inhibit binding of DNA to Nacylurea transferrins to varying extents. The N-acyl urea transferrins have been shown to bind certain specific restriction endonuclease cleavage sites on pBR322 DNA. The N-acylurea transferrin-DNA complexes would thus be suitable for experiments in cell transfections using cells which have transferrin receptors. / Thesis (M.Sc.)-University of Durban-Westville, 1986.

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Copy of author's previously published article in pocket on back end-paper. / Includes bibliography (leaves 135-185). / 185, [94] leaves, [32] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to characterise the expression of PETA-3 (Platelet Endothelial Tetraspan Antigen-3), CD9, CD63 and ?gb?s1 integrins in normal human tissue ; to determine the subcellular localisation in endothilial cells and platelets ; to investigate protein-protein interactions involving PETA-3 ; and to examine the effects of anti-PETA-3 monoclonial antibodies on platelet and endothilial cell function. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1999

Thesis (M.S.)--West Virginia University, 2007. / Title from document title page. Document formatted into pages; contains vii, 124 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 116-124).

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 129-141). Also available for download via the World Wide Web; free to University of Oregon users.

Thesis (Ph. D.)--University of Oregon, 2007. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 81-88). Also available for download via the World Wide Web; free to University of Oregon users.