Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Darla J. Sherrier, Dept. of Plant and Soil Science. Includes bibliographical references.

No description available.

Plant cells resistant to specific amino acid analogs have been reported to accumulate the corresponding free amino acids. The purpose of this study was to determine the concentrations of fifteen free amino acids: alanine, valine, leucine, isoleucine, glutamate, proline, arginine, aspartate, threonine, methionine, lysine, serine, glycine, tryptophan and phenylalanine in Daucus carota cell lines, resistant either to the proline analog azetidine-2 carboxylic acid (A2C), or to the tryptophan analog 5-methyltryptophan (5-MT), or to both the analogs combined. This study also intended to determine if these analogs influence the biosynthesis of the above-mentioned fifteen amino acids in the cell line resistant to A2C and 5-MT. Carrot cell lines resistant to 5-MT, to A2C, or to both the analogs were selected by incubating carrot cells in liquid growth media containing either 0.3 mM 5-MT, or 0.5 mM A2C for 6 to 16 weeks. Free amino acid concentrations were then determined in the extracts of the cells. Resistance to 5-MT resulted in significant increases in the intracellular concentrations of tryptophan, phenylalanine, leucine, valine, isoleucine, and proline. Resistance to A2C resulted in significant increase in proline only. Resistance to both the analogs caused increases in proline, lysine, phenylalanine, and tryptophan concentrations. In the cell line resistant to both the analogs, the treatment with 5-MT caused increases in leucine, proline, aspartate, threonine, lysine, and tryptophan. The treatment with A2C caused increases in isoleucine, arginine, threonine, methionine, lysine, and glycine, whereas treatment with both the analogs caused increases in threonine, lysine, phenylalanine, and tryptophan. These results indicate the possibility of a common biosynthetic control of a number of amino acids in carrot cells, resembling that found in microorganisms. It is also evident from the results that the analogs play an active role in the biosynthesis of amino acids in the resistant cell lines.

Soyabean meal (SBM), the major protein source in feeds in sub-Saharan Africa, is in short supply. The shortage is a major constraint to intensified animal production to meet increased demand hence the dire need to search for alternatives. Kigelia africana, Mumisops zeyheri, Terminalia sericea and Ximenia caffra are indigenous fruit bearing trees (IFBTs) whose seeds’ potential as alternative protein sources in feeds were evaluated. The evaluation consisted of an initial physico-chemical characterization of the seeds followed by determining in vitro the safety of seed oils on cell lines. Based on the physico-chemical and in vitro evaluation, the most suitable seed was selected, defatted and its meal used as a dietary substitute to SBM in the in vivo trials using adult and weanling male Sprague Dawley rats. The T. sericea seed yield was not viable. Chemically K. africana and X. caffra seed demonstrated potential as protein sources in feeds. M. zeyheri seed demonstrated potential as an energy source. The IFBTs seeds oil yield surpassed that of some traditional oilseed crops. Oleic and linoleic acid were the major fatty acids contained in the oils. In vitro, K. africana, M. zeyheri and X. caffra seed oils suppressed Caco-2 and HEK-293 cell proliferation without causing cell death. X. caffra seed, deemed the most suitable, was defatted and its seed meal used in the in vivo trials. In mature rats, dietary substitution of SBM with the defatted X. caffra seed meal did not affect (P > 0.05) dry matter intake, apparent digestibility of nutrients and nitrogen absorption and retention. In weanling rats, the defatted X. caffra seed meal had no effect on termination (body mass at the end of the feeding trial) and empty carcass mass and linear growth of the rats. Metabolic substrate storage, fasting blood glucose concentration and the general health profile of the growing rats were not altered by dietary X. caffra seed meal. The defatted X. caffra seed meal increased the mass of the stomach and small intestine (P = 0.0071; P = 0.0001) of rats on the test diet where a 100% dietary crude protein (CP) from SBM was substituted by CP from the defatted X. caffra seed meal. Defatted X. caffra seed meal could substitute SBM in rat and possibly monogastrics feeds without compromising digestibility, nitrogen balance, growth and general health.

Shen, Jinbo. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 110-119). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Plant cell walls determine cellular shape and provide structural support for the entire plant. Polysaccharides, comprising the major components of the wall, are actively remodelled throughout development. Xyloglucan endotransglucosylase (XET)/hydrolase (XTH, EC 2.4.1.207) cleaves xyloglucan (XyG), the donor substrate, and attaches a portion to another XyG chain, the acceptor substrate. Recently, a novel transglucosylase called mixed-linkage β-glucan (MLG) : XyG endotransglucosylase (MXE) was discovered in horsetails (Equisetum spp.) that could attach a portion of MLG to XyG, resulting in a hetero-polymer product. My aims were to further investigate the nature of this activity, biochemically characterize the enzyme, and explore its physiological role. MXE activity was attributable to an enzyme unlike Equisetum XTHs. MXE had a p1 of 4.1 (XTHs were 6.6-9), a pH optimum of 6.3 (XTHs preferred 5.5), and had higher activity using smaller oligosaccharide acceptor substrates like XXXGol (XTHs were more active using XLLGol). Importantly, the MXE protein was shown to utilize both MLG and XyG as donor substrates, and therefore have both MXE and XET activity. Also, the enzyme was capable of using various glucan oligosaccharides (O) as substrates, including MLGO, XyGO, and cello-O, but not laminari-O. By using a novel ex vivo approach, the proportion of extractable MXE product to XET product was found to increase in older tissues. Transglucosylase products were localized in sclerenchyma and structural parenchyma by in situ assays, implying a strenghening function for MXE. Surprisingly, another novel activity was discovered that could covalently attach cellulose to XyG, and termed cellulose : xyloglucan endotransglucosylase (CXE). This activity was attributed to the MXE enzyme, implying that the protein is a promiscuous endotransglucosylase. The presence of CXE in other plants has not yet been tested. Besides being a novel discovery in plant cell biology, the modification of cellulose has applications in a number of industries.

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

Published works [representing] original research conducted during the various phases of [his] academic development--Pref / Includes references / 1 v. (various pagings) : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (D. Sc.)--Faculty of Science, University of Adelaide, 1990

A common problem observed in large-scale cell cultivation is reduced culture performance compared with small-scale processes due to the existence of concentration gradients caused by poor mixing. Small-scale simulations using microbial cell suspensions have shown that circulation of cells through concentration gradients of oxygen, pH and glucose can result in reduction of cell growth and product formation similar to the effects observed in large-scale bioreactors. This study was aimed at using scale-down studies to investigate poor mixing in large-scale bioreactors used for suspended plant and animal cell culture. Two plant cell suspensions and a hybridoma cell line were used in this work. The range of oxygen transfer coefficients achieved in the hybridoma and plant suspensions were about 50???20 h-1 and 12???6 h-1, respectively. One-vessel simulation was developed to induce fluctuations of dissolved oxygen tension in a 2-L bioreactor using intermittent sparging of air and nitrogen. The effect of dissolved oxygen fluctuations on the cells was examined by comparing the performance of the cultures with those operated at constant dissolved oxygen tension. In the hybridoma suspension culture, only slight effects on cell growth were observed at circulation times above 300 s. No effect on the specific glucose uptake rate or antibody production was observed at the circulation times tested. Analysis of gene expression for selected hypoxia-related genes also suggested that the overall effect was limited. In plant cell suspensions, the specific growth rates and biomass yields on total sugar in the cultures under fluctuating dissolved oxygen tension were essentially the same as those at constant dissolved oxygen tension for both transgenic Nicotiana tabacum and Thalictrum minus. Under fluctuating dissolved oxygen tension, no effect on antibody accumulation was observed in transgenic N. tabacum suspensions, but a decrease in berberine accumulation was observed in T. minus. From the results, it can be concluded that only minimal effects due to the development of concentration gradients would be expected in large-scale bioreactors used for the cultivation of the hybridoma and plant cell suspensions tested in this work.

The thesis begins with an introduction to signal transduction and an analysis of current understanding of plant signal transduction. There are similarities between plants and animals, but also key differences, including lack of protein kinase C and of a cAMP signaling pathway in plants, and presence in plants of calcium dependent protein kinase (CDPK), which has a kinase catalytic domain contiguous with a C-terminal calmodulin-like domain. The next section examines protein kinase activity in the plasma membrane (PM) of zucchini hypocotyls. Zucchini PM contains four or more polypeptides with calcium-requiring protein kinase activity. The enzymes appear to be tightly associated with the PM, and at least three are recognized by monoclonal antibody to soybean soluble CDPK. Total proteins from several different organs of zucchini seedlings contain kinases with molecular weights similar to the hypocotyl PM enzymes. In the third section details of partial purification of the solubilized PM kinases are presented. Kinases which do not crossreact with anti-CDPK monoclonal antibody were resolved by anion exchange from ones which do crossreact. Peptide mapping was used to test the relationship between the kinases. Results of peptide mapping suggest that at least three types of protein kinase are present in zucchini PM, two of which are immunologically similar to CDPK and one of which is not. The last section concerns the potential for testing interactions between PM protein kinases and plasma membrane auxin binding proteins (ABP's) by use of photoaffinity labeling of ABP's. Causes of variable photoaffinity labeling by an azido-IAA are considered. Labeling of both the tomato mutant diageotropica and the parent VFN membranes was inexplicably inconsistent. / Graduation date: 1994