The mechanisms of substrate recognition by regulatory proteases are not well understood. Presently, two opposing models have arisen to describe E. coil Lon's ability to discriminate between substrates: one suggests the N-terminus involvement while the second suggests the C-terminus involvement. In this project, the role of the C-terminal domain as it relates to the recognition of Lon's normal physiological substrates RcsA, an activator of colanic acid capsular polysaccharide, and SulA, an inhibitor of cell division, was addressed. Using site-directed mutagenesis, five mutations in Lon (R537G, E538A, GS40W, R542G, R542P) were isolated. Their phenotypic impact was either similar in character to wildtype Lon (R537G, E538A) or ��lon cells (G540W, R542G, R542P). The stabilization of both RcsA and SulA based on phenotypic assays and immunological detection of lon* strains (G540W, R542G, R542P) suggests the C-terminal domain may be involved in substrate degradation as opposed to discriminator activity. / Graduation date: 2002

Graduation date: 1998

Thesis (Ph. D.)--Oregon State University, 1971. / Typescript (photocopy). Includes bibliographical references. Also available online.

Thesis (Ph. D.)--University of Hong Kong, 2006. / Title proper from title frame. Also available in printed format.

Herein is presented the collective experimental record of research performed in the Laboratory for Biological Surface Science. These investigations are generally classified under the category of bioanalytical surface science and include the following projects. Chapters III and IV describe the creation of a microfluidic device capable of generating fixed arrays of concentration gradients. Experimental results were matched with computational fluid dynamics simulations to predict analyte distributions in these systems. Chapters V and VI demonstrate the discovery and utility of photobleaching fluorophores for micropatterning applications. Bleached fluorophores were found to rapidly attach to electron rich surfaces and this property was used to pattern enzymes inside microfluidic channels in situ. Finally, Chapter VII exhibits a method by which solid supported lipid bilayers can be dried and preserved by specifically bound proteins. The intrinsic property of lateral lipid mobility was maintained during this process and a mechanism by which the protein protects the bilayer was suggested.

<p>Alternative sources of fuel are required worldwide, and bio-ethanol is the leading candidate. Lignocellulosic biomass, a waste component of the agricultural industry, is a promising renewable source. Due to its complex structure it is highly recalcitrant, requiring the synergistic action of a battery of enzymes to achieve complete digestion. These enzymes include cellulases, hemicellulase and the accessory enzymes acetyl xylan esterase (AXE) and ferulic acid esterase (FAE). Thermpohilic Actinomyces isolates with the ability to hydrolyze xylan were screened for esterase activity. Two isolates (ORS10 and GSIV1), identified as Streptomyces spp, were positive for AXE activity. A cosmid library representative of isolate ORS10 was composed and screened for AXE activity using -naphthyl acetate as substrate. An 18 kb cosmid clone, 18D7, tested positive for AXE activity. Intracellular fractions extracted from ORS10 were precipitated with ammonium sulphate and partially purified 161-fold. Specific activity was measured after dialysis and ion-exchange chromatography. Overall yield of the partially purified enzyme was 34 %. Two protein bands of molecular masses 40 kDa and 60 kDa have been subjected to trypsin digestion and MALDI-TOF mass spectrometry analysis. The partially purified AXE displayed optimum activity at pH 9 and at 50&deg / C. AXE activity was stable for at least 1.5 hours between 30&deg / C and 40&deg / C, and for 24 hours between pH 6-9. The kM and Vmax values were 16.93 mg/ml and 1645 units/mg enzyme, respectively. The stability of the partially purified AXE at 30&deg / C-40&deg / C suggests potential for industrial applications that utilise mesophilic fermentations.</p>

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sans-serif&quot / mso-bidi-font-family:Helvetica">that flavonoids have important effects on cancer chemoprevention and therapy.</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">Flavonoids may interfere in several steps that lead to cancer development but may</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">also lead to toxicity as the inhibition of carcinogen-activating enzymes may also cause</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">potential toxic flavonoid-drug interactions. However, the potential toxicity of these</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">dietary components has not been well studied. The use of polyphenol-enriched</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">supplements prepared from South African herbal teas, rooibos (</span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">Aspalathus linearis</span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">)</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">and honeybush (</span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">Cyclopia </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">spp.) are being marketed to alleviate symptoms that are</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">known to be &ldquo / cured&rdquo / by the herbal teas. However, there is a lack of information</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">regarding the possible health promoting effects of these polyphenol-enriched extracts</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">on xenobiotic metabolism. In the present study, the modulating effects of aspalathinenriched</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">rooibos and mangiferin-enriched C. </span><i><span style="font-size:11.5pt / font-family: Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">genistoides </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">and </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">C. subternata </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">extracts on</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">the gene expression of xenobiotic metabolising enzymes (XMEs) were investigated </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">in</span></i> <i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">vivo </span></i><span style="font-size:11.5pt / font-family: &quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">in the rat liver and kidneys. An </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">in vitro </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">study, utilising a primary rat hepatocyte cell</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">model, was included to further evaluate changes in the expression of selected XMEs</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">by the herbal tea extracts, including their major polyphenolic constituents, aspalathin</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">and mangiferin. The use of the </span><i><span style="font-size:11.5pt / font-family: Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">in vitro </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">primary hepatocytes assay as a predictive cell</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">model for the modulation of the expression of XMEs genes by the herbal tea extracts</span> <i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">in vivo </span></i><span style="font-size:11.5pt / font-family: &quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">was critically evaluated.</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">In the liver and kidneys, the </span><i><span style="font-size:11.5pt / font-family: Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">C. subternata </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">polyphenol-enriched herbal tea extract</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">effected the majority of changes regarding the expression of XMEs genes when</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">compared to the rooibos and </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">C. genistoides</span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">. Variations in the modulation of gene</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">expression of the XMEs by the herbal tea extracts were related to differences in their</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">polyphenol constituents, although non-polyphenolic constituent could also be involved.</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">Overall the herbal teas regulated alcohol, energy, drug and steroid metabolism in the</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">liver, whereas in the kidneys the gene expression of phase I, phase II, steroid</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">metabolising enzymes, as well as drug transporters were modulated. It would appear</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">that the herbal teas are likely to exhibit both beneficial and adverse effects </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">in vivo</span></i><span style="font-size:11.5pt / font-family: &quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">, depending on the specific organ, the xenobiotic and/or drug that are involved. The</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">primary rat hepatocytes model display varying effects with respect to modulating gene</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">expression of specific XMEs by the polyphenol-enriched herbal tea extracts. Apart</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">from the reduction in 17<span style="font-size: 11.5pt / font-family:TT61t00 / mso-bidi-font-family:TT61t00">&beta / </span>-hydroxysteroid dehydrogenase gene expression care should</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">be taken to directly extrapolate the </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">in vitro </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">findings to changes that prevail </span><i><span style="font-size: 11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">in vivo</span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">.</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">However, interesting results regarding the masking effect of complex mixture on the</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">modulation of XME gene expression of individual polyphenols were encountered. In</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">addition differences in the dose and duration of exposure between the </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">in vitro </span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">and </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">in</span></i> <i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">vivo </span></i><span style="font-size:11.5pt / font-family: &quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">studies were not comparable and should be further explored to validate the </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family: Helvetica-Oblique">in vitro</span></i> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">primary hepatocytes model to predict changes </span><i><span style="font-size:11.5pt / font-family:Helvetica-Oblique / mso-bidi-font-family:Helvetica-Oblique">in vivo</span></i><span style="font-size:11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family: Helvetica">. Future studies should</span> <span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica">investigate the effects of the herbal tea extracts, its polyphenols and etabolites on</span><span style="font-size: 11.5pt / font-family:&quot / Helvetica&quot / ,&quot / sans-serif&quot / mso-bidi-font-family:Helvetica"> ME induction at a protein level as well as varying herb-drug-enzyme interactions.</span></p> <p>&nbsp / </p>

Thymidine kinase is a key enzyme in the nucleotide salvage pathway, catalyzing the production of dTMP from thymidine and ATP. In order to identify the structural features of the TK protein and/or primary amino acid sequences which contribute to the catalytic and regulatory activities of this enzyme, an in vitro transcription and translation system has been used in concert with protein engineering techniques for the production and phenotypic characterization of mutant and wild-type TK enzymes. Because of discrepancies in the literature regarding the quaternary structure of the VVTK, the native molecular weight and quaternary structure was determined to be an 80kDa homotetrameric enzyme by glycerol gradient fractionation, gel filtration and glutaraldehyde cross-linking analyses. Computer-assisted alignment of the predicted amino acid sequences derived from cellular and poxvirus TK genes identified seven highly-conserved domains distributed throughout the VVTK polypeptide (domains I-VII). Domain I (amino acid residues 11-18 ) exhibits a high degree of similarity to both ATP and GTP binding site consensus sequences, although the VVTK utilizes only ATP as a phosphate donor. Site directed mutagenesis and ATP-agarose affinity chromatography techniques were employed to confirm that this region was responsible for ATP binding and to determine which amino acids were essential for efficient binding. The TK gene (tdk) from E. coli was isolated and sequenced to serve as a prokaryotic enzyme with which to compare VVTK. The alignment revealed only 23% shared identity with VVTK and, interestingly, the identical and similar residues were clustered into three of the seven domains identified previously (domains I, III and VII). Preliminary evidence supports domain III (residues 78-90) as a putative magnesium binding site and that a highly conserved cysteine residue (cysteine 170) within domain VII (residues 168-171) may be a component of the catalytic site. Secondary structure alignment between Herpes Simplex Virus (HSV) TK and monkeypox TK (a close relative of VVTK) revealed that the putative nucleoside binding site of HSVTK aligns with residues within domain IV. Replacement of a VVTK residue (Q114) with the corresponding residue of HSVTK (an aspartic acid) greatly alters the substrate specificity and dTTP sensitivity of VVTK. / Graduation date: 1991

No description available.

No description available.