No description available.

The occurrence of multiple adjacent G-quadruplex-forming motifs in the human genome, notably in the telomere, has promoted interest in how one adjacently positioned quadruplex affects another. A range of oligonucleotides were designed to feature one, two or three quadruplex-forming sequences and their biophysical properties were compared. CD, DSC, thermal denaturation experiments and supporting molecular simulations were the main techniques used. Sequences were based on the human telomeric sequence dAGGG(TTAGGG)3 and a parallel quadruplex-forming sequence dTGGGTGGGTTTGGGTGGG. Single and multiple telomeric quadruplexes formed similar topologies in equivalent quantities. Decreased thermal stability was observed for tandem quadruplexes. Divergence from a generally adequate two-state melting process was attributed to interaction between quadruplex subunits and domain-asymmetry. DSC analysis suggested that changes in heat capacity on folding were non-trivial and that quadruplex stabilities based on Van't Hoff enthalpy may be overestimated. Melting behaviour showed that topology of adjacent quadruplexes affects their interaction. Inter-quadruplex linker-length significantly affected topology and could induce domain asymmetry with 5' and 3' quadruplexes differing significantly in T m' Mixed telomeric and parallel quadruplex-forming sequences suggested that 5' quadruplexes were significantly destabilised by a 3' partner. Steric hindrance was a possible explanation. Quadruplex-binding ligands in the context of tandem quadruplexes were also investigated. Acridine derivative, RHPS4, increased thermal stability and induced topological changes in longer telomeric repeat sequences. A changed pattern of thermal stabilities hinted at an indirect effect on mutual quadruplex interactions via topological selection.

Chapter One introduces metalloenzymes and focusses on hydrogenase enzymes, which catalyse hydrogen production. Specific attention is given to the [NiFe] hydrogenases including their structures, redox states and possible catalytic mechanisms. Synthetic analogues of the active site of this enzyme reported in the literature are reviewed, including Ni thiolate complexes, [NiFe] complexes and macrocyclic complexes. The aims of this project conclude the chapter.

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CTCF is a highly conserved, ubiquitously expressed zinc-finger protein with diverse regulatory functions, binding to tens of thousands of the CTCF target sites (CTS) in the genome. CTCF interacts with a number of proteins including the largest subunit of RNA Polymerase H (LS Pol II), previously studied in our laboratory. Two sites within the CTCF Cterminal domain (Site 1 and Site 2) were shown to be involved in the CTCF-LS Pol II binding. The main aim of this study is to further characterize CTCF-LS Pol II interaction and its role in the regulation of CTCF function in transcription. To achieve this aim, seven mutant variants of CTCF, designed to be deficient for binding with the LS Pol II, were generated and characterized. They were then employed in functional assays to assess the implications of the CTCF-LS Pol II interaction in the regulation of CTCF known functions. The interaction of the wild type CTCF (wt CTCF) and the mutant variants with LS Pol II in vivo was investigated using an imaging technique, AP-FRET. All CTCF mutant variants were verified by sequencing and revealed similar stability, localization and molecular weights as the wt CTCF. The CTCF-LS Pol II interactions appeared to be involved in the regulation of the promoteriess luciferase gene fused to the CTSs (alone or in the context of the p14ARF promoter); mutants in the CTCF Site 2 or both sites interfered most with the luciferase activity. However, the effects of this interaction were not confirmed in the context of the endogenous genes, where the role of CTCF-LS Pol II association may be obscured by other influences. In AP-FRET experiments, two mutant variants of Site 1 (lA and IB) and one mutant variant of Site 2 (2A2B), were found to most likely be deficient for interaction with the LS Pol II.

No description available.

The present study investigated the in vitro antioxidant and ACE-inhibitory activities of egg albumen hydrolysate (EAR) prepared with pepsin and pancreatin enzymes. The EAR and peptides were purified by ultrafiltration (UF), gel filtration (GF), and High Performance Liquid Chromatography (HPLC) and tested for" antioxidant and ACE-inhibitory activities. Antioxidant activity was assessed by lipid peroxidation inhibition in a linoleic acid system using the ferric thiocyanate (FTC) and thiobarbituric acid reactive species (TBARS) methods. The EAR, and 2, 5 and 10 kDa UP fractions, as well as the GF26 peptide fractions (I mg/ml) inhibited linoleic acid autoxidation, by 40, 76, 63, 53 and 79 % respectively, which was inversely related to peptide fraction size. However, 0.01 % butylated hydroxytoluene (BHT) and 0.01 % trolox had higher activity (95 and 82 %, respectively) compared with the peptide fractions (p<0.05). Similarly, inhibition of TBARS was in the order 29, 39, 27, 17, 70 and 78 % for EAR, 2 kDa, ' 5 kDa, 10 kDa, trolox and BHT respectively. The putative antioxidant mechanism of EAR involved scavenging activity based on the I, l-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl (OH") and superoxide anion (02") radical scavenging assays, and ferric (Fe3+) reducing and ferrous (Fe2+) ion chelating activities in a dose-dependent manner. All peptide fractions exhibited ACE-inhibitory activity, based on the ACE-catalyzed liberation of hippuric acid from the hippuryl-L-histidyl-L-Ieucine residue, which improved on further GF and HPLC purification. The 2, 5 and 10 kDa peptides exhibited % ACE inhibitory activities of 78, 71 and 62 %, respectively, compared to the positive control captopril (96 %). The 2, 5 and 10 kDa peptides had ICso values of 6.01, 6.86 and 7.93 mg/ml respectively. Further, GF and HPLC purification of the 2 kDa peptides improved the ICso values to 5.76 and 5.13 mg/ml, respectively. Cell viability of human colon carcinoma mono-layer (caco-2) cell line, assayed by the tetrazolium dye (MTT) colorimetric assay confIrmed that the 2 kDa peptides were not toxic. The 2 kDa peptides (0.1 mg/ml) reduced most of the endogenous antioxidant enzyme activities in a dose-dependent manner, indicating scavenging of ROS. It was evident that a significant proportion of the 2 kDa peptides were resistant to cellular aminopeptidases present in caco-2 cell epithelium and were therefore transported in their intact forms across the caco-2 cell epithelium. In addition, The 2 kDa peptides exhibited significant ROS scavenging activity evidenced by enhanced viability of the Ea.hy926 (HUVECs) cell lines, using the lucigenin-enhanced chemiluminescent and fluorescence methods. The results confirm that bioactive peptides derived from the EAH have significant antioxidant and ACE·inhibitory activities and potentially useful neutraceutical applications.

No description available.

No description available.

Negative cooperativity refers to the mechanism whereby the sequential binding of ligands to a protein decreases in affinity. A protein's native state ensemble consists of numerous conformers and is dynamic. A ligand which induces negative cooperativity causes a shift in the distribution of this ensemble such that the subsequent molecules bind with less affinity; this requires a physically linked pathway through which the change is propagated. Positive cooperativity has been well documented but the mechanisms and advantages of negative cooperativity are less understood. To this end, the research presented here focuses on the mechanism of negative cooperativity in human NAD(P)H quinone oxidoreductase 1 (NQ01), human NRH quinone oxidoreductase (NQ02), MdaB from Escherichia coli and Lot6p from Saccharomyces cerevisiae. This thesis presents evidence to support the presence of a pathway linking the active sites in NQ01, through which, negative cooperativity towards the inhibitor dicoumarol propagates. Glycine 151 within this pathway has been identified as pivotal to this mechanism. Two cancer-associated variants of NQ01, p.P187S and p.R139W, have been characterised. Neither change affected negative cooperativity; the proline to serine change was the most severe in terms of structure and function. Two variants of human NQ02 have been compared. NQ02-47L is overall more flexible and exists as an ensemble containing three metastable states; NQ02-47F exhibits negative cooperativity. MdaB has been characterised and novel inhibitors identified. An alteration which decreased steric hindrance in the active site allowed for enhanced dicoumarol inhibition; however, unlike NQ01 , MdaB did not exhibit negative cooperativity towards this inhibitor. An additional alteration to increase flexibility did not enable the enzyme to exhibit negative cooperativity. Lot6p has been characterised and novel inhibitors identified some of which, induced negative cooperativity. An interconnecting pathway and a specific glycine within it have been identified as central to the mechanism of negative cooperativity.