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No description available.

The effects of structurally diverse agonists were assessed on receptor-mediated activation of G-proteins using in Chinese hamster ovary cells stably expressing similar densities of M1 and M3 mACh receptors. Using a total [35S]-GTPgammaS binding protocol both receptor subtypes were shown to couple to both pertussis toxin-sensitive and -insensitive G-proteins. M1 mACh receptors coupled with greater potency and intrinsic efficacy than M3 receptors, indicating that in this cell-line that M1 mACh receptor may be more efficiently coupled to its complement of G-proteins. Total [36S]-GTPgammaS binding concentration-response curves for both receptor subtypes were shallow and, in the case of the M1 receptor, could be readily resolved into high and low affinity components. The heterogeneity of Galpha subunits activated was further investigated using an immunoprecipitation technique. This strategy revealed that agonist binding to M1 receptor caused the activation of a heterogeneous population of G-proteins, which was directly related to agonist efficacy. Full agonists were able to activate both Galphaq/11 and Galphail-3 subtypes, whereas partial agonists activated only the most efficiently coupled G-protein Galpha q/11. In contrast, the complement of G-proteins activated after M3 receptor stimulation was not related to the efficacy of the agonist and suggested that agonists may form active conformations of the M 3 receptor that possess different G-protein coupling profiles.;To investigate the G-protein coupling of M1 and M3 receptors further, homologous point mutations were introduced in both subtypes, which conferred agonist-independent constitutive activity. All of the antagonists tested were able to concentration-dependently inhibit basal [3H]-inositol phosphate accumulation and were therefore classified as 'inverse agonists'. However, subtle differences in the results obtained for different inverse agonists via different functional and binding readouts suggested the existence of more than one conformation of inactive receptor.

Typically, levels of inhibitor of DNA binding 1 (IdI) are high in stem cells and decrease upon senescence or differentiation. Defining how IdI expression is regulated is critical to understanding what happens at the molecular level as cells differentiate during development and age in the adult organism. It has been proposed that zinc finger E-box binding protein (Zeb)l is a repressor of the pluripotency regulator Nanog and that IdI maintains embryonic stem (ES) cell pluripotency by inhibiting Zebl expression. However, ablation of Id1 in mouse ES cells did not alter Zebl protein levels. Somatic cells can be reprogrammed to pluripotency. The initiation of reprogramming without feeder cells was impaired in Id1 -/- and Id3 -/- MEFs. However, no difference was observed between WT and Id1 -/- in the presence of feeder cells. As MEFs were maintained in vitro and approached senescence, Idl protein, but not transcript, decreased. microRNAs are short, non-coding RNAs that inhibit mRNA expression by binding to the 3' untranslated region (UTR), resulting in either degradation or translational repression of the transcript. The 3' UTRs of mouse Idl - Id4 were cloned into a reporter vector. Reporter transcripts were repressed by the Idl 3' UTR, unlike other family members. A 67 base region of the IdI 3' UTR that was necessary for repression contained a predicted miR-381 / miR-539-3p binding site. A single point mutation at this site relieved repression by the Idl 3' UTR in a reporter vector. However, attempts to modulate activity of these microRNAs did not alter repression by the IdI 3' UTR in a reporter vector, or Id l protein levels. The present work has identified a previously unknown regulatory element located in a highly conserved region of the 3' UTR of Idl. Whereas the mechanism of regulation is unclear, tools have been generated that will allow manipulation of this site in a variety of cell lines.

The bioactive lipid sphingosine-1-phosphate (SIP) has the unique characteristic of being capable of engaging in both paracrine/autocrine signalling and intracrine signalling pathways. Research into the latter has been limited by the failure to identify downstream targets for intracellular S1P. The majority of cellular responses to S1P are mediated by a family of G-protein coupled receptors (GPCR) known as S1P receptors: SIP₁₋₅. The current study demonstrates that SIP₅ and both sphingosine kinases (SPHK1 and SPHK2) exhibit a unique intracellular localization in the centrosome of mammalian cells.

Minor Groove Binders are a group of compound that bind to the minor grooves of DNA. Some of these compounds are able to bind to specific sequences on DNA, causing toxicity to cells, and this has generated an interest in their use as antimicrobial or anticancer agents. They are generally characterised by their recognisable crescent shape. The focus of this research is the effect of two minor groove binders AIK 19/56/2 and AIK 20/25/1 on the survival of various bacterial cell types as well as mammalian and fungal cells. This has been examined using growth curves, killing curves, fluorescent microscopy, HPLC spectra and topoisomerase I inhibition have been used. These experiments have given an insight into the mechanism of action of these compounds.

Telomeres are solely responsible for the protection of linear chromosome ends. Each is a nucleoprotein complex that prevents chromosome end-to-end fusions and solves the end replication problem. Six different proteins types are involved and form a complex called Shelterin. Three of these specifically recognise the 5'-TTAGGG-3' tandem repeat DNA sequence: TRFl and TRF2 recognise the double-stranded region and POTl binds to the 3' single strand overhang. Characterisation of DNA recognition by these proteins and subsequent behaviour once associated is currently limited and conflicting. This is due in part to the incapability of conventional methods to capture detailed dynamic information at the atomistic level. In this thesis molecular modelling is employed to more fully explore the potential energy surface of each telomeric protein-DNA complex. An unprecedented level of recognition detail is accessed by molecular dynamics, which reconciles previous characterisations. New interactions emerge and time-dependent analysis predicts that some previously proposed contacts are not biologically significant. Hydrophobic and water-mediated interactions are measured, together with direct and indirect readout. This ensemble of contacts I I I I , is the first complete model for TRF-DNA recognition. TRFl and TRF2 are shown to differ, both in terms of recognition and DNA remodelling behaviour. TRF2 relies much less on indirect readout suggesting it is better suited to unusual DNA structures than TRF 1. Difference in sequence means that TRF2 develops a strong hydrophobic patch not seen in TRF 1 and this causes a change in binding mode resulting in DNA distortion. This distortion may contribute to the explanation for functional differences observed for TRF 1 and TRF2. Single strand binder POTl is shown by molecular dynamics to be able to disrupt the quadruplex structure adopted by single-stranded telomeric DNA to minimise nucleoside solvent exposure. An initial recognition site for quadruplex DNA on the surface of POTl is also determined using molecular fitting. The unfolding mechanism involves a protein groove flanked by lysines. Once accepted into the groove, the quadruplex is uncoiled by lysine penetrating the electron rich central channel, ejecting stabilising potassium IOns.

The spindle assembly checkpoint is a cell-cycle surveillance system which ensures the mother cell equally and faithfully separates its replicated DNA contents into two daughter cells before the onset of anaphase. Mad2, BubRl and Cdc20 (Fzy in Drosophila melanogaster) are all kinetochore proteins which dynamically turnover on and off the mitotic kinetochores. It is believed that these proteins are forming a diffusible kinetochore anaphase inhibitory signal for spindle checkpoint function, inhibiting APC/C activity in order to delay the metaphase-anaphase transition (Musacchio and Salmon, 2007). APC/C is an E3 ubiquitin ligase. It ubiquitinates numbers of cell cycle regulating proteins, such as Cyclin B and Securin, and allows them to be targeted by proteasomes for destruction (Peters, 2006). However, the mechanism of the spatial and temporal interaction of these checkpoint proteins (Mad2, BubRl and Cdc20/Fzy) on kinetochores, and how they assemble to form this inhibitory signal remains unclear.