Selective recognition of cyclic GMP using a fluorescence-based molecularly imprinted polymer

Guanosine 3′,5′-cyclic monophosphate (cGMP) plays a role as a second messenger in many different biological systems. Given the ubiquitous nature of cGMP, a simple method of detecting cGMP is of interest. To that end a fluorescent polymer with recognition sites for cGMP has been prepared. Its selectivity and sensitivity were investigated and a dose-dependant decrease in fluorescence of the polymer in the presence of cGMP was observed. In contrast, virtually no effect was detected upon application of the structurally similar molecules, guanosine 5′-monophosphate (GMP) and adenosine 3′,5′-cyclic monophosphate (cAMP), thus demonstrating the high selectivity of this polymer. The association constant for the binding of cGMP to the imprinted polymer was determined in order of 3 × 10 5 M -1. A fluorescent, molecularly imprinted polymer that selectively recognises cGMP may have a useful application as a fluorescent chemosensor for cGMP detection in biological samples.

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology."

Microarray analysis of expression of cell death-associated genes in rat spinal cord cells exposed to cyclic tensile stresses in vitro

The application of mechanical insults to the spinal cord results in profound cellular and molecular changes, including the induction of neuronal cell death and altered gene expression profiles. Previous studies have described alterations in gene expression following spinal cord injury, but the specificity of this response to mechanical stimuli is difficult to investigate in vivo. Therefore, we have investigated the effect of cyclic tensile stresses on cultured spinal cord cells from E15 Sprague-Dawley rats, using the FX3000 Flexercell Strain Unit. We examined cell morphology and viability over a 72 hour time course. Microarray analysis of gene expression was performed using the Affymetrix GeneChip System, where categorization of identified genes was performed using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) systems. Changes in expression of 12 genes were validated with quantitative real-time reverse transcription polymerase chain reaction (RT-PCR).

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

Organic reactions in ionic liquids: N-alkylation of cyclic imides with alkyl halides promoted by potassium fluoride

An ionic liquid based on 1-butyl-3-methylimidazolium hexafluorophosphate is used as an efficient reusable reaction medium in the N-alkylation of cyclic imides with alkyl halides promoted by fluoride ion.

The cationic ring-opening polymerization of cyclic ethers

The kinetics and mechanisms of ring opening polymerization and copolymerizntion of different cyclic ethers were studied using mainly a cationic system of iinitiation. BF30Et2/ethanediol. The cyclic ethers reacted differently showing that ring strain and basicity are the main driving forces in cationic ring opening polymerizaion. In most cases it was found that the degree of polymerization is controlled kinetically via terminations with the counterion and the monomers, and that the contribution of each type of reaction to the overall termination differs markedly. The Gel permeation chromatography studies showed that the molecular weight distribution of the samples of polyoxetanes were bimodal. This was in accordance with previous work establishing that the cyclic tetramer is found in much higher proportions than any of the other cyclic oligomers. However the molecular weight distribution of the copolymers made from oxetane and THF or from oxetane and oxepane were shown to be unimodal. These observations could be explained by a change in the structure of the growing end involved in the cationic polymerization. In addition crown ethers like dibenzo-crown-6 and compounds such as veratrole are believed to stabilise the propagating end and promote the formation of living polymers from oxetane.

Studies of the cationic polymerisation of vinylic and energetic cyclic ether monomers

The cationic polymerisation of various monomers, including cyclic ethers bearing energetic nitrate ester (-ON02) groups, substituted styrenes and isobutylene has been investigated. The main reaction studied has been the ring-opening polymerisation of 3- (nitratomethyl)-3-methyl oxetane (NIMMO) using the alcohol/BF3.0Et2 binary initiator system. A series of di-, tri- and tetrafunctional telechelic polymers has been synthesised. In order to optimise the system, achieve controlled molecular weight polymers and understand the mechanism of polymerisation the effects of certain parameters on the molecular weight distribution, as determined by Size Exclusion Chromatography, have been examined. This shows the molecular weight achieved depends on a combination of factors including -OH concentration, addition rate of monomer and, most importantly, temperature. A lower temperature and OH concentration tends to produce higher molecular weight, whereas, slower addition rates of monomer, either have no significant effect or produce a lower molecular weight polymer. These factors were used to increase the formation of a cyclic oligomer, by a side reaction, and suggest, that the polymerisation of NIMMO is complicated with endbiting and back biting reactions, along with other transfer/termination processes. These observations appear to fit the model of an active-chain end mechanism. Another cyclic monomer, glycidyl nitrate (GLYN), has been polymerised by the activated monomer mechanism. Various other monomers have been used to end-cap the polymer chains to produce hydroxy ends which are expected to form more stable urethane links, than the glycidyl nitrate ends, when cured with isocyanates. A novel monomer, butadiene oxide dinitrate (BODN), has been prepared and its homopolymerisation and copolymerisation with GL YN studied. In concurrent work the carbocationic polymerisations of isobutylene or substituted styrenes have been studied. Materials with narrow molecular weight distributions have been prepared using the diphenyl phosphate/BCl3 initiator. These systems and monomers are expected to be used in the synthesis of thermoplastic elastomers.

The ring opening polymerization of ring strained cyclic ethers

The kinetics and mechanisms of the ring-opening polymerization of oxetane were studied using cationic and coordinated anionic catalysts. The cationic initiators used were BF30Et2!/ethanol, BF30Et2!/ethanediol and BF30Et2/propantriol. Kinetic determinations with the BF30Et2/diol system indicated that a 1: 1 BF3:0H ratio gave the maximum rate of polymerization and this ratio was employed to detenmne the overall rates of polymerization. An overall second-order dependence was obtained when the system involved ethanediol or propantriol as co-catalyst and a 3/2-order dependence with ethanol, in each case the monomer gave a first-order relationship. This suggested that two mechanisms accounted for the cationic polymerization. These mechanisms were investigated and further evidence for these was obtained from the study of the complex formation of BF30Et2 and the co-catalysts by 1H NMR. Molecular weight studies (using size-exclusion chromatography) indicated that the hydroxyl ion acted as a chain transfer reagent when the [OH] > [BF3]. A linear relationship was observed when the number average molecular weight was plotted against [oxetane] at constant [BF3:0H], and similarly a linear dependency was observed on the BF3:0H 1:1 adduct at constant oxetane concentration. Copolymerization of oxetane and THF was carried out using BF30Et2/ethanol system. The reactivity ratios were calculated as rOXT = 1.2 ± 0.30 and rTHF = 0.14 ± 0.03. These copolymers were random copolymers with no evidence of oligomer formation. The coordinated anionic catalyst, porphinato-aluminium chloride [(TPP)AICl], was used to produce a living polymerization of oxetane. An overall third-order kinetics was obtained, with a second-order with respect to the [(TPP)AICl] and a first-order with respect to the [oxetane] and a mechanism was postulated using these results. The stereochemistry of [(TPP)AlCl] catalyst was investigated using cyclohexene and cyclopentene oxide monomers, using extensive 1H NMR, 2-D COSY and decoupling NMR techniques it was concluded that [(TPP)AlCl] gave rise to stereoregular polymers.

Hydrogels containing linear and cyclic polyethers

Hydrogels are a unique class of polymer which swell, but do not dissolve in, water. A range of 2-hydroxyethyl methacrylate based copolymer hydrogels containing both cyclic and linear polyethers have been synthesised and are described in this thesis. Initially, cyclic polyethers were occluded within the polymer matrix and the transport properties investigated. The results indicated that the presence of an ionophore can be used to modulate ion transport and that ion transport is described by a dual-sorption mechanism. However, these studies were limited due to ionophore loss during hydration. Hence, the synthesis of a range of acrylate based crown ether monomers was considered. A pure sample of 4-acryolylaminobenzo-15-crown-5 was obtained and a terpolymer containing this monomer was prepared. Transport studies illustrated that the presence of a `bound' ionophore modulates ion transport in a similar way to the occluded systems. The transport properties of a series of terpolymers containing linear polyethers were then investigated. The results indicated that the dual-sorption mechanism is observed for these systems with group II metal cations while the transport of group I metal cations, with the exception of sodium, is enhanced. Finally, the equilibrium water contents (EWC) surface and mechanical properties of these terpolymers containing linear polyethers were examined. Although subtle variations in EWC are observed as the structure of the polyether side chain varies, generally EWC is enhanced due to the hydrophilicity of the polyether side chain. The macroscopic surface properties were investigated using a sessile drop technique and FTIR spectroscopy. At a molecular level surface properties were probed using an in vitro ocular spoilation model and preliminary cell adhesion studies. The results indicate that the polyethylene oxide side chains are expressed at the polymer surface thus reducing the adhesion of biological species.