Biomedical subjects
Publications and source records attributed to Christopher P Palmer.
This review concerns the introduction, characterization, and application of polymeric pseudostationary phases (PSPs) for EKC since 2004. Achiral and chiral polymers and separations are reviewed, as is the application of polymeric PSPs for the combination of EKC with mass spectrometric detection.
Prostate is a unique organ that produces and releases large amounts of citrate. This is reduced significantly in cancer and it is possible that citrate is (re)taken up and used as a metabolite to enhance cellular activity. The main purpose of this study was to determine how cytosolic citrate might affect in vitro metastatic cell behaviours (lateral motility, endocytosis and adhesion). Normal (PNT2-C2) and metastatic (PC-3M) human prostate cancer cells were used in a comparative approach. As regards intermediary metabolic enzymes, aconitase and fatty acid synthase, already implicated in prostate cancer, were evaluated. The level of intracellular citrate was significantly higher in PNT2-C2 cells under both control conditions and following preincubation in extracellular citrate. Supply of exogenous citrate enhanced endocytosis, lateral motility, decreased cell adhesion of PC-3M cells but failed to produce any effect on normal cells. Real-time PCR measurements showed that the mRNA levels of mitochondrial and cytosolic aconitases and fatty acid synthase were significantly higher in PC-3M cells. Correspondingly, aconitase activity was also higher in PC-3M cells. Using cerulenin (an inhibitor of fatty acid synthase), oxalomalate and fluorocitrate (inhibiting aconitases), we investigated the dependence of citrate-induced down-regulation of cellular adhesion on aconitase and fatty acid synthase activities. It was concluded: (1) that strongly metastatic PC-3M cells stored less/utilised more cytosolic citrate than the normal PNT2-C2 cells and (2) that cancer cells could metabolise cytoplasmic citrate via aconitase and fatty acid synthase to enhance their metastatic behaviour.
Sigma (sigma) receptors have been implicated in cancer. However, to date there is little molecular data demonstrating the role of sigma1 receptors in cancer. Expression of sigma1 receptors in various human cancer cell lines in comparison to non-cancerous cell lines was investigated, using real time RT-PCR and by western blotting with a sigma1 receptor specific antibody. Our results indicate that cancer cells express higher levels of sigma1 receptors than corresponding non-cancerous cells. Localization of the sigma1 receptor was investigated in MDA-MB-231 cells by immunocytochemistry and confocal microscopy, expression was visualized predominantly at the cell periphery. We have tested the effect of sigma1 and sigma2 drugs and a sigma1 receptor silencing construct on various aspects of the metastatic process on two breast cell lines of different metastatic potential and a normal breast cell line. Both sigma1 and sigma2 drugs and the sigma1 receptor silencing construct had effects on proliferation and adhesion for breast cancer cell lines, compared to a non-cancerous breast cell line. This data suggests sigma1 receptor plays a role in proliferation and adhesion of breast cancer cells. Therefore, it is likely to be a potential target for the diagnosis and therapy of breast cancer.
MEKC and linear solvation free energy relationships (LSFERs) have been used to characterize the solute distribution between water and self-assemblies formed from ionic liquid type mono-chain cationic surfactants containing a cyclic pyrrolidinium head group. Several features of the solvation environment afforded by these micellar solutions were found to be quite different from that of CTAB, a structurally analogous cationic surfactant with a conventional, acyclic quaternary ammonium head group. None of the LSFER coefficients were found to vary in any systematic way with increasing alkyl chain length for these unique surfactants. Overall, however, these surfactants display different behavior than do all known cationic detergents such as CTAB. In chemical terms, pseudo-phases formed by these N-alkyl-N-methylpyrrolidinium bromides interact more strongly with polar compounds and less strongly with compounds having nonbonding or pi-electrons and are more cohesive compared to the well-studied CTAB.
Separation selectivity in electrokinetic chromatography (EKC) is directly affected by the chemistry and solvent characteristics of the pseudostationary phase (PSP). The chemical selectivity of micellar PSPs has been previously demonstrated to vary significantly between anionic and cationic surfactants as well as between hydrocarbon and fluorocarbon surfactants. Polymeric PSPs have also been demonstrated to provide unique selectivity. In the current study, four cationic polymeric pseudo-stationary phases, two of which have perfluorinated pendant groups, are introduced and characterized as PSPs in EKC. Their performance and selectivity is compared to conventional micellar PSPs with similar structure. The solvation characteristics and selectivity of the four polymers most closely resemble those observed for cationic micelles. The polymers are all more cohesive and more polar than their hydrocarbon micellar counterparts. The fluorocarbon PSPs did show preferential interaction with fluorocarbon solutes, were somewhat more cohesive, and were stronger hydrogen bond donors. However, the presence of fluorocarbon moieties did not have as dramatic an effect on selectivity as was observed and published previously for fluorocarbon micelles. This may result from the selectivity being dominated by the presence of the cationic head groups or from the fluorocarbon character of the pendant groups being moderated by the presence of hydrocarbon functionality on the polymer backbones.
Ion channel genes have been discovered in many microbial organisms. We have investigated a microbial TRP (transient receptor potential) ion channel gene which has most similarity to polycystic-kidney-disease-related ion channel genes. We have shown that this gene (pkd2) is essential for cellular viability, and is involved in cell growth and cell wall synthesis. Expression of this gene increases following damage to the cell wall. This fission yeast pkd2 gene, orthologues of which are found in all eukaryotic cells, appears to be a key signalling component in the regulation of cell shape and cell wall synthesis in yeast through an interaction with a Rho1-GTPase. A model for the mode of action of this Schizosaccharomyces pombe protein in a Ca2+ signalling pathway is hypothesized.
Two polymeric pseudostationary phases, one an acrylamide polymer and the second a siloxane polymer, have been investigated for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized amino acids and small peptides. The dervatized amino acids were detected by UV absorbance and laser-induced fluorescence (LIF) detection. The polymers provided very high efficiency and good selectivity for the separation of the amino acids. The separation selectivity using the polymers was significantly different from that of SDS micelles, and there were subtle differences in selectivities between the polymers. Although very good detection limits were obtained with LIF detection, a significant background signal was observed when the polymers were not washed to remove fluorescent impurities. The polymers did not separate the peptides very well. It is postulated that the fixed covalent structure of the polymers prevents them from interacting strongly or efficiently with the peptides, which are large in relation to the analytes typically separated by electrokinetic chromatography using polymers.
The effect of the conformation of a polymeric pseudostationary phase on performance and selectivity in electrokinetic chromatography was studied using an amphiphilic pH-responsive polymer that forms compact intramolecular aggregates (unimer micelles) at low pH and a more open conformation at high pH. The change in conformation was found to affect the electrophoretic mobility, retention, selectivity, and separation efficiency. The low-pH conformer has higher electrophoretic mobility and greater affinity for most solutes. The unimer micelle conformation was also found to provide a solvation environment more like that of micelles and other amphiphilic self-associative polymers studied previously. It was not possible to fully characterize the effect of conformation on efficiency, but very hydrophobic solutes with long alkyl chains appeared to migrate with better efficiency when the unimer micelle conformation was employed. The results imply that polymers with a carefully optimized lipophilic-hydrophilic balance that allow self-association will perform better as pseudostationary phases. In addition, the results show that electrokinetic chromatography is a useful method for determining the changes in solvation environment provided by stimuli-responsive polymers with changes in the conditions.
Prostate is a unique organ which synthesizes and releases large amounts of citrate. It has been shown that in metastatic prostate cancer, the amount of citrate in prostatic fluid is significantly reduced, approaching the level normally found in blood. In our previous study, we characterized electrophysiologically the mechanism of citrate transport in a normal prostatic epithelial (PNT2-C2) cell line. It was concluded that the cells expressed a novel transporter carrying 1 citrate3- together with 4 K+, primarily out of cells. In the present study, we aimed similarly to characterize the mechanism(s) of citrate transport in a strongly metastatic human prostate cancer (PC-3M) cell line and to compare this with the previous data. Citrate transport in PC-3M cells was found to be both Na+ and K+ dependent. Intracellular application of citrate produced an outward current that was primarily K+ dependent whilst extracellular citrate elicited an inward current that was mainly Na+ dependent. The electrophysiological and pharmacological characteristics of the citrate outward current were similar to the K+-dependent citrate transporter found in the PNT2-C2 cells. On the other hand, the inward citrate current had a markedly different reversal potential, ionic characteristics, inhibitor profile and pH sensitivity. Preincubation of the PC-3M cells (24 or 48 h) with the voltage-gated Na+ channel (VGSC) blocker tetrodotoxin (TTX) significantly reduced the Na+ sensitivity of the citrate current, up-regulated VGSC mRNA expression but did not change the partial permeability of the membrane to Na+. It was concluded (a) that PC-3M cells express a K+-dependent transporter (carrying citrate outward), similar to that found in normal prostate epithelial cells, as well as (b) a Na+-dependent transporter (carrying citrate inward). The molecular nature of the latter was investigated by RT-PCR; the three known Na+-dependent citrate/dicarboxylate transporters could not be detected. VGSC activity, which itself has been associated with metastatic prostate cancer, had a differential effect on the two citrate transporters, down-regulating the expression of the Na+-dependent component whilst enhancing the K+-dependent citrate transporter.
The sigma (sigma) receptor and its agonists have been implicated in a myriad of cellular functions, biological processes and diseases. Whereas the precise molecular mechanism(s) of sigma receptors and their involvement in cancer cell biology have not been elucidated, recent work has started to shed some light on these issues. A molecular model has been proposed for the cloned sigma1 receptor; the precise molecular nature of the sigma2 receptor remains unknown. sigma receptors have been found to be frequently up-regulated in human cancer cells and tissues. sigma2 receptor drugs particularly have been shown to have antiproliferative effects. An interesting possibility is that sigma and/or sigma1 drugs could produce anticancerous effects by modulating ion channels. As well as proliferation, a variety of other metastatic cellular behaviors such as adhesion, motility, and secretion may also be affected. Other mechanisms of sigma receptor action may involve interaction with ankyrin and modulation of intracellular Ca(2+) and sphingolipid levels. Although more research is needed to further define the molecular physiology of sigma receptors, their involvement in the cellular pathophysiology of cancer raises the possibility that sigma drugs could be useful as novel therapeutic agents.
This article reviews the development, characterization and application of soluble ionic polymeric materials as pseudo-stationary phases for electrokinetic chromatography and as stationary phases for electrochromatography since 1997. Polymeric pseudo-stationary phases for electrokinetic chromatography, including cationic polymers, anionic siloxane and acrylamide polymers, polymerized surfactants (micelle polymers), and chiral polymers are reviewed. Also reviewed are suspended molecularly imprinted polymer micro-particles. Application of polymeric pseudo-stationary phases with electrospray ionization mass spectrometric detection is presented. Recent progress in the development and characterization of physically adsorbed stationary phases for electrochromatography using polymers of the same or similar chemistry is also reviewed.
This review concerns the development, characterization, and application of soluble ionic polymeric materials as pseudostationary phases for electrokinetic chromatography since 2002. Cationic polymers, anionic siloxanes, polymerized surfactants (micelle polymers), and chiral polymers are considered. The use of stable suspensions of polymer nanoparticles in electrokinetic chromatography is also reviewed.
Solubilization environment afforded by several of the novel allyl glycidyl ether-modified methylhydrosiloxane polymers are investigated using a common polycyclic aromatic hydrocarbon fluorescence probe, pyrene. The backbone of the polymer has been modified by the addition of an alkyl chain of varying length (either C8, C12, or C18) and to differing degrees of substitution. The nomenclature adopted for the purposes of these studies is as follows: "AGENT" represents the backbone polymer with no alkyl substitution, and "OAGENT", "DAGENT", and "SAGENT" are substituted with n-octyl, n-dodecyl, and n-octadecyl, respectively. The percentage of alkyl substitution is designated as 10, 15, and 20%. The pyrene polarity scale (defined as the ratio of the intensity of peak I to peak III) was used to determine the relative dipolarity of the cybotactic region provided by approximately 1 w/w% aqueous polymer solutions compared to 10 mM sodium dodecylsulfate (SDS) micellar solution. Results indicate that 10-15% DAGENT afforded the most hydrophobic solubilization site, followed by 15% OAGENT and 15% SAGENT. In addition, as the degree of alkyl substitution of DAGENT increased from 10 to 20%, the cybotactic region appeared to become more hydrophobic. Furthermore, a deeper investigation into the relative size of the solubilization site revealed that all alkyl-substituted polymers promoted excimer formation at relatively low pyrene concentrations, indicating the possibility of localized concentration enhancement within the solvation pockets and/or compartmentalization of the solute molecules. The pyrene fluorescence excitation data strongly indicates ground-state heterogeneity that is most prominent in AGENT and decreases as the alkyl chain length is increased. This provides a relative sense of the size and shape of the solvation pockets afforded by each polymer solution. An overall analysis of the collected data indicated that these alkyl-substituted polymers may provide a more selective and efficient pseudostationary phase in electrokinetic chromatography with better solvation capacity for hydrophobic compounds compared to SDS.
Anionic siloxane polymers with novel linker arm structures have been synthesized and characterized with respect to their performance and selectivity as pseudophases for electrokinetic chromatography. The linker arm between the siloxane backbone and the sulfonate head group is shorter and does not have the tertiary amine structure found in the siloxane pseudophases studied previously. This change in the linker arm structure and chemistry has dramatic effects on the chemical selectivity of the pseudophases. Linear solvation energy relationship (LSER) studies show that the greatest contributor to the difference in selectivity is that the new polymers are not as nonpolar as those previously studied. This result indicates that siloxane polymers are not by their nature more nonpolar or hydrophobic than other pseudophases. The LSER studies also demonstrate that siloxane pseudophases have a strong tendency to accept hydrogen bonds that cannot be attributed to the presence of the tertiary amine in the linker arm.
Anionic water-soluble siloxanes modified with different amounts of alkyl chains have been used as pseudostationary phases in electrokinetic chromatography. Ionic siloxane polymers with attached alkyl chains of C8 and C12 and having different alkyl chain densities have been employed previously to achieve selective and efficient separations with a range of electrophoretic mobilities and methylene selectivities. In this study, the performance of three alkyl-modified siloxanes is examined in different organic-modified buffers and at differing amounts of organic modifier. The organic modifiers used are acetonitrile and methanol. The siloxanes are stable in these organic solvents and show good mobility and good methylene selectivities even at high concentration of organic solvent. Siloxanes have also been used to separate a mixture of 14 polynuclear aromatic hydrocarbons in an acetonitrile-modified buffer.
The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.
Amphiphilic copolymers of 2-acrylamido-2-methyl-1-propane sulfonic acid (AMPS) with dihydrocholesteryl acrylate (pDHCHAt) or tert-octyl acrylamide (ptOAm) as comonomers were synthesized, characterized and used as new polymeric pseudostationary phases in electrokinetic chromatography. High-efficiency separations with theoretical plates around 500,000 to 870,000/m were achieved. Linear solvation energy relationships analysis of 20 solutes was performed to investigate the retention mechanism. Polycyclic aromatic hydrocarbons (PAHs) were separated under 30% v/v acetonitrile. The separation of nine aromatic model solutes by pDHCHAt showed significantly different selectivity from that of sodium dodecyl sulfate. Although only minor difference in selectivity was found for small aromatic compounds on pDHCHAt and poly(AMPS-lauryl acrylate), significantly different retention mechanism was found for PAHs and n-dodecanophenone comparing both copolymers. Significant chemical selectivity difference was found for selected small aromatic compounds on ptOAm and poly(AMPS-stearyl acrylamide).
This review article details the development, characterization and application of polymeric materials as pseudostationary phases for electrokinetic chromatography over the past two years. Recent developments in cationic polymers and anionic siloxane, acrylamide and polymerized surfactants (micelle polymers) are reviewed. Also reviewed is recent progress in the development and characterization of chiral polymeric phases for chiral separations by electrokinetic chromatography, and application of a polymeric pseudophase with electrospray ionization mass spectrometric detection.