Biomedical subjects

Paul O'Shea

Publications and source records attributed to Paul O'Shea.

13 recordsLinked to original sources

PubMed2006

Visualizing the solubilization of supported lipid bilayers by an amphiphilic peptide.

The effect of the presequence peptide of cytochrome c oxidase subunit IV (p25) on supported phospholipid bilayers (SPBs) was visualized using atomic force microscopy (AFM). The presequence was found to cause the complete disruption of supported bilayers containing neutral lipids. At relatively low concentrations of presequence, the peptide was found to bind to the membrane, coalescing to form microdomains within the liquid-crystalline bilayer that were located predominantly at bilayer-mica boundaries. Further increases in peptide concentration resulted in the formation of holes within the SPB that were spanned by an interpenetrating network of narrower regions of the bilayer, which, at higher applied peptide concentrations, were observed to disappear through a budding process, ultimately leading to the formation of spherical structures at yet higher peptide concentrations. Within this paper, the impact the presequence has upon the structure and order of the membrane is discussed, as is the potential implication of this apparent solubilization process on the translocation of cytochrome c oxidase into the inner mitochondrial membrane.

Electron Transport Complex IV↗

PubMed2006

N-acylhomoserine lactones antagonize virulence gene expression and quorum sensing in Staphylococcus aureus.

Many gram-negative bacteria employ N-acylhomoserine lactone (AHL)-mediated quorum sensing to control virulence. To determine whether gram-positive bacteria such as Staphylococcus aureus respond to AHLs, we used a growth-dependent lux reporter fusion. Exposure of S. aureus to different AHLs revealed that 3-oxo-substituted AHLs with C10 to C14 acyl chains inhibited light output and growth in a concentration-dependent manner, while short-chain AHLs had no effect. N-(3-Oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) inhibited the production of exotoxins and cell wall fibronectin-binding proteins but enhanced protein A expression. Since these processes are reciprocally regulated via the S. aureus agr quorum-sensing system, which in turn, is regulated via sar, we examined the effect of AHLs on sarA and agr. At sub-growth-inhibitory concentrations of 3-oxo-C12-HSL, both sarA expression and agr expression were inhibited, indicating that the action of 3-oxo-C12-HSL is mediated at least in part through antagonism of quorum sensing in S. aureus. Spent culture supernatants from Pseudomonas aeruginosa, which produces both 3-oxo-C12-HSL and N-butanoyl-homoserine lactone (C4-HSL), also inhibited agr expression, although C4-HSL itself was inactive in this assay. Since quorum sensing in S. aureus depends on the activities of membrane-associated proteins, such as AgrB, AgrC, and AgrD, we investigated whether AHLs perturbed S. aureus membrane functionality by determining their influence on the membrane dipole potential. From the binding curves obtained, a dissociation constant of 7 muM was obtained for 3-oxo-C12-HSL, indicating the presence of a specific saturable receptor, whereas no binding was observed for C4-HSL. These data demonstrate that long-chain 3-oxo-substituted AHLs, such as 3-oxo-C12-HSL, are capable of interacting with the S. aureus cytoplasmic membrane in a saturable, specific manner and at sub-growth-inhibitory concentrations, down-regulating exotoxin production and both sarA and agr expression.

4-Butyrolactone↗

PubMed2005

Modeling the absorption spectrum of tryptophan in proteins.

We have applied time-dependent density functional theory (TDDFT) to study the valence pi-pi* excited states of the tryptophan chromophore in the environment of the proteins barnase and human serum albumin. The chromophore is represented by indole. Due to the approximate nature of TDDFT, in the gas phase the calculated vertical transition energies to the 1L valence states are reordered with respect to experiment. The 1L(a) state responds more than the 1L(b) state to the local environment, described fully at the TDDFT level, and to bulk environment, described by a set of point charges. Nevertheless, the vertical transitions are readily identified. For human serum albumin, our calculations predict distinct spectral characteristics between structures with different tryptophan side chain torsion angles. The computational tractability of TDDFT relative to more accurate ab initio methods allows a large part of the surrounding protein environment (up to 100 atoms) to be explicitly included in the TDDFT calculations.

Bacillus↗

PubMed2005

Physical landscapes in biological membranes: physico-chemical terrains for spatio-temporal control of biomolecular interactions and behaviour.

The evolving complexities of biological membranes are discussed from the point of view of potential roles of the physical constitution of the membrane. These include features of the surface and dipole potentials and membrane 'rafts'. These properties are outlined; they emphasize that protein-lipid and specific lipid environments are influential parameters in how biomolecular interactions may take place with and within membranes. Several fluorescence detection technologies directed towards measurement of these properties are also outlined that permit high-resolution experimental determination of intermolecular interactions with membranes by measuring small changes of these potentials. These point to the possibility that the membrane dipole potential in particular is enormously influential in determining the behaviour of receptor and signalling systems within membrane rafts, and offers the means of a novel mechanism for biological control.

Cell Membrane↗

PubMed2005

All subtypes of the cytotoxin VacA adsorb to the surface of Helicobacter pylori post-secretion.

The major secreted virulence factor of Helicobacter pylori, the vacuolating cytotoxin VacA, is known to insert into eukaryotic membranes and has been observed in association with the surface of H. pylori cells that are actively producing it. Here, it is demonstrated that VacA is capable of interacting with the surface of H. pylori and Escherichia coli after secretion. It is shown that this interaction is resistant to disruption of electrostatic and hydrophobic forces, and that it appears to occur despite truncation of LPS and the removal of trypsin-accessible surface proteins. Adsorption to bacterial cell surfaces was independent of the VacA subtype, suggesting that it is not mediated through recognition of a known receptor by the VacA p58 subunit. Similarly, adsorption to bacterial cell surfaces is unlikely to be instigated by the extreme N-terminus of VacA, since a hydrophilic extension at this location that is known to disrupt VacA-induced vacuolation did not interfere with adsorption to H. pylori cells.

Adsorption↗

PubMed2004

Fibronectin interactions with osteoblasts: identification of a non-integrin-mediated binding mechanism using a real-time fluorescence binding assay.

Fibronectin (Fn) is an extracellular matrix protein that interacts with specific integrins on the cell surface, initiating signal transduction processes that lead to a reorganization of the cytoskeleton and the assembly of focal adhesions. Cell surface proteoglycans or glycosaminoglycans (GAGs) such as heparan sulfate are also known to participate in the interaction of Fn with the cell surface by binding to two different heparin-binding domains. The influence of Fn and GAGs on the spreading and differentiation of human osteoblasts was also previously described. In the current work, a method developed in our laboratory is established to evaluate the interaction between Fn and human osteoblasts and the influence of GAGs on such interactions. This technique makes use of fluoresceinphosphatidylethanolamine (FPE) such that when inserted into the lipidic bilayer, it acts as a fluorescent indicator of membrane interactions. The results indicate that the binding profile of Fn with the osteoblast cell surface is best represented by a hyperbolic single binding site model with a membrane affinity of 120 nM. Removal of cell surface heparan sulfate by treatment with heparitinase indicates that the cell surface moiety is directly involved in the binding process. Studies directed to assess the influence of heparin on the interaction of Fn with osteoblasts reveal that although it does not hamper Fn binding to the cell surface, it blocks the initial attachment to Fn-coated surfaces, indicating that binding to the integrin receptor alone is not enough to promote cell attachment but that the participation of the cell-surface GAGs is also a necessary condition.

Calcium↗

PubMed2003

Estimating domain orientation of two human antibody IgG4 chimeras by crystallohydrodynamics.

A modified crystallohydrodynamic approach introduced in 2001 is applied to two human IgG4 constructs from mouse IgG1. The constructs were point mutants of the chimeric antibody molecule cB72.3(gamma4): cB72.3(gamma4A), devoid of inter-chain disulfide bridging, and cB72.3(gamma4P), which has full inter-chain bridging. As before, the known crystallographic structures for the Fab and Fc domains were combined with the measured translational frictional ratios to obtain an estimate for the apparent time-averaged hydration of the domains and hence for that of the intact molecule. The original approach was modified with the hydrated dimensions of the domains being applied, rather than the anhydrous crystallographic dimensions, for assessing the inter-domain orientations using the algorithms HYDROSUB and SOLPRO. Both chimeric IgG4 molecules were found to have open, rather than compact, structures, in agreement with the previous study on wild-type human IgG4. The insertion of a frictionless connector between the domains was necessary, however, for representing the cB72.3(gamma4A) chimera. It therefore appears that the inter-chain disulfide bonds act as physical constraints in the cB72.3(gamma4P) chimera, forcing the antibody domains together and producing a less elongated structure than that of cB72.3(gamma4A). The open structures produced for the two IgG4 chimeras showed similarity to those structures identified for murine IgG1 and IgG2a molecules through X-ray crystallography.

Animals↗

PubMed2003

Practical asymmetric synthesis of a potent Cathepsin K inhibitor. Efficient palladium removal following Suzuki coupling.

A large-scale, chromatography-free synthesis of a potent and selective Cathepsin K inhibitor 1 is reported. The key asymmetric center was installed by addition of (R)-pantolactone to the in situ-generated ketene 4a. The final step of the convergent synthesis of 1 was completed via Suzuki coupling of aryl bromide 7a with unprotected aryl piperazine boronic acid 13. Residual palladium and iron generated in the Suzuki coupling were efficiently removed from crude 1 via a simple extractive workup using lactic acid.

Catalysis↗

PubMed2003

Influence of molecular dipoles on human skin permeability: Use of 6-ketocholestanol to enhance the transdermal delivery of bacitracin.

In the present work, we report the possibility of modifying the electrostatic properties of the skin by treating human epidermis with compounds whose structures possess a large molecular dipole moment. Data are presented showing that such a modification can be used to enhance dermal drug delivery. Inclusion of such compounds in biological membranes affects the so-called membrane dipole potential, an electrical potential originating from molecular dipoles present on the lipid molecules. Modifications in the magnitude of this potential are known to affect the interaction of hydrophobic ions and peptides with model membranes. Using fluorescein-labeled bacitracin and confocal microscopy, we show that the penetration of the antibiotic peptide bacitracin into the epidermis is enhanced when the skin has been pretreated with liposomes loaded with 30 mol % 6-ketocholestanol, a compound known to increase the magnitude of the membrane dipole potential. Studies using the fluorescent indicators fluoresceinphosphatidylethanolamine and 1-(3-sulfonatopropyl)-4-[beta [2-(di-n-octylamino)-6-naphthyl] vinyl] pyridinium betaine show that the interaction of bacitracin with model membranes is also enhanced by the presence of 6-ketocholestanol in the bilayer and offers some indication to the mechanism of penetration enhancement.

Adjuvants, Pharmaceutic↗

PubMed2003

Electrostatic sensor for identifying interactions between peptides and bacterial membranes.

The use of the membrane probe fluorescein phosphatidylethanolamine (FPE) to investigate membrane binding is well established. However, until now, its use has been restricted to studies involving peptides and eukaryotic membranes. This useful tool has been developed to interrogate peptide:prokaryotic membrane interactions by introducing novel methodology to incorporate FPE into the membranes of UV killed, whole bacterial cells. The electrostatic potential of the membrane in the immediate vicinity of the probe affects the protonation state of the xanthene ring system in the fluorescein head group, which is held close to the membrane surface. When altered, e.g. by peptide binding and insertion, a change in fluorescence results, which can be measured spectrophotometrically. Applicability of this technique to bacterial surface interactions was confirmed by production of a binding curve for both a synthetic peptide and a 37kDa protein. Future investigations are anticipated to utilize this technology to characterize interactions of other toxins plus antimicrobial peptides such as lactoferricin and defensins with their target membranes.

Bacteria↗

PubMed2003

A communication protocol to help patients with sensory impairment.

Patients with sight, hearing and speech impairment are entitled in law to equal access to health care according to the Disabilities Discrimination Act. More and more patients experience these problems as the population grows older. This paper reports on how a group of staff in one trust helped set up a protocol to ensure that this group's communication needs are tackled.

Communication↗

PubMed2002

Measurement of the lateral diffusion of human MHC class I molecules on HeLa cells by fluorescence recovery after photobleaching using a phycoerythrin probe.

The mobility of cell surface MHC class I molecules on HeLa cells was measured by fluorescence recovery after photobleaching (FRAP). The probe used for these studies was the phycobiliprotein R-phycoerythrin coupled to Fab fragments of a monoclonal antibody specific for human monomorphic MHC class I molecules. It was found that the recovery curves could be equally well fitted by either a random diffusion model with an immobile component or by an anomalous diffusion model. In the latter case, the anomalous diffusion exponent was consistent with that previously determined by single-particle tracking (SPT) experiments using the same probe (P. R. Smith, I. E. G. Morrison, K. M. Wilson, N. Fernandez, and R. J. Cherry. 1999. Biophys. J. 76:3331-3344). The FRAP experiments, however, yielded a considerably higher value of D(0), the diffusion coefficient for a time interval of 1 s. To determine whether the results were probe dependent, FRAP measurements were also performed with the same monoclonal antibody labeled with Oregon Green. These experiments gave similar results to those obtained with the phycoerythrin probe. FRAP experiments with the lipid probe 5-N-(octadecanoyl) aminofluoroscein (ODAF) bound to HeLa cells gave typical results for lipid diffusion. Overall, our observations and analysis are consistent with anomalous diffusion of MHC class I diffusion on HeLa cells, but quantitative differences between FRAP and SPT data remain to be explained.

Antibodies, Monoclonal↗

PubMed1995

Hydrophobic properties of the cell surface of Candida albicans: a role in aggregation.

The ability of Candida albicans to aggregate and adhere to biological surfaces is a topic of major biological and medical importance. One factor which has been implicated in such properties is the hydrophobic nature of the cell surface. Two simple spectroscopic techniques are described which permit the rapid determination of this property. The first involves the use of arylnaphthalenesulfonate, the fluorescence emission maximum of which was shown to be a sensitive indicator of dielectric polarity. This was used to identify the hydrophobic characteristics of the cell surface of C. albicans. The second technique involves the use of 90 degrees Rayleigh-Debye light scattering as an indicator of the aggregation state of a fungal suspension. These techniques were then used to compare the surface properties of three different strains of C. albicans and the effects of culture conditions: the hydrophobicity of the strains varied, and galactose-based culture media promoted the greatest degree of cell surface hydrophobicity.

Anilino Naphthalenesulfonates↗