PubMed Health⌕ Search

Biomedical subjects

P O'Shea

Publications and source records attributed to P O'Shea.

At least 19 recordsLinked to original sources
PubMed2005

Two-photon fluorescence surface wave microscopy.

This paper demonstrates the principle of two-photon surface wave microscopy with a view to applications on biological samples. We describe a modified scanning optical microscope, which uses specially prepared coverslips. These coverslips are designed to support the propagation of surface waves capable of large field enhancements. We also discuss the beam conditioning necessary to ensure efficient use of the available illumination. Two-photon surface wave fluorescent excitation is demonstrated on fluorescent nanospheres, demonstrating a point spread function width of approximately 220 nm at an illumination wavelength of 925 nm. The potential of non-linear surface wave excitation for both fluorescence and harmonic imaging microscopy is discussed.

Membrane Microdomains↗
PubMed2003

Analysis of thyroid hormone responsive gene expression in osteoblastic cells.

Thyroid hormones regulate gene expression to influence the development and metabolism of many tissues including bone. The identification of genes that are regulated by thyroid hormones during skeletal development requires sensitive and quantitative techniques that are not limited by small amounts of available tissue and RNA. We have compared the efficiencies of differential display and poly A PCR subtraction hybridisation methods for the detection of thyroid hormone responsive genes expressed in osteoblastic cells. The utility of each technique was evaluated with respect to its sensitivity, specificity, cost and ability to identify novel genes. Subtraction hybridisation was rapid and more efficient in all categories. Poly A PCR facilitates quantitative and representative global amplification of cDNAs from low concentrations of RNA extracted from small tissue samples. The method, in combination with microarray analyses, may prove useful as an additional, complementary strategy to subtraction hybridisation for the analysis of differential gene expression in tissues where sample size is limiting.

Animals↗
PubMed2003

The effects of anabolic-androgenic steroids upon resting and peak exercise left ventricular heart wall motion kinetics in male strength and power athletes.

Previous investigations reported alterations in myocardial fibres and systolic function associated with anabolic-androgenic steroid consumption by athletes. Advances in bio-medical technology have allowed further investigation in assessing the possible effects of anabolic-androgenic steroids on gross left ventricular kinetics. Twenty-three male strength and power athletes with a past and current history of anabolic-androgenic steroid consumption (x 46 days, range 28 days to 70 days), were compared to 23 controls. Testing consisted of resting and immediate post-exercise transthoracic left ventricular wall cardiokymograms. Statistical results identified no difference over time between groups or condition. Cardiokymographic waveform analysis found 32.61% of all (n =184) waveforms to be abnormal (Type II, n = 56 or Type III, n = 4). There were 14 treatment subjects (60.87%) who demonstrated an abnormal waveform as compared to 9 controls (39.13%). A significant difference (p < or = 0.01) in the overall proportions of waveform types was identified where the treatment group exhibited 41.30% abnormal waveforms, compared to 23.91% by controls. Additionally, two athletes (1 treatment, 1 control) demonstrated abnormal left ventricular wall motions (Type III) analogous to impaired left ventricular performance. The results indicated: (a) highly strength trained athletes with no history of anabolic-androgenic steroid usage exhibited an unexpected high incidence of Type II waveforms (28.26% pre/23.91% post); (b) a comparable group of strength trained athletes using anabolic-androgenic steroids exhibited a significantly higher percentage of abnormal waveforms as compared to controls (34.78% pre/37.21% post). Based on these results, high intensity strength training with and without anabolic-androgenic steroid supplementation induced alterations in the left ventricular wall motion.

Adult↗
PubMed2003

Intermolecular interactions with/within cell membranes and the trinity of membrane potentials: kinetics and imaging.

The interactions of (macro-)molecules with biological membranes underlies much of cell biology. This paper outlines many of the factors that must be taken into account in order to understand fully the nature of these interactions. These include some roles of the membrane potentials including features of the surface and dipole potentials. Several fluorescence detection technologies directed towards these are outlined that offer high-resolution experimental determination of the intermolecular interactions by measuring small changes of these potentials resulting from specific interactions of many kinds of molecular species. The possibilities for making single-cell spatial imaging measurements of such interactions is also described. Examples are used to indicate the feasibility of identifying and tracking localized interactions on the membrane surface in real-time. Some of this work points to the possibility that the membrane dipole potential spatially varies about the cell surface, particularly within membrane microdomains such as 'rafts'. Such variation is suggested to underlie the altered behaviour of signalling systems within rafts and offer the means of an additional level of biological control.

Animals↗
PubMed2003

Measuring and visualizing single molecular interactions in biology.

In recent years, considerable attention has focused upon the biological applications of the atomic force microscope (AFM), and in particular in its ability to explore biomolecular interaction events at the single molecule level. Such measurements can provide considerable advantages, as they remove the data averaging inherent in other biophysical/biochemical approaches that record measurements over large ensembles of molecules. To this end AFM has been used for both the high-resolution imaging of a range of individual biological molecules and their complexes, and to record interaction forces between single interacting molecules. In a recently initiated project we have begun to utilize these approaches to explore the interactions of a range of biologically important peptides with model and cell membrane surfaces. In this review, the potential value of AFM for the investigation of a range of biomolecular interaction events will be discussed, but highlighting in particular its potential for the study of interactions of peptides/proteins with biological membranes.

Animals↗
PubMed2001

Effects of the membrane dipole potential on the interaction of saquinavir with phospholipid membranes and plasma membrane receptors of Caco-2 cells.

The combined use of the membrane surface potential fluorescent sensor fluorescein phosphatidylethanolamine (FPE) and the membrane dipole potential fluorescent sensor di-8-ANEPPS to characterize the interaction of molecules with model and cellular membranes and to asses the influence of the dipole potential on the interaction is reported. The study of the human immunodeficiency virus protease inhibitor saquinavir with Caco-2 cells and phospholipid membranes reveals that the compound interacts with the lipidic bilayer of model membranes with a simple hyperbolic binding profile but with Caco-2 cells in a cooperative way involving membrane receptors. Additional studies indicated that colchicine acts as a competitor ligand to saquinavir and suggests, in agreement with other reports, that the identity of the saquinavir "receptor" could be P-glycoprotein or the multiple drug resistance-associated protein. The modification of the magnitude of the membrane dipole potential using compounds such as cholesterol, phloretin, and 6-ketocholestanol influences the binding capacity of saquinavir. Furthermore, removal of cholesterol from the cell membrane using methyl-beta-cyclodextrin significantly decreases the binding capacity of saquinavir. Because removal of cholesterol from the cell membrane has been reported to disrupt membrane domains known as "rafts," our observations imply that the membrane dipole potential plays an important role as a modulator of molecule-membrane interactions in these membrane structures. Such a role is suggested to contribute to the altered behavior of receptor-mediated signaling systems in membrane rafts.

Binding, Competitive↗
PubMed2001

The fusion domain of HIV gp41 interacts specifically with heparan sulfate on the T-lymphocyte cell surface.

Studies of the interaction of the 16 residue fusion peptide domain of human immunodeficiency virus glycoprotein gp41 (gp41(FD)) with T lymphocytes are outlined. Fluorescence measurements of changes in the electrostatic surface and dipole potentials of the plasma membrane following the interaction with gp41(FD) are described. The results show that gp41(FD) interacts with heparan sulfate located on the cell surface. This interaction is blocked by interleukin-8 and abolished by pre-treating the cells with heparitinase. The specificity of the reaction was also assessed by observations that soluble heparan sulfate competes with the cell membrane interaction whereas soluble heparin (at the levels utilized) does not. Following binding to heparan sulfate, the interaction with the membrane seems to take place in a cooperative manner with the formation of gp41(FD) trimers. In simpler phospholipid membranes, however, a trimeric complex does not appear to be the dominant mode of interaction. Finally, by repeating some of these studies within an imaging regime, it appears that the gp41(FD)-T-cell interaction takes place within specific domains on the cell surface to similarly localized heparan sulfate moieties.

Binding Sites↗
PubMed2001

Effect of phloretin on the percutaneous absorption of lignocaine across human skin.

The potential use of phloretin, a polyphenolic compound, as a penetration enhancer in the transdermal delivery of lignocaine hydrochloride (L-HCl) has been investigated. Standard in vitro skin permeation methods, using excised human skin, were used to characterize the percutaneous absorption of L-HCl. Initially, phloretin was applied to the skin surface as a methanolic solution. The skin samples were treated 12 h prior to application of the lignocaine donor solution, which was buffered at pH 4.0 and 7.0. The data obtained from the methanolic solutions at pH 4.0 show a 3.2-fold increase of the cumulative amount permeated after 24 h compared with the control. A second series of experiments were conducted using unilamellar phosphatidylcholine liposomes instead of methanol as a vehicle for the phloretin. The L-HCl amount permeated from liposomal-pretreated skin was 5.4-fold (p < 0.05) higher than the control within 24 h. In addition to the diffusion experiments, pressure area isotherms were recorded on a Langmuir-Blodgett trough using the model skin lipid ceramide-2. They showed a slight increase in the area occupied per lipid molecule of 1.04 nm(2) at constant surface pressure. This result indicates an interaction between the model lipid and phloretin. The results suggest the potential use of phloretin as penetration enhancer in the delivery of L-HCl through skin.

Chromatography, High Pressure Liquid↗
PubMed2000

Effect of sequential transdermal progesterone cream on endometrium, bleeding pattern, and plasma progesterone and salivary progesterone levels in postmenopausal women.

BACKGROUND: Transdermal progesterone is being used in some countries as a purported treatment for menopausal symptoms, either alone or prescribed in conjunction with estrogen, but little information exists regarding the biological activity and effectiveness of this method of delivery of progesterone in protecting the endometrium from excess proliferation. This study was designed to evaluate the use of sequential transdermal progesterone. End-points evaluated included endometrial cellular response and bleeding pattern as well as plasma hormone levels and salivary progesterone estimations. METHOD: Twenty-seven postmenopausal women were treated with continuous transdermal estrogen (28-day cycle) and a cream containing 16, 32 or 64 mg of progesterone in each 4-cm extrusion from a tube of Pro-Feme administered daily in a sequential (days 15-28 of cycle) regimen. Blood and endometrial samples were analyzed for progesterone response prior to therapy, after the first 14 days of unopposed transdermal estrogen and following 14 days of transdermal progesterone. Saliva samples were taken during the last 14 days of the 84-day study, when the final progesterone cream therapy was being applied. RESULTS: Hormone assay indicated that physiological levels of estradiol were achieved, but progesterone levels were insufficient to induce any detectable change in the endometrium. Only one patient experienced bleeding during the study period. Levels of salivary progesterone were so variable as to be considered completely unreliable in determining the potential influence on biological activity. INTERPRETATION: Pro-Feme transdermal progesterone administered in a 16-, 32- or 64-mg daily dose for 14 days in a sequential regimen does not appear to be effective in inducing a secretory change in a proliferative endometrium. Salivary progesterone levels were not of value in managing the therapy of postmenopausal women.

Administration, Cutaneous↗
PubMed1999

Characterization of the sequence of interactions of the fusion domain of the simian immunodeficiency virus with membranes. Role of the membrane dipole potential.

The simian immunodeficiency virus fusion peptide constitutes a 12-residue N-terminal segment of the gp32 protein that is involved in the fusion between the viral and cellular membranes, facilitating the penetration of the virus in the host cell. Simian immunodeficiency virus fusion peptide is a hydrophobic peptide that in Me(2)SO forms aggregates that contain beta-sheet pleated structures. When added to aqueous media the peptide forms large colloidal aggregates. In the presence of lipidic membranes, however, the peptide interacts with the membranes and causes small changes of the membrane electrostatic potential as shown by fluorescein phosphatidylethanolamine fluorescence. Thioflavin T fluorescence and Fourier transformed infrared spectroscopy measurements reveal that the interaction of the peptide with the membrane bilayer results in complete disassembly of the aggregates originating from an Me(2)SO stock solution. Above a lipid/peptide ratio of about 5, the membrane disaggregation and water precipitation processes become dependent on the absolute peptide concentration rather than on the lipid/peptide ratio. A schematic mechanism is proposed, which sheds light on how peptide-peptide interactions can be favored with respect to peptide-lipid interactions at various lipid/peptide ratios. These studies are augmented by the use of the fluorescent dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6-naphthyl]vinyl ] pyridinium betaine that shows the interaction of the peptide with the membranes has a clear effect on the magnitude of the so-called dipole potential that arises from dipolar groups located on the lipid molecules and oriented water molecules at the membrane-water interface. It is shown that the variation of the membrane dipole potential affects the extent of the membrane fusion caused by the peptide and implicates the dipolar properties of membranes in their fusion.

Amino Acid Sequence↗
PubMed1999

Ligand-dependent conformational equilibria of serum albumin revealed by tryptophan fluorescence quenching.

Ligand-dependent structural changes in serum albumin are suggested to underlie its role in physiological solute transport and receptor-mediated cellular selection. Evidence of ligand-induced (oleic acid) structural changes in serum albumin are shown in both time-resolved and steady-state fluorescence quenching and anisotropy measurements of tryptophan 214 (Trp214). These studies were augmented with column chromatography separations. It was found that both the steady-state and time-resolved Stern-Volmer collisional quenching studies of Trp214 with acrylamide pointed to the existence of an oleate-dependent structural transformation. The bimolecular quenching rate constant of defatted human serum albumin, 1.96 x 10(9) M-1 s-1, decreased to 0.94 x 10(9) M-1 s-1 after incubation with oleic acid (9:1). Furthermore, Stern-Volmer quenching studies following fractionation of the structural forms by hydrophobic interaction chromatography were in accordance with this interpretation. Time-resolved fluorescence anisotropy measurements of the Trp214 residue yielded information of motion within the protein together with the whole protein molecule. Characteristic changes in these motions were observed after the binding of oleate to albumin. The addition of oleate was accompanied by an increase in the rotational diffusion time of the albumin molecule from approximately 22 to 33.6 ns. Within the body of the protein, however, the rotational diffusion time for Trp214 exhibited a slight decrease from 191 to 182 ps and was accompanied by a decrease in the extent of the angular motion of Trp214, indicating a transition after oleate binding to a more spatially restricted but less viscous environment.

Acrylamide↗
PubMed1998

Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide.

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.

Animals↗
PubMed1997

Pathology of mucormycosis of cane toads in Australia.

The gross and microscopic pathology of a fungal septicaemia caused by the zygomycete. Mucor amphibiorum in 27 free-ranging cane toads, Bufo marinus, in Australia is described. Seven of the 27 toads had clinical signs of illness when discovered and five of these seven were moribund. Multiple granulomas were found in many organs, and in massive infections granulomas tended to coalesce. Liver, spleen, kidneys, urinary bladder, heart and lung were most commonly involved, but granulomas also occurred in subcutaneous lymph spaces, skin, gastro-intestinal tract, voluntary muscle, bone, cranial cavity and the oral cavity. Single lesions appeared grossly as a lemon coloured nodule < or = 5 mm in diameter. Histologically, the primary lesion was a granuloma composed of multinucleate giant cells, macrophages, occasional lymphocytes and eosinophils surrounding the distinctive sphaerules of M. amphibiorum. Fibroblasts occurred in greater numbers at the periphery and collagen formed a dense fibrous capsule around some nodules. A less common lesion resembled a microabscess and consisted of mononuclear cells, neutrophils and eosinophils surrounded by macrophages. Many of the centrally placed mixed inflammatory cells appeared necrotic. This reaction appeared to be more acute. Both types of lesions sometimes occurred concurrently, but the latter was less common. The pattern of lesions and natural history of M. amphibiorum suggested that ingestion of contaminated soil may have been the route of infection.

Animals↗
PubMed1996

Time resolution of binding and membrane insertion of a mitochondrial signal peptide: correlation with structural changes and evidence for cooperativity.

Utilizing a recently developed novel fluorescence technique [Wall et al. (1995) Mol. Membr. Biol. 12, 183-192], it is shown that the interactions of p25, the leader peptide of subunit IV of cytochrome c oxidase, with phospholipid membranes can be identified in real time. p25 is observed to bind following stopped-flow mixing of the peptide with phospholipid membranes with rate constants up to about 700 s-1 and then insert into the membrane with rate constants on the order of 0.4 s-1. Comparison of these processes with similarly time-resolved experiments performed with a stopped-flow CD spectrometer revealed that p25 does not become alpha-helical upon binding to the membrane. Following membrane insertion, however, p25 was observed to adopt an alpha-helical configuration. The temperature dependency of these processes was then found to yield activation energies for the respective components of the p25-membrane interaction.

Cell Membrane↗
PubMed1996

Fibromatoses of childhood: the spectrum of radiographic findings.

Fibrous tumor of childhood include several disorders with variable biologic behavior. In the review by Coffin and Dehner [1] of 190 soft-tissue neoplasms in 183 children, 27% were fibroblastic or myofibroblastic in origin. Although nearly all fibrous lesions are benign, they may be locally aggressive. The purpose of this essay is to describe clinical characteristics and to illustrate radiologic features of commonly encountered fibrous lesions of childhood. Familiarity with the presentation and variable appearance may aid the radiologist in suggesting the diagnosis of fibromatosis.

Adolescent↗
PubMed1995

Serum neuron-specific enolase and immunohistochemical markers of neuroendocrine differentiation in lung cancer.

An enzyme immunoassay for serum neuron-specific enolase (NSE) was evaluated with respect to analytical performance and clinical utility and compared with immunohistochemical evaluation of neuroendocrine differentiation. Values obtained agreed well with values obtained using a radioimmunoassay method giving a correlation coefficient of 0.934. Analytical performance of the enzyme immunoassay was good but the diagnostic sensitivity of 82% in extensive and 67% in limited disease was insufficient for serum NSE to be of value in the diagnosis of small cell lung cancer (SCLC). Serum NSE decreased significantly in 11 of 15 patients with SCLC following institution of chemotherapy. Classification of lung cancers into SCLC and non small cell lung cancer (NSCLC) types is largely based on tumour morphology. Neuroendocrine differentiation may not be morphologically evident. Immunohistochemical staining of tumour tissue with markers of neuroendocrine differentiation, i.e. NSE (both monoclonal and polyclonal antibodies) Leu 7, Chromogranin A and P G P 9.5 was performed in both patients with SCLC and NSCLC. 38 per cent of patients with NSCLC had both raised serum NSE and positive NSE (polyclonal) immunoperoxidase staining of lung tissue. A further 35 per cent of patients showed a raised serum NSE or positive immunohistochemistry but not both. The presence of two positive immunoperoxidase markers in lung tissue has been suggested as an indicator of responsiveness to chemotherapy in NSCLC patients. A number of factors may affect immunohistochemical positivity in tissue sections and the additional use of a serum marker may better define chemotherapy responsive groups.

Adult↗