Biomedical subjects

G O'Beirne

Publications and source records attributed to G O'Beirne.

10 recordsLinked to original sources

PubMed2004

Functional genomics and proteomics: application in neurosciences.

The sequencing of the complete genome for many organisms, including man, has opened the door to the systematic understanding of how complex structures such as the brain integrate and function, not only in health but also in disease. This blueprint, however, means that the piecemeal analysis regimes of the past are being rapidly superseded by new methods that analyse not just tens of genes or proteins at any one time, but thousands, if not the entire repertoire of a cell population or tissue under investigation. Using the most appropriate method of analysis to maximise the available data therefore becomes vital if a complete picture is to be obtained of how a system or individual cell is affected by a treatment or disease. This review examines what methods are currently available for the large scale analysis of gene and protein expression, and what are their limitations.

Brain↗

PubMed1999

Intracellular measurement of prostaglandin E2: effect of anti-inflammatory drugs on cyclooxygenase activity and prostanoid expression.

Cyclooxygenase (COX) converts arachidonic acid to prostaglandin (PG) H2, which is further metabolized to various prostaglandins, prostacyclin and thromboxane A2. COX exists in at least two different isoforms. COX-1 is constitutively expressed, whereas COX-2 is induced by proinflammatory stimuli. Prostaglandin E2 is a major metabolite of COX activation. In order to compare the activity of target ligands and COX inhibitors on PGE2 synthesis and release, the responsiveness of several cell lines to the calcium ionophore A23187, bacterial lipopolysaccharide (LPS), nonsteroidal anti-inflammatory drugs (NSAIDs), and the glucocorticoid, dexamethasone, were investigated. For intracellular measurements, the culture supernatant was aspirated, and the cells were thoroughly washed and lysed with dodecyltrimethylammonium bromide. Intracellular and secreted PGE2 were measured with an enzyme immunoassay. A23187 and LPS increased intracellular PGE2 in a dose-dependent manner. Kinetic experiments with A23187-stimulated mouse 3T3 fibroblast cells revealed a distinct biphasic response in COX activity. In the presence of NSAIDs or dexamethasone, there was a dose-dependent inhibition in intracellular PGE2 with A23187-stimulated 3T3 cells. Inhibitory studies demonstrated an apparent increased sensitivity of COX activity to the action of inhibitors when measuring intracellular PGE2 compared with using cell culture supernatants. Indeed, intracellular PGE2 levels were comprehensively reduced in the presence of low concentrations of inhibitor. The utilization of cell culture lysates and, in particular, measurement of intracellular PGE2 should prove useful for identifying new COX inhibitors.

3T3 Cells↗

PubMed1999

Interactions between inositol trisphosphate receptors and fluorescent Ca2+ indicators.

Fura-2 and BAPTA were previously shown to be competitive antagonists of inositol trisphosphate (InsP3) receptors, but for practical reasons the analyses were performed at pH 8.3. We recently developed a scintillation proximity assay (SPA) for pure cerebellar InsP3 receptors which allows low affinity interactions to be characterized and is readily applicable to scarce or expensive ligands. In the present study, we use SPA to demonstrate that at pH 7.2, many of the commonly used fluorescent Ca2+ indicators reversibly displace 3H-InsP3 from its receptor and that they differ substantially in their affinities for the InsP3 receptor (IC50 = 6.5-137 microM). Recombinant type 1 InsP3 receptors expressed in Sf9 cells were used to examine 3H-InsP3 binding in cytosol-like medium: both fura-2 (IC50 = 796 +/- 86 microM) and Ca Green-5N (IC50 = 62 +/- 7 microM) completely inhibited the binding, but only in their Ca(2+)-free forms. Similar results were obtained with type 3 InsP3 receptors. We conclude that many Ca2+ indicators in their Ca(2+)-free forms compete with InsP3 for binding to its receptor, and that for Ca Green-5N the interaction occurs with sufficient affinity to significantly perturb physiological responses.

Animals↗

PubMed1998

Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.

Acrylic Resins↗

PubMed1997

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: redistribution of calmodulin as a possible means of regulating Ca2+ mobilization.

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP3), and pure cerebellar InsP3 receptors were characterized by using a scintillation proximity assay. In the absence of Ca2+, 125I-labeled calmodulin reversibly bound to multiple sites on InsP3 receptors and Ca2+ increased the binding by 190% +/- 10%; the half-maximal effect occurred when the Ca2+ concentration was 184 +/- 14 nM. In the absence of Ca2+, calmodulin caused a reversible, concentration-dependent (IC50 = 3.1 +/- 0.2 microM) inhibition of [3H]InsP3 binding by decreasing the affinity of the receptor for InsP3. This effect was similar at all Ca2+ concentrations, indicating that the site through which calmodulin inhibits InsP3 binding has similar affinities for calmodulin and Ca2+-calmodulin. Calmodulin (10 microM) inhibited the Ca2+ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP3. Tonic inhibition of InsP3 receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP3 and limit spontaneous activation of InsP3 receptors in the dendritic spines. Inhibition of InsP3 receptors by calmodulin at all cytosolic Ca2+ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca2+, suggests that calmodulin may also allow both feedback control of InsP3 receptors and integration of inputs from other signaling pathways.

Animals↗

PubMed1997

An homogeneous assay for measuring the uptake and efflux of radiolabelled drugs in adherent cells.

We have developed an homogeneous assay to measure the uptake and efflux of [14C]adriamycin (doxorubicin hydrochloride) in human squamous lung carcinoma cells (SKMES-1), using 96 well scintillating microplates. The assay was also used to examine the effect of inhibitors of multidrug resistance in adriamycin resistant cells (SKMES-1/ADR). The effect of adriamycin on cell growth and viability was examined by continuous monitoring of the uptake of [14C]thymidine. The non-invasive nature of these assays, and the ease of use of the microplates, suggests a role in screens for, and characterisation of, novel chemotherapeutic or chemosensitizing agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Noninvasive, real-time method for the examination of thymidine uptake events--application of the method to V-79 cell synchrony studies.

[14C]Thymidine uptake into V-79 hamster lung fibroblasts has been successfully demonstrated using a noninvasive, real-time method utilizing Cytostar-T scintillating microplates. These plates are standard format, tissue culture-treated, 96-well microplates with an integral scintillating base. The microplates permit the culture and observation of adherent cell monolayers. Biological activities of the cells can be studied by the provision of specific radiolabeled compounds. The biological activities of the adherent monolayer bring the specific radiolabel into proximity with the scintillating base and a scintillation signal is thereby generated. [14C]Thymidine incorporation on the microplates can be used to examine cell proliferation and cell cycle events. Using a combined mitotic shake-off/aphidicolin treatment to achieve synchronization, the thymidine incorporation activities of V-79 cells have been examined on Cytostar-T plates and correlated to traditional methods of determining incorporation. The method was further used to examine the effects of colcemid and olomoucine, both chemical inhibitors of cell proliferation, on synchronous populations of cells. The homogeneous detection format and the microplate nature of the method suggest a role for scintillating microplates in cell biology research and drug discovery.

Animals↗

PubMed1996

Kinetic analysis of inositol trisphosphate binding to pure inositol trisphosphate receptors using scintillation proximity assay.

Inositol 1,4,5-triphosphate (InsP3) receptors are regulated by many intracellular signals including proteins and small messengers. By linking purified cerebellar InsP3 receptors to scintillation proximity assay beads, binding of radioligands can be measured without separation of bound from free ligand. InsP3 receptors assayed by scintillation proximity assay bound heparin with high affinity and stereoselectively bound InsP3 with similar affinity to cerebellar membranes. By rapidly freezing scintillation proximity assay reactions and then counting the frozen samples, both fast and slow components of [3H] InsP3 association and dissociation were identified. Our novel freeze-quench method in combination with conventional stopped-quench equipment and scintillation proximity assay allows the rapid kinetics of the interactions of pure receptors with their ligands to be resolved.

Calcium Channels↗

PubMed1996

Efficacy of a sonic toothbrush on inflammation and probing depth in adult periodontitis.

This single-blind, 8-week study compared the efficacy of a sonic toothbrush and a manual brush in 40 patients with adult periodontitis. Qualitative clinical indices and quantitative laboratory methods were used to monitor the periodontal status of 3 pockets 5 to 7 mm deep in each subject. Patients were randomly assigned either a sonic or manual toothbrush. The two groups were comparable with respect to age, gender, and anatomical location of the test sites. Data were collected from all sites at baseline and at 2, 4, and 8 weeks. Over the 8-week period, both groups showed significant improvements in the clinical indices used. Descriptive statistics indicated the sonic brush group had greater improvement than the manual group in the clinical parameters (gingival index, bleeding index, probing depth, and clinical attachment level). Gingival crevicular fluid (GCF) flow was significantly lower in the sonic brush group (P = 0.018). Considerable variation was present in the levels detected for both inflammatory cytokines tested, however, concentration of interleukin-1 beta was significantly lower in the GCF of sonic group patients (P = 0.05), while concentration of interleukin-6 was significantly reduced in both groups (P < or = 0.05) (t tests). Under these conditions, there is some evidence to suggest that the sonic toothbrush is more beneficial in resolving inflammation in patients with moderate periodontal disease.

Adolescent↗

PubMed1991

Shade selection for porcelain restorations.

This short paper describes the fundamental qualities of colour, applies these to the difficulties of choosing the correct shades for porcelain restorations and suggests a practical procedure for achieving a satisfactory shade match.

Color↗