Biomedical subjects

J E Swatton

Publications and source records attributed to J E Swatton.

5 recordsLinked to original sources

PubMed2004

Protein profiling of human postmortem brain using 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE).

Two-dimensional gel electrophoresis (2-D GE) is a key tool for comparative proteomics research. With its ability to separate complex protein mixtures with high resolution, 2-D GE is a technique commonly employed for protein profiling studies. Significant improvements have been made in 2-D GE technology with the development of two-dimensional fluorescence difference gel electrophoresis (2-D DIGE), where proteins are first labelled with one of three spectrally resolvable fluorescent cyanine dyes before being separated over first and second dimensions according to their charge and size, respectively. When used in conjunction with automated analysis packages, this multiplexing approach can accurately and reproducibly quantify protein expression for control and experimental groups. Differentially expressed proteins can be subsequently identified by mass spectrometric methods. Here, we describe the successful application and optimisation of 2-D DIGE technology for human postmortem brain studies. This technology, especially when coupled with other functional genomics approaches, such as transcriptomics and metabolomics studies, will enhance our current understanding of human disease and lead to new therapeutic and diagnostic possibilities.

Brain Chemistry↗

PubMed2004

Mitochondrial dysfunction in schizophrenia: evidence for compromised brain metabolism and oxidative stress.

The etiology and pathophysiology of schizophrenia remain unknown. A parallel transcriptomics, proteomics and metabolomics approach was employed on human brain tissue to explore the molecular disease signatures. Almost half the altered proteins identified by proteomics were associated with mitochondrial function and oxidative stress responses. This was mirrored by transcriptional and metabolite perturbations. Cluster analysis of transcriptional alterations showed that genes related to energy metabolism and oxidative stress differentiated almost 90% of schizophrenia patients from controls, while confounding drug effects could be ruled out. We propose that oxidative stress and the ensuing cellular adaptations are linked to the schizophrenia disease process and hope that this new disease concept may advance the approach to treatment, diagnosis and disease prevention of schizophrenia and related syndromes.

Brain↗

PubMed2001

Functional properties of Drosophila inositol trisphosphate receptors.

The functional properties of the only inositol trisphosphate (IP(3)) receptor subtype expressed in Drosophila were examined in permeabilized S2 cells. The IP(3) receptors of S2 cells bound (1,4,5)IP(3) with high affinity (K(d)=8.5+/-1.1 nM), mediated positively co-operative Ca(2+) release from a thapsigargin-sensitive Ca(2+) store (EC(50)=75+/-4 nM, Hill coefficient=2.1+/-0.2), and they were recognized by an antiserum to a peptide conserved in all IP(3) receptor subtypes in the same way as mammalian IP(3) receptors. As with mammalian IP(3) receptors, (2,4,5)IP(3) (EC(50)=2.3+/-0.3 microM) and (4,5)IP(2) (EC(50) approx. 10 microM) were approx. 20- and 100-fold less potent than (1,4,5)IP(3). Adenophostin A, which is typically approx. 10-fold more potent than IP(3) at mammalian IP(3) receptors, was 46-fold more potent than IP(3) in S2 cells (EC(50)=1.67+/-0.07 nM). Responses to submaximal concentrations of IP(3) were quantal and IP(3)-evoked Ca(2+) release was biphasically regulated by cytosolic Ca(2+). Using rapid superfusion to examine the kinetics of IP(3)-evoked Ca(2+) release from S2 cells, we established that IP(3) (10 microM) maximally activated Drosophila IP(3) receptors within 400 ms. The activity of the receptors then slowly decayed (t(1/2)=2.03+/-0.07 s) to a stable state which had 47+/-1% of the activity of the maximally active state. We conclude that the single subtype of IP(3) receptor expressed in Drosophila has similar functional properties to mammalian IP(3) receptors and that analyses of IP(3) receptor function in this genetically tractable organism are therefore likely to contribute to understanding the roles of mammalian IP(3) receptors.

Adenosine↗

PubMed1999

Type 3 inositol trisphosphate receptors in RINm5F cells are biphasically regulated by cytosolic Ca2+ and mediate quantal Ca2+ mobilization.

There are three subtypes of mammalian Ins(1,4,5)P(3) (InsP(3)) receptor, each of which forms an intracellular Ca(2+) channel. Biphasic regulation of InsP(3) receptors by cytosolic Ca(2+) is well documented in cells expressing predominantly type 1 or type 2 InsP(3) receptors and might contribute to the regenerative recruitment of Ca(2+) release events and to limiting their duration in intact cells. The properties of type 3 receptors are less clear. Bilayer recording from InsP(3) receptors of RIN-5F cells, cells in which the InsP(3) receptors are likely to be largely type 3, recently suggested that the receptors are not inhibited by Ca(2+) [Hagar, Burgstahler, Nathanson and Ehrlich (1998) Nature (London) 296, 81-84]. By using antipeptide antisera that either selectively recognized each InsP(3) receptor subtype or interacted equally well with all subtypes, together with membranes from Spodoptera frugiperda (Sf9) cells expressing only single receptor subtypes to calibrate the immunoblotting, we quantified the relative levels of expression of type 1 (17%) and type 3 (77%) InsP(3) receptors in RINm5F cells. In unidirectional (45)Ca(2+) efflux experiments from permeabilized RINm5F cells, submaximal concentrations of InsP(3) released only a fraction of the InsP(3)-sensitive Ca(2+) stores, indicating that responses to InsP(3) are quantal. Increasing the cytosolic free [Ca(2+)] ([Ca(2+)](i)) from approx. 4 to 186 nM increased the sensitivity of the Ca(2+) stores to InsP(3): the EC(50) decreased from 281+/-15 to 82+/-2 nM. Further increases in [Ca(2+)](i) massively decreased the sensitivity of the stores to InsP(3), by almost 10-fold when [Ca(2+)](i) was 2.4 microM, and by more than 3000-fold when it was 100 microM. The inhibition caused by 100 microM Ca(2+) was fully reversed within 60 s of the restoration of [Ca(2+)](i) to 186 nM. The effect of submaximal InsP(3) concentrations on Ca(2+) mobilization from permeabilized RINm5F cells is therefore biphasically regulated by cytosolic Ca(2+). We conclude that type 3 InsP(3) receptors of RINm5F cells mediate quantal Ca(2+) release and they are biphasically regulated by cytosolic Ca(2+), either because a single type 1 subunit within the tetrameric receptor confers the Ca(2+) inhibition or because the type 3 subtype is itself directly inhibited by Ca(2+).

Animals↗

PubMed1999

Inhibition of fibril formation in beta-amyloid peptide by a novel series of benzofurans.

A series of benzofuran derivatives have been identified as inhibitors of fibril formation in the beta-amyloid peptide. The activity of these compounds has been assessed by a novel fibril-formation-specific immunoassay and for their effects on the production of a biologically active fibril product. The inhibition afforded by the compounds seems to be associated with their binding to beta-amyloid, as identified by scintillation proximity binding assay. Binding assays and NMR studies also indicate that the inhibition is associated with self-aggregation of the compounds. There is a close correlation between the activity of the benzofurans as inhibitors of fibril formation and their ability to bind to beta-amyloid. Non-benzofuran inhibitors of the fibril formation process do not seem to bind to the same site on the beta-amyloid molecule as the benzofurans. Thus a specific recognition site might exist for benzofurans on beta-amyloid, binding to which seems to interfere with the ability of the peptide to form fibrils.

Amyloid beta-Peptides↗