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Biomedical subjects

R J Ward

Publications and source records attributed to R J Ward.

At least 37 records · Page 2Linked to original sources
PubMed2001

New 8-hydroxyquinoline and catecholate iron chelators: influence of their partition coefficient on their biological activity.

Four new hexadendate chelators, three hydroxyquinoline-based, Csox, O-Trensox, Cox750, and one catecholate-based CacCam-which have comparable skeletal structures and pFe, but widely different partition coefficients, (Kpart), 0.01, 0.02, 1 and 3.2 respectively, have been tested for their iron chelating efficacy in vitro by two methods. First, by their ability to remove iron from ferritin in solution or second, to remove iron from iron-loaded hepatocytes in vitro. Our objective was to ascertain the importance of Kpart and pFe, on the biological efficiency of the molecule. Previous studies proposed that an ideal value of Kpart of 1 should give maximum biological activity. Mobilization of iron by Csox and CacCAM from ferritin was similar and furthermore more efficient than desferrioxamine B. In the iron-loaded hepatocyte cultures, the three hydroxyquinoline chelators, although showing diversity in terms of lipophilicity, appeared to be very similar in their capacity to chelate iron. CacCAM, the unique catecholate, was the most efficient of the molecules tested, as well as being the least toxic in the cellular model despite having the lowest value of pFe. In conclusion, the use of the partition coefficient and pFe, as tools for predicting biological activity of iron chelators should be not generalized. Further studies are required in order to understand the influence of the structure on the biological activity of the molecule.

Animals↗
PubMed2001

The effect of resonance energy homotransfer on the intrinsic tryptophan fluorescence emission of the bothropstoxin-I dimer.

Bothopstoxin-I (BthTX-I) is a homodimeric Lys49-PLA2 homologue from the venom of Bothrops jararacussu in which a single Trp77 residue is located at the dimer interface. Intrinsic tryptophan fluorescence emission (ITFE) quenching by iodide and acrylamide has confirmed that a dimer to monomer transition occurs on reducing the pH from 7.0 to 5.0. Both the monomer and the dimer showed an excitation wavelength-dependent increase in the fluorescence emission maximum, however the excitation curve of the dimer was blue-shifted with respect to the monomeric form. No differences in the absorption or circular dichroism spectra between pH 5.0 and 7.0 were observed, suggesting that this curve shift is due neither to altered electronic ground states nor to exciton coupling of the Trp residues. We suggest that fluorescence resonance energy homotransfer between Trp77 residues at the BthTX-I dimer interface results in excitation of an acceptor Trp population which demonstrates a red-shifted fluorescence emission.

Animals↗
PubMed2001

A pH-induced dissociation of the dimeric form of a lysine 49-phospholipase A2 abolishes Ca2+-independent membrane damaging activity.

The hydrolysis of phospholipids by class II phospholipase A2 (PLA2) involves a Ca2+ ion cofactor bound to the Asp49 residue in the active site region. In the lysine 49 phospholipase A2 homologues (Lys49-PLA2), the Asp49 residue is substituted by Lys, and consequently the Lys49-PLA2s show no Ca2+ binding and no detectable phospholipid hydrolysis. Nevertheless, the Lys49-PLA2s demonstrate membrane damaging activity by an incompletely understood Ca2+-independent mechanism of action. Using a combination of steady-state and time-resolved fluorescence techniques, we have examined the effect of pH on the monomer-dimer equilibrium of bothropstoxin I (BthTX-I), a Lys49-PLA2 from the venom of Bothrops jararacussu which contains a single Trp77 residue located at the dimer interface. At pH 5.0, we observe a decreased quantum yield, a decreased rotational correlation time, and an increased bimolecular quenching rate constant with iodide. These results are consistent with a pH-induced dissociation of the BthTX-I dimer, with the consequent exposure of the Trp77 residue to aqueous solvent. In the presence of liposomes, membrane damaging activity is observed only under conditions in which the dimeric form of the BthTX-I is favored. These results demonstrate that the dimeric form of the protein is essential for the initiation of the Ca2+-independent membrane damaging activity.

Animals↗
PubMed2001

Dissociation of enzymatic and pharmacological properties of piratoxins-I and -III, two myotoxic phospholipases A2 from Bothrops pirajai snake venom.

Piratoxins (PrTX) I and III are phospholipases A2 (PLA2s) or PLA2 homologue myotoxins isolated from Bothrops pirajai snake venom, which also induce myonecrosis, bactericidal activity against Escherichia coli, disruption of artificial membranes, and edema. PrTX-III is a catalytically active hemolytic and anticoagulant Asp49 PLA2, while PrTX-I is a Lys49 PLA2 homologue, which is catalytically inactive on artificial substrates, but promotes blockade of neuromuscular transmission. Chemical modifications of His, Lys, Tyr, and Trp residues of PrTX-I and PrTX-III were performed, together with cleavage of the N-terminal octapeptide by CNBr and inhibition by heparin and EDTA. The lethality, bactericidal activity, myotoxicity, neuromuscular effect, edema inducing effect, catalytic and anticoagulant activities, and the liposome-disruptive activity of the modified toxins were evaluated. A complex pattern of functional differences between the modified and native toxins was observed. However, in general, chemical modifications that significantly affected the diverse pharmacological effects of the toxins did not influence catalytic or membrane disrupting activities. Analysis of structural changes by circular dichroism spectroscopy demonstrated significant changes in the secondary structure only in the case of N-terminal octapeptide cleavage. These data indicate that PrTX-I and PrTX-III possess regions other than the catalytic site, which determine their toxic and pharmacological activities.

Acetophenones↗
PubMed2001

Refolding and purification of Bothropstoxin-I, a Lys49-phospholipase A2 homologue, expressed as inclusion bodies in Escherichia coli.

Hydrolysis of phospholipids by Group II phospholipase A2 enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region. Bothropstoxin-I (BthTX-I) is a PLA(2) variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E. coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes.

Amino Acid Substitution↗
PubMed2001

Changes in plasma and urinary taurine and amino acids in runners immediately and 24h after a marathon.

Changes in urinary and plasma taurine and amino acids have been evaluated in trained runners competing in the Rotterdam Marathon, 1998, both immediately after completing the event and 24h after recovery. There were significant changes in the urinary amino acids excretion, the majority showing a significant decrease both immediately at the completion of the Marathon and after 24h recovery. In contrast urinary taurine excretion increased immediately post Marathon, although not significantly as the range of results was wide. Such changes in urinary taurine correlated with percentage changes in plasma creatine kinase both immediately post race, (r = 0.972, P < 0.001), and 24h later (r = 0.872, P < 0.001), possibly indicating that the source of the taurine was muscle. Significant correlations between the individual values for urinary and plasma amino acids in all of the athletes were calculated for taurine (r = 0.528), glycine (r = 0.853), threonine (r = 0.749), alanine (r = 0.747), serine (r = 0.620), glutamine (0.614), arginine (r = 0.507), histidine (r = 0.470) and valine (r = 0.486). Changes in the mean plasma concentrations of amino acids were comparable to our previously published data (Ward et al., 1999) the majority showing significant decreases immediately and 24h post Marathon, such an adaptation being due primarily to their utilisation for gluconeogenesis. However, in contrast, the mean taurine concentrations were significantly elevated both post race, P < 0.01 and after 24h, P < 0.05. The physiological response by the muscle to exhaustive exercise, particularly with regard to changes in plasma and urinary taurine concentrations remain to be elucidated, but is probably related to muscle function impairment. The increase in taurine urinary excretion could be used as an indicator of muscle damage occurring during exhaustive exercise. Whether taurine supplementation would minimise such changes is an interesting scientific question and merits investigation.

Amino Acids↗
PubMed2001

Aluminium toxicity and iron homeostasis.

In an animal model of aluminum overload, (aluminium gluconate), the increases in tissue aluminium content were paralleled by elevations of tissue iron in the kidney, liver heart and spleen as well as in various brain regions, frontal, temporal and parietal cortex and hippocampus. Despite such increases in iron content there were no significant changes in the activities of a wide range of cytoprotective enzymes apart from an increase in superoxide dismutase in the frontal cortex of the aluminium loaded rats. Such increases in tissue iron content may be attributed to the stabilisation of IRP-2 by aluminium thereby promoting transferrin receptor synthesis while blocking ferritin synthesis. Using the radioactive tracer (26)Al less than 1% of the injected dose was recovered in isolated ferritin, supporting previous studies which also found little evidence for aluminium storage within ferritin. The increases in brain iron may well be contributory to neurodegeneration, although the pathogenesis by which iron exerts such an effect is unclear.

Aluminum↗
PubMed2001

Differential capacities of the RGS1, RGS16 and RGS-GAIP regulators of G protein signaling to enhance alpha2A-adrenoreceptor agonist-stimulated GTPase activity of G(o1)alpha.

Recombinant RGS1, RGS16 and RGS-GAIP, but not RGS2, were able to substantially further stimulate the maximal GTPase activity of G(o1)alpha promoted by agonists at the alpha2A-adrenoreceptor in a concentration-dependent manner. Kinetic analysis of the regulation of an alpha2A-adrenoreceptor-G(o1)alpha fusion protein by all three RGS proteins revealed that they had similar affinities for the receptor-G protein fusion. However, their maximal effects on GTP hydrolysis varied over threefold with RGS16 > RGS1 > RGS-GAIP. Both RGS1 and RGS16 reduced the potency of the alpha2A-adrenoreceptor agonist adrenaline by some 10-fold. A lower potency shift was observed for the partial agonist UK14304 and the effect was absent for the weak partial agonist oxymetazoline. Each of these RGS proteins altered the intrinsic activity of both UK14304 and oxymetazoline relative to adrenaline. Such results require the RGS interaction with G(o1)alpha to alter the conformation of the alpha2A-adrenoreceptor and are thus consistent with models invoking direct interactions between RGS proteins and receptors. These studies demonstrate that RGS1, RGS16 and RGS-GAIP show a high degree of selectivity to regulate alpha2A-adrenoreceptor-activated G(o1)alpha rather than G(i1)alpha, G(i2)alpha or G(i3)alpha and different capacities to inactivate this G protein.

Adrenergic alpha-Agonists↗
PubMed2001

MR imaging of bone oedema: mechanisms and interpretation.

Widespread use of MRI now allows the routine identification of previously unevaluated traumatic bone marrow oedema and haemorrhage. Similar marrow oedema is identified in patients with tumours, hyperaemia and medullary congestion. Patterns and extent of traumatic bone marrow oedema and haemorrhage are dictated by mechanism. Diffusion techniques may allow precise evaluation of severity of injury. Illustrative examples and discussion are presented. Eustace, S. (2001). Clinical Radiology56, 4-12.

Adult↗
PubMed2001

Neuroreceptors and ion channels as targets of alcohol.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Kinya Kuriyama. The presentations were (1) Modulation of neuroreceptors and ion channels by alcohol, by T. Narahashi; (2) Inhibition by ethanol of NMDA and AMPA receptor-channels, by P. Illes, K. Wirkner, W. Fischer, K. Mühlberg, P. Scheibler, and C. Allgaier; (3) Effects of ethanol on metabotropic glutamate receptors, by K. Minami; (4) Acute alcohol actions on the 5-HT3 ligand-gated ion channel, by D. Lovinger; (5) Inhibition of NMDA receptors by MK801 attenuates ethanol-induced taurine release from the hippocampus, by F. Lallemand, R.J. Ward, and P. DeWitte; and (6) Effect of ethanol on voltage-operated Ca2+ channels in hepatic stellate cells, by T. Itatsu, Y. Takei, H. Oide, M. Hirose, X. E. Wang, S. Watanabe, M. Tateyama, R. Ochi, and N. Sato.

Animals↗
PubMed2000

Does the haemosiderin iron core determine its potential for chelation and the development of iron-induced tissue damage?

Haemosiderin, the major iron storage protein in tissues of iron-loaded tissues shows heterogeneity with respect to both its iron mineralisation product and associated protein. Such mineralisation products have been characterised by a variety of physical techniques including Mössbauer spectroscopy, electron diffraction and EXAFS, and are closely related to the mineral ferrihydrite. A wide range of iron chelators are being developed for the treatment of abnormal haemoglobinopathies, predominantly beta-thalassaemia, which may show greater chelator efficacy for particular mineralisation products of haemosiderin. Even though the tissue iron loadings achieved in different iron-loading syndromes are similar, e.g. naturally occurring iron loading, genetic haemochromatosis and thalassaemia, it is clear that the iron loading in thalassaemic causes extensive damage. The explanation for this could relate to the distribution of iron within different cell types, predominantly reticuloendothelial, its rate of deposition and the mineralisation product of its haemosiderin iron core, goethite.

Animals↗
PubMed2000

The structural failure of a multi-hole collimator.

During measurements of the extrinsic spatial resolution of a gamma camera, it was noted that there was a variation with the position in the field of view. The problem was traced to the collimator, and this was confirmed by inspection of the parallel hole structure. Removal of the cover showed that the lead foil honeycomb had become detached from its surround. The fault could have had serious implications for the quality of patient investigations and could also have caused damage to the crystal if it had gone undetected. As a result of this investigation, the quality assurance procedures within this department have been updated to include a visual check of all collimator cores.

Gamma Cameras↗
PubMed2000

Effect of inhaled fluticasone propionate on airway responsiveness in treatment-naive individuals--a lesser benefit in females.

A randomized double-blind placebo-controlled parallel group study with inhaled fluticasone propionate over 6 weeks, designed to quantify the beneficial effect on airway responsiveness, and so assess whether short pulses of intermittent prophylactic treatment might serve as an alternative means of managing mild asthma, is reported. The 20-50-yr-old participants, who were recruited from an epidemiological study of the general population, had never knowingly received any regular treatment for asthma. Fluticasone propionate at the maximum recommended dose level (2,000 microg daily) and placebo were administered via metered-dose inhalers, and airway responsiveness was quantified conventionally by the provocative dose of methacholine causing a 20% fall in forced expiratory volume in one second (FEV1) (PD20) at 2-week intervals during the treatment phase and at various intervals subsequently. Compared with placebo fluticasone propionate was associated with a highly significant decrease in airway responsiveness (1.9 doublings of the geometric mean PD20), which was maximal at the end of the 6-week treatment period. No persisting benefit was detectable at the next measurement 2 weeks later, or thereafter. Multiple linear regression analysis showed that the magnitude of the fluticasone propionate effect was significantly greater in males than in females (3.2 versus 1.2 doublings respectively of the geometric mean PD20), but was uninfluenced by current smoking, age or FEV1. In conclusion, in the absence of any possibility of tachyphylaxis, inhaled fluticasone propionate at this dose causes a steadily increasing improvement in airway responsiveness over a 6-week period, which is modified by sex but lost almost immediately on treatment cessation. Short pulses of intermittent prophylactic treatment would not, therefore, be useful as a means of managing mild asthma.

Administration, Inhalation↗
PubMed2000

Ovine adipose tissue monounsaturated fat content is correlated to depot-specific expression of the stearoyl-CoA desaturase gene.

The basis for the variation in fatty acid composition in different ovine adipose tissue depots was investigated. The proportion of stearic (C18:0) and oleic (C18:1) acids vary in a site-specific fashion; abdominal depots (omental and perirenal) contain relatively more C18:0 than C18:1, and carcass depots, especially sternum, have a markedly higher proportion of C18:1. Additionally, expression of a number of lipogenic enzyme genes (stearoyl-CoA desaturase [SCD], acetyl-CoA carboxylase-alpha [ACC-alpha], lipoprotein lipase [LPL]) and the cytoskeletal protein gene alpha-tubulin vary among depots, although the pattern of variation differs for each mRNA. When these expression data were related to the mean cell volume of adipocytes pooled from all depots, a significant pattern emerged: expression of the ACC-alpha, LPL, and alpha-tubulin genes was highly correlated with the size of adipocytes. In contrast, when the expression of SCD mRNA was assessed as a function of mean cell volume, two populations of adipocytes emerged: no significant correlation was found between the expression of SCD mRNA per adipocyte and mean cell volume for the abdominal depots, although a highly significant correlation was observed between SCD gene expression and mean cell volume for the carcass and epicardial depots. Similarly, a highly significant correlation was found for the amount of C18:1 per adipocyte and the abundance of SCD mRNA per adipocyte for the carcass and epicardial depots, whereas no significant correlation was observed for these traits for the omental and perirenal depots. Thus, the SCD gene seems to be regulated in a depot-specific fashion and in a manner distinct from that of the ACC and LPL genes.

Acetyl-CoA Carboxylase↗
PubMed1999

An Asp79Asn mutation of the alpha2A-adrenoceptor interferes equally with agonist activation of individual Gialpha-family G protein subtypes.

The quantitative effects of an Asp79Asn mutation in the porcine alpha2A-adrenoceptor on adrenaline-mediated stimulation of the alpha subunit of individual members of the Gi family of G proteins were assessed by measuring GTP turnover number for fusion proteins between the wild type or mutated receptor and pertussis toxin-resistant forms of each of Gi1, Gi2 and Gi3. In each case the receptor mutation limited activation of the G protein to 8-14% of that produced by the wild type receptor. Previous demonstration that in a single cell this mutation selectively interferes with alpha2A-adrenoceptor regulation of distinct effector end points transduced by Gi family members must therefore reflect differential requirements for amplification or the cellular location of individual, co-expressed, G proteins.

Amino Acid Substitution↗