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Biomedical subjects

P Eaton

Publications and source records attributed to P Eaton.

At least 19 recordsLinked to original sources
PubMed2001

Lipid hydroperoxide modification of proteins during myocardial ischaemia.

OBJECTIVE: Lipid hydroperoxides (LOOH) are lipid peroxidation products formed during oxidative stress. A component of their cytotoxicity is mediated by the direct modification of proteins. OBJECTIVES: (i) To assess whether ischaemia and reperfusion in the isolated rat heart generates LOOH-protein (ii) to characterise the extent, time-course and subcellular localization of any protein adducts formed. METHODS: Using a well-characterised antibody which binds to LOOH-modified proteins and densitometry of Western blots, we quantified the amounts of LOOH-protein in control aerobically perfused rat hearts and those subjected to ischaemia with and without reperfusion. RESULTS: Hearts (n=3/4 group), analysed after various periods (0, 5, 10, 20 and 30 min) of zero-flow global ischaemia, exhibited a time-dependent increase in the LOOH-mediated modification of a number of proteins. In hearts subjected to 30 min of ischaemia and then reperfused for various times (0, 5, 10, 20, 30 or 60 min) no changes in LOOH-protein content achieved during the proceeding ischaemic episode were detected. Reperfusion after short periods of ischaemia (5 or 10 min) also did not result in reperfusion-induced LOOH-protein formation. Administration of mercaptopropionylglycine (1 mM) to hearts for 5 min before the onset of 30 min ischaemia efficiently attenuated the formation of LOOH-protein, maintaining the modified proteins at control levels. These Western immunoblot results were confirmed by additional in situ immunofluorescent studies which showed marked LOOH-protein immunostaining in ischaemic tissue around the sarcolemmal membrane. CONCLUSIONS: We conclude that the modification of proteins (particularly those associated with sarcolemmal membranes) by LOOH during ischaemia may contribute to the pathophysiology of ischaemic injury. In addition, these modifications may be initiators of oxidant-induced signal transduction pathways. These findings are consistent with an oxidant stress occurring during ischaemia which is not exacerbated or reduced during the first 60 min of reperfusion.

Animals↗
PubMed2001

Differential centrifugation separates cardiac sarcolemmal and endosomal membranes from Langendorff-perfused rat hearts.

The application of subcellular fractionation protocols developed in soft tissues to fibrous organs such as the heart is unsuitable given the substantial differences in subcellular structure these tissues exhibit. The purpose of this study was to develop a simple method for the separation of sarcolemma and endosomes from isolated Langendorff-perfused rat hearts. Hearts were homogenized with either an Ultra-Turrax homogenizer or a hand-held glass tissue grinder. Quantitative immunoblots assessed the enrichment of the sarcolemmal proteins caveolin 3 and the sodium potassium ATPase and the endosomal proteins rab4 and GLUT4 in different membrane fractions. Application of homogenates to sucrose and Percoll density gradients failed to resolve membranes differentially enriched in sarcolemmal or endosomal marker proteins, indicating little difference in density between the sarcolemma and endosomes. However, successive spins of homogenates from a hand-held glass tissue grinder successfully separated the endosomes from the sarcolemma, indicating differences in masses between the two membrane fractions. Approximately 70% of total caveolin 3 and sodium potassium ATPase immunoreactivity was in membrane pellets up to 20,000g and approximately 85% of rab4 and GLUT4 in pellets from 20,000-100,000g. In addition, 86% of ouabain-sensitive ATPase activity (sodium potassium ATPase activity) was in membrane pellets up to 20,000g. Therefore, sarcolemmal membranes were pelleted up to 20,000g, and endosomal membranes between 20,000 and 100,000g. Regional ischemia (40 min) followed by reperfusion (60 min) caused the translocation of GLUT4 (but not rab4) from the endosomal membranes to the sarcolemma in the area of the heart subjected to ischemia.

Animals↗
PubMed2001

AlphaB crystallin translocation and phosphorylation: signal transduction pathways and preconditioning in the isolated rat heart.

In this program of studies we have characterized in detail the translocation (assessed by Triton-insolubility) and phosphorylation (using serine-45 or -59 phosphospecific antibodies) of alphaB crystallin during myocardial ischemia [both with or without ischemic preconditioning (IPC)]. Pharmacological activators and inhibitors allowed us to characterize the signaling pathways involved in alphaB crystallin phosphorylation during ischemia. Ischemic preconditioning alone caused 30% of the heart's alphaB crystallin pool to translocate, providing a significant translocation 'head-start' in protected tissue. This enhanced translocation is coupled with increased (3-fold) alphaB crystallin phosphorylation at both serine residues. The possible role of alphaB crystallin in the protection afforded by ischemic preconditioning is supported by the signal transduction data; which showed preconditioning-induced alphaB crystallin phosphorylation can be blocked by tyrosine kinase inhibition (using genistein) and by p38 MAP kinase or PKC inhibition (using SB203580 or bisindolylmaleimide, respectively). The activation of both p38 MAP kinase and PKC are recognized requirements for the induction of preconditioning and their inhibition is known to block protection. Western immunoblotting analysis after isoelectric focusing electrophoresis, confirmed the observations made with the phosphospecific antibodies; but also showed that 27+/-4% of total cardiac crystallin was phosphorylated after 30 min of ischemia. AlphaB crystallin exists as large polymeric aggregates in cardiac tissue under basal conditions (approximately 1 MDa as determined by gel filtration chromatography). We induced phosphorylation of alphaB crystallin during aerobic perfusion by the administration of phenylephrine. However this treatment did not alter the molecular aggregate size of alphaB crystallin. It appears that alphaB crystallin molecular aggregate size is not simply regulated by phosphorylation. AlphaB crystallin may have a role to play in the myocardial protection induced by ischemic preconditioning, as both translocation and phosphorylation are both accelerated and enhanced by ischemic preconditioning.

Adrenergic alpha-Agonists↗
PubMed2000

Ischemic preconditioning: a potential role for constitutive low molecular weight stress protein translocation and phosphorylation?

We have investigated whether translocation of constitutive low molecular weight stress proteins (alphaB-crystallin and HSP27) to the myofilament/cytoskeletal compartment occurs during ischemic preconditioning and assessed if this is causally associated with cardioprotection. Triton-insoluble preparations from fresh or aerobically perfused rat hearts (n=4/group) contained relatively little alphaB-crystallin (96 +/- 43 and 43 +/- 36 units respectively) or HSP27 (177 +/- 32 and 101 +/- 26 units respectively). Three preconditioning cycles of (5 min ischemia + 5 min reperfusion) increased the Triton-insoluble crystallin to 864 +/- 61 units (P<0.05) and HSP27 to 1353 +/- 53 units (P<0.05). Two hours of aerobic perfusion following the preconditioning protocol resulted the return of alphaB-crystallin and HSP27 to near control levels (189 +/- 14 units and 252 +/- 24 units, respectively). Stress protein translocation, comparable to that achieved by the IPC protocol was induced by aerobic perfusion with hypercarbic (pH 6.8) perfusion. Thus, three cycles of 5 min hypercarbia + 5 min normocarbia increased alphaB-crystallin to 628 +/- 30 units (P<0.05) and HSP27 to 1353 +/- 53 units. In parallel functional studies, the recovery of LVDP after 35 min ischemia and 60 min of reperfusion was 43 +/- 7% in the ischemic control group, 61 +/- 3% (P<0.05) in the preconditioned group and 42 +/- 6% in the hypercarbic group. Thus, translocation of alphaB-crystallin and/or is not of-itself sufficient to induce cardioprotection. Using a phospho-specific antibody, we have demonstrated that preconditioning not only translocates alphaB-crystallin but also increases its phosphorylation at Ser-59 by 9.7-fold compared to aerobic controls (1616 +/- 402 v 166 +/- 28 units respectively). In contrast, hypercarbia while eliciting a comparable translocation, failed to alter the phosphorylation state of alphaB-crystallin. Preconditioning-induced phosphorylation was significantly attenuated by 50 microM genistein (by 61%), 10 microM SB203580 (by 91%) and 10 microM bisindolylmaleimide (by 68%), but not by 10 microM PD98059 (by 4%). Our findings are consistent with the possibility that ischemic preconditioning may be mediated by phosphorylation and translocation of constitutive low molecular weight stress proteins, particularly alphaB-crystallin.

Animals↗
PubMed1999

Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart.

4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion.

Aldehydes↗
PubMed1998

The origins, fate, and ecological significance of free amino compounds released by freshwater pulmonate snails.

The mass-specific accumulation rates (MSAR) of both total (TFAC) and individual free amino compounds (FAC) in conditioned media were measured by HPLC, using the orthophthaldialdehyde (OPA) methods, in the following cases: (a) laboratory-reared freshwater snails (B. glabrata) with chemosterilized shells; (b) Biomphalaria glabrata with non-chemosterilized shells; (c) B. glabrata faeces; (d) isolated shells of B. glabrata; and (e) 10 other species of freshwater gastropods from the Lewes Brooks, East Sussex, U.K. The MSAR values for B. glabrata show that 95% of the TFAC's (predominantly ethanolamine, phosphoserine, and the amino acids leucine, isoleucine, valine, aspartic acid, and glycine/threonine) originated from the snails themselves as the faeces and shells contributed only 5.0 and 0%, respectively. In contrast, epizootic organisms on the shells of all 10 snail species from the Lewes Brooks released significant amounts of FAC with the two smallest species (Planorbis vortex and Planorbis contortus) having the highest MSAR values. The MSAR for isolated B. glabrata mucus was 42.45 micromol x g(-1)h(-1). As 500 mg snails can release 16.67 mg of mucus daily, this could potentially result in the daily loss of 707.5 micromol of FAC. The cost/benefits of mucus secretion and the various anatomical, physiological, biochemical, and ecological mechanisms which allow freshwater snails to recover FAC's lost as a result of a high rate of urine production in their hypotonic environment, are discussed.

Amines↗
PubMed1988

Contamination of a monoclonal antibody with LDH-virus causes interferon induction.

Interferon induction occurred unexpectedly during an in vivo study using a mouse monoclonal antibody. The interferon was typed alpha/beta and the titer reached a maximum at 24 hours in contrast with other inducers. Similar results were obtained with a virus pool derived from the antibody and with a LDV reference strain. MAP-testing of the monoclonal antibody revealed contamination with lactate dehydrogenase virus (LDV). The production of IFN seems to be controlled genetically. This experimental error demonstrates the importance of an appropriate quality control of biological materials.

Animals↗
PubMed1986

Multiple attempters of suicide presenting at an emergency department.

As part of a study involving seven hundred and forty-seven (747) consecutive cases of self-injury presenting at the emergency department of an inner city hospital, data pertaining to a multiple attempt group of ninety-nine subjects were compared with the data pertaining to first attempters. All subjects were rated on Beck's Lethality Scales, Intent Scale and Hopelessness Scale. In addition, a standard interview was conducted to provide psychosocial information. Of the total suicide attempt population, 14% can be considered chronic. The multiple attempters were found to show more chronicity in their symptomatology, poorer coping histories, a positive family history of suicidal behaviour and a positive history of alcohol and drug abuse, all at a statistically significant level compared with first-time attempters. The multiple attempters also tend to show higher lethality and depression ratings, less impulsiveness and are significantly more often admitted to the in-patient unit following the attempt, than first attempters. Their intent scores were significantly related to their sense of hopelessness, which may be a reflection of their basic feelings of inadequacy. It was concluded that the multiple or "chronic" attempter differed sufficiently from the first-time attempter to warrant special intervention strategies.

Adolescent↗
PubMed1985

Suicide attempters presenting at an emergency department.

Seven hundred and forty-seven consecutive cases of deliberate self-injury presenting at the Emergency Department of an inner city hospital were rated on Beck's Medical Lethality Scales, the Intent Scale and Poldinger's Risk Scale. In addition, demographic data were collected on each patient. The results pertaining to the demographic characteristics of the subjects were found to be similar to those reported elsewhere. However, although the overall intent scores were not found to be predictive of the lethality of the method employed, the "objective" circumstances surrounding the attempt were found to be highly related to the risk factor assigned to each patient. The recommendation is made that specific sub-groups of attempters need to be identified and may warrant specialized intervention strategies.

Adolescent↗
PubMed1980

Enterotoxaemia involving Clostridium perfringens iota toxin in a hysterectomy-derived rabbit colony.

During an explosive outbreak of fatal enteropathic disease involving Clostridium perfringens iota (i) toxin, a total of 183 deaths occurred in 18 weeks. The clinical signs and post-mortem findings are reported. Examinations for virus, Bacillus piliformis and coccidia were negative. Clostridium perfringens i toxin was detected in 22 of 27 animals examined (81.5%), but clostridia were not isolated. Various treatments were attempted. It is concluded that i toxin and the syndrome described are closely related.

Animals↗