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David Sheehan

Publications and source records attributed to David Sheehan.

18 recordsLinked to original sources
PubMed2006

Detection of redox-based modification in two-dimensional electrophoresis proteomic separations.

Oxidative stress arises when cellular defenses against molecular oxygen and its by-products (reactive oxygen species; ROS) are overcome leading to covalent modification of lipids, DNA, and protein. Redox-based modification of proteins can be conveniently studied by proteomic analysis using two-dimensional electrophoresis (2D SDS-PAGE). Despite some technical shortcomings, this technique allows rapid and quantitative analysis of paired samples, the visualization of discrete protein spots, and provides a robust platform for subsequent analysis and identification of specific proteins. Exposure to oxidative stress introduces a wide range of reversible or irreversible alterations to amino acid side chains. These include carbonylation, glutathionylation, formation of mixed disulphides, effects on disulphide bridge patterns, ubiquitinylation, and racemization. Identification of proteins targeted for specific modification adds a deeper dimension to the dissection of effects of oxidative stress on the proteome with potentially far-reaching implications. This article describes key methodologies now available for identification of redox-based modifications in proteins separated by 2D SDS-PAGE.

Animals↗
PubMed2006

Redox proteomics in the blue mussel Mytilus edulis: carbonylation is not a pre-requisite for ubiquitination in acute free radical-mediated oxidative stress.

Mytilus edulis was exposed under controlled conditions to a panel of model pro-oxidants (H(2)O(2), CdCl(2) and menadione) for 24h. Protein extracts of gill, mantle and digestive gland were analysed by immunoblotting in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) separations. Immunoblotting revealed extensive and comparable levels of protein carbonylation across the pro-oxidant panel with approximately 1.5-fold higher levels in gill than digestive gland. Ubiquitination in gill was modest in response to H(2)O(2), but increased in response to menadione and CdCl(2). High ubiquitination levels were found for all pro-oxidants in digestive gland with levels comparable to the highest found in gill. Two-dimensional (2D) SDS-PAGE confirmed specific targeting of individual proteins by ubiquitin against a generally stable protein expression signature. Spot matching suggested that carbonylation is not a pre-requisite for ubiquitination. While gill showed consistently higher constitutive levels of glutathione transferase, glucose 6-phosphate dehydrogenase and glutathione reductase activity, pro-oxidant treatment had only modest effect on these enzymes and on the ratio of reduced/oxidised glutathione. In digestive gland, this latter ratio was higher than in gill and increased in response to menadione and CdCl(2). Ubiquitination may provide a marker for acute onset of oxidative stress in bivalves.

Animals↗
PubMed2006

Chemical modification at subunit 1 of rat kidney Alpha class glutathione transferase with 2,3,5,6-tetrachloro-1,4-benzoquinone: close structural connectivity between glutathione conjugation activity and non-substrate ligand binding.

2, 3, 5, 6-Tetrachloro-1, 4-benzoquinone (TCBQ) is a metabolite of pentachlorophenol known to react with cysteines of glutathione transferases (GSTs). TCBQ treatment of rat kidney rGSTA1-2 and rGSTA1-1 abolishes 70-80% conjugation of glutathione (GSH) to 1-chloro-2, 4-dinitrobenzene and results in strongly correlated quenching of intrinsic fluorescence of Trp-20 (R>0.96). rGSTA2-2 is only inhibited by 25%. Approximately 70% (rGSTA1-1) and 60% (rGSTA1-2) conjugation activity is abolished at TCBQ: GST stoichiometries near 1:1. The inactivation follows a Kitz/Wilson model with K(D) of 4.77+/-2.5microM for TCBQ and k(3) for inactivation of 0.036+/-0.01min(-1). A single tryptic peptide labelled with TCBQ was isolated from kidney rGSTA1-2 containing Cys-17 which we identify as the site of modification. Treatment with more than stoichiometric amounts of TCBQ modified other residues but resulted in only modest further inhibition of catalysis. We interpret these findings in terms of localised steric effects on the relatively rigid alpha-helix 1 adjacent to the catalytic site of subunit 1 possibly affecting the Alpha class-specific alpha-helix 9 which acts as a "lid" on the hydrophobic part of the active site. Homology modelling of rGSTA1-1 modified at Cys-17 of one subunit revealed only modest structural perturbations in the second subunit and tends to exclude global structural effects.

Animals↗
PubMed2006

Proteomics as a route to identification of toxicity targets in environmental toxicology.

Ecotoxicology describes a three-way relationship between ecosystems, chemical pollutants and living organisms. It is predicated on the fact that chemical pollution can exert toxic effects on organisms at the individual and population levels. These toxic effects may provide important information to supplement chemical analysis of environmental samples and aid in assessing the environmental quality of specific ecosystems. Traditionally, effects have been detected by means of biomarkers which, of necessity, were often molecules or processes known to be affected by pollutants. Proteomics provides a means of achieving high-throughput analysis of effects on protein populations and sub-populations with the potential to identify novel biomarkers. This review summarises the main approaches currently used in this area and assesses the potential of proteomics for identification of novel toxicity targets.

Chromatography↗
PubMed2006

A two-species biomarker model for the assessment of sediment toxicity in the marine and estuarine environment using the comet assay.

Sediments frequently cause damage to biota due to the accumulation of toxic compounds and the bioavailability of sediment-associated contaminants. Damage can be assessed using biomarkers, such as the degree of genotoxic impact following in vivo exposure to contaminants. Genotoxic damage, expressed as single-strand DNA breaks, was measured in cells isolated from haemolymph/blood, gill and digestive gland/liver from the clam Tapes semidecussatus and turbot Scophthalmus maximus, using the single cell gel electrophoresis (Comet Assay). Both animals were exposed for three weeks to sediment samples collected from a polluted site and a 'clean' reference site. The level of DNA damage was assessed using an image analysis package and expressed as % tail DNA. Throughout the study, significant differences in DNA damage were recorded for each tissue type, in both species, between animals exposed to the two sediment samples. However, turbot appeared to be a more sensitive indicator species, because, due to lower background levels, they were able to detect a significant difference between reference site and background values. This suggests that turbot, rather than clams, are more suitable as a sentinel species for the assessment of genotoxic impact of low-level contamination in aquatic sediments and highlights the need for a two- or multi-species approach.

Animals↗
PubMed2005

Protein carbonylation and heat shock response in Ruditapes decussatus following p,p'-dichlorodiphenyldichloroethylene (DDE) exposure: a proteomic approach reveals that DDE causes oxidative stress.

Protein carbonylation and levels of heat shock proteins (hsp; 60, 70 and 90 kDa) were measured in gill, mantle and digestive gland of Ruditapes decussatus following exposure to p,p'-dichlorodiphenyldichloroethylene (DDE). Heat shock response was measured by immunoblotting using antibodies specific to heat shock proteins (hsps). Densitometry analysis of individual bands revealed no difference between control and treated samples except appearance of hsp90 in DDE-treated mantle. Carbonylated protein content was determined following 2,4-dinitrophenylhydrazine derivatization and two-dimensional electrophoresis coupled with western blotting. Immunoblotting with dinitrophenol-specific antibody revealed extensive differences in both extent and number of carbonylated proteins in mantle and digestive gland in response to DDE while gill was unaffected. These results demonstrate for the first time that DDE causes tissue-specific formation of reactive oxygen species in clams.

Animals↗
PubMed2005

Cellular responses in primary epidermal cultures from rainbow trout exposed to zinc chloride.

In this study, we examined the effects of zinc chloride (ZnCl(2)) (0-200mg/L) on primary epidermal cultures from Oncorhynchus mykiss. Increases in the rate and amount of mucus released were detected post-exposure, as was a dose-dependent increase in the synthesis of acidic glycoproteins. The cytotoxicity of ZnCl(2) to the cultures was significantly increased (P< or =0.05) when exposures were conducted in serum-free medium as opposed to medium containing serum. Significant increases in the levels of apoptosis and necrosis were observed with increasing exposure concentration, but these were seen to decrease over time. The in vitro cytological and pathological changes observed in this study were found to be in accordance with previously published in vivo studies on the effects of heavy metals on the integument. This model system may help to further elucidate the effects of ecotoxicants on the external innate immune system of fish.

Animals↗
PubMed2005

The 110kDa glutathione transferase of Yarrowia lipolytica is encoded by a homologue of the TEF3 gene from Saccharomyces cerevisiae: cloning, expression, and homology modeling of the recombinant protein.

The TEF4 gene of the non-saccharomyces yeast Yarrowia lipolytica encodes an EF1Bgamma protein with structural similarity to the glutathione transferases (GSTs). This 1203bp gene was cloned, over-expressed in Escherichia coli, and the recombinant protein characterized. DNA sequencing of the cloned gene agreed with the recently completed Y. lipolytica genome and showed 100% identity to a previously reported 30-residue N-terminal sequence for a 110kDa Y. lipolytica GST, except that it encoded two additional N-terminal residues (N-Met-Ser-). The recombinant protein (subunit M(r) 52kDa) was found not to possess GST activity with 1-chloro-2,4-dinitrobenzene. Partial tryptic digestion released two fragments of M(r) 22 and 18kDa, which we interpret as N- and C-terminal domains. Homology modeling confirmed that the N-terminal domain of Y. lipolytica TEF4 encodes a GST-like protein.

Cloning, Molecular↗
PubMed2005

Carbonylation and glutathionylation of proteins in the blue mussel Mytilus edulis detected by proteomic analysis and Western blotting: Actin as a target for oxidative stress.

Protein expression profiles (PEPs) were generated by two-dimensional electrophoresis (2-D SDS-PAGE) for gill and digestive glands of Mytilus edulis sampled from a polluted and reference site in Cork Harbour, Ireland. Similar patterns and expression levels were found for both sites in silver stained gels. However, Western blotting for carbonylated proteins demonstrated higher levels of specific carbonylation of proteins in tissues from animals in the polluted site. Animals from the reference site were acclimated in holding tanks, exposed to 1 mM H2O2 for 24 h, dissected and analysed by 2-D SDS-PAGE. Again, generally similar PEPs were found in control and exposed animals for gill and digestive gland but carbonylation was more pronounced in polluted and exposed animals. Western blotting of extracts after one-dimensional electrophoresis with antibodies to glutathione and actin revealed that gill proteins are glutathionylated more strongly than digestive gland and that this process is more pronounced in polluted animals than in controls. We conclude that carbonylation and glutathionylation can occur in gill and digestive gland in response to oxidative stress in M. edulis. Actin is a major target for both glutathionylation and carbonylation under oxidative stress conditions.

Actins↗
PubMed2005

Identification of a multixenobiotic resistance mechanism in primary cultured epidermal cells from Oncorhynchus mykiss and the effects of environmental complex mixtures on its activity.

Multixenobiotic resistance (MXR) is a mechanism analogous to the mammalian multidrug resistance (MDR) phenotype, whereby, simultaneous resistance is conferred against the intracellular accumulation of structurally and functionally diverse, natural, endogenous and environmental toxicants. Expression of P-glycoproteins (P-gp), ATP-dependent transporters encoded for by the mdr1 gene that have been implicated in this xenobiotic efflux mechanism, have previously been detected in normal teleost tissues involved in a secretory, absorption or a barrier function. The presence of these proteins in the epidermis of fish species has not to our knowledge previously been investigated. In the present study, primary cultures of epidermis from the rainbow trout Oncorhynchus mykiss were employed to investigate whether an MXR mechanism is functional in the epidermis of fish. The efflux of the fluorescent mdr1 substrate rhodamine 123 from the cells was significantly inhibited by verapamil, a compound known to interfere with P-gp mediated transport. The cultured epidermal cells were also observed to accumulate this fluorescent dye in a verapamil sensitive manner, thus indicating the presence of an mdr1-like mechanism. Immunocytochemical analysis, using a monoclonal antibody (JSB1) directed against a conserved cytoplasmic P-gp epitope, also demonstrated the presence of P-gp-like proteins. Sediment elutriate extracts were employed as models of environmental complex mixtures to evaluate the potential of the epidermal cultures to discriminate between samples of varying contaminant burden using MXR activity as an endpoint. The induction of P-gp expression was found to be in accordance with the level of contamination detected in the sediments from which the elutriates were extracted. The findings of the functional study also demonstrated that environmental pollutants, which interfere with P-gp function, could be identified using this model.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2005

Glutathione transferase-like proteins encoded in genomes of yeasts and fungi: insights into evolution of a multifunctional protein superfamily.

Most fungal glutathione transferases (GSTs) do not fit easily into any of the previously characterised classes by immunological, sequence or catalytic criteria. In contrast to the paucity of studies on GSTs cloned or isolated from fungal sources, a screen of databases revealed 67 GST-like sequences from 21 fungal species. Comparison by multiple sequence alignment generated a dendrogram revealing five clusters of GST-like proteins designated clusters 1, 2, EFIBgamma, Ure2p and MAK16, the last three of which have previously been related to the GST superfamily. Surprisingly, a relatively small number of fungal GSTs belong to mainstream classes and the previously-described fungal Gamma class is not widespread in the 21 species studied. Representative crystal structures are available for the EFIBgamma and Ure2p classes and the domain structures of representative sequences are compared with these. In addition, there are some "orphan" sequences that do not fit into any previously-described class, but show similarity to genes implicated in fungal biosynthetic gene clusters. We suggest that GST-like sequences are widespread in fungi, participating in a wide range of functions. They probably evolved by a process similar to domain "shuffling".

Amino Acid Sequence↗
PubMed2004

Redox modulation of integrin [correction of integin] alpha IIb beta 3 involves a novel allosteric regulation of its thiol isomerase activity.

The molecular mechanisms involved in regulating the activation-dependent conformational switch in integrins are not known although recent evidence suggests that integrins are a direct target for redox modulation. We have identified an endogenous integrin thiol isomerase activity that may be responsible for regulating integrin activation states. The purpose of this study was to examine the effects of redox conditions elicited by nitric oxide and glutathione on the thiol isomerase activity of the platelet integrin alphaIIbbeta3 and also on the activation status of this integrin in intact platelets. The universal integrin activator, Mn2+, stimulates the thiol isomerase activity in purified alphaIIbbeta3. Kinetic analysis reveals that alphaIIbbeta3 is an allosteric enzyme which displays positive cooperativity in the presence of Mn2+ with an apparent Hill coefficient of 1.9. Also, addition of Mn2+ to platelets results solely in activation of the integrin as demonstrated by the binding of the antibody PAC-1. The addition of the nitric oxide donors SNP, SIN-1, and SNOAC in combination with glutathione can directly reverse the activation state of the platelet integrin induced by Mn2+. These compounds have no effect on platelet secretory responses indicating a direct effect on the integrin. In the presence of nitric oxide and glutathione, the enzymatic activity of alphaIIbbeta3 also displays positive cooperativity (apparent Hill coefficient of 1.9), and a significant increase in the saturability of the enzyme was observed. Thus, redox agents simultaneously modulate the thiol isomerase activity of purified alphaIIbbeta3 and its active conformation in intact platelets, suggesting a molecular mechanism for integrin regulation.

Allosteric Regulation↗
PubMed2003

Biochemical properties of phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides.

Phosphorus-modified phosphonoacetate and thiophosphonoacetate oligodeoxyribonucleotides were chemically synthesized and their biochemical properties evaluated. Under physiological pH, these DNA analogs possess negative charge and form stable, complementary A-like DNA:RNA heteroduplexes when analyzed via circular dichroism spectroscopy. Phosphonoacetate and thiophosphonoacetate oligomers were found to stimulate RNase H activity and to be completely resistant to degradation by snake venom phosphodiesterase, DNase I and HeLa cell nuclear extract. Further research has demonstrated that neutral, esterified forms of these analogs can be taken up by cells. Phosphonoacetate and thiophosphonoacetate oligomers therefore represent a new class of oligodeoxyribonucleotide analogs having phosphorus- carbon bonds with considerable potential for use in biological research.

Chromatography, High Pressure Liquid↗
PubMed2003

Variability of heat shock proteins and glutathione S-transferase in gill and digestive gland of blue mussel, Mytilus edulis.

Glutathione S-transferase (GST) and heat shock proteins (hsps) 40, 60, 70 and 90 were determined by immunoblotting using actin as an internal control in Mytilus edulis from one station outside (site1) and three stations within (sites 2-4) Cork Harbour, Ireland. Comparisons were made between gill and digestive gland and between sites. Gill shows generally higher hsp 60, 70 and 90 while digestive gland has higher hsp 40. Site 1 showed higher gill hsps 40 and 70 than sites 2-4 while gill GST was higher in sites 3 and 4 than 1 and 2. Comparison with sites in the North Sea (site 5: outside Tjärnö in The Koster archipelago in the Skagerack) and Baltic Sea (site 6: Askö island) also revealed lower hsps 40 and 70 in site 6 (low salinity) than site 5 (high salinity) although hsps 60, 70 and 90 were detectable in digestive gland unlike sites 1-4. Previously, only hsp 70 had been studied at these sites [Mar. Environ. Res. 39. (1995), 181]. At the mRNA level, gill hsp 70 is 80-fold higher at Tjärnö than Askö. These data suggest that, while salinity may slightly decrease hsp 40 and 70, both hsp 70 and GST are selectively up-regulated by approx. 10- and 3-fold, respectively, at Tjärnö compared to the other sites which we attribute to exposure to more widely fluctuating pollution levels.

Animals↗
PubMed2003

Paroxetine treatment of generalized anxiety disorder: a double-blind, placebo-controlled study.

OBJECTIVE: This study assessed the efficacy of two fixed doses of paroxetine in the treatment of generalized anxiety disorder. METHOD: Outpatients (N=566) with generalized anxiety disorder and no other axis I disorder were eligible if they scored >/=20 on the Hamilton Rating Scale for Anxiety (with a score of 2 or higher on the anxious mood and tension items). Following a 1-week placebo run-in phase, patients were randomly assigned to 8 weeks of treatment with paroxetine, 20 or 40 mg/day, or placebo. The primary outcome measure was the change from baseline in total score on the Hamilton anxiety scale. Response was defined as a rating of "very much improved" or "much improved" on the Clinical Global Impression global improvement measure; remission was defined as a Hamilton anxiety scale score </=7. Change in functional impairment was measured with the Sheehan Disability Scale. RESULTS: At 8 weeks, reductions in total score on the Hamilton anxiety scale were significantly greater for both paroxetine groups. Response was achieved by 62% and 68% of the patients receiving 20 and 40 mg of paroxetine, respectively, compared with a 46% response rate in the placebo group. Remission was achieved by 30% and 36% of patients in the 20- and 40-mg paroxetine groups, respectively, compared with 20% given placebo. For all three domains of the Sheehan Disability Scale, significantly greater improvement was seen with paroxetine than placebo. Both doses of paroxetine were well tolerated. CONCLUSIONS: This study demonstrates that paroxetine is an efficacious and well-tolerated treatment for generalized anxiety disorder.

Adult↗
PubMed2003

The Tourette's Disorder Scale (TODS): development, reliability, and validity.

To address the lack of a simple and standardized instrument to assess overall illness severity of Tourette's disorder (TD), the authors developed and tested a 15-item scale to measure a broad range of common symptoms including tics, inattention, hyperactivity, obsessions, compulsions, aggression, and emotional symptoms. Independent investigators used the 15-item Tourette's Disorder Scale (TODS) to assess 60 TD patients who were taking part in a double-blind placebo-controlled multicenter 8-week treatment study. Interrater reliability, internal consistency, convergent and discriminant validity, and sensitivity to change were examined. The TODS was associated with good interrater reliability, excellent internal consistency, and favorable levels of validity and sensitivity to change. Individual TODS items showed good convergent and discriminant validity against other measures. The TODS is a simple, efficient way for clinicians and parents to rate the severity of multiple symptoms commonly found in patients with Tourette's disorder.

Adolescent↗
PubMed2002

Generalized anxiety disorder: comorbidity, comparative biology and treatment.

Generalized anxiety disorder (GAD) is a severe and chronic anxiety disorder characterized by uncontrollable worrying and somatic anxiety (tension, insomnia and hypervigilance). It is a common condition, with lifetime prevalence rates for DSM-IV GAD in the general population of approx. 5-6% being reported. In addition, like other anxiety disorders, GAD also shows comorbidity with depression and most of the other anxiety disorders. This article reviews data on the prevalence of GAD, its comorbidity with depression, and its social and economic impact. Proposed neurobiological mechanisms for GAD are discussed, since an understanding of these may help in the development of future therapies. Finally, current pharmacological and non-pharmacological treatment options for GAD are reviewed, with particular attention being paid to published clinical-trial data.

Anti-Anxiety Agents↗