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D Sheehan

Publications and source records attributed to D Sheehan.

At least 55 records · Page 3Linked to original sources
PubMed1995

Characterization of a glutathione S-transferase and a related glutathione-binding protein from gill of the blue mussel, Mytilus edulis.

The major isoenzyme of glutathione S-transferase (GST 1) was purified to homogeneity from cytosolic extracts of Mytilus edulis gill tissue by GSH-agarose affinity chromatography followed by Mono Q ion-exchange f.p.l.c. This enzyme was particularly active with 1-chloro-2,4-dinitrobenzene, ethacrynic acid and cumene hydroperoxide as substrates. Immunoblotting and amino acid sequencing studies indicate that the enzyme belongs to the Pi class of GSTs. A related protein which binds to GSH-agarose was also purified. This GSH-binding protein did not immunoblot with GST antisera and showed no detectable catalytic activity with GST substrates although its N-terminal sequence was similar to Mu-class GSTs. Gel-filtration chromatography indicated that GST 1 is a dimer and the GSH-binding protein a monomer. Mass spectrometry and SDS/PAGE indicate subunit molecular masses of 24 kDa (GST 1) and 25 kDa (GSH-binding protein), respectively. Both proteins have amino acid compositions typical of GSTs.

Amino Acid Sequence↗
PubMed1995

Drug metabolizing enzymes of mussels as bioindicators of chemical pollution.

The GSTs of M. edulis provide an easily assayed activity which would be expected to respond to changes in pollution status. The main GST and a related GSH-binding protein have been purified and biochemically characterized. The former protein is most similar to the Pi class while the latter is a catalytically inactive monomer which appears to be related to the Mu class. This enzyme activity has been assessed as a potential indicator of exposure to chemical pollutants in both Cork Harbour and Venice Lagoon (using the closely related species, M. galloprovincialis). Controlled exposure studies with mussels in holding tanks have indicated that the herbicide aldicarb gives a slight but significant increase in GST activity consistent with the inducibility of these enzymes by xenobiotics in this bivalve. At present, we are studying samples which have been deliberately exposed to PAH and PCB compounds. Studies of this type are important in helping to understand the effects and fate of chemical pollutants released into estuarine environments.

Animals↗
PubMed1995

Methylation profile and amplification of proto-oncogenes in rat pancreas induced with phytoestrogens.

Specific gene hypermethylation has been shown in DNA from neonatal rats exposed to the phytoestrogens, coumestrol, and equol. The pancreas is an organ in which estrogen receptors have been shown to be present. Studies have correlated the development of acute pancreatitis with rising levels of human estrogen binding proteins. Neonatal rats were dosed with 10 or 100 micrograms of coumestrol or equol on postnatal day (PND) 1-10. The animals were sacrificed at Day 15. The pancreas was excised and pancreatic acinar cells isolated for molecular analysis. DNA was isolated from the cells by lysis in TEN-9 buffer supplemented with proteinase K and 0.1% SDS. High molecular weight (HMW) DNA was digested with the methylated DNA specific restriction enzymes, Hpa II and Msp I, for determination of methylation profiles. Both coumestrol and equol at high doses caused hypermethylation of the c-H-ras proto-oncogene. No hypermethylation or hypomethylation was observed in the proto-oncogenes, c-myc or c-fos. Methylation is thought to be an epigenetic mechanism involved in the activation (hypomethylation) or inactivation (hypermethylation) of cellular genes which are known to play a role in carcinogenesis. Epidemiology studies have shown that equol may have anti-carcinogenic effects on some hormone-dependent cancers. Additional studies are needed to further understand the role of phytoestrogens and methylation in relation to pancreatic disorders.

Animals↗
PubMed1994

Nucleotide and deduced amino acid sequences of Rhizobium meliloti 102F34 lacZ gene: comparison with prokaryotic beta-galactosidases and human beta-glucuronidase.

The nucleotide (nt) sequence of a 2.57-kb Sau3A fragment carrying the Rhizobium meliloti beta-galactosidase (beta Gal)-encoding gene (RmlacZ) was determined. An open reading frame (ORF) of 2.26 kb was identified which encoded a 755-amino-acid (aa) polypeptide with a calculated molecular mass of 84,141 Da, in fair agreement with the value of 88 kDa determined by SDS-PAGE. The deduced N-terminal aa sequence was confirmed by direct sequencing of electrophoretically purified R. meliloti beta Gal. The size of the native R. meliloti beta Gal was approx. 174 kDa. Similarities were found between the aa sequence of the R. meliloti beta Gal and those from Clostridium thermosulfurogenes EM1 and Agrobacterium radiobacter, as well as human beta-glucuronidase (beta Glu). Comparisons with beta Gal from Escherichia coli, Klebsiella pneumoniae, Lactobacillus bulgaricus and Kluyveromyces lactis found only weak similarities; however, the putative active site residues appear to be conserved. The RmlacZ sequence is flanked by two partially sequenced ORFs, which show aa sequence and organisational similarities to the previously reported lac operon in A. radiobacter.

Amino Acid Sequence↗
PubMed1993

Evidence for alpha and Mu class glutathione S-transferases in a number of fungal species.

1. The glutathione S-transferases of the following fungal species have been affinity purified on glutathione-Sepharose; Yarrowia lipolytica (NCYC825), Sporotrichum thermophile (ATCC16479), Talaromyces emersonii (ATCC26914) and Issatchenkia orientalis (CBS5147 and an isolate provided by Prof. Kumagai, Kyoto University). 2. SDS-PAGE analysis of affinity purified fungal extracts, followed by Western blots with polyclonal antisera to rat GSTs, was performed and indicated the presence of Alpha and Mu class glutathione S-transferases in the fungi immunologically and electrophoretically similar to the rat enzymes. 3. Each extract gave a characteristic affinity-gradient elution pattern on glutathione-agarose.

Animals↗
PubMed1993

Separation of multiple forms of glutathione S-transferase from the blue mussel, Mytilus edulis.

1. Glutathione S-transferase isoenzymes from Mytilus edulis and M. galloprovincialis have been partially purified by glutathione-sepharose affinity chromatography followed by Mono Q anion exchange fast protein liquid chromatography (f.p.l.c.). 2. The tissue distribution of glutathione S-transferase in M. edulis has been studied. Using 1-chloro-2,4-dinitrobenzene as substrate, highest specific activity is observed in the gill, the main feeding organ. Affinity-purified extracts of this organ give a characteristic f.p.l.c. profile. A similar profile is obtained with affinity-purified extracts of the digestive gland of M. galloprovincialis. 3. The subunit structure of the purified isoenzymes has been studied by SDS polyacrylamide gel electrophoresis and reversed-phase h.p.l.c. The subunits have similar molecular weights and h.p.l.c. retention times to rat glutathione S-transferases.

Animals↗
PubMed1992

A nuclear protein associated with lethal heat shock of HL-60 cells.

The responses to stress in living cells are well known. Thermal stress causes decreased protein synthesis as well as rapid induction of heat shock proteins (hsps), or alternately termed stress proteins (sps). The exposure of cultured promyelocytic leukemia cells (HL-60) to a 45 degrees C lethal heat shock for 1 h elicited synthesis and phosphorylation of a polypeptide M(r) 48,000 and pI 7.5 (p 48) as visualized by two-dimensional polyacrylamide gel ultra-microelectrophoresis. p 48, which was not observed at sublethal temperatures (39 and 41 degrees C), was synthesized during all phases of the cell cycle but was phosphorylated only in G0 + G1 and S-phases. The appearance of p 48 was marked by a concomitant and reciprocal reduction in hsps or sps 70 and 90. Distinct protease V8 fragment maps of p 48, hsps 70 and 90 in conjunction with immunochemical determination indicated vast differences in their primary structures. These facts suggest that p 48 was not formed from coalesced breakdown products of hsps 70 or 90. Western blotting showed that p 48 possessed the same immunochemical determinants as two other proteins with the same molecular mass but different isoelectric points. In an association assay, p 48 was shown to bind with actins and hsp 90 from HL-60 nuclei.

Cell Cycle↗
PubMed1992

The effect of paroxetine on anxiety and agitation associated with depression.

To assess the effects of treatment on symptoms of anxiety and agitation associated with depression, a data base of 2963 paroxetine treated patients was compared with 554 who received placebo and 1151 on active control. Paroxetine and active control both reduced baseline psychic anxiety more effectively than placebo. Both pharmacological treatments were effective in treating somatic anxiety with active control demonstrating an earlier onset of activity. Neither paroxetine nor active control induced new anxiety symptoms. Paroxetine was superior to placebo in the treatment of agitation at Weeks 4 and 6 and to active control at Week 4 only. Both paroxetine and active control were more protective against emergent (new) agitation than placebo. There was no difference between the three groups in the incidence of spontaneously reported adverse events indicative of anxiety.

Adolescent↗