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S J Gaskell

Publications and source records attributed to S J Gaskell.

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

Measurement of platelet-activating factor in a canine model of coronary thrombosis and in endarterectomy samples from patients with advanced coronary artery disease.

Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent phospholipid mediator of numerous inflammatory and thrombotic responses. The purpose of this study was to determine if PAF synthesis is elevated in damaged coronary arteries after a sustained period of cyclic flow variation (CFV), a phenomenon caused by alternating periods of thrombosis and reperfusion at sites of endothelial injury. Cyclic flow was established and maintained in the left anterior descending coronary arteries (LADs) of 10 dogs. After 8 hours of CFV, the section of damaged LAD containing the thrombus and control sections of the circumflex artery, carotid artery, and saphenous vein was excised, and the total lipids were extracted. The PAF was then purified by silica column chromatography and high-performance liquid chromatography and assayed by both a rabbit platelet bioassay and a PAF radioimmunoassay. With the platelet bioassay, PAF levels of 8.9 +/- 4.0 (range, 4.8 to 15.5) pg/mg wet wt were found in the damaged LADs from the 10 dogs. This PAF bioactivity was completely inhibited by a PAF receptor antagonist. When the radioimmunoassay was used, slightly higher PAF levels of 16.3 +/- 12.9 (range, 4.5 to 41.8) pg/mg wet wt were observed in the LADs. Overall, these PAF levels were 3- to 64-fold higher than in the control vessels when either assay method was used. Although increases in PAF were observed in the damaged LADs, measurements of PAF in blood samples taken from the LAD and the aorta (control) failed to demonstrate any site-specific increase of PAF in the blood. In related experiments, PAF was also measured in 23 endarterectomy samples taken from the coronary arteries of 16 patients with severe atherosclerosis. The PAF levels in these samples were highly variable (2.9 +/- 2.2 [range, 0.3 to 8.5] pg/mg wet wt) and showed no correlation with tissue mass, suggesting that PAF is affected by factors other than the simple presence of atherosclerotic tissue in the vessel. These findings provide direct evidence that PAF is synthesized locally at the site of endothelial injury during thrombosis and that PAF accumulates in the atherosclerotic plaque of some patients with advanced coronary artery disease.

Animals↗
PubMed1994

Structure of human apolipoprotein D: locations of the intermolecular and intramolecular disulfide links.

We have determined the primary structure of human apolipoprotein D (apoD) by aligning peptides derived from digestions by cyanogen bromide, trypsin, and chymotrypsin. Our results confirm the primary structure derived from cDNA [Drayna et al. (1986) J. Biol. Chem. 261, 16535-16539]. ApoD consists of 169 amino acid residues, including 5 cysteines. Tryptic peptide analysis indicated that Cys41 and Cys16 are joined by a disulfide bridge. Using a combination of manual Edman degradations and mass spectrometric analysis on a purified cluster of chymotryptic fragments, we identified an intramolecular disulfide bridge between Cys8 and Cys114 and an intermolecular bridge between Cys116 of apoD and Cys6 of apoA-II. In addition, sites of N-glycosylation were found at Asn45 and Asn78. Because apoD contains two intramolecular disulfide linkages and has a high content of proline to disrupt alpha-helical structures, formation of the amphipathic helical regions that characterize the other soluble apolipoproteins is unlikely. We conclude that apoD binds to lipoprotein surfaces through structures other than alpha-helices, such as disulfide links.

Amino Acid Sequence↗
PubMed1994

Mass spectrometry.

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Carbohydrates↗
PubMed1993

5-Lipoxygenase metabolites of arachidonic acid stimulate isolated osteoclasts to resorb calcified matrices.

Bone resorption requires cooperation between osteoclasts and mononuclear accessory cells by mechanisms which have not been elucidated. Since multinucleated cells in giant cell tumors of bone have many phenotypic and functional characteristics of normal osteoclasts, we have examined the interaction between the bone-resorbing multinucleated cells and the distinct mononuclear stromal cells from these tumors. We have found that these mononuclear cells produce an activity which stimulates both giant cells from giant cell tumors and rodent osteoclasts to resorb bone in vitro. We have identified the activity and found that it represents several products of the 5-lipoxygenase pathway of arachidonic acid metabolism, namely 5-hydroxyeicosatetraenoic acid and the leukotrienes. These data indicate that 5-lipoxygenase metabolites stimulate isolated osteoclasts to resorb bone in vitro and may represent a mechanism by which mononuclear stromal cells in human giant cell tumors communicate with the giant cells. In addition, these results may explain a possible mechanism for communication between accessory cells and osteoclasts involved in normal bone resorption.

Acid Phosphatase↗
PubMed1992

Mass spectrometric quantification of cysteine-containing leukotrienes in rat bile using 13C-labeled internal standards.

Leukotriene C4 and N-acetyl leukotriene E4 were determined in rat bile using a procedure based on high-performance liquid chromatographic fractionation, hydrogenation to 5-hydroxyeicosanoic acid, and gas chromatographic/mass spectrometric selected ion monitoring analysis of the pentafluorobenzyl ester, trimethylsilyl ether derivatives. 13C4-Labeled analogs of the leukotrienes were synthesized and used as internal standards. The concentrations of both leukotrienes in rat bile were markedly elevated after administration of endotoxin to anesthetized animals; N-acetyl leukotriene E4 was the more abundant product. The presence of leukotriene C4 in rat bile after endotoxin challenge was confirmed by fast atom bombardment/tandem mass spectrometry with precursor ion scanning. Quantitative determination of leukotriene C4 using the tandem mass spectrometric procedure was consistent with the gas chromatographic/mass spectrometric data but the latter procedure gave a substantially lower detection limit.

Animals↗
PubMed1992

Charge promotion of low-energy fragmentations of peptide ions.

We have examined the hypothesis that structural features which predispose to localization of charge at a strongly favored site are not conducive to the low-energy fragmentation of peptide ions via a multiplicity of pathways. Consistent with this proposal, it is demonstrated that the formation of N- or C-terminal pre-charged derivatives is detrimental to the formation of sequence-specific product ions following low-energy collisional activation. Protonation of pre-charged derivatives (yielding doubly charged ions) restores favorable fragmentation properties; the effect is attributed to the fragmentation-directing properties of the proton which may occupy one of several sites. Similarly, a doubly protonated peptide which incorporates a C-terminal arginine residue as a single strongly favored site of protonation exhibits favored low-energy fragmentations attributable to location of the second proton at one of several sites remote from the C-terminus.

Mass Spectrometry↗
PubMed1992

Serum complement mediates endotoxin-induced cysteinyl leukotriene formation in rats in vivo.

To investigate potential mediators responsible for cysteinyl leukotriene formation during endotoxemia, male Fischer rats received an intravenous bolus injection of 5 mg/kg Salmonella enteritidis endotoxin and cysteinyl leukotrienes were measured by gas chromatography-mass spectrometry. The biliary excretion of leukotriene (LT) C4 (0.20 +/- 0.02 pmol.min-1.g liver-1) and N-acetyl-LTE4 (0.37 +/- 0.07 pmol.min-1.g-1) was increased by 190 and 1,000%, respectively, during the first 30 min after endotoxin injection. Endotoxin also caused a temporary reduction of hepatic ATP levels by 84%. Depletion of serum complement almost completely abolished the endotoxin-induced increase of cysteinyl leukotrienes in bile without affecting the biliary excretion mechanism. Intravenous injection of activated complement factors caused cysteinyl leukotriene formation and reduced the hepatic ATP content similar to endotoxin. Depletion of glutathione in the liver prevented cysteinyl leukotriene formation and the complement-induced ATP depletion. It is concluded that endotoxin-induced cysteinyl leukotriene generation in vivo is mediated predominantly through activation of complement. The vasoconstrictive cysteinyl leukotrienes are then responsible for ATP depletion in the liver.

Adenosine Triphosphate↗
PubMed1991

Tandem mass spectrometry of peptides using hybrid and four-sector instruments: a comparative study.

Product-ion spectra produced by high- and low-energy collisionally activated dissociation (CAD) of [M + H]+ ions of a series of peptides (Mr 550-2500) have been compared on four-sector and hybrid tandem mass spectrometers, respectively. The fast atom bombardment product-ion spectra obtained for the smallest peptide analyzed (methionine-enkephalin) were remarkably similar, but substantial differences in fragmentation were observed for the heavier analytes. For peptides with Mr greater than 1000, more complete sequence information was obtained from high-energy CAD on the four-sector instrument. Nevertheless, low-energy CAD on the hybrid mass spectrometer was able to produce partial sequence information even for the largest of the peptides compared. Limits of analysis, defined as the least quantities of analyte for which product-ion spectra of essentially uncompromised quality could be obtained, were similar (ca. 15 pmol) for small peptides, but lower limits were achieved for larger peptides (Mr greater than 1000) with the four-sector instrument. High-energy CAD spectra were found to be highly reproducible, with qualitatively similar spectra obtained over a wide range of operating conditions. In contrast, it was necessary to carefully control collision gas pressures and collision energies in order to obtain good reproducible data for low-energy CAD. Experimental procedures for obtaining reproducible spectra with good sensitivity for peptides on the hybrid instrument are presented.

Adrenocorticotropic Hormone↗
PubMed1991

Oligosaccharide sequence determination using B/E linked field scanning or tandem mass spectrometry of phosphatidylethanolamine derivatives.

Product ion mass spectral data of [M + H]+ ions of oligosaccharides, mainly tetra- and pentasaccharides, as their dipalmitoyl phosphatidylethanolamine derivatives were obtained using both liquid secondary ion mass spectrometry with B/E linked scanning and fast atom bombardment ionization with collision-induced dissociation/tandem mass spectrometry. Both methods give similar positive product ion spectra of equivalent high sensitivity (detection limits of approximately 50 pmol) that principally contain glycosidic cleavage ions retaining the reducing end of the molecule from which monosaccharide sequence can be deduced. A series of ions from fission of the phosphate ester bond together with glycosidic cleavage are present in the tandem mass spectra and B/E linked scan spectra when helium collision gas is used. Monosaccharide linkage position of isomeric molecules is reflected in the intensity of glycosidic fragmentation, without retention of the oxygen atom, with decreasing cleavage in the order 1-3 greater than 1-4 greater than 1-6 linkage. Fucose and N-acetylhexosamines show an increased degree of fragmentation over hexose sugars. The application of product ion spectra of derivatized oligosaccharides is demonstrated for characterizing mixed samples and also the acquisition of spectra directly from the silica surface of high-performance thin-layer chromatography plates.

Mass Spectrometry↗
PubMed1991

Isotopic exchange during derivatization of platelet activating factor for gas chromatography-mass spectrometry.

One approach to the quantitative analysis of platelet activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycerol-3-phosphocholine; also referred to as AGEPC, alkyl glyceryl ether phosphocholine) is hydrolytic removal of the phosphocholine group and conversion to an electron-capturing derivative for gas chromatography-negative ion mass spectrometry. [2H3]Acetyl-AGEPC has been commonly employed as an internal standard. When 1-hexadecyl-2-[2H3]acetyl glycerol (obtained by enzymatic hydrolysis of [2H3]-C16:0 AGEPC) is treated with pentafluorobenzoyl chloride at 120 degrees C, the resulting 3-pentafluorobenzoate derivative shows extensive loss of the deuterium label. This exchange is evidently acid-catalyzed since derivatization of 1-hexadecyl-2-acetyl glycerol under the same conditions in the presence of a trace of 2HCl results in the incorporation of up to three deuterium atoms. Isotope exchange can be avoided if the reaction is carried out at low temperature in the presence of base. Direct derivatization of [2H3]-C16:0 AGEPC by treatment with pentafluorobenzoyl chloride or heptafluorobutyric anhydride also results in loss of the deuterium label. The use of [13C2]-C16:0 AGEPC as an internal standard is recommended for rigorous quantitative analysis.

Carbon Isotopes↗
PubMed1990

The fruitfly Drosophila melanogaster contains a novel charged adipokinetic-hormone-family peptide.

A member of the RPCH/AKH (red-pigment-concentrating hormone/adipokinetic hormone) family of arthropod neuropeptides was identified in the fruitfly Drosophila melanogaster, and its structure was determined by automated Edman degradation and m.s. using fast-atom-bombardment ionization and a tandem hybrid instrument capable of high sensitivity. The sequence of this peptide, which we call 'DAKH', is pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2 (where pGlu is pyroglutamic acid and Trp-NH2 is tryptophan carboxyamide). H.p.l.c. analyses of extracts of the three body segments revealed that more than 80% of the peptide is contained in the thorax. Although DAKH is typical of family members in its general structure and distribution in the animal, it is unique in containing a residue which is charged under physiological conditions. The evolutionary significance of this change is considered.

Amino Acid Sequence↗
PubMed1990

Preparation and tandem mass spectrometric analyses of deuterium-labeled cysteine-containing leukotrienes.

Leukotrienes (LT) C4, E4 and N-acetyl-E4, their respective monomethyl esters and 14,15-2H2 analogs have been synthesized. The collisionally activated decompositions of the [M + H]+ and [M - H]- ions formed by fast atom bombardment (FAB) have been studied by tandem mass spectrometry using a hybrid sector/quadrupole instrument. Structurally informative product ion spectra were obtained for each analyte; the fragmentation pathways proposed are consistent with the parallel data obtained for labeled and derivatized species. Fragmentation of [M + H]+ ions occurs prominently via cleavage of the thioether linkage with charge retention on the cysteine-containing (predominant for LTC4) or lipid-derived (predominant for LTE4) moieties. More pronounced differences were observed between the fragmentations of [M - H]- ions derived from LTC4 and LTE4; the preference for charge retention, however, parallels that observed for the fragmentation of [M + H]+ ions. Selected ion monitoring during continuous-flow FAB mass spectrometric analysis of authentic LTC4 indicated a low-picogram detection limit.

Cysteine↗
PubMed1990

Quantification of steroid conjugates using fast atom bombardment mass spectrometry.

Fast atom bombardment/mass spectrometry or liquid secondary ion mass spectrometry provides the capability for direct analysis of steroid conjugates (sulfates, glucuronides) without prior hydrolysis or derivatization. During the analysis of biologic extracts, limitations on the sensitivity of detection arise from the presence of co-extracted material which may suppress or obscure the analyte signal. A procedure is described for the quantitative determination of dehydroepiandrosterone sulfate in serum which achieved selective isolation of the analyte using immunoadsorption extraction and highly specific detection using tandem mass spectrometry. A stable isotope-labeled analog [( 2H2]dehydroepiandrosterone sulfate) was used as internal standard. Fast atom bombardment of dehydroepiandrosterone sulfate yielded abundant [M-H]- ions that fragmented following collisional activation to give HSO4-; m/z 97. During fast atom bombardment/tandem mass spectrometry of serum extracts, a scan of precursor ions fragmenting to give m/z 97 detected dehydroepiandrosterone sulfate and the [2H2]-labeled analog with a selectivity markedly superior to that observed using conventional mass spectrometry detection. Satisfactory agreement was observed between quantitative data obtained in this way and data obtained by gas chromatography/mass spectrometry of the heptafluorobutyrates of dehydroepiandrosterone sulfate and [2H2]dehydroepiandrosterone sulfate obtained by direct derivatization.

Animals↗
PubMed1989

Quantitative analysis of platelet activating factor using fast atom bombardment/tandem mass spectrometry.

A procedure is described for the quantitative determination of platelet activating factor (PAF) using stable isotope dilution and fast atom bombardment/tandem mass spectrometry. Low-energy collisional activation of the [M + H]+ ion of PAF yields a single daughter ion of m/z 184, characteristic of phosphocholine derivatives. For precise and accurate quantification, the internal standard is (2H3)acetyl-hexadecyl PAF, which yields an analogous daughter ion of m/z 185. Quantitative analyses are based on limited mass-range parent ion scanning with transmission of daughters of m/z 184 and 185 during alternate scans; all scans are accumulated into a single data file to facilitate determination of the analyte/internal standard response ratio. Analysis of authentic hexadecyl PAF indicates a low-picogram detection limit. The method has been applied to the determination of PAF in preparations of human neutrophils stimulated by addition of a calcium ionophore. Concentrations of PAF of 7-17 ng/10(6) cells were observed, in keeping with earlier reports. The method has been validated by standard addition and dilution experiments. Comparison of data obtained by the new procedure and those obtained by a method involving gas chromatography/electron capture mass spectrometry of dephosphorylated and derivatized PAF showed excellent agreement.

Calcimycin↗
PubMed1989

Elucidation of some fragmentations of small peptides using sequential mass spectrometry on a hybrid instrument.

Sequential daughter-ion-scanning analyses of small peptides have been performed using a hybrid tandem instrument of BEqQ configuration. Precursor ions are selected by B and allowed or induced (by high-energy collisional activation) to decompose in the region preceding E. Decoupling of E from the accelerating voltage permits the selection of the first-generation daughter ion whilst retaining appropriate float voltages for the quadrupole assemblies. The daughter ion selected by E is further subjected to low-energy collisional-activation dissociation (CAD) in q and the fragment-ion spectrum is obtained by scanning Q. The sequential daughter-ion-scanning technique has been used to establish that 'internal' fragments of the types, (AY') and (BY'), are formed via initial Y-type cleavage. Fragmentation of a protonated peptide (angiotensin III) by loss of the C-terminal amino acid residue, yielding a (B'n + OH) ion, is reported for the first time. This process is analogous to that previously described for metal-cationized peptides.

Angiotensin II↗
PubMed1989

Characterization of impurities in a synthetic renin substrate peptide by fast-atom bombardment mass spectrometry and hybrid tandem mass spectrometry.

Fast-atom bombardment mass spectrometry of a synthetic renin substrate decapeptide (Pro-His-Pro-Phe-His-Leu-Val-Ile-His-D-Lys) indicated the presence of several side-products, including a component 12 Da higher in mass. Low-energy collisionally activated decomposition analyses were performed using a hybrid tandem instrument and demonstrated that the heavier side product had two components, in which the structural modification was either at the N- or the C-terminus. Additional analyses of the N-acetyl derivative indicated that for each component the structural modification blocked a site of N-acetylation. It is suggested that the formation of these side products is attributable to the generation of formaldehyde, during removal of the histidine protecting group (benzyloxymethyl), which reacts with the N-terminus of the peptide to give an imidazolidinone structure or with the D-lysine epsilon-amine group to yield an imine. While the precise genesis of the side-products remains speculative, it is clear that the combined strategy of derivatization and tandem mass spectrometry has allowed structural conclusions concerning individual components of an isobaric mixture.

Angiotensinogen↗