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Biomedical subjects

D G Watson

Publications and source records attributed to D G Watson.

At least 19 recordsLinked to original sources
PubMed2000

Myristoylation alters retinoic acid-induced down-regulation of MARCKS in immortalized hippocampal cells.

The myristoylated alanine-rich C kinase substrate (MARCKS) is a prominent PKC-substrate in the brain, which has been implicated in brain development, cytoskeletal remodeling, calcium/calmodulin signaling, and neuroplasticity. The sequence of the Macs gene codes for a protein that has three highly conserved domains including a 5' myristoylation region and a 25-amino-acid phosphorylation site domain (PSD), which are involved in anchoring MARCKS to the cellular membrane. In this study, we examined the role of the myristoylation signal in the regulation of MARCKS in transfected rat hippocampal cells (H19-7) following retinoic acid (RA) treatment. A mutant MARCKS lacking the myristoylation signal was engineered by substitution of alanine for glycine at position 2 of the Macs gene and was found to be exclusively expressed in the cytosol fraction of transfected cells. Exposure of the wild-type MARCKS-transfected cells to RA resulted in an apparent shift of MARCKS from the membrane to the cytosol, while the total protein of wild-type MARCKS was not significantly changed. In contrast, RA-exposed cells transfected with the mutant MARCKS revealed a dramatic reduction of expression of MARCKS protein in both cytosol and total protein fractions. These data suggest that the absence of the myristoyl moiety may not only alter the anchoring of the protein to the membrane but also play a novel role in modulating cellular levels of MARCKS protein in response to RA.

Animals↗
PubMed2000

In vitro glucuronidation of kaempferol and quercetin by human UGT-1A9 microsomes.

Flavonoids are important polyphenolic substances with widespread occurrence in plants and therefore in the human diet. Although considerable work has been done on the pharmacology of flavonoids, the understanding of their metabolism is still incomplete. In this work, the in vitro glucuronidation of the common dietary flavonoids quercetin and kaempferol by human UDP-glucuronosyltransferase microsomes (UGT-1A9) was investigated using HPLC and LC-MS. The two flavonoids were extensively metabolised by this enzyme with four monoglucuronides of quercetin and two of kaempferol being detected after incubation. The presence of a quercetin monoglucuronide in the urine of a volunteer after consumption of Ginkgo biloba tablets was demonstrated.

Chromatography, Liquid↗
PubMed2000

Liquid chromatography-mass spectrometry in the study of the metabolism of drugs and other xenobiotics.

The application of liquid chromatography-mass spectrometry (LC/MS) to the study of metabolism of drugs and other xenobiotics is reviewed. Original research papers covering the period from 1998 to early 2000 and concerning the use of LC/MS in the study of xenobiotic metabolism in humans and other mammalian species are reviewed. LC/MS interfaces, sample preparation steps, column types, mobile phases and additives, and the type of metabolites detected are summarized and discussed in an attempt to identify the current and future trends in the use of LC/MS for metabolism studies. Applications are listed according to the parent xenobiotic type and include substances used in therapeutics, drug candidates, compounds being evaluated in clinical trials, environmental pollutants, adulterants and naturally occurring substances.

Animals↗
PubMed2000

The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties.

The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units.

Allergens↗
PubMed2000

The compatibility and stability of octreotide acetate in the presence of diamorphine hydrochloride in polypropylene syringes.

Varying concentrations of octreotide acetate (Sandostatin) and diamorphine hydrochloride were prepared and stored in polypropylene syringes at 37 degrees C in the dark. The solutions were analysed for octreotide acetate content using a validated HPLC method at regular intervals over a 48-h period. The results indicate that octreotide acetate remains stable in the presence of diamorphine hydrochloride at 37 degrees C for 24 h. In addition, the solutions prepared maintained their clarity, with no signs of precipitation upon visual examination under normal light conditions.

Analgesics, Opioid↗
PubMed2000

Isolation and identification of three potential impurities of pholcodine bulk drug substance.

Three previously unreported manufacturing impurities were isolated from a pholcodine mother liquor using preparative reversed-phase HPLC. The liquor was the residue remaining after recrystallisation of a production batch of pholcodine. The impurities, which are structurally related to pholcodine, were initially detected by thin-layer chromatography (TLC). Their structures were determined after separation by preparative HPLC (Econo-Prep 5 microm C18 column, 30 cm x 21.2 mm i.d.). Structure elucidation was carried out using nuclear magnetic resonance (NMR) spectroscopy, mass spectroscopy (MS) and ultra violet (UV) spectroscopy. The impurities were identified as alkylated derivatives of pholcodine possessing second 2-morpholinoethyl substituents at various positions.

Antitussive Agents↗
PubMed2000

Agonistic behaviour and biogenic amines in shore crabs Carcinus maenas.

To investigate the role of certain neurohormones in agonistic behaviour, fights were staged between pairs of size-matched male shore crabs Carcinus maenas, and blood samples were taken immediately after the contests had been resolved. Samples were also taken from these crabs at rest (before and after fighting) and after walking on a treadmill. A control group of crabs also had samples taken on each experimental day. Concentrations of tyramine, dopamine, octopamine, serotonin (5-HT) and norepinephrine were determined in each blood sample using a gas chromatography/mass spectrometry (GC-MS) system. Norepinephrine was not detectable in any of the samples, but the standards were recovered. Tyramine values were not significantly different between the control group and the fought group, so tyramine does not appear to be important in agonistic behaviour. A comparison between the control and fought groups shows that fighting had an effect on the concentrations of octopamine, dopamine and 5-HT, but exercise only had an effect on octopamine levels, which showed a reduction from resting values in both winners and losers. Resting and post-fight concentrations of octopamine, dopamine and 5-HT were higher in winners than in losers. 5-HT concentration increased in the blood of fought crabs from resting values, whereas dopamine concentration decreased. In winners, octopamine concentrations decreased from resting values, but in losers octopamine levels increased from resting concentrations. The escalatory behaviour or intensity of fighting performed by winners and losers was related to dopamine levels but not to those of octopamine or 5-HT. Therefore, there appears to be a link between relative concentrations of these three amines (dopamine, octopamine and 5-HT) and fighting ability; the effects are not simply a result of activity. The better competitors have higher concentrations of these three amines at rest and after fighting.

Agonistic Behavior↗
PubMed2000

Visual marking: evidence for inhibition using a probe-dot detection paradigm.

Watson and Humphreys (1997, 1998) have recently demonstrated that new objects can be prioritized for visual attentional processing by the top-down attentional inhibition of old objects already in the field, a mechanism they called visual marking. The experiments reported here show that the detection of a dim probe dot is impaired when it falls at the location of an old object (Experiments 1 and 3) but that this occurs only in conditions in which it is advantageous for subjects to mark (inhibit) old objects (Experiment 2). These results further support previous work showing that visual marking is based on the inhibition of the locations of old objects and that visual marking can be flexibly applied (or withheld), depending on the goals of current behavior.

Adult↗
PubMed1999

The in vitro evaluation of polyethylene glycol esters of hydrocortisone 21-succinate as ocular prodrugs.

The rate of hydrolysis of polyethylene glycol esters of hydrocortisone 21-succinate (H-PEGs) rate by a commercial esterase and by enzymes from ovine cornea was studied. Both the commercial esterase and the corneal enzymes rapidly hydrolysed the H-PEGs. Both lipophilicity and the PEG chain length had an influence on the rate of enzymatic hydrolysis, a significant decrease in enzymatic hydrolysis rate being associated with an increase in the polymer chain length. The in vitro penetration of H-PEGs across ovine cornea and sclera was assessed. In corneal penetration studies no prodrug was detected in the receiver phase while in the sclera penetration study both drug and prodrug were detected. Results from in vitro corneal and scleral absorption studies showed that the use of H-PEG400 increased the rate at which hydrocortisone diffused through excised ovine cornea compared to the use of hydrocortisone itself, particularly when the donor phase was removed after 15 min in order to mimic the situation in vivo. In addition, H-PEG200, H-PEG400, H-PEG600, H-PEG900 and H-PEG2000 gave a significant increase in the rate of diffusion of hydrocortisone+H-PEG through the sclera compared to hydrocortisone itself. Our results on ovine eye show that the rate of diffusion of hydrocortisone across the sclera is about six times higher than that in cornea and for the H-PEGs the scleral diffusion was 10-100 times higher than that observed in the cornea.

Animals↗
PubMed1999

Solid-phase extraction and gas chromatography-mass spectrometry determination of kaempferol and quercetin in human urine after consumption of Ginkgo biloba tablets.

A method was developed for the quantification of the flavonoids quercetin and kaempferol in human urine using a solid-phase extraction procedure followed by gas chromatography-mass spectrometry. Deuterated internal standards of the analytes were spiked into the samples prior to extraction. The limit of detection of the method was ca. 10 pg on column and precision of the method for quantification in a sample of urine was +/-9.40% for kaempferol and +/-7.34% for quercetin (n = 6). The levels of quercetin and kaempferol found in urine samples were only a small fraction of the amount ingested. The treatment of urine samples with beta-glucuronidase markedly increased the levels of flavonoids detected, supporting the view that kaempferol and quercetin are eliminated in the urine as glucuronides.

Calibration↗
PubMed1999

Analysis of a residual diamine in a pharmaceutical polymer using solid phase extraction with analysis by gas chromatography mass spectrometry.

A method was developed for the analysis of 4,4'-methylenebiscyclohexylamine (DMDA) and 4,4'-methylenedicyclohexylisocyanate (DMDI) in a pharmaceutical polymer. The DMDA was extracted from the polymer with either buffer (0.1 M potassium phosphate pH 3.1) and the extract was passed through a SCX solid phase extraction cartridge. It was eluted from the cartridge with methanolic ammonia and then converted to its heptafluorobutyramide (HFB) derivative prior to analysis by gas chromatography-negative chemical ionisation mass spectrometry (GC-MS) in the negative ion chemical ionisation (NICI) mode. It was not possible to directly measure DMDI and it was thus analysed by selecting extraction conditions such that it would decompose to DMDA. The quantification of the residues in the polymer was based on the method of standard additions since this gave a better indication of the recovery from the complex matrix.

Cyclohexylamines↗
PubMed1999

LC determination of octreotide acetate in compound formulations of Sandostatin and diamorphine hydrochloride.

The determination of octreotide acetate in compound formulations of Sandostatin and diamorphine hydrochloride by RP-LC is described. Octreotide acetate, diamorphine hydrochloride and their respective degradants, [des-Thr-ol8]-octreotide and 6-O-acetylmorphine, were baseline resolved using a Lichrospher-60 RP-select B column with a mobile phase composition of acetonitrile/phosphate buffer (pH 7.4, 20 mM) (35:65 v/v) with UV detection at 210 nm. The method is simple, selective, precise and suitable for the determination of octreotide acetate in admixture.

Buffers↗
PubMed1999

On the efficiency of drawing computer-generated line stimuli: rectangles can be faster than lines.

A simple method is presented for optimizing the efficiency of drawing line stimuli in Turbo Pascal. It involves the substitution of Rectangle and Bar commands for Line commands and results in time savings of up to 90%. The savings are most consistent for horizontal lines, although some savings may also be made for vertical lines. The effects of different graphics libraries, graphics modes, and hardware configurations are discussed. The method proves especially useful in improving the spatiotemporal resolution of on-line-generated motion displays.

Algorithms↗
PubMed1998

Analysis of flavonoids in tablets and urine by gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry.

A method was developed for the analysis and characterization of quercetin and kaempferol in urine following ingestion of Ginkgo biloba tablets. The method utilized gas chromatography/negative ion chemical ionization mass spectrometry of the trimethysilyl derivatives of the flavonoids. Limits of detection for these compounds using this method were ca. 20 pg on column. Liquid chromatography with electrospray mass spectrometry in the negative ion mode was utilized to characterize the complex mixture of glycosides present in the G. biloba tablets, the limit of detection with this technique was ca. 10 ng on column.

Chromatography, High Pressure Liquid↗
PubMed1998

The effect of praziquantel treatment on glutathione concentration in Schistosoma mansoni.

A fluorescent dye monochlorobimane (MCB) that binds glutathione (GSH) was used as a tool for measuring the concentration of GSH in skin and mechanically-transformed schistosomula. The specificity of MCB binding to GSH was confirmed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The MCB binding to GSH is an energy-dependent process since no labelling could be seen at low temperature. When 24-h-old schistosomula were depleted of GSH by buthionine sulfoximine (a specific inhibitor of GSH synthesis) for 18 h, a significant decrease (P < 0.001) in fluorescence was observed. PZQ treatment of the schistosomula after first labelling the parasites with MCB did not greatly affect MCB binding to GSH. However, when the 24-h-old schistosomula were first PZQ treated and afterwards labelled with MCB, the pattern of labelling was identical to that of those of the non-labelled parasites. When 24-h-old schistosomula were first PZQ treated, washed and labelled in the presence of 1 mM GSH, the level of fluorescence was recovered. These results suggest that PZQ depletes GSH from schistosomula, and may render them susceptible to the host's immune system.

Animals↗
PubMed1998

Visual marking of moving objects: a role for top-down feature-based inhibition in selection.

Recently, the authors presented evidence that new items can be prioritized for selection by the top-down attentional inhibition of old stimuli already in the field (visual marking; D. G. Watson & G. W. Humphreys, 1997). In this article the authors assess whether this inhibition extends to moving old items and test an alternative account of visual marking. Six experiments showed that old moving items could be inhibited provided they did not undergo abrupt property changes. Further, and in contrast to effects with static stimuli, the marking of old moving stimuli was based on inhibition applied at the level of a whole feature map, rather than at their locations. The results also rule out an alternative account of visual marking based on the top-down weighting of dynamic or static processing pathways.

Adolescent↗
PubMed1998

Sodium valproate down-regulates the myristoylated alanine-rich C kinase substrate (MARCKS) in immortalized hippocampal cells: a property of protein kinase C-mediated mood stabilizers.

Sodium valproate (VPA) is a short-chain fatty acid with well-established anticonvulsant properties and apparent clinical efficacy in the treatment of bipolar disorder (manic-depressive illness). Little is known regarding the mechanism of action of VPA in the brain that could account for this clinical therapeutic profile. Lithium has been the standard treatment for bipolar disorder, and it is known to be an uncompetitive inhibitor of inositol monophosphatase in the phosphoinositide (PI) signaling cascade at clinically relevant concentrations. Recent studies have provided data in support of a role for protein kinase C and the down-regulation of expression of the myristoylated alanine-rich C kinase substrate (MARCKS) in the long-term therapeutic action of lithium in the brain, which is dependent on both the relative activity of receptor-coupled PI signaling and the concentration of myo-inositol. Our current results demonstrated that valproate induces a concentration- and time-dependent reduction of MARCKS in immortalized hippocampal cells that appears to be independent of both the level of muscarinic receptor-activated PI signaling as well as the concentration of myo-inositol. In CHO-K1 cells transfected with the human m1 muscarinic receptor, unlike lithium, there is no evidence for receptor-mediated accumulation of CMP-PA in the presence of VPA, providing more direct data for its lack of interaction within the PI signaling cascade. The action of VPA on MARCKS occurs within the therapeutic concentrations and time course observed in clinical studies of patients with bipolar disorder. Furthermore, the effect on MARCKS protein is additive in the presence of therapeutic concentrations of both lithium and valproate, consistent with clinical observations regarding the enhanced efficacy of the combination treatment. Finally, in studies examining acute and chronic effects of a variety of psychotropic compounds and VPA structural analogs, it is evident that the property of regulation of MARCKS is shared by the mood-stabilizers lithium and VPA, which may be specific to a class of drugs effective in the treatment of bipolar disorder.

Animals↗