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M W Duncan

Publications and source records attributed to M W Duncan.

At least 37 records · Page 2Linked to original sources
PubMed1995

Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium.

A protein map of the smallest known self-replicating organism, Mycoplasma genitalium (Class: Mollicutes), revealed a high proportion of acidic proteins. Amino acid composition was used to putatively identify, or provide unique parameters, for 50 gene products separated by two-dimensional gel electrophoresis. A further 19 proteins were subjected to peptide-mass fingerprinting using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry and 4 were subjected to N-terminal Edman degradation. The majority of M. genitalium proteins remain uncharacterised. However, the combined approach of amino acid analysis and peptide-mass fingerprinting allowed gene products to be linked to homologous genes in a variety of organisms. This has allowed proteins to be identified prior to detection of their respective genes via the M. genitalium sequencing initiative. The principle of 'hierarchical' analysis for the mass screening of proteins and the analysis of microbial genomes via their protein complement or 'proteome' is detailed. Here, characterisation of gene products depends upon the quickest and most economical technologies being employed initially, so as to determine if a large number of proteins are already present in both homologous and heterologous species databases. Initial screening, which lends itself to automation and robotics, can then be followed by more time and cost intensive procedures, when necessary.

Amino Acids↗
PubMed1994

The stereoselective disposition of the enantiomers of ibuprofen in blood, blister and synovial fluid.

1. A sensitive, stereospecific assay using gas chromatography-mass spectrometry (GC/MS) was established to measure the concentrations of the enantiomers of ibuprofen in small volumes (50 microliters) of blister fluid. 2. The concentrations of the enantiomers in blister fluid, assessed in eight patients, were similar to those in synovial fluid, both fluids behaving as peripheral compartments with respect to plasma. 3. The mean rate constants of transfer of R-ibuprofen into (0.14 +/- 0.06 h-1) and out of (0.20 +/- 0.04 h-1) blister fluid were not significantly different from those for synovial fluid (0.19 +/- 0.12 h-1, 0.34 +/- 0.11 h-1, respectively). Similarly, the mean rate constants of transfer of S-ibuprofen into (0.22 +/- 0.07 h-1) and out of (0.27 +/- 0.08 h-1) blister fluid were not significantly different from those for synovial fluid (0.29 +/- 0.10, 0.36 +/- 0.11 h-1). However, the correlations were poor between the transfer constants for each of the enantiomers between plasma, and both blister and synovial fluid (P > 0.2). 4. The complex rate constant of transfer of S-ibuprofen into blister fluid (0.22 +/- 0.07 h-1) was greater than that of R-ibuprofen (0.14 +/- 0.07 h-1), which may be explained by the lesser protein binding of the S-enantiomer.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption↗
PubMed1993

Protein-bound 3,4-dihydroxyphenylalanine is a major reductant formed during hydroxyl radical damage to proteins.

Proteins and aromatic amino acids previously exposed to hydroxyl radicals reduced cytochrome c, free iron, and copper ions. A major product of hydroxyl radical addition to tyrosine is 3,4-dihydroxyphenylalanine (DOPA), which has these reducing properties. The reduction of nitro blue tetrazolium by radical-damaged protein was consistent with the generation of quinones in the protein. By acid hydrolysis followed by high-performance C18 reversed-phase liquid chromatography we have shown that hydroxyl radical-damaged proteins contain significant amounts of protein-bound DOPA (PB-DOPA). The authenticity of the DOPA measured was confirmed by gas chromatography-mass spectrometry. PB-DOPA was also generated enzymatically using mushroom tyrosinase, which catalyzes the hydroxylation of tyrosine residues. By comparing the levels of DOPA in radical-damaged or enzyme-treated protein with that of cytochrome c reduction, we show that PB-DOPA is a major source of the observed reducing activity. PB-DOPA may have a role in the replenishment of reduced transition metal ions involved in free radical generating systems in vivo.

Catechols↗
PubMed1993

Quantitative analysis of low molecular weight compounds of biological interest by matrix-assisted laser desorption ionization.

Internal standards were used to demonstrate that matrix-assisted laser desorption/ionization (MALDI) mass spectrometry can be applied to the quantitative analysis of low molecular weight polar compounds. Three examples were tested: a standard curve for 3,4-dihydroxyphenylalanine (DOPA) was prepared using a stable isotope analogue (i.e., [13C6]DOPA) as an internal standard; [2H16]-acetylcholine was employed as an internal standard for the quantification of acetylcholine; and in the final example, the peptide Ac-Ser-Ile-Arg-His-Tyr-NH2 was used as an internal standard for the quantification of the peptide H-Ser-Ala-Leu-Arg-His-Tyr-NH2. In each instance, straight line fits (r2 > 0.95) demonstrate that MALDI is a viable approach for the quantitative analysis of low molecular weight analytes.

Acetylcholine↗
PubMed1992

Formation of proline thiohydantoin with ammonium thiocyanate: progress towards a viable C-terminal amino-acid-sequencing procedure.

Pure amino acid thiohydantoins are required as reference standards for development of C-terminal-sequencing procedures based on thiohydantoin formation of the C-terminal amino acids of peptides and proteins. Proline thiohydantoin was prepared using a straightforward method involving reaction of acetylproline with ammonium thiocyanate. It was characterized by UV spectrophotometry, mass spectrometry and back-hydrolysis to the free amino acid. These data establish unequivocally that the thiocyanate procedure is applicable to proline as well as to the other common amino acids. This work also validates earlier claims that proline thiohydantoin can be prepared by reaction with thiocyanic acid.

Amino Acid Sequence↗
PubMed1992

Facilitated transport of the neurotoxin, beta-N-methylamino-L-alanine, across the blood-brain barrier.

beta-N-Methylamino-L-alanine (BMAA) is a neurotoxic plant amino acid that has been implicated in the pathogenesis of the high incidence amyotrophic lateral sclerosis and related parkinsonism dementia of the western Pacific. Previous studies have demonstrated that BMAA is taken up into brain following intravenous or oral administration. To examine the kinetics and mechanism of brain transfer, BMAA influx across the blood-brain barrier was measured in rats using an in situ brain perfusion technique. BMAA influx was found to be saturable with a maximal transfer rate (Vmax) of 1.6 +/- 0.3 x 10(-3) mumol/s/g and a half-saturation constant (Km) of 2.9 +/- 0.7 mM based on total perfusate BMAA concentration. Uptake was sodium independent and inhibitable by excess L-leucine, but not by L-lysine, L-glutamate, or methylaminoisobutyric acid, indicative of transfer by the cerebrovascular large neutral amino acid carrier. L-BMAA competitively reduced brain influx of L-[14C]leucine, as expected for cross-inhibition. The results demonstrate that BMAA is taken up into brain by the large neutral amino acid carrier of the blood-brain barrier and suggest that uptake may be sensitive to the same factors that affect neutral amino acid transport, such as diet, metabolism, disease, and age.

Amino Acid Transport Systems↗
PubMed1992

Zinc, a neurotoxin to cultured neurons, contaminates cycad flour prepared by traditional guamanian methods.

We have used cultured ventral mesencephalic and cerebellar granule cells to test the toxicity of extracts of cycad seeds (genus Cycas) and cycad-derived flours traditionally prepared in Guam. There was no significant difference in the toxicity of extracts prepared from the female gametophyte tissue of C. circinalis, C. revoluta, and C. media, common wheat flour, and 13 of 17 cycad flour samples. However, extracts prepared from 4 of 17 Guamanian flour samples exhibited marked dose-dependent neurotoxicity to mesencephalic and granule cell cultures. There was no correlation between toxicity and 2-amino-3-(methylamino)-propanoic acid (BMAA) content, and the concentration of BMAA in the medium arising from these extracts was far below that required to be neurotoxic. Toxicity of extracts was not blocked by the NMDA receptor antagonist MK-801 or the non-NMDA receptor antagonist 6-cyano-7-dinitroquinoxaline-2,3-dione, indicating that toxicity was not mediated by excitatory amino acid receptors. Analysis of the four toxic processed flour samples indicated high zinc content. Zinc produced a concentration-dependent neurotoxic response in mesencephalic and granule cell cultures that paralleled the calculated concentrations of zinc in the cultures derived from the four toxic flour samples. When sliced C. circinalis gametophyte tissue was "processed" in our laboratory by soaking in a galvanized container, there was a time-dependent increase in zinc content.

Animals↗
PubMed1991

2-amino-3-(methylamino)-propanoic acid (BMAA) pharmacokinetics and blood-brain barrier permeability in the rat.

2-Amino-3-(methylamino)-propanoic acid (BMAA) is a neurotoxic, excitatory amino acid which has been linked through cycad use and consumption with the onset of a variant of amyotrophic lateral sclerosis occurring with high incidence in the western Pacific region. We have studied BMAA pharmacokinetics, oral bioavailability and blood-brain barrier permeability in the rat in an attempt to better define the possible role for BMAA in this disease. To evaluate its kinetics and uptake, BMAA (25-400 mg/kg) was administered to rats, either acutely or chronically, and then plasma and brain concentrations were determined at various times thereafter by combined gas chromatography mass spectrometry. After single dose i.v. injection, BMAA was cleared from plasma in a rapid distribution phase (Vd approximately 16 liters/kg) followed by a slower elimination phase (t1/2 approximately 1 day). Brain uptake was limited by a low blood-brain barrier permeability-surface area product of 2 to 5 x 10(-5) ml/sed/g. Brain BMAA levels peaked within 8 hr after injection, and then declined with a t1/2 similar to that of plasma. After two weeks of continuous infusion (100 mg/kg/day), steady-state brain concentrations equalled 10 to 30 micrograms/g, and only moderately exceeded those in plasma. The results suggest that BMAA may reach potentially toxic levels in brain (i.e., greater than 250 microM) after large doses (greater than 100 mg/kg). However, such doses are orders of magnitude greater than those available from dietary or medicinal use of cycads.

Administration, Oral↗
PubMed1990

2-Amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour: an unlikely cause of amyotrophic lateral sclerosis and parkinsonism-dementia of Guam.

We conducted an investigation of the levels of the neurotoxin 2-amino-3-(methylamino)-propanoic acid (BMAA) in cycad flour. Analysis of 30 flour samples processed from the endosperm of Cycas circinalis seeds collected on Guam indicated that more than 87% of the total BMAA content was removed during processing. Furthermore, in 1/2 the samples almost all (greater than 99%) of the total BMAA was removed. We found no significant regional differences in the BMAA content of flour prepared from cycad seeds collected from several villages on Guam. Testing of different samples prepared by the same Chamorro woman over 2 years suggests that the washing procedure probably varies in thoroughness from preparation to preparation but is routinely efficient in removing at least 85% of the total BMAA from all batches. Analysis of a flour sample that had undergone only 24 hours of soaking indicated that this single wash removed 90% of the total BMAA. We conclude that processed cycad flour as prepared by the Chamorros of Guam and Rota contains extremely low levels of BMAA, which are in the order of only 0.005% by weight (mean values for all samples). Thus, even when cycad flour is a dietary staple and eaten regularly, it seems unlikely that these low levels could cause the delayed and widespread neurofibrillary degeneration of nerve cells observed in amyotrophic lateral sclerosis and the parkinsonism-dementia complex of Guam (ALS-PD).

Amino Acids, Diamino↗
PubMed1989

Quantitative analysis of low molecular weight polar compounds by continuous flow liquid secondary ion tandem mass spectrometry.

Quantitative analyses of low molecular weight (100-200) polar compounds [1-methyl-4-phenylpyridine (MPP+), 2-amino-3-(methylamino)propanoic acid (synonyms, beta-(methyl-amino)-L-alanine or BMAA), and tryptophan] were conducted on a triple-stage quadrupole mass spectrometer configured for continuous flow liquid secondary ion mass spectrometry ionization (CF L-SIMS). It is shown that quantification by CF L-SIMS at subnanogram sensitivity can be precise (correlation coefficients greater than 0.99), accurate, specific, and routine for compounds not measurable by static L-SIMS. Successful analyses, however, are strongly dependent upon the stability of the film formed by the mobile phase on the probe tip. In our system, film stability is affected by mobile phase composition and flow rate, ion source and probe tip temperature, probe-tip and capillary alignment, film thickness, and sample composition.

1-Methyl-4-phenylpyridinium↗
PubMed1988

Measurement of norepinephrine and 3,4-dihydroxyphenylglycol in urine and plasma for the diagnosis of pheochromocytoma.

We compared the value of plasma samples with that of 24-hour urine samples in identifying patients with pheochromocytoma among those with hypertension. We employed specific gas chromatographic-mass spectrometric analysis of both urine and plasma for simultaneous assay of norepinephrine and its neuronal metabolite 3,4-dihydroxyphenylglycol (DHPG). The study population consisted of 1086 patients with hypertension, among them 25 patients with proved pheochromocytoma. Reference ranges for free norepinephrine and DHPG in plasma and urine were established. Measurement of free norepinephrine in 24-hour urine samples provided the best index of a pheochromocytoma. This technique had 100 percent sensitivity and 98 percent specificity among 1192 urine samples, as compared with 82 percent sensitivity and 95 percent specificity among 358 plasma samples. Simultaneous measurement of norepinephrine and DHPG in urine further improved specificity (to 99 percent), but the use of the ratio of norepinephrine to DHPG reduced sensitivity (to 95 percent), since some patients with pheochromocytoma secrete large amounts of DHPG. We therefore recommend measurement of 24-hour urinary levels of free norepinephrine for the diagnosis of pheochromocytoma and suggest that simultaneous analysis for DHPG may sometimes prove useful in reducing the rate of false positive results.

Adrenal Gland Neoplasms↗
PubMed1985

Dopamine and salsolinol levels in rat hypothalami and striatum after schedule-induced self-injection (SISI) of ethanol and acetaldehyde.

Endogenous levels of salsolinol and dopamine were measured by a gas chromatography/mass spectrometry (GC/MS)--selected ion monitoring technique using deuterated internal standards in rats allowed to self-inject either acetaldehyde, ethanol or saline control solution over 20 days for 1 h/day. Significant increases in medial basal hypothalamic (MBH) and striatal salsolinol concentrations were found in animals exposed to acetaldehyde but not ethanol, whereas dopamine concentrations for these animals did not differ significantly from rats exposed to control conditions. The data provides further support for the in vivo formation of salsolinol following acetaldehyde exposure in experimental animals.

Acetaldehyde↗
PubMed1985

Salsolinol and dopamine in rat medial basal hypothalamus after chronic ethanol exposure.

Endogenous levels of salsolinol and dopamine were measured by a gas chromatography/mass spectrometry (GC/MS) - selected ion monitoring technique using deuterated internal standards in Long Evans rats chronically exposed to ethanol for ten months. Chronic ethanol exposure produced significant increases of dopamine and salsolinol concentrations in the medial basal hypothalamus but not striatum. The data suggest that the occurrence of salsolinol in rat brain tissue is a consequence of an in vivo Pictet-Spengler cyclization.

Alcoholism↗
PubMed1985

Gas chromatographic/mass spectrometric methodology for simultaneous assay of salsolinol, dopamine, norepinephrine, dihydroxyphenylacetic acid and dihydroxyphenylethanol.

Synthesis of deuterated (2H4)salsolinol from (2H4)dopamine via a Pictet-Spengler condensation is described. This (2H4)salsolinol is an ideal internal standard to determine picomole (ng) amounts of salsolinol (SAL) in a variety of sample types including urine, plasma, beverages and fruits. The deuterated standard is completely free of contamination by the non-deuterated species. The extraction procedure described is fast, highly efficient and does not lead to artifactual salsolinol formation even in the face of high dopamine concentrations. As well as SAL the method described allows simultaneous determination of norepinephrine (NE), dopamine (DA) and its two metabolites dihydroxyphenylacetic acid (DOPAC) and dihydroxyphenylethanol (DOPET). Each of the analytes is measured as its trifluoroacetyl derivative. Using trifluoroacetic anhydride in conjunction with trifluoroethanol allows simultaneous one-step derivatization of the acid function of DOPAC. All compounds were measured in the single ion monitoring (SIM) mode and quantified using appropriate deuterated internal standards. SAL, DA, DOPET, DOPAC and NE have been quantified in a variety of food and beverage sources. Soy sauce and dried banana have been identified as rich dietary sources of SAL. These data suggest diet should be considered a potentially important source of 'mammalian alkaloids' such as SAL, and the presence of SAL in mammalian systems is not necessarily evidence for an in vivo Pictet-Spengler condensation.

3,4-Dihydroxyphenylacetic Acid↗