Search PubMed⌕ Search

Biomedical subjects

D A Cowan

Publications and source records attributed to D A Cowan.

At least 73 records · Page 4Linked to original sources
PubMed1988

Two assays for dihydrocodeine in plasma and in urine and their use to determine the bioavailability of a controlled-release product.

A GC method (20 ng/mL quantitation limit) and an HPLC method (37.5 ng/mL quantitation limit) for the analysis of dihydrocodeine in plasma and an HPLC method for dihydrocodeine in urine (2 micrograms/mL quantitation limit) are described. These methods have been used to compare the bioavailability in four subjects of a controlled-release formulation of dihydrocodeine bitartrate (equivalent to 90 mg of base) with that of a solution (equivalent to 3 x 30 mg of base). With each preparation, the mean urinary recovery of dihydrocodeine was approximately 30 mg, and both had a comparable bioavailability. A further study with 12 subjects, each taking the equivalent of 30 mg of base at 12 hourly intervals for 4 d, indicated that the controlled-release formulation produced effective steady-state concentrations of drug whether administered before or after meals, with no accumulation being observed.

Administration, Oral↗
PubMed1987

An extremely thermostable extracellular proteinase from a strain of the archaebacterium Desulfurococcus growing at 88 degrees C.

An organism growing at 88 degrees C that closely resembles Desulfurococcus mucosus produced a single extracellular proteinase. We have purified this enzyme and carried out a preliminary characterization. The proteinase, which is a serine-type enzyme, had a molecular mass of 52,000 Da by SDS/polyacrylamide-gel electrophoresis, but only 10,000-13,000 Da by gel-permeation chromatography. Molecular mass values from sucrose-gradient centrifugation were of the same order as those from SDS/polyacrylamide-gel electrophoresis. It had an isoelectric point of 8.7, and was inhibited by di-isopropyl phosphorofluoridate, phenylmethanesulphonyl fluoride and chymostatin. Substrate-specificity studies suggested a possible preference for hydrophobic residues on the C-terminal side of the splitting point. The thermostability of this enzyme is probably greater than any other reported proteinase (t1/2 at 95 degrees C, 70-90 min; t1/2 at 105 degrees C, 8-9 min). Ca2+ chelation does not appear to be implicated in stabilization of the protein structure. The stability of the Desulfurococcus proteinase was not greatly affected by the presence of reducing reagents (e.g. dithiothreitol), some chaotropic agents (e.g. NaSCN) and some detergents, but activity was lost rapidly at 95 degrees C in the presence of the oxidizing agent NaBO3. Proteolytic activity was readily detected at temperatures up to and including 125 degrees C, although denaturation was very rapid above 115 degrees C. A number of Figures supporting some of the findings reported in this paper have been deposited in supplement SUP 50137 (14 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies may be obtained on the terms indicated in Biochem. J. (1987) 241, 5.

Archaea↗
PubMed1987

Some observations on the inhibition and activation of a thermophilic protease.

The activity of Caldolysin, the extracellular protease from the extreme thermophile Thermus aquaticus strain T351, was reduced in the presence of high protein concentrations. The absence of this effect after enzyme immobilization, or when using chromogenic substrates, suggests that a steric mechanism is involved. The apparent activation of caldolysin under certain conditions was shown to be related to both temperature and the ionic strength of the aqueous environment. The effects on activity, substrate affinity and thermostability of chemical modification with various reagents are also discussed.

Endopeptidases↗
PubMed1987

The specific activities of mesophilic and thermophilic proteinases.

1. Most enzymes from extreme thermophiles do not possess higher specific activities than similar enzymes from mesophiles (measured at their respective growth temperatures). 2. However, using protein substrates, the specific activities of thermophilic proteinases are considerably higher than those of most microbial and eukaryotic proteinases. 3. This property could be attributed to purely kinetic influences on the enzyme, to some specific "design" feature of the proteinase, or to the effects of temperature on the substrate. 4. Comparisons of the rates of hydrolysis of large and small substrates by both mesophilic and thermophilic proteinases suggest that temperature-induced changes in substrate susceptibility are a major factor.

Bacillus↗
PubMed1985

Phenolic metabolites of thioridazine in man.

After oral administration of thioridazine or mesoridazine to man, faecal extracts contained mainly unconjugated 7-hydroxy-thioridazine and its demethylated analogue, together with some 3-hydroxy-thioridazine, and 7-hydroxy derivatives of sulphoridazine and desmethyl sulphoridazine. In contrast, urine contained free 7-hydroxy-mesoridazine, and no free 7-hydroxy-thioridazine was detectable. Conjugates of all these phenols were also found. After oral sulphoridazine administration (thioridazine side-chain sulphone), urine contained mainly 7-hydroxy-sulphoridazine and faeces both 7-hydroxy-sulphoridazine and its desmethyl analogue. Sulphoridazine metabolism is therefore much simpler than that of thioridazine. This different elimination pattern in faeces and urine may be due to reduction of sulphoxides but not sulphones by intestinal bacteria.

Administration, Oral↗
PubMed1984

Interactions of calcium and other metal ions with caldolysin, the thermostable proteinase from Thermus aquaticus strain T351.

Caldolysin, the extracellular proteinase from the extreme thermophile Thermus aquaticus strain T351, is stabilized by Ca2+. A variety of metal ions were able to substitute for Ca2+. Most were unable to confer as much stability as Ca2+, with the exception of the lanthanide ions, which increased the half-life at 95 degrees C from 1 h to more than 4 h. Results from a variety of separation methods indicated that caldolysin binds 6 Ca2+ ions/molecule of enzyme. The presence of non-linear Ca2+ titration plots, and the removal of 4 Ca2+ ions/molecule by treatment with a cationic ion-exchange gel suggested that caldolysin possesses at least two different types of Ca2+-binding sites, with different affinities. Average binding constants of the two types of binding sites were 2.8 X 10(4)M-1 (for the low-affinity sites) and 7.5 X 10(5) M-1 (for the high-affinity sites). The total Ca2+-binding free energy for caldolysin was shown to be greater than for either thermolysin or Bacillus subtilis neutral proteinase. It appears that the higher thermostability of caldolysin is due to the presence of 6 Ca2+ ions rather than 4 Ca2+ ions/molecule.

Apoenzymes↗
PubMed1982

A correlation between protein thermostability and resistance to proteolysis.

The loss of activity due to proteolysis of purified L-asparaginase and beta-galactosidase from different sources correlates with the thermal instability of the enzymes. A similar correlation is found when populations of soluble proteins from micro-organisms grown at different temperatures are compared for proteolytic susceptibility and thermal stability. It is proposed that there is a general correlation between the thermostability of proteins and their resistance to proteolysis.

Asparaginase↗
PubMed1982

Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile.

An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351. Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration. It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx. 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5. It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin. No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed. A specificity for small aliphatic amino acids on either side of the splitting point was indicated. Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms. Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h). The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min). None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin. Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days. Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h. At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx. 1 h, 1 h and more than 5 h, respectively.

Carbohydrates↗
PubMed1982

Metabolism of 4-substituted-N-ethyl-N-methylanilines: chromatographic and mass spectrometric identification of N-oxidation metabolic products formed in vitro.

The electron impact and isobutane and ammonia chemical ionization mass spectral characteristics of the N-oxidation products of a series of 4-substituted-N-ethyl-N-methylanilines are described. The thermolability and the relative non-volatility of N-oxides of this type of tertiary aromatic amine makes their unequivocal identification by gas chromatography and electron impact mass spectrometry difficult. Mass spectral identification is facilitated by employing CI mass spectrometry where, under appropriate conditions, the N-oxides give intense protonated molecular ions. Chromatographic, electron impact and chemical ionization mass spectrometric evidence is presented which shows that these 4-substituted tertiary anilines undergo metabolic N-demethylation, N-de-ethylation and N-oxidation in vitro. The suitability of these tertiary aromatic amines as model substrates for mechanistic studies on C- and N-oxidation is discussed.

Aniline Compounds↗
PubMed1982

A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases.

A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases. Because many proteases are thermostable, assay at higher temperatures is possible. The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g. Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C). A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement. Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g. 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K. (1970) Acta Pathol. Microbiol. Scand. Sect. B 78, 350-361)). The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.

Agar↗
PubMed1982

Bone marrow processing and cryopreservation.

Three different closed procedures for concentrating bone marrow progenitor cells prior to cryopreservation have been compared. These were by a manual double centrifugation method, a Hemonetics 30 cell separator and an Aminco Celltrifuge. The best results were achieved using the paediatric pheresis set on the Hemonetics model 30. Marrow was frozen in 120 ml aliquots in a programmed freezer with rapid cooling of the freezing chamber during the phase change from the liquid to the solid state. After freeze-thawing the average nucleated cell recovery was approximately 50% and the progenitor cell recovery 80%.

Bone Marrow Cells↗
PubMed1981

Metabolism of metyrapone. 2-Chromatographic and mass spectral properties of the N-oxides of metyrapone and metyrapol.

Electron impact mass spectral studies on the mono- and di-N-oxides of metyrapone and metyrapol revealed that the technique readily differentiates between the mono-N-oxides of metyrapol, but not of metyrapone. The unequivocal mass spectral differentiation between the isomeric N-oxide metabolites of metyrapone was achieved by the selective and rapid reduction of the keto group in these compounds, with sodium borohydride, to yield the corresponding metyrapol-N-oxides. Chromatographic methods (thin-layer chromatography, gas-liquid chromatography and high-performance liquid chromatography) have been developed for the separation and identification of potential in vitro metabolites of metyrapone. Incubation of metyrapone, under oxidative conditions, with rat or mouse hepatic microsomal preparations, afforded metyrapol (keto reduction) and the isomeric mono-N-oxides of metyrapone. Similar incubation with the hepatic soluble fraction yielded, in addition to metyrapol, an alpha-pyridone metabolite.

Animals↗