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Biomedical subjects

D A Cowan

Publications and source records attributed to D A Cowan.

At least 19 recordsLinked to original sources
PubMed1997

Thermophilic proteins: stability and function in aqueous and organic solvents.

The molecular stability of thermophilic and hyperthermophilic enzymes generally reflects the growth temperatures of the parent organisms. Extracellular enzymes from the hyperthermophilic Archaea typically show very high levels of thermal stability and a number of enzymes with Tm values of greater than 100 degrees C have been reported. The mechanisms responsible for high molecular stability are typically intrinsic characteristics of the protein, as shown by the comparative stabilities of many native and recombinant proteins. However, some extrinsic stabilisation mechanisms have been demonstrated. High levels of thermal stability are positively correlated with stability in the presence of other denaturing agents, including detergents and organic solvents. This correlation suggests a common denaturation pathway where molecular mobility/flexibility is the prime determinant of susceptibility to irreversible denaturation. In single phase organic-aqueous solvents, protein destabilisation occurs via solvent-induced alteration to the protein hydration shell. However, correlations between protein stability and solvent hydrophobicity are unreliable. In two-phase organic-aqueous systems, interfacial denaturation predominates and is a function of both interfacial tension and interfacial surface area. Intracellular enzymes are protected from interfacial denaturation but are potentially susceptible to direct organic solvent effects, possibly depending on the role of the cell wall and cell membrane in the partitioning of the organic solvent into the cell cytoplasm. Immobilisation of thermophilic enzymes provides a method for enhancing both the thermal and solvent stabilities of thermophilic and mesophilic enzymes. Multi-point covalent immobilisation to glyoxal-agarose enhances thermal stability and limits protein-protein inactivation mechanisms. Miscible organic solvents have a profound influence on the specificities of enzyme reactions. The presence of high concentrations of miscible organic solvents may induce gross changes in substrate specificity and/or more subtle alterations in chiral selectivity. Correlations between the variation in enantioselectivity and both solvent hydrophobicity and solvent dielectric constant have been demonstrated although some recent studies implicate the formation of specific solvent-enzyme complexes which directly affect reaction kinetics.

Archaea↗
PubMed1997

Serum IGF-I and IGF binding proteins 2 and 3 as potential markers of doping with human GH.

OBJECTIVE: IGF-I and IGF binding protein (IGFBP)-3 levels in man are positively regulated by GH status; in contrast, evidence suggests an inverse relationship between GH status and IGFBP-2. We investigated the effects of somatropin administration on the serum concentrations of these analytes, together with serum and urinary concentrations of GH, to evaluate their potential as markers in the development of a test for detecting doping with GH in sports competitors. DESIGN: Somatropin was administered subcutaneously at a dose of 0.15 U/kg bodyweight/day at 1000 h for 3 days to eight healthy men (20-32 years old). MEASUREMENTS: Serum concentrations of GH, IGF-I, IGFBP-2 and -3 were determined in blood samples collected at 1600 h on the days prior to (day -1), during (days 0, 1 and 2), and following administration (days 3 and 7). Urine was collected continuously from days -2 to 3 and then on day 7. RESULTS: Serum and urinary concentrations of GH were only raised on the days of administration whereas, following cessation of somatropin, the increases in the serum concentrations of IGF-I and IGFBP-3 were sustained for at least 1 day (30 h). Serum IGFBP-2 decreased during the period of administration and was still suppressed on day 3. The concentration ratios of IGFBP-3 to IGFBP-2 and IGF-I to IGFBP-2 increased markedly with administration and both ratios were still significantly augmented compared with basal values 30 h after the last administration. CONCLUSION: With acute administration of somatropin to healthy men the serum concentration of IGFBP-2 decreases and the ratios of serum IGF-I/ IGFBP-2 and IGFBP-3/IGFBP-2 increase. These ratios should be considered in the development of a test for detecting somatropin administration in sport.

Adult↗
PubMed1997

Cortisol concentrations in post competition horse urine: a French and British survey.

The purpose of the present report was to estimate the population parameters of cortisol concentrations in urine, an endogenous hormone used as a 'doping' agent and for which an international threshold (1.0 micrograms/ml) has been proposed. Two data bases (French and UK) corresponding to 112 and 142 samples, respectively were considered. Urine was collected under specific post competition conditions. Cortisol concentrations were obtained by validated methods (HPLC for the French samples, and GC-MS for UK samples). No difference was observed between the 2 data sets and statistical analyses were carried out on the two merged files. The overall geometric mean cortisol concentration was 48 ng/ml. Distribution was not Gaussian. A log-normal distribution was not rejected (for P > 0.05). Using the log-normal distribution, it was calculated that the probability of exceeding a cortisol concentration in urine of 1.0 micrograms/ml was 1.1 x 10(-4). It was concluded that the actual international threshold is specific i.e. robust with regard to the risk of erroneously declaring an unmedicated horse as positive.

Animals↗
PubMed1997

Intramuscular administration of 5 alpha-dihydrotestosterone heptanoate: changes in urinary hormone profile.

A recommended confirmatory procedure for detecting 5 alpha-dihydrotestosterone (DHT) doping in male athletes proposed the use of the urinary concentration ratio of DHT to epitestosterone (EpiT) as the primary marker and those of 5 alpha-androstane-3 alpha,17 beta-diol (5 alpha-Adiol) to EpiT, luteinizing hormone (LH), and 5 beta-androstane-3 alpha,17 beta-diol (5 beta-Adiol) as secondary markers. Here we investigate the effects on these markers of intramuscular administration of DHT heptanoate (250 mg) to six healthy men. Within 24 h of administration all four markers greatly exceeded the published discrimination limits, remaining above these limits for 10-14 days. All ratios returned to basal values by day 28. In contrast to results after percutaneous administration, 5 beta-Adiol excretion decreased, probably as a consequence of greater suppression of testicular steroidogenesis. Results were largely in agreement with those obtained after percutaneous administration, although probably augmented by the larger dose and the different route of delivery.

Adult↗
PubMed1996

Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms.

Hyperthermophiles exist in conditions which present an increased threat to the informational integrity of their DNA, particularly by hydrolytic damage. As in mesophilic organisms, specific activities must exist to restore and protect this template function of DNA. In this study we have demonstrated the presence of thermally stable uracil-DNA glycosylase activities in seven hyperthermophiles; one bacterial: Thermotoga maritima, and six archaeal: Sulfolobus solfataricus, Sulfolobus shibatae, Sulfolobus acidocaldarius, Thermococcus litoralis, Pyrococcus furiosus and Pyrobaculum islandicum. Uracil-DNA glycosylase inhibitor protein of the Bacillus subtilis bacteriophage PBS1 shows activity against all of these, suggesting a highly conserved tertiary structure between hyperthermophilic and mesophilic uracil-DNA glycosylases.

Archaea↗
PubMed1996

Rapid purification of two thermophilic proteinases using dye-ligand chromatography.

Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures. Single step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained. Elution studies suggested that the mode of binding involved the enzyme active sites. The method was readily scaleable to 600 1 volume.

Bacillus↗
PubMed1996

Urinary testosterone (T) to epitestosterone (E) ratios by GC/MS. I. Initial comparison of uncorrected T/E in six international laboratories.

Six laboratories in six countries collaborated to investigate the analytical method for estimating the testosterone to epitestosterone ratio (T/E) in urine by gas chromatography/mass spectrometry in the context of detecting the application of T as a doping agent in sport. The protocol specified many but not all details of reagents and instrument conditions. The design included the distribution and analysis of four urines with different T/E values, three replicates per value, and one standard. The ranges of mean T/E values for the four urines estimated by peak area (PA) were 0.32-0.42, 0.72-0.94, 0.91-1.14 and 3.19-5.48. The analyses of variance for these data and for the peak height (PH) data were significant for the laboratory factor (p < 0.0001). In addition there was a significant interaction between the urine factor and the laboratory factor which indicates the complexity of the analysis. T/E calculated using PA was not significantly different from that using PH. For within-laboratory precision all values for PH and PA were < 8.3%, and for between-laboratory precision all values were < 11.7% except for one (20.1%). The data represent a baseline for future experiments designed to elucidate the sources of within-and between-laboratory variance, and to harmonize estimates of T/E.

Analysis of Variance↗
PubMed1996

Rapid purification of two thermophilic proteinases using dye-ligand chromatography.

Dye-ligand chromatography has been used successfully for the purification of extracellular thermostable proteinases from thermophilic Bacillus and Thermus cultures. Single-step purification factors of up to 115-fold (for Thermus protease) and 2195-fold (for Bacillus protease) were obtained. Elution studies suggested that the mode of binding involved the enzyme active sites. The method was readily scalable to 600 1 volume.

Bacillus↗
PubMed1996

Complementation of a pgk deletion mutation in Saccharomyces cerevisiae with expression of the phosphoglycerate-kinase gene from the hyperthermophilic Archaeon Sulfolobus solfataricus.

The gene encoding phosphoglycerate kinase (PGK) from the Archaeon Sulfolobus solfataricus, an organism growing optimally at 87 degrees C, was inserted into a yeast expression vector under the control of the galactose-inducible GAL1 yeast promoter. This vector was then transformed into a pgk::TRP1 yeast mutant, a strain inhibited for growth on galactose or glucose due to its lack of PGK enzyme. Slow-growing transformants were obtained on galactose plates at 37 degrees C, but not 28 degrees C. These transformants contained low levels of transcripts of the heterologous gene and low amounts of thermostable PGK activity. Weak expression of the hyperthermophile gene in yeast, a mesophile, therefore enabled complementation of the yeast pgk defect at 37 degrees C but not at 28 degrees C.

Base Sequence↗
PubMed1995

The phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase genes from the thermophilic archaeon Sulfolobus solfataricus overlap by 8-bp. Isolation, sequencing of the genes and expression in Escherichia coli.

The overlapping genes encoding phosphoglycerate kinase (PGK) and glyceraldehyde-3-phosphate dehydrogenase (GraP-DH) from the hyperthermophilic archaeon Sulfolobus solfataricus have been cloned and sequenced. PCR primers based on highly conserved regions of different PGK sequences were used to isolate an internal region of the pgk gene. This was then used to screen a genomic library to isolate the full length pgk gene. A 2.5-kb BglII fragment of S. solfataricus DNA contained both the pgk gene and the gap gene immediately downstream. Unexpectedly, the pgk and gap genes were found to overlap by 8 bp, with the initiation codon of the gap gene preceding the termination codon of the pgk gene. Evidence that the two genes are co-transcribed was obtained by Northern-blot analysis. The S. solfataricus PGK amino acid sequence shows 43% and 45% identity to the PGK sequences of the Archaea Methanobacterium bryantii and Methanothermus fervidus, respectively. High level expression of the S. solfataricus PGK and GraP-DH in Escherichia coli was achieved, with heat treatment at 80 degrees C proving an effective first step in the purification of these recombinant enzymes from extracts of the E. coli host. Purified recombinant S. solfataricus PGK and GraP-DH showed half lives of 39 min and 17 h, respectively, at 80 degrees C. Unlike bacterial GraP-DH enzymes, S. solfataricus GraP-DH was able to use both NAD+ and NADP+ as cofactors, but exhibited a marked preference for NADP+.

Amino Acid Sequence↗
PubMed1995

Cloning and sequencing of a gene from the archaeon Pyrococcus furiosus with high homology to a gene encoding phosphoenolpyruvate synthetase from Escherichia coli.

A gene from the hyperthermophilic archaeon Pyrococcus furiosus, strain Vc1 (DSM 3638), contains an 817-amino-acid open reading frame which shows 42% identity to the phosphoenolpyruvate (PEP) synthetase of Escherichia coli. This putative P. furiosus PEP synthetase is slightly larger than the E. coli enzyme, the region between residues 58 and 89 being absent from the latter.

Amino Acid Sequence↗
PubMed1995

Tryptic mapping of human chorionic gonadotropin by matrix-assisted laser desorption/ionization mass spectrometry.

The potential of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for use in the identification of human chorionic gonadotropin (hCG) seized by law enforcement agencies is investigated. Analysis of untreated hCG revealed signals corresponding to the molecular ions of the intact hCG heterodimer and both its non-covalently linked subunits. Unfortunately, due to carbohydrate heterogeneity, the peaks are broad which makes accurate mass assignment, and consequently identification, difficult. Peptide mapping by MALDI-TOF following tryptic digestion gave sequence coverage of 59% and 52% for the alpha- and beta-subunits respectively. Nevertheless, the tryptic map was considered to provide unambiguous identification of hCG. This was confirmed by searching peptide-mass databases with the experimentally determined masses. Our data suggest that peptide mapping by proteolytic digestion followed by MALDI-TOF mass spectrometry is a suitable analytical technique for the identification of hCG seized by the legal authorities.

Amino Acid Sequence↗
PubMed1995

Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity.

An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery. The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C. The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration. Native polyacrylamide gels stained for esterase activity showed three bands. The isoelectric points were estimated to be 5.7, 5.8, and 6.0. Forty amino acid residues were sequenced at the N-terminus. The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids. The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0. At this pH and 60 degrees C the half-life was 170 h. Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid. The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent. The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths).

Amino Acid Sequence↗
PubMed1995

Proposed confirmatory procedure for detecting 5 alpha-dihydrotestosterone doping in male athletes.

Currently, there is no recommended confirmatory procedure for detecting doping with the anabolic steroid 5 alpha-dihydrotestosterone (DHT) in sportsmen. To develop a method, we determined ratios of hormone concentrations in urine from 120 healthy men and used these to set discrimination limits. These limits were then applied to results for urine specimens from 10 volunteers given DHT percutaneously (125 mg twice daily for 4 days). The ratio of DHT to epitestosterone (EpiT) was chosen as the primary marker of DHT administration, and ratios of 5 alpha-androstane-3 alpha, 17 beta-diol (5 alpha-ADIOL) to EpiT, 5 alpha-ADIOL to luteinizing hormone, and 5 alpha-ADIOL to 5 beta-androstane-3 alpha, 17 beta-diol were proposed as secondary markers. To evaluate method suitability, we analyzed 194 samples from sports competitors; results for 193 samples were negative, but the ratios in 1 sample greatly exceeded all the chosen limits. In conclusion, we propose that a test scheme based on our recommendations be considered for implementation in all Olympic drug-testing laboratories.

Adolescent↗
PubMed1995

Protein stability at high temperatures.

The enzymology of hyperthermophilic micro-organisms is a growing field. As increasing numbers of novel high-temperature organisms are isolated and made available through culture collections, and, as biomass becomes more readily available, more laboratories will undoubtedly expand their research interests into this area. The prospect of totally novel enzyme systems and of new approaches to the investigation of fundamental molecular properties will continue to stimulate interest in this field. Studies of thermostable enzymes have already provided valuable data on the relationships between protein stability and activity. The subtle molecular mechanisms which have evolved to stabilize these proteins provide the clues needed for the intelligent design of stabilized mesophilic enzymes, an important target where a combination of high activity at 'low' temperatures and resistance to denaturation is required. The current role of hyperthermophilic enzymes in biotechnology is relatively minor, despite these enzymes having a high 'profile'. While early over-enthusiastic predictions that these enzymes would revolutionize biotechnology should be disregarded, it can reasonably be assumed that, where functional and economic criteria are suitable, thermophilic enzymes will be readily incorporated into current and future biotechnology.

Bacteria↗
PubMed1994

Effect on sports drug tests of ingesting meat from steroid (methenolone)-treated livestock.

Anabolic-androgenic steroids are widely misused in human sports and are also used as growth promoters in livestock. Athletes who consume meat containing such hormone residues may risk failing a sports drug test. Prompted by an athlete's defense case, we questioned whether the consumption of small livestock given doses of anabolic steroid, orally or intramuscularly, could generate positive results in samples tested by our analytical procedures. We analyzed urine from eight men who consumed chickens that had been either fed with methenolone acetate (1 mg/day) from day 0 to 21 or injected with methenolone heptanoate depot (1 mg/intramuscular injection) on days 0, 7, and 14 and slaughtered on day 22. No methenolone or characteristic major metabolite was detected in samples from subjects who ate meat from the orally dosed chickens. However, 50% of the samples collected 24 h after consumption of the intramuscularly dosed chickens were confirmed positive. Hence, eating meat containing small amounts of injected hormone may constitute a serious liability to the athlete.

Administration, Oral↗
PubMed1994

New decision limits and quality-control material for detecting human chorionic gonadotropin misuse in sports.

Male athletes may administer human chorionic gonadotropin (hCG) to enhance performance. Despite imposing a ban on hCG doping, the International Olympic Committee has no recommended procedure to confirm the presence of hCG. Our aim was to establish a limit for hCG in urine above which a sample may be considered positive. We measured hCG concentrations in urine samples from 1400 men with the Serono MAIAclone IRMA. Statistical evaluation of the results gave a "far outside" value [75th percentile + (3 x interquartile range)] of 5.0 IU/L; greater values are extremely unusual. Immunoreactive material in urine samples from a reference group of 120 noncompeting individuals was concentrated about sevenfold by centrifugal ultrafiltration; all these concentrated samples gave assay values < 5.0 IU/L. To ensure with the greatest possible degree of certainty that no false-positive result is reported, we propose a decision limit of 10 IU/L in nonconcentrated ultrafiltered samples. We also prepared quality-control material to contain hCG at 10 IU/L. This material provides a decision limit above which, after confirmatory procedures, a sample should be considered positive. This should help establish comparability of results among laboratories testing for sports drugs.

Adolescent↗