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Biomedical subjects

D A Cowan

Publications and source records attributed to D A Cowan.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1993

Potential use of ketoconazole in a dynamic endocrine test to differentiate between biological outliers and testosterone use by athletes.

Determination of the ratio of testosterone to epitestosterone (T/E) in urine is used to detect testosterone administration in athletes, with a ratio > 6 considered as evidence of an offense. We show that administration of ketoconazole, which inhibits testosterone biosynthesis, may be useful for differentiating between an athlete who is using testosterone and one who naturally gives a ratio > 6. In a control subject pretreated with testosterone, ketoconazole caused the ratio to increase; conversely, it caused a decrease in the ratio in an athlete under investigation. Repeated administration of ketoconazole to two normal men caused a decrease in the ratio due to a large decrease in the urinary excretion rate of testosterone relative to epitestosterone. Stimulation with human chorionic gonadotropin exacerbated the differences in excretion rates. A single administration of ketoconazole to six normal men caused the T/E ratios to decrease significantly within 8 h, a suitable time scale for use in a dynamic test.

Adult↗
PubMed1992

Biotechnology of the Archaea.

The Archaea, designated since 1979 as a separate Super-Kingdom (the highest taxonomic order), are a highly novel group of microorganisms which look much like bacteria but have many molecular and genetic characteristics that are more typical of eukaryotes. These unusual organisms can be conveniently divided according to their 'extreme' environmental niche, into three broad phenotypes: the thermophiles, methanogens and extreme halophiles. Each group has unique biochemical features which can be exploited for use in the biotechnological industries. The extreme molecular stability of thermophile enzymes, the novel C1 pathways of the methanogens and the synthesis of organic polymers by some halophiles are all currently or potentially valuable examples of the biotechnology of the Archaea.

Archaea↗
PubMed1992

Possible indices for the detection of the administration of dihydrotestosterone to athletes.

Dihydrotestosterone (DHT) can be used by an athlete as an anabolic steroid to evade the current International Olympic Committee approved drug tests. To investigate the possibility of a method for its detection, the heptanoate ester of DHT was administered to two male subjects (150 mg i.m.). Urine samples, collected before and after the injection, were subjected to enzymatic hydrolysis and the excretion rates of DHT, 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and testosterone (T) were determined by radioimmunoassay. Relative changes in the excretion of DHT, 3 alpha-diol, 5 alpha-androstane-3 beta,17 beta-diol (3 beta-diol), 5 beta-androstane-3 alpha, 17 beta-diol (5 beta-diol), T and epitestosterone (17 alpha hydroxyandrost-4-en-3-one; Epi-T) were determined by gas chromatography-mass spectrometry (GC-MS). Following administration of DHT, the urinary excretion rates of DHT, 3 alpha-diol and 3 beta-diol increased when compared to those of T, Epi-T, 5 beta-diol and luteinizing hormone (LH). Concentrations of DHT in the plasma increased whereas those of T, LH and follicle stimulating hormone decreased. The changes following such modest doses of DHT suggest that these ratios of urinary hormones may be used for the detection of doping with DHT.

Adult↗
PubMed1992

Peptide hormones and sport: misuse and detection.

In 1989 the Medical Commission of the International Olympic Committee (IOC) introduced the new doping class of 'peptide hormones and analogues,' which include human chorionic gonadotrophin (hCG) and related compounds, adrenocorticotrophic hormone (ACTH), human growth hormone (hGH), all the releasing factors of these listed hormones, and erythropoietin (Epo). Currently there are no IOC approved definitive tests for these hormones but highly specific immunoassays combined with suitable purification techniques may be sufficient to warrant IOC approval. The importance of measuring hCG and luteinizing hormone (LH) in the control of testosterone misuse is discussed and strategies for the detection of hGH, ACTH and Epo administration are suggested.

Adrenocorticotropic Hormone↗
PubMed1992

Enzymes from thermophilic archaebacteria: current and future applications in biotechnology.

The one guaranteed property of enzymes isolated from extremely thermophilic micro-organisms is their thermostability. Most significantly, almost any such enzyme will be more thermostable than the functionally similar enzyme from a lower temperature source. Thermostability is not an isolated property: resistance to heat denaturation imparts stability to a number of other denaturing influences (detergents, organic solvents, etc). These characteristics of hyperthermophilic enzymes are the most likely basis for the development of new biotechnological applications. A limited number of hyperthermophilic enzymes have found application in specialist biotechnological applications; others have visible potential in growing areas of biotechnology. Existing and potential applications are discussed using DNA manipulation enzymes, dehydrogenases, and esterases as examples.

Archaea↗
PubMed1991

Human chorionic gonadotrophin and sport.

Human chorionic gonadotrophin (hCG) is a glycoprotein hormone which is produced in large amounts during pregnancy and also by certain types of tumour. The biological action of hCG is identical to that of luteinizing hormone, although the former has a much longer plasma half-life. Some male athletes use pharmaceutical preparations of hCG to stimulate testosterone production before competition and/or to prevent testicular shutdown and atrophy during and after prolonged courses of androgen administration. Testosterone administration can be detected by measuring the ratio of concentrations of testosterone to epitestosterone (T/E). An athlete is often considered to have failed a drug test if the urinary T/E ratio is greater than 6. In contrast, hCG administration stimulates the endogenous production of both testosterone and epitestosterone without increasing the urinary T/E ratio above normal values. Although the administration of hCG was banned by the International Olympic Committee (IOC) in 1987, no definitive test for hCG has been approved by the IOC. Currently, the only way of measuring small concentrations of hCG is by immunoassay, and this does not have a discriminating power as great as gas-liquid chromatography with mass-spectrometry which is necessary to satisfy IOC requirements. Extraction procedures and chromatographic steps could be introduced before using a selected immunoassay for hCG to meet these requirements.

Chorionic Gonadotropin↗
PubMed1991

Effect of administration of human chorionic gonadotrophin on criteria used to assess testosterone administration in athletes.

Abnormal ratios of testosterone to epitestosterone (T/E) and testosterone to LH (T/LH) in the urine of male athletes are indicative of testosterone administration. The T/E ratio has been adopted by the International Olympic Committee as the sole criterion used in the detection of testosterone administration. An athlete is usually considered to have failed a drug test if the urinary T/E ratio is greater than 6. Human chorionic gonadotrophin (hCG) has been used by some male athletes to stimulate testicular secretion of testosterone. The purpose of this investigation was to examine whether the urinary T/E ratio can remain unaffected by administration of hCG to normal adult males. Administration of hCG resulted in large increases in serum testosterone concentrations and urinary T/LH ratios but small changes in urinary T/E ratios of two subjects (maximum T/E values observed were 0.8 and 1.2 respectively). These observations suggest that the urinary T/LH ratio is a valuable indicator of hCG as well as of testosterone administration. This study is the first to measure urinary T/LH ratios using the technique of gas chromatography-mass spectrometry for quantification of testosterone, and highly specific monoclonal antibodies for the measurement of LH. An ultrafiltration method is proposed as part of a confirmatory procedure to be adopted in the measurement of urinary gonadotrophins for drug control in sport.

Chorionic Gonadotropin↗
PubMed1990

Criteria to indicate testosterone administration.

A detection method for testosterone administration was developed using radioimmunoassay to measure the urinary ratios of testosterone (T) to epitestosterone (E) and to luteinizing hormone (LH). A comparative study of the effect on these ratios of a single intramuscular injection of testosterone heptanoate followed by stimulation with human chorionic gonadotrophin (HCG) in three normal men was undertaken. To allow immediate investigation, a commercially supplied epitestosterone antiserum was used. This study showed that both T/E and T/LH ratios could be used to detect testosterone administration, the latter also being an indicator of HCG use due to cross-reactivity with the LH antiserum. Subsequently, an epitestosterone antiserum of superior specificity was raised and used in a study to demonstrate the insignificant effect of exercise on these ratios. Finally, an intramuscular injection of a combined preparation of testosterone/epitestosterone heptanoates resulted in raised ratios of T/LH but not of T/E. This demonstrated the importance of the T/LH ratio in circumstances where the T/E ratio can be easily circumvented.

Adult↗
PubMed1989

Caldolase, a chelator-insensitive extracellular serine proteinase from a Thermus spp.

An extracellular alkaline serine proteinase from Thermus strain ToK3 was isolated and purified to homogeneity by (NH4)2SO4 precipitation followed by ion-exchange chromatography on DEAE-cellulose and QAE-Sephadex, affinity chromatography on N alpha-benzyloxycarbonyl-D-phenylalanyl-triethylenetetraminyl-Sepha rose 4B and gel-filtration chromatography on Sephadex G-75. The purified enzyme had a pI of 8.9 and an Mr determined by gel-permeation chromatography of 25,000. The specific activity was about 37,700 proteolytic units/mg with casein as substrate, and the pH optimum was 9.5. Proteolytic activity was inhibited by low concentrations of di-isopropyl phosphorofluoridate and phenylmethanesulphonyl fluoride, but was unaffected by EDTA, EGTA, o-phenanthroline, N-ethyl-5-phenylisoxazolium-3'-sulphonate, N alpha-p-tosyl-L-phenylalanylchloromethane, N alpha-p-tosyl-L-lysylchloromethane, trypsin inhibitors and pepstatin A. The enzyme contained approx. 10% carbohydrate and four disulphide bonds. No Ca2+, Zn2+ or free thiol groups were detected. It hydrolysed several native and dye-linked proteins and synthetic chromogenic peptides and esters. The enzyme was very thermostable (half-life values were 840 min at 80 degrees C, 45 min at 90 degrees C and 5 min at 100 degrees C). The enzyme was unstable at low ionic strength: after 60 min at 75 degrees C in 0.1 M-Tris/acetate buffer, pH 8, only 20% activity remained, compared with no loss in 0.1 M-Tris/acetate buffer, pH 8, containing 0.4 M-NaCl.

Enzyme Stability↗