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K J Stevenson

Publications and source records attributed to K J Stevenson.

At least 19 recordsLinked to original sources
PubMed2004

No deterioration after 13 years in a stability study of a rabbit brain, plain, thromboplastin, RBT 1010, in rubber-stoppered ampoules.

RBT 1010, a rabbit brain thromboplastin, plain, was prepared at the Thrombosis Reference Centre, Withington Hospital, Manchester, in 1989. The batch has been stored at -20 degrees C, in rubber-stoppered ampoules, for 13 years. The material was unchanged after this time. This was confirmed in a stability study, in which the reagent was used to test the prothrombin times of a panel of plasmas stored in liquid nitrogen, one from a normal volunteer and two from stable anticoagulated patients. RBT 1010 was also tested in calibrations, according to World Health Organization recommendations, against the reference thromboplastin preparations CRM 149S and RBT 90.

Animals↗
PubMed1998

Ozone levels in Chongqing: a potential threat to crop plants commonly grown in the region?

Chongqing, a city with a population of >2.5 million, constitutes the biggest industrial and commercial centre in southwest China. Recent industrialization has led to an increasing air pollutant problem which is exacerbated by the topography and prevailing climate of the region. To date, interest has remained firmly focused on the levels of traditional air pollutants (sulphur dioxide [SO2], oxides of nitrogen [NOx], smoke and suspended particulate matter [SPM]), with little attention paid to photochemical oxidants such as ozone (O3). This paper reports the first assessment of ambient O3 levels in southwest China. Measurements were made in and around Chongqing using a combination of UV-absorption (at a site located in the northern sector of the city) and passive samplers (at 20 sites located in and around the city) between 1993 and 1996. The 7-h daily mean O3 concentrations ranged between 2 and 16 ppb (x10(9)) during the winter months, increasing to 18-41 ppb during the summer (June-August), when peak hourly mean O3 concentrations of 93 ppb were attained. Ozone exposures across the region commonly approached (or exceeded) UN-ECE and WHO short-term guidelines for the protection of crops. In addition, controlled chamber studies, in which 11 cultivars of Chinese crops commonly grown in the Chongqing region were screened for relative O3 sensitivity, indicated the potential for subtle effects on the growth of a number of crop plants, if ambient O3 levels continue to rise in the region. Employing ozone exposures somewhat higher than those experienced in the field, several cultivars of commonly grown Chinese cereal, vegetable and salad crops were found to be sensitive to O3 in terms of growth, but this was not always associated with the appearance of visible symptoms of injury and, in contrast to what was generally expected, only three species showed significant O3-induced reductions in root:shoot partitioning. There is a clear and urgent need for a comprehensive study of ambient air quality and its impact on crops and natural vegetation in this, as in other, rapidly developing regions of China.

Journal Article↗
PubMed1998

The determination of INR in stored whole blood.

AIMS: To examine the reliability of international normalised ratio (INR) determination on samples stored as whole blood for up to two days at room temperature. METHODS: The INR of 40 patients receiving oral anticoagulants was determined on fresh blood and on samples stored for 24 and 48 hours, using five locally calibrated prothrombin time systems. These incorporated Manchester reagent, Recombiplastin, IL PT Fibrinogen HS Plus, Manchester combined capillary prothrombin time reagent, and a freeze dried in-house reference rabbit brain thromboplastin, RBT 1010. In addition, factors II, V, VII, and X were determined on samples obtained from 18 of these patients before and after incubation at room temperature. RESULTS: The INR of the samples changed by differing amounts during storage, depending on which system was employed. Although the mean change after 24 hours storage was relatively small, there were individual samples that changed by > 0.5 INR with all systems. These changes would lead to adjustment in dosage of certain patients. After 48 hours these effects were greater with all systems except that employing Recombiplastin. There were only small reduction in the measured factors by 48 hours. CONCLUSIONS: After storage of samples for only 24 hours, some patients' INR changed sufficiently to affect dosage. In view of these observations, the practice of storing whole blood samples for INR determination cannot be recommended.

Anticoagulants↗
PubMed1997

System ISI calibration: a universally applicable scheme is possible only when coumarin plasma calibrants are used.

Coagulometer-induced errors in International Normalized Ratios (INR) may be corrected by assigning an International Sensitivity Index (ISI) specific for the local combination of coagulometer and thromboplastin reagent (System ISI). In the present study, 57 laboratories determined the INR of eight frozen coumarin plasmas. There was considerable variation in INR reported at the different centres, with coefficients of variation (CV) ranging from 11.30% to 17.29%. In addition, their consensus mean INR values were consistently higher than those obtained manually. The overall mean percentage deviation was 20.06%, indicating that correction was required to reduce the over-estimation of INR that occurred even in those Systems incorporating a thromboplastin reagent with an instrument-specific ISI. Local correction using coumarin plasma calibrants reduced this value to < or = 7% with current reference thromboplastins BCT 441, OBT 79 and RBT 90 and also lowered the CV to < 5%. In contrast, when adsorbed plasma calibrants were employed, mean percentage deviations were 7.87% with BCT 441, 12.47% with OBT 79 and 77.37% with RBT 90 as reference respectively. This difference may be due, in part, to the difficulty in assigning an INR to adsorbed plasma calibrants, which contain no protein induced by vitamin K antagonists (PIVKA). This study demonstrates that coumarin plasma calibrants are superior in System ISI calibration.

Blood Coagulation Tests↗
PubMed1997

Purification and sequence analysis of a novel NADP(H)-dependent type III alcohol dehydrogenase from Thermococcus strain AN1.

An NADP(H)-dependent alcohol dehydrogenase was isolated from the hyperthermophilic archaeon Thermococcus strain AN1. This enzyme is a homotetramer with a subunit molecular weight of 46,700. The enzyme oxidizes a series of primary linear alcohols but not methanol. The pH and temperature optima with ethanol as the substrate are 6.8 to 7.0 and 85 degrees C, respectively. The enzyme readily reduced acetaldehyde with NADPH as the cofactor. The gene encoding this enzyme has been cloned and sequenced. An open reading frame of 1,218 bp, starting with ATG and ending with TGA, was identified and corresponded to 406 amino acids. Sequence comparisons show that this Thermococcus strain AN1 enzyme has significant homologies with enzymes from the newly defined type III alcohol dehydrogenase family. Thermococcus strain AN1 alcohol dehydrogenase is the first archaeal enzyme belonging to this family.

Alcohol Oxidoreductases↗
PubMed1997

Calculation of System International Sensitivity Index: how many calibrant plasmas are required?

AIMS: To determine the minimum number of calibrant plasmas required to assign an accurate System International Sensitivity Index (ISI). METHODS: Eighteen lyophilised adsorbed and eight frozen coumarin plasmas were despatched to 57 participating laboratories. Each centre determined the prothrombin time of all of the plasmas using their routine system of coagulometer and thromboplastin reagent and provided their system mean normal prothrombin time (MNPT). For each laboratory, the slope of the line relating local system results, including MNPT, to calibrated values obtained with BCT 441, RBT 1303 or RBT 90 was derived by orthogonal regression. The number of adsorbed calibrant plasmas included in the calculation was reduced, two at a time, from 18 to two. The validity of the System ISI was tested using the frozen coumarin plasmas. RESULTS: All systems tested gave higher International Normalised Ratios (INR) than those obtained manually with the reference thromboplastins. All systems therefore required correction. No changes were seen in the System ISI as the number of calibrant plasmas was reduced from 18 to 12. Below 12, inconsistent results were obtained with some systems and were dependent on plasma selection. CONCLUSIONS: Although System ISI could be assigned using as few as two calibrant plasmas, to avoid imprecise calibration, a minimum of 12 calibrant plasmas is recommended.

Calibration↗
PubMed1997

Local INR correction: justification for a simplified approach.

AIMS: Errors in reporting International Normalised Ratios (INR) may be corrected by assignment of a System International Sensitivity Index (System ISI). This 57 centre study tests the validity of several procedures for INR correction. METHODS: Prothrombin times of eight lyophilised coumarin calibrants, a lyophilised normal pool calibrant, and eight frozen coumarin plasmas were determined at each centre. The calibrants were calibrated using international reference preparations. The eight frozen coumarin plasmas were calibrated in a four centre international exercise. The relations tested were: (a) the logarithm of local prothrombin time against the logarithm of reference prothrombin time; (b) reference INR against local prothrombin time; and (c) logarithm of reference INR against logarithm of local prothrombin time. These methods were analysed by both linear and orthogonal regression. RESULTS: All system groups required correction, the mean percentage deviation of the uncorrected data from the calibrated values was 19.0%. There was also considerable variation in INR, with the coefficient of variance (CV) ranging from 11.30 to 17.29%. Correction of INR was possible with all methods (CV reduced to < 7%). However, only when a plot of the logarithm of local prothrombin time against the logarithm of reference prothrombin time was fitted by orthogonal regression, or a plot of logarithm of reference INR against logarithm of local prothrombin time was fitted by either type of regression analysis, did the best fit line through the calibrant plasmas also pass close to the local mean normal prothrombin time. CONCLUSIONS: While INR correction may be achieved by all the above methods, that relating log reference INR to log local prothrombin time by linear regression analysis is the simplest to perform.

Anticoagulants↗
PubMed1994

The role of lipids in the detection of lupus anticoagulant by the dilute Russell Viper venom test: are platelets or reagents containing hexagonal HII phases necessary?

Liposomes prepared from rabbit brain extracts (RBE) and individual pure lipids (high phosphatidyl serine content, HIPS) were compared with frozen-thawed platelets (PLTS) in the dilute Russell Viper venom time (dRVVt). While all three preparations demonstrated sensitivity to lupus anticoagulant (LA) the highest detection rate was seen with RBE. For confirmation of LA, high concentration RBE achieved the most efficient correction of the defect. Electron microscopy and particle sizing showed RBE to be small, discrete liposomes, whereas HIPS and PLTS were aggregates of larger diameter particles. Low-angle X-ray diffraction showed no evidence of hexagonal HII phase. There appears to be no specific requirement either for platelets or for reagents containing hexagonal HII phases in the dRVVt. The dRVVt can be optimized by incorporating a simple dilute rabbit brain lipid mixture for detection of LA and a concentrated mixture as a correcting reagent.

Animals↗
PubMed1994

Activated partial thromboplastin time for automated techniques: a comparison of two commonly used reagents.

In a comparison between two widely used activated partial thromboplastin time reagents (Instrumentation Laboratories, Manchester low-opacity), using one popular instrument (Automated Coagulation Laboratory, model 300R), a wide range of defects and 40 normals were tested. There was generally good agreement between methods, although for samples from patients on high heparin dosages the agreement was poor. The manufacturer's recommendation for the therapeutic range for heparin for the Instrumentation Laboratory reagent was lower than the range in current general use. The Manchester reagent was more sensitive to the lupus anticoagulant.

Heparin↗
PubMed1994

Long-term stability studies on the WHO IRP for thromboplastin (human plain BCT/253).

Long-term stability studies on the WHO second primary International Reference Preparation (IRP) for thromboplastin (human plain BCT/253) stored at -20 degrees C have been conducted for ten years. Three centres took part in the exercise using frozen normal and coumarinised plasma samples which were tested throughout. There has been no measurable change in the prothrombin time performance of the human plain IRP over the ten-year period. It can therefore be concluded that new IRP may continue to be calibrated against this preparation in accord with WHO recommendations.

Analysis of Variance↗
PubMed1992

Aromatic trivalent arsenicals: covalent yet reversible reagents for the agonist binding site of nicotinic receptors.

The agonist binding site of nicotinic acetylcholine receptors (AChRs) includes a disulfide bond that is easily reduced with dithiothreitol to a pair of thiols, and can be then either reoxidized with dithiobis(nitrobenzoic acid) (DTNB) or irreversibly alkylated with bromoacetylcholine (BAC). Aromatic trivalent arsenicals form stable complexes with pairs of appropriately-spaced thiols, but not single thiols. Furthermore, once complexed in proteins, trivalent arsenicals can be removed with dimercaptans, such as 2,3-dimercaptopropanesulfonic acid (DMPS). In an effort to develop reagents that will covalently, yet reversibly label AChRs, we investigated the effects of two model arsenicals, p-aminophenyldichloroarsine (APA) and 4-bromoacetyl-aminophenylarsenoxide (BAPA) on two types of nicotinic receptors: AChRs from Torpedo electroplax and neuronal receptors from chick retina. APA and BAPA significantly decrease the number of 125I-alpha-bungarotoxin binding sites in reduced Torpedo AChRs. Furthermore, arsenylation of neuronal and Torpedo receptors with APA or BAPA (1) prevents reoxidation with DTNB, (2) is reversible with DMPS, and (3) protects against irreversible alkylation by BAC. In Torpedo receptors, the EC50 of protection against BAC alkylation with APA or BAPA is approximately 30 nM. APA arsenylation of Torpedo receptors persists up to 20 h, but can be reversed at any time with DMPS. These results suggest that heterobifunctional arsenicals could anchor labeling groups in the agonist binding site in order to map the agonist binding site, quantitate receptors, or purify and reconstitute functional receptors.

Acetylcholinesterase↗
PubMed1992

Dihydrolipoamide dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.

Dihydrolipoamide dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.

Amino Acid Sequence↗
PubMed1992

Dihydrolipoamide dehydrogenase from Haloferax volcanii: gene cloning, complete primary structure, and comparison to other dihydrolipoamide dehydrogenases.

We used the N-terminal amino acid sequence of dihydrolipoamide dehydrogenase from Haloferax volcanii, to design and synthesize two oligonucleotide probes that were used to identify and clone a 4.3 kilobase pair (kbp) fragment from MboI restriction endonuclease digestion of Hf. volcanii genomic DNA. The nucleotide sequence of a 1.5-kbp region of this clone was determined and this revealed an open reading frame that translated into a protein with good homology to dihydrolipoamide dehydrogenase from other sources. The first 48 amino acids were identical with the N-terminal sequence data obtained from the purified protein. The complete primary structure of the halophilic dihydrolipoamide dehydrogenase was analyzed in terms of its homologies to dihydrolipoamide dehydrogenases from other sources and its molecular adaptations to high intracellular ionic strength.

Amino Acid Sequence↗
PubMed1991

Production of plasma selectively depleted in fibrinogen by affinity chromatography.

An affinity chromatography column was prepared using a polyclonal anti-fibrinogen antibody bound to Sephacryl S 1000. Normal human plasma was depleted of fibrinogen by passage through this column. The fibrinogen free plasma corrected the prolonged clotting times of plasmas deficient in factors V, VII, VIII and IX. Clottable fibrinogen was recovered from the column. An immunodepleted fibrinogen free plasma could fulfil a useful role in internal and external quality control alone, or in combination with other test materials; it could also be used in research.

Antibodies↗
PubMed1990

Subcellular localisation of dihydrolipoamide dehydrogenase and detection of lipoic acid in bloodstream forms of Trypanosoma brucei.

In the long-slender bloodstream form of Trypanosoma brucei, the enzyme dihydrolipoamide dehydrogenase exists in the absence of the 2-oxo-acid dehydrogenase complexes of which it is normally a component, and appears to be associated with the plasma membrane of the organism [Danson, M. J., Conroy, K., McQuattie, A. & Stevenson, K. J. (1987) Biochem. J. 243, 661-665]. In the present paper, a complete subcellular fractionation of T. brucei has been carried out and, by comparison with marker enzymes, it is confirmed that the dihydrolipoamide dehydrogenase is indeed associated with the plasma membrane. In addition, we now provide evidence that the distribution of the enzyme is over the whole surface of the membrane, including the flagellar pocket region, and that the enzyme is not found in any other cellular fraction. A study of the latency of the enzyme suggests that it is located on the cytoplasmic surface of the plasma membrane. The discovery of the presumed substrate of dihydrolipoamide dehydrogenase, lipoic acid, is reported for T. brucei. Using a biological assay involving a strain of Escherichia coli that requires lipoic acid for growth, we have found that acid-hydrolysed extracts of T. brucei contain 1.7 (+/- 0.2) ng of the cofactor/mg protein. The chemical nature of the lipoic acid was confirmed by gas chromatography/mass spectrometry.

Animals↗
PubMed1989

Detection of bacterial lipoic acid. A modified gas-chromatographic-mass-spectrometric procedure.

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

Bacterial Proteins↗