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D Perrett

Publications and source records attributed to D Perrett.

At least 73 records · Page 4Linked to original sources
PubMed1992

Sulphydryl reactivity of the HLA-B27 epitope: accessibility of the free cysteine studied by flow cytometry.

HLA-B27 has an unpaired cysteine on or near its serologically defined spondylitis associated epitope, and it has been argued that its sulphydryl side chain may be chemically reactive. In a previous study it was shown that chemical treatment of HLA-B27 cells with the sulphydryl binding agent p-chloromercuriphenylsulphonic acid (pCMPSA) specifically reduced binding of antibodies to HLA-B27 by up to 80%, as measured in a cellular enzyme linked immunosorbent assay (CELISA). The effect of sulphydryl blockade on intact B27 cells was investigated using flow cytometry. Compared with the CELISA, inhibition required higher concentrations of pCMPSA, and the degree of inhibition produced by a greater than or equal to 30 microM solution of pCMPSA as measured by flow cytometry (median 28.9%) was significantly lower than that measured by CELISA (median 73.6%; p = 1.6 x 10(-6)). Analysis of unfixed, cell surface HLA-B27 by flow cytometry suggests that on most B27 molecules the unpaired sulphydryl site is not available. On the basis of this evidence for modification after translation, a new 'altered self' hypothesis is proposed for the part which HLA-B27 plays in inflammatory disease.

4-Chloromercuribenzenesulfonate↗
PubMed1991

Comparative performance of ion exchange and ion-paired reversed phase high performance liquid chromatography for the determination of nucleotides in biological samples.

We have compared anion exchange chromatography on APS-Hypersil (4.6 x 100 mm) eluted with a phosphate gradient with reversed phase chromatography on ODS-Hypersil (4.6 x 100 mm) in the presence of either tetrabutylammonium (TBA) or triethylammonium (TEA) ions with a methanol gradient. The systems have been compared both for ease of operation and for their resolving power with standard mixtures and acid extracts of both normal red cells (RBC) and ischaemic tissues. The two chromatographic modes exhibited similar separating efficiencies for standard mixtures of nucleotides but retention times were most stable using reversed phase liquid chromatography (RPLC) with TEA. Anion exchange columns slowly lost ion exchange capacity but selectivity was unchanged. RPLC in the presence of TBA gave reproducibile capacity factors only when operated isocratically due to irreversible changes to the silica surface. For RBCs the RPLC with TEA and anion exchange systems resolved 17 and 15 peaks, respectively, and for the ischaemic samples 22 and 14 peaks, respectively. However, nucleosides and bases were also resolved by the ODS column causing chromatographic crowding and uncertain peak identification.

Animals↗
PubMed1990

Rapid assay for hard tissue collagen cross-links using isocratic ion-pair reversed-phase liquid chromatography.

Following a detailed study, a rapid and sensitive assay for the naturally fluorescent collagen cross-links pyridinoline and deoxypyridinoline has been developed using ion-pair reversed-phase high-performance liquid chromatography in the presence of 1-octanesulphonic acid (OSA). Pyridinoline and deoxypyridinoline were separated on an Exsil 100 ODS, 5-microns column (100 mm X 4.6 mm I.D.) using 25 mM sodium formate, 5 mM OSA and 1 mM ethylenediaminetetraacetic acid adjusted to pH 3.25, containing 20% (v/v) methanol. The mobile phase flow-rate was 1.5 ml/min. Compounds were detected by their natural fluorescence (xenon lamp; excitation wavelength 290 nm, emission wavelength 400 nm). Peak areas were linear to 25 pmol injected for pyridinoline and 20 pmol injected for deoxypyridinoline (r = 0.99). Intra-assay coefficients of variation for urinary extracts were 7.65 and 9.07% (n = 10), respectively. Limit of detection (signal-to-noise ratio = 5) was 200 fmol injected. Quantification of the cross-links in acid hydrolysates and human urine samples was possible in under 15 min.

Alkanesulfonates↗
PubMed1990

The high performance liquid chromatography of enzyme systems relating to purine and pyrimidine metabolism: an overview.

Purines and pyrimidines are now routinely separated by HPLC. By careful selection of chromatographic conditions which match the expected changes in hydrophobicity and/or ionic nature of the substrate and products most enzymes of the purine and pyrimidine salvage pathways can be routinely and accurately determined. Ion-paired reversed-phase systems are often the most advantageous. The relevance of such assays to biomedical analysis including their role in the diagnosis of inborn errors of metabolism is stressed.

Chromatography, High Pressure Liquid↗
PubMed1989

Determination of serum cytidine deaminase activity using ion-pair reversed-phase liquid chromatography.

A rapid and sensitive assay for serum cytidine deaminase has been developed utilising ion-pair reversed-phase high-performance liquid chromatography. The addition of 1-octanesulphonic acid (OSA) caused the retention of cytidine and uridine to reverse and uridine, the minor component in the assay, to elute first. Cytidine, uridine and allopurinol (internal standard) were separated on a 5-micron Hypersil ODS column using 100 mM ammonium acetate with 1% (v/v) methanol and 1 mM OSA adjusted to pH 5.0. Detection was at 262 nm. Peak areas were linear from 7 pmol to 6 nmol injected (r = 0.99). Intra-assay variation was 7.8% (n = 10) and the correlation with a colorimetric assay was r = 0.78 (p less than 0.001).

Arthritis, Rheumatoid↗
PubMed1989

Fragmented fibronectin and other synovial fluid proteins in chronic arthritis: their relation to immune complexes.

Fibronectin, an opsonic glycoprotein has been shown to exist in fragmented forms in serum and synovial fluid. Some fragments in synovial fluid appear to be polyethylene glycol (PEG) precipitable, suggesting incorporation into immune complexes (IC). PEG precipitation, SDS-PAGE and immunoblotting were used to determine whether PEG precipitable fragments are real or artefactual. Disease specificity of fragmentation and IC incorporation of fibronectin and other proteins were also studied using these techniques. PEG precipitable fragments do not appear to be artefactual, although some fibronectin fragments are cryoprecipitable. Protein fragments showed similar distributions in whole serum and synovial fluid, disease specific differences being confined to PEG precipitates. Rheumatoid arthritis (RA) synovial fluid PEG precipitates displayed the greatest array of fragmented immunoglobulins and fibronectin. No PEG precipitates contained albumin fragments. Protein fragments in IC may impair their effective removal from RA joints. Accumulated IC could lead to tissue damage via complement activation.

Adult↗
PubMed1989

Determination of cytidine deaminase activity in synovial fluid by HPLC.

A reversed-phase high-performance liquid chromatographic method for the determination of cytidine deaminase activity in synovial fluid is described. Diluted synovial fluid was incubated for 10 min at 56 degrees C with 0.4 mM cytidine. The protein in 5 vol of incubate was then precipitated using 1 vol of trichloroacetic acid (20% m/v) and the substrate, cytidine, and the product, uridine, were determined in the resultant supernatant. These substances were separated by reversed-phase HPLC using 0.05 M potassium dihydrogen orthophosphate (pH 6.5) containing methanol (3% v/v) and were detected at 280 nm. The enzyme activity was determined by measuring uridine formation. The effects of substrate concentration, pH and reaction temperature on uridine formation are described.

Ammonia↗
PubMed1989

Effect of sustained exercise on plasma amino acid concentrations and on 5-hydroxytryptamine metabolism in six different brain regions in the rat.

Sustained exercise to fatigue elicits no major differences either in plasma amino acid levels or in brain 5-hydroxytryptamine (5-HT) metabolism between sedentary and endurance-trained animals. Furthermore, 11 weeks of endurance training did not influence the maximal activity of the enzyme monoamine oxidase in the brain areas which were studied. In both sedentary and endurance-trained rats, sustained running to fatigue caused an increase in the plasma concentration ratio of free tryptophan/other large neutral amino acids and an increase in the concentration of tryptophan in the six brain areas that were studied. The increase was similar in the different regions of the brain and averaged 36%. Exercise caused an increase in the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) in the brain stem (14 and 44% respectively) and hypothalamus (16 and 17% respectively) and an increase in the level of 5-HIAA in the hippocampus (21%) and striatum (28%). Exercise also caused an increase in the level of dopamine in the brain stem (56%) and hypothalamus (46%) and of nor adrenaline in the striatum (59%). Since the levels of 5-HT and dopamine were both increased in the brain stem and hypothalamus, it is possible that these changes may play important roles in the central effects of exercise, including both physical and mental fatigue and effects on mood.

Amino Acids, Branched-Chain↗
PubMed1989

Development of a new radiographic scoring system using digital image analysis.

A scoring system using computerised analysis of digital stored images of knee radiographs has been developed. Measurement is based on the assessment of joint space size. It is sensitive, rapid, and reproducible. Plain radiographs are positioned on an acetate grid; a computer generated grid is superimposed on a digital image of the radiograph viewed on a closed circuit television monitor and the joint space measured automatically. Area and distance have been assessed; area measurements are more reproducible. Application of microcomputer based digital image analysis to radiological scoring systems is an important step in understanding the nature and progression of arthritis.

Arthritis↗
PubMed1989

Status of an unpaired thiol group on the HLA-B27 epitope.

Sequence analysis and site-directed mutagenesis of HLA-B27 indicate an unpaired cysteine at position 67 of the hypervariable region corresponding to its serologically defined, disease-associated epitope. We investigated whether chemical modification of this thiol group affected the serological reactions of B27. B27-positive cells were treated with thiol-blocking agents and then tested for recognizable B27 expression. Anti-HLA-B27 alloantisera and monoclonal antibodies were used in cytotoxicity, absorption, and cellular ELISA (cELISA). The semi-quantitative cytotoxicity-based assays showed some decrease in both B27 and controls. However, cELISA indicated that the inhibition of B27 was significantly greater than control antigens, and dependent on thiol-blocker concentration. This suggests that a proportion of HLA-B27 molecules have a free, reactive thiol at the antibody-defined epitope. Incomplete inhibition by thiol-blocking agents indicates that the remainder are inaccessible.

Antibodies, Monoclonal↗