Biomedical subjects
A J Lawrence
Publications and source records attributed to A J Lawrence.
Signal response transduction in rabbit neutrophil leucocytes. The effects of exogenous phospholipase A2 suggest two pathways exist.
Rabbit neutrophils stimulated by chemotactic peptide (fMLP) or phorbol ester (PMA) respond with a metabolic burst which can be assayed by following luminol-enhanced chemiluminescence. Depending upon the agonist used, exogenous bee-venom phospholipase A2 (PLA2) will enhance or inhibit the response. Neutrophil activation by fMLP is enhanced by PLA2 or by the addition of arachidonic acid, but unaffected by lysophosphatide. The cellular response to PMA is markedly inhibited by PLA2 or by lysophosphatide, though not completely abrogated, but is enhanced by arachidonic acid. The lysophosphatide inhibition overrides the arachidonic acid potentiation of the PMA-induced response. Neither PLA2 nor arachidonic acid alone will activate the cells; it seems that agonist is essential. We interpret these results to mean that at least two signal-response transduction systems are involved in agonist-induced metabolic activation of rabbit neutrophil leucocytes.
Efficacy of enzyme-linked immunosorbent assay for rapid diagnosis of Bordetella pertussis infection.
We examined the diagnostic efficacy of an enzyme-linked immunosorbent assay (ELISA) for class-specific antibodies to Bordetella pertussis in acute-phase sera collected from 1,240 patients with suspected pertussis. A total of 833 serum specimens (67%) yielded positive results. The proportion of positive results increased to 77% if a second (convalescent-phase) serum was also tested. By comparison, a bacterial agglutination test for B. pertussis antibodies was positive in only 21% of acute-phase specimens and 50% of paired specimens. The high proportion of acute-phase sera which were ELISA positive indicates that a measurable serologic response has usually occurred by the time the diagnosis is suspected. Thus, the ELISA is potentially the most rapid means of laboratory confirmation of B. pertussis infection.
Activation of bee venom phospholipase A2 by oleoyl imidazolide produces a thiol- and proteinase-resistant conformation.
Assay methods for bee venom phospholipase A2 are presented which respond to different aspects of enzymic behaviour and which allow basal activity, fatty acid activation and acyl-group activation to be distinguished. The stability of the enzyme to thiols and proteinases is dramatically increased by activation with the selective acylating agent, oleoyl imidazolide. These results support the model of activation by conformation change. Limited-fixation studies indicate that enzyme conformation is determined by interaction with the substrate. The oleoyl-enzyme is partially inactivated by trypsin, but its electrophoretic mobility is unchanged. This protective effect is highly selective and only one other component of the venom is protected against trypsin by oleoyl imidazolide. Combination of trypsin and thiol treatment produces a large fragment of the activated enzyme which could be used for structural studies of the activation site.
The purification and characterisation of hyaluronidase from the venom of the honey bee, Apis mellifera.
Hyaluronidase has been purified from the venom of the honey bee, Apis mellifera. The purification proved remarkably difficult, requiring a large number of chromatographic steps culminating in the removal of traces of phospholipase A2 with an affinity purified rabbit anti-phospholipase A2 immunosorbent column. The purified enzyme showed a 1143-fold increase in specific activity and was homogeneous. Electrophoresis in polyacrylamide gels (12%) containing sodium dodecyl sulphate (pH 8.9) or urea (pH 2.8) and electrofocusing in polyacrylamide (5%) gave a single band. The final product contained less than 0.1% phospholipase A2 and less than 1.5% acid phosphatase and gave a single line of precipitation against rabbit anti-hyaluronidase but was not precipitated by rabbit anti-phospholipase A2. Previous reports of instability were not confirmed, and we found the enzyme to be highly stable over a wide range of temperature and pH, and to denaturing agents. Purified hyaluronidase was found to be 'sticky' when highly pure and at low concentration, and adhered strongly to Sephadex G-75. The relative molecular mass was estimated at 35 000-37 000 by gel filtration, and at 41 000 by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. A value of 50 000 was obtained by ultracentrifugation assuming a partial specific volume of 0.73 cm3/g. Hyaluronidase was found to be a minor allergen in bee venom allergic patients.
Quantities of Clostridium botulinum organisms and toxin in feces and presence of Clostridium botulinum toxin in the serum of an infant with botulism.
A 7-week-old boy presented with symptoms and signs characteristic of infant botulism, and the diagnosis was confirmed by the detection of Clostridium botulinum type A organisms and toxin in the feces. The levels of organisms and toxin in the feces were measured throughout the 81-day period in hospital. The maximum levels detected were 2.46 x 10(8) C. botulinum type A colony-forming units and 64,000 mouse 100% lethal doses of type A toxin per g (wet weight) of feces. C. botulinum toxin was also detected in two samples of the patient's serum, collected 3 and 10 days after admission. Improvement in the patient's clinical condition occurred before the levels of organisms and toxin in the feces reached their maxima. A slight improvement may also have occurred while toxin was still present in the serum.
Quantitation of Clostridium botulinum organisms and toxin in the feces of an infant with botulism.
A 4-month-old boy presented with symptoms and signs characteristic of infant botulism. Examination of feces revealed Clostridium botulinum type B and type B toxin. The numbers of C. botulinum and the amount of toxin in feces were measured throughout the 4-week period in hospital. The maximum numbers and amounts were detected in a fecal specimen collected 16 days after admission: this contained 8.4 X 10(6) C. botulinum type B colony-forming units and 61,440 mouse 100% lethal doses of type B toxin per g (wet weight) of feces. This latter figure is the highest fecal toxin titer reported yet for a case of infant botulism. By day 16, however, substantial improvement in the patient's clinical condition had occurred. This suggests that initiation of recovery from infant botulism is not necessarily preceded by a reduction in the numbers of C. botulinum organisms and the quantity of toxin in the gut.
Mechanisms for albumin-mediated membrane damage.
1. Bee venom phospholipase A2 causes a small increase in the sublytic leakiness of rabbit erythrocytes which is strongly inhibited by lysolecithin and potentiated by albumin. 2. Albumin extracts long-chain fatty acids from the erythrocyte membrane by a process which is faster than diffusion-determined efflux of fatty acid and during the process it greatly increases the sublytic leakiness of the membrane. In contrast, albumin rapidly terminates sublytic responses to lysolecithin. 3. These actions of albumin are abolished only when albumin is preloaded with four molecules of oleic acid per protein molecule. 4. A minor proportion of the response of phospholipase-A2-treated cells to albumin is due to accumulated fatty acid products; the combination of active enzyme and accumulated fatty acids is much more effective than either alone. Membrane partially depleted of phospholipids by enzyme attack is not highly leaky. 5. Albumin may, in part, act by removing lysolecithin, the inhibitory reaction product. A model is proposed in which susceptibility to phospholipase A2 attack is not uniform on the cell surface and is used to explain the inhibitory action of lysolecithin and the high sensitivity of the membrane in the presence of albumin to damage by newly released fatty acids.
Albumin mediates lysis of erythrocytes by bee venom phospholipase A2 activated with oleoyl imidazolide.
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Activation of bee venom phospholipase A2 by oleoyl imidazolide.
Imidazolide derivatives of long-chain fatty acids are shown to be potent irreversible activators of bee venom phospholipase A2. Activation corresponds to the addition of a single acyl residue to the protein molecule. The rate of activation increases with the acyl chain length of the activator, but the highest activation factors are given by the oleoyl and linoleoyl residues. Acyl group activation of the enzyme substitutes completely for activation by free fatty acids (but produces 3--4-fold higher activities) indicating that fatty acids are allosteric activators of the enzyme. The degree of activation is calcium dependent and exceeds 100-fold at low calcium concentration. Activation is extremely sensitive to substrate structure, but modification of the substrate surface by intercalated activator does not form the basis of this specificity.
The preparation of activated bee venom phospholipase A2.
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Lysolecithin inhibits an action of bee venom phospholipase A2 in erythrocyte membrane.
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Activation of bee venom phospholipase A2 by fatty acids, aliphatic anhydrides and glutaraldehyde.
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A conductimetric study of erythrocyte lysis by tysolecithin and linoleic acid.
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Measurement of gastric acid secretion by conductivity.
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Conductimetry in enzyme studies.
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Conductimetric enzyme assays.
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