Biomedical subjects
Publications and source records attributed to M J Green.
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The market for decentralized clinical testing is undergoing expansion. Electrochemical biosensors represent one approach to the different demands of this market. A range of sensing systems are described which use electrochemical techniques for the measurement of various analytes and which have been demonstrated to be applicable to the manufacturing methods required for single-use disposable tests.
The six histidines of the channel-forming protein aerolysin have been replaced one at a time with asparagine by site-directed mutagenesis, and each of the modified proteins has been purified. Three proteins had the same hemolytic activity as native toxin, but the others, those changed at His107, His132, or His332, were less able to disrupt both human and rat erythrocytes. The largest reduction in activity, more than 100-fold, was observed with the His132 mutant protein. Studies with radioiodinated samples showed that it had approximately the same affinity as native aerolysin for the rat erythrocyte receptor. However, once bound to either rat or human erythrocytes, it was much less able to carry out the next essential step in hole formation, aggregation to form a stable oligomer. Aggregation was also reduced by replacing His107, but the contrast with native aerolysin and the effect on hemolytic activity were less pronounced. The protein modified at His332 behaved in a different way from those substituted at positions 107 and 132. Its affinity for the rat erythrocyte receptor was considerably lower than the affinity of the wild-type protein, but when bound it appeared to aggregate normally. The results suggest that His132 and perhaps His107 are involved in the aggregation of aerolysin whereas His332 may be at or near the receptor binding site.
A method is proposed for the determination of paracetamol in whole undiluted blood, based on the enzymatic hydrolysis of the drug to p-aminophenol, which is then measured by chronoamperometry at a glassy carbon electrode. Hydrolysis of the paracetamol prior to electro-oxidation is shown to alleviate problems that arise from high background currents in the whole blood and so ensures a good linear correlation (r greater than 0.99) between the current and the paracetamol concentration. Recovery experiments and comparison with a reference method based on spectrophotometry suggest that the electrochemical assay only measures that proportion of paracetamol that is not bound to serum albumin.
The Polycomb (Pc) gene is required from the extended germ band stage onwards, to maintain spatially restricted patterns of homeotic gene expression. It has been thought to be involved in the 'stable inheritance of the determined state'. In this paper, we have tested the notion that the Pc gene is required specifically during or after DNA replication to enable the stable transmission of states of gene activity. We found that arresting cell division using the string mutation or blocking DNA replication with aphidicolin failed to prevent ectopic expression of the homeotic gene Ultrabithorax in Pc mutants. Thus, even in the absence of DNA replication, Pc is required to maintain spatially restricted patterns of homeotic gene expression. The role of the Pc gene product in the stable repression of homeotic gene transcription is discussed.
The promoterless structural genes for aerolysin and the extracellular phospholipase of Aeromonas hydrophila were inserted into a multi-host-range expression vector and transferred into Aeromonas salmonicida and Escherichia coli. In both species, gene expression was under the control of the inducible tac promoter of the vector. Neither the phospholipase nor the aerolysin was released by intact E. coli. Instead, both proteins accumulated in the periplasm, leading to reduced growth and eventual cell death. When the aerolysin gene inserted into the vector contained its own promoter, the toxin was expressed constitutively by A. salmonicida but not by E. coli. Production of aerolysin and the phospholipase by A. salmonicida did not affect cell growth, and the proteins were correctly processed and exported by intact cells. Both proteins could also be detected in the periplasm, where their concentrations were considerably higher then they were outside the cells. Periplasmic aerolysin was rapidly released when cells were transferred to fresh medium, indicating that this compartment is part of the normal export pathway and that the protein is not shunted there as a consequence of overproduction. Plasmid-coded aerolysin did not appear to compete with the cell proteins for export components, as even when very large quantities of aerolysin were being exported by A. salmonicida, there was no effect on chromosomal protease release and only a modest reduction in the export of chromosomal phospholipase.
Immunoassays are routinely used to detect, specifically, low levels of many antigens. The trend away from the use of radioisotopic labels has resulted in a proliferation of alternative labels, many of which have electrochemical activity. The more successful of these assays have used enzyme labels, coupled with amperometric or potentiometric methods of detection of the products. A number of assays have also been designed which are specifically electrochemical in origin, not simply adaptations of currently used spectrophotometric methods. Much effort has been expended in developing a potentiometric immunoassay that measures the change in potential that should occur when an antibody binds to its antigen. The use of electroactive labels has resulted in a number of assays for drugs. The advantages of an enzyme-linked mediated assay for lidocaine, an antiarrhythmic drug, are discussed.
The respiratory burst of neutrophils was measured as a function of the ratio of the opsonised beads to neutrophils. The rate of oxygen uptake was found to be linear up to a bead:neutrophil ratio of 25. The production of the superoxide anion, as measured by the rate of reduction of cytochrome c, was negligible until a certain 'critical' value of the bead:neutrophil ratio was reached. The rate of oxygen consumption and superoxide release above the critical value varies linearly with the bead:neutrophil ratio. Both the rate of oxygen consumption and of superoxide release vary with the square of the radius of the ingested particle. It is suggested that this depends on the surface area of neutrophil membrane, activated by contact with the antagonist.
Uncertainty concerning the use, efficacy and possible adverse effects of clostridial vaccination in goats prompted a study of the injection site reactions and antibody responses in 40 goats and 40 sheep. The vaccines used were Covexin 8, Heptavac and Tasvax 8. In all the animals swellings averaging 2.5 cm in diameter were present at the injection site seven days after vaccination and were still apparent 28 days after vaccination. The injection site reactions could not be attributed to faulty vaccination technique because they did not occur in a control group injected with sterile water. By 14 days the reactions were significantly larger in sheep than in goats and by 28 days the reactions to Covexin 8 were larger than those to the other vaccines in sheep and goats. Serum antibody was present in all groups before vaccination and, with the exception of the goats vaccinated with Heptavac, increased 14 days after vaccination. The increase was greater in sheep than in goats. By 28 days antibody levels had declined in all but the sheep vaccinated with Heptavac in which a further increase occurred. At that time, the antibody levels in vaccinated sheep were still higher than in the unvaccinated sheep whereas the antibody levels in vaccinated goats were no longer different from those in the control goats. These results suggest that there is a difference between the vaccines used and between the responses of the two species and support the clinical observation that the protection afforded to goats by multivalent clostridial vaccines is poorer than that afforded to sheep.
The gene for the hole-forming toxin aerolysin from Aeromonas hydrophila was sequenced. Although most of the sequence seems unrelated to that of Staphylococcus aureus alpha-toxin, both proteins are very hydrophilic, and they each contain a nearly identical string of 10 amino acids.
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Veratridine inhibited angiotensin binding to its receptors in bovine adrenal glomerulosa cells and vascular smooth muscle. Fifty percent inhibition of adrenal receptors required about 2 X 10(-5) M veratridine. Receptors from vascular tissue were less sensitive. Graphic analysis showed that inhibition resulted primarily from a reduction of receptor number. Angiotensin stimulation of phosphatidylinositol turnover and of aldosterone production was inhibited by veratridine. Analogues of veratridine varied in their receptor inhibitory potency, but these variations did not correlate with the potencies of analogues as hypotensive agents or inhibitors of aldosteronogenesis. Very low extracellular sodium concentrations inhibited the adrenal effects of angiotensin. Neither veratridine nor grayanotoxin, both of which open sodium channels in excitable tissues, had a detectable effect on sodium fluxes in adrenal cells. Inhibition of aldosteronogenesis by veratridine is more likely the result of receptor and post-receptor effects than an alteration of the sodium channel.
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A pyrolytic graphite electrode was surface modified with human IgG and used as a stimulus to elicit a respiratory burst from human neutrophils. The oxidation current observed was shown to be due to re-oxidation of superoxide produced by the neutrophils. Both superoxide dismutase and N-ethylmaleimide were effective inhibitors of the oxidation current.
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