Biomedical subjects
Publications and source records attributed to M K Johnson.
The comparative inhibitory power of organophosphorus esters in vitro against hen brain acetylcholinesterase and neurotoxic esterase correlates with their comparative effects (death or delayed neuropathy) in vivo. Further comparisons of the in vitro effects seen with hen and human enzymes facilitates extrapolations to the human in vivo situation.
Several dimethyl phosphate behave anomalously in tests for delayed neurotoxicity. Doses given to hens caused high inhibition of brain neurotoxic esterase (NTE) but no ataxia. Less inhibition of NTE was seen in spinal cord than in brain. Di-isopropyl phosphorofluoridate caused equal inhibition of NTE in brain and cord. When dosing with dimethyl phosphates was repeated NTE inhibition in cord increased and pair-dosed birds became ataxic. In vitro brain and cord NTE were indistinguishable but the in vivo discrepancy between inhibition of brain and cord NTE was matched by a similar discrepancy in inhibition of AChE. It appears that ataxia arises from inhibition of spinal cord NTE and that only in the present cases (among about 200) was the effect in brain not a perfect biochemical monitor.
Progressive neuropathy developed in a man during 2--8 weeks after acute poisoning by a pesticide said to contain trichlorphon. The neuropathy was typical of that caused by organophosphorus esters in the delay and in the maintenance of normal conduction velocity in surviving nerve fibres. A sample alleged to be typical of the ingested material was not more active against hen brain neurotoxic esterase (NTE) than was pure trichlorphon. Delayed neuropathy has never been produced in hens by a single dose of trichlorphon. This incident and studies of human brain in vitro suggest that the ratio neurotoxicity/lethality for trichlorphon is higher in man than in the hen. Suggestion is made of laboratory tests to improve neurotoxicity screening.
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Cultures of Pseudomonas aeruginosa considered to be of proven virulence were found to have higher titers of extracellular hemolysin than cultures of lesser virulence. Intracorneal injection of purified hemolysin produced extensive corneal opacification with extensive leukocytic infiltration of the tissue. It is suggested that the hemolysin plays a role in the pathogenesis of P. aeruginosa infections by effecting lysis of host cells and/or subcellular organelles, leading to the release of enzymes destructive to corneal tissue.
The assay of neurotoxic esterase (NTE) in brains taken from dosed hens enables potential neurotoxicity of organophosphate pesticides, plasticers, etc. to be assessed. The original assay [Johnson, M.K. Biochem. J. 114, 711-717 (1969)] has been simplified to eliminate centrifugation and transfer steps and both the selectivity and the sensitivity have been increased. The procedures necessary to obtain stable reagent stocks are described.
The sensitivities of the binding step and the lytic step of haemolysis by pneumolysin to the action of various inhibitors and to variations in the assay conditions were studied. Binding was inhibited by HgCl2 and N-ethylmaleimide. Lysis by previously fixed lysin was insensitive to HgCl2 and only slightly sensitive to N-ethylmaleimide. Binding of pneumolysin was independent of ionic strength. Binding of pneumolysin and streptolysin O decreased above pH 8-0 and 8-4 respectively. These results suggest that binding requires a non-ionized unsubstituted sulphydryl group. Incubation of erythrocytes with NaF caused inhibition of pneumolysin, indicating that some metabolic function of the cell may be involved in lysis. The action of streptolysin O was not affected by NaF.
Delayed neurotoxic effects arise as a result of two distinct biochemical events: 1) inhibiton of neurotoxic esterase, and 2) 'aging' of inhibited enzyme. Protective agents undergo reaction 1 but cannot undergo 2. Reaction 2 may interfere with a normal physiological control mechanism.
Evidence is reviewed that the initial biochemical event leading to delayed neurotoxicity is phosphorylation of the active site of a specific enzyme called Neurotoxic Esterase. This is followed by a bondcleavage (? hydrolytic) leading to formation of a mono-substituted phosphoric acid residue on the protein. The mechanism by which some phosphinates protect hens against neurotoxic compounds is explained. Screening Assay. Assay of effects of compounds on Neurotoxic Esterase activity of hen brain in vitro and in vivo provides a quick biochemical screen to supplement the 3-week clinical test. This test provides an estimate of safety margin for compounds which give negative results in the clinical test and are currently used as pesticides, plasticisers, etc. Simplified assay procedures are being developed. Structure/Activity Studies. Data is now available for the biochemical and neurotoxic activity of many compounds. This provides a basis for structure/activity predictions; neurotoxicity data published since 1930 has been assessed in this light.
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A highly purified preparation of pneumococcal cytolysin was responsible for the corneal damage that ensued upon intra-corneal injection of crude cell extract into the rabbit eye. The lysin may produce this pathologic finding by effecting activation or release of corneal degradative enzymes.
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A methionyl-specific dipeptidase from Streptococcus pneumoniae has been described. This enzyme and the pneumococcal tripeptidase have been shown to be intracellular, soluble, and constitutive. In addition to their function in cleavage of peptide nutrients, these peptidases may play a role in protein synthesis and turnover.
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