Biomedical subjects
Publications and source records attributed to E Heilbronn.
The possibility to use organs, organelle preparations and biologically active chemicals in toxicity tests and in toxicology will be reviewed. Examples are perfused liver preparations, tissue slices and homogenates, isolated nerve preparations, nerve-muscle preparations, membrane preparations, microsomes, mitochondria, synaptosomes, antibodies and isolated chemical compounds (receptors, enzymes).
A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na+ + K+)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C12E8). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K+-phosphatase or (Na+ + K+)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the alpha- and beta-subunits. The (Na+ + K+)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis alpha-toxin-Sepharose columns. The data suggest that the alpha-subunit of (Na+ + K+)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.
Release of [3H]-ACh from [3H]-Ch loaded nerve endings from rat hippocampus is dependent on Ca2+ and K+-concentration. [3H]-ACh release, evoked by a depolarizing K+ concentration is decreased in the presence of IgG isolated from the serum of some myasthenic patients but not in that of IgG from controls. Myasthenic IgG contains antibodies to the nicotinic acetylcholine receptor (nAChR). Isolated nerve endings were shown not to be leaky. Curare-sensitive alpha-bgt receptor was found at a concentration of 60-90 fmol/mg protein. It is discussed if a presynaptic nAChR exists in the membrane of hippocampal nerve endings and is involved in an autoregulation of ACh-release or if myasthenic IgG in a manner independent of the receptor.
Beta-Neurotoxins from certain snake venoms are highly specific toxins acting at the presynaptic side of the neuromuscular junction. In this study biochemical aspects of this high specificity have been investigated. When toxins (notexin and Naja nigricollis basic phospholipase) act on a mixture of subcellular fractions obtained from brain cortex (synaptosomes, myelin, and mitochondria), the synaptosomal fraction is preferentially attacked and shows the highest release of membrane protein. As seen from isolated fractions, however, even the mitochondria are rapidly and strongly attached. Examining the phospholipase A2 activity of the toxin instead of the release of proteins reveals that synaptosomes represent the best substrate. In contrast to nonneurotoxic phospholipases A2, that from neurotoxin preferentially uses synaptosomal phosphatidylcholine as a substrate when pure phospholipids isolated from subcellular fractions are used. A relationship between the cholesterol/phospholipid ratio and the sensitivity to toxin action in the various subcellular fractions was found. These data suggest that the neurotoxic effect is mainly due to the substrate specificity of the beta-neurotoxins. It is suggested that synaptosomal phosphatidylcholine, embedded in a membrane containing a low amount of cholesterol, is a highly specific substrate for beta-neurotoxins.
A summary is given of the present state of the use of tissue cultures in toxicology, as it emerged from the workshop. The need for better education in in vitro toxicology and for extensive comparison of results with those from in vivo studies is pointed out. Some of the important difficulties in the application of tissue cultures to toxicological problems are highlighted, as are the needs for basic research on tissue cultures and those for better communication between scientists, legislators, money sources and laymen.
Using the mouse passive transfer model, the effects of purified human myasthenic immunoglobulin G and of the monovalent Fab fragment on neuromuscular junctions were investigated. Treatment with IgG markedly reduced amplitudes of miniature endplate potentials. When Fab fragments were transferred alone or with subsequent addition of IgG no neuromuscular transmission block was induced. Myasthentic IgG and Fab were specifically demonstrated at the neuromuscular junctions by immunohistochemistry. On electronmicroscopy endplate structure was normal in transfer experiments using IgG for up to 30 days. It is suggested that Fab fragments bind to acetylcholine receptors without affecting transmission and protect them from the attack of complete IgG antibodies.
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Rabbits were immunized with cholinergic synaptic vesicles isolated from the electric organ of Torpedo marmorata. The resultant antiserum had one major antibody activity against an antigen called the Torpedo vesicle antigen. This antigen could not be demonstrated in muscle, liver or blood and is therefore, suggested to be nervous-tissue specific. The vesicle antigen was quantified in various parts of the nervous system and in subcellular fractions of the electric organ of Torpedo marmorata and was found to be highly enriched in synaptic vesicle membranes. The antigen bound to concanavalin A, thereby demonstrating the presence of a carbohydrate moiety. By means of charge-shift electrophoresis, amphiphilicity was demonstrated, indicating that the Torpedo vesicle antigen is an intrinsic membrane protein. The antigen was immunochemically unrelated to other brain specific proteins such as 14-3-2, S-100, the glial fibrillary acidic protein and synaptin. Furthermore, it was unrelated to two other membrane proteins, the nicotinic acetylcholine receptor and acetylcholinesterase, present in Torpedo electric organ. The antiserum against Torpedo synaptic vesicles did not react with preparations of rat brain synaptic vesicles or ox adrenal medullary chromaffin granules.
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The lipids from the electric organ of the ray, Torpedo marmorata, have been isolated and characterized. The major lipids were cholesterol, choline phospholipids, ethanolamine phospholipids, and sphingomyelins. The major fatty acids of ethanolamine phospholipids were 18:1, 18:0, 22:6, and 20:4. More than 50% of the acids in choline phospholipids were 16:0. The sphingomyelins consisted of five major ceramide species, all with sphingosine and the fatty acids 14:0, 15:0, 16:0, 22:1, and 24:1. The fatty acid 15:0 was mostly branched (n-2), a fatty acid earlier identified in sphingomyelins of the rectal gland of spiny dogfish. All long-chain bases were dihydroxy bases with a small percentage of branched chains. Sulfatides (cerebroside sulfate) made up the largest glycolipid fraction. The polar moiety wase galactose-3-sulfate. The fatty acids were normal and 2-hydroxy; the homologue 24:1 was the most abundant in both types of fatty acids. Most fatty acids were higher homologues of mono-unsaturated acids, but normal 18:0 fatty acid was also found. The long-chain bases were both dihydroxy and trihydroxy, with very small amounts of branched chains. The two major ceramide species of sulfatides were sphingosine combined with normal and hydroxy 24:1 fatty acids, respectively. Smaller amounts of trihydroxy base (18:0) were found linked to hydroxy 24:1 fatty acid, but not to its normal homologue. The cerebrosides contained the two major species mentioned above but lacked the trihydroxy base-hydroxy fatty acid species. The ratio of the activity of Na+-K+-dependent ATPase (EC 3.6.1.3) and the concentration of sulfatides was similar to ratios found for other tissues with normal and increased Na+ and K+ transporting capacity. The significance of this finding is discussed.
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Two-phase systems consisting of water, dextran and poly(ethylene glycol) have been used for partition of membranes obtained from Torpedo marmorata electric organ. The partition behaviour of the membranes could be adjusted by using a polymer with covalently-bound charged groups. By using this method, the membranes were divided into several fractions which were analyzed for nicotinic acetylcholine receptor and acetylcholinesterase content. It was found that nicotinic receptor-enriched membranes were separated from those containing esterase in a single partition step. Receptor-enriched membranes obtained by gradient centrifugation could be further separated into two receptor fractions by the two-phase technique. The results also reveal at least two types of acetylcholinesterase-rich membranes.
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We have investigated a chloroform--methanol extracted material from electric organ of Torpedo marmorata with respect to binding of [3H]-acetyl-alpha-neurotoxin and [3H]-acetylcholine. No binding was observed which is in contrast to observations with detergent extracted receptor. Further, no crossreactivity could be demonstrated between the chloroform--methanol extracted proteolipid and antibodies against the detergent extracted receptor. Labelling in the membrane with [3H]-acetyl-alpha-neurotoxin before extraction with chloroform--methanol gave a very low yield of extracted labelled compound. The protein yield is about 0.3% of the total in the homogenate and only 0.04% of the specifically bound neurotoxin. Using the alkylating affinity label [3H]-N-propylbenzilylcholine mustard (1) and also [3H]-atropine, which both bind, at low concentrations, specifically to the muscarinic acetylcholine receptor, binding was studied to the chloroform--methanol extracted proteolipid from rat brain cortex. No specific binding could, however, be demonstrated. Prelabelling in the membrane with 1 and subsequent extraction with chloroform--methanol gave a protein yield of about 1% and an extraction of 26% of the specifically bound label.
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