Biomedical subjects
Publications and source records attributed to C A Lewis.
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An organ culture system devised for studying the development of the secondary palate was modified so that it retained high partial pressures of oxygen and supported total anterior and posterior palatal elevation. The cultured tissues appeared healthy as judged by histological examination. Medium was continuously recirculated through the culture system in which Day 13 embryonic mouse heads, with the brain and tongue removed, were totally submerged and suspended. The medium was constantly gassed via hollow fiber devices. A motor-driven stirrer, run at a low rate, agitated the medium so that the boundary layer surrounding the tissue was dispersed. Embryonic mouse heads were cultured in each of four media: Eagle's basal medium, Ham's F-12 medium, Fitton-Jackson's modified BGJb medium, and Waymouth's MB 752/1 medium. Elevation of the palate in both anterior and posterior regions with excellent tissue viability was achieved in all heads grown in BGJb medium.
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1. The acetylcholine-sensitive ionic channels at the neuromuscular junction were studied in voltage-clamped single muscle fibres from a monolayer preparation of the cutaneous pectoris muscle from Rana pipiens. The experimental observations were of three types: (a) reversal potential as a function of external Na and Ca concentrations, (b) the single channel conductance (gamma) from noise analysis as a function of these same concentrations, and (c) gamma as a function of membrane potential. 2. The reversal potential in normal Na Ringer was -3.8 +/- 0.5 mV (+/- S.E. of mean, n = 22) and decreased approximately linearly as the logarithm of the outside Na activity as this activity decreased to 10% of normal. 3. The single channel conductance in normal Na Ringer was 27.5 +/- 0.7 pS (n = 28) and reached a limiting value close to 10 pS as Na was replaced with sucrose. 4. Increasing [Ca]o from 2 to 10 mM made the reversal potential more positive and decreased the single channel conductance. Mg caused similar effects. 5. Various theories that have been used to describe the mechanism of ion permeation through e.p.c. channels were tested. Constant field theory (eqns. (3), (4) and (5)), a modified Takeuchi approach (eqn. (6)), and a single barrier theory (eqns. (8), (9) and (10)) could not account for all of the experimental observations. 6. In particular, constant field theory, with no surface charge density, could account for the following: (a) the reversal potential measurements for solutions containing 2 mM-Ca (with PK/PNa = 1.2 and PCa/PNa = 1.02), (b) the single channel conductance values for solutions containing 2 mM-Ca and Na concentrations down to 20% of normal, (c) that gamma has little voltage dependence. 7. However, constant field theory, with no assumed surface charge density, could not account for the following: (a) the reversal potential observed for Ringer containing 80 mM-Ca, (b) the gamma values observed for very low Na concentrations, (c) the observation that increasing Ca from 2 to 10 mM in a solution containing 75% normal Na results in a decrease in gamma. 8. From the failure of the Takeuchi approach (eqn. (6)), it is argued that ion interactions must occur at e.p.c. channels because ion flux independence is the only asumption in the derivation of eqn. (6) without experimental verification. 9. The ion interactions at e.p.c. channels probably include both surface charge effects and competition for a binding site.
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Equilibrium constants for hydration of ketones, in dilute D2O solution at 34 degrees C, observed by proton magnetic resonance under conditions of slow exchange, were acetone 0.002, chloroacetone 0.08, 1,3-dichloroacetone 4.17, bromoacetone 0.07, and 1,3-dibromoacetone (an inhibitor of papain) 1.85. Neither acetamidoacetone nor N,N-diacetylaminoacetone showed evidence of appreciable hydration in dilute aqueous solution, nor was any hydrate detectable in solutions of tosylglycine chloromethyl ketone. Substitution of acetaldehyde with acylamido substituents, as in several potent reversible inhibitors of papain, was found to enhance its equilibrium constant for covalent hydration by an order of magnitude; these inhibitors are about 90% hydrated in dilute aqueous solution, and their affinity for proteases may have been underestimated accordingly. The effects of deuterium substitution at C-1 of acetaldehyde, on equilibrium addition of oxygen and sulfur nucleophiles, are substantial and vary with the nature of the nucleophile. These isotope effects may be useful as a mean of distinguishing between alternative structures of complexes formed between enzymes and aldehydes.
Papain is strongly inhibited by aldehydes resembling carboxylic acids, released by hydrolysis of specific substrates (Westerik, J. O'C., and Wolfenden, R. (1972), J. Biol. Chem. 247, 8195-8197). Inhibitory complexes might involve binding of the aldehyde intact or as a covalent hydrate, or the aldehyde might undergo covalent addition of an active site sulfhydryl group to form a thiohemiacetal derivative. In an attempt to distinguish between these possibilities, benzamidoacetaldehyde-1-d has been synthesized, and its properties compared with those of the undeuterated inhibitor. After correction for differences in hydration, the observed effect on inhibition is found to be compatible with formation of a thiohemiacetal. In keeping with this conclusion, benzamidoethanol (a partial analogue of the covalent hydrate) and benzamide, N-methylbenzamide and N-ethylbenzamide (somewhat similar to the free aldehyde in size and hydrophobic character) are found to exhibit negligible affinity for the active site.
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The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD(+) and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and alpha-oxobutyrate were weak inhibitors, a number of other alpha-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.
Low concentrations of acetaldehyde, similar to those that can occur in the brain of ethanol-treated animals, effectively inhibit the pyruvate dehydrogenase complex purified from ox brain or from ox kidney, although the precise mechanism of this inhibition remains to be defined.