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Biomedical subjects

C A Lewis

Publications and source records attributed to C A Lewis.

At least 109 records · Page 6Linked to original sources
PubMed1985

Deuterium oxide and temperature effects on the properties of endplate channels at the frog neuromuscular junction.

The effects of deuterium oxide (D2O) and temperature on the properties of endplate channels were studied in voltage-clamped muscle fibers from the frog Rana pipiens. Studies were performed at temperatures of 8, 12, 16, and 20 degrees C. The single channel conductance (gamma) and mean channel lifetime (tau) were calculated from fluctuation analysis of the acetylcholine-induced end-plate currents. The reversal potential was determined by interpolation of the acetylcholine-induced current-voltage relation. The mean reversal potential was slightly more negative in D2O Ringer's (-7.9 +/- 0.1 mV [+/- SEM]) compared with H2O Ringer's (-5.2 +/- 0.6 mV, P less than 0.01). The single channel conductance was decreased in D2O. This decrease was greater than could be accounted for by the increased viscosity of D2O solutions, and the amount of the decrease was greater at higher temperatures. For example, gamma was 38.4 +/- 1.3 pS (+/- SEM) in H2O Ringer's and 25.7 +/- 1.0 pS in D2O Ringer's for a holding potential of -70 mV at 12 degrees C. The mean channel lifetime was significantly shorter in D2O, and the effect was greater at lower temperatures. There was not a strong effect of solvent on the temperature dependence of gamma. On the other hand, the temperature dependence of the reciprocal mean channel lifetime, alpha (where alpha = 1/tau), was strongly dependent upon the solvent. The single channel conductances showed no demonstrable voltage dependence over the range of -90 to -50 mV in both solvents. The reciprocal mean channel lifetime showed a voltage dependence, which could be described by the relation alpha = B exp(AV). The slope A was not strongly affected by either temperature or the solvent. On the other hand, the intercept B was a strong function of temperature and was weakly dependent upon the solvent, with most values greater in D2O. The D2O effects on alpha were what would be expected if they were due to the properties of D2O as a solvent (solvent isotope effects), while the D2O effects on gamma must also include the exchange of D for H in the vicinity of the selectivity filter (primary and/or secondary kinetic isotope effects).

Animals↗
PubMed1985

Regional distribution of pulmonary epithelial permeability in normal subjects and patients with asbestosis.

The overall and regional clearance of an inhaled isotope labelled solute from the lungs was examined on the basis of a 15 minute period of data collection, for which a technique was developed that does not require intravenous injection to correct for blood-tissue background activity. The technique was applied to 52 normal subjects (31 non-smokers and 21 smokers) and to 37 patients with asbestosis (21 non-smokers and 16 smokers). In normal smokers solute clearance was faster in the upper and middle zones, with a mean ratio of T1/2 LB (half time solute clearance from lungs to blood) in the upper two thirds to the lower one third of the lungs of 0.66 (0.28-1.33), compared with 1.24 (0.43-2.77) in normal non-smokers (p less than 0.002). In patients with asbestosis solute clearance was accelerated throughout the lungs even though radiographic abnormalities were limited to lower or lower to middle zones. Regional distribution of clearance was not affected by posture in normal subjects. Overall solute clearance was significantly faster in normal current smokers and in patients with asbestosis than in normal non-smokers (p less than 0.001 respectively). Among patients with asbestosis, smokers had faster overall clearance than non-smokers (p less than 0.01). Among normal non-smokers T1/2 LB was not significantly different between those who had never smoked and ex-smokers. Regional abnormalities in pulmonary epithelial permeability may offer insight into the pathogenesis of interstitial lung diseases and smoking related disorders.

Adult↗
PubMed1984

Characterization of basal and methylxanthine-stimulated Ca2+ transport in abalone spermatozoa.

Methylxanthines, such as 1-methyl-3-isobutylxanthine (MIX) and theophylline, stimulate abalone sperm 45Ca2+ uptake in a time- and concentration-dependent manner. MIX is the most potent compound tested, and the ability of these compounds to alter 45Ca2+ uptake resides with methyl or isobutyl substitution of the xanthine nucleus at multiple sites. Methylxanthine-stimulated 45Ca2+ uptake does not occur as a secondary consequence of cyclic nucleotide phosphodiesterase inhibition, and added cyclic nucleotides are also without effect. The dramatic elevation of intracellular cAMP concentrations and induction of the acrosome reaction of sperm incubated with methylxanthines in the presence of Ca2+ is mediated by a primary effect of methylxanthines on Ca2+ transport. Basal 45Ca2+ uptake occurs through a verapamil-insensitive site that obeys the properties of a simple diffusion-mediated process. MIX-stimulated 45Ca2+ uptake occurs through a carrier-mediated transport site that has low affinity for Ca2+ (Km = 19.9 mM) and is verapamil sensitive. 45Ca2+ uptake through both basal and MIX-stimulated sites is enhanced by low extracellular Na+ concentrations (less than or equal to 15 mM) and is not affected by either extracellular Mg2+ or K+ X 45Ca2+ uptake through both sites is pH sensitive, but this sensitivity is different for each site. These data suggest that methylxanthines can affect sperm function via primary effects on Ca2+ transport, which occur through a specific carrier-mediated site(s). It is possible that many of the previously described effects of methylxanthines on sperm function are mediated via such changes in Ca2+ conductance.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Divalent cation effects on acetylcholine-activated channels at the frog neuromuscular junction.

The effects of the divalent cations Ca and Mg on the properties of ACh-activated channels at the frog neuromuscular junction were studied using a two-microelectrode voltage clamp. The divalent cation concentration was varied from 2 to 40 mM in solutions containing 50% normal Na. The reversal potential was determined by interpolation of the acetylcholine (ACh)-induced current versus voltage relationship. The single-channel conductance and the mean channel lifetime were calculated from fluctuation analysis of the ACh-induced end-plate current. Extracellular Na and/or divalent cations affected the reversal potential of endplate channels in a way that cannot be described by the Goldman-Hodgkin-Katz equation or by a simple two-barrier, one-binding site model of the channel if the assumption was made that permeability ratios were constant and not a function of ion concentrations. Increasing the divalent cation concentration decreased the single-channel conductance to approximately 10 pS in solutions with 50% Na and 40 mM divalent cation concentrations. The effect of the divalent cations Ca and Mg on the mean channel lifetime was complex and dependent on whether the divalent cation was Ca or Mg. The mean channel lifetime was not significantly changed in most solutions with increased Ca concentration, while it was slightly prolonged by increased Mg concentration.

Acetylcholine↗
PubMed1984

Seasonal changes in the properties of frog. End-plate channels.

The properties of the acetylcholine (ACh)-activated channel at the frog neuromuscular junction were studied using a two-microelectrode voltage clamp. The reversal potential was determined by interpolation of the ACh-induced current vs. voltage relation, while the single-channel conductance and the mean channel lifetime were calculated from fluctuation analysis of the mean ACh-induced end-plate current. Seasonal changes were observed in some of the measured parameters. While the reversal potential and the mean channel lifetime remained constant throughout the year, the single-channel conductance did not. The single-channel conductance was on the average 35% higher in the winter than in the summer. This effect could have survival value for hibernating frogs.

Acclimatization↗
PubMed1983

Correction for light absorption in fluorescence studies of protein-ligand interactions.

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.

Ligands↗
PubMed1983

Methylxanthines stimulate calcium transport and inhibit cyclic nucleotide phosphodiesterases in abalone sperm.

Experiments were designed to determine the mechanism by which methylxanthines elevate abalone sperm cAMP concentrations and induce the acrosome reaction (AR). Theophylline or, more effectively, 1-methyl-3-isobutylxanthine (MIX) inhibit the cyclic nucleotide phosphodiesterase activities of abalone sperm homogenates. 45Ca2+ uptake by sperm is also stimulated by theophylline, and more effectively by MIX, and this stimulatory effect is blocked by KCN. Verapamil, a compound known to antagonize Ca2+ conductance, has no effect on the Ca2+ or MIX-induced cAMP elevation at concentrations up to 200 microM. However, verapamil reduces the sperm cAMP elevation caused by the addition of Ca2+ plus MIX. This inhibition is not complete, even at 200 microM verapamil. The AR induced by Ca2+ plus MIX is completely inhibited by 200 microM verapamil. The data suggest that these methylxanthines elevate abalone sperm cyclic nucleotide concentrations by inhibiting cyclic nucleotide phosphodiesterase activities. Furthermore, since sperm cAMP metabolism is modulated by Ca2+ flux, methylxanthines also appear to elevate abalone sperm cAMP concentrations by their effects on Ca2+ transport. The Ca2+-induced cAMP elevation occurs through a verapamil-insensitive mechanism, whereas the potentiation by MIX of the Ca2+ effect to elevate cAMP occurs through both verapamil-insensitive and -sensitive mechanisms. The methylxanthine-induced AR is mediated by a primary effect on Ca2+ transport and occurs through a verapamil-sensitive mechanism. Cyclic AMP may play a role in the methylxanthine-induced AR, but does not appear to act as the primary mediator of this exocytotic event.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Regulation of abalone sperm cyclic AMP concentrations and the acrosome reaction by calcium and methylxanthines.

When Ca2+ is added to abalone sperm (Haliotis rufescens) in Ca2+-free artificial seawater (CaFASW) to a final concentration of 9.6 mM a 4-fold elevation in sperm cAMP occurs within 15-30 sec. The methylxanthines, theophylline and 1-methyl-3-isobutylxanthine (MIX), also elevate sperm cAMP concentrations. In CaFASW, either compound causes up to a 3-fold increase in cAMP within 15-30 sec. MIX (150 microM), added to sperm in the presence of 9.6 mM Ca2+, elevates sperm cAMP 100-fold within 15-30 sec and also triggers the acrosome reaction (AR) in the same period. Under identical conditions theophylline (1.67 mM) is much less potent at elevating cAMP and inducing the AR. The effects of methylxanthines on cAMP of sperm incubated in the presence of Ca2+ appear to represent a potentiation by these compounds of the action of Ca2+. Neither compound induces the AR in the absence of Ca2+. All of the observed effects on sperm cAMP and the AR are dependent on Ca2+ and methylxanthine concentrations. Added cyclic nucleotides or their derivatives do not induce the AR in either the absence or presence of Ca2+. Experiments with isolated sperm heads and flagella indicate that the dramatic stimulatory response of sperm cAMP to Ca2+ plus MIX is present in the head region (acrosome, nucleus, midpiece) of the cell. The data suggest that the dramatic elevation of cAMP by MIX in the presence of Ca2+ may occur directly by an inhibition of phosphodiesterase activity and indirectly by an increase in cellular Ca2+. A strong temporal correlation between the cAMP elevation and the abalone AR exists, although cAMP elevation by itself does not act as the primary mediator of this exocytotic event.

1-Methyl-3-isobutylxanthine↗
PubMed1983

Acetylcholine receptor channel ionic selectivity: ions experience an aqueous environment.

Alkali metal and alkaline earth cations pass readily through the acetylcholine receptor channel. Monovalent cations with larger crystal radii are more permeant than ones with smaller radii. For divalent ions, this selectivity sequence is reversed: smaller ions are more permeant than larger ones. This reversal in selectivity sequence with change of valence from 1 to 2 can be naturally accounted for by electrostatic interactions between the ion and its environment in the selectivity region of the channel. For monovalent ions, ion-dipole interactions dominate, and, for divalent ions, ion-induced dipole interactions are more important. The sign of these two types of effects is opposite and produces the reversal in the selectivity sequence. The magnitude of electrostatic interactions can be estimated from experimental data and suggests that the permeating ion's environment in the selectivity region of the channel is essentially like that in free water.

Animals↗
PubMed1982

Carbon 13 nuclear magnetic resonance studies of Lactobacillus casei thymidylate synthetase containing biosynthetically incorporated [guanidino-13C]arginine.

Thymidylate synthetase, containing [guanidino-13C]arginine, was obtained from amethopterin-resistant Lactobacillus casei grown on a defined medium containing [guanidino-13C]arginine. Carbon 13 nuclear magnetic resonance spectroscopy was used to investigate the native enzyme and the response of 13C-enriched arginyl residues to binary and ternary complex formation and to chemical modification by 2,3-butanedione. Native enzyme exhibited four resonances (A, 158.3 ppm; B, 157.4 ppm; C, 156.9 ppm; and D, 156.1 ppm) in a chemical shift range of 2.2 ppm. The 12 arginyl residues per subunit of this enzyme were found in a 1:5:5:1 distribution in the four resonances. Carbon 13 NMR spectra revealed that of the four resonances, resonance C was most perturbed by binary and ternary complex formation. The ternary complex formed with [guanidino-13C]arginine-enriched enzyme, 5-fluoro-deoxyuridylate, and 5,10-methylenetetrahydrofolate exhibited two new resonances at 157.9 and 156.5 ppm. Carbon 13 NMR analysis of enzyme inactivated with butanedione yielded three new single-carbon resonances, one from resonance B and two from resonance C, demonstrating that three arginyl residues per subunit were derivatized. The results of the binary and ternary complex studies and the chemical modification experiment suggest that the active site arginyl residue is represented in resonance C of the native enzyme.

Arginine↗
PubMed1982

Comparison of the photodynamic action of Rose Bengal and tetrachlorosalicylanilide on isolated porcine erythrocyte membranes.

The light-mediated effects of Rose Bengal and 3,3',4',5-tetrachlorosalicylanilide (T4CS) on porcine erythrocyte membranes have been investigated. Irradiation in the presence of Rose Bengal and oxygen causes extensive destruction of the unsaturated fatty acids of the erythrocyte membrane. This results in a decrease in the membrane flexibility as measured by a nitroxide spin probe. Irradiation in the presence of T4CS and oxygen had no measurable effect on the unsaturated fatty acids or on the membrane flexibility. Irradiation of erythrocyte membranes both in the presence of Rose Bengal and oxygen and of T4CS gave rise to polymerisation of the membrane proteins. This was evident by polyacrylamide gel electrophoresis and by freeze-fracture electron microscopy. Aggregation of membrane proteins could be observed with low levels of Rose Bengal and short irradiation times at which no loss of unsaturated fatty acids could be detected. Irradiation of the n-butanol-extracted apoprotein of porcine erythrocyte membranes both in the presence of Rose Bengal and of T4CS resulted in polymerisation of the protein as measured by gel electrophoresis and electron microscopy. The results obtained from the two photodynamic compounds are compared in terms of their different mechanisms of action on the membrane. The implications of the results in determining the molecular events leading to photohaemolysis are discussed.

Allergens↗
PubMed1981

Light spectroscopic studies of the binary complex of Lactobacillus casei thymidylate synthetase and 5-fluoro-2'-deoxyuridylate.

In contrast to previous reports, fluorescence and ultraviolet difference spectroscopy are shown to reflect the interaction of the inhibitor, 5-fluoro-2'-deoxyuridylate, with thymidylate synthetase isolated from amethopterin-resistant Lactobacillus casei. Analysis of the quenching of protein fluorescence upon nucleotide binding yields a value of 2.1 X 10(5) M-1 for the association constant for binary complex formation. The ultraviolet difference spectrum for the nucleotide-enzyme complex exhibited a broad trough with the greatest loss of absorbance centered at 275 nm. Results of previous circular dichroic studies of nucleotide-enzyme binary complexes showed apparently parallel changes in ellipticity in the 267-269-nm and 290-nm regions, which were interpreted to reflect coordinated alteration in nucleotide and enzyme structure, respectively. When examined by difference circular dichroic spectroscopy, the nucleotide-enzyme interaction is accompanied by a substantial loss in ellipticity from 250 to 300 nm which is greatest at 280 nm. We interpret the results obtained from the three light spectroscopic techniques as indications of subtle alterations in the environments of certain tyrosine and tryptophan residues in the enzyme which are caused by the association of 5-fluoro-2'-deoxyuridylate with the enzyme.

Circular Dichroism↗
PubMed1981

Fluorine-19 nuclear magnetic resonance characterization of ternary complexes of folate derivatives, 5-fluorodeoxyuridylate and Lactobacillus.

Numerous biochemical techniques have been employed to characterize the covalent inhibitory ternary complex of thymidylate synthetase consisting of enzyme, 5-fluorodeoxyuridylate, and 5,10-methylenetetrahydrofolate. 19F NMR studies of this covalent ternary complex reveal a single, broad resonance centered at 12.7 ppm to higher shielding of free nucleotide, while the 5-fluorodexyuridylate-enzyme binary complex exhibits two resonances to higher shielding of free nucleotide, one at 1.4 ppm representing noncovalently bound ligand and the other at 34.5 ppm indicative of covalently bound 5,6-dihydro-5-fluorodeoxyuridylate. In order to follow the transformation of the latter binary complex to a ternary complex, we have employed 19F NMR to profile changes in the environment of the nucleotide which result from the interaction of folates with the coenzyme binding site. At low molar excesses of folates (5-fold), the effects observed in the 19F NMR spectrum fall into three major classes. (1) 5-Methyltetrahydrofolate exhibited a weak interaction with the binary complex. (2) Methotrexate and aminopterin, antifolate drugs, were observed to increase the exchange rate among the species detected in the 19F NMR spectrum of the binary complex. (3) Folate, dihydrofolate, and a series of tetrahydrofolate derivatives were found to shift the equilibrium of the binary complex toward the covalent 5,6-dihydro-5-fluorodeoxyuridylate-enzyme complex. With the latter folates the chemical shifts for the covalent species of these ternary complexes were found in the range of 35-40 ppm to higher shielding and are interpreted to reflect subtle differences in the strength and steric nature of the interaction of the folate ligand with the binary complex. These data illustrate that the latter folates promote the conversion of the enzyme-bound nucleotide to a species which would be poised to form the second covalent bond of the ternary complex, namely the linkage of the methylene group of the coenzyme with carbon 5 of the nucleotide.

Aminopterin↗