Cystic fibrosis transmembrane conductance regulator. Physical basis for lyotropic anion selectivity patterns.

The cystic fibrosis transmembrane conductance regulator (CFTR) Cl channel exhibits lyotropic anion selectivity. Anions that are more readily dehydrated than Cl exhibit permeability ratios (P(S)/P(Cl)) greater than unity and also bind more tightly in the channel. We compared the selectivity of CFTR to that of a synthetic anion-selective membrane [poly(vinyl chloride)-tridodecylmethylammonium chloride; PVC-TDMAC] for which the nature of the physical process that governs the anion-selective response is more readily apparent. The permeability and binding selectivity patterns of CFTR differed only by a multiplicative constant from that of the PVC-TDMAC membrane; and a continuum electrostatic model suggested that both patterns could be understood in terms of the differences in the relative stabilization of anions by water and the polarizable interior of the channel or synthetic membrane. The calculated energies of anion-channel interaction, derived from measurements of either permeability or binding, varied as a linear function of inverse ionic radius (1/r), as expected from a Born-type model of ion charging in a medium characterized by an effective dielectric constant of 19. The model predicts that large anions, like SCN, although they experience weaker interactions (relative to Cl) with water and also with the channel, are more permeant than Cl because anion-water energy is a steeper function of 1/r than is the anion-channel energy. These large anions also bind more tightly for the same reason: the reduced energy of hydration allows the net transfer energy (the well depth) to be more negative. This simple selectivity mechanism that governs permeability and binding acts to optimize the function of CFTR as a Cl filter. Anions that are smaller (more difficult to dehydrate) than Cl are energetically retarded from entering the channel, while the larger (more readily dehydrated) anions are retarded in their passage by "sticking" within the channel.

CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.

The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

Interarch tooth size relationships of 3 populations: "does Bolton's analysis apply?".

This study evaluates whether Bolton's interarch ratios extend across populations and genders. The data were derived from systematically collected preorthodontic casts of 180 patients, including 30 males and 30 females from each of 3 populations (black, Hispanic, and white). Forty-eight mesiodistal contact points were digitized on each model, and the lengths of the anterior, posterior, and overall arch segments were calculated. The results showed significant (P <.05) ethnic group differences in all 6 arch segment lengths and in all 3 interarch ratios. Whites displayed the lowest overall ratio (92.3%), followed by Hispanics (93.1%), and blacks (93.4%). The group differences were due primarily to the relationships between the posterior segments. The arch segments of males were significantly larger than females; the overall and posterior ratios were also significantly larger in males than in females. Multiple regression analyses showed that individual differences in the overall ratio were most closely associated with the size of the lower second premolar, followed by the upper lateral incisors, upper second premolars, and the lower central incisors. In combination, these 4 teeth explained approximately 50% of the variation in the overall ratio between subjects. We conclude that interarch tooth size relationships are population and gender specific. Bolton ratios apply to white females only; the ratios should not be indiscriminately applied to white males, blacks, or Hispanics.

Purification of human DNA (cytosine-5-)-methyltransferase.

We have developed a facile procedure for the purification of DNA methyltransferase activity from human placenta. The procedure avoids the isolation of nuclei and the dialysis and chromatography of large volumes. A purification of 38,000-fold from the whole cell extract has been achieved. The procedure employs ion exchange, affinity, and hydrophobic interaction chromatography coupled with preparative glycerol gradient centrifugation. A protein of 126,000 daltons was found to copurify with the activity and was the major band seen in the most highly purified material after SDS gel electrophoresis. This observation, coupled with an observed sedimentation coefficient of 6.3S, suggests that the enzyme is composed of a single polypeptide chain of this molecular weight. Hemimethylated DNA was found to be the preferred substrate for the enzyme at each stage in the purification. The ratio of the activity of the purified product on hemimethylated to that on unmethylated M13 duplex DNA was about 12 to 1. Thus, the purified activity has the properties postulated for a maintenance methyltransferase. The availability of highly purified human DNA methyltransferase should facilitate many studies on the structure, function, and expression of these activities in both normal and transformed cells.

Effects of famotidine on gastric pH and residual volume in pediatric surgery.

Aspiration pneumonitis is a severe complication of anesthesia. The objectives of this study were to determine if preoperative famotidine, a new histamine2-receptor antagonist, given by mouth either the evening before or the morning of elective surgery, reduced gastric residual volume and increased gastric pH in pediatric patients. Either famotidine or placebo (or both) were orally administered to 58 children (aged 2-17 years). The patients were randomly assigned to four groups: Famotidine-Famotidine, Placebo-Placebo, Placebo-Famotidine, and Famotidine-Placebo; subjects in the Famotidine-Famotidine group received two doses of famotidine (0.5 mg.kg-1 per dose), those in the Placebo-Placebo group, two doses of placebo, those in the Placebo-Famotidine and Famotidine-Placebo group, one dose of each by mouth. The Famotidine-Famotidine group received one dose of famotidine at 22:00 the evening before surgery and a second dose 60-90 min before the scheduled time of surgery. The Placebo-Placebo group received two doses of placebo at the same times as the Famotidine-Famotidine group. The Placebo-Famotidine group received a dose of placebo the night before surgery and a dose of famotidine the morning of surgery; the Famotidine-Placebo group received famotidine the night before surgery and placebo the morning of surgery. The administration of famotidine on the morning of surgery significantly increased gastric pH (4.8 vs. 1.3) in comparison with placebo, as did two doses of famotidine (6.6). Famotidine failed to reduce gastric residual volume significantly in any group. The administration of famotidine significantly reduced the number of pediatric patients considered at higher risk for aspiration pneumonitis, despite not decreasing gastric residual volume.