We have explored the mechanisms of uncatalyzed membrane ion permeation using atomistic simulations and electrophysiological recordings. The solubility-diffusion mechanism of membrane charge transport has prevailed since the 1960s, despite inconsistencies in experimental observations and its lack of consideration for the flexible response of lipid bilayers. We show that direct lipid bilayer translocation of alkali metal cations, Cl(-), and a charged arginine side chain analog occurs via an ion-induced defect mechanism. Contrary to some previous suggestions, the arginine analog experiences a large free-energy barrier, very similar to those for Na(+), K(+), and Cl(-). Our simulations reveal that membrane perturbations, due to the movement of an ion, are central for explaining the permeation process, leading to both free-energy and diffusion-coefficient profiles that show little dependence on ion chemistry and charge, despite wide-ranging hydration energies and the membrane's dipole potential. The results yield membrane permeabilities that are in semiquantitative agreement with experiments in terms of both magnitude and selectivity. We conclude that ion-induced defect-mediated permeation may compete with transient pores as the dominant mechanism of uncatalyzed ion permeation, providing new understanding for the actions of a range of membrane-active peptides and proteins.

Animals inheriting the slick hair gene have a short, sleek, and sometimes glossy coat. The objective of the present study was to determine whether slick-haired Holstein cows regulate body temperature more effectively than wild-type Holstein cows when exposed to an acute increase in heat stress. Lactating slick cows (n = 10) and wild-type cows (n = 10) were placed for 10 h in an indoor environment with a solid roof, fans, and evaporative cooling or in an outdoor environment with shade cloth and no fans or evaporative cooling. Cows were exposed to both environments in a single reversal design. Vaginal temperature, respiration rate, surface temperature, and sweating rate were measured at 1200, 1500, 1800, and 2100 h (replicate 1) or 1200 and 1500 h (replicate 2), and blood samples were collected for plasma cortisol concentration. Cows in the outdoor environment had higher vaginal and surface temperatures, respiration rates, and sweating rates than cows in the indoor environment. In both environments, slick-haired cows had lower vaginal temperatures (indoor: 39.0 vs. 39.4 degrees C; outdoor 39.6 vs. 40.2 degrees C; SEM = 0.07) and respiration rate (indoor: 67 vs. 79 breaths/ min; outdoor 97 vs. 107 breaths/min; SEM = 5.5) than wild-type cows and greater sweating rates in unclipped areas of skin (indoor: 57 vs. 43 g x h(-1)/m(2); outdoor 82 vs. 61 g x h(-1)/m(2); SEM = 8). Clipping the hair at the site of sweating measurement eliminated the difference between slick-haired and wild-type cows. Results indicate that slick-haired Holstein cows can regulate body temperature more effectively than wild-type cows during heat stress. One reason slick-haired animals are better able to regulate body temperature is increased sweating rate.

Activation of prothrombin to alpha-thrombin generates not only the catalytic site and associated regions but also an independent site (an exosite) which binds anionic substances, such as Amberlite CG-50 resin [cross-linked poly(methylacrylic acid)]. Like human alpha-thrombin with high fibrinogen clotting activity (peak elution at I = 0.40 +/- 0.01 M, pH 7.4, approximately 23 degrees C), catalytically inactivated forms (e.g., i-Pr2P-alpha- and D-Phe-Pro-Arg-CH2-alpha-thrombins) were eluted with only slightly lower salt concentrations (I = 0.36-0.39 M), while gamma-thrombin with very low clotting activity was eluted with much lower concentrations (I = 0.29 M) and the hirudin complex of alpha-thrombin was not retained by the resin. In a similar manner, hirudin complexes of alpha-, i-Pr2P-alpha-, and gamma-thrombin were not retained by nonpolymerized fibrin-agarose resin. Moreover, the ionic strengths for the elution from the CG-50 resin of seven thrombin forms were directly correlated with those from the fibrin resin (y = 0.15 + 0.96x, r = 0.95). In other experiments, the 17 through 27 synthetic peptide of the human fibrinogen A alpha chain was not an inhibitor of alpha-thrombin, while the NH2-terminal disulfide knot (NDSK) fragment was a simple competitive inhibitor of alpha-thrombin with a Ki approximately 3 microM (0.15 M NaCl, pH 7.3, approximately 23 degrees C). These data suggest that alpha-thrombin recognizes fibrin(ogen) by a negatively charged surface, noncontiguous with the A alpha cleavage site but found within the NDSK fragment. Such interaction involving an anion-binding exosite may explain the exceptional specificity of alpha-thrombin for the A alpha cleavage in fibrinogen and alpha-thrombin incorporation into fibrin clots.

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We tested whether resistance of lymphocytes to heat stress is modified by breed, intracellular glutathione content, and extracellular antioxidants. In the first experiment, lymphocytes from Angus (Bos taurus, non-heat-tolerant), Brahman (B. indicus, heat-tolerant), and Senepol (B. taurus, heat-tolerant) heifers (12 heifers per breed) were cultured at 45 degrees C for 3 h to evaluate thermal killing, at 42 degrees C for 12 h in a 60-h phytohemagglutinin-induced proliferation test, and at 42 degrees C for 1 h to measure induction of heat shock protein 70 (HSP70). Killing at 45 degrees C was affected by breed x temperature (P < .01); the decrease in viability caused by a temperature of 45 degrees C was greater for Angus than for Brahman or Senepol. For phytohemagglutinin-stimulated lymphocytes, heating to 42 degrees C reduced [3H]thymidine incorporation equally for all breeds. Viability at the end of culture was affected (P < .001) by a breed x temperature interaction because the decrease in viability caused by culture at 42 degrees C was greatest for lymphocytes from Angus heifers. Heat shock for 1 h at 42 degrees C caused a two- to threefold increase in intracellular concentrations of HSP70, but there was no interaction of temperature with breed. In another experiment (with lymphocytes harvested from three Holstein cows), buthionine sulfoximine, a glutathione synthesis inhibitor, inhibited (P < .01) proliferation of phytohemagglutinin-stimulated lymphocytes at 38.5 and 42 degrees C. Addition of the antioxidants glutathione or thioredoxin to culture did not reduce the effects of heating to 42 degrees C on proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)