Differential polarized phase fluorometric investigations of diphenylhexatriene in lipid bilayers. Quantitation of hindered depolarizing rotations.

Differential polarized phase fluorometry has been used to investigate the depolarizing rotations of 1,6-diphenyl-1,3,5-hexatriene (DPH) in isotropic solvents and in lipid bilayers. For DPH dissolved in isotropic solvents, there is a precise agreement between the observed and predicted values for maximum differential tangents, indicating that in these media DPH is a free isotropic rotator. In lipid bilayers the tangent defects (i.e., the differences between the calculated and the observed maximum differential tangents) are too large to be explained by anisotropy in the depolarizing rotations but are accounted for by hindered isotropic torsional motions for the fluorophore [Weber, G (1978) Acta Phys. Pol A 54, 173]. This theory describes the depolarizing rotations of the fluorophore by its rotational rate R (in radians/second) and the limiting fluorescence anisotropy (r) at times long compared with the fluorescence lifetime. Through the combined use of both steady-state anisotropy measurements and differential phase measurements, we have demonstrated that one may obtain unique solutions for both R and r. For DPH embedded in vesicles prepared from dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholines, the depolarizing motions are highly hindered at temperatures below the transition temperature (Tc) but are unhindered above Tc. The apparent rotational rates of the probe do not change significantly at Tc. These data suggest that the changes observed in the steady-state anisotropy near Tc derive primarily from changes in the degree to which the probe's rotations are hindered, and only to a small extent from changes in rotational rate. For DPH embedded in bilayers that contained 25 mol % cholesterol, no clear transition occurred and the rotations appeared to be hindered at all temperatures. The rotational motions of DPH embedded in dioleolyphosphatidylcholine were found to be far less hindered, but the rotational rates were similar to those obtained in the saturated phosphatidylcholines. Finally, the data show that in an anisotropic environment, such as that of a lipid bilayer, steady-state fluorescence anisotropy measurements alone cannot yield quantitatively meaningful rotational rates. Extrapolation of steady-state aniosotropy data to the quantitation of membrane viscosity is therefore difficult, if not invalid; however, qualitative comparisons can be useful.     

Liposomal membranes XI. A suggestion to structural characteristics of acido-thermophilic bacterial membranes

To understand the role of ω-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1,2-di-11-cyclohexylundecanoyl-l-α-phosphatidylcholine (11_CYPC), 1,2-di-13-cyclohexyltridecanoyl-l-α-phosphatidylcholine (13_CYPC), and 1–13-cyclohexyltridecanoyl-2–11-cyclohexylundecanoyl-l-α-phosphatidylcholine (1–13_CY-2–11_CYPC) were prepared and the steady-state fluorescence anisotropy of 1,6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined. Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the ω-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures. The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations. Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.     

GUVs Melt Like LUVs: The Large Heat Capacity of MLVs Is Not Due to Large Size or Small Curvature

The excess heat capacity functions (ΔC_p) associated with the main phase transition of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) are very different. Two explanations are possible. First, the difference in vesicle size (curvature) results in different gel-fluid interactions in the membrane; those interactions have a large effect on the cooperativity of the phase transition. Second, there is communication between the bilayers in an MLV when they undergo the gel-fluid transition; this communication results in thermodynamic coupling of the phase transitions of the bilayers in the MLV and, consequently, in an apparent increase in the cooperativity of the transition. To test these hypotheses, differential scanning calorimetry was performed on giant unilamellar vesicles (GUVs) of pure dipalmitoylphosphatidylcholine. The ΔC_p curve of GUVs was found to resemble that of the much smaller LUVs. The transition in GUVs and LUVs is much broader (half-width ∼1.5°C) than in MLVs (∼0.1°C). This similarity in GUVs and LUVs indicates that their size has little effect on gel-fluid interactions in the phase transition. The result suggests that coupling between the transitions in the bilayers of an MLV is responsible for their apparent higher cooperativity in melting.     

The static and dynamic behaviour of fluorescent probe molecules in lipid bilayers

Angle-resolved fluorescence depolarization experiments were carried out on 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-(trimethylammonium)phenyl]-6-phenyl-1,3,5-hexatriene (TMA-DPH) molecules embedded in multibilayers of dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC) above their respective phase transitions. The finding that the order parameter 〈P₂〉 of the absorption moment is significantly higher than that for the emission moment for each probe is shown to arise from a tilt of the emission moment relative to the molecular symmetry axis. It is further shown that while the order parameter 〈P₂〉 is the same for both probes in DMPC bilayers, it is higher for TMA-DPH than for DPH molecules in POPC bilayers. Considerations of the order parameters 〈P₄〉, however, show that this difference can be ascribed solely to the higher fraction of DPH molecules lying with their axes parallel to the bilayer surface. Furthermore it is found that TMA-DPH molecules undergo slower reorientational motions than DPH molecules in the same bilayer system. Nevertheless the motion of both probe molecules is faster in DMPC than in POPC bilayers. The results indicate that TMA-DPH is a more useful probe than DPH in the systems investigated.     

Analysis of the bilayer phase transition temperatures of phosphatidylcholines with mixed chains

The analysis of the chain-length dependence of the chain-melting transition temperatures of bilayers composed of lipids with identical chains (Marsh, D. 1991. Biochim. Biophys. Acta. 1062: 1-6) is extended to include lipids with chains of unequal length. The bilayer transition temperatures of saturated asymmetrical phosphatidylcholines are interpreted by assuming that the transition enthalpy and transition entropy are linearly related to the absolute value of the difference in chain length between the sn-1 and sn-2 chains, with constant end contributions. Such an assumption is supported by calorimetric data on phosphatidylcholines of constant mean chainlength and varying chain asymmetry. In particular, a symmetrical linear dependence is observed on the chain asymmetry, Δn, which is centered around a value Δn° that corresponds to the conformational inequivalence of the sn-1 and sn-2 chains. The transition temperature then takes the form: T_t = T_t^∞(n - n_H - h′ | Δn + Δn° |)/(n - n_s - s′ | Δn + Δn°) where n_H, n_s are the end contributions, and h′, s′ are fractional deficits in the incremental transition enthalpy and entropy, respectively, arising from the overlapping regions of the longer chains. Optimization on the transition temperature data for the dependence on chain asymmetry of three series of phosphatidylcholines with constant mean chainlength, n, yields parameters that are capable of predicting the dependence of the transition temperatures on chain asymmetry for other mean chainlengths. The dependence of the transition temperature on mean chainlength for phosphatidylcholines in which the chain asymmetry is maintained constant, as well as the dependence on both mean chain length and chain asymmetry for phosphatidylcholines in which one of the two chains is maintained of constant length, are also described with high accuracy by using the same parameters.     

Bioenergetic Conditions of Butyrate Metabolism by a Syntrophic, Anaerobic Bacterium in Coculture with Hydrogen-Oxidizing Methanogenic and Sulfidogenic Bacteria

The butyrate-oxidizing, proton-reducing, obligately anaerobic bacterium NSF-2 was grown in batch cocultures with either the hydrogen-oxidizing bacterium Methanospirillum hungatei PM-1 or Desulfovibrio sp. strain PS-1. Metabolism of butyrate occurred in two phases. The first phase exhibited exponential growth kinetics (phase a) and had a doubling time of 10 h. This value was independent of whether NSF-2 was cultured with a methanogen or a sulfate reducer and likely represents the maximum specific growth rate of NSF-2. This exponential growth phase was followed by a second phase with a nearly constant rate of degradation (phase b) which dominated the time course of butyrate degradation. The specific activity of H₂ uptake by the hydrogen-oxidizing bacterium controlled the bioenergetic conditions of metabolism in phase b. During this phase both the Gibbs free energy (ΔG′) and the butyrate degradation rate (v) were greater for NSF-2-Desulfovibrio sp. strain PS-1 (ΔG′ = −17.0 kJ/mol; v = 0.20 mM/h) than for NSF-2-M. hungatei PM-1 (ΔG′ = −3.8 kJ/mol, v = 0.12 mM/h). The ΔG′ value remained stable and characteristic of the two hydrogen oxidizers during phase b. The stable ΔG′ resulted from the close coupling of the rates of butyrate and H₂ oxidation. The addition of 2-bromoethanesulfonate to a NSF-2-methanogen coculture resulted in the total inhibition of butyrate degradation; the inhibition was relieved when Desulfovibrio sp. strain PS-1 was added as a new H₂ sink. When the specific activity of H₂ consumption was increased by adding higher densities of the Desulfovibrio sp. to 2-bromoethanesulfonate-inhibited NSF-2-methanogen cocultures, lower H₂ pool sizes and higher rates of butyrate degradation resulted. Thus, it is the kinetic parameters of H₂ consumption, not the type of H₂ consumer per se, that establishes the thermodynamic conditions which in turn control the rate of fatty acid degradation. The bioenergetic homeostasis we observed in phase b was a result of the kinetics of the coculture members and the feedback inhibition by hydrogen which prevents butyrate degradation rates from reaching their theoretical V_max.     

Dynamic motions of 1,6-diphenyl-1,3,5-hexatriene in interdigitated C(18):C(10)phosphatidylcholine bilayers.

This study investigates the dynamic behavior of 1,6-diphenyl-1,3,5-hexatriene (DPH) in C(18):C(10)phosphatidylcholine [C(18):C(10)PC] bilayers. C(18):C(10)PC is an asymmetric mixed-chain phosphatidylcholine known to form mixed-interdigitated structures below the transition temperature and form partially interdigitated bilayers above the transition temperature. The rotation of DPH in C(18):C(10)PC has been described in terms of the thermal coefficient of rotation using the modified Y-plot method which takes into account the limiting anisotropy value. During the phase transition of C(18):C(10)PC, DPH exhibits a thermal coefficient b2M = 0.41 - 0.51 degrees C-1 which is similar to the b2M values obtained with noninterdigitated phosphatidylcholine bilayers. Differential polarized phase-modulation fluorometry has also been employed to study the dynamic behavior of DPH in C(18):C(10)PC in real time. The data show that DPH contains considerable motion in the highly ordered mixed interdigitated bilayers. The DPH motion steadily increases with an increase in temperature as shown by the rotational correlation time, and the wobbling diffusion constant. However, the limiting anisotropy, the order parameter, and the width of the lifetime distribution undergo an abrupt decrease, and a corresponding abrupt increase in the cone angle, at approximately 16 degrees C. This temperature range is near the onset temperature of the phase transition as determined by differential scanning calorimetry. The rotational parameters show strong hysteresis on heating and cooling. All the rotational parameters derived from DPH fluorescence in mixed interdigitated C(18):C(10)PC exhibit magnitudes similar to those obtained from noninterdigitated gel phases of symmetric diacylphosphatidylcholines.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparative Characterization of Complete and Truncated Forms of Lactobacillus amylovorus α-Amylase and Role of the C-Terminal Direct Repeats in Raw-Starch Binding

Two constructs derived from the α-amylase gene (amyA) of Lactobacillus amylovorus were expressed in Lactobacillus plantarum, and their expression products were purified, characterized, and compared. These products correspond to the complete (AmyA) and truncated (AmyAΔ) forms of α-amylase; AmyAΔ lacks the 66-kDa carboxyl-terminal direct-repeating-unit region. AmyA and AmyAΔ exhibit similar amylase activities towards a range of soluble substrates (amylose, amylopectin and α-cyclodextrin, and soluble starch). The specific activities of the enzymes towards soluble starch are similar, but the K_M and V_max values of AmyAΔ were slightly higher than those of AmyA, whereas the thermal stability of AmyAΔ was lower than that of AmyA. In contrast to AmyA, AmyAΔ is unable to bind to β-cyclodextrin and is only weakly active towards glycogen. More striking is the fact that AmyAΔ cannot bind or hydrolyze raw starch, demonstrating that the carboxyl-terminal repeating-unit domain of AmyA is required for raw-starch binding activity.     

Estimating Finite Rate of Population Increase for Sharks Based on Vital Parameters

The vital parameter data for 62 stocks, covering 38 species, collected from the literature, including parameters of age, growth, and reproduction, were log-transformed and analyzed using multivariate analyses. Three groups were identified and empirical equations were developed for each to describe the relationships between the predicted finite rates of population increase (λ’) and the vital parameters, maximum age (T_max), age at maturity (T_m), annual fecundity (f/R_c)), size at birth (L_b), size at maturity (L_m), and asymptotic length (L_∞). Group (1) included species with slow growth rates (0.034 yr^-1 < k < 0.103 yr^-1) and extended longevity (26 yr < T_max < 81 yr), e.g., shortfin mako Isurus oxyrinchus, dusky shark Carcharhinus obscurus, etc.; Group (2) included species with fast growth rates (0.103 yr^-1 < k < 0.358 yr^-1) and short longevity (9 yr < T_max < 26 yr), e.g., starspotted smoothhound Mustelus manazo, gray smoothhound M. californicus, etc.; Group (3) included late maturing species (L_m/L_∞ ≧ 0.75) with moderate longevity (T_max < 29 yr), e.g., pelagic thresher Alopias pelagicus, sevengill shark Notorynchus cepedianus. The empirical equation for all data pooled was also developed. The λ’ values estimated by these empirical equations showed good agreement with those calculated using conventional demographic analysis. The predictability was further validated by an independent data set of three species. The empirical equations developed in this study not only reduce the uncertainties in estimation but also account for the difference in life history among groups. This method therefore provides an efficient and effective approach to the implementation of precautionary shark management measures.     

Differential polarized phase fluorometric studies of phospholipid bilayers under high hydrostatic pressure

Differential polarized phase fluorometry was used to quantify the rotational rate ($\text{R}$) and limiting anisotropy ($\text{r}{}_{\mathrm{\infty }}$) of the membrane probe diphenylhexatriene (DPH) in solvents and lipid vesicles exposed to hydrostatic pressures ranging from 1 bar to 2 kbar. These measurements reveal the effect of pressure on the phase-transition temperatures of the phosphatidylcholine vesicles, and the effects of pressure on order parameter of the acyl side-chain region of the membranes, the latter as indicated by $\text{r}{}_{\mathrm{\infty }}$. In addition to the well-known elevation of the transition temperature ($\text{T}{}_{\mathrm{<mtext>c</mtext>}}$) with pressure, our results demonstrate that increased pressure restores the order of the bilayers to that representative of temperatures below the transition temperature. We also found that solvents which allowed free isotropic rotation of DPH at 1 bar no longer allowed free rotation when sufficiently compressed; moreover, the apparent DPH rotational rate increased with $\text{r}{}_{\mathrm{\infty }}$. Pressure studies using both DPH and the charged DPH analogue, trimethylammonium DPH (TMA-DPH) indicated that the $\text{T}{}_{\mathrm{<mtext>c</mtext>}}$ of dipalmitoylphosphatidylcholine vesicles increased 23 K/kbar and an apparent volume change of 0.036 ml/mol lipid at the phase transition. Assuming, as has been proposed, that TMA-DPH is localized near the glycerol backbone region of the bilayers, these results indicate a similar temperature- and pressure-dependent phase transition in this region and the acyl side-chain region of the membrane.     

Uncoupling of Bacterioplankton and Phytoplankton Production in Fresh Waters Is Affected by Inorganic Nutrient Limitation

Pelagic bacterial production is often positively correlated, or coupled, with primary production through utilization of autotrophically produced dissolved organic carbon. Recent studies indicate that inorganic N or P can directly limit both bacterial and phytoplanktonic growth. Our mesocosm experiments, with whole communities from mesotrophic Calder Lake, test whether this apparent bacterial-algal coupling may be the result of independent responses to limiting inorganic nutrients. In systems without N additions, numbers of bacteria but not phytoplankton increased 2- to 2.5-fold in response to P fertilization (0 to 2.0 μmol of P per liter); this resulted in uncoupled production patterns. In systems supplemented with 10 μmol of NH₄NO₃ per liter, P addition resulted in up to threefold increases in bacteria and two- to fivefold increases in total phytoplankton biomass (close coupling). P limitation of pelagic bacteria occurred independently of phytoplankton dynamics, and regressions between bacterial abundance and phytoplankton chlorophyll a were nonsignificant in all systems without added N. We describe a useful and simple coupling index which predicts that shifts in phytoplankton and bacterioplankton growth will be unrelated (Δ bacteria/Δ phytoplankton → either + ∞ or - ∞) in systems with inorganic N/P (molar) ratios of <~40. In systems with higher N/P ratios (>40), the coupling index will approach 1.0 and close coupling between bacteria and phytoplankton is predicted to occur.     

Pharmacokinetics and Bioequivalence of Two Formulations of Febuxostat 40-Mg and 80-Mg Tablets: A Randomized,Open-Label, 4-Way Crossover Study in Healthy Chinese Male Volunteers

The present study aimed to investigate the pharmacokinetic properties of febuxostat in healthy Chinese male volunteers and evaluate whether the two formulations of febuxostat 40-mg and 80-mg tablets are bioequivalent. A randomized, open-label, 4-way crossover study was conducted in healthy Chinese male volunteers under fasting conditions. 24 eligible subjects were randomized in a 1:1:1:1 ratio to receive a single dose of test or reference formulation of febuxostat 40-mg or 80-mg tablet. The washout period between each administration was 1 week. Plasma febuxostat was quantified by a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Tolerability was evaluated by monitoring adverse events, physical examinations, 12-lead ECG and laboratory tests. After single-dosing of 1 tablet of 40-mg febuxostat, the pharmacokinetic parameters of test and reference formulations were: T_max 1.22±0.87 and 1.85±1.03 h, C_max 1689.16±461.31 and 1613.80±608.43 ng·mL^-1, AUC_0-t 5139.87±1349.28 and 5517.91±2024.26 ng·mL^-1·h, AUC_0−∞ 5263.06±1339.16 and 5640.48±2040.22 ng·mL^-1·h, t_1/2 4.82±2.61 and 4.85±1.78 h, respectively. After single-dosing of 1 tablet of 80-mg febuxostat, the pharmacokinetic parameters of test and reference formulations were: T_max 1.71±1.21 and 2.23±1.55 h, C_max 2744.47±1157.44 and 2998.17±1200.13 ng·mL^-1, AUC_0-t 9634.03±2768.25 and 10467.95±3501.65 ng·mL^-1·h, AUC_0−∞ 9834.32±2730.51 and 10626.63±3504.08 ng·mL^-1·h, t_1/2 6.25±2.44 and 5.46±1.65 h, respectively. For single-dosing of 1 tablet of 40-mg febuxostat, 90% CIs for the test/reference ratio of AUC_0-t, AUC_0−∞ and C_max were 89.79 to 102.55, 90.14 to 102.56 and 93.99 to 129.63, respectively. For single-dosing of 1 tablet of 80-mg febuxostat, 90% CIs for the test/reference ratio of AUC_0-t, AUC_0−∞ and C_max were 86.67 to 100.00, 87.50 to 100.51 and 79.48 to 105.99, respectively. This single dose study revealed similar pharmacokinetic properties in healthy Chinese male volunteers as those found in Caucasic population. The test and reference febuxostat tablets formulations met the regulatory criteria for bioequivalence at 40-mg and 80-mg strengths in fasting healthy Chinese male volunteers.Trial Registration: Chictr.org ChiCTR-TTRCC-14004288     

Evaluation of the Stability,Bioavailability, and Hypersensitivity of the Omega-3 Derived Anti-Leukemic Prostaglandin: Δ12-Prostaglandin J3

Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ¹²-PGJ₃, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ¹²-PGJ₃. The stability of Δ¹²-PGJ₃ was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ¹²-PGJ₃ in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ¹²-PGJ₃ being a downstream metabolite of PGD₃ was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD₂ receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ¹²-PGJ₃ was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ¹²-PGJ₃ was bioavailable and well absorbed into systemic circulation with a C_max of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ¹²-PGJ₃ did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ¹²-PGJ₃ and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ¹²-PGJ₃ was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ¹²-PGJ₃ failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ¹²-PGJ₃ to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.     

Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans

Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the V_o domain (V_oa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V₁V_o assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pH_vph1Δ_/Δ = 6.8 versus pH_vph1Δ_/Δ_R = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.     

Random Coil to Globular Thermal Response of a Protein (H3.1) with Three Knowledge-Based Coarse-Grained Potentials

The effect of temperature on the conformation of a histone (H3.1) is studied by a coarse-grained Monte Carlo simulation based on three knowledge-based contact potentials (MJ, BT, BFKV). Despite unique energy and mobility profiles of its residues, the histone H3.1 undergoes a systematic (possibly continuous) structural transition from a random coil to a globular conformation on reducing the temperature. The range over which such a systematic response in variation of the radius of gyration (R_g) with the temperature (T) occurs, however, depends on the potential, i.e. ΔT_MJ ≈ 0.013–0.020, ΔT_BT ≈ 0.018–0.026, and ΔT_BFKV ≈ 0.006–0.013 (in reduced unit). Unlike MJ and BT potentials, results from the BFKV potential show an anomaly where the magnitude of R_g decreases on raising the temperature in a range ΔT_A ≈ 0.015–0.018 before reaching its steady-state random coil configuration. Scaling of the structure factor, S(q) ∝ q^−1/ν, with the wave vector, q = 2π/λ, and the wavelength, λ, reveals a systematic change in the effective dimension (D_e∼1/ν) of the histone with all potentials (MJ, BT, BFKV): D_e∼3 in the globular structure with D_e∼2 for the random coil. Reproducibility of the general yet unique (monotonic) structural transition of the protein H3.1 with the temperature (in contrast to non-monotonic structural response of a similar but different protein H2AX) with three interaction sets shows that the knowledge-based contact potential is viable tool to investigate structural response of proteins. Caution should be exercise with the quantitative comparisons due to differences in transition regimes with these interactions.     

Charges and Potentials at the Nerve Surface : Divalent ions and pH

The voltage dependence of the voltage clamp responses of myelinated nerve fibers depends on the concentration of divalent cations and of hydrogen ions in the bathing medium. In general, increases of the [Ca], [Ni], or [H] increase the depolarization needed to elicit a given response of the nerve. An e-fold increase of the [Ca] produces the following shifts of the voltage dependence of the parameters in the Hodgkin-Huxley model: m_∞, 8.7 mv; h_∞, 6.5 mv; τ_n, 0.0 mv. The same increase of the [H], if done below pH 5.5, produces the following shifts: m_∞, 13.5 mv; h_∞, 13.5 mv; τ_n, 13.5 mv; and if done above pH 5.5: m_∞, 1.3 mv; h_∞, 1.3 mv; τ_n, 4.0 mv. The voltage shifts are proportional to the logarithm of the concentration of the divalent ions and of the hydrogen ion. The observed voltage shifts are interpreted as evidence for negative fixed charges near the sodium and potassium channels. The charged groups are assumed to comprise several types, of varying affinity for divalent and hydrogen ions. The charges near the sodium channels differ from those near the potassium channels. As the pH is lowered below pH 6, the maximum sodium conductance decreases quickly and reversibly in a manner that suggests that the protonation of an acidic group with a pK_a of 5.2 blocks individual sodium channels.     

Alterations in Membrane Lipid Dynamics of Leukemic Cells Undergoing Growth Arrest and Differentiation: Dependency on the Inducing Agent

The effect of various differentiation inducers on membrane cell dynamics was studied using HL-60 and K562 leukemic cell lines. Membrane lipid dynamics was measured by the steady-state fluorescence polarization (P) method utilizing either 1,6-diphenyl-1,3,5-hexatriene (DPH) or the trimethyl ammonium derivative of DPH (TMA-DPH), which ascertains anchorage of the label to the membrane–water–lipid interface. Decrease in membrane microfluidity was observed in HL-60 cells undergoing differentiation into macrophages by 1,25-dihydroxyvitamin D₃and by K562 cells induced to differentiate by DMSO. Sodium butyrate caused an increase in membrane fluidity in K562 cells undergoing differentiation into erythroid-like cells while in HL-60 cells a dual effect was observed. At 0.4 mM concentration, in which the cells were induced to differentiate along the monocyte pathway, a decrease in membrane fluidity was observed, while at 1 mM concentration an increase in membrane fluidity occurred. Interferon-γ (IFN-γ) induced an increase in membrane fluidity in both cell lines. Using HL-60 cells fluorescently labeled by TMA-DPH, similar results indicating fluidization of the membrane following IFN-γ treatment were obtained. Advanced fluorescence lifetime measurements, evaluated either by phase modulation spectrofluorometry or by single photon correlation fluorometry confirmed that the decrease in fluorescence polarization by IFN-γ resulted from membrane fluidization and not from elongation of the probe's excited state lifetime. It is suggested that the inducer mode of action, and not the differentiation route, determine the outcome of changes in membrane microviscosity.     

The Deletion of Several Amino Acid Stretches of Escherichia coli Alpha-Hemolysin (HlyA) Suggests That the Channel-Forming Domain Contains Beta-Strands

Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71–110, 158–167, 180–203, and 264–286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyA_Δ71–110 and HlyA_Δ264–286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyA_Δ158–167 and HlyA_Δ180–203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyA_Δ71–110 and HlyA_Δ264–286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyA_Δ71–110, and HlyA_Δ264–286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.     

Pharmacokinetics of oral moxidectin in individuals with Onchocerca volvulus infection

BackgroundOnchocerciasis (“river blindness”), is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus and transmitted to humans through repeated bites by infective blackflies of the genus Simulium. Moxidectin was approved by the United States Food and Drug Administration in 2018 for the treatment of onchocerciasis in people at least 12 years of age. The pharmacokinetics of orally administered moxidectin in 18- to 60-year-old men and women infected with Onchocerca volvulus were investigated in a single-center, ivermectin-controlled, double-blind, randomized, single-ascending-dose, ascending severity of infection study in Ghana.Methodology/Principal findingsParticipants were randomized to either a single dose of 2, 4 or 8 mg moxidectin or ivermectin. Pharmacokinetic samples were collected prior to dosing and at intervals up to 12 months post-dose from 33 and 34 individuals treated with 2 and 4 mg moxidectin, respectively and up to 18 months post-dose from 31 individuals treated with 8 mg moxidectin. Moxidectin plasma concentrations were determined using high-performance liquid chromatography with fluorescence detection. Moxidectin plasma AUC_0-∞ (2 mg: 26.7–31.7 days*ng/mL, 4 mg: 39.1–60.0 days*ng/mL, 8 mg: 99.5–129.0 days*ng/mL) and C_max (2mg, 16.2 to17.3 ng/mL, 4 mg: 33.4 to 35.0 ng/mL, 8 mg: 55.7 to 74.4 ng/mL) were dose-proportional and independent of severity of infection. Maximum plasma concentrations were achieved 4 hours after drug administration. The mean terminal half-lives of moxidectin were 20.6, 17.7, and 23.3 days at the 2, 4 and 8 mg dose levels, respectively.Conclusion/SignificanceWe found no relationship between severity of infection (mild, moderate or severe) and exposure parameters (AUC_0-∞ and C_max), T_1/2 and T_max for moxidectin. T_max, volume of distribution (V/F) and oral clearance (CL/F) are similar to those in healthy volunteers from Europe. From a pharmacokinetic perspective, moxidectin is an attractive long-acting therapeutic option for the treatment of human onchocerciasis.     

Improved Inhibitor Screening Experiments by Comparative Analysis of Simulated Enzyme Progress Curves

A difficulty associated with high throughput screening for enzyme inhibitors is to establish reaction conditions that maximize the sensitivity and resolution of the assay. Deduction of information from end-point assays at single concentrations requires a detailed understanding of the time progress of the enzymatic reaction, an essential but often difficult process to model. A tool to simulate the time progress of enzyme catalyzed reactions and allows adjustment of reactant concentrations and parameters (initial concentrations, K _m, k _cat, K _i values, enzyme half-life, product•enzyme dissociation constant, and the rate constant for the reversed reaction) has been developed. This tool provides comparison of the progress of uninhibited versus inhibited reactions for common inhibitory mechanisms, and guides the tuning of reaction conditions. Possible applications include: analysis of substrate turnover, identification of the point of maximum difference in product concentration (Δ_max[P]) between inhibited and uninhibited reactions, determination of an optimal observation window unbiased for inhibitor mechanisms or potency, and interpretation of observed inhibition in terms of true inhibition. An important observation that can be utilized to improve assay signal strength and resolution is that Δ_max[P] occurs at a high degree of substrate consumption (commonly >75%) and that observation close to this point does not adversely affect observed inhibition or IC₅₀ values.