Fluctuations of the red blood cell membrane: relation to mechanical properties and lack of ATP dependence

We have analyzed the fluctuations of the red blood cell membrane in both the temporal ((ω(s⁻¹)) and spatial (q(m⁻¹)) frequency domains. The cells were examined over a range of osmolarities leading to cell volumes from 50% to 170% of that in the isotonic state. The fluctuations of the isotonic cell showed an ∼q⁻³-dependence, indicative of a motion dominated by bending, with an inferred bending modulus of ∼9 × 10⁻¹⁹J. When the cells were osmotically swollen to just below the point of lysis (166% of physiological volume), a q⁻¹-dependence of the fluctuations supervened, implying that the motion was now dominated by membrane tension; estimated as ∼1.3 × 10⁻⁴ nm⁻¹. When, on the other hand, the cells were osmotically dehydrated, the fluctuation amplitude progressively decreased. This was caused by a rise in internal viscosity, as shown by measurements on resealed ghosts containing a reduced hemoglobin concentration, which displayed no such effect. We examined, in addition, cells depleted of ATP, before the onset of echinocytosis, and could observe no change in fluctuation amplitude. We conclude that the membrane fluctuations of the red cell are governed by bending modulus, membrane tension, and cytosolic viscosity, with little or no dependence on the presence or absence of ATP.     

Fluctuation in the electric conductivity of lipid bilayers after one-sided application of polyene antibiotics

It is shown that the fluctuation spectrum of the lipid bilayer conductance induced by one-sided application of polyene antibiotics has a profound l/f component with a magnitude being proportional to the single-channel conductance. The observed component is thought to be caused by the channel mobility increasing at one-sided polyene application in a comparison with two-sided application where a Lorentzian spectrum of fluctuations was found earlier.     

Softening of lipid bilayers

The softening of wet lipid bilayer membranes during their gel-to-fluid first-order phase transition is studied by computer simulation of a family of two-dimensional microscopic interaction models. The models include a variable number, q, of lipid chain conformational states, where 2q10. Results are presented as functions of q and temperature for a number of bulk properties, such as internal energy, specific heat, and lateral compressibility. A quantitative account is given of the statistics of the lipid clusters which are found to form in the neighborhood of the transition. The occurrence of these clusters is related to the softening and the strong thermal density fluctuations which dominate the specific heat and the lateral compressibility for the high-q models. The cluster distributions and the fluctuations behave in a manner reminiscent of critical phenomena and percolation. The findings of long-lived metastable states and extremely slow relaxational behavior in the transition region are shown to be caused by the presence of intermediate lipid chain conformational states which kinetically stabilize the cluster distribution and the effective phase coexistence. This has as its macroscopic consequence that the first-order transition apperas as a continuous transition, as invariably observed in all experiments on uncharged lecithin bilayer membranes. The results also suggest an explanation of the non-horizontal isotherms of lipid monolayers. Possible implications of lipid bilayer softening and enhanced passive permeability for the functioning of biological membranes are discussed.Abbreviations PC phosphatidvlcholine - DMPC dimyristoyl PC - DPPC dipalmitoyl PC - ac alternating current - DSC differential scanning calorimetry - T _m lipid gel-to-fluid phase transition temperature - TEMPO 2,2,6,6-tetramethylpiperidine-N-oxyl Supported by the Danish Natural Science Research Council and A/S De Danske Spritfabrikkers JubilæumslegatSupported in part by the NSERC of Canada and Le FCAC du Quebec     

Solid-State H NMR Shows Equivalence of Dehydration and Osmotic Pressures in Lipid Membrane Deformation

Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S_CD) of DMPC approach very large values of ≈0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10−100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.     

Relationship between water regime and hummock-building by Melaleuca ericifolia and Phragmites australis in a brackish wetland

We investigated the relationship between hummock height and depth of inundation in a permanently inundated wetland in south-eastern Australia. Our survey of 318 hummocks, in water ranging from 0 to 70 cm depth, revealed a significant positive linear relationship and strong correlation between hummock height and water depth (r² = 0.53 and 0.79 for Melaleuca ericifolia and Phragmites australis hummocks respectively). We also investigated whether water regime affects the decomposition rate of litter on hummocks; specifically, whether constant inundation slows decomposition to an extent that would promote accumulation of litter and hummock-building. On the contrary, we found that constantly submerged M. ericifolia litter decomposed faster than dry litter, but at a similar rate to litter that experienced intermittent inundation (decay rates (k) 0.0015 d⁻¹, 0.0010 d⁻¹ and 0.0008 d⁻¹ for submerged, intermittent and dry treatments respectively). Submerged P. australis litter also decomposed faster (k = 0.0024 d⁻¹) than dry litter (k = 0.0011 d⁻¹). We discuss the interaction of water regime and decomposition of organic material and implications for the maintenance of hummock and hollow topography.     

Neutron Spin-Echo Studies of Hemoglobin and Myoglobin: Multiscale Internal Dynamics

Neutron spin-echo spectroscopy was used to study structural fluctuations that occur in hemoglobin (Hb) and myoglobin (Mb) in solution. Using neutron spin-echo data up to a very high momentum transfer q (∼ 0.62 Å^− 1), we characterized the internal dynamics of these proteins at the levels of dynamic pair correlation function and self-correlation function in the time range of several picoseconds to a few nanoseconds. In the same protein solution, data transition from pair correlation motion to self-correlation motion as the momentum transfer q increases. At low q, coherent scattering dominates; at high q, observations are largely due to incoherent scattering. The low q data were interpreted in terms of an effective diffusion coefficient; on the other hand, the high q data were interpreted in terms of mean square displacements. Comparison of data from the two homologous proteins collected at different temperatures and protein concentrations was used to assess the contributions made by translational and rotational diffusion and internal modes of motion to the data. The temperature dependence of decay times can be attributed to changes in the viscosity and temperature of the solvent, as predicted by the Stokes-Einstein relationship. This is true for contributions from both diffusive and internal modes of motion, indicating an intimate relationship between the internal dynamics of the proteins and the viscosity of the solvent. Viscosity change associated with protein concentration can account for changes in diffusion observed at different concentrations, but is apparently not the only factor involved in the changes in internal dynamics observed with change in protein concentration. Data collected at high q indicate that internal modes in Mb are generally faster than those in Hb, perhaps due to the greater surface-to-volume ratio of Mb and the fact that surface groups tend to exhibit faster motion than buried groups. Comparison of data from Hb and data from Mb at low q indicates an unexpectedly rapid motion of Hb αβ dimers relative to one another. Dynamic motion of subunits is increasingly perceived as important to the allosteric behavior of Hb. Our data demonstrate that this motion is highly sensitive to protein concentration, temperature, and solvent viscosity, indicating that great care needs to be exercised in interpreting its effect on protein function.     

Thermodynamics of partitioning of substance P in isotropic bicelles

The temperature dependence of the partition of a neuropeptide, substance P (SP), in isotropic (q = 0.5) bicelles was investigated by using pulsed field gradient NMR diffusion technique. The partition coefficient decreases as the temperature is increased from 295 to 325 K, indicating a favorable (negative) enthalpy change upon partitioning of the peptide. Thermodynamic analysis of the data shows that the partitioning of SP at 300 K is driven by the enthalpic term (ΔH) with the value of ? 4.03 kcal mol^?1, while it is opposed by the entropic term (?TΔS) by approximately 1.28 kcal mol^?1 with a small negative change in heat capacity (ΔC_p). The enthalpy‐driven process for the partition of SP in bicelles is the same as in dodecylphosphocholine (DPC) micelles, however, the negative entropy change in bicelles of flat bilayer surface is in sharp contrast with the positive entropy change in DPC micelles of highly curved surface, indicating that the curvature of the membrane surface might play a significant role in the partitioning of peptides. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Effect of dust charge fluctuations on dust acoustic structures in magnetized dusty plasma containing nonextensive electrons and two-temperature isothermal ions

Effect of dust electrical charge fluctuations on the nature of dust acoustic solitary waves (DASWs) in a four-species magnetized dusty plasma containing nonextensive electrons and two-temperature isothermal ions has been investigated. In this model, the negative dust electric charge is considered to be proportional to the plasma space potential. The nonlinear Zakharov–Kuznetsov (ZK) and modified Zakharov–Kuznetsov (mZK) equations are derived for DASWs by using the standard reductive perturbation method. The combined effects of electron nonextensivity and dust charge fluctuations on the DASW profile are analyzed. The different ranges of the nonextensive q-parameter are considered. The results show that solitary waves the amplitude and width of which depend sensitively on the nonextensive q-parameter can exist. Due to the electron nonextensivity and dust charge fluctuation rate, our dusty plasma model can admit both positive and negative potential solitons. The results show that the amplitude of the soliton increases with increasing electron nonextensivity, but its width decreases. Increasing the electrical charge fluctuations leads to a decrease in both the amplitude and width of DASWs.     

A new method for analysis of heart rate variability: counting statistics of 1/f fluctuations

1/f fluctuations in heart rate are usually measured by spectral analysis using Fast Fourier Transform. Here, we introduce a new method based on counting the number of QRS complexes in time intervals t of various length. For a 1/f ^b spectrum the variance of counts in t follows a power-law as gDt ^1+b. This method is applied to analyze long term fluctuations in heart rate variability in 10 healthy men. The results show periods of 1/f fluctuations with interpolated periods of white noise during night hours.     

Redundant and dosage sensitive requirements for Fgf3 and Fgf10 in cardiovascular development

Heart development requires contributions from, and coordinated signaling interactions between, several cell populations, including splanchnic and pharyngeal mesoderm, postotic neural crest and the proepicardium. Here we report that Fgf3 and Fgf10, which are expressed dynamically in and near these cardiovascular progenitors, have redundant and dosage sensitive requirements in multiple aspects of early murine cardiovascular development. Embryos with Fgf3^−/+;Fgf10^−/−, Fgf3^−/−;Fgf10^−/+ and Fgf3^−/−;Fgf10^−/− genotypes formed an allelic series of increasing severity with respect to embryonic survival, with double mutants dead by E11.5. Morphologic analysis of embryos with three mutant alleles at E11.5–E13.5 and double mutants at E9.5–E11.0 revealed multiple cardiovascular defects affecting the outflow tract, ventricular septum, atrioventricular cushions, ventricular myocardium, dorsal mesenchymal protrusion, pulmonary arteries, epicardium and fourth pharyngeal arch artery. Assessment of molecular markers in E8.0–E10.5 double mutants revealed abnormalities in each progenitor population, and suggests that Fgf3 and Fgf10 are not required for specification of cardiovascular progenitors, but rather for their normal developmental coordination. These results imply that coding or regulatory mutations in FGF3 or FGF10 could contribute to human congenital heart defects.     

Stiffening Effect of Cholesterol on Disordered Lipid Phases: A Combined Neutron Spin Echo + Dynamic Light Scattering Analysis of the Bending Elasticity of Large Unilamellar Vesicles

In this study, the center-of-mass diffusion and shape fluctuations of large unilamellar 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylcholine vesicles prepared by extrusion are studied by means of neutron spin echo in combination with dynamic light scattering. The intermediate scattering functions were measured for several different values of the momentum transfer, q, and for different cholesterol contents in the membrane. The combined analysis of neutron spin echo and dynamic light scattering data allows calculation of the bending elastic constant, κ, of the vesicle bilayer. A stiffening effect monitored as an increase of κ with increasing cholesterol molar ratio is demonstrated by these measurements.     

Factors affecting akinete differentiation in Cylindrospermopsis raciborskii (Nostocales, Cyanobacteria)

1. Cylindrospermopsis raciborskii is a potentially toxic freshwater cyanobacterium which can produce akinetes (reproductive spores) that on germinating can contribute to future populations. To further understand factors controlling the formation of these specialised cells, the effects of diurnal temperature fluctuations (magnitude and frequency), in combination with different light intensities and phosphorus concentrations were investigated under laboratory conditions. 2. Akinete differentiation was affected by the frequency of temperature fluctuations. Maximum akinete concentrations were observed in cultures that experienced multiple diurnal temperature fluctuations. 3. Akinete concentrations increased with increasing magnitude of temperature fluctuation. A maximum akinete concentration was achieved under multiple diurnal temperature fluctuations with a magnitude of 10 °C (25 °C to 15 °C). 4. A fourfold increase in light intensity (25–100 μmol m^?2 s^?1) resulted in an approximate 14‐fold increase in akinete concentration. 5. High filterable reactive phosphorus (FRP) concentrations (>70 μg L^?1) in the medium, combined with a multiple diurnal temperature fluctuation of 10 °C, supported the development of the highest akinete concentration.     

Pressure-Dependent Structure Changes in Barnase on Ligand Binding Reveal Intermediate Rate Fluctuations

In this work we measured ¹H NMR chemical shifts for the ribonuclease barnase at pressures from 3 MPa to 200 MPa, both free and bound to d(CGAC). Shift changes with pressure were used as restraints to determine the change in structure with pressure. Free barnase is compressed by ∼0.7%. The largest changes are on the ligand-binding face close to Lys-27, which is the recognition site for the cleaved phosphate bond. This part of the protein also contains the buried water molecules. In the presence of d(CGAC), the compressibility is reduced by ∼70% and the region of structural change is altered: the ligand-binding face is now almost incompressible, whereas changes occur at the opposite face. Because compressibility is proportional to mean square volume fluctuation, we conclude that in free barnase, volume fluctuation is largest close to the active site, but when the inhibitor is bound, the fluctuations become much smaller and are located mainly on the opposite face. The timescale of the fluctuations is nanoseconds to microseconds, consistent with the degree of ordering required for the fluctuations, which are intermediate between rapid uncorrelated side-chain dynamics and slow conformational transitions. The high-pressure technique is therefore useful for characterizing motions on this relatively inaccessible timescale.     

Cell protrusions and tethers: a unified approach

Low pulling forces applied locally to cell surface membranes produce viscoelastic cell surface protrusions. As the force increases, the membrane can locally separate from the cytoskeleton and a tether forms. Tethers can grow to great lengths exceeding the cell diameter. The protrusion-to-tether transition is known as the crossover. Here we propose a unified approach to protrusions and tethers providing, to our knowledge, new insights into their biomechanics. We derive a necessary and sufficient condition for a crossover to occur, a formula for predicting the crossover time, conditions for a tether to establish a dynamic equilibrium (characterized by constant nonzero pulling force and tether extension rate), a general formula for the tether material after crossover, and a general modeling method for tether pulling experiments. We introduce two general protrusion parameters, the spring constant and effective viscosity, valid before and after crossover. Their first estimates for neutrophils are 50 pN μm⁻¹ and 9 pN s μm⁻¹, respectively. The tether elongation after crossover is described as elongation of a viscoelastic-like material with a nonlinearly decaying spring (NLDs-viscoelastic material). Our model correctly describes the results of the published protrusion and tether pulling experiments, suggesting that it is universally applicable to such experiments.     

Clathrin triskelia show evidence of molecular flexibility

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or “basket”. Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket. We extend the earlier solution studies by performing small-angle neutron scattering (SANS) experiments on isolated triskelia, allowing us to examine a higher q range than that probed by static light scattering. Results of the SANS measurements are consistent with the light scattering measurements, but show a shoulder in the scattering function at intermediate q values (0.016 Å⁻¹), just beyond the Guinier regime. This feature can be accounted for by Brownian dynamics simulations based on flexible bead-spring models of a triskelion, which generate time-averaged scattering functions. Calculated scattering profiles are in good agreement with the experimental SANS profiles when the persistence length of the assumed semiflexible triskelion is close to that previously estimated from the analysis of electron micrographs.     

Instability of C60 fullerene interacting with lipid bilayer

Due to the large number of possible applications of nanoparticles in cosmetic and medical products, the possible hazards of nanoparticles in the human body are a major concern. A worst-case scenario is that nanoparticles might cause health issues such as skin damage or even induce cancer. As a first step to study the toxicity of nanoparticles, we investigate the energy behaviour of a C₆₀ fullerene interacting with a lipid bilayer. Using the 6–12 Lennard-Jones potential function and the continuous approximation, the equilibrium spacing between the two layers of a bilayer is predicted to be 3.36 ?. On assuming that there is a circular hole in the lipid bilayer, a relation for the molecular interaction energy is determined, involving the circular radius b of the hole and the perpendicular distance Z of the spherical fullerene from the hole. A graph of the minimum energy location Z _min verses the hole radius b shows that a C₆₀ fullerene first penetrates through a lipid bilayer when b > 6.81 ?, and shows a simple circular relation \textZ_min² + b² = 6.81² {\text{Z}}_{{\min }}^2 + {b^{{2}}} = {6}.{8}{{1}^{{2}}} for Z _min positive and b ≤ 6.81 ?. For b > 6.81, the fullerene relocates from the surface of the bilayer to the interior, and as the hole radius increases further it moves to the centre of the bilayer and remains there for increasing hole radii. Accordingly, our modelling indicates that at least for the system with no external forces, the C₆₀ fullerene will not penetrate through the lipid bilayer but rather remains encased between the two layers at the mid-plane location.     

Metabolic quotient of the soil microflora in relation to plant succession

Summary In this study we propose the hypothesis that ecosystem succession is accompanied by a decrease in the metabolic quotient qCO₂ (respiration-to-biomass ratio) of the soil microflora. The qCO₂ is calculated from basal respiration (CO₂-C·h^-1) per unit microbial biomass carbon (C_mier). The hypothesis was tested by studying two primary successions on recessional moraines of the Rotmoos Ferner (Austria) and the Athabasca Glacier (Canada). For both soil seres (0->200 years) it was shown that the qCO₂ decreased with time, which corroborated the hypothesis. In addition, the short term development of the qCO₂ was demonstrated with a revegetation trial. We observed a rise in qCO₂ for the first two years after reclamation, followed by a subsequent decrease.     

Reformulation of BMI and Percent Body Fat to Remove the Height Bias in 8‐year‐olds

BMI and percent body fat (%BF) are both related to height (Ht) in prepubertal children, so may misrepresent childhood adiposity, especially in tall or short children. We sought to construct replacement functions for BMI and %BF that are independent of Ht. Fat mass (FM) was measured using dual‐energy X‐ray absorptiometry, together with Ht and body mass (BM) in 746 healthy boys and girls aged 8 years (0.34 s.d.). Relationships between BM, FM, and Ht were measured and values of p and q derived such that the functions BM. Ht^?p and FM.BM^?q were unrelated to Ht. BM was not directly proportional to Ht², BMI being significantly related to Ht in both boys and girls (P < 0.001). BM was proportional to Ht³, BM. Ht^?3 being independent of Ht. Similarly, FM was not directly proportional to BM and %BF was significantly related to Ht (P < 0.001). While FM was proportional to BM², FM.BM^?1.5 was the function found to be independent of Ht. Using the 85th and 95th percentiles as the cutoffs for overweight and obesity respectively, 6.4% of the boys and 6.8% of the girls were classified differently by BMI and the Ht independent measure BM. Ht^?3. Similarly, 10.1% boys and 13.7% girls were classified differently by %BF and the Ht independent measure FM.BM^?1.5. We propose that improved diagnostic accuracy of body composition in 8‐year‐olds is provided by the BM function (BMF, BM. Ht^?3) and FM function (FMF, FM.BM^?1.5) replacing BMI and %BF, which both overestimate the adiposity of taller children and underestimate it in shorter children.     

Culture–area relation in Axelrod’s model for culture dissemination

Axelrod’s model for culture dissemination offers a nontrivial answer to the question of why there is cultural diversity given that people’s beliefs have a tendency to become more similar to each other’s as they interact repeatedly. The answer depends on the two control parameters of the model, namely, the number F of cultural features that characterize each agent, and the number q of traits that each feature can take on, as well as on the size A of the territory or, equivalently, on the number of interacting agents. Here, we investigate the dependence of the number C of distinct coexisting cultures on the area A in Axelrod’s model, the culture–area relationship, through extensive Monte Carlo simulations. We find a non-monotonous culture–area relation, for which the number of cultures decreases when the area grows beyond a certain size, provided that q is smaller than a threshold value q _c = q _c (F) and F ≥ 3. In the limit of infinite area, this threshold value signals the onset of a discontinuous transition between a globalized regime marked by a uniform culture (C = 1), and a completely polarized regime where all C = q ^F possible cultures coexist. Otherwise, the culture–area relation exhibits the typical behavior of the species–area relation, i.e., a monotonically increasing curve the slope of which is steep at first and steadily levels off at some maximum diversity value.     

DISTRIBUTION OF DIURNAL ACID METABOLISM IN THE GENUS ISOETES

Previous studies have revealed a crassulacean acid metabolism-like pathway in the submerged aquatic Isoetes howellii (Isoetaceae). A survey of 11 other species in this largely aquatic genus showed all had diurnal fluctuations in titratable acidity and malic acid concentration. The magnitude of the diurnal fluctuation in titratable acidity varied from 119 (μeq g^–1 fresh weight) for I. bolanderi to 44 for I. engelmanni. Although these species were grown under similar conditions in a greenhouse, it is questionable whether or not any significance can be attached to observed species specific differences in magnitude of diurnal acid fluctuation. This is suggested by a single species comparison of plants in the field with greenhouse grown plants which indicated nearly as much variation in magnitude of acid fluctuations as observed across all species in the greenhouse. Much of this variation may be due to seasonal differences. Substrate appears to have very little effect on magnitude of acid fluctuations. Since this survey covered most of the range of the latitudinal, elevational, habitat, and morphological variation in the genus, it is likely that diurnal acid metabolism will prove to be common in the genus Isoetes.