On the Role of Curved Membrane Nanodomains, and Passive and Active Skeleton Forces in the Determination of Cell Shape and Membrane Budding.

Biological membranes are composed of isotropic and anisotropic curved nanodomains. Anisotropic membrane components, such as Bin/Amphiphysin/Rvs (BAR) superfamily protein domains, could trigger/facilitate the growth of membrane tubular protrusions, while isotropic curved nanodomains may induce undulated (necklace-like) membrane protrusions. We review the role of isotropic and anisotropic membrane nanodomains in stability of tubular and undulated membrane structures generated or stabilized by cyto- or membrane-skeleton. We also describe the theory of spontaneous self-assembly of isotropic curved membrane nanodomains and derive the critical concentration above which the spontaneous necklace-like membrane protrusion growth is favorable. We show that the actin cytoskeleton growth inside the vesicle or cell can change its equilibrium shape, induce higher degree of segregation of membrane nanodomains or even alter the average orientation angle of anisotropic nanodomains such as BAR domains. These effects may indicate whether the actin cytoskeleton role is only to stabilize membrane protrusions or to generate them by stretching the vesicle membrane. Furthermore, we demonstrate that by taking into account the in-plane orientational ordering of anisotropic membrane nanodomains, direct interactions between them and the extrinsic (deviatoric) curvature elasticity, it is possible to explain the experimentally observed stability of oblate (discocyte) shapes of red blood cells in a broad interval of cell reduced volume. Finally, we present results of numerical calculations and Monte-Carlo simulations which indicate that the active forces of membrane skeleton and cytoskeleton applied to plasma membrane may considerably influence cell shape and membrane budding.

Sensitivity of quantitative relaxometry and susceptibility mapping to microscopic iron distribution.

PURPOSE: Determine the impact of the microscopic spatial distribution of iron on relaxometry and susceptibility-based estimates of iron concentration. METHODS: Monte Carlo simulations and in vitro experiments of erythrocytes were used to create different microscopic distributions of iron. Measuring iron with intact erythrocyte cells created a heterogeneous distribution of iron, whereas lysing erythrocytes was used to create a homogeneous distribution of iron. Multi-echo spin echo and spoiled gradient echo acquisitions were then used to estimate relaxation parameters ( R2 and R2* ) and susceptibility. RESULTS: Simulations demonstrate that R2 and R2* measurements depend on the spatial distribution of iron even for the same iron concentration and volume susceptibility. Similarly, in vitro experiments demonstrate that R2 and R2* measurements depend on the microscopic spatial distribution of iron whereas the quantitative susceptibility mapping (QSM) susceptibility estimates reflect iron concentration without sensitivity to spatial distribution. CONCLUSIONS: R2 and R2* for iron quantification depend on the spatial distribution or iron. QSM-based estimation of iron concentration is insensitive to the microscopic spatial distribution of iron, potentially providing a distribution independent measure of iron concentration.

Active Forces of Myosin Motors May Control Endovesiculation of Red Blood Cells.

By using Monte Carlo (MC) simulations, we have shown that the active forces generated by (NMIIA) motor domains bound to F-actin may partially control the endovesiculation of the red blood cell (RBC) membrane. The myosin generated active forces favor pancake-like (torocyte) RBC endovesicles with a large flat central membrane region and a bulby periphery. We suggest that the myosin generated active forces acting on the RBC membrane in the direction perpendicular to the membrane surface towards the interior of the RBC may influence also other RBC shape transformations and the stability of different types of RBC shapes and should be therefore considered in the future theoretical studies of the RBC vesiculation and shape transformations.

Highly unsaturated n-3 fatty acids status of Canadian Inuit: International Polar Year Inuit Health Survey, 2007-2008.

OBJECTIVES: Previous studies suggest that dietary patterns and the extent of reliance upon traditional food vary among Inuit communities. Inuit traditional foods are an important source of nutrients such as highly unsaturated n-3 fatty acids (HUFA n-3), whose beneficial effects include protection against ischemic heart disease. Dietary transition is occurring with younger generations consuming less traditional foods and more market foods with low nutrient density. Utilizing erythrocyte membrane fatty acid composition as an indicator of body HUFA n-3 status, which reflects dietary intake levels of traditional Inuit foods, we explored the regional and age variability of highly unsaturated n-3 fatty acids (HUFA n-3) in the International Polar Year Inuit Health Survey. STUDY DESIGN: Cross-sectional health survey. METHODS: Participants were recruited through random sampling of households. Fatty acid data were available among 2,200 adults (≥18 yr). RESULTS: HUFA n-3 levels in the Eastern Arctic were significantly higher than in the Western Arctic, with Nunatsiavut (northern Labrador) and Baffin showing the highest HUFA n-3 status compared to Kivalliq, Kitikmeot and Inuvialuit Settlement Region (ISR) (p<0.0001). Fatty acid proportion in erythrocyte membranes showed pronounced differences between coastal communities and inland communities, including a higher HUFA n-3 status among the coastal communities (p<0.0001). Additionally, the HUFA n-3 status showed a strong positive association with age, particularly in Baffin and Kivalliq. HUFA n-3 were inversely associated with saturated (ß=-0.98 [SE=0.03], R2=0.36, p<0.0001) and trans fatty acids (ß=-0.06 [SE=0.004], R2=0.07, p<0.0001). CONCLUSIONS: The present study results provided biochemical support for varying dietary patterns and dietary transition among Inuit across the Canadian Arctic. The analyses also suggested multifactorial determinants of HUFA n-3 status among Canadian Arctic Inuit. A nutritional intervention strategy with multiple approaches may be needed to improve and maintain their HUFA n-3 status.

Erythrocyte membrane fluidity and indices of plasmatic oxidative damage after acute physical exercise in humans.

Optimal levels of membrane fluidity are essential for numerous cell functions including cell growth, solute transport and signal transduction. Since exercise enhances free radical production, our aim was to evaluate in healthy male subjects the effects of an acute bout of maximal and submaximal exercise on the erythrocyte membrane fluidity and its possible relation to the oxidative damage overproduction due to exercise. Subjects (n = 34) performed three cycloergometric tests: a continuous progressive exercise, a strenuous exercise until exhaustion and an acute bout of exercise at an intensity corresponding to 70% of maximal work capacity for 30 min. Venous blood samples were collected before and immediately after these exercises. Erythrocyte membrane fluidity was assessed by fluorescence spectroscopy. Plasma malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations and carbonyl content of plasmatic proteins were used as an index of lipid and protein oxidation, respectively. Exercise produced a dramatic drop in the erythrocyte membrane fluidity as compared to resting time, but this was not accompanied by significant changes in the plasmatic MDA and 4-HDA concentrations. The highest erythrocyte membrane rigidity was detected immediately after strenuous exercise until exhaustion was performed. Protein carbonyl levels were higher after exhaustive exercises than at rest. Continuous progressive and strenuous exercises until exhaustion, but not submaximal workload, resulted in a significant enhanced accumulation of carbonylated proteins in the plasma. These findings are consistent with the idea that exercise exaggerates oxidative damage, which may contribute, at least partially, to explain the rigidity in the membrane of the erythrocytes due to acute exercise.

Effect of toluene on erythrocyte membrane stability under in vivo and in vitro conditions with assessment of oxidant/antioxidant status.

Toluene, an organic solvent used widely in the industry, is highly lipophilic and accumulates in the cell membrane impeding transport through it. Its metabolites cause oxygen radical formation that react with unsaturated fatty acids and proteins in erythrocytes leading to lipid peroxidation and protein breakdown. In this study, we aimed to investigate the membrane stabilizing and the oxidative stress-inducing effects of toluene in human erythrocytes. Measurements of osmotic fragility, mean corpuscular volume (MCV), oxidative stress parameters and antioxidant enzyme activities were performed simultaneously both in individuals exposed to toluene professionally (in vivo) and human erythrocytes treated with toluene (in vitro). To measure osmotic fragility, erythrocytes were placed in NaCl solutions at various concentrations (0.1% [blank], 0.38%, 0.40%, 0.42%, 0.44%, 0.46%, 0.48% and 1% [stock]). Percentage of haemolysis in each solution was calculated with respect to the 100% haemolysis in the blank solution. The erythrocyte packs prepared at the day of the above-mentioned measurements were kept at -80 degrees C until the time for determination of malonyldialdehyde and protein carbonyl levels, and catalase (CAT) and glutathione peroxidase activities as indicators of oxidative stress. Toluene increased oxidative stress parameters significantly both in vivo and in vitro; it also caused a significant decrease in the activities of antioxidant enzymes. Osmotic fragility was altered only in the case of in vitro exposure. In conclusion, toluene exposure resulted in increased lipid peroxidation and protein damage both in vivo and in vitro. Although, it is natural to expect increased osmotic fragility due to oxidative properties of toluene, its membrane-stabilizing effect overcame the oxidative properties leading to decreased osmotic fragility or preventing its deterioration in vitro and in vivo toluene exposures, respectively, in the present study.

Cytoskeleton mediated effective elastic properties of model red blood cell membranes.

The plasma membrane of human red blood cells consists of a lipid bilayer attached to a regular network of underlying cytoskeletal polymers. We model this system at a dynamic coarse-grained level, treating the bilayer as an elastic sheet and the cytoskeletal network as a series of phantom entropic springs. In contrast to prior simulation efforts, we explicitly account for dynamics of the cytoskeletal network, both via motion of the protein anchors that attach the cytoskeleton to the bilayer and through breaking and reconnection of individual cytoskeletal filaments. Simulation results are explained in the context of a simple mean field percolation model and comparison is made to experimental measurements of red blood cell fluctuation amplitudes.

[Study of the sorption capacity of red blood cell membranes for the assessment of the pattern of endogenous intoxication in dermatoses].

The parameters of the sorption capacity of red blood cell membranes and the level of small and medium molecular weight substances were studied in 53 patients with chronic dermatoses. The plasma accumulation of small and medium molecular weight substances contributes to the altered sorption capacity of red blood cell membranes and to the occurrence of resistance to long used drugs. Biological blood clearance should be performed to enhance the efficiency of therapeutic measures.

[Assessment of the effects of sodium oxybutyrate and normal pressure hyperoxia on human blood plasma and erythrocyte membrane lipids during antiorthostatic hypokinesia].

Benefits from an antihypoxic agent (sodium oxibutyrate) and normal pressure hyperoxia to the lipid composition of erythrocyte membranes and blood plasma were assessed in 9 subjects on the thirtieth day of head-down tilting at -8(0). The greatest variations in HDT occurred in polyunsaturated fatty acids in erythrocyte membranes. After infusion of sodium oxibutyrate a marked reduction in polyunsaturated fatty acids was mainly in consequence of expressed decreases in linoleic and, particularly, arachidonic acids. However, ensuing hyperoxic treatment led to their steady rise. Sequential sodium oxibutyrate infusion to the tilted subjects breathing air and then exposed to normal pressure hyperoxia seemed to have compensated, as judged by the fatty acids in erythrocyte membranes, for the activity of free radical oxidation in hypoperfusated regional tissues.

Dynamics of fluid vesicles in shear flow: effect of membrane viscosity and thermal fluctuations.

The dynamical behavior of vesicles is investigated in simple shear flow. A simulation technique is presented that combines a three-dimensional particle-based mesoscopic model (multiparticle collision dynamics) for the solvent with a dynamically triangulated surface model for the membrane. In this model, thermal fluctuations of the solvent and of the membrane are consistently taken into account. The membrane viscosity can be varied by changing the bond-flip rate of the dynamically triangulated surface. Vesicles are found to transit from steady tank-treading to unsteady tumbling motion with increasing membrane viscosity. At small reduced volumes, the shear induces a transformation from a discocyte to a prolate shape at low membrane viscosity. On the other hand, at high membrane viscosity, the shear induces a transformation from prolate to discocyte, or tumbling motion accompanied by shape oscillations between these two states. Thermal fluctuations induce intermittent tumbling and smooth out the transitions. This effect can be understood from a simplified stochastic model.

Dynamic simulations of membranes with cytoskeletal interactions.

We describe a simulation algorithm for the dynamics of elastic membrane sheets over long length and time scales. Our model includes implicit hydrodynamic coupling between membrane and surrounding solvent and allows for arbitrary external forces acting on the membrane surface. In particular, the methodology is well suited to studying membranes in interaction with cytoskeletal filaments. We present results for the thermal undulations of a lipid bilayer attached to a regular network of spectrin filaments as a model for the red blood cell membrane. The dynamic fluctuations of the bilayer over the spectrin network are quantified and used to predict the macroscopic diffusion constant of band 3 on the surface of the red blood cell. We find that thermal undulations likely play a role in the mobility of band 3 in the plane of the erythrocyte membrane.

[A new approach to assessment of biological consequences of exposure to low-dose radiation]. / Novye podkhody k otsenke biologicheskikh posledstvii vozdeistviia radiatsii v malykh dozakh.

A high sensitivity of characteristics of the lipid metabolism in erythrocytes to exposure to low doses of gamma- and X-rays was found; the changes in lipid peroxidation (LPO) of blood components were persistent, which substantially influenced the development of biological consequences of low doses of radiation. The effect of low doses of radiation on the interrelation between the LPO intensity in blood plasma and the lymphocyte DNA structural integrity or the cell-free DNA content in animal blood plasma was found. Different sensitivity of the examined parameters (neither normalization nor linear dependence on a dose rate was found) implies the transition of the LPO regulatory system to another level of functioning due to scale changes of interrelations between the examined parameters under influence of low doses of radiation. These findings make possible to assess biological consequences of radiation factor for animal groups by the scale changes of interrelations between the examined characteristics in blood plasma.

Spontaneous transfer of phospholipid and cholesterol hydroperoxides between cell membranes and low-density lipoprotein: assessment of reaction kinetics and prooxidant effects.

Under oxidative pressure in the vascular circulation, erythrocytes and phagocytic cells may accumulate membrane lipid hydroperoxides (LOOHs), including cholesterol- and phospholipid-derived species (ChOOHs, PLOOHs). LOOH translocation from cells to low-density lipoprotein (LDL) might sensitize the latter to free radical-mediated oxidative modification, an early event associated with atherogenesis. To test this, we examined the spontaneous transfer kinetics of various ChOOH species (5 alpha-OOH, 6 alpha-OOH, 6 beta-OOH, 7 alpha/7 beta-OOH) and various PLOOH groups (PCOOH, PEOOH, PSOOH, SMOOH) using photoperoxidized erythrocyte ghosts as model donors and freshly prepared LDL as an acceptor. LOOH departure or uptake was monitored by reverse-phase HPLC with reductive electrochemical detection. Mildly peroxidized ghost membranes transferred overall ChOOH and PLOOH to LDL with apparent first-order rate constants approximately 60 and approximately 35 times greater than those of the respective parent lipids. Individual ChOOH rate constants decreased in the following order: 7 alpha/7 beta-OOH > 5 alpha-OOH > 6 alpha-OOH > 6 beta-OOH. Kinetics for reverse transfer from LDL to ghosts followed the same trend, but rates were significantly higher for all species and their combined activation energy was lower (41 vs 85 kJ/mol). PLOOH transfer rate constants ranged from 4- to 15-fold lower than the composite ChOOH constant, their order being as follows: PCOOH approximately PEOOH approximately PSOOH > SMOOH. Similar PLOOH transfer kinetics were observed when LDL acceptor was replaced by unilamellar liposomes, consistent with desorption from the donor membrane being the rate-limiting step. The susceptibility of transfer LOOH-enriched LDL to Cu2+-induced chain peroxidative damage was assessed by monitoring the accumulation of conjugated dienes and products of free radical-mediated cholesterol oxidation. In both cases, transfer-acquired LOOHs significantly reduced the lag time for chain initiation relative to that observed using nonperoxidized ghosts. These findings are consistent with the idea that LDL can acquire significant amounts of "seeding" LOOHs via translocation from various donors in the circulation.

Erythrocyte consumption of nitric oxide: competition experiment and model analysis.

It is generally believed that the erythrocyte membrane is highly permeable to nitric oxide (NO). To prevent NO from freely entering and being scavenged by the red blood cell (RBC), it has been suggested that NO consumption is limited by the mass transfer resistance of the diffusion layer adjacent to the erythrocyte membrane. Recently, we (Vaughn et al. (2000). J. Biol. Chem. 275, 2342) presented an experimental technique that overcomes experimental diffusional limitations and showed that RBCs also possess a mechanism to slow nitric oxide uptake. Here, we present a mathematical analysis of this technique by modeling the NO uptake of a single cell. We obtain additional data (n = 33, total) by use of the competition experiment and, through application of the model, show that either the RBC membrane permeability to NO or the intracellular reaction rate between NO and hemoglobin (Hb) is at least 2000-fold lower than previously thought. As a result, RBCs react with NO at a rate three orders of magnitude slower than free oxyHb. This phenomena may play an important role in NO bioavailability.

Assessment of bilirubin toxicity to erythrocytes. Implication in neonatal jaundice management.

BACKGROUND: Neonatal hyperbilirubinaemia remains one of the most common clinical conditions requiring therapeutic intervention. Nevertheless, reliable indicators of bilirubin toxicity are still missing. This prompted us to investigate (a) the progression of cytotoxic events produced by increasing concentrations of bilirubin; (b) the relevance of the membrane lipid package on bilirubin binding to erythrocytes; and (c) the reliability of chloroform extraction compared with albumin extraction to evaluate erythrocyte-bound bilirubin and cytotoxicity. MATERIALS AND METHODS: Morphological alterations, free bilirubin, erythrocyte-bound bilirubin (albumin- and chloroform-extractable), haemolysis and membrane-released lipids, were determined in human erythrocytes at 4 degrees C or 37 degrees C, after 4 h incubation at pH 7.4, with increasing molar ratios of bilirubin to albumin (0.5-5). The reversibility of cytotoxicity by albumin washing was assessed by morphological analysis. RESULTS: Decreased free bilirubin, lower erythrocyte-bound bilirubin concentration by albumin extraction (superficial/non-aggregated bilirubin) and higher values by chloroform extraction (deep/aggregated bilirubin) were observed for 37 degrees C vs. 4 degrees C, at molar ratios > 1. Echinocytosis increased with bilirubin concentration and temperature and was not fully reversed by albumin washing. Haemolysis was already significant at a molar ratio of 1, and was enhanced by temperature at molar ratios 3 and 5 (P < 0.01). The loss of membrane lipids was remarkable at molar ratios > or = 0.5, both at 4 degrees C and 37 degrees C (P < 0.01), although correlation with bilirubin concentration was only significant at 37 degrees C (r = 0.971; P < 0.01). CONCLUSIONS: These results suggest that increased lipid fluidity and high bilirubin concentrations promote membrane bilirubin translocation and toxicity. They also show that albumin is not able to displace the bilirubin located deeply or aggregated within the membrane, which in turn is removed by chloroform. Accordingly, chloroform-extractable rather than albumin-extractable bilirubin is a more accurate parameter to assess erythrocyte-bound bilirubin during severe hyperbilirubinaemia.

Assessment of the efficacious dose of arachidonic and docosahexaenoic acids in preterm infant formulas: fatty acid composition of erythrocyte membrane lipids.

The nutritional requirements of preterm infants for the long chain polyunsaturated essential fatty acids, arachidonic acid (AA) and docosahexaenoic acid (DHA), have not been clearly defined. The present study evaluated preterm infants of less than 2.3 kg birth weight fed a commercial formula (Preemie SMA) devoid of AA and DHA and compared this control group with similar infant groups fed one of three formulas containing a range of 0.32 to 1.1% AA and 0.24 to 0.76% DHA. An analogous group of infants fed their mothers' breast milk and a breast milk fortifier (when indicated) was also studied. Erythrocyte membrane phospholipids were isolated from blood samples collected at 12 d of age and after a further 4 wk of feeding. Infants fed the formula without AA and DHA showed a reduction in AA level in erythrocyte phosphatidylcholine, and a reduced level of DHA in phosphatidylethanolamine in comparison with infants fed breast milk or infant formula containing AA and DHA. Supplementing infant formula with increasing levels of AA and DHA produced a clear dose response in the levels of AA and DHA found in erythrocyte membrane phospholipids. From comparison of membrane phospholipid fatty acid composition it appears that a formula level of 0.32-1.1% AA and 0.24-0.76% DHA provides sufficient levels of these fatty acids to achieve a similar fatty acid composition to that of infants fed human milk for most of the lipid fractions examined.

[Indicator of adrenoreceptors in cell membranes: reference values and informative validity in the assessment of the functional state of the cardiovascular system]. / Pokazatel' adrenoretseptsii kletochnykh membran: referentnye velichiny i informativnost' v otsenke funktsional'nogo sostoianiia serdechno-sosudistoi sistemy.

A new method of individual determination of sympathoadrenal activity (SAA) uses the parameter of beta-adrenoreception in cell membranes (beta-ARM) based on the erythrocyte model. Correlation of an individual reference beta-ARM value with specific features of hypertension states, and CVS responses to psychoemotional stress was studied. Described are the method, and normal distribution of individual beta-ARM values in healthy flyers, aviation students, non-flyers, and aviators with diagnosed the neurocirculatory dystonia of hypertensive type and patients with the hypertonic disease of 2nd stage (the overall numbers of subjects = 221). Another group of healthy subjects and patients with the hypertonic disease of 1st stage and different reference beta-ARM values (n = 39) participated in determination of HR and BP levels during the verbal count test. On the evidence of individual beta-ARM distribution in the test groups, the upper limit of the beta-ARM physiological norm (16.0 arbitrary units) was suggested to be a critical parameter in diagnosis of a hyperadrenergic state. Analysis of hemodynamics and the quality of verbal count during performance of a psychoemotional test by subjects with varying reference beta-ARM values revealed hyperreactive cardiovascular systems in and less successful fulfillment of the count test by subjects with high beta-ARM.

Sodium transport in hypertension: assessment of membrane-associated defects in South African black and white hypertensives.

Sodium transport was studied in red cells taken from four groups of patients, whites or blacks, and normotensive or hypertensive, with approximately ten patients in each group. There were no differences between normotensive and hypertensive whites. Normotensive blacks had higher rates of sodium-proton exchange than did the other groups, and hypertensive blacks had higher rates of efflux via the sodium-potassium ATPase than did other groups. These data support proposals that hypertension in black subjects is linked to abnormalities of control of intracellular sodium.

Lateral diffusion in a mixture of mobile and immobile particles. A Monte Carlo study.

The lateral diffusion coefficient for mixtures of mobile and immobile particles is obtained from Monte Carlo calculations of random walks by mobile tracers in the presence of immobile obstacles on a triangular lattice. The diffusion coefficient of the mobile species is obtained as a function of the area fractions of mobile and immobile species. The results are applied to diffusion of band 3 in the erythrocyte membrane, and indicate that obstruction of diffusion of mobile band 3 by band 3 and glycophorin attached to the membrane skeleton is not sufficient to explain the observed diffusion coefficient.