Segregation into separate rouleaux of erythrocytes from different species. Evidence against the agglomerin hypothesis of rouleaux formation.

Erythrocytes from one species were labelled with fluorescein isothiocyanate and mixed with unlabelled erythrocytes from another species. Albumin polymers were added to generate rouleaux. The species of origin of erythrocytes in rouleaux was determined by fluorescence microscopy. Erythrocytes from different species segregated into independent rouleaux. However, fluorescent and non-fluorescent erythrocytes from one individual were mixed randomly in rouleaux. These results confirm, using a novel experimental approach, previous observations of Sewchand & Canham [(1976) Can. J. Physiol. Pharmacol. 54, 437-442]. Since rouleaugenic agents are not species-specific, under the 'agglomerin' hypothesis of rouleau formation they would be expected to form bridges between cells from different species. It follows that either the agglomerin hypothesis is incorrect, or additional species-specific surface components are involved in the aggregation of agglomerin-cross-bridged cells.     

Effect of binding on melting transitions in polymer–diluent systems

An expression is derived for the melting point of a polymer when in equilibrium with a solution in which binding of low molecular weight compounds to the polymer takes place. Allowance is made for the possibility that the crystalline polymer itself is a complex. The argument is a purely thermodynamic one and is based on a consideration of the change in free energy as a result of a change in binding. Allowance is made also for non-specific polymer–solvent interactions, in which the mixture of low molecular weight solvents is treated as a single solvent. Special attention is paid to “inverted” melting transitions, i.e., cases in which the melting point increases with increasing dilution of the polymer. It is shown that as a rule this is accompanied by a corresponding, inverted effect of the solvent composition on the melting point. It is further shown that-in the absence of binding, “normal” behavior at the critical point (i.e., phase separation is induced by lowering the temperature) is always accompanied by “normal” melting behavior (i.e., a decrease in melting point when the polymer is diluted). Also, “inverted” melting always implies that phase separation at the critical point is induced by heating, but the reverse is not necessarily true.     

Influence of fibrinogen and haematocrit on erythrocyte sedimentation kinetics

This study investigates the influence of haematocrit, fibrinogen concentration and fibrinogen availability (amount of fibrinogen per red blood cell) on erythrocyte sedimentation. The Westergren technique was applied to blood samples from 36 subjects and to their blood manipulated to haematocrits of 10, 20, 30 and 40%. Readings were taken every 10 minutes for 300 minutes. Previous studies indicate that erythrocyte sedimentation occurs in three phases. In this study, we show that haematocrit has little influence on either the rate of fall of particles in the first phase (m1) or the duration of the first phase. This is also true for fibrinogen availability and for fibrinogen concentration at low haematocrits. At high haematocrits m1 increases with fibrinogen concentration. The rate of fall of rouleaux during phase 2 (m2) and ESR60 both decrease exponentially with haematocrit and increase linearly with fibrinogen concentration. While m2 is more closely correlated to fibrinogen availability than to fibrinogen concentration or to haematocrit, this is not the case for ESR60. Thus haematocrit, fibrinogen concentration and fibrinogen availability are more important to the velocity of sedimentation in the second phase than to the sedimenting velocity during phase 1 or to the duration of phase 1.     

Why do Polyethylene Glycol-Coated Liposomes Circulate So Long?: Molecular Mechanism of Liposome Steric Protection with Polyethylene Glycol: Role of Polymer Chain Flexibility

Abstract

The hypothesis is suggested describing the molecular mechanism of protective action of poly(ethylene glycol) on liposomes in vivo on the basis of polymer properties in solvent. The protective layer of polymer on the liposome surface is considered as a “cloud” of possible conformations of macromolecules. If polymer is water-soluble and has flexible main chain, the density of this cloud is high enough to prevent the interaction of opsonins with liposome. At the same time, certain optimal concentration of the protective polymer can be found, when more loose areas in polymeric “clouds” can be used for the immobilization of antibodies on liposomes. As a result, long-circulating targeted liposomes can be obtained.     

Electrophoretic and aggregation behavior of bovine, horse and human red blood cells in plasma and in polymer solutions

The electrophoretic mobility of native and glutaraldehyde-fixed bovine, human, and horse red blood cells (RBC) was investigated as a function of ionic strength (5-150 mM) and concentration of 464 kDa dextran (2 and 3 g/dl); RBC aggregation in autologous plasma and in dextran solutions was also measured. In agreement with previous observations, human and horse RBC form stable rouleaux whereas bovine RBC do not aggregate in either plasma or in dextran 464 kDa solutions. Electrophoretic measurements showed a species-dependent adsorption and depletion of dextran that can be theoretically evaluated. Adsorption of polymer is not a prerequisite for RBC aggregation (bovine RBC show the highest amount of adsorbed dextran yet do not aggregate). Aggregate formation thus occurs as long as the Gibbs free energy difference, given by the osmotic pressure difference between the bulk phase and the polymer-depleted region between two RBC, is larger than the steric and electrostatic repulsive energy contributed by the macromolecules present on the RBC surface. With increasing bulk-phase polymer concentration the depletion layer thickness decreases and the amount of adsorbed macromolecules increases, thereby resulting in an increase of the repulsive component of the interaction energy and decreased aggregation. We thus view electrophoretic measurements of RBC in various media as an important tool for understanding polymer behavior near the red cell surface and hence the mechanisms involved in RBC aggregation.     

Modulated growth,stability and interactions of liquid-like coacervate assemblies of elastin

Elastin self-assembles from monomers into polymer networks that display elasticity and resilience. The first major step in assembly is a liquid–liquid phase separation known as coacervation. This process represents a continuum of stages from initial phase separation to early growth of droplets by coalescence and later “maturation” leading to fiber formation. Assembly of tropoelastin-rich globules is on pathway for fiber formation in vivo. However, little is known about these intermediates beyond their size distribution. Here we investigate the contribution of sequence and structural motifs from full-length tropoelastin and a set of elastin-like polypeptides to the maturation of coacervate assemblies, observing their growth, stability and interaction behavior, and polypeptide alignment within matured globules. We conclude that maturation is driven by surface properties, leading to stabilization of the interface between the hydrophobic interior and aqueous solvent, potentially through structural motifs, and discuss implications for droplet interactions in fiber formation.     

Estrogen receptor 1 modulates circadian rhythms in adult female mice

Estradiol influences the level and distribution of daily activity, the duration of the free-running period, and the behavioral phase response to light pulses. However, the mechanisms by which estradiol regulates daily and circadian rhythms are not fully understood. We tested the hypothesis that estrogens modulate daily activity patterns via both classical and “non-classical” actions at the estrogen receptor subtype 1 (ESR1). We used female transgenic mice with mutations in their estrogen response pathways; ESR1 knock-out (ERKO) mice and “non-classical” estrogen receptor knock-in (NERKI) mice. NERKI mice have an ESR1 receptor with a mutation in the estrogen-response-element binding domain, allowing only actions via “non-classical” genomic and second messenger pathways. Ovariectomized female NERKI, ERKO, and wildtype (WT) mice were given a subcutaneous capsule with low- or high-dose estradiol and compared with counterparts with no hormone replacement. We measured wheel-running activity in a light:dark cycle and constant darkness, and the behavioral phase response to light pulses given at different points during the subjective day and night. Estradiol increased average daily wheel-running, consolidated activity to the dark phase, and shortened the endogenous period in WT, but not NERKI and ERKO mice. The timing of activity onset during entrainment was advanced in all estradiol-treated animals regardless of genotype suggesting an ESR1-independent mechanism. We propose that estradiol modifies period, activity level, and distribution of activity via classical actions of ESR1 whereas an ESR1 independent mechanism regulates the phase of rhythms.     

Dependence of dielectrophoretic force on the size of cylindrical particles by the example of a suspension of erythrocytes

For the modeling of erythrocyte rouleaux (linear cell aggregations), we propose an approximation procedure for the dipole moment in short cylinders, which contains the case of ellipsoidal bodies only as a first approximation. The method allows one to introduce corrections that are more representative for these particles. Depending on the number of erythrocytes forming an aggregation, i.e., on different but discrete measures of rouleaux lengths, the dielectrophoretic force is calculated and represented against the frequency of the applied a.c. field. As a result, frequency regions in the upper MHz range appear in which the strength and direction of DEP forces are different for different rouleaux sizes. This property is suitable for the detection and spatial separation of rouleaux populations of different length in blood samples by means of a microelectronic array.     

Pendular activity of human upper limbs during slow and normal walking

When walking at normal and fast speeds, humans swing their upper limbs in alternation, each upper limb swinging in phase with the contralateral lower limb. However, at slow and very slow speeds, the upper limbs swing forward and back in unison, at twice the stride frequency of the lower limbs. The change from “single swinging” (in alternation) to “double swinging” (in unison) occurs consistently at a certain stride frequency for agiven individual, though different individuals may change at different stride frequencies. To explain this change in the way we use our upper limbs and individual variations in the occurrence of the change, the upper limb is modelled as a compound pendulum. Based on the kinematic properties of pendulums, we hypothesize that the stride frequency at which the change from “single swinging” to “double swinging” occurs will be at or slightly below the natural pendular frequency (NPF) of the upper limbs. Twenty-seven subjects were measured and then filmed while walking at various speeds. The mathematically derived NPF of each subject's upper limbs was compared to the stride frequency at which the subject changed from “single swinging” to “double swinging.” The results of the study conform very closely to the hypothesis, even when the NPF is artificially altered by adding weights to the subjects' hands. These results indicate that the pendulum model of the upper limb will be useful in further investigations of the function of the upper limbs in human walking. © 1994 Wiley-Liss, Inc.     

Dependence of dielectrophoretic force on the size of linear erythrocyte aggregates in suspension

For modeling of erythrocyte rouleaux (linear cell aggregates) we develop an approximation procedure for the dipole moment in short cylinders, which contains the case of ellipsoidal bodies as a first approximation, but allows corrections for short cylinders, more representative for such particles. In dependence on the number of erythrocytes in an aggregation, i.e., on different but discrete rouleaux lengths, the dielectrophoretic force is calculated and represented against the frequency of the applied AC field. Predictions are made for frequency regions in the 107–108 Hz range where the magnitude and the direction of dielectrophoretic forces is different for different rouleaux sizes. This property can be used for the detection and spatial separation of rouleaux populations of different length in a microelectronic array.     

Kinetics of rouleau formation. I. A mass action approach with geometric features.

In the presence of certain macromolecules, such as fibrinogen, immunoglobulin, dextran, and polylysine, erythrocytes tend to aggregate and form cylindrical clusters called "rouleaux" in which cells resemble coins in a stack. The aggregates may remain cylindrical or they may branch, forming tree, and networklike structures. Using the law of mass action and notions from polymer chemistry, we derive expressions describing the kinetics of the early phase of aggregation. Our models generalize work initiated by Ponder in 1927 who used the Smoluchowski equation to predict the concentration of rouleaux of different sizes. There are two novel features to our generalization. First, we allow erythrocytes that collide near the end of a stack of cells to move to the end of the cylinder and elongate it. Second, we incorporate geometric information into our models and describe the kinetics of branched rouleau formation. From our models we can predict the concentration of rouleaux with n cells and b branches, the mean number of cells per rouleau, the mean number of branches per rouleau, and the average length of a branch. Comparisons are made with the available experimental data.     

Volume change on formation of native collagen aggregate

The volume change which occurs in dilute tropocollagen solution as a result of the phase transition producing the “native” form of collagen aggregate has been measured dilatometrically. A volume increase of 0.8 × 10^?3 ml./g. collagen in phosphate buffer (pH 7–7.5) was determined. The volume expansion is attributed to a reduction in the organization of water molecules around nonpolar surfaces of the individual tropocollagen units. This volume expansion is consistent with a previous hypothesis that hydro-phobic bonding is the driving force in this collagen aggregation.     

A model for rouleaux pattern formation of red blood cells

Human red blood cells (RBCs) in a solution form rouleaux patterns under various conditions. The degree of rouleaux formation depends on, for example, the concentration and molecular weight of added large molecules. We present a two-dimensional discrete cellular space model in which an RBC is represented by a rectangle and differential adhesion is assumed among the longer (a-site), the shorter (b-site) sides of the rectangle and the solvent. The total sum of the adhesion energy is assumed to guide the step-by-step change of the model cell configuration and also define absolutely stable patterns. We compare the set of absolutely stable patterns and cell aggregate patterns for both actual and computer-simulated cases to obtain the basic validity of our framework. Then we proceed to assess the effects of added high polymers to the adhesion parameters. We first note that under suitable conditions, decrease in a-site-solvent affinity is necessary to have complex patterns rather than increase of a-a affinity. The hypothesis that addition of high polymers reduce the a-site-solvent affinity is concomitant with a newly proposed osmotic stress theory. The parameter fitting results for the experimental phase change curves can also be interpreted as supporting more the new theory than existing traditional explanations.     

A covalently bonded teicoplanin chiral stationary phase for HPLC enantioseparations

A macrocyclic glycopeptide antibiotic containing a hydrophobic “tail” is covalently attached to silica gel via linkage chains. This material is extensively evaluated as a chiral stationary phase (CSP) for HPLC. The relevant structural features of the teicoplanin molecule which make it an effective chiral selector are discussed. The teicoplanin CSP appears to have excellent enantioselectivity for native amino acids, peptides, α-hydroxycarboxylic acids, and a variety of neutral analytes including cyclic amides and amines. Enantio-separations can be achieved in the reversed phase, normal phase, and “polar-organic” modes. This chiral selector is stable and the integrity of the CSP is excellent in all separation modes. Hence it can be considered a highly effective multimodal column. Optimization of these separations is discussed in terms of both selectivity and efficiency. Results indicate that the surface loading of the chiral selector affects all relevant separation parameters. A hypothesis is proposed to explain the enhanced efficiency obtained when using teicoplanin CSPs with higher surface coverage. It appears that teicoplanin is a widely applicable, highly effective chiral selector for HPLC enantioseparations. © 1995 Wiley-Liss, Inc.     

肠淋巴液引流对失血性休克大鼠红细胞流变性的影响

目的:观察肠淋巴液引流对失血性休克大鼠红细胞流变性指标以及血液黏度的作用。方法:Wistar雄性大鼠均分为假休克组、休克组(复制失血性休克模型)、引流组(复制失血性休克模型,自低血压1 h引流休克肠淋巴液)。在低血压3 h或相应时间,经腹主动脉取血,检测红细胞参数、红细胞电泳、红细胞沉降率(ESR)以及血液黏度,计算红细胞聚集指数、红细胞变形指数。结果:与假休克组比较,休克组红细胞数量、红细胞比积(HCT)、血红蛋白(Hb)、平均红细胞血红蛋白浓度(MCHC)、红细胞电泳率与迁移率、红细胞变形指数、全血黏度、全血低切与高切相对黏度和还原黏度显著降低,休克组平均红细胞体积、红细胞电泳时间、ESR、血沉方程K值与校正K值、红细胞聚集性指数、血浆黏度显著升高;引流组MCHC、红细胞电泳率与迁移率、全血黏度、全血低切与高切还原黏度均显著降低,引流组红细胞体积分布宽度(RDW-SD)显著增加。同时,引流组HCT、RDW-SD、红细胞变形指数、全血黏度、全血低切与高切相对黏度显著高于休克组;ESR、血沉方程K值与校正K值、红细胞聚集性指数、血浆黏度显著低于休克组。结论:休克肠淋巴液引流可改善失血性休克大鼠红细胞流变行为,从而改善血液流变性。     

Maintenance and mobility of hemoglobin and water within the human erythrocyte after detergent disruption of the plasma membrane.

Is an intact plasma membrane responsible for keeping hemoglobin and water within the human erythrocyte? If not, what is responsible? How free is Hb to move about within the erythrocyte? To answer these questions erythrocytes were taken for phase contrast microscopy, transmission electron microscopy (TEM), determination of water-holding capacity, and proton NMR studies both before and after membrane disruption with a nonionic detergent (Brij 58). Addition of 0.2% Brij to a D₂O saline solution of hemoglobin (Hb) caused particles of Hb to appear and to aggregate. This aggregation of Hb caused the amplitude of the Hb proton NMR spectra to decrease. Thus, the less mobile the Hb the lower the Hb proton spectra amplitude. Erythrocytes washed in D₂O saline showed proton NMR spectra of relatively low amplitude. Addition of Brij (0.2%) to these erythrocytes caused increased Hb mobility within these erythrocytes. The TEM of fixed and thin-sectioned erythrocytes treated with Brij showed disruption of the plasma membrane of all erythrocytes regardless of whether or not they had lost Hb. Brij-permeabilized erythrocytes washed in D₂O saline or in a D₂O K buffer maintained a higher heavy water-holding capacity upon centrifugation as compared to nonpermeabilized erythrocytes. The TEM of Brij-treated and washed erythrocyte “shells” revealed a continuous submembrane lamina but no other evidence of cytoskeletal elements. The water-holding capacity of the erythrocyte can be accounted for by the water-holding capacity of hemoglobin. The evidence favors a relatively immobile state of Hb and of water in the erythrocyte that is not immediately dependent on an intact plasma membrane but is attributed to interactions between Hb molecules and the submembrane lamina.     

Mesoscale organization of domains in the plasma membrane – beyond the lipid raft

The plasma membrane is compartmentalized into several distinct regions or domains, which show a broad diversity in both size and lifetime. The segregation of lipids and membrane proteins is thought to be driven by the lipid composition itself, lipid–protein interactions and diffusional barriers. With regards to the lipid composition, the immiscibility of certain classes of lipids underlies the “lipid raft” concept of plasmalemmal compartmentalization. Historically, lipid rafts have been described as cholesterol and (glyco)sphingolipid-rich regions of the plasma membrane that exist as a liquid-ordered phase that are resistant to extraction with non-ionic detergents. Over the years the interest in lipid rafts grew as did the challenges with studying these nanodomains. The term lipid raft has fallen out of favor with many scientists and instead the terms “membrane raft” or “membrane nanodomain” are preferred as they connote the heterogeneity and dynamic nature of the lipid-protein assemblies. In this article, we will discuss the classical lipid raft hypothesis and its limitations. This review will also discuss alternative models of lipid-protein interactions, annular lipid shells, and larger membrane clusters. We will also discuss the mesoscale organization of plasmalemmal domains including visible structures such as clathrin-coated pits and caveolae.     

Structural and functional changes in erythrocyte membranes in experimental atherosclerosis

Structural and functional characteristics of erythrocyte membranes were studied in rabbits with experimental atherosclerosis. In animals with single lipid spots in the aorta, a significant rise of the plasma cholesterol level was associated with the increased cholesterol/phospholipid (CS/PL) ratio and diminished activity of erythrocyte membrane Na+, K+-ATPase. EPMR spin probe data point to changes in structural membrane characteristics--an increase in order parameter for fatty acid chains of lipids and expansion of the temperature interval of the transition phase in the membranes. In rabbits with total aorta injury, a further increase both in the plasma cholesterol concentration and in the CS/PL ratio as well as in structural changes in erythrocyte membranes does not lead to another decrease in the enzymatic activity. In aorta homogenates of the experimental animals, the activity of Na+, K+-ATPase correlated with that in the erythrocyte membrane. This suggests the existence of similar chemical and structural changes in aorta cell membranes. The data may provide an indirect evidence in favour of the hypothesis of the involvement of smooth muscle cells and membrane enzymatic activity alterations in atherosclerosis.     

Kinetics of rouleau formation. II. Reversible reactions.

Red blood cells aggregate face-to-face to form long, cylindrical, straight chains and sometimes branched structures called rouleaux. Here we extend a kinetic model developed by R. W. Samsel and A. S. Perelson (1982, Biophys. J. 37:493-514) to include both the formation and dissociation of rouleaux. We examine thermodynamic constraints on the rate constants of the model imposed by the principle of detailed balance. Incorporation of reverse reactions allows us to compute mean sizes of rouleaux and straight chain segments within rouleaux, as functions of time and at equilibrium. Using the Flory - Stockmayer method from polymer chemistry, we obtain a closed-form solution for the size distribution of straight chain segments within rouleaux at any point in the evolution of the reaction. The predictions of our theory compare favorably with data collected by D. Kernick , A.W.L. Jay , S. Rowlands , and L. Skibo (1973, Can. J. Physiol. Pharmacol. 51:690-699) on the kinetics of rouleau formation. When rouleaux grow large, they may contain rings or loops and take on the appearance of a network. We demonstrate the importance of including the kinetics of ring closure in the development of realistic models of rouleaux formation.     

Effects of a homogeneous magnetic field on erythrocyte sedimentation and aggregation

Effects of a homogeneous static magnetic field on erythrocyte sedimentation rate (ESR) have been assessed by using the standard Westergren method. A magnetic field of 6.3 T in the vertical direction only slightly enhanced ESR in saline solution, which was consistent with an effect on cell orientation. On the other hand, the magnetic field greatly enhanced ESR in plasma. It took a long time (about 20 min) for an ESR change to occur in plasma in response to the magnetic field. The effects in plasma were too large to originate only from cell orientation and were clearly distinct from a magnetic field-induced Boycott effect under an inhomogeneous magnetic field. A morphological examination and the nonlinear time course of the sedimentation in plasma indicated that the magnetic field increased cell aggregation and thereby enhanced ESR in plasma. Bioelectromagnetics 18:215–222, 1997. © 1997 Wiley-Liss, Inc.