Kinetics of linear rouleaux formation studied by visual monitoring of red cell dynamic organization

Red blood cells (RBCs) in the presence of plasma proteins or other macromolecules may form aggregates, normally in rouleaux formations, which are dispersed with increasing blood flow. Experimental observations have suggested that the spontaneous aggregation process involves the formation of linear rouleaux (FLR) followed by formation of branched rouleaux networks. Theoretical models for the spontaneous rouleaux formation were formulated, taking into consideration that FLR may involve both "polymerization," i.e., interaction between two single RBCs (e + e) and the addition of a single RBC to the end of an existing rouleau (e + r), as well as "condensation" between two rouleaux by end-to-end addition (r + r). The present study was undertaken to experimentally examine the theoretical models and their assumptions, by visual monitoring of the spontaneous FLR (from singly dispersed RBC) in plasma, in a narrow gap flow chamber. The results validate the theoretical model, showing that FLR involves both polymerization and condensation, and that the kinetic constants for the above three types of intercellular interactions are the same, i.e., k(ee) = k(er) = k(rr) = k, and for all tested hematocrits (0.625-6%) k < 0.13 +/- 0.03 s(-1).     

Protein-ligand binding studies with a table-top,air-driven,high-speed centrifuge

Procedures developed earlier for the ultracentrifuge in order to study the binding of low molecular weight ligands to proteins have been adapted for use with a relatively inexpensive, table-top, air-driven centrifuge known as the Airfuge. This instrument, which holds six plastic tubes with a total capacity of 1 ml, generates such high centrifugal fields (up to 160,000 times that of gravity) that proteins are readily sedimented to the bottom of the tubes, leaving unbound ligand in the supernatant. Hence, direct sampling and analysis of the solution at the conclusion of the centrifuge experiment and knowledge of the total concentration of ligand permit a quantitative determination of the amount of ligand bound to the protein. The method depends on the use of dextran in the solution in order to provide density stabilization and prevent serious convective stirring of the contents of the tubes during the deceleration of the rotor. Two systems were studied as a test of the technique and it was found that the centrifuge method yields results comparable to those obtained by equilibrium dialysis. With aspartate transcarbamoylase and CTP, conditions were obtained (100,000 rpm for 30 min) such that the enzyme and enzyme-CTP complexes were sedimented rapidly to the bottom of the tubes, leaving free CTP distributed throughout the solution. In contrast to this sedimentation velocity experiment, studies were also made with RNase and 5′-AMP. The procedure for this system involves sedimentation equilibrium and the protein, although not completely removed from the top of the solution, is distributed predominantly at the bottom of the tubes as unbound ligand remains in the supernatant. For such systems, it is possible to estimate theoretically the effects of the size of the ligand and it is shown that re-equilibration causes only minor complications for ligands of molecular weight less than 1000. The method is simple, uses only small amounts of proteins and ligands, requires only short times for proteins of molecular weight about 10⁵, and shows promise of providing binding data with an accuracy comparable to other techniques.     

Crystallization of biological macromolecules from flash frozen samples on the Russian Space Station Mir

One hundred eighty-three flash frozen, liquid-liquid diffusion and batch method protein and virus crystallization samples were launched aboard the Space Shuttle Discovery on June 27 (STS-71) and transferred to the Russian Space Station Mir on July 1, 1995. They were returned to earth November 20, 1995 (STS-74). Subsequent examination showed that of the 19 types of proteins and viruses investigated, 17 were crystallized during the period on Mir. The experiment demonstrates the utility of this very simple and inexpensive approach for the crystallization of biological macromolecules in space over extended time periods. The distribution of crystals among the three types of containers used indicated small samples yielded results equal or better than larger samples and that long diffusion path lengths were clearly better. Distribution of crystals within the container tubes showed a striking gradient of quality and size that indicated long, narrow tubes yield superior crystals, as predicted from other work based on crystallization in capillaries.     

Execution of "ARC" experiment on space shuttle "Discovery" STS 51-C: some results on aggregation of red blood cells under zero gravity

A project on "Aggregation of Red Cells" has been accepted by NASA in 1977. An automated slit-capillary photo-viscometer has been designed during 1979-1984, and its last version met NASA's space hazards requirements. The 'heart' of instrument is a set of two highly polished glass plates, spaced by a gap of 12.5 micrometers. An original drum-like infusion pump allows utilization of up to eight blood samples. During a sequential process, blood flows through the slit, and then stops to allow formation of aggregates. Micro- and macro-photography is carried out, and 500 photographs are obtained. Blood from normal donors and patients with history of ischaemic heart disease, colon cancer, juvenile-onset diabetes, hyperlipidaemia, etc., is anticoagulated and adjusted to haematocrit of 0.30 using native plasma. Samples are divided, and infused into the 'flight' and 'ground' instruments. Prior to experiment temp. is 5 degrees C; temp. during experiment is 25 degrees C. Experiments took place on 24-25 January 1985, on the middeck of space shuttle 'Discovery'. Subsequent results showed that red blood cells do not change shape under zero gravity; that aggregation of red cells does take place; that aggregates in pathologic blood show morphology of normal rouleaux under zero gravity, while identical blood shows clumps of red cells on the ground. The latter observation suggests that zero gravity might affect cell-to-cell interaction, and perhaps microstructure of the cell membrane. These aspects must remain however tentative till a confirmation by subsequent experiments can be obtained.     

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 shear rate on rouleau formation in simple shear flow

A kinetic equation for rouleau formation in a simple shear flow is derived, based on several assumptions. These are (a) colliding rouleaux stick to one another with a certain probability to form a single rouleau; (b) simultaneous collisions between more than two rouleaux are negligible; (c) rouleaux are broken by a viscous force exerted by the suspending fluid on the surfaces of rouleaux; (d) when a rouleau is broken by viscous forces, only two fragments are formed. Based on a simple mathematical model, collision rate, sticking probability and degradation rate are obtained as functions of applied shear rate. From the solution of the kinetic equation, the average size of rouleaux is obtained as a function of time with shear rate as a parameter. It is shown that the average size of rouleaux increases monotonically with increasing time and tends to an equilibrium size. The average size of rouleaux in a dynamical equilibrium decreases monotonically with increasing shear rate and tends to one cell as shear rate approaches infinity. It is also found that the initial rate of rouleau formation increases with increasing shear rate at very low shear rate, but this trend is reversed at higher shear rates. The theoretical results are compared quantitatively with experimental data.     

Theory of long-range coherence in biological systems. I. the anomalous behaviour of human erythrocytes

In this paper, an explicit expression of the interaction potential has been obtained, based on the Fröhlich model of long-range coherence in biological cells. These theoretical expressions are used to correlate with the experimental data obtained from the light scattering and the Brownian motion for the red blood cells (human erythrocytes). The necessary conditions are derived for the formation of rouleaux in human erythrocytes in the presence of long range interactions. The kinetics of rouleaux formation in terms of the properties of the diffusion coefficient has been investigated. It is concluded that both types of experimental data can be interpreted satisfactorily by this model.     

Geotaxis in protozoa I. A propulsion-gravity model for Tetrahymena (Ciliata)

A propulsion-based model for negative geotaxis of ciliated protozoa is presented which views geotaxic reorientation as the unbalancing of gyrational torque by a sedimentation torque. The balanced gyrational torque results from the location of the propulsive center of effort forward of the body center of mass. When gravity is ignored, the propulsive forces generating the gyrational moments may be confined to an envelope surrounding the cell. The effect of gravity is to induce sedimentation of the body-plus-envelope system. Viscous resistance to this sedimentation at the envelope “surface” is transmitted to the beating cilia whose net constant energy output must now deal with a new source of dissipation (not “present” when gravity was ignored) which is maximal in the downswing portion of the gyration cycle. In such a manner sedimentation resistance acts as a counter torque to the downswing gyrational moment of force and an enhancing torque to the upswing moment thereby generating a net upward reorientation of the gyrational axis. Upon addition of the translational component of propulsion, the negative geotaxis behavior pattern is completed. The forward location of the center of effort which provides the basic validity indicator for the model is verified by observations from the ciliate Tetrahymena.     

Effects of hematocrit on thixotropic properties of human blood

The rheological properties of whole human blood exhibit thixotropic behavior at low shear rates up to about ten reciprocal seconds (1). The accepted cause of this shear rate-dependent and time-dependent behavior is the progressive breakdown of rouleaux into individual red cells. Huang developed a rheological equation which incorporates the kinetics of rouleau breakdown in his models (2). This five-parameter equation was used successfully to represent the hysteresis loop and the torque-decay curve of whole human blood. Numerical values of these five thixotropic parameters, which characterize the rheological behavior of the blood from apparently healthy human subjects, were established (3). In this communication, we examined the effect of hematocrit on each of the above mentioned parameters. The results show that the following parameters will increase their values with an increase in hematocrit: the yield stress, Newtonian contribution of viscosity, non-Newtonian contribution of viscosity, apparent viscosity and the equilibrium value of the structural parameter which indicates the relative amount of rouleaux in blood. Mathematical equations were developed to give the relationship between parameters and hematocrit. Two other thixotropic parameters, viz. the kinetic rate constant of rouleaux breakdown into individual red cells and the order of the breakdown reaction, were found to be independent of the hematocrit. It is consistent with reaction kinetic theory that the rate constant and the order of reaction are independent of the concentration of reactants.     

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.     

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.     

The kinesin-immunoreactive homologue from Nicotiana tabacum pollen tubes: Biochemical properties and subcellular localization

In plant cells, microtubule-based motor proteins have not been characterized to the same degree as in animal cells; therefore, it is not yet clear whether the movement of organelles and vesicles is also dependent on the microtubular cytoskeleton. In this work the kinesinimmunoreactive homologue from pollen tubes of Nicotiana tabacum L. has been purified and biochemically characterized. The protein preparation mainly contained a polypeptide with a relative molecular weight of approx. 100 kDa. This polypeptide bound to animal microtubules in an ATP-dependent manner and it further copurified with an ATPase activity fourfold-stimulated by the presence of microtubules. In addition, the sedimentation coefficient (approx. 9S) was similar to those previously shown for other kinesins. Immunofluorescence analyses revealed a partial co-distribution of the protein with microtubules in the pollen tube. These data clearly indicate that several properties of the kinesin-immunoreactive homologue are similar to those of kinesin proteins, and suggest that molecular mechanisms analogous to those of animal cells may drive the microtubule-based motility of organelles and vesicles in plants.Abbreviations AE-LPLC anion-exchange low-pressure liquid chromatography - AMPPNP 5-adenylylimidodiphosphate - PKH pollen kinesin homologue - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Characterization of a CV-1 cell cycle. V. An assessment of velocity sedimentation for cell separation and synchrony.

Velocity sedimentation at unit gravity has been used to enrich populations of logarithmically growing cells in different cell cycle phases. In order to evaluate the degree of synchrony obtained by this method of cell separation, synchronous populations of CV-1 cells, initially obtained by the selective detachment of mitotic cells from roller cultures, were separated by velocity sedimentation. It was found that although the mean cell volume increased linearly, the cells remained heterogeneous with respect to size during all phases of the cell cycle. Since the velocity sedimentation technique depends upon discrimination of cell size, the size heterogeneity of cells throughout the cycle limits the degree of synchrony which can be obtained by this method.     

Joining forces: The interface of gravitropism and plastid protein import

In flowering plants, gravity perception appears to involve the sedimentation of starch-filled plastids, called amyloplasts, within specialized cells (the statocytes) of shoots (endodermal cells) and roots (columella cells). Unfortunately, how the physical information derived from amyloplast sedimentation is converted into a biochemical signal that promotes organ gravitropic curvature remains largely unknown. Recent results suggest an involvement of the Translocon of the Outer Envelope of (Chloro) plastids (TOC) in early phases of gravity signal transduction within the statocytes. This review summarizes our current knowledge of the molecular mechanisms that govern gravity signal transduction in flowering plants and summarizes models that attempt to explain the contribution of TOC proteins in this important behavioral plant growth response to its mechanical environment.Key words: gravitropism, root, amyloplast, TOC complex, TOC132, TOC75     

Biomechanical and Histological Evaluation of Roughened Surface Titanium Screws Fabricated by Electron Beam Melting

Background

Various fabrication methods are used to improve the stability and osseointegration of screws within the host bone. The aim of this study was to investigate whether roughened surface titanium screws fabricated by electron beam melting can provide better stability and osseointegration as compared with smooth titanium screws in sheep cervical vertebrae.

Methods

Roughened surface titanium screws, fabricated by electron beam melting, and conventional smooth surface titanium screws were implanted into sheep for 6 or 12 weeks (groups A and B, respectively). Bone ingrowth and implant stability were assessed with three-dimensional imaging and reconstruction, as well as histological and biomechanical tests.

Results

No screws in either group showed signs of loosening. Fibrous tissue formation could be seen around the screws at 6 weeks, which was replaced with bone at 12 weeks. Bone volume/total volume, bone surface area/bone volume, and the trabecular number were significantly higher for a define region of interest surrounding the roughened screws than that surrounding the smooth screws at 12 weeks. Indeed, for roughened screws, trabecular number was significantly higher at 12 weeks than at 6 weeks. On mechanical testing, the maximum pullout strength was significantly higher at 12 weeks than at 6 weeks, as expected; however, no significant differences were found between smooth and roughened screws at either time point. The maximum torque to extract the roughened screws was higher than that required for the smooth screws.

Conclusions

Electron beam melting is a simple and effective method for producing a roughened surface on titanium screws. After 12 weeks, roughened titanium screws demonstrated a high degree of osseointegration and increased torsional resistance to extraction over smooth titanium screws.     

Rouleaux formation as a measure of the phase separating ability of plasma

The increased erythrocyte sedimentation rate (ESR) occurring in various diseases reflects a change in certain plasma proteins which causes erythrocytes to aggregate into rapidly sedimenting “piles of coins”, or rouleaux. In spite of some 300 years of study, the mechanism of the phenomenon and the adaptive significance, if any, of the change in properties of plasma, remain elusive. One hypothesis on the mechanism is that rouleaugenic agents in plasma act as multivalent “agglomerins”, which react with the erythrocyte surface and cross-link cells. Alternatively, a “phase separation” hypothesis can be derived by analogy with the phase separation of high molecular weight polymers from polymer mixtures at high polymer concentrations. Considering the cell surface to resemble that of a large polymer, it is postulated that there should be a separation of a cell phase from a solution phase in the presence of high concentrations of a high molecular weight polymer. In keeping with the available data, both hypotheses predict that rouleaux formation should increase with increasing polymer size. In addition, the phase separation hypothesis predicts (i) no need for reaction of a rouleaugenic agent with the erythrocyte surface, and (ii) that cells from different species, when mixed in the presence of a rouleaugenic agent, will form independent rouleaux. Current evidence is consistent with both predictions. We suggest that an increased ESR reflects a change in the physical properties of plasma such that particulate material (e.g. viruses, tumour cells), will tend to spontaneously aggregate in regions of stagnant blood flow (such as the sinusoids of the spleen). This change in the physical properties of plasma, tending to favour phase separations, will also favour antigen-antibody and other intermolecular reactions.     

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.     

Dielectric approach to investigation of erythrocyte aggregation. II. Kinetics of erythrocyte aggregation-disaggregation in quiescent and flowing blood

A method based on dielectric properties of dispersed systems was applied to investigate the kinetics of RBC aggregation and the break-up of the aggregates. Experimentally, this method consists of measuring the capacitance at a frequency in the beginning of the beta-dispersion. Two experimental protocols were used to investigate the aggregation process. In the first case, blood samples were fully dispersed and then the flow was decreased or stopped to promote RBC aggregation. It was found that the initial phases of RBC aggregation are not affected by the shear rate. This finding indicates that RBC aggregation is a slow coagulation process. In the second case, RBCs aggregated under flow conditions at different shear rates and after the capacitance reached plateau levels, the flow was ceased. The steady-state capacitance of the quiescent blood and the kinetics of RBC aggregation after stoppage of shearing depend on the prior shear rate. To clarify the reasons for this effect, the kinetics of the disaggregation process was studied. In these experiments, time courses of the capacitance were recorded under different flow conditions and then a higher shear stress was applied to break up RBC aggregates. It was found that the kinetics of the disaggregation process depend on both the prior and current shear stresses. Results obtained in this study and their analysis show that the kinetics of RBC aggregation in stasis consists of two consecutive phases: At the onset, red blood cells interact face-to-face to form linear aggregates and then, after an accumulation of an appropriate concentration of these aggregates, branched rouleaux are formed via reactions of ends of the linear rouleaux with sides of other rouleaux (face-to-side interactions). Branching points are broken by low shear stresses whereas dispersion of the linear rouleaux requires significantly higher energy.