Investigation of surface and shape changes accompanying the membrane alteration responsible for the heat-induced lysis of human erythrocytes

Heat induces several successive events in erythrocyte membrane; the denaturation of spectrin at about 50°C, thermoporation at 62°C and denaturation of the anion channel at 67°C. The heat denaturation of major membrane proteins, spectrin and the anion channel, is not needed for the thermoporation which is involved in thermohemolysis. This study reports about the surface and shape changes which are specific for thermoporated membranes with spectrin and anion channel preserved intact. Thermoporation was produced exposing human erythrocytes to 39.5°C for 3 min in isotonic medium containing 18% (v/v) ethanol as membrane fluidizer and sucrose as osmotic protectant which prevents hemolysis (Ivanov, J. Therm. Biol. 1996). The control cells were processed similarly except that they were incubated at 23°C, thus avoiding thermoporation. In control and porated membranes the overall structure of spectrin and the anion channel was retained inasmuch as their enthalpies and denaturation temperatures were microcalorimetrically found preserved. Nevertheless, irregular shape, grainy surface and asymmetric spicules were apparent in porated cells through scanning electron microscopy. A decrease in the number of binding sites for Alcian blue and an increased binding of eosine was established in the membranes of porated cells. After poration the hexane/aqueous partition coefficient K_d of cells increased from 5 to about 220 and the electrophoretic mobility of cells decreased by about 25% indicating marked increase in cell surface hydrophobicity and a decrease in surface charge, respectively. In addition, adhesivity to hydrophobic interfaces and aggregability in low ionic media strongly increased after poration. In contrast to intact and control cells, the porated ones (all prefixed with 0.2% glutaraldehyde) made molecular contacts with inclined hydrophobic interfaces at low (5 mM NaCl) but not at high (150 mM NaCl) ionic media. Thus, the microtopological shape changes and exposure of new hydrophobic and charge groups over the outer cell surface, without major thermal unfolding, possibly indicates an irreversible redistribution of membrane material and disturbed lipid–protein complementation during thermoporation.     

Spherocyte shape transformation and release of tubular nanovesicles in human erythrocytes

We have studied dodecylmaltoside-induced echinocyte-spheroechincyte-spherocyte shape transformation and membrane vesiculation using transmission electron microscopy (TEM) on freeze-fracture replicas. It is indicated that spherical erythrocyte shape at higher dodecylmaltoside concentration is formed due to loss of membrane in the process where small, mostly tubular nanovesicles are released predominantly from the top of echinocyte and spheroechinocyte spicules.     

Relationship between melting behavior and morphological changes of semicrystalline polymers

The present study on the case of poly(hexamethylene succinate) is to provide a basis for a better understanding of the subtle relationship between melting behavior and morphological changes of semicrystalline polymers. The melting behavior and morphological changes of poly(hexamethylene succinate) during both isothermal secondary crystallization and annealing processes were investigated by DSC and SAXS. DSC results showed that, with increasing crystallization time or annealing time, the melting endotherm continuously shifted to higher temperature, which suggested that some minor structural or morphological changes must occur. However, almost no changes at all on the crystal thickness were observed from SAXS measurements. The observed evidence confirmed that the increase in the melting temperature is not attributed to crystal thickening but crystal perfection. More exactly, the rearrangement and smoothing of tie molecules at the folding surface result in the reduction of the fold surface free energy, which dominantly contributes to the increase in the melting peak temperature. The origin of the new endothermic peak observed after annealing at elevated temperature was also discussed. TMDSC results indicated that the annealing peak resulted from the enthalpy relaxation and devitrification transition of rigid amorphous fraction formed by the driving force of thermodynamic nonequilibrium, rather than usually regarded as the melting of thin lamellae or imperfect crystals formed by annealing secondary crystallization.     

Effect of the shape of human erythrocytes on the evaluation of the passive electrical properties of the cell membrane

The possible influence of the cell shape on the derivation of the passive electrical parameters of a biological cell membrane is discussed in light of two different models which describe the cell as a shelled ellipsoidal particle and as a biconcave disk obtained by the revolution of the Cassini oval, respectively. Whereas within the first model, the Laplace equation can be solved analytically, in the second one a numerical algorithm based on the boundary element method has been employed. We have compared the results obtained by these two different models in the case of normal human erythrocyte cell membrane, using radiowave dielectric spectroscopy measurements. Our findings show that, although in principle the cell shape might deeply affect the evaluation of the passive electrical parameters of the cell membrane, in the case of the erythrocyte shape modelled by the Cassini curve, only small deviations are evidenced in comparison to the values derived, as usually done in the dielectric spectroscopy of biological cell suspensions, from an ellipsoidal model analysis. This result gives further support to the reliability of the data reported in the literature based on an ellipsoidal shape erythrocyte model.     

Interaction of liposomes with human erythrocytes

Interaction of liposomes (phospholipid vesicles) with human erythrocytes was studied by means of a spectroscopic method. Transfer of hemoglobin between liposomes and erythrocytes was observed. This transfer was mediated by a migration of band 3 proteins. In this case, a transfer of band 4.5 also was observed by means of electrophoresis. An interaction of lipid monomers from the liposomes with the erythrocyte membranes seemed to be closely correlated to the transfer of these proteins. It was presumed that this interaction induced some changes in the molecular organization of the cell membranes around band 3, resulting in release of the proteins from the erythrocyte membranes.     

煤灰熔融特性与灰成分之间关系的矿物变化研究

通过在一种真实煤灰中添加不同的氧化物或直接用氧化物配制合成灰,探究了不同灰成分对灰熔融特性的影响规律。利用Fact Sage 7.0对不同灰分的熔融过程进行了热力学模拟,通过熔融过程中的矿物质变化为各种灰成分对熔融特性的影响规律提供理论依据。结果表明,氧化钠对灰熔点的降低作用源于钠长石和霞石对钙长石的取代;氧化镁含量的增加对灰熔点起先降低后升高的作用,当氧化镁含量超过一定时,产生的镁橄榄石能够升高灰熔点;硫对灰熔点的升高作用源于镁橄榄石和硫酸钙对透辉石的取代;氧化钙含量的增加对灰熔点起到先降低后升高的作用,当氧化钙含量超过一定时,硅从熔点较低的矿物质迁移到熔点较高的矿物质中,升高了灰熔点。在与硅氧单元体结合的过程中,氧化钠优先于氧化钙;与氧化钙和硅氧单元体结合的氧化物的优先级为:氧化铝氧化镁氧化铁。     

Relationship between collagen autofluorescence of the human cervix and menopausal status

The goal of this study was to evaluate the effect of different menopausal states (pre- and post-) on the endogenous fluorescence of normal cervical tissues. In particular, the average fluorescence as well as the interpatient and intrasample variability in the average fluorescence of the epithelium and stroma were evaluated as a function of pre- and postmenopausal states. High-resolution fluorescence images at excitation-emission wavelengths of 440, 520 nm and 365, 465 nm were obtained from epithelia and stroma of freeze-trapped cervical tissue blocks maintained at -196 degrees C. The fluorescence images were recorded using a low temperature optical scanner. Fluorescence images from a normal sample population (n = 27) were quantitatively analyzed, and the average epithelial and stromal fluorescence intensities were obtained. Data grouped according to menopausal status (pre- vs post-) showed statistically significant differences (P < 0.002) in stromal fluorescence. In particular, the cervical stroma of postmenopausal women showed (1) significantly greater average fluorescence and (2) greater interpatient and intrasample variability in the fluorescence, relative to that of premenopausal women. These results provide evidence for changes in collagen cross-linking with menopause.     

Transketolase from human erythrocytes. Purification and properties

Human erythrocyte transketolase (sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehyde-transferase) was purified 8200-fold by adsorption onto hydroxylapatite, DEAE-cellulose treatment, acetone fractionation, and chromatography on Sephadex G-100. The purified transketolase could not be separated from glyceraldehyde-3-phosphate dehydrogenase, whereas the latter enzyme could be isolated in a pure state. Its homogeneity is suggested by sedimentation velocity, sedimentation equilibrium, and acrylamide electrophoresis. A molecular weight of 136000 was found. The physicochemical properties of glyceraldehyde-3-phosphate dehydrogenase and transketolase are very similar. A molecular weight of 136000 is suggested for transketolase, although gel filtration with Sephadex G-100 gave only 104000 ± 10%. This discrepancy is a reflection of an interaction of transketolase with the gel filtration medium. The isoelectric point for transketolase as well as for glyceraldehyde-3-phosphate dehydrogenase, as determined by isoelectric focussing, was found to be around 8.5. The activity of the enzyme is close to the maximum for pH 7.5 to pH 8.6. Additions of thiamine pyrophosphate or other cofactors do not influence the activity. Several divalent cations were tested. Sulfate and phosphate inhibit transketolase approximately to 50% between 50 and 100 mM concentration. Thiamine was present in transketolase, as shown by a microbiological assay and by the thiochrome reaction. The activation energy for the formation of sedoheptulose-7-phosphate from xylulose-5-phosphate was estimated from rate measurements to be 11.2 kcal/mole in the temperature range from 5° to 55°.     

Induction of transient radioresistance in human erythrocytes

Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%), were irradiated with γ-rays with single and split doses under air or N₂O in order to determine the physicochemical changes caused by the dose inducing an increase in resistance to radiation-induced hemolysis.The obtained results showed that under the applied irradiation conditions, the dose of 0.4 kGy induced changes in erythrocytes, which were responsible for temporary resistance of erythrocytes to hemolysis. We concluded that the observed resistance is caused mainly by the structural changes in proteins.     

Analysis of molecular shape changes along reaction paths

In this work, a detailed discussion and illustrative example are given for a new conceptual approach to the notion of similarity between nuclear configurations. We analyze the changes in molecular shape, as described by a topological characterization of molecular envelope surfaces, along reaction paths. This approach provides an independent definition of formal species along the path, defined in terms of molecular shape, rather than based on the curvature properties of the potential energy function. The two approaches are compared in this work, using the [1,3] hydrogen-shift in formic acid as an illustrative example. The reaction is studied at SCF and MP 2 ab initio levels. Although the actual reaction paths are different, the molecular shape changes encountered along them are similar, indicating that the essential shape changes provide chemical information on a more fundamental level than do detailed reaction paths.     

Photochemically driven shape changes of crystalline organic nanorods

Nanorods composed of 9-tert-butylanthroate (9-TBAE) are synthesized using an Al2O3 template and solvent annealing. The rods consist of micron-scale crystalline domains, and UV light induces a [4 + 4] photodimerization that results in a uniform 15% expansion along the rod axis. This is in contrast to random 9-TBAE crystals, which disintegrate under the same conditions. Transmission electron microscopy, atomic force microscopy, and comparison of the X-ray crystal structures of the monomer and photodimer all provide evidence for a mechanism based on a crystal-to-crystal photoreaction leading to an increase in molecular volume. It is likely that the high surface-to-volume ratio in the nanorods provides a strain relief pathway that is absent in larger crystals. Preliminary attempts to reverse the reaction using shorter wavelength light to photodissociate the dimers were only partly successful. These results suggest that crystalline organic nanostructures may provide an efficient way to transform photochemical energy into mechanical motion on the nanometer scale.     

Ionophoretic activities of oligopeptide lactones and resin-glycosides in human erythrocytes

Oligopeptide lactones (theonellapeptolides) isolated from the Okinawan marine sponge Theonella swinhoei and resin-glycosides (merremosides) from the tuber of an Indonesian medicinal plant Merremia mammosa were examined regarding their activities in transporting Na+, K+, and Ca++ ions into human erythrocytes. Each of these lactones, which had been shown, using a supported liquid membrane, to have an ionophoretic effect on the alkali metal ions, transported the ions to a different extent. The ion transporting activities of these compounds were completely lost when the macrocyclic lactone structures were cleaved by sodium methylate. Resin-glycosides with an additional branched glycosyl residue showed much greater ion transporting activities than those without it.     

A chip calorimetry-based method for the real-time investigation of metabolic activity changes in human erythrocytes caused by cell sickling

Journal of Thermal Analysis and Calorimetry - A new calorimetric technique is described which allows the measurement of metabolic heat rates in biological materials which are triggered by changes...     

Hollow polyelectrolyte multilayer tubes: mechanical properties and shape changes

In this paper, novel hollow polyelectrolyte multilayer tubes from poly(diallyldimethylammonium chloride) (PDADMAC), poly(styrene sulfonate) (PSS), and poly(allylamine hydrochloride) (PAH) were prepared: Readily available glass fiber templates are coated with polyelectrolytes using the layer-by-layer technique, followed by subsequent fiber dissolution. Depending on the composition of the polymeric multilayer, stable hollow tubes or tubes showing a pearling instability are observed. This instability corresponds to the Rayleigh instability and is a consequence of an increased mobility of the polyelectrolyte chains within the multilayer. The well-defined stable tubes were characterized with fluorescence microscopy, confocal laser scanning microscopy, and atomic force microscopy (AFM). The tubes were found to be remarkably free of defects, which results in an impermeable tube wall for even low molecular weight molecules. The mechanical properties of the tubes were determined with AFM force spectroscopy in water, and because continuum mechanical models apply, the Young's modulus of the wall material was determined. Additionally, scaling relations for the dependency of tube stiffness on diameter and wall thickness were validated. Because both parameters can be experimentally controlled by our approach, the deformability of the tubes can be varied over a broad range and adjusted for the particular needs.     

Electrocatalytic determination of reduced glutathione in human erythrocytes

The determination of reduced glutathione (GSH) in human erythrocytes using a simple, fast and sensitive method employing a glassy carbon electrode modified with cobalt tetrasulfonated phthalocyanine (CoTSPc) immobilized in poly(l-lysine) (PLL) film was investigated. This modified electrode showed very efficient electrocatalytic activity for anodic oxidation of GSH, decreasing substantially the anodic overpotentials for 0.2 V versus Ag/AgCl. The modified electrode presented better performance in 0.1 mol l⁻¹ piperazine-N,N′-bis(2-ethanesulfonic acid) buffer at pH 7.4. The other experimental parameters, such as the concentration of CoTSPc and PLL in the membrane preparation, pH, type of buffer solution and applied potential, were optimized. Under optimized operational conditions, a linear response from 50 to 2,160 nmol l⁻¹ was obtained with a high sensitivity of 1.5 nA l nmol⁻¹ cm⁻². The detection limit for GSH determination was 15 nmol l⁻¹. The proposed sensor presented good repeatability, evaluated in terms of the relative standard deviation (1.5%) for n = 10. The modified electrode was applied for determination of GSH in erythrocyte samples and the results were in agreement with those obtained by a comparative method described in the literature The average recovery for these fortified samples was 100 ± 1)%. Applying a paired Student’s-t test to compare these methods, we could observe that, at the 95% confidence level, there was no statistical difference between the reference and the proposed methods.     

Insulin binding and degradation by human erythrocytes and erythrocyte membranes

Conclusions By the current results there is evidence that the insulin degrading enzyme activity of the erythrocytes is not located on the inner or outer surface of the plasma membrane but is exclusively cytosolic. On the other hand, specific insulin binding to erythrocytes and erythrocyte membranes has been demonstrated and it could be supposed that the insulin binding region of the plasma membrane is not associated with an insulin degrading activity.In comparison to erythrocytes unsealed and sealed ghosts bound an equal amount of ¹²⁵I-insulin. Since there is binding but not degradation of insulin the membranes of erythrocytes might be a useful tool for the investigation of insulin internalization.

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Insulin-Bindung und -Abbau durch menschliche Erythrocyten und Erythrocyten-Membranen