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Introduction Heavy metals pose a serious environmental problem and progressive industrialization has led to widespread global metal pollution. Most heavy metals show cumulative effect, especially in aquatic animals due to [...], Cadmium chloride (Cd[Cl.sub.2]) is a widely used industrial compound that exhibits multiple organ toxicity. Cadmium is transported through blood where erythrocytes are exposed to its action. Here the effect of Cd[Cl.sub.2] on human erythrocytes was examined under in vitro conditions. Human erythrocytes were treated with 0.01-0.5 mM Cd[Cl.sub.2] for 24 h at 37 [degrees]C. Lysates were made from Cd[Cl.sub.2] treated and untreated (control) cells and used for further analysis. Cd[Cl.sub.2] treatment resulted in marked hemolysis of erythrocytes and oxidation of hemoglobin to methemoglobin. This will result in anemia and also reduce the oxygen carrying ability of erythrocytes. Hemoglobin oxidation was accompanied by degradation of heme and release of free ferrous iron moiety. Further analysis showed elevated lipid hydroperoxides and formation of advanced oxidation protein products along with reduction in total sulfhydryl content, indicating the generation of oxidative stress condition in the cell. Incubation of erythrocytes with Cd[Cl.sub.2] enhanced generation of reactive oxygen and nitrogen species, decreased the antioxidant power and inhibited pathways of glucose metabolism. Plasma membrane was damaged as indicated by enhanced osmotic fragility and inhibition of membrane bound enzymes. This was confirmed by electron microscopy which showed formation of echinocytes. These results show that Cd[Cl.sub.2] generates reactive species which impair the antioxidant system resulting in oxidative damage to erythrocytes. Key words: cadmium chloride, erythrocytes, reactive oxygen species, hemoglobin, antioxidant power, oxidative stress Le chlorure de cadmium (Cd[Cl.sub.2]) est un compose industriel largement utilise qui presente une toxicite pour plusieurs organes. Le cadmium est transports par le sang ou les erythrocytes sont exposes a son action. Ici, l'effet du Cd[Cl.sub.2] sur les erythrocytes humains a ete examine dans des conditions in vitro. Des erythrocytes humains ont ete traites avec 0,01 a 0,5 mM de Cd[Cl.sub.2] pendant 24 h a 37 [degrees]C. Des lysats ont ete prepares a partir de cellules traitees au Cd[Cl.sub.2] et non traitees (temoins) et utilises pour une analyse plus approfondie. Le traitement au Cd[Cl.sub.2] a entraine une hemolyse marquee des erythrocytes et une oxydation de l'hemoglobine en methemoglobine. Cela entrainera une anemie et reduira egalement la capacite de transport d'oxygene des erythrocytes. L'oxydation de l'hemoglobine s'est accompagnee d'une degradation de l'heme et d'une liberation de fraction de fer ferreux libre. Une analyse plus approfondie a montre des hydroperoxydes lipidiques eleves et la formation de produits proteiques d'oxydation avances ainsi qu'une reduction de la teneur totale en sulfhydryle, indiquant la generation d'un etat de stress oxydatif dans la cellule. L'incubation des erythrocytes avec Cd[Cl.sub.2] a ameliore la generation d'especes reactives d'oxygene et d'azote, a diminue le pouvoir antioxydant et inhibe les voies du metabolisme du glucose. La membrane plasmique a ete endommagee comme indique par une fragilite osmotique accrue et une inhibition des enzymes liees a la membrane. Ceci a ete confirme par microscopie electronique qui a montre la formation d'echinocytes. Ces resultats montrent que le Cd[Cl.sub.2] genere des especes reactives qui alterent le systeme antioxydant entrainant des dommages oxydatifs aux erythrocytes. [Traduit par la Redaction] Mots-cles : chlorure de cadmium, erythrocytes, les especes reactives de l'oxygene, hemoglobine, pouvoir antioxydant, stress oxydatif

Introduction Red blood cell transfusion, the most common procedure performed on hospitalized patients (1), is an indispensable component of modern medicine. Establishing an adequate blood supply depends on the ability [...], BACKGROUND. Some countries have limited the maximum allowable storage duration for red cells to 5 weeks before transfusion. In the US, red blood cells can be stored for up to 6 weeks, but randomized trials have not assessed the effects of this final week of storage on clinical outcomes. METHODS. Sixty healthy adult volunteers were randomized to a single standard, autologous, leukoreduced, packed red cell transfusion after 1, 2, 3, 4, 5, or 6 weeks of storage (n = 10 per group). 51-Chromium posttransfusion red cell recovery studies were performed and laboratory parameters measured before and at defined times after transfusion. RESULTS. Extravascular hemolysis after transfusion progressively increased with increasing storage time (P < 0.001 for linear trend in the AUC of serum indirect bilirubin and iron levels). Longer storage duration was associated with decreasing posttransfusion red cell recovery (P = 0.002), decreasing elevations in hematocrit (P = 0.02), and increasing serum ferritin (P < 0.0001). After 6 weeks of refrigerated storage, transfusion was followed by increases in AUC for serum iron (P < 0.01), transferrin saturation (P < 0.001), and nontransferrin-bound iron (P < 0.001) as compared with transfusion after 1 to 5 weeks of storage. CONCLUSIONS. After 6 weeks of refrigerated storage, transfusion of autologous red cells to healthy human volunteers increased extravascular hemolysis, saturated serum transferrin, and produced circulating nontransferrin-bound iron. These outcomes, associated with increased risks of harm, provide evidence that the maximal allowable red cell storage duration should be reduced to the minimum sustainable by the blood supply, with 35 days as an attainable goal. REGISTRATION. ClinicalTrials.gov NCT02087514. FUNDING. NIH grant HL115557 and UL1 TR000040.

Author(s): Anna Petherick Author Affiliations: Blood boosts bids to unpick membrane science Illustration by Michelle Thompson; Photos: Getty, Shutterstock Illustration When Liangfang Zhang thinks of blood, he doesn't think of [...], Fresh techniques are opening the world of the cell up to a range of disciplines. Fresh techniques are opening the world of the cell up to a range of disciplines.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.forsciint.2008.04.007 Byline: Sylvain Giraud, Neil Robinson, Patrice Mangin, Martial Saugy Keywords: Transfusion; Doping; Validation; Flow cytometry; ISO-17025 Abbreviations: RBCs, red blood cells; FITC, fluorescein isothiocyanate; SD, standard deviation; SEM, standard error of the mean; S/N, signal to noise ratio Abstract: Since the introduction in 2001 of a urine-based detection method for recombinant erythropoietin (rHuEPO), transfusion-doping practices have regained interest. To address this problem, an efficient antidoping test designed to obtain direct proof of allogeneic blood transfusion was developed and validated. This test, based on flow cytometry analysis of red blood cell (RBCs) phenotypes, was used to determine the absence or the presence of numerous RBCs populations in a blood sample. A such, it may constitute a direct proof of an abnormal blood population resulting from homologous transfusion. Single-blind and single-site studies were carried out to validate this method as a forensic quality standard analysis and to allow objective interpretation of real cases. The analysis of 140 blood samples containing different percentages (0-5%) of a minor RBCs population were carried on by four independent analysts. Robustness, sensitivity, specificity, precision and stability were assessed. ISO-accredited controls samples were used to demonstrate that the method was robust, stable and precise. No false positive results were observed, resulting in a 100% specificity of the method. Most samples containing a 1.5% minor RBCs population were unambiguously detected, yielding a 78.1% sensitivity. These samples mimicked blood collected from an athlete 3 months after a homologous blood transfusion event where 10% of the total RBCs present in the recipient originated in the donor. The observed false negative results could be explained by differences in antigen expression between the donor and the recipient. False negatives were more numerous with smaller minor RBCs populations. The method described here fulfils the ISO-17025 accreditation and validation requirements. The controls and the methodology are solid enough to determine with certainty whether a sample contains one or more RBCs populations. This variable is currently the best indicator for homologous blood transfusion doping. Author Affiliation: Laboratoire Suisse d'Analyse du Dopage, Institut Universitaire de Medecine Legale, Chemin des Croisettes 22, CH-1066 Epalinges, Switzerland Article History: Received 4 December 2006; Revised 7 January 2008; Accepted 12 April 2008

Moderate hemolytic anemia, abnormal erythrocyte morphology (spherocytosis), and decreased membrane stability are observed in mice with complete deficiency of all erythroid protein 4.1 protein isoforms ([4.1.sup.-/-]; Shi TS et al. J Clin Invest 103: 331, 1999). We have examined the effects of erythroid protein 4.1 (4.1R) deficiency on erythrocyte cation transport and volume regulation. [4.1.sup.-/-] mice exhibited erythrocyte dehydration that was associated with reduced cellular K and increased Na content. Increased Na permeability was observed in these mice, mostly mediated by Na/H exchange with normal Na-K pump and Na-K-2Cl cotransport activities. The Na/H exchange of [4.1.sup.-/-] erythrocytes was markedly activated by exposure to hypertonic conditions (18.2 [+ or -] 3.2 in [4.1.sup.-/-] vs. 9.8 [+ or -] 1.3 mmol/[10.sup.13] cell x h in control mice), with an abnormal dependence on osmolality ([EC.sub.50] = 417 [+ or -] 42 in [4.1.sup.-/-] vs. 460 [+ or -] 35 mosmol/kg[H.sub.2]O in control mice), suggestive of an upregulated functional state. While the affinity for internal protons was not altered ([K.sub.0.5] = 489.7 [+ or -] 0.7 vs. 537.0 [+ or -] 0.56 nM in control mice), the [V.sub.max] of the H-induced Na/H exchange activity was markedly elevated in [4.1.sup.-/-] erythrocytes ([V.sub.max] 91.47 [+ or -] 7.2 compared with 46.52 [+ or -] 5.4 mmol/[10.sup.13] cell x h in control mice). Na/H exchange activation by okadaic acid was absent in [4.1.sup.-/-] erythrocytes. Altogether, these results suggest that erythroid protein 4.1 plays a major role in volume regulation and physiologically down-regulates Na/H exchange in mouse erythrocytes. Upregulation of the Na/H exchange is an important contributor to the elevated cell Na content of [4.1.sup.-/-] erythrocytes. spherocytosis; cell Na; Na/H exchange

A study is conducted to evaluate reticulocyte hemoglobin content (CHr) for detecting iron deficiency without anemia in healthy 9- to 12-month-old infants and to compare CHr with hemoglobin in screening for iron deficiency in the population. The results indicate that a CHr of less than 27.5 pg is a more accurate hematological indicator of iron deficiency compared with hemoglobin of less than 11 g/dL in these healthy 9- to 12-month-old infants.

The ability of nitric oxide to relax smooth muscle cells surrounding resistance vessels in vivo is well documented. Here, we describe a series of studies designed to quantify amounts of adenosine triphosphate (ATP), a known stimulus of NO production in endothelial cells, released from erythrocytes that are mechanically deformed as these cells traverse microbore channels in lithographically patterned microchips. Results indicate that micromolar amounts of ATP are released from erythrocytes flowing through channels having cross sectional dimensions of 60 x 38 [micro]m (2.22 [+ or -] 0.50 [micro]M ATP). Microscopic images indicate that erythrocytes, when being pumped through the microchip channels, migrate toward the center of the channels, leaving a cell-free or skimming layer at the walls of the channel, a profile known to exist in circulatory vessels in vivo. A comparison of the amounts of ATP released from RBCs mechanically deformed in microbore tubing (2.54 [+ or 1] 0.15 [micro]M) vs a microchip (2.59 [+ or -] 0.32 [micro]M) suggests that channels in microchips may serve as functional biomimics of the microvasculature. Control studies involving diamide, a membrane-stiffening agent, suggest that the RBC-derived ATP is not due to cell lysis but rather physical deformation.

Capillary electrophoresis/electrospray ionization-mass spectrometry (CE/ESI-MS) was applied to the analysis of human red blood cells (RBCs) using the split-flow technique for interfacing CE to MS. By using a long (~125cm) and narrow (~15-[micro]m-i.d.) capillary, the four major proteins of the RBC, which are hemoglobin (Hb, [alpha]- and [beta]-chains, 900 amol/chain), carbonic anhydrase I (CAI, ~7 amog/cell), and carbonic anhydrase II (CAII, ~0.8 amol/cell), were separated from each other and detected at low-attomole levels in one run and minimal sample preparation. Under these conditions, the detection limits for CAI and CAII in lysed RBCs were ~20 and ~44 amol, respectively. The ~20-amol detection limit of CAI was confirmed by the CE/ESI-MS analysis of three intact RBCs that had been drawn into the capillary under a microscope. A shorter capillary (~55 cm long) provided faster analysis time but did not separate CAII from the [beta]-chain of hemoglobin, causing the CAII signal to be masked by the background chemical noise generated by the ~1000 x molar excess of the [beta]-chain. Under this condition, the CAII detection limit increased to ~500 amol. From three methods of sample introduction (injection of lysed blood, injection of intact cells under microscope, and injection of intact cells suspended in saline solution), injection of lysed blood provided the optimum sensitivity. It was found that a background electrolyte (BGE) containing 0.1% acetic acid in water worked best for the analysis of intact cells, while a BGE containing 0.1% acetic acid in water+ acetonitrile (50/50 by volume) worked best for the analysis of lysed blood.

Research has been conducted on the human erythrocytes alpha and beta spectrin. The use of the recombinant spectrin peptides to model alpha and beta spectrin in studying their associations at the tetramerization site is described.

Research has been conducted on the human thiol-specific antioxidant protein 1 (hTSA1). The involvement of the C-terminal region of hTSA1 in membrane binding has been investigated.

A totally new biosorption concept has been tested. It involves the use of a biological membrane with given transport characteristics that encloses a reaction compartment chosen to bind incorporated substances irreversibly. Such a system is an erythrocyte ghost: erythrocytes that are hypotonically lysed can be filled with the desired media and resealed by warming up for 1 h. The transport system exploited is the predominant one in erythrocytes, i.e., the anion transport system with [10.sup.6] channels/cell. Erythrocyte ghosts (in the following simply called ghosts) filled with ascorbate or cysteine as intracellular reductants were compared to unmodified human erythrocytes with regard to their ability to accumulate chromate specifically. Furthermore, the lifetime and behavior of the ghosts with respect to the influence of extracellular Cr(III) were tested. The great similarity found between the ghosts tested and unmodified erythrocytes clearly shows the general applicability of this new biosorption concept which offers possibilities for the biosorption of analytes that cannot be accumulated by unmodified erythrocytes.

A study was conducted to investigate the transport and redox cycling of vitamin C in human erythrocytes using (super 13)C nuclear magnetic resonance. (super 13)C-labeled compounds were combined with thermally equilibrated red blood cell (RBC) suspensions while the effect of different incubation media was represented as a function on the time dependence of RBC lysis. Results showed that the transport and recyling of dehydroascorbic acid were correlated on glycolysis in the RBC.

The electromagnetic far field, as well as the near field, originating from light interaction with a red blood cell (RBC) volume-equivalent spheroid, was analyzed by utilizing the T-matrix theory. This method is a powerful tool that makes it possible to study the influence of cell shape on the angular distribution of scattered light. General observations were that the three-dimensional shape, as well as the optical thickness apparent to the incident field, affects the forward scattering. The backscattering was influenced by the shape of the surface facing the incident beam. Furthermore sphering as well as elongation of an oblate RBC into a volume-equivalent sphere or a prolate spheroid, respectively, was theoretically modeled to imitate physiological phenomena caused, e.g., by heat or the increased shear stress of flowing blood. Both sphering and elongation were shown to decrease the intensity of the forward-directed scattering, thus yielding lower g factors. The sphering made the scattering pattern independent of azimuthal scattering angle [[Phi].sub.s], whereas the elongation induced more apparent [[Phi].sub.s]-dependent patterns. The light scattering by a RBC volume-equivalent spheroid was thus found to be highly influenced by the shape of the scattering object. A near-field radius [r.sub.nf] was evaluated as the distance to which the maximum intensity of the total near field had decreased to 2.5 times that of the incident field. It was estimated to 2-24.5 times the maximum radius of the scattering spheroid, corresponding to 12-69 [[micro]meter]. Because the near-field radius was shown to be larger than a simple estimation of the distance between the RBC's in whole blood, the assumption of independent scattering, frequently employed in optical measurements on whole blood, seems inappropriate. This also indicates that one cannot extrapolate the results obtained from diluted blood to whole blood by multiplying with a simple concentration factor.

An analysis was conducted to determine whether the redistribution of intracelluar cations and the movement of phosphatidylserine could be related to the amount of PIP2 in the bilayer. Human erythrocyte suspension were prepared from fresh human venous blood. Results revealed that phosphatidylserine exposure could be seen in the red blood cells which showed an increased cytosolic cation concentration and a mechanism for PIP2-induced loss of phospholipid asymmetry in the in the red cell mebrane existed.

Research was conducted to examine the impact of alkaline pH 6-13 on the conformational, self-association and formation of spectrin. Purified spectrin was dialyzed against sodium phosphate and concentrated by dialysis against Aquacide II. An analytical ultracentrifuge was also utilized to carry out sedimentation velocity experiments. Results indicated that the spectrin oligomers dissociated into individual alpha and beta-subunits above pH 9.5 which was coupled with a loss of alpha-helix.

Research was conducted to describe a technique for isolating the alpha and beta-subunits of human erythrocyte spectrin. Moles of alpha and beta-subunits were mixed in buffers to reconstitute heterodimer from the isolated subunits while a spectropolarimeter was utilized to gather CD spectra. Results showed that the secondary structure of the purified alpha and beta-subunits and the reconstituted heterodimer were similar to the native spectrin.

Research was conducted to examine the hepatic uptake of hippuric acid (HA), an organocarboxylic acid, in the single-pass perfused rat liver. The multiple indicator dilution method was utilized to study the sinusoidal transfer constants for HA while the distribution of HA into red blood cells was determined by mixing plasma perfusate with varying HA concentrations. Results showed that hippuric acid exhibited poor binding properties to albumin and does not distribute red blood cells.

Research was conducted to test whether the exchange of amino acids between plasma and fetal red blood cells is slow enough to permit the use of plasma concentrations and plasma flows for estimating umbilical amino acid uptakes. The umbilical uptake of each amino acid was compared with its normal fetal accretion rate while hemoglobin and O2 saturation were measured spectrophotometrically. Results showed that uterine and umbilical uptakes of amino acids can be determined on either metabolic pathways or transport systems.

In a Hungarian family with triosephosphate isomerase (TPI) deficiency, two compound heterozygote brothers were found with the same severe decrease in TPI activity, but only one of them had the classical symptoms. In search for the pathogenesis of the differing phenotype of the same genotypic TPI deficiency, an increase in red cell membrane fluidity was found. There were roughly 100% and 30% more 16:0/20:4 and 18:0/20:4 diacyl-phosphatidylcholine species in erythrocytes from the two TPI-deficient brothers than in the probes from healthy controls. The activities of acethyicholinesterase and calmodulin induced [Ca.sup.2+] ATPase were significantly enhanced in erythrocytes from the propositus as compared with those of the neurologically symptom-free brother and other members of the TPI-deficient family as well as to those from healthy controls. Both enzymes are crucially involved in the function of nerve cells. The observed differences in membrane fluidity and enzyme activities between the erythrocytes from the phenotypically differing TPI-deficient brothers underline the importance of investigations into the effect of biophysical changes in the lipid environment of the membrane proteins on the development of disseminated focal neurological disorders of unknown pathogenic origin.

The importance of ankyrin and band 4.1 in restricting band 3 rotational mobility was determined through fluorescence binding assay of fluorescein isothiocyanate-labeled ankyrin. Results have shown that ankyrin induces the association of band 3 into more slowly rotating complexes autonomous of any other components of the erythrocyte membrane. Furthermore, membrane fluidity and protein concentration also determine the band 3 reconstitution.

The function of ankyrin-mediated skeleton-bilayer linkage in maintaining red cell mechanical stability was established following treatment of nb/nb spectrin with nonionic detergent octaethylene glycol n-dodecyl monoether (C12E8). Results have shown that nb/nb spectrin and related proteins aggregate into a highly ordered and mechanically stable hexagonal structure slightly distinct from the normal skeleton. It was also found that band 3 would be less associated with the skeleton in ankyrin-deficient nb/nb red cells than in normal red cells.

Inhibition of bacterial adhesion was demonstrated in a study conducted on di-, tri-, and tetravalent dendritic galabiosides that inhibit hemagglutination by Streptococcus suis at nanomolar concentration. To form such compounds, coupling of galabiose derivatives to carrier molecules was initiated. Findings revealed the relationship between the number of galabiose units found in an inhibitor and inhibitory efficacy.

The difficulty in controlling Plasmodium vivax, the most common cause of human malaria, has been complicated by growing drug resistance. We have established a method to cycle parasite generations in continuous culture using human blood cells. Chesson strain parasites were passaged from owl monkey erythrocytes to human reticulocytes in McCoy's 5A medium modified with L-glutamine with 25 mM Hepes buffer supplemented with 20% A[B.sup.+] human serum. Reticulocytes were separated by differential centrifugation in homologous plasma from the peripheral blood of a hemochromatosis patient. Parasites were grown during each 48-hr cycle in a static candle jar environment until the beginning of schizogony, at about 36-40 hr, when reticulocytes were added and cultures transferred to a shaker for 10-12 hr. The addition of a concentration of 10% reticulocytes resulted in stabilizing parasite densities between 0.28 and 0.57 after cycle 3 and increasing the total number of parasites at least 2-fold with each generational cycle. Cultured parasites successfully infected an owl monkey. The morphology of cultured parasites was typical of P. vivax, with highly ameboid trophozoites evident; however, infected erythrocytes were enlarged and distorted on thin film preparations. The species identity of cultivated parasites was confirmed by analysis of the A and C 18S rRNA genes from genomic DNA and expression of only the A gene during erythrocytic asexual growth. The ability to culture P. vivax opens new opportunities to develop vaccines, test drugs, and clone parasites for genome sequencing.

An expression relating the red blood cell (RBC) volume and membrane surface area to the pore minimum radius/maximum length which the cell is able to traverse is derived. The application of this analytical model to design/specification of filters for RBC deformability evaluation is presented. The passage of the RBC's through relatively long (10 [[micro]meter] or more) submicron radii pores, which the developed model demonstrates, is also discussed. Index Terms - Cylindrical pore filter, RBC, red blood cell.

The polymerization of the microbial membrane-damaging streptolysin O (SLO) protein was analyzed to determine mechanisms of SLO pore formation. The temperature-dependent polymerization of SLO molecules leads to the formation of ring-shaped structures which forms SLO rings at 37 degrees centigrade. Proteins from the erythrocyte membranes were not detected from the SLO spores which are composed mostly of SLO fractions.

1-chloro-2,4-dinitrobenzene (CDNB) causes small biphasic effect on K-Cl cotransport and prevents K-Cl flux stimulation by N-ethylmaleimide, diamide, and MMTS. CDNB treatment also stops stimulatory effect of intracellular Mg reduction, independent of cellular glutathione removal. Glutathione removal reveals kinases as the common targets for K-Cl cotransport stimulation in sheep erythrocytes.

An in vitro study of the parasitized erythrocyte adherence to postcapillary venule walls through an interaction between parasite-derived erythrocyte surface ligands and endothelial cell surface receptors in Plasmodium falciparum using a parasite population expressing at least two different ligands demonstrated that parasitized cells may coexpress ligands that have a preference for multiple receptors. Single parasitized erythrocytes bound to three distinct receptors through two separate ligands. A trypsin-sensitive molecule-induced cytoadherence to CD36 and intercellular adhesion molecule 1 and a trypsin-insensitive molecule were responsible for the binding to a third receptor on the melanoma cell surface.

A particle-counting immunoassay system for ultrasensitive analysis of proteins in a capillary environment has been developed. The assay is based on the agglutination of antibody-coated particles in the presence of an antigen (usually a protein). The particles were electrophoretically migrated in a 20-micrometer-i.d. capillary past a detection window where a laser beam irradiates continuously. The light scattering events generated by the agglutinated particles were counted while those produced by unreacted particles were electronically rejected. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as a test compound for the off-column as well as for the on-column versions of this method. A limit of detection of 620 molecules of G6PDH (1 zmol) was found in the on-column assay. The standard deviation between runs was approximately 6%, which is comparable to that of standard immunoassay methods. The application to the determination of G6PDH levels in individual human erythrocytes is presented. A 14-fold cell-to-cell variation was found which can be explained by the age distribution in the red blood cells.

Mouse periotoneal macrophages bind and phagocytose oxidized human erythrocytes in the absence of opsonizing antibodies. Untreated erythrocytes exhibit marginal or no binding. Lipid-proteins conjugate, linked to a few conjugates observed in oxidized low density lipoprotein, result from oxidative damage of erythrocytes.

Time-resolved fluorescence anisotropy provided insights into the segmental dynamics of the cytoplasmic domain of erythrocyte band 3, cdb3. Ligand binding significantly affects cdb3 conformation, confirming a related longe-range structural communication. Fluorescein maleimide was used to preferantially label band 3 where pH-dependent conformational shifts occurs.

Previous studies have shown that ultrasonic backscattering from red blood cells suspended in saline is proportional to the fourth power of frequency for frequencies below 15 MHz, as predicted by Rayleigh scattering theory. Recently, we have extended the measurements up to 30 MHz, because scattering of ultrasound by red blood cells may no longer be negligible at these frequencies and can affect, to a great degree, the operation of intravascular imaging devices. The experimental results show that the fourth power dependence on frequency of the backscattering coefficient for porcine erythrocytes suspended in saline solution appears to be valid up to 30 MHz. To confirm this, backscattering cross-section of porcine red cells was computed as a function of frequency using the T-matrix method. Since at higher frequencies the shape of the scatterers may also play a significant role, its effect was investigated by treating the red cell as a sphere, a disc, and a biconcave disc of the same volume. Good agreement was obtained between the experimental and theoretical results.

The influence of peptide P thionin, which becomes cationic on dispersal of phospholipids and formation of domains in native erythrocyte and ghost membranes, was examined. Phospholipid regions increase when peptides are added to erythrocyte membranes and vesicles produced from phospholipids which are separated from erythrocyte membranes. These increases can be seen when fluorescence digital imaging microscopy along with fluorescent derivatives of phosphatidylserine and phosphatidylcholine are used.

Human red blood cells, CHIP28, yields CHIP, a 28-kDa channel-forming integral membrane protein, which is identified to determine the molecular properties of membrane water channels. Osmosis, seen in CHIP, causes water-selective pore movement. Enhancement in osmotic water permeability is seen in Xenopus oocytes due to CHIP expression. Transmembrane movement of ions or other minute molecules is not permitted by CHIP. Permeability of unit water is determined by the recreation of distilled CHIP into proteoliposomes.

The administration of urea in dog red blood cells caused a decline in the set point volume for shrinkage stimulated Na-H exchange, swelling stimulated K-Cl cotransport and Ca-Na exchange in dog red blood cells. The analyses of mechanisms, whereby a coordination between the three different ion flux pathways is achieved during the set point volume decline, indicate a regulatory-mechanism consisting of membrane transporters, volume sensor and signaling system, responsible for the cell's responses observed during the altered levels of cell water content. Activation of K-Cl cotransport and Ca-Na exchange at normal and below normal levels of volume occurs in cells equilibrated with urea. Na-H exchange in cells treated with urea requires a stronger stimulus provided by a higher degree of shrinkage when compared to cells not exposed to urea. The response to swelling and shrinkage makes the cell water content in urea-treated cells to seem greater than it is.

A study assessed the accessibility of polyphosphoinositide (PPI) to be venom phospholipase A2 (PLA2) in native erythrocyte membranes. After treatments meant to remove peripheral proteins and cytoplasmic domains of integral proteins, preferential interactions with proteins were tested to see if they caused the metabolic segregation of PPI.

The functional role of cations on erythrocyte spectrin was studied. It was observed that spectrin contains two types of calcium-binding sites. Higher affinity binding is calcium-specific, whereas lower affinity binding occurs with both calcium and magnesium. In both cases, binding produces structural changes in the protein. The results indicate that divalent cation binding to erythrocyte spectrin is the basis for the flexibility of the erythrocyte membrane skeleton.

We present in detail our impedance measurement method, the cell embodding technique, for human erythrocytes, and an accurate calibration procedure for a true four-electrode impedance measurement system. This technique with the calibration procedure gives reliable impedance measurements over a wide frequency range - 1 Hz to 10 MHz. To achieve high sensitivity in this frequency range, we embed the cells in the pores of a Nuclepore filter. The calibration procedure assumes that the measurement system is linear, and requires measurement of three reference impedances. The reliability of this procedure is demonstrated with various RC circuits, and application of it to the bio-impedance measurement system eliminates a quasi-dispersion in the high-frequency range, and increases the bandwidth at both the low- and high-frequency ends of the range by about a decade. We model the impedance of the cells embedded within the filter with an equivalent circuit that is consistent with the geometry and interfaces present. The experimental data are fitted to this model by means of a complex nonlinear least squares (CNLS) fit, which simultaneously fits the real and imaginary parts of the impedance with the Levenberg-Marquardt algorithm. The impedance spectra of human erythrocytes are found to display constant-phase-angle (CPA) characteristics. A CPA element is an impedance of the form Z = A/(j-Omega)(super Alpha), where A is a constant, j = square root of -1, Omega is angular frequency, and 0 < Alpha < 1, and has been used to describe the ac response of the interface between the cell surface and the external electrolyte solution. Such a CPA element may be related to fractal character of the interface.

The interactions between the two domains of human erythrocyte band 3, which is the chloride/bicarbonate exchange protein, as a consequence of covalent binding of anion transport inhibitors to the intramonomeric stilbenedisulfonate (ISD) site were investigated. Band 3 protein is a glycoprotein of two parts, an N-terminal cytoplasmic domain and a C-terminal integral domain. The study protocol involved the use of near-UV circular dichroism spectrum analysis of band 3. The results indicated that the attached cytoplasmic domain inhibits some aspects of the conformational response of the integral domain.