Intact Erythrocytes Research Articles

MODEL BIOMEMBRANES AS TEST OBJECTS FOR THE DETERMINATION OF CONCENTRATION RANGES OF HARMFUL CHEMICAL SUBSTANCES IN BIOLOGICAL MEDIUMS AND OBJECTS OF EXTERNAL ENVIRONMENT

The paper presents data on changes in model biomembranes (liposomes, erythrocyte shadows, erythrocytes) used as test objects to determine those ranges of concentrations of biologically active substances in which there is no violation of the structure or function of experimental objects. Melaphene, plant growth regulator used in small doses in seed pre-treatment, and antioxidant phenosan derivatives, phenoxane and IHFANs, have been used as biologically active substances. It was shown by DSC that phenosan derivatives at concentrations equal to 10-5 M and higher destroy the microdomain organization in the bilayers of phospholipid multilamellar liposomes and reshape protein microdomains in the shadows of red blood cells. Melaphene in small and large concentrations changes polymodal the microdomain organization in the bilayers of phospholipid multilamellar liposomes without destroying the structure and does not affect the protein microdomains in the shadows. An increase in the membrane permeability in isolated intact erythrocytes in the presence of melaphene in large and small concentrations has been revealed by means of spectral anaslysis. The method of small-angle diffraction scattering showed the absence of the effect of melaphene in a wide range of concentrations on the thickness of phospholipid bilayers and the order of their packaging in multilamellar liposomes.

Lysed Erythrocyte Membranes Promote Vascular Calcification.

Intraplaque hemorrhage promotes atherosclerosis progression, and erythrocytes may contribute to this process. In this study we examined the effects of red blood cells on smooth muscle cell mineralization and vascular calcification and the possible mechanisms involved. Erythrocytes were isolated from human and murine whole blood. Intact and lysed erythrocytes and their membrane fraction or specific erythrocyte components were examined in vitro using diverse calcification assays, ex vivo by using the murine aortic ring calcification model, and in vivo after murine erythrocyte membrane injection into neointimal lesions of hypercholesterolemic apolipoprotein E-deficient mice. Vascular tissues (aortic valves, atherosclerotic carotid artery specimens, abdominal aortic aneurysms) were obtained from patients undergoing surgery. The membrane fraction of lysed, but not intact human erythrocytes promoted mineralization of human arterial smooth muscle cells in culture, as shown by Alizarin red and van Kossa stain and increased alkaline phosphatase activity, and by increased expression of osteoblast-specific transcription factors (eg, runt-related transcription factor 2, osterix) and differentiation markers (eg, osteopontin, osteocalcin, and osterix). Erythrocyte membranes dose-dependently enhanced calcification in murine aortic rings, and extravasated CD235a-positive erythrocytes or Perl iron-positive signals colocalized with calcified areas or osteoblast-like cells in human vascular lesions. Mechanistically, the osteoinductive activity of lysed erythrocytes was localized to their membrane fraction, did not involve membrane lipids, heme, or iron, and was enhanced after removal of the nitric oxide (NO) scavenger hemoglobin. Lysed erythrocyte membranes enhanced calcification to a similar extent as the NO donor diethylenetriamine-NO, and their osteoinductive effects could be further augmented by arginase-1 inhibition (indirectly increasing NO bioavailability). However, the osteoinductive effects of erythrocyte membranes were reduced in human arterial smooth muscle cells treated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide or following inhibition of NO synthase or the NO receptor soluble guanylate cyclase. Erythrocytes isolated from endothelial NO synthase-deficient mice exhibited a reduced potency to promote calcification in the aortic ring assay and after injection into murine vascular lesions. Our findings in cells, genetically modified mice, and human vascular specimens suggest that intraplaque hemorrhage with erythrocyte extravasation and lysis promotes osteoblastic differentiation of smooth muscle cells and vascular lesion calcification, and also support a role for erythrocyte-derived NO.

Determination of the pressure of damage to the spleen histological method in mechanical injury in forensic medical practice

Introduction. The trauma of the spleen in the minds of the minds should be accomplished often by that middle of the body of the empty light of a loan from one of the other countries. The frequency of spleens injuries according to the literature ranges from 15 to 33%. Also, it is often necessary to postpone a long time about the injuries of the spleen. One of the methods used in the long-term follow-up injuries spleens histological method. The aim of this work was to study the dynamic changes of histological parameters injured spleens tissues in various types of mechanical injury, depending on the period of injury. Material and methods. The material for the present study, there are tissues of the spleens 78 people male and female, aged 20 to 60 years, who died at injury of spleens and subjected to autopsy in Thanatology Department Office of Forensic medical examination. In research conducted used: histological, histochemical techniques to detect dynamic processes of regeneration histological parameters injured tissues of the spleens and performed statistical analysis of the results. Results and discussion. As a result of research we have found that the mechanical injury of spleen ruptures often developed capsule and parenchyma with the presence of hemorrhage in the area of injury. Our records show that during the first 6 hours after injury, histological examination of the spleens is damaged hematoma in a cluster of erythrocytes orange with clear contours, center pieces hematoma observed hemolysis of erythrocytes; closer to the edge of the hematoma determined by single leukocytes, fibrin strands. Note that the edge of the hematoma increases perifocal edema. In vessels were observed lekocytic stasis and perifocal accumulation of leukocytes. Within 6-12 hours after injury most damage red blood cells in the center were vague outlines, notes their common hemolysis, sometimes intact erythrocytes are just on the edge of the hematoma, the number of granulocytes in the cell increases bleeding and begin their destruction. Within 12-24 hours watched hemolyzed erythrocytes, granulocytes completely destroyed. Limit shaft is formed, represented destroyed granulocytes, macrophages with hemosiderin intracellular grains, fibrin strands on the edge of the hematoma intact tissues, blood clots in the blood vessels are. Further, for 2-3 days, beginning resorption products of destruction of red blood cells and start forming siderophage. On the edge of hematoma, on the border with intact tissue proliferation begins histio-fibroblastic cells, platelets organization in the vessels. Continuing formation demarcation shaft as fuzzy mass of fibrin strands in which grow in histio-fibroblastic cells and collagen fibers form a delicate including many hemosiderophages. With the emergence of damage limitation period 4-6 days observed prevalence of connective tissue elements (lymphocytes, histiocytes, plasma cells), there is a massive collapse of granulocytes, signs of blood clots in the vessels of the spleen. After 6 days continues capsules of hematoma formation. Hematoma presented erythrocytes are hemolyzed completely, a large number of leukocytes nuclear detritus, the integrity of which is broken, compacted fibrin strands, which clearly demarcates hematoma of damaged tissue by parenchymal hematoma to grow multiple strands of histofibroblastic cellular elements, delicate collagen fibers, which are beginning to form a capsule. Conclusions. As a result of the above, the morphological picture could differ from those explained earlier that you must consider when installing old injury. Thus, the results indicate that in the injured tissue legitimate spleen observed histological changes that are directly dependent on the time elapsed since the infliction of harm. Using histological methods to determine the time of causing harm, as in the case of isolated spleens injury, and in the case of the United trauma of the abdomen, is appropriate because it allows you to increase the objectivity of the results establish limitations causing injury. Summary. As a result of the analysis of literary sources and a statistical analysis of our own research on the nature, mechanism and prescription of spleen injury, it was possible to determine the duration of damage to the spleen in case of mechanical injury by the dynamics of changes in its histological parameters, which is important in forensic practice.

Evidence for three populations of the glucose transporter in the human erythrocyte membrane

Glucose transporter 1 (GLUT1) is one of 13 members of the human equilibrative glucose transport protein family and the only glucose transporter thought to be expressed in human erythrocyte membranes. Although GLUT1 has been shown to be anchored to adducin at the junctional spectrin-actin complex of the membrane through interactions with multiple proteins, whether other populations of GLUT1 also exist in the human erythrocyte membrane has not been examined. Because GLUT1 plays such a critical role in erythrocyte biology and since it comprises 10% of the total membrane protein, we undertook to evaluate the subpopulations of erythrocyte GLUT1 using single particle tracking. By monitoring the diffusion of individual AlexaFluor 488-labeled GLUT1 molecules on the surfaces of intact erythrocytes, we are able to identify three distinct subpopulations of GLUT1. While the mobility of the major subpopulation is similar to that of the anion transporter, band 3, both a more mobile and more anchored subpopulation also exist. From these studies, we conclude that ~65% of GLUT1 resides in similar or perhaps the same protein complex as band 3, while the remaining 1/3rd are either freely diffusing or interacting with other cytoskeletally anchored membrane protein complexes.

Rhodamine-Hoechst positional isomers for highly efficient staining of heterochromatin.

Hoechst conjugates to fluorescent dyes are popular DNA stains for live-cell imaging, but the relationship between their structure and performance remains elusive. This study of carboxyrhodamine-Hoechst 33258 conjugates reveals that a minimal change in the attachment point of the dye has dramatic effects on the properties of the final probe. All tested 6'-carboxyl dye-containing probes exhibited dual-mode binding to DNA and formed a dimmer complex at high DNA concentrations. The 5'-carboxyl dye-containing probes exhibited single-mode binding to DNA which translated into increased brightness and lower cytotoxicity. Up to 10-fold brighter nuclear staining by the newly developed probes allowed acquisition of stimulated emission depletion (STED) nanoscopy images of outstanding quality in living and fixed cells. Therefore we were able to estimate a diameter of ∼155 nm of the heterochromatin exclusion zones in the nuclear pore region in living cells and intact chicken erythrocytes and to localize telomeres relative to heterochromatin in living U-2 OS cells. Employing the highly efficient probes for two-color STED allowed visualization of DNA and tubulin structures in intact nucleated erythrocytes - a system where imaging is greatly hampered by high haemoglobin absorbance.

Hemolysis, tocopherol, and lipid oxidation in erythrocytes and muscle tissue in chickens, ducks, and turkeys

Muscle from turkeys is more sensitive to lipid oxidation during post mortem storage compared with that of chicken and duck which may involve increased lysis of turkey erythrocytes that releases hemoglobin oxidant. Three separate experiments were conducted to study characteristics of chicken, duck, and turkey erythrocytes in which dietary tocopherols were standardized. In Experiment I, tocopherol, fatty acid composition, and lipid oxidation capacity were measured in erythrocytes from chickens, ducks, and turkeys. Tocopherol content was greater in chicken erythrocytes compared with that of duck and turkey (P < 0.05). Oleic and linoleic acid content was higher in chicken erythrocytes compared with that of turkey (P < 0.05). Lipid oxidation capacity of erythrocytes in washed turkey muscle (WTM) at pH 5.8 ranked chicken > duck > turkey (P < 0.05). In Experiment II, hemolysis was measured in erythrocytes from turkeys and chickens. Detergent-induced hemolysis (pH 7.4) was on average 12-fold greater for turkey erythrocytes compared with that of chicken (P < 0.05). In Experiment III, the ability of lysed and non-lysed erythrocytes to promote lipid oxidation was examined. Lysed erythrocytes promoted lipid oxidation in WTM more effectively than intact erythrocytes (P < 0.05). Reasons that turkey erythrocytes were more labile to detergent-induced hemolysis whereas chicken erythrocytes more effectively promoted lipid oxidation in the WTM model system are discussed. These studies describe variation in chemical and physical properties of erythrocytes from chickens, ducks, and turkeys that can influence progression of lipid oxidation in poultry muscle.

Nitrosyl-hemoglobin formation in rodent and human venous erythrocytes reflects NO formation from the vasculature in vivo.

Reduced bioavailability of nitric oxide (NO) is a major feature of endothelial dysfunction characteristic of cardiovascular and metabolic diseases but the short half-life of NO precludes its easy quantification in circulating blood for early diagnosis. In erythrocytes, NO can react with hemoglobin to form an iron-nitrosyl complex (5-coordinate-α-HbNO) directly quantifiable by Electron Paramagnetic Resonance spectroscopy (EPR) in mouse, rat and human venous blood ex vivo. However, the sources of the nitrosylating species in vivo and optimal conditions of HbNO preservation for diagnostic use in human erythrocytes are unknown. Using EPR spectroscopy, we found that HbNO stability was significantly higher under hypoxia (equivalent to venous pO2; 12.0±0.2% degradation of HbNO at 30 minutes) than at room air (47.7±0.2% degradation) in intact erythrocytes; at 20°C (15.2±0.3% degradation after 30 min versus 29.6±0.1% at 37°C) and under acidic pH (31.7±0.8% versus 62.2±0.4% degradation after 30 min at physiological pH) at 50% of haematocrit. We next examined the relative contribution of NO synthase (NOS) from the vasculature or in erythrocytes themselves as a source of nitrosylating NO. We detected a NOS activity (and eNOS expression) in human red blood cells (RBC), and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice, as measured by HbNO formation and nitrite/nitrate accumulation. NO formation was increased after inhibition of arginase but abrogated upon NOS inhibition in human RBC and in RBCs from eNOS(+/+) (but not eNOS(-/-)) mice. However, the HbNO signal from freshly drawn venous RBCs was minimally sensitive to the inhibitors ex vivo, while it was enhanced upon caveolin-1 deletion in vivo, suggesting a minor contribution of erythrocyte NOS to HbNO complex formation compared with vascular endothelial NOS or other paracrine NO sources. We conclude that HbNO formation in rodent and human venous erythrocytes is mainly influenced by vascular NO sources despite the erythrocyte NOS activity, so that its measurement by EPR could serve as a surrogate for NO-dependent endothelial function.

A Study of the Mechanism of the FeCl3-Induced Aggregation of Human Erythrocytes

Abstract—Aggregation of intact and ATP-depleted human erythrocytes, which was induced by FeCl3 at a final concentration of 200 μM, was studied. The shape of erythrocytes and the stability of their cytoskeleton, the effect of shear flow on erythrocyte deformation, and the content of anionic sites in cell membranes were investigated. It is shown that the ATP depletion of erythrocytes led to a change in their shape: discocytes were transformed into echinocytes. This was accompanied by a decrease in the membrane cytoskeleton stability, disturbance in the deformation of erythrocytes, and reduction in the content of anionic sites in the membrane of ATP-depleted erythrocytes. Aggregation of such erythrocytes significantly decreased. A possible mechanism of FeCl3-induced aggregation of human erythrocytes is discussed.

EFFECT OF NITRIC OXIDE METABOLITES ON THE FORMATION OF MEMBRANEBOUND HEMOGLOBIN UNDER CARBONYL STRESS

Hemoglobin (Hb) in intact erythrocytes exists both in soluble and membrane-bound states. Hemoglobin transition to the membrane-bound state (MBHb) can be physiologically significant. We can presume that it provokes an adaptive reaction aimed to change different parameters connected with erythrocyte biochemistry and membrane properties. Nitric oxide (NO) is known to affect hb-membrane binding. We have shown that the addition of NaNO 2 , сysNO and GSNO to the suspension of erythrocytes results in changes in MBHb concentration. hemolytic stability depends on MBhb level: even small MBHb fluctuations change the level of erythrocytes hemolysis. NaNO 2 triggered the highestb amplitude of MBHb changes due to the formation of free-radical products in the system (oxyHb − NaNO 2 ). Methylglyoxal (MG) provoked a dose-dependent increase of MBHb, whereas the Hb oxidative-reductive state was not affected. The level of MBhb was negatively correlated with the amount of reduced Sh-groups of membrane proteins. The addition of NO metabolites to erythrocytes suspension in the presence of MG induced various effects: NaNO 2 reduced MBhb by 50 %, сysNO by 20 %, while GS-NO, on the contrary, slightly promoted the MBHb level. The effect of nitrosothiols can be explained by freeradical products being formed in the system (MG−Nh 3 −Pr−RSNO), which promote Hb binding with membrane components by inducing lipid and protein oxidation processes. It was shown that even insignificant MBHb fluctuations resulted in changes of erythrocyte hemolysis rates.

Exploitation of a newly-identified entry pathway into the malaria parasite-infected erythrocyte to inhibit parasite egress

While many parasites develop within host cells to avoid antibody responses and to utilize host cytoplasmic resources, elaborate egress processes have evolved to minimize the time between escaping and invading the next cell. In human erythrocytes, malaria parasites perforate their enclosing erythrocyte membrane shortly before egress. Here, we show that these pores clearly function as an entry pathway into infected erythrocytes for compounds that inhibit parasite egress. The natural glycosaminoglycan heparin surprisingly inhibited malaria parasite egress, trapping merozoites within infected erythrocytes. Labeled heparin neither bound to nor translocated through the intact erythrocyte membrane during parasite development, but fluxed into erythrocytes at the last minute of the parasite lifecycle. This short encounter was sufficient to significantly inhibit parasite egress and dispersion. Heparin blocks egress by interacting with both the surface of intra-erythrocytic merozoites and the inner aspect of erythrocyte membranes, preventing the rupture of infected erythrocytes but not parasitophorous vacuoles, and independently interfering with merozoite disaggregation. Since this action of heparin recapitulates that of neutralizing antibodies, membrane perforation presents a brief opportunity for a new strategy to inhibit parasite egress and replication.

Extranodal Rosai–Dorfman disease arising in the heart: clinical course and review of literature

Rosai-Dorfman disease (RDD), also known as sinus histiocytosis with massive lymphadenopathy, is a nonmalignant disease of unknown etiology. It may present as bilateral, massive, painless cervical lymphadenopathy; however, one third of the cases have extranodal involvement. Extranodal sites include soft tissue, skin, upper respiratory and gastrointestinal tracts, and central nervous system, with head and neck being the most common. Here we report a rare case of intracardiac RDD in a 46-year-old, African-American female who presented with worsening shortness of breath and fatigue for 7 weeks. Comprehensive physical examination was within normal limits; the patient had no lymphadenopathy. Echocardiogram revealed a large mass in the left atrium extending into the mitral valve. The mass was resected under the pretext of clinical diagnosis of myxoma, and the patient underwent a radical extended interatrial septectomy with bovine pericardial patch reconstruction. The 8-cm resected mass consisted of areas of fibrosis alternating with sheets of histiocytes enmeshed in a mixed inflammatory infiltrate. Histiocytes contained intact lymphocytes, plasma cells, neutrophils, and erythrocytes suggesting emperipolesis. The diagnosis of RDD was confirmed by immunohistochemistry. This case highlights the extranodal manifestation of RDD in the heart which could be easily mistaken for a myxoma on clinical and radiologic evaluation. RDD must be considered in the differential diagnosis of any patient who presents with a cardiac mass.

Urate hydroperoxide oxidizes human peroxiredoxin 1 and peroxiredoxin 2

Urate hydroperoxide is a product of the oxidation of uric acid by inflammatory heme peroxidases. The formation of urate hydroperoxide might be a key event in vascular inflammation, where there is large amount of uric acid and inflammatory peroxidases. Urate hydroperoxide oxidizes glutathione and sulfur-containing amino acids and is expected to react fast toward reactive thiols from peroxiredoxins (Prxs). The kinetics for the oxidation of the cytosolic 2-Cys Prx1 and Prx2 revealed that urate hydroperoxide oxidizes these enzymes at rates comparable with hydrogen peroxide. The second-order rate constants of these reactions were 4.9 × 105 and 2.3 × 106 m-1 s-1 for Prx1 and Prx2, respectively. Kinetic and simulation data suggest that the oxidation of Prx2 by urate hydroperoxide occurs by a three-step mechanism, where the peroxide reversibly associates with the enzyme; then it oxidizes the peroxidatic cysteine, and finally, the rate-limiting disulfide bond is formed. Of relevance, the disulfide bond formation was much slower in Prx2 (k3 = 0.31 s-1) than Prx1 (k3 = 14.9 s-1). In addition, Prx2 was more sensitive than Prx1 to hyperoxidation caused by both urate hydroperoxide and hydrogen peroxide. Urate hydroperoxide oxidized Prx2 from intact erythrocytes to the same extent as hydrogen peroxide. Therefore, Prx1 and Prx2 are likely targets of urate hydroperoxide in cells. Oxidation of Prxs by urate hydroperoxide might affect cell function and be partially responsible for the pro-oxidant and pro-inflammatory effects of uric acid.

A novel in vitro model reveals distinctive modulatory roles of Plasmodium falciparum and Plasmodium vivax on na\xefve cell-mediated immunity

BackgroundTo date, human peripheral blood mononuclear cells (PBMCs) have been used mainly in immune stimulation assays and the interpretation of data can be influenced by the previous immunological history of donors and cross reactivity with other infectious agents. Resolving these limitations requires an alternative in vitro model to uncover the primary response profiles.MethodsA novel in vitro model of mononuclear cells (MNCs) generated from haematopoietic stem cells (HSCs) was developed and these cells were then co-cultured with various antigens from Plasmodium falciparum and Plasmodium vivax to investigate the response of naïve immune cells to malaria antigens by flow cytometry.ResultsIn vitro stimulation of naïve lymphocytes showed that CD4+ and CD8+ T lymphocytes were significantly reduced (P < 0.01) by exposure to lysates of infected erythrocytes or intact erythrocytes infected with P. falciparum. The depletion was associated with the expression of CD95 (Fas receptor) on the surface of T lymphocytes. Maturation of T lymphocytes was affected differently, showing elevated CD3+CD4+CD8+ and CD3+CD4−CD8− T lymphocytes after stimulation with cell lysates of P. falciparum and P. vivax, respectively. In addition, antigen presenting monocytes and dendritic cells derived from haematopoietic stem cells showed impaired HLA-DR expression as a consequence of exposure to different species of malaria parasites.ConclusionThese results suggest that naïve mononuclear cells differentiated in vitro from HSCs could provide a valid model for the assessment of immunity. P. falciparum and P. vivax malaria parasites could modulate various populations of immune cells starting from newly differentiated mononuclear cells.

Mapping of hemoglobin in erythrocytes and erythrocyte ghosts using two photon excitation fluorescence microscopy.

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

Atomic force microscopy of swelling and hardening of intact erythrocytes fixed on substrate

Peak force measurements with the aid of atomic force microscopy are used to quantitatively map nanomechanical properties of intact erythrocytes of rats under conditions that are close to physiological conditions. Erythrocytes that are immobilized on the substrate preliminary processed using poly-L-lysine predominantly exhibit plane shape. However, cells may also exhibit stepwise transformation to semispherical objects with an increase in volume and hardening. Possible reasons for such transformations are discussed.

Full-Length Anion Exchanger 1 Structure and Interactions with Ankyrin-1 Determined by Zero Length Crosslinking of Erythrocyte Membranes

Anion exchanger 1 (AE1) is a critical transporter and the primary structural scaffold for large macromolecular complexes responsible for erythrocyte membrane flexibility and integrity. We used zero-length crosslinking and mass spectrometry to probe AE1 structures and interactions in intact erythrocyte membranes. An experimentally verified full-length model of AE1 dimers was developed by combining crosslink-defined distance constraints with homology modeling. Previously unresolved cytoplasmic loops in the AE1 C-terminal domain are packed at the domain-domain interface on the cytoplasmic face of the membrane where they anchor the N-terminal domain's location and prevent it from occluding the ion channel. Crosslinks between AE1 dimers and ankyrin-1 indicate the likely topology for AE1 tetramers and suggest that ankyrin-1 wraps around AE1 tetramers, which may stabilize this oligomer state. This interaction and interactions of AE1 with other major erythrocyte membrane proteins show that protein-protein contacts are often substantially more extensive than previously reported.

Effects of Thimerosal on Lipid Bilayers and Human Erythrocytes: An In Vitro Study.

Thimerosal (THI, ethyl-mercury thiosalicylate) is added to vaccines as a preservative; as a consequence, infants may have been exposed to bolus doses of Hg that collectively added up to nominally 200µgHg during the first 6months of life. While several studies report an association between THI-containing vaccines and neurological disorders, other studies do not support the causal relation between THI and autism. With the purpose to understand the molecular mechanisms of the toxic effect of THI it was assayed on human red cells and in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), classes of phospholipids found in the outer and inner monolayers of the human erythrocyte membrane, respectively. The capacity of THI to interact with DMPC and DMPE was determined by X-ray diffraction and differential scanning calorimetry, whereas intact human erythrocytes were observed by optical, defocusing and scanning electron microscopy. The experimental findings of this study demonstrated that THI interacted in a concentration-dependent manner with DMPC and DMPE bilayers, and in vitro interacted with erythrocytes inducing morphological changes. However, concentrations were considerable higher than those present in vaccines.

Single Molecule Studies of the Diffusion of Band 3 in Sickle Cell Erythrocytes.

Sickle cell disease (SCD) is caused by an inherited mutation in hemoglobin that leads to sickle hemoglobin (HbS) polymerization and premature HbS denaturation. Previous publications have shown that HbS denaturation is followed by binding of denatured HbS (a.k.a. hemichromes) to band 3, the consequent clustering of band 3 in the plane of the erythrocyte membrane that in turn promotes binding of autologous antibodies to the clustered band 3, and removal of the antibody-coated erythrocytes from circulation. Although each step of the above process has been individually demonstrated, the fraction of band 3 that is altered by association with denatured HbS has never been determined. For this purpose, we evaluated the lateral diffusion of band 3 in normal cells, reversibly sickled cells (RSC), irreversibly sickled cells (ISC), and hemoglobin SC erythrocytes (HbSC) in order to estimate the fraction of band 3 that was diffusing more slowly due to hemichrome-induced clustering. We labeled fewer than ten band 3 molecules per intact erythrocyte with a quantum dot to avoid perturbing membrane structure and we then monitored band 3 lateral diffusion by single particle tracking. We report here that the size of the slowly diffusing population of band 3 increases in the sequence: normal cells<HbSC<RSC<ISC. We also demonstrate that the size of the compartment in which band 3 is free to diffuse decreases roughly in the same order, with band 3 diffusing in two compartments of sizes 35 and 71 nm in normal cells, but only a single compartment in HbSC cells (58 nm), RSC (45 nm) and ISC (36 nm). These data suggest that the mobility of band 3 is increasingly constrained during SCD progression, suggesting a global impact of the mutated hemoglobin on erythrocyte membrane properties.

Diffusion of glycophorin A in human erythrocytes

Several lines of evidence suggest that glycophorin A (GPA) interacts with band 3 in human erythrocyte membranes including: i) the existence of an epitope shared between band 3 and GPA in the Wright b blood group antigen, ii) the fact that antibodies to GPA inhibit the diffusion of band 3, iii) the observation that expression of GPA facilitates trafficking of band 3 from the endoplasmic reticulum to the plasma membrane, and iv) the observation that GPA is diminished in band 3 null erythrocytes. Surprisingly, there is also evidence that GPA does not interact with band 3, including data showing that: i) band 3 diffusion increases upon erythrocyte deoxygenation whereas GPA diffusion does not, ii) band 3 diffusion is greatly restricted in erythrocytes containing the Southeast Asian Ovalocytosis mutation whereas GPA diffusion is not, and iii) most anti-GPA or anti-band 3 antibodies do not co-immunoprecipitate both proteins. To try to resolve these apparently conflicting observations, we have selectively labeled band 3 and GPA with fluorescent quantum dots in intact erythrocytes and followed their diffusion by single particle tracking. We report here that band 3 and GPA display somewhat similar macroscopic and microscopic diffusion coefficients in unmodified cells, however perturbations of band 3 diffusion do not cause perturbations of GPA diffusion. Taken together the collective data to date suggest that while weak interactions between GPA and band 3 undoubtedly exist, GPA and band 3 must have separate interactions in the membrane that control their lateral mobility.

The role of the erythrocyte cytoskeleton protein spectrin during invasion by Francisella tularensis.

Francisella tularensis is classified as a Category A bioterrorism agent by the Centers for Disease Control and Prevention. Inhaling as few as ten of these bacteria can cause acute, lethal pneumonia. Studies using mouse models of infection have shown that erythrocyte invasion is a novel feature of F. tularensis during infection; however, the mechanism by which this bacterium enters red blood cells is unknown. Cytoskeletal rearrangement of other host cells such as macrophages and hepatocytes is required for entry of F. tularensis. Therefore, in this study, we evaluated the role of the major erythrocyte cytoskeletal component, spectrin, in red blood cell invasion. To test this, human erythrocytes were treated with venom from the Blue-bellied black snake (Pseudechis guttatus), which disrupts the spectrin in the cytoskeleton. Treatment with this venom significantly reduced the number of intra-erythrocytic bacteria suggesting that spectrin is involved in erythrocyte invasion. Importantly, the concentrations of venom used in this study did not reduce the number of intact erythrocytes. This is the first study showing the involvement of an erythrocyte protein during invasion by F. tularensis.