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Sample records for red cell proteins

  1. Functions of red cell surface proteins.

    PubMed

    Daniels, G

    2007-11-01

    The external membrane of the red cell contains numerous proteins that either cross the lipid bilayer one or more times or are anchored to it through a lipid tail. Many of these proteins express blood group activity. The functions of some of these proteins are known; in others their function can only be surmised from the protein structure or from limited experimental evidence. They are loosely divided into four categories based on their functions: membrane transporters; adhesion molecules and receptors; enzymes; and structural proteins that link the membrane with the membrane skeleton. Some of the proteins carry out more than one of these functions. Some proteins may complete their major functions during erythropoiesis or may only be important under adverse physiological conditions. Furthermore, some might be evolutionary relics and may no longer have significant functions. Polymorphisms or rare changes in red cell surface proteins are often responsible for blood groups. The biological significance of these polymorphisms or the selective pressures responsible for their stability within populations are mostly not known, although exploitation of the proteins by pathogenic micro-organisms has probably played a major role.

  2. Dysferlin and other non-red cell proteins accumulate in the red cell membrane of Diamond-Blackfan Anemia patients.

    PubMed

    Pesciotta, Esther N; Sriswasdi, Sira; Tang, Hsin-Yao; Speicher, David W; Mason, Philip J; Bessler, Monica

    2014-01-01

    Diamond Blackfan Anemia (DBA) is a congenital anemia usually caused by diverse mutations in ribosomal proteins. Although the genetics of DBA are well characterized, the mechanisms that lead to macrocytic anemia remain unclear. We systematically analyzed the proteomes of red blood cell membranes from multiple DBA patients to determine whether abnormalities in protein translation or erythropoiesis contribute to the observed macrocytosis or alterations in the mature red blood cell membrane. In depth proteome analysis of red cell membranes enabled highly reproducible identification and quantitative comparisons of 1100 or more proteins. These comparisons revealed clear differences between red cell membrane proteomes in DBA patients and healthy controls that were consistent across DBA patients with different ribosomal gene mutations. Proteins exhibiting changes in abundance included those known to be increased in DBA such as fetal hemoglobin and a number of proteins not normally found in mature red cell membranes, including proteins involved in the major histocompatibility complex class I pathway. Most striking was the presence of dysferlin in the red blood cell membranes of DBA patients but absent in healthy controls. Immunoblot validation using red cell membranes isolated from additional DBA patients and healthy controls confirmed a distinct membrane protein signature specific to patients with DBA.

  3. Cloning of intronic sequence within DsRed2 increased the number of cells expressing red fluorescent protein.

    PubMed

    Pisal, Rishikaysh V; Hrebikova, Hana; Chvatalova, Jana; Soukup, Tomas; Stanislav, Filip; Mokry, Jaroslav

    2017-08-24

    Cloning of artificial intronic sequence within the open reading frame (ORF) of DsRed2 gene. Splice prediction software was used to analyze DsRed2 sequence to find an ideal site for cloning artificial intronic sequence. Intron was cloned within DsRed2 using cyclic ligation assembly. Flow cytometry was used to quantify the number of cells expressing red fluorescence. Sequencing data confirmed precise cloning of intron at the desired position using cyclic ligation assembly. Successful expression of red fluorescence after cloning of intron confirmed successful intron recognition and splicing by host cell line. Cloning of intron increased the number of cells expressing red fluorescent protein. Cloning of intronic sequence within DsRed2 has helped to increase the number of cells expressing red fluorescence by approximately four percent.

  4. The novel role of peroxiredoxin-2 in red cell membrane protein homeostasis and senescence.

    PubMed

    Matté, Alessandro; Pantaleo, Antonella; Ferru, Emanuela; Turrini, Franco; Bertoldi, Mariarita; Lupo, Francesca; Siciliano, Angela; Ho Zoon, Chae; De Franceschi, Lucia

    2014-11-01

    Peroxiredoxin-2 (Prx2), a typical two-cysteine peroxiredoxin, is the third most abundant protein in red cells. Although progress has been made in the functional characterization of Prx2, its role in red cell membrane protein homeostasis is still under investigation. Here, we studied Prx2(-/-) mouse red cells. The absence of Prx2 promotes (i) activation of the oxidative-induced Syk pathway; (ii) increased band 3 Tyr phosphorylation, with clustered band 3; and (iii) increased heat shock protein (HSP27 and HSP70) membrane translocation. This was associated with enhanced in vitro erythrophagocytosis of Prx2(-/-) red cells and reduced Prx2(-/-) red cell survival, indicating the possible role of Prx2 membrane recruitment in red cell aging and in the clearance of oxidized hemoglobin and damaged proteins through microparticles. Indeed, we observed an increased release of microparticles from Prx2(-/-) mouse red cells. The mass spectrometric analysis of erythroid microparticles found hemoglobin chains, membrane proteins, and HSPs. To test these findings, we treated Prx2(-/-) mice with antioxidants in vivo. We observed that N-acetylcysteine reduced (i) Syk activation, (ii) band 3 clusterization, (iii) HSP27 membrane association, and (iv) erythroid microparticle release, resulting in increased Prx2(-/-) mouse red cell survival. Thus, we propose that Prx2 may play a cytoprotective role in red cell membrane protein homeostasis and senescence.

  5. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    PubMed

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  6. Proteomic analysis identifies differentially expressed proteins after red propolis treatment in Hep-2 cells.

    PubMed

    Frozza, Caroline Olivieri da Silva; Ribeiro, Tanara da Silva; Gambato, Gabriela; Menti, Caroline; Moura, Sidnei; Pinto, Paulo Marcos; Staats, Charley Christian; Padilha, Francine Ferreira; Begnini, Karine Rech; de Leon, Priscila Marques Moura; Borsuk, Sibele; Savegnago, Lucielli; Dellagostin, Odir; Collares, Tiago; Seixas, Fabiana Kömmling; Henriques, João Antonio Pêgas; Roesch-Ely, Mariana

    2014-01-01

    Here we investigated alterations in the protein profile of Hep-2 treated with red propolis using two-dimensional electrophoresis associated to mass spectrometry and apoptotic rates of cells treated with and without red propolis extracts through TUNEL and Annexin-V assays. A total of 325 spots were manually excised from the two-dimensional gel electrophoresis and 177 proteins were identified using LC-MS-MS. Among all proteins identified that presented differential expression, most were down-regulated in presence of red propolis extract at a concentration of 120 μg/mL (IC50): GRP78, PRDX2, LDHB, VIM and TUBA1A. Only two up-regulated proteins were identified in this study in the non-cytotoxic (6 μg/mL) red propolis treated group: RPLP0 and RAD23B. TUNEL staining assay showed a markedly increase in the mid- to late-stage apoptosis of Hep-2 cells induced by red propolis at concentrations of 60 and 120 μg/mL when compared with non-treated cells. The increase of late apoptosis was confirmed by in situ Annexin-V analysis in which red propolis extract induced late apoptosis in a dose-dependent manner. The differences in tumor cell protein profiles warrant further investigations including isolation of major bioactive compounds of red propolis in different cell lines using proteomics and molecular tests to validate the protein expression here observed. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Isolation of a Kell-reactive protein from red cell membranes.

    PubMed

    Wallas, C; Simon, R; Sharpe, M A; Byler, C

    1986-01-01

    A red cell membrane protein which exhibits Kell blood group antigen activity has been identified with a purified anti-Kell bound to a Protein-A agarose column and eluting with lithium diiodosalicylate (LIS). Although anti-Kell as well as the Kell-reactive membrane protein were eluted from the column, the eluate was capable of reducing the titer of added anti-Kell from 64 to 4. In addition, the eluate was shown to possess Kell reactivity by binding I125 Protein A after incubation with anti-Kell. Electrophoresis (SDS gel polyacrylamide 5-20% gradient) showed a band at approximately 90,000 daltons when solubilized membranes from Kell-positive red cells were used but not when membranes from dithiothreitol- and papain-treated Kell-positive red cells or Kell-negative red cells were used. A band isolated with unreduced conditions was capable of neutralizing anti-Kell.

  8. Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging.

    PubMed

    Hense, Anika; Prunsche, Benedikt; Gao, Peng; Ishitsuka, Yuji; Nienhaus, Karin; Nienhaus, G Ulrich

    2015-12-09

    The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M(-1)cm(-1), mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths.

  9. The application of KillerRed for acute protein inactivation in living cells

    PubMed Central

    Jarvela, Timothy S.; Linstedt, Adam D.

    2017-01-01

    Generating loss of protein function is a powerful investigatory tool particularly if carried out at a physiologically relevant timescale in a live-cell fluorescent imaging experiment. KillerRed mediated chromophore assisted light inactivation (CALI) uses genetic encoding for specificity and light for acute inactivation that can also be spatially restricted. This unit provides protocols for setting up and carrying out properly controlled KillerRed experiments during live-cell imaging of cultured cells. PMID:24984963

  10. Detection of IgG sensitization of red cells with /sup 125/I staphylococcal protein A

    SciTech Connect

    Yam, P.; Petz, L.D.; Spath, P.

    1982-06-01

    Most cases of immune hemolytic anemia are associated with a positive direct antiglobulin test. However, in some cases, the antiglobulin test is not sensitive enough to detect low levels of red-cell bound antibodies. This report describes a method using radiolabelled purified staphylococcal protein A which is capable of detecting IgG sensitization of red cells beyond the threshold of serologic techniques. It is less cumbersome than previously described methods and does not require antibody purification procedures. Its effectiveness was demonstrated for the detection of red-cell alloantibodies and in evaluation of patients with acquired hemolytic anemias associated with a negative direct antiglobulin test.

  11. The relationship between in vivo generated hemoglobin skeletal protein complex and increased red cell membrane rigidity.

    PubMed

    Fortier, N; Snyder, L M; Garver, F; Kiefer, C; McKenney, J; Mohandas, N

    1988-05-01

    In vitro induced oxidative damage to normal human RBCs has previously been shown to result in increased membrane rigidity as a consequence of the generation of a protein complex between hemoglobin and spectrin. In order to determine if in vivo generated hemoglobin-spectrin complexes may play a role in increased membrane rigidity of certain pathologic red cells, we measured both these parameters in membranes prepared from hereditary xerocytosis (Hx), sickle cell disease (Sc), and red cells from thalassemia minor (beta thal). Membranes were prepared from density-fractionated red cells, and membrane deformability was measured using an ektacytometer. Hemoglobin-spectrin complex was determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel analysis, as well as by Western blot analysis using a monoclonal antibody against the beta-subunit of hemoglobin. For these three types of pathologic red cells, progressive cellular dehydration was associated with increased membrane rigidity and increased content of hemoglobin-spectrin complex. Moreover, the increase in membrane rigidity appeared to be directly related to the quantity of hemoglobin-spectrin complex associated with the membrane. Our findings imply that hemoglobin-spectrin complex is generated in vivo, and this in turn results in increased membrane rigidity of certain pathologic red cells. The data further suggest that oxidative crosslinking may play an important role in the pathophysiology of certain red cell disorders.

  12. Tissue distribution of blood group membrane proteins beyond red cells: evidence from cDNA libraries.

    PubMed

    Rojewski, Markus T; Schrezenmeier, Hubert; Flegel, Willy A

    2006-08-01

    The proteins of blood group systems are expressed on red blood cells (RBC) by definition. We searched nucleotide databases of human expressed sequence tags (EST) to collate the distribution of 22 distinct membrane proteins in cells and tissues other than RBC. The documented blood group genes are: MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, John-Milton-Hagen and Gill. The genes were grouped according to their overall and their relative expression in embryo and adults. We describe the distribution of EST in cells, tissues and cell lines with a focus on non-RBC tissues.

  13. Monomeric Garnet, a far-red fluorescent protein for live-cell STED imaging

    PubMed Central

    Hense, Anika; Prunsche, Benedikt; Gao, Peng; Ishitsuka, Yuji; Nienhaus, Karin; Ulrich Nienhaus, G.

    2015-01-01

    The advancement of far-red emitting variants of the green fluorescent protein (GFP) is crucially important for imaging live cells, tissues and organisms. Despite notable efforts, far-red marker proteins still need further optimization to match the performance of their green counterparts. Here we present mGarnet, a robust monomeric marker protein with far-red fluorescence peaking at 670 nm. Thanks to its large extinction coefficient of 95,000 M−1cm−1, mGarnet can be efficiently excited with 640-nm light on the red edge of its 598-nm excitation band. A large Stokes shift allows essentially the entire fluorescence emission to be collected even with 640-nm excitation, counterbalancing the lower fluorescence quantum yield of mGarnet, 9.1%, that is typical of far-red FPs. We demonstrate an excellent performance as a live-cell fusion marker in STED microscopy, using 640 nm excitation and 780 nm depletion wavelengths. PMID:26648024

  14. Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions.

    PubMed

    Lee, Kisung; Kinnunen, Matti; Khokhlova, Maria D; Lyubin, Evgeny V; Priezzhev, Alexander V; Meglinski, Igor; Fedyanin, Andrey A

    2016-03-01

    Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

  15. Optical tweezers study of red blood cell aggregation and disaggregation in plasma and protein solutions

    NASA Astrophysics Data System (ADS)

    Lee, Kisung; Kinnunen, Matti; Khokhlova, Maria D.; Lyubin, Evgeny V.; Priezzhev, Alexander V.; Meglinski, Igor; Fedyanin, Andrey A.

    2016-03-01

    Kinetics of optical tweezers (OT)-induced spontaneous aggregation and disaggregation of red blood cells (RBCs) were studied at the level of cell doublets to assess RBC interaction mechanics. Measurements were performed under in vitro conditions in plasma and fibrinogen and fibrinogen + albumin solutions. The RBC spontaneous aggregation kinetics was found to exhibit different behavior depending on the cell environment. In contrast, the RBC disaggregation kinetics was similar in all solutions qualitatively and quantitatively, demonstrating a significant contribution of the studied proteins to the process. The impact of the study on assessing RBC interaction mechanics and the protein contribution to the reversible RBC aggregation process is discussed.

  16. Red cell membrane disorders.

    PubMed

    Narla, J; Mohandas, N

    2017-05-01

    Significant advances have been made in our understanding of the structural basis for altered cell function in various inherited red cell membrane disorders with reduced red cell survival and resulting hemolytic anemia. The current review summarizes these advances as they relate to defining the molecular and structural basis for disorders involving altered membrane structural organization (hereditary spherocytosis [HS] and hereditary elliptocytosis [HE]) and altered membrane transport function (hereditary overhydrated stomatocytosis and hereditary xerocytosis). Mutations in genes encoding membrane proteins that account for these distinct red cell phenotypes have been identified. These molecular insights have led to improved understanding of the structural basis for altered membrane function in these disorders. Weakening of vertical linkage between the lipid bilayer and spectrin-based membrane skeleton leads to membrane loss in HS. In contrast, weakening of lateral linkages among different skeletal proteins leads to membrane fragmentation and decreased surface area in HE. The degrees of membrane loss and resultant increases in cell sphericity determine the severity of anemia in these two disorders. Splenectomy leads to amelioration of anemia by increasing the circulatory red cell life span of spherocytic red cells that are normally sequestered by the spleen. Disordered membrane cation permeability and resultant increase or decrease in red cell volume account for altered cellular deformability of hereditary overhydrated stomatocytosis and hereditary xerocytosis, respectively. Importantly, splenectomy is not beneficial in these two membrane transport disorders and in fact contraindicated due to severe postsplenectomy thrombotic complications. © 2017 John Wiley & Sons Ltd.

  17. Microfluidic cell sorter for use in developing red fluorescent proteins with improved photostability.

    PubMed

    Davis, Lloyd M; Lubbeck, Jennifer L; Dean, Kevin M; Palmer, Amy E; Jimenez, Ralph

    2013-06-21

    This paper presents a novel microfluidic cytometer for mammalian cells that rapidly measures the irreversible photobleaching of red fluorescent proteins expressed within each cell and achieves high purity (>99%) selection of individual cells based on these measurements. The selection is achieved by using sub-millisecond timed control of a piezo-tilt mirror to steer a focused 1064-nm laser spot for optical gradient force switching following analysis of the fluorescence signals from passage of the cell through a series of 532-nm laser beams. In transit through each beam, the fluorescent proteins within the cell undergo conversion to dark states, but the microfluidic chip enables the cell to pass sufficiently slowly that recovery from reversible dark states occurs between beams, thereby enabling irreversible photobleaching to be quantified separately from the reversible dark-state conversion. The microfluidic platform achieves sorting of samples down to sub-millilitre volumes with minimal loss, wherein collected cells remain alive and can subsequently proliferate. The instrument provides a unique first tool for rapid selection of individual mammalian cells on the merits of photostability and is likely to form the basis of subsequent lab-on-a-chip platforms that combine photobleaching with other spectroscopic measurements for on-going research to develop advanced red fluorescent proteins by screening of genetic libraries.

  18. Plasmolysis, red blood cell partitioning, and plasma protein binding of etofibrate, clofibrate, and their degradation products.

    PubMed

    Altmayer, P; Garrett, E R

    1983-11-01

    Etofibrate (I), the ethylene glycol diester of clofibric and nicotinic acids, degrades almost equally through both half-esters with half-lives of approximately 10 and 1 min in fresh dog and human plasma, respectively. The nicotinate V degrades with half-lives of approximately 12 hr and 50 min in fresh dog and human plasma, respectively. Ester III and clofibrate VI degrade by saturable Michaelis-Menten kinetics in fresh human plasma, with similar maximum initial rates and respective terminal first-order half-lives of 12 and 26 min. Tetraethyl pyrophosphate at 100 micrograms/ml inhibited human plasma and red blood cell esterases permitting plasma protein binding and red blood cell partitioning studies. The red blood cell-plasma water partition coefficient was 5.4 for 0.2-80 micrograms/ml of I. Clofibrate (VI) showed a saturable erythrocyte partitioning that decreased from 7.8 (10 micrograms/ml) to 1 (50 micrograms/ml). The strong binding of I and VI to ultrafiltration membranes necessitated the determination of their plasma protein binding by the method of variable plasma concentrations of erythrocyte suspensions to give 96.6% (0.2-80 micrograms/ml) and 98.2% (13.6-108.4 micrograms/ml) binding, respectively. Methods for the determination of the parameters of saturable and nonsaturable plasma protein binding for unstable and membrane-binding drugs by the method of variable plasma concentrations in partitioning erythrocyte suspensions are presented.

  19. p-Chloromercuribenzoate-induced dissociation of cytoskeletal proteins in red blood cells of rats.

    PubMed

    Kunimoto, M; Shibata, K; Miura, T

    1987-12-11

    Effects of p-chloromercuribenzoate (PCMB) on the cytoskeletal organization of rat red blood cells were studied. Upon incubation with 50 microM PCMB in 10 mM Tris-HCl (pH 7.4) at 37 degrees C for 30 min, 80% of actin and 45% of spectrin were released from the ghosts, resulting in the fragmentation of ghost membranes. Addition of 2 mM Mg2+ or 0.1 M KCl, or lowering incubation temperature to 0 degree C substantially inhibited the solubilization of the cytoskeletal proteins and the fragmentation of ghost membranes, which enable to examine the effects of PCMB on the interaction between transmembrane proteins and the peripheral cytoskeletal network. Decreased recoveries of transmembrane proteins, such as band 3 and glycophorin, in Triton shell fraction were observed in the ghosts incubated with PCMB either in the presence of Mg2+ or at 0 degree C. PCMB also inhibited the in vitro association of purified spectrin with spectrin-depleted inside-out vesicles through interaction with proteins in the vesicle, such as bands 2.1 and 3. In the PCMB-treated ghosts, intramembrane particles were highly aggregated, which further supports the PCMB-induced dissociation of the transmembrane proteins from the cytoskeletal network. The decreased recovery of glycophorin in the Triton shell fraction also observed in intact red blood cells upon incubation with PCMB. These results suggest that the main action of PCMB on red cell membranes under physiological condition, at higher ionic strength and in the presence of Mg2+, is to dissociate transmembrane proteins from the peripheral cytoskeletal network, which may modify functions of these proteins.

  20. Multiphoton photochemistry of red fluorescent proteins in solution and live cells.

    PubMed

    Drobizhev, Mikhail; Stoltzfus, Caleb; Topol, Igor; Collins, Jack; Wicks, Geoffrey; Mikhaylov, Alexander; Barnett, Lauren; Hughes, Thomas E; Rebane, Aleksander

    2014-08-07

    Genetically encoded fluorescent proteins (FPs), and biosensors based on them, provide new insights into how living cells and tissues function. Ultimately, the goal of the bioimaging community is to use these probes deep in tissues and even in entire organisms, and this will require two-photon laser scanning microscopy (TPLSM), with its greater tissue penetration, lower autofluorescence background, and minimum photodamage in the out-of-focus volume. However, the extremely high instantaneous light intensities of femtosecond pulses in the focal volume dramatically increase the probability of further stepwise resonant photon absorption, leading to highly excited, ionizable and reactive states, often resulting in fast bleaching of fluorescent proteins in TPLSM. Here, we show that the femtosecond multiphoton excitation of red FPs (DsRed2 and mFruits), both in solution and live cells, results in a chain of consecutive, partially reversible reactions, with individual rates driven by a high-order (3-5 photon) absorption. The first step of this process corresponds to a three- (DsRed2) or four-photon (mFruits) induced fast isomerization of the chromophore, yielding intermediate fluorescent forms, which then subsequently transform into nonfluorescent products. Our experimental data and model calculations are consistent with a mechanism in which ultrafast electron transfer from the chromophore to a neighboring positively charged amino acid residue triggers the first step of multiphoton chromophore transformations in DsRed2 and mFruits, consisting of decarboxylation of a nearby deprotonated glutamic acid residue.

  1. Dynamics of Pinned Membranes with Application to Protein Diffusion on the Surface of Red Blood Cells

    PubMed Central

    Lin, Lawrence C.-L.; Brown, Frank L. H.

    2004-01-01

    We present a theoretical treatment and simulation algorithm for the dynamics of Helfrich elastic membrane surfaces in the presence of general harmonic perturbations and hydrodynamic coupling to the surrounding solvent. In the limit of localized and strong interactions, this harmonic model can be used to pin the membrane to intracellular/intercellular structures. We consider the case of pinning to the cytoskeleton and use such a model to estimate the macroscopic diffusion constant for band 3 protein on the surface of human erythrocytes. Comparison to experimental results suggests that thermal undulations of the membrane surface should play a significant role in protein mobility on the red blood cell. PMID:14747313

  2. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    SciTech Connect

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

  3. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    PubMed Central

    Liu, Daniel S.; Nivón, Lucas G.; Richter, Florian; Goldman, Peter J.; Deerinck, Thomas J.; Yao, Jennifer Z.; Richardson, Douglas; Phipps, William S.; Ye, Anne Z.; Ellisman, Mark H.; Drennan, Catherine L.; Baker, David; Ting, Alice Y.

    2014-01-01

    Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies. PMID:25313043

  4. Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

    DOE PAGES

    Liu, Daniel S.; Nivon, Lucas G.; Richter, Florian; ...

    2014-10-13

    In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of themore » intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.« less

  5. Red blood cell production

    MedlinePlus

    ... to one part of the body or another. Red blood cells are an important element of blood. Their job ... is carried to and eliminated by the lungs. Red blood cells are formed in the red bone marrow of ...

  6. Integral protein linkage and the bilayer-skeletal separation energy in red blood cells.

    PubMed

    Butler, James; Mohandas, Narla; Waugh, Richard E

    2008-08-01

    Stabilization of the lipid bilayer membrane in red blood cells by its association with an underlying membrane-associated cytoskeleton has long been recognized as critical for proper red blood cell function. One of the principal connections between skeleton and bilayer is via linkages between band 3, the integral membrane protein that transports anions across the cell surface, and membrane skeletal elements including ankyrin, adducin, spectrin, and the junctional complex of the skeleton. Here, we use membrane tether formation coupled with fluorescent labeling of membrane components to examine the importance of band 3 in stabilizing the bilayer-skeletal association. In membranes from a patient deficient in band 3, the energy associated with the bilayer skeleton is approximately zero, whereas when band 3 is immobilized by ligation with the monoclonal antibody R10, the energy of association approximately doubles. Fluorescence images of tethers reveal that approximately 40% of the band 3 on the normal cell surface can be pulled into the tether, confirming a lateral segregation of membrane components during tether formation. These results validate a critical role for band 3 in stabilizing the bilayer-skeletal association in red cells.

  7. Novel embryonic stem cells expressing tdKaede protein photoconvertible from green to red fluorescence.

    PubMed

    Shigematsu, Yoko; Yoshida, Naoki; Miwa, Yoshihiro; Mizobuti, Atsushi; Suzuki, Yuko; Tanimoto, Yoko; Takahashi, Satoru; Kunita, Satoshi; Sugiyama, Fumihiro; Yagami, Ken-Ichi

    2007-10-01

    Kaede protein is a photoconvertible tracer that emits green fluorescence after synthesis, which changes to stable red fluorescence upon irradiation with violet or UV illumination. This color-change characteristic is a very effective means of optically marking living cells of interest. We established novel embryonic stem (ES) cell lines, B6KED-1 and -2, from C57BL/6J transgenic mouse blastocysts ubiquitously expressing tandem dimeric Kaede (tdKaede) protein. Undifferentiated B6KED-1 and -2 cells showed bright green fluorescence and mRNAs of pluripotent marker genes. Photoconversion of tdKaede protein in undifferentiated and differentiated B6KED cells in vitro occurred upon short-term UV irradiation. B6KED cells completely generated ES cell-derived females on transfer into tetraploid blastomeres. All organs showed strong green emission in the females derived completely from B6KED cells. These novel ES cell lines ubiquitously expressing photoconvertible Kaede protein, B6KED-1 and -2, are useful for basic research in developmental biology and regenerative medicine.

  8. Generation of transgenic Wuzhishan miniature pigs expressing monomeric red fluorescent protein by somatic cell nuclear transfer.

    PubMed

    Lu, Yue; Kang, Jin-Dan; Li, Suo; Wang, Wei; Jin, Jun-Xue; Hong, Yu; Cui, Cheng-du; Yan, Chang-Guo; Yin, Xi-Jun

    2013-08-01

    Red fluorescent protein and its variants enable researchers to study gene expression, localization, and protein-protein interactions in vitro in real-time. Fluorophores with higher wavelengths are usually preferred since they efficiently penetrate tissues and produce less toxic emissions. A recently developed fluorescent protein marker, monomeric red fluorescent protein (mRFP1), is particularly useful because of its rapid maturation and minimal interference with green fluorescent protein (GFP) and GFP-derived markers. We generated a pCX-mRFP1-pgk-neoR construct and evaluated the ability of mRFP1 to function as a fluorescent marker in transgenic Wuzhishan miniature pigs. Transgenic embryos were generated by somatic cell nuclear transfer (SCNT) of nuclei isolated from ear fibroblasts expressing mRFP1. Embryos generated by SCNT developed into blastocysts in vitro (11.65%; 31/266). Thereafter, a total of 685 transgenic embryos were transferred into the oviducts of three recipients, two of which became pregnant. Of these, one recipient had six aborted fetuses, whereas the other recipient gave birth to four offspring. All offspring expressed the pCX-mRFP1-pgk-neoR gene as shown by PCR and fluorescence in situ hybridization analysis. The transgenic pigs expressed mRFP1 in all organs and tissues at high levels. These results demonstrate that Wuzhishan miniature pigs can express mRFP1. To conclude, this transgenic animal represents an excellent model with widespread applications in medicine and agriculture.

  9. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows.

    PubMed

    Brust, M; Aouane, O; Thiébaud, M; Flormann, D; Verdier, C; Kaestner, L; Laschke, M W; Selmi, H; Benyoussef, A; Podgorski, T; Coupier, G; Misbah, C; Wagner, C

    2014-03-11

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.

  10. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

    PubMed Central

    Brust, M.; Aouane, O.; Thiébaud, M.; Flormann, D.; Verdier, C.; Kaestner, L.; Laschke, M. W.; Selmi, H.; Benyoussef, A.; Podgorski, T.; Coupier, G.; Misbah, C.; Wagner, C.

    2014-01-01

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects. PMID:24614613

  11. The plasma protein fibrinogen stabilizes clusters of red blood cells in microcapillary flows

    NASA Astrophysics Data System (ADS)

    Brust, M.; Aouane, O.; Thiébaud, M.; Flormann, D.; Verdier, C.; Kaestner, L.; Laschke, M. W.; Selmi, H.; Benyoussef, A.; Podgorski, T.; Coupier, G.; Misbah, C.; Wagner, C.

    2014-03-01

    The supply of oxygen and nutrients and the disposal of metabolic waste in the organs depend strongly on how blood, especially red blood cells, flow through the microvascular network. Macromolecular plasma proteins such as fibrinogen cause red blood cells to form large aggregates, called rouleaux, which are usually assumed to be disaggregated in the circulation due to the shear forces present in bulk flow. This leads to the assumption that rouleaux formation is only relevant in the venule network and in arterioles at low shear rates or stasis. Thanks to an excellent agreement between combined experimental and numerical approaches, we show that despite the large shear rates present in microcapillaries, the presence of either fibrinogen or the synthetic polymer dextran leads to an enhanced formation of robust clusters of red blood cells, even at haematocrits as low as 1%. Robust aggregates are shown to exist in microcapillaries even for fibrinogen concentrations within the healthy physiological range. These persistent aggregates should strongly affect cell distribution and blood perfusion in the microvasculature, with putative implications for blood disorders even within apparently asymptomatic subjects.

  12. Alterations of membrane protein expression in red blood cells of Alzheimer's disease patients.

    PubMed

    Várady, György; Szabó, Edit; Fehér, Ágnes; Németh, Adrienn; Zámbó, Boglárka; Pákáski, Magdolna; Janka, Zoltán; Sarkadi, Balázs

    2015-09-01

    Preventive measures, prognosis, or selected therapy in multifactorial maladies, including Alzheimer's disease (AD), require the application of a wide range of diagnostic assays. There is a large unmet need for relatively simple, blood-based biomarkers in this regard. We have recently developed a rapid and reliable flow cytometry and antibody-based method for the quantitative measurement of various red blood cell (RBC) membrane proteins from a drop of blood. Here, we document that the RBC expression of certain membrane proteins, especially that of the GLUT1 transporter and the insulin receptor (INSR), is significantly higher in AD patients than in age-matched healthy subjects. The observed differences may reflect long-term metabolic alterations relevant in the development of AD. These findings may pave the way for a diagnostic application of RBC membrane proteins as relatively stable and easily accessible personalized biomarkers in AD.

  13. Bioinformatic prediction of the exportome of Babesia bovis and identification of novel proteins in parasite-infected red blood cells.

    PubMed

    Gohil, Sejal; Kats, Lev M; Seemann, Torsten; Fernandez, Kate M; Siddiqui, Ghizal; Cooke, Brian M

    2013-04-01

    Babesia bovis is a pathogen of considerable economic significance to the livestock industry worldwide but the precise mechanisms by which this parasite causes disease in susceptible cattle remain poorly understood. It is clear, however, that alterations to the structure and function of red blood cells in which the parasites reside and replicate play an important role in pathogenesis and that these are secondary to the export of numerous, currently unknown and uncharacterised parasite-encoded proteins. Using a rational bioinformatic approach, we have identified a set of 362 proteins (117 of which are hypothetical) that we predict encompasses the B. bovis exportome. These exported proteins are likely to be trafficked to various cellular locations, with a subset destined for the red blood cell cytosol or the red blood cell cytoskeleton. These proteins are likely to play important roles in mediating the pathogenesis of babesiosis. We have selected three novel proteins and confirmed their predicted export and localisation within the host red blood cell by immunofluorescence using specific antibodies raised against these proteins. Complete characterisation of these novel exported parasite proteins will help elucidate their function within the host red blood cell and assist in identification of new therapeutic targets for babesiosis.

  14. Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application.

    PubMed

    He, Huining; Ye, Junxiao; Wang, Yinsong; Liu, Quan; Chung, Hee Sun; Kwon, Young Min; Shin, Meong Cheol; Lee, Kyuri; Yang, Victor C

    2014-02-28

    Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab-low molecular weight protamine (LMWP). l-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different l-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed.

  15. Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application

    PubMed Central

    Wang, Yinsong; Liu, Quan; Chung, Hee Sun; Kwon, Young Min; Shin, Meong Cheol; Lee, Kyuri; Yang, Victor C

    2014-01-01

    Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab—low molecular weight protamine (LMWP). L-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different L-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed. PMID:24374002

  16. Extraction methods of red blood cell membrane proteins for Multidimensional Protein Identification Technology (MudPIT) analysis.

    PubMed

    De Palma, Antonella; Roveri, Antonella; Zaccarin, Mattia; Benazzi, Louise; Daminelli, Simone; Pantano, Giorgia; Buttarello, Mauro; Ursini, Fulvio; Gion, Massimo; Mauri, Pier Luigi

    2010-08-13

    Since red blood cells (RBCs) lack nuclei and organelles, cell membrane is their main load-bearing component and, according to a dynamic interaction with the cytoskeleton compartment, plays a pivotal role in their functioning. Even if erythrocyte membranes are available in large quantities, the low abundance and the hydrophobic nature of cell membrane proteins complicate their purification and detection by conventional 2D gel-based proteomic approaches. So, in order to increase the efficiency of RBC membrane proteome identification, here we took advantage of a simple and reproducible membrane sub-fractionation method coupled to Multidimensional Protein Identification Technology (MudPIT). In addition, the adoption of a stringent RBC filtration strategy from the whole blood, permitted to remove exhaustively contaminants, such as platelets and white blood cells, and to identify a total of 275 proteins in the three RBC membrane fractions collected and analysed. Finally, by means of software for the elaboration of the great quantity of data obtained and programs for statistical analysis and protein classification, it was possible to determine the validity of the entire system workflow and to assign the proper sub-cellular localization and function for the greatest number of the identified proteins.

  17. Evidence that the red cell skeleton protein 4.2 interacts with the Rh membrane complex member CD47.

    PubMed

    Mouro-Chanteloup, Isabelle; Delaunay, Jean; Gane, Pierre; Nicolas, Virginie; Johansen, Mette; Brown, Eric J; Peters, Luanne L; Van Kim, Caroline Le; Cartron, Jean Pierre; Colin, Yves

    2003-01-01

    Rh(null) red cells are characteristically stomato-spherocytic. This and other evidence suggest that the Rh complex represents a major attachment site between the membrane lipid bilayer and the erythroid skeleton. As an attempt to identify the linking protein(s) between the red cell skeleton and the Rh complex, we analyzed the expression of Rh, RhAG, CD47, LW, and glycophorin B proteins in red cells from patients with hereditary spherocytosis associated with complete protein 4.2 deficiency but normal band 3 (4.2(-)HS). Flow cytometric and immunoblotting analysis revealed a severe reduction of CD47 (up to 80%) and a slower mobility of RhAG on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, possibly reflecting an overglycosylation state. Unexpectedly, 4.2(-/-) mice, which are anemic, displayed a normal red cell expression of CD47 and RhAG. These results suggest that human protein 4.2, through interaction with CD47, is involved in the skeleton linkage and/or membrane translocation of the Rh complex. However, these potential role(s) of protein 4.2 might be not conserved across species. Finally, the absence or low expression of red cell CD47 in CD47(-/-) mice and in some humans carrying RHCE gene variants (D--, D., and R(N)), respectively, had no detectable effect on protein 4.2 and RhAG expression. Since these cells are morphologically normal with no sign of hemolysis, it is assumed that CD47 deficiency per se is not responsible for the cell shape abnormalities and for the compensated hemolytic anemia typical of 4.2(-) and Rh(null) red cells.

  18. Cytosolic protein concentration is the primary volume signal in dog red cells

    PubMed Central

    1991-01-01

    It is not known whether the activation of Na/H exchange by shrinkage in dog red cells is due to the packing of cell contents or a change in cell configuration. To make this distinction we prepared resealed ghosts that resembled intact cells in hemoglobin concentration and surface area, but had one-third their volume. A shrinkage-induced, amiloride-sensitive Na flux in the ghosts was activated at a much smaller volume in the ghosts than in the intact cells, but at the same concentration (by weight) of dry solids in both preparations. Na/H exchange in ghosts containing a mixture of 40% albumin and 60% hemoglobin (weight/weight) was activated by osmotic shrinkage at a dry solid concentration similar to that of intact cells or of ghosts containing only hemoglobin. We conclude that the process of Na/H exchange activation by cell shrinkage originates with an increase in the concentration of intracellular protein and not with a change in membrane configuration or tension. The macromolecular crowding that accompanies the reduction in cell volume probably alters the activities of key enzymes that in turn modulate the Na/H exchanger. PMID:1662684

  19. Deciphering the nuclear import pathway for the cytoskeletal red cell protein 4.1R.

    PubMed

    Gascard, P; Nunomura, W; Lee, G; Walensky, L D; Krauss, S W; Takakuwa, Y; Chasis, J A; Mohandas, N; Conboy, J G

    1999-06-01

    The erythroid membrane cytoskeletal protein 4.1 is the prototypical member of a genetically and topologically complex family that is generated by combinatorial alternative splicing pathways and is localized at diverse intracellular sites including the nucleus. To explore the molecular determinants for nuclear localization, we transfected COS-7 cells with epitope-tagged versions of natural red cell protein 4.1 (4.1R) isoforms as well as mutagenized and truncated derivatives. Two distant topological sorting signals were required for efficient nuclear import of the 4.1R80 isoform: a basic peptide, KKKRER, encoded by alternative exon 16 and acting as a weak core nuclear localization signal (4.1R NLS), and an acidic peptide, EED, encoded by alternative exon 5. 4.1R80 isoforms lacking either of these two exons showed decreased nuclear import. Fusion of various 4.1R80 constructs to the cytoplasmic reporter protein pyruvate kinase confirmed a requirement for both motifs for full NLS function. 4.1R80 was efficiently imported in the nuclei of digitonin-permeabilized COS-7 cells in the presence of recombinant Rch1 (human importin alpha2), importin beta, and GTPase Ran. Quantitative analysis of protein-protein interactions using a resonant mirror detection technique showed that 4.1R80 bound to Rch1 in vitro with high affinity (KD = 30 nM). The affinity decreased at least 7- and 20-fold, respectively, if the EED motif in exon 5 or if 4.1R NLS in exon 16 was lacking or mutated, confirming that both motifs were required for efficient importin-mediated nuclear import of 4.1R80.

  20. Ruthenium red-induced bundling of bacterial cell division protein, FtsZ.

    PubMed

    Santra, Manas Kumar; Beuria, Tushar K; Banerjee, Abhijit; Panda, Dulal

    2004-06-18

    The assembly of FtsZ plays a major role in bacterial cell division, and it is thought that the assembly dynamics of FtsZ is a finely regulated process. Here, we show that ruthenium red is able to modulate FtsZ assembly in vitro. In contrast to the inhibitory effects of ruthenium red on microtubule polymerization, we found that a substoichiometric concentration of ruthenium red strongly increased the light-scattering signal of FtsZ assembly. Further, sedimentable polymer mass was increased by 1.5- and 2-fold in the presence of 2 and 10 microm ruthenium red, respectively. In addition, ruthenium red strongly reduced the GTPase activity and prevented dilution-induced disassembly of FtsZ polymers. Electron microscopic analysis showed that 4-10 microm of ruthenium red produced thick bundles of FtsZ polymers. The significant increase in the light-scattering signal and pelletable polymer mass in the presence of ruthenium red seemed to be due to the bundling of FtsZ protofilaments into larger polymers rather than the actual increase in the level of polymeric FtsZ. Furthermore, ruthenium red was found to copolymerize with FtsZ, and the copolymerization of substoichiometric amounts of ruthenium red with FtsZ polymers promoted cooperative assembly of FtsZ that produced large bundles. Calcium inhibited the binding of ruthenium red to FtsZ. However, a concentration of calcium 1000-fold higher than that of ruthenium red was required to produce similar effects on FtsZ assembly. Ruthenium red strongly modulated FtsZ polymerization, suggesting the presence of an important regulatory site on FtsZ and suggesting that a natural ligand, which mimics the action of ruthenium red, may regulate the assembly of FtsZ in bacteria.

  1. Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria

    PubMed Central

    Beeson, James G.; Drew, Damien R.; Boyle, Michelle J.; Feng, Gaoqian; Fowkes, Freya J.I.; Richards, Jack S.

    2016-01-01

    Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. PMID:26833236

  2. Targeting recombinant thrombomodulin fusion protein to red blood cells provides multifaceted thromboprophylaxis.

    PubMed

    Zaitsev, Sergei; Kowalska, M Anna; Neyman, Michael; Carnemolla, Ronald; Tliba, Samira; Ding, Bi-Sen; Stonestrom, Aaron; Spitzer, Dirk; Atkinson, John P; Poncz, Mortimer; Cines, Douglas B; Esmon, Charles T; Muzykantov, Vladimir R

    2012-05-17

    Thrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombin's substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.

  3. Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria.

    PubMed

    Beeson, James G; Drew, Damien R; Boyle, Michelle J; Feng, Gaoqian; Fowkes, Freya J I; Richards, Jack S

    2016-05-01

    Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. © FEMS 2016.

  4. Modeling of band-3 protein diffusion in the normal and defective red blood cell membrane.

    PubMed

    Li, He; Zhang, Yihao; Ha, Vi; Lykotrafitis, George

    2016-04-21

    We employ a two-component red blood cell (RBC) membrane model to simulate lateral diffusion of band-3 proteins in the normal RBC and in the RBC with defective membrane proteins. The defects reduce the connectivity between the lipid bilayer and the membrane skeleton (vertical connectivity), or the connectivity of the membrane skeleton itself (horizontal connectivity), and are associated with the blood disorders of hereditary spherocytosis (HS) and hereditary elliptocytosis (HE) respectively. Initially, we demonstrate that the cytoskeleton limits band-3 lateral mobility by measuring the band-3 macroscopic diffusion coefficients in the normal RBC membrane and in a lipid bilayer without the cytoskeleton. Then, we study band-3 diffusion in the defective RBC membrane and quantify the relation between band-3 diffusion coefficients and percentage of protein defects in HE RBCs. In addition, we illustrate that at low spectrin network connectivity (horizontal connectivity) band-3 subdiffusion can be approximated as anomalous diffusion, while at high horizontal connectivity band-3 diffusion is characterized as confined diffusion. Our simulations show that the band-3 anomalous diffusion exponent depends on the percentage of protein defects in the membrane cytoskeleton. We also confirm that the introduction of attraction between the lipid bilayer and the spectrin network reduces band-3 diffusion, but we show that this reduction is lower than predicted by the percolation theory. Furthermore, we predict that the attractive force between the spectrin filament and the lipid bilayer is at least 20 times smaller than the binding forces at band-3 and glycophorin C, the two major membrane binding sites. Finally, we explore diffusion of band-3 particles in the RBC membrane with defects related to vertical connectivity. We demonstrate that in this case band-3 diffusion can be approximated as confined diffusion for all attraction levels between the spectrin network and the lipid bilayer

  5. Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure.

    PubMed

    Fernandes, João C; Eaton, Peter; Nascimento, Henrique; Belo, Luís; Rocha, Susana; Vitorino, Rui; Amado, Francisco; Gomes, Joana; Santos-Silva, Alice; Pintado, Manuela E; Malcata, F Xavier

    2008-12-01

    Recent studies of chitosan have increased the interest in its conversion to chitooligosaccharides (COSs) because these compounds are water-soluble and have potential use in several biomedical applications. Furthermore, such oligomers may be more advantageous than chitosans because of their much higher absorption profiles at the intestinal level, which permit their facilitated access to systemic circulation and potential distribution throughout the entire human body. In that perspective, it is important to clarify their effect on blood further, namely, on human red blood cells (RBCs). The aim of this work was thus to study the effect of two COS mixtures with different molecular weight (MW) ranges, <3 and <5 kDa, at various concentrations (5.0-0.005 mg/mL) on human RBCs. The interactions of these two mixtures with RBC membrane proteins and with hemoglobin were assessed, and the RBC morphology and surface structure were analyzed by optical microscopy (OM) and atomic force microscopy (AFM). In the presence of either COS mixture, no significant hemolysis was observed; however, at COS concentrations >0.1 mg/mL, changes in membrane binding hemoglobin were observed. Membrane protein changes were also observed with increasing COS concentration, including a reduction in both alpha- and beta-spectrin and in band 3 protein, and the development of three new protein bands: peroxiredoxin 2, calmodulin, and hemoglobin chains. Morphologic evaluation by OM showed that at high concentrations COSs interact with RBCs, leading to RBC adhesion, aggregation, or both. An increase in the roughness of the RBC surface with increasing COS concentration was observed by AFM. Overall, these findings suggest that COS damage to RBCs was dependent on the COS MW and concentration, and significant damage resulted from either a higher MW or a greater concentration (>0.1 mg/mL).

  6. Use of Kaede and Kikume green-red fusions for live cell imaging of G protein-coupled receptors.

    PubMed

    Schmidt, Antje; Wiesner, Burkhard; Schülein, Ralf; Teichmann, Anke

    2014-01-01

    The fusion of fluorescent proteins to G protein-coupled receptors (GPCRs) is an important tool to study, e.g., trafficking and protein interactions of these important drug targets. In the past, the green fluorescent protein and its derivatives have been widely used as fluorescent tags. More recently, it was reported that photoconvertible fluorescent proteins (PCFPs) such as Kaede or Kikume green-red protein could also be used as fluorescent tags for GPCRs. These proteins have the obvious advantage that their fluorescence can be switched once the GPCR of interest has reached a specific subcellular compartment. Here, we summarize the recent progress for live cell imaging of GPCRs using these PCFPs for trafficking, biosynthesis, and protein/protein interaction studies.

  7. High Red Blood Cell Count

    MedlinePlus

    Symptoms High red blood cell count By Mayo Clinic Staff A high red blood cell count is an increase in oxygen-carrying cells in your bloodstream. Red blood cells transport oxygen from your lungs to tissues throughout ...

  8. Some physico-chemical and serological properties of isolated protein components of red cell membranes

    PubMed Central

    Poulik, M. D.; Lauf, P. K.

    1969-01-01

    Exposure of human red cell ghosts to low pH (pH 2–2·2) for 20–24 hr and extraction with a biphasic system (butanol–water) at acid pH yielded a water-phase material which was separated by gel filtration into two major fractions. Fraction A is of glycoprotein nature having a molecular weight of approximately 200,000 and exhibiting A, M and N antigen activity. Fraction B is primarily protein in nature having a molecular weight of about 50,000 to 60,000, and is practically devoid of A and MN antigens. The materials isolated were found heterogeneous by electrophoresis in urea starch gels. Peak A and peak B differed significantly with respect to number, mobility, and intensity of the separated components. The physico-chemical and serological properties of the water phase and its sub-fractions are discussed with respect to structural make-up of erythrocyte membrane. ImagesFIG. 2FIG. 3 PMID:5786811

  9. The red cell band 3 protein: its role in anion transport.

    PubMed

    Rothstein, A; Ramjeesingh, M

    1982-12-01

    Studies of anion transport across the red blood cell membrane fall generally into two categories: (1) those concerned with the operational characterization of the transport system, largely by kinetic analysis and inhibitor studies; and (2) those concerned with the structure of band 3, a transmembrane peptide identified as the transport protein. The kinetics are consistent with a ping-pong model in which positively charged anion-binding sites can alternate between exposure to the inside and outside compartments but can only shift one position to the other when occupied by an anion. The structural studies on band 3 indicate that only 60% of the peptide is essential for transport. That particular portion is in the form of a dimer consisting of an assembly of membrane-crossing strands (each monomer appears to cross at least five times). The assembly presents its hydrophobic residues toward the interior of the bilayer, but its hydrophilic residues provide an aqueous core. The transport involves a small conformational change in which an anion-binding site (involving positively charged residues) can alternate between positions that are topologically in and topologically out.

  10. Development of a bifunctional filter for prion protein and leukoreduction of red blood cell components.

    PubMed

    Yokomizo, Tomo; Kai, Takako; Miura, Morikazu; Ohto, Hitoshi

    2015-02-01

    Leukofiltration of blood components is currently implemented worldwide as a precautionary measure against white blood cell-associated adverse effects and the potential transmission of variant Creutzfeldt-Jakob disease (vCJD). A newly developed bifunctional filter (Sepacell Prima, Asahi Kasei Medical) was assessed for prion removal, leukoreduction (LR), and whether the filter significantly affected red blood cells (RBCs). Sepacell Prima's postfiltration effects on RBCs, including hemolysis, complement activation, and RBC chemistry, were compared with those of a conventional LR filter (Sepacell Pure RC). Prion removal was measured by Western blot after spiking RBCs with microsomal fractions derived from scrapie-infected hamster brain homogenate. Serially diluted exogenous prion solutions (0.05 mL), with or without filtration, were injected intracerebrally into Golden Syrian hamsters. LR efficiency of 4.44 log with the Sepacell Prima was comparable to 4.11 log with the conventional LR filter. There were no significant differences between the two filters in hemoglobin loss, hemolysis, complement activation, and RBC biomarkers. In vitro reduction of exogenously spiked prions by the filter exceeded 3 log. The titer, 6.63 (log ID50 /mL), of prefiltration infectivity of healthy hamsters was reduced to 2.52 (log ID50 /mL) after filtration. The reduction factor was calculated as 4.20 (log ID50 ). With confirmed removal efficacy for exogenous prion protein, this new bifunctional prion and LR filter should reduce the residual risk of vCJD transmission through blood transfusion without adding complexity to component processing. © 2014 AABB.

  11. OpenRBC: A Fast Simulator of Red Blood Cells at Protein Resolution

    NASA Astrophysics Data System (ADS)

    Tang, Yu-Hang; Lu, Lu; Li, He; Evangelinos, Constantinos; Grinberg, Leopold; Sachdeva, Vipin; Karniadakis, George Em

    2017-05-01

    We present OpenRBC, a coarse-grained molecular dynamics code, which is capable of performing an unprecedented in silico experiment --- simulating an entire mammal red blood cell lipid bilayer and cytoskeleton as modeled by 4 million mesoscopic particles --- using a single shared memory commodity workstation. To achieve this, we invented an adaptive spatial-searching algorithm to accelerate the computation of short-range pairwise interactions in an extremely sparse 3D space. The algorithm is based on a Voronoi partitioning of the point cloud of coarse-grained particles, and is continuously updated over the course of the simulation. The algorithm enables the construction of the key spatial searching data structure in our code, i.e. a lattice-free cell list, with a time and space cost linearly proportional to the number of particles in the system. The position and shape of the cells also adapt automatically to the local density and curvature. The code implements OpenMP parallelization and scales to hundreds of hardware threads. It outperforms a legacy simulator by almost an order of magnitude in time-to-solution and more than 40 times in problem size, thus providing a new platform for probing the biomechanics of red blood cells.

  12. High affinity interactions between red blood cell receptors and synthetic Plasmodium thrombospondin-related apical merozoite protein (PTRAMP) peptides.

    PubMed

    Calderón, Juan Carlos; Curtidor, Hernando; González, Oscar; Cifuentes, Gladys; Reyes, Claudia; Patarroyo, Manuel E

    2008-05-01

    Plasmodium falciparum thrombospondin-related apical merozoite protein (PTRAMP) has a thrombospondin related (TSR) domain which in many proteins has been reported as a fragment involved in pathogen-host and cell-interactions. Receptor-ligand studies using eighteen non-overlapping 20-aminoacid-long synthetic peptides from this protein were carried out to determine regions involved in parasite invasion of red blood cells (RBC). Two high activity binding peptides (HABPs) were determined, 33405 (21YISSNDLTSTNLKVRNNWEH40) and 33413 (180LEGPIQFSLGKSSGAFRINY199), presenting high dissociation constants and positive cooperativity. One of the HABPs displayed a modified Plasmodium export element (PEXEL), suggesting that this protein could be involved in the merozoite cytoplasmic reticulum, parasitophorous vacuole, red blood cell (RBC) cytosol, and probably infected RBC (iRBC) membrane transport of some other molecules and nutrients. Enzymatic treatment of RBCs increased HABP 33405 binding to them whilst it decreased HABP 33413 binding. Merozoite invasion assays revealed that HABPs have around 57% ability to inhibit new RBC invasion. Circular dichroism revealed the presence of possible alpha-helical elements in both HABPs structures. RBC binding interaction specificity and the presence of a PEXEL motif make these 2 HABPs good candidates for being included in further studies to develop a new multi-antigenic, multi-stage, subunit-based, chemically-synthesised, anti-malarial vaccine.

  13. In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins.

    PubMed

    Wegner, Waja; Ilgen, Peter; Gregor, Carola; van Dort, Joris; Mott, Alexander C; Steffens, Heinz; Willig, Katrin I

    2017-09-18

    The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

  14. Red blood cells, multiple sickle cells (image)

    MedlinePlus

    Sickle cell anemia is an inherited disorder in which abnormal hemoglobin (the red pigment inside red blood cells) is produced. The abnormal hemoglobin causes red blood cells to assume a sickle shape, like the ones seen in this photomicrograph.

  15. Giant Host Red Blood Cell Membrane Mimicking Polymersomes Bind Parasite Proteins and Malaria Parasites.

    PubMed

    Najer, Adrian; Thamboo, Sagana; Palivan, Cornelia G; Beck, Hans-Peter; Meier, Wolfgang

    2016-01-01

    Malaria is an infectious disease that needs to be addressed using innovative approaches to counteract spread of drug resistance and to establish or optimize vaccination strategies. With our approach, we aim for a dual action with drug- and 'vaccine-like' activity against malaria. By inhibiting entry of malaria parasites into host red blood cells (RBCs) - using polymer vesicle-based (polymersome) nanomimics of RBC membranes - the life cycle of the parasite is interrupted and the exposed parasites are accessible to the host immune system. Here, we describe how host cell-sized RBC membrane mimics, formed with the same block copolymers as nanomimics, also bind the corresponding malaria parasite ligand and whole malaria parasites, similar to nanomimics. This was demonstrated using fluorescence imaging techniques and confirms the suitability of giant polymersomes (GUVs) as simple mimics for RBC membranes.

  16. Protein Structure-Function Correlation in Living Human Red Blood Cells Probed by Isotope Exchange-based Mass Spectrometry.

    PubMed

    Narayanan, Sreekala; Mitra, Gopa; Muralidharan, Monita; Mathew, Boby; Mandal, Amit K

    2015-12-01

    To gain insight into the underlying mechanisms of various biological events, it is important to study the structure-function correlation of proteins within cells. Structural probes used in spectroscopic tools to investigate protein conformation are similar across all proteins. Therefore, structural studies are restricted to purified proteins in vitro and these findings are extrapolated in cells to correlate their functions in vivo. However, due to cellular complexity, in vivo and in vitro environments are radically different. Here, we show a novel way to monitor the structural transition of human hemoglobin upon oxygen binding in living red blood cells (RBCs), using hydrogen/deuterium exchange-based mass spectrometry (H/DX-MS). Exploiting permeability of D2O across cell membrane, the isotope exchange of polypeptide backbone amide hydrogens of hemoglobin was carried out inside RBCs and monitored using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). To explore the conformational transition associated with oxygenation of hemoglobin in vivo, the isotope exchange kinetics was simplified using the method of initial rates. RBC might be considered as an in vivo system of pure hemoglobin. Thus, as a proof-of-concept, the observed results were correlated with structural transition of hemoglobin associated with its function established in vitro. This is the first report on structural changes of a protein upon ligand binding in its endogenous environment. The proposed method might be applicable to proteins in their native state, irrespective of location, concentration, and size. The present in-cell approach opens a new avenue to unravel a plethora of biological processes like ligand binding, folding, and post-translational modification of proteins in living cells.

  17. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting.

    PubMed

    Bajar, Bryce T; Wang, Emily S; Lam, Amy J; Kim, Bongjae B; Jacobs, Conor L; Howe, Elizabeth S; Davidson, Michael W; Lin, Michael Z; Chu, Jun

    2016-02-16

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.

  18. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting

    PubMed Central

    Bajar, Bryce T.; Wang, Emily S.; Lam, Amy J.; Kim, Bongjae B.; Jacobs, Conor L.; Howe, Elizabeth S.; Davidson, Michael W.; Lin, Michael Z.; Chu, Jun

    2016-01-01

    Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude. PMID:26879144

  19. Effect of Peumus boldus on the labeling of red blood cells and plasma proteins with technetium-99m.

    PubMed

    Reiniger, I W; de Oliveira, J F; Caldeira-de-Araújo, A; Bernardo-Filho, M

    1999-08-01

    Peumus boldus is used in popular medicine in Brazil. The influence of Peumus boldus on the labeling of red blood cells and plasma proteins with 99mTc was studied. Stannous chloride and 99mTc pertechnetate were incubated with blood and a tincture of Peumus boldus. Aliquots of plasma and blood cells were isolated from the mixture and treated with trichloroacetic acid (TCA). After separation, analysis of the soluble and insoluble fractions showed a rapid uptake of the radioactivity by blood cells in the presence of the drug, whereas there was a slight decrease in the amount of 99mTc radioactivity in the TCA-insoluble fraction of plasma.

  20. Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes.

    PubMed

    Baker, Susan M; Buckheit, Robert W; Falk, Matthias M

    2010-02-22

    Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered. Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching. We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (alpha-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this technique promises to further

  1. Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes

    PubMed Central

    2010-01-01

    Background Green fluorescent protein (GFP) and other FP fusions have been extensively utilized to track protein dynamics in living cells. Recently, development of photoactivatable, photoswitchable and photoconvertible fluorescent proteins (PAFPs) has made it possible to investigate the fate of discrete subpopulations of tagged proteins. Initial limitations to their use (due to their tetrameric nature) were overcome when monomeric variants, such as Dendra, mEos, and mKikGR were cloned/engineered. Results Here, we report that by closing the field diaphragm, selective, precise and irreversible green-to-red photoconversion (330-380 nm illumination) of discrete subcellular protein pools was achieved on a wide-field fluorescence microscope equipped with standard DAPI, Fluorescein, and Rhodamine filter sets and mercury arc illumination within 5-10 seconds. Use of a DAPI-filter cube with long-pass emission filter (LP420) allowed the observation and control of the photoconversion process in real time. Following photoconversion, living cells were imaged for up to 5 hours often without detectable phototoxicity or photobleaching. Conclusions We demonstrate the practicability of this technique using Dendra2 and mEos2 as monomeric, photoconvertible PAFP representatives fused to proteins with low (histone H2B), medium (gap junction channel protein connexin 43), and high (α-tubulin; clathrin light chain) dynamic cellular mobility as examples. Comparable efficient, irreversible green-to-red photoconversion of selected portions of cell nuclei, gap junctions, microtubules and clathrin-coated vesicles was achieved. Tracking over time allowed elucidation of the dynamic live-cycle of these subcellular structures. The advantage of this technique is that it can be performed on a standard, relatively inexpensive wide-field fluorescence microscope with mercury arc illumination. Together with previously described laser scanning confocal microscope-based photoconversion methods, this

  2. Cryopreservative effects of the recombinant ice-binding protein from the arctic yeast Leucosporidium sp. on red blood cells.

    PubMed

    Lee, Sung Gu; Koh, Hye Yeon; Lee, Jun Hyuck; Kang, Sung-Ho; Kim, Hak Jun

    2012-06-01

    Antifreeze proteins (AFPs) have important functions in many freeze-tolerant organisms. The proteins non-colligatively lower the freezing point and functionally inhibit ice recrystallization in frozen solutions. In our previous studies, we found that the Arctic yeast Leucosporidium sp. produces an AFP (LeIBP), and that the protein could be successfully produced in Pichia expression system. The present study showed that recombinant LeIBP possesses the ability to reduce the damage induced to red blood cells (RBCs) by freeze thawing. In addition to 40 % glycerol, both 0.4 and 0.8 mg/ml LeIBPs significantly reduced freeze-thaw-induced hemolysis at either rapid- (45 °C) or slow-warming (22 °C) temperatures. Post-thaw cell counts of the cryopreserved RBCs were dramatically enhanced, in particular, in 0.8 mg/ml LeIBP. Interestingly, the cryopreserved cells in the presence of LeIBP showed preserved cell size distribution. These results indicate that the ability of LeIBP to inhibit ice recrystallization helps the RBCs avoid critically damaging electrolyte concentrations, which are known as solution effects. Considering all these data, LeIBP can be thought of as a key component in improving RBC cryopreservation efficiency.

  3. Peptides from the Plasmodium falciparum STEVOR putative protein bind with high affinity to normal human red blood cells.

    PubMed

    García, Javier E; Puentes, Alvaro; Curtidor, Hernando; Vera, Ricardo; Rodriguez, Luis; Valbuena, John; López, Ramses; Ocampo, Marisol; Cortés, Jimena; Vanegas, Magnolia; Rosas, Jaiver; Reyes, Claudia; Patarroyo, Manuel E

    2005-07-01

    Synthetic 20-mer long non-overlapped peptides, from STEVOR protein, were tested in RBC binding assays for identifying STEVOR protein regions having high RBC binding activity and evaluating whether these regions inhibit Plasmodium falciparum in vitro invasion. Affinity constants, binding site number per cell and Hill coefficients were determined by saturation assay with high activity binding peptides (HABPs). HABP binding assays using RBCs previously treated with enzymes were carried out to study the nature of the receptor. The molecular weight of RBC surface proteins interacting with HABPs was determined by cross-linking assays and SDS-PAGE analysis. RBC binding assays revealed that peptides 30561 (41MKSRRLAEIQLPKCPHYNND60), 30562 (61PELKKIIDKLNEERIKKYIE80) and 30567 (161ASCCKVHDNYLDNLKKGCFG180) bound saturably and with high binding activity, presenting nanomolar affinity constants. HABP binding activity to RBCs previously treated with neuraminidase and trypsin decreased, suggesting that these peptides bound to RBC surface proteins and that such binding could be sialic acid dependent. Cross-linking and SDS-PAGE assays showed that the three HABPs specifically bound to 30 and 40 kDa molecular weight RBC membrane proteins. Peptides 30561, 30562 and 30567 inhibited P. falciparum in vitro invasion of red blood cells in a concentration-dependent way. Goat sera having STEVOR protein polymeric peptides antibodies inhibit parasite in vitro invasion depending on concentration. Three peptides localized in STEVOR N-terminal and central regions had high, saturable, binding activity to 30 and 40 kDa RBC membrane proteins. These peptides inhibited the parasite's in vitro invasion, suggesting that STEVOR protein regions are involved in P. falciparum invasion processes during intra-erythrocyte stage.

  4. Spectroscopic Analysis of Red Fluorescent Proteins and Development of a Microfluidic Cell Sorter for the Generation of Improved Variants

    NASA Astrophysics Data System (ADS)

    Lubbeck, Jennifer L.

    The discovery of the green fluorescent protein (GFP) launched the development of a wide variety of fluorescent protein (FP) mutants whose spectral and photophysical diversity revolutionized in vivo imaging. The excitation and emission spectra of red fluorescent proteins (RFPs), in particular, have been ideally tuned to a window optically favorable for in vivo work. However, their quantum yields, photostabilities and fluorescence intermittency properties require improvement if they are to be broadly employed for low-copy or single-molecule measurements. Attempts to engineer improved RFPs often result in optimization of one photophysical property at the expense of others. We developed a microfluidic-based cytometer for screening HeLa cell-based genetic RFP-libraries simultaneously on the basis of fluorescence lifetime (a proxy for quantum yield), photostability, and brightness. Ten 532 nm excitation beams interrogate each cell in flow. The first is electro-optically modulated (30 MHz) to enable lifetime measurement with phase fluorimetry. The remaining beams act as a pulse sequence for isolating the irreversible photobleaching time constant. Optical-force switching is employed to sort cells based on any combination of the photophysical parameters. Screening with this instrument enables identification of regions of the structure that synergistically affect quantum yield and photostability and the sorting capability provides a new tool for accelerating the development of next generation RFPs.

  5. Evidence that red blood cell protein p55 may participate in the skeleton-membrane linkage that involves protein 4.1 and glycophorin C.

    PubMed

    Alloisio, N; Dalla Venezia, N; Rana, A; Andrabi, K; Texier, P; Gilsanz, F; Cartron, J P; Delaunay, J; Chishti, A H

    1993-08-15

    Human erythrocyte p55 is a peripheral membrane protein that contains three distinct domains in its primary structure: an N-terminal domain, an SH3 motif, and a C-terminal guanylate kinase domain. We used naturally mutated red blood cells (RBCs) with primary genetic defects resulting in the absence of protein 4.1 (4.1[-] hereditary elliptocytosis) or glycophorin C (Leach elliptocytosis). The absence of either protein was associated with the absence of p55. On a stoichiometric basis, the reduction in glycophorin C (about 80%) was concomitant to the lack of p55 in RBCs devoid of protein 4.1. Similarly, the reduction of protein 4.1 (about 20%) was equivalent to the absence of p55 in RBCs devoid of glycophorin C. These correlations suggest that p55 is associated, in precise proportions, with the protein 4.1-glycophorin-C complex, linking the skeleton and the membrane. The protein 4.1-glycophorin-C cross-bridge is known to be critically important for the stability and mechanical properties of human RBC plasma membrane. Because isoforms of protein 4.1, glycophorin C, and p55 exist in many tissues, these results provide evidence of a linkage between the skeleton and the membrane that may have implications in many nonerythroid cells.

  6. Red cell DAMPs and inflammation.

    PubMed

    Mendonça, Rafaela; Silveira, Angélica A A; Conran, Nicola

    2016-09-01

    Intravascular hemolysis, or the destruction of red blood cells in the circulation, can occur in numerous diseases, including the acquired hemolytic anemias, sickle cell disease and β-thalassemia, as well as during some transfusion reactions, preeclampsia and infections, such as those caused by malaria or Clostridium perfringens. Hemolysis results in the release of large quantities of red cell damage-associated molecular patterns (DAMPs) into the circulation, which, if not neutralized by innate protective mechanisms, have the potential to activate multiple inflammatory pathways. One of the major red cell DAMPs, heme, is able to activate converging inflammatory pathways, such as toll-like receptor signaling, neutrophil extracellular trap formation and inflammasome formation, suggesting that this DAMP both activates and amplifies inflammation. Other potent DAMPs that may be released by the erythrocytes upon their rupture include heat shock proteins (Hsp), such as Hsp70, interleukin-33 and Adenosine 5' triphosphate. As such, hemolysis represents a major inflammatory mechanism that potentially contributes to the clinical manifestations that have been associated with the hemolytic diseases, such as pulmonary hypertension and leg ulcers, and likely plays a role in specific complications of sickle cell disease such as endothelial activation, vaso-occlusive processes and tissue injury.

  7. Malaria Parasite Proteins and Their Role in Alteration of the Structure and Function of Red Blood Cells.

    PubMed

    Proellocks, Nicholas I; Coppel, Ross L; Mohandas, Narla; Cooke, Brian M

    2016-01-01

    Malaria, caused by Plasmodium spp., continues to be a major threat to human health and a significant cause of socioeconomic hardship in many countries. Almost half of the world's population live in malaria-endemic regions and many of them suffer one or more, often life-threatening episodes of malaria every year, the symptoms of which are attributable to replication of the parasite within red blood cells (RBCs). In the case of Plasmodium falciparum, the species responsible for most malaria-related deaths, parasite replication within RBCs is accompanied by striking alterations to the morphological, biochemical and biophysical properties of the host cell that are essential for the parasites' survival. To achieve this, the parasite establishes a unique and extensive protein export network in the infected RBC, dedicating at least 6% of its genome to the process. Understanding the full gamut of proteins involved in this process and the mechanisms by which P. falciparum alters the structure and function of RBCs is important both for a more complete understanding of the pathogenesis of malaria and for development of new therapeutic strategies to prevent or treat this devastating disease. This review focuses on what is currently known about exported parasite proteins, their interactions with the RBC and their likely pathophysiological consequences.

  8. Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells.

    PubMed

    Kats, Lev M; Proellocks, Nicholas I; Buckingham, Donna W; Blanc, Lionel; Hale, John; Guo, Xinhua; Pei, Xinhong; Herrmann, Susann; Hanssen, Eric G; Coppel, Ross L; Mohandas, Narla; An, Xiuli; Cooke, Brian M

    2015-07-01

    During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined sub-domains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process.

  9. Interactions between Plasmodium falciparum skeleton-binding protein 1 and the membrane skeleton of malaria-infected red blood cells

    PubMed Central

    Buckingham, Donna W.; Blanc, Lionel; Hale, John; Guo, Xinhua; Pei, Xinhong; Herrmann, Susann; Hanssen, Eric G.; Coppel, Ross L.; Mohandas, Narla; An, Xiuli; Cooke, Brian M.

    2015-01-01

    During development inside red blood cells (RBCs), Plasmodium falciparum malaria parasites export proteins that associate with the RBC membrane skeleton. These interactions cause profound changes to the biophysical properties of RBCs that underpin the often severe and fatal clinical manifestations of falciparum malaria. P. falciparum erythrocyte membrane protein 1 (PfEMP1) is one such exported parasite protein that plays a major role in malaria pathogenesis since its exposure on the parasitised RBC surface mediates their adhesion to vascular endothelium and placental syncytioblasts. En route to the RBC membrane skeleton, PfEMP1 transiently associates with Maurer's clefts (MCs), parasite-derived membranous structures in the RBC cytoplasm. We have previously shown that a resident MC protein, skeleton-binding protein 1 (SBP1), is essential for the placement of PfEMP1 onto the RBC surface and hypothesised that the function of SBP1 may be to target MCs to the RBC membrane. Since this would require additional protein interactions, we set out to identify binding partners for SBP1. Using a combination of approaches, we have defined the region of SBP1 that binds specifically to defined subdomains of two major components of the RBC membrane skeleton, protein 4.1R and spectrin. We show that these interactions serve as one mechanism to anchor MCs to the RBC membrane skeleton, however, while they appear to be necessary, they are not sufficient for the translocation of PfEMP1 onto the RBC surface. The N-terminal domain of SBP1 that resides within the lumen of MCs clearly plays an essential, but presently unknown role in this process. PMID:25883090

  10. Red cell membrane protein deficiencies in Mexican patients with hereditary spherocytosis.

    PubMed

    Sánchez-López, J Yoaly; Camacho, Ana L; Magaña, Maria Teresa; Ibarra, Bertha; Perea, F Javier

    2003-01-01

    Twenty-seven families and four individual patients with hereditary spherocytosis (HS) from the northwestern region of Mexico were studied. An autosomal dominant inheritance pattern was identified in 59% of 22 families. Densitometric analysis of erythrocyte membrane proteins revealed individual protein deficiencies in 39% of the patients studied, in whom the principal altered proteins were the alpha spectrins (13%), band 3 protein (10%), ankyrin (6%), 4.2 protein (6%), and the beta spectrins (3%). A predominant deficiency of spectrins has also been observed in other Latin American and Mediterranean countries. However, it is well known that deficiencies in these proteins are heterogeneous across different ethnic groups. A combined protein deficiency was observed in 52% of patients, most frequently involving the spectrins, band 3 protein, 4.2 protein, and 4.1 protein. In three subjects, no abnormalities were detected (10%). We conclude that, despite the observed heterogeneity, the principal affected proteins are essentially similar to those observed in other ethnic groups.

  11. LC-MS analysis of Hep-2 and Hek-293 cell lines treated with Brazilian red propolis reveals differences in protein expression.

    PubMed

    da Silva Frozza, Caroline O; da Silva Brum, Emyle; Alving, Anjali; Moura, Sidnei; Henriques, João A P; Roesch-Ely, Mariana

    2016-08-01

    Red propolis, an exclusive variety of propolis found in the northeast of Brazil has shown to present antitumour activity, among several other biological properties. This article aimed to help to evaluate the underlying molecular mechanisms of the potential anticancer effects of red propolis on tumour, Hep-2, and non-tumour cells, Hek-293. Differentially expressed proteins in human cell lines were identified through label-free quantitative MS-based proteomic platform, and cells were stained with Giemsa to show morphological changes. A total of 1336 and 773 proteins were identified for Hep-2 and Hek-293, respectively. Among the proteins here identified, 16 were regulated in the Hep-2 cell line and 04 proteins in the Hek-293 line. Over a total of 2000 proteins were identified under MS analysis, and approximately 1% presented differential expression patterns. The GO annotation using Protein Analysis THrough Evolutionary Relationships classification system revealed predominant molecular function of catalytic activity, and among the biological processes, the most prominent was associated to cell metabolism. The proteomic profile here presented should help to elucidate further molecular mechanisms involved in inhibition of cancer cell proliferation by red propolis, which remain unclear to date. © 2016 Royal Pharmaceutical Society.

  12. Photoconversion in orange and red fluorescent proteins

    PubMed Central

    Kremers, Gert-Jan; Hazelwood, Kristin L.; Murphy, Christopher S.; Davidson, Michael W.; Piston, David W.

    2009-01-01

    We report that photoconversion is fairly common among orange and red fluorescent proteins, as a screen of 12 variants yielded 8 that exhibit photoconversion. Specifically, three red fluorescent proteins can be switched into a green state, and two orange variants can be photoconverted to the far red. The orange highlighters are ideal for dual-probe highlighter applications, and they exhibit the most red-shifted excitation of all fluorescent protein described to date. PMID:19363494

  13. Integrin-associated protein (CD47) is a putative mediator for soluble fibrinogen interaction with human red blood cells membrane.

    PubMed

    De Oliveira, S; Vitorino de Almeida, V; Calado, A; Rosário, H S; Saldanha, C

    2012-03-01

    Fibrinogen is a multifunctional plasma protein that plays a crucial role in several biological processes. Elevated fibrinogen induces erythrocyte hyperaggregation, suggesting an interaction between this protein and red blood cells (RBCs). Several studies support the concept that fibrinogen interacts with RBC membrane and this binding, due to specific and non-specific mechanisms, may be a trigger to RBC hyperaggregation in inflammation. The main goals of our work were to prove that human RBCs are able to specifically bind soluble fibrinogen, and identify membrane molecular targets that could be involved in this process. RBCs were first isolated from blood of healthy individuals and then separated in different age fractions by discontinuous Percoll gradients. After isolation RBC samples were incubated with human soluble fibrinogen and/or with a blocking antibody against CD47 followed by fluorescence confocal microscopy, flow cytometry acquisitions and zeta potential measurements. Our data show that soluble fibrinogen interacts with the human RBC membrane in an age-dependent manner, with younger RBCs interacting more with soluble fibrinogen than the older cells. Importantly, this interaction is abrogated in the presence of a specific antibody against CD47. Our results support a specific and age-dependent interaction of soluble fibrinogen with human RBC membrane; additionally we present CD47 as a putative mediator in this process. This interaction may contribute to RBC hyperaggregation in inflammation. Copyright © 2011 Elsevier B.V. All rights reserved.

  14. Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells

    NASA Astrophysics Data System (ADS)

    Diez-Silva, Monica; Park, Yongkeun; Huang, Sha; Bow, Hansen; Mercereau-Puijalon, Odile; Deplaine, Guillaume; Lavazec, Catherine; Perrot, Sylvie; Bonnefoy, Serge; Feld, Michael S.; Han, Jongyoon; Dao, Ming; Suresh, Subra

    2012-08-01

    Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host.

  15. Pf155/RESA protein influences the dynamic microcirculatory behavior of ring-stage Plasmodium falciparum infected red blood cells

    PubMed Central

    Diez-Silva, Monica; Park, YongKeun; Huang, Sha; Bow, Hansen; Mercereau-Puijalon, Odile; Deplaine, Guillaume; Lavazec, Catherine; Perrot, Sylvie; Bonnefoy, Serge; Feld, Michael S.; Han, Jongyoon; Dao, Ming; Suresh, Subra

    2012-01-01

    Proteins exported by Plasmodium falciparum to the red blood cell (RBC) membrane modify the structural properties of the parasitized RBC (Pf-RBC). Although quasi-static single cell assays show reduced ring-stage Pf-RBCs deformability, the parameters influencing their microcirculatory behavior remain unexplored. Here, we study the dynamic properties of ring-stage Pf-RBCs and the role of the parasite protein Pf155/Ring-Infected Erythrocyte Surface Antigen (RESA). Diffraction phase microscopy revealed RESA-driven decreased Pf-RBCs membrane fluctuations. Microfluidic experiments showed a RESA-dependent reduction in the Pf-RBCs transit velocity, which was potentiated at febrile temperature. In a microspheres filtration system, incubation at febrile temperature impaired traversal of RESA-expressing Pf-RBCs. These results show that RESA influences ring-stage Pf-RBCs microcirculation, an effect that is fever-enhanced. This is the first identification of a parasite factor influencing the dynamic circulation of young asexual Pf-RBCs in physiologically relevant conditions, offering novel possibilities for interventions to reduce parasite survival and pathogenesis in its human host. PMID:22937223

  16. Malaria and Human Red Blood Cells

    PubMed Central

    Mohandas, Narla; An, Xiuli

    2013-01-01

    Invasion by the malaria parasite, P. falciparum brings about extensive changes in the host red cells. These include loss of the normal discoid shape, increased rigidity of the membrane, elevated permeability to a wide variety of ionic and other species, and increased adhesiveness, most notably to endothelial surfaces. These effects facilitate survival of the parasite within the host cell and tend to increase the virulence of disease that include cerebral malaria and anemia. Numerous proteins secreted by the internalized parasite and interaction with red cell membrane proteins are responsible for the changes occurring to the host cell. Anemia a serious clinical manifestation of malaria is due to increased destruction of both infected and uninfected red cells due to membrane alterations, as well as ineffective erythropoiesis. There is very good evidence that various red cell disorders including hemoglobinopathies and hereditary ovalocytosis decrease the virulence of disease following parasite infection. A number of mechanism(s) are likely responsible for the protective effect of various red cell abnormalities including decreased invasion, impaired intraerythrocytic development of the parasites and altered interaction between exported parasite proteins and the red cell membrane skeleton. PMID:22965173

  17. Malaria and human red blood cells.

    PubMed

    Mohandas, Narla; An, Xiuli

    2012-11-01

    Invasion by the malaria parasite, Plasmodium falciparum, brings about extensive changes in the host red cells. These include loss of the normal discoid shape, increased rigidity of the membrane, elevated permeability to a wide variety of ionic and other species and increased adhesiveness, most notably to endothelial surfaces. These effects facilitate survival of the parasite within the host cell and tend to increase the virulence of disease that includes cerebral malaria and anemia. Numerous proteins secreted by the internalized parasite and interacting with red cell membrane proteins are responsible for the changes occurring to the host cell. Anemia, a serious clinical manifestation of malaria, is due to increased destruction of both infected and uninfected red cells due to membrane alterations, as well as ineffective erythropoiesis. There is very good evidence that various red cell disorders including hemoglobinopathies and hereditary ovalocytosis decrease the virulence of disease following parasite infection. A number of mechanism(s) are likely responsible for the protective effect of various red cell abnormalities including decreased invasion, impaired intraerythrocytic development of the parasites and altered interaction between exported parasite proteins and the red cell membrane skeleton.

  18. Red blood cells, sickle cell (image)

    MedlinePlus

    ... is an inherited blood disease in which the red blood cells produce abnormal pigment (hemoglobin). The abnormal hemoglobin causes deformity of the red blood cells into crescent or sickle-shapes, as seen in ...

  19. Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet.

    PubMed

    Oliva, Laia; Baron, Cristian; Fernández-López, José-Antonio; Remesar, Xavier; Alemany, Marià

    2015-01-01

    Background and Objectives. Glucose, an aldose, spontaneously reacts with protein amino acids yielding glycosylated proteins. The compounds may reorganize to produce advanced glycosylation products, which regulatory importance is increasingly being recognized. Protein glycosylation is produced without the direct intervention of enzymes and results in the loss of function. Glycosylated plasma albumin, and glycosylated haemoglobin are currently used as index of mean plasma glucose levels, since higher glucose availability results in higher glycosylation rates. In this study we intended to detect the early changes in blood protein glycosylation elicited by an obesogenic diet. Experimental Design. Since albumin is in constant direct contact with plasma glucose, as are the red blood cell (RBC) membranes, we analyzed their degree or glycosylation in female and male rats, either fed a standard diet or subjected to a hyper-energetic self-selected cafeteria diet for 30 days. This model produces a small increase in basal glycaemia and a significant increase in body fat, leaving the animals in the initial stages of development of metabolic syndrome. We also measured the degree of glycosylation of hemoglobin, and the concentration of glucose in contact with this protein, that within the RBC. Glycosylation was measured by colorimetric estimation of the hydroxymethylfurfural liberated from glycosyl residues by incubation with oxalate. Results. Plasma glucose was higher in cafeteria diet and in male rats, both independent effects. However, there were no significant differences induced by sex or diet in either hemoglobin or plasma proteins. Purified RBC membranes showed a marked effect of diet: higher glycosylation in cafeteria rats, which was more marked in females (not in controls). In any case, the number of glycosyl residues per molecule were higher in hemoglobin than in plasma proteins (after correction for molecular weight). The detected levels of glucose in RBC were lower

  20. Synthetic peptides from conserved regions of the Plasmodium falciparum early transcribed membrane and ring exported proteins bind specifically to red blood cell proteins.

    PubMed

    Garcia, Jeison; Curtidor, Hernando; Obando-Martinez, Ana Z; Vizcaíno, Carolina; Pinto, Martha; Martinez, Nora L; Patarroyo, Manuel A; Patarroyo, Manuel E

    2009-11-16

    Severe malaria pathology is directly associated with cytoadherence of infected red blood cells (iRBCs) to healthy RBCs and/or endothelial cells occurring during the intraerythrocytic development of Plasmodium falciparum. We synthesized, as 20-mer long peptides, the members of the ring exported (REX) protein family encoded in chromosome 9, as well as the early transcribed membrane proteins (E-TRAMP) 10.2 and 4, to identify specific RBC binding regions in these proteins. Twelve binding peptides were identified (designated as HABPs): three were identified in REX1, two in REX2, one in REX3, two in REX4 and four in E-TRAMP 10.2. The majority of these HABPs was conserved among different P. falciparum strains, according to sequence analysis. No HABPs were found in E-TRAMP 4. Bindings of HABPs were saturable and sensitive to the enzymatic treatment of RBCs and HABPs had different structural features, according to circular dichroism studies. Our results suggest that the REX and E-TRAMP families participate in relevant interactions with RBC membrane proteins, which highlight these proteins as potential targets for the development of fully effective immunoprophylactic methods.

  1. In vitro cytotoxicity assays: comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride.

    PubMed

    Fotakis, George; Timbrell, John A

    2006-01-05

    The aim of this study was to compare four in vitro cytotoxicity assays and determine their ability to detect early cytotoxic events. Two hepatoma cell lines, namely HTC and HepG2 cells, were exposed to cadmium chloride (0-300 microM) for 3, 5 and 8 h. Following exposure to the toxic metal cytotoxicity was determined with the lactate dehydrogenase leakage assay (LDH), a protein assay, the neutral red assay and the methyl tetrazolium (MTT) assay. In HTC cells no toxicity was observed for any incubation period when the LDH leakage, the MTT and the protein assay were employed whereas the neutral red assay revealed early cytotoxicity starting after incubation of HTC cells with CdCl(2) for 3 h. In the case of HepG2 cells the MTT assay reveals cytotoxicity due to CdCl(2) exposure after 3 h whereas no such effect is seen with the other three assays. Following 5 h exposure of HepG2 cells to CdCl(2), toxicity is observed with the MTT assay at lower concentrations compared to the ones required for detection of toxicity with the LDH leakage and the neutral red assay. In conclusion different sensitivity was observed for each assay with the neutral red and the MTT assay being the most sensitive in detecting cytotoxic events compared to the LDH leakage and the protein assay.

  2. Red cell membrane: past, present, and future

    PubMed Central

    Gallagher, Patrick G.

    2008-01-01

    As a result of natural selection driven by severe forms of malaria, 1 in 6 humans in the world, more than 1 billion people, are affected by red cell abnormalities, making them the most common of the inherited disorders. The non-nucleated red cell is unique among human cell type in that the plasma membrane, its only structural component, accounts for all of its diverse antigenic, transport, and mechanical characteristics. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. Structural and functional characterization of the many constituents of the red cell membrane, in conjunction with biophysical and physiologic studies, has led to detailed description of the way in which the remarkable mechanical properties and other important characteristics of the red cells arise, and of the manner in which they fail in disease states. Current studies in this very active and exciting field are continuing to produce new and unexpected revelations on the function of the red cell membrane and thus of the cell in health and disease, and shed new light on membrane function in other diverse cell types. PMID:18988878

  3. Red cell membrane: past, present, and future.

    PubMed

    Mohandas, Narla; Gallagher, Patrick G

    2008-11-15

    As a result of natural selection driven by severe forms of malaria, 1 in 6 humans in the world, more than 1 billion people, are affected by red cell abnormalities, making them the most common of the inherited disorders. The non-nucleated red cell is unique among human cell type in that the plasma membrane, its only structural component, accounts for all of its diverse antigenic, transport, and mechanical characteristics. Our current concept of the red cell membrane envisions it as a composite structure in which a membrane envelope composed of cholesterol and phospholipids is secured to an elastic network of skeletal proteins via transmembrane proteins. Structural and functional characterization of the many constituents of the red cell membrane, in conjunction with biophysical and physiologic studies, has led to detailed description of the way in which the remarkable mechanical properties and other important characteristics of the red cells arise, and of the manner in which they fail in disease states. Current studies in this very active and exciting field are continuing to produce new and unexpected revelations on the function of the red cell membrane and thus of the cell in health and disease, and shed new light on membrane function in other diverse cell types.

  4. Dog Red Blood Cells

    PubMed Central

    Parker, John C.

    1973-01-01

    Dog red blood cells (RBC) lack a ouabain-sensitive sodium pump, and yet they are capable of volume regulation in vivo. The present study was designed to find in vitro conditions under which dog RBC could transport sodium outward, against an electrochemical gradient. Cells were first loaded with sodium chloride and water by preincubation in hypertonic saline. They were then incubated at 37°C in media containing physiologic concentrations of sodium, potassium, chloride, bicarbonate, glucose, and calcium. The cells returned to a normal salt and water content in 16–20 h. Without calcium in the medium the cells continued slowly to accumulate sodium. Removal of glucose caused rapid swelling and lysis, whether or not calcium was present. The net efflux of sodium showed a close relationship to medium calcium over a concentration range from 0 to 5 mM. Extrusion of salt and water was also demonstrated in fresh RBC (no hypertonic preincubation) when calcium levels in the media were sufficiently raised. The ion and water movements in these experiments were not influenced by ouabain or by removal of extracellular potassium. Magnesium could not substitute for calcium. It is concluded that dog RBC have an energy-dependent mechanism for extruding sodium chloride which requires external calcium and is quite distinct from the sodium-potassium exchange pump. PMID:4722565

  5. ALTERATIONS OF PROPERTIES OF RED BLOOD CELLS MEMBRANES PROTEINS OF DIFFERENT AGE AND SEX VOLUNTEERS.

    PubMed

    Pruidze, N; Khetsuriani, R; Sujashvili, R; Ioramashvili, I; Arabuli, M; Sanikidze, T

    2015-01-01

    Considering the age and sex-dependent trend in the manifestation of various diseases, as well as an important pathogenic role of circulatory disorders, we decided to study the age-dependent changes in the physical properties of RBCs membrane proteins (their electric charge and molecular weight) in healthy people of different sex (males and females) and age. Blood of 56 healthy volunteers (Tbilisi, Georgia) of different sex and gender was studied (the patients were divided in 8 groups (7 patients in each groups): 1 - 18-25 years old male, 2 - 18-25 years old female, 3 - 25-44 years old male, 4 - 25-44 years old female, 5 - 44-60 years old male, 6 - 44-60 years old female; 7 - 60-80 years old male, 8 - 70-80 years old female). In groups 6 and 8 were women in menopause was determined according 12 months of amenorrhea. Individuals often consume alcohol addicts, pregnant women and patients with chronic diseases were excluded from the study. The study protocol was approved by Ethical Committee of the Tbilisi State Medical University. RBCs membrane proteins have been extracted from human heparinized blood and their mobility was studied by electrophoretic method. The electrophoretic mobility of RBCs membrane proteins decreases with age of healthy volunteers, that indicates decrease of total charge of proteins, depending on the electrically charged amino acids content. In female patients the electrophoretic mobility of the RBCs membrane proteins especially intensively decreases in period of menopause. Increase of molecular weight of proteins (100-200 kDa) from RBCs' membranes of alder age group was manifested. Intensively decrease electrophoretic mobility of erythrocytes membrane proteins from female patients in period of menopause indicates on estrogen related mechanism of the regulation of membrane protein conformation and composition in females. Increased content of high molecular weight proteins in the RBCs membranes from patients of older age groups may be caused to

  6. The calcium-dependent protease of Loxosceles gaucho venom acts preferentially upon red cell band 3 transmembrane protein.

    PubMed

    Barretto, O C de O; Satake, M; Nonoyama, K; Cardoso, J L C

    2003-03-01

    Eighty micrograms red blood cell (RBC) ghosts from patients who had previously exhibited the cutaneous form of loxoscelism (presenting localized dermonecrosis) and the viscerocutaneous form of loxoscelism (presenting dermonecrosis, hemoglobinuria, hematuria, and jaundice) and from controls were incubated with 2.5 microg crude Loxosceles gaucho venom in 5 mM phosphate buffer, pH 7.4, at 37 C. Among all membrane proteins, quantitative proteolysis of the important integral transmembrane protein 3 increased with venom dose and with incubation time from 30 to 120 min, as demonstrated by gel densitometry. Similar quantitative data were obtained for RBC ghosts from patients and from control subjects, a fact that argues against the possibility of genetic factors favoring the hemolytic viscerocutaneous form. These data suggest that the clinical forms may be different types of the same disease, with the viscerocutaneous form being the result of large amounts of intravascularly injected venom and the superficial form being the result of in situ venom action. Since protein 3 is a housekeeping integral membrane protein, whose genetic deficiency leads to hemolytic anemia, it is reasonable to relate it to the hemolysis which occurs in the viscerocutaneous form of loxoscelism. The venom protease responsible for the process was not inhibited after 120-min incubation by 0.2 mM paramethylsulfonyl fluoride or by 0.2 mM N-ethylmaleimide but was inhibited by 25 mM ethylenediaminetetraacetic acid (a calcium-chelating agent) in 5 mM phosphate buffer at pH 7.4, which suggests that the enzyme is a calcium-dependent metalloprotease.

  7. Protein 4.1R-dependent multiprotein complex: New insights into the structural organization of the red blood cell membrane

    PubMed Central

    Salomao, Marcela; Zhang, Xihui; Yang, Yang; Lee, Soohee; Hartwig, John H.; Chasis, Joel Anne; Mohandas, Narla; An, Xiuli

    2008-01-01

    Protein 4.1R (4.1R) is a multifunctional component of the red cell membrane. It forms a ternary complex with actin and spectrin, which defines the nodal junctions of the membrane-skeletal network, and its attachment to the transmembrane protein glycophorin C creates a bridge between the protein network and the membrane bilayer. We now show that deletion of 4.1R in mouse red cells leads to a large diminution of actin accompanied by extensive loss of cytoskeletal lattice structure, with formation of bare areas of membrane. Whereas band 3, the preponderant transmembrane constituent, and proteins known to be associated with it are present in normal or increased amounts, glycophorin C is missing and XK, Duffy, and Rh are much reduced in the 4.1R-deficient cells. The inference that these are associated with 4.1R was borne out by the results of in vitro pull-down assays. Furthermore, whereas Western blot analysis showed normal levels of band 3 and Kell, flow cytometric analysis using an antibody against the extracellular region of band 3 or Kell revealed reduction of these two proteins, suggesting a conformational change of band 3 and Kell epitopes. Taken together, we suggest that 4.1R organizes a macromolecular complex of skeletal and transmembrane proteins at the junctional node and that perturbation of this macromolecular complex not only is responsible for the well characterized membrane instability but may also remodel the red cell surface. PMID:18524950

  8. Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine.

    PubMed

    Barber, Latorya A; Palascak, Mary B; Qi, Xiaoyang; Joiner, Clinton H; Franco, Robert S

    2015-11-01

    Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.

  9. [Band 3 protein as a metabolic sensor--CO2 regulates the amount of oxygen delivered to tissues from red blood cells].

    PubMed

    Hamasaki, Naotaka

    2006-03-01

    Oxygen is essential for most forms of life, but too much oxygen is harmful and can induce tissue damage. Living creatures therefore have a tightly regulated system to deliver the necessary amount of oxygen to specific tissues at the right time. CO2 is not simply waste matter from tissues, but regulates the amount of oxygen delivered to tissues from red blood cells, utilizing the synergistic effects of hemoglobin, carbonic anhydrase and the anion exchange activity of band 3 protein. Red blood cells play an important role in this system and provide an ideal vehicle for delivering oxygen to tissues, depending on their metabolic activity.

  10. Hereditary spherocytosis, elliptocytosis, and other red cell membrane disorders.

    PubMed

    Da Costa, Lydie; Galimand, Julie; Fenneteau, Odile; Mohandas, Narla

    2013-07-01

    Hereditary spherocytosis and elliptocytosis are the two most common inherited red cell membrane disorders resulting from mutations in genes encoding various red cell membrane and skeletal proteins. Red cell membrane, a composite structure composed of lipid bilayer linked to spectrin-based membrane skeleton is responsible for the unique features of flexibility and mechanical stability of the cell. Defects in various proteins involved in linking the lipid bilayer to membrane skeleton result in loss in membrane cohesion leading to surface area loss and hereditary spherocytosis while defects in proteins involved in lateral interactions of the spectrin-based skeleton lead to decreased mechanical stability, membrane fragmentation and hereditary elliptocytosis. The disease severity is primarily dependent on the extent of membrane surface area loss. Both these diseases can be readily diagnosed by various laboratory approaches that include red blood cell cytology, flow cytometry, ektacytometry, electrophoresis of the red cell membrane proteins, and mutational analysis of gene encoding red cell membrane proteins.

  11. Sickle red blood cells accumulate in tumor.

    PubMed

    Brown, S L; Ewing, J R; Nagaraja, T N; Swerdlow, P S; Cao, Y; Fenstermacher, J D; Kim, J H

    2003-12-01

    The preferential accumulation of sickle blood cells in tumor vasculature is demonstrated noninvasively using MRI and sickle red blood cells loaded with Gd-DTPA and invasively by two other techniques. The distribution of red blood cells in rat brain tumors relative to normal brains were measured using three separate techniques: MRI of Gd-DTPA loaded cells, fluorescent microscopy detection of Oregon Green 488 fluorescence conjugated to a streptavidin-biotin complex that binds to red blood cell surface proteins, and autoradiography using a technetium (99m)Tc-labeling kit. Labeled red cells were infused intravenously in rats with brain tumors. Sickle cells preferentially accumulated in tumor relative to normal brain, with highest concentrations near the tumor / normal tissue boundary, whereas control normal red cells did not preferentially aggregate at the tumor periphery. This demonstrates the potential of sickle red blood cells to accumulate in the abnormal tumor vessel network, and the ability to detect their aggregation noninvasively and at high spatial resolution using MRI. The application of the noninvasive measurement of sickle cells for imaging tumor neovasculature, or as a delivery tool for therapy, requires further study. Copyright 2003 Wiley-Liss, Inc.

  12. Molecular behavior in living mitotic cells of human centromere heterochromatin protein HPLalpha ectopically expressed as a fusion to red fluorescent protein.

    PubMed

    Sugimoto, K; Tasaka, H; Dotsu, M

    2001-12-01

    We constructed stable mammalian cell lines in which human heterochromatin protein HP1alpha and kinetochore protein CENP-A were differentially expressed as fusions to red (RFP-HP1) and green fluorescent proteins (GFP-CENP-A). Heterochromatin localization of RFP-HP1 was clearly shown in mouse and Indian muntjac cells. By preparing mitotic chromosome spreads, the inner centromere localization of RFP-HP1 was observed in human and Indian muntjac cells. To characterize its molecular behavior in living mitotic cells, time-lapse images of RFP-HP1 were obtained by computer-assisted image analyzing system, mainly with mouse cells. In G2 phase, a significant portion of RFP-HP1 diffused homogeneously in the nucleus and further dispersed into the cytoplasm soon after the nuclear membrane breakdown, while some remained in the centromeric region. Simultaneous observations with GFP-CENP-A in human cells showed that RFP-HP1 was located just between the sister kinetochores and then aligned to the spindle midzone. With the onset of anaphase, once it was released from there, it moved to the centromeres of segregating chromosomes or returned to the spindle equator. As cytokinesis proceeded, HP1alpha was predominantly found in the newly formed daughter nuclei and again displayed a heterochromatin-like distribution. These results suggested that, although the majority of HP1alpha diffuses into the cytoplasm, some populations are retained in the centromeric region and involved in the association and segregation of sister kinetochores during mitosis.

  13. Generation of red fluorescent protein transgenic dogs.

    PubMed

    Hong, So Gun; Kim, Min Kyu; Jang, Goo; Oh, Hyun Ju; Park, Jung Eun; Kang, Jung Taek; Koo, Ok Jae; Kim, Teoan; Kwon, Mo Sun; Koo, Bon Chul; Ra, Jeong Chan; Kim, Dae Yong; Ko, CheMyong; Lee, Byeong Chun

    2009-05-01

    Dogs (Canis familiaris) share many common genetic diseases with humans and development of disease models using a transgenic approach has long been awaited. However, due to the technical difficulty in obtaining fertilizable eggs and the unavailability of embryonic stem cells, no transgenic dog has been generated. Canine fetal fibroblasts were stably transfected with a red fluorescent protein (RFP) gene-expressing construct using retrovirus gene delivery method. Somatic cell nuclear transfer was then employed to replace the nucleus of an oocyte with the nucleus of the RFP-fibroblasts. Using this approach, we produced the first generation of transgenic dogs with four female and two male expressing RFP.

  14. Oxygen delivery from red cells.

    PubMed Central

    Clark, A; Federspiel, W J; Clark, P A; Cokelet, G R

    1985-01-01

    This paper deals with the theoretical analysis of the unloading of oxygen from a red cell. A scale analysis of the governing transport equations shows that the solutions have a boundary layer structure near the red-cell membrane. The boundary layer is a region of chemical nonequilibrium, and it owes its existence to the fact that the kinetic time scales are shorter than the diffusion time scales in the red cell. The presence of the boundary layer allows an analytical solution to be obtained by the method of matched asymptotic expansions. A very useful result from the analysis is a simple, lumped-parameter description of the oxygen delivery from a red cell. The accuracy of the lumped-parameter description has been verified by comparing its predictions with results obtained by numerical integration of the full equations for a one-dimensional slab. As an application, we calculate minimum oxygen unloading times for red cells. PMID:3978198

  15. Protein disulfide isomerase may facilitate the efflux of nitrite derived S-nitrosothiols from red blood cells.

    PubMed

    Kallakunta, Vasantha Madhuri; Slama-Schwok, Anny; Mutus, Bulent

    2013-01-01

    Protein disulfide isomerase (PDI) is an abundant protein primarily found in the endoplasmic reticulum and also secreted into the blood by a variety of vascular cells. The evidence obtained here, suggests that PDI could directly participate in the efflux of NO(+) from red blood cells (RBC). PDI was detected both in RBC membranes and in the cytosol. PDI was S-nitrosylated when RBCs were exposed to nitrite under ∼50% oxygen saturation but not under ∼100% oxygen saturation. Furthermore, it was observed that hemoglobin (Hb) could promote PDI S-nitrosylation in the presence of ∼600 nM nitrite. In addition, three lines of evidence were obtained for PDI-Hb interactions: (1) Hb co-immunoprecipitated with PDI; (2) Hb quenched the intrinsic PDI fluorescence in a saturable manner; and (3) Hb-Fe(II)-NO absorption spectrum decreased in a [PDI]-dependent manner. Finally, PDI was detected on the surface RBC under ∼100% oxygen saturation and released as soluble under ∼50% oxygen saturation. The soluble PDI detected under ∼50% oxygen saturation was S-nitrosylated. Based on these data it is proposed that PDI is taken up by RBC and forms a complex with Hb. Hb-Fe(II)-NO that is formed from nitrite reduction under ∼50% O2, then transfers NO(+) to either Hb-Cys β93 or directly to PDI resulting in S-nitroso-PDI which transverses the RBC membrane and attaches to the RBC surface. When RBCs enter tissues the S-nitroso-PDI is released from the RBC-surface into the blood where its NO(+) is transferred into the endothelium thereby inducing vasodilation, suggesting local oxygen-dependent dynamic interplays between nitrite, NO and S-nitrosylation.

  16. Albumin modulates S1P delivery from red blood cells in perfused microvessels: mechanism of the protein effect.

    PubMed

    Adamson, R H; Clark, J F; Radeva, M; Kheirolomoom, A; Ferrara, K W; Curry, F E

    2014-04-01

    Removal of plasma proteins from perfusates increases vascular permeability. The common interpretation of the action of albumin is that it forms part of the permeability barrier by electrostatic binding to the endothelial glycocalyx. We tested the alternate hypothesis that removal of perfusate albumin in rat venular microvessels decreased the availability of sphingosine-1-phosphate (S1P), which is normally carried in plasma bound to albumin and lipoproteins and is required to maintain stable baseline endothelial barriers (Am J Physiol Heart Circ Physiol 303: H825-H834, 2012). Red blood cells (RBCs) are a primary source of S1P in the normal circulation. We compared apparent albumin permeability coefficients [solute permeability (Ps)] measured using perfusates containing albumin (10 mg/ml, control) and conditioned by 20-min exposure to rat RBCs with Ps when test perfusates were in RBC-conditioned protein-free Ringer solution. The control perfusate S1P concentration (439 ± 46 nM) was near the normal plasma value at 37 °C and established a stable baseline Ps (0.9 ± 0.4 × 10(-6) cm/s). Ringer solution perfusate contained 52 ± 8 nM S1P and increased Ps more than 10-fold (16.1 ± 3.9 × 10(-6) cm/s). Consistent with albumin-dependent transport of S1P from RBCs, S1P concentrations in RBC-conditioned solutions decreased as albumin concentration, hematocrit, and temperature decreased. Protein-free Ringer solution perfusates that used liposomes instead of RBCs as flow markers failed to maintain normal permeability, reproducing the "albumin effect" in these mammalian microvessels. We conclude that the albumin effect depends on the action of albumin to facilitate the release and transport of S1P from RBCs that normally provide a significant amount of S1P to the endothelium.

  17. Production of transgenic cashmere goat embryos expressing red fluorescent protein and containing IGF1 hair-follicle-cell specific expression cassette by somatic cell nuclear transfer.

    PubMed

    Guo, XuDong; Yang, DongShan; Ao, XuDong; Wu, Xia; Li, GuangPeng; Wang, LingLing; Bao, Ming-Tao; Xue, Lian; Bou, ShorGan

    2009-04-01

    In the present study, cashmere goat fetal fibroblasts were transfected with pCDsR-KI, a hair-follicle-cell specific expression vector for insulin-like growth factor 1 (IGF1) that contains two markers for selection (red fluorescent protein gene and neomycin resistant gene). The transgenic fibroblasts cell lines were obtained after G418 selection. Prior to the somatic cell nuclear transfer (SCNT), the maturation rate of caprine cumulus oocytes complexes (COCs) was optimized to an in vitro maturation time of 18 h. Parthenogenetic ooctyes were used as a model to investigate the effect of two activation methods, one with calcium ionophore IA23187 plus 6-DMAP and the other with ethanol plus 6-DMAP. The cleavage rates after 48 h were respectively 88.7% and 86.4%, with no significant difference (P>0.05). There was no significant difference between the cleavage rate and the blastocyst rate in two different media (SO-Faa and CR1aa; 86.3% vs 83.9%, P>0.05 and 23.1% vs 17.2%, P>0.05). The fusion rate of a 190 V/mm group (62.4%) was significantly higher than 130 V/mm (32.8%) and 200 V/mm (42.9%), groups (P>0.05). After transgenic somatic cell nuclear transfer (TSCNT) manipulation, 203 reconstructed embryos were obtained in which the cleavage rate after in vitro development (IVD) for 48 h was 79.3% (161/203). The blastocyst rate after IVD for 7 to 9 d was 15.3% (31/203). There were 17 embryos out of 31 strongly expressing red fluorescence. Two of the red fluorescent blastocysts were randomly selected to identify transgene by polymerase chain reaction. Both were positive. These results showed that: (i) RFP and Neo ( r ) genes were correctly expressed indicating that transgenic somatic cell lines and positive transgenic embryos were obtained; (ii) one more selection at the blastocyst stage was necessary although the donor cells were transgenic positive, because only partially transgenic embryos expressing red fluorescence were obtained; and (iii) through TSCNT manipulation and

  18. Production of rhesus monkey cloned embryos expressing monomeric red fluorescent protein by interspecies somatic cell nuclear transfer

    SciTech Connect

    Zhu, Hai-Ying; Kang, Jin-Dan; Li, Suo; Jin, Jun-Xue; Hong, Yu; Jin, Long; Guo, Qing; Gao, Qing-Shan; Yan, Chang-Guo; Yin, Xi-Jun

    2014-02-21

    Highlights: • Rhesus monkey cells were electroporated with a plasmid containing mRFP1, and an mRFP1-expressing cell line was generated. • For the first time, mRFP1-expressing rhesus monkey cells were used as donor cells for iSCNT. • The effect of VPA on the development of embryos cloned using iSCNT was determined. - Abstract: Interspecies somatic cell nuclear transfer (iSCNT) is a promising method to clone endangered animals from which oocytes are difficult to obtain. Monomeric red fluorescent protein 1 (mRFP1) is an excellent selection marker for transgenically modified cloned embryos during somatic cell nuclear transfer (SCNT). In this study, mRFP-expressing rhesus monkey cells or porcine cells were transferred into enucleated porcine oocytes to generate iSCNT and SCNT embryos, respectively. The development of these embryos was studied in vitro. The percentage of embryos that underwent cleavage did not significantly differ between iSCNT and SCNT embryos (P > 0.05; 71.53% vs. 80.30%). However, significantly fewer iSCNT embryos than SCNT embryos reached the blastocyst stage (2.04% vs. 10.19%, P < 0.05). Valproic acid was used in an attempt to increase the percentage of iSCNT embryos that developed to the blastocyst stage. However, the percentages of embryos that underwent cleavage and reached the blastocyst stage were similar between untreated iSCNT embryos and iSCNT embryos treated with 2 mM valproic acid for 24 h (72.12% vs. 70.83% and 2.67% vs. 2.35%, respectively). These data suggest that porcine-rhesus monkey interspecies embryos can be generated that efficiently express mRFP1. However, a significantly lower proportion of iSCNT embryos than SCNT embryos reach the blastocyst stage. Valproic acid does not increase the percentage of porcine-rhesus monkey iSCNT embryos that reach the blastocyst stage. The mechanisms underling nuclear reprogramming and epigenetic modifications in iSCNT need to be investigated further.

  19. High-protein-PUFA supplementation, red blood cell membranes, and plasma antioxidant activity in volleyball athletes.

    PubMed

    Malaguti, Marco; Baldini, Marta; Angeloni, Cristina; Biagi, Pierluigi; Hrelia, Silvana

    2008-06-01

    The authors evaluated the role of a high-protein, low-calorie, polyunsaturated fatty-acid (PUFA) -supplemented diet on anthropometric parameters, erythrocyte-membrane fatty-acid composition, and plasma antioxidant defenses of nonprofessional volleyball athletes. The athletes were divided in two groups: One (n = 5) followed the Mediterranean diet, and the other (n = 6) followed a high-protein, low-calorie diet with a 3-g/day fish-oil supplementation. All the athletes had anthropometric measurements taken, both at the beginning and at the end of the study, which lasted for 2 months. Body-mass index and total body fat were significantly diminished in the second group, while they remained unchanged in the first. Plasma total antioxidant activity (TAA) was significantly increased in the plasma of both groups, with no differences between the groups, suggesting that physical activity, not the different diets, is the main contributor to the increase of plasma TAA. The second group showed a significant increase in erythrocyte-membrane PUFA content and in the unsaturation index value (UI) because of the fish-oil supplementation.A high-protein, low-carbohydrate, fish-oil-supplemented diet seems to be useful only when the aim of the diet is to obtain weight loss in a short-term period. The significant increase in the UI of erythrocyte membranes indicates the potential for harm, because a high intake of PUFA might increase susceptibility to lipid peroxidation not counterbalanced by a higher increase in TAA. Adherence to the Mediterranean diet seems to be the better choice.

  20. Regulation of Na+-K+-2Cl− cotransport in turkey red cells: the role of oxygen tension and protein phosphorylation

    PubMed Central

    Muzyamba, M C; Cossins, A R; Gibson, J S

    1999-01-01

    Na+-K+-2Cl− cotransport (NKCC) was studied in turkey red cells using Na+ dependence or bumetanide sensitivity of 86Rb+ influx to monitor activity of the transporter. Deoxygenation was the major physiological stimulus for NKCC activity: oxygen tensions (PO2) over the physiological range modulated the transporter, with a PO2 for half-maximal activation of about 41 mmHg (n = 3). In air, activity of NKCC was also stimulated by shrinkage and isoproteronol (isoprenaline, 5 μm). By contrast, in deoxygenated cells, although the transporter activity was markedly elevated, it was no longer sensitive to volume or β-adrenergic stimulation. Calyculin A, a protein phosphatase inhibitor, stimulated cotransport with a lag of about 5 min. N-Ethylmaleimide (NEM) inhibited cotransport and also blocked the stimulatory effect of calyculin A if administered before calyculin A. Stimulation by calyculin A and deoxygenation were not additive. Staurosporine (2 μm) inhibited deoxygenated-stimulated K+ influxes, but not those stimulated by calyculin A. NEM added during calyculin A stimulation, i.e. during the 5 min lag, caused transport activity to be clamped at levels intermediate between maximal (calyculin A alone) and control. Cells treated with calyculin A alone or with calyculin A followed by NEM were no longer sensitive to volume, isoproteronol or PO2. The results have characterized the interaction between deoxygenation and other stimuli of NKCC activity. They have also shown that it is possible to manipulate the transporter in a reciprocal way to that shown previously for K+-Cl− cotransport. PMID:10332092

  1. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells

    PubMed Central

    Baldwin, Michael R.; Li, Xuerong; Hanada, Toshihiko; Liu, Shih-Chun

    2015-01-01

    Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA– band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA–band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs. PMID:25778531

  2. Merozoite surface protein 1 recognition of host glycophorin A mediates malaria parasite invasion of red blood cells.

    PubMed

    Baldwin, Michael R; Li, Xuerong; Hanada, Toshihiko; Liu, Shih-Chun; Chishti, Athar H

    2015-04-23

    Plasmodium falciparum invasion of human red blood cells (RBCs) is an intricate process requiring a number of distinct ligand-receptor interactions at the merozoite-erythrocyte interface. Merozoite surface protein 1 (MSP1), a highly abundant ligand coating the merozoite surface in all species of malaria parasites, is essential for RBC invasion and considered a leading candidate for inclusion in a multiple-subunit vaccine against malaria. Our previous studies identified an interaction between the carboxyl-terminus of MSP1 and RBC band 3. Here, by employing phage display technology, we report a novel interaction between the amino-terminus of MSP1 and RBC glycophorin A (GPA). Mapping of the binding domains established a direct interaction between malaria MSP1 and human GPA within a region of MSP1 known to potently inhibit P falciparum invasion of human RBCs. Furthermore, a genetically modified mouse model lacking the GPA- band 3 complex in RBCs is completely resistant to malaria infection in vivo. These findings suggest an essential role of the MSP1-GPA-band 3 complex during the initial adhesion phase of malaria parasite invasion of RBCs.

  3. Structural aspects of graphitic carbon modified SBA-15 mesoporous silica and biological interactions with red blood cells and plasma proteins.

    PubMed

    Martinez, Diego S T; Damasceno, João Paulo V; Franqui, Lidiane S; Bettini, Jefferson; Mazali, Italo O; Strauss, Mathias

    2017-09-01

    Functional mesoporous materials have been worldwide studied for different applications. Mesoporous silicas are highlighted due to the synthetic possibilities for the preparation of such materials with different particle sizes and morphologies, and controlled pores sizes and structures. Moreover, the silica superficial silanol groups are explored in several chemical modifications, leading to functional materials with tuned functionalities and properties. In this work, an organo-functionalization and pyrolysis synthetic procedure is used to obtain graphitic carbon modified mesoporous SBA-15 silica. The carbon content was tuned during the functionalization step, and the graphitic nanodomains were formed in the pores surface and particles outer surface. Textural and small angle X-ray diffraction analysis accessed the presence of the carbon nanostructures inside the SBA-15 mesopores. Advanced microanalysis using electron energy loss spectroscopy coupled to a transmission electron microscope had confirmed the carbon distribution along the silica pores, which gives higher hydrophobicity and changed the interaction of the mesoporous material with biological systems. Finally, the influence of the surface modification with graphitic carbon species over the interaction with human red blood cells (hemolysis) and human blood plasma (protein corona formation) was elucidated for the very first time for this kind of functional materials. It was observed that the graphitic carbon species considerably reduced the hemolytic effect of the silica particles, and was responsible for modulating the loading and composition of the hard corona plasma proteins. This work deepness the fundamental knowledge on the interaction between such nanomaterials and biological systems, one step further the use of these modified silicas in biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Mitochondria-targeted DsRed2 protein expression during the early stage of bovine somatic cell nuclear transfer embryo development.

    PubMed

    Park, Hyo-Jin; Min, Sung-Hun; Choi, Hoonsung; Park, Junghyung; Kim, Sun-Uk; Lee, Seunghoon; Lee, Sang-Rae; Kong, Il-Keun; Chang, Kyu-Tae; Koo, Deog-Bon; Lee, Dong-Seok

    2016-09-01

    Somatic cell nuclear transfer (SCNT) has been widely used as an efficient tool in biomedical research for the generation of transgenic animals from somatic cells with genetic modifications. Although remarkable advances in SCNT techniques have been reported in a variety of mammals, the cloning efficiency in domestic animals is still low due to the developmental defects of SCNT embryos. In particular, recent evidence has revealed that mitochondrial dysfunction is detected during the early development of SCNT embryos. However, there have been relatively few or no studies regarding the development of a system for evaluating mitochondrial behavior or dynamics. For the first time, in mitochondria of bovine SCNT embryos, we developed a method for the visualization of mitochondria and expression of fluorescence proteins. To express red fluorescence in mitochondria of cloned embryos, bovine ear skin fibroblasts, nuclear donor, were stably transfected with a vector carrying mitochondria-targeting DsRed2 gene tagged with V5 epitope (mito-DsRed2-V5 tag) using lentivirus-mediated gene transfer because of its ability to integrate in the cell genome and the potential for long-term transgene expression in the transduced cells and their dividing cells. From western blotting analysis of V5 tag protein using mitochondrial fraction and confocal microscopy of red fluorescence using SCNT embryos, we found that the mitochondrial expression of the mito-DsRed2 protein was detected until the blastocyst stage. In addition, according to image analysis, it may be suggested possible use of the system for visualization of mitochondrial localization and evaluation of mitochondrial behaviors or dynamics in early development of bovine SCNT embryos.

  5. Pediatric red cell disorders and pure red cell aplasia.

    PubMed

    Perkins, Sherrie L

    2004-12-01

    Anemia in children may arise from a wide variety of pathogenetic mechanisms that include congenital and acquired disorders. Often the diagnostic considerations include disorders that are not seen commonly in adults and lifelong disorders that arise in children and persist throughout life. Consideration of diverse causes of anemia such as red cell membrane disorders, red cell enzymopathies, congenital dyserythropoietic anemias, congenital sideroblastic anemias, and hereditary pure red cell aplasia (Diamond-Blackfan anemia), as well as infectious causes such as parvovirus B19 infection, often is required when diagnosing anemia in an infant or young child. Knowledge of these entities that are important causes of anemia in the pediatric population, including clinical manifestations and laboratory workup, will aid in recognition of the specific disease entities and effective workup of pediatric red cell disorders.

  6. Effect of Thuya occidentalis on the labeling of red blood cells and plasma proteins with technetium-99m.

    PubMed Central

    Oliveira, J. F.; Braga, A. C.; Avila, A. S.; Fonseca, L. M.; Gutfilen, B.; Bernardo-Filho, M.

    1996-01-01

    Thuya occidentalis is used in popular medicine in the treatment of condyloma and has antibacterial action. Red blood cells (RBC) labeled with technetium-99m (99mTc) are used for several evaluations in nuclear medicine. This labeling depends on a reducing agent, usually stannous ion. Any drug which alters the labeling of the tracer could be expected to modify the disposition of the radiopharmaceutical. We have evaluated the influence of T. occidentalis extract on the labeling of RBC and plasma proteins with 99mTc. Blood was withdrawn and incubated with T. occidentalis (0.25; 2.5; 20.5; and 34.1 percent v/v). Stannous chloride (1.2 micrograms/ml) was added and then 99mTc was added. Plasma (P) and blood cells (BC) were isolated, also precipitated with trichloroacetic acid and soluble (SF) and insoluble fractions (IF) separated. The analysis of the results shows that there is a decrease in radioactivity (from 97.64 to 75.89 percent) in BC with 34.1 percent of the drug. In the labeling process of RBC with 99mTc, the stannous and pertechnetate ions pass through the membrane, so we suggest that the T. occidentalis effect can be explained (i) by an inhibition of the transport of these ions, (ii) by damage in membrane, (iii) by competition with the cited ions for the same binding sites, or (iv) by possible generation of reactive oxygen species that could oxidize the stannous ion. PMID:9436292

  7. Therapy with hydroxyurea is associated with reduced adhesion molecule gene and protein expression in sickle red cells with a concomitant reduction in adhesive properties.

    PubMed

    Gambero, Sheley; Canalli, Andreia A; Traina, Fabiola; Albuquerque, Dulcinéia M; Saad, Sara T O; Costa, Fernando F; Conran, Nicola

    2007-02-01

    Propagation of the vaso-occlusive process in sickle cell anaemia (SCA) is a complex process involving the adhesion of steady-state SCA patients red cells and reticulocytes to the vascular endothelium. The effect of hydroxyurea therapy (HUT) on the adhesive properties of sickle cells and the expression of adhesion molecule genes by erythroid cells of SCA individuals is not yet fully understood. The expressions of the CD36 gene and the VLA-4-integrin subunit genes, CD49d (alpha-subunit) and CD29 (beta-subunit), were compared in the reticulocytes of steady-state SCA patients and patients on HUT using real-time PCR. Basal adhesion of red cells from these subjects was also compared using static adhesion assays, as was surface protein expression, using flow cytometry. Basal sickle red cell adhesion to fibronectin was significantly greater than that of normal cells (P < 0.01); in contrast, HUT was associated with significantly lower levels (P < 0.01) of red cell adhesion that were similar to those of control cells; this decrease could not be justified solely by altered reticulocyte numbers in this population. Accordingly, flow cytometry demonstrated that reticulocytes from patients on HUT had significantly lower CD36 and CD49d surface expressions (P < 0.01) and, importantly, significantly lower expressions of the CD36, CD49d and CD29 genes (P < 0.05) than reticulocytes of SCA patients not on HUT. Taken together, data support the hypothesis that HUT reduces the adhesive properties of sickle cells and that this decrease appears to be mediated, at least in part, by a decrease in the gene and, consequently, surface protein expression of adhesion molecules such as VLA-4 and CD36.

  8. Localization, Dynamics, and Function of Survivin Revealed by Expression of Functional SurvivinDsRed Fusion Proteins in the Living Cell

    PubMed Central

    Temme, Achim; Rieger, Michael; Reber, Friedemann; Lindemann, Dirk; Weigle, Bernd; Diestelkoetter-Bachert, Petra; Ehninger, Gerhard; Tatsuka, Masaaki; Terada, Yasuhiko; Rieber, Ernst Peter

    2003-01-01

    Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein. PMID:12529428

  9. Plasma protein induced clustering of red blood cells in micro capillaries

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Brust, Mathias; Aouane, Othmane; Flormann, Daniel; Thiebaud, Marine; Verdier, Claude; Coupier, Gwennou; Podgorski, Thomas; Misbah, Chaouqi; Selmi, Hassib

    2013-11-01

    The plasma molecule fibrinogen induces aggregation of RBCs to clusters, the so called rouleaux. Higher shear rates in bulk flow can break them up which results in the pronounced shear thinning of blood. This led to the assumption that rouleaux formation does not take place in the microcapillaries of the vascular network where high shear rates are present. However, the question is of high medical relevance. Cardio vascular disorders are still the main cause of death in the western world and cardiac patients have often higher fibrinogen level. We performed AFM based single cell force spectroscopy to determine the work of separation. Measurements at low hematocrit in a microfluidic channel show that the number of size of clusters is determined by the adhesion strength and we found that cluster formation is strongly enhanced by fibrinogen at physiological concentrations, even at shear rate as high as 1000 1/s. Numerical simulations based on a boundary integral method confirm our findings and the clustering transition takes place both in the experiments and in the simulations at the same interaction energies. In vivo measurements with intravital fluorescence microscopy in a dorsal skin fold chamber in a mouse reveal that RBCs indeed form clusters in the micrcapillary flow. This work was supported by the German Science Foundation research imitative SFB1027.

  10. Endothelial progenitor and mesenchymal stem cell-derived cells persist in tissue-engineered patch in vivo: application of green and red fluorescent protein-expressing retroviral vector.

    PubMed

    Sales, Virna L; Mettler, Bret A; Lopez-Ilasaca, Marco; Johnson, John A; Mayer, John E

    2007-03-01

    An unresolved question regarding tissue-engineered (TE) cardiac valves and vessels is the fate of the transplanted cells in vivo. We have developed a strategy to track the anatomic location of seeded cells within TE constructs and neighboring tissues using a retroviral vector system encoding green and red fluorescent proteins (GFPs and RFPs, respectively) in ovine circulating endothelial progenitor cells (EPCs) and bone marrow-derived mesenchymal stem cells (BMSCs). We demonstrate that stable transduction ex vivo with high-titer Moloney murine leukemia virus-based retroviral vector yields transduction efficiency of greater than 97% GFP(+) EPC- and RFP(+) mesenchymal stem cell (MSC)-derived cells. Cellular phenotype and transgene expression were also maintained through 25 subsequent passages. Using a retroviral vector system to distinguish our pre-seeded cells from tissue-resident progenitor cells and circulating endothelial and marrow-derived precursors, we simultaneously co-seeded 2 x 10(6) GFP(+) EPCs and 2 x 10(5) RFP(+) MSCs onto the TE patches. In a series of ovine pulmonary artery patch augmentation studies, transplanted GFP(+) EPC- and RFP(+) MSC-derived cells persisted within the TE patch 7 to 14 days after implantation, as identified using immunofluorescence. Analysis showed 81% luminal coverage of the TE patches before implantation with transduced cells, increasing to 96% at day 7 and decreasing to 67% at day 14 post-implantation. This suggests a temporal association between retroviral expression of progenitor cells and mediating effects of these cells on the physiological remodeling and maturation of the TE constructs. To our knowledge, this is the first cardiovascular tissue-engineering in vivo study using a double-labeling method to demonstrate a direct evidence of the source, persistence, and incorporation into a TE vascular patch of co-cultured and simultaneously pre-seeded adult progenitor cells.

  11. Red blood cells, spherocytosis (image)

    MedlinePlus

    Spherocytosis is a hereditary disorder of the red blood cells (RBCs), which may be associated with a mild anemia. Typically, the affected RBCs are small, spherically shaped, and lack the light centers seen ...

  12. Red Blood Cell Antibody Identification

    MedlinePlus

    ... name: Red Blood Cell Antibody Identification Related tests: Direct Antiglobulin Test ; RBC Antibody Screen ; Blood Typing ; Type ... a positive RBC antibody screen or a positive direct antiglobulin test (DAT) . It is used to identify ...

  13. Effects of a diet high in plant sterols, vegetable proteins, and viscous fibers (dietary portfolio) on circulating sterol levels and red cell fragility in hypercholesterolemic subjects.

    PubMed

    Jones, Peter J; Raeini-Sarjaz, Mahmoud; Jenkins, David J A; Kendall, Cyril W C; Vidgen, Edward; Trautwein, Elke A; Lapsley, Karen G; Marchie, Augustine; Cunnane, Stephen C; Connelly, Philip W

    2005-02-01

    Plant sterols, soy proteins, viscous fibers, and nuts are advised for cholesterol reduction, but their combined effect on plant sterol absorption has never been tested. We assessed their combined action on serum sterols in hyperlipidemic subjects who were following low-saturated fat diets before starting the study and who returned to these diets post-test. The 1-mon test (combination) diet was high in plant sterols (1 g/1,000 kcal), soy protein (23 g/1,000 kcal), viscous fiber (9 g/1,000 kcal), and almonds (14 g/1000 kcal). Fasting blood was obtained for serum lipids and sterols, and erythrocytes were obtained for fragility prior to and at 2-wk intervals during the study. The combination diet raised serum campesterol concentrations by 50% and beta-sitosterol by 27%, although these changes were not significant after Bonferroni correction; near-maximal rises were found by the end of the first week, but no change was found in red cell fragility despite a 29% reduction in the LDL cholesterol level. No significant associations were observed between changes in red cell fragility and blood lipids or sterols. We conclude that plant sterols had a minimal impact on serum sterol concentrations or red cell fragility in hyperlipidemic subjects on diets that greatly reduced their serum lipids.

  14. Red cell metabolism studies on Skylab

    NASA Technical Reports Server (NTRS)

    Mengel, C. E.

    1977-01-01

    Blood samples from Spacelab crewmembers were studied for possible environment effects on red cell components. Analysis involved peroxidation of red cell lipids, enzymes of red cell metabolism, and levels of 2,3-diphosphoglyceric acid and adenosine triphosphate. Results show that there is no evidence of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage. Changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

  15. Red cell metabolism studies on Skylab

    NASA Technical Reports Server (NTRS)

    Mengel, C. E.

    1977-01-01

    Blood samples from Spacelab crewmembers were studied for possible environment effects on red cell components. Analysis involved peroxidation of red cell lipids, enzymes of red cell metabolism, and levels of 2,3-diphosphoglyceric acid and adenosine triphosphate. Results show that there is no evidence of lipid peroxidation, that biochemical effect known to be associated with irreversible red cell damage. Changes observed in glycolytic intermediates and enzymes cannot be directly implicated as indicating evidence of red cell damage.

  16. Reactive oxygen species in photochemistry of the red fluorescent protein "Killer Red".

    PubMed

    Vegh, Russell B; Solntsev, Kyril M; Kuimova, Marina K; Cho, Soohee; Liang, Yue; Loo, Bernard L W; Tolbert, Laren M; Bommarius, Andreas S

    2011-05-07

    The fluorescent protein aptly named "Killer Red" (KRed) is capable of killing transfected cells and inactivating fused proteins upon exposure to visible light in the presence of oxygen. We have investigated the source of the bioactive species through a variety of photophysical and photochemical techniques. Our results indicate a Type I (electron transfer mediated) photosensitizing mechanism.

  17. Evaluation of haemoglobin, haematocrit, haemolysis, residual protein content and leucocytes in 345 red blood cell concentrates used for the treatment of patients with β-thalassaemia

    PubMed Central

    Mancini, Roberta; Marinelli, Leonardo; Mirante, Nadia; Gallo, Assunta; Matteocci, Antonella; Terlizzi, Filomena; Palange, Maria; Fioravanti, Daniela; Donnini, Lorella; Pierelli, Luca

    2012-01-01

    Background The aim of this study was to evaluate the quality of red blood cell concentrates obtained from donated whole blood, selected for transfusion therapy of thalassaemic patients, by measuring the following parameters: haemoglobin, haematocrit, percentage haemolysis, residual leucocyte count and residual protein content. Materials and methods Overall 345 red cell concentrates were evaluated, of which 205 had been filtered in-line pre-storage and washed and 140 were buffy coat-depleted and used within 2 days of collection. Of the buffy coat-depleted concentrates, 62 were leucodepleted and 78 washed and leucodepleted post-storage all within 2 days of collection. The off-line filters used for the leucodepletion were gamma-irradiated polyester with a pore size of 200 μm. The washing procedure was automated (Haemonetics ACP 215, Braintree, MA, USA). The haematological parameters were evaluated by a blood cell counter (Coulter, Ramsey, IL, USA) and the white blood cell count by cytofluorimetry (FACScan). Results Ninety-five percent (194/205) of the red cell concentrates that had been filtered pre-storage and washed, 92% (57/62) of the red cell concentrates that had been leucodepleted post-storage and 94% (73/78) of the those subjected to both treatments had normal values of haemoglobin (>40 g/unit), haematocrit (between 50–70%), percentage haemolysis (<0.8/unit), white cell count (<1×106) and residual protein content (<0.5 g/L). Five percent (11/205) of the red cell concentrates that had been filtered pre-storage and washed, 8% (5/62) of those leucodepleted post-storage after 2 days and 6% (5/78) of those that underwent both procedures had a haemoglobin content <40 g/unit and a haematocrit <50%. Conclusions The preparation procedures had been carried out satisfactorily; nevertheless, transfusion therapy with some “low dose” normal units could be less effective and might, therefore, result in greater transfusion requirements in patients receiving such units

  18. Study of the influence of laser of low potency to label red blood cells and plasmatic protein with 99mTc in vitro

    NASA Astrophysics Data System (ADS)

    Rolim, Ricardo Q.; Carvalho, Elaine F.; Nascimento, Edgar V.; Souza, Grace M.; Magnata, Simey S.; Bernardo-Filho, Mario; Catanho, Maria T.

    2004-09-01

    This work aims to verify the effect of laser of low potency to label red blood cells and plasmatic protein with 99mTc in vitro. The experiments were carried out by incubating of anticoagulant whole blood. Differents doses of gallium arsenide laser with energy density of 3j/cm2, 6j/cm2, 9 j/cm2, 18 j/cm2 and 804nm wavelength was applied. A stannous chloride solution of 1,2μg/mL was added the incubation for 60 minutes. After this the 99mTc was added and the incubation was continued for another 10 minutes. Those were centrifuged, precipitated with thrichoroacetic acid 5% and mensured in a counter. The results shows that there is a significant decrease in the fixation of 99mTc in red blood cells when the concentration of 3j/ cm2 and (from 93 to 32.2%) and in occurs an increase %ATI from 12 to 54,6% of plasma protein. The laser promote modification on some properties of the red blood cellular membrane, probably, due to the metabolization of cellular that could be capable to generation the active metabolites.

  19. Identification of a Role for the PfEMP1 Semi-Conserved Head Structure in Protein Trafficking to the Surface of Plasmodium falciparum Infected Red Blood Cells

    PubMed Central

    Melcher, Martin; Muhle, Rebecca A.; Henrich, Philipp P.; Kraemer, Susan M.; Avril, Marion; Vigan-Womas, Ines; Mercereau-Puijalon, Odile; Smith, Joseph D.; Fidock, David A.

    2010-01-01

    Summary Transport of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants to the red blood cell (RBC) surface enables malarial parasite evasion of host immunity by modifying the antigenic and adhesive properties of infected RBCs. In this study, we applied the Bxb1 integrase system to integrate transgenes encoding truncated PfEMP1-GFP fusions into cytoadherent A4 parasites and characterize their surface transport requirements. Our studies revealed that the semi-conserved head structure of PfEMP1 proteins, in combination with the predicted transmembrane region and cytoplasmic tail, encodes sufficient information for RBC surface display. In contrast, miniPfEMP1 proteins with truncated head structures were exported to the RBC cytoplasm but were not detected at the RBC surface by flow cytometry or immuno-electron microscopy. We demonstrated the absence of a mechanistic barrier to having native and miniPfEMP1 proteins displayed simultaneously at the RBC surface. However, surface exposed miniPfEMP1 proteins did not convey cytoadherence properties to their host cells, implicating potential steric considerations in host-receptor interactions or the need for multiple domains to mediate cell binding. This study establishes a new system to investigate PfEMP1 transport and demonstrates that the PfEMP1 semi-conserved head structure is under selection for protein transport, in addition to its known roles in adhesion. PMID:20438573

  20. Identification of a role for the PfEMP1 semi-conserved head structure in protein trafficking to the surface of Plasmodium falciparum infected red blood cells.

    PubMed

    Melcher, Martin; Muhle, Rebecca A; Henrich, Philipp P; Kraemer, Susan M; Avril, Marion; Vigan-Womas, Ines; Mercereau-Puijalon, Odile; Smith, Joseph D; Fidock, David A

    2010-10-01

    Transport of Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP1) variants to the red blood cell (RBC) surface enables malarial parasite evasion of host immunity by modifying the antigenic and adhesive properties of infected RBCs. In this study, we applied the Bxb1 integrase system to integrate transgenes encoding truncated PfEMP1-GFP fusions into cytoadherent A4 parasites and characterize their surface transport requirements. Our studies revealed that the semi-conserved head structure of PfEMP1 proteins, in combination with the predicted transmembrane region and cytoplasmic tail, encodes sufficient information for RBC surface display. In contrast, miniPfEMP1 proteins with truncated head structures were exported to the RBC cytoplasm but were not detected at the RBC surface by flow cytometry or immuno-electron microscopy. We demonstrated the absence of a mechanistic barrier to having native and miniPfEMP1 proteins displayed simultaneously at the RBC surface. However, surface-exposed miniPfEMP1 proteins did not convey cytoadherence properties to their host cells, implicating potential steric considerations in host-receptor interactions or the need for multiple domains to mediate cell binding. This study establishes a new system to investigate PfEMP1 transport and demonstrates that the PfEMP1 semi-conserved head structure is under selection for protein transport, in addition to its known roles in adhesion.

  1. High-throughput living cell-based optical biosensor for detection of bacterial lipopolysaccharide (LPS) using a red fluorescent protein reporter system

    PubMed Central

    Jiang, Hui; Jiang, Donglei; Shao, Jingdong; Sun, Xiulan; Wang, Jiasheng

    2016-01-01

    Due to the high toxicity of bacterial lipopolysaccharide (LPS), resulting in sepsis and septic shock, two major causes of death worldwide, significant effort is directed toward the development of specific trace-level LPS detection systems. Here, we report sensitive, user-friendly, high-throughput LPS detection in a 96-well microplate using a transcriptional biosensor system, based on 293/hTLR4A-MD2-CD14 cells that are transformed by a red fluorescent protein (mCherry) gene under the transcriptional control of an NF-κB response element. The recognition of LPS activates the biosensor cell, TLR4, and the co-receptor-induced NF-κB signaling pathway, which results in the expression of mCherry fluorescent protein. The novel cell-based biosensor detects LPS with specificity at low concentration. The cell-based biosensor was evaluated by testing LPS isolated from 14 bacteria. Of the tested bacteria, 13 isolated Enterobacteraceous LPSs with hexa-acylated structures were found to increase red fluorescence and one penta-acylated LPS from Pseudomonadaceae appeared less potent. The proposed biosensor has potential for use in the LPS detection in foodstuff and biological products, as well as bacteria identification, assisting the control of foodborne diseases. PMID:27841364

  2. Red Blood Cell Magnetophoresis

    PubMed Central

    Zborowski, Maciej; Ostera, Graciela R.; Moore, Lee R.; Milliron, Sarah; Chalmers, Jeffrey J.; Schechter, Alan N.

    2003-01-01

    The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 × 10−6 mm3 s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 × 10−6 mm3 s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from −0.2 × 10−6 mm3 s/kg to +0.30 × 10−6 mm3 s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules. PMID:12668472

  3. Red blood cell magnetophoresis.

    PubMed

    Zborowski, Maciej; Ostera, Graciela R; Moore, Lee R; Milliron, Sarah; Chalmers, Jeffrey J; Schechter, Alan N

    2003-04-01

    The existence of unpaired electrons in the four heme groups of deoxy and methemoglobin (metHb) gives these species paramagnetic properties as contrasted to the diamagnetic character of oxyhemoglobin. Based on the measured magnetic moments of hemoglobin and its compounds, and on the relatively high hemoglobin concentration of human erythrocytes, we hypothesized that differential migration of these cells was possible if exposed to a high magnetic field. With the development of a new technology, cell tracking velocimetry, we were able to measure the migration velocity of deoxygenated and metHb-containing erythrocytes, exposed to a mean magnetic field of 1.40 T and a mean gradient of 0.131 T/mm, in a process we call cell magnetophoresis. Our results show a similar magnetophoretic mobility of 3.86 x 10(-6) mm(3) s/kg for erythrocytes with 100% deoxygenated hemoglobin and 3.66 x 10(-6) mm(3) s/kg for erythrocytes containing 100% metHb. Oxygenated erythrocytes had a magnetophoretic mobility of from -0.2 x 10(-6) mm(3) s/kg to +0.30 x 10(-6) mm(3) s/kg, indicating a significant diamagnetic component relative to the suspension medium, in agreement with previous studies on the hemoglobin magnetic susceptibility. Magnetophoresis may open up an approach to characterize and separate cells for biochemical analysis based on intrinsic and extrinsic magnetic properties of biological macromolecules.

  4. A lysine-rich membrane-associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum-infected red blood cells.

    PubMed

    Proellocks, Nicholas I; Herrmann, Susann; Buckingham, Donna W; Hanssen, Eric; Hodges, Emma K; Elsworth, Brendan; Morahan, Belinda J; Coppel, Ross L; Cooke, Brian M

    2014-07-01

    The genomes of malaria parasites (Plasmodium spp.) contain a family of genes encoding proteins with a Plasmodium helical interspersed subtelomeric (PHIST) domain, most of which are predicted to be exported into the parasite-infected human red blood cell (iRBC). Here, using transgenic parasites and a combination of cellular, biochemical, and biophysical assays, we have characterized and determined the function of a novel member of the PHIST protein family in Plasmodium falciparum, termed lysine-rich membrane-associated PHISTb (LyMP). LyMP was shown to associate directly with the cytoskeleton of iRBCs where it plays a role in their abnormal ability to adhere to a protein expressed on vascular endothelial cells, resulting in sequestration. Deletion of LyMP dramatically reduced adhesion of iRBCs to CD36 by 55%, which was completely restored to wild-type levels on complementation. Intriguingly, in the absence of LyMP, formation of RBC membrane knobs and the level of surface exposure of the parasites' major cytoadhesive ligand, PfEMP1, were identical to those for the parental parasite line, demonstrating for the first time an additional mechanism that enhances cytoadherence of iRBCs beyond those already recognized. Our findings identify LyMP as a previously unknown RBC cytoskeletal-binding protein that is likely to be of major significance in the complex pathophysiology of falciparum malaria.-Proellocks, N. I., Herrmann, S., Buckingham, D. W., Hanssen, E., Hodges, E. K., Elsworth, B., Morahan, B. J., Coppel, R. L., Cooke, B. M. A lysine-rich membrane-associated PHISTb protein involved in alteration of the cytoadhesive properties of Plasmodium falciparum infected red blood cells.

  5. Performance of a novel sieving matrix of poly(vinyl alcohol)/acrylamide copolymer in electrophoretic separations of high molecular weight proteins from red cell membrane.

    PubMed

    Matte, Alessandro; Sola, Laura; Chiari, Marcella; Tomelleri, Carlo; Consonni, Roberto; Turrini, Franco; Franceschi, Lucia De

    2014-04-01

    The analysis of high molecular weight (HMW) proteins from complex mixtures is still a challenge in proteomics. This work introduces a novel hydrogel obtained by the copolymerization of an allyl-PVA derivative with acrylamide and bisacrylamide and applies this matrix to the electrophoretic separation of HMW proteins. By inducing gelation of polyacrylamide in the presence of variable amounts of allyl-PVA, it is possible to control and vary the average gel porosity. This gel is easy to produce and handle and offers the advantage of being highly mechanically resistant and macroporous. The new matrix was tested in mono-dimensional separations of complex protein mixtures extracted from red cell membranes with different detergents. The improved performance of this macroporous matrix allowed to identify new proteins by MS and immunoblot analysis using specific antibodies. In particular, the resolution of proteins ranging in size between 97 and 279 kDa was greatly improved here compared to standard polyacrylamide gels, suggesting that this matrix can be a useful tool in routine analysis of HMW proteins in cell biology.

  6. Extraction and Enrichment of Protein from Red and Green Macroalgae.

    PubMed

    Harnedy, Pádraigín A; FitzGerald, Richard J

    2015-01-01

    Macroalgae, in particular red and green species, are gaining interest as protein-rich foods for human consumption and sources of proteinaceous biofunctional peptide ingredients. During protein extraction the starting raw material, the cell disruption method utilized and the reagents employed have a major effect on the yield of protein recovered. A method is described herein for extraction and semi-purification of food-grade aqueous and alkaline soluble proteins from red and green macroalgae. Dried milled macroalgae are disrupted by osmotic shock with subsequent removal of aqueous soluble proteins by centrifugation. Alkaline soluble proteins are removed following consecutive treatment of the resultant pellet with an alkaline solution. Aqueous and alkaline soluble proteins are then enriched from the crude extracts by isoelectric precipitation.

  7. Red cell distribution width and nonalcoholic steatohepatitis

    PubMed Central

    Gulcan Kurt, Yasemin; Cayci, Tuncer; Aydin, Fevzi Nuri; Agilli, Mehmet

    2014-01-01

    Red cell distribution width is a measure of deviation of the volume of red blood cells. It is a marker of anisocytosis and often used to evaluate the possible causes of anemia. Elevated red cell distribution width levels are also associated with acute and chronic inflammatory responses. In nonalcoholic steatohepatitis, inflammation is accompanied with steatosis. For assuming red cell distribution width as a marker of nonalcoholic steatohepatitis, intervening factors such as levels of inflammatory markers should also be evaluated. PMID:25473202

  8. A G protein-coupled receptor (GPCR) in red: live cell imaging of the kappa opioid receptor-tdTomato fusion protein (KOPR-tdT) in neuronal cells.

    PubMed

    Huang, Peng; Chiu, Yi-Ting; Chen, Chongguang; Wang, Yujun; Liu-Chen, Lee-Yuan

    2013-01-01

    In contrast to green fluorescent protein and variants (GFPs), red fluorescent proteins (RFPs) have rarely been employed for the generation of GPCR fusion proteins, likely because of formation of aggregates and cell toxicity of some RFPs. Among all the RFPs, tdTomato (tdT), one of the non-aggregating RFP, has the highest brightness score (about 3 times that of eGFP) and unsurpassed photostability. We fused tdT to the KOPR C-terminus. The KOPR-tdT cDNA construct was transfected into a Neuro2A mouse neuroblastoma cell line (Neuro2A cells) and rat cortical primary neurons for characterization of pharmacological properties and imaging studies on KOPR trafficking. KOPR-tdT retained KOPR properties (cell surface expression, ligand binding, agonist-induced signaling and internalization) when expressed in Neuro2A cells and rat primary cortical neurons. Live cell imaging of KOPR-tdT enables visualization of the time course of agonist-induced internalization of KOPR in real time for 60 min, without photobleaching and apparent cell toxicity. U50,488H-induced KOPR internalization occurred as early as 4min and plateaued at about 30 min. A unique pattern of internalized KOPR in processes of primary neurons was induced by U50,488H. tdT is an alternative to, or even a better tool than, GFPs for fusion to GPCR for trafficking studies, because tdT has higher brightness and thus better resolution and less photobleaching problems due to the reduced laser power used. It also has advantages associated with its longer-wavelength emission including spectral separation from autofluorescence and GFPs, reduced cell toxicity that the laser may impose, and greater tissue penetration. These advantages of tdT over GPFs may be critical for live cell imaging studies of GPCRs in vitro and for studying GPCRs in vivo because of their low abundance. © 2013.

  9. Metabolic dependence of red cell deformability

    PubMed Central

    Weed, Robert I.; LaCelle, Paul L.; Merrill, Edward W.

    1969-01-01

    The contribution of the metabolic state of human erythrocytes to maintenance of cellular deformability was studied during and after in vitro incubation in serum for periods up to 28 hr. An initial loss of membrane deformability became apparent between 4 and 6 hr when cellular adenosine triphosphate (ATP) levels were approximately 70% of initial values. Membrane deformability then remained stable between 6 and 10 hr. After 10 hr, when cellular ATP had decreased to < 15% of initial values, progressive parallel changes occurred in red cell calcium which increased 400% by 24 hr and in the viscosity of red cell suspensions which had risen 500-750% at 24 hr. A further progressive decrease in membrane deformability also occurred and was reflected by a 1000% increase in negative pressure required to deform the membrane. Red cell filterability decreased to zero as the disc-sphere shape transformation ensued. These changes were accompanied by an increase in ghost residual hemoglobin and nonhemoglobin protein. Regeneration of ATP in depleted cells by incubation with adenosine produced significant reversal of these changes, even in the presence of ouabain. Introduction of calcium into reconstituted ghosts prepared from fresh red cells mimicked the depleted state, and introduction of ATP, ethylenediamine tetraacetate (EDTA), and magnesium into depleted cells mimicked the adenosine effects in intact depleted cells. ATP added externally to 24-hr depleted cells was without effect. Simultaneous introduction of EDTA, ATP, or magnesium along with calcium into reconstituted ghosts prevented the marked decrease in deformability produced by calcium alone. Incorporation of adenosine diphosphate (ADP), nicotinamide adenine dinucleotide (NAD), NAD phosphate (NADP), NADP, reduced form (NADPH), glutatione, reduced form (GSH), inosine triphosphate (ITP), guanosine triphosphate (GTP), and uridine triphosphate (UTP) was without effect. These data suggest that a major role of ATP in maintenance

  10. Identification of peptides with high red blood cell and hepatocyte binding activity in the Plasmodium falciparum multi-stage invasion proteins: PfSPATR and MCP-1.

    PubMed

    Curtidor, Hernando; García, Jeison; Vanegas, Magnolia; Puentes, Fabian; Forero, Martha; Patarroyo, Manuel Elkin

    2008-01-01

    Plasmodium falciparum multi-stage proteins are involved in vital processes for parasite survival, which turns them into attractive targets for studies aimed at developing a fully effective antimalarial vaccine. MCP-1 and PfSPATR are both found in sporozoite and merozoite forms, and have been associated respectively with invasion of hepatocytes and red blood cells (RBCs). Binding assays with synthetic peptides derived from these two important proteins have enabled identifying those sequences binding with high specific activity (named High activity binding peptides-HABPs) to hepatoma-derived HepG2 cells and human RBCs. Twelve RBC HABPs were identified within the MCP-1 amino acid sequence, most of them in the C-terminal region. The MCP-1 HABPs 33387 and 33397 also presented high activity binding to HepG2 cells. PfSPATR presented four RBC HABPs and two HepG2 HABPs, but only one (32686) could bind to both cell types. RBC binding assays evidenced that binding of all HABPs was saturable and differentially affected by the enzymatic treatment of target cells. Moreover, all HABPs inhibited in vitro invasion of merozoites at 200 microM and had particular structural features when analyzed by circular dichroism. The results suggest that these synthetic peptides capable of binding to the two P. falciparum target cells could be potentially included in the design of a multi-stage, subunit-based, chemically synthesized antimalarial vaccine.

  11. Nitric oxide scavenging by red cell microparticles.

    PubMed

    Liu, Chen; Zhao, Weixin; Christ, George J; Gladwin, Mark T; Kim-Shapiro, Daniel B

    2013-12-01

    Red cell microparticles form during the storage of red blood cells and in diseases associated with red cell breakdown and asplenia, including hemolytic anemias such as sickle cell disease. These small phospholipid vesicles that are derived from red blood cells have been implicated in the pathogenesis of transfusion of aged stored blood and hemolytic diseases, via activation of the hemostatic system and effects on nitric oxide (NO) bioavailability. Red cell microparticles react with the important signaling molecule NO almost as fast as cell-free hemoglobin, about 1000 times faster than red-cell-encapsulated hemoglobin. The degree to which this fast reaction with NO by red cell microparticles influences NO bioavailability depends on several factors that are explored here. In the context of stored blood preserved in ADSOL, we find that both cell-free hemoglobin and red cell microparticles increase as a function of duration of storage, and the proportion of extra erythrocytic hemoglobin in the red cell microparticle fraction is about 20% throughout storage. Normalized by hemoglobin concentration, the NO-scavenging ability of cell-free hemoglobin is slightly higher than that of red cell microparticles as determined by a chemiluminescence NO-scavenging assay. Computational simulations show that the degree to which red cell microparticles scavenge NO will depend substantially on whether they enter the cell-free zone next to the endothelial cells. Single-microvessel myography experiments performed under laminar flow conditions demonstrate that microparticles significantly enter the cell-free zone and inhibit acetylcholine, endothelial-dependent, and NO-dependent vasodilation. Taken together, these data suggest that as little as 5 μM hemoglobin in red cell microparticles, an amount formed after the infusion of one unit of aged stored packed red blood cells, has the potential to reduce NO bioavailability and impair endothelial-dependent vasodilation.

  12. Non-Invasive In Vivo Imaging and Quantification of Tumor Growth and Metastasis in Rats Using Cells Expressing Far-Red Fluorescence Protein.

    PubMed

    Christensen, Jon; Vonwil, Daniel; Shastri, V Prasad

    2015-01-01

    Non-invasive in vivo imaging is emerging as an important tool for basic and preclinical research. Near-infrared (NIR) fluorescence dyes and probes have been used for non-invasive optical imaging since in the NIR region absorption and auto fluorescence by body tissue is low, thus permitting for greater penetration depths and high signal to noise ratio. Currently, cell tracking systems rely on labeling cells prior to injection or administering probes targeting the cell population of choice right before imaging. These approaches do not enable imaging of tumor growth, as the cell label is diluted during cell division. In this study we have developed cell lines stably expressing the far-red fluorescence protein E2-Crimson, thus enabling continuous detection and quantification of tumor growth. In a xenograft rat model, we show that E2-Crimson expressing cells can be detected over a 5 week period using optical imaging. Fluorescence intensities correlated with tumor volume and weight and allowed for a reliable and robust quantification of the entire tumor compartment. Using a novel injection regime, the seeding of MDA-MB-231 breast cancer cells in the lungs in a rat model was established and verified.

  13. Reversibility of red blood cell deformation

    NASA Astrophysics Data System (ADS)

    Zeitz, Maria; Sens, P.

    2012-05-01

    The ability of cells to undergo reversible shape changes is often crucial to their survival. For red blood cells (RBCs), irreversible alteration of the cell shape and flexibility often causes anemia. Here we show theoretically that RBCs may react irreversibly to mechanical perturbations because of tensile stress in their cytoskeleton. The transient polymerization of protein fibers inside the cell seen in sickle cell anemia or a transient external force can trigger the formation of a cytoskeleton-free membrane protrusion of μm dimensions. The complex relaxation kinetics of the cell shape is shown to be responsible for selecting the final state once the perturbation is removed, thereby controlling the reversibility of the deformation. In some case, tubular protrusion are expected to relax via a peculiar “pearling instability.”

  14. Reversibility of red blood cell deformation.

    PubMed

    Zeitz, Maria; Sens, P

    2012-05-01

    The ability of cells to undergo reversible shape changes is often crucial to their survival. For red blood cells (RBCs), irreversible alteration of the cell shape and flexibility often causes anemia. Here we show theoretically that RBCs may react irreversibly to mechanical perturbations because of tensile stress in their cytoskeleton. The transient polymerization of protein fibers inside the cell seen in sickle cell anemia or a transient external force can trigger the formation of a cytoskeleton-free membrane protrusion of μm dimensions. The complex relaxation kinetics of the cell shape is shown to be responsible for selecting the final state once the perturbation is removed, thereby controlling the reversibility of the deformation. In some case, tubular protrusion are expected to relax via a peculiar "pearling instability."

  15. Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes

    PubMed Central

    Wirth, Christine C; Glushakova, Svetlana; Scheuermayer, Matthias; Repnik, Urska; Garg, Swati; Schaack, Dominik; Kachman, Marika M; Weißbach, Tim; Zimmerberg, Joshua; Dandekar, Thomas; Griffiths, Gareth; Chitnis, Chetan E; Singh, Shailja; Fischer, Rainer; Pradel, Gabriele

    2014-01-01

    Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(−)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(−) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(−) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(−) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(−) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(−) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm. PMID:24602217

  16. Ratchets, red cells, and metastability.

    PubMed

    Ferrone, Frank A; Aprelev, Alexey

    2013-06-01

    Sickle cell disease is a genetic disorder in which a negatively charged glutamic acid is replaced by a hydrophobic valine on the surface of the hemoglobin molecule, leading to polymerization of the deoxygenated form, and resulting in microvascular obstruction. Because of the high volume occupancy under which polymerization occurs physiologically, this process has been an exemplar in the study of excluded volume effects on assembly. More recently, we have identified yet another type of crowding effect involving the obstruction of the ends at which the polymers grow as a consequence of the dense arrays in which these polymers form. This makes such solutions metastable, and leads to Brownian ratchet behavior in which pressure is exerted outward when the gel occupies a finite volume, as in an emulsion or red cell. Such behavior is capable of holding sickled cells in place in the microcirculation against weak pressure differentials (hundreds of Pa), but not against the typical pressures found in vivo.

  17. Hypoalbuminemia causes high blood viscosity by increasing red cell lysophosphatidylcholine.

    PubMed

    Joles, J A; Willekes-Koolschijn, N; Koomans, H A

    1997-09-01

    Albumin deficiency is accompanied by a reduction in red cell deformability and blood hyperviscosity. Albumin deficiency increases plasma fibrinogen and triglyceride levels and may alter red cell membrane lipid composition. These options, which could all contribute to reduced red cell deformability (RCD) and hyperviscosity, were studied in the Nagase analbuminemic rat (NAR), a mutant Sprague Dawley rat (CON), characterized by normal total protein levels, with an absolute deficiency of albumin, but elevated levels of non-albumin proteins and hyperlipidemia. Plasma protein-binding of the polar phopholipid lysophosphatidylcholine (LPC) was markedly decreased. LPC comprised only 26 +/- 1% of total plasma phospholipids as compared to 42 +/- 2% in CON. NAR red cells in CON plasma had a viscosity that was similar to CON red cells in CON plasma. Conversely, CON red cells in NAR plasma show an increased viscosity as compared to CON red cells in CON plasma. The maximum deformation index of both NAR and CON red cells was markedly decreased in NAR plasma as compared to either NAR or CON cells in CON plasma (0.04 +/- 0.03 and 0.02 +/- 0.02 vs. 0.22 +/- 0.06 and 0.15 +/- 0.04, respectively; P < 0.05). Thus, plasma composition causes hyperviscosity and reduced RCD in NAR. Fibrinogen is not responsible since red cells in serum and red cells in plasma had a similar viscosity and differences in viscosity and RCD between NAR and CON were maintained. Plasma triglycerides are also not responsible since the viscosity of red cells in serum with a 50% reduction in triglycerides was not reduced. LPC levels in red cells were increased in NAR (8.7 +/- 0.2 vs. 5.5 +/- 0.3% of total phospholipids; P < 0.01). Adding albumin to NAR blood dose-dependently decreased whole blood viscosity, despite marked increases in plasma viscosity, and increased RCD of NAR cells (from 0.04 +/- 0.03 to 0.21 +/- 0.01; P < 0.05). There was also some effect on CON RCD of similar albumin addition to CON blood (from 0

  18. The human Kell blood group binds the erythroid 4.1R protein: new insights into the 4.1R-dependent red cell membrane complex

    PubMed Central

    Azouzi, Slim; Collec, Emmanuel; Mohandas, Narla; An, Xiuli; Colin, Yves; Le Van Kim, Caroline

    2015-01-01

    Summary Protein 4.1R plays an important role in maintaining the mechanical properties of the erythrocyte membrane. We analysed the expression of Kell blood group protein in erythrocytes from a patient with hereditary elliptocytosis associated with complete 4.1R deficiency (4.1(−) HE). Flow cytometry and Western blot analyses revealed a severe reduction of Kell. In vitro pull down and co-immunoprecipitation experiments from erythrocyte membranes showed a direct interaction between Kell and 4.1R. Using different recombinant domains of 4.1R and the cytoplasmic domain of Kell, we demonstrated that the R46R motif in the juxta-membrane region of Kell binds to lobe B of the 4.1R FERM domain. We also observed that 4.1R deficiency is associated with a reduction of XK and DARC (also termed ACKR1) proteins, the absence of the glycosylated form of the urea transporter B and a slight decrease of band 3. The functional alteration of the 4.1(−) HE erythrocyte membranes was also determined by measuring various transport activities. We documented a slower rate of HCO3−/Cl− exchange, but normal water and ammonia transport across erythrocyte membrane in the absence of 4.1. These findings provide novel insights into the structural organization of blood group antigen proteins into the 4.1R complex of the human red cell membrane. PMID:26455906

  19. Monomeric red fluorescent proteins with a large Stokes shift.

    PubMed

    Piatkevich, Kiryl D; Hulit, James; Subach, Oksana M; Wu, Bin; Abdulla, Arian; Segall, Jeffrey E; Verkhusha, Vladislav V

    2010-03-23

    Two-photon microscopy has advanced fluorescence imaging of cellular processes in living animals. Fluorescent proteins in the blue-green wavelength range are widely used in two-photon microscopy; however, the use of red fluorescent proteins is limited by the low power output of Ti-Sapphire lasers above 1,000 nm. To overcome this limitation we have developed two red fluorescent proteins, LSS-mKate1 and LSS-mKate2, which possess large Stokes shifts with excitation/emission maxima at 463/624 and 460/605 nm, respectively. These LSS-mKates are characterized by high pH stability, photostability, rapid chromophore maturation, and monomeric behavior. They lack absorbance in the green region, providing an additional red color to the commonly used red fluorescent proteins. Substantial overlap between the two-photon excitation spectra of the LSS-mKates and blue-green fluorophores enables multicolor imaging using a single laser. We applied this approach to a mouse xenograft model of breast cancer to intravitally study the motility and Golgi-nucleus alignment of tumor cells as a function of their distance from blood vessels. Our data indicate that within 40 mum the breast cancer cells show significant polarization towards vessels in living mice.

  20. Osmotic properties of human red cells.

    PubMed

    Solomon, A K; Toon, M R; Dix, J A

    1986-01-01

    When an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of "nonosmotic water" which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol. 52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (ZHb). A number of investigators, particularly Freedman and Hoffman (1979, J. Gen. Physiol. 74:157) have been unable to confirm Gary-Bobo and Solomon's experimental evidence for this concentration dependence of ZHb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5 +/- 0.8% of the total isosmotic cell water (P much less than 0.0005, t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.

  1. Physico-Chemical Characterization of the Interaction of Red Fluorescent Protein-DsRed With Thin Silica Layers.

    PubMed

    Soumbo, Marvine; Pugliara, Alessandro; Monje, Marie-Carmen; Roques, Christine; Despax, Bernard; Bonafos, Caroline; Carles, Robert; Mlayah, Adnen; Makasheva, Kremena

    2016-07-01

    The Discosoma recombinant red fluorescent (DsRed) protein is the latest member of the family of fluorescent proteins. It holds great promise for applications in biotechnology and cell biology. However, before being used for rational engineering, knowledge on the behavior of DsRed and the underlying mechanisms relating its structural stability and adsorption properties on solid surfaces is highly demanded. The physico-chemical analysis performed in this study reveals that the interaction of DsRed with SiO2 surfaces does not lead to complete protein denaturation after adsorption and dehydration. Nevertheless, the photoluminescence emission of dehydrated DsRed small droplets was found to be slightly red-shifted, peaking at 590 nm. The measured contact angles of droplets containing different concentration of DsRed proteins determine the interaction as hydrophilic one, however with larger contact angles for larger DsRed concentrations. The DsRed protein behavior is not pH-dependent with respect of the contact angle measurements, in agreement with previously reported studies.

  2. Use of red fluorescent protein from Discosoma sp. (dsRED) as a reporter for plant gene expression.

    PubMed

    Jach, G; Binot, E; Frings, S; Luxa, K; Schell, J

    2001-11-01

    The suitability of the recently described red fluorescent protein dsRED from reef corals for use as a reporter in plant molecular biology was investigated. Based on the clone pDSRED (Clontech), plant expression vectors were constructed for constitutive dsRED expression in the cytosol, the endoplasmic reticulum and the vacuole. Fluorescence microscopy of tobacco BY2 suspension culture cells transiently expressing the plant vectors generated proved that cytosolic expression of the dsRED gives rise to readily detectable levels of red fluorescence, whereas expression in the ER was poor. Vacuolar dsRED expression did not result in any significant fluorescence. dsRED transgenic tobacco SR1 plants were generated to test the sensitivity of dsRED as a reporter in an autofluorescent background, and to identify the possible impact of the introduced fluorescent protein on morphogenesis, plant development and fertility. During the transformation and regeneration phase plants did not show any abnormalities, indicating that dsRED is not interfering with plant development and morphogenesis. Regenerated plants were analysed by PCR, Western blot and fluorescence microscopy for the presence and expression of the transferred genes. The filter sets chosen for fluorescence microscopy proved to be able to block the red chlorophyll fluorescence completely, allowing specific dsRED detection. Best expression levels were obtained with dsRED targeted to the cytosol or chloroplasts. ER-targeted expression of dsRED also gave rise to readily detectable fluorescence levels, whereas vacuolar expression yielded no fluorescence. dsRED transgenic plant lines expressing the protein in the cytosol, ER or chloroplast proved to be fertile. Seed set and germination were normal, except that the seeds and seedlings maintained the red fluorescence phenotype.

  3. The Use of Living Cancer Cells Expressing Green Fluorescent Protein in the Nucleus and Red Fluorescence Protein in the Cytoplasm for Real-time Confocal Imaging of Chromosome and Cytoplasmic Dynamics During Mitosis.

    PubMed

    Suetsugu, Atsushi; Jiang, Ping; Yang, Meng; Yamamoto, Norio; Moriwaki, Hisataka; Saji, Shigetoyo; Hoffman, Robert M

    2015-05-01

    A library of dual-color fluorescent cancer cells with green fluorescent protein (GFP), linked to histone H2B, expressed in the nucleus and red fluorescent protein (RFP) expressed in the cytoplasm was previously genetically engineered. The aim of the current study was to use the dual-color cancer cells to visualize chromosome and cytoplasmic dynamics during mitosis. Using an Olympus FV1000 confocal microscope, a library of dual-color cells from the major cancer types was cultured on plastic. The cells were imaged by confocal microscopy to demonstrate chromosome and cytoplasmic dynamics during mitosis. Nuclear GFP expression enabled visualization of chromosomes behavior, whereas simultaneous cytoplasmic RFP expression enabled visualization of cytoplasmic behavior during mitosis. Thus, total cellular dynamics can be visualized at high resolution, including individual chromosomes in some cases, in living dual-color cells in real time. Dual-color cancer cells expressing H2B-GFP in the nucleus and RFP in the cytoplasm provide unique tools for visualizing subcellular nuclear and cytoplasm dynamics, including the behavior of individual chromosomes during mitosis. The dual-color cells can be used to evaluate chromosomal loss or gain in real time during treatment with a variety of agents or as the cells are selected for increased or decreased malignancy in culture or in vivo. The dual color cells will be a useful tool to discover and evaluate novel strategies for killing cancer cells. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  4. Chemical toxicity of red cells.

    PubMed Central

    Piomelli, S

    1981-01-01

    Exposure to toxic chemicals may result in alterations of red cell function. In certain cases, the toxic effect requires a genetic predisposition and thus affects only a restricted number of individuals; in other instances, the toxic effect is exerted on the hematopoietic system of every person. Glucose-6-phosphate dehydrogenase deficiency is probably the most widespread genetic disorder. It is observed at highest frequency in populations from subtropical countries as a result of its selective advantage vis à vis falciparum malaria. The gene controlling this enzyme is located on the X-chromosome; thus, the defect is sex-linked. Individuals with a genetic defect of this enzyme are extremely susceptible to hemolysis, when exposed to oxidant drugs (such as certain antimalarials and sulfonamides) because of the inability of their red cells to regenerate NADPH. Lead poisoning result in profound effects on the process of heme synthesis. Among the steps most sensitive to lead toxicity are the enzyme delta-aminolevulinic acid dehydratase and the intramitochondrial step that leads to the incorporation of iron into protoporphyrin. By these mechanisms, in severe lead intoxication there is an accumulation of large amounts of delta-aminolevulinic acid (a compound with inherent neurotoxicity), and there are abnormalities of mitochondrial function in all cells of the body. Individuals living in an industrialized society are unavoidably exposed to some environmental lead. Recent evidence indicates that, even at levels of exposure which do not increase the blood lead level above values presently considered normal, abnormalities of heme synthesis are clearly detectable. PMID:7016524

  5. Red cell transfusion "trigger": a review.

    PubMed

    Petrides, Marian

    2003-07-01

    Despite the publication of several consensus guidelines that set forth recommendations for the transfusion of red cells, actual clinical practice continues to vary widely. Animal data and studies in human volunteers and patients support a red cell transfusion threshold of 7 to 8 g/dl in most patients. However, conflicting data, particularly in cardiac patients and in the elderly, suggest that it may be impossible to define a single red cell "trigger" for all patients. A well-designed, randomized, controlled trial is still needed to establish a safe threshold for red cell transfusion in adults with coronary artery disease.

  6. Endothelial Lu/BCAM glycoproteins are novel ligands for red blood cell alpha4beta1 integrin: role in adhesion of sickle red blood cells to endothelial cells.

    PubMed

    El Nemer, Wassim; Wautier, Marie-Paule; Rahuel, Cécile; Gane, Pierre; Hermand, Patricia; Galactéros, Frédéric; Wautier, Jean-Luc; Cartron, Jean-Pierre; Colin, Yves; Le Van Kim, Caroline

    2007-04-15

    The Lutheran (Lu) blood group and basal cell adhesion molecule (BCAM) antigens are both carried by 2 glycoprotein isoforms of the immunoglobulin superfamily representing receptors for the laminin alpha(5) chain. In addition to red blood cells, Lu/BCAM proteins are highly expressed in endothelial cells. Abnormal adhesion of red blood cells to the endothelium could potentially contribute to the vaso-occlusive episodes in sickle cell disease. Considering the presence of integrin consensus-binding sites in Lu/BCAM proteins, we investigated their potential interaction with integrin alpha(4)beta(1), the unique integrin expressed on immature circulating sickle red cells. Using cell adhesion assays under static and flow conditions, we demonstrated that integrin alpha(4)beta(1) expressed on transfected cells bound to chimeric Lu-Fc protein. We showed that epinephrine-stimulated sickle cells, but not control red cells, adhered to Lu-Fc via integrin alpha(4)beta(1) under flow conditions. Antibody-mediated activation of integrin alpha(4)beta(1) induced adhesion of sickle red cells to primary human umbilical vein endothelial cells; this adhesion was inhibited by soluble Lu-Fc and vascular cell adhesion molecule-1 (VCAM-1)-Fc proteins. This novel interaction between integrin alpha(4)beta(1) in sickle red cells and endothelial Lu/BCAM proteins could participate in sickle cell adhesion to endothelium and potentially play a role in vaso-occlusive episodes.

  7. Deoxygenation affects tyrosine phosphoproteome of red cell membrane from patients with sickle cell disease.

    PubMed

    Siciliano, Angela; Turrini, Franco; Bertoldi, Mariarita; Matte, Alessandro; Pantaleo, Antonella; Olivieri, Oliviero; De Franceschi, Lucia

    2010-04-15

    Sickle cell disease (SCD) is a worldwide distributed hereditary red cell disorder related to the production of a defective form of hemoglobin, hemoglobin S (HbS). One of the hallmarks of SCD is the presence of dense, dehydrate highly adhesive sickle red blood cells (RBCs) that result from persistent membrane damage associated with HbS polymerization, abnormal activation of membrane cation transports and generation of distorted and rigid red cells with membrane perturbation and cytoskeleton dysfunction. Although modulation of phosphorylation state of the proteins from membrane and cytoskeleton networks has been proposed to participate in red cell homeostasis, much still remains to be investigated in normal and diseased red cells. Here, we report that tyrosine (Tyr-) phosphoproteome of sickle red cells was different from normal controls and was affected by deoxygenation. We found proteins, p55 and band 4.1, from the junctional complex, differently Tyr-phosphorylated in SCD RBCs compared to normal RBCs under normoxia and modulated by deoxygenation, while band 4.2 was similarly Tyr-phosphorylated in both conditions. In SCD RBCs we identified the phosphopeptides for protein 4.1R located in the protein FERM domain (Tyr-13) and for alpha-spectrin located near or in a linker region (Tyr-422 and Tyr-1498) involving protein areas crucial for their functions in the context of red cell membrane properties, suggesting that Tyr-phosphorylation may be part of the events involved in maintaining membrane mechanical stability in SCD red cells.

  8. Modelling the structure of the red cell membrane.

    PubMed

    Burton, Nicholas M; Bruce, Lesley J

    2011-04-01

    The red cell membrane has long been the focus of extensive study. The macromolecules embedded within the membrane carry the blood group antigens and perform many functions including the vital task of gas exchange. Links between the intramembrane macromolecules and the underlying cytoskeleton stabilize the biconcave morphology of the red cell and allow deformation during microvascular transit. Much is now known about the proteins of the red cell membrane and how they are organised. In many cases we have an understanding of which proteins are expressed, the number of each protein per cell, their oligomeric state(s), and how they are collected in large multi-protein complexes. However, our typical view of these structures is as cartoon shapes in schematic figures. In this study we have combined knowledge of the red cell membrane with a wealth of protein structure data from crystallography, NMR, and homology modelling to generate the first, tentative models of the complexes which link the membrane to the cytoskeleton. Measurement of the size of these complexes and comparison with known cytoskeletal distance parameters suggests the idea of interaction between the membrane complexes, which may have profound implications for understanding red cell function and deformation.

  9. Red cell membrane response to hydrogen peroxide-sensitivity in hereditary xerocytosis and in other abnormal red cells.

    PubMed

    Snyder, L M; Sauberman, N; Condara, H; Dolan, J; Jacobs, J; Szymanski, I; Fortier, N L

    1981-07-01

    Osmotically resistant red cells associated with some haemolytic anaemias, including hereditary xerocytosis, sickle-cell disease and beta thalassaemia minor, are more sensitive than normal red cells to exogenous in vitro hydrogen peroxide (H2O2). This sensitivity is manifested by a rapid loss of intracellular potassium, shape change, protein aggregation, and methaemoglobin formation at lower concentrations of H2O2 (225 microM) than are required to induce similar effects in normal red cells (450 microM). Malonyldialdehyde (MDA) formation occurs later than the other effects and can be inhibited by the antioxidant, butylated hydroxytoluene (BHT), without affecting protein aggregation or potassium leak. Incubation of normal red cells directly with MDA induces protein aggregation, but only after 1 h of incubation. Although nystatin-sucrose treated normal cells which are dehydrated with altered cation content, and therefore osmotically resistant, do not display abnormal H2O2 hypersensitivity as manifested by excessive potassium permeability, they do show an increase in methaemoglobin formation and protein aggregation similar to xerocytes. These data indicate that membrane protein cross-linking occurring immediately following H2O2 exposure seems independent of either the sulfhydryl or MDA mechanisms, and that the membrane permeability of the abnormal red cells predisposes them to oxidative damage.

  10. Inflight Assay of Red Blood Cell Deformability

    NASA Technical Reports Server (NTRS)

    Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.

    1985-01-01

    Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

  11. Inflight Assay of Red Blood Cell Deformability

    NASA Technical Reports Server (NTRS)

    Ingram, M.; Paglia, D. E.; Eckstein, E. C.; Frazer, R. E.

    1985-01-01

    Studies on Soviet and American astronauts have demonstrated that red blood cell production is altered in response to low gravity (g) environment. This is associated with changes in individual red cells including increased mean cell volume and altered membrane deformability. During long orbital missions, there is a tendency for the red cell mass deficit to be at least partly corrected although the cell shape anomalies are not. Data currently available suggest that the observed decrease in red cell mass is the result of sudden suppression of erythropoieses and that the recovery trend observed during long missions reflects re-establishment of erythropoietic homeostasis at a "set point" for the red cell mass that is slightly below the normal level at 1 g.

  12. Conserved regions of the Plasmodium falciparum rhoptry-associated protein 3 mediate specific host-pathogen interactions during invasion of red blood cells.

    PubMed

    García, Jeison; Curtidor, Hernando; Vanegas, Magnolia; Arévalo-Pinzon, Gabriela; Patarroyo, Manuel A; Patarroyo, Manuel E

    2010-12-01

    Invasion of red blood cells (RBCs) by the Plasmodium falciparum malaria merozoite is mediated by parasite surface molecules and proteins contained within apical organelles that are capable of recognizing receptors on the membrane of RBCs. The identification and characterization of these P. falciparum invasion-associated proteins is the first step for unveiling potential new drug and vaccine target molecules to eradicate this deadly disease. Among the exclusive set of malarial vaccine candidates, the members of the rhoptry-associated protein (RAP) family have been associated with the parasite's binding to and invasion of RBCs. Remarkably, the third member of this family (named RAP-3) has been recently detected on the surface of non-infected RBCs exposed to free merozoites, therefore suggesting the participation of this protein during RBC infection. In this study, the sequence of RAP-3 was finely mapped using synthetic peptides in order to identify which are the specific binding regions involved in RAP3-RBC interactions. Two high-activity binding peptides (HABPs) established high affinity interactions with RBC surface molecules of about 27-90 kDa, which were differentially affected by different enzymatic treatments. RAP-1 and RAP-2 HABPs inhibited binding of RAP-3 HABPs to different extents, thus suggesting the recognition of similar binding sites on RBC membrane, as well as ability of RAP-3 HABPs to inhibit P. falciparum infection in vitro. Altogether, these functional analyses of RAP-3 HABPs strongly suggest a potential role for this protein in RBC invasion, and highlight its HABPs as potential targets to develop a fully protective minimal subunit-based malarial vaccine. Copyright © 2010 Elsevier Inc. All rights reserved.

  13. The human malaria parasite Plasmodium falciparum exports the ATP-binding cassette protein PFGCN20 to membrane structures in the host red blood cell.

    PubMed

    Bozdech, Z; VanWye, J; Haldar, K; Schurr, E

    1998-11-30

    PFGCN20 is a member of the ATP-binding cassette family of proteins that is closely related to the yeast translational regulator Gcn20p. We have generated a polyclonal antibody against the N-terminal region of PFGCN20 and studied the cellular localization of PFGCN20 throughout the erythrocytic life cycle of Plasmodium falciparum. PFGCN20 was found to be present at all stages and a pronounced export of PFGCN20 into the erythrocyte was observed in the trophozoite and schizont stages. In the indirect immunofluorescence assay, PFGCN20 was found to display significant colocalization with antigens detected by the monoclonal antibody 41E11. In contrast, there was only a minimal overlap of PFGCN20 localization with EMP2 and HRP2. Immunoelectron microscopy demonstrated a pronounced accumulation of PFGCN20 in the lumen of the parasitophorous vacuole and deconvolution fluorescence microscopy showed membrane association with selective regions of a tubovesicular network in the red cell. We also observed a concentration of PFGCN20 in electron-dense plaques just underneath the parasite's plasma membrane and an association of PFGCN20 with cytoplasmic vesicular structures within the parasite. The observed export of PFGCN20 and its association with the tubovesicular network in host red cells, may be indicative of the fact that PFGCN20 functions as ATP-binding subunit of an unknown multimeric ABC-transporter. The cytoplasmic localization of PFGCN20 in the parasite, however, suggests that the involvement of PFGCN20 in translational regulation or other cytoplasmic biological functions cannot be ruled out.

  14. Pure red cell hypoplasia secondary to isoniazid.

    PubMed Central

    Lewis, C. R.; Manoharan, A.

    1987-01-01

    We describe a 77 year old man who developed pure red cell aplasia while receiving antituberculous therapy including isoniazid. Prompt recovery occurred following cessation of isoniazid. In this paper we also review previously described case reports of isoniazid-induced pure red cell aplasia. PMID:3120168

  15. Generation of cloned transgenic cats expressing red fluorescence protein.

    PubMed

    Yin, Xi Jun; Lee, Hyo Sang; Yu, Xian Feng; Choi, Eugene; Koo, Bon Chul; Kwon, Mo Sun; Lee, Young S; Cho, Su Jin; Jin, Guang Zhen; Kim, Lyoung Hyo; Shin, Hyoung Doo; Kim, Teoan; Kim, Nam Hyung; Kong, Il Keun

    2008-03-01

    A method for engineering and producing genetically modified cats is important for generating biomedical models of human diseases. Here we describe the use of somatic cell nuclear transfer to produce cloned transgenic cats that systemically express red fluorescent protein. Immature oocytes were collected from superovulating cat ovaries. Donor fibroblasts were obtained from an ear skin biopsy of a white male Turkish Angora cat, cultured for one to two passages, and subjected to transduction with a retrovirus vector designed to transfer and express the red fluorescent protein (RFP) gene. A total of 176 RFP cloned embryos were transferred into 11 surrogate mothers (mean = 16 +/- 7.5 per recipient). Three surrogate mothers were successfully impregnated (27.3%) and delivered two liveborn and one stillborn kitten at 65 to 66 days of gestation. Analysis of nine feline-specific microsatellite loci confirmed that the cloned cats were genetically identical to the donor cat. Presence of the RFP gene in the transgenic cat genome was confirmed by PCR and Southern blot analyses. Whole-body red fluorescence was detected 60 days after birth in the liveborn transgenic (TG) cat but not in the surrogate mother cat. Red fluorescence was detected in tissue samples, including hair, muscle, brain, heart, liver, kidney, spleen, bronchus, lung, stomach, intestine, tongue, and even excrement of the stillborn TG cat. These results suggest that this nuclear transfer procedure using genetically modified somatic cells could be useful for the efficient production of transgenic cats.

  16. Red blood cell decreases of microgravity

    NASA Technical Reports Server (NTRS)

    Johnson, P. C.

    1985-01-01

    Postflight decreases in red blood cell mass (RBCM) have regularly been recorded after exposure to microgravity. These 5-25 percent decreases do not relate to the mission duration, workload, caloric intake or to the type of spacecraft used. The decrease is accompanied by normal red cell survivals, increased ferritin levels, normal radioactive iron studies, and increases in mean red blood cell volume. Comparable decreases in red blood cell mass are not found after bed rest, a commonly used simulation of the microgravity state. Inhibited bone marrow erythropoiesis has not been proven to date, although reticulocyte numbers in the peripheral circulation are decreased about 50 percent. To date, the cause of the microgravity induced decreases in RBCM is unknown. Increased splenic trapping of circulating red blood cells seem the most logical way to explain the results obtained.

  17. Red blood cell decreases of microgravity

    NASA Technical Reports Server (NTRS)

    Johnson, P. C.

    1985-01-01

    Postflight decreases in red blood cell mass (RBCM) have regularly been recorded after exposure to microgravity. These 5-25 percent decreases do not relate to the mission duration, workload, caloric intake or to the type of spacecraft used. The decrease is accompanied by normal red cell survivals, increased ferritin levels, normal radioactive iron studies, and increases in mean red blood cell volume. Comparable decreases in red blood cell mass are not found after bed rest, a commonly used simulation of the microgravity state. Inhibited bone marrow erythropoiesis has not been proven to date, although reticulocyte numbers in the peripheral circulation are decreased about 50 percent. To date, the cause of the microgravity induced decreases in RBCM is unknown. Increased splenic trapping of circulating red blood cells seem the most logical way to explain the results obtained.

  18. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  19. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  20. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  1. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  2. 21 CFR 640.10 - Red Blood Cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Red Blood Cells. 640.10 Section 640.10 Food and... ADDITIONAL STANDARDS FOR HUMAN BLOOD AND BLOOD PRODUCTS Red Blood Cells § 640.10 Red Blood Cells. The proper name of this product shall be Red Blood Cells. The product is defined as red blood cells...

  3. Abnormal Red Cell Structure and Function in Neuroacanthocytosis

    PubMed Central

    Cluitmans, Judith C. A.; Tomelleri, Carlo; Yapici, Zuhal; Dinkla, Sip; Bovee-Geurts, Petra; Chokkalingam, Venkatachalam; De Franceschi, Lucia; Brock, Roland; Bosman, Giel J. G. C. M.

    2015-01-01

    Background Panthothenate kinase-associated neurodegeneration (PKAN) belongs to a group of hereditary neurodegenerative disorders known as neuroacanthocytosis (NA). This genetically heterogeneous group of diseases is characterized by degeneration of neurons in the basal ganglia and by the presence of deformed red blood cells with thorny protrusions, acanthocytes, in the circulation. Objective The goal of our study is to elucidate the molecular mechanisms underlying this aberrant red cell morphology and the corresponding functional consequences. This could shed light on the etiology of the neurodegeneration. Methods We performed a qualitative and semi-quantitative morphological, immunofluorescent, biochemical and functional analysis of the red cells of several patients with PKAN and, for the first time, of the red cells of their family members. Results We show that the blood of patients with PKAN contains not only variable numbers of acanthocytes, but also a wide range of other misshapen red cells. Immunofluorescent and immunoblot analyses suggest an altered membrane organization, rather than quantitative changes in protein expression. Strikingly, these changes are not limited to the red blood cells of PKAN patients, but are also present in the red cells of heterozygous carriers without neurological problems. Furthermore, changes are not only present in acanthocytes, but also in other red cells, including discocytes. The patients’ cells, however, are more fragile, as observed in a spleen-mimicking device. Conclusion These morphological, molecular and functional characteristics of red cells in patients with PKAN and their family members offer new tools for diagnosis and present a window into the pathophysiology of neuroacanthocytosis. PMID:25933379

  4. Target analysis studies of red cell water and urea transport.

    PubMed

    Dix, J A; Ausiello, D A; Jung, C Y; Verkman, A S

    1985-12-05

    Radiation inactivation was used to determine the nature and molecular weight of water and urea transporters in the human red cell. Red cells were frozen to -50 degrees C in a cryoprotectant solution, irradiated with 1.5 MeV electrons, thawed, washed and assayed for osmotic water and urea permeability by stopped-flow light scattering. The freezing and thawing process did not affect the rates of water or urea transport or the inhibitory potency of p-chloromercuribenzenesulfonate (pCMBS) on water transport and of phloretin on urea transport. Red cell urea transport inactivated with radiation (0-4 Mrad) with a single target size of 469 +/- 36 kDa. 40 microM phloretin inhibited urea flux by approx. 50% at each radiation dose, indicating that urea transporters surviving radiation were inhibitable. Water transport did not inactivate with radiation; however, the inhibitory potency of 2.5 mM pCMBS decreased from 86 +/- 1% to 4 +/- 9% over a 0-2 Mrad dose range. These studies suggest that red cell water transport either required one or more low-molecular-weight proteins, or is lipid-mediated, and that the pCMBS-binding site which regulates water flow inactivates with radiation. These results also suggest that red cell urea transport is mediated by a specific, high-molecular-weight protein. These results do not support the hypothesis that a band 3 dimer (190 kDa) mediates red cell osmotic water and urea transport.

  5. Plasmodium falciparum rhoptry neck protein 5 peptides bind to human red blood cells and inhibit parasite invasion.

    PubMed

    Curtidor, Hernando; Patiño, Liliana C; Arévalo-Pinzón, Gabriela; Vanegas, Magnolia; Patarroyo, Manuel E; Patarroyo, Manuel A

    2014-03-01

    Plasmodium falciparum malaria parasite invasion of erythrocytes is an essential step in host infection and the proteins involved in such invasion are the main target in developing an antimalarial vaccine. Secretory organelle-derived proteins (micronemal AMA1 protein and the RON2, 4, and 5 rhoptry neck proteins) have been recently described as components of moving junction complex formation allowing merozoites to move into a newly created parasitophorous vacuole. This study led to identifying RON5 regions involved in binding to human erythrocytes by using a highly robust, sensitive and specific receptor-ligand interaction assay; it is further shown that the RON5 protein remains highly conserved throughout different parasite strains. It is shown that the binding peptide-erythrocyte interaction is saturable and sensitive to chymotrypsin and trypsin. Invasion inhibition assays using erythrocyte binding peptides showed that the RON5-erythrocyte interaction could be critical for merozoite invasion of erythrocytes. This work provides evidence (for the first time) suggesting a fundamental role for RON5 in erythrocyte invasion. Copyright © 2013 Elsevier Inc. All rights reserved.

  6. Uptake of carnitine by red blood cells

    SciTech Connect

    Campa, M.; Borum, P.

    1986-05-01

    A significant amount of blood carnitine (70% of cord blood and 40% of blood from healthy adults) is partitioned into the red blood cell compartment of whole blood. Data indicate that the plasma compartment and the red blood cell compartment of whole blood represent different metabolic pools of carnitine. There are no data to indicate that red blood cells synthesize carnitine, but our understanding of the uptake of carnitine by red blood cells is negligible. Red blood cells were obtained from healthy adults, washed twice with normal saline, and used for uptake experiments. When the cells were incubated at 37/sup 0/C in the presence of /sup 14/C-carnitine, radioactivity was found both in the soluble cytosolic and membrane fractions of the cells following lysis. The uptake was dependent upon the time of incubation, temperature of incubation, and carnitine concentration in the incubation medium. Washed red blood cell membranes incubated with /sup 14/C-carnitine showed specific binding of radioactivity. These data are consistent with the hypothesis that red blood cells have an uptake mechanism for L-carnitine.

  7. The accumulation of lipids and proteins during red blood cell storage: the roles of leucoreduction and experimental filtration

    PubMed Central

    Silliman, Christopher C.; Burke, Timothy; Kelher, Marguerite R.

    2017-01-01

    Pre-storage leucoreduction has been universally adopted in most developed countries in Asia, Europe and the Americas. It decreases febrile transfusion reactions, alloimmunisation to HLA antigens, cytomegalovirus exposure, the accumulation of a number of pro-inflammatory mediators in the supernatant, including the accumulation of platelet-and leucocyte-derived proteins and metabolites during routine storage. This review will highlight the lipids and proteins, biological response modifiers (BRMs) that accumulate, their clinical effects in transfused hosts, and methods of mitigation. PMID:28263170

  8. Human spleen and red blood cells

    NASA Astrophysics Data System (ADS)

    Pivkin, Igor; Peng, Zhangli; Karniadakis, George; Buffet, Pierre; Dao, Ming

    2016-11-01

    Spleen plays multiple roles in the human body. Among them is removal of old and altered red blood cells (RBCs), which is done by filtering cells through the endothelial slits, small micron-sized openings. There is currently no experimental technique available that allows us to observe RBC passage through the slits. It was previously noticed that people without a spleen have less deformable red blood cells, indicating that the spleen may play a role in defining the size and shape of red blood cells. We used detailed RBC model implemented within the Dissipative Particle Dynamics (DPD) simulation framework to study the filter function of the spleen. Our results demonstrate that spleen indeed plays major role in defining the size and shape of the healthy human red blood cells.

  9. Ultrafast Nonlinear Spectroscopy of Red Fluorescent Proteins

    NASA Astrophysics Data System (ADS)

    Konold, Patrick Eugene

    Red-emitting homologues (RFPs) of the native Green Fluorescent Protein (GFP) with emission wavelengths beyond 650 nm are desirable probes for in vivo imaging experiments. They offer the potential for deeper tissue penetration and lower background scatter given a cleaner spectral window. However, bioimaging applications are hindered by poor photophysics ( e.g. low fluorescence quantum yield, high photobleaching), which limits experimental resolution and represents a significant obstacle towards utilization for low copy-number, long-duration imaging applications. In this thesis, a variety of femtosecond nonlinear electronic spectroscopies were employed jointly with site-directed mutagenesis to investigate the photophysical properties of RFPs. In one study, the molecular mechanism of red emission was pursued in two notable RFPs, mPlum and TagRFP675. Solvation dynamics observed with time-resolved transient grating spectroscopy were interpreted with the aid of molecular dynamics simulations to indicate that their red-emission is correlated with the ability of specific chromophore-sidechain hydrogen-bonding interactions to interconvert between direct and water-mediated states. In a second set of studies, two-dimensional double quantum coherence spectroscopy was used to probe the electronic transitions of mPlum. It was discovered that it displayed a response distinctly different from an organic dye in bulk solvent. Modeling indicate of these spectra indicate the spectral features may be attributed to the existence of multiple high-lying (n>1) excited states. The results provide new insight into the electronic structure of these widely used fluorescent probes.

  10. Associations of obesity with triglycerides and C-reactive protein are attenuated in adults with high red blood cell eicosapentaenoic and docosahexaenoic acids

    USDA-ARS?s Scientific Manuscript database

    Background:N-3 fatty acids are associated with favorable, and obesity with unfavorable, concentrations of chronic disease risk biomarkers.Objective:We examined whether high eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid intakes, measured as percentages of total red blood cell (RBC) fatty acid...

  11. Quantification of Depletion-Induced Adhesion of Red Blood Cells

    NASA Astrophysics Data System (ADS)

    Steffen, P.; Verdier, C.; Wagner, C.

    2013-01-01

    Red blood cells (RBCs) are known to form aggregates in the form of rouleaux due to the presence of plasma proteins under physiological conditions. The formation of rouleaux can also be induced in vitro by the addition of macromolecules to the RBC suspension. Current data on the adhesion strength between red blood cells in their natural discocyte shapes mostly originate from indirect measurements such as flow chamber experiments, but data is lacking at the single cell level. Here, we present measurements on the dextran-induced aggregation of red blood cells using atomic force microscopy-based single cell force spectroscopy. The effects of dextran concentration and molecular weight on the interaction energy of adhering RBCs were determined. The results on adhesion energy are in excellent agreement with a model based on the depletion effect and previous experimental studies. Furthermore, our method allowed to determine the adhesion force, a quantity that is needed in theoretical investigations on blood flow.

  12. Quantification of depletion-induced adhesion of red blood cells.

    PubMed

    Steffen, P; Verdier, C; Wagner, C

    2013-01-04

    Red blood cells (RBCs) are known to form aggregates in the form of rouleaux due to the presence of plasma proteins under physiological conditions. The formation of rouleaux can also be induced in vitro by the addition of macromolecules to the RBC suspension. Current data on the adhesion strength between red blood cells in their natural discocyte shapes mostly originate from indirect measurements such as flow chamber experiments, but data is lacking at the single cell level. Here, we present measurements on the dextran-induced aggregation of red blood cells using atomic force microscopy-based single cell force spectroscopy. The effects of dextran concentration and molecular weight on the interaction energy of adhering RBCs were determined. The results on adhesion energy are in excellent agreement with a model based on the depletion effect and previous experimental studies. Furthermore, our method allowed to determine the adhesion force, a quantity that is needed in theoretical investigations on blood flow.

  13. Anatomy of the red cell membrane skeleton: unanswered questions.

    PubMed

    Lux, Samuel E

    2016-01-14

    The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3-containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

  14. Photoswitchable red fluorescent protein with a large Stokes shift

    PubMed Central

    Piatkevich, Kiryl D.; English, Brian P.; Malashkevich, Vladimir N.; Xiao, Hui; Almo, Steven C.; Singer, Robert H.; Verkhusha, Vladislav V.

    2014-01-01

    SUMMARY Subclass of fluorescent proteins, large Stokes shift fluorescent proteins, is characterized by their increased spread between the excitation and emission maxima. Here we report a photoswitchable variant of a red fluorescent protein with a large Stokes shift, PSLSSmKate, which initially exhibits excitation/emission at 445/622 nm, but irradiation with violet light photoswitches PSLSSmKate into a common red form with excitation/emission at 573/621 nm. We characterize spectral, photophysical and biochemical properties of PSLSSmKate in vitro and in mammalian cells, and determine its crystal structure in the large Stokes shift form. Mass-spectrometry, mutagenesis and spectroscopic analysis of PSLSSmKate allow us to propose molecular mechanisms for the large Stokes shift, pH dependence and light-induced chromophore transformation. We demonstrate applicability of PSLSSmKate to superresolution PALM microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects. PMID:25242289

  15. Freeze-Dried Human Red Blood Cells.

    DTIC Science & Technology

    1992-07-15

    instructed not to consume any alcohol -containing beverage during the period from seven days prior to phlebotomy to the completion of the follow-up...over the heart, spleen and liver at 4 hours and 24 hours post-infusion to determine the organ distribution of the radiolabeled reconstituted red blood...cells. 4. External probe counts taken over the heart, liver and spleen to determine the distribution of the lyophilized red blood cells at 4 hours and

  16. Autoimmune Lymphoproliferative Syndrome with Red Cell Aplasia.

    PubMed

    Meena, K R; Bisht, Supriya; Tamaria, K C

    2015-12-01

    Autoimmune Lymphoproliferative Syndrome (ALPS) is a rare inherited disorder of abnormal lymphocyte apoptosis, leading to chronic lymphoproliferation. It presents as lymphadenopathy, hepatosplenomegaly and autoimmune phenomena. Pure red cell aplasia is characterized by normochromic normocytic anemia, reticulocytopenia, and absence of erythroblasts from a normal bone marrow. Only few lymphoproliferative disorders have been associated with erythroid aplasia. The authors are reporting a case of ALPS associated with red cell aplasia in a 7-y-old girl.

  17. Diphenylhydantoin-induced pure red cell aplasia.

    PubMed

    Rusia, Usha; Malhotra, Purnima; Joshi, Panul

    2006-01-01

    Pure red cell aplasia is an uncommon complication of diphenylhydantoin therapy. It has not been reported in Indian literature. Awareness of the entity helps in establishing the cause of anaemia in these patients and alerts the physicians to the need of comprehensive haematological monitoring in these patients. A case of 58-year-old male who developed pure red cell aplasia following three months of diphenylhydantoin therapy is reported here.

  18. Home improvements: malaria and the red blood cell.

    PubMed

    Foley, M; Tilley, L

    1995-11-01

    In real-estate agent's terms, the red blood cell is a renovator's dream. The mature human erythrocyte has no internal organelles, no protein synthesis machinery and no infrastructure for protein trafficking. The malaria parasite invades this empty shell and effectively converts the erythrocyte back into a fully functional eukaryotic cell. In this article, Michael Foley and Leann Tilley examine the Plasmodium falciparum proteins that interact with the membrane skeleton at different stages of the infection and speculate on the roles of these proteins in the remodelling process.

  19. Genomic Typing of Red Cell Antigens

    DTIC Science & Technology

    2011-09-01

    Antigen‐Matched  Red  Cells   for  Sickle   Cell   Anemia  Patients  Using  Molecular Typing to Augment Testing: Meghan Delaney, Prashant Gaur, Askale...H, Constans J, Quilici JC, Lefevre‐Witier P, Sevin J, Stevens M: Study of red blood  cell  and serum enzymes in  five  Pyrenean communities and in a...Antigen‐Matched Red  Cells  for  Sickle   Cell  Anemia Patients  Using Molecular Typing to Augment Testing: AABB (poster) 2009.  Background: Patients with  sickle

  20. Phosphatidylserine exposure and red cell viability in red cell aging and in hemolytic anemia

    PubMed Central

    Boas, Franz Edward; Forman, Linda; Beutler, Ernest

    1998-01-01

    Phosphatidylserine (PS) normally localizes to the inner leaflet of cell membranes but becomes exposed in abnormal or apoptotic cells, signaling macrophages to ingest them. Along similar lines, it seemed possible that the removal of red cells from circulation because of normal aging or in hemolytic anemias might be triggered by PS exposure. To investigate the role of PS exposure in normal red cell aging, we used N-hydroxysuccinimide-biotin to tag rabbit red cells in vivo, then used phycoerythrin-streptavidin to label the biotinylated cells, and annexin V-fluorescein isothiocyanate (FITC) to detect the exposed PS. Flow cytometric analysis of these cells drawn at 10-day intervals up to 70 days after biotinylation indicated that older, biotinylated cells expose more PS. Furthermore, our data match a simple model of red cell senescence that assumes both an age-dependent destruction of senescent red cells preceded by several hours of PS exposure and a random destruction of red cells without PS exposure. By using this model, we demonstrated that the exposure of PS parallels the rate at which biotinylated red cells are removed from circulation. On the other hand, using an annexin V-FITC label and flow cytometry demonstrates that exposed PS does not cause the reduced red cell life span of patients with hemolytic anemia, with the possible exception of those with unstable hemoglobins or sickle cell anemia. Thus, in some cases PS exposure on the cell surface may signal the removal of red cells from circulation, but in other cases some other signal must trigger the sequestration of cells. PMID:9501218

  1. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for...

  2. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

  3. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for...

  4. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

  5. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

  6. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red cell indices device. 864.5300 Section 864.5300....5300 Red cell indices device. (a) Identification. A red cell indices device, usually part of a larger... corpuscular hemoglobin (MCH), and the mean corpuscular hemoglobin concentration (MCHC). The red cell indices...

  7. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for...

  8. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent. (a) Identification. A red cell lysing reagent is a device used to lyse (destroy) red blood cells for...

  9. Characterization of dsRed2-positive cells in the doublecortin-dsRed2 transgenic adult rat retina.

    PubMed

    Trost, A; Schroedl, F; Marschallinger, J; Rivera, F J; Bogner, B; Runge, C; Couillard-Despres, S; Aigner, L; Reitsamer, H A

    2014-12-01

    Doublecortin (DCX) is predominantly expressed in neuronal precursor cells and young immature neurons of the developing and adult brain, where it is involved in neuronal differentiation, migration and plasticity. Moreover, its expression pattern reflects neurogenesis, and transgenic DCX promoter-driven reporter models have been previously used to investigate adult neurogenesis. In this study, we characterize dsRed2 reporter protein-expressing cells in the adult retina of the transgenic DCX promoter-dsRed2 rat model, with the aim to identify cells with putative neurogenic activity. Additionally, we confirmed the expression of the dsRed2 protein in DCX-expressing cells in the adult hippocampal dentate gyrus. Adult DCX-dsRed2 rat retinas were analyzed by immunohistochemistry for expression of DCX, NF200, Brn3a, Sox2, NeuN, calbindin, calretinin, PKC-a, Otx2, ChAT, PSA-NCAM and the glial markers GFAP and CRALBP, followed by confocal laser-scanning microscopy. In addition, brain sections of transgenic rats were analyzed for dsRed2 expression and co-localization with DCX, NeuN, GFAP and Sox2 in the cortex and dentate gyrus. Endogenous DCX expression in the adult retina was confined to horizontal cells, and these cells co-expressed the DCX promoter-driven dsRed2 reporter protein. In addition, we encountered dsRed2 expression in various other cell types in the retina: retinal ganglion cells (RGCs), a subpopulation of amacrine cells, a minority of bipolar cells and in perivascular cells. Since also RGCs expressed dsRed2, the DCX-dsRed2 rat model might offer a useful tool to study RGCs in vivo under various conditions. Müller glial cells, which have previously been identified as cells with stem cell features and with neurogenic potential, did express neither endogenous DCX nor the dsRed2 reporter. However, and surprisingly, we identified a perivascular glial cell type expressing the dsRed2 reporter, enmeshed with the glia/stem cell marker GFAP and colocalizing with the

  10. Hereditary red cell membrane disorders and laboratory diagnostic testing.

    PubMed

    King, M-J; Zanella, A

    2013-06-01

    This overview describes two groups of nonimmune hereditary hemolytic anemias caused by defects in membrane proteins located in distinct layers of the red cell membrane. Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP) represent disorders of the red cell cytoskeleton. Hereditary stomatocytoses represents disorders of cation permeability in the red cell membrane. The current laboratory screening tests for HS are the osmotic fragility test, acid glycerol lysis time test (AGLT), cryohemolysis test, and eosin-5'-maleimide (EMA)-binding test. For atypical HS, SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins is carried out to confirm the diagnosis. The diagnosis of HE/HPP is based on abnormal red cell morphology and the detection of protein 4.1R deficiency or spectrin variants using gel electrophoresis. None of screening tests can detect all HS cases. Some testing centers (a survey of 25 laboratories) use a combination of tests (e.g., AGLT and EMA). No specific screening test for hereditary stomatocytoses is available. The preliminary diagnosis is based on presenting a compensated hemolytic anemia, macrocytosis, and a temperature or time dependent pseudohyperkalemia in some patients. Both the EMA-binding test and the osmotic fragility test may help in differential diagnosis of HS and hereditary stomatocytosis.

  11. Red blood cell storage and cell morphology.

    PubMed

    Blasi, B; D'Alessandro, A; Ramundo, N; Zolla, L

    2012-04-01

    In this study, we performed weekly assessment of morphology-related parameters through monitoring of CPD-SAGM leuco-filtered erythrocyte concentrates from blood withdrawal until the 42nd day of storage. Liquid storage of red blood cells (RBCs) delivers a blood-derived therapeutic, which is safe, available, effective and affordable for most patients who need transfusion therapy in developed countries. However, a growing body of accumulating controversial evidences, from either biochemical or retrospective clinical studies, prompted safety concerns about longer stored RBCs. Statistical image analysis through scanning electron microscope was coupled to osmotic fragility and erythrocyte sedimentation rate. We could observe that by day 21 more than 50% of RBCs displayed non-discocyte phenotypes. This observation was related to an increase in osmotic fragility, which was totally overlapped in day 0 controls and day 7 RBCs while only slightly augmented in day 14 samples. Cation dysregulation (pH internal/external alteration and potassium) might both reflect and trigger a negative feedback loop with metabolic fluxes and membrane cation pumps. Morphology parameters suggest that significant alterations to RBC morphology over storage duration occur soon after the 14th day of storage, as to become significant enough within the 21st day. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

  12. Polypeptide multilayer nanofilm artificial red blood cells.

    PubMed

    Palath, Naveen; Bhad, Sujaykumar; Montazeri, Reza; Guidry, Christopher A; Haynie, Donald T

    2007-04-01

    Reliable encapsulation of hemoglobin (Hb) within polypeptide multilayer nanofilms has been achieved by a template-based approach, and protein functionality has been demonstrated postencapsulation. The method is general in scope and could be useful for many other encapsulants. Met-Hb was adsorbed onto 5 microm-diameter CaCO3 microparticles, and the Hb-coated particles were encapsulated within a multilayer nanofilm of poly(L-glutamic acid) (PLGA) and poly(L-lysine) (PLL) by layer-by-layer assembly. The CaCO3 templates were then dissolved within the PLGA/PLL nanofilms by addition of ethylenediaminetetraacetic acid. Encapsulation of Hb was proved by fluorescence microscopy, the pH-dependence of retention of Hb was determined by visible wavelength absorbance, and conversion of the encapsulated met-Hb to deoxy-Hb and oxy-Hb was demonstrated by spectroscopic analysis of the Soret absorption peak under various conditions. It thus has been shown that control of Hb oxygenation within polypeptide multilayer nanofilm artificial cells is possible, and that Hb thus encapsulated can bind, release, and subsequently rebind molecular oxygen. This work therefore represents an advance in the development of polypeptide multilayer film artificial red blood cells. (c) 2006 Wiley Periodicals, Inc.

  13. Photoswitchable red fluorescent protein with a large Stokes shift.

    PubMed

    Piatkevich, Kiryl D; English, Brian P; Malashkevich, Vladimir N; Xiao, Hui; Almo, Steven C; Singer, Robert H; Verkhusha, Vladislav V

    2014-10-23

    A subclass of fluorescent proteins (FPs), large Stokes shift (LSS) FP, are characterized by increased spread between excitation and emission maxima. We report a photoswitchable variant of a red FP with an LSS, PSLSSmKate, which initially exhibits excitation and emission at 445 and 622 nm, but violet irradiation photoswitches PSLSSmKate into a common red form with excitation and emission at 573 and 621 nm. We characterize spectral, photophysical, and biochemical properties of PSLSSmKate in vitro and in mammalian cells and determine its crystal structure in the LSS form. Mass spectrometry, mutagenesis, and spectroscopy of PSLSSmKate allow us to propose molecular mechanisms for the LSS, pH dependence, and light-induced chromophore transformation. We demonstrate the applicability of PSLSSmKate to superresolution photoactivated localization microscopy and protein dynamics in live cells. Given its promising properties, we expect that PSLSSmKate-like phenotype will be further used for photoactivatable imaging and tracking multiple populations of intracellular objects.

  14. Interaction of ruthenium red with Ca2(+)-binding proteins

    SciTech Connect

    Charuk, J.H.; Pirraglia, C.A.; Reithmeier, R.A. )

    1990-07-01

    The interaction of ruthenium red, ((NH3)5Ru-O-Ru(NH3)4-O-Ru(NH3)5)Cl6.4H2O, with various Ca2(+)-binding proteins was studied. Ruthenium red inhibited Ca2+ binding to the sarcoplasmic reticulum protein, calsequestrin, immobilized on Sepharose 4B. Furthermore, ruthenium red bound to calsequestrin with high affinity (Kd = 0.7 microM; Bmax = 218 nmol/mg protein). The dye stained calsequestrin in sodium dodecyl sulfate-polyacrylamide gels or on nitrocellulose paper and was displaced by Ca2+ (Ki = 1.4 mM). The specificity of ruthenium red staining of several Ca2(+)-binding proteins was investigated by comparison with two other detection methods, 45Ca2+ autoradiography and the Stains-all reaction. Ruthenium red bound to the same proteins detected by the 45Ca2+ overlay technique. Ruthenium red stained both the erythrocyte Band 3 anion transporter and the Ca2(+)-ATPase of skeletal muscle sarcoplasmic reticulum. Ruthenium red also stained the EF hand conformation Ca2(+)-binding proteins, calmodulin, troponin C, and S-100. This inorganic dye provides a simple, rapid method for detecting various types of Ca2(+)-binding proteins following electrophoresis.

  15. Theory of non-Newtonian viscosity of red blood cell suspension: effect of red cell deformation.

    PubMed

    Murata, T

    1983-01-01

    The effects of the deformation of red blood cells on non-Newtonian viscosity of a concentrated red cell suspension are investigated theoretically. To simplify the problem an elastic spherical shell filled with an incompressible Newtonian fluid is considered as a model of a normal red cell. The equation of the surface of the shell suspended in a steady simple shear flow is calculated on the assumption that the deformation from a spherical shape is very small. The relative viscosity of a concentrated suspension of such particles is obtained based on the "free surface cell" method proposed by Happel. It is shown that the relative viscosity decreases as the shear rate increases.

  16. Red cell metabolism studies on Skylab

    NASA Technical Reports Server (NTRS)

    Mengel, C. E.

    1974-01-01

    On the basis of these background data, metabolic studies were performed on humans involved in space flight. These studies included the Skylab experiences. The primary purpose of the investigations was to study red cells for: (1) evidences of lipid peroxidation, or (2) changes at various points in the glycolytic pathway. The Skylab missions were an opportunity to study blood samples before, during, and after flight and to compare results with simultaneous controls. No direct evidence that lipid peroxidation had occurred in the red blood cells was apparent in the studies.

  17. Associations of obesity with triglycerides and C-reactive protein are attenuated in adults with high red blood cell eicosapentaenoic and docosahexaenoic acids

    PubMed Central

    Makhoul, Zeina; Kristal, Alan R.; Gulati, Roman; Luick, Bret; Bersamin, Andrea; O'Brien, Diane; Hopkins, Scarlett E.; Stephensen, Charles B.; Stanhope, Kimber L.; Havel, Peter J.; Boyer, Bert

    2011-01-01

    Background N-3 fatty acids are associated with favorable, and obesity with unfavorable, concentrations of chronic disease risk biomarkers. Objective We examined whether high eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid intakes, measured as percentages of total red blood cell (RBC) fatty acids, modify associations of obesity with chronic disease risk biomarkers. Methods In a cross-sectional study of 330 Yup'ik Eskimos, generalized additive models (GAM) and linear and quadratic regression models were used to examine associations of BMI with biomarkers across RBC EPA and DHA categories. Results Median (5th–95th percentile) RBC EPA and DHA were 2.6% (0.5–5.9%) and 7.3% (3.3–8.9%), respectively. In regression models, associations of BMI with triglycerides, glucose, insulin, C-reactive protein (CRP) and leptin differed significantly by RBC EPA and DHA. The GAM confirmed regression results for triglycerides and CRP: At low RBC EPA and RBC DHA, the predicted increases in triglycerides and CRP concentrations associated with a BMI increase from 25 to 35 were 99.5±45.3 mg/dl (106%) and 137.8±71.0 mg/dl (156%), respectively, for triglycerides and 1.2±0.7 mg/l (61%) and 0.8±1.0 mg/l (35%), respectively, for CRP. At high RBC EPA and RBC DHA, these predicted increases were 13.9±8.1 mg/dl (23%) and 12.0±12.3 mg/dl (18%), respectively, for triglycerides and 0.5±0.5 mg/l (50%) and −0.5±0.6 mg/l (−34%), respectively, for CRP. Conclusions In this population, high RBC EPA and DHA were associated with attenuated dyslipidemia and low-grade systemic inflammation among overweight and obese persons. This may help inform recommendations for n-3 fatty acid intakes in the reduction of obesity-related disease risk. PMID:21427737

  18. Adhesion of platelets to artificial surfaces: effect of red cells.

    PubMed

    Brash, J L; Brophy, J M; Feuerstein, I A

    1976-05-01

    Adhesion of platelets to several polymer- and protein-coated glass surfaces has been studied in vitro. The apparatus consists of a cylindrical probe rotating in a test tube containing the platelet medium and allows close control of fluid shear and mass transport. Suspensions of washed pig platelets constitute the basic platelet medium, and can be modified by adding back red cells and plasma proteins. Adhesion is measured via 51Cr-labeling of platelets. In the absence of red cells, identical low levels of adhesion were seen on all surfaces and saturation was reached within 2 min. In the presence of red cells, adhesion was greater. Saturation on all surfaces except fibrinogen and collagen again occurred within 2 min. The adhesion levels on polymer surfaces and glass were indistinguishable, while those on albumin were lower and those on fibrinogen were higher. Collagen was the most reactive surface. It did not equilibrate within 15 min., and kinetic data indicated a platelet diffusivity strongly dependent on hematocrit. These effects were attributed to rotational and translational motion of the red cells causing increased diffusion and surface-platelet collision energy.

  19. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  20. Using Green and Red Fluorescent Proteins to Teach Protein Expression, Purification, and Crystallization

    ERIC Educational Resources Information Center

    Wu, Yifeng; Zhou, Yangbin; Song, Jiaping; Hu, Xiaojian; Ding, Yu; Zhang, Zhihong

    2008-01-01

    We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or…

  1. Storage Lesion. Role of Red Cell Breakdown

    PubMed Central

    Kim-Shapiro, Daniel B.; Lee, Janet; Gladwin, Mark T.

    2011-01-01

    As stored blood ages intraerythrocytic energy sources are depleted resulting in reduced structural integrity of the membrane. Thus, stored red cells become less deformable and more fragile as they age. This fragility leads to release of cell-free hemoglobin and formation of microparticles, sub-micron hemoglobin-containing vesicles. Upon transfusion, it is likely that additional hemolysis and microparticle formation occurs due to breakdown of fragile red blood cells. Release of cell-free hemoglobin and microparticles leads to increased consumption of nitric oxide (NO), an important signaling molecule that modulates blood flow, and may promote inflammation. Stored blood may also be deficient in recently discovered blood nitric oxide synthase activity. We hypothesize that these factors play a potential role in the blood storage lesion. PMID:21496045

  2. Freeze-Dried Human Red Blood Cells

    DTIC Science & Technology

    1992-01-15

    outlined in our November progress report had been successfully used to repair damage incurred by RBC during hypotonic lysis ( referencesl and 2). The...reconstitution. Figures IA-IF show the osmotic deformability profiles of our thawed red cell preparation. Note the well defined hypotonic and hypertonic...upon rehydration. The osmotic deformability profiles of the reconstituted lyophilized cells are also nearly normal with well defined hypotonic and

  3. Red cell investigations: art and artefacts.

    PubMed

    Minetti, Giampaolo; Egée, Stephane; Mörsdorf, Daniel; Steffen, Patrick; Makhro, Asya; Achilli, Cesare; Ciana, Annarita; Wang, Jue; Bouyer, Guillaume; Bernhardt, Ingolf; Wagner, Christian; Thomas, Serge; Bogdanova, Anna; Kaestner, Lars

    2013-03-01

    Red blood cell research is important for both, the clinical haematology, such as transfusion medicine or anaemia investigations, and the basic research fields like exploring general membrane physiology or rheology. Investigations of red blood cells include a wide spectrum of methodologies ranging from population measurements with a billion cells evaluated simultaneously to single-cell approaches. All methods have a potential for pitfalls, and the comparison of data achieved by different technical approaches requires a consistent set of standards. Here, we give an overview of common mistakes using the most popular methodologies in red blood cell research and how to avoid them. Additionally, we propose a number of standards that we believe will allow for data comparison between the different techniques and different labs. We consider biochemical analysis, flux measurements, flow cytometry, patch-clamp measurements and dynamic fluorescence imaging as well as emerging single-cell techniques, such as the use of optical tweezers and atomic force microscopy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Challenges for red blood cell biomarker discovery through proteomics.

    PubMed

    Barasa, Benjamin; Slijper, Monique

    2014-05-01

    Red blood cells are rather unique body cells, since they have lost all organelles when mature, which results in lack of potential to replace proteins that have lost their function. They maintain only a few pathways for obtaining energy and reducing power for the key functions they need to fulfill. This makes RBCs highly sensitive to any aberration. If so, these RBCs are quickly removed from circulation, but if the RBC levels reduce extremely fast, this results in hemolytic anemia. Several causes of HA exist, and proteome analysis is the most straightforward way to obtain deeper insight into RBC functioning under the stress of disease. This should result in discovery of biomarkers, typical for each source of anemia. In this review, several challenges to generate in-depth RBC proteomes are described, like to obtain pure RBCs, to overcome the wide dynamic range in protein expression, and to establish which of the identified/quantified proteins are active in RBCs. The final challenge is to acquire and validate suited biomarkers unique for the changes that occur for each of the clinical questions; in red blood cell aging (also important for transfusion medicine), for thalassemias or sickle cell disease. Biomarkers for other hemolytic anemias that are caused by dysfunction of RBC membrane proteins (the RBC membrane defects) or RBC cytosolic proteins (the enzymopathies) are sometimes even harder to discover, in particular for the patients with RBC rare diseases with unknown cause. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge.

  5. Interferometric phase microscopy of red blood cells

    NASA Astrophysics Data System (ADS)

    Xue, Liang; Sun, Nan; Tang, Xian; Wang, Yin; Wang, Shouyu

    2013-12-01

    Quantitative phase imaging of cells with high accuracy in a completely noninvasive manner is a challenging task. To provide a proper solution to this important need, interferometric phase microscopy is described which relies on the off-axis interferometry, confocal microscopy and high-speed image capture technology. Phase retrieval from the single interferogram is done by algorithms based on the fast Fourier transform, traditional Hilbert transform and two-step Hilbert transform, respectively. Furthermore, a phase aberrations compensation approach is applied to correct the phase distribution of the red blood cells obtained via the three methods mentioned before without the pre-known knowledge for removing the wave front curvature introduced by the microscope objectives, off-axis imaging, etc., which otherwise hinders the phase reconstruction. The improved results reveal the better inner structures of the red blood cells. The development of quantitative phase imaging technique is shedding light on their future directions and applications for basic and clinical research.

  6. Coupled selection of protein solubility in E. coli using uroporphyrinogen III methyltransferase as red fluorescent reporter.

    PubMed

    Wang, Zhenzhen; Yan, Hanwei; Li, Si; Zhang, Kuanliang; Cheng, Beijiu; Fan, Jun

    2014-09-30

    Uroporphyrinogen III methyltransferase (UMT) is a novel reporter owing to the catalytic products accumulated in cells emitting red florescence. Overexpression of UMT confers resistance of the Escherichia coli cells to potassium tellurite that inhibits cell growth. In this study, we applied UMT reporter for monitoring protein solubility of MBP or TEV protease variants under different expression conditions, as well as 12 maize proteins with either the designed linker or N-terminal SUMO tag. Effects of five enzymes involved in heme and siroheme biosynthesis on the reporter were also investigated. With increasing concentrations of potassium tellurite, colony numbers of the mixed cells expressing the selected five proteins with different solubility were decreased, but colonies displaying red fluorescence was identified to be produced the protein with relatively high solubility. The developed UMT reporter system is sensitive for monitoring protein solubility based on coupled fluorescence and chemical selection. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Quenching of red cell tryptophan fluorescence by mercurial compounds.

    PubMed

    Verkman, A S; Lukacovic, M F; Tinklepaugh, M S; Dix, J A

    1986-01-01

    Intrinsic tryptophan fluorescence in red cell ghost membranes labeled with N-ethylmaleimide (N-EM) is quenched in a dose-dependent manner by the organic mercurial p-chloromercuribenzene sulfonate (p-CMBS). Fluorescence lifetime analysis shows that quenching occurs by a static mechanism. Binding of p-CMBS occurs by a rapid (less than 5 s) biomolecular association (dissociation constant K1 = 1.8 mM) followed by a slower unimolecular transition with forward rate constant k2 = 0.015 s-1 and reverse rate constant k-2 = 0.0054 s-1. Analysis of the temperature dependence of k2 gives delta H = 6.5 kcal/mol and delta S = -21 eu. The mercurial compounds p-chloromercuribenzoic acid, p-aminophenylmercuric acetate, and mercuric chloride quench red cell tryptophan fluorescence by the same mechanism as p-CMBS does; the measured k2 value was the same for each compound, whereas K1 varied. p-CMBS also quenches the tryptophan fluorescence in vesicles reconstituted with purified band 3, the red cell anion exchange protein, in a manner similar to that in ghost membranes. These experiments define a mercurial binding site on band 3 in ghosts treated with N-EM and establish the binding mechanism to this site. The characteristics of this p-CMBS binding site on band 3 differ significantly from those of the p-CMBS binding site involved in red cell water and urea transport inhibition.

  8. A General Mechanism of Photoconversion of Green-to-Red Fluorescent Proteins Based on Blue and Infrared Light Reduces Phototoxicity in Live-Cell Single-Molecule Imaging.

    PubMed

    Turkowyd, Bartosz; Balinovic, Alexander; Virant, David; Carnero, Haruko G Gölz; Caldana, Fabienne; Endesfelder, Marc; Bourgeois, Dominique; Endesfelder, Ulrike

    2017-09-11

    Photoconversion of fluorescent proteins by blue and complementary near-infrared light, termed primed conversion (PC), is a mechanism recently discovered for Dendra2. We demonstrate that controlling the conformation of arginine at residue 66 by threonine at residue 69 of fluorescent proteins from Anthozoan families (Dendra2, mMaple, Eos, mKikGR, pcDronpa protein families) represents a general route to facilitate PC. Mutations of alanine 159 or serine 173, which are known to influence chromophore flexibility and allow for reversible photoswitching, prevent PC. In addition, we report enhanced photoconversion for pcDronpa variants with asparagine 116. We demonstrate live-cell single-molecule imaging with reduced phototoxicity using PC and record trajectories of RNA polymerase in Escherichia coli cells. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments intended...

  10. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments intended...

  11. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments intended...

  12. 21 CFR 864.8185 - Calibrator for red cell and white cell counting.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Calibrator for red cell and white cell counting... Calibrator for red cell and white cell counting. (a) Identification. A calibrator for red cell and white cell counting is a device that resembles red or white blood cells and that is used to set instruments intended...

  13. Increased phorbol 12,13-dibutyrate (PDBu) receptor function associated with sickle red cell membrane ghosts

    SciTech Connect

    Ramachandran, M.; Nair, C.N.; Abraham, E.C.

    1987-05-01

    The biological receptor for tumor-promoting phorbol esters has been identified as the CaS /phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular CaS is allowed to rise. Since cellular CaS in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of TH-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attempts to assay the enzyme directly as phospholipid-activated TSP incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition.

  14. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 7 2011-04-01 2010-04-01 true Reagent Red Blood Cells. 660.30 Section 660.30 Food... ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be Reagent...

  15. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  16. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 7 2012-04-01 2012-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  17. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 7 2014-04-01 2014-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  18. 21 CFR 660.30 - Reagent Red Blood Cells.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 7 2013-04-01 2013-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall...

  19. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

  20. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

  1. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

  2. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Red blood cell enzyme assay. 864.7100 Section 864...) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood cell enzyme assay. (a) Identification. Red blood cell enzyme assay is a device used to measure the activity in...

  3. Red Cell Immunohematology Research Conducted in China.

    PubMed

    Zhu, Ziyan; Ye, Luyi; Li, Qin; Gao, Hongwei; Tan, Yinxia; Cai, Wei

    2017-04-01

    ABO subtypes and RhD variants are the most studied blood groups in China. Some of the polymorphisms in these two blood groups have direct clinical relevance. Molecular diagnosis of blood group polymorphisms is underway in China. In addition, research groups have developed methods such as screening for blood group mimetic peptides using phage display technology. New reagents, akin to antibodies directed against RhD and ABO, are being investigated using aptamer-based techniques. Progress is also being made in the development of synthetic exoglycosidases for conversion of group A and/or B antigens to group O. Development of methoxy-polyethylene-glycol modified red cells has been successful in vitro but has not reached clinical application. In this paper, we summarize red cell immunohematology research that has been conducted in China. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  4. Red cell distribution width and cancer

    PubMed Central

    Danese, Elisa

    2016-01-01

    Red cell distribution width (RDW) is an index which primarily reflects impaired erythropoiesis and abnormal red blood cell survival. In last years the interest in this marker has considerably grown and now a lot of data are available indicating that this simple and inexpensive parameter is a strong and independent risk factor for death in the general population. Moreover, several investigations have been performed to investigate the role of RDW in cardiovascular and thrombotic disorders. Contrarily, there are relatively few reports focusing on RDW in the area of oncology and to date none review have been performed in this specific field. As such, the aim of this narrative review is to summarize some interesting results obtained in studies performed in patients affected by solid and hematological tumors. Even if larger studies are needed before these preliminary findings can be generalized, it seems plausible to affirm that RDW can be useful by adding prognostic information in patients with oncologic disease. PMID:27867951

  5. Multiscale simulation of red blood cell aggregation

    NASA Astrophysics Data System (ADS)

    Bagchi, P.; Popel, A. S.

    2004-11-01

    In humans and other mammals, aggregation of red blood cells (RBC) is a major determinant to blood viscosity in microcirculation under physiological and pathological conditions. Elevated levels of aggregation are often related to cardiovascular diseases, bacterial infection, diabetes, and obesity. Aggregation is a multiscale phenomenon that is governed by the molecular bond formation between adjacent cells, morphological and rheological properties of the cells, and the motion of the extra-cellular fluid in which the cells circulate. We have developed a simulation technique using front tracking methods for multiple fluids that includes the multiscale characteristics of aggregation. We will report the first-ever direct computer simulation of aggregation of deformable cells in shear flows. We will present results on the effect of shear rate, strength of the cross-bridging bonds, and the cell rheological properties on the rolling motion, deformation and subsequent breakage of an aggregate.

  6. Freeze-Dried Human Red Blood Cells

    DTIC Science & Technology

    1992-04-15

    chromium labelled mRBC via an intravenous catheter in the peripheral saphenous vein . Following administration of labelled mRBC into each monkey, 0.2ml...with a single dose of autologous , lyophilized and reconstituted, 5 1Cr-labelled packed erythrocytes via an intravenous catheter in a peripheral vein ...study site, at which time a 25 ml aliquot of reconstituted 51-Cr labeled autologous red blood cells will be infused intravenously into a large arm vein

  7. From Red Cells to Soft Porous Lubrication

    NASA Astrophysics Data System (ADS)

    Wu, Qianhong; Gacka, Thomas; Nathan, Rungun; Crawford, Robert; Vucbmss Team

    2014-11-01

    Biological scientists have wondered, since the motion of red cells was first observed in capillaries, how the highly flexible red cell can move with so little friction in tightly fitting microvessels without being damaged or undergoing hemolysis. Theoretical studies (Feng and Weinbaum, 2000, JFM; Wu et al., 2004, PRL) attributed this frictionless motion to the dramatically enhanced hydrodynamic lifting force generated inside the soft, porous, endothelial surface layer (ESL) covering the inner surfaces of our capillaries, as a red blood cell glides over it. Herein we report the first experimental examination of this concept. The results conclusively demonstrate that significant fraction of the overall lifting force generated in a soft porous layer as a planing surface glides over it, is contributed by the pore fluid pressure, and thus frictional loss is reduced significantly. Moreover, the experimental predictions showed excellent agreement with the experimental data. This finding has the potential of dramatically changing existing lubrication approaches, and can result in substantial savings in energy consumption and thus reduction in greenhouse gas emissions.

  8. Red blood cell transfusion in newborn infants

    PubMed Central

    Whyte, Robin K; Jefferies, Ann L

    2014-01-01

    Red blood cell transfusion is an important and frequent component of neonatal intensive care. The present position statement addresses the methods and indications for red blood cell transfusion of the newborn, based on a review of the current literature. The most frequent indications for blood transfusion in the newborn are the acute treatment of perinatal hemorrhagic shock and the recurrent correction of anemia of prematurity. Perinatal hemorrhagic shock requires immediate treatment with large quantities of red blood cells; the effects of massive transfusion on other blood components must be considered. Some guidelines are now available from clinical trials investigating transfusion in anemia of prematurity; however, considerable uncertainty remains. There is weak evidence that cognitive impairment may be more severe at follow-up in extremely low birth weight infants transfused at lower hemoglobin thresholds; therefore, these thresholds should be maintained by transfusion therapy. Although the risks of transfusion have declined considerably in recent years, they can be minimized further by carefully restricting neonatal blood sampling. PMID:24855419

  9. Glycolate kinase activity in human red cells.

    PubMed

    Fujii, S; Beutler, E

    1985-02-01

    Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

  10. Red Blood Cell Polymorphism and Susceptibility to Plasmodium vivax

    PubMed Central

    Zimmerman, Peter A.; Ferreira, Marcelo U.; Howes, Rosalind E.; Mercereau-Puijalon, Odile

    2013-01-01

    Resistance to Plasmodium vivax blood-stage infection has been widely recognised to result from absence of the Duffy (Fy) blood group from the surface of red blood cells (RBCs) in individuals of African descent. Interestingly, recent studies from different malaria-endemic regions have begun to reveal new perspectives on the association between Duffy gene polymorphism and P. vivax malaria. In Papua New Guinea and the Americas, heterozygous carriers of a Duffy-negative allele are less susceptible to P. vivax infection than Duffy-positive homozygotes. In Brazil, studies show that the Fya antigen, compared to Fyb, is associated with lower binding to the P. vivax Duffy-binding protein and reduced susceptibility to vivax malaria. Additionally, it is interesting that numerous studies have now shown that P. vivax can infect RBCs and cause clinical disease in Duffy-negative people. This suggests that the relationship between P. vivax and the Duffy antigen is more complex than customarily described. Evidence of P. vivax Duffy-independent red cell invasion indicates that the parasite must be evolving alternative red cell invasion pathways. In this chapter, we review the evidence for P. vivax Duffy-dependent and Duffy-independent red cell invasion. We also consider the influence of further host gene polymorphism associated with malaria endemicity on susceptibility to vivax malaria. The interaction between the parasite and the RBC has significant potential to influence the effectiveness of P. vivax-specific vaccines and drug treatments. Ultimately, the relationships between red cell polymorphisms and P. vivax blood-stage infection will influence our estimates on the population at risk and efforts to eliminate vivax malaria. PMID:23384621

  11. Guide to Red Fluorescent Proteins and Biosensors for Flow Cytometry

    PubMed Central

    Piatkevich, Kiryl D.; Verkhusha, Vladislav V.

    2014-01-01

    Since the discovery of the first red fluorescent protein (RFP), named DsRed, 12 years ago, a wide pallet of red-shifted fluorescent proteins has been cloned and biotechnologically developed into monomeric fluorescent probes for optical microscopy. Several new types of monomeric RFPs that change the emission wavelength either with time, called fluorescent timers, or after a brief irradiation with violet light, known as photoactivatable proteins, have been also engineered. Moreover, RFPs with a large Stokes shift of fluorescence emission have been recently designed. Because of their distinctive excitation and fluorescence detection conditions developed specifically for microscopy, these fluorescent probes can be suboptimal for flow cytometry. Here, we have selected and summarized the advanced orange, red, and far-red fluorescent proteins with the properties specifically required for the flow cytometry applications. Their effective brightness was calculated for the laser sources available for the commercial flow cytometers and sorters. Compatibility of the fluorescent proteins of different colors in a multiparameter flow cytometry was determined. Novel FRET pairs, utilizing RFPs, RFP-based intracellular biosensors, and their application to a high-throughput screening, are also discussed. PMID:21704849

  12. THE SPECIFIC HEAT OF RED CELLS, WITH SPECIAL REFERENCE TO THE PARACRYSTALLINE RAT RED CELL

    PubMed Central

    Ponder, Eric

    1953-01-01

    The specific heat of the rat red cell, kept in cold sodium citrate, changes in the neighborhood of 6°C., the temperature near which the cell passes from its paracrystalline state to a state of greater disorder. The change in the specific heat is from 0.74 with a standard deviation of ±0.022 (paracrystalline state) to 0.87 with a standard deviation of ±0.021 (normal state). Although it has been looked for, no evidence of a change in specific heat has been found, between 1°C. and 15°C., in the case of the human red cell or of the fresh rat red cell in saline or plasma. PMID:13035066

  13. Osmotic water permeability of human red cells

    PubMed Central

    1981-01-01

    The osmotic water permeability of human red cells has been reexamined with a stopped-flow device and a new perturbation technique. Small osmotic gradients are used to minimize the systematic error caused by nonlinearities in the relationship between cell volume and light scattering. Corrections are then made for residual systematic error. Our results show that the hydraulic conductivity, Lp, is essentially independent of the direction of water flow and of osmolality in the range 184-365 mosM. the mean value of Lp obtained obtained was 1.8 +/- 0.1 (SEM) X 10-11 cm3 dyne -1 s-1. PMID:7229611

  14. Red Blood Cell Membrane-Cloaked Nanoparticles For Drug Delivery

    NASA Astrophysics Data System (ADS)

    Carpenter, Cody Westcott

    Herein we describe the development of the Red Blood Cell coated nanoparticle, RBC-NP. Purified natural erythrocyte membrane is used to coat drug-loaded poly(lacticco-glycolic acid) (PLGA). Synthetic PLGA co-polymer is biocompatible and biodegradable and has already received US FDA approval for drug-delivery and diagnostics. This work looks specifically at the retention of immunosuppressive proteins on RBC-NPs, right-sidedness of natural RBC membranes interfacing with synthetic polymer nanoparticles, sustained and retarded drug release of RBC-NPs as well as further surface modification of RBC-NPs for increased targeting of model cancer cell lines.

  15. Monoclonal gammopathy-associated pure red cell aplasia.

    PubMed

    Korde, Neha; Zhang, Yong; Loeliger, Kelsey; Poon, Andrea; Simakova, Olga; Zingone, Adriana; Costello, Rene; Childs, Richard; Noel, Pierre; Silver, Samuel; Kwok, Mary; Mo, Clifton; Young, Neal; Landgren, Ola; Sloand, Elaine; Maric, Irina

    2016-06-01

    Pure red cell aplasia (PRCA) is a rare disorder characterized by inhibition of erythroid precursors in the bone marrow and normochromic, normocytic anaemia with reticulocytopenia. Among 51 PRCA patients, we identified 12 (24%) patients having monoclonal gammopathy, monoclonal gammopathy of undetermined significance or smouldering multiple myeloma, with presence of monoclonal protein or abnormal serum free light chains and atypical bone marrow features of clonal plasmacytosis, hypercellularity and fibrosis. Thus far, three patients treated with anti-myeloma based therapeutics have responded with reticulocyte recovery and clinical transfusion independence, suggesting plasma cells play a key role in the pathogenesis of this specific monoclonal gammopathy-associated PRCA.

  16. Two-plasmid vector system for independently controlled expression of green and red fluorescent fusion proteins in Staphylococcus aureus.

    PubMed

    Brzoska, Anthony J; Firth, Neville

    2013-05-01

    We have constructed a system for the regulated coexpression of green fluorescent protein (GFP) and red fluorescent protein (RFP) fusions in Staphylococcus aureus. It was validated by simultaneous localization of cell division proteins FtsZ and Noc and used to detect filament formation by an actin-like ParM plasmid partitioning protein in its native coccoid host.

  17. Neocytolysis: physiological down-regulator of red-cell mass

    NASA Technical Reports Server (NTRS)

    Alfrey, C. P.; Rice, L.; Udden, M. M.; Driscoll, T. B.

    1997-01-01

    It is usually considered that red-cell mass is controlled by erythropoietin-driven bone marrow red-cell production, and no physiological mechanisms can shorten survival of circulating red cells. In adapting to acute plethora in microgravity, astronauts' red-cell mass falls too rapidly to be explained by diminished red-cell production. Ferrokinetics show no early decline in erythropolesis, but red cells radiolabelled 12 days before launch survive normally. Selective destruction of the youngest circulating red cells-a process we call neocytolysis-is the only plausible explanation. A fall in erythropoietin below a threshold is likely to initiate neocytolysis, probably by influencing surface-adhesion molecules. Recognition of neocytolysis will require re-examination of the pathophysiology and treatment of several blood disorders, including the anaemia of renal disease.

  18. Neocytolysis: physiological down-regulator of red-cell mass

    NASA Technical Reports Server (NTRS)

    Alfrey, C. P.; Rice, L.; Udden, M. M.; Driscoll, T. B.

    1997-01-01

    It is usually considered that red-cell mass is controlled by erythropoietin-driven bone marrow red-cell production, and no physiological mechanisms can shorten survival of circulating red cells. In adapting to acute plethora in microgravity, astronauts' red-cell mass falls too rapidly to be explained by diminished red-cell production. Ferrokinetics show no early decline in erythropolesis, but red cells radiolabelled 12 days before launch survive normally. Selective destruction of the youngest circulating red cells-a process we call neocytolysis-is the only plausible explanation. A fall in erythropoietin below a threshold is likely to initiate neocytolysis, probably by influencing surface-adhesion molecules. Recognition of neocytolysis will require re-examination of the pathophysiology and treatment of several blood disorders, including the anaemia of renal disease.

  19. Freeze-Dried Human Red Blood Cells

    DTIC Science & Technology

    1991-11-08

    which we thought may improve the resealing and phospholipid packing of the cell membranes. Neither treatment appeared to significantly improve the cell...cells were experiencing subtle membrane damage. This hypothesis was based on our model of transient lysis- resealing of the cells during the shock of...appreciable rates of chemical reactions. This theory has already found general application in the food and pharmaceuticals (i.e., lyophilized protein

  20. Protein concentration by precipitation with pyrogallol red prior to electrophoresis.

    PubMed

    Marshall, T; Abbott, N J; Fox, P; Williams, K M

    1995-01-01

    The pyrogallol red protein assay (Clinical Chemistry 1986, 32, 1551-1554) is based upon formation of a blue protein-dye complex in the presence of molybdate under acidic conditions. However, centrifugation of the assay mixture results in loss of color yield and precipitation of the protein-dye complex which can be recovered and resolubilized to achieve protein concentration prior to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The method has been evaluated relative to trichloroacetic acid (TCA) precipitation for recovery and electrophoresis of commercial protein and peptide molecular weight markers. Precipitation with pyrogallol red-molybdate (PRM) gives better and more uniform recovery of both proteins and peptides as compared to TCA. The lower limit of PRM precipitation is similar to TCA and corresponds to 1 microgram protein per mL assay mixture. This is equivalent to 100 microL of 10 micrograms/mL protein using the standard protein assay or 1 microgram/mL protein using a modified assay incorporating a fivefold concentrate of the dye reagent. Application of the method is demonstrated by concentration of urinary proteins. The method is simple and economic and useful for conserving trace amounts of precious sample as it allows recovery of protein for electrophoresis following protein assay.

  1. Metabolic profiling of hematopoietic stem and progenitor cells during proliferation and differentiation into red blood cells.

    PubMed

    Daud, Hasbullah; Browne, Susan; Al-Majmaie, Rasoul; Murphy, William; Al-Rubeai, Mohamed

    2016-01-25

    An understanding of the metabolic profile of cell proliferation and differentiation should support the optimization of culture conditions for hematopoietic stem and progenitor cell (HSPC) proliferation, differentiation, and maturation into red blood cells. We have evaluated the key metabolic parameters during each phase of HSPC culture for red blood cell production in serum-supplemented (SS) and serum-free (SF) conditions. A simultaneous decrease in growth rate, total protein content, cell size, and the percentage of cells in the S/G2 phase of cell cycle, as well as an increase in the percentage of cells with a CD71(-)/GpA(+) surface marker profile, indicates HSPC differentiation into red blood cells. Compared with proliferating HSPCs, differentiating HSPCs showed significantly lower glucose and glutamine consumption rates, lactate and ammonia production rates, and amino acid consumption and production rates in both SS and SF conditions. Furthermore, extracellular acidification was associated with late proliferation phase, suggesting a reduced cellular metabolic rate during the transition from proliferation to differentiation. Under both SS and SF conditions, cells demonstrated a high metabolic rate with a mixed metabolism of both glycolysis and oxidative phosphorylation (OXPHOS) in early and late proliferation, an increased dependence on OXPHOS activity during differentiation, and a shift to glycolytic metabolism only during maturation phase. These changes indicate that cell metabolism may have an important impact on the ability of HSPCs to proliferate and differentiate into red blood cells.

  2. Role molecular signaling pathways in changes of red blood cell deformability.

    PubMed

    Muravyov, Alexei V; Tikhomirova, Irina A

    2013-01-01

    This study was designed to investigate the dependency of the red blood cell deformability upon activation of extra- and intracellular signaling pathways. Exposures of red blood cells (RBCs) to catecholamines and to insulin led to positive change in the RBC deformability. When forskolin, a stimulator of adenylyl cyclase (AC), was added to RBC suspension, the RBC deformability was increased. Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. The inhibitors of phosphodiesterase (PDE) activity increased red cell deformability. These results revealed a considerable role of the AC-cAMP signaling system in the regulation of red blood cell deformability. The rise of the red blood cell Ca(2+) influx, stimulated by mechanical loading or A23187 was accompanied by a marked lowering of RBC deformability. At the same time blocking of Ca(2+) entry into RBC by verapamil or Ca(2+) chelating by EGTA led to significant deformability rise. The comparison of the effect of the different protein kinases on the red blood cell deformability showed that it was altered more considerable under PKA activation by forskolin or dB-cAMP than by other protein kinases. There was a lesser but quite statistically significant effect of tyrosine protein kinase (TPK) on RBC microrheology. Whereas the microrheological effect of PKC was not so considerable. The problem of the short-term regulation of red blood cell microrheology is examined. The latter includes: the modes of activation of extra- and intracellular molecular signaling pathways, ligand - receptor interaction, second messengers, membrane protein phosphorylation. On the whole the total data clearly show that the red cell deformability changes are connected with activation of different extra - and intracellular signaling pathways. It seems reasonable to suppose that red blood cell deformability changes were mainly associated with activation of the AC-cAMP-PKA pathway, and with decrease of Ca(2+) entry into

  3. Spectroscopy detection of green and red fluorescent proteins in genetically modified plants using a fiber optics system

    NASA Astrophysics Data System (ADS)

    Liew, Oi Wah; Asundi, Anand K.; Chen, Jun-Wei; Chew, Yiwen; Yu, Shangjuan; Yeo, Gare H.

    2001-05-01

    In this paper, fiber optic spectroscopy is developed to detect and quantify recombinant green (EGFP) and red (DsRED) fluorescent proteins in vitro and in vivo. The bacterial expression vectors carrying the coding regions of EGFP and DsRED were introduced into Escherichia coli host cells and fluorescent proteins were produced following induction with IPTG. Soluble EGFP and DsRED proteins were isolated from lysed bacterial cells and serially diluted for quantitative analysis by fiber optic spectroscopy. Fluorescence at the appropriate emission wavelengths could be detected up to 64X dilution for EGFP and 40X dilution for DsRED. To determine the capability of spectroscopy detection in vivo, transgenic potato hairy roots expressing EGFP and DsRED were regenerated. This was achieved by cloning the EGFP and DsRED genes into the plant binary vector, pTMV35S, to create the recombinant vectors pGLOWGreen and pGLOWRed. These latter binary vectors were introduced into Agrobacterium rhizogenes strain A4T. Infection of potato cells with transformed agrobacteria was used to insert the fluorescent protein genes into the potato genome. Genetically modified potato cells were then regenerated into hairy roots. A panel of transformed hairy roots expressing varying levels of fluorescent proteins was selected by fluorescence microscopy. We are now assessing the capability of spectroscopic detection system for in vivo quantification of green and red fluorescence levels in transformed roots.

  4. Cryopreserved red blood cells are superior to standard liquid red blood cells.

    PubMed

    Hampton, David A; Wiles, Connor; Fabricant, Loïc J; Kiraly, Laszlo; Differding, Jerome; Underwood, Samantha; Le, Dinh; Watters, Jennifer; Schreiber, Martin A

    2014-07-01

    Liquid preserved packed red blood cell (LPRBC) transfusions are used to treat anemia and increase end-organ perfusion. Throughout their storage duration, LPRBCs undergo biochemical and structural changes collectively known as the storage lesion. These changes adversely affect perfusion and oxygen off-loading. Cryopreserved RBCs (CPRBC) can be stored for up to 10 years and potentially minimize the associated storage lesion. We hypothesized that CPRBCs maintain a superior biochemical profile compared with LPRBCs. This was a prospective, randomized, double-blinded study. Adult trauma patients with an Injury Severity Score (ISS) greater than 4 and an anticipated 1-U to 2-U transfusion of PRBCs were eligible. Enrolled patients were randomized to receive either CPRBCs or LPRBCs. Serum proteins (haptoglobin, serum amyloid P, and C-reactive protein), proinflammatory and anti-inflammatory cytokines, d-dimer, nitric oxide, and 2,3-DPG concentrations were analyzed. Mann-Whitney U-test and Wilcoxon rank sum test were used to assess significance (p < 0.05). Fifty-seven patients were enrolled (CPRBC, n = 22; LPRBC, n = 35). The LPRBC group's final interleukin 8, tumor necrosis factor α, and d-dimer concentrations were elevated compared with their pretransfusion values (p < 0.05). After the second transfused units, 2,3-DPG was higher in the patients receiving CPRBCs (p < 0.05); this difference persisted throughout the study. Finally, serum protein concentrations were decreased in the transfused CPRBC units compared with LPRBC (p < 0.01). CPRBC transfusions have a superior biochemical profile: an absent inflammatory response, attenuated fibrinolytic state, and increased 2,3-DPG. A blood banking system using both storage techniques will offer the highest-quality products to critically injured patients virtually independent of periodic changes in donor availability and transfusion needs. Therapeutic study, level II.

  5. Impact of glycocalyx structure on red cell-red cell affinity in polymer suspensions.

    PubMed

    Rad, Samar; Meiselman, Herbert J; Neu, Björn

    2014-11-01

    A theoretical framework based on macromolecular depletion has been utilized in order to examine the energetics of red blood cell interactions. Three different glycocalyx structures are considered and cell-cell affinities are calculated by superposition of depletion, steric and electrostatic interactions. The theoretical model predicts a non-monotonic dependence of the interaction energies on polymer size. Further, our results indicate that the glycocalyx segment distribution has a large impact on adhesion energies between cells: a linear segment distribution induces the strongest adhesion between cells followed by pseudo-tail and uniform distributions. Our approach confirms the concept of a depletion mechanism for RBC aggregation, and also provides new insights that may eventually help to understand and quantify cellular factors that control red blood cell interactions in health and disease.

  6. [Genetically encoded FRET-pair on the basis of terbium-binding peptide and red fluorescent protein].

    PubMed

    Arslanbaeva, L R; Zherdeva, V V; Ivashina, T V; Vinokurov, L M; Rusanov, A L; Savitskiĭ, A P

    2010-01-01

    The genetically encoded FRET-pair was developed on the basis of terbium-binding peptide and red fluorescent protein DsRed2. To study fluorescence resonance energy transfer within FRET-pair, the gene-engineered construction was obtained, where sequences of terbium-binding peptide and red fluorescent protein DsRed2 were fused in single reading frame. The expression of this construction in strain E. coli BL21 (DE3) was studied and conditions of synthesis, isolation and purification of recombinant protein were optimized. The hydrodynamic radius of hybrid protein was determined by the method of dynamic light scattering. Energy transfer between sensitized terbium and red fluorescent protein was confirmed by the methods of phosporescent spectroscopy. The obtained FRET-pair can be used both for studies in vitro and as a reporter in living cells.

  7. High protein-high red meat versus high carbohydrate weight loss diets do not differ in effect on genome stability and cell death in lymphocytes of overweight men.

    PubMed

    Benassi-Evans, Bianca; Clifton, Peter M; Noakes, Manny; Keogh, Jennifer B; Fenech, Michael

    2009-05-01

    The importance of diet in DNA damage prevention is well established; however, the comparison of weight loss diets with different micronutrient and macronutrient profiles on genome stability in peripheral blood lymphocytes (PBLs) has not been studied. This study tested the hypothesis that genome stability in PBLs of overweight men who consume a high protein-high red meat (HP) weight loss diet is different from that of overweight men who consume a high carbohydrate (HC) weight loss diet. Thirty-three male subjects were randomly assigned to an HP or HC isocaloric energy-restricted dietary intervention for 12 weeks intensive weight loss and weight maintenance up to 52 weeks. Blood samples were collected at 0, 12 and 52 weeks. DNA damage in PBLs was assessed using the cytokinesis-block micronucleus cytome (CBMN-Cyt) assay. Average weight loss after 12 weeks was 9.3 +/- 0.7 kg for both diets, with no further change at 52 weeks. Two-way analysis of variance showed no time or diet effect on micronucleus frequency (chromosome loss/breaks). There was a significant trend with time (P = 0.03) but not diet, for reduction of nuclear buds (gene amplification). There was a positive trend with time for increased nucleoplasmic bridges (chromosome rearrangement) (P = 0.051). Necrosis and apoptosis both significantly decreased with time (P = 0.037 and P = 0.007, respectively) with no diet effect. There was no significant effect of time or diet for nuclear division index, a biomarker of immune response. The results suggest that the effect of the HP weight loss diet on DNA damage measured using the CBMN-Cyt assay in PBLs was not different from that observed for the HC weight loss diet.

  8. Vibrational modes of hemoglobin in red blood cells.

    PubMed

    Martel, P; Calmettes, P; Hennion, B

    1991-02-01

    Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective.

  9. Vibrational modes of hemoglobin in red blood cells.

    PubMed Central

    Martel, P; Calmettes, P; Hennion, B

    1991-01-01

    Equine red blood cells were washed in saline heavy water (2H2O) to exchange the hydrogen atoms of the non-hemoglobin components with deuterons. This led to novel neutron scattering measurements of protein vibrations within a cellular system and permitted a comparison with inelastic neutron scattering measurements on purified horse hemoglobin, either dry or wetted with 2H2O. As a function of wavevector transfer Q and the frequency transfer v the neutron response typified by the dynamic structure factor S(Q, v) was found to be similar for extracted and cellular hemoglobin at low and high temperatures. At 77 K, in the cells, a peak in S(Q, v) due to the protein was found near 0.7 THz, approximately half the frequency of a strong peak in the aqueous medium. Measurements at higher temperatures (170 and 230 K) indicated similar small shifts downwards in the peak frequencies of both components. At 260 K the low frequency component became predominantly quasielastic, but a significant inelastic component could still be ascribed to the aqueous scattering. Near 295 K the frequency responses of both components were similar and centered near zero. When scattering due to water is taken into account it appears that the protein neutron response in, or out of, red blood cells is little affected by hydration in the low frequency regime where Van der Waals forces are thought to be effective. PMID:1849028

  10. Argon laser radiation of human clots: differential photoabsorption in red cell rich and red cell poor clots

    SciTech Connect

    Lee, G.; Chan, M.C.; Seckinger, D.L.; Vazquez, A.; Rosenthal, P.K.; Lee, K.K.; Ikeda, R.M.; Reis, R.L.; Hanna, E.S.; Mason, D.T.

    1985-06-01

    Since argon laser radiation (454-514 nm) can vaporize human clots, the authors determined whether the absorption of laser energies can differ among different types of blood clots. Thus, they performed spectrophotometric studies and examined the ability of this laser to penetrate red cell rich and red cell poor clots. Fifty-four red cell rich and red cell poor clot samples, varying in depth from 1.8 to 5.0 mm, were subjected to 3, 5 and 7 watts from an argon laser beam. At a given power intensity, the deeper the red cell rich clot, the longer was the time needed to penetrate the clot. The higher the power used, the shorter was the red clot penetration time. In contrast, all power levels used up to 5 minutes did not penetrate any of the varying depths of red cell poor clots. Spectrophotometrically, the red cell rich clot had an absorption curve typical of hemoglobin pigment while the red cell poor clot, in the absence of hemoglobin, had poor absorption between 350 and 600 nm and was unable to absorb argon laser energies. Thus, the argon laser provides a therapeutic modality for human red cell rich clot dissolution but the present approach does not appear to be effective against red cell poor clots.

  11. Anesthetics and red blood cell rheology

    NASA Astrophysics Data System (ADS)

    Aydogan, Burcu; Aydogan, Sami

    2014-05-01

    There are many conditions where it is useful for anesthetists to have a knowledge of blood rheology. Blood rheology plays an important role in numerous clinical situations. Hemorheologic changes may significantly affect the induction and recovery times with anesthetic agents. But also, hemorheologic factors are directly or indirectly affected by many anesthetic agents or their metabolites. In this review, the blood rheology with special emphasis on its application in anesthesiology, the importance hemorheological parameters in anesthesiology and also the effect of some anesthetic substances on red blood cell rheology were presented.

  12. Inhibition of anion permeability by amphiphilic compounds in human red cell: evidence for an interaction of niflumic acid with the band 3 protein.

    PubMed

    Cousin, J L; Motais, R

    1979-04-20

    In human erythrocyte, permeability to the anion is instantaneously, reversibly, and noncompetitively inhibited by the nonsteroidal anti-inflammatory drug, niflumic acid. The active form of this powerful inhibitor (I50 = 6 X 10(-7) M) is the ionic form. We demonstrated that: (i) The binding of niflumic acid to the membrane of unsealed ghosts show one saturable and one linear component over the concentration range studied. The saturable component vanishes when chloride transport is fully inhibited by covalently bound 4-acetamido-4'-isothiocyano stilbene-2,2'-disulfonic acid (SITS). Our estimate of these SITS protectable niflumate binding sites (about 9 x 10(5) per cell) agrees with the number of protein molecules per cell in band 3. These sites are half-saturated with 10(-6) M niflumic acid, a concentration very close to I50. (ii) Niflumic acid inhibits the binding reaction of SITS with anion controlling transport sites. These results indicate that niflumic acid and SITS are mutually exclusive inhibitors, suggesting that niflumic acid interacts with the protein in band 3. Niflumic acid also decreases glucose and ouabain-insensitive sodium permeabilities. However, these effects are produced at a very high concentration of niflumic acid (in millimolar range), suggesting unspecific action, possibly through lipid phase.

  13. Image analysis of nucleated red blood cells.

    PubMed

    Zajicek, G; Shohat, M; Melnik, Y; Yeger, A

    1983-08-01

    Bone marrow smears stained with Giemsa were scanned with a video camera under computer control. Forty-two cells representing the six differentiation classes of the red bone marrow were sampled. Each cell was digitized into 70 X 70 pixels, each pixel representing a square area of 0.4 micron2 in the original image. The pixel gray values ranged between 0 and 255. Zero stood for white, 255 represented black, while the numbers in between stood for the various shades of gray. After separation and smoothing the images were processed with a Sobel operator outlining the points of steepest gray level change in the cell. These points constitute a closed curve denominated as inner cell boundary, separating the cell into an inner and an outer region. Two types of features were extracted from each cell: form features, e.g., area and length, and gray level features. Twenty-two features were tested for their discriminative merit. After selecting 16, the discriminant analysis program classified correctly all 42 cells into the 6 classes.

  14. DNA unmasked in the red rain cells of Kerala.

    PubMed

    Gangappa, Rajkumar; Hogg, Stuart I

    2013-01-01

    Extraordinary claims have been made for the biological properties of the red rain cells of Kerala, including a suggestion that they lack DNA. We have investigated the fluorescence properties of red rain cells, and the solubility of the red pigment in a variety of solvents. Extraction of the pigment with DMSO allowed successful demonstration of DNA using DAPI staining. Cellular impermeability to staining reagents due to the red pigment is the likely explanation for the failure of previous efforts to demonstrate DNA in red rain cells.

  15. Single-Molecule Specific Mislocalization of Red Fluorescent Proteins in Live Escherichia coli.

    PubMed

    Ghodke, Harshad; Caldas, Victor E A; Punter, Christiaan M; van Oijen, Antoine M; Robinson, Andrew

    2016-07-12

    Tagging of individual proteins with genetically encoded fluorescent proteins (FPs) has been used extensively to study localization and interactions in live cells. Recent developments in single-molecule localization microscopy have enabled the dynamic visualization of individual tagged proteins inside living cells. However, tagging proteins with FPs is not without problems: formation of insoluble aggregates and inhibition of native functions of the protein are well-known issues. Previously reported artifacts manifest themselves at all expression levels of the FP-tagged proteins, making the design of control experiments relatively straightforward. Here, we describe a previously uncharacterized mislocalization artifact of Entacmaea quadricolor red fluorescent protein variants that is detectable at the single-molecule level in live Escherichia coli cells. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Poly-N-acetyl glucosamine-mediated red blood cell interactions.

    PubMed

    Thatte, Hemant S; Zagarins, Sofija E; Amiji, Mansoor; Khuri, Shukri F

    2004-07-01

    Investigations were performed to assess the effect of poly-N-acetyl glucosamine (p-GlcNAc) fiber slurry-mediated hemostasis by interactions with red blood cells. Red blood cell aggregation studies were performed using test material-coated microscope slides and multiphoton microscopic measurements. Enzymatic removal of red blood cell surface proteins was achieved using trypsin and neuraminidase treatment. Zeta-potential measurements (surface charge) were performed. Red blood cells interact directly with poly-N-acetyl glucosamine polymers through ionic interactions and cell-surface proteins. The effective concentration of poly-N-acetyl glucosamine fiber material for 50% red blood cell aggregation was 0.28 mg/mL. The p-GlcNAc beta-configuration fibers and an alpha-configuration structural modification of the fibers both produced maximal responses because of their zeta-potentials, whereas other chemically modified p-GlcNAcs and chitosans were ineffective. Poly-N-acetyl glucosamine-induced red blood cell aggregation is mediated by interactions with red blood cell surface charges.

  17. Interactions of mefloquine with ABC proteins, MRP1 (ABCC1) and MRP4 (ABCC4), that are present in human red cell membranes

    PubMed Central

    Wu, Chung-Pu; Klokouzas, Antonios; Hladky, Stephen B.; Ambudkar, Suresh V.; Barrand, Margery A.

    2005-01-01

    Human erythrocyte membranes express the multidrug resistance-associated proteins, MRP1, MRP4 and MRP5, that collectively can efflux oxidised glutathione, glutathione conjugates and cyclic nucleotides. It is already known that the quinoline derivative, MK-571, is a potent inhibitor of MRP-mediated transport. We here examine whether the quinoline-based antimalarial drugs, amodiaquine, chloroquine, mefloquine, primaquine, quinidine and quinine, also interact with erythrocyte MRPs with consequences for their access to the intracellular parasites or for efflux of oxidised glutathione from infected cells. Using inside-out vesicles prepared from human erythrocytes we have shown that mefloquine and MK-571 inhibit transport of 3 μM [3H]DNP-SG known to be mediated by MRP1 (IC50 127 μM and 1.1 μM respectively) and of 3.3 μM [3H]cGMP thought but not proven to be mediated primarily by MRP4 (IC50 21 μM and 0.41 μM). They also inhibited transport in membrane vesicles prepared from tumour cells expressing MRP1 or MRP4 and blocked calcein efflux from MRP1 overexpressing cells and BCECF efflux from MRP4 overexpressing cells. Both stimulated ATPase activity in membranes prepared from MRP1 and MRP4 overexpressing cells and inhibited activity stimulated by quercetin or PGE1 respectively. Neither inhibited [α-32P]8-azidoATP binding confirming that the interactions are not at the ATP binding site. These results demonstrate that mefloquine and MK-571 both inhibit transport of other substrates and stimulate ATPase activity and thus may themselves be substrates for transport. But at concentrations achieved clinically mefloquine is unlikely to affect the MRP1-mediated transport of GSSG across the erythrocyte membrane. PMID:16004972

  18. Red cell antigens: Structure and function

    PubMed Central

    Pourazar, Abbasali

    2007-01-01

    Landsteiner and his colleagues demonstrated that human beings could be classified into four groups depending on the presence of one (A) or another (B) or both (AB) or none (O) of the antigens on their red cells. The number of the blood group antigens up to 1984 was 410. In the next 20 years, there were 16 systems with 144 antigens and quite a collection of antigens waiting to be assigned to systems, pending the discovery of new information about their relationship to the established systems. The importance of most blood group antigens had been recognized by immunological complications of blood transfusion or pregnancies; their molecular structure and function however remained undefined for many decades. Recent advances in molecular genetics and cellular biochemistry resulted in an abundance of new information in this field of research. In this review, we try to give some examples of advances made in the field of ‘structure and function of the red cell surface molecules.’ PMID:21938229

  19. Red blood cell volume in preterm neonates

    SciTech Connect

    Quaife, M.A.; Dirksen, J.W.; Paxson, C.L. Jr.; McIntire, R.H. Jr.

    1981-10-01

    In the high-risk neonate, the direct determination of the red cell volume by radionuclide dilution technique appears to be the singularly definitive method of defining treatment efficacy, and is thus a useful evaluation and management tool for the pediatrician. For effective patient management, the red blood cell(RBC) volume of 69 preterm and term neonates was determined. The method utilized, Tc-99m-labeled RBCs, provided a fast and accurate answer with a large reduction in the absorbed radiation dose. In the population studied within a high-risk newborn ICU, the mean RBC volumes between the preterm and term neonates were without significant difference. Grouping and analysis of the RBC volume data with respect to birth weight, gestational ages, and 1- and 5-minute Apgar scores revealed on statistical difference. The mean value found in our population, 32.2 +/- 9.2 ml/kg, however, does differ from those previously reported in which the determinations were made using an indirect estimation from the plasma compartment.

  20. Mechanosensing Dynamics of Red blood Cells

    NASA Astrophysics Data System (ADS)

    Wan, Jiandi

    2015-11-01

    Mechanical stress-induced deformation of human red blood cells (RBCs) plays important physiopathological roles in oxygen delivery, blood rheology, transfusion, and malaria. Recent studies demonstrate that, in response to mechanical deformation, RBCs release adenosine-5'-triphosphate (ATP), suggesting the existence of mechanotransductive pathways in RBCs. Most importantly, the released ATP from RBCs regulates vascular tone and impaired release of ATP from RBCs has been linked to diseases such as type II diabetes and cystic fibrosis. To date, however, the mechanisms of mechanotransductive release of ATP from RBCs remain unclear. Given that RBCs experience shear stresses continuously during the circulation cycle and the released ATP plays a central role in vascular physiopathology, understanding the mechanotransductive release of ATP from RBCs will provide not only fundamental insights to the role of RBCs in vascular homeostasis but also novel therapeutic strategies for red cell dysfunction and vascular disease. This talk describes the main research in my group on integrating microfluidic-based approaches to study the mechanosensing dynamics of RBCs. Specifically, I will introduce a micro?uidic approach that can probe the dynamics of shear-induced ATP release from RBCs with millisecond resolution and provide quantitative understandings of the mechanosensitive ATP release processes in RBCs. Furthermore, I will also describe our recent findings about the roles of the Piezo1 channel, a newly discovered mechanosensitive cation channel in the mechanotransductive ATP release in RBCs. Last, possible functions of RBCs in the regulation of cerebral blood flow will be discussed.

  1. Skeleton deformation of red blood cells during tank treading motions

    NASA Astrophysics Data System (ADS)

    Zhu, Qiang; Peng, Zhangli

    2012-11-01

    By coupling a fluid-structure interaction algorithm with a three-level multiscale structural model, we simulate the tank treading responses of erythrocytes (red blood cells, or RBC) in shear flows. The fluid motion is depicted within the Stokes-flow framework, and is mathematically formulated with the boundary integral equations. The structural model takes into account the flexible connectivity between the lipid bilayer and the protein skeleton as well as the viscoelastic responses. The concentration of this study is on the transient process involving the development of the local area deformation of the protein skeleton. Under the assumption that the protein skeleton is stress-free in the natural biconcave configuration, our simulations indicate the following properties: (1) During tank treading motions it takes long time for significant area deformations to establish. For cells with diminished connectivity between the lipid bilayer and the protein skeleton (e.g. cells with mutations or defects), the relaxation time will be greatly reduced; (2) Deformations of the skeleton depend on the initial orientation of the cell with respect to the incoming flow; (3) The maximum area expansion occurs around the regions corresponding to the dimples in the original biconcave state; (4) Oscillations in cell geometry (breathing) and orientation (e.g. swinging) are observed. This work was supported by the National Heart, Lung, and Blood Institute under award number R01HL092793.

  2. Altered red blood cell surface area in hereditary xerocytosis.

    PubMed

    Sauberman, N; Fairbanks, G; Lutz, H U; Fortier, N L; Snyder, L M

    1981-08-10

    Hereditary xerocytes appear larger than normal red cells in scanning electron micrographs and exhibit a higher ghost packing volume. The major chemical components--protein, phosphorus, cholesterol and sialic acid--are increased uniformly, as are all polypeptides visible on gel electrophoresis patterns of xerocyte membranes. These data are consistent with a xerocyte surface area 15 to 25% above normal. Certain clinical anomalies common to this disorder, including unexpectedly low reticulocyte count and 2,3-diphosphoglycerate level, are discussed in the light of the present findings.

  3. Evaluation of an autologous red cell agglutination test, VetRED FIV, for the presence of FIV antibody in cats.

    PubMed

    del Fierro, G M; Bundesen, P; Martin, S; Jones, S; Beetson, S; Robinson, W F

    1995-05-01

    A commercially available whole blood agglutination test, VetRED FIV, used for the detection of antibodies to feline immunodeficiency virus (FIV), was evaluated. The test is based on the use of a synthetic peptide conjugated to a non-agglutinating anti-feline red blood cell monoclonal antibody. The amino acid sequence of the synthetic peptide was derived from the predicted sequence of the transmembrane protein of FIV. The sensitivity and specificity of VetRED FIV was 100% when 34 known FIV-positive and 15 known FIV-negative cats were tested. These cats were part of studies on experimentally induced FIV infection, with their FIV status confirmed by virus isolation. Further, VetRED FIV was compared with another commercially available test for FIV antibody, PetChek in a field trial on 548 feline blood samples received by a diagnostic laboratory. Of the test results 94.2% (516/548) were in agreement: 112 were positive by VetRED FIV and PetChek; 404 were negative by both tests and 32 were discordant. These 5.8% discordant samples producing VetRED FIV-positive/PetChek-negative or VetRED FIV-negative/PetChek-positive were further assessed by Western blot assay. In the field trial, the sensitivity and specificity of VetRED FIV was 97% and 97%, respectively, comparable to the 98% sensitivity and 99% specificity for PetChek. The results from the trial also confirm the relatively high overall prevalence of FIV in Australian cats predominantly among mature male cats in the 9-12 year age group. Given the simplicity of the VetRED FIV procedure, it is concluded that VetRED FIV is a useful addition to the available commercial tests for FIV infection.

  4. Functional 20S proteasomes in mature human red blood cells.

    PubMed

    Neelam, Sudha; Kakhniashvili, David G; Wilkens, Stephan; Levene, Stephen D; Goodman, Steven R

    2011-05-01

    The purpose of the present study was to investigate whether functional 20S and/or 26S proteasomes are present within mature human red blood cells (RBCs; depleted of reticulocytes and leukocytes). Double-immunofluorescence confocal microscopy showed the presence of immunoreactive 20S and 19S proteasomal subunit proteins and their partial co-localization within mature RBCs. Proteasomes isolated from mature RBCs displayed 20S activity in vitro; atomic-force and transmission electron microscopy of isolated proteasomes revealed abundant 20S core particles and very few 26S particles. A two-dimensional differential in-gel electrophoresis (2D-DIGE) approach was used to determine if proteasome-dependent protein degradation occurs within mature RBCs. Twenty-eight proteins were identified with altered protein content in response to lactacystin. Seven cytosolic proteins showed an increase and 16 showed a decrease; five membrane proteins showed a decrease. We conclude that the proteins showing increased abundance are either primary or secondary targets of the 20S proteasome and that putatively degraded proteins are secondary targets. Therefore, functional 20S proteasomes exist within mature RBCs. Our study did not detect 26S proteasome activity using the 2D-DIGE approach.

  5. Microconfined flow behavior of red blood cells.

    PubMed

    Tomaiuolo, Giovanna; Lanotte, Luca; D'Apolito, Rosa; Cassinese, Antonio; Guido, Stefano

    2016-01-01

    Red blood cells (RBCs) perform essential functions in human body, such as gas exchange between blood and tissues, thanks to their ability to deform and flow in the microvascular network. The high RBC deformability is mainly due to the viscoelastic properties of the cell membrane. Since an impaired RBC deformability could be found in some diseases, such as malaria, sickle cell anemia, diabetes and hereditary disorders, there is the need to provide further insight into measurement of RBC deformability in a physiologically relevant flow field. Here, RBCs deformability has been studied in terms of the minimum apparent plasma-layer thickness by using high-speed video microscopy of RBCs flowing in cylindrical glass capillaries. An in vitro systematic microfluidic investigation of RBCs in micro-confined conditions has been performed, resulting in the determination of the RBCs time recovery constant, RBC volume and surface area and RBC membrane shear elastic modulus and surface viscosity. It has been noticed that the deformability of RBCs induces cells aggregation during flow in microcapillaries, allowing the formation of clusters of cells. Overall, our results provide a novel technique to estimate RBC deformability and also RBCs collective behavior, which can be used for the analysis of pathological RBCs, for which reliable quantitative methods are still lacking.

  6. Red Blood Cell Count Automation Using Microscopic Hyperspectral Imaging Technology.

    PubMed

    Li, Qingli; Zhou, Mei; Liu, Hongying; Wang, Yiting; Guo, Fangmin

    2015-12-01

    Red blood cell counts have been proven to be one of the most frequently performed blood tests and are valuable for early diagnosis of some diseases. This paper describes an automated red blood cell counting method based on microscopic hyperspectral imaging technology. Unlike the light microscopy-based red blood count methods, a combined spatial and spectral algorithm is proposed to identify red blood cells by integrating active contour models and automated two-dimensional k-means with spectral angle mapper algorithm. Experimental results show that the proposed algorithm has better performance than spatial based algorithm because the new algorithm can jointly use the spatial and spectral information of blood cells.

  7. A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids.

    PubMed

    Sturm, Sabine; Engelken, Johannes; Gruber, Ansgar; Vugrinec, Sascha; Kroth, Peter G; Adamska, Iwona; Lavaud, Johann

    2013-07-30

    Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages. Here, we demonstrate that the recently discovered "red lineage chlorophyll a/b-binding-like proteins" (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities. The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light shifts as regularly experienced by

  8. Mechanochemistry of single red blood cells monitored using Raman tweezers.

    PubMed

    Raj, Saurabh; Marro, Mónica; Wojdyla, Michal; Petrov, Dmitri

    2012-04-01

    Two microparticles were biochemically attached to a red blood cell at diametrically opposite parts and held by optical traps allowing to impose deformations. The cell deformation was monitored from the microscopy images. Raman spectra of the cell under tunable deformations were studied. Vibrational spectra analysis at different stretching states was supported with two statistical methods. Principal Component Analysis distinguishes the most prominent changes in spectra while 2D correlation technique monitors the evolution of Raman bands during stretching. The measurements show significant changes in the cell chemical structure with stretching however the changes saturate above 20% of cell deformation. Mechanical deformation of the cell mainly affects the bands corresponding to hemoglobin but contributions from spectrin and membrane proteins can not be excluded. The saturation of bands at higher deformations suggests some structural relaxation that RBC has to undergo to bear extra load. The results confirm widely accepted belief that spectrin released from membrane proteins allows for significant shape changes of the cells. We therefore tentatively suggest that interaction between membrane and cytoskeleton during deformation can be efficiently probed by confocal Raman spectroscopy, in particular via the peak around 1035 cm(-1).

  9. Optical analysis of red blood cell suspension

    NASA Astrophysics Data System (ADS)

    Szołna, Alicja A.; Grzegorzewski, Bronisław

    2008-12-01

    The optical properties of suspensions of red blood cells (RBCs) were studied. Fresh human venues blood was obtained from adult healthy donors. RBCs were suspended in isotonic salt solution, and in autologous plasma. Suspensions with haematocrit 0.25 - 3% were investigated. Novel technique was proposed to determine the scattering coefficient μs for the suspensions. The intensity of He-Ne laser light transmitted through a wedge-shape container filled with a suspension was recorded. To find the dependence of the intensity on the thickness of the sample the container was moved horizontally. The dependence of μs on the haematocrit was determined for RBCs suspended in the isotonic salt solution. RBCs suspended in plasma tend to form rouleaux. For the RBCs suspended in plasma, the scattering coefficient as a function of time was obtained. It is shown that this technique can be useful in the study of rouleaux formation.

  10. Fluorometric assay for red blood cell antibodies

    SciTech Connect

    Schreiber, A.B.; Lambermont, M.; Strosberg, A.D.; Wybran, J.

    1981-03-01

    A fluorometric assay is described for the detection of red blood cell antibodies. The assay reveals as little as 600 molecules of bound, fluoroesceinated rabbit anti-human IgG antibodies per erythrocyte. Eleven patients with possible autoimmune erythrocyte disorder and negative direct antiglobulin test were studied by the fluorometric assay. The outcome of the fluorometric assay was compared with that of the human allogeneic rosette test. Results obtained by the two methods were in complete agreement. Five of the patients were shown to possess unexpectedly high levels of erythrocyte-bound IgG in spite of a negative, direct antiglobulin test. These findings and the validity of the fluorometric assay are discussed.

  11. Depletion induced clustering of red blood cells in microchannels

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Brust, Mathias; Podgorski, Thomas; Coupier, Gwennou

    2012-11-01

    The flow properties of blood are determined by the physical properties of its main constituents, the red blood cells (RBC's). At low shear rates RBC's form aggregates, so called rouleaux. Higher shear rates can break them up and the viscosity of blood shows a shear thinning behavior. The physical origin of the rouleaux formation is not yet fully resolved and there are two competing models available. One predicts that the adhesion is induced by bridging of the plasma (macromolecular) proteins in-between two RBC's. The other is based on the depletion effect and thus predicts the absence of macromolecules in-between the cells of a rouleaux. Recent single cell force measurements by use of an AFM support strongly the depletion model. By varying the concentration of Dextran at different molecular weights we can control the adhesions strength. Measurements at low hematocrit in a microfluidic channel show that the number of size of clusters is determined by the depletion induced adhesion strength.

  12. Mechanisms of immune red cell destruction, and red cell compatibility testing

    SciTech Connect

    Garratty, G.

    1983-03-01

    The immune destruction of red cells can occur as a complement-mediated intravascular process, or extravascularly, where the red cells are destroyed by macrophages following interaction with cell-bound IgG1, IgG3, and/or C3b. Many of the factors that affect this in vivo destruction are not taken into account during in vitro pretransfusion compatibility testing. At present, even by use of more elaborate tests, it is difficult to accurately predict the fate of a transfused unit of blood. By using some simple information, such as antibody specificity and thermal range, it is sometimes possible to predict the outcome of transfusing a unit of blood that is incompatible in vitro. At other times it may be necessary to utilize /sup 51/Cr-labeled red cells to determine the risk of transfusing such units. Because of the paucity of reported clinical correlations, macrophage/monocyte monolayer assays are of little practical value at present.

  13. A novel type of light-harvesting antenna protein of red algal origin in algae with secondary plastids

    PubMed Central

    2013-01-01

    Background Light, the driving force of photosynthesis, can be harmful when present in excess; therefore, any light harvesting system requires photoprotection. Members of the extended light-harvesting complex (LHC) protein superfamily are involved in light harvesting as well as in photoprotection and are found in the red and green plant lineages, with a complex distribution pattern of subfamilies in the different algal lineages. Results Here, we demonstrate that the recently discovered “red lineage chlorophyll a/b-binding-like proteins” (RedCAPs) form a monophyletic family within this protein superfamily. The occurrence of RedCAPs was found to be restricted to the red algal lineage, including red algae (with primary plastids) as well as cryptophytes, haptophytes and heterokontophytes (with secondary plastids of red algal origin). Expression of a full-length RedCAP:GFP fusion construct in the diatom Phaeodactylum tricornutum confirmed the predicted plastid localisation of RedCAPs. Furthermore, we observed that similarly to the fucoxanthin chlorophyll a/c-binding light-harvesting antenna proteins also RedCAP transcripts in diatoms were regulated in a diurnal way at standard light conditions and strongly repressed at high light intensities. Conclusions The absence of RedCAPs from the green lineage implies that RedCAPs evolved in the red lineage after separation from the the green lineage. During the evolution of secondary plastids, RedCAP genes therefore must have been transferred from the nucleus of the endocytobiotic alga to the nucleus of the host cell, a process that involved complementation with pre-sequences allowing import of the gene product into the secondary plastid bound by four membranes. Based on light-dependent transcription and on localisation data, we propose that RedCAPs might participate in the light (intensity and quality)-dependent structural or functional reorganisation of the light-harvesting antennae of the photosystems upon dark to light

  14. Growth and replication of red rain cells at 121°C and their red fluorescence

    NASA Astrophysics Data System (ADS)

    Gangappa, Rajkumar; Wickramasinghe, Chandra; Wainwright, Milton; Kumar, A. Santhosh; Louis, Godfrey

    2010-09-01

    We have shown that the red cells found in the Red Rain (which fell on Kerala, India, in 2001) survive and grow after incubation for periods of up to two hours at 121°C . Under these conditions daughter cells appear within the original mother cells and the number of cells in the samples increases with length of exposure to 121°C. No such increase in cells occurs at room temperature, suggesting that the increase in daughter cells is brought about by exposure of the Red Rain cells to high temperatures. This is an independent confirmation of results reported earlier by two of the present authors, claiming that the cells can replicate under high pressure at temperatures upto 300°C. The flourescence behaviour of the red cells is shown to be in remarkable correspondence with the extended red emission observed in the Red Rectagle planetary nebula and other galactic and extragalactic dust clouds, suggesting, though not proving an extraterrestrial origin.

  15. Red blood cell membrane fragments but not intact red blood cells promote calcium oxalate monohydrate crystal growth and aggregation.

    PubMed

    Chutipongtanate, Somchai; Thongboonkerd, Visith

    2010-08-01

    Cell membranes are thought to promote calcium oxalate kidney stone formation but to our knowledge the modulating effect of red blood cell membranes on calcium oxalate crystals has not been previously investigated. Thus, we examined the effects of red blood cell membrane fragments on calcium oxalate monohydrate and calcium oxalate dihydrate crystal growth and aggregation. Calcium oxalate monohydrate and calcium oxalate dihydrate crystals were treated with red blood cell membrane fragments or intact red blood cells from a healthy donor. Phase contrast microscopy was performed to evaluate crystal morphology and aggregation. We used ImageMaster 2D Platinum software to evaluate crystal size and spectrophotometric oxalate depletion assay to monitor crystal growth. Red blood cell membrane fragments had significant promoting activity on calcium oxalate monohydrate crystal growth with an approximately 75% increase in size and aggregation with an approximately 2.5-fold increase in aggregate number compared to the control without membrane fragments or cells. Approximately 50% of calcium oxalate monohydrate crystals were adhered by red blood cell membrane fragments. Intact red blood cells had no significant effect on calcium oxalate monohydrate crystal growth or aggregation but they could transform calcium oxalate monohydrate to calcium oxalate dihydrate crystals. Red blood cell membrane fragments and intact red blood cells had no effect on calcium oxalate dihydrate crystals. The promoting activity of red blood cell membrane fragments on calcium oxalate monohydrate crystal growth was successfully confirmed by spectrophotometric oxalate depletion assay. To our knowledge our data provide the first direct evidence that red blood cell membrane fragments are a promoting factor for calcium oxalate monohydrate crystal growth and aggregation. Thus, they may aggravate calcium oxalate stone formation. Copyright (c) 2010 American Urological Association Education and Research, Inc

  16. Kinematics of red cell aspiration by fluorescence-imaged microdeformation.

    PubMed Central

    Discher, D E; Mohandas, N

    1996-01-01

    Maps of fluorescing red cell membrane components on a pipette-aspirated projection are quantitated in an effort to elucidate and unify the heterogeneous kinematics of deformation. Transient gradients of diffusing fluorescent lipid first demonstrate the fluidity of an otherwise uniform-density bilayer and corroborate a "universal" calibration scale for relative surface density. A steep but smooth and stable gradient in the densities of the skeleton components spectrin, actin, and protein 4.1 is used to estimate large elastic strains along the aspirated skeleton. The deformation fields are argued to be an unhindered response to loading in the surface normal direction. Density maps intermediate to those of the compressible skeleton and fluid bilayer are exhibited by particular transmembrane proteins (e.g., Band 3) and yield estimates for the skeleton-connected fractions. Such connected proteins appear to occupy a significant proportion of the undeformed membrane surface and can lead to steric exclusion of unconnected integral membrane proteins from regions of network condensation. Consistent with membrane repatterning kinematics in reversible deformation, final vesiculation of the projection tip produces a cell fragment concentrated in freely diffusing proteins but depleted of skeleton. Images FIGURE 1 FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 FIGURE 9 FIGURE 10 FIGURE 11 PMID:8889146

  17. Destruction of newly released red blood cells in space flight

    NASA Technical Reports Server (NTRS)

    Alfrey, C. P.; Udden, M. M.; Huntoon, C. L.; Driscoll, T.

    1996-01-01

    Space flight results in a rapid change in total blood volume, plasma volume, and red blood cell mass because the space to contain blood is decreased. The plasma volume and total blood volume decreases during the first hours in space and remain at a decreased level for the remainder of the flight. During the first several hours following return to earth, plasma volume and total blood volume increase to preflight levels. During the first few days in space recently produced red blood cells disappear from the blood resulting in a decrease in red blood cell mass of 10-15%. Red cells 12 d old or older survive normally and production of new cells continues at near preflight levels. After the first few days in space, the red cell mass is stable at the decreased level. Following return to earth the hemoglobin and red blood cell mass concentrations decrease reflecting the increase in plasma volume. The erythropoietin levels increase responding to "postflight anemia"; red cell production increases, and the red cell mass is restored to preflight levels after several weeks.

  18. Destruction of newly released red blood cells in space flight

    NASA Technical Reports Server (NTRS)

    Alfrey, C. P.; Udden, M. M.; Huntoon, C. L.; Driscoll, T.

    1996-01-01

    Space flight results in a rapid change in total blood volume, plasma volume, and red blood cell mass because the space to contain blood is decreased. The plasma volume and total blood volume decreases during the first hours in space and remain at a decreased level for the remainder of the flight. During the first several hours following return to earth, plasma volume and total blood volume increase to preflight levels. During the first few days in space recently produced red blood cells disappear from the blood resulting in a decrease in red blood cell mass of 10-15%. Red cells 12 d old or older survive normally and production of new cells continues at near preflight levels. After the first few days in space, the red cell mass is stable at the decreased level. Following return to earth the hemoglobin and red blood cell mass concentrations decrease reflecting the increase in plasma volume. The erythropoietin levels increase responding to "postflight anemia"; red cell production increases, and the red cell mass is restored to preflight levels after several weeks.

  19. Development of redox-sensitive red fluorescent proteins for imaging redox dynamics in cellular compartments.

    PubMed

    Fan, Yichong; Ai, Hui-wang

    2016-04-01

    We recently reported a redox-sensitive red fluorescent protein, rxRFP1, which is one of the first genetically encoded red-fluorescent probes for general redox states in living cells. As individual cellular compartments have different basal redox potentials, we hereby describe a group of rxRFP1 mutants, showing different midpoint redox potentials for detection of redox dynamics in various subcellular domains, such as mitochondria, the cell nucleus, and endoplasmic reticulum (ER). When these redox probes were expressed and subcellularly localized in human embryonic kidney (HEK) 293 T cells, they responded to membrane-permeable oxidants and reductants. In addition, a mitochondrially localized rxRFP1 mutant, Mito-rxRFP1.1, was used to detect mitochondrial oxidative stress induced by doxorubicin-a widely used cancer chemotherapy drug. Our work has expanded the fluorescent protein toolkit with new research tools for studying compartmentalized redox dynamics and oxidative stress under various pathophysiological conditions.

  20. Red blood cell nitric oxide synthase modulates red blood cell deformability in sickle cell anemia.

    PubMed

    Mozar, Anaïs; Connes, Philippe; Collins, Bianca; Hardy-Dessources, Marie-Dominique; Romana, Marc; Lemonne, Nathalie; Bloch, Wilhelm; Grau, Marijke

    2016-11-04

    Sickle cell anemia (SCA) is an inherited red blood cells (RBC) disorder characterized by significantly decreased RBC deformability. The present study aimed to assess whether modulation of RBC Nitric Oxide Synthase (RBC-NOS) activation could affect RBC deformability in SCA.Blood of twenty-five SCA patients was treated for 1 hour at 37°C with Phosphate Buffered Saline (PBS) or PBS containing 1% of Dimethylsulfoxyde as control, L-arginine or N(5)-(1-Iminoethyl)-L-ornithine (L-NIO) to directly stimulate or inhibit RBC-NOS, insulin or wortmannin to indirectly stimulate or inhibit RBC-NOS through their effects on the PI3 Kinase/Akt pathway, and sodium nitroprusside (SNP) and 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) as NO donor and NO scavenger, respectively. RBC deformability was measured by ektacytometry at 3 Pa.RBC deformability significantly increased after insulin treatment and significantly decreased after L-NIO and wortmannin incubation. The other conditions did not affect deformability. Significantly increased nitrotyrosine levels, a marker of enhanced free radical generation, were detected by immunohistochemistry in SNP and insulin treated samples.These data suggest that RBC deformability of SCA can be modulated by RBC-NOS activity but also that oxidative stress may impair effectiveness of RBC-NOS produced NO.

  1. Red blood cell alloimmunization among sickle cell Kuwaiti Arab patients who received red blood cell transfusion.

    PubMed

    Ameen, Reem; Al Shemmari, Salem; Al-Bashir, Abdulaziz

    2009-08-01

    Sickle cell disease (SCD) is common in the Arabian Gulf region. Most cases require a red blood cell (RBC) transfusion, increasing the potential for RBC alloantibody development. The incidence of RBC alloimmunization among Kuwaiti Arab SCD patients is not yet known. This study retrospectively assessed the effect of using two different matching protocols on the incidence of alloimmunization among multiply transfused Kuwaiti Arab SCD patients. A total of 233 Kuwaiti Arab SCD patients were divided into two groups: Group 1 (n = 110) received RBC transfusion through standard ABO- and D-matched nonleukoreduced blood; Group 2 (n = 123) received RBCs matched for ABO, Rh, and K1 poststorage-leukoreduced blood. Multivariate analysis was performed on the factors associated with RBC alloimmunization and antibody specificity. Sixty-five percent of patients in Group 1 developed clinically significant RBC alloantibody with an increased prevalence in females; in patients in Group 2, 23.6% developed RBC alloantibodies (p = 0.01). In Group 1, 72 patients (65.5%) had alloantibodies directed against Rh and Kell systems (p = 0.01). Multivariate analysis further confirmed the results, showing that blood transfusion type and sex have significant effects on the rate of alloimmunizations. This study confirms the importance of selecting RBCs matched for Rh and Kell to reduce the risk of alloimmunizations among Kuwaiti Arab SCD patients.

  2. Vesicles, capsules and red blood cells under flow

    NASA Astrophysics Data System (ADS)

    Misbah, Chaouqi

    2012-12-01

    Blood flow is dictated by the dynamics of red blood cells (RBCs), which constitute by far the major component. RBCs are made of a a two dimensional fluid bilayer of phospholipids, having underneath a network of proteins conferring to them shear elasticity, and they possess many membrane and transmembrane proteins (like ion channels). Simplified systems, like vesicles (made of a pure bilayer of phospholipid) and capsules (made of an extensible polymer shell) are used as models for RBCs. Both systems reproduce several features known for RBCs under flow. Their interest lies, besides some simplicity, in the fact that they can be fabricated in the laboratory, and their properties (size, stiffness, internal content....) can be varied in a wide range allowing thus to explore a quite significant parameter space that is essential to test predictions and discriminate between different models. We shall review the main recent achievement in this field, both for a single entity, collective effects and the impact on rheology.

  3. 21 CFR 864.8540 - Red cell lysing reagent.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing...

  4. Efflux of red cell water into buffered hypertonic solutions.

    PubMed

    OLMSTEAD, E G

    1960-03-01

    Buffered NaCl solutions hypertonic to rabbit serum were prepared and freezing point depressions of each determined after dilution with measured amounts of water. Freezing point depression of these dilutions was a linear function of the amount of water added. One ml. of rabbit red cells was added to each 4 ml. of the hypertonic solutions and after incubation at 38 degrees C. for 30 minutes the mixture was centrifuged and a freezing point depression determined on the supernatant fluid. The amount of water added to the hypertonic solutions by the red cells was calcuated from this freezing point depression. For each decrease in the freezing point of -0.093 degrees C. of the surrounding solution red cells gave up approximately 5 ml. of water per 100 ml. of red cells in the range of -0.560 to -0.930 degrees C. Beyond -0.930 degrees C. the amount of water given up by 100 ml. of red cells fits best a parabolic equation. The maximum of this equation occurred at a freezing point of the hypertonic solution of -2.001 degrees C. at which time the maximum amount of water leaving the red cells would be 39.9 ml. per 100 ml. of red cells. The data suggest that only about 43 per cent of the red cell water is available for exchange into solutions of increasing tonicity.

  5. Control of red blood cell mass during spaceflight

    NASA Technical Reports Server (NTRS)

    Lane, H. W.; Alfrey, C. P.; Driscoll, T. B.; Smith, S. M.; Nyquist, L. E.

    1996-01-01

    Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.

  6. Control of red blood cell mass during spaceflight

    NASA Technical Reports Server (NTRS)

    Lane, H. W.; Alfrey, C. P.; Driscoll, T. B.; Smith, S. M.; Nyquist, L. E.

    1996-01-01

    Data are reviewed from twenty-two astronauts from seven space missions in a study of red blood cell mass. The data show that decreased red cell mass in all astronauts exposed to space for more than nine days, although the actual dynamics of mass changes varies with flight duration. Possible mechanisms for these changes, including alterations in erythropoietin levels, are discussed.

  7. Role Ca(2+) in mechanisms of the red blood cells microrheological changes.

    PubMed

    Muravyov, Alexei; Tikhomirova, Irina

    2012-01-01

    To assess the physiological role of intracellular Ca(2+) in the changes of microrheological red blood cell (RBC) properties (RBC deformability and aggregation), we employed several types of chemicals that can increase and decrease of the intracellular Ca(2+) concentration. The rise of Ca(2+) influx, stimulated by mechanical loading, A23187, thrombin, prostaglandin F(2α) was accompanied by a moderate red cell deformability lowering and an increase of their aggregation. In contrast, Ca(2+) entry blocking into the red cells by verapamil led to a significant RBC aggregation decrease and deformability rise. Similar microrheological changes were observed in the red blood cells treated with phosphodiesterase inhibitors IBMX, vinpocetine, rolipram, pentoxifylline. When forskolin (10 μM), an AC stimulator was added to RBC suspension, the RBC deformability was increased (p <0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP. Red cell aggregation was significantly decreased under these conditions (p<0.01). On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of both intracellular signaling pathways: Ca(2+) regulatory mechanism and Gs-protein/adenylyl-cyclase-cAMP system. And the final red cell microrheological regulatory effect is connected with the crosstalk between these systems.

  8. Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells.

    PubMed

    Mairbäurl, Heimo

    2013-01-01

    During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood's buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called "sports anemia." This is not anemia in a clinical sense, because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume (PV). The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g., in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise.

  9. Red blood cells in sports: effects of exercise and training on oxygen supply by red blood cells

    PubMed Central

    Mairbäurl, Heimo

    2013-01-01

    During exercise the cardiovascular system has to warrant substrate supply to working muscle. The main function of red blood cells in exercise is the transport of O2 from the lungs to the tissues and the delivery of metabolically produced CO2 to the lungs for expiration. Hemoglobin also contributes to the blood's buffering capacity, and ATP and NO release from red blood cells contributes to vasodilation and improved blood flow to working muscle. These functions require adequate amounts of red blood cells in circulation. Trained athletes, particularly in endurance sports, have a decreased hematocrit, which is sometimes called “sports anemia.” This is not anemia in a clinical sense, because athletes have in fact an increased total mass of red blood cells and hemoglobin in circulation relative to sedentary individuals. The slight decrease in hematocrit by training is brought about by an increased plasma volume (PV). The mechanisms that increase total red blood cell mass by training are not understood fully. Despite stimulated erythropoiesis, exercise can decrease the red blood cell mass by intravascular hemolysis mainly of senescent red blood cells, which is caused by mechanical rupture when red blood cells pass through capillaries in contracting muscles, and by compression of red cells e.g., in foot soles during running or in hand palms in weightlifters. Together, these adjustments cause a decrease in the average age of the population of circulating red blood cells in trained athletes. These younger red cells are characterized by improved oxygen release and deformability, both of which also improve tissue oxygen supply during exercise. PMID:24273518

  10. Red blood cell vesiculation in hereditary hemolytic anemia

    PubMed Central

    Alaarg, Amr; Schiffelers, Raymond M.; van Solinge, Wouter W.; van Wijk, Richard

    2013-01-01

    Hereditary hemolytic anemia encompasses a heterogeneous group of anemias characterized by decreased red blood cell survival because of inherited membrane, enzyme, or hemoglobin disorders. Affected red blood cells are more fragile, less deformable, and more susceptible to shear stress and oxidative damage, and show increased vesiculation. Red blood cells, as essentially all cells, constitutively release phospholipid extracellular vesicles in vivo and in vitro in a process known as vesiculation. These extracellular vesicles comprise a heterogeneous group of vesicles of different sizes and intracellular origins. They are described in literature as exosomes if they originate from multi-vesicular bodies, or as microvesicles when formed by a one-step budding process directly from the plasma membrane. Extracellular vesicles contain a multitude of bioactive molecules that are implicated in intercellular communication and in different biological and pathophysiological processes. Mature red blood cells release in principle only microvesicles. In hereditary hemolytic anemias, the underlying molecular defect affects and determines red blood cell vesiculation, resulting in shedding microvesicles of different compositions and concentrations. Despite extensive research into red blood cell biochemistry and physiology, little is known about red cell deformability and vesiculation in hereditary hemolytic anemias, and the associated pathophysiological role is incompletely assessed. In this review, we discuss recent progress in understanding extracellular vesicles biology, with focus on red blood cell vesiculation. Also, we review recent scientific findings on the molecular defects of hereditary hemolytic anemias, and their correlation with red blood cell deformability and vesiculation. Integrating bio-analytical findings on abnormalities of red blood cells and their microvesicles will be critical for a better understanding of the pathophysiology of hereditary hemolytic anemias. PMID

  11. Red blood cell vesiculation in hereditary hemolytic anemia.

    PubMed

    Alaarg, Amr; Schiffelers, Raymond M; van Solinge, Wouter W; van Wijk, Richard

    2013-12-13

    Hereditary hemolytic anemia encompasses a heterogeneous group of anemias characterized by decreased red blood cell survival because of inherited membrane, enzyme, or hemoglobin disorders. Affected red blood cells are more fragile, less deformable, and more susceptible to shear stress and oxidative damage, and show increased vesiculation. Red blood cells, as essentially all cells, constitutively release phospholipid extracellular vesicles in vivo and in vitro in a process known as vesiculation. These extracellular vesicles comprise a heterogeneous group of vesicles of different sizes and intracellular origins. They are described in literature as exosomes if they originate from multi-vesicular bodies, or as microvesicles when formed by a one-step budding process directly from the plasma membrane. Extracellular vesicles contain a multitude of bioactive molecules that are implicated in intercellular communication and in different biological and pathophysiological processes. Mature red blood cells release in principle only microvesicles. In hereditary hemolytic anemias, the underlying molecular defect affects and determines red blood cell vesiculation, resulting in shedding microvesicles of different compositions and concentrations. Despite extensive research into red blood cell biochemistry and physiology, little is known about red cell deformability and vesiculation in hereditary hemolytic anemias, and the associated pathophysiological role is incompletely assessed. In this review, we discuss recent progress in understanding extracellular vesicles biology, with focus on red blood cell vesiculation. Also, we review recent scientific findings on the molecular defects of hereditary hemolytic anemias, and their correlation with red blood cell deformability and vesiculation. Integrating bio-analytical findings on abnormalities of red blood cells and their microvesicles will be critical for a better understanding of the pathophysiology of hereditary hemolytic anemias.

  12. Hemodynamic effects of red blood cell aggregation.

    PubMed

    Baskurt, Oguz K; Meiselman, Herbert J

    2007-01-01

    The influence of red blood cell (RBC) aggregation on blood flow in vivo has been under debate since early 1900's, yet a full understanding has still has not been reached. Enhanced RBC aggregation is well known to increase blood viscosity measured in rotational viscometers. However, it has been demonstrated that RBC aggregation may decrease flow resistance in cylindrical tubes, due to the formation of a cell-poor zone near the tube wall which results from the enhanced central accumulation of RBC. There is also extensive discussion regarding the effects of RBC aggregation on in vivo blood flow resistance. Several groups have reported increased microcirculatory flow resistance with enhanced RBC aggregation in experiments that utilized intravital microscopy. Alternatively, whole organ studies revealed that flow resistance may be significantly decreased if RBC aggregation is enhanced. Recently, new techniques have been developed to achieve well-controlled, graded alterations in RBC aggregation without influencing suspending phase properties. Studies using this technique revealed that the effects of RBC aggregation are determined by the degree of aggregation changes, and that this relationship can be explained by different hemodynamic mechanisms.

  13. Developmental Plasticity of Red Blood Cell Homeostasis

    PubMed Central

    Golub, Mari S.; Hogrefe, Casey E.; Malka, Roy; Higgins, John M.

    2014-01-01

    Most human physiologic set points like body temperature are tightly regulated and show little variation between healthy individuals. Red blood cell (RBC) characteristics such as hematocrit (HCT) and mean cell volume (MCV) are stable within individuals but can vary by 20% from one healthy person to the next. The mechanisms for the majority of this inter-individual variation are unknown and do not appear to involve common genetic variation. Here we show that environmental conditions present during development, namely in utero iron availability, can exert long-term influence on a set point related to the RBC life cycle. In a controlled study of rhesus monkeys and a retrospective study of humans, we use a mathematical model of in vivo RBC population dynamics to show that in utero iron deficiency is associated with a lowered threshold for RBC clearance and turnover. This in utero effect is plastic, persisting at least two years after birth and after the cessation of iron deficiency. Our study reports a rare instance of developmental plasticity in the human hematologic systems and also shows how mathematical modeling can be used to identify cellular mechanisms involved in the adaptive control of homeostatic set points. PMID:24415575

  14. Red blood cells in retinal vascular disorders.

    PubMed

    Agrawal, Rupesh; Sherwood, Joseph; Chhablani, Jay; Ricchariya, Ashutosh; Kim, Sangho; Jones, Philip H; Balabani, Stavroula; Shima, David

    2016-01-01

    Microvascular circulation plays a vital role in regulating physiological functions, such as vascular resistance, and maintaining organ health. Pathologies such as hypertension, diabetes, or hematologic diseases affect the microcirculation posing a significant risk to human health. The retinal vasculature provides a unique window for non-invasive visualisation of the human circulation in vivo and retinal vascular image analysis has been established to predict the development of both clinical and subclinical cardiovascular, metabolic, renal and retinal disease in epidemiologic studies. Blood viscosity which was otherwise thought to play a negligible role in determining blood flow based on Poiseuille's law up to the 1970s has now been shown to play an equally if not a more important role in controlling microcirculation and quantifying blood flow. Understanding the hemodynamics/rheology of the microcirculation and its changes in diseased states remains a challenging task; this is due to the particulate nature of blood, the mechanical properties of the cells (such as deformability and aggregability) and the complex architecture of the microvasculature. In our review, we have tried to postulate a possible role of red blood cell (RBC) biomechanical properties and laid down future framework for research related to hemorrheological aspects of blood in patients with retinal vascular disorders.

  15. Red blood cell transfusion in clinical practice.

    PubMed

    Klein, Harvey G; Spahn, Donat R; Carson, Jeffrey L

    2007-08-04

    Every year, about 75 million units of blood are collected worldwide. Red blood cell (RBC) transfusion is one of the few treatments that adequately restore tissue oxygenation when oxygen demand exceeds supply. Although the respiratory function of blood has been studied intensively, the trigger for RBC transfusion remains controversial, and doctors rely primarily on clinical experience. Laboratory assays that indicate failing tissue oxygenation would be ideal to guide the need for transfusion, but none has proved easy, reproducible, and sensitive to regional tissue hypoxia. The clinical importance of the RBCs storage lesion (ie, the time-dependent metabolic, biochemical, and molecular changes that stored blood cells undergo) is poorly understood. RBCs can be filtered, washed, frozen, or irradiated for specific indications. Donor screening and testing have dramatically reduced infectious risks in the developed world, but infection remains a major hazard in developing countries, where 13 million units of blood are not tested for HIV or hepatitis viruses. Pathogen inactivation techniques are in clinical trials for RBCs, but none is available for use. Despite serious immunological and non-immunological complications, RBC transfusion holds a therapeutic index that exceeds that of many common medications.

  16. Red blood cell storage duration and trauma.

    PubMed

    Sparrow, Rosemary L

    2015-04-01

    Numerous retrospective clinical studies suggest that transfusion of longer stored red blood cells (RBCs) is associated with an independent risk of poorer outcomes for certain groups of patients, including trauma, intensive care, and cardiac surgery patients. Large multicenter randomized controlled trials are currently underway to address the concern about RBC storage duration. However, none of these randomized controlled trials focus specifically on trauma patients with hemorrhage. Major trauma, particularly due to road accidents, is the leading cause of critical injury in the younger-than-40-year-old age group. Severe bleeding associated with major trauma induces hemodynamic dysregulation that increases the risk of hypoxia, coagulopathy, and potentially multiorgan failure, which can be fatal. In major trauma, a multitude of stress-associated changes occur to the patient's RBCs, including morphological changes that increase cell rigidity and thereby alter blood flow hemodynamics, particularly in the microvascular vessels, and reduce RBC survival. Initial inflammatory responses induce deleterious cellular interactions, including endothelial activation, RBC adhesion, and erythrophagocytosis that are quickly followed by profound immunosuppressive responses. Stored RBCs exhibit similar biophysical characteristics to those of trauma-stressed RBCs. Whether transfusion of RBCs that exhibit storage lesion changes exacerbates the hemodynamic perturbations already active in the trauma patient is not known. This article reviews findings from several recent nonrandomized studies examining RBC storage duration and clinical outcomes in trauma patients. The rationale for further research on RBC storage duration in the trauma setting is provided. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Understanding red blood cell alloimmunization triggers.

    PubMed

    Hendrickson, Jeanne E; Tormey, Christopher A

    2016-12-02

    Blood group alloimmunization is "triggered" when a person lacking a particular antigen is exposed to this antigen during transfusion or pregnancy. Although exposure to an antigen is necessary for alloimmunization to occur, it is not alone sufficient. Blood group antigens are diverse in structure, function, and immunogenicity. In addition to red blood cells (RBCs), a recipient of an RBC transfusion is exposed to donor plasma, white blood cells, and platelets; the potential contribution of these elements to RBC alloimmunization remains unclear. Much attention in recent years has been placed on recipient factors that influence RBC alloantibody responses. Danger signals, identified in murine and human studies alike as being risk factors for alloimmunization, may be quite diverse in nature. In addition to exogenous or condition-associated inflammation, autoimmunity is also a risk factor for alloantibody formation. Triggers for alloimmunization in pregnancy are not well-understood beyond the presence of a fetal/maternal bleed. Studies using animal models of pregnancy-induced RBC alloimmunization may provide insight in this regard. A better understanding of alloimmunization triggers and signatures of "responders" and "nonresponders" is needed for prevention strategies to be optimized. A common goal of such strategies is increased transfusion safety and improved pregnancy outcomes.

  18. Quantum refinement of protein structures: implementation and application to the red fluorescent protein DsRed.M1.

    PubMed

    Hsiao, Ya-Wen; Sanchez-Garcia, Elsa; Doerr, Markus; Thiel, Walter

    2010-11-25

    Quantum refinement is an improvement upon the molecular mechanics (MM)-based crystallographic refinement. In the latter, X-ray data are supplemented with additional chemical information through MM force fields, whereas quantum refinement describes crucial regions of interest in the macromolecule by quantum mechanics (QM) instead of MM. In this paper, we report the implementation of quantum refinement in the ChemShell QM/MM framework and its application in an investigation of the chromophore structure of the red fluorescent protein DsRed.M1. Both mechanical and electrostatic QM/MM embedding schemes are implemented and tested. In the quantum refinement of DsRed.M1, the anionic red acylimine chromophore adopts a nearly orthogonal arrangement (rather than a cis or trans form), and the bond lengths in the acylimine moiety are more consistent with a phenolate (rather than a quinoid) structure. These findings are in contrast to the structure deduced from a standard crystallographic refinement (PDB: 2VAD), but in agreement with our earlier results from a purely theoretical QM/MM study. On the other hand, the quantum refinement of the anionic acylimine form of DsRed.M1 yields a hydrogen bonding network around the chromophore, especially with regard to the arrangement of the water molecules and the Glu148 residue, that is closer to the 2VAD structure than to the previously optimized QM/MM structure. In our earlier study the initial classical molecular dynamics (MD) simulations during QM/MM setup apparently exaggerated the mobility of the water molecules around the chromophore. On the basis of the present results, it seems likely that the Glu148 residue is protonated in the DsRed.M1 protein. The calculation of electronic excitation energies allows for further assessment of the proposed structures, especially in the chromophore region. Using a combination of density functional theory and multireference configuration interaction (DFT/MRCI), we find excellent agreement between

  19. Post-mortem re-cloning of a transgenic red fluorescent protein dog

    PubMed Central

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo

    2011-01-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification. PMID:22122908

  20. Post-mortem re-cloning of a transgenic red fluorescent protein dog.

    PubMed

    Hong, So Gun; Koo, Ok Jae; Oh, Hyun Ju; Park, Jung Eun; Kim, Minjung; Kim, Geon-A; Park, Eun Jung; Jang, Goo; Lee, Byeong-Chun

    2011-12-01

    Recently, the world's first transgenic dogs were produced by somatic cell nuclear transfer. However, cellular senescence is a major limiting factor for producing more advanced transgenic dogs. To overcome this obstacle, we rejuvenated transgenic cells using a re-cloning technique. Fibroblasts from post-mortem red fluorescent protein (RFP) dog were reconstructed with in vivo matured oocytes and transferred into 10 surrogate dogs. One puppy was produced and confirmed as a re-cloned dog. Although the puppy was lost during birth, we successfully established a rejuvenated fibroblast cell line from this animal. The cell line was found to stably express RFP and is ready for additional genetic modification.

  1. Atomic force microscopy: From red blood cells to immunohaematology.

    PubMed

    Yeow, Natasha; Tabor, Rico F; Garnier, Gil

    2017-05-11

    Atomic force microscopy (AFM) offers complementary imaging modes that can provide morphological and structural details of red blood cells (RBCs), and characterize interactions between specific biomolecules and RBC surface antigen. This review describes the applications of AFM in determining RBC health by the observation of cell morphology, elasticity and surface roughness. Measurement of interaction forces between plasma proteins and antibodies against RBC surface antigen using the AFM also brought new information to the immunohaematology field. With constant improvisation of the AFM in resolution and imaging time, the reaction of RBC to changes in the physico-chemistry of its environment and the presence of RBC surface antigen specific-biomolecules is achievable. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. A new material concept for the red cell membrane.

    PubMed

    Evans, E A

    1973-09-01

    The proposition is made that the red cell membrane is a two-dimensional, incompressible material and a general stress-strain law is developed for finite deformations. In the linear form, the character of such a material is analogous to a two-dimensional Mooney material (e.g., rubber), indicating that the molecular structure in the plane of the membrane would consist of long chains, randomly kinked and cross-linked in the natural state. The loose network could be provided by the protein component and the lipid phase could exist interstitially as a liquid bilayer, giving the membrane its two-dimensional incompressibility. The material provides the capability of large deformations exhibited by the discocyte and yet the rigidity associated with the osmotic spherocyte state. It is demonstrated that a membrane of this type can form a sphere at constant area. An illustrative example of the application to single cell discocyte-to-osmotic spherocyte transformations is presented.

  3. Active elastic network: cytoskeleton of the red blood cell.

    PubMed

    Gov, Nir S

    2007-01-01

    In red blood cells there is a cortical cytoskeleton; a two-dimensional elastic network of membrane-attached proteins. We describe, using a simple model, how the metabolic activity of the cell, through the consumption of ATP, controls the stiffness of this elastic network. The unusual mechanical property of active strain softening is described and compared to experimental data. As a by-product of this activity there is also an active contribution to the amplitude of membrane fluctuations. We model this membrane as a field of independent "curvature motors," and calculate the spectrum of active fluctuations. We find that the active cytoskeleton contributes to the amplitude of the membrane height fluctuations at intermediate wavelengths, as observed experimentally.

  4. Anti-galactose antibodies do not bind to normal human red cells

    SciTech Connect

    Kay, M.M.B.; Bosman, G.J.C.G.M.

    1986-03-01

    The authors investigated the possibility that senescent cell IgG might have an anti-galactose (anti-gal) specificity as suggested by others. Anti-gal was isolated from normal human serum with ..cap alpha.. melibiose-agarose. The assays used were hemagglutination, rosetting, phagocytosis, and /sup 125/I protein A binding assay, immunoblotting, and glycine/HCL, pH 2.3, versus sugar elutions. Results revealed binding of anti-gal to rabbit but not human RBC. Immunoblotting of anti-gal revealed labeling of approx.29 bands in rabbit red cell membranes and no labeling of autologous human red cell membranes. The authors attempted to inhibit binding of anti-gal with various sugars. Melibiose caused enhancement rather than inhibition of agglutination when used at concentrations reported by previous investigators to cause inhibition. Neither ..cap alpha.. melibiose or galactose caused inhibition of phagocytosis of senescent cells. Senescent cell IgG was not displaced from freshly isolated old red cells by incubation with melibiose or galactose as determined by an /sup 125/I protein A binding assay. The authors were also unable to elute IgG from stored red cells with galactose. The authors conclude that senescent cell IgG does not have an anti-galactose specificity. The authors were unable to demonstrate an anti-gal antibody to normal human red cells.

  5. Photochemical decontamination of red cell concentrates with the silicon phthalocyanine Pc 4 and red light

    NASA Astrophysics Data System (ADS)

    Ben-Hur, Ehud; Zuk, Maria M.; Oetjen, Joyce; Chan, Wai-Shun; Lenny, Leslie; Horowitz, Bernard

    1999-07-01

    Virus inactivation in red blood cells concentrates (RBCC) is being studied in order to increase the safety of the blood supply. For this purpose we have been studying the silicon phthalocyanine (Pc 4), a photosensitizer activated with red light. Two approaches were used to achieve enhanced selectivity of Pc 4 for virus inactivation. One was formulation of Pc 4 in liposomes that reduce its binding to red cells. The other was the use of a light emitting diode (LED) array emitting at 700 nm. Vesicular stomatitis virus (VSV) infectivity served as an endpoint for virus kill in treated RBCC. Red cell hemolysis and circulatory survival in rabbits served as measures for red cell damage. Treatment of small aliquots of human RBCC with 2 (mu) M Pc 4 in liposomes and 10 J/cm2 of 700 nm LED light in the presence of the quenches of reactive oxygen species glutathione and trolox resulted in 6 log10 inactivation of VSV. Under these conditions hemolysis of treated red cells stored at 4 degree(s)C for 21 days was only slightly above that of control cells. Rabbit RBCC similarly treated circulated with a half life of 7.5 days compared with 10.5 days of control. It is concluded that Pc 4 used as described here may be useful for viral decontamination of RBCC, pending toxicological and clinical studies.

  6. [Regularity of sugar-uptake in human red blood cells].

    PubMed

    Quan, Guo-Bo; Lü, Cui-Cui; Liu, Min-Xia; Hu, Wen-Bo; Wang, Yan; Han, Ying

    2006-06-01

    Lyophilization of human red blood cells has important significance in clinical application. Some sugars, especially trehalose, can be more tolerant of some organism or cells to dry environments, But, how to bring sugars into cells is a challenge. This study was aimed to investigate the regularity of sugar-uptake in human red blood cells. The absorption rate of trehalose and glucose in red blood cells, free hemoglobin level and erythrocyte deformation index were determined at different incubation temperature (4, 25 and 37 degrees C), different sugar concentration (0, 0.2, 0.4, 0.6, 0.8 and 1 mol/L) and different incubation time (1, 3, 5, 7 and 9 hours). The results showed that with increase of temperature and extracellular sugar concentration, the uptake of sugar in red blood cells also increased, the intracellular trehalose and glucose concentrations were over 30 mmol/L and 40 mmol/L respectively. The effects of incubation time on uptake of trehalose and glucose were different. With prolonging of incubation time, the uptake of trehalose showed firstly increase and then decrease, however, the uptake of glucose showed a constant increase. But the loading process had side-effect on free hemoglobin and maximum deformation index (MAXDI) of red blood cells, especially for trehalose, which mainly come from high osmotic pressure. It is concluded that the uptake of sugars in red blood cells is closely dependent on incubation temperature, extracellular sugar concentration and incubation time. In certain condition, the efficiency of sugar uptake is very high, but this process also damages red blood cells so as to affect the application of sugars in lyophilization of red blood cells. The research in the future should focus on how to deal with the relation between cell injury and uptake efficiency of sugar in red blood cells.

  7. Genetically engineered red cells expressing single domain camelid antibodies confer long-term protection against botulinum neurotoxin.

    PubMed

    Huang, Nai-Jia; Pishesha, Novalia; Mukherjee, Jean; Zhang, Sicai; Deshycka, Rhogerry; Sudaryo, Valentino; Dong, Min; Shoemaker, Charles B; Lodish, Harvey F

    2017-09-04

    A short half-life in the circulation limits the application of therapeutics such as single-domain antibodies (VHHs). We utilize red blood cells to prolong the circulatory half-life of VHHs. Here we present VHHs against botulinum neurotoxin A (BoNT/A) on the surface of red blood cells by expressing chimeric proteins of VHHs with Glycophorin A or Kell. Mice whose red blood cells carry the chimeric proteins exhibit resistance to 10,000 times the lethal dose (LD50) of BoNT/A, and transfusion of these red blood cells into naive mice affords protection for up to 28 days. We further utilize an improved CD34+ culture system to engineer human red blood cells that express these chimeric proteins. Mice transfused with these red blood cells are resistant to highly lethal doses of BoNT/A. We demonstrate that engineered red blood cells expressing VHHs can provide prolonged prophylactic protection against bacterial toxins without inducing inhibitory immune responses and illustrates the potentially broad translatability of our strategy for therapeutic applications.The therapeutic use of single-chain antibodies (VHHs) is limited by their short half-life in the circulation. Here the authors engineer mouse and human red blood cells to express VHHs against botulinum neurotoxin A (BoNT/A) on their surface and show that an infusion of these cells into mice confers long lasting protection against a high dose of BoNT/A.

  8. Neutral red uptake assay for the estimation of cell viability/cytotoxicity.

    PubMed

    Repetto, Guillermo; del Peso, Ana; Zurita, Jorge L

    2008-01-01

    The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.

  9. Dynamic insight into protein structure utilizing red edge excitation shift.

    PubMed

    Chattopadhyay, Amitabha; Haldar, Sourav

    2014-01-21

    Proteins are considered the workhorses in the cellular machinery. They are often organized in a highly ordered conformation in the crowded cellular environment. These conformations display characteristic dynamics over a range of time scales. An emerging consensus is that protein function is critically dependent on its dynamics. The subtle interplay between structure and dynamics is a hallmark of protein organization and is essential for its function. Depending on the environmental context, proteins can adopt a range of conformations such as native, molten globule, unfolded (denatured), and misfolded states. Although protein crystallography is a well established technique, it is not always possible to characterize various protein conformations by X-ray crystallography due to transient nature of these states. Even in cases where structural characterization is possible, the information obtained lacks dynamic component, which is needed to understand protein function. In this overall scenario, approaches that reveal information on protein dynamics are much appreciated. Dynamics of confined water has interesting implications in protein folding. Interfacial hydration combines the motion of water molecules with the slow moving protein molecules. The red edge excitation shift (REES) approach becomes relevant in this context. REES is defined as the shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of absorption spectrum. REES arises due to slow rates (relative to fluorescence lifetime) of solvent relaxation (reorientation) around an excited state fluorophore in organized assemblies such as proteins. Consequently, REES depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. In the case of a protein, the confined water in the protein creates a dipolar field that acts as the solvent for a fluorophore

  10. Light scattering by aggregated red blood cells

    NASA Astrophysics Data System (ADS)

    Tsinopoulos, Stephanos V.; Sellountos, Euripides J.; Polyzos, Demosthenes

    2002-03-01

    In low flow rates, red blood cells (RBCs) fasten together along their axis of symmetry and form a so-called rouleaux. The scattering of He-Ne laser light by a rouleau consisting of n (2 less-than-or-equal n less-than-or-equal 8) average-sized RBCs is investigated. The interaction problem is treated numerically by means of an advanced axisymmetric boundary element--fast Fourier transform methodology. The scattering problem of one RBC was solved first, and the results showed that the influence of the RBC's membrane on the scattering patterns is negligible. Thus the rouleau is modeled as an axisymmetric, homogeneous, low-contrast dielectric cylinder, on the surface of which appears, owing to aggregated RBCs, a periodic roughness along the direction of symmetry. The direction of the incident laser light is considered to be perpendicular to the scatterer's axis of symmetry. The differential scattering cross sections in both perpendicular and parallel scattering planes and for all the scattering angles are calculated and presented in detail.

  11. Effects of red blood cells on hemostasis.

    PubMed

    Roeloffzen, Wilfried W H; Kluin-Nelemans, Hanneke C; Bosman, Lotte; de Wolf, Joost Th M

    2010-07-01

    Currently there is no sensitive laboratory test to establish the influence of red blood cells (RBCs) on hemostasis. As thromboelastography (TEG) measures hemostasis in whole blood, taking into account the interactions of all cellular elements, we used this instrument to investigate the role that RBCs play in hemostasis. In 29 patients with chemotherapy-induced anemia we studied the effect of progressive anemia on the coagulation profile. In 24 patients with chronic anemia we studied the effect of transfusion of RBCs on coagulation. Finally, in 18 patients we evaluated whether storage time of RBCs has additional effects on hemostasis. We observed a significant negative correlation between hemoglobin and TEG variables related to both clot strength and elasticity (p < 0.05). Moreover, anemia was associated with a delay in the initiation of the coagulation cascade. Correction of anemia by RBC transfusion resulted in significant shortening of this initiation phase with now the opposite effect on clot strength and elasticity. The negative effects on clot quality were significantly worse when fresh RBCs were transfused compared to longer-stored RBCs. Furthermore, in contrast to the longer-stored RBCs, fresh RBCs did not enhance initial fibrin formation. In this study we found that anemia was associated with a delay in the initiation of the coagulation cascade with a finally formed clot with superior strength and viscoelastic properties. Transfusion of RBCs was associated with impaired clot quality, with even worse effects on the initial fibrin build-up and clot quality by fresh RBCs.

  12. Malaria parasites and red cell variants: when a house is not a home

    PubMed Central

    Taylor, Steve M.; Fairhurst, Rick M.

    2014-01-01

    Purpose of review Multiple red cell variants are known to confer protection from malaria. Here we review advances in identifying new variants that modulate malaria risk and in defining molecular mechanisms that mediate malaria protection. Recent findings New red cell variants, including an innate variant in the red cell’s major Ca2+ pump and the acquired state of iron deficiency, have been associated with protection from clinical falciparum malaria. The hemoglobin (Hb) mutants HbC and HbS – known to protect carriers from severe falciparum malaria – enhance parasite passage to mosquitoes and may promote malaria transmission. At the molecular level, substantial advances have been made in understanding the impact of HbS and HbC upon the interactions between host microRNAs and Plasmodium falciparum protein translation; remodeling of red cell cytoskeletal components and transport of parasite proteins to the red cell surface; and chronic activation of the human innate immune system which induces tolerance to blood-stage parasites. Several polymorphisms have now been associated with protection from clinical vivax malaria or reduced P. vivax density, including Southeast Asian ovalocytosis and two common forms of glucose-6-phosphate dehydrogenase deficiency. Summary Red cell variants that modulate malaria risk can serve as models to identify clinically relevant mechanisms of pathogenesis, and thus define parasite and host targets for next-generation therapies. PMID:24675047

  13. Quantification of red blood cells using atomic force microscopy.

    PubMed

    O'Reilly, M; McDonnell, L; O'Mullane, J

    2001-01-01

    For humans the sizes and shapes of their red blood cells are important indicators of well being. In this study, the feasibility of using the atomic force microscope (AFM) to provide the sizes and shapes of red blood cells has been investigated. An immobilisation procedure has been developed that enabled red blood cells to be reliably imaged by contact AFM in air. The shapes of the red blood cells were readily apparent in the AFM images. Various cell quantification parameters were investigated, including thickness, width, surface area and volume. Excellent correlation was found between the AFM-derived immobilised mean cell volume (IMCV) parameter and the mean cell volume (MCV) parameter used in current haematological practice. The correlation between MCV and IMCV values has validated the immobilisation procedure by demonstrating that the significant cell shrinkage that occurs during immobilisation and drying does not introduce quantification artifacts. Reliable IMCV values were obtained by quantifying 100 red blood cells and this typically required 3-5 AFM images of 100 microm x 100 microm area. This work has demonstrated that the AFM can provide in a single test the red blood cell size and shape data needed in the assessment of human health.

  14. Lack of Erythropoietic Inhibitory Effect of Serum From Patients with Congenital Pure Red Cell Aplasia

    ERIC Educational Resources Information Center

    Geller, Gary; And Others

    1975-01-01

    Serum of five children ages 1 to 19 months with congenital pure red cell aplasia (incomplete or defective development of red blood cells) was injected in normal mice to determine possible inhibition of red blood cell formulating stimulants. (CL)

  15. Lack of Erythropoietic Inhibitory Effect of Serum From Patients with Congenital Pure Red Cell Aplasia

    ERIC Educational Resources Information Center

    Geller, Gary; And Others

    1975-01-01

    Serum of five children ages 1 to 19 months with congenital pure red cell aplasia (incomplete or defective development of red blood cells) was injected in normal mice to determine possible inhibition of red blood cell formulating stimulants. (CL)

  16. Effects of helicopter transport on red blood cell components

    PubMed Central

    Otani, Taiichi; Oki, Ken-ichi; Akino, Mitsuaki; Tamura, Satoru; Naito, Yuki; Homma, Chihiro; Ikeda, Hisami; Sumita, Shinzou

    2012-01-01

    Background There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate. Materials and methods Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points. Results During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells. Discussion These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance. PMID:22153688

  17. Effects of helicopter transport on red blood cell components.

    PubMed

    Otani, Taiichi; Oki, Ken-ichi; Akino, Mitsuaki; Tamura, Satoru; Naito, Yuki; Homma, Chihiro; Ikeda, Hisami; Sumita, Shinzou

    2012-01-01

    There are no reported studies on whether a helicopter flight affects the quality and shelf-life of red blood cells stored in mannitol-adenine-phosphate. Seven days after donation, five aliquots of red blood cells from five donors were packed into an SS-BOX-110 container which can maintain the temperature inside the container between 2 °C and 6 °C with two frozen coolants. The temperature of an included dummy blood bag was monitored. After the box had been transported in a helicopter for 4 hours, the red blood cells were stored again and their quality evaluated at day 7 (just after the flight), 14, 21 and 42 after donation. Red blood cell quality was evaluated by measuring adenosine triphosphate, 2,3-diphosphoglycerate, and supernatant potassium, as well as haematocrit, intracellular pH, glucose, supernatant haemoglobin, and haemolysis rate at the various time points. During the experiment the recorded temperature remained between 2 and 6 °C. All data from the red blood cells that had undergone helicopter transportation were the same as those from a control group of red blood cell samples 7 (just after the flight), 14, 21, and 42 days after the donation. Only supernatant Hb and haemolysis rate 42 days after the donation were slightly increased in the helicopter-transported group of red blood cell samples. All other parameters at 42 days after donation were the same in the two groups of red blood cells. These results suggest that red blood cells stored in mannitol-adenine-phosphate are not significantly affected by helicopter transportation. The differences in haemolysis by the end of storage were small and probably not of clinical significance.

  18. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo.

    PubMed

    Burrill, Devin R; Vernet, Andyna; Collins, James J; Silver, Pamela A; Way, Jeffrey C

    2016-05-10

    The design of cell-targeted protein therapeutics can be informed by natural protein-protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics.

  19. Red oil A5 inhibits proliferation and induces apoptosis in pancreatic cancer cells.

    PubMed

    Dong, Mi-Lian; Ding, Xian-Zhong; Adrian, Thomas E

    2004-01-01

    To study the effect of red oil A5 on pancreatic cancer cells and its possible mechanisms. Effect of different concentrations of red oil A5 on proliferation of three pancreatic cancer cell lines, AsPC-1, MiaPaCa-2 and S2013, was measured by (3)H-methyl thymidine incorporation. Time-dependent effects of 1:32 000 red oil A5 on proliferation of three pancreatic cancer cell lines, were also measured by (3)H-methyl thymidine incorporation, and Time-course effects of 1:32 000 red oil A5 on cell number. The cells were counted by Z1-Coulter Counter. Flow-cytometric analysis of cellular DNA content in the control and red oil A5 treated AsPC-1, MiaPaCa-2 and S2013 cells, were stained with propidium iodide. TUNEL assay of red oil A5-induced pancreatic cancer cell apoptosis was performed. Western blotting of the cytochrome c protein in AsPC-1, MiaPaCa-2 and S2013 cells treated 24 hours with 1:32 000 red oil A5 was performed. Proteins in cytosolic fraction and in mitochondria fraction were extracted. Proteins extracted from each sample were electrophoresed on SDS-PAGE gels and then were transferred to nitrocellulose membranes. Cytochrome c was identified using a monoclonal cytochrome c antibody. Western blotting of the caspase-3 protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was carried out. Proteins in whole cellular lysates were electrophoresed on SDS-PAGE gels and then transferred to nitrocellulose membranes. Caspase-3 was identified using a specific antibody. Western blotting of poly-ADP ribose polymerase (PARP) protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was performed. Proteins in whole cellular lysates were separated by electrophoresis on SDS-PAGE gels and then transferred to nitrocellulose membranes. PARP was identified by using a monoclonal antibody. Red oil A5 caused dose- and time-dependent inhibition of pancreatic cancer cell proliferation. Propidium iodide DNA staining showed

  20. Diffusion pathways of oxygen species in the phototoxic fluorescent protein KillerRed.

    PubMed

    Roy, Arijit; Carpentier, Philippe; Bourgeois, Dominique; Field, Martin

    2010-10-28

    The fluorescent protein KillerRed generates reactive oxygen species through the CALI effect. This property paves the way for the design of genetically encoded photosensitizers for use in cell killing and cancer photodynamic therapy. In this article, we have investigated the diffusion pathways of di-oxygen and the superoxide radical in KillerRed, using molecular dynamics simulations. Our results suggest that, by comparison to the Ser-65-Thr mutant of GFP, diffusion of molecular oxygen (and singlet oxygen) is greatly facilitated in KillerRed, mostly due to the presence of a unique water-filled channel. In contrast, due to their negative charge, superoxide radical ions putatively produced inside the chromophore pocket are unable to escape the protein. These results are consistent with the hypothesis that superoxide generation, if it occurs, proceeds via light-induced photoreduction of the chromophore followed by long-range electron transfer, a mechanism in which the long hydrogen bond network through the channel could play a key role. Alternatively, the facilitated diffusion of di-oxygen through the channel suggests that singlet di-oxygen could be the principal cause of specific CALI of fused proteins. The entry of di-oxygen through the channel probably also accounts for the high susceptibility of KillerRed to photobleaching.

  1. Numerical analysis on cell-cell interaction of red blood cells during sedimentation

    NASA Astrophysics Data System (ADS)

    Shi, Xing

    2017-07-01

    The long-range hydrodynamic interaction among red blood cells plays an important role on the macroscopic behaviors, however, the molecular interaction at such scale is much weaker. In this paper, the sedimentations under external body force of two red blood cells are numerical simulated to investigate the hydrodynamic interaction between cells. The flow is solved by lattice Boltzmann method and the membrane of red blood cell is model by the spring model where the fluid-membrane interaction is coupled by fictitious domain method. It is found that the cells have the tendency to aggregate and may be aligned in a line along the sediment direction. Compared to the properties of a single cell under the same conditions, the sediment velocity of red blood cell group is larger; the leading cell deforms less and the following cell endures larger deformation.

  2. Red cell exchange: special focus on sickle cell disease.

    PubMed

    Kim, Haewon C

    2014-12-05

    The primary function of red blood cells (RBCs) is to deliver oxygen from the lungs to tissues. Tissue hypoxia occurs when the oxygen-carrying capacity of RBCs is compromised due primarily to 3 causes: (1) a reduction in circulating RBC mass, (2) an increase in circulating RBC mass, or (3) abnormal hemoglobin (Hb) that either does not sufficiently release oxygen to tissues (high-oxygen-affinity hemoglobin) or occludes the microvasculature due to deformed RBCs (sickled RBCs). To improve oxygenation in patients with reduced or increased RBC mass, RBC administration (simple transfusion) or RBC removal (RBC depletion) is performed, respectively. However, for patients with abnormal Hb, RBCs containing abnormal Hb are removed and replaced by healthy volunteer donor RBCs by red cell exchange (RCE). RCE can be performed by manual exchange or by automated exchange using a blood cell separator (erythrocytapheresis). In this review, indications for RCE in sickle cell disease using the evidence-based American Society for Apheresis categories(1) are presented and the rationale for RCE in each disorder are discussed. Simple transfusion versus RCE and manual RCE versus automated RCE are compared. Finally, this review briefly presents some of the challenges of performing erythrocytapheresis in small children and discusses various choices for central venous access during RCE.(2.)

  3. 21 CFR 864.7100 - Red blood cell enzyme assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red blood cell enzyme assay. 864.7100 Section 864.7100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7100 Red blood...

  4. Preclinical investigation of the pharmacokinetics, metabolism, and protein and red blood cell binding of DRDE-07: a prophylactic agent against sulphur mustard.

    PubMed

    Verma, Pankaj; Vijayaraghavan, Rajagopalan

    2014-10-01

    DRDE-07, a newly synthesized amifostine analog currently under clinical investigation in a phase I trial, is a potent antidote against sulfur mustard toxicity. The purpose of this research was to evaluate the pharmacokinetic profile of DRDE-07 in female Swiss Albino mice after a single oral dose of 400 or 600 mg/kg. The physicochemical properties of DRDE-07, including solubility, pK a, Log P, plasma protein binding and plasma/blood partitioning, were determined to support the pharmacokinetic characterization. DRDE-07 concentration was determined by an HPLC-UV method. The profile of plasma concentration versus time was analyzed using a non-compartmental model. Plasma protein binding was assessed using ultrafiltration. DRDE-07 appeared rapidly in plasma after oral administration with peak plasma levels (C max) observed in less than 15 min. There was a rapid decline in the plasma levels followed by a smaller second peak about 90 min after dosing. The plasma protein binding of DRDE-07 was found to be less than 25% at all concentrations studied. Plasma clearance of DRDE-07 is expected to be ~1.5 fold higher than the blood clearance of DRDE-07. The probable metabolite of DRDE-07 was identified as phenyl-S-ethyl amine.

  5. Nitric Oxide Scavenging by Red Cell Microparticles and Cell Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion

    PubMed Central

    Donadee, Chenell; Raat, Nicolaas J.H.; Kanias, Tamir; Tejero, Jesús; Lee, Janet S.; Kelley, Eric E.; Zhao, Xuejun; Liu, Chen; Reynolds, Hannah; Azarov, Ivan; Frizzell, Sheila; Meyer, E Michael; Donnenberg, Albert D.; Qu, Lirong; Triulzi, Darrel; Kim-Shapiro, Daniel B.; Gladwin, Mark T.

    2013-01-01

    Background Intravascular red cell hemolysis impairs NO-redox homeostasis, producing endothelial dysfunction, platelet activation and vasculopathy. Red blood cell storage under standard conditions results in reduced integrity of the erythrocyte membrane, with formation of exocytic microvesicles or “microparticles” and hemolysis, which we hypothesized could impair vascular function and contribute to the putative “storage lesion” of banked blood. Methods and Results We now find that storage of human red blood cells under standard blood banking conditions results in the accumulation of cell free and microparticle-encapsulated hemoglobin which, despite 39 days of storage, remains in the reduced ferrous oxyhemoglobin redox state and stoichiometrically reacts with and scavenges the vasodilator nitric oxide (NO). Using stopped-flow spectroscopy and laser triggered NO release from a caged NO compound we found that both free hemoglobin and microparticles react with NO about 1000 times faster than with intact erythrocytes. In complementary in vivo studies we show that hemoglobin, even at concentrations below 10 μM (in heme), produces potent vasoconstriction when infused into the rat circulation, while controlled infusions of methemoglobin and cyanomethemoglobin, which do not consume NO, have substantially reduced vasoconstrictor effects. Infusion of the plasma from stored human red cell units into the rat circulation produces significant vasoconstriction related to the magnitude of storage related hemolysis. Conclusions The results of these studies suggest new mechanisms for endothelial injury and impaired vascular function associated with the most fundamental of storage lesions, hemolysis. PMID:21747051

  6. Single molecule spectroscopic characterization of a far-red fluorescent protein (HcRed) from the Anthozoa coral Heteractis crispa

    NASA Astrophysics Data System (ADS)

    Cotlet, Mircea; Habuchi, Satoshi; Whitier, Jennifer E.; Werner, James H.; De Schryver, Frans C.; Hofkens, Johan; Goodwin, Peter M.

    2006-02-01

    We report on the photophysical properties of a far-red intrinsic fluorescent protein by means of single molecule and ensemble spectroscopic methods. The green fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent marker with genetically encoded fluorescence and which can be fused to any biological structure without affecting its function. GFP and its variants provide emission colors from blue to yellowish green. Red intrinsic fluorescent proteins from Anthozoa species represent a recent addition to the emission color palette provided by GFPs. Red intrinsic fluorescent markers are on high demand in protein-protein interaction studies based on fluorescence-resonance energy transfer or in multicolor tracking studies or in cellular investigations where autofluorescence possesses a problem. Here we address the photophysical properties of a far-red fluorescent protein (HcRed), a mutant engineered from a chromoprotein cloned from the sea anemone Heteractis crispa, by using a combination of ensemble and single molecule spectroscopic methods. We show evidence for the presence of HcRed protein as an oligomer and for incomplete maturation of its chromophore. Incomplete maturation results in the presence of an immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow chromophore is involved in a fast resonance-energy transfer with the mature (purple) chromophore. The mature chromophore of HcRed is found to adopt two conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in solution and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These two forms co-exist in solution in thermal equilibrium. Excitation-power dependence fluorescence correlation spectroscopy of HcRed shows evidence for singlet-triplet transitions in the microseconds time scale and for cis-trans isomerization occurring in a time scale of tens of microseconds. Single molecule fluorescence data recorded from immobilized HcRed proteins, all

  7. The RhopH complex is transferred to the host cell cytoplasm following red blood cell invasion by Plasmodium falciparum.

    PubMed

    Vincensini, Laetitia; Fall, Gamou; Berry, Laurence; Blisnick, Thierry; Braun Breton, Catherine

    2008-08-01

    The high-molecular mass rhoptry protein complex (PfRhopH), which comprises three distinct gene products, RhopH1, RhopH2, and RhopH3, is known to be secreted and transferred to the parasitophorous vacuole membrane upon invasion of a red blood cell by the malaria parasite Plasmodium falciparum. Here we show that the merozoite-acquired RhopH complex is also transferred to defined domains of the red blood cell cytoplasm, and possibly transiently associated with Maurer's clefts. This is the first report of trafficking in the host cell cytoplasm for P. falciparum rhoptry proteins secreted upon red blood cell invasion. Based on its newly identified sub-cellular location and the phenotype of RhopH1 mutants, we propose that the RhopH complex participate in the assembly of the cytoadherence complex.

  8. Models for the Red Blood Cell Lifespan

    PubMed Central

    Shrestha, Rajiv P.; Horowitz, Joseph; Hollot, Christopher V.; Germain, Michael J.; Widness, John A.; Mock, Donald M.; Veng-Pedersen, Peter; Chait, Yossi

    2016-01-01

    The lifespan of red blood cells (RBCs) plays an important role in the study and interpretation of various clinical conditions. Yet, confusion about the meanings of fundamental terms related to cell survival and their quantification still exists in the literature. To address these issues, we started from a compartmental model of RBC populations based on an arbitrary full lifespan distribution, carefully defined the residual lifespan, current age, and excess lifespan of the RBC population, and then derived the distributions of these parameters. For a set of residual survival data from biotin-labeled RBCs, we fit models based on Weibull, gamma, and lognormal distributions, using nonlinear mixed effects (NLME) modeling and parametric bootstrapping. From the estimated Weibull, gamma, and lognormal parameters we computed the respective population mean full lifespans (95% confidence interval): 115.60 (109.17–121.66), 116.71 (110.81–122.51), and 116.79 (111.23–122.75) days together with the standard deviations of the full lifespans: 24.77 (20.82–28.81), 24.30 (20.53–28.33), and 24.19 (20.43–27.73). We then estimated the 95th percentiles of the lifespan distributions (a surrogate for the maximum lifespan): 153.95 (150.02–158.36), 159.51 (155.09–164.00), and 160.40 (156.00–165.58) days, the mean current ages (or the mean residual lifespans): 60.45 (58.18–62.85), 60.82 (58.77–63.33), and 57.26 (54.33–60.61) days, and the residual half-lives: 57.97 (54.96–60.90), 58.36 (55.45–61.26), and 58.40 (55.62–61.37) days, for the Weibull, gamma, and lognormal models respectively. Corresponding estimates were obtained for the individual subjects. The three models provide equally excellent goodness-of-fit, reliable estimation, and physiologically plausible values of the directly interpretable RBC survival parameters. PMID:27039311

  9. Models for the red blood cell lifespan.

    PubMed

    Shrestha, Rajiv P; Horowitz, Joseph; Hollot, Christopher V; Germain, Michael J; Widness, John A; Mock, Donald M; Veng-Pedersen, Peter; Chait, Yossi

    2016-06-01

    The lifespan of red blood cells (RBCs) plays an important role in the study and interpretation of various clinical conditions. Yet, confusion about the meanings of fundamental terms related to cell survival and their quantification still exists in the literature. To address these issues, we started from a compartmental model of RBC populations based on an arbitrary full lifespan distribution, carefully defined the residual lifespan, current age, and excess lifespan of the RBC population, and then derived the distributions of these parameters. For a set of residual survival data from biotin-labeled RBCs, we fit models based on Weibull, gamma, and lognormal distributions, using nonlinear mixed effects modeling and parametric bootstrapping. From the estimated Weibull, gamma, and lognormal parameters we computed the respective population mean full lifespans (95 % confidence interval): 115.60 (109.17-121.66), 116.71 (110.81-122.51), and 116.79 (111.23-122.75) days together with the standard deviations of the full lifespans: 24.77 (20.82-28.81), 24.30 (20.53-28.33), and 24.19 (20.43-27.73). We then estimated the 95th percentiles of the lifespan distributions (a surrogate for the maximum lifespan): 153.95 (150.02-158.36), 159.51 (155.09-164.00), and 160.40 (156.00-165.58) days, the mean current ages (or the mean residual lifespans): 60.45 (58.18-62.85), 60.82 (58.77-63.33), and 57.26 (54.33-60.61) days, and the residual half-lives: 57.97 (54.96-60.90), 58.36 (55.45-61.26), and 58.40 (55.62-61.37) days, for the Weibull, gamma, and lognormal models respectively. Corresponding estimates were obtained for the individual subjects. The three models provide equally excellent goodness-of-fit, reliable estimation, and physiologically plausible values of the directly interpretable RBC survival parameters.

  10. A label-free proteome analysis strategy for identifying quantitative changes in erythrocyte membranes induced by red cell disorders.

    PubMed

    Pesciotta, Esther N; Sriswasdi, Sira; Tang, Hsin-Yao; Mason, Philip J; Bessler, Monica; Speicher, David W

    2012-12-05

    Red blood cells have been extensively studied but many questions regarding membrane properties and pathophysiology remain unanswered. Proteome analysis of red cell membranes is complicated by a very wide dynamic range of protein concentrations as well as the presence of proteins that are very large, very hydrophobic, or heterogeneously glycosylated. This study investigated the removal of other blood cell types, red cell membrane extraction, differing degrees of fractionation using 1-D SDS gels, and label-free quantitative methods to determine optimized conditions for proteomic comparisons of clinical blood samples. The results showed that fractionation of red cell membranes on 1-D SDS gels was more efficient than low-ionic-strength extractions followed by 1-D gel fractionation. When gel lanes were sliced into 30 uniform slices, a good depth of analysis that included the identification of most well-characterized, low-abundance red cell membrane proteins including those present at 500 to 10,000 copies per cell was obtained. Furthermore, the size separation enabled detection of changes due to proteolysis or in vivo protein crosslinking. A combination of Rosetta Elucidator quantitation and subsequent statistical analysis enabled the robust detection of protein differences that could be used to address unresolved questions in red cell disorders. This article is part of a Special Issue entitled: Integrated omics.

  11. Increased red cell calcium, decreased calcium adenosine triphosphatase, and altered membrane proteins during fava bean hemolysis in glucose-6-phosphate dehydrogenase-deficient (Mediterranean variant) individuals.

    PubMed

    Turrini, F; Naitana, A; Mannuzzu, L; Pescarmona, G; Arese, P

    1985-08-01

    RBCs from four glucose-6-phosphate dehydrogenase (G6PD)-deficient (Mediterranean variant) subjects were studied during fava bean hemolysis. In the density-fractionated RBC calcium level, Ca2+-ATPase activity, reduced glutathione level, and ghost protein pattern were studied. In the bottom fraction, containing most heavily damaged RBCs, calcium level ranged from 143 to 244 mumol/L RBCs (healthy G6PD-deficient controls: 17 +/- 5 mumol/L RBCs). The Ca2+-ATPase activity ranged from 0.87 to 1.84 mumol ATP consumed/g Hb/min (healthy G6PD-deficient controls: 2.27 +/- 0.4). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of ghosts showed: (1) the presence of high mol wt aggregates (in three cases they were reduced by dithioerythritol; in one case, only partial reduction was possible); (2) the presence of multiple, scattered new bands; and (3) the reduction of band 3. Oxidant-mediated damage to active calcium extrusion, hypothetically associated with increased calcium permeability, may explain the large increase in calcium levels. They, in turn, could activate calcium-dependent protease activity, giving rise to the profound changes in the ghost protein pattern.

  12. The curious genomic path from leaky red cell to nephrotic kidney.

    PubMed

    Stewart, G W; Fricke, B

    2003-01-01

    The human red cell has proved to be an invaluable model cell for the study of many aspects of membrane structure and function. It has a series of transport pathways which mediate the movements of the univalent cations Na and K, which are either identical or similar to systems in other human tissues, including the human kidney. The balance between the energy-consuming NaK pump and a 'passive leak' component maintains a net deficit of cations within the cell, which defends the cell volume against osmotic swelling. There exist a series of dominantly inherited human red cell conditions, gathered under the generic title 'hereditary stomatocytoses', in which the so-called 'passive leak' to Na and K is pathologically increased. In the more severe variants this compromises the integrity of the cell and the patients suffer haemolytic anaemia. Some less severe variants present with pseudohyperkalaemia caused by loss of K from red cells on storage of blood at room temperature. The most severe variants show a deficiency in a widely distributed 'raft' protein known as stomatin. The stomatin protein is homologous to the 'podocin' protein, the gene for which is mutated in a recessively inherited form of nephrotic syndrome. Among other possible functions, both proteins could be involved in the trafficking of membrane proteins to and from the plasma membrane.

  13. Technetium-99m-labeled red blood cell imaging

    SciTech Connect

    Front, D.; Israel, O.; Groshar, D.; Weininger, J.

    1984-07-01

    Red blood cells labeled with 99mTc constitute a suitable intravascular agent for imaging of vascular abnormalities. Hemangiomas are characterized by low perfusion and a high blood pool. This ''perfusion blood-pool mismatch,'' not encountered in other lesions, may help in the specific diagnosis of this tumor. This is particularly so in cavernous hemangiomas of the liver where three-phase 99mTc-labeled red blood cell scintigraphy should precede liver biopsy. Red cell scintigraphy also is useful for establishing the vascular nature of hemangiomas of the head and neck and the skin and for diagnosis of venous occlusion. Heat-damaged red blood cells provide a specific spleen imaging agent. This should be used when patients with suspected splenic pathology have equivocal colloid scintigraphy.

  14. Astringency reduction in red wine by whey proteins.

    PubMed

    Jauregi, Paula; Olatujoye, Jumoke B; Cabezudo, Ignacio; Frazier, Richard A; Gordon, Michael H

    2016-05-15

    Whey is a by-product of cheese manufacturing and therefore investigating new applications of whey proteins will contribute towards the valorisation of whey and hence waste reduction. This study shows for the first time a detailed comparison of the effectiveness of gelatin and β-lactoglobulin (β-LG) as fining agents. Gelatin was more reactive than whey proteins to tannic acid as shown by both the astringency method (with ovalbumin as a precipitant) and the tannins determination method (with methylcellulose as a precipitant). The two proteins showed similar selectivity for polyphenols but β-LG did not remove as much catechin. The fining agent was removed completely or to a trace level after centrifugation followed by filtration which minimises its potential allergenicity. In addition, improved understanding of protein-tannin interactions was obtained by fluorescence, size measurement and isothermal titration calorimetry (ITC). Overall this study demonstrates that whey proteins have the potential of reducing astringency in red wine and can find a place in enology.

  15. Biophotonic techniques for the study of malaria-infected red blood cells.

    PubMed

    Mauritz, Jakob M A; Esposito, Alessandro; Tiffert, Teresa; Skepper, Jeremy N; Warley, Alice; Yoon, Young-Zoon; Cicuta, Pietro; Lew, Virgilio L; Guck, Jochen R; Kaminski, Clemens F

    2010-10-01

    Investigation of the homeostasis of red blood cells upon infection by Plasmodium falciparum poses complex experimental challenges. Changes in red cell shape, volume, protein, and ion balance are difficult to quantify. In this article, we review a wide range of optical techniques for quantitative measurements of critical homeostatic parameters in malaria-infected red blood cells. Fluorescence lifetime imaging and tomographic phase microscopy, quantitative deconvolution microscopy, and X-ray microanalysis, are used to measure haemoglobin concentration, cell volume, and ion contents. Atomic force microscopy is briefly reviewed in the context of these optical methodologies. We also describe how optical tweezers and optical stretchers can be usefully applied to empower basic malaria research to yield diagnostic information on cell compliance changes upon malaria infection. The combined application of these techniques sheds new light on the detailed mechanisms of malaria infection providing potential for new diagnostic or therapeutic approaches.

  16. Targeted erythropoietin selectively stimulates red blood cell expansion in vivo

    PubMed Central

    Burrill, Devin R.; Vernet, Andyna; Collins, James J.; Silver, Pamela A.; Way, Jeffrey C.

    2016-01-01

    The design of cell-targeted protein therapeutics can be informed by natural protein–protein interactions that use cooperative physical contacts to achieve cell type specificity. Here we applied this approach in vivo to the anemia drug erythropoietin (EPO), to direct its activity to EPO receptors (EPO-Rs) on red blood cell (RBC) precursors and prevent interaction with EPO-Rs on nonerythroid cells, such as platelets. Our engineered EPO molecule was mutated to weaken its affinity for EPO-R, but its avidity for RBC precursors was rescued via tethering to an antibody fragment that specifically binds the human RBC marker glycophorin A (huGYPA). We systematically tested the impact of these engineering steps on in vivo markers of efficacy, side effects, and pharmacokinetics. huGYPA transgenic mice dosed with targeted EPO exhibited elevated RBC levels, with only minimal platelet effects. This in vivo selectivity depended on the weakening EPO mutation, fusion to the RBC-specific antibody, and expression of huGYPA. The terminal plasma half-life of targeted EPO was ∼28.3 h in transgenic mice vs. ∼15.5 h in nontransgenic mice, indicating that huGYPA on mature RBCs acted as a significant drug sink but did not inhibit efficacy. In a therapeutic context, our targeting approach may allow higher restorative doses of EPO without platelet-mediated side effects, and also may improve drug pharmacokinetics. These results demonstrate how rational drug design can improve in vivo specificity, with potential application to diverse protein therapeutics. PMID:27114509

  17. Ex-vivo generation of human red cells for transfusion.

    PubMed

    Anstee, David J; Gampel, Alexandra; Toye, Ashley M

    2012-05-01

    The present article reviews the recent data concerning the generation of red blood cells from haematopoietic stem cells using laboratory culture and discusses the potential for generating cultured red cells in sufficient quantity for use in transfusion practice. Functional human reticulocytes have been generated from adult peripheral blood haematopoietic stem cells in laboratory culture without the use of heterologous feeder cells and their viability was demonstrated in vivo. Human erythroid progenitor cells lines have been produced from cord and human induced pluripotent stem cell (hiPSC) haematopoietic progenitors. Availability of cultured human red cells from haematopoietic stem cells in the quantities required for transfusion therapy would have a major impact on healthcare provision worldwide. Recent studies provide cause for optimism that this ambitious goal is achievable. Functional adult reticulocytes have been made in culture and shown to survive in vivo. Erythroid progenitor cell lines have been derived from cord blood and from human induced pluripotent stem cells, suggesting that large-scale culture of erythroid cell lines and their differentiation to reticulocytes will be possible. Significant problems remain. More efficient enucleation and induction of maturation to an adult phenotype will be required in order to exploit high proliferative capacity of human embryonic stem cells and hiPSCs. Novel bioengineering solutions will be required to generate cultured red cells in the large quantities required, and in this context, use of synthetic three-dimensional scaffolds to mimic the bone marrow niche holds great promise for the future.

  18. Reflectance confocal microscopy of red blood cells: simulation and experiment.

    PubMed

    Zeidan, Adel; Yelin, Dvir

    2015-11-01

    Measuring the morphology of red blood cells is important for clinical diagnosis, providing valuable indications on a patient's health. In this work, we have simulated the appearance of normal red blood cells under a reflectance confocal microscope and discovered unique relations between the morphological parameters and the resulting characteristic interference patterns of the cell. The simulation results showed good agreement with in vitro reflectance confocal images of red blood cells, acquired using spectrally encoded flow cytometry that imaged the cells in a linear flow without artificial staining. By matching the simulated patterns to confocal images of the cells, this method could be used for measuring cell morphology in three dimensions and for studying their physiology.

  19. Contamination of ribosome inactivating proteins with ribonucleases, separated by affinity chromatography on red sepharose.

    PubMed

    Wang, H X; Ng, T B; Cheng, C H K; Fong, W P

    2003-05-01

    Three preparations of type 1 ribosome inactivating proteins (RIPs), namely, agrostin, saporin, and luffin, were subjected to affinity chromatography on Red Sepharose and eluted with a linear concentration gradient of NaCl in 10 mM Tris-HCl buffer (pH 7.4). The eluate was assayed for ability to inhibit translation in a cell-free rabbit reticulocyte lysate system which measures RIP activity, and for ability to hydrolyze yeast transfer RNA which measures RNase activity. It was found that, in all three RIP preparations, the peak of RIP activity, which coincided with the peak of absorbance at 280 nm, was eluted earlier than the peak of RNase activity. It appears that RNase is a possible contaminant of ribosome inactivating protein preparations and that this contamination can be minimized by using Red Sepharose.

  20. Multi-omics Evidence for Inheritance of Energy Pathways in Red Blood Cells.

    PubMed

    Weisenhorn, Erin M M; van T Erve, Thomas J; Riley, Nicholas M; Hess, John R; Raife, Thomas J; Coon, Joshua J

    2016-12-01

    Each year over 90 million units of blood are transfused worldwide. Our dependence on this blood supply mandates optimized blood management and storage. During storage, red blood cells undergo degenerative processes resulting in altered metabolic characteristics which may make blood less viable for transfusion. However, not all stored blood spoils at the same rate, a difference that has been attributed to variable rates of energy usage and metabolism in red blood cells. Specific metabolite abundances are heritable traits; however, the link between heritability of energy metabolism and red blood cell storage profiles is unclear. Herein we performed a comprehensive metabolomics and proteomics study of red blood cells from 18 mono- and di-zygotic twin pairs to measure heritability and identify correlations with ATP and other molecular indices of energy metabolism. Without using affinity-based hemoglobin depletion, our work afforded the deepest multi-omic characterization of red blood cell membranes to date (1280 membrane proteins and 330 metabolites), with 119 membrane protein and 148 metabolite concentrations found to be over 30% heritable. We demonstrate a high degree of heritability in the concentration of energy metabolism metabolites, especially glycolytic metabolites. In addition to being heritable, proteins and metabolites involved in glycolysis and redox metabolism are highly correlated, suggesting that crucial energy metabolism pathways are inherited en bloc at distinct levels. We conclude that individuals can inherit a phenotype composed of higher or lower concentrations of these proteins together. This can result in vastly different red blood cells storage profiles which may need to be considered to develop precise and individualized storage options. Beyond guiding proper blood storage, this intimate link in heritability between energy and redox metabolism pathways may someday prove useful in determining the predisposition of an individual toward metabolic

  1. Method for determining properties of red blood cells

    SciTech Connect

    Gourley, Paul L.

    2001-01-01

    A method for quantifying the concentration of hemoglobin in a cell, and indicia of anemia, comprises determining the wavelength of the longitudinal mode of a liquid in a laser microcavity; determining the wavelength of the fundamental transverse mode of a red blood cell in the liquid in the laser microcavity; and determining if the cell is anemic from the difference between the wavelength of the longitudinal mode and the fundamental transverse mode. In addition to measuring hemoglobin, the invention includes a method using intracavity laser spectroscopy to measure the change in spectra as a function of time for measuring the influx of water into a red blood cell and the cell's subsequent rupture.

  2. Red blood cell-derived microparticles: An overview.

    PubMed

    Westerman, Maxwell; Porter, John B

    2016-07-01

    The red blood cell (RBC) is historically the original parent cell of microparticles (MPs). In this overview, we describe the discovery and the early history of red cell-derived microparticles (RMPs) and present an overview of the evolution of RMP. We report the formation, characteristics, effects of RMP and factors which may affect RMP evaluation. The review examines RMP derived from both normal and pathologic RBC. The pathologic RBC studies include sickle cell anemia (SCA), sickle cell trait (STr), thalassemia intermedia (TI), hereditary spherocytosis (HS), hereditary elliptocytosis (HE), hereditary stomatocytosis (HSt) and glucose-6-phosphate dehydrogenase deficiency (G6PD). Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Bright and stable near infra-red fluorescent protein for in vivo imaging

    PubMed Central

    Filonov, Grigory S.; Piatkevich, Kiryl D.; Ting, Li-Min; Zhang, Jinghang; Kim, Kami; Verkhusha, Vladislav V.

    2011-01-01

    The ability of non-invasive monitoring of deep-tissue developmental, metabolic, and pathogenic processes will advance modern biotechnology. Imaging of live mammals using fluorescent probes is more feasible within a “near-infrared optical window” (NIRW)1. Here we report a phytochrome-based near infra-red fluorescent protein (iRFP) with the excitation/emission maxima at 690/713 nm. Bright fluorescence in a living mouse proved iRFP to be a superior probe for non-invasive imaging of internal mammalian tissues. Its high intracellular stability, low cytotoxicity, and lack of the requirement to add external biliverdin-chromophore makes iRFP as easy to use as conventional GFP-like proteins. Compared to earlier phytochrome-derived fluorescent probes, the iRFP protein has better in vitro characteristics and performs well in cells and in vivo, having greater effective brightness and photostability. Compared to the far-red GFP-like proteins, iRFP has substantially higher signal to background ratio in a mouse model owing to its infra-red shifted spectra. PMID:21765402

  4. [Effects of infusion media on human red blood cell morphology].

    PubMed

    Burova, O O; Gusev, A A; Petrikov, S S; Gusev, S A; Basyreva, L Iu

    2006-01-01

    The effect of various infusion media on the structure of human red blood cells was evaluated in vitro and in vivo. The in vitro experiments used 10% sodium chloride (NaCl) solution, 10% glucose solution, 20% albumin solution, Rheopolyglucin, HyperHAES solution (18 g of NaCl in combination with 60 g of hydroxyethylstarch (HES), 200/0.5), Voluven (HES 130/0.4/9:1), and a combination of hypertensive NaCl solution and Rheopolyglucin. The morphofunctional response of red blood cells was studied in the clinical setting when 6% Voluven solution (HES 130/0.4/ 9:1) and hypertensive NaCl and glucose solutions were used. It was established that 10% NaCl solution caused considerable changes in the morphology of red blood cells both in the experiment and in patients with severe brain injury. The magnitude of structural changes increased as blood NaCl concentrations became higher. 10% glucose solution, Voluven, Rheopolyglucin, and albumin did not virtually affect the structure of red blood cells. Infusion of Voluven (500 ml of 6% solution for 40 minutes) induced no changes in the morphology of red blood cells in the clinical setting. Among the test solutions used to correct intracranial hypertension (HyperHAES, 10% NaCl, a combination of rheopolyglucin and 10% NaCl), HyperHAES exerted the least effect on the morphology of red blood cells.

  5. Red blood cells serve as intravascular carriers of myeloperoxidase.

    PubMed

    Adam, Matti; Gajdova, Silvie; Kolarova, Hana; Kubala, Lukas; Lau, Denise; Geisler, Anne; Ravekes, Thorben; Rudolph, Volker; Tsao, Philip S; Blankenberg, Stefan; Baldus, Stephan; Klinke, Anna

    2014-09-01

    Myeloperoxidase (MPO) is a heme enzyme abundantly expressed in polymorphonuclear neutrophils. MPO is enzymatically capable of catalyzing the generation of reactive oxygen species (ROS) and the consumption of nitric oxide (NO). Thus MPO has both potent microbicidal and, upon binding to the vessel wall, pro-inflammatory properties. Interestingly, MPO - a highly cationic protein - has been shown to bind to both endothelial cells and leukocyte membranes. Given the anionic surface charge of red blood cells, we investigated binding of MPO to erythrocytes. Red blood cells (RBCs) derived from patients with elevated MPO plasma levels showed significantly higher amounts of MPO by flow cytometry and ELISA than healthy controls. Heparin-induced MPO-release from patient-derived RBCs was significantly increased compared to controls. Ex vivo experiments revealed dose and time dependency for MPO-RBC binding, and immunofluorescence staining as well as confocal microscopy localized MPO-RBC interaction to the erythrocyte plasma membrane. NO-consumption by RBC-membrane fragments (erythrocyte "ghosts") increased with incrementally greater concentrations of MPO during incubation, indicating preserved catalytic MPO activity. In vivo infusion of MPO-loaded RBCs into C57BL/6J mice increased local MPO tissue concentrations in liver, spleen, lung, and heart tissue as well as within the cardiac vasculature. Further, NO-dependent relaxation of aortic rings was altered by RBC bound-MPO and systemic vascular resistance significantly increased after infusion of MPO-loaded RBCs into mice. In summary, we find that MPO binds to RBC membranes in vitro and in vivo, is transported by RBCs to remote sites in mice, and affects endothelial function as well as systemic vascular resistance. RBCs may avidly bind circulating MPO, and act as carriers of this leukocyte-derived enzyme. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. Vitamin E nanoemulsion activity on stored red blood cells.

    PubMed

    Silva, C A L; Azevedo Filho, C A; Pereira, G; Silva, D C N; Castro, M C A B; Almeida, A F; Lucena, S C A; Santos, B S; Barjas-Castro, M L; Fontes, A

    2017-06-01

    Stored red blood cells (RBCs) undergo numerous changes that have been termed RBC storage lesion, which can be related to oxidative damage. Vitamin E is an important antioxidant, acting on cell lipids. Thus, this study aimed to investigate vitamin E activity on stored RBCs. We prepared a vitamin E nanoemulsion that was added to RBC units and stored at 4 °C. Controls, without vitamin E, were kept under the same conditions. Reactive oxygen species (ROS) production was monitored for up to 35 days of storage. RBC elasticity was also evaluated using an optical tweezer system. Vitamin E-treated samples presented a significant decrease in ROS production. Additionally, the elastic constant for vitamin E-treated RBCs did not differ from the control. Vitamin E decreased the amount of ROS in stored RBCs. Because vitamin E acts on lipid oxidation, results suggest that protein oxidation should also be considered a key factor for erythrocyte elastic properties. Thus, further studies combining vitamin E with protein antioxidants deserve attention, aiming to better preserve overall stored RBC properties. © 2017 British Blood Transfusion Society.

  7. Hemoglobin dynamics in red blood cells: correlation to body temperature.

    PubMed

    Stadler, A M; Digel, I; Artmann, G M; Embs, J P; Zaccai, G; Büldt, G

    2008-12-01

    A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9 degrees C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature.

  8. Diamond Blackfan anemia: a disorder of red blood cell development.

    PubMed

    Ellis, Steven R; Lipton, Jeffrey M

    2008-01-01

    Diamond Blackfan anemia (DBA) is an inherited hypoplastic anemia that typically presents in the first year of life. The genes identified to date that are mutated in DBA encode ribosomal proteins, and in these cases ribosomal protein haploinsufficiency gives rise to the disease. The developmental timing of DBA presentation suggests that the changes in red blood cell production that occur around the time of birth trigger a pathophysiological mechanism, likely linked to defective ribosome synthesis, which precipitates the hematopoietic phenotype. Variable presentation of other clinical phenotypes in DBA patients indicates that other developmental pathways may also be affected by ribosomal protein haploinsufficiency and that the involvement of these pathways is influenced by modifier genes. Understanding the molecular basis for the developmental timing of DBA presentation promises to shed light on a number of baffling features of this disease. This chapter also attempts to demonstrate how the marriage of laboratory and clinical science may enhance each and permit insights into human disease that neither alone can accomplish.

  9. Hemoglobin Dynamics in Red Blood Cells: Correlation to Body Temperature

    PubMed Central

    Stadler, A. M.; Digel, I.; Artmann, G. M.; Embs, J. P.; Zaccai, G.; Büldt, G.

    2008-01-01

    A transition in hemoglobin behavior at close to body temperature has been discovered recently by micropipette aspiration experiments on single red blood cells (RBCs) and circular dichroism spectroscopy on hemoglobin solutions. The transition temperature was directly correlated to the body temperatures of a variety of species. In an exploration of the molecular basis for the transition, we present neutron scattering measurements of the temperature dependence of hemoglobin dynamics in whole human RBCs in vivo. The data reveal a change in the geometry of internal protein motions at 36.9°C, at human body temperature. Above that temperature, amino acid side-chain motions occupy larger volumes than expected from normal temperature dependence, indicating partial unfolding of the protein. Global protein diffusion in RBCs was also measured and the findings compared favorably with theoretical predictions for short-time self-diffusion of noncharged hard-sphere colloids. The results demonstrated that changes in molecular dynamics in the picosecond time range and angstrom length scale might well be connected to a macroscopic effect on whole RBCs that occurs at body temperature. PMID:18708462

  10. Co-ordinated stage-dependent enhancement of Plasmodium falciparum antioxidant enzymes and heat shock protein expression in parasites growing in oxidatively stressed or G6PD-deficient red blood cells

    PubMed Central

    Akide-Ndunge, Oscar Bate; Tambini, Elisa; Giribaldi, Giuliana; McMillan, Paul J; Müller, Sylke; Arese, Paolo; Turrini, Francesco

    2009-01-01

    Background Plasmodium falciparum-parasitized red blood cells (RBCs) are equipped with protective antioxidant enzymes and heat shock proteins (HSPs). The latter are only considered to protect against thermal stress. Important issues are poorly explored: first, it is insufficiently known how both systems are expressed in relation to the parasite developmental stage; secondly, it is unknown whether P. falciparum HSPs are redox-responsive, in view of redox sensitivity of HSP in eukaryotic cells; thirdly, it is poorly known how the antioxidant defense machinery would respond to increased oxidative stress or inhibited antioxidant defense. Those issues are interesting as several antimalarials increase the oxidative stress or block antioxidant defense in the parasitized RBC. In addition, numerous inhibitors of HSPs are currently developed for cancer therapy and might be tested as anti-malarials. Thus, the joint disruption of the parasite antioxidant enzymes/HSP system would interfere with parasite growth and open new perspectives for anti-malaria therapy. Methods Stage-dependent mRNA expression of ten representative P. falciparum antioxidant enzymes and hsp60/70–2/70–3/75/90 was studied by quantitative real-time RT-PCR in parasites growing in normal RBCs, in RBCs oxidatively-stressed by moderate H2O2 generation and in G6PD-deficient RBCs. Protein expression of antioxidant enzymes was assayed by Western blotting. The pentosephosphate-pathway flux was measured in isolated parasites after Sendai-virus lysis of RBC membrane. Results In parasites growing in normal RBCs, mRNA expression of antioxidant enzymes and HSPs displayed co-ordinated stage-dependent modulation, being low at ring, highest at early trophozoite and again very low at schizont stage. Additional exogenous oxidative stress or growth in antioxidant blunted G6PD-deficient RBCs indicated remarkable flexibility of both systems, manifested by enhanced, co-ordinated mRNA expression of antioxidant enzymes and HSPs

  11. In vivo red blood cell compatibility testing using indium-113m tropolone-labeled red blood cells

    SciTech Connect

    Morrissey, G.J.; Gravelle, D.; Dietz, G.; Driedger, A.A.; King, M.; Cradduck, T.D.

    1988-05-01

    In vivo radionuclide crossmatch is a method for identifying compatible blood for transfusion when allo- or autoantibodies preclude the use of conventional crossmatching techniques. A technique for labeling small volumes of donor red blood cells with (/sup 113m/In)tropolone is reported. The use of /sup 113m/In minimizes the accumulation of background radioactivity and the radiation dose especially so when multiple crossmatches are performed. Labeling red cells with (/sup 113m/In)tropolone is faster and easier to perform than with other radionuclides. Consistently high labeling efficiencies are obtained and minimal /sup 113m/In activity elutes from the labeled red blood cells. A case study involving 22 crossmatches is presented to demonstrate the technique. The radiation dose equivalent from /sup 113m/In is significantly less than with other radionuclides that may be used to label red cells.

  12. Born approximation model for light scattering by red blood cells.

    PubMed

    Lim, Joonoh; Ding, Huafeng; Mir, Mustafa; Zhu, Ruoyu; Tangella, Krishnarao; Popescu, Gabriel

    2011-10-01

    The primary role of a red blood cell (RBC) is delivering oxygen throughout our body. Abnormalities of this basic function lead to anemia and are caused by numerous diseases such as malaria and sickle cell anemia. As prompt and inexpensive tests for blood screening are in demand, we have developed a faster and reliable way to measure morphological parameters associated with the structure of red blood cells and the size distribution of the cells in a whole blood smear. Modeling the RBC shape under Born approximation, we are able to determine parameters of clinical relevance, such as the diameter, thickness and dimple size. From a measured quantitative phase image of a blood smear, we can determine the average and standard deviation of the red blood cell volume simultaneously, i.e., without analyzing each cell individually. This approach may open the door for a new generation of label-free, high-throughput blood testing.

  13. Modification of red blood cells for laboratory quality control use.

    PubMed

    Henry, Stephen M

    2009-11-01

    This review describes the current state-of-the-art with respect to the modification of red blood cells for creating quality controls for use in immunohaematology. The author has identified five technologies able to create modified red blood cells potentially suitable for use in quality control. Two of the technologies use enzymes, glycosidases or glycosyltransferases, to modify red blood cells and create ABO quality control cells. A third technology uses polyethylene glycol to reduce antigen expression by masking epitopes, whereas a fourth technology is speculative and involves the in-vitro generation of genetically modified erythroid cells. None of these four technologies are in routine use to make commercially available quality controls. A fifth commercially available technology creates quality controls by adding synthetic blood group A and B antigens (FSLs) to group O red blood cells, creating what are referred to as 'kodecytes'. This technology is also being used to add blood group peptides onto red cells for use in the future in a range of diagnostic applications. Transducing cell-derived erythroid populations with blood group encoding or silencing vectors, and the use of FSLs to create kodecytes, are two technologies with the potential to provide quality controls for laboratory use.

  14. Red cell exchange to mitigate a delayed hemolytic transfusion reaction in a patient transfused with incompatible red blood cells.

    PubMed

    Irani, Mehraboon S; Karafin, Matthew S; Ernster, Luke

    2017-02-01

    A red cell exchange was performed to prevent a potentially fatal hemolytic transfusion reaction in a patient with anti-e who was transfused with e-antigen unscreened red blood cells during liver transplant surgery. A 64-year-old woman with cirrhosis due to hepatitis C was scheduled to receive a liver transplant. She had a previously documented anti-e, an antibody to the Rh(e)-antigen that is known to cause delayed hemolytic transfusion reactions. Pre-operatively and intra-operatively, she had massive hemorrhage which required transfusion of 34 e-antigen unscreened red blood cells (RBCs) most of which were incompatible. The hemoglobin dropped from 9.1 g/dL on post-operative day (POD)1 to 6.6 g/dL on POD6, with no evidence of blood loss. The bilirubin also increased from 5.0 mg/dL on POD 1 to 11.0 mg/dL on POD 6. As she was also becoming more hemodynamically unstable, a red cell exchange with 10 units of e-negative RBCs was performed on POD 6. She improved clinically and was extubated the following day. A few residual transfused e-positive red cells were detected after the red cell exchange until POD 13. This case illustrates how a red cell exchange can mitigate the potentially harmful effects of a delayed hemolytic transfusion reaction caused by red cell antibodies. With massive intraoperative blood loss it may not be possible to have antigen-negative RBCs immediately available, particularly for the e-antigen, which is present in 98% of the donor population. The ability to perform such a procedure may be life-saving in such patients. J. Clin. Apheresis 32:59-61, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  15. Membrane rigidity of red blood cells parasitized by different strains of Plasmodium falciparum.

    PubMed

    Paulitschke, M; Nash, G B

    1993-11-01

    Changes in the structure of parasitized red blood cells may influence their ability to circulate. We have used a micropipette technique to examine the effects of invasion and maturation of Plasmodium falciparum on the membrane rigidity of red blood cells. In the presence of immature, ring form parasites from different laboratory strains, membrane rigidity remained unchanged as compared with uninfected red cells. However, development of more mature pigmented trophozoites caused a marked increase in membrane rigidity. Parasites from knobless strains caused a less-pronounced increase than parasites from knob-positive strains. Using closely synchronized cultures, the dependence of membrane rigidity on parasite maturation was studied in more detail for selected knob-positive and knobless strains. Over a period of 12 hours, while trophozoites developed into schizonts, no further rigidification of the red cell membrane occurred. The increase in membrane rigidity, occurring with the initial development of pigmented trophozoites, may be related to insertion of neoantigens into the red cell surface or modification of native membrane proteins that also occur at this time. In contrast to others, we found no effect of parasite-culture supernatant, harvested at different stages, on the rigidity of uninfected cells exposed to it. Interstrain variation of membrane rigidity could influence pathophysiology in several ways: by promoting margination and cytoadherence of knob-positive strains in the microcirculation, by modulating clearance of parasitized cells by the reticuloendothelial system, and by influencing ischemic complications of severe falciparum malaria.

  16. Aggregation of mononuclear and red blood cells through an {alpha}4{beta}1-Lu/basal cell adhesion molecule interaction in sickle cell disease.

    PubMed

    Chaar, Vicky; Picot, Julien; Renaud, Olivier; Bartolucci, Pablo; Nzouakou, Ruben; Bachir, Dora; Galactéros, Frédéric; Colin, Yves; Le Van Kim, Caroline; El Nemer, Wassim

    2010-11-01

    Abnormal interactions between red blood cells, leukocytes and endothelial cells play a critical role in the occurrence of the painful vaso-occlusive crises associated with sickle cell disease. We investigated the interaction between circulating leukocytes and red blood cells which could lead to aggregate formation, enhancing the incidence of vaso-occlusive crises. Blood samples from patients with sickle cell disease (n=25) and healthy subjects (n=5) were analyzed by imaging and classical flow cytometry after density gradient separation. The identity of the cells in the peripheral blood mononuclear cell layer was determined using antibodies directed specifically against white (anti-CD45) or red (anti-glycophorin A) blood cells. Aggregates between red blood cells and peripheral blood mononuclear cells were visualized in whole blood from patients with sickle cell disease. The aggregation rate was 10-fold higher in these patients than in control subjects. Both mature red blood cells and reticulocytes were involved in these aggregates through their interaction with mononuclear cells, mainly with monocytes. The size of the aggregates was variable, with one mononuclear cell binding to one, two or several red blood cells. Erythroid Lu/basal cell adhesion molecule and α(4)β(1) integrin were involved in aggregate formation. The aggregation rate was lower in patients treated with hydroxycarbamide than in untreated patients. Our study gives visual evidence of the existence of circulating red blood cell-peripheral blood mononuclear cell aggregates in patients with sickle cell disease and shows that these aggregates are decreased during hydroxycarbamide treatment. Our results strongly suggest that erythroid Lu/basal cell adhesion molecule proteins are implicated in these aggregates through their interaction with α(4)β(1) integrin on peripheral blood mononuclear cells.

  17. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders.

    PubMed

    King, M-J; Garçon, L; Hoyer, J D; Iolascon, A; Picard, V; Stewart, G; Bianchi, P; Lee, S-H; Zanella, A

    2015-06-01

    Hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary stomatocytosis (HSt) are inherited red cell disorders caused by defects in various membrane proteins. The heterogeneous clinical presentation, biochemical and genetic abnormalities in HS and HE have been well documented. The need to raise the awareness of HSt, albeit its much lower prevalence than HS, is due to the undesirable outcome of splenectomy in these patients. The scope of this guideline is to identify the characteristic clinical features, the red cell parameters (including red cell morphology) for these red cell disorders associated, respectively, with defective cytoskeleton (HS and HE) and abnormal cation permeability in the lipid bilayer (HSt) of the red cell. The current screening tests for HS are described, and their limitations are highlighted. An appropriate diagnosis can often be made when the screening test result(s) is reviewed together with the patient's clinical/family history, blood count results, reticulocyte count, red cell morphology, and chemistry results. SDS-polyacrylamide gel electrophoresis of erythrocyte membrane proteins, monovalent cation flux measurement, and molecular analysis of membrane protein genes are specialist tests for further investigation. Specialist tests provide additional evidence in supporting the diagnosis and that will facilitate the management of the patient. In the case of a patient's clinical phenotype being more severe than the affected members within the immediate family, molecular testing of all family members is useful for confirming the diagnosis and allows an insight into the molecular basis of the abnormality such as a recessive mode of inheritance or a de novo mutation. © 2015 John Wiley & Sons Ltd.

  18. The buckling instability of aggregating red blood cells.

    PubMed

    Flormann, Daniel; Aouane, Othmane; Kaestner, Lars; Ruloff, Christian; Misbah, Chaouqi; Podgorski, Thomas; Wagner, Christian

    2017-08-11

    Plasma proteins such as fibrinogen induce the aggregation of red blood cells (RBC) into rouleaux, which are responsible for the pronounced shear thinning behavior of blood, control the erythrocyte sedimentation rate (ESR) - a common hematological test - and are involved in many situations of physiological relevance such as structuration of blood in the microcirculation or clot formation in pathological situations. Confocal microscopy is used to characterize the shape of RBCs within rouleaux at equilibrium as a function of macromolecular concentration, revealing the diversity of contact zone morphology. Three different configurations that have only been partly predicted before are identified, namely parachute, male-female and sigmoid shapes, and quantitatively recovered by numerical simulations. A detailed experimental and theoretical analysis of clusters of two cells shows that the deformation increases nonlinearly with the interaction energy. Models indicate a forward bifurcation in which the contacting membrane undergoes a buckling instability from a flat to a deformed contact zone at a critical value of the interaction energy. These results are not only relevant for the understanding of the morphology and stability of RBC aggregates, but also for a whole class of interacting soft deformable objects such as vesicles, capsules or cells in tissues.

  19. Radiolabeled red blood cells: status, problems, and prospects

    SciTech Connect

    Srivastava, S.C.

    1983-01-01

    Radionuclidic labels for red cells can be divided into two main categories - cohort or pulse labels, and random labels. The random labels are incorporated into circulating cells of all ages and the labeling process is usually carried out in vitro. The red cell labels in predominant use involve random labeling and employ technetium-99m, chromium-51, indium-111, and gallium-68, roughly in that order. The extent of usefulness depends on the properties of the label such as the half-life, decay mode, and in-vivo stability, etc. Labeled cells can be used for red cell survival measurements when the half-life of the radionuclide is sufficiently long. The major portion of this article deals with random labels.

  20. Non-invasive intravital imaging of cellular differentiation with a bright red-excitable fluorescent protein

    PubMed Central

    Chu, Jun; Haynes, Russell D; Corbel, Stéphane Y; Li, Pengpeng; González-González, Emilio; Burg, John S; Ataie, Niloufar J; Lam, Amy J; Cranfill, Paula J; Baird, Michelle A; Davidson, Michael W; Ng, Ho-Leung; Garcia, K Christopher; Contag, Christopher H; Shen, Kang; Blau, Helen M; Lin, Michael Z

    2014-01-01

    A method for non-invasive visualization of genetically labelled cells in animal disease models with micron-level resolution would greatly facilitate development of cell-based therapies. Imaging of fluorescent proteins (FPs) using red excitation light in the “optical window” above 600 nm is one potential method for visualizing implanted cells. However, previous efforts to engineer FPs with peak excitation beyond 600 nm have resulted in undesirable reductions in brightness. Here we report three new red-excitable monomeric FPs obtained by structure-guided mutagenesis of mNeptune, previously the brightest monomeric FP when excited beyond 600 nm. Two of these, mNeptune2 and mNeptune2.5, demonstrate improved maturation and brighter fluorescence, while the third, mCardinal, has a red-shifted excitation spectrum without reduction in brightness. We show that mCardinal can be used to non-invasively and longitudinally visualize the differentiation of myoblasts and stem cells into myocytes in living mice with high anatomical detail. PMID:24633408

  1. DNA mimics of red fluorescent proteins (RFP) based on G-quadruplex-confined synthetic RFP chromophores.

    PubMed

    Feng, Guangfu; Luo, Chao; Yi, Haibo; Yuan, Lin; Lin, Bin; Luo, Xingyu; Hu, Xiaoxiao; Wang, Honghui; Lei, Chunyang; Nie, Zhou; Yao, Shouzhuo

    2017-09-13

    Red fluorescent proteins (RFPs) have emerged as valuable biological markers for biomolecule imaging in living systems. Developing artificial fluorogenic systems that mimic RFPs remains an unmet challenge. Here, we describe the design and synthesis of six new chromophores analogous to the chromophores in RFPs. We demonstrate, for the first time, that encapsulating RFP chromophore analogues in canonical DNA G-quadruplexes (G4) can activate bright fluorescence spanning red and far-red spectral regions (Em = 583-668 nm) that nearly match the entire RFP palette. Theoretical calculations and molecular dynamics simulations reveal that DNA G4 greatly restricts radiationless deactivation of chromophores induced by a twisted intramolecular charge transfer (TICT). These DNA mimics of RFP exhibit attractive photophysical properties comparable or superior to natural RFPs, including high quantum yield, large Stokes shifts, excellent anti-photobleaching properties, and two-photon fluorescence. Moreover, these RFP chromophore analogues are a novel and distinctive type of topology-selective G4 probe specific to parallel G4 conformation. The DNA mimics of RFP have been further exploited for imaging of target proteins. Using cancer-specific cell membrane biomarkers as targets, long-term real-time monitoring in single live cell and two-photon fluorescence imaging in tissue sections have been achieved without the need for genetic coding. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Prolonged red cell storage before transfusion increases extravascular hemolysis

    PubMed Central

    Rapido, Francesca; Brittenham, Gary M.; Bandyopadhyay, Sheila; La Carpia, Francesca; L’Acqua, Camilla; McMahon, Donald J.; Rebbaa, Abdelhadi; Wojczyk, Boguslaw S.; Netterwald, Jane; Wang, Hangli; Schwartz, Joseph; Eisenberger, Andrew; Soffing, Mark; Yeh, Randy; Divgi, Chaitanya; Ginzburg, Yelena Z.; Shaz, Beth H.; Sheth, Sujit; Francis, Richard O.; Spitalnik, Steven L.; Hod, Eldad A.

    2016-01-01

    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. PMID:27941245

  3. Cutting Management Effects on Yield, Fiber, and Protein Degradability of Red Clover Conserved as Silage.

    USDA-ARS?s Scientific Manuscript database

    With adequate soil moisture, forage yields of red clover can equal or exceed alfalfa in many temperate regions. Red clover also boasts superior fiber digestibility and greater rumen-undegradable protein than alfalfa, but milk yields from cattle fed red clover diets are often below expectations. Coup...

  4. [Relations between red cell structure, function and immune homeostasis in prostatic diseases].

    PubMed

    Shatokhin, M N; Teodorovich, O V; Konoplia, A I; Dolgareva, S A; Gavriliuk, V P; Krasnov, L V; Mavrin, M Iu

    2012-01-01

    Patients with chronic prostatitis alone and in combination with prostatic adenoma have changes in the activity of the complement system, neutrophil function and content of pro- and anti-inflammatory cytokines. Abnormal representation of the proteins of the red cell membrane in patients with prostatic diseases affects structural and functional activity of erythrocytes in these patients. Dynamic changes in immune status of patients with chronic prostatitis and prostatic adenoma correlate with changes in functional red cell activity. This fact helps better understanding of pathogenesis of chronic prostatitis and prostatic adenoma.

  5. From green to red--To more dead? Autofluorescent proteins as photosensitizers.

    PubMed

    Mueller, Gabriele; Waldeck, Waldemar; Braun, Klaus

    2010-01-21

    Inactive compounds like autofluorescent proteins can absorb visible daylight (around 500-700 nm) and can emit active electrons producing reactive oxygen species (ROS) leading to an increase in photokilling processes in bacteria. The endogenously originated ROS create single strand breaks in the cells DNA. These various types of breaks can be partially repaired by different cellular repair systems but a high number of breaks leads to cell death. A dramatic increase in cell killing can be observed from green, via yellow to red color emission. This was tested by colony forming ability. The generation of ROS and the bacterial protection mechanisms are discussed. We outline some possibilities for use the protein's properties for treatment of antibiotic multi-resistant and difficult to treat bacteria like the methicillin-resistant Staphylococcus aureus (MRSA).

  6. Photoactivation in green to red converting EosFP and other fluorescent proteins from the GFP family

    NASA Astrophysics Data System (ADS)

    Wiedenmann, Jörg; Nienhaus, G. Ulrich

    2006-02-01

    The green fluorescent protein (GFP) from the hydromedusa Aequorea victoria and its derivatives have become indispensable imaging devices in cell biology. In previous years, a wide variety of GFP-like proteins were discovered in non-bioluminescent anthozoa. Some of them displayed exciting new properties, including photoactivated changes of the fluorescence emission intensity and wavelength. Photoactivatable proteins offer a high potential as tools for regional optical marking in live cells and tissues. This review aims to give an overview of photoactivatable marker proteins, focusing on the molecular basis of light-induced green to red photoconversion in EosFP.

  7. Image classification of unlabeled malaria parasites in red blood cells.

    PubMed

    Zheng Zhang; Ong, L L Sharon; Kong Fang; Matthew, Athul; Dauwels, Justin; Ming Dao; Asada, Harry

    2016-08-01

    This paper presents a method to detect unlabeled malaria parasites in red blood cells. The current "gold standard" for malaria diagnosis is microscopic examination of thick blood smear, a time consuming process requiring extensive training. Our goal is to develop an automate process to identify malaria infected red blood cells. Major issues in automated analysis of microscopy images of unstained blood smears include overlapping cells and oddly shaped cells. Our approach creates robust templates to detect infected and uninfected red cells. Histogram of Oriented Gradients (HOGs) features are extracted from templates and used to train a classifier offline. Next, the ViolaJones object detection framework is applied to detect infected and uninfected red cells and the image background. Results show our approach out-performs classification approaches with PCA features by 50% and cell detection algorithms applying Hough transforms by 24%. Majority of related work are designed to automatically detect stained parasites in blood smears where the cells are fixed. Although it is more challenging to design algorithms for unstained parasites, our methods will allow analysis of parasite progression in live cells under different drug treatments.

  8. Red blood cell dynamics: from cell deformation to ATP release.

    PubMed

    Wan, Jiandi; Forsyth, Alison M; Stone, Howard A

    2011-10-01

    The mechanisms of red blood cell (RBC) deformation under both static and dynamic, i.e., flow, conditions have been studied extensively since the mid 1960s. Deformation-induced biochemical reactions and possible signaling in RBCs, however, were proposed only fifteen years ago. Therefore, the fundamental relationship between RBC deformation and cellular signaling dynamics i.e., mechanotransduction, remains incompletely understood. Quantitative understanding of the mechanotransductive pathways in RBCs requires integrative studies of physical models of RBC deformation and cellular biochemical reactions. In this article we review the physical models of RBC deformation, spanning from continuum membrane mechanics to cellular skeleton dynamics under both static and flow conditions, and elaborate the mechanistic links involved in deformation-induced ATP release.

  9. Quadruple 9-mer-based protein binding microarray with DsRed fusion protein

    PubMed Central

    Kim, Min-Jeong; Lee, Tae-Ho; Pahk, Yoon-Mok; Kim, Yul-Ho; Park, Hyang-Mi; Choi, Yang Do; Nahm, Baek Hie; Kim, Yeon-Ki

    2009-01-01

    Background The interaction between a transcription factor and DNA motif (cis-acting element) is an important regulatory step in gene regulation. Comprehensive genome-wide methods have been developed to characterize protein-DNA interactions. Recently, the universal protein binding microarray (PBM) was introduced to determine if a DNA motif interacts with proteins in a genome-wide manner. Results We facilitated the PBM technology using a DsRed fluorescent protein and a concatenated sequence of oligonucleotides. The PBM was designed in such a way that target probes were synthesized as quadruples of all possible 9-mer combinations, permitting unequivocal interpretation of the cis-acting elements. The complimentary DNA strands of the features were synthesized with a primer and DNA polymerase on microarray slides. Proteins were labeled via N-terminal fusion with DsRed fluorescent protein, which circumvents the need for a multi-step incubation. The PBM presented herein confirmed the well-known DNA binding sequences of Cbf1 and CBF1/DREB1B, and it was also applied to elucidate the unidentified cis-acting element of the OsNAC6 rice transcription factor. Conclusion Our method demonstrated PBM can be conveniently performed by adopting: (1) quadruple 9-mers may increase protein-DNA binding interactions in the microarray, and (2) a one-step incubation shortens the wash and hybridization steps. This technology will facilitate greater understanding of genome-wide interactions between proteins and DNA. PMID:19761621

  10. Mechanisms linking red blood cell disorders and cardiovascular diseases.

    PubMed

    Mozos, Ioana

    2015-01-01

    The present paper aims to review the main pathophysiological links between red blood cell disorders and cardiovascular diseases, provides a brief description of the latest studies in this area, and considers implications for clinical practice and therapy. Anemia is associated with a special risk in proatherosclerotic conditions and heart disease and became a new therapeutic target. Guidelines must be updated for the management of patients with red blood cell disorders and cardiovascular diseases, and targets for hemoglobin level should be established. Risk scores in several cardiovascular diseases should include red blood cell count and RDW. Complete blood count and hemorheological parameters represent useful, inexpensive, widely available tools for the management and prognosis of patients with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke. Hypoxia and iron accumulation cause the most important cardiovascular effects of sickle cell disease and thalassemia. Patients with congenital chronic hemolytic anemia undergoing splenectomy should be monitored, considering thromboembolic and cardiovascular risk.

  11. Mechanisms Linking Red Blood Cell Disorders and Cardiovascular Diseases

    PubMed Central

    2015-01-01

    The present paper aims to review the main pathophysiological links between red blood cell disorders and cardiovascular diseases, provides a brief description of the latest studies in this area, and considers implications for clinical practice and therapy. Anemia is associated with a special risk in proatherosclerotic conditions and heart disease and became a new therapeutic target. Guidelines must be updated for the management of patients with red blood cell disorders and cardiovascular diseases, and targets for hemoglobin level should be established. Risk scores in several cardiovascular diseases should include red blood cell count and RDW. Complete blood count and hemorheological parameters represent useful, inexpensive, widely available tools for the management and prognosis of patients with coronary heart disease, heart failure, hypertension, arrhythmias, and stroke. Hypoxia and iron accumulation cause the most important cardiovascular effects of sickle cell disease and thalassemia. Patients with congenital chronic hemolytic anemia undergoing splenectomy should be monitored, considering thromboembolic and cardiovascular risk. PMID:25710019

  12. Red Blood Cell Docosapentaenoic Acid (DPA n-3) is Inversely Associated with Triglycerides and C-reactive Protein (CRP) in Healthy Adults and Dose-Dependently Increases Following n-3 Fatty Acid Supplementation

    PubMed Central

    Skulas-Ray, Ann C.; Flock, Michael R.; Richter, Chesney K.; Harris, William S.; West, Sheila G.; Kris-Etherton, Penny M.

    2015-01-01

    The role of the long-chain omega-3 (n-3) fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in lipid metabolism and inflammation has been extensively studied; however, little is known about the relationship between docosapentaenoic acid (DPA, 22:5 n-3) and inflammation and triglycerides (TG). We evaluated whether n-3 DPA content of red blood cells (RBC) was associated with markers of inflammation (interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and C-reactive protein (CRP) and fasting TG prior to n-3 supplementation in two studies (Study 1: n = 115, aged 20–44 years, body mass index (BMI) 20–30 kg/m2, TG = 34–176 mg/dL; Study 2: n = 28, aged 22–65 years, BMI 24–37 kg/m2, TG = 141–339 mg/dL). We also characterized the dose-response effects of n-3 fatty acid supplementation on RBC n-3 DPA after five months of supplementation with fish oil (Study 1: 0, 300, 600, 900, and 1800 mg/day EPA + DHA) and eight weeks of prescription n-3 ethyl esters (Study 2: 0, 850, and 3400 mg/day EPA + DHA). In Study 1, RBC n-3 DPA was inversely correlated with CRP (R2 = 36%, p < 0.001) and with fasting TG (r = −0.30, p = 0.001). The latter finding was replicated in Study 2 (r = −0.33, p = 0.04). In both studies, n-3 supplementation significantly increased RBC n-3 DPA dose-dependently. Relative increases were greater for Study 1, with increases of 29%–61% vs. 14%–26% for Study 2. The associations between RBC n-3 DPA, CRP, and fasting TG may have important implications for the prevention of atherosclerosis and chronic inflammatory diseases and warrant further study. PMID:26247967

  13. Multimarker approach with cystatin C, N-terminal pro-brain natriuretic peptide, C-reactive protein and red blood cell distribution width in risk stratification of patients with acute coronary syndromes.

    PubMed

    Vieira, Catarina; Nabais, Sérgio; Ramos, Vítor; Braga, Carlos; Gaspar, António; Azevedo, Pedro; Álvares Pereira, Miguel; Salomé, Nuno; Correia, Adelino

    2014-03-01

    Biomarkers have emerged as interesting predictors of risk in patients with acute coronary syndromes (ACS). The aim of this study was to determine the utility of the combined measurement of cystatin C (CysC), C-reactive protein (CRP), N-terminal pro-brain natriuretic peptide (NT-proBNP) and red blood cell distribution width (RDW) in the risk stratification of patients with ACS. In this prospective study including 682 patients consecutively admitted to a coronary care unit for ACS, baseline measurements of CysC, CRP, NT-proBNP and RDW were performed. Patients were categorized on the basis of the number of elevated biomarkers at presentation. The primary outcome was 6-month mortality. The number of biomarkers elevated on admission (study score) was an independent predictor of 6-month mortality; patients with four biomarkers elevated on admission had a significantly higher risk of 6-month mortality compared with patients with none or one. In addition, in patients with high risk defined by the GRACE score, our multimarker score was able to further categorize their risk of 6-month mortality. A multimarker approach using CysC, NT-proBNP, CRP and RDW was an independent predictor of 6-month mortality and added prognostic information to the GRACE risk score in patients with ACS and high risk defined by GRACE, with increasing mortality in patients with a higher number of elevated biomarkers on admission. Copyright © 2013 Sociedade Portuguesa de Cardiologia. Published by Elsevier España. All rights reserved.

  14. Red blood cell ghosts and intact red blood cells as complementary models in photodynamic cell research.

    PubMed

    Kaestner, Lars

    2004-05-01

    Recent research on erythrocytes as model cells for photodynamic therapy showed differing behaviour of certain photosensitisers in erythrocytes compared to other cells. Differences of dye accumulation in the cell membrane were proposed to be the reason for the distinct photodynamic effects. Using pheophorbide a as an example, the combination of erythrocyte ghosts as models to follow the dye accumulation in the cell membrane and intact erythrocytes as model cells to show the photodynamic damage is provided. Evidence for the correctness of the combination of erythrocyte ghosts and intact erythrocytes as a functioning model system in photodynamic cell research is provided using the confocal laser scanning microscopy on intact, pheophorbide a loaded erythrocytes.

  15. Red blood cell aggregate flux in a bifurcating microchannel.

    PubMed

    Kaliviotis, E; Pasias, D; Sherwood, J M; Balabani, S

    2017-10-01

    Red blood cell aggregation plays a key role in microcirculatory flows, however, little is known about the transport characteristics of red blood cell aggregates in branching geometries. This work reports on the fluxes of red blood cell aggregates of various sizes in a T-shaped microchannel, aiming to clarify the effects of different flow conditions in the outlet branches of the channel. Image analysis techniques, were utilised, and moderately aggregating human red blood cell suspensions were tested in symmetric (∼50-50%) and asymmetric flow splits through the two outlet (daughter) branches. The results revealed that the flux decreases with aggregate size in the inlet (parent) and daughter branches, mainly due to the fact that the number of larger structures is significantly smaller than that of smaller structures. However, when the flux in the daughter branches is examined relative to the aggregate size flux in the parent branch an increase with aggregate size is observed for a range of asymmetric flow splits. This increase is attributed to size distribution and local concentration changes in the daughter branches. The results show that the flow of larger aggregates is not suppressed downstream of a bifurcation, and that blood flow is maintained, for physiological levels of red blood cell aggregation. Copyright © 2017 IPEM. Published by Elsevier Ltd. All rights reserved.

  16. Observation of dynamic subdomains in red blood cells.

    PubMed

    Popescu, Gabriel; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

    2006-01-01

    We quantify the nanoscale structure and low-frequency dynamics associated with live red blood cells. The membrane displacements are measured using quantitative phase images provided by Fourier phase microscopy, with an average path-length stability of 0.75 nm over 45 min. The results reveal the existence of dynamic, independent subdomains across the cells that fluctuate at various dominant frequencies.

  17. Computational Biomechanics of Human Red Blood Cells in Hematological Disorders.

    PubMed

    Li, Xuejin; Li, He; Chang, Hung-Yu; Lykotrafitis, George; Em Karniadakis, George

    2017-02-01

    We review recent advances in multiscale modeling of the biomechanical characteristics of red blood cells (RBCs) in hematological diseases, and their relevance to the structure and dynamics of defective RBCs. We highlight examples of successful simulations of blood disorders including malaria and other hereditary disorders, such as sickle-cell anemia, spherocytosis, and elliptocytosis.

  18. Advances in understanding the pathogenesis of the red cell volume disorders.

    PubMed

    Badens, Catherine; Guizouarn, Hélène

    2016-09-01

    Genetic defects of erythrocyte transport proteins cause disorders of red blood cell volume that are characterized by abnormal permeability to the cations Na(+) and K(+) and, consequently, by changes in red cell hydration. Clinically, these disorders are associated with chronic haemolytic anaemia of variable severity and significant co-morbidities, such as iron overload. This review provides an overview of recent insights into the molecular basis of this group of rare anaemias involving cation channels and transporters dysfunction. To date, a total of 5 different membrane proteins have been reported to be responsible for volume homeostasis alteration when mutated, 3 of them leading to overhydrated cells (AE1 [also termed SLC4A1], RHAG and GLUT1 [also termed SCL2A1) and 2 others to dehydrated cells (PIEZO1 and the Gardos Channel). These findings are not only of basic scientific interest, but also of direct clinical significance for improving diagnostic procedures and identify potential approaches for novel therapeutic strategies.

  19. Perturbation of red blood cell membrane rigidity by extracellular ligands.

    PubMed

    Paulitschke, M; Nash, G B; Anstee, D J; Tanner, M J; Gratzer, W B

    1995-07-01

    It is known that binding of extracellular antibodies against the major sialoglycoprotein, glycophorin A, reduced the deformability of the red blood cell membrane. This has been taken to result from new or altered interactions between the glycophorin A and the membrane skeleton. We have shown by means of the micropipette aspiration technique that antibodies against the preponderant transmembrane protein, band 3, induce similar effects. A definite but much smaller reduction in elasticity of the membrane is engendered by univalent Fab fragments of the anti-band 3 antibodies. By examining cells genetically devoid of glycophorin A or containing a variant of this constituent, truncated at the inner membrane surface, we have shown that the anti-band 3 antibodies do not act through the band 3-associated glycophorin A. We examined the effect of anti-glycophorin A antibodies on homozygous Wr(a+b-) cells, in which an amino acid replacement in band 3 annihilates the Wright b (Wrb) epitope (comprising sequence elements of glycophorin A and band 3) and thus, by implication disrupts or perturbs the band 3-glycophorin A interaction; these cells show a much smaller response to an anti-glycophorin A antibody than do normal controls. We infer that in this case anti-glycophorin A antibodies exert their rigidifying effect through the associated band 3. Another anti-glycophorin A antibody, directed against an epitope remote from the membrane surface, however, increases the rigidity of both Wr(a+b-) and normal cells. This implies that not all antibodies act in the same manner in modifying the membrane mechanical properties. The effect exerted by anti-band 3 antibodies appears not to be transmitted through the band 3-ankyrin-spectrin pathway because the rigidifying effect of the intact antibody persists at alkaline pH, at which there is evidence that the ankyrin-band 3 link is largely dissociated. The large difference between the effects of saturating concentrations of the divalent and

  20. Reduction of prion infectivity in packed red blood cells

    SciTech Connect

    Morales, Rodrigo; Buytaert-Hoefen, Kimberley A.; Gonzalez-Romero, Dennisse; Castilla, Joaquin; Hansen, Eric T.; Hlavinka, Dennis; Goodrich, Raymond P.; Soto, Claudio

    2008-12-12

    The link between a new variant form of Creutzfeldt-Jakob disease (vCJD) and the consumption of prion contaminated cattle meat as well as recent findings showing that vCJD can be transmitted by blood transfusion have raised public health concerns. Currently, a reliable test to identify prions in blood samples is not available. The purpose of this study was to evaluate the possibility to remove scrapie prion protein (PrP{sup Sc}) and infectivity from red blood cell (RBC) suspensions by a simple washing procedure using a cell separation and washing device. The extent of prion removal was assessed by Western blot, PMCA and infectivity bioassays. Our results revealed a substantial removal of infectious prions ({>=}3 logs of infectivity) by all techniques used. These data suggest that a significant amount of infectivity present in RBC preparations can be removed by a simple washing procedure. This technology may lead to increased safety of blood products and reduce the risk of further propagation of prion diseases.

  1. Lytic resistance of fibrin containing red blood cells.

    PubMed

    Wohner, Nikolett; Sótonyi, Péter; Machovich, Raymund; Szabó, László; Tenekedjiev, Kiril; Silva, Marta M C G; Longstaff, Colin; Kolev, Krasimir

    2011-10-01

    Arterial thrombi contain variable amounts of red blood cells (RBCs), which interact with fibrinogen through an eptifibatide-sensitive receptor and modify the structure of fibrin. In this study, we evaluated the modulator role of RBCs in the lytic susceptibility of fibrin. If fibrin is formed at increasing RBC counts, scanning electron microscopy evidenced a decrease in fiber diameter from 150 to 96 nm at 40% (v/v) RBCs, an effect susceptible to eptifibatide inhibition (restoring 140 nm diameter). RBCs prolonged the lysis time in a homogeneous-phase fibrinolytic assay with tissue plasminogen activator (tPA) by up to 22.7±1.6%, but not in the presence of eptifibatide. Confocal laser microscopy using green fluorescent protein-labeled tPA and orange fluorescent fibrin showed that 20% to 40% (v/v) RBCs significantly slowed down the dissolution of the clots. The fluorescent tPA variant did not accumulate on the surface of fibrin containing RBCs at any cell count above 10%. The presence of RBCs in the clot suppressed the tPA-induced plasminogen activation, resulting in 45% less plasmin generated after 30 minutes of activation at 40% (v/v) RBCs. RBCs confer lytic resistance to fibrin resulting from modified fibrin structure and impaired plasminogen activation through a mechanism that involves eptifibatide-sensitive fibrinogen-RBC interactions.

  2. Characterization of Microvesicles Released from Human Red Blood Cells.

    PubMed

    Nguyen, Duc Bach; Ly, Thi Bich Thuy; Wesseling, Mauro Carlos; Hittinger, Marius; Torge, Afra; Devitt, Andrew; Perrie, Yvonne; Bernhardt, Ingolf

    2016-01-01

    Extracellular vesicles (EVs) are spherical fragments of cell membrane released from various cell types under physiological as well as pathological conditions. Based on their size and origin, EVs are classified as exosome, microvesicles (MVs) and apoptotic bodies. Recently, the release of MVs from human red blood cells (RBCs) under different conditions has been reported. MVs are released by outward budding and fission of the plasma membrane. However, the outward budding process itself, the release of MVs and the physical properties of these MVs have not been well investigated. The aim of this study is to investigate the formation process, isolation and characterization of MVs released from RBCs under conditions of stimulating Ca2+ uptake and activation of protein kinase C. Experiments were performed based on single cell fluorescence imaging, fluorescence activated cell sorter/flow cytometer (FACS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and dynamic light scattering (DLS). The released MVs were collected by differential centrifugation and characterized in both their size and zeta potential. Treatment of RBCs with 4-bromo-A23187 (positive control), lysophosphatidic acid (LPA), or phorbol-12 myristate-13 acetate (PMA) in the presence of 2 mM extracellular Ca2+ led to an alteration of cell volume and cell morphology. In stimulated RBCs, exposure of phosphatidylserine (PS) and formation of MVs were observed by using annexin V-FITC. The shedding of MVs was also observed in the case of PMA treatment in the absence of Ca2+, especially under the transmitted bright field illumination. By using SEM, AFM and DLS the morphology and size of stimulated RBCs, MVs were characterized. The sizes of the two populations of MVs were 205.8 ± 51.4 nm and 125.6 ± 31.4 nm, respectively. Adhesion of stimulated RBCs and MVs was observed. The zeta potential of MVs was determined in the range from - 40 mV to - 10 mV depended on the solutions and buffers used. An

  3. Contribution of whole cell and cytoplasmic polypeptides to apparent red cell membrane alterations.

    PubMed

    Sauberman, N; Snyder, L M

    1980-11-04

    We have compared densitometric tracings of whole cell, cytoplasmic and membrane polypeptide electrophoretic patterns in an attempt to distinguish atypical partitioning from intrinsic membrane polypeptide changes occurring as a result of reticulocyte enrichment, metabolic depletion, N-ethylmaleimide treatment and hereditary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in tary xerocytosis. We report that membrane alterations seen in a reticulocyte-enriched population of normal cells are present in the whole cells prior to membrane isolation. Some of the membrane alterations in metabolically depleted cells and all of those in N-ethylmaleimide-treated cells are traced to modifications in the partitioning of polypeptides between membranes and supernatant (cytoplasm) at hemolysis. The power of this approach in resolving the sources of apparent red cell membrane protein alterations is demonstrated in studies with hereditary xerocytes. Suggested altered partitioning of these cells described earlier (Sauberman, N., Fortier, N.L., Fairbanks, G., O'Connor, R.J. and Snyder, L.M. (1979) Biochim. Biophys. Acta 556, 292-313) is further documented and found to be unrelated to the younger cell population or slight metabolic depletion that occurs during the washing of xerocytes prior to hemolysis.

  4. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease

    PubMed Central

    ALAPAN, YUNUS; KIM, CEONNE; ADHIKARI, ANIMA; GRAY, KAYLA E.; GURKAN-CAVUSOGLU, EVREN; LITTLE, JANE A.; GURKAN, UMUT A.

    2016-01-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  5. Structure-guided wavelength tuning in far-red fluorescent proteins

    PubMed Central

    Ng, Ho-Leung; Lin, Michael Z.

    2017-01-01

    In recent years, protein engineers have succeeded in tuning the excitation spectra of natural fluorescent proteins from green wavelengths into orange and red wavelengths, resulting in the creation of a series of fluorescent proteins with emission in the far-red portions of the optical spectrum. These results have arisen from the synergistic combination of structural knowledge of fluorescent proteins, chemical intuition, and high-throughput screening methods. Here we review structural features found in autocatalytic far-red fluorescent proteins, and discuss how they add to our understanding of the biophysical mechanisms of wavelength tuning in biological chromophores. PMID:27468111

  6. Prolonged storage of packed red blood cells for blood transfusion.

    PubMed

    Martí-Carvajal, Arturo J; Simancas-Racines, Daniel; Peña-González, Barbra S

    2015-07-14

    A blood transfusion is an acute intervention, used to address life- and health-threatening conditions on a short-term basis. Packed red blood cells are most often used for blood transfusion. Sometimes blood is transfused after prolonged storage but there is continuing debate as to whether transfusion of 'older' blood is as beneficial as transfusion of 'fresher' blood. To assess the clinical benefits and harms of prolonged storage of packed red blood cells, in comparison with fresh, on recipients of blood transfusion. We ran the search on 1st May 2014. We searched the Cochrane Injuries Group Specialized Register, Cochrane Central Register of Controlled Trials (CENTRAL, The Cochrane Library), MEDLINE (OvidSP), Embase (OvidSP), CINAHL (EBSCO Host) and two other databases. We also searched clinical trials registers and screened reference lists of the retrieved publications and reviews. We updated this search in June 2015 but these results have not yet been incorporated. Randomised clinical trials including participants assessed as requiring red blood cell transfusion were eligible for inclusion. Prolonged storage was defined as red blood cells stored for ≥ 21 days in a blood bank. We did not apply limits regarding the duration of follow-up, or country where the study took place. We excluded trials where patients received a combination of short- and long-stored blood products, and also trials without a clear definition of prolonged storage. We independently performed study selection, risk of bias assessment and data extraction by at least two review authors. The major outcomes were death from any cause, transfusion-related acute lung injury, and adverse events. We estimated relative risk for dichotomous outcomes. We measured statistical heterogeneity using I(2). We used a random-effects model to synthesise the findings. We identified three randomised clinical trials, involving a total of 120 participants, comparing packed red blood cells with ≥ 21 days storage

  7. In vivo red cell destruction by anti-Lu6

    SciTech Connect

    Issitt, P.D.; Valinsky, J.E.; Marsh, W.L.; DiNapoli, J.; Gutgsell, N.S. )

    1990-03-01

    An example is presented of an IgG1, anti-Lu6, that reacted by indirect antiglobulin test and was capable of destroying antigen-positive red cells in vivo. Two methods for the measurement of red cell survival, {sup 51}Cr labeling and flow cytometry, gave the same result: 20 percent of the test dose of Lu:6 red cells was destroyed in the first hour after injection and 80 percent in the first 24 hours. The clinical relevance of the antibody was correctly predicted by an in vitro monocyte monolayer assay. The finding that this example of anti-Lu6 was clinically significant should not be taken to mean that all antibodies directed against high-incidence Lutheran and Lutheran system-related antigens will behave similarly. When such antibodies are encountered, in vivo and/or in vitro studies to assess their clinical significance are necessary before rare blood is used for transfusion.

  8. Structure and deformation properties of red blood cells: concepts and quantitative methods.

    PubMed

    Evans, E A

    1989-01-01

    The lamellar configuration of the red cell membrane includes a (liquid) superficial bilayer of amphiphilic molecules supported by a (rigid) subsurface protein meshwork. Because of this composite structure, the red cell membrane exhibits very large resistance to changes in surface density or area with very low resistance to in-plane extension and bending deformations. The primary extrinsic factor in cell deformability is the surface area-to-volume ratio which establishes the minimum-caliber vessel into which a cell can deform (without rupture). Within the restriction provided by surface area and volume, the intrinsic properties of the membrane and cytoplasm determine the deformability characteristics of the red cell. Since the cytoplasm is liquid, the static rigidity of the cell is determined by membrane elastic constants. These include an elastic modulus for area compressibility in the range of 300-600 dyn/cm, an elastic modulus for in-plane extension or shear (at constant area) of 5-7 X 10(-3) dyn/cm, and a curvature or bending elastic modulus on the order of 10(-12) dyn.cm. Even though small, the surface rigidity of the cell membrane is sufficient to return the membrane capsule to a discoid shape after deformation by external forces. Viscous dissipation in the peripheral protein structure (cytoskeleton) dominates the dynamic response of the cell to extensional forces. Based on a time constant for recovery after extensional deformation on the order of 0.1 sec, the coefficient of surface viscosity is on the order of 10(-3) dyn.sec/cm. On the other hand, the dynamic resistance to folding of the cell appears to be limited by viscous dissipation in the cytoplasmic and external fluid phases. Dynamic rigidities for both extensional and folding deformations are important factors in the distribution of flow in the small microvessels. Although the red cell membrane normally behaves as a resilient viscoelastic shell, which recovers its conformation after deformation

  9. Caught red-handed: Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice.

    PubMed

    Sweeney, Megan T; Thomson, Michael J; Pfeil, Bernard E; McCouch, Susan

    2006-02-01

    Rc is a domestication-related gene required for red pericarp in rice (Oryza sativa). The red grain color is ubiquitous among the wild ancestors of O. sativa, in which it is closely associated with seed shattering and dormancy. Rc encodes a basic helix-loop-helix (bHLH) protein that was fine-mapped to an 18.5-kb region on rice chromosome 7 using a cross between Oryza rufipogon (red pericarp) and O. sativa cv Jefferson (white pericarp). Sequencing of the alleles from both mapping parents as well as from two independent genetic stocks of Rc revealed that the dominant red allele differed from the recessive white allele by a 14-bp deletion within exon 6 that knocked out the bHLH domain of the protein. A premature stop codon was identified in the second mutant stock that had a light red pericarp. RT-PCR experiments confirmed that the Rc gene was expressed in both red- and white-grained rice but that a shortened transcript was present in white varieties. Phylogenetic analysis, supported by comparative mapping in rice and maize (Zea mays), showed that Rc, a positive regulator of proanthocyanidin, is orthologous with INTENSIFIER1, a negative regulator of anthocyanin production in maize, and is not in the same clade as rice bHLH anthocyanin regulators.

  10. Unique interactions between the chromophore and glutamate 16 lead to far-red emission in a red fluorescent protein

    PubMed Central

    Shu, Xiaokun; Wang, Lei; Colip, Leslie; Kallio, Karen; Remington, S James

    2009-01-01

    mPlum is a far-red fluorescent protein with emission maximum at ∼650 nm and was derived by directed evolution from DsRed. Two residues near the chromophore, Glu16 and Ile65, were previously revealed to be indispensable for the far-red emission. Ultrafast time-resolved fluorescence emission studies revealed a time dependent shift in the emission maximum, initially about 625 nm, to about 650 nm over a period of 500 ps. This observation was attributed to rapid reorganization of the residues solvating the chromophore within mPlum. Here, the crystal structure of mPlum is described and compared with those of two blue shifted mutants mPlum-E16Q and-I65L. The results suggest that both the identity and precise orientation of residue 16, which forms a unique hydrogen bond with the chromophore, are required for far-red emission. Both the far-red emission and the time dependent shift in emission maximum are proposed to result from the interaction between the chromophore and Glu16. Our findings suggest that significant red shifts might be achieved in other fluorescent proteins using the strategy that led to the discovery of mPlum. PMID:19165727

  11. Optical trapping and propulsion of red blood cells on waveguide surfaces.

    PubMed

    Ahluwalia, Balpreet Singh; McCourt, Peter; Huser, Thomas; Hellesø, Olav Gaute

    2010-09-27

    We have studied optical trapping and propulsion of red blood cells in the evanescent field of optical waveguides. Cell propulsion is found to be highly dependent on the biological medium and serum proteins the cells are submerged in. Waveguides made of tantalum pentoxide are shown to be efficient for cell propulsion. An optical propulsion velocity of up to 
6 µm/s on a waveguide with a width of ~6 µm is reported. Stable optical trapping and propulsion of cells during transverse flow is also reported.

  12. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein

    PubMed Central

    Rodriguez, Erik A.; Tran, Geraldine N.; Gross, Larry A.; Crisp, Jessica L.; Shu, Xiaokun; Lin, John Y.; Tsien, Roger Y.

    2016-01-01

    Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because less light is scattered, absorbed, or reemitted by endogenous biomolecules. A new class of FP was developed from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, named small Ultra-Red FP (smURFP), covalently attaches biliverdin (BV) without a lyase, and has 642/670 nm excitation/emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and comparable photostability to eGFP. SmURFP has significantly increased BV incorporation rate and protein stability compared to the bacteriophytochrome (BPH) FPs. BV supply is limited by membrane permeability, so expression of heme oxygenase-1 with heme precursors increases fluorescence of BPH/APCα FPs. SmURFP (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence in situ comparable to FPs from jellyfish/coral. A far-red/near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP. PMID:27479328

  13. Metabolic imaging of the tumor treated by KillerRed fluorescent protein-based photodynamic therapy in mice

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Qin, Lingsong; Wang, Anle; Liu, Zheng; Yang, Fei; Jin, Honglin; Zhang, Zhihong

    2014-02-01

    KillerRed is a unique red fluorescent protein exhibiting excellent phototoxic properties. It has the ability to produce reactive oxygen species (ROS), for killing tumor cells in vitro upon laser irradiation and has the potential to act as a photosensitizer in the application of tumor therapy. Here, we investigated the effects of KillerRed-based photodynamic therapy (PDT) on tumor growth in vivo and examined the subsequent tumor metabolic states including the changes of pyridine nucleotide (PN) and flavoprotein (Fp), two important metabolic coenzymes of tumor cells. Results showed that the tumor was scabbed in response to 561 nm laser irradiation at 80 mV for 3 min, and the tumor growth had been significantly inhibited by KillerRed-based PDT treatment compared to control groups. More importantly, a home-made cryo-imaging redox scanner was used to measure intrinsic fluorescence and exogenous KillerRed fluorescence signals in tumors. The flavoprotein was remarkable elevated and the PN was seldom increased with concomitant photobleaching of KillerRed fluorescence after irradiation, suggesting that flavoprotein and PN were oxidized in the course of KillerRed-based PDT.

  14. Two-Dimensional Computational Flow Analysis and Frictional Characteristics Model for Red Blood Cell under Inclined Centrifuge Microscopy

    NASA Astrophysics Data System (ADS)

    Funamoto, Kenichi; Hayase, Toshiyuki; Shirai, Atsushi

    Simplified two-dimensional flow analysis is performed in order to simulate frictional characteristics measurement of red blood cells moving on a glass plate in a medium with an inclined centrifuge microscope. Computation under various conditions reveals the influences of parameters on lift, drag, and moment acting on a red blood cell. Among these forces, lift appears only when the cell is longitudinally asymmetric. By considering the balance of forces, the frictional characteristics of the red blood cell are modeled as the sum of Coulomb friction and viscous drag. The model describes the possibility that the red blood cell deforms to expand in the front side in response to the inclined centrifugal force. When velocity exceeds some critical value, the lift overcomes the normal centrifugal force component, and the thickness of the plasma layer between the cell and the glass plate increases from the initial value of the plasma protein thickness.

  15. Photoacoustic response of suspended and hemolyzed red blood cells

    NASA Astrophysics Data System (ADS)

    Saha, Ratan K.; Karmakar, Subhajit; Roy, Madhusudan

    2013-07-01

    The effect of confinement of hemoglobin molecules on photoacoustic (PA) signal is studied experimentally. The PA amplitudes for samples with suspended red blood cells (SRBCs) and hemolyzed red blood cells (HRBCs) were found to be comparable at each hematocrit for 532 nm illumination. The difference between the corresponding amplitudes increased with increasing hematocrit for 1064 nm irradiation. For example, the PA amplitude for the SRBCs was about 260% higher than that of the HRBCs at 40% hematocrit. This observation may help to develop a PA method detecting hemolysis noninvasively.

  16. Fibrinogen, red blood cells, and factor XIII in venous thrombosis.

    PubMed

    Walton, B L; Byrnes, J R; Wolberg, A S

    2015-06-01

    Cardiovascular disease is the leading cause of death and disability worldwide. Among cardiovascular causes of death, venous thrombosis (VT) is ranked third most common in the world. Venous thrombi have high red blood cell and fibrin content; however, the pathophysiologic mechanisms that contribute to venous thrombus composition and stability are still poorly understood. This article reviews biological, biochemical, and biophysical contributions of fibrinogen, factor XIII, and red blood cells to VT, and new evidence suggesting interactions between these components mediate venous thrombus composition and size.

  17. Generation of monomeric reversibly switchable red fluorescent proteins for far-field fluorescence nanoscopy.

    PubMed

    Stiel, Andre C; Andresen, Martin; Bock, Hannes; Hilbert, Michael; Schilde, Jessica; Schönle, Andreas; Eggeling, Christian; Egner, Alexander; Hell, Stefan W; Jakobs, Stefan

    2008-09-15

    Reversibly switchable fluorescent proteins (RSFPs) are GFP-like proteins that may be repeatedly switched by irradiation with light from a fluorescent to a nonfluorescent state, and vice versa. They can be utilized as genetically encodable probes and bear large potential for a wide array of applications, in particular for new protein tracking schemes and subdiffraction resolution microscopy. However, the currently described monomeric RSFPs emit only blue-green or green fluorescence; the spectral window for their use is thus rather limited. Using a semirational engineering approach based on the crystal structure of the monomeric nonswitchable red fluorescent protein mCherry, we generated rsCherry and rsCherryRev. These two novel red fluorescent RSFPs exhibit fluorescence emission maxima at approximately 610 nm. They display antagonistic switching modes, i.e., in rsCherry irradiation with yellow light induces the off-to-on transition and blue light the on-to-off transition, whereas in rsCherryRev the effects of the switching wavelengths are reversed. We demonstrate time-lapse live-cell subdiffraction microscopy by imaging rsCherryRev targeted to the endoplasmic reticulum utilizing the switching and localization of single molecules.

  18. Molecular maps of red cell deformation: Hidden elasticity and in situ connectivity

    SciTech Connect

    Discher, D.E.; Mohandas, N.; Evans, E.A.

    1994-11-11

    Fluorescence-imaged micropipette aspiration was used to map redistribution of the proteins and lipids in highly extended human red blood cell membranes. Whereas the fluid bilayer distributed uniformly ({+-} 10 percent), the underlying, solidlike cytoskeleton of spectrin, actin, and protein 4.1 exhibited a steep gradient in density along the aspirated projection, which was reversible on release from deformation. Quantitation of the cytoskeletal protein density gradients showed that skeletal elasticity is well represented by a grafted polymer network with a ratio of surface dilation modulus to shear modulus of approximately 2:1. Fractionally mobile integral proteins, such as band 3, and highly mobile receptors, such as CD59 as well as glycophorin C in protein 4.1-deficient cells, appeared to be squeezed out of areas dense in the underlying network and enriched in areas of network dilation. This complementary segregation demonstrates patterning of cell surface components by cytoskeletal dilation.

  19. A Transgenic Mouse Line Expressing the Red Fluorescent Protein tdTomato in GABAergic Neurons

    PubMed Central

    Besser, Stefanie; Sicker, Marit; Marx, Grit; Winkler, Ulrike; Eulenburg, Volker; Hülsmann, Swen; Hirrlinger, Johannes

    2015-01-01

    GABAergic inhibitory neurons are a large population of neurons in the central nervous system (CNS) of mammals and crucially contribute to the function of the circuitry of the brain. To identify specific cell types and investigate their functions labelling of cell populations by transgenic expression of fluorescent proteins is a powerful approach. While a number of mouse lines expressing the green fluorescent protein (GFP) in different subpopulations of GABAergic cells are available, GFP expressing mouse lines are not suitable for either crossbreeding to other mouse lines expressing GFP in other cell types or for Ca2+-imaging using the superior green Ca2+-indicator dyes. Therefore, we have generated a novel transgenic mouse line expressing the red fluorescent protein tdTomato in GABAergic neurons using a bacterial artificial chromosome based strategy and inserting the tdTomato open reading frame at the start codon within exon 1 of the GAD2 gene encoding glutamic acid decarboxylase 65 (GAD65). TdTomato expression was observed in all expected brain regions; however, the fluorescence intensity was highest in the olfactory bulb and the striatum. Robust expression was also observed in cortical and hippocampal neurons, Purkinje cells in the cerebellum, amacrine cells in the retina as well as in cells migrating along the rostral migratory stream. In cortex, hippocampus, olfactory bulb and brainstem, 80% to 90% of neurons expressing endogenous GAD65 also expressed the fluorescent protein. Moreover, almost all tdTomato-expressing cells coexpressed GAD65, indicating that indeed only GABAergic neurons are labelled by tdTomato expression. This mouse line with its unique spectral properties for labelling GABAergic neurons will therefore be a valuable new tool for research addressing this fascinating cell type. PMID:26076353

  20. Squeezing red blood cells on an optical waveguide to monitor cell deformability during blood storage.

    PubMed

    Ahluwalia, Balpreet Singh; McCourt, Peter; Oteiza, Ana; Wilkinson, James S; Huser, Thomas R; Hellesø, Olav Gaute

    2015-01-07

    Red blood cells squeeze through micro-capillaries as part of blood circulation in the body. The deformability of red blood cells is thus critical for blood circulation. In this work, we report a method to optically squeeze red blood cells using the evanescent field present on top of a planar waveguide chip. The optical forces from a narrow waveguide are used to squeeze red blood cells to a size comparable to the waveguide width. Optical forces and pressure distributions on the cells are numerically computed to explain the squeezing process. The proposed technique is used to quantify the loss of blood deformability that occurs during blood storage lesion. Squeezing red blood cells using waveguides is a sensitive technique and works simultaneously on several cells, making the method suitable for monitoring stored blood.

  1. Red Raspberry Phenols Inhibit Angiogenesis: A Morphological and Subcellular Analysis Upon Human Endothelial Cells.

    PubMed

    Sousa, M; Machado, V; Costa, R; Figueira, M E; Sepodes, B; Barata, P; Ribeiro, L; Soares, R

    2016-07-01

    Polyphenols are a class of natural compounds whose potential as antioxidant, anti-inflammatory, and anti-angiogenesis has been reported in many pathological conditions. Red raspberry extract, rich in polyphenols, has been reported to exert anti-inflammatory effects and prevent cell proliferation in distinct animal models. However, the signaling pathways involved remain unknown. Herein, we used human microvascular endothelial cells (HMVECs) to determine the influence of red raspberry phenolic compound extract concentrations, ranging from 10 to 250 µg gallic acid equivalents (GAE)/mL, on endothelium viability (MTS assay), proliferation (BrdU incorporation), migration (injury assay), and capillary-like structures formation (Matrigel assay). Protein expression in cell lysates was determined by Western blot analysis. We showed that red raspberry extracts reduced cell viability (GI50  = 87,64 ± 6,59 μg GAE/mL) and proliferation in a dose-dependent manner. A significant abrogation of cells ability to migrate to injured areas, even at low concentrations, was observed by injury assay. Cell assembly into capillary-like structures on Matrigel also decreased in a dose dependent-manner for higher extract concentrations, as well as the number of branching points per unit of area. Protein expression analysis showed a dose-dependent decrease in Phospho-VEGFR2 expression, implying abrogation of VEGF signaling activity. We also showed for the first time that red raspberry phenolic compounds induce the rearrangement of filamentous actin cytoskeleton, with an isotropy increase found for higher testing concentrations. Taken together, our findings corroborate the anti-angiogenic potential of red raspberry phenolic compounds and provide new insights into their mode of action upon endothelium. J. Cell. Biochem. 117: 1604-1612, 2016. © 2015 Wiley Periodicals, Inc.

  2. Red light of the visual spectrum attenuates cell death in culture and retinal ganglion cell death in situ.

    PubMed

    Del Olmo-Aguado, Susana; Núñez-Álvarez, Claudia; Osborne, Neville N

    2016-09-01

    To ascertain whether red light, known to enhance mitochondrial function, can blunt chemical insults to cell cultures and ischaemic insults to the rat retina. Raised intraocular pressure (IOP, 140 mmHg, 60 min) or ischaemia was delivered in complete darkness or in the presence of low intensity red light (16.5 watts/m(2) , 3000 lux, 625-635 nm) to one eye of each rat. Animals were killed at specific times after ischemia and retinas analysis for ganglion cell numbers, the localization of specific antigens or for changes in defined RNAs. RGC-5 cell cultures were also exposed to various chemical insults in the presence or absence of red light. Significant differences were determined by t-test and anova. Elevation of IOP causes changes in the localization of glial fibrillary acid protein (GFAP), calretinin, calbindin, choline acetyltransferase, ganglion cell numbers and an elevation (GFAP, vimentin, HO-1 and mTORC1) or reduction (Thy-1 and Brn3a) of mRNAs in the rat retina. These negative effects to the rat retina caused by ischaemia are reduced by red light. Moreover, chemical insults to cell cultures are blunted by red light. Low, non-toxic levels of red light focussed on the retina for a short period of time are sufficient to attenuate an insult of raised IOP to the rat retina. Since mitochondrial dysfunctions are thought to play a major role in ganglion cell death in glaucoma, we propose the potential use of red light therapy for the treatment of the disease. © 2016 Acta Ophthalmologica Scandinavica Foundation. Published by John Wiley & Sons Ltd.

  3. Red Blood Cell Dysfunction Induced by High-Fat Diet

    PubMed Central

    Unruh, Dusten; Srinivasan, Ramprasad; Benson, Tyler; Haigh, Stephen; Coyle, Danielle; Batra, Neil; Keil, Ryan; Sturm, Robert; Blanco, Victor; Palascak, Mary; Franco, Robert S.; Tong, Wilson; Chatterjee, Tapan; Hui, David Y.; Davidson, W. Sean; Aronow, Bruce J.; Kalfa, Theodosia; Manka, David; Peairs, Abigail; Blomkalns, Andra; Fulton, David J.; Brittain, Julia E.; Weintraub, Neal L.; Bogdanov, Vladimir Y.

    2015-01-01

    Background High-fat diet (HFD) promotes endothelial dysfunction and proinflammatory monocyte activation, which contribute to atherosclerosis in obesity. We investigated whether HFD also induces the dysfunction of red blood cells (RBCs), which serve as a reservoir for chemokines via binding to Duffy antigen receptor for chemokines (DARC). Methods and Results A 60% HFD for 12 weeks, which produced only minor changes in lipid profile in C57/BL6 mice, markedly augmented the levels of monocyte chemoattractant protein-1 bound to RBCs, which in turn stimulated macrophage migration through an endothelial monolayer. Levels of RBC-bound KC were also increased by HFD. These effects of HFD were abolished in DARC−/− mice. In RBCs from HFD-fed wild-type and DARC−/− mice, levels of membrane cholesterol and phosphatidylserine externalization were increased, fostering RBC-macrophage inflammatory interactions and promoting macrophage phagocytosis in vitro. When labeled ex vivo and injected into wild-type mice, RBCs from HFD-fed mice exhibited ≈3-fold increase in splenic uptake. Finally, RBCs from HFD-fed mice induced increased macrophage adhesion to the endothelium when they were incubated with isolated aortic segments, indicating endothelial activation. Conclusions RBC dysfunction, analogous to endothelial dysfunction, occurs early during diet-induced obesity and may serve as a mediator of atherosclerosis. These findings may have implications for the pathogenesis of atherosclerosis in obesity, a worldwide epidemic. PMID:26467254

  4. Fermented red ginseng extract inhibits cancer cell proliferation and viability.

    PubMed

    Oh, Jisun; Jeon, Seong Bin; Lee, Yuri; Lee, Hyeji; Kim, Ju; Kwon, Bo Ra; Yu, Kang-Yeol; Cha, Jeong-Dan; Hwang, Seung-Mi; Choi, Kyung-Min; Jeong, Yong-Seob

    2015-04-01

    Red ginseng (Panax ginseng C.A. Meyer) is the most widely recognized medicinal herb due to its remedial effects in various disorders, such as cancers, diabetes, and heart problems. In this study, we investigated the anticancer effect of fermented red ginseng extract (f-RGE; provided by Jeonju Biomaterials Institute, Jeonju, South Korea) in a parallel comparison with the effect of nonfermented red ginseng extract (nf-RGE; control) on several cancer cell lines--MCF-7 breast cancer cells, HepG2 hepatocellular carcinoma cells, and reprogrammed MCF-7 cells (mimicking cancer stem cells). Cells were cultured at various concentrations of RGE (from 0.5 up to 5 mg/mL) and their viabilities and proliferative properties were examined. Our data demonstrate the following: (1) nf-RGE inhibited cell viability at ≥1 mg/mL for MCF-7 cells and ≥2 mg/mL for HepG2 cells, (2) in the presence of a carcinogenic agent, 12-O-tetradecanoylphorbol-13-acetate (TPA), nf-RGE treatment in combination with paclitaxel synergistically decreased MCF-7 as well as HepG2 cell viability, (3) f-RGE (which contained a greater level of Rg3 content) more effectively decreased the viability of MCF-7 and HepG2 cells compared to nf-RGE, and (4) f-RGE appeared more potent for inhibiting cancerous differentiation of reprogrammed MCF-7 cells in a synergistic fashion with paclitaxel, especially in the presence of TPA, compared to nf-RGE. These findings suggest that f-RGE treatment may be more effective for decreasing cancer cell survival by inducing apoptotic cell death and also presumably for preventing cancer stem cell differentiation compared to nf-RGE.

  5. Artificial Red Cells with Polyhemoglobin Membranes.

    DTIC Science & Technology

    1981-09-01

    preparing emulsions and ejecting cells from the oil phase. IX. REFERENCES 1. Wallace, H. W., Asher, W. J., and Li, N. N. Liquid - liquid oxygenation: a...1S. KEY WORDS (Continue, an reverse side if naceoay mnd identify by block number) Artificial Blood, Hemoglobin, Polyhemoglobin, Biotonometry Liquid ...cell-size microdroplets containing 30% of hemoglobin were held in liquid membrane capsules and treated with glutaralddhyde that cross linked the

  6. Chronic red blood cell exchange to prevent clinical complications in sickle cell disease.

    PubMed

    Cabibbo, Sergio; Fidone, Carmelo; Garozzo, Giovanni; Antolino, Agostino; Manenti, Giovanna Oriella; Bennardello, Francesco; Licitra, Vincenzo; Calabrese, Salvatore; Costantino, Francesco; Travali, Simone; Distefano, Roberto; Bonomo, Pietro

    2005-06-01

    We tracked the results of 394 manual or automatic red blood cell exchanges done with a cell separator in 20 sickle cell patients at high risk for recurrent complications. Over an average of 6 years, none of the patients developed complications related to the procedure or to the increased blood use. It was safe and effective in preventing complications of sickle cell disease, and if done automatically, reduced iron overload. Ferritin levels also decreased in patients treated with automatic red blood cell exchange. Furthermore, using Single Donor Red Blood Cell units (SDRC) we reduced the potential exposure to transfusion transmitted infectious diseases (TTI).

  7. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology.

    PubMed

    Fens, Marcel H A M; van Wijk, Richard; Andringa, Grietje; van Rooijen, Karlijn L; Dijstelbloem, Hilde M; Rasmussen, Jan T; de Vooght, Karen M K; Schiffelers, Raymond M; Gaillard, Carlo A J M; van Solinge, Wouter W

    2012-04-01

    Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and α(v)-integrin. Phagocytosis via the phosphatidylserine-lactadherin-α(v)-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes. Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity. Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape ('rounding up'). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects. Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to vasopathological effects as seen, for instance, in sickle cell

  8. A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

    PubMed Central

    Fens, Marcel H.A.M.; van Wijk, Richard; Andringa, Grietje; van Rooijen, Karlijn L.; Dijstelbloem, Hilde M.; Rasmussen, Jan T.; de Vooght, Karen M.K.; Schiffelers, Raymond M.; Gaillard, Carlo A.J.M.; van Solinge, Wouter W.

    2012-01-01

    Background Phosphatidylserine exposure by red blood cells is acknowledged as a signal that initiates phagocytic removal of the cells from the circulation. Several disorders and conditions are known to induce phosphatidylserine exposure. Removal of phosphatidylserine-exposing red blood cells generally occurs by macrophages in the spleen and liver. Previously, however, we have shown that endothelial cells are also capable of erythrophagocytosis. Key players in the erythrophagocytosis by endothelial cells appeared to be lactadherin and αv-integrin. Phagocytosis via the phosphatidylserine-lactadherin-αv-integrin pathway is the acknowledged route for removal of apoptotic innate cells by phagocytes. Design and Methods Endothelial cell phagocytosis of red blood cells was further explored using a more (patho)physiological approach. Red blood cells were exposed to oxidative stress, induced by tert-butyl hydroperoxide. After opsonization with lactadherin, red blood cells were incubated with endothelial cells to study erythrophagocytosis and examine cytotoxicity. Results Red blood cells exposed to oxidative stress show alterations such as phosphatidylserine exposure and loss of deformability. When incubated with endothelial cells, marked erythrophagocytosis occurred in the presence of lactadherin under both static and flow conditions. As a consequence, intracellular organization was disturbed and endothelial cells were seen to change shape (‘rounding up’). Increased expression of apoptotic markers indicated that marked erythrophagocytosis has cytotoxic effects. Conclusions Activated endothelial cells show significant phagocytosis of phosphatidylserine-exposing and rigid red blood cells under both static and flow conditions. This results in a certain degree of cytotoxicity. We postulate that activated endothelial cells play a role in red blood cell clearance in vivo. Significant erythrophagocytosis can induce endothelial cell loss, which may contribute to

  9. 21 CFR 864.5300 - Red cell indices device.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell indices device. 864.5300 Section 864.5300 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Automated and Semi-Automated Hematology Devices §...

  10. Spatial Distributions of Red Blood Cells Significantly Alter Local Haemodynamics

    PubMed Central

    Sherwood, Joseph M.; Holmes, David; Kaliviotis, Efstathios; Balabani, Stavroula

    2014-01-01

    Although bulk changes in red blood cell concentration between vessels have been well characterised, local distributions are generally overlooked. Red blood cells aggregate, deform and migrate within vessels, forming heterogeneous distributions which have considerable effect on local haemodynamics. The present study reports data on the local distribution of human red blood cells in a sequentially bifurcating microchannel, representing the branching geometry of the microvasculature. Imaging methodologies with simple extrapolations are used to infer three dimensional, time-averaged velocity and haematocrit distributions under a range of flow conditions. Strong correlation between the bluntness of the velocity and haematocrit profiles in the parent branch of the geometry is observed and red blood cell aggregation has a notable effect on the observed trends. The two branches of the first bifurcation show similar characteristics in terms of the shapes of the profiles and the extent of plasma skimming, despite the difference in geometric configuration. In the second bifurcation, considerable asymmetry between the branches in the plasma skimming relationship is observed, and elucidated by considering individual haematocrit profiles. The results of the study highlight the importance of considering local haematocrit distributions in the analysis of blood flow and could lead to more accurate computational models of blood flow in microvascular networks. The experimental approaches developed in this work provide a foundation for further examining the characteristics of microhaemodynamics. PMID:24950214

  11. Shape of red blood cells in contact with artificial surfaces.

    PubMed

    Grzhibovskis, Richards; Krämer, Elisabeth; Bernhardt, Ingolf; Kemper, Björn; Zanden, Carl; Repin, Nikolay V; Tkachuk, Bogdan V; Voinova, Marina V

    2017-03-01

    The phenomenon of physical contact between red blood cells and artificial surfaces is considered. A fully three-dimensional mathematical model of a bilayer membrane in contact with an artificial surface is presented. Numerical results for the different geometries and adhesion intensities are found to be in agreement with experimentally observed geometries obtained by means of digital holographic microscopy.

  12. Full dynamics of a red blood cell in shear flow.

    PubMed

    Dupire, Jules; Socol, Marius; Viallat, Annie

    2012-12-18

    At the cellular scale, blood fluidity and mass transport depend on the dynamics of red blood cells in blood flow, specifically on their deformation and orientation. These dynamics are governed by cellular rheological properties, such as internal viscosity and cytoskeleton elasticity. In diseases in which cell rheology is altered genetically or by parasitic invasion or by changes in the microenvironment, blood flow may be severely impaired. The nonlinear interplay between cell rheology and flow may generate complex dynamics, which remain largely unexplored experimentally. Under simple shear flow, only two motions, "tumbling" and "tank-treading," have been described experimentally and relate to cell mechanics. Here, we elucidate the full dynamics of red blood cells in shear flow by coupling two videomicroscopy approaches providing multidirectional pictures of cells, and we analyze the mechanical origin of the observed dynamics. We show that contrary to common belief, when red blood cells flip into the flow, their orientation is determined by the shear rate. We discuss the "rolling" motion, similar to a rolling wheel. This motion, which permits the cells to avoid energetically costly deformations, is a true signature of the cytoskeleton elasticity. We highlight a hysteresis cycle and two transient dynamics driven by the shear rate: an intermittent regime during the "tank-treading-to-flipping" transition and a Frisbee-like "spinning" regime during the "rolling-to-tank-treading" transition. Finally, we reveal that the biconcave red cell shape is highly stable under moderate shear stresses, and we interpret this result in terms of stress-free shape and elastic buckling.

  13. Protein digestion in red aak borer larvae, Enaphalodes rufulus

    USDA-ARS?s Scientific Manuscript database

    In the Ozark Mountains of Arkansas, Oklahoma, and Missouri, a recent outbreak of red oak borer, Enaphalodes rufulus (Haldeman), contributed to the death of tens of thousands of red oaks. To better understand nutrient digestion in E. rufulus larvae, biochemical analyses were used to characterize dige...

  14. THE AGGLUTINATION OF RED BLOOD CELLS

    PubMed Central

    Northrop, John H.; Freund, Jules

    1924-01-01

    1. Unsensitized sheep cells suspended in sugar solutions are agglutinated by electrolytes whenever the potential is depressed to 6 millivolts or less, except in the case of MgCl2 or CaCl2. 2. With these salts no agglutination occurs although there is practically no potential. The presence of these salts prevents acid agglutination. This is presumably due to a decrease in the "cohesion" between the cells. 3. Cells which have been sensitized with specific antibody, ricin, colloidal stannic hydroxide, or paraffin oil, are agglutinated whenever the potential is decreased below about 12 millivolts. 4. The agglutination by electrolytes is therefore primarily due to a decrease in the potential whereas agglutination by immune serum, ricin, etc., is due primarily to an increase in the critical potential. PMID:19872099

  15. Red blood cell replacement, or nanobiotherapeutics with enhanced red blood cell functions?

    PubMed

    Chang, Thomas Ming Swi

    2015-06-01

    Why is this important? Under normal circumstances, donor blood is the best replacement for blood. However, there are exceptions: During natural epidemics (e.g., HIV, Ebola, etc.) or man-made epidemics (terrorism, war, etc.), there is a risk of donor blood being contaminated, and donors being disqualified because they have contracted disease. Unlike red blood cells (RBCs), blood substitutes can be sterilized to remove infective agents. Heart attack and stroke are usually caused by obstruction of arterial blood vessels. Unlike RBCs, which are particulate, blood substitutes are in the form of a solution that can perfuse through obstructed vessels with greater ease to reach the heart and brain, as has been demonstrated in animal studies. Severe blood loss from injuries sustained during accidents, disasters, or war may require urgent blood transfusion that cannot wait for transportation to the hospital for blood group testing. Unlike RBCs, blood substitutes do not have specific blood groups, and can be administered on the spot. RBCs have to be stored under refrigeration for up to 42 days, and are thus difficult to transport and store in times of disaster and at the battlefront. Blood substitutes can be stored at room temperature for more than 1 year, compared to the RBC shelf life of 1 day, at room temperature. In cases of very severe hemorrhagic shock, there is usually a safety window of 60 min for blood replacement, beyond which there could be problems related to irreversible shock. Animal studies show that a particular type of blood substitute, with enhanced RBC enzymes, may be able to prolong the duration of the safety window.

  16. Red photon treatment inhibits apoptosis via regulation of bcl-2 proteins and ROS levels, alleviating hypoxic-ischemic brain damage.

    PubMed

    Jiang, W; Chen, L; Zhang, X J; Chen, J; Li, X C; Hou, W S; Xiao, N

    2014-05-30

    Therapeutic options for hypoxic-ischemic brain damage (HIBD) are scarce and inefficient. Recently, many studies have demonstrated that red photon plays an important role in anti-inflammatory processes as well as apoptosis, the main trait of HIBD. In this study, we investigated whether red photon can protect from HIBD in SD rats and oxygen-glucose deprivation (OGD) in PC12 cells. Apoptosis, mitochondrial transmembrane potential (MMP), and reactive oxygen species (ROS) rates were assessed in PC12 cells. We found that 6-h irradiation resulted in decreased MMP, ROS and apoptosis rates, although these changes were reversible with prolonged irradiation. Importantly, these effects were sustained for 2-8h upon quenching of the red photon. Similar trends were observed for protein and mRNA expression of bax and bcl-2, with short-term irradiation (6h) inhibiting apoptosis in PC12 Cells. However, long-term (>6h) irradiation caused cell damage. In vivo experiments, bax mRNA and protein levels were reduced after 7days in HIBD model rats treated with red photon, in contrast to bcl-2. Furthermore, we found that bax and bcl-2 were mainly expressed in pyramidal cells of the hippocampus CA1 and CA3. Importantly, Morris Water Maze test results revealed an improvement in learning ability and spatial memory in rats after irradiation. Overall, our data showed that short-term irradiation with red photon in the acute phase inhibits the mitochondrial apoptotic pathway via regulation of bcl-2-related proteins and reduction of ROS levels, thereby decreasing apoptosis in nerve cells and improving the neurological prognosis of HIBD.

  17. Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota.

    PubMed

    Cian, Raúl E; Drago, Silvina R; de Medina, Fermín Sánchez; Martínez-Augustin, Olga

    2015-08-20

    Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain "cellulose binding domains", phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and "dl-hybrid") and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function.

  18. Proteins and Carbohydrates from Red Seaweeds: Evidence for Beneficial Effects on Gut Function and Microbiota

    PubMed Central

    Cian, Raúl E.; Drago, Silvina R.; Sánchez de Medina, Fermín; Martínez-Augustin, Olga

    2015-01-01

    Based on their composition, marine algae, and namely red seaweeds, are good potential functional foods. Intestinal mucosal barrier function refers to the capacity of the intestine to provide adequate containment of luminal microorganisms and molecules. Here, we will first outline the component of seaweeds and will summarize the effects of these on the regulation of mucosal barrier function. Special attention will be paid to unique components of red seaweeds: proteins and derived peptides (e.g., phycobiliproteins, glycoproteins that contain “cellulose binding domains”, phycolectins and the related mycosporine-like amino acids) together with polysaccharides (e.g., floridean starch and sulfated galactans, such as carrageenans, agarans and “dl-hybrid”) and minerals. These compounds have been shown to exert prebiotic effects, to regulate intestinal epithelial cell, macrophage and lymphocyte proliferation and differentiation and to modulate the immune response. Molecular mechanisms of action of peptides and polysaccharides are starting to be elucidated, and evidence indicating the involvement of epidermal growth factor receptor (EGFR), insulin-like growth factor receptor (IGFR), Toll-like receptors (TLR) and signal transduction pathways mediated by protein kinase B (PKB or AKT), nuclear factor-κB (NF-κB) and mitogen activated protein kinases (MAPK) will also be summarized. The need for further research is clear, but in vivo experiments point to an overall antiinflammatory effect of these algae, indicating that they can reinforce membrane barrier function. PMID:26308006

  19. Aggregation of red blood cells: From rouleaux to clot formation

    NASA Astrophysics Data System (ADS)

    Wagner, Christian; Steffen, Patrick; Svetina, Saša

    2013-06-01

    Red blood cells are known to form aggregates in the form of rouleaux. This aggregation process is believed to be reversible, but there is still no full understanding on the adhesion mechanism. There are at least two competing models, based either on bridging or on depletion. We review recent experimental results on the single cell level and theoretical analyses of the depletion model and of the influence of the cell shape on the adhesion strength. Another important aggregation mechanism is caused by activation of platelets. This leads to clot formation which is life-saving in the case of wound healing, but also a major cause of death in the case of a thrombus induced stroke. We review historical and recent results on the participation of red blood cells in clot formation.

  20. Calcium movements across the membrane of human red cells

    PubMed Central

    Schatzmann, H. J.; Vincenzi, F. F.

    1969-01-01

    1. A study has been made of the cellular content and movement of Ca across the membrane of human red blood cells. 2. The [Ca] in the cellular contents of fresh red cells is 4·09 × 10-2 mM. The intracellular concentration of free ionic Ca ([Ca2+]) is considered to be less than this value and therefore less than extracellular [Ca2+] under normal conditions. 3. Observation of unidirectional Ca fluxes with 45Ca confirms previous reports of low permeability of the red cell membrane for Ca. After nearly 1 week of loading in the cold, intracellular 45Ca content is 1·8% of extracellular 45Ca content. Appearance in extracellular fluid of 45Ca from coldloaded cells can be considered to arise from two compartments. Efflux of 45Ca from the `slower compartment' is accelerated by the addition of glucose. 4. Starved red cells, incubated at 37° C, after reversible haemolysis for loading with Ca and Mg-ATP, exhibit an outward net transport of Ca against an electrochemical gradient. The transport is associated with the appearance of inorganic phosphate (Pi). Cells treated similarly, but without ATP show no transport and no appearance of Pi. 5. During the initial phase of transport, 1·3 mole Pi appear per mole Ca transported. 6. The transport of Ca from ATP-loaded cells is highly temperature-dependent, with a Q10 of 3·5. 7. Cell membrane adenosine triphosphatase (ATPase) activity of reversibly haemolysed cells is stimulated only by intracellular, and not by extracellular Ca. 8. Neither Ca transport in reversibly haemolysed cells, nor the Ca-Mg activated ATPase of isolated cell membranes is sensitive to Na, K, ouabain or oligomycin. 9. Mg is not transported under the conditions which reveal Ca transport, but Mg appears to be necessary for Ca transport. 10. Sr is transported from reversibly haemolysed Mg-ATP-loaded cells. Sr also can substitute for Ca, but not for Mg, in the activation of membrane ATPase. 11. It is concluded that, in addition to a low passive permeability, an

  1. Regulation of red blood cell deformability is independent of red blood cell-nitric oxide synthase under hypoxia.

    PubMed

    Grau, Marijke; Lauten, Alexander; Hoeppener, Steffen; Goebel, Bjoern; Brenig, Julian; Jung, Christian; Bloch, Wilhelm; Suhr, Frank

    2016-09-12

    The aim was to study impacts of mild to severe hypoxia on human red blood cell (RBC)-nitric oxide synthase (NOS)-dependent NO production, protein S-nitrosylation and deformability.Ambient air oxygen concentration of 12 healthy subjects was step-wisely reduced from 20.95% to 16.21%, 12.35%, 10% and back to 20.95%. Additional in vitro experiments involved purging of blood (±sodium nitrite) with gas mixtures corresponding to in vivo intervention.Vital and hypoxia-associated parameters showed physiological adaptation to changing demands. Activation of RBC-NOS decreased with increasing hypoxia. RBC deformability, which is influenced by RBC-NOS activation, decreased under mild hypoxia, but surprisingly increased at severe hypoxia in vivo and in vitro. This was causatively induced by nitrite reduction to NO which increased S-nitrosylation of RBC α- and β-spectrins -a critical step to improve RBC deformability. The addition of sodium nitrite prevented decreases of RBC deformability under hypoxia by sustaining S-nitrosylation of spectrins suggesting compensatory mechanisms of non-RBC-NOS-produced NO.The results first time indicate a direct link between maintenance of RBC deformability under severe hypoxia by non-enzymatic NO production because RBC-NOS activation is reduced. These data improve our understanding of physiological mechanisms supporting adequate blood and, thus, oxygen supply to different tissues under severe hypoxia.

  2. Red blood cells transfusion in intensive care unit.

    PubMed

    Volpato, Solange Emanuelle; Ferreira, Jovino Dos Santos; Ferreira, Vera Lúcia Paes Cavalcanti; Ferreira, David Cavalcanti

    2009-12-01

    The anemia is a common problem upon admission of the patients in the intensive care unit being the red blood cell transfusion a frequent therapeutic. The causes of anemia in critical patients who under go red blood cell transfusion are several: acute loss of blood after trauma, gastrointestinal hemorrhage, surgery amongst others. Currently, few studies are available regarding the use of blood components in patients at intensive care unit. Although blood transfusions are frequent in intensive care unit, the optimized criteria for handling are not clearly defined, with no available guidelines. To analyze the clinical indications of the use of the red blood cell in the intensive care unit. The clinical history of the patients admitted in the intensive care unit were analyzed, revisiting which had have red blood cell transfusion in the period between January 1st 2005 and December 31 2005. The study was accepted by the Research Ethics Committee - Comitê de Ética em Pesquisa (CEP) - of the University of South of Santa Catarina (UNISUL). The transfusion rate was 19,33, and the majority of the patients were of the male gender. Their age prevalence was of 60 years old or older. The mortality rate among patients who under went red blood cell transfusion died was of 38,22%. The transfusions criterias were low serum hemoglobin (78%) and the hemoglobin pre - transfusion was 8,11 g/dL. Politrauma and sepsis/sepsis chock were the pre diagnosis criteria. A low hemoglobin level is the main clinical criteria with average hemoglobin pre - transfusion was 8,11 g/dL.

  3. Red blood cell and iron metabolism during space flight

    NASA Technical Reports Server (NTRS)

    Smith, Scott M.

    2002-01-01

    Space flight anemia is a widely recognized phenomenon in astronauts. Reduction in circulating red blood cells and plasma volume results in a 10% to 15% decrement in circulatory volume. This effect appears to be a normal physiologic adaptation to weightlessness and results from the removal of newly released blood cells from the circulation. Iron availability increases, and (in the few subjects studied) iron stores increase during long-duration space flight. The consequences of these changes are not fully understood.

  4. Red blood cell and iron metabolism during space flight

    NASA Technical Reports Server (NTRS)

    Smith, Scott M.

    2002-01-01

    Space flight anemia is a widely recognized phenomenon in astronauts. Reduction in circulating red blood cells and plasma volume results in a 10% to 15% decrement in circulatory volume. This effect appears to be a normal physiologic adaptation to weightlessness and results from the removal of newly released blood cells from the circulation. Iron availability increases, and (in the few subjects studied) iron stores increase during long-duration space flight. The consequences of these changes are not fully understood.

  5. [Familial transient red cell aplasia from parvovirus B-19 infection].

    PubMed

    Salvini, F; Tonella, M; Carpani, G; Scaglioni, S; Zuccotti, G V

    2002-01-01

    In our Paediatric Clinic we observed a case of transient aplastic crisis caused by Parvovirus B19 in a child and his mother, both affected by spherocytic haemolytic anemia. Anti-Parvovirus IgM antibody titre and viral search by PCR were positive. Anemia was treated with transfusion of concentrated red blood cells. In case of a family onset of hyperacute anemia it is necessary to consider a bone marrow aplastic crisis of the red series, induced by Parvovirus B19, especially if there is notice of an ongoing outbreak of erythema infectiosum.

  6. Freeze-Dried Human Red Blood Cells

    DTIC Science & Technology

    1991-07-12

    cells with 5 1Cr sodium chromate, collection of urine and peripheral blood samples, 5 1Cr organ uptake, whole body gamma imaging, and calculation of whole...7. Subject does not have a history of: - Renal disorders or BUN, creatinine, uric acid, sodium , potassium or chloride values outside the...uric acid, calcium, inorganic phosphorus, glucose, sodium , potassium, chloride, bicarbonate. Coagulation: prothrombin time (subject and control) and

  7. Effects of aluminum in red spruce (Picea rubens) cell cultures: Cell growth and viability, mitochondrial activity, ultrastructure and potential sites of intracellular aluminum accumulation

    Treesearch

    Rakesh Minocha; Carolyn McQuattie; Wayne Fagerberg; Stephanie Long; Eun Woon Noh

    2001-01-01

    The effects of Al on red spruce (Picea rubens Sarg.) cell suspension cultures were examined using biochemical, stereo-logical and microscopic methods. Exposure to Al for 24-48 h resulted in a loss of cell viability, inhibition of growth and a significant decrease in mitochondrial activity. Soluble protein content increased in cells treated with Al....

  8. Mechanical interactions between ice crystals and red blood cells during directional solidification.

    PubMed

    Ishiguro, H; Rubinsky, B

    1994-10-01

    Experiments in which red blood cells were frozen on a directional solidification stage under a microscope show that there is a mechanical interaction between ice crystals and cells in which cells are pushed and deformed by the ice crystals. The mechanical interaction occurs during freezing of cells in physiological saline and is significantly inhibited by the addition of 20% v/v glycerol to the solution. The addition of osmotically insignificant quantities of antifreeze proteins from the winter flounder or ocean pout to the physiological saline with 20% v/v glycerol generates strong mechanical interactions between the ice and the cells. The cells were destroyed during freezing in physiological saline, survived freezing in physiological saline with glycerol, and were completely destroyed by the addition of antifreeze proteins to the solution with glycerol. The difference in cell survival through freezing and thawing appears to be related, in part, to the habit of ice crystal growing in the suspension of red blood cells and the nature of mechanical interaction between the ice crystal and the cells. This suggests that mechanical damage may be a factor during cryopreservation of cells.

  9. The red cell storage lesion(s): of dogs and men

    PubMed Central

    Klein, Harvey G.

    2017-01-01

    The advent of preservative solutions permitted refrigerated storage of red blood cells. However, the convenience of having red blood cell inventories was accompanied by a disadvantage. Red cells undergo numerous physical and metabolic changes during cold storage, the “storage lesion(s)”. Whereas controlled clinical trials have not confirmed the clinical importance of such changes, ethical and operational issues have prevented careful study of the oldest stored red blood cells. Suggestions of toxicity from meta-analyses motivated us to develop pre-clinical canine models to compare the freshest vs the oldest red blood cells. Our model of canine pneumonia with red blood cell transfusion indicated that the oldest red blood cells increased mortality, that the severity of pneumonia is important, but that the dose of transfused red blood cells is not. Washing the oldest red blood cells reduces mortality by removing senescent cells and remnants, whereas washing fresher cells increases mortality by damaging the red blood cell membrane. An opposite effect was found in a model of haemorrhagic shock with reperfusion injury. Physiological studies indicate that release of iron from old cells is a primary mechanism of toxicity during infection, whereas scavenging of cell-free haemoglobin may be beneficial during reperfusion injury. Intravenous iron appears to have toxicity equivalent to old red blood cells in the pneumonia model, suggesting that intravenous iron and old red blood cells should be administered with caution to infected patients. PMID:28263166

  10. The red cell storage lesion(s): of dogs and men.

    PubMed

    Klein, Harvey G

    2017-03-01

    The advent of preservative solutions permitted refrigerated storage of red blood cells. However, the convenience of having red blood cell inventories was accompanied by a disadvantage. Red cells undergo numerous physical and metabolic changes during cold storage, the "storage lesion(s)". Whereas controlled clinical trials have not confirmed the clinical importance of such changes, ethical and operational issues have prevented careful study of the oldest stored red blood cells. Suggestions of toxicity from meta-analyses motivated us to develop pre-clinical canine models to compare the freshest vs the oldest red blood cells. Our model of canine pneumonia with red blood cell transfusion indicated that the oldest red blood cells increased mortality, that the severity of pneumonia is important, but that the dose of transfused red blood cells is not. Washing the oldest red blood cells reduces mortality by removing senescent cells and remnants, whereas washing fresher cells increases mortality by damaging the red blood cell membrane. An opposite effect was found in a model of haemorrhagic shock with reperfusion injury. Physiological studies indicate that release of iron from old cells is a primary mechanism of toxicity during infection, whereas scavenging of cell-free haemoglobin may be beneficial during reperfusion injury. Intravenous iron appears to have toxicity equivalent to old red blood cells in the pneumonia model, suggesting that intravenous iron and old red blood cells should be administered with caution to infected patients.

  11. Backward elastic light scattering of malaria infected red blood cells

    NASA Astrophysics Data System (ADS)

    Lee, Seungjun; Lu, Wei

    2011-08-01

    We investigated the backward light scattering pattern of healthy and malaria (Plasmodium falciparum) parasitized red blood cells. The spectrum could clearly distinguish between predominant ring stage infected blood cells and healthy blood cells. Further, we found that infected samples mixed with different stages of P. falciparum showed different signals, suggesting that even variance in parasite stages could also be detected by the spectrum. These results together with the backward scattering technique suggest the potential of non-invasive diagnosis of malaria through light scattering of blood cells near the surface of human body, such as using eyes or skin surface.

  12. Development of Pseudorabies Virus Strains Expressing Red Fluorescent Proteins: New Tools for Multisynaptic Labeling Applications

    PubMed Central

    Banfield, Bruce W.; Kaufman, Jessica D.; Randall, Jessica A.; Pickard, Gary E.

    2003-01-01

    The transsynaptic retrograde transport of the pseudorabies virus Bartha (PRV-Bartha) strain has become an important neuroanatomical tract-tracing technique. Recently, dual viral transneuronal labeling has been introduced by employing recombinant strains of PRV-Bartha engineered to express different reporter proteins. Dual viral transsynaptic tracing has the potential of becoming an extremely powerful method for defining connections of single neurons to multiple neural circuits in the brain. However, the present use of recombinant strains of PRV expressing different reporters that are driven by different promoters, inserted in different regions of the viral genome, and detected by different methods limits the potential of these recombinant virus strains as useful reagents. We previously constructed and characterized PRV152, a PRV-Bartha derivative that expresses the enhanced green fluorescent protein. The development of a strain isogenic to PRV152 and differing only in the fluorescent reporter would have great utility for dual transsynaptic tracing. In this report, we describe the construction, characterization, and application of strain PRV614, a PRV-Bartha derivative expressing a novel monomeric red fluorescent protein, mRFP1. In contrast to viruses expressing DsRed and DsRed2, PRV614 displayed robust fluorescence both in cell culture and in vivo following transsynaptic transport through autonomic circuits afferent to the eye. Transneuronal retrograde dual PRV labeling has the potential to be a powerful addition to the neuroanatomical tools for investigation of neuronal circuits; the use of strain PRV614 in combination with strain PRV152 will eliminate many of the pitfalls associated with the presently used pairs of PRV recombinants. PMID:12941921

  13. Tumor redox metabolism correlation with the expression level of red fluorescent protein

    NASA Astrophysics Data System (ADS)

    Sha, Shuang; Wang, Anle; Lin, Qiaoya; Zhang, Zhihong

    2015-03-01

    The redox metabolism is variable and complicated with the progress of tumor development. Whether the tumor redox state will affect the exogenous gene expression or not, are still not clear now . To investigate the relationship between tumor endogenous redox state and the exogenous gene expression level, a far red fluorescent protein fRFP was used to monitor tumor cells proliferation and as an exogenous protein expression in tumors. NADH (nicotinamide adenine dinucleotide) and Fp (flavin protein) are two important coenzymes in the mitochondria respiratory chain, which can be as a standard representation for redox metabolism state. Three tumor subcutaneous models (melanoma, human pancreatic carcinoma and nasopharyngeal carcinoma) were used to observe their redox state and protein expression by our home-made redox scanner. The results showed that the distribution of fRFP fluorescent protein expression in the inner tumor regions are heterogeneous, and the fluorescent intensity of fRFP and the fluorescent intensity of NADH have high correlation. In addition, we also found the linear coefficient in three tumors are different, the value of coefficient is (R2 = 0.966 and R2 = 0.943) in melanoma, (R2 = 0.701 and R2 = 0.942) in human pancreatic carcinoma, and (R2 = 0.994) in nasopharyngeal carcinoma, respectively. From these results, we consider that the exogenous protein expression of fRFP in tumor had some relationship with the tumor redox state of NADH.

  14. Familial distal renal tubular acidosis is associated with mutations in the red cell anion exchanger (Band 3, AE1) gene.

    PubMed Central

    Bruce, L J; Cope, D L; Jones, G K; Schofield, A E; Burley, M; Povey, S; Unwin, R J; Wrong, O; Tanner, M J

    1997-01-01

    All affected patients in four families with autosomal dominant familial renal tubular acidosis (dRTA) were heterozygous for mutations in their red cell HCO3-/Cl- exchanger, band 3 (AE1, SLC4A1) genes, and these mutations were not found in any of the nine normal family members studied. The mutation Arg589--> His was present in two families, while Arg589--> Cys and Ser613--> Phe changes were found in the other families. Linkage studies confirmed the co-segregation of the disease with a genetic marker close to AE1. The affected individuals with the Arg589 mutations had reduced red cell sulfate transport and altered glycosylation of the red cell band 3 N-glycan chain. The red cells of individuals with the Ser613--> Phe mutation had markedly increased red cell sulfate transport but almost normal red cell iodide transport. The erythroid and kidney isoforms of the mutant band 3 proteins were expressed in Xenopus oocytes and all showed significant chloride transport activity. We conclude that dominantly inherited dRTA is associated with mutations in band 3; but both the disease and its autosomal dominant inheritance are not related simply to the anion transport activity of the mutant proteins. PMID:9312167

  15. Partitioning of red blood cell aggregates in bifurcating microscale flows

    NASA Astrophysics Data System (ADS)

    Kaliviotis, E.; Sherwood, J. M.; Balabani, S.

    2017-03-01

    Microvascular flows are often considered to be free of red blood cell aggregates, however, recent studies have demonstrated that aggregates are present throughout the microvasculature, affecting cell distribution and blood perfusion. This work reports on the spatial distribution of red blood cell aggregates in a T-shaped bifurcation on the scale of a large microvessel. Non-aggregating and aggregating human red blood cell suspensions were studied for a range of flow splits in the daughter branches of the bifurcation. Aggregate sizes were determined using image processing. The mean aggregate size was marginally increased in the daughter branches for a range of flow rates, mainly due to the lower shear conditions and the close cell and aggregate proximity therein. A counterintuitive decrease in the mean aggregate size was apparent in the lower flow rate branches. This was attributed to the existence of regions depleted by aggregates of certain sizes in the parent branch, and to the change in the exact flow split location in the T-junction with flow ratio. The findings of the present investigation may have significant implications for microvascular flows and may help explain why the effects of physiological RBC aggregation are not deleterious in terms of in vivo vascular resistance.

  16. Partitioning of red blood cell aggregates in bifurcating microscale flows

    PubMed Central

    Kaliviotis, E.; Sherwood, J. M.; Balabani, S.

    2017-01-01

    Microvascular flows are often considered to be free of red blood cell aggregates, however, recent studies have demonstrated that aggregates are present throughout the microvasculature, affecting cell distribution and blood perfusion. This work reports on the spatial distribution of red blood cell aggregates in a T-shaped bifurcation on the scale of a large microvessel. Non-aggregating and aggregating human red blood cell suspensions were studied for a range of flow splits in the daughter branches of the bifurcation. Aggregate sizes were determined using image processing. The mean aggregate size was marginally increased in the daughter branches for a range of flow rates, mainly due to the lower shear conditions and the close cell and aggregate proximity therein. A counterintuitive decrease in the mean aggregate size was apparent in the lower flow rate branches. This was attributed to the existence of regions depleted by aggregates of certain sizes in the parent branch, and to the change in the exact flow split location in the T-junction with flow ratio. The findings of the present investigation may have significant implications for microvascular flows and may help explain why the effects of physiological RBC aggregation are not deleterious in terms of in vivo vascular resistance. PMID:28303921

  17. Alterations of Red Cell Membrane Properties in Nneuroacanthocytosis

    PubMed Central

    Siegl, Claudia; Hamminger, Patricia; Jank, Herbert; Ahting, Uwe; Bader, Benedikt; Danek, Adrian; Gregory, Allison; Hartig, Monika; Hayflick, Susan; Hermann, Andreas; Prokisch, Holger; Sammler, Esther M.; Yapici, Zuhal; Prohaska, Rainer; Salzer, Ulrich

    2013-01-01

    Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington’s disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an “acanthocytic state” of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration. PMID:24098554

  18. Rapid, Specific, No-wash, Far-red Fluorogen Activation in Subcellular Compartments by Targeted Fluorogen Activating Proteins

    PubMed Central

    2015-01-01

    Live cell imaging requires bright photostable dyes that can target intracellular organelles and proteins with high specificity in a no-wash protocol. Organic dyes possess the desired photochemical properties and can be covalently linked to various protein tags. The currently available fluorogenic dyes are in the green/yellow range where there is high cellular autofluorescence and the near-infrared (NIR) dyes need to be washed out. Protein-mediated activation of far-red fluorogenic dyes has the potential to address these challenges because the cell-permeant dye is small and nonfluorescent until bound to its activating protein, and this binding is rapid. In this study, three single chain variable fragment (scFv)-derived fluorogen activating proteins (FAPs), which activate far-red emitting fluorogens, were evaluated for targeting, brightness, and photostability in the cytosol, nucleus, mitochondria, peroxisomes, and endoplasmic reticulum with a cell-permeant malachite green analog in cultured mammalian cells. Efficient labeling was achieved within 20–30 min for each protein upon the addition of nM concentrations of dye, producing a signal that colocalized significantly with a linked mCerulean3 (mCer3) fluorescent protein and organelle specific dyes but showed divergent photostability and brightness properties dependent on the FAP. These FAPs and the ester of malachite green dye (MGe) can be used as specific, rapid, and wash-free labels for intracellular sites in live cells with far-red excitation and emission properties, useful in a variety of multicolor experiments. PMID:25650487

  19. Omics markers of the red cell storage lesion and metabolic linkage

    PubMed Central

    D’Alessandro, Angelo; Nemkov, Travis; Reisz, Julie; Dzieciatkowska, Monika; Wither, Matthew J.; Hansen, Kirk C.

    2017-01-01

    The introduction of omics technologies in the field of Transfusion Medicine has significantly advanced our understanding of the red cell storage lesion. While the clinical relevance of such a lesion is still a matter of debate, quantitative and redox proteomics approaches, as well quantitative metabolic flux analysis and metabolic tracing experiments promise to revolutionise our understanding of the role of blood processing strategies, inform the design and testing of novel additives or technologies (such as pathogen reduction), and evaluate the clinical relevance of donor and recipient biological variability with respect to red cell storability and transfusion outcomes. By reviewing existing literature in this rapidly expanding research endeavour, we highlight for the first time a correlation between metabolic markers of the red cell storage age and protein markers of haemolysis. Finally, we introduce the concept of metabolic linkage, i.e. the appreciation of a network of highly correlated small molecule metabolites which results from biochemical constraints of erythrocyte metabolic enzyme activities. For the foreseeable future, red cell studies will advance Transfusion Medicine and haematology by addressing the alteration of metabolic linkage phenotypes in response to stimuli, including, but not limited to, storage additives, enzymopathies (e.g. glucose 6-phosphate dehydrogenase deficiency), hypoxia, sepsis or haemorrhage. PMID:28263171

  20. Visualization of nuclear localization of transcription factors with cyan and green fluorescent proteins in the red alga Porphyra yezoensis.

    PubMed

    Uji, Toshiki; Takahashi, Megumu; Saga, Naotsune; Mikami, Koji

    2010-04-01

    Transcription factors play a central role in expression of genomic information in all organisms. The objective of our study is to analyze the function of transcription factors in red algae. One way to analyze transcription factors in eukaryotic cells is to study their nuclear localization, as reported for land plants and green algae using fluorescent proteins. There is, however, no report documenting subcellular localization of transcription factors from red algae. In the present study, using the marine red alga Porphyra yezoensis, we confirmed for the first time successful expression of humanized fluorescent proteins (ZsGFP and ZsYFP) from a reef coral Zoanthus sp. and land plant-adapted sGFP(S65T) in gametophytic cells comparable to expression of AmCFP. Following molecular cloning and characterization of transcription factors DP-E2F-like 1 (PyDEL1), transcription elongation factor 1 (PyElf1) and multiprotein bridging factor 1 (PyMBF1), we then demonstrated that ZsGFP and AmCFP can be used to visualize nuclear localization of PyElf1 and PyMBF1. This is the first report to perform visualization of subcellular localization of transcription factors as genome-encoded proteins in red algae.

  1. Forage Management Effects on Protein and Fiber Fractions, Protein Degradability, and Dry Matter Yield of Red Clover Conserved as Silage

    USDA-ARS?s Scientific Manuscript database

    Due to the action of o-quinones formed via polyphenol oxidase, conserved red clover (Trifolium pratense L.) contains abundant rumen undegradable protein (RUP), but inadequate rumen degradable protein (RDP) for dairy cattle. This study examined how forage management influences RDP, RUP, crude protein...

  2. Light inactivation of water transport and protein–protein interactions of aquaporin–Killer Red chimeras

    PubMed Central

    Baumgart, Florian; Rossi, Andrea

    2012-01-01

    Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling–induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (∼10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein–protein interactions as well as other membrane transporters and receptors. PMID:22200949

  3. Production of transgenic cloned pigs expressing the far-red fluorescent protein monomeric Plum

    PubMed Central

    WATANABE, Masahito; KOBAYASHI, Mirina; NAGAYA, Masaki; MATSUNARI, Hitomi; NAKANO, Kazuaki; MAEHARA, Miki; HAYASHIDA, Gota; TAKAYANAGI, Shuko; SAKAI, Rieko; UMEYAMA, Kazuhiro; WATANABE, Nobuyuki; ONODERA, Masafumi; NAGASHIMA, Hiroshi

    2015-01-01

    Monomeric Plum (Plum), a far-red fluorescent protein with photostability and photopermeability, is potentially suitable for in vivo imaging and detection of fluorescence in body tissues. The aim of this study was to generate transgenic cloned pigs exhibiting systemic expression of Plum using somatic cell nuclear transfer (SCNT) technology. Nuclear donor cells for SCNT were obtained by introducing a Plum-expression vector driven by a combination of the cytomegalovirus early enhancer and chicken beta-actin promoter into porcine fetal fibroblasts (PFFs). The cleavage and blastocyst formation rates of reconstructed SCNT embryos were 81.0% (34/42) and 78.6% (33/42), respectively. At 36–37 days of gestation, three fetuses systemically expressing Plum were obtained from one recipient to which 103 SCNT embryos were transferred (3/103, 2.9%). For generation of offspring expressing Plum, rejuvenated PFFs were established from one cloned fetus and used as nuclear donor cells. Four cloned offspring and one stillborn cloned offspring were produced from one recipient to which 117 SCNT embryos were transferred (5/117, 4.3%). All offspring exhibited high levels of Plum fluorescence in blood cells, such as lymphocytes, monocytes and granulocytes. In addition, the skin, heart, kidney, pancreas, liver and spleen also exhibited Plum expression. These observations demonstrated that transfer of the Plum gene did not interfere with the development of porcine SCNT embryos and resulted in the successful generation of transgenic cloned pigs that systemically expressed Plum. This is the first report of the generation and characterization of transgenic cloned pigs expressing the far-red fluorescent protein Plum. PMID:25739316

  4. Red blood cells and thrombin generation in sickle cell disease.

    PubMed

    Whelihan, Matthew F; Lim, Ming Y; Key, Nigel S

    2014-05-01

    The prothrombotic nature of sickle cell disease (SCD) is evidenced by the chronically elevated levels of almost all coagulation activation biomarkers, and an increased incidence of certain thrombotic events, including venous thromboembolism. Numerous studies have attempted to define the extent and elucidate the mechanism of the observed increase in thrombin generation in SCD patients in vivo. In general, these studies were performed using thrombin generation assays in platelet poor or platelet rich plasma and showed little difference in endogenous thrombin potential between the SCD cohort and healthy matched controls. In SCD, erythrocytes and monocytes have been demonstrated to exhibit procoagulant characteristics. Thus, the absence of these cellular components in standard thrombin generation assays may fail to reflect global hypercoagulability in the whole blood of patients with SCD. We were therefore surprised to see no difference in net thrombin generation in tissue factor-initiated initiated clotting of whole blood from patients with SCD. However, we are continuing to reconcile these seemingly disparate observations by slight modifications of the whole blood model that include alternative coagulation triggers and a re-examination of the net thrombin generation when the protein/protein S system is simultaneously interrogated.

  5. Blood volume and red cell life span (M113), part C

    NASA Technical Reports Server (NTRS)

    Johnson, P. C., Jr.

    1973-01-01

    Prechamber, in-chamber, and postchamber blood samples taken from Skylab simulation crewmembers did not indicate significant shortening of the red cell life span during the mission. This does not suggest that the space simulation environment could not be associated with red cell enzyme changes. It does show that any changes in enzymes were not sufficiently great to significantly shorten red cell survival. There was no evidence of bone marrow erythropoetic suppression nor was there any evidence of increased red cell destruction.

  6. Blood volume and red cell life span (M113), part C

    NASA Technical Reports Server (NTRS)

    Johnson, P. C., Jr.

    1973-01-01

    Prechamber, in-chamber, and postchamber blood samples taken from Skylab simulation crewmembers did not indicate significant shortening of the red cell life span during the mission. This does not suggest that the space simulation environment could not be associated with red cell enzyme changes. It does show that any changes in enzymes were not sufficiently great to significantly shorten red cell survival. There was no evidence of bone marrow erythropoetic suppression nor was there any evidence of increased red cell destruction.

  7. Quantitative measurement of red blood cell central pallor and hypochromasia.

    PubMed

    Bacus, J W

    1980-06-01

    A quantitataive definition and techniques of measurement for central pallor of red blood cells are proposed. These are based on high-resolution measurements of absorbance across the center of the cell. Thus, the measurements reflect both variations in cell thickness and hemoglobin concentration. Although contributions of thickness and concentration may differ in individual cells, to a first approximation, a specific cell may be considered as having a similar concentration of hemoglobin throughout, and thus the major contribution to the central pallor is that due to the difference in thickness between the edges of the cell and the center. The definition proposed expresses central pallor as the percentage volume of indentation, comparing the red cell to a disc of uniform absorbance equal to the maximum found at the cell edges. Population distributions of central pallor then provide a basis for quantitation of hypochromasia. The mean and standard deviation of such distributions are proposed as quantitative descriptors. Sample distributions from 27 normal persons, 8 patients with spherocytic anemia and 26 patients with iron deficiency anemia were studied.

  8. Expression of blood group antigens on red cell microvesicles.

    PubMed

    Oreskovic, R T; Dumaswala, U J; Greenwalt, T J

    1992-01-01

    The purpose of this study was to determine whether epitopes of the A, B, D, Fya, M, N, S, s, and K blood group antigens are present on microvesicle membranes shed by red cells during storage. Vesicles were isolated from outdated units of blood having and lacking the specified antigens. Diluted antisera were absorbed with fixed quantities of vesicles from red cells with the test antigen and red cells lacking that antigen (controls). The adsorbed and unadsorbed antisera were titrated and scored by using panel cells from persons known to be heterozygous for all the non-AB antigens. The mean titration scores following adsorption with the vesicles from A, B, D, M+N-, M-N+, S+s-, S-s+, and Fy(a+b-) units were appreciably lower than the control scores (0, 0, 3, 2, 2, 0, 4, and 4 vs. 19, 23, 34, 13, 12, 16, 18, and 29, respectively), which indicated the presence of these epitopes on the membrane of shed vesicles. The results following adsorption with K:1,2 vesicles were equivocal.

  9. Nanomechanical characterization of red blood cells using optical tweezers.

    PubMed

    Li, Chuan; Liu, K K

    2008-04-01

    Deformation behaviours of red blood cells (RBCs) have been studied by applying stretching forces via optical tweezers. Combined with finite-element analyses (FEA), the RBCs' mechanical properties are determined quantitatively based on a best fitting between the experimental deformed geometries and the simulated counterparts. Experimentally, a silica beads attached erythrocyte is optical-mechanically stretched to different lengths. On the theoretical front, a large deformation model with Mooney-Rivlin constitutive equations has been simulated by using FEA to predict the cell deformation geometries. The numerically simulated transverse and longitudinal strains which are in a good agreement with the experimental measurements facilitate the determination of elastic constants of the cells.

  10. Multiscale Modeling of Red Blood Cells Squeezing through Submicron Slits

    NASA Astrophysics Data System (ADS)

    Peng, Zhangli; Lu, Huijie

    2016-11-01

    A multiscale model is applied to study the dynamics of healthy red blood cells (RBCs), RBCs in hereditary spherocytosis, and sickle cell disease squeezing through submicron slits. This study is motivated by the mechanical filtration of RBCs by inter-endothelial slits in the spleen. First, the model is validated by comparing the simulation results with experiments. Secondly, the deformation of the cytoskeleton in healthy RBCs is investigated. Thirdly, the mechanisms of damage in hereditary spherocytosis are investigated. Finally, the effects of cytoplasm and membrane viscosities, especially in sickle cell disease, are examined. The simulations results provided guidance for future experiments to explore the dynamics of RBCs under extreme deformation.

  11. Photoacoustic tomography of unlabelled red blood cell at the nanoscale

    NASA Astrophysics Data System (ADS)

    Samant, Pratik; Chen, Jian; Xiang, Liangzhong

    2016-09-01

    In this letter, we present the principle behind nanoscale photoacoustic tomography (nPAT), in addition to simulation results demonstrating the thermal safety and the diagnostic potential of such a modality. Nanoscale photoacoustic tomography is a novel biomedical imaging modality that can allow for the 3D imaging of cells at nanometer resolutions. This modality also allows for the imaging of single red blood cells (RBCs) such that the hemoglobin concentration quantities can be visualized within the cell. As a result, we believe that nPAT can allow for diagnostic information at unprecedented resolutions and enable the visualization of previously unseen phenomenon in RBCs.

  12. Is pure red cell aplasia (PRCA) a clonal disorder?

    PubMed

    Sivakumaran, M; Bhavnani, M; Stewart, A; Roberts, B E; Geary, G C

    1993-01-01

    Pure red cell aplasia (PRCA) is an uncommon disorder, many cases lacking a well defined aetiology. This report describes three cases of PRCA (two idiopathic and one associated with B-CLL) who were investigated to assess the possibility of their PRCA being associated with a clonal proliferation of T-lymphocytes. The results show that one patient had evidence of T-cell receptor (TCR) gamma chain rearrangement, and the other had a TCR delta chain rearrangement. These two cases raise the possibility of PRCA being associated with a clonal proliferation of T-cells and further studies are warranted.

  13. Method using CO for extending the useful shelf-life of refrigerated red blood cells

    DOEpatents

    Bitensky, Mark W.

    1995-01-01

    Method using CO for extending the useful shelf-life of refrigerated red blood cells. Carbon monoxide is utilized for stabilizing hemoglobin in red blood cells to be stored at low temperature. Changes observed in the stored cells are similar to those found in normal red cell aging in the body, the extent thereof being directly related to the duration of refrigerated storage. Changes in cell buoyant density, vesiculation, and the tendency of stored cells to bind autologous IgG antibody directed against polymerized band 3 IgG, all of which are related to red blood cell senescence and increase with refrigerated storage time, have been substantially slowed when red blood cells are treated with CO. Removal of the carbon monoxide from the red blood cells is readily and efficiently accomplished by photolysis in the presence of oxygen so that the stored red blood cells may be safely transfused into a recipient.

  14. Method using CO for extending the useful shelf-life of refrigerated red blood cells

    DOEpatents

    Bitensky, M.W.

    1995-12-19

    A method is disclosed using CO for extending the useful shelf-life of refrigerated red blood cells. Carbon monoxide is utilized for stabilizing hemoglobin in red blood cells to be stored at low temperature. Changes observed in the stored cells are similar to those found in normal red cell aging in the body, the extent thereof being directly related to the duration of refrigerated storage. Changes in cell buoyant density, vesiculation, and the tendency of stored cells to bind autologous IgG antibody directed against polymerized band 3 IgG, all of which are related to red blood cell senescence and increase with refrigerated storage time, have been substantially slowed when red blood cells are treated with CO. Removal of the carbon monoxide from the red blood cells is readily and efficiently accomplished by photolysis in the presence of oxygen so that the stored red blood cells may be safely transfused into a recipient. 5 figs.

  15. Photodynamic treatment of red blood cell concentrates for virus inactivation enhances red blood cell aggregation: protection with antioxidants.

    PubMed

    Ben-Hur, E; Barshtein, G; Chen, S; Yedgar, S

    1997-10-01

    Photodynamic treatment (PDT) using phthalocyanines and red light appears to be a promising procedure for decontamination of red blood cell (RBC) concentrates for transfusion. A possible complication of this treatment may be induced aggregation of RBC. The production of RBC aggregates was measured with a novel computerized cell flow properties analyzer (CFA). The PDT of RBC concentrates with sulfonated aluminum phthalocyanine (AIPcS4) and the silicon phthalocyanine Pc 4 under virucidal conditions markedly enhanced RBC aggregation and higher shear stress was required to disperse these aggregates. The clusters of cells were huge and abnormally shaped, unlike the rouleaux formed by untreated RBC. This aggregation was prevented when a mixture of antioxidants was included during PDT. Addition of the antioxidants after PDT reduced aggregation only partially. It is concluded that inclusion of antioxidants during PDT of RBC concentrates prior to transfusion may reduce or eliminate the hemodynamic risk that the virucidal treatment may present to the recipient.

  16. Vesiculation of healthy and defective red blood cells

    NASA Astrophysics Data System (ADS)

    Li, He; Lykotrafitis, George

    2015-07-01

    Vesiculation of mature red blood cells (RBCs) contributes to removal of defective patches of the erythrocyte membrane. In blood disorders, which are related to defects in proteins of the RBC membrane, vesiculation of the plasma membrane is intensified. Several hypotheses have been proposed to explain RBC vesiculation but the exact underlying mechanisms and what determines the sizes of the vesicles are still not completely understood. In this work, we apply a two-component coarse-grained molecular dynamics RBC membrane model to study how RBC vesiculation is controlled by the membrane spontaneous curvature and by lateral compression of the membrane. Our simulation results show that the formation of small homogeneous vesicles with a diameter less than 40 nm can be attributed to a large spontaneous curvature of membrane domains. On the other hand, compression on the membrane can cause the formation of vesicles with heterogeneous composition and with sizes comparable with the size of the cytoskeleton corral. When spontaneous curvature and lateral compression are simultaneously considered, the compression on the membrane tends to facilitate formation of vesicles originating from curved membrane domains. We also simulate vesiculation of RBCs with membrane defects connected to hereditary elliptocytosis (HE) and to hereditary spherocytosis (HS). When the vertical connectivity between the lipid bilayer and the membrane skeleton is elevated, as in normal RBCs, multiple vesicles are shed from the compressed membrane with diameters similar to the cytoskeleton corral size. In HS RBCs, where the connectivity between the lipid bilayer and the cytoskeleton is reduced, larger-size vesicles are released under the same compression ratio as in normal RBCs. Lastly, we find that vesicles released from HE RBCs can contain cytoskeletal filaments due to fragmentation of the membrane skeleton while vesicles released from the HS RBCs are depleted of cytoskeletal filaments.

  17. What is antibody-mediated pure red cell aplasia (PRCA)?

    PubMed

    Casadevall, Nicole

    2005-05-01

    Antibody (Ab)-mediated pure red cell aplasia (PRCA) is an immunological pathology associated with the production of neutralizing Abs that inhibit the erythropoietic activity of endogenous erythropoietin (EPO) and recombinant erythropoiesis-stimulating agents (ESAs). Although this disorder occurs very rarely, the number of reported cases has increased dramatically in recent years, predominantly in patients with chronic kidney disease (CKD)-associated anaemia receiving subcutaneous (s.c.) injections of one particular formulation of recombinant epoetin-alpha. This disorder is differentiated from classic forms of PRCA that are caused by chemical toxaemia (i.e. erythroblastopenia induced by chemical compounds), lymphoproliferative neoplasms, thymoma, human parvovirus B19 and certain autoimmune disorders. Patients with Ab-mediated PRCA develop resistance to EPO and severe anaemia that follows a period of successful erythropoietic response, and exhibit characteristic decreases in blood haemoglobin (Hb) level and in the number of circulating reticulocytes. However, it is not yet possible to predict which patients will develop PRCA or when in the course of their treatments PRCA may develop. Laboratory confirmation of Ab-mediated PRCA requires bone marrow examination demonstrating few or no erythroid precursors and the presence of serum anti-EPO Abs using a validated assay. These neutralizing anti-EPO Abs recognize the protein core of the EPO molecule; carbohydrate groups on EPO can affect the binding of Abs but are themselves not immunological determinants. Animal models are being developed to increase further our understanding of the immunological mechanisms underlying the onset and progression of Ab-mediated PRCA.

  18. Deep sequencing and proteomic analysis of the microRNA-induced silencing complex in human red blood cells.

    PubMed

    Azzouzi, Imane; Moest, Hansjoerg; Wollscheid, Bernd; Schmugge, Markus; Eekels, Julia J M; Speer, Oliver

    2015-05-01

    During maturation, erythropoietic cells extrude their nuclei but retain their ability to respond to oxidant stress by tightly regulating protein translation. Several studies have reported microRNA-mediated regulation of translation during terminal stages of erythropoiesis, even after enucleation. In the present study, we performed a detailed examination of the endogenous microRNA machinery in human red blood cells using a combination of deep sequencing analysis of microRNAs and proteomic analysis of the microRNA-induced silencing complex. Among the 197 different microRNAs detected, miR-451a was the most abundant, representing more than 60% of all read sequences. In addition, miR-451a and its known target, 14-3-3ζ mRNA, were bound to the microRNA-induced silencing complex, implying their direct interaction in red blood cells. The proteomic characterization of endogenous Argonaute 2-associated microRNA-induced silencing complex revealed 26 cofactor candidates. Among these cofactors, we identified several RNA-binding proteins, as well as motor proteins and vesicular trafficking proteins. Our results demonstrate that red blood cells contain complex microRNA machinery, which might enable immature red blood cells to control protein translation independent of de novo nuclei information. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  19. Sensitive red protein calcium indicators for imaging neural activity

    PubMed Central

    Dana, Hod; Mohar, Boaz; Sun, Yi; Narayan, Sujatha; Gordus, Andrew; Hasseman, Jeremy P; Tsegaye, Getahun; Holt, Graham T; Hu, Amy; Walpita, Deepika; Patel, Ronak; Macklin, John J; Bargmann, Cornelia I; Ahrens, Misha B; Schreiter, Eric R; Jayaraman, Vivek; Looger, Loren L; Svoboda, Karel; Kim, Douglas S

    2016-01-01

    Genetically encoded calcium indicators (GECIs) allow measurement of activity in large populations of neurons and in small neuronal compartments, over times of milliseconds to months. Although GFP-based GECIs are widely used for in vivo neurophysiology, GECIs with red-shifted excitation and emission spectra have advantages for in vivo imaging because of reduced scattering and absorption in tissue, and a consequent reduction in phototoxicity. However, current red GECIs are inferior to the state-of-the-art GFP-based GCaMP6 indicators for detecting and quantifying neural activity. Here we present improved red GECIs based on mRuby (jRCaMP1a, b) and mApple (jRGECO1a), with sensitivity comparable to GCaMP6. We characterized the performance of the new red GECIs in cultured neurons and in mouse, Drosophila, zebrafish and C. elegans in vivo. Red GECIs facilitate deep-tissue imaging, dual-color imaging together with GFP-based reporters, and the use of optogenetics in combination with calcium imaging. DOI: http://dx.doi.org/10.7554/eLife.12727.001 PMID:27011354

  20. THE BIREFRINGENCE OF THE HUMAN RED CELL GHOSTS

    PubMed Central

    Ponder, Eric; Barreto, Delia

    1956-01-01

    The type of birefringence described by Mitchison, which extends some 0.5 µ in from the surface of the human red cell ghost in glycerol and which shows a maximum retardation of about 7 A, is only found in ghosts which are sufficiently well hemoglobinised to be seen with the ordinary microscope. Ghosts from which all hemoglobin has been lost are not visible with the ordinary microscope and are not birefringent, although they are clearly visible with phase contrast. About 90 per cent of the ghosts in glycerol preparations are of the latter type, the exact percentage being a function of time. Mitchison's measurements of birefringence, although reproducible, accordingly apply only to ghosts in which some hemoglobin still remains complexed with the lipoprotein layers of the red cell ultrastructure, and do not enable one to draw conclusions as to the thickness and orientation of the lipoprotein surface layers. PMID:13286451

  1. Patterns of Nonelectrolyte Permeability in Human Red Blood Cell Membrane

    PubMed Central

    Naccache, P.; Sha'afi, R. I.

    1973-01-01

    The permeability of human red cell membrane to 90 different molecules has been measured. These solutes cover a wide spectrum of nonelectrolytes with varying chemical structure, chain length, lipid solubility, chemical reactive group, ability to form hydrogen bonds, and other properties. In general, the present study suggests that the permeability of red cell membrane to a large solute is determined by lipid solubility, its molecular size, and its hydrogen-bonding ability. The permeability coefficient increases with increasing lipid solubility and decreasing ability to form hydrogen bonds, whereas it decreases with increasing molecular size. In the case of small solutes, the predominant diffusion factor is steric hindrance augmented by lipid solubility. It is also found that replacement of a hydroxyl group by a carbonyl group or an ether linkage tends to increase permeability. On the other hand, replacement of a hydroxyl group by an amide group tends to decrease the permeability coefficient. PMID:4804758

  2. Online biomedical resources for malaria-related red cell disorders.

    PubMed

    Piel, Frédéric B; Howes, Rosalind E; Nyangiri, Oscar A; Moyes, Catherine L; Williams, Thomas N; Weatherall, David J; Hay, Simon I

    2013-07-01

    Warnings about the expected increase of the global public health burden of malaria-related red cell disorders are accruing. Past and present epidemiological data are necessary to track spatial and temporal changes in the frequencies of these genetic disorders. A number of open access biomedical databases including data on malaria-related red cell disorders have been launched over the last two decades. Here, we review the content of these databases, most of which focus on genetic diversity, and we describe a new epidemiological resource developed by the Malaria Atlas Project. To tackle upcoming public health challenges, the integration of epidemiological and genetic data is important. As many countries are considering implementing national screening programs, strategies to make such data more accessible are also needed.

  3. Automation of cross-matching and red cell antibody screening.

    PubMed

    Wattar, B; Lambermont, M; Govaerts, A

    1982-01-01

    This automatic system combines the major cross-match with screening for allo- and autoantibodies. Moreover, the detected antibodies can be identified on a panel of frozen and thawed red blood cells (RBC). The system is made up of two connected samplers, three channels working, respectively, with bromelin PVP, LISP and saline PVP at 4 degrees C, three colorimeters or three red cell autocounters and their recorders. The optimal speed is 50 samples/h and one whole test requires 19 min. Our experience indicates that this automatic system is appreciably more sensitive and much more rapid and efficient than manual techniques. In spite of increased sensitivity, the ratio of rejected bags does not exceed 2.7%.

  4. Low frequency electrorotation of fixed red blood cells.

    PubMed Central

    Georgieva, R; Neu, B; Shilov, V M; Knippel, E; Budde, A; Latza, R; Donath, E; Kiesewetter, H; Bäumler, H

    1998-01-01

    Electrorotation of fixed red blood cells has been investigated in the frequency range between 16 Hz and 30 MHz. The rotation was studied as a function of electrolyte conductivity and surface charge density. Between 16 Hz and 1 kHz, fixed red blood cells undergo cofield rotation. The maximum of cofield rotation occurs between 30 and 70 Hz. The position of the maximum depends weakly on the bulk electrolyte conductivity and surface charge density. Below 3.5 mS/m, the cofield rotation peak is broadened and shifted to higher frequencies accompanied by a decrease of the rotation speed. Surface charge reduction leads to a decrease of the rotation speed in the low frequency range. These observations are consistent with the recently developed electroosmotic theory of low frequency electrorotation. PMID:9545070

  5. Online Biomedical Resources for Malaria-Related Red Cell Disorders

    PubMed Central

    Piel, Frédéric B; Howes, Rosalind E; Nyangiri, Oscar A; Moyes, Catherine L; Williams, Thomas N; Weatherall, David J; Hay, Simon I

    2013-01-01

    Warnings about the expected increase of the global public health burden of malaria-related red cell disorders are accruing. Past and present epidemiological data are necessary to track spatial and temporal changes in the frequencies of these genetic disorders. A number of open access biomedical databases including data on malaria-related red cell disorders have been launched over the last two decades. Here, we review the content of these databases, most of which focus on genetic diversity, and we describe a new epidemiological resource developed by the Malaria Atlas Project. To tackle upcoming public health challenges, the integration of epidemiological and genetic data is important. As many countries are considering implementing national screening programs, strategies to make such data more accessible are also needed. PMID:23568771

  6. The Proteome of the Red Blood Cell: An Auspicious Source of New Insights into Membrane-Centered Regulation of Homeostasis

    PubMed Central

    Bosman, Giel J. C. G. M.

    2016-01-01

    During the past decade, the hand-in-hand development of biotechnology and bioinformatics has enabled a view of the function of the red blood cell that surpasses the supply of oxygen and removal of carbon dioxide. Comparative proteomic inventories have yielded new clues to the processes that regulate membrane–cytoskeleton interactions in health and disease, and to the ways by which red blood cells communicate with their environment. In addition, proteomic data have revealed the possibility that many, hitherto unsuspected, metabolic processes are active in the red blood cell cytoplasm. Recent metabolomic studies have confirmed and expanded this notion. Taken together, the presently available data point towards the red blood cell membrane as the hub at which all regulatory processes come together. Thus, alterations in the association of regulatory proteins with the cell membrane may be a sine qua non for the functional relevance of any postulated molecular mechanism. From this perspective, comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red blood cell homeostasis. Additionally, this perspective provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood. PMID:28248245

  7. The Proteome of the Red Blood Cell: An Auspicious Source of New Insights into Membrane-Centered Regulation of Homeostasis.

    PubMed

    Bosman, Giel J C G M

    2016-11-25

    During the past decade, the hand-in-hand development of biotechnology and bioinformatics has enabled a view of the function of the red blood cell that surpasses the supply of oxygen and removal of carbon dioxide. Comparative proteomic inventories have yielded new clues to the processes that regulate membrane-cytoskeleton interactions in health and disease, and to the ways by which red blood cells communicate with their environment. In addition, proteomic data have revealed the possibility that many, hitherto unsuspected, metabolic processes are active in the red blood cell cytoplasm. Recent metabolomic studies have confirmed and expanded this notion. Taken together, the presently available data point towards the red blood cell membrane as the hub at which all regulatory processes come together. Thus, alterations in the association of regulatory proteins with the cell membrane may be a sine qua non for the functional relevance of any postulated molecular mechanism. From this perspective, comparative proteomics centered on the red blood cell membrane constitute a powerful tool for the identification and elucidation of the physiologically and pathologically relevant pathways that regulate red blood cell homeostasis. Additionally, this perspective provides a focus for the interpretation of metabolomic studies, especially in the development of biomarkers in the blood.

  8. Recovery of Red Fluorescent Protein Chromophore Maturation Deficiency through Rational Design

    PubMed Central

    Moore, Matthew M.; Oteng-Pabi, Samuel K.; Pandelieva, Antonia T.; Mayo, Stephen L.; Chica, Roberto A.

    2012-01-01

    Red fluorescent proteins (RFPs) derived from organisms in the class Anthozoa have found widespread application as imaging tools in biological research. For most imaging experiments, RFPs that mature quickly to the red chromophore and produce little or no green chromophore are most useful. In this study, we used rational design to convert a yellow fluorescent mPlum mutant to a red-emitting RFP without reverting any of the mutations causing the maturation deficiency and without altering the red chromophore’s covalent structure. We also created an optimized mPlum mutant (mPlum-E16P) that matures almost exclusively to the red chromophore. Analysis of the structure/function relationships in these proteins revealed two structural characteristics that are important for efficient red chromophore maturation in DsRed-derived RFPs. The first is the presence of a lysine residue at position 70 that is able to interact directly with the chromophore. The second is an absence of non-bonding interactions limiting the conformational flexibility at the peptide backbone that is oxidized during red chromophore formation. Satisfying or improving these structural features in other maturation-deficient RFPs may result in RFPs with faster and more complete maturation to the red chromophore. PMID:23285050

  9. Color contrast of red blood cells on solid substrate

    NASA Astrophysics Data System (ADS)

    Paiziev, Adkham A.

    2013-02-01

    In present study we developed the new method of colour visualization of red blood cells without using any chemical staining. The method based on physical phenomena a white light interference on thin transparent films. It is shown that in the case of thin human blood smears colour interference contrast occurs on solid polished substrates. The best contrast shows substrates with maximal refractive index (Mo, W, Si). These materials have been selected as substrate instead of ordinary microscopic slide in reflected light microscopy. It is shown that reflection of incident white light from blood cell surface and boundary cell-substrate generate two coherent lights. The second one (object signal) after passing through red blood cell gathers additional phase and after interference interaction with reference signal (light reflected from outer cell surface) enables cell image in colour. Number of blood smears of healthy persons (control) and patients who were diagnosed with cancer are presented. It is concluded that the offered method may be used as an effective diagnostic tool to detect early stage blood cells lesion by its interference painting in white light. Offered method may be used in research laboratories, hospitals, diagnostic centres, emergency medicine and other as complementary diagnostic tool to present convenient optical and electron microscopy technique.

  10. Characterization of red blood cells (RBCs) using dual Brillouin/Raman micro-spectroscopy

    NASA Astrophysics Data System (ADS)

    Meng, Zhaokai; Bustamante-Lopez, Sandra C.; Yakovlev, Vladislav V.; Meissner, Kenith E.

    2016-04-01

    Erythrocytes, or red blood cells, transport oxygen to and carbon dioxide from the body's tissues and organs. Red blood cell mechanical properties are altered in a number of diseases such as sickle cell anaemia and malaria. Additionally, mechanically modified red blood cell ghosts are being considered as a long-term, biocompatible carrier for drug delivery and for blood analyte sensing. Brillouin spectroscopy enables viscoelastic characterization of samples at the microscale. In this report, Brillouin spectroscopy is applied to characterize the mechanical properties of red blood cells and red blood cell ghosts.

  11. Role of Complement in Red Cell Dysfunction in Trauma

    DTIC Science & Technology

    2012-12-01

    3 Introduction Trauma-induced changes in the red blood cells ( RBC ) contribute to the reduction of blood flow to distant...The expression of C4d on the surface of RBCs was measured by flow cytometry and expressed as mean fluorescence intensity. (A) Representative data. (B...measured by flow cytometry. Fluorescence levels of RBCs were acquired for 30 seconds using FACScan flow cytometer to establish a baseline for intra- 8

  12. Pure Red Cell Aplasia Associated with Good Syndrome

    PubMed Central

    Okui, Masayuki; Yamamichi, Takashi; Asakawa, Ayaka; Harada, Masahiko; Horio, Hirotoshi

    2017-01-01

    Pure red cell aplasia (PRCA) and hypogammaglobulinemia are paraneoplastic syndromes that are rarer than myasthenia gravis in patients with thymoma. Good syndrome coexisting with PRCA is an extremely rare pathology. We report the case of a 50-year-old man with thymoma and PRCA associated with Good syndrome who achieved complete PRCA remission after thymectomy and postoperative immunosuppressive therapy, and provide a review of the pertinent literature. PMID:28382272

  13. Red blood cells flows in rectilinear microfluidic chip.

    PubMed

    Anandan, P; Ortiz, D; Intaglietta, M; Cabrales, P J; Bucolo, M

    2015-01-01

    The red blood cells flow in a controlled environment as a microfluidic chip with a rectilinear geometry was investigated. The optical monitoring performed by an automatic Particle Image Velocimetry procedure has allowed a quantitative analysis on flow features. Various parameters such as velocity, shear rate, strain rate, vorticity, divergence were extracted. The comparisons of the results obtained from the different experiments was used for the overall understanding of the RBC movements in different conditions and the establishment of the analysis procedure.

  14. Research on red cell membrane permeability in arterial hypertension.

    PubMed

    Gatina, R; Balta, N; Moisin, C; Burtea, C; Botea, S; Ioan, M; Teleianu, C

    1998-01-01

    Arterial hypertension, including the elucidation of hypertension pathogenic mechanisms involving elements in the composition of the blood, continues to represent a topical research area. Recent work, such as nuclear magnetic resonance studies looking into red cell permeability, illustrates the presence of modifications of red cell permeability to water (RCPW) related to the stage of arterial hypertension. The identification of a significant increase of RCPW compared to that present in the population with normal arterial pressure values can be useful both in early diagnosis and in warning about a possible predisposition for this condition. At the same time, the dynamic investigation of protonic relaxation time of both intra- and extra-erythrocytic water, the assessment of proton exchange time across the red cell and the calculation of permeability to water enable one not only to diagnose arterial hypertension but also to ascertain the evolution of the disease, its complications and the effectiveness of anti-hypertensive medication. Our studies have also proven the existence of a correlation between the values of systolic arterial pressure and red cell permeability to water. The curve describing the interdependence of the two values has the shape of a bell, in the case of males. The peak of the curve is reached for a systolic pressure of 160 mmHg and gets below the values of the control group in the case of systolic pressures above 200 mmHg. The RCPW test can also be considered a valuable indicator in evaluating the risk of stroke in hypertensive patients. In the chronic therapy of arterial hypertension with various types of anti-hypertensive drugs, one can note differences in the RCPW values related to the effectiveness of the respective medication, to the clinical form and stage of the disease, the sex of the patient as well as to the existence of cerebro-vascular complications.

  15. My passion and passages with red blood cells.

    PubMed

    Hoffman, Joseph F

    2008-01-01

    This article mainly presents, in sequential panels of time, an overview of my professional involvements and laboratory experiences. I became smitten with red blood cells early on, and this passion remains with me to this day. I highlight certain studies, together with those who performed the work, recognizing that it was necessary to limit the details and the topics chosen for discussion. I am uncertain of the interest a personal account has for others, but at least it's here for the record.

  16. Two-color RESOLFT nanoscopy with green and red fluorescent photochromic proteins.

    PubMed

    Lavoie-Cardinal, Flavie; Jensen, Nickels A; Westphal, Volker; Stiel, Andre C; Chmyrov, Andriy; Bierwagen, Jakob; Testa, Ilaria; Jakobs, Stefan; Hell, Stefan W

    2014-03-17

    Up to now, all demonstrations of reversible saturable optical fluorescence transitions (RESOLFT) superresolution microscopy of living cells have relied on the use of reversibly switchable fluorescent proteins (RSFP) emitting in the green spectral range. Here we show RESOLFT imaging with rsCherryRev1.4, a new red-emitting RSFP enabling a spatial resolution up to four times higher than the diffraction barrier. By co-expressing green and red RSFPs in living cells we demonstrate two-color RESOLFT imaging both for single ("donut") beam scanning and for parallelized versions of RESOLFT nanoscopy where an array of >23,000 "donut-like" minima are scanned simultaneously. ©2014 The Authors. Published by Wiley-VCH Verlag GmbH&Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

  17. Progress in improving the pathogen safety of red