Science.gov

Sample records for human erythrocytes induced

  1. Ritonavir-Induced Suicidal Death of Human Erythrocytes.

    PubMed

    Waibel, Sabrina; Bissinger, Rosi; Bouguerra, Ghada; Abbès, Salem; Lang, Florian

    2016-07-01

    The antiviral drug ritonavir has been shown to trigger suicidal death or apoptosis of tumour cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca(2+) entry with increase in cytosolic Ca(2+) activity ([Ca(2+) ]i ), oxidative stress and ceramide. The present study explored whether and how ritonavir induces eryptosis. To this end, flow cytometry was employed to estimate cell volume from forward scatter, phosphatidylserine exposure at the cell surface from Annexin-V binding, [Ca(2+) ]i from Fluo3 fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence and ceramide abundance utilizing specific antibodies. As a result, a 48-hr exposure of human erythrocytes to ritonavir significantly increased the percentage of Annexin-V-binding cells (≥5 μg/ml), significantly decreased forward scatter (≥5 μg/ml), significantly increased Fluo3 fluorescence (20 μg/ml), slightly, but significantly increased DCFDA fluorescence (20 μg/ml) and slightly, but significantly increased ceramide abundance (20 μg/ml). The effect of ritonavir on Annexin-V binding was significantly blunted, but not fully abolished by the removal of extracellular Ca(2+) . In conclusion, ritonavir triggers erythrocyte shrinkage and phosphatidylserine translocation at the erythrocyte cell membrane, an effect in part due to the stimulation of Ca(2+) entry, oxidative stress and ceramide. PMID:27264208

  2. Influence of Cocoa Flavanols and Procyanidins on Free Radical-induced Human Erythrocyte Hemolysis

    PubMed Central

    Zhu, Qin Yan; Schramm, Derek D.; Gross, Heidrun B.; Holt, Roberta R.; Kim, Sun H.; Yamaguchi, Tomoko; Kwik-Uribe, Catherine L.; Keen, Carl L.

    2005-01-01

    Cocoa can be a rich source of antioxidants including the flavan-3-ols, epicatechin and catechin, and their oligomers (procyanidins). While these flavonoids have been reported to reduce the rate of free radical-induced erythrocyte hemolysis in experimental animal models, little is known about their effect on human erythrocyte hemolysis. The major objective of this work was to study the effect of a flavonoid-rich cocoa beverage on the resistance of human erythrocytes to oxidative stress. A second objective was to assess the effects of select purified cocoa flavonoids, epicatechin, catechin, the procyanidin Dimer B2 and one of its major metabolites, 3ʹ-O-methyl epicatechin, on free radical-induced erythrocyte hemolysis in vitro. Peripheral blood was obtained from 8 healthy subjects before and 1, 2, 4 and 8 h after consuming a flavonoid-rich cocoa beverage that provided 0.25 g/kg body weight (BW), 0.375 or 0.50 g/kg BW of cocoa. Plasma flavanol and dimer concentrations were determined for each subject. Erythrocyte hemolysis was evaluated using a controlled peroxidation reaction. Epicatechin, catechin, 3ʹ-O-methyl epicatechin and (-)-epicatechin-(4β > 8)epicatechin (Dimer B2) were detected in the plasma within 1 h after the consumption of the beverage. The susceptibility of erythrocytes to hemolysis was reduced significantly following the consumption of the beverages. The duration of the lag time, which reflects the capacity of cells to buffer free radicals, was increased. Consistent with the above, the purified flavonoids, epicatechin, catechin, Dimer B2 and the metabolite 3ʹ-O-methyl epicatechin, exhibited dose-dependent protection against AAPH-induced erythrocyte hemolysis at concentrations ranging from 2.5 to 20 μM. Erythrocytes from subjects consuming flavonoid-rich cocoa show reduced susceptibility to free radical-induced hemolysis (p < 0.05). PMID:15712596

  3. Insulin Inhibits Low Oxygen-Induced ATP Release from Human Erythrocytes: Implication for Vascular Control

    PubMed Central

    Hanson, Madelyn S.; Ellsworth, Mary L.; Achilleus, David; Stephenson, Alan H.; Bowles, Elizabeth A.; Sridharan, Meera; Adderley, Shaquria; Sprague, Randy S.

    2010-01-01

    Objective ATP released from human erythrocytes in response to reduced oxygen tension (pO2) participates in the matching of oxygen (O2) supply with need in skeletal muscle by stimulating increases in blood flow to areas with increased O2 demand. Here we investigated the hypothesis that hyperinsulinemia inhibits ATP release from erythrocytes and impairs their ability to stimulate dilation of isolated arterioles exposed to decreased extra-luminal pO2. Methods Erythrocyte ATP release was stimulated pharmacologically (mastoparan 7) and physiologically (reduced pO2) in the absence or presence of insulin. We also examined the ability of isolated skeletal muscle arterioles perfused with buffer containing erythrocytes treated with insulin or its vehicle (saline) to dilate in response to decreased extra-luminal pO2. Results Insulin significantly attenuated mastoparan 7– and reduced pO2–induced ATP release. In vessels perfused with untreated erythrocytes, low extra-luminal pO2 resulted in an increase in vessel diameter. In contrast, when erythrocytes were treated with insulin, no vasodilation occurred. Conclusions These studies demonstrate that insulin inhibits ATP release from erythrocytes in response to reduced pO2 and impairs their ability to stimulate dilation of skeletal muscle arterioles. These results suggest that hyperinsulinemia could hinder the matching of O2 supply with need in skeletal muscle. PMID:19412833

  4. Pressure-induced hemolysis of in vivo aged human erythrocytes is enhanced by inhibition of water transport via aquaporin-1

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Takeo; Miyauchi, Shin; Isahara, Yasuyuki

    2013-06-01

    Human erythrocytes are fractionated into young, intermediate, and old cells according to their densities. Pressure-induced hemolysis reflects sensitively membrane perturbations. Therefore, the hemolysis of erythrocytes at 200 MPa was examined using fractionated cells. Pressure-induced hemolysis of old (or in vivo aged) erythrocytes was enhanced, compared with those of young and intermediate cells which showed the same hemolytic values. Flow cytometric analysis showed less fragmentation of old erythrocytes under pressure. Moreover, the water transport through the membrane was suppressed in old erythrocytes than intermediate ones. The low permeability of water in old erythrocytes was confirmed by osmotic hemolysis using a hypotonic buffer. These results suggest that water transport via aquaporin-1 (AQP1) is inhibited in old erythrocytes. As the number of AQP1 molecules remained constant in old erythrocytes, the function of AQP1 may be reduced.

  5. Potassium bromate causes cell lysis and induces oxidative stress in human erythrocytes.

    PubMed

    Ahmad, Mir Kaisar; Amani, Samreen; Mahmood, Riaz

    2014-02-01

    In the present study, we have studied the effect of KBrO3 on human erythrocytes under in vitro conditions. Erythrocytes were isolated from the blood of healthy nonsmoking volunteers and incubated with different concentrations of KBrO3 at 37°C for 60 min. This resulted in marked hemolysis in a KBrO3 -concentration dependent manner. Lysates were prepared from KBrO3 -treated and control erythrocytes and assayed for various parameters. KBrO3 treatment caused significant increase in protein oxidation, lipid peroxidation, hydrogen peroxide levels, and decrease in total sulfhydryl content, which indicates induction of oxidative stress in human erythrocytes. Methemoglobin levels and methemoglobin reductase activity were significantly increased while the total antioxidant power of lysates was greatly reduced upon KBrO3 treatment. Intracellular production of reactive oxygen species increased in a dose dependent manner. Exposure of erythrocytes to KBrO3 also caused decrease in the activities of catalase, glutathione peroxidase, thioredoxin reductase, glucose 6-phosphate dehydrogenase and glutathione reductase whereas the activities of Cu-Zn superoxide dismutase and glutathione-S-transferase were increased. These results show that KBrO3 induces oxidative stress in human erythrocytes through the generation of reactive oxygen species and alters the cellular antioxidant defense system. PMID:22012894

  6. Study of the effect of dose-rate on radiation-induced damage to human erythrocytes

    NASA Astrophysics Data System (ADS)

    Krokosz, Anita; Koziczak, Renata; Gonciarz, Marta; Szweda-Lewandowska, Zofia

    2006-01-01

    Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%) were irradiated with γ-rays at three dose-rates of 66.7, 36.7, 25 Gy min -1 in order to investigate the influence of the dose-rate on radiation-induced membrane damage, hemoglobin oxidation and loss of reduced glutathione. The obtained results showed that such processes as erythrocyte hemolysis, lipid and protein destruction depend on the radiation dose-rate. The parameter values describing these processes showed an inverse dose-rate effect.

  7. H2O2-Induced Oxidative Stress Affects SO4= Transport in Human Erythrocytes

    PubMed Central

    Morabito, Rossana; Romano, Orazio; La Spada, Giuseppa; Marino, Angela

    2016-01-01

    The aim of the present investigation was to verify the effect of H2O2-induced oxidative stress on SO4= uptake through Band 3 protein, responsible for Cl-/HCO3- as well as for cell membrane deformability, due to its cross link with cytoskeletal proteins. The role of cytoplasmic proteins binding to Band 3 protein has been also considered by assaying H2O2 effects on hemoglobin-free resealed ghosts of erythrocytes. Oxidative conditions were induced by 30 min exposure of human erythrocytes to different H2O2 concentrations (10 to 300 μM), with or without GSH (glutathione, 2 mM) or curcumin (10 μM), compounds with proved antioxidant properties. Since SO4= influx through Band 3 protein is slower and better controllable than Cl- or HCO3- exchange, the rate constant for SO4= uptake was measured to prove anion transport efficiency, while MDA (malondialdehyde) levels and –SH groups were estimated to quantify the effect of oxidative stress. H2O2 induced a significant decrease in rate constant for SO4= uptake at both 100 and 300 μM H2O2. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in –SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H2O2-induced changes in erythrocytes shape. Our results show that: i) 30 min exposure to 300 μM H2O2 reduced SO4= uptake in human erythrocytes; ii) oxidative damage was revealed by the reduction in rate constant for SO4= uptake, but not by MDA or –SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury; v) SO4= uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions possibly deriving from alcohol consumption, use of drugs, radiographic contrast media administration, hyperglicemia or

  8. H2O2-Induced Oxidative Stress Affects SO4= Transport in Human Erythrocytes.

    PubMed

    Morabito, Rossana; Romano, Orazio; La Spada, Giuseppa; Marino, Angela

    2016-01-01

    The aim of the present investigation was to verify the effect of H2O2-induced oxidative stress on SO4= uptake through Band 3 protein, responsible for Cl-/HCO3- as well as for cell membrane deformability, due to its cross link with cytoskeletal proteins. The role of cytoplasmic proteins binding to Band 3 protein has been also considered by assaying H2O2 effects on hemoglobin-free resealed ghosts of erythrocytes. Oxidative conditions were induced by 30 min exposure of human erythrocytes to different H2O2 concentrations (10 to 300 μM), with or without GSH (glutathione, 2 mM) or curcumin (10 μM), compounds with proved antioxidant properties. Since SO4= influx through Band 3 protein is slower and better controllable than Cl- or HCO3- exchange, the rate constant for SO4= uptake was measured to prove anion transport efficiency, while MDA (malondialdehyde) levels and -SH groups were estimated to quantify the effect of oxidative stress. H2O2 induced a significant decrease in rate constant for SO4= uptake at both 100 and 300 μM H2O2. This reduction, observed in erythrocytes but not in resealed ghosts and associated to increase in neither MDA levels nor in -SH groups, was impaired by both curcumin and GSH, whereas only curcumin effectively restored H2O2-induced changes in erythrocytes shape. Our results show that: i) 30 min exposure to 300 μM H2O2 reduced SO4= uptake in human erythrocytes; ii) oxidative damage was revealed by the reduction in rate constant for SO4= uptake, but not by MDA or -SH groups levels; iii) the damage was produced via cytoplasmic components which cross link with Band 3 protein; iv) the natural antioxidant curcumin may be useful in protecting erythrocytes from oxidative injury; v) SO4= uptake through Band 3 protein may be reasonably suggested as a tool to monitor erythrocytes function under oxidative conditions possibly deriving from alcohol consumption, use of drugs, radiographic contrast media administration, hyperglicemia or neurodegenerative

  9. Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity

    PubMed Central

    Gupta, Vivek Kumar; Pal, Rajnish; Siddiqi, Nikhat Jamal; Sharma, Bechan

    2015-01-01

    Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at −20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte's AChE exhibited Km for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type (Ki value, 3.6 mM) which negatively influenced both the Vmax and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead. PMID:26600946

  10. Acetylcholinesterase from Human Erythrocytes as a Surrogate Biomarker of Lead Induced Neurotoxicity.

    PubMed

    Gupta, Vivek Kumar; Pal, Rajnish; Siddiqi, Nikhat Jamal; Sharma, Bechan

    2015-01-01

    Lead induced neurotoxicity in the people engaged in different occupations has received wide attention but very little studies have been carried out to monitor occupational neurotoxicity directly due to lead exposure using biochemical methods. In the present paper an endeavour has been made in order to assess the lead mediated neurotoxicity by in vitro assay of the activity of acetylcholinesterase (AChE) from human erythrocytes in presence of different concentrations of lead. The results suggested that the activity of this enzyme was localized in membrane bound fraction and it was found to be highly stable up to 30 days when stored at -20°C in phosphate buffer (50 mM, pH 7.4) containing 0.2% Triton X-100. The erythrocyte's AChE exhibited K m for acetylcholinesterase to be 0.1 mM. Lead caused sharp inhibition of the enzyme and its IC50 value was computed to be 1.34 mM. The inhibition of the enzyme by lead was found to be of uncompetitive type (K i value, 3.6 mM) which negatively influenced both the V max and the enzyme-substrate binding affinity. Taken together, these results indicate that AChE from human erythrocytes could be exploited as a surrogate biomarker of lead induced neurotoxicity particularly in the people occupationally exposed to lead. PMID:26600946

  11. Effect of ethanol on nitrite- and 1-naphthol-induced oxidant stress in human and sheep erythrocytes

    SciTech Connect

    Calabrese, E.J.; Yang, J.H.; Horton, H.M.

    1988-01-01

    The enhancement by ethanol of nitrite- and 1-naphthol-induced oxidant stress was assessed in vitro in human and Dorset sheep erythrocytes as measured by changes in methemoglobin (MetHb) and glutathione (GSH) levels. The human and sheep erythrocytes treated with nitrite (0.5, 1.0 and 2.0 mM), 1-naphthol (1.0, 2.0 and 3.0 mM) or ethanol (0.1, 0.5, 1.0 and 5.0%) alone revealed significant increases in MetHb and no significant decreases in GSH except for sheep erythrocytes exposed to 1-naphthol and ethanol. The combined nitrite-ethanol treatment resulted in greater than additive increases in MetHb levels in both species; however, a protective effect occurred in sheep erythrocytes at the lowest combined treatment levels. The joint naphthol-ethanol treatment also resulted in synergistic increases in MetHb levels in both species. No synergistic decreases in GSH levels were detected for either of the combined treatments. These results suggest that ethanol combined with nitrite or 1-naphthol exposure in vitro synergistically increases MetHb levels of human and sheep erythrocytes.

  12. Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins.

    PubMed

    Lepock, J R; Frey, H E; Bayne, H; Markus, J

    1989-04-14

    Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably

  13. Iloprost- and isoproterenol-induced increases in cAMP are regulated by different phosphodiesterases in erythrocytes of both rabbits and humans

    PubMed Central

    Adderley, Shaquria P.; Dufaux, Eileen A.; Sridharan, Meera; Bowles, Elizabeth A.; Hanson, Madelyn S.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

    2009-01-01

    Activation of the G protein Gs results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (Ilo), a PGI2 analog, and isoproterenol (Iso), a β-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways. PMID:19252089

  14. Damascenine induced hepatotoxicity and nephrotoxicity in mice and in vitro assessed human erythrocyte toxicity

    PubMed Central

    Khettal, Bachra; Tir, Lydia; Boudrioua, Souad

    2015-01-01

    Nigella damascena seed is characterized by the presence of the major alkaloid, damascenine and its related metabolites. To our knowledge, no detailed subchronic toxicological assessment of damascenine (DA) has been reported. The present study evaluated the potential toxicity of DA in vivo after sub-chronic intraperitoneal (i.p) administration in mice and in vitro following human erythrocyte hemolysis. In vivo, a total of 48 adult male and female Swiss albino mice were used in a sub-chronic toxicity study. The mice received intraperitoneally two doses of DA (20 and 100 mg/kg) for 28 days. Food intake, body weight and central body temperature were measured during the experiment. After completion of drug treatment, biochemical and histological analyses were performed. No mortality was observed in any of the treatment groups of mice, showing no toxic effects during the study. Neither were biochemical parameters altered; no significant differences were observed concerning glucose, bilirubin, aspartate transaminase (AST), alanine aminotransferase (ALT), urea, and creatinine parameters. No histopathological alterations were found in kidney and liver structures. In vitro, we focused on the human erythrocyte hemolytic process in the presence of several concentrations of DA. High level concentration of 1 000 μg/ml of DA revealed normal cell shapes and absence of hemolysis and deformation. PMID:27486370

  15. Damascenine induced hepatotoxicity and nephrotoxicity in mice and in vitro assessed human erythrocyte toxicity.

    PubMed

    Bouguezza, Yacine; Khettal, Bachra; Tir, Lydia; Boudrioua, Souad

    2015-09-01

    Nigella damascena seed is characterized by the presence of the major alkaloid, damascenine and its related metabolites. To our knowledge, no detailed subchronic toxicological assessment of damascenine (DA) has been reported. The present study evaluated the potential toxicity of DA in vivo after sub-chronic intraperitoneal (i.p) administration in mice and in vitro following human erythrocyte hemolysis. In vivo, a total of 48 adult male and female Swiss albino mice were used in a sub-chronic toxicity study. The mice received intraperitoneally two doses of DA (20 and 100 mg/kg) for 28 days. Food intake, body weight and central body temperature were measured during the experiment. After completion of drug treatment, biochemical and histological analyses were performed. No mortality was observed in any of the treatment groups of mice, showing no toxic effects during the study. Neither were biochemical parameters altered; no significant differences were observed concerning glucose, bilirubin, aspartate transaminase (AST), alanine aminotransferase (ALT), urea, and creatinine parameters. No histopathological alterations were found in kidney and liver structures. In vitro, we focused on the human erythrocyte hemolytic process in the presence of several concentrations of DA. High level concentration of 1 000 μg/ml of DA revealed normal cell shapes and absence of hemolysis and deformation. PMID:27486370

  16. Protection of human erythrocyte using Crinumasiaticum extract and lycorine from oxidative damage induced by 2-amidinopropane

    PubMed Central

    Ilavenil, S.; Kaleeswaran, B.; Sumitha, P.; Tamilvendan, D.; Ravikumar, S.

    2010-01-01

    The intention of this investigation was to evaluate the free radical scavenging activity and erythrocyte protective activity of ethanolic extract of Crinumasiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were found to have different levels of antioxidant properties in the test models. Both ethanolic extract of C. asiaticum (L) (0.5–2.5 mg/ml) and lycorine (0.010 mg–0.050 mg/ml) increases the percentage of lipid peroxidation inhibition (26.25 ± 0.23% and 19.25 ± 0.23%) and enhances the free radical scavenging activity (20.92 ± 0.22% and 20.52 ± 0.22%), scavenging of hydrogen peroxide (25.67 ± 0.17% and 23.07 ± 0.3%) superoxide anion scavenging activity (27.69 ± 0.16% and 16.09 ± 0.7%) at concentration of 2.5 and 0.050 mg of C. asiaticum (L) and lycorine, respectively. But compared with tocopherol (P < 0.05) less activity was observed by C. asiaticum (L) and lycorine. The ethanolic extract of C. asiaticum (L) and lycorine were normalized to reduce the level of glutathione and also to sustain the status of protein in erythrocytes during the peroxyl radical [2,2-azobis (2-amidinopropane) dihydrochloride (AAPH)] induced oxidative damage in ex vivo model. The present results of the investigations demonstrated that protective nature of the C. asiaticum (L) and lycorine will be considered as a significant natural antioxidant source. PMID:23961122

  17. Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death

    PubMed Central

    Lang, Elisabeth; Gatidis, Sergios; Freise, Noemi F; Bock, Hans; Kubitz, Ralf; Lauermann, Christian; Orth, Hans Martin; Klindt, Caroline; Schuier, Maximilian; Keitel, Verena; Reich, Maria; Liu, Guilai; Schmidt, Sebastian; Xu, Haifeng C; Qadri, Syed M; Herebian, Diran; Pandyra, Aleksandra A; Mayatepek, Ertan; Gulbins, Erich; Lang, Florian; Häussinger, Dieter; Lang, Karl S; Föller, Michael; Lang, Philipp A

    2015-01-01

    Hepatic failure is commonly associated with anemia, which may result from gastrointestinal bleeding, vitamin deficiency, or liver-damaging diseases, such as infection and alcohol intoxication. At least in theory, anemia during hepatic failure may result from accelerated clearance of circulating erythrocytes. Here we show that bile duct ligation (BDL) in mice leads to severe anemia despite increased reticulocyte numbers. Bilirubin stimulated suicidal death of human erythrocytes. Mechanistically, bilirubin triggered rapid Ca2+ influx, sphingomyelinase activation, formation of ceramide, and subsequent translocation of phosphatidylserine to the erythrocyte surface. Consistent with our in vitro and in vivo findings, incubation of erythrocytes in serum from patients with liver disease induced suicidal death of erythrocytes in relation to their plasma bilirubin concentration. Consistently, patients with hyperbilirubinemia had significantly lower erythrocyte and significantly higher reticulocyte counts compared to patients with low bilirubin levels. Conclusion: Bilirubin triggers suicidal erythrocyte death, thus contributing to anemia during liver disease. (Hepatology 2015;61:275–284) PMID:25065608

  18. Micropipette aspiration of human erythrocytes induces echinocytes via membrane phospholipid translocation.

    PubMed Central

    Artmann, G M; Sung, K L; Horn, T; Whittemore, D; Norwich, G; Chien, S

    1997-01-01

    When a discocytic erythrocyte (RBC) was partially aspirated into a 1.5-microns glass pipette with a high negative aspiration pressure (delta P = -3.9 kPa), held in the pipette for 30 s (holding time, th), and then released, it underwent a discocyte-echinocyte shape transformation. The degree of shape transformation increased with an increase in th. The echinocytes recovered spontaneously to discocytes in approximately 10 min, and there was no significant difference in recovery time at 20.9 degrees C, 29.5 degrees C, and 37.4 degrees C, respectively. At 11 degrees C the recovery time was significantly elevated to 40.1 +/- 6.7 min. At 20.9 degrees C the shape recovery time varied directly with the isotropic RBC tension induced by the pipetting. Sodium orthovanadate (vanadate, 200 microM), which inhibits the phospholipid translocase, blocks the shape recovery. Chlorpromazine (CP, 25 microM) reversed the pipette-induced echinocytic shape to discocytic in < 2 min, and the RBC became a spherostomatocyte-II after another 30 min. It was hypothesized that the increase in cytosolic pressure during the pipette aspiration induced an isotropic tension in the RBC membrane followed by a net inside-to-outside membrane lipid translocation. After a sudden release of the aspiration pressure the cytosolic pressure and the membrane tension normalized immediately, but the translocated phospholipids remained temporarily "trapped" in the outer layer, causing an area excess and hence the echinocytic shape. The phospholipid translocase activity, when not inhibited by vanadate, caused a gradual return of the translocated phospholipids to the inner layer, and the RBC shape recovered with time. Images FIGURE 1 FIGURE 4 FIGURE 5 FIGURE 7 FIGURE 8 PMID:9138589

  19. Ceranib-2-induced suicidal erythrocyte death.

    PubMed

    Signoretto, Elena; Zierle, Jens; Bhuyan, Abdulla Al Mamun; Castagna, Michela; Lang, Florian

    2016-07-01

    Ceramide is known to trigger apoptosis of nucleated cells and eryptosis of erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Besides ceramide, stimulators of eryptosis include increase of cytosolic Ca(2+) -activity ([Ca(2+) ]i ) and oxidative stress. Ceramide is degraded by acid ceramidase and inhibition of the enzyme similarly triggers apoptosis. The present study explored, whether ceramidase inhibitor Ceranib-2 induces eryptosis. Flow cytometry was employed to quantify phosphatidylserine-exposure at the cell surface from annexin-V-binding, cell volume from forward scatter, [Ca(2+) ]i from Fluo3-fluorescence, reactive oxygen species (ROS) from DCF dependent fluorescence, and ceramide abundance utilizing specific antibodies. Hemolysis was estimated from hemoglobin concentration in the supernatant. A 48 h exposure of human erythrocytes to Ceranib-2 significantly increased the percentage of annexin-V-binding cells (≥50 μM) and the percentage of hemolytic cells (≥10 μM) without significantly modifying forward scatter. Ceranib-2 significantly increased Fluo3-fluorescence, DCF fluorescence and ceramide abundance. The effect of Ceranib-2 on annexin-V-binding was not significantly blunted by removal of extracellular Ca(2+) . Ceranib-2 triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to increase of ceramide abundance and induction of oxidative stress, but not dependent on Ca(2+) entry. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27291470

  20. Effects of ascorbic acid on copper-induced oxidative changes in human erythrocytes: example of a biphasic dose-response relationship

    SciTech Connect

    Kemp, J.; Calabrese, E.J.

    1985-01-01

    In an in vitro study, ascorbic acid reduced the occurrence of copper acetate-induced oxidative stress in human erythrocytes at biologically relevant concentrations (0.06 - 0.25 mM) while enhancing oxidative changes (i.e., changes in methemoglobin (METHB) and reduced glutathione (GSH)) at higher levels of exposure (>1.0 mM).

  1. Quantitative determination of native proteins in individual human erythrocytes by capillary zone electrophoresis with laser-induced fluorescence detection

    SciTech Connect

    Lee, T.T.; Yeung, E.S. Iowa State Univ., Ames )

    1992-12-01

    Intracellular fluid within single human erythrocytes is analyzed by capillary electrophoresis with laser-excited native protein fluorescence. Good signal-to-noise is achieved, allowing even minor components to be quantified. Non-Gaussian distributions were found for total protein, fraction carbonic anhydrase, fraction hemoglobin A[sub 0], and an unidentified component. Variations among a group of 29 cells for each quantity are as much as 1 order of magnitude, even though erythrocytes are known to be fairly homogeneous in size distribution. Variations in fraction hemoglobin A[sub 0] reflect differences in in vitro oxidation rates to methemoglobin. A positive correlation was observed between carbonic anhydrase and hemoglobin A[sub 0] for individual cells. This is consistent with the presence of erythrocytes of different ages within the population, with the older cells being less capable of maintaining enzyme activity and preventing oxidative damage. 35 refs., 10 figs., 1 tab.

  2. Protective Role of Catechin and Quercetin in Sodium Benzoate-Induced Lipid Peroxidation and the Antioxidant System in Human Erythrocytes In Vitro

    PubMed Central

    Yetuk, Gamze; Pandir, Dilek; Bas, Hatice

    2014-01-01

    The aim of this study was to evaluate the protective effect of catechin and quercetin in sodium benzoate- (SB-) induced oxidative stress in human erythrocytes in vitro. For this, the effects of SB (6.25, 12.5, 25, 50, and 100 μg/mL), catechin (10 μM), and quercetin (10 μM) on lipid peroxidation (LPO) and the activities of SOD, CAT, GPx, and GST were studied. Significantly higher LPO and lower activities of antioxidant enzymes were observed with the increasing concentrations of SB. Catechin or quercetin protected the erythrocytes against SB-induced toxicity only at low concentrations of SB. The presence of catechin or quercetin at 10 μM have no effect on SB-induced toxicity at high concentrations of SB (50 and 100 μg/mL). In conclusion, SB may cause oxidative stress as food additive in human erythrocytes in vitro. So, it appears that our findings provide evidence for the protection of erythrocytes from SB that could be considered for further studies. PMID:24693251

  3. Hydroxytyrosol inhibits phosphatidylserine exposure and suicidal death induced by mercury in human erythrocytes: Possible involvement of the glutathione pathway.

    PubMed

    Officioso, Arbace; Alzoubi, Kousi; Lang, Florian; Manna, Caterina

    2016-03-01

    Hydroxytyrosol (HT) is a phenolic antioxidant naturally occurring in virgin olive oil. In this study, we investigated the possible protective effects of HT on programmed suicidal death (eryptosis) induced by mercury (Hg) treatment in intact human erythrocytes (RBC). Our study confirms that the Hg-eryptosis is characterized by phosphatidylserine (PS) exposure at the cell surface, with cell shrinkage and ATP and glutathione depletion; calcium influx is also a key event that triggers eryptosis. Here we report that cell preconditioning with an optimal dose (1-5 μM) of HT prior to exposure to 2.5 μM HgCl2 causes a noteworthy decrease in PS-exposing RBC, almost restoring ATP and GSH content. Conversely, HT shows no effect against decrease in cell volume nor against influx of extracellular calcium. Taken together our data provide the first experimental evidence of the efficacy of HT in modulating the programmed suicidal death in non nucleated cells; the reported findings also confirm that the prevention of Hg toxicity should be regarded as an additional mechanism responsible for the health-promoting potential of this dietary phenol. Finally, virgin olive oil would appear to be a promising healthy food to reduce the adverse effects of chronic mercury exposure in humans. PMID:26774912

  4. Vanadate-induced suicidal erythrocyte death.

    PubMed

    Föller, Michael; Sopjani, Mentor; Mahmud, Hasan; Lang, Florian

    2008-01-01

    Vanadium, a trace element, as vanadate (VO4(3-)) is known to interfere with a wide variety of enzymes including Ca2+ ATPase and Na+/+ ATPase. VO4(3-) is excreted mainly via the kidney. In renal insufficiency, the impaired VO4(3-) excretion leads to VO4(3-) accumulation in blood.The present study explored the effect of VO4(3-) on eryptosis, the suicidal death of erythrocytes. Eryptosis is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptotic cells are phagocytosed and thus rapidly cleared from circulating blood. Stimulators of eryptosis include an increase of the cytosolic Ca2+ concentration. Erythrocyte Ca2+ activity was estimated from Fluo-3 fluorescence, phosphatidylserine exposure from annexin V-binding, and erythrocyte volume from forward scatter in FACS analysis. Exposure of erythrocytes to VO4(3-) increased cytosolic Ca2+ concentration, enhanced the percentage of annexin V-binding erythrocytes, decreased erythrocyte forward scatter, and lowered the intracellular ATP concentration. In conclusion, VO4(3-) induces eryptosis at least partially through increase of cytosolic Ca2+ concentration, an effect presumably contributing to the development of anemia in chronic renal failure. PMID:18319605

  5. Effects of ascorbic acid supplementation on copper-induced oxidative changes in human erythrocytes

    SciTech Connect

    Calabrese, E.J.; Kemp, J.

    1985-01-01

    A previously reported study indicated that ascorbic acid reduces the occurrence of copper acetate-induced methemoglobin (METHB) formation in vitro. The present study was designed to evaluate these findings in an in vivo exposure of ascorbic acid (1 gm/day) for up to four weeks with an in vitro copper acetate incubation stress at baseline (just prior to supplementation) and at two and four weeks after initiation of treatment. The results indicated that the ascorbic acid supplementation had no significant effects on the occurrence of copper acetate induced oxidant stress (i.e. METHB increase and GSH decrease). Possible explanations for this apparent discrepancy are provided.

  6. [Lysophosphatidic acid and human erythrocyte aggregation].

    PubMed

    Sheremet'ev, Iu A; Popovicheva, A N; Levin, G Ia

    2014-01-01

    The effects of lysophosphatidic acid on the morphology and aggregation of human erythrocytes has been studied. Morphology of erythrocytes and their aggregates were studied by light microscopy. It has been shown that lysophosphatidic acid changes the shape of red blood cells: diskocyte become echinocytes. Aggregation of red blood cells (rouleaux) was significantly reduced in autoplasma. At the same time there is a strong aggregation of echinocytes. This was accompanied by the formation of microvesicles. Adding normal plasma to echinocytes restores shape and aggregation of red blood cells consisting of "rouleaux". A possible mechanism of action of lysophosphatidic acid on erythrocytes is discussed. PMID:25509147

  7. Mechanisms of human erythrocytic bioactivation of nitrite.

    PubMed

    Liu, Chen; Wajih, Nadeem; Liu, Xiaohua; Basu, Swati; Janes, John; Marvel, Madison; Keggi, Christian; Helms, Christine C; Lee, Amber N; Belanger, Andrea M; Diz, Debra I; Laurienti, Paul J; Caudell, David L; Wang, Jun; Gladwin, Mark T; Kim-Shapiro, Daniel B

    2015-01-01

    Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO2 are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood. PMID:25471374

  8. Protective Effects of Ferulic Acid on High Glucose-Induced Protein Glycation, Lipid Peroxidation, and Membrane Ion Pump Activity in Human Erythrocytes

    PubMed Central

    Sompong, Weerachat; Cheng, Henrique; Adisakwattana, Sirichai

    2015-01-01

    Ferulic acid (FA) is the ubiquitous phytochemical phenolic derivative of cinnamic acid. Experimental studies in diabetic models demonstrate that FA possesses multiple mechanisms of action associated with anti-hyperglycemic activity. The mechanism by which FA prevents diabetes-associated vascular damages remains unknown. The aim of study was to investigate the protective effects of FA on protein glycation, lipid peroxidation, membrane ion pump activity, and phosphatidylserine exposure in high glucose-exposed human erythrocytes. Our results demonstrated that FA (10-100 μM) significantly reduced the levels of glycated hemoglobin (HbA1c) whereas 0.1-100 μM concentrations inhibited lipid peroxidation in erythrocytes exposed to 45 mM glucose. This was associated with increased glucose consumption. High glucose treatment also caused a significant reduction in Na+/K+-ATPase activity in the erythrocyte plasma membrane which could be reversed by FA. Furthermore, we found that FA (0.1-100 μM) prevented high glucose-induced phosphatidylserine exposure. These findings provide insights into a novel mechanism of FA for the prevention of vascular dysfunction associated with diabetes. PMID:26053739

  9. Drug-induced erythrocyte membrane internalization

    PubMed Central

    Ben-Bassat, Isaac; Bensch, Klaus G.; Schrier, Stanley L.

    1972-01-01

    In vitro erythrocyte membrane internalization, resulting in the formation of membrane-lined vacuoles, can be quantified by a radioisotopic method. A complex of 37Co-labeled vitamin B12 and its plasma protein binders is first adsorbed to the cell surface, and after vacuoles are formed, the noninternalized label is removed by washing and trypsin treatment. The residual radioactivity represents trapped label and can be used to measure the extent of membrane internalization. Using this method, it was found that in addition to primaquine, a group of membrane-active drugs, specifically hydrocortisone, vinblastine, and chlorpromazine can induce membrane internalization in erythrocytes. This is a metabolic process dependent on drug concentration, temperature, and pH. Vacuole formation by all agents tested can be blocked by prior depletion of endogenous substrates or by poisoning the erythrocytes with sodium fluoride and sulfhydryl blocking agents. This phenomenon resembles in some respects the previously reported membrane internalization of energized erythrocyte ghosts. It is suggested that membrane internalization is dependent on an ATP-energized state and is influenced by the balance between the concentrations of magnesium and calcium in the membrane. This study provides a basis for proposing a unifying concept of the action of some membrane-active drugs, and for considering the role of erythrocyte membrane internalization in pathophysiologic events. Images PMID:4555785

  10. Dietary indicaxanthin from cactus pear (Opuntia ficus-indica L. Mill) fruit prevents eryptosis induced by oxysterols in a hypercholesterolaemia-relevant proportion and adhesion of human erythrocytes to endothelial cell layers.

    PubMed

    Tesoriere, Luisa; Attanzio, Alessandro; Allegra, Mario; Livrea, Maria A

    2015-08-14

    Toxic oxysterols in a hypercholesterolaemia-relevant proportion cause suicidal death of human erythrocytes or eryptosis. This process proceeds through early production of reactive oxygen species (ROS), release of prostaglandin (PGE2) and opening of PGE2-dependent Ca channels, membrane phosphatidylserine (PS) externalisation, and cell shrinkage. The present study was the first to reveal that a bioavailable phytochemical, indicaxanthin (Ind) from cactus pear fruit, in a concentration range (1.0-5.0 μM) consistent with its plasma level after a fruit meal, prevents PS externalisation and cell shrinkage in a dose-dependent manner when incubated with isolated healthy human erythrocytes exposed to an oxysterol mixture for 48 h. Dietary Ind inhibited ROS production, glutathione (GSH) depletion, PGE2 release and Ca2+ entry. Ind alone did not modify the erythrocyte redox environment or affect other parameters. Ex vivo spiking of normal human blood with the oxysterol mixture for 48 h induced eryptosis, resulting in the production of ROS and decreased levels of GSH, which was prevented by concurrent exposure to 5 μm-Ind. The adherence of eryptotic erythrocytes to the endothelium causes vascular tissue injury. Erythrocytes isolated from blood incubated with the oxysterol mixture plus 5 μm-Ind did not adhere to endothelial cell monolayers. Eryptotic erythrocytes may contribute to thrombotic complications in hypercholesterolaemia. Our findings suggest the positive effects of diets containing Ind on erythrocytes in hypercholesterolaemic subjects. PMID:26169206

  11. Metabolism of acetylcholine in human erythrocytes

    SciTech Connect

    Chapman, E.S.

    1990-01-01

    In order to examine the possible role of erythrocyte acetylcholinesterase in the maintenance of membrane phospholipid content and membrane fluidity, experiments were performed to monitor the activity of the enzyme and follow the fate of one of its hydrolytic products, choline. Intact human erythrocytes were incubated with acetylcholine (choline methyl-{sup 14}C). The incubation resulted in the hydrolysis of acetylcholine to acetate and choline; the reaction was catalyzed by membrane acetylcholinesterase. The studies demonstrate the further metabolism of choline. Experiments were carried out to determine rate of hydrolysis of acetylcholine, uptake of choline, identification of intracellular metabolites of choline, and identification of radiolabeled membrane components. Erythrocytes at a 25% hematocrit were incubated in an isoosmotic bicarbonate buffer pH 7.4, containing glucose, adenosine, streptomycin and penicillin with 0.3 {mu}Ci of acetylcholine (choline methyl-{sup 14}C), for 24 hours. Aliquots of the erythrocyte suspension were taken throughout for analysis. Erythrocytes were washed free of excess substrate, lysed, and the hemolysate was extracted for choline and its metabolites. Blank samples containing incubation buffer and radiolabeled acetylcholine only, and erythrocyte hemolysate extracts were analyzed for choline content, the difference between blank samples and hemolysate extracts was the amount of choline originating from acetylcholine and attributable to acetylcholinesterase activity. The conversion of choline to {sup 14}C-betaine is noted after several minutes of incubation; at 30 minutes, more than 80% of {sup 14}C-choline is taken up and after several hours, detectable levels of radiolabeled S-adenosylmethionine were present in the hemolysate extract.

  12. Red wine activates plasma membrane redox system in human erythrocytes.

    PubMed

    Tedesco, Idolo; Moccia, Stefania; Volpe, Silvestro; Alfieri, Giovanna; Strollo, Daniela; Bilotto, Stefania; Spagnuolo, Carmela; Di Renzo, Massimo; Aquino, Rita P; Russo, Gian Luigi

    2016-05-01

    In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13-73 μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70-100 μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity. PMID:26866566

  13. Uncaria tomentosa extracts protect human erythrocyte catalase against damage induced by 2,4-D-Na and its metabolites.

    PubMed

    Bukowska, Bożena; Bors, Milena; Gulewicz, Krzysztof; Koter-Michalak, Maria

    2012-06-01

    The effect of ethanolic and aqueous extracts from leaves and bark of Uncaria tomentosa was studied, with particular attention to catalase activity (CAT - EC. 1.11.1.6). We observed that all tested extracts, at a concentration of 250 μg/mL were not toxic to erythrocyte catalase because they did not decreased its activity. Additionally, we investigated the protective effect of extracts on changes in CAT activity in the erythrocytes incubated with sodium salt of 2,4-dichlorophenoxyacetic acid (2,4-D-Na) and its metabolites i.e., 2,4-dichlorophenol (2,4-DCP) and catechol. Previous investigations showed that these chemicals decreased activity of erythrocyte catalase (Bukowska et al., 2000; Bukowska and Kowalska, 2004). The erythrocytes were divided into two portions. The first portion was incubated for 1 and 5h at 37°C with 2,4-D-Na, 2,4-DCP and catechol, and second portion was preincubated with extracts for 10 min and then incubated with xenobiotics for 1 and 5h. CAT activity was measured in the first and second portion of the erythrocytes. We found a protective effect of the extracts from U. tomentosa on the activity of catalase incubated with xenobiotics studied. Probably, phenolic compounds contained in U. tomentosa scavenged free radicals, and therefore protected active center (containing -SH groups) of catalase. PMID:22426356

  14. Recombinant human erythrocyte cytochrome b5.

    PubMed

    Lloyd, E; Ferrer, J C; Funk, W D; Mauk, M R; Mauk, A G

    1994-09-27

    The gene encoding the human erythrocyte form of cytochrome b5 (97 residues in length) has been prepared by mutagenesis of an expression vector encoding lipase-solubilized bovine liver microsomal cytochrome b5 (93 residues in length) (Funk et al., 1990). Efficient expression of this gene in Escherichia coli has provided the first opportunity to obtain this protein in quantities sufficient for physical and functional characterization. Comparison of the erythrocytic cytochrome with the trypsin-solubilized bovine liver cytochrome b5 by potentiometric titration indicates that the principal electrostatic difference between the two proteins results from two additional His residues present in the human erythrocytic protein. The midpoint reduction potential of this protein determined by direct electrochemistry is -9 +/- 2 mV vs SHE at pH 7.0 (mu = 0.10 M, 25.0 degrees C), and this value varies with pH in a fashion that is consistent with the presence of a single ionizable group that changes pKa from 6.0 +/- 0.1 in the ferricytochrome to 6.3 +/- 0.1 in the ferrocytochrome with delta H degrees = -3.2 +/- 0.1 kcal/mol and delta S degrees = -11.5 +/- 0.3 eu (pH 7.0, mu = 0.10). The 1D 1H NMR spectrum of the erythrocytic ferricytochrome indicates that 90% of the protein binds heme in the "major" orientation and 10% of the protein binds heme in the "minor" orientation (pH 7.0, 25 degrees C) with delta H degrees = -2.9 +/- 0.3 kcal/mol and delta S degrees = -5.4 +/- 0.9 eu for this equilibrium. PMID:7918357

  15. Reduced sodium concentration and increased sodium-potassium pump activity of erythrocytes in human hypertension

    SciTech Connect

    Simon, G.; Engel, C.R.

    1987-06-01

    Erythrocyte Nai, Nai/Ki and ouabain-sensitive and ouabain-insensitive /sup 86/Rb uptake (K transport) were measured in whole blood of 16 normotensive and 19 hypertensive white male subjects, within seconds or minutes after withdrawal of blood. Erythrocyte Nai and Nai/Ki were reduced (p less than 0.05), and ouabain-sensitive /sup 86/Rb uptake was increased (p less than 0.01) in hypertensive subjects. In a separate group of hypertensive white male subjects, an inverse correlation was found between erythrocyte Nai/Ki and ouabain-binding sites per erythrocyte (r = 0.85, p less than 0.01, n = 9). The abnormalities of erythrocyte cation fluxes in hypertensive subjects are similar to those induced by aldosterone in vascular smooth muscle cells and by glucocorticoid administration in the erythrocytes of human subjects, suggesting similarities in pathogenesis.

  16. Volume-dependent regulation of ion transport and membrane phosphorylation in human and rat erythrocytes.

    PubMed

    Orlov, S N; Pokudin, N I; Kotelevtsev, Y V; Gulak, P V

    1989-02-01

    Osmotic swelling of human and rat erythrocytes does not induce regulatory volume decrease. Regulatory volume increase was observed in shrunken erythrocytes of rats only. This reaction was blocked by the inhibitors of Na+/H+ exchange. Cytoplasmic acidification in erythrocytes of both species increases the amiloride-inhibited component of 22Na influx by five- to eight-fold. Both the osmotic and isosmotic shrinkage of rat erythrocytes results in the 10- to 30-fold increase of amiloride-inhibited 22Na influx and a two-fold increase of furosemide-inhibited 86Rb influx. We failed to indicate any significant changes of these ion transport systems in shrunken human erythrocytes. The shrinking of quin 2-loaded human and rat erythrocytes results in the two- to threefold increase of the rate of 45Ca influx, which is completely blocked by amiloride. The dependence of volume-induced 22Na influx in rat erythrocytes and 45Ca influx in human erythrocytes on amiloride concentration does not differ. The rate of 45Ca influx in resealed ghosts was reduced by one order of magnitude when intravesicular potassium and sodium were replaced by choline. It is assumed that the erythrocyte shrinkage increases the rate of a nonselective Cao2+/(Nai+, Ki+) exchange. Erythrocyte shrinking does not induce significant phosphorylation of membrane protein but increases the 32P incorporation in diphosphoinositides. The effect of shrinkage on the 32P labeling of phosphoinositides is diminished after addition of amiloride. It is assumed that volume-induced phosphoinositide response plays an essential role in the mechanism of the activation of transmembrane ion movements. PMID:2541247

  17. Variability of intracellular lactate dehydrogenase isoenzymes in single human erythrocytes

    SciTech Connect

    Xue, Q.; Yeung, E.S. Iowa State Univ., Ames, IA )

    1994-04-01

    Trace amounts of enzymes within single human erythrocytes can be quantified by a combination of on-column reaction and capillary electrophoresis. A detection limit of 1.3 x 10[sup [minus]21] mol of LDH was achieved with laser-induced fluorescence by monitoring the product of the enzyme-catalyzed reaction between lactate and NAD[sup +]. Single erythrocyte analysis clearly isolates the major forms of LDH. The variation of total LDH activity in a population of cells from a single individual is large, but the relative activities of the isoenzymes LDH-1 and LDH-2 are fairly constant. This can be explained by the distribution of cell age in the population. A lower enzyme activity is indicative of senescence. The efficient separation of different LDH forms and the high detection sensitivity opens up the possibility of multiple-enzyme assays with a single mammalian cell. 41 refs., 5 figs.

  18. Radical scavenging activities of Tyr-, Trp-, Cys- and Met-Gly and their protective effects against AAPH-induced oxidative damage in human erythrocytes.

    PubMed

    Zheng, Lin; Dong, Hongzhu; Su, Guowan; Zhao, Qiangzhong; Zhao, Mouming

    2016-04-15

    Radical scavenging activities of Tyr-, Trp-, Cys- and Met-Gly and their protective effects against AAPH-induced oxidative damage in erythrocytes were evaluated in this study. This damage includes hemolysis, oxidation of hemoglobin, formation of MDA and the depletion of glutathione (GSH) and catalase (CAT). Results showed that Tyr- and Trp-Gly could quench the radicals effectively in ABTS and ORAC assays with TE (Trolox equivalent) values of more than 1.0 μmol TE/μmol, followed by Cys- and Met-Gly. All these dipeptides could protect erythrocytes against AAPH-induced hemolysis in a dose-dependent manner. They could also significantly (p<0.05) retard the oxidation of hemoglobin and depletion of GSH in erythrocytes. The protective effects of these dipeptides decreased in the following order: Trp-Gly>Tyr-Gly>Met-Gly>Cys-Gly, which were consistent with their peroxyl radical scavenging activities. It suggested that these dipeptides might protect erythrocytes against AAPH-induced oxidative damage, mainly by acting as the direct radical scavengers. PMID:26617020

  19. Comparative study on thiol drugs' effect on tert-butyl hydroperoxide induced luminol chemiluminescence in human erythrocyte lysate and hemoglobin oxidation.

    PubMed

    Sajewicz, Waldemar; Zalewska, Marta; Milnerowicz, Halina

    2015-02-01

    The current studies have investigated the effect of heterocyclic drugs with the single thiol group (thiamazole, mercaptopurine) and dithiol aliphatic drugs (dimercaptosuccinic acid, dithiothreitol) under oxidative stress conditions, using tert-butyl hydroperoxide (t-BuOOH), in human erythrocyte lysate with the luminol-enhanced chemiluminescence technique. Knowing that oxidative processes induced by t-BuOOH are triggered by (oxy)hemoglobin (Hb), the effect of different thiol drugs (RSH) on isolated human Hb oxidation to methemoglobin (MHb) and hemichromes (HChr) was further considered. Three types of chemiluminescence curves, fitting to logistic-exponential model, have been revealed under influence of RSH. Structure of the data (MHb and HChr production, and free radical activity of RSH) in Principal Component Analysis visualization and kinetic profiles of chemiluminescence integrate information in terms of the diversity of RSH reaction mechanisms depending on the specific molecular context of the given thiol: aliphatic or aromatic nature as well as the number and position of the -SH groups in the molecule. The study conducted in presented in vitro systems indicates the potential role of thiol drugs mediated toxicity in an oxidative stress dependent mechanism. PMID:25308193

  20. Alteration of human erythrocyte membrane properties by complement fixation.

    PubMed Central

    Durocher, J R; Gockerman, J P; Conrad, M E

    1975-01-01

    Erythrocyte survival studies of complement-coated radiolabeled erythrocytes have shown rapid removal of these cells from the peripheral blood with a return of these cells into the circulation within a few hours. We studied complement-coated human erythrocytes and measured surface charge and deformability, two parameters believed to be important in erythrocyte survival. Erythrocytes were coated with complement by two in vitro techniques: the addition of (a) low ionic strength sucrose, and (b) IgM cold agglutinins. Erythrocytes obtained from three patients with cold agglutinin disease were used as a source of in vivo complement-coated cells. No difference was found in surface charge as measured by electrophoretic mobility between erythrocytes from normal subjects and complement-coated erythrocytes from any of the three sources. When deformability was measured by filtration through 3-mum polycarbonate sieves, marked decreases in deformability were found in complement-coated erythrocytes. The filtration returned toward control levels by incubating the complement-coated erythrocytes in serum for 1 h and correlated with decreases in immune adherence. Using screen filtration pressure as a measure of deformability, a positive correlation between number of C3 molecules per erythrocyte and decreased deformability was found. C3b appeared responsible for the decreased deformability of the erythrocytes, since conversion of C3b to C3d resulted in a return of deformability toward normal. The data suggested that the sequestration of complement-coated human erythrocytes in the microvasculature can be explained in part by decreased deformability and changes in immune adherence. PMID:1120777

  1. Effects of phenylpropanolamine (PPA) on in vitro human erythrocyte membranes and molecular models

    SciTech Connect

    Suwalsky, Mario; Zambrano, Pablo; Mennickent, Sigrid; Villena, Fernando; Sotomayor, Carlos P.; Aguilar, Luis F.; Bolognin, Silvia

    2011-03-18

    Research highlights: {yields} PPA is a common ingredient in cough-cold medication and appetite suppressants. {yields} Reports on its effects on human erythrocytes are very scarce. {yields} We found that PPA induced in vitro morphological changes to human erythrocytes. {yields} PPA interacted with isolated unsealed human erythrocyte membranes. {yields} PPA interacted with class of lipid present in the erythrocyte membrane outer monolayer. -- Abstract: Norephedrine, also called phenylpropanolamine (PPA), is a synthetic form of the ephedrine alkaloid. After reports of the occurrence of intracranial hemorrhage and other adverse effects, including several deaths, PPA is no longer sold in USA and Canada. Despite the extensive information about PPA toxicity, reports on its effects on cell membranes are scarce. With the aim to better understand the molecular mechanisms of the interaction of PPA with cell membranes, ranges of concentrations were incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), and molecular models of cell membranes. The latter consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, respectively. The capacity of PPA to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). This study presents evidence that PPA affects human red cell membranes as follows: (a) in SEM studies on human erythrocytes it was observed that 0.5 mM PPA induced shape changes; (b) in IUM PPA induced a sharp decrease in the fluorescence anisotropy in the lipid bilayer acyl chains in a concentration range lower than 100 {mu}M; (c) X-ray diffraction studies showed that PPA in the 0.1-0.5 m

  2. Evidence for anionic cation transport of lithium, sodium and potassium across the human erythrocyte membrane induced by divalent anions.

    PubMed Central

    Becker, B F; Duhm, J

    1978-01-01

    1. The passive net transport of Li+ and Na+ across the human red cell membrane was accelerated by the divalent anions carbonate, sulphite, oxalate, phosphite and malonate. Phthalate, maleate, sulphate and succinate were found additionally to stimulate downhill transport of K+. Marked differences in anion efficacy and selectivity were observed. 2. The effects of these 'carbonate type' anions were reversible and fully blocked by SITS, dipyridamole and other inhibitors of anion transfer. 3. Cation transport acceleration induced by the monovalent anions salicylate, benzoate, thiocyanate and 2,4-dinitrophenol were inhibited by dipyridamole, but not affected by SITS. A great number of mono- and polyvalent anions were without detectable influence on Li+ transport. 4. Li+ net uptake induced by oxalate exhibited a pH dependence similar to that reported for halide self exchange. 5. Transport acceleration by carbonate type anions displayed a linear, 1:1 dependence on the concentrations of both the anion and the cation and was symmetric with respect to the two sides of the membrane. 6. It is concluded that the divalent carbonate type anions form singly charged, negative 1:1 ion pairs with the respective alkali metal cations, the ion pairs traversing the red cell membrane via the anion exchange pathway. This concept of anionic formation of some of the ion pairs considered. The relative efficacies and cation selectivities of polyvalent anions can largely be explained on the basis of electrostatic interactions governing ion pair formation. However, the chelating properties, structural flexibility, polarizability of the anions and the accessibility of the ion pairs to the anion exchange pathway need also be considered. 7. An exchange of NaCO-3 ion pairs for internal HCO-3 or Cl- is discussed as a possible mode of cellular pH regulation. PMID:31458

  3. [AGGREGATION OF METABOLICALLY DEPLETED HUMAN ERYTHROCYTES].

    PubMed

    Sheremet'ev, Yu A; Popovicheva, A N; Rogozin, M M; Levin, G Ya

    2016-01-01

    An aggregation of erythrocytes in autologous plasma after blood storage for 14 days at 4 °C was studied using photometry and light microscopy. The decrease of ATP content, the formation of echinocytes and spheroechinocytes, the decrease of rouleaux form of erythrocyte aggregation were observed during the storage. On the other hand the aggregates of echinocytes were formed in the stored blood. The addition of plasma from the fresh blood didn't restore the normal discocytic shape and aggregation of erythrocytes in the stored blood. The possible mechanisms of erythrocytes and echinocytes aggregation are discussed. PMID:27220249

  4. Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte.

    PubMed

    Huber, Stephan M; Duranton, Christophe; Henke, Guido; Van De Sand, Claudia; Heussler, Volker; Shumilina, Ekaterina; Sandu, Ciprian D; Tanneur, Valerie; Brand, Verena; Kasinathan, Ravi S; Lang, Karl S; Kremsner, Peter G; Hübner, Christian A; Rust, Marco B; Dedek, Karin; Jentsch, Thomas J; Lang, Florian

    2004-10-01

    Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival. PMID:15272009

  5. Site-Specific GlcNAcylation of Human Erythrocyte Proteins

    PubMed Central

    Wang, Zihao; Park, Kyoungsook; Comer, Frank; Hsieh-Wilson, Linda C.; Saudek, Christopher D.; Hart, Gerald W.

    2009-01-01

    OBJECTIVE—O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals. RESEARCH DESIGN AND METHODS—GlcNAcylated erythrocyte proteins or GlcNAcylated peptides were tagged and selectively enriched by a chemoenzymatic approach and identified by mass spectrometry. The enrichment approach was combined with solid-phase chemical derivatization and isotopic labeling to detect O-GlcNAc modification sites and to compare site-specific O-GlcNAc occupancy levels between normal and diabetic erythrocyte proteins. RESULTS—The enzymes that catalyze the cycling (addition and removal) of O-GlcNAc were detected in human erythrocytes. Twenty-five GlcNAcylated erythrocyte proteins were identified. Protein expression levels were compared between diabetic and normal erythrocytes. Thirty-five O-GlcNAc sites were reproducibly identified, and their site-specific O-GlcNAc occupancy ratios were calculated. CONCLUSIONS—GlcNAcylation is differentially regulated at individual sites on erythrocyte proteins in response to glycemic status. These data suggest not only that site-specific O-GlcNAc levels reflect the glycemic status of an individual but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as a diagnostic tool for the early detection of diabetes. PMID:18984734

  6. Identification of the phorbol ester receptor in human and avian erythrocytes

    SciTech Connect

    Kramer, C.M.; Sando, J.J.; Speizer, L.A.

    1986-05-01

    The ability of phorbol esters to inhibit the uptake of a fluorescent glucose analogue in goose but not human erythrocytes is consistent with earlier reports that the human red blood cell lacks the phorbol ester receptor. However, they have located specific phorbol 12,13-dibutyrate binding sites in both human and goose erythrocytes. Human and goose red blood cells contain 2 classes of phorbol ester receptors with similar affinities, however the human erythrocyte contains 1/3 as many phorbol ester receptors as does the goose red blood cell. An additional contrast in the binding of phorbol esters to human and goose red blood cells is the temperature-induced enhancement of binding to goose, but not human erythrocytes. Equilibrium phorbol ester binding to goose red blood cells at 37/sup 0/C is enhanced 3.3 +/- 0.4 times that amount bound at 4/sup 0/C. Equilibrium binding of phorbol esters to human erythrocytes is identical at both temperatures. In vivo and in vitro phosphorylation profiles of C-kinase substrates also differ between the human and goose erythrocyte.

  7. Protective activity of the Uncaria tomentosa extracts on human erythrocytes in oxidative stress induced by 2,4-dichlorophenol (2,4-DCP) and catechol.

    PubMed

    Bors, Milena; Bukowska, Bożena; Pilarski, Radosław; Gulewicz, Krzysztof; Oszmiański, Jan; Michałowicz, Jaromir; Koter-Michalak, Maria

    2011-09-01

    The purpose of this study was to evaluate the effect of the ethanolic and aqueous extracts of Uncaria tomentosa on human erythrocytes and additionally the assessment of protective effect of these extracts on hemolysis induction, hemoglobin oxidation, and changes in the level of reactive oxygen species (ROS) and lipid peroxidation, which were provoked by selected xenobiotics, i.e. 2,4-dichlorophenol (2,4-DCP) and catechol. All tested extracts, even at a very high concentration of 500 μg/ml were not toxic to the erythrocytes because they did not cause lipid peroxidation, increase methemoglobin and ROS levels nor provoked hemolysis. The results of this study also revealed protective effect of extracts of U. tomentosa. The extracts studied depleted the extent of hemoglobin oxidation and lipid peroxidation as well as decreased the level of ROS and hemolysis, which was provoked by 2,4-DCP. No protective activity of the extracts against catechol action, which is a precursor of semiquinones in cell was found. A difference in the effect of the extracts studied was observed. Ethanol-based extracts revealed more pronounced ability to inhibit oxidation processes in human erythrocytes. PMID:21712061

  8. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  9. Human erythrocytes and neuroblastoma cells are affected in vitro by Au(III) ions

    SciTech Connect

    Suwalsky, Mario; Gonzalez, Raquel; Villena, Fernando; Aguilar, Luis F.; Sotomayor, Carlos P.; Bolognin, Silvia; Zatta, Paolo

    2010-06-25

    Gold compounds are well known for their neurological and nephrotoxic implications. However, haematological toxicity is one of the most serious toxic and less studied effects. The lack of information on these aspects of Au(III) prompted us to study the structural effects induced on cell membranes, particularly that of human erythrocytes. AuCl{sub 3} was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of multibilayers of dimyristoylphosphatidylcholine and dimyristoylphosphatidylethanolamine, phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence that Au(III) interacts with red cell membranes as follows: (a) in scanning electron microscopy studies on human erythrocytes it was observed that Au(III) induced shape changes at a concentration as low as 0.01 {mu}M; (b) in isolated unsealed human erythrocyte membranes Au(III) induced a decrease in the molecular dynamics and/or water content at the glycerol backbone level of the lipid bilayer polar groups in a 5-50 {mu}M concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 {mu}m-1 mM range induced increasing structural perturbation only to dimyristoylphosphatidylcholine bilayers. Additional experiments were performed in human neuroblastoma cells SH-SY5Y. A statistically significant decrease of cell viability was observed with Au(III) ranging from 0.1 {mu}M to 100 {mu}M.

  10. Human erythrocytes analyzed by generalized 2D Raman correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Wesełucha-Birczyńska, Aleksandra; Kozicki, Mateusz; Czepiel, Jacek; Łabanowska, Maria; Nowak, Piotr; Kowalczyk, Grzegorz; Kurdziel, Magdalena; Birczyńska, Malwina; Biesiada, Grażyna; Mach, Tomasz; Garlicki, Aleksander

    2014-07-01

    The most numerous elements of the blood cells, erythrocytes, consist mainly of two components: homogeneous interior filled with hemoglobin and closure which is the cell membrane. To gain insight into their specific properties we studied the process of disintegration, considering these two constituents, and comparing the natural aging process of human healthy blood cells. MicroRaman spectra of hemoglobin within the single RBC were recorded using 514.5, and 785 nm laser lines. The generalized 2D correlation method was applied to analyze the collected spectra. The time passed from blood donation was regarded as an external perturbation. The time was no more than 40 days according to the current storage limit of blood banks, although, the average RBC life span is 120 days. An analysis of the prominent synchronous and asynchronous cross peaks allow us to get insight into the mechanism of hemoglobin decomposition. Appearing asynchronous cross-peaks point towards globin and heme separation from each other, while synchronous shows already broken globin into individual amino acids. Raman scattering analysis of hemoglobin “wrapping”, i.e. healthy erythrocyte ghosts, allows for the following peculiarity of their behavior. The increasing power of the excitation laser induced alterations in the assemblage of membrane lipids. 2D correlation maps, obtained with increasing laser power recognized as an external perturbation, allows for the consideration of alterations in the erythrocyte membrane structure and composition, which occurs first in the proteins. Cross-peaks were observed indicating an asynchronous correlation between the senescent-cell antigen (SCA) and heme or proteins vibrations. The EPR spectra of the whole blood was analyzed regarding time as an external stimulus. The 2D correlation spectra points towards participation of the selected metal ion centers in the disintegration process.

  11. Reduction of hydrogen peroxide-induced erythrocyte damage by Carica papaya leaf extract

    PubMed Central

    Okoko, Tebekeme; Ere, Diepreye

    2012-01-01

    Objective To investigate the in vitro antioxidant potential of Carica papaya (C. papaya) leaf extract and its effect on hydrogen peroxide-induced erythrocyte damage assessed by haemolysis and lipid peroxidation. Methods Hydroxyl radical scavenging activities, hydrogen ion scavenging activity, metal chelating activity, and the ferrous ion reducing ability were assessed as antioxidant indices. In the other experiment, human erythrocytes were treated with hydrogen peroxide to induce erythrocyte damage. The extract (at various concentrations) was subsequently incubated with the erythrocytes and later analysed for haemolysis and lipid peroxidation as indices for erythrocyte damage. Results Preliminary investigation of the extract showed that the leaf possessed significant antioxidant and free radical scavenging abilities using in vitro models in a concentration dependent manner (P<0.05). The extract also reduced hydrogen peroxide induced erythrocyte haemolysis and lipid peroxidation significantly when compared with ascorbic acid (P<0.05). The IC50 values were 7.33 mg/mL and 1.58 mg/mL for inhibition of haemolysis and lipid peroxidation, respectively. In all cases, ascorbic acid (the reference antioxidant) possessed higher activity than the extract. Conclusions The findings show that C. papaya leaves possess significant bioactive potential which is attributed to the phytochemicals which act in synergy. Thus, the leaves can be exploited for pharmaceutical and nutritional purposes. PMID:23569948

  12. Human erythrocytes inhibit complement-mediated solubilization of immune complexes by human serum

    SciTech Connect

    Dorval, B.L.

    1987-01-01

    The aim of this study was to develop an autologus human system to evaluate the effects of human erythrocytes on solubilization of immune complex precipitates (IC) by human serum. Incubation of IC with fresh human serum or guinea pig serum resulted in solubilization of IC. When packed erythrocytes were added to human serum or guinea pig serum binding of IC to the erythrocyte occurred and IC solubilization was inhibited significantly (p <.025). Sheep erythrocytes did not bind IC or inhibit IC solubilization. To evaluate the role of human erythrocyte complement receptor (CR1) on these findings, human erythrocytes were treated with trypsin or anti-CR1 antibodies. Both treatments abrogated IC binding to human erythrocytes but did not affect the ability of the human erythrocyte to inhibit IC solubilization. Radioimmunoassay was used to measure C3, C4 and C5 activation in human serum after incubation with IC, human erythrocytes, human erythrocytes plus IC, whole blood or in whole blood plus IC.

  13. Mechanism of erythrocyte death in human population exposed to arsenic through drinking water

    SciTech Connect

    Biswas, Debabrata; Banerjee, Mayukh; Sen, Gargi; Das, Jayanta K.; Banerjee, Apurba; Sau, T.J.; Pandit, Sudipta; Giri, A.K. Biswas, Tuli

    2008-07-01

    Arsenic contamination in drinking water is one of the biggest natural calamities, which has become an imperative threat to human health throughout the world. Abbreviation of erythrocyte lifespan leading to the development of anemia is a common sequel in arsenic exposed population. This study was undertaken to explore the mechanism of cell death in human erythrocytes during chronic arsenic exposure. Results revealed transformation of smooth discoid red cells into evaginated echinocytic form in the exposed individuals. Further distortion converted reversible echinocytes to irreversible spheroechinocytes. Arsenic toxicity increased membrane microviscosity along with an elevation of cholesterol/phospholipid ratio, which hampered the flexibility of red cell membrane and made them less deformable. Significant increase in the binding of merocyanine 540 with erythrocyte membrane due to arsenic exposure indicated disruption of lipid packing in the outer leaflet of the cell membrane resulting from altered transbilayer phospholipid asymmetry. Arsenic induced eryptosis was characterized by cell shrinkage and exposure of phosphatidylserine at the cell surface. Furthermore, metabolic starvation with depletion of cellular ATP triggered apoptotic removal of erythrocytes from circulation. Significant decrease in reduced glutathione content indicating defective antioxidant capacity was coupled with enhancement of malondialdehyde and protein carbonyl levels, which pointed to oxidative damage to erythrocyte membrane. Arsenic toxicity intervened into red cell membrane integrity eventually leading to membrane destabilization and hemoglobin release. The study depicted the involvement of both erythrophagocytosis and hemolysis in the destruction of human erythrocytes during chronic arsenic exposure.

  14. Automatic Identification of Human Erythrocytes in Microscopic Fecal Specimens.

    PubMed

    Liu, Lin; Lei, Haoting; Zhang, Jing; Yuan, Yang; Zhang, Zhenglong; Liu, Juanxiu; Xie, Yu; Ni, Guangming; Liu, Yong

    2015-11-01

    Traditional fecal erythrocyte detection is performed via a manual operation that is unsuitable because it depends significantly on the expertise of individual inspectors. To recognize human erythrocytes automatically and precisely, automatic segmentation is very important for extraction of characteristics. In addition, multiple recognition algorithms are also essential. This paper proposes an algorithm based on morphological segmentation and a fuzzy neural network. The morphological segmentation process comprises three operational steps: top-hat transformation, Otsu's method, and image binarization. Following initial screening by area and circularity, fuzzy c-means clustering and the neural network algorithms are used for secondary screening. Subsequently, the erythrocytes are screened by combining the results of five images obtained at different focal lengths. Experimental results show that even when the illumination, noise pollution, and position of the erythrocytes are different, they are all segmented and labeled accurately by the proposed method. Thus, the proposed method is robust even in images with significant amounts of noise. PMID:26349804

  15. Comparative sensitivity of human and bovine erythrocytes to sonolysis by 1-MHz ultrasound.

    PubMed

    Miller, M W; Sherman, T A; Brayman, A A

    2000-10-01

    This project tested the hypothesis that human erythrocytes, being larger than bovine erythrocytes, would be the more sensitive to sonolysis induced by inertial cavitation. The rationale behind this hypothesis was an earlier demonstration that, among sized populations of erythrocytes, an inverse relation existed between erythrocyte volume and mechanically-induced shear forces in the surrounding medium; viz, the larger the cell, the less shear force required to rupture the cell's membrane. At low erythrocyte densities (i.e., approximately 5% hematocrit) the hypothesis was supported; at high cell densities (i.e., approximately 35% hematocrit) it was not supported. The data are consistent with an ultrasound (US)-induced symmetric implosion of affected gas nuclei as causing the effect at low cell densities; under such conditions there is ample spacing among cells for US-induced symmetric growth and collapse of gas nuclei and the concomitant production of radially-expanding shock waves (which lyse the cells); at high cell densities there is not sufficient spacing among cells for US-induced symmetric growth and collapse of bubbles and an alternative mechanism, possibly asymmetric bubble collapse, becomes operational. PMID:11120370

  16. Expression of senescent antigen on erythrocytes infected with a knobby variant of the human malaria parasite Plasmodium falciparum

    SciTech Connect

    Winograd, E.; Greenan, J.R.T.; Sherman, I.W.

    1987-04-01

    Erythrocytes infected with a knobby variant of Plasmodium falciparum selectively bind IgG autoantibodies in normal human serum. Quantification of membrane-bound IgG, by use of /sup 125/I-labeled protein A, revealed that erythrocytes infected with the knobby variant bound 30 times more protein A than did noninfected erythrocytes; infection with a knobless variant resulted in less than a 2-fold difference compared with noninfected erythrocytes. IgG binding to knobby erythrocytes appeared to be related to parasite development, since binding of /sup 125/I-labeled protein A to cells bearing young trophozoites (less than 20 hr after parasite invasion) was similar to binding to uninfected erythrocytes. By immunoelectron microscopy, the membrane-bound IgG on erythrocytes infected with the knobby variant was found to be preferentially associated with the protuberances (knobs) of the plasma membrane. The removal of aged or senescent erythrocytes from the peripheral circulation is reported to involve the binding of specific antibodies to an antigen (senescent antigen) related to the major erythrocyte membrane protein band 3. Since affinity-purified autoantibodies against band 3 specifically bound to the plasma membrane of erythrocytes infected with the knobby variant of P. falciparum, it is clear that the malaria parasite induces expression of senescent antigen.

  17. Catalase and glutathione peroxidase are equally active in detoxification of hydrogen peroxide in human erythrocytes

    SciTech Connect

    Gaetani, G.F.; Galiano, S.; Canepa, L.; Ferraris, A.M.; Kirkman, H.N.

    1989-01-01

    Genetic deficiencies of glucose-6-phosphate dehydrogenase (G6PD) and NADPH predispose affected erythrocytes to destruction from peroxides. Conversely, genetic deficiencies of catalase do not predispose affected erythrocytes to peroxide-induced destruction. These observations have served to strengthen the assumption that the NADPH/glutathione/glutathione peroxidase pathway is the principal means for disposal of H/sub 2/O/sub 2/ in human erythrocytes. Recently, however, mammalian catalase was found to have tightly bound NADPH and to require NADPH for the prevention and reversal of inactivation by its toxic substrate (H/sub 2/O/sub 2/). Since both catalase and the glutathione pathway are dependent on NADPH for function, this finding raises the possibility that both mechanisms destroy H/sub 2/O/sub 2/ in human erythrocytes. A comparison of normal and acatalasemic erythrocytes in the present study indicated that catalase accounts for more than half of the destruction of H/sub 2/O/sub 2/ when H/sub 2/O/sub 2/ is generated at a rate comparable to that which leads to hemolysis in G6PD- deficient erythrocytes.

  18. Protective effects of a 21-aminosteroid against copper-induced erythrocyte and plasma lipid peroxidation.

    PubMed

    Fernandes, A C; Filipe, P M; Manso, C F

    1992-09-22

    The 21-aminosteroids, or lazaroids, are a novel class of antioxidant drugs designed to inhibit iron-dependent lipid peroxidation in biological lipid environments. They have been shown to be of therapeutic value in several animal models of traumatic, ischemic and hemorrhagic injury of the central nervous system. Our purpose was to evaluate the ability of 21-aminosteroids to protect human erythrocytes and plasma against oxidative damage in vitro. We found that the 21-aminosteroid U74500A inhibited erythrocyte and plasma lipid peroxidation. U74500A at 1 microM significantly reduced copper-induced and hydrogen peroxide-induced erythrocyte lipid peroxidation by 76.5 and 27.6%, respectively. The inhibition of erythrocyte lipid peroxidation was accompanied by an inhibition of hemolysis. Copper-induced plasma lipid peroxidation was also significantly reduced by as little as 1 microM U74500A. These results suggest that 21-aminosteroids may prove useful in preventive or therapeutic interventions in situations where erythrocyte or plasma components are subjected to oxidative stress and in situations related to copper-induced oxidative damage. PMID:1425992

  19. Immunological identification of the human erythrocyte glucose transporter.

    PubMed Central

    Sogin, D C; Hinkle, P C

    1980-01-01

    A rabbit antibody against the human erythrocyte glucose transporter was purified by affinity chromatography and used to determine the distribution of transporter on polyacrylamide gels after electrophoresis in sodium dodecyl sulfate. Fresh erythrocyte ghosts showed transporter only at the broad 55,000 Mr band, as did the isolated transporter. HeLa cell plasma membranes showed a similar band of crossreacting material at Mr 55,000. The amount of crossreacting material in human erythrocyte ghosts and in plasma membranes from human HeLa cells and mouse L-1210 cells was determined in an enzyme-linked immunosorbent assay which gave results consistent with the extent of glucose-reversible binding of cytochalasin B. PMID:6934506

  20. Erythrocyte stiffness during morphological remodeling induced by carbon ion radiation.

    PubMed

    Zhang, Baoping; Liu, Bin; Zhang, Hong; Wang, Jizeng

    2014-01-01

    The adverse effect induced by carbon ion radiation (CIR) is still an unavoidable hazard to the treatment object. Thus, evaluation of its adverse effects on the body is a critical problem with respect to radiation therapy. We aimed to investigate the change between the configuration and mechanical properties of erythrocytes induced by radiation and found differences in both the configuration and the mechanical properties with involving in morphological remodeling process. Syrian hamsters were subjected to whole-body irradiation with carbon ion beams (1, 2, 4, and 6 Gy) or X-rays (2, 4, 6, and 12 Gy) for 3, 14 and 28 days. Erythrocytes in peripheral blood and bone marrow were collected for cytomorphological analysis. The mechanical properties of the erythrocytes were determined using atomic force microscopy, and the expression of the cytoskeletal protein spectrin-α1 was analyzed via western blotting. The results showed that dynamic changes were evident in erythrocytes exposed to different doses of carbon ion beams compared with X-rays and the control (0 Gy). The magnitude of impairment of the cell number and cellular morphology manifested the subtle variation according to the irradiation dose. In particular, the differences in the size, shape and mechanical properties of the erythrocytes were well exhibited. Furthermore, immunoblot data showed that the expression of the cytoskeletal protein spectrin-α1 was changed after irradiation, and there was a common pattern among its substantive characteristics in the irradiated group. Based on these findings, the present study concluded that CIR could induce a change in mechanical properties during morphological remodeling of erythrocytes. According to the unique characteristics of the biomechanical categories, we deduce that changes in cytomorphology and mechanical properties can be measured to evaluate the adverse effects generated by tumor radiotherapy. Additionally, for the first time, the current study provides a new

  1. Erythrocyte Stiffness during Morphological Remodeling Induced by Carbon Ion Radiation

    PubMed Central

    Zhang, Baoping; Liu, Bin; Zhang, Hong; Wang, Jizeng

    2014-01-01

    The adverse effect induced by carbon ion radiation (CIR) is still an unavoidable hazard to the treatment object. Thus, evaluation of its adverse effects on the body is a critical problem with respect to radiation therapy. We aimed to investigate the change between the configuration and mechanical properties of erythrocytes induced by radiation and found differences in both the configuration and the mechanical properties with involving in morphological remodeling process. Syrian hamsters were subjected to whole-body irradiation with carbon ion beams (1, 2, 4, and 6 Gy) or X-rays (2, 4, 6, and 12 Gy) for 3, 14 and 28 days. Erythrocytes in peripheral blood and bone marrow were collected for cytomorphological analysis. The mechanical properties of the erythrocytes were determined using atomic force microscopy, and the expression of the cytoskeletal protein spectrin-α1 was analyzed via western blotting. The results showed that dynamic changes were evident in erythrocytes exposed to different doses of carbon ion beams compared with X-rays and the control (0 Gy). The magnitude of impairment of the cell number and cellular morphology manifested the subtle variation according to the irradiation dose. In particular, the differences in the size, shape and mechanical properties of the erythrocytes were well exhibited. Furthermore, immunoblot data showed that the expression of the cytoskeletal protein spectrin-α1 was changed after irradiation, and there was a common pattern among its substantive characteristics in the irradiated group. Based on these findings, the present study concluded that CIR could induce a change in mechanical properties during morphological remodeling of erythrocytes. According to the unique characteristics of the biomechanical categories, we deduce that changes in cytomorphology and mechanical properties can be measured to evaluate the adverse effects generated by tumor radiotherapy. Additionally, for the first time, the current study provides a new

  2. Specific binding of beta-endorphin to normal human erythrocytes

    SciTech Connect

    Chenet, B.; Hollis, V. Jr.; Kang, Y.; Simpkins, C.

    1986-03-05

    Beta-endorphin (BE) exhibits peripheral functions which may not be mediated by interactions with receptors in the brain. Recent studies have demonstrated binding of BE to both opioid and non-opioid receptors on lymphocytes and monocytes. Abood has reported specific binding of /sup 3/H-dihydromorphine in erythrocytes. Using 5 x 10/sup -11/M /sup 125/I-beta-endorphin and 10/sup -5/M unlabeled BE, they have detected 50% specific binding to human erythrocytes. This finding is supported by results from immunoelectron microscopy using rabbit anti-BE antibody and biotinylated secondary antibody with avidin-biotin complexes horseradish peroxidase. Binding is clearly observed and is confined to only one side of the cells. Conclusions: (1) BE binding to human erythrocytes was demonstrated by radioreceptor assay and immunoelectron microscopy, and (2) BE binding sites exist on only one side of the cells.

  3. Retention of radiolead by human erythrocytes in vitro

    SciTech Connect

    Barton, J.C.

    1989-06-15

    An in vitro method was developed to assess human erythrocyte lead uptake and release directly, rapidly, and reproducibly; the technique requires small aliquots of blood and uses silicone fluid to separate erythrocytes from their suspending media. Uptake occurred rapidly and was directly related to temperature. Increasing quantities of available elemental lead were associated with increasing absolute quantities but decreasing percentages of uptake. Low values of pH diminished the uptake and enhanced the release of radiolead by erythrocytes, and could be correlated with diminished lead-hemoglobin binding para-Chloromecuribenzoate increased and dithiothreitol inhibited radiolead uptake but neither compound affected lead release, suggesting that sulfhydryl groups are important for lead binding to the erythrocyte. Cyanamide and N-ethylmaleimide did not significantly affect the net uptake or release of radiolead. Calcium disodium EDTA, penicillamine, and dimercaprol significantly reduced lead uptake, although only incubation with dimercaprol resulted in a net removal of lead from erythrocytes. Iron and ceruloplasmin significantly decreased radiolead uptake, but inorganic metal cations other than iron, hyperosmolarity, human serum albumin, cholesterol, and transferrin had no significant effect on uptake or release.

  4. Antioxidant status of erythrocytes and their response to oxidative challenge in humans with argemone oil poisoning

    SciTech Connect

    Babu, Challagundla K.; Khanna, Subhash K.; Das, Mukul

    2008-08-01

    Oxidative damage of biomolecules and antioxidant status in erythrocytes of humans from an outbreak of argemone oil (AO) poisoning in Kannauj (India) and AO intoxicated experimental animals was investigated. Erythrocytes of the dropsy patients and AO treated rats were found to be more susceptible to 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) induced peroxidative stress. Significant decrease in RBC glutathione (GSH) levels (46, 63%) with concomitant enhancement in oxidized glutathione (172, 154%) levels was noticed in patients and AO intoxicated animals. Further, depletion of glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G-6-PDH) and glutathione-S-transferase (GST) (42-52%) was observed in dropsy patients. Oxidation of erythrocyte membrane lipids and proteins was increased (120-144%) in patients and AO treated animals (112-137%) along with 8-OHdG levels in whole blood (180%) of dropsy patients. A significant reduction in {alpha}-tocopherol content (68%) was noticed in erythrocytes of dropsy patients and hepatic, plasma and RBCs of AO treated rats (59-70%) thereby indicating the diminished antioxidant potential to scavenge free radicals or the limited transport of {alpha}-tocopherol from liver to RBCs leading to enhanced oxidation of lipids and proteins in erythrocytes. These studies implicate an important role of erythrocyte degradation in production of anemia and breathlessness in epidemic dropsy.

  5. Effect of safeners on damage of human erythrocytes treated with chloroacetamide herbicides.

    PubMed

    Bernasinska, Joanna; Duchnowicz, Piotr; Koter-Michalak, Maria; Koceva-Chyla, Aneta

    2013-09-01

    Chloroacetamides are used as pre-emergent substances for growth control of annual grasses and weeds. Since they can be harmful for crop plants, protective compounds (safeners) are used along with herbicides. So far, their effects on human blood cells have not been evaluated, and this study is the very first one devoted to this subject. We examined the harmful effects of chloroacetamides, their metabolites and safeners, used alone or in combination with herbicides, on human erythrocytes measuring the extent of hemolysis, lipid peroxidation and catalase activity. Higher impact of herbicides than their metabolites on all of the investigated parameters was found. Safeners alone did not produce any damage to erythrocytes and did not elicit any changes in oxidative stress parameters. Combination of safener with herbicide did not attenuate hemolysis of erythrocytes compared to the herbicide alone. Safeners reduced lipid peroxidation induced by herbicides, which suggest the role of safeners as antioxidants. PMID:23732483

  6. In vitro protective effects of resveratrol against oxidative damage in human erythrocytes.

    PubMed

    Suwalsky, M; F Villena; Gallardo, M J

    2015-01-01

    Resveratrol (RV) is a potent antioxidant, anticancer and anti-inflammatory agent. Its main target of action is the cell membrane; however, its effect on that of human erythrocytes has been scarcely investigated. With the aim to better understand the molecular mechanisms of the interaction of RV with cell membranes both human erythrocytes and molecular models of its membrane have been utilized. The latter consisted in bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Results by X-ray diffraction showed that RV produced a significant structural perturbation on DMPC bilayers, but no effects were observed in DMPE. Scanning electron (SEM) and defocusing microscopy (DM) observations showed that RV induced morphological alterations to the red cells from the normal discoid shape to echinocytes. These results imply that RV was located in the outer monolayer of the erythrocyte membrane. Results of its antioxidant properties showed that RV neutralized the oxidative capacity of HClO on DMPC and DMPE bilayers. On the other hand, SEM and DM observations as well as hemolysis assays demonstrated the protective effect of RV against the deleterious effects of HClO upon human erythrocytes. PMID:25268679

  7. Antioxidant capacity of Ugni molinae fruit extract on human erythrocytes: an in vitro study.

    PubMed

    Suwalsky, Mario; Avello, Marcia

    2014-08-01

    Ugni molinae is an important source of molecules with strong antioxidant activity widely used as a medicinal plant in Southern Chile-Argentina. Total phenol concentration from its fruit extract was 10.64 ± 0.04 mM gallic acid equivalents. Analysis by means of HPLC/MS indicated the presence of the anthocyanins cyanidin and peonidin, and the flavonol quercitin, all in glycosylated forms. Its antioxidant properties were assessed in human erythrocytes in vitro exposed to HClO oxidative stress. Scanning electron microscopy showed that HClO induced an alteration in erythrocytes from a normal shape to echinocytes; however, this change was highly attenuated in samples containing U. molinae extracts. It also had a tendency in order to reduce the hemolytic effect of HClO. In addition, X-ray diffraction experiments were performed in dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine bilayers, classes of lipids preferentially located in the outer and inner monolayers, respectively, of the human erythrocyte membrane. It was observed that U. molinae only interacted with DMPC. Results by fluorescence spectroscopy on DMPC large unilamellar vesicles and isolated unsealed human erythrocyte membranes also showed that it interacted with the erythrocyte membrane and DMPC. It is possible that the location of U. molinae components into the membrane outer monolayer might hinder the diffusion of HClO and of free radicals into cell membranes and the consequent decrease of the kinetics of free radical reactions. PMID:24928227

  8. Human erythrocytes are affected in vitro by flavonoids of Aristotelia chilensis (Maqui) leaves.

    PubMed

    Suwalsky, Mario; Vargas, Pedro; Avello, Marcia; Villena, Fernando; Sotomayor, Carlos P

    2008-11-01

    Aristotelia chilensis (Mol.) Stuntz (A. chilensis), also known as maqui, is a plant of the Elaeocarpaceae family that grows in central and southern Chile as well as southwestern Argentina. Infusions of its leaves have long been used in the traditional native herbal medicine to treat different ailments. Phytochemical studies of the plant's chemical composition of the plant indicate the presence of indolic alkaloids, flavonoids, cianidine glucosides, delfidine, malvidine, petunidine, cumarines and triterpenes. These compounds, particularly the flavonoids, have antioxidant properties. In order to evaluate the mechanisms of its toxicity and their antioxidant properties, the leaves' aqueous extracts were induced to interact with human red cells, their isolated unsealed membranes (IUM), and molecular models of the human erythrocyte membrane. These consisted of multibilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), representative of phospholipids classes located in the outer and inner monolayers of the human erythrocyte membrane, and large unilamellar vesicles (LUV) of DMPC. The capacity of A. chilensis aqueous extracts to perturb the bilayer structure of DMPC and DMPE was evaluated by X-ray diffraction, DMPC LUV and IUM were studied by fluorescence spectroscopy, and intact human erythrocytes were observed by scanning electron microscopy (SEM). Results of the present study indicate that aqueous extracts of A. chilensis induced an alteration of human erythrocyte morphology from the normal discoid shape to an echinocytic form, changes that are explained in terms of the extract interaction with the membrane's outer phospholipid monolayer. PMID:18687390

  9. Effects of airborne dust collected from Kuwait on human erythrocytes

    SciTech Connect

    Siddiqui, S.M.; Khan, S.A.; Ahmad, S.; Beg, M.U.

    1992-01-01

    Air borne dust as deposited on air conditioner's filter was collected from most polluted regions of Kuwait and for comparison also from Dubai. Kuwait dust samples were found to contain high concentrations of Ni, Mn and Pb and a number of organic compounds different from the oil samples collected from the oil pool in the oil fields. Toxicity evaluation against human erythrocytes showed strong hemolytic nature of the dust. Treatment of erythrocytes with the dust exhibited peroxidative damage of the membrane. The dust collected from Dubai was innocuous. The present data suggest that erythrocyte damaging potential of the dust can be used as a marker of toxicity and provide information about the dissipation of toxic factors from airborne dust with time. 15 refs., 2 figs., 3 tabs.

  10. Determination of somatic mutations in human erythrocytes by cytometry

    SciTech Connect

    Jensen, R.H.; Langlois, R.G.; Bigbee, W.L.

    1985-06-21

    Flow cytometric assays of human erythrocytes labeled with monoclonal antibodies specific for glycophorin A were used to enumerate variant cells that appear in peripheral blood as a result of somatic gene-loss mutations in erythrocyte precursor cells. The assay was performed on erythrocytes from 10 oncology patients who had received at least one treatment from radiation or mutagenic chemotherapy at least 3 weeks before being assayed. The patients were suffering from many different malignancies (e.g., breast, renal, bone, colon and lung), and were treated with several different mutagenic therapeutics (e.g., cisplatinum, adriamycin, daunomycin, or cyclophosphamide). The frequency of these variant cells is an indication of the amount of mutagenic damage accumulated in the individual's erythropoietic cell population. Comparing these results to HPRT clonogenic assays, we find similar baseline frequencies of somatic mutation as well as similar correlation with mutagenic exposures. 9 refs., 3 figs., 1 tab.

  11. Robust inducible Cre recombinase activity in the human malaria parasite Plasmodium falciparum enables efficient gene deletion within a single asexual erythrocytic growth cycle.

    PubMed

    Collins, Christine R; Das, Sujaan; Wong, Eleanor H; Andenmatten, Nicole; Stallmach, Robert; Hackett, Fiona; Herman, Jean-Paul; Müller, Sylke; Meissner, Markus; Blackman, Michael J

    2013-05-01

    Asexual blood stages of the malaria parasite, which cause all the pathology associated with malaria, can readily be genetically modified by homologous recombination, enabling the functional study of parasite genes that are not essential in this part of the life cycle. However, no widely applicable method for conditional mutagenesis of essential asexual blood-stage malarial genes is available, hindering their functional analysis. We report the application of the DiCre conditional recombinase system to Plasmodium falciparum, the causative agent of the most dangerous form of malaria. We show that DiCre can be used to obtain rapid, highly regulated site-specific recombination in P. falciparum, capable of excising loxP-flanked sequences from a genomic locus with close to 100% efficiency within the time-span of a single erythrocytic growth cycle. DiCre-mediated deletion of the SERA5 3' UTR failed to reduce expression of the gene due to the existence of alternative cryptic polyadenylation sites within the modified locus. However, we successfully used the system to recycle the most widely used drug resistance marker for P. falciparum, human dihydrofolate reductase, in the process producing constitutively DiCre-expressing P. falciparum clones that have broad utility for the functional analysis of essential asexual blood-stage parasite genes. PMID:23489321

  12. Ultraviolet irradiation induces autofluorescence enhancement via production of reactive oxygen species and photodecomposition in erythrocytes

    SciTech Connect

    Wu, Xian; Pan, Leiting; Wang, Zhenhua; Liu, Xiaoli; Zhao, Dan; Zhang, Xinzheng; Rupp, Romano A.; Xu, Jingjun

    2010-06-11

    Ultraviolet (UV) light has a significant influence on human health. In this study, human erythrocytes were exposed to UV light to investigate the effects of UV irradiation (UVI) on autofluorescence. Our results showed that high-dose continuous UVI enhanced erythrocyte autofluorescence, whereas low-dose pulsed UVI alone did not have this effect. Further, we found that H{sub 2}O{sub 2}, one type of reactive oxygen species (ROS), accelerated autofluorescence enhancement under both continuous and pulsed UVI. In contrast, continuous and pulsed visible light did not result in erythrocyte autofluorescence enhancement in the presence or absence of H{sub 2}O{sub 2}. Moreover, NAD(P)H had little effect on UVI-induced autofluorescence enhancement. From these studies, we conclude that UVI-induced erythrocyte autofluorescence enhancement via both UVI-dependent ROS production and photodecomposition. Finally, we present a theoretical study of this autofluorescence enhancement using a rate equation model. Notably, the results of this theoretical simulation agree well with the experimental data further supporting our conclusion that UVI plays two roles in the autofluorescence enhancement process.

  13. Is Peroxiredoxin II's peroxidase activity strongly inhibited in human erythrocytes?

    PubMed

    Benfeitas, Rui; Selvaggio, Gianluca; Antunes, Fernando; Coelho, Pedro; Salvador, Armindo

    2014-10-01

    H2O2 elimination in human erythrocytes is mainly carried out by catalase (Cat), glutathione peroxidase (GPx1) and the more recently discovered peroxiredoxin 2 (Prx2). However, the contribution of Prx2 to H2O2 consumption is still unclear. Prx2's high reactivity with H2O2 (kPrx2=10×10(7) M(-1)s(-1), kCat =7×10(7) M(-1)s(-1), kGPx1 =4×10(7) M(-1)s(-1)) and high abundance ([Prx2]= 570µM, [Cat]= 32µM, [GPx1]= 1µM) suggest that under low H2O2 supply rates it should consume >99% of the H2O2. However, extensive evidence indicates that in intact erythrocytes Prx2 contributes no more than Cat to H2O2 consumption. In order for this to be attained, Prx2's effective rate constant with H2O2would have to be just ~10(5) M(-1)s(-1), much lower than that determined in multiple experiments with the purified proteins. Nevertheless, nearly all Prx2 is oxidized within 1min of exposing erythrocytes to a H2O2 bolus, which is inconsistent with an irreversible inhibition. A mathematical model of the H2O2 metabolism in human erythrocytes [Benfeitas et al. (2014) Free Radic. Biol. Med.] where Prx2 either has a low kPrx2 or is subject to a strong (>99%) but readily reversible inhibition achieves quantitative agreement with detailed experimental observations of the responses of the redox status of Prx2 in human erythrocytes and suggests functional advantages of this design (see companion abstract). By contrast, a variant where Prx2 is fully active with kPrx2=10(8) M(-1)s(-1) shows important qualitative discrepancies. Altogether, these results suggest that Prx2's peroxidase activity is strongly inhibited in human erythrocytes. We acknowledge fellowship SFRH/BD/51199/2010, grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, PEst-OE/QUI/UI0313/2014, and FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) co-financed by FEDER through the COMPETE program and by FCT. PMID:26461310

  14. Evaluation of the effect of Uncaria tomentosa extracts on the size and shape of human erythrocytes (in vitro).

    PubMed

    Bors, Milena; Sicińska, Paulina; Michałowicz, Jaromir; Wieteska, Paulina; Gulewicz, Krzysztof; Bukowska, Bożena

    2012-03-01

    In this study, we continued our investigations concerning the interaction of Uncaria tomentosa extracts with the human erythrocytes. The analysis of the size and shape of the erythrocytes by means of flow cytometry and phase contrast microscopy was performed. We executed our experiments using ethanolic and aqueous extracts from the leaves and bark of U. tomentosa. Disturbances were observed in the size and shape of the erythrocytes incubated with ethanolic and aqueous extracts at the concentrations of 100 μg/mL and 250 μg/mL, respectively. The observed changes were probably related to the entry of polyphenolic compounds contained in U. tomentosa extracts into erythrocyte membrane. Externalization of phosphatidylserine on the erythrocytic surfaces was also noticed during incubation with extracts at concentration of 250 μg/mL. We concluded that all of the extracts examined induced changes in the erythrocyte membrane properties, whereas ethanolic extracts from bark induced the most significant changes. The possible binding of polyphenols to the erythrocyte surface may have accounted for the protective properties of extracts against haemolysis of RBCs, which was observed in our previous study (Bors et al., 2011), but considerable incorporation of polyphenols into cell membranes can result in disturbance of phosphatidylserine transport and changes in erythrocyte shape. Nevertheless the results of the investigations showed that considerable morphological changes appear only as a result of erythrocyte exposure to high concentrations (50 ppm and 100 ppm) of the extracts studied, thus they should not lead to clinical erythrocytic damage if recommended doses of U. tomentosa preparations are administrated. PMID:22217608

  15. Dimethyl sulfoxide at high concentrations inhibits non-selective cation channels in human erythrocytes.

    PubMed

    Nardid, Oleg A; Schetinskey, Miroslav I; Kucherenko, Yuliya V

    2013-03-01

    Dimethyl sulfoxide (DMSO), a by-product of the pulping industry, is widely used in biological research, cryobiology and medicine. On cellular level DMSO was shown to suppress NMDA-AMPA channels activation, blocks Na+ channel activation and attenuates Ca2+ influx (Lu and Mattson 2001). In the present study we explored the whole-cell patch-clamp to examine the acute effect of high concentrations of DMSO (0.1-2 mol/l) on cation channels activity in human erythrocytes. Acute application of DMSO (0.1-2 mol/l) dissolved in Cl--containing saline buffer solution significantly inhibited cation conductance in human erythrocytes. Inhibition was concentration-dependent and had an exponential decay profile. DMSO (2 mol/l) induced cation inhibition in Cl-- containing saline solutions of: 40.3 ± 3.9% for K+, 35.4 ± 3.1% for Ca2+ and 47.4 ± 1.9% for NMDG+. Substitution of Cl- with gluconate- increased the inhibitory effect of DMSO on the Na+ current. Inhibitory effect of DMSO was neither due to high permeability of erythrocytes to DMSO nor to an increased tonicity of the bath media since no effect was observed in 2 mol/l glycerol solution. In conclusion, we have shown that high concentrations of DMSO inhibit the non-selective cation channels in human erythrocytes and thus protect the cells against Na+ and Ca2+ overload. Possible mechanisms of DMSO effect on cation conductance are discussed. PMID:23531832

  16. Stoichiometry of compounds bound to human erythrocytes in relation to morphology.

    PubMed

    Lovrien, R; Tisel, W; Pesheck, P

    1975-04-25

    Most work on human erythrocyte interaction with drugs and other compounds has been reported on the basis of total concentrations. Total concentrations alone do not reveal numbers of molecules bound per cell, v. This paper emphasizes determination of v and of binding isotherms, in conjunction with changes in cell morphologies and in hypotonic shock behavior as v is varied. Four drugs and five other compounds were studied, with fresh erythrocytes. The principal findings are: (1) the intact erythrocyte engages in two kinds of binding mechanisms, statistical binding and cooperative binding, depending on the compound. In the case of a detergent, dodecylbenzene sulfonate, the binding is nearly quantitative. (2) The compounds often induce considerable protection against hypotonic hemolysis. However, the binding levels at which maximum protection occurs are rather close to the levels, vL, that occur upon complete conversion to the first distorted morphology. Therefore, the maximally protected erythrocyte may be a distorted erythrocyte. (3) The value n is the apparent total number of sites from Scatchard plotting for compounds which bind in a statistical manner. Levels vp and vw characterize maxima in cooperative binding behavior, also from Scatchard plotting of the data. Despite the wide diversity of over-all levels at which compounds exert their effects, the critical binding levels of and numbers of sites fall into a narrow range:n, vL, vP, and vw are all between 1 and 8 times 10-7 molecules or sites per cell. Most of our data, and that from some other laboratories, indicate that about 2 plus and minus 1 times 10-7 sites per erythrocyte are available for compound binding by the intact cell. Beyond that level, the cell in suspension almost always will be forced into the first obvious morphology change, as seen by phase contrast microscopy. (4) Once stoichiometries are established, the total binding capacity of erythrocytes for such compounds, in blood, can be estimated. An

  17. Piperlongumine-Induced Phosphatidylserine Translocation in the Erythrocyte Membrane

    PubMed Central

    Bissinger, Rosi; Malik, Abaid; Warsi, Jamshed; Jilani, Kashif; Lang, Florian

    2014-01-01

    Background: Piperlongumine, a component of Piper longum fruit, is considered as a treatment for malignancy. It is effective by inducing apoptosis. Mechanisms involved in the apoptotic action of piperlongumine include oxidative stress and activation of p38 kinase. In analogy to apoptosis of nucleated cells, erythrocytes may undergo eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i), formation of ceramide, oxidative stress and activation of p38 kinase. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3 fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to piperlongumine (30 µM) was followed by significant decrease of forward scatter and increase of annexin-V-binding. Piperlongumine did not significantly modify [Ca2+]i and the effect was not dependent on presence of extracellular Ca2+. Piperlongumine significantly increased ROS formation and ceramide abundance. Conclusions: Piperlongumine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca2+ but at least partially due to ROS and ceramide formation. PMID:25317837

  18. A comparison of erythrocyte glutathione S-transferase activity from human foetuses and adults.

    PubMed Central

    Strange, R C; Johnston, J D; Coghill, D R; Hume, R

    1980-01-01

    Glutathione S-transferase activity was measured in partially purified haemolysates of erythrocytes from human foetuses and adults. Enzyme activity was present in erythrocytes obtained between 12 and 40 weeks of gestation. The catalytic properties of the enzyme from foetal cells were similar to those of the enzyme from adult erythrocytes, indicating that probably only one form of the erythrocytes enzyme exists throughout foetal and adult life. PMID:7396875

  19. Abscisic Acid Transport in Human Erythrocytes*

    PubMed Central

    Vigliarolo, Tiziana; Guida, Lucrezia; Millo, Enrico; Fresia, Chiara; Turco, Emilia; De Flora, Antonio; Zocchi, Elena

    2015-01-01

    Abscisic acid (ABA) is a plant hormone involved in the response to environmental stress. Recently, ABA has been shown to be present and active also in mammals, where it stimulates the functional activity of innate immune cells, of mesenchymal and hemopoietic stem cells, and insulin-releasing pancreatic β-cells. LANCL2, the ABA receptor in mammalian cells, is a peripheral membrane protein that localizes at the intracellular side of the plasma membrane. Here we investigated the mechanism enabling ABA transport across the plasmamembrane of human red blood cells (RBC). Both influx and efflux of [3H]ABA occur across intact RBC, as detected by radiometric and chromatographic methods. ABA binds specifically to Band 3 (the RBC anion transporter), as determined by labeling of RBC membranes with biotinylated ABA. Proteoliposomes reconstituted with human purified Band 3 transport [3H]ABA and [35S]sulfate, and ABA transport is sensitive to the specific Band 3 inhibitor 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. Once inside RBC, ABA stimulates ATP release through the LANCL2-mediated activation of adenylate cyclase. As ATP released from RBC is known to exert a vasodilator response, these results suggest a role for plasma ABA in the regulation of vascular tone. PMID:25847240

  20. Attempts to validate a possible predictive animal model for human erythrocyte G-6-PD deficiency

    SciTech Connect

    Horton, H.M.; Calabrese, E.J.

    1986-01-01

    The use of Dorset sheep erythrocytes as a model for human G-6-PD deficient erythrocytes was investigated. Seven pharmaceuticals were examined for oxidant stressor effects using a liver microsomal enzyme system to generate metabolites of the drugs. The pharmaceuticals examined were salicyclic acid, dapsone, naphthalene, B-naphtol, p-aminobenzoic acid, sulfanilamide and sulfapyridine. The test compounds were incubated with Dorset sheep erythrocytes and oxidant stressor effects were measured through reduced glutathione (GSH) levels and methemaglobin formation. The response of the Dorset sheep erythrocytes to the seven agents was compared to previous studies revealing the response of human G-6-PD deficient erythrocytes to these agents. The results indicated that metabolites of the pharmaceuticals, B-naphthol, dapsone, and sulfanilamide, are oxidant stressor agents towards sheep G-6-PD deficient erythrocytes. These results agreed with studies on the response of human G-6-PD deficient erythrocytes. The metabolized naphthalene and sulfapyridine did not cause oxidant stress in the sheep erythrocytes, despite the fact that these two agents caused oxidizing effects in human G-6-PD deficient erythrocytes in previous studies. None of the non-metabolized parent compounds caused oxidant stress in the sheep erythrocytes, which agreed with the responses of human G-6-PD deficient erythrocytes.

  1. 7Li NMR study of normal human erythrocytes

    NASA Astrophysics Data System (ADS)

    Pettegrew, J. W.; Post, J. F. M.; Panchalingam, K.; Withers, G.; Woessner, D. E.

    The biological action of lithium is of great interest because of the therapeutic efficacy of the cation in manic-depressive illness. To investigate possible molecular interactions of lithium, 7Li NMR studies were conducted on normal human erythrocytes which had been incubated with lithium chloride. The uptake of lithium ions was followed by 7Li NMR, using a dysprosium, tripolyphosphate shift reagent. Lithium uptake followed single-exponential kinetics with a time constant of 14.7 h. The intracellular lithium relaxation times were T 1 ⋍ 5 s and T 2 ⋍ 0.15 s, which implies a lengthening of the lithium correlation time. It was found that lithium does not interact significantly with hemoglobin, the erythrocyte membrane, or artificial phospholipid membranes. Based on measurements of lithium T1 and T2 in concentrated agar gels, the large difference between T1 and T2 for intracellular lithium ions may be due to diffusion of the hydrated lithium ion through heterogeneous electrostatic field gradients created by the erythrocyte membrane-associated cytoskeletal network. Lithium binding to the membrane-associated cytoskeleton, however, cannot be ruled out. Because of the large differences between T1 and T2 of intracellular lithium ions, 1Li NMR may be a sensitive and promising noninvasive method to probe the intracellular environment.

  2. The free heme concentration in healthy human erythrocytes

    PubMed Central

    Aich, Anupam; Freundlich, Melissa; Vekilov, Peter G.

    2016-01-01

    Heme, the prosthetic group of hemoglobin, may be released from its host due to an intrinsic instability of hemoglobin and accumulate in the erythrocytes. Free heme is in the form of hematin (Fe3+ protoporphyrin IX OH) and follows several pathways of biochemical toxicity to tissues, cells, and organelles since it catalyzes the production of reactive oxygen species. To determine concentration of soluble free heme in human erythrocytes, we develop a new method. We lyse the red blood cells and isolate free heme from hemoglobin by dialysis. We use the heme to reconstitute horseradish peroxidase (HRP) from an excess of the apoenzyme and determine the HRP reaction rate from the evolution of the emitted luminescence. We find that in a population of five healthy adults the average free heme concentration in the erythrocytes is 21 ± 2 μM, ca. 100× higher than previously determined. Tests suggest that the lower previous value was due to the use of elevated concentrations of NaCl, which drive hematin precipitation and re-association with apoglobin. We show that the found hematin concentration is significantly higher than estimates based on equilibrium release and the known hematin dimerization. The factors that lead to enhanced heme release remain an open question. PMID:26460266

  3. Transport of 3-bromopyruvate across the human erythrocyte membrane.

    PubMed

    Sadowska-Bartosz, Izabela; Soszyński, Mirosław; Ułaszewski, Stanisław; Ko, Young; Bartosz, Grzegorz

    2014-06-01

    3-Bromopyruvic acid (3-BP) is a promising anticancer compound because it is a strong inhibitor of glycolytic enzymes, especially glyceraldehyde 3-phosphate dehydrogenase. The Warburg effect means that malignant cells are much more dependent on glycolysis than normal cells. Potential complications of anticancer therapy with 3-BP are side effects due to its interaction with normal cells, especially erythrocytes. Transport into cells is critical for 3-BP to have intracellular effects. The aim of our study was the kinetic characterization of 3-BP transport into human erythrocytes. 3-BP uptake by erythrocytes was linear within the first 3 min and pH-dependent. The transport rate decreased with increasing pH in the range of 6.0-8.0. The Km and Vm values for 3-BP transport were 0.89 mM and 0.94 mmol/(l cells x min), respectively. The transport was inhibited competitively by pyruvate and significantly inhibited by DIDS, SITS, and 1-cyano-4-hydroxycinnamic acid. Flavonoids also inhibited 3-BP transport: the most potent inhibition was found for luteolin and quercetin. PMID:24715475

  4. Agglutination of human erythrocytes by the interaction of Zn(2+)ion with histidine-651 on the extracellular domain of band 3.

    PubMed

    Kiyotake, Kento; Ochiai, Hideharu; Yamaguchi, Takeo

    2016-05-01

    Clustering of band 3, chloride/bicarbonate exchanger, has been reported in Zn(2+)-treated human erythrocytes. However, the agglutination of human erythrocytes is also induced by the interaction of Zn(2+)ion with histidine on band 3. Identification of histidine that interacts with Zn(2+)ion remains to be determined. The Zn(2+)-induced agglutination of human erythrocytes was unaffected by chymotrypsin cleavage of the small loop region containing His-547 in the extracellular domain of band 3. On the other hand, papain digestion of the large loop region containing His-651 in band 3 inhibited such Zn(2+)-induced agglutination. Moreover, Zn(2+)-induced erythrocyte agglutination was inhibited by the peptide (ARGWVIHPLG) containing His-651, but not by the peptide such as ARGWVIRPLG, which His-651 was substituted by arginine. Among 10 kinds of animal erythrocytes tested, interestingly, no agglutination by Zn(2+)ions was observed in cow cells only that the forth amino acid in the upstream from His-669 on the large loop of cow band 3 is aspartate (Asp-665) instead of glycine. As expected, the agglutination of human erythrocytes by Zn(2+) ions was inhibited in the presence of aspartate. These data indicate that the interaction of Zn(2+) ion with His-651 residue of band 3 plays an important role in the Zn(2+)-induced agglutination of human erythrocytes. PMID:26859120

  5. Tyrosine phosphorylation of band 3 protein in Ca2+/A23187-treated human erythrocytes.

    PubMed Central

    Minetti, G; Piccinini, G; Balduini, C; Seppi, C; Brovelli, A

    1996-01-01

    Human erythrocytes were induced to release membrane vesicles by treatment with Ca2+ and ionophore A23187. In addition to the biochemical changes already known to accompany loading of human erythrocytes with Ca2+, the present study reveals that tyrosine phosphorylation of the anion exchanger band 3 protein also occurs. The relationship between tyrosine phosphorylation of band 3 and membrane vesiculation was analysed using quinine (a non-specific inhibitor of the Ca(2+)-activated K+ channel, and the only known inhibitor of Ca(2+)-induced vesiculation) and charybdotoxin, a specific inhibitor of the apamin-insensitive K(+)-channel. Both inhibitors suppressed tyrosine phosphorylation of band 3. In the presence of quinine, membrane vesiculation was also suppressed. In contrast, at the concentration of charybdotoxin required to suppress tyrosine phosphorylation of band 3, membrane vesiculation was only mildly inhibited (16-23% inhibition), suggesting that tyrosine phosphorylation of band 3 is not necessary for membrane vesiculation. Phosphorylation of band 3 was in fact observed when erythrocytes were induced to shrink in a Ca(2+)-independent manner, e.g. by treatment with the K+ ionophore valinomycin or with hypertonic solutions. These observations suggest that band 3 tyrosine phosphorylation occurs when cell volume regulation is required. PMID:8973551

  6. Phosphatidylserine externalization and procoagulant activation of erythrocytes induced by Pseudomonas aeruginosa virulence factor pyocyanin.

    PubMed

    Qadri, Syed M; Donkor, David A; Bhakta, Varsha; Eltringham-Smith, Louise J; Dwivedi, Dhruva J; Moore, Jane C; Pepler, Laura; Ivetic, Nikola; Nazi, Ishac; Fox-Robichaud, Alison E; Liaw, Patricia C; Sheffield, William P

    2016-04-01

    The opportunistic pathogen Pseudomonas aeruginosa causes a wide range of infections in multiple hosts by releasing an arsenal of virulence factors such as pyocyanin. Despite numerous reports on the pleiotropic cellular targets of pyocyanin toxicity in vivo, its impact on erythrocytes remains elusive. Erythrocytes undergo an apoptosis-like cell death called eryptosis which is characterized by cell shrinkage and phosphatidylserine (PS) externalization; this process confers a procoagulant phenotype on erythrocytes as well as fosters their phagocytosis and subsequent clearance from the circulation. Herein, we demonstrate that P. aeruginosa pyocyanin-elicited PS exposure and cell shrinkage in erythrocyte while preserving the membrane integrity. Mechanistically, exposure of erythrocytes to pyocyanin showed increased cytosolic Ca(2+) activity as well as Ca(2+) -dependent proteolytic processing of μ-calpain. Pyocyanin further up-regulated erythrocyte ceramide abundance and triggered the production of reactive oxygen species. Pyocyanin-induced increased PS externalization in erythrocytes translated into enhanced prothrombin activation and fibrin generation in plasma. As judged by carboxyfluorescein succinimidyl-ester labelling, pyocyanin-treated erythrocytes were cleared faster from the murine circulation as compared to untreated erythrocytes. Furthermore, erythrocytes incubated in plasma from patients with P. aeruginosa sepsis showed increased PS exposure as compared to erythrocytes incubated in plasma from healthy donors. In conclusion, the present study discloses the eryptosis-inducing effect of the virulence factor pyocyanin, thereby shedding light on a potentially important mechanism in the systemic complications of P. aeruginosa infection. PMID:26781477

  7. An in vitro study on the antioxidant capacity of usnic acid on human erythrocytes and molecular models of its membrane.

    PubMed

    Suwalsky, M; Jemiola-Rzeminska, M; Astudillo, C; Gallardo, M J; Staforelli, J P; Villena, F; Strzalka, K

    2015-11-01

    Usnic acid (UA) has been associated with chronic diseases through its antioxidant action. Its main target is the cell membrane; however, its effect on that of human erythrocytes has been scarcely investigated. To gain insight into the molecular mechanisms of the interaction between UA and cell membranes human erythrocytes and molecular models of its membrane have been utilized. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. Results by X-ray diffraction showed that UA produced structural perturbations on DMPC and DMPE bilayers. DSC studies have indicated that thermotropic behavior of DMPE was most strongly distorted by UA than DMPC, whereas the latter is mainly affected on the pretransition. Scanning electron (SEM) and defocusing microscopy (DM) showed that UA induced alterations to erythrocytes from the normal discoid shape to echinocytes. These results imply that UA molecules were located in the outer monolayer of the erythrocyte membrane. Results of its antioxidant properties showed that UA neutralized the oxidative capacity of HClO on DMPC and DMPE bilayers; SEM, DM and hemolysis assays demonstrated the protective effect of UA against the deleterious oxidant effects of HClO upon human erythrocytes. PMID:26299817

  8. Decrease in erythrocyte glycophorin sialic acid content is associated with increased erythrocyte aggregation in human diabetes.

    PubMed

    Rogers, M E; Williams, D T; Niththyananthan, R; Rampling, M W; Heslop, K E; Johnston, D G

    1992-03-01

    1. Sialic acid moieties of erythrocyte membrane glycoproteins are the principal determinants of the negative charge on the cell surface. The resultant electrostatic repulsion between the cells reduces erythrocyte aggregation and hence the low shear rate viscosity and yield stress of blood. 2. Using g.c.-m.s., a decrease in sialic acid content has been observed in the major erythrocyte membrane glycoprotein, glycophorin A, obtained from nine diabetic patients compared with that from seven normal control subjects [median (range): 3.30 (0.01-11.90) versus 18.60 (3.20-32.60) micrograms/100 micrograms of protein, P less than 0.02]. 3. Erythrocyte aggregation, measured by viscometry as the ratio of suspension viscosity to supernatant viscosity (LS/S) in fibrinogen solution, was increased in ten diabetic patients compared with ten normal control subjects (mean +/- SEM, 37.6 +/- 1.3 versus 33.8 +/- 0.6, P less than 0.02). 4. In the patients in whom both viscometry and carbohydrate analysis were performed, the decrease in erythrocyte glycophorin sialylation and the increase in erythrocyte aggregation in fibrinogen solution were related statistically (LS/S correlated negatively with glycophorin sialic acid content, r = 0.73, P less than 0.05). 5. Decreased glycophorin sialylation provides an explanation at the molecular level for increased erythrocyte aggregation and it may be important in the pathogenesis of vascular disease in diabetes. PMID:1312416

  9. Evaluation of hemagglutination activity of chitosan nanoparticles using human erythrocytes.

    PubMed

    de Lima, Jefferson Muniz; Sarmento, Ronaldo Rodrigues; de Souza, Joelma Rodrigues; Brayner, Fábio André; Feitosa, Ana Paula Sampaio; Padilha, Rafael; Alves, Luiz Carlos; Porto, Isaque Jerônimo; Batista, Roberta Ferreti Bonan Dantas; de Oliveira, Juliano Elvis; de Medeiros, Eliton Souto; Bonan, Paulo Rogério Ferreti; Castellano, Lúcio Roberto

    2015-01-01

    Chitosan is a polysaccharide composed of randomly distributed chains of β-(1-4) D-glucosamine and N-acetyl-D-glucosamine. This compound is obtained by partial or total deacetylation of chitin in acidic solution. The chitosan-based hemostatic agents have been gaining much attention in the management of bleeding. The aim of this study was to evaluate in vitro hemagglutination activity of chitosan nanoparticles using human erythrocytes. The preparation of nanoparticles was achieved by ionotropic gelification technique followed by neutralization with NaOH 1 mol/L(-1). The hemagglutination activity was performed on a solution of 2% erythrocytes (pH 7.4 on PBS) collected from five healthy volunteers. The hemolysis determination was made by spectrophotometric analysis. Chitosan nanoparticle solutions without NaOH addition changed the reddish colour of the wells into brown, suggesting an oxidative reaction of hemoglobin and possible cell lysis. All neutralized solutions of chitosan nanoparticles presented positive haemagglutination, without any change in reaction color. Chitosan nanoparticles presented hemolytic activity ranging from 186.20 to 223.12%, while neutralized solutions ranged from 2.56 to 72.54%, comparing to distilled water. Results highlight the need for development of new routes of synthesis of chitosan nanoparticles within human physiologic pH. PMID:25759815

  10. Evaluation of Hemagglutination Activity of Chitosan Nanoparticles Using Human Erythrocytes

    PubMed Central

    de Lima, Jefferson Muniz; Sarmento, Ronaldo Rodrigues; de Souza, Joelma Rodrigues; Brayner, Fábio André; Feitosa, Ana Paula Sampaio; Padilha, Rafael; Alves, Luiz Carlos; Porto, Isaque Jerônimo; Batista, Roberta Ferreti Bonan Dantas; de Oliveira, Juliano Elvis; de Medeiros, Eliton Souto; Bonan, Paulo Rogério Ferreti; Castellano, Lúcio Roberto

    2015-01-01

    Chitosan is a polysaccharide composed of randomly distributed chains of β-(1-4) D-glucosamine and N-acetyl-D-glucosamine. This compound is obtained by partial or total deacetylation of chitin in acidic solution. The chitosan-based hemostatic agents have been gaining much attention in the management of bleeding. The aim of this study was to evaluate in vitro hemagglutination activity of chitosan nanoparticles using human erythrocytes. The preparation of nanoparticles was achieved by ionotropic gelification technique followed by neutralization with NaOH 1 mol/L−1. The hemagglutination activity was performed on a solution of 2% erythrocytes (pH 7.4 on PBS) collected from five healthy volunteers. The hemolysis determination was made by spectrophotometric analysis. Chitosan nanoparticle solutions without NaOH addition changed the reddish colour of the wells into brown, suggesting an oxidative reaction of hemoglobin and possible cell lysis. All neutralized solutions of chitosan nanoparticles presented positive haemagglutination, without any change in reaction color. Chitosan nanoparticles presented hemolytic activity ranging from 186.20 to 223.12%, while neutralized solutions ranged from 2.56 to 72.54%, comparing to distilled water. Results highlight the need for development of new routes of synthesis of chitosan nanoparticles within human physiologic pH. PMID:25759815

  11. The osmotic response of human erythrocytes and the membrane cytoskeleton.

    PubMed

    Heubusch, P; Jung, C Y; Green, F A

    1985-02-01

    The volumes of human erythrocytes suspended in solutions of varying concentrations of sodium chloride and sucrose were measured by a Coulter Channelyzer Model H4 with appropriate corrections. The cells showed greatly restricted volume changes at osmolarities between 200-700 mOsm. At osmolarities outside this limit, on the other hand, the cells showed nonrestricted volume changes following essentially the predictions of an ideal osmometer. This unexpected volume response was not spuriously due to changes in shape or to a changing orientation of the cells as they traversed the aperture. The restricted volume change observed was abolished when the cells had previously been treated with diamide or had been heated for 60 minutes at 50 degrees C, conditions that are known to disturb the spectrin-actin network. The possibility must be considered that the osmotic behavior of human erythrocytes may be nonideal and that this nonideal behavior is primarily due to mechanical restriction provided by the spectrin-actin network of the membrane cytoskeleton. PMID:3918046

  12. The Protective Effect of Epigallocatechin-3-gallate on Paraquat-induced Haemolysis of Erythrocyte Membrane

    PubMed Central

    Moses, K; Pepple, D; Singh, P

    2015-01-01

    ABSTRACT Epigallocatechin-3-gallate (EGCG) is a major ingredient present in green tea, which has a high anti-oxidant activity. In this study, the effect of EGCG was investigated on paraquat-induced haemolysis of erythrocyte membrane. Erythrocytes were incubated in 0.03, 0.3, 3.0 and 30 mg/mL EGCG, respectively and exposed to 30 mg/mL of paraquat for 10 minutes. The effect of paraquat was determined by an analysis of the osmotic fragility of the erythrocytes. The results showed that EGCG (30 mg/mL) significantly (p < 0.05) reduced the haemolysis of erythrocytes exposed to paraquat (5.0 mg/mL). This suggests that EGCG may have a protective effect on paraquat-induced erythrocyte membrane haemolysis and that consumption of green tea, with high EGCG concentration, could ameliorate the deleterious effect of paraquat toxicity on the haemolysis of erythrocyte membrane. PMID:26426167

  13. Human erythrocytes are affected in vitro by extracts of Ugni molinae leaves.

    PubMed

    Suwalsky, M; Orellana, P; Avello, M; Villena, F; Sotomayor, C P

    2006-08-01

    Ugni molinae Turcz, also known as "Murtilla", is a plant that grows in the south of Chile. Infusions of their leaves have long been used in traditional native herbal medicine. The chemical composition of the leaves indicates the presence of polyphenols, which have antioxidant properties. In order to evaluate the mechanisms of their antioxidant properties and the toxicity of the aqueous extracts of leaves, the extracts were induced to interact with human red cells, their isolated unsealed membranes (IUM) and large unilamellar vesicles (LUV) of dimyristoylphosphatidyltidylcholine (DMPC), representative of phospholipid classes located in the outer monolayer of the erythrocyte membrane. Scanning electron microscopy (SEM) observations indicated that the extracts achieved a significant alteration in the shape of the erythrocytes as they changed their discoid shape to echinocytes. According to the bilayer couple hypothesis, the shape change indicates that the polyphenols were located in the outer moiety of the red cell membrane. This conclusion was confirmed by the fluorescence experiments performed in IUM and DMPC LUV. In fact, the extracts produced slight initial increases followed by sharp decreases at higher concentrations in the anisotropy and general polarization parameters. These results imply that the extracts induced structural perturbations in the acyl chain and polar group packing arrangements of the erythrocyte IUM and DMPC LUV lipid bilayers: first ordering and afterwards disordering them as the extract concentration increased. PMID:16716480

  14. In Vitro Effect of Sodium Fluoride on Malondialdehyde Concentration and on Superoxide Dismutase, Catalase, and Glutathione Peroxidase in Human Erythrocytes

    PubMed Central

    Gutiérrez-Salinas, José; García-Ortíz, Liliana; Morales González, José A.; Hernández-Rodríguez, Sergio; Ramírez-García, Sotero; Núñez-Ramos, Norma R.; Madrigal-Santillán, Eduardo

    2013-01-01

    The aim of this paper was to describe the in vitro effect of sodium fluoride (NaF) on the specific activity of the major erythrocyte antioxidant enzymes, as well as on the membrane malondialdehyde concentration, as indicators of oxidative stress. For this purpose, human erythrocytes were incubated with NaF (0, 7, 28, 56, and 100 μg/mL) or NaF (100 μg/mL) + vitamin E (1, 2.5, 5 and 10 μg/mL). The malondialdehyde (MDA) concentration on the surface of the erythrocytes was determined, as were the enzymatic activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GlPx). Our results demonstrated that erythrocytes incubated with increasing NaF concentrations had an increased MDA concentration, along with decreased activity of antioxidant enzymes. The presence of vitamin E partially reversed the toxic effects of NaF on erythrocytes. These findings suggest that NaF induces oxidative stress in erythrocytes in vitro, and this stress is partially reversed by the presence of vitamin E. PMID:24223512

  15. Uric acid protects erythrocytes from ozone-induced changes

    SciTech Connect

    Meadows, J.; Smith, R.C.

    1987-08-01

    Uric acid effectively reduced hemolysis and methemoglobin formation in bovine and swine erythrocytes bubbled with ozone in vitro. In bovine erythrocytes, formation of thiobarbituric acid-reactive material was inhibited by uric acid, but there was little immediate protection for the swine cells. Antioxidant protection was due to preferential degradation of the uric acid by ozone. These results provide evidence to support the hypothesis that in plasma, uric acid can provide antioxidant protection for erythrocytes.

  16. Effect of hydration on the water content of human erythrocytes.

    PubMed

    Levin, R L; Cravalho, E G; Huggins, C E

    1976-12-01

    An ideal, hydrated, nondilute pseudobinary salt-protein-water solution model of the RBC intracellular solution has been developed to describe the osmotic behavior of human erythrocytes during freezing and thawing. Because of the hydration of intracellular solutes (mostly cell proteins), our analytical results predict that at least 16.65% of the isotonic cell water content will be retained within RBCs placed in hypertonic solutions. These findings are consistent not only with the experimental measurements of the amount of isotonic cell water retained within RBCs subjected to nonisotonic extracellular solutions (20-32%) but also with the experimental evidence that all of the water within RBCs is solvent water. By modeling the RBC intracellular solution as a hydrated salt-protein-water solution, no anomalous osmotic behavior is apparent. PMID:990394

  17. Effect of glycyrrhetinic acid on membrane band 3 in human erythrocytes.

    PubMed

    Fiore, Cristina; Bordin, Luciana; Pellati, Donatella; Armanini, Decio; Clari, Giulio

    2008-11-01

    Glycyrrhetinic acid (GA) is a hydrolytic product of the triterpene glycoside of glycyrrhizic acid, one of the main constituents of licorice root, which has long been studied, due to its several biological and endocrine properties. In this paper, GA was tested on human erythrocytes, and GA-induced alterations were compared with those caused by diamide, a mild oxidant inducing well-characterized cell/membrane alterations, and n-ethylmaleimide (NEM), as alkylating agent. In order to verify the biochemical steps underlying the action of GA, band 3 Tyr-phosphorylation level, enzyme recruitment and band 3 clustering in cells pre-incubated with GA before diamide treatment were all examined. Results show that GA, in a dose-dependent manner, prevents both diamide and NEM-induced band 3 Tyr-phosphorylation, but not GSH decrease caused by both compounds. In addition, diamide-induced band 3 clustering and IgG binding to altered cells were also completely reversed by GA pre-treatment. Also, when membrane sensitivity toward proteolytic digestion was tested, GA-treated cells showed high resistance to proteolysis. In conclusion, in human erythrocytes, GA is proposed to strengthen membrane integrity against both oxidative and proteolytic damage. PMID:18778682

  18. Quantitative evaluation of respiration induced metabolic oscillations in erythrocytes.

    PubMed

    Hald, Bjørn; Madsen, Mads F; Danø, Sune; Quistorff, Bjørn; Sørensen, Preben G

    2009-04-01

    The changes in the partial pressures of oxygen and carbon dioxide (P(O(2)) and P(CO(2))) during blood circulation alter erythrocyte metabolism, hereby causing flux changes between oxygenated and deoxygenated blood. In the study we have modeled this effect by extending the comprehensive kinetic model by Mulquiney and Kuchel [P.J. Mulquiney, and P.W. Kuchel. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement, Biochem. J. 1999, 342, 581-596.] with a kinetic model of hemoglobin oxy-/deoxygenation transition based on an oxygen dissociation model developed by Dash and Bassingthwaighte [R. Dash, and J. Bassingthwaighte. Blood HbO(2) and HbCO(2) dissociation curves at varied O(2), CO(2), pH, 2,3-DPG and temperature levels, Ann. Biomed. Eng., 2004, 32(12), 1676-1693.]. The system has been studied during transitions from the arterial to the venous phases by simply forcing P(O(2)) and P(CO(2)) to follow the physiological values of venous and arterial blood. The investigations show that the system passively follows a limit cycle driven by the forced oscillations of P(O(2)) and is thus inadequately described solely by steady state consideration. The metabolic system exhibits a broad distribution of time scales. Relaxations of modes with hemoglobin and Mg(2+) binding reactions are very fast, while modes involving glycolytic, membrane transport and 2,3-BPG shunt reactions are much slower. Incomplete slow mode relaxations during the 60 s period of the forced transitions cause significant overshoots of important fluxes and metabolite concentrations - notably ATP, 2,3-BPG, and Mg(2+). The overshoot phenomenon arises in consequence of a periodical forcing and is likely to be widespread in nature - warranting a special consideration for relevant systems. PMID:19162390

  19. Insulin inhibits human erythrocyte cAMP accumulation and ATP release: role of PDE3 and PI3K

    PubMed Central

    Hanson, Madelyn S.; Stephenson, Alan H.; Bowles, Elizabeth A.; Sprague, Randy S.

    2010-01-01

    In non – erythroid cells, insulin stimulates a signal transduction pathway that results in the activation of phosphoinositide 3 – kinase (PI3K) and phosphorylation of phosphodiesterase 3 (PDE3). Erythrocytes possess insulin receptors, PI3K, and PDE3B. These cells release ATP via a signaling pathway that requires activation of the G protein, Gi, as well as increases in cAMP. Although insulin inhibits ATP release from human erythrocytes in response to Gi activation with mastoparan 7 (Mas 7), no effect on cAMP was described. Here, we investigated the hypothesis that insulin activates PDE3 in human erythrocytes via a PI3K – mediated mechanism resulting in cAMP hydrolysis and inhibition of ATP release. We show that insulin attenuates Mas 7 – induced increases in cAMP and that selective inhibitors of PDE3 (cilostazol) or PI3K (LY294002) rescue this effect of insulin. In addition, we demonstrated that both cilostazol and LY294002 prevent insulin – induced attenuation of Mas 7 – induced ATP release. These results provide support for the hypothesis that insulin activates PDE3 in erythrocytes via a PI3K – dependent mechanism. Once activated, PDE3 limits Mas 7 – induced increases in intracellular cAMP. This effect of insulin leads, ultimately, to decreased ATP release in response to Mas 7. Since the activation of Gi is required for reduced O2 tension – induced ATP release from erythrocytes, and insulin has been shown to inhibit that release, these results suggest a novel mechanism by which supraphysiological levels of plasma insulin, such as those reported in humans with prediabetes, could inhibit ATP release from erythrocytes. Erythrocyte – derived ATP has been shown to participate in the matching of O2 supply with demand in skeletal muscle. Thus, pathological increases in circulating insulin could, via activation of PDE3, inhibit ATP release from erythrocytes depriving the peripheral circulation of a mechanism that regulates delivery of O2 to meet tissue

  20. PHOSPHINE-MEDIATED HEINZ BODY FORMATION AND HEMOGLOBIN OXIDATION IN HUMAN ERYTHROCYTES

    EPA Science Inventory

    Exposure of hen erythrocytes in vitro to phosphine (PH 3) induces the development of Heinz body lesions. he lower limit for phosphine mediated Heinz body formation is 1.25 ppm for a four hour exposure. t exposure levels of 3.0 ppm or higher all erythrocytes are observed to contai...

  1. Lysophosphatidylcholine hydrolases of human erythrocytes, lymphocytes, and brain: Sensitive targets of conserved specificity for organophosphorus delayed neurotoxicants

    SciTech Connect

    Vose, Sarah C.; Holland, Nina T.; Eskenazi, Brenda; Casida, John E.

    2007-10-01

    Brain neuropathy target esterase (NTE), associated with organophosphorus (OP)-induced delayed neuropathy, has the same OP inhibitor sensitivity and specificity profiles assayed in the classical way (paraoxon-resistant, mipafox-sensitive hydrolysis of phenyl valerate) or with lysophosphatidylcholine (LysoPC) as the substrate. Extending our earlier observation with mice, we now examine human erythrocyte, lymphocyte, and brain LysoPC hydrolases as possible sensitive targets for OP delayed neurotoxicants and insecticides. Inhibitor profiling of human erythrocytes and lymphocytes gave the surprising result of essentially the same pattern as with brain. Human erythrocyte LysoPC hydrolases are highly sensitive to OP delayed neurotoxicants, with in vitro IC{sub 50} values of 0.13-85 nM for longer alkyl analogs, and poorly sensitive to the current OP insecticides. In agricultural workers, erythrocyte LysoPC hydrolyzing activities are similar for newborn children and their mothers and do not vary with paraoxonase status but have high intersample variation that limits their use as a biomarker. Mouse erythrocyte LysoPC hydrolase activity is also of low sensitivity in vitro and in vivo to the OP insecticides whereas the delayed neurotoxicant ethyl n-octylphosphonyl fluoride inhibits activity in vivo at 1-3 mg/kg. Overall, inhibition of blood LysoPC hydrolases is as good as inhibition of brain NTE as a predictor of OP inducers of delayed neuropathy. NTE and lysophospholipases (LysoPLAs) both hydrolyze LysoPC, yet they are in distinct enzyme families with no sequence homology and very different catalytic sites. The relative contributions of NTE and LysoPLAs to LysoPC hydrolysis and clearance from erythrocytes, lymphocytes, and brain remain to be defined.

  2. Geometry of the human erythrocyte. I. Effect of albumin on cell geometry.

    PubMed Central

    Jay, A W

    1975-01-01

    The effects of albumin on the geometry of human erythrocytes have been studied. Individual red cells, hanging on edge from coverslips were photographed. Enlarged cell profiles were digitized using a Gradicon digitizer (Instronics Ltd., Stittsville, Ontario). Geometric parameters including diameter, area, volume, minimum cylindrical diameter, sphericity index, swelling index, maximum and minimum cell thickness, were calculated for each cell using a CDC 6400 computer. Maximum effect of human serum albumin was reached at about 1 g/liter. Studies of cell populations showed decreases in mean cell diameter of up to 6%, area 6%, and volume 15%, varying from sample to sample. The thickness of the rim was increased while that at the dimple was decreased. Studies of single cells showed that area and volume changes do not occur equally in all cells. Cells with lower sphericity indices showed larger effects. In the presence of albumin, up to 50% of the cells assumed cup-shapes (stomatocytes). These cells had smaller volumes but the same area as biconcave cells. Mechanical agitation could reversibly induce biconcave cells to assume cup shapes without area or volume changes. Experiments with de-fatted human albumins showed that the presence of bound fatty acids in varying concentrations does not alter the observed effects. Bovine serum albumin has similar effects on human erythrocytes as human serum albumin. Images FIGURE 2 FIGURE 6 FIGURE 9 PMID:1122337

  3. Protective Effect of Selenium-Based Medicines on Toxicity of Three Common Organophosphorus Compounds in Human Erythrocytes In Vitro

    PubMed Central

    Mostafalou, Sara; Navaei-Nigjeh, Mona; Baeeri, Maryam; Mohammadirad, Azadeh; Abdollahi, Mohammad

    2016-01-01

    Objective Organophosphorus (OP) compounds are used to control pests, however they can reach the food chain and enter the human body causing serious health problems by means of acetylcholinesterase (AChE) inhibition and oxidative stress (OS). Among the OPs, chlorpyrifos (CHP), malathion (MAL), and diazinon (DIA) are commonly used for commercial extermination purposes, in addition to veterinary practices, domestic, agricul- ture and public health applications. Two new recently registered medicines that contain selenium and other antioxidants, IMOD and angipars (ANG), have shown beneficial ef- fects for OS related disorders. This study examines the effect of selenium-based medi- cines on toxicity of three common OP compounds in erythrocytes. Materials and Methods In the present experimental study, we determined the ef- ficacy of IMOD and ANG on OS induced by three mentioned OP pesticides in human erythrocytes in vitro. After dose-response studies, AChE, lipid peroxidation (LPO), total antioxidant power (TAP) and total thiol molecules (TTM) were measured in eryth- rocytes after exposure to OPs alone and in combined treatment with IMOD or ANG. Results AChE activity, TAP and TTM reduced in erythrocytes exposed to CHP, MAL and DIA while they were restored in the presence of ANG and IMOD. ANG and IMOD reduced the OPs-induced elevation of LPO. Conclusion The present study shows the positive effects of IMOD and ANG in re- duction of OS and restoration of AChE inhibition induced by CHP, MAL and DIA in erythrocytes in vitro. PMID:26862533

  4. Influence of osmolarity on the optical properties of human erythrocytes

    NASA Astrophysics Data System (ADS)

    Friebel, Moritz; Helfmann, Jürgen; Meinke, Martina C.

    2010-09-01

    Plasma osmolarity influences the volume and shape of red blood cells (RBCs). The volume change is inversely related to the hemoglobin concentration and as a consequence to the complex refractive index within the cell. These morphological changes can be linked to changes in the optical behavior of the cells. The optical parameters, absorption coefficient μa, scattering coefficient μs, and effective scattering phase function of red blood cells are investigated in dependence on osmolarity in the spectral range from 250 to 1100 nm. Integrating sphere measurements of light transmittance and reflectance in combination with inverse Monte-Carlo simulations are carried out for osmolarities from 225 to 400 mosmol/L. Osmolarity changes have a significant influence on the optical parameters, which can in part be explained by changes in the complex refractive index, cell shape, and cell volume. Spherical forms of RBCs induced by low osmolarity show reduced scattering effects compared to the normal RBC biconcave disk shape. Spinocytes, which are crenated erythrocytes induced by high osmolarity, show the highest scattering effects. Even only a 10% change in osmolarity has a drastic influence on the optical parameters, which appears to be of the same order as for 10% hematocrit and oxygen saturation changes.

  5. Morphological Effects and Antioxidant Capacity of Solanum crispum (Natre) In Vitro Assayed on Human Erythrocytes.

    PubMed

    Suwalsky, Mario; Ramírez, Patricia; Avello, Marcia; Villena, Fernando; Gallardo, María José; Barriga, Andrés; Manrique-Moreno, Marcela

    2016-06-01

    In order to gain insight into the molecular mechanism of the antioxidant properties of Solanum crispum, aqueous extracts of its leaves were assayed on human erythrocytes and molecular models of its membrane. Phenolics and alkaloids were detected by HPLC-MS. Scanning electron and defocusing microscopy showed that S. crispum changed erythrocytes from the normal shape to echinocytes. These results imply that molecules present in the aqueous extracts were located in the outer monolayer of the erythrocyte membrane. Dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were chosen as representative of phospholipid classes located in the outer and inner monolayers of the erythrocyte membrane, respectively. X-ray diffraction showed that S. crispum preferentially interacted with DMPC bilayers. Experiments regarding its antioxidant properties showed that S. crispum neutralized the oxidative capacity of HClO on DMPE bilayers; defocusing microscopy and hemolysis assays demonstrated the protective effect of S. crispum against the oxidant effects of HClO on human erythrocytes. PMID:26809653

  6. Comparative study of the effect of BPA and its selected analogues on hemoglobin oxidation, morphological alterations and hemolytic changes in human erythrocytes.

    PubMed

    Maćczak, Aneta; Bukowska, Bożena; Michałowicz, Jaromir

    2015-01-01

    Bisphenol A (BPA) has been shown to provoke many deleterious impacts on human health, and thus it is now successively substituted by BPA analogues, whose effects have been poorly investigated. Up to now, only one study has been realized to assess the effect of BPA on human erythrocytes, which showed its significant hemolytic and oxidative potential. Moreover, no study has been conducted to evaluate the effect of BPA analogues on red blood cells. The purpose of the present study was to compare the impact of BPA and its selected analogues such as bisphenol F (BPF), bisphenol S (BPS) and bisphenol AF (BPAF) on hemolytic and morphological changes and hemoglobin oxidation (methemoglobin formation) of human erythrocytes. The erythrocytes were incubated with different bisphenols concentrations ranging from 0.5 to 500μg/ml for 1, 4 and 24h. The compounds examined caused hemolysis in human erythrocytes with BPAF exhibiting the strongest effect. All bisphenols examined caused methemoglobin formation with BPA inducing the strongest oxidative potential. Flow cytometry analysis showed that all bisphenols (excluding BPS) induced significant changes in erythrocytes size. Changes in red blood cells shape were conducted using phase contrast microscopy. It was noticed that BPA and BPAF induced echinocytosis, BPF caused stomatocytosis, while BPS did not provoke significant changes in shape of red blood cells. Generally, the results showed that BPS, which is the main substituent of bisphenol A in polymers and thermal paper production, exhibited significantly lower disturbance of erythrocyte functions than BPA. PMID:26232583

  7. Effects of Iron Overload on the Activity of Na,K-ATPase and Lipid Profile of the Human Erythrocyte Membrane

    PubMed Central

    Sousa, Leilismara; Garcia, Israel J. P.; Costa, Tamara G. F.; Silva, Lilian N. D.; Renó, Cristiane O.; Oliveira, Eneida S.; Tilelli, Cristiane Q.; Santos, Luciana L.; Cortes, Vanessa F.; Santos, Herica L.; Barbosa, Leandro A.

    2015-01-01

    Iron is an essential chemical element for human life. However, in some pathological conditions, such as hereditary hemochromatosis type 1 (HH1), iron overload induces the production of reactive oxygen species that may lead to lipid peroxidation and a change in the plasma-membrane lipid profile. In this study, we investigated whether iron overload interferes with the Na,K-ATPase activity of the plasma membrane by studying erythrocytes that were obtained from the whole blood of patients suffering from iron overload. Additionally, we treated erythrocytes of normal subjects with 0.8 mM H2O2 and 1 μM FeCl3 for 24 h. We then analyzed the lipid profile, lipid peroxidation and Na,K-ATPase activity of plasma membranes derived from these cells. Iron overload was more frequent in men (87.5%) than in women and was associated with an increase (446%) in lipid peroxidation, as indicated by the amount of the thiobarbituric acid reactive substances (TBARS) and an increase (327%) in the Na,K-ATPase activity in the plasma membrane of erythrocytes. Erythrocytes treated with 1 μM FeCl3 for 24 h showed an increase (132%) in the Na,K-ATPase activity but no change in the TBARS levels. Iron treatment also decreased the cholesterol and phospholipid content of the erythrocyte membranes and similar decreases were observed in iron overload patients. In contrast, erythrocytes treated with 0.8 mM H2O2 for 24 h showed no change in the measured parameters. These results indicate that erythrocytes from patients with iron overload exhibit higher Na,K-ATPase activity compared with normal subjects and that this effect is specifically associated with altered iron levels. PMID:26197432

  8. Modifications of Superoxide Dismutase (SOD1) in Human Erythrocytes

    PubMed Central

    Wilcox, Kyle C.; Zhou, Li; Jordon, Joshua K.; Huang, Yi; Yu, Yanbao; Redler, Rachel L.; Chen, Xian; Caplow, Michael; Dokholyan, Nikolay V.

    2009-01-01

    Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the Kd. This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nm rather than 212 nm at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. PMID:19299510

  9. Effects of aggregation and solvent on the toxicity of amphotericin B to human erythrocytes.

    PubMed Central

    Legrand, P; Romero, E A; Cohen, B E; Bolard, J

    1992-01-01

    In aqueous suspensions of amphotericin B (AmB), a polyene antibiotic and antifungal agent, three forms of AmB coexist: monomers, water-soluble oligomers, and non-water-soluble aggregates. The toxicity of the water-soluble self-associated form of AmB compared with that of the non-water-soluble self-associated form was tested by measuring induction of K+ leakage from human erythrocytes, using different suspensions containing the antibiotic and phosphate-buffered saline. These suspensions were obtained from various stock solutions of the antibiotic in dimethyl formamide or dimethyl sulfoxide. Their circular dichroism spectra around 340 nm, indicative of the degree of AmB self-association, were strongly dependent on the concentration of organic solvent in the suspensions. The nonsoluble self-associated form was separated from the water-soluble form by centrifugation. The nonsoluble form was favored by a high concentration of AmB of the stock solution. The kinetics of AmB-induced K+ leakage from human erythrocytes also appeared to be strongly dependent on the AmB concentration of the stock solution being much weaker with concentrated stock solutions. It was concluded that the only form of AmB toxic to human erythrocytes is the water-soluble self-associated form (in contrast with fungal cells on which the monomeric form is also active). This result may be important in the design of new less toxic AmB derivatives and in the understanding of the mechanism of action of liposomal AmB. PMID:1489196

  10. Diminution of Oxidative Damage to Human Erythrocytes and Lymphocytes by Creatine: Possible Role of Creatine in Blood

    PubMed Central

    Qasim, Neha; Mahmood, Riaz

    2015-01-01

    Creatine (Cr) is naturally produced in the body and stored in muscles where it is involved in energy generation. It is widely used, especially by athletes, as a staple supplement for improving physical performance. Recent reports have shown that Cr displays antioxidant activity which could explain its beneficial cellular effects. We have evaluated the ability of Cr to protect human erythrocytes and lymphocytes against oxidative damage. Erythrocytes were challenged with model oxidants, 2, 2'-azobis(2-amidinopropane) dihydrochloride (AAPH) and hydrogen peroxide (H2O2) in the presence and absence of Cr. Incubation of erythrocytes with oxidant alone increased hemolysis, methemoglobin levels, lipid peroxidation and protein carbonyl content. This was accompanied by decrease in glutathione levels. Antioxidant enzymes and antioxidant power of the cell were compromised while the activity of membrane bound enzyme was lowered. This suggests induction of oxidative stress in erythrocytes by AAPH and H2O2. However, Cr protected the erythrocytes by ameliorating the AAPH and H2O2 induced changes in these parameters. This protective effect was confirmed by electron microscopic analysis which showed that oxidant-induced cell damage was attenuated by Cr. No cellular alterations were induced by Cr alone even at 20 mM, the highest concentration used. Creatinine, a by-product of Cr metabolism, was also shown to exert protective effects, although it was slightly less effective than Cr. Human lymphocytes were similarly treated with H2O2 in absence and presence of different concentrations of Cr. Lymphocytes incubated with oxidant alone had alterations in various biochemical and antioxidant parameters including decrease in cell viability and induction of DNA damage. The presence of Cr attenuated all these H2O2-induced changes in lymphocytes. Thus, Cr can function as a blood antioxidant, protecting cells from oxidative damage, genotoxicity and can potentially increase their lifespan. PMID

  11. Relationship between erythrocyte volume and cell age in humans and baboons. Technical report

    SciTech Connect

    Thompson, C.B.; Galli, R.L.; Melaragno, A.J.; Valeri, C.R.

    1983-03-30

    The relationship of red blood cell size to age during steady-state hematopoiesis has been studied using erythrocytes separated on the basis of size using counterflow centrifugation. The ratio of the age-related enzyme, erythrocyte glutamic oxaloacetic transferase (EGOT), to hemoglobin (Hb) increased progressively through the fractions, suggesting a correlation between erythrocyte volume and age. Reticulocytes, while present in all fractions, were selectively enriched in the larger subpopulations. To verify the biochemical evidence that erythrocytes decrease in volume with aging, in vivo cohort labeling of red blood cells with 59Fe was performed in baboons. A similar relationship of EGOT to Hb was observed to that in the human subpopulations. While a certain amount of erythrocyte volume heterogeneity seems to be present as a result of erythropoeisis, our data support the hypothesis that red blood cells decrease in volume as they age.

  12. Diffusion properties of band 3 in human erythrocytes

    NASA Astrophysics Data System (ADS)

    Spector, Jeffrey O.

    The plasma membrane of the human erythrocyte (RBC) is a six fold symmetric network held together at various pinning points by several multi-protein complexes. This unique architecture is what gives the RBC its remarkable material properties and any disruptions to the network can have severe consequences for the cell. Band 3 is a major transmembrane protein that plays the role of linking the fluid lipid bilayer to the cytoskeletal network. To interrogate the structural integrity of the RBC membrane we have tracked individual band 3 molecules in RBCs displaying a variety of pathologies that are all a consequence of membrane or network related defects. These diseases are spherocytosis, elliptocytosis, and pyropokilocytosis. We have also investigated the protein related diseases sickle cell, and south east asian ovalocytosis. To assess the impact that the network has on the dynamic organization of the cell we have also studied the mobility of band 3 in RBC progenitor cells. Individual band 3 molecules were imaged at 120 frames/second and their diffusion coefficients and compartment sizes recorded. The distributions of the compartment sizes combined with the information about the short and long time diffusion of band 3 has given us insight into the architecture of the membrane in normal and diseased cells. The observation that different membrane pathologies can be distinguished, even to the point of different molecular origins of the same disease, implies that the mobility of transmembrane proteins may be a useful tool for characterizing the "health" of the membrane.

  13. Activated and unactivated forms of human erythrocyte aldose reductase.

    PubMed Central

    Srivastava, S K; Hair, G A; Das, B

    1985-01-01

    Aldose reductase (alditol:NADP+ 1-oxidoreductase, EC 1.1.1.21) has been partially purified from human erythrocytes by DEAE-cellulose (DE-52) column chromatography. This enzyme is activated severalfold upon incubation with 10 microM each glucose 6-phosphate, NADPH, and glucose. The activation of the enzyme was confirmed by following the oxidation of NADPH as well as the formation of sorbitol with glucose as substrate. The activated form of aldose reductase exhibited monophasic kinetics with both glyceraldehyde and glucose (Km of glucose = 0.68 mM and Km of glyceraldehyde = 0.096 mM), whereas the native (unactivated) enzyme exhibited biphasic kinetics (Km of glucose = 9.0 and 0.9 mM and Km of glyceraldehyde = 1.1 and 0.14 mM). The unactivated enzyme was strongly inhibited by aldose reductase inhibitors such as sorbinil, alrestatin, and quercetrin, and by phosphorylated intermediates such as ADP, glycerate 3-phosphate, glycerate 1,3-bisphosphate, and glycerate 2,3-trisphosphate. The activated form of the enzyme was less susceptible to inhibition by aldose reductase inhibitors and phosphorylated intermediates. PMID:3933003

  14. Membrane flickering of the human erythrocyte: physical and chemical effectors.

    PubMed

    Puckeridge, Max; Chapman, Bogdan E; Conigrave, Arthur D; Kuchel, Philip W

    2014-05-01

    Recent studies suggest a link between adenosine triphosphate (ATP) concentration and the amplitude of cell membrane flickering (CMF) in the human erythrocyte (red blood cell; RBC). Potentially, the origin of this phenomenon and the unique discocyte shape could be active processes that account for some of the ATP turnover in the RBC. Active flickering could depend on several factors, including pH, osmolality, enzymatic rates and metabolic fluxes. In the present work, we applied the data analysis described in the previous article to study time courses of flickering RBCs acquired using differential interference contrast light microscopy in the presence of selected effectors. We also recorded images of air bubbles in aqueous detergent solutions and oil droplets in water, both of which showed rapid fluctuations in image intensity, the former showing the same type of spectral envelope (relative frequency composition) to RBCs. We conclude that CMF is not directly an active process, but that ATP affects the elastic properties of the membrane that flickers in response to molecular bombardment in a manner that is described mathematically by a constrained random walk. PMID:24668224

  15. Plasmodiumfalciparum infection induces dynamic changes in the erythrocyte phospho-proteome.

    PubMed

    Bouyer, Guillaume; Reininger, Luc; Ramdani, Ghania; D Phillips, Lee; Sharma, Vikram; Egee, Stephane; Langsley, Gordon; Lasonder, Edwin

    2016-05-01

    The phosphorylation status of red blood cell proteins is strongly altered during the infection by the malaria parasite Plasmodium falciparum. We identify the key phosphorylation events that occur in the erythrocyte membrane and cytoskeleton during infection, by a comparative analysis of global phospho-proteome screens between infected (obtained at schizont stage) and uninfected RBCs. The meta-analysis of reported mass spectrometry studies revealed a novel compendium of 495 phosphorylation sites in 182 human proteins with regulatory roles in red cell morphology and stability, with about 25% of these sites specific to infected cells. A phosphorylation motif analysis detected 7 unique motifs that were largely mapped to kinase consensus sequences of casein kinase II and of protein kinase A/protein kinase C. This analysis highlighted prominent roles for PKA/PKC involving 78 phosphorylation sites. We then compared the phosphorylation status of PKA (PKC) specific sites in adducin, dematin, Band 3 and GLUT-1 in uninfected RBC stimulated or not by cAMP to their phosphorylation status in iRBC. We showed cAMP-induced phosphorylation of adducin S59 by immunoblotting and we were able to demonstrate parasite-induced phosphorylation for adducin S726, Band 3 and GLUT-1, corroborating the protein phosphorylation status in our erythrocyte phosphorylation site compendium. PMID:27067487

  16. Characterization of Human Erythrocytes as Potential Carrier for Pravastatin: An In Vitro Study

    PubMed Central

    Harisa, Gamal El-din I.; Ibrahim, Mohamed F.; Alanazi, Fars K.

    2011-01-01

    Drug delivery systems including chemical, physical and biological agents that enhance the bioavailability, improve pharmacokinetics and reduce toxicities of the drugs. Carrier erythrocytes are one of the most promising biological drug delivery systems investigated in recent decades. The bioavailability of statin drugs is low due the effects of P-glycoprotein in the gastro-intestinal tract as well as the first-pass metabolism. Therefore in this work we study the effect of time, temperature as well as concentration on the loading of pravastatin in human erythrocytes to be using them as systemic sustained release delivery system for this drug. After the loading process is performed the carriers' erythrocytes were physically and cellulary characterized. Also, the in vitro release of pravastatin from carrier erythrocytes was studied over time interval. Our results revealed that, human erythrocytes have been successfully loaded with pravastatin using endocytosis method either at 25oC or at 37oC. The loaded amount at 10 mg/ml is 0.32mg/0.1 ml and 0.69 mg/0.1 ml. Entrapment efficiency is 34% and 94% at 25oC and 37oC respectively at drug concentration 4 mg/ml. Moreover the percent of cells recovery is 87-93%. Hematological parameters and osmotic fragility behavior of pravastatin loaded erythrocytes were similar that of native erythrocytes. Scanning electron microscopy demonstrated that the pravastatin loaded cells has no change in the morphology. Pravastatin releasing from carrier cell was 83% after 23 hours in phosphate buffer saline and decreased to 72% by treatment of carrier cells with glutaraldehyde. The releasing pattern of the drug from loaded erythrocytes obeyed first order kinetics. It concluded that pravastatin is successfully entrapped into erythrocytes with acceptable loading parameters and moderate morphological changes, this suggesting that erythrocytes can be used as prolonged release for pravastatin. PMID:21448309

  17. A GBP 130 derived peptide from Plasmodium falciparum binds to human erythrocytes and inhibits merozoite invasion in vitro.

    PubMed

    Suarez, J E; Urquiza, M; Curtidor, H; Rodriguez, L E; Ocampo, M; Torres, E; Guzman, F; Patarroyo, M E

    2000-01-01

    The malarial GBP 130 protein binds weakly to intact human erythrocytes; the binding sites seem to be located in the repeat region and this region's antibodies block the merozoite invasion. A peptide from this region (residues from 701 to 720) which binds to human erythrocytes was identified. This peptide named 2220 did not bind to sialic acid; the binding site on human erythrocyte was affected by treatment with trypsin but not by chymotrypsin. The peptide was able to inhibit Plasmodium falciparum merozoite invasion of erythrocytes. The residues F701, K703, L705, T706, E713 (FYKILTNTDPNDEVERDNAD) were found to be critical for peptide binding to erythrocytes. PMID:10904405

  18. Sodium nitrite enhances generation of reactive oxygen species that decrease antioxidant power and inhibit plasma membrane redox system of human erythrocytes.

    PubMed

    Ansari, Fariheen Aisha; Mahmood, Riaz

    2016-08-01

    Nitrite/nitrate salts are used in fertilizers and as food preservatives. Human exposure to high levels of nitrite results in its uptake and subsequent entry into blood where it can interact with erythrocytes. We show that treatment of human erythrocytes with sodium nitrite (NaNO2 ) results in a dose-dependent increase in the production of reactive oxygen species. This was accompanied by a decrease in the antioxidant power which lowered the free radical quenching and metal-reducing ability. NaNO2 treatment also inhibited plasma membrane redox system (PMRS) of erythrocytes. These changes increase the susceptibility of erythrocytes to oxidative damage, decrease the antioxidant power of whole blood, and can be a major cause of nitrite-induced cellular toxicity. PMID:27214747

  19. Aluminum Trichloride Induces Hypertension and Disturbs the Function of Erythrocyte Membrane in Male Rats.

    PubMed

    Zhang, Qiuyue; Cao, Zheng; Sun, Xudong; Zuang, Cuicui; Huang, Wanyue; Li, Yanfei

    2016-05-01

    Aluminum (Al) is the most abundant metal in the earth's crust. Al accumulates in erythrocyte and causes toxicity on erythrocyte membrane. The dysfunction of erythrocyte membrane is a potential risk to hypertension. The high Al content in plasma was associated with hypertension. To investigate the effect of AlCl3 on blood pressure and the function of erythrocyte membrane, the rats were intragastrically exposed to 0, 64(1/20 LD50), 128(1/10 LD50), and 256(1/5 LD50) mg/kg body weight AlCl3 in double distilled water for 120 days, respectively. Then, we determined the systolic and mean arterial blood pressures of rats, the osmotic fragility, the percentage of membrane proteins, the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-pX), and malondialdehyde (MDA) content of the erythrocyte membrane in this experiment. The results showed that AlCl3 elevated the systolic and mean arterial blood pressure of rats, increased the osmotic fragility, decreased the percentage of membrane protein, inhibited the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, Ca(2+)-ATPase, CAT, SOD and GSH-pX, and increased the MDA content of erythrocyte membrane. These results indicate that AlCl3 may induce hypertension by disturbing the function of erythrocyte membrane. PMID:26354416

  20. Fosmidomycin Uptake into Plasmodium and Babesia-Infected Erythrocytes Is Facilitated by Parasite-Induced New Permeability Pathways

    PubMed Central

    Reichenberg, Armin; Hintz, Martin; Bietz, Sven; Harb, Omar S.; Roos, David S.; Kordes, Maximilian; Friesen, Johannes; Matuschewski, Kai; Lingelbach, Klaus; Jomaa, Hassan; Seeber, Frank

    2011-01-01

    Background Highly charged compounds typically suffer from low membrane permeability and thus are generally regarded as sub-optimal drug candidates. Nonetheless, the highly charged drug fosmidomycin and its more active methyl-derivative FR900098 have proven parasiticidal activity against erythrocytic stages of the malaria parasite Plasmodium falciparum. Both compounds target the isoprenoid biosynthesis pathway present in bacteria and plastid-bearing organisms, like apicomplexan parasites. Surprisingly, the compounds are inactive against a range of apicomplexans replicating in nucleated cells, including Toxoplasma gondii. Methodology/Principal Findings Since non-infected erythrocytes are impermeable for FR90098, we hypothesized that these drugs are taken up only by erythrocytes infected with Plasmodium. We provide evidence that radiolabeled FR900098 accumulates in theses cells as a consequence of parasite-induced new properties of the host cell, which coincide with an increased permeability of the erythrocyte membrane. Babesia divergens, a related parasite that also infects human erythrocytes and is also known to induce an increase in membrane permeability, displays a similar susceptibility and uptake behavior with regard to the drug. In contrast, Toxoplasma gondii-infected cells do apparently not take up the compounds, and the drugs are inactive against the liver stages of Plasmodium berghei, a mouse malaria parasite. Conclusions/Significance Our findings provide an explanation for the observed differences in activity of fosmidomycin and FR900098 against different Apicomplexa. These results have important implications for future screens aimed at finding new and safe molecular entities active against P. falciparum and related parasites. Our data provide further evidence that parasite-induced new permeability pathways may be exploited as routes for drug delivery. PMID:21573242

  1. Intracellular free calcium concentration and calcium transport in human erythrocytes of lead-exposed workers

    SciTech Connect

    Quintanar-Escorza, M.A.; Gonzalez-Martinez, M.T.; Navarro, L.; Maldonado, M.; Arevalo, B.; Calderon-Salinas, J.V. . E-mail: jcalder@cinvestav.mx

    2007-04-01

    Erythrocytes are the route of lead distribution to organs and tissues. The effect of lead on calcium homeostasis in human erythrocytes and other excitable cells is not known. In the present work we studied the effect of lead intoxication on the uptake and efflux (measured as (Ca{sup 2+}-Mg{sup 2+})-ATPase activity) of calcium were studied in erythrocytes obtained from lead-exposed workers. Blood samples were taken from 15 workers exposed to lead (blood lead concentration 74.4 {+-} 21.9 {mu}g/dl) and 15 non-exposed workers (9.9 {+-} 2 {mu}g/dl). In erythrocytes of lead-exposed workers, the intracellular free calcium was 79 {+-} 13 nM, a significantly higher concentration (ANOVA, P < 0.01) than the one detected in control (30 {+-} 9 nM). The enhanced intracellular free calcium was associated with a higher osmotic fragility and with important modifications in erythrocytes shape. The high intracellular free calcium in lead-exposed workers was also related to a 100% increase in calcium incorporation and to 50% reduction of (Ca{sup 2+}-Mg{sup 2+})-ATPase activity. Lipid peroxidation was 1.7-fold higher in erythrocytes of lead-exposed workers as compared with control. The alteration on calcium equilibrium in erythrocytes is discussed in light of the toxicological effects in lead-exposed workers.

  2. Oxidative damage increases intracellular free calcium [Ca2+]i concentration in human erythrocytes incubated with lead.

    PubMed

    Quintanar-Escorza, M A; González-Martínez, M T; del Pilar, Intriago-Ortega Ma; Calderón-Salinas, J V

    2010-08-01

    One important effect of lead toxicity in erythrocytes consists of increasing [Ca(2+)](i) which in turn may cause alterations in cell shape and volume and it is associated with cellular rigidity, hemolysis, senescence and apoptosis. In this work, we proposed the use of erythrocytes incubated with Pb(2+) to assess association of the mechanisms of lead erythrocyte oxidative damage and calcium homeostasis. Lead incubation produced an increase in [Ca(2+)](i) dose- and time-dependent, which mainly involved Ca(2+) entry mechanism. Additionally, in this in vitro model alterations similar to erythrocytes of lead-exposed workers were produced: Increase in Ca(2+) influx, decrease in (Ca(2+)-Mg(2+))-ATPase activity and GSH/GSGG ratio; increase in lipoperoxidation, protein carbonylation and osmotic fragility accompanied of dramatic morphological changes. Co-incubation with trolox, a soluble vitamin-E analog is able to prevent these alterations indicating that lead damage mechanism is strongly associated with oxidative damage with an intermediate toxic effect via [Ca(2+)](i) increase. Furthermore, erythrocytes oxidation induced with a free radical generator (APPH) showed effects in [Ca(2+)](i) and oxidative damage similar to those found in erythrocytes incubated with lead. Co-incubation with trolox prevents the oxidative effects induced by AAPH in erythrocytes. These results suggest that increase of [Ca(2+)](i) depends on the oxidative status of the erythrocytes incubated with lead. We consider that this model contributes in the understanding of the relation between oxidative damage induced by lead exposure and Ca(2+) homeostasis, the consequences related to these phenomena and the molecular basis of lead toxicity in no excitable cells. PMID:20460147

  3. Chlorella is an effective dietary source of lutein for human erythrocytes.

    PubMed

    Miyazawa, Taiki; Nakagawa, Kiyotaka; Kimura, Fumiko; Nakashima, Yuya; Maruyama, Isao; Higuchi, Ohki; Miyazawa, Teruo

    2013-01-01

    Chlorella contains a high amount of carotenoids, especially lutein, and has received attention as a possible dietary source for improving carotenoid levels in human blood. In the present study, we performed a 2-month single arm human study, and investigated the efficacy of Chlorella supplementation (9 g Chlorella/day; equivalent to 32 mg lutein/day) on lutein and other carotenoid concentrations in plasma as well as erythrocytes of 12 healthy subjects. Following Chlorella supplementation, lutein was the predominant carotenoid in erythrocytes, showing a 4-fold increase (from 14 to 54 pmol/mL packed cells). After the one month without Chlorella ingestion, erythrocyte lutein then decreased to a basal level (17 pmol/mL packed cells). Erythrocyte carotenoid (lutein, zeaxanthin, α-carotene, and β-carotene) levels were proportional to plasma carotenoid levels. The results suggest the transfer of Chlorella carotenoids, especially lutein, from plasma lipoprotein particles to the erythrocyte membrane. Chlorella intake would be effective for improving and maintaining lutein concentrations in human erythrocytes. PMID:24088514

  4. Phosphorylation of intact erythrocytes in human muscular dystrophy

    SciTech Connect

    Johnson, R.M.; Nigro, M.

    1986-04-01

    The uptake of exogenous /sup 32/Pi into the membrane proteins of intact erythrocytes was measured in 8 patients with Duchenne muscular dystrophy. No abnormalities were noted after autoradiographic analysis. This contrasts with earlier results obtained when isolated membranes were phosphorylated with gamma-(/sup 32/P)ATP, and suggests a possible reinterpretation of those experiments.

  5. Effect of high glucose concentrations on human erythrocytes in vitro

    PubMed Central

    Viskupicova, Jana; Blaskovic, Dusan; Galiniak, Sabina; Soszyński, Mirosław; Bartosz, Grzegorz; Horakova, Lubica; Sadowska-Bartosz, Izabela

    2015-01-01

    Exposure to high glucose concentrations in vitro is often employed as a model for understanding erythrocyte modifications in diabetes. However, effects of such experiments may be affected by glucose consumption during prolonged incubation and changes of cellular parameters conditioned by impaired energy balance. The aim of this study was to compare alterations in various red cell parameters in this type of experiment to differentiate between those affected by glycoxidation and those affected by energy imbalance. Erythrocytes were incubated with 5, 45 or 100 mM glucose for up to 72 h. High glucose concentrations intensified lipid peroxidation and loss of activities of erythrocyte enzymes (glutathione S-transferase and glutathione reductase). On the other hand, hemolysis, eryptosis, calcium accumulation, loss of glutathione and increase in the GSSG/GSH ratio were attenuated by high glucose apparently due to maintenance of energy supply to the cells. Loss of plasma membrane Ca2+-ATPase activity and decrease in superoxide production were not affected by glucose concentration, being seemingly determined by processes independent of both glycoxidation and energy depletion. These results point to the necessity of careful interpretation of data obtained in experiments, in which erythrocytes are subject to treatment with high glucose concentrations in vitro. PMID:26141922

  6. Effect of 3-bromopyruvic acid on human erythrocyte antioxidant defense system.

    PubMed

    Sadowska-Bartosz, Izabela; Bartosz, Grzegorz

    2013-12-01

    3-Bromopyruvate (3-BP) is a promising compound for anticancer therapy, its main mode of action being the inhibition of glycolytic enzymes, but this compound also induces oxidative stress. This study aimed at characterisation of the effect of 3-BP on the antioxidant defense system of erythrocytes. Suspensions of erythrocytes in PBS containing 5 mM glucose were treated with different concentration of 3-BP at 37°C for 1 h. Activities of antioxidant enzymes were estimated by standard colorimetric methods. The antioxidant capacity of erythrocytes was estimated using the 2,2'-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS(•+)) decolorisation assay and ferricyanide reduction. The content of reduced and oxidized glutathione was estimated fluorimetrically with o-phtalaldehyde. 3-BP did not affect the integrity of the erythrocyte membrane (lack of changes in the osmotic fragility). However, it induced oxidative stress in erythrocytes, as evidenced by the decrease in the content of acid-soluble thiols and reduced glutathione (GSH). Superoxide dismutase (SOD) and glutathione S-transferase (GST) activities were significantly decreased. 3-BP also decreased the transmembrane reduction of ferricyanide. Thus induction of oxidative stress in erythrocytes by 3-BP is due to depletion of glutathione and inhibition of antioxidant enzymes. PMID:23881849

  7. Magnetic measurements on human erythrocytes: Normal, beta thalassemia major, and sickle

    NASA Astrophysics Data System (ADS)

    Sakhnini, Lama

    2003-05-01

    In this article magnetic measurements were made on human erythrocytes at different hemoglobin states (normal and reduced hemoglobin). Different blood samples: normal, beta thalassemia major, and sickle were studied. Beta thalassemia major and sickle samples were taken from patients receiving lifelong blood transfusion treatment. All samples examined exhibited diamagnetic behavior. Beta thalassemia major and sickle samples showed higher diamagnetic susceptibilities than that for the normal, which was attributed to the increase of membrane to hemoglobin volume ratio of the abnormal cells. Magnetic measurements showed that the erythrocytes in the reduced state showed less diamagnetic response in comparison with erythrocytes in the normal state. Analysis of the paramagnetic component of magnetization curves gave an effective magnetic moment of μeff=7.6 μB per reduced hemoglobin molecule. The same procedure was applied to sickle and beta thalassemia major samples and values for μeff were found to be comparable to that of the normal erythrocytes.

  8. Recruitment of human aquaporin 3 to internal membranes in the Plasmodium falciparum infected erythrocyte.

    PubMed

    Bietz, Sven; Montilla, Irine; Külzer, Simone; Przyborski, Jude M; Lingelbach, Klaus

    2009-09-01

    The molecular mechanisms underlying the formation of the parasitophorous vacuolar membrane in Plasmodium falciparum infected erythrocytes are incompletely understood, and the protein composition of this membrane is still enigmatic. Although the differentiated mammalian erythrocyte lacks the machinery required for endocytosis, some reports have described a localisation of host cell membrane proteins at the parasitophorous vacuolar membrane. Aquaporin 3 is an abundant plasma membrane protein of various cells, including mammalian erythrocytes where it is found in distinct oligomeric states. Here we show that human aquaporin 3 is internalized into infected erythrocytes, presumably during or soon after invasion. It is integrated into the PVM where it is organized in novel oligomeric states which are not found in non-infected cells. PMID:19393693

  9. Biomarkers of oxidative stress in erythrocytes as a function of human age.

    PubMed

    Maurya, Pawan Kumar; Kumar, Prabhanshu; Chandra, Pranjal

    2015-12-26

    Despite more than 300 theories to explain the aging process, oxidative stress theory offers the best mechanism to explain aging and age related disorders. Several studies has shown the importance of oxidative stress during aging. PubMed, Science Direct and Springer online data bases are taken into consideration to write this mini-review. Human erythrocytes are most abundant and specialized cells in the body. Erythrocytes were extensively studied due to their metabolism and gas transport functions. Recent studies on erythrocytes have provided us detailed information of cell membrane and its structural organization that may help in studying the aging and age associated changes. The susceptibility of an organism is associated with the antioxidant potential of the body. Erythrocytes have potent antioxidant protection consisting of enzymatic and non-enzymatic pathways that counteract with reactive oxygen species, thus maintaining the redox regulation in the body. The non-enzymatic and enzymatic antioxidants and other biomarkers associated with erythrocyte membrane transport functions are the main content of this review. Biomarkers of oxidative stress in erythrocytes and its membrane were taken into the consideration during human aging that will be the main subject of this mini- review. PMID:26713282

  10. Biomarkers of oxidative stress in erythrocytes as a function of human age

    PubMed Central

    Maurya, Pawan Kumar; Kumar, Prabhanshu; Chandra, Pranjal

    2015-01-01

    Despite more than 300 theories to explain the aging process, oxidative stress theory offers the best mechanism to explain aging and age related disorders. Several studies has shown the importance of oxidative stress during aging. PubMed, Science Direct and Springer online data bases are taken into consideration to write this mini-review. Human erythrocytes are most abundant and specialized cells in the body. Erythrocytes were extensively studied due to their metabolism and gas transport functions. Recent studies on erythrocytes have provided us detailed information of cell membrane and its structural organization that may help in studying the aging and age associated changes. The susceptibility of an organism is associated with the antioxidant potential of the body. Erythrocytes have potent antioxidant protection consisting of enzymatic and non-enzymatic pathways that counteract with reactive oxygen species, thus maintaining the redox regulation in the body. The non-enzymatic and enzymatic antioxidants and other biomarkers associated with erythrocyte membrane transport functions are the main content of this review. Biomarkers of oxidative stress in erythrocytes and its membrane were taken into the consideration during human aging that will be the main subject of this mini- review. PMID:26713282

  11. Effect of Copper on l-Cysteine/l-Cystine Influx in Normal Human Erythrocytes and Erythrocytes of Wilson's Disease.

    PubMed

    Mandal, Nabarun; Bhattacharjee, Debojyoti; Rout, Jayanta Kumar; Dasgupta, Anindya; Bhattacharya, Gorachand; Sarkar, Chandan; Gangopadhyaya, Prasanta Kumar

    2016-10-01

    Wilson's disease is a disease of abnormal copper metabolism in which free serum copper level is raised. The objective of the study was to determine, whether in Wilson disease, l-cysteine/l-cystine influx into RBC was decreased or not and the specific amino acid transporter affected by copper in normal human RBC. For l-cysteine/l-cystine influx, ten untreated cases, ten treated cases and ten age and sex matched healthy controls were recruited. To study the effect of copper on l-cysteine/l-cystine influx in RBC, 15 healthy subjects were selected. RBC GSH and l-cysteine/l-cystine influx were estimated by Beautler's and Yildiz's method respectively. In untreated cases, l-cysteine/l-cystine influx and erythrocyte GSH level were decreased showing that elevated level of free copper in serum or media decreased l-cysteine/l-cystine influx in human RBC. Copper treatment inhibited L amino acid transporter in normal RBC specifically. PMID:27605746

  12. Comparative study of oxime-induced reactivation of erythrocyte and muscle AChE from different animal species following inhibition by sarin or paraoxon.

    PubMed

    Herkert, Nadja M; Aurbek, Nadine; Eyer, Peter; Thiermann, Horst; Worek, Franz

    2010-05-01

    Standard treatment of acute poisoning by organophosphorus compounds (OP) includes administration of an antimuscarinic (e.g. atropine) and of an oxime-based reactivator of OP-inhibited acetylcholinesterase (AChE). A recently introduced dynamically working in vitro model with real-time determination of membrane-bound AChE activity was shown to be a very versatile and promising model to investigate oxime-induced reactivation kinetics of OP-inhibited enzyme. In this assay, human AChE from erythrocytes or muscle tissue was immobilized on a particle filter. This bioreactor was continuously perfused with substrate and chromogen and AChE activity was analyzed on-line in a flow-through detector. The model has been successfully adopted to Rhesus monkey, swine and guinea pig erythrocytes and intercostal muscle AChE. In addition, the basic kinetic constants of inhibition, aging, spontaneous- and oxime-induced-reactivation of erythrocyte AChE from these species were determined with a standard static model. The major findings were, in part substantial species differences in the inhibition (sarin, paraoxon) and reactivation kinetics (obidoxime, HI 6) of erythrocyte AChE, but comparable kinetics of inhibition and reactivation between erythrocyte and muscle AChE. Hence, these data provide further support of the assumption that erythrocyte AChE is an adequate surrogate of muscle (synaptic) AChE and admonish that major species differences have to be considered for the design and evaluation of therapeutic animal models. PMID:20156534

  13. A modelling study of feedforward activation in human erythrocyte glycolysis.

    PubMed

    Bali, M; Thomas, S R

    2001-03-01

    Though feedforward activation (FA) is a little known principle of control in metabolic networks, there is one well-known example; namely, the activation of pyruvate kinase (PK) by fructose-1,6-biphosphate (FBP) in glycolysis. The effects of this activation on the enzyme's kinetics are well characterised, but its possible role in glycolytic control has not been determined, and, experimentally, there is as yet no direct way of modifying the enzyme to remove just the FBP activation without affecting other aspects of the enzyme's kinetics. Given this limitation, we used a detailed numerical simulation of human erythrocyte glycolysis to simulate the effects of selective removal of the activation of PK by FBP on steady-state metabolite concentrations and on the dynamic response of glycolytic flux to a sudden increase of the cell's demand for ATP. Our modelling results predict that in the absence of FA steady-state levels of metabolites within the activation loop, i.e. from FBP to phosphoenolpyruvate, would be four- to thirteen-fold higher than normal, whereas levels of ATP and metabolites outside the loop, i.e. glucose-6-phosphate, fructose-6-phosphate and pyruvate, would be lower than normal. Existing clinical evidence in a patient with haemolytic anaemia, correlated with a lack of activation of PK by FBP (Paglia D.E., Valentine W.N., Holbrook C.T., Brockway R., Blood (1983) 62 972-979), is consistent with this prediction. In response to changing demand for ATP, the model predicts that the corresponding change of glycolytic flux would entail changes of metabolite concentrations in the absence of FA, but that in its presence the levels of metabolites within the activation loop remain essentially unperturbed. Thus, our results suggest that by stabilising metabolite pools in the face of variable glycolytic flux, FA may serve to avoid perturbations of the oxygen affinity of haemoglobin (sensitive to the levels of 2,3-phosphoglycerate) and of cell osmolality that would

  14. Oleic acid may be the key contributor in the BAMLET-induced erythrocyte hemolysis and tumoricidal action.

    PubMed

    Hoque, Mehboob; Dave, Sandeep; Gupta, Pawan; Saleemuddin, Mohammed

    2013-01-01

    A chance discovery of the tumoricidal action of a human milk fraction led to the characterization of the active component as oleic acid complex of the α-lactalbumin, which was given the acronym HAMLET. We report in this study that the oleic acid complex of bovine α-lactalbumin (BAMLET) is hemolytic to human erythrocytes as well as to those derived from some other mammals. Indirect immunofluorescence analysis suggested binding of BAMLET to erythrocytes prior to induction of hemolysis. Free OA was hemolytic albeit at higher concentrations, while sodium oleate caused hemolysis at far lower concentrations. Amiloride and BaCl2 offered protection against BAMLET-induced hemolysis suggesting the involvement of a cation leak channel in the process. BAMLET coupled to CNBr-activated Sepharose was not only hemolytic but also tumoricidal to Jurkat and MCF-7 cells in culture. The Sepharose-linked preparation was however not toxic to non-cancerous peritoneal macrophages and primary adipocytes. The tumoricidal action was studied using the MTT-assay while apoptosis induction measured by the annexin V-propidium iodide assay. Repeated incubation of the immobilized BAMLET with erythrocytes depleted oleic acid and decreased the hemolytic activity of the complex. Incubation of MCF-7 and Jurkat cells with OA, soluble or immobilized BAMLET resulted in increase in the uptake of Lyso Tracker Red and Nile red by the cells. The data presented support the contention that oleic acid plays the key role, both in BAMLET-induced hemolysis and tumoricidal action. PMID:24039698

  15. Protective effect of Ugni molinae Turcz against oxidative damage of human erythrocytes.

    PubMed

    Suwalsky, M; Orellana, P; Avello, M; Villena, F

    2007-01-01

    Ugni molinae Turcz, also known as "Murtilla", is a plant that grows in the south of Chile. Infusions of its leaves have long been used in traditional native herbal medicine. The chemical composition of the leaves indicates the presence of polyphenols, which have antioxidant properties. In the present work, the antioxidant properties of U. molinae were evaluated in human erythrocytes exposed in vitro to oxidative stress induced by HClO. The experiments were carried out by scanning electron microscopy (SEM) and hemolysis measurements. The SEM observations showed that HClO induced a morphological alteration in the red blood cells from a discoid to an echinocytic form. According to the bilayer couple hypothesis, the formation of echinocytes indicates that HClO was inserted in the outer leaflet of the erythrocyte membrane. However, a concentration as low as 10 microM gallic acid equivalents (GAE) U. molinae aqueous extract neutralized the shape change effect of HClO applied in a concentration as high as 0.25 mM. The significant protection of U. molinae aqueous extract was also shown in the hemolysis experiments. In fact, very low concentrations of the extract considerably reduced the deleterious capacity of HClO to induce hemolysis in red blood cells. It is concluded that the location of the extract components into the membrane bilayer and the resulting restriction on its fluidity might hinder the diffusion of HClO and its consequent damaging effects. This conclusion can also imply that this restriction could apply to the diffusion of free radicals into cell membranes and the subsequent decrease of the kinetics of free radical reactions. PMID:17030381

  16. Effects of lead chloride on human erythrocyte membranes and on kinetic anion sulphate and glutathione concentrations.

    PubMed

    Gugliotta, Tiziana; De Luca, Grazia; Romano, Pietro; Rigano, Caterina; Scuteri, Adriana; Romano, Leonardo

    2012-12-01

    Our study concerns the effects of exposure to lead chloride on the morphology, K(+) efflux, SO(4)(-) influx and GSH levels of the human erythrocyte. Blood was collected in heparinized tubes and washed three times. The cells were suspended at 3% hematocrit and incubated for 1 h at 25°C in a medium containing increasing concentrations of lead chloride (0, 0.3, 0.5 and 1 μM). After incubation, the suspensions were centrifuged and the erythrocyte pellets were divided into three aliquots for testing. The results show: an increase in the permeability of erythrocytes treated with lead chloride with consequent damage and cellular death, especially in the presence of high concentrations; an increase in potassium ion efflux; alterations in the morphology and membrane structure of the red blood cells; and a decrease in sulphate uptake, due either to the oxidative effect of this compound on the band 3 protein, which loses its biological valence as a carrier of sulphate ions, or to a decrease in the ATP erythrocyte concentration. In conclusion, the exposure of erythrocytes to Pb(2+) ions leads to a reduction in the average lifetime of the erythrocytes and the subsequent development of anemia. These data are discussed in terms of the possible effect of lead on the reduction-oxidation systems of the cell. Oxidant agents, such as lead, are known to cross-link integral membrane proteins, leading to K/Cl-cotransport. The increased K(+) efflux affects the altered redox state. PMID:22941203

  17. Release of PAF by human polymorphonuclear leucocytes stimulated by immune complexes bound to Sepharose particles and human erythrocytes.

    PubMed Central

    Virella, G; Lopes-Virella, M F; Shuler, C; Sherwood, T; Espinoza, G A; Winocour, P; Colwell, J A

    1983-01-01

    Human polymorphonuclear leucocytes (PMN) incubated with surface-bound immune complexes (IC) release a substance that induces platelet aggregation and serotonin-release. This substance was identified as platelet-activating factor (PAF) on the basis of its sensitivity to phospholipase A2 and of its purification by thin-layer chromatography in identical conditions to those used to purify zymosan-induced PAF. We used two types of substrates to absorb our IC:Sepharose particles to which we coupled human serum albumin, and which were later incubated with specific rabbit antiserum to form surface-bound immune complexes, and human erythrocytes, to which soluble IC can be passively adsorbed. Both types of surface-bound IC were found to stimulate the release of PAF by human PMN in the absence of complement. These results suggest that PMN may play a central role in the early stages of IC-induced inflammation: they recognize IC adsorbed to red cells or to any other cell able to adsorb IC, and they induce the activation of platelets and release of vasoactive amines, which leads to the increase of vascular permeability believed to be essential for extravascular IC deposition. PMID:6885111

  18. Gender differences in alcohol-induced oxidative stress and altered membrane properties in erythrocytes of rats.

    PubMed

    Reddy, Kindinti Rameshwar; Reddy, Vaddi Damodara; Padmavathi, Pannuru; Kavitha, Godugu; Saradamma, Bulle; Varadacharyulu, N C

    2013-02-01

    Alcohol-induced oxidative stress leads to imbalance between reactive oxygen species (ROS) and the antioxidant defense system, resulting in oxidative damage to membrane components such as lipids and proteins, ultimately altering membrane properties. In this study, we assessed oxidative stress status and alterations in erythrocyte membrane properties in alcohol-administered rats with respect to gender difference. Alcohol (20% v/v) administered rats of both genders showed significant changes in plasma lipid profile with elevated nitrite/nitrate levels. Furthermore, alcohol-administration significantly decreased erythrocyte antioxidant enzymes and enhanced erythrocyte membrane lipid peroxidation, cholesterol/phospholipid (C/P) ratio and Na+/K(+)-ATPase activity in both males and females. Besides, anisotropic studies revealed that alcohol-administration significantly decreased erythrocyte membrane fluidity. In conclusion, alcohol-administration significantly increased oxidative stress by decreasing antioxidant status, and subsequent generation of ROS altered membrane properties by altering fluidity and Na+/K(+)-ATPase activity. Female rats were more vulnerable to alcohol-induced biochemical and biophysical changes in plasma and erythrocyte including oxidative stress than male rats. PMID:23617072

  19. Diet-induced hypercholesterolemia imparts structure-function changes to erythrocyte chondroitin sulphate/dermatan sulphate.

    PubMed

    Kiran, G; Srikanth, C B; Salimath, P V; Nandini, C D

    2015-09-01

    Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulphate/dermatan sulphate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. This study was carried out to determine whether diet-induced hypercholesterolemia had any effect on erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulphated and 4-O-sulphated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding. PMID:25862809

  20. Protective effect of quince (Cydonia oblonga Miller) fruit against oxidative hemolysis of human erythrocytes.

    PubMed

    Magalhães, Ana S; Silva, Branca M; Pereira, José A; Andrade, Paula B; Valentão, Patrícia; Carvalho, Márcia

    2009-06-01

    The aim of this study was to determine the phenolic content and evaluate the antioxidant activity of quince (Cydonia oblonga) fruit. For this purpose, fruits were separated into pulps, peels and seeds and methanolic extracts were prepared. The phenolic profiles were determined by HPLC/UV and antioxidant properties were studied for their ability to quench the stable free radical 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and to inhibit the 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced oxidative hemolysis of human erythrocytes. The main phenolic compounds were 5-O-caffeoylquinic acid for pulp and peel (57% and 29%, respectively) and stellarin-2 for seed (18%). Total phenolics content was 2.5, 6.3 and 0.4g/kg of methanolic extract for pulp, peel and seed, respectively. Pulp and peel extracts showed similar DPPH free radical scavenging activities (EC(50) of 0.6 and 0.8 mg/ml, respectively), while seed extract presented much lower antioxidant potential (EC(50) of 12.2mg/ml). Under the oxidative action of AAPH, pulp and peel extracts showed significant protection of the erythrocyte membrane from hemolysis, in a time- and concentration-dependent manner. Seed extracts by themselves induced extensive hemolysis. These results indicate higher antioxidant activity for certain parts of quince fruit, namely pulp and peel, that may therefore represent accessible sources of natural antioxidants with potential application in nutritional/pharmaceutical fields, as preventive or therapeutic agents in diseases in which free radicals are implicated. PMID:19306906

  1. Solubilization of native actin monomers from human erythrocyte membranes.

    PubMed

    Tilley, L; Dwyer, M; Ralston, G B

    1986-01-01

    Up to 50% of the actin in erythrocyte membranes can be solubilized at low ionic strength in a form capable of inhibiting DNAse I, in the presence of 0.4 mM ATP and 0.05 mM calcium. In the absence of calcium and ATP, actin is released but is apparently rapidly denatured. Solubilization of G-actin increases with temperature up to 37 degrees C. At higher temperatures, actin is released rapidly but quickly loses its ability to inhibit DNAse I. PMID:3789986

  2. Ameliorative effect of Opuntia ficus indica juice on ethanol-induced oxidative stress in rat erythrocytes.

    PubMed

    Alimi, Hichem; Hfaeidh, Najla; Bouoni, Zouhour; Sakly, Mohsen; Rhouma, Khémais Ben

    2013-05-01

    The aim of the present study was to investigate the efficacy of Opuntia ficus indica f. inermis fruit juice (OFIj) on reversing oxidative damages induced by chronic ethanol intake in rat erythrocytes. OFIj was firstly analyzed with HPLC for phenolic and flavonoids content. Secondly, 40 adult male Wistar rats were equally divided into five groups and treated for 90 days as follows: control (C), ethanol-only 3 g/kg body weight (b.w) (E), low dose of OFIj 2 ml/100 g b.w+ethanol (Ldj+E), high dose of OFIj 4 ml/100 g b.w+ethanol (Hdj+E), and only a high dose of OFIj 4 ml/100g b.w (Hdj). HPLC analysis indicated high concentrations of phenolic acids and flavonoids in OFIj. Ethanol treatment markedly decreased the activities of erythrocyte superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), and the level of reduced glutathione (GSH). Changes in the erythrocyte's antioxidant ability were accompanied by enhanced oxidative modification of lipids (increase of malondialdeyde level) and proteins (increase in carbonyl groups). Interestingly, pre-administration of either 2 ml/100 g b.w or 4 ml/100 g b.w of OFIj to ethanol-intoxicated rats significantly reversed decreases in enzymatic as well as non enzymatic antioxidants parameters in erythrocytes. Also, the administration of OFIj significantly protected lipids and proteins against ethanol-induced oxidative modifications in rat erythrocytes. The beneficial effect of OFIj can result from the inhibition of ethanol-induced free radicals chain reactions in rat erythrocytes or from the enhancement of the endogenous antioxidants activities. PMID:22285760

  3. Diffractomery analysis of human and rat erythrocytes deformability under ischemia

    NASA Astrophysics Data System (ADS)

    Lugovtsov, Andrei E.; Priezzhev, Alexander V.; Nikitin, Sergei Y.; Koshelev, Vladimir B.

    2007-07-01

    In this work, the analysis of human and rat red blood cells (RBC) deformability, internal viscosity and yield stress of RBC in norm and ischemia was performed by means of laser diffractometry - a modern technique allowing for measuring the flexibility of RBC, which determines the blood flow parameters in vessels. Ischemic diseases of people and animals are accompanied with deterioration of microrheologic properties of their blood, in particular, with impairing the RBC deformability. Human RBCs were obtained from the blood of healthy individuals and from patients suffering from ischemic diseases. The RBC deformability indices from both groups of individuals were measured. Rat RBCs were obtained from a control group of animals and from a group with experimentally induced ischemia (EII). This animal model is frequently used for studying the response of an organism to ischemia. The effect of semax, a medication that is frequently used for therapeutic treatments of human brain diseases in clinical practice, on RBC deformability was studied with its application in vitro and in vivo. It is shown that in human ischemic patients, the deformability index of RBC was lower than that from healthy individuals. Both in vivo and in vitro applied semax positively influences the impaired deformability properties of RBCs of ischemic rats.

  4. Recognition and invasion of human erythrocytes by malarial parasites: contribution of sialoglycoproteins to attachment and host specificity

    SciTech Connect

    Friedman, M.J.; Blankenberg, T.; Sensabaugh, G.; Tenforde, T.S.

    1984-05-01

    The receptivity of human erythrocytes to invasion by Plasmodium falciparum merozoites can be decreased by neuraminidase or trypsin treatment, an observation that supports a role for the erythrocyte sialoglycoproteins (glycophorins) in invasion. We have found that ..cap alpha../sub 1/-acid glycoprotein (AGP), added to in vitro cultures, can restore invasion of enzyme-treated human erythrocytes. AGP is structurally different from the glycophorins although it does carry 12% sialic acid. Its ability to restore receptivity to desialylated cells is dependent on its sialic acid complement, its concentration, and its binding to the erythrocyte surface. We present evidence that AGP forms a bridge between the merozoite and the enzyme-treated erythrocyte that allows the stronger and more complex interactions of invasion to proceed. We suggest that the glycophorins play the same role on the surface of the intact erythrocyte. 31 references, 3 figures, 6 tables.

  5. Recognition of Human Erythrocyte Receptors by the Tryptophan-Rich Antigens of Monkey Malaria Parasite Plasmodium knowlesi

    PubMed Central

    Tyagi, Kriti; Gupta, Deepali; Saini, Ekta; Choudhary, Shilpa; Jamwal, Abhishek; Alam, Mohd. Shoeb; Zeeshan, Mohammad; Tyagi, Rupesh K.; Sharma, Yagya D.

    2015-01-01

    Background The monkey malaria parasite Plasmodium knowlesi also infect humans. There is a lack of information on the molecular mechanisms that take place between this simian parasite and its heterologous human host erythrocytes leading to this zoonotic disease. Therefore, we investigated here the binding ability of P. knowlesi tryptophan-rich antigens (PkTRAgs) to the human erythrocytes and sharing of the erythrocyte receptors between them as well as with other commonly occurring human malaria parasites. Methods Six PkTRAgs were cloned and expressed in E.coli as well as in mammalian CHO-K1 cell to determine their human erythrocyte binding activity by cell-ELISA, and in-vitro rosetting assay, respectively. Results Three of six PkTRAgs (PkTRAg38.3, PkTRAg40.1, and PkTRAg67.1) showed binding to human erythrocytes. Two of them (PkTRAg40.1 and PkTRAg38.3) showed cross-competition with each other as well as with the previously described P.vivax tryptophan-rich antigens (PvTRAgs) for human erythrocyte receptors. However, the third protein (PkTRAg67.1) utilized the additional but different human erythrocyte receptor(s) as it did not cross-compete for erythrocyte binding with either of these two PkTRAgs as well as with any of the PvTRAgs. These three PkTRAgs also inhibited the P.falciparum parasite growth in in-vitro culture, further indicating the sharing of human erythrocyte receptors by these parasite species and the biological significance of this receptor-ligand interaction between heterologous host and simian parasite. Conclusions Recognition and sharing of human erythrocyte receptor(s) by PkTRAgs with human parasite ligands could be part of the strategy adopted by the monkey malaria parasite to establish inside the heterologous human host. PMID:26393350

  6. Effect of human plasma on the reactivation of sarin-inhibited human erythrocyte acetylcholinesterase.

    PubMed

    Worek, F; Eyer, P; Kiderlen, D; Thiermann, H; Szinicz, L

    2000-03-01

    The reactivation of organophosphate-inhibited acetylcholinesterase (AChE) by oximes inevitably results in the formation of highly reactive phosphoryloximes (POX), which are able to re-inhibit the enzyme. In this study, the dependence of POX formation on AChE concentration was investigated with sarin-inhibited human erythrocyte AChE (EryAChE). A marked dependence was found with obidoxime but not with the experimental oxime HI 6, suggesting great differences in the decomposition rates of the respective POXs. At a physiological erythrocyte content the reactivation of EryAChE was markedly affected by POX with obidoxime and pralidoxime (2-PAM) but not with the newer oximes HI 6 and HLö 7. Addition of extensively dialysed, sarin-treated human plasma reduced the reactivation by obidoxime and 2-PAM even more. Obidoxime and 2-PAM were superior to HI 6 and HLö 7 in reactivating butyrylcholinesterase (BChE). This effect was pronounced in diluted plasma, but was obscured in concentrated plasma, probably because of re-inhibition by the generated POX. Addition of native erythrocytes to sarin-treated plasma resulted in marked inhibition of EryAChE in the presence of obidoxime, suggesting a higher affinity of the POX for EryAChE. The results indicate that obidoxime and 2-PAM may reactivate sarin-inhibited AChE insufficiently due to re-inhibition by the POX formed. In addition, the re-inhibition of Ery-AChE may be aggravated by the POX that is produced during BChE reactivation. These reactions must be regarded as therapeutically detrimental and disqualify those oximes which are capable of forming stable POX by reactivation of BChE. PMID:10817663

  7. Effects of temperature on water diffusion in human erythrocytes and ghosts--nuclear magnetic resonance studies.

    PubMed

    Benga, G; Pop, V I; Popescu, O; Hodârnău, A; Borza, V; Presecan, E

    1987-12-11

    The temperature-dependence of water diffusion across human erythrocyte membrane was studied on isolated erythrocytes and resealed ghosts by a doping nuclear magnetic resonance technique. The conclusions are the following: (1) The storage of suspended erythrocytes at 2 degrees C up to 24 h or at 37 degrees C for 30 min did not change the water exchange time significantly, even if Mn2+ was present in the medium. This indicates that no significant penetration of Mn2+ is taking place under such conditions. (2) In case of cells previously incubated at 37 degrees C for longer than 30 min with concentrations of p-chloromercuribenzene sulfonate (PCMBS) greater than 0.5 mM, the water-exchange time gradually decreased if the cells were stored in the presence of Mn2+ for more than 10 min at 37 degrees C. (3) When the Arrhenius plot of the water-exchange time was calculated on the basis of measurements performed in such a way as to avoid a prolonged exposure of erythrocytes to Mn2+ no discontinuity occurred, regardless of the treatment with PCMBS. (4) No significant differences between erythrocytes and resealed ghosts regarding their permeability and the activation energy of water diffusion (Ea,d) were noticed. The mean value of Ea,d obtained on erythrocytes from 35 donors was 24.5 kJ/mol. (5) The value of Ea,d increased after treatment with PCMBS, in parallel with the percentage inhibition of water diffusion. A mean value of 41.3 kJ/mol was obtained for Ea,d of erythrocytes incubated with 1 mM PCMBS for 60 min at 37 degrees C and 28.3 kJ/mol for ghosts incubated with 0.1 mM PCMBS for 15 min, the values of inhibition being 46% and 21% respectively. PMID:2825782

  8. Chlorodinitrobenzene-mediated damage in the human erythrocyte membrane leads to haemolysis.

    PubMed

    Zou, Cheng-Gang; Agar, Nihal S; Jones, Graham Lloyd

    2002-07-01

    1-chloro-2,4-dinitrobenzene (CDNB), an intracellular glutathione-depleting agent, has been shown to have an adverse effect on erythrocyte membrane integrity. In the current study, we have demonstrated that CDNB caused haemolysis of human red blood cells (RBC) at higher concentrations (>or= 5 mM). The haemolysis induced by CDNB was preceded by the leakage of K(+) from the cells suggesting the colloid-osmotic nature of this lysis. The inclusion of molecules of increasing size in the extracellular media inhibited both the rate and extent of haemolysis thus supporting the proposal of CDNB-induced pore formation. The size of membrane lesions increased with an increase in the concentration of CDNB. SDS-PAGE demonstrated that CDNB causes the polymerisation and/or fragmentation of membrane proteins. Although CDNB has been shown to cause a drastic reduction in membrane thiols, our data suggest that the CDNB-induced formation of membrane disulfide bonds as a prima facie cause of permeability enhancement is unlikely. PMID:12074932

  9. [Raman spectra of single human living erythrocyte with the effect of pH and serum albumin].

    PubMed

    Wu, Zheng-Jie; Wang, Cheng; Lin, Zheng-Chun; Jiao, Qing-Ze

    2014-05-01

    In the present work, a cell environment which mimicked the real body environment according to the concentration radio between serum albumin and hemoglobin was built, and the cell morphology, the membrane deformation capacity, and the structure of intracellular hemoglobin of single human living erythrocyte under the effect of pH and serum albumin were studied. It was found that at different suspension pH, the magnitude of variations in cell shape and membrane deformation capacity changes with the structural changes of the intracellular hemoglobin. At pH 4. 14, 4. 76 and 10. 18, the loss of helical structure for hemoglobin, exposing of the hydrophobic amino acid in the globin chains, and changing of the combination of heme and globin, would completely destroy the stability of hemoglobin's structure, which seriously changes RBC's morphology and membrane deformation capacity. While at pH 6. 51 and 7. 80, the Raman spectra of erythrocytes are found to have no such changes, indicating that the structure of intracellular hemoglobin was not varied, thus the cell morphology and membrane deformation capacity are quite close to the normal values. At pH 5. 49 and 8. 76, RBC's morphology and membrane deformation capacity have different degrees of variation, but the structure of intracellular hemoglobin has not changed, suggesting that the cell morphology and membrane deformation capacity may be reversible. The results suggest that in the suspension solution containing serum albumin, erythrocytes have better ability to regulate and control the variation of the extracellular pH. In summary, upon building an environment which contains the same concentration radio of serum albumin to hemoglobin in the blood, this work performed systematic studies on the effect of pH on human erythrocytes. It can not only help to solve the problems about the mechanism of the structural and functional changes of erythrocytes induced by environmental pH, but also elucidates the possible variation of

  10. Inclusion bodies in loggerhead erythrocytes are associated with unstable hemoglobin and resemble human Heinz bodies.

    PubMed

    Basile, Filomena; Di Santi, Annalisa; Caldora, Mercedes; Ferretti, Luigi; Bentivegna, Flegra; Pica, Alessandra

    2011-08-01

    The aim of this study was to clarify the role of the erythrocyte inclusions found during the hematological screening of loggerhead population of the Mediterranean Sea. We studied the erythrocyte inclusions in blood specimens collected from six juvenile and nine adult specimens of the loggerhead turtle, Caretta caretta, from the Adriatic and Tyrrhenian Seas. Our study indicates that the percentage of mature erythrocytes containing inclusions ranged from 3 to 82%. Each erythrocyte contained only one round inclusion body. Inclusion bodies stained with May Grünwald-Giemsa show that their cytochemical and ultrastructure characteristics are identical to those of human Heinz bodies. Because Heinz bodies originate from the precipitation of unstable hemoglobin (Hb) and cause globular osmotic resistance to increase, we analyzed loggerhead Hb using electrophoresis and high-performance liquid chromatography to detect and quantitate Hb fractions. We also tested the resistance of Hb to alkaline pH, heat, isopropanol denaturation, and globular osmosis. Our hemogram results excluded the occurrence of any infection, which could be associated with an inclusion body, in all the specimens. Negative Feulgen staining indicated that the inclusion bodies are not derived from DNA fragmentation. We hypothesize that amino acid substitutions could explain why loggerhead Hb precipitates under normal physiologic conditions, forming Heinz bodies. The identification of inclusion bodies in loggerhead erythrocytes allow us to better understand the haematological characteristics and the physiology of these ancient reptiles, thus aiding efforts to conserve such an endangered species. PMID:21538919

  11. Purification of homologous protein carboxyl methyltransferase isozymes from human and bovine erythrocytes

    SciTech Connect

    Gilbert, J.M.; Fowler, A.; Bleibaum, J.; Clarke, S.

    1988-07-12

    The authors have purified the two major isozymes of the L-isoaspartyl/D-aspartyl protein methyltransferase from both human and bovine erythrocytes. These four enzymes all have polypeptide molecular weights of approximately 26,500 and appear to be monomers in solution. Each of these enzymes cross-reacts with antibodies directed against protein carboxyl methyltransferase I from bovine brain. Their structures also appear to be similar when analyzed by dodecyl sulfate gel electrophoresis for the large fragments produced by digestion with Staphylococcus aureus protease V8 or when analyzed by high-performance liquid chromatography (HPLC) for tryptic peptides. The structural relatedness of these enzymes was confirmed by sequence analysis of a total of 433 residues in 32 tryptic fragments of the human erythrocyte isozymes I and II and of the bovine erythrocyte isozyme II. They found sequence identity or probable identity in 111 out of 112 residues when they compared the human isozymes I and II and identities in 127 out of 134 residues when the human and bovine isozymes II were compared. These results suggest that the erythrocyte isozymes from both organisms may have nearly identical structures and confirm the similarities in the function of these methyltransferases that have been previously demonstrated.

  12. Biological Activity of Japanese Quince Extract and Its Interactions with Lipids, Erythrocyte Membrane, and Human Albumin.

    PubMed

    Strugała, Paulina; Cyboran-Mikołajczyk, Sylwia; Dudra, Anna; Mizgier, Paulina; Kucharska, Alicja Z; Olejniczak, Teresa; Gabrielska, Janina

    2016-06-01

    The aim of the study was to determine in vitro biological activity of fruit ethanol extract from Chaenomeles speciosa (Sweet) Nakai (Japanese quince, JQ) and its important constituents (-)-epicatechin (EC) and chlorogenic acid (CA). The study also investigated the structural changes in phosphatidylcholine (PC) liposomes, dipalmitoylphosphatidylcholine liposomes, and erythrocyte membranes (RBC) induced by the extract. It was found that the extract effectively inhibits oxidation of RBC, induced by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), and PC liposomes, induced by UVB radiation and AAPH. Furthermore, JQ extract to a significant degree inhibited the activity of the enzymes COX-1 and COX-2, involved in inflammatory reactions. The extract has more than 2 times greater activity in relation to COX-2 than COX-1 (selectivity ratio 0.48). JQ extract stimulated growth of the beneficial intestinal bacteria Lactobacillus casei and Lactobacillus plantarum. In the fluorimetric method by means of the probes Laurdan, DPH and TMA-DPH, and (1)H-NMR, we examined the structural changes induced by JQ and its EC and CA components. The results show that JQ and its components induce a considerable increase of the packing order of the polar heads of lipids with a slight decrease in mobility of the acyl chains. Lipid membrane rigidification could hinder the diffusion of free radicals, resulting in inhibition of oxidative damage induced by physicochemical agents. JQ extract has the ability to quench the intrinsic fluorescence of human serum albumin through static quenching. This report thus could be of huge significance in the food industry, pharmacology, and clinical medicine. PMID:26861057

  13. Age-dependent effects of He-Ne laser irradiation on the membrane fluidity of human erythrocytes

    NASA Astrophysics Data System (ADS)

    Kovacs, Eugenia; Savopol, Tudor; Pologea-Moraru, Roxana; Makropoulou, Mersini I.; Serafetinides, Alexander A.

    1997-12-01

    The low power He-Ne laser radiation has been extensively used in past decades as medical device to relieve pain, accelerate wound healing as well as aiming beam in invisible laser beam in invisible laser beam applications. It is not known however if there are any secondary, undesirable effects of He-Ne laser radiation on the irradiated tissue. In this paper we investigate the changes induced in membrane fluidity of human erythrocyte during/upon the interaction with the He-Ne laser beam having the parameters currently used for target aiming in laser surgery.

  14. Modulation of human erythrocyte shape and fatty acids by diet.

    PubMed

    Parsons, H G; Hill, R; Pencharz, P; Kuksis, A

    1986-08-21

    A semi-synthetic diet (Vivonex) was administered via nasogastric tube to three cystic fibrosis patients with pancreatic exocrine deficiency for 14 days to gain weight. Dietary essential fatty acids were provided as safflower oil, which constituted 1.3% of total calories. Plasma and red blood cells were analyzed for the content and composition of lipids at the start of the diet and at days 7 and 14 of the dietary period, and the results were correlated with the morphology of the cells. Feeding Vivonex to the patients led to an essential fatty acid deficiency, which was manifested in a 50% decrease in the linoleic acid content of the phosphatidylcholine of plasma and red blood cells at days 7 and 14 and in a 20% decrease in the linoleic acid content of red cell phosphatidylethanolamine at day 14. There was no significant alteration in the levels or composition of the other phospholipid classes and in the free cholesterol/phospholipid ratio. The decrease in the linoleic acid content of the erythrocytes was accompanied by a dramatic increase in the proportion of cells as echinocytes. We conclude that restricted linoleic acid availability in cystic fibrosis patients causes a change in red blood cell shape either directly by decreasing the linoleoylphosphatidylcholine content of the membrane or indirectly by affecting enzyme activity. PMID:3741859

  15. Identification of high affinity folate binding proteins in human erythrocyte membranes.

    PubMed

    Antony, A C; Kincade, R S; Verma, R S; Krishnan, S R

    1987-09-01

    Mature human erythrocyte membranes contained specific, high affinity (Kd 3.3 X 10(-11) M) folate binding moieties. Folate binding was pH, time- and temperature-dependent, saturable, and was much greater for pteroylmonoglutamate and 5-methyltetrahydrofolate than 5-formyltetrahydrofolate and amethopterin. On detergent solubilization of membranes, two peaks of specific folate binding with Mr greater than or equal to 200,000 and 160,000 were identified on Sephacryl S-200 gel filtration chromatography in Triton X-100, and this corresponded to two similar peaks of immunoprecipitated material when solubilized iodinated membranes were probed with anti-human placental folate receptor antiserum. Age-dependent separation of erythrocytes by Stractan density gradients revealed a sevenfold greater folate binding capacity in membranes purified from younger compared with aged erythrocytes. Since this difference was not reflected in proportionately higher immunoreactive folate binding protein, (as determined by a specific radioimmunoassay for these proteins), or differences in affinity in younger than aged cells, these findings indicate that erythrocyte folate binding proteins become progressively nonfunctional at the onset of red cell aging. PMID:3624486

  16. Derivativation of the human erythrocyte glucose transporter using a novel forskolin photoaffinity label

    SciTech Connect

    Wadzinski, B.; Shanahan, M.; Ruoho, A.

    1987-05-01

    An iodinated photoaffinity label for the glucose transporter, 3-iodo-4-azidophenethylamido-7-0-succinyldeacetyl-forskolin (IAPS-Fsk), has been synthesized, purified, and characterized. The K/sub i/ for inhibition of 3-0-methylglucose transport by TAPS-Fsk in human erythrocytes was found to be 0.1 uM. The carrier-free radioiodinated label has been shown to be a highly specific photoaffinity label for the human erythrocyte glucose transporter. Photolysis of erythrocyte membranes with 1-10 nM (I-125)IAPS-Fsk and analysis by SDS-PAGE showed specific derivatization of a broad band with an apparent molecular weight of 40-70 kDa. Photoincorporation using 2 nM (I-125)IAPS-Fsk was protected with D-glucose, cytochalasin B, and forskolin. No protection was observed with L-glucose. Endo-B-galactosidase digestion and trypsinization of (I-125)IAPS-Fsk labelled erythrocytes reduced the specifically radiolabelled transporter to 40 kDa and 18 kDa respectively. (I-125)-IAPS-Fsk will be used to study the structural aspects of the glucose transporter.

  17. Spontaneous and forced oscillations of cell membrane of normal human erythrocytes: absence of resonance frequencies in a range of 0.03-500 Hz.

    PubMed

    Kononenko, V L; Shimkus, J K

    2000-01-01

    The spectra of natural oscillations of human erythrocyte cell membranes were studied experimentally and theoretically. The measurements were carried out at room temperature for both single normal cells and erythrocyte rouleaux in a range of 0.03-500 Hz. The spectra were measured at a resolution better than 1% using two techniques: registration of spontaneous membrane oscillations induced by thermal agitation in the surrounding medium and registration of the amplitude-frequency characteristics of the forced oscillations of erythrocyte elongation induced by a high-frequency electric field with the amplitude harmonic modulation. The spectra measured by both techniques had no resonance frequencies and decreased monotonically with the frequency increase. These results are confirmed by the theory developed for the extracellular excitation mechanisms of membrane oscillations. The spectra of active oscillatory biomechanical processes were measured for comparison. These processes are ciliary beating of human bronchial epithelium and ciliary beating and artificial periodic contractions of the cytoplasm of the ciliate Spirostomum ambiguum. The quality of the resonance lines of the order of 10-20 registered may serve as estimates for the line width in search of the resonance oscillations in erythrocytes induced by active cell processes. PMID:11368497

  18. ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia

    PubMed Central

    Arashiki, Nobuto; Takakuwa, Yuichi; Mohandas, Narla; Hale, John; Yoshida, Kenichi; Ogura, Hiromi; Utsugisawa, Taiju; Ohga, Shouichi; Miyano, Satoru; Ogawa, Seishi; Kojima, Seiji; Kanno, Hitoshi

    2016-01-01

    Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca2+ concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient’s senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca2+-activated scrambling in both control erythrocytes and the patient’s erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes. PMID:26944472

  19. ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia.

    PubMed

    Arashiki, Nobuto; Takakuwa, Yuichi; Mohandas, Narla; Hale, John; Yoshida, Kenichi; Ogura, Hiromi; Utsugisawa, Taiju; Ohga, Shouichi; Miyano, Satoru; Ogawa, Seishi; Kojima, Seiji; Kanno, Hitoshi

    2016-05-01

    Phosphatidylserine is localized exclusively to the inner leaflet of the membrane lipid bilayer of most cells, including erythrocytes. This asymmetric distribution is critical for the survival of erythrocytes in circulation since externalized phosphatidylserine is a phagocytic signal for splenic macrophages. Flippases are P-IV ATPase family proteins that actively transport phosphatidylserine from the outer to inner leaflet. It has not yet been determined which of the 14 members of this family of proteins is the flippase in human erythrocytes. Herein, we report that ATP11C encodes a major flippase in human erythrocytes, and a genetic mutation identified in a male patient caused congenital hemolytic anemia inherited as an X-linked recessive trait. Phosphatidylserine internalization in erythrocytes with the mutant ATP11C was decreased 10-fold compared to that of the control, functionally establishing that ATP11C is a major flippase in human erythrocytes. Contrary to our expectations phosphatidylserine was retained in the inner leaflet of the majority of mature erythrocytes from both controls and the patient, suggesting that phosphatidylserine cannot be externalized as long as scramblase is inactive. Phosphatidylserine-exposing cells were found only in the densest senescent cells (0.1% of total) in which scramblase was activated by increased Ca(2+) concentration: the percentage of these phosphatidylserine-exposing cells was increased in the patient's senescent cells accounting for his mild anemia. Furthermore, the finding of similar extents of phosphatidylserine exposure by exogenous Ca(2+)-activated scrambling in both control erythrocytes and the patient's erythrocytes implies that suppressed scramblase activity rather than flippase activity contributes to the maintenance of phosphatidylserine in the inner leaflet of human erythrocytes. PMID:26944472

  20. Nanoscale Surface Characterization of Human Erythrocytes by Atomic Force Microscopy: A Critical Review.

    PubMed

    Mukherjee, Rashmi; Saha, Monjoy; Routray, Aurobinda; Chakraborty, Chandan

    2015-09-01

    Erythrocytes (red blood cells, RBCs), the most common type of blood cells in humans are well known for their ability in transporting oxygen to the whole body through hemoglobin. Alterations in their membrane skeletal proteins modify shape and mechanical properties resulting in several diseases. Atomic force microscopy (AFM), a new emerging technique allows non-invasive imaging of cell, its membrane and characterization of surface roughness at micrometer/nanometer resolution with minimal sample preparation. AFM imaging provides direct measurement of single cell morphology, its alteration and quantitative data on surface properties. Hence, AFM studies of human RBCs have picked up pace in the last decade. The aim of this paper is to review the various applications of AFM for characterization of human RBCs topology. AFM has been used for studying surface characteristics like nanostructure of membranes, cytoskeleton, microstructure, fluidity, vascular endothelium, etc., of human RBCs. Various modes of AFM imaging has been used to measure surface properties like stiffness, roughness, and elasticity. Topological alterations of erythrocytes in response to different pathological conditions have also been investigated by AFM. Thus, AFM-based studies and application of image processing techniques can effectively provide detailed insights about the morphology and membrane properties of human erythrocytes at nanoscale. PMID:25935044

  1. Cytochrome P{sub 450}-dependent toxic effects of primaquine on human erythrocytes

    SciTech Connect

    Ganesan, Shobana; Tekwani, Babu L.; Sahu, Rajnish; Tripathi, Lalit M.; Walker, Larry A.

    2009-11-15

    Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(-)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(-) erythrocytes, microsomes or recombinant cytochrome P{sub 450} (CYP) isoforms has allowed in situ generation of potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(-) and normal human erythrocytes. However, G6PD(-) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.

  2. Attenuation of erythrocyte membrane oxidative stress by Sesbania grandiflora in streptozotocin-induced diabetic rats.

    PubMed

    Sureka, Chandrabose; Ramesh, Thiyagarajan; Begum, Vavamohaideen Hazeena

    2015-08-01

    The aim of the present study was to investigate the protective effects of Sesbania grandiflora flower (SGF) extract on erythrocyte membrane in Streptozotocin (STZ)-induced diabetic rats. Adult male albino rats of Wistar strain, weighing 190-220 g, were made diabetic by an intraperitonial administration of STZ (45 mg/kg). Normal and diabetic rats were treated with SGF, and diabetic rats were also treated with glibenclamide as drug control, for 45 days. In this study plasma insulin and haemoglobin levels were decreased and blood glucose, glycosylated haemoglobin, protein oxidation, lipid peroxidation markers, and osmotic fragility levels were increased in diabetic rats. Moreover, erythrocytes antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxide, glutathione reductase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase activities and non-enzymatic antioxidants such as vitamin C, vitamin E, reduced glutathione (GSH), and oxidized glutathione (GSSG) levels were altered. Similarly, the activities of total ATPases, Na(+)/K(+)-ATPase, Ca(2+)-ATPase, and Mg(2+)-ATPase were also decreased in the erythrocytes of diabetic rats. Administration of SGF to STZ-induced diabetic rats reduced blood glucose and glycosylated haemoglobin levels with increased levels of insulin and haemoglobin. Moreover, SGF reversed the protein and lipid peroxidation markers, osmotic fragility, membrane-bound ATPases activities, and antioxidant status in STZ-induced diabetic rats. These results suggest that SGF could provide a protective effect on diabetes by decreasing oxidative stress-associated diabetic complications. PMID:26176361

  3. Radiation-induced micronuclei in peripheral erythrocytes of Rana catesbeiana: an aquatic animal model for in vivo genotoxicity studies

    SciTech Connect

    Krauter, P.W.; Anderson, S.L.; Harrison, F.L.

    1987-01-01

    An in vivo micronucleus assay for peripheral erythrocytes of Rana catesbeiana tadpoles was developed and evaluated. The assay was used to determine the spontaneous frequency of micronuclei in circulating erythrocytes in tadpoles from two different populations, to define the time from administering the clastogen to the maximum micronucleus frequency in peripheral erythrocytes, and to determine the response to radiation. The spontaneous frequency of micronuclei in circulating erythrocytes of early-stage tadpoles was low (3.6 +/- 2.8 micronuclei per 1,000 erythrocytes, MN o/oo), but higher than that of late-stage tadpoles (1.7 +/- 0.7 MN o/oo). The time from the exposure of early-stage tadpoles to radiation (2.1 Gy) to the maximum micronucleus frequency was about 2 wk. The increase in frequency of micronuclei in peripheral erythrocytes of late-stage tadpoles receiving doses ranging from 0.5 to 3.0 Gy was linear with dose; a 3-fold increase was obtained with a dose of 3.0 Gy. The spontaneous frequency of micronuclei in erythrocytes and the increase in frequency induced by radiation appeared to differ in tadpoles from different populations. Quantification of micronuclei in the peripheral erythrocytes of R catesbeiana tadpoles provides a promising whole-animal system for studies of genotoxicity in aquatic environments.

  4. Facilitated uptake of a bioactive metabolite of maritime pine bark extract (pycnogenol) into human erythrocytes.

    PubMed

    Kurlbaum, Max; Mülek, Melanie; Högger, Petra

    2013-01-01

    Many plant secondary metabolites exhibit some degree of biological activity in humans. It is a common observation that individual plant-derived compounds in vivo are present in the nanomolar concentration range at which they usually fail to display measurable activity in vitro. While it is debatable that compounds detected in plasma are not the key effectors of bioactivity, an alternative hypothesis may take into consideration that measurable concentrations also reside in compartments other than plasma. We analysed the binding of constituents and the metabolite δ-(3,4-dihydroxy-phenyl)-γ-valerolactone (M1), that had been previously detected in plasma samples of human consumers of pine bark extract Pycnogenol, to human erythrocytes. We found that caffeic acid, taxifolin, and ferulic acid passively bind to red blood cells, but only the bioactive metabolite M1 revealed pronounced accumulation. The partitioning of M1 into erythrocytes was significantly diminished at higher concentrations of M1 and in the presence of glucose, suggesting a facilitated transport of M1 via GLUT-1 transporter. This concept was further supported by structural similarities between the natural substrate α-D-glucose and the S-isomer of M1. After cellular uptake, M1 underwent further metabolism by conjugation with glutathione. We present strong indication for a transporter-mediated accumulation of a flavonoid metabolite in human erythrocytes and subsequent formation of a novel glutathione adduct. The physiologic role of the adduct remains to be elucidated. PMID:23646194

  5. The Cell Adhesion Molecule “CAR” and Sialic Acid on Human Erythrocytes Influence Adenovirus In Vivo Biodistribution

    PubMed Central

    Wodrich, Harald; Billet, Olivier; Perreau, Matthieu; Hippert, Claire; Mennechet, Franck; Schoehn, Guy; Lortat-Jacob, Hugues; Dreja, Hanna; Ibanes, Sandy; Kalatzis, Vasiliki; Wang, Jennifer P.; Finberg, Robert W.; Cusack, Stephen; Kremer, Eric J.

    2009-01-01

    Although it has been known for 50 years that adenoviruses (Ads) interact with erythrocytes ex vivo, the molecular and structural basis for this interaction, which has been serendipitously exploited for diagnostic tests, is unknown. In this study, we characterized the interaction between erythrocytes and unrelated Ad serotypes, human 5 (HAd5) and 37 (HAd37), and canine 2 (CAV-2). While these serotypes agglutinate human erythrocytes, they use different receptors, have different tropisms and/or infect different species. Using molecular, biochemical, structural and transgenic animal-based analyses, we found that the primary erythrocyte interaction domain for HAd37 is its sialic acid binding site, while CAV-2 binding depends on at least three factors: electrostatic interactions, sialic acid binding and, unexpectedly, binding to the coxsackievirus and adenovirus receptor (CAR) on human erythrocytes. We show that the presence of CAR on erythrocytes leads to prolonged in vivo blood half-life and significantly reduced liver infection when a CAR-tropic Ad is injected intravenously. This study provides i) a molecular and structural rationale for Ad–erythrocyte interactions, ii) a basis to improve vector-mediated gene transfer and iii) a mechanism that may explain the biodistribution and pathogenic inconsistencies found between human and animal models. PMID:19119424

  6. Proteomic Profiling of Nonenzymatically Glycated Proteins in Human Plasma and Erythrocyte Membrane

    SciTech Connect

    Zhang, Qibin; Tang, Ning; Schepmoes, Athena A.; Phillips, Lawrence S.; Smith, Richard D.; Metz, Thomas O.

    2008-05-01

    Non-enzymatic glycation of peptides and proteins by D-glucose has important implications in the pathogenesis of diabetes mellitus, particularly in the development of diabetic complications. In this report, a thorough proteomic profiling of glycated proteins was attempted by using phenylboronate affinity chromatography to enrich glycated proteins and glycated, tryptic peptides from human plasma and erythrocyte membranes. Enriched peptides were subsequently analyzed by liquid chromatography coupled with electron transfer dissociation tandem mass spectrometry, and 76 and 31 proteins were confidently identified as glycated from human plasma and erythrocyte membrane, respectively. It was observed that most of the glycated proteins can be identified in samples from individuals with normal glucose tolerance, although samples from individuals with impaired glucose tolerance and type 2 diabetes mellitus have slightly higher numbers of glycated proteins and more glycation sites identified.

  7. Some relationships between membrane phospholipid domains, conformational order, and cell shape in intact human erythrocytes.

    PubMed

    Moore, D J; Gioioso, S; Sills, R H; Mendelsohn, R

    1999-01-01

    A novel method developed in this laboratory [D.J. Moore et al., Biochemistry 35 (1996) 229-235; D.J. Moore et al., Biochemistry 36 (1997) 660-664] to study the conformational order and the propensity for domain formation of specific phospholipids in intact human erythrocytes is extended to two additional species. Acyl chain perdeuterated 1,2-dilauroylphosphatidylethanolamine (diC12PE-d46) was incorporated preferentially (in separate experiments) into the inner leaflet of stomatocytic erythrocytes and into the outer leaflet of echinocytic erythrocytes, while acyl chain perdeuterated 1,2-dipentadecanoylphosphatidylcholine (diC15PC-d58) was incorporated into the outer leaflet of echinocytic erythrocytes. The conformational order and phase behavior of the incorporated molecules were monitored through FT-IR studies of the temperature dependence of the CD2 stretching vibrations. For both diC12PE-d46 and diC15PC-d58, the gel-->liquid crystal phase transition persisted when these lipids were located in the outer leaflet of echinocytic cells, a result indicative of the persistence of phospholipid domains. In each case, the transition widths were broadened compared to the pure lipids, suggestive of either small domains or the presence of additional molecular components within the domains. The conformational order of diC12PE-d46 differed markedly depending on its location and the morphology of the cells. When located predominantly in the inner membrane of stomatocytes, the phase transition of this species was abolished and the conformational order compared with pure lipid vesicles at the same temperature was much lower. The current results along with our previous studies provide a sufficient experimental basis to deduce some general principles of phospholipid conformational order and organization in both normal and shape-altered erythrocytes. PMID:9889394

  8. Modulatory role of Emblica officinalis against alcohol induced biochemical and biophysical changes in rat erythrocyte membranes.

    PubMed

    Reddy, Vaddi Damodara; Padmavathi, Pannuru; Paramahamsa, Maturu; Varadacharyulu, Nallanchakravarthula

    2009-08-01

    This study investigated the protective effect of Emblica officinalis against alcohol-induced biochemical and biophysical changes in rat erythrocyte membranes. Thirty-two male rats were divided into four groups (n=8 in each group): control (C), alcohol (A), alcohol plus Emblica fruit extract (A+EFE) and Emblica fruit extract (EFE) alone. Administration of twenty percent alcohol (5 g/kg body weight) to rats significantly increased cholesterol/phospholipid (C/P) ratio, lipid peroxidation and the activities of Na(+)/K(+) and Mg(2+) ATPases in erythrocyte membranes as well as augmented nitric oxide (NO) levels. However, membrane fluidity studies using the fluorescent probe DPH (1,6 diphenyl 1,3 hexatriene) reveals that alcohol administration significantly (p<0.05) increased membrane anisotropic values and altered membrane individual phospholipid content. Administration of EFE (250 mg/kg body weight) to alcoholic rats resulted in significant (p<0.05) reduction of NO levels, erythrocyte membrane lipid peroxidation, C/P ratio, activities of Na(+)/K(+) and Mg(2+) ATPases and fluorescent anisotropic values. Further, EFE administration to alcoholic rats beneficially modulated membrane properties as evidenced from the contents of total phospholipids as well individual phospholipid classes. The tannoid principles present in Emblica offers protection against alcohol induced adverse effects in rats. PMID:19454300

  9. Facilitated transport of amino acids across organic phases and the human erythrocyte membrane.

    PubMed Central

    Hider, R C; McCormack, W

    1980-01-01

    1. An artificial facilitated amino-acid-transfer process operating across a chloroform phase is reported. 2. This process utilizes a family of bis(salicylamidato)copper(II) complexes. 3. A mechanism is proposed for this process and for its sensitivity towards cyanide and bathophenanthroline sulphonate. 4. Facilitated transfer of L-leucine in human erythrocytes has been shown to be inhibited by bathophenanthroline sulphonate. PMID:7396879

  10. Carbamazepine-induced hemolytic and aplastic crises associated with reduced glutathione peroxidase activity of erythrocytes.

    PubMed

    Yamamoto, Masaki; Suzuki, Nobuhiro; Hatakeyama, Naoki; Kubo, Noriaki; Tachi, Nobutada; Kanno, Hitoshi; Fujii, Hisaichi; Tsutsumi, Hiroyuki

    2007-11-01

    Although pure red cell aplasia is a well-known side effect of carbamazepine treatment, intravascular hemolytic anemia is rare. We describe a 5-year-old boy who developed concurrent intravascular hemolytic anemia and erythroblastopenia, probably due to carbamazepine. Carbamazepine treatment was subsequently discontinued, and the patient was treated with red blood cell transfusions, haptoglobin, and methylprednisolone. His hematologic abnormalities were almost fully recovered within 2 weeks. Examination of the patient's and mother's erythrocyte enzyme activities revealed mildly decreased erythrocyte glutathione peroxidase (GSH-Px) activity. We speculate that patients with reduced GSH-Px activity are at a high risk of developing carbamazepine-induced hemolytic crisis and/or aplastic crisis. PMID:18055338

  11. Glycophorin and the concanavalin A receptor of human erythrocytes: their receptor function in lipid bilayers.

    PubMed Central

    Sharom, F J; Barratt, D G; Grant, C W

    1977-01-01

    Two integral glycoproteins from the human erythrocyte have been studied after their incorporation into lipid bilayer systems. Glycophorin (which is the M/N blood group determinant) and the concanavalin A receptor were isolated and purified prior to incorporation into model membranes by dialytic removal of detergent from lipid/protein solutions. Under the conditions described, glycoprotein receptors maintain their function in that they bind external agents specific for them, such as concanavalin A and immunoglobulins. So-called intramembranous particles are a feature of freeze-fractured preparations of lipid bilayers containing either (or both) glycoprotein(s), and to some extent each has a characteristic particle appearance. Liposomes containing the concanavalin A receptor (with or without glycophorin) are agglutinable by concanavalin A, whereas human erythrocytes are normally considered to be nonagglutinable by this lectin. Liposomes containing glycophorin alone are readily agglutinable by the appropriate glycophorin-directed M/N antiserum, as are human erythrocytes. The added presence of concanavalin A receptor in the liposomes can markedly inhibit agglutination by M/N antiserum without preventing immunoglobulin binding. Images PMID:268624

  12. LC-MS/MS analysis of carboxymethylated and carboxyethylated phosphatidylethanolamines in human erythrocytes and blood plasma[S

    PubMed Central

    Shoji, Naoki; Nakagawa, Kiyotaka; Asai, Akira; Fujita, Ikuko; Hashiura, Aya; Nakajima, Yasushi; Oikawa, Shinichi; Miyazawa, Teruo

    2010-01-01

    An amino group of phosphatidylethanolamine (PE) is considered as a target for nonenzymatic glycation, and the potential involvement of lipid glycation in the pathogenesis of diabetic complications has generated interest. However, unlike an early glycation product of PE (Amadori-PE), the occurrence and roles of advanced glycation end products of PE (AGE-PE) in vivo have been unclear. Here, we developed an LC-MS/MS method for the analysis of AGE-PE [carboxymethyl-PE (CM-PE) and carboxyethyl-PE (CE-PE)]. Collision-induced dissociation of CM-PE and CE-PE produced characteristic ions, permitting neutral loss scanning (NLS) and multiple reaction monitoring (MRM) of AGE-PE. By NLS analysis, a series of AGE-PE molecular species was detected in human erythrocytes and blood plasma. In LC-MS/MS analysis, MRM enabled the separation and determination of the predominant AGE-PE species. Between healthy subjects and diabetic patients, no significant differences were observed in AGE-PE concentrations in erythrocytes and plasma, whereas Amadori-PE concentrations were higher in diabetic patients. These results provide direct evidence for the presence of AGE-PE in human blood, and indicated that, compared with Amadori-PE, AGE-PE is less likely to be accumulated in diabetic blood. The presently developed LC-MS/MS method appears to be a powerful tool for understanding in vivo lipid glycation and its pathophysiological consequence. PMID:20386060

  13. Mathematical modeling of electro-rotation spectra of small particles in liquid solutions: application to human erythrocyte aggregates.

    PubMed

    Zehe, A; Ramírez, A; Starostenko, O

    2004-02-01

    Electro-rotation can be used to determine the dielectric properties of cells, as well as to observe dynamic changes in both dielectric and morphological properties. Suspended biological cells and particles respond to alternating-field polarization by moving, deforming or rotating. While in linearly polarized alternating fields the particles are oriented along their axis of highest polarizability, in circularly polarized fields the axis of lowest polarizability aligns perpendicular to the plane of field rotation. Ellipsoidal models for cells are frequently applied, which include, beside sphere-shaped cells, also the limiting cases of rods and disks. Human erythrocyte cells, due to their particular shape, hardly resemble an ellipsoid. The additional effect of rouleaux formation with different numbers of aggregations suggests a model of circular cylinders of variable length. In the present study, the induced dipole moment of short cylinders was calculated and applied to rouleaux of human erythrocytes, which move freely in a suspending conductive medium under the effect of a rotating external field. Electro-rotation torque spectra are calculated for such aggregations of different length. Both the maximum rotation speeds and the peak frequencies of the torque are found to depend clearly on the size of the rouleaux. While the rotation speed grows with rouleaux length, the field frequency nu(p) is lowest for the largest cell aggregations where the torque shows a maximum. PMID:14762571

  14. Morphological and Molecular Descriptors of the Developmental Cycle of Babesia divergens Parasites in Human Erythrocytes

    PubMed Central

    Rossouw, Ingrid; Maritz-Olivier, Christine; Niemand, Jandeli; van Biljon, Riette; Smit, Annel; Olivier, Nicholas A.; Birkholtz, Lyn-Marie

    2015-01-01

    Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries. PMID:25955414

  15. Erythrocyte Sialic Acid Content during Aging in Humans: Correlation with Markers of Oxidative Stress

    PubMed Central

    Mehdi, Mohammad Murtaza; Singh, Prabhakar; Rizvi, Syed Ibrahim

    2012-01-01

    Sialic acids are substituted neuraminic acid derivatives which are typically found at the outermost end of glycan chains on the membrane in all cell types. The role of erythrocyte membrane sialic acids during aging has been established however the relationship between sialic acid and oxidative stress is not fully understood. The present work was undertaken to analyze the relationship between erythrocyte membrane sialic acid with its plasma level, membrane and plasma lipid hydroperoxide levels and plasma total antioxidant capacity. Results show that sialic acid content decreases significantly (P < 0.001) in RBC membrane (r = −0.901) and increases in plasma (r = 0.860) as a function of age in humans. Lipid peroxidation measured in the form of hydroperoxides increases significantly (P < 0.001) in plasma (r = 0.830) and RBC membranes (r = 0.875) with age in humans. The Trolox Equivalent Total Antioxidant Capacity (TETAC) of plasma was found to be significantly decreased (P < 0.001, r = −0.844). We observe significant correlations between decrease of erythrocyte membrane sialic acid and plasma lipid hydroperoxide and TETAC. Based on the observed correlations, we hypothesize that increase in oxidative stress during aging may influence the sialic acid decomposition from membrane thereby altering the membrane configuration affecting many enzymatic and transporter activities. Considering the importance of plasma sialic acid as a diagnostic parameter, it is important to establish age-dependent reference. PMID:22377734

  16. Morphological and Molecular Descriptors of the Developmental Cycle of Babesia divergens Parasites in Human Erythrocytes.

    PubMed

    Rossouw, Ingrid; Maritz-Olivier, Christine; Niemand, Jandeli; van Biljon, Riette; Smit, Annel; Olivier, Nicholas A; Birkholtz, Lyn-Marie

    2015-05-01

    Human babesiosis, especially caused by the cattle derived Babesia divergens parasite, is on the increase, resulting in renewed attentiveness to this potentially life threatening emerging zoonotic disease. The molecular mechanisms underlying the pathophysiology and intra-erythrocytic development of these parasites are poorly understood. This impedes concerted efforts aimed at the discovery of novel anti-babesiacidal agents. By applying sensitive cell biological and molecular functional genomics tools, we describe the intra-erythrocytic development cycle of B. divergens parasites from immature, mono-nucleated ring forms to bi-nucleated paired piriforms and ultimately multi-nucleated tetrads that characterizes zoonotic Babesia spp. This is further correlated for the first time to nuclear content increases during intra-erythrocytic development progression, providing insight into the part of the life cycle that occurs during human infection. High-content temporal evaluation elucidated the contribution of the different stages to life cycle progression. Moreover, molecular descriptors indicate that B. divergens parasites employ physiological adaptation to in vitro cultivation. Additionally, differential expression is observed as the parasite equilibrates its developmental stages during its life cycle. Together, this information provides the first temporal evaluation of the functional transcriptome of B. divergens parasites, information that could be useful in identifying biological processes essential to parasite survival for future anti-babesiacidal discoveries. PMID:25955414

  17. Influence of magnesium sulfate on HCO3/Cl transmembrane exchange rate in human erythrocytes.

    PubMed

    Chernyshova, Ekaterina S; Zaikina, Yulia S; Tsvetovskaya, Galina A; Strokotov, Dmitry I; Yurkin, Maxim A; Serebrennikova, Elena S; Volkov, Leonid; Maltsev, Valeri P; Chernyshev, Andrei V

    2016-03-21

    Magnesium sulfate (MgSO4) is widely used in medicine but molecular mechanisms of its protection through influence on erythrocytes are not fully understood and are considerably controversial. Using scanning flow cytometry, in this work for the first time we observed experimentally (both in situ and in vitro) a significant increase of HCO3(-)/Cl(-) transmembrane exchange rate of human erythrocytes in the presence of MgSO4 in blood. For a quantitative analysis of the obtained experimental data, we introduced and verified a molecular kinetic model, which describes activation of major anion exchanger Band 3 (or AE1) by its complexation with free intracellular Mg(2+) (taking into account Mg(2+) membrane transport and intracellular buffering). Fitting the model to our in vitro experimental data, we observed a good correspondence between theoretical and experimental kinetic curves that allowed us to evaluate the model parameters and to estimate for the first time the association constant of Mg(2+) with Band 3 as KB~0.07mM, which is in agreement with known values of the apparent Mg(2+) dissociation constant (from 0.01 to 0.1mM) that reflects experiments on enrichment of Mg(2+) at the inner erythrocyte membrane (Gunther, 2007). Results of this work partly clarify the molecular mechanisms of MgSO4 action in human erythrocytes. The method developed allows one to estimate quantitatively a perspective of MgSO4 treatment for a patient. It should be particularly helpful in prenatal medicine for early detection of pathologies associated with the risk of fetal hypoxia. PMID:26780645

  18. Radiation-induced micronuclei in peripheral erythrocytes of Rana catesbeiana: an aquatic animal model for in vivo genotoxicity studies

    SciTech Connect

    Krauter, P.W.; Anderson, S.L.; Harrison, F.L.

    1987-01-01

    An in vivo micronucleus assay for peripheral erythrocytes of Rana catesbeiana tadpoles was developed and evaluated. The assay was used to determine the spontaneous frequency of micronuclei in circulating erythrocytes in tadpoles from two different populations, to define the time from administering the clastogen to the maximum micronucleus frequency in peripheral erythrocytes, and to determine the response to radiation. The spontaneous frequency of micronuclei in circulating erythrocytes of early-stage tadpoles was low, but higher than that of late-stage tadpoles. The time from the exposure of early-stage tadpoles to radiation (2.1 Gy) to the maximum micronucleus frequency was about 2 wk. The increase in frequency of micronuclei in peripheral erythrocytes of late-stage tadpoles receiving doses ranging from 0.5 to 3.0 Gy was linear with dose; a 3-fold increase was obtained with a dose of 3.0 Gy. The spontaneous frequency of micronuclei in erythrocytes and the increase in frequency induced by radiation appeared to differ in tadpoles from different populations. Quantification of micronuclei in the peripheral erythrocytes of R castesbeiana tadpoles provides a promising whole-animal system for studies of genotoxicity in aquatic environments.

  19. Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes.

    PubMed

    Udomsangpetch, Rachanee; Pipitaporn, Busaba; Silamut, Kamolrat; Pinches, Robert; Kyes, Sue; Looareesuwan, Sornchai; Newbold, Christopher; White, Nicholas J

    2002-09-01

    In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40 degrees C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37 degrees C did not. Heating to 40 degrees C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37 degrees C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance. PMID:12177447

  20. Neisseria meningitidis and Escherichia coli are protected from leukocyte phagocytosis by binding to erythrocyte complement receptor 1 in human blood.

    PubMed

    Brekke, Ole-Lars; Hellerud, Bernt Christian; Christiansen, Dorte; Fure, Hilde; Castellheim, Albert; Nielsen, Erik Waage; Pharo, Anne; Lindstad, Julie Katrine; Bergseth, Grethe; Leslie, Graham; Lambris, John D; Brandtzaeg, Petter; Mollnes, Tom Eirik

    2011-09-01

    The initial interaction of Gram-negative bacteria with erythrocytes and its implications on leukocyte phagocytosis and oxidative burst in human whole blood were examined. Alexa-labeled Escherichia coli, wild-type H44/76 N. meningitidis and the H44/76lpxA lipopolysaccharide (LPS)-deficient mutant were incubated with whole blood using lepirudin as anticoagulant which has no adverse effects on complement. Bacteria free in plasma, bound to erythrocytes or phagocytized by granulocytes and monocytes were quantified using flow cytometry. The effects of the C3 inhibitor compstatin, a C5a receptor antagonist (C5aRa) and a complement receptor 1 (CR1)-blocking antibody (3D9) were examined. Most bacteria (80%) immediately bound to erythrocytes. The binding gradually declined over time, with a parallel increase in phagocytosis. Complement inhibition with compstatin reduced erythrocyte binding and bacterial C3 opsonization. In contrast, the C5aRa efficiently reduced phagocytosis, but did not affect the binding of bacteria to erythrocytes. The anti-CR1 blocking mAb dose-dependently reduced bacterial binding to erythrocytes to nil, with subsequent increased phagocytosis and oxidative burst. LPS had no effect on these processes since similar results were obtained using an LPS-deficient N. meningitidis mutant. In vivo experiments in a pig model of sepsis showed limited binding of bacteria to erythrocytes, consistent with the facts that erythrocyte CR1 receptors are absent in non-primates and that the bacteria were mainly found in the lungs. In conclusion, complement-dependent binding of Gram-negative bacteria to erythrocyte CR1 decreases phagocytosis and oxidative burst by leukocytes in human whole blood. PMID:21839519

  1. Plasmodium falciparum Adhesins Play an Essential Role in Signalling and Activation of Invasion into Human Erythrocytes

    PubMed Central

    Tham, Wai-Hong; Lim, Nicholas T. Y.; Weiss, Greta E.; Lopaticki, Sash; Ansell, Brendan R. E.; Bird, Megan; Lucet, Isabelle; Dorin-Semblat, Dominique; Doerig, Christian; Gilson, Paul R.; Crabb, Brendan S.; Cowman, Alan F.

    2015-01-01

    The most severe form of malaria in humans is caused by the protozoan parasite Plasmodium falciparum. The invasive form of malaria parasites is termed a merozoite and it employs an array of parasite proteins that bind to the host cell to mediate invasion. In Plasmodium falciparum, the erythrocyte binding-like (EBL) and reticulocyte binding-like (Rh) protein families are responsible for binding to specific erythrocyte receptors for invasion and mediating signalling events that initiate active entry of the malaria parasite. Here we have addressed the role of the cytoplasmic tails of these proteins in activating merozoite invasion after receptor engagement. We show that the cytoplasmic domains of these type 1 membrane proteins are phosphorylated in vitro. Depletion of PfCK2, a kinase implicated to phosphorylate these cytoplasmic tails, blocks P. falciparum invasion of red blood cells. We identify the crucial residues within the PfRh4 cytoplasmic domain that are required for successful parasite invasion. Live cell imaging of merozoites from these transgenic mutants show they attach but do not penetrate erythrocytes implying the PfRh4 cytoplasmic tail conveys signals important for the successful completion of the invasion process. PMID:26694741

  2. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes

    PubMed Central

    Clafshenkel, William P.; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Russell, Alan J.

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system. PMID:27331401

  3. Antibodies against a Plasmodium falciparum antigen PfMSPDBL1 inhibit merozoite invasion into human erythrocytes.

    PubMed

    Sakamoto, Hirokazu; Takeo, Satoru; Maier, Alexander G; Sattabongkot, Jetsumon; Cowman, Alan F; Tsuboi, Takafumi

    2012-03-01

    One approach to develop a malaria blood-stage vaccine is to target proteins that play critical roles in the erythrocyte invasion of merozoites. The merozoite surface proteins (MSPs) and the erythrocyte-binding antigens (EBAs) are considered promising vaccine candidates, for they are known to play important roles in erythrocyte invasion and are exposed to host immune system. Here we focused on a Plasmodium falciparum antigen, PfMSPDBL1 (encoded by PF10_0348 gene) that is a member of the MSP3 family and has both Duffy binding-like (DBL) domain and secreted polymorphic antigen associated with merozoites (SPAM) domain. Therefore, we aimed to characterize PfMSPDBL1 as a vaccine candidate. Recombinant full-length protein (rFL) of PfMSPDBL1 was synthesized by a wheat germ cell-free system, and rabbit antiserum was raised against rFL. We show that rabbit anti-PfMSPDBL1 antibodies inhibited erythrocyte invasion of wild type parasites in vitro in a dose dependent manner, and the specificity of inhibitory activity was confirmed using PfMSPDBL1 knockout parasites. Pre-incubation of the anti-PfMSPDBL1 antibodies with the recombinant SPAM domain had no effect on the inhibitory activity suggesting that antibodies to this region were not involved. In addition, antibodies to rFL were elicited by P. falciparum infection in malaria endemic area, suggesting the PfMSLDBL1 is immunogenic to humans. Our results suggest that PfMSPDBL1 is a novel blood-stage malaria vaccine candidate. PMID:22248820

  4. Protection of Clitoria ternatea flower petal extract against free radical-induced hemolysis and oxidative damage in canine erythrocytes.

    PubMed

    Phrueksanan, Wathuwan; Yibchok-anun, Sirinthorn; Adisakwattana, Sirichai

    2014-10-01

    The present study assessed the antioxidant activity and protective ability of Clitoria ternatea flower petal extract (CTE) against in vitro 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH)-induced hemolysis and oxidative damage of canine erythrocytes. From the phytochemical analysis, CTE contained phenolic compounds, flavonoids, and anthocyanins. In addition, CTE showed antioxidant activity as measured by oxygen radical absorbance capacity (ORAC) method and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. CTE (400 µg/ml) remarkably protected erythrocytes against AAPH-induced hemolysis at 4 h of incubation. Moreover, CTE (400 µg/ml) reduced membrane lipid peroxidation and protein carbonyl group formation and prevented the reduction of glutathione concentration in AAPH-induced oxidation of erythrocytes. The AAPH-induced morphological alteration of erythrocytes from a smooth discoid to an echinocytic form was effectively protected by CTE. The present results contribute important insights that CTE may have the potential to act as a natural antioxidant to prevent free radical-induced hemolysis, protein oxidation and lipid peroxidation in erythrocytes. PMID:25241390

  5. Dynamic Regulation of Cell Volume and Extracellular ATP of Human Erythrocytes

    PubMed Central

    Leal Denis, M. Florencia; Alvarez, H. Ariel; Lauri, Natalia; Alvarez, Cora L.; Chara, Osvaldo; Schwarzbaum, Pablo J.

    2016-01-01

    Introduction The peptide mastoparan 7 (MST7) triggered in human erythrocytes (rbcs) the release of ATP and swelling. Since swelling is a well-known inducer of ATP release, and extracellular (ATPe), interacting with P (purinergic) receptors, can affect cell volume (Vr), we explored the dynamic regulation between Vr and ATPe. Methods and Treatments We made a quantitative assessment of MST7-dependent kinetics of Vr and of [ATPe], both in the absence and presence of blockers of ATP efflux, swelling and P receptors. Results In rbcs 10 μM MST7 promoted acute, strongly correlated changes in [ATPe] and Vr. Whereas MST7 induced increases of 10% in Vr and 190 nM in [ATPe], blocking swelling in a hyperosmotic medium + MST7 reduced [ATPe] by 40%. Pre-incubation of rbcs with 10 μM of either carbenoxolone or probenecid, two inhibitors of the ATP conduit pannexin 1, reduced [ATPe] by 40–50% and swelling by 40–60%, while in the presence of 80 U/mL apyrase, an ATPe scavenger, cell swelling was prevented. While exposure to 10 μM NF110, a blocker of ATP-P2X receptors mediating sodium influx, reduced [ATPe] by 48%, and swelling by 80%, incubation of cells in sodium free medium reduced swelling by 92%. Analysis and Discussion Results were analyzed by means of a mathematical model where ATPe kinetics and Vr kinetics were mutually regulated. Model dependent fit to experimental data showed that, upon MST7 exposure, ATP efflux required a fast 1960-fold increase of ATP permeability, mediated by two kinetically different conduits, both of which were activated by swelling and inactivated by time. Both experimental and theoretical results suggest that, following MST7 exposure, ATP is released via two conduits, one of which is mediated by pannexin 1. The accumulated ATPe activates P2X receptors, followed by sodium influx, resulting in cell swelling, which in turn further activates ATP release. Thus swelling and P2X receptors constitute essential components of a positive feedback loop

  6. Physical modeling of human skin optical properties using milk and erythrocytes mixtures

    NASA Astrophysics Data System (ADS)

    Pravdin, Alexander B.; Utz, Sergei R.; Kochubey, Vyacheslav I.

    1995-12-01

    We offer a two-layer dermis-like model phantom with controlled concentration of absorbers and scatterers; namely, the mixture of whole milk diluted with isotonic solution and suspension of washed human erythrocytes - as one layer, and teflon base - as another. The optimum milk dilution was determined, and wavelength dependencies of phantom remittance (R) over the range 480 - 680 nm were obtained. We use the mixtures with physiological concentrations of erythrocytes which corresponded to 2 - 18% per volume blood content in human papillary dermis and upper blood plexus. Phantom remittance spectra for the cases of 2% and 4% blood content virtually coincide with remittance spectra of normal and erythema human skin. Collimated transmittance (T) of blood-milk layer was also measured. At 500 nm we estimated the range of linearity of D and D' (D equals $min1gT, D' equals -1gR) dependence on blood content: offset from linearity was observed near 5 - 6% and 10% of blood, respectively.

  7. Binding of rabbit muscle aldolase to band 3, the predominant polypeptide of the human erythrocyte membrane.

    PubMed

    Strapazon, E; Steck, T L

    1976-04-01

    Aldolase is a trace protein in isolated human red cell membrane preparations. Following total elution of the endogenous enzyme by a saline wash, the interaction of this membrane with rabbit muscle aldolase was studied. At saturation, exogenous aldolase constituted over 40% of the repleted membrane protein. Scatchard analysis revealed two classes of sites, each numbering approximately 7 X 10(5) per ghost. Specificity was suggested by the exclusive binding of the enzyme to the membrane's inner (cytoplasmic) surface. Furthermore, milimolar levels of fructose 1,6-bisphosphate eluted the enzyme from ghosts, while fructose 6-phosphate and NADH (a metabolite which elutes human erythrocyte glyceraldehyde-3-phosphate dehydrogenase (G3PD) from its binding site) were ineffectuve. Removing peripheral membrane proteins with EDTA and lithium 3,5-diiodosalicylate did not diminish the binding capacity of the membranes. An aldolase-band 3 complex, dissociable by high ionic strength or fructose 1,6-bisphosphate treatment, was demonstrated in Triton X-100 extracts of repleted membranes by rate zonal sedimentation analysis on sucrose gradients. We conclude that the association of rabbit muscle aldolase with isolated human erythrocyte membranes reflects its specific binding to band 3 at the cytoplasmic surface, as is also true of G3PD. PMID:1259946

  8. Human erythrocytes as a system for evaluating the antioxidant capacity of vegetable extracts.

    PubMed

    Arbos, Kettelin A; Claro, Ligia M; Borges, Lucielly; Santos, Cid A M; Weffort-Santos, Almeriane M

    2008-07-01

    Free radicals are fairly unstable and highly reactive substances, able of causing oxidation and sometimes-irreversible damage to cells, compromising their function. The Brassicaceae family has many important species for the regular human diet as they provide several antioxidant constituents. In this study, the antioxidant potential of the hydroethanolic extracts prepared from the edible parts of kale, broccoli, and radish was investigated in vitro using human erythrocytes under oxidative stress imposed by phenylhydrazine as an experimental model, in which the methemoglobin levels were measured. When the results were compared with the antioxidant capacity shown by the traditional 2,2-diphenyl-2-picrylhydrazyl hydrate free radical and phosphomolybdenum complex methods, the extracts tested showed significant and correspondent antioxidant activity. Broccoli extract presented the highest antioxidant activity, followed closely by the kale, whereas the radish extract occupied the lowest position. The results derived from the human erythrocyte system have shown it as an alternative method for evaluating the antioxidant properties of vegetable extracts. PMID:19083446

  9. Physicochemical characterization of C3b receptors isolated from human erythrocytes by immunoprecipitation.

    PubMed Central

    Gerdes, J; Stein, H

    1980-01-01

    A high yield of active C3b receptors was obtained by solubilizing human erythrocyte membranes with 2 M KBr, whereas other solubilization agents yielded no, or significantly less activity. Gel filtration of the KBr lysates revealed that the apparent molecular wieght of biologically active C3b receptor molecules was greater than 1 x 10(6). Immunoprecipitates prepared with radio-iodinated KBr lysates and anti-C3 receptor sera (AC3RS) were subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) or sodium dodecyl gel filtration. Unreduced SDS-PAGE and gel filtration profiles showed three predominant peaks with apparent mol. wts of 1--1.3 x 10(6), 80,000 and 60,000. Whereas the high mol. wt component decreased only slightly after reduction, the 80,000 and 60,000 mol. wt components disappeared and two new peaks with apparent mol. wts of 38,000 and 18,000 appeared in SDS-PAGE profiles. Although the high mol. wt component present in reduced SDS-PAGE profiles was detectable in some of the control experiments, none of the other peaks could be precipitated with control sera, and these components could be demonstrated only when KBr lysates of C3b receptor-positive erythrocytes and AC3RS that were able to inhibit ligand binding of the C3b receptors were used for precipitation. These findings suggest that (a) the C3b receptor of human erythrocytes in its biologically active state is a macromolecule with an apparent mol. wt higher than 1 x 10(6) and (b) the protein moiety consists predominantly of non-covalently linked protein molecules with apparent mol wts of 80,000 and 60,000. These protein molecules are composed of disulphide-bridged polypeptide chains with apparent mol. wts of 38,000 and 18,000. PMID:7461716

  10. Protective Effect of Sundakai (Solanum torvum) Seed Protein (SP) Against Oxidative Membrane Damage in Human Erythrocytes.

    PubMed

    Sivapriya, M; Gowda, S S Thammanna; Srinivas, Leela

    2015-12-01

    Lipid peroxidation by ROS at the membrane level disturbs the inherit integrity of components activating subsequent alterations in the function. In this study, the protective effect of purified Sundakai (Solanum torvum) seed protein (SP) was tested against oxidative membrane damage in erythrocyte membrane. SP prevented oxidative RBC lysis induced by pro-oxidants; Fe:As (2:20 μmol), periodate (0.4 mM), and t-BOOH (1 mM) up to 86, 81, and 86 %, respectively. Further, SP prevented the Fe:As-induced K(+) leakage up to the tune of 95 %. The inhibition offered by SP on K(+) leakage was comparable to inhibition offered by quinine sulfate, a known K(+) channel blocker. SP dose dependently restored Na(+)K(+) ATPase and Ca(2+)Mg(2+) ATPase activities in erythrocyte membrane. The restoration of ATPase activity by SP was two times more than standard antioxidants BHA and α-tocopherol. Besides, SP at 1.6 μmol restored the membrane proteins over Fe:As induction when analyzed by SDS-PAGE, which was comparable to protection offered by BHA. In conclusion, SP is an effective antioxidant in preventing oxidative membrane damage and associated functions mediated by ROS. As SP is non-toxic, it can be used as an effective bioprotective antioxidant agent to cellular components. PMID:26374653

  11. Use of steady-state laurdan fluorescence to detect changes in liquid ordered phases in human erythrocyte membranes.

    PubMed

    Vest, Rebekah; Wallis, Rachel; Jensen, Lauren B; Haws, Andrea C; Callister, Joseph; Brimhall, Brent; Judd, Allan M; Bell, John D

    2006-05-01

    In artificial phospholipid bilayers, dual measurements of laurdan steady-state anisotropy and emission spectra can be used to identify the presence of liquid ordered phases. Human erythrocytes were used as a model to test whether similar measurements could be applied to biological samples. Specifically, laurdan anisotropy and emission spectra were obtained from native erythrocytes before and after treatment with calcium ionophore and from the microvesicles (known to be enriched in liquid ordered domains) shed from the cells during calcium entry. Spectral and anisotropy data were consistent with an increased order and reduced fluidity of erythrocyte membrane lipids upon ionophore treatment. Microvesicle membranes appeared more ordered than native erythrocytes and similar to ionophore-treated cells based on laurdan emission. In contrast, the anisotropy value was lower in microvesicles compared to ionophore-treated cells, suggesting greater probe mobility. Parallel measurements of diphenylhexatriene anisotropy corroborated the laurdan data. These results were consistent with the liquid ordered property of microvesicle membranes based on comparisons to behavior in artificial membranes. Two-photon microscopy was used to examine the distribution of laurdan fluorescence along the surface of erythrocyte membranes before and after ionophore treatment. A dual spatial analysis of laurdan anisotropy, as revealed by the distribution of laurdan emission spectra, and intensity excited by polarized light suggested that the plasma membranes of ionophore-treated erythrocytes may also exhibit elevated numbers of liquid ordered domains. PMID:16988865

  12. Membrane permeation characteristics of abacavir in human erythrocytes and human T-lymphoblastoid CD4+ CEM cells: comparison with (-)-carbovir.

    PubMed

    Mahony, William B; Domin, Barbara A; Daluge, Susan M; Zimmerman, Thomas P

    2004-11-01

    Abacavir, (-)-(1S,4R)-4-[2-amino-6-(cyclopropylamino)-9H-purin-9-yl]-2-cyclopentene-1-methanol, is a novel purine carbocyclic nucleoside analogue that has been approved by the FDA for the treatment of HIV (as Ziagen trade mark [abacavir sulfate]). Chemically, abacavir and (-)-carbovir (CBV) differ only at the 6-position of the purine ring; abacavir contains a cyclopropylamino moiety in place of the 6-lactam functionality of CBV. Intracellularly both are ultimately metabolized to CBV triphosphate. We compared the membrane permeation characteristics of these two compounds at 20 degrees C in human erythrocytes and in human T-lymphoblastoid CD4+ CEM cells, using a "papaverine-stop" assay. In erythrocytes, abacavir influx was rapid, nonsaturable (rate constant=200 pmol/s/mM/microl cell water), and unaffected by inhibitors of nucleoside or nucleobase transport. CBV influx was slow, saturable, strongly inhibited by adenine or hypoxanthine, and occurred via both the nucleobase carrier (Vmax=0.67 pmol/s/microl cell water; Km=50 microM) and the nucleoside carrier (Vmax=0.47 pmol/s/microl cell water; Km=440 microM). Similar qualitative results were obtained with CD4+ CEM cells, although CBV influx rates were somewhat higher and abacavir influx rates lower, compared to the corresponding rates in erythrocytes. Equilibrium studies further revealed that both compounds are concentrated intracellularly, but nonmetabolically, in both cell types, apparently due to cytosolic protein binding (absent in erythrocyte ghosts). We conclude that, in both cell types, while CBV influx is slow and carrier-dependent, abacavir influx occurs rapidly by nonfacilitated diffusion. The membrane permeation characteristics of abacavir are consistent with its superior oral bioavailability and its impressive ability to penetrate the central nervous system. PMID:15450945

  13. Impact of p53 status on heavy-ion radiation-induced micronuclei in circulating erythrocytes

    NASA Technical Reports Server (NTRS)

    Chang, P. Y.; Torous, D.; Lutze-Mann, L.; Winegar, R.

    2000-01-01

    Transgenic mice that differed in their p53 genetic status were exposed to an acute dose of highly charged and energetic (HZE) iron particle radiation. Micronuclei (MN) in two distinct populations of circulating peripheral blood erythrocytes, the immature reticulocytes (RETs) and the mature normochromatic erythrocytes (NCEs), were measured using a simple and efficient flow cytometric procedure. Our results show significant elevation in the frequency of micronucleated RETs (%MN-RETs) at 2 and 3 days post-radiation. At 3 days post-irradiation, the magnitude of the radiation-induced MN-RET was 2.3-fold higher in the irradiated p53 wild-type animals compared to the unirradiated controls, 2.5-fold higher in the p53 hemizygotes and 4.3-fold higher in the p53 nullizygotes. The persistence of this radiation-induced elevation of MN-RETs is dependent on the p53 genetic background of the animal. In the p53 wild-type and p53 hemizygotes, %MN-RETs returned to control levels by 9 days post-radiation. However, elevated levels of %MN-RETs in p53 nullizygous mice persisted beyond 56 days post-radiation. We also observed elevated MN-NCEs in the peripheral circulation after radiation, but the changes in radiation-induced levels of MN-NCEs appear dampened compared to those of the MN-RETs for all three strains of animals. These results suggest that the lack of p53 gene function may play a role in the iron particle radiation-induced genomic instability in stem cell populations in the hematopoietic system.

  14. Is the Peroxiredoxin 2/Thioredoxin/Thioredoxin Reductase system in human erythrocytes designed for redox signaling?

    PubMed

    Benfeitas, Rui; Selvaggio, Gianluca; Antunes, Fernando; Coelho, Pedro; Salvador, Armindo

    2014-10-01

    In human erythrocytes H2O2 is mainly consumed by glutathione peroxidase, catalase and peroxiredoxin 2 (Prx2). Our previous analyses indicate that Prx2's peroxidase activity is subjected to a strong but quickly reversible inhibition (see companion abstract). If this activity is inhibited then the main role of Prx2 cannot be to eliminate H2O2. What functional advantages could then such an inhibition confer?We set up and validated a kinetic model of H2O2 metabolism human erythrocytes that shows quantitative agreement with extensive experimental observations. We then applied it to analyze the behavior of Prx2 and Trx under the H2O2 exposure dynamics that erythrocytes face in circulation. The significance of Prx2 inhibition was assessed by comparing the behavior of this model with that of an otherwise identical model lacking inhibition.Our analysis shows that Prx2 inhibition leads to 25-40% lower NADPH consumption under low to moderately high H2O2 supply (<0.8µM H2O2/s). Further, the inhibition extends the range where the concentrations of potential redox signaling readouts - H2O2, Prx2 sulfenic acid, Prx2 disulfide and Trx disulfide- show a proportional response to changes in H2O2 supply, covering practically the whole physiological range of the latter. This is desirable for analogic signal transduction and allows the Prx2/Trx/TrxR system to reliably transduce changes in H2O2 supply as changes in thiol oxidation. Finally, the inhibition allows other less abundant peroxiredoxins in the erythrocyte to be oxidized by H2O2 at physiological H2O2 supplies.Altogether, these results suggest that the postulated reversible inhibition of Prx2's peroxidase facilitates signal transduction by the peroxiredoxins and spares NADPH.We acknowledge: fellowship SFRH/BD/51199/2010, grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, PEst-OE/QUI/UI0313/2014, and FCOMP-01-0124-FEDER-020978 co-financed by FEDER through the COMPETE program and by FCT (project PTDC/QUI-BIQ/119657

  15. Nitrosylated Hemoglobin Levels in Human Venous Erythrocytes Correlate with Vascular Endothelial Function Measured by Digital Reactive Hyperemia

    PubMed Central

    Lobysheva, Irina I.; Biller, Pauline; Gallez, Bernard; Beauloye, Christophe; Balligand, Jean-Luc

    2013-01-01

    Impaired nitric oxide (NO)–dependent endothelial function is associated with the development of cardiovascular diseases. We hypothesized that erythrocyte levels of nitrosylated hemoglobin (HbNO-heme) may reflect vascular endothelial function in vivo. We developed a modified subtraction method using Electron Paramagnetic Resonance (EPR) spectroscopy to identify the 5-coordinate α-HbNO (HbNO) concentration in human erythrocytes and examined its correlation with endothelial function assessed by peripheral arterial tonometry (PAT). Changes in digital pulse amplitude were measured by PAT during reactive hyperemia following brachial arterial occlusion in a group of healthy volunteers (50 subjects). Erythrocyte HbNO levels were measured at baseline and at the peak of hyperemia. We digitally subtracted an individual model EPR signal of erythrocyte free radicals from the whole EPR spectrum to unmask and quantitate the HbNO EPR signals. Results Mean erythrocyte HbNO concentration at baseline was 219+/−12 nmol/L (n = 50). HbNO levels and reactive hyperemia (RH) indexes were higher in female (free of contraceptive pills) than male subjects. We observed a dynamic increase of HbNO levels in erythrocytes isolated at 1–2 min of post-occlusion hyperemia (120+/−8% of basal levels); post-occlusion HbNO levels were correlated with basal levels. Both basal and post-occlusion HbNO levels were significantly correlated with reactive hyperemia (RH) indexes (r = 0.58; P<0.0001 for basal HbNO). Conclusion The study demonstrates quantitative measurements of 5-coordinate α-HbNO in human venous erythrocytes, its dynamic physiologic regulation and correlation with endothelial function measured by tonometry during hyperemia. This opens the way to further understanding of in vivo determinants of NO bioavailability in human circulation. PMID:24130774

  16. Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: an in vitro study.

    PubMed

    Senturk, Murat; Irfan Kufrevioglu, O; Ciftci, Mehmet

    2009-04-01

    Inhibitory effects of some analgesic and anaesthetic drugs on human erythrocyte glutathione reductase were investigated. For this purpose, human erythrocyte glutathione reductase was initially purified 2139-fold in a yield of 29% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis confirmed the purity of the enzyme by sharing a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Diclofenac sodium, ketoprofen, lornoxicam, tenoxicam, etomidate, morphine and propofol exhibited inhibitory effects on the enzyme in vitro using the Beutler assay method. K(i) constants and IC(50) values for drugs were determined from Lineweaver-Burk graphs and plotting activity % versus [I] graphs, respectively. The IC(50) values of diclofenac sodium, ketoprofen, lornoxicam, propofol, tenoxicam, etomidate and morphine were 7.265, 6.278, 0.3, 0.242, 0.082, 0.0523 and 0.0128 mM and the K(i) constants were 23.97 +/- 2.1, 22.14 +/- 7.6, 0.42 +/- 0.18, 0.418 +/- 0.056, 0.13 +/- 0.025, 0.0725 +/- 0.0029 and 0.0165 +/- 0.0013 mM, respectively. While diclofenac sodium, ketoprofen, lornoxicam, tenoxicam etomidate and morphine showed competitive inhibition, propofol displayed noncompetitive inhibition. PMID:18608753

  17. Adaptive Optics-Assisted Identification of Preferential Erythrocyte Aggregate Pathways in the Human Retinal Microvasculature

    PubMed Central

    Arichika, Shigeta; Uji, Akihito; Ooto, Sotaro; Miyamoto, Kazuaki; Yoshimura, Nagahisa

    2014-01-01

    Purpose To characterize human parafoveal blood flow using adaptive optics scanning laser ophthalmoscopy (AO-SLO). Methods In 5 normal subjects, erythrocyte aggregate distributions were analyzed on 3 different days. Erythrocyte aggregates were described as a “dark tail” in AO-SLO. The characteristics of the pathways with dark tail flow in the parafovea were measured. Additionally, the tendency for dark tail flow before and after bifurcations was analyzed to study the blood flow in detail. Results Average velocity in parent vessels with dark tail flow was 1.30±0.27 mm/s. Average velocity in daughter vessels with dark tail flow was 1.12±0.25 mm/s, and the average velocity of plasma gaps in daughter vessels without dark tail flow was 0.64±0.11 mm/s. Downstream from the bifurcations, the velocity in vessels with dark tail flow was higher than that in those without it (p<0.001), and the branching angles of vessels with dark tail flow were smaller than those of vessels without it (p<0.001). Conclusions Images from the AO-SLO noninvasively revealed pathways with and without dark tail flow in the human parafovea. Pathways with dark tail flow in the daughter vessels generally had faster flow and smaller bifurcation angles than daughter vessels without dark tail flow. Thus, AO-SLO is an instructive tool for analyzing retinal microcirculatory hemodynamics. PMID:24586959

  18. Direct visualization and characterization of erythrocyte flow in human retinal capillaries

    PubMed Central

    Bedggood, Phillip; Metha, Andrew

    2012-01-01

    Imaging the retinal vasculature offers a surrogate view of systemic vascular health, allowing noninvasive and longitudinal assessment of vascular pathology. The earliest anomalies in vascular disease arise in the microvasculature, however current imaging methods lack the spatiotemporal resolution to track blood flow at the capillary level. We report here on novel imaging technology that allows direct, noninvasive optical imaging of erythrocyte flow in human retinal capillaries. This was made possible using adaptive optics for high spatial resolution (1.5 μm), sCMOS camera technology for high temporal resolution (460 fps), and tunable wavebands from a broadband laser for maximal erythrocyte contrast. Particle image velocimetry on our data sequences was used to quantify flow. We observed marked spatiotemporal variability in velocity, which ranged from 0.3 to 3.3 mm/s, and changed by up to a factor of 4 in a given capillary during the 130 ms imaging period. Both mean and standard deviation across the imaged capillary network varied markedly with time, yet their ratio remained a relatively constant parameter (0.50 ± 0.056). Our observations concur with previous work using less direct methods, validating this as an investigative tool for the study of microvascular disease in humans. PMID:23243576

  19. Extraction of Phospholipids from Human Erythrocyte Membranes by Hemoglobin Oxidation Products.

    PubMed

    Brunauer, Linda S; Chen, James Y; Koontz, M Zachary; Davis, Kathryn K; O'Brien, Laura E; Wright, Emily M; Huestis, Wray H

    2016-06-01

    This investigation examines oxidation conditions under which hemoglobin extracts membrane phospholipid from erythrocytes and model membranes. In erythrocytes made echinocytic with exogenous phospholipid, addition of hemoglobin oxidized with hydrogen peroxide (H2O2) or Vitamin C (conditions that result in the formation of significant quantities of choleglobin), but not ferricyanide (which produces predominantly methemoglobin), induced dose-dependent shape reversion to less echinocytic forms, consistent with extraction of phospholipids from the exofacial side of the membrane. Erythrocytes preloaded with radiolabeled phosphatidylcholine or NBD-labeled phosphatidylcholine, phosphatidylglycerol or phosphatidic acid, exhibited greatest extraction of radiolabel or fluorescence signal with exogenous hemoglobin oxidized via H2O2 or Vitamin C, but not ferricyanide. However, with NBD-phosphatidylserine (a preferential inner monolayer intercalator), significantly less extraction of labeled lipid occurred with oxidized hemoglobin prepared under all three oxidizing conditions. In dimyristoylphosphatidylcholine liposomes containing radiolabeled phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine, subsequent addition of hemoglobin oxidized with H2O2 or Vitamin C extracted radiolabeled lipid with significantly greater efficiency than ferricyanide-treated hemoglobin, with enhanced extraction detectable at levels approaching physiological plasma oxidant concentrations. Radiolabeled lipid extraction was comparable for phospholipids containing saturated acyl chains between 12 and 18 carbons but diminished significantly for oleoyl-containing phospholipids. Hemoglobin dimerization occurred at very low levels with H2O2 treatment, and even lower levels with Vitamin C treatment, and thus did not correlate to the high efficiency and consistent levels of lipid extraction observed with these treatments. These findings indicate that choleglobin extracts lipids from cell

  20. Exercise effects on erythrocyte deformability in exercise-induced arterial hypoxemia.

    PubMed

    Alis, R; Sanchis-Gomar, F; Ferioli, D; La Torre, A; Blesa, J R; Romagnoli, M

    2015-04-01

    Exercise-induced arterial hypoxemia (EIAH) is often found in endurance-trained subjects at high exercise intensity. The role of erythrocyte deformability (ED) in EIAH has been scarcely explored. We aimed to explore the role of erythrocyte properties and lactate accumulation in the response of ED in EIAH. ED was determined in 10 sedentary and in 16 trained subjects, both before and after a maximal incremental test, and after recovery, along with mean corpuscular volume (MCV) and red blood cell lactate concentrations. EIAH was found in 6 trained subjects (∆SaO2=-8.25±4.03%). Sedentary and non-EIAH trained subjects showed reduced ED after exercise, while no effect on ED was found in EIAH trained subjects. After exercise, lactate concentrations rose and MCV increased equally in all groups. ED is strongly driven by cell volume, but the different ED response to exercise in EIAH shows that other cellular mechanisms may be implicated. Interactions between membrane and cytoskeleton, which have been found to be O2-regulated, play a role in ED. The drop in SaO2 in EIAH subjects can improve ED response to exercise. This can be an adaptive mechanism that enhances muscular and pulmonary perfusion, and allows the achievement of high exercise intensity in EIAH despite lower O2 arterial transport. PMID:25429547

  1. Highly Selective Fluorescence Determination of the Hematin Level in Human Erythrocytes with No Need for Separation from Bulk Hemoglobin.

    PubMed

    Ji, Lijuan; Chen, Li; Wu, Ping; Gervasio, Dominic F; Cai, Chenxin

    2016-04-01

    Hematin-induced fluorescence quenching of boron-doped graphene quantum dots (BGQDs) allows for determination of hematin concentration in human erythrocytes with no need for separating hematin from hemoglobin before performing the assay. The BGQDs are made by oxidizing a graphite anode by holding the voltage between a graphite rod and a Pt cathode at 3 V for 2 h in an aqueous borax solution at pH 7; then, the borate solution was filtered with BGQDs, and the borate was dialyzed from the filtrate, leaving a solution of BGQDs in water. The fluorescence intensity of BGQDs is measurable in real time, and its quenching is very sensitive to the concentration of hematin in the system but not to other coexisting biological substances. The analytical signal is defined as ΔF = 1 - F/F0, where F0 and F are the fluorescence intensities of the BGQDs before and after interaction with hematin, respectively. There is a good linear relationship between ΔF and hematin concentration, ranging from 0.01 to 0.92 μM, with the limit of detection (LOD) being ∼0.005 ± 0.001 μM at a signal-to-noise ratio of 3. This new method is sensitive, label-free, simple, and inexpensive, and many tedious procedures related to sample separation and preparation can be omitted, implying that this method has potential for applications in clinical examinations and disease diagnoses. For example, the determination of the hematin levels in two kind of red blood cell samples, healthy human and sickle cell erythrocytes, gives average concentrations of hematin of ∼(23.1 ± 4.9) μM (average of five samples) for healthy red cell cytosols and ∼(52.5 ± 9.5) μM (average of two samples) for sickle red cell cytosols. PMID:26942664

  2. Isolation and characterization of a serine proteinase specific to human C3b from human erythrocyte membranes

    SciTech Connect

    Charriaut, C.; Krikorian, L.; Barel, M.; Frade, R.

    1986-03-05

    In a previous report, they have shown that human C3b bound through CR1 to human erythrocytes is cleaved by a membrane proteinase activity. Following the molecular analysis of this proteinase activity, they have purified it by a four step procedure: ammonium sulfate precipitation, biogel filtration, fluid phase electrophoresis and hydroxylapatite chromatography. The highly purified proteinase was labeled by /sup 125/I iodine or /sup 3/H-DFP and analyzed by gel electrophoresis: a single band membrane component was characterized by its apparent molecular weight of 57 K or 60 K, under non reducing or reducing conditions respectively and was called p 57. Its reactivity with /sup 3/H-DFP and the inhibition by PMSF of the proteinase activity indicate that p 57 is a serine proteinase. Moreover, it is sensitive to aprotinin and ..gamma..1-antitrypsin. This membrane proteinase presents a higher activity in the presence of detergent and cleaves both alpha and beta chains of human C3b. Polyclonal antibody prepared against this purified proteinase inhibits its activity. On the basis of its structure and its functions, i.e. molecular weight, antigenic properties, proteinase properties and proteinases inhibitors sensitivity, p57 is not related to CR1 or DAF, two others membrane components which react with human C3b and identified by others on human erythrocytes. These specific antibodies allow to analyze the presence of p57 on human cells.

  3. Location of brown recluse venom attachment sites on human erythrocytes by the firritin-labeled antibody technique.

    PubMed Central

    Futrell, J. M.; Morgan, P. N.; Su, S. P.; Roth, S. I.

    1979-01-01

    Brown recluse spider (loxosceles reclusa) venom has been demonstrated by a ferritin-labeled antibody technique to attach to human erythrocyte cell membranes. The number of individual attachment sites per cell is proportional to the concentration of the venom used to sensitize the erythrocytes. Structural changes in the red cell membrane are associated with the venom attachment. These sites may be related to the red cell hemolysis which sometimes occurs in the human as a result of the spider bite. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:377995

  4. [Research advances on directional induction and differentiation in vitro from human pluripotent stem cells into erythrocytes].

    PubMed

    Liu, Sen-Quan; Zhang, Li-Fei; Wang, Ye-Bo; Huang, He

    2014-02-01

    Red blood cell transfusion is an effective method to treat acute hemorrhage and severe anemia. However, blood source from donors is very limited, and transfusion-transmitted diseases occurred frequently, thus threatening human health. Therefore, the safe, abundant and functional blood source is needed. Generation of blood cells from human pluripotent stem cells(hPSC) will offer alternative approach. Lots of studies have been focused on erythroid cell differentiation in vitro, including how to enhance efficiency and improve their function. In this review, the research advances on differentiation methods and the regulatory mechanism are summarized. In addition, the progress in PSC differentiation into erythrocytes and the problems to be solved are discussed briefly. PMID:24598681

  5. Microviscosity of human erythrocytes studied using hypophosphite two-spin order relaxation.

    PubMed Central

    Price, W S; Perng, B C; Tsai, C L; Hwang, L P

    1992-01-01

    A new 31P NMR method is used to probe the cytoplasmic viscosity of human erythrocytes. The method is based on observing two-spin order relaxation of the 31P atom of the hypophosphite ion. This method is superior to our previous method, using the longitudinal relaxation time of the ion, because random field effects such as intermolecular dipole-dipole relaxation can be separated from intramolecular relaxation. This allows a more accurate determination of the effective reorientational correlation time from the measured intramolecular relaxation because it is now unaffected by random field effects. The new method also provides a means by which to estimate the random field effects. Both two-spin order and proton-decoupled T1 measurements were conducted on hypophosphite in water solutions at various temperatures, glycerol solutions of various viscosities, and in erythrocyte samples of various cell volumes. The results show that the effective reorientational correlation time of the hypophosphite ion varies from 7.2 to 15.2 ps in the cytoplasm of cells ranging in volume from 102 to 56 fl cells. PMID:1504239

  6. Nigella sativa oil reduces aluminium chloride-induced oxidative injury in liver and erythrocytes of rats.

    PubMed

    Bouasla, Ihcene; Bouasla, Asma; Boumendjel, Amel; Messarah, Mahfoud; Abdennour, Cherif; Boulakoud, Mohamed Salah; El Feki, Abdelfattah

    2014-12-01

    The present study was planned to investigate the protective effects of Nigella sativa oil (NSO) supplementation against aluminium chloride (AlCl3)-induced oxidative damage in liver and erythrocytes of rats. Simultaneously, a preliminary phytochemical study was affected in order to characterize the bioactive components containing in the NSO using chemical assays. The antioxidant capacities of NSO were evaluated by DPPH assay. The results showed that NSO was found to contain large amounts of total phenolics, flavonoids and tannins. Twenty-four rats were equally divided into two groups, in which group A received standard diet, whereas group B treated daily with an oral gavage dose of 2 ml NSO/kg body weight. After 5 weeks pretreatment, both groups were divided again into two subgroups (A and B) of six animals each and treated for other 3 weeks. Therefore, subgroup A1 was served as a control which received standard diet, but subgroup A2 received AlCl3 (34 mg/kg bw mixed with food). Subgroup B1 received both AlCl3 and NSO; however, subgroup B2 received NSO only. Results showed that AlCl3 exhibited an increase in white blood cell counts and a marked decrease in erythrocyte counts and haemoglobin content. Plasma aspartate transaminase, alanine transaminase, alkaline phosphatase and lactate dehydrogenase activities and total bilirubin concentration were higher in AlCl3 group than those of the control, while albumin and total protein concentration were significantly lower. Compared to the control, a significant raise of hepatic and erythrocyte malondialdehyde level associated with a decrease in reduced glutathione content, glutathione peroxidase, superoxide dismutase and catalase, activities of AlCl3 treated rats. However, the administration of NSO alone or combined with AlCl3 has improved the status of all parameters studied. It can be concluded that AlCl3 has induced the oxidative stress, altered the biochemical parameters and the hepatic histological profile, but the

  7. Oxidative stress induced by cadmium in the plasma, erythrocytes and lymphocytes of rats: Attenuation by grape seed proanthocyanidins.

    PubMed

    Nazima, B; Manoharan, V; Miltonprabu, S

    2016-04-01

    The present study has been designed to investigate the ameliorative effect of grape seed proanthocyanidins (GSP) on cadmium (Cd)-induced oxidative damage in rat erythrocytes. Twenty four male Wistar rats were divided into four groups: control, GSP-treated group (100 mg kg(-1) body weight (BW)), Cd-treated group (cadmium chloride, 5 mg kg(-1) BW), and GSP + Cd-treated group in which GSP was orally pre-administered 90 min before Cd intoxication for 4 weeks. At the end of the experimental period, blood samples were collected by cardiac puncture and were processed for various biochemical estimations. The extent of oxidative damage in isolated rat erythrocyte membrane was assessed by measuring lipid peroxidation, enzymatic and non-enzymatic content, calcium ion (Ca(2+))/magnesium ion (Mg(2+))-ATPase and sodium ion (Na(+))/potassium ion (K(+))-ATPase activities, free iron, calcium, hydrogen peroxide (H2O2) concentration, and osmotic fragility. Our results unveiled that Cd intoxication significantly increased the erythrocyte lipid peroxidation markers and decreased the activity of enzymatic and non-enzymatic markers in erythrocytes. Conversely, GSP pretreatment significantly prevented the decrease in the activities of antioxidant enzymes and membrane-bound ATPases. GSP also restored the levels of iron, calcium, and H2O2 in Cd-treated rats. Conformational changes in erythrocytes of various groups were also determined using morphological and ultrastructural electron microscopic analysis. The findings of our study clearly revealed that GSP affords superior protection against Cd-induced reactive oxygen species generation, lipid peroxidation, and free radical generation in Cd-treated rats, which presumably reflects the ability of this flavonoid to protect erythrocytes and lymphocytes of rats from the toxic effects of Cd. PMID:26089033

  8. Identification of oxidized protein hydrolase of human erythrocytes as acylpeptide hydrolase.

    PubMed

    Fujino, T; Watanabe, K; Beppu, M; Kikugawa, K; Yasuda, H

    2000-03-16

    Partial amino acid sequence of 80 kDa oxidized protein hydrolase (OPH), a serine protease present in human erythrocyte cytosol (Fujino et al., J. Biochem. 124 (1998) 1077-1085) that is adherent to oxidized erythrocyte membranes and preferentially degrades oxidatively damaged proteins (Beppu et al., Biochim. Biophys. Acta 1196 (1994) 81-87; Fujino et al., Biochim. Biophys. Acta 1374 (1998) 47-55) was determined. The N-terminal amino acid of diisopropyl fluorophosphate (DFP)-labeled OPH was suggested to be masked. Six peptide fragments of OPH obtained by digestion of DFP-labeled OPH with lysyl endopeptidase were isolated by use of reverse-phase high-performance liquid chromatography, and the sequence of more than eight amino acids from the N-terminal position of each peptide was determined. Results of homology search of amino acid sequence of each peptide strongly suggested that the protein was identical with human liver acylpeptide hydrolase (ACPH). OPH showed ACPH activity when N-acetyl-L-alanine p-nitroanilide and N-acetylmethionyl L-alanine were used as substrates. Glutathione S-transferase (GST)-tagged recombinant ACPH (rACPH) was prepared by use of baculovirus expression system as a 107-kDa protein from cDNA of human erythroleukemic cell line K-562. rACPH reacted with anti-OPH antiserum from rabbit. rACPH showed OPH activity when hydrogen peroxide-oxidized or glycated bovine serum albumin was used as substrates. As well as the enzyme activities of OPH, those of rACPH were inhibited by DFP. The results clearly demonstrate that ACPH, whose physiological function has not yet been well characterized, can play an important role as OPH in destroying oxidatively damaged proteins in living cells. PMID:10719179

  9. Glutathione-dependent reduction of arsenate in human erythrocytes--a process independent of purine nucleoside phosphorylase.

    PubMed

    Németi, Balázs; Gregus, Zoltán

    2004-12-01

    Reduction of arsenate (AsV) to the more toxic arsenite (AsIII) is toxicologically important, yet its mechanism is unknown. To clarify this, AsV reduction was investigated in human red blood cells (RBC), as they possess a simple metabolism. RBC were incubated with AsV in gluconate buffer, and the formed AsIII was quantified by high performance liquid chromatography-hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). The observations are compatible with the following conclusions. (1) Human RBC reduce AsV intracellularly, because 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS, inhibitor of the chloride-bicarbonate exchanger, which also mediates phosphate and AsV uptake), as well as chloride and phosphate, countered AsIII formation. (2) Purine nucleoside phosphorylase (PNP), whose AsV reductase activity has been directly demonstrated, cannot be a physiologically relevant AsV reductase, because its inhibitor (BCX-1777) failed to decrease the basal erythrocytic AsV reduction, although it prevented the increase in AsIII formation caused by artificial activation of PNP with inosine and dithiothreitol. (3) The basal (PNP-independent) AsV reduction requires glutathione (GSH), because the GSH depletor diethylmaleate strongly diminished AsIII formation. (4) The erythrocytic AsV reduction apparently depends on NAD(P) supply, because oxidants of NAD(P)H (i.e., pyruvate, ferricyanide, methylene blue, nitrite, tert-butylhydroperoxide, dehydroascorbate, 4-dimethylaminophenol) enhanced AsIII formation from AsV. The oxidant-stimulated AsV reduction is PNP-independent, because BCX-1777 failed to affect it, but is GSH-dependent, because diethylmaleate impaired it. (5) Pyruvate-induced glucose depletion, which causes NAD enrichment in the erythrocytes at the expense of NADH, enhanced AsV reduction. This suggests that the erythrocytic AsV reduction requires both NAD supply and operation of the lower part of the glycolytic pathway starting from glyceraldehyde-3

  10. Triggering of Programmed Erythrocyte Death by Alantolactone

    PubMed Central

    Alzoubi, Kousi; Calabrò, Salvatrice; Egler, Jasmin; Faggio, Caterina; Lang, Florian

    2014-01-01

    The sesquiterpene alantolactone counteracts malignancy, an effect at least in part due to stimulation of suicidal death or apoptosis of tumor cells. Signaling of alantolactone induced apoptosis involves altered gene expression and mitochondrial depolarization. Erythrocytes lack mitochondria and nuclei but may enter suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the erythrocyte surface. Cellular mechanisms involved in triggering of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i) and oxidative stress. The present study explored, whether alantolactone stimulates eryptosis. To this end, erythrocyte volume was estimated from forward scatter, phosphatidylserine-exposure at the erythrocyte surface from FITC-annexin-V-binding, [Ca2+]i from Fluo3-fluorescence, ceramide abundance from binding of fluorescent antibodies, and oxidative stress from 2',7'-dichlorodihydrofluorescein-diacetate (DCFDA) fluorescence. As a result, a 48 h exposure of human erythrocytes to alantolactone (≥20 μM) significantly decreased erythrocyte forward scatter and increased the percentage of annexin-V-binding cells. Alantolactone significantly increased Fluo3 fluorescence (60 μM), ceramide abundance (60 μM) and DCFDA fluorescence (≥40 μM). The effect of alantolactone (60 μM) on annexin-V-binding was not significantly modified by removal of extracellular Ca2+. In conclusion, alantolactone stimulates suicidal erythrocyte death or eryptosis, an effect paralleled by increase of [Ca2+]i, ceramide abundance and oxidative stress. PMID:25533522

  11. Human erythrocyte nucleoside transporter ENT1 functions at ice-cold temperatures.

    PubMed

    Takano, Mikihisa; Kimura, Eri; Suzuki, Satoshi; Nagai, Junya; Yumoto, Ryoko

    2010-01-01

    The functionality of human erythrocyte nucleoside transporter ENT1 was examined at ice-cold temperatures (ICT; measured temperature, 0.5-0.7 degrees C) using rightside-out membrane vesicles (ROVs). The uptake of uridine, an ENT1 substrate, showed saturation kinetics and was inhibited by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), a specific ENT1 inhibitor, at both 23 degrees C and ICT. [3H]Uridine uptake was markedly trans-stimulated by preloading ROVs with unlabeled uridine or ribavirin, another ENT1 substrate, and the overshoot phenomenon was observed at ICT. Similarly, [3H]ribavirin uptake was markedly trans-stimulated by unlabeled ribavirin or uridine at ICT. The trans-stimulated uptake of [3H]uridine at ICT was inhibited by ENT1 inhibitors/substrates such as NBMPR, dipyridamole, adenosine, and ribavirin in a concentration-dependent manner. The inhibition of [3H]uridine uptake by NBMPR and dipyridamole at ICT was also observed in intact red blood cells. Like uridine uptake, [3H]D-glucose uptake by ROVs, which is mediated by facilitative glucose transporter GLUT1, was trans-stimulated by unlabeled D-glucose at ICT, and the overshoot phenomenon was observed. In contrast, the ability of ATP-dependent transport of 5-(and-6)-carboxy-2',7'-dichlorofluorescein via multidrug resistance-associated protein 5 in inside-out membrane vesicles disappeared at ICT. These results clearly indicate that human erythrocyte transporters such as ENT1 function even at very low temperatures near 0 degrees C. The significance of these findings in transporter research is discussed. PMID:20814156

  12. Inhibition of Sendai virus fusion with phospholipid vesicles and human erythrocyte membranes by hydrophobic peptides

    SciTech Connect

    Kelsey, D.R.; Flanagan, T.D.; Young, J.E.; Yeagle, P.L. )

    1991-06-01

    Hydrophobic di- and tripeptides which are capable of inhibiting enveloped virus infection of cells are also capable of inhibiting at least three different types of membrane fusion events. Large unilamellar vesicles (LUV) of N-methyl dioleoylphosphatidylethanolamine (N-methyl DOPE), containing encapsulated 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS) and/or p-xylene bis(pyridinium bromide) (DPX), were formed by extrusion. Vesicle fusion and leakage were then monitored with the ANTS/DPX fluorescence assay. Sendai virus fusion with lipid vesicles and Sendai virus fusion with human erythrocyte membranes were measured by following the relief of fluorescence quenching of virus labeled with octadecylrhodamine B chloride (R18). This study found that the effectiveness of the peptides carbobenzoxy-L-Phe-L-Phe (Z-L-Phe-L-Phe), Z-L-Phe, Z-D-Phe, and Z-Gly-L-Phe-L-Phe in inhibiting N-methyl DOPE LUV fusion or fusion of virus with N-methyl DOPE LUV also paralleled their reported ability to block viral infectivity. Furthermore, Z-D-Phe-L-PheGly and Z-Gly-L-Phe inhibited Sendai virus fusion with human erythrocyte membranes with the same relative potency with which they inhibited vesicle-vesicle and virus-vesicle fusion. The evidence suggests a mechanism by which these peptides exert their inhibition of plaque formation by enveloped viruses. This class of inhibitors apparently acts by inhibiting fusion of the viral envelope with the target cell membrane, thereby preventing viral infection. The physical pathway by which these peptides inhibit membrane fusion was investigated. {sup 31}P nuclear magnetic resonance (NMR) of proposed intermediates in the pathway for membrane fusion in LUV revealed that the potent fusion inhibitor Z-D-Phe-L-PheGly selectively altered the structure (or dynamics) of the hypothesized fusion intermediates and that the poor inhibitor Z-Gly-L-Phe did not.

  13. Proteinase-resistant factors in human erythrocyte membranes mediate CD4-dependent fusion with cells expressing human immunodeficiency virus type 1 envelope glycoproteins.

    PubMed Central

    Dragic, T; Picard, L; Alizon, M

    1995-01-01

    Murine CD4+ cells are resistant to human immunodeficiency virus type 1 (HIV-1) entry and to fusion with cells expressing HIV-1 envelope glycoproteins (Env). The role of human-specific factors in Env/CD4-mediated fusion is shown by the ability of transient cell hybrids formed between CD4+ murine cells and human HeLa cells to fuse with Env+ cells. Fusion events were observed when other human cells, including erythrocytes, were substituted for HeLa cells in the hybrids. Experiments with erythrocyte ghosts showed that the factors allowing Env/CD4-mediated fusion are located in the plasma membrane. These factors were fully active after extensive digestion of erythrocytes with proteinase K or pronase. Nonprotein components of human plasma membranes, possibly glycolipids, could therefore be required for Env/CD4-mediated fusion and virus entry. PMID:7815477

  14. Hydroxychloroquine binding to cytoplasmic domain of Band 3 in human erythrocytes: Novel mechanistic insights into drug structure, efficacy and toxicity.

    PubMed

    Nakagawa, Mizuki; Sugawara, Kotomi; Goto, Tatsufumi; Wakui, Hideki; Nunomura, Wataru

    2016-05-13

    Hydroxychloroquine (HCQ) is a widely used drug in the treatment of autoimmune diseases, such as arthritis and systemic lupus erythematosus. It has also been prescribed for the treatment of malaria owing to its lower toxicity compared to its closely related compound chloroquine (CQ). However, the mechanisms of action of HCQ in erythrocytes (which bind preferentially this drug) have not been documented and the reasons underlying the lower side effects of HCQ compared to CQ remain unclear. Here we show that, although the activity of erythrocyte lactate dehydrogenase (LDH), but not GAPDH, was inhibited by both HCQ and CQ in vitro, LDH activity in erythrocytes incubated with 20 mM HCQ was not significantly reduced within 5 h in contrast to CQ did. Using HCQ coupled Sepharose chromatography (HCQ-Sepharose), we identified Band 3, spectrin, ankyrin, protein 4.1R and protein 4.2 as HCQ binding proteins in human erythrocyte plasma membrane. Recombinant cytoplasmic N-terminal 43 kDa domain of Band 3 bound to HCQ-Sepharose and was eluted with 40 mM (but not 20 mM) HCQ. Band 3 transport activity was reduced by only 23% in the presence of 20 mM HCQ. Taken together, these data demonstrate that HCQ binds to the cytoplasmic N-terminal domain of Band 3 in human erythrocytes but does not inhibit dramatically its transport activity. We hypothesize that the trapping of HCQ on Band 3 contributes to the lower side effects of the drug on energy production in erythrocytes. PMID:27049308

  15. Protective effects of boldine against free radical-induced erythrocyte lysis.

    PubMed

    Jiménez, I; Garrido, A; Bannach, R; Gotteland, M; Speisky, H

    2000-08-01

    Boldine, an aporphine alkaloid extracted from the leaves and bark of boldo (Peumus boldus Mol.), has been shown to exhibit strong free-radical scavenger and antioxidant properties. Here, we report the in vitro ability of boldine to protect intact red cells against the haemolytic damage induced by the free radical initiator 2, 2'-azobis-(2-amidinopropane) (AAPH). Boldine concentration-dependently prevented the AAPH-induced leakage of haemoglobin into the extracellular medium. Substantial and similar cyto-protective effects of boldine were observed whether the antioxidant was added 1 h prior to, or simultaneously with, the azo-compound. The delayed addition of boldine, by 1 h relative to AAPH, diminished but did not abolish its cytoprotective effect. However, negligible effects of boldine were observed after its addition to erythrocytes previously incubated with AAPH for 2 h. The data presented demonstrate that, in addition to its well-established antioxidant effects, boldine also displays time-dependently strong cytoprotective properties against chemically induced haemolytic damage. PMID:10925398

  16. Alterations of hemorheological parameters and tubulin content in erythrocytes from diabetic subjects.

    PubMed

    Nigra, Ayelén D; Monesterolo, Noelia E; Rivelli, Juan F; Amaiden, Marina R; Campetelli, Alexis N; Casale, Cesar H; Santander, Verónica S

    2016-05-01

    Treatment of human erythrocytes with high glucose concentrations altered the content and distributions of three tubulin isotypes, with consequent reduction of erythrocyte deformability and osmotic resistance. In erythrocytes from diabetic subjects (D erythrocytes), (i) tubulin in the membrane-associated fraction (Mem-Tub) was increased and tubulin in the sedimentable fraction (Sed-Tub) was decreased, (ii) deformability was lower than in erythrocytes from normal subjects (N erythrocytes), and (iii) detyrosinated/acetylated tubulin content was higher in the Mem-Tub fraction and tyrosinated/acetylated tubulin content was higher in the Sed-Tub fraction, in comparison with N erythrocytes. Similar properties were observed for human N erythrocytes treated with high glucose concentrations, and for erythrocytes from rats with streptozotocin-induced diabetes. In N erythrocytes, high-glucose treatment caused translocation of tubulin from the Sed-Tub to Mem-Tub fraction, thereby reducing deformability and inducing acetylation/tyrosination in the Sed-Tub fraction. The increased tubulin acetylation in these cells resulted from inhibition of deacetylase enzymes. Increased tubulin acetylation and translocation of this acetylated tubulin to the Mem-Tub fraction were both correlated with reduced osmotic resistance. Our findings suggest that (i) high glucose concentrations promote tubulin acetylation and translocation of this tubulin to the membrane, and (ii) this tubulin is involved in regulation of erythrocyte deformability and osmotic fragility. PMID:26923290

  17. Plasmodium falciparum STEVOR proteins impact erythrocyte mechanical properties.

    PubMed

    Sanyal, Sohini; Egée, Stéphane; Bouyer, Guillaume; Perrot, Sylvie; Safeukui, Innocent; Bischoff, Emmanuel; Buffet, Pierre; Deitsch, Kirk W; Mercereau-Puijalon, Odile; David, Peter H; Templeton, Thomas J; Lavazec, Catherine

    2012-01-12

    Infection of erythrocytes with the human malaria parasite, Plasmodium falciparum, results in dramatic changes to the host cell structure and morphology. The predicted functional localization of the STEVOR proteins at the erythrocyte surface suggests that they may be involved in parasite-induced modifications of the erythrocyte membrane during parasite development. To address the biologic function of STEVOR proteins, we subjected a panel of stevor transgenic parasites and wild-type clonal lines exhibiting different expression levels for stevor genes to functional assays exploring parasite-induced modifications of the erythrocyte membrane. Using this approach, we show that stevor expression impacts deformability of the erythrocyte membrane. This process may facilitate parasite sequestration in deep tissue vasculature. PMID:22106347

  18. Preferential Elimination of Older Erythrocytes in Circulation and Depressed Bone Marrow Erythropoietic Activity Contribute to Cadmium Induced Anemia in Mice

    PubMed Central

    Chatterjee, Sreoshi; Saxena, Rajiv K.

    2015-01-01

    Feeding cadmium chloride (50 or 1000 ppm CdCl2 in drinking water, ad libitum) to C57BL/6 mice resulted in a significant and sustained fall in blood erythrocyte count and hemoglobin levels that started 4 and 3 weeks after the start of 50 and 1000 ppm cadmium doses respectively. A transient yet significant reticulocytosis occurred during the first 4 weeks of cadmium treatment. Using the recently developed double in vivo biotinylation (DIB) technique, turnover of erythrocyte cohorts of different age groups was simultaneously monitored in control and cadmium treated mice. A significant accumulation of younger erythrocytes and a concomitant decline in the relative proportions of older erythrocytes in circulation was observed in both 50 and 1000 ppm cadmium groups indicating that older erythrocytes were preferentially eliminated in cadmium induced anemia. A significant increase in the erythropoietin levels in plasma was seen in mice exposed to 1000 ppm cadmium. Levels of inflammatory cytokines (IL1A, IL6, TNFα, IFNγ) were however not significantly altered in cadmium treated mice. A significant increase in cellular levels of reactive oxygen species (ROS) was observed in older erythrocytes in circulation but not in younger erythrocytes. Erythropoietic activity in the bone marrows and spleens of cadmium treated mice was examined by monitoring the relative proportion of cells belonging to the erythroid line of differentiation in these organs. Erythroid cells in bone marrow declined markedly (about 30%) in mice in the 1000 ppm cadmium group but the decline was not significant in the 50 ppm cadmium group. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Decline of erythroid cells was essentially confined to pro-erythroblast and erythroblast-A, along with a concurrent increase in the splenic erythroid

  19. The merozoite surface protein 1 complex is a platform for binding to human erythrocytes by Plasmodium falciparum.

    PubMed

    Lin, Clara S; Uboldi, Alessandro D; Marapana, Danushka; Czabotar, Peter E; Epp, Christian; Bujard, Hermann; Taylor, Nicole L; Perugini, Matthew A; Hodder, Anthony N; Cowman, Alan F

    2014-09-12

    Plasmodium falciparum is the causative agent of the most severe form of malaria in humans. The merozoite, an extracellular stage of the parasite lifecycle, invades erythrocytes in which they develop. The most abundant protein on the surface of merozoites is merozoite surface protein 1 (MSP1), which consists of four processed fragments. Studies indicate that MSP1 interacts with other peripheral merozoite surface proteins to form a large complex. Successful invasion of merozoites into host erythrocytes is dependent on this protein complex; however, the identity of all components and its function remain largely unknown. We have shown that the peripheral merozoite surface proteins MSPDBL1 and MSPDBL2 are part of the large MSP1 complex. Using surface plasmon resonance, we determined the binding affinities of MSPDBL1 and MSPDBL2 to MSP1 to be in the range of 2-4 × 10(-7) m. Both proteins bound to three of the four proteolytically cleaved fragments of MSP1 (p42, p38, and p83). In addition, MSPDBL1 and MSPDBL2, but not MSP1, bound directly to human erythrocytes. This demonstrates that the MSP1 complex acts as a platform for display of MSPDBL1 and MSPDBL2 on the merozoite surface for binding to receptors on the erythrocyte and invasion. PMID:25074930

  20. Variations in the distribution of selenium between erythrocyte glutathione peroxidase and hemoglobin in different human populations

    SciTech Connect

    Whanger, P.D.; Robinson, M.F.; Feldman, E.B.; Beilstein, M.A.; Butler, J.A.

    1986-03-01

    The majority of erythrocyte (RBC) selenium (Se) is associated with glutathione peroxidase (GPx) in animals, but most of it is with hemoglobin (Hb) in human RBCs. Dietary forms of Se may influence this distribution since a rat study showed that selenite promoted the deposition of Se in GPx but selenomethionine (SeMet) resulted in greater amounts with Hb. Three different populations of people were chosen to investigate some possible reasons for the Se distribution in human RBC proteins. An average of 12% of the RBC Se (0.71 ng Se/mg Hb) was associated with GPx in people living in Oregon, but nearly 30% of the Se was associated with GPx in RBC (0.26 ng Se/mg Hb) from New Zealanders. Georgia residents with low RBC Se levels (0.35 ng Se/mg Hb) had 38% of the Se associated with GPx as compared to 29% for those with higher RBC levels (0.56 ng Se/mg Hb). In a third group of people the amount of Se tended to be higher in RBC GPx from non-vegetarian OSU students than from vegetarians. The predominant form of Se in meat appears to be selenocysteine, which is metabolized similarly to selenite, and presumably contributes to this difference since many plant foods contain Se as SeMet. These are examples of many possible factors affecting the relative distribution of Se in human RBC proteins.

  1. Isolation and characterization of cDNA clones for human erythrocyte. beta. -spectrin

    SciTech Connect

    Prchal, J.T.; Morley, B.J.; Yoon, S.H.; Coetzer, T.L.; Palek, J.; Conboy, J.G.; Kan, Y.W.

    1987-11-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical ..cap alpha.. (M/sub r/ 240,000) and ..beta.. (M/sub r/ 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. The authors report here the isolation and characterization of a human erythroid-specific ..beta..-spectrin cDNA clone that encodes parts of the ..beta..-9 through ..beta..-12 repeat segments. This cDNA was used as a hybridization probe to assign the ..beta..-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte ..beta..-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the ..beta..-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

  2. The effects of phorbol ester, diacylglycerol, phospholipase C and Ca2+ ionophore on protein phosphorylation in human and sheep erythrocytes.

    PubMed Central

    Raval, P J; Allan, D

    1985-01-01

    Treatment of human or sheep erythrocytes with PMA (phorbol myristate acetate) enhanced [32P]phosphate labelling of membrane polypeptides of approx. 100, 80 and 46 kDa. The 80 kDa and 46 kDa polypeptides coincided with bands 4.1 and 4.9 respectively on Coomassie-Blue-stained gels. Similar but smaller effects were obtained by treating human cells with 1-oleoyl-2-acetyl-rac-glycerol (OAG), exogenous bacterial phospholipase C or ionophore A23187 + Ca2+, each of which treatments would be expected to raise the concentration of membrane diacylglycerol. In contrast, sheep cells, which do not increase their content of diacylglycerol when treated with phospholipase C or A23187 + Ca2+, only showed enhanced phosphorylation with OAG. Neither human nor sheep cells showed any enhanced [32P]phosphate labelling of phosphoproteins when treated with 1-mono-oleoyl-rac-glycerol. It is concluded that diacylglycerol from a variety of sources can activate erythrocyte protein kinase C, but that the most effective diacylglycerol is that derived from endogenous polyphosphoinositides. In contrast with bacterial phospholipase C and A23187, which stimulate synthesis of phosphatidate by increasing the cell-membrane content of diacylglycerol in human erythrocytes, PMA, OAG or 1-mono-oleoyl-rac-glycerol caused no change in phospholipid metabolism. Images PMID:4084238

  3. Inhibition of erythrocyte invasion and Plasmodium falciparum merozoite surface protein 1 processing by human immunoglobulin G1 (IgG1) and IgG3 antibodies.

    PubMed

    Lazarou, Maria; Guevara Patiño, José A; Jennings, Richard M; McIntosh, Richard S; Shi, Jianguo; Howell, Steven; Cullen, Eilish; Jones, Tarran; Adame-Gallegos, Jaime R; Chappel, Jonathan A; McBride, Jana S; Blackman, Michael J; Holder, Anthony A; Pleass, Richard J

    2009-12-01

    Antigen-specific antibodies (Abs) to the 19-kDa carboxy-terminal region of Plasmodium falciparum merozoite surface protein 1 (MSP1(19)) play an important role in protective immunity to malaria. Mouse monoclonal Abs (MAbs) 12.10 and 12.8 recognizing MSP1(19) can inhibit red cell invasion by interfering with MSP1 processing on the merozoite surface. We show here that this ability is dependent on the intact Ab since Fab and F(ab')(2) fragments derived from MAb 12.10, although capable of binding MSP1 with high affinity and competing with the intact antibody for binding to MSP1, were unable to inhibit erythrocyte invasion or MSP1 processing. The DNA sequences of the variable (V) regions of both MAbs 12.8 and 12.10 were obtained, and partial amino acid sequences of the same regions were confirmed by mass spectrometry. Human chimeric Abs constructed by using these sequences, which combine the original mouse V regions with human gamma1 and gamma3 constant regions, retain the ability to bind to both parasites and recombinant MSP1(19), and both chimeric human immunoglobulin G1s (IgG1s) were at least as good at inhibiting erythrocyte invasion as the parental murine MAbs 12.8 and 12.10. Furthermore, the human chimeric Abs of the IgG1 class (but not the corresponding human IgG3), induced significant NADPH-mediated oxidative bursts and degranulation from human neutrophils. These chimeric human Abs will enable investigators to examine the role of human Fcgamma receptors in immunity to malaria using a transgenic parasite and mouse model and may prove useful in humans for neutralizing parasites as an adjunct to antimalarial drug therapy. PMID:19805526

  4. Triggering of Erythrocyte Death by Triparanol

    PubMed Central

    Officioso, Arbace; Manna, Caterina; Alzoubi, Kousi; Lang, Florian

    2015-01-01

    The cholesterol synthesis inhibitor Triparanol has been shown to trigger apoptosis in several malignancies. Similar to the apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress which may activate erythrocytic Ca2+ permeable unselective cation channels with subsequent Ca2+ entry and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether and how Triparanol induces eryptosis. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, hemolysis from hemoglobin release, [Ca2+]i from Fluo3-fluorescence, and ROS formation from 2’,7’-dichlorodihydrofluorescein diacetate (DCFDA) dependent fluorescence. As a result, a 48 h exposure of human erythrocytes to Triparanol (20 µM) significantly increased DCFDA fluorescence and significantly increased Fluo3-fluorescence. Triparanol (15 µM) significantly increased the percentage of annexin-V-binding cells, and significantly decreased the forward scatter. The effect of Triparanol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. In conclusion, Triparanol leads to eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane. Triparanol is at least in part effective by stimulating ROS formation and Ca2+ entry. PMID:26305256

  5. Structurally Similar but Functionally Diverse ZU5 Domains in Human Erythrocyte Ankyrin

    SciTech Connect

    Yasunaga, Mai; Ipsaro, Jonathan J.; Mondragón, Alfonso

    2014-10-02

    The metazoan cell membrane is highly organized. Maintaining such organization and preserving membrane integrity under different conditions are accomplished through intracellular tethering to an extensive, flexible protein network. Spectrin, the principal component of this network, is attached to the membrane through the adaptor protein ankyrin, which directly bridges the interaction between {beta}-spectrin and membrane proteins. Ankyrins have a modular structure that includes two tandem ZU5 domains. The first domain, ZU5A, is directly responsible for binding {beta}-spectrin. Here, we present a structure of the tandem ZU5 repeats of human erythrocyte ankyrin. Structural and biophysical experiments show that the second ZU5 domain, ZU5B, does not participate in spectrin binding. ZU5B is structurally similar to the ZU5 domain found in the netrin receptor UNC5b supramodule, suggesting that it could interact with other domains in ankyrin. Comparison of several ZU5 domains demonstrates that the ZU5 domain represents a compact and versatile protein interaction module.

  6. Structural basis of human erythrocyte glucose transporter function in proteoliposome vesicles: circular dichroism measurements.

    PubMed Central

    Chin, J J; Jung, E K; Chen, V; Jung, C Y

    1987-01-01

    The secondary structural compositions of the human erythrocyte glucose transporter in proteoliposome vesicles were assessed on the basis of circular dichroism (CD) spectra measured in the absence and in the presence of D-glucose or an inhibitor, cytochalasin B. We designed and used a scattered-light-collecting device, which corrects CD spectra for optical artifacts originating from light scattering. Relative contents of eight types of secondary structure were estimated by using basis spectra generated by the eigenvector method based on CD spectra of 15 proteins of known structure. Results indicate that the glucose transporter is composed of approximately 82% alpha-helices, 10% beta-turns, and 8% other random structure, with no beta-strands. In the presence of an excess of D-glucose, the alpha-helical content is reduced by more than 10% and there is a significant increase in the random structure content. Cytochalasin B does not appear to affect the secondary structural composition of the transporter to any significant degree. PMID:3473495

  7. Laser-based particle-counting microimmunoassay for the analysis of single human erythrocytes

    SciTech Connect

    Rosenzweig, Z.; Yeung, E.S. Ames Lab., IA )

    1994-05-15

    A particle-counting immunoassay system for ultrasensitive analysis of proteins in a capillary environment has been developed. The assay is based on the agglutination of antibody-coated particles in the presence of an antigen (usually a protein). The particles were electrophoretically migrated in a 20-[mu]m-i.d. capillary past a detection window where a laser beam irradiates continuously. The light scattering events generated by the agglutinated particles were counted while those produced by unreacted particles were electronically rejected. Glucose-6-phosphate dehydrogenase (G6PDH) was chosen as a test compound for the off-column as well as for the on-column versions of this method. A limit of detection of 620 molecules of G6PDH (1 zmol) was found in the on-column assay. The standard deviation between runs was approximately 6%, which is comparable to that of standard immunoassay methods. The application to the determination of G6PDH levels in individual human erythrocytes is presented. A 14-fold cell-to-cell variation was found which can be explained by the age distribution in the red blood cells. 42 refs., 5 figs.

  8. Transmembrane Exchange of Hyperpolarized 13C-Urea in Human Erythrocytes: Subminute Timescale Kinetic Analysis

    PubMed Central

    Pagès, Guilhem; Puckeridge, Max; Liangfeng, Guo; Tan, Yee Ling; Jacob, Chacko; Garland, Marc; Kuchel, Philip W.

    2013-01-01

    The rate of exchange of urea across the membranes of human erythrocytes (red blood cells) was quantified on the 1-s to 2-min timescale. 13C-urea was hyperpolarized and subjected to rapid dissolution and the previously reported (partial) resolution of 13C NMR resonances from the molecules inside and outside red blood cells in suspensions was observed. This enabled a stopped-flow type of experiment to measure the (initially) zero-trans transport of urea with sequential single-pulse 13C NMR spectra, every second for up to ∼2 min. Data were analyzed using Bayesian reasoning and a Markov chain Monte Carlo method with a set of simultaneous nonlinear differential equations that described nuclear magnetic relaxation combined with transmembrane exchange. Our results contribute to quantitative understanding of urea-exchange kinetics in the whole body; and the methodological approach is likely to be applicable to other cellular systems and tissues in vivo. PMID:24209840

  9. [A structure-activity study of a catalytic antiidiotypic antibody to the human erythrocyte acetylcholinesterase].

    PubMed

    Aleksandrova, E S; Koralevski, F; Titov, M I; Demin, A V; Kozyr', A V; Kolesnikov, A V; Tramontano, A; Paul, S; Thomas, D; Gabibov, A G; Gnuchev, N V; Friboulet, A

    2002-01-01

    The catalytic monoclonal antibody 9A8 (MA 9A8), antiidiotypic to the antibody AE-2 (MA AE2) produced to the active site of acetyl cholinesterase from human erythrocytes, was subjected to a structure-function study. The specific binding of MA 9A8 to MA AE2 (K 2.26 x 10(9) M-1) was shown by the method of surface plasmon resonance, and the functional activity of MA 9A8 was demonstrated. Unlike acetyl cholinesterase, this antibody specifically reacted with the irreversible phosphonate inhibitors of esterases. A peptide map of MA 9A8 was analyzed by MALDI mass spectrometry. The Ser99 residue of its heavy chain was shown to be within the active site of the catalytic antibody. A computer modeling of the MA 9A8 active site suggested the existence of a catalytic dyad formed by Ser99 and His35. A comparison of the tertiary structures of the MA 9A8 and the 17E8 monoclonal antibody, which also exhibited an esterase activity and was produced to the stable analogue of the reaction transition state, indicated a practically complete coincidence of the structures of their presumed active sites. PMID:11962233

  10. The first case of a complete deficiency of diphosphoglycerate mutase in human erythrocytes.

    PubMed Central

    Rosa, R; Prehu, M O; Beuzard, Y; Rosa, J

    1978-01-01

    An inherited and complete deficiency of diphosphoglycerate mutase was discovered in the erythrocytes of a 42-yr-old man of French origin whose blood hemoglobin concentration was 19.0 g/dl. Upon physical examination he was normal with the exception of a ruddy cyanosis. The morphology of his erythrocytes was also normal and there was no evidence of hemolysis. The erythrocyte 2,3-diphosphoglycerate level was below 3% of normal values and, as a consequence, the affinity of the cells for oxygen was increased. Diphosphoglycerate mutase activity was undetectable in erythrocytes as was that of diphosphoglycerate phosphatase. The activities of all the other erythrocyte enzymes that were tested were normal except for nomophosphoglycerate mutase which was diminished to 50% of the normal value. The levels of reduced glutathione, ATP, fructose 1,6-diphosphate, and of triose phosphates were elevated, whereas those of glucose 6-phosphate and fructose 6-phosphate were decreased. This report sheds new light on the role of diphosphoglycerate mutase in the metabolism of erythrocytes. Images PMID:152321

  11. Functional and structural changes in human erythrocyte surface after irradiation by uv waves of various wavelengths. Report 1: expression of ABO and Rhesus system antigen

    SciTech Connect

    Samoylova, K.A.; Klimova, K.N.; Priyezzheva, L.S.; Artsishevskaya, R.A.

    1985-01-01

    The effect of shortwave ultraviolet (SUV) radiation ad causes change in the external surface of human erythrocytes, modifying the expression of the ABO and Rh system antigens which are related to the surface of the cells was investigated. Erythrocytes in a structurally prepared erythrocyte mass from 23 donors stabilized by glugicir or heparin were examined. Three series of experiments were performed: (1) isolated erythrocytes, before irradiation thrice washed to remove plasma with isotonic NaC1 0.9%, erythrocytes diluted to 5 x s10 to the 7th power cells per milliliter and erythrocytes on the undiluted erythrocyte mass about 7 x 5 x 109 to the 9th power cells power milliliter. The agglutinating activity of the ABO and Rh antigens was studied. Two to three hours after exposure to 248, 620, 1240 and 2480 J/m2, the degree of hemolysis of isolated erythrocytes increased by 5,10,18 and 28%. Changes were also observed in agglutinating activity of ABO antigens. The agglutinating activity of A and B antigens increased by an average factor of 2 minus H antigens by a factor of 4. The SUV radiation did not cause any activation of the Rh antigen.

  12. Malaria Induces Anemia through CD8+ T Cell-Dependent Parasite Clearance and Erythrocyte Removal in the Spleen

    PubMed Central

    Safeukui, Innocent; Gomez, Noé D.; Adelani, Aanuoluwa A.; Burte, Florence; Afolabi, Nathaniel K.; Akondy, Rama; Velazquez, Peter; Tewari, Rita; Buffet, Pierre; Brown, Biobele J.; Shokunbi, Wuraola A.; Olaleye, David; Sodeinde, Olugbemiro; Kazura, James; Ahmed, Rafi; Mohandas, Narla; Fernandez-Reyes, Delmiro

    2015-01-01

    ABSTRACT  Severe malarial anemia (SMA) in semi-immune individuals eliminates both infected and uninfected erythrocytes and is a frequent fatal complication. It is proportional not to circulating parasitemia but total parasite mass (sequestered) in the organs. Thus, immune responses that clear parasites in organs may trigger changes leading to anemia. Here, we use an outbred-rat model where increasing parasite removal in the spleen escalated uninfected-erythrocyte removal. Splenic parasite clearance was associated with activated CD8+ T cells, immunodepletion of which prevented parasite clearance. CD8+ T cell repletion and concomitant reduction of the parasite load was associated with exacerbated (40 to 60%) hemoglobin loss and changes in properties of uninfected erythrocytes. Together, these data suggest that CD8+ T cell-dependent parasite clearance causes erythrocyte removal in the spleen and thus anemia. In children infected with the human malaria parasite Plasmodium falciparum, elevation of parasite biomass (not the number of circulating parasites) increased the odds ratio for SMA by 3.5-fold (95% confidence intervals [CI95%], 1.8- to 7.5-fold). CD8+ T cell expansion/activation independently increased the odds ratio by 2.4-fold (CI95%, 1.0- to 5.7-fold). Concomitant increases in both conferred a 7-fold (CI95%, 1.9- to 27.4-fold)-greater risk for SMA. Together, these data suggest that CD8+-dependent parasite clearance may predispose individuals to uninfected-erythrocyte loss and SMA, thus informing severe disease diagnosis and strategies for vaccine development. Importance  Malaria is a major global health problem. Severe malaria anemia (SMA) is a complex disease associated with partial immunity. Rapid hemoglobin reductions of 20 to 50% are commonly observed and must be rescued by transfusion (which can carry a risk of HIV acquisition). The causes and risk factors of SMA remain poorly understood. Recent studies suggest that SMA is linked to parasite biomass

  13. Induction of Suicidal Erythrocyte Death by Nelfinavir

    PubMed Central

    Bissinger, Rosi; Waibel, Sabrina; Lang, Florian

    2015-01-01

    The HIV protease inhibitor, nelfinavir, primarily used for the treatment of HIV infections, has later been shown to be effective in various infectious diseases including malaria. Nelfinavir may trigger mitochondria-independent cell death. Erythrocytes may undergo eryptosis, a mitochondria-independent suicidal cell death characterized by cell shrinkage and phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include oxidative stress and increase of cytosolic Ca2+-activity ([Ca2+]i). During malaria, accelerated death of infected erythrocytes may decrease parasitemia and thus favorably influence the clinical course of the disease. In the present study, phosphatidylserine abundance at the cell surface was estimated from annexin V binding, cell volume from forward scatter, reactive oxidant species (ROS) from 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and [Ca2+]i from Fluo3-fluorescence. A 48 h treatment of human erythrocytes with nelfinavir significantly increased the percentage of annexin-V-binding cells (≥5µg/mL), significantly decreased forward scatter (≥2.5µg/mL), significantly increased ROS abundance (10 µg/mL), and significantly increased [Ca2+]i (≥5 µg/mL). The up-regulation of annexin-V-binding following nelfinavir treatment was significantly blunted, but not abolished by either addition of the antioxidant N-acetylcysteine (1 mM) or removal of extracellular Ca2+. In conclusion, exposure of erythrocytes to nelfinavir induces oxidative stress and Ca2+ entry, thus leading to suicidal erythrocyte death characterized by erythrocyte shrinkage and erythrocyte membrane scrambling. PMID:26008229

  14. Human erythrocyte Band 3 functions as a receptor for the sialic acid-independent invasion of Plasmodium falciparum. Role of the RhopH3-MSP1 complex

    PubMed Central

    Baldwin, Michael; Yamodo, Innocent; Ranjan, Ravi; Li, Xuerong; Mines, Gregory; Marinkovic, Marina; Hanada, Toshihiko; Oh, Steven S.; Chishti, Athar H.

    2014-01-01

    Plasmodium falciparum takes advantage of two broadly defined alternate invasion pathways when infecting human erythrocytes: one that depends on and the other that is independent of host sialic acid residues on the erythrocyte surface. Within the sialic acid-dependent (SAD) and sialic acid-independent (SAID) invasion pathways, several alternate host receptors are used by Plasmodium falciparum based on its particular invasion phenotype. Earlier, we reported that two putative extracellular regions of human erythrocyte band 3 termed 5C and 6A function as host invasion receptor segments binding parasite proteins MSP1 and MSP9 via a SAID mechanism. In this study, we developed two mono-specific anti-peptide chicken IgY antibodies to demonstrate that the 5C and 6A regions of band 3 are exposed on the surface of human erythrocytes. These antibodies inhibited erythrocyte invasion by the Plasmodium falciparum 3D7 and 7G8 strains (SAID invasion phenotype), and the blocking effect was enhanced in sialic acid-depleted erythrocytes. In contrast, the IgY antibodies had only a marginal inhibitory effect on FCR3 and Dd2 strains (SAD invasion phenotype). A direct biochemical interaction between erythrocyte band 3 epitopes and parasite RhopH3, identified by the yeast two-hybrid screen, was established. RhopH3 formed a complex with MSP119 and 5ABC region of band 3, and a recombinant segment of RhopH3 inhibited parasite invasion in human erythrocytes. Together, these findings provide evidence that erythrocyte band 3 functions as a major host invasion receptor in the SAID invasion pathway by assembling a multi-protein complex composed of parasite ligands RhopH3 and MSP1. PMID:25157665

  15. Erythrocyte sedimentation rate of human blood exposed to low-level laser.

    PubMed

    Al Musawi, Mustafa S; Jaafar, M S; Al-Gailani, B; Ahmed, Naser M; Suhaimi, Fatanah M; Bakhsh, Muhammad

    2016-08-01

    This study is designed to investigate in vitro low-level laser (LLL) effects on rheological parameter, erythrocyte sedimentation rate (ESR), of human blood. The interaction mechanism between LLL radiation and blood is unclear. Therefore, research addresses the effects of LLL irradiation on human blood and this is essential to understanding how laser radiation interacts with biological cells and tissues. The blood samples were collected through venipuncture into EDTA-containing tubes as an anticoagulant. Each sample was divided into two equal aliquots to be used as a non-irradiated sample (control) and an irradiated sample. The aliquot was subjected to doses of 36, 54, 72 and 90 J/cm(2) with wavelengths of 405, 589 and 780 nm, with a radiation source at a fixed power density of 30 mW/cm(2). The ESR and red blood cell count and volume are measured after laser irradiation and compared with the non-irradiated samples. The maximum reduction in ESR is observed with radiation dose 72 J/cm(2) delivered with a 405-nm wavelength laser beam. Moreover, no hemolysis is observed under these irradiation conditions. In a separate protocol, ESR of separated RBCs re-suspended in irradiated plasma (7.6 ± 2.3 mm/h) is found to be significantly lower (by 51 %) than their counterpart re-suspended in non-irradiated plasma (15.0 ± 3.7 mm/h). These results indicate that ESR reduction is mainly due to the effects of LLL on the plasma composition that ultimately affect whole blood ESR. PMID:27250712

  16. Identification of a human erythrocyte receptor for colonization factor antigen I pili expressed by H10407 enterotoxigenic Escherichia coli.

    PubMed Central

    Pieroni, P; Worobec, E A; Paranchych, W; Armstrong, G D

    1988-01-01

    We have identified a receptor for colonization factor antigen I (CFA/I) pili in human erythrocyte membranes. Erythrocyte binding assays, using whole organisms, suggested that the CFA/I receptor was a glycoprotein containing important sialic acid moieties. Subsequently, human erythrocyte membranes were extracted with lithium diiodosalicylate to obtain a soluble glycoprotein fraction from which to isolate receptors. The extracted material caused agglutination of the CFA/I+ but not the CFA/I- organisms at a protein concentration of 0.5 mg/ml. The CFA/I receptor was identified in iodinated extract by an affinity isolation procedure, using whole bacterial cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of the washed, extract-coated H10407 CFA/I+ organisms revealed a band with an apparent molecular weight of 26,000 which was present in the original extract but was not observed on extract-coated H10407 CFA/I- bacteria. The addition of purified CFA/I pili reduced binding of the 26,000-molecular-weight receptor to CFA/I+ bacteria. The CFA/I-specific receptor species also bound to wheat germ agglutinin-agarose. This observation supported the suggestion that the CFA/I receptor identified in this report is a sialoglycoprotein. Images PMID:2895745

  17. Real-time study of shape and thermal fluctuations in the echinocyte transformation of human erythrocytes using defocusing microscopy.

    PubMed

    Etcheverry, Sebastián; Gallardo, María José; Solano, Pablo; Suwalsky, Mario; Mesquita, Oscar N; Saavedra, Carlos

    2012-10-01

    We present a real-time method to measure the amplitude of thermal fluctuations in biological membranes by means of a new treatment of the defocusing microscopy (DM) optical technique. This approach was also applied to study the deformation of human erythrocytes to its echinocyte structure. This was carried out by making three-dimensional shape reconstructions of the cell and measuring the thermal fluctuations of its membrane, as the cell is exposed to the anti-inflammatory drug naproxen and as it recovers its original shape, when it is subsequently cleansed of the drug. The results showed biomechanical changes in the membrane even at low naproxen concentration (0.2 mM). Also, we found that when the cell recovered its original shape, the membrane properties were different compared to the nondrugged initial erythrocyte, indicating that the drug administration-recovery process is not completely reversible. PMID:23224012

  18. Distribution of sodium and potassium within individual human erythrocytes by pulsed-laser vaporization in a sheath flow

    SciTech Connect

    Cheung, N.H.; Yeung, E.S. )

    1994-04-01

    Simultaneous determination of the amounts of Na and K inside single human erythrocytes was accomplished by laser vaporization and monitoring the atomic emission produced. By using a modified sheath-flow arrangement, detection of 8 fg (0.35 fmol) of Na is possible with one laser pulse. By using Poisson statistics, one can obtain single-cell information even when multiple cells are vaporized per laser pulse. The intracellular contents for a given individual were found to vary significantly. The [+-]55% and [+-]155% variations for Na and K, respectively, cannot be explained by changes in cell volume. There is only a weak correlation between the Na and K contents in single cells. The results reflect the age distribution of erythrocytes in the sample. Presumably, the enzymes regulating ion transport lose their activities in the older cells. 28 refs., 11 figs., 2 tabs.

  19. Functional topography of band 3: specific structural alteration linked to function aberrations in human erythrocytes

    SciTech Connect

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

    1988-01-01

    Band 3 is the major anion transport polypeptide of erythrocytes. It appears to be the binding site of several glycolytic enzymes. Structurally, band 3 is the major protein spanning the erythrocyte membrane and connects the plasma membrane to band 2.1, which binds to the cytoskeleton. In the present study, the authors report an alteration of band 3 molecule that is associated with the following changes: erythrocyte shape change from discoid to thorny cells (acanthocytes), restriction of rotational diffusion of band 3 in the membrane, increase in anion transport, and decrease in the number of high-affinity ankyrin-binding sites. Changes in erythrocyte IgG binding, glyceraldehyde-3-phosphate dehydrogenase, fluorescence polarization (indicative of membrane fluidity), and other membrane proteins as determined by polyacrylamide gel electrophoresis were not detected. Cells containing the altered band 3 polypeptide were obtained from individuals with abnormal erythrocyte morphology. Two-dimensional peptide maps revealed differences in the M/sub r/ 17,000 anion transport segment of band 3 consistent with additions of tyrosines or tyrosine-containing peptides. The data suggest that (i) this alteration of band 3 does not result in accelerated aging as does cleavage and (ii) structural changes in the anion transport region result in alterations in anion transport.

  20. Homology-Based Prediction of Potential Protein–Protein Interactions between Human Erythrocytes and Plasmodium falciparum

    PubMed Central

    Ramakrishnan, Gayatri; Srinivasan, Narayanaswamy; Padmapriya, Ponnan; Natarajan, Vasant

    2015-01-01

    Plasmodium falciparum, a causative agent of malaria, is a well-characterized obligate intracellular parasite known for its ability to remodel host cells, particularly erythrocytes, to successfully persist in the host environment. However, the current levels of understanding from the laboratory experiments on the host–parasite interactions and the strategies pursued by the parasite to remodel host erythrocytes are modest. Several computational means developed in the recent past to predict host–parasite/pathogen interactions have generated testable hypotheses on feasible protein–protein interactions. We demonstrate the utility of protein structure-based protocol in the recognition of potential interacting proteins across P. falciparum and host erythrocytes. In concert with the information on the expression and subcellular localization of host and parasite proteins, we have identified 208 biologically feasible interactions potentially brought about by 59 P. falciparum and 30 host erythrocyte proteins. For selected cases, we have evaluated the physicochemical viability of the predicted interactions in terms of surface complementarity, electrostatic complementarity, and interaction energies at protein interface regions. Such careful inspection of molecular and mechanistic details generates high confidence on the predicted host–parasite protein–protein interactions. The predicted host–parasite interactions generate many experimentally testable hypotheses that can contribute to the understanding of possible mechanisms undertaken by the parasite in host erythrocyte remodeling. Thus, the key protein players recognized in P. falciparum can be explored for their usefulness as targets for chemotherapeutic intervention. PMID:26740742

  1. Development and validation of HPLC method for the determination of alpha-tocopherol in human erythrocytes for clinical applications.

    PubMed

    Solichová, Dagmar; Korecká, Lucie; Svobodová, Iveta; Musil, Frantisek; Bláha, Vladimír; Zdánský, Petr; Zadák, Zdenek

    2003-06-01

    In this work, a simple isocratic reversed-phase HPLC method for determination of alpha-tocopherol in human erythrocytes has been developed and validated. After separation of plasma the erythrocytes were washed three times with 0.9% sodium chloride containing 0.01% butylated hydroxytoluene (BHT) as antioxidant and then were diluted 1:1 (v/v) with the same solution. In the liquid-liquid extraction (LLE) procedure, 2500 microL of n-hexane was added to 500 microL of erythrocytes. After 2 min this mixture was deproteinized by addition of cool ethanol (500 microL, 5 min) denatured with 5% methanol containing alpha-tocopherol acetate (20 micromol L(-1)), as internal standard, and then extracted for 5 min by vortex mixing. After centrifugation (10 min, 1600xg) an aliquot (2000 microL) of the clean extract was separated and evaporated under nitrogen. The residue was dissolved in 400 microL methanol and analysed by reversed-phase HPLC on a 4.6 mmx150 mm, 5 microm Pecosphere C18 column; the mobile phase was 100% methanol, flow rate 1.2 mL min(-1). The volume injected was 100 microL and detection was by diode-array detector at a wavelength of 295 nm. The extraction recovery of alpha-tocopherol from human erythrocytes was 100.0+/-2.0%. The detection limit was 0.1 micromol L(-1) and a linear calibration plot was obtained in the concentration range 0.5-20.0 micromol L(-1). Within determination precision was 5.2% RSD (n=10), between determination precision was 6.1% RSD (n=10). The method was applied successfully in a clinical study of patients with acute pancreatitis and for determination of the reference values in the healthy Czech population. PMID:12719955

  2. RhD Specific Antibodies Are Not Detectable in HLA-DRB1(*)1501 Mice Challenged with Human RhD Positive Erythrocytes.

    PubMed

    Bernardo, Lidice; Denomme, Gregory A; Shah, Kunjlata; Lazarus, Alan H

    2014-01-01

    The ability to study the immune response to the RhD antigen in the prevention of hemolytic disease of the fetus and newborn has been hampered by the lack of a mouse model of RhD immunization. However, the ability of transgenic mice expressing human HLA DRB1(*)1501 to respond to immunization with purified RhD has allowed this question to be revisited. In this work we aimed at inducing anti-RhD antibodies by administering human RhD(+) RBCs to mice transgenic for the human HLA DRB1(*)1501 as well as to several standard inbred and outbred laboratory strains including C57BL/6, DBA1/J, CFW(SW), CD1(ICR), and NSA(CF-1). DRB1(*)1501 mice were additionally immunized with putative extracellular immunogenic RhD peptides. DRB1(*)1501 mice immunized with RhD(+) erythrocytes developed an erythrocyte-reactive antibody response. Antibodies specific for RhD could not however be detected by flow cytometry. Despite this, DRB1(*)1501 mice were capable of recognizing immunogenic sequences of Rh as injection with Rh peptides induced antibodies reactive with RhD sequences, consistent with the presence of B cell repertoires capable of recognizing RhD. We conclude that while HLA DRB1(*)1501 transgenic mice may have the capability of responding to immunogenic sequences within RhD, an immune response to human RBC expressing RhD is not directly observed. PMID:25628657

  3. RhD Specific Antibodies Are Not Detectable in HLA-DRB1*1501 Mice Challenged with Human RhD Positive Erythrocytes

    PubMed Central

    Bernardo, Lidice; Denomme, Gregory A.; Shah, Kunjlata; Lazarus, Alan H.

    2014-01-01

    The ability to study the immune response to the RhD antigen in the prevention of hemolytic disease of the fetus and newborn has been hampered by the lack of a mouse model of RhD immunization. However, the ability of transgenic mice expressing human HLA DRB1*1501 to respond to immunization with purified RhD has allowed this question to be revisited. In this work we aimed at inducing anti-RhD antibodies by administering human RhD+ RBCs to mice transgenic for the human HLA DRB1*1501 as well as to several standard inbred and outbred laboratory strains including C57BL/6, DBA1/J, CFW(SW), CD1(ICR), and NSA(CF-1). DRB1*1501 mice were additionally immunized with putative extracellular immunogenic RhD peptides. DRB1*1501 mice immunized with RhD+ erythrocytes developed an erythrocyte-reactive antibody response. Antibodies specific for RhD could not however be detected by flow cytometry. Despite this, DRB1*1501 mice were capable of recognizing immunogenic sequences of Rh as injection with Rh peptides induced antibodies reactive with RhD sequences, consistent with the presence of B cell repertoires capable of recognizing RhD. We conclude that while HLA DRB1*1501 transgenic mice may have the capability of responding to immunogenic sequences within RhD, an immune response to human RBC expressing RhD is not directly observed. PMID:25628657

  4. Erythrocyte Protoporphyrin Fluorescence as a Biomarker to Monitor the Anticancer Effect of Semecarpus Anacardium in DMBA Induced Mammary Carcinoma Rat Model.

    PubMed

    Khan, Haseena Banu Hedayathullah; Vani, S; Palanivelu, Shanthi; Panchanadham, Sachdanandam

    2015-07-01

    Endogenous fluorescence has been proposed as a means of aiding the diagnosis of various malignancies. It has been suggested that erythrocytes may be the carriers of fluorophors that accumulate in cancer tissue and may be useful in the diagnosis and treatment of malignancies. Hence, the present study was designed to explore the spectrofluorimetric analysis of blood components as a marker for the analysis of mammary carcinoma treatment and also to bring about the protective effect of the drug Semecarpus anacardium on oxidative stress mediated damage of erythrocytes. Fluorescence spectra of the blood components were studied and also the level of lipid per oxides and antioxidant enzymes status in erythrocytes were determined in DMBA induced mammary carcinoma rats treated with Semecarpus anacardium Linn nut milk extract. Fluorescence emission spectroscopy of blood components are altered under cancer conditions and the drug effectively ameliorated these alterations in mammary carcinoma induced rats. The drug also effectively reduced the oxidative stress induced erythrocyte damage thereby restoring the erythrocytes antioxidant status. These results suggest that erythrocytes may be the carriers of fluorophors that accumulate in cancer tissue and hence acts as new biomarkers for the diagnosis and treatment. PMID:25943985

  5. The orientation of eosin-5-maleimide on human erythrocyte band 3 measured by fluorescence polarization microscopy.

    PubMed Central

    Blackman, S M; Cobb, C E; Beth, A H; Piston, D W

    1996-01-01

    The dominant motional mode for membrane proteins is uniaxial rotational diffusion about the membrane normal axis, and investigations of their rotational dynamics can yield insight into both the oligomeric state of the protein and its interactions with other proteins such as the cytoskeleton. However, results from the spectroscopic methods used to study these dynamics are dependent on the orientation of the probe relative to the axis of motion. We have employed polarized fluorescence confocal microscopy to measure the orientation of eosin-5-maleimide covalently reacted with Lys-430 of human erythrocyte band 3. Steady-state polarized fluorescence images showed distinct intensity patterns, which were fit to an orientation distribution of the eosin absorption and emission dipoles relative to the membrane normal axis. This orientation was found to be unchanged by trypsin treatment, which cleaves band 3 between the integral membrane domain and the cytoskeleton-attached domain. this result suggests that phosphorescence anisotropy changes observed after trypsin treatment are due to a rotational constraint change rather than a reorientation of eosin. By coupling time-resolved prompt fluorescence anisotropy with confocal microscopy, we calculated the expected amplitudes of the e-Dt and e-4Dt terms from the uniaxial rotational diffusion model and found that the e-4Dt term should dominate the anisotropy decay. Delayed fluorescence and phosphorescence anisotropy decays of control and trypsin-treated band 3 in ghosts, analyzed as multiple uniaxially rotating populations using the amplitudes predicted by confocal microscopy, were consistent with three motional species with uniaxial correlation times ranging from 7 microseconds to 1.4 ms. Images FIGURE 4 FIGURE 8 FIGURE 9 PMID:8804603

  6. The metabolites of glutamine prevent hydroxyl radical-induced apoptosis through inhibiting mitochondria and calcium ion involved pathways in fish erythrocytes.

    PubMed

    Li, Huatao; Jiang, Weidan; Liu, Yang; Jiang, Jun; Zhang, Yongan; Wu, Pei; Zhao, Juan; Duan, Xudong; Zhou, Xiaoqiu; Feng, Lin

    2016-03-01

    The present study explored the apoptosis pathways in hydroxyl radicals ((∙)OH)-induced carp erythrocytes. Carp erythrocytes were treated with the caspase inhibitors in physiological carp saline (PCS) or Ca(2+)-free PCS in the presence of 40μM FeSO4/20μM H2O2. The results showed that the generation of reactive oxygen species (ROS), the release of cytochrome c and DNA fragmentation were caspase-dependent, and Ca(2+) was involved in calpain activation and phosphatidylserine (PS) exposure in (∙)OH-induced carp erythrocytes. Moreover, the results suggested that caspases were involved in PS exposure, and Ca(2+) was involved in DNA fragmentation in (∙)OH-induced fish erythrocytes. These results demonstrated that there might be two apoptosis pathways in fish erythrocytes, one is the caspase and cytochrome c-dependent apoptosis that is similar to that in mammal nucleated cells, the other is the Ca(2+)-involved apoptosis that was similar to that in mammal non-nucleated erythrocytes. So, fish erythrocytes may be used as a model for studying oxidative stress and apoptosis in mammal cells. Furthermore, the present study investigated the effects of glutamine (Gln)'s metabolites [alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala10Pro4Cit1)] on the pathways of apoptosis in fish erythrocytes. The results displayed that Ala, Cit, Pro and Ala10Pro4Cit1 effectively suppressed ROS generation, cytochrome c release, activation of caspase-3, caspase-8 and caspase-9 at the physiological concentrations, prevented Ca(2+) influx, calpain activation, PS exposure, DNA fragmentation and the degradation of the cytoskeleton and oxidation of membrane and hemoglobin (Hb) and increased activity of anti-hydroxyl radical (AHR) in (∙)OH-induced carp erythrocytes. Ala10Pro4Cit1 produced a synergistic effect of inhibited oxidative stress and apoptosis in fish erythrocytes. These results demonstrated that Ala, Cit, Pro and their combination can protect mammal erythrocytes

  7. Expression and purification of recombinant cytoplasmic domain of human erythrocyte band 3 with hexahistidine tag or chitin-binding tag in Escherichia coli.

    PubMed

    Ding, Yu; Jiang, Weihua; Su, Yang; Zhou, Hanqing; Zhang, Zhihong

    2004-04-01

    The cytoplasmic domain of erythrocyte band 3 (cdb3) serves as a center of membrane organization in the erythrocytes by its interaction with multiple proteins including ankyrin, protein 4.1, protein 4.2, hemoglobin, and several glycolytic enzymes. In this paper, human cdb3 was cloned into three different expression vectors controlled by T7 polymerase promoter and induced with isopropyl beta-D-thiogalactopyranoside in Escherichia coli. Two of the fusion proteins containing hexahistidine tag in the N-terminal or C-terminal were purified by immobilized metal affinity column chromatography. The third recombinant cdb3 containing the affinity chitin-binding tag was purified using chitin beads followed by specific self-cleavage, which released cdb3 according to the mechanism of protein splicing. The molecular weights of purified recombinant proteins were verified by mass spectrometry. The pH-dependent properties of the intrinsic tryptophan fluorescence of the three kinds of recombinant cdb3 were compared with that of the cdb3 extracted from the erythrocytes, showing that there were no significant differences between them. Using pull-down assay combined with Western blot analysis, the interaction between recombinant cdb3 and protein 4.2 C3 fragment was verified. These demonstrated that the recombinant proteins were both structurally and functionally active. The typical yields of cdb3 purified with hexahistidine tag in the N-terminal, C-terminal, and cleaved from chitin bead were 10.6, 9.6, and 1.5 mg from 1L culture medium, respectively. PMID:15003247

  8. Engineered binding to erythrocytes induces immunological tolerance to E. coli asparaginase

    PubMed Central

    Lorentz, Kristen M.; Kontos, Stephan; Diaceri, Giacomo; Henry, Hugues; Hubbell, Jeffrey A.

    2015-01-01

    Antigen-specific immune responses to protein drugs can hinder efficacy and compromise safety because of drug neutralization and secondary clinical complications. We report a tolerance induction strategy to prevent antigen-specific humoral immune responses to therapeutic proteins. Our modular, biomolecular approach involves engineering tolerizing variants of proteins such that they bind erythrocytes in vivo upon injection, on the basis of the premise that aged erythrocytes and the payloads they carry are cleared tolerogenically, driving the deletion of antigen-specific T cells. We demonstrate that binding the clinical therapeutic enzyme Escherichia coli l-asparaginase to erythrocytes in situ antigen-specifically abrogates development of antibody titers by >1000-fold and extends the pharmacodynamic effect of the drug 10-fold in mice. Additionally, a single pretreatment dose of erythrocyte-binding asparaginase tolerized mice to multiple subsequent doses of the wild-type enzyme. This strategy for reducing antigen-specific humoral responses may enable more effective and safer treatment with therapeutic proteins and drug candidates that are hampered by immunogenicity. PMID:26601215

  9. Effect of ethanol intake on human erythrocyte membrane fluidity and lipid composition.

    PubMed

    Hrelia, S; Lercker, G; Biagi, P L; Bordoni, A; Stefanini, F; Zunarelli, P; Rossi, C A

    1986-05-01

    Erythrocyte membrane fluidity was evaluated in chronic alcoholic patients without any liver alteration, assuming different daily ethanol amounts, and in normal subjects and related to ghost fatty acid and total lipid composition obtained by high resolution gas chromatography. Erythrocyte membrane fluidity was significantly increased in a dose dependent manner in chronic alcoholic patients respect to normal subjects. This real fluidizing effect of ethanol "in vivo" was attributed mainly to a significant increase in the polyunsaturated fatty acids amount in patient ghosts in comparison with control subjects. On the other hand the cholesterol/phospholipid ratio was not significantly affected by chronic ethanol assumption. PMID:3729966

  10. Radiation damage to human erythrocytes: Influence of the composition of medium

    NASA Astrophysics Data System (ADS)

    Komorowska, Magdalena; Krokosz, Anita; Szweda-Lewandowska, Zofia

    2007-10-01

    The erythrocyte suspensions in PBS (Na-phosphate buffered isotonic NaCl solution) or PB (Na-phosphate isotonic buffer) (hematocrit 1%) were irradiated with the dose of 400 Gy in aerobic conditions. The level of damage to cells was estimated after incubation in different media. A higher level of destruction of cells irradiated in PBS than in PB was observed. The same level of MetHb and lipid peroxidation determined right after irradiation was detected. However, the loss of reduced glutathione was higher in PB than in PBS. We discussed the contribution of hydroxyl and chloride radicals in the initiation of erythrocyte damage.

  11. Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11-7082, parthenolide and dimethyl fumarate.

    PubMed

    Ghashghaeinia, Mehrdad; Giustarini, Daniela; Koralkova, Pavla; Köberle, Martin; Alzoubi, Kousi; Bissinger, Rosi; Hosseinzadeh, Zohreh; Dreischer, Peter; Bernhardt, Ingolf; Lang, Florian; Toulany, Mahmoud; Wieder, Thomas; Mojzikova, Renata; Rossi, Ranieri; Mrowietz, Ulrich

    2016-01-01

    In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11-7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11-7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11-7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach "Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target" (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity. PMID:27353740

  12. Pharmacological targeting of glucose-6-phosphate dehydrogenase in human erythrocytes by Bay 11–7082, parthenolide and dimethyl fumarate

    PubMed Central

    Ghashghaeinia, Mehrdad; Giustarini, Daniela; Koralkova, Pavla; Köberle, Martin; Alzoubi, Kousi; Bissinger, Rosi; Hosseinzadeh, Zohreh; Dreischer, Peter; Bernhardt, Ingolf; Lang, Florian; Toulany, Mahmoud; Wieder, Thomas; Mojzikova, Renata; Rossi, Ranieri; Mrowietz, Ulrich

    2016-01-01

    In mature erythrocytes, glucose-6-phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase (6PGDH) yield NADPH, a crucial cofactor of the enzyme glutathione reductase (GR) converting glutathione disulfide (GSSG) into its reduced state (GSH). GSH is essential for detoxification processes in and survival of erythrocytes. We explored whether the anti-inflammatory compounds Bay 11–7082, parthenolide and dimethyl fumarate (DMF) were able to completely deplete a common target (GSH), and to impair the function of upstream enzymes of GSH recycling and replenishment. Treatment of erythrocytes with Bay 11–7082, parthenolide or DMF led to concentration-dependent eryptosis resulting from complete depletion of GSH. GSH depletion was due to strong inhibition of G6PDH activity. Bay 11–7082 and DMF, but not parthenolide, were able to inhibit the GR activity. This approach “Inhibitors, Detection of their common target that is completely depleted or inactivated when pharmacologically relevant concentrations of each single inhibitor are applied, Subsequent functional analysis of upstream enzymes for this target” (IDS), can be applied to a broad range of inhibitors and cell types according to the selected target. The specific G6PDH inhibitory effect of these compounds may be exploited for the treatment of human diseases with high NADPH and GSH consumption rates, including malaria, trypanosomiasis, cancer or obesity. PMID:27353740

  13. Binding specificities of eight monoclonal antibodies to human glycophorin A - studies with M/sup c/M, and M/sub k/En(UK) variant human erythrocytes and M- and MN/sup V/-type chimpanzee erythrocytes

    SciTech Connect

    Bigbee, W.L.; Langlois, R.G.; Vanderlaan, M.; Jensen, R.H.

    1984-12-01

    Four newly derived mouse monoclonal antibodies to human glycophorin A are described. Three of these antibodies bind preferentially to the N form of glycophorin A; the fourth recognizes a shared determinant of the M and N forms. All four antibodies are directed toward the 39 amino acid, amino-terminal portion of the protein, and the N-specific antibodies require for binding the presence of N-acetyl-neuraminic acid on the glycosidically linked oligosaccharides. Cross-reaction of the N-specific antibodies to homozygous MM erythrocytes appears to result from binding to glycophorin B. In addition, these antibodies together with four previously reported glycophorin monoclonal antibodies, including two that specifically recognize the M form of glycophorin A, were tested for binding to M/sup c/M and M/sup k/En(UK) variant human erythrocytes. Results obtained for five of the six M- or N-specific monoclonal antibodies point to the general immunodominance of the amino-terminal serine-leucine polymorphism and the requirement for sialic acid. The epitopes for all three N-specific monoclonal antibodies include the amino terminal leucine that occurs in the N form of glycophorin A and may also include the glutamic acid that occurs at position five. Their studies support the proposed Lepore-type glycophorin A-B hybrid gene rearrangement for the En(UK) allele found in the English En(a-) family. The data also confirm the expression of the M-like glycoprotein on chimpanzee erythrocytes and the presence of a human glycophorin B-like antigen on the MN/sup V/-type cells.

  14. Human Erythrocyte PIG-A Assay: An Easily Monitored Index of Gene Mutation Requiring Low Volume Blood Samples

    PubMed Central

    Dertinger, Stephen D.; Avlasevich, Svetlana L.; Bemis, Jeffrey C.; Chen, Yuhchyau; MacGregor, James T.

    2015-01-01

    This laboratory has previously described a method for scoring the incidence of rodent blood Pig-a mutant phenotype erythrocytes using immunomag-netic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends this approach to human blood. The frequencies of CD59- and CD55-negative reticulo-cytes (RETCD59−/CD55−) and erythrocytes (RBCCD59−/CD55−) seve as phenotypic reporters of PIG-A gene mutation. Immunomagnetic separation was found to provide an effective means of increasing the number of reticulocytes and erythro-cytes evaluated. Technical replicates were utilized to provide a sufficient number of cells for precise scoring while at the same time controlling for procedural accuracy by allowing comparison of replicate values. Cold whole blood samples could be held for at least one week without affecting reticulo-cyte, RETCD59−/CD55− or RBCCD59−/CD55− frequencies. Specimens from a total of 52 nonsmoking, self-reported healthy adult subjects were evaluated. The mean frequency of RETCD59−/CD55− and RBCCD592−/CD55− were 6.0 × 10−6 and 2.9 × 10−6, respectively. The difference is consistent with a modest selective pressure against mutant phenotype erythrocytes in the circulation, and suggests advantages of studying both populations of erythrocytes. Whereas intra-subject variability was low, inter-subject variability was relatively high, with RETCD59−/CD55− frequencies differing by more than 30-fold. There was an apparent correlation between age and mutant cell frequencies. Taken together, the results indicate that the frequency of human PIG-A mutant phenotype cells can be efficiently and reliably estimated using a labeling and analysis protocol that is well established for rodent-based studies. The applicability of the assay across species, its simplicity and statistical power, and the relatively non-invasive nature of the assay should benefit myriad research areas involving DNA damage

  15. Association of human erythrocyte membrane glycoproteins with blood-group Cad specificity.

    PubMed Central

    Cartron, J P; Blanchard, D

    1982-01-01

    Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of erythrocyte membranes from a blood-group-B individual with the rare Cad phenotype indicates a lower-than-normal mobility of the main sialoglycoproteins, suggesting an increase in apparent molecular mass of 3kDa and 2kDa respectively for glycoprotein alpha (synonym glycophorin A) and glycoprotein delta (synonym glycophorin B). Since the chief structural determinant of Cad specificity is N-acetylgalactosamine, the membrane receptors have been isolated by affinity binding on immobilized Dolichos biflorus (horse gram) lectin. The predominant species eluted from the gel was the abnormal glycoprotein alpha, whereas in control experiments no material could be recovered from the adsorbent incubated with group-B Cad-negative erythrocyte membranes. After partition of the membranes with organic solvents, the blood-group-Cad activity was found in aqueous phases containing the sialoglycoproteins, but not in the organic phases containing simple or complex glycolipids, which, however, retained the blood-group-B activity. The carbohydrate composition of highly purified lipid-free glycoprotein alpha molecules prepared from Cad and control erythrocytes was determined. Interestingly the molar ratio of N-acetylneuraminic acid to N-acetylgalactosamine was equal to 2:1 in the case of controls and equal to 1:1 in the case of Cad erythrocytes. Taken together these results suggest that Cad specificity is defined by N-acetylgalactosamine residues carried by the alkali-labile oligosaccharide chains attached to the erythrocyte membrane sialo-glycoproteins. Images Fig. 1. Fig. 2. PMID:6187337

  16. A critical evaluation of the proposal that serum apolipoproteins are the major constituents of the human erythrocyte membrane.

    PubMed

    Carey, C; Wang, C S; Alaupovic, P

    1975-08-01

    1. The EDTA and Triton X-100 extracts of human erythrocyte ghosts gave no precipitin lines in double diffusion analyses with antibodies to either lipoprotein A, lipoprotein B, lipoprotein C, lipoprotein D, Lp(a) lipoprotein or arginine-rich apolipoprotein of normal human serum (for nomenclature for serum lipoprotein families and apolipoptoteins, see Alaupovic, P., Kostner, G., Lee, D. M., McConathy, W.J. and Magnani, HN. (1972) Expo. Annu. Biochem. Med. 31, 145-160 and Alaupovic, P., Lee, D.M. and McConathy, W.J., (1972) Biochim. Biophys. Acta 260, 689-707.) These membrane preparations also reacted negatively with commercially available antisera to alpha- and beta-lipoproteins. 2. The normal serum very low density, low density and high density lipoproteins formed no precipitin lines with antibodies to either intact or EDTA-extracted ghosts. 3. The serum apolipoproteins and their constitutive polypeptides (A-I, A-II, B, C-I, C-II, C-III, D and arginine-rich apolipoprotein) reacted negatively with antibodies to intact or EDTA-extracted ghosts. The EDTA and Triton X-100 extracts of erythrocyte ghosts gave no reaction with monospecific antibodies to serum apolipoproteins and their constitutive polypeptides. 4. Ghosts dissolved 2% sodium dodecyl sulfate gave positive immunoprecipitin lines with antisera to alpha- and beta-lipoproteins. However, the sodium dodecyl sulfate solution in concentrations greater than 0.1% also formed precipitin lines with antisera to the same lipoproteins. 5. These results do not support the suggestion (Langdon, R.G. (1974) Biochim. Biophys. Acta 342, 213-228) that serum apolipoptoteins are integral protein constituents of human erythrocyte ghosts. The immunoprecipitin lines observed in the latter study might have been due to the presence of trace amounts of serum lipoproteins loosely attached to the cellular surfaces or, more probably, resulted from nonspecific interactions between the proteins and the sodium dodecyl sulfate used as the

  17. Hereditary Hemolytic Anemia with Human Erythrocyte Pyrimidine 5′-Nucleotidase Deficiency

    PubMed Central

    Valentine, William N.; Fink, Kay; Paglia, Donald E.; Harris, Susan R.; Adams, William S.

    1974-01-01

    A severe deficiency of a red cell pyrimidine 5′-nucleotidase was found to be associated with hereditary hemolytic anemia in four members of three kindreds. The syndrome was characterized by marked increases above normal in red cell basophilic stippling, total nucleotides, and GSH and by a fairly severe deficiency of ribosephosphate pyrophosphokinase (EC 2.7.6.1.). Patient erythrocytes uniquely contained large amounts of pyrimidine 5′-ribonucleotides. In earlier studies, these were erroneously considered to be adenosine phosphates, since all previous investigations of the nucleotides of human red cells and reticulocytes have shown 97% or more to contain adenine. Total nucleotides in patient cells were present in amounts 3-6 times greater than normal, and approximately 80% contained pyrimidine. The ultraviolet spectral curves of deproteinized red cell extracts exhibited a shift in maximum absorbance from the usual 256-257 nm to approximately 266-270 nm, and absorbance at 250, 270, 280, and 290 nm, expressed as a ratio of that at 260 nm, differed greatly from normal. The spectral characteristics of extracts provide the basis of a readily performed screening procedure, which does not require enzyme assay. The nucleotidase activity in deficient red cells assayed less than 14%, and usually less than 10%, of normal and much less in terms of reticulocyte-rich blood, where it was consistently found to be increased. The enzyme has a pH optimum of 7.5-8.0, is inhibited by EDTA, and does not utilize purine 5′-ribonucleotides or β-glycerophosphate as substrates. While comparatively few family members have been available thus far for study, initial data are compatible with an autosomal, recessive mode of transmission of the deficiency. The pyrimidine 5′-ribonucleotides are presumably derived from RNA degradation and, not being diffusible, accumulate when the enzyme catalyzing their dephosphorylation is deficient. It is postulated that the prominent basophilic stippling

  18. Annotating N Termini for the Human Proteome Project: N Termini and Nα-Acetylation Status Differentiate Stable Cleaved Protein Species from Degradation Remnants in the Human Erythrocyte Proteome

    PubMed Central

    2014-01-01

    A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as “missing”, this is challenging. Moreover, proteolytic processing generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier . PMID:24555563

  19. Comparative cytotoxic and genotoxic effects of permethrin and its nanometric form on human erythrocytes and lymphocytes in vitro.

    PubMed

    Sundaramoorthy, Rajiv; Velusamy, Yuvaraj; Balaji, A P B; Mukherjee, Amitava; Chandrasekaran, Natarajan

    2016-09-25

    The research on the novel pesticides such as nanopesticides has become inevitable to control the mosquito population. Nanopermethrin (NP), one of such kind was formulated in pesticide loaded oil-in-water (o/w) microemulsion by rapid evaporation. Even though NP possess improved efficacy against the target pests, the toxicological investigation on the human or mammalian system remains unexplored. So, the present study focused on a comparative investigation of the cytotoxic and genotoxic effects of NP in vitro and its commercial parental bulk form of permethrin (BP) on human peripheral erythrocyte/lymphocyte by erythrocyte morphology analysis, cell viability assay, and cytokinesis-block micronucleus (CBMN) assay. The NP and BP concentrations (10, 25, 50 and 100 μg/ml) interacted with human blood cells, and the morphological changes were observed using a phase contrast microscope. The drastic increase of echinocyte was observed at 24, 48 and 72 h treatment as compared with the control. The cell viability studies have shown the significant decrease with increase in NP and BP concentration. CBMN study showed a series correlation in the number of micronuclei, bridge, bud, trinucleated and tetranucleated when interacted with different levels of NP and BP, as comparative to control *p < 0.05, **p < 0.001, ***p < 0.0001. PMID:27502151

  20. Quantification of L-ergothioneine in human plasma and erythrocytes by liquid chromatography-tandem mass spectrometry.

    PubMed

    Wang, Ling-Zhi; Thuya, Win-Lwin; Toh, Dorothy Su-Lin; Lie, Michael George-Limenta; Lau, Jie-Ying Amelia; Kong, Li-Ren; Wan, Seow-Ching; Chua, Kian-Ngiap; Lee, Edmund Jon-Deoon; Goh, Boon-Cher

    2013-03-01

    A sensitive analytical method has been developed and validated for the quantification of L-ergothioneine in human plasma and erythrocytes by liquid chromatography-tandem mass spectrometry. A commercially available isotope-labeled L-ergothioneine-d9 is used as the internal standard. A simple protein precipitation with acetonitrile is utilized for bio-sample preparation prior to analysis. Chromatographic separation of L-ergothioneine is conducted using gradient elution on Alltime C18 (150 mm × 2.1 mm, 5 µ). The run time is 6 min at a constant flow rate of 0.45 ml/min. The mass spectrometer is operated under a positive electrospray ionization condition with multiple reaction monitoring mode. The mass transitions of L-ergothioneine and L-ergothioneine-d9 are m/z 230 > 127 and m/z 239 > 127, respectively. Excellent linearity [coefficient of determination (r(2)) ≥ 0.9998] can be achieved for L-ergothioneine quantification at the ranges of 10 to 10,000 ng/ml, with the intra-day and inter-day precisions at 0.9-3.9% and 1.3-5.7%, respectively, and the accuracies for all quality control samples between 94.5 and 101.0%. This validated analytical method is suitable for pharmacokinetic monitoring of L-ergothioneine in human and erythrocytes. Based on the determination of bio-samples from five healthy subjects, the mean concentrations of L-ergothioneine in plasma and erythrocytes are 107.4 ± 20.5 ng/ml and 1285.0 ± 1363.0 ng/ml, respectively. PMID:23494799

  1. Two-Component Coarse-Grained Molecular-Dynamics Model for the Human Erythrocyte Membrane

    PubMed Central

    Li, He; Lykotrafitis, George

    2012-01-01

    We present a two-component coarse-grained molecular-dynamics model for simulating the erythrocyte membrane. The proposed model possesses the key feature of combing the lipid bilayer and the erythrocyte cytoskeleton, thus showing both the fluidic behavior of the lipid bilayer and the elastic properties of the erythrocyte cytoskeleton. In this model, three types of coarse-grained particles are introduced to represent clusters of lipid molecules, actin junctions, and band-3 complexes, respectively. The proposed model facilitates simulations that span large length scales (approximately micrometers) and timescales (approximately milliseconds). By tuning the interaction potential parameters, we were able to control the diffusivity and bending rigidity of the membrane model. We studied the membrane under shearing and found that at a low shear strain rate, the developed shear stress was due mainly to the spectrin network, whereas the viscosity of the lipid bilayer contributed to the resulting shear stress at higher strain rates. In addition, we investigated the effects of a reduced spectrin network connectivity on the shear modulus of the membrane. PMID:22225800

  2. Photoaffinity labeling of the human erythrocyte glucose transporter with /sup 4/H-labelled forskolin

    SciTech Connect

    Shanahan, M.F.; Edwards, B.M.; Morris, D.P.

    1986-05-01

    Forskolin, a potent activator of adenylate cyclase, is also known to inhibit glucose transport in a number of cells. The authors have investigated photoincorporation of (/sup 3/H)forskolin into erythrocyte membrane proteins using a technique they previously developed for photolabeling the erythrocyte glucose transporter with cytochalasin B (CB). A 30-40s irradiation of erythrocyte ghosts in the presence of (/sup 3/H)forskolin resulted in a concentration-dependent, covalent incorporation of radiolabel into all of the major membrane protein bands. However, most of the incorporation occurred in only three regions of the gel. Peak 1 was a sharp peak near the top of the gel in the region corresponding to spectrin, peak 2 appeared to be associated with band 3 (approx. 90kDa), and the third region labeled was between 41-60 kDa which corresponds to the region of the glucose transporter. This region appeared to contain several overlapping peaks with the largest incorporation of label occurring around 45 kDa in the area of red cell actin. When photolabeling was performed in the presence of 400 ..mu..M cytochalasin B (8.0 ..mu..M forskolin) the labeling in the 41-60 kDa region was totally inhibited while labeling of the 90 kDa peak was partially blocked. CB had no effect on the photolabeling of peak 1 by forskolin.

  3. Concentration-dependent effects of resveratrol and metabolites on the redox status of human erythrocytes in single-dose studies.

    PubMed

    Pignitter, Marc; Schueller, Katharina; Burkon, Alexander; Knorr, Verena; Esefelder, Laura; Doberer, Daniel; Wolzt, Michael; Somoza, Veronika

    2016-01-01

    Dietary trans-resveratrol (RES) is rapidly metabolized into sulfated and glucuronated conjugates in humans. This study focused on the in vitro determination of the antioxidant capacity of RES and its main physiological metabolites and on its relevance in vivo. In vitro, RES, RES-3-O-sulfate (R3S) and 3-O-glucuronide (R3G) showed antioxidant activities at a concentration of 1mM when compared to Trolox using an assay in which the antioxidant inhibits iron-induced linoleic acid oxidation: 0.87±0.08mM Trolox equivalents (TE) for RES, 0.52±0.01mM TE for R3S and 0.36±0.02mM TE for R3G. At a concentration of 1μM, compounds promoted linoleic acid peroxidation (RES -0.30±0.09mM TE, R3S -0.48±0.05mM TE and R3G -0.57±0.07mM TE). To elucidate whether these effects were reflected in vivo, total antioxidant capacity, reactive oxygen species (ROS), conjugated fatty acid dienes (CD), superoxide dismutase (SOD) and catalase (CAT) activities were determined in human plasma and erythrocytes over 24h, after oral intake of either 0.05g RES as piceid or 5g RES. Oral administration of RES did not show an impact on total antioxidant capacity, ROS or CD. However, enzymatic activities of ROS scavenging SOD and CAT were significantly lower after high-dose compared to low-dose administration of RES (P<.03 and P<.01). In conclusion, in healthy subjects, neither 0.05g nor 5g RES changed blood oxidative state, although our in vitro data point to a prooxidative activity of low concentrations of RES and its metabolites, which could be important in vivo for individuals with compromised antioxidant defense capacity. PMID:26454510

  4. Antioxidant Capacity and Radical Scavenging Effect of Polyphenol Rich Mallotus philippenensis Fruit Extract on Human Erythrocytes: An In Vitro Study

    PubMed Central

    Gautam, Manish Kumar; Sharma, Amit Kumar; Tripathi, Yamini B.; Goel, R. K.; Nath, Gopal

    2014-01-01

    Mallotus philippinensis is an important source of molecules with strong antioxidant activity widely used medicinal plant. Previous studies have highlighted their anticestodal, antibacterial, wound healing activities, and so forth. So, present investigation was designed to evaluate the total antioxidant activity and radical scavenging effect of 50% ethanol fruit glandular hair extract (MPE) and its role on Human Erythrocytes. MPE was tested for phytochemical test followed by its HPLC analysis. Standard antioxidant assays like DPPH, ABTS, hydroxyl, superoxide radical, nitric oxide, and lipid peroxidation assay were determined along with total phenolic and flavonoids content. Results showed that MPE contains the presence of various phytochemicals, with high total phenolic and flavonoid content. HPLC analysis showed the presence of rottlerin, a polyphenolic compound in a very rich quantity. MPE exhibits significant strong scavenging activity on DPPH and ABTS assay. Reducing power showed dose dependent increase in concentration absorption compared to standard, Quercetin. Superoxide, hydroxyl radical, lipid peroxidation, nitric oxide assay showed a comparable scavenging activity compared to its standard. Our finding further provides evidence that Mallotus fruit extract is a potential natural source of antioxidants which have a protective role on human Erythrocytes exhibiting minimum hemolytic activity and this justified its uses in folklore medicines. PMID:25525615

  5. Antioxidant capacity and radical scavenging effect of polyphenol rich Mallotus philippenensis fruit extract on human erythrocytes: an in vitro study.

    PubMed

    Gangwar, Mayank; Gautam, Manish Kumar; Sharma, Amit Kumar; Tripathi, Yamini B; Goel, R K; Nath, Gopal

    2014-01-01

    Mallotus philippinensis is an important source of molecules with strong antioxidant activity widely used medicinal plant. Previous studies have highlighted their anticestodal, antibacterial, wound healing activities, and so forth. So, present investigation was designed to evaluate the total antioxidant activity and radical scavenging effect of 50% ethanol fruit glandular hair extract (MPE) and its role on Human Erythrocytes. MPE was tested for phytochemical test followed by its HPLC analysis. Standard antioxidant assays like DPPH, ABTS, hydroxyl, superoxide radical, nitric oxide, and lipid peroxidation assay were determined along with total phenolic and flavonoids content. Results showed that MPE contains the presence of various phytochemicals, with high total phenolic and flavonoid content. HPLC analysis showed the presence of rottlerin, a polyphenolic compound in a very rich quantity. MPE exhibits significant strong scavenging activity on DPPH and ABTS assay. Reducing power showed dose dependent increase in concentration absorption compared to standard, Quercetin. Superoxide, hydroxyl radical, lipid peroxidation, nitric oxide assay showed a comparable scavenging activity compared to its standard. Our finding further provides evidence that Mallotus fruit extract is a potential natural source of antioxidants which have a protective role on human Erythrocytes exhibiting minimum hemolytic activity and this justified its uses in folklore medicines. PMID:25525615

  6. DNA content determination of micronucleated polychromatic erythrocytes induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood

    SciTech Connect

    Grawe, J.; Amneus, H. Uppsala Univ. ); Zetterberg, G. )

    1993-01-01

    The frequencies and DNA distributions of micronuclei in polychromatic erythrocytes from the bone marrow and peripheral blood of mice after four different treatments were determined by flow cytometry. Polychromatic erthrocytes were detected using the fluorescent RNA stain thiazole orange, while micronuclei were detected with the DNA stain Hoechst 33342. The treatments were X-irradiation (1 Gy), cyclophosphamide (30 mg/kg), vincristine sulfphate (0.08 mg/kg), and cochicine (1 mg/kg). All treatments showed increased frequencies of micronucleated polychromatic erythrocytes at 30h after treatment in the bone marrow (colchicine 50h) and at 50h in the peripheral blood. The clostogenic agents X-irradiation and cyclophosphamide and the spindle poisons vincristine sulphate and cochicine could be grouped according to the fluorescent characteristics of the induced micronuclei as well as the relative frequency of small (0.5-2% if the diploid G1 DNA content) and large (2-10%) micronuclei. In the peripheral blood the relative frequency of large micronuclei was lower than in the bone marrow, indicating that they were partly eliminated before entrance into the peripheral circulation. The nature of presumed micronuclei was verified by sorting. The potential of this approach to give information on the mechanism of induction of micronuclei is discussed.

  7. Laser diffraction analysis of shear deformability of human and rat erythrocytes in norm and ischemia

    NASA Astrophysics Data System (ADS)

    Lugovtsov, A. E.; Priezzhev, A. V.; Nikitin, S. Y.; Koshelev, V. B.

    2007-05-01

    Ischemic diseases of people and animals are accompanied with deterioration of microrheologic properties of their blood, in particular, with impairing red blood cells (RBC) deformability. In this work, the analysis of human and rat RBC deformability in norm and ischemia was performed by means of the laser diffractometry - a modern technique allowing for measuring the flexibility of RBC, which determines the blood flow parameters in vessels. Human RBC were obtained from the blood of healthy individuals and from patients suffering from ischemic diseases. Human RBC deformability from both groups of individuals was measured. Rat RBC were obtained from a control group of animals and from a group with experimentally induced ischemia (EII). This animal model is frequently used for studying the response of an organism to ischemia. The effect of Semax, a medication that is frequently used for therapeutic treatments of human brain diseases in clinical practice, on RBC deformability was studied with its application in vitro and in vivo. It is shown that in human ischemic patients, the deformability of RBC was lower than that from healthy individuals. Both in vivo and in vitro applied semax positively influences the impaired deformability properties of RBC of ischemic rats.

  8. Inhibition of lactate transport in Ehrlich ascites tumor cells and human erythrocytes by a synthetic anhydride of lactic acid.

    PubMed

    Johnson, J H; Belt, J A; Dubinsky, W P; Zimniak, A; Racker, E

    1980-08-01

    The synthesis and some of the physical and biological characteristics of a new inhibitor of lactate transport are described. The inhibitor is isobutylcarbonyl lactayl anhydride (iBCLA). It is formed by the condensation of lactic acid and isobutylchloroformate. It inhibits lactate transport 50% at 0.5 microgram/mg of protein in both Ehrlich ascites tumor cells and human erythrocytes. In contrast, 15 microgram of iBCLA/mg of protein is required for 50% inhibition of phosphate transport in erythrocytes, and phosphate transport in Ehrlich ascites tumor cells is unaffected at levels as high as 50 microgram of iBCLA/mg of protein. A time-dependent and concentration-dependent reversal of lactate transport inhibition took place on exposure of iBCLA-treated Ehrlich ascites cells to hydroxylamine or dithiothreitol. These data, along with the observed sensitivity of the lactate transporter to sulfhydryl reagents [Spencer, T. L., & Lehninger, A. L. (1976) Biochem. J. 154, 405-414], suggest that iBCLA acylates an essential sulfhydryl group on the transporter. When glycolyzing Ehrlich ascites tumor cells were treated with concentrations of iBCLA sufficient for complete inhibition of lactate transport, intracellular lactate levels increased, intracellular pH and extra-cellular lactate levels decreased, and overall lactate production was inhibited. PMID:7407072

  9. Interaction of Plasmodium vivax Tryptophan-rich Antigen PvTRAg38 with Band 3 on Human Erythrocyte Surface Facilitates Parasite Growth*

    PubMed Central

    Alam, Mohd. Shoeb; Choudhary, Vandana; Zeeshan, Mohammad; Tyagi, Rupesh K.; Rathore, Sumit; Sharma, Yagya D.

    2015-01-01

    Plasmodium tryptophan-rich proteins are involved in host-parasite interaction and thus potential drug/vaccine targets. Recently, we have described several P. vivax tryptophan-rich antigens (PvTRAgs), including merozoite expressed PvTRAg38, from this noncultivable human malaria parasite. PvTRAg38 is highly immunogenic in humans and binds to host erythrocytes, and this binding is inhibited by the patient sera. This binding is also affected if host erythrocytes were pretreated with chymotrypsin. Here, Band 3 has been identified as the chymotrypsin-sensitive erythrocyte receptor for this parasite protein. Interaction of PvTRAg38 with Band 3 has been mapped to its three different ectodomains (loops 1, 3, and 6) exposed at the surface of the erythrocyte. The binding region of PvTRAg38 to Band3 has been mapped to its sequence, KWVQWKNDKIRSWLSSEW, present at amino acid positions 197–214. The recombinant PvTRAg38 was able to inhibit the parasite growth in in vitro Plasmodium falciparum culture probably by competing with the ligand(s) of this heterologous parasite for the erythrocyte Band 3 receptor. In conclusion, the host-parasite interaction at the molecular level is much more complicated than known so far and should be considered during the development of anti-malarial therapeutics. PMID:26149684

  10. Protein 4.1: its association with the human erythrocyte membrane.

    PubMed Central

    Shiffer, K A; Goodman, S R

    1984-01-01

    125I-labeled protein 4.1a and 4.1b have equal ability to reassociate with inside-out erythrocyte vesicles that were depleted of protein 4.1 in addition to other peripheral membrane proteins. The reassociation of 125I-labeled protein 4.1 to protein 4.1-depleted vesicles at 4 degrees C is salt dependent, pH dependent, and saturable with a Kd of 42-50 nM and an extrapolated maximal binding capacity of 120-140 micrograms of protein 4.1 bound per mg of vesicle protein or 60-70 micrograms of protein 4.1 bound per mg of ghost protein, correlating with the protein 4.1 content in the erythrocyte membrane (6-7% of the total membrane protein). Selective proteolytic cleavage of these vesicles with papain (5 micrograms/ml at 4 degrees C) eliminates greater than 60% of the high-affinity binding sites; therefore, we conclude that the interaction of protein 4.1 with the cytoplasmic membrane surface is through a specific high-affinity protein-protein association. Images PMID:6589603

  11. Advanced Glycation-Modified Human Serum Albumin Evokes Alterations in Membrane and Eryptosis in Erythrocytes.

    PubMed

    Awasthi, Saurabh; Gayathiri, S K; Ramya, R; Duraichelvan, R; Dhason, A; Saraswathi, N T

    2015-11-01

    Increased burden of advanced glycation end-products (AGEs) in case of hyperglycemic conditions leads to the development of retinopathy, nephropathy, and cardiovascular and neurological disorders such as Alzheimer's disease. AGEs are considered as pro-oxidants, and their accumulation increases the oxidative stress. The prolonged exposure to these AGEs is the fundamental cause of chronic oxidative stress. Abnormal morphology of red blood cells (RBCs) and excessive eryptosis has been observed in diabetes, glomerulonephritis, dyslipidemia, and obesity, but yet the contribution of extracellular AGEs remains undefined. In this study, we investigated the effect of AGEs on erythrocytes to determine their impact on the occurrence of different pathological forms of these blood cells. Specifically, carboxymethyllysine (CML), carboxyethyllysine (CEL), and Arg-pyrimidine (Arg-P) which have been reported to be the most pre-dominant AGEs formed under in vivo conditions were used in this study. Results suggested the eryptotic properties of CML, CEL, and Arg-P for RBCs, which were evident from the highly damaged cell membrane and occurrence of abnormal morphologies. Methylglyoxal-modified albumin showed more severe effects, which can be attributed to the high reactivity and pro-oxidant nature of glycation end products. These findings suggest the possible role of AGE-modified albumin towards the morphological changes in erythrocyte's membrane associated with diabetic conditions. PMID:26276445

  12. Carnitine palmitoyltransferase in human erythrocyte membrane. Properties and malonyl-CoA sensitivity.

    PubMed Central

    Ramsay, R R; Mancinelli, G; Arduini, A

    1991-01-01

    Carnitine palmitoyltransferase located in the erythrocyte plasma membrane is sensitive to inhibition by malonyl-CoA and 2-bromopalmitoyl-CoA plus carnitine. Although this inhibition and other properties suggest similarities to the intracellular enzymes in other tissues, no cross-reaction was observed with antisera to the peroxisomal or to the mitochondrial inner-membrane enzyme. The activity was solubilized by and was stable in Triton X-100, which destroys the enzymes found in microsomes and in the mitochondrial outer membrane. The substrate specificity is broader than for the intracellular enzymes, the activities with stearoyl-CoA (114%) and arachidonoyl-CoA (97%) being equal to that with palmitoyl-CoA, and the activities with linoleoyl-CoA (44%) and erucoyl-CoA (46%) about half that with palmitoyl-CoA. The function of this carnitine palmitoyltransferase is probably to buffer the acyl-CoA present in the erythrocyte for turnover of the fatty acyl groups of the membrane lipids. PMID:2039446

  13. Zinc ions and alkaline pH alter the phosphorylation state of human erythrocyte membrane proteins

    SciTech Connect

    Fennell, R.L. Jr.

    1988-01-01

    Since the phosphorylation state of the red cell membrane proteins in vitro is likely to be regulated by phosphorylation and dephosphorylation, this research was carried out to investigate the possible role of membrane-bound phosphatase activities. These studies were conducted with red blood cell ghosts and IOVs from normal individuals and from an individual with hereditary spherocytosis. In vitro phosphorylation with ({gamma}-{sup 32}P) ATP was conducted in the presence and the absence of Zn{sup ++}, or erythrocyte ghosts and IOVs were pretreated for 30 minutes at 37{degree}C and pH 7-11 in the presence and the absence of calf intestine alkaline phosphatase. The resulting phosphoproteins were analyzed by SDS-polyacrylamide gel electrophoresis, stained with Coomassie blue, and fluorographed. In the presence of Zn{sup ++}, the red blood ghosts, with or without pretreatment, demonstrated enhanced phosphorylation of membrane proteins, including band 4.2. Preincubation at pH 10 in the presence of absence of exogenous phosphatase further stimulates phosphorylation of these proteins. Under similar conditions, the erythrocyte membranes also demonstrated the ability to hydrolyze p-nitrophenyl phosphate and to remove {sup 32}P from red blood cell phosphoproteins.

  14. Development and behavior of cultured Schistosoma mansoni fed on human erythrocyte ghosts.

    PubMed

    Basch, P F

    1984-09-01

    Schistosomula and adults of Schistosoma mansoni were grown from cercariae in cultures differing only in the treatment of the red blood cells fed to the organisms. "Pink ghosts," containing about 5% of the original hemoglobin, were produced by hemolysis in water; "white ghosts" with no detectable hemoglobin were made in 5 mM phosphate buffer, pH 8. Early growth and development were more rapid and vigorous, and pairs formed more readily when pink ghosts, rather than intact erythrocytes were fed. Schistosomula remained stunted and undeveloped when fed with white ghosts. Attempts at reconstitution of the latter by addition of hemoglobin, concentrated erythrocyte lysate, or pressure-liquefied pink ghosts did not restore growth-promoting activity. Pink ghost-fed worms, particularly paired males, attached to the dish bottom by their acetabulum and oral sucker and travelled by an active looping motion. Substrates of collagen or fibrin or a mammalian cell monolayer did not affect this behavior. Such attachment and locomotion are interpreted as instinctive migratory behavior of schistosomes. PMID:6486300

  15. Effects of Pistacia Atlantica Extract on Erythrocyte Membrane Rigidity, Oxidative Stress, and Hepatotoxicity Induced by CCl4 in Rats

    PubMed Central

    Tolooei, Mohsen; Mirzaei, Ali

    2015-01-01

    Background: Previous findings have suggested that antioxidants may reduce the levels of free radicals, which induce oxidative damage and play a key role in various diseases. Thus, we evaluated the protective activity of a Pistacia atlantica extract on erythrocyte membrane rigidity, oxidative stress, and hepatotoxicity induced by carbon tetrachloride (CCl4) in rats. Materials and Methods: Fresh leaves of P. atlantica were collected from the mountains in Yasuj, Iran. Acute oral toxicity (LD50) was evaluated in Wistar rats (200–230 g). Animals were randomly divided into 4 groups, out of which the negative and plant control groups received distilled water and P. atlantica extracts (500 mg/kg), respectively. The toxic rat group received CCl4, while the treatment group received CCl4 + P. atlantica extract. Blood plasma was utilized for the estimation of enzyme markers and lipid peroxidation, whereas hemolysate was applied for the determination of superoxide dismutase (SOD) and catalase activities. The levels of cholesterol and phospholipids in erythrocyte membranes were also determined. Rats were killed under anesthesia by cervical dislocation; liver was isolated from each rat and tissues homogenization was prepared for biochemical parameters such as malondialdehyde (MDA) and reduced glutathione (GSH) levels. Results: LD50 values were determined for doses >3000 mg/kg (p.o.). The activities of glutamic pyruvate transaminase (GPT), glutamic oxaloacetate transaminase (GOT), alkaline phosphatase (ALP) and GSH in the protected group were significantly (p < 0.001) reduced compared with those of toxic rats. In addition, we observed a decrease in the cholesterol level and an increase in red blood cell membrane phospholipids, SOD, and catalase activities (p < 0.001) in the protected group, as compared with toxic rats. Administration of Pistacia atlantica extract normalized liver tissue MDA level (p < 0. 01) when compared to CCl4 treated group. Conclusion: The P. atlantica

  16. Hemoglobin-sulfhydryls from tortoise (Geochelone carbonaria) can reduce oxidative damage induced by organic hydroperoxide in erythrocyte membrane.

    PubMed

    Torsoni, M A; Ogo, S H

    2000-08-01

    Sulfhydryl groups are important to avoid oxidative damage to the cell. In RBC, tert-butyl hydroperoxide (tert-BOOH) and hydrogen peroxide (H2O2) are capable of oxidizing heme and promoting lipid peroxidation. H2O2 caused greater oxidation of heme than tert-BOOH, although the oxidation of sulfhydryl groups was similar. Geochelone carbonaria Hb, a rich sulfhydryl protein, inhibited the TBA-reactive substances formation of human erythrocytes exposed to tert-BOOH by about 30%; this decrease was smaller with Geochelone denticulata Hb. Sulfhydryl reagents diminished the number of reactive sulfhydryl groups in the G. carbonaria Hb resulting in a decrease of its antioxidant power, suggesting the involvement of sulfhydryls of Hb in the protection against lipid peroxidation. PMID:11026669

  17. Ophthalmic manifestations of induced sickling of erythrocytes in Japanese sika deer (Cervus nippon).

    PubMed

    Vainisi, S J; Parshall, C J; Goldberg, M F; Wolf, E D

    1975-06-01

    Nine female Japanese sika deer (Cervus nippon) were used in a total of 25 experiments in which sickling was chemically induced. During these experiments, color fundic and color fluorescein photographs were taken. Fundic changes included retinal vascular attenuation, blood column pallor, and decreased tapetal reflectivity. These changes were most likely directly associated with a decreased hematocrit and a generalized shocklike condition. Three deer had a congested appearance in retinal blood vessels and tapetum lucidum. Two of the 3 deer developed serous detachment of the retina. These changes seemingly were associated with severe venous statis; all 3 deer died shortly after the experiment was terminated. These experiments yielded data only for the acutely affected deer. None of the ocular changes could be considered the result of chronic sickling because of the reversal of sickling that occurred despite continued intravenous administration of bicarbonate. None of the deer developed ocular changes characteristic of sickle cell retinopathy in human beings. The changes in human beings probably result from continued stress and prolongation of sickling, and especially from a multiplicity of repeated severe episodes of sickling occurring over many years. PMID:1147329

  18. Protection against oxidative damage in human erythrocytes and preliminary photosafety assessment of Punica granatum seed oil nanoemulsions entrapping polyphenol-rich ethyl acetate fraction.

    PubMed

    Baccarin, Thaisa; Mitjans, Montserrat; Lemos-Senna, Elenara; Vinardell, Maria Pilar

    2015-12-25

    The main purpose of the present study is to evaluate the ability of nanoemulsion entrapping pomegranate peel polyphenol-rich ethyl acetate fraction (EAF) prepared from pomegranate seed oil and medium chain triglyceride to protect human erythrocyte membrane from oxidative damage and to assess preliminary in vitro photosafety. In order to evaluate the phototoxic effect of nanoemulsions, human red blood cells (RBCs) are used as a biological model and the rate of haemolysis and photohaemolysis (5 J cm(-2) UVA) is assessed in vitro. The level of protection against oxidative damage caused by the peroxyl radical generator AAPH in human RBCs as well as its effects on bilayer membrane characteristics such as fluidity, protein profile and RBCs morphology are determined. EAF-loaded nanoemulsions do not promote haemolysis or photohaemolysis. Anisotropy measurements show that nanoemulsions significantly retrain the increase in membrane fluidity caused by AAPH. SDS-PAGE analysis reveals that AAPH induced degradation of membrane proteins, but that nanoemulsions reduce the extension of degradation. Scanning electron microscopy examinations corroborate the interaction between AAPH, nanoemulsions and the RBC membrane bilayer. Our work demonstrates that Punica granatum nanoemulsions are photosafe and protect RBCs against oxidative damage and possible disturbance of the lipid bilayer of biomembranes. Moreover it suggests that these nanoemulsions could be promising new topical products to reduce the effects of sunlight on skin. PMID:26407526

  19. Quantitative non-invasive intracellular imaging of Plasmodium falciparum infected human erythrocytes

    NASA Astrophysics Data System (ADS)

    Edward, Kert; Farahi, Faramarz

    2014-05-01

    Malaria is a virulent pathological condition which results in over a million annual deaths. The parasitic agent Plasmodium falciparum has been extensively studied in connection with this epidemic but much remains unknown about its development inside the red blood cell host. Optical and fluorescence imaging are among the two most common procedures for investigating infected erythrocytes but both require the introduction of exogenous contrast agents. In this letter, we present a procedure for the non-invasive in situ imaging of malaria infected red blood cells. The procedure is based on the utilization of simultaneously acquired quantitative phase and independent topography data to extract intracellular information. Our method allows for the identification of the developmental stages of the parasite and facilitates in situ analysis of the morphological changes associated with the progression of this disease. This information may assist in the development of efficacious treatment therapies for this condition.

  20. Identical kinetics of human erythrocyte and muscle acetylcholinesterase with respect to carbamate pre-treatment, residual activity upon soman challenge and spontaneous reactivation after withdrawal of the inhibitors.

    PubMed

    Herkert, Nadja M; Eckert, Saskia; Eyer, Peter; Bumm, Rudolf; Weber, Georg; Thiermann, Horst; Worek, Franz

    2008-04-18

    The efficacy of oxime treatment in soman poisoning is limited due to rapid aging of inhibited acetylcholinesterase (AChE). Pre-treatment with carbamates was shown to improve antidotal treatment substantially. Recently, by using a dynamically working in vitro model with real-time determination of membrane-bound AChE activity, we were able to demonstrate that pre-inhibition of human erythrocyte AChE with pyridostigmine or physostigmine resulted in a markedly higher residual AChE activity after inhibition by soman or paraoxon than in the absence of reversible inhibitors. The purpose of the present study was to compare the effect of carbamate pre-treatment and soman challenge with human erythrocyte and muscle homogenate AChE. Both enzyme sources were immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. AChE activity was continuously analyzed in a flow-through detector. Pre-inhibition of AChE with pyridostigmine or physostigmine resulted in a concentration-dependent increase in carbamylation, residual activity after soman inhibition and fraction of decarbamylation AChE after discontinuation of the inhibitors without differences between human erythrocyte and muscle AChE. This data support the view that human erythrocyte AChE is an adequate surrogate marker for synaptic AChE in OP poisoning. PMID:18304715

  1. EFFECT OF METHYL LINOLEATE HYDROPEROXIDE (MLHP), A POSSIBLE TOXIC INTERMEDIATE OF OZONE, ON HUMAN NORMAL AND GLUCOSE-6-PHOSPHATE DEHYDROGENASE (G-6-PD) DEFICIENT ERYTHROCYTES

    EPA Science Inventory

    Erythrocytes of both normal and G-6-PD deficient humans responded in a dose-dependent manner to the oxidant stress of MLHP as measured by decreases in G-6-PD activity, increases in methemoglobin (METHB) levels and decreases in reduced glutahione (GSH). The G-6-PD deficient erythr...

  2. Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes.

    PubMed Central

    Beddell, C. R.; Goodford, P. J.; Kneen, G.; White, R. D.; Wilkinson, S.; Wootton, R.

    1984-01-01

    Substituted benzaldehydes have been designed to bind preferentially to the oxy conformation of human haemoglobin at a site between the amino terminal residues of the alpha-subunits. Such compounds should stabilize the oxygenated form of haemoglobin and thereby increase its oxygen affinity. The compounds produce the expected effect, left-shifting the oxygen saturation curve of dilute haemoglobin solutions and of whole blood, although the binding pattern to haemoglobin is more complex than envisaged by the design hypothesis. The predicted best compound is also a potent inhibitor, at low oxygen pressure, of the sickling of erythrocytes from patients homozygous for sickle cell disease, and may prove to be a clinically useful anti-sickling agent. PMID:6733364

  3. Changes in the Fatty Acid Profile and Phospholipid Molecular Species Composition of Human Erythrocyte Membranes after Hybrid Palm and Extra Virgin Olive Oil Supplementation.

    PubMed

    Pacetti, D; Gagliardi, R; Balzano, M; Frega, N G; Ojeda, M L; Borrero, M; Ruiz, A; Lucci, P

    2016-07-13

    This work aims to evaluate and compare, for the first time, the effects of extra virgin olive oil (EVOO) and hybrid palm oil (HPO) supplementation on the fatty acid profile and phospholipid (PL) molecular species composition of human erythrocyte membranes. Results supported the effectiveness of both HPO and EVOO supplementation (3 months, 25 mL/day) in decreasing the lipophilic index of erythrocytes with no significant differences between HPO and EVOO groups at month 3. On the other hand, the novel and rapid ultraperformance liquid chromatography-tandem mass spectrometry method used for PL analysis reveals an increase in the levels of phosphatidylcholine and phosphatidylethanolamine species esterified with polyunsaturated fatty acids. This work demonstrates the ability of both EVOO and HPO to increase the degree of unsaturation of erythrocyte membrane lipids with an improvement in membrane fluidity that could be associated with a lower risk of developing cardiovascular diseases. PMID:27315139

  4. Erythrocyte-dependent regulation of human skeletal muscle blood flow: role of varied oxyhemoglobin and exercise on nitrite, S-nitrosohemoglobin, and ATP.

    PubMed

    Dufour, Stéphane P; Patel, Rakesh P; Brandon, Angela; Teng, Xinjun; Pearson, James; Barker, Horace; Ali, Leena; Yuen, Ada H Y; Smolenski, Ryszard T; González-Alonso, José

    2010-12-01

    The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O(2)-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O(2) saturation (O(2)Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O(2)Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O(2)Hb (r(2) = 0.86-0.98; P = 0.01), decreases in venous plasma nitrite (r(2) = 0.93; P < 0.01), increases in venous erythrocyte nitroso species (r(2) = 0.74; P < 0.05), and to a lesser extent increases in erythrocyte SNO (r(2) = 0.59; P = 0.07). No relationship was observed with plasma ATP (r(2) = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature. PMID:20852046

  5. Erythrocyte-dependent regulation of human skeletal muscle blood flow: role of varied oxyhemoglobin and exercise on nitrite, S-nitrosohemoglobin, and ATP

    PubMed Central

    Patel, Rakesh P.; Brandon, Angela; Teng, Xinjun; Pearson, James; Barker, Horace; Ali, Leena; Yuen, Ada H. Y.; Smolenski, Ryszard T.; González-Alonso, José

    2010-01-01

    The erythrocyte is proposed to play a key role in the control of local tissue perfusion via three O2-dependent signaling mechanisms: 1) reduction of circulating nitrite to vasoactive NO, 2) S-nitrosohemoglobin (SNO-Hb)-dependent vasodilatation, and 3) release of the vasodilator and sympatholytic ATP; however, their relative roles in vivo remain unclear. Here we evaluated each mechanism to gain insight into their roles in the regulation of human skeletal muscle blood flow during hypoxia and hyperoxia at rest and during exercise. Arterial and femoral venous hemoglobin O2 saturation (O2Hb), plasma and erythrocyte NO and ATP metabolites, and leg and systemic hemodynamics were measured in 10 healthy males exposed to graded hypoxia, normoxia, and graded hyperoxia both at rest and during submaximal one-legged knee-extensor exercise. At rest, leg blood flow and NO and ATP metabolites in plasma and erythrocytes remained unchanged despite large alterations in O2Hb. During exercise, however, leg and systemic perfusion and vascular conductance increased in direct proportion to decreases in arterial and venous O2Hb (r2 = 0.86–0.98; P = 0.01), decreases in venous plasma nitrite (r2 = 0.93; P < 0.01), increases in venous erythrocyte nitroso species (r2 = 0.74; P < 0.05), and to a lesser extent increases in erythrocyte SNO (r2 = 0.59; P = 0.07). No relationship was observed with plasma ATP (r2 = 0.01; P = 0.99) or its degradation compounds. These in vivo data indicate that, during low-intensity exercise and hypoxic stress, but not hypoxic stress alone, plasma nitrite consumption and formation of erythrocyte nitroso species are associated with limb vasodilatation and increased blood flow in the human skeletal muscle vasculature. PMID:20852046

  6. The unexpected effect of PEGylated gold nanoparticles on the primary function of erythrocytes

    NASA Astrophysics Data System (ADS)

    He, Zeng; Liu, Jiaxin; Du, Libo

    2014-07-01

    Polyethylene glycol-functionalized gold nanoparticles (PEGylated AuNPs) have been widely used as nanocarriers for the delivery of various drugs. However, little attention has been paid to whether the PEGylated AuNPs could affect the primary function of human erythrocytes, which is the main cellular component in the blood. In the current study, we show that both the deformability and oxygen-delivering ability of erythrocytes are decreased when treated with PEGyalted AuNPs of various sizes, which can be attributed to the interaction between PEGylated AuNPs and erythrocyte membranes. It is observed that the PEGylated AuNPs could also induce the aggregation of band-3 and the ATP decrease of erythrocytes. In addition, the PEGylated AuNPs can accelerate the loss of CD47 on erythrocyte membranes, possibly enhancing the senescent process of erythrocytes and the following clearance by SIRPα-expressing leukocytes in bloodstream. The results suggested that PEGylated AuNPs have the potential to affect the primary function of human erythrocytes, which should be considered when using them as drug carriers.Polyethylene glycol-functionalized gold nanoparticles (PEGylated AuNPs) have been widely used as nanocarriers for the delivery of various drugs. However, little attention has been paid to whether the PEGylated AuNPs could affect the primary function of human erythrocytes, which is the main cellular component in the blood. In the current study, we show that both the deformability and oxygen-delivering ability of erythrocytes are decreased when treated with PEGyalted AuNPs of various sizes, which can be attributed to the interaction between PEGylated AuNPs and erythrocyte membranes. It is observed that the PEGylated AuNPs could also induce the aggregation of band-3 and the ATP decrease of erythrocytes. In addition, the PEGylated AuNPs can accelerate the loss of CD47 on erythrocyte membranes, possibly enhancing the senescent process of erythrocytes and the following clearance by

  7. Attenuation of Oxidative Stress of Erythrocytes by Plant-Derived Flavonoids, Orientin and Luteolin

    PubMed Central

    An, Fang; Wang, Shulin; Yuan, Danhua; Gong, Yuewen; Wang, Shuhua

    2016-01-01

    Erythrocytes are easy to be injured by oxidative stress in their lifespan. Although there are several chemicals such as vitamin C (VC) that would be able to reduce oxidative stress, natural herbal products still remain an interesting research area. The current study investigated the effects of two plant-derived flavonoids, orientin and luteolin, on erythrocytes and their possible mechanisms. This experiment was divided into nine groups, which were normal group, model group, VC control group, and treated groups with different doses of orientin and luteolin (10, 20, and 40 μg/mL), respectively. Hemolysis rate was determined by spectrophotometry. Antioxidative enzyme and products were evaluated by different methods. Erythrocyte cell surface and cellular structure were observed with scanning or transmission electron microscope, respectively. Oxidative stress induced significant increase in hemolysis rate of erythrocytes. Orientin or luteolin ameliorated hemolysis of erythrocytes in oxidative stress in a dose-dependent manner. Both orientin and luteolin reduced oxidative products and increased antioxidative enzyme activities. Moreover, orientin and luteolin attenuated oxidative stress induced damage of erythrocyte cell surface morphology and cellular structure. In conclusion, orientin and luteolin could protect human erythrocytes from oxidative damage by attenuating oxidative stress, protecting antioxidative enzyme activities, and preserving integrity of erythrocyte structure. PMID:26966458

  8. Inflammation-associated changes in lipid composition and the organization of the erythrocyte membrane

    PubMed Central

    Dinkla, Sip; van Eijk, Lucas T.; Fuchs, Beate; Schiller, Jürgen; Joosten, Irma; Brock, Roland; Pickkers, Peter; Bosman, Giel J.C.G.M.

    2016-01-01

    Background Reduced erythrocyte survival and deformability may contribute to the so-called anemia of inflammation observed in septic patients. Erythrocyte structure and function are affected by both the membrane lipid composition and the organization. We therefore aimed to determine whether these parameters are affected during systemic inflammation. Methods A sensitive matrix-assisted laser desorption and ionization time-of-flight mass spectrometric method was used to investigate the effect of plasma components of 10 patients with septic shock and of 10 healthy volunteers subjected to experimental endotoxemia on erythrocyte membrane lipid composition. Results Incubation of erythrocytes from healthy control donors with plasma from patients with septic shock resulted in membrane phosphatidylcholine hydrolysis into lysophosphatidylcholine (LPC). Plasma from volunteers undergoing experimental human endotoxemia did not induce LPC formation. The secretory phospholipase A2 IIA concentration was enhanced up to 200-fold in plasma of septic patients and plasma from endotoxin-treated subjects, but did not correlate with the ability of these plasmas to generate LPC. Erythrocyte phosphatidylserine exposure increased up to two-fold during experimental endotoxemia. Conclusions Erythrocyte membrane lipid remodeling as reflected by LPC formation and/or PS exposure occurs during systemic inflammation in a secretory phospholipase A2 IIA-independent manner. General significance Sepsis-associated inflammation induces a lipid remodeling of the erythrocyte membrane that is likely to affect erythrocyte function and survival, and that is not fully mimicked by experimental endotoxemia. PMID:27200268

  9. Comparison of the oxime-induced reactivation of erythrocyte and muscle acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity.

    PubMed

    Eckert, Saskia; Eyer, Peter; Herkert, Nadja; Bumm, Rudolf; Weber, Georg; Thiermann, Horst; Worek, Franz

    2008-02-01

    The purpose of these experiments was to compare oxime-induced reactivation rate constants of acetylcholinesterase from different human tissue sources inhibited by organophosphorus compounds. To this end, preliminary testing was necessary to generate a stable system both for working with erythrocytes and musculature. We established a dynamically working in vitro model with a fixed enzyme source in a bioreactor that was perfused with acetylthiocholine, Ellman's reagent and any agent of interest (e.g. nerve agents, oximes) and analyzed in a common HPLC flow-through detector. The enzyme reactor was composed of a particle filter (Millex-GS, 0.22 microm) containing a thin layer of membrane-bound acetylcholinesterase and was kept at constant temperature in a water bath. At constant flow the height of absorbance was directly proportional to the enzyme activity. To start with, we applied this system to human red cell membranes and then adapted the system to acetylcholinesterase of muscle tissue. Homogenate (Ultra-Turrax and Potter-Elvehjem homogenizer) of human muscle tissue (intercostal musculature) was applied to the same particle filter and perfused in a slightly modified way, as done with human red cell membranes. We detected no decrease of acetylcholinesterase activity within 2.5h and we reproducibly determined reactivation rate constants for reactivation with obidoxime (10 microM) or HI 6 (30 microM) of sarin-inhibited human muscle acetylcholinesterase (0.142+/-0.004 min(-1) and 0.166+/-0.008 min(-1), respectively). The reactivation rate constants of erythrocyte and muscular acetylcholinesterase differed only slightly, highlighting erythrocyte acetylcholinesterase as a proper surrogate marker. PMID:17977518

  10. Zinc inhibits glycation induced structural, functional modifications in albumin and protects erythrocytes from glycated albumin toxicity.

    PubMed

    Tupe, Rashmi; Kulkarni, Amruta; Adeshara, Krishna; Sankhe, Neena; Shaikh, Shamim; Dalal, Sayli; Bhosale, Siddharth; Gaikwad, Sushama

    2015-08-01

    The present work aims to investigate the concentration and time dependant effect of zinc on the in vitro non enzymatic modifications of albumin by diabetic levels of glucose. Further, preventive and curative effect of zinc was studied by adding zinc before and after initiation of glycation respectively. Glycation of albumin was done at different concentrations of zinc (125, 250 and 500 μM) at different time intervals (21, 28 and 35 days) with appropriate controls. The antiglycation potential of zinc was assessed by estimating different markers of albumin glycation (fructosamines, carbonyls, bound sugar, AGEs), structural modifications (free amino, thiol group, β amyloid, native PAGE, ANS binding, fluorescence lifetime decay and CD analysis) and functional properties (antioxidant activity, hemolysis). Zinc at highest concentration (500 μM) significantly reduced modifications of albumin which was comparable to aminoguanidine and also protected secondary and tertiary structure of albumin after 28 days of incubation. Zinc exhibited significant protective effect on erythrocytes by inhibiting hemolysis. Thus the present study indicate preventive mode of albumin glycation inhibition by zinc. PMID:26027608

  11. In vitro study on methemoglobin formation in erythrocytes following hexyl-aminolevulinate induced photodynamic therapy

    NASA Astrophysics Data System (ADS)

    Larsen, Eivind L. P.; Randeberg, Lise L.; Gederaas, Odrun A.; Krokan, Hans E.; Hjelme, Dag R.; Svaasand, Lars O.

    2007-02-01

    Photodynamic therapy (PDT) is a treatment modality which has been shown to be effective for both malignant and non-malignant diseases. New photosensitizers such as hexyl-aminolevulinate (HAL) may increase the efficiency of PDT. HAL penetrates into the cell where the photosensitizer protoporphyrin IX (PPIX) is produced endogenously. In a previous study on HAL based PDT treatment of rat bladder cancer (AY-27 transitional cell carcinoma), a depression of the optical reflectance spectra after treatment was observed in some of the animals. This depression of the spectra was caused by metHemoglobin (metHb). MetHb is an indication of oxidative stress, and can be formed as a result of for instance UV-radiation and heating of blood. The aim of this study was to identify if metHb can be formed in vitro as a result of oxidative stress caused by singlet oxygen and ROS produced during PDT. Methemoglobin formed during PDT might thus be used as an indirect measure of the photochemical processes. This may help predict the PDT treatment outcome. Red blood cells mixed with AY-27 cells exposed to HAL, or PPIX received light treatment, and the changes in the absorption spectra were measured spectrophotometrically. The methemoglobin absorbance spectrum was also studied, and found to be strongly dependant on pH. Hemolysis of erythrocytes by PDT was found, however no metHb was formed in vitro.

  12. Insect cells are superior to Escherichia coli in producing malaria proteins inducing IgG targeting PfEMP1 on infected erythrocytes

    PubMed Central

    2010-01-01

    Background The PFD1235w Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) antigen is associated with severe malaria in children and can be expressed on the surface of infected erythrocytes (IE) adhering to ICAM1. However, the exact three-dimensional structure of this PfEMP1 and its surface-exposed epitopes are unknown. An insect cell and Escherichia coli based system was used to express single and double domains encoded by the pfd1235w var gene. The resulting recombinant proteins have been evaluated for yield and purity and their ability to induce rat antibodies, which react with the native PFD1235w PfEMP1 antigen expressed on 3D7PFD1235w-IE. Their recognition by human anti-malaria antibodies from previously infected Tanzanian donors was also analysed. Methods The recombinant proteins were run on SDS-PAGE and Western blots for quantification and size estimation. Insect cell and E. coli-produced recombinant proteins were coupled to a bead-based Luminex assay to measure the plasma antibody reactivity of 180 samples collected from Tanzanian individuals. The recombinant proteins used for immunization of rats and antisera were also tested by flow cytometry for their ability to surface label 3D7PFD1235w-IE. Results All seven pAcGP67A constructs were successfully expressed as recombinant protein in baculovirus-infected insect cells and subsequently produced to a purity of 60-97% and a yield of 2-15 mg/L. By comparison, only three of seven pET101/D-TOPO constructs expressed in the E. coli system could be produced at all with purity and yield ranging from 3-95% and 6-11 mg/L. All seven insect cell, but only two of the E. coli produced proteins induced antibodies reactive with native PFD1235w expressed on 3D7PFD1235w-IE. The recombinant proteins were recognized in an age- and transmission intensity-dependent manner by antibodies from 180 Tanzanian individuals in a bead-based Luminex assay. Conclusions The baculovirus based insect cell system was distinctly

  13. Production of 1,2-diacylglycerol and phosphatidate in human erythrocytes treated with calcium ions and ionophore A23187.

    PubMed Central

    Allan, D; Watts, R; Michell, R H

    1976-01-01

    1. When the ionophore A23187 and Ca2+ were added to normal human erythrocytes, the incorporation of 32P into phosphatidate was enhanced within 1 min, but there was only slight labelling of other phospholipids. 2. Labelling of phosphatidate in these cells did not continue to increase after about 20min at 37 degrees C; by this time, radioactivity in phosphatidate was about ten times higher inionophore A23187-treated cells than in controls. A net synthesis of phosphatidate was measured in response to the increase in intracellular Ca2+ concentration; the content of this phospholipid in the cell was increased by approximately 50%. 3. In the presence of 2.5 mM-Ca2+ a maximum effect was seen with about 0.5 mug of ionophore/ml. 4. The concentration of Ca2+ giving half-maximal labelling of phosphatidate in the presence of 10 mug of ionophore A23187/ml was about 10 muM. 5. A rapid decrease of ATP content in the cell occurred in ionophore-treated cells. 6. Labelling of phosphatidate appeared to be secondary to the production of 1,2-diacylglycerol in the cells; accumulation of 1,2-diacylglycerol was only seen after about 15 min. After 60 min, the 1,2-diacylglycerol content of the cells was five to seven times that of untreated control cells. 7. The change in the shape of erythrocytes treated with Ca2+ and ionophore appeared to be related to accumulation of 1,2-diacylglycerol. 8. The source of 1,2-diacylglycerol has not been definitely identified, but its fatty acid compositon was similar to that of phosphatidylcholine. However, it has an unusually high content of hexadecenoic acid, a fatty acid not common in the major erythrocyte phospholipids. 9. Accumulation of 1,2-diacyglycerol also occurred in energy-starved cells, even in the absence of calcium; in this case it appeared to be produced by phosphatidate breakdown. PMID:821476

  14. Treatment with garlic restores membrane thiol content and ameliorates lead induced early death of erythrocytes in mice.

    PubMed

    Sarkar, Avik; Sengupta, Dipanwita; Mandal, Samir; Sen, Gargi; Dutta Chowdhury, Kaustav; Chandra Sadhukhan, Gobinda

    2015-04-01

    Sequelae of chronic lead (Pb(2+) ) toxicity includes anemia that is partially due to early death of erythrocytes characterized by excess accumulation of ROS and downregulation of antioxidant system causing oxidative stress and externalization of phosphatidylserine. In this study, pathophysiological based therapeutic application of garlic was evaluated against erythrocyte death. Results suggest that garlic administration prevents oxidative stress, restored the antioxidant balance in erythrocytes of Pb(2+) exposed mice. Moreover, in vitro studies revealed that activity of both scramblase and aminophospholipid translocase could be changed by modifying the critical sulfhydryl groups in presence of dithiothreitol during Pb(2+) exposure. Data also indicated that garlic treatment in Pb(2+) exposed mice exhibited sharp decline in PS exposure and increase in erythrocyte membrane thiol group followed by increase in aminophospholipid translocase activity and decline in scramblase activity. Findings indicated that garlic has the ability to restore the lifespan of erythrocytes during Pb(2+) exposure. PMID:23997012

  15. Site-specific antibodies as probes of the topology and function of the human erythrocyte glucose transporter.

    PubMed Central

    Davies, A; Ciardelli, T L; Lienhard, G E; Boyle, J M; Whetton, A D; Baldwin, S A

    1990-01-01

    Antibodies were raised against synthetic peptides corresponding to most of the regions of the human erythrocyte glucose transporter predicted to be extramembranous in the model of Mueckler, Caruso, Baldwin, Panico, Blench, Morris, Lienhard, Allard & Lodish [(1985) Science 229, 941-945]. Most of the antibodies (17 out of a total of 19) recognized the intact denatured protein on Western blots. However, only seven of the antibodies recognized the native membrane-bound protein, even after its deglycosylation. These antibodies, against peptides encompassing residues 217-272 and 450-492 in the hydrophilic central and C-terminal regions of the transporter, bound to the cytoplasmic surface of the erythrocyte membrane. This finding is in agreement with the prediction of the model that these regions of the sequence are cytoplasmic. Antibodies against peptides from the central cytoplasmic loop of the transporter were found to inhibit the binding of cytochalasin B to the membrane-bound protein, whereas antibodies against the C-terminal region had no effect. The anti-peptide antibodies were then used to map the sequence locations of fragments of the transporter arising from tryptic digestion of the membrane-bound protein. This in turn enabled the epitopes for a number of anti-transporter monoclonal antibodies to be located within either the central cytoplasmic loop or the C-terminal region of the protein. Of those monoclonal antibodies which inhibited cytochalasin B binding to the protein, all but one were found to have epitopes within the central region of the sequence. In conjunction with the results of the anti-peptide antibody studies, these findings indicate the importance of this part of the protein for transporter function. Images Fig. 7. PMID:1691633

  16. Phosphatidylinositol-4,5-biphosphate (PIP2) differentially regulates the interaction of human erythrocyte protein 4.1 (4.1R) with membrane proteins.

    PubMed

    An, Xiuli; Zhang, Xihui; Debnath, Gargi; Baines, Anthony J; Mohandas, Narla

    2006-05-01

    Human erythrocyte protein 4.1 (4.1R) participates in organizing the plasma membrane by linking several surface-exposed transmembrane proteins to the internal cytoskeleton. In the present study, we characterized the interaction of 4.1R with phosphatidylinositol-4,5-bisphosphate (PIP2) and assessed the effect of PIP2 on the interaction of 4.1R with membrane proteins. We found that 4.1R bound to PIP2-containing liposomes through its N-terminal 30 kDa membrane-binding domain and PIP2 binding induced a conformational change in this domain. Phosphatidylinositol-4-phosphate (PIP) was a less effective inducer of this conformational change, and phosphatidylinositol (PI) and inositol-1,4,5-phosphate (IP3) induced no change. Replacement of amino acids K63,64 and K265,266 by alanine abolished the interaction of the membrane-binding domain with PIP2. Importantly, binding of PIP2 to 4.1R selectively modulated the ability of 4.1R to interact with its different binding partners. While PIP2 significantly enhanced the binding of 4.1R to glycophorin C (GPC), it inhibited the binding of 4.1R to band 3 in vitro. PIP2 had no effect on 4.1R binding to p55. Furthermore, GPC was more readily extracted by Triton X-100 from adenosine triphosphate (ATP)-depleted erythrocytes, implying that the GPC-4.1R interaction may be regulated by PIP2 in situ. These findings define an important role for PIP2 in regulating the function of 4.1R. Because 4.1R and its family members (4.1R, 4.1B, 4.1G, and 4.1N) are widely expressed and the PIP2-binding motifs are highly conserved, it is likely that the functions of other 4.1 proteins are similarly regulated by PIP2 in many different cell types. PMID:16669616

  17. Hypobaric hypoxia-reoxygenation diminishes band 3 protein functions in human erythrocytes.

    PubMed

    González, Gustavo; Celedón, Gloria; Sandoval, Mario; González, Gabriela E; Ferrer, Verónica; Astete, Rodrigo; Behn, Claus

    2002-12-01

    We have previously shown that subjects exposed to acute hypobaric hypoxia display an erythrocyte membrane protein band 3 with an increased susceptibility to proteolytic degradation. We suggested it was due to an oxidative damage of band 3. We now report that exposure to hypobaric hypoxia followed by reoxygenation affects protein band 3 functions such as anion transport and binding of glyceraldehyde-3P-dehydrogenase. Transport capacity was assessed with the fluorescent probe 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] ethanesulfonate (NBD-taurine). Binding capacity was evaluated from the activity of the membrane-associated enzyme. Healthy young men were exposed for 20 min to hypobaric hypoxia, simulating an altitude of 4,500 m above sea level and after recompression band 3 function was assessed. An inhibition of band 3 anion transport function and a decrease in the binding of glyceraldehyde-3P-dehydrogenase to band 3 were observed. Evidence is given supporting the hypothesis that functional alteration of band 3 is due to its oxidative modification originated as a consequence of the exposure to hypobaric hypoxia and further reoxygenation. PMID:12466935

  18. Preferential targeting of human erythrocytes infected with the malaria parasite Plasmodium falciparumvia hexose transporter surface proteins.

    PubMed

    Heikham, Kajal Devi; Gupta, Ankit; Kumar, Ambrish; Singh, Chandan; Saxena, Juhi; Srivastava, Kumkum; Puri, Sunil K; Dwivedi, Anil K; Habib, Saman; Misra, Amit

    2015-04-10

    Glucose uptake by Plasmodium-infected erythrocytes (RBC) is higher compared to uninfected RBC. Glucose is transported across the cell membrane by transporter proteins. Particles of median size 146.3±18.7 nm, containing anti-malarial agents in corn starch were prepared for investigating: (a) whether the glucose moiety in starch targets RBC via hexose transporter(s), (b) whether there are differences in the extent of targeting to uninfected RBC versus infected RBC (iRBC) in view of higher cell surface density of these proteins on iRBC and (c) whether targeting provides enhanced efficacy against P. falciparum in comparison to drugs in solution. Binding of these particles to RBC was target-specific, since it could be blocked by phloretin, an inhibitor of glucose transporters (GLUT), or competed out in a dose-dependent manner with d-glucose in a flow cytometry assay. Significant (P=0.048, t-test) differences in extent of targeting to iRBC versus RBC were observed in flow cytometry. CDRI 97/63 incorporated in particles was 63% more efficacious than its solution at 250 ng/ml, while quinine was 20% more efficacious at 6.25 ng/ml in a SYBR Green incorporation assay. Preferential targeting of iRBC using an inexpensive excipient promises advantages in terms of dose reduction and toxicity alleviation. PMID:25666024

  19. Growth of plasmodium falciparum in human erythrocytes containing abnormal membrane proteins

    SciTech Connect

    Schulman, S. City Univ. of New York, NY ); Roth, E.F. Jr.; Cheng, B.; Rybicki, A.C.; Sussman, I.I.; Wong, M.; Nagel, R.L.; Schwartz, R.S. ); Wang, W. ); Ranney, H.M. )

    1990-09-01

    To evaluate the role of erythrocyte (RBC) membrane proteins in the invasion and maturation of Plasmodium falciparum, the authors have studied, in culture, abnormal RBCs containing quantitative or qualitative membrane protein defects. These defects included hereditary spherocytosis (HS) due to decreases in the content of spectrin (HS(Sp{sup +})), hereditary elliptocytosis (HE) due to protein 4.1 deficiency (HE(4.1{sup 0})), HE due to a spectrin {alpha}I domain structural variant that results in increased content of spectrin dimers (HE(Sp{alpha}{sup I/65})), and band 3 structural variants. Parasite invasion, measured by the initial uptake of ({sup 3}H)hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp{sup +}) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. The extent of decreased parasite growth in HS(Sp{sup +}) RBCs closely correlated with the extent of RBC spectrin deficiency. Homogeneous subpopulations of dense HS RBCs exhibited decreased parasite growth to the same extent as did HS whole blood. RBCs from four HE subjects showed marked parasite growth and development.

  20. Prophylactic effects of pomegranate (Punica granatum) juice on sodium fluoride induced oxidative damage in liver and erythrocytes of rats.

    PubMed

    Bouasla, Asma; Bouasla, Ihcène; Boumendjel, Amel; Abdennour, Cherif; El Feki, Abdelfattah; Messarah, Mahfoud

    2016-07-01

    The objective of this study was to investigate the protective effects of pomegranate (Punica granatum) juice (PGJ) on oxidative damages in liver tissue and erythrocytes of rats intoxicated by sodium fluoride (NaF). Rats were randomly divided into two groups: group I received standard diet and group II received orally 1 mL of PGJ. After 5 weeks of pretreatment, each group was divided again into two subgroups and treated for another 3 weeks as follows: group I was subdivided into a control group and a group that was treated with 100 ppm of NaF (in drinking water); group II was subdivided into one group that was treated daily with both 100 ppm NaF and PGJ (1 mL orally) and one that received daily 1 mL of pomegranate juice. Exposure to NaF decreased hematological parameters, changed the total protein, albumin, bilirubin levels, and increased the activities of hepatic marker enzymes. We also noted an increase in lipid peroxidation contents, accompanied by a decrease of reduced glutathione levels. Antioxidant enzyme activities in both tissues were modified in the NaF group compared with the control group. However, the administration of PGJ juice caused an amelioration of the previous parameters. Our results indicated the potential effects of NaF to induce oxidative damage in tissues and the ability of PGJ to attenuate NaF-induced oxidative injury. PMID:27124270

  1. Plasmodium-infected erythrocytes (pRBC) induce endothelial cell apoptosis via a heme-mediated signaling pathway

    PubMed Central

    Liu, Mingli; Dickinson-Copeland, Carmen; Hassana, Salifu; Stiles, Jonathan K

    2016-01-01

    Heme is cytotoxic to the plasmodium parasite, which converts it to an insoluble crystalline form called hemozoin (malaria pigment) in erythrocytes during replication. The increased serum levels of free heme cause tissue damage, activation of microvascular endothelial and glial cells, focal inflammation, activation of apoptotic pathways, and neuronal tissue damage. Several hypotheses have been proposed to explain how these causative factors exacerbate fatal malaria. However, none of them fully explain the detailed mechanisms leading to the high morbidity and mortality associated with malaria. We have previously reported that heme-induced brain microvascular endothelial cell (HBVEC) apoptosis is a major contributor to severe malaria pathogenesis. Here, we hypothesized that heme (at clinically relevant levels) induces inflammation and apoptosis in HBVEC, a process that is mediated by independent proinflammatory and proapoptotic signaling pathways. In this study, we determined the key signaling molecules associated with heme-mediated apoptosis in HBVEC in vitro using RT2 profiler polymerase chain reaction array technology and confirmed results using immunostaining techniques. While several expressed genes in HBVEC were altered upon heme stimulation, we determined that the apoptotic effects of heme were mediated through p73 (tumor protein p73). The results provide an opportunity to target heme-mediated apoptosis therapeutically in malaria-infected individuals. PMID:27042002

  2. Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes

    SciTech Connect

    Lutz, H.U.; Bussolino, F.; Flepp, R.; Fasler, S.; Stammler, P.; Kazatchkine, M.D.; Arese, P.

    1987-11-01

    Treatment of erythrocytes with the thiol-specific oxidant azodicarboxylic acid bis(dimethylamide) (diamide) enhances their phagocytosis by adherent monocytes. Phagocytosis of diamide-treated erythrocytes required that the cells were opsonized with whole serum, since complement inactivation abolished phagocytosis. Opsonization with whole serum containing 20-100 times the physiological concentration of naturally occurring anti-band-3- antibodies enhanced phagocytosis of diamide-treated erythrocytes. High inputs of anti-band-3 also restored phagocytosis of erythrocytes that had been incubated with complement-inactivated serum. Elevated concentrations of anti-spectrin antibodies were ineffective in whole and complement-inactivated serum. Specific recognition of diamide-treated erythrocytes by anti-band-3 antibodies may be due to generation of anti-band-3 reactive protein oligomers on intact diamide-treated erythrocytes. Generation of such oligomers was dose-dependent with respect to diamide. Bound anti-band-3 alone was not sufficient to mediate phagocytosis. It resulted in deposition of complement component C3b on the cells through activation of the alternative complement pathway in amounts exceeding that of bound antibodies by two orders of magnitude. Thus, anti-band-3 and complement together mediate phagocytosis of oxidatively stressed erythrocytes, which simulate senescent erythrocytes with respect to bound antibody and complement.

  3. Volume regulatory potassium transport in rabbit and human sickle erythrocytes in vitro

    SciTech Connect

    Al-Rohil, N.S.

    1988-01-01

    One approach to the therapy of sickle cell anemia is to decrease the hemoglobin concentration by inducing a slight swelling of the cell to retard the rate of hemoglobin polymerization. We found that a prolonged incubation of rabbit or human SS red cell in hypotonic medium caused an inactivation of the inactivation of swelling-stimulated potassium transport. The inactivation may have important practical consequences for the therapy of sickle cell anemia. Large cytoskeleton-free vesicles were prepared in order to study the possible role of the spectrin-actin membrane skeleton in the swelling-stimulated and N-ethylmaleimide (NEM)-stimulated transport. NEM pretreatment stimulated {sup 86}Rb efflux in vesicles by a factor of 2.4 + 0.55 (mean {plus minus} S.D.). The NEM effect on {sup 86}Rb efflux was specific in that the {sup 22}Na efflux into a Na medium was not stimulated but actually inhibited. The {sup 86}Rb efflux from the vesicles was not stimulated by hypotonic media. This finding is consistent with a role of the membrane skeleton in the detection and/or transduction of the signal by which cell swelling activates the transport.

  4. Potent radioprotective effects of combined regimens of famotidine and vitamin C against radiation-induced micronuclei in mouse bone marrow erythrocytes.

    PubMed

    Zangeneh, M; Mozdarani, H; Mahmoudzadeh, A

    2015-05-01

    To investigate the radioprotective effect of the combination of famotidine and vitamin C against radiation-induced micronucleus formation in mouse bone marrow erythrocytes, various doses of famotidine or vitamin C or combinations thereof were administered intraperitoneally to adult male NMRI mice 2 h before 2 and 4 Gy γ-irradiation. The frequency of micronucleated polychromatic erythrocytes (MnPCEs) was scored in 5,000 polychromatic erythrocytes (PCEs), and the cell proliferation ratio [PCE/(PCE + NCE); NCE = normochromatic erythrocytes] was also calculated for each treatment group. Data were statistically evaluated using one-way ANOVA test. The results show that pretreatment with various doses of famotidine and vitamin C before γ-irradiation significantly reduced the frequency of MnPCEs with a protection factor (PF) of 2 and 1.7, respectively. Pretreatment with vitamin C also significantly increased the cell proliferation ratio, while famotidine had no effect. Combination of famotidine and vitamin C was more effective in reducing MnPCEs than each compound alone, leading to a PF of 4.3 after irradiation. Cell proliferation ratio was also significantly improved by the combination compared with the irradiated control groups. Both famotidine and vitamin C are potent scavengers of free radicals and reactive oxygen species, especially OH(·). The combination of the two compounds probably further enhances this activity, thus leading to high bone marrow protection. PMID:25634516

  5. Functional analysis of erythrocyte determinants of Plasmodium infection

    PubMed Central

    Bei, Amy K.; Duraisingh, Manoj T.

    2012-01-01

    The Plasmodium falciparum parasite is an obligate intracellular pathogen whose invasion and remodeling of the human erythrocyte results in the clinical manifestations of malarial disease. The functional analysis of erythrocyte determinants of invasion and growth is a relatively unexplored frontier in malaria research, encompassing studies of natural variation of the erythrocyte, as well as genomic, biochemical and chemical biological and transgenic approaches. These studies have allowed the functional analysis of the erythrocyte in vitro, resulting in the discovery of critical erythrocyte determinants of Plasmodium infection. Here, we will focus on the varied approaches used for the study of the erythrocyte in Plasmodium infection, with a particular emphasis on erythrocyte invasion. PMID:22726752

  6. Stem bark and flower extracts of Vismia cauliflora: modulation of oxidative burst in human neutrophils' and inhibition of oxidative damage in human erythrocytes.

    PubMed

    Ribeiro, Alessandra Braga; Berto, Alessandra; Ribeiro, Daniela; Freitas, Marisa; Chisté, Renan Campos; Visentainer, Jesuí Vergílio; Fernandes, Eduarda

    2014-10-01

    Vismia cauliflora is an Amazonian plant traditionally used to treat dermatosis and inflammatory processes of the skin by indigenous population. Our research group showed that stem bark and flower extracts of V. cauliflora are efficient in vitro scavengers of reactive oxygen and nitrogen species. In this study, we determined the activity of stem bark and flower extracts of V. cauliflora plant on the modulation of in vitro oxidative burst in human neutrophils and their potential to inhibit the oxidative damage in human erythrocytes. The oxidative burst in activated neutrophils were monitored by specific probes to detect the oxidizing effect of superoxide anion radical (MCLA), hydrogen peroxide (amplex red) and hypochlorous acid (APF), and both extracts were efficient to neutralize the oxidative burst (IC50 from 3 to 15µg/mL). These same extracts were also effective against oxidative damage in erythrocytes by inhibiting hemoglobin oxidation (IC50=18µg/mL) and lipid peroxidation (IC50=2.7 and 7.5µg/mL, flower and stem bark, respectively). In addition, stem bark extract (100µg/mL) inhibited the depletion of glutathione by 13%. These extracts have similar phenolic composition, but flower presents quercetin (14%) in its composition. Therefore, these results reinforce the potential therapeutic of stem bark and flower extracts of V. cauliflora to heal topical skin disease and requires further research targeted effectively to develop phytopharmaceutical drug based on this plant. PMID:26461382

  7. In vitro evaluation of cell death induced by cadmium, lead and their binary mixtures on erythrocytes of Common buzzard (Buteo buteo).

    PubMed

    Hernández-García, A; Romero, D; Gómez-Ramírez, P; María-Mojica, P; Martínez-López, E; García-Fernández, A J

    2014-03-01

    Cadmium and lead are persistent and ubiquitous metals that can cause several deleterious effects in living beings. Apoptosis and necrosis are two types of cell death that can be found after in vivo and in vitro exposure to these metals. In this study, isolated red blood cells from living captive Common buzzard (Buteo buteo) were exposed in vitro to different concentrations of lead, cadmium, and the mixture lead-cadmium in a proportion of 1:10 (similar to that found in previous field studies). Data obtained from dose-response curves were used to evaluate the interactive effects of metal mixtures on cell viability. In general, except for the exposure to NOEC, additivity was the most frequently observed response. As described in human, after in vitro exposure, lead was highly accumulated in buzzard erythrocytes, while cadmium accumulation was scarce. Finally, the type of cell death (apoptosis or necrosis) induced by the exposure to different concentrations of these heavy metals and their mixtures was evaluated in the red blood cells. Apoptosis was found to be the main type of cell death observed after cadmium and/or lead exposure. However, this exposure caused an increase in lysis or necrosis, especially if red blood cells were exposed to high doses. PMID:24287112

  8. Environmentally Relevant Concentrations of Atrazine and Ametrine Induce Micronuclei Formation and Nuclear Abnormalities in Erythrocytes of Fish.

    PubMed

    Botelho, R G; Monteiro, S H; Christofoletti, C A; Moura-Andrade, G C R; Tornisielo, V L

    2015-11-01

    A rapid and sensitive method using liquid chromatography coupled with mass spectrometry triple quadrupole direct aqueous injection for analysis of atrazine and ametrine herbicides in surface waters was developed. According to the validation method, water samples from six different locations in the Piracicaba River were collected monthly from February 2011 to January 2012 and injected into a liquid chromatographer/dual mass spectrometer without the need for sample extraction. The method was validated and shown to be precise and accurate; limits of detection and quantification were 0.07 and 0.10 µg L(-1) for atrazine and 0.09 and 0.14 µg L(-1) for ametrine. During the sampling period, concentrations of atrazine ranged from 0.11 to 1.92 µg L(-1) and ametrine from 0.25 to 1.44 µg L(-1). After analysis of the herbicides, Danio rerio were exposed a range of concentrations found in the river water to check the induction of micronuclei and nuclear abnormalities (NAs) in erythrocytes. Concentrations of atrazine and ametrine >1.0 and 1.5 µg L(-1), respectively, induced MN formation in D. rerio. Ametrine was shown to be more genotoxic to D. rerio because a greater incidence of NAs was observed compared with atrazine. Therefore, environmentally relevant concentrations of atrazine and ametrine found in the Piracicaba River are dangerous to the aquatic biota. PMID:26081367

  9. A Sequence in Subdomain 2 of DBL1α of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies

    PubMed Central

    Blomqvist, Karin; Albrecht, Letusa; Quintana, Maria del Pilar; Angeletti, Davide; Joannin, Nicolas; Chêne, Arnaud; Moll, Kirsten; Wahlgren, Mats

    2013-01-01

    Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1α previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1α-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1α antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface. PMID:23335956

  10. A sequence in subdomain 2 of DBL1α of Plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies.

    PubMed

    Blomqvist, Karin; Albrecht, Letusa; Quintana, Maria del Pilar; Angeletti, Davide; Joannin, Nicolas; Chêne, Arnaud; Moll, Kirsten; Wahlgren, Mats

    2013-01-01

    Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1α previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1α-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1α antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface. PMID:23335956

  11. An in vitro spectrometric method for determining the partition coefficients of non-steroidal anti-inflammatory drugs into human erythrocyte ghost membranes

    NASA Astrophysics Data System (ADS)

    Omran, Ahmed A.

    2013-03-01

    Usefulness of second derivative spectrophotometry for determining the partition coefficients (Kps) of four non-steroidal anti-inflammatory drugs (NSAIDs) between human erythrocyte ghost (HEG) membranes and buffer at simulated physiological conditions (pH = 7.4, 37 °C) has been adequately emphasized. In the absorption spectra for each of the investigated NSAIDs, λmax was red-shifted in presence of HEG membranes, indicating that NSAIDs have the nature of metachromasy between lipid bilayer and water. Further quantitative spectral data for calculating Kps could not be obtained from the absorption spectra because of the presence of background signal impacts of HEG lipid bilayers. Second derivative spectra were calculated from absorption spectra and fortunately showed three isosbestic derivative points for each NSAID, indicating without doubt that the background signals were entirely eliminated. From the relation between the derivative intensity change (ΔD) induced by addition of HEG membranes, Kps were calculated and obtained with RSD of below 6%. Fractions of partitioned NSAIDs are in well-harmony with that derived from the experimental values. Moreover, validity of the proposed method was confirmed. Conclusively, the second derivative spectrometry has proven to be a facile, reliable and more expeditious method to obtain in vitro Kps of drugs to HEG without previous separation.

  12. Diagnosis of myocardial infarction based on lectin-induced erythrocyte agglutination: a feasibility study

    NASA Astrophysics Data System (ADS)

    Bocsi, József; Nieschke, Kathleen; Mittag, Anja; Reichert, Thomas; Laffers, Wiebke; Marecka, Monika; Pierzchalski, Arkadiusz; Piltz, Joachim; Esche, Hans-Jürgen; Wolf, Günther; Dähnert, Ingo; Baumgartner, Adolf; Tarnok, Attila

    2014-03-01

    Myocardial infarction (MI) is an acute life-threatening disease with a high incidence worldwide. Aim of this study was to test lectin-carbohydrate binding-induced red blood cell (RBC) agglutination as an innovative tool for fast, precise and cost effective diagnosis of MI. Five lectins (Ricinus communis agglutinin (RCA), Phaseolus vulgaris erythroagglutinin (PHA), Datura stramonium agglutinin (DSA), Artocarpus agglutinin (ArA), Triticum agglutinin (TA)) were tested for ability to differentiate between agglutination characteristics in patients with MI (n = 101) or angina pectoris without MI (AP) (n = 34) and healthy volunteers (HV) as control (n =68) . RBC agglutination was analyzed by light absorbance of a stirred RBC suspension in the green to red light spectrum in an agglutimeter (amtec, Leipzig, Germany) for 15 min after lectin addition. Mean cell count in aggregates was estimated from light absorbance by a mathematical model. Each lectin induced RBC agglutination. RCA led to the strongest RBC agglutination (~500 RBCs/aggregate), while the others induced substantially slower agglutination and lead to smaller aggregate sizes (5-150 RBCs/aggregate). For all analyzed lectins the lectin-induced RBC agglutination of MI or AP patients was generally higher than for HV. However, only PHA induced agglutination that clearly distinguished MI from HV. Variance analysis showed that aggregate size after 15 min. agglutination induced by PHA was significantly higher in the MI group (143 RBCs/ aggregate) than in the HV (29 RBC-s/aggregate, p = 0.000). We hypothesize that pathological changes during MI induce modification of the carbohydrate composition on the RBC membrane and thus modify RBC agglutination. Occurrence of carbohydrate-lectin binding sites on RBC membranes provides evidence about MI. Due to significant difference in the rate of agglutination between MI > HV the differentiation between these groups is possible based on PHA-induced RBC-agglutination. This novel assay

  13. Longxuetongluo Capsule Improves Erythrocyte Function against Lipid Peroxidation and Abnormal Hemorheological Parameters in High Fat Diet-Induced ApoE−/− Mice

    PubMed Central

    Zheng, Jiao; Liu, Binglin; Lun, Qixing; Yao, Weijuan; Zhao, Yunfang; Xiao, Wei; Huang, Wenzhe; Wang, Yonghua; Li, Jun; Tu, Pengfei

    2016-01-01

    Chinese dragon's blood, the red resin of Dracaena cochinchinensis, one of the renowned traditional medicines, has been used to facilitate blood circulation and disperse blood stasis for thousands of years. Phenolic compounds are considered to be responsible for its main biological activities. In this study, total phenolic compounds of Chinese dragon's blood were made into capsule (Longxuetongluo Capsule, LTC) and their effects on the abnormal hemorheological properties were examined by high fat diet (HFD) induced ApoE−/− mice. Compared to the model group, LTC recovered the abnormal hemorheological parameters in HFD-induced ApoE−/− mice by reducing whole blood viscosity (WBV) at high rate and improving erythrocyte function. In conclusion, LTC could ameliorate erythrocyte deformability and osmotic fragility through the reduction of lipid peroxidation on plasma and erythrocyte membranes in HFD-induced ApoE−/− mice, which supported the traditional uses of Chinese dragon's blood as an effective agent for improving blood microcirculation in hypercholesterolemia. PMID:26649134

  14. Stable-isotope dilution GC-MS approach for nitrite quantification in human whole blood, erythrocytes, and plasma using pentafluorobenzyl bromide derivatization: nitrite distribution in human blood.

    PubMed

    Schwarz, Alexandra; Modun, Darko; Heusser, Karsten; Tank, Jens; Gutzki, Frank-Mathias; Mitschke, Anja; Jordan, Jens; Tsikas, Dimitrios

    2011-05-15

    Previously, we reported on the usefulness of pentafluorobenzyl bromide (PFB-Br) for the simultaneous derivatization and quantitative determination of nitrite and nitrate in various biological fluids by GC-MS using their (15)N-labelled analogues as internal standards. As nitrite may be distributed unevenly in plasma and blood cells, its quantification in whole blood rather than in plasma or serum may be the most appropriate approach to determine nitrite concentration in the circulation. So far, GC-MS methods based on PFB-Br derivatization failed to measure nitrite in whole blood and erythrocytes because of rapid nitrite loss by oxidation and other unknown reactions during derivatization. The present article reports optimized and validated procedures for sample preparation and nitrite derivatization which allow for reliable quantification of nitrite in human whole blood and erythrocytes. Essential measures for stabilizing nitrite in these samples include sample cooling (0-4°C), hemoglobin (Hb) removal by precipitation with acetone and short derivatization of the Hb-free supernatant (5 min, 50°C). Potassium ferricyanide (K(3)Fe(CN)(6)) is useful in preventing Hb-caused nitrite loss, however, this chemical is not absolutely required in the present method. Our results show that accurate GC-MS quantification of nitrite as PFB derivative is feasible virtually in every biological matrix with similar accuracy and precision. In EDTA-anticoagulated venous blood of 10 healthy young volunteers, endogenous nitrite concentration was measured to be 486±280 nM in whole blood, 672±496 nM in plasma (C(P)), and 620±350 nM in erythrocytes (C(E)). The C(E)-to-C(P) ratio was 0.993±0.188 indicating almost even distribution of endogenous nitrite between plasma and erythrocytes. By contrast, the major fraction of nitrite added to whole blood remained in plasma. The present GC-MS method is useful to investigate distribution and metabolism of endogenous and exogenous nitrite in blood

  15. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasite Plasmodium knowlesi.

    PubMed

    Moon, Robert W; Sharaf, Hazem; Hastings, Claire H; Ho, Yung Shwen; Nair, Mridul B; Rchiad, Zineb; Knuepfer, Ellen; Ramaprasad, Abhinay; Mohring, Franziska; Amir, Amirah; Yusuf, Noor A; Hall, Joanna; Almond, Neil; Lau, Yee Ling; Pain, Arnab; Blackman, Michael J; Holder, Anthony A

    2016-06-28

    The dominant cause of malaria in Malaysia is now Plasmodium knowlesi, a zoonotic parasite of cynomolgus macaque monkeys found throughout South East Asia. Comparative genomic analysis of parasites adapted to in vitro growth in either cynomolgus or human RBCs identified a genomic deletion that includes the gene encoding normocyte-binding protein Xa (NBPXa) in parasites growing in cynomolgus RBCs but not in human RBCs. Experimental deletion of the NBPXa gene in parasites adapted to growth in human RBCs (which retain the ability to grow in cynomolgus RBCs) restricted them to cynomolgus RBCs, demonstrating that this gene is selectively required for parasite multiplication and growth in human RBCs. NBPXa-null parasites could bind to human RBCs, but invasion of these cells was severely impaired. Therefore, NBPXa is identified as a key mediator of P. knowlesi human infection and may be a target for vaccine development against this emerging pathogen. PMID:27303038

  16. Characterization of Precursor PfHsp60 in Plasmodium falciparum Cytosol during Its Asexual Development in Human Erythrocytes

    PubMed Central

    Padma Priya, P.; Grover, Manish; Tatu, Utpal S.; Natarajan, Vasant

    2015-01-01

    Mitochondrial heat shock protein 60 (Hsp60) is a nuclear encoded gene product that gets post-translationally translocated into the mitochondria. Using multiple approaches such as immunofluorescence experiments, isoelectric point analysis with two-dimensional gel electrophoresis, and mass spectrometric identification of the signal peptide, we show that Hsp60 from Plasmodium falciparum (PfHsp60) accumulates in the parasite cytoplasm during the ring, trophozoite, and schizont stages of parasite development before being imported into the parasite mitochondria. Using co-immunoprecipitation experiments with antibodies specific to cytoplasmic PfHsp90, PfHsp70-1, and PfHsp60, we show association of precursor PfHsp60 with cytoplasmic chaperone machinery. Metabolic labeling involving pulse and chase indicates translocation of the precursor pool into the parasite mitochondrion during chase. Analysis of results obtained with Geldanamycin treatment confirmed precursor PfHsp60 to be one of the clients for PfHsp90. Cytosolic chaperones bind precursor PfHsp60 prior to its import into the mitochondrion of the parasite. Our data suggests an inefficient co-ordination in the synthesis and translocation of mitochondrial PfHsp60 during asexual growth of malaria parasite in human erythrocytes. PMID:26317863

  17. Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro

    SciTech Connect

    Herkert, N.M.; Schulz, S.; Wille, T.; Thiermann, H.; Hatz, R.A.; Worek, F.

    2011-05-15

    Standard treatment of organophosphorus (OP) poisoning includes administration of an antimuscarinic (e.g., atropine) and of an oxime-based reactivator. However, successful oxime treatment in soman poisoning is limited due to rapid aging of phosphylated acetylcholinesterase (AChE). Hence, the inability of standard treatment procedures to counteract the effects of soman poisoning resulted in the search for alternative strategies. Recently, results of an in vivo guinea pig study indicated a therapeutic effect of physostigmine given after soman. The present study was performed to investigate a possible pre- and post-treatment effect of physostigmine on soman-inhibited human AChE given at different time intervals before or after perfusion with soman by using a well-established dynamically working in vitro model for real-time analysis of erythrocyte and muscle AChE. The major findings were that prophylactic physostigmine prevented complete inhibition of AChE by soman and resulted in partial spontaneous recovery of the enzyme by decarbamylation. Physostigmine given as post-treatment resulted in a time-dependent reduction of the protection from soman inhibition and recovery of AChE. Hence, these date indicate that physostigmine given after soman does not protect AChE from irreversible inhibition by the OP and that the observed therapeutic effect of physostigmine in nerve agent poisoning in vivo is probably due to other factors.

  18. Helical image reconstruction of the outward-open human erythrocyte band 3 membrane domain in tubular crystals.

    PubMed

    Yamaguchi, Tomohiro; Fujii, Takashi; Abe, Yoshito; Hirai, Teruhisa; Kang, Dongchon; Namba, Keiichi; Hamasaki, Naotaka; Mitsuoka, Kaoru

    2010-03-01

    The C-terminal membrane domain of erythrocyte band 3 functions as an anion exchanger. Here, we report the three-dimensional (3D) structure of the membrane domain in an inhibitor-stabilized, outward-open conformation at 18A resolution. Unstained, frozen-hydrated tubular crystals containing the membrane domain of band 3 purified from human red blood cells (hB3MD) were examined using cryo-electron microscopy and iterative helical real-space reconstruction (IHRSR). The 3D image reconstruction of the tubular crystals showed the molecular packing of hB3MD dimers with dimensions of 60 x 110 A in the membrane plane and a thickness of 70A across the membrane. Immunoelectron microscopy and carboxyl-terminal digestion demonstrated that the intracellular surface of hB3MD was exposed on the outer surface of the tubular crystal. A 3D density map revealed that hB3MD consists of at least two subdomains and that the outward-open form is characterized by a large hollow area on the extracellular surface and continuous density on the intracellular surface. PMID:20005958

  19. Architecture of Human IgM in Complex with P. falciparum Erythrocyte Membrane Protein 1.

    PubMed

    Akhouri, Reetesh Raj; Goel, Suchi; Furusho, Hirotoshi; Skoglund, Ulf; Wahlgren, Mats

    2016-02-01

    Plasmodium falciparum virulence is associated with sequestration of infected erythrocytes. Microvascular binding mediated by PfEMP1 in complex with non-immune immunoglobulin M (IgM) is common among parasites that cause both severe childhood malaria and pregnancy-associated malaria. Here, we present cryo-molecular electron tomography structures of human IgM, PfEMP1 and their complex. Three-dimensional reconstructions of IgM reveal that it has a dome-like core, randomly oriented Fab2s units, and the overall shape of a turtle. PfEMP1 is a C- shaped molecule with a flexible N terminus followed by an arc-shaped backbone and a bulky C terminus that interacts with IgM. Our data demonstrate that the PfEMP1 binding pockets on IgM overlap with those of C1q, and the bulkiness of PfEMP1 limits the capacity of IgM to interact with PfEMP1. We suggest that P. falciparum exploits IgM to cluster PfEMP1 into an organized matrix to augment its affinity to host cell receptors. PMID:26776517

  20. Changes in the activities of some membrane-associated enzymes during in vivo ageing of the normal human erythrocyte.

    PubMed

    Kadlubowski, M; Agutter, P S

    1977-09-01

    Human erythrocytes from healthy male donors were fractionated with respect to in vivo age by simple centrifugation in order to characterize changes in the functional integrity of the membrane during the life-span of the cell. The three enzymes, Na/K-ATPase, glyceraldehyde-3-phosphate dehydrogenase and NADH-ferricyanide reductase, were found not to change with age, but significant age-dependent decreases were observed in the cases of acetylcholinesterase, phosphoglycerate kinase, purine nucleoside phosphorylase, adenylate kinase, Mg-ATPase and alkaline phosphatase. The possibility that these changes were attributable to mechanisms other than age-related inactivation, such as reticulocyte contamination, differential resealing and crypticity, was investigated. Only the decrease in acetylcholinesterase could be explained wholly in terms of reticulocyte contamination. A decrease in membrane integrity on ageing was observed, which accounted for approximately half the change in alkaline phosphatase and may have contributed to the other enzyme activity changes. This membrane integrity effect masked a real decrease in the highly cryptic NADH-ferricyanide reductase, this decrease being apparent only after total disaggregation of the membrane with nonionic surfactant. PMID:145240

  1. Correlation of the internal microviscosity of human erythrocytes to the cell volume and the viscosity of hemoglobin solutions.

    PubMed

    Herrmann, A; Müller, P

    1986-01-23

    The microviscosity of the cytoplasm of human erythrocytes as well as of membrane-free hemoglobin solutions was investigated measuring the rotation of the small spin-label molecule, Tempone. The dependence of the intracellular microviscosity on the extracellular pH and osmotic pressure which was varied by NaCl or sucrose was sufficiently explained on the basis of alterations of the red blood cell volume. The intracellular microviscosity depended exclusively on the hemoglobin concentration. It did not differ from that of comparable membrane-free hemoglobin solutions. It was not necessary to take into account long-range interactions between hemoglobin molecules. The conclusion therefore was that the intracellular viscosity is not modified by cytoplasmic structures or the cell membrane. Above a hemoglobin concentration of 6 mM the viscosity of hemoglobin solutions increased much faster than the microviscosity. From measurements obtained with different spin-labels it followed that also the charge of these molecules is of importance. PMID:3002490

  2. Site on the human erythrocyte glucose transporter phosphorylated by protein kinase C resides on the protein's hydrophilic domain

    SciTech Connect

    Deziel, M.R.; McReynolds, J.H.; Lippes, H.A.; Jung, C.Y.

    1986-05-01

    A recently published model of the human erythrocyte hexose transporter deduced from the protein's primary structure proposes that the transporter is organized into two membrane domains comprising 77% of the protein's mass and three hydrophilic domains, a short segment that includes the polypeptide's N-terminus and two larger segments, one lying between the membrane domains and the other at the protein's C-terminus. Limited tryptic digestion of the transporter produces two membrane-bound fragments corresponding to the proposed membrane domains and releases a number of soluble peptides. Fast Atom Bombardment Mass Spectroscopic analysis of the released peptides and comparison of the peptide's masses with the transporter's amino acid sequence revealed that tryptic peptides corresponding to at least 63% of the hydrophilic domains' mass were recovered. The site of phosphorylation by protein kinase C, tagged using (/sup 32/P)-ATP, was also released from the transporter under these conditions, (in contrast to sites located within the protein's membrane domains), indicating that this site is located within one of the hydrophilic domains. Tryptic digestion at elevated ionic strength or cleavage with S. Aureus V8 protease results in the recovery of the /sup 32/P label on the carbohydrate-bearing membrane domain that is located near the protein's N-terminus, thus eliminating the C-terminal hydrophilic segment as a possible site of phosphorylation.

  3. Monitoring the uptake of glycosphingolipids in Plasmodium falciparum-infected erythrocytes using both fluorescence microscopy and capillary electrophoresis with laser-induced fluorescence detection

    PubMed Central

    Essaka, David C.; White, John; Rathod, Pradip; Whitmore, Colin D.; Hindsgaul, Ole; Palcic, Monica M.

    2010-01-01

    The metabolism of glycosphingolipids by the malaria-causing parasite Plasmodium falciparum plays an important role in the progression of the disease. We report a new and highly sensitive method to monitor the uptake of glycosphingolipids in infected red blood cells (iRBCs). A tetramethylrhodamine-labeled glycosphingolipid (GM1-TMR) was used as a substrate. Uptake was demonstrated by fluorescence microscopy. The iRBCs were lysed with a 15% solution of saponin and washed with phosphate buffered saline to release intact parasites. The parasites were further lysed and the resulting homogenates were analyzed by capillary electrophoresis with laser-induced fluorescence detection. The lysate from erythrocytes infected at 1% parasitemia generated a signal twenty standard deviations larger than uninfected erythrocytes, which suggests that relatively low infection levels can be studied with this technique. PMID:21043509

  4. Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

    PubMed Central

    Bissinger, Rosi; Lang, Elisabeth; Ghashghaeinia, Mehrdad; Singh, Yogesh; Zelenak, Christine; Fehrenbacher, Birgit; Honisch, Sabina; Chen, Hong; Fakhri, Hajar; Umbach, Anja T.; Liu, Guilai; Rexhepaj, Rexhep; Liu, Guoxing; Schaller, Martin; Mack, Andreas F.; Lupescu, Adrian; Birnbaumer, Lutz; Lang, Florian; Qadri, Syed M.

    2016-01-01

    Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death. PMID:27499046

  5. Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2.

    PubMed

    Bissinger, Rosi; Lang, Elisabeth; Ghashghaeinia, Mehrdad; Singh, Yogesh; Zelenak, Christine; Fehrenbacher, Birgit; Honisch, Sabina; Chen, Hong; Fakhri, Hajar; Umbach, Anja T; Liu, Guilai; Rexhepaj, Rexhep; Liu, Guoxing; Schaller, Martin; Mack, Andreas F; Lupescu, Adrian; Birnbaumer, Lutz; Lang, Florian; Qadri, Syed M

    2016-01-01

    Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca(2+) activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2(-/-)) and corresponding wild-type mice (Gαi2(+/+)). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2(-/-) and Gαi2(+/+) mice but the mean corpuscular volume was significantly larger in Gαi2(-/-) mice. Spontaneous PS exposure of circulating Gαi2(-/-) erythrocytes was significantly lower than that of circulating Gαi2(+/+) erythrocytes. PS exposure was significantly lower in Gαi2(-/-) than in Gαi2(+/+) erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca(2+) activity and cell shrinkage. Moreover, Gαi2(-/-) erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2(+/+) erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death. PMID:27499046

  6. Comparison of the oxime-induced reactivation of rhesus monkey, swine and guinea pig erythrocyte acetylcholinesterase following inhibition by sarin or paraoxon, using a perfusion model for the real-time determination of membrane-bound acetylcholinesterase activity.

    PubMed

    Herkert, Nadja M; Lallement, Guy; Clarençon, Didier; Thiermann, Horst; Worek, Franz

    2009-04-28

    Recently, a dynamically working in vitro model with real-time determination of membrane-bound human acetylcholinesterase (AChE) activity was shown to be a versatile model to investigate oxime-induced reactivation kinetics of organophosphate- (OP) inhibited enzyme. In this assay, AChE was immobilized on particle filters which were perfused with acetylthiocholine, Ellman's reagent and phosphate buffer. Subsequently, AChE activity was continuously analyzed in a flow-through detector. Now, it was an intriguing question whether this model could be used with erythrocyte AChE from other species in order to investigate kinetic interactions in the absence of annoying side reactions. Rhesus monkey, swine and guinea pig erythrocytes were a stable and highly reproducible enzyme source. Then, the model was applied to the reactivation of sarin- and paraoxon-inhibited AChE by obidoxime or HI 6 and it could be shown that the derived reactivation rate constants were in good agreement to previous results obtained from experiments with a static model. Hence, this dynamic model offers the possibility to investigate highly reproducible interactions between AChE, OP and oximes with human and animal AChE. PMID:19428926

  7. Differences in Rat and Human Erythrocytes Following Blood Component Manufacturing: The Effect of Additive Solutions

    PubMed Central

    da SilveiraCavalcante, Luciana; Acker, Jason P.; Holovati, Jelena L.

    2015-01-01

    Background Small animal models have been previously used in transfusion medicine studies to evaluate the safety of blood transfusion products. Although there are multiple studies on the effects of blood banking practices on human red blood cells (RBCs), little is known about the effect of blood component manufacturing on the quality of rat RBCs. Methods Blood from Sprague-Dawley rats and human volunteers (n = 6) was collected in CPD anticoagulant, resuspended in SAGM or AS3, and leukoreduced. In vitro quality was analyzed, including deformability, aggregation, microvesiculation, phosphatidylserine (PS) expression, percent hemolysis, ATP, 2,3-DPG, osmotic fragility, and potassium concentrations. Results Compared to human RBCs, rat RBCs had decreased deformability, membrane rigidity, aggregability, and microvesiculation after component manufacturing process. Rat RBCs in SAGM showed higher hemolysis compared to human RBCs in SAGM (rat 4.70 ± 0.83% vs. human 0.34 ± 0.07%; p = 0.002). Rat RBCs in AS3 had greater deformability and rigidity than in SAGM. The number of microparticles/µl and the percentage PS expression were lower in rat RBCs in AS3 than in rat RBCs in SAGM. Hemolysis was also significantly lower in AS3 compared to SAGM (2.21 ± 0.68% vs. 0.87 ± 0.39%; p = 0.028). Conclusion Rat RBCs significantly differ from human RBCs in metabolic and membrane-related aspects. SAGM, which is commonly used for human RBC banking, causes high hemolysis and is not compatible with rat RBCs. PMID:26195928

  8. Hypoxia-mediated impaired erythrocyte Lands’ Cycle is pathogenic for sickle cell disease

    PubMed Central

    Wu, Hongyu; Bogdanov, Mikhail; Zhang, Yujin; Sun, Kaiqi; Zhao, Shushan; Song, Anren; Luo, Renna; Parchim, Nicholas F.; Liu, Hong; Huang, Aji; Adebiyi, Morayo G.; Jin, Jianping; Alexander, Danny C.; Milburn, Michael V.; Idowu, Modupe; Juneja, Harinder S.; Kellems, Rodney E.; Dowhan, William; Xia, Yang

    2016-01-01

    Although Lands’ cycle was discovered in 1958, its function and cellular regulation in membrane homeostasis under physiological and pathological conditions remain largely unknown. Nonbiased high throughput metabolomic profiling revealed that Lands’ cycle was impaired leading to significantly elevated erythrocyte membrane lysophosphatidylcholine (LysoPC) content and circulating and erythrocyte arachidonic acid (AA) in mice with sickle cell disease (SCD), a prevalent hemolytic genetic disorder. Correcting imbalanced Lands’ cycle by knockdown of phospholipase 2 (cPLA2) or overexpression of lysophosphatidycholine acyltransferase 1 (LPCAT1), two key enzymes of Lands’ cycle in hematopoietic stem cells, reduced elevated erythrocyte membrane LysoPC content and circulating AA levels and attenuated sickling, inflammation and tissue damage in SCD chimeras. Human translational studies validated SCD mouse findings and further demonstrated that imbalanced Lands’ cycle induced LysoPC production directly promotes sickling in cultured mouse and human SCD erythrocytes. Mechanistically, we revealed that hypoxia-mediated ERK activation underlies imbalanced Lands’ cycle by preferentially inducing the activity of PLA2 but not LPCAT in human and mouse SCD erythrocytes. Overall, our studies have identified a pathological role of imbalanced Lands’ cycle in SCD erythrocytes, novel molecular basis regulating Lands’ cycle and therapeutic opportunities for the disease. PMID:27436223

  9. Hypoxia-mediated impaired erythrocyte Lands' Cycle is pathogenic for sickle cell disease.

    PubMed

    Wu, Hongyu; Bogdanov, Mikhail; Zhang, Yujin; Sun, Kaiqi; Zhao, Shushan; Song, Anren; Luo, Renna; Parchim, Nicholas F; Liu, Hong; Huang, Aji; Adebiyi, Morayo G; Jin, Jianping; Alexander, Danny C; Milburn, Michael V; Idowu, Modupe; Juneja, Harinder S; Kellems, Rodney E; Dowhan, William; Xia, Yang

    2016-01-01

    Although Lands' cycle was discovered in 1958, its function and cellular regulation in membrane homeostasis under physiological and pathological conditions remain largely unknown. Nonbiased high throughput metabolomic profiling revealed that Lands' cycle was impaired leading to significantly elevated erythrocyte membrane lysophosphatidylcholine (LysoPC) content and circulating and erythrocyte arachidonic acid (AA) in mice with sickle cell disease (SCD), a prevalent hemolytic genetic disorder. Correcting imbalanced Lands' cycle by knockdown of phospholipase 2 (cPLA2) or overexpression of lysophosphatidycholine acyltransferase 1 (LPCAT1), two key enzymes of Lands' cycle in hematopoietic stem cells, reduced elevated erythrocyte membrane LysoPC content and circulating AA levels and attenuated sickling, inflammation and tissue damage in SCD chimeras. Human translational studies validated SCD mouse findings and further demonstrated that imbalanced Lands' cycle induced LysoPC production directly promotes sickling in cultured mouse and human SCD erythrocytes. Mechanistically, we revealed that hypoxia-mediated ERK activation underlies imbalanced Lands' cycle by preferentially inducing the activity of PLA2 but not LPCAT in human and mouse SCD erythrocytes. Overall, our studies have identified a pathological role of imbalanced Lands' cycle in SCD erythrocytes, novel molecular basis regulating Lands' cycle and therapeutic opportunities for the disease. PMID:27436223

  10. Erythrocyte G Protein as a Novel Target for Malarial Chemotherapy

    PubMed Central

    Murphy, Sean C; Harrison, Travis; Hamm, Heidi E; Lomasney, Jon W; Mohandas, Narla; Haldar, Kasturi

    2006-01-01

    Background Malaria remains a serious health problem because resistance develops to all currently used drugs when their parasite targets mutate. Novel antimalarial drug targets are urgently needed to reduce global morbidity and mortality. Our prior results suggested that inhibiting erythrocyte Gs signaling blocked invasion by the human malaria parasite Plasmodium falciparum. Methods and Findings We investigated the erythrocyte guanine nucleotide regulatory protein Gs as a novel antimalarial target. Erythrocyte “ghosts” loaded with a Gs peptide designed to block Gs interaction with its receptors, were blocked in β-adrenergic agonist-induced signaling. This finding directly demonstrates that erythrocyte Gs is functional and that propranolol, an antagonist of G protein–coupled β-adrenergic receptors, dampens Gs activity in erythrocytes. We subsequently used the ghost system to directly link inhibition of host Gs to parasite entry. In addition, we discovered that ghosts loaded with the peptide were inhibited in intracellular parasite maturation. Propranolol also inhibited blood-stage parasite growth, as did other β2-antagonists. β-blocker growth inhibition appeared to be due to delay in the terminal schizont stage. When used in combination with existing antimalarials in cell culture, propranolol reduced the 50% and 90% inhibitory concentrations for existing drugs against P. falciparum by 5- to 10-fold and was also effective in reducing drug dose in animal models of infection. Conclusions Together these data establish that, in addition to invasion, erythrocyte G protein signaling is needed for intracellular parasite proliferation and thus may present a novel antimalarial target. The results provide proof of the concept that erythrocyte Gs antagonism offers a novel strategy to fight infection and that it has potential to be used to develop combination therapies with existing antimalarials. PMID:17194200

  11. Effect of anionic amphophiles on erythrocyte properties.

    PubMed

    McMillan, D E; Utterback, N G; Wujek, J J

    1983-01-01

    This preliminary study describes effects of two pharmacologic agents on erythrocyte behavior. Increased erythrocyte aggregation has been proposed as important in the pathogenesis of a number of disorders, but the exact mechanism by which it plays a role in disease production remains unclear. Several anionic amphophiles have been reported to benefit diabetic vascular disease and atherosclerosis. If anionic amphophiles enter the erythrocyte plasma membrane they can increase its negative charge, reducing the energy of attraction between red blood cells and diminishing erythrocyte aggregation. Erythrocytes were studied after suspension in phosphate-buffered saline containing dextran as an aggregation-promoting agent. A marginal reduction of the suspension's viscosity was found at low shear rate when 2,5- dihydroxybenzene sulfonate was added. Additionally, erythrocyte sedimentation rate was marginally influenced. Both dihydroxybenzene sulfonate and acetylsalicylate protected human erythrocytes from hemolysis at concentrations from 10(-3) to 10(-5) M. The removal of erythrocyte sialic acid using neuraminidase to reduce surface negative charge led to unequivocal interference with aggregation (MAI technique of CHIEN et al., J. Gen. Physiol., 1973) by both anionic amphophiles were studied. Dihydroxybenzene sulfonate and actylsalicylate reduced the aggregation propensity of sialic-free erythrocytes, suggesting that the effect on the low shear rate viscosity of sialic acid-containing erythrocytes, though modest, is real. PMID:6587820

  12. Biophysical characterization of genistein-membrane interaction and its correlation with biological effect on cells - The case of EYPC liposomes and human erythrocyte membranes.

    PubMed

    Pawlikowska-Pawlęga, Bożena; Misiak, Lucjan E; Jarosz-Wilkołazka, Anna; Zarzyka, Barbara; Paduch, Roman; Gawron, Antoni; Gruszecki, Wieslaw I

    2014-08-01

    With application of EPR and (1)H NMR techniques genistein interaction with liposomes formed with egg yolk lecithin and with erythrocyte membranes was assessed. The present study addressed the problem of genistein localization and its effects on lipid membrane fluidity and protein conformation. The range of microscopic techniques was employed to study genistein effects on HeLa cells and human erythrocytes. Moreover, DPPH bioassay, superoxide anion radical test and enzymatic measurements were performed in HeLa cells subjected to genistein. The gathered results from both EPR and NMR techniques indicated strong ordering effect of genistein on the motional freedom of lipids in the head group region and the adjacent hydrophobic zone in liposomal as well as in red blood cell membranes. EPR study of human ghost showed also the changes in the erythrocyte membrane protein conformation. The membrane effects of genistein were correlated with the changes in internal membranes arrangement of HeLa cells as it was noticed using transmission electron microscopic and fluorescent techniques. Scanning electron and light microscopy methods showed that one of the aftermaths of genistein incorporation into membranes was creation of echinocytic form of the red blood cells with reduced diameter. Genistein improved redox status of HeLa cells treated with H2O2 by lowering radicals' level. In conclusion, the capacity of genistein to incorporate, to affect membrane organization and to change its biophysical properties is correlated with the changes inside the cells. PMID:24813834

  13. Uptake of hexakis(t-butylisonitrile) technetium (I) and hexakis(isopropylisonitrile) technetium (I) by neonatal rat myocytes and human erythrocytes

    SciTech Connect

    Sands, H.; Delano, M.L.; Gallagher, B.M.

    1986-03-01

    The uptake mechanism of two potential cardiac imaging agents (/sup 99m/Tc)hexakis(t-butylisonitrile) technetium (I) (TBI) and (/sup 99m/Tc)hexakis(isopropylisonitrile) technetium (I) (IPI) has been studied using neonatal rat myocytes and human erythrocytes. Uptake of these complexes was rapid, of greater magnitude than seen previously for 42K, and was unaffected by either 0.15 mM ouabain or 10 mM KCI. Both (/sup 99m/Tc)isonitrile complexes had a high affinity for the membranes of the myocytes and erythrocytes. The data suggest that the uptake is not dependent on the membrane Na/sup +//K/sup +/ ATPase but may be related to the lipophilicity of these agents as evidenced by the rapidity, tenacity, and quantity of the binding observed.

  14. The gene for human erythrocyte membrane protein band 7. 2 (EPB72) maps to 9q33-q34 centromeric to the Philadelphia chromosome translocation breakpoint region

    SciTech Connect

    Gallagher, P.G.; Upender, M.; Ward, D.C.; Forget, B.G. )

    1993-10-01

    Erthrocyte band 7.2b is a 31-kDa integral phosphoprotein absent from the erythrocytes of many patients with hereditary stomatocytosis (HSt). HSt is a heterogeneous group of disorders characterized by mouth-shaped erythrocyte morphology on peripheral blood smears. The clinical severity of HSt is variable; some patients experience hemolysis and anemia while others are asymptomatic. The red cell membranes of these patients usually exhibit abnormal permeability to sodium and potassium with resultant modification of intracellular water content. The band 7.2b protein has been purified and the cDNA cloned. The approved gene name and symbol are erythrocyte membrane protein band 7.2 and EPB72, respectively, as assigned by the Human Gene Nomenclature Committee. Using a human reticulocyte cDNA library as template, a 491-bp fragment corresponding to the 3' end of the coding region of the EPB72 cDNA was amplified. Three overlapping phase DNA clones were isolated using this probe. Four genomic DNA fragments of 2.0, 2.5, 4.5, and 5.0 kb, respectively, were isolated from these clones. To localize the EPB72 gene by fluorescence in situ hybridization, these genomic DNA fragments were labeled with biotin-11-dUTP and hybridized to metaphase chromosomes as described. Probes were preannealed to C[sub 0]t1-fractionated DNA to block repetitive sequences. Experiments were analyzed and digitally imaged using a cooled CCD camera. The probes, in combination, gave specific hybridization signals only in chromosome 9q. The gene for erythrocyte membrane protein 7.2 localized to 9q33-q34.

  15. Respiratory alkalosis does not alter NOx concentrations in human plasma and erythrocytes.

    PubMed

    Ishibashi, T; Kubota, K; Himeno, M; Matsubara, T; Hori, T; Ozaki, K; Yamozoe, M; Aizawa, Y; Yoshida, J; Nishio, M

    2001-12-01

    To test the hypothesis that NOx (NO and NO, metabolites of NO) accumulates in red blood cells (RBC) in response to changes in PCO(2) and bicarbonate (HCO) concentration in blood, we examined the effect of changes in PCO(2) and HCO induced by hyperventilation in healthy adults on partitioning of NOx in whole blood. NOx in hemolysate was measured by a high-performance liquid chromatography-Griess system equipped with a C(18) reverse phase column to trap hemoglobin, which enables determination of whole blood NOx concentration and calculation of NOx concentration in RBC with high accuracy and reproducibility. NOx concentration in RBC was lower than that in plasma, and equilibrium between plasma and RBC was achieved rapidly after addition of NO. Changes in PCO(2) and HCO by hyperventilation failed to influence NOx concentrations in both plasma and RBC. Plasma NOx concentrations correlated with whole blood NOx and RBC NOx concentrations. Our results indicate that changes in PCO(2) or HCO induced by hyperventilation do not influence NOx compartmentalization in plasma and RBC. PMID:11709445

  16. Erythrocyte /sup 3/H-ouabain binding and digitalis treatment in ethanol addicted patients

    SciTech Connect

    Battaini, F.; Govoni, S.; Mauri, A.; Civelli, L.; Trabucchi, M.

    1987-06-29

    The binding of /sup 3/H-ouabain to human erythrocytes was analyzed in a population of hospitalized male ethanol addicted patients under long term digitalis treatment. In the non-alcoholic patient group the long term digitalis treatment induced an increase in Bmax and Kd values; such modification was not observed in the alcoholic patients. Chronic alcohol intake itself induced an increase in /sup 3/H-ouabain kinetic parameters. These observations confirm that ouabain binding to human erythrocytes is subject to pharmacological and toxicological regulation and that adaptive changes in peripheral tissues can be useful in predicting possible parallel modifications in other less accessible tissues. 22 references, 1 table.

  17. Contribution of ankyrin-band 3 complexes to the organization and mechanical properties of the membrane skeleton of human erythrocyte

    SciTech Connect

    Shen, B.W.

    1995-02-01

    To understand the role of ankyrin-band 3 complexes in the organization of the spectrin-based membrane skeleton and its contribution to the mechanical properties of human erythrocytes, intact skeletons and single-layered skeleton leaflets were prepared from intact and physically sheared membrane ghosts, expanded in low salt buffer, and examined by transmission electron microscopy. While the structures of intact skeletons and single-layered skeleton leaflets shared many common features, including rigid junctional complexes of spectrin, actin, and band 4.1; short stretches ({approximately}50 {angstrom}) of flexible spectrin filaments; and globular masses of ankyrin-band 3 complexes situated close to the middle of the spectrin filaments, the definition of structural units in the intact skeleton is obscured by the superposition of the two layers. However, the spatial disposition of structural elements can be clearly defined in the images of the single-layered skeleton leaflets. Partially expanded skeletal leaflets contain conglomerates of ankyrin-band 3 complexes arranged in a circular or clove-leaf configuration that straddles multiple strands of thick spectrin cables, presumably reflecting the association of ankyrin-band 3 complexes on neighboring spectrin tetramers as well as the lateral association of the spectrin filaments. Hyperexpansion of the skeleton leaflets led to dissociation of the conglomerates of ankyrin-band 3 complexes, full-extension of the spectrin tetramers, and separation of the individual strands of spectrin tetramers. Clearly defined stands of spectrin tetramers in the hyperexpanded single-layered skeletal leaflets often contained two sets of globular protein masses that divided the spectrin tetramers into three segments of approximately equal length.

  18. Inositol phosphates influence the membrane bound Ca/sup 2 +//Mg/sup 2 +/ stimulated ATPase from human erythrocyte membranes

    SciTech Connect

    Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

    1986-03-01

    The modulation by exogenous inositol phosphates of the membrane Ca/sup 2 +//Mg/sup 2 +/ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl/sub 2/, 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na/sub 2/ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl/sub 2/ and EGTA. The ATPase assay was linear with time at 44/sup 0/C. The inositol phosphates were commercially obtained and were also prepared from /sup 32/P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP/sub 3/) elevated the Ca/sup 2 +//Mg/sup 2 +/ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na/sup +//K/sup +/-ATPase and a Mg/sup 2 +/ ATPase were not effected by IP/sub 3/. Ca/sup 2 +//Mg/sup 2 +/APTase activity with IP/sub 2/ or IP/sub 3/ could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP/sub 3/ was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP/sub 3/ stimulated Ca/sup 2 +//Mg/sup 2 +/ ATPase.

  19. Effect of Scoparia dulcis extract on insulin receptors in streptozotocin induced diabetic rats: studies on insulin binding to erythrocytes.

    PubMed

    Pari, Leelavinothan; Latha, Muniappan; Rao, Chippada Appa

    2004-01-01

    We investigated the insulin-receptor-binding effect of Scoparia dulcis plant extract in streptozotocin (STZ)-induced male Wistar rats, using circulating erythrocytes (ER) as a model system. An aqueous extract of S dulcis plant (SPEt) (200 mg/kg body weight) was administered orally. We measured blood levels of glucose and plasma insulin and the binding of insulin to cell-membrane ER receptors. Glibenclamide was used as standard reference drug. The mean specific binding of insulin to ER was significantly lower in diabetic control rats (DC) (55.0 +/- 2.8%) than in SPEt-treated (70.0 +/- 3.5%)- and glibenclamide-treated (65.0 +/- 3.3%) diabetic rats, resulting in a significant decrease in plasma insulin. Scatchard plot analysis demonstrated that the decrease in insulin binding was accounted for by a lower number of insulin receptor sites per cell in DC rats when compared with SPEt- and glibenclamide-treated rats. High-affinity (Kd1), low-affinity (Kd2), and kinetic analysis revealed an increase in the average receptor affinity in ER from SPEt and glibenclamide treated diabetic rats having 2.5 +/- 0.15 x 10(10) M(-1) (Kd1); 17.0 +/- 1.0 x 10(-8) M(-1) (Kd2), and 2.0 +/- 0.1 x 10(-10) M(-1) (Kd1); 12.3 +/- 0.9 x 10(-8) M(-1) (Kd2) compared with 1.0 +/- 0.08 x 10(-10) M(-1) (Kd1); 2.7 +/- 0.25 x 10(-8) M(-1) (Kd2) in DC rats. The results suggest an acute alteration in the number of insulin receptors on ER membranes in STZ-induced diabetic rats. Treatment with SPEt and glibenclamide significantly improved specific insulin binding, with receptor number and affinity binding (p < 0.001) reaching almost normal non-diabetic levels. The data presented here show that SPEt and glibenclamide increase total ER membrane insulin binding sites with a concomitant significant increase in plasma insulin. PMID:15803960

  20. The detection of clinically significant erythrocyte alloantibodies using a human mononuclear phagocyte assay

    SciTech Connect

    Schanfield, M.S.; Stevens, J.O.; Bauman, D.

    1981-01-01

    Red blood cell (RBC) allo- or autoantibodies, which markedly reduce the survival of transfused or autologous RBC, are considered to be clinically significant antibodies. The occurrence of antibodies against high-incidence antigens, which are occasionally associated with clinically significant RBC destruction or are of unknown clinical significance, often creates delays in providing blood to patients. In the majority of cases these antibodies are benign; however, clinically significant examples of these antibodies have been reported. An in vitro homologous human mononuclear phagocyte assay (MPA) was used to study antibodies directed against specificities associated with variable clinical significance. Two antibodies reported to be clinically significant and 25 antibodies known to be clinically insignificant were tested by MPA. The results indicate that clinically significant antibodies have a significantly higher score than do clinically insignificant antibodies, with no overlap observed between the two groups. An additional eight antibodies with unknown clinical significance were tested. None of these antibodies had scores in the clinically significant range.

  1. 31P MAS-NMR of human erythrocytes: independence of cell volume from angular velocity.

    PubMed

    Kuchel, P W; Bubb, W A; Ramadan, S; Chapman, B E; Philp, D J; Coen, M; Gready, J E; Harvey, P J; McLean, A J; Hook, J

    2004-09-01

    31P magic angle spinning NMR (MAS-NMR) spectra were obtained from suspensions of human red blood cells (RBCs) that contained the cell-volume-sensitive probe molecule, dimethyl methylphosphonate (DMMP). A mathematical representation of the spectral-peak shape, including the separation and width-at-half-height in the 31P NMR spectra, as a function of rotor speed, enabled us to explore the extent to which a change in cell volume would be reflected in the spectra if it occurred. We concluded that a fractional volume change in excess of 3% would have been detected by our experiments. Thus, the experiments indicated that the mean cell volume did not change by this amount even at the highest spinning rate of 7 kHz. The mean cell volume and intracellular 31P line-width were independent of the packing density of the cells and of the initial cell volume. The relationship of these conclusions to other non-NMR studies of pressure effects on cells is noted. PMID:15334588

  2. Cellular effects of the pulsed tunable dye laser at 577 nanometers on human endothelial cells, fibroblasts, and erythrocytes: an in vitro study

    SciTech Connect

    Glassberg, E.; Lask, G.P.; Tan, E.M.; Uitto, J.

    1988-01-01

    The 577-nm flashlamp-pumped tunable dye laser pulsed at 450 microseconds is rapidly becoming the treatment of choice for removal of portwine stains and other vascular ectasias. In this study, we examined the mechanisms of vessel destruction by determining the effects of laser irradiation on three types of primary target cells--erythrocytes, endothelial cells, and fibroblasts. Human endothelial cells and fibroblasts in microwell plates were irradiated at various energy densities with the laser, after which several aspects of cellular biology were determined, including 1) viability of cells by trypan blue exclusion test; 2) cell proliferation by (3H)thymidine incorporation; and 3) rate of protein synthesis using (3H)leucine incorporation as a marker. In endothelial cell cultures, both (3H)thymidine and (3H)leucine incorporations were inhibited at energy levels of 5-12 J/cm2 (P less than 0.01). In fibroblast cultures, cell proliferation was similarly inhibited, while supratherapeutic energy density (greater than or equal to 12 J/cm2) was required for inhibition of protein synthesis. The laser energy in the range of 5-8.5 J/cm2 had no effect on cell viability. Erythrocytes as target cells for laser energy demonstrated rapid, dose-dependent lysis, as determined by release of free hemoglobin into culture medium. Addition of erythrocytes into a coculture with endothelial cells abolished the direct inhibitory effect noted in cultures when endothelial cells were present alone. The results of the latter experiment imply that erythrocytes are the primary target cell absorbing the laser energy at 577 nm. However, direct laser effects on endothelial cells may also contribute to the mechanisms of ablation of the vascular ectasias by the tunable dye laser at 577 nm.

  3. Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with sup 125 I-derivatives of cymarin and digitoxigenin

    SciTech Connect

    Lowndes, J.M.

    1988-01-01

    NaK-ATPase is an enzyme which maintains Na{sup +} and K{sup +} gradients across the plasma membrane of eukaryotic cells, and is specifically inhibited by cardiac glycosides. The cardiac glycoside binding site is located primarily on the catalytic {alpha} subunit but the glycoprotein {beta} and proteolipid-{gamma} subunits may also contribute to the structure of the site. In order to label the cardiac glycoside binding site of human erythrocytes, four photoaffinity ligands with very high specific radioactivity were synthesized. The compounds, which are abbreviated ({sup 125}I)AISC, ({sup 125}I)AIPP-GluD, ({sup 125}I)AIPP-GalD and ({sup 125}I)IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the {alpha}, {beta}, and proteolipid-{gamma} subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, ({sup 125}I)I-TASC, was synthesized; this compound had the same structure as ({sup 125}I)AISC except that a light-sensitive azide group was replaced with a hydroxyl group. Competitive binding assays with cymarin against 0.2 nM ({sup 125}I)I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct ({sup 125}I)I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K{sub D} of 54 pM and a B{sub max} of 23 fmol/mg protein.

  4. Band 3/complement-mediated recognition and removal of normally senescent and pathological human erythrocytes.

    PubMed

    Arese, Paolo; Turrini, Franco; Schwarzer, Evelin

    2005-01-01

    Band 3 modifications that normally occur during physiological red blood cell (RBC) senescence in humans, and occasionally in pathological conditions are described in the context of their role in enhancing RBC recognition and phagocytic removal. Band 3 modifications are mostly due to oxidative insults that gradually accumulate during the RBC lifespan or impact massively in a shorter time period in pathological conditions. The oxidative insults that impact on the RBC, the protective mechanisms that counteract those damages and the phenotypic modifications that accumulate during the RBC lifespan are described. It is shown how specific oxidative as well as non-oxidative band 3 modifications enhance RBC membrane affinity for normally circulating anti-band 3 antibodies, and how membrane-bound anti-band 3 antibodies bring about a limited complement activation and membrane deposition of complement C3 fragments. The partially covalent complexes between anti-band 3 antibodies and complement C3 fragments are very powerful opsonins readily recognized by the CR1 complement receptor on the phagocyte. Band 3 modifications typically encountered in old RBCs have crystallized to a number of band 3-centered models of RBC senescence. One of those band 3-centered models, the so-called 'band 3/complement RBC removal model' first put up by Lutz et al. is discussed in more detail. Finally, it is shown how the genetic deficiency of glucose-6-phosphate dehydrogenase (G6PD) plus fava bean consumption, and a widespread RBC parasitic disease, P. falciparum malaria, may lead to massive and rapid destruction of RBCs by a mechanism comparable to a dramatic, time-compressed enhancement of normal RBC senescence. PMID:16301814

  5. Cytotoxicity of graphene oxide and graphene in human erythrocytes and skin fibroblasts.

    PubMed

    Liao, Ken-Hsuan; Lin, Yu-Shen; Macosko, Christopher W; Haynes, Christy L

    2011-07-01

    Two-dimensional carbon-based nanomaterials, including graphene oxide and graphene, are potential candidates for biomedical applications such as sensors, cell labeling, bacterial inhibition, and drug delivery. Herein, we explore the biocompatibility of graphene-related materials with controlled physical and chemical properties. The size and extent of exfoliation of graphene oxide sheets was varied by sonication intensity and time. Graphene sheets were obtained from graphene oxide by a simple (hydrazine-free) hydrothermal route. The particle size, morphology, exfoliation extent, oxygen content, and surface charge of graphene oxide and graphene were characterized by wide-angle powder X-ray diffraction, atomic force microscopy, X-ray photoelectron spectroscopy, dynamic light scattering, and zeta-potential. One method of toxicity assessment was based on measurement of the efflux of hemoglobin from suspended red blood cells. At the smallest size, graphene oxide showed the greatest hemolytic activity, whereas aggregated graphene sheets exhibited the lowest hemolytic activity. Coating graphene oxide with chitosan nearly eliminated hemolytic activity. Together, these results demonstrate that particle size, particulate state, and oxygen content/surface charge of graphene have a strong impact on biological/toxicological responses to red blood cells. In addition, the cytotoxicity of graphene oxide and graphene sheets was investigated by measuring mitochondrial activity in adherent human skin fibroblasts using two assays. The methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, a typical nanotoxicity assay, fails to predict the toxicity of graphene oxide and graphene toxicity because of the spontaneous reduction of MTT by graphene and graphene oxide, resulting in a false positive signal. However, appropriate alternate assessments, using the water-soluble tetrazolium salt (WST-8), trypan blue exclusion, and reactive oxygen species assay reveal that the compacted graphene

  6. Characteristics of prolinase against various iminodipeptides in erythrocyte lysates from a normal human and a patient with prolidase deficiency.

    PubMed

    Wang, Weifang; Liu, Gang; Yamashita, Koichi; Manabe, Masanobu; Kodama, Hiroyuki

    2004-01-01

    The effect of various amino acids and MnCl2 on prolinase activity in erythrocyte lysates from a healthy individual and a patient with prolidase deficiency was investigated. A concentration of 0.1 mM MnCl2 increased prolinase activity in normal erythrocytes against pro-gly, pro-glu, pro-leu, pro-ser and pro-phe, but inhibited that against pro-ala, pro-val, pro-met and pro-asp. However, prolinase activity against these iminodipeptides was enhanced by pre-incubation with glycine, independent of MnCl2. The same studies on erythrocytes from a prolidase-deficient patient showed almost the same results as the normal control, except that prolinase activity against pro-gly and pro-ser was slightly inhibited by adding 0.1 mM MnCl2. Some amino acids, glutamic acid and glutamine, slightly enhanced prolinase activity against pro-gly in erythrocytes from both the normal control and the prolidase-deficient patient, but N-acetyl-L-glutamic acid, gamma-aminobutyric acid (GABA) and beta-alanine showed no effect. Branched amino acids, L-valine, L-leucine and L-isoleucine strongly inhibited the prolinase activity against pro-gly. However, conversely, their isomers, D-valine, D-leucine and D-isoleucine, enhanced it. The kinetics of prolinase activity in the erythrocytes from both the normal individual and the prolidasedeficient patient were also studied. Their Km values were changed by adding glycine or 0.1 mM MnCl2, but Vmax values were almost the same. PMID:15552267

  7. Identification and Quantification of Flavonoids from Two Southern Italian Cultivars of Allium cepa L., Tropea (Red Onion) and Montoro (Copper Onion), and Their Capacity to Protect Human Erythrocytes from Oxidative Stress.

    PubMed

    Tedesco, Idolo; Carbone, Virginia; Spagnuolo, Carmela; Minasi, Paola; Russo, Gian Luigi

    2015-06-01

    Onions (Allium cepa) are consumed worldwide and represent an important source of dietary phytochemicals with proven antioxidant properties, such as phenolic acids, flavonoids, thiosulfinates, and anthocyanins. Epidemiological and experimental data suggest that regular consumption of onions is associated with a reduced risk of degenerative disorders. Therefore, it is of interest to investigate the biological properties of different varieties of onions. Here, we characterized for the first time a variety of onion, called Ramata di Montoro (coppery onion from Montoro), grown in a niche area in southern Italy, and compared its phenolic profile and antioxidant properties to a commercial ecotype of red onion, Tropea, also present in southern Italy. An analytical method based on high-performance liquid chromatography coupled with UV detection and mass spectrometry was used to separate and characterize the phenolic fraction (anthocyanins and flavonols) extracted from both coppery and red types. The main compounds detected in the two ecotypes were quercetin and quercetin glucosides, isorhamnetin glucosides, kaempferol glucoside, and, among anthocyanins, cyanidin glucosides. Tropea ecotype onion showed a higher content of flavonols (632.82 mg/kg fresh weight) than Montoro type onion (252.91 mg/kg fresh weight). Accordingly, the antioxidant activity of the former was 2.8-fold higher compared to the latter. More pronounced were the differences existing between the four anthocyanins detected in the two ecotypes, with those in the Tropea ecotype onion present at concentrations 20-230-fold higher than in the Montoro type onion. Both extracts reduced LDL oxidation about 6-fold and protected human erythrocytes from oxidative damage induced by HClO by about 40%. In addition, as a consequence of HClO treatment, glutathione concentration in erythrocytes was reduced about 50% and pretreatment with onion extracts induced a recovery of glutathione level by about 15-22%. Qualitative

  8. Differences in the toxicity of six Gambierdiscus (Dinophyceae) species measured using an in vitro human erythrocyte lysis assay.

    PubMed

    Holland, William C; Litaker, R Wayne; Tomas, Carmelo R; Kibler, Steven R; Place, Allen R; Davenport, Erik D; Tester, Patricia A

    2013-04-01

    This study examined the toxicity of six Gambierdiscus species (Gambierdiscus belizeanus, Gambierdiscus caribaeus, Gambierdiscus carolinianus, Gambierdiscus carpenteri, Gambierdiscus ribotype 2 and Gambierdiscus ruetzleri) using a human erythrocyte lysis assay. In all, 56 isolates were tested. The results showed certain species were significantly more toxic than others. Depending on the species, hemolytic activity consistently increased by ∼7-40% from log phase growth to late log - early stationary growth phase and then declined in mid-stationary growth phase. Increasing growth temperatures from 20 to 31 °C for clones of G. caribaeus showed only a slight increase in hemolytic activity between 20 and 27 °C. Hemolytic activity in the G. carolinianus isolates from different regions grown over the same 20-31 °C range remained constant. These data suggest that growth temperature is not a significant factor in modulating the inter-isolate and interspecific differences in hemolytic activity. The hemolytic activity of various isolates measured repeatedly over a 2 year period remained constant, consistent with the hemolytic compounds being constitutively produced and under strong genetic control. Depending on species, greater than 60-90% of the total hemolytic activity was initially associated with the cell membranes but diffused into solution over a 24 h assay incubation period at 4 °C. These findings suggest that hemolytic compounds produced by Gambierdiscus isolates were held in membrane bound vesicles as reported for brevetoxins produced by Karenia brevis. Gambierdiscus isolates obtained from other parts of the world exhibited hemolytic activities comparable to those found in the Caribbean and Gulf of Mexico confirming the range of toxicities is similar among Gambierdiscus species worldwide. Experiments using specific inhibitors of the MTX pathway and purified MTX, Gambierdiscus whole cell extracts, and hydrophilic cell extracts containing MTX, were consistent with

  9. Protection of DNA and erythrocytes from free radical induced oxidative damage by black gram (Vigna mungo L.) husk extract.

    PubMed

    Girish, Talakatta K; Vasudevaraju, Padmaraju; Prasada Rao, Ummiti J S

    2012-05-01

    Antioxidants present in various plant tissues exhibit health benefits by scavenging reactive oxygen species generated under various pathophysiological conditions. In the present study, bioactive compounds from black gram husk were extracted with water and the protection of black gram husk (BGH) extract against oxidative damage in DNA and erythrocytes were studied. BGH extract had total polyphenol content of 59 mg of gallic acid equivalents (GAE). The phenolic acids identified in the extract using RP-HPLC were gallic, protocatechuic, gentisic and ferulic acids. The extract showed good antioxidant properties. The IC(50) value for DPPH radical scavenging activity was found to be 3.92 μg of GAE. The BGH extract also showed α-glucosidase inhibition and the IC(50) value was found to be 2.78 μg of GAE. The oxidative hemolysis caused by hydrogen peroxide in rat erythrocytes was inhibited by BGH extract in a dose dependent manner. The IC(50) values for BGH extract and BHA for hemolysis were 11.5 and 14 μg of GAE, respectively. Morphological changes in erythrocyte membrane caused by hydrogen peroxide were protected by BGH extract. As BGH extract exhibited various antioxidant properties in different systems, it could be used as a functional food or nutraceutical product for health benefits. PMID:22330200

  10. Glycosylation of Erythrocyte Spectrin and Its Modification in Visceral Leishmaniasis

    PubMed Central

    Samanta, Sajal; Dutta, Devawati; Ghoshal, Angana; Mukhopadhyay, Sumi; Saha, Bibhuti; Sundar, Shyam; Jarmalavicius, Saulius; Forgber, Michael; Mandal, Chhabinath; Walden, Peter; Mandal, Chitra

    2011-01-01

    Using a lectin, Achatinin-H, having preferential specificity for glycoproteins with terminal 9-O-acetyl sialic acid derivatives linked in α2-6 linkages to subterminal N-acetylgalactosamine, eight distinct disease-associated 9-O-acetylated sialoglycoproteins was purified from erythrocytes of visceral leishmaniaisis (VL) patients (RBCVL). Analyses of tryptic fragments by mass spectrometry led to the identification of two high-molecular weight 9-O-acetylated sialoglycoproteins as human erythrocytic α- and β-spectrin. Total spectrin purified from erythrocytes of VL patients (spectrinVL) was reactive with Achatinin-H. Interestingly, along with two high molecular weight bands corresponding to α- and β-spectrin another low molecular weight 60 kDa band was observed. Total spectrin was also purified from normal human erythrocytes (spectrinN) and insignificant binding with Achatinin-H was demonstrated. Additionally, this 60 kDa fragment was totally absent in spectrinN. Although the presence of both N- and O-glycosylations was found both in spectrinN and spectrinVL, enhanced sialylation was predominantly induced in spectrinVL. Sialic acids accounted for approximately 1.25 kDa mass of the 60 kDa polypeptide. The demonstration of a few identified sialylated tryptic fragments of α- and β-spectrinVL confirmed the presence of terminal sialic acids. Molecular modelling studies of spectrin suggest that a sugar moiety can fit into the potential glycosylation sites. Interestingly, highly sialylated spectrinVL showed decreased binding with spectrin-depleted inside-out membrane vesicles of normal erythrocytes compared to spectrinN suggesting functional abnormality. Taken together this is the first report of glycosylated eythrocytic spectrin in normal erythrocytes and its enhanced sialylation in RBCVL. The enhanced sialylation of this cytoskeleton protein is possibly related to the fragmentation of spectrinVL as evidenced by the presence of an additional 60 kDa fragment, absent in

  11. Flavonoid mixture ameliorates increase in erythrocyte osmotic fragility and malondialdehyde concentration induced by Trypanosoma brucei brucei-infection in Wistar rats.

    PubMed

    Kobo, Patricia I; Ayo, Joseph O; Aluwong, Tagang; Zezi, Abdulkadir U; Maikai, Victor; Ambali, Suleiman F

    2014-02-01

    The experiment was performed with the aim of investigating the effect of a flavonoid mixture, Daflon® 500 mg (DF) on the erythrocyte fragility and lipoperoxidative changes, induced by Trypanosoma brucei brucei infection in Wistar rats. Fifty adult male rats randomly divided into five groups of 10 animals each were used. Rats in the control group were administered (1 mL/kg) distilled water only, while the other groups were infected with T. brucei brucei and treated with Daflon® 500 mg and/or Diminazene aceturate. At the end of 5 weeks, EDTA-blood samples and serum samples were collected from the rats, and were used to determine erythrocyte osmotic fragility (EOF) and serum malondialdehyde (MDA) concentration respectively. The results showed that EOF and MDA concentration significantly (P<0.05) increased in the infected untreated group when compared to the treatment groups. Treatment with Daflon® 500 mg and Diminazene aceturate significantly (P<0.05) reduced trypanosome-induced increases in EOF and lipoperoxidative changes, suggesting possible antioxidant properties of Daflon® 500 mg and its therapeutic value in trypanosomosis. PMID:24332272

  12. The structural and functional effects of Hg(II) and Cd(II) on lipid model systems and human erythrocytes: A review.

    PubMed

    Payliss, Brandon J; Hassanin, Mohamed; Prenner, Elmar J

    2015-12-01

    The anthropogenic mobilization of mercury and cadmium into the biosphere has led to an increased and ineludible entry of these metals into biological systems. Here we discuss the impact of Hg(II) and Cd(II) on lipid model systems and human erythrocytes from a biophysical perspective. After a brief introduction to their implications on human health, studies that have investigated the effects of Hg(II) and Cd(II) on lipid model systems and human erythrocytes are discussed. In terms of lipids as toxicological target sites, predominantly variations in lipid head groups have been the source of investigation. However, as research in this field progresses, the effects of Hg(II) and Cd(II) on other structural features, such as acyl chain length and unsaturation, and other important lipid components and complex biomimetic lipid mixtures, will require further examinations. This review provides an analysis of what has been learned collectively from the diverse methodologies and experimental conditions used thus far. Consequently, there is a need for more comprehensive and thorough investigations into the effects of Hg(II) and Cd(II) on lipid membranes under consistent experimental conditions such as pH, ionic strength, temperature, and choice of lipid model system. PMID:26455331

  13. Erythrocyte ion channels in regulation of apoptosis.

    PubMed

    Lang, Florian; Birka, Christina; Myssina, Svetlana; Lang, Karl S; Lang, Philipp A; Tanneur, Valerie; Duranton, Christophe; Wieder, Thomas; Huber, Stephan M

    2004-01-01

    Erythrocytes lack mitochondria and nuclei, key organelles in the regulation of apoptosis. Until recently, erythrocytes were thus not considered subject to this type of cell death. However, exposure of erythrocytes to the Ca2+ ionophore ionomycin was shown to induce cell shrinkage, cell membrane blebbing and breakdown of phosphatidylserine asymmetry with subsequent phosphatidylserine exposure at the cell surface, all typical features of apoptosis. Further studies revealed the participation of ion channels in the regulation of erythrocyte "apoptosis." Osmotic shock, oxidative stress and energy depletion all activate a Ca2(+)-permeable non-selective cation channel in the erythrocyte cell membrane. The subsequent increase of Ca2+ concentration stimulates a scramblase leading to breakdown of cell membrane phosphatidylserine asymmetry and activates Ca2+ sensitive K+ (Gardos) channels leading to KCl loss and (further) cell shrinkage. Phosphatidylserine exposure and cell shrinkage are blunted in the nominal absence of extracellular Ca2+, in the presence of the cation channel inhibitors amiloride or ethylisopropylamiloride, at increased extracellular K+ or in the presence of the Gardos channel inhibitors clotrimazole or charybdotoxin. Thus, increase of cytosolic Ca2+ and cellular loss of K+ participate in the triggering of erythrocyte scramblase. Nevertheless, phosphatidylserine exposure is not completely abrogated in the nominal absence of Ca2+, pointing to additional Ca2(+)-independent pathways. One of those is activation of sphingomyelinase with subsequent formation of ceramide which in turn leads to stimulation of erythrocyte scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Erythropoietin inhibits the non-selective cation channel and thus

  14. Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane.

    PubMed

    Nunomura, W; Takakuwa, Y; Parra, M; Conboy, J; Mohandas, N

    2000-08-11

    Three binary protein-protein interactions, glycophorin C (GPC)-4.1R, GPC-p55, and p55-4.1R, constitute the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little is known regarding the molecular basis for the interaction of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R constitute the binding interfaces for GPC and p55, respectively. We further show that 4.1R increases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaction between p55 and GPC. Finally, we document that binding of calmodulin to 4.1R decreases the affinity of 4.1R interactions with both p55 and GPC in a Ca(2+)-dependent manner, implying that the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation in the erythrocyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. PMID:10831591

  15. [Studies of the blood antioxidant system and oxygen-transporting properties of human erythrocytes during 105-day isolation].

    PubMed

    Brazhe, N A; Baĭzhumanov, A A; Parshina, E Iu; Iusipovich, A I; Akhalaia, M Ia; Iarlykova, Iu V; Labetskaia, O I; Ivanova, S M; Morukov, B V; Maksimov, G V

    2011-01-01

    Effects of strict 105-d isolation on blood antioxidant status, erythrocyte membrane processes and oxygen-binding properties of hemoglobin were studied in 6 male volunteers (25 to 40 y.o.) in ground-based simulation of a mission to Mars (experiment Mars-105). The parameters were measured using venous blood samples collected during BDC, on days 35, 70 and 105 of the experiment and on days 7 and 14-15 after its completion. Methods of biochemistry (determination of enzyme activity and thin-layer chromatography) and biophysical (laser interference microscopy, Raman spectroscopy) showed changes in relative content of lipid and phospholipid fractions suggesting growth of membrane microviscosity and increase in TBA-AP (active products of lipids peroxidation interacting with thiobarbituric acid). A significant increase in glucose-6-phosphate dehydrogenase and superoxide dismutase activities against reduction of catalase activity points to both reparative processes in erythrocytes and disbalance between the number of evolving active forms of oxygen and antioxidant protection mechanisms in cells. Hemoglobin sensitivity of oxygen and blood level of oxyhemoglobin were found to increase, too. It is presumed that adaptation of organism to stresses experienced during and after the experiment may destroy balance of the antioxidant protection systems which is conducive to oxidation of membrane phospholipids, alteration of their content, increase of membrane microviscosity and eventual failure of the gas-exchange function of erythrocytes. PMID:21675192

  16. Articulation and flexibility in the physical linkage between band 3 and the cytoskeleton in the human erythrocyte

    NASA Astrophysics Data System (ADS)

    Sawyer, William H.; Tilley, Leann M.

    1992-04-01

    The erythrocyte membrane with its underlying cytoskeleton undergoes gross deformation during passage of the cell through fine capillary networks in the circulation. The physical linkages between the cytoskeleton and the membrane must involve substantial internal flexibility or points of articulation within and between molecules in the system. We have used time-resolved phosphorescence spectroscopy to examine three aspects of this dynamic flexibility on the microsecond to millisecond time scale: (1) the rotational diffusion and internal flexibility of F-actin, (2) the intrinsic flexibility of spectrin in solution and when reconstituted to erythrocyte membranes, and (3) internal flexibility in the cytoplasmic domain of band three. The results show that the spectrin dimer displays substantial flexibility on the microsecond time scale: this flexibility is largely retained when spectrin is reassociated with the erythrocyte membrane. There is also evidence that the cytoplasmic domain of band three itself is flexible since the rotational correlation time for band three labeled through this domain (via a phosphorescently labeled monoclonal Fab) is significantly shorter than when the protein is labeled directly at the external face of its transmembrane domain. Short actin filaments possess limited torsional motion on the submicrosecond time scale and are unlikely to contribute to the flexibility of the cytoskeleton.

  17. Reduced cellular redox status induces 4-hydroxynonenal-mediated caspase 3 activation leading to erythrocyte death during chronic arsenic exposure in rats

    SciTech Connect

    Biswas, Debabrata; Sen, Gargi; Biswas, Tuli

    2010-05-01

    Chronic exposure to arsenic in rats led to gradual accumulation of the toxicant in erythrocytes causing oxidative stress in these cells. 4-Hydroxynonenal (4-HNE), a major aldehyde product of lipid peroxidation, contributed significantly to the cytopathological events observed during oxidative stress in the erythrocytes of exposed rats. 4-HNE triggered death signal cascade that was initiated with the formation of HNE-protein adducts in cytosol. HNE-protein adduct formation resulted in depletion of cytosolic antioxidants followed by increased generation of ROS. Results showed accumulation of hydrogen peroxide (H{sub 2}O{sub 2}) from the early stages of arsenic exposure, while superoxide (O{sub 2}{sup c}entre dot{sup -}) and hydroxyl radical ({sup c}entre dotOH) also contributed to the oxidative stress during longer period of exposure. Suppression of antioxidant system coupled with increased generation of ROS eventually led to activation of caspase 3 during arsenic exposure. Attenuation of HNE-mediated activation of caspase 3 in presence of N-acetylcysteine (NAC) indicated the involvement of GSH in the process. Prevention of HNE-mediated degradation of membrane proteins in presence of Z-DEVD-FMK identified caspase 3 as the principal mediator of HNE-induced cellular damage during arsenic exposure. Degradation of band 3 followed by its aggregation on the red cell surface promoted immunologic recognition of redistributed band 3 by autologous IgG with subsequent attachment of C3b. Finally, the formation of C3b-IgG-band 3 immune complex accelerated the elimination of affected cells from circulation and led to the decline of erythrocyte life span during chronic arsenic toxicity.

  18. Erythrocyte Shrinkage and Cell Membrane Scrambling after Exposure to the Ionophore Nonactin.

    PubMed

    Peter, Thomas; Bissinger, Rosi; Lang, Florian

    2016-02-01

    The ionophore antibiotic nonactin permeabilizes cell membranes to NH4+ and K(+) . Treatment of erythrocytes with nonactin is expected to trigger cellular K(+) loss with subsequent cell shrinkage, which in turn is known to trigger suicidal death of a wide variety of cells including erythrocytes. This study explored whether nonactin exposure induces eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signalling of eryptosis includes increase in cytosolic Ca(2+) activity [(Ca(2+) )i ] and stimulation of protein kinase C (PKC) as well as p38 mitogen-activated protein kinase. Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter (FSC) and (Ca(2+) )i from Fluo3-fluorescence. A 48-hr treatment of human erythrocytes with nonactin significantly decreased FSC (≥10 ng/ml) and significantly increased the percentage of annexin-V-binding cells (≥10 ng/ml), effects paralleled by increase in (Ca(2+) )i (≥50 ng/ml) and virtually abrogated by increase in extracellular K(+) concentration to 120 mM at the expense of Na(+) . The up-regulation of annexin-V-binding after nonactin treatment was significantly blunted but not abolished by the removal of extracellular Ca(2+) and by addition of either PKC inhibitor staurosporine (0.4 μM) or p38 kinase inhibitor SB203580 (2 μM). In conclusion, exposure of erythrocytes to the K(+) ionophore nonactin induces erythrocyte shrinkage and subsequent erythrocyte membrane scrambling, effects involving cellular K(+) loss, Ca(2+) entry and activation of staurosporine as well as SB203580-sensitive kinases. PMID:26280658

  19. Use of heteropolymeric monoclonal antibodies to attach antigens to the C3b receptor of human erythrocytes: A potential therapeutic treatment

    SciTech Connect

    Taylor, R.P.; Sutherland, W.M.; Reist, C.J.; Webb, D.J.; Wright, E.L.; Labuguen, R.H. )

    1991-04-15

    The authors prepared bispecific, cross-linked monoclonal antibodies (heteropolymers) with specificity for both targeted antigens and the human erythrocyte (RBC) complement receptor. These heteropolymers facilitate binding of target antigens (human IgG and dinitrophenylated bovine {gamma} globulin) to human RBCs under conditions that either allow or preclude complement activation. Radioimmuno-assay analyses of this binding agree well with the number of complement receptors per RBC. In vitro whole-blood model experiments indicate heteropolymer-facilitated binding of antigens to RBCs is rapid and stable at 37C. It may be possible to extend these prototype experiments to the in vivo situation and use heteropolymer-attached RBCs for the safe and rapid binding, neutralization, and removal from the circulation of pathogenic antigens associated with infectious disease.

  20. Induction of Suicidal Erythrocyte Death by Cantharidin

    PubMed Central

    Alzoubi, Kousi; Egler, Jasmin; Briglia, Marilena; Fazio, Antonella; Faggio, Caterina; Lang, Florian

    2015-01-01

    The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca2+-activity ([Ca2+]i), ceramide, oxidative stress and dysregulation of several kinases. Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 μg/mL), significantly decreased forward scatter (≥25 μg/mL), significantly increased [Ca2+]i (≥25 μg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca2+ but was abolished by kinase inhibitor staurosporine (1 μM) and slightly decreased by p38 inhibitor skepinone (2 μM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone. PMID:26226001

  1. Enhancement of erythrocyte antioxidants by green and black tea polyphenols during 7,12-dimethylbenz[a]anthracene-induced hamster buccal pouch carcinogenesis.

    PubMed

    Chandra Mohan, K V P; Subapriya, R; Hara, Y; Nagini, S

    2006-01-01

    We evaluated the comparative chemopreventive efficacy of green tea polyphenols (polyphenon-E) and black tea polyphenols (polyphenon-B) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. Lipid peroxidation, reduced and oxidized glutathione (GSH and GSSG, respectively), and the GSH-dependent enzymes glutathione peroxidase and glutathione S-transferase in the erythrocytes were used as biomarkers of chemoprevention. Enhanced lipid peroxidation in erythrocytes of DMBA-treated animals was accompanied by a significant decrease in the antioxidant status. Dietary administration of polyphenon-E and -B to DMBA-treated animals significantly decreased the extent of lipid peroxidation and enhanced the levels of GSH, GSH/GSSG ratio, and activities of GSH-dependent enzymes. Our study provides evidence that polyphenon-B is more effective in inhibiting HBP carcinogenesis than polyphenon-E by enhancing the antioxidant status, suggesting that polyphenon-B may have a major impact in the chemoprevention of oral cancer. PMID:17004901

  2. Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

    PubMed Central

    Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V.; Moore, Lee R.; Chalmers, Jeffrey J.; Zborowski, Maciej

    2012-01-01

    Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. PMID:22952572

  3. Ameliorative properties of aqueous extract of Ficus thonningii on erythrocyte osmotic fragility induced by acetaminophen in Rattus norvegicus

    PubMed Central

    Ahur, Victor Masekaven; Adenkola, Yahaya Adenkola; Saganuwan, Saganuwan Alhaji; Ikye-Tor, Job Terungwa

    2013-01-01

    In vitro antioxidant and erythrocyte protecting activities by aqueous extract of Ficus thonningii leaves on blood cells were studied in acetaminophen treated rats. The extract was safe at limit dose of 5000 mg kg-1 body weight. The extract demonstrated dose dependent antihemolytic effect at dose levels between 50 and 200 mg kg-1 body weight. The lowest antihemolytic effect was observed at dose level of 200 mg kg-1 body given the lowest percentage hemolysis of 10.53 ± 1.76%, whereas the highest percentage hemolysis at dose level of 50 mg kg-1 was 29.02 ± 7.45%. Hematology revealed erythrocytosis at dose levels of 100 and 200 mg kg-1 body weight. Hyperglobinemia and lymphocytopenia were observed at dose levels of 100 mg kg-1 and 200 mg kg-1, respectively. The extract effectively showed scavenging activity on a stable oxidative radical diphenylpicrylhydrazyl (DPPH) and a significant ferric reducing antioxidant power (FRAP) activity. The plausible erythrocyte membrane protective effect may be due to its free radical scavenging activity and hence the extract can be used to improve hematological parameters and ameliorate oxidative stress. PMID:25568673

  4. Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease.

    PubMed

    van Beers, Eduard J; Samsel, Leigh; Mendelsohn, Laurel; Saiyed, Rehan; Fertrin, Kleber Y; Brantner, Christine A; Daniels, Mathew P; Nichols, James; McCoy, J Philip; Kato, Gregory J

    2014-06-01

    In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient's disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged "sickled" and "normal" erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = -0.558, P = 0.027), negatively with pH (R = -0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = -0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development. PMID:24585634

  5. Peptide-induced membrane leakage by lysine derivatives of gramicidin A in liposomes, planar bilayers, and erythrocytes.

    PubMed

    Sorochkina, Alexandra I; Kovalchuk, Sergei I; Omarova, Elena O; Sobko, Alexander A; Kotova, Elena A; Antonenko, Yuri N

    2013-11-01

    Introducing a charged group near the N-terminus of gramicidin A (gA) is supposed to suppress its ability to form ion channels by restricting its head-to-head dimerization. The present study dealt with the activity of [Lys1]gA, [Lys3]gA, [Glu1]gA, [Glu3]gA, [Lys2]gA, and [Lys5]gA in model membrane systems (planar lipid bilayers and liposomes) and erythrocytes. In contrast to the Glu-substituted peptides, the lysine derivatives of gA caused non-specific liposomal leakage monitored by fluorescence dequenching of lipid vesicles loaded with carboxyfluorescein or other fluorescent dyes. Measurements of electrical current through a planar lipid membrane revealed formation of giant pores by Lys-substituted analogs, which depended on the presence of solvent in the bilayer lipid membrane. The efficacy of unselective pore formation in liposomes depended on the position of the lysine residue in the amino acid sequence, increasing in the row: [Lys2]gA<[Lys5]gA<[Lys1]gA<[Lys3]gA. The similar series of potency was exhibited by the Lys-substituted gA analogs in facilitating erythrocyte hemolysis, whereas the Glu-substituted analogs showed negligible hemolytic activity. Oligomerization of the Lys-substituted peptides is suggested to be involved in the process of nonselective pore formation. PMID:23806648

  6. Viscosity of concentrated solutions and of human erythrocyte cytoplasm determined from NMR measurement of molecular correlation times. The dependence of viscosity on cell volume.

    PubMed

    Endre, Z H; Kuchel, P W

    1986-08-01

    Metabolically active human erythrocytes were incubated with [alpha-13C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using 13C-NMR in which the longitudinal relaxation times (T1) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. The T1 values of labelled glycine were also determined in various-concentration solutions of bovine serum albumin and glycerol and also of the natural abundance 13C of glycerol in glycerol solutions. From the T1 estimates the rotational correlation time (tau r) was calculated using a formula based on a model of an isotropic spherical rotor or that of a symmetrical ellipsoidal rotor; for glycine the differences in estimates of tau r obtained using the two models were not significant. From the correlation times and by use of the Stokes-Einstein equations viscosity and translational diffusion coefficients were calculated; thus comment can be made on the likelihood of diffusion control of certain enzyme-catalysed reactions in the erythrocyte. Bulk viscosities of the erythrocyte cytoplasm and the above-mentioned solutions were measured using Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T1 measurements. We derived an equation from the theory of the viscosity of concentrated solutions which contains two phenomenological interaction parameters, a 'shape' factor and a 'volume' factor; it was fitted to data relating to the concentration dependence of viscosity measured by both methods. We showed, by using the equation and interaction-parameter estimates for a particular probe molecule in a particular solution, that it was possible to correlate NMR viscosity and bulk viscosity; in other words, given an estimate of the bulk viscosity, it was possible to calculate the NMR 'micro' viscosity or vice versa. However, the values of the interaction parameters depend upon the relative sizes of

  7. The approximate entropy of the electromyographic signals of tremor correlates with the osmotic fragility of human erythrocytes

    PubMed Central

    2010-01-01

    Background The main problem of tremor is the damage caused to the quality of the life of patients, especially those at more advanced ages. There is not a consensus yet about the origins of this disorder, but it can be examined in the correlations between the biological signs of aging and the tremor characteristics. Methods This work sought correlations between the osmotic fragility of erythrocytes and features extracted from electromyographic (EMG) activity resulting from physiological tremor in healthy patients (N = 44) at different ages (24-87 years). The osmotic fragility was spectrophotometrically evaluated by the dependence of hemolysis, provided by the absorbance in 540 nm (A54o), on the concentration of NaCl. The data were adjusted to curves of sigmoidal regression and characterized by the half transition point (H50), amplitude of lysis transition (dx) and values of A540 in the curve regions that characterize the presence of lysed (A1) and preserved erythrocytes (A2). The approximate entropy was estimated from EMG signals detected from the extensor carpi ulnaris muscle during the movement of the hand of subjects holding up a laser pen towards an Archimedes spiral, fixed in a whiteboard. The evaluations were carried out with the laser pen at rest, at the center of the spiral, and in movement from the center to the outside and from outside to the center. The correlations among the parameters of osmotic fragility, tremor and age were tested. Results Negative correlations with age were found for A1 and dx. With the hand at rest, a positive correlation with H50 was found for the approximate entropy. Negative correlations with H50 were found for the entropy with the hand in movement, as from the center to the outside or from the outside to the center of the spiral. Conclusion In healthy individuals, the increase in the erythrocyte osmotic fragility was associated with a decrease in the approximate entropy for rest tremor and with an increase of the entropy for

  8. In Vitro Protective Effect and Antioxidant Mechanism of Resveratrol Induced by Dapsone Hydroxylamine in Human Cells

    PubMed Central

    Albuquerque, Rosyana V.; Malcher, Nívea S.; Amado, Lílian L.; Coleman, Michael D.; dos Santos, Danielle C.; Borges, Rosivaldo Sa.; Valente, Sebastião Aldo S.; Valente, Vera C.; Monteiro, Marta Chagas

    2015-01-01

    Dapsone (DDS) hydroxylamine metabolites cause oxidative stress- linked adverse effects in patients, such as methemoglobin formation and DNA damage. This study evaluated the ameliorating effect of the antioxidant resveratrol (RSV) on DDS hydroxylamine (DDS-NHOH) mediated toxicity in vitro using human erythrocytes and lymphocytes. The antioxidant mechanism was also studied using in-silico methods. In addition, RSV provided intracellular protection by inhibiting DNA damage in human lymphocytes induced by DDS-NHOH. However, whilst pretreatment with RSV (10–1000 μM significantly attenuated DDS-NHOH-induced methemoglobinemia, but it was not only significantly less effective than methylene blue (MET), but also post-treatment with RSV did not reverse methemoglobin formation, contrarily to that observed with MET. DDS-NHOH inhibited catalase (CAT) activity and reactive oxygen species (ROS) generation, but did not alter superoxide dismutase (SOD) activity in erythrocytes. Pretreatment with RSV did not alter these antioxidant enzymes activities in erythrocytes treated with DDS-NHOH. Theoretical calculations using density functional theory methods showed that DDS-NHOH has a pro-oxidant effect, whereas RSV and MET have antioxidant effect on ROS. The effect on methemoglobinemia reversion for MET was significantly higher than that of RSV. These data suggest that the pretreatment with resveratrol may decrease heme-iron oxidation and DNA damage through reduction of ROS generated in cells during DDS therapy. PMID:26284371

  9. Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains

    PubMed Central

    Rungaldier, Stefanie; Oberwagner, Walter; Salzer, Ulrich; Csaszar, Edina; Prohaska, Rainer

    2013-01-01

    The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins. PMID:23219802

  10. Erythrocyte Lysis and Xenopus laevis Oocyte Rupture by Recombinant Plasmodium falciparum Hemolysin III

    PubMed Central

    Moonah, Shannon; Sanders, Natalie G.; Persichetti, Jason K.

    2014-01-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia. PMID:25148832

  11. Erythrocyte lysis and Xenopus laevis oocyte rupture by recombinant Plasmodium falciparum hemolysin III.

    PubMed

    Moonah, Shannon; Sanders, Natalie G; Persichetti, Jason K; Sullivan, David J

    2014-10-01

    Malaria kills more than 1 million people per year worldwide, with severe malaria anemia accounting for the majority of the deaths. Malaria anemia is multifactorial in etiology, including infected erythrocyte destruction and decrease in erythrocyte production, as well as destruction or clearance of noninfected erythrocytes. We identified a panspecies Plasmodium hemolysin type III related to bacterial hemolysins. The identification of a hemolysin III homologue in Plasmodium suggests a potential role in host erythrocyte lysis. Here, we report the first characterization of Plasmodium falciparum hemolysin III, showing that the soluble recombinant P. falciparum hemolysin III is a pore-forming protein capable of lysing human erythrocytes in a dose-, time-, and temperature-dependent fashion. The recombinant P. falciparum hemolysin III-induced hemolysis was partially inhibited by glibenclamide, a known channel antagonist. Studies with polyethylene glycol molecules of different molecular weights indicated a pore size of approximately 3.2 nm. Heterologous expression of recombinant P. falciparum hemolysin III in Xenopus oocytes demonstrated early hypotonic lysis similar to that of the pore-forming aquaporin control. Live fluorescence microscopy localized transfected recombinant green fluorescent protein (GFP)-tagged P. falciparum hemolysin III to the essential digestive vacuole of the P. falciparum parasite. These transfected trophozoites also possessed a swollen digestive vacuole phenotype. Native Plasmodium hemolysin III in the digestive vacuole may contribute to lysis of the parasitophorous vacuole membrane derived from the host erythrocyte. After merozoite egress from infected erythrocytes, remnant P. falciparum hemolysin III released from digestive vacuoles could potentially contribute to lysis of uninfected erythrocytes to contribute to severe life-threatening anemia. PMID:25148832

  12. The mechanics of malaria parasite invasion of the human erythrocyte - towards a reassessment of the host cell contribution.

    PubMed

    Koch, Marion; Baum, Jake

    2016-03-01

    Despite decades of research, we still know little about the mechanics of Plasmodium host cell invasion. Fundamentally, while the essential or non-essential nature of different parasite proteins is becoming clearer, their actual function and how each comes together to govern invasion are poorly understood. Furthermore, in recent years an emerging world view is shifting focus away from the parasite actin-myosin motor being the sole force responsible for entry to an appreciation of host cell dynamics and forces and their contribution to the process. In this review, we discuss merozoite invasion of the erythrocyte, focusing on the complex set of pre-invasion events and how these might prime the red cell to facilitate invasion. While traditionally parasite interactions at this stage have been viewed simplistically as mediating adhesion only, recent work makes it apparent that by interacting with a number of host receptors and signalling pathways, combined with secretion of parasite-derived lipid material, that the merozoite may initiate cytoskeletal re-arrangements and biophysical changes in the erythrocyte that greatly reduce energy barriers for entry. Seen in this light Plasmodium invasion may well turn out to be a balance between host and parasite forces, much like that of other pathogen infection mechanisms. PMID:26663815

  13. The mechanics of malaria parasite invasion of the human erythrocyte – towards a reassessment of the host cell contribution

    PubMed Central

    Koch, Marion

    2016-01-01

    Summary Despite decades of research, we still know little about the mechanics of Plasmodium host cell invasion. Fundamentally, while the essential or non‐essential nature of different parasite proteins is becoming clearer, their actual function and how each comes together to govern invasion are poorly understood. Furthermore, in recent years an emerging world view is shifting focus away from the parasite actin–myosin motor being the sole force responsible for entry to an appreciation of host cell dynamics and forces and their contribution to the process. In this review, we discuss merozoite invasion of the erythrocyte, focusing on the complex set of pre‐invasion events and how these might prime the red cell to facilitate invasion. While traditionally parasite interactions at this stage have been viewed simplistically as mediating adhesion only, recent work makes it apparent that by interacting with a number of host receptors and signalling pathways, combined with secretion of parasite‐derived lipid material, that the merozoite may initiate cytoskeletal re‐arrangements and biophysical changes in the erythrocyte that greatly reduce energy barriers for entry. Seen in this light Plasmodium invasion may well turn out to be a balance between host and parasite forces, much like that of other pathogen infection mechanisms. PMID:26663815

  14. In silico-screening approaches for lead generation: identification of novel allosteric modulators of human-erythrocyte pyruvate kinase.

    PubMed

    Tripathi, Ashutosh; Safo, Martin K

    2012-01-01

    Identification of allosteric binding site modulators have gained increased attention lately for their potential to be developed as selective agents with a novel chemotype and targeting perhaps a new and unique binding site with probable fewer side effects. Erythrocyte pyruvate kinase (R-PK) is an important glycolytic enzyme that can be pharmacologically modulated through its allosteric effectors for the treatment of hemolytic anemia, sickle-cell anemia, hypoxia-related diseases, and other disorders arising from erythrocyte PK malfunction. An in-silico screening approach was applied to identify novel allosteric modulators of pyruvate kinase. A small-molecules database of the National Cancer Institute (NCI), was virtually screened based on structure/ligand-based pharmacophore. The virtual screening campaign led to the identification of several compounds with similar pharmacophoric features as fructose-1,6-bisphosphate (FBP), the natural allosteric activator of the kinase. The compounds were subsequently docked into the FBP-binding site using the programs FlexX and GOLD, and their interactions with the protein were analyzed with the energy-scoring function of HINT. Seven promising candidates were obtained from the NCI and subjected to kinetics analysis, which revealed both activators and inhibitors of the R-isozyme of PK (R-PK). PMID:22052500

  15. Anti-oxidant activity of holo- and apo-c-phycocyanin and their protective effects on human erythrocytes.

    PubMed

    Pleonsil, Pornthip; Soogarun, Suphan; Suwanwong, Yaneenart

    2013-09-01

    This study was conducted to investigate the anti-oxidant activity of the recombinant apo-c-phycocyanin (c-PC) β-subunit compared to native c-PC purified from Spirulina sp. The gene encoding the β-subunit of c-PC was successfully cloned and expressed in Escherichia coli. The anti-oxidant capacities of recombinant apo-c-PC(β) and native c-PC were evaluated by measuring their Trolox equivalent antioxidant capacities and examining their protective effects on erythrocytes from normal and homozygous haemoglobin E individuals against peroxyl radicals and hydrogen peroxide. The results demonstrated that the anti-oxidant capacities are native c-PC≫Trolox>recombinant apo-c-PC(β). Both anti-oxidant proteins can potentially protect erythrocytes from oxidative damage. Expression of c-PC in bacteria reduces the cost and time for protein production, and the recombinant protein could be further developed to obtain a more efficient protein for therapeutic purposes. PMID:23806319

  16. Release of vitamin B12 from carrier erythrocytes in vitro.

    PubMed

    Eichler, H G; Raffesberg, W; Gasic, S; Korn, A; Bauer, K

    1985-01-01

    Resealed erythrocyte ghosts (carrier erythrocytes) are potential in vivo carriers for exogenous enzymes or drugs, but data on carrier erythrocyte survival and clearance rate in humans are not available. We have measured the in vitro efflux of vitamin B12 encapsulated in human red cell by hypo-osmotic dialysis, as a preliminary for its use as a marker for in vivo human studies. Vitamin B12 was encapsulated into erythrocytes at a relative incorporation efficiency of 60%. In vitro hemolysis of carrier erythrocytes was minimal over 40 h, but vitamin B12 was rapidly lost from the cells, efflux t/2 was 5 h, presumably by diffusion through the intact cell membrane. Vitamin B12 (Vit B12) may, nevertheless, be a suitable marker for short-term human studies on carrier erythrocyte splanchnic clearance. PMID:4048655

  17. Imaging flow cytometry for automated detection of hypoxia-induced erythrocyte shape change in sickle cell disease

    PubMed Central

    van Beers, Eduard J.; Samsel, Leigh; Mendelsohn, Laurel; Saiyed, Rehan; Fertrin, Kleber Y.; Brantner, Christine A.; Daniels, Mathew P.; Nichols, James; McCoy, J. Philip; Kato, Gregory J.

    2014-01-01

    In preclinical and early phase pharmacologic trials in sickle cell disease, the percentage of sickled erythrocytes after deoxygenation, an ex vivo functional sickling assay, has been used as a measure of a patient’s disease outcome. We developed a new sickle imaging flow cytometry assay (SIFCA) and investigated its application. To perform the SIFCA, peripheral blood was diluted, deoxygenated (2% oxygen) for 2 hr, fixed, and analyzed using imaging flow cytometry. We developed a software algorithm that correctly classified investigator tagged “sickled” and “normal” erythrocyte morphology with a sensitivity of 100% and a specificity of 99.1%. The percentage of sickled cells as measured by SIFCA correlated strongly with the percentage of sickle cell anemia blood in experimentally admixed samples (R = 0.98, P ≤ 0.001), negatively with fetal hemoglobin (HbF) levels (R = −0.558, P = 0.027), negatively with pH (R = −0.688, P = 0.026), negatively with pretreatment with the antisickling agent, Aes-103 (5-hydroxymethyl-2-furfural) (R = −0.766, P = 0.002), and positively with the presence of long intracellular fibers as visualized by transmission electron microscopy (R = 0.799, P = 0.002). This study shows proof of principle that the automated, operator-independent SIFCA is associated with predictable physiologic and clinical parameters and is altered by the putative antisickling agent, Aes-103. SIFCA is a new method that may be useful in sickle cell drug development. PMID:24585634

  18. Probable macrophage origin of the lipopolysaccharide-induced cytostatic effect on intra-erythrocytic malarial parasites (Plasmodium vinckei)

    PubMed Central

    Rzepczyk, Christine M.

    1982-01-01

    This study showed that intra-erythrocytic Plasmodium vinckei parasites taken from either normal, irradiated, nude or splenectomized mice 7–8 hr after the injection of a small dose of bacterial lipopolysaccharide (LPS) incorporate hypoxanthine more slowly in an in vitro assay than parasites from saline-treated controls. The incorporation by parasites of isoleucine, which was also measured in some experiments, was similarly affected. However, this cytostatic effect on parasite metabolism was found to be markedly reduced in experiments with mice which had received an intravenous injection of silica dust 28–30 hr before being injected with LPS. These findings indicate that macrophages, being radioresistant and silica-sensitive, are the source of the cytostatic effect. The present results also imply that T cells are not required in the response, and they show that the host cells mediating this response are not restricted to the spleen. It was also shown that an intravenous injection of a small dose of LPS into mice infected with P. vinckei 24 hr previously, could temporarily arrest the rise in parasitaemia in these animals, thereby prolonging their survival. This protection afforded by LPS was also found to be radioresistant and T-independent. It is suggested that the effect on parasitaemia seen in vivo and the cytostatic effect in vitro are both due to the release of a soluble factor from macrophages which is ultimately capable of causing intra-erythrocytic parasite death. P. vinckei-infected mice exhibited symptoms of endotoxaemia following the injection of LPS. However, no clear relationship was noted between the severity of the illness in the host and the cytostatic effect on the parasites. PMID:6282737

  19. Protein kinases A and C regulate receptor-mediated increases in cAMP in rabbit erythrocytes

    PubMed Central

    Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Ellsworth, Mary L.; Sprague, Randy S.

    2010-01-01

    Activation of the β-adrenergic receptor (β-AR) or the prostacyclin receptor (IPR) results in increases in cAMP and ATP release from erythrocytes. cAMP levels depend on a balance between synthesis via adenylyl cyclase and hydrolysis by phosphodiesterases (PDEs). Previously, we reported that cAMP increases associated with activation of the β-AR and IPR in rabbit and human erythrocytes are tightly regulated by distinct PDEs (1). Importantly, inhibitors of these PDEs potentiated both increases in cAMP and ATP release. It has been shown that increases in protein kinase (PK) activity can activate PDE3 and PDE4. Both PKA and PKC are present in the erythrocyte and can phosphorylate and activate these PDEs. Here we investigate the hypothesis that PKA regulates PDE activity associated with the β-AR and both PKA and PKC regulate the PDE activity associated with the IPR in rabbit erythrocytes. Pretreatment of erythrocytes with the PKA inhibitor, H89 (10 μM), in the presence of the PDE4 inhibitor, rolipram (10 μM), augmented isoproterenol (1 μM)-induced cAMP increases. In contrast, in the presence of the PDE3 inhibitor, cilostazol (10 μM), pretreatment of erythrocytes with either H89 (1 μM) or two chemically dissimilar inhibitors of PKC, calphostin C (1 μM) or GFX109203X (1 μM), potentiated iloprost (1 μM)-induced cAMP increases. Furthermore, pretreatment of erythrocytes with both H89 and GFX109203X in the presence of cilostazol augmented the iloprost-induced increases in cAMP to a greater extent than either PK inhibitor individually. These results support the hypothesis that PDEs associated with receptor-mediated increases in cAMP in rabbit erythrocytes are regulated by kinases specific to the receptor's signaling pathway. PMID:20008267

  20. Characterization of lipid domains in erythrocyte membranes.

    PubMed Central

    Rodgers, W; Glaser, M

    1991-01-01

    Fluorescence digital imaging microscopy was used to study the lateral distribution of the lipid components in erythrocyte membranes. Intact erythrocytes labeled with phospholipids containing a fluorophore attached to one fatty acid chain showed an uneven distribution of the phospholipids in the membrane thereby demonstrating the presence of membrane domains. The enrichment of the lipotropic compound chlor-promazine in domains in intact erythrocytes also suggested that the domains are lipid-enriched regions. Similar membrane domains were present in erythrocyte ghosts. The phospholipid enrichment was increased in the domains by inducing membrane protein aggregation. Double-labeling experiments were done to determine the relative distributions of different phospholipids in the membrane. Vesicles made from extracted lipids did not show the presence of domains consistent with the conclusion that membrane proteins were responsible for creating the domains. Overall, it was found that large domains exist in the red blood cell membrane with unequal enrichment of the different phospholipid species. Images PMID:1996337

  1. Isolation and partial characterization of antibody- and globin-enriched complexes from membranes of dense human erythrocytes.

    PubMed Central

    Kannan, R; Yuan, J; Low, P S

    1991-01-01

    In previous studies we have described a process whereby an erythrocyte in biochemical distress can initiate its own removal by macrophages of the reticuloendothelial system. This process involves the clustering of the integral membrane protein band 3 by denatured haemoglobin and the subsequent recognition of the exofacial poles of clustered band 3 and associated proteins by autologous antibodies. To determine whether this clearance pathway might mediate normal cell turnover, the fraction of normal erythrocytes containing the 0.5% densest cells, which are known to be destined for immediate removal, was isolated and characterized biochemically. This densest fraction was found to contain 6 times more membrane-bound globin (haemichromes) and 10 times more surface-bound autologous IgG than the other fractions containing cells of lower density. To determine whether the autologous IgG was physically associated with the haemichrome-stabilized membrane protein clusters, a procedure was developed for isolation and characterization of the microscopic aggregates. The isolated aggregates were found to contain a disulphide-cross-linked mixture of several membrane proteins, predominantly haemichromes, spectrin and band 3. Although the aggregates constituted only 0.09% of the total membrane protein, they still contained approximately 55% of the total cell-surface IgG. Since in control studies anti-(blood group A) antibodies, which are distributed randomly over the surface of type A cells, could not be recovered in the aggregate, we conclude that the autologous cell-surface IgGs were physically associated with the membrane protein clusters when they were co-isolated with them in our procedure. Thus the 640-fold enrichment of autologous IgG in the aggregates compared with regions of the membrane devoid of tightly clustered protein suggests that sites of integral protein clustering either are non-specifically sticky to IgG or are viewed as foreign or 'non-self' by the immune system

  2. Opioids and rat erythrocyte deformability.

    PubMed

    Rhoads, D L; Wei, L X; Lin, E T; Rezvani, A; Way, E L

    1986-01-01

    In previous studies from this laboratory, it was noted that opioids in vitro reduced human red blood cell deformability. The effect was found to be dose-dependent, naloxone reversible and preferentially selective kappa ligands exhibited the highest potency. To extend these findings studies were carried out using rat erythrocytes. The time required for erythrocytes to pass through a 5.0 um pore membrane was determined and used as an index of deformability. Opioids added in vitro produced inhibition of deformability in a dose-dependent, naloxone reversible manner. Injecting naive animals with morphine or nalbuphine also produced dose related reductions in red cell deformability. The degree of inhibition produced by nalbuphine correlated well with its plasma concentrations as measured by high performance liquid chromatography (HPLC). Chronic morphine treatment by pellet implantation resulted in the development of tolerance as evidenced by a loss in the ability of morphine in vitro to inhibit red cell deformability. Addition of naloxone resulted in a decrease in filtration time. Thus, the data confirm and extend previous findings on human red blood cells. In as much as previous data from this laboratory demonstrated that opioids inhibit calcium flux from erythrocytes by inhibiting calcium-ATPase and calcium efflux is necessary for normal deformability, it is concluded that opioids act to reduce red cell deformability by inhibition of the calcium pump. PMID:3123933

  3. A dynamic and stationary rheological study of erythrocytes incubated in a glucose medium.

    PubMed

    Riquelme, Bibiana; Foresto, Patricia; D'Arrigo, Mabel; Valverde, Juana; Rasia, Rodolfo

    2005-02-28

    A higher than normal glucose concentration in a suspending medium may produce non-enzymatic glycosylation of erythrocyte proteins. This process can modify the viscoelastic properties of erythrocytes. In this paper, we studied the possible relationship between glucose concentration in a suspending medium and erythrocyte rheological parameters. Human venous blood was obtained from the antecubital veins of 10 healthy volunteers. Blood samples were anticoagulated with EDTA and centrifuged. Red blood cells (RBCs) were washed and subsequently divided in aliquots, which were incubated in vitro with glucose solutions of different concentrations. Dynamic and stationary viscoelastic parameters of RBCs were determined by laser diffractometry in an Erythrodeformeter. Aggregate shape parameter (ASP) of the RBCs was determined by digital image processing. Significant changes were observed both in ASP and in rheological parameters when the glucose concentration in the medium was increased, demonstrating that a glucose concentration as low as 1% induces alterations in the mechanical properties of RBCs. PMID:15680283

  4. Antioxidant activity and protective effect of banana peel against oxidative hemolysis of human erythrocyte at different stages of ripening.

    PubMed

    Sundaram, Shanthy; Anjum, Shadma; Dwivedi, Priyanka; Rai, Gyanendra Kumar

    2011-08-01

    Phytochemicals such as polyphenols and carotenoids are gaining importance because of their contribution to human health and their multiple biological effects such as antioxidant, antimutagenic, anticarcinogenic, and cytoprotective activities and their therapeutic properties. Banana peel is a major by-product in pulp industry and it contains various bioactive compounds like polyphenols, carotenoids, and others. In the present study, effect of ripening, solvent polarity on the content of bioactive compounds of crude banana peel and the protective effect of peel extracts of unripe, ripe, and leaky ripe banana fruit on hydrogen peroxide-induced hemolysis and their antioxidant capacity were investigated. Banana (Musa paradisica) peel at different stages of ripening (unripe, ripe, leaky ripe) were treated with 70% acetone, which were partitioned in order of polarity with water, ethyl acetate, chloroform (CHCl₃), and hexane sequentially. The antioxidant activity of the samples was evaluated by the red cell hemolysis assay, free radical scavenging (1,1-diphenyl-2-picrylhydrazyl free radical elimination) and superoxide dismutase activities. The Folin-Ciocalteu's reagent assay was used to estimate the phenolic content of extracts. The findings of this investigation suggest that the unripe banana peel sample had higher antioxidant potency than ripe and leaky ripe. Further on fractionation, ethyl acetate and water soluble fractions of unripe peel displayed high antioxidant activity than CHCl₃ and hexane fraction, respectively. A positive correlation between free radical scavenging capacity and the content of phenolic compound were found in unripe, ripe, and leaky ripe stages of banana peel. PMID:21369778

  5. Triggering of Suicidal Erythrocyte Death by the Antibiotic Ionophore Nigericin.

    PubMed

    Bissinger, Rosi; Malik, Abaid; Bouguerra, Ghada; Zhou, Yuetao; Singh, Yogesh; Abbès, Salem; Lang, Florian

    2016-05-01

    The K(+) ,H(+) ionophore and antibiotic nigericin has been shown to trigger apoptosis and is thus considered for the treatment of malignancy. Cellular mechanisms involved include induction of oxidative stress, which is known to activate erythrocytic Ca(2+) -permeable unselective cation channels leading to Ca(2+) entry, increase in cytosolic Ca(2+) activity ([Ca(2+) ]i ) and subsequent stimulation of eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. This study explored whether and how nigericin induces eryptosis. Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, [Ca(2+) ]i from Fluo3 fluorescence, pHi from BCECF fluorescence, ceramide abundance utilizing antibodies and reactive oxygen species (ROS) formation from DCFDA-dependent fluorescence. A 48-hr exposure of human erythrocytes to nigericin significantly increased the percentage of annexin-V-binding cells (0.1-10 nM), significantly decreased forward scatter (0.1-1 nM), significantly decreased cytosolic pH (0.1-1 nM) and significantly increased Fluo3 fluorescence (0.1-10 nM). Nigericin (1 nM) slightly, but significantly, increased ROS, but did not significantly modify ceramide abundance. The effect of nigericin on annexin V binding was significantly blunted, but not abolished by removal of extracellular Ca(2+) . The nigericin-induced increase in [Ca(2+) ]i and annexin V binding was again significantly blunted but not abolished by the Na(+) /H(+) exchanger inhibitor cariporide (10 μM). Nigericin triggers eryptosis, an effect paralleled by ROS formation, in part dependent on stimulation of Ca(2+) entry, and involving the cariporide-sensitive Na(+) /H(+) exchanger. PMID:26458067

  6. Mechanism of induction of complement susceptibility of erythrocytes by spider and bacterial sphingomyelinases

    PubMed Central

    Tambourgi, Denise V; De Sousa Da Silva, Marcelo; Billington, Stephen J; Gonçalves De Andrade, Rute M; Magnoli, Fábio C; Songer, J Glenn; Van Den Berg, Carmen W

    2002-01-01

    We have recently shown that the sphingomyelinase toxins P1 and P2 from the venom of the spider Loxosceles intermedia induce complement (C)-dependent lysis of autologous erythrocytes by induction of the cleavage of cell surface glycophorins through activation of an endogenous metalloproteinase facilitating the activation of the alternative pathway of C. Phospholipase D (PLD) from Corynebacterium pseudotuberculosis shows some degree of homology with the spider sphingomyelinases and can induce similar clinical symptoms to those observed after spider envenomation. The aim of this study was to investigate if the bacterial PLD-induced haemolysis of human erythrocytes was C dependent and if cleavage of glycophorins occurred. We show here that haemolysis of both PLD- and P1-treated human erythrocytes was C dependent, but while PLD-mediated haemolysis was dependent on activation of the classical pathway of C, P1 induced lysis via both the classical and alternative pathways. P1, but not PLD, induced cleavage of glycophorins and no change in expression of complement regulators was induced by either of the toxins. In both cases, annexin V binding sites were exposed, suggesting that the membrane asymmetry had been disturbed causing exposure of phosphatidylserine to the cell surface. Our results suggest that C susceptibility induced by L. intermedia and C. pseudotuberculosis PLD is a result of exposure of phosphatidylserine, and the higher potency of P1 toxin can be explained by its additional effect of cleavage of glycophorins. PMID:12225367

  7. Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes.

    PubMed

    Zhu, A; Leng, L; Monahan, C; Zhang, Z; Hurst, R; Lenny, L; Goldstein, J

    1996-03-15

    Alpha-Galactosidase (alpha-GAL) purified from green coffee bean cleaves the terminal galactose residues from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells. Such enzymatically converted red cells have been transfused into group A and O recipients as part of the first phase of FDA-approved clinical trials. Recently we expressed the recombinant alpha-GAL (r)alpha-GAL) in large quantities in a methylotrophic yeast strain Pichia pastoris and purified the protein to apparent homogeneity by chromatography on a macro prep S50 column. Purified (r)alpha-GAL, migrating as a single band of 41 kDa on a SDS-PAGE, appears to be identical to its native counterpart in specific activity (32 U/mg) and kinetic parameters (K(m) =0.363 mM and V(max) = 46.9 U/mg). Both enzymes demonstrate the same pH profile in the pH range from 2 to 9, with an optimal pH at 6.4 when tested with the substrate p-nitrophenol-alpha-D-galactopyranoside. Furthermore, as with its native counterpart, (r)alpha-GAL specifically cleaves alpha-linked terminal galactose residues from group B red cells without affecting other major antigens on the red cell surface. In addition, we developed a method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns for in vivo studies. Thus, with a simple procedure for over-expression and purification of (r)alpha-GAL from P. pastoris culture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells. PMID:8619622

  8. Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes.

    PubMed

    Shimo, Hanae; Arjunan, Satya Nanda Vel; Machiyama, Hiroaki; Nishino, Taiko; Suematsu, Makoto; Fujita, Hideaki; Tomita, Masaru; Takahashi, Koichi

    2015-06-01

    Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs. PMID:26046580

  9. Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

    PubMed Central

    Shimo, Hanae; Arjunan, Satya Nanda Vel; Machiyama, Hiroaki; Nishino, Taiko; Suematsu, Makoto; Fujita, Hideaki; Tomita, Masaru; Takahashi, Koichi

    2015-01-01

    Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs. PMID:26046580

  10. Increased calcium deposits and decreased Ca2+ -ATPase in erythrocytes of ascitic broiler chickens.

    PubMed

    Li, Kai; Zhao, Lihong; Geng, Guangrui; Ma, Liqin; Dong, Shishan; Xu, Tong; Wang, Jianlin; Wang, Huiyu; Tian, Yong; Qiao, Jian

    2011-06-01

    The decrease of erythrocyte deformability may be one of the predisposing factors for pulmonary hypertension and ascites in broiler chickens. In mammals, the cytoplasmic calcium is a major regulator of erythrocyte deformability. In this study, the erythrocyte deformability was measured, and the precise locations of Ca2+ and Ca2+ -ATPase in the erythrocytes were investigated in chickens with ascites syndrome induced by low ambient temperature. The results showed that ascitic broilers had higher filtration index of erythrocyte compared with control groups, indicating a decrease in erythrocyte deformability in ascitic broilers. The more calcium deposits were observed in the erythrocytes of ascitic broilers compared with those of the age-matched control birds. The Ca2+ -ATPase reactive grains were significantly decreased on the erythrocyte membranes of ascitic broilers. Our data suggest that accumulation of intracellular calcium and inhibition of Ca2+ -ATPase might be important factors for the reduced deformability of the erythrocytes of ascitic broilers. PMID:20728193

  11. Detection of anti-H5 responses in human sera by HI using horse erythrocytes following MF59-adjuvanted influenza A/Duck/Singapore/97 vaccine.

    PubMed

    Stephenson, I; Wood, J M; Nicholson, K G; Charlett, A; Zambon, M C

    2004-07-01

    Haemagglutination-inhibition (HI) tests are a simple method used to assess immune responses to influenza haemagglutinin. However, HI tests are insensitive at detection of antibody responses to avian haemagglutinin after vaccination or natural infection, even in the presence of high titres of neutralising antibody or virus isolation. Avian influenza viruses preferentially bind to sialic acid receptors that contain N-acetylneuraminic acid alpha2,3-galactose (alpha2,3Gal) linkages while human viruses preferentially bind to those containing N-acetylneuraminic acid alpha2,6-galactose (alpha2,6Gal) linkages. By using horse erythrocytes in the HI test and thereby increasing the proportion of alpha2,3Gal linkages available for binding, we are able to demonstrate improved detection of antibody to avian H5 in human sera following vaccination with MF59-adjuvanted A/Duck/Singapore/97 surface antigen vaccine. This modified HI test was more sensitive in detection of anti-H5 antibody evoked by revaccination of primed subjects and may be useful in assessing potential avian HA vaccine candidates. PMID:15163495

  12. Comparison of inhibition kinetics of several organophosphates, including some nerve agent surrogates, using human erythrocyte and rat and mouse brain acetylcholinesterase.

    PubMed

    Coban, Alper; Carr, Russell L; Chambers, Howard W; Willeford, Kenneth O; Chambers, Janice E

    2016-04-25

    Because testing of nerve agents is limited to only authorized facilities, our laboratory developed several surrogates that resemble nerve agents because they phosphylate the acetylcholinesterase (AChE) with the same moiety as the actual nerve agents. The inhibition kinetic parameters were determined for AChE by surrogates of cyclosarin (NCMP), sarin (NIMP, PIMP and TIMP) and VX (NEMP and TEMP) and other organophosphorus compounds derived from insecticides. All compounds were tested with rat brain and a subset was tested with mouse brain and purified human erythrocyte AChE. Within the compounds tested on all AChE sources, chlorpyrifos-oxon had the highest molecular rate constant followed by NCMP and NEMP. This was followed by NIMP then paraoxon and DFP with rat and mouse brain AChE but DFP was a more potent inhibitor than NIMP and paraoxon with human AChE. With the additional compounds tested only in rat brain, TEMP was slightly less potent than NEMP but more potent than PIMP which was more potent than NIMP. Methyl paraoxon was slightly less potent than paraoxon but more potent than TIMP which was more potent than DFP. Overall, this study validates that the pattern of inhibitory potencies of our surrogates is comparable to the pattern of inhibitory potencies of actual nerve agents (i.e., cyclosarin>VX>sarin), and that these are more potent than insecticidal organophosphates. PMID:26965078

  13. Data in the activities of caspases and the levels of reactive oxygen species and cytochrome c in the •OH-induced fish erythrocytes treated with alanine, citrulline, proline and their combination

    PubMed Central

    Li, Huatao; Jiang, Weidan; Liu, Yang; Jiang, Jun; Zhang, Yongan; Wu, Pei; Zhao, Juan; Duan, Xudong; Zhou, Xiaoqiu; Feng, Lin

    2016-01-01

    The present study explored the effects of alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala10Pro4Cit1) on the activities of caspases and levels of reactive oxygen species (ROS) and cytochrome c in hydroxyl radicals (•OH)-induced carp erythrocytes. The data displayed that •OH induced the increases in the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c in carp erythrocytes. However, Ala, Cit, Pro and Ala10Pro4Cit1 effectively suppressed the •OH-induced increases in the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c in carp erythrocytes. Furthermore, the activities of caspase−3, caspase−8 and caspase−9 and the levels of ROS and cytochrome c were gradually decreased with increasing concentrations of Ala, Cit, Pro and Ala10Pro4Cit1 (0.175−1.400 mM) in the •OH-induced carp erythrocytes. These data demonstrated that the 50% inhibitory doses (ID50) of Ala10Pro4Cit1 on the activities of caspase−8, caspase−9 and caspase−3 and levels of ROS and cytochrome c were respectively estimated to be the minimum values among amino acids examined so far. The 5% inhibitory doses (ID5) of Ala, Cit, Pro and Ala10Pro4Cit1 on the activities of caspase−8, caspase−9 and caspase−3 and levels of ROS and cytochrome c were estimated to be at their physiological concentrations in mammalian. Our research article for further interpretation and discussion from these data in Li et al. (2016) [1]. PMID:26952131

  14. Data in the activities of caspases and the levels of reactive oxygen species and cytochrome c in the •OH-induced fish erythrocytes treated with alanine, citrulline, proline and their combination.

    PubMed

    Li, Huatao; Jiang, Weidan; Liu, Yang; Jiang, Jun; Zhang, Yongan; Wu, Pei; Zhao, Juan; Duan, Xudong; Zhou, Xiaoqiu; Feng, Lin

    2016-06-01

    The present study explored the effects of alanine (Ala), citrulline (Cit), proline (Pro) and their combination (Ala10Pro4Cit1) on the activities of caspases and levels of reactive oxygen species (ROS) and cytochrome c in hydroxyl radicals (•OH)-induced carp erythrocytes. The data displayed that •OH induced the increases in the activities of caspase-3, caspase-8 and caspase-9 and the levels of ROS and cytochrome c in carp erythrocytes. However, Ala, Cit, Pro and Ala10Pro4Cit1 effectively suppressed the •OH-induced increases in the activities of caspase-3, caspase-8 and caspase-9 and the levels of ROS and cytochrome c in carp erythrocytes. Furthermore, the activities of caspase-3, caspase-8 and caspase-9 and the levels of ROS and cytochrome c were gradually decreased with increasing concentrations of Ala, Cit, Pro and Ala10Pro4Cit1 (0.175-1.400 mM) in the •OH-induced carp erythrocytes. These data demonstrated that the 50% inhibitory doses (ID50) of Ala10Pro4Cit1 on the activities of caspase-8, caspase-9 and caspase-3 and levels of ROS and cytochrome c were respectively estimated to be the minimum values among amino acids examined so far. The 5% inhibitory doses (ID5) of Ala, Cit, Pro and Ala10Pro4Cit1 on the activities of caspase-8, caspase-9 and caspase-3 and levels of ROS and cytochrome c were estimated to be at their physiological concentrations in mammalian. Our research article for further interpretation and discussion from these data in Li et al. (2016) [1]. PMID:26952131

  15. [Detection of stimulator-induced cytotoxicity of human mononuclear cells in short-term culture].

    PubMed

    von Baehr, R; Timm, M; Fröbe, I

    1983-01-01

    A new test modification for the detection of stimulant-induced cytotoxicity of human mononuclear blood cells is described. Papain-treated human erythrocytes were used as indicator cells. The effector cells were lymphocytes. The ratio of target cells to effector cells was 10/1. Haemoglobin as a marker of lysis of the erythrocytes, released in the supernatant, was measured quantitatively in form of its pseudoperoxidase-activity. PHA, ConA and tannic acid were ascertained and tested as stimulants of cytotoxicity. The reaction was inhibitable by anti-human-lymphocyte-globulin. The test conditions were optimized in regard to incubation time, -temperature, -vessels, culture medium and target cells. The technique is easy to manipulate, has only slight pretensions to the equipment of the laboratory and appears to be very effective. We recommend to apply this method of stimulant-induced cytotoxicity within the detection of the immune state, especially in the progress of immunopathological diseases and the analysis of efficiency of immunosuppressive therapy. PMID:6224413

  16. Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress.

    PubMed

    Ingrosso, D; D'angelo, S; di Carlo, E; Perna, A F; Zappia, V; Galletti, P

    2000-07-01

    Protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT; EC 2. 1.1.77) catalyses the methyl esterification of the free alpha-carboxyl group of abnormal L-isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age-damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of L-isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t-butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane-cytoskeleton network stability. We conclude that the increased formation/exposure of L-isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl

  17. Antioxidant-mediated protective effect of potato peel extract in erythrocytes against oxidative damage.

    PubMed

    Singh, Nandita; Rajini, P S

    2008-05-28

    Potato peels are waste by-product of the potato processing industry. They are reportedly rich in polyphenols. Our earlier studies have shown that extracts derived from potato peel (PPE) possess strong antioxidant activity in chemical and biological model systems in vitro, attributable to its polyphenolic content. The main objective of this study was to investigate the ability of PPE to protect erythrocytes against oxidative damage, in vitro. The protection rendered by PPE in erythrocytes was studied in terms of resistance to oxidative damage, morphological alterations as well as membrane structural alterations. The total polyphenolic content in PPE was found to be 3.93 mg/g powder. The major phenolic acids present in PPE were predominantly: gallic acid, caffeic acid, chlorogenic acid and protocatechuic acid. We chose the experimental prooxidant system: FeSO(4) and ascorbic acid to induce lipid peroxidation in rat RBCs and human RBC membranes. PPE was found to inhibit lipid peroxidation with similar effectiveness in both the systems (about 80-85% inhibition by PPE at 2.5 mg/ml). While PPE per se did not cause any morphological alteration in the erythrocytes, under the experimental conditions, PPE significantly inhibited the H(2)O(2)-induced morphological alterations in rat RBCs as revealed by scanning electron microscopy. Further, PPE was found to offer significant protection to human erythrocyte membrane proteins from oxidative damage induced by ferrous-ascorbate. In conclusion, our results indicate that PPE is capable of protecting erythrocytes against oxidative damage probably by acting as a strong antioxidant. PMID:18452909

  18. Physicochemical Aspects of the Plasmodium chabaudi-Infected Erythrocyte

    PubMed Central

    Hayakawa, Eri H.; Kobayashi, Seiki; Matsuoka, Hiroyuki

    2015-01-01

    Membrane electrochemical potential is a feature of the molecular profile of the cell membrane and the two-dimensional arrangement of its charge-bearing molecules. Plasmodium species, the causative agents of malaria, are intracellular parasites that remodel host erythrocytes by expressing their own proteins on erythrocyte membranes. Although various aspects of the modifications made to the host erythrocyte membrane have been extensively studied in some human Plasmodium species (such as Plasmodium falciparum), details of the structural and molecular biological modifications made to host erythrocytes by nonhuman Plasmodium parasites have not been studied. We employed zeta potential analysis of erythrocytes parasitized by P. chabaudi, a nonhuman Plasmodium parasite. From these measurements, we found that the surface potential shift was more negative for P. chabaudi-infected erythrocytes than for P. falciparum-infected erythrocytes. However, electron microscopic analysis of the surface of P. chabaudi-infected erythrocytes did not reveal any modifications as compared with nonparasitized erythrocytes. These results suggest that differences in the membrane modifications found herein represent unique attributes related to the pathogenesis profiles of the two different malaria parasite species in different host animals and that these features have been acquired through parasite adaptations acquired over long evolutionary time periods. PMID:26557685

  19. Erythrocyte binding preference of avian influenza H5N1 viruses.

    PubMed

    Louisirirotchanakul, Suda; Lerdsamran, Hatairat; Wiriyarat, Witthawat; Sangsiriwut, Kantima; Chaichoune, Kridsda; Pooruk, Phisanu; Songserm, Taweesak; Kitphati, Rungrueng; Sawanpanyalert, Pathom; Komoltri, Chulaluk; Auewarakul, Prasert; Puthavathana, Pilaipan

    2007-07-01

    Five erythrocyte species (horse, goose, chicken, guinea pig, and human) were used to agglutinate avian influenza H5N1 viruses by hemagglutination assay and to detect specific antibody by hemagglutination inhibition test. We found that goose erythrocytes confer a greater advantage over other erythrocyte species in both assays. PMID:17522271

  20. The fusion of erythrocytes by fatty acids, esters, retinol and alpha-tocopherol.

    PubMed

    Ahkong, Q F; Fisher, D; Tampion, W; Lucy, J A

    1973-09-01

    1. The ability of a number of carboxylic acids, their esters, retinol and alpha-tocopherol to induce fusion of hen erythrocytes in vitro was investigated. 2. Some 30 different fat-soluble substances (100mug/ml) were found to cause the formation of multinucleated erythrocytes with a suspension of 3x10(8) erythrocytes/ml. The most effective agents induced fusion within 5-10min at 37 degrees C; some substances required about 1h. 3. Inclusion of Dextran 60C in the test medium minimized colloid osmotic lysis caused by exogenous lipids that induce cell fusion. 4. Cell swelling, followed by cell adhesion, was then seen to precede cell fusion. 5. Fusion occurred with C(10)-C(14) saturated carboxylic acids, with unsaturated, longer-chain carboxylic acids and their mono-esters; retinol, and to a lesser extent alpha-tocopherol, also caused cell fusion. 6. C(6)-C(9), C(15), C(16) and C(18) saturated carboxylic acids did not induce fusion within 4h; glyceryl dioleate was only weakly active, and glyceryl trioleate was inactive in the test system. 7. Fusion was facilitated by a high ratio of chemical agents to cell number and by incubation between pH5 and 6. It was inhibited by EDTA and by serum albumin. 8. Glyceryl mono-oleate caused both a similar fusion of several species of mammalian erythrocyte and the interspecific fusion of human and chicken erythrocytes. 9. The term ;fusogenic' is proposed to describe chemical, viral and physical agents that cause membranes to fuse. 10. The biochemical mechanisms involved and the possible biological significance of membrane fusion by fusogenic lipids are discussed. PMID:4204034

  1. Protective Effect of Quercetin on Oxidative Stress in Glucose-6-Phosphate Dehydrogenase-Deficient Erythrocytes in Vitro

    PubMed Central

    Jamshidzadeh, Akram; Rezaeian Mehrabadi, Abbas

    2010-01-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficient subjects are vulnerable to oxidative stress. Quercetin, a flavonoids, has been employed as a potent oxygen-free radical scavenger in order to assess the protective effects of quercetin against H2O2-induced oxidative damage in G6PD-deficient and normal human erythrocytes. Erythrocytes of G6PD-deficient (n = 10) and normal (n = 10) subjects were incubated with different concentrations of quercetin. The produced thiobarbituric acid reactive substance (TBARS) and glutathione (GSH) level of erythrocytes were then subsequently measured. Different concentrations of quercetin showed no significant hemolysis, compared with the phosphate buffer solution. Upon challenge with H2O2, there was a significant (p < 0.005) decrease in GSH and an increase in TBARS level in G6PD-deficient erythrocytes. With quercetin, it managed to preserve concentrations of 15 to 75 mM preserved GSH and TBARS levels of normal and G6PD-deficient erythrocytes against H2O2-induced oxidative damage. In addition to its well-established antioxidant effects, quercetin was also found to have cytoprotective properties. PMID:24363724

  2. Trilinolein improves erythrocyte deformability during cardiopulmonary bypass.

    PubMed Central

    Tsai, S K; Chan, P; Lee, T Y; Yung, J M; Hong, C Y

    1994-01-01

    The in vitro effect of trilinolein, a triglyceride with linoleic acid as the major fatty acid residue in the esterified positions of glycerol, on erythrocyte deformability was studied in blood samples collected from 12 patients before and after cardiopulmonary bypass (CPB). Erythrocyte deformability was measured with a filtration method and expressed as red cell filtration rate (RFR). RFR was reduced after CPB and the reduction was time dependent. Trilinolein at a concentration of 10(-7) M significantly reversed the CPB-induced reduction of RFR when it was mixed with blood samples collected 30, 60 and 90 min from the start of CPB. This study confirmed the effect of CPB on erythrocyte deformability and showed that this damage could be significantly improved by mixing blood with trilinolein. PMID:8054252

  3. Influence of the ionophore A23187 on the plastic behavior of normal erythrocytes.

    PubMed

    Kuettner, J F; Dreher, K L; Rao, G H; Eaton, J W; Blackshear, P L; White, J G

    1977-07-01

    Previous studies have demonstrated that A23187, an ionophore which selectively transports divalent cations across cell membranes, has profound effects on human erythrocytes: it causes red cells to take up calcium; lose potassium, water, and ATP; convert from biconcave discs to echinocytes and spheroechinocytes; and become more rigid. The present study has explored the influence of calcium uptake induced by the ionophore on the behavior of individual erythrocyte membranes by the micropipette aspiration technique. Exposure of erythrocytes to calcium and A23187 for intervals of up to 30 minutes resulted in marked changes in membrane viscoelastic properties, including the development of increased resistance to aspiration. The most striking manifestation of altered membrane mechanics was apparent after 10 minutes on incubation. Cells pulled into the pipette for a few seconds and the extruded back into the medium retained the deformity imposed by the pipette for several seconds to a few minutes before regaining the form they manifested prior to initial aspiration. The calcium-induced changes in erythrocyte behavior observed in this study strongly support the concept that extrinsic proteins located inside the membrane provide mechanical support to the cell wall, and that increased levels of calcium cause precipitation or cross-linking of the proteins responsible for the increased resistence to deformation and recoil observed after aspiration into micropipettes. PMID:327824

  4. A Boronate Affinity-Assisted SERS Tag Equipped with a Sandwich System for Detection of Glycated Hemoglobin in the Hemolysate of Human Erythrocytes.

    PubMed

    Usta, Duygu Deniz; Salimi, Kouroush; Pinar, Asli; Coban, İlknur; Tekinay, Turgay; Tuncel, Ali

    2016-05-18

    Phenylboronic acid-functionalized, Ag shell-coated, magnetic, monodisperse polymethacrylate microspheres equipped with a glycoprotein-sensitive sandwich system were proposed as a surface-enhanced Raman scattering (SERS) substrate for quantitative determination of glycated hemoglobin (HbA1c). The magnetization of the SERS tag and the formation of the Ag shell on the magnetic support were achieved using the bifunctional reactivity of newly synthesized polymethacrylate microspheres. The hemolysate of human red blood cells containing both HbA1c and nonglycated hemoglobin was used for determination of HbA1c. The working principle of the proposed SERS tag is based on the immobilization of HbA1c by cyclic boronate ester formation between glycosyl residues of HbA1c and boronic acid groups of magnetic polymethacrylate microspheres and the binding of p-aminothiophenol (PATP)-functionalized Ag nanoparticles (Ag NPs) carrying another boronic acid ligand via cyclic boronate ester formation via unused glycosyl groups of bound HbA1c. Then, in situ formation of a Raman reporter, 4,4'-dimercaptoazobenzene from PATP under 785 nm laser irradiation allowed for the quantification of HbA1c bound onto the magnetic SERS tag, which was proportional to the HbA1c concentration in the hemolysate of human erythrocytes. The sandwich system provided a significant enhancement in the SERS signal intensity due to the plasmon coupling between Ag NPs and Ag shell-coated magnetic microspheres, and low HbA1c concentrations down to 50 ng/mL could be detected. The calibration curve obtained with a high correlation coefficient between the SERS signal intensity and HbA1c level showed the usability of the SERS protocol for the determination of the HbA1c level in any person. PMID:27149109

  5. Full-length CD4 electroinserted in the erythrocyte membrane as a long-lived inhibitor of infection by human immunodeficiency virus

    SciTech Connect

    Zeira, M.; Volsky, D.J. ); Tosi, P.F.; Mouneimne, Y.; Lazarte, J.; Sneed, L.; Nicolau, C. )

    1991-05-15

    Recombinant full-length CD4 expressed in Spodoptera frugiperda 9 cells with the baculovirus system was electroinserted in erythrocyte (RBC) membranes. Of the inserted CD4, 70% was correctly oriented as shown by fluorescence quenching experiments with fluorescein-labeled CD4. The inserted CD4 displayed the same epitopes as the naturally occurring CD4 in human T4 cells. Double-labeling experiments ({sup 125}I-CD4 and {sup 51}Cr-RBC) showed that the half-life of CD4 electroinserted in RBC membrane in rabbits was approximately 7 days. Using the fluorescence dequenching technique with octadecylrhodamine B-labeled human immunodeficiency virus (HIV)-1, the authors showed fusion of the HIV envelope with the plasma membrane of RBC-CD4, whereas no such fusion could be detected with RBC. The dequenching efficiency of RBC-CD4 is the same as that of CEM cells. Exposure to anti-CD4 monoclonal antibody OKT4A, which binds to the CD4 region that attaches to envelope glycoprotein gp120, caused a significant decrease in the dequenching of fluorescence. In vitro infectivity studies showed that preincubation of HIV-1 with RBC-CD4 reduced by 80-90% the appearance of HIV antigens in target cells, the amount of viral reverse transcriptase, and the amount of p24 core antigen produced by the target cells. RBC-CD4, but not RBCs, aggregated with chronically HIV-1-infected T cells and caused formation of giant cells. These data show that the RBC-CD4 reagent is relatively long lived in circulation and efficient in attaching to HIV-1 and HIV-infected cells, and thus it may have value as a therapeutic agent against AIDS.

  6. P2X Receptor-Dependent Erythrocyte Damage by α-Hemolysin from Escherichia coli Triggers Phagocytosis by THP-1 Cells

    PubMed Central

    Fagerberg, Steen K.; Skals, Marianne; Leipziger, Jens; Praetorius, Helle A.

    2013-01-01

    The pore-forming exotoxin α-hemolysin from E. coli causes a significant volume reduction of human erythrocytes that precedes the ultimate swelling and lysis. This shrinkage results from activation of Ca2+-sensitive K+ (KCa3.1) and Cl− channels (TMEM16A) and reduced functions of either of these channels potentiate the HlyA-induced hemolysis. This means that Ca2+-dependent activation of KCa3.1 and TMEM16A protects the cells against early hemolysis. Simultaneous to the HlyA-induced shrinkage, the erythrocytes show increased exposure of phosphatidylserine (PS) in the outer plasma membrane leaflet, which is known to be a keen trigger for phagocytosis. We hypothesize that exposure to HlyA elicits removal of the damaged erythrocytes by phagocytic cells. Cultured THP-1 cells as a model for erythrocytal phagocytosis was verified by a variety of methods, including live cell imaging. We consistently found the HlyA to very potently trigger phagocytosis of erythrocytes by THP-1 cells. The HlyA-induced phagocytosis was prevented by inhibition of KCa3.1, which is known to reduce PS-exposure in human erythrocytes subjected to both ionomycin and HlyA. Moreover, we show that P2X receptor inhibition, which prevents the cell damages caused by HlyA, also reduced that HlyA-induced PS-exposure and phagocytosis. Based on these results, we propose that erythrocytes, damaged by HlyA-insertion, are effectively cleared from the blood stream. This mechanism will potentially reduce the risk of intravascular hemolysis. PMID:23462688

  7. Defining the Erythrocyte Binding Domains of Plasmodium vivax Tryptophan Rich Antigen 33.5

    PubMed Central

    Bora, Hema; Tyagi, Rupesh Kumar; Sharma, Yagya Dutta

    2013-01-01

    Tryptophan-rich antigens play important role in host-parasite interaction. One of the Plasmodium vivax tryptophan-rich antigens called PvTRAg33.5 had earlier been shown to be predominantly of alpha helical in nature with multidomain structure, induced immune responses in humans, binds to host erythrocytes, and its sequence is highly conserved in the parasite population. In the present study, we divided this protein into three different parts i.e. N-terminal (amino acid position 24–106), middle (amino acid position 107–192), and C-terminal region (amino acid position 185–275) and determined the erythrocyte binding activity of these fragments. This binding activity was retained by the middle and C-terminal fragments covering 107 to 275 amino acid region of the PvTRAg33.5 protein. Eight non-overlapping peptides covering this 107 to 275 amino acid region were then synthesized and tested for their erythrocyte binding activity to further define the binding domains. Only two peptides, peptide P4 (at 171–191 amino acid position) and peptide P8 (at 255–275 amino acid position), were found to contain the erythrocyte binding activity. Competition assay revealed that each peptide recognizes its own erythrocyte receptor. These two peptides were found to be located on two parallel helices at one end of the protein in the modelled structure and could be exposed on its surface to form a suitable site for protein-protein interaction. Natural antibodies present in the sera of the P. vivax exposed individuals or the polyclonal rabbit antibodies against this protein were able to inhibit the erythrocyte binding activity of PvTRAg33.5, its fragments, and these two synthetic peptides P4 and P8. Further studies on receptor-ligand interaction might lead to the development of the therapeutic reagent. PMID:23638151

  8. Oxidative Hemolysis of Erythrocytes

    ERIC Educational Resources Information Center

    Wlodek, Lidia; Kusior, Dorota

    2006-01-01

    This exercise for students will allow them to simultaneously observe lipid peroxidation and consequent hemolysis of rat erythrocytes and the effect of sodium azide, a catalase inhibitor, on these processes. It will also demonstrate a protective action of antioxidants, the therapeutically used N-acetylcysteine and albumins present in plasma.

  9. INCREASED ERYTHROCYTE C4D IS ASSOCIATED WITH KNOWN ALLOANTIBODY AND AUTOANTIBODY MARKERS OF ANTIBODY MEDIATED REJECTION IN HUMAN LUNG TRANSPLANT RECIPIENTS

    PubMed Central

    Golocheikine, Angali; Nath, Dilip S.; Basha, Haseeb Ilias; Saini, Deepti; Phelan, Donna; Aloush, Aviva; Trulock, Elbert P.; Hachem, Ramsey R.; Patterson, G.Alexander; Ahearn, Joseph M.; Mohanakumar, Thalachallour

    2009-01-01

    Background Immune responses to mismatched donor HLA antigens play a significant role in the pathogenesis of chronic rejection. The study objective was to evaluate whether erythrocyte bound C4d (E-C4d) is associated with known alloimmune and autoimmune markers of antibody mediated rejection (AMR) following human lung transplantation (LTx). Methods 22 LTx recipients and 15 normal subjects were analyzed for E-C4d using flow cytometry. Development of antibodies (Abs) to donor mismatched HLA (DSA) and Abs to HLA were determined using solid phase method by Luminex. Development of Abs to self-antigens, K-alpha-1-tubulin (KA1T) and collagen V (Col-V) were measured by ELISA. C3d deposition in lung biopsies was determined by immunohistochemical staining. Results Percent E-C4d (%E-C4d) levels in LTx patients were higher compared to normal subjects (19.9% vs. 3.7%, p = 0.02). DSA+ patients had higher E-C4d levels compared to DSA- patients (34.1% vs. 16.7%, p = 0.02). In 5 patients with preformed anti-HLA, E-C4d levels were not significantly different compared to 13 patients with no detectable anti-HLA (p=0.1). Higher E-C4d levels were noted in patients who developed Abs to KA1T (p = 0.02) and Col-V (p = 0.03). Recipients with C3d tissue deposition had higher E-C4d levels compared to patients with C3d negative biopsy results (p = 0.01). Conclusions Increased % E-C4d levels are found in patients with positive DSA, high Abs titers to KA1T and Col-V, and have C3d positive lung biopsy findings. Therefore, % E-C4d can serve as a potential marker for AMR following LTx. PMID:20022265

  10. [Immune response to sheep erythrocytes fixed in formaldehyde].

    PubMed

    Kostadinov, D

    1978-01-01

    Treatment of sheep erythrocytes with 1.5% of formaldehyde alter their immunogenic properties. On the background of immune response to nonfixed erythrocytes, the performed studies showed the following peculiarities in the reaction of mice to fixed erythrocytes: 1) the primary response of animals, in ected with fixed erythrocytes, was rather weak after its determination by the number of rosette forming cells (RFC), antibody forming cells (AFC) in the spleen and by the of hemagglutinins (HA) in serum; 2) in contrast to the primary response the fixed erythrocytes induced a good secondary response, which was considerably higher/according to the number of RFC and titre of HA/in mice sensibilized by nonfixed erytrocytes in advance than in those presensibilized by fixed cells. Therefore the fixed erythrocytes were weaker inducers of immunologic memory than the nonfixed under equal other conditions. 3) In contrast to the nonfixed the fixed erythrocytes did not induce the formation of large amounts of direct AFC during the secondary response as well even then when the animals were presensibilized by nonfixed form of the antigen. PMID:77759

  11. Effect of solution non-ideality on erythrocyte volume regulation.

    PubMed

    Levin, R L; Cravalho, E G; Huggins, C E

    1977-03-01

    A non-ideal, hydrated, non-dilute pseudo-binary salt-protein-water solution model of the erythrocyte intracellular solution is presented to describe the osmotic behavior of human erythrocytes. Existing experimental activity data for salts and proteins in aqueous solutions are used to formulate van Laar type expressions for the solvent and solute activity coefficients. Reasonable estimates can therefore be made of the non-ideality of the erythrocyte intracellular solution over a wide range of osmolalities. Solution non-ideality is shown to affect significantly the degree of solute polarization within the erythrocyte intracellular solution during freezing. However, the non-ideality has very little effect upon the amount of water retained within erythrocytes cooled at sub-zero temperatures. PMID:16250333

  12. Changes of lipofuscin-like pigments in erythrocytes and spleen after whole-body gamma irradiation of rats

    SciTech Connect

    Wilhelm, J.; Brzak, P.; Rejholcova, M. )

    1989-11-01

    Whole-body gamma irradiation of rats induced the formation of lipofuscin-like pigments in erythrocytes. Erythrocytes that were damaged by oxidation were scavenged in the spleen, and lipofuscin-like pigments were transferred from erythrocytes to the spleen during this process. The time course of lipofuscin-like pigments in erythrocytes and spleen indicates that the pigments were not induced by the action of free radicals produced by ionizing radiation but rather were a sequela of postirradiation metabolic changes.

  13. Brucella melitensis Invades Murine Erythrocytes during Infection

    PubMed Central

    Vitry, Marie-Alice; Hanot Mambres, Delphine; Deghelt, Michaël; Hack, Katrin; Machelart, Arnaud; Lhomme, Frédéric; Vanderwinden, Jean-Marie; Vermeersch, Marjorie; De Trez, Carl; Pérez-Morga, David; Letesson, Jean-Jacques

    2014-01-01

    Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature. PMID:25001604

  14. Brucella melitensis invades murine erythrocytes during infection.

    PubMed

    Vitry, Marie-Alice; Hanot Mambres, Delphine; Deghelt, Michaël; Hack, Katrin; Machelart, Arnaud; Lhomme, Frédéric; Vanderwinden, Jean-Marie; Vermeersch, Marjorie; De Trez, Carl; Pérez-Morga, David; Letesson, Jean-Jacques; Muraille, Eric

    2014-09-01

    Brucella spp. are facultative intracellular Gram-negative coccobacilli responsible for brucellosis, a worldwide zoonosis. We observed that Brucella melitensis is able to persist for several weeks in the blood of intraperitoneally infected mice and that transferred blood at any time point tested is able to induce infection in naive recipient mice. Bacterial persistence in the blood is dramatically impaired by specific antibodies induced following Brucella vaccination. In contrast to Bartonella, the type IV secretion system and flagellar expression are not critically required for the persistence of Brucella in blood. ImageStream analysis of blood cells showed that following a brief extracellular phase, Brucella is associated mainly with the erythrocytes. Examination by confocal microscopy and transmission electron microscopy formally demonstrated that B. melitensis is able to invade erythrocytes in vivo. The bacteria do not seem to multiply in erythrocytes and are found free in the cytoplasm. Our results open up new areas for investigation and should serve in the development of novel strategies for the treatment or prophylaxis of brucellosis. Invasion of erythrocytes could potentially protect the bacterial cells from the host's immune response and hamper antibiotic treatment and suggests possible Brucella transmission by bloodsucking insects in nature. PMID:25001604

  15. Contributions of unfrozen fraction and of salt concentration to the survival of slowly frozen human erythrocytes: influence of warming rate

    SciTech Connect

    Mazur, P.; Rigopoulos, N.

    1983-01-01

    The general belief is that slow freezing injury is either the result of exposure to high salt concentrations or the result of excessive cell shrinkage. Ordinarily, salt concentration and the amount of liquid in the unfrozen channels are reciprocally related; but they can be separated within limits by varying the total concentration of solutes in the suspending medium while holding the mass ratio of additive to salt constant, and by then slowly freezing samples to various subzero temperatures. The authors have recently reported that when human red cells are frozen under these conditions and thawed rapidly, survival is more dependent on the unfrozen water fraction than it is on the salt concentration in that fraction. The present work compares these results with those obtained with slow thawing. While the general conclusion remains unaltered, slowly thawed cells were able to survive the freezing of a higher fraction of extracellular water than were rapidly thawed cells. Calculations were made of the changes in cell volume during the equilibration with glycerol and the subsequent freezing involved in these experiments.

  16. Cation transport in oxidant-stressed human erythrocytes: heightened N-ethylmaleimide activation of passive K+ influx after mild peroxidation.

    PubMed

    Sheerin, H E; Snyder, L M; Fairbanks, G

    1989-07-24

    Normal and chronically dehydrated (hereditary xerocytosis) human red cells were subjected to mild peroxidative treatment (315 microM hydrogen peroxide (H2O2), 15 min) in the presence of azide. The subsequent expression of passive (ouabain-resistant) K+ transport activities was analyzed by measurement of 86Rb+ influx. Peroxidation of normal red cells did not affect basal K+ transport activity, but the increment in K+ influx elicited by 0.5 mM N-ethylmaleimide (NEM) was increased 3-fold. The enhanced K+ influx was chloride-dependent, but only partially inhibited by 0.1 mM furosemide. Stimulated activity declined progressively after NEM activation, but could be restored by a second NEM treatment. Prior conversion of hemoglobin to the carbonmonoxy form abolished the response to peroxide, while 200 microM butylated hydroxytoluene (BHT) exerted only partial inhibition, suggesting that the effect of H2O2 requires interaction of activated, unstable hemoglobin species with the membrane, but that lipid peroxidation is not sufficient. Peroxidation following NEM treatment also enhanced NEM activation, indicating that enhancement does not require altered NEM reactions with stimulatory or inhibitory sites. Passive K+ transport in hereditary xerocytosis red cells was not activated by NEM, with or without H2O2 pretreatment. The results demonstrate that modest peroxidative damage to red cells can heighten the activation of a transport system that is thought to be capable of mediating net K+ efflux and volume reduction in cells that express it. Models are proposed in which the effects of NEM, H2O2, cell swelling and other factors are mediated by conformational changes in a postulated subpopulation of anion channel (Band 3) molecules that bind the K+ transporter. PMID:2758051

  17. P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes

    PubMed Central

    Valton, Emeline; Amblard, Christian; Wawrzyniak, Ivan; Penault-Llorca, Frederique; Bamdad, Mahchid

    2013-01-01

    Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous “membrane detoxification proteins” implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality. PMID:24305632

  18. Attachment of killed Mycoplasma gallisepticum cells and membranes to erythrocytes

    SciTech Connect

    Banai, M.; Kahane, I.; Feldner, J.; Razin, S.

    1981-11-01

    To correlate viability with attachment capacity, Mycoplasma gallisepticum cells harvested at different growth phases and treated by various agents were tested for their capacity to attach to human erythrocytes. The results show that viability per se is not essential for M. gallisepticum attachment to erythrocytes, as cells killed by ultraviolet irradiation and membranes isolated by lysing M. gallisepticum cells by various means retained attachment capacity. However, treatment of the mycoplasmas by protein-denaturing agents, such as heart, glutaraldehyde, or prolonged exposure to low pH, drastically affected or even abolished attachment, supporting the protein nature of the mycoplasma membrane components responsible for specific binding to the sialoglycoprotein receptors on the erythrocytes.

  19. Electrophoretic mobilities of erythrocytes in various buffers

    NASA Technical Reports Server (NTRS)

    Plank, L. D.; Kunze, M. E.; Todd, P. W.

    1985-01-01

    The calibration of space flight equipment depends on a source of standard test particles, this test particle of choice is the fixed erythrocyte. Erythrocytes from different species have different electrophoretic mobilities. Electrophoretic mobility depends upon zeta potential, which, in turn depends upon ionic strength. Zeta potential decreases with increasing ionic strength, so cells have high electrophoretic mobility in space electrophoresis buffers than in typical physiological buffers. The electrophoretic mobilities of fixed human, rat, and rabbit erythrocytes in 0.145 M salt and buffers of varying ionic strength, temperature, and composition, to assess the effects of some of the unique combinations used in space buffers were characterized. Several effects were assessed: glycerol or DMSO (dimethylsulfoxide) were considered for use as cryoprotectants. The effect of these substances on erythrocyte electrophoretic mobility was examined. The choice of buffer depended upon cell mobility. Primary experiments with kidney cells established the choice of buffer and cryoprotectant. A nonstandard temperature of EPM in the suitable buffer was determined. A loss of ionic strength control occurs in the course of preparing columns for flight, the effects of small increases in ionic strength over the expected low values need to be evaluated.

  20. Erythrocyte survival in sheep exposed to ozone

    SciTech Connect

    Moore, G.S.; Calabrese, E.J.; Labato, F.J.

    1981-07-01

    Erythrocyte survival studies in the Dorset ewe using chromium 51 were performed. The purpose of the study was to determine if ozone exposure produces decreased cell survival which may be the result of premature erythrocyte aging. This strain of sheep has an erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity that is very low, being comparable to human A-variants with G6PD deficiency. Ozone exposure may produce hemolytic effects in G6PD deficients more readily than in erythrocytes with normal activity. A decrease in hematocrit was observed in the ozone exposed groups. With respect to red cell destruction, ozone does not appear to act immediately, but rather there appears to be a delayed effect. At 0.25 ppM ozone, the group reached the 50% remaining level an average of 1 day sooner than the control group. There was no significant difference between control and exposed groups at the 0.50 ppM and 0.70 ppM levels. Also, the results demonstrate a net decrease in hematocrit which is greater for 0.25 ppM ozone than any other exposure level. (RJC)

  1. Adhesion of Annexin 7 Deficient Erythrocytes to Endothelial Cells

    PubMed Central

    Abed, Majed; Balasaheb, Siraskar; Towhid, Syeda Tasneem; Daniel, Christoph; Amann, Kerstin; Lang, Florian

    2013-01-01

    Annexin 7 deficiency has previously been shown to foster suicidal death of erythrocytes or eryptosis, which is triggered by increase of intracellular Ca2+ concentration ([Ca2+]i) and characterized by cell shrinkage and cell membrane scrambling with subsequent phosphatidylserine exposure at the cell surface. Eryptosis following increase of [Ca2+]i by Ca2+ ionophore ionomycin, osmotic shock or energy depletion was more pronounced in erythrocytes from annexinA7-deficient mice (anxA7−/−) than in erythrocytes from wild type mice (anxA7+/+). As phosphatidylserine exposure is considered to mediate adhesion of erythrocytes to the vascular wall, the present study explored adhesion of erythrocytes from anx7−/− and anx7+/+-mice following increase of [Ca2+]i by Ca2+ ionophore ionomycin (1 µM for 30 min), hyperosmotic shock (addition of 550 mM sucrose for 2 hours) or energy depletion (removal of glucose for 12 hours). Phosphatidylserine exposing erythrocytes were identified by annexin V binding, cell volume estimated from forward scatter in FACS analysis and adhesion to human umbilical vein endothelial cells (HUVEC) utilizing a flow chamber. As a result, ionomycin, sucrose addition and glucose removal all triggered phosphatidylserine-exposure, decreased forward scatter and enhanced adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC), effects significantly more pronounced in anx7−/− than in anx7+/+-erythrocytes. Following ischemia, morphological renal injury was significantly higher in anx7−/− than in anx7+/+-mice. The present observations demonstrate that enhanced eryptosis of annexin7 deficient cells is paralleled by increased adhesion of erythrocytes to the vascular wall, an effect, which may impact on microcirculation during ischemia. PMID:23437197

  2. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement.

    PubMed

    Mulquiney, P J; Kuchel, P W

    1999-09-15

    Over the last 25 years, several mathematical models of erythrocyte metabolism have been developed. Although these models have identified the key features in the regulation and control of erythrocyte metabolism, many important aspects remain unexplained. In particular, none of these models have satisfactorily accounted for 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is an important modulator of haemoglobin oxygen affinity, and hence an understanding of the regulation of 2,3-BPG concentration is important for understanding blood oxygen transport. A detailed, comprehensive, and hence realistic mathematical model of erythrocyte metabolism is presented that can explain the regulation and control of 2,3-BPG concentration and turnover. The model is restricted to the core metabolic pathways, namely glycolysis, the 2,3-BPG shunt and the pentose phosphate pathway (PPP), and includes membrane transport of metabolites, the binding of metabolites to haemoglobin and Mg(2+), as well as pH effects on key enzymic reactions and binding processes. The model is necessarily complex, since it is intended to describe the regulation and control of 2,3-BPG metabolism under a wide variety of physiological and experimental conditions. In addition, since H(+) and blood oxygen tension are important external effectors of 2,3-BPG concentration, it was important that the model take into account the large array of kinetic and binding phenomena that result from changes in these effectors. Through an iterative loop of experimental and simulation analysis many values of enzyme-kinetic parameters of the model were refined to yield close conformity between model simulations and 'real' experimental data. This iterative process enabled a single set of parameters to be found which described well the metabolic behaviour of the erythrocyte under a wide variety of conditions. PMID:10477269

  3. Model of 2,3-bisphosphoglycerate metabolism in the human erythrocyte based on detailed enzyme kinetic equations: equations and parameter refinement.

    PubMed Central

    Mulquiney, P J; Kuchel, P W

    1999-01-01

    Over the last 25 years, several mathematical models of erythrocyte metabolism have been developed. Although these models have identified the key features in the regulation and control of erythrocyte metabolism, many important aspects remain unexplained. In particular, none of these models have satisfactorily accounted for 2,3-bisphosphoglycerate (2,3-BPG) metabolism. 2,3-BPG is an important modulator of haemoglobin oxygen affinity, and hence an understanding of the regulation of 2,3-BPG concentration is important for understanding blood oxygen transport. A detailed, comprehensive, and hence realistic mathematical model of erythrocyte metabolism is presented that can explain the regulation and control of 2,3-BPG concentration and turnover. The model is restricted to the core metabolic pathways, namely glycolysis, the 2,3-BPG shunt and the pentose phosphate pathway (PPP), and includes membrane transport of metabolites, the binding of metabolites to haemoglobin and Mg(2+), as well as pH effects on key enzymic reactions and binding processes. The model is necessarily complex, since it is intended to describe the regulation and control of 2,3-BPG metabolism under a wide variety of physiological and experimental conditions. In addition, since H(+) and blood oxygen tension are important external effectors of 2,3-BPG concentration, it was important that the model take into account the large array of kinetic and binding phenomena that result from changes in these effectors. Through an iterative loop of experimental and simulation analysis many values of enzyme-kinetic parameters of the model were refined to yield close conformity between model simulations and 'real' experimental data. This iterative process enabled a single set of parameters to be found which described well the metabolic behaviour of the erythrocyte under a wide variety of conditions. PMID:10477269

  4. Erythrocyte hemodynamics in stenotic microvessels: A numerical investigation

    NASA Astrophysics Data System (ADS)

    Wang, T.; Xing, Z. W.

    2013-10-01

    This paper presents a two-dimensional numerical investigation of deformation and motion of erythrocytes in stenotic microvessels using the immersed boundary-fictitious domain method. The erythrocytes were modeled as biconcave-shaped closed membranes filled with cytoplasm. We studied the biophysical characteristics of human erythrocytes traversing constricted microchannels with the narrowest cross-sectional diameter as small as 3 μm. The effects of essential parameters, namely, stenosis severity, shape of the erythrocytes, and erythrocyte membrane stiffness, were simulated and analyzed in this study. Moreover, simulations were performed to discuss conditions associated with the shape transitions of the cells along with the relative effects of radial position and initial orientation of erythrocytes, membrane stiffness, and plasma environments. The simulation results were compared with existing experiment findings whenever possible, and the physical insights obtained were discussed. The proposed model successfully simulated rheological behaviors of erythrocytes in microscale flow and thus is applicable to a large class of problems involving fluid flow with complex geometry and fluid-cell interactions. Our study would be helpful for further understanding of pathology of malaria and some other blood disorders.

  5. Erythrocyte hemodynamics in stenotic microvessels: A numerical investigation

    NASA Astrophysics Data System (ADS)

    Wang, Tong; Xing, Zhongwen

    2014-03-01

    This paper presents a two-dimensional numerical investigation of deformation and motion of erythrocytes in stenotic microvessels using the immersed boundary-fictitious domain method. The erythrocytes were modeled as biconcave-shaped closed membranes filled with cytoplasm. We studied the biophysical characteristics of human erythrocytes traversing constricted microchannels with the narrowest cross-sectional diameter as small as 3 μm. The effects of essential parameters, namely, stenosis severity, shape of the erythrocytes, and erythrocyte membrane stiffness, were simulated and analyzed in this study. Moreover, simulations were performed to discuss conditions associated with the shape transitions of the cells along with the relative effects of radial position and initial orientation of erythrocytes, membrane stiffness, and plasma environments. The simulation results were compared with existing experiment findings whenever possible, and the physical insights obtained were discussed. The proposed model successfully simulated rheological behaviors of erythrocytes in microscale flow and thus is applicable to a large class of problems involving fluid flow with complex geometry and fluid-cell interactions. Our study would be helpful for further understanding of pathology of malaria and some other blood disorders.

  6. Environmental Factors Inducing Human Cancers

    PubMed Central

    Parsa, N

    2012-01-01

    Background An explosion of research has been done in discovering how human health is affected by environmental factors. I will discuss the impacts of environmental cancer causing factors and how they continue to cause multiple disruptions in cellular networking. Some risk factors may not cause cancer. Other factors initiate consecutive genetic mutations that would eventually alter the normal pathway of cellular proliferations and differentiation. Genetic mutations in four groups of genes; (Oncogenes, Tumor suppressor genes, Apoptosis genes and DNA repairing genes) play a vital role in altering the normal cell division. In recent years, molecular genetics have greatly increased our understanding of the basic mechanisms in cancer development and utilizing these molecular techniques for cancer screening, diagnosis, prognosis and therapies. Inhibition of carcinogenic exposures wherever possible should be the goal of cancer prevention programs to reduce exposures from all environmental carcinogens. PMID:23304670

  7. Interrelationships between maternal DHA in erythrocytes, milk and adipose tissue. Is 1 wt% DHA the optimal human milk content? Data from four Tanzanian tribes differing in lifetime stable intakes of fish.

    PubMed

    Luxwolda, Martine F; Kuipers, Remko S; Koops, Jan-Hein; Muller, Stefan; de Graaf, Deti; Dijck-Brouwer, D A Janneke; Muskiet, Frits A J

    2014-03-14

    Little is known about the interrelationships between maternal and infant erythrocyte-DHA, milk-DHA and maternal adipose tissue (AT)-DHA contents. We studied these relationships in four tribes in Tanzania (Maasai, Pare, Sengerema and Ukerewe) differing in their lifetime intakes of fish. Cross-sectional samples were collected at delivery and after 3 d and 3 months of exclusive breast-feeding. We found that intra-uterine biomagnification is a sign of low maternal DHA status, that genuine biomagnification occurs during lactation, that lactating mothers with low DHA status cannot augment their infants' DHA status, and that lactating mothers lose DHA independent of their DHA status. A maternal erythrocyte-DHA content of 8 wt% was found to correspond with a mature milk-DHA content of 1·0 wt% and with subcutaneous and abdominal (omentum) AT-DHA contents of about 0·39 and 0·52 wt%, respectively. Consequently, 1 wt% DHA might be a target for Western human milk and infant formula that has milk arachidonic acid, EPA and linoleic acid contents of 0·55, 0·22 and 9·32 wt%, respectively. With increasing DHA status, the erythrocyte-DHA content reaches a plateau of about 9 wt%, and it plateaus more readily than milk-DHA and AT-DHA contents. Compared with the average Tanzanian-Ukerewe woman, the average US woman has four times lower AT-DHA content (0·4 v. 0·1 wt%) and five times lower mature milk-DHA output (301 v. 60 mg/d), which contrasts with her estimated 1·8-2·6 times lower mobilisable AT-DHA content (19 v. 35-50 g). PMID:24175990

  8. Platelet Inhibition by Nitrite Is Dependent on Erythrocytes and Deoxygenation

    PubMed Central

    Srihirun, Sirada; Sriwantana, Thanaporn; Unchern, Supeenun; Kittikool, Dusadee; Noulsri, Egarit; Pattanapanyasat, Kovit; Fucharoen, Suthat; Piknova, Barbora; Schechter, Alan N.; Sibmooh, Nathawut

    2012-01-01

    Background Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. Methodology/Finding Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 µM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. Conclusion Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia. PMID:22276188

  9. Hyperbaric hyperoxia reversibly inhibits erythrocyte phospholipid fatty acid turnover

    NASA Technical Reports Server (NTRS)

    Dise, Craig A.; Clark, James M.; Lambersten, Christian J.; Goodman, David B. P.

    1987-01-01

    The effect of hyperbaric hyperoxia on the acylation of membrane phospholipid was studied by measuring the rates of activation of exogenous tritiated oleic acid to acyl thioester and of transesterification of the thioester into membrane phospholipids in intact human erythrocytes obtained 1 h after an exposure of the subjects to a hyperbaric oxygen atmosphere (3.5 h, 100 pct O2, 3 ATA). Exposure to pure oxygen was found to inhibit both the acylation and transesterification reactions by more than 30 percent, with partial recovery detected 24 h later. On the other hand, no rate changes were observed when isolated membranes from the same batches of cells were used in similar experiments. It is suggested that the decrease in the incorporation of tritiated oleic acid after hyperbaric hyperoxia may reflect an early event in the pathogenesis of oxygen-induced cellular injury and that it may be a useful index for the assessment of the tolerance of tissues to hyperoxia.

  10. Lipid diffusibility in the intact erythrocyte membrane.

    PubMed Central

    Bloom, J A; Webb, W W

    1983-01-01

    The lateral diffusion of fluorescent lipid analogues in the plasma membrane of intact erythrocytes from man, mouse, rabbit, and frog has been measured by fluorescence photobleaching recovery (FPR). Intact cells from dystrophic, normoblastic, hemolytic, and spherocytotic mouse mutants; from hypercholesterolemic rabbits and humans; and from prenatal, neonatal, and juvenile mice have been compared with corresponding normals. The lateral diffusion coefficient (D) for 3,3'-dioctadecylindodicarbocyanine iodide (DiI[5]) in intact normal human erythrocytes is D = 8.2 +/- 1.2 X 10(-9) cm2/s at 25 degrees C and D = 2.1 +/- 0.4 X 10(-8) cm2/s at 37 degrees C, and varies approximately 50-fold between 1 degree and 42 degrees C. The diffusion constants of lipid analogue rhodamine-B phosphatidylethanolamine (RBPE) are about twice those of DiI[5]. The temperature dependence and magnitude of D vary by up to a factor of 3 between species and are only influenced by donor age in prenatals. DiI[5] diffusibility is not perturbed by the presence of calcium or local anesthetics or by spectrin depletion (via mutation). However, lipid-analogue diffusibility in erythrocyte ghosts may differ from intact cells. Dietary hypercholesterolemia in rabbits reduces the diffusion coefficient and eliminates the characteristic break in Arrhenius plots of D found in all other cells studied except frog. PMID:6603237

  11. A Wet Chemical Method for Rendering Scanning Electron Microscopy Samples Conductive and Observations on the Surface Morphology of Human Erythrocytes and Ehrlich Ascites Cells*

    PubMed Central

    Goldman, Mark A.; Leif, Robert C.

    1973-01-01

    An alternate method to the common technique of evaporating a metallic coating on cells to render them conductive for scanning electron microscopy is described. This wet chemical technique is less expensive and easier to use, but is not as widely applicable as the evaporative technique. It should prove invaluable in the identification of artifacts by comparison of micrographs of material prepared in both manners. The first step in our application of this wet chemical method is to use the Centrifugal Cytology bucket to deposit the cells onto conductive polyethylene. After centrifugation and fixation with 4% glutaraldehyde, the sample is placed in a 50% glutaraldehyde solution. After a rinse in distilled water it is treated with ammoniacal silver nitrate. The reduced silver renders the sample conductive and, thus, for erythrocytes, eliminates artifacts due to charging and reduces these artifacts to virtually acceptable levels for larger cells. The surfaces of erythrocytes appear smooth with this technique while those coated by conventional vapor deposition of Au-Pd alloys often appear slightly wrinkled. Ehrlich ascites cells apparently can be divided into two classes by surface morphology. The surface structure of Ehrlich ascites cells rendered conductive by the wet method appears to be finer than conventionally prepared ones. Images PMID:4202847

  12. A novel human erythrocyte glycosylphosphatidylinositol (GPI)-anchored glycoprotein ACA. Isolation, purification, primary structure determination, and molecular parameters of its lipid structure.

    PubMed

    Becker Kojić, Zorica A; Terness, Peter

    2002-10-25

    A method has been elaborated to isolate and purify up to homogeneity a novel membrane glycoprotein containing a glycosyl-phosphatidylinositol (GPI) anchor by means of salting out with ammonium sulfate (40-80% saturation), followed by preparative SDS-PAGE, chromatography and acetone precipitation. The preparation obtained was homogeneous upon electrophoresis in the presence of 0.1% SDS after reduction with 2-mercaptoethanol. It is protein-soluble at its isoelectrical point (pH 5.5) with molecular mass of 65,000 daltons. The isolated protein is linked to the membrane via glycosyl-phosphatidylinositol susceptible to cleavage by purified phospholipase C. The hydrophobic portion of the glycolipid membrane anchor of the protein was radiolabeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[(125)I]iodophenyl)diazirine and hydrolyzed with glycosyl-phosphatidylinositol-specific phospholipase C, followed by enzymatic deacetylation of the remaining lipid. Thin-layer chromatography showed that the generated radiolabeled fragment migrates with the same mobility as that of variant surface glycoprotein (VSG), obtained in the same manner. In this study we describe a novel erythrocyte membrane GPI-linked protein with the structural feature of an anchor that, in contrast to other GPI-linked erythrocyte proteins, has a non-acetylated inositol ring and diacylglycerol rather than alkyl-acyl glycerol as a lipid tail of the anchor. PMID:12167612

  13. Disorders of Erythrocyte Volume Homeostasis

    PubMed Central

    Glogowska, Edyta; Gallagher, Patrick G.

    2015-01-01

    Inherited disorders of erythrocyte volume homeostasis are a heterogeneous group of rare disorders with phenotypes ranging from dehydrated to overhydrated erythrocytes. Clinical, laboratory, physiologic, and genetic heterogeneity characterize this group of disorders. A series of recent reports have provided novel insights into our understanding of the genetic bases underlying some of these disorders of red cell volume regulation. This report reviews this progress in understanding determinants that influence erythrocyte hydration and how they have yielded a better understanding of the pathways that influence cellular water and solute homeostasis. PMID:25976965

  14. Protective effects of kaempferol against reactive oxygen species-induced hemolysis and its antiproliferative activity on human cancer cells.

    PubMed

    Liao, Wenzhen; Chen, Luying; Ma, Xiang; Jiao, Rui; Li, Xiaofeng; Wang, Yong

    2016-05-23

    The protective effects of kaempferol against reactive oxygen species (ROS)-induced hemolysis and its antiproliferative activity on human cancer cells were evaluated in this study. Kaempferol exhibited strong cellular antioxidant ability (CAA) with a CAA value of 59.80 ± 0.379 μM of quercetin (QE)/100 μM (EC50 = 7.74 ± 0.049 μM). Pretreatment with kaempferol significantly attenuated the ROS-induced hemolysis of human erythrocyte (87.4% hemolysis suppressed at 100 μg/mL) and reduced the accumulation of toxic lipid peroxidation product malondialdehyde (MDA). The anti-hemolytic activity of kaempferol was mainly through scavenging excessive ROS and preserving the intrinsic antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; and glutathione peroxidase, GPx) activities in normal levels. Additionally, kaempferol showed significant antiproliferative activity on a panel of human cancer cell lines including human breast carcinoma (MCF-7) cells, human stomach carcinoma (SGC-7901) cells, human cervical carcinoma (Hela) cells and human lung carcinoma (A549) cells. Kaemperol induced apoptosis of MCF-7 cells accompanied with nuclear condensation and mitochondria dysfunction. PMID:26974372

  15. Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

    PubMed Central

    Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

    2007-01-01

    Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

  16. Human cytomegalovirus induces JC virus DNA replication in human fibroblasts.

    PubMed Central

    Heilbronn, R; Albrecht, I; Stephan, S; Bürkle, A; zur Hausen, H

    1993-01-01

    JC virus, a human papovavirus, is the causative agent of the demyelinating brain disease progressive multifocal leucoencephalopathy (PML). PML is a rare but fatal disease which develops as a complication of severe immunosuppression. Latent JC virus is harbored by many asymptomatic carriers and is transiently reactivated from the latent state upon immunosuppression. JC virus has a very restricted host range, with human glial cells being the only tissue in which it can replicate at reasonable efficiency. Evidence that latent human cytomegalovirus is harbored in the kidney similar to latent JC virus led to the speculation that during episodes of impaired immunocompetence, cytomegalovirus might serve as helper virus for JC virus replication in otherwise nonpermissive cells. We show here that cytomegalovirus infection indeed leads to considerable JC virus DNA replication in cultured human fibroblasts that are nonpermissive for the replication of JC virus alone. Cytomegalovirus-mediated JC virus replication is dependent on the JC virus origin of replication and T antigen. Ganciclovir-induced inhibition of cytomegalovirus replication is associated with a concomitant inhibition of JC virus replication. These results suggest that reactivation of cytomegalovirus during episodes of immunosuppression might lead to activation of latent JC virus, which would enhance the probability of subsequent PML development. Ganciclovir-induced repression of both cytomegalovirus and JC virus replication may form the rational basis for the development of an approach toward treatment or prevention of PML. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8248262

  17. Fusion of Erythrocytes by Sendai Virus Studied by Immuno-Freeze-Etching

    PubMed Central

    Bächi, Thomas; Aguet, Michel; Howe, Calderon

    1973-01-01

    Extensive fusion of human erythrocytes agglutinated by Sendai virus was observed after 30 s of incubation at 37 C. Electron microscopy of thin sections failed to reveal the presence of virions, viral fragments, or discrete viral antigens reactive with ferritin-labeled antibody at the sites of fusion. Immuno-freezeetching of membrane surfaces demonstrated the dispersal of viral envelope antigens from what appeared to be original sites of viral attachment. Virus-induced clustering of membrane glycoproteins was interpreted as resulting from interaction of viral antigens with membrane receptor proteins and forming the structural basis for fusion of membranes with one another. Images PMID:4351454

  18. Atomic force microscopy study of erythrocyte shape and membrane structure after treatment with a dihydropyridinic drug

    NASA Astrophysics Data System (ADS)

    Girasole, M.; Cricenti, A.; Generosi, R.; Congiu-Castellano, A.; Boffi, F.; Arcovito, A.; Boumis, G.; Amiconi, G.

    2000-06-01

    The overall shape and membrane surface of human erythrocytes (RBCs) in the presence of nifedipine (a dihydropyridinic drug used in the clinical treatment of hypertension and angina pectoris) were imaged by contact-mode atomic force microscopy. Nifedipine induces in RBCs relevant morphological changes the extent of which increases as a function of drug concentration and incubation time. The modifications have been interpreted as mainly due to insertion of nifedipine into the outer layer of the RBC membrane. The potential effect of nifedipine as a hemoglobin denaturant has been ruled out by x-ray absorption near-edge structure and optical spectroscopies.

  19. Inhibition of ATP release from Erythrocytes: A role for EPACs and PKC

    PubMed Central

    Adderley, Shaquria P.; Sridharan, Meera; Bowles, Elizabeth A.; Stephenson, Alan H.; Sprague, Randy S.; Ellsworth, Mary L.

    2010-01-01

    Objective Here we demonstrate that, in human erythrocytes, increases in cAMP that are not localized to a specific receptor-mediated signaling pathway for ATP release can activate effector proteins resulting in inhibition of ATP release. Specifically we sought to establish that exchange proteins activated by cAMP (EPACs) inhibit ATP release via activation of protein kinase C (PKC). Methods ATP release stimulated by iloprost (ILO), or isoproterenol (ISO), was determined in the absence and presence of selective phosphodiesterase inhibitors and/or the EPAC activator, 8CPT2OMecAMP (8CPT). To determine whether EPACs inhibit ATP release via activation of PKC, erythrocytes were incubated with phorbol 12-myristate 13-acetate (PMA) prior to either forskolin or ILO in the absence and presence of a PKC inhibitor, calphostin C (CALC). Results Selective inhibition of PDEs in one pathway inhibited ATP release in response to activation of the other cAMP-dependent pathway. 8CPT and PMA inhibited both ILO- and ISO-induced ATP release. Inhibition of ATP release with 8CPT was rescued by CALC. Conclusion These results support the hypothesis that cAMP not localized to a specific signaling pathway can activate EPACs which inhibit ATP release via activation of PKC and suggest a novel role for EPACs in erythrocytes. PMID:21166931

  20. Role of erythrocyte-released ATP in the regulation of microvascular oxygen supply in skeletal muscle.

    PubMed

    Ellsworth, M L; Ellis, C G; Sprague, R S

    2016-03-01

    In a 1914 book entitled The Respiratory Function of the Blood, Joseph Barcroft stated that 'the cell takes what it needs and leaves the rest'. He postulated that there must be both a 'call for oxygen' and a 'mechanism by which the call elicits a response...' In the past century, intensive investigation has provided significant insights into the haemodynamic and biophysical mechanisms involved in supplying oxygen to skeletal muscle. However, the identification of the mechanism by which tissue oxygen needs are sensed and the affector responsible for altering the upstream vasculature to enable the need to be appropriately met has been a challenge. In 1995, Ellsworth et al. proposed that the oxygen-carrying erythrocyte, by virtue of its capacity to release the vasoactive mediator ATP in response to a decrease in oxygen saturation, could serve both roles. Several in vitro and in situ studies have established that exposure of erythrocytes to reduced oxygen tension induces the release of ATP which does result in a conducted arteriolar vasodilation with a sufficiently rapid time course to make the mechanism physiologically relevant. The components of the signalling pathway for the controlled release of ATP from erythrocytes in response to exposure to low oxygen tension have been determined. In addition, the implications of defective ATP release on human pathological conditions have been explored. This review provides a perspective on oxygen supply and the role that such a mechanism plays in meeting the oxygen needs of skeletal muscle. PMID:26336065

  1. Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death.

    PubMed

    Villamizar, Olga; Chambers, Christopher B; Mo, Yin-Yuan; Torry, Donald S; Hofstrand, Reese; Riberdy, Janice M; Persons, Derek A; Wilber, Andrew

    2016-05-01

    Long noncoding RNAs (lncRNAs) interact with other RNAs, DNA and/or proteins to regulate gene expression during development. Erythropoiesis is one developmental process that is tightly controlled throughout life to ensure accurate red blood cell production and oxygen transport to tissues. Thus, homeostasis is critical and maintained by competitive outcomes of pro- and anti-apoptotic pathways. LncRNAs are expressed during blood development; however, specific functions are largely undefined. Here, a culture model of human erythropoiesis revealed that lncRNA Fas-antisense 1 (Fas-AS1 or Saf) was induced during differentiation through the activity of essential erythroid transcription factors GATA-1 and KLF1. Saf was also negatively regulated by NF-κB, where decreasing NF-κB activity levels tracked with increasing transcription of Saf. Furthermore, Saf over-expression in erythroblasts derived from CD34(+) hematopoietic stem/progenitor cells of healthy donors reduced surface levels of Fas and conferred protection against Fas-mediated cell death signals. These studies reveal a novel lncRNA-regulated mechanism that modulates a critical cell death program during human erythropoiesis. PMID:27067490

  2. Lymphocyte cytotoxicity and erythrocytic abnormalities induced in broiler chicks by fumonisins B1 and B2 and moniliformin from Fusarium proliferatum.

    PubMed

    Dombrink-Kurtzman, M A; Javed, T; Bennett, G A; Richard, J L; Cote, L M; Buck, W B

    1993-10-01

    Peripheral blood lymphocytes were isolated from broiler chicks that had ingested feed amended with autoclaved Fusarium proliferatum culture material containing fumonisin B1 (FB1), fumonisin B2 (FB2) and moniliformin. Lymphocyte viability was determined for birds that were placed on amended rations at day 1 or day 7 of age at three different levels of mycotoxins, ranging from 61-546 ppm FB1, 14-94 ppm FB2 and 66-367 ppm moniliformin. Reduction of the tetrazolium salt, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], to yield MTT formazan, based on mitochondrial metabolic activity, was used to assess cell viability. Lymphocyte cytotoxic effects were observed in all treatment groups on day 21; chicks that started on amended feed at day 1 of age were affected more than those that started at day 7. Abnormal erythrocytes resembling early stages of erythroblasts were observed in peripheral blood from test chicks. Abnormally shaped red cells (poikilocytes) having a spindle-shape with one or both ends pointed were present. Some red cells appeared to be undergoing mitosis. Both reduced lymphocyte viability and abnormal erythrogenesis occurred in chicks given feed amended with F. proliferatum culture material containing FB1, FB2 and moniliformin. PMID:8159217

  3. Helium-ion-induced human cataractogenesis

    NASA Technical Reports Server (NTRS)

    Blakely, E. A.; Daftari, I. K.; Meecham, W. J.; Alonso, L. C.; Collier, J. M.; Kroll, S. M.; Gillette, E. L.; Lee, A. C.; Lett, J. T.; Cox, A. B.

    1994-01-01

    Retrospective and ongoing analyses of clinical records from 347 primary intraocular melanoman patients treated with helium ions at Lawrence Berkeley Laboratory (LBL) will allow examination of the exposure-response data for human cataract; which is a complication of the therapy from incidental exposure of the lens. Direct particle beam traversal of at least a portion of the lens usually is unavoidable in treatment of posterior intraocular tumors. The precise treatment planned for each patient permits quantitative assessment of the lenticular dose and its radiation quality. We are reporting our preliminary results on the development of helium-ion-induced lens opacifications and cataracts in 54 of these patients who had 10% or less of their lens in the treatment field. We believe these studies will be relevant to estimating the human risk for cataract in space flight.

  4. Cation channels, cell volume and the death of an erythrocyte.

    PubMed

    Lang, Florian; Lang, Karl S; Wieder, Thomas; Myssina, Svetlana; Birka, Christina; Lang, Philipp A; Kaiser, Stephanie; Kempe, Daniela; Duranton, Christophe; Huber, Stephan M

    2003-11-01

    Similar to a variety of nucleated cells, human erythrocytes activate a non-selective cation channel upon osmotic cell shrinkage. Further stimuli of channel activation include oxidative stress, energy depletion and extracellular removal of Cl-. The channel is permeable to Ca2+ and opening of the channel increases cytosolic [Ca2+]. Intriguing evidence points to a role of this channel in the elimination of erythrocytes by apoptosis. Ca2+ entering through the cation channel stimulates a scramblase, leading to breakdown of cell membrane phosphatidylserine asymmetry, and stimulates Ca(2+)-sensitive K+ channels, thus leading to KCl loss and (further) cell shrinkage. The breakdown of phosphatidylserine asymmetry is evidenced by annexin binding, a typical feature of apoptotic cells. The effects of osmotic shock, oxidative stress and energy depletion on annexin binding are mimicked by the Ca2+ ionophore ionomycin (1 microM) and blunted in the nominal absence of extracellular Ca2+. Nevertheless, the residual annexin binding points to additional mechanisms involved in the triggering of the scramblase. The exposure of phosphatidylserine at the extracellular face of the cell membrane stimulates phagocytes to engulf the apoptotic erythrocytes. Thus, sustained activation of the cation channels eventually leads to clearance of affected erythrocytes from peripheral blood. Susceptibility to annexin binding is enhanced in several genetic disorders affecting erythrocyte function, such as thalassaemia, sickle-cell disease and glucose-6-phosphate dehydrogenase deficiency. The enhanced vulnerability presumably contributes to the shortened life span of the affected erythrocytes. Beyond their role in the limitation of erythrocyte survival, cation channels may contribute to the triggering of apoptosis in nucleated cells exposed to osmotic shock and/or oxidative stress. PMID:12905029

  5. Thrombospondin-induced adhesion of human platelets.

    PubMed Central

    Tuszynski, G P; Kowalska, M A

    1991-01-01

    Washed human unactivated platelets attached and spread on thrombospondin (TSP)-coated microtiter plates. Platelet adhesion was promoted by divalent cations Mn2+, Mg2+, and Ca2+ as compared to buffer having all divalent cations complexed with EDTA. TSP-dependent adhesion was inhibited by anti-TSP fab fragments, an anti-TSP monoclonal antibody, an RGD-containing peptide, complex-specific anti-glycoprotein (GP)IIb-IIIa monoclonal antibodies (A2A9 or AP-2) and anti-VLA-2 monoclonal antibodies (6F1 and Gi9), but not by rabbit preimmune fab fragments, mouse IgG, an anti-GPIIIa monoclonal antibody, or monoclonal antibodies against either the human vitronectin receptor, glycocalicin, or GPIV. At saturating concentrations, anti-GPIIb-IIIa inhibited adhesion by 40-60%. Glanzman's thrombasthenic platelets, which lack GPIIb-IIIa, adhered to TSP to the same extent as anti-GPIIb-IIIa-treated normal platelets or 40-60% as well as untreated normal platelets. Antibody 6F1 (5-10 micrograms/ml) inhibited platelet adhesion of both normal and thrombasthenic platelets by 84-100%. Both VLA-2 antibodies also inhibited collagen-induced platelet adhesion, but had no effect on fibronectin-induced adhesion of normal platelets. These data indicate that platelets specifically adhere to TSP and that this adhesion is mediated through GPIIb-IIIa and/or VLA-2. Images PMID:2010551

  6. Rapid and Highly Sensitive Detection of Malaria-Infected Erythrocytes Using a Cell Microarray Chip

    PubMed Central

    Yamaguchi, Yuka; Shinohara, Yasuo; Tamiya, Eiichi; Horii, Toshihiro; Baba, Yoshinobu; Kataoka, Masatoshi

    2010-01-01

    Background Malaria is one of the major human infectious diseases in many endemic countries. For prevention of the spread of malaria, it is necessary to develop an early, sensitive, accurate and conventional diagnosis system. Methods and Findings A cell microarray chip was used to detect for malaria-infected erythrocytes. The chip, with 20,944 microchambers (105 µm width and 50 µm depth), was made from polystyrene, and the formation of monolayers of erythrocytes in the microchambers was observed. Cultured Plasmodium falciparum strain 3D7 was used to examine the potential of the cell microarray chip for malaria diagnosis. An erythrocyte suspension in a nuclear staining dye, SYTO 59, was dispersed on the chip surface, followed by 10 min standing to allow the erythrocytes to settle down into the microchambers. About 130 erythrocytes were accommodated in each microchamber, there being over 2,700,000 erythrocytes in total on a chip. A microarray scanner was employed to detect any fluorescence-positive erythrocytes within 5 min, and 0.0001% parasitemia could be detected. To examine the contamination by leukocytes of purified erythrocytes from human blood, 20 µl of whole blood was mixed with 10 ml of RPMI 1640, and the mixture was passed through a leukocyte isolation filter. The eluted portion was centrifuged at 1,000×g for 2 min, and the pellet was dispersed in 1.0 ml of medium. SYTO 59 was added to the erythrocyte suspension, followed by analysis on a cell microarray chip. Similar accommodation of cells in the microchambers was observed. The number of contaminating leukocytes was less than 1 on a cell microarray chip. Conclusion The potential of the cell microarray chip for the detection of malaria-infected erythrocytes was shown, it offering 10–100 times higher sensitivity than that of conventional light microscopy and easy operation in 15 min with purified erythrocytes. PMID:20967248

  7. Simulation of dielectric spectra of erythrocytes with various shapes

    NASA Astrophysics Data System (ADS)

    Asami, Koji

    2009-07-01

    Dielectric spectra of erythrocyte suspensions were numerically simulated over a frequency range from 1 kHz to 100 MHz to study the effects of erythrocyte shape on the dielectric spectra. First, a biconcave-discoid model for normal erythrocytes or discocytes was compared with an equivalent oblate spheroid model. The two models showed similar dielectric spectra to each other, suggesting that the oblate spheroid model can be approximately used for discocytes. Second, dielectric spectra were simulated for discocytes deformed by osmotic cell swelling. The deformation resulted in the increase in relaxation intensity and the sharpening of spectrum shape. Finally, dielectric spectra were simulated for echinocytes, stomatocytes and sickle cells that are induced by chemical agents and diseases. The dielectric spectra of echinocytes and stomatocytes were similar to each other, being distinguishable from that of discocytes and quite different from that of sickle cells.

  8. Erythrocyte-derived optical nano-vesicles as theranostic agents

    NASA Astrophysics Data System (ADS)

    Mac, Jenny T.; Nunez, Vicente; Bahmani, Baharak; Guerrero, Yadir; Tang, Jack; Vullev, Valentine I.; Anvari, Bahman

    2015-07-01

    We have engineered nano-vesicles, derived from erythrocytes, which can be doped with various near infrared (NIR) organic chromophores, including the FDA-approved indocyanine green (ICG). We refer to these vesicles as NIR erythrocyte-mimicking transducers (NETS) since in response to NIR photo-excitation they can generate heat or emit fluorescent light. Using biochemical methods based on reduction amination, we have functionalized the surface of NET with antibodies to target specific biomolecules. We present results that demonstrate the effectiveness of NETs in targeted imaging of cancer cells that over-express the human epidermal growth factor receptor-2 (HER2).

  9. Atomic Force Microscopy of Asymmetric Membranes from Turtle Erythrocytes

    PubMed Central

    Tian, Yongmei; Cai, Mingjun; Xu, Haijiao; Ding, Bohua; Hao, Xian; Jiang, Junguang; Sun, Yingchun; Wang, Hongda

    2014-01-01

    The cell membrane provides critical cellular functions that rely on its elaborate structure and organization. The structure of turtle membranes is an important part of an ongoing study of erythrocyte membranes. Using a combination of atomic force microscopy and single-molecule force spectroscopy, we characterized the turtle erythrocyte membrane structure with molecular resolution in a quasi-native state. High-resolution images both leaflets of turtle erythrocyte membranes revealed a smooth outer membrane leaflet and a protein covered inner membrane leaflet. This asymmetry was verified by single-molecule force spectroscopy, which detects numerous exposed amino groups of membrane proteins in the inner membrane leaflet but much fewer in the outer leaflet. The asymmetric membrane structure of turtle erythrocytes is consistent with the semi-mosaic model of human, chicken and fish erythrocyte membrane structure, making the semi-mosaic model more widely applicable. From the perspective of biological evolution, this result may support the universality of the semi-mosaic model. PMID:25134535

  10. 21 CFR 864.6700 - Erythrocyte sedimentation rate test.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Erythrocyte sedimentation rate test. 864.6700 Section 864.6700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6700...

  11. 21 CFR 864.6700 - Erythrocyte sedimentation rate test.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Erythrocyte sedimentation rate test. 864.6700 Section 864.6700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6700...

  12. 21 CFR 864.6700 - Erythrocyte sedimentation rate test.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Erythrocyte sedimentation rate test. 864.6700 Section 864.6700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6700...

  13. 21 CFR 864.6700 - Erythrocyte sedimentation rate test.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocyte sedimentation rate test. 864.6700 Section 864.6700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6700...

  14. 21 CFR 864.6700 - Erythrocyte sedimentation rate test.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Erythrocyte sedimentation rate test. 864.6700 Section 864.6700 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Manual Hematology Devices § 864.6700...

  15. Osmotic and diffusive properties of intracellular water in camel erythrocytes: effect of hemoglobin crowdedness.

    PubMed

    Bogner, Peter; Miseta, Attila; Berente, Zoltan; Schwarcz, Attila; Kotek, Gyula; Repa, Imre

    2005-09-01

    Camel erythrocytes have exceptional osmotic resistance and is believed to be due to augmented water-binding associated with the high hydrophilicity of camel hemoglobin. In practical terms this means that the proportion of osmotically non-removable water in camel erythrocytes is nearly 3-fold greater than that in human erythrocytes (approximately 65 vs approximately 20%). The relationship between water diffusion and the osmotic characteristics of intracellular water is the subject of this report. The amount of osmotically inactive water is 2-fold greater in camel hemoglobin solution in vitro compared to that of human, but water diffusion does not differ in camel and human hemoglobin solutions. However, the evaluation of water diffusion by magnetic resonance measurements in camel erythrocytes revealed approximately 15% lower apparent diffusion coefficient (ADC) compared with human erythrocytes. When human erythrocytes were dehydrated to the level of camel erythrocytes, their osmotic and water diffusion properties were similar. These results show that a lower ADC is associated with a more pronounced increase in osmotically inactive water fraction. It is proposed that increased hemoglobin hydrophilicity allows not only augmented water-binding, but also a closer hemoglobin packaging in vivo, which in turn is associated with slower ADC and increased osmotic resistance. PMID:15951204

  16. Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum.

    PubMed Central

    Brockelman, C R; Wongsattayanont, B; Tan-ariya, P; Fucharoen, S

    1987-01-01

    Blood specimens from 100 thalassemic patients were screened in vitro for inhibitory effects on growth and multiplication of Plasmodium falciparum. The culture medium mixt