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Sample records for erythrocytes inhibit human

  1. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    International Nuclear Information System (INIS)

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

    1990-01-01

    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

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

    International Nuclear Information System (INIS)

    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

  3. Inhibition by nucleosides of glucose-transport activity in human erythrocytes.

    OpenAIRE

    Jarvis, S M

    1988-01-01

    The interaction of nucleosides with the glucose carrier of human erythrocytes was examined by studying the effect of nucleosides on reversible cytochalasin B-binding activity and glucose transport. Adenosine, inosine and thymidine were more potent inhibitors of cytochalasin B binding to human erythrocyte membranes than was D-glucose [IC50 (concentration causing 50% inhibition) values of 10, 24, 28 and 38 mM respectively]. Moreover, low concentrations of thymidine and adenosine inhibited D-glu...

  4. The osmotic fragility of human erythrocytes is inhibited by laser irradiation

    International Nuclear Information System (INIS)

    Habodaszova, D.; Sikurova, L.; Waczulikova, I.

    2004-01-01

    In this study we investigated the influence of green laser irradiation (532 nm, 30 mW, 31,7 J/cm 2 ) on the membrane integrity of human erythrocytes and compared the results with the effect of infrared laser irradiation (810 nm, 50 mW, 31,3 J/cm 2 ). To evaluate the membrane integrity of erythrocytes, one clinical parameter, the osmotic fragility, was investigated. We observed a decrease in osmotic fragility of the erythrocytes after irradiation by the green laser light as well as by the infrared laser compared to non-irradiated controls (Authors)

  5. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

    Science.gov (United States)

    Naftalin, Richard J; Cunningham, Philip; Afzal-Ahmed, Iram

    2004-01-01

    Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide galanin in human erythrocytes in vitro. The potencies of nootropic drugs in opposing scopolamine-induced memory loss correlate with their potencies in antagonising pentobarbital inhibition of erythrocyte glucose transport in vitro (Pnootropics, D-levetiracetam and D-pyroglutamate, have higher antagonist Ki's against pentobarbital inhibition of glucose transport than more potent L-stereoisomers (Pnootropics, like aniracetam and levetiracetam, while antagonising pentobarbital action, also inhibit glucose transport. Analeptics like bemigride and methamphetamine are more potent inhibitors of glucose transport than antagonists of hypnotic action on glucose transport. There are similarities between amino-acid sequences in human glucose transport protein isoform 1 (GLUT1) and the benzodiazepine-binding domains of GABAA (gamma amino butyric acid) receptor subunits. Mapped on a 3D template of GLUT1, these homologies suggest that the site of diazepam and piracetam interaction is a pocket outside the central hydrophilic pore region. Nootropic pyrrolidone antagonism of hypnotic drug inhibition of glucose transport in vitro may be an analogue of TRH antagonism of galanin-induced narcosis. PMID:15148255

  6. Pre- and post-treatment effect of physostigmine on soman-inhibited human erythrocyte and muscle acetylcholinesterase in vitro

    International Nuclear Information System (INIS)

    Herkert, N.M.; Schulz, S.; Wille, T.; Thiermann, H.; Hatz, R.A.; Worek, F.

    2011-01-01

    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.

  7. Human antibodies fix complement to inhibit Plasmodium falciparum invasion of erythrocytes and are associated with protection against malaria.

    Science.gov (United States)

    Boyle, Michelle J; Reiling, Linda; Feng, Gaoqian; Langer, Christine; Osier, Faith H; Aspeling-Jones, Harvey; Cheng, Yik Sheng; Stubbs, Janine; Tetteh, Kevin K A; Conway, David J; McCarthy, James S; Muller, Ivo; Marsh, Kevin; Anders, Robin F; Beeson, James G

    2015-03-17

    Antibodies play major roles in immunity to malaria; however, a limited understanding of mechanisms mediating protection is a major barrier to vaccine development. We have demonstrated that acquired human anti-malarial antibodies promote complement deposition on the merozoite to mediate inhibition of erythrocyte invasion through C1q fixation and activation of the classical complement pathway. Antibody-mediated complement-dependent (Ab-C') inhibition was the predominant invasion-inhibitory activity of human antibodies; most antibodies were non-inhibitory without complement. Inhibitory activity was mediated predominately via C1q fixation, and merozoite surface proteins 1 and 2 were identified as major targets. Complement fixation by antibodies was very strongly associated with protection from both clinical malaria and high-density parasitemia in a prospective longitudinal study of children. Ab-C' inhibitory activity could be induced by human immunization with a candidate merozoite surface-protein vaccine. Our findings demonstrate that human anti-malarial antibodies have evolved to function by fixing complement for potent invasion-inhibitory activity and protective immunity. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  8. Ebselen exhibits glycation-inhibiting properties and protects against osmotic fragility of human erythrocytes in vitro.

    Science.gov (United States)

    Soares, Julio C M; Folmer, Vanderlei; Da Rocha, João B T; Nogueira, Cristina W

    2014-05-01

    Diabetic status is associated with an increase on oxidative stress markers in humans and animal models. We have investigated the in vitro effects of high concentrations of glucose on the profile of oxidative stress and osmotic fragility of blood from control and diabetic patients; we considered whether its antioxidant properties could afford some protection against glucose-induced osmotic fragility, and whether ebselen could act as an inhibitor of hemoglobin glycation. Raising blood glucose to 5-100 mmol/L resulted in a concentration-dependent increase of glycated hemoglobin (HbA1c; P Ebselen significantly reduced the glucose-induced increase in osmotic fragility and inhibited HbA1c formation (P < 0.0001). These results indicate that blood from patients with uncontrolled diabetes are more sensitive to osmotic shock than from patients with controlled diabetes and control subjects in relation to increased production of free radicals in vivo. © 2014 International Federation for Cell Biology.

  9. [Ceruloplasmin receptor on human erythrocytes].

    Science.gov (United States)

    Saenko, E L; Basevich, V V; Iaropolov, A I

    1988-08-01

    The structural fragments of the human ceruloplasmin (CP) molecule and of erythrocyte receptors which provide for the specific interaction of CP with erythrocytes were identified, and their properties were investigated. The interaction of CP with erythrocytes, both intact and treated with neuroaminidase and proteolytic enzymes (trypsin, chymotrypsin, papaine, pronase E) is described. Experiments with CP reception were performed at 4 degrees C, using [125I]CP and [125I]asialo-CP. The parameters of binding were determined in Scatchard plots. It was demonstrated that the specific binding of CP to erythrocyte receptors is determined by its interaction with two structural sites of the carbohydrate moiety of the CP molecule, i.e., the terminal residues of sialic acids and a site, (formula; see text) located at a large distance from the chain terminus.

  10. Induction of transient radioresistance in human erythrocytes

    International Nuclear Information System (INIS)

    Krokosz, Anita; 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 with single and split doses under air or N 2 O in order to determine the physicochemical changes caused by the dose inducing an increase in resistance to radiation-induced hemolysis. The obtained results showed that under the applied irradiation conditions, the dose of 0.4 kGy induced changes in erythrocytes, which were responsible for temporary resistance of erythrocytes to hemolysis. We concluded that the observed resistance is caused mainly by the structural changes in proteins

  11. Piracetam and TRH analogues antagonise inhibition by barbiturates, diazepam, melatonin and galanin of human erythrocyte D-glucose transport

    OpenAIRE

    Naftalin, Richard J; Cunningham, Philip; Afzal-Ahmed, Iram

    2004-01-01

    Nootropic drugs increase glucose uptake into anaesthetised brain and into Alzheimer's diseased brain. Thyrotropin-releasing hormone, TRH, which has a chemical structure similar to nootropics increases cerebellar uptake of glucose in murine rolling ataxia. This paper shows that nootropic drugs like piracetam (2-oxo 1 pyrrolidine acetamide) and levetiracetam and neuropeptides like TRH antagonise the inhibition of glucose transport by barbiturates, diazepam, melatonin and endogenous neuropeptide...

  12. Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.

    Directory of Open Access Journals (Sweden)

    Sirada Srihirun

    Full Text Available 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.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.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.

  13. NMR studies of transmembrane electron transport in human erythrocytes

    International Nuclear Information System (INIS)

    Kennett, E.C.; Bubb, W.A.; Kuchel, P.W.

    2002-01-01

    Full text: Electron transport systems exist in the plasma membranes of all cells. These systems appear to play a role in cell growth and proliferation, intracellular signalling, hormone responses, apoptotic events, cell defence and perhaps most importantly they enable the cell to respond to changes in the redox state of both the intra- and extracellular environments. Previously, 13 C NMR has been used to study transmembrane electron transport in human erythrocytes, specifically the reduction of extracellular 13 C-ferricyanide. NMR is a particularly useful tool for studying such systems as changes in the metabolic state of the cell can be observed concomitantly with extracellular reductase activity. We investigated the oxidation of extracellular NADH by human erythrocytes using 1 H and 31 P NMR spectroscopy. Recent results for glucose-starved human erythrocytes indicate that, under these conditions, extracellular NADH can be oxidised at the plasma membrane with the electron transfer across the membrane resulting in reduction of intracellular NAD + . The activity is inhibited by known trans-plasma membrane electron transport inhibitors (capsaicin and atebrin) and is unaffected by inhibition of the erythrocyte Band 3 anion transporter. These results suggest that electron import from extracellular NADH allows the cell to re-establish a reducing environment after the normal redox balance is disturbed

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

    Science.gov (United States)

    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.

  15. Clofazimine Induced Suicidal Death of Human Erythrocytes

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    Arbace Officioso

    2015-08-01

    Full Text Available Background/Aims: The antimycobacterial riminophenazine clofazimine has previously been shown to up-regulate cellular phospholipase A2 and to induce apoptosis. In erythrocytes phospholipase A2 stimulates eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Phospholipase A2 is in part effective by fostering formation of prostaglandin E2, which triggers Ca2+ entry. Stimulators of Ca2+ entry and eryptosis further include oxidative stress and energy depletion. The present study tested, whether and how clofazimine induces eryptosis. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin V binding, cell volume from forward scatter, hemolysis from hemoglobin release, cytosolic Ca2+ activity ([Ca2+]i from Fluo3-fluorescence, reactive oxygen species (ROS from 2′, 7′-dichlorodihydrofluorescein diacetate (DCFDA fluorescence, and cytosolic ATP level utilizing a luciferin-luciferase assay kit. Results: A 24-48 hours exposure of human erythrocytes to clofazimine (≥1.5 µg/ml significantly increased the percentage of annexin-V-binding cells without appreciably modifying forward scatter. Clofazimine significantly increased [Ca2+]i, significantly decreased cytosolic ATP, but did not significantly modify ROS. The effect of clofazimine on annexin-V-binding was significantly blunted, but not fully abolished by removal of extracellular Ca2+, and by phospholipase A2 inhibitor quinacrine (25 µM. Clofazimine further augmented the effect of Ca2+ ionophore ionomycin (0.1 µM on eryptosis. The clofazimine induced annexin-V-binding was, however, completely abrogated by combined Ca2+ removal and addition of quinacrine. Conclusion: Clofazimine stimulates phospholipid scrambling of the erythrocyte cell membrane, an effect in part dependent on entry of extracellular Ca2+, paralleled by cellular energy depletion and sensitive to

  16. Fucoxanthin Induced Suicidal Death of Human Erythrocytes

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    Marilena Briglia

    2015-12-01

    Full Text Available Background/Aims: Fucoxanthin, a carotenoid isolated from brown seaweeds, induces suicidal death or apoptosis of tumor cells and is thus considered for the treatment or prevention of malignancy. In analogy to apoptosis of nucleated cell, 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 Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i, oxidative stress and activation of p38 kinase or protein kinase C. The present study explored, whether and how fucoxanthin induces eryptosis. Methods: 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 abundance of reactive oxygen species (ROS from DCFDA dependent fluorescence and lipid peroxidation using BODIPY fluoresence. Results: A 48 hours exposure of human erythrocytes to fucoxanthin significantly increased the percentage of annexin-V-binding cells (≥ 50 µM, significantly decreased average forward scatter (≥ 25 µM, significantly increased hemolysis (≥ 25 µM, significantly increased Fluo3-fluorescence (≥ 50 µM, significantly increased lipid peroxidation, but did not significantly modify DCFDA fluorescence. The effect of fucoxanthin on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+, and was insensitive to p38 kinase inhibitor skepinone (2 µM and to protein kinase C inhibitor calphostin (100 nM. Conclusion: Fucoxanthin triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of Ca2+ entry.

  17. Inhibition of Suicidal Erythrocyte Death by Indirubin-3’-Monoxime

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    Chunqiu Liu

    2018-02-01

    Full Text Available Background/Aims: Qing Dai is a prized traditional Chinese medicine whose major component, indirubin, and its derivative, indirubin-3’-monoxime (IDM, have inhibitory effects on the growth of many human tumor cells and pronounced anti-leukemic activities. However, the effects of IDM on mature human erythrocytes are unclear. This study aimed to evaluate the potential impact of IDM on erythrocytes and the mechanisms underlying that impact. Methods: Utilizing flow cytometry and confocal laser scanning microscopy, phosphatidylserine exposure at the cell surface was estimated by annexin V-fluorescein isothiocyanate (FITC. The relative cell size, expressed in arbitrary units, was evaluated by forward scatter in a flow cytometer. Fluo-3 fluorescence was used to bewrite changes in cytosolic Ca2+ activity, reactive oxygen species (ROS formation was assessed by 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA fluorescence, and ceramide abundance was evaluated by FITC-conjugated specific antibodies. Results: The 24-h exposure of human erythrocytes to IDM (12 µM significantly decreased the percentage of annexin V-binding erythrocytes and the intracellular calcium concentration ([Ca2+]i. IDM (3-12 µM did not significantly modify the ceramide level or DCFH-DA fluorescence. Energy depletion (removal of glucose for 24 hours significantly increased annexin V binding and Fluo-3 fluorescence and diminished forward scatter, and these effects were significantly mitigated by IDM (12 µM. Moreover, the Ca2+ ionophore ionomycin (1 µM, 60 min and oxidative stress (30 min exposure to 0.05 mM tert-butyl hydroperoxide, t-BHP similarly triggered eryptosis, which was also significantly suppressed by IDM. Conclusions: IDM is a novel inhibitor of suicidal erythrocyte death following ionomycin treatment, t-BHP treatment and energy depletion. Thus, IDM may counteract anemia and impairment of microcirculation, at least in part, by inhibition of Ca2+ entry into erythrocytes.

  18. Proton NMR studies of creatine in human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kuchel, P W; Chapman, B E [Sydney Univ. (Australia). Dept. of Biochemistry

    1983-09-01

    Proton spin-echo nuclear magnetic resonance spectroscopy was used to measure the relative levels of some metabolites in intact human erythrocytes that had been fractionated by density gradient centrifugation. Age dependent changes in the concentrations of free glycine, choline and ergothioneine were seen for the first time, while glutathione was essentially invariant. In addition, there was a 10-fold decrease in creatine levels from the youngest to oldest cells. This confirms earlier reports and provides a simple explanation for the variable creatine resonance intensities seen in spectra obtained from different erythrocyte samples prepared from the same donor. The different chemical shifts of the methylene resonances of creatine and creatine phosphate was demonstrated and hence confirmed that the bulk of the creatine in intact erythrocytes is not phosphorylated. The chemical shift difference enabled the monitoring of the creatine phosphokinase catalysed reaction in lysates to which the rabbit muscle enzyme had been added. This experiment indicated that the enzyme is not significantly inhibited by factors in the lysates, and introduced a new means of assaying the in situ activity of the enzyme.

  19. Proton NMR studies of creatine in human erythrocytes

    International Nuclear Information System (INIS)

    Kuchel, P.W.; Chapman, B.E.

    1983-01-01

    Proton spin-echo nuclear magnetic resonance spectroscopy was used to measure the relative levels of some metabolites in intact human erythrocytes that had been fractionated by density gradient centrifugation. Age dependent changes in the concentrations of free glycine, choline and ergothioneine were seen for the first time, while glutathione was essentially invariant. In addition, there was a 10-fold decrease in creatine levels from the youngest to oldest cells. This confirms earlier reports and provides a simple explanation for the variable creatine resonance intensities seen in spectra obtained from different erythrocyte samples prepared from the same donor. The different chemical shifts of the methylene resonances of creatine and creatine phosphate was demonstrated and hence confirmed that the bulk of the creatine in intact erythrocytes is not phosphorylated. The chemical shift difference enabled the monitoring of the creatine phosphokinase catalysed reaction in lysates to which the rabbit muscle enzyme had been added. This experiment indicated that the enzyme is not significantly inhibited by factors in the lysates, and introduced a new means of assaying the in situ activity of the enzyme. (author)

  20. Erythrocyte metallothionein as an index of zinc status in humans

    International Nuclear Information System (INIS)

    Grider, A.; Bailey, L.B.; Cousins, R.J.

    1990-01-01

    Metallothionein concentrations in erythrocyte lysates derived from human subjects were measured by an ELISA procedure. IgG obtained from serum of sheep injected with human metallothionein 1 was used in this competitive assay. Subjects were fed a semipurified zinc-deficient diet for an 8-day depletion period after 3 days of acclimation. Fasting plasma zinc concentrations were reduced ∼7%. Metallothionein in the erythrocyte lysates was significantly decreased to 59% of the initial level by the end of the depletion period. Supplementation of these depleted subjects with zinc did not increase erythrocyte metallothionein levels within 24 hr. Daily supplementation of control subjects with zinc increased erythrocyte metallothionein to a 7-fold maximum within 7 days. These levels were reduced by 61% within 14 days after zinc supplementation was terminated. Incubation of rat [ 35 S]metallothionein with human erythrocyte lysate showed a time-dependent increase in 35 S soluble in 20% trichloroacetic acid, indicating degradation of the labeled protein, presumably via protease activity in the lysate. It is proposed that zinc supplementation induces erythrocyte metallothionein during erythropoiesis and that low zinc intake decreases synthesis and/or accelerates degradation of the protein in reticulocytes/erythrocytes. Metallothionein levels in erythrocytes may provide a useful index upon which to assess zinc status in humans

  1. Loading of erythrocyte membrane with pentacyclic triterpenes inhibits Plasmodium falciparum invasion

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staalsø, Trine; Jaroszewski, Jerzy W

    2006-01-01

    Lupeol and betulinic acid inhibit the proliferation of Plasmodium falciparum parasites by inhibition of the invasion of merozoites into erythrocytes. This conclusion is based on experiments employing parasite cultures synchronized by magnetic cell sorting (MACS). Identical inhibitory effects were...

  2. Spin labelling of human erythrocytes with nitroxide radicals

    International Nuclear Information System (INIS)

    Chagalj, C.; DePaoli, T.C.P.; Hager, A.A.; Palaoro, L.A.; Rubin de Celis, E.; Farach, H.A.; Poole, C.P. jr

    1984-01-01

    Human erythrocytes were labelled with nitroxide, the spin label SYNVAR 101, under various experimantal conditions. A study was made of the influence of antireductants on the labelling efficiency and the kinetics of the radical decay during the labelling process

  3. Blood-group-Ii-active gangliosides of human erythrocyte membranes

    International Nuclear Information System (INIS)

    Feizi, T.; Childs, R.A.; Hakomori, S.-I.; Powell, M.E.

    1978-01-01

    More than ten new types of gangliosides, in addition to haematoside and sialosylparagloboside, were isolated from human erythrocyte membranes. These were separated by successive chromatographies on DAEA-Sephadex, on porous silica-gel columns and on thin-layer silica gel as acetylated compounds. Highly potent blood-group-Ii and moderate blood-group-H activities were demonstrated in some of the ganglioside fractions. The gangliosides incorporated into chlolesterol/phosphatidylcholine liposomes stoicheiometrically inhibited binding of anti-(blood-group-I and i) antibodies to a radioiodinated blood-group-Ii-active glycoprotein. The fraction with the highest blood-group-I activity, I(g) fraction, behaved like sialosyl-deca- to dodeca-glycosylceramides on t.l.c. Certain blood-group-I and most of the i-determinants were in partially or completely cryptic form and could be unmasked by sialidase treatment. Thus the I and i antigens, which are known to occur on internal structures of blood-group-ABH-active glycoproteins in secretions, also occur in the interior of the carbohydrate chains of erythrocyte gangliosides. (author)

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

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    Kriti Tyagi

    Full Text Available 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.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.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.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.

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

    Science.gov (United States)

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

    2015-01-01

    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. 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. 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. 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.

  6. Grape (Vitis vinifera) extracts protects against radiation-induced oxidative stress in human erythrocyte (RBC)

    International Nuclear Information System (INIS)

    Ghosh, Subhashis

    2016-01-01

    Ionizing radiation (IR) causes oxidative stress through the overwhelming generation of reactive oxygen species (ROS) in the living cells leading further to the oxidative damage to biomolecules. Grapes (Vitis vinifera) contain several bioactive phytochemicals and are the richest source of antioxidant. In this study, we investigated the radioprotective actions of the grape extracts of two different cultivars, including the Thompson seedless (green) and Kishmish chorni (black) in human erythrocytes. Pretreatment with grape extracts attenuates oxidative stress induced by 4 Gy-radiation in human erythrocytes in vitro. These results suggest that grape extract serve as a potential source of natural antioxidants against the IR-induced oxidative stress and also inhibit apoptosis. Furthermore, the protective action of grape depends on the source of extract (seed, skin or pulp) and type of the cultivars. Effects of grape extracts of different cultivars on protein content, Thiobarbituric acid reactive substances (TBARS) level, reduced glutathione (GSH) content and activities of Catalase, Nitrite, GST, GR in human erythrocytes against -radiation exposure at a dose of 4 Gy are investigated. The grape extracts did not appear to alter the viability of human erythrocytes. Exposure of erythrocytes to the -irradiation at a dose of 4 Gy significantly increased the extent of formation of TBARS, while decreased the level of GSH and activities of CAT, GSSG , GST, GR in the erythrocytes as compared to the non-irradiated control counterparts. This was significantly attenuated by the pretreatment with the grape seed extracts (p<0.001) and significantly with the skin extracts (p<0.05) compared to the ionizing radiation exposed group. Moreover, protection offered by the seed extracts was found significantly better than that was offered by the pulp extract of the same cultivar. In conclusion, our results suggested that the grape extracts significantly attenuated IR induced oxidative stress and

  7. Aquaporin-1-Mediated Effects of Low Level He-Ne Laser Irradiation on Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Gang-Yue Luo

    2012-01-01

    Full Text Available The role of membrane aquaporin-1 (APQ-1 in the photobiomodulation (PBM on erythrocyte deformability will be studied in this paper with human dehydrated erythrocytes as echinocytic shape alterations lead to decreased cellular deformability. Human dehydrated erythrocytes were irradiated with low intensity He-Ne laser irradiation (LHNL at 0.9, 1.8, 2.7, and 4.4 mW/cm2 for 5, 15, and 30 min, respectively, and APQ-1 inhibitor, 0.2 μmol/L HgCl2, was used to study the role of APQ-1 in mediating PBM with LHNL at 4.4 mW/cm2 for 5 min. Comprehensive morphological parameters of an intact cell such as contact area, perimeter, roundness and erythrocyte elongation index (EEI were measured to characterize erythrocyte deformability with fast micro multi-channel spectrophotometer. It was observed that the dosage of LHNL improvement of the morphological parameters of dehydrated erythrocytes was morphological-parameter-dependent, but the Bunsen-Roscoe rule did not hold for roundness. The LHNL at 4.4 mW/cm2 for 5 min significantly improved the contact area (P<0.05 and EEI (P<0.05 of the dehydrated erythrocytes, but the improvement was significantly inhibited by 0.2 μmol/L HgCl2 (P<0.05. It was concluded that AQP-1 might mediate the effects of LHNL on erythrocyte deformability, which supports the membranotropic mechanism of PBM.

  8. Functional and structural changes of human erythrocyte catalase induced by cimetidine: proposed model of binding.

    Science.gov (United States)

    Yazdi, Fatemeh; Minai-Tehrani, Dariush; Jahngirvand, Mahboubeh; Almasirad, Ali; Mousavi, Zahra; Masoud, Masoudeh; Mollasalehi, Hamidreza

    2015-06-01

    In erythrocyte, catalase plays an important role to protect cells from hydrogen peroxide toxicity. Hydrogen peroxide is a byproduct compound which is produced during metabolic pathway of cells. Cimetidine, a histamine H2 receptor antagonist, is used for gastrointestinal tract diseases and prevents the extra release of gastric acid. In this study, the effect of cimetidine on the activity of human erythrocyte catalase was investigated. Erythrocytes were broken by hypotonic solution. The supernatant was used for catalase assay and kinetics study. Lineweaver-Burk plot was performed to determine the type of inhibition. The kinetics data revealed that cimetidine inhibited the catalase activity by mixed inhibition. The IC50 (1.54 μM) and Ki (0.45 μM) values of cimetidine determined that the drug was bound to the enzyme with high affinity. Circular dichroism and fluorescence measurement showed that the binding of cimetidine to the enzyme affected the content of secondary structure of the enzyme as well as its conformational changes. Docking studies were carried out to detect the site in which the drug was bound to the enzyme. Molecular modeling and energy calculation of the binding showed that the cyanoguanidine group of the drug connected to Asp59 via two hydrogen bonds, while the imidazole group of the drug interacted with Phe64 in the enzyme by a hydrophobic interaction. In conclusion, cimetidine could bind to human erythrocyte catalase, and its interaction caused functional and conformational changes in the enzyme.

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

    DEFF Research Database (Denmark)

    Brekke, O. L.; Hellerud, B. C.; Christiansen, D.

    2011-01-01

    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...... 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...

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  11. Alcohol and the calcium-dependent potassium transport of human erythrocytes

    International Nuclear Information System (INIS)

    Harris, R.A.; Caldwell, K.K.

    1985-01-01

    In vitro exposure of human red blood cells to ethanol (100 and 400 mM) was found to increase the initial rate of calcium-dependent potassium efflux through the red cell membrane. This effect of ethanol was apparently not due to an elevation of the intracellular free calcium but rather to a direct action of the drug on the transport process as, (1) intracellular calcium concentrations were tightly buffered with EGTA, (2) ethanol did not alter the efflux of 45 Ca from the cells, and (3) dantrolene, which has been proposed to counteract the effect of ethanol on intracellular calcium levels in the erythrocyte, did not inhibit the stimulatory action of ethanol. The efflux of potassium from erythrocytes obtained from chronic alcoholics was not different from that of erythrocytes from non-alcoholic individuals. The relationship of these findings to neuronal potassium transport is discussed

  12. The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase.

    Science.gov (United States)

    Haggarty, N W; Dunbar, B; Fothergill, L A

    1983-01-01

    The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase, comprising 239 residues, was determined. The sequence was deduced from the four cyanogen bromide fragments, and from the peptides derived from these fragments after digestion with a number of proteolytic enzymes. Comparison of this sequence with that of the yeast glycolytic enzyme, phosphoglycerate mutase, shows that these enzymes are 47% identical. Most, but not all, of the residues implicated as being important for the activity of the glycolytic mutase are conserved in the erythrocyte diphosphoglycerate mutase. PMID:6313356

  13. Determination of somatic mutations in human erythrocytes by cytometry

    International Nuclear Information System (INIS)

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

    1985-01-01

    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

  14. Determination of somatic mutations in human erythrocytes by cytometry

    Energy Technology Data Exchange (ETDEWEB)

    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.

  15. Metallic mercury uptake by catalase Part 1 In Vitro metallic mercury uptake by various kind of animals' erythrocytes and purified human erythrocyte catalase

    OpenAIRE

    劒持,堅志

    1980-01-01

    The uptake of metallic mercury was studied using erythrocytes with different catalase activities taken from various kind of animals. The results were: 1) The uptake of metallic mercury by erythrocytes paralleled the activity of catalase in the erythrocytes with and without hydrogen peroxide, suggesting that the erythrocyte catalase activity is related to the uptake of metallic mercury. 2) The uptake of metallic mercury occurred not only with purified human erythrocyte catalase but also with h...

  16. The role of inorganic phosphate in intact human erythrocytes

    International Nuclear Information System (INIS)

    Nishiguchi, Eiko; Umeda, Masahiro.

    1988-01-01

    The role of inorganic phosphate in intact human erythrocytes was investigated by phosphorus-31 nuclear magnetic resonance ( 31 P NMR). When erythrocytes stored for 5 weeks were incubated at 37 deg C, pH 7.4, in medium containing 2 mM adenine and 10 mM inosine, with or without 5 mM glucose, a substance of around 4 ppm, as assessed by 31 P NMR chemical shift, was detected in the mixture. However, this substance disappeared by the addition of inorganic phosphate. When erythrocytes stored for 4 weeks in acid citrate dextrose (ACD) solution were incubated with 2 mM adenine, 10 mM inosine, 5 mM glucose, 50 mM inorganic phosphate and 10 mM pyruvate at 37 deg C, pH 7.4, the 2,3-DPG level increased gradually, whereas the ATP level initially increased and then decreased. Intracellular inorganic phosphate appeared to be used for the synthesis of ATP and 2,3-DPG during the first 30 min. of the reaction. These results suggests that the inorganic phosphate accelerates glycolysis by increasing the activity of glycolytic enzymes rather than its direct involvement in synthesizing organic phosphorus compounds in stored erythrocytes. The results also suggests that the reserve energy from ATP synthesis is not sufficient for the synthesis of 2,3-DPG. (author)

  17. Role of aminotransferases in glutamate metabolism of human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Ellinger, James J. [University of Wisconsin-Madison, Department of Biochemistry (United States); Lewis, Ian A. [Princeton University, Lewis-Sigler Institute for Integrative Genomics (United States); Markley, John L., E-mail: markley@nmrfam.wisc.edu [University of Wisconsin-Madison, Department of Biochemistry (United States)

    2011-04-15

    Human erythrocytes require a continual supply of glutamate to support glutathione synthesis, but are unable to transport this amino acid across their cell membrane. Consequently, erythrocytes rely on de novo glutamate biosynthesis from {alpha}-ketoglutarate and glutamine to maintain intracellular levels of glutamate. Erythrocytic glutamate biosynthesis is catalyzed by three enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and glutamine aminohydrolase (GA). Although the presence of these enzymes in RBCs has been well documented, the relative contributions of each pathway have not been established. Understanding the relative contributions of each biosynthetic pathway is critical for designing effective therapies for sickle cell disease, hemolytic anemia, pulmonary hypertension, and other glutathione-related disorders. In this study, we use multidimensional {sup 1}H-{sup 13}C nuclear magnetic resonance (NMR) spectroscopy and multiple reaction mode mass spectrometry (MRM-MS) to measure the kinetics of de novo glutamate biosynthesis via AST, ALT, and GA in intact cells and RBC lysates. We show that up to 89% of the erythrocyte glutamate pool can be derived from ALT and that ALT-derived glutamate is subsequently used for glutathione synthesis.

  18. Bifenthrin-induced oxidative stress in human erythrocytes in vitro and protective effect of selected flavonols.

    Science.gov (United States)

    Sadowska-Woda, Izabela; Popowicz, Diana; Karowicz-Bilińska, Agata

    2010-03-01

    Bifenthrin is a synthetic pyrethroid with a broad spectrum of insecticidal and acaricidal activity used to control wide range of insect pests in a variety of applications. This investigation was designed to examine (1) bifenthrin as an inducer of oxidative stress in human erythrocytes in vitro through effects on catalase (CAT) and superoxide dismutase (SOD) activities, and (2) the role of the flavonoids quercetin (Q, 40 and 80microM) and rutin (R, 80microM) in alleviating the effects of bifenthrin. Erythrocytes were divided into portions. The first portion was incubated for 4h at 37 degrees C with different concentrations (0, 42.2, 211, 1055ppm) of bifenthrin. The other portions were preincubated with Q or R for 30min, followed incubation with bifenthrin for 4h. The influence of solvent (ethanol) was also checked on the parameters studied. Malondialdehyde (MDA) concentrations, CAT and SOD activities were measured in all treatment portions of erythrocytes. Our results demonstrated that bifenthrin-induced oxidative stress causes enhanced lipid peroxidation and decreased antioxidative enzyme activities in human peripheral blood. R pretreated erythrocytes were protected against the increase of MDA induced by bifenthrin. Q (80microM) and R pretreated erythrocytes were protected against the inhibition of CAT activity induced by bifenthrin. The protective action against the inhibition of SOD activity of Q was greater than that of R at the same concentration. These results suggest that Q and R may play a role in reducing bifenthrin-induced oxidative stress in vitro. Copyright (c) 2009 Elsevier Ltd. All rights reserved.

  19. Purification and properties of enolase of human erythrocytes

    NARCIS (Netherlands)

    Hoorn, R.K.J.; Flikweert, J.P.; Staal, Gerard E.J.

    1974-01-01

    1. 1. Human erythrocyte enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was purified I000-fold. 2. 2. The pH-optimum was at pH 6.5. The molecular weight, estimated by gel filtration, was found to be 95,000 ± 5,000. 3. 3. Electrophoresis on agar-agarose at pH 8.5 and 6.4 showed only one

  20. The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase.

    OpenAIRE

    Haggarty, N W; Dunbar, B; Fothergill, L A

    1983-01-01

    The complete amino acid sequence of human erythrocyte diphosphoglycerate mutase, comprising 239 residues, was determined. The sequence was deduced from the four cyanogen bromide fragments, and from the peptides derived from these fragments after digestion with a number of proteolytic enzymes. Comparison of this sequence with that of the yeast glycolytic enzyme, phosphoglycerate mutase, shows that these enzymes are 47% identical. Most, but not all, of the residues implicated as being important...

  1. The erythrocyte membrane in human muscular dystrophy

    NARCIS (Netherlands)

    W. Ruitenbeek (Willem)

    1979-01-01

    textabstractMore than 250 different forms of human neuromuscular diseases are known. They differ in age of onset, severity of weakness, rate of progression, type of inheritance, groups of muscles affected, frequency of incidence. Sometimes the clinical symptoms are not restricted to nervous

  2. The effects of chemical and radioactive properties of Tl-201 on human erythrocyte glucose 6-phosphate dehydrogenase activity

    International Nuclear Information System (INIS)

    Sahin, Ali; Senturk, Murat; Ciftci, Mehmet; Varoglu, Erhan; Kufrevioglu, Omer Irfan

    2010-01-01

    Aim: The inhibitory effects of thallium-201 ( 201 Tl) solution on human erythrocyte glucose 6-phosphate dehydrogenase (G6PD) activity were investigated. Methods: For this purpose, erythrocyte G6PD was initially purified 835-fold at a yield of 41.7% using 2',5'-Adenosine diphosphate sepharose 4B affinity gel chromatography. The purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which showed a single band for the final enzyme preparation. The in vitro and in vivo effects of the 201 Tl solution including Tl + , Fe +3 and Cu +2 metals and the in vitro effects of the radiation effect of the 201 Tl solution and non-radioactive Tl + , Fe +3 and Cu +2 metals on human erythrocyte G6PD enzyme were studied. Enzyme activity was determined with the Beutler method at 340 nm using a spectrophotometer. All purification procedures were carried out at +4 deg. C. Results: 201 Tl solution and radiation exposure had inhibitory effects on the enzyme activity. IC 50 value of 201 Tl solution was 36.86 μl ([Tl + ]: 0.0036 μM, [Cu +2 ]: 0.0116 μM, [Fe +3 ]: 0.0132 μM), of human erythrocytes G6PD. Seven human patients were also used for in vivo studies of 201 Tl solution. Furthermore, non-radioactive Tl + , Fe +3 and Cu +2 were found not to have influenced the enzyme in vitro. Conclusion: Human erythrocyte G6PD activity was inhibited by exposure for up to 10 minutes to 0.057 mCi/kg 201 Tl solution. It was detected in in vitro and in vivo studies that the human erythrocyte G6PD enzyme is inhibited due to the radiation effect of 201 Tl solution.

  3. Human erythrocyte electrofusion kinetics monitored by aqueous contents mixing.

    OpenAIRE

    Stenger, D A; Hui, S W

    1988-01-01

    The kinetics of electrically induced fusion of human erythrocyte ghosts were monitored by the Tb/DPA and ANTS/DPX fluorescence fusion assays. Ghosts were aligned by dielectrophoresis using a 3-MHz 350-V/cm alternating field and were fused by single 15- or 50-microseconds electric field pulses of amplitude 2.5-5.0 kV/cm. Fusion was detected immediately after the pulse. The peak fluorescence change due to fusion was always obtained within 7 s of pulse application, and was highest for a 5.0 kV/c...

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

    International Nuclear Information System (INIS)

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

    2007-01-01

    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 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

  5. Effects of an angelica extract on human erythrocyte aggregation, deformation and osmotic fragility.

    Science.gov (United States)

    Wang, X; Wei, L; Ouyang, J P; Muller, S; Gentils, M; Cauchois, G; Stoltz, J F

    2001-01-01

    In Chinese traditional medicine, angelica is widely used for its known clinical effects of ameliorating blood microcirculation. But the mechanism of these beneficial effects still remains unclear. In this work the rheological behaviour of human erythrocytes treated by angelica was studied in vitro. Normal RBCs incubated with an angelica extract at different concentrations (5, 10 or 20 mg/ml) for 60 min at 37 degrees C and then their aggregation, deformation and osmotic fragility were measured with different recently developed optical techniques, namely Erythroaggregometer (Regulest, Florange, France), LORCA (Mechatronics, Amsterdam) and Fragilimeter (Regulest, Florange, France). Experimental results show that angelica (20 mg/ml) significantly decreased normal RBCs' aggregation speed (p<0.01) and could inhibit the hyperaggregability caused by dextran 500. However, the strength of normal RBCs aggregates were not influenced by angelica. When a calcium ionophore A23187 (1.9 microM) was used to harden cell membrane, angelica (20 mg/ml) could significantly (p<0.01) protect erythrocytes against the loss of their deformability even it had no effects on normal RBCs deformation. Finally angelica (5 and 10 mg/ml) decreased significantly (p<0.01) normal RBCs osmotic fragility. In conclusion angelica plays a rheologically active role on human erythrocytes, and this study suggests a possible mechanism for angelica's positive effects against certain cardiovascular diseases.

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

    Directory of Open Access Journals (Sweden)

    Jefferson Muniz de Lima

    2015-01-01

    Full Text Available 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.

  7. Erythrocytes L-arginine y+ transporter inhibition by N-ethylmaleimide in ice-bath.

    Science.gov (United States)

    Pinheiro da Costa, Bartira Ercília; de Almeida, Priscilla Barcellos; Conceição, Ioná Rosine; Antonello, Ivan Carlos Ferreira; d'Avila, Domingos O; Poli-de-Figueiredo, Carlos Eduardo

    2010-11-01

    Erythrocytes L: -arginine uptake is conveyed by y+ and y+L membrane transport systems. Pre-incubation with N-ethylmaleimide for 10 min at 37°C inhibits the y+ system. The aim of this study was to determine the ideal pre-incubation temperature in evaluating y+ and y+L systems. Cells were pre-incubated with or without N-ethylmaleimide for 10 min at 4°C and 37°C. L: -Arginine uptake was quantified by radioisotope and standard erythrocytes membrane flux methodology. Results demonstrate that erythrocytes L: -arginine content is depleted by pre-incubation at 37°C for 10 min, thus changing the V (max) measurement. The inhibitory effect of N-ethylmaleimide pre-incubation was temperature independent and already complete after 1 min of incubation. No significant difference in kinetic parameters was detected between cells pre-incubated at 37°C or 4°C, under zero-trans conditions. In conclusion, we suggest that measurement of erythrocytes L: -arginine uptake by y+ and y+L systems could be carried out without N-ethylmaleimide pre-incubation at 37°C.

  8. Acute dark chocolate ingestion is beneficial for hemodynamics via enhancement of erythrocyte deformability in healthy humans.

    Science.gov (United States)

    Radosinska, Jana; Horvathova, Martina; Frimmel, Karel; Muchova, Jana; Vidosovicova, Maria; Vazan, Rastislav; Bernatova, Iveta

    2017-03-01

    Erythrocyte deformability is an important property of erythrocytes that considerably affects blood flow and hemodynamics. The high content of polyphenols present in dark chocolate has been reported to play a protective role in functionality of erythrocytes. We hypothesized that chocolate might influence erythrocytes not only after repeated chronic intake, but also immediately after its ingestion. Thus, we determined the acute effect of dark chocolate and milk (with lower content of biologically active substances) chocolate intake on erythrocyte deformability. We also focused on selected factors that may affect erythrocyte deformability, specifically nitric oxide production in erythrocytes and total antioxidant capacity of plasma. We determined posttreatment changes in the mentioned parameters 2hours after consumption of chocolate compared with their levels before consumption of chocolate. In contrast to milk chocolate intake, the dark chocolate led to a significantly higher increase in erythrocyte deformability. Nitric oxide production in erythrocytes was not changed after dark chocolate intake, but significantly decreased after milk chocolate. The plasma total antioxidant capacity remained unaffected after ingestion of both chocolates. We conclude that our hypothesis was confirmed. Single ingestion of dark chocolate improved erythrocyte deformability despite unchanged nitric oxide production and antioxidant capacity of plasma. Increased deformability of erythrocytes may considerably improve rheological properties of blood and thus hemodynamics in humans, resulting in better tissue oxygenation. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Trifluoperazine-Induced Suicidal Erythrocyte Death and S-Nitrosylation Inhibition, Reversed by the Nitric Oxide Donor Sodium Nitroprusside

    Directory of Open Access Journals (Sweden)

    Mehrdad Ghashghaeinia

    2017-08-01

    Full Text Available Background and Purpose: The high potency antipsychotic drug trifluoperazine (10-[3-(4-methyl-1-piperazinyl-propyl]-2-(trifluoromethyl-(10H-phenothiazine dihydrochloride; TFP may either counteract or promote suicidal cell death or apoptosis. Similar to apoptosis, erythrocytes may enter eryptosis, characterized by phosphatidylserine exposure at the cell surface and cell shrinkage. Eryptosis can be stimulated by an increase in cytoplasmic Ca2+ concentration ([Ca2+]i and inhibited by nitric oxide (NO. We explored whether TFP treatment of erythrocytes induces phosphatidylserine exposure, cell shrinkage, and calcium influx, whether it impairs S-nitrosylation and whether these effects are inhibited by NO. Methods: Phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, and protein nitrosylation from fluorescence switch of the Bodipy-TMR/Sypro Ruby signal. Results: Exposure of human erythrocytes to TFP significantly enhanced the percentage of annexin-V-binding cells, raised [Ca2+]i, and decreased S-nitrosylation. The effect of TFP on annexin-V-binding was not affected by removal of extracellular Ca2+ alone, but was significantly inhibited by pre-treatment with sodium nitroprusside (SNP, an effect significantly augmented by additional removal of extracellular Ca2+. A 3 hours treatment with 0.1 µM Ca2+ ionophore ionomycin triggered annexin-V-binding and cell shrinkage, effects fully reversed by removal of extracellular Ca2+. Conclusions: TFP induces eryptosis and decreases protein S-nitrosylation, effects blunted by nitroprusside. The effect of nitroprusside is attenuated in the presence of extracellular Ca2+.

  10. Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

    Science.gov (United States)

    Vandorpe, David H; Xu, Chang; Shmukler, Boris E; Otterbein, Leo E; Trudel, Marie; Sachs, Frederick; Gottlieb, Philip A; Brugnara, Carlo; Alper, Seth L

    2010-01-15

    Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca(2+)] ([Ca(2+)](i)) and subsequent activation of K(Ca) 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+)- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM), dipyridamole (100 microM), DIDS (100 microM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca(2+)](i), in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+)](i) in mouse sickle erythrocytes did not require KCa3.1 activity. The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+)-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+) for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

  11. Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

    Directory of Open Access Journals (Sweden)

    David H Vandorpe

    2010-01-01

    Full Text Available Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle, leading to elevated intracellular [Ca(2+] ([Ca(2+](i and subsequent activation of K(Ca 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration.We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca(2+- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 microM, dipyridamole (100 microM, DIDS (100 microM, and carbon monoxide (25 ppm pretreatment. Deoxygenation also elevates sickle erythrocyte [Ca(2+](i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca(2+](i in mouse sickle erythrocytes did not require KCa3.1 activity.The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca(2+-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca(2+ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Inc. All rights reserved.

  13. Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

    OpenAIRE

    Matteucci, Elena; Giampietro, Ottavio

    2007-01-01

    Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+ exchange and HC3 -/Cl- anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore dama...

  14. Monoclonal antibody OKM5 inhibits the in vitro binding of Plasmodium falciparum-infected erythrocytes to monocytes, endothelial, and C32 melanoma cells

    International Nuclear Information System (INIS)

    Barnwell, J.W.; Ockenhouse, C.F.; Knowles, D.M. II

    1985-01-01

    Plasmodium falciparum-infected erythrocytes bind in vitro to human endothelial cells, monocytes, and a certain melanoma cell line. Evidence suggests that this interaction is mediated by similar mechanisms which lead to the sequestration of parasitized erythrocytes in vivo through their attachment to endothelial cells of small blood vessels. They show here the monoclonal antibody OKM5, previously shown to react with the membranes of endothelial cells, monocyte,s and platelets, also reacts with the C32 melanoma cell line which also binds P. falciparum-infected erythrocytes. At relatively low concentrations, OKM5 inhibits and reverses the in vitro adherence of infected erythrocytes to target cells. As with monocytes, OKM5 antibody recognizes an 125 I-labeled protein of approximately 88 Kd on the surface of C32 melanoma cells. It seems likely, therefore, that the 88 Kd polypeptide plays a role in cytoadherence, possibly as the receptor or part of a receptor for a ligand on the surface of infected erythrocytes

  15. Solubilization of human erythrocyte membranes by ASB detergents

    Directory of Open Access Journals (Sweden)

    C.C. Domingues

    2008-09-01

    Full Text Available Understanding the membrane solubilization process and finding effective solubilizing agents are crucial challenges in biochemical research. Here we report results on the interaction of the novel linear alkylamido propyl dimethyl amino propanosulfonate detergents, ASB-14 and ASB-16, with human erythrocyte membranes. An estimation of the critical micelle concentration of these zwitterionic detergents (ASB-14 = 100 µM and ASB-16 = 10 µM was obtained using electron paramagnetic resonance. The amount of proteins and cholesterol solubilized from erythrocytes by these detergents was then determined. The hemolytic activities of the ASB detergents were assayed and the detergent/lipid molar ratios for the onset of hemolysis (Re sat and total lysis (Re sol were calculated, allowing the determination of the membrane binding constants (Kb. ASB-14 presented lower membrane affinity (Kb = 7050 M-1 than ASB-16 (Kb = 15610 M-1. The amount of proteins and cholesterol solubilized by both ASB detergents was higher while Re sat values (0.22 and 0.08 detergent/lipid for ASB-14 and ASB-16, respectively were smaller than those observed with the classic detergents CHAPS and Triton X-100. These results reveal that, besides their well-known use as membrane protein solubilizers to enhance the resolution of two dimensional electrophoresis/mass spectrometry, ASB-14 and ASB-16 are strong hemolytic agents. We propose that the physicochemical properties of ASB detergents determine their membrane disruption efficiency and can help to explain the improvement in the solubilization of membrane proteins, as reported in the literature.

  16. Comparative studies on osmosis based encapsulation of sodium diclofenac in porcine and outdated human erythrocyte ghosts.

    Science.gov (United States)

    Bukara, Katarina; Drvenica, Ivana; Ilić, Vesna; Stančić, Ana; Mišić, Danijela; Vasić, Borislav; Gajić, Radoš; Vučetić, Dušan; Kiekens, Filip; Bugarski, Branko

    2016-12-20

    The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials - porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process - gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  18. Grape extract protects against γ-radiation-induced membrane damage strains of human erythrocytes

    International Nuclear Information System (INIS)

    Das, Subir Kumar

    2017-01-01

    The membrane integrity of circulating red blood cells (RBCs) is compromised by the deleterious actions of γ-radiation in humans. Grapes are the richest source of antioxidants due to presence of potentially bioactive phytochemicals. The objective of the present study was to assess the radioprotective actions of grape extracts against the γ-radiation-induced membrane permeability of human erythrocytes. The scavenging activities in seeds of grape in DPPH, hydrogen peroxide and hydroxyl radicals, were higher than skin or pulp of different cultivars. Grape extracts also showed appreciable extent of total antioxidant capacity and effective antihemolytic action. Grape extracts significantly ameliorated the γ-radiation-induced increase of the levels of thiobarbituric acid-reactive substances (TBARS, an index of lipid peroxidation) in the RBC membrane ghosts. Stored blood showed higher levels of K + ion as compared to the normal blood which was elevated by γ-radiation. Membrane ATPase was inhibited by the exposure to γ-radiation.Treatment of RBCs with the grape extracts prior to the exposure of γ-radiation significantly mitigated these changes in the erythrocyte membranes caused by the lower dose of radiation (4 Gy). (author)

  19. Isolation of low-molecular-weight lead-binding protein from human erythrocytes

    International Nuclear Information System (INIS)

    Raghavan, S.R.V.; Gonick, H.C.

    1977-01-01

    In blood, lead is mainly associated with erythrocytes and only a very small amount is found in plasma. Previously it was thought that the lead was bound to the erythrocyte cell membrane but more recently it has been observed that lead is bound primarily to the cell contents, ostensibly hemoglobin. In examining the lead-binding properties of normal human erythrocytes and those of lead-exposed industrial workers, we have found that, whereas lead binds only to hemoglobin in normal erythrocytes, there is also appreciable binding of lead to a low-molecular weight-protein in erythrocytes from lead-exposed workers. The synthesis of this protein may be induced by lead exposure. The 10,000 molecular weight protein may act as a storage site and mechanism for segregating lead in a non-toxic form

  20. Diffusion properties of band 3 in human erythrocytes

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Suwalsky, Mario, E-mail: msuwalsk@udec.cl [Faculty of Chemical Sciences, University of Concepcion, Concepcion (Chile); Zambrano, Pablo; Mennickent, Sigrid [Faculty of Pharmacy, University of Concepcion, Concepcion (Chile); Villena, Fernando [Faculty of Biological Sciences, University of Concepcion, Concepcion (Chile); Sotomayor, Carlos P.; Aguilar, Luis F. [Instituto de Quimica, Pontificia Universidad Catolica de Valparaiso, Valparaiso (Chile); Bolognin, Silvia [CNR-Institute for Biomedical Technologies, University of Padova, Padova (Italy)

    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. PIXE elemental analysis of erythrocyte and blood plasma samples from human pregnancies

    International Nuclear Information System (INIS)

    Borbely-Kiss, I.; Koltay, E.; Laszlo, S.; Szabo, Gy.

    1984-01-01

    Elemental concentrations of P, S, Cl, K, Ca, Fe, Ni, Cu, Zn, Br, Rb have been determined in erythrocyte and blood plasma samples from normal and diabetic human pregnancies. Average values, the dependence of the concentrations on the time during gestation period, the correlation coefficients for pairs of elements as well as for the same elements in plasma and erythrocyte samples are given. A marked difference appeared in a number of cases between normal and diabetic pregnancies. (author)

  3. PIXE elemental analysis of erythrocyte and blood plasma samples from human pregnancies

    Energy Technology Data Exchange (ETDEWEB)

    Borbely-Kiss, I; Koltay, E; Laszlo, S; Szabo, Gy [Magyar Tudomanyos Akademia, Debrecen. Atommag Kutato Intezete; Goedeny, S [Orvostudomanyi Egyetem, Szeged (Hungary). Szueleszeti es Noegyogyaszati Klinika; Seif El-Nasr, S [Teachers' Coll. for Women, Samia (Kuwait)

    1984-07-01

    Elemental concentrations of P, S, Cl, K, Ca, Fe, Ni, Cu, Zn, Br, Rb have been determined in erythrocyte and blood plasma samples from normal and diabetic human pregnancies. Average values, the dependence of the concentrations on the time during gestation period, the correlation coefficients for pairs of elements as well as for the same elements in plasma and erythrocyte samples are given. A marked difference appeared in a number of cases between normal and diabetic pregnancies. 11 refs.

  4. Fragmentation of Human Erythrocyte Actin following Exposure to Hypoxia

    Czech Academy of Sciences Publication Activity Database

    Risso, A.; Santamaria, B.; Pistarino, E.; Cosulich, M. E.; Pompach, Petr; Bezouška, Karel; Antonutto, G.

    2010-01-01

    Roč. 123, č. 1 (2010), s. 6-13 ISSN 0001-5792 Institutional research plan: CEZ:AV0Z50200510 Keywords : beta-Actin * Erythrocytes * Hypoxia Subject RIV: EE - Microbiology, Virology Impact factor: 1.316, year: 2010

  5. Phosphorylation of intact erythrocytes in human muscular dystrophy

    International Nuclear Information System (INIS)

    Johnson, R.M.; Nigro, M.

    1986-01-01

    The uptake of exogenous 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-[ 32 P]ATP, and suggests a possible reinterpretation of those experiments

  6. Erratum Detergent-resistant membranes in human erythrocytes and ...

    Indian Academy of Sciences (India)

    Unknown

    Figure 3. Immunodetection of flotillin-2 and band 3 in DRMs isolated from erythrocyte ghosts by various treatments. Flotillin-2. (left) and band 3 (right) Western blotting in ten fractions of 0⋅5 ml each, obtained from the sucrose gradients described in figure 2 and numbered from top to bottom. Flotillin-2 is enriched in DRMs ...

  7. Mercury chloride-induced oxidative stress in human erythrocytes ...

    African Journals Online (AJOL)

    ONOS

    2010-01-25

    Jan 25, 2010 ... Mercury can exist in the environment as metal, as monovalent and divalent salts and as organomercurials, one of the most important of which is mercuric chloride (HgCl2). It has been shown to induce oxidative stress in erythrocytes through the generation of free radicals and alteration of the.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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 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 μM; (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 μM concentration range, and (c) X-ray diffraction studies showed that Au(III) in the 10 μm-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 μM to 100 μM.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Qin Yan Zhu

    2005-01-01

    Full Text Available 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 > 8epicatechin (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.

  12. The Plasmodium falciparum erythrocyte invasion ligand Pfrh4 as a target of functional and protective human antibodies against malaria.

    Directory of Open Access Journals (Sweden)

    Linda Reiling

    Full Text Available BACKGROUND: Acquired antibodies are important in human immunity to malaria, but key targets remain largely unknown. Plasmodium falciparum reticulocyte-binding-homologue-4 (PfRh4 is important for invasion of human erythrocytes and may therefore be a target of protective immunity. METHODS: IgG and IgG subclass-specific responses against different regions of PfRh4 were determined in a longitudinal cohort of 206 children in Papua New Guinea (PNG. Human PfRh4 antibodies were tested for functional invasion-inhibitory activity, and expression of PfRh4 by P. falciparum isolates and sequence polymorphisms were determined. RESULTS: Antibodies to PfRh4 were acquired by children exposed to P. falciparum malaria, were predominantly comprised of IgG1 and IgG3 subclasses, and were associated with increasing age and active parasitemia. High levels of antibodies, particularly IgG3, were strongly predictive of protection against clinical malaria and high-density parasitemia. Human affinity-purified antibodies to the binding region of PfRh4 effectively inhibited erythrocyte invasion by P. falciparum merozoites and antibody levels in protected children were at functionally-active concentrations. Although expression of PfRh4 can vary, PfRh4 protein was expressed by most isolates derived from the cohort and showed limited sequence polymorphism. CONCLUSIONS: Evidence suggests that PfRh4 is a target of antibodies that contribute to protective immunity to malaria by inhibiting erythrocyte invasion and preventing high density parasitemia. These findings advance our understanding of the targets and mechanisms of human immunity and evaluating the potential of PfRh4 as a component of candidate malaria vaccines.

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

    International Nuclear Information System (INIS)

    Quintanar-Escorza, M.A.; Gonzalez-Martinez, M.T.; Navarro, L.; Maldonado, M.; Arevalo, B.; Calderon-Salinas, J.V.

    2007-01-01

    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 2+ -Mg 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 μg/dl) and 15 non-exposed workers (9.9 ± 2 μg/dl). In erythrocytes of lead-exposed workers, the intracellular free calcium was 79 ± 13 nM, a significantly higher concentration (ANOVA, P 2+ -Mg 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

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

    International Nuclear Information System (INIS)

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

    2008-01-01

    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 α-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 α-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

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

    Science.gov (United States)

    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.

  16. Caffeine inhibits erythrocyte membrane derangement by antioxidant activity and by blocking caspase 3 activation.

    Science.gov (United States)

    Tellone, Ester; Ficarra, Silvana; Russo, Annamaria; Bellocco, Ersilia; Barreca, Davide; Laganà, Giuseppina; Leuzzi, Ugo; Pirolli, Davide; De Rosa, Maria Cristina; Giardina, Bruno; Galtieri, Antonio

    2012-02-01

    The aim of this research was to investigate the effect of caffeine on band 3 (the anion exchanger protein), haemoglobin function, caspase 3 activation and glucose-6-phosphate metabolism during the oxygenation-deoxygenation cycle in human red blood cells. A particular attention has been given to the antioxidant activity by using in vitro antioxidant models. Caffeine crosses the erythrocyte membrane and interacts with the two extreme conformational states of haemoglobin (the T and the R-state within the framework of the simple two states allosteric model) with different binding affinities. By promoting the high affinity state (R-state), the caffeine-haemoglobin interaction does enhance the pentose phosphate pathway. This is of benefit for red blood cells since it leads to an increase of NADPH availability. Moreover, caffeine effect on band 3, mediated by haemoglobin, results in an extreme increase of the anion exchange, particularly in oxygenated erythrocytes. This enhances the transport of the endogenously produced CO(2) thereby avoiding the production of dangerous secondary radicals (carbonate and nitrogen dioxide) which are harmful to the cellular membrane. Furthermore caffeine destabilizes the haeme-protein interactions within the haemoglobin molecule and triggers the production of superoxide and met-haemoglobin. However this damaging effect is almost balanced by the surprising scavenger action of the alkaloid with respect to the hydroxyl radical. These experimental findings are supported by in silico docking and molecular dynamics studies and by what we may call the "caspase silence"; in fact, there is no evidence of any caspase 3 activity enhancement; this is likely due to the promotion of positive metabolic conditions which result in an increase of the cellular reducing power. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  17. Photoaffinity labeling of the human erythrocyte monosaccharide transporter with an aryl azide derivative of D-glucose

    International Nuclear Information System (INIS)

    Shanahan, M.F.; Wadzinski, B.E.; Lowndes, J.M.; Ruoho, A.E.

    1985-01-01

    A photoreactive, radioiodinated derivative of glucose, N-(4-iodoazidosalicyl)-6-amido-6-deoxyglucopyranose (IASA-glc), has been synthesized and used as a photoaffinity label for the human erythrocyte monosaccharide transporter. Photoinactivation and photoinsertion are both light-dependent and result in a marked decrease in the absorption spectra of the compound. When [ 125 I]IASA-glc was photolyzed with erythrocyte ghost membranes, photoinsertion of radiolabel was observed in three major regions, spectrin, band 3, and a protein of 58,000 daltons located in the zone 4.5 region. Of the three regions which were photolabeled, only labeling of polypeptides in the zone 4.5 region was partially blocked by D-glucose. In the non-iodinated form, N-(4-azidosalicyl)-6-amido-6-deoxy-glucopyranose inhibited the labeling of the transporter by [ 125 I]IASA-glc more effectively than D-glucose. The ability to synthesize this [ 125 I]containing photoprobe for the monosaccharide transporter at carrier-free levels offers several new advantages for investigating the structure of this transport protein in the erythrocyte

  18. The influence of split doses of γ-radiation on human erythrocytes

    International Nuclear Information System (INIS)

    Koziczak, R.; Gonciarz, M.; Krokosz, A.; Szweda-Lewandowska, Z.

    2003-01-01

    Human erythrocyte suspensions in an isotonic Na-phosphate buffer, pH 7.4, of hematocrit of 2% were exposed under air to gamma radiation at a dose rate of 2.2 kGy. Erythrocytes were irradiated with single doses, and identical doses split into two fractions with an interval time of 3.5 h between following exposures. The obtained results indicated that the irradiation of enucleated human erythrocytes with split doses caused a reduction of hemolysis (2.4 times), a decrease in the level of damage to membrane lipids and the contents of MetHb, compared with identical single doses. However, the splitting of radiation doses did not change the level of damage to the membrane proteins, as was estimated with a maleimide spin label. The obtained results suggest that a decrease in the level of damage to lipids was related to a decrease in hemolysis. (author)

  19. Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with 125I-derivatives of cymarin and digitoxigenin

    International Nuclear Information System (INIS)

    Lowndes, J.M.

    1988-01-01

    NaK-ATPase is an enzyme which maintains Na + and K + 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 α subunit but the glycoprotein β and proteolipid-γ 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 [ 125 I]AISC, [ 125 I]AIPP-GluD, [ 125 I]AIPP-GalD and [ 125 I]IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the α, β, and proteolipid-γ subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, [ 125 I]I-TASC, was synthesized; this compound had the same structure as [ 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 [ 125 I]I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct [ 125 I]I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K D of 54 pM and a B max of 23 fmol/mg protein

  20. Regulation of hemoglobin AIc formation in human erythrocytes in vitro. Effects of physiologic factors other than glucose.

    OpenAIRE

    Smith, R J; Koenig, R J; Binnerts, A; Soeldner, J S; Aoki, T T

    1982-01-01

    The formation of hemoglobin AIc was studied in intact human erythrocytes in vitro. Satisfactory methods were developed for maintaining erythrocytes under physiologic conditions for greater than 8 d with less than 10% hemolysis. Hemoglobin AIc levels were determined chromatographically on erythrocyte hemolysates after removal of reversible components by incubation for 6 h at 37 degree C. Hemoglobin AIc concentration was found to increase linearly with time during 8 d of incubation. The rate of...

  1. Labelling malaria-infected human erythrocytes with Tc-99m

    International Nuclear Information System (INIS)

    Garmelius-Larsson, B.; Pettersson, F.; Vogt, A.; Jonsson, C.

    2002-01-01

    Aim: Malaria is an old and a very common disease, especially in undeveloped countries. The malaria parasites infect the erythrocytes and the aim of this work was to label infected cells for future studies of their distribution and life span. Material and Method: With a commercial kit containing stannous fluoride and sodium medronate, which is used to label erythrocytes in vivo, in vitro and in vivo/vitro methods, we labelled the cells by using a modified method and a small volume, 5 - 50 microlitre, of packed cells. The cells were labelled with Tc-99m in the range of 60 - 1500 MBq. The kit was reconstituted with saline and the pH was adjusted to 7.0. The cells were incubated with 1 ml of the kitsolution in 37 0 C for 5 min. The remaining Sn-ions were reduced by adding NaOCl and then the solution was centrifuged.The supernantant was discarded and the Tc-99m was added to the precipitate and incubated 37 0 C for 20 min and then washed 3 times. This labelling procedure was performed on both infected and on non-infected cells. Results: Ten samples of cells have been labelled. The best labelling result was obtained using 7 - 20 MBq per 10 microlitre of packed cells. The labelling efficiency was, on average, 35%. Conclusion: It is possible to label both infected and non-infected cells in very small volumes. The cells were visually inspected in a microscope and were viable after labelling. Furthermore, the cell distribution was traced in vivo in an animal model by a gamma camera

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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 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 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

  3. Clotrimazole enhances lysis of human erythrocytes induced by t-BHP.

    Science.gov (United States)

    Lisovskaya, Irene L; Shcherbachenko, Irina M; Volkova, Rimma I; Ataullakhanov, Fazoil I

    2009-08-14

    Clotrimazole (CLT) is an antifungal and antimalarial agent also effective as a Gardos channel inhibitor. In addition, CLT possesses antitumor properties. Recent data provide evidence that CLT forms a complex with heme (hemin), which produces a more potent lytic effect than heme alone. This study addressed the effect of CLT on the lysis of normal human erythrocytes induced by tert-butyl hydroperoxide (t-BHP). For the first time, it was shown that 10 microM CLT significantly enhanced the lytic effect of t-BHP on erythrocytes in both Ca(2+)-containing and Ca(2+)-free media, suggesting that the effect is not related to Gardos channels. CLT did not affect the rate of free radical generation, the kinetics of GSH degradation, methemoglobin formation and TBARS generation; therefore, we concluded that CLT does not cause additional oxidative damage to erythrocytes treated with t-BHP. It is tempted to speculate that CLT enhances t-BHP-induced changes in erythrocyte volume and lysis largely by forming a complex with hemin released during hemoglobin oxidation in erythrocytes: the CLT-hemin complex destabilizes the cell membrane more potently than hemin alone. If so, the effect of CLT on cell membrane damage during free-radical oxidation may be used to increase the efficacy of antitumor therapy.

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

    Science.gov (United States)

    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. Copyright © 2011 Wiley-Liss, Inc., A Wiley Company.

  5. Curcumin Protects -SH Groups and Sulphate Transport after Oxidative Damage in Human Erythrocytes

    Directory of Open Access Journals (Sweden)

    Rossana Morabito

    2015-05-01

    Full Text Available Background/Aims: Erythrocytes, continuously exposed to oxygen pressure and toxic compounds, are sensitive to oxidative stress, namely acting on integral Band 3 protein, with consequences on cell membranes deformability and anion transport efficiency. The aim of the present investigation, conducted on human erythrocytes, is to verify whether curcumin (1 or 10µM, a natural compound with proved antioxidant properties, may counteract Band 3-mediated anion transport alterations due to oxidative stress. Methods: Oxidative conditions were induced by exposure to, alternatively, either 2 mM N-ethylmaleimide (NEM or pH-modified solutions (6.5 and 8.5. Rate constant for SO4= uptake and -SH groups estimation were measured to verify the effect of oxidative stress on anion transport efficiency and erythrocyte membranes. Results: After the exposure of erythrocytes to, alternatively, NEM or pH-modified solutions, a significant decrease in both rate constant for SO4= uptake and -SH groups was observed, which was prevented by curcumin, with a dose-dependent effect. Conclusions: Our results show that: i the decreased efficiency of anion transport may be due to changes in Band 3 protein structure caused by cysteine -SH groups oxidation, especially after exposure to NEM and pH 6.5; ii 10 µM Curcumin is effective in protecting erythrocytes from oxidative stress events at level of cell membrane transport.

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

    Energy Technology Data Exchange (ETDEWEB)

    Krokosz, Anita [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, Lodz (Poland)]. E-mail: krokosz@biol.uni.lodz.pl; Koziczak, Renata [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, Lodz (Poland); Gonciarz, Marta [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, Lodz (Poland); Szweda-Lewandowska, Zofia [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, Lodz (Poland)

    2006-01-15

    Human erythrocytes suspended in an isotonic Na-phosphate buffer, pH 7.4 (hematocrit of 2%) were irradiated with {gamma}-rays at three dose-rates of 66.7, 36.7, 25 Gy min{sup -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. Study of the effect of dose-rate on radiation-induced damage to human erythrocytes

    International Nuclear Information System (INIS)

    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

  8. The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

    NARCIS (Netherlands)

    Roelofsen, B.; Sibenius Trip, M.; Verheij, H.M.; Zevenbergen, J.L.

    1980-01-01

    1. 1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine

  9. Plasmodium falciparum avoids change in erythrocytic surface expression of phagocytosis markers during inhibition of nitric oxide synthase activity

    DEFF Research Database (Denmark)

    Hempel, Casper; Kohnke, Hannes; Maretty, Lasse

    2014-01-01

    Nitric oxide (NO) accumulates in Plasmodium falciparum-infected erythrocytes. It may be produced by a parasite NO synthase (NOS) or by nitrate reduction. The parasite's benefit of NO accumulation is not understood. We investigated if inhibiting the P. falciparum NOS with specific and unspecific NOS...... increased the fraction of phosphatidyl serine exposing cells significantly. The infection did not change the level of expression of neither total CD47 nor its oxidized form. Unrelated to NOS inhibition, incubation with caveolin-1 scaffolding domain peptide lead to a decrease in oxidized CD47. In conclusion...

  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.

    Science.gov (United States)

    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.

  11. Resistance of human erythrocytes containing elevated levels of vitamin E to radiation-induced hemolysis

    International Nuclear Information System (INIS)

    Brown, M.A.

    1983-01-01

    Human erythrocytes were isolated from the blood of healthy donors and then incubated in the presence of suspensions of alpha-tocopherol for 30 min at 37 degrees C. Unabsorbed tocopherol was removed by centrifugation using several washes of isotonic phosphate-buffered saline. Washed erythrocytes were resuspended to 0.05%. Hct and exposed to hemolyzing doses of 60 Co gamma radiation, and hemolysis was monitored continuously by light scattering at 700 nm in a recording spectrophotometer. The extent of hemolysis with time was sigmoid and data analysis was carried out on the time taken for 50% hemolysis to occur (t50%). The vitamin E content of erythrocytes was significantly elevated by the incubation procedure and resulted in the cells exhibiting a significantly increased resistance to hemolysis as reflected by the extended t50% values. Oral supplementation of 500 IU of vitamin E per day to eight normal human subjects for a period of 16 days also resulted in their washed erythrocytes exhibiting a significant increase in resistance to radiation-induced hemolysis. When comparing vitamin E incubated cells with control cells, both the dose-reducing factor (DRF) and the time for 50% hemolysis quotient (Qt50%) were observed to increase with increasing radiation dose

  12. Enzymatic methylation of band 3 anion transporter in intact human erythrocytes

    International Nuclear Information System (INIS)

    Lou, L.L.; Clarke, S.

    1987-01-01

    Band 3, the anion transport protein of erythrocyte membranes, is a major methyl-accepting substrate of the intracellular erythrocyte protein carboxyl methyltransferase (S-adenosyl-L-methionine: protein-D-aspartate O-methyltransferase; EC 2.1.1.77). The localization of methylation sites in intact cells by analysis of proteolytic fragments indicated that sites were present in the cytoplasmic N-terminal domain as well as the membranous C-terminal portion of the polypeptide. The amino acid residues that serve as carboxyl methylation sites of the erythrocyte anion transporter were also investigated. 3 H-Methylated band 3 was purified from intact erythrocytes incubated with L-[methyl- 3 H]methionine and from trypsinized and lysed erythrocytes incubated with S-adenosyl-L-[methyl- 3 H]methionine. After proteolytic digestion with carboxypeptidase Y, D-aspartic acid beta-[ 3 H]methyl ester was isolated in low yields (9% and 1%, respectively) from each preparation. The bulk of the radioactivity was recovered as [ 3 H]methanol, and the amino acid residue(s) originally associated with these methyl groups could not be determined. No L-aspartic acid beta-[ 3 H]methyl ester or glutamyl gamma-[ 3 H]methyl ester was detected. The formation of D-aspartic acid beta-[ 3 H]methyl esters in this protein in intact cells resulted from protein carboxyl methyltransferase activity since it was inhibited by adenosine and homocysteine thiolactone, which increases the intracellular concentration of the potent product inhibitor S-adenosylhomocysteine, and cycloleucine, which prevents the formation of the substrate S-adenosyl-L-[methyl- 3 H]methionine

  13. Membrane-Wrapping Contributions to Malaria Parasite Invasion of the Human Erythrocyte

    Science.gov (United States)

    Dasgupta, Sabyasachi; Auth, Thorsten; Gov, Nir S.; Satchwell, Timothy J.; Hanssen, Eric; Zuccala, Elizabeth S.; Riglar, David T.; Toye, Ashley M.; Betz, Timo; Baum, Jake; Gompper, Gerhard

    2014-01-01

    The blood stage malaria parasite, the merozoite, has a small window of opportunity during which it must successfully target and invade a human erythrocyte. The process of invasion is nonetheless remarkably rapid. To date, mechanistic models of invasion have focused predominantly on the parasite actomyosin motor contribution to the energetics of entry. Here, we have conducted a numerical analysis using dimensions for an archetypal merozoite to predict the respective contributions of the host-parasite interactions to invasion, in particular the role of membrane wrapping. Our theoretical modeling demonstrates that erythrocyte membrane wrapping alone, as a function of merozoite adhesive and shape properties, is sufficient to entirely account for the first key step of the invasion process, that of merozoite reorientation to its apex and tight adhesive linkage between the two cells. Next, parasite-induced reorganization of the erythrocyte cytoskeleton and release of parasite-derived membrane can also account for a considerable energetic portion of actual invasion itself, through membrane wrapping. Thus, contrary to the prevailing dogma, wrapping by the erythrocyte combined with parasite-derived membrane release can markedly reduce the expected contributions of the merozoite actomyosin motor to invasion. We therefore propose that invasion is a balance between parasite and host cell contributions, evolved toward maximal efficient use of biophysical forces between the two cells. PMID:24988340

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

    Directory of Open Access Journals (Sweden)

    Max Kurlbaum

    Full Text Available 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.

  15. Physical and Chemical Processes and the Morphofunctional Characteristics of Human Erythrocytes in Hyperglycaemia

    Directory of Open Access Journals (Sweden)

    Victor V. Revin

    2017-08-01

    Full Text Available Background: This study examines the effect of graduated hyperglycaemia on the state and oxygen-binding ability of hemoglobin, the correlation of phospholipid fractions and their metabolites in the membrane, the activity of proteolytic enzymes and the morphofunctional state of erythrocytes.Methods: Conformational changes in the molecule of hemoglobin were determined by Raman spectroscopy. The structure of the erythrocytes was analyzed using laser interference microscopy (LIM. To determine the activity of NADN-methemoglobinreductase, we used the P.G. Board method. The degree of glycosylation of the erythrocyte membranes was determined using a method previously described by Felkoren et al. Lipid extraction was performed using the Bligh and Dyer method. Detection of the phospholipids was performed using V. E. Vaskovsky method.Results: Conditions of hyperglycaemia are characterized by a low affinity of hemoglobin to oxygen, which is manifested as a parallel decrease in the content of hemoglobin oxyform and the growth of deoxyform, methemoglobin and membrane-bound hemoglobin. The degree of glycosylation of membrane proteins and hemoglobin is high. For example, in the case of hyperglycaemia, erythrocytic membranes reduce the content of all phospholipid fractions with a simultaneous increase in lysoforms, free fatty acids and the diacylglycerol (DAG. Step wise hyperglycaemia in incubation medium and human erythrocytes results in an increased content of peptide components and general trypsin-like activity in the cytosol, with a simultaneous decreased activity of μ-calpain and caspase 3.Conclusions: Metabolic disorders and damage of cell membranes during hyperglycaemia cause an increase in the population of echinocytes and spherocytes. The resulting disorders are accompanied with a high probability of intravascular haemolysis.

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

    Directory of Open Access Journals (Sweden)

    Ingrid Rossouw

    2015-05-01

    Full Text Available 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.

  17. 31P-NMR study of human pyrimidine 5'-nucleotidase deficient erythrocytes

    International Nuclear Information System (INIS)

    Higaki, Tsuyoshi; Kagimoto, Tadashi; Nagata, Koichi; Tanase, Sumio; Morino, Yoshimasa; Takatsuki, Kiyoshi

    1982-01-01

    Metabolic disorder of nucleotides in human pyrimidine 5'-nucleotidase (P5N) deficient erythrocytes was studied by 31 P-NMR with high resolution. Identification by combination of high-speed liquid chromatography revealed two-fold increases from the normal in the spectra in the α-, β- and γ-zones of nucleoside triphosphates of P5N deficient erythrocytes, 2,3-diphosphoglycerate shifted to the 0.3 ppm low magnetic field and signals of NAD and UDP-sugars(s) in the diphosphodiester zone. These results were obtained from the 31 P-NMR spectrum about one hour after blood sampling, indicating the high utility of this NMR for the diagnosis of P5N deficiency. (Chiba, N.)

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

    Science.gov (United States)

    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. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. In vitro effects of the anti-Alzheimer drug memantine on the human erythrocyte membrane and molecular models

    International Nuclear Information System (INIS)

    Zambrano, Pablo; Suwalsky, Mario; Villena, Fernando; Jemiola-Rzeminska, Malgorzata; Strzalka, Kazimierz

    2017-01-01

    Memantine is a NMDA antagonist receptor clinically used for treating Alzheimer's disease. NMDA receptors are present in the human neurons and erythrocyte membranes. The aim of the present study was to investigate the effects of memantine on human erythrocytes. With this purpose, the drug was developed to in vitro interact with human red cells and bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE). The latter represent lipids respectively present in both outer and inner monolayers of the red cell membrane. Results obtained by scanning electron microscopy (SEM) showed that memantine changed the normal biconcave shape of red cells to cup-shaped stomatocytes. According to the bilayer-couple hypothesis the drug intercalated into the inner monolayer of the erythrocyte membrane. Experimental results obtained by X-ray diffraction on multibilayers of DMPC and DMPE, and by differential scanning calorimetry on multilamellar vesicles indicated that memantine preferentially interacted with DMPC in a concentration-dependent manner. Thus, it can be concluded that in the low therapeutic plasma concentration of circa 1 μM memantine is located in NMDA receptor channel without affecting the erythrocyte shape. However, at higher concentrations, once the receptors became saturated excess of memantine molecules (20 μM) would interact with phosphoinositide lipids present in the inner monolayer of the erythrocyte membrane inducing the formation of stomatocytes. However, 40–50 μM memantine was required to interact with isolated phosphatidylcholine bilayers. - Highlights: • The interaction of memantine with human erythrocytes and lipid bilayers were assessed. • Memantine induced morphological changes to human erythrocytes. • Memantine interacted with classes of phospholipids present in the erythrocyte membrane. • Results support the hypothesis that memantine interacts with NMDA receptors.

  20. Effects of diethylene glycol butyl ether and butoxyethoxyacetic acid on rat and human erythrocytes.

    Science.gov (United States)

    Udden, M M

    2005-03-28

    The toxicity of diethylene glycol butyl ether (DGBE), and its principal metabolite, butoxyethoxyacetic acid (BEAA), were assessed in vitro for rat and human red blood cells. Rat erythrocytes showed evidence of mild hemolysis when exposed to BEAA at concentrations of 5 or 10 mM for 4 h. BEAA treated rat red blood cells also showed evidence of sub-hemolytic damage: increased spherocytosis, a shift in distribution of cell size to larger cells, a significant increase in mean cellular volume, and a decrease in cellular deformability. However, DGBE had no effect on rat red blood cell morphology, cell size, hemolysis or deformability. There was no hemolysis when human red blood cells were exposed to DGBE or BEAA at the same concentrations. No changes in mean cellular volume, distribution of cell size, or morphologic appearance of human red blood cells were observed. No evidence for decreased deformability of human red blood cells exposed to DGBE or BEAA was found. In conclusion, BEAA has weak hemolytic activity and sub-hemolytic effects in vitro on rat erythrocytes, which is consistent with the finding of mild hemolysis when the parent compound DGBE is administered to rats by gavage. The absence of hemolysis or sub-hemolytic damage when human red blood cells were exposed to BEAA or DGBE in vitro indicates that it is unlikely that hemolysis will occur as a result of human exposure to DGBE.

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

    Directory of Open Access Journals (Sweden)

    Wai-Hong Tham

    2015-12-01

    Full Text Available 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.

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

    Science.gov (United States)

    Clafshenkel, William P; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Koepsel, Richard R; 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.

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

    Directory of Open Access Journals (Sweden)

    William P Clafshenkel

    Full Text Available 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(sulfosuccinimidylsuberate (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.

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Ultrastructure changes produced by the action of uranyl acetate on the human erythrocyte in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wyatt, J H

    1975-06-01

    Human erythrocytes exposed in vitro to low concentrations of uranyl ions are immediately changed in shape to stomatocytes. Electron microscope examination demonstrates that cellular damage is confined to the plasma membrane. Endocytosis of the cell membrane produces groups of inside out membrane-lined vesicles within the cell; lipid from the membrane enters the cell, giving rise to intracellular myelin figures, and breaks are seen in the cell membrane. It is proposed that the lipid fraction of the cell membrane is the primary target for damage by uranyl ions.

  6. Ultrastructure changes produced by the action of uranyl acetate on the human erythrocyte in vitro

    International Nuclear Information System (INIS)

    Wyatt, J.H.

    1975-06-01

    Human erythrocytes exposed in vitro to low concentrations of uranyl ions are immediately changed in shape to stomatocytes. Electron microscope examination demonstrates that cellular damage is confined to the plasma membrane. Endocytosis of the cell membrane produces groups of inside out membrane-lined vesicles within the cell; lipid from the membrane enters the cell, giving rise to intracellular myelin figures, and breaks are seen in the cell membrane. It is proposed that the lipid fraction of the cell membrane is the primary target for damage by uranyl ions. (author)

  7. Application of human erythrocytes to a radioimmune assay of immune complexes in serum. [Lupus erythematosus, type B hepatitis

    Energy Technology Data Exchange (ETDEWEB)

    Tsuda, F; Miyakawa, Y; Mayumi, M [Tokyo Metropolitan Lab. of Public Health (Japan)

    1979-07-01

    An immune adherence receptor exists on the surface of primate erythrocytes, and has been characterized as a receptor for the activated third component of complement (C3b). Human red blood cells (RCBs, blood group O) were applied to a sensitive determination of complement-fixing, soluble immune complexes in serum. The method involved the binding of immune complexes with RBCs in the presence of complement and the detection of cell-bound IgG molecules by radiolabelled anti-human IgG antibodies. Since the binding of RBCs with monomeric IgG was minimal, cell bound IgG molecules were taken as representing immune complexes. When aggregated human gammaglobulin (AHG) was used as a model of immune complexes, as little as 5 ..mu..g dissolved in 1 ml of normal human serum were detected. The binding of RBCs with AHG was inhibited in EDTA solution where the classical complement pathway could not be activated. The RBC radioimmune assay was successfully applied to the determination of soluble immune complexes in pathological serum samples obtained from the patients with systemic lupus erythematosus and those with fulminant Type B hepatitis. False-positive results by autoantibodies against RBCs could be excluded by a Coombs test and by comparing the binding in the presence of complement with that in EDTA solution. The ubiquitous availability of RBCs coupled with a high sensitivity would allow the RBC radioimmune assay to be used as a further method of determining immune complexes in the serum.

  8. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

    KAUST Repository

    Moon, Robert

    2012-12-24

    Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.

  9. Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

    KAUST Repository

    Moon, Robert; Hall, Joanna M.; Rangkuti, Farania; Ho, YungShwen; Almond, Neil M.; Mitchell, Graham Howard; Pain, Arnab; Holder, Anthony A.; Blackman, Michael J.

    2012-01-01

    Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Human-adapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as species-specific features of an emerging pathogen.

  10. Effects of cobalt-60 ionizing radiation on human erythrocyte and its membrane proteins

    International Nuclear Information System (INIS)

    Amancio, Francisco Fernandes

    1998-01-01

    Ionizing radiation has several uses, as sterilization and radiotherapy, by its effects on living beings. recently, it has been used, at relatively lower doses (25 Gy), on blood for transfusions, mainly to eliminate undesirable graft host reactions, for use in multi transfused or immunocompromised patients. Here, we study the effect of larger doses of cobalt-60 ionizing radiation (25-1600 Gy) on human erythrocytes, by cytometric, physiologic, biochemical and immunological methods, looking for its effects and its detection. The red cells presented a clear dose-dependent increase in this volume, when irradiated in doses higher than 200 Gy, more significant in stored blood, but without hemolysis. Osmotic fragility was increased only after irradiation of more than 400 Gy. By ektacytometry, there was a lower deformability of irradiated red cells, at low stress (0.3 Pa), similar to capillary flow, but without alteration in higher stress (3 Pa), found in cardiac chambers. By SDS-PAGE, it was demonstrated that irradiated isolated erythrocyte membranes had aggregation of spectrin molecules, and decay of bands with lower molecular mass. This effect could be attributed to the radiation-induced hydroxyl radical, by specific scavenger studies. Those modifications were both antigenic and immunogenic in experimental animals, and the induced antibodies recognizes, by ELISA and immunoblot, both native or irradiated membrane proteins. They recognize rather irradiated whole erythrocyte than native ones, by hemagglutination, indirect immunofluorescence or flow cytometry assays. Our data suggests that human red cells could be irradiated at higher doses than those usually employed, with possible effect on other contaminant pathogens, without loss of viability of its use in transfusions. After improvements, irradiation induced epitopes detection could be a new tool in biological dosimetry. (author)

  11. Nitrosylated hemoglobin levels in human venous erythrocytes correlate with vascular endothelial function measured by digital reactive hyperemia.

    Directory of Open Access Journals (Sweden)

    Irina I Lobysheva

    Full Text Available 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.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.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.

  12. Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

    International Nuclear Information System (INIS)

    Roberts, W.L.; Rosenberry, T.L.

    1986-01-01

    The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[ 125 I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase

  13. Inhibition of lipid oxidation in foods and feeds and hydroxyl radical-treated fish erythrocytes: A comparative study of Ginkgo biloba leaves extracts and synthetic antioxidants

    Directory of Open Access Journals (Sweden)

    Huatao Li

    2016-09-01

    Full Text Available This study explored the effects of butylated hydroxytoluene (BHT and ethoxyquin (EQ and ethyl ether extracts, ethyl acetate extracts (EAE, acetone extracts, ethanol extracts and aqueous extracts of Ginkgo biloba leaves (EGbs on lipid oxidation in a linoleic acid emulsion, fish flesh and fish feed and in hydroxyl radical (·OH-treated carp erythrocytes. The linoleic acid, fish flesh and fish feed were incubated with BHT, EQ and EGbs at 45°C for 8 d, respectively, except for the control group. The lipid oxidation in the linoleic acid emulsion, fish flesh and fish feed was then measured by the ferric thiocyanate method or thiobarbituric acid method. The carp erythrocytes were treated with BHT, EQ or EGbs in the presence of 40 μmol/L FeSO4 and 20 μmol/L H2O2 at 37°C for 6 h, except for the control group. Oxidative stress and apoptosis parameters in carp erythrocytes were then evaluated by the commercial kit. The results showed that BHT, EQ and EGbs inhibited lipid oxidation in the linoleic acid emulsion, fish flesh and fish feed and ·OH-induced phosphatidylserine exposure and DNA fragmentation (the biomarkers of apoptosis in carp erythrocytes. Furthermore, BHT, EQ and EGbs decreased the generation of reactive oxygen species (ROS, inhibited the oxidation of cellular components and restored the activities of enzymatic antioxidants in ·OH-treated carp erythrocytes. Of all examined EGbs, EAE showed the strongest effects. The effects of EAE on lipid oxidation in the linoleic acid emulsion and on superoxide anion and malonaldehyde levels, catalase activity and apoptosis in ·OH-treated carp erythrocytes were equivalent to or stronger than those of BHT. Moreover, these results indicated that the inhibition order of EGbs on the generation of ROS and oxidation of cellular components in fish erythrocytes approximately agreed with that for the food and feed materials tested above. And, the antioxidative and anti-apoptotic effects of EGbs were

  14. An Improved 2-Dimensional Gel Electrophoresis Method for Resolving Human Erythrocyte Membrane Proteins.

    Science.gov (United States)

    Kumar, Manoj; Singh, Rajendra; Meena, Anil; Patidar, Bhagwan S; Prasad, Rajendra; Chhabra, Sunil K; Bansal, Surendra K

    2017-01-01

    The 2-dimensional gel electrophoresis (2-DE) technique is widely used for the analysis of complex protein mixtures extracted from biological samples. It is one of the most commonly used analytical techniques in proteomics to study qualitative and quantitative protein changes between different states of a cell or an organism (eg, healthy and diseased), conditionally expressed proteins, posttranslational modifications, and so on. The 2-DE technique is used for its unparalleled ability to separate thousands of proteins simultaneously. The resolution of the proteins by 2-DE largely depends on the quality of sample prepared during protein extraction which increases results in terms of reproducibility and minimizes protein modifications that may result in artifactual spots on 2-DE gels. The buffer used for the extraction and solubilization of proteins influences the quality and reproducibility of the resolution of proteins on 2-DE gel. The purification by cleanup kit is another powerful process to prevent horizontal streaking which occurs during isoelectric focusing due to the presence of contaminants such as salts, lipids, nucleic acids, and detergents. Erythrocyte membrane proteins serve as prototypes for multifunctional proteins in various erythroid and nonerythroid cells. In this study, we therefore optimized the selected major conditions of 2-DE for resolving various proteins of human erythrocyte membrane. The modification included the optimization of conditions for sample preparation, cleanup of protein sample, isoelectric focusing, equilibration, and storage of immobilized pH gradient strips, which were further carefully examined to achieve optimum conditions for improving the quality of protein spots on 2-DE gels. The present improved 2-DE analysis method enabled better detection of protein spots with higher quality and reproducibility. Therefore, the conditions established in this study may be used for the 2-DE analysis of erythrocyte membrane proteins for

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

    Directory of Open Access Journals (Sweden)

    Weerachat Sompong

    Full Text Available 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.

  16. Prostaglandin A1 metabolism and inhibition of cyclic AMP extrusion by avian erythrocytes

    International Nuclear Information System (INIS)

    Heasley, L.E.; Brunton, L.L.

    1985-01-01

    Prostaglandins (PG) inhibit active cyclic AMP export from pigeon red cells, PGA1 and PGA2 most potently. To probe the mechanism of this action of PGA1, the authors have studied the interaction of [ 3 H]PGA1 with suspensions of pigeon red cells. The interaction of PGA1 with pigeon red cells is a multistep process of uptake, metabolism, and secretion. [ 3 H] PGA1 rapidly enters red cells and is promptly metabolized to a compound(s) that remains in the aqueous layer after ethylacetate extraction. The glutathione-depleting agent, diamide, inhibits formation of the PGA1 metabolite. The red cells secrete the polar metabolite of PGA1 by a saturable mechanism that lowered temperatures inhibit. Because uptake and metabolism progress with much greater rates than metabolite secretion, red cells transiently concentrate the polar compound intracellularly. Onset and reversal of inhibition of cyclic AMP export by PGA1 coincide with accumulation and secretion of PGA1 metabolite, suggesting that the polar metabolite acts at an intracellular site to inhibit cyclic AMP efflux

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

    Directory of Open Access Journals (Sweden)

    Leilismara Sousa

    Full Text Available 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.

  18. Influence of high energy electron irradiation and gamma irradiation on the osmotic resistance of human erythrocyte membranes

    International Nuclear Information System (INIS)

    Catana, D.; Hategan, Alina; Moraru, Rodica; Popescu, Alina; Morariu, V. V.

    1998-01-01

    The effects of 5 MeV electrons and of gamma irradiation at 0 deg. C on the osmotic fragility of human erythrocyte membranes are presented. Both electron and gamma radiation in the range 0-400 Gy induced no hemolysis indicating that the membrane modifications due to radiation interaction do not reach a critical point as to cause swelling of the cells and subsequent lysis. The osmotic stress experiments performed after irradiation showed that the gamma irradiated erythrocytes exhibited an almost similar sigmoidal behavior for all irradiation doses, whereas the electron irradiated samples showed a much larger increase in hemolysis degree and, in the case of a given electron dose (100 Gy), the hemolysis was found much smaller than for the control sample (a similar behavior of the erythrocytes was found in the case of microwave irradiation at temperatures under 0 deg. C). Our experimental data suggest that electron radiation and gamma radiation have different impacts on the erythrocyte membrane fluidity, involving, probably, the different rate of energy deposition in the samples and the direct interaction of electrons with the erythrocyte membranes. (authors)

  19. Microcalorimetric measurements of heat production in human erythrocytes. IV. Comparison between different calorimetric techniques, suspension media, and preparation methods.

    Science.gov (United States)

    Monti, M; Wadsö, I

    1976-10-01

    Heat production in human erythrocytes from healthy subjects has been measured under different experimental conditions. Simultaneous measurements were made on the same samples using different types of microcalorimeters: a static ampoule calorimeter, an air perfusion calorimeter, and a flow calorimeter. Obtained heat effect values for specified standard conditions, P degrees, were within uncertainty limits the same for the different calorimeters. Cells were suspended either in autologous plasma or in a phosphate buffer. P degrees values for buffer suspensions were significantly higher than those for plasma suspensions. Erythrocyte samples prepared by the column adsorption technique gave higher P degrees values than those obtained by a conventional centrifugation procedure.

  20. Effect of some radiosensitising drugs on human erythrocyte membrane - - spin label study

    Energy Technology Data Exchange (ETDEWEB)

    Mishra, K P [Bhabha Atomic Research Centre, Bombay (India). Biology and Agriculture Div.

    1982-02-01

    Electron spin resonance and spin label techniques have been employed to study the effects of local anaesthetic drugs, procaine and tetracaine, on human erythrocyte membrane. Both the drugs altered the protein and lipid arrangements in the membrane and these changes were reversible. Procaine had greater effect on the labels attached to proteins while tetracaine fluidized interior of lipid bilayer to a greater extent. The differential effects of these drugs on the protein and lipid labels have been interpreted in terms of their relative penetrability in the membrane. Present results have explained that radiation induced enhanced killing of cells in the presence of these drugs might be due to the alterations in membrane, particularly proteins both structural and enzymatic. In addition, these results indicate a possible relationship between drug-induced structural changes in membrane and their anaesthetic potency.

  1. Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

    Science.gov (United States)

    Schröter, W; Neuvians, M

    1970-12-01

    Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

  2. Study of kinetics of 2,3-diphosphoglycerate degradation by 31P-NMR technique in depleted human erythrocytes

    International Nuclear Information System (INIS)

    Ataullakhanov, F.I.; Vitvitskii, V.M.; Dubinskaya, E.I.; Dubinskii, V.Z.

    1986-01-01

    The kinetics of 2,3-diphosphoglycerate degradation in depleted human erythrocytes was studied by the high-resolution 31 P-NMR technique. A plateau was found on the kinetic curve in the first 1.5-2 h after the beginning of depletion. The mechanisms that may be responsible for the existence of such a plateau are discussed

  3. Demonstration of specific binding sites for 3H-RRR-alpha-tocopherol on human erythrocytes

    International Nuclear Information System (INIS)

    Kitabchi, A.E.; Wimalasena, J.

    1982-01-01

    Previous work from our laboratory demonstrated specific binding sites for 3 H-RRR-alpha-tocopherol ( 3 H-d alpha T) in membranes of rat adrenal cells. As tocopherol deficiency is associated with increased susceptibility of red blood cells to hemolysis, we investigated tocopherol binding sites in human RBCs. Erythrocytes were found to have specific binding sites for 3 H-d alpha T that exhibited saturability and time and cell-concentration dependence as well as reversibility of binding. Kinetic studies of binding demonstrated two binding sites--one with high affinity (Ka of 2.6 x 10(7) M-1), low capacity (7,600 sites per cell) and the other with low affinity (1.2 x 10(6) M-1), high capacity (150,000 sites per cell). In order to localize the binding sites further, RBCs were fractionated and greater than 90% of the tocopherol binding was located in the membranes. Similar to the findings in intact RBCs, the membranes exhibited two binding sites with a respective Ka of 3.3 x 10(7) M-1 and 1.5 x 10(6) M-1. Specificity data for binding demonstrated 10% binding for RRR-gamma-tocopherol, but not other tocopherol analog exhibited competition for 3 H-d alpha T binding sites. Instability data suggested a protein nature for these binding sites. Preliminary studies on Triton X-100 solubilized fractions resolved the binding sites to a major component with an Mr of 65,000 and a minor component with an Mr of 125,000. We conclude that human erythrocyte membranes contain specific binding sites for RRR-alpha-tocopherol. These sites may be of physiologic significance in the function of tocopherol on the red blood cell membrane

  4. Grape (Vitis vinifera) extracts protect against radiation-induced oxidative stress in human erythrocyte (red blood cell)

    International Nuclear Information System (INIS)

    Singha, Indrani; Das, Subir Kumar; Gautam, S.

    2016-01-01

    Ionizing radiation (IR) causes oxidative stress through the overwhelming generation of reactive oxygen species (ROS) in the living cells leading further to the oxidative damage to biomolecules. Grapes (Vitis vinifera) contain several bioactive phytochemicals and are the richest source of antioxidant. In this study, we investigated and compared in vitro antioxidant activity and DNA damage protective property of the grape extracts of four different cultivars, including the Thompson seedless, Flame seedless, Kishmish chorni and Red globe. The activities of ascorbic acid oxidase and catalase significantly (p<0.01) differed among extracts within the same cultivar, while that of peroxidase and polyphenol oxidase did not differ significantly among extracts of any cultivar. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay and ABTS. The superoxide radical-scavenging activity was higher in the seed as compared to the skin or pulp of the same cultivar. Pretreatment with grape extracts attenuates oxidative stress induced by 4 Gy γ-radiation in human erythrocytes in vitro. These results suggest that grape extract serve as a potential source of natural antioxidants against the IR-induced oxidative stress and also inhibit apoptosis. Furthermore, the protective action of grape depends on the source of extract (seed, skin or pulp) and type of the cultivars. (author)

  5. Membrane polypeptide in rabbit erythrocytes associated with the inhibition of L-lactate transport by a synthetic anhydride of lactic acid

    International Nuclear Information System (INIS)

    Donovan, J.A.; Jennings, M.L.

    1985-01-01

    The synthetic lactyl anhydride isobutylcarbonyl lactyl anhydride (iBCLA), a selective and potent inhibitor of L-(+)-lactate transport in rabbit erythrocytes, reduces the chemical labeling of a 40-50-kdalton polypeptide by tritiated 4,4'-diisothiocyanato-2,2'-dihydrostilbenedisulfonate ([ 3 H]H 2 DIDS). iBCLA does so in a dose-dependent manner at concentrations that strongly inhibit lactate-lactate exchange but not chloride-phosphate exchange. These labeling experiments and inhibition reversal studies using iBCLA, p-(chloro-mercuri)benzenesulfonic acid (pCMBS), and dithiothreitol (DDT) suggest that iBCLA does not act at sulfhydryl groups but at or near an amino group that is near a disulfide linkage in the polypeptide which catalyzes lactate transport. These experiments support the association between specific monocarboxylate transport and a 40-50-kdalton membrane-bound polypeptide of the rabbit erythrocyte

  6. Extracellular Adenosine Triphosphate Associated with Amphibian Erythrocytes: Inhibition of ATP Release by Anion Channel Blockers.

    Science.gov (United States)

    1986-01-01

    Paddle and Burnstock (326), Williams and Forrester (463), Forrester and Williams (151) and Clemens and Forrester (82) provide evidence that hypoxia may...an ATp4 - receptor. Fed. Proc. 45:208, 1986. (abstr) 99. Dahlen , S.E. and Hedqvist, P. ATP, B,y-methylene ATP andN adenosine inhibit non-cholinergic...regulation of skeletal muscle blood low. Circ Res. 29:375-384, 1971. 117. Dodd, J., Jahr, C.E., Hamilton, P.N., Heath, M.J., Matthew , W.P., and Jessell, T.M

  7. Active site of tripeptidyl peptidase II from human erythrocytes is of the subtilisin type

    Energy Technology Data Exchange (ETDEWEB)

    Tomkinson, B.; Wernstedt, C.; Hellman, U.; Zetterqvist, Oe.

    1987-11-01

    The present report presents evidence that the amino acid sequence around the serine of the active site of human tripeptidyl peptidase II is of the subtilisin type. The enzyme from human erythrocytes was covalently labeled at its active site with (/sup 3/H)diisopropyl fluorophosphate, and the protein was subsequently reduced, alkylated, and digested with trypsin. The labeled tryptic peptides were purified by gel filtration and repeated reversed-phase HPLC, and their amino-terminal sequences were determined. Residue 9 contained the radioactive label and was, therefore, considered to be the active serine residue. The primary structure of the part of the active site (residues 1-10) containing this residue was concluded to be Xaa-Thr-Gln-Leu-Met-Asx-Gly-Thr-Ser-Met. This amino acid sequence is homologous to the sequence surrounding the active serine of the microbial peptidases subtilisin and thermitase. These data demonstrate that human tripeptidyl peptidase II represents a potentially distinct class of human peptidases and raise the question of an evolutionary relationship between the active site of a mammalian peptidase and that of the subtilisin family of serine peptidases.

  8. Effects of flaxseed oil on anti-oxidative system and membrane deformation of human peripheral blood erythrocytes in high glucose level.

    Science.gov (United States)

    Yang, Wei; Fu, Juan; Yu, Miao; Huang, Qingde; Wang, Di; Xu, Jiqu; Deng, Qianchun; Yao, Ping; Huang, Fenghong; Liu, Liegang

    2012-07-08

    The erythrocyte membrane lesion is a serious diabetic complication. A number of studies suggested that n-3 fatty acid could reduce lipid peroxidation and elevate α- or γ-tocopherol contents in membrane of erythrocytes. However, evidence regarding the protective effects of flaxseed oil, a natural product rich in n-3 fatty acid, on lipid peroxidation, antioxidative capacity and membrane deformation of erythrocytes exposed to high glucose is limited. Human peripheral blood erythrocytes were isolated and treated with 50 mM glucose to mimic hyperglycemia in the absence or presence of three different doses of flaxseed oil (50, 100 or 200 μM) in the culture medium for 24 h. The malondialdehyde (MDA) and L-glutathione (GSH) were measured by HPLC and LC/MS respectively. The phospholipids symmetry and membrane fatty acid composition of human erythrocytes were detected by flow cytometry and gas chromatograph (GC). The morphology of human erythrocyte was illuminated by ultra scanning electron microscopy. Flaxseed oil attenuated hyperglycemia-induced increase of MDA and decrease of GSH in human erythrocytes. Human erythrocytes treated with flaxseed oil contained higher C22:5 and C22:6 than those in the 50 mM glucose control group, indicating that flaxseed oil could reduce lipid asymmetric distribution and membrane perturbation. The ultra scanning electron microscopy and flow cytometer have also indicated that flaxseed oil could protect the membrane of human erythrocytes from deformation at high glucose level. The flaxseed oil supplementation may prevent lipid peroxidation and membrane dysfunction of human erythrocytes in hyperglycemia.

  9. Inhibition of Glutathione Synthesis Induced by Exhaustive Running Exercise via the Decreased Influx Rate of L-Cysteine in Rat Erythrocytes.

    Science.gov (United States)

    Xiong, Yanlian; Xiong, Yanlei; Zhou, Shuai; Yu, Zhenhai; Zhao, Dongmei; Wang, Zhiqiang; Li, Yuling; Yan, Jingtong; Cai, Yu; Zhang, Wenqian

    2016-01-01

    The main purpose of this study was to investigate the effect of exhaustive exercise on L-cysteine uptake and its effect on erythrocyte glutathione (GSH) synthesis and metabolism. Rats were divided into three groups: sedentary control (C), exhaustive running exercise (ERE) and moderate running exercise (MRE) (n=12 rats/group). We determined the L-cysteine efflux and influx in vitro in rat erythrocytes and its relationship with GSH synthesis. Total anti-oxidant potential of plasma was measured in terms of the ferric reducing ability of plasma (FRAP) values for each exercise group. In addition, the glucose metabolism enzyme activity of erythrocytes was also measured under in vitro incubation conditions. Biochemical studies confirmed that exhaustive running exercise significantly increased oxidative damage parameters in thiobarbituric acid reactive substances (TBARS) and methemoglobin levels. Pearson correlation analysis suggested that L-cysteine influx was positively correlated with erythrocyte GSH synthesis and FRAP values in both the control and exercise groups. In vitro oxidation incubation significantly decreased the level of glucose metabolism enzyme activity in the control group. We presented evidence of the exhaustive exercise-induced inhibition of GSH synthesis due to a dysfunction in L-cysteine transport. In addition, oxidative stress-induced changes in glucose metabolism were the driving force underlying decreased L-cysteine uptake in the exhaustive exercise group. © 2016 The Author(s) Published by S. Karger AG, Basel.

  10. Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Momand, J.; Clarke, S.

    1987-12-01

    The authors have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Try-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater that the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by (/sup 3/H) diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.

  11. Growth of plasmodium falciparum in human erythrocytes containing abnormal membrane proteins

    International Nuclear Information System (INIS)

    Schulman, S.; 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.

    1990-01-01

    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 + )], hereditary elliptocytosis (HE) due to protein 4.1 deficiency [HE(4.1 0 )], HE due to a spectrin αI domain structural variant that results in increased content of spectrin dimers [HE(Spα I/65 )], and band 3 structural variants. Parasite invasion, measured by the initial uptake of [ 3 H]hypoxanthine 18 hr after inoculation with merozoites, was normal in all of the pathologic RBCs. In contrast, RBCs from six HS(Sp + ) subjects showed marked growth inhibition that became apparent after the first or second growth cycle. The extent of decreased parasite growth in HS(Sp + ) 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

  12. Isolation and characterization of cDNA clones for human erythrocyte β-spectrin

    International Nuclear Information System (INIS)

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

    1987-01-01

    Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical α (M/sub r/ 240,000) and β (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 β-spectrin cDNA clone that encodes parts of the β-9 through β-12 repeat segments. This cDNA was used as a hybridization probe to assign the β-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 β-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 β-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

  13. Detection of antibodies in human serum using trimellityl-erythrocytes: direct and indirect haemagglutination and haemolysis.

    Science.gov (United States)

    Turner, E S; Pruzansky, J J; Patterson, R; Zeiss, C R; Roberts, M

    1980-02-01

    Utilizing trimellityl-erythrocytes (TM-E), antibodies were detected in sera of seven workers with trimellitic anhydride (TMA) induced airway syndromes by direct haemagglutination, indirect haemagglutination with anti-human IgG, IgA or IgM or by haemolysis. Detectable levels of antibody were obtained with all three methods. The most sensitive technique was indirect haemagglutination using anti-IgG. When added as an inhibitor, TM-human serum albumin produced a 10- to 800-fold reduction in titres. TM-ovalbumin of similar epitope density was less inhibitory and sodium trimellitate the least inhibitory on a molar basis. All of the assays using haptenized human red cells were also capable of detecting anti-TM antibodies in Rhesus monkeys whose airways had been exposed to TMA. These assays are useful for detecting anti-TM antibodies and may also be adapted to demonstrate antibodies induced against other inhaled haptens in sera of environmentally exposed individuals or in animal models of such exposure.

  14. 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.

    Science.gov (United States)

    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.

  15. Bio-field array: a dielectrophoretic electromagnetic toroidal excitation to restore and maintain the golden ratio in human erythrocytes.

    Science.gov (United States)

    Purnell, Marcy C; Butawan, Matthew B A; Ramsey, Risa D

    2018-06-01

    Erythrocytes must maintain a biconcave discoid shape in order to efficiently deliver oxygen (O 2 ) molecules and to recycle carbon dioxide (CO 2 ) molecules. The erythrocyte is a small toroidal dielectrophoretic (DEP) electromagnetic field (EMF) driven cell that maintains its zeta potential (ζ) with a dielectric constant (ԑ) between a negatively charged plasma membrane surface and the positively charged adjacent Stern layer. Here, we propose that zeta potential is also driven by both ferroelectric influences (chloride ion) and ferromagnetic influences (serum iron driven). The Golden Ratio, a function of Phi φ, offers a geometrical mathematical measure within the distinct and desired curvature of the red blood cell that is governed by this zeta potential and is required for the efficient recycling of CO 2 in our bodies. The Bio-Field Array (BFA) shows potential to both drive/fuel the zeta potential and restore the Golden Ratio in human erythrocytes thereby leading to more efficient recycling of CO 2 . Live Blood Analyses and serum CO 2 levels from twenty human subjects that participated in immersion therapy sessions with the BFA for 2 weeks (six sessions) were analyzed. Live Blood Analyses (LBA) and serum blood analyses performed before and after the BFA immersion therapy sessions in the BFA pilot study participants showed reversal of erythrocyte rheological alterations (per RBC metric; P = 0.00000075), a morphological return to the Golden Ratio and a significant decrease in serum CO 2 (P = 0.017) in these participants. Immersion therapy sessions with the BFA show potential to modulate zeta potential, restore this newly defined Golden Ratio and reduce rheological alterations in human erythrocytes. © 2018 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  16. Inhibition of the pentose phosphate shunt by 2,3-diphosphoglycerate in erythrocyte pyruvate kinase deficiency.

    Science.gov (United States)

    Tomoda, A; Lachant, N A; Noble, N A; Tanaka, K R

    1983-07-01

    Pentose phosphate shunt activity was studied by the release of 14CO2 from 14C-1-glucose and 14C-2-glucose in the red cells of five patients with pyruvate kinase deficiency and found to be significantly decreased after new methylene blue stimulation when compared to high reticulocyte controls. Incubated Heinz body formation was increased and the ascorbate cyanide test was positive in blood from these patients. The activity of glucose-6-phosphate dehydrogenase (G6PD) as well as that of 6-phosphogluconate dehydrogenase (6PGD) was inhibited to 20% of baseline in normal red cell haemolysate by 4 mM 2,3-diphosphoglycerate at pH 7.1. 2,3-Diphosphoglycerate was a competitive inhibitor with 6-phosphogluconate (Ki=1.05 mM) and a noncompetitive inhibitor with NADP (Ki=3.3 mM) for 6PGD. Since the intracellular concentrations of glucose-6-phosphate, 6-phosphogluconate and NADP are below their Kms for G6PD and 6PGD, the kinetic data suggest that increased concentrations of 2,3-diphosphoglycerate in pyruvate kinase deficient red cells are sufficiently high to suppress pentose phosphate shunt activity. This suppression may be an additional factor contributing to the haemolytic anaemia of pyruvate kinase deficiency, particularly during periods of infection or metabolic stress.

  17. Molecular cloning of human protein 4.2: A major component of the erythrocyte membrane

    International Nuclear Information System (INIS)

    Sung, L.A.; Chien, Shu; Lambert, K.; Chang, Longsheng; Bliss, S.A.; Bouhassira, E.E.; Nagel, R.L.; Schwartz, R.S.; Rybicki, A.C.

    1990-01-01

    Protein 4.2 (P4.2) comprises ∼5% of the protein mass of human erythrocyte (RBC) membranes. Anemia occurs in patients with RBCs deficient in P4.2, suggesting a role for this protein in maintaining RBC stability and integrity. The authors now report the molecular cloning and characterization of human RBC P4.2 cDNAs. By immunoscreening a human reticulocyte cDNA library and by using the polymerase chain reaction, two cDNA sequences of 2.4 and 2.5 kilobases (kb) were obtained. These cDNAs differ only by a 90-base-air insert in the longer isoform located three codons downstream from the putative initiation site. The 2.4- and 2.5-kb cDNAs predict proteins of ∼77 and ∼80 kDa, respectively, and the authenticity was confirmed by sequence identity with 46 amino acids of three cyanogen bromide-cleaved peptides of P4.2. Northern blot analysis detected a major 2.4-kb RNA species in reticulocytes. Isolation of two P4.2 cDNAs implies existence of specific regulation of P4.2 expression in human RBCs. Human RBC P4.2 has significant homology with human factor XIII subunit a and guinea pig liver transglutaminase. Sequence alignment of P4.2 with these two transglutaminases, however, revealed that P4.2 lacks the critical cysteine residue required for the enzymatic crosslinking of substrates

  18. Unexpected role of the L-domain of calpastatin during the autoproteolytic activation of human erythrocyte calpain.

    Science.gov (United States)

    De Tullio, Roberta; Franchi, Alice; Martines, Antonino; Averna, Monica; Pedrazzi, Marco; Melloni, Edon; Sparatore, Bianca

    2018-04-26

    Autoproteolysis of human erythrocyte calpain-1 proceeds in vitro at high [Ca 2+ ], through the conversion of the 80-kDa catalytic subunit into a 75-kDa activated enzyme that requires lower [Ca 2+ ] for catalysis. Importantly, here we detect a similar 75 kDa calpain-1 form also in vivo , in human meningiomas. Although calpastatin is so far considered the specific inhibitor of calpains, we have previously identified in rat brain a calpastatin transcript truncated at the end of the L-domain (cast110, L-DOM), coding for a protein lacking the inhibitory units. Aim of the present study was to characterize the possible biochemical role of the L-DOM during calpain-1 autoproteolysis in vitro , at high (100 µM) and low (5 µM) [Ca 2+ ]. Here we demonstrate that the L-DOM binds the 80 kDa proenzyme in the absence of Ca 2+ Consequently, we have explored the ability of the 75 kDa activated protease to catalyze at 5 µM Ca 2+ the intermolecular activation of native calpain-1 associated with the L-DOM. Notably, this [Ca 2+ ] is too low to promote the autoproteolytic activation of calpain-1 but enough to support the catalysis of the 75 kDa calpain. We show for the first time that the L-DOM preserves native calpain-1 from the degradation mediated by the 75 kDa form. Taken together, our data suggest that the free L-domain of calpastatin is a novel member of the calpain/calpastatin system endowed with a function alternative to calpain inhibition. For this reason, it will be crucial to define the intracellular relevance of the L-domain in controlling calpain activation/activity in physiopathological conditions having altered Ca 2+ homeostasis. © 2018 The Author(s).

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

    Directory of Open Access Journals (Sweden)

    Neha Qasim

    Full Text Available 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

  20. The 10 kDa domain of human erythrocyte protein 4.1 binds the Plasmodium falciparum EBA-181 protein

    Directory of Open Access Journals (Sweden)

    Coetzer Theresa L

    2006-11-01

    Full Text Available Abstract Background Erythrocyte invasion by Plasmodium falciparum parasites represents a key mechanism during malaria pathogenesis. Erythrocyte binding antigen-181 (EBA-181 is an important invasion protein, which mediates a unique host cell entry pathway. A novel interaction between EBA-181 and human erythrocyte membrane protein 4.1 (4.1R was recently demonstrated using phage display technology. In the current study, recombinant proteins were utilized to define and characterize the precise molecular interaction between the two proteins. Methods 4.1R structural domains (30, 16, 10 and 22 kDa domain and the 4.1R binding region in EBA-181 were synthesized in specific Escherichia coli strains as recombinant proteins and purified using magnetic bead technology. Recombinant proteins were subsequently used in blot-overlay and histidine pull-down assays to determine the binding domain in 4.1R. Results Blot overlay and histidine pull-down experiments revealed specific interaction between the 10 kDa domain of 4.1R and EBA-181. Binding was concentration dependent as well as saturable and was abolished by heat denaturation of 4.1R. Conclusion The interaction of EBA-181 with the highly conserved 10 kDa domain of 4.1R provides new insight into the molecular mechanisms utilized by P. falciparum during erythrocyte entry. The results highlight the potential multifunctional role of malaria invasion proteins, which may contribute to the success of the pathogenic stage of the parasite's life cycle.

  1. NMR studies of human blood cells in health and disease. I. Alterations of the plasma membrane water permeability of erythrocytes

    International Nuclear Information System (INIS)

    Katona, Eva; Doaga, I. O.; Radulet, Diana; Caplanusi, A.; Negreanu, Cezarina; Mihele, Denisa

    1999-01-01

    Alterations in pathological cases of the human erythrocyte membrane water permeability were investigated by using a Mn 2+ -doping 1 H nuclear magnetic resonance (NMR) technique. The temperature dependence of the apparent water diffusional exchange through erythrocyte membranes in chronic hepatitis, diabetes, dyslipidemia and essential hypertension was measured and compared to healthy controls. Using moderate manganese concentrations (9-18 mM) and Carr-Purcell-Meiboom-Gill pulse sequences with a large number of refocusing π pulses and short interpulse delay (100 μs) our values of the water exchange times (τ e ) across erythrocyte membranes, obtained within a 10 min time period following the moment of doping, were independent of the actual manganese concentration and the Arrhenius plot for water exchange was linear over the range of 22-42 deg C. A marked increase of the water exchange times values was observed in all studied disease states. In case of chronic hepatitis, diabetes and dyslipidemia the changes observed in transmembrane water exchange time were associated with significant increase in the apparent activation energy of the diffusional water permeability thus, pointing out alterations in the function of the erythrocyte water channel. (author)

  2. NMR studies of human blood cells in health and disease. I. Alterations of the plasma membrane water permeability of erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Katona, Eva; Doaga, I O; Radulet, Diana [Department of Biophysics, Carol Davila University of Medicine and Pharmaceutics, 8 Blvd. Eroilor Sanitari, POB 15-205, RO-76241 Bucharest (Romania); Caplanusi, A [Medical Biochemistry Department, Carol Davila University of Medicine and Pharmaceutics, 8 Blvd. Eroilor Sanitari, POB 15-205, RO-76241 Bucharest (Romania); Negreanu, Cezarina [Division of New Energy Conversion Methods, Institute of Research and Design for Thermoenergetic Equipment, ICPET-CERCETARE, Bucharest (Romania); Mihele, Denisa [Clinical Laboratory Department, Carol Davila University of Medicine and Pharmaceutics, 8 Blvd. Eroilor Sanitari, POB 15-205, RO-76241 Bucharest (Romania)

    1999-07-01

    Alterations in pathological cases of the human erythrocyte membrane water permeability were investigated by using a Mn{sup 2+}-doping {sup 1}H nuclear magnetic resonance (NMR) technique. The temperature dependence of the apparent water diffusional exchange through erythrocyte membranes in chronic hepatitis, diabetes, dyslipidemia and essential hypertension was measured and compared to healthy controls. Using moderate manganese concentrations (9-18 mM) and Carr-Purcell-Meiboom-Gill pulse sequences with a large number of refocusing {pi} pulses and short interpulse delay (100 {mu}s) our values of the water exchange times ({tau}{sub e}) across erythrocyte membranes, obtained within a 10 min time period following the moment of doping, were independent of the actual manganese concentration and the Arrhenius plot for water exchange was linear over the range of 22-42 deg C. A marked increase of the water exchange times values was observed in all studied disease states. In case of chronic hepatitis, diabetes and dyslipidemia the changes observed in transmembrane water exchange time were associated with significant increase in the apparent activation energy of the diffusional water permeability thus, pointing out alterations in the function of the erythrocyte water channel. (author)

  3. SO4= uptake and catalase role in preconditioning after H2O2-induced oxidative stress in human erythrocytes.

    Science.gov (United States)

    Morabito, Rossana; Remigante, Alessia; Di Pietro, Maria Letizia; Giannetto, Antonino; La Spada, Giuseppina; Marino, Angela

    2017-02-01

    Preconditioning (PC) is an adaptive response to a mild and transient oxidative stress, shown for the first time in myocardial cells and not described in erythrocytes so far. The possible adaptation of human erythrocytes to hydrogen peroxide (H 2 O 2 )-induced oxidative stress has been here verified by monitoring one of band 3 protein functions, i.e., Cl - /HCO 3 - exchange, through rate constant for SO 4 = uptake measurement. With this aim, erythrocytes were exposed to a mild and transient oxidative stress (30 min to either 10 or 100 μM H 2 O 2 ), followed by a stronger oxidant condition (300- or, alternatively, 600-μM H 2 O 2 treatment). SO 4 = uptake was measured by a turbidimetric method, and the possible role of catalase (CAT, significantly contributing to the anti-oxidant system in erythrocytes) in PC response has been verified by measuring the rate of H 2 O 2 degradation. The preventive exposure of erythrocytes to 10 μM H 2 O 2 , and then to 300 μM H 2 O 2 , significantly ameliorated the rate constant for SO 4 = uptake with respect to 300 μM H 2 O 2 alone, showing thus an adaptive response to oxidative stress. Our results show that (i) SO 4 = uptake measurement is a suitable model to monitor the effects of a mild and transient oxidative stress in human erythrocytes, (ii) band 3 protein anion exchange capability is retained after 10 μM H 2 O 2 treatment, (iii) PC response induced by the 10 μM H 2 O 2 pretreatment is clearly detected, and (iv) PC response, elicited by low-concentrated H 2 O 2 , is mediated by CAT enzyme and does not involve band 3 protein tyrosine phosphorylation pathways. Erythrocyte adaptation to a short-term oxidative stress may serve as a basis for future studies about the impact of more prolonged oxidative events, often associated to aging, drug consumption, chronic alcoholism, hyperglycemia, or neurodegenerative diseases.

  4. 31P-NMR measurements of ATP, ADP, 2,3-diphosphoglycerate and Mg2+ in human erythrocytes.

    Science.gov (United States)

    Petersen, A; Kristensen, S R; Jacobsen, J P; Hørder, M

    1990-08-17

    Absolute 31P-NMR measurements of ATP, ADP and 2,3-diphosphoglycerate (2,3-DPG) in oxygenated and partly deoxygenated human erythrocytes, compared to measurements by standard assays after acid extraction, show that ATP is only 65% NMR visible, ADP measured by NMR is unexpectedly 400% higher than the enzymatic measurement and 2,3-DPG is fully NMR visible, regardless of the degree of oxygenation. These results show that binding to hemoglobin is unlikely to cause the decreased visibility of ATP in human erythrocytes as deoxyhemoglobin binds the phosphorylated metabolites more tightly than oxyhemoglobin. The high ADP visibility is unexplained. The levels of free Mg2+ [( Mg2+]free) in human erythrocytes are 225 mumol/l at an oxygen saturation of 98.6% and instead of the expected increase, the level decreased to 196 mumol/l at an oxygen saturation of 38.1% based on the separation between the alpha- and beta-ATP peaks. [Mg2+]free in the erythrocytes decreased to 104 mumol/l at a high 2,3-DPG concentration of 25.4 mmol/l red blood cells (RBC) and a normal ATP concentration of 2.05 mmol/l RBC. By increasing the ATP concentration to 3.57 mmol/l RBC, and with a high 2,3-DPG concentration of 24.7 mmol/l RBC, the 31P-NMR measured [Mg2+]free decreased to 61 mumol/l. These results indicate, that the 31P-NMR determined [Mg2+]free in human erythrocytes, based solely on the separation of the alpha- and beta-ATP peaks, does not give a true measure of intracellular free Mg2+ changes with different oxygen saturation levels. Furthermore the measurement is influenced by the concentration of the Mg2+ binding metabolites ATP and 2,3-DPG. Failure to take these factors into account when interpreting 31P-NMR data from human erythrocytes may explain some discrepancies in the literature regarding [Mg2+]free.

  5. Phage display used for gene cloning of human recombinant antibody against the erythrocyte surface antigen, rhesus D

    DEFF Research Database (Denmark)

    Dziegiel, M; Nielsen, L K; Andersen, P S

    1995-01-01

    A novel phage display system has been developed for PCR amplification and cloning of the Fab fragments of human immunoglobulin genes. Using this system, we have cloned an antibody from a mouse-human hybridoma cell line directed against the erythrocyte antigen rhesus D. Intact erythrocytes were used...... for absorption of the Fab phages. Soluble Fab fragments produced from the cloned material showed identical performance to the parental antibody in agglutination assays. Gel filtration confirmed that the Fab fragment consists of a kappa-Fd heterodimer. The successful use of intact cells for selection of specific...... Fab phages demonstrates that it is possible to by-pass purification of the antigen of interest. Comparison with published germline sequences demonstrated that the immunoglobulin coding regions had the highest homology to the VH 1.9III and V kappa Hum kappa v325 germline genes, respectively....

  6. Characterization of human placental glycosaminoglycans and regional binding to VAR2CSA in malaria infected erythrocytes

    DEFF Research Database (Denmark)

    Beaudet, Julie M; Mansur, Leandra; Joo, Eun Ji

    2014-01-01

    expressing VAR2CSA on the erythrocyte surface. This protein adheres to a low-sulfated chondroitin sulfate-A found in placental tissue causing great harm to both mother and developing fetus. In rare cases, the localization of infected erythrocytes to the placenta can even result in the vertical transmission...... placental tissue accessible to parasites in the bloodstream, suggesting it is the primary receptor for parasite infected red blood cells....

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    1978-11-01

    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.

  9. Haemolytic effect of saponin extract from Vernonia amygdalina (bitter leaf) on human erythrocyte

    International Nuclear Information System (INIS)

    Oboh, G.

    2001-09-01

    Leaves of Veronia amygdalina were extracted using ethanol and aqueous extraction respectively. The physico-chemical analysis of the extracts revealed that both extracts had darkish brown colour, sweetish bitter taste, pungent smell, positive froth and haemolytic test, this indicated the presence of saponin in both extracts. The result of the haemolytic assay revealed that blood group-O had the highest susceptibility to the saponin-induced haemolysis, while blood group-A had the least susceptibility to haemolysis among the blood groups tested. Genotype-AA had the highest resistant to haemolysis by Vernonia amygdalina saponin induced haemolysis, while genotype-SS had the least resistant to haemolysis among the genotype tested. Furthermore the ethanol extract had a higher haemolytic activity than the aqueous extract on the various human erythrocyte analysed. This study revealed that Vernonia amygdalina had haemolytic substance, this substance had a high haemolytic effect on blood group-O and genotype-SS. The active haemolytic substance in both extracts was identified to be saponin. (author)

  10. Synthesis, characterization, and cytotoxicity in human erythrocytes of multifunctional, magnetic, and luminescent nanocrystalline rare earth fluorides

    Science.gov (United States)

    Grzyb, Tomasz; Mrówczyńska, Lucyna; Szczeszak, Agata; Śniadecki, Zbigniew; Runowski, Marcin; Idzikowski, Bogdan; Lis, Stefan

    2015-10-01

    Multifunctional nanoparticles exhibiting red or green luminescence properties and magnetism were synthesized and thoroughly analyzed. The hydrothermal method was used for the synthesis of Eu3+- or Tb3+-doped GdF3-, NaGdF4-, and BaGdF5-based nanocrystalline materials. The X-ray diffraction patterns of the samples confirmed the desired compositions of the materials. Transmission electron microscope images revealed the different morphologies of the products, including the nanocrystal sizes, which varied from 12 nm in the case of BaGdF5-based nanoparticles to larger structures with dimensions exceeding 300 nm. All of the samples presented luminescence under ultraviolet irradiation, as well as when the samples were in the form of water colloids. The highest luminescence was observed for BaGdF5-based materials. The obtained nanoparticles exhibited paramagnetism along with probable evidence of superparamagnetic behavior at low temperatures. The particles' magnetic characteristics were also preserved for samples in the form of a suspension in distilled water. The cytotoxicity studies against the human erythrocytes indicated that the synthesized nanoparticles are non-toxic because they did not cause the red blood cells shape changes nor did they alter their membrane structure and permeabilization.

  11. Synthesis, characterization, and cytotoxicity in human erythrocytes of multifunctional, magnetic, and luminescent nanocrystalline rare earth fluorides

    International Nuclear Information System (INIS)

    Grzyb, Tomasz; Mrówczyńska, Lucyna; Szczeszak, Agata; Śniadecki, Zbigniew; Runowski, Marcin; Idzikowski, Bogdan; Lis, Stefan

    2015-01-01

    Multifunctional nanoparticles exhibiting red or green luminescence properties and magnetism were synthesized and thoroughly analyzed. The hydrothermal method was used for the synthesis of Eu 3+ - or Tb 3+ -doped GdF 3 -, NaGdF 4 -, and BaGdF 5 -based nanocrystalline materials. The X-ray diffraction patterns of the samples confirmed the desired compositions of the materials. Transmission electron microscope images revealed the different morphologies of the products, including the nanocrystal sizes, which varied from 12 nm in the case of BaGdF 5 -based nanoparticles to larger structures with dimensions exceeding 300 nm. All of the samples presented luminescence under ultraviolet irradiation, as well as when the samples were in the form of water colloids. The highest luminescence was observed for BaGdF 5 -based materials. The obtained nanoparticles exhibited paramagnetism along with probable evidence of superparamagnetic behavior at low temperatures. The particles’ magnetic characteristics were also preserved for samples in the form of a suspension in distilled water. The cytotoxicity studies against the human erythrocytes indicated that the synthesized nanoparticles are non-toxic because they did not cause the red blood cells shape changes nor did they alter their membrane structure and permeabilization

  12. Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

    Directory of Open Access Journals (Sweden)

    Douglas R Boettner

    2008-01-01

    Full Text Available Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK, was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i incubation of ameba with anti-PATMK antibodies; (ii PATMK mRNA knock-down using a novel shRNA expression system; and (iii expression of a carboxy-truncation of PATMK (PATMK(delta932. Expression of the carboxy-truncation of PATMK(delta932 also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

  13. Entamoeba histolytica phagocytosis of human erythrocytes involves PATMK, a member of the transmembrane kinase family.

    Science.gov (United States)

    Boettner, Douglas R; Huston, Christopher D; Linford, Alicia S; Buss, Sarah N; Houpt, Eric; Sherman, Nicholas E; Petri, William A

    2008-01-01

    Entamoeba histolytica is the cause of amebic colitis and liver abscess. This parasite induces apoptosis in host cells and utilizes exposed ligands such as phosphatidylserine to ingest the apoptotic corpses and invade deeper into host tissue. The purpose of this work was to identify amebic proteins involved in the recognition and ingestion of dead cells. A member of the transmembrane kinase family, phagosome-associated TMK96 (PATMK), was identified in a proteomic screen for early phagosomal proteins. Anti-peptide affinity-purified antibody produced against PATMK demonstrated that it was a type I integral membrane protein that was expressed on the trophozoite surface, and that co-localized with human erythrocytes at the site of contact. The role of PATMK in erythrophagocytosis in vitro was demonstrated by: (i) incubation of ameba with anti-PATMK antibodies; (ii) PATMK mRNA knock-down using a novel shRNA expression system; and (iii) expression of a carboxy-truncation of PATMK (PATMK(delta932)). Expression of the carboxy-truncation of PATMK(delta932) also caused a specific reduction in the ability of E. histolytica to establish infection in the intestinal model of amebiasis, however these amebae retained the ability to cause hepatic abscesses when directly injected in the liver. In conclusion, PATMK was identified as a member of the TMK family that participates in erythrophagocytosis and is uniquely required for intestinal infection.

  14. Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

    International Nuclear Information System (INIS)

    Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

    1986-01-01

    The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 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 + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

  15. Calorimetric study on human erythrocyte glycolysis. Heat production in various metabolic conditions.

    Science.gov (United States)

    Minakami, S; de Verdier, C H

    1976-06-01

    The heat production of human erythrocytes was measured on a flow microcalorimeter with simultaneous analyses of lactate and other metabolites. The heat production connected with the lactate formation was about 17 kcal (71 kJ) per mol lactate formed which corresponded to the sum of heat production due to the formation of lactate from glucose and the heat production due to neutralization. The heat production rate increased as the pH of the suspension increased, corresponding to the increase in lactate formation. Glycolytic inhibitors such as fluoride and monoiodoacetate caused a decrease in the rate of heat production, whereas arsenate induced a large transient increase in heat production associated with a transient increase in lactate formation. Decrease in pyruvate concentration was usually associated with increase in heat production, although the decreased pyruvate concentration was coupled with formation of 2,3-bisphosphoglycerate. When inosine, dihydroxyacetone or D-glyceraldehyde was used as a substrate, an increase in the heat production rate was observed. Addition of methylene blue caused an oxygen uptake which was accompanied by a remarkable increase in heat production rate corresponding to about 160 kcal (670 kJ) per mol oxygen consumed. The value for heat production in red cells in the above-mentioned metabolic conditions was considered in relation to earlier known data on free energy and enthalpy changes of the different metabolic steps in the glycolytic pathway.

  16. New human erythrocyte protein with binding sites for both spectrin and calmodulin

    International Nuclear Information System (INIS)

    Gardner, K.; Bennett, V.

    1986-01-01

    A new cytoskeletal protein that binds calmodulin has been purified to greater than 95% homogeneity from human erythrocyte cytoskeletons. The protein is a heterodimer with subunits of 103,000 and 97,000 and M/sub r/ = 197,000 calculated from its Stokes radius of 6.9 nm and sedimentation coefficient of 6.8. A binding affinity of this protein for calmodulin has been estimated to be 230 nM by displacement of two different concentrations of 125 I-azidocalmodulin with increasing concentrations of unmodified calmodulin followed by Dixon plot analysis. This protein is present in red cells at approximately 30,000 copies per cell and contains a very tight binding site(s) on cytoskeletons. The protein can be only partially solubilized from isolated cytoskeletons in buffers containing high salt, but can be totally solubilized from red cell ghost membranes by extraction in low ionic strength buffers. Affinity purified IgG against this calmodulin-binding protein identifies crossreacting polypeptide(s) in brain, kidney, testes and retina. Visualization of the calmodulin-binding protein by negative staining, rotary shadowing and unidirectional shadowing indicate that it is a flattened circular molecule with molecular height of 5.4 nm and a diameter of 12.4 nm. Preliminary cosedimentation studies with purified spectrin and F-actin indicate that the site of interaction of this calmodulin-binding protein with the cytoskeleton resides on spectrin

  17. In situ assembly states of (Na+,K+)-pump ATPase in human erythrocytes. Radiation target size analyses

    International Nuclear Information System (INIS)

    Hah, J.; Goldinger, J.M.; Jung, C.Y.

    1985-01-01

    The in situ assembly state of the (Na+,K+)-pump ATPase of human erythrocytes was studied by applying the classical target theory to radiation inactivation data of the ouabain-sensitive sodium efflux and ATP hydrolysis. Erythrocytes and their extensively washed white ghosts were irradiated at -45 to -50 degrees C with an increasing dose of 1.5-MeV electron beam, and after thawing, the Na+-pump flux and/or enzyme activities were assayed. Each activity measured was reduced as a simple exponential function of radiation dose, from which a radiation sensitive mass (target size) was calculated. When intact cells were used, the target sizes for the pump and for the ATPase activities were equal and approximately 620,000 daltons. The target size for the ATPase activity was reduced to approximately 320,000 daltons if the cells were pretreated with digitoxigenin. When ghosts were used, the target size for the ATPase activity was again approximately 320,000 daltons. Our target size measurements together with other information available in literature suggest that (Na+,K+)-pump ATPase may exist in human erythrocytes either as a tetramer of alpha beta or as a dimer of alpha beta in tight association with other protein mass, probably certain glycolytic enzymes, and that this tetrameric or heterocomplex association is dissociable by digitoxigenin treatment or by extensive wash during ghost preparation

  18. Decreased calcium pump expression in human erythrocytes is connected to a minor haplotype in the ATP2B4 gene.

    Science.gov (United States)

    Zámbó, Boglárka; Várady, György; Padányi, Rita; Szabó, Edit; Németh, Adrienn; Langó, Tamás; Enyedi, Ágnes; Sarkadi, Balázs

    2017-07-01

    Plasma membrane Ca 2+ -ATPases are key calcium exporter proteins in most tissues, and PMCA4b is the main calcium transporter in the human red blood cells (RBCs). In order to assess the expression level of PMCA4b, we have developed a flow cytometry and specific antibody binding method to quantitatively detect this protein in the erythrocyte membrane. Interestingly, we found several healthy volunteers showing significantly reduced expression of RBC-PMCA4b. Western blot analysis of isolated RBC membranes confirmed this observation, and indicated that there are no compensatory alterations in other PMCA isoforms. In addition, reduced PMCA4b levels correlated with a lower calcium extrusion capacity in these erythrocytes. When exploring the potential genetic background of the reduced PMCA4b levels, we found no missense mutations in the ATP2B4 coding regions, while a formerly unrecognized minor haplotype in the predicted second promoter region closely correlated with lower erythrocyte PMCA4b protein levels. In recent GWA studies, SNPs in this ATP2B4 haplotype have been linked to reduced mean corpuscular hemoglobin concentrations (MCHC), and to protection against malaria infection. Our data suggest that an altered regulation of gene expression is responsible for the reduced RBC-PMCA4b levels that is probably linked to the development of human disease-related phenotypes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the 'Sinton and Mulligan' Stipplings in the Cytoplasm of Monkey and Human Erythrocytes.

    Science.gov (United States)

    Lucky, Amuza Byaruhanga; Sakaguchi, Miako; Katakai, Yuko; Kawai, Satoru; Yahata, Kazuhide; Templeton, Thomas J; Kaneko, Osamu

    2016-01-01

    The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as 'Sinton and Mulligan' stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum.

  20. Plasmodium knowlesi Skeleton-Binding Protein 1 Localizes to the ‘Sinton and Mulligan’ Stipplings in the Cytoplasm of Monkey and Human Erythrocytes

    Science.gov (United States)

    Lucky, Amuza Byaruhanga; Sakaguchi, Miako; Katakai, Yuko; Kawai, Satoru; Yahata, Kazuhide; Templeton, Thomas J.

    2016-01-01

    The malaria parasite, Plasmodium, exports protein products to the infected erythrocyte to introduce modifications necessary for the establishment of nutrient acquisition and surface display of host interaction ligands. Erythrocyte remodeling impacts parasite virulence and disease pathology and is well documented for the human malaria parasite Plasmodium falciparum, but has been less described for other Plasmodium species. For P. falciparum, the exported protein skeleton-binding protein 1 (PfSBP1) is involved in the trafficking of erythrocyte surface ligands and localized to membranous structures within the infected erythrocyte, termed Maurer's clefts. In this study, we analyzed SBP1 orthologs across the Plasmodium genus by BLAST analysis and conserved gene synteny, which were also recently described by de Niz et al. (2016). To evaluate the localization of an SBP1 ortholog, we utilized the zoonotic malaria parasite, Plasmodium knowlesi. Immunofluorescence assay of transgenic P. knowlesi parasites expressing epitope-tagged recombinant PkSBP1 revealed a punctate staining pattern reminiscent of Maurer's clefts, following infection of either monkey or human erythrocytes. The recombinant PkSBP1-positive puncta co-localized with Giemsa-stained structures, known as ‘Sinton and Mulligan’ stipplings. Immunoelectron microscopy also showed that recombinant PkSBP1 localizes within or on the membranous structures akin to the Maurer's clefts. The recombinant PkSBP1 expressed in P. falciparum-infected erythrocytes co-localized with PfSBP1 at the Maurer's clefts, indicating an analogous trafficking pattern. A member of the P. knowlesi 2TM protein family was also expressed and localized to membranous structures in infected monkey erythrocytes. These results suggest that the trafficking machinery and induced erythrocyte cellular structures of P. knowlesi are similar following infection of both monkey and human erythrocytes, and are conserved with P. falciparum. PMID:27732628

  1. Peripheral erythrocytes decrease upon specific respiratory challenge with grass pollen allergen in sensitized mice and in human subjects.

    Directory of Open Access Journals (Sweden)

    Galateja Jordakieva

    Full Text Available BACKGROUND AND AIMS: Specific hyper-responsiveness towards an allergen and non-specific airway hyperreactivity both impair quality of life in patients with respiratory allergic diseases. We aimed to investigate cellular responses following specific and non-specific airway challenges locally and systemically in i sensitized BALB/c mice challenged with grass pollen allergen Phl p 5, and in ii grass pollen sensitized allergic rhinitis subjects undergoing specific airway challenge in the Vienna Challenge Chamber (VCC. METHODS AND RESULTS: BALB/c mice (n = 20 were intraperitoneally immunized with grass pollen allergen Phl p 5 and afterwards aerosol challenged with either the specific allergen Phl p 5 (n = 10 or the non-specific antigen ovalbumin (OVA (n = 10. A protocol for inducing allergic asthma as well as allergic rhinitis, according to the united airway concept, was used. Both groups of exposed mice showed significantly reduced physical activity after airway challenge. Specific airway challenge further resulted in goblet cell hyperplasia, enhanced mucous secretion, intrapulmonary leukocyte infiltration and lymphoid follicle formation, associated with significant expression of IL-4, IL-5 and IL-13 in splenocytes and also partially in lung tissue. Concerning circulating blood cell dynamics, we observed a significant drop of erythrocyte counts, hemoglobin and hematocrit levels in both mouse groups, challenged with allergen or OVA. A significant decrease in circulating erythrocytes and hematocrit levels after airway challenges with grass pollen allergen was also found in grass pollen sensitized human rhinitis subjects (n = 42 at the VCC. The effects on peripheral leukocyte counts in mice and humans however were opposed, possibly due to the different primary inflammation sites. CONCLUSION: Our data revealed that, besides significant leukocyte dynamics, particularly erythrocytes are involved in acute hypersensitivity reactions to respiratory allergens

  2. [Mechanism of changes in the rate of glycolysis and levels of ATP and 2,3-diphosphoglycerate in human erythrocytes during aging].

    Science.gov (United States)

    Bogatskaia, L N; Pisaruk, A V

    1987-01-01

    Reasons which have induced changes in the glycolysis rate, ATP and 2,3-diphosphoglycerate content in human erythrocytes with ageing are studied. A fall of the hexokinase activity is shown to be one of the reasons of a significant decrease in the glycolysis rate. The total ATPase activity in erythrocytes does not change with the age. At the same time the decay rate of 2,3-diphosphoglycerate increases, that, evidently, is one of the reasons of the 2,3-diphosphoglycerate content decrease in erythrocytes with ageing.

  3. In vitro Plasmodium falciparum drug sensitivity assay: inhibition of parasite growth by incorporation of stomatocytogenic amphiphiles into the erythrocyte membrane

    DEFF Research Database (Denmark)

    Ziegler, Hanne L; Staerk, Dan; Christensen, Jette

    2002-01-01

    Lupeol, which shows in vitro inhibitory activity against Plasmodium falciparum 3D7 strain with a 50% inhibitory concentration (IC50) of 27.7 +/- 0.5 microM, was shown to cause a transformation of the human erythrocyte shape toward that of stomatocytes. Good correlation between the IC50 value...... culture continued to grow well in untreated erythrocytes. Thus, the antiplasmodial activity of lupeol appears to be indirect, being due to stomatocytic transformation of the host cell membrane and not to toxic effects via action on a drug target within the parasite. A number of amphiphiles that cause...... for development of new antimalarial drugs, care must be exercised in the interpretation of results of screening of plant extracts and natural product libraries by an in vitro Plasmodium toxicity assay....

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

    Science.gov (United States)

    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

  5. Viscosity of concentrated solutions and of human erythrocyte cytoplasm determined from NMR measurement of molecular correlation times

    International Nuclear Information System (INIS)

    Endre, Z.H.; Kuchel, P.W.

    1986-01-01

    Metabolically active human erythrocytes were incubated with [α- 13 C]glycine which led to the specific enrichment of intracellular glutathione. The cells were then studied using 13 C-NMR in which the longitudinal relaxation times (T 1 ) and nuclear Overhauser enhancements of the free glycine and glutathione were measured. Bulk viscosities of the erythrocyte cytoplasm were measured using Ostwald capillary viscometry. Large differences existed between the latter viscosity estimates and those based upon NMR-T 1 measurements. The authors 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. Under various conditions of extracellular osmotic pressure, erythrocytes change volume and thus the viscosity of the intracellular milieu is altered. The volume changes resulted in changes in the T 1 of [α- 13 C]glycine. Conversely, the authors showed that alterations in T 1 , when appropriately calibrated, could be used for monitoring changes in volume of metabolically active cells. (Auth.)

  6. The Duffy binding protein (PkDBPαII) of Plasmodium knowlesi from Peninsular Malaysia and Malaysian Borneo show different binding activity level to human erythrocytes.

    Science.gov (United States)

    Lim, Khai Lone; Amir, Amirah; Lau, Yee Ling; Fong, Mun Yik

    2017-08-11

    The zoonotic Plasmodium knowlesi is a major cause of human malaria in Malaysia. This parasite uses the Duffy binding protein (PkDBPαII) to interact with the Duffy antigen receptor for chemokines (DARC) receptor on human and macaque erythrocytes to initiate invasion. Previous studies on P. knowlesi have reported distinct Peninsular Malaysia and Malaysian Borneo PkDBPαII haplotypes. In the present study, the differential binding activity of these haplotypes with human and macaque (Macaca fascicularis) erythrocytes was investigated. The PkDBPαII of Peninsular Malaysia and Malaysian Borneo were expressed on the surface of COS-7 cells and tested with human and monkey erythrocytes, with and without anti-Fy6 (anti-Duffy) monoclonal antibody treatment. Binding activity level was determined by counting the number of rosettes formed between the transfected COS-7 cells and the erythrocytes. Anti-Fy6 treatment was shown to completely block the binding of human erythrocytes with the transfected COS-7 cells, thus verifying the specific binding of human DARC with PkDBPαII. Interestingly, the PkDBPαII of Peninsular Malaysia displayed a higher binding activity with human erythrocytes when compared with the Malaysian Borneo PkDBPαII haplotype (mean number of rosettes formed = 156.89 ± 6.62 and 46.00 ± 3.57, respectively; P < 0.0001). However, no difference in binding activity level was seen in the binding assay using M. fascicularis erythrocytes. This study is the first report of phenotypic difference between PkDBPαII haplotypes. The biological implication of this finding is yet to be determined. Therefore, further studies need to be carried out to determine whether this differential binding level can be associated with severity of knowlesi malaria in human.

  7. Human milk glycoconjugates that inhibit pathogens.

    Science.gov (United States)

    Newburg, D S

    1999-02-01

    Breast-fed infants have lower incidence of diarrhea, respiratory disease, and otitis media. The protection by human milk has long been attributed to the presence of secretory IgA. However, human milk contains large numbers and amounts of complex carbohydrates, including glycoproteins, glycolipids, glycosaminoglycans, mucins, and especially oligosaccharides. The oligosaccharides comprise the third most abundant solid constituent of human milk, and contain a myriad of structures. Complex carbohydrate moieties of glycoconjugates and oligosaccharides are synthesized by the many glycosyltransferases in the mammary gland; those with homology to cell surface glycoconjugate pathogen receptors may inhibit pathogen binding, thereby protecting the nursing infant. Several examples are reviewed: A fucosyloligosaccharide inhibits the diarrheagenic effect of stable toxin of Escherichia coli. A different fucosyloligosaccharide inhibits infection by Campylobacter jejuni. Binding of Streptococcus pneumoniae and of enteropathogenic E. coli to their respective receptors is inhibited by human milk oligosaccharides. The 46-kD glycoprotein, lactadherin, inhibits rotavirus binding and infectivity. Low levels of lactadherin in human milk are associated with a higher incidence of symptomatic rotavirus in breast-fed infants. A mannosylated glycopeptide inhibits binding by enterohemorrhagic E. coli. A glycosaminoglycan inhibits binding of gp120 to CD4, the first step in HIV infection. Human milk mucin inhibits binding by S-fimbriated E. coli. The ganglioside, GM1, reduces diarrhea production by cholera toxin and labile toxin of E. coli. The neutral glycosphingolipid, Gb3, binds to Shigatoxin. Thus, many complex carbohydrates of human milk may be novel antipathogenic agents, and the milk glycoconjugates and oligosaccharides may be a major source of protection for breastfeeding infants.

  8. The reactivation effect of pralidoxime in human blood on parathion and paraoxon–induced cholinesterase inhibition

    Directory of Open Access Journals (Sweden)

    Mahvash Jafari

    2006-03-01

    Full Text Available In this investigation the reactivation of cholinesterases by pralidoxime in parathion and paraoxon intoxication in plasma and erythrocytes were studied. For this purpose, human plasma and erythrocytes were incubated with various concentrations of parathion (0.1-10 µM and paraoxon (0.03-0.3 µM at 37 oC for 10 min. Then, pralidoxime (10-300 µM was added to the samples and incubated for 10 min before cholinesterases assay. The results showed that effects of parathion and paraoxon were dose dependent. These agents inhibited more than 85% of butyrylcholinesterase (BChE and acetylcholinesterase (AChE activity and the inhibitory effect of paraoxon was 10 times more than parathion. BChE activity was significantly higher than the control at 100 µM of pralidoxime and it reduced inhibitory effects of parathion to less than 50% and of paraoxon to 42% of control. When pralidoxime (10 µM was added to erythrocytes, the inhibitory effects of two organophosphates were reduced to less than 15%. At higher concentrations of pralidoxime (>100 µM, both BChE and AChE activities were inhibited.

  9. Quality control of insulin radioreceptor assay for human erythrocytes. Effect of ageing of mono-125I-Tyr-A14-insulin preparation

    International Nuclear Information System (INIS)

    Marttinen, A.; Pasternack, A.; Koivula, T.; Jokela, H.; Lehtinen, M.

    1984-01-01

    The quality control of insulin radioreceptor assay for human erythrocytes is based on the storage of erythrocyte preparations in Hepes buffer of pH 8.0, containing 10 g/l of albumin and 20 mmol/l of glucose. The change of erythrocytes into spherocytes and crenated cells reduces the apparent number of insulin receptors in a relatively constant way by less than 8% a week after 10 days of storage. At the same time the dissociation constants of the insulin-receptor complex increase rapidly. Thus the use of a preparation must be limited to controlling the determination of the insulin binding sites of erythrocytes, and not to the measurement of the affinities of the receptors. When mono- 125 I-Tyr-A14-insulin gets old, a slow decrease in the insulin binding sites can be measured, but the dissociation constants of the insulin receptor complex are not affected. (author)

  10. Quality control of insulin radioreceptor assay for human erythrocytes. Effect of ageing of mono-/sup 125/I-Tyr-A14-insulin preparation

    Energy Technology Data Exchange (ETDEWEB)

    Marttinen, A; Pasternack, A [Tampere Univ. (Finland). Dept. of Clinical Sciences; Koivula, T; Jokela, H; Lehtinen, M [Tampere Univ. Central Hospital (Finland). Dept. of Clinical Chemistry

    1984-09-01

    The quality control of insulin radioreceptor assay for human erythrocytes is based on the storage of erythrocyte preparations in Hepes buffer of pH 8.0, containing 10 g/l of albumin and 20 mmol/l of glucose. The change of erythrocytes into spherocytes and crenated cells reduces the apparent number of insulin receptors in a relatively constant way by less than 8% a week after 10 days of storage. At the same time the dissociation constants of the insulin-receptor complex increase rapidly. Thus the use of a preparation must be limited to controlling the determination of the insulin binding sites of erythrocytes, and not to the measurement of the affinities of the receptors. When mono-/sup 125/I-Tyr-A14-insulin gets old, a slow decrease in the insulin binding sites can be measured, but the dissociation constants of the insulin receptor complex are not affected.

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

    Science.gov (United States)

    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.

  12. A murine model of falciparum-malaria by in vivo selection of competent strains in non-myelodepleted mice engrafted with human erythrocytes.

    Directory of Open Access Journals (Sweden)

    Iñigo Angulo-Barturen

    Full Text Available To counter the global threat caused by Plasmodium falciparum malaria, new drugs and vaccines are urgently needed. However, there are no practical animal models because P. falciparum infects human erythrocytes almost exclusively. Here we describe a reliable falciparum murine model of malaria by generating strains of P. falciparum in vivo that can infect immunodeficient mice engrafted with human erythrocytes. We infected NOD(scid/beta2m-/- mice engrafted with human erythrocytes with P. falciparum obtained from in vitro cultures. After apparent clearance, we obtained isolates of P. falciparum able to grow in peripheral blood of engrafted NOD(scid/beta2m-/- mice. Of the isolates obtained, we expanded in vivo and established the isolate Pf3D7(0087/N9 as a reference strain for model development. Pf3D7(0087/N9 caused productive persistent infections in 100% of engrafted mice infected intravenously. The infection caused a relative anemia due to selective elimination of human erythrocytes by a mechanism dependent on parasite density in peripheral blood. Using this model, we implemented and validated a reproducible assay of antimalarial activity useful for drug discovery. Thus, our results demonstrate that P. falciparum contains clones able to grow reproducibly in mice engrafted with human erythrocytes without the use of myeloablative methods.

  13. Expression of Plasmodium falciparum erythrocyte membrane protein 1 in experimentally infected humans

    DEFF Research Database (Denmark)

    Lavstsen, Thomas; Magistrado, Pamela; Hermsen, Cornelus C

    2005-01-01

    -encoded Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, which is expressed on the surface of infected erythrocytes where it mediates binding to endothelial receptors. Thus, severe malaria may be caused by parasites expressing PfEMP1 variants that afford parasites optimal sequestration...... in immunologically naive individuals and high effective multiplication rates. METHODS: var gene transcription was analysed using real time PCR and PfEMP1 expression by western blots as well as immune plasma recognition of parasite cultures established from non-immune volunteers shortly after infection with NF54...... compared to parasites expressing other var genes. The differential expression of PfEMP1 was confirmed at the protein level by immunoblot analysis. In addition, serological typing showed that immune sera more often recognized second and third generation parasites than first generation parasites. CONCLUSION...

  14. Pharmacologic manipulation of human erythrocyte 2,3-diphosphoglycerate levels by prednisone administration.

    Science.gov (United States)

    Silken, A B

    1975-02-01

    Erythrocyte 2,3-diphosphoglycerate (2,3-DPG) concentrations in 10 patients with acute lymphoblastic leukemia rose 21.3%(P smaller than 0.02) after 3 weeks of prednisone and vincristine therapy, and returned to pretreatment level 2 weeks after therapy had been discontinued. The mean 2,3-DPG level of three patients on vincristine alone did not vary significantly from the control level of the leukemia patients on prednisone and vincristine. No significant change in serum inorganic phosphate level was observed. The mean erythrocyte 2,3-DPG concentration of 17 nephrotic syndrome patients being treated with chronic prednisone therapy was 14.0% higher than a control group of 20 nephrotic syndrome patients not being treated with prednisone (P small than 0.01). A significant positive correlation was observed between the dose of prednisone and 2,3-DPG level.

  15. Investigations into the binding of 125I-calmodulin to CA++ transport ATPase of human erythrocytes

    International Nuclear Information System (INIS)

    Sterk, V.

    1983-01-01

    The study described was carried out in order to investigate the binding of 125 I-calmodulin to Ca ++ transport ATPase using different Ca ++ concentrations and temperatures. The data obtained from these experiments were subsequently analysed in such as a way as to yield meaningful information relating to the mechanisms underlying the attachment of calmodulin to Ca ++ transport ATPase, the % proportion of membrane protein that was attributable to the enzyme as well as the number of calmodulin receptor sites on the individual erythrocytes, etc. Comparisons with data from the relevant literature permitted conclusions to be drawn concerning the mode of Ca ++ transport at the level of the erythrocytes. A new methodology and processing technique had to be developed prior to the beginning of the experiments. (orig./MG) [de

  16. Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum*

    Science.gov (United States)

    Cassera, María B.; Hazleton, Keith Z.; Riegelhaupt, Paul M.; Merino, Emilio F.; Luo, Minkui; Akabas, Myles H.; Schramm, Vern L.

    2008-01-01

    Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum. PMID:18799466

  17. 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.

    Science.gov (United States)

    Lange, Philipp F; Huesgen, Pitter F; Nguyen, Karen; Overall, Christopher M

    2014-04-04

    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 .

  18. Effect of Lidocaine and Epinephrine on Human Erythrocyte Shape and Vesiculability of Blood Cells

    Directory of Open Access Journals (Sweden)

    Tanja Slokar

    2015-01-01

    Full Text Available The effect of local anesthetic composed of lidocaine and epinephrine on vesiculability of blood cells and erythrocyte shape was studied. Whole blood and plasma were incubated with lidocaine/epinephrine. Extracellular vesicles were isolated by centrifugation and washing and counted by flow cytometry. Lidocaine/epinephrine and each component alone were added to diluted blood. Shape changes were recorded by micrographs. An ensemble of captured frames was analyzed for populations of discocytes, echinocytes, and stomatocytes by using statistical methods. Incubation of whole blood and blood plasma with lidocaine/epinephrine considerably increased concentration of extracellular vesicles in isolates (for an average factor 3.4 in blood and 2.8 in plasma. Lidocaine/epinephrine caused change of erythrocyte shape from mainly discocytic to mainly stomatocytic (higher than 50%. Lidocaine alone had even stronger stomatocytic effect (the percent of stomatocytes was higher than 95% while epinephrine had echinocytic effect (the percent of echinocytes was higher than 80%. The differences were highly statistically significant p<10-8 with statistical power P=1. Lidocaine/epinephrine induced regions of highly anisotropically curved regions indicating that lidocaine and epinephrine interact with erythrocyte membrane. It was concluded that lidocaine/epinephrine interacts with cell membranes and increases vesiculability of blood cells in vitro.

  19. [Inhibition rate of gamma-aminolevulinic acid dehydratase activity in erythrocytes as a reliable index for individual workers of low lead exposure].

    Science.gov (United States)

    Hirano, H; Omichi, M; Ohishi, H; Ishikawa, K; Hirashima, N

    1983-09-01

    As the delta-aminolevulinic acid dehydratase (ALAD) activity in erythrocytes is decreased by lead exposure, we considered that a net reduction of ALAD activity by lead in blood should be the difference between the activity fully activated with zinc (Zn2+) and dithiothreitol (DTT) and that without activation. The optimal condition of activation of ALAD was found by addition of 0.25 mM of Zn2+ and 10 mM of DTT in the reaction mixture. Judging from our previous results that the amount of inhibition of ALAD activity can be represented as the rate of inhibition and is closely correlated with the dose of lead administered to rabbits, the inhibition rate of ALAD activity and lead content in blood (Pb-B) of lead workers were measured. The scatter diagram obtained from the inhibition rate and lead content in blood has two groups being divided at 50 micrograms/ml of Pb-B. In one group less than 50 micrograms/100 ml of Pb-B, the inhibition rate has been closely related to Pb-B., the regression equation being Y = 1.82 X + 11.7, and the correlation coefficient + 0.926. In another group more than 50 micrograms/100 ml of Pb-B the inhibition rate remained constant at the 90% level. Measurement of the inhibition rate suggests to have practical validity for monitoring lead exposure in workers, and by means of a nomograph lead content in blood can be estimated from the inhibition rate.

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

    Science.gov (United States)

    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.

  1. Inhibition in the Human Auditory Cortex.

    Directory of Open Access Journals (Sweden)

    Koji Inui

    Full Text Available Despite their indispensable roles in sensory processing, little is known about inhibitory interneurons in humans. Inhibitory postsynaptic potentials cannot be recorded non-invasively, at least in a pure form, in humans. We herein sought to clarify whether prepulse inhibition (PPI in the auditory cortex reflected inhibition via interneurons using magnetoencephalography. An abrupt increase in sound pressure by 10 dB in a continuous sound was used to evoke the test response, and PPI was observed by inserting a weak (5 dB increase for 1 ms prepulse. The time course of the inhibition evaluated by prepulses presented at 10-800 ms before the test stimulus showed at least two temporally distinct inhibitions peaking at approximately 20-60 and 600 ms that presumably reflected IPSPs by fast spiking, parvalbumin-positive cells and somatostatin-positive, Martinotti cells, respectively. In another experiment, we confirmed that the degree of the inhibition depended on the strength of the prepulse, but not on the amplitude of the prepulse-evoked cortical response, indicating that the prepulse-evoked excitatory response and prepulse-evoked inhibition reflected activation in two different pathways. Although many diseases such as schizophrenia may involve deficits in the inhibitory system, we do not have appropriate methods to evaluate them; therefore, the easy and non-invasive method described herein may be clinically useful.

  2. Inhibitin: a specific inhibitor of sodium/sodium exchange in erythrocytes.

    OpenAIRE

    Morgan, K; Brown, R C; Spurlock, G; Southgate, K; Mir, M A

    1986-01-01

    An inhibitor of ouabain-insensitive sodium/sodium exchange in erythrocytes has been isolated from leukemic promyelocytes. To explore the specific effects of this inhibitor, named inhibitin, sodium transport experiments were carried out in human erythrocytes. Inhibitin reduced ouabain-insensitive bidirectional sodium transport. It did not change net sodium fluxes, had no significant effect on rubidium influx, and did not inhibit sodium-potassium-ATPase activity. The inhibitory effect of inhibi...

  3. Luteoloside Inhibits Proliferation of Human Chronic Myeloid ...

    African Journals Online (AJOL)

    Purpose: To investigate the effects of luteoloside on the proliferation of human chronic ..... Zhang N, Wang D, Zhu Y, Wang J, Lin H. Inhibition ... Han X. Protection of Luteolin-7-O-Glucoside Against ... Hwang YJ, Lee EJ, Kim HR, Hwang KA.

  4. Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

    Directory of Open Access Journals (Sweden)

    Prabhanshu Kumar

    2014-10-01

    Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

  5. L-cysteine efflux in erythrocytes as a function of human age: correlation with reduced glutathione and total anti-oxidant potential.

    Science.gov (United States)

    Kumar, Prabhanshu; Maurya, Pawan Kumar

    2013-06-01

    Thiol compounds such as cysteine (Cys) and reduced glutathione (GSH) play an important role in human aging and age-related diseases. In erythrocytes, GSH is synthesized by glutamic acid, cysteine, and glycine, but the rate of GSH synthesis is determined only by the availability of L-cysteine. Cysteine supplementation has been shown to ameliorate several parameters that are known to degenerate during human aging. We have studied L-cysteine efflux in vitro in human erythrocytes as a function of age by suspending cells in solution containing 10 mM L-cysteine for uptake; later cells were re-suspended in phosphate-buffered saline (PBS)-glucose to allow efflux. Change in the free sulfhydryl (-SH) concentration was then measured to calculate the rate of efflux. The GSH/oxidized glutathione (GSSG) ratio was taken as a control to study the oxidation/reduction state of the erythrocyte. The total anti-oxidant potential of plasma was measured in terms of ferric reducing ability of plasma (FRAP) values. We have shown a significant (pL-cysteine in erythrocytes during human aging, and the GSH/GSSG ratio decreases as a function of human age. The decline in L-cysteine efflux during aging correlates with the decrease in GSH and the FRAP value. This finding may help to explain the shift in the redox status and low GSH concentration that might determine the rate of L-cysteine efflux observed in erythrocytes and an important factor in the development of oxidative stress in erythrocytes during aging.

  6. Retention of Plasmodium falciparum ring-infected erythrocytes in the slow, open microcirculation of the human spleen.

    Science.gov (United States)

    Safeukui, Innocent; Correas, Jean-Michel; Brousse, Valentine; Hirt, Déborah; Deplaine, Guillaume; Mulé, Sébastien; Lesurtel, Mickael; Goasguen, Nicolas; Sauvanet, Alain; Couvelard, Anne; Kerneis, Sophie; Khun, Huot; Vigan-Womas, Inès; Ottone, Catherine; Molina, Thierry Jo; Tréluyer, Jean-Marc; Mercereau-Puijalon, Odile; Milon, Geneviève; David, Peter H; Buffet, Pierre A

    2008-09-15

    The current paradigm in Plasmodium falciparum malaria pathogenesis states that young, ring-infected erythrocytes (rings) circulate in peripheral blood and that mature stages are sequestered in the vasculature, avoiding clearance by the spleen. Through ex vivo perfusion of human spleens, we examined the interaction of this unique blood-filtering organ with P falciparum-infected erythrocytes. As predicted, mature stages were retained. However, more than 50% of rings were also retained and accumulated upstream from endothelial sinus wall slits of the open, slow red pulp microcirculation. Ten percent of rings were retained at each spleen passage, a rate matching the proportion of blood flowing through the slow circulatory compartment established in parallel using spleen contrast-enhanced ultrasonography in healthy volunteers. Rings displayed a mildly but significantly reduced elongation index, consistent with a retention process, due to their altered mechanical properties. This raises the new paradigm of a heterogeneous ring population, the less deformable subset being retained in the spleen, thereby reducing the parasite biomass that will sequester in vital organs, influencing the risk of severe complications, such as cerebral malaria or severe anemia. Cryptic ring retention uncovers a new role for the spleen in the control of parasite density, opening novel intervention opportunities.

  7. Effects of Three Kinds of Curcuminoids on Anti-Oxidative System and Membrane Deformation of Human Peripheral Blood Erythrocytes in High Glucose Levels

    Directory of Open Access Journals (Sweden)

    Wei Yang

    2015-01-01

    Full Text Available Background/Aims: Curcuminoids are the main bioactive constituents of the rhizome of turmeric. Erythrocytes lesions in diabetes are probably related to hyperglycemia and protein glycation. It has been reported that curcumin prevent lipid peroxidation. However, reports on the effects of demethoxycurcumin and bis-demethoxycurcumin on human erythrocytes at high glucose levels are scarce. Our aim is to investigate the effect of curcuminoids on oxidative stress and membrane of erythrocytes exposed to hyperglycemic condition. Methods: In this study, the different blood samples were treated with two doses of glucose (10 or 30 mM to mimic hyperglycemia in the presence or absence of three kinds of curcuminoids (5 or 10 μM in a medium at 37 °C for 24 h (Each experiment consists of 20 blood samples from 10 male and 10 female volunteers. The malondialdehyde was checked by HPLC, antioxidase (GSH and GSSG were measured by LC/MS, SOD was checked by WST-1 kit, morphology and phospholipid symmetry were detected by flow cytometry, confocal scanning microscope and scanning electron microscope. Results: The results illustrated that all three curcuminoids reduce oxidative stress damage on the membrane and maintain a better profile for erythrocytes. Furthermore, three curcuminoids had benefit effects on antioxidase. Conclusion: The three kinds of curcuminoids supplementation may prevent lipid peroxidation at different intensity and membrane dysfunction of human erythrocytes in hyperglycemia.

  8. Peptide inhibition of human cytomegalovirus infection

    Directory of Open Access Journals (Sweden)

    Morris Cindy A

    2011-02-01

    Full Text Available Abstract Background Human cytomegalovirus (HCMV is the most prevalent congenital viral infection in the United States and Europe causing significant morbidity and mortality to both mother and child. HCMV is also an opportunistic pathogen in immunocompromised individuals, including human immunodeficiency virus (HIV- infected patients with AIDS, and solid organ and allogeneic stem cell transplantation recipients. Current treatments for HCMV-associated diseases are insufficient due to the emergence of drug-induced resistance and cytotoxicity, necessitating novel approaches to limit HCMV infection. The aim of this study was to develop therapeutic peptides targeting glycoprotein B (gB, a major glycoprotein of HCMV that is highly conserved across the Herpesviridae family, that specifically inhibit fusion of the viral envelope with the host cell membrane preventing HCMV entry and infection. Results Using the Wimley-White Interfacial Hydrophobicity Scale (WWIHS, several regions within gB were identified that display a high potential to interact with lipid bilayers of cell membranes and hydrophobic surfaces within proteins. The ability of synthetic peptides analogous to WWIHS-positive sequences of HCMV gB to inhibit viral infectivity was evaluated. Human foreskin fibroblasts (HFF were infected with the Towne-GFP strain of HCMV (0.5 MOI, preincubated with peptides at a range of concentrations (78 nm to 100 μM, and GFP-positive cells were visualized 48 hours post-infection by fluorescence microscopy and analyzed quantitatively by flow cytometry. Peptides that inhibited HCMV infection demonstrated different inhibitory concentration curves indicating that each peptide possesses distinct biophysical properties. Peptide 174-200 showed 80% inhibition of viral infection at a concentration of 100 μM, and 51% and 62% inhibition at concentrations of 5 μM and 2.5 μM, respectively. Peptide 233-263 inhibited infection by 97% and 92% at concentrations of 100

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

    International Nuclear Information System (INIS)

    Edward, Kert; Farahi, Faramarz

    2014-01-01

    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. (letters)

  10. A study on the complexes between human erythrocyte enzymes participating in the conversions of 1,3-diphosphoglycerate.

    Science.gov (United States)

    Fokina, K V; Dainyak, M B; Nagradova, N K; Muronetz, V I

    1997-09-15

    The ability of D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzing the reaction of 1,3-diphosphoglycerate synthesis in human erythrocytes to form complexes with enzymes which use this metabolite as substrate (3-phosphoglycerate kinase (3-PGK) or 2,3-diphosphoglycerate mutase (2,3-DPGM)) was studied. It was found that highly active 2,3-DPGM can be extracted from human erythrocyte hemolysates in a complex with GAPDH adsorbed on Sepharose-bound anti-GAPDH antibodies at pH 6.5, the molar ratio being one 2,3-GPGM subunit per subunit of GAPDH. No complexation was, however, detected at pH 8.0. The opposite was true for the interaction between GAPDH and 3-PGK, which could be observed at pH 8.0. In experiments carried out at pH 7.4, both GAPDH x 2,3-DPGM and GAPGH x 3-PGK complexes were detected. The Kd values of the complexes determined with purified enzyme preparations were in the range 2.40-2.48 microM for both the GAPDH x 2,3-DPGM and GAPGH x 3-PGK enzyme pairs, when titrations of GAPDH covalently bound to CNBr-activated Sepharose were performed by the soluble 2,3-DPGM or 3-PGK. If, however, GAPDH adsorbed on the specific antibodies covalently bound to Sepharose was used in the titration experiments, the Kd for the GAPDH x 2,3-DPGM complex was found to be 0.54 microM, and the Kd for the GAPDH x 3-PGK complex was 0.49 microM. The concentration of 2,3-diphosphoglycerate determined after 1 h of incubation of erythrocytes in the presence of glucose was found to increase 1.5-fold if the incubation was carried out at pH 6.5, but did not change upon incubation at pH 8.0. On the other hand, the concentration of 3-phosphoglycerate after incubation at pH 8.0 was twice as large as that found after incubation at pH 6.5. The results are interpreted on the hypothesis that specific protein-protein interactions between GAPDH and 2,3-DPGM or between GAPDH and 3-PGK may play a role in determining the fate of 1,3-diphosphoglycerate produced in the GAPDH-catalyzed reaction.

  11. The influence of Bauhinia forficata Link subsp. pruinosa tea on lipid peroxidation and non-protein SH groups in human erythrocytes exposed to high glucose concentrations.

    Science.gov (United States)

    Salgueiro, Andréia C F; Leal, Carina Q; Bianchini, Matheus C; Prado, Ianeli O; Mendez, Andreas S L; Puntel, Robson L; Folmer, Vanderlei; Soares, Félix A; Avila, Daiana S; Puntel, Gustavo O

    2013-06-21

    Bauhinia forficata (BF) has been traditionally used as tea in folk medicine of Brazil for treatment of Diabetes mellitus (DM). To evaluate the effects of BF leaf tea on markers of oxidative damage and antioxidant levels in an experimental model of hyperglycemia in human erythrocytes in vitro. Human erythrocytes were incubated with high glucose concentrations or glucose and BF tea for 24h and 48h. After incubation lipid peroxidation and non-protein SH levels were analyzed. Moreover, quantification of polyphenols and flavonoids, iron chelating property, scavenging of DPPH, and prevention of lipid peroxidation in isolated lipids were also assessed. A significant amount of polyphenols and flavonoids was observed. The main components found by LC-MS analysis were quercetin-3-O-(2-rhamnosyl) rutinoside, kaempferol-3-O-(2-rhamnosyl) rutinoside, quercetin-3-O-rutinoside and kaempferol-3-O-rutinoside. BF tea presents important antioxidant and chelating properties. Moreover, BF tea was effective to increase non-protein SH levels and reduce lipid peroxidation induced by high glucose concentrations in human erythrocytes. The antioxidant effects of BF tea could be related to the presence of different phenolic and flavonoids components. We believe that these components can be responsible to protect human erythrocytes exposed to high glucose concentrations against oxidative damage. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

  13. Equilibrium thermodynamics of the partitioning of non-steroidal anti-inflammatory drugs into human erythrocyte ghost membranes

    International Nuclear Information System (INIS)

    Omran, Ahmed A.

    2013-01-01

    Graphical abstract: Bar diagram representing thermodynamic parameters obtained for the partitioning of NSAIDs into human erythrocyte ghost membranes at physiological pH; 7.4. Highlights: • Partition coefficients of NSAIDs into HEG membranes were determined. • Thermodynamic parameters were evaluated and successfully analyzed. • Partitioning of NSAIDs into HEG membranes was exothermic. • Partitioning of NSAIDs into HEG is spontaneous with negative free energy values. • Identical partitioning enthalpy–entropy driven compensation mechanism was shown. -- Abstract: In this work,second derivative spectrophotometry was applied for determining the partition coefficients (K p s) of four non-steroidal anti-inflammatory drugs (NSAIDs; flufenamic, meclofenamic, mefenamic and niflumic acids) into human erythrocyte ghost (HEG) membranes over a temperature range from (283.2 to 313.2) K. The proposed method allowed the evaluation and direct analyses of thermodynamic parameters; enthalpy (ΔH W→M ), Gibbs energy (ΔG W→M ) and entropy (ΔS W→M ) changes of the partitioning of NSAIDs into HEG membranes. The partitioning of NSAIDs between polar aqueous phase and non-polar lipid bilayer HEG membrane phase was exothermic with negative (ΔH W→M ) which compensated for the changes in (ΔS W→M ). The negative values of (ΔG W→M ) revealed that the partitioning of NSAIDs into HEG, owing to their transfer from polar aqueous phase and non-polar HEG phase is spontaneous. The enthalpy–entropy correlation analysis resulted in a good linearity that suggests an identical partitioning enthalpy–entropy driven compensation mechanism for the studied NSAIDs

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

    Science.gov (United States)

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

    2016-04-05

    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.

  15. Loss of the clock protein PER2 shortens the erythrocyte life span in mice.

    Science.gov (United States)

    Sun, Qi; Zhao, Yue; Yang, Yunxia; Yang, Xiao; Li, Minghui; Xu, Xi; Wen, Dan; Wang, Junsong; Zhang, Jianfa

    2017-07-28

    Cell proliferation and release from the bone marrow have been demonstrated to be controlled by circadian rhythms in both humans and mice. However, it is unclear whether local circadian clocks in the bone marrow influence physiological functions and life span of erythrocytes. Here, we report that loss of the clock gene Per2 significantly decreased erythrocyte life span. Mice deficient in Per2 were more susceptible to acute stresses in the erythrocytes, becoming severely anemic upon phenylhydrazine, osmotic, and H 2 O 2 challenges. 1 H NMR-based metabolomics analysis revealed that the Per2 depletion causes significant changes in metabolic profiles of erythrocytes, including increased lactate and decreased ATP levels compared with wild-type mice. The lower ATP levels were associated with hyperfunction of Na + /K + -ATPase activity in Per2 -null erythrocytes, and inhibition of Na + /K + -ATPase activity by ouabain efficiently rescued ATP levels. Per2 -null mice displayed increased levels of Na + /K + -ATPase α1 (ATP1A1) in the erythrocyte membrane, and transfection of Per2 cDNA into the erythroleukemic cell line TF-1 inhibited Atp1a1 expression. Furthermore, we observed that PER2 regulates Atp1a1 transcription through interacting with trans-acting transcription factor 1 (SP1). Our findings reveal that Per2 function in the bone marrow is required for the regulation of life span in circulating erythrocytes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. A single point in protein trafficking by Plasmodium falciparum determines the expression of major antigens on the surface of infected erythrocytes targeted by human antibodies.

    Science.gov (United States)

    Chan, Jo-Anne; Howell, Katherine B; Langer, Christine; Maier, Alexander G; Hasang, Wina; Rogerson, Stephen J; Petter, Michaela; Chesson, Joanne; Stanisic, Danielle I; Duffy, Michael F; Cooke, Brian M; Siba, Peter M; Mueller, Ivo; Bull, Peter C; Marsh, Kevin; Fowkes, Freya J I; Beeson, James G

    2016-11-01

    Antibodies to blood-stage antigens of Plasmodium falciparum play a pivotal role in human immunity to malaria. During parasite development, multiple proteins are trafficked from the intracellular parasite to the surface of P. falciparum-infected erythrocytes (IEs). However, the relative importance of different proteins as targets of acquired antibodies, and key pathways involved in trafficking major antigens remain to be clearly defined. We quantified antibodies to surface antigens among children, adults, and pregnant women from different malaria-exposed regions. We quantified the importance of antigens as antibody targets using genetically engineered P. falciparum with modified surface antigen expression. Genetic deletion of the trafficking protein skeleton-binding protein-1 (SBP1), which is involved in trafficking the surface antigen PfEMP1, led to a dramatic reduction in antibody recognition of IEs and the ability of human antibodies to promote opsonic phagocytosis of IEs, a key mechanism of parasite clearance. The great majority of antibody epitopes on the IE surface were SBP1-dependent. This was demonstrated using parasite isolates with different genetic or phenotypic backgrounds, and among antibodies from children, adults, and pregnant women in different populations. Comparisons of antibody reactivity to parasite isolates with SBP1 deletion or inhibited PfEMP1 expression suggest that PfEMP1 is the dominant target of acquired human antibodies, and that other P. falciparum IE surface proteins are minor targets. These results establish SBP1 as part of a critical pathway for the trafficking of major surface antigens targeted by human immunity, and have key implications for vaccine development, and quantifying immunity in populations.

  17. Influence of polar and non-polar digoxin and digitoxin metabolites on the /sup 86/Rb-uptake of human erythrocytes and the contractility of guinea pig papillary muscles

    Energy Technology Data Exchange (ETDEWEB)

    Belz, G G; Heinz, N [Bundeswehr-Zentralkrankenhaus, Koblenz (Germany, F.R.). Medizinische Abt.; Beiersdorf A G, Hamburg Pharma-Forschung [Germany, F.R.

    1977-01-01

    The potency of various digoxigenin and digitoxigenin derivatives with different polarity was tested in two biological systems: First, in an /sup 86/Rb-erythrocyte assay which allows to determine the influence on active cation transport (measured as the glycoside concentration exerting half maximal inhibition of /sup 86/Rb-uptake of human erythrocytes = IC/sub 50/). Second, with isolated guinea pig papillary muscle, which allows to determine glycoside effects on contractile force (measured as the glycoside concentration exerting a 100% increase of contractile force = C+/sub 100/B). The IC/sub 50/ of the substances covered a range from 3.2 to 4800 x 10/sup -9/M, the C+/sub 100/B from 0.7 to 978 x 10/sup -6/ M. In both assay systems the glucuronides of glycosides and genins were between 1.4 and 11 times less potent than the original substances. A highly significant correlation (p < 0.0001) was found between IC/sub 50/ and C+/sub 100/B (r = 0.9996) and between log IC/sub 50/ and log C+/sub 100/B (r = 0.9819), the slope for the latter correlation being nearly unity (= 0.9912). The results support the hypothesis that inhibition of active cation transport is an important step in glycoside induced positive-inotropic effect. (orig.) 891 VJ 892 AP.

  18. 2-Hexadecynoic acid inhibits plasmodial FAS-II enzymes and arrests erythrocytic and liver stage Plasmodium infections.

    Science.gov (United States)

    Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H; Brun, Reto; Carballeira, Néstor M

    2010-11-01

    Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of Plasmodium yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC(50) value 6.6 μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC(50) value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescence analysis (IC(50) 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory activity against the PfFAS-II enzymes PfFabI and PfFabZ with IC(50) values of 0.38 and 0.58 μg/ml (IC(50) control drugs 14 and 30 ng/ml), respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC(50) values 3.7-31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC(50) 20.2 μg/ml), and Leishmania donovani (IC(50) values 4.1-13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature, and calculated pharmacokinetic properties suggests that 2-HDA could be a useful compound to

  19. 2-Hexadecynoic Acid Inhibits Plasmodial FAS-II Enzymes and Arrest Erythrocytic and Liver Stage Plasmodium Infections

    Science.gov (United States)

    Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L.; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H.; Brun, Reto; Carballeira, Néstor M.

    2010-01-01

    Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of P. yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC50 value 6.6. μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC50 value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescense analysis (IC50 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory against the PfFAS-II enzymes PfFabI and PfFabZ with IC50 values of 0.38 and 0.58 μg/ml (IC50 control drugs 14 and 30 ng/ml) respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC50 values 3.7–31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC50 20.2 μg/ml), and Leishmania donovani (IC50 values 4.1–13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature and calculated pharmacokinetic properties suggest that 2-HDA could be a useful compound to study the interaction of fatty

  20. MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

    Directory of Open Access Journals (Sweden)

    N. G. Zemlianskykh

    2016-10-01

    Full Text Available The aim of the work was to study the effect of polyethylene glycol PEG-1500 on the Ca2+-ATPase activity and changes in CD44 surface marker expression in human erythrocyte membranes. Determination of the Ca2+-ATPase activity was carried out in sealed erythrocyte ghosts by the level of accumulation of inorganic phosphorus. Changes in the expression of CD44 and amount of CD44+-erythrocytes were evaluated by flow cytometry. The inhibition of Ca2+-ATPase activity and a reduction in the level of CD44 expression and also the decrease in the amount CD44+-cells were found, reflecting a fairly complex restructuring in the membrane-cytoskeleton complex of erythrocytes under the influence of PEG-1500. Effect of PEG-1500 on the surface CD44 marker could be mediated by modification of proteins of membrane-cytoskeleton complex, as indicated by accelerated loss of CD44 in erythrocyte membranes after application of protein cross-linking reagent diamide. Reduced activity of Ca2+-ATPase activity may contribute to the increase in intracellular Ca2+ level and thus leads to a modification of interactions of integral proteins with cytoskeletal components that eventually could result in membrane vesiculation and decreasing in expression of the CD44 marker, which is dynamically linked to the cytoskeleton.

  1. 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.

    Science.gov (United States)

    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

  2. INHIBITION OF HUMAN ERYTHROCYTE CA2+-ATPASE BY ZN2+. (R826104)

    Science.gov (United States)

    The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

  3. Towards a multiscale description of microvascular flow regulation: O2-dependent release of ATP from human erythrocytes and the distribution of ATP in capillary networks

    Directory of Open Access Journals (Sweden)

    Daniel eGoldman

    2012-07-01

    Full Text Available Integration of the numerous mechanisms that have been suggested to contribute to optimization of O2 supply to meet O2 need in skeletal muscle requires a systems biology approach which permits quantification of these physiological processes over a wide range of length scales. Here we describe two individual computational models based on in vivo and in vitro studies which, when incorporated into a single robust multiscale model, will provide information on the role of erythrocyte-released ATP in perfusion distribution in skeletal muscle under both physiological and pathophysiological conditions. Healthy human erythrocytes exposed to low O2 tension release ATP via a well characterized signaling pathway requiring activation of the G-protein, Gi, and adenylyl cyclase leading to increases in cAMP. This cAMP then activates PKA and subsequently CFTR culminating in ATP release via pannexin 1. A critical control point in this pathway is the level of cAMP which is regulated by pathway-specific phosphodiesterases. Using time constants (~100ms that are consistent with measured erythrocyte ATP release, we have constructed a dynamic model of this pathway. The model predicts levels of ATP release consistent with measurements obtained over a wide range of hemoglobin O2 saturations (sO2. The model further predicts how insulin, at concentrations found in prediabetes, enhances the activity of PDE3 and reduces intracellular cAMP levels leading to decreased low O2-induced ATP release from erythrocytes. The second model, which couples O2 and ATP transport in capillary networks, shows how intravascular ATP and the resulting conducted vasodilation are affected by local sO2, convection and ATP degradation. This model also predicts network-level effects of decreased ATP release resulting from elevated insulin levels. Taken together, these models lay the groundwork for investigating the systems biology of the regulation of microvascular perfusion distribution by

  4. Discriminating the hemolytic risk of blood type A plasmas using the complement hemolysis using human erythrocytes (CHUHE) assay.

    Science.gov (United States)

    Cunnion, Kenji M; Hair, Pamela S; Krishna, Neel K; Sass, Megan A; Enos, Clinton W; Whitley, Pamela H; Maes, Lanne Y; Goldberg, Corinne L

    2017-03-01

    The agglutination-based cross-matching method is sensitive for antibody binding to red blood cells but is only partially predictive of complement-mediated hemolysis, which is important in many acute hemolytic transfusion reactions. Here, we describe complement hemolysis using human erythrocytes (CHUHE) assays that directly evaluate complement-mediated hemolysis between individual serum-plasma and red blood cell combinations. The CHUHE assay is used to evaluate correlations between agglutination titers and complement-mediated hemolysis as well as the hemolytic potential of plasma from type A blood donors. Plasma or serum from each type A blood donor was incubated with AB or B red blood cells in the CHUHE assay and measured for free hemoglobin release. CHUHE assays for serum or plasma demonstrate a wide, dynamic range and high sensitivity for complement-mediated hemolysis for individual serum/plasma and red blood cell combinations. CHUHE results suggest that agglutination assays alone are only moderately predictive of complement-mediated hemolysis. CHUHE results also suggest that plasma from particular type A blood donors produce minimal complement-mediated hemolysis, whereas plasma from other type A blood donors produce moderate to high-level complement-mediated hemolysis, depending on the red blood cell donor. The current results indicate that the CHUHE assay can be used to assess complement-mediated hemolysis for plasma or serum from a type A blood donor, providing additional risk discrimination over agglutination titers alone. © 2016 AABB.

  5. Radiation-induced structural changes in membrane proteins of human erythrocytes and ghosts and the relation to cellular morphology

    Energy Technology Data Exchange (ETDEWEB)

    Schuurhuis, G.J.; Hommes, J.; Vos, J.; Molenaar, I.; Konings, A.W.T. (Rijksuniversiteit Groningen (Netherlands))

    1984-02-01

    Isolated human erythrocytes and ghosts were irradiated with X-rays under different experimental conditions and the effect examined with regard to the structure of membrane proteins and morphology of whole cells and ghosts. From sodium dodecyl sulphate/polyacrylamide gel electrophoresis it is concluded that spectrin (band 1 and 2) is the most radiosensitive of the membrane proteins examined. X-irradiation of cells and ghosts induced covalent cross-linking of a small fraction of membrane proteins. In the protein aggregates thus formed spectrin was found to be the major component. Molecular disulphide (-SS-) bridges seemed to account for part of the cross-links observed. Some nondisulphide cross-links were found, especially when ghosts were irradiated. Significant amounts of spectrin aggregates were formed during post-irradiation incubation at 37/sup 0/C but not at 4/sup 0/C. In the intact cell a transformation in shape from discocyte to echinocyte accompanied the process of post-irradiation spectrin aggregation. The characteristics of both processes, such as their reversibility with adenosine, point to a metabolic involvement. It is shown that there is no causal relationship between the two phenomena observed. Possible causes of the post-irradiation effects and the parallelism with similar processes in non-irradiated metabolically depleted cells are discussed.

  6. Radiation-induced structural changes in membrane proteins of human erythrocytes and ghosts and the relation to cellular morphology

    International Nuclear Information System (INIS)

    Schuurhuis, G.J.; Hommes, J.; Vos, J.; Molenaar, I.; Konings, A.W.T.

    1984-01-01

    Isolated human erythrocytes and ghosts were irradiated with X-rays under different experimental conditions and the effect examined with regard to the structure of membrane proteins and morphology of whole cells and ghosts. From sodium dodecyl sulphate/polyacrylamide gel electrophoresis it is concluded that spectrin (band 1 and 2) is the most radiosensitive of the membrane proteins examined. X-irradiation of cells and ghosts induced covalent cross-linking of a small fraction of membrane proteins. In the protein aggregates thus formed spectrin was found to be the major component. Molecular disulphide (-SS-) bridges seemed to account for part of the cross-links observed. Some nondisulphide cross-links were found, especially when ghosts were irradiated. Significant amounts of spectrin aggregates were formed during post-irradiation incubation at 37 0 C but not at 4 0 C. In the intact cell a transformation in shape from discocyte to echinocyte accompanied the process of post-irradiation spectrin aggregation. The characteristics of both processes, such as their reversibility with adenosine, point to a metabolic involvement. It is shown that there is no causal relationship between the two phenomena observed. Possible causes of the post-irradiation effects and the parallelism with similar processes in non-irradiated metabolically depleted cells are discussed. (author)

  7. Local anesthetics: interaction with human erythrocyte membranes as studied by 1H and 31P nuclear magnetic resonance

    International Nuclear Information System (INIS)

    Fraceto, Leonardo Fernandes; Paula, Eneida de

    2004-01-01

    The literature carries many theories about the mechanism of action of local anesthetics (LA). We can highlight those focusing the direct effect of LA on the sodium channel protein and the ones that consider the interaction of anesthetic molecules with the lipid membrane phase. The interaction between local anesthetics and human erythrocyte membranes has been studied by 1 H and 31 P nuclear magnetic resonance spectroscopy. It was found that lidocaine (LDC) and benzocaine (BZC) bind to the membranes, increase the mobility of the protons of the phospholipids acyl chains, and decrease the mobility and/or change the structure of the polar head groups. The results indicate that lidocaine molecules are inserted across the polar and liquid interface of the membrane, establishing both electrostatic (charged form) and hydrophobic (neutral form) interactions. Benzocaine locates itself a little deeper in the bilayer, between the interfacial glycerol region and the hydrophobic core. These changes in mobility or conformation of membrane lipids could affect the Na + -channel protein insertion in the bilayer, stabilizing it in the inactivated state, thus causing anesthesia. (author)

  8. Stimulation of Suicidal Erythrocyte Death by Garcinol

    Directory of Open Access Journals (Sweden)

    Antonella Fazio

    2015-09-01

    Full Text Available Background/Aims: The benzophenone garcinol from dried fruit rind of Garcinia indica counteracts malignancy, an effect at least in part due to stimulation of apoptosis. The proapototic effect of garcinol is attributed in part to inhibition of histone acetyltransferases and thus modification of gene expression. Moreover, garcinol triggers mitochondrial depolarisation. Erythrocytes lack gene expression and mitochondria but are nevertheless able to enter apoptosis-like suicidal death or eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, energy depletion and Ca2+ entry with increase of cytosolic Ca2+ activity ([Ca2+]i. The present study explored, whether and how garcinol induces eryptosis. Methods: 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, ROS formation from DCFDA dependent fluorescence and cytosolic ATP levels utilizing a luciferin-luciferase-based assay. Results: A 24 hours exposure of human erythrocytes to garcinol (2.5 or 5 µM significantly increased the percentage of annexin-V-binding cells. Garcinol decreased (at 1 µM and 2.5 µM or increased (at 5 µM forward scatter. Garcinol (5 µM further increased Fluo3-fluorescence, increased DCFDA fluorescence, and decreased cytosolic ATP levels. The effect of garcinol on annexin-V-binding was significantly blunted, but not abolished by removal of extracellular Ca2+. Conclusions: Garcinol triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect in part due to stimulation of ROS formation, energy depletion and Ca2+ entry.

  9. The treatment of Plasmodium falciparum-infected erythrocytes with chloroquine leads to accumulation of ferriprotoporphyrin IX bound to particular parasite proteins and to the inhibition of the parasite's 6-phosphogluconate dehydrogenase

    Directory of Open Access Journals (Sweden)

    Famin O.

    2003-03-01

    Full Text Available Ferriprotoporphyrin IX (FPIX is a potentially toxic product of hemoglobin digestion by intra-erythrocytic malaria parasites. It is detoxified by biomineralization or through degradation by glutathione. Both processes are inhibited by the antimalarial drug chloroquine, leading to the accumulation of FPIX in the membranes of the infected cell and their consequent permeabilization. It is shown here that treatment of Plasmodium falciparum-infected erythrocytes with chloroquine also leads to the binding of FPIX to a subset of parasite proteins. Parasite enzymes such as aldolase, pyrimidine nucleoside monophosphate kinase and pyrimidine 5'- nucleotidase were inhibited by FPIX in vitro, but only the activity of 6-phosphogluconate dehydrogenase was reduced significantly in cells after drug treatment. Additional proteins were extracted from parasite cytosol by their ability to bind FPIX. Sequencing of these proteins identified heat shock proteins 90 and 70, enolase, elongation factor 1-α, phoshoglycerate kinase, glyceraldehyde 3- phosphate dehydrogenase, L-lactate dehydrogenase and gametocytogenesis onset-specific protein. The possible involvement of these proteins in the antimalarial mode of action of chloroquine is discussed. It is concluded that drug-induced binding of FPIX to parasite glycolytic enzymes could underlie the demonstrable inhibition of glycolysis by chloroquine. The inhibition of 6- phosphogluconate dehydrogenase could explain the reduction of the activity of the hexose monophosphate shunt by the drug. Inhibition of both processes is deleterious to parasite survival. Binding of FPIX to other proteins is probably inconsequential to the rapid killing of the parasite by chloroquine.

  10. Modeling hysteresis observed in the human erythrocyte voltage-dependent cation channel

    DEFF Research Database (Denmark)

    Flyvbjerg, Henrik; Gudowska-Nowak, Ewa; Christophersen, Palle

    2012-01-01

    The non-selective voltage-activated cation channel from human red cells, which is activated at depolarizing potentials, has been shown to exhibit counter-clockwise gating hysteresis. Here, we analyze this phenomenon with the simplest possible phenomenological models. Specifically, the hysteresis ...

  11. Spice phenolics inhibit human PMNL 5-lipoxygenase.

    Science.gov (United States)

    Prasad, N Satya; Raghavendra, R; Lokesh, B R; Naidu, K Akhilender

    2004-06-01

    A wide variety of phenolic compounds and flavonoids present in spices possess potent antioxidant, antimutagenic and anticarcinogenic activities. We examined whether 5-lipoxygenase (5-LO), the key enzyme involved in biosynthesis of leukotrienes is a possible target for the spices. Effect of aqueous extracts of turmeric, cloves, pepper, chili, cinnamon, onion and also their respective active principles viz., curcumin, eugenol, piperine, capsaicin, cinnamaldehyde, quercetin, and allyl sulfide were tested on human PMNL 5-LO activity by spectrophotomeric and HPLC methods. The formation of 5-LO product 5-HETE was significantly inhibited in a concentration-dependent manner with IC(50) values of 0.122-1.44 mg for aqueous extracts of spices and 25-83 microM for active principles, respectively. The order of inhibitory activity was of quercetin>eugenol>curcumin>cinnamaldehyde>piperine>capsaicin>allyl sulfide. Quercetin, eugenol and curcumin with one or more phenolic ring and methoxy groups in their structure showed high inhibitory effect, while the non-phenolic spice principle allyl sulfide showed least inhibitory effect on 5-LO. The inhibitory effect of quercetin, curcumin and eugenol was similar to that of synthetic 5-LO inhibitors-phenidone and NDGA. Moreover, the inhibitory potency of aqueous extracts of spice correlated with the active principles of their respective spices. The synergistic or antagonistic effect of mixtures of spice active principles and spice extracts were investigated and all the combinations of spice active principles/extracts exerted synergistic effect in inhibiting 5-LO activity. These findings clearly suggest that phenolic compounds present in spices might have physiological role in modulating 5-LO pathway.

  12. The role of electrostatic interactions in the Streptococcus thermophilus adhesion on human erythrocytes in media with different 1:1 electrolyte concentration

    Directory of Open Access Journals (Sweden)

    О. І. Гордієнко

    2015-10-01

    Full Text Available The process of bacterial adhesion is usually discussed in terms of the two-stage sorption model. According to the model, at the first stage the bacteria fastly attaches to the surface by weak physical interactions, while at the second stage irreversible molecular and cellular adhesion process takes place. An important factor, influencing the adhesion processes, is physical-chemical characteristics of the medium, in particular, the presence of monovalent cations therein. The aim of this work is to assess the role of electrostatic component of the intercellular interactions at the first reversible stage of adhesion. Comparison of experimental data of adhesion of lactobacilli S. thermophilus on human erythrocytes and theoretical definition of the Debye radius and the erythrocytes surface potential in the experimental solutions showed that with decreasing ionic strength of the solution the change in the adhesion index in our experiments is fully in line with the theory DLVO predictions.

  13. Radiation damage to human erythrocytes. Relative contribution of hydroxyl and chloride radicals in N2O-saturated buffers

    International Nuclear Information System (INIS)

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

    2008-01-01

    The erythrocyte suspensions in Na-phosphate buffered isotonic NaCl solution (PBS) or Na-phosphate isotonic buffer (PB) (hematocrit 1%) were irradiated with the dose of 400 Gy under N 2 O. Erythrocytes were incubated in the medium in which the cells were irradiated or in fresh PBS. The level of damage to cells was estimated on the basis of the course of post-radiation hemolysis and hemoglobin (Hb) oxidation. The medium in which the cells were irradiated and incubated influenced the course of the post-radiation hemolysis and Hb oxidation as well as some other parameters. We discussed the contribution of hydroxyl and chloride radicals in the initiation of erythrocyte damage and oxygen modification of these processes

  14. Computed aided system for separation and classification of the abnormal erythrocytes in human blood

    Science.gov (United States)

    Wąsowicz, Michał; Grochowski, Michał; Kulka, Marek; Mikołajczyk, Agnieszka; Ficek, Mateusz; Karpieńko, Katarzyna; Cićkiewicz, Maciej

    2017-12-01

    The human peripheral blood consists of cells (red cells, white cells, and platelets) suspended in plasma. In the following research the team assessed an influence of nanodiamond particles on blood elements over various periods of time. The material used in the study consisted of samples taken from ten healthy humans of various age, different blood types and both sexes. The markings were leaded by adding to the blood unmodified diamonds and oxidation modified. The blood was put under an impact of two diamond concentrations: 20μl and 100μl. The amount of abnormal cells increased with time. The percentage of echinocytes as a result of interaction with nanodiamonds in various time intervals for individual specimens was scarce. The impact of the two diamond types had no clinical importance on red blood cells. It is supposed that as a result of longlasting exposure a dehydratation of red cells takes place, because of the function of the cells. The analysis of an influence of nanodiamond particles on blood elements was supported by computer system designed for automatic counting and classification of the Red Blood Cells (RBC). The system utilizes advanced image processing methods for RBCs separation and counting and Eigenfaces method coupled with the neural networks for RBCs classification into normal and abnormal cells purposes.

  15. Apolipoprotein J/Clusterin is a novel structural component of human erythrocytes and a biomarker of cellular stress and senescence.

    Directory of Open Access Journals (Sweden)

    Marianna H Antonelou

    Full Text Available BACKGROUND: Secretory Apolipoprotein J/Clusterin (sCLU is a ubiquitously expressed chaperone that has been functionally implicated in several pathological conditions of increased oxidative injury, including aging. Nevertheless, the biological role of sCLU in red blood cells (RBCs remained largely unknown. In the current study we identified sCLU as a component of human RBCs and we undertook a detailed analysis of its cellular topology. Moreover, we studied the erythrocytic membrane sCLU content during organismal aging, in conditions of increased organismal stress and accelerated RBCs senescence, as well as during physiological in vivo cellular senescence. METHODOLOGY/PRINCIPAL FINDINGS: By using a combination of molecular, biochemical and high resolution microscopical methods we found that sCLU is a novel structural component of RBCs extra- and intracellular plasma membrane and cytosol. We observed that the RBCs membrane-associated sCLU decreases during organismal aging or exposure to acute stress (e.g. smoking, in patients with congenital hemolytic anemia, as well as during RBCs in vivo senescence. In all cases, sCLU reduction paralleled the expression of typical cellular senescence, redox imbalance and erythrophagocytosis markers which are also indicative of the senescence- and oxidative stress-mediated RBCs membrane vesiculation. CONCLUSIONS/SIGNIFICANCE: We propose that sCLU at the mature RBCs is not a silent remnant of the erythroid precursors, but an active component being functionally implicated in the signalling mechanisms of cellular senescence and oxidative stress-responses in both healthy and diseased organism. The reduced sCLU protein levels in the RBCs membrane following cell exposure to various endogenous or exogenous stressors closely correlates to the levels of cellular senescence and redox imbalance markers, suggesting the usefulness of sCLU as a sensitive biomarker of senescence and cellular stress.

  16. Contribution of ankyrin-band 3 complexes to the organization and mechanical properties of the membrane skeleton of human erythrocyte

    Energy Technology Data Exchange (ETDEWEB)

    Shen, B.W. [Argonne National Lab., IL (United States). Biological and Medical Research Div.

    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.

  17. Examination of the calcium-erythrocyte membrane interactions

    International Nuclear Information System (INIS)

    Gardos, Gy.; Szasz, I.; Sarkadi, B.

    1979-01-01

    A review of the cation-transport mechanisms of human erythrocytes is given. The following experimental methods were applied: measurement of 45 Ca influx, 45 Ca efflux, 42 K influx, 42 K efflux, 22 Na efflux and determination of the activity of the Ca-ATP-ase enzyme. The increase of the intracellular Ca-level opens some specific K-channels, through which K is leaking out passively. The kinetics and the chemical nature of this K-transport are given in detail. On the other hand, Ca ions taken up are removed by active transport. Detailed data are given on the activity and specific inhibition of this Ca-pump. In human erythrocytes the pump is working with the stoichiometry of Ca:ATP=2. (L.E.)

  18. An incubation medium for the elevation of adenosine triphosphate and 2,3-diphosphoglycerate in fresh and long-preserved human erythrocytes.

    Science.gov (United States)

    Rubinstein, D; Warrendorf, E

    1975-06-01

    The levels of adenosine triphosphate (ATP) and 2,3-diphosphoglycerate in freshly drawn human erythrocytes can be tripled by a 2 h incubation at 37 degrees C in a medium containing 21 mM glucose, 1.8 mM adenine, 5 mM pyruvate, 10 mM inosine, and 96 mM phosphate. Similar incubation conditions will restore the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood levels preserved for 12 and 15 weeks, respectively, to those of fresh cells. Omission of pyruvate from the incubation medium further increases the level of ATP slightly, but there is little elevation of 2,3-diphosphoglycerate. Under these conditions labelled pyruvate and lactate production from [14-C]glucose or [14-C]inosine is not diminished, but labelled fructose 1,6-diphosphate, rather than 2,3-diphosphoglycerate, accumulates. In addition, omission of pyruvate from the incubation medium, with a concomitant decrease in accumulation of 2,3-diphosphoglycerate, diminishes the concentration of inorganic phosphate required for optimal ATP elevation. A 5 h incubation in the glucose-adenine-pyruvate-inosine-phosphate medium elevates the levels of ATP and 2,3-diphosphoglycerate in erythrocytes from blood preserved in the cold for 15 weeks to twice that of fresh cells, indicating that the cells retain their metabolic potential even after prolonged storage at 2 degrees C. The medium may provide a method of rejuvenating 10-12 week cold-preserved erythrocytes for transfusion purposes, by a 1 h incubation at 37 degrees C.

  19. Physical-chemical basis of the protection of slowly frozen human erythrocytes by glycerol

    Energy Technology Data Exchange (ETDEWEB)

    Rall, W.F.; Mazur, P.; Souzu, H.

    1978-07-01

    One theory of freezing damage suggests that slowly cooled cells are killed by being exposed to increasing concentrations of electrolytes as the suspending medium freezes. A corollary to this view is that protective additives such as glycerol protect cells by acting colligatively to reduce the electrolyte concentration at any subzero temperature. Recently published phase-diagram data for the ternary system glycerol-NaCl-water by M.L. Shepard et al. (Cryobiology, 13: 9-23, 1976), in combination with the data on human red cell survival vs. subzero temperature presented here and in the companion study of Souzu and Mazur (Biophys. J., 23: 89-100), permit a precise test of this theory. Appropriate liquidus phase-diagram information for the solutions used in the red cell freezing experiments was obtained by interpolation of liquidus data of Shepard and his co-workers. The results of phase-diagram analysis of red cell survival indicate that the correlation between the temperature that yields 50% hemolysis (LT/sub 50/) and the electrolyte concentration attained at that temperature in various concentrations of glycerol is poor. With increasing concentrations of glycerol, the cells were killed at progressively lower concentrations of NaCl. For example, the LT/sub 50/ for cells frozen in the absence of glycerol corresponds to a NaCl concentration of 12 weight percent (2.4 molal), while for cells frozen in 1.75 M glycerol in buffered saline the LT/sub 50/ corresponds to 3.0 weight percent NaCl (1.3 molal). The data, in combination with other findings, lead to two conclusions: (a) The protection from glycerol is due to its colligative ability to reduce the concentration of sodium chloride in the external medium, but (b) the protection is less than that expected from colligative effects; apparently glycerol itself can also be a source of damage, probably because it renders the red cells susceptible to osmotic shock during thawing.

  20. Human erythrocytes bind and inactivate type 5 adenovirus by presenting Coxsackie virus-adenovirus receptor and complement receptor 1

    Czech Academy of Sciences Publication Activity Database

    Carlisle, R. C.; Di, Y.; Cerny, A. M.; Sonnen, A. F. P.; Sim, R. B.; Green, N. K.; Šubr, Vladimír; Ulbrich, Karel; Gilbert, R. J. C.; Fisher, K. D.; Finberg, R. W.; Seymour, L. W.

    2009-01-01

    Roč. 113, č. 9 (2009), s. 1909-1918 ISSN 0006-4971 EU Projects: European Commission(XE) 512087 - GIANT Institutional research plan: CEZ:AV0Z40500505 Keywords : adenovirus * erythrocyte * complement receptor 1 Subject RIV: CD - Macromolecular Chemistry Impact factor: 10.555, year: 2009

  1. Actividad hemolítica de la ortovainillina y la isovainillina sobre eritrocitos humanos Haemolytic activity of orthovanillin and isovanillin on human erythrocytes

    Directory of Open Access Journals (Sweden)

    Yamirka Alonso Geli

    2005-04-01

    Full Text Available Los eritrocitos portadores de hemoglobina S ( b 6 glu ® val , son menos flexibles que los eritrocitos normales, lo que los hace más frágiles y se hemolizan con mayor facilidad. La ortovainillina y la isovainillina, isómeros químicos de la vainillina, pueden inhibir la polimerización de la desoxihemoglobina S (actividad antipolimerizante y evitar la falciformación de los eritrocitos. Se determinó la actividad citotóxica de estos compuestos sobre eritrocitos normales y SS, a razones molares 1:1, 1:4, 1:8 y 1:10, por espectrofotometría midiendo la absorbancia a una longitud de onda l =545 nm de la hemoglobina libre en el sobrenadante, después de incubar la solución de eritrocitos con los compuestos, y se determinó el porcentaje de hemólisis. Los resultados muestran que el porcentaje de hemólisis promedio calculado fue inferior al 1 %. No se detectaron diferencias estadísticamente significativas entre las medias por razón molar en una misma clase de eritrocitos (p=0,05 ni una dependencia entre la concentración y la actividad hemolítica. Se compararon las medias entre ambos tipos de eritrocitos, para todas las relaciones molares, y no se observaron diferencias estadísticamente significativas. Se compararon, además, los resultados de trabajos anteriores sobre la actividad hemolítica de la vainillina con la de sus isómeros estructurales, y se obtuvo que la isovainillina y la ortovainillina provocaron porcentajes de hemólisis inferiores a los provocados por la vainillina. La baja actividad hemolítica de estos aldehídos aromáticos potencia su actividad antipolimerizante.The erythrocytes carriers of haemoglobin S ( b6 glu®val are less flexible than the normal erythrocytes, which makes them more fragile and allow them to haemolyse easier. The orthovanillin and the isovanillin, chemical isomers of vanillin, may inhibit the polymerisation of desoxohaemoglobin S (antipolimerizing activity and prevent the falciformation of

  2. Factors influencing erythrocyte choline concentrations.

    Science.gov (United States)

    Miller, B L; Jenden, D J; Tang, C; Read, S

    1989-01-01

    Choline concentrations in human erythrocytes increase after freezing and thawing, during incubation in Krebs-phosphate for 30 min or on storage at 0 degrees C for 3-24 hr. The increase is prevented by protein precipitation by 10% perchloric acid, 10% zinc hydroxide, 10% sodium tungstate or boiling in water. It is not prevented by EDTA (10 mM) and is increased by oleate (5 mM). We suggest that the increase is due to the action of phospholipase D on erythrocyte phospholipids.

  3. RhD Specific Antibodies Are Not Detectable in HLA-DRB11501* Mice Challenged with Human RhD Positive Erythrocytes

    Directory of Open Access Journals (Sweden)

    Lidice Bernardo

    2014-01-01

    Full Text Available 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 DRB11501* 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 DRB11501* as well as to several standard inbred and outbred laboratory strains including C57BL/6, DBA1/J, CFW(SW, CD1(ICR, and NSA(CF-1. DRB11501* mice were additionally immunized with putative extracellular immunogenic RhD peptides. DRB11501* 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, DRB11501* 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 DRB11501* 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.

  4. Inhibition of human aromatase complex (CYP19) by antiepileptic drugs

    DEFF Research Database (Denmark)

    Jacobsen, Naja Wessel; Halling-Sørensen, Bent; Birkved, Franziska Maria A Kramer

    2008-01-01

    of 1.4-49.7 mM. Carbamazepine, gabapentin, primidone, topiramate and vigabatrin showed no inhibition. Additionally, binary drug combinations were tested to investigate if combination therapy could potentiate the aromatase inhibition. Additive inhibition was seen in combination experiments...... with valproate and phenobarbital. When adding carbamazepine to a range of valproate concentrations no additional inhibition was seen. The data for some of the AEDs show that side effects on steroid synthesis in humans due to inhibition of aromatase should be considered....

  5. Synthesis and evaluation of the potential deleterious effects of ZnO nanomaterials (nanoneedles and nanoflowers) on blood components, including albumin, erythrocytes and human isolated primary neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Pastrello, Bruna [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil); Paracatu, Luana Chiquetto [São Paulo State University (UNESP), Department of Clinical Analysis, School of Pharmaceutical Sciences (Brazil); Carvalho Bertozo, Luiza de [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil); Paino, Iêda Maria Martinez [University of São Paulo (USP), Nanomedicine and Nanotoxicology Group, Physics Institute of São Carlos (IFSC) (Brazil); Lisboa-Filho, Paulo Noronha [São Paulo State University (UNESP), Department of Physics, Faculty of Sciences (Brazil); Ximenes, Valdecir Farias, E-mail: vfximenes@fc.unesp.br [São Paulo State University (UNESP), Department of Chemistry, Faculty of Sciences (Brazil)

    2016-07-15

    The application of zinc oxide (ZnO) nanoparticles in biomaterials has increased significantly in the recent years. Here, we aimed to study the potential deleterious effects of ZnO on blood components, including human serum albumin (HSA), erythrocytes and human isolated primary neutrophils. To test the influence of the morphology of the nanomaterials, ZnO nanoneedles (ZnO-nn) and nanoflowers (ZnO-nf) were synthesized. The zeta potential and mean size of ZnO-nf and ZnO-nn suspensions in phosphate-buffered saline were −10.73 mV and 3.81 nm and −5.27 mV and 18.26 nm, respectively. The incubation of ZnO with HSA did not cause its denaturation as verified by the absence of significant alterations in the intrinsic and extrinsic fluorescence and in the circular dichroism spectrum of the protein. The capacity of HSA as a drug carrier was not affected as verified by employing site I and II fluorescent markers. Neither type of ZnO was able to provoke the activation of neutrophils, as verified by lucigenin- and luminol-dependent chemiluminescence and by the extracellular release of hydrogen peroxide. ZnO-nf, but not ZnO-nn, induced the haemolysis of erythrocytes. In conclusion, our results reinforce the concept that ZnO nanomaterials are relatively safe for usage in biomaterials. A potential exception is the capacity of ZnO-nf to promote the lysis of erythrocytes, a discovery that shows the importance of the morphology in the toxicity of nanoparticles.

  6. A simple in vitro method of radiolabelling human erythrocytes in whole blood with 113mIn-tropolonate

    International Nuclear Information System (INIS)

    Osman, S.; Danpure, H.J.

    1987-01-01

    A simple and rapid in vitro procedure has been developed for selectively radiolabelling erythrocytes in whole blood using 113m In-tropolonate. A maximum labelling efficiency of 97% was achieved, of which 95.5% was on the erythrocytes after only 5 min incubation of whole blodd at room temperature. The optimum amount of tropolone for labelling whole blood was 10 μg of tropolone per ml of blood using acid-citrate dextrose (ACD) as the anticoagulant and 50 μg of tropolone per ml of blood using heparin. Under these optimim conditions, only 2.5% of the cell-bound 113m In was released from the labelled cells during a 1 h in vitro incubation in cell-free plasma, irrespective of the anticoagulant used. These results suggest that 113m In-tropolonate may prove to be useful in vitro agent for labelling erythrocytes for short-term clinical investigations, especially at centres where 99m Tc and 111 In are unavailable. (author)

  7. Interaction of complement-solubilized immune complexes with CR1 receptors on human erythrocytes. The binding reaction

    DEFF Research Database (Denmark)

    Jepsen, H H; Svehag, S E; Jarlbaek, L

    1986-01-01

    showed no binding. IC solubilized in 50% human serum in the presence of autologous RBC bound rapidly to RBC-CR1, with maximal binding within less than 1 min at 37 degrees C. Release of CR1-bound IC under these conditions occurred slowly, requiring more than 30 min. Only binding of 'partially' solubilized...... of an intact classical pathway in preparing the IC for binding to RBC-CR1. C-solubilized IC could be absorbed to solid-phase conglutinin or antibody to C3c and C4c, and these ligands were able to inhibit the binding of solubilized IC to RBC. Heparin also exerted a marked, dose-dependent inhibitory effect...

  8. Intracellular Erythrocyte Platelet-activating Factor Acetylhydrolase I Inactivates Aspirin in Blood*

    Science.gov (United States)

    Zhou, Gang; Marathe, Gopal K.; Willard, Belinda; McIntyre, Thomas M.

    2011-01-01

    Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A2 with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A2 synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood. PMID:21844189

  9. Intracellular erythrocyte platelet-activating factor acetylhydrolase I inactivates aspirin in blood.

    Science.gov (United States)

    Zhou, Gang; Marathe, Gopal K; Willard, Belinda; McIntyre, Thomas M

    2011-10-07

    Aspirin (acetylsalicylic acid) prophylaxis suppresses major adverse cardiovascular events, but its rapid turnover limits inhibition of platelet cyclooxygenase activity and thrombosis. Despite its importance, the identity of the enzyme(s) that hydrolyzes the acetyl residue of circulating aspirin, which must be an existing enzyme, remains unknown. We find that circulating aspirin was extensively hydrolyzed within erythrocytes, and chromatography indicated these cells contained a single hydrolytic activity. Purification by over 1400-fold and sequencing identified the PAFAH1B2 and PAFAH1B3 subunits of type I platelet-activating factor (PAF) acetylhydrolase, a phospholipase A(2) with selectivity for acetyl residues of PAF, as a candidate for aspirin acetylhydrolase. Western blotting showed that catalytic PAFAH1B2 and PAFAH1B3 subunits of the type I enzyme co-migrated with purified erythrocyte aspirin hydrolytic activity. Recombinant PAFAH1B2, but not its family member plasma PAF acetylhydrolase, hydrolyzed aspirin, and PAF competitively inhibited aspirin hydrolysis by purified or recombinant erythrocyte enzymes. Aspirin was hydrolyzed by HEK cells transfected with PAFAH1B2 or PAFAH1B3, and the competitive type I PAF acetylhydrolase inhibitor NaF reduced erythrocyte hydrolysis of aspirin. Exposing aspirin to erythrocytes blocked its ability to inhibit thromboxane A(2) synthesis and platelet aggregation. Not all individuals or populations are equally protected by aspirin prophylaxis, the phenomenon of aspirin resistance, and erythrocyte hydrolysis of aspirin varied 3-fold among individuals, which correlated with PAFAH1B2 and not PAFAH1B3. We conclude that intracellular type I PAF acetylhydrolase is the major aspirin hydrolase of human blood.

  10. The approximate entropy of the electromyographic signals of tremor correlates with the osmotic fragility of human erythrocytes

    Directory of Open Access Journals (Sweden)

    Penha-Silva Nilson

    2010-06-01

    Full Text Available Abstract 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

  11. Synthesis and In Vitro Inhibition Effect of New Pyrido[2,3-d]pyrimidine Derivatives on Erythrocyte Carbonic Anhydrase I and II

    Directory of Open Access Journals (Sweden)

    Hilal Kuday

    2014-01-01

    Full Text Available In vitro inhibition effects of indolylchalcones and new pyrido[2,3-d]pyrimidine derivatives on purified human carbonic anhydrase I and II (hCA I and II were investigated by using CO2 as a substrate. The results showed that all compounds inhibited the hCA I and hCA II enzyme activities. Among all the synthesized compounds, 7e (IC50=6.79 µM was found to be the most active compound for hCA I inhibitory activity and 5g (IC50=7.22 µM showed the highest hCA II inhibitory activity. Structure-activity relationships study showed that indolylchalcone derivatives have higher inhibitory activities than pyrido[2,3-d]pyrimidine derivatives on hCA I and hCA II. Additionally, methyl group bonded to uracil ring increases inhibitory activities on both hCA I and hCA II.

  12. Uptake of Eudragit Retard L (Eudragit® RL Nanoparticles by Human THP-1 Cell Line and Its Effects on Hematology and Erythrocyte Damage in Rats

    Directory of Open Access Journals (Sweden)

    Mosaad A. Abdel-Wahhab

    2014-02-01

    Full Text Available The aim of this study was to prepare Eudragit Retard L (Eudragit RL nanoparticles (ENPs and to determine their properties, their uptake by the human THP-1 cell line in vitro and their effect on the hematological parameters and erythrocyte damage in rats. ENPs showed an average size of 329.0 ± 18.5 nm, a positive zeta potential value of +57.5 ± 5.47 mV and nearly spherical shape with a smooth surface. THP-1 cell lines could phagocyte ENPs after 2 h of incubation. In the in vivo study, male Sprague-Dawley rats were exposed orally or intraperitoneally (IP with a single dose of ENP (50 mg/kg body weight. Blood samples were collected after 4 h, 48 h, one week and three weeks for hematological and erythrocytes analysis. ENPs induced significant hematological disturbances in platelets, red blood cell (RBC total and differential counts of white blood cells (WBCs after 4 h, 48 h and one week. ENP increased met-Hb and Co-Hb derivatives and decreased met-Hb reductase activity. These parameters were comparable to the control after three weeks when administrated orally. It could be concluded that the route of administration has a major effect on the induction of hematological disturbances and should be considered when ENPs are applied for drug delivery systems.

  13. Enrichment of antioxidants in black garlic juice using macroporous resins and their protective effects on oxidation-damaged human erythrocytes.

    Science.gov (United States)

    Zou, Ying; Zhao, Mouming; Yang, Kun; Lin, Lianzhu; Wang, Yong

    2017-08-15

    The black garlic juice is popular for its nutritive value. Enrichment of antioxidants is needed to make black garlic extract an effective functional ingredient. Five macroporous resins were evaluated for their capacity in adsorbing antioxidants in black garlic juice. XAD-16 resin was chosen for further study due to its high adsorption and desorption ratios. Pseudo-second-order kinetics (q e =625μmol Trolox equiv/g dry resin, k 2 =0.0001463) and Freundlich isotherm models (ΔH=-10.1547kJ/mol) were suitable for describing the whole exothermic and physical adsorption processes of the antioxidants from black garlic juice on XAD-16 resin. The antioxidants and phenolics were mostly enriched in 40% ethanol fraction by XAD-16 resin column chromatography. The black garlic extract and its fractions could protect erythrocytes against AAPH-induced hemolysis in dose-dependent manners. The pretreatment of AAPH-damaged erythrocytes with 40% ethanol fractions (2.5mg/mL) significantly decreased the hemolysis ratios from 53.58% to 3.79%. The 40% ethanol fraction possessing strong intracellular antioxidant activity could be used as a functional food ingredient. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Screening of plant extracts for human tyrosinase inhibiting effects.

    Science.gov (United States)

    Kim, M; Park, J; Song, K; Kim, H G; Koh, J-S; Boo, Y C

    2012-04-01

    Screening for tyrosinase (TYR) inhibitors potentially useful for control of skin pigmentation has been hampered by the limited availability of human TYR. To overcome this hurdle, we have established human embryonic kidney (HEK293)-TYR cells that constitutively express human TYR. In the current study, we assayed human TYR inhibition activities of 50 plant extracts using the lysates of transformed HEK293-TYR cells. The strongest inhibition of human TYR was shown by the extract of Vaccinium bracteatum Thunberg, followed by the extract of Morus bombycis Koidzumi. The former extract did not inhibit mushroom TYR activity whereas significant inhibition was observed with the latter extract, demonstrating the importance of using human TYR in the screening for human TYR inhibitors. Upon liquid-liquid partitioning of the extract from V. bracteatum, the active constituents were enriched in the ethyl acetate fraction, and the subsequent preparatory thin-layer chromatography identified p-coumaric acid (PCA) as the main active constituent. The hypo-pigmentation of PCA was verified in the MelanoDerm™ Skin Model. This study demonstrates that transformed HEK293-TYR cells could expedite the discovery of human TYR-specific inhibitors from natural sources which might be useful in the control of skin pigmentation. © 2012 The Authors. ICS © 2012 Society of Cosmetic Scientists and the Société Française de Cosmétologie.

  15. Fluconazole inhibits human adrenocortical steroidogenesis in vitro

    NARCIS (Netherlands)

    R. van der Pas (Rob); L.J. Hofland (Leo); J. Hofland (Johannes); A.E. Taylor (A.); W. Arlt (Wiebke); J. Steenbergen (Jacobie); P.M. van Koetsveld (Peter); W.W. de Herder (Wouter); F.H. de Jong (Frank); R.A. Feelders (Richard)

    2012-01-01

    textabstractThe antifungal agent ketoconazole is often used to suppress cortisol production in patients with Cushing's syndrome (CS). However, ketoconazole has serious side effects and is hepatotoxic. Here, the in vitro effects of ketoconazole and fluconazole, which might be less toxic, on human

  16. Luteoloside Inhibits Proliferation of Human Chronic Myeloid ...

    African Journals Online (AJOL)

    Purpose: To investigate the effects of luteoloside on the proliferation of human chronic myeloid leukemia K562 cells and whether luteoloside induces cell cycle arrest and apoptosis in K562 cells. Methods: Luteoloside's cytotoxicity was assessed using a cell counting kit. Cell cycle distribution was analysed by flow cytometry ...

  17. The action of blocking agents applied to the inner face of Ca(2+)-activated K+ channels from human erythrocytes.

    Science.gov (United States)

    Dunn, P M

    1998-09-15

    The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate conductance calcium-activated potassium (IKCa) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba2+ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed by TEA at its binding site (delta) of 0.18 and a Kd at 0 mV of 20.5 mM. Ba2+ was a very potent blocker of the channel, breaking the single channel activity up into bursts, inter-spersed with silent periods lasting several seconds. The effect of Ba2+ was very voltage sensitive, delta = 0.44, and a Kd at 0 mV of 0.15 microM. Clotrimazole applied to the inner face of the membrane at a concentration block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the channel. A high concentration of cetiedil (100 microM) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular site on the channel.

  18. Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death

    Science.gov (United States)

    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

  19. Influence of some DNA-alkylating drugs on thermal stability, acid and osmotic resistance of the membrane of whole human erythrocytes and their ghosts.

    Science.gov (United States)

    Ivanov, I T; Gadjeva, V

    2000-09-01

    Human erythrocytes and their resealed ghosts were alkylated under identical conditions using three groups of alkylating antitumor agents: mustards, triazenes and chloroethyl nitrosoureas. Osmotic fragility, acid resistance and thermal stability of membranes were changed only in alkylated ghosts in proportion to the concentration of the alkylating agent. All the alkylating agents decreased acid resistance in ghosts. The clinically used drugs sarcolysine, dacarbazine and lomustine all decreased osmotic fragility and thermal stability of ghost membranes depending on their lipophilicity. DM-COOH did not decrease osmotic fragility and thermal stability of ghost membranes, while NEM increased thermal stability of membranes. The preliminary but not subsequent treatment of ghosts with DM-COOH fully abolished the alkylation-induced thermal labilization of ghost membrane proteins while NEM had a partial effect only. The present study gives direct evidence that alkylating agents, having a high therapeutic activity against malignant growth, bind covalently to proteins of cellular membranes.

  20. Effect of changes in dietary sodium on active electrolyte transport by erythrocytes at different stages of human pregnancy.

    Science.gov (United States)

    Gallery, E D; Rowe, J; Brown, M A; Ross, M

    1988-02-01

    1. Active electrolyte transport was examined in erythrocytes from women in the second and third trimesters of pregnancy and post partum, and compared with that in ovulating women. 2. There was a significant reduction in intracellular sodium ([Na]i) and increase in intracellular potassium ([K]i) in pregnancy with a return towards normal values in the post-partum period. 3. Maximum specific ouabain binding [number of Na+,K+-adenosine triphosphatase (Na+, K+-ATPase) units] was increased by 70% in pregnancy and returned slowly towards normal values post partum. 4. Na+,K+-ATPase activity as determined by ouabain-sensitive 86Rb influx in artificial media was also increased in pregnancy by 13%. It returned towards normal post partum. 5. The increases in Na+,K+-ATPase in pregnancy were not closely related to the concomitant increases in aldosterone or cholesterol nor to reticulocytosis and were not affected by 7 days of high (greater than 250 mmol/day) or low (less than 50 mmol/day) sodium intake.

  1. Lipid-Lowering Pharmaceutical Clofibrate Inhibits Human Sweet Taste

    Science.gov (United States)

    Kochem, Matthew

    2017-01-01

    T1R2-T1R3 is a heteromeric receptor that binds sugars, high potency sweeteners, and sweet taste blockers. In rodents, T1R2-T1R3 is largely responsible for transducing sweet taste perception. T1R2-T1R3 is also expressed in non-taste tissues, and a growing body of evidence suggests that it helps regulate glucose and lipid metabolism. It was previously shown that clofibric acid, a blood lipid-lowering drug, binds T1R2-T1R3 and inhibits its activity in vitro. The purpose of this study was to determine whether clofibric acid inhibits sweetness perception in humans and is, therefore, a T1R2-T1R3 antagonist in vivo. Fourteen participants rated the sweetness intensity of 4 sweeteners (sucrose, sucralose, Na cyclamate, acesulfame K) across a broad range of concentrations. Each sweetener was prepared in solution neat and in mixture with either clofibric acid or lactisole. Clofibric acid inhibited sweetness of every sweetener. Consistent with competitive binding, inhibition by clofibric acid was diminished with increasing sweetener concentration. This study provides in vivo evidence that the lipid-lowering drug clofibric acid inhibits sweetness perception and is, therefore, a T1R carbohydrate receptor inhibitor. Our results are consistent with previous in vitro findings. Given that T1R2-T1R3 may in part regulate glucose and lipid metabolism, future studies should investigate the metabolic effects of T1R inhibition. PMID:27742692

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

    Science.gov (United States)

    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. Copyright © 2015 Elsevier Ltd. All rights reserved.

  3. Physiological markers of motor inhibition during human behavior

    Science.gov (United States)

    Duque, Julie; Greenhouse, Ian; Labruna, Ludovica; Ivry, Richard B.

    2017-01-01

    Transcranial magnetic stimulation (TMS) studies in humans have shown that many behaviors engage processes that suppress excitability within the corticospinal tract. Inhibition of the motor output pathway has been extensively studied in the context of action stopping, where a planned movement needs to be abruptly aborted. Recent TMS work has also revealed markers of motor inhibition during the preparation of movement. Here, we review the evidence for motor inhibition during action stopping and action preparation, focusing on studies that have used TMS to monitor changes in the excitability of the corticospinal pathway. We discuss how these physiological results have motivated theoretical models of how the brain selects actions, regulates movement initiation and execution, and switches from one state to another. PMID:28341235

  4. Metformin and Its Sulfenamide Prodrugs Inhibit Human Cholinesterase Activity

    Directory of Open Access Journals (Sweden)

    Magdalena Markowicz-Piasecka

    2017-01-01

    Full Text Available The results of epidemiological and pathophysiological studies suggest that type 2 diabetes mellitus (T2DM may predispose to Alzheimer’s disease (AD. The two conditions present similar glucose levels, insulin resistance, and biochemical etiologies such as inflammation and oxidative stress. The diabetic state also contributes to increased acetylcholinesterase (AChE activity, which is one of the factors leading to neurodegeneration in AD. The aim of this study was to assess in vitro the effects of metformin, phenformin, and metformin sulfenamide prodrugs on the activity of human AChE and butyrylcholinesterase (BuChE and establish the type of inhibition. Metformin inhibited 50% of the AChE activity at micromolar concentrations (2.35 μmol/mL, mixed type of inhibition and seemed to be selective towards AChE since it presented low anti-BuChE activity. The tested metformin prodrugs inhibited cholinesterases (ChE at nanomolar range and thus were more active than metformin or phenformin. The cyclohexyl sulfenamide prodrug demonstrated the highest activity towards both AChE (IC50 = 890 nmol/mL, noncompetitive inhibition and BuChE (IC50 = 28 nmol/mL, mixed type inhibition, while the octyl sulfenamide prodrug did not present anti-AChE activity, but exhibited mixed inhibition towards BuChE (IC50 = 184 nmol/mL. Therefore, these two bulkier prodrugs were concluded to be the most selective compounds for BuChE over AChE. In conclusion, it was demonstrated that biguanides present a novel class of inhibitors for AChE and BuChE and encourages further studies of these compounds for developing both selective and nonselective inhibitors of ChEs in the future.

  5. Inhibition of autophagy induced by proteasome inhibition increases cell death in human SHG-44 glioma cells.

    Science.gov (United States)

    Ge, Peng-Fei; Zhang, Ji-Zhou; Wang, Xiao-Fei; Meng, Fan-Kai; Li, Wen-Chen; Luan, Yong-Xin; Ling, Feng; Luo, Yi-Nan

    2009-07-01

    The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Recent studies suggest that proteasome inhibitors may reduce tumor growth and activate autophagy. Due to the dual roles of autophagy in tumor cell survival and death, the effect of autophagy on the destiny of glioma cells remains unclear. In this study, we sought to investigate whether inhibition of the proteasome can induce autophagy and the effects of autophagy on the fate of human SHG-44 glioma cells. The proteasome inhibitor MG-132 was used to induce autophagy in SHG-44 glioma cells, and the effect of autophagy on the survival of SHG-44 glioma cells was investigated using an autophagy inhibitor 3-MA. Cell viability was measured by MTT assay. Apoptosis and cell cycle were detected by flow cytometry. The expression of autophagy related proteins was determined by Western blot. MG-132 inhibited cell proliferation, induced cell death and cell cycle arrest at G(2)/M phase, and activated autophagy in SHG-44 glioma cells. The expression of autophagy-related Beclin-1 and LC3-I was significantly up-regulated and part of LC3-I was converted into LC3-II. However, when SHG-44 glioma cells were co-treated with MG-132 and 3-MA, the cells became less viable, but cell death and cell numbers at G(2)/M phase increased. Moreover, the accumulation of acidic vesicular organelles was decreased, the expression of Beclin-1 and LC3 was significantly down-regulated and the conversion of LC3-II from LC3-I was also inhibited. Inhibition of the proteasome can induce autophagy in human SHG-44 glioma cells, and inhibition of autophagy increases cell death. This discovery may shed new light on the effect of autophagy on modulating the fate of SHG-44 glioma cells.Acta Pharmacologica Sinica (2009) 30: 1046-1052; doi: 10.1038/aps.2009.71.

  6. IL-17 inhibits chondrogenic differentiation of human mesenchymal stem cells.

    Directory of Open Access Journals (Sweden)

    Masahiro Kondo

    Full Text Available OBJECTIVE: Mesenchymal stem cells (MSCs can differentiate into cells of mesenchymal lineages, such as osteoblasts and chondrocytes. Here we investigated the effects of IL-17, a key cytokine in chronic inflammation, on chondrogenic differentiation of human MSCs. METHODS: Human bone marrow MSCs were pellet cultured in chondrogenic induction medium containing TGF-β3. Chondrogenic differentiation was detected by cartilage matrix accumulation and chondrogenic marker gene expression. RESULTS: Over-expression of cartilage matrix and chondrogenic marker genes was noted in chondrogenic cultures, but was inhibited by IL-17 in a dose-dependent manner. Expression and phosphorylation of SOX9, the master transcription factor for chondrogenesis, were induced within 2 days and phosphorylated SOX9 was stably maintained until day 21. IL-17 did not alter total SOX9 expression, but significantly suppressed SOX9 phosphorylation in a dose-dependent manner. At day 7, IL-17 also suppressed the activity of cAMP-dependent protein kinase A (PKA, which is known to phosphorylate SOX9. H89, a selective PKA inhibitor, also suppressed SOX9 phosphorylation, expression of chondrogenic markers and cartilage matrix, and also decreased chondrogenesis. CONCLUSIONS: IL-17 inhibited chondrogenesis of human MSCs through the suppression of PKA activity and SOX9 phosphorylation. These results suggest that chondrogenic differentiation of MSCs can be inhibited by a mechanism triggered by IL-17 under chronic inflammation.

  7. 21 CFR 864.6700 - Erythrocyte sedimentation rate test.

    Science.gov (United States)

    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 Erythrocyte...

  8. Python erythrocytes are resistant to α-hemolysin from Escherichia coli.

    Science.gov (United States)

    Larsen, Casper K; Skals, Marianne; Wang, Tobias; Cheema, Muhammad U; Leipziger, Jens; Praetorius, Helle A

    2011-12-01

    α-Hemolysin (HlyA) from Escherichia coli lyses mammalian erythrocytes by creating nonselective cation pores in the membrane. Pore insertion triggers ATP release and subsequent P2X receptor and pannexin channel activation. Blockage of either P2X receptors or pannexin channels reduces HlyA-induced hemolysis. We found that erythrocytes from Python regius and Python molurus are remarkably resistant to HlyA-induced hemolysis compared to human and Trachemys scripta erythrocytes. HlyA concentrations that induced maximal hemolysis of human erythrocytes did not affect python erythrocytes, but increasing the HlyA concentration 40-fold did induce hemolysis. Python erythrocytes were more resistant to osmotic stress than human erythrocytes, but osmotic stress tolerance per se did not confer HlyA resistance. Erythrocytes from T. scripta, which showed higher osmotic resistance than python erythrocytes, were as susceptible to HlyA as human erythrocytes. Therefore, we tested whether python erythrocytes lack the purinergic signalling known to amplify HlyA-induced hemolysis in human erythrocytes. P. regius erythrocytes increased intracellular Ca²⁺ concentration and reduced cell volume when exposed to 3 mM ATP, indicating the presence of a P2X₇-like receptor. In addition, scavenging extracellular ATP or blocking P2 receptors or pannexin channels reduced the HlyA-induced hemolysis. We tested whether the low HlyA sensitivity resulted from low affinity of HlyA to the python erythrocyte membrane. We found comparable incorporation of HlyA into human and python erythrocyte membranes. Taken together, the remarkable HlyA resistance of python erythrocytes was not explained by increased osmotic resistance, lack of purinergic hemolysis amplification, or differences in HlyA affinity.

  9. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.; Sharaf, Hazem; Hastings, Claire H.; Ho, Yung Shwen; Nair, Mridul; 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.

    2016-01-01

    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.

  10. Normocyte-binding protein required for human erythrocyte invasion by the zoonotic malaria parasitePlasmodium knowlesi

    KAUST Repository

    Moon, Robert W.

    2016-06-15

    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.

  11. High efficacy of anti DBL4e-VAR2CSA antibodies in inhibition of CSA-binding Plasmodium falciparum-infected erythrocytes from pregnant women

    DEFF Research Database (Denmark)

    Magistrado, Pamela A; Minja, Daniel; Doritchamou, Justin

    2011-01-01

    Malaria during pregnancy is a major cause of intra-uterine growth-retardation and infant death in sub-Saharan Africa. Ideally, this could be prevented by a vaccine delivered before the first pregnancy. Antibodies against domain DBL4¿ from VAR2CSA has been shown to inhibit adhesion of laboratory i...

  12. Influence of erythrocyte oxygenation and intravascular ATP on resting and exercising skeletal muscle blood flow in humans with mitochondrial myopathy

    DEFF Research Database (Denmark)

    Jeppesen, Tina D; Vissing, John; González-Alonso, José

    2012-01-01

    Oxygen (O(2)) extraction is impaired in exercising skeletal muscle of humans with mutations of mitochondrial DNA (mtDNA), but the muscle hemodynamic response to exercise has never been directly investigated. This study sought to examine the extent to which human skeletal muscle perfusion can incr...

  13. Hypoxia activates a Ca2+-permeable cation conductance sensitive to carbon monoxide and to GsMTx-4 in human and mouse sickle erythrocytes.

    OpenAIRE

    David H Vandorpe; Chang Xu; Boris E Shmukler; Leo E Otterbein; Marie Trudel; Frederick Sachs; Philip A Gottlieb; Carlo Brugnara; Seth L Alper

    2010-01-01

    Background: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca2+] ([Ca2+]i) and subsequent activation of KCa 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded...

  14. Plasmodium falciparum-infected erythrocytes and IL-12/IL-18 induce diverse transcriptomes in human NK cells: IFN-α/β pathway versus TREM signaling.

    Directory of Open Access Journals (Sweden)

    Elisandra Grangeiro de Carvalho

    Full Text Available The protective immunity of natural killer (NK cells against malarial infections is thought to be due to early production of type II interferon (IFN and possibly direct NK cell cytotoxicity. To better understand this mechanism, a microarray analysis was conducted on NK cells from healthy donors PBMCs that were co-cultured with P. falciparum 3D7-infected erythrocytes. A very similar pattern of gene expression was observed among all donors for each treatment in three replicas. Parasites particularly modulated genes involved in IFN-α/β signaling as well as molecules involved in the activation of interferon regulatory factors, pathways known to play a role in the antimicrobial immune response. This pattern of transcription was entirely different from that shown by NK cells treated with IL-12 and IL-18, in which IFN-γ- and TREM-1-related genes were over-expressed. These results suggest that P. falciparum parasites and the cytokines IL-12 and IL-18 have diverse imprints on the transcriptome of human primary NK cells. IFN-α-related genes are the prominent molecules induced by parasites on NK cells and arise as candidate biomarkers that merit to be further investigated as potential new tools in malaria control.

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

    Science.gov (United States)

    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. © 2013 John Wiley & Sons Ltd.

  16. Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B

    International Nuclear Information System (INIS)

    Kurokawa, T.; Tillotson, L.G.; Chen, C.C.; Isselbacher, K.J.

    1986-01-01

    The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [ 3 H]cytochalasin B and solubilized with n-octyl β-D-glucopyranoside (octyl glucoside). [ 3 H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [ 3 H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [ 3 H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. These results indicate the [ 3 H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of the conformational changes of the protein as a result of covalent binding with [ 3 H]cytochalasin B by photoaffinity labeling

  17. Inhibition of endogenous lactate turnover with lactate infusion in humans

    International Nuclear Information System (INIS)

    Searle, G.L.; Feingold, K.R.; Hsu, F.S.; Clark, O.H.; Gertz, E.W.; Stanley, W.C.

    1989-01-01

    The extent to which lactate infusion may inhibit endogenous lactate production, though previously considered, has never been critically assessed. To examine this proposition, single injection tracer methodology (U- 14 C Lactate) has been used for the estimation of lactate kinetics in 12 human subjects under basal conditions and with the infusion of sodium lactate. The basal rate of lactate turnover was measured on a day before the study with lactate infusion, and averaged 63.7 + 5.5 mg/kg/h. Six of these individuals received a stable lactate infusion at an approximate rate of 160 mg/kg/h, while the remaining six individuals were infused at the approximate rate of 100 mg/kg/h. It has been found that stable lactate infused at rates approximating 160 mg/kg/h consistently produced a complete inhibition of endogenous lactate production. Infusion of lactate at 100 mg/kg/h caused a lesser and more variable inhibition of endogenous lactate production (12% to 64%). In conclusion, lactate infusion significantly inhibits endogenous lactate production

  18. The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

    Directory of Open Access Journals (Sweden)

    Deanna M. Schmitt

    2017-05-01

    Full Text Available Francisella tularensis is an extremely virulent bacterium that can be transmitted naturally by blood sucking arthropods. During mammalian infection, F. tularensis infects numerous types of host cells, including erythrocytes. As erythrocytes do not undergo phagocytosis or endocytosis, it remains unknown how F. tularensis invades these cells. Furthermore, the consequence of inhabiting the intracellular space of red blood cells (RBCs has not been determined. Here, we provide evidence indicating that residing within an erythrocyte enhances the ability of F. tularensis to colonize ticks following a blood meal. Erythrocyte residence protected F. tularensis from a low pH environment similar to that of gut cells of a feeding tick. Mechanistic studies revealed that the F. tularensis type VI secretion system (T6SS was required for erythrocyte invasion as mutation of mglA (a transcriptional regulator of T6SS genes, dotU, or iglC (two genes encoding T6SS machinery severely diminished bacterial entry into RBCs. Invasion was also inhibited upon treatment of erythrocytes with venom from the Blue-bellied black snake (Pseudechis guttatus, which aggregates spectrin in the cytoskeleton, but not inhibitors of actin polymerization and depolymerization. These data suggest that erythrocyte invasion by F. tularensis is dependent on spectrin utilization which is likely mediated by effectors delivered through the T6SS. Our results begin to elucidate the mechanism of a unique biological process facilitated by F. tularensis to invade erythrocytes, allowing for enhanced colonization of ticks.

  19. Recombinant human prion protein inhibits prion propagation in vitro.

    Science.gov (United States)

    Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

    2013-10-09

    Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

  20. Inhibition of fatty acid metabolism reduces human myeloma cells proliferation.

    Directory of Open Access Journals (Sweden)

    José Manuel Tirado-Vélez

    Full Text Available Multiple myeloma is a haematological malignancy characterized by the clonal proliferation of plasma cells. It has been proposed that targeting cancer cell metabolism would provide a new selective anticancer therapeutic strategy. In this work, we tested the hypothesis that inhibition of β-oxidation and de novo fatty acid synthesis would reduce cell proliferation in human myeloma cells. We evaluated the effect of etomoxir and orlistat on fatty acid metabolism, glucose metabolism, cell cycle distribution, proliferation, cell death and expression of G1/S phase regulatory proteins in myeloma cells. Etomoxir and orlistat inhibited β-oxidation and de novo fatty acid synthesis respectively in myeloma cells, without altering significantly glucose metabolism. These effects were associated with reduced cell viability and cell cycle arrest in G0/G1. Specifically, etomoxir and orlistat reduced by 40-70% myeloma cells proliferation. The combination of etomoxir and orlistat resulted in an additive inhibitory effect on cell proliferation. Orlistat induced apoptosis and sensitized RPMI-8226 cells to apoptosis induction by bortezomib, whereas apoptosis was not altered by etomoxir. Finally, the inhibitory effect of both drugs on cell proliferation was associated with reduced p21 protein levels and phosphorylation levels of retinoblastoma protein. In conclusion, inhibition of fatty acid metabolism represents a potential therapeutic approach to treat human multiple myeloma.

  1. ITE inhibits growth of human pulmonary artery endothelial cells.

    Science.gov (United States)

    Pang, Ling-Pin; Li, Yan; Zou, Qing-Yun; Zhou, Chi; Lei, Wei; Zheng, Jing; Huang, Shi-An

    2017-10-01

    Pulmonary arterial hypertension (PAH), a deadly disorder is associated with excessive growth of human pulmonary artery endothelial (HPAECs) and smooth muscle (HPASMCs) cells. Current therapies primarily aim at promoting vasodilation, which only ameliorates clinical symptoms without a cure. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an endogenous aryl hydrocarbon receptor (AhR) ligand, and mediates many cellular function including cell growth. However, the roles of ITE in human lung endothelial cells remain elusive. Herein, we tested a hypothesis that ITE inhibits growth of human pulmonary artery endothelial cells via AhR. Immunohistochemistry was performed to localize AhR expression in human lung tissues. The crystal violet method and MTT assay were used to determine ITE's effects on growth of HPAECs. The AhR activation in HPAECs was confirmed using Western blotting and RT-qPCR. The role of AhR in ITE-affected proliferation of HPAECs was assessed using siRNA knockdown method followed by the crystal violet method. Immunohistochemistry revealed that AhR was present in human lung tissues, primarily in endothelial and smooth muscle cells of pulmonary veins and arteries, as well as in bronchial and alveolar sac epithelia. We also found that ITE dose- and time-dependently inhibited proliferation of HPAECs with a maximum inhibition of 83% at 20 µM after 6 days of treatment. ITE rapidly decreased AhR protein levels, while it increased mRNA levels of cytochrome P450 (CYP), family 1, member A1 (CYP1A1) and B1 (CYP1B1), indicating activation of the AhR/CYP1A1 and AhR/CYP1B1 pathways in HPAECs. The AhR siRNA significantly suppressed AhR protein expression, whereas it did not significantly alter ITE-inhibited growth of HPAECs. ITE suppresses growth of HPAECs independent of AhR, suggesting that ITE may play an important role in preventing excessive growth of lung endothelial cells.

  2. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    Energy Technology Data Exchange (ETDEWEB)

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-03-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10/sup -7/ to 10/sup -4/M), naloxone inhibited (p < .001) the release of superoxide (O/sub 2//sup -/) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O/sub 2//sup -/ released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of /sup 3/H FMLP to HN. Using /sup 3/H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10/sup -5/) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED/sub 50/ for naloxone inhibition of O/sub 2//sup -/ (1 x 10/sup -5/M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D/sub 2/ or E/sub 2/. Conclusions: (1) naloxone inhibits FMLP-stimulated O/sub 2/ but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN.

  3. Naloxone inhibits superoxide but not enzyme release by human neutrophils

    International Nuclear Information System (INIS)

    Simpkins, C.; Alailima, S.; Tate, E.

    1986-01-01

    The release of toxic oxygen metabolites and enzymes by phagocytic cells is thought to play a role in the multisystemic tissue injury of sepsis. Naloxone protects septic animals. We have found that at concentrations administered to animals (10 -7 to 10 -4 M), naloxone inhibited (p 2 - ) by human neutrophils (HN), stimulated with N-formyl methionyl leucyl phenylalanine (FMLP). Naloxone had no effect on cell viability. Maximum inhibition was 65% of the total O 2 - released (13.1 nMoles/8 min/320,000 cells). FMLP-stimulated release of beta-glucoronidase or lysozyme was not altered by naloxone. Naloxone had no effect on the binding of 3 H FMLP to HN. Using 3 H naloxone and various concentrations of unlabeled naloxone higher affinity (K/sub D/ = 12nM) and lower affinity (K/sub D/ = 4.7 x 10 -5 ) binding sites were detected. The K/sub D/ of the low affinity site corresponded to the ED 50 for naloxone inhibition of O 2 - (1 x 10 -5 M). Binding to this low affinity site was decreased by (+) naloxone, beta-endorphin and N acetyl beta-endorphin, but not by leu-enkephalin, thyrotropin releasing factor, prostaglandin D 2 or E 2 . Conclusions: (1) naloxone inhibits FMLP-stimulated O 2 but not enzyme release, (2) this inhibition is not due to alteration of FMLP receptor binding, (3) naloxone may act via a low affinity binding site which is ligand specific, and (4) a higher affinity receptor is present on HN

  4. Erythrocyte membrane stabilization effect and antioxidant activity of methyl methacrylate

    International Nuclear Information System (INIS)

    Popov, B.

    2004-01-01

    Methyl methacrylate (MMK) is a synthetic product with mild impact on human health that is not well studied on cellular basis. Here, human erythrocytes were used to investigate the effects MMK exerts on acid and heat-induced hemolysis. Biphasic effect of MMK was observed for acid-induced hemolysis; i.e., protection at low (0 - 0.05% v/v) and stimulation at higher (0.1- 0.4% v/v) concentrations. The maximal protective effect was produced at 0.03% (v/v). At this concentration MMK increased the temperatures of heat denaturation of erythrocyte membrane proteins, spectrin and integral proteins, by about 2 0 C and inhibited the heat-induced hemolysis by 20 %. This membrane stabilization effect of MMK is similar to that produced by some anti-inflammatory and antirheumatic drugs. The increased acid resistance possibly indicated anti-oxidant properties of MMK. The nonenzymatic antioxidant activity test evidenced that MMK has no superoxide dismutase-like activity but demonstrates strong catalase-like activity (about 900 kU/mmol at 0.05-0.1 mmol/l concentration). The results indicate that at low concentration MMK exerts benign effect on cellular membrane that could find therapeutic usage. (author)

  5. Host-parasite interaction: selective Pv-fam-a family proteins of Plasmodium vivax bind to a restricted number of human erythrocyte receptors.

    Science.gov (United States)

    Zeeshan, Mohammad; Tyagi, Rupesh Kumar; Tyagi, Kriti; Alam, Mohd Shoeb; Sharma, Yagya Dutta

    2015-04-01

    Plasmodium vivax synthesizes the largest number of 36 tryptophan-rich proteins belonging to the Pv-fam-a family. These parasite proteins need to be characterized for their biological function because tryptophan-rich proteins from other Plasmodium species have been proposed as vaccine candidates. Recombinant P. vivax tryptophan-rich antigens (PvTRAgs) were used to determine their erythrocyte-binding activity by a cell-based enzyme-linked immunosorbent assay, flow cytometry, and a rosetting assay. Only 4 (PvTRAg26.3, PvTRAg34, PvTRAg36, and PvTRAg36.6) of 21 PvTRAgs bind to host erythrocytes. The cross-competition data indicated that PvTRAg36 and PvTRAg34 share their erythrocyte receptors with previously described proteins PvTRAg38 and PvTRAg33.5, respectively. On the other hand, PvTRAg26.3 and PvTRAg36.6 cross-compete with each other and not with any other PvTRAg, indicating that these 2 proteins bind to the same but yet another set of erythrocyte receptor(s). Together, 10 of 36 PvTRAgs possess erythrocyte-binding activity in which each protein recognizes >1 erythrocyte receptor. Further, each erythrocyte receptor is shared by >1 PvTRAg. This redundancy may be useful for the parasite to invade red blood cells and cause disease pathogenesis, and it can be exploited to develop therapeutics against P. vivax malaria. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  6. Erythrocyte Membrane Failure by Electromechanical Stress

    Directory of Open Access Journals (Sweden)

    E Du

    2018-01-01

    Full Text Available We envision that electrodeformation of biological cells through dielectrophoresis as a new technique to elucidate the mechanistic details underlying membrane failure by electrical and mechanical stresses. Here we demonstrate the full control of cellular uniaxial deformation and tensile recovery in biological cells via amplitude-modified electric field at radio frequency by an interdigitated electrode array in microfluidics. Transient creep and cyclic experiments were performed on individually tracked human erythrocytes. Observations of the viscoelastic-to-viscoplastic deformation behavior and the localized plastic deformations in erythrocyte membranes suggest that electromechanical stress results in irreversible membrane failure. Examples of membrane failure can be separated into different groups according to the loading scenarios: mechanical stiffening, physical damage, morphological transformation from discocyte to echinocyte, and whole cell lysis. These results show that this technique can be potentially utilized to explore membrane failure in erythrocytes affected by other pathophysiological processes.

  7. Human mesenchymal stem cells inhibit osteoclastogenesis through osteoprotegerin production.

    Science.gov (United States)

    Oshita, Koichi; Yamaoka, Kunihiro; Udagawa, Nobuyuki; Fukuyo, Shunsuke; Sonomoto, Koshiro; Maeshima, Keisuke; Kurihara, Ryuji; Nakano, Kazuhisa; Saito, Kazuyoshi; Okada, Yosuke; Chiba, Kenji; Tanaka, Yoshiya

    2011-06-01

    Mesenchymal stem cells (MSCs) have been proposed to be a useful tool for treatment of rheumatoid arthritis (RA), not only because of their multipotency but also because of their immunosuppressive effect on lymphocytes, dendritic cells, and other proinflammatory cells. Since bone destruction caused by activated osteoclasts occurs in RA, we undertook the present study to investigate the effect of MSCs on osteoclast function and differentiation in order to evaluate their potential use in RA therapy. Human MSCs and peripheral blood mononuclear cells were cultured under cell-cell contact-free conditions with osteoclast induction medium. Differentiation into osteoclast-like cells was determined by tartrate-resistant acid phosphatase staining and expression of osteoclast differentiation markers. The number of osteoclast-like cells was decreased and expression of cathepsin K and nuclear factor of activated T cells c1 (NF-ATc1) was down-regulated by the addition of either MSCs or a conditioned medium obtained from MSCs. Osteoprotegerin (OPG) was constitutively produced by MSCs and inhibited osteoclastogenesis. However, osteoclast differentiation was not fully recovered upon treatment with either anti-OPG antibody or OPG small interfering RNA, suggesting that OPG had only a partial role in the inhibitory effect of MSCs. Moreover, bone-resorbing activity of osteoclast-like cells was partially recovered by addition of anti-OPG antibody into the conditioned medium. The present results indicate that human MSCs constitutively produce OPG, resulting in inhibition of osteoclastogenesis and expression of NF-ATc1 and cathepsin K in the absence of cell-cell contact. Therefore, we conclude that human MSCs exert a suppressive effect on osteoclastogenesis, which may be beneficial in inhibition of joint damage in RA. Copyright © 2011 by the American College of Rheumatology.

  8. Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes

    DEFF Research Database (Denmark)

    Reinholdt, Jesper; Poulsen, Knud; Brinkmann, Christel Rothe

    2013-01-01

    Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear...

  9. Oxidative Hemolysis of Erythrocytes

    Science.gov (United States)

    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.

  10. Human Diversity in a Cell Surface Receptor that Inhibits Autophagy.

    Science.gov (United States)

    Chaudhary, Anu; Leite, Mara; Kulasekara, Bridget R; Altura, Melissa A; Ogahara, Cassandra; Weiss, Eli; Fu, Wenqing; Blanc, Marie-Pierre; O'Keeffe, Michael; Terhorst, Cox; Akey, Joshua M; Miller, Samuel I

    2016-07-25

    Mutations in genes encoding autophagy proteins have been associated with human autoimmune diseases, suggesting that diversity in autophagy responses could be associated with disease susceptibility or severity. A cellular genome-wide association study (GWAS) screen was performed to explore normal human diversity in responses to rapamycin, a microbial product that induces autophagy. Cells from several human populations demonstrated variability in expression of a cell surface receptor, CD244 (SlamF4, 2B4), that correlated with changes in rapamycin-induced autophagy. High expression of CD244 and receptor activation with its endogenous ligand CD48 inhibited starvation- and rapamycin-induced autophagy by promoting association of CD244 with the autophagy complex proteins Vps34 and Beclin-1. The association of CD244 with this complex reduced Vps34 lipid kinase activity. Lack of CD244 is associated with auto-antibody production in mice, and lower expression of human CD244 has previously been implicated in severity of human rheumatoid arthritis and systemic lupus erythematosus, indicating that increased autophagy as a result of low levels of CD244 may alter disease outcomes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Apple juice inhibits human low density lipoprotein oxidation.

    Science.gov (United States)

    Pearson, D A; Tan, C H; German, J B; Davis, P A; Gershwin, M E

    1999-01-01

    Dietary phenolic compounds, ubiquitous in vegetables and fruits and their juices possess antioxidant activity that may have beneficial effects on human health. The phenolic composition of six commercial apple juices, and of the peel (RP), flesh (RF) and whole fresh Red Delicious apples (RW), was determined by high performance liquid chromatography (HPLC), and total phenols were determined by the Folin-Ciocalteau method. HPLC analysis identified and quantified several classes of phenolic compounds: cinnamates, anthocyanins, flavan-3-ols and flavonols. Phloridzin and hydroxy methyl furfural were also identified. The profile of phenolic compounds varied among the juices. The range of concentrations as a percentage of total phenolic concentration was: hydroxy methyl furfural, 4-30%; phloridzin, 22-36%; cinnamates, 25-36%; anthocyanins, n.d.; flavan-3-ols, 8-27%; flavonols, 2-10%. The phenolic profile of the Red Delicious apple extracts differed from those of the juices. The range of concentrations of phenolic classes in fresh apple extracts was: hydroxy methyl furfural, n.d.; phloridzin, 11-17%; cinnamates, 3-27%; anthocyanins, n.d.-42%; flavan-3-ols, 31-54%; flavonols, 1-10%. The ability of compounds in apple juices and extracts from fresh apple to protect LDL was assessed using an in vitro copper catalyzed human LDL oxidation system. The extent of LDL oxidation was determined as hexanal production using static headspace gas chromatography. The apple juices and extracts, tested at 5 microM gallic acid equivalents (GAE), all inhibited LDL oxidation. The inhibition by the juices ranged from 9 to 34%, and inhibition by RF, RW and RP was 21, 34 and 38%, respectively. Regression analyses revealed no significant correlation between antioxidant activity and either total phenolic concentration or any specific class of phenolics. Although the specific components in the apple juices and extracts that contributed to antioxidant activity have yet to be identified, this study

  12. Monomethylarsonous acid inhibited endogenous cholesterol biosynthesis in human skin fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Lei [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Xiao, Yongsheng [Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States); Wang, Yinsheng, E-mail: yinsheng.wang@ucr.edu [Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403 (United States); Department of Chemistry, University of California, Riverside, CA 92521-0403 (United States)

    2014-05-15

    Human exposure to arsenic in drinking water is a widespread public health concern, and such exposure is known to be associated with many human diseases. The detailed molecular mechanisms about how arsenic species contribute to the adverse human health effects, however, remain incompletely understood. Monomethylarsonous acid [MMA(III)] is a highly toxic and stable metabolite of inorganic arsenic. To exploit the mechanisms through which MMA(III) exerts its cytotoxic effect, we adopted a quantitative proteomic approach, by coupling stable isotope labeling by amino acids in cell culture (SILAC) with LC-MS/MS analysis, to examine the variation in the entire proteome of GM00637 human skin fibroblasts following acute MMA(III) exposure. Among the ∼ 6500 unique proteins quantified, ∼ 300 displayed significant changes in expression after exposure with 2 μM MMA(III) for 24 h. Subsequent analysis revealed the perturbation of de novo cholesterol biosynthesis, selenoprotein synthesis and Nrf2 pathways evoked by MMA(III) exposure. Particularly, MMA(III) treatment resulted in considerable down-regulation of several enzymes involved in cholesterol biosynthesis. In addition, real-time PCR analysis showed reduced mRNA levels of select genes in this pathway. Furthermore, MMA(III) exposure contributed to a distinct decline in cellular cholesterol content and significant growth inhibition of multiple cell lines, both of which could be restored by supplementation of cholesterol to the culture media. Collectively, the present study demonstrated that the cytotoxicity of MMA(III) may arise, at least in part, from the down-regulation of cholesterol biosynthesis enzymes and the resultant decrease of cellular cholesterol content. - Highlights: • MMA(III)-induced perturbation of the entire proteome of GM00637 cells is studied. • Quantitative proteomic approach revealed alterations of multiple cellular pathways. • MMA(III) inhibits de novo cholesterol biosynthesis. • MMA

  13. Diminished ability of erythrocytes from patients with systemic lupus erythematosus to limit opsonized immune complex deposition on leukocytes and activation of granulocytes

    DEFF Research Database (Denmark)

    Nielsen, C H; Rasmussen, J M; Voss, A

    1998-01-01

    OBJECTIVE: To compare the ability of normal erythrocytes and erythrocytes from systemic lupus erythematosus (SLE) patients to bind immune complexes (IC), thereby inhibiting IC deposition on polymorphonuclear leukocytes (PMN) and the consequent induction of a PMN respiratory burst (RB). METHODS...

  14. Acute inhibition of iron bioavailability by zinc: studies in humans.

    Science.gov (United States)

    Olivares, Manuel; Pizarro, Fernando; Ruz, Manuel; de Romaña, Daniel López

    2012-08-01

    Iron (Fe) and zinc (Zn) deficiencies constitute two of the most important nutritional and public health problems affecting developing countries. Combined supplementation or fortification with Zn and Fe are strategies that can be used to improve the Zn and Fe status of a population. However, there is concern about potential negative interactions between these two micronutrients due to a competitive binding to DMT1 and Zip14 transporter. Studies performed in humans have shown an inhibitory effect of Zn on Fe absorption when both minerals are given together as a solution in fasting conditions. We found that at low doses of iron (0.5 mg) the threshold for the inhibition of iron bioavailability was at a Zn:Fe wt/wt ratio ≥5.9:1, whereas at higher doses of Fe (10 mg) this inhibition occurred at 1:1 Zn:Fe wt/wt ratio. This differential response could be explained by the variation in the abundance of both cations as they compete for a limited number of shared transporters at the enterocyte. Conflicting results have been obtained when this interaction was studied in different food matrices. A negative interaction was not observed when Fe and Zn were provided in a composite hamburger meal, premature formula, human milk, or cow milk. A decrease on Fe absorption was observed in only 1 of 3 studies when Fe and Zn were supplied in wheat flour. The possibility of a negative interaction should be considered for supplementation or fortification programs with both microminerals.

  15. Aliphatic alcohols in spirits inhibit phagocytosis by human monocytes.

    Science.gov (United States)

    Pál, László; Árnyas, Ervin M; Bujdosó, Orsolya; Baranyi, Gergő; Rácz, Gábor; Ádány, Róza; McKee, Martin; Szűcs, Sándor

    2015-04-01

    A large volume of alcoholic beverages containing aliphatic alcohols is consumed worldwide. Previous studies have confirmed the presence of ethanol-induced immunosuppression in heavy drinkers, thereby increasing susceptibility to infectious diseases. However, the aliphatic alcohols contained in alcoholic beverages might also impair immune cell function, thereby contributing to a further decrease in microbicidal activity. Previous research has shown that aliphatic alcohols inhibit phagocytosis by granulocytes but their effect on human monocytes has not been studied. This is important as they play a crucial role in engulfment and killing of pathogenic microorganisms and a decrease in their phagocytic activity could lead to impaired antimicrobial defence in heavy drinkers. The aim of this study was to measure monocyte phagocytosis following their treatment with those aliphatic alcohols detected in alcoholic beverages. Monocytes were separated from human peripheral blood and phagocytosis of opsonized zymosan particles by monocytes treated with ethanol and aliphatic alcohols individually and in combination was determined. It was shown that these alcohols could suppress the phagocytic activity of monocytes in a concentration-dependent manner and when combined with ethanol, they caused a further decrease in phagocytosis. Due to their additive effects, it is possible that they may inhibit phagocytosis in a clinically meaningful way in alcoholics and episodic heavy drinkers thereby contribute to their increased susceptibility to infectious diseases. However, further research is needed to address this question.

  16. No effect of human serum and erythrocytes enriched in n-3 fatty acids by oral intake on Plasmodium falciparum blood stage parasites in vitro

    DEFF Research Database (Denmark)

    Abu-Zeid, Y A; Hansen, H S; Jakobsen, P H

    1993-01-01

    -s) and pre-intake erythrocyte (pre-e). Also the effect of EPA and arachidonic acid (AA, 20:4n-6) on the erythrocytic growth of P. falciparum was tested using in vitro assays. The results show that both post-s and post-e had no antimalarial activity on P. falciparum. No differential antimalarial effect...... acid (EPA, 20:5n-3) of 3.5 g/d and docosahexaenoic acid (DHA, 22:6n-3) of 2.5 g/d and 24 mg/d of total tocopherol. Post-intake fish oil serum (post-s) and erythrocytes (post-e) were tested in vitro for inhibitory activity against blood stages of P. falciparum compared with pre-intake serum (pre...

  17. No effect of human serum and erythrocytes enriched in n-3 fatty acids by oral intake on Plasmodium falciparum bloodstage parasites in vitro

    DEFF Research Database (Denmark)

    Abu-Zeid, Y.A.; Hansen, Harald S.; Jakobsen, P.H.

    1993-01-01

    -s) and pre-intake erythrocyte (pre-e). Also the effect of EPA and arachidonic acid (AA, 20:4n-6) on the erythrocytic growth of P. falciparum was tested using in vitro assays. The results show that both post-s and post-e had no antimalarial activity on P. falciparum. No differential antimalarial effect...... acid (EPA, 20:5n-3) of 3.5g/d and docosahexaenoic acid (DHA, 22:6n-3) of 2.5 g/d and 24 mg/d of total tocopherol. Post-intake fish oil serum (post-s) and erythrocytes (post-e) were tested in vitro for inhibitory activity against blood stages of P. falciparum compared with pre-intake serum (pre...

  18. Radiation damage to human erythrocytes. Relative contribution of hydroxyl and chloride radicals in N{sub 2}O-saturated buffers

    Energy Technology Data Exchange (ETDEWEB)

    Krokosz, Anita [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90 237 Lodz (Poland)], E-mail: krokosz@biol.uni.lodz.pl; Komorowska, Magdalena A.; Szweda-Lewandowska, Zofia [Department of Molecular Biophysics, University of Lodz, Banacha 12/16, 90 237 Lodz (Poland)

    2008-06-15

    The erythrocyte suspensions in Na-phosphate buffered isotonic NaCl solution (PBS) or Na-phosphate isotonic buffer (PB) (hematocrit 1%) were irradiated with the dose of 400 Gy under N{sub 2}O. Erythrocytes were incubated in the medium in which the cells were irradiated or in fresh PBS. The level of damage to cells was estimated on the basis of the course of post-radiation hemolysis and hemoglobin (Hb) oxidation. The medium in which the cells were irradiated and incubated influenced the course of the post-radiation hemolysis and Hb oxidation as well as some other parameters. We discussed the contribution of hydroxyl and chloride radicals in the initiation of erythrocyte damage and oxygen modification of these processes.

  19. Inhibition of canonical WNT signaling attenuates human leiomyoma cell growth

    Science.gov (United States)

    Ono, Masanori; Yin, Ping; Navarro, Antonia; Moravek, Molly B.; Coon, John S.; Druschitz, Stacy A.; Gottardi, Cara J.; Bulun, Serdar E.

    2014-01-01

    Objective Dysregulation of WNT signaling plays a central role in tumor cell growth and progression. Our goal was to assess the effect of three WNT/β-catenin pathway inhibitors, Inhibitor of β-Catenin And TCF4 (ICAT), niclosamide, and XAV939 on the proliferation of primary cultures of human uterine leiomyoma cells. Design Prospective study of human leiomyoma cells obtained from myomectomy or hysterectomy. Setting University research laboratory. Patient(s) Women (n=38) aged 27–53 years undergoing surgery. Intervention(s) Adenoviral ICAT overexpression or treatment with varying concentrations of niclosamide or XAV939. Main Outcome Measure(s) Cell proliferation, cell death, WNT/β-catenin target gene expression or reporter gene regulation, β-catenin levels and cellular localization. Result(s) ICAT, niclosamide, or XAV939 inhibit WNT/β-catenin pathway activation and exert anti-proliferative effects in primary cultures of human leiomyoma cells. Conclusion(s) Three WNT/β-catenin pathway inhibitors specifically block human leiomyoma growth and proliferation, suggesting that the canonical WNT pathway may be a potential therapeutic target for the treatment of uterine leiomyoma. Our findings provide rationale for further preclinical and clinical evaluation of ICAT, niclosamide, and XAV939 as candidate anti-tumor agents for uterine leiomyoma. PMID:24534281

  20. Erythrocytes for Drug Delivery and their Applications: A Review ...

    African Journals Online (AJOL)

    , dogs, rabbits, rats and mice. Encapsulation in erythrocytes drastically changes the pharmacokinetic properties of drugs in both animals and humans, enhancing liver and spleen uptake and targeting the reticulo-endothelial system (RES).

  1. Antioxidant activity and protective effect of banana peel against oxidative hemolysis of human erythrocyte at different stages of ripening.

    Science.gov (United States)

    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.

  2. Automated three-dimensional morphology-based clustering of human erythrocytes with regular shapes: stomatocytes, discocytes, and echinocytes

    Science.gov (United States)

    Ahmadzadeh, Ezat; Jaferzadeh, Keyvan; Lee, Jieun; Moon, Inkyu

    2017-07-01

    We present unsupervised clustering methods for automatic grouping of human red blood cells (RBCs) extracted from RBC quantitative phase images obtained by digital holographic microscopy into three RBC clusters with regular shapes, including biconcave, stomatocyte, and sphero-echinocyte. We select some good features related to the RBC profile and morphology, such as RBC average thickness, sphericity coefficient, and mean corpuscular volume, and clustering methods, including density-based spatial clustering applications with noise, k-medoids, and k-means, are applied to the set of morphological features. The clustering results of RBCs using a set of three-dimensional features are compared against a set of two-dimensional features. Our experimental results indicate that by utilizing the introduced set of features, two groups of biconcave RBCs and old RBCs (suffering from the sphero-echinocyte process) can be perfectly clustered. In addition, by increasing the number of clusters, the three RBC types can be effectively clustered in an automated unsupervised manner with high accuracy. The performance evaluation of the clustering techniques reveals that they can assist hematologists in further diagnosis.

  3. Retinoid inhibition of in vitro invasion of human amnion basement membrane by human tumor cells

    International Nuclear Information System (INIS)

    Fazely, F.; Ledinko, N.; Smith, D.J.

    1986-01-01

    The biological activity of retinoids was assayed in an in vitro quantitative assay of human tumor cell invasion using human amnion basement membrane (BM). The effects measured were the inhibition of tumor cell migration through the BM and tumor cell degradative enzyme activity on 14 C-proline labeled collagenous and noncollagenous components of the BM. The human lung carcinoma A549 or the human Ewing's sarcoma TC-106 cell lines treated with retinoids for two days were incubated on the BM in the absence of retinoids. A dose-dependent inhibition of cell invasion was produced by retinoids. Among the retinoids tested, the most powerful was retinol acetate which inhibited invasion by 50% of A549 cells at a concentration of 0.009 μg/mL, and of TC-106 cells at 0.07 μg/mL. Retinol acetate inhibited A549 and TC-106 cell growth by approximately 50% at levels over 100-fold higher than those needed for antiinvasive activity. Retinol acetate was about 20 times more potent than retinoic acid and 30 times more potent than retinol palmitate. The model system will be useful for investigating antiinvasive activity of other retinoids as well as other compounds

  4. Erythrocyte nanovesicles: Biogenesis, biolo

    Directory of Open Access Journals (Sweden)

    Gamaleldin I. Harisa

    2017-01-01

    Full Text Available Nanovesicles (NVs represent a novel transporter for cell signals to modify functions of target cells. Therefore, NVs play many roles in both physiological and pathological processes. This report highlights biogenesis, composition and biological roles of erythrocytes derived nanovesicles (EDNVs. Furthermore, we address utilization of EDNVs as novel drug delivery cargo as well as therapeutic target. EDNVs are lipid bilayer vesicles rich in phospholipids, proteins, lipid raft, and hemoglobin. In vivo EDNVs biogenesis is triggered by an increase of intracellular calcium levels, ATP depletion and under effect of oxidative stress conditions. However, in vitro production of EDNVs can be achieved via hypotonic treatment and extrusion of erythrocyte. NVs can be used as biomarkers for diagnosis, monitoring of therapy and drug delivery system. Many therapeutic agents are suggested to decrease NVs biogenesis.

  5. The Effect in Vitro of Ionizing Irradiation and Small Rises in Temperature on the Uptake and Release of Labelled Lipids by the Human Erythrocyte Membrane

    DEFF Research Database (Denmark)

    Hansen, Heinz Johs. Max; Karle, H.; Stender, S.

    1978-01-01

    1. The effect of X-irradiation (50 000 rad) and an increase in temperature from 37 to 42° C on the synthesis, uptake and release of labelled lipids by erythrocytes was studied in plasma incubations in vitro. 2. Both irradiation and a rise in temperature resulted in an enhanced synthesis of [32P]phosphatidic...

  6. ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium falciparum-infected Erythrocytes

    Science.gov (United States)

    Branch, Donald R.; Hult, Annika K.; Olsson, Martin L.; Liles, W. Conrad; Cserti-Gazdewich, Christine M.; Kain, Kevin C.

    2012-01-01

    Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A1, A2, and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria. PMID:23071435

  7. Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes.

    Science.gov (United States)

    Yang, Jin-ting; Tang, Li-hui; Liu, Yun-qing; Wang, Yin; Wang, Lie-ju; Zhang, Feng-jiang; Yan, Min

    2015-05-01

    The safe use of intraoperative blood salvage (IBS) in cancer surgery remains controversial. Here, we investigated the killing effect of cisplatin combined with hyperthermia on human hepatocarcinoma (HepG2) cells and erythrocytes from IBS in vitro. HepG2 cells were mixed with concentrated erythrocytes and pretreated with cisplatin (50, 100, and 200 μg/ml) alone at 37 °C for 60 min and cisplatin (25, 50, 100, and 200 μg/ml) combined with hyperthermia at 42 °C for 60 min. After pretreatment, the cell viability, colony formation and DNA metabolism in HepG2 and the Na(+)-K(+)-ATPase activity, 2,3-diphosphoglycerate (2,3-DPG) concentration, free hemoglobin (Hb) level, osmotic fragility, membrane phosphatidylserine externalization, and blood gas variables in erythrocytes were determined. Pretreatment with cisplatin (50, 100, and 200 μg/ml) combined with hyperthermia (42 °C) for 60 min significantly decreased HepG2 cell viability, and completely inhibited colony formation and DNA metabolism when the HepG2 cell concentration was 5×10(4) ml(-1) in the erythrocyte (P2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 μg/ml) (P0.05). In conclusion, pretreatment with cisplatin (50 μg/ml) combined with hyperthermia (42 °C) for 60 min effectively eliminated HepG2 cells from IBS but did not significantly affect erythrocytes in vitro.

  8. DMSO inhibits human platelet activation through cyclooxygenase-1 inhibition. A novel agent for drug eluting stents?

    International Nuclear Information System (INIS)

    Asmis, Lars; Tanner, Felix C.; Sudano, Isabella; Luescher, Thomas F.; Camici, Giovanni G.

    2010-01-01

    Background: DMSO is routinely infused together with hematopoietic cells in patients undergoing myeloablative therapy and was recently found to inhibit smooth muscle cells proliferation and arterial thrombus formation in the mouse by preventing tissue factor (TF), a key activator of the coagulation cascade. This study was designed to investigate whether DMSO prevents platelet activation and thus, whether it may represent an interesting agent to be used on drug eluting stents. Methods and results: Human venous blood from healthy volunteers was collected in citrated tubes and platelet activation was studied by cone and platelet analyzer (CPA) and rapid-platelet-function-assay (RPFA). CPA analysis showed that DMSO-treated platelets exhibit a lower adherence in response to shear stress (-15.54 ± 0.9427%, n = 5, P < 0.0001 versus control). Additionally, aggregometry studies revealed that DMSO-treated, arachidonate-stimulated platelets had an increased lag phase (18.0% ± 4.031, n = 9, P = 0.0004 versus control) as well as a decreased maximal aggregation (-6.388 ± 2.212%, n = 6, P = 0.0162 versus control). Inhibitory action of DMSO could be rescued by exogenous thromboxane A2 and was mediated, at least in part, by COX-1 inhibition. Conclusions: Clinically relevant concentrations of DMSO impair platelet activation by a thromboxane A2-dependent, COX-1-mediated effect. This finding may be crucial for the previously reported anti-thrombotic property displayed by DMSO. Our findings support a role for DMSO as a novel drug to prevent not only proliferation, but also thrombotic complications of drug eluting stents.

  9. Isocyanides inhibit human heme oxygenases at the verdoheme stage.

    Science.gov (United States)

    Evans, John P; Kandel, Sylvie; Ortiz de Montellano, Paul R

    2009-09-22

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides, isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 microM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design.

  10. Isocyanides Inhibit Human Heme Oxygenases at the Verdoheme Stage†

    Science.gov (United States)

    Evans, John P.; Kandel, Sylvie; Ortiz de Montellano, Paul R.

    2010-01-01

    Heme oxygenases (HO) catalyze the oxidative cleavage of heme to generate biliverdin, CO, and free iron. In humans, heme oxygenase-1 (hHO-1) is overexpressed in tumor tissues, where it helps to protect cancer cells from anticancer agents, while HOs in fungal pathogens, such as Candida albicans, function as the primary means of iron acquisition. Thus, HO can be considered a potential therapeutic target for certain diseases. In this study, we have examined the equilibrium binding of three isocyanides; isopropyl, n-butyl, and benzyl, to the two major human HO isoforms (hHO-1 and hHO-2), Candida albicans HO (CaHmx1), and human cytochrome P450 CYP3A4 using electronic absorption spectroscopy. Isocyanides coordinate to both ferric and ferrous HO-bound heme, with tighter binding by the more hydrophobic isocyanides, and 200-300-fold tighter binding to the ferrous form. Benzyl isocyanide was the strongest ligand to ferrous heme in all the enzymes. Because the dissociation constants (KD) of the ligands for ferrous heme-hHO-1 were below the limit of accuracy for equilibrium titrations, stopped-flow kinetic experiments were used to measure the binding parameters of the isocyanides to ferrous hHO-1. Steady-state activity assays showed that benzyl isocyanide was the most potent uncompetitive inhibitor with respect to heme with a KI = 0.15 μM for hHO-1. Importantly, single turnover assays revealed that the reaction was completely stopped by coordination of the isocyanide to the verdoheme intermediate rather than to the ferric heme complex. Much tighter binding of the inhibitor to the verdoheme intermediate differentiates it from inhibition of, for example, CYP3A4 and offers a possible route to more selective inhibitor design. PMID:19694439

  11. Increase in the amount of erythrocyte delta-aminolevulinic acid dehydratase in workers with moderate lead exposure

    International Nuclear Information System (INIS)

    Fujita, H.; Sano, S.

    1982-01-01

    The amount of ALA-D in human erythrocytes was determined directly by radioimmunoassay or calculated from the restored activity assayed in the presence of zinc and dithiothreitol, and a good correlation was observed between the RIA-based and the restored activity-based amounts. The RIA-based amount of ALA-D in the blood of 10 normal individuals (blood lead levels of 5.6 +- 2.3 μg/100 ml: mean +- SD) and 19 lead-exposed workers (blood lead levels of 41.2 +- 10.2 μg/100 ml) was 54.1 +- 11.8 μg/ml blood and 92.3 +- 20.6 μg/ml blood, respectively, indicating an apparent increase of the enzyme amount in lead-exposed workers. A significant increase in the amount of erythrocyte ALA-D calculated from the restored activity in lead-exposed workers was observed even in the low blood lead level of 10-20 μg/100 ml, resulting in the range of blood lead level 20-40 μg/100 ml. No significant difference was observed in hematocrit and hemoglobin content between lead-exposed and non-exposed groups. These observations suggested that the increase of erythrocyte ALA-D in lead exposure was not due to anemia, which might result in the increase of young erythrocytes in peripheral blood. This increase in the amount of ALA-D in human erythrocytes might be a result of the function to overcome the inhibition of the enzyme in bone marrow cells during lead exposure, and these findings may throw light on the danger to human health of low-level lead toxicity. (orig.)

  12. Plasmodium falciparum-infected erythrocytes do not adhere well to C32 melanoma cells or CD36 unless rosettes with uninfected erythrocytes are first disrupted.

    OpenAIRE

    Handunnetti, S M; Hasler, T H; Howard, R J

    1992-01-01

    Plasmodium falciparum malaria parasites modify the human erythrocytes in which they grow so that some parasitized erythrocytes (PE) can cytoadhere (C+) to host vascular endothelial cells or adhere in rosettes (R+) to uninfected erythrocytes. These C+ and R+ adherence properties of PE appear to mediate much of the pathogenesis of severe malaria infections, in part by blocking blood flow in microvessels. From one parasite strain, PE were selected in vitro for C+ R+ or C+ R- adherence properties...

  13. Retinoid inhibition of in vitro invasion of human amnion basement membrane by human tumor cells

    International Nuclear Information System (INIS)

    Fazely, F.

    1988-01-01

    The effects measured were the inhibition of tumor cell migration through the basement membrane (BM) and tumor cell degradative enzyme activity on 3 H-proline labeled collagenous and non collagenous components of the BM. The human lung carcinoma A549 or the human Ewing's sarcoma TC-106 cell lines treated with retinoids for two days were incubated on the BM in the absence of retinoids. A dose-dependent inhibition of cell invasion was produced by retinoids. Among the retinoids tested the most powerful was retinol acetate which inhibited invasion by 50% of A549 cells at a concentration of 0.09 μg/ml, and TC-106 cells at 0.08 μg/ml. Retinol acetate inhibited A549 and TC-106 cell growth by approximately 50% at levels almost 100-fold higher than those needed for antiinvasive activity. Retinol acetate was about 20 times more potent than retinoic acid and 30 times more than retinol palmitate. Furthermore, A549 cells treated with retinol acetate, under conditions whereby an anti-invasive state was induced,showed an increase in the number of cellular retinoic acid binding proteins (CRABP), a decrease in the activity of type IV collagenase and ectosialyltransferase, and no change in the activity of transglutaminase

  14. Harman inhibits the removal of pyrimidine dimers from the DNA of human cells

    International Nuclear Information System (INIS)

    Castellani, A.; Setlow, R.B.

    1981-01-01

    Normal human fibroblasts were UV-irradiated and incubated for 6 hr with harman. The losses of sites, in the extracted DNA, sensitive to a UV specific endonuclease were determined as precision measures of the excision of UV-induced pyrimidine dimers. Harman inhibited excision, rising from approx. 30% inhibition at 200 μM to 75% inhibition at 500 μM

  15. Polybrene inhibits human mesenchymal stem cell proliferation during lentiviral transduction.

    Directory of Open Access Journals (Sweden)

    Paul Lin

    Full Text Available Human mesenchymal stem cells (hMSCs can be engineered to express specific genes, either for their use in cell-based therapies or to track them in vivo over long periods of time. To obtain long-term expression of these genes, a lentivirus- or retrovirus-mediated cell transduction is often used. However, given that the efficiency with these viruses is typically low in primary cells, additives such as polybrene are always used for efficient viral transduction. Unfortunately, as presented here, exposure to polybrene alone at commonly used concentratons (1-8 µg/mL negatively impacts hMSC proliferation in a dose-dependent manner as measured by CyQUANT, EdU incorporation, and cell cycle analysis. This inhibition of proliferation was observable in culture even 3 weeks after exposure. Culturing the cells in the presence of FGF-2, a potent mitogen, did not abrogate this negative effect of polybrene. In fact, the normally sharp increase in hMSC proliferation that occurs during the first days of exposure to FGF-2 was absent at 4 µg/mL or higher concentrations of polybrene. Similarly, the effect of stimulating cell proliferation under simulated hypoxic conditions was also decreased when cells were exposed to polybrene, though overall proliferation rates were higher. The negative influence of polybrene was, however, reduced when the cells were exposed to polybrene for a shorter period of time (6 hr vs 24 hr. Thus, careful evaluation should be done when using polybrene to aid in lentiviral transduction of human MSCs or other primary cells, especially when cell number is critical.

  16. Inhibition of EGF processing in responsive and nonresponsive human fibroblasts

    International Nuclear Information System (INIS)

    Schaudies, R.P.; Wray, H.L.

    1988-01-01

    We have examined the proteolytic processing of radiolabeled epidermal growth factor (EGF) in EGF growth-responsive human foreskin fibroblasts (HFF) versus EGF nonresponsive human fetal lung fibroblasts (HFL). Previous studies have shown that both cell lines demonstrate similar binding affinities and numbers of binding sites, as well as similar rates of internalization and degradation of the bound, radiolabeled hormone. We have used nondenaturing electrophoresis to compare how these two cell lines process EGF at its carboxy terminus. EGF lacking either one [des-(53)-EGF] or six [des (48-53)-EGF] carboxy terminal amino acids could be distinguished by this method. Chloroquine or leupeptin were added to the incubation system in an attempt to accentuate potential differences in hormonal processing between the responsive and nonresponsive cell lines. In the absence of inhibitors, the responsive and nonresponsive cells generated similar distributions of processed forms of EGF after 30-minutes incubation. However, after 4-hours incubation in the constant presence of 125I-EGF, the electrophoretic profiles of extracted hormone were substantially different. The radiolabel within the responsive cells, as well as that released from them, migrated predominantly at the dye front, indicating complete degradation of EGF. In contrast, the majority of the radiolabel within the nonresponsive cells migrated as partially processed forms of hormone, while the released radiolabel migrated at the dye front. Addition of chloroquine to either cell line inhibited processing of EGF beyond removal of the carboxyl terminal arginine residue. Both intact 125I-EGF, and 125I-EGF lacking the carboxyl terminal arginine were released from chloroquine-treated cells in a ratio equal to that present in the intact cells

  17. Temperature-dependent binding of cyclosporine to an erythrocyte protein

    International Nuclear Information System (INIS)

    Agarwal, R.P.; Threatte, G.A.; McPherson, R.A.

    1987-01-01

    In this competitive binding assay to measure endogenous binding capacity for cyclosporine (CsA) in erythrocyte lysates, a fixed amount of [ 3 H]CsA plus various concentrations of unlabeled CsA is incubated with aliquots of a test hemolysate. Free CsA is then adsorbed onto charcoal and removed by centrifugation; CsA complexed with a cyclosporine-binding protein (CsBP) remains in the supernate. We confirmed the validity of this charcoal-separation mode of binding analysis by comparison with equilibrium dialysis. Scatchard plot analysis of the results at 4 degrees C yielded a straight line with slope corresponding to a binding constant of 1.9 X 10(7) L/mol and a saturation capacity of approximately 4 mumol per liter of packed erythrocytes. Similar analysis of binding data at 24 degrees C and 37 degrees C showed that the binding constant decreased with increasing temperature, but the saturation capacity did not change. CsBP was not membrane bound but appeared to be freely distributed within erythrocytes. 125 I-labeled CsA did not complex with the erythrocyte CsBP. Several antibiotics and other drugs did not inhibit binding between CsA and CsBP. These findings may explain the temperature-dependent uptake of CsA by erythrocytes in whole blood and suggest that measurement of CsBP in erythrocytes or lymphocytes may help predict therapeutic response or toxicity after administration of CsA

  18. Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

    International Nuclear Information System (INIS)

    Wu Xiaofeng; Fan Jia; Wang Xiaoying; Zhou Jian; Qiu Shuangjian; Yu Yao; Liu Yinkun; Tang Zhaoyou

    2007-01-01

    CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment

  19. Flow cytometric determination of osmotic behaviour of animal erythrocytes toward their engineering for drug delivery

    Directory of Open Access Journals (Sweden)

    Kostić Ivana T.

    2015-01-01

    Full Text Available Despite the fact that the methods based on the osmotic properties of the cells are the most widely used for loading of drugs in human and animal erythrocytes, data related to the osmotic properties of erythrocytes derived from animal blood are scarce. This work was performed with an aim to investigate the possibility of use the flow cytometry as a tool for determination the osmotic behaviour of porcine and bovine erythrocytes, and thus facilitate the engineering of erythrocytes from animal blood to be drug carriers. The method of flow cytometry successfully provided the information about bovine and porcine erythrocyte osmotic fragility, and made the initial steps in assessment of erythrocyte shape in a large number of erythrocytes. Although this method is not able to confirm the swelling of pig erythrocytes, it indicated to the differences in pig erythrocytes that had basic hematological parameters inside and outside the reference values. In order to apply/use the porcine and bovine erythrocytes as drug carriers, the method of flow cytometry, confirming the presence of osmotically different fractions of red blood cells, indicated that various amounts of the encapsulated drug in porcine and bovine erythrocytes can be expected.

  20. Dielectric inspection of erythrocyte morphology

    International Nuclear Information System (INIS)

    Hayashi, Yoshihito; Oshige, Ikuya; Katsumoto, Yoichi; Omori, Shinji; Yasuda, Akio; Asami, Koji

    2008-01-01

    We performed a systematic study of the sensitivity of dielectric spectroscopy to erythrocyte morphology. Namely, rabbit erythrocytes of four different shapes were prepared by precisely controlling the pH of the suspending medium, and their complex permittivities over the frequency range from 0.1 to 110 MHz were measured and analyzed. Their quantitative analysis shows that the characteristic frequency and the broadening parameter of the dielectric relaxation of interfacial polarization are highly specific to the erythrocyte shape, while they are insensitive to the cell volume fraction. Therefore, these two dielectric parameters can be used to differentiate erythrocytes of different shapes, if dielectric spectroscopy is applied to flow-cytometric inspection of single blood cells. In addition, we revealed the applicability and limitations of the analytical theory of interfacial polarization to explain the experimental permittivities of non-spherical erythrocytes

  1. Dielectric inspection of erythrocyte morphology

    Energy Technology Data Exchange (ETDEWEB)

    Hayashi, Yoshihito; Oshige, Ikuya; Katsumoto, Yoichi; Omori, Shinji; Yasuda, Akio [Life Science Laboratory, Materials Laboratories, Sony Corporation, Sony Bioinformatics Center, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510 (Japan); Asami, Koji [Laboratory of Molecular Aggregation Analysis, Division of Multidisciplinary Chemistry, Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011 (Japan)], E-mail: Yoshihito.Hayashi@jp.sony.com

    2008-05-21

    We performed a systematic study of the sensitivity of dielectric spectroscopy to erythrocyte morphology. Namely, rabbit erythrocytes of four different shapes were prepared by precisely controlling the pH of the suspending medium, and their complex permittivities over the frequency range from 0.1 to 110 MHz were measured and analyzed. Their quantitative analysis shows that the characteristic frequency and the broadening parameter of the dielectric relaxation of interfacial polarization are highly specific to the erythrocyte shape, while they are insensitive to the cell volume fraction. Therefore, these two dielectric parameters can be used to differentiate erythrocytes of different shapes, if dielectric spectroscopy is applied to flow-cytometric inspection of single blood cells. In addition, we revealed the applicability and limitations of the analytical theory of interfacial polarization to explain the experimental permittivities of non-spherical erythrocytes.

  2. Telomerase inhibition effectively targets mouse and human AML stem cells and delays relapse following chemotherapy

    DEFF Research Database (Denmark)

    Bruedigam, Claudia; Bagger, Frederik Otzen; Heidel, Florian H.

    2014-01-01

    (-/-) LSCs express a specific gene expression signature that can be identified in human AML patient cohorts and is positively correlated with patient survival following chemotherapy. In xenografts of primary human AML, genetic or pharmacological inhibition of telomerase targets LSCs, impairs leukemia...... progression, and delays relapse following chemotherapy. Altogether, these results establish telomerase inhibition as an effective strategy for eliminating AML LSCs....

  3. Erythrocyte signal transduction pathways, their oxygenation dependence and functional significance.

    Science.gov (United States)

    Barvitenko, Nadezhda N; Adragna, Norma C; Weber, Roy E

    2005-01-01

    Erythrocytes play a key role in human and vertebrate metabolism. Tissue O2 supply is regulated by both hemoglobin (Hb)-O2 affinity and erythrocyte rheology, a key determinant of tissue perfusion. Oxygenation-deoxygenation transitions of Hb may lead to re-organization of the cytoskeleton and signalling pathways activation/deactivation in an O2-dependent manner. Deoxygenated Hb binds to the cytoplasmic domain of the anion exchanger band 3, which is anchored to the cytoskeleton, and is considered a major mechanism underlying the oxygenation-dependence of several erythrocyte functions. This work discusses the multiple modes of Hb-cytoskeleton interactions. In addition, it reviews the effects of Mg2+, 2,3-diphosphoglycerate, NO, shear stress and Ca2+, all factors accompanying the oxygenation-deoxygenation cycle in circulating red cells. Due to the extensive literature on the subject, the data discussed here, pertain mainly to human erythrocytes whose O2 affinity is modulated by 2,3-diphosphoglycerate, ectothermic vertebrate erythrocytes that use ATP, and to bird erythrocytes that use inositol pentaphosphate. Copyright 2005 S. Karger AG, Basel.

  4. Inhibiting DNA-PKCS radiosensitizes human osteosarcoma cells

    International Nuclear Information System (INIS)

    Mamo, Tewodros; Mladek, Ann C.; Shogren, Kris L.; Gustafson, Carl; Gupta, Shiv K.; Riester, Scott M.; Maran, Avudaiappan; Galindo, Mario; Wijnen, Andre J. van; Sarkaria, Jann N.; Yaszemski, Michael J.

    2017-01-01

    Osteosarcoma survival rate has not improved over the past three decades, and the debilitating side effects of the surgical treatment suggest the need for alternative local control approaches. Radiotherapy is largely ineffective in osteosarcoma, indicating a potential role for radiosensitizers. Blocking DNA repair, particularly by inhibiting the catalytic subunit of DNA-dependent protein kinase (DNA-PK CS ), is an attractive option for the radiosensitization of osteosarcoma. In this study, the expression of DNA-PK CS in osteosarcoma tissue specimens and cell lines was examined. Moreover, the small molecule DNA-PK CS inhibitor, KU60648, was investigated as a radiosensitizing strategy for osteosarcoma cells in vitro. DNA-PK CS was consistently expressed in the osteosarcoma tissue specimens and cell lines studied. Additionally, KU60648 effectively sensitized two of those osteosarcoma cell lines (143B cells by 1.5-fold and U2OS cells by 2.5-fold). KU60648 co-treatment also altered cell cycle distribution and enhanced DNA damage. Cell accumulation at the G2/M transition point increased by 55% and 45%, while the percentage of cells with >20 γH2AX foci were enhanced by 59% and 107% for 143B and U2OS cells, respectively. These results indicate that the DNA-PK CS inhibitor, KU60648, is a promising radiosensitizing agent for osteosarcoma. - Highlights: • DNA-PKcs is consistently expressed in human osteosarcoma tissue and cell lines. • The DNA-PKcs inhibitor, KU60648, effectively radiosensitizes osteosarcoma cells. • Combining KU60648 with radiation increases G2/M accumulation and DNA damage.

  5. Stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes by human monocytes Estágios da fagocitose in vitro por monócitos humanos de eritrócitos infectados por Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Maria Imaculada Muniz-Junqueira

    2009-04-01

    Full Text Available Monocytes/macrophages play a critical role in the defense mechanisms against malaria parasites, and are the main cells responsible for the elimination of malaria parasites from the blood circulation. We carried out a microscope-aided evaluation of the stages of in vitro phagocytosis of Plasmodium falciparum-infected erythrocytes, by human monocytes. These cells were obtained from healthy adult individuals by means of centrifugation through a cushion of Percoll density medium and were incubated with erythrocytes infected with Plasmodium falciparum that had previously been incubated with a pool of anti-plasmodial immune serum. We described the stages of phagocytosis, starting from adherence of infected erythrocytes to the phagocyte membrane and ending with their destruction within the phagolisosomes of the monocytes. We observed that the different erythrocytic forms of the parasite were ingested by monocytes, and that the process of phagocytosis may be completed in around 30 minutes. Furthermore, we showed that phagocytosis may occur continuously, such that different phases of the process were observed in the same phagocyte.Monócitos/macrófagos desempenham uma função crítica nos mecanismos de defesa antiplasmódio e constituem as principais células responsáveis pela eliminação das formas eritrocitárias do plasmódio da circulação sangüínea. Realizamos uma avaliação microscópica dos estágios da fagocitose in vitro de eritrócitos infectados por Plasmodium falciparum por monócitos humanos. Essas células foram obtidas de indivíduos adultos sadios por centrifugação em Percoll e incubadas com eritrócitos infectados por Plasmodium falciparum previamente incubados com um pool de soro imune contra plasmódio. Descrevemos os estágios da fagocitose, desde a aderência dos eritrócitos infectados até sua destruição nos fagolisossomas dos monócitos. Observou-se que eritrócitos infectados por todos os diferentes est

  6. Cytoplasmic free Ca2+ is essential for multiple steps in malaria parasite egress from infected erythrocytes

    Directory of Open Access Journals (Sweden)

    Glushakova Svetlana

    2013-01-01

    Full Text Available Abstract Background Egress of Plasmodium falciparum, from erythrocytes at the end of its asexual cycle and subsequent parasite invasion into new host cells, is responsible for parasite dissemination in the human body. The egress pathway is emerging as a coordinated multistep programme that extends in time for tens of minutes, ending with rapid parasite extrusion from erythrocytes. While the Ca2+ regulation of the invasion of P. falciparum in erythrocytes is well established, the role of Ca2+ in parasite egress is poorly understood. This study analysed the involvement of cytoplasmic free Ca2+ in infected erythrocytes during the multistep egress programme of malaria parasites. Methods Live-cell fluorescence microscopy was used to image parasite egress from infected erythrocytes, assessing the effect of drugs modulating Ca2+ homeostasis on the egress programme. Results A steady increase in cytoplasmic free Ca2+ is found to precede parasite egress. This increase is independent of extracellular Ca2+ for at least the last two hours of the cycle, but is dependent upon Ca2+ release from internal stores. Intracellular BAPTA chelation of Ca2+ within the last 45 minutes of the cycle inhibits egress prior to parasitophorous vacuole swelling and erythrocyte membrane poration, two characteristic morphological transformations preceding parasite egress. Inhibitors of the parasite endoplasmic reticulum (ER Ca2+-ATPase accelerate parasite egress, indicating that Ca2+ stores within the ER are sufficient in supporting egress. Markedly accelerated egress of apparently viable parasites was achieved in mature schizonts using Ca2+ ionophore A23187. Ionophore treatment overcomes the BAPTA-induced block of parasite egress, confirming that free Ca2+ is essential in egress initiation. Ionophore treatment of immature schizonts had an adverse effect inducing parasitophorous vacuole swelling and killing the parasites within the host cell. Conclusions The parasite egress

  7. Inhibition of human lung adenocarcinoma growth using survivint34a ...

    Indian Academy of Sciences (India)

    Prakash

    knockdown, survivin-directed vaccines (Pisarev et al. 2003), ... Endotoxin levels of the plasmid DNA prepared were determined by .... power fields/slide). ... inhibition of angiogenesis and directly increase apoptosis of .... α-induced apoptosis.

  8. Stimulation of Suicidal Erythrocyte Death by the Antimalarial Drug Mefloquine

    Directory of Open Access Journals (Sweden)

    Rosi Bissinger

    2015-07-01

    Full Text Available Background: The antimalarial drug mefloquine has previously been shown to stimulate apoptosis of nucleated cells. Similar to apoptosis, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane with phosphatidylserine translocation to the erythrocyte surface. Stimulators of eryptosis include oxidative stress, increase of cytosolic Ca2+-activity ([Ca2+]i, and ceramide. Methods: 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, [Ca2+]i from Fluo3-fluorescence, and ceramide abundance from specific antibody binding. Results: A 48 h treatment of human erythrocytes with mefloquine significantly increased the percentage of annexin-V-binding cells (≥5 µg/ml, significantly decreased forward scatter (≥5 µg/ml, significantly increased ROS abundance (5 µg/ml, significantly increased [Ca2+]i (7.5 µg/ml and significantly increased ceramide abundance (10 µg/ml. The up-regulation of annexin-V-binding following mefloquine treatment was significantly blunted but not abolished by removal of extracellular Ca2+. Even in the absence of extracellular Ca2+, mefloquine significantly increased annexin-V-binding. Conclusions: Mefloquine treatment leads to erythrocyte shrinkage and erythrocyte membrane scrambling, effects at least partially due to induction of oxidative stress, increase of [Ca2+]i and up-regulation of ceramide abundance.

  9. Triggers, Inhibitors, Mechanisms, and Significance of Eryptosis: The Suicidal Erythrocyte Death

    Directory of Open Access Journals (Sweden)

    Elisabeth Lang

    2015-01-01

    Full Text Available Suicidal erythrocyte death or eryptosis is characterized by erythrocyte shrinkage, cell membrane blebbing, and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include Ca2+ entry, ceramide formation, stimulation of caspases, calpain activation, energy depletion, oxidative stress, and dysregulation of several kinases. Eryptosis is triggered by a wide variety of xenobiotics. It is inhibited by several xenobiotics and endogenous molecules including NO and erythropoietin. The susceptibility of erythrocytes to eryptosis increases with erythrocyte age. Phosphatidylserine exposing erythrocytes adhere to the vascular wall by binding to endothelial CXC-Motiv-Chemokin-16/Scavenger-receptor for phosphatidylserine and oxidized low density lipoprotein (CXCL16. Phosphatidylserine exposing erythrocytes are further engulfed by phagocytosing cells and are thus rapidly cleared from circulating blood. Eryptosis eliminates infected or defective erythrocytes thus counteracting parasitemia in malaria and preventing detrimental hemolysis of defective cells. Excessive eryptosis, however, may lead to anemia and may interfere with microcirculation. Enhanced eryptosis contributes to the pathophysiology of several clinical disorders including metabolic syndrome and diabetes, malignancy, cardiac and renal insufficiency, hemolytic uremic syndrome, sepsis, mycoplasma infection, malaria, iron deficiency, sickle cell anemia, thalassemia, glucose 6-phosphate dehydrogenase deficiency, and Wilson’s disease. Facilitating or inhibiting eryptosis may be a therapeutic option in those disorders.

  10. 3-Bromopyruvate inhibits human gastric cancer tumor growth in nude mice via the inhibition of glycolysis.

    Science.gov (United States)

    Xian, Shu-Lin; Cao, Wei; Zhang, Xiao-Dong; Lu, Yun-Fei

    2015-02-01

    Tumor cells primarily depend upon glycolysis in order to gain energy. Therefore, the inhibition of glycolysis may inhibit tumor growth. Our previous study demonstrated that 3-bromopyruvate (3-BrPA) inhibited gastric cancer cell proliferation in vitro . However, the ability of 3-BrPA to suppress tumor growth in vivo, and its underlying mechanism, have yet to be elucidated. The aim of the present study was to investigate the inhibitory effect of 3-BrPA in an animal model of gastric cancer. It was identified that 3-BrPA exhibited strong inhibitory effects upon xenograft tumor growth in nude mice. In addition, the antitumor function of 3-BrPA exhibited a dose-effect association, which was similar to that of the chemotherapeutic agent, 5-fluorouracil. Furthermore, 3-BrPA exhibited low toxicity in the blood, liver and kidneys of the nude mice. The present study hypothesized that the inhibitory effect of 3-BrPA is achieved through the inhibition of hexokinase activity, which leads to the downregulation of B-cell lymphoma 2 (Bcl-2) expression, the upregulation of Bcl-2-associated X protein expression and the subsequent activation of caspase-3. These data suggest that 3-BrPA may be a novel therapy for the treatment of gastric cancer.

  11. Triggering of Suicidal Erythrocyte Death by Regorafenib

    Directory of Open Access Journals (Sweden)

    Jens Zierle

    2016-01-01

    Full Text Available Background/Aims: The multikinase inhibitor regorafenib is utilized for the treatment of malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Side effects of regorafenib include anemia. At least in theory, regorafenib induced anemia could result from stimulated suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i, oxidative stress and ceramide. The present study explored, whether regorafenib induces eryptosis and, if so, whether it is effective up- and/or downstream of Ca2+. Methods: To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: A 48 hours exposure of human erythrocytes to regorafenib (≥ 0.5 µg/ml significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 1.25 µg/ml, but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of regorafenib on annexin-V-binding and forward scatter was not significantly blunted by removal of extracellular Ca2+. Regorafenib (5 µg/ml significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin. Conclusions: Regorafenib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+.

  12. Triggering of Suicidal Erythrocyte Death by Regorafenib.

    Science.gov (United States)

    Zierle, Jens; Bissinger, Rosi; Bouguerra, Ghada; Abbès, Salem; Lang, Florian

    2016-01-01

    The multikinase inhibitor regorafenib is utilized for the treatment of malignancy. The substance is effective in part by triggering suicidal death or apoptosis of tumor cells. Side effects of regorafenib include anemia. At least in theory, regorafenib induced anemia could result from stimulated suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress and ceramide. The present study explored, whether regorafenib induces eryptosis and, if so, whether it is effective up- and/or downstream of Ca2+. To this end, phosphatidylserine exposure at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. A 48 hours exposure of human erythrocytes to regorafenib (≥ 0.5 µg/ml) significantly increased the percentage of annexin-V-binding cells, significantly decreased forward scatter (≥ 1.25 µg/ml), but did not significantly increase Fluo3-fluorescence, DCFDA fluorescence or ceramide abundance. The effect of regorafenib on annexin-V-binding and forward scatter was not significantly blunted by removal of extracellular Ca2+. Regorafenib (5 µg/ml) significantly augmented the increase of annexin-V-binding, but significantly blunted the decrease of forward scatter following treatment with the Ca2+ ionophore ionomycin. Regorafenib triggers cell shrinkage and phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part downstream of Ca2+. © 2016 The Author(s) Published by S. Karger AG, Basel.

  13. INHIBITION OF SPONTANEOUS APOPTOSIS IN HUMAN BREAST CANCER

    Institute of Scientific and Technical Information of China (English)

    邵志敏; 江明; 吴炅; 余黎民; 韩企夏; 张延璆; 沈镇宙

    1996-01-01

    Breast tumorigenesis proceeds through an accumulation of specific genetic alteration. Breast malignant transformation is dependent on not only the rate of cell production but also on apoptcsis,a genetically prograined process of autonomous ceil death. We investigated whether breast tumorigenesis involved an altered susceptibility to apoptosis and proliferation by examining normal breast epithelium and breast cancer sampies. We found there is a great inhibition of spontaneous apoptosis in breast cancer ceils compared with normal breast epithelium. The inhibition of apoptosis in breast cancer may contribute to neoplastic transformation.

  14. The inhibition of the Human Immunodeficiency Virus type 1 activity by crude and purified human pregnancy plug mucus and mucins in an inhibition assay

    Directory of Open Access Journals (Sweden)

    Schoeman Leann

    2008-05-01

    Full Text Available Abstract Background The female reproductive tract is amongst the main routes for Human Immunodeficiency Virus (HIV transmission. Cervical mucus however is known to protect the female reproductive tract from bacterial invasion and fluid loss and regulates and facilitates sperm transport to the upper reproductive tract. The purpose of this study was to purify and characterize pregnancy plug mucins and determine their anti-HIV-1 activity in an HIV inhibition assay. Methods Pregnancy plug mucins were purified by caesium chloride density-gradient ultra-centrifugation and characterized by Western blotting analysis. The anti-HIV-1 activities of the crude pregnancy plug mucus and purified pregnancy plug mucins was determined by incubating them with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells. Results The pregnancy plug mucus had MUC1, MUC2, MUC5AC and MUC5B. The HIV inhibition assay revealed that while the purified pregnancy plug mucins inhibit HIV-1 activity by approximately 97.5%, the crude pregnancy plug mucus failed to inhibit HIV-1 activity. Conclusion Although it is not clear why the crude sample did not inhibit HIV-1 activity, it may be that the amount of mucins in the crude pregnancy plug mucus (which contains water, mucins, lipids, nucleic acids, lactoferrin, lysozyme, immunoglobulins and ions, is insufficient to cause viral inhibition or aggregation.

  15. Acute effects of cocaine and cannabis on response inhibition in humans: an ERP investigation

    NARCIS (Netherlands)

    Spronk, D.B.; De Bruijn, E.R.; van Wel, J.H.; Ramaekers, J.G.; Verkes, R.J.

    2016-01-01

    Substance abuse has often been associated with alterations in response inhibition in humans. Not much research has examined how the acute effects of drugs modify the neurophysiological correlates of response inhibition, or how these effects interact with individual variation in trait levels of

  16. Inhibition of rat, mouse, and human glutathione S-transferase by eugenol and its oxidation products

    NARCIS (Netherlands)

    Rompelberg, C.J.M.; Ploemen, J.H.T.M.; Jespersen, S.; Greef, J. van der; Verhagen, H.; Bladeren, P.J. van

    1996-01-01

    The irreversible and reversible inhibition of glutathione S-transferases (GSTs) by eugenol was studied in rat, mouse and man. Using liver cytosol of human, rat and mouse, species differences were found in the rate of irreversible inhibition of GSTs by eugenol in the presence of the enzyme

  17. Hemispheric asymmetry and somatotopy of afferent inhibition in healthy humans.

    NARCIS (Netherlands)

    Helmich, R.C.G.; Baumer, T.; Siebner, H.R.; Bloem, B.R.; Munchau, A.

    2005-01-01

    A conditioning electrical stimulus to a digital nerve can inhibit the motor-evoked potentials (MEPs) in adjacent hand muscles elicited by transcranial magnetic stimulation (TMS) to the contralateral primary motor cortex (M1) when given 25-50 ms before the TMS pulse. This is referred to as

  18. Local anesthetic-induced inhibition of human neutrophil priming: the influence of structure, lipophilicity, and charge

    NARCIS (Netherlands)

    Picardi, Susanne; Cartellieri, Sibylle; Groves, Danja; Hahnenekamp, Klaus; Gerner, Peter; Durieux, Marcel E.; Stevens, Markus F.; Lirk, Philipp; Hollmann, Markus W.

    2013-01-01

    Local anesthetics (LAs) are widely known for inhibition of voltage-gated sodium channels underlying their antiarrhythmic and antinociceptive effects. However, LAs have significant immunomodulatory properties and were shown to affect human neutrophil functions independent of sodium-channel blockade.

  19. Inhibition of human pancreatic and biliary output but not intestinal motility by physiological intraileal lipid loads

    DEFF Research Database (Denmark)

    Keller, Jutta; Holst, Jens Juul; Layer, Peter

    2005-01-01

    Lipid perfusion into the distal ileal lumen at supraphysiological loads inhibits pancreatic exocrine secretion and gastrointestinal motility in humans. In the present study, we sought to determine the effects of physiological postprandial intraileal lipid concentrations on endogenously stimulated...

  20. Design and evaluation of antimalarial peptides derived from prediction of short linear motifs in proteins related to erythrocyte invasion.

    Directory of Open Access Journals (Sweden)

    Alessandra Bianchin

    Full Text Available The purpose of this study was to investigate the blood stage of the malaria causing parasite, Plasmodium falciparum, to predict potential protein interactions between the parasite merozoite and the host erythrocyte and design peptides that could interrupt these predicted interactions. We screened the P. falciparum and human proteomes for computationally predicted short linear motifs (SLiMs in cytoplasmic portions of transmembrane proteins that could play roles in the invasion of the erythrocyte by the merozoite, an essential step in malarial pathogenesis. We tested thirteen peptides predicted to contain SLiMs, twelve of them palmitoylated to enhance membrane targeting, and found three that blocked parasite growth in culture by inhibiting the initiation of new infections in erythrocytes. Scrambled peptides for two of the most promising peptides suggested that their activity may be reflective of amino acid properties, in particular, positive charge. However, one peptide showed effects which were stronger than those of scrambled peptides. This was derived from human red blood cell glycophorin-B. We concluded that proteome-wide computational screening of the intracellular regions of both host and pathogen adhesion proteins provides potential lead peptides for the development of anti-malarial compounds.

  1. The inhibition of repair in UV irradiated human cells

    International Nuclear Information System (INIS)

    Collins, A.R.S.; Schor, S.L.; Johnson, R.T.

    1977-01-01

    Three different assay procedures are used to determine the effects of hydroxyurea on excision repair in UV-irradiated HeLa cells. At the cytological level, incubation of UV-irradiated metaphase cells with hydroxyurea caused chromosome decondensation. Using a modified alkaline sucrose gradient sedimentation technique involving minimal lysis before centrifugation, a marked retardation was found in the sedimentation of DNA from UV-irradiated cells incubated for a short period with hydroxyurea. The effect of hydroxyurea on the incorporation of [ 3 H]thymidine by UV-irradiated G1 cells was found to depend on the concentration of thymidine present in the medium. The results point to an inhibition of repair DNA synthesis by hydroxyurea (or deoxyadenosine), at the level of the supply of DNA precursors, i.e. in the same way that these agents inhibit semiconservative DNA synthesis. In the presence of these inhibitors, single-strand gaps accumulate in the DNA

  2. Ebselen Reversibly Inhibits Human Glutamate Dehydrogenase at the Catalytic Site.

    Science.gov (United States)

    Jin, Yanhong; Li, Di; Lu, Shiying; Zhao, Han; Chen, Zhao; Hou, Wei; Ruan, Benfang Helen

    Human glutamate dehydrogenase (GDH) plays an important role in neurological diseases, tumor metabolism, and hyperinsulinism-hyperammonemia syndrome (HHS). However, there are very few inhibitors known for human GDH. Recently, Ebselen was reported to crosslink with Escherichia coli GDH at the active site cysteine residue (Cys321), but the sequence alignment showed that the corresponding residue is Ala329 in human GDH. To investigate whether Ebselen could be an inhibitor for human GDH, we cloned and expressed an N-terminal His-tagged human GDH in E. coli. The recombinant human GDH enzyme showed expected properties such as adenosine diphosphate activation and nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate dual recognition. Further, we developed a 2-(3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-tetrazol-3-ium-5-yl) benzenesulfonate sodium salt (EZMTT)-based assay for human GDH, which was highly sensitive and is suitable for high-throughput screening for potent GDH inhibitors. In addition, ForteBio binding assays demonstrated that Ebselen is a reversible active site inhibitor for human GDH. Since Ebselen is a multifunctional organoselenium compound in Phase III clinical trials for inflammation, an Ebselen-based GDH inhibitor might be valuable for future drug discovery for HHS patients.

  3. Erythrocyte fluorescence and lead intoxication.

    Science.gov (United States)

    Clark, K G

    1976-01-01

    Blood samples from people exposed to inorganic lead were examined by fluorescence microscopy for excess erythrocyte porphyrin. With continued lead absorption, fluorescent erythrocytes appeared in the circulation of workers handling this metal or its compounds, and they progressively increased in number and brilliance. These changes ensued if the blood lead concentration was maintained above 2-42 mumol/l (50 mug/100 ml), and preceded any material fall in the haemoglobin value. At one factory, 62-5% of 81 symptomless workers showed erythrocyte fluorescence attributable to the toxic effects of lead. Excess fluorocytes were found in blood samples from a child with pica and three of her eight siblings. These four were subsequently shown to have slightly increased blood lead concentrations (2-03 to 2-32 mumol/l). Fluorescence microscopy for excess erythrocyte porphyrin is a sensitive method for the detection of chronic lead intoxication. A relatively slight increase in the blood lead is associated with demonstrabel changes in erythrocyte porphyrin content. The procedure requires little blood, and may be performed upon stored samples collected for lead estimation. The results are not readily influenced by contamination, and provide good confirmatory evidence for the absorption of biochemically active lead. PMID:963005

  4. Lipid-Lowering Pharmaceutical Clofibrate Inhibits Human Sweet Taste

    OpenAIRE

    Kochem, Matthew; Breslin, Paul A.S.

    2016-01-01

    T1R2-T1R3 is a heteromeric receptor that binds sugars, high potency sweeteners, and sweet taste blockers. In rodents, T1R2-T1R3 is largely responsible for transducing sweet taste perception. T1R2-T1R3 is also expressed in non-taste tissues, and a growing body of evidence suggests that it helps regulate glucose and lipid metabolism. It was previously shown that clofibric acid, a blood lipid-lowering drug, binds T1R2-T1R3 and inhibits its activity in vitro. The purpose of this study was to dete...

  5. Enzymatic oxidation of mercury vapor by erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Halbach, S; Clarkson, T W

    1978-01-01

    The formation of glutathione radicals, the evolution of nascent oxygen or the peroxidatic reaction with catalase complex I are considered as possible mechanisms for the oxidation of mercury vapor by red blood cells. To select among these, the uptake of atomic mercury by erythrocytes from different species was studied and related to their various activities of catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) and glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9). A slow and continuouus infusion of diluted H/sub 2/O/sub 2/ was used to maintain steady concentrations of complex I. 1% red cell suspensions were found most suitable showing high rates of Hg uptake and yielding still enough cells for subsequent determinations. The results indicate that the oxidation of mercury depends upon the H/sub 2/O/sub 2/-generation rate and upon the specific acticity of red-cell catalase. The oxidation occurred in a range of the catalase-H/sub 2/O/sub 2/ reaction where the evolution of oxygen could be excluded. Compounds reacting with complex I were shown to be effective inhibitors of the mercury uptake. GSH-peroxidase did not participate in the oxidation but rather, was found to inhibit it by competing with catalase for hydrogen peroxide. These findings support the view that elemental mercury is oxidized in erythrocytes by a peroxidatic reaction with complex I only.

  6. Human milk oligosaccharides inhibit growth of group B Streptococcus

    NARCIS (Netherlands)

    Lin, Ann E; Autran, Chloe A; Szyszka, Alexandra; Escajadillo, Tamara; Huang, Mia; Godula, Kamil; Prudden, Anthony R; Boons, Geert-Jan; Lewis, Amanda L; Doran, Kelly S; Nizet, Victor; Bode, Lars

    2017-01-01

    Streptococcus agalactiae (group B Streptococcus, GBS) is a leading cause of invasive bacterial infections in newborns, typically acquired vertically during childbirth secondary to maternal vaginal colonization. Human milk oligosaccharides (HMOs) have important nutritional and biological activities

  7. Inhibition of human polymorphonuclear leukocyte function by components of human colostrum and mature milk.

    Science.gov (United States)

    Pickering, L K; Cleary, T G; Caprioli, R M

    1983-04-01

    To compare the effect of human colostrum (days 1 to 3 postpartum) and mature milk (days 170 +/- 24 postpartum) on the function of polymorphonuclear leukocytes (PMNL), Ficoll-Hypaque-separated PMNL from the blood of 60 healthy volunteers were incubated with whole colostrum, colostral lipid, and colostral aqueous phase from 30 mothers, or with mature whole milk and its separated components from 30 mothers, and tested for resting and zymosan-stimulated oxidative metabolism, functional activity, and the presence of Fc receptors. Stimulated oxygen consumption, quantitative nitroblue tetrazolium dye reduction, [1-(14)C]glucose utilization, and Fc receptors were significantly (P cells or cells exposed to the aqueous phase of colostrum. In contrast, PMNL exposed to whole mature milk or to its lipid or aqueous phase caused no significant decrease in any of these parameters when compared to nonexposed cells. In assays of phagocytosis, colostral PMNL or blood PMNL exposed to colostral lipid had a significant (P < 0.001) decrease in their ability to ingest [methyl-(3)H]thymidine-labeled Staphylococcus aureus when compared to non-lipid-exposed PMNL. Blood PMNL exposed to lipid from mature milk had no decrease in ability to ingest S. aureus. Analysis of total lipid and total and individual fatty acid content revealed a uniform increase in all components in mature milk when compared to colostrum. Lipid or lipid-soluble material present in human colostrum but not mature milk causes inhibition of phagocytosis and respiratory burst-related activities of PMNL.

  8. Bovine Lactoferrampin, Human Lactoferricin, and Lactoferrin 1-11 Inhibit Nuclear Translocation of HIV Integrase.

    Science.gov (United States)

    Wang, Winston Yan; Wong, Jack Ho; Ip, Denis Tsz Ming; Wan, David Chi Cheong; Cheung, Randy Chifai; Ng, Tzi Bun

    2016-08-01

    This study aimed to investigate fragments derived from human and bovine lactoferrins for ability to inhibit nuclear translocation of HIV-1 integrase. It was shown that human lactoferricin, human lactoferrin 1-11, and bovine lactoferrampin reduced nuclear distribution of HIV-1 integrase. Bovine lactoferrampin could inhibit both the activity and nuclear translocation of HIV-1 integrase. Human lactoferrampin, bovine lactoferricin, and bovine lactoferrin 1-11 had no effect on HIV-1 integrase nuclear translocation. Human lactoferrampin which inhibited the activity of integrase did not prevent its nuclear translocation. Human lactoferricin and lactoferrin 1-11 did not inhibit HIV-1 integrase nuclear translocation despite their ability to attenuate the enzyme activity. The discrepancy between the findings on reduction of HIV-1 activity and inhibition of nuclear translocation of HIV-1 integrase was due to the different mechanisms involved. A similar reasoning can also be applied to the different inhibitory potencies of the milk peptides on different HIV enzymes, i.e., nuclear translocation.

  9. Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

    International Nuclear Information System (INIS)

    Kaufmann, W.K.; Cleaver, J.E.

    1981-01-01

    The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher cytotoxic fluences inhibited DNA synthesis in operating replicons. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences, indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation despite their ability to remove pyrimidine dimers. The analysis suggested that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery. (author)

  10. Inhibition of phospholipase A2 from human plasma by sodium bisulfite

    International Nuclear Information System (INIS)

    Wiggins, C.W.; Franson, R.C.

    1987-01-01

    The anti-oxidant sodium bisulfite has been shown to inhibit acid active(lysosomal), non-Ca ++ -dependent phospholipase A 2 (PLA 2 ), and to interact reversibly with unsaturated fatty acids, altering their chromatographic mobility. The authors examined the effect of bisulfite on neutral active, Ca ++ -dependent PLA 2 from human plasma. Using [1- 14 C]oleate-labelled autoclaved E. coli as substrate, PLA 2 activity was inhibited in a dose-dependent manner by bisulfite. Maximal inhibition occurred at 100μM bisulfite. Preincubation of plasma for 0-30 minutes with bisulfite resulted in a time-dependent increase in PLA 2 inhibition. Preincubation of substrate with bisulfite had no such effect. When the plasma PLA 2 was purified 25-fold by SP-Sephadex chromatography it was no longer inhibited by bisulfite. The SP-Sephadex wash through fraction, which contained greater than 95% of the applied protein but not PLA 2 activity, did not inhibit the purified enzyme. When incubated with bisulfite however, the SP-wash through fraction produced dose-dependent inhibition of the purified enzyme. These results indicate that sodium bisulfite inhibits human plasma PLA 2 , in vitro, indirectly by interaction with a factor(s) present in plasma and suggests that anti-oxidants may similarly influence expression of extracellular PLA 2 in vivo

  11. In vitro effects of estrogen and progesterone containing drugs on human erythrocyte carbonic anhydrase I and II isozymes in women smokers and nonsmokers

    Directory of Open Access Journals (Sweden)

    Mine Islimye Taskin

    2015-09-01

    Conclusion: The results of the current study provide important information to clinicians about how to consider the possible adverse effects of these drugs which are produced as a result of inhibition of CA I and CA II enzyme. Clinicians should take into consideration the side effects caused by CA I and CA II enzyme inhibition when prescribing these drugs in the treatment of different clinical conditions, especially in women who smoke.

  12. Gastric inhibitory polypeptide does not inhibit gastric emptying in humans

    DEFF Research Database (Denmark)

    Meier, Juris J; Goetze, Oliver; Anstipp, Jens

    2004-01-01

    ) = 0.15, P = 0.15 for intact GIP; r(2) = 0.21, P = 0.086 for total GIP). We conclude that gastric emptying does not appear to be influenced by GIP. The secretion of GIP after meal ingestion is not suppressed by its exogenous administration. The lack of effect of GIP on gastric emptying underlines......The insulinotropic gut hormone gastric inhibitory polypeptide (GIP) has been demonstrated to inhibit gastric acid secretion and was proposed to possess "enterogastrone" activity. GIP effects on gastric emptying have not yet been studied. Fifteen healthy male volunteers (23.9 +/- 3.3 yr, body mass....... Gastric emptying was calculated from the (13)CO(2) exhalation rates in breath samples collected over 360 min. Venous blood was drawn in 30-min intervals for the determination of glucose, insulin, C-peptide, and GIP (total and intact). Statistical calculations were made by use of repeated-measures ANOVA...

  13. Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity

    International Nuclear Information System (INIS)

    Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki

    2005-01-01

    Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion

  14. Inhibition of proliferation, migration and invasion of human non ...

    African Journals Online (AJOL)

    Purpose: To determine the effect of phlomisoside F (PMF) on the proliferation, migration and invasion of human non-small cell lung cancer cell line A549 and explore the possible mechanisms. Methods: The anti-proliferative effect of PMF on A549 cells was determined by CCK-8. Subsequently, migration and invasion were ...

  15. Inhibition of diabetes in NOD mice by human pregnancy factor

    NARCIS (Netherlands)

    Khan, N.A.; Khan, A.; Savelkoul, H.F.J.; Benner, R.

    2001-01-01

    Clinical symptoms of Th1 mediated autoimmune diseases regress in many patients during pregnancy. A prominent feature of pregnancy is the presence of human chorionic gonadotrophin hormone (hCG) in blood and urine. In this report we tested the effect of clinical grade hCG (c-hCG) on the development of

  16. Milk Oligosaccharides Inhibit Human Rotavirus Infectivity in MA104 Cells.

    Science.gov (United States)

    Laucirica, Daniel R; Triantis, Vassilis; Schoemaker, Ruud; Estes, Mary K; Ramani, Sasirekha

    2017-09-01

    Background: Oligosaccharides in milk act as soluble decoy receptors and prevent pathogen adhesion to the infant gut. Milk oligosaccharides reduce infectivity of a porcine rotavirus strain; however, the effects on human rotaviruses are less well understood. Objective: In this study, we determined the effect of specific and abundant milk oligosaccharides on the infectivity of 2 globally dominant human rotavirus strains. Methods: Four milk oligosaccharides-2'-fucosyllactose (2'FL), 3'-sialyllactose (3'SL), 6'-sialyllactose (6'SL), and galacto-oligosaccharides-were tested for their effects on the infectivity of human rotaviruses G1P[8] and G2P[4] through fluorescent focus assays on African green monkey kidney epithelial cells (MA104 cells). Oligosaccharides were added at different time points in the infectivity assays. Infections in the absence of oligosaccharides served as controls. Results: When compared with infections in the absence of glycans, all oligosaccharides substantially reduced the infectivity of both human rotavirus strains in vitro; however, virus strain-specific differences in effects were observed. Compared with control infections, the maximum reduction in G1P[8] infectivity was seen with 2'FL when added after the onset of infection (62% reduction, P rotaviruses in MA104 cells, primarily through an effect on the virus. Although breastfed infants are directly protected, the addition of specific oligosaccharides to infant formula may confer these benefits to formula-fed infants. © 2017 American Society for Nutrition.

  17. Comparison of oxime reactivation and aging of nerve agent-inhibited monkey and human acetylcholinesterases.

    Science.gov (United States)

    Luo, Chunyuan; Tong, Min; Maxwell, Donald M; Saxena, Ashima

    2008-09-25

    Non-human primates are valuable animal models that are used for the evaluation of nerve agent toxicity as well as antidotes and results from animal experiments are extrapolated to humans. It has been demonstrated that the efficacy of an oxime primarily depends on its ability to reactivate nerve agent-inhibited acetylcholinesterase (AChE). If the in vitro oxime reactivation of nerve agent-inhibited animal AChE is similar to that of human AChE, it is likely that the results of an in vivo animal study will reliably extrapolate to humans. Therefore, the goal of this study was to compare the aging and reactivation of human and different monkey (Rhesus, Cynomolgus, and African Green) AChEs inhibited by GF, GD, and VR. The oximes examined include the traditional oxime 2-PAM, two H-oximes HI-6 and HLo-7, and the new candidate oxime MMB4. Results indicate that oxime reactivation of all three monkey AChEs was very similar to human AChE. The maximum difference in the second-order reactivation rate constant between human and three monkey AChEs or between AChEs from different monkey species was 5-fold. Aging rate constants of GF-, GD-, and VR-inhibited monkey AChEs were very similar to human AChE except for GF-inhibited monkey AChEs, which aged 2-3 times faster than the human enzyme. The results of this study suggest that all three monkey species are suitable animal models for nerve agent antidote evaluation since monkey AChEs possess similar biochemical/pharmacological properties to human AChE.

  18. Interaction of lectins with membrane receptors on erythrocyte surfaces.

    Science.gov (United States)

    Sung, L A; Kabat, E A; Chien, S

    1985-08-01

    The interactions of human genotype AO erythrocytes (red blood cells) (RBCs) with N-acetylgalactosamine-reactive lectins isolated from Helix pomatia (HPA) and from Dolichos biflorus (DBA) were studied. Binding curves obtained with the use of tritium-labeled lectins showed that the maximal numbers of lectin molecules capable of binding to human genotype AO RBCs were 3.8 X 10(5) and 2.7 X 10(5) molecules/RBC for HPA and DBA, respectively. The binding of one type of lectin may influence the binding of another type. HPA was found to inhibit the binding of DBA, but not vice versa. The binding of HPA was weakly inhibited by a beta-D-galactose-reactive lectin isolated from Ricinus communis (designated RCA1). Limulus polyphemus lectin (LPA), with specificity for N-acetylneuraminic acid, did not influence the binding of HPA but enhanced the binding of DBA. About 80% of LPA receptors (N-acetylneuraminic acid) were removed from RBC surfaces by neuraminidase treatment. Neuraminidase treatment of RBCs resulted in increases of binding of both HPA and DBA, but through different mechanisms. An equal number (7.6 X 10(5) of new HPA sites were generated on genotypes AO and OO RBCs by neuraminidase treatment, and these new sites accounted for the enhancement (AO cells) and appearance (OO cells) of hemagglutinability by HPA. Neuraminidase treatment did not generate new DBA sites, but increased the DBA affinity for the existing receptors; as a result, genotype AO cells increased their hemagglutinability by DBA, while OO cells remained unagglutinable. The use of RBCs of different genotypes in binding assays with 3H-labeled lectins of known specificities provides an experimental system for studying cell-cell recognition and association.

  19. Effects of copper, organic mercury and a mixture of the two on glycerol lysis of erythrocytes.

    OpenAIRE

    宮地,芳之

    1987-01-01

    The effects of copper, organic mercury and a mixture of the two on glycerol lysis of erythrocytes were examined. Copper ion and organic mercury (EMP; ethylmercury phosphate, and PCMB; sodium p-chloromercuricbenzoate) inhibited glycerol lysis of erythrocytes. The inhibitory effects was dependent on the incubation period. An equimolor solution of copper ion and EMP showed between copper ion and EMP. Similar results were obtained with copper and PCMB.

  20. Red not dead: signaling in and from erythrocytes.

    Science.gov (United States)

    Sprague, Randy S; Stephenson, Alan H; Ellsworth, Mary L

    2007-11-01

    The oxygen required to meet metabolic needs of all tissues is delivered by the erythrocyte, a small, flexible cell which, in mammals, is devoid of a nucleus and mitochondria. Despite its simple appearance, this 'bag of hemoglobin' has an important role in its own distribution, enabling the delivery of oxygen to precisely meet localized metabolic need. When an erythrocyte enters an area in which tissue oxygen demand exceeds supply, a signaling pathway is activated resulting in the release of adenosine 5'-triphosphate (ATP). This ATP acts in a paracrine fashion to increase vascular caliber resulting in increased oxygen delivery. Defects in this pathway are found in erythrocytes of humans with type 2 diabetes (DM2) and could contribute to the perfusion abnormalities in skeletal muscle associated with this disease.

  1. Changes in haematology, plasma biochemistry and erythrocyte ...

    African Journals Online (AJOL)

    The results suggest that maintaining wild birds in captivity for a prolonged period could be stressful as shown by the heterophil/lymphocytes ratio and reduced erythrocyte osmotic resistance, and could lead to decreases in erythrocyte parameters and muscle wasting. Keywords: Haematological parameters, erythrocyte ...

  2. Inhibiting the Ca2+ Influx Induced by Human CSF

    Directory of Open Access Journals (Sweden)

    Anna Drews

    2017-12-01

    Full Text Available One potential therapeutic strategy for Alzheimer’s disease (AD is to use antibodies that bind to small soluble protein aggregates to reduce their toxic effects. However, these therapies are rarely tested in human CSF before clinical trials because of the lack of sensitive methods that enable the measurement of aggregate-induced toxicity at low concentrations. We have developed highly sensitive single vesicle and single-cell-based assays that detect the Ca2+ influx caused by the CSF of individuals affected with AD and healthy controls, and we have found comparable effects for both types of samples. We also show that an extracellular chaperone clusterin; a nanobody specific to the amyloid-β peptide (Aβ; and bapineuzumab, a humanized monoclonal antibody raised against Aβ, could all reduce the Ca2+ influx caused by synthetic Aβ oligomers but are less effective in CSF. These assays could be used to characterize potential therapeutic agents in CSF before clinical trials.

  3. Serum protein inhibition of thyrotropin binding to human thyroid tissue

    International Nuclear Information System (INIS)

    Beall, G.N.; Chopra, I.J.; Solomon, D.H.; Kruger, S.R.

    1978-01-01

    We used a modificaton of the TSH radioreceptor assay to detect TSH-binding inhibition (TBI) activity in serum and serum fractions from normal subjects and patients with Graves' disease. TBI activity is present in normal IgG prepared by DEAE-Sephadex chromatography and in normal globulins prepared by precipitation at 1.6 M ammonium sulfate. Other normal serum proteins also had TBI activity when large concentrations were tested. Gel filtration chromatography and powder block electrophoresis were used to prepare fractions of normal and Graves' disease sera. In these fractions from normal serum, TBI activity was found in both γ-globulin and α-globulin-albumin fractions electrophoretically and in both 7S and 4S peaks from gel filtration. TBI activity from Graves' disease patients' sera was similarly distributed, but relatively more TBI accompanied the electrophoretic γ-globulins. Sepharose Protein-A and anti-IgG were used as immunoabsorbents to isolate and purify IgG from normal and Graves' disease sera. TBI activity in IgG was proportional to the IgG concentration, indicating that the TBI which migrates as a γ-globulin electrophoretically is an IgG and thus may possibly be an antibody. Inhibitory activity found in normal serum globulins and in the non-IgG fractions of both normal and abnormal sera seriously interferes with attempts to use the TSH radioreceptor assay to study the hypothesized anti-TSH receptor antibody in the serum of patients with Graves' disease

  4. Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

    Energy Technology Data Exchange (ETDEWEB)

    Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won (Toronto); (IBC-Belarus)

    2010-09-22

    The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B{prime} helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

  5. Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D

    Directory of Open Access Journals (Sweden)

    Yuan Li

    2017-02-01

    Full Text Available We aimed to investigate the effect of advanced glycation end products (AGEs on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs. Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT assay, real-time cell analyzer and 5-Ethynyl-2′-deoxyuridine (EdU staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3 II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

  6. Advanced Glycation End Products Inhibit the Proliferation of Human Umbilical Vein Endothelial Cells by Inhibiting Cathepsin D.

    Science.gov (United States)

    Li, Yuan; Chang, Ye; Ye, Ning; Dai, Dongxue; Chen, Yintao; Zhang, Naijin; Sun, Guozhe; Sun, Yingxian

    2017-02-17

    We aimed to investigate the effect of advanced glycation end products (AGEs) on the proliferation and migration ability of human umbilical vein endothelial cells (HUVECs). Cell proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay, real-time cell analyzer and 5-Ethynyl-2'-deoxyuridine (EdU) staining. Cell migration was detected by wound-healing and transwell assay. AGEs significantly inhibited the proliferation and migration of HUVECs in a time-and dose-dependent way. Western blotting revealed that AGEs dramatically increased the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I and p62. Immunofluorescence of p62 and acridine orange staining revealed that AGEs significantly increased the expression of p62 and the accumulation of autophagic vacuoles, respectively. Chloroquine (CQ) could further promote the expression of LC3 II/I and p62, increase the accumulation of autophagic vacuoles and promote cell injury induced by AGEs. In addition, AGEs reduced cathepsin D (CTSD) expression in a time-dependent way. Overexpression of wild-type CTSD significantly decreased the ratio of LC 3 II/I as well as p62 accumulation induced by AGEs, but overexpression of catalytically inactive mutant CTSD had no such effects. Only overexpression of wild-type CTSD could restore the proliferation of HUVECs inhibited by AGEs. However, overexpression of both wild-type CTSD and catalytically inactive mutant CTSD could promote the migration of HUVECs inhibited by AGEs. Collectively, our study found that AGEs inhibited the proliferation and migration in HUVECs and promoted autophagic flux, which in turn played a protective role against AGEs-induced cell injury. CTSD, in need of its catalytic activity, may promote proliferation in AGEs-treated HUVECs independent of the autophagy-lysosome pathway. Meanwhile, CTSD could improve the migration of AGEs-treated HUVECs regardless of its enzymatic activity.

  7. Why Do SGLT2 inhibitors inhibit only 30-50% of renal glucose reabsorption in humans?

    Science.gov (United States)

    Liu, Jiwen Jim; Lee, TaeWeon; DeFronzo, Ralph A

    2012-09-01

    Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30-50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible.

  8. Why Do SGLT2 Inhibitors Inhibit Only 30–50% of Renal Glucose Reabsorption in Humans?

    Science.gov (United States)

    Liu, Jiwen (Jim); Lee, TaeWeon; DeFronzo, Ralph A.

    2012-01-01

    Sodium glucose cotransporter 2 (SGLT2) inhibition is a novel and promising treatment for diabetes under late-stage clinical development. It generally is accepted that SGLT2 mediates 90% of renal glucose reabsorption. However, SGLT2 inhibitors in clinical development inhibit only 30–50% of the filtered glucose load. Why are they unable to inhibit 90% of glucose reabsorption in humans? We will try to provide an explanation to this puzzle in this perspective analysis of the unique pharmacokinetic and pharmacodynamic profiles of SGLT2 inhibitors in clinical trials and examine possible mechanisms and molecular properties that may be responsible. PMID:22923645

  9. Inhibition of histamine and eicosanoid release from dispersed human lung cells in vitro by quinotolast.

    Science.gov (United States)

    Okayama, Y; Hiroi, J; Lau, L C; Church, M K

    1995-12-01

    We have examined the effects of a new anti-allergic drug, quinotolast [sodium 5-(4-oxo-1-phenoxy-4H-quinolizine-3-carboxamido) yetrazolate monohydrate], in inhibiting the release of histamine and the generation of leukotriene (LT) C4 and prostaglandin (PG) D2 from dispersed human lung cells and compared this with those of its active metabolite in the rat, hydroxy quinotolast, and reference drugs, tranilast and sodium cromoglycate (SCG). Quinotolast in the concentration range of 1-100 micrograms/ml inhibited histamine and LTC4 release in a concentration-dependent manner. The inhibitory effect of quinotolast on histamine release from dispersed lung cells was largely independent of the preincubation period, no tachyphylaxis being observed. Hydroxy quinotolast and tranilast showed a weak inhibition of histamine release only when the drugs were added to the cells simultaneously with anti-IgE challenge. Quinotolast, 100 micrograms/ml, and SCG, 1 mM, significantly inhibited PGD2 and LTC4 release. Quinotolast inhibited PGD2 release by 100% and LTC4 release by 54%, whereas SCG inhibited PDG2 release by 33% and LTC4 release by 100%. No cross-tachyphylaxis between quinotolast and SCG was observed. The results demonstrated that quinotolast showed a significant inhibition of inflammatory mediators from human dispersed lung cells, suggesting that quinotolast is a good candidate for a clinical anti-allergic drug.

  10. Brassinosteroids inhibit in vitro angiogenesis in human endothelial cells

    Czech Academy of Sciences Publication Activity Database

    Rárová, L.; Zahler, S.; Liebl, J.; Kryštof, Vladimír; Sedlák, David; Bartůněk, Petr; Kohout, Ladislav; Strnad, Miroslav

    2012-01-01

    Roč. 77, č. 13 (2012), s. 1502-1509 ISSN 0039-128X R&D Projects: GA MŠk(CZ) LC06077 Grant - others:GA MŠk(CZ) ED0007/01/01 Program:ED Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50520514; CEZ:AV0Z40550506 Keywords : Angiogenesis * Human umbilical vein endothelial cells * Migration Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.803, year: 2012

  11. Decreased erythrocyte superoxide dismutase in elderly men with early nuclear cataract

    Directory of Open Access Journals (Sweden)

    Rose Rose

    2015-12-01

    Full Text Available BACKGROUND Imbalance between oxidative processes and antioxidant defenses has been considered to play a role in cataractogenesis, particularly in diabetes patients. Superoxide dismutase (SOD is an important precursor for oxidative stress in the human lens, and its activity is mainly dependent on the copper and zinc levels in the body. The aim of this study was to compare erythrocyte SOD, erythrocyte zinc and total serum testosterone levels in male patients with early senile nuclear cataract and evaluate the correlations between the parameters in all subjects. METHODS A community-based study of cross-sectional design was conducted at Cilandak District Primary Health Center where 52 adult and 17 elderly men with early senile nuclear cataract were chosen as the study subjects. Erythrocyte SOD, erythrocyte zinc, serum testosterone, and fasting blood glucose (FBG levels were measured in all subjects. Nuclear cataract stage was assessed with the Pentacam® instrument (Oculus, Germany. Independent Student t test and Pearson’s correlation were used to analyze the results. RESULTS Erythrocyte SOD level was significantly decreased in elderly men compared to adult men (p=0.014. Erythrocyte zinc, serum testosterone and FBG did not differ significantly in adult and elderly males (at p=0.304; p=0.145;and p=0.376, respectively. Erythrocyte SOD activity was significantly associated with erythrocyte zinc level (r=0.486; p=0.048. CONCLUSIONS Lower erythrocyte SOD activity was found in elderly males than in adult males with early nuclear cataract. There was a relationship between erythrocyte SOD and erythrocyte zinc level in elderly males with early nuclear cataract.

  12. Decreased erythrocyte superoxide dismutase in elderly men with early nuclear cataract

    Directory of Open Access Journals (Sweden)

    Rose

    2014-04-01

    Full Text Available BACKGROUND Imbalance between oxidative processes and antioxidant defenses has been considered to play a role in cataractogenesis, particularly in diabetes patients. Superoxide dismutase (SOD is an important precursor for oxidative stress in the human lens, and its activity is mainly dependent on the copper and zinc levels in the body. The aim of this study was to compare erythrocyte SOD, erythrocyte zinc and total serum testosterone levels in male patients with early senile nuclear cataract and evaluate the correlations between the parameters in all subjects. METHODS A community-based study of cross-sectional design was conducted at Cilandak District Primary Health Center where 52 adult and 17 elderly men with early senile nuclear cataract were chosen as the study subjects. Erythrocyte SOD, erythrocyte zinc, serum testosterone, and fasting blood glucose (FBG levels were measured in all subjects. Nuclear cataract stage was assessed with the Pentacam® instrument (Oculus, Germany. Independent Student t test and Pearson’s correlation were used to analyze the results. RESULTS Erythrocyte SOD level was significantly decreased in elderly men compared to adult men (p=0.014. Erythrocyte zinc, serum testosterone and FBG did not differ significantly in adult and elderly males (at p=0.304; p=0.145;and p=0.376, respectively. Erythrocyte SOD activity was significantly associated with erythrocyte zinc level (r=0.486; p=0.048. CONCLUSIONS Lower erythrocyte SOD activity was found in elderly males than in adult males with early nuclear cataract. There was a relationship between erythrocyte SOD and erythrocyte zinc level in elderly males with early nuclear cataract.

  13. Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation.

    Science.gov (United States)

    Campos, Elisa; Moura, Teresa F; Oliva, Abel; Leandro, Paula; Soveral, Graça

    2011-05-13

    In general, erythrocytes are highly permeable to water, urea and glycerol. However, expression of aquaporin isoforms in erythrocytes appears to be species characteristic. In the present study, human (hRBC) and bovine (bRBC) erythrocytes were chosen for comparative studies due to their significant difference in membrane glycerol permeability. Osmotic water permeability (P(f)) at 23°C was (2.89 ± 0.37) × 10(-2) and (5.12 ± 0.61) × 10(-2)cms(-1) for human and bovine cells, respectively, with similar activation energies for water transport. Glycerol permeability (P(gly)) for human ((1.37 ± 0.26) × 10(-5)cms(-1)) differed in three orders of magnitude from bovine erythrocytes ((5.82 ± 0.37) × 10(-8)cms(-1)) that also showed higher activation energy for glycerol transport. When compared to human, bovine erythrocytes showed a similar expression pattern of AQP1 glycosylated forms on immunoblot analysis, though in slight higher levels, which could be correlated with the 1.5-fold larger P(f) found. However, AQP3 expression was not detectable. Immunofluorescence analysis confirmed the absence of AQP3 expression in bovine erythrocyte membranes. In conclusion, lack of AQP3 in bovine erythrocytes points to the lipid pathway as responsible for glycerol permeation and explains the low glycerol permeability and high E(a) for transport observed in ruminants. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

    Directory of Open Access Journals (Sweden)

    Surasak Jittavisutthikul

    2015-04-01

    Full Text Available There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN. Human hepatic (Huh7 cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β, indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.

  15. Inhibition of Human Arginase I by Substrate adn Product Analogues

    Energy Technology Data Exchange (ETDEWEB)

    L Di Costanzo; M Ilies; K Thorn; D Christianson

    2011-12-31

    Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.

  16. Inhibition of nitric oxide synthesis enhances leukocyte rolling and adhesion in human microvasculature

    Directory of Open Access Journals (Sweden)

    Hossain Mokarram

    2012-07-01

    Full Text Available Abstract Background Nitric oxide (NO is a multifunctional signaling molecule that regulates important cellular events in inflammation including leukocyte recruitment. Previous studies have shown that pharmacological inhibition of NO synthesis induces leukocyte recruitment in various in vitro and animal models. However, it is not known whether NO modulation has similar effects on leukocyte-endothelial cell interactions within the human microvasculature. The present study explored the effect of systemic L-NAME treatment on leukocyte recruitment in the SCID-hu mouse model. Methods Human skin xenografts were transplanted in SCID mice to study human leukocyte dynamics in human vasculature. Early events of human leukocyte recruitment in human vasculature were studied using intravital microscopy. NO synthesis was pharmacologically inhibited using NG-nitro-L-arginine methyl ester (L-NAME. Immunohistochemical analysis was performed to elucidate E-selectin expression in human xenograft skin. Human neutrophil-endothelial cell interactions were also studied in an in vitro flow chamber assay system. P- and E-selectin expression on cultured human umbilical vein endothelial cells (HUVECs was measured using ELISA. Platelet-activating factor (PAF synthesis was detected using a TLC-based assay. Results L-NAME treatment significantly enhanced the rolling and adhesion of human leukocytes to the human vasculature. Functional blocking of P- and E-selectins significantly inhibited rolling but not adhesion induced by inhibition of NO synthesis. Systemic L-NAME treatment enhanced E-selectin expression in human xenograft skin. L-NAME treatment significantly enhanced P- and E-selectin expression on HUVECs. L-NAME treatment did not significantly modify neutrophil rolling or adhesion to HUVECs indicating that L-NAME−induced subtle P- and E-selectin expression was insufficient to elicit dynamic neutrophil-HUVEC interactions in vitro. Moreover, synthesis of endothelial

  17. Influence of Erythrocyte Membrane Stability in Atherosclerosis.

    Science.gov (United States)

    da Silva Garrote-Filho, Mario; Bernardino-Neto, Morun; Penha-Silva, Nilson

    2017-04-01

    The purpose of this study is to show how an excess of cholesterol in the erythrocyte membrane contributes stochastically to the progression of atherosclerosis, leading to damage in blood rheology and O 2 transport, deposition of cholesterol (from trapped erythrocytes) in an area of intraplaque hemorrhage, and local exacerbation of oxidative stress. Cholesterol contained in the membrane of erythrocytes trapped in an intraplaque hemorrhage contributes to the growth of the necrotic nucleus. There is even a relationship between the amount of cholesterol in the erythrocyte membrane and the severity of atherosclerosis. In addition, the volume variability among erythrocytes, measured by RDW, is predictive of a worsening of this disease. Erythrocytes contribute to the development of atherosclerosis in several ways, especially when trapped in intraplate hemorrhage. These erythrocytes are oxidized and phagocytosed by macrophages. The cholesterol present in the membrane of these erythrocytes subsequently contributes to the growth of the atheroma plaque. In addition, when they rupture, erythrocytes release hemoglobin, which leads to the generation of free radicals. Finally, increased RDW may predict the worsening of atherosclerosis, due to the effects of inflammation and oxidative stress on erythropoiesis and erythrocyte volume. A better understanding of erythrocyte participation in atherosclerosis may contribute to the improvement of the prevention and treatment strategies of this disease.

  18. Hemolitic action of Naja naja atra cardiotoxin on erythrocytes from different animals

    Directory of Open Access Journals (Sweden)

    J. C. Troiano

    2006-01-01

    Full Text Available A comparative study on the sensitivity of erythrocytes from different vertebrate species (avian, mammalian and reptilian to the hemolytic action caused by cardiotoxin isolated from Naja naja atra venom was carried out. Cardiotoxin was able to induce direct hemolysis in washed erythrocytes from several animals, except for llama. The EC50 values from hemolysis of the most sensitive (cat and the most resistant (snake animal varied approximately tenfold. According to the cell behavior, it was possible to characterize four types of behavior: The first was observed in cat, horse and human cells; the second in rat, rabbit and dog erythrocytes; and the third only in llama erythrocytes, which were resistant to cardiotoxin concentrations up to 300 µg/ml. Finally, avian and reptilian erythrocytes were more resistant to cardiotoxin III-induced hemolysis than those of the mammalian species.

  19. Diterpenoids from Tetraclinis articulata that inhibit various human leukocyte functions.

    Science.gov (United States)

    Barrero, Alejandro F; Quílez del Moral, José F; Lucas, Rut; Payá, Miguel; Akssira, Mohamed; Akaad, Said; Mellouki, Fouad

    2003-06-01

    Ten new compounds, eight of them pimarane derivatives (1-8), together with a menthane dimer (9) and a totarane diterpenoid (10), were isolated from the leaves and wood of Tetraclinis articulata. The structures of 1-10 were established by using spectroscopic techniques, including 2D NMR spectra. Pimaranes 1-5 were found to possess an unusual cis interannular union of the B and C rings, which, from a biogenetic perspective, could be derived from the hydration of a carbocation at C-8. Compounds 4-6 and a mixture of 7 and 11 modulated different human leukocyte functions at a concentration of 10 microM, mainly the degranulation process measured as myeloperoxidase release and, to a lesser extent, the superoxide production measured by chemiluminescence.

  20. Plasmodium falciparum secretome in erythrocyte and beyond

    Directory of Open Access Journals (Sweden)

    Rani eSoni

    2016-02-01

    Full Text Available Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for development of novel anti-malarial therapies.

  1. Human Neutralizing Monoclonal Antibody Inhibition of Middle East Respiratory Syndrome Coronavirus Replication in the Common Marmoset.

    Science.gov (United States)

    Chen, Zhe; Bao, Linlin; Chen, Cong; Zou, Tingting; Xue, Ying; Li, Fengdi; Lv, Qi; Gu, Songzhi; Gao, Xiaopan; Cui, Sheng; Wang, Jianmin; Qin, Chuan; Jin, Qi

    2017-06-15

    Middle East respiratory syndrome coronavirus (MERS-CoV) infection in humans is highly lethal, with a fatality rate of 35%. New prophylactic and therapeutic strategies to combat human infections are urgently needed. We isolated a fully human neutralizing antibody, MCA1, from a human survivor. The antibody recognizes the receptor-binding domain of MERS-CoV S glycoprotein and interferes with the interaction between viral S and the human cellular receptor human dipeptidyl peptidase 4 (DPP4). To our knowledge, this study is the first to report a human neutralizing monoclonal antibody that completely inhibits MERS-CoV replication in common marmosets. Monotherapy with MCA1 represents a potential alternative treatment for human infections with MERS-CoV worthy of evaluation in clinical settings. © Crown copyright 2017.

  2. 4-Pyridone-3-carboxamide-1-β-d-ribonucleoside Triphosphate (4PyTP, a Novel NAD+ Metabolite Accumulating in Erythrocytes of Uremic Children: A Biomarker for a Toxic NAD+ Analogue in Other Tissues?

    Directory of Open Access Journals (Sweden)

    Elizabeth A. Carrey

    2011-06-01

    Full Text Available We have identified a novel nucleotide, 4-pyridone 3/5-carboxamide ribonucleoside triphosphate (4PyTP, which accumulates in human erythrocytes during renal failure. Using plasma and erythrocyte extracts obtained from children with chronic renal failure we show that the concentration of 4PyTP is increased, as well as other soluble NAD+ metabolites (nicotinamide, N1-methylnicotinamide and 4Py-riboside and the major nicotinamide metabolite N1-methyl-2-pyridone-5-carboxamide (2PY, with increasing degrees of renal failure. We noted that 2PY concentration was highest in the plasma of haemodialysis patients, while 4PyTP was highest in erythrocytes of children undergoing peritoneal dialysis: its concentration correlated closely with 4Py-riboside, an authentic precursor of 4PyTP, in the plasma. In the dialysis patients, GTP concentration was elevated: similar accumulation was noted previously, as a paradoxical effect in erythrocytes during treatment with immunosuppressants such as ribavirin and mycophenolate mofetil, which deplete GTP through inhibition of IMP dehydrogenase in nucleated cells such as lymphocytes. We predict that 4Py-riboside and 4Py-nucleotides bind to this enzyme and alter its activity. The enzymes that regenerate NAD+ from nicotinamide riboside also convert the drugs tiazofurin and benzamide riboside into NAD+ analogues that inhibit IMP dehydrogenase more effectively than the related ribosides: we therefore propose that the accumulation of 4PyTP in erythrocytes during renal failure is a marker for the accumulation of a related toxic NAD+ analogue that inhibits IMP dehydrogenase in other cells.

  3. Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yunjun [The Military General Hospital of Beijing PLA, Affiliated Bayi Brain Hospital (China); Zhang, Jinqian, E-mail: jingwanghou@yahoo.com.cn [Capital Medical University, Institute of Infectious Diseases, Beijing Ditan Hospital (China); Zhao, Ming [Peking University, Department of Chemical Biology, School of Pharmaceutical Sciences (China); Shi, Zujin [Peking University, Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Rare Earth Materials Chemistry and Applications, College of Chemistry and Molecular Engineering (China); Chen, Xin; He, Xihui; Han, Nanyin, E-mail: jingwanghou@sina.com [Peking University, Department of Chemical Biology, School of Pharmaceutical Sciences (China); Xu, Ruxiang, E-mail: everbright999@163.com [The Military General Hospital of Beijing PLA, Affiliated Bayi Brain Hospital (China)

    2013-08-15

    Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

  4. Single-wall carbon nanohorns (SWNHs) inhibited proliferation of human glioma cells and promoted its apoptosis

    Science.gov (United States)

    Li, Yunjun; Zhang, Jinqian; Zhao, Ming; Shi, Zujin; Chen, Xin; He, Xihui; Han, Nanyin; Xu, Ruxiang

    2013-08-01

    Although single-wall carbon nanohorns (SWNHs) have been demonstrated to accumulate to cytotoxic levels within organs of various animal models and cell types, they have been exploited for cancer therapies. The role of SWNHs in human glioma cell lines was unclear. To address this question, the research about direct role of SWNHs on the growth, proliferation, and apoptosis of human glioma cell lines (U87, U251, and U373) had been performed. Our results indicate that particle size of SWNHs in water is between 342 and 712 nm, the films of SEM show that SWNHs on PS surface are individual particles. SWNHs significantly delayed mitotic entry of human glioma cell lines cells, and inhibited its proliferation in a time- and dose-dependent manner. SWNHs induced a significant increase in G1 phase and inhibition of S phase followed the gradually increasing concentrations. SWNHs in human glioma cell lines cells significantly induced apoptosis followed by their gradually increasing concentrations. The TEM images showed that individual spherical SWNHs particles smaller than 100 nm in diameters were localized inside lysosomes of human glioma cell lines. SWNHs inhibited mitotic entry, growth, and proliferation of human glioma cell lines, and promoted its apoptosis. SWNHs may be a novel opportunity or method for the research on treatment of human glioma.

  5. Inhibition of human UDP-glucuronosyltransferase enzymes by lapatinib, pazopanib, regorafenib and sorafenib: Implications for hyperbilirubinemia.

    Science.gov (United States)

    Miners, John O; Chau, Nuy; Rowland, Andrew; Burns, Kushari; McKinnon, Ross A; Mackenzie, Peter I; Tucker, Geoffrey T; Knights, Kathleen M; Kichenadasse, Ganessan

    2017-04-01

    Kinase inhibitors (KIs) are a rapidly expanding class of drugs used primarily for the treatment of cancer. Data relating to the inhibition of UDP-glucuronosyltransferase (UGT) enzymes by KIs is sparse. However, lapatinib (LAP), pazopanib (PAZ), regorafenib (REG) and sorafenib (SOR) have been implicated in the development of hyperbilirubinemia in patients. This study aimed to characterise the role of UGT1A1 inhibition in hyperbilirubinemia and assess the broader potential of these drugs to perpetrate drug-drug interactions arising from UGT enzyme inhibition. Twelve recombinant human UGTs from subfamilies 1A and 2B were screened for inhibition by LAP, PAZ, REG and SOR. IC 50 values for the inhibition of all UGT1A enzymes, except UGT1A3 and UGT1A4, by the four KIs were enzyme identified to date. In vitro-in vivo extrapolation indicates that inhibition of UGT1A1 contributes significantly to the hyperbilirubinemia observed in patients treated with REG and SOR, but not with LAP and PAZ. Inhibition of other UGT1A1 substrates in vivo is likely. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Modulation of recurrent inhibition from knee extensors to ankle motoneurones during human walking

    DEFF Research Database (Denmark)

    Lamy, Jean-Charles; Iglesias, Caroline; Lackmy, Alexandra

    2008-01-01

    The neural control for muscle coordination during human locomotion involves spinal and supraspinal networks, but little is known about the exact mechanisms implicated. The present study focused on modulation of heteronymous recurrent inhibition from knee extensors to ankle motoneurones at different...... times in the gait cycle, when quadriceps (Quad) muscle activity overlaps that in tibialis anterior (TA) and soleus (Sol). The effects of femoral nerve stimulation on ankle motoneurones were investigated during treadmill walking and during tonic co-contraction of Quad and TA/Sol while standing. Recurrent...... inhibition of TA motoneurones depended on the level of background EMG, and was similar during walking and standing for matched background EMG levels. On the other hand, recurrent inhibition in Sol was reduced in early stance, with respect to standing, and enhanced in late stance. Reduced inhibition in Sol...

  7. Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

    Science.gov (United States)

    Crans, D C; Simone, C M; Saha, A K; Glew, R H

    1989-11-30

    A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

  8. Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum

    Science.gov (United States)

    2010-06-17

    Sciences, Bethesda, MD, ...... 14. ABSTRACT Plasmodium falciparum is a highly lethal malaria parasite of humans. A major portion of its life cycle is...parasite of humans. A major portion of its life cycle is dedicated to invading and multiplying inside erythrocytes. The molecular mechanisms of...Complement Receptor 1 Is a Sialic Acid-Independent Erythrocyte Receptor of Plasmodium falciparum Carmenza Spadafora1,2,3, Gordon A. Awandare4

  9. Contribution of the major and minor subunits to fimbria-mediated adherence of Haemophilus influenzae to human epithelial cells and erythrocytes

    NARCIS (Netherlands)

    van Ham, S. M.; van Alphen, L.; Mooi, F. R.; van Putten, J. P.

    1995-01-01

    Fimbriae are colonization factors of the human pathogen Haemophilus influenzae in that they mediate bacterial adherence to human eukaryotic cells. The contribution of the major (HifA) and putative minor (HifD and HifE) subunits of H. influenzae fimbriae to fimbria-specific adherence was studied by

  10. Apolipoprotein M mediates sphingosine-1-phosphate efflux from erythrocytes

    DEFF Research Database (Denmark)

    Christensen, Pernille M.; Bosteen, Markus H.; Hajny, Stefan

    2017-01-01

    Sphingosine-1-phosphate (S1P) is a bioactive lipid implicated in e.g. angiogenesis, lymphocyte trafficking, and endothelial barrier function. Erythrocytes are a main source of plasma S1P together with platelets and endothelial cells. Apolipoprotein M (apoM) in HDL carries 70% of plasma S1P, whereas...... 30% is carried by albumin. The current aim was to investigate the role of apoM in export of S1P from human erythrocytes. Erythrocytes exported S1P more efficiently to HDL than to albumin, particularly when apoM was present in HDL. In contrast, export of sphingosine to HDL was unaffected...... by the presence of apoM. The specific ability of apoM to promote export of S1P was independent of apoM being bound in HDL particles. Treatment with MK-571, an inhibitor of the ABCC1 transporter, effectively reduced export of S1P from human erythrocytes to apoM, whereas the export was unaffected by inhibitors...

  11. Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR

    Directory of Open Access Journals (Sweden)

    Zheng Wang

    2016-09-01

    Full Text Available Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

  12. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    Energy Technology Data Exchange (ETDEWEB)

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R., E-mail: mundy.william@epa.gov

    2011-11-15

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  13. Comparative sensitivity of human and rat neural cultures to chemical-induced inhibition of neurite outgrowth

    International Nuclear Information System (INIS)

    Harrill, Joshua A.; Freudenrich, Theresa M.; Robinette, Brian L.; Mundy, William R.

    2011-01-01

    There is a need for rapid, efficient and cost-effective alternatives to traditional in vivo developmental neurotoxicity testing. In vitro cell culture models can recapitulate many of the key cellular processes of nervous system development, including neurite outgrowth, and may be used as screening tools to identify potential developmental neurotoxicants. The present study compared primary rat cortical cultures and human embryonic stem cell-derived neural cultures in terms of: 1) reproducibility of high content image analysis based neurite outgrowth measurements, 2) dynamic range of neurite outgrowth measurements and 3) sensitivity to chemicals which have been shown to inhibit neurite outgrowth. There was a large increase in neurite outgrowth between 2 and 24 h in both rat and human cultures. Image analysis data collected across multiple cultures demonstrated that neurite outgrowth measurements in rat cortical cultures were more reproducible and had higher dynamic range as compared to human neural cultures. Human neural cultures were more sensitive than rat cortical cultures to chemicals previously shown to inhibit neurite outgrowth. Parallel analysis of morphological (neurite count, neurite length) and cytotoxicity (neurons per field) measurements were used to detect selective effects on neurite outgrowth. All chemicals which inhibited neurite outgrowth in rat cortical cultures did so at concentrations which did not concurrently affect the number of neurons per field, indicating selective effects on neurite outgrowth. In contrast, more than half the chemicals which inhibited neurite outgrowth in human neural cultures did so at concentrations which concurrently decreased the number of neurons per field, indicating that effects on neurite outgrowth were secondary to cytotoxicity. Overall, these data demonstrate that the culture models performed differently in terms of reproducibility, dynamic range and sensitivity to neurite outgrowth inhibitors. While human neural

  14. Quinones: reactions with hemoglobin, effects within erythrocytes and potential for antimalarial development

    International Nuclear Information System (INIS)

    Denny, B.J.

    1986-01-01

    The focus of this research was to characterize the interactions of some simple quinone like compounds with purified hemoglobin and to study the effects of these compounds within erythrocytes. It is proposed that these sorts of agents can have an antimalarial effect. The simplest compounds chosen for study were benzoquinone, methylquinone (toluquinone) and hydroquinone. When 14 C-quinone was reacted with purified hemoglobin (Hb) there was rapid binding of the first two moles of substrate per Hb molecule. An unusual property of the modified Hb's is that in the presence of a redox sensitive agent such as cytochrome c they are capable of generating superoxide anions. Within erythrocytes, quinone and toluquinone which differ only by a single methyl group have completely different effects. Toluquinone causes the cells to hemolyse and the effect was enhanced when the erythrocyte superoxide dismutase was inhibited; the effect was diminished when scavengers of activated oxygen such as histidine, mannitol and vital E were present. Benzoquinone on the other hand did not cause the cells to hemolyse and instead appeared to protect the cells from certain hemolytic stresses. Growth of malaria parasites in erythrocytes has been shown to be inhibited by activated forms of oxygen, also some quinone like agents in the past have been shown to inhibit the parasite's metabolism. An initial experiment with erythrocytes infected with malaria parasites showed that quinone and toluquinone could both inhibit the growth rate of parasites

  15. Probenecid inhibits α-adrenergic receptor-mediated vasoconstriction in the human leg vasculature

    DEFF Research Database (Denmark)

    Nyberg, Michael Permin; Piil, Peter Bergmann; Kiehn, Oliver Thistrup

    2018-01-01

    to α1- and α2-adrenergic receptor stimulation in the human forearm and leg vasculature of young healthy male subjects (23±3 years). By use of immunolabeling and confocal microscopy, Panx1 channels were found to be expressed in vascular smooth muscle cells of arterioles in human leg skeletal muscle....... Probenecid treatment increased (Padrenergic receptor stimulation) by ≈15%, whereas the response to the α1-agonist phenylephrine was unchanged. Inhibition...

  16. Alpha-tocopherol transfer factor (aTTF) from rat liver mediates the transfer of d-alpha-[3H]-tocopherol from liposomes to human erythrocyte ghosts and exhibits saturation kinetics

    International Nuclear Information System (INIS)

    Verdon, C.P.; Blumberg, J.B.

    1986-01-01

    aTTF was observed to transfer d-alpha-[ 3 H]-tocopherol ( 3 HaT) from egg lecithin liposomes to human erythrocyte ghosts (EG). aTTF may be associated with the 32,000-35,000 MW alpha-Tocopherol Binding Protein previously described to transfer 3 HaT from liposomes to rat liver microsomes and mitochondria prepared by ammonium sulfate precipitation of rat liver cytosol followed by dialysis against 50 mM TRIS-HCl/1 mM EDTA buffer, pH 7.4. Assay for aTTF activity consisted of incubating liposomal 3 HaT and EG in the presence of aTTF or buffer blank for various time periods at 37 0 C, then counting the resulting radioactivity in washed EG after pelleting by centrifugation. Liposomes were prelabeled-with non-exchangable glycerol-[ 14 C]-trioleate to correct for liposomes adhering to pelleted EG. Greater than 50% of the tritium found with the EG pellet was recovered by HPLC as 3 HaT. aTTF activity increased with increasing liposomal 3 HaT concentration before reaching a plateau. aTTF activity was similarly saturated by increasing EG concentrations. The same assay conditions with buffer blank along resulted in negligible transfer activity

  17. 51Cr - erythrocyte survival curves

    International Nuclear Information System (INIS)

    Paiva Costa, J. de.

    1982-07-01

    Sixteen patients were studied, being fifteen patients in hemolytic state, and a normal individual as a witness. The aim was to obtain better techniques for the analysis of the erythrocytes, survival curves, according to the recommendations of the International Committee of Hematology. It was used the radiochromatic method as a tracer. Previously a revisional study of the International Literature was made in its aspects inherent to the work in execution, rendering possible to establish comparisons and clarify phonomena observed in cur investigation. Several parameters were considered in this study, hindering both the exponential and the linear curves. The analysis of the survival curves of the erythrocytes in the studied group, revealed that the elution factor did not present a homogeneous answer quantitatively to all, though, the result of the analysis of these curves have been established, through listed programs in the electronic calculator. (Author) [pt

  18. MiR-1254 inhibits proliferation, migration and invasion of human ...

    African Journals Online (AJOL)

    MiR-1254 inhibits proliferation, migration and invasion of human brain tumour cell lines. ... The transcripts were analysed by real-time polymerase chain reaction (RT-PCR) ... Over-expression of miR- 1254 also led to significant decrease in cell ...

  19. Inhibition of uropathogenic biofilm growth on silicone rubber in human urine by lactobacilli - a teleologic approach

    NARCIS (Netherlands)

    Velraeds, MMC; van de Belt-Gritter, B; Busscher, HJ; Reid, G; van der Mei, HC

    2000-01-01

    The ability of three Lactobacillus strains to inhibit the adhesion and growth of naturally occurring uropathogens on silicone rubber was investigated in human urine. The importance of biosurfactant production by Lactobacillus in discouraging uropathogen growth was determined in relation to the

  20. Inhibition of ultraviolet irradiation response of human skin by topical phlogostatic compounds

    International Nuclear Information System (INIS)

    Weirich, E.G.; Lutz, U.C.

    1977-01-01

    By adaption of the model of UV dermatitis in human skin a test procedure has been developed which facilitates realistic assessment of topical contra-inflammatory activity of steroidal as well as non-steroidal compounds. Sixt typical skin drug agents were tested according to their reaction inhibition effect. (orig./MG) [de

  1. Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

    Directory of Open Access Journals (Sweden)

    Xiyuan Bai

    Full Text Available Nuclear factor-kappa B (NFκB is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB. However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

  2. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    Science.gov (United States)

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Adhesion of Plasmodium falciparum infected erythrocytes in ex vivo perfused placental tissue

    DEFF Research Database (Denmark)

    Pehrson, Caroline; Mathiesen, Line; Heno, Kristine K

    2016-01-01

    placental tissue. RESULTS: The ex vivo placental perfusion model was modified to study adhesion of infected erythrocytes binding to CSA, endothelial protein C receptor (EPCR) or a transgenic parasite where P. falciparum erythrocyte membrane protein 1 expression had been shut down. Infected erythrocytes......, such as binding to immunoglobulins. Furthermore, other parasite antigens have been associated with placental malaria. These findings have important implications for placental malaria vaccine design. The objective of this study was to adapt and describe a biologically relevant model of parasite adhesion in intact...... expressing VAR2CSA accumulated in perfused placental tissue whereas the EPCR binding and the transgenic parasite did not. Soluble CSA and antibodies specific against VAR2CSA inhibited binding of infected erythrocytes. CONCLUSION: The ex vivo model provides a novel way of studying receptor-ligand interactions...

  4. The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes.

    Directory of Open Access Journals (Sweden)

    Muriel Vayssier-Taussat

    2010-06-01

    Full Text Available Bacterial pathogens typically infect only a limited range of hosts; however, the genetic mechanisms governing host-specificity are poorly understood. The alpha-proteobacterial genus Bartonella comprises 21 species that cause host-specific intraerythrocytic bacteremia as hallmark of infection in their respective mammalian reservoirs, including the human-specific pathogens Bartonella quintana and Bartonella bacilliformis that cause trench fever and Oroya fever, respectively. Here, we have identified bacterial factors that mediate host-specific erythrocyte colonization in the mammalian reservoirs. Using mouse-specific Bartonella birtlesii, human-specific Bartonella quintana, cat-specific Bartonella henselae and rat-specific Bartonella tribocorum, we established in vitro adhesion and invasion assays with isolated erythrocytes that fully reproduce the host-specificity of erythrocyte infection as observed in vivo. By signature-tagged mutagenesis of B. birtlesii and mutant selection in a mouse infection model we identified mutants impaired in establishing intraerythrocytic bacteremia. Among 45 abacteremic mutants, five failed to adhere to and invade mouse erythrocytes in vitro. The corresponding genes encode components of the type IV secretion system (T4SS Trw, demonstrating that this virulence factor laterally acquired by the Bartonella lineage is directly involved in adherence to erythrocytes. Strikingly, ectopic expression of Trw of rat-specific B. tribocorum in cat-specific B. henselae or human-specific B. quintana expanded their host range for erythrocyte infection to rat, demonstrating that Trw mediates host-specific erythrocyte infection. A molecular evolutionary analysis of the trw locus further indicated that the variable, surface-located TrwL and TrwJ might represent the T4SS components that determine host-specificity of erythrocyte parasitism. In conclusion, we show that the laterally acquired Trw T4SS diversified in the Bartonella lineage

  5. The aryl hydrocarbon receptor ligand ITE inhibits TGFβ1-induced human myofibroblast differentiation.

    Science.gov (United States)

    Lehmann, Geniece M; Xi, Xia; Kulkarni, Ajit A; Olsen, Keith C; Pollock, Stephen J; Baglole, Carolyn J; Gupta, Shikha; Casey, Ann E; Huxlin, Krystel R; Sime, Patricia J; Feldon, Steven E; Phipps, Richard P

    2011-04-01

    Fibrosis can occur in any human tissue when the normal wound healing response is amplified. Such amplification results in fibroblast proliferation, myofibroblast differentiation, and excessive extracellular matrix deposition. Occurrence of these sequelae in organs such as the eye or lung can result in severe consequences to health. Unfortunately, medical treatment of fibrosis is limited by a lack of safe and effective therapies. These therapies may be developed by identifying agents that inhibit critical steps in fibrotic progression; one such step is myofibroblast differentiation triggered by transforming growth factor-β1 (TGFβ1). In this study, we demonstrate that TGFβ1-induced myofibroblast differentiation is blocked in human fibroblasts by a candidate endogenous aryl hydrocarbon receptor (AhR) ligand 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE). Our data show that ITE disrupts TGFβ1 signaling by inhibiting the nuclear translocation of Smad2/3/4. Although ITE functions as an AhR agonist, and biologically persistent AhR agonists, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, cause severe toxic effects, ITE exhibits no toxicity. Interestingly, ITE effectively inhibits TGFβ1-driven myofibroblast differentiation in AhR(-/-) fibroblasts: Its ability to inhibit TGFβ1 signaling is AhR independent. As supported by the results of this study, the small molecule ITE inhibits myofibroblast differentiation and may be useful clinically as an antiscarring agent. Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  6. Na+/K+-ATPase: Activity and inhibition

    Science.gov (United States)

    Čolović, M.; Krstić, D.; Krinulović, K.; Momić, T.; Savić, J.; Vujačić, A.; Vasić, V.

    2009-09-01

    The aim of the study was to give an overview of the mechanism of inhibition of Na+/K+-ATPase activity induced by some specific and non specific inhibitors. For this purpose, the effects of some ouabain like compounds (digoxin, gitoxin), noble metals complexes ([PtCl2DMSO2], [AuCl4]-, [PdCl4]2-, [PdCl(dien)]+, [PdCl(Me4dien)]+), transition metal ions (Cu2+, Zn2+, Fe2+, Co2+), and heavy metal ions (Hg2+, Pb2+, Cd2+) on the activity of Na+/K+-ATPase from rat synaptic plasma membranes (SPM), porcine cerebral cortex and human erythrocytes were discussed.

  7. BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

    International Nuclear Information System (INIS)

    Goupille, Olivier; Penglong, Tipparat; Lefèvre, Carine; Granger, Marine; Kadri, Zahra; Fucharoen, Suthat; Maouche-Chrétien, Leila; Leboulch, Philippe; Chrétien, Stany

    2012-01-01

    Highlights: ► UT7 erythroleukemia cells are known to be refractory to differentiate. ► Brief JQ1 treatment initiates the first steps of erythroid differentiation program. ► Engaged UT7 cells then maturate in the presence of erythropoietin. ► Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

  8. BET bromodomain inhibition rescues erythropoietin differentiation of human erythroleukemia cell line UT7

    Energy Technology Data Exchange (ETDEWEB)

    Goupille, Olivier [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Penglong, Tipparat [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Thalassemia Research Center and Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Thailand); Lefevre, Carine; Granger, Marine; Kadri, Zahra [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Fucharoen, Suthat [Thalassemia Research Center and Department of Clinical Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University (Thailand); Maouche-Chretien, Leila [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Leboulch, Philippe [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France); Genetics Division, Department of Medicine, Brigham and Women' s Hospital and Harvard Medical School, Boston, MA (United States); Chretien, Stany, E-mail: stany.chretien@cea.fr [CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay-aux-Roses (France); UMR INSERM U.962, University Paris XI, CEA, Fontenay-aux-Roses (France)

    2012-12-07

    Highlights: Black-Right-Pointing-Pointer UT7 erythroleukemia cells are known to be refractory to differentiate. Black-Right-Pointing-Pointer Brief JQ1 treatment initiates the first steps of erythroid differentiation program. Black-Right-Pointing-Pointer Engaged UT7 cells then maturate in the presence of erythropoietin. Black-Right-Pointing-Pointer Sustained JQ1 treatment inhibits both proliferation and erythroid differentiation. -- Abstract: Malignant transformation is a multistep process requiring oncogenic activation, promoting cellular proliferation, frequently coupled to inhibition of terminal differentiation. Consequently, forcing the reengagement of terminal differentiation of transformed cells coupled or not with an inhibition of their proliferation is a putative therapeutic approach to counteracting tumorigenicity. UT7 is a human leukemic cell line able to grow in the presence of IL3, GM-CSF and Epo. This cell line has been widely used to study Epo-R/Epo signaling pathways but is a poor model for erythroid differentiation. We used the BET bromodomain inhibition drug JQ1 to target gene expression, including that of c-Myc. We have shown that only 2 days of JQ1 treatment was required to transitory inhibit Epo-induced UT7 proliferation and to restore terminal erythroid differentiation. This study highlights the importance of a cellular erythroid cycle break mediated by c-Myc inhibition before initiation of the erythropoiesis program and describes a new model for BET bromodomain inhibitor drug application.

  9. Mechanism of inhibition of human secretory phospholipase A2 by flavonoids: rationale for lead design

    Science.gov (United States)

    Lättig, Jens; Böhl, Markus; Fischer, Petra; Tischer, Sandra; Tietböhl, Claudia; Menschikowski, Mario; Gutzeit, Herwig O.; Metz, Peter; Pisabarro, M. Teresa

    2007-08-01

    The human secretory phospholipase A2 group IIA (PLA2-IIA) is a lipolytic enzyme. Its inhibition leads to a decrease in eicosanoids levels and, thereby, to reduced inflammation. Therefore, PLA2-IIA is of high pharmacological interest in treatment of chronic diseases such as asthma and rheumatoid arthritis. Quercetin and naringenin, amongst other flavonoids, are known for their anti-inflammatory activity by modulation of enzymes of the arachidonic acid cascade. However, the mechanism by which flavonoids inhibit Phospholipase A2 (PLA2) remained unclear so far. Flavonoids are widely produced in plant tissues and, thereby, suitable targets for pharmaceutical extractions and chemical syntheses. Our work focuses on understanding the binding modes of flavonoids to PLA2, their inhibition mechanism and the rationale to modify them to obtain potent and specific inhibitors. Our computational and experimental studies focused on a set of 24 compounds including natural flavonoids and naringenin-based derivatives. Experimental results on PLA2-inhibition showed good inhibitory activity for quercetin, kaempferol, and galangin, but relatively poor for naringenin. Several naringenin derivatives were synthesized and tested for affinity and inhibitory activity improvement. 6-(1,1-dimethylallyl)naringenin revealed comparable PLA2 inhibition to quercetin-like compounds. We characterized the binding mode of these compounds and the determinants for their affinity, selectivity, and inhibitory potency. Based on our results, we suggest C(6) as the most promising position of the flavonoid scaffold to introduce chemical modifications to improve affinity, selectivity, and inhibition of PLA2-IIA by flavonoids.

  10. Potent inhibition of human neutrophil activations by bractelactone, a novel chalcone from Fissistigma bracteolatum

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Yang-Chang [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Graduate Institute of Integrated Medicine, College of Chinese Medicine, China Medical University, Taichung 404, Taiwan (China); Sureshbabu, Munisamy; Fang, Yao-Ching; Wu, Yi-Hsiu [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Lan, Yu-Hsuan [School of Pharmacy, China Medical University, Taichung 404, Taiwan (China); Chang, Fang-Rong [Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung 807, Taiwan (China); Chang, Ya-Wen [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Hwang, Tsong-Long, E-mail: htl@mail.cgu.edu.tw [Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan (China); Chinese Herbal Medicine Research Team, Healthy Aging Research Center, Chang Gung University, Kweishan, Taoyuan 333, Taiwan (China)

    2013-02-01

    Fissistigma bracteolatum is widely used in traditional medicine to treat inflammatory diseases. However, its active components and mechanisms of action remain unclear. In this study, (3Z)-6,7-dihydroxy-4-methoxy-3-(phenylmethylidene)-5-(3-phenylpropanoyl) -1-benzofuran-2(3H) (bractelactone), a novel chalcone from F. bracteolatum, showed potent inhibitory effects against superoxide anion (O{sub 2}{sup ·−}) production, elastase release, and CD11b expression in formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-induced human neutrophils. However, bractelactone showed only weak inhibition of phorbol myristate acetate-caused O{sub 2}{sup ·−} production. The peak cytosolic calcium concentration ([Ca{sup 2+}]{sub i}) was unaltered by bractelactone in FMLP-induced neutrophils, but the decay time of [Ca{sup 2+}]{sub i} was significantly shortened. In a calcium-free solution, changes in [Ca{sup 2+}]{sub i} caused by the addition of extracellular Ca{sup 2+} were inhibited by bractelactone in FMLP-activated cells. In addition, bractelactone did not alter the phosphorylation of p38 MAPK, ERK, JNK, or AKT or the concentration of cAMP. These results suggest that bractelactone selectively inhibits store-operated calcium entry (SOCE). In agreement with this concept, bractelactone suppressed sustained [Ca{sup 2+}]{sub i} changes in thapsigargin-activated neutrophils. Furthermore, bractelactone did not alter FMLP-induced formation of inositol 1,4,5-triphosphate. Taken together, our results demonstrate that the anti-inflammatory effects of bractelactone, an active ingredient of F. bracteolatum, in human neutrophils are through the selective inhibition of SOCE. Highlights: ► Bractelactone isolated from Fissistigma bracteolatum. ► Bractelactone inhibited FMLP-induced human neutrophil activations. ► Bractelactone had no effect on IP3 formation. ► Bractelactone did not alter MAPKs, AKT, and cAMP pathways. ► Bractelactone inhibited store-operated calcium entry.

  11. Combined inhibition of nitric oxide and prostaglandins reduces human skeletal muscle blood flow during exercise

    DEFF Research Database (Denmark)

    Boushel, Robert Christopher; Langberg, Henning; Gemmer, Carsten

    2002-01-01

    The vascular endothelium is an important mediator of tissue vasodilatation, yet the role of the specific substances, nitric oxide (NO) and prostaglandins (PG), in mediating the large increases in muscle perfusion during exercise in humans is unclear. Quadriceps microvascular blood flow......, respectively (P exercise in humans. These findings demonstrate an important synergistic role of NO and PG for skeletal muscle vasodilatation and hyperaemia during muscular contraction....... was quantified by near infrared spectroscopy and indocyanine green in six healthy humans during dynamic knee extension exercise with and without combined pharmacological inhibition of NO synthase (NOS) and PG by L-NAME and indomethacin, respectively. Microdialysis was applied to determine interstitial release...

  12. Fundamental studies on the insulin receptor in rabbit erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shinomiya, Y; Kagawa, S; Konishi, Y; Morimoto, H; Tsumura, Y [Hyogo Medical Coll. (Japan)

    1975-09-01

    The authors studied the binding of insulin to rabbit erythrocytes as a mode case in the hope of characterizing the physiologic role of the binding of insulin to receptor in both normal adults and patients. Specific binding sites for insulin were detected in rabbit erythrocytes. The characteristics of the binding were similar to those observed in other target tissues. The specific binding of /sup 125/I-labeled insulin was competitively inhibited by a small amount of unlabeled insulin and was completely inhibited by 1,000 ng/ml of unlabeled insulin. Glucagon, however, had no effect on the insulin binding to fat cells or liver membranes nor had it any effect on the binding of insulin to rabbit erythrocytes. Scatchard analysis of this binding reaction indicated two different binding sites with Ksub(aff)=3.2 x 10/sup 8//M, Ksub(diss)=3.1 x 10/sup -9/M; Ksub(aff)=1.4 x 10/sup 8//M, Ksub(diss)=7.1 x 10/sup -9/M respectively, and the binding capacities of each site were estimated at 0.011 ng/4 x 10/sup 8/ cells and 0.138 ng/4 x 10/sup 8/ cells. The binding of /sup 125/I-insulin to rabbit erythrocytes was a saturable function of the insulin concentration and was a linear function of cell concentration. The pH optimum for the reaction was 7.4 at 0/sup 0/C, the amount of insulin binding increased continuously under the reaction and this binding reaction reached a steady state after 10 to 15hr. On the other hand, the specific binding of insulin at higher temperatures showed maximal amounts after 20 to 30 min. and subsequently fell off at later time points.

  13. Affinity of hemoglobin for the cytoplasmic fragment of human erythrocyte membrane band 3. Equilibrium measurements at physiological pH using matrix-bound proteins: the effects of ionic strength, deoxygenation and of 2,3-diphosphoglycerate.

    Science.gov (United States)

    Chétrite, G; Cassoly, R

    1985-10-05

    The cytoplasmic fragment of band 3 protein isolated from the human erythrocyte membrane was linked to a CNBr-activated Sepharose matrix in an attempt to measure, in batch experiments, its equilibrium binding constant with oxy- and deoxyhemoglobin at physiological pH and ionic strength values and in the presence or the absence of 2,3-diphosphoglycerate. All the experiments were done at pH 7.2, and equilibrium constants were computed on the basis of one hemoglobin tetramer bound per monomer of fragment. In 10 mM-phosphate buffer, a dissociation constant KD = 2 X 10(-4)M was measured for oxyhemoglobin and was shown to increase to 8 X 10(-4)M in the presence of 50 mM-NaCl. Association could not be demonstrated at higher salt concentrations. Diphosphoglycerate-stripped deoxyhemoglobin was shown to associate more strongly with the cytoplasmic fragment of band 3. In 10 mM-bis-Tris (pH 7.2) and in the presence of 120 mM-NaCl, a dissociation constant KD = 4 X 10(-4)M was measured. Upon addition of increasing amounts of 2,3-diphosphoglycerate, the complex formed between deoxyhemoglobin and the cytoplasmic fragment of band 3 was dissociated. On the reasonable assumption that the hemoglobin binding site present on band 3 fragment was not modified upon linking the protein to the Sepharose matrix, the results indicated that diphosphoglycerate-stripped deoxyhemoglobin or partially liganded hemoglobin tetramers in the T state could bind band 3 inside the intact human red blood cell.

  14. Inhibition of platelet [3H]- imipramine binding by human plasma protein fractions

    International Nuclear Information System (INIS)

    Strijewski, A.; Chudzik, J.; Tang, S.W.

    1988-01-01

    Inhibition of high-affinity [ 3 H]-imipramine binding to platelet membranes by human plasma fractions and isolated plasma proteins was investigated. Several plasma proteins were found to contribute to the observed apparent inhibition and this contribution was assessed in terms of inhibitor units. Alpha 1 acid glycoprotein, high density and low density lipoprotein, IgG and α 1 -antitrypsin were identified as effective non-specific inhibitors. Alpha-1-acid glycoprotein was confirmed to be the most potent plasma protein inhibitor. Cohn fractions were evaluated for the presence of the postulated endocoid of [ 3 H]-imipramine binding site

  15. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    Energy Technology Data Exchange (ETDEWEB)

    Hintzpeter, Jan, E-mail: hintzpeter@toxi.uni-kiel.de [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Seliger, Jan Moritz [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Hofman, Jakub [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Martin, Hans-Joerg [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany); Wsol, Vladimir [Department of Biochemical Sciences, Faculty of Pharmacy in Hradec Kralove, Charles University in Prague, Heyrovskeho 1203, 50005 Hradec Kralove (Czech Republic); Maser, Edmund [Institute of Toxicology and Pharmacology for Natural Scientists, University Medical School Schleswig-Holstein, Campus Kiel, Brunswiker Str. 10, 24105 Kiel (Germany)

    2016-02-15

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC{sub 50}- and K{sub i}-values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin

  16. Inhibition of human anthracycline reductases by emodin — A possible remedy for anthracycline resistance

    International Nuclear Information System (INIS)

    Hintzpeter, Jan; Seliger, Jan Moritz; Hofman, Jakub; Martin, Hans-Joerg; Wsol, Vladimir; Maser, Edmund

    2016-01-01

    The clinical application of anthracyclines, like daunorubicin and doxorubicin, is limited by two factors: dose-related cardiotoxicity and drug resistance. Both have been linked to reductive metabolism of the parent drug to their metabolites daunorubicinol and doxorubicinol, respectively. These metabolites show significantly less anti-neoplastic properties as their parent drugs and accumulate in cardiac tissue leading to chronic cardiotoxicity. Therefore, we aimed to identify novel and potent natural inhibitors for anthracycline reductases, which enhance the anticancer effect of anthracyclines by preventing the development of anthracycline resistance. Human enzymes responsible for the reductive metabolism of daunorubicin were tested for their sensitivity towards anthrachinones, in particular emodin and anthraflavic acid. Intense inhibition kinetic data for the most effective daunorubicin reductases, including IC 50 - and K i -values, the mode of inhibition, as well as molecular docking, were compiled. Subsequently, a cytotoxicity profile and the ability of emodin to reverse daunorubicin resistance were determined using multiresistant A549 lung cancer and HepG2 liver cancer cells. Emodin potently inhibited the four main human daunorubicin reductases in vitro. Further, we could demonstrate that emodin is able to synergistically sensitize human cancer cells towards daunorubicin at clinically relevant concentrations. Therefore, emodin may yield the potential to enhance the therapeutic effectiveness of anthracyclines by preventing anthracycline resistance via inhibition of the anthracycline reductases. In symphony with its known pharmacological properties, emodin might be a compound of particular interest in the management of anthracycline chemotherapy efficacy and their adverse effects. - Highlights: • Natural and synthetic compounds were identified as inhibitors for human daunorubicin reductases. • Emodin is a potent inhibitor for human daunorubicin reductases.

  17. In vitro inhibition of human papillomavirus following use of a carrageenan-containing vaginal gel.

    Science.gov (United States)

    Novetsky, Akiva P; Keller, Marla J; Gradissimo, Ana; Chen, Zigui; Morgan, Stephanie L; Xue, Xiaonan; Strickler, Howard D; Fernández-Romero, José A; Burk, Robert; Einstein, Mark H

    2016-11-01

    To assess in vitro efficacy of Divine 9, a carrageenan-based vaginal lubricant that is being studied as a microbicide to inhibit HPV16 pseudovirus (PsV) infection. Sexually active US women between 19 and 35years without prior HPV vaccination or cervical intraepithelial neoplasia were instructed to use Divine 9 vaginally with an applicator either before sex only or before and after intercourse. Women who applied a single dose of gel returned for cervicovaginal lavage (CVL) collection 1, 4 or 8-12h after intercourse versus those who applied gel before and after intercourse returned 1, 4 or 8-12h after the second gel dose. Carrageenan concentrations were assessed using an ELISA assay and the inhibitory activity was assessed using a PsV-based neutralization assay against HPV16 infection. Carrageenan concentrations and the percentage of PsV16 inhibition were compared using the Wilcoxon rank sum test. Thirteen women were enrolled and thirty specimens from different time-points were assessed. 87% of CVL samples had detectable carrageenans with levels decreasing over time from intercourse. 93% of CVL samples had detectable PsV16 inhibition with median inhibition of 97.5%. PsV16 inhibition decreased over time, but remained high, with median inhibition of 98.1%, 97.4% and 83.4% at 1, 4 and 8-12h, respectively. Higher carrageenan concentrations were associated with higher levels of PsV16 inhibition (rho=0.69). This is the first report of a human study investigating in vitro HPV inhibition of a carrageenan-based vaginal lubricant with CVL collected after sexual intercourse. We demonstrate excellent efficacy in preventing PsV16 infection. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Encapsulation of interleukin-2 in murine erythrocytes and subsequent deposition in mice receiving a subcutaneous injection

    International Nuclear Information System (INIS)

    DeLoach, J.R.; Andrews, K.; Sheffield, C.L.

    1988-01-01

    Radiolabeled recombinant human interleukin-2 (IL-2) was successfully encapsulated in both mouse and sheep erythrocytes. Of the added IL-2, 70% was recovered bound to or encapsulated within the carrier cells. Erythrocytes containing IL-2 were stable in vitro and most of the IL-2 remained associated with the cells following a 16-h incubation at 37 degrees C. When carrier erythrocytes containing IL-2 were injected subcutaneously into mice, intact [ 35 S]IL-2 was detectable in a number of tissues 3 days after injection

  19. Evaluation of in vitro effects of some analgesic drugs on erythrocyte and recombinant carbonic anhydrase I and II.

    Science.gov (United States)

    Gökçe, Başak; Gençer, Nahit; Arslan, Oktay; Turkoğlu, Sumeyye Aydogan; Alper, Meltem; Köçkar, Feray

    2012-02-01

    The in vitro effects of the injectable form of analgesic drugs, dexketoprofen trometamol, dexamethasone sodium phosphate, metamizole sodium, diclofenac sodium, thiocolchicoside, on the activity of purified human carbonic anhydrase I and II were evaluated. The effect of these drugs on erythrocyte hCA I and hCA II was compared to recombinant hCA I and hCA II expressed in Ecoli. IC(50) values of the drugs that caused inhibition were determined by means of activity percentage diagrams. The IC(50) concentrations of dexketoprofen trometamol and dexamethasone sodium phosphate on hCA I were 683 μM and 4250 μM and for hCA II 950 μM and 6200 μM respectively. Conversely, the enzyme activity was increased by diflofenac sodium. In addition, thiocolchicoside has not any affect on hCA I and hCA II. The effect of these drugs on erythrocyte hCA I and hCA II were consistent with the inhibition of recombinant enzymes.

  20. Inhibiting actin depolymerization enhances osteoblast differentiation and bone formation in human stromal stem cells

    DEFF Research Database (Denmark)

    Chen, Li; Shi, Kaikai; Frary, Charles

    2015-01-01

    Remodeling of the actin cytoskeleton through actin dynamics is involved in a number of biological processes, but its role in human stromal (skeletal) stem cells (hMSCs) differentiation is poorly understood. In the present study, we demonstrated that stabilizing actin filaments by inhibiting gene...... expression of the two main actin depolymerizing factors (ADFs): Cofilin 1 (CFL1) and Destrin (DSTN) in hMSCs, enhanced cell viability and differentiation into osteoblastic cells (OB) in vitro, as well as heterotopic bone formation in vivo. Similarly, treating hMSC with Phalloidin, which is known to stabilize...... polymerized actin filaments, increased hMSCs viability and OB differentiation. Conversely, Cytocholasin D, an inhibitor of actin polymerization, reduced cell viability and inhibited OB differentiation of hMSC. At a molecular level, preventing Cofilin phosphorylation through inhibition of LIM domain kinase 1...

  1. Inhibition of human copper trafficking by a small molecule significantly attenuates cancer cell proliferation

    Science.gov (United States)

    Wang, Jing; Luo, Cheng; Shan, Changliang; You, Qiancheng; Lu, Junyan; Elf, Shannon; Zhou, Yu; Wen, Yi; Vinkenborg, Jan L.; Fan, Jun; Kang, Heebum; Lin, Ruiting; Han, Dali; Xie, Yuxin; Karpus, Jason; Chen, Shijie; Ouyang, Shisheng; Luan, Chihao; Zhang, Naixia; Ding, Hong; Merkx, Maarten; Liu, Hong; Chen, Jing; Jiang, Hualiang; He, Chuan

    2015-12-01

    Copper is a transition metal that plays critical roles in many life processes. Controlling the cellular concentration and trafficking of copper offers a route to disrupt these processes. Here we report small molecules that inhibit the human copper-trafficking proteins Atox1 and CCS, and so provide a selective approach to disrupt cellular copper transport. The knockdown of Atox1 and CCS or their inhibition leads to a significantly reduced proliferation of cancer cells, but not of normal cells, as well as to attenuated tumour growth in mouse models. We show that blocking copper trafficking induces cellular oxidative stress and reduces levels of cellular ATP. The reduced level of ATP results in activation of the AMP-activated protein kinase that leads to reduced lipogenesis. Both effects contribute to the inhibition of cancer cell proliferation. Our results establish copper chaperones as new targets for future developments in anticancer therapies.

  2. Cardiac glycosides induce cell death in human cells by inhibiting general protein synthesis.

    Directory of Open Access Journals (Sweden)

    Andrea Perne

    2009-12-01

    Full Text Available Cardiac glycosides are Na(+/K(+-pump inhibitors widely used to treat heart failure. They are also highly cytotoxic, and studies have suggested specific anti-tumor activity leading to current clinical trials in cancer patients. However, a definitive demonstration of this putative anti-cancer activity and the underlying molecular mechanism has remained elusive.Using an unbiased transcriptomics approach, we found that cardiac glycosides inhibit general protein synthesis. Protein synthesis inhibition and cytotoxicity were not specific for cancer cells as they were observed in both primary and cancer cell lines. These effects were dependent on the Na(+/K(+-pump as they were rescued by expression of a cardiac glycoside-resistant Na(+/K(+-pump. Unlike human cells, rodent cells are largely resistant to cardiac glycosides in vitro and mice were found to tolerate extremely high levels.The physiological difference between human and mouse explains the previously observed sensitivity of human cancer cells in mouse xenograft experiments. Thus, published mouse xenograft models used to support anti-tumor activity for these drugs require reevaluation. Our finding that cardiac glycosides inhibit protein synthesis provides a mechanism for the cytotoxicity of CGs and raises concerns about ongoing clinical trials to test CGs as anti-cancer agents in humans.

  3. Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

    Science.gov (United States)

    Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

    2017-06-01

    To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

  4. Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles

    International Nuclear Information System (INIS)

    Teimoori, Salma; Seesuay, Watee; Jittavisutthikul, Surasak; Chaisri, Urai; Sookrung, Nitat; Densumite, Jaslan; Saelim, Nawannaporn; Chulanetra, Monrat; Maneewatch, Santi; Chaicumpa, Wanpen

    2016-01-01

    A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials. - Highlights: • Cell penetrable human scFvs (transbodies) to Ebolavirus (EBOV) VP40 were produced. • The transbodies inhibited egress of EBOV-like particles (VLPs) from human hepatocytes. • They interacted with VP40 CTD basic residues important for plasma membrane binding. • And hence interfere with viral matrix assembly and viral progeny budding. • This is the first report on human antibodies that target intracellular EBOV VP40.

  5. Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

    Directory of Open Access Journals (Sweden)

    Barretto O.C. de O.

    2006-01-01

    Full Text Available In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 ± 38 IU g-1 Hb-1 min-1 at 37ºC, compared to the human erythrocyte activity of 12 ± 2 IU g-1 Hb-1 min-1 at 37ºC. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa. The Michaelis-Menten constants (Km: 55 µM for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 µM were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively. A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.

  6. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    International Nuclear Information System (INIS)

    Fan, Rong-hui; Zhu, Xiu-mei; Sun, Yao-wen; Peng, Hui-zi; Wu, Hang-li; Gao, Wen-jie

    2016-01-01

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  7. CTRP6 inhibits fibrogenesis in TGF-β1-stimulated human dermal fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Fan, Rong-hui, E-mail: fan_ronghuixa@163.com [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Zhu, Xiu-mei; Sun, Yao-wen [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China); Peng, Hui-zi [Department of Cosmetology Plastic Surgery, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi 710061 (China); Wu, Hang-li; Gao, Wen-jie [Department of Burn and Plastic Surgery, Shaanxi Provincial People’s Hospital, Xi’an 710068 (China)

    2016-07-08

    Skin fibrosis is characterized by excessive proliferation of fibroblasts and overproduction of extracellular matrix (ECM). C1q/tumor necrosis factor-related protein 6 (CTRP6), a member of CTRPs, has been involved in the development of cardiac fibrosis. However, the function and detailed regulatory mechanism of CTRP6 in skin fibrosis remain unclear. The aim of this study was to investigate the effect of CTRP6 on the activation of human dermal fibroblasts. Our results showed that CTRP6 was lowly expressed in scar tissues and transforming growth factor-β1 (TGF-β1)-treated dermal fibroblasts. CTRP6 overexpression significantly inhibited the proliferation of dermal fibroblasts, as well as suppressed the expression of ECM in TGF-β1-treated dermal fibroblasts. Furthermore, CTRP6 overexpression markedly inhibited TGF-β1-induced phosphorylation of Smad3 in dermal fibroblasts. In conclusion, the data reported here demonstrate that CTRP6 is able to inhibit the proliferation and ECM expression in human dermal fibroblasts through suppressing the TGF-β1/Smad3 signaling pathway. These findings suggest that CTRP6 may be a potential therapeutic target for the prevention of skin fibrosis. -- Highlights: •CTRP6 expression was decreased in scar tissues and TGF-β1-treated dermal fibroblasts. •CTRP6 inhibits TGF-β1-induced the proliferation of dermal fibroblasts. •CTRP6 inhibits expression of collagen type I and α-SMA. •CTRP6 inhibits the activation of TGF-β1/Smad3 signaling pathway in dermal fibroblasts.

  8. Prolyl oligopeptidase inhibition-induced growth arrest of human gastric cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Suzuki, Kanayo [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Sakaguchi, Minoru, E-mail: sakaguti@gly.oups.ac.jp [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Tanaka, Satoshi [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan); Yoshimoto, Tadashi [Department of Life Science, Setsunan University, 17-8 Ikeda-Nakamachi, Neyagawa, Osaka 572-8508 (Japan); Takaoka, Masanori [Laboratory of Cell Biology, Osaka University of Pharmaceutical Sciences, 4-20-1 Nasahara, Takatsuki, Osaka 569-1094 (Japan)

    2014-01-03

    Highlights: •We examined the effects of prolyl oligopeptidase (POP) inhibition on p53 null gastric cancer cell growth. •POP inhibition-induced cell growth suppression was associated with an increase in a quiescent G{sub 0} state. •POP might regulate the exit from and/or reentry into the cell cycle. -- Abstract: Prolyl oligopeptidase (POP) is a serine endopeptidase that hydrolyzes post-proline peptide bonds in peptides that are <30 amino acids in length. We recently reported that POP inhibition suppressed the growth of human neuroblastoma cells. The growth suppression was associated with pronounced G{sub 0}/G{sub 1} cell cycle arrest and increased levels of the CDK inhibitor p27{sup kip1} and the tumor suppressor p53. In this study, we investigated the mechanism of POP inhibition-induced cell growth arrest using a human gastric cancer cell line, KATO III cells, which had a p53 gene deletion. POP specific inhibitors, 3-((4-[2-(E)-styrylphenoxy]butanoyl)-L-4-hydroxyprolyl)-thiazolidine (SUAM-14746) and benzyloxycarbonyl-thioprolyl-thioprolinal, or RNAi-mediated POP knockdown inhibited the growth of KATO III cells irrespective of their p53 status. SUAM-14746-induced growth inhibition was associated with G{sub 0}/G{sub 1} cell cycle phase arrest and increased levels of p27{sup kip1} in the nuclei and the pRb2/p130 protein expression. Moreover, SUAM-14746-mediated cell cycle arrest of KATO III cells was associated with an increase in the quiescent G{sub 0} state, defined by low level staining for the proliferation marker, Ki-67. These results indicate that POP may be a positive regulator of cell cycle progression by regulating the exit from and/or reentry into the cell cycle by KATO III cells.

  9. Erythrocytes in alternating electric fields

    International Nuclear Information System (INIS)

    Morariu, V.V.; Chifu, A.; Simplaceanu, T.; Frangopol, P.T.

    1983-02-01

    The elastic and inelastic deformation of erythrocytes induced by alternating fields and the suggestion that moderate field intensities (1.2 kV/cm) when continuously applied can cause lysis by a different mechanism compared to the action of short intense field pulses is presented. The different experimental conditions can be used to approach various properties of the membrane such as those related to the dielectric polarization of the membrane or to the interfacial polarization, leading to the inelastic deformation of the cells. (authors)

  10. 3-Bromopyruvate inhibits cell proliferation and induces apoptosis in CD133+ population in human glioma.

    Science.gov (United States)

    Xu, Dong-Qiang; Tan, Xiao-Yu; Zhang, Bao-Wei; Wu, Tao; Liu, Ping; Sun, Shao-Jun; Cao, Yin-Guang

    2016-03-01

    The study was aimed to investigate the role of 3-bromopyruvate in inhibition of CD133+ U87 human glioma cell population growth. The results demonstrated that 3-bromopyruvate inhibited the viability of both CD133+ and parental cells derived from U87 human glioma cell line. However, the 3-bromopyruvate-induced inhibition in viability was more prominent in CD133+ cells at 10 μM concentration after 48 h. Treatment of CD133+ cells with 3-bromopyruvate caused reduction in cell population and cell size, membrane bubbling, and degradation of cell membranes. Hoechst 33258 staining showed condensation of chromatin material and fragmentation of DNA in treated CD133+ cells after 48 h. 3-Bromopyruvate inhibited the migration rate of CD133+ cells significantly compared to the parental cells. Flow cytometry revealed that exposure of CD133+ cells to 3-bromopyruvate increased the cell population in S phase from 24.5 to 37.9 % with increase in time from 12 to 48 h. In addition, 3-bromopyruvate significantly enhanced the expression of Bax and cleaved caspase 3 in CD133+ cells compared to the parental cells. Therefore, 3-bromopyruvate is a potent chemotherapeutic agent for the treatment of glioma by targeting stem cells selectively.

  11. Inhibition of the differentiation of monocyte-derived dendritic cells by human gingival fibroblasts.

    Directory of Open Access Journals (Sweden)

    Sylvie Séguier

    Full Text Available We investigated whether gingival fibroblasts (GFs can modulate the differentiation and/or maturation of monocyte-derived dendritic cells (DCs and analyzed soluble factors that may be involved in this immune modulation. Experiments were performed using human monocytes in co-culture with human GFs in Transwell® chambers or using monocyte cultures treated with conditioned media (CM from GFs of four donors. The four CM and supernatants from cell culture were assayed by ELISA for cytokines involved in the differentiation of dendritic cells, such as IL-6, VEGF, TGFβ1, IL-13 and IL-10. The maturation of monocyte-derived DCs induced by LPS in presence of CM was also studied. Cell surface phenotype markers were analyzed by flow cytometry. In co-cultures, GFs inhibited the differentiation of monocyte-derived DCs and the strength of this blockade correlated with the GF/monocyte ratio. Conditioned media from GFs showed similar effects, suggesting the involvement of soluble factors produced by GFs. This inhibition was associated with a lower stimulatory activity in MLR of DCs generated with GFs or its CM. Neutralizing antibodies against IL-6 and VEGF significantly (P<0.05 inhibited the inhibitory effect of CM on the differentiation of monocytes-derived DCs and in a dose dependent manner. Our data suggest that IL-6 is the main factor responsible for the inhibition of DCs differentiation mediated by GFs but that VEGF is also involved and constitutes an additional mechanism.

  12. Inhibition of isolated human myometrium contractility by minoxidil and reversal by glibenclamide.

    Science.gov (United States)

    Prabhakaran, S S; Dhanasekar, K R; Thomas, E; Jose, R; Peedicayil, J; Samuel, P

    2010-03-01

    This study investigated the ability of the antihypertensive drug minoxidil to inhibit potassium chloride (KCl)-induced contractility of the isolated human myometrium. Twelve strips of myometrium obtained from 12 patients who underwent hysterectomy were triggered to contract with 55 mM KCl before and after incubation with 3 concentrations (1, 3 and 10 microM) of minoxidil. The percent inhibition by minoxidil on the extent of contraction, and the area under the contractile curve of KCl-induced contraction of the myometrial strips was determined. Furthermore, the effect of 10 microM glibenclamide on the inhibition generated by 3 microM minoxidil on KCl-induced contractility was studied. It was found that minoxidil produced a concentration-dependent inhibition of KCl-induced contractility of the myometrium and that glibenclamide reversed this inhibitory effect. These results suggest that the inhibitory effect of minoxidil on isolated human myometrium contractility may prove useful in clinical conditions requiring relaxation of the myometrium. 2010 Prous Science, S.A.U. or its licensors. All rights reserved.

  13. Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity

    Science.gov (United States)

    Farb, Melissa G.; Tiwari, Stephanie; Karki, Shakun; Ngo, Doan TM; Carmine, Brian; Hess, Donald T.; Zuriaga, Maria A.; Walsh, Kenneth; Fetterman, Jessica L.; Hamburg, Naomi M.; Vita, Joseph A.; Apovian, Caroline M.; Gokce, Noyan

    2013-01-01

    Objective The purpose of this study was to determine whether cyclooxygenase inhibition improves vascular dysfunction of adipose microvessels from obese humans. Design and Methods In 20 obese subjects (age 37±12 yrs, BMI 47±8 kg/m2) we collected subcutaneous and visceral fat during bariatric surgery and characterized adipose depot-specific gene expression, endothelial cell phenotype, and microvascular function. Vasomotor function was assessed in response to endothelium-dependent agonists using videomicroscopy of small arterioles from fat. Results Arterioles from visceral fat exhibited impaired endothelium-dependent, acetylcholine-mediated vasodilation, compared to the subcutaneous depot (p<0.001). Expression of mRNA transcripts relevant to the cyclooxygenase pathway were upregulated in visceral compared to subcutaneous fat. Pharmacological inhibition of cyclooxygenase with indomethacin improved endothelium-dependent vasodilator function of arterioles from visceral fat by 2-fold (p=0.01), whereas indomethacin had no effect in the subcutaneous depot. Indomethacin increased activation via serine-1177 phosphorylation of endothelial nitric oxide synthase in response to acetylcholine in endothelial cells from visceral fat. Inhibition of endothelial nitric oxide synthase with Nω-nitro-L-arginine methyl ester abrogated the effects of cyclooxygenase-inhibition suggesting that vascular actions of indomethacin were related to increased nitric oxide bioavailability. Conclusions Our findings suggest that cyclooxygenase-mediated vasoconstrictor prostanoids partly contribute to endothelial dysfunction of visceral adipose arterioles in human obesity. PMID:23640904

  14. Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea

    International Nuclear Information System (INIS)

    Francis, A.A.; Carrier, W.L.; Smith, D.P.; Regan, J.D.; Blevins, R.D.

    1979-01-01

    The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect on hydroxyurea was observed at the concentration of 2mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65-70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well. (Auth.)

  15. Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light

    International Nuclear Information System (INIS)

    Painter, R.B.

    1985-01-01

    The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion. (orig.)

  16. Inhibition of DNA repair in ultraviolet-irradiated human cells by hydroxyurea

    Energy Technology Data Exchange (ETDEWEB)

    Francis, A.A. (Oak Ridge National Lab., TN); Blevins, R.D.; Carrier, W.L.; Smith, D.P.; Regan, J.D.

    1979-01-01

    The effect on DNA repair in ultraviolet-irradiated human skin fibroblasts by hydroxyurea has been examined in this study using three independent methods for measuring DNA repair: the 5-bromodeoxyuridine photolysis assay which measures DNA repair replication, chromatographic measurement of thymine-containing dimers, and measurement of specific ultraviolet-endonuclease-sensitive sites in irradiated DNA. Little effect of hydroxyurea was observed at the concentration of 2 mM, which is often used to inhibit semiconservative DNA synthesis; however, 10 mM hydroxyurea resulted in marked inhibition (65 to 70%) of excision repair. This inhibition was accompanied by a possible doubling in the size of the repaired region. The accumulation of large numbers of single-strand breaks following ultraviolet irradiation and hydroxyurea incubation seen by other investigators was not observed with the normal skin fibroblasts used in this study. A comparison of hydroxyurea effects on the different DNA repair assays indicates inhibition of one step in DNA repair also results in varying degrees of inhibition of other steps as well.

  17. [The 2,3-diphosphoglycerate shunt and stabilization of the ATP level in mammalian erythrocytes].

    Science.gov (United States)

    Ataullakhanov, A I; Ataullakhanov, F I; Vitvitskiĭ, V M; Zhabotinskiĭ, A M; Pichugin, A V

    1985-06-01

    The mechanisms of regulation of energy metabolism in erythrocytes of various mammalian species were investigated. In native erythrocytes of man, sheep, cow, dog and mouse the dependencies of the rates of glucose uptake on ATP concentration (i.e., regulatory parameters of glycolysis) were measured. These parameters plotted in normalized coordinates are not species-specific (invariant). The dependence of the rate of ATP-consuming processes on ATP concentration has been studied for the first time in intact mammalian erythrocytes. This dependence was found to be linear only in the species, in whose erythrocytes the activity of 2,3-diphosphoglycerate shunt is practically zero. In all species under study, the stabilization of ATP level is provided for mainly by the hexokinase-phosphofructokinase system. A comparison of regulatory mechanisms of energy metabolism in mammalian (sheep, cow) erythrocytes, in which the 2,3-diphosphoglycerate shunt is absent, with human and animal erythrocytes, in which this pathway is active, points to the important role of the 2,3-diphosphoglycerate shunt in regulation of energy conversion in erythrocytes. This shunt operates as an additional stabilizer protecting the cell from extremal influences.

  18. PPARγ ligand ciglitazone inhibits TNFα-induced ICAM-1 in human airway smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Chien-Da Huang

    2014-08-01

    Full Text Available Background: Modification of human airway smooth muscle (ASM function by proinflammatory cytokines has been regarded as a potential mechanism underlying bronchial hyperresponsiveness in asthma. Human ASM cells express intercellular adhesion molecule (ICAM-1 in response to cytokines. Synthetic ligands for peroxisome proliferator-activated receptor (PPARγ reportedly possess anti-inflammatory and immunomodulatory properties. In this study, we examined whether ciglitazone, a synthetic PPARγ ligand, can modulate the basal and tumor necrosis factor (TNFα-induced ICAM1 gene expression in human ASM cells. Methods: Human ASM cells were treated with TNFα. ICAM-1 expression was assessed by flow cytometry and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. PPARγ activity was inhibited by target-specific small interfering (si RNA targeting PPARγ and GW9662, a PPARγ antagonist. Activity of nuclear factor (NF-κB was assessed by using immunoblot analysis, immune-confocal images, and electrophoretic mobility shift assay (EMSA. Results: By flow cytometry, ciglitazone alone had no effect on ICAM-1 expression in ASM cells, but inhibited ICAM-1 expression in response to TNFα (10 ng/ml in a dose-dependent manner (1-10 μM. It also inhibited TNFα-induced ICAM1 gene expression by RT-PCR analysis. Knockdown of PPARγ gene by target-specific siRNA targeting PPARγ enhanced ICAM-1 expression and the inhibitory effect of ciglitazone on TNFα-induced ICAM-1 expression was reversed by PPARγ siRNA and GW9662. SN-50 (10 μg/ml, an inhibitor for nuclear translocation of NF-κB, inhibited TNFα-induced ICAM-1 expression. Ciglitazone did not prevent TNFα-induced degradation of the cytosolic inhibitor of NF-κB (IκB, but inhibited the nuclear translocation of p65 induced by TNFα and suppressed the NF-κB/DNA binding activity. Conclusion: These findings suggest that ciglitazone inhibits TNFα-induced ICAM1 gene expression in human ASM cells through

  19. Fatty acid synthase inhibition in human breast cancer cells leads to malonyl-CoA-induced inhibition of fatty acid oxidation and cytotoxicity.

    Science.gov (United States)

    Thupari, J N; Pinn, M L; Kuhajda, F P

    2001-07-13

    Inhibition of fatty acid synthase (FAS) induces apoptosis in human breast cancer cells in vitro and in vivo without toxicity to proliferating normal cells. We have previously shown that FAS inhibition causes a rapid increase in malonyl-CoA levels identifying malonyl-CoA as a potential trigger of apoptosis. In this study we further investigated the role of malonyl-CoA during FAS inhibition. We have found that: [i] inhibition of FAS with cerulenin causes carnitine palmitoyltransferase-1 (CPT-1) inhibition and fatty acid oxidation inhibition in MCF-7 human breast cancer cells likely mediated by elevation of malonyl-CoA; [ii] cerulenin cytotoxicity is due to the nonphysiological state of increased malonyl-CoA, decreased fatty acid oxidation, and decreased fatty acid synthesis; and [iii] the cytotoxic effect of cerulenin can be mimicked by simultaneous inhibition of CPT-1, with etomoxir, and fatty acid synthesis with TOFA, an acetyl-CoA carboxylase (ACC) inhibitor. This study identifies CPT-1 and ACC as two new potential targets for cancer chemotherapy. Copyright 2001 Academic Press.

  20. Triggering of Erythrocyte Cell Membrane Scrambling by Emodin

    Directory of Open Access Journals (Sweden)

    Morena Mischitelli

    2016-11-01

    Full Text Available Background/Aims: The natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone is a component of several Chinese medicinal herbal preparations utilized for more than 2000 years. The substance has been used against diverse disorders including malignancy, inflammation and microbial infection. The substance is effective in part by triggering suicidal death or apoptosis. Similar to apoptosis of nucleated cells erythrocytes may enter suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling involved in the triggering of eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i, oxidative stress and ceramide. The present study aimed to test, whether emodin induces eryptosis and, if so, to elucidate underlying cellular mechanisms. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ROS formation from DCFDA dependent fluorescence, and ceramide abundance utilizing specific antibodies. Results: Exposure of human erythrocytes for 48 hours to emodin (≥ 10 µM significantly increased the percentage of annexin-V-binding cells, and at higher concentrations (≥ 50 µM significantly increased forward scatter. Emodin significantly increased Fluo3-fluorescence (≥ 10 µM, DCFDA fluorescence (75 µM and ceramide abundance (75 µM. The effect of emodin on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Conclusions: Emodin triggers phospholipid scrambling of the erythrocyte cell membrane, an effect at least in part due to stimulation of Ca2+ entry and paralleled by oxidative stress and ceramide appearance at the erythroctye surface.

  1. Semicarbazone EGA Inhibits Uptake of Diphtheria Toxin into Human Cells and Protects Cells from Intoxication

    Directory of Open Access Journals (Sweden)

    Leonie Schnell

    2016-07-01

    Full Text Available Diphtheria toxin is a single-chain protein toxin that invades human cells by receptor-mediated endocytosis. In acidic endosomes, its translocation domain inserts into endosomal membranes and facilitates the transport of the catalytic domain (DTA from endosomal lumen into the host cell cytosol. Here, DTA ADP-ribosylates elongation factor 2 inhibits protein synthesis and leads to cell death. The compound 4-bromobenzaldehyde N-(2,6-dimethylphenylsemicarbazone (EGA has been previously shown to protect cells from various bacterial protein toxins which deliver their enzymatic subunits from acidic endosomes to the cytosol, including Bacillus anthracis lethal toxin and the binary clostridial actin ADP-ribosylating toxins C2, iota and Clostridium difficile binary toxin (CDT. Here, we demonstrate that EGA also protects human cells from diphtheria toxin by inhibiting the pH-dependent translocation of DTA across cell membranes. The results suggest that EGA might serve for treatment and/or prevention of the severe disease diphtheria.

  2. Theophylline prevents NAD+ depletion via PARP-1 inhibition in human pulmonary epithelial cells

    International Nuclear Information System (INIS)

    Moonen, Harald J.J.; Geraets, Liesbeth; Vaarhorst, Anika; Bast, Aalt; Wouters, Emiel F.M.; Hageman, Geja J.

    2005-01-01

    Oxidative DNA damage, as occurs during exacerbations in chronic obstructive pulmonary disease (COPD), highly activates the nuclear enzyme poly(ADP-ribose)polymerase-1 (PARP-1). This can lead to cellular depletion of its substrate NAD + , resulting in an energy crisis and ultimately in cell death. Inhibition of PARP-1 results in preservation of the intracellular NAD + pool, and of NAD + -dependent cellular processes. In this study, PARP-1 activation by hydrogen peroxide decreased intracellular NAD + levels in human pulmonary epithelial cells, which was found to be prevented in a dose-dependent manner by theophylline, a widely used compound in the treatment of COPD. This enzyme inhibition by theophylline was confirmed in an ELISA using purified human PARP-1 and was found to be competitive by nature. These findings provide new mechanistic insights into the therapeutic effect of theophylline in oxidative stress-induced lung pathologies

  3. Mucus and Mucins: do they have a role in the inhibition of the human immunodeficiency virus?

    OpenAIRE

    Mall, Anwar Suleman; Habte, Habtom; Mthembu, Yolanda; Peacocke, Julia; de Beer, Corena

    2017-01-01

    Background Mucins are large O-linked glycosylated proteins which give mucus their gel-forming properties. There are indications that mucus and mucins in saliva, breast milk and in the cervical plug inhibit the human immunodeficiency virus (HIV-1) in an in vitro assay. Main body of abstract Crude mucus gels form continuous layers on the epithelial surfaces of the major internal tracts of the body and protect these epithelial surfaces against aggressive luminal factors such as hydrochloric acid...

  4. Vaginal Lactobacillus Inhibits HIV-1 Replication in Human Tissues Ex Vivo

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    Rogers A. Ñahui Palomino

    2017-05-01

    Full Text Available Lactobacillus species, which dominate vaginal microbiota of healthy reproductive-age women, lower the risks of sexually transmitted infections, including the risk of human immunodeficiency virus (HIV acquisition. The exact mechanisms of this protection remain to be understood. Here, we investigated these mechanisms in the context of human cervico-vaginal and lymphoid tissues ex vivo. We found that all six Lactobacillus strains tested in these systems significantly suppressed HIV type-1 (HIV-1 infection. We identified at least three factors that mediated this suppression: (i Acidification of the medium. The pH of the undiluted medium conditioned by lactobacilli was between 3.8 and 4.6. Acidification of the culture medium with hydrochloric acid (HCl to this pH in control experiments was sufficient to abrogate HIV-1 replication. However, the pH of the Lactobacillus-conditioned medium (CM diluted fivefold, which reached ∼6.9, was also suppressive for HIV-1 infection, while in control experiments HIV-1 infection was not abrogated when the pH of the medium was brought to 6.9 through the use of HCl. This suggested the existence of other factors responsible for HIV-1 inhibition by lactobacilli. (ii Lactic acid. There was a correlation between the concentration of lactic acid in the Lactobacillus-CM and its ability to suppress HIV-1 infection in human tissues ex vivo. Addition of lactic acid isomers D and L to tissue culture medium at the concentration that corresponded to their amount released by lactobacilli resulted in HIV-1 inhibition. Isomer L was produced in higher quantities than isomer D and was mostly responsible for HIV-1 inhibition. These results indicate that lactic acid, in particular its L-isomer, inhibits HIV-1 independently of lowering of the pH. (iii Virucidal effect. Incubation of HIV-1 in Lactobacillus-CM significantly suppressed viral infectivity for human tissues ex vivo. Finally, lactobacilli adsorb HIV-1, serving as a sink

  5. Fatty acid synthase inhibition triggers apoptosis during S phase in human cancer cells.

    Science.gov (United States)

    Zhou, Weibo; Simpson, P Jeanette; McFadden, Jill M; Townsend, Craig A; Medghalchi, Susan M; Vadlamudi, Aravinda; Pinn, Michael L; Ronnett, Gabriele V; Kuhajda, Francis P

    2003-11-01

    C75, an inhibitor of fatty acid synthase (FAS), induces apoptosis in cultured human cancer cells. Its proposed mechanism of action linked high levels of malonyl-CoA after FAS inhibition to potential downstream effects including inhibition of carnitine palmitoyltransferase-1 (CPT-1) with resultant inhibition of fatty acid oxidation. Recent data has shown that C75 directly stimulates CPT-1 increasing fatty acid oxidation in MCF-7 human breast cancer cells despite inhibitory concentrations of malonyl-CoA. In light of these findings, we have studied fatty acid metabolism in MCF7 human breast cancer cells to elucidate the mechanism of action of C75. We now report that: (a) in the setting of increased fatty acid oxidation, C75 inhibits fatty acid synthesis; (b) C273, a reduced form of C75, is unable to inhibit fatty acid synthesis and is nontoxic to MCF7 cells; (c) C75 and 5-(tetradecyloxy)-2-furoic acid (TOFA), an inhibitor of acetyl-CoA carboxylase, both cause a significant reduction of fatty acid incorporation into phosphatidylcholine, the major membrane phospholipid, within 2 h; (d) pulse chase studies with [(14)C]acetate labeling of membrane lipids show that both C75 and TOFA accelerate the decay of (14)C-labeled lipid from membranes within 2 h; (e) C75 also promotes a 2-3-fold increase in oxidation of membrane lipids within 2 h; and (f) because interference with phospholipid synthesis during S phase is known to trigger apoptosis in cycling cells, we performed double-labeled terminal deoxynucleotidyltransferase-mediated nick end labeling and BrdUrd analysis with both TOFA and C75. C75 triggered apoptosis during S phase, whereas TOFA did not. Moreover, application of TOFA 2 h before C75 blocked the C75 induced apoptosis, whereas etomoxir did not. Taken together these data indicate that FAS inhibition and its downstream inhibition of phospholipid production is a necessary part of the mechanism of action of C75. CPT-1 stimulation does not likely play a role in the

  6. Metal inhibition of human alkylpurine-DNA-N-glycosylase activityin base excision repair

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Ping; Guliaev, Anton B.; Hang, Bo

    2006-02-28

    Cadmium (Cd{sup 2+}), nickel (Ni{sup 2+}) and cobalt (Co{sup 2+}) are human and/or animal carcinogens. Zinc (Zn{sup 2+}) is not categorized as a carcinogen, and rather an essential element to humans. Metals were recently shown to inhibit DNA repair proteins that use metals for their function and/or structure. Here we report that the divalent ions Cd{sup 2+}, Ni{sup 2+}, and Zn{sup 2+} can inhibit the activity of a recombinant human N-methylpurine-DNA glycosylase (MPG) toward a deoxyoligonucleotide with ethenoadenine (var epsilonA). MPG removes a variety of toxic/mutagenic alkylated bases and does not require metal for its catalytic activity or structural integrity. At concentrations starting from 50 to 1000 {micro}M, both Cd{sup 2+} and Zn{sup 2+} showed metal-dependent inhibition of the MPG catalytic activity. Ni{sup 2+} also inhibited MPG, but to a lesser extent. Such an effect can be reversed with EDTA addition. In contrast, Co{sup 2+} and Mg{sup 2+} did not inhibit the MPG activity in the same dose range. Experiments using HeLa cell-free extracts demonstrated similar patterns of inactivation of the var epsilonA excision activity by the same metals. Binding of MPG to the substrate was not significantly affected by Cd{sup 2+}, Zn{sup 2+}, and Ni{sup 2+} at concentrations that show strong inhibition of the catalytic function, suggesting that the reduced catalytic activity is not due to altered MPG binding affinity to the substrate. Molecular dynamics (MD) simulations with Zn{sup 2+} showed that the MPG active site has a potential binding site for Zn{sup 2+}, formed by several catalytically important and conserved residues. Metal binding to such a site is expected to interfere with the catalytic mechanism of this protein. These data suggest that inhibition of MPG activity may contribute to metal genotoxicity and depressed repair of alkylation damage by metals in vivo.

  7. [Comparative investigation of the non-histone proteins of chromatin from pigeon erythroblasts and erythrocytes].

    Science.gov (United States)

    Fedina, A B; Gazarian, G G

    1976-01-01

    Chromosomal non-histone proteins are obtained from nuclei of two types of pigeon erythroid cells: erythroblasts (cells active in RNA synthesis) and erythrocytes (cells with repressed RNA synthesis). They are well soluble in solutions of low ionic strength. Electrophoretic separation of the obtained non-histone proteins in polyacrylamide gels with urea and SDS shows the presence of qualitative differences in the pattern of non-histone proteins of chromatine from erythroblasts and erythrocytes. By electrophoresis in urea some protein bands of non-histone proteins of chromatine from erythroblasts were found which disappear with the aging of cells. At the same time two protein fractions were observed in chromatine from erythrocytes which were absent in that of erythroblasts. Disappearance of some high molecular weight protein fractions from erythrocyte chromatine as compared to erythroblasts was observed by separation of the non-histone proteins in the presence of SDS. These fractions of the non-histone proteins disappearing during aging of cells are well extractable from erythroblast chromatine by 0.35 M NaCl solution. In the in vitro system with E. coli RNA polymerase addition of non-histone proteins of chromatine from erythroblasts to chromatine from erythrocytes increases RNA synthesis 2--3 times. At the same time addition of non-histone proteins from erythrocytes is either without any influence on this process or somewhat inhibiting.

  8. Saw palmetto extracts potently and noncompetitively inhibit human alpha1-adrenoceptors in vitro.

    Science.gov (United States)

    Goepel, M; Hecker, U; Krege, S; Rübben, H; Michel, M C

    1999-02-15

    We wanted to test whether phytotherapeutic agents used in the treatment of lower urinary tract symptoms have alpha1-adrenoceptor antagonistic properties in vitro. Preparations of beta-sitosterol and extracts of stinging nettle, medicinal pumpkin, and saw palmetto were obtained from several pharmaceutical companies. They were tested for their ability to inhibit [3H]tamsulosin binding to human prostatic alpha1-adrenoceptors and [3H]prazosin binding to cloned human alpha1A- and alpha1B-adrenoceptors. Inhibition of phenylephrine-stimulated [3H]inositol phosphate formation by cloned receptors was also investigated. Up to the highest concentration which could be tested, preparations of beta-sitosterol, stinging nettle, and medicinal pumpkin were without consistent inhibitory effect in all assays. In contrast, all tested saw palmetto extracts inhibited radioligand binding to human alpha1-adrenoceptors and agonist-induced [3H]inositol phosphate formation. Saturation binding experiments in the presence of a single saw palmetto extract concentration indicated a noncompetitive antagonism. The relationship between active concentrations in vitro and recommended therapeutic doses for the saw palmetto extracts was slightly lower than that for several chemically defined alpha1-adrenoceptor antagonists. Saw palmetto extracts have alpha1-adrenoceptor-inhibitory properties. If bioavailability and other pharmacokinetic properties of these ingredients are similar to those of the chemically defined alpha1-adrenoceptor antagonists, alpha1-adrenoceptor antagonism might be involved in the therapeutic effects of these extracts in patients with lower urinary tract symptoms suggestive of benign prostatic obstruction.

  9. Inhibition of human dendritic cell activation by hydroethanolic but not lipophilic extracts of turmeric (Curcuma longa).

    Science.gov (United States)

    Krasovsky, Joseph; Chang, David H; Deng, Gary; Yeung, Simon; Lee, Mavis; Leung, Ping Chung; Cunningham-Rundles, Susanna; Cassileth, Barrie; Dhodapkar, Madhav V

    2009-03-01

    Turmeric has been extensively utilized in Indian and Chinese medicine for its immune-modulatory properties. Dendritic cells (DCs) are antigen-presenting cells specialized to initiate and regulate immunity. The ability of DCs to initiate immunity is linked to their activation status. The effects of turmeric on human DCs have not been studied. Here we show that hydroethanolic (HEE) but not lipophilic "supercritical" extraction (SCE) of turmeric inhibits the activation of human DCs in response to inflammatory cytokines. Treatment of DCs with HEE also inhibits the ability of DCs to stimulate the mixed lymphocyte reaction (MLR). Importantly, the lipophilic fraction does not synergize with the hydroethanolic fraction for the ability of inhibiting DC maturation. Rather, culturing of DCs with the combination of HEE and SCE leads to partial abrogation of the effects of HEE on the MLR initiated by DCs. These data provide a mechanism for the anti-inflammatory properties of turmeric. However, they suggest that these extracts are not synergistic and may contain components with mutually antagonistic effects on human DCs. Harnessing the immune effects of turmeric may benefit from specifically targeting the active fractions.

  10. Inhibition of adipocytogenesis by canonical WNT signaling in human mesenchymal stem cells

    International Nuclear Information System (INIS)

    Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu

    2011-01-01

    The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPARγ2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of β-catenin protein, inhibited induction of WNT4 and 11 and inhibited adipocytogenesis. In contrast, knockdown of β-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.

  11. Antiviral activity of human lactoferrin: inhibition of alphavirus interaction with heparan sulfate

    International Nuclear Information System (INIS)

    Waarts, Barry-Lee; Aneke, Onwuchekwa J.C.; Smit, Jolanda M.; Kimata, Koji; Bittman, Robert; Meijer, Dirk K.F.; Wilschut, Jan

    2005-01-01

    Human lactoferrin is a component of the non-specific immune system with distinct antiviral properties. We used alphaviruses, adapted to interaction with heparan sulfate (HS), as a tool to investigate the mechanism of lactoferrin's antiviral activity. Lactoferrin inhibited infection of BHK-21 cells by HS-adapted, but not by non-adapted, Sindbis virus (SIN) or Semliki Forest virus (SFV). Lactoferrin also inhibited binding of radiolabeled HS-adapted viruses to BHK-21 cells or liposomes containing lipid-conjugated heparin as a receptor analog. On the other hand, low-pH-induced fusion of the viruses with liposomes, which occurs independently of virus-receptor interaction, was unaffected. Studies involving preincubation of virus or cells with lactoferrin suggested that the protein does not bind to the virus, but rather blocks HS-moieties on the cell surface. Charge-modified human serum albumin, with a net positive charge, had a similar antiviral effect against HS-adapted SIN and SFV, suggesting that the antiviral activity of lactoferrin is related to its positive charge. It is concluded that human lactoferrin inhibits viral infection by interfering with virus-receptor interaction rather than by affecting subsequent steps in the viral cell entry or replication processes

  12. Opposite responses of rabbit and human globin mRNAs to translational inhibition by cap analogues

    International Nuclear Information System (INIS)

    Shakin, S.H.; Liebhaber, S.A.

    1987-01-01

    The translational efficiency of an mRNA may be determined at the step of translational initiation by the efficiency of its interaction with the cap binding protein complex. To further investigate the role of these interactions in translational control, the authors compare in vitro the relative sensitivities of rabbit and human α- and β-globin mRNAs to translational inhibition by cap analogues. They find that rabbit β-globin mRNA is more resistant to translational inhibition by cap analogues than rabbit α-globin mRNA, while in contrast, human β-globin mRNA is more sensitive to cap analogue inhibition than human α- and β-globin mRNAs is unexpected as direct in vivo and in vitro comparisons of polysome profiles reveal parallel translational handling of the α- and β-globin mRNAs from these two species. This discordance between the relative translational sensitivities of these mRNAs to cap analogues and their relative ribosome loading activities suggests that cap-dependent events may not be rate limiting in steady-state globin translation

  13. Inhibition of radio cobalt uptake by human bone powder using Mg and Ni

    International Nuclear Information System (INIS)

    Abdel Fattah, A.T.A.; Mohamed, S.A.

    1992-01-01

    Human bone powder samples of 30 - 40 Μ in diameter were prepared from human bone femurs as fat free (FFB), protein free (PEB) or left untreated as a raw bone powder (RB). The uptake of 60 Co by these types of bone powder took the sequence : PFB > FFB> RB. Stable ions of magnesium and nickel exhibit an inhibition or competing effect on the uptake process of 60 Co. The competing effect did not disturb the uptake sequence. The competing effect of nickel was higher than magnesium

  14. Exercise and IL-6 infusion inhibit endotoxin-induced TNF-alpha production in humans

    DEFF Research Database (Denmark)

    Starkie, Rebecca; Ostrowski, Sisse Rye; Jauffred, Sune

    2003-01-01

    and atherosclerosis. To test this hypothesis, we performed three experiments in which eight healthy males either rested (CON), rode a bicycle for 3 h (EX), or were infused with recombinant human IL-6 (rhIL-6) for 3 h while they rested. After 2.5 h, the volunteers received a bolus of Escherichia coli...... exercise and rhIL-6 infusion at physiological concentrations inhibit endotoxin-induced TNF-alpha production in humans. Hence, these data provide the first experimental evidence that physical activity mediates antiinflammatory activity and suggest that the mechanism include IL-6, which is produced...

  15. Tuning SERS for living erythrocytes

    DEFF Research Database (Denmark)

    Brazhe, Nadezda; Parshina, E.Y.; Khabanova, V.V.

    2013-01-01

    Surface-enhanced Raman spectroscopy (SERS) is a unique technique to study submembrane hemoglobin (Hbsm) in erythrocytes. We report the detailed design of SERS experiments on living erythrocytes to estimate dependence of the enhancemen t factor for main Raman bands of Hbsm on silver nanoparticle (Ag......NP) properties. We demonstrate that the enhancement factor for 4/A1g, 10/B1g and A2g Raman bands of Hbsm varies from 105 to 107 under proposed experimental conditions with 473 nm laser excitation. For the first time we show that the enhancement of Raman scattering increases with the increase in the relative...... between small AgNPs and Hbsm and, consequently, leads to the higher enhancement of Raman scattering of Hbsm. The enhancement of higher wavenumber bands 10/B1g and A2g is more sensitive to AgNPs' size and the relative amount of small AgNPs than the enhancement of the lower wavenumber band 4/A1g. This can...

  16. Invasion of erythrocytes by Babesia bovis

    NARCIS (Netherlands)

    Gaffar, Fasila Razzia

    2004-01-01

    In this thesis we investigated the invasion of erythrocytes taking place during the asexual erythrocytic blood stage of the apicomplexan parasites Babesia bovis parasite. Host cell invasion by apicomplexan parasites is a complex process requiring multiple receptor-ligand interactions, involving

  17. Enzymatic assay for methotrexate in erythrocytes

    DEFF Research Database (Denmark)

    Schrøder, H; Heinsvig, E M

    1985-01-01

    Methotrexate (MTX) accumulates in erythrocytes in MTX-treated patients. We present a modified enzymatic assay measuring MTX concentrations between 10 and 60 nmol/l in erythrocytes, adapted for a centrifugal analyser (Cobas Bio). About 40 patient's samples could be analysed within 1 h. The detection...

  18. Molecular mechanisms of erythrocyte photo-irradiation

    International Nuclear Information System (INIS)

    Ferreira, W.T.; Souza, M.C.

    1985-01-01

    The role of singlet oxygen and the lipid peroxidation of erythrocyte membrane are studied. The irradiation of erythrocytes with visible light in the presence of a photodynamic mediator (toluidine blue) is reported. A system of light application by optical fiber, connected to a catheter is suggested for local instillation of the photosensitizing agent. (M.A.C.) [pt

  19. Restoration of adenosine deaminase-deficient human thymocyte development in vitro by inhibition of deoxynucleoside kinases.

    Science.gov (United States)

    Joachims, Michelle L; Marble, Patrick A; Laurent, Aletha B; Pastuszko, Peter; Paliotta, Marco; Blackburn, Michael R; Thompson, Linda F

    2008-12-01

    Mutations in the gene encoding adenosine deaminase (ADA), a purine salvage enzyme, lead to immunodeficiency in humans. Although ADA deficiency has been analyzed in cell culture and murine models, information is lacking concerning its impact on the development of human thymocytes. We have used chimeric human/mouse fetal thymic organ culture to study ADA-deficient human thymocyte development in an "in vivo-like" environment where toxic metabolites accumulate in situ. Inhibition of ADA during human thymocyte development resulted in a severe reduction in cellular expansion as well as impaired differentiation, largely affecting mature thymocyte populations. Thymocyte differentiation was not blocked at a discrete stage; rather, the paucity of mature thymocytes was due to the induction of apoptosis as evidenced by activation of caspases and was accompanied by the accumulation of intracellular dATP. Inhibition of adenosine kinase and deoxycytidine kinase prevented the accumulation of dATP and restored thymocyte differentiation and proliferation. Our work reveals that multiple deoxynucleoside kinases are involved in the phosphorylation of deoxyadenosine when ADA is absent, and suggests an alternate therapeutic strategy for treatment of ADA-deficient patients.

  20. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    Directory of Open Access Journals (Sweden)

    Yongjun Yin

    2016-05-01

    Full Text Available Activating mutations in fibroblast growth factor receptor 3 (FGFR3 have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9, a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11 with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3.

  1. FLAX OIL FROM TRANSGENIC LINUM USITATISSIMUM SELECTIVELY INHIBITS IN VITRO PROLIFERATION OF HUMAN CANCER CELL LINES.

    Science.gov (United States)

    Gebarowski, Tomasz; Gebczak, Katarzyna; Wiatrak, Benita; Kulma, Anna; Pelc, Katarzyna; Czuj, Tadeusz; Szopa, Jan; Gasiorowski, Kazimierz

    2017-03-01

    Emulsions made of oils from transgenic flaxseeds significantly decreased in vitro proliferation of six tested human cancer cell lines in 48-h cultures, as assessed with the standard sulforhodamine assay. However, the emulsions also increased proliferation rate of normal human dermal fibroblasts and, to a lower extend, of keratinocytes. Both inhibition of in vitro proliferation of human cancer cell lines and stimulation of proliferation of normal dermal fibroblasts and keratinocytes were especially strong with the emulsion type B and with emulsion type M. Oils from seeds of transgenic flax type B and M should be considered as valuable adjunct to standard cytostatic therapy of human cancers and also could be applied to improve the treatment of skin lesions in wound healing.

  2. Oxytetracycline Inhibits Mucus Secretion and Inflammation in Human Airway Epithelial Cells.

    Science.gov (United States)

    Shah, Said Ahmad; Ishinaga, Hajime; Takeuchi, Kazuhiko

    2017-01-01

    Oxytetracycline is a broad-spectrum antibiotic, but its nonantibacterial effects in the human respiratory tract are unknown. In this study, the effects of oxytetracycline on mucus secretion and inflammation were examined by PCR and ELISA in the human airway epithelial cell line NCI-H292. Oxytetracycline (10 μg/mL) significantly inhibited TNF-α-induced MUC5AC gene expression and MUC5AC protein levels in NCI-H292 cells. It also downregulated IL-8 and IL-1β gene expression and IL-1β protein levels. Our findings demonstrated that oxytetracycline suppressed mucus production and inflammation in human respiratory epithelial cells, providing further evidence for the usefulness of oxytetracycline for human airway inflammatory diseases. © 2017 S. Karger AG, Basel.

  3. Biophysics of malarial parasite exit from infected erythrocytes.

    Science.gov (United States)

    Chandramohanadas, Rajesh; Park, YongKeun; Lui, Lena; Li, Ang; Quinn, David; Liew, Kingsley; Diez-Silva, Monica; Sung, Yongjin; Dao, Ming; Lim, Chwee Teck; Preiser, Peter Rainer; Suresh, Subra

    2011-01-01

    Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.

  4. An evaluation of the inhibition of human butyrylcholinesterase and acetylcholinesterase by the organophosphate chlorpyrifos oxon

    International Nuclear Information System (INIS)

    Shenouda, Josephine; Green, Paula; Sultatos, Lester

    2009-01-01

    Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) are enzymes that belong to the superfamily of α/β-hydrolase fold proteins. While they share many characteristics, they also possess many important differences. For example, whereas they have about 54% amino acid sequence identity, the active site gorge of acetylcholinesterase is considerably smaller than that of butyrylcholinesterase. Moreover, both have been shown to display simple and complex kinetic mechanisms, depending on the particular substrate examined, the substrate concentration, and incubation conditions. In the current study, incubation of butyrylthiocholine in a concentration range of 0.005-3.0 mM, with 317 pM human butyrylcholinesterase in vitro, resulted in rates of production of thiocholine that were accurately described by simple Michaelis-Menten kinetics, with a K m of 0.10 mM. Similarly, the inhibition of butyrylcholinesterase in vitro by the organophosphate chlorpyrifos oxon was described by simple Michaelis-Menten kinetics, with a k i of 3048 nM -1 h -1 , and a K D of 2.02 nM. In contrast to inhibition of butyrylcholinesterase, inhibition of human acetylcholinesterase by chlorpyrifos oxon in vitro followed concentration-dependent inhibition kinetics, with the k i increasing as the inhibitor concentration decreased. Chlorpyrifos oxon concentrations of 10 and 0.3 nM gave k i s of 1.2 and 19.3 nM -1 h -1 , respectively. Although the mechanism of concentration-dependent inhibition kinetics is not known, the much smaller, more restrictive active site gorge of acetylcholinesterase almost certainly plays a role. Similarly, the much larger active site gorge of butyrylcholinesterase likely contributes to its much greater reactivity towards chlorpyrifos oxon, compared to acetylcholinesterase.

  5. [RITA combined with temozolomide inhibits the proliferation of human glioblastoma U87 cells].

    Science.gov (United States)

    He, Xiao-Yan; Feng, Xiao-Li; Song, Xin-Pei; Zeng, Huan-Chao; Cao, Zhong-Xu; Xiao, Wei-Wei; Zhang, Bao; Wu, Qing-Hua

    2016-10-20

    To observe the effect of RITA, a small molecule that targets p53, combined with temozolomide (TMZ) on proliferation, colony formation and apoptosis of human glioblastoma U87 cells and explore the underlying mechanism. Cultured U87 cells were treated with RITA (1, 5, 10, 20 µmol/L), TMZ, or RITA+TMZ (half dose) for 24, 48 or 72 h. MTS assay were used to detect the cell proliferation, and the cell proliferation rate and inhibitory rate were calculated. The effect of combined treatments was evaluated by the q value. The expressions of p53, p21 and other apoptosis-associated genes were detected by qRT-PCR and Western blotting; cell apoptosis was assayed using flow cytometry with Annexin V/PI double staining; colony formation of the cells was detected with crystal violet staining. MTS assay showed that RITA at the 4 doses more potently inhibited U87 cell viability than TMZ at 72 h (P=0.000) with inhibitory rates of 25.94%-41.38% and 3.84%-8.20%, respectively. RITA combined with TMZ caused a more significant inhibition of U87 cells (29.21%-52.11%) than RITA (PRITA+TMZ for 48 h resulted in q values exceeding 1.2 and showed an obvious synergistic effect of the drugs. Both RITA and TMZ, especially the latter, significantly increased the expressions of p53, p21, puma, and other apoptosis-associated genes to accelerate apoptosis and inhibit the growth and colony formation of U87 cells, and the effect was more obvious with a combined treatment. RITA inhibits the growth of human glioblastoma cells and enhance their sensitivity to TMZ by up-regulating p53 expression, and when combined, RITA and TMZ show a synergistic effect to cause a stronger cell inhibition.

  6. Inhibition of Human Cytomegalovirus pUL89 Terminase Subunit Blocks Virus Replication and Genome Cleavage.

    Science.gov (United States)

    Wang, Yan; Mao, Lili; Kankanala, Jayakanth; Wang, Zhengqiang; Geraghty, Robert J

    2017-02-01

    The human cytomegalovirus terminase complex cleaves concatemeric genomic DNA into unit lengths during genome packaging and particle assembly. This process is an attractive drug target because cleavage of concatemeric DNA is not required in mammalian cell DNA replication, indicating that drugs targeting the terminase complex could be safe and selective. One component of the human cytomegalovirus terminase complex, pUL89, provides the endonucleolytic activity for genome cleavage, and the domain responsible is reported to have an RNase H-like fold. We hypothesize that the pUL89 endonuclease activity is inhibited by known RNase H inhibitors. Using a novel enzyme-linked immunosorbent assay (ELISA) format as a screening assay, we found that a hydroxypyridonecarboxylic acid compound, previously reported to be an inhibitor of human immunodeficiency virus RNase H, inhibited pUL89 endonuclease activity at low-micromolar concentrations. Further characterization revealed that this pUL89 endonuclease inhibitor blocked human cytomegalovirus replication at a relatively late time point, similarly to other reported terminase complex inhibitors. Importantly, this inhibitor also prevented the cleavage of viral genomic DNA in infected cells. Taken together, these results substantiate our pharmacophore hypothesis and validate our ligand-based approach toward identifying novel inhibitors of pUL89 endonuclease. Human cytomegalovirus infection in individuals lacking a fully functioning immune system, such as newborns and transplant patients, can have severe and debilitating consequences. The U.S. Food and Drug Administration-approved anti-human cytomegalovirus drugs mainly target the viral polymerase, and resistance to these drugs has appeared. Therefore, anti-human cytomegalovirus drugs from novel targets are needed for use instead of, or in combination with, current polymerase inhibitors. pUL89 is a viral ATPase and endonuclease and is an attractive target for anti-human cytomegalovirus

  7. Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Heldens, Genoveva T H; Blaney Davidson, Esmeralda N; Vitters, Elly L; Schreurs, B Willem; Piek, Ester; van den Berg, Wim B; van der Kraan, Peter M

    2012-01-01

    Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.

  8. Inhibition of interferon production in human fibroblasts by a tumor promoting phorbol ester

    International Nuclear Information System (INIS)

    Frankfort, H.M.; Vilcek, J.

    1982-01-01

    The effect of 12-0-tetradecanoylphorbol-13-acetate (TPA) on the induction of interferon in cultures of human fibroblasts was examined. TPA was found to inhibit polyinosinate-polycytidylate [poly(I) X poly(C)]-induced interferon production when added either before or with the inducer. A 3-hour pretreatment of FS-4 cells with TPA produced the greatest ihibitory effect. Partially inhibitory treatments with TPA caused a delay in interferon production. On the other hand, interferon yields were slightly enhanced by TPA added at 1 1/2 or 3 hours postinduction. No gross metabolic perturbations (e.g., inhibition of cellular protein or RNA synthesis) were detected which would explain the phenomenon. The inhibition of interferon production was a stereospecific event: biologically inactive derivatives of TPA (4-0-methyl TPA, 4-α-phorbol-12, 13-didecanoate and phorbol-12, 13-diacetate) had no effect on interferon production. Cellular proteases or nucleases did not appear to be involved in this process. The binding of labeled poly(I) X poly(C) to FS-4 cells was unaltered in TPA-treated cultures. In superinduced cultures (i.e., after enhancement of interferon yields by actinomycin D and cycloheximide), interferon production was generally less inhibited by TPA than after simple induction. Newcastle disease virus (NDV)-induced interferon synthesis in GM-258 cells was also inhibited by the phorbol ester. Both α (leukocyte) and β (fibroblast) interferon production was inhibited to a similar degree in TPA-treated cells inoculated with 0.1 or 1 plaque forming unit (PFU) of NDV per cell. Increasing the multiplicity of infection with NDV to 10 PFU per cell overcame the inhibitory action of TPA. We conclude that the site of TPA action is either the triggering (generation of the hypothetical inducing signal) or transcription of the interferom mRNA. (Author)

  9. Baicalein inhibits the migration and invasive properties of human hepatoma cells

    International Nuclear Information System (INIS)

    Chiu, Yung-Wei; Lin, Tseng-Hsi; Huang, Wen-Shih; Teng, Chun-Yuh; Liou, Yi-Sheng; Kuo, Wu-Hsien; Lin, Wea-Lung; Huang, Hai-I; Tung, Jai-Nien; Huang, Chih-Yang; Liu, Jer-Yuh; Wang, Wen-Hung; Hwang, Jin-Ming

    2011-01-01

    Flavonoids have been demonstrated to exert health benefits in humans. We investigated whether the flavonoid baicalein would inhibit the adhesion, migration, invasion, and growth of human hepatoma cell lines, and we also investigated its mechanism of action. The separate effects of baicalein and baicalin on the viability of HA22T/VGH and SK-Hep1 cells were investigated for 24 h. To evaluate their invasive properties, cells were incubated on matrigel-coated transwell membranes in the presence or absence of baicalein. We examined the effect of baicalein on the adhesion of cells, on the activation of matrix metalloproteinases (MMPs), protein kinase C (PKC), and p38 mitogen-activated protein kinase (MAPK), and on tumor growth in vivo. We observed that baicalein suppresses hepatoma cell growth by 55%, baicalein-treated cells showed lower levels of migration than untreated cells, and cell invasion was significantly reduced to 28%. Incubation of hepatoma cells with baicalein also significantly inhibited cell adhesion to matrigel, collagen I, and gelatin-coated substrate. Baicalein also decreased the gelatinolytic activities of the matrix metalloproteinases MMP-2, MMP-9, and uPA, decreased p50 and p65 nuclear translocation, and decreased phosphorylated I-kappa-B (IKB)-β. In addition, baicalein reduced the phosphorylation levels of PKCα and p38 proteins, which regulate invasion in poorly differentiated hepatoma cells. Finally, when SK-Hep1 cells were grown as xenografts in nude mice, intraperitoneal (i.p.) injection of baicalein induced a significant dose-dependent decrease in tumor growth. These results demonstrate the anticancer properties of baicalein, which include the inhibition of adhesion, invasion, migration, and proliferation of human hepatoma cells in vivo. - Highlight: → Baicalein inhibits several essential steps in the onset of metastasis.

  10. Erythrocytes in muscular dystrophy. Investigation with 31P nuclear magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Sarpel, G.; Lubansky, H.J.; Danon, M.J.; Omachi, A.

    1981-01-01

    Phosphorus 31 nuclear magnetic resonance ( 31 P NMR) signals were recorded from intact human erythrocytes for 16 hours. Total phosphate concentration, which was estimated as the sum of the individual 31 P signals, was 25% lower in erythrocytes from men with myotonic dystrophy than in control erythrocytes. The inorganic-phosphate fraction contained the highest average phosphate concentration over the 16-hour period, and made the major contribution to the difference in total phosphate between the two groups. This result was not observed in erythrocytes from either women with myotonic dystrophy or patients with Duchenne's dystrophy and may be due to a change in cell membrane permeability to inorganic phosphate, which leads to lower steady-state concentrations of the intracellular phosphates

  11. Erythrocytes in muscular dystrophy. Investigation with 31P nuclear magnetic resonance spectroscopy

    International Nuclear Information System (INIS)

    Sarpel, G.; Lubansky, H.J.; Danon, M.J.; Omachi, A.

    1981-01-01

    Phosphorus 31 nuclear magnetic resonance (31P NMR) signals were recorded from intact human erythrocytes for 16 hours. Total phosphate concentration, which was estimated as the sum of the individual 31P signals, was 25% lower in erythrocytes from men with myotonic dystrophy than in control erythrocytes. The inorganic-phosphate fraction contained the highest average phosphate concentration over the 16-hour period, and made the major contribution to the difference in total phosphate between the two groups. This result was not observed in erythrocytes from either women with myotonic dystrophy or patients with Duchenne's dystrophy and may be due to a change in cell membrane permeability to inorganic phosphate, which lead to lower steady-state concentrations of the intracellular phosphates

  12. miR-141-3p inhibits human stromal (mesenchymal) stem cell proliferation and differentiation

    DEFF Research Database (Denmark)

    Qiu, Weimin; Kassem, Moustapha

    2014-01-01

    Wnt signaling determines human stromal (mesenchymal) stem cell (hMSC) differentiation fate into the osteoblast or adipocyte lineage. microRNAs (miRNAs) are small RNA molecules of 21-25 nucleotides that regulate many aspects of osteoblast biology. Thus, we examined miRNAs regulated by Wnt signaling...... in hMSC. We identified miRNA (miR)-141-3p as a Wnt target which in turn inhibited Wnt signaling. Moreover, miR-141-3p inhibited hMSC proliferation by arresting cells at the G1 phase of the cell cycle. miR-141-3p inhibited osteoblast differentiation of hMSC as evidenced by reduced alkaline phosphatase...... activity, gene expression and in vitro mineralized matrix formation. Bioinformatic studies, Western blot analysis and 3'UTR reporter assay demonstrated that cell division cycle 25A (CDC25A) is a direct target of miR-141-3p. siRNA-mediated knock-down of CDC25A inhibited hMSC proliferation and osteoblast...

  13. Luteolin, a flavonoid, inhibits CD40 ligand expression by activated human basophils.

    Science.gov (United States)

    Hirano, Toru; Arimitsu, Junsuke; Higa, Shinji; Naka, Tetsuji; Ogata, Atsushi; Shima, Yoshihito; Fujimoto, Minoru; Yamadori, Tomoki; Ohkawara, Tomoharu; Kuwabara, Yusuke; Kawai, Mari; Kawase, Ichiro; Tanaka, Toshio

    2006-01-01

    We have previously shown that flavonoids such as luteolin, apigenin and fisetin inhibit interleukin 4 and interleukin 13 production. In this study, we investigated whether luteolin can suppress CD40 ligand expression by basophils. A human basophilic cell line, KU812, was stimulated with A23187 and phorbol myristate acetate (PMA) with or without various concentrations of luteolin or other flavonoids for 12 h, and CD40 ligand expression was analyzed by FACS. The effect of luteolin on CD40 ligand mRNA expression was studied by semiquantitative reverse transcription PCR analysis. In addition, CD40 ligand expression was also measured in purified basophils that had been stimulated for 12 h with A23187 plus PMA with or without various concentrations of luteolin. CD40 ligand expression by KU812 cells was enhanced noticeably in response to A23187 and even more strikingly augmented by A23187 plus PMA. The expression was significantly suppressed by 10 or 30 microM of luteolin, whereas myricetin failed to inhibit. Reverse transcription PCR analyses demonstrated that luteolin inhibited CD40 ligand mRNA expression by stimulated KU812 cells. Of the six flavonoids examined, luteolin, apigenin, fisetin and quercetin at 30 microM showed a significant inhibitory effect on CD40 ligand expression. The incubation of purified basophils with A23187 plus PMA significantly enhanced CD40 ligand expression, and the presence of luteolin again had an inhibitory effect. Luteolin inhibits CD40 ligand expression by activated basophils.

  14. Aminomethylphosphonic acid inhibits growth and metastasis of human prostate cancer in an orthotopic xenograft mouse model.

    Science.gov (United States)

    Parajuli, Keshab Raj; Zhang, Qiuyang; Liu, Sen; You, Zongbing

    2016-03-01

    Aminomethylphosphonic acid (AMPA) has been shown to inhibit prostate cancer cell growth in vitro. The purpose of the present study was to determine if AMPA could inhibit growth and metastasis of prostate cancer in vivo. Human prostate cancer PC-3-LacZ-luciferase cells were implanted into the ventral lateral lobes of the prostate in 39 athymic Nu/Nu nude male mice. Seven days later, mice were randomized into the control group (n = 14, treated intraperitoneally with phosphate buffered saline), low dose group (n = 10, treated intraperitoneally with AMPA at 400 mg/kg body weight/day), and high dose group (n = 15, treated intraperitoneally with AMPA at 800 mg/kg body weight/day). Tumor growth and metastasis were examined every 4-7 days by bioluminescence imaging of live mice. We found that AMPA treatment significantly inhibited growth and metastasis of orthotopic xenograft prostate tumors and prolonged the survival time of the mice. AMPA treatment decreased expression of BIRC2 and activated caspase 3, leading to increased apoptosis in the prostate tumors. AMPA treatment decreased expression of cyclin D1. AMPA treatment also reduced angiogenesis in the prostate tumors. Taken together, these results demonstrate that AMPA can inhibit prostate cancer growth and metastasis, suggesting that AMPA may be developed into a therapeutic agent for the treatment of prostate cancer.

  15. Susceptibility of human head and neck cancer cells to combined inhibition of glutathione and thioredoxin metabolism.

    Directory of Open Access Journals (Sweden)

    Arya Sobhakumari

    Full Text Available Increased glutathione (GSH and thioredoxin (Trx metabolism are mechanisms that are widely implicated in resistance of cancer cells to chemotherapy. The current study determined if simultaneous inhibition of GSH and Trx metabolism enhanced cell killing of human head and neck squamous cell carcinoma (HNSCC cells by a mechanism involving oxidative stress. Inhibition of GSH and Trx metabolism with buthionine sulfoximine (BSO and auranofin (AUR, respectively, induced significant decreases in clonogenic survival compared to either drug alone in FaDu, Cal-27 and SCC-25 HNSCC cells in vitro and in vivo in Cal-27 xenografts. BSO+AUR significantly increased glutathione and thioredoxin oxidation and suppressed peroxiredoxin activity in vitro. Pre-treatment with N-acetylcysteine completely reversed BSO+AUR-induced cell killing in FaDu and Cal-27 cells, while catalase and selenium supplementation only inhibited BSO+AUR-induced cell killing in FaDu cells. BSO+AUR decreased caspase 3/7 activity in HNSCC cells and significantly reduced the viability of both Bax/Bak double knockout (DKO and DKO-Bax reconstituted hematopoietic cells suggesting that necrosis was involved. BSO+AUR also significantly sensitized FaDu, Cal-27, SCC-25 and SQ20B cells to cell killing induced by the EGFR inhibitor Erlotinib in vitro. These results support the conclusion that simultaneous inhibition of GSH and Trx metabolism pathways induces oxidative stress and clonogenic killing in HNSCCs and this strategy may be useful in sensitizing HNSCCs to EGFR inhibitors.

  16. L-sorbose but not D-tagatose induces hemolysis of dog erythrocytes in vitro

    NARCIS (Netherlands)

    Bär, A.; Leeman, W.R.

    1999-01-01

    Previous investigations have demonstrated that L-sorbose induces hemolysis of dog erythrocytes. This effect is probably the consequence of an ATP depletion of the red blood cells subsequent to inhibition of hexokinase, and thus the glycolytic pathway, by sorbose 1-phosphate. In the present study,

  17. Rapamycin causes growth arrest and inhibition of invasion in human chondrosarcoma cells.

    Science.gov (United States)

    Song, Jian; Wang, Xiaobo; Zhu, Jiaxue; Liu, Jun

    2016-01-01

    Chondrosarcoma is a highly malignant tumor that is characterized by a potent capacity to invade locally and cause distant metastasis and notable for its lack of response to conventional chemotherapy or radiotherapy. Rapamycin, the inhibitor of mammalian target of rapamycin (mTOR), is a valuable drug with diverse clinical applications and regulates many cellular processes. However, the effects of rapamycin on cell growth and invasion of human chondrosarcoma cells are not well known. We determined the effect of rapamycin on cell proliferation, cell cycle arrest and invasion by using MTS, flow cytometry and invasion assays in two human chondrosarcoma cell lines, SW1353 and JJ012. Cell cycle regulatory and invasion-related genes' expression analysis was performed by quantitative RT-PCR (qRT-PCR). We also evaluated the effect of rapamycin on tumor growth by using mice xenograph models. Rapamycin significantly inhibited the cell proliferation, induced cell cycle arrest and decreased the invasion ability of human chondrosarcoma cells. Meanwhile, rapamycin modulated the cell cycle regulatory and invasion-related genes' expression. Furthermore, the tumor growth of mice xenograph models with human chondrosarcoma cells was significantly inhibited by rapamycin. These results provided further insight into the role of rapamycin in chondrosarcoma. Therefore, rapamycin targeted therapy may be a potential treatment strategy for chondrosarcoma.

  18. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    Science.gov (United States)

    2011-01-01

    Background Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT. PMID:21435270

  19. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes.

    Science.gov (United States)

    Tsai, Wei-Jern; Chang, Chu-Ting; Wang, Guei-Jane; Lee, Tzong-Huei; Chang, Shwu-Fen; Lu, Shao-Chun; Kuo, Yuh-Chi

    2011-03-25

    Arctium lappa (Niubang), a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC), isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2) and interferon-γ (IFN-γ) production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

  20. Arctigenin from Arctium lappa inhibits interleukin-2 and interferon gene expression in primary human T lymphocytes

    Directory of Open Access Journals (Sweden)

    Chang Shwu-Fen

    2011-03-01

    Full Text Available Abstract Background Arctium lappa (Niubang, a Chinese herbal medicine, is used to treat tissue inflammation. This study investigates the effects of arctigenin (AC, isolated from A. lappa, on anti-CD3/CD28 Ab-stimulated cell proliferation and cytokine gene expression in primary human T lymphocytes. Methods Cell proliferation was determined with enzyme immunoassays and the tritiated thymidine uptake method. Cytokine production and gene expression were analyzed with reverse transcription-polymerase chain reaction. Results AC inhibited primary human T lymphocytes proliferation activated by anti-CD3/CD28 Ab. Cell viability test indicated that the inhibitory effects of AC on primary human T lymphocyte proliferation were not due to direct cytotoxicity. AC suppressed interleukin-2 (IL-2 and interferon-γ (IFN-γ production in a concentration-dependent manner. Furthermore, AC decreased the IL-2 and IFN-γ gene expression in primary human T lymphocytes induced by anti-CD3/CD28 Ab. Reporter gene analyses revealed that AC decreased NF-AT-mediated reporter gene expression. Conclusion AC inhibited T lymphocyte proliferation and decreased the gene expression of IL-2, IFN-γ and NF-AT.

  1. A-Type Cranberry Proanthocyanidins Inhibit the RANKL-Dependent Differentiation and Function of Human Osteoclasts

    Directory of Open Access Journals (Sweden)

    Amy B. Howell

    2011-03-01

    Full Text Available This study investigated the effect of A-type cranberry proanthocyanidins (AC-PACs on osteoclast formation and bone resorption activity. The differentiation of human pre-osteoclastic cells was assessed by tartrate-resistant acid phosphatase (TRAP staining, while the secretion of interleukin-8 (IL-8 and matrix metalloproteinases (MMPs was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen helical peptides. AC-PACs up to 100 µg/mL were atoxic for osteoclastic cells. TRAP staining evidenced a dose-dependent inhibition of osteoclastogenesis. More specifically, AC-PACs at 50 µg/mL caused a 95% inhibition of RANKL-dependent osteoclast differentiation. This concentration of AC-PACs also significantly increased the secretion of IL-8 (6-fold and inhibited the secretion of both MMP-2 and MMP-9. Lastly, AC-PACs (10, 25, 50 and 100 µg/ml affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. This study suggests that AC-PACs can interfere with osteoclastic cell maturation and physiology as well as prevent bone resorption. These compounds may be considered as therapeutic agents for the prevention and treatment of periodontitis.

  2. Effects of Mitochondrial Uncoupling Protein 2 Inhibition by Genipin in Human Cumulus Cells

    Directory of Open Access Journals (Sweden)

    Hongshan Ge

    2015-01-01

    Full Text Available UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality.

  3. Descriptive parameters of the erythrocyte aggregation phenomenon using a laser transmission optical chip

    Science.gov (United States)

    Toderi, Martín A.; Castellini, Horacio V.; Riquelme, Bibiana D.

    2017-01-01

    The study of red blood cell (RBC) aggregation is of great interest because of its implications for human health. Altered RBC aggregation can lead to microcirculatory problems as in vascular pathologies, such as hypertension and diabetes, due to a decrease in the erythrocyte surface electric charge and an increase in the ligands present in plasma. The process of erythrocyte aggregation was studied in stasis situation (free shear stresses), using an optical chip based on the laser transmission technique. Kinetic curves of erythrocyte aggregation under different conditions were obtained, allowing evaluation and characterization of this process. Two main characteristics of blood that influence erythrocyte aggregation were analyzed: the erythrocyte surface anionic charge (EAC) after digestion with the enzyme trypsin and plasmatic protein concentration in suspension medium using plasma dissolutions in physiological saline with human albumin. A theoretical approach was evaluated to obtain aggregation and disaggregation ratios by syllectograms data fitting. Sensible parameters (Amp100, t) regarding a reduced erythrocyte EAC were determined, and other parameters (AI, M-Index) resulted that are representative of a variation in the plasmatic protein content of the suspension medium. These results are very useful for further applications in biomedicine.

  4. Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

    International Nuclear Information System (INIS)

    Di Consiglio, Emma; Meneguz, Annarita; Testai, Emanuela

    2005-01-01

    The drug-toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2-400 μM) and OPTs (≤10 μM) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25-2.5 μM). The OPTs, having lower K m values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7-1.1 μM) or with HLM rich in 1A2-related activity (2-10.8 μM). The K i values (2-14 μM), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs)

  5. Quantitative evaluation of respiration induced metabolic oscillations in erythrocytes

    DEFF Research Database (Denmark)

    Hald, Bjørn; Madsen, Mads F; Danø, Sune

    2009-01-01

    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...... 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...

  6. Inhibition of acrolein-stimulated MUC5AC production by fucoidan in human bronchial epithelial cells.

    Science.gov (United States)

    Pokharel, Yuba Raj; Yoon, Se Young; Kim, Sang Kyum; Li, Jian-Dong; Kang, Keon Wook

    2008-10-01

    Fucoidan, a marine sulfated polysaccharide has both antithrombotic and anti-inflammatory effects. We determined the effect of fucoidan on MUC5AC expression in a human bronchial epithelial cell line, NCI-H292. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that fucoidan inhibited MUC5AC expression and protein secretion in cells stimulated with acrolein, a toxic aldehyde present in tobacco smoke. The activation of both nuclear factor-kappa B (NF-kappa B) and activator protein 1 (AP-1) are key steps in the transcriptional activation of MUC5AC. We found that the acrolein-mediated transactivation of MUC5AC was selectively dependent on AP-1 activation and was suppressed by fucoidan. Fucoidan-induced AP-1 inhibition and MUC5AC repression might be associated with fucoidan's protective effects against respiratory diseases.

  7. Progranulin Inhibits Human T Lymphocyte Proliferation by Inducing the Formation of Regulatory T Lymphocytes

    Directory of Open Access Journals (Sweden)

    Kyu Hwan Kwack

    2017-01-01

    Full Text Available We have examined the effect of progranulin (PGRN on human T cell proliferation and its underlying mechanism. We show that PGRN inhibits the PHA-induced multiplication of T lymphocytes. It increases the number of iTregs when T lymphocytes are activated by PHA but does not do so in the absence of PHA. PGRN-mediated inhibition of T lymphocyte proliferation, as well as the induction of iTregs, was completely reversed by a TGF-β inhibitor or a Treg inhibitor. PGRN induced TGF-β secretion in the presence of PHA whereas it did not in the absence of PHA. Our findings indicate that PGRN suppresses T lymphocyte proliferation by enhancing the formation of iTregs from activated T lymphocytes in response to TGF-β.

  8. Human-Phosphate-Binding-Protein inhibits HIV-1 gene transcription and replication

    Directory of Open Access Journals (Sweden)

    Candolfi Ermanno

    2011-07-01

    Full Text Available Abstract The Human Phosphate-Binding protein (HPBP is a serendipitously discovered lipoprotein that binds phosphate with high affinity. HPBP belongs to the DING protein family, involved in various biological processes like cell cycle regulation. We report that HPBP inhibits HIV-1 gene transcription and replication in T cell line, primary peripherical blood lymphocytes and primary macrophages. We show that HPBP is efficient in naïve and HIV-1 AZT-resistant strains. Our results revealed HPBP as a new and potent anti HIV molecule that inhibits transcription of the virus, which has not yet been targeted by HAART and therefore opens new strategies in the treatment of HIV infection.

  9. Lycorine reduces mortality of human enterovirus 71-infected mice by inhibiting virus replication

    Directory of Open Access Journals (Sweden)

    Qin Chuan

    2011-10-01

    Full Text Available Abstract Human enterovirus 71 (EV71 infection causes hand, foot and mouth disease in children under 6 years old and this infection occasionally induces severe neurological complications. No vaccines or drugs are clinical available to control EV71 epidemics. In present study, we show that treatment with lycorine reduced the viral cytopathic effect (CPE on rhabdomyosarcoma (RD cells by inhibiting virus replication. Analysis of this inhibitory effect of lycorine on viral proteins synthesis suggests that lycorine blocks the elongation of the viral polyprotein during translation. Lycorine treatment of mice challenged with a lethal dose of EV71 resulted in reduction of mortality, clinical scores and pathological changes in the muscles of mice, which were achieved through inhibition of viral replication. When mice were infected with a moderate dose of EV71, lycorine treatment was able to protect them from paralysis. Lycorine may be a potential drug candidate for the clinical treatment of EV71-infected patients.

  10. Pertussis toxin inhibits somatostatin-induced K+ conductance in human pituitary tumor cells

    International Nuclear Information System (INIS)

    Yamashita, N.; Kojima, I.; Shibuya, N.; Ogata, E.

    1987-01-01

    The effect of pertussis toxin on somatostatin-induced K + current was examined in dissociated human pituitary tumor cells obtained from two acromegalic patients. Somatostatin-induced hyperpolarization or K + current was observed in 20 of 23 cells in adenoma 1 and 10 of 11 cells in adenoma 2. After treatment with pertussis toxin for 24 h, these responses were completely suppressed (0/14 in adenoma, 1, 0/10 in adenoma 2). Spontaneous action potentials, K + , Na + , and Ca 2+ currents were well preserved after pertussis toxin treatment. When crude membrane fraction was incubated with [ 32 P]NAD, a 41K protein was ADP-ribosylated by pertussis toxin. Hormone release was inhibited by somatostatin and this inhibition was blocked by pertussis toxin treatment

  11. Memory consolidation in human sleep depends on inhibition of glucocorticoid release.

    Science.gov (United States)

    Plihal, W; Born, J

    1999-09-09

    Early sleep dominated by slow-wave sleep has been found to be particularly relevant for declarative memory formation via hippocampo-neocortical networks. Concurrently, early nocturnal sleep is characterized by an inhibition of glucocorticoid release from the adrenals. Here, we show in healthy humans that this inhibition serves to support declarative memory consolidation during sleep. Elevating plasma glucocorticoid concentration during early sleep by administration of cortisol impaired consolidation of paired associate words, but not of non-declarative memory of visuomotor skills. Since glucocorticoid concentration was enhanced only during retention sleep, but not during acquisition or retrieval, a specific effect on the consolidation process is indicated. Blocking mineralocorticoid receptors by canrenoate did not affect memory, suggesting inactivation of glucocorticoid receptors to be the essential prerequisite for memory consolidation during early sleep.

  12. Extracellular histones induce erythrocyte fragility and anemia.

    Science.gov (United States)

    Kordbacheh, Farzaneh; O'Meara, Connor H; Coupland, Lucy A; Lelliott, Patrick M; Parish, Christopher R

    2017-12-28

    Extracellular histones have been shown to play an important pathogenic role in many diseases, primarily through their cytotoxicity toward nucleated cells and their ability to promote platelet activation with resultant thrombosis and thrombocytopenia. In contrast, little is known about the effect of extracellular histones on erythrocyte function. We demonstrate in this study that histones promote erythrocyte aggregation, sedimentation, and using a novel in vitro shear stress model, we show that histones induce erythrocyte fragility and lysis in a concentration-dependent manner. Furthermore, histones impair erythrocyte deformability based on reduced passage of erythrocytes through an artificial spleen. These in vitro results were mirrored in vivo with the injection of histones inducing anemia within minutes of administration, with a concomitant increase in splenic hemoglobin content. Thrombocytopenia and leukopenia were also observed. These findings suggest that histones binding to erythrocytes may contribute to the elevated erythrocyte sedimentation rates observed in inflammatory conditions. Furthermore, histone-induced increases in red blood cell lysis and splenic clearance may be a significant factor in the unexplained anemias seen in critically ill patients. © 2017 by The American Society of Hematology.

  13. Reactivation of organophosphate-inhibited human acetylcholinesterase by isonitrosoacetone (MINA): a kinetic analysis.

    Science.gov (United States)

    Worek, Franz; Thiermann, Horst

    2011-11-15

    Treatment of poisoning by highly toxic organophosphorus compounds (OP) with atropine and an acetylcholinesterase (AChE) reactivator (oxime) is of limited effectiveness in case of different nerve agents and pesticides. One challenge is the reactivation of OP-inhibited brain AChE which shows inadequate success with charged pyridinium oximes. Recent studies with high doses of the tertiary oxime isonitrosoacetone (MINA) indicated a beneficial effect on central and peripheral AChE and on survival in nerve agent poisoned guinea pigs. Now, an in vitro study was performed to determine the reactivation kinetics of MINA with tabun-, sarin-, cyclosarin-, VX- and paraoxon-inhibited human AChE. MINA showed an exceptionally low affinity to inhibited AChE but, with the exception of tabun-inhibited AChE, a moderate to high reactivity. In comparison to the pyridinium oximes obidoxime, 2-PAM and HI-6 the affinity and reactivity of MINA was in most cases lower and in relation to the most effective reactivators, the second order reactivation constant of MINA was 500 to 3400-fold lower. Hence, high in vivo MINA concentrations would be necessary to achieve at least partial reactivation. This assumption corresponds to in vivo data showing a dose-dependent effect on reactivation and survival in animals. In view, of the toxic potential of MINA in animals human studies would be necessary to determine the tolerability and pharmacokinetics of MINA in order to enable a proper assessment of the value of this oxime as an antidote in OP poisoning. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  14. Inhibition of human lung cancer cell proliferation and survival by wine

    Science.gov (United States)

    2014-01-01

    Background Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events. Methods Human NSCLC adenocarcinoma A549 and H1299 cells were used. Cell proliferation was assessed by thymidine incorporation. Clonogenic assays were used to assess cell survival. Immunoblotting was used to examine total and phosphorylated levels of Akt, Erk and p53. Results In A549 cells red wine inhibited cell proliferation and reduced clonogenic survival at doses as low as 0.02%. Red wine significantly reduced basal and EGF-stimulated Akt and Erk phosphorylation while it increased the levels of total and phosphorylated p53 (Ser15). Control experiments indicated that the anti-proliferative effects of wine were not mediated by the associated contents of ethanol or the polyphenol resveratrol and were independent of glucose transport into cancer cells. White wine also inhibited clonogenic survival, albeit at a higher doses (0.5-2%), and reduced Akt phosphorylation. The effects of both red and white wine on Akt phosphorylation were also verified in H1299 cells. Conclusions Red wine inhibits proliferation of lung cancer cells and blocks clonogenic survival at low concentrations. This is associated with inhibition of basal and EGF-stimulated Akt and Erk signals and enhancement of total and phosphorylated levels of p53. White wine mediates similar effects albeit at higher concentrations. Our data suggest that wine may have considerable anti-tumour and chemoprevention properties in lung cancer and deserves further

  15. Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

    International Nuclear Information System (INIS)

    Xue, Gang; Zou, Xi; Zhou, Jin-Yong; Sun, Wei; Wu, Jian; Xu, Jia-Li; Wang, Rui-Ping

    2013-01-01

    Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medic