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

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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

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.

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

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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.

In-Depth, Label-Free Analysis of the Erythrocyte Cytoplasmic Proteome in Diamond Blackfan Anemia Identifies a Unique Inflammatory Signature.

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Esther N Pesciotta

Full Text Available Diamond Blackfan Anemia (DBA is a rare, congenital erythrocyte aplasia that is usually caused by haploinsufficiency of ribosomal proteins due to diverse mutations in one of several ribosomal genes. A striking feature of this disease is that a range of different mutations in ribosomal proteins results in similar disease phenotypes primarily characterized by erythrocyte abnormalities and macrocytic anemia, while most other cell types in the body are minimally affected. Previously, we analyzed the erythrocyte membrane proteomes of several DBA patients and identified several proteins that are not typically associated with this cell type and that suggested inflammatory mechanisms contribute to the pathogenesis of DBA. In this study, we evaluated the erythrocyte cytosolic proteome of DBA patients through in-depth analysis of hemoglobin-depleted erythrocyte cytosols. Simple, reproducible, hemoglobin depletion using nickel columns enabled in-depth analysis of over 1000 cytosolic erythrocyte proteins with only moderate total analysis time per proteome. Label-free quantitation and statistical analysis identified 29 proteins with significantly altered abundance levels in DBA patients compared to matched healthy control donors. Proteins that were significantly increased in DBA erythrocyte cytoplasms included three proteasome subunit beta proteins that make up the immunoproteasome and proteins induced by interferon-γ such as n-myc interactor and interferon-induced 35 kDa protein [NMI and IFI35 respectively]. Pathway analysis confirmed the presence of an inflammatory signature in erythrocytes of DBA patients and predicted key upstream regulators including mitogen activated kinase 1, interferon-γ, tumor suppressor p53, and tumor necrosis factor. These results show that erythrocytes in DBA patients are intrinsically different from those in healthy controls which may be due to an inflammatory response resulting from the inherent molecular defect of ribosomal

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

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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

On the sulfation of O-desmethyltramadol by human cytosolic sulfotransferases.

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Rasool, Mohammed I; Bairam, Ahsan F; Kurogi, Katsuhisa; Liu, Ming-Cheh

2017-10-01

Previous studies have demonstrated that sulfate conjugation is involved in the metabolism of the active metabolite of tramadol, O-desmethyltramadol (O-DMT). The current study aimed to systematically identify the human cytosolic sulfotransferases (SULTs) that are capable of mediating the sulfation of O-DMT. The sulfation of O-DMT under metabolic conditions was demonstrated using HepG2 hepatoma cells and Caco-2 human colon carcinoma cells. O-DMT-sulfating activity of thirteen known human SULTs and four human organ specimens was examined using an established sulfotransferase assay. pH-Dependency and kinetic parameters were also analyzed using, respectively, buffers at different pHs and varying O-DMT concentrations in the assays. Of the thirteen human SULTs tested, only SULT1A3 and SULT1C4 were found to display O-DMT-sulfating activity, with different pH-dependency profiles. Kinetic analysis revealed that SULT1C4 was 60 times more catalytically efficient in mediating the sulfation of O-DMT than SULT1A3 at respective optimal pH. Of the four human organ specimens tested, the cytosol prepared from the small intestine showed much higher O-DMT-sulfating activity than cytosols prepared from liver, lung, and kidney. Both cultured HepG2 and Caco-2 cells were shown to be capable of sulfating O-DMT and releasing sulfated O-DMT into cultured media. SULT1A3 and SULT1C4 were the major SULTs responsible for the sulfation of O-DMT. Collectively, the results obtained provided a molecular basis underlying the sulfation of O-DMT and contributed to a better understanding about the pharmacokinetics and pharmacodynamics of tramadol in humans. Copyright © 2017 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

Triggering of Erythrocyte Cell Membrane Scrambling by Emodin

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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.

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

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)

Copper, Zinc Superoxide Dismutase is Primarily a Cytosolic Protein in Human Cells

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Crapo, James D.; Oury, Tim; Rabouille, Catherine; Slot, Jan W.; Chang, Ling-Yi

1992-11-01

The intracellular localization of human copper, zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.

Triggering of Suicidal Erythrocyte Death by Regorafenib

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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+.

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.

Role of aminotransferases in glutamate metabolism of human erythrocytes

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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.

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

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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.

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)

Proton NMR studies of creatine in human erythrocytes

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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.

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)

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

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.

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

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

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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.

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

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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.

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

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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.

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

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

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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.

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

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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.

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

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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.

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

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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.

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...

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

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)

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.

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

International Nuclear Information System (INIS)

Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

1987-01-01

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

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

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

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.

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)

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.

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.

Induction of Suicidal Erythrocyte Death by Novobiocin

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Adrian Lupescu

2014-03-01

Full Text Available Background: Novobiocin, an aminocoumarin antibiotic, interferes with heat shock protein 90 and hypoxia inducible factor dependent gene expression and thus compromises cell survival. Similar to survival of nucleated cells, erythrocyte survival could be disrupted by eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and by phospholipd scrambling of the cell membrane with phosphatidylserine translocation to the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i. The Ca2+ sensitivity of phospholipid scrambling is enhanced by ceramide. The present study explored, whether novobiocin elicits eryptosis. Methods: [Ca2+]i was estimated from Fluo3-fluorescence, ceramide abundance utilizing fluorescent antibodies, cell volume from forward scatter, phosphatidylserine-exposure from annexin V binding. Results: A 48 hours exposure to novobiocin (500 µM was followed by a significant increase of [Ca2+]i, decrease of forward scatter, increase of annexin-V-binding and enhanced ceramide formation. Removal of extracellular Ca2+ virtually abrogated the increase of annexin-V-binding following novobiocin exposure. Conclusions: Novobiocin stimulates eryptosis, an effect at least in part due to entry of extracellular Ca2+ and formation of ceramide.

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.

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.

Stimulation of Suicidal Erythrocyte Death by Increased Extracellular Phosphate Concentrations

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Jakob Voelkl

2014-02-01

Full Text Available Background/Aim: Anemia in renal insufficiency results in part from impaired erythrocyte formation due to erythropoietin and iron deficiency. Beyond that, renal insufficiency enhances eryptosis, the suicidal erythrocyte death characterized by phosphatidylserine-exposure at the erythrocyte surface. Eryptosis may be stimulated by increase of cytosolic Ca2+-activity ([Ca2+]i. Several uremic toxins have previously been shown to stimulate eryptosis. Renal insufficiency is further paralleled by increase of plasma phosphate concentration. The present study thus explored the effect of phosphate on erythrocyte death. Methods: Cell volume was estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, and [Ca2+]i from Fluo3-fluorescence. Results: Following a 48 hours incubation, the percentage of phosphatidylserine exposing erythrocytes markedly increased as a function of extracellular phosphate concentration (from 0-5 mM. The exposure to 2 mM or 5 mM phosphate was followed by slight but significant hemolysis. [Ca2+]i did not change significantly up to 2 mM phosphate but significantly decreased at 5 mM phosphate. The effect of 2 mM phosphate on phosphatidylserine exposure was significantly augmented by increase of extracellular Ca2+ to 1.7 mM, and significantly blunted by nominal absence of extracellular Ca2+, by additional presence of pyrophosphate as well as by presence of p38 inhibitor SB203580. Conclusion: Increasing phosphate concentration stimulates erythrocyte membrane scrambling, an effect depending on extracellular but not intracellular Ca2+ concentration. It is hypothesized that suicidal erythrocyte death is triggered by complexed CaHPO4.

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.

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.

Stimulation of erythrocyte phosphatidylserine exposure by mercury ions

International Nuclear Information System (INIS)

Eisele, Kerstin; Lang, Philipp A.; Kempe, Daniela S.; Klarl, Barbara A.; Niemoeller, Olivier; Wieder, Thomas; Huber, Stephan M.; Duranton, Christophe; Lang, Florian

2006-01-01

The sequelae of mercury intoxication include induction of apoptosis. In nucleated cells, Hg 2+ -induced apoptosis involves mitochondrial damage. The present study has been performed to elucidate effects of Hg 2+ in erythrocytes which lack mitochondria but are able to undergo apoptosis-like alterations of the cell membrane. Previous studies have documented that activation of a Ca 2+ -sensitive erythrocyte scramblase leads to exposure of phosphatidylserine at the erythrocyte surface, a typical feature of apoptotic cells. The erythrocyte scramblase is activated by osmotic shock, oxidative stress and/or energy depletion which increase cytosolic Ca 2+ activity and/or activate a sphingomyelinase leading to formation of ceramide. Ceramide sensitizes the scramblase to Ca 2+ . The present experiments explored the effect of Hg 2+ ions on erythrocytes. Phosphatidylserine exposure after mercury treatment was estimated from annexin binding as determined in FACS analysis. Exposure to Hg 2+ (1 μM) indeed significantly increased annexin binding from 2.3 ± 0.5% (control condition) to 23 ± 6% (n = 6). This effect was paralleled by activation of a clotrimazole-sensitive K + -selective conductance as measured by patch-clamp recordings and by transient cell shrinkage. Further experiments revealed also an increase of ceramide formation by ∼66% (n = 7) after challenge with mercury (1 μM). In conclusion, mercury ions activate a clotrimazole-sensitive K + -selective conductance leading to transient cell shrinkage. Moreover, Hg 2+ increases ceramide formation. The observed mechanisms could similarly participate in the triggering of apoptosis in nucleated cells by Hg 2+

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

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...

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.)

Cloning and characterization of human liver cytosolic beta-glycosidase

NARCIS (Netherlands)

De Graaf, M; Van Veen, IC; Van Der Meulen-Muileman, IH; Gerritsen, WR; Pinedo, HM; Haisma, HJ

2001-01-01

Cytosolic beta -glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta -D-glycosides. including beta -D-glucoside acid beta -D-galactoside. We therefore refer to this enzyme as cytosolic beta

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

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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.

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

Lipopeptide-Induced Suicidal Erythrocyte Death Correlates with the Degree of Acylation

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Abdulla Al Mamun Bhuyan

2017-01-01

Full Text Available Background/Aims: Consequences of bacterial infection include anemia, which could result from stimulation of suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Bacterial components known to stimulate eryptosis include lipopeptides. Signaling mediating the triggering of eryptosis include increased cytosolic Ca2+ activity ([Ca2+]i, oxidative stress and cellular accumulation of ceramide. The present study aimed to define the molecular requirements for lipopeptide-induced cell membrane scrambling. Methods: Human erythrocytes were incubated for 48 hours in the absence and presence of 1 or 5 µg/ml of the synthetic lipopeptides Pam1 (lipopeptide with one fatty acid, Pam2 (lipopeptide with two fatty acids, or Pam3 (lipopeptide with three fatty acids. In the following 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 DCF dependent fuorescence, and ceramide abundance utilizing specific antibodies. Results: Pam1 (5 µg/ml, Pam2 (5 µg/ml and Pam3 (1 and 5 µg/ml significantly increased the percentage of annexin-V-binding to erythrocytes in a dose dependent manner, which was largely independent of Ca2+. Pam1-3 increased the percentage of both, swollen and shrunken erythrocytes without significantly modifying the average forward scatter. They also increased reactive oxygen species (ROS and ceramide abundance. In all assays the effect on eryptosis increased with increasing number of fatty acids, with Pam3 showing always the strongest effect. In contrast, a comparison of the effect of Pam1-3 on TLR2 dependent immune stimulation showed that not Pam3 but Pam2 displayed the strongest activity, and that immune stimulation was triggered at much lower concentrations than eryptosis. Conclusions: Lipopeptides are not only important

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

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

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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.

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

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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.

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

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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.

Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members.

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Currà, Chiara; Pace, Tomasino; Franke-Fayard, Blandine M D; Picci, Leonardo; Bertuccini, Lucia; Ponzi, Marta

2012-03-01

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions. © 2011 John Wiley & Sons A/S.

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

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

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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.

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

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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.

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)

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.

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

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

Evaluation of Hemagglutination Activity of Chitosan Nanoparticles Using Human Erythrocytes

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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.

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.

Interaction of Plasmodium falciparum knob-associated histidine-rich protein (KAHRP) with erythrocyte ankyrin R is required for its attachment to the erythrocyte membrane.

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Weng, Haibo; Guo, Xinhua; Papoin, Julien; Wang, Jie; Coppel, Ross; Mohandas, Narla; An, Xiuli

2014-01-01

The malaria parasite Plasmodium falciparum exports a large number of proteins into the erythrocyte cytoplasm during the asexual intraerythrocytic stage of its life cycle. A subset of these proteins interacts with erythrocyte membrane skeletal proteins and grossly alters the structure and function of the membrane. Several of the exported proteins, such as PfEMP1, PfEMP3, RESA and KAHRP, interact with the preponderant erythrocyte skeleton protein, spectrin. Here we have searched for possible interaction of these four malaria proteins with another major erythrocyte skeleton protein, ankyrin R. We have shown that KAHRP, but none of the other three, binds to ankyrin R. We have mapped the binding site for ankyrin R to a 79-residue segment of the KAHRP sequence, and the reciprocal binding site for KAHRP in ankyrin R to a subdomain (D3) of the 89kDa ankyrin R membrane-binding domain. Interaction of intact ankyrin R with KAHRP was inhibited by the free D3 subdomain. When, moreover, red cells loaded with the soluble D3 subdomain were infected with P. falciparum, KAHRP secreted by the intraerythrocytic parasite no longer migrated to the host cell membrane, but remained diffusely distributed throughout the cytosol. Our findings suggest a potentially important role for interaction of KAHRP with red cell membrane skeleton in promoting the adhesion of malaria-infected red cells to endothelial surfaces, a central element in the pathophysiology of malaria. © 2013.

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...

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

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.

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.

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...

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.

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.

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.

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.

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

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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.

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.

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

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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

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

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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.

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)

Chloride concentrations in human hepatic cytosol and mitochondria are a function of age.

Science.gov (United States)

Jahn, Stephan C; Rowland-Faux, Laura; Stacpoole, Peter W; James, Margaret O

2015-04-10

We recently reported that, in a concentration-dependent manner, chloride protects hepatic glutathione transferase zeta 1 from inactivation by dichloroacetate, an investigational drug used in treating various acquired and congenital metabolic diseases. Despite the importance of chloride ions in normal physiology, and decades of study of chloride transport across membranes, the literature lacks information on chloride concentrations in animal tissues other than blood. In this study we measured chloride concentrations in human liver samples from male and female donors aged 1 day to 84 years (n = 97). Because glutathione transferase zeta 1 is present in cytosol and, to a lesser extent, in mitochondria, we measured chloride in these fractions by high-performance liquid chromatography analysis following conversion of the free chloride to pentafluorobenzylchloride. We found that chloride concentration decreased with age in hepatic cytosol but increased in liver mitochondria. In addition, chloride concentrations in cytosol, (105.2 ± 62.4 mM; range: 24.7-365.7 mM) were strikingly higher than those in mitochondria (4.2 ± 3.8 mM; range 0.9-22.2 mM). These results suggest a possible explanation for clinical observations seen in patients treated with dichloroacetate, whereby children metabolize the drug more rapidly than adults following repeated doses, and also provide information that may influence our understanding of normal liver physiology. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells*

Science.gov (United States)

VanLinden, Magali R.; Dölle, Christian; Pettersen, Ina K. N.; Kulikova, Veronika A.; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E.; Palmieri, Ferdinando; Nikiforov, Andrey A.; Tronstad, Karl Johan; Ziegler, Mathias

2015-01-01

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD+ biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD+ in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD+ content, we have expressed plant and yeast mitochondrial NAD+ carriers in human cells and observed a profound increase in mitochondrial NAD+. None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD+ content. Surprisingly, constitutive redistribution of NAD+ from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD+ transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD+ levels. These results suggest that a mitochondrial NAD+ transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD+ synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. PMID:26432643

The tobacco carcinogen NNK is stereoselectively reduced by human pancreatic microsomes and cytosols.

Science.gov (United States)

Trushin, Neil; Leder, Gerhard; El-Bayoumy, Karam; Hoffmann, Dietrich; Beger, Hans G; Henne-Bruns, Doris; Ramadani, Marco; Prokopczyk, Bogdan

2008-07-01

Cigarette smoking increases the risk of cancer of the pancreas. The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is the only known environmental compound that induces pancreatic cancer in laboratory animals. Concentrations of NNK are significantly higher in the pancreatic juice of smokers than in that of nonsmokers. The chiral NNK metabolite, (R,S)-4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is itself a potent pancreatic carcinogen in rats. The carcinogenicity of NNAL is related to its stereochemistry; (S)-NNAL is a more potent lung tumorigen in the A/J mouse than is (R)-NNAL. In this study, we determined the potential of the human pancreas to convert NNK into NNAL. Human pancreatic microsomes and cytosols were incubated with [5-(3)H]NNK, and the metabolic products were determined by high-performance liquid chromatography (HPLC). (S)-NNAL was the predominant isomer formed in all cytosolic incubations. In ten microsomal samples, NNAL was formed at an average rate of 3.8 +/- 1.6 pmol/mg/min; (R)-NNAL was the predominant isomer in this group. The average rate of NNAL formation in 18 other microsomal samples was significantly lower, 0.13 +/- 0.12 pmol/mg/min (p < 0.001); (S)-NNAL was the predominant isomer formed in this group. In human pancreatic tissues, there is intraindividual variability regarding the capacity for, and stereoselectivity of, carbonyl reduction of NNK.

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.

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.

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

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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.

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...

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.

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

Science.gov (United States)

VanLinden, Magali R; Dölle, Christian; Pettersen, Ina K N; Kulikova, Veronika A; Niere, Marc; Agrimi, Gennaro; Dyrstad, Sissel E; Palmieri, Ferdinando; Nikiforov, Andrey A; Tronstad, Karl Johan; Ziegler, Mathias

2015-11-13

The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death

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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

Cigarette smoke toxicants as substrates and inhibitors for human cytosolic SULTs

International Nuclear Information System (INIS)

Yasuda, Shin; Idell, Steven; Fu Jian; Carter, Glendora; Snow, Rhodora; Liu, M.-C.

2007-01-01

The current study was designed to examine the role of sulfation in the metabolism of cigarette smoke toxicants and clarify whether these toxicants, by serving as substrates for the cytosolic sulfotransferases (SULTs), may interfere with the sulfation of key endogenous compounds. By metabolic labeling, [ 35 S]sulfated species were found to be generated and released into the media of HepG2 human hepatoma cells and primary human lung endothelial cells labeled with [ 35 S]sulfate in the presence of cigarette smoke extract (CSE). Concomitantly, several [ 35 S]sulfated metabolites observed in the medium in the absence of CSE either decreased or disappeared. Eleven previously prepared human cytosolic SULTs were tested for sulfating activity with CSE and known cigarette smoke toxicants as substrates. Activity data revealed SULT1A1, SULT1A2, SULT1A3, and SULT1C2 as major enzymes responsible for their sulfation. To examine their inhibitory effects on the sulfation of 17β-estradiol by SULT1A1, enzymatic assays were performed in the presence of three representative toxicant compounds, namely N-hydroxy-4-aminobiphenyl (N-OH-4-ABP), 4-aminobiphenyl (4-ABP) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). IC 50 values determined for the sulfation of 17β-estradiol by SULT1A1 were 11.8 μM, 28.2 μM, and 500 μM, respectively, for N-OH-4-ABP, 4-ABP and PhIP. Kinetic analyses indicated that the mechanism underlying the inhibition of 17β-estradiol sulfation by these cigarette smoke toxicants is of a mixed competitive-noncompetitive type. Metabolic labeling experiments clearly showed inhibition of the production of [ 35 S]sulfated 17β-estradiol by N-OH-4-ABP in a concentration-dependent manner in HepG2 cells. Taken together, these results suggest that sulfation plays a significant role in the metabolism of cigarette smoke compounds. By serving as substrates for SULTs, cigarette smoke toxicants may interfere with the metabolism of 17β-estradiol and other endogenous

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.

[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.

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.

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.

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.

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

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)

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.

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.

Stereoselective sulfate conjugation of racemic 4-hydroxypropranolol by human and rat liver cytosol

Energy Technology Data Exchange (ETDEWEB)

Walle, T.; Walle, U.K. (Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, Charleston (USA))

1991-03-01

The objective of this study was to determine the stereochemistry of sulfoconjugation of a chiral phenolic amine drug, 4-hydroxypropranolol (HOP), by the human liver. The reaction was catalyzed by the 100,000 g cytosol as the phenolsulfotransferase (PST) enzyme source with PAP35S as the co-substrate. The enantiomers of the intact sulfate conjugate formed, (+)-HOP35S and (-)-HOP35S, were separated by HPLC and measured by liquid scintillation spectrometry. Complex velocity vs. substrate concentration curves were obtained with two peaks of activity, one at 3 microM (high affinity) and one at 500 microM (low affinity). The high-affinity reaction demonstrated a high degree of stereoselectivity. Whereas the affinity of the enantiomers for this reaction was identical, with a very low apparent KM value of 0.59 microM, the apparent Vmax value for (+)-HOPS formation was 4.6-fold higher than for (-)-HOPS. In sharp contrast, the low-affinity reaction, with an apparent KM of 65 microM, was not stereoselective. Inhibition of the high-affinity reaction by elevated temperature, but not by dichloronitrophenol, indicated that this activity was due to a monoamine form of PST. Inhibition of the low-affinity reaction by dichloronitrophenol, but not by elevated temperature, indicated that this activity was due to a phenol form of PST. As a comparison, experiments with the rat liver cytosol demonstrated only one activity, with apparent KM values of 50 microM for both enantiomers and opposite stereoselectivity in maximum velocity compared to humans, {plus minus}-HOPS ratio 0.72. The results of this study demonstrate stereoselectivity in human hepatic sulfation of a chiral phenolic amine, with clear differences between PST isoenzymes.

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...

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.

Enzymatic sulfation of tocopherols and tocopherol metabolites by human cytosolic sulfotransferases.

Science.gov (United States)

Hashiguchi, Takuyu; Kurogi, Katsuhisa; Sakakibara, Yoichi; Yamasaki, Masao; Nishiyama, Kazuo; Yasuda, Shin; Liu, Ming-Cheh; Suiko, Masahito

2011-01-01

Tocopherols are essential micronutrients for mammals widely known as potent lipid-soluble antioxidants that are present in cell membranes. Recent studies have demonstrated that most of the carboxychromanol (CEHC), a tocopherol metabolite, in the plasma exists primarily in sulfate- and glucuronide-conjugated forms. To gain insight into the enzymatic sulfation of tocopherols and their metabolites, a systematic investigation was performed using all 14 known human cytosolic sulfotransferases (SULTs). The results showed that the members of the SULT1 family displayed stronger sulfating activities toward tocopherols and their metabolites. These enzymes showed a substrate preference for γ-tocopherol over α-tocopherol and for γ-CEHC over other CEHCs. Using A549 human lung epithelial cells in a metabolic labeling study, a similar trend in the sulfation of tocopherols and CEHCs was observed. Collectively, the results obtained indicate that SULT-mediated enzymatic sulfation of tocopherols and their metabolites is a significant pathway for regulation of the homeostasis and physiological functions of these important compounds.

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.

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)

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.

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

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.

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....

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

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...

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

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

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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.

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

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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.

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...

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)

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 .

Cellular processing of gold nanoparticles: CE-ICP-MS evidence for the speciation changes in human cytosol.

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Legat, Joanna; Matczuk, Magdalena; Timerbaev, Andrei R; Jarosz, Maciej

2018-01-01

The cellular uptake of gold nanoparticles (AuNPs) may (or may not) affect their speciation, but information on the chemical forms in which the particles exist in the cell remains obscure. An analytical method based on the use of capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry (ICP-MS) has been proposed to shed light on the intracellular processing of AuNPs. It was observed that when being introduced into normal cytosol, the conjugates of 10-50 nm AuNPs with albumin evolved in human serum stayed intact. On the contrary, under simulated cancer cytosol conditions, the nanoconjugates underwent decomposition, the rate of which and the resulting metal speciation patterns were strongly influenced by particle size. The new peaks that appeared in ICP-MS electropherograms could be ascribed to nanosized species, as upon ultracentrifugation, they quantitatively precipitated whereas the supernatant showed only trace Au signals. Our present study is the first step to unravel a mystery of the cellular chemistry for metal-based nanomedicines.

[Effects of cytosolic bacteria on cyclic GMP-AMP synthase expression in human gingival tissues and periodontal ligament cells].

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Xiaojun, Yang; Yongmei, Tan; Zhihui, Tian; Ting, Zhou; Wanghong, Zhao; Jin, Hou

2017-04-01

This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues. The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry. P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues. Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns.
.

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

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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.

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

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

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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.

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.)

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

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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.

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

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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.

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.

Genome-Derived Cytosolic DNA Mediates Type I Interferon-Dependent Rejection of B Cell Lymphoma Cells

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Yu J. Shen

2015-04-01

Full Text Available The DNA damage response (DDR induces the expression of type I interferons (IFNs, but the underlying mechanisms are poorly understood. Here, we show the presence of cytosolic DNA in different mouse and human tumor cells. Treatment of cells with genotoxic agents increased the levels of cytosolic DNA in a DDR-dependent manner. Cloning of cytosolic DNA molecules from mouse lymphoma cells suggests that cytosolic DNA is derived from unique genomic loci and has the potential to form non-B DNA structures, including R-loops. Overexpression of Rnaseh1, which resolves R-loops, reduced the levels of cytosolic DNA, type I Ifn transcripts, and type I IFN-dependent rejection of lymphoma cells. Live-cell imaging showed a dynamic contact of cytosolic DNA with mitochondria, an important organelle for innate immune recognition of cytosolic nucleotides. In summary, we found that cytosolic DNA is present in many tumor cells and contributes to the immunogenicity of tumor cells.

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

Erythrocyte Membrane Failure by Electromechanical Stress

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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.

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.

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

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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

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.)

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

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.

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)

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)

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

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

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.

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

The Trw type IV secretion system of Bartonella mediates host-specific adhesion to erythrocytes.

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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

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.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane; Demangel, Caroline; Van Ingen, Jakko; Perez, Jorge; Baldeó n, Lucy R.; Abdallah, Abdallah; Caleechurn, Laxmee; Bottai, Daria; Van Zon, Maaike; De Punder, Karin; Van Der Laan, Tridia; Kant, Arie; Bossers-De Vries, Ruth; Willemsen, Peter Th J; Bitter, Wilbert M.; Van Soolingen, Dick; Brosch, Roland; Van Der Wel, Nicole N.; Peters, Peter J.

2012-01-01

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

ESX-1-mediated translocation to the cytosol controls virulence of mycobacteria

KAUST Repository

Houben, Diane

2012-05-08

Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, are among the most potent human bacterial pathogens. The discovery of cytosolic mycobacteria challenged the paradigm that these pathogens exclusively localize within the phagosome of host cells. As yet the biological relevance of mycobacterial translocation to the cytosol remained unclear. In this current study we used electron microscopy techniques to establish a clear link between translocation and mycobacterial virulence. Pathogenic, patient-derived mycobacteria species were found to translocate to the cytosol, while non-pathogenic species did not. We were further able to link cytosolic translocation with pathogenicity by introducing the ESX-1 (type VII) secretion system into the non-virulent, exclusively phagolysosomal Mycobacterium bovis BCG. Furthermore, we show that translocation is dependent on the C-terminus of the early-secreted antigen ESAT-6. The C-terminal truncation of ESAT-6 was shown to result in attenuation in mice, again linking translocation to virulence. Together, these data demonstrate the molecular mechanism facilitating translocation of mycobacteria. The ability to translocate from the phagolysosome to the cytosol is with this study proven to be biologically significant as it determines mycobacterial virulence. © 2012 Blackwell Publishing Ltd.

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 ...

Decreased erythrocyte superoxide dismutase in elderly men with early nuclear cataract

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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.

Decreased erythrocyte superoxide dismutase in elderly men with early nuclear cataract

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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.

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

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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.

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

Human cytosolic thymidine kinase: purification and physical characterization of the enzyme from HeLa cells

International Nuclear Information System (INIS)

Sherley, J.L.; Kelly, T.J.

1988-01-01

The mammalian cytosolic thymidine kinase is one of a number of enzymes involved in DNA replication whose activities increase dramatically during S phase of the cell cycle. As a first step in defining the mechanisms that control the S phase induction of thymidine kinase activity, the authors have purified the human enzyme from HeLa cells and raised a specific immune serum against the purified protein. The enzyme was isolated from cells arrested in S phase by treatment with methotrexate and purified to near homogeneity by ion-exchange and affinity chromatography. Stabilization of the purified enzyme was achieved by the addition of digitonin. An electrophoretic R/sub m/ of 0.2 in nondenaturing gels characterizes the purified enzyme activity as cytosolic thymidine kinase. The enzyme has a Stoke's radius of 40 A determined by gel filtration and a sedimentation coefficient of 5.5 S determined by glycerol gradient sedimentation. Based on these hydrodynamic values, a native molecular weight of 96,000 was calculated for the purified enzyme. When electrophoresed in denaturing sodium dodecyl sulfate-polyacrylamide gels under reducing conditions, the most purified enzyme fraction was found to contain one predominant polypeptide of M/sub r/ = 24,000. Several lines of evidence indicate that this polypeptide is responsible for thymidine kinase enzymatic activity

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.

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

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

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

Life cycle-dependent cytoskeletal modifications in Plasmodium falciparum infected erythrocytes.

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Hui Shi

Full Text Available Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM. We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM-based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.

Life cycle-dependent cytoskeletal modifications in Plasmodium falciparum infected erythrocytes.

Science.gov (United States)

Shi, Hui; Liu, Zhuo; Li, Ang; Yin, Jing; Chong, Alvin G L; Tan, Kevin S W; Zhang, Yong; Lim, Chwee Teck

2013-01-01

Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM). We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM-based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.

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...

Detection of Cytosolic Shigella flexneri via a C-Terminal Triple-Arginine Motif of GBP1 Inhibits Actin-Based Motility

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Anthony S. Piro

2017-12-01

Full Text Available Dynamin-like guanylate binding proteins (GBPs are gamma interferon (IFN-γ-inducible host defense proteins that can associate with cytosol-invading bacterial pathogens. Mouse GBPs promote the lytic destruction of targeted bacteria in the host cell cytosol, but the antimicrobial function of human GBPs and the mechanism by which these proteins associate with cytosolic bacteria are poorly understood. Here, we demonstrate that human GBP1 is unique among the seven human GBP paralogs in its ability to associate with at least two cytosolic Gram-negative bacteria, Burkholderia thailandensis and Shigella flexneri. Rough lipopolysaccharide (LPS mutants of S. flexneri colocalize with GBP1 less frequently than wild-type S. flexneri does, suggesting that host recognition of O antigen promotes GBP1 targeting to Gram-negative bacteria. The targeting of GBP1 to cytosolic bacteria, via a unique triple-arginine motif present in its C terminus, promotes the corecruitment of four additional GBP paralogs (GBP2, GBP3, GBP4, and GBP6. GBP1-decorated Shigella organisms replicate but fail to form actin tails, leading to their intracellular aggregation. Consequentially, the wild type but not the triple-arginine GBP1 mutant restricts S. flexneri cell-to-cell spread. Furthermore, human-adapted S. flexneri, through the action of one its secreted effectors, IpaH9.8, is more resistant to GBP1 targeting than the non-human-adapted bacillus B. thailandensis. These studies reveal that human GBP1 uniquely functions as an intracellular “glue trap,” inhibiting the cytosolic movement of normally actin-propelled Gram-negative bacteria. In response to this powerful human defense program, S. flexneri has evolved an effective counterdefense to restrict GBP1 recruitment.

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.

Hemolitic action of Naja naja atra cardiotoxin on erythrocytes from different animals

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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.

Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis

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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.

In vitro host erythrocyte specificity and differential morphology of Babesia divergens and a zoonotic Babesia sp. from eastern cottontail rabbits (Sylvilagus floridanus).

Science.gov (United States)

Spencer, Angela M; Goethert, Heidi K; Telford, Samuel R; Holman, Patricia J

2006-04-01

A Babesia sp. isolated from eastern cottontail rabbits (Sylvilagus floridanus) is morphologically similar and genetically identical, based on SSU rRNA gene comparisons, to 2 agents responsible for human babesiosis in the United States. This zoonotic agent is closely related to the European parasite, Babesia divergens. The 2 organisms were characterized by in vitro comparisons. In vitro growth of the rabbit Babesia sp. was supported in human and cottontail rabbit erythrocytes, but not in bovine cells. Babesia divergens was supported in vitro in bovine and human erythrocytes, but not in cottontail rabbit cells. Morphometric analysis classifies B. divergens as a small babesia in bovine erythrocytes, but the parasite exceeds this size in human erythrocytes. The rabbit Babesia sp. is large, the same size in both human or rabbit erythrocytes, and is significantly larger than B. divergens. Eight or more rabbit Babesia sp. parasites may occur within a single erythrocyte, sometimes in a floret array, unlike B. divergens. The erythrocyte specificity and morphological differences reported in this study agree with previous in vivo results and validate the use of in vitro methods for characterization of Babesia species.

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

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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.

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

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

Cytosolic adenylate changes during exercise in prawn muscle

International Nuclear Information System (INIS)

Thebault, M.T.; Raffin, J.P.; Pichon, R.

1994-01-01

31 P NMR and biochemical analysis were used to assess the effect of heavy exercise on cytosolic adenylate levels in Palaemon serratus abdominal muscle. At rest, the MgATP level corresponded to 85.5% of the total ATP content. The cytosolic adenylate concentrations of the prawn muscle are considerably different from that of vertebrates. The percentage of ADP bound to myofilaments was lower in the prawn muscle. Consequently, the level of free cytosolic AMP was greatly higher (thirty fold higher) than in vertebrate muscle. During vigorous work, the concentration of MgATP dropped and the cytosolic AMP accumulated, while the cytosolic adenine nucleotide pool decreased significantly. The phosphorylation potential value and the ATP/ADP ratio, calculated from the cytosolic adenylate, dropped acutely during the whole period of muscular contractions. On the contrary, the adenylate energy charge calculated from the cytosolic adenylate decreased slightly. Therefore, even in muscle displaying no AMP deamination, the adenylate charge is stabilized during exercise by the dynamic changes between cytosolic and bound adenylate species. (author). 21 refs., 2 tabs

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

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

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.

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.

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)

Breakdown of Phosphatidylserine Asymmetry Following Treatment of Erythrocytes with Lumefantrine

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Kousi Alzoubi

2014-02-01

Full Text Available Background: Lumefantrine, a commonly used antimalarial drug, inhibits hemozoin formation in parasites. Several other antimalarial substances counteract parasitemia by triggering suicidal death or eryptosis of infected erythrocytes. Eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine-exposure at the erythrocyte surface. Signaling involved in eryptosis include increase of cytosolic Ca2+-activity ([Ca2+]i, formation of ceramide, oxidative stress and/or activation of p38 kinase, protein kinase C (PKC, or caspases. The present study explored, whether lumefantrine stimulates eryptosis. Methods: Cell volume has been estimated from forward scatter, phosphatidylserine-exposure from annexin V binding, [Ca2+]i from Fluo3-fluorescence, reactive oxygen species from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, content of reduced glutathione (GSH from mercury orange fluorescence, and ceramide abundance from binding of fluorescent antibodies in flow cytometry. Results: A 48 h exposure to lumefantrine (3 µg/mL was followed by a significant increase of annexin-V-binding without significantly altering forward scatter, [Ca2+]i, ROS formation, reduced GSH, or ceramide abundance. The annexin-V-binding following lumefantrine treatment was not significantly modified by p38 kinase inhibitors SB203580 (2 μM and p38 Inh III (1 μM, PKC inhibitor staurosporine (1 µM or pancaspase inhibitor zVAD (1 or 10 µM. Conclusions: Lumefantrine triggers cell membrane scrambling, an effect independent from entry of extracellular Ca2+, ceramide formation, ROS formation, glutathione content, p38 kinase, PKC or caspases.

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

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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.

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).

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.

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

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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.

MODIFICATION OF ERYTHROCYTE MEMBRANE PROTEINS WITH POLYETHYLENE GLYCOL 1500

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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.

Surface-Enhanced Raman Spectroscopy (SERS Tracking of Chelerythrine, a Na+/K+ Pump Inhibitor, into Cytosol and Plasma Membrane Fractions of Human Lens Epithelial Cell Cultures

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Kevin M. Dorney

2013-12-01

Full Text Available Background/Aims: The quaternary benzo-phenanthridine alkaloid (QBA chelerythrine (CET is a pro-apoptotic drug and Na+/K+ pump (NKP inhibitor in human lens epithelial cells (HLECs. In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS. Methods: Silver nanoparticles (AgNPs prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm-1 marker band as a function of CET concentration. Results: SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Conclusion: Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET+ accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect.

Surface-enhanced Raman spectroscopy (SERS) tracking of chelerythrine, a Na(+)/K(+) pump inhibitor, into cytosol and plasma membrane fractions of human lens epithelial cell cultures.

Science.gov (United States)

Dorney, Kevin M; Sizemore, Ioana E P; Alqahtani, Tariq; Adragna, Norma C; Lauf, Peter K

2013-01-01

The quaternary benzo-phenanthridine alkaloid (QBA) chelerythrine (CET) is a pro-apoptotic drug and Na(+)/K(+) pump (NKP) inhibitor in human lens epithelial cells (HLECs). In order to obtain further insight into the mechanism of NKP inhibition by CET, its sub-cellular distribution was quantified in cytosolic and membrane fractions of HLEC cultures by surface-enhanced Raman spectroscopy (SERS). Silver nanoparticles (AgNPs) prepared by the Creighton method were concentrated, and size-selected using a one-step tangential flow filtration approach. HLECs cultures were exposed to 50 μM CET in 300 mOsM phosphate-buffered NaCl for 30 min. A variety of cytosolic extracts, crude and purified membranes, prepared in lysing solutions in the presence and absence of a non-ionic detergent, were incubated with AgNPs and subjected to SERS analysis. Determinations of CET were based on a linear calibration plot of the integrated CET SERS intensity at its 659 cm(-1) marker band as a function of CET concentration. SERS detected chemically unaltered CET in both cytosol and plasma membrane fractions. Normalized for protein, the CET content was some 100 fold higher in the crude and purified plasma membrane fraction than in the soluble cytosolic extract. The total free CET concentration in the cytosol, free of membranes or containing detergent-solubilized membrane material, approached that of the incubation medium of HLECs. Given a negative membrane potential of HLECs the data suggest, but do not prove, that CET may traverse the plasma membrane as a positively charged monomer (CET(+)) accumulating near or above passive equilibrium distribution. These findings may contribute to a recently proposed hypothesis that CET binds to and inhibits the NKP through its cytosolic aspect. © 2014 S. Karger AG, Basel.

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.

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

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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.

The cytosolic exonuclease TREX1 inhibits the innate immune response to HIV-1

Science.gov (United States)

Yan, Nan; Regalado-Magdos, Ashton D.; Stiggelbout, Bart; Lee-Kirsch, Min Ae; Lieberman, Judy

2010-01-01

Viral infection triggers innate immune sensors to produce type I interferons (IFN). However, HIV infection of T cells and macrophages does not trip these alarms. How HIV avoids activating nucleic acid sensors is unknown. The cytosolic exonuclease TREX1 suppressed IFN triggered by HIV. In Trex1−/− mouse cells and human CD4+ T cells and macrophages in which TREX1 was inhibited by RNA interference, cytosolic HIV DNA accumulated, and HIV infection induced type I IFN that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate IFN expression via a TBK1, STING and IRF3 dependent pathway. HIV-stimulated IFN production in cells deficient in TREX1 did not involve known nucleic acid sensors. PMID:20871604

Dielectric response of biconcave erythrocyte membranes to D- and L-Glucose

International Nuclear Information System (INIS)

Livshits, L; Caduff, A; Talary, M S; Feldman, Y

2007-01-01

In this paper, we report on the influence of D- and L-glucose on the dielectric properties of native shaped (biconcave) human erythrocytes using time domain dielectric spectroscopy. The dielectric spectra of biconcave cells were analysed using a modified form of the model originally reported for spheroid particle suspensions (Asami and Yonezawa 1995 Biochim. Biophys. Acta. 1245 317-24) The observed increase in the specific membrane capacitance of the biconcave erythrocytes was correlated with an increase in the concentration of D-glucose. In contrast, no associated correlation was found to changes in the membrane capacitance with increasing concentrations of L-glucose. A similar analysis of the dielectric response of osmotically swollen erythrocytes to changes in D-glucose concentration revealed a significantly different calculated specific cell membrane capacitance at elevated (>12 mM) D-glucose concentrations. The paper outlines and discusses the possible biochemical mechanisms that could be responsible for the measured dielectric properties of the erythrocyte membrane capacitances

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

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.

THE NANOSTRUCTURE OF ERYTHROCYTE MEMBRANES UNDER BLOOD INTOXICATION: AN ATOMIC FORCE MICROSCOPY STUDY

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V. A. Sergunova

2016-01-01

Full Text Available Background: The effects of toxins on nanostructure of blood cells are one of the key problems of biophysics and medicine. Erythrocyte morphology and membrane structure are recognized as the main parameters of blood quality. Therefore, analysis of membrane defects under toxin effects seems an urgent issue. Aim: To identify characteristic features and patterns of changes in membrane nanostructure under hemin intoxication and during extended storage of erythrocyte suspension. Materials and methods: The study was done in vitro in human whole blood with addition of hemin, аnd in erythrocyte suspension with a CPD blood preservative stored at 4 °С for 30 days. The nanostructure of erythrocyte membrane was assessed by atomic force microscopy. Results: Characteristic size of space periods between “granules” was from 120 to 200 nm. “Granule” numbers within a topological defect varied from 4 to 5 and to several dozens. Such domains arose virtually on all cells in erythrocyte suspension, as well as after hemin addition to the blood. An increase in hemin intoxication and an increase in a storage time were associated by increases in echinocyte numbers that subsequently transformed into spherical echinocytes. Both under hemin and during the storage of erythrocyte suspension for 9 to 12 days, a specific abnormality in nanostructure of erythrocyte membrane was observed: structural clusters, i.e., domains with granular structure, were formed. Conclusion: The experiments showed that both hemin and oxidative processes in the blood can specifically affect the nanostructure of erythrocyte membranes with formation of domains on their surface. The specific size of granular structures in the domains is from 100 to 200 nm that coincides with a  specific size of spectrin matrix. These results can be used in basic and applied medicine, in blood transfusion, for the analysis of a toxin effects in the human body. The biophysical mechanisms of domain

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.

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.

The Arabidopsis cytosolic proteome

DEFF Research Database (Denmark)

Ito, Jun; Parsons, Harriet Tempé; Heazlewood, Joshua L.

2014-01-01

compartments. However, a detailed study of enriched cytosolic fractions from Arabidopsis cell culture has been performed only recently, with over 1,000 proteins reproducibly identified by mass spectrometry. The number of proteins allocated to the cytosol nearly doubles to 1,802 if a series of targeted...

[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.

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.

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

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.

X-ray microscopy of human malaria

International Nuclear Information System (INIS)

Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W.

1997-01-01

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease

X-ray microscopy of human malaria

Energy Technology Data Exchange (ETDEWEB)

Magowan, C.; Brown, J.T.; Mohandas, N.; Meyer-Ilse, W. [Ernest Orlando Lawrence Berkeley National Lab., CA (United States)

1997-04-01

Associations between intracellular organisms and host cells are complex and particularly difficult to examine. X-ray microscopy provides transmission images of subcellular structures in intact cells at resolutions superior to available methodologies. The spatial resolution is 50-60nm with a 1 micron depth of focus, superior to anything achievable with light microscopy. Image contrast is generated by differences in photoelectric absorption by the atoms in different areas (i.e. subcellular structures) throughout the full thickness of the sample. Absorption due to carbon dominates among all the elements in the sample at 2.4 nm x-ray wavelength. Thus images show features or structures, in a way not usually seen by other types of microscopy. The authors used soft x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes, and in infected erythrocytes treated with compounds that have anti-malarial effects. X-ray microscopy showed newly elaborated structures in the cytosol of unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. Better understanding of the process of intracellular parasite maturation and the interactions between the parasite and its host erythrocyte can help define new approaches to the control of this deadly disease.

Biochemical responses to cadmium exposure in Oncorhynchus mykiss erythrocytes.

Science.gov (United States)

Orlando, Patrick; Silvestri, Sonia; Ferlizza, Enea; Andreani, Giulia; Carpenè, Emilio; Falcioni, Giancarlo; Tiano, Luca; Isani, Gloria

2017-11-01

Cd is known for its carcinogenic effects, however its mechanism of toxicity and in particular its ability to promote oxidative stress is debated. In fact, although it is considered a redox-inactive metal, at high concentration Cd was shown to promote indirectly oxidative stress. In this study we investigated metal accumulation in ex vivo exposed trout (Oncorhynchus mykiss) erythrocytes and Cd dose-dependent effect in terms of RBC viability, cytosolic and mitochondrial ROS levels as well as its effects on mitochondrial membrane depolarization, hemoglobin stability and precipitation. In the concentration range used, Cd did not affect cell viability. However, metal accumulation was associated with an increase in all oxidative indexes evaluated, except mitochondrial superoxide anion production that, on the contrary, was significantly decreased, probably due to a lowered respiration rate associated with interference of Cd with complex I, II and III, as suggested by the observed Cd-dependent mitochondrial membrane depolarization. On the other hand, hemoglobin destabilisation seems to be the major trigger of oxidative stress in this cell type. Copyright © 2017. Published by Elsevier Inc.

Erythrocyte 3H-ouabain binding and digitalis treatment in ethanol addicted patients

International Nuclear Information System (INIS)

Battaini, F.; Govoni, S.; Mauri, A.; Civelli, L.; Trabucchi, M.

1987-01-01

The binding of 3 H-ouabain to human erythrocytes was analyzed in a population of hospitalized male ethanol addicted patients under long term digitalis treatment. In the non-alcoholic patient group the long term digitalis treatment induced an increase in Bmax and Kd values; such modification was not observed in the alcoholic patients. Chronic alcohol intake itself induced an increase in 3 H-ouabain kinetic parameters. These observations confirm that ouabain binding to human erythrocytes is subject to pharmacological and toxicological regulation and that adaptive changes in peripheral tissues can be useful in predicting possible parallel modifications in other less accessible tissues. 22 references, 1 table

Detection of metalloproteins in human liver cytosol by synchrotron radiation X-ray fluorescence after sodium dodecyl sulphate polyacrylamide gel electrophoresis

International Nuclear Information System (INIS)

Gao Yuxi; Chen Chunying; Zhang Peiqun; Chai Zhifang; He Wei; Huang Yuying

2003-01-01

An improved method of analysis of metals in protein bands with synchrotron radiation X-ray fluorescence (SRXRF) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation is introduced and applied to human liver cytosol. Through a step of drying the gel before SRXRF determination, the continuous background resulting mainly from the Compton-scattering of X-rays by the gel matrix was substantially reduced, and the detection of biological trace elements, such as Cu, Fe, and Zn in protein bands was thereby made possible. With the new procedure, six Zn-containing proteins with molecular weights (MWs) of 17.5, 20.5, 27, 35, 55, and 63 kDa, respectively were found in human liver cytosol, among which the 63 kDa Zn-containing band was shown to be the dominant form of zinc. In addition, at least four Fe containing proteins with MWs of 20, 23, 43, and 83.5 kDa, respectively, were present in the samples. The metal contents in some metalloproteins, such as the 63 kDa Zn-containing protein, the 23 and 83.5 kDa Fe-containing proteins, and a 22 kDa Cu-containing protein were more closely related to the metal level in the sample. It is demonstrated that the procedure could be widely used to further investigate metal-binding proteins in biological samples

Effect of gamma irradiation on membranes of normal and pathological erythrocytes (beta-thalassemia)

International Nuclear Information System (INIS)

Sportelli, L.; Bonincontro, A.; Cametti, C.; Consiglio Nazionale delle Ricerche, Rome

1987-01-01

The influence of ionizing radiation on the membrane of human normal erythrocytes has extensively been studied and a variety of effects including changes in the cation fluxes or in non-electrolytes permeability, in membrane fluidity, in peroxidation of unsaturated lipids as well as chemical composition or structural modifications has been observed. However, only few studies deal with the effects of ionizing radiation on pathological red blood cells. In this work, we have investigated by means of electron spin resonance (ESR) spectroscopy the effects of 60 Co γ-radiation on the normal and homozygous β-thalassemic human erythrocyte membranes. (orig.)

P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes.

Science.gov (United States)

Valton, Emeline; Amblard, Christian; Wawrzyniak, Ivan; Penault-Llorca, Frederique; Bamdad, Mahchid

2013-12-05

Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous "membrane detoxification proteins" implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality.

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

Effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice

International Nuclear Information System (INIS)

Meng Qingyong; Chen Shali; Liu Shuzheng

2003-01-01

Objective: To the effect of low dose radiation on thymocyte cytosol and nuclei protein synthesis in mice. Methods: The expression of proteins was analyzed by gel filtration with Sephadex G-100 and HPLC based on separation of proteins on thymocyte cytosol and nuclei after whole-body irradiation with 75 mGy X-rays and sham-irradiation, and their biological activity was examined by mouse splenocyte proliferation and chromosome aberration of human peripheral blood lymphocytes. Results: HPLC analysis showed that there was a marked increase in expression of 61.4 kD protein in the extract of thymocyte cytosol and 30.4 kD protein in the extract of thymocyte nuclei in comparison with the corresponding fractions from the sham-irradiated control mice. These protein fractions from the thymocyte cytosol and nuclei of the irradiated mice showed both stimulating effect on normal T cell proliferation and protective effect on chromosome damage induced by high dose radiation. Conclusion: These findings might have implications in study of mechanism of immunoenhancement and cytogenetic adaptive response induced by low dose radiation

LeftyA sensitive cytosolic pH regulation and glycolytic flux in Ishikawa human endometrial cancer cells

Energy Technology Data Exchange (ETDEWEB)

Salker, Madhuri S.; Zhou, Yuetao; Singh, Yogesh [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany); Brosens, Jan [Division of Reproductive Health, Warwick Medical School, Clinical Sciences Research Laboratories, University Hospital, Coventry CV2 2DX (United Kingdom); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tuebingen, 72076 Tuebingen (Germany)

2015-05-08

Objective: LeftyA, a powerful regulator of stemness, embryonic differentiation, and reprogramming of cancer cells, counteracts cell proliferation and tumor growth. Key properties of tumor cells include enhanced glycolytic flux, which is highly sensitive to cytosolic pH and thus requires export of H{sup +} and lactate. H{sup +} extrusion is in part accomplished by Na{sup +}/H{sup +} exchangers, such as NHE1. An effect of LeftyA on transport processes has, however, never been reported. The present study thus explored whether LeftyA modifies regulation of cytosolic pH (pHi) in Ishikawa cells, a well differentiated endometrial carcinoma cell model. Methods: NHE1 transcript levels were determined by qRT-PCR, NHE1 protein abundance quantified by Western blotting, pH{sub i} estimated utilizing (2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein [BCECF] fluorescence, Na{sup +}/H{sup +} exchanger activity from Na{sup +} dependent realkalinization after an ammonium pulse, and lactate concentration in the supernatant utilizing an enzymatic assay and subsequent colorimetry. Results: A 2 h treatment with LeftyA (8 ng/ml) significantly decreased NHE1 transcript levels (by 99.6%), NHE1 protein abundance (by 71%), Na{sup +}/H{sup +} exchanger activity (by 55%), pHi (from 7.22 ± 0.02 to 7.05 ± 0.02), and lactate release (by 41%). Conclusions: LeftyA markedly down-regulates NHE1 expression, Na{sup +}/H{sup +} exchanger activity, pHi, and lactate release in Ishikawa cells. Those effects presumably contribute to cellular reprogramming and growth inhibition. - Highlights: • LeftyA, an inhibitor of tumor growth, reduces Na{sup +}/H{sup +}-exchanger activity by 55%. • LeftyA decreases NHE1 transcripts by 99.6% and NHE1 protein by 71%. • LeftyA decreases cytosolic pH from 7.22 ± 0.02 to 7.05 ± 0.02. • Cytosolic acidification by Lefty A decreases glycolysis by 41%. • Cytosolic acidification by Lefty A compromises energy production of tumor cells.

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.

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

Role of erythrocyte tropomodulin in the biomechanics and topology of the erythrocyte membrane skeletal network

OpenAIRE

Green, Terrell Ann

2010-01-01

The erythrocyte membrane skeleton is a multi-protein complex providing mechanical properties and stability to erythrocytes. Defects in the skeleton can manifest in dysfunction and disease such as hemolytic anemia. Erythrocyte tropomodulin (E-Tmod) is a slow-growing end actin-capping protein and has been proposed that together with tropomyosin 5 or 5b they form a "molecular ruler" which dictates protofilament length of 37 nm in the network. In this study, the role for E-Tmod in the network org...

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.

Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation

Science.gov (United States)

Kirkby, Nicholas S.; Reed, Daniel M.; Edin, Matthew L.; Rauzi, Francesca; Mataragka, Stefania; Vojnovic, Ivana; Bishop-Bailey, David; Milne, Ginger L.; Longhurst, Hilary; Zeldin, Darryl C.; Mitchell, Jane A.; Warner, Timothy D.

2016-01-01

Eicosanoids are important vascular regulators, but the phospholipase A2 (PLA2) isoforms supporting their production within the cardiovascular system are not fully understood. To address this, we have studied platelets, endothelial cells, and leukocytes from 2 siblings with a homozygous loss-of-function mutation in group IVA cytosolic phospholipase A2 (cPLA2α). Chromatography/mass spectrometry was used to determine levels of a broad range of eicosanoids produced by isolated vascular cells, and in plasma and urine. Eicosanoid release data were paired with studies of cellular function. Absence of cPLA2α almost abolished eicosanoid synthesis in platelets (e.g., thromboxane A2, control 20.5 ± 1.4 ng/ml vs. patient 0.1 ng/ml) and leukocytes [e.g., prostaglandin E2 (PGE2), control 21.9 ± 7.4 ng/ml vs. patient 1.9 ng/ml], and this was associated with impaired platelet activation and enhanced inflammatory responses. cPLA2α-deficient endothelial cells showed reduced, but not absent, formation of prostaglandin I2 (prostacyclin; control 956 ± 422 pg/ml vs. patient 196 pg/ml) and were primed for inflammation. In the urine, prostaglandin metabolites were selectively influenced by cPLA2α deficiency. For example, prostacyclin metabolites were strongly reduced (18.4% of control) in patients lacking cPLA2α, whereas PGE2 metabolites (77.8% of control) were similar to healthy volunteer levels. These studies constitute a definitive account, demonstrating the fundamental role of cPLA2α to eicosanoid formation and cellular responses within the human circulation.—Kirkby, N. S., Reed, D. M., Edin, M. L., Rauzi, F., Mataragka, S., Vojnovic, I., Bishop-Bailey, D., Milne, G. L., Longhurst, H., Zeldin, D. C., Mitchell, J. A., Warner, T. D. Inherited human group IVA cytosolic phospholipase A2 deficiency abolishes platelet, endothelial, and leucocyte eicosanoid generation. PMID:26183771

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.

The mechanism of erythrocyte sedimentation. Part 2: The global collapse of settling erythrocyte network.

Science.gov (United States)

Pribush, A; Meyerstein, D; Meyerstein, N

2010-01-01

Results reported in the companion paper showed that erythrocytes in quiescent blood are combined into a network followed by the formation of plasma channels within it. This study is focused on structural changes in the settling dispersed phase subsequent to the channeling and the effect of the structural organization on the sedimentation rate. It is suggested that the initial, slow stage of erythrocyte sedimentation is mainly controlled by the gravitational compactness of the collapsed network. The lifetime of RBC network and hence the duration of the slow regime of erythrocyte sedimentation decrease with an increase in the intercellular pair potential and with a decrease in Hct. The gravitational compactness of the collapsed network causes its rupture into individual fragments. The catastrophic collapse of the network transforms erythrocyte sedimentation from slow to fast regime. The size of RBC network fragment is insignificantly affected by Hct and is mainly determined by the intensity of intercellular attractive interactions. When cells were suspended in the weak aggregating medium, the Stokes radius of fragments does not differ measurably from that of individual RBCs. The proposed mechanism provides a reasonable explanation of the effects of RBC aggregation, Hct and the initial height of the blood column on the delayed erythrocyte sedimentation.

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

Redox characteristics of the eukaryotic cytosol

DEFF Research Database (Denmark)

López-Mirabal, H Reynaldo; Winther, Jakob R

2007-01-01

The eukaryotic cytoplasm has long been regarded as a cellular compartment in which the reduced state of protein cysteines is largely favored. Under normal conditions, the cytosolic low-molecular weight redox buffer, comprising primarily of glutathione, is highly reducing and reactive oxygen species...... (ROS) and glutathionylated proteins are maintained at very low levels. In the present review, recent progress in the understanding of the cytosolic thiol-disulfide redox metabolism and novel analytical approaches to studying cytosolic redox properties are discussed. We will focus on the yeast model...... organism, Saccharomyces cerevisiae, where the combination of genetic and biochemical approaches has brought us furthest in understanding the mechanisms underlying cellular redox regulation. It has been shown in yeast that, in addition to the enzyme glutathione reductase, other mechanisms may exist...

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...

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.

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.

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

EFFECT OF PESTICIDES ON RAT (Rattus norvegicus ERYTHROCYTES ANTIOXIDANT ENZYMES IN VITRO

Directory of Open Access Journals (Sweden)

Jasna Friščić

2014-10-01

Full Text Available Abstract: In the last century, maximum in herbicide production was achieved. Growing use of herbicides initiated the need for continuous evaluation of damaging effects of herbicides on human health and environment. Paraquat is the trade name for N,N′-dimethyl-4,4′-bipyridinium dichloride and one of the most widely used herbicides in the world. Although mechanism of paraquat toxicity remains undefined, a great portion of toxicity is attributed to the process of redox cycling. In this research, rat erythrocytes were exposed to various paraquat concentrations (0, 0.25, 0.5, 0.75, 1.25 mM. Changes in antioxidant enzymes activity, catalase and superoxide dismutase were determined, and also the activity of erythrocyte acetylcholinesterase. Obtained results show damaging effects of paraquat on erythrocytes due to oxidative stress.

Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models

Science.gov (United States)

Wen, Li; Voronina, Svetlana; Javed, Muhammad A.; Awais, Muhammad; Szatmary, Peter; Latawiec, Diane; Chvanov, Michael; Collier, David; Huang, Wei; Barrett, John; Begg, Malcolm; Stauderman, Ken; Roos, Jack; Grigoryev, Sergey; Ramos, Stephanie; Rogers, Evan; Whitten, Jeff; Velicelebi, Gonul; Dunn, Michael; Tepikin, Alexei V.; Criddle, David N.; Sutton, Robert

2015-01-01

Background & Aims Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release–activated calcium modulator ORAI1 is the most abundant Ca2+ entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice. Methods Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects. Results GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca2+ currents after Ca2+ release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis. Conclusions Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis. ORAI1 inhibitors might be developed

Cytosolic delivery of materials with endosome-disrupting colloids

Science.gov (United States)

Helms, Brett A.; Bayles, Andrea R.

2016-03-15

A facile procedure to deliver nanocrystals to the cytosol of live cells that is both rapid and general. The technique employs a unique cationic core-shell polymer colloid that directs nanocrystals to the cytosol of living cells within a few hours of incubation. The present methods and compositions enable a host of advanced applications arising from efficient cytosolic delivery of nanocrystal imaging probes: from single particle tracking experiments to monitoring protein-protein interactions in live cells for extended periods.

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.

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

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

International Nuclear Information System (INIS)

Kaperonis, A.A.; Chien, S.

1989-01-01

Plasma 125 I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the 125 I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies

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 ...

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

Cytosolic PrP Can Participate in Prion-Mediated Toxicity

Science.gov (United States)

Thackray, Alana M.; Zhang, Chang; Arndt, Tina

2014-01-01

ABSTRACT Prion diseases are characterized by a conformational change in the normal host protein PrPC. While the majority of mature PrPC is tethered to the plasma membrane by a glycosylphosphatidylinositol anchor, topological variants of this protein can arise during its biosynthesis. Here we have generated Drosophila transgenic for cytosolic ovine PrP in order to investigate its toxic potential in flies in the absence or presence of exogenous ovine prions. While cytosolic ovine PrP expressed in Drosophila was predominantly detergent insoluble and showed resistance to low concentrations of proteinase K, it was not overtly detrimental to the flies. However, Drosophila transgenic for cytosolic PrP expression exposed to classical or atypical scrapie prion inocula showed a faster decrease in locomotor activity than similar flies exposed to scrapie-free material. The susceptibility to classical scrapie inocula could be assessed in Drosophila transgenic for panneuronal expression of cytosolic PrP, whereas susceptibility to atypical scrapie required ubiquitous PrP expression. Significantly, the toxic phenotype induced by ovine scrapie in cytosolic PrP transgenic Drosophila was transmissible to recipient PrP transgenic flies. These data show that while cytosolic PrP expression does not adversely affect Drosophila, this topological PrP variant can participate in the generation of transmissible scrapie-induced toxicity. These observations also show that PrP transgenic Drosophila are susceptible to classical and atypical scrapie prion strains and highlight the utility of this invertebrate host as a model of mammalian prion disease. IMPORTANCE During prion diseases, the host protein PrPC converts into an abnormal conformer, PrPSc, a process coupled to the generation of transmissible prions and neurotoxicity. While PrPC is principally a glycosylphosphatidylinositol-anchored membrane protein, the role of topological variants, such as cytosolic PrP, in prion-mediated toxicity and

Erythrocyte Glutathione S-transferase Activity of Non-Malarious Male ...

African Journals Online (AJOL)

Hilaire

Cameroon Journal of Experimental Biology 2009 Vol. 05 N° 02, 112-116. ... GST activity from the inhibitory action of the drugs. The results of these findings suggested the capability of these drugs to bind to the human erythrocyte GST, accompanied with ... of the five antimalarial drugs constituted the control sample analysis.

Tirilazad mesylate protects stored erythrocytes against osmotic fragility.

Science.gov (United States)

Epps, D E; Knechtel, T J; Bacznskyj, O; Decker, D; Guido, D M; Buxser, S E; Mathews, W R; Buffenbarger, S L; Lutzke, B S; McCall, J M

1994-12-01

The hypoosmotic lysis curve of freshly collected human erythrocytes is consistent with a single Gaussian error function with a mean of 46.5 +/- 0.25 mM NaCl and a standard deviation of 5.0 +/- 0.4 mM NaCl. After extended storage of RBCs under standard blood bank conditions the lysis curve conforms to the sum of two error functions instead of a possible shift in the mean and a broadening of a single error function. Thus, two distinct sub-populations with different fragilities are present instead of a single, broadly distributed population. One population is identical to the freshly collected erythrocytes, whereas the other population consists of osmotically fragile cells. The rate of generation of the new, osmotically fragile, population of cells was used to probe the hypothesis that lipid peroxidation is responsible for the induction of membrane fragility. If it is so, then the antioxidant, tirilazad mesylate (U-74,006f), should protect against this degradation of stored erythrocytes. We found that tirilazad mesylate, at 17 microM (1.5 mol% with respect to membrane lecithin), retards significantly the formation of the osmotically fragile RBCs. Concomitantly, the concentration of free hemoglobin which accumulates during storage is markedly reduced by the drug. Since the presence of the drug also decreases the amount of F2-isoprostanes formed during the storage period, an antioxidant mechanism must be operative. These results demonstrate that tirilazad mesylate significantly decreases the number of fragile erythrocytes formed during storage in the blood bank.

Reduced Plasmodium vivax erythrocyte infection in PNG Duffy-negative heterozygotes.

Science.gov (United States)

Kasehagen, Laurin J; Mueller, Ivo; Kiniboro, Benson; Bockarie, Moses J; Reeder, John C; Kazura, James W; Kastens, Will; McNamara, David T; King, Charles H; Whalen, Christopher C; Zimmerman, Peter A

2007-03-28

Erythrocyte Duffy blood group negativity reaches fixation in African populations where Plasmodium vivax (Pv) is uncommon. While it is known that Duffy-negative individuals are highly resistant to Pv erythrocyte infection, little is known regarding Pv susceptibility among heterozygous carriers of a Duffy-negative allele (+/-). Our limited knowledge of the selective advantages or disadvantages associated with this genotype constrains our understanding of the effect that interventions against Pv may have on the health of people living in malaria-endemic regions. We conducted cross-sectional malaria prevalence surveys in Papua New Guinea (PNG), where we have previously identified a new Duffy-negative allele among individuals living in a region endemic for all four human malaria parasite species. We evaluated infection status by conventional blood smear light microscopy and semi-quantitative PCR-based strategies. Analysis of a longitudinal cohort constructed from our surveys showed that Duffy heterozygous (+/-) individuals were protected from Pv erythrocyte infection compared to those homozygous for wild-type alleles (+/+) (log-rank tests: LM, p = 0.049; PCR, p = 0.065). Evaluation of Pv parasitemia, determined by semi-quantitative PCR-based methods, was significantly lower in Duffy +/- vs. +/+ individuals (Mann-Whitney U: p = 0.023). Overall, we observed no association between susceptibility to P. falciparum erythrocyte infection and Duffy genotype. Our findings provide the first evidence that Duffy-negative heterozygosity reduces erythrocyte susceptibility to Pv infection. As this reduction was not associated with greater susceptibility to Pf malaria, our in vivo observations provide evidence that Pv-targeted control measures can be developed safely.

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

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.

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....

NMR studies of the fate of adenine nucleotides in glucose-starved erythrocytes

International Nuclear Information System (INIS)

Bubb, W.A.; Mulquiney, P.J.; Kuchel, P.W.; Rohwer, J.; De Atauri, P.

2002-01-01

Full text: As a consequence of many refinements during the past 30 years, we now have a detailed understanding of the glycolytic pathway in human erythrocytes. By comparison, and notwithstanding their central importance to four key steps in erythrocyte glycolysis, our knowledge of the catabolism of adenine nucleotides remains relatively limited. In particular, the mechanism for the degradation of AMP, whose concentration rises under conditions of oxidative stress or glucose deprivation, remains poorly understood, AMP degradation may proceed via two possible pathways which converge in the production of inosine. Analysis of the key intermediates for the respective pathways, adenosine and AMP, as well as determination of end products is not straightforward. High-resolution NMR spectroscopy affords a potentially simple analytical solution to this problem but is complicated by spectral overlap and the sensitivity of key resonances to variations in pH and the concentrations of cations such as Mg 2+ . We describe a multinuclear NMR approach towards characterising the intermediates and end-products of adenine nucleotide metabolism in glucose-starved human erythrocytes. Assignments based on homo- and heteronuclear correlation experiments for both 13 C and 31 P are presented

Cytosolic iron chaperones: Proteins delivering iron cofactors in the cytosol of mammalian cells.

Science.gov (United States)

Philpott, Caroline C; Ryu, Moon-Suhn; Frey, Avery; Patel, Sarju

2017-08-04

Eukaryotic cells contain hundreds of metalloproteins that are supported by intracellular systems coordinating the uptake and distribution of metal cofactors. Iron cofactors include heme, iron-sulfur clusters, and simple iron ions. Poly(rC)-binding proteins are multifunctional adaptors that serve as iron ion chaperones in the cytosolic/nuclear compartment, binding iron at import and delivering it to enzymes, for storage (ferritin) and export (ferroportin). Ferritin iron is mobilized by autophagy through the cargo receptor, nuclear co-activator 4. The monothiol glutaredoxin Glrx3 and BolA2 function as a [2Fe-2S] chaperone complex. These proteins form a core system of cytosolic iron cofactor chaperones in mammalian cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

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.

21 CFR 864.7360 - Erythrocytic glucose-6-phosphate dehydrogenase assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Erythrocytic glucose-6-phosphate dehydrogenase assay. 864.7360 Section 864.7360 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages...

New insights into the posttranslational regulation of human cytosolic thioredoxin by S-palmitoylation

Energy Technology Data Exchange (ETDEWEB)

Xu, Zhiyu; Zhong, Liangwei, E-mail: liazho@ucas.ac.cn

2015-05-15

High level of palmitate is associated with metabolic disorders. We recently showed that enhanced level of S-palmitoylated cytosolic thioredoxin (Trx1) in mouse liver was new characteristic feature of insulin resistance. However, our understanding of the effect of S-palmitoylation on Trx1 is limited, and the tissue specificity of Trx1 S-palmitoylation is unclear. Here we show that S-palmitoylation also occurs at Cys73 of Trx1 in living endothelial cells, and the level of S-palmitoylated Trx1 undergoes regulation by insulin signaling. Trx1 prefers thiol-thioester exchange with palmitoyl-CoA to acetyl-CoA. S-palmitoylation alters conformation or secondary structure of Trx1, as well as decreases the ability of Trx1 to transfer electrons from thioredoxin reductase to S-nitrosylated protein–tyrosine phosphatase 1B and S-nitroso-glutathione. Our results demonstrate that S-palmitoylation is an important post-translational modification of human Trx1. - Highlights: • S-palmitoylation occurs at Cys73 of Trx1 in living endothelial cells. • Insulin signaling may regulate level of S-palmitoylated Trx1 in the cells. • S-palmitoylation plays significant effects on Trx1 structure and functions.

Allosensibilisation to erythrocyte antigens (literature review

Directory of Open Access Journals (Sweden)

N. V. Mineeva

2015-01-01

Full Text Available In this article literature review of the causes of allosensibilisation to erythrocyte antigens are presented. It is shown that the ability to produce antierythrocyte antibodies is affected by many factors, principal of whom it is difficult to identify. For the allosensibilisation development requires genetically determined differences in erythrocyte antigens phenotypes of donor and recipient, mother and fetus, which can lead to immune response and antibodies production. The biochemical nature of erythrocyte antigens, antigen dose (the amount of transfused doses, the number of antigens determinants on donor and fetus erythrocytes, the number of pregnancies are important. Individual patient characteristics: age, gender, diseases, the use of immunosuppressive therapy and the presence of inflammatory processes, are also relevant. Note that antibody to one erythrocyte antigens have clinical value, and to the other – have no. The actual data about frequency of clinically significant antibodies contribute to the development of post-transfusion hemolytic complications prophylaxis as well as the improvement of laboratory diagnosis of hemolytic disease of the newborn in the presence of maternal antierythrocyte antibodies.

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.

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.)

Effects of centrifugation on transmembrane water loss from normal and pathologic erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Kaperonis, A.A.; Chien, S.

1989-02-01

Plasma /sup 125/I-albumin was used as a marker of extracellular dilution in order to study the effect of high-speed centrifugation on transmembrane water distribution in several types of human red cells, including normal (AA), hemoglobin variants (beta A, AS, SC, beta S, and SS), and those from patients with hereditary spherocytosis. SS and AA erythrocytes were also examined for changes in intracellular hemoglobin concentration of three different density fractions and with increasing duration of spin. The minimum force and duration of centrifugation required to impair water permeability were found to vary with the red cell type, the anticoagulant used (heparin or EDTA), the initial hematocrit of the sample centrifuged, as well as among the individual erythrocyte fractions within the same sample. When subjecting pathologic erythrocytes to high-speed centrifugation, the /sup 125/I-albumin dilution technique can be used to determine whether the centrifugation procedure has led to an artifactual red cell water loss and to correct for this when it does occur. An abnormal membrane susceptibility to mechanical stress was demonstrated in erythrocytes from patients with hereditary spherocytosis and several hemoglobinopathies.

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)

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.

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

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

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

Cytosolic cholesterol ester hydrolase in adrenal cortex

OpenAIRE

Tocher, Douglas R.

1983-01-01

Cholesterol ester hydrolase (CEH) in adrenocortical cytosol was known to be phosphorylated and activated, in response to ACTH in a cAMPdependent protein kinase mediated process. The purification of CEH from bovine adrenocortical cytosol was attempted. The use of detergents to solubilise the enzyme from lipid-rich aggregates was investigated and sodium cholate was found to be effective. A purification procedure using cholate solubilised enzyme was developed. The detergent int...

The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling

Energy Technology Data Exchange (ETDEWEB)

Bernard, S.M.; Habash, D.Z.

2009-07-02

Glutamine synthetase assimilates ammonium into amino acids, thus it is a key enzyme for nitrogen metabolism. The cytosolic isoenzymes of glutamine synthetase assimilate ammonium derived from primary nitrogen uptake and from various internal nitrogen recycling pathways. In this way, cytosolic glutamine synthetase is crucial for the remobilization of protein-derived nitrogen. Cytosolic glutamine synthetase is encoded by a small family of genes that are well conserved across plant species. Members of the cytosolic glutamine synthetase gene family are regulated in response to plant nitrogen status, as well as to environmental cues, such as nitrogen availability and biotic/abiotic stresses. The complex regulation of cytosolic glutamine synthetase at the transcriptional to post-translational levels is key to the establishment of a specific physiological role for each isoenzyme. The diverse physiological roles of cytosolic glutamine synthetase isoenzymes are important in relation to current agricultural and ecological issues.

The malaria parasite RhopH protein complex interacts with erythrocyte calmyrin identified from a comprehensive erythrocyte protein library.

Science.gov (United States)

Miura, Toyokazu; Takeo, Satoru; Ntege, Edward H; Otsuki, Hitoshi; Sawasaki, Tatsuya; Ishino, Tomoko; Takashima, Eizo; Tsuboi, Takafumi

2018-06-02

Malaria merozoite apical organelles; microneme and rhoptry secreted proteins play functional roles during and following invasion of host erythrocytes. Among numerous proteins, the rhoptries discharge high molecular weight proteins known as RhopH complex. Recent reports suggest that the RhopH complex is essential for growth and survival of the malaria parasite within erythrocytes. However, an in-depth understanding of the host-parasite molecular interactions is indispensable. Here we utilized a comprehensive mouse erythrocyte protein library consisting of 443 proteins produced by a wheat germ cell-free system, combined with AlphaScreen technology to identify mouse erythrocyte calmyrin as an interacting molecule of the rodent malaria parasite Plasmodium yoelii RhopH complex (PyRhopH). The PyRhopH interaction was dependent on the calmyrin N-terminus and divalent cation capacity. The finding unveils a recommendable and invaluable usefulness of our comprehensive mouse erythrocyte protein library together with the AlphaScreen technology in investigating a wide-range of host-parasite molecular interactions. Copyright © 2018 Elsevier Inc. All rights reserved.

Paired Chicken and Mammalian Erythrocyte Indicator Systems for ...

African Journals Online (AJOL)

Three levels of erythrocytes suspensions, 1.5%, 1% and 0.5% respectively from goat and guinea pig, were compared to conventional 0.5% chicken erythrocytes, in an attempt to investigate the suitability for the two sources of mammalian erythrocytes as indicators for Newcastle disease virus haemagglutination (HA) tests.

Effects of dietary fat on lipid composition of serum and erythrocytes of the swine and in vitro incorporation of fatty acids into erythrocyte membranes

International Nuclear Information System (INIS)

Sato, Hiroaki

1974-01-01

Changes in ftty acid patterns of lipids in serum and erythrocytes induced by dietary fats and in vitro incorporation of fatty acids into erythrocyte membranes were investigated with pigs. On feeding various diets, it was found that fatty acid composition of serum and erythrocytes could be modified and altered toward the fatty acid pattern of the diet. In vitro, the incorporation of labelled fatty acids into erythrocyte membranes was accelerated by the addition of cofactors such as lysolecithin, CoA and ATP. Dietary fats also had certain effects on the incorporation of fatty acids into erythrocyte membranes. Erythrocytes, collected from the blood of pigs fed corn oil, incorporated and also released more labelled linoleate than those of pigs fed hydrogenated soybean oil. Palmitic acid was more slowly incorporated into erythrocyte membranes than linoleic acid in the pigs fed both a commercial chow and scheduled meals, indicating selective esterification of fatty acids in the erythrocyte membranes. (author)

Impact of Ficoll density gradient centrifugation on major and trace element concentrations in erythrocytes and blood plasma.

Science.gov (United States)

Lu, Ying; Ahmed, Sultan; Harari, Florencia; Vahter, Marie

2015-01-01

Ficoll density gradient centrifugation is widely used to separate cellular components of human blood. We evaluated the suitability to use erythrocytes and blood plasma obtained from Ficoll centrifugation for assessment of elemental concentrations. We determined 22 elements (from Li to U) in erythrocytes and blood plasma separated by direct or Ficoll density gradient centrifugation, using inductively coupled plasma mass spectrometry. Compared with erythrocytes and blood plasma separated by direct centrifugation, those separated by Ficoll had highly elevated iodine and Ba concentration, due to the contamination from the Ficoll-Paque medium, and about twice as high concentrations of Sr and Mo in erythrocytes. On the other hand, the concentrations of Ca in erythrocytes and plasma were markedly reduced by the Ficoll separation, to some extent also Li, Co, Cu, and U. The reduced concentrations were probably due to EDTA, a chelator present in the Ficoll medium. Arsenic concentrations seemed to be lowered by Ficoll, probably in a species-specific manner. The concentrations of Mg, P, S, K, Fe, Zn, Se, Rb, and Cs were not affected in the erythrocytes, but decreased in plasma. Concentrations of Mn, Cd, and Pb were not affected in erythrocytes, but in plasma affected by EDTA and/or pre-analytical contamination. Ficoll separation changed the concentrations of Li, Ca, Co, Cu, As, Mo, I, Ba, and U in erythrocytes and blood plasma, Sr in erythrocytes, and Mg, P, S, K, Fe, Zn, Se, Rb and Cs in blood plasma, to an extent that will invalidate evaluation of deficiencies or excess intakes. Copyright © 2014 Elsevier GmbH. All rights reserved.

Erythrocyte enrichment in hematopoietic progenitor cell cultures based on magnetic susceptibility of the hemoglobin.

Directory of Open Access Journals (Sweden)

Xiaoxia Jin

Full Text Available Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A, hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes.

Erythrocyte Saturation with IgG Is Required for Inducing Antibody-Mediated Immune Suppression and Impacts Both Erythrocyte Clearance and Antigen-Modulation Mechanisms.

Science.gov (United States)

Cruz-Leal, Yoelys; Marjoram, Danielle; Lazarus, Alan H

2018-02-15

Anti-D prevents hemolytic disease of the fetus and newborn, and this mechanism has been referred to as Ab-mediated immune suppression (AMIS). Anti-D, as well as other polyclonal AMIS-inducing Abs, most often induce both epitope masking and erythrocyte clearance mechanisms. We have previously observed that some Abs that successfully induce AMIS effects could be split into those that mediate epitope masking versus those that induce erythrocyte clearance, allowing the ability to analyze these mechanisms separately. In addition, AMIS-inducing activity has recently been shown to induce Ag modulation (Ag loss from the erythrocyte surface). To assess these mechanisms, we immunized mice with transgenic murine RBCs expressing a single Ag protein comprising a recombinant Ag composed of hen egg lysozyme, OVA sequences comprising aa 251-349, and the human Duffy transmembrane protein (HOD-Ag) with serial doses of polyclonal anti-OVA IgG as the AMIS-inducing Ab. The anti-OVA Ab induced AMIS in the absence of apparent epitope masking. AMIS occurred only when the erythrocytes appeared saturated with IgG. This Ab was capable of inducing HOD-RBC clearance, as well as loss of the OVA epitope at doses of Ab that caused AMIS effects. HOD-RBCs also lost reactivity with Abs specific for the hen egg lysozyme and Duffy portions of the Ag consistent with the initiation of Ag modulation and/or trogocytosis mechanisms. These data support the concept that an AMIS-inducing Ab that does not cause epitope masking can induce AMIS effects in a manner consistent with RBC clearance and/or Ag modulation. Copyright © 2018 by The American Association of Immunologists, Inc.

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...

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...

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.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

Energy Technology Data Exchange (ETDEWEB)

Wang, Hui; Klein, Michael G.; Snell, Gyorgy; Lane, Weston; Zou, Hua; Levin, Irena; Li, Ke; Sang, Bi-Ching (Takeda Cali)

2016-07-01

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

International Nuclear Information System (INIS)

Ebner, Andreas; Hinterdorfer, Peter; Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann; Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika

2008-01-01

CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl - ) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients

Determination of CFTR densities in erythrocyte plasma membranes using recognition imaging

Energy Technology Data Exchange (ETDEWEB)

Ebner, Andreas; Hinterdorfer, Peter [Institute for Biophysics, University of Linz, A-4040 Linz (Austria); Nikova, Dessy; Lange, Tobias; Bruns, Reimer; Oberleithner, Hans; Schillers, Hermann [Institute of Physiology II, University of Muenster, D-48149 Muenster (Germany); Haeberle, Johannes; Falk, Sabine; Duebbers, Angelika [Department of Pediatrics, University Hospitals of Muenster, D-48149 Muenster (Germany)], E-mail: schille@uni-muenster.de

2008-09-24

CFTR (cystic fibrosis transmembrane conductance regulator) is a cAMP-regulated chloride (Cl{sup -}) channel that plays an important role in salt and fluid movement across epithelia. Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR. The most predominant mutation, F508del, disturbs CFTR protein trafficking, resulting in a reduced number of CFTR in the plasma membrane. Recent studies indicate that CFTR is not only found in epithelia but also in human erythrocytes. Although considerable attempts have been made to quantify CFTR in cells, conclusions on numbers of CFTR molecules localized in the plasma membrane have been drawn indirectly. AFM has the power to provide the needed information, since both sub-molecular spatial resolution and direct protein recognition via antibody-antigen interaction can be observed. We performed a quantification study of the CFTR copies in erythrocyte membranes at the single molecule level, and compared the difference between healthy donors and CF patients. We detected that the number of CFTR molecules is reduced by 70% in erythrocytes of cystic fibrosis patients.

Cytosolic antibody delivery by lipid-sensitive endosomolytic peptide

Science.gov (United States)

Akishiba, Misao; Takeuchi, Toshihide; Kawaguchi, Yoshimasa; Sakamoto, Kentarou; Yu, Hao-Hsin; Nakase, Ikuhiko; Takatani-Nakase, Tomoka; Madani, Fatemeh; Gräslund, Astrid; Futaki, Shiroh

2017-08-01

One of the major obstacles in intracellular targeting using antibodies is their limited release from endosomes into the cytosol. Here we report an approach to deliver proteins, which include antibodies, into cells by using endosomolytic peptides derived from the cationic and membrane-lytic spider venom peptide M-lycotoxin. The delivery peptides were developed by introducing one or two glutamic acid residues into the hydrophobic face. One peptide with the substitution of leucine by glutamic acid (L17E) was shown to enable a marked cytosolic liberation of antibodies (immunoglobulins G (IgGs)) from endosomes. The predominant membrane-perturbation mechanism of this peptide is the preferential disruption of negatively charged membranes (endosomal membranes) over neutral membranes (plasma membranes), and the endosomolytic peptide promotes the uptake by inducing macropinocytosis. The fidelity of this approach was confirmed through the intracellular delivery of a ribosome-inactivation protein (saporin), Cre recombinase and IgG delivery, which resulted in a specific labelling of the cytosolic proteins and subsequent suppression of the glucocorticoid receptor-mediated transcription. We also demonstrate the L17E-mediated cytosolic delivery of exosome-encapsulated proteins.

The Aotus nancymaae erythrocyte proteome and its importance for biomedical research.

Science.gov (United States)

Moreno-Pérez, D A; García-Valiente, R; Ibarrola, N; Muro, A; Patarroyo, M A

2017-01-30

The Aotus nancymaae species has been of great importance in researching the biology and pathogenesis of malaria, particularly for studying Plasmodium molecules for including them in effective vaccines against such microorganism. In spite of the forgoing, there has been no report to date describing the biology of parasite target cells in primates or their biomedical importance. This study was thus designed to analyse A. nancymaae erythrocyte protein composition using MS data collected during a previous study aimed at characterising the Plasmodium vivax proteome and published in the pertinent literature. Most peptides identified were similar to those belonging to 1189 Homo sapiens molecules; >95% of them had orthologues in New World primates. GO terms revealed a correlation between categories having the greatest amount of proteins and vital cell function. Integral membrane molecules were also identified which could be possible receptors facilitating interaction with Plasmodium species. The A. nancymaae erythrocyte proteome is described here for the first time, as a starting point for more in-depth/extensive studies. The data reported represents a source of invaluable information for laboratories interested in carrying out basic and applied biomedical investigation studies which involve using this primate. An understanding of the proteomics characteristics of A. nancymaae erythrocytes represents a fascinating area for research regarding the study of the pathogenesis of malaria since these are the main target for Plasmodium invasion. However, and even though Aotus is one of the non-human primate models considered most appropriate for biomedical research, knowledge of its proteome, particularly its erythrocytes, remains unknown. According to the above and bearing in mind the lack of information about the A. nancymaae species genome and transcriptome, this study involved a search for primate proteins for comparing their MS/MS spectra with the available information for

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.

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)

Characterization of the cytosolic distribution of priority pollutant metals and metalloids in the digestive gland cytosol of marine mussels: seasonal and spatial variability.

Science.gov (United States)

Strižak, Zeljka; Ivanković, Dušica; Pröfrock, Daniel; Helmholz, Heike; Cindrić, Ana-Marija; Erk, Marijana; Prange, Andreas

2014-02-01

Cytosolic profiles of several priority pollutant metals (Cu, Cd, Zn, Pb) and metalloid As were analyzed in the digestive gland of the mussel (Mytilus galloprovincialis) sampled at locations with different environmental pollution levels along the Croatian coast in the spring and summer season. Size-exclusion chromatography (SEC) connected to inductively coupled plasma mass spectrometry (ICP-MS) was used to determine selected elements bound to cytosolic biomolecules separated based on their molecular size. Copper, cadmium and zinc eluted mostly associated with high molecular weight (HMW) and medium molecular weight (MMW) biomolecules, but with a more prominent elution in the MMW peak at polluted locations which were probably associated with the 20 kDa metallothionein (MT). Elution of all three metals within this peak was also strongly correlated with cytosolic Cd as strong inducer of MT. Lead mostly eluted in HMW biomolecule range, but in elevated cytosolic Pb concentrations, significant amount eluted in low molecular weight (LMW) biomolecules. Arsenic, on the other hand eluted almost completely in LMW range, but we could not distinguish specific molecular weight biomolecules which would be predominant in detoxification mechanism. Seasonal variability in element abundance within specific peaks was present, although not in the same extent, for all elements and locations, especially for As. The results confirm the suitability of the distribution of selected metals/metalloids among different cytosolic ligands as potential indicator for metal exposure. Obtained findings can also serve as guidelines for further separation and characterization of specific cytosolic metal-binding biomolecules. © 2013.

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.

Liver cytosolic 1 antigen-antibody system in type 2 autoimmune hepatitis and hepatitis C virus infection.

Science.gov (United States)

Lenzi, M; Manotti, P; Muratori, L; Cataleta, M; Ballardini, G; Cassani, F; Bianchi, F B

1995-01-01

Within the multiform liver/kidney microsomal (LKM) family, a subgroup of sera that reacts with a liver cytosolic (LC) protein has been isolated and the new antigen-antibody system is called LC1. Unlike LKM antibody type 1 (anti-LKM1), anti-LC1 is said to be unrelated to hepatitis C virus (HCV) infection and has therefore been proposed as a marker of 'true' autoimmune hepatitis type 2. Altogether 100 LKM1 positive sera were tested by immunodiffusion (ID). Twenty five gave a precipitation line with human liver cytosol; 17 of the 25 also reacted with rat liver cytosol. Thirteen of the 25 sera were anti-HCV positive by second generation ELISA: anti-HCV positive patients were significantly older (p LKM1, and that it is an additional marker of juvenile autoimmune hepatitis type 2. It does not, however, discriminate between patients with and without HCV infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7797126

Chromosomal instability drives metastasis through a cytosolic DNA response.

Science.gov (United States)

Bakhoum, Samuel F; Ngo, Bryan; Laughney, Ashley M; Cavallo, Julie-Ann; Murphy, Charles J; Ly, Peter; Shah, Pragya; Sriram, Roshan K; Watkins, Thomas B K; Taunk, Neil K; Duran, Mercedes; Pauli, Chantal; Shaw, Christine; Chadalavada, Kalyani; Rajasekhar, Vinagolu K; Genovese, Giulio; Venkatesan, Subramanian; Birkbak, Nicolai J; McGranahan, Nicholas; Lundquist, Mark; LaPlant, Quincey; Healey, John H; Elemento, Olivier; Chung, Christine H; Lee, Nancy Y; Imielenski, Marcin; Nanjangud, Gouri; Pe'er, Dana; Cleveland, Don W; Powell, Simon N; Lammerding, Jan; Swanton, Charles; Cantley, Lewis C

2018-01-25

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Interferon-γ, a valuable surrogate marker of Plasmodium falciparum pre-erythrocytic stages protective immunity

Directory of Open Access Journals (Sweden)

BenMohamed Lbachir

2011-02-01

Full Text Available Abstract Immunity against the pre-erythrocytic stages of malaria is the most promising, as it is strong and fully sterilizing. Yet, the underlying immune effectors against the human Plasmodium falciparum pre-erythrocytic stages remain surprisingly poorly known and have been little explored, which in turn prevents any rational vaccine progress. Evidence that has been gathered in vitro and in vivo, in higher primates and in humans, is reviewed here, emphasizing the significant role of IFN-γ, either as a critical immune mediator or at least as a valuable surrogate marker of protection. One may hope that these results will trigger investigations in volunteers immunized either by optimally irradiated or over-irradiated sporozoites, to quickly delineate better surrogates of protection, which are essential for the development of a successful malaria vaccine.

BAF is a cytosolic DNA sensor that leads to exogenous DNA avoiding autophagy.

Science.gov (United States)

Kobayashi, Shouhei; Koujin, Takako; Kojidani, Tomoko; Osakada, Hiroko; Mori, Chie; Hiraoka, Yasushi; Haraguchi, Tokuko

2015-06-02

Knowledge of the mechanisms by which a cell detects exogenous DNA is important for controlling pathogen infection, because most pathogens entail the presence of exogenous DNA in the cytosol, as well as for understanding the cell's response to artificially transfected DNA. The cellular response to pathogen invasion has been well studied. However, spatiotemporal information of the cellular response immediately after exogenous double-stranded DNA (dsDNA) appears in the cytosol is lacking, in part because of difficulties in monitoring when exogenous dsDNA enters the cytosol of the cell. We have recently developed a method to monitor endosome breakdown around exogenous materials using transfection reagent-coated polystyrene beads incorporated into living human cells as the objective for microscopic observations. In the present study, using dsDNA-coated polystyrene beads (DNA-beads) incorporated into living cells, we show that barrier-to-autointegration factor (BAF) bound to exogenous dsDNA immediately after its appearance in the cytosol at endosome breakdown. The BAF(+) DNA-beads then assembled a nuclear envelope (NE)-like membrane and avoided autophagy that targeted the remnants of the endosome membranes. Knockdown of BAF caused a significant decrease in the assembly of NE-like membranes and increased the formation of autophagic membranes around the DNA-beads, suggesting that BAF-mediated assembly of NE-like membranes was required for the DNA-beads to evade autophagy. Importantly, BAF-bound beads without dsDNA also assembled NE-like membranes and avoided autophagy. We propose a new role for BAF: remodeling intracellular membranes upon detection of dsDNA in mammalian cells.

The lateral distribution of intramembrane particles in the erythrocyte membrane and recombinant vesicles

NARCIS (Netherlands)

Gerritsen, A.; Verkleij, A.J.; Deenen, L.L.M. van

1979-01-01

Triton X-100 (in concentrations which did not cause a significant solubilization of membrane material) caused aggregation of the intramembrane particles of human erythrocyte ghosts. Ghosts from which the extrinsic proteins had been removed by alkali treatment showed a temperature-induced

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

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.

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

Erythrocyte and platelet fatty acids in retinitis pigmentosa.

Science.gov (United States)

Stanzial, A M; Bonomi, L; Cobbe, C; Olivieri, O; Girelli, D; Trevisan, M T; Bassi, A; Ferrari, S; Corrocher, R

1991-05-01

The fatty acid composition and the glutathione-peroxidase activity (GSH-Px) of erythrocytes and platelets, the production of malondialdehyde (MDA) by platelets and the activity of the main systems of transmembrane cation transport in erythrocyte have been studied in 12 patients (5 males and 7 females) affected by retinitis pigmentosa (RP). A remarkable increase of saturated fatty acids (SFA), particularly of stearic acid (C18:0), has been noted in these patients. The reduced unsaturated/saturated fatty acids ratio (PUFA/SFA) observed in both erythrocytes and platelets and the decrease of arachidonic acid in platelets may depend by an active peroxidation process as documented by the increase of MDA. Platelet glutathione-peroxidase (PTL-GSH-PX) and plasma retinol were in the normal range, whereas erythrocyte glutathione-peroxidase (E-GSH-PX), MDA and plasma alfa-toco-pherol were increased in patients with RP. The activities of Na(+)-K+ pump, cotransport and Na(+)-Li+ countertransport were normal in RP erythrocytes.

Hepatic or splenic targeting of carrier erythrocytes: a murine model

International Nuclear Information System (INIS)

Zocchi, E.; Guida, L.; Benatti, U.; Canepa, M.; Borgiani, L.; Zanin, T.; De Flora, A.

1987-01-01

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H 2 O 2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51 Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved

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

Kinetics of viral load and erythrocytic inclusion body formation in pacific herring artificially infected with erythrocytic necrosis virus

Science.gov (United States)

Glenn, Jolene A.; Emmenegger, Eveline J.; Grady, Courtney A.; Roon, Sean R.; Gregg, Jacob L.; Conway, Carla M.; Winton, James R.; Hershberger, Paul K.

2012-01-01

Viral erythrocytic necrosis (VEN) is a condition that affects marine and anadromous fish species, including herrings and salmonids, in the Atlantic and Pacific oceans. Infection is frequently associated with severe anemia and causes episodic mortality among wild and hatchery fish when accompanied by additional stressors; VEN can be presumptively diagnosed by (1) light microscopic identification of a single characteristic—a round, magenta-colored, 0.8-μm-diameter inclusion body (IB) within the cytoplasm of erythrocytes and their precursors on Giemsa-stained blood films; or (2) observation (via transmission electron microscopy [TEM]) of the causative iridovirus, erythrocytic necrosis virus (ENV), within erythrocytes or their precursors. To better understand the kinetics of VEN, specific-pathogen-free Pacific herring Clupea pallasii were infected with ENV by intraperitoneal injection. At 1, 4, 7, 10, 14, 21, and 28 d postexposure, samples of blood, spleen, and kidney were collected and assessed (1) via light microscopy for the number of intracytoplasmic IBs in blood smears and (2) via TEM for the number of virions within erythrocytes. The mean prevalence of intracytoplasmic IBs in the blood cells increased from 0% at 0–4 d postexposure to 94% at 28 d postexposure. Viral load within circulating red blood cells peaked at 7 d postexposure, fell slightly, and then reached a plateau. However, blood cells observed within the kidney and spleen tissues demonstrated high levels of ENV between 14 and 28 d postexposure. The results indicate that the viral load within erythrocytes does not correlate well with IB prevalence and that the virus can persist in infected fish for more than 28 d.

Cyclic GMP-AMP Synthase Is the Cytosolic Sensor of Plasmodium falciparum Genomic DNA and Activates Type I IFN in Malaria.

Science.gov (United States)

Gallego-Marin, Carolina; Schrum, Jacob E; Andrade, Warrison A; Shaffer, Scott A; Giraldo, Lina F; Lasso, Alvaro M; Kurt-Jones, Evelyn A; Fitzgerald, Katherine A; Golenbock, Douglas T

2018-01-15

Innate immune receptors have a key role in the sensing of malaria and initiating immune responses. As a consequence of infection, systemic inflammation emerges and is directly related to signs and symptoms during acute disease. We have previously reported that plasmodial DNA is the primary driver of systemic inflammation in malaria, both within the phagolysosome and in the cytosol of effector cells. In this article, we demonstrate that Plasmodium falciparum genomic DNA delivered to the cytosol of human monocytes binds and activates cyclic GMP-AMP synthase (cGAS). Activated cGAS synthesizes 2'3'-cGAMP, which we subsequently can detect using liquid chromatography-tandem mass spectrometry. 2'3'-cGAMP acts as a second messenger for STING activation and triggers TBK1/IRF3 activation, resulting in type I IFN production in human cells. This induction of type I IFN was independent of IFI16. Access of DNA to the cytosolic compartment is mediated by hemozoin, because incubation of purified malaria pigment with DNase abrogated IFN-β induction. Collectively, these observations implicate cGAS as an important cytosolic sensor of P. falciparum genomic DNA and reveal the role of the cGAS/STING pathway in the induction of type I IFN in response to malaria parasites. Copyright © 2018 by The American Association of Immunologists, Inc.

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)

Erythrocyte endogenous proteinase activity during blood bank storage.

Science.gov (United States)

de Angelis, V; de Matteis, M C; Orazi, B M; Santarossa, L; Della Toffola, L; Raineri, A; Vettore, L

1990-01-01

We studied proteolytic alterations of membrane proteins in ghosts derived from human red blood cells, preserved up to 35 days in the liquid state either as whole blood or with additive solution. The study was carried out by performing sodium dodecyl sulfate polyacrylamide gel electrophoresis of stromal proteins from erythrocytes, either previously treated with proteinase inhibitors or previously incubated in conditions promoting proteolysis. To differentiate the effect of erythrocyte from granulocyte proteinases, the investigation was also carried out in leukocyte-free red cell preparations. The results show: (1) the effects of endogenous proteinases on membrane proteins derived from red cells stored under blood bank conditions; (2) a decrease of proteolytic effects in ghosts derived from red cells which have been submitted to a longer storage; (3) a relevant influence of the red cell resuspending medium before lysis on the time-dependent onset and exhaustion of proteolysis in ghosts. The presence of increased proteolysis in ghosts could be regarded as a marker of molecular lesions induced in red cells by storage under blood bank conditions.

Mechanistic logic underlying the axonal transport of cytosolic proteins

Science.gov (United States)

Scott, David A.; Das, Utpal; Tang, Yong; Roy, Subhojit

2011-01-01

Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors. PMID:21555071

Effect of fluorozis on the erythrocyte antioxidant enzyme activity levels

International Nuclear Information System (INIS)

Akdogan, M.; YiImaz, D.; Yontem, M.; Kalei, S.; Kilic, I.

2011-01-01

While the flourine level of (drinking) water was higher than normal ranges in the center of Isparta region before 1995 year, this problematic situation is solved in later years. (However) the individuals who are staying in Yenice district are still expose to high levels of fluorine because of the usage of Andik spring water (3.8 mg/L flour level) as drinking water. In this study we aimed to investigate the harmful effect of floride on human erythrocytes via antioxidant defence system and lipid peroxidation. Therefore, we studied the activities of erythrocyte antioxidant enzymes such as Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Catalase (CAT), and the level of erythrocyte Glutathione (GSH), thiobarbituric acid reactive substance (TBARS) and the level of urine floride in high floride exposed people (children, adult and elderly). The activities of SOD, GSH-Px and CAT and the level of GSH, TBARS and urine floride were higher in 3.8 mg/L floride exposed children (Group II) than 0.8 mg/L floride exposed control children (Group I) (p 0.05). The activities of SOD, GSH-Px and CAT were lower and the levels of TBARS and urine floride were higher in 3.8 mg/L floride exposed elderly people (Group VI) than 0.8 mg/L floride exposed control elderly people (Group V) (p 0.05). As a result we thought that increased SOD, GSH-Px and CAT activities in floride exposed children and adult people, decreased activities of these enzymes in floride exposed elderly people, and increased TBARS in all groups may indicate floride caused oxidative damage in erythrocytes. (author)

Transcriptomic Analysis of Young and Old Erythrocytes of Fish

Directory of Open Access Journals (Sweden)

Miriam Götting

2017-12-01

Full Text Available Understanding gene expression changes over the lifespan of cells is of fundamental interest and gives important insights into processes related to maturation and aging. This study was undertaken to understand the global transcriptome changes associated with aging in fish erythrocytes. Fish erythrocytes retain their nuclei throughout their lifetime and they are transcriptionally and translationally active. However, they lose important functions during their lifespan in the circulation. We separated rainbow trout (Oncorhynchus mykiss erythrocytes into young and old fractions using fixed angle-centrifugation and analyzed transcriptome changes using RNA sequencing (RNA-seq technology and quantitative real-time PCR. We found 930 differentially expressed between young and old erythrocyte fractions; 889 of these showed higher transcript levels in young, while only 34 protein-coding genes had higher transcript levels in old erythrocytes. In particular genes involved in ion binding, signal transduction, membrane transport, and those encoding various enzyme classes are affected in old erythrocytes. The transcripts with higher levels in old erythrocytes were associated with seven different GO terms within biological processes and nine within molecular functions and cellular components, respectively. Our study furthermore found several highly abundant transcripts as well as a number of differentially expressed genes (DEGs for which the protein products are currently not known revealing the gaps of knowledge in most non-mammalian vertebrates. Our data provide the first insight into changes involved in aging on the transcriptional level and thus opens new perspectives for the study of maturation processes in fish erythrocytes.

Evidence for coordinate genetic control of Na,K pump density in erythrocytes and lymphocytes

International Nuclear Information System (INIS)

DeLuise, M.; Flier, J.S.

1985-01-01

The erythrocyte is widely used as a model cell for studies of the Na,K pump in health and disease. However, little is known about the factors that control the number of Na,K pumps expressed on the erythrocytes of a given individual, nor about the extent to which erythrocytes can be used to validly assess the pump density on other cell types. In this report, the authors have compared the interindividual variance of Na,K pump density in erythrocytes of unrelated individuals to that seen with identical twins. Unlike unrelated individuals, in whom pump parameters, i.e., ouabain binding sites, 86 Rb uptake, and cell Na concentration vary widely, identical twin pairs showed no significant intrapair variation for these values. Thus, a role for genetic factors is suggested. In addition, the authors established and validated a method for determining Na,K pump density and pump-mediated 86 Rb uptake in human peripheral lymphocytes. Using this method, they show that whereas Na,K pump density differs markedly between erythrocytes (mean of 285 sites per cell) and lymphocytes (mean 40,600 sites per cell), there is a strong and highly significant correlation (r = 0.79, P less than 0.001) between the pump density in these cell types in any given individual. Taken together, these studies suggest that genetic factors are important determinants of Na,K pump expression, and that pump density appears to be coordinately regulated in two cell types in healthy individuals

DMPD: Cytosolic DNA recognition for triggering innate immune responses. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 18280611 Cytosolic DNA recognition for triggering innate immune responses. Takaoka ...A, Taniguchi T. Adv Drug Deliv Rev. 2008 Apr 29;60(7):847-57. Epub 2007 Dec 31. (.png) (.svg) (.html) (.csml) Show Cytosol...ic DNA recognition for triggering innate immune responses. PubmedID 18280611 Title Cytosolic D

Erythrocyte zinc levels in children with bronchial asthma.

Science.gov (United States)

Arik Yilmaz, E; Ozmen, S; Bostanci, I; Misirlioglu, E Dibek; Ertan, U

2011-12-01

Zinc deficiency may be suspected to play a role in the pathogenesis, control, and severity of asthma because of its antioxidant, antiapoptotic, and anti-inflammatory effects. We aimed to investigate whether there was any relationship between erythrocyte zinc levels and childhood asthma. The erythrocyte zinc levels of 67 asthmatic and 45 healthy children were analyzed in this case-control study. The mean concentrations of erythrocyte zinc were 1215.8 ± 145.1 µg/dl in asthma patients and 1206.9 ± 119.5 µg/dl in controls with no significant difference (P = 0.472). The erythrocyte zinc level was below 1,000 µg/dl in 6 asthmatic patients (8.9%) and 2 control group patients (4.4%). There was no relationship between erythrocyte zinc levels and duration of follow-up, severity, and control of the asthma (P > 0.05). On the other hand, patients hospitalized for an asthma attack had significantly lower erythrocyte zinc levels compared with nonhospitalized patients and the control group (P = 0.000 and P = 0.004 respectively). This study's findings indicate that asthmatic children are not a risk group for zinc deficiency. We emphasize that checking zinc levels in children who are hospitalized for an asthma attack may be useful. Copyright © 2011 Wiley Periodicals, Inc.

Altitude Acclimatization and Blood Volume: Effects of Exogenous Erythrocyte Volume Expansion

National Research Council Canada - National Science Library

Sawka, M

1996-01-01

...: (a) altitude acclimatization effects on erythrocyte volume and plasma volume; (b) if exogenous erythrocyte volume expansion alters subsequent erythrocyte volume and plasma volume adaptations; (c...

Deformability of Erythrocytes and Oxidative Damage in Alzheimer Disease

Directory of Open Access Journals (Sweden)

Mukerrem Betul Yerer

2012-04-01

Full Text Available Purpose: A lowered cerebral perfusion as a consequence of hemodynamic microcirculatory insufficiency is one of the factors underlying in Alzheimer's disease, which is a neurodegenerative disorder leading to progressive cognitive impairment. Erythrocyte deformability is one of the major factors affecting the microcirculatory hemodynamics which is closely related to the oxidative damage. The aim of this study is to investigate the relationship between the erythrocyte deformability, nitric oxide levels and oxidative stress in Alzheimer's disease. Methods: The blood samples of 30 elderly people in three groups consisting of healthy control and different severities of the disease (low and severe were used. Then the erythrocytes were isolated and the deformability of erythrocytes was determined by Rheodyne SSD evaluating the elongation indexes of the erythrocytes under different shear stress. The catalase, glutathione peroxidase and plasma nitric oxide levels were measured spectrophotometric ally. Results: The plasma nitric oxide levels, catalase activities were found significantly higher and glutathione peroxidase activity was significantly lower in severe Alzheimer's disease patients compared to the control group. However, the deformability of erythrocytes was not significantly affected from these alterations. Conclusion: the oxidant-antioxidant status is dramatically changed in Alzheimer's disease patients with the severity of the disease and similar alterations were seen in the nitric oxide levels without any significant change in erythrocyte deformability. [Cukurova Med J 2012; 37(2.000: 65-75

Well-defined polypeptide-based systems as non-viral vectors for cytosolic delivery

OpenAIRE

Niño Pariente, Amaya

2017-01-01

A convenient cytosolic drug delivery constitutes a very powerful tool for the treatment and/or prevention of several relevant human diseases. Along with recent advances in therapeutic technologies based on biomacromolecules (e.g. oligonucleotides or proteins), we also require the development of technologies which improve the transport of therapeutic molecules to the cell of choice. This has led to the emergence of a variety of promising methods over the last 20 years. Despite significant prog...

Cytosol cathepsin-D content and proliferative activity of human breast cancer. The Comitato Italiano per il Controllo di Qualita del Laboratorio in Oncologia.

Science.gov (United States)

Paradiso, A; Mangia, A; Correale, M; Abbate, I; Ferri, G; Piffanelli, A; Catozzi, L; Amadori, D; Riccobon, A; De Lena, M

1992-01-01

Mitogenic properties have been demonstrated in vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p < 0.03). When classified in low, intermediate or high tumor cath-D content and slow or fast proliferative activity (cut-off: median values of the series), no significant agreement was found between the two variables. Statistical analysis, however, showed that a significant inverse correlation existed in node positive tumors between cath-D and 3H-Tdr-LI values which was even more evident in N-positive high estrogen receptor-positive (ER+) cases (coefficient of correlation = 0.6828; p = 0.0001). Cytosol cath-D content cannot be generally proposed as a direct marker of proliferative activity for operable breast cancer.

Ferrokinetic and erythrocyte survival studies in healthy and anemic cats

Energy Technology Data Exchange (ETDEWEB)

Madewell, B.R.; Holmes, P.H.; Onions, D.E.

1983-03-01

Erythrocyte survival and ferrokinetic studies were adapted to the cat. For 5 clinically healthy 4- to 9-month-old cats, mean /sup 51/Cr-labeled erythrocyte survival was 144 hours, and mean plasma /sup 59/Fe-labeled transferrin disappearance halftime was 51 minutes. Erythrocyte use of radioiron was rapid and efficient, with 50% to 80% of labeled iron incorporated into the erythron by 100 hours after injection into the cat. Six cats with feline leukemia virus infection were studied. For 2 cats with erythroid aplasia associated with C subgroup of feline leukemia virus, erythrocyte survival times were similar to those determined for the healthy cats, but plasma radioiron disappearance half time and erythrocyte use of radioiron were markedly diminished.

Ferrokinetic and erythrocyte survival studies in healthy and anemic cats

International Nuclear Information System (INIS)

Madewell, B.R.; Holmes, P.H.; Onions, D.E.

1983-01-01

Erythrocyte survival and ferrokinetic studies were adapted to the cat. For 5 clinically healthy 4- to 9-month-old cats, mean 51 Cr-labeled erythrocyte survival was 144 hours, and mean plasma 59 Fe-labeled transferrin disappearance halftime was 51 minutes. Erythrocyte use of radioiron was rapid and efficient, with 50% to 80% of labeled iron incorporated into the erythron by 100 hours after injection into the cat. Six cats with feline leukemia virus infection were studied. For 2 cats with erythroid aplasia associated with C subgroup of feline leukemia virus, erythrocyte survival times were similar to those determined for the healthy cats, but plasma radioiron disappearance half time and erythrocyte use of radioiron were markedly diminished

Morphological characteristics of urine erythrocytes in children with erythrocyturia

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V.A. Minakova

2017-09-01

Full Text Available Background. Nephropathies with erythrocyturia make up about 1/3 of all diseases of the kidneys and the urinary system, and they have some difficulties in differential diagnostics. Quite often, erythrocyturia is the only symptom of these diseases. In connection with this, determination of its origin is an important task in forming the correct diagnosis. Erythrocyturia in most diseases of the lower urinary tract is not accompanied by proteinuria or the presence of cylinders in the urine. The presence of proteinuria (more than 0.3 g/l or 1 g protein in urine per day, along with the appearance of erythrocytic cylinder in the urine sediment, raises suspicion in favor of glomerular or tubular diseases. Glomerular erythrocytes may be detected by means of urea concentration factor (UCF in the urinary sediment as a preliminary test for the determination of the erythrocyturia site. Erythrocytes that pass through the glomerular membrane have a changed form (dysmorphic. Determination of acanthocytes in the urine (ring-shaped erythrocytes with one or several bulges in the form of bubbles of different sizes and types is a more precise criterion of glomerular nephropathy than the presence of dysmorphic erythrocytes. The purpose of the study was to determine the morphological characteristics of urine erythrocytes in children with erythrocyturia, to improve the quality of differential diagnosis. Materials and methods. Determination of the morphological characteristics of urinary erythrocytes using UCF in 73 patients aged 1 to 18 years, of which 45 (61.6 % are patients with hematuric form of glomerulonephritis, 23 (31.5 % — with hereditary nephritis, and 5 (6.8 % — with dysmetabolic nephropathy. Detection of 50 to 80 % of dysmorphic erythrocytes in the urine sediment and finding in urine of more than 5 % of acanthocytes is a highly sensitive and specific diagnostic criterion for glomerular hematuria. Results. In children with a clinical diagnosis �

Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes

International Nuclear Information System (INIS)

Safarik, I. Ivo; Safarikova, Mirka

2001-01-01

New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states.

Science.gov (United States)

Bordbar, Aarash; Jamshidi, Neema; Palsson, Bernhard O

2011-07-12

The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in the functional capabilities of human erythrocyte metabolism.

Morphometric analysis of erythrocytes from patients with thalassemia using tomographic diffractive microscopy

Science.gov (United States)

Lin, Yang-Hsien; Huang, Shin-Shyang; Wu, Shang-Ju; Sung, Kung-Bin

2017-11-01

Complete blood count is the most common test to detect anemia, but it is unable to obtain the abnormal shape of erythrocytes, which highly correlates with the hematologic function. Tomographic diffractive microscopy (TDM) is an emerging technique capable of quantifying three-dimensional (3-D) refractive index (RI) distributions of erythrocytes without labeling. TDM was used to characterize optical and morphological properties of 172 erythrocytes from healthy volunteers and 419 erythrocytes from thalassemic patients. To efficiently extract and analyze the properties of erythrocytes, we developed an adaptive region-growing method for automatically delineating erythrocytes from 3-D RI maps. The thalassemic erythrocytes not only contained lower hemoglobin content but also showed doughnut shape and significantly lower volume, surface area, effective radius, and average thickness. A multi-indices prediction model achieved perfect accuracy of diagnosing thalassemia using four features, including the optical volume, surface-area-to-volume ratio, sphericity index, and surface area. The results demonstrate the ability of TDM to provide quantitative, hematologic measurements and to assess morphological features of erythrocytes to distinguish healthy and thalassemic erythrocytes.

Dynamin-Related Protein 1 Translocates from the Cytosol to Mitochondria during UV-Induced Apoptosis

Science.gov (United States)

Zhang, Zhenzhen; Wu, Shengnan; Feng, Jie

2011-01-01

Mitochondria are dynamic structures that frequently divide and fuse with one another to form interconnecting network. This network disintegrates into punctiform organelles during apoptosis. However, the mechanisms involved in these processes are still not well characterized. In this study, we investigate the role of dynamin-related protein 1 (Drp1), a large GTPase that mediates outer mitochondrial membrane fission, in mitochondrial dynamics in response to UV irradiation in human lung adenocarcinoma cells (ASTC-α-1) and HeLa cells. Using time-lapse fluorescent imaging, we find that Drp1 primarily distributes in cytosol under physiological conditions. After UV treatment, Drp1 translocates from cytosol to mitochondria, indicating the enhancement of Drp1 mitochondrial accumulation. Our results suggest that Drp1 is involved in the regulation of transition from an interconnecting network to a punctiform mitochondrial phenotype during UV-induced apoptosis.

Experiment study and FEM simulation on erythrocytes under linear stretching of optical micromanipulation

Science.gov (United States)

Liu, Ying; Song, Huadong; Zhu, Panpan; Lu, Hao; Tang, Qi

2017-08-01

The elasticity of erythrocytes is an important criterion to evaluate the quality of blood. This paper presents a novel research on erythrocytes' elasticity with the application of optical tweezers and the finite element method (FEM) during blood storage. In this work, the erythrocytes with different in vitro times were linearly stretched by trapping force using optical tweezers and the time dependent elasticity of erythrocytes was investigated. The experimental results indicate that the membrane shear moduli of erythrocytes increased with the increasing in vitro time, namely the elasticity was decreasing. Simultaneously, an erythrocyte shell model with two parameters (membrane thickness h and membrane shear modulus H) was built to simulate the linear stretching states of erythrocytes by the FEM, and the simulations conform to the results obtained in the experiment. The evolution process was found that the erythrocytes membrane thicknesses were decreasing. The analysis assumes that the partial proteins and lipid bilayer of erythrocyte membrane were decomposed during the in vitro preservation of blood, which results in thin thickness, weak bending resistance, and losing elasticity of erythrocyte membrane. This study implies that the FEM can be employed to investigate the inward mechanical property changes of erythrocyte in different environments, which also can be a guideline for studying the erythrocyte mechanical state suffered from different diseases.

Influence of styryl dyes on blood erythrocytes

Science.gov (United States)

Nizomov, Negmat; Barakaeva, Mubaro; Kurtaliev, Eldar N.; Rahimov, Sherzod I.; Khakimova, Dilorom P.; Khodjayev, Gayrat; Yashchuk, Valeriy N.

2008-08-01

It was studied the influence of F, Sbt, Sil, Sbo monomer and homodimer Dst-5, Dst-10, Dbt-5, Dbt-10, Dil-10, Dbo-10 styryl dyes on blood erythrocytes of white rats. It was shown that the homodimer styryl dyes Dst-5, Dbt-5 and Dbo-10 decrease the erythrocytes quantity by 1.5-2 times more as compared with monomer dyes Sbt and Sbo. The main cause of dyes different action is the different oxidation degree of intracellular hemoglobin evoked by these dyes. It was established that the observed effects was connected with different penetration of these dyes through membrane of erythrocytes and with interaction of these dyes with albumin localized in membranes of cells.

The Role and Mechanism of Erythrocyte Invasion by Francisella tularensis

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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.

Cytosolic Pellino-1-Mediated K63-Linked Ubiquitination of IRF5 in M1 Macrophages Regulates Glucose Intolerance in Obesity

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Donghyun Kim

2017-07-01

Full Text Available IRF5 is a signature transcription factor that induces M1 macrophage polarization. However, little is known regarding cytosolic proteins that induce IRF5 activation for M1 polarization. Here, we report the interaction between ubiquitin E3 ligase Pellino-1 and IRF5 in the cytoplasm, which increased nuclear translocation of IRF5 by K63-linked ubiquitination in human and mouse M1 macrophages. LPS and/or IFN-γ increased Pellino-1 expression, and M1 polarization was attenuated in Pellino-1-deficient macrophages in vitro and in vivo. Defective M1 polarization in Pellino-1-deficient macrophages improved glucose intolerance in mice fed a high-fat diet. Furthermore, macrophages in adipose tissues from obese humans exhibited increased Pellino-1 expression and IRF5 nuclear translocation compared with nonobese subjects, and these changes are associated with insulin resistance index. This study demonstrates that cytosolic Pellino-1-mediated K63-linked ubiquitination of IRF5 in M1 macrophages regulates glucose intolerance in obesity, suggesting a cytosolic mediator function of Pellino-1 in TLR4/IFN-γ receptor-IRF5 axis during M1 polarization.

Endosomolytic Nano-Polyplex Platform Technology for Cytosolic Peptide Delivery To Inhibit Pathological Vasoconstriction.

Science.gov (United States)

Evans, Brian C; Hocking, Kyle M; Kilchrist, Kameron V; Wise, Eric S; Brophy, Colleen M; Duvall, Craig L

2015-06-23

A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.

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

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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.

Increase of a Calcium Independent Transglutaminase Activity in the Erythrocyte during the Infection with Plasmodium falciparum

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Wasserman Moisés

1999-01-01

Full Text Available We have studied the activity of a calcium dependent transglutaminase (EC 2.3.2.13 during the growth of the parasite Plasmodium falciparum inside the infected human erythrocyte. There is only one detectable transglutaminase in the two-cell-system, and its origin is erythrocytic. No activity was detected in preparations of the parasite devoid of erythrocyte cytoplasm. The Michaelis Menten constants (Km of the enzyme for the substrates N'N'dimethylcaseine and putrescine were undistinguishable whether the cell extracts used in their determination were obtained from normal or from infected red cells. The total activity of transglutaminase in stringently synchronized cultures, measured at 0.5mM Ca2+, decreased with the maturation of the parasite. However, a fraction which became irreversibly activated and independent of calcium concentration was detected. The proportion of this fraction grew with maturation; it represented only 20% of the activity in 20 hr-old-trophozoites while in 48-hr-schizonts it was more than 85% of the total activity. The activation of this fraction of transglutaminase did not depend on an increase in the erythrocyte cytoplasmic calcium, since most of the calcium was shown to be located in the parasite.

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.

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

Paired Chicken and Mammalian Erythrocyte Indicator Systems for ...

African Journals Online (AJOL)

A retrospective flock health analysis revealed that the higher titres were associated with confirmable Newcastle Disease (ND) outbreaks in the affected flocks. These findings therefore suggested that the use of standardised guinea pig erythrocytes in parallel with chicken erythrocytes as indicators, might facilitate field ND ...

iAB-RBC-283: A proteomically derived knowledge-base of erythrocyte metabolism that can be used to simulate its physiological and patho-physiological states

Directory of Open Access Journals (Sweden)

Palsson Bernhard O

2011-07-01

Full Text Available Abstract Background The development of high-throughput technologies capable of whole cell measurements of genes, proteins, and metabolites has led to the emergence of systems biology. Integrated analysis of the resulting omic data sets has proved to be hard to achieve. Metabolic network reconstructions enable complex relationships amongst molecular components to be represented formally in a biologically relevant manner while respecting physical constraints. In silico models derived from such reconstructions can then be queried or interrogated through mathematical simulations. Proteomic profiling studies of the mature human erythrocyte have shown more proteins present related to metabolic function than previously thought; however the significance and the causal consequences of these findings have not been explored. Results Erythrocyte proteomic data was used to reconstruct the most expansive description of erythrocyte metabolism to date, following extensive manual curation, assessment of the literature, and functional testing. The reconstruction contains 281 enzymes representing functions from glycolysis to cofactor and amino acid metabolism. Such a comprehensive view of erythrocyte metabolism implicates the erythrocyte as a potential biomarker for different diseases as well as a 'cell-based' drug-screening tool. The analysis shows that 94 erythrocyte enzymes are implicated in morbid single nucleotide polymorphisms, representing 142 pathologies. In addition, over 230 FDA-approved and experimental pharmaceuticals have enzymatic targets in the erythrocyte. Conclusion The advancement of proteomic technologies and increased generation of high-throughput proteomic data have created the need for a means to analyze these data in a coherent manner. Network reconstructions provide a systematic means to integrate and analyze proteomic data in a biologically meaning manner. Analysis of the red cell proteome has revealed an unexpected level of complexity in

Erythrocyte survival studies in a rat myelogenous leukemia

International Nuclear Information System (INIS)

Derelanko, M.J.; Meagher, R.C.; Lobue, J.; Khouri, J.A.; Gordon, A.S.

1982-01-01

To determine the extent intrinsic erythrocyte defects and/or extrinsic factors were involved in anemia of rats bearing Shay chloroleukemia (SCL), survival of 3 H-DFP labeled erythrocytes was studied in leukemic and nonleukemic hosts. Red blood cells labeled before induction of leukemia, were rapidly lost from the peripheral circulation of SCL rats in terminal stages of disease. However, labeled erythrocytes from terminal SCL animals displayed normal lifespans when transfused into nonleukemic controls. Thus the anemia of this leukemia probably resulted from extrinsic factors associated with the leukemic process. Hemorrhage appeared to be primarily responsible for the anemia of this disease

Hsp70 facilitates trans-membrane transport of bacterial ADP-ribosylating toxins into the cytosol of mammalian cells.

Science.gov (United States)

Ernst, Katharina; Schmid, Johannes; Beck, Matthias; Hägele, Marlen; Hohwieler, Meike; Hauff, Patricia; Ückert, Anna Katharina; Anastasia, Anna; Fauler, Michael; Jank, Thomas; Aktories, Klaus; Popoff, Michel R; Schiene-Fischer, Cordelia; Kleger, Alexander; Müller, Martin; Frick, Manfred; Barth, Holger

2017-06-02

Binary enterotoxins Clostridium (C.) botulinum C2 toxin, C. perfringens iota toxin and C. difficile toxin CDT are composed of a transport (B) and a separate non-linked enzyme (A) component. Their B-components mediate endocytic uptake into mammalian cells and subsequently transport of the A-components from acidic endosomes into the cytosol, where the latter ADP-ribosylate G-actin resulting in cell rounding and cell death causing clinical symptoms. Protein folding enzymes, including Hsp90 and peptidyl-prolyl cis/trans isomerases facilitate transport of the A-components across endosomal membranes. Here, we identified Hsp70 as a novel host cell factor specifically interacting with A-components of C2, iota and CDT toxins to facilitate their transport into the cell cytosol. Pharmacological Hsp70-inhibition specifically prevented pH-dependent trans-membrane transport of A-components into the cytosol thereby protecting living cells and stem cell-derived human miniguts from intoxication. Thus, Hsp70-inhibition might lead to development of novel therapeutic strategies to treat diseases associated with bacterial ADP-ribosylating toxins.

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

A membrane model for cytosolic calcium oscillations. A study using Xenopus oocytes.

OpenAIRE

Jafri, M S; Vajda, S; Pasik, P; Gillo, B

1992-01-01

Cytosolic calcium oscillations occur in a wide variety of cells and are involved in different cellular functions. We describe these calcium oscillations by a mathematical model based on the putative electrophysiological properties of the endoplasmic reticulum (ER) membrane. The salient features of our membrane model are calcium-dependent calcium channels and calcium pumps in the ER membrane, constant entry of calcium into the cytosol, calcium dependent removal from the cytosol, and buffering ...

Inward cholesterol gradient of the membrane system in P. falciparum-infected erythrocytes involves a dilution effect from parasite-produced lipids

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Fuyuki Tokumasu

2014-05-01

Full Text Available Plasmodium falciparum (Pf infection remodels the human erythrocyte with new membrane systems, including a modified host erythrocyte membrane (EM, a parasitophorous vacuole membrane (PVM, a tubulovesicular network (TVN, and Maurer's clefts (MC. Here we report on the relative cholesterol contents of these membranes in parasitized normal (HbAA and hemoglobin S-containing (HbAS, HbAS erythrocytes. Results from fluorescence lifetime imaging microscopy (FLIM experiments with a cholesterol-sensitive fluorophore show that membrane cholesterol levels in parasitized erythrocytes (pRBC decrease inwardly from the EM, to the MC/TVN, to the PVM, and finally to the parasite membrane (PM. Cholesterol depletion of pRBC by methyl-β-cyclodextrin treatment caused a collapse of this gradient. Lipid and cholesterol exchange data suggest that the cholesterol gradient involves a dilution effect from non-sterol lipids produced by the parasite. FLIM signals from the PVM or PM showed little or no difference between parasitized HbAA vs HbS-containing erythrocytes that differed in lipid content, suggesting that malaria parasites may regulate the cholesterol contents of the PVM and PM independently of levels in the host cell membrane. Cholesterol levels may affect raft structures and the membrane trafficking and sorting functions that support Pf survival in HbAA, HbAS and HbSS erythrocytes.

In Vitro Protective Effect of Phikud Navakot Extraction on Erythrocyte

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Kanchana Kengkoom

2016-01-01

Full Text Available Phikud Navakot (PN, Thai herbal remedy in National List of Essential Medicines, has been claimed to reduce many cardiovascular symptoms especially dizziness and fainting. Apart from blood supply, erythrocyte morphology, in both shape and size, is one of the main consideration factors in cardiovascular diseases and may be affected by vascular oxidative stress. However, little is known about antioxidative property of PN on erythrocyte to preserve red blood cell integrity. In this study, 1,000 μM hydrogen peroxide-induced oxidative stress was conducted on sheep erythrocyte. Three doses of PN (1, 0.5, and 0.25 mg/mL and 10 μM of ascorbic acid were compared. The released hemoglobin absorbance was measured to demonstrate hemolysis. Electron microscopic and immunohistochemical studies were also performed to characterize dysmorphic erythrocyte and osmotic ability in relation to aquaporin- (AQP- 1 expression, respectively. The results revealed that all doses of PN and ascorbic acid decreased the severity of dysmorphic erythrocyte, particularly echinocyte, acanthocyte, knizocyte, codocyte, clumping, and other malformations. However, the most effective was 0.5 mg/mL PN dosage. In addition, hydrostatic pressure may be increased in dysmorphic erythrocyte in association with AQP-1 upregulation. Our results demonstrated that PN composes antioxidative effect to maintain the integrity and osmotic ability on sheep erythrocyte.

Fluctuations in Cytosolic Calcium Regulate the Neuronal Malate-Aspartate NADH Shuttle

DEFF Research Database (Denmark)

Satrústegui, Jorgina; Bak, Lasse K

2015-01-01

that MAS is regulated by fluctuations in cytosolic Ca(2+) levels, and that this regulation is required to maintain a tight coupling between neuronal activity and mitochondrial respiration and oxidative phosphorylation. At cytosolic Ca(2+) fluctuations below the threshold of the mitochondrial calcium...

Erythrocyte nanovesicles: Biogenesis, biolo

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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.

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae

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Sasnauskas Kęstutis

2011-05-01

Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of

Subcutaneous administration of carrier erythrocytes: slow release of entrapped agent

International Nuclear Information System (INIS)

DeLoach, J.R.; Corrier, D.E.

1988-01-01

Carrier erythrocytes administered subcutaneously in mice release encapsulated molecules at the injection site and through cells that escape the injection site. One day postinjection, the efflux of encapsulated [ 14 C]sucrose, [ 3 H]inulin, and 51 Cr-hemoglobin from the injection site was 45, 55, and 65%, respectively. Intact carrier erythrocytes escaped the injection site and entered the blood circulation carrying with them the encapsulated molecules. Most of the encapsulated [ 3 H]inulin that reached whole blood circulated within erythrocytes. Small but measurable numbers of encapsulated molecules were trapped within lymph nodes. Subcutaneous injection of carrier erythrocytes may allow for limited extravascular tissue targeting of drugs

Intraspecific variation in erythrocyte sizes among populations of Hypsiboas cordobas (Anura: Hylidae

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Mariana Baraquet

2013-12-01

Full Text Available We studied the morphology and size of erythrocytes of H. cordobae, and analysed the geographic variation of this character along the distribution of the species, in relation to the latitudinal and altitudinal distances. Erythrocyte shape of the H. cordobae is ellipsoidal and the nuclei are also ellipsoidal and centrally oriented. Erythrocyte and nuclear size showed significant differences among populations, with the highest mean size corresponding to the population of Achiras (low altitude site and the lowest mean size to Los Linderos (high altitude site. There was no significant relationship between the latitude of each population and the both erythrocyte and nuclear size. The altitudinal variation in erythrocyte cell size may be attributable to the surface available for gas exchange; a small erythrocyte offers a possibility of greater rate of exchange than a larger one. Our results are consistent with studies of other amphibians, where intraspecific comparisons of populations at different altitudes show that individuals at higher altitudes are characterized by smaller erythrocytes.

Hematology and erythrocyte osmotic fragility of the Franquet's fruit bat (Epomops franqueti).

Science.gov (United States)

Ekeolu, Oyetunde Kazeem; Adebiyi, Olamide Elizabeth

2018-03-15

Hematological parameters are vital diagnostic tools for understanding health dynamics of humans and animals. Franquet's fruit bat (Epomops franqueti) is host to several parasites such as protozoa, bacteria, viruses and mites. Yet, studies exploring the values of its blood components with interest for research or food purposes are scarce. Thus, this study was carried out to investigate the hematological values of the adult E. franqueti. Seventeen (nine female and eight male) apparently healthy adult E. franqueti were captured from their roosting colony. Blood samples were collected for determination of erythrocyte indices [red blood cell count (RBC), packed cell volume (PCV), hemoglobin (Hb) concentration, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC)] and leukocyte indices [total white blood cell counts (WBC), lymphocytes, eosinophil, monocytes, neutrophil count and erythrocytes osmotic fragility]. There were no significant (p≥0.05) sex-related differences in RBC, PCV, Hb concentration, MCV, MCH, MCHC and total and differential WBC of E. franqueti. Erythrocyte osmotic fragility was significantly higher in female than in male E. franqueti at 0.1% NaCl. These considerations are critical in establishing reference ranges of blood parameters for E. franqueti and may provide insight to why they serve as reservoir hosts for several microorganisms.

Decreased erythrocyte CCS content is a biomarker of copper overload in rats.

Science.gov (United States)

Bertinato, Jesse; Sherrard, Lindsey; Plouffe, Louise J

2010-07-02

Copper (Cu) is an essential trace metal that is toxic in excess. It is therefore important to be able to accurately assess Cu deficiency or overload. Cu chaperone for Cu/Zn superoxide dismutase (CCS) protein expression is elevated in tissues of Cu-deficient animals. Increased CCS content in erythrocytes is particularly sensitive to decreased Cu status. Given the lack of a non-invasive, sensitive and specific biomarker for the assessment of Cu excess, we investigated whether CCS expression in erythrocytes reflects Cu overload. Rats were fed diets containing normal or high levels of Cu for 13 weeks. Diets contained 6.3 +/- 0.6 (Cu-N), 985 +/- 14 (Cu-1000) or 1944 +/- 19 (Cu-2000) mg Cu/kg diet. Rats showed a variable response to the high Cu diets. Some rats showed severe Cu toxicity, while other rats showed no visible signs of toxicity and grew normally. Also, some rats had high levels of Cu in liver, whereas others had liver Cu concentrations within the normal range. Erythrocyte CCS protein expression was 30% lower in Cu-2000 rats compared to Cu-N rats (P CCS (47% reduction, P CCS content is associated with Cu overload in rats and should be evaluated further as a potential biomarker for assessing Cu excess in humans.

Automation of metabolic stability studies in microsomes, cytosol and plasma using a 215 Gilson liquid handler.

Science.gov (United States)

Linget, J M; du Vignaud, P

1999-05-01

A 215 Gilson liquid handler was used to automate enzymatic incubations using microsomes, cytosol and plasma. The design of automated protocols are described. They were based on the use of 96 deep well plates and on HPLC-based methods for assaying the substrate. The assessment of those protocols was made with comparison between manual and automated incubations, reliability and reproducibility of automated incubations in microsomes and cytosol. Examples of the use of those programs in metabolic studies in drug research, i.e. metabolic screening in microsomes and plasma were shown. Even rapid processes (with disappearance half lives as low as 1 min) can be analysed. This work demonstrates how stability studies can be automated to save time, render experiments involving human biological media less hazardous and may be improve inter-laboratory reproducibility.

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

Transbilayer distribution and mobility of phosphatidylcholine in intact erythrocyte membranes. A study with phosphatidylcholine exchange protein

NARCIS (Netherlands)

van Meer, G.|info:eu-repo/dai/nl/068570368; Poorthuis, B.J.H.M.; Wirtz, K.W.A.|info:eu-repo/dai/nl/068427956; op den Kamp, J.A.F.; van Deenen, L.L.M.

1980-01-01

The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles

Detection of Occult Erythrocytic Membrane Damages upon Pharmacological Exposures

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P. Yu. Alekseyeva

2007-01-01

Full Text Available Blood administration of pharmaceuticals may cause occult effects of these agents on erythrocytic membranes. These effects may damage and cause additional membrane defects, but may strengthen. The type and degree of the effects of an agent were detected by calibrated irreversible electroporation with a pulsed electric field (PEF. The paper considers the erythrocytic membranous effects of a wide concentration range of agents used in anesthesiology, such as esmerone, tracrium, and mar-caine-adrenaline. Under the action of PEF and esmerone at the normal concentration N, the rate of erythrocytic hemolysis increased by several times as compared with the control. The similar effect also occurred when esmerone was added at the concentration C=10N. Tracrium exerted a fixing effect on erythrocytic membranes. Upon a combined exposure to PEF and tracrium in the normal concentration C=N; erythrocytic hemolysis was slow. So was with the concentration C=10N. The rate of hemolysis of the red blood cells subjected to a combined action of marcaine adrenaline at the normal concentration C=N and even at the concentration C=10N and PEF was comparable with the hemolytic rate of the reference suspension. 

Cryo scanning electron microscopy of Plasmodium falciparum-infected erythrocytes

DEFF Research Database (Denmark)

Hempel, Casper

2017-01-01

Plasmodium falciparum invades erythrocytes as an essential part of their life cycle. While living inside erythrocytes, the parasite remodels the cell's intracellular organization as well as its outer surface. Late trophozoite-stage parasites and schizonts introduce numerous small protrusions...

Bilayer/cytoskeleton interactions in lipid-symmetric erythrocytes assessed by a photoactivable phospholipid analogue

International Nuclear Information System (INIS)

Pradhan, D.; Schlegel, R.A.; Williamson, P.

1991-01-01

Two mechanisms have been proposed for maintenance of transbilayer phospholipid asymmetry in the erythrocyte plasma membrane, one involving specific interactions between the aminophospholipids of the inner leaflet of the bilayer and the cytoskeleton, particularly spectrin, and the other involving the aminophospholipid translocase. If the former mechanism is correct, then erythrocytes which have lost their asymmetric distribution of phospholipids should display altered bilayer/cytoskeleton interactions. To test this possibility, normal erythrocytes, erythrocytes from patients with chronic myelogenous leukemia or sickle disease, and lipid-symmetric and -asymmetric erythrocyte ghosts were labeled with the radioactive photoactivable analogue of phosphatidylethanolamine, 2-(2-azido-4-nitrobenzoyl)-1-acyl-sn-glycero-3-phospho[ 14 C] ethanolamine ([ 14 C]AzPE), previously shown to label cytoskeletal proteins from the bilayer. The labeling pattern of cytoskeletal proteins in pathologic erythrocytes and lipid-asymmetric erythrocyte ghosts was indistinguishable from normal erythrocytes, indicating that the probe detects no differences in bilayer/cytoskeleton interactions in these cells. In contrast, in lipid-symmetric erythrocyte ghosts, labeling of bands 4.1 and 4.2 and actin, and to a lesser extent ankyrin, by [ 14 C]AzPE was considerably reduced. Significantly, however, labeling of spectrin was unaltered in the lipid-symmetric cells. These results do not support a model in which spectrin is involved in the maintenance of an asymmetric distribution of phospholipids in erythrocytes

Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites

International Nuclear Information System (INIS)

Doinel, C.; Ropars, C.; Salmon, C.

1978-01-01

Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)

Delayed effects of radiation on enzymes in erythrocytes

International Nuclear Information System (INIS)

Li Jinying; Zhang Weiping; Liu Benti

1998-01-01

Objective: To study the delayed effects of radiation on the enzymes in erythrocytes. Methods: The activity of 8 enzymes, related glycolysis, hexose monophosphate shunt, nucleotide metabolism, redox reaction and esterase in erythrocytes of five patients with bone marrow form of acute radiation sickness (ARS) were assayed at 1,2,3 and 6 years after exposure to 60 Co radiation. Results: The decreased activities of glucose-6-phosphate dehydrogenase (G6PD), pyruvate kinase (PK), NADH-methemoglobin reductase (MR) during the stage of crisis and of acetylcholinesterase (ACE) during the stage of convalescence were recovered to varying extent, whereas the lowered activities of the first three enzymes in some cases remained unchanged. There was no correlation between the enzyme activity and the radiation dose as well as the age of the patients. Conclusion: It is demonstrated that the delayed effects of radiation damage to erythrocyte enzymes are most significant in PK of glycolysis, G6PD of hexose monophosphate shunt and MR of redox reaction. It is suggested that the genes related to the synthesis of erythrocyte enzymes may be damaged by radiation

Metabolism of Citrate and Other Carboxylic Acids in Erythrocytes As a Function of Oxygen Saturation and Refrigerated Storage

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Travis Nemkov

2017-10-01

Full Text Available State-of-the-art proteomics technologies have recently helped to elucidate the unanticipated complexity of red blood cell metabolism. One recent example is citrate metabolism, which is catalyzed by cytosolic isoforms of Krebs cycle enzymes that are present and active in mature erythrocytes and was determined using quantitative metabolic flux analysis. In previous studies, we reported significant increases in glycolytic fluxes in red blood cells exposed to hypoxia in vitro or in vivo, an observation relevant to transfusion medicine owing to the potential benefits associated with hypoxic storage of packed red blood cells. Here, using a combination of steady state and quantitative tracing metabolomics experiments with 13C1,2,3-glucose, 13C6-citrate, 13C515N2-glutamine, and 13C1-aspartate via ultra-high performance liquid chromatography coupled on line with mass spectrometry, we observed that hypoxia in vivo and in vitro promotes consumption of citrate and other carboxylates. These metabolic reactions are theoretically explained by the activity of cytosolic malate dehydrogenase 1 and isocitrate dehydrogenase 1 (abundantly represented in the red blood cell proteome, though moonlighting functions of additional enzymes cannot be ruled out. These observations enhance understanding of red blood cell metabolic responses to hypoxia, which could be relevant to understand systemic physiological and pathological responses to high altitude, ischemia, hemorrhage, sepsis, pulmonary hypertension, or hemoglobinopathies. Results from this study will also inform the design and testing of novel additive solutions that optimize red blood cell storage under oxygen-controlled conditions.

Adenosine deaminase activity of erythrocytes in hyperuricemia

International Nuclear Information System (INIS)

Krueger, W.; Richter, V.; Beenken, O.; Weinhold, D.; Hirschberg, K.; Rotzsch, W.; Akademie der Wissenschaften der DDR, Leipzig. Zentralinstitut fuer Isotopen- und Strahlenforschung)

1982-01-01

Erythrocytic adenosine deaminase (ADA) activity was determined in 55 patients with primary hyperuricemia and in 37 healthy control persons. Unlike the controls, the ADA activity in the patient group showed a two-peak response. Hyperuricemia patients with high ADA activity also exhibited increased uric acid excretion and elevated 15 N incorporation into uric acid. High activity values of erythrocytic ADA can be interpreted as an uric acid overproduction, giving hints for a therapeutic plan. (author)

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.

Transbilayer distribution and mobility of phosphatidylcholine in intact erythrocyte membranes. A study with phosphatidylcholine exchange protein

NARCIS (Netherlands)

van Meer, G.; Poorthuis, B. J.; Wirtz, K. W.; Op den Kamp, J. A.; van Deenen, L. L.

1980-01-01

1. The exchange of phosphatidylcholine between intact human or rat erythrocytes and rat liver microsomes was greatly stimulated by phosphatidylcholine-specific exchange proteins from rat liver and beef liver. It was found, however, that compared to the exchange reaction between phospholipid vesicles

Null-point titration measurements of free magnesium in stored erythrocytes

International Nuclear Information System (INIS)

Bock, J.L.; Yusuf, Y.; Puntillo, E.

1987-01-01

Free intracellular magnesium concentration (Mg/sub i/) was measured in stored human erythrocytes, using null-point titration with ionophore A23187. For cells stored 31 P NMR spectroscopy, which showed a decrease in Mg/sub i/ with storage. However, the NMR measurements are performed with no pretreatment of the cells, while the null-point method requires an initial washing step, which alters pH/sub i/ and may also alter Mg/sub i/. The titration-measured Mg/sub i/ values are still surprisingly low for long-stored cells, considering that depletion of ATP and 2,3-DPG should release bound Mg. Using the titration-measured Mg/sub i/ values along with measurements of total Mg, ATP, and 2,3-DPG, they estimate that an additional buffer contains about 47% of total Mg in cells stored 21 days. Mg/sub i/ determinations by both 31 P NMR and null-point titration thus indicate that erythrocyte Mg is largely bound to a high-capacity, low-affinity buffer whose relative importance increases during cell storage. Discrepancies between the methods require further investigation

The modulation of erythrocyte Na+/K+-ATPase activity by curcumin

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Prabhakar Singh

2015-11-01

Full Text Available Curcumin, an active biphenolic molecule present in turmeric (Curcuma longa, has been reported to elicit plethora of health protective effects. The present study was carried out in vitro, in vivo and in silico to investigate the modulatory effects of curcumin on erythrocyte membrane Na+/K+-ATPase activity. In vitro curcumin (10−5 M to 10−8 M was incubated with human erythrocytes membrane. In vivo curcumin (340 mg/kg b.w. and 170 mg/kg b.w. was supplemented to wistar rats for 21 days. In silico, catalytic unit α of Na+/K+-ATPase (3b8e.pdb protein was used as a receptor for the natural ligand ATP to study curcumin-mediated docking simulation using AutoDock4. The in vitro effect of curcumin on the Na+/K+-ATPase activity in human erythrocytes was biphasic. An inhibitory response was observed at 10−5 M (p < 0.001. An activation of the Na+/K+-ATPase activity was observed at 10−7 and 10−8 M (p < 0.001 and p < 0.01. In vivo, curcumin supplementation to rats increased the Na+/K+-ATPase activity at doses 340 mg/kg b.w. (p < 0.001 as well as at 170 mg/kg b.w., (p < 0.01. AutoDock4 docking simulation study showed that both ligands curcumin and ATP actively interacted with amino acids Glu214, Ser215, Glu216, Thr371, Asn377, Arg378, Met379, Arg438, Val440, Ala444, Lys451 and Asp586 at the catalytic cavity of Na+/K+-ATPase. ATP had more H bonding and hydrophobic interaction with active site amino acid residues compared to curcumin. These finding may explain some of the health beneficial properties of curcumin associated with deregulated Na+/K+-ATPase activity or ions homeostasis.

Dynamics of morphofunctional erythrocyte properties during intravenous glucose injection in patients with coronary heart disease

Science.gov (United States)

Malinova, Lidia I.; Simonenko, Georgy V.; Denisova, Tatyana P.; Tuchin, Valery V.

2007-02-01

Dynamics of glucose concentration in human organism is an important diagnostic characteristic for it's parameters correlate significantly with the severity of metabolic, vessel and perfusion disorders. 36 patients with stable angina pectoris of II and III functional classes were involved in this study. All of them were men in age range of 45-59 years old. 7 patients hospitalized with acute myocardial infarction (aged from 49 to 59 years old) form the group of compare. Control group (n = 5) was of practically healthy men in comparable age. To all patients intravenous glucose solution (40%) in standard loading dose was injected. Capillary and vein blood samples were withdrawn before, and 5, 60, 120, 180 and 240 minutes after glucose load. At these time points blood pressure and glucose concentration were measured. In prepared blood smears shape, deformability and sizes of erythrocytes, quantity and degree of shear stress resistant erythrocyte aggregates were studied. Received data were approximated by polynomial of high degree to receive concentration function of studied parameters, which first derivative elucidate velocity characteristics of morphofunctional erythrocyte properties during intravenous glucose injection in patients with coronary heart disease and practically healthy persons. Received data show principle differences in dynamics of morphofunctional erythrocyte properties during intravenous glucose injection in patients with coronary heart disease as a possible mechanism of coronary blood flow destabilization.

Physiological variations in levels of 2,3-diphosphoglycerate in horse erythrocytes.

Science.gov (United States)

Lewis, I M; McLan, J G

1975-03-01

The levels of 2,3-diphosphoglycerate (2,3-DPG), which affects the transport of oxygen by haemoglobin, were examined in horse blood. Resting levels of erythrocyte 2,3-DPG were established in thoroughbred horses, and levels of 2,3-DPG together with haemoglobin levels, were examined in a variety of conditions. A negative correlation was observed between erythrocyte 2,3-DPG and haemoglobin levels. Mares had higher erythrocyte 2,3-DPG levels was observed during training, and this variation may have a significant effect on haemoglobin oxygen transport. Erythrocyte 2,3-DPG levels were not affected by age or exercise.

Specific binding of [alpha-32P]GTP to cytosolic and membrane-bound proteins of human platelets correlates with the activation of phospholipase C

International Nuclear Information System (INIS)

Lapetina, E.G.; Reep, B.R.

1987-01-01

We have assessed the binding of [alpha- 32 P]GTP to platelet proteins from cytosolic and membrane fractions. Proteins were separated by NaDodSO 4 /PAGE and electrophoretically transferred to nitrocellulose. Incubation of the nitrocellulose blots with [alpha- 32 P]GTP indicated the presence of specific and distinct GTP-binding proteins in cytosol and membranes. Binding was prevented by 10-100 nM GTP and by 100 nM guanosine 5'-[gamma-thio]triphosphate (GTP[gamma S]) or GDP; binding was unaffected by 1 nM-1 microM ATP. One main GTP-binding protein (29.5 kDa) was detected in the membrane fraction, while three others (29, 27, and 21 kDa) were detected in the soluble fraction. Two cytosolic GTP-binding proteins (29 and 27 kDa) were degraded by trypsin; another cytosolic protein (21 kDa) and the membrane-bound protein (29.5 kDa) were resistant to the action of trypsin. Treatment of intact platelets with trypsin or thrombin, followed by lysis and fractionation, did not affect the binding of [alpha- 32 P]GTP to the membrane-bound protein. GTP[gamma S] still stimulated phospholipase C in permeabilized platelets already preincubated with trypsin. This suggests that trypsin-resistant GTP-binding proteins might regulate phospholipase C stimulated by GTP[gamma S

Desickling of Sickle Cell Erythrocytes by Pulsed RF Fields.

Science.gov (United States)

1986-09-16

spectrophotometery. Field induced menbrane potential which causes the L partyl breakdown of the memrbrane and the formation of pores was calculated... plasma . Fig.5 shows the photographs of sickled and desickled SS erythrocytes which are suspended in Hank’s solution. As shown, desickled erythrocytes

Retargeting the Clostridium botulinum C2 toxin to the neuronal cytosol.

Science.gov (United States)

Pavlik, Benjamin J; Hruska, Elizabeth J; Van Cott, Kevin E; Blum, Paul H

2016-03-30

Many biological toxins are known to attack specific cell types, delivering their enzymatic payloads to the cytosol. This process can be manipulated by molecular engineering of chimeric toxins. Using toxins with naturally unlinked components as a starting point is advantageous because it allows for the development of payloads separately from the binding/translocation components. Here the Clostridium botulinum C2 binding/translocation domain was retargeted to neural cell populations by deleting its non-specific binding domain and replacing it with a C. botulinum neurotoxin binding domain. This fusion protein was used to deliver fluorescently labeled payloads to Neuro-2a cells. Intracellular delivery was quantified by flow cytometry and found to be dependent on artificial enrichment of cells with the polysialoganglioside receptor GT1b. Visualization by confocal microscopy showed a dissociation of payloads from the early endosome indicating translocation of the chimeric toxin. The natural Clostridium botulinum C2 toxin was then delivered to human glioblastoma A172 and synchronized HeLa cells. In the presence of the fusion protein, native cytosolic enzymatic activity of the enzyme was observed and found to be GT1b-dependent. This retargeted toxin may enable delivery of therapeutics to peripheral neurons and be of use in addressing experimental questions about neural physiology.

The role of the erythrocyte in antitumour drug transport

NARCIS (Netherlands)

Dumez, Herlinde

2005-01-01

The area of research on the substance-carrier capacity of the erythrocyte is rather limited and it remains difficult to estimate the impact of erythrocyte drug level monitoring in the clinic. Although equilibrium between blood and tissues based on the dissolution of compounds in the plasma water

Insulin radioreceptor assay on murine splenic leukocytes and peripheral erythrocytes

International Nuclear Information System (INIS)

Shimizu, F.; Kahn, R.

1982-01-01

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH 4 Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [ 125 I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 x 10 7 /ml) consisted of incubation with [ 125 I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [ 125 I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 x 10 9 /ml) were incubated with [ 125 ]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [ 125 I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner

Partial recovery of erythrocyte glycogen in diabetic rats treated with phenobarbital

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da-Silva C.A.

1997-01-01

Full Text Available Erythrocytes may play a role in glucose homeostasis during the postprandial period. Erythrocytes from diabetic patients are defective in glucose transport and metabolism, functions that may affect glycogen storage. Phenobarbital, a hepatic enzyme inducer, has been used in the treatment of patients with non-insulin-dependent diabetes mellitus (NIDDM, increasing the insulin-mediated glucose disposal. We studied the effects of phenobarbital treatment in vivo on glycemia and erythrocyte glycogen content in control and alloxan-diabetic rats during the postprandial period. In control rats (blood glucose, 73 to 111 mg/dl in femoral and suprahepatic veins the erythrocyte glycogen content was 45.4 ± 1.1 and 39.1 ± 0.8 µg/g Hb (mean ± SEM, N = 4-6 in the femoral artery and vein, respectively, and 37.9 ± 1.1 in the portal vein and 47.5 ± 0.9 in the suprahepatic vein. Diabetic rats (blood glucose, 300-350 mg/dl presented low (P<0.05 erythrocyte glycogen content, i.e., 9.6 ± 0.1 and 7.1 ± 0.7 µg/g Hb in the femoral artery and vein, respectively, and 10.0 ± 0.7 and 10.7 ± 0.5 in the portal and suprahepatic veins, respectively. After 10 days of treatment, phenobarbital (0.5 mg/ml in the drinking water did not change blood glucose or erythrocyte glycogen content in control rats. In diabetic rats, however, it lowered (P<0.05 blood glucose in the femoral artery (from 305 ± 18 to 204 ± 45 mg/dl and femoral vein (from 300 ± 11 to 174 ± 48 mg/dl and suprahepatic vein (from 350 ± 10 to 174 ± 42 mg/dl, but the reduction was not sufficient for complete recovery. Phenobarbital also stimulated the glycogen synthesis, leading to a partial recovery of glycogen stores in erythrocytes. In treated rats, erythrocyte glycogen content increased to 20.7 ± 3.8 µg/g Hb in the femoral artery and 30.9 ± 0.9 µg/g Hb in the suprahepatic vein (P<0.05. These data indicate that phenobarbital activated some of the insulin-stimulated glucose metabolism steps which were

The effects of polymeric plutonium on erythrocyte survival in mice, (1)

International Nuclear Information System (INIS)

Joshima, Hisamasa; Kashima, Masatoshi; Matsuoka, Osamu

1976-01-01

The changes in erythrocyte counts, hematocrit, hemoglobin, reticulocyte counts and erythrocyte survival following an intravenous injection of polymeric 239 Pu at the dose level of 15 μCi/kg, 10 μCi/kg and 5 μCi/kg were studied in CF no. 1 male mice in order to investigate the possible pathogenesis of anemia produced by irradiation of polymeric plutonium. The administration of 15 μCi/kg and 10 μCi/kg of polymeric plutonium produced anemia but 5 μCi/kg had no significant effect. Studies with 51 Cr labelled erythrocyte showed a moderate reduction in survival of erythrocyte following a single intraveneous injection of polymeric plutonium. Not only the intracorpuscular effect but also extracorpuscular effect of polymeric plutonium was considered to lead to a reduction in erythrocyte survival, but no clear dose relationship could be observed between the reduction of survival and either intracorpuscular effect or extracorpuscular effect. Although the most important pathogenesis of anemia produced by polymeric plutonium is supposed to be a decreased erythropoiesis, it was believed that both qualitatively impaired erythropoiesis and abnormal erythrocyte destruction might also play some role in the occurrence of anemia. (auth.)

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

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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.

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.

A new view of the bacterial cytosol environment.

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Benjamin P Cossins

2011-06-01

Full Text Available The cytosol is the major environment in all bacterial cells. The true physical and dynamical nature of the cytosol solution is not fully understood and here a modeling approach is applied. Using recent and detailed data on metabolite concentrations, we have created a molecular mechanical model of the prokaryotic cytosol environment of Escherichia coli, containing proteins, metabolites and monatomic ions. We use 200 ns molecular dynamics simulations to compute diffusion rates, the extent of contact between molecules and dielectric constants. Large metabolites spend ∼80% of their time in contact with other molecules while small metabolites vary with some only spending 20% of time in contact. Large non-covalently interacting metabolite structures mediated by hydrogen-bonds, ionic and π stacking interactions are common and often associate with proteins. Mg(2+ ions were prominent in NIMS and almost absent free in solution. Κ(+ is generally not involved in NIMSs and populates the solvent fairly uniformly, hence its important role as an osmolyte. In simulations containing ubiquitin, to represent a protein component, metabolite diffusion was reduced owing to long lasting protein-metabolite interactions. Hence, it is likely that with larger proteins metabolites would diffuse even more slowly. The dielectric constant of these simulations was found to differ from that of pure water only through a large contribution from ubiquitin as metabolite and monatomic ion effects cancel. These findings suggest regions of influence specific to particular proteins affecting metabolite diffusion and electrostatics. Also some proteins may have a higher propensity for associations with metabolites owing to their larger electrostatic fields. We hope that future studies may be able to accurately predict how binding interactions differ in the cytosol relative to dilute aqueous solution.

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...

Effect of rare-earth-based nanoparticles on the erythrocyte osmotic adaptation

OpenAIRE

О. К. Пакулова; В. К. Kлочков; Н. С. Кавок; И. А. Костина; А. С. Сопотова; В. А. Бондаренко

2017-01-01

Rare-earth-based nanoparticles (REB NPs) have been employed in molecular and cell biology due to their unique features. However, their interaction with biosystems and the influence on cell functioning are poorly understood. In this study effect of REB NPs (composed of dielectric nanocrystalls of cerium dioxide and orthovanadates of gadolinium and yttrium) with different form-factor as well as REB NPs-cholesterol complexes on the adaptation of human erythrocytes to hypertonic lysis (4 M NaCl) ...

 Oxidative stress modulates the organization of erythrocyte membrane cytoskeleton

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Maria Olszewska

2012-07-01

Full Text Available  Background:Apart from their main role in transporting oxygen and carbon dioxide, erythrocytes play also an important role in organism antioxidative defence. Direct exposure to reactive oxygen species (ROS results in shortening of their half-life, even by 50�20The presence of glucose, being the substrate in pentose phosphate pathway (PPP cycle, is one of the factors that can have influence on the level of oxidative stress. The activity of PPP increases during oxidative stress. Glucose guarantees normal PPP functioning with the production of reductive equivalents in the amounts necessary to reproduction of glutathione – nonenzymatic free radical scavenger. In available literature there are no reports regarding the changes in protein contents of erythrocyte cytoskeleton exposed to t-butyl hydroperoxide in relation to glucose presence in incubation medium.Material/methods:Erythrocytes taken from 10 healthy subjects were used to assess the influence of generated free radicals on erythrocyte proteins and chosen parameters of oxidative stress. Erythrocytes were incubated in the solutions containing deferent concentrations of t-butyl hydroperoxide and glucose. Electrophoresis was performed on polyacrylamide gel in denaturating conditions. The contents of tryptophan in membranes was evaluated spectrofluorometrically.Results/conclusions:In vitro conditions oxidative stress leads to protein damage in erythrocyte cytoskeleton, both in proteins inside the cell as well as having contact with extracellular environment. In consequence, the amount of low-molecular proteins – mainly globin, which bind to cytoskeleton, increases. This process takes place independently of glucose presence in incubation medium. One of the element of protein cytoskeleton, tryptophan, also undergoes degradation. The decrease of its contents is higher during erythrocyte exposure to t-BOOH in environment containing glucose, what can suggest prooxidative influence of glucose in

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation.

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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.

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.

Methods Employed in Cytofluorometric Assessment of Eryptosis, the Suicidal Erythrocyte Death

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Mohamed Jemaà

2017-09-01

Full Text Available Suicidal erythrocyte death or eryptosis contributes to or even accounts for anemia in a wide variety of clinical conditions, such as iron deficiency, dehydration, hyperphosphatemia, vitamin D excess, chronic kidney disease (CKD, hemolytic-uremic syndrome, diabetes, hepatic failure, malignancy, arteriitis, sepsis, fever, malaria, sickle-cell disease, beta-thalassemia, Hb-C and G6PD-deficiency, Wilsons disease, as well as advanced age. Moreover, eryptosis is triggered by a myriad of xenobiotics and endogenous substances including cytotoxic drugs and uremic toxins. Eryptosis is characterized by cell membrane scrambling with phosphatidylserine exposure to the erythrocyte surface. Triggers of eryptosis include oxidative stress, hyperosmotic shock, and energy depletion. Signalling involved in the regulation of eryptosis includes Ca2+ entry, ceramide, caspases, calpain, p38 kinase, protein kinase C, Janus-activated kinase 3, casein kinase 1α, cyclin-dependent kinase 4, AMP-activated kinase, p21-activated kinase 2, cGMP-dependent protein kinase, mitogen- and stress-activated kinase MSK1/2, and ill-defined tyrosine kinases. Inhibitors of eryptosis may prevent anaemia in clinical conditions associated with enhanced eryptosis and stimulators of eryptosis may favourably influence the clinical course of malaria. Additional experimentation is required to uncover further clinical conditions with enhanced eryptosis, as well as further signalling pathways, further stimulators, and further inhibitors of eryptosis. Thus, a detailed description of the methods employed in the analysis of eryptosis may help those, who enter this exciting research area. The present synopsis describes the experimental procedures required for the analysis of phosphatidylserine exposure at the cell surface with annexin-V, cell volume with forward scatter, cytosolic Ca2+ activity ([Ca2+]i with Fluo3, oxidative stress with 2′,7′-dichlorodihydrofuorescein diacetate (DCFDA, glutathione

Circadian Rhythm of Hepatic Cytosolic and Nuclear Estrogen and Androgen Receptors

Science.gov (United States)

FRANCAVILLA, ANTONIO; EAGON, PATRICIA K.; DiLEO, ALFREDO; VAN THIEL, DAVID H.; PANELLA, CARMINE; POLIMENO, LORENZO; AMORUSO, CINZIA; INGROSSO, MARCELLO; AQUILINO, A. MARIA; STARZL, THOMAS E.

2010-01-01

Mammalian liver is a sex steroid-responsive tissue. The effects of these hormones presumably are mediated by hepatic estrogen receptors (ER) and androgen receptors (AR). Serum levels of sex hormones display circadian rhythms. Further, estrogens and androgens are commonly administered; administration of these agents is associated frequently with liver disease. Therefore, we investigated whether the cytosolic and nuclear sex steroid receptors also display a similar circadian rhythm, and whether variations occurred in the distribution of receptors between cytosolic and nuclear compartments. Animals were killed every 4 h from midnight till the following midnight; cytosolic and nuclear levels of both ER and AR were measured. Cytosolic ER reached a maximum level at 4 AM, and a minimum at 8 PM and midnight of both days. Nuclear ER was highest at 8 AM and lowest at 4 PM and 8 PM, a pattern which parallels variations in serum estradiol levels. Cytosolic AR was highest at 8 PM and lowest at midnight and 4 AM. Nuclear AR was highest at 4 AM and lowest at 4 PM and 8 PM. The highest level of nuclear AR does not correspond to the maximum serum testosterone level, which occurred at 4 PM. The total hepatic content of both ER and AR was not constant over the 24-h period, but varied considerably with time of day. These studies suggest that both ER and AR show a distinct circadian rhythm in subcellular compartmentalization, and that total hepatic content of ER and AR varies significantly during a 24-h period. PMID:3710067

Limits to anaerobic energy and cytosolic concentration in the living cell

Science.gov (United States)

Paglietti, A.

2015-11-01

For many physical systems at any given temperature, the set of all states where the system's free energy reaches its largest value can be determined from the system's constitutive equations of internal energy and entropy, once a state of that set is known. Such an approach is fraught with complications when applied to a living cell, because the cell's cytosol contains thousands of solutes, and thus thousands of state variables, which makes determination of its state impractical. We show here that, when looking for the maximum energy that the cytosol can store and release, detailed information on cytosol composition is redundant. Compatibility with cell's life requires that a single variable that represents the overall concentration of cytosol solutes must fall between defined limits, which can be determined by dehydrating and overhydrating the cell to its maximum capacity. The same limits are shown to determine, in particular, the maximum amount of free energy that a cell can supply in fast anaerobic processes, starting from any given initial state. For a typical skeletal muscle in normal physiological conditions this energy, i.e., the maximum anaerobic capacity to do work, is calculated to be about 960 J per kg of muscular mass. Such energy decreases as the overall concentration of solutes in the cytosol is increased. Similar results apply to any kind of cell. They provide an essential tool to understand and control the macroscopic response of single cells and multicellular cellular tissues alike. The applications include sport physiology, cell aging, disease produced cell damage, drug absorption capacity, to mention the most obvious ones.

Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties

DEFF Research Database (Denmark)

Munch-Petersen, Birgitte; Munch-Petersen, Sune; Berenstein, Dvora

2006-01-01

Thymidine kinase (TK1) is a key enzyme in the salvage pathway of nucleotide metabolism and catalyzes the first rate-limiting step in the synthesis of dTTP, transfer of a gamma-phosphate group from a nucleoside triphosphate to the 5′-hydroxyl group of thymidine, thus forming dTMP. TK1 is cytosolic...

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

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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

Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein

DEFF Research Database (Denmark)

Liu, Xiangdong; Martens, Helle; Schulz, Alexander

Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein.......Developmental changes in cytosolic coupling between epidermis cells as visualized by photoactivation of fluorescein....

Monocyte-mediated erythrocyte destruction. A comparative study of current methods

International Nuclear Information System (INIS)

Hunt, J.S.; Beck, M.L.; Wood, G.W.

1981-01-01

Three assay systems-EAIgG rosette formation, 51Cr release, and erythrophagocytosis-were used to quantitate interaction between antibody-coated human erythrocytes and normal blood monocytes. The three methods were compared in terms of time requirements and sensitivity. Erythrophagocytosis required more time to perform (2 hours) than did rosette tests (30 minutes) but less than minimum 51Cr release assays (5.5 hours). Erythrophagocytosis was 20-fold more sensitive than either of the other two procedures. Results obtained with purified IgG anti-D and with antibodies induced by transfusion or pregnancy were similar

Erythrocyte antioxidant protection of rose hips (Rosa spp.).

Science.gov (United States)

Widén, C; Ekholm, A; Coleman, M D; Renvert, S; Rumpunen, K

2012-01-01

Rose hips are popular in health promoting products as the fruits contain high content of bioactive compounds. The aim of this study was to investigate whether health benefits are attributable to ascorbic acid, phenols, or other rose-hip-derived compounds. Freeze-dried powder of rose hips was preextracted with metaphosphoric acid and the sample was then sequentially eluted on a C(18) column. The degree of amelioration of oxidative damage was determined in an erythrocyte in vitro bioassay by comparing the effects of a reducing agent on erythrocytes alone or on erythrocytes pretreated with berry extracts. The maximum protection against oxidative stress, 59.4 ± 4.0% (mean ± standard deviation), was achieved when incubating the cells with the first eluted meta-phosphoric extract. Removal of ascorbic acid from this extract increased the protection against oxidative stress to 67.9 ± 1.9%. The protection from the 20% and 100% methanol extracts was 20.8 ± 8.2% and 5.0 ± 3.2%, respectively. Antioxidant uptake was confirmed by measurement of catechin by HPLC-ESI-MS in the 20% methanol extract. The fact that all sequentially eluted extracts studied contributed to protective effects on the erythrocytes indicates that rose hips contain a promising level of clinically relevant antioxidant protection.

Spray drying for preservation of erythrocytes: effect of atomization on hemolysis.

Science.gov (United States)

McLean, Mary; Han, Xiao-Yue; Higgins, Adam Z

2013-04-01

Spray drying has the potential to enable storage of erythrocytes at room temperature in the dry state. The spray drying process involves atomization of a liquid into small droplets and drying of the droplets in a gas stream. In this short report, we focus on the atomization process. To decouple atomization from drying, erythrocyte suspensions were sprayed with a two-fluid atomizer nozzle using humid nitrogen as the atomizing gas. The median droplet size was less than 100 μm for all of the spray conditions investigated, indicating that the suspensions were successfully atomized. Hemolysis was significantly affected by the hematocrit of the erythrocyte suspension, the suspension flow rate, and the atomizing gas flow rate (pspray drying may be a feasible option for erythrocyte biopreservation.

Ammodytoxin, a neurotoxic secreted phospholipase A2, can act in the cytosol of the nerve cell

International Nuclear Information System (INIS)

Petrovic, Uros; Sribar, Jernej; Paris, Alenka; Rupnik, Marjan; Krzan, Mojca; Vardjan, Nina; Gubensek, Franc; Zorec, Robert; Krizaj, Igor

2004-01-01

Recent identification of intracellular proteins that bind ammodytoxin (calmodulin, 14-3-3 proteins, and R25) suggests that this snake venom presynaptically active phospholipase A 2 acts intracellularly. As these ammodytoxin acceptors are cytosolic and mitochondrial proteins, the toxin should be able to enter the cytosol of a target cell and remain stable there to interact with them. Using laser scanning confocal microscopy we show here that Alexa-labelled ammodytoxin entered the cytoplasm of the rat hippocampal neuron and subsequently also its nucleus. The transport of proteins into the nucleus proceeds via the cytosol of a cell, therefore, ammodytoxin passed the cytosol of the neuron on its way to the nucleus. Although it is not yet clear how ammodytoxin is translocated into the cytosol of the neuron, our results demonstrate that its stability in the cytosol is not in question, providing the evidence that the toxin can act in this cellular compartment

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...

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...

Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae) Possess Functional Mitochondria.

Science.gov (United States)

Di Giacomo, Giuseppina; Campello, Silvia; Corrado, Mauro; Di Giambattista, Livia; Cirotti, Claudia; Filomeni, Giuseppe; Gentile, Gabriele

2015-01-01

Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm)-sensitive probe MitoTracker Red indicated that mitochondria i) widely occur in erythrocytes, ii) are polarized, and iii) seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

Radioprotection on nucleated and anucleated erythrocytes by oxide-reduction coenzymes

International Nuclear Information System (INIS)

Fernandez, M.; Tomicic, I.; Rojo, I.

1981-01-01

The protective effects of NAD, FAD and quinone and mixtures of these compounds were studied on gamma irradiated rabbit and chicken erythrocytes. The dose relative factor (DRF 37) was evaluated by visible absorbancy measurements of liberated hemoglobin. The DRF 37 obtained on rabbit erythrocytes were: NAD+FAD+quinone mixture: 11,1; NAD+ quinone mixture: 6,1; FAD+quinone mixture: 6,1; NAD: 1,6; FAD: 5,5; quinone: 5,1. The DRF 37 obtained with the mixture NAD+FAD+quinone on chicken erythrocytes was 3,9. The high efficiency of the radioprotective mixture NAD+FAD+ quinone is discussed. (author)

Differential effect of extracellular calcium on the Na(+)-K+ pump activity in intact polymorphonuclear leucocytes and erythrocytes

DEFF Research Database (Denmark)

Petersen, R H; Knudsen, T; Johansen, Torben

1991-01-01

The effect of extracellular calcium on the Na(+)-K+ pump activity in human polymorphonuclear leucocytes and erythrocytes was studied and compared with the activity in mixed peritoneal leucocytes from rats. While there was maximal decrease in the pump activity (25-30%) of leucocytes from both rat ...

Organizers and activators: Cytosolic Nox proteins impacting on vascular function.

Science.gov (United States)

Schröder, Katrin; Weissmann, Norbert; Brandes, Ralf P

2017-08-01

NADPH oxidases of the Nox family are important enzymatic sources of reactive oxygen species (ROS) in the cardiovascular system. Of the 7 members of the Nox family, at least three depend for their activation on specific cytosolic proteins. These are p47phox and its homologue NoxO1 and p67phox and its homologue NoxA1. Also the Rho-GTPase Rac is important but as this protein has many additional functions, it will not be covered here. The Nox1 enzyme is preferentially activated by the combination of NoxO1 with NoxA1, whereas Nox2 gains highest activity with p47phox together with p67phox. As p47phox, different to NoxO1 contains an auto inhibitory region it has to be phosphorylated prior to complex formation. In the cardio-vascular system, all cytosolic Nox proteins are expressed but the evidence for their contribution to ROS production is not well established. Most data have been collected for p47phox, whereas NoxA1 has basically not yet been studied. In this article the specific aspects of cytosolic Nox proteins in the cardiovascular system with respect to Nox activation, their expression and their importance will be reviewed. Finally, it will be discussed whether cytosolic Nox proteins are suitable pharmacological targets to tamper with vascular ROS production. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Does the chocolate interfere with the radiolabelling of erythrocytes with 99 mTc?; Le chocolat interfere-t-il avec le radiomarquage des erythrocytes au 99 mTc?

Energy Technology Data Exchange (ETDEWEB)

Bustani, H.; Colavolpe, C.; Imbert-Joscht, I.; Moubarik, C.; Havlik, P.; Pisano, P.; Guillet, B. [Service de medecine nucleaire, CHU Nord, Marseille, (France)

2009-05-15

We present in this work the case of a failure of erythrocytes labelling with {sup 99m}Tc for a twenty five years old patient making the examination not interpretable. The patient reported that she drank a chocolate drink the morning before the examination. it is the first observation of a such interaction between the chocolate consumption. We do not have any explanation at this date, some compounds in the cocoa can be considered with caution. Some flavonoids (catechins and pro-cyanidins) modify the plasmatic and intra-erythrocyte oxido-reducer statute and could interact with the labelling (specific reduction of the {sup 99m}Tc pertechnetate in the middle of erythrocyte by the tin pyrophosphate. These compounds, as well as the methyl-xanthine-theobromine, seem modify the membrane permeability of erythrocytes and could interfere with the input of pyrophosphate or {sup 99m}Tc-pertechnetate in the middle of erythrocyte. these observations, in spite of preliminary ones, lead us to recommend near the patients to avoid the chocolate foods in the twenty four hours before this type of examinations. (N.C.)

Targeted disruption of py235ebp-1: Invasion of erythrocytes by Plasmodium yoelii using an alternative py235 erythrocyte binding protein

KAUST Repository

Ogun, Solabomi A.

2011-02-17

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional

Targeted disruption of py235ebp-1: Invasion of erythrocytes by Plasmodium yoelii using an alternative py235 erythrocyte binding protein

KAUST Repository

Ogun, Solabomi A.; Tewari, Rita; Otto, Thomas D.; Howell, Steven A.; Knuepfer, Ellen; Cunningham, Deirdre A.; Xu, Zhengyao; Pain, Arnab; Holder, Anthony A.

2011-01-01

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional

Targeted disruption of py235ebp-1: invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein.

Directory of Open Access Journals (Sweden)

Solabomi A Ogun

2011-02-01

Full Text Available Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2 is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this

In vivo survival of [14C]sucrose-loaded porcine carrier erythrocytes

International Nuclear Information System (INIS)

DeLoach, J.R.

1983-01-01

Porcine carrier erythrocyte survival was measured in adult pigs. [14C]Sucrose-loaded erythrocytes had a biphasic survival curve, with as much as 50% of the cells removed from circulation in the first 24 hours. The remaining cells had a 35-day half-life. Encapsulation values were measured for porcine erythrocytes and entrapment of [14C]sucrose was greater than 45%. Addition of inosine and glucose to the dialyzed cells and to the final wash buffer before reinjection of autologous cells did not improve their survival

DMPD: Regulation of arachidonic acid release and cytosolic phospholipase A2activation. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 10080535 Regulation of arachidonic acid release and cytosolic phospholipase A2activ...on of arachidonic acid release and cytosolic phospholipase A2activation. PubmedID 10080535 Title Regulation ...of arachidonic acid release and cytosolic phospholipase A2activation. Authors Gij

Mature Erythrocytes of Iguana iguana (Squamata, Iguanidae Possess Functional Mitochondria.

Directory of Open Access Journals (Sweden)

Giuseppina Di Giacomo

Full Text Available Electron microscopy analyses of Iguana iguana blood preparations revealed the presence of mitochondria within erythrocytes with well-structured cristae. Fluorescence microscopy analyses upon incubation with phalloidin-FITC, Hoechst 33342 and mitochondrial transmembrane potential (Δψm-sensitive probe MitoTracker Red indicated that mitochondria i widely occur in erythrocytes, ii are polarized, and iii seem to be preferentially confined at a "perinuclear" region, as confirmed by electron microscopy. The analysis of NADH-dependent oxygen consumption showed that red blood cells retain the capability to consume oxygen, thereby providing compelling evidence that mitochondria of Iguana erythrocytes are functional and capable to perform oxidative phosphorylation.

Quantitative assessment of cellular uptake and cytosolic access of antibody in living cells by an enhanced split GFP complementation assay

Energy Technology Data Exchange (ETDEWEB)

Kim, Ji-sun; Choi, Dong-Ki; Park, Seong-wook; Shin, Seung-Min; Bae, Jeomil [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Dong-Myung [Department of Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yoo, Tae Hyeon [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of); Kim, Yong-Sung, E-mail: kimys@ajou.ac.kr [Department of Molecular Science and Technology, Ajou University, Suwon 443-749 (Korea, Republic of)

2015-11-27

Considering the number of cytosolic proteins associated with many diseases, development of cytosol-penetrating molecules from outside of living cells is highly in demand. To gain access to the cytosol after cellular uptake, cell-penetrating molecules should be released from intermediate endosomes prior to the lysosomal degradation. However, it is very challenging to distinguish the pool of cytosolic-released molecules from those trapped in the endocytic vesicles. Here we describe a method to directly demonstrate the cytosolic localization and quantification of cytosolic amount of a cytosol-penetrating IgG antibody, TMab4, based on enhanced split GFP complementation system. We generated TMab4 genetically fused with one GFP fragment and separately established HeLa cells expressing the other GFP fragment in the cytosol such that the complemented GFP fluorescence is observed only when extracellular-treated TMab4 reaches the cytosol after cellular internalization. The high affinity interactions between streptavidin-binding peptide 2 and streptavidin was employed as respective fusion partners of GFP fragments to enhance the sensitivity of GFP complementation. With this method, cytosolic concentration of TMab4 was estimated to be about 170 nM after extracellular treatment of HeLa cells with 1 μM TMab4 for 6 h. We also found that after cellular internalization into living cells, nearly 1.3–4.3% of the internalized TMab4 molecules escaped into the cytosol from the endocytic vesicles. Our enhanced split GFP complementation assay provides a useful tool to directly quantify cytosolic amount of cytosol-penetrating agents and allows cell-based high-throughput screening for cytosol-penetrating agents with increased endosomal-escaping activity.

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

Ca2+-mobilizing agonists increase mitochondrial ATP production to accelerate cytosolic Ca2+ removal: aberrations in human complex I deficiency.

NARCIS (Netherlands)

Visch, H.J.; Koopman, W.J.H.; Zeegers, D.; Emst-de Vries, S.E. van; Kuppeveld, F.J.M. van; Heuvel, L.W. van den; Smeitink, J.A.M.; Willems, P.H.G.M.

2006-01-01

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic Ca2+ removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial

Optical Assay of Erythrocyte Function in Banked Blood

Science.gov (United States)

Bhaduri, Basanta; Kandel, Mikhail; Brugnara, Carlo; Tangella, Krishna; Popescu, Gabriel

2014-09-01

Stored red blood cells undergo numerous biochemical, structural, and functional changes, commonly referred to as storage lesion. How much these changes impede the ability of erythrocytes to perform their function and, as result, impact clinical outcomes in transfusion patients is unknown. In this study we investigate the effect of the storage on the erythrocyte membrane deformability and morphology. Using optical interferometry we imaged red blood cell (RBC) topography with nanometer sensitivity. Our time-lapse imaging quantifies membrane fluctuations at the nanometer scale, which in turn report on cell stiffness. This property directly impacts the cell's ability to transport oxygen in microvasculature. Interestingly, we found that cells which apparently maintain their normal shape (discocyte) throughout the storage period, stiffen progressively with storage time. By contrast, static parameters, such as mean cell hemoglobin content and morphology do not change during the same period. We propose that our method can be used as an effective assay for monitoring erythrocyte functionality during storage time.

Unusual bone marrow visualization with sup(99m)Tc-damaged erythrocytes

Energy Technology Data Exchange (ETDEWEB)

Dolgova-Korubin, V; Simova, N

1982-05-01

A visualization of the bone marrow and of the spleen was obtained with sup(99m)Tc-labeled N-ethylmaleimide (NEM) damaged erythrocytes in a patient with systemic lupus erythematosus. This phenomenon could be produced both with autologous and homologous red cells. The patient's NEM-damaged erythrocytes given to a healthy patient did not produce a visualization of the marrow. Such an observation was not present in a series of healthy persons and patients with different disorders to whom labeled and damaged erythrocytes were administered, and has not been reported in the literature so far.

New Insight into Erythrocyte through In Vivo Surface-Enhanced Raman Spectroscopy

DEFF Research Database (Denmark)

Brazhe, Nadezda A.; Abdali, Salim; Brazhe, Alexey R.

2009-01-01

containing erythrocytes in their normal physiological environment in a mixture of colloid solution with silver nanoparticles and the procedure for the optimization of SERS conditions to achieve high signal enhancement without affecting the properties of living erythrocytes. By means of three independent...... techniques, we demonstrate that under the proposed conditions a colloid solution of silver nanoparticles does not affect the properties of erythrocytes. For the first time to our knowledge, we describe how to use the SERS-RS approach to study two populations of hemoglobin molecules inside an intact living...

Investigating the function of F_c -specific binding of IgM to Plasmodium falciparum erythrocyte membrane protein 1 mediating erythrocyte rosetting

DEFF Research Database (Denmark)

Stevenson, Liz; Huda, Pie; Jeppesen, Anine

2015-01-01

of opsonized infected erythrocytes (IEs) without compromising the placental IE adhesion mediated by this PfEMP1 type. IgM also binds via Fc to several other PfEMP1 proteins, where it has been proposed to facilitate rosetting (binding of uninfected erythrocytes to a central IE). To further dissect...

Spatial modeling of the membrane-cytosolic interface in protein kinase signal transduction.

Directory of Open Access Journals (Sweden)

Wolfgang Giese

2018-04-01

Full Text Available The spatial architecture of signaling pathways and the interaction with cell size and morphology are complex, but little understood. With the advances of single cell imaging and single cell biology, it becomes crucial to understand intracellular processes in time and space. Activation of cell surface receptors often triggers a signaling cascade including the activation of membrane-attached and cytosolic signaling components, which eventually transmit the signal to the cell nucleus. Signaling proteins can form steep gradients in the cytosol, which cause strong cell size dependence. We show that the kinetics at the membrane-cytosolic interface and the ratio of cell membrane area to the enclosed cytosolic volume change the behavior of signaling cascades significantly. We suggest an estimate of average concentration for arbitrary cell shapes depending on the cell volume and cell surface area. The normalized variance, known from image analysis, is suggested as an alternative measure to quantify the deviation from the average concentration. A mathematical analysis of signal transduction in time and space is presented, providing analytical solutions for different spatial arrangements of linear signaling cascades. Quantification of signaling time scales reveals that signal propagation is faster at the membrane than at the nucleus, while this time difference decreases with the number of signaling components in the cytosol. Our investigations are complemented by numerical simulations of non-linear cascades with feedback and asymmetric cell shapes. We conclude that intracellular signal propagation is highly dependent on cell geometry and, thereby, conveys information on cell size and shape to the nucleus.

A Lectin-Like Receptor is Involved in Invasion of Erythrocytes by Plasmodium falciparum

Science.gov (United States)

Jungery, M.; Pasvol, G.; Newbold, C. I.; Weatherall, D. J.

1983-02-01

Glycophorin both in solution and inserted into liposomes blocks invasion of erythrocytes by the malaria parasite Plasmodium falciparum. Furthermore, one sugar, N-acetyl-D-glucosamine (GlcNAc), completely blocks invasion of the erythrocyte by this parasite. GlcNAc coupled to bovine serum albumin to prevent the sugar entering infected erythrocytes was at least 100,000 times more effective than GlcNAc alone. Bovine serum albumin coupled to lactose or bovine serum albumin alone had no effect on invasion. These results suggest that the binding of P. falciparum to erythrocytes is lectin-like and is determined by carbohydrates on glycophorin.

Pressurized liquid extraction-assisted mussel cytosol preparation for the determination of metals bound to metallothionein-like proteins

International Nuclear Information System (INIS)

Santiago-Rivas, Sandra; Moreda-Pineiro, Antonio; Bermejo-Barrera, Pilar; Moreda-Pineiro, Jorge; Alonso-Rodriguez, Elia; Muniategui-Lorenzo, Soledad; Lopez-Mahia, Purificacion; Prada-Rodriguez, Dario

2007-01-01

The possibilities of pressurized liquid extraction (PLE) have been novelty tested to assist the cytosol preparation from wet mussel soft tissue before the determination of metals bound to metallothionein-like proteins (MLPs). Results obtained after PLE were compared with those obtained after a classical blending procedure for mussel cytosolic preparation. Isoforms MLP-1 (retention time of 4.1 min) and MLP-2 (retention time of 7.4 min) were separated by anion exchange high-performance liquid chromatography (HPLC) and the concentrations of Ba, Cu, Mn, Sr and Zn bound to MLP isoforms were directly measured by inductively coupled plasma-atomic emission spectrometry (ICP-OES) as a multi-element detector. The optimized PLE-assisted mussel cytosol preparation has consisted of one extraction cycle at room temperature and 1500 psi for 2 min. Since separation between the solid mussel residue and the extract (cytosol) is performed by the PLE system, the cytosol preparation method is faster than conventional cytosol preparation methods by cutting/blending using Ultraturrax or Stomacher devices

Viral erythrocytic necrosis: Chapter 2.2.7

Science.gov (United States)

Winton, James R.; Hershberger, Paul K.

2014-01-01

Viral erythrocytic necrosis (VEN), originally termed piscine erythrocytic necrosis, is a condition that has been reported to affect the red blood cells (RBCs) of many species of marine and anadromous fishes in both the Atlantic and Pacific Oceans (Nicholson and Reno 1981; Smail 1982; Wolf 1988; Dannevig and Thorud 1999). Fish with VEN may develop a severe anemia that can reduce their stamina, predispose them to other infections or increase the impact of other stressors (MacMillan et al. 1980; Nicholson and Reno 1981; Meyers et al. 1986; Haney et al. 1992) resulting in population-scale impacts in susceptible species (Hershberger et al. 2009).

The effect of centrifugation at various g force levels on rheological properties of rat, dog, pig and human red blood cells.

Science.gov (United States)

Kiss, Ferenc; Toth, Eniko; Miszti-Blasius, Kornel; Nemeth, Norbert

2016-01-01

Laboratory investigations often require centrifugation of blood samples for various erythrocyte tests. Although there is a lack of data about the effect of centrifugation at various g force levels on erythrocyte rheological properties. We aimed to investigate the effect of a 10-minute centrifugation at 500, 1000 or 1500 g at 15°C of rat, dog, pig and human venous (K3-EDTA, 1.5 mg/ml) blood samples. Hematological parameters, erythrocyte deformability, cell membrane stability, osmotic gradient ektacytometry (osmoscan) and erythrocyte aggregation were determined. Hematological and erythrocyte deformability parameters showed interspecies differences, centrifugation caused no significant alterations. Cell membrane stability for human erythrocytes centrifuged at higher g level showed less decrease in deformability. Osmoscan O min parameter showed slight elevation in dog centrifuged aliquots. Erythrocyte aggregation parameters changed unexpectedly. Rat and dog erythrocyte aggregation indices significantly dropped in centrifuged aliquots. Pig erythrocyte aggregation indices increased significantly after centrifugation. Human erythrocyte aggregation was the most stable one among the investigated species. The used centrifugation protocols caused the largest alterations in erythrocyte aggregation in a controversial way among the investigated species. On the other hand, erythrocyte deformability parameters were stable, cell membrane stability and osmoscan data show minor shifts.

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...

Characterization of the methotrexate transport pathway in murine L1210 leukemia cells: Involvement of a membrane receptor and a cytosolic protein

International Nuclear Information System (INIS)

Price, E.M.; Ratnam, M.; Rodeman, K.M.; Freisheim, J.H.

1988-01-01

A radioiodinated photoaffinity analogue of methotrexate, N α -(4-amino-4-deoxy-10-methyl-pteroyl)-N ε -(4-azidosalicylyl)-L-lysine (APA-ASA-Lys), was recently used to identify the plasma membrane derived binding protein involved in the transport of this folate antagonist into murine L1210 cells. The labeled protein has an apparent molecular weight of 46K-48K when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but no such labeling occurs in a methotrexate transport-defective cell line (L1210/R81). Labeling of the total cytosolic protein from disrupted cells, followed by electrophoresis and autoradiography, showed, among other proteins, a 21K band, corresponding to dihydrofolate reductase (DHFR), in both the parent and R81 cells and a 38K band only in the parent cells. However, when whole cells were UV irradiated at various times at 37 degree C following addition of radiolabeled APA-ASA-Lys, the 38K protein and DHFR were the only cytosolic proteins labeled in the parent cells, while the intact R81 cells showed no labeled cytosolic protein, since the photoprobe is not transported. Further, when the parent cells were treated with a pulse of radiolabeled photoprobe, followed by UV irradiation at different times at 37 degree C, the probe appeared sequentially on the 48K membrane protein and both the 38K cytosolic protein and dihydrofolate reductase. A 48K protein could be detected in both parent L1210 cells and the R81 cells on Western blots using antisera to a membrane folate binding protein from human placenta. These results suggest a vectorial transport of APA-ASA-Lys or methotrexate and reduced folate coenzymes into murine L1210 cells mediated by a 48K integral membrane protein and a 38K cytosolic or peripheral membrane protein. The 38K protein may help in the trafficking of reduced folate coenzymes, shuttling them to various cytosolic targets

Change of properties of erythrocytes membranes in UV-irradiated blood

International Nuclear Information System (INIS)

Gromov, A.E.; Vetosh, A.N.; Nikonchuk, N.P.; Perelygin, V.G.; Ruzanov, I.B.

1986-01-01

An increase in erythrocyte membrane permeability for gases, decrease in erythrocyte thermal stability and activation of membrane transport systems after autotransfusion of UV-irradiated blood are ascertained. The data obtained testify to the fact that the greatest changes in membranes take place not directly under irradiation but after the introduction of irradiated blood to the organism

Exo-erythrocytic development of avian malaria and related haemosporidian parasites.

Science.gov (United States)

Valkiūnas, Gediminas; Iezhova, Tatjana A

2017-03-03

Avian malaria parasites (Plasmodium spp.) and related haemosporidians (Haemosporida) are responsible for diseases which can be severe and even lethal in avian hosts. These parasites cause not only blood pathology, but also damage various organs due to extensive exo-erythrocytic development all over the body, which is not the case during Plasmodium infections in mammals. However, exo-erythrocytic development (tissue merogony or schizogony) remains the most poorly investigated part of life cycle in all groups of wildlife haemosporidian parasites. In spite of remarkable progress in studies of genetic diversity, ecology and evolutionary biology of avian haemosporidians during the past 20 years, there is not much progress in understanding patterns of exo-erythrocytic development in these parasites. The purpose of this review is to overview the main information on exo-erythrocytic development of avian Plasmodium species and related haemosporidian parasites as a baseline for assisting academic and veterinary medicine researchers in morphological identification of these parasites using tissue stages, and to define future research priorities in this field of avian malariology. The data were considered from peer-reviewed articles and histological material that was accessed in zoological collections in museums of Australia, Europe and the USA. Articles describing tissue stages of avian haemosporidians were included from 1908 to the present. Histological preparations of various organs infected with the exo-erythrocytic stages of different haemosporidian parasites were examined. In all, 229 published articles were included in this review. Exo-erythrocytic stages of avian Plasmodium, Fallisia, Haemoproteus, Leucocytozoon, and Akiba species were analysed, compared and illustrated. Morphological characters of tissue stages that can be used for diagnostic purposes were specified. Recent molecular studies combined with histological research show that avian haemosporidians are more

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

Insulin Induces an Increase in Cytosolic Glucose Levels in 3T3-L1 Cells with Inhibited Glycogen Synthase Activation

Directory of Open Access Journals (Sweden)

Helena H. Chowdhury

2014-10-01

Full Text Available Glucose is an important source of energy for mammalian cells and enters the cytosol via glucose transporters. It has been thought for a long time that glucose entering the cytosol is swiftly phosphorylated in most cell types; hence the levels of free glucose are very low, beyond the detection level. However, the introduction of new fluorescence resonance energy transfer-based glucose nanosensors has made it possible to measure intracellular glucose more accurately. Here, we used the fluorescent indicator protein (FLIPglu-600µ to monitor cytosolic glucose dynamics in mouse 3T3-L1 cells in which glucose utilization for glycogen synthesis was inhibited. The results show that cells exhibit a low resting cytosolic glucose concentration. However, in cells with inhibited glycogen synthase activation, insulin induced a robust increase in cytosolic free glucose. The insulin-induced increase in cytosolic glucose in these cells is due to an imbalance between the glucose transported into the cytosol and the use of glucose in the cytosol. In untreated cells with sensitive glycogen synthase activation, insulin stimulation did not result in a change in the cytosolic glucose level. This is the first report of dynamic measurements of cytosolic glucose levels in cells devoid of the glycogen synthesis pathway.

Plasmodium falciparum glutamate dehydrogenase a is dispensable and not a drug target during erythrocytic development

LENUS (Irish Health Repository)

Storm, Janet

2011-07-14

Abstract Background Plasmodium falciparum contains three genes encoding potential glutamate dehydrogenases. The protein encoded by gdha has previously been biochemically and structurally characterized. It was suggested that it is important for the supply of reducing equivalents during intra-erythrocytic development of Plasmodium and, therefore, a suitable drug target. Methods The gene encoding the NADP(H)-dependent GDHa has been disrupted by reverse genetics in P. falciparum and the effect on the antioxidant and metabolic capacities of the resulting mutant parasites was investigated. Results No growth defect under low and elevated oxygen tension, no up- or down-regulation of a number of antioxidant and NADP(H)-generating proteins or mRNAs and no increased levels of GSH were detected in the D10Δgdha parasite lines. Further, the fate of the carbon skeleton of [13C] labelled glutamine was assessed by metabolomic studies, revealing no differences in the labelling of α-ketoglutarate and other TCA pathway intermediates between wild type and mutant parasites. Conclusions First, the data support the conclusion that D10Δgdha parasites are not experiencing enhanced oxidative stress and that GDHa function may not be the provision of NADP(H) for reductive reactions. Second, the results imply that the cytosolic, NADP(H)-dependent GDHa protein is not involved in the oxidative deamination of glutamate but that the protein may play a role in ammonia assimilation as has been described for other NADP(H)-dependent GDH from plants and fungi. The lack of an obvious phenotype in the absence of GDHa may point to a regulatory role of the protein providing glutamate (as nitrogen storage molecule) in situations where the parasites experience a limiting supply of carbon sources and, therefore, under in vitro conditions the enzyme is unlikely to be of significant importance. The data imply that the protein is not a suitable target for future drug development against intra-erythrocytic

Very Deep Convolutional Neural Networks for Morphologic Classification of Erythrocytes.

Science.gov (United States)

Durant, Thomas J S; Olson, Eben M; Schulz, Wade L; Torres, Richard

2017-12-01

Morphologic profiling of the erythrocyte population is a widely used and clinically valuable diagnostic modality, but one that relies on a slow manual process associated with significant labor cost and limited reproducibility. Automated profiling of erythrocytes from digital images by capable machine learning approaches would augment the throughput and value of morphologic analysis. To this end, we sought to evaluate the performance of leading implementation strategies for convolutional neural networks (CNNs) when applied to classification of erythrocytes based on morphology. Erythrocytes were manually classified into 1 of 10 classes using a custom-developed Web application. Using recent literature to guide architectural considerations for neural network design, we implemented a "very deep" CNN, consisting of >150 layers, with dense shortcut connections. The final database comprised 3737 labeled cells. Ensemble model predictions on unseen data demonstrated a harmonic mean of recall and precision metrics of 92.70% and 89.39%, respectively. Of the 748 cells in the test set, 23 misclassification errors were made, with a correct classification frequency of 90.60%, represented as a harmonic mean across the 10 morphologic classes. These findings indicate that erythrocyte morphology profiles could be measured with a high degree of accuracy with "very deep" CNNs. Further, these data support future efforts to expand classes and optimize practical performance in a clinical environment as a prelude to full implementation as a clinical tool. © 2017 American Association for Clinical Chemistry.

Live cell imaging of cytosolic NADH/NAD+ ratio in hepatocytes and liver slices.

Science.gov (United States)

Masia, Ricard; McCarty, William J; Lahmann, Carolina; Luther, Jay; Chung, Raymond T; Yarmush, Martin L; Yellen, Gary

2018-01-01

Fatty liver disease (FLD), the most common chronic liver disease in the United States, may be caused by alcohol or the metabolic syndrome. Alcohol is oxidized in the cytosol of hepatocytes by alcohol dehydrogenase (ADH), which generates NADH and increases cytosolic NADH/NAD + ratio. The increased ratio may be important for development of FLD, but our ability to examine this question is hindered by methodological limitations. To address this, we used the genetically encoded fluorescent sensor Peredox to obtain dynamic, real-time measurements of cytosolic NADH/NAD + ratio in living hepatocytes. Peredox was expressed in dissociated rat hepatocytes and HepG2 cells by transfection, and in mouse liver slices by tail-vein injection of adeno-associated virus (AAV)-encoded sensor. Under control conditions, hepatocytes and liver slices exhibit a relatively low (oxidized) cytosolic NADH/NAD + ratio as reported by Peredox. The ratio responds rapidly and reversibly to substrates of lactate dehydrogenase (LDH) and sorbitol dehydrogenase (SDH). Ethanol causes a robust dose-dependent increase in cytosolic NADH/NAD + ratio, and this increase is mitigated by the presence of NAD + -generating substrates of LDH or SDH. In contrast to hepatocytes and slices, HepG2 cells exhibit a relatively high (reduced) ratio and show minimal responses to substrates of ADH and SDH. In slices, we show that comparable results are obtained with epifluorescence imaging and two-photon fluorescence lifetime imaging (2p-FLIM). Live cell imaging with Peredox is a promising new approach to investigate cytosolic NADH/NAD + ratio in hepatocytes. Imaging in liver slices is particularly attractive because it allows preservation of liver microanatomy and metabolic zonation of hepatocytes. NEW & NOTEWORTHY We describe and validate a new approach for measuring free cytosolic NADH/NAD + ratio in hepatocytes and liver slices: live cell imaging with the fluorescent biosensor Peredox. This approach yields dynamic, real

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

[Age-related change in the alpha-tocopherolquinone/alpha-tocopherol ratio in the rat erythrocyte membrane].

Science.gov (United States)

Yanagawa, K; Takeda, H; Matsumiya, T; Takasaki, M

1999-05-01

alpha-Tocopherol (alpha-Toc), a lipophilic phenolic antioxidant that is localized mainly in the biomembrane, protects cells against oxidation-associated cytotoxicity by prevention of membrane lipid peroxidation, maintenance of the redox balance intracellular thiols and stabilization of the membrane structure. We investigated the age-related changes in redox dynamics of alpha-Toc in plasma and erythrocyte membrane of an elderly (66 weeks old) and young group (10 weeks old). Total, alpha-, beta + gamma-, delta-Toc and alpha-tocopherolquinone (alpha-TocQ) in plasma and erythrocyte membrane were determined by high-performance liquid chromatography (HPLC) with a series of multiple coulometric working electrodes (CWE). Rat venous blood sample was divided into plasma and erythrocyte layers by centrifugation, and then erythrocyte membrane sample was prepared according to the method of Dodge et al. under a stream of nitrogen. In plasma, total and alpha-Toc concentrations were increased, and beta + gamma-, delta-Toc and alpha-TocQ concentrations were decreased age-dependently. In the erythrocyte membrane, total, alpha-TocQ concentrations and three fractions of tocopherols decreased age-dependently. Also, a decrease in the alpha-TocQ/alpha-Toc ratio in erythrocyte membrane was observed in the elderly group. These findings suggest that the alpha-Toc uptake in erythrocyte membrane and utilization rate of alpha-Toc in erythrocyte membrane decline age-dependently. This decline may promote membrane lipid peroxidation. alpha-Toc redox dynamics in erythrocyte membrane were useful to investigate the pathophysiology of aging mechanisms related to oxidative stress.

Effect of dietary zinc deficiency on the endogenous phosphorylation and dephosphorylation of rat erythrocyte membrane

International Nuclear Information System (INIS)

Paterson, P.G.; Allen, O.B.; Bettger, W.J.

1987-01-01

The effect of dietary zinc deficiency on patterns of phosphorylation and dephosphorylation of rat erythrocyte membrane proteins and erythrocyte filterability was examined. Weanling male Wistar rats were fed an egg white-based diet containing less than 1.1 mg zinc/kg diet ad libitum for 3 wk. Control rats were either pair-fed or ad libitum-fed the basal diet supplemented with 100 mg zinc/kg diet. Net phosphorylation and dephosphorylation of erythrocyte membrane proteins were carried out by an in vitro assay utilizing [gamma- 32 P]ATP. The membrane proteins were subsequently separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the 32 P content of gel slices was counted by Cerenkov counting. Erythrocyte filterability was measured as the filtration time of suspensions of erythrocytes, both untreated and preincubated with diamide, under constant pressure. Erythrocyte ghosts from zinc-deficient rats demonstrated greater dephosphorylation of protein bands R1 plus R2 and R7 than pair-fed rats and greater net phosphorylation of band R2.2 than pair-fed or ad libitum-fed control rats (P less than 0.05). Erythrocytes from ad libitum-fed control rats showed significantly longer filtration times than those from zinc-deficient or pair-fed control rats. In conclusion, dietary zinc deficiency alters in vitro patterns of erythrocyte membrane protein phosphorylation and dephosphorylation, whereas the depression in food intake associated with the zinc deficiency increases erythrocyte filterability. 71 references

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...

Biophysical Properties of Irradiated Erythrocytes and Role of Antioxidants

International Nuclear Information System (INIS)

Eman Mohammed Elbakrawy, E.M.

2010-01-01

Irradiation of blood and blood components with gamma-irradiation is recommended for the inactivation of T-lymphocytes and prevention of transfusion-associated graft versus host diseases. The aim of the present work is to ensure that the currently applied irradiation dose 25 Gy is a safe dose based on the study of the electrical behavior, rheological properties, membrane solubilization, membrane hemolysis and scanning electron microscope of stored erythrocytes. In addition it aims to study the possibility of increasing the irradiation doses to 50 and 100 Gy. Moreover Alpha lipoic acid (a potent natural antioxidant) was added to the stored erythrocytes before irradiation for radioprotection. Irradiation of erythrocytes with 25 Gy did not show significant changes for the calculated dielectric parameters. However, increasing the irradiation doses resulted in significant decrease in the calculated dielectric parameters reflecting the damaging effects of radiation on the membrane structure. The obtained results showed that α lipoic acid can play an important role in minimizing the radiation-induced damage to the erythrocytes and conserve their electrical properties. There were non-significant changes in viscosity after exposure to 25 Gy, while a significant increase at 50 Gy and a significant decrease at 100 Gy were observed. The study found that α lipoic acid (ALA) resulted in non-significant change in viscosity after exposure to 50 Gy and 100 Gy. A significant increase in the yield stress was observed after exposure to 25 and 50 Gy while at 100 Gy, the yield stress showed a remarkable decrease. Addition of .-lipoic acid before irradiation resulted in non-significant changes in the yield stress at doses 25, 50, 100 Gy.The obtained results of the average membrane solubilization (D 50 ) and the average membrane hemolysis (H 50 ) showed non-significant change at 25 Gy; while an observable decrease was observed at 50 Gy and 100 Gy. The addition of lipoic acid did not

Splenic Trapping of Heat-Treated Erythrocytes in Leukaemia and Allied Conditions

Energy Technology Data Exchange (ETDEWEB)

Badrawi, H. S.; Razzak, M. A.; Guirgis, B. [Department of Medicine and Division of Nuclear Medicine, Faculty of Medicine, Cairo University, Cairo, United Arab Republic (Egypt)

1971-02-15

In a trial to find whether or not the enlarged spleen plays a role in the production of the form of anaemia commonly encountered in leukaemias and allied conditions, 44 patients suffering from these disease states were studied using {sup 51}Cr-labelled erythrocytes heated at 50 Degree-Sign C for 60 min. Cells altered in this manner have been shown by various workers to be selectively sequestered by the spleen. As a control, the test was performed on 24 normal subjects. In these normals, the disappearance half-time of radioactivity from the circulation (T{sub Vulgar-Fraction-One-Half} amounted to 172 {+-} 69 min (mean {+-} 1 S.D.), the lowest limit being 74 min. Accordingly, patients with less than 74 min were considered to have an abnormally rapid disappearance of heat-treated erythrocytes from the circulation and consequently exaggerated splenic sequestration of these altered cells. Splenic trapping of heat-treated erythrocytes was most marked in acute leukaemia (four out of six patients). However, three had associated normoblastic hypoplasia of the sternal marrow. Corticosteroids induced a remission with reversion of both processes responsible for the anaemia in two out of the four patients. In chronic myeloid leukaemia, exaggerated splenic sequestration of altered cells was seen in four of the 15 cases examined. This condition was of extra-erythrocytic origin, since repetition of the test using normal donor heat-treated erythrocytes did not significantly alter the disappearance half-time. However, there was no correlation between the size of the spleen and its avidity for trapping the altered cells. Follow-up studies showed that therapy caused prolongation of the half-time of heat-treated erythrocytes, the effect being more apparent after corticosteroids than with X-rays or Endoxan, In Hodgkin's disease, increased red cell trapping was observed in two out of the seven patients studied. In contrast, five cases of chronic lymphatic leukaemia, six lymphosarcoma and

Cytosolic 5'-nucleotidase II interacts with the leucin rich repeat of NLR family member Ipaf.

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Federico Cividini

Full Text Available IMP/GMP preferring cytosolic 5'-nucleotidase II (cN-II is a bifunctional enzyme whose activities and expression play crucial roles in nucleotide pool maintenance, nucleotide-dependent pathways and programmed cell death. Alignment of primary amino acid sequences of cN-II from human and other organisms show a strong conservation throughout the entire vertebrata taxon suggesting a fundamental role in eukaryotic cells. With the aim to investigate the potential role of this homology in protein-protein interactions, a two hybrid system screening of cN-II interactors was performed in S. cerevisiae. Among the X positive hits, the Leucin Rich Repeat (LRR domain of Ipaf was found to interact with cN-II. Recombinant Ipaf isoform B (lacking the Nucleotide Binding Domain was used in an in vitro affinity chromatography assay confirming the interaction obtained in the screening. Moreover, co-immunoprecipitation with proteins from wild type Human Embryonic Kidney 293 T cells demonstrated that endogenous cN-II co-immunoprecipitated both with wild type Ipaf and its LRR domain after transfection with corresponding expression vectors, but not with Ipaf lacking the LRR domain. These results suggest that the interaction takes place through the LRR domain of Ipaf. In addition, a proximity ligation assay was performed in A549 lung carcinoma cells and in MDA-MB-231 breast cancer cells and showed a positive cytosolic signal, confirming that this interaction occurs in human cells. This is the first report of a protein-protein interaction involving cN-II, suggesting either novel functions or an additional level of regulation of this complex enzyme.

Improved Human Erythropoiesis and Platelet Formation in Humanized NSGW41 Mice

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Susann Rahmig

2016-10-01

Full Text Available Human erythro-megakaryopoiesis does not occur in humanized mouse models, preventing the in vivo analysis of human hematopoietic stem cell (HSC differentiation into these lineages in a surrogate host. Here we show that stably engrafted KIT-deficient NOD/SCID Il2rg−/− KitW41/W41 (NSGW41 mice support much improved human erythropoiesis and platelet formation compared with irradiated NSG recipients. Considerable numbers of human erythroblasts and mature thrombocytes are present in the bone marrow and blood, respectively. Morphology, composition, and enucleation capacity of de novo generated human erythroblasts in NSGW41 mice are comparable with those in human bone marrow. Overexpression of human erythropoietin showed no further improvement in human erythrocyte output, but depletion of macrophages led to the appearance of human erythrocytes in the blood. Human erythropoiesis up to normoblasts and platelet formation is fully supported in NSGW41 mice, allowing the analysis of human HSC differentiation into these lineages, the exploration of certain pathophysiologies, and the evaluation of gene therapeutic approaches.

Stimulation of eryptosis by aluminium ions

International Nuclear Information System (INIS)

Niemoeller, Olivier M.; Kiedaisch, Valentin; Dreischer, Peter; Wieder, Thomas; Lang, Florian

2006-01-01

Aluminium salts are utilized to impede intestinal phosphate absorption in chronic renal failure. Toxic side effects include anemia, which could result from impaired formation or accelerated clearance of circulating erythrocytes. Erythrocytes may be cleared secondary to suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and exposure of phosphatidylserine (PS) at the erythrocyte surface. As macrophages are equipped with PS receptors, they bind, engulf and degrade PS-exposing cells. The present experiments have been performed to explore whether Al 3+ ions trigger eryptosis. The PS exposure was estimated from annexin binding and cell volume from forward scatter in FACS analysis. Exposure to Al 3+ ions (≥ 10 μM Al 3+ for 24 h) indeed significantly increased annexin binding, an effect paralleled by decrease of forward scatter at higher concentrations (≥ 30 μM Al 3+ ). According to Fluo3 fluorescence Al 3+ ions (≥ 30 μM for 3 h) increased cytosolic Ca 2+ activity. Al 3+ ions (≥ 10 μM for 24 h) further decreased cytosolic ATP concentrations. Energy depletion by removal of glucose similarly triggered annexin binding, an effect not further enhanced by Al 3+ ions. The eryptosis was paralleled by release of hemoglobin, pointing to loss of cell membrane integrity. In conclusion, Al 3+ ions decrease cytosolic ATP leading to activation of Ca 2+ -permeable cation channels, Ca 2+ entry, stimulation of cell membrane scrambling and cell shrinkage. Moreover, Al 3+ ions lead to loss of cellular hemoglobin, a feature of hemolysis. Both effects are expected to decrease the life span of circulating erythrocytes and presumably contribute to the development of anemia during Al 3+ intoxication

Complement and Antibody-Mediated Enhancement of Erythrocyte Invasion by Plasmodium Falciparum

Science.gov (United States)

2016-04-01

Haynes, J.D., Moch , J.K., Smoot, D.S., 2002. Erythrocytic malaria growth or invasion inhibi- tion assays with emphasis on suspension culture GIA... Moch , J.K., Finberg, R.W., Tsokos, G.C., Stoute, J.A., 2010. Complement receptor 1 is a sialic acid-independent erythrocyte receptor of Plasmodium

A novel approach for assessments of erythrocyte sedimentation rate.

Science.gov (United States)

Pribush, A; Hatskelzon, L; Meyerstein, N

2011-06-01

Previous studies have shown that the dispersed phase of sedimenting blood undergoes dramatic structural changes: Discrete red blood cell (RBC) aggregates formed shortly after a settling tube is filled with blood are combined into a continuous network followed by its collapse via the formation of plasma channels, and finally, the collapsed network is dispersed into individual fragments. Based on this scheme of structural transformation, a novel approach for assessments of erythrocyte sedimentation is suggested. Information about erythrocyte sedimentation is extracted from time records of the blood conductivity measured after a dispersion of RBC network into individual fragments. It was found that the sedimentation velocity of RBC network fragments correlates positively with the intensity of attractive intercellular interactions, whereas no effect of hematocrit (Hct) was observed. Thus, unlike Westergren erythrocyte sedimentation rate, sedimentation data obtained by the proposed method do not require correction for Hct. © 2010 Blackwell Publishing Ltd.

Apoptotic DNA Degradation into Oligonucleosomal Fragments, but Not Apoptotic Nuclear Morphology, Relies on a Cytosolic Pool of DFF40/CAD Endonuclease*

Science.gov (United States)

Iglesias-Guimarais, Victoria; Gil-Guiñon, Estel; Gabernet, Gisela; García-Belinchón, Mercè; Sánchez-Osuna, María; Casanelles, Elisenda; Comella, Joan X.; Yuste, Victor J.

2012-01-01

Apoptotic cell death is characterized by nuclear fragmentation and oligonucleosomal DNA degradation, mediated by the caspase-dependent specific activation of DFF40/CAD endonuclease. Here, we describe how, upon apoptotic stimuli, SK-N-AS human neuroblastoma-derived cells show apoptotic nuclear morphology without displaying concomitant internucleosomal DNA fragmentation. Cytotoxicity afforded after staurosporine treatment is comparable with that obtained in SH-SY5Y cells, which exhibit a complete apoptotic phenotype. SK-N-AS cell death is a caspase-dependent process that can be impaired by the pan-caspase inhibitor q-VD-OPh. The endogenous inhibitor of DFF40/CAD, ICAD, is correctly processed, and dff40/cad cDNA sequence does not reveal mutations altering its amino acid composition. Biochemical approaches show that both SH-SY5Y and SK-N-AS resting cells express comparable levels of DFF40/CAD. However, the endonuclease is poorly expressed in the cytosolic fraction of healthy SK-N-AS cells. Despite this differential subcellular distribution of DFF40/CAD, we find no differences in the subcellular localization of both pro-caspase-3 and ICAD between the analyzed cell lines. After staurosporine treatment, the preferential processing of ICAD in the cytosolic fraction allows the translocation of DFF40/CAD from this fraction to a chromatin-enriched one. Therefore, the low levels of cytosolic DFF40/CAD detected in SK-N-AS cells determine the absence of DNA laddering after staurosporine treatment. In these cells DFF40/CAD cytosolic levels can be restored by the overexpression of their own endonuclease, which is sufficient to make them proficient at degrading their chromatin into oligonucleosome-size fragments after staurosporine treatment. Altogether, the cytosolic levels of DFF40/CAD are determinants in achieving a complete apoptotic phenotype, including oligonucleosomal DNA degradation. PMID:22253444

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)

Cytosolic Cl- Affects the Anticancer Activity of Paclitaxel in the Gastric Cancer Cell Line, MKN28 Cell

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Sachie Tanaka

2017-05-01

Full Text Available Background/Aims: Our previous study revealed that cytosolic Cl- affected neurite elongation promoted via assembly of microtubule in rat pheochromocytoma PC12D cells and Cl-–induced blockade of intrinsic GTPase enhanced tubulin polymerization in vitro. Paclitaxel (PTX is a microtubule-targeted chemotherapeutic drug and stabilizes microtubules resulting in mainly blockade of mitosis at the metaphase-anaphase transition and induction of apoptosis. In the present study, we tried to clarify whether the cytosolic Cl- affected PTX ability to inhibit cell growth in the gastric cancer cell line, MKN28. Methods: To clarify the cytosolic Cl- action on PTX-induced cell death and metaphase-anaphase transition in the gastric cancer cell line, MKN28 cell, and PTX-induced tubulin polymerization, we performed cell proliferation assay, cytosolic Cl- concentration measurement, immunofluorescence microscopy, and in vitro tubulin polymerization assay. Results: The decline of cytosolic Cl- weakened the cytotoxic effect of PTX on cell proliferation of MKN28 cells, which could pass through the metaphase-anaphase transition. Moreover, in vitro PTX-induced tubulin polymerization was diminished under the low Cl- condition. Conclusions: Our results strongly suggest that the upregulation of cytosolic Cl- concentration would enhance the antitumor effect of PTX, and that the cytosolic Cl- would be one of the key targets for anti-cancer therapy.

Phenotypic variations in osmotic lysis of Sahel goat erythrocytes in non-ionic glucose media.

Science.gov (United States)

Igbokwe, Nanacha Afifi; Igbokwe, Ikechukwu Onyebuchi

2016-03-01

Erythrocyte osmotic lysis in deionised glucose media is regulated by glucose influx, cation efflux, and changes in cell volume after water diffusion. Transmembrane fluxes may be affected by varied expression of glucose transporter protein and susceptibility of membrane proteins to glucose-induced glycosylation and oxidation in various physiologic states. Variations in haemolysis of Sahel goat erythrocytes after incubation in hyposmotic non-ionic glucose media, associated with sex, age, late pregnancy, and lactation, were investigated. The osmotic fragility curve in glucose media was sigmoidal with erythrocytes from goats in late pregnancy (PRE) or lactation (LAC) or from kid (KGT) or middle-aged (MGT) goats. Non-sigmoidal phenotype occurred in yearlings (YGT) and old (OGT) goats. The composite fragility phenotype for males and non-pregnant dry (NPD) females was non-sigmoidal. Erythrocytes with non-sigmoidal curves were more stable than those with sigmoidal curves because of inflectional shift of the curve to the left. Erythrocytes tended to be more fragile with male than female sex, KGT and MGT than YGT and OGT, and LAC and PRE than NPD. Thus, sex, age, pregnancy, and lactation affected the haemolytic pattern of goat erythrocytes in glucose media. The physiologic state of the goat affected the in vitro interaction of glucose with erythrocytes, causing variations in osmotic stability with variants of fragility phenotype. Variations in the effect of high extracellular glucose concentrations on the functions of membrane-associated glucose transporter, aquaporins, and the cation cotransporter were presumed to be relevant in regulating the physical properties of goat erythrocytes under osmotic stress.

Direct measurement of erythrocyte deformability in diabetes mellitus with a transparent microchannel capillary model and high-speed video camera system.

Science.gov (United States)

Tsukada, K; Sekizuka, E; Oshio, C; Minamitani, H

2001-05-01

To measure erythrocyte deformability in vitro, we made transparent microchannels on a crystal substrate as a capillary model. We observed axisymmetrically deformed erythrocytes and defined a deformation index directly from individual flowing erythrocytes. By appropriate choice of channel width and erythrocyte velocity, we could observe erythrocytes deforming to a parachute-like shape similar to that occurring in capillaries. The flowing erythrocytes magnified 200-fold through microscopy were recorded with an image-intensified high-speed video camera system. The sensitivity of deformability measurement was confirmed by comparing the deformation index in healthy controls with erythrocytes whose membranes were hardened by glutaraldehyde. We confirmed that the crystal microchannel system is a valuable tool for erythrocyte deformability measurement. Microangiopathy is a characteristic complication of diabetes mellitus. A decrease in erythrocyte deformability may be part of the cause of this complication. In order to identify the difference in erythrocyte deformability between control and diabetic erythrocytes, we measured erythrocyte deformability using transparent crystal microchannels and a high-speed video camera system. The deformability of diabetic erythrocytes was indeed measurably lower than that of erythrocytes in healthy controls. This result suggests that impaired deformability in diabetic erythrocytes can cause altered viscosity and increase the shear stress on the microvessel wall. Copyright 2001 Academic Press.

The effects of erythrocyte deformability upon hematocrit assessed by the conductance method

International Nuclear Information System (INIS)

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

2009-01-01

A comparative study of centrifugation and conductance methods for the estimation of cell volume fraction (φ) was performed to examine whether the strong forces exerted upon erythrocytes during centrifugation affect their volume, and the results are discussed in terms of erythrocyte deformability. Rabbit erythrocytes of four shapes (spherocytes, echinocytes, stomatocyte-like enlarged erythrocytes and discocytes) were prepared by controlling the pH of the suspending media. The packed cell volumes of the suspensions were measured by standard hematocrit determination methods using centrifugation in capillary tubes. Simultaneously, the same suspensions and their supernatants were used in dielectric spectroscopy measurements, and the low-frequency limits of their conductivities were used for the numerical estimation of φ. The hematocrit values of spherocytes and echinocytes were markedly less than the volume fractions obtained by the conductance method. Namely, the centrifugation reduced the cell volume. For enlarged erythrocytes and discocytes, however, the reduction of cell volume was not observed. These findings showed that φ obtained by the centrifugation method can be greatly affected by the deformability of the cells, but the level of the effect depends on the cell types. Consequently, φ obtained by the centrifugation method should be carefully interpreted.

The effects of erythrocyte deformability upon hematocrit assessed by the conductance method

Energy Technology Data Exchange (ETDEWEB)

Hayashi, Yoshihito; Katsumoto, Yoichi; Oshige, Ikuya; Omori, Shinji; Yasuda, Akio [Life Science Laboratory, Advanced 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

2009-04-21

A comparative study of centrifugation and conductance methods for the estimation of cell volume fraction ({phi}) was performed to examine whether the strong forces exerted upon erythrocytes during centrifugation affect their volume, and the results are discussed in terms of erythrocyte deformability. Rabbit erythrocytes of four shapes (spherocytes, echinocytes, stomatocyte-like enlarged erythrocytes and discocytes) were prepared by controlling the pH of the suspending media. The packed cell volumes of the suspensions were measured by standard hematocrit determination methods using centrifugation in capillary tubes. Simultaneously, the same suspensions and their supernatants were used in dielectric spectroscopy measurements, and the low-frequency limits of their conductivities were used for the numerical estimation of {phi}. The hematocrit values of spherocytes and echinocytes were markedly less than the volume fractions obtained by the conductance method. Namely, the centrifugation reduced the cell volume. For enlarged erythrocytes and discocytes, however, the reduction of cell volume was not observed. These findings showed that {phi} obtained by the centrifugation method can be greatly affected by the deformability of the cells, but the level of the effect depends on the cell types. Consequently, {phi} obtained by the centrifugation method should be carefully interpreted.

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.

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.

Identification and characterization of a novel Plasmodium falciparum adhesin involved in erythrocyte invasion.

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Nidhi Hans

Full Text Available Malaria remains a major health problem worldwide. All clinical symptoms of malaria are attributed to the asexual blood stages of the parasite life cycle. Proteins resident in apical organelles and present on the surface of P. falciparum merozoites are considered promising candidates for the development of blood stage malaria vaccines. In the present study, we have identified and characterized a microneme associated antigen, PfMA [PlasmoDB Gene ID: PF3D7_0316000, PFC0700c]. The gene was selected by applying a set of screening criteria such as transcriptional upregulation at late schizogony, inter-species conservation and the presence of signal sequence or transmembrane domains. The gene sequence of PfMA was found to be conserved amongst various Plasmodium species. We experimentally demonstrated that the transcript for PfMA was expressed only in the late blood stages of parasite consistent with a putative role in erythrocyte invasion. PfMA was localized by immunofluorescence and immuno-electron microscopy to be in the micronemes, an apical organelle of merozoites. The functional role of the PfMA protein in erythrocyte invasion was identified as a parasite adhesin involved in direct attachment with the target erythrocyte. PfMA was demonstrated to bind erythrocytes in a sialic acid independent, chymotrypsin and trypsin resistant manner and its antibodies inhibited P. falciparum erythrocyte invasion. Invasion of erythrocytes is a complex multistep process that involves a number of redundant ligand-receptor interactions many of which still remain unknown and even uncharacterized. Our work has identified and characterized a novel P. falciparum adhesin involved in erythrocyte invasion.

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes[S

Science.gov (United States)

Kobayashi, Naoki; Otsuka, Masato; Yamaguchi, Akihito; Nishi, Tsuyoshi

2016-01-01

Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be useful for the high-throughput screening of S1P transporter inhibitors using conventional fluorometers. PMID:27655910

Sulfation of fulvestrant by human liver cytosols and recombinant SULT1A1 and SULT1E1