Science.gov

Sample records for reticulocytes

  1. Reticulocyte count

    MedlinePlus

    Anemia - reticulocyte ... A higher than normal reticulocytes count may indicate: Anemia due to red blood cells being destroyed earlier than normal ( hemolytic anemia ) Bleeding Blood disorder in a fetus or newborn ( ...

  2. Reticulocyte Count Test

    MedlinePlus

    ... Reticulocyte Count Related tests: Red Blood Cell Count ; Hemoglobin ; Hematocrit ; Complete Blood Count ; Blood Smear ; Erythropoietin ; Vitamin ... on a complete blood count (CBC) , RBC count , hemoglobin or hematocrit , to help determine the cause To ...

  3. Membrane remodeling during reticulocyte maturation

    PubMed Central

    Liu, Jing; Guo, Xinhua; Mohandas, Narla; Chasis, Joel A.

    2010-01-01

    The transition of reticulocytes into erythrocytes is accompanied by extensive changes in the structure and properties of the plasma membrane. These changes include an increase in shear resistance, loss of surface area, and acquisition of a biconcave shape. The processes by which these changes are effected have remained largely undefined. Here we examine how the expression of 30 distinct membrane proteins and their interactions change during murine reticulocyte maturation. We show that tubulin and cytosolic actin are lost, whereas the membrane content of myosin, tropomyosin, intercellular adhesion molecule-4, glucose transporter-4, Na-K-ATPase, sodium/hydrogen exchanger 1, glycophorin A, CD47, Duffy, and Kell is reduced. The degradation of tubulin and actin is, at least in part, through the ubiquitin-proteasome degradation pathway. In regard to the protein-protein interactions, the formation of membrane-associated spectrin tetramers from dimers is unperturbed, whereas the interactions responsible for the formation of the membrane-skeletal junctions are weaker in reticulocytes, as is the attachment of transmembrane proteins to these structures. This weakness, in part, results from the elevated phosphorylation of 4.1R in reticulocytes, which leads to a decrease in shear resistance by reducing its interaction with spectrin and actin. These observations begin to unravel the mechanistic basis of crucial changes accompanying reticulocyte maturation. PMID:20038785

  4. Manual and automated reticulocyte counts.

    PubMed

    Simionatto, Mackelly; de Paula, Josiane Padilha; Chaves, Michele Ana Flores; Bortoloso, Márcia; Cicchetti, Domenic; Leonart, Maria Suely Soares; do Nascimento, Aguinaldo José

    2010-12-01

    Manual reticulocyte counts were examined under light microscopy, using the property whereby supravital stain precipitates residual ribosomal RNA versus the automated flow methods, with the suggestion that in the latter there is greater precision and an ability to determine both mature and immature reticulocyte fractions. Three hundred and forty-one venous blood samples of patients were analyzed of whom 224 newborn and the rest adults; 51 males and 66 females, with ages between 0 and 89 years, as part of the laboratory routine for hematological examinations at the Clinical Laboratory of the Hospital Universitário do Oeste do Paraná. This work aimed to compare manual and automated methodologies for reticulocyte countings and evaluate random and systematic errors. The results obtained showed that the difference between the two methods was very small, with an estimated 0·4% systematic error and 3·9% random error. Thus, it has been confirmed that both methods, when well conducted, can reflect precisely the reticulocyte counts for adequate clinical use.

  5. [Reticulocytes in the diagnosis of anaemia].

    PubMed

    Heiligers-Duckers, Connie; Werner, Philo T; van Drunen, Marlea E P

    2013-01-01

    Reticulocytes are immature erythrocytes; the number of reticulocytes in the peripheral blood reflects erythropoietic activity. Two cases are described to illustrate the use of the reticulocyte count in the diagnostic workup of anaemia. The first patient was a 62-year-old woman presenting with fatigue. Laboratory evaluation showed severe macrocytic anaemia, thrombocytopaenia and the presence of schistocytes. A low reticulocyte count suggested decreased erythropoiesis underlying the anaemia; this led to the diagnosis of vitamin B12 deficiency. The second patient, a 52-year-old woman, also presented with fatigue and macrocytic anaemia. A high reticulocyte count indicated increased erythrocyte degradation, and the patient was eventually diagnosed with autoimmune haemolytic anaemia. The role of reticulocytes in the differential diagnostic workup of anaemia was explored on the basis of these case descriptions. The test methodology, analytical performance, reference values and pitfalls were discussed, as well as the reticulocyte indices and their use in monitoring therapy.

  6. Reticulocytes in sports medicine: an update.

    PubMed

    Lombardi, Giovanni; Colombini, Alessandra; Lanteri, Patrizia; Banfi, Giuseppe

    2013-01-01

    Reticulocytes are young red blood cells which develop from erythroblasts and circulate in the bloodstream for about 1-4 days before maturing into erythrocytes. With the introduction of reticulocyte count in equations and statistical models for detecting suspected blood doping, its application to sports medicine has attracted growing interest in reticulocyte behavior during training and competition seasons in athletes and experimental blood doping treatment in healthy volunteers. An update on recent publications is therefore needed to improve the interpretation of reticulocyte analysis and its variability in sportsmen. Reticulocyte count constitutes a robust parameter during the preanalytical phase, but cell stability can be assured only if blood samples are kept at constantly cold temperatures (4 degrees C) and test results will differ depending on the blood analyzer system used. Marked intraindividual variability is the principal finding to be evaluated when exercise-induced changes are observed or illicit procedures suspected. Furthermore, reticulocyte variability is greater than that of other hematological parameters such as hemoglobin or hematocrit. Ideally, any variation should be interpreted against long-term time series for the individual athlete: values obtained from large athlete cohorts ought to be used only for extrapolating outliers that deserve further examination. Reticulocyte distribution in athletes is similar to that found in the general population, and a gender effect in some sports disciplines or selected athlete groups may be seen. Reticulocyte variability is strongly influenced by seasonal factors linked to training and competition schedules and by the type of sports discipline. Published experimental data have confirmed the high sensitivity of reticulocyte analysis in identifying abnormal bone marrow stimulation by either erythropoietin administration or blood withdrawal and reinfusion.

  7. Automated reticulocyte parameters for hereditary spherocytosis screening.

    PubMed

    Lazarova, Elena; Pradier, Olivier; Cotton, Frédéric; Gulbis, Béatrice

    2014-11-01

    The laboratory diagnosis of hereditary spherocytosis (HS) is based on several screening and confirmatory tests; our algorithm includes clinical features, red blood cell morphology analysis and cryohaemolysis test, and, in case of positive screening, sodium dodecyl sulphate polyacrylamide gel electrophoresis as a diagnostic test. Using the UniCel DxH800 (Beckman Coulter) haematology analyser, we investigated automated reticulocyte parameters as HS screening tool, i.e. mean reticulocyte volume (MRV), immature reticulocyte fraction (IRF) and mean sphered cell volume (MSCV). A total of 410 samples were screened. Gel electrophoresis was applied to 159 samples that were positive for the screening tests. A total of 48 patients were diagnosed as HS, and seven were diagnosed as acquired autoimmune haemolytic anaemia (AIHA). Some other 31 anaemic conditions were also studied. From the receiver operating characteristic (ROC) curve analysis, both delta (mean cell volume (MCV)-MSCV) and MRV presented an area under the curve (AUC) of 0.98. At the diagnostic cut-off of 100 % sensitivity, MRV showed the best specificity of 88 % and a positive likelihood ratio of 8.7. The parameters IRF, MRV and MSCV discriminated HS not only from controls and other tested pathologies but also from AIHA contrary to the cryohaemolysis test. In conclusion, automated reticulocyte parameters might be helpful for haemolytic anaemia diagnostic orientation even for general laboratories. In combination with cryohaemolysis, they ensure an effective and time-saving screening for HS for more specialised laboratories.

  8. Pig Reticulocytes. III. Glucose permeability in naturally occurring reticulocytes and red cells from newborn piglets

    PubMed Central

    Kim, HD; Luthra, MG

    1977-01-01

    The loss of facilitated glucose transport of red cells occurring in the newborn pig was monitored in 11 density-separated cells from birth to a 4 wk of age. At birth there was a threefold increase in glucose permeability from the lightest cells to the most dense, suggesting that cells having progressively less glucose permeability are released into the circulation as gestation proceeds. Because of extraordinary stimulation of erythropoietic activity, the uppermost top fraction constituting 2-3 percent of the total cells is composed purely of reticulocytes in the growing animal. The glucose permeability of these reticulocytes which at birth has a slow but significant rate of 3.7 μmol/ml cell x min at 25 degrees C is rapidly decreased within 3-4 days to the level of reticulocytes produced in the adult in response to phenylhydrazine assault. Moreover, reticulocytes themselves discard their membrane permeability to glucose in the course of maturation to red cells. Thus, even though reticulocytes at birth are permeable to glucose, they will become red cells practically impervious to glucose within a few days. These findings suggest that the transition from a glucose- permeable fetal state to a glucose-impermeable postnatal state is brought about by two mechanisms: (a) dilution of fetal cells by glucose-impervious cells produced coincidentally with or shortly after birth; and (b) elimination of fetal cells, which have a shorter half-life, from the circulation. PMID:894256

  9. Proteolipidic composition of exosomes changes during reticulocyte maturation.

    PubMed

    Carayon, Kévin; Chaoui, Karima; Ronzier, Elsa; Lazar, Ikrame; Bertrand-Michel, Justine; Roques, Véronique; Balor, Stéphanie; Terce, François; Lopez, André; Salomé, Laurence; Joly, Etienne

    2011-09-30

    During the orchestrated process leading to mature erythrocytes, reticulocytes must synthesize large amounts of hemoglobin, while eliminating numerous cellular components. Exosomes are small secreted vesicles that play an important role in this process of specific elimination. To understand the mechanisms of proteolipidic sorting leading to their biogenesis, we have explored changes in the composition of exosomes released by reticulocytes during their differentiation, in parallel to their physical properties. By combining proteomic and lipidomic approaches, we found dramatic alterations in the composition of the exosomes retrieved over the course of a 7-day in vitro differentiation protocol. Our data support a previously proposed model, whereby in reticulocytes the biogenesis of exosomes involves several distinct mechanisms for the preferential recruitment of particular proteins and lipids and suggest that the respective prominence of those pathways changes over the course of the differentiation process.

  10. Flow cytometric analysis of leukocytes and reticulocytes stained with proflavine.

    PubMed

    Sagawa, H; Tatsumi, N

    1997-12-01

    Proflavine, an acridine analog for industrial use, was used to stain blood cells. A drop of blood treated with ethylenediaminetetraacetic acid-2K was mixed with a 0.00001% solution of the dye and observed immediately by fluorescence microscopy with a green filter. Leukocytes, platelets, and reticulocytes were stained but mature red blood cells were not. Chromatin in the nuclei of all leukocytes and nucleoli of lymphocytes and monocytes had greenish-yellow fluorescence, and the kind of cell could be identified by the tone and intensity of this color. Granules in granulocytes were in green. Reticular fine-granular or granulofibrous structures in the reticulocytes were brownish. The proflavine could be used routinely in clinical laboratories because this single stain makes possible simultaneous differentiation of leukocytes and counting of reticulocytes.

  11. Reticulocyte quantification by flow cytometry, image analysis, and manual counting.

    PubMed

    Schimenti, K J; Lacerna, K; Wamble, A; Maston, L; Iaffaldano, C; Straight, M; Rabinovitch, A; Lazarus, H M; Jacobberger, J W

    1992-01-01

    Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty-nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty-nine samples from bone marrow transplant patients were compared during the period before and through chemo-irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methylene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay as measured. The rank order of precision from high to low was flow cytometry > image analysis > manual counting.

  12. Sensitivity of reticulocyte indices to iron therapy in an intensely training athlete.

    PubMed

    Ashenden, M J; Dobson, G P; Hahn, A G

    1998-09-01

    Iron deficiency anaemia, and its debilitating effect on performance, is an area of concern for many female athletes. Automated technologies that analyse individual reticulocytes may provide a sensitive measure of bone marrow response to iron supplementation. The reticulocyte characteristics of a female volleyball player with frank iron deficiency anaemia, and her subsequent response to oral iron therapy, are reported.

  13. Plasmodium vivax: restricted tropism and rapid remodeling of CD71-positive reticulocytes.

    PubMed

    Malleret, Benoit; Li, Ang; Zhang, Rou; Tan, Kevin S W; Suwanarusk, Rossarin; Claser, Carla; Cho, Jee Sun; Koh, Esther Geok Liang; Chu, Cindy S; Pukrittayakamee, Sasithon; Ng, Mah Lee; Ginhoux, Florent; Ng, Lai Guan; Lim, Chwee Teck; Nosten, François; Snounou, Georges; Rénia, Laurent; Russell, Bruce

    2015-02-19

    Plasmodium vivax merozoites only invade reticulocytes, a minor though heterogeneous population of red blood cell precursors that can be graded by levels of transferrin receptor (CD71) expression. The development of a protocol that allows sorting reticulocytes into defined developmental stages and a robust ex vivo P vivax invasion assay has made it possible for the first time to investigate the fine-scale invasion preference of P vivax merozoites. Surprisingly, it was the immature reticulocytes (CD71(+)) that are generally restricted to the bone marrow that were preferentially invaded, whereas older reticulocytes (CD71(-)), principally found in the peripheral blood, were rarely invaded. Invasion assays based on the CD71(+) reticulocyte fraction revealed substantial postinvasion modification. Thus, 3 to 6 hours after invasion, the initially biomechanically rigid CD71(+) reticulocytes convert into a highly deformable CD71(-) infected red blood cell devoid of host reticular matter, a process that normally spans 24 hours for uninfected reticulocytes. Concurrent with these changes, clathrin pits disappear by 3 hours postinvasion, replaced by distinctive caveolae nanostructures. These 2 hitherto unsuspected features of P vivax invasion, a narrow preference for immature reticulocytes and a rapid remodeling of the host cell, provide important insights pertinent to the pathobiology of the P vivax infection.

  14. Enrichment of reticulocytes from whole blood using aqueous multiphase systems of polymers.

    PubMed

    Kumar, Ashok A; Lim, Caeul; Moreno, Yovany; Mace, Charles R; Syed, Abeer; Van Tyne, Daria; Wirth, Dyann F; Duraisingh, Manoj T; Whitesides, George M

    2015-01-01

    This paper demonstrates the enrichment of reticulocytes by centrifuging whole blood through aqueous multiphase systems (AMPSs)-immiscible phases of solutions of polymers that form step-gradients in density. The interfaces of an AMPS concentrate cells; this concentration facilitates the extraction of blood enriched for reticulocytes. AMPS enrich reticulocytes from blood from both healthy and hemochromatosis donors. Varying the osmolality and density of the phases of AMPS provides different levels of enrichment and yield of reticulocytes. A maximum enrichment of reticulocytemia of 64 ± 3% was obtained from donors with hemochromatosis. When used on peripheral blood from normal donors, AMPS can provide a higher yield of enriched reticulocytes and a higher proportion of reticulocytes expressing CD71 than differential centrifugation followed by centrifugation over Percoll. Blood enriched for reticulocytes by AMPS could be useful for research on malaria. Several species of malaria parasites show a preference to invade young erythrocytes and reticulocytes; this preference complicates in vitro cultivation of these species in human blood. Plasmodium knowlesi malaria parasites invade normal human blood enriched for reticulocytes by AMPSs at a rate 2.2 times greater (P < 0.01) than they invade unenriched blood. Parasite invasion in normal blood enriched by AMPS was 1.8 times greater (P < 0.05) than in blood enriched to a similar reticulocytemia by differential centrifugation followed by centrifugation over Percoll. The enrichment of reticulocytes that are invaded by malaria parasites demonstrates that AMPSs can provide a label-free method to enrich cells for biological research.

  15. Clinical utility of Beckman-Coulter Gen's reticulocyte analysis in the study of anemia of chronic disease (ACD).

    PubMed

    Rodrigues, Ana; Ortega, Carlos; Santos, Luís; Teixeira, Alexandre; Dinis, Maria Joáo; Vasconcelos, Iponina; Lacerda, Jorge; Fonseca, Elisa

    2007-01-01

    The aim of this study is to analyze the reticulocyte parameters (classical and research parameters) acquired by the Beckman Coulter GEN'S blood counter (GEN'S; Brea, CA, USA) to establish the "reticulocyte profile" characteristics of patients with anemia of chronic disease (ACD). The reticulocyte parameters and profile provided by the GEN'S were studied in 38 anemic patients who fulfilled the diagnostic criteria for the ACD, and the results were compared with those of 38 healthy controls in a multivariate statistical analysis using the Student t-test and the receiver operating characteristics (ROC) curve analysis.Statistically significant (P <.05) differences between the 2 population groups were observed in several of the reticulocyte parameters provided by the GEN'S: mean volume of the reticulocyte population (MRV) (high), percentage of high light scatter reticulocytes HLR% (high), mean volume of the whole sphered red cell and reticulocyte population (MSCV) (high) MCV-MSCV (low) with the highest diagnostic value, as measured by the area under the ROC curve (>0.9), for the IRF (high) and the reticulocyte population data: mean channel scatter retics (high) and the mean channel conductivity retics (high). This study establishes the reticulocyte parameters and reticulocyte profile as provided by the GEN'S and characteristic of patients with ACD as compared with normal subjects. This should provide the basis for further studies comparing the reticulocyte profile of patients with ACD with those found in other types of anemia.

  16. The RNA in reticulocytes is not just debris: it is necessary for the final stages of erythrocyte formation.

    PubMed

    Lee, EunMi; Choi, Hye Sook; Hwang, Jung Hye; Hoh, Jeong Kyu; Cho, Youl-Hee; Baek, Eun Jung

    2014-01-01

    Reticulocytes contain both RNA and micro-organelles and represent the last stage of erythropoiesis before full maturation to red blood cells (RBCs). Even though there is continuing synthesis of hemoglobin and membrane-bound proteins in reticulocytes, the small amount of RNA that they contain has been regarded as non-functional residual material. Here we show that this residual RNA is both functional and essential for further reticulocyte maturation. Reticulocytes from which the remnant RNA had been removed by exposure to RNase did not survive or mature into RBCs in either humans or mice. Conversely, reticulocytes treated with an RNase Inhibitor were able to form normal biconcave cells. Similarly, poor survival was also seen in reticulocytes in which protein synthesis had been blocked. To identify the signaling pathways involved we isolated RNAs in reticulocytes versus those present in fully matured erythroblasts cultured from hematopoietic stem cells. RNAs found in erythroblasts were related to exocytosis, metabolism, and signal transduction all of which are critical for maturation through reticulocyte and into a fully mature, biconcave erythrocyte. Our results suggest that the mRNA in reticulocytes has to be translated into novel proteins that act to preserve mitochondria and maintain cell membrane integrity as reticulocytes mature. These results enhance our understanding of the final stage of erythropoiesis and may clarify why in vitro-generated reticulocytes for transfusion purposes survive poorly.

  17. Sensitivity of reticulocyte indices to iron therapy in an intensely training athlete

    PubMed Central

    Ashenden, M. J.; Dobson, G. P.; Hahn, A. G.

    1998-01-01

    Iron deficiency anaemia, and its debilitating effect on performance, is an area of concern for many female athletes. Automated technologies that analyse individual reticulocytes may provide a sensitive measure of bone marrow response to iron supplementation. The reticulocyte characteristics of a female volleyball player with frank iron deficiency anaemia, and her subsequent response to oral iron therapy, are reported. 




 PMID:9773180

  18. [Automated measurement of reticulocyte count by flow cytometry. II: Analysis of the blood containing abnormal erythrocytes or giant platelets].

    PubMed

    Oyamatsu, T; Shimizu, N; Takeuchi, K; Yamamoto, M; Kawai, Y; Watanabe, K; Iri, H

    1989-07-01

    We have examined the influence of erythrocytes containing inclusion bodies, nucleated red cells or giant platelets on the measurement of reticulocyte count by automated machine, R-1000. Correlation of the reticulocyte count between automated and conventional method was extremely good in the blood containing red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling . However, correlation was poor when the sample contained the nucleated red cells. Reticulocyte count was decreased in the blood with significant amounts of nucleated red cells. Since nucleated red cells themselves are not counted as reticulocytes in the machine, this was considered to be due to increased young reticulocytes which frequently appeared with nucleated red cells. Both cold agglutinated red cells and giant platelets apparently influenced the reticulocyte count by the R-1000. These results suggest that red cells with Jolly bodies, Pappenheimer bodies or basophilic stippling do not influence the automatic counting of reticulocytes. Although nucleated red cells, cold agglutinated red cells and giant platelets affected the reticulocyte count, the machine shows abnormal flags in most of above cases (except highly agglutinated red cells), so that one can recount reticulocytes by conventional method. We conclude the machine can safely count the reticulocytes even in the blood containing abnormal red cells or platelets.

  19. VLA-4 blockade by natalizumab inhibits sickle reticulocyte and leucocyte adhesion during simulated blood flow.

    PubMed

    White, Jennell; Krishnamoorthy, Sriram; Gupta, Dipti; Lancelot, Moira; Moore, Nancy; Sarnaik, Sharada; Hobbs, William E; Light, David R; Hines, Patrick

    2016-09-01

    Very Late Antigen-4 (VLA-4, α4β1-integrin, ITGA4) orchestrates cell-cell and cell-endothelium adhesion. Given the proposed role of VLA-4 in sickle cell disease (SCD) pathophysiology, we evaluated the ability of the VLA-4 blocking antibody natalizumab to inhibit SCD blood cell adhesion. Natalizumab recognized surface VLA-4 on leucocytes and reticulocytes in whole blood from SCD subjects. SCD reticulocytes were positive for VLA-4, while VLA-4 staining of non-SCD reticulocytes was undetectable. Titrations with natalizumab revealed the presence of saturable levels of VLA-4 on both SCD reticulocytes and leucocytes similar to healthy subject leucocytes. Under physiological flow conditions, the adhesion of SCD whole blood cells and isolated SCD leucocytes to immobilized vascular cell adhesion molecule 1 (VCAM-1) was blocked by natalizumab in a dose-dependent manner, which correlated with cell surface receptor binding. Natalizumab also inhibited >50% of whole blood cell binding to TNF-α activated human umbilical vein endothelial cell monolayers under physiological flow at clinically relevant concentrations (10 to 100 μg/ml). This indicates that VLA-4 is the dominant receptor that drives SCD reticulocyte and mononuclear cell adhesion to VCAM-1 and that the VLA-4 adhesion to VCAM-1 is a significant contributor to SCD blood cell adhesion to endothelium. Thus, VLA-4 blockade may be beneficial in sickle cell disease.

  20. Quantification of pathways of glucose utilization and balance of energy metabolism of rabbit reticulocytes.

    PubMed

    Siems, W; Müller, M; Dumdey, R; Holzhütter, H G; Rathmann, J; Rapoport, S M

    1982-06-01

    In this work it is demonstrated that glucose constitutes the main substrate of energy metabolism of rabbit reticulocytes under aerobic conditions in the presence of 5 mM glucose. Amino acids and fatty acids are minor sources of energy. The shares of processes utilizing glucose in reticulocytes were estimated from tracer experiments. A new mathematical technique used permits the derivation of closed terms for the specific radioactivity of single positions of C atoms of the metabolites of the citrate cycle. By means of regression analysis, the undetermined flux rates in the citrate cycle were calculated. On the basis of the data an overall balance sheet of glucose utilization and of ATP generation is given. About 45% of the glucose of reticulocytes is catabolized via the citrate cycle, about the same percentage yields lactate. Only 2% of the glucose was oxidized in the oxidative pentose pathway whereas the remainder is used for the formation of serine and glycine required for hemoglobin synthesis. These results are related to knowledge about the main processes utilizing ATP in reticulocytes, i.e. the synthesis of hemoglobin and the energy-dependent proteolysis. Our approach to the investigation of metabolic relations in the reticulocytes can be applied to other tissues in which equilibria between large metabolite pools play a role.

  1. Hexokinase microheterogeneity in rabbit red blood cells and its behaviour during reticulocytes maturation.

    PubMed

    Stocchi, V; Magnani, M; Piccoli, G; Fornaini, G

    1988-02-01

    Hexokinase in rabbit reticulocytes is present in two molecular forms (hexokinase Ia and Ib) separable by ion-exchange chromatography on DE-52 columns. By the use of ion-exchange HPLC we have been able to show that the isozymic form we previously called hexokinase Ia can be resolved into two peaks of activity one of which is (Ia) soluble, the other (Ia*) particulate. Hexokinase Ia* can be solubilized by detergents like saponine and Triton X-100 and disappears during 'in vivo' reticulocytes maturation. This new hexokinase microheterogeneity is not caused by different oxidized forms of the enzyme nor influenced by the presence of proteolytic inhibitors during lysate preparation.

  2. Effect of Lead Exposure on the Status of Reticulocyte Count Indices among Workers from Lead Battery Manufacturing Plant

    PubMed Central

    Kalahasthi, Ravibabu; Barman, Tapu

    2016-01-01

    Earlier studies conducted on lead-exposed workers have determined the reticulocyte count (RC) (%), but the parameters of Absolute Reticulocyte Count (ARC), Reticulocyte Index (RI), and Reticulocyte Production Index (RPI) were not reported. This study assessed the effect of lead (Pb) exposure on the status of reticulocyte count indices in workers occupied in lead battery plants. The present cross-sectional study was carried out on 391 male lead battery workers. The blood lead levels (BLL) were determined by using an Atomic Absorption Spectrophotometer. The RC (%) was estimated by using the supravital staining method. The parameters, such as ARC, RI, and RPI, were calculated by using the RC (%) with the red cell indices (RBC count and hematocrit). The levels of RBC count and hematocrit were determined by using an ABX Micros ES-60 hematology analyzer. The levels of reticulocyte count indices - RC (%), ARC, RI, and RPI significantly increased with elevated BLL. The association between BLL and reticulocyte count indices was positive and significant. The results of linear multiple regression analysis showed that the reticulocyte count (β = 0.212, P < 0.001), ARC (β = 0.217, P < 0.001), RI (β = 0.194, P < 0.001), and RPI (β = 0.208, P < 0.001) were positively associated with BLL. The variable, smoking habits, showed a significant positive association with reticulocyte count indices: RC (%) (β = 0.188, P < 0.001), ARC (β = 0.174, P < 0.001), RI (β = 0.200, P < 0.001), and RPI (β = 0.151, P < 0.005). The study results revealed that lead exposure may cause reticulocytosis with an increase of reticulocyte count indices. PMID:27818730

  3. Effect of Lead Exposure on the Status of Reticulocyte Count Indices among Workers from Lead Battery Manufacturing Plant.

    PubMed

    Kalahasthi, Ravibabu; Barman, Tapu

    2016-10-01

    Earlier studies conducted on lead-exposed workers have determined the reticulocyte count (RC) (%), but the parameters of Absolute Reticulocyte Count (ARC), Reticulocyte Index (RI), and Reticulocyte Production Index (RPI) were not reported. This study assessed the effect of lead (Pb) exposure on the status of reticulocyte count indices in workers occupied in lead battery plants. The present cross-sectional study was carried out on 391 male lead battery workers. The blood lead levels (BLL) were determined by using an Atomic Absorption Spectrophotometer. The RC (%) was estimated by using the supravital staining method. The parameters, such as ARC, RI, and RPI, were calculated by using the RC (%) with the red cell indices (RBC count and hematocrit). The levels of RBC count and hematocrit were determined by using an ABX Micros ES-60 hematology analyzer. The levels of reticulocyte count indices - RC (%), ARC, RI, and RPI significantly increased with elevated BLL. The association between BLL and reticulocyte count indices was positive and significant. The results of linear multiple regression analysis showed that the reticulocyte count (β = 0.212, P < 0.001), ARC (β = 0.217, P < 0.001), RI (β = 0.194, P < 0.001), and RPI (β = 0.208, P < 0.001) were positively associated with BLL. The variable, smoking habits, showed a significant positive association with reticulocyte count indices: RC (%) (β = 0.188, P < 0.001), ARC (β = 0.174, P < 0.001), RI (β = 0.200, P < 0.001), and RPI (β = 0.151, P < 0.005). The study results revealed that lead exposure may cause reticulocytosis with an increase of reticulocyte count indices.

  4. Erythrocyte antigen and reticulocyte engraftment after allogeneic hematopoietic stem cell transplantation.

    PubMed

    Yildirim, I; Ozer, Y; Yüksel, M K; Arat, M; Arslan, O

    2004-08-01

    The aim of this study was to study the usefulness of erythrocyte antigen (EA) measurement to study engraftment after allogeneic HSCT. In all, 31 consecutive patients receiving HLA-identical bone marrow (BM) (n=13) or peripheral blood stem cells (n=18) were investigated. Apart from the ABO group, 15 EAs representing six minor blood groups were followed by the simple tube agglutination technique. A total of 20 (64.5%) patients received ABO-identical, eight (25.8%) received ABO minor and three (9.7%) received ABO major mismatched grafts. In all, 29 patients were followed for a median of 12 (6-16) months; 65% of the patients expressed donor type EA 1 month and almost all did so 6 months after transplant. Reticulocyte engraftment was significantly shorter than EA engraftment (median 18 vs 35 days) (P=0.001). Patients who received PB stem cells showed significantly faster EA and reticulocyte engraftment than patients who received BM stem cells (P=0.038 and 0.025). ABO compatibility did not have an impact on reticulocyte and EA engraftment (P=0.4 and 0.55). The earliest donor type EA detected was from the Rh and Kidd system. These data suggest that EA and reticulocyte assays are useful in monitoring engraftment.

  5. ATP-dependent degradation of /sup 125/I-bovine serum albumin by rabbit reticulocytes does not need repression of an endogenous inhibitor

    SciTech Connect

    Saus, J.; Timoneda, J.

    1987-01-01

    The ubiquitin-dependent proteolysis of /sup 125/I-bovine serum albumin in rabbit reticulocytes has been investigated. Using various reticulocyte fractions (reticulocyte protease, inhibitor-free protease, ubiquitin and inhibitor) in the presence or absence of ATP, we found that the repression of an endogenous inhibitor, as suggested by others for alpha-casein proteolysis, is unlikely for bovine serum albumin. Therefore, differences exist in the ATP-dependent proteolytic pathway of rabbit reticulocytes depending on the substrate. Fractionation of the reticulocyte ATP-dependent proteolytic system revealed at least two proteolytic and two inhibitory fractions involved in the proteolysis of bovine serum albumin.

  6. Giemsa-stained wet mount based method for reticulocyte quantification: a viable alternative in resource limited or malaria endemic settings.

    PubMed

    Lee, Wenn-Chyau; Russell, Bruce; Lau, Yee-Ling; Fong, Mun-Yik; Chu, Cindy; Sriprawat, Kanlaya; Suwanarusk, Rossarin; Nosten, Francois; Renia, Laurent

    2013-01-01

    The quantity of circulating reticulocytes is an important indicator of erythropoietic activity in response to a wide range of haematological pathologies. While most modern laboratories use flow cytometry to quantify reticulocytes, most field laboratories still rely on 'subvital' staining. The specialist 'subvital' stains, New Methylene Blue (NMB) and Brilliant Crésyl Blue are often difficult to procure, toxic, and show inconsistencies between batches. Here we demonstrate the utility of Giemsa's stain (commonly used microbiology and parasitology) in a 'subvital' manner to provide an accurate method to visualize and count reticulocytes in blood samples from normal and malaria-infected individuals.

  7. Clinical utility of the reticulocyte hemoglobin content in the diagnosis of iron deficiency.

    PubMed

    Mast, Alan E; Blinder, Morey A; Lu, Qing; Flax, Sherri; Dietzen, Dennis J

    2002-02-15

    Determination of the reticulocyte hemoglobin content (CHr) provides an early measure of functional iron deficiency because reticulocytes are the earliest erythrocytes released into blood and circulate for only 1 to 2 days. The CHr in 78 patients undergoing bone marrow examination was measured to assess its clinical utility for the diagnosis of iron deficiency. Twenty-eight patients were iron deficient, based on the lack of stainable iron in the aspirate. The diagnostic power of CHr is limited in patients with high mean cellular volume (MCV) or red cell disorders such as thalassemia. However, when patients with MCV more than 100 fL are excluded, receiver operator curve analysis of CHr, ferritin, transferrin saturation, and MCV demonstrates that CHr has the highest overall sensitivity and specificity of these peripheral blood tests for predicting the absence of bone marrow iron stores.

  8. Automated analysis of mature red blood cells and reticulocytes in SS and SC disease.

    PubMed

    Maier-Redelsperger, Micheline; Flahault, Antoine; Neonato, Maria Grazia; Girot, Robert; Labie, Dominique

    2004-01-01

    Phenotypic expression of sickle cell disease (SCD) is highly variable. We investigated red blood cells (RBCs) and reticulocytes using a laser light scattering method (ADVIA120, Bayer Diagnostics, Tarrytown, NY) in a series of patients with either sickle cell anemia (SS) or compound SC heterozygosity (SC), both groups with or without alpha thalassemia. Results were compared with those of a series of patients without hematological disease. Known data were consistently confirmed, namely heterogeneity in cell volume and hemoglobin (Hb) concentration, as well as the premature exit of "stress" reticulocytes from the bone marrow, mostly in SS patients. Specific changes were observed during maturation, including decreases in macrocytic and hypodense cells. Simultaneous viewing of the indices of the different RBC populations provided information on erythropoietic maturation by a rapid, reproducible, and cost-effective method.

  9. Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins

    PubMed Central

    Hietanen, Jenni; Chim-ong, Anongruk; Chiramanewong, Thanprakorn; Gruszczyk, Jakub; Roobsoong, Wanlapa; Sattabongkot, Jetsumon

    2015-01-01

    Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion. PMID:26712206

  10. [Analysis of reticulocytes: manual microscopy, flow analyzers or image analyzers? (analytical review)].

    PubMed

    Piatnitskiĭ, A M; Medovyĭ, V S; Parpara, A A

    2007-10-01

    Three procedures for determining the quantity and composition of reticulocytes (visual analysis during manual microscopy; flow cytometry, semi-automatic analysis using a computerized microscopy unit) are compared. The errors characteristics for each procedure and the reasons why manual analysis still remains most used are discussed. The paper points out that it is impossible to draw a conclusion on the accuracy of the procedures, by using the values of the correlation coefficient. Evidence is provided for the thesis that the statistical accuracy may be achieved only when the image analyzer of a computerized microscopy unit is employed, therefore this procedure is recommended for use as a reference one. The characteristics of a procedure for analyzing reticulocytes by means of a MEKOS-Ts2 computerized microscopy unit are presented.

  11. Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: results of a multicentre study.

    PubMed

    Mullier, François; Lainey, Elodie; Fenneteau, Odile; Da Costa, Lydie; Schillinger, Françoise; Bailly, Nicolas; Cornet, Yvan; Chatelain, Christian; Dogne, Jean-Michel; Chatelain, Bernard

    2011-07-01

    Hereditary spherocytosis (HS) is characterised by weakened vertical linkages between the membrane skeleton and the red blood cell's lipid bilayer, leading to the release of microparticles. All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters like percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, and percentage of immature reticulocytes. The levels of haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, reticulocytes (Ret), immature reticulocytes fraction (IRF), hypochromic erythrocytes (Hypo-He) and microcytic erythrocytes (MicroR) were determined on EDTA samples on Sysmex instruments from a cohort of 45 confirmed SH. The HS group was then compared with haemolytical disorders, microcytic anaemia, healthy individuals and routine samples (n = 1,488). HS is characterised by a high Ret count without an equally elevated IRF. All 45 HS have Ret >80,000/μl and Ret(10(9)/L)/IRF (%) greater than 7.7 (rule 1). Trait and mild HS had a Ret/IRF ratio greater than 19. Moderate and severe HS had increased MicroR and MicroR/Hypo-He (rule 2). Combination of both rules gave predictive positive value and negative predictive value of respectively 75% and 100% (n=1,488), which is much greater than single parameters or existing rules. This simple and fast diagnostic method could be used as an excellent screening tool for HS. It is also valid for mild HS, neonates and ABO incompatibilities and overcomes the lack of sensitivity of electrophoresis in ankyrin deficiencies.

  12. Autophagic vesicles on mature human reticulocytes explain phosphatidylserine-positive red cells in sickle cell disease.

    PubMed

    Mankelow, Tosti J; Griffiths, Rebecca E; Trompeter, Sara; Flatt, Joanna F; Cogan, Nicola M; Massey, Edwin J; Anstee, David J

    2015-10-08

    During maturation to an erythrocyte, a reticulocyte must eliminate any residual organelles and reduce its surface area and volume. Here we show this involves a novel process whereby large, intact, inside-out phosphatidylserine (PS)-exposed autophagic vesicles are extruded. Cell surface PS is a well-characterized apoptotic signal initiating phagocytosis. In peripheral blood from patients after splenectomy or in patients with sickle cell disease (SCD), the number of circulating red cells exposing PS on their surface is elevated. We show that in these patients PS is present on the cell surface of red cells in large (∼1.4 µm) discrete areas corresponding to autophagic vesicles. The autophagic vesicles found on reticulocytes are identical to those observed on red cells from splenectomized individuals and patients with SCD. Our data suggest the increased thrombotic risk associated with splenectomy, and patients with hemoglobinopathies is a possible consequence of increased levels of circulating mature reticulocytes expressing inside-out PS-exposed autophagic vesicles because of asplenia.

  13. Reticulocyte hemoglobin content in the evaluation of iron status of hemodialysis patients.

    PubMed

    Fishbane, S; Galgano, C; Langley, R C; Canfield, W; Maesaka, J K

    1997-07-01

    The assessment of iron status for hemodialysis patients has been hindered by the inaccuracy of commonly used diagnostic tests. A novel assay, the reticulocyte hemoglobin content (CHr), has recently been found to sensitively detect functional iron deficiency among nonuremic patients treated with recombinant erythropoietin (rHuEPO). The purpose of this study was to evaluate the CHr for the assessment of iron status in hemodialysis patients. One hundred sixty-four stable hemodialysis patients had a mean CHr of 27.5 +/- 2.8 pg with a normal distribution of values. The mean CH (mature red cell hemoglobin content) was 26.4 +/- 2.4 pg. There was a close correlation between CHr and CH (r = 0.86, P < 0.0001). A significant subgroup of patients (12.2%) had CHr values < CH. These patients had recent increases in rHuEPO dose, and a lower mean transferrin saturation and hematocrit, suggesting the recent onset of functional iron deficiency due to the increase in rHuEPO dose. In the second phase of the study, 32 patients were randomly selected to receive treatment with a single dose infusion of 1,000 mg of intravenous iron dextran (IVFe). Patients were classified as iron deficient (N = 7) if they responded with a significant reticulocytosis (sustained 1 basis point increase in corrected reticulocyte index within 2 weeks). All other patients were classified as iron replete (N = 25). A CHr < 26 pg at baseline predicted iron deficiency with a sensitivity of 100%, specificity of 80%. The serum ferritin, transferrin saturation and percentage of hypochromic red blood cells all were less accurate. The time to correction of iron deficiency at the level of the reticulocyte was found to be within 48 hours as measured by correction of the mean CHr to > 26 pg, and by the shift of the vast majority of the reticulocyte population to CHr > 26 pg within this time span. We conclude that CHr < 26 pg is an accurate measure of iron status in hemodialysis patients, that a CHr value < CH indicates the

  14. Mean reticulocyte volume: a specific parameter to screen for hereditary spherocytosis.

    PubMed

    Xu, Yuchan; Yang, Wang; Liao, Lin; Deng, Zengfu; Qiu, Yuling; Chen, Wenqiang; Lin, Faquan

    2016-02-01

    This study assessed the value of mean reticulocyte volume (MRV) for differential diagnosis of hereditary spherocytosis (HS) so as to develop conventional and new specific screen indexes. Subjects in this study were divided into three groups: 53 cases in HS group, 217 cases in hemolytic anemia control group (109 cases of thalassemia (THAL), 56 cases of glucose-6-phosphate dehydrogenase G6PD deficiency anemia, and 52 cases of autoimmune hemolytic anemia (AIHA)), and 100 cases in healthy control group. We analyzed erythrocyte and reticulocyte parameters including MRV, mean sphered corpuscular volume, mean corpuscular hemoglobin concentration, and immature reticulocyte fraction. Results demonstrated that MRV was significantly lower in the HS group but significantly higher in the AIHA and G6PD deficiency anemia groups than that in the healthy control group (P = 0.000). MRV was not significantly different between the AIHA and G6PD deficiency anemia groups (P = 0.977) and between the healthy control and THAL groups (P = 0.168). The area under the ROC curve of MRV for diagnosis of HS was 0.942, with a standard error of 0.019, 95% confidence interval of 0.905-0.979, and optimal critical diagnosis point of 95.77 fL. When the MRV was ≤95.77 fL, the sensitivity and specificity for diagnosis of HS were 86.80% and 91.20%, respectively. Therefore, MRV is a general and specific new index for screening HS and important for differential diagnosis of different types of hemolytic anemia.

  15. Gamma Radiation Induces Micronucleated Reticulocytes in 3-D Bone Marrow Bioreactors in Vitro

    PubMed Central

    Sun, Hongliang; Dertinger, Stephen D.; Hyrien, Ollivier; David Wu, J. H.; Chen, Yuhchyau

    2009-01-01

    Radiation injury to the bone marrow is potentially lethal due to the potent DNA-damaging effects on cells of the hematopoietic system, including bone marrow stem cell, progenitor, and the precursor cell populations. Investigation of radiation genotoxic effects on bone marrow progenitor/precursor cells has been challenged by the lack of optimal in vitro surrogate organ culture systems, and the overall difficulty to sustain lineage-specific proliferation and differentiation of hematopoiesis in vitro. We report the investigation of radiation genotoxic effects in bone marrow cultures of C57Bl/6 mice established in 3-D bioreactors, which sustain long-term bone marrow cultures. For these studies, genotoxicity is measured by the induction of micronucleated reticulocytes (MN-RET). The kinetics and dose-response relationship of MN-RET induction in response to gamma-radiation of bioreactor-maintained bone marrow cultures are presented. Our data showed that 3-D long-term bone marrow cultures had sustained erythropoiesis capable of generating reticulocytes up to 8 weeks. The peak time-interval of viable cell output and percentage of reticulocytes increased steadily and reached the initial peak between the 14th to 21st days after inoculations. This was followed by a rebound or staying relatively constant until week 8. The percentage of MN-RET reached the maximum between 24 and 32 hours post 1 Gy gamma-ray. There was a near linear MN-RET induction by gamma radiation from 0 Gy to 1.0 Gy, followed by an attenuated increase to 1.5 – 2.0 Gy. The MN-RET response showed a downtrend beyond 2 Gy. Our data suggest that bone marrow culture in the 3-D bioreactor may be a useful organ culture system for the investigation of radiation genotoxic effect in vitro. PMID:19786117

  16. Electron microscopic evidence for externalization of the transferrin receptor in vesicular form in sheep reticulocytes

    PubMed Central

    1985-01-01

    Using ferritin-labeled protein A and colloidal gold-labeled anti-rabbit IgG, the fate of the sheep transferrin receptor has been followed microscopically during reticulocyte maturation in vitro. After a few minutes of incubation at 37 degrees C, the receptor is found on the cell surface or in simple vesicles of 100-200 nm, in which the receptor appears to line the limiting membrane of the vesicles. With time (60 min or longer), large multivesicular elements (MVEs) appear whose diameter may reach 1-1.5 micron. Inside these large MVEs are round bodies of approximately 50-nm diam that bear the receptor at their external surfaces. The limiting membrane of the large MVEs is relatively free from receptor. When the large MVEs fuse with the plasma membrane, their contents, the 50-nm bodies, are released into the medium. The 50-nm bodies appear to arise by budding from the limiting membrane of the intracellular vesicles. Removal of surface receptor with pronase does not prevent exocytosis of internalized receptor. It is proposed that the exocytosis of the approximately 50-nm bodies represents the mechanism by which the transferrin receptor is shed during reticulocyte maturation. PMID:2993317

  17. Discovery of Potent and Selective Inhibitors of Human Reticulocyte 15- Lipoxygenase-1

    PubMed Central

    Rai, Ganesha; Kenyon, Victor; Jadhav, Ajit; Schultz, Lena; Armstrong, Michelle; Jameson, J Brian; Hoobler, Eric; Leister, William; Simeonov, Anton; Holman, Theodore R.; Maloney, David J.

    2010-01-01

    There are a variety of lipoxygenases in the human body (hLO), each having a distinct role in cellular biology. Human reticulocyte 15-Lipoxygenase-1 (15-hLO-1), which catalyzes the dioxygenation of 1,4-cis,cis-pentadiene-containing polyunsaturated fatty acids, is implicated in a number of diseases including cancer, atherosclerosis, and neurodegenerative conditions. Despite the potential therapeutic relevance of this target, few inhibitors have been reported that are both potent and selective. To this end, we have employed a quantitative high-throughput (qHTS) screen against ~74,000 small molecules in search of reticulocyte 15-hLO-1 selective inhibitors. This screen led to the discovery of a novel chemotype for 15-hLO-1 inhibition, which displays nM potency and is >7,500-fold selective against the related isozymes, 5-hLO, platelet 12-hLO, epithelial 15-hLO-2, ovine cyclooxygenase-1 and human cyclooxygenase-2. In addition, kinetic experiments were performed which indicate that this class of inhibitor is tight binding, reversible, and appears not to reduce the active-site ferric ion. PMID:20866075

  18. Hemolysis Is Associated with Low Reticulocyte Production Index and Predicts Blood Transfusion in Severe Malarial Anemia

    PubMed Central

    Rudat, Annika; Kreidenweiss, Andrea; Steur, Claudia; Appelmann, Iris; Ruehe, Bettina; Schröder, Paul; Berdel, Wolfgang E.; Kremsner, Peter G.; Mordmüller, Benjamin

    2010-01-01

    Background Falciparum Malaria, an infectious disease caused by the apicomplexan parasite Plasmodium falciparum, is among the leading causes of death and morbidity attributable to infectious diseases worldwide. In Gabon, Central Africa, one out of four inpatients have severe malarial anemia (SMA), a life-threatening complication if left untreated. Emerging drug resistant parasites might aggravate the situation. This case control study investigates biomarkers of enhanced hemolysis in hospitalized children with either SMA or mild malaria (MM). Methods and Findings Ninety-one children were included, thereof 39 SMA patients. Strict inclusion criteria were chosen to exclude other causes of anemia. At diagnosis, erythrophagocytosis (a direct marker for extravascular hemolysis, EVH) was enhanced in SMA compared to MM patients (5.0 arbitrary units (AU) (interquartile range (IR): 2.2–9.6) vs. 2.1 AU (IR: 1.3–3.9), p<0.01). Furthermore, indirect markers for EVH, (i.e. serum neopterin levels, spleen size enlargement and monocyte pigment) were significantly increased in SMA patients. Markers for erythrocyte ageing, such as CD35 (complement receptor 1), CD55 (decay acceleration factor) and phosphatidylserine exposure (annexin-V-binding) were investigated by flow cytometry. In SMA patients, levels of CD35 and CD55 on the red blood cell surface were decreased and erythrocyte removal markers were increased when compared to MM or reconvalescent patients. Additionally, intravascular hemolysis (IVH) was quantified using several indirect markers (LDH, α-HBDH, haptoglobin and hemopexin), which all showed elevated IVH in SMA. The presence of both IVH and EVH predicted the need for blood transfusion during antimalarial treatment (odds ratio 61.5, 95% confidence interval (CI): 8.9–427). Interestingly, this subpopulation is characterized by a significantly lowered reticulocyte production index (RPI, p<0.05). Conclusions Our results show the multifactorial pathophysiology of SMA

  19. A Novel Erythrocyte Binding Protein of Plasmodium vivax Suggests an Alternate Invasion Pathway into Duffy-Positive Reticulocytes

    PubMed Central

    Thomson-Luque, Richard; Torres, Letícia de Menezes; Gunalan, Karthigayan; Carvalho, Luzia H.

    2016-01-01

    ABSTRACT Erythrocyte invasion by malaria parasites is essential for blood-stage development and an important determinant of host range. In Plasmodium vivax, the interaction between the Duffy binding protein (DBP) and its cognate receptor, the Duffy antigen receptor for chemokines (DARC), on human erythrocytes is central to blood-stage infection. Contrary to this established pathway of invasion, there is growing evidence of P. vivax infections occurring in Duffy blood group-negative individuals, suggesting that the parasite might have gained an alternative pathway to infect this group of individuals. Supporting this concept, a second distinct erythrocyte binding protein (EBP2), representing a new member of the DBP family, was discovered in P. vivax and may be the ligand in an alternate invasion pathway. Our study characterizes this novel ligand and determines its potential role in reticulocyte invasion by P. vivax merozoites. EBP2 binds preferentially to young (CD71high) Duffy-positive (Fy+) reticulocytes and has minimal binding capacity for Duffy-negative reticulocytes. Importantly, EBP2 is antigenically distinct from DBP and cannot be functionally inhibited by anti-DBP antibodies. Consequently, our results do not support EBP2 as a ligand for invasion of Duffy-negative blood cells, but instead, EBP2 may represent a novel ligand for an alternate invasion pathway of Duffy-positive reticulocytes. PMID:27555313

  20. Proteomics: a review and an example using the reticulocyte membrane proteome.

    PubMed

    Prenni, Jessica E; Avery, Anne C; Olver, Christine S

    2007-03-01

    Proteomics is a rapidly expanding field of scientific study that combines techniques in protein solubilization and separation, mass spectrometry, and genome and protein database searching. The proteome is most commonly defined as the entire complement of proteins expressed in a given cell type or tissue under a given condition. A proteomics experiment may be as simple as identifying a single protein or as complex as identifying thousands of proteins in a cell lysate. In this review, we describe the general principles of proteomics and its analytic methods and present an example of an experiment to characterize the murine reticulocyte membrane proteome. A brief summary of proteomics applications and their clinical potential and relevance to clinical pathology is also presented.

  1. Anticlastogenic effects of galangin against mitomycin C-induced micronuclei in reticulocytes of mice.

    PubMed

    Heo, M Y; Jae, L H; Jung, S S; Au, W W

    1996-05-17

    We investigated the suppressive effect of galangin on the induction of micronucleated reticulocytes (MNRETs) by mitomycin C (MMC) in mouse peripheral blood. When galangin was given to mice 24 h before the intraperitoneal injection of MMC (1 mg/kg), a more marked decrease in the frequency of MNRETs was observed than in mice with simultaneous and post-treatment of galangin. On the other hand, when galangin was given to mice for 7 consecutive days before MMC injection, galangin showed potent anticlastogenic effects, even at the lowest dose level of 0.1 mg/kg. Results from our in vivo studies indicate that galangin is capable of suppressing the clastogenic activity of the direct acting MMC. Together with our earlier observations, it appears that galangin is capable of protecting cells from the toxic effects of a variety of hazardous chemicals. Therefore, galangin may be an useful chemopreventive compound.

  2. Reticulocyte profile in top-level alpine skiers during four consecutive competitive seasons.

    PubMed

    Banfi, Giuseppe; Tavana, Rodolfo; Freschi, Marco; Lundby, Carsten

    2010-06-01

    The role of reticulocytes (Ret) in sports medicine became clear when the count of immature erythrocytes was introduced in protocols used for anti-doping purposes. Because specific research regarding seasonal variations in Ret is lacking, we assessed Ret (and [Hb]) in top-level male and female skiers during four consecutive competitive seasons. A difference (P < 0.05) between males and females was found for [Hb] and Ret values: [Hb] was lower and Ret was higher in females. The difference was maintained across all four competitive seasons. Marked within-subject differences in [Hb], Ret and immature reticulocyte fraction values were noted; the within-subject variability was greater than the between-subject variability in both genders. For instance, a difference for Ret was consistently shown between first and second blood drawings, i.e. between basal value, before the start of training and competition, and the value at middle of season, when training workload was at highest level. Unlike Ret%, the analysis of variance showed significant changes in [Hb] values across competitive seasons for both genders. Comparison between consecutive seasons (e.g., 2005-2006 vs. 2006-2007) showed significant differences for both parameters. The behaviour of [Hb] and Ret during the various seasons was parallel in females, whereas a discrepancy existed in males. In general, inter-individual variability is quite high, thus, Ret and [Hb] modifications should be referred only to the single athlete. We confirm the validity of the use of Ret counts for anti-doping purposes.

  3. Effects of artemisinins on reticulocyte count and relationship to possible embryotoxicity in confirmed and unconfirmed malarial patients.

    PubMed

    Clark, Robert L

    2012-02-01

    Rat studies suggest that artemisinin-induced decreases in reticulocyte count are a marker for embryotoxicity (in one study, r = 0.82; p < 0.05). In clinical studies, therapeutic doses of artemisinins induced decreases in reticulocyte count that were larger in five of six groups of healthy volunteers (mean decreases of 47-75%) than in 12 groups of patients with malaria (mean decreases of 0-34% and incidences of low reticulocyte count of 0.6-18%). Malaria causes hypoferremia and drug concentrates in infected red cells so, among the explanations for the lesser decreases in patients, is that malaria protects against artemisinin-induced decreases in reticulocyte count by reducing the target tissue levels of active drug and/or ferrous iron which activates the drug to toxic free radicals. The disease could also protect against embryotoxicity in which case pregnant women without malaria would be at greater risk of artemisinin-induced embryotoxicity. Malaria protection against artesunate toxicity has been observed in rats. No artemisinin-induced embryotoxicity has been identified in limited numbers of women with confirmed malaria in the first trimester. However, in large parts of tropical Africa, malaria treatment is based on fever rather than confirmation of parasitemia and many pregnant women without malaria are exposed to antimalarials. No clinical studies have been conducted on uninfected women for whom pregnancy was identified and then an artemisinin was administered subsequently. Testing in rats and/or humans is needed to determine if malaria protects against reticulocytopenia and embryotoxicity and whether the parasite is a more or less sensitive target than the embryo and reticulocyte.

  4. Reticulocyte hemoglobin content predicts functional iron deficiency in hemodialysis patients receiving rHuEPO.

    PubMed

    Mittman, N; Sreedhara, R; Mushnick, R; Chattopadhyay, J; Zelmanovic, D; Vaseghi, M; Avram, M M

    1997-12-01

    Early detection of iron sufficiency at the level of the erythropoietic cell is necessary to optimize management of uremic anemia with recombinant human erythropoietin (rHuEPO). "Absolute" and "functional" iron deficiency are the most important factors causing resistance to administered rHuEPO. Transferrin saturation and serum ferritin measurements have been noted to be insensitive and inaccurate measures to detect functional iron deficiency. Recently, the reticulocyte hemoglobin content (CHr) has been shown to be a sensitive and specific indicator of functional iron deficiency in nondialysis patients treated with rHuEPO. The purpose of this study is to compare CHr with currently used indices of iron sufficiency in rHuEPO-treated hemodialysis (HD) patients. In study 1, 364 stable HD patients were studied at two outpatient dialysis centers. CHr was normally distributed, with a mean value of 28.3 pg, and was consistent over two consecutive monthly samples in each center. CHr was weakly but consistently correlated with transferrin saturation and serum ferritin. CHr and reticulocyte number were inversely correlated with red blood cell (RBC) number, suggesting that the erythropoietic stimulus of routinely administered rHuEPO may have resulted in functional iron deficiency. Month-to-month changes in CHr correlated weakly with changes in serum iron and percent transferrin saturation, but not at all with changes in serum ferritin. When we analyzed those patients with baseline CHr less than 26 pg, a level strongly suggestive of functional iron deficiency, these correlations strengthened, and in addition, month-to-month changes in CHr correlated strongly and directly with concomitant changes in RBC count, hemoglobin, and hematocrit, suggesting that rising CHr was indicative of an erythropoietic response. In study 2, 79 patients received a single-dose infusion of 500 mg iron dextran. After intravenous iron, CHr rose within 48 hours, peaked at 96 hours, and then fell toward

  5. Diagnosis of iron deficiency anaemia in hospital patients: Use of the reticulocyte haemoglobin content to differentiate iron deficiency anaemia from anaemia of chronic disease.

    PubMed

    Schapkaitz, Elise; Buldeo, Suvarna; Mahlangu, Johnny Ndoni

    2015-11-20

    The diagnosis of iron deficiency anaemia in hospital patients with chronic infections and inflammation presents a challenge. Recently laboratory tests such as the reticulocyte haemoglobin content, which are independent of infection and inflammation, have become available for routine diagnostic use.

  6. Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening.

    PubMed

    Han, Jin-Hee; Li, Jian; Wang, Bo; Lee, Seong-Kyun; Nyunt, Myat Htut; Na, Sunghun; Park, Jeong-Hyun; Han, Eun-Taek

    2015-08-01

    Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.

  7. Novel hematological parameters for the evaluation of patients with myeloproliferative neoplasms: the immature platelet and reticulocyte fractions.

    PubMed

    Strati, Paolo; Bose, Prithviraj; Lyle, Lindsey; Gaw, Katie; Zhou, Lingsha; Pierce, Sherry A; Huynh-Lu, Julie; Hirsch-Ginsberg, Cheryl F; Bueso-Mendoza, Daniel E; Bueso-Ramos, Carlos E; Verstovsek, Srdan

    2017-02-28

    New automated hematology analyzers have led to the availability of novel hematological parameters, including the immature platelet fraction (IPF) and the immature reticulocyte fraction (IRF), both of potential interest in patients with myeloproliferative neoplasms (MPNs). We performed a prospective analysis of 217 patients with MPN, including 32 (15%) with essential thrombocythemia (ET), 43 (20%) with polycythemia vera (PV), and 142 (65%) with myelofibrosis (MF); the IPF and IRF were measured by the Sysmex XN analyzer. As compared to patients with ET, both a higher IPF and IRF were observed among patients with PV and MF. Factors associated with high IPF among patients with PV/ET were male sex, thrombocytopenia, and diagnosis of PV; among patients with MF, they were elevated peripheral blasts, low platelet count, JAK2 V617F mutation, and previous therapy. Factors associated with high IRF among patients with PV/ET were low hemoglobin, high reticulocyte count, and PV diagnosis; among patients with MF, they were peripheral blasts and elevated reticulocytes. The IPF and IRF represent novel parameters in patients with MPN with potential relevant clinical implications. Comparison with healthy subjects and those with secondary polycythemia is needed to confirm our preliminary findings.

  8. Evaluation of erythrocyte and reticulocyte parameters as indicative of iron deficiency in patients with anemia of chronic disease

    PubMed Central

    Torino, Ana Beatriz Barbosa; Gilberti, Maria de Fátima Pererira; da Costa, Edvilson; de Lima, Gisélia Aparecida Freire; Grotto, Helena Zerlotti Wolf

    2015-01-01

    Objective The aim of this study was to evaluate the effectiveness of mature red cell and reticulocyte parameters to identify three conditions: iron deficiency anemia, anemia of chronic disease, and anemia of chronic disease associated with absolute iron deficiency. Methods Peripheral blood cells from 117 adult patients with anemia were classified according to iron status, inflammation, and hemoglobinopathies as: iron deficiency anemia (n = 42), anemia of chronic disease (n = 28), anemia of chronic disease associated with iron deficiency anemia (n = 22), and heterozygous β-thalassemia (n = 25). The percentage of microcytic erythrocytes, hypochromic erythrocytes, and the levels of hemoglobin in both reticulocytes and mature red cells were determined. Receiver operating characteristic analysis was used to evaluate the accuracy of the parameters in differentiating anemia. Results There was no difference between the groups of iron deficiency and anemia of chronic disease associated with absolute iron deficiency for any of the parameters. The percentage of hypochromic erythrocytes was the best parameter to identify absolute iron deficiency in patients with anemia of chronic disease (area under curve = 0.785; 95% confidence interval: 0.661–0.909 with sensitivity of 72.7%, and specificity of 70.4%; cut-off value 1.8%). The formula microcytic erythrocyte count minus hypochromic erythrocyte count was very accurate to differentiate iron deficiency anemia from heterozygous β-thalassemia (area under curve = 0.977; 95% confidence interval: 0.950–1.005 with a sensitivity of 96.2%, and specificity of 92.7%; cut-off value 13.8). Conclusion The erythrocyte and reticulocyte indices are moderately good to identify absolute iron deficiency in patients with anemia of chronic disease. PMID:25818816

  9. Evaluation of red cell and reticulocyte parameters as indicative of iron deficiency in patients with anemia of chronic disease

    PubMed Central

    Torino, Ana Beatriz Barbosa; Gilberti, Maria de Fátima Pererira; da Costa, Edvilson; de Lima, Gisélia Aparecida Freire; Grotto, Helena Zerlotti Wolf

    2014-01-01

    Objective The purpose of this study was to evaluate the effectiveness of mature red cell and reticulocyte parameters under three conditions: iron deficiency anemia, anemia of chronic disease, and anemia of chronic disease associated with absolute iron deficiency. Methods Peripheral blood cells from 117 adult patients with anemia were classified according to iron status, and inflammatory activity, and the results of a hemoglobinopathy investigation as: iron deficiency anemia (n = 42), anemia of chronic disease (n = 28), anemia of chronic disease associated with iron deficiency anemia (n = 22), and heterozygous β thalassemia (n = 25). The percentage of microcytic red cells, hypochromic red cells, and levels of hemoglobin content in both reticulocytes and mature red cells were determined. Receiver operating characteristic analysis was used to evaluate the accuracy of the parameters in differentiating between the different types of anemia. Results There was no significant difference between the iron deficient group and anemia of chronic disease associated with absolute iron deficiency in respect to any parameter. The percentage of hypochromic red cells was the best parameter to discriminate anemia of chronic disease with and without absolute iron deficiency (area under curve = 0.785; 95% confidence interval: 0.661–0.909, with sensitivity of 72.7%, and specificity of 70.4%; cut-off value 1.8%). The formula microcytic red cells minus hypochromic red cells was very accurate in differentiating iron deficiency anemia and heterozygous β thalassemia (area under curve = 0.977; 95% confidence interval: 0.950–1.005; with sensitivity of 96.2%, and specificity of 92.7%; cut-off value 13.8). Conclusion The indices related to red cells and reticulocytes have a moderate performance in identifying absolute iron deficiency in patients with anemia of chronic disease. PMID:25453653

  10. Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T cell Subsets' Distribution.

    PubMed

    Martín-Jaular, Lorena; de Menezes-Neto, Armando; Monguió-Tortajada, Marta; Elizalde-Torrent, Aleix; Díaz-Varela, Míriam; Fernández-Becerra, Carmen; Borras, Francesc E; Montoya, Maria; Del Portillo, Hernando A

    2016-01-01

    Reticulocyte-derived exosomes (rex) are 30-100 nm membrane vesicles of endocytic origin released during the maturation of reticulocytes to erythrocytes upon fusion of multivesicular bodies with the plasma membrane. Combination of CpG-ODN with rex obtained from BALB/c mice infected with the reticulocyte-prone non-lethal P. yoelii 17X malaria strain (rexPy), had been shown to induce survival and long lasting protection. Here, we show that splenectomized mice are not protected upon rexPy+CpG inmunizations and that protection is restored upon passive transfer of splenocytes obtained from animals immunized with rexPy+CpG. Notably, rexPy immunization of mice induced changes in PD1(-) memory T cells with effector phenotype. Proteomics analysis of rexPy confirmed their reticulocyte origin and demonstrated the presence of parasite antigens. Our studies thus prove, for what we believe is the first time, that rex from reticulocyte-prone malarial infections are associated with splenic long-lasting memory responses. To try extrapolating these data to human infections, in vitro experiments with spleen cells of human transplantation donors were performed. Plasma-derived exosomes from vivax malaria patients (exPv) were actively uptaken by human splenocytes and stimulated spleen cells leading to changes in T cell subsets.

  11. Spleen-Dependent Immune Protection Elicited by CpG Adjuvanted Reticulocyte-Derived Exosomes from Malaria Infection Is Associated with Changes in T cell Subsets' Distribution

    PubMed Central

    Martín-Jaular, Lorena; de Menezes-Neto, Armando; Monguió-Tortajada, Marta; Elizalde-Torrent, Aleix; Díaz-Varela, Míriam; Fernández-Becerra, Carmen; Borras, Francesc E.; Montoya, Maria; del Portillo, Hernando A.

    2016-01-01

    Reticulocyte-derived exosomes (rex) are 30–100 nm membrane vesicles of endocytic origin released during the maturation of reticulocytes to erythrocytes upon fusion of multivesicular bodies with the plasma membrane. Combination of CpG-ODN with rex obtained from BALB/c mice infected with the reticulocyte-prone non-lethal P. yoelii 17X malaria strain (rexPy), had been shown to induce survival and long lasting protection. Here, we show that splenectomized mice are not protected upon rexPy+CpG inmunizations and that protection is restored upon passive transfer of splenocytes obtained from animals immunized with rexPy+CpG. Notably, rexPy immunization of mice induced changes in PD1− memory T cells with effector phenotype. Proteomics analysis of rexPy confirmed their reticulocyte origin and demonstrated the presence of parasite antigens. Our studies thus prove, for what we believe is the first time, that rex from reticulocyte-prone malarial infections are associated with splenic long-lasting memory responses. To try extrapolating these data to human infections, in vitro experiments with spleen cells of human transplantation donors were performed. Plasma-derived exosomes from vivax malaria patients (exPv) were actively uptaken by human splenocytes and stimulated spleen cells leading to changes in T cell subsets. PMID:27900319

  12. Analysis of reticulocyte parameters on the Sysmex XE 5000 and LH 750 analyzers in the diagnosis of inefficient erythropoiesis.

    PubMed

    Urrechaga, E; Borque, L; Escanero, J F

    2011-02-01

    The reticulocyte hemoglobin equivalent (Ret He) represents an indirect measure of the functional iron available for erythropoiesis over the previous 2-3 days. Only the analyzers of a single manufacturer, Sysmex (Sysmex Corporation, Kobe, Japan), include Ret He. Red blood cell size factor (RSf) is a new parameter provided by Beckman Coulter, which joins together the volume of the erythrocytes and the volume of reticulocytes. The aims of the study were to investigate the clinical usefulness of RSf in the study of erythropoiesis status and to assess its concordance with Ret He values. Samples from 417 patients were run on both LH 780 (Beckman Coulter) and Sysmex XE 5000 analyzers. Independent samples t-test, Pearson correlation, receiver operating characteristic (ROC) analysis and inter-rater reliability (κ index) were applied. Good correlation between RSf and Ret He was observed, r = 0.8184. Significant differences (P < 0.001) were detected when groups with inefficient erythropoiesis were compared with patients undergoing therapy and healthy subjects. ROC analysis for RSf in the diagnosis of inefficient erythropoiesis, cutoff 91.1 fl, area under curve 0.963, sensitivity 91.7%, specificity 88.5%. Concordance between RSf and Ret He κ = 0.68. RSf and Ret He are suitable parameters for the assessment of erythropoiesis status.

  13. From Rabbit Reticulocytes to Clam Oocytes: In Search of the System That Targets Mitotic Cyclins for Degradation

    PubMed Central

    2010-01-01

    By the late 1980s, the basic biochemistry of ubiquitin-mediated protein degradation had already been elucidated by studies that used reticulocyte lysates. However, the scope and biological functions of this system remained largely obscure. Therefore, I became interested at that time in the mechanisms by which mitotic cyclins are degraded in exit from mitosis. Using a cell-free system from clam oocytes that faithfully reproduced cell cycle stage–specific degradation of cyclins, we identified in 1995 a large ubiquitin ligase complex that targets mitotic cyclins for degradation. Subsequent studies in many laboratories showed that this ubiquitin ligase, now called the anaphase-promoting complex/cyclosome, has centrally important roles in many aspects of cell cycle control. PMID:20335505

  14. Quantitation of CD55 and CD59 expression on reticulocytes and mature erythrocytes in paroxysmal nocturnal hemoglobinuria, aplastic anemia, and healthy control subjects.

    PubMed

    Kim, Yeongsic; Lim, Jihyang; Kim, Myungshin; Kim, Yonggoo; Lee, Jong-Wook; Han, Kyungja

    2010-01-01

    Since PNH occasionally results in bone marrow failure, it is difficult to differentiate PNH from AA with small numbers of CD(55) (-)CD(59) (-) erythrocytes. We quantified CD55 and CD59 molecules expressed on normal reticulocytes and mature erythrocytes of paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA), and normal individuals in order to determine their usefulness for differentiation between PNH and AA. A total of 56 patients (AA 39 patients, PNH 17 patients) and 10 healthy volunteers were enrolled. Two-color flow cytometric analyses were conducted using thiazole orange, anti-CD55 and CD59 monoclonal antibodies to identify CD(55) (-)CD(59) (-) reticulocytes and mature erythrocytes. Mean fluorescence level of CD(55) (+) mature erythrocytes was lowest in the PNH patients (13.2 x 10(3) MESF), and was significantly lower than in normal controls (16.7 x 10(3) MESF, p <0.05), and highest in the AA subjects (22.8 x 10(3) MESF). Mean fluorescence level of CD(59) (+) mature erythrocytes was lowest in the PNH subjects (24.5 x 10(3) MESF), significantly less than in normal controls (39.0 x 10(3) MESF, p <0.05), and highest in the AA subjects (49.2 x 10(3) MESF). Total reticulocyte counts correlated strongly with the CD(55) (-)CD(59) (-) reticulocytes. Comparative quantitative analysis of CD55 and CD59 molecules expressed on normal erythrocytes may be useful in differentiating PNH from AA patients with small numbers of PNH-phenotype erythrocytes, and the total reticulocyte count may prove useful as a marker for PNH clone size.

  15. Evaluation of reticulocyte haemoglobin content as marker of iron deficiency and predictor of response to intravenous iron in haemodialysis patients.

    PubMed

    Kim, J M; Ihm, C H; Kim, H J

    2008-02-01

    Because serum ferritin and transferrin saturation (TS) have a limitation in estimating iron status in haemodialysis patients, the reticulocyte haemoglobin content (CHr) has been proposed as a new tool. We investigate the accuracy of CHr in comparison with conventional tests and the relationship between changes in CHr and haemoglobin levels after therapy. We selected 140 haemodialysis patients receiving rHuEPO and intravenous iron supplementation and measured their complete blood count, CHr and iron parameters. Iron deficiency was defined as a ferritin <100 microg/l and/or a TS <20%. Hb, CHr, ferritin and TS levels were determined 1 month after therapy. Fifty-three patients were iron deficient. CHr were distributed with 33.7 +/- 1.4 pg in the iron sufficient group and with 29.9 +/- 1.9 pg in the iron deficient group (P = 0.001). The cutoff value of CHr for detecting iron deficiency was <32.4 pg. In iron deficient patients, a significant correlation was found between CHr and TS. The change in CHr after therapy was significantly larger in iron-deficient patients, and a lower baseline CHr is associated with a greater haemoglobin change. CHr is useful in screening iron status in dialysis patients, and a CHr cut-off value of 32 pg is appropriate for the assessment of iron deficiency. Moreover, CHr may serve as a predictor of the response to anaemia treatment.

  16. [Automated hematology analysers and spurious counts Part 3. Haemoglobin, red blood cells, cell count and indices, reticulocytes].

    PubMed

    Godon, Alban; Genevieve, Franck; Marteau-Tessier, Anne; Zandecki, Marc

    2012-01-01

    Several situations lead to abnormal haemoglobin measurement or to abnormal red blood cells (RBC) counts, including hyperlipemias, agglutinins and cryoglobulins, haemolysis, or elevated white blood cells (WBC) counts. Mean (red) cell volume may be also subject to spurious determination, because of agglutinins (mainly cold), high blood glucose level, natremia, anticoagulants in excess and at times technological considerations. Abnormality related to one measured parameter eventually leads to abnormal calculated RBC indices: mean cell haemoglobin content is certainly the most important RBC parameter to consider, maybe as important as flags generated by the haematology analysers (HA) themselves. In many circumstances, several of the measured parameters from cell blood counts (CBC) may be altered, and the discovery of a spurious change on one parameter frequently means that the validity of other parameters should be considered. Sensitive flags allow now the identification of several spurious counts, but only the most sophisticated HA have optimal flagging, and simpler ones, especially those without any WBC differential scattergram, do not share the same capacity to detect abnormal results. Reticulocytes are integrated into the CBC in many HA, and several situations may lead to abnormal counts, including abnormal gating, interference with intraerythrocytic particles, erythroblastosis or high WBC counts.

  17. TOP mRNAs are translationally inhibited by a titratable repressor in both wheat germ extract and reticulocyte lysate.

    PubMed

    Biberman, Y; Meyuhas, O

    1999-08-13

    Vertebrate TOP mRNAs contain a 5' terminal oligopyrimidine tract (5' TOP), which is subject to selective translational repression in non-growing cells or in cell-free translation systems. In the present study, we monitored in vitro the effect of increasing amounts of a 16 nucleotides long oligoribonucleotide representing the 5' terminus of mouse ribosomal protein S16 mRNA on the translation of TOP and non-TOP mRNAs. Our results demonstrate that the wild-type sequence (but not its mutant counterparts) derepresses the translation of mRNAs containing 5' TOP motifs, but failed to stimulate the translation of non-TOP mRNAs, even if the latter differed only by a single nucleotide from their 5' TOP-containing counterparts. Similar results have been obtained with both wheat germ extract and rabbit reticulocyte lysate. It appears, therefore, that translational repression of TOP mRNAs is achieved in vitro by the accumulation of a titratable repressor rather than by the loss of an activator and that this repressor recognizes multiple TOP mRNAs with a diverse set of 5' TOP motifs.

  18. Reticulocyte haemoglobin content vs. soluble transferrin receptor and ferritin index in iron deficiency anaemia accompanied with inflammation.

    PubMed

    Marković, M; Majkić-Singh, N; Ignjatović, S; Singh, S

    2007-10-01

    Ferritin concentration, as a parameter of iron status that is commonly used in the diagnosis of iron deficiency anaemia (IDA), often has limited values if the iron deficiency is accompanied by inflammatory disease. This study evaluated the value of reticulocyte haemoglobin content (CHr) and soluble transferrin receptor-ferritin index (sTfR/F) in the diagnosis of IDA and differential diagnosis of IDA and anaemia of chronic disease. The study included 66 nonanaemic individuals as controls, 86 patients with IDA divided into noninflammatory and inflammatory subgroups, and 32 patients with anaemia of chronic disease. Blood count, iron, transferrin saturation, total iron binding capacity, ferritin, C-reactive protein, sTfR and CHr were determined. Receiver operator characteristic curve analysis showed very high discriminating power for CHr, soluble transferrin receptor (sTfR) and sTfR/F in the diagnosis of IDA. In patients with anaemia of chronic disease these parameters showed no significant difference from the control. CHr and sTfR enabled recognition of iron deficiency and were not affected by acute phase reaction. They are sensitive markers of body iron status with additional value to conventional tests for the detection of iron deficiency.

  19. Unwinding protein specific for mRNA translation fractionated together with rabbit reticulocyte initiation factor 3 complex

    PubMed Central

    Ilan, Joseph; Ilan, Judith

    1977-01-01

    Experiments with a rabbit reticulocyte cell-free system dependent on the addition of initiation factor 3 (eIF-3) and mRNA were carried out. In this system, using ribosomal subunits, AUG(U)n can direct polyphenylalanine synthesis in the absence of eIF-3 at 3 mM MgCl2. Globin mRNA was not translated under similar conditions; its translation requires the addition of eIF-3. Moreover, the maximal rate of globin synthesis was achieved when the molar ratio of eIF-3 to ribosomes was approximately 1. This was taken to indicate that some ribosomal proteins were fractionated with eIF-3 and functioned in reconstitution of salt-washed ribosomes. In our system, almost all ribosomes were active, as evident from the fact that all were found in polysomes when analyzed at the time of linear incorporation, and the molar ratio of ribosomes to mRNA was maintained at 4:1. When AUG(U)n was hybridized with poly(A), it could not direct polyphenylalanine synthesis with or without eIF-3 and was a potent inhibitor of the translation of globin mRNA in the presence of eIF-3. When poly(A) containing 10% U was hybridized with AUG(U)n and added to the cell-free system, addition of eIF-3 promoted polyphenylalanine synthesis to about 80% of control. Moreover, eIF-3 was seen to shift significantly the melting temperature of globin and synthetic double-stranded RNA. These observations suggest that extraction of ribosomes with 0.5 M KCl may release a ribosomal protein that fractionates with eIF-3. This protein may function in unwinding or melting the secondary structure of mRNA and thus facilitate translation. PMID:267926

  20. Characterization of ribonuclease H activities present in two cell-free protein synthesizing systems, the wheat germ extract and the rabbit reticulocyte lysate.

    PubMed

    Cazenave, C; Frank, P; Büsen, W

    1993-01-01

    Experimental evidence accumulated to date by several research groups indicates that antisense oligodeoxynucleotides targeted against messenger RNA (mRNA) sequences located downstream of the initiation codon fail to inhibit the translation of this mRNA unless the hybrid is cleaved by RNase H. It has previously been shown that exogenous RNase H has to be added to rabbit reticulocyte lysate to obtain translational arrest (unless freshly prepared lysates are used). In contrast there is no need of exogenous RNase H by using wheat germ extract for translation because the level of endogenous RNase H is high enough to ensure cleavage of the hybrid formed between the antisense oligodeoxyribonucleotide and its complementary sequence on the mRNA. Surprisingly, we found that these two cell-free translation systems display similar amounts of RNase H activities when tested under standard conditions (extract diluted 500 times in the RNase H reaction mix). The RNase H activity of the rabbit reticulocyte lysate has a divalent cation requirement and sensitivity to inhibitors similar to class I ribonuclease H, whereas the activity of the wheat germ extract shows similarities to class II ribonuclease H. However, when these activities were assayed under conditions similar to those used for translation experiments, only highly reduced levels of activity were found in comparison to the standard assays. This reduction is due in part to sub-optimal ionic conditions for the endogenous RNase H activities in these extracts, and, for the other part, likely due to interactions with other proteins present in the lysates. In these conditions, however, the remaining activity found in the wheat germ extract was three times higher than the activity found in the rabbit reticulocyte lysate. Whether this difference can by itself explain the indicated differences in the two systems observed in hybrid-arrest of translation experiments remains open to discussion.

  1. A nontetrameric species is the major soluble form of keratin in Xenopus oocytes and rabbit reticulocyte lysates

    PubMed Central

    1996-01-01

    Inside the interphase cell, approximately 5% of the total intermediate filament protein exists in a soluble form. Past studies using velocity gradient sedimentation (VGS) indicate that soluble intermediate filament protein exists as an approximately 7 S tetrameric species. While studying intermediate filament assembly dynamics in the Xenopus oocyte, we used both VGS and size-exclusion chromatography (SEC) to analyze the soluble form of keratin. Previous studies (Coulombe, P. A., and E. Fuchs. 1990. J. Cell Biol. 111:153) report that tetrameric keratins migrate on SEC with an apparent molecular weight of approximately 150,000; the major soluble form of keratin in the oocyte, in contrast, migrates with an apparent molecular weight of approximately 750,000. During oocyte maturation, the keratin system disassembles into a soluble form (Klymkowsky, M. W., L. A. Maynell, and C. Nislow. 1991. J. Cell Biol. 114:787) and the amount of the 750-kD keratin complex increases dramatically. Immunoprecipitation analysis of soluble keratin from matured oocytes revealed the presence of type I and type II keratins, but no other stoichiometrically associated polypeptides, suggesting that the 750-kD keratin complex is composed solely of keratin. To further study the formation of the 750-kD keratin complex, we used rabbit reticulocyte lysates (RRL). The 750-kD keratin complex was formed in RRLs contranslating type I and type II Xenopus keratins, but not when lysates translated type I or type II keratin RNAs alone. The 750-kD keratin complex could be formed posttranslationally in an ATP-independent manner when type I and type II keratin translation reactions were mixed. Under conditions of prolonged incubation, such as occur during VGS analysis, the 750-kD keratin complex disassembled into a 7 S (by VGS), 150-kD (by SEC) form. In urea denaturation studies, the 7 S/150-kD form could be further disassembled into an 80-kD species that consists of cofractionating dimeric and monomeric

  2. Reticulocyte count is the most important predictor of acute cerebral ischemia and high-risk transcranial Doppler in a newborn cohort of 395 children with sickle cell anemia.

    PubMed

    Belisário, André Rolim; Sales, Rahyssa Rodrigues; Toledo, Nayara Evelin; Muniz, Maristela Braga de Sousa Rodrigues; Velloso-Rodrigues, Cibele; Silva, Célia Maria; Viana, Marcos Borato

    2016-10-01

    Stroke is a severe clinical manifestation of sickle cell anemia (SCA). Despite the prognostic relevance of transcranial Doppler (TCD), more accurate tools to assess stroke risk in children with SCA are required. Here, we describe the effect of clinical, laboratory, and molecular features on the risk of stroke and high-risk TCD in children from the newborn cohort of Minas Gerais, Brazil. Outcomes studied were acute cerebral ischemia and high-risk TCD. Clinical and hematological data were retrieved from children's records. Genetic markers, which were known for their association with stroke risk, were genotyped by polymerase chain reaction/restriction fragment length polymorphism and sequencing. The cumulative incidence of acute cerebral ischemia by the age of 8 years was 7.4 % and that of high-risk TCD by the age of 11.5 years was 14.2 %. The final multivariate model for acute cerebral ischemia risk included high white blood cell count and reticulocyte count, acute chest syndrome rate, and the single nucleotide polymorphisms (SNPs) TEK rs489347 and TNF-α rs1800629. The model for high-risk TCD included high reticulocyte count and the SNPs TEK rs489347 and TGFBR3 rs284875. Children with risk factors should be considered for intensive risk monitoring and for intervention therapy.

  3. Hydroxycarbamide decreases sickle reticulocyte adhesion to resting endothelium by inhibiting endothelial lutheran/basal cell adhesion molecule (Lu/BCAM) through phosphodiesterase 4A activation.

    PubMed

    Chaar, Vicky; Laurance, Sandrine; Lapoumeroulie, Claudine; Cochet, Sylvie; De Grandis, Maria; Colin, Yves; Elion, Jacques; Le Van Kim, Caroline; El Nemer, Wassim

    2014-04-18

    Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α4β1 integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.

  4. Pig-a gene mutation and micronucleated reticulocyte induction in rats exposed to tumorigenic doses of the leukemogenic agents chlorambucil, thiotepa, melphalan, and 1,3-propane sultone.

    PubMed

    Dertinger, Stephen D; Phonethepswath, Souk; Avlasevich, Svetlana L; Torous, Dorothea K; Mereness, Jared; Cottom, John; Bemis, Jeffrey C; Macgregor, James T

    2014-05-01

    To evaluate whether blood-based genotoxicity endpoints can provide temporal and dose-response data within the low-dose carcinogenic range that could contribute to carcinogenic mode of action (MoA) assessments, we evaluated the sensitivity of flow cytometry-based micronucleus and Pig-a gene mutation assays at and below tumorigenic dose rate 50 (TD50) levels. The incidence of micronucleated reticulocytes (MN-RET) was used to evaluate chromosomal damage, and the frequency of CD59-negative reticulocytes (RET(CD59-) ) and erythrocytes (RBC(CD59-) ) served as phenotypic reporters of mutation at the X-linked Pig-a gene. Several leukemogenic agents with a presumed genotoxic MoA were studied. Specifically, male Sprague Dawley rats were treated via oral gavage for 28 days with chlorambucil, thiotepa, melphalan, and 1,3-propane sultone at doses corresponding to 0.33x, 1x, and 3x TD50, as well as at the maximum tolerated dose. Frequencies of MN-RET were determined at Days 4 and 29, and RET(CD59-) and RBC(CD59-) data were collected pretreatment as well as Days 15/16, 29, and 56/57. Dose-related increases were observed for each endpoint, and time to maximal effect was consistently: MN-RET < RET(CD59-)  < RBC(CD59-) . For each of the chemicals studied, the genotoxic events occurred long before tumors or preneoplastic lesions would be expected. Furthermore, in the case of Pig-a gene mutation, the responses were observed at or below the TD50 dose for three out of the four chemicals studied. These data illustrate the potential for quantitative blood-based analyses to provide dose-response and temporality information that relates genetic damage to cancer induction.

  5. Efficiency of cell-free protein synthesis based on a crude cell extract from Escherichia coli, wheat germ, and rabbit reticulocytes.

    PubMed

    Hino, Mami; Kataoka, Masatoshi; Kajimoto, Kazuaki; Yamamoto, Takenori; Kido, Jun-Ichi; Shinohara, Yasuo; Baba, Yoshinobu

    2008-01-20

    The efficiency of protein synthesis for glyceraldehyde-3-phosphate dehydrogenase (G3PDH) was examined with several in vitro coupled transcription/translation protein synthesis systems based on Escherichia coli lysate, wheat germ, or reticulocyte lysate, and an in vitro translation system based on wheat germ extract. A significant amount of protein synthesis was observed only in systems based on E. coli using pET/G3PDH as the expression vector. A remarkable increase of protein synthesis was obtained in wheat germ using a pT(N)T expression vector which contains a 5'-globin leader sequence and a synthetic poly(A)(30) tail instead of pET. A significant difference of T7 RNA polymerase presence by Western blot analysis was not observed in the first four systems, and the difference of total RNA presence in each reaction mixture by Northern blot analysis seemed unrelated to protein synthesis. Although a small amount of protein was synthesized using RNA-encoding G3PDH transcribed in vitro with pET/G3PDH by an in vitro translation system, an extreme increase was observed using transcribed RNA with pEU/G3PDH, which contains T7 RNA promoter and a translation enhancer, Omega sequence. These results suggest that the presence of an enhancer sequence for translation is one of the critical steps for protein synthesis by a eukaryotic cell-free protein synthesis system.

  6. Back to basics: the untreated rabbit reticulocyte lysate as a competitive system to recapitulate cap/poly(A) synergy and the selective advantage of IRES-driven translation.

    PubMed

    Soto Rifo, Ricardo; Ricci, Emiliano P; Décimo, Didier; Moncorgé, Olivier; Ohlmann, Théophile

    2007-01-01

    Translation of most eukaryotic mRNAs involves the synergistic action between the 5' cap structure and the 3' poly(A) tail at the initiation step. The poly(A) tail has also been shown to stimulate translation of picornavirus internal ribosome entry sites (IRES)-directed translation. These effects have been attributed principally to interactions between eIF4G and poly(A)-binding protein (PABP) but also to the participation of PABP in other steps during translation initiation. As the rabbit reticulocyte lysate (RRL) does not recapitulate this cap/poly(A) synergy, several systems based on cellular cell-free extracts have been developed to study the effects of poly(A) tail in vitro but they generally exhibit low translational efficiency. Here, we describe that the non-nuclease-treated RRL (untreated RRL) is able to recapitulate the effects of poly(A) tail on translation in vitro. In this system, translation of a capped/polyadenylated RNA was specifically inhibited by either Paip2 or poly(rA), whereas translation directed by HCV IRES remained unaffected. Moreover, cleavage of eIF4G by FMDV L protease strongly stimulated translation directed by the EMCV IRES, thus recapitulating the competitive advantage that the proteolytic processing of eIF4G confers to IRES-driven RNAs.

  7. Under-filled blood collection tubes containing K2EDTA as anticoagulant are acceptable for automated complete blood counts, white blood cell differential, and reticulocyte count.

    PubMed

    Xu, M; Robbe, V A; Jack, R M; Rutledge, J C

    2010-10-01

    Current laboratory standards from Clinical Laboratory Standards Institute (CLSI) and manufacturer's (Becton Dickinson) data indicate that under-filling K(2)EDTA blood collection tubes can result in erroneous hematology values. To accommodate under-filled tubes and reduce collection volumes while optimizing our automation, we explored the acceptable limit of under-filled tubes for hematology values. We collected 8.0 ml of blood from 30 normal adult volunteers. Each donation was aliquoted in the following volumes: 4.0, 2.0, 1.0, 0.5 ml x 2. These samples were analyzed within 1 h of blood collection on Sysmex XE-2100 (Sysmex America Inc., Mundelein, IL, USA) for complete blood count, reticulocyte, and white blood cell differentials. Results of the under-filled tubes were compared to those of the standard volume. The Deming regression analysis show excellent correlation for all parameters between each under-filled blood collection volume compared to a standard 4 ml volume. The Bland and Altman analysis shows good agreement between both 1.0 and 2.0 ml compared to a 4.0 ml volume. The 0.5 ml compared to a 4.0 ml volume, however, shows increased variation on many parameters. In addition all three collection volumes show negative bias compared to the standard volume for platelet count, but the difference is considered insignificant with a percent difference of 5.5%, 3.2%, and 1.5% for 0.5, 1.0, and 2.0 ml collection volume respectively. Finally for 0.5 ml collection volume we noticed a low level of false positive flagging rate for white blood cell. Acceptable complete blood count values of under-filled powdered K(2)EDTA tubes can be obtained with as little as 1.0 ml of blood.

  8. Comparison of three-colour flow cytometry and slide-based microscopy for the scoring of micronucleated reticulocytes in rat bone-marrow and peripheral blood.

    PubMed

    Zhou, Changhui; Wang, Qingli; Wang, Zheng; Chang, Yan

    2013-12-12

    The aim of this study was to perform the first transferability assessment in China of the micronucleus (MN) scoring method based on three-colour flow cytometry (FCM). This was accomplished for both rat bone-marrow and peripheral blood specimens following exposure to a variety of genotoxic and non-genotoxic chemicals, whereby micronucleus induction was measured both with FCM and with traditional microscopy. In an initial study, rats were treated with vehicle or cyclophosphamide (CP) for 2 consecutive days by oral gavage, and blood and bone marrow were sampled at 24 h after the second treatment. Staining with acridine orange (AO) of methanol-fixed slides was used for microscopical analysis and 2000 reticulocytes (RET) or polychromatic erythrocytes (PCE) were scored for MN frequency. The erythrocytes in the remaining bone-marrow cell suspensions were eluted on cellulose columns. The eluted bone marrow as well as the peripheral blood cells was fixed, incubated and analyzed by FCM. In another experiment, the performance of the FCM-MN method was further evaluated with five clastogens (urethane, 5-fluorouracil, mitomycin C, methylmethane sulfonate and 6-thioguanine), two aneugens (vincristine sulfate and colchicine) and two non-genotoxic new drugs (compounds A and B), whose results were negative in the routine mouse-micronucleus test (MNT). The MN frequencies in rat peripheral blood induced by the positive chemicals were found to be lower than the frequencies in rat bone-marrow by both scoring methods. However, a high level of agreement for the MN frequencies in both compartments was obtained. Good correspondence between the two analysis methods was also achieved. These data provide support that the three-colour FCM method is more rapid and objective than manual microscopy, while yielding comparable data. It further supports the premise that rat peripheral blood may be an alternative to rat bone marrow in the MNT.

  9. In vivo erythrocyte micronucleus assay III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test.

    PubMed

    Hayashi, Makoto; MacGregor, James T; Gatehouse, David G; Blakey, David H; Dertinger, Stephen D; Abramsson-Zetterberg, Lilianne; Krishna, Gopala; Morita, Takeshi; Russo, Antonella; Asano, Norihide; Suzuki, Hiroshi; Ohyama, Wakako; Gibson, Dave

    2007-02-03

    The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test. Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation. In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed

  10. Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems.

    PubMed

    Komar, A A; Lesnik, T; Cullin, C; Guillemet, E; Ehrlich, R; Reiss, C

    1997-09-22

    The Ure2p yeast prion-like protein was translated in vitro in the presence of labeled [35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31-34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93-95]. Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.

  11. The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

    PubMed

    Kimoto, Takafumi; Horibata, Katsuyoshi; Miura, Daishiro; Chikura, Satsuki; Okada, Yuki; Ukai, Akiko; Itoh, Satoru; Nakayama, Shiho; Sanada, Hisakazu; Koyama, Naomi; Muto, Shigeharu; Uno, Yoshifumi; Yamamoto, Mika; Suzuki, Yuta; Fukuda, Takayuki; Goto, Ken; Wada, Kunio; Kyoya, Takahiro; Shigano, Miyuki; Takasawa, Hironao; Hamada, Shuichi; Adachi, Hideki; Uematsu, Yasuaki; Tsutsumi, Eri; Hori, Hisako; Kikuzuki, Ryuta; Ogiwara, Yosuke; Yoshida, Ikuma; Maeda, Akihisa; Narumi, Kazunori; Fujiishi, Yohei; Morita, Takeshi; Yamada, Masami; Honma, Masamitsu

    2016-11-15

    The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×10(6) RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

  12. Evaluation of the automated hematology analyzer Sysmex XT-2000iV ™ compared to the ADVIA ® 2120 for its use in dogs, cats, and horses. Part II: Accuracy of leukocyte differential and reticulocyte count, impact of anticoagulant and sample aging.

    PubMed

    Bauer, Natali; Nakagawa, Julia; Dunker, Cathrin; Failing, Klaus; Moritz, Andreas

    2012-01-01

    The automated laser-based hematology analyzer Sysmex XT-2000iV™ provides a 5-part differential count and specific cytograms that are of great interest for large veterinary laboratories. The aim of the study was to validate the Sysmex XT-2000iV compared to the laser-based hematology analyzer ADVIA® 2120 and manual differential in dogs, cats, and horses as well as the impact of anticoagulant (heparin, ethylenediamine tetra-acetic acid [EDTA], and citrate) and storage at 22°C and 4°C. Consecutive fresh K(3)-EDTA blood samples from 216 cats, 314 dogs, and 174 horses were included. The impact of anticoagulant and sample storage was assessed in specimens obtained from an additional 9 cats, 10 dogs, and 10 horses. Agreement between both analyzers was excellent to good except for monocytes and canine reticulocytes. Spearman rank correlation coefficients (r (s)) between Sysmex XT-2000iV and manual differential were good to fair and ranged from 0.91 (cat lymphocytes) to 0.44 (cat monocytes). Hematocrit value (Hct), mean corpuscular hemoglobin (MCH), MCH concentration (MCHC; all: P < 0.001), and mean corpuscular volume (MCV; P < 0.01) were higher in canine citrated blood compared to heparin and EDTA. In cats, lymphocytes and monocytes were lower in heparinized blood compared to EDTA (P < 0.05), whereas in horses no significant effect was seen. Regarding storage time and temperature, white and red blood cell counts, hemoglobin, and MCH were stable. Hct, MCV, and MCHC were influenced by erythrocyte swelling. Differential count remained stable for 24 hr (22°C) and nearly 72 hr (4°C) except for monocytes. The overall performance of the Sysmex XT-2000iV was excellent and compared favorably with that of the ADVIA 2120. A special strength was the excellent detection of feline eosinophils.

  13. Absolute Reticulocyte Count and Reticulocyte Hemoglobin Content as Predictors of Early Response to Exclusive Oral Iron in Children with Iron Deficiency Anemia

    PubMed Central

    Parodi, Emilia; Giraudo, Maria Teresa; Ricceri, Fulvio; Aurucci, Maria Luigia; Mazzone, Raffaela; Ramenghi, Ugo

    2016-01-01

    We report data regarding kinetic of response to oral iron in 34 iron deficiency anemia children. Twenty-four/34 patients (70.5%) reached reference value of hemoglobin (Hb) concentration for age and sex at day + 30 from the beginning of treatment (complete early responders (CERs)), and 4/34 (12%) reached an Hb concentration at least 50% higher than the original (partial early responders (PERs)). CHr at T1 (within 7 days from the beginning of treatment) was significantly different in the different groups (22.95 in CERs versus 18.41 in other patients; p = 0.001; 22.42 in early responders versus 18.07 in NERs; p = 0.001). Relative increase of CHr from T0 to T1 resulted significantly higher in CERs than in other patients (0.21 versus 0.11, p = 0.042) and in early responders than in NERs (0.22 versus 0.004, p = 0.006). Multivariate logistic models revealed a higher probability of being a complete early responder due to relative increase of ARC from T0 to T1 [OR (95% CI) = 44.95 (1.54–1311.98)] and to CHr at T1 [OR (95% CI) =3.18 (1.24–8.17)]. Our preliminary data confirm CHr as early and accurate predictor of hematological response to oral iron. PMID:27092272

  14. Surface Area and Volume Changes During Maturation of Reticulocytes in the Circulation of the Baboon

    DTIC Science & Technology

    2007-11-02

    authors thank Jane Trainor, Gina Ragno and Jane and George Cassidy for technical support, and Dr. Charles Kiefer for insightful scientific discussion...donations with or without recombinant erythropoietin. Br J Haematol 1993;84:118-30. 10. Valeri CR, Lindbergh JR, Contreras TJ, et ah Freeze-preserved

  15. Potent and Selective Inhibitors of Human Reticulocyte 12/15-Lipoxygenase as Anti-Stroke Therapies

    PubMed Central

    2015-01-01

    A key challenge facing drug discovery today is variability of the drug target between species, such as with 12/15-lipoxygenase (12/15-LOX), which contributes to ischemic brain injury, but its human and rodent isozymes have different inhibitor specificities. In the current work, we have utilized a quantitative high-throughput (qHTS) screen to identify compound 1 (ML351), a novel chemotype for 12/15-LOX inhibition that has nanomolar potency (IC50 = 200 nM) against human 12/15-LOX and is protective against oxidative glutamate toxicity in mouse neuronal HT22 cells. In addition, it exhibited greater than 250-fold selectivity versus related LOX isozymes, was a mixed inhibitor, and did not reduce the active-site ferric ion. Lastly, 1 significantly reduced infarct size following permanent focal ischemia in a mouse model of ischemic stroke. As such, this represents the first report of a selective inhibitor of human 12/15-LOX with demonstrated in vivo activity in proof-of-concept mouse models of stroke. PMID:24684213

  16. THE IRON TRANSPORTER DMT1 (OR NRAMP2 OR DCT1) IS LOCATED MOSTLY IN ENDOSOMES IN NORMAL AND BELGRADE RAT RETICULOCYTES. (R826248)

    EPA Science Inventory

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

  17. RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

    PubMed

    Rivers, Angela; Vaitkus, Kestis; Ruiz, Maria Armila; Ibanez, Vinzon; Jagadeeswaran, Ramasamy; Kouznetsova, Tatiana; DeSimone, Joseph; Lavelle, Donald

    2015-07-01

    Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.

  18. Polychromatophilia

    MedlinePlus

    ... cells (RBCs) called reticulocytes. These cells have a blue-colored center. Too many reticulocytes are the result of the bone marrow making more red blood cells than normal. This can be caused by ...

  19. Hematocrit

    MedlinePlus

    ... Crit; Packed Cell Volume; PCV; H and H (Hemoglobin and Hematocrit) Formal name: Hematocrit Related tests: Complete Blood Count , Hemoglobin , RBC Count , Blood Smear , Iron Tests , Reticulocyte Count , ...

  20. Clinical utility of the IRF: assessment of erythroid regeneration following parvo B19 infection.

    PubMed

    Wyrick-Glatzel, Janis; Conway-Klaassen, Janice

    2002-01-01

    Parvo B19 (Fifth disease) is an erythrotropic virus which attaches through the 'P' globoside receptor on the surface of human red blood cells and precursors. This typically benign viral infection can cause a transient aplastic anemia in patients with underlying red cell disorders. In this case, a two-year-old child presents with severe aplastic anemia without evidence of underlying disease. Erythroid regeneration is monitored through the use of the immature reticulocyte fraction (IRF) and is demonstrated by the presence of high and medium fluorescence reticulocytes in the peripheral blood three to five days prior to the peak in absolute reticulocytes.

  1. Immune hemolytic anemia

    MedlinePlus

    ... Absolute reticulocyte count Direct or indirect Coombs test Hemoglobin in the urine LDH (level of this enzyme ... of tissue damage) Red blood cell count (RBC), hemoglobin, and hematocrit Serum bilirubin level Serum free hemoglobin ...

  2. Bilirubin Test

    MedlinePlus

    ... Also known as: Total Bilirubin; TBIL; Neonatal Bilirubin; Direct Bilirubin; Conjugated Bilirubin; Indirect Bilirubin; Unconjugated Bilirubin Formal ... Hepatitis B ; Hepatitis C ; Complete Blood Count ; Urinalysis ; Direct Antiglobulin Test ; Haptoglobin ; Reticulocyte Count All content on ...

  3. Hematology of healthy Florida manatees (Trichechus manatus)

    USGS Publications Warehouse

    Harvey, J.W.; Harr, K.E.; Murphy, D.; Walsh, M.T.; Nolan, E.C.; Bonde, R.K.; Pate, M.G.; Deutsch, C.J.; Edwards, H.H.; Clapp, W.L.

    2009-01-01

    Background: Hematologic analysis is an important tool in evaluating the general health status of free-ranging manatees and in the diagnosis and monitoring of rehabilitating animals. Objectives: The purpose of this study was to evaluate diagnostically important hematologic analytes in healthy manatees (Trichechus manatus) and to assess variations with respect to location (free ranging vs captive), age class (small calves, large calves, subadults, and adults), and gender. Methods: Blood was collected from 55 free-ranging and 63 captive healthy manatees. Most analytes were measured using a CELL-DYN 3500R; automated reticulocytes were measured with an ADVIA 120. Standard manual methods were used for differential leukocyte counts, reticulocyte and Heinz body counts, and plasma protein and fibrinogen concentrations. Results: Rouleaux, slight polychromasia, stomatocytosis, and low numbers of schistocytes and nucleated RBCs (NRBCs) were seen often in stained blood films. Manual reticulocyte counts were higher than automated reticulocyte counts. Heinz bodies were present in erythrocytes of most manatees. Compared with free-ranging manatees, captive animals had slightly lower MCV, MCH, and eosinophil counts and slightly higher heterophil and NRBC counts, and fibrinogen concentration. Total leukocyte, heterophil, and monocyte counts tended to be lower in adults than in younger animals. Small calves tended to have higher reticulocyte counts and NRBC counts than older animals. Conclusions: Hematologic findings were generally similar between captive and free-ranging manatees. Higher manual reticulocyte counts suggest the ADVIA detects only reticulocytes containing large amounts of RNA. Higher reticulocyte and NRBC counts in young calves probably reflect an increased rate of erythropoiesis compared with older animals. ?? 2009 American Society for Veterinary Clinical Pathology.

  4. Quantitative analysis of mechanisms that govern red blood cell age structure and dynamics during anaemia.

    PubMed

    Savill, Nicholas J; Chadwick, William; Reece, Sarah E

    2009-06-01

    Mathematical modelling has proven an important tool in elucidating and quantifying mechanisms that govern the age structure and population dynamics of red blood cells (RBCs). Here we synthesise ideas from previous experimental data and the mathematical modelling literature with new data in order to test hypotheses and generate new predictions about these mechanisms. The result is a set of competing hypotheses about three intrinsic mechanisms: the feedback from circulating RBC concentration to production rate of immature RBCs (reticulocytes) in bone marrow, the release of reticulocytes from bone marrow into the circulation, and their subsequent ageing and clearance. In addition we examine two mechanisms specific to our experimental system: the effect of phenylhydrazine (PHZ) and blood sampling on RBC dynamics. We performed a set of experiments to quantify the dynamics of reticulocyte proportion, RBC concentration, and erythropoietin concentration in PHZ-induced anaemic mice. By quantifying experimental error we are able to fit and assess each hypothesis against our data and recover parameter estimates using Markov chain Monte Carlo based Bayesian inference. We find that, under normal conditions, about 3% of reticulocytes are released early from bone marrow and upon maturation all cells are released immediately. In the circulation, RBCs undergo random clearance but have a maximum lifespan of about 50 days. Under anaemic conditions reticulocyte production rate is linearly correlated with the difference between normal and anaemic RBC concentrations, and their release rate is exponentially correlated with the same. PHZ appears to age rather than kill RBCs, and younger RBCs are affected more than older RBCs. Blood sampling caused short aperiodic spikes in the proportion of reticulocytes which appear to have a different developmental pathway than normal reticulocytes. We also provide evidence of large diurnal oscillations in serum erythropoietin levels during anaemia.

  5. Structural analysis of the messenger RNA cap-binding protein: presence of phosphate, sulfhydryl, and disulfide groups

    SciTech Connect

    Not Available

    1986-01-05

    The messenger-RNA cap-binding protein (CBP) was isolated from human erythrocyte, rabbit erythrocyte, and rabbit reticulocyte lysate by affinity chromatography on 7-methylguanosine 5'-triphosphate-Sepharose. The specific activity of binding to capped oligonucleotides was similar for the human erythrocyte and rabbit reticulocyte CBPs. Isoelectric focusing of human and rabbit preparations revealed that each was composed of up to five species. The pI values of human and rabbit CBPs ranged from 5.7 to 6.5. The predominant form in erythrocytes had a pI of 6.3 while in reticulocytes, two major species, having pI values of 5.9 and 6.3, were present. Labeling of rabbit reticulocytes with (/sup 32/P)orthophosphate revealed that the pI 5.9 but not the pI 6.3 form contained phosphate. All of the phosphate was found in phosphoserine residues. The amino acid compositions of human erythrocyte and rabbit reticulocyte CBPs were quite similar. Both proteins had 7 tryptophanyl and 6 cysteinyl residues. Labeling with (1-/sup 14/C)iodoacetic acid under native and denaturing conditions provided evidence that 2 of the cysteinyl residues are present in the reduced form and 4 in disulfide bridges.

  6. How to approach chronic anemia.

    PubMed

    Koury, Mark J; Rhodes, Melissa

    2012-01-01

    We present herein an approach to diagnosing the cause of chronic anemia based on a patient's history and complete blood cell count (CBC). Four patterns that are encountered frequently in CBCs associated with chronic anemias are considered: (1) anemia with abnormal platelet and/or leukocyte counts, (2) anemia with increased reticulocyte counts, (3) life-long history of chronic anemia, and (4) anemia with inappropriately low reticulocytes. The pathophysiologic bases for some chronic anemias with low reticulocyte production are reviewed in terms of the bone marrow (BM) events that reduce normal rates of erythropoiesis. These events include: apoptosis of erythroid progenitor and precursor cells by intrinsic and extrinsic factors, development of macrocytosis when erythroblast DNA replication is impaired, and development of microcytosis due to heme-regulated eIF2α kinase inhibition of protein synthesis in iron-deficient or thalassemic erythroblasts.

  7. Cord blood transferrin receptors to assess fetal iron status

    PubMed Central

    Sweet, D.; Savage, G.; Tubman, R.; Lappin, T.; Halliday, H.

    2001-01-01

    OBJECTIVE—To study iron status at different gestational ages using cord blood serum transferrin receptors (STfRs).
METHODS—STfRs, iron, ferritin, total iron binding capacity, haemoglobin, and reticulocytes were measured in 144 cord blood samples. The babies were divided into three groups according to gestation (26 very preterm (24-29 weeks); 50 preterm (30-36 weeks); 68 term (37-41 weeks)).
RESULTS—Serum iron, ferritin, and total iron binding capacity were highest at term, whereas reticulocytes were highest in the very preterm. STfR levels were not influenced by gestation. Haemoglobin (r = 0.46; p < 0.0001) and reticulocytes (r = 0.42; p < 0.0001) were the only indices that independently correlated with STfR levels.
CONCLUSIONS—STfR levels in cord blood are not directly influenced by gestation and probably reflect the iron requirements of the fetus for erythropoiesis.

 PMID:11420322

  8. Do biomarkers of exposure and effect correlate with internal exposure to PAHs in swine?

    PubMed

    Peters, Rachel E; Wickstrom, Mark; Siciliano, Steven D

    2016-01-01

    Humans are commonly exposed to polycyclic aromatic hydrocarbons (PAHs), a family of compounds present as mixtures in the environment. This study exposed swine to PAH mixtures in single and subacute dose regimens and collected liver and ileum tissue to measure cytochrome P450 mRNA expression and enzyme activity as biomarkers of exposure and DNA adducts and oxidized proteins as biomarkers of effect. Micronucleated reticulocytes were measured as systemic biomarkers of effect. Duration of exposure did not influence biomarkers of exposure, though exposure duration produced significant increases in DNA adducts and oxidative stress. Micronucleated reticulocyte numbers were not affected by exposure length.

  9. Kinetics of erythrogenesis after bone marrow transplantation.

    PubMed

    Lazarus, H M; Chahine, A; Lacerna, K; Wamble, A; Iaffaldano, C; Straight, M; Rabinovitch, A; Schimenti, K J; Jacobberger, J

    1992-04-01

    To determine the kinetics of bone marrow erythrogenesis after bone marrow transplantation, the authors counted reticulocytes (by blood smear and flow cytometry) and compared those data with neutrophil and platelet recovery in 23 consecutive bone marrow transplant patients. The earliest indication of marrow recovery after allogeneic and autologous bone marrow transplantation was defined as the second increasing cell count after the lowest recorded count, provided that the trend continued upward. Recovery of marrow function was detected earlier in 10 of 23 patients using reticulocyte counts than by either neutrophil or platelet count alone. Specifically, in 8 of these 10 patients, recovery of erythropoiesis was determined earlier by flow cytometric examination than by the blood smear method. On the other hand, combining the data using the earliest value of platelet, neutrophil, and reticulocyte counts indicated that the mean day of recovery in our patient population was determined to be 12.1 +/- 4 days after marrow infusion. In patients undergoing autologous and allogeneic bone marrow transplantation, serial neutrophil and reticulocyte count determinations are complementary in early clinical detection of successful engraftment.

  10. Double diagnostic meaning of serum transferrin receptor in hemodialysis patients: two case reports.

    PubMed

    Passanante, S; Diquattro, M; Greco, V; Li Cavoli, G; Bono, L; Palma, B; Scola, S; Faraci, C; Rotolo, U; Menozzi, I

    2004-01-01

    Hemodialysis patients on maintenance erythropoietin need an adequate supply of iron to optimize therapy and achieve and maintain target levels of hemoglobin. Evaluation of iron stores and early detection of iron deficiency are essential for management of erythropoiesis in chronic renal failure, but there is still no single biochemical or hematological parameter that is sensitive or specific enough to completely describe the distribution of iron in the body. Serum transferrin receptor (sTfR) is a marker of iron that is available for erythropoiesis. We selected 2 clinical cases in which hemodialysis patients were receiving maintenance erythropoietin. To suggest how sTfR can be used in its double diagnostic meaning according to the clinical context of the patient, sTfR was evaluated in one case as a marker of iron deficiency and in the other as a marker of erythropoiesis. The association of sTfR with hematological parameters of iron-deficient erythropoiesis (reticulocyte hemoglobin content, percentage of hypochromic erythrocytes, ratio of reticulocyte hemoglobin content to hemoglobin content) and parameters of stimulated erythropoiesis (absolute reticulocyte count, immature reticulocyte fraction) increases the accuracy of sTfR in its double diagnostic power.

  11. Hereditary Spherocytosis and Hereditary Elliptocytosis: Aberrant Protein Sorting during Erythroblast Enucleation

    SciTech Connect

    Salomao, Marcela; Chen, Ke; Villalobos, Jonathan; Mohandas, Narla; An, Xiuli; Chasis, Joel Anne

    2010-02-08

    During erythroblast enucleation, membrane proteins distribute between extruded nuclei and reticulocytes. In hereditary spherocytosis (HS) and hereditary elliptocytosis (HE), deficiencies of membrane proteins, in addition to those encoded by the mutant gene, occur. Elliptocytes, resulting from protein 4.1R gene mutations, lack not only 4.1R but also glycophorin C, which links the cytoskeleton and bilayer. In HS resulting from ankyrin-1 mutations, band 3, Rh-associated antigen, and glycophorin A are deficient. The current study was undertaken to explore whether aberrant protein sorting, during enucleation, creates these membrane-spanning protein deficiencies. We found that although glycophorin C sorts to reticulocytes normally, it distributes to nuclei in 4.1R-deficient HE cells. Further, glycophorin A and Rh-associated antigen, which normally partition predominantly to reticulocytes, distribute to both nuclei and reticulocytes in an ankyrin-1-deficient murine model of HS. We conclude that aberrant protein sorting is one mechanistic basis for protein deficiencies in HE and HS.

  12. Examination of Reticulocytosis among Chronically Transfused Children with Sickle Cell Anemia

    PubMed Central

    Kaushal, Megha; Byrnes, Colleen; Khademian, Zarir; Duncan, Natalie; Luban, Naomi L. C.; Miller, Jeffery L.; Fasano, Ross M.; Meier, Emily Riehm

    2016-01-01

    Sickle cell anemia (SCA) is an inherited hemolytic anemia with compensatory reticulocytosis. Recent studies have shown that increased levels of reticulocytosis during infancy are associated with increased hospitalizations for SCA sequelae as well as cerebrovascular pathologies. In this study, absolute reticulocyte counts (ARC) measured prior to transfusion were analysed among a cohort of 29 pediatric SCA patients receiving chronic transfusion therapy (CTT) for primary and secondary stroke prevention. A cross-sectional flow cytometric analysis of the reticulocyte phenotype was also performed. Mean duration of CTT was 3.1 ± 2.6 years. Fifteen subjects with magnetic resonance angiography (MRA) -vasculopathy had significantly higher mean ARC prior to initiating CTT compared to 14 subjects without MRA-vasculopathy (427.6 ± 109.0 K/μl vs. 324.8 ± 109.2 K/μl, p<0.05). No significant differences in hemoglobin or percentage sickle hemoglobin (HbS) were noted between the two groups at baseline. Reticulocyte phenotyping further demonstrated that the percentages of circulating immature [CD36(+), CD71(+)] reticulocytes positively correlated with ARC in both groups. During the first year of CTT, neither group had significant reductions in ARC. Among this group of children with SCA, cerebrovasculopathy on MRA at initiation of CTT was associated with increased reticulocytosis, which was not reduced after 12 months of transfusions. PMID:27116614

  13. p,p'-DDT induces microcytic anemia in rats.

    PubMed

    Tomita, Mariko; Yoshida, Toshinori; Fukumori, Junko; Yamaguchi, Satoru; Kojima, Sayuri; Fukuyama, Tomoki; Ohnuma-Koyama, Aya; Takahashi, Naofumi; Takeuchi-Kashimoto, Yukiko; Kuwahara, Maki; Nakashima, Nobuaki; Ohtsuka, Ryoichi; Takeda, Makio; Kosaka, Tadashi; Harada, Takanori

    2013-01-01

    Emerging evidence suggests that chronic exposure to DDT and its derivatives is associated with a variety of human disorders such as anemia. The present study demonstrated that p,p'-DDT caused microcystic anemia in a dose-dependent manner (0, 5, 50, and 500 ppm) in the long-term study up to 2 years. To elucidate the mechanism(s) by which p,p'-DDT induces anemia, certain hematological parameters were assessed in rats fed specific doses of p,p'-DDT for 2 weeks, and the effect of lipopolysaccharide on anemia of inflammation was also examined in p,p'-DDT-treated rats. The parameters included the content of hemoglobin per reticulocyte, mean corpuscular volume of reticulocytes and mature erythrocytes, corpuscular hemoglobin concentration mean of mature erythrocytes, and saturation levels of transferrin and iron. During the 2-week treatment period, hypochromic microcytic reticulocytes and hypochromic normocytic mature erythrocytes were observed in p,p'-DDT-treated rats, with no evidence of alteration in plasma iron levels. p,p'-DDT enhanced microcytosis of reticulocytes, as well as mature erythrocytes, which occurred due to severe hypoferremia resulting from anemia of inflammation; however, plasma iron levels were attenuated probably through the inhibition of interleukin-6. Our data suggests that long-term treatment with p,p'-DDT induces microcytic anemia, possibly because of the impairment of iron utility in erythrocytes.

  14. Globin chain synthesis ratios in sideroblastic anaemia.

    PubMed

    Peters, R E; May, A; Jacobs, A

    1983-02-01

    Globin synthesis ratios were measured on reticulocytes from nine patients with primary acquired sideroblastic anaemia (SA), four patients with hereditary or congenital SA, two patients with secondary acquired SA and three patients with iron deficiency (ID). Ten of the samples from patients with SA and all the samples from patients with ID had normal ratios. Samples from three patients had significantly abnormal ratios, one from a patient with SA and acquired Hb H disease (alpha/beta 0 X 26), one from a patient with secondary acquired SA (alpha/beta 0 X 88), and one from a patient who went on to develop acute myeloblastic leukaemia (alpha/beta 1 X 36). Globin synthesis was stimulated by 100 microM haem similarly in normal, SA and ID reticulocytes. Any limitation of globin synthesis in SA and ID is therefore not easily reversible by adding haem. Inhibition of haem synthesis in nonsideroblastic reticulocytes using 4 mM isonicotinic acid hydrazide for 1 h incubation affected neither total globin synthesis nor the alpha/beta ratio. These results contradict the view that decreased haem synthesis decreases globin chain synthesis and decreases the alpha/beta globin chain synthesis ratios in human reticulocytes. Previously reported findings that haem could reverse globin chain synthesis inhibition in SA were good evidence for a primary deficiency of haem synthesis in the erythroblasts of these patients. Our inability to substantiate these findings emphasizes the need for a re-evaluation of the aetiology of sideroblastic anaemia.

  15. Biochemical Comparisons of Two-Hour Exposures to Hydrogen-Oxygen, Helium-Oxygen and Nitrogen-Oxygen Atmospheres at 200 FSWG

    DTIC Science & Technology

    1982-09-23

    chemistry. *: PLAT = platelets, 103/mm3; HGBN = hemoglobin, g/dl; HCRT = hemato - crit, %; RBCS = red blood ceUs, 10°/mmm3; RTIC = reticulocytes, % RBCS...avoidance ses- sions in the monkey. Psychom Med 40: 710-720. 32. Melton, C.E., J.M. McKenzie , J. T. Salvidar, Jr., and S.M. Hoffman. 1974. A

  16. 40 CFR 799.9310 - TSCA 90-day oral toxicity in rodents.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ..., respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects... (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea... hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other...

  17. 40 CFR 799.9310 - TSCA 90-day oral toxicity in rodents.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ..., respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects... (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea... hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other...

  18. 40 CFR 799.9310 - TSCA 90-day oral toxicity in rodents.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ..., respiratory and circulatory effects, autonomic effects such as salivation, central nervous system effects... (retina, optic nerve). (C) Glandular system—adrenals, parathyroid, thyroid. (D) Respiratory system—trachea... hematopoietic system, reticulocyte counts and bone marrow cytology may be indicated. (D) Other...

  19. Predicting erythroid response to recombinant erythropoietin plus granulocyte colony-stimulating factor therapy following a single subcutaneous bolus in patients with myelodysplasia.

    PubMed

    Bowen, David; Hyslop, Ann; Keenan, Norene; Groves, Michael; Culligan, Dominic; Johnson, Peter; Shaw, Ann; Geddes, Fiona; Evans, Patricia; Porter, John; Cavill, Ivor

    2006-05-01

    We randomized 21 patients with low-risk myelodysplastic syndromes (MDS) to receive a single subcutaneous bolus of recombinant erythropoietin (epoietin) +/- granulocyte-colony stimulating factor (G-CSF), or placebo and monitored erythropoietic response over 7 days. In this small study, the reticulocyte response at day 7 was highly predictive of subsequent response to a therapeutic trial of epoietin + G-CSF.

  20. [Hemopoietic response to americium and plutonium exposure in nuclear industry workers].

    PubMed

    Gasteva, G N; Ivanova, T A; Gordeeva, A A; Suvorova, L A; Molokanov, A A; Bad'in, V I; Kasymova, O A; Mezhakova, E V

    2004-01-01

    The examinees demonstrated definite effects caused by total influence of Am-241 and Pu-239: chronic radiation sickness with diffuse pneumosclerosis, chronic toxic radiation bronchitis and peripheral blood changes (reactive alterations and stable lower platelets and reticulocytes count), microfocal hypoplasia, osteodysplasia, reactive hepatopathy. Frequency of these effects increased with higher radiation exposure and did not depend on the age.

  1. Department of Clinical Investigation, Annual Research Progress Report, Fiscal Year 2006 (Madigan Army Medical Center)

    DTIC Science & Technology

    2006-09-30

    Findings in Bariatric Surgery Specimens DEPARTMENT OF PEDIATRICS Cason RL #203014 T L Clinical Use of Reticulocyte Hemoglobin to Detect Iron...Status: Ongoing Title: Incidental Anatomic and Histologic Findings in Bariatric Surgery Specimens Principal Investigator: MAJ Anne L. Champeaux, MC...Collection, review and analysis of bariatric surgery specimen reports generated by the MAMC Anatomic Pathology service from 1994 through 2004 to

  2. Mouse Pig-a and micronucleus assays respond to N-ethyl-N-nitrosourea, benzo[a]pyrene, and ethyl carbamate, but not pyrene or methyl carbamate.

    PubMed

    Labash, Carson; Avlasevich, Svetlana L; Carlson, Kristine; Berg, Ariel; Torous, Dorothea K; Bryce, Steven M; Bemis, Jeffrey C; MacGregor, James T; Dertinger, Stephen D

    2016-01-01

    This laboratory previously described a method for scoring the incidence of peripheral blood Pig-a mutant phenotype rat erythrocytes using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). The current work extends the method to mouse blood, using the frequency of CD24-negative reticulocytes (RET(CD24-)) and erythrocytes (RBC(CD24-)) as phenotypic reporters of Pig-a gene mutation. Following assay optimization, reconstruction experiments demonstrated the ability of the methodology to return expected values. Subsequently, the responsiveness of the assay to the genotoxic carcinogens N-ethyl-N-nitrosourea, benzo[a]pyrene, and ethyl carbamate was studied in male CD-1 mice exposed for 3 days to several dose levels via oral gavage. Blood samples were collected on Day 4 for micronucleated reticulocyte analyses, and on Days 15 and 30 for determination of RET(CD24-) and RBC(CD24-) frequencies. The same design was used to study pyrene, with benzo[a]pyrene as a concurrent positive control, and methyl carbamate, with ethyl carbamate as a concurrent positive control. The three genotoxicants produced marked dose-related increases in the frequencies of Pig-a mutant phenotype cells and micronucleated reticulocytes. Ethyl carbamate exposure resulted in moderately higher micronucleated reticulocyte frequencies relative to N-ethyl-N-nitrosourea or benzo[a]pyrene (mean ± SEM = 3.0 ± 0.36, 2.3 ± 0.17, and 2.3 ± 0.49%, respectively, vs. an aggregate vehicle control frequency of 0.18 ± 0.01%). However, it was considerably less effective at inducing Pig-a mutant cells (e.g., Day 15 mean no. RET(CD24-) per 1 million reticulocytes = 7.6 ± 3, 150 ± 9, and 152 ± 43 × 10(-6), respectively, vs. an aggregate vehicle control frequency of 0.6 ± 0.13 × 10(-6)). Pyrene and methyl carbamate, tested to maximum tolerated dose or limit dose levels, had no effect on mutant cell or micronucleated reticulocyte frequencies. Collectively, these results

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

    PubMed

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

    2007-02-01

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

  4. Artesunate and artelinic acid: association of embryotoxicity, reticulocytopenia, and delayed stimulation of hematopoiesis in pregnant rats.

    PubMed

    Clark, Robert L; Brannen, Kimberly C; Sanders, James E; Hoberman, Alan M

    2011-02-01

    The artemisinin antimalarials cause embryo death and malformations in animals by killing embryonic erythroblasts. Groups of pregnant rats (N = 4) were administered 35 and 48 µmol/kg artesunate and 17.2, 28.7, 48, 96, and 191 µmol/kg artelinic acid as a single oral dose on gestational day (GD) 12. Litters were examined on GD21. The ED(50) for embryo death with artelinic acid (23.4 µmol/kg) was just slightly lower than that for decreased reticulocyte count at 24 hr postdose (33.5 µmol/kg) and both had similarly steep dose responses (maximal effects of total litter loss and ∼60% decreases in reticulocyte count at 48 µmol/kg). Results with artesunate were similar. The correlation coefficient between embryo death and decreased reticulocyte count was 0.82 (p<0.01). The close relationship between embryotoxicity and reticulocytopenia is suggestive of a common mechanism-artemisinin-induced mitochondrial damage leading to cell death. At 9 days postdose, treatment with artesunate and artelinic acid also caused increases in counts of reticulocytes, lymphocytes, basophils, and monocytes (up to 3.7 ×, 1.7 ×, 4.7 ×, and 1.7 × control, respectively). This stimulation of hematopoiesis may have been mediated by the direct oxidative conversion of artesunate or artelinic acid to the artemisininyl hydroperoxide within the bone marrow cells or by an indirect increase in reactive oxygen species. The high correlation between embryotoxicity and reticulocytopenia further supports the assertion that therapeutic dosage regimens of artemisinins that cause decreases in reticulocyte count in pregnant women during the putative critical period (approximately postconception wk 3 to 9) are at risk of also causing adverse effects on the embryo.

  5. Effects of thyroid status on glycated hemoglobin

    PubMed Central

    Bhattacharjee, Rana; Thukral, Anubhav; Chakraborty, Partha Pratim; Roy, Ajitesh; Goswami, Soumik; Ghosh, Sujoy; Mukhopadhyay, Pradip; Mukhopadhyay, Satinath; Chowdhury, Subhankar

    2017-01-01

    Introduction: Glycated hemoglobin (HbA1c) can be altered in different conditions. We hypothesize that HbA1c levels may change due to altered thyroid status, possibly due to changes in red blood cell (RBC) turnover. Objectives: The objective of this study was to determine the effects of altered thyroid status on HbA1c levels in individuals without diabetes, with overt hyper- and hypo-thyroidism, and if present, whether such changes in HbA1c are reversed after achieving euthyroid state. Methods: Euglycemic individuals with overt hypo- or hyper-thyroidism were selected. Age- and sex-matched controls were recruited. Baseline HbA1c and reticulocyte counts (for estimation of RBC turnover) were estimated in all the patients and compared. Thereafter, stable euthyroidism was achieved in a randomly selected subgroup and HbA1c and reticulocyte count was reassessed. HbA1c values and reticulocyte counts were compared with baseline in both the groups. Results: Hb A1c in patients initially selected was found to be significantly higher in hypothyroid group. HbA1c values in hyperthyroid patients were not significantly different from controls. HbA1c reduction and rise in reticulocyte count were significant in hypothyroid group following treatment without significant change in glucose level. Hb A1c did not change significantly following treatment in hyperthyroid group. The reticulocyte count, however, decreased significantly. Conclusion: Baseline HbA1c levels were found to be significantly higher in hypothyroid patients, which reduced significantly after achievement of euthyroidism without any change in glucose levels. Significant baseline or posttreatment change was not observed in hyperthyroid patients. Our study suggests that we should be cautious while interpreting HbA1c data in patients with hypothyroidism. PMID:28217494

  6. The effects of superoxide dismutase knockout on the oxidative stress parameters and survival of mouse erythrocytes.

    PubMed

    Grzelak, Agnieszka; Kruszewski, Marcin; Macierzyńska, Ewa; Piotrowski, Łukasz; Pułaski, Łukasz; Rychlik, Błazej; Bartosz, Grzegorz

    2009-01-01

    The erythrocytes of 12-month old Sod1 (-/-) mice showed an increased level of reactive oxygen species (ROS), as estimated by the degree of dihydroethidine and dihydrorhodamine oxidation, and the increased level of Heinz bodies. No indices of severe oxidative stress were found in the red blood cells and blood plasma of Sod1 (-/-) mice as judged from the lack of significant changes in the levels of erythrocyte and plasma glutathione, plasma protein thiol and carbonyl groups and thiobarbituric-acid reactive substances in the blood plasma. However, a decreased erythrocyte lifespan, increased reticulocyte count and splenomegaly were noted, indicating the importance of superoxide dismutase for maintaining erythrocyte viability. The levels of erythrocyte ROS and Heinz bodies and the reticulocyte count were indistinguishable in Sod1 (+/+) and Sod1 (+/-) mice, suggesting that a superoxide dismutase activity decrease to half of its normal value may be sufficient to secure the protective effects of the enzyme.

  7. Dianthin 30 and 32 from Dianthus caryophyllus: two inhibitors of plant protein synthesis and their tissue distribution.

    PubMed

    Reisbig, R R; Bruland, O

    1983-07-15

    The ability of dianthin 30 and 32 to inhibit translation in reticulocyte lysates and wheat germ extracts has been studied. The dianthins, like the A chains of the toxins abrin and ricin, inhibited protein synthesis in reticulocyte lysates by inactivating the 60S ribosomal subunit. They also inhibited, at concentrations of 10 ng/ml, a protein-synthesizing system from wheat germ and inactivated isolated wheat germ ribosomes. The concentration of the dianthins in different tissues of the plant was determined by rocket immunoelectrophoresis and by the dianthin's ability to inhibit protein synthesis. Dianthin 32 was found only in the leaves and in growing shoots, while dianthin 30 was present throughout the plant. In the older parts of the plant, the dianthins constituted between 1 and 3% of the total extractable protein whereas much less was found in the younger parts.

  8. Evaluation of a novel haematology analyser for use with feline blood.

    PubMed

    Weissenbacher, S; Riond, B; Hofmann-Lehmann, R; Lutz, H

    2011-03-01

    A novel haematology analyser was evaluated for its use with feline samples. Complete blood cell counts, a five-part differential count, and reticulocyte numbers were determined, and the results compared with reference data. Coefficients of correlation, Passing-Bablok regression analysis and Bland-Altmann difference plots with biases and 95% limits of agreement are reported. Precision and linearity were also studied. The instrument demonstrated very low imprecision, and the tested range of linearity exceeded the reference ranges provided by the manufacturer. For all parameters except monocytes (r = 0.65), the analyser results correlated well with reference methods. Compared with the manual count of aggregated reticulocytes, the instrument showed good agreement with a positive bias. The optical platelet count correlated well with the manual chamber count. In conclusion the analyser was found to be highly reliable for the analysis of feline blood samples in a large veterinary laboratory.

  9. Conjugated Bilirubin Triggers Anemia by Inducing Erythrocyte Death

    PubMed Central

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

    2015-01-01

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

  10. Evaluation of macrocytosis.

    PubMed

    Kaferle, Joyce; Strzoda, Cheryl E

    2009-02-01

    Macrocytosis, generally defined as a mean corpuscular volume greater than 100 fL, is frequently encountered when a complete blood count is performed. The most common etiologies are alcoholism, vitamin B12 and folate deficiencies, and medications. History and physical examination, vitamin B12 level, reticulocyte count, and a peripheral smear are helpful in delineating the underlying cause of macrocytosis. When the peripheral smear indicates megaloblastic anemia (demonstrated by macro-ovalocytes and hyper-segmented neutrophils), vitamin B12 or folate deficiency is the most likely cause. When the peripheral smear is non-megaloblastic, the reticulocyte count helps differentiate between drug or alcohol toxicity and hemolysis or hemorrhage. Of other possible etiologies, hypothyroidism, liver disease, and primary bone marrow dysplasias (including myelodysplasia and myeloproliferative disorders) are some of the more common causes.

  11. [Seasonal changes in mechanical resistance of erythrocytes of the long-tailed ground squirrel (Citellus undulatus)].

    PubMed

    Gulevsky, A K; Shchenyavsky, I I

    2014-01-01

    Seasonal changes of relative blood reticulocyte counts, free plasma hemoglobin and mechanical erythrocyte resistance were studied in the long-tailed ground squirrel (Citellus undulatus), under different functional conditions (winter hibernation, forced arousal in winter, and summer activity). A significant increase in reticulocyte counts in the ground squirrel blood was observed in April and October, indicating enhancement of erythropoeisis. This conclusion is also confirmed by a significant increase in free plasma hemoglobin at these periods. It has been also shown that erythrocytes of hibernating and forcibly awaken winter ground squirrels have a significantly lower mechanical resistance in comparison with those of active summer animals. The obtained data indicate that during the periods preceding the onset of winter hibernation and transition to summer activity there occurs a seasonal replacement of the erythrocyte pool by a pool more adapted to performance of functions in the novel functional state of the animal-hibernator.

  12. The sorting of blood group active proteins during enucleation.

    PubMed

    Satchwell, Timothy J; Bell, Amanda J; Toye, Ashley M

    2015-04-01

    Enucleation represents the critical stage during red blood cell development when the nucleus is extruded from an orthochromatic erythroblast in order to generate a nascent immature reticulocyte. Extrusion of the nucleus results in loss of a proportion of the erythroblast plasma membrane, which surrounds the nucleus, the bulk of the endoplasmic reticulum and a small region of cytoplasm. For this reason enucleation provides an important point in erythroblast differentiation at which proteins not required for the function of the erythrocyte can be lost, whilst those that are important for the structure-function properties of the mature erythrocyte must be efficiently retained in the reticulocyte plasma membrane. Disturbances in protein distribution during enucleation are envisaged to occur during human diseases such as Hereditary Spherocytosis. This article will discuss the current knowledge of erythroblast enucleation in the context of retention and loss of proteins that display antigenic blood group sites and that exist within multiprotein complexes within the erythrocyte membrane.

  13. [Risk factors for children's population health in stressed environmental conditions of lead pollition].

    PubMed

    Baidaulet, I O; Namazbaeva, Z I; Dasybayeva, G N; Bazeluk, L T; Sabirov, Zh V; Kusainova, D S

    2013-01-01

    Adverse environmental conditions in Shymkent significantly increase the risk of accumulation of lead in the bodies of the children of the third generation of the population residing in the contaminated areas, cause deteriorations of antioxidant defense in the respiratory system, greatly decline barrier-protective properties of cellular systems of the local immunity, disturb the process of hematopoiesis. Performed statistical analysis of the data permitted to identify a correlation relationship between the accumulation of lead in the soil and the change in the functional activity of the cells of buccal cheek epithelium, catalase activity in expired breath condensate. Haematological signs of lead poisoning include not only the number of reticulocytes, but also the correction (RPI) for the alteration with allowances made for the maturation of reticulocytes in peripheral blood circulation as early criterion for toxic anemia.

  14. Critical role for NAD glycohydrolase in regulation of erythropoiesis by hematopoietic stem cells through control of intracellular NAD content.

    PubMed

    Nam, Tae-Sik; Park, Kwang-Hyun; Shawl, Asif Iqbal; Kim, Byung-Ju; Han, Myung-Kwan; Kim, Youngho; Moss, Joel; Kim, Uh-Hyun

    2014-06-06

    NAD glycohydrolases (NADases) catalyze the hydrolysis of NAD to ADP-ribose and nicotinamide. Although many members of the NADase family, including ADP-ribosyltransferases, have been cloned and characterized, the structure and function of NADases with pure hydrolytic activity remain to be elucidated. Here, we report the structural and functional characterization of a novel NADase from rabbit reticulocytes. The novel NADase is a glycosylated, glycosylphosphatidylinositol-anchored cell surface protein exclusively expressed in reticulocytes. shRNA-mediated knockdown of the NADase in bone marrow cells resulted in a reduction of erythroid colony formation and an increase in NAD level. Furthermore, treatment of bone marrow cells with NAD, nicotinamide, or nicotinamide riboside, which induce an increase in NAD content, resulted in a significant decrease in erythroid progenitors. These results indicate that the novel NADase may play a critical role in regulating erythropoiesis of hematopoietic stem cells by modulating intracellular NAD.

  15. Role of Myelofibrosis in Hematotoxicity of Munition RDX Environmental Degradation Product MNX

    DTIC Science & Technology

    2008-09-01

    consequence of anemia in the absence of sufficient regeneration from bone marrow erythroid precursors is recruitment of extramedullatory sites of...Another event associated with a successful regeneration of an anemic episode is increased reticulocytes in the peripheral blood. We quantitated...planned. Statistics: Effects of MNX and RDX on endpoints with normally distributed data ( EPO , BUN, liver weights) were determined by ANOVA with post

  16. [Research proceeding on the effect of microgravity on erythropoiesis].

    PubMed

    Dong, Q; Shen, X Y

    2001-08-01

    The reduction of red blood cell mass is a main change of hematology under microgravity. It is considered that the decrease of erythroid cell production is the key cause. The studies on reticulocytes, Price-Jones curve, ferrum turn over, cytology of bone marrow and Erythropoietin (Epo) level suggested the suppression of erythropoiesis was due to microgravity. The mechanisms about it and the aspects of research in the future were discussed briefly.

  17. Rice embryos can express heat-shock genes under anoxia.

    PubMed

    Mocquot, B; Ricard, B; Pradet, A

    1987-01-01

    Heat-shock proteins (hsps) are induced by a number of oxidative stresses. The proposal that the reduction products of oxygen initiate hsp induction was tested in rice embryos, capable of coleoptile growth under oxygen-free conditions. In such embryos, hsps could be detected by both in vivo labeling and in vitro translation of RNA using the reticulocyte lysate system. It is therefore improbable that the mechanism for hsp induction involves oxygen.

  18. Antisense Oligodeoxynucleotide Inhibition of HIV Gene Expression

    DTIC Science & Technology

    1989-03-20

    of HtL -60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examined and compared with Me2 SO. Differentiation of HL-60...reticulocyte eIF4B binds specifically to AUG (36). Both eIF4F, which includes eIF4A, eIF4E, and a 220 kD subunit, and eIF4B may be crosslinked to the

  19. Thirteen Week Oral Toxicity Study of WR242511 in Rats. Volume 1

    DTIC Science & Technology

    1994-01-14

    Nucleated RBCs Platelet count Reticulocyte count ’Measured with a Co-oximeter (Instrumentation Laboratory Model 282). The assay was performed...Part I: Part II: Part III: Part IV: Identity and Purity Study of WR242511 Assay Precision and Accuracy for the Quantitation of WR2425U Stability...standard deviation - 0.02%, terminal 99.59% ± 0.02%. The assay results are presented in Tables 1 and 2. FIGURE 1 MASS SPECTRUM OF WR2425U SAMPLE 8

  20. Problem Definition Study: Lead Beta-Resorcylate

    DTIC Science & Technology

    1979-02-01

    Preteperphyrtn IX (PROTO 9) • Hamt tynthttaw Ft T Mama 7Pb«—e* Iron utilization Fa transferrin (»arum) Into reticulocytes Pb Fb GlObin...in their study of tumor induction by lead acetate. 4. Teratogenicty and Mutagenicity Several studies have been conducted on the teratogenicity of... tumors in experimental animals has been reported by several authors (Van Each et al.t 1962; Van Esch and Kroes, 1969; Waszynski, 1977). In their

  1. Hemolytic disease of the fetus and newborn with late-onset anemia due to anti-M: a case report and review of the Japanese literature.

    PubMed

    Yasuda, Hiroyasu; Ohto, Hitoshi; Nollet, Kenneth E; Kawabata, Kinuyo; Saito, Shunnichi; Yagi, Yoshihito; Negishi, Yutaka; Ishida, Atsushi

    2014-01-01

    Hemolytic disease of the fetus and newborn (HDFN) attributed to M/N-incompatibility varies from asymptomatic to lethally hydropic. Case reports are rare, and the clinical significance of anti-M is not completely understood. A challenging case of HDFN due to anti-M prompted an investigation of the Japanese literature, in order to characterize the clinical spectrum of M/N-incompatibility pregnancies in Japan and report results to English-language readers. Japanese reports of HDFN attributed to M/N incompatibility were compiled. Abstracted data include maternal antibody titers at delivery, fetal direct antiglobulin test, hemoglobin, total bilirubin, reticulocyte count at birth, and therapeutic interventions. We investigated characteristics of HDFN due to M/N-incompatible pregnancies in Japan after encountering a case of severe HDFN along with late-onset anemia in an infant born to a woman carrying IgG anti-M with a titer of 1. In total, thirty-three babies with HDFN due to anti-M and one due to anti-N have been reported in Japan since 1975. The median maternal antibody titer was 64 at delivery and was 16 or less in 10 of 34 women (29%). Five of 34 babies (15%) were stillborn or died as neonates. Twenty-one of 29 survivors (72%) had severe hemolytic anemia and/or hydrops fetalis. The reticulocyte count of neonates with anemia stayed below the reference interval. Sixteen (55%) developed late-onset anemia and 14 (48%) were transfused with M-negative RBCs. Significant positive correlation (P < .05) between the hemoglobin value and the reticulocyte count within 4 days of birth was obtained in 16 babies with anti-M HDFN. In the Japanese population, 21 of 34 cases of M/N-incompatible HDFN (72%) have manifested as severe hemolytic anemia and/or hydrops fetalis. Low reticulocyte count in neonates with late-onset anemia is consistent with suppressed erythropoiesis due to anti-M.

  2. Analytic errors in Sysmex-generated hematology results in blood from a dog with chronic lymphocytic leukemia.

    PubMed

    Novacco, Marilisa; Martini, Valeria; Grande, Carmen; Comazzi, Stefano

    2015-09-01

    A blood sample from a 14-year-old dog was submitted to the veterinary diagnostic laboratory of the University of Milan for marked leukocytosis with atypical cells. A diagnosis of chronic T-cell lymphocytic leukemia (CLL) was made based on blood smear evaluation and flow cytometric phenotyping. A CBC by Sysmex XT-2000iV revealed a moderate normocytic normochromic anemia. Red blood cells counted by optic flow cytometry (RBC-O) resulted in a higher value than using electrical impedance (RBC-I). The relative reticulocyte count based on RNA content and size was 35.3%, while the manual reticulocyte count was < 1%. The WBC count of 1,562,680 cells/μL was accompanied by a flag. Manual counts for RBC and WBC using the Bürker chamber confirmed the Sysmex impedance results. Finally the manual PCV was lower than HCT by Sysmex. While Sysmex XT can differentiate between RBC and WBC by impedance, even in the face of extreme lymphocytosis due to CLL, RBC-O can be affected by bias, resulting in falsely increased RBC and reticulocyte numbers. Overestimation of RBC-O may be due to incorrect Sysmex classification of leukemic cells or their fragments as reticulocytes. This phenomenon is known as pseudoreticulocytosis and can lead to misinterpretation of regenerative anemia. On the other side PCV can be affected by bias in CLL due to the trapping of RBC in the buffy coat, resulting in a pink hue in the separation area. As HGB concentration is not affected by flow cytometric or other cell-related artifacts it may represent the most reliable variable to assess the degree of anemia in cases of CLL.

  3. In vivo lability of glucose-6-phosphate dehydrogenase in GdA- and Gdmediterranean deficiency

    PubMed Central

    Piomelli, Sergio; Corash, Laurence M.; Davenport, Deatra D.; Miraglia, Janet; Amorosi, Edward L.

    1968-01-01

    A decreased level of glucose-6-phosphate dehydrogenase might result from decreased rate of synthesis, synthesis of an enzyme of lower catalytic efficiency, increased lability, or a combined mechanism. To test the hypothesis of increased lability, the rate of decline of the enzyme in vivo was measured in three groups of individuals, controls, Gd(—),A-males, and Gd(—), Mediterranean males, by the slope of decline of activity in fractions containing erythrocytes of progressively increasing mean age. These fractions were obtained by ultracentrifugation on a discontinuous density gradient of erythrocyte suspensions free of contaminating platelets and leukocytes. The rate of in vivo decline of pyruvate kinase (another age-dependent enzyme) was also measured and found very similar in the three groups. The in vivo decline of glucose-6-phosphate dehydrogenase was found to follow an exponential rate, with a half-life of 62 days for controls and 13 days for Gd(—),A- erythrocytes. The activity in normal reticulocytes was estimated at 9.7 U and in Gd(—),A- reticulocytes at 8.8 U. These estimates were confirmed by direct measurements in reticulocytes isolated from patients with extreme reticulocytosis. In Gd(—),Mediterranean erythrocytes activity could be demonstrated only in reticulocytes, which were estimated to average 1.4 U. The rate of decline is so extreme that no activity could be detected in mature erythrocytes. These data suggest that the glucose-6-phosphate dehydrogenase deficiency of both the GdA- and the GdMediterranean variant results from different degrees of in vivo instability of the abnormal enzyme. PMID:5641629

  4. Induction of hyperchromic microcytic anaemia by repeated oral administration of methotrexate in rats.

    PubMed

    Kojima, Sayuri; Yoshida, Toshinori; Sasaki, Junya; Takahashi, Naofumi; Kuwahara, Maki; Shutoh, Yasufumi; Saka, Machiko; Nakashima, Nobuaki; Kosaka, Tadashi; Harada, Takanori

    2012-01-01

    Anaemia is a significant prognostic factor in cancer patients receiving anticancer drugs such as methotrexate (MTX). This study focuses on the effects of toxicological changes on the hematopoietic systems in male and female Wistar Hannover rats when MTX is orally administered at a dose of 0, 0.05, 0.15, or 0.45 mg/(kg·day) for a period of 28 days. Both male and female rats receiving 0.45 mg/kg MTX showed a decrease in the haemoglobin concentration (Hb), haematocrit, and erythrocyte count. Female rats showed a decrease in mean corpuscular volume (MCV) and an increase in cell mean Hb (CHCM) in total erythrocytes, including the mature erythrocytes. These results indicate that MTX causes the production of small, mature erythrocytes that contain a high concentration of Hb. MTX reduced the number of peripheral reticulocytes but produced the cells with a large size and a high concentration of Hb, as demonstrated by the reticulocyte MCV and CHCM as well as the content of haemoglobin per reticulocyte (CHr). Consistent with these findings, bone marrow haematopoiesis was impaired by MTX, as there was a reduction in erythroid count in rats of both sexes. The number of cells of the myeloid lineage reduced in female rats, followed by a reduction in the total leukocyte and neutrophil counts in peripheral blood. Thrombocytopenia was detected in a small population of rats. These results indicate that MTX induces hyperchromic microcytic anaemia and pancytopenia, and the use of MCV and CHCM in mature erythrocytes and reticulocytes, along with the CHr, gives a better understanding of the development and nature of anaemia.

  5. Thirteen Week Oral (Diet) Toxicity Study of Trinitrotoluene (TNT), Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) and TNT/RDX Mixtures in the Fischer 344 Rat

    DTIC Science & Technology

    1981-09-01

    differential Mean corpuscular hemoglobin (MCH) Reticulocyte count Mean corpuscular hemoglobin concentration (MCHC) Methemoglobin lIT RIESEARCH INSTITUTE 15 L...the deqenerative tubule. The SertolV cells and spermatogonia appeared to lie unaffected. Lesions graded as marked in severity consisted of atrophic...sepiiniferou. tubules lined with a few Sertoli cells and spermatogonia . Diffuse intertubu’lar edema was also observed in the testes of these animals. In

  6. Erythropoietic drive is the strongest predictor of hepcidin level in adults with sickle cell disease.

    PubMed

    Karafin, Matthew S; Koch, Kathryn L; Rankin, Amy B; Nischik, Debora; Rahhal, Ghady; Simpson, Pippa; Field, Joshua J

    2015-12-01

    Levels of hepcidin, a key modulator of iron metabolism, are influenced by erythropoiesis, iron, and inflammation, all of which may be increased in patients with sickle cell disease (SCD). The objectives of this study were to determine: 1) the variation in hepcidin level, and 2) the relative contribution of erythropoietic drive, iron, and inflammation to differences in hepcidin level in an adult cohort with SCD. In a prospective study, cross-sectional measurements of hepcidin, reticulocyte percentage, erythropoietin, ferritin, and high-sensitivity CRP were obtained. A regression tree analysis was used to measure the association between these interacting factors and hepcidin level. The cohort was comprised of 40 adults with SCD. Median age was 26years, 68% were female, and all had HbSS. Hepcidin values ranged from 30ng/ml to 326ng/ml, with a median of 87ng/ml. Regression tree analysis demonstrated that reticulocyte percentage, erythropoietin, ferritin and hs-CRP all were associated with hepcidin. The highest hepcidin values were found in subjects with low reticulocyte percentage and erythropoietin. In conclusion, erythropoietic drive, iron status, and inflammation all contribute to variation in hepcidin level. The strongest contributor is erythropoietic drive. Future studies could determine whether suppression of erythropoiesis with chronic transfusion influences hepcidin level.

  7. An Electrophoretically Silent Polymorphism for the Beta Chains of Rabbit Hemoglobin and Associated Polyribosome Patterns

    PubMed Central

    Garrick, Michael D.; Bricker, J.; Garrick, Laura M.

    1974-01-01

    The β chain of rabbit (Oryctolagus caniculus) hemoglobin has previously been reported to contain a single residue of isoleucine at β112. We have detected other rabbits with either zero isoleucyl residues or half a residue per β chain. This character is polymorphic and inherited as a simple mendelian autosomal codominant.—Normally the modal number of ribosomes per polyribosome is 4 to 6 in reticulocyte lysates; but incubation of rabbit reticulocytes prior to lysis with L-o-methylthreonine (OMT), an isostere of isoleucine, leads to a bimodal distribution in lysates with 2–3 and 8–12 ribosomes as modes. This alteration has been attributed to ribosomal traffic jams caused by starvation for ile-tRNA at mRNA codons corresponding to the locations of isoleucyl residues at positions α10, α17, α55 and β112. We have confirmed this interpretation by incubating OMT with reticulocytes from rabbits with integral, half integral and nil values for isoleucyl residues per β chain to show that formation of the larger clusters of polyribosomes requires that β112 = ile. PMID:4818267

  8. Patients with sickle cell disease taking hydroxyurea in the Hemocentro Regional de Montes Claros

    PubMed Central

    Santos, Fernanda Kelle de Souza; Maia, Caroline Nogueira

    2011-01-01

    Background The development of therapies for sickle cell disease has received special attention, particularly those that reduce the polymerization of hemoglobin S. Hydroxyurea is a commonly used medication because it has the ability to raise levels of fetal hemoglobin, decrease the frequency of vaso-occlusive episodes and thus improve the clinical course of sickle cell disease patients. Objective To study hematological data and the clinical profile of sickle cell disease patients taking hydroxyurea in a regional blood center. Methods From the charts of 20 patients with sickle cell anemia, the clinical outcomes and a number of hematological variables were analyzed before and during treatment with hydroxyurea. Results The patients' ages ranged from 6 to 41 years old, most were dark skinned and there was a predominance of women. The main symptom that defined whether patients were prescribed hydroxyurea was painful crises followed by hospitalizations. During treatment with hydroxyurea there were significant increases in hemoglobin, fetal hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin. The reticulocyte and white blood cell counts dropped significantly with treatment. A positive correlation was found between fetal hemoglobin and mean corpuscular volume before and during treatment. Additionally, a correlation was found between the white blood cell and reticulocyte counts before treatment with hydroxyurea. Conclusion Most patients showed improvements with treatment as demonstrated by increases in hemoglobin, fetal hemoglobin and mean corpuscular volume, as well as by reductions in the reticulocyte and white blood cell counts. Clinically, more than 50% of patients had a significant reduction of events. PMID:23284256

  9. Selection of AUG initiation codons differs in plants and animals.

    PubMed Central

    Lütcke, H A; Chow, K C; Mickel, F S; Moss, K A; Kern, H F; Scheele, G A

    1987-01-01

    The influence of the nucleotide at position -3 relative to the AUG initiation codon on the initiation of protein synthesis was studied in two different in vitro translation systems using synthetic mRNAs. The four mRNAs, transcribed from cDNAs directed by an SP6 promoter, were identical except for mutations at nucleotide -3. In each case, translation of mRNAs produced a single protein of Mr = 12,600. Relative translational efficiencies showed a hierarchy in the reticulocyte lysate system (100, 85, 61 and 38% for A, G, U and C in position -3, respectively) but no differences in the wheat germ system. Differential mRNA degradation or polypeptide chain elongation were excluded as causes of the differences observed in translation in the reticulocyte lysate. mRNA competition increased the differences observed in translational efficiencies in reticulocyte lysate but showed no effect in wheat germ. Analysis of 61 plant and 209 animal mRNA sequences revealed qualitative and quantitative differences between the consensus sequences surrounding AUG initiation codons. Whereas the consensus sequence for animals was CACCAUG that for plants was AACAAUGGC. Both the structural and functional findings suggest that the factors which select AUG initiation codons in plants and animals differ significantly. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3556162

  10. Complementary markers for the clinical severity classification of hereditary spherocytosis in unsplenectomized patients.

    PubMed

    Rocha, Susana; Costa, Elísio; Rocha-Pereira, Petronila; Ferreira, Fátima; Cleto, Esmeralda; Barbot, José; Quintanilha, Alexandre; Belo, Luís; Santos-Silva, Alice

    2011-02-15

    Hereditary spherocytosis (HS) is usually classified as mild, moderate or severe using conventional features, namely, hemoglobin (Hb) concentration, reticulocyte count and bilirubin levels, which do not always contribute to an adequate clinical classification. The aim of our study was to establish the importance of some laboratory routine parameters, as markers of HS clinical outcome, by studying a control group (n=26) and unsplenectomized HS patients (n=82) presenting mild, moderate or severe HS. We performed a basic hematologic study and evaluated the reticulocyte count, bilirubin, erythropoietin (EPO) and soluble transferrin receptor (sTfR) levels; the osmotic fragility (OFT) and criohemolysis tests (CHT); the ratios Hb/MCHC (mean cell hemoglobin concentration), Hb/RDW (red cell distribution width) and MCHC/RDW, were calculated. Hb changed significantly in accordance with HS severity, but not reticulocytes or bilirubin. We found that MCHC, RDW, EPO, sTfR, OFT, CHT and the calculated ratios were significantly changed in patients, and, therefore, were valuable as complementary diagnostic tools for HS. Moreover, RDW, Hb/MCHC, Hb/RDW and MCHC/RDW changed significantly with worsening of HS; thus, they are also good markers for the clinical outcome of HS. In conclusion, we propose the use of these routine parameters as useful to complement the analysis of HS severity.

  11. Elevated erythrocyte adenosine deaminase activity in a patient with primary acquired sideroblastic anemia.

    PubMed

    Kanno, H; Fujii, H; Tani, K; Morisaki, T; Takahashi, K; Horiuchi, N; Kizaki, M; Ogawa, T; Miwa, S

    1988-03-01

    We report a case of primary acquired sideroblastic anemia (PASA) associated with elevated erythrocyte adenosine deaminase (ADA) activity. The patient was an 85-year-old Japanese male. Analysis of the peripheral blood revealed pancytopenia, and the bone marrow findings showed marked ringed sideroblasts and chromosomal deletion (46XY, 11q-). The erythrocyte ADA activity was 17 times higher than that of normal control, the leukocyte ADA activity was within the normal range, and the plasma ADA activity was 2 times higher than the normal mean. The adenine nucleotides in the patient's erythrocytes were within normal range. According to starch gel electrophoresis, ADA isozyme of the patient was ADA 1. Western blotting showed an increased amount of ADA protein in the patient's erythrocytes. Southern blotting revealed no gene amplification or large structural change. Dot blot analysis of the reticulocyte mRNA showed no increase in the amount of ADA mRNA in the patient's reticulocytes compared with those of reticulocyte-rich controls. We considered that the mechanism of elevated ADA activity in this acquired defect was similar to that found in hereditary hemolytic anemia associated with ADA overproduction.

  12. Erythroid Adhesion Molecules in Sickle Cell Anaemia Infants: Insights Into Early Pathophysiology.

    PubMed

    Brousse, Valentine; Colin, Yves; Pereira, Catia; Arnaud, Cecile; Odièvre, Marie Helene; Boutemy, Anne; Guitton, Corinne; de Montalembert, Mariane; Lapouméroulie, Claudine; Picot, Julien; Le Van Kim, Caroline; El Nemer, Wassim

    2015-01-01

    Sickle cell anaemia (SCA) results from a single mutation in the β globin gene. It is seldom symptomatic in the first semester of life. We analysed the expression pattern of 9 adhesion molecules on red blood cells, in a cohort of 54 SCA and 17 non-SCA very young infants of comparable age (median 144 days, 81-196). Haemoglobin F (HbF) level was unsurprisingly elevated in SCA infants (41.2% ± 11.2) and 2-4 fold higher than in non-SCA infants, yet SCA infants presented significantly decreased Hb level and increased reticulocytosis. Cytometry analysis evidenced a specific expression profile on reticulocytes of SCA infants, with notably an increased expression of the adhesion molecules Lu/BCAM, ICAM-4 and LFA-3, both in percentage of positive cells and in surface density. No significant difference was found on mature red cells. Our findings demonstrate the very early onset of reticulocyte membrane modifications in SCA asymptomatic infants and allow an insight into the first pathological changes with the release of stress reticulocytes expressing a distinctive profile of adhesion molecules.

  13. Height indicates hematopoietic capacity in elderly Japanese men

    PubMed Central

    Shimizu, Yuji; Sato, Shimpei; Koyamatsu, Jun; Yamanashi, Hirotomo; Nagayoshi, Mako; Kadota, Koichiro; Maeda, Takahiro

    2016-01-01

    Previously, we reported that height is an indicator of the capacity of vascular repair in elderly men, especially hypertensive men. On the other hand, hemoglobin could act as a possible biochemical index of hypertension-induced vascular damage. However, no studies have clarified the correlation between height and hematopoietic activity. We conducted a cross-sectional study of 249 men aged 65-69 undergoing a general health check-up. Reticulocyte was used to evaluate hematopoietic activity. Because hemoglobin concentration should influence hematopoietic activity, analyses stratified by hemoglobin level were performed. Independent of known cardiovascular risk factors and other hematological parameters (white blood cell count), a significant positive correlation was seen between height and reticulocytes for total subjects and subjects with a high hemoglobin concentration (≥14.5 g/dL), but not in subjects with a low hemoglobin concentration (<14.5 g/dL). The standardized parameter estimates (β) were β=0.18, p=0.003 for total subjects, β=0.28, p=0.001 for subjects with a high hemoglobin concentration, and β=0.03, p=0.717 for subjects with low hemoglobin. Independently, height is significantly positively correlated with reticulocyte in elderly Japanese men, particularly in men with a high hemoglobin concentration. These results indicate that subjects with a short stature might have lower hematopoietic capacity than those with a high stature. PMID:27705902

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

    PubMed

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

    2015-11-01

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

  15. Influence of counting methodology on erythrocyte ratios in the mouse micronucleus test.

    PubMed

    LeBaron, Matthew J; Schisler, Melissa R; Torous, Dorothea K; Dertinger, Stephen D; Gollapudi, B Bhaskar

    2013-04-01

    The mammalian erythrocyte micronucleus test is widely used to investigate the potential interaction of a test substance with chromosomes or mitotic apparatus of replicating erythroblasts. In addition to the primary endpoint, micronucleated erythrocyte frequency, the proportion of immature erythrocytes is measured to assess the influence of treatment on erythropoiesis. The guideline recommendation for an acceptable limit of the immature erythrocyte fraction of not < 20% of the controls was based on traditional scoring methods that consider RNA content. Flow-based sample analysis (e.g., MicroFlow®) characterizes a subpopulation of RNA-containing reticulocytes (RETs) based on CD71 (transferrin receptor) expression. As CD71+ cells represent a younger cohort of RETs, we hypothesized that this subpopulation may be more responsive than the RNA+ fraction for acute exposures. This study evaluated RET population in the peripheral blood of two strains of mice treated by oral gavage with three clastogens (cyclophosphamide, N-ethyl-N-nitrosourea, and methyl methanesulfonate). Although CD71+ frequencies correlated with RNA-based counts, the relative treatment-related reductions were substantially greater. Accordingly, when using the flow cytometry-based CD71+ values for scoring RETs in an acute treatment design, it is suggested that a target value ≥ 5% CD71+ reticulocytes (i.e., 95% depression in reticulocytes proportion) be considered as acceptable for a valid assay.

  16. Diagnosing anemia in inflammatory bowel disease: beyond the established markers.

    PubMed

    Oustamanolakis, Pantelis; Koutroubakis, Ioannis E; Kouroumalis, Elias A

    2011-10-01

    The main types of anemia in inflammatory bowel disease (IBD) are iron deficiency anemia (IDA) and anemia of inflammatory etiology, or anemia of chronic disease (ACD). In the management of IBD patients with anemia it is essential for the physician to diagnose the type of anemia in order to decide in an evidence-based manner for the appropriate treatment. However, the assessment of iron status in IBD in many cases is rather difficult due to coexistent inflammation. For this assessment several indices and markers have been suggested. Ferritin, seems to play a central role in the definition and diagnosis of anemia in IBD and transferrin, transferrin saturation (Tsat), and soluble transferrin receptors are also valuable markers. All these biochemical markers have several limitations because they are not consistently reliable indices, since they are influenced by factors other than changes in iron balance. In this review, in addition to them, we discuss the newer alternative markers for iron status that may be useful when serum ferritin and Tsat are not sufficient. The iron metabolism regulators, hepcidin and prohepcidin, are still under investigation in IBD. Erythrocytes parameters like the red cell distribution width (RDW) and the percentage of hypochromic red cells as well as reticulocyte parameters such as hemoglobin concentration of reticulocytes, red blood cell size factor and reticulocyte distribution width could be useful markers for the evaluation of anemia in IBD.

  17. The encephalomyocarditis virus 3C protease is a substrate for the ubiquitin-mediated proteolytic system.

    PubMed

    Lawson, T G; Gronros, D L; Werner, J A; Wey, A C; DiGeorge, A M; Lockhart, J L; Wilson, J W; Wintrode, P L

    1994-11-11

    The encephalomyocarditis virus 3C protease has been shown to be rapidly degraded in infected cells and in vitro in rabbit reticulocyte lysate. The in vitro degradation, at least, is accomplished by a virus-independent, ATP-dependent proteolytic system. Here we identify this proteolytic system as the ubiquitin-mediated system. Incubation of the 3C protease in rabbit reticulocyte or cultured mouse cell lysate preparations, alone or in the presence of added ubiquitin or methylated ubiquitin, resulted in the generation of new higher molecular weight species. These new products were shown to be 3C protease-ubiquitin conjugates by their ability to bind antibodies against both the 3C protease and ubiquitin. Supplemental ubiquitin also stimulated the degradation of the 3C protease in these preparations. Large 3C protease-polyubiquitin conjugates were observed to accumulate in reticulocyte lysate in the presence of adenosine 5'-O-(3-thiotriphosphate), an inhibitor of the 26 S multicatalytic protease. This, combined with the fact that the proteolytic activity could be removed from the lysate by sedimentation, implicates the multicatalytic protease in the degradation of the 3C protease-ubiquitin conjugates. It was also found that the slow rate of degradation of a model polyprotein, which resembles the stable viral 3CD diprotein produced in vivo, is likely due to the fact that the polyprotein is a poor substrate for the ubiquitin-conjugating system.

  18. Erythrocyte-based Pig-a gene mutation assay: demonstration of cross-species potential.

    PubMed

    Phonethepswath, Souk; Bryce, Steven M; Bemis, Jeffrey C; Dertinger, Stephen D

    2008-12-08

    Glycosylphosphatidylinositol (GPI) anchors attach specific proteins to the cell surface of hematopoietic cells. Of the genes required to form GPI anchors, only Pig-a is located on the X-chromosome. Prior work with rats suggests that the GPI anchor deficient phenotype is a reliable indicator of Pig-a mutation [Bryce et al., Environ. Mol. Mutagen., 49 (2008) 256-264]. The current report extends this line of investigation by describing simplified blood handling procedures, and by testing the assay principle in a second species, Mus musculus. With this method, erythrocytes are isolated, incubated with anti-CD24-PE, and stained with SYTO 13. Flow cytometric analyses quantify GPI anchor-deficient erythrocytes and reticulocytes. After reconstruction experiments with mutant-mimicking cells demonstrated that the analytical performance of the method is high, CD-1 mice were treated on three occasions with 7,12-dimethyl-1,2-benz[a]anthracene (DMBA, 75 mg/kg/day) or ethyl-N-nitrosourea (ENU, 40 mg/kg/day). Two weeks after the final treatment, DMBA-treated mice were found to exhibit markedly elevated frequencies of GPI anchor deficient erythrocytes and reticulocytes. For the ENU experiment, blood specimens were collected at weekly intervals over a 5-week period. Whereas the frequencies of mutant reticulocytes were significantly elevated 1 week after the last administration, the erythrocyte population was unchanged until the second week. Thereafter, both populations exhibited persistently elevated frequencies for the duration of the experiment (mean frequency at termination=310x10(-6) and 523x10(-6) for erythrocyte and reticulocyte populations, respectively). These data provide evidence that Pig-a mutation does not convey an appreciable positive or negative cell survival advantage to affected erythroid progenitors, although they do suggest that affected erythrocytes have a reduced lifespan in circulation. Collectively, accumulated data support the hypothesis that flow cytometric

  19. In vivo flow cytometric Pig-a and micronucleus assays: highly sensitive discrimination of the carcinogen/noncarcinogen pair benzo(a)pyrene and pyrene using acute and repeated-dose designs.

    PubMed

    Torous, Dorothea K; Phonethepswath, Souk; Avlasevich, Svetlana L; Mereness, Jared; Bryce, Steven M; Bemis, Jeffrey C; Weller, Pamela; Bell, Sara; Gleason, Carol; Custer, Laura L; MacGregor, James T; Dertinger, Stephen D

    2012-07-01

    Combining multiple genetic toxicology endpoints into a single in vivo study, and/or integrating one or more genotoxicity assays into general toxicology studies, is attractive because it reduces animal use and enables comprehensive comparative analysis using toxicity, metabolism, and pharmacokinetic information from the same animal. This laboratory has developed flow cytometric scoring techniques for monitoring two blood-based genotoxicity endpoints-micronucleated reticulocyte frequency and gene mutation at the Pig-a locus-thereby making combination and integration studies practical. The ability to effectively monitor these endpoints in short-term and repeated dosing schedules was investigated with the carcinogen/noncarcinogen pair benzo(a)pyrene (BP) and pyrene (Pyr). Male Sprague-Dawley rats were treated via oral gavage for 3 or 28 consecutive days with several dose levels of Pyr, including maximum tolerated doses. BP exposure was administered by the same route but at one dose level, 250 or 125 mg/kg/day for 3-day and 28-day studies, respectively. Serial blood samples were collected up to Day 45, and were analyzed for Pig-a mutation with a dual labeling method (SYTO 13 in combination with anti-CD59-PE) that facilitated mutant cell frequency measurements in both total erythrocytes and the reticulocyte subpopulation. A mutant cell enrichment step based on immunomagnetic column separation was used to increase the statistical power of the assay. BP induced robust mutant reticulocyte responses by Day 15, and elevated frequencies persisted until study termination. Mutant erythrocyte responses lagged mutant reticulocyte responses, with peak incidences observed on Day 30 of the 3-day study (43-fold increase) and on Day 42 of the 28-day study (171-fold increase). No mutagenic effects were apparent for Pyr. Blood samples collected on Day 4, and Day 29 for the 28-day study, were evaluated for micronucleated reticulocyte frequency. Significant increases in micronucleus

  20. Application of Innovative Hemocytometric Parameters and Algorithms for Improvement of Microcytic Anemia Discrimination.

    PubMed

    Schoorl, Margreet; Schoorl, Marianne; van Pelt, Johannes; Bartels, Piet C M

    2015-06-03

    Hemocytometric parameters like red blood cell (RBC) count, mean red blood cell volume (MCV), reticulocyte count, red blood cell distribution width (RDW-SD) and zinc protoporphyrin (ZPP) are frequently established for discrimination between iron-deficiency anemia and thalassemia in subjects with microcytic erythropoiesis. However, no single marker or combination of tests is optimal for discrimination between iron-deficiency anemia and thalassemia. This is the reason why many algorithms have been introduced. However, application of conventional algorithms, only resulted in appropriate classification of 30-40% of subjects. In this mini-review the efficacy of innovative hematological parameters for detection of alterations in RBCs has been considered. It refers to parameters concerning hemoglobinization of RBCs and reticulocytes and the percentages microcytic and hypochromic RBCs, for discrimination between subjects with iron-deficiency anemia (IDA) or thalassemia as well as a combination of both. A new discriminating tool including the above mentioned parameters was developed, based on two precondition steps and discriminating algorithms. The percentage microcytic RBCs is considered in the first precondition step. MCV, RDW-SD and RBC count are applied in the second precondition step. Subsequently, new algorithms, including conventional as well as innovative hematological parameters, were assessed for subgroups with microcytic erythropoiesis. The new algorithms for IDA discrimination yielded results for sensitivity of 79%, specificity of 97%, positive and negative predictive values of 74% and 98% respectively. The algorithms for β-thalassemia discrimination revealed similar results (74%, 98%, 75% and 99% respectively). We advocate that innovative algorithms, including parameters reflecting hemoglobinization of RBCs and reticulocytes, are integrated in an easily accessible software program linked to the hematology equipment to improve the discrimination between IDA and

  1. CNTO 530 increases expression of HbA and HbF in murine models of β-thalassemia and sickle cell anemia.

    PubMed

    Makropoulos, Dorie A; Achuthanandam, Ram; Avery, Justin; Wilson, Krista; Brosnan, Kerry; Miller, Andrew; Nesspor, Thomas; Chroscinski, Denise; Walker, Mindi; Egenolf, Devon; Huang, ChiChi; Bugelski, Peter J

    2013-01-01

    CNTO 530 is an erythropoietin receptor agonist MIMETIBODYTM construct. CNTO 530 has been shown to be active in a number of rodent models of acquired anemia (e.g. renal insufficiency and chemotherapy induced anemia). We investigated the efficacy of CNTO 530 in murine models of β-thalassemia and sickle cell anemia (Berkeley mice). β- thalassemic mice are deficient in expression of α-globin chain and heterozygous mice are characterized by a clinical syndrome similar to the human β-thalassemia intermedia. Berkeley mice are knocked out for murine alpha and beta globin and are transgenic for human alpha, beta (sickle) and gamma globin genes. Berkeley mice thus express human sickle hemoglobin A (HbS) and can also express human fetal hemoglobin. These mice express a severe compensated hypochromic microcytic anemia and display the sickle cell phenotype. To test the effectiveness of CNTO 530, mice from both genotypes received a single subcutaneous (s.c.) dose of CNTO 530 or darbepoetin-α (as a comparator) at 10,000 U/kg, a dose shown to cause a similar increase in reticulocytes and hemoglobin in normal mice. Hematologic parameters were evaluated over time. CNTO 530, but not darbepoetin-α, increased reticulocytes, red blood cells and total hemoglobin in β- thalassemic mice. In Berkeley mice CNTO 530 showed an increase in reticulocytes, red blood cells, F-cells, total hemoglobin and fetal hemoglobin. In conclusion, CNTO 530 is effective in murine models of β-thalassemia and sickle cell anemia. These data suggest that CNTO 530 may have beneficial effects in patients with genetically mediated hemoglobinopathies.

  2. Severe Ankyrin-R deficiency results in impaired surface retention and lysosomal degradation of RhAG in human erythroblasts

    PubMed Central

    Satchwell, Timothy J.; Bell, Amanda J.; Hawley, Bethan R.; Pellegrin, Stephanie; Mordue, Kathryn E.; van Deursen, Cees Th. B. M.; Braak, Nicole Heitink-ter; Huls, Gerwin; Leers, Mathie P.G; Overwater, Eline; Tamminga, Rienk Y. J.; van der Zwaag, Bert; Fermo, Elisa; Bianchi, Paola; van Wijk, Richard; Toye, Ashley M.

    2016-01-01

    Ankyrin-R provides a key link between band 3 and the spectrin cytoskeleton that helps to maintain the highly specialized erythrocyte biconcave shape. Ankyrin deficiency results in fragile spherocytic erythrocytes with reduced band 3 and protein 4.2 expression. We use in vitro differentiation of erythroblasts transduced with shRNAs targeting ANK1 to generate erythroblasts and reticulocytes with a novel ankyrin-R ‘near null’ human phenotype with less than 5% of normal ankyrin expression. Using this model, we demonstrate that absence of ankyrin negatively impacts the reticulocyte expression of a variety of proteins, including band 3, glycophorin A, spectrin, adducin and, more strikingly, protein 4.2, CD44, CD47 and Rh/RhAG. Loss of band 3, which fails to form tetrameric complexes in the absence of ankyrin, alongside GPA, occurs due to reduced retention within the reticulocyte membrane during erythroblast enucleation. However, loss of RhAG is temporally and mechanistically distinct, occurring predominantly as a result of instability at the plasma membrane and lysosomal degradation prior to enucleation. Loss of Rh/RhAG was identified as common to erythrocytes with naturally occurring ankyrin deficiency and demonstrated to occur prior to enucleation in cultures of erythroblasts from a hereditary spherocytosis patient with severe ankyrin deficiency but not in those exhibiting milder reductions in expression. The identification of prominently reduced surface expression of Rh/RhAG in combination with direct evaluation of ankyrin expression using flow cytometry provides an efficient and rapid approach for the categorization of hereditary spherocytosis arising from ankyrin deficiency. PMID:27247322

  3. Cessation of intensive treatment with recombinant human erythropoietin is followed by secondary anemia.

    PubMed

    Piron, M; Loo, M; Gothot, A; Tassin, F; Fillet, G; Beguin, Y

    2001-01-15

    Little information is available on the evolution of erythropoiesis after interruption of recombinant human erythropoietin (rHuEpo) therapy. Iron-overloaded rats received 20 daily injections of rHuEpo. During treatment, reticulocytes, soluble transferrin receptor (sTfR), and hematocrit increased progressively. This was accompanied by a substantial expansion of spleen erythropoiesis but a decrease in the bone marrow. Five weeks after treatment, rats developed a significant degree of a regenerative anemia. Erythropoietic activity, as assessed by reticulocytes, sTfR, erythroid cellularity, iron incorporation into heme, and the number of erythroid colonies, was severely depressed 3 weeks after cessation of rHuEpo. This was followed by regeneration of erythroblasts and reticulocytes at weeks 6 to 7 post-Epo, but erythroid progenitors recovered only partially by that time. The anemia was definitely corrected 2 months after cessation of rHuEpo treatment. Serum Epo levels remained elevated for several weeks, but the sensitivity of marrow erythroid precursors to Epo was preserved. No rat antibodies to rHuEpo were detected, and serum from post-Epo animals did not exert any inhibitory activity on erythropoiesis. In conclusion, after cessation of intensive rHuEpo therapy, there was a strong inhibition of erythropoietic activity with secondary anemia followed by late recovery. This was not due to antibodies or other soluble inhibitory factors, a defect in endogenous Epo production, or a loss of sensitivity to Epo. This may rather represent intrinsic erythroid marrow exhaustion, mostly at the level of erythroid progenitors but also at later stages of erythropoiesis.

  4. Incidence of megaloblastic anaemia and its correction in leishmaniasis--a prospective study at BPKIHS hospital, Nepal.

    PubMed

    Sinha, Arvind K; Rijal, Suman; Karki, Prahlad; Majhi, S

    2006-10-01

    Fifty (50) cases of Leishmaniasis were included in a prospective study after making the diagnosis based on the demonstration of L D Bodies on bone marrow aspirate. Hemoglobin (Hb) and total leucocyte count (TLC) were less than 10 gm/dl and 4000 per/ml respectively in 22 out of 50 cases. Among 50 cases, 30 showed macrocytic red cell and hypersegmented neutrophils on peripheral blood smear as features of megaloblastic anemia. Out of 30 cases of leishmaniasis with megaloblastic anemia, 10 cases showed decreased reticulocyte count. Bone marrow aspiration cytology showed megaloblastic maturation in erythroid series and giant metamyelocytes in myeloid series in all 30 cases of leishmaniasis with megaloblastic changes. Bone marrow iron was also increased in these cases. However megakaryocyte was normal in all. Among 30 cases of leishmaniasis with megaloblastic changes, 15 cases were treated with sodium antimony gluconate (SAG) only and other 15 with SAG, folic acid and Vit. B12. Rest 20 cases of leishmaniasis without megaloblastic changes were also treated with SAG only. After treatment with SAG alone as well as with SAG, folic acid and vit. B12, hemoglobin and TLC improved in all 50 cases. Of 15 cases of leishmaniasis with megaloblastic changes, who were treated with SAG, folic acid and vit. B12, the reticulocyte count reached at peak on the 7th day and normalized at the end of 4th week. Red blood cells (RBC) also became normocytic normochromic and neutrophils became normal on peripheral blood smear. But no change was observed in reticulocyte count, RBC and white blood cell (WBC) morphology on peripheral blood smear in cases of leishmaniasis without megaloblastic changes and other 15 cases of leishmaniasis with megaloblastic changes, who received SAG only.

  5. Application of Innovative Hemocytometric Parameters and Algorithms for Improvement of Microcytic Anemia Discrimination

    PubMed Central

    Schoorl, Margreet; Schoorl, Marianne; van Pelt, Johannes; Bartels, Piet C.M.

    2015-01-01

    Hemocytometric parameters like red blood cell (RBC) count, mean red blood cell volume (MCV), reticulocyte count, red blood cell distribution width (RDW-SD) and zinc protoporphyrin (ZPP) are frequently established for discrimination between iron-deficiency anemia and thalassemia in subjects with microcytic erythropoiesis. However, no single marker or combination of tests is optimal for discrimination between iron-deficiency anemia and thalassemia. This is the reason why many algorithms have been introduced. However, application of conventional algorithms, only resulted in appropriate classification of 30-40% of subjects. In this mini-review the efficacy of innovative hematological parameters for detection of alterations in RBCs has been considered. It refers to parameters concerning hemoglobinization of RBCs and reticulocytes and the percentages microcytic and hypochromic RBCs, for discrimination between subjects with iron-deficiency anemia (IDA) or thalassemia as well as a combination of both. A new discriminating tool including the above mentioned parameters was developed, based on two precondition steps and discriminating algorithms. The percentage microcytic RBCs is considered in the first precondition step. MCV, RDW-SD and RBC count are applied in the second precondition step. Subsequently, new algorithms, including conventional as well as innovative hematological parameters, were assessed for subgroups with microcytic erythropoiesis. The new algorithms for IDA discrimination yielded results for sensitivity of 79%, specificity of 97%, positive and negative predictive values of 74% and 98% respectively. The algorithms for β-thalassemia discrimination revealed similar results (74%, 98%, 75% and 99% respectively). We advocate that innovative algorithms, including parameters reflecting hemoglobinization of RBCs and reticulocytes, are integrated in an easily accessible software program linked to the hematology equipment to improve the discrimination between IDA and

  6. Changes in haematology measurements with the Sysmex XT-2000iV during storage of feline blood sampled in EDTA or EDTA plus CTAD.

    PubMed

    Granat, Fanny; Geffré, Anne; Bourgès-Abella, Nathalie; Braun, Jean-Pierre; Trumel, Catherine

    2013-06-01

    In veterinary medicine a complete blood cell count (CBC) cannot always be performed within 24 h as usually recommended, particularly for specimens shipped to a reference laboratory. This raises the question of the stability of the variables, especially in ethylenediamine tetra-acetic acid (EDTA) feline blood specimens, known to be prone to in vitro platelet aggregation. Citrate, theophylline, adenosine and dipyridamole (CTAD) has been reported to limit platelet aggregation in feline blood specimens. The aim of this study was to measure the stability of the haematological variables and the platelet aggregation score in EDTA and EDTA plus CTAD (EDCT) feline blood specimens during 48 h of storage at room temperature. Forty-six feline EDTA and EDCT blood specimens were analysed with a Sysmex XT-2000iV analyser, and the platelet count and score of platelet aggregation were estimated immediately and after 24 and 48 h of storage. A significant increase in mean corpuscular volume, haematocrit, reticulocyte and eosinophil counts, and a significant decrease in mean corpuscular haemoglobin concentration and monocyte count were observed. Haemoglobin, mean corpuscular haemoglobin, and red blood cell, white blood cell, neutrophil and lymphocyte counts remained stable. Changes in reticulocyte indexes with time (low fluorescence ratio, medium fluorescence ratio, high fluorescence ratio and immature reticulocyte fraction) were not significant. Changes were generally more pronounced in EDTA than in EDCT. Platelet aggregation decreased markedly in initially highly aggregated EDTA specimens, and increased slightly in initially non- or mildly-aggregated EDTA or EDCT specimens. Platelet counts increased and decreased, or remained stable, respectively. CTAD can reduce storage-induced changes of the haematological variables in feline samples, thus improving the reliability of a CBC and limiting clinical misinterpretations.

  7. Rat Pig-a mutation assay responds to the genotoxic carcinogen ethyl carbamate but not the non-genotoxic carcinogen methyl carbamate.

    PubMed

    Bemis, Jeffrey C; Labash, Carson; Avlasevich, Svetlana L; Carlson, Kristine; Berg, Ariel; Torous, Dorothea K; Barragato, Matthew; MacGregor, James T; Dertinger, Stephen D

    2015-05-01

    Determination of the mode of action of carcinogenic agents is an important factor in risk assessment and regulatory practice. To assess the ability of the erythrocyte-based Pig-a mutation assay to discriminate between genotoxic and non-genotoxic modes of action, the mutagenic response of Sprague Dawley rats exposed to methyl carbamate (MC) or ethyl carbamate (EC) was investigated. EC, a potent carcinogen, is believed to induce DNA damage through the formation of a DNA-reactive epoxide group, whereas the closely structurally related compound, MC, cannot form this epoxide and its weaker carcinogenic activity is thought to be secondary to inflammation and promotion of cell proliferation. The frequency of Pig-a mutant phenotype cells was monitored before, during, and after 28 consecutive days of oral gavage exposure to either MC (doses ranging from 125 to 500 mg/kg/day) or EC (250 mg/kg/day). Significant increases in the frequency of mutant reticulocytes were observed from Days 15 through 43, with a peak mean frequency of 19.9×10(-6) on Day 29 (i.e. 24.9-fold increase relative to mean vehicle control across all four sampling times). As expected, mutant erythrocyte responses lagged behind mutant reticulocyte responses, with a maximal mean frequency of 8.2×10(-6) on Day 43 (i.e. 16.4-fold increase). No mutagenic effects were observed with MC. A second indicator of in vivo genotoxicity, peripheral blood micronucleated reticulocytes, was also studied. This endpoint was responsive to EC (3.3-fold mean increase), but not to MC. These results support the hypothesis that genotoxicity contributes to the carcinogenicity of EC but not of MC, and illustrates the value of the Pig-a assay for discriminating between genotoxic and non-genotoxic modes of action.

  8. Doppler-Defined Pulmonary Hypertension in Sickle Cell Anemia in Kurdistan, Iraq.

    PubMed

    Al-Allawi, Nasir; Mohammad, Ameen M; Jamal, Shakir

    2016-01-01

    To determine the frequency, clinical and laboratory associations of pulmonary hypertension in Iraqi Kurds with sickle cell anemia, a total of ninety four such patients attending a major hemoglobinopathy center in Iraqi Kurdistan were enrolled. All patients were re-evaluated clinically and had their blood counts, HbF, serum ferritin, LDH, renal and liver function assessed. Transthoracic Doppler echocardiography with measurement of tricuspid valve regurgitant jet velocity (TRV) was performed. A TRV in excess of 2.8 m/s was considered for the purposes of this study as indicative of pulmonary hypertension (PH). The prevalence of TRV in excess of 2.8m/s was 10.6%. By univariate analysis: significantly higher reticulocyte count, more frequent blood transfusions and pain episodes were encountered in the PH group as compared to the non-PH group (p = 0.001, 0.045 and 0.02 respectively). Moreover, PH patients had significantly higher mean right atrial area, left atrial size, E wave/A wave ratio and ejection fraction by echocardiography (p = 0.027, 0.037, <0.001 and 0.008 respectively). Except for reticulocyte count none of the other parameters remained significant by multivariate analysis (p = 0.024). In conclusion the current study revealed that pulmonary hypertension is rather frequent among Iraqi Kurds with sickle cell anemia, and identified reticulocyte count as an independently associated parameter with PH in this population. Future prospective studies including right heart catheterization and appropriate medical intervention are warranted.

  9. Correlation between the Lactate Dehydrogenase Levels with Laboratory Variables in the Clinical Severity of Sickle Cell Anemia in Congolese Patients

    PubMed Central

    Mikobi, Tite Minga; Lukusa Tshilobo, Prosper; Aloni, Michel Ntetani; Mvumbi Lelo, Georges; Akilimali, Pierre Zalagile; Muyembe-Tamfum, Jean Jacques; Race, Valérie; Matthijs, Gert; Mbuyi Mwamba, Jean Marie

    2015-01-01

    Background Sickle cell anemia is an inflammatory disease and is characterized by chronic hemolysis. We sought to evaluate the association of lactate dehydrogenase levels with specific clinical phenotypes and laboratory variables in patients with sickle cell anemia. Methods The present cross-sectional study was conducted in Sickle Cell Centre of Yolo in Kinshasa, the Democratic Republic of Congo. Two hundred and eleven patients with Sickle Cell Anemia in steady state were recruited. Seventy-four participants with normal Hb (Hb-AA) were selected as a control group. Results The average rates of hemoglobin, hematocrit, and red blood cells tended to be significantly lower in subjects with Hb-SS (p<0.001). The average rates of white blood cells, platelets, reticulocytes and serum LDH were significantly higher in subjects with Hb-SS (p<0.001). The average rates of Hb, HbF, hematocrit and red blood cells of Hb-SS patients with asymptomatic clinical phenotype were significantly higher than those of the two other phenotypes. However, the average rates of white blood cells, platelets, reticulocytes, and LDH of Hb-SS patients with the severe clinical phenotype are higher than those of two other clinical phenotypes. Significant correlations were observed between Hb and white blood cell in severe clinical phenotype (r3 = -0.37 *) between Hb and red blood cells in the three phenotypes (r1 = 0.69 * r2 * = 0.69, r3 = 0.83 *), and finally between Hb and reticulocytes in the asymptomatic clinical phenotype and severe clinical phenotype (r1 = -0.50 * r3 = 0.45 *). A significant increase in LDH was observed in patients with leg ulcer, cholelithiasis and aseptic necrosis of the femoral head. Conclusion The increase in serum LDH is accompanied by changes in hematological parameters. In our midst, serum LDH may be considered as an indicator of the severity of the disease. PMID:25946088

  10. Dysregulated Erythropoietin, Hepcidin, and Bone Marrow Iron Metabolism Contribute to Interferon-Induced Anemia in Hepatitis C.

    PubMed

    van Rijnsoever, Marius; Galhenage, Sumedha; Mollison, Lindsay; Gummer, Joel; Trengove, Robert; Olynyk, John K

    2016-11-01

    Anemia is a complication of interferon-containing hepatitis C treatments. We characterized effects of interferon-based therapy on hepcidin and erythropoietin (EPO) production, iron metabolism, hemolysis, and hematopoiesis. Standard hemopoiesis [reticulocyte hemoglobin (Hb), reticulocyte production index (RPI), free Hb, and haptoglobin], iron biochemistry, hepcidin, and EPO levels were measured in 10 subjects over 12 weeks. There was a rapid decline in Hb during treatment, from a mean pretreatment (t = 0 weeks) Hb of 158.6 to 125.2 g/L at week 4 (P = 0.003) and 122.8 g/L at week 12 (P = 0.005). Paradoxically, the RPI (a measure of bone marrow responsiveness to EPO) decreased on initiation of hepatitis C virus treatment from 0.78% to 0.53% (P = 0.04). Despite worsening anemia, there was no significant increase in EPO levels. Hepcidin levels increased to >20 nM in 3 subjects from 5.8 to 27.5 nM (P = 0.009) compared with 9.6 to 12.3 nM (P = 0.5) for the remainder of subjects. Hepcidin levels peaked at week 1 before returning to baseline levels at week 4. Subjects who responded with a rise in serum hepcidin levels to >20 nM had a significantly greater drop in Hb (27.2 g/L, P = 0.008) and reticulocyte Hb (-1.4 g/L, P = 0.013) compared with the subjects who did not exhibit any change in hepcidin production. In conclusion, 30% of subjects treated with interferon exhibited significant transient increase in serum hepcidin levels, which was associated with more extreme anemia and decreased iron availability as evidenced by decreased reticulocyte Hb. In addition, there was a failure to upregulate EPO production in response to anemia and hemolysis ( https://clinicaltrials.gov trial NCT01726400).

  11. [Aregenerative anemia and erythrocytes hemighosts: a case report].

    PubMed

    Bibi-Triki, Tewfik; Bélien, Valérie; Chapelon, Emeline; Sauvion, Sylvie; Gaudelus, Joel; Laurian, Yves; de Pontual, Loïc

    2011-01-01

    An 11 year old African boy without previous history was hospitalised for fever and a severe anaemia (haemoglobin = 55 g/L) with low reticulocyte count. Blood smear showed more than 35% of ghost red blood cells which allows the diagnosis of G6PD deficiency (< 1% of normal level). Anaemia was demonstrated as haemolytic and was associated with a drepanocytosis trait. Aspect of red blood cells on blood smear remains important for the diagnosis of congenital or acquired red blood cell diseases, even abnormalities are often of low specificity.

  12. [A comparative study of the anti-anemic effect of new Bulgarian iron-dextran complexes with miofer-100 and ferroglucin-75 in rats and swine].

    PubMed

    Dilov, P

    1975-01-01

    It was found that the A-100 preparation (obtained from oxidized dextrane) produces highest antianaemic effects. It is to be used advantageously in cases of pigs born with negligible supplies of irod. Studied were the possibilities of inducing iron deficiency anemia in young rats through feeding with cow milk and macaroni having no eggs. The reticulocyte count rise and the drop in the haemoglobin level in the blood were shown to demonstrate most clearly the iron deficiency anaemia in albino rats. The anemia induced is said to be instrumental in the study of ferrodextrane preparations.

  13. Monoclonal gammopathy-associated pure red cell aplasia.

    PubMed

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

    2016-06-01

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

  14. Anemia for the Primary Care Physician.

    PubMed

    Powell, Darryl J; Achebe, Maureen Okam

    2016-12-01

    Anemia denotes a reduced red blood cell (RBC) mass from any cause. The causes of anemia are numerous and due to decreased (or abnormal) erythropoesis, shortened RBC life span, or blood loss. The most common etiology of anemia is iron deficiency. A judicious work up of anemia includes evaluating the reticulocyte count and peripheral smear. The severity of illness of a patient with anemia is determined by the degree of anemia and the seriousness of the underlying disorder. Management of patients with hereditary and hemolytic anemias should involve a hematologist.

  15. Anemia: Evaluation and Diagnostic Tests.

    PubMed

    Cascio, Michael J; DeLoughery, Thomas G

    2017-03-01

    Anemia is among the most common medical problems and clinical and laboratory evaluation need to be approached logically. The complete blood count with red cell indices offers clues to diagnosis. Many anemias have characteristic red cell morphology. The reticulocyte count serves as a useful screen for hemolysis or blood loss. Testing for specific causes of the anemia is performed. Occasionally, examination of the bone marrow is required for diagnosis. Molecular testing is increasingly being use to aid the diagnostic process. This article reviews diagnostic tests for anemia and suggests a rational approach to determining the etiology of a patient's anemia.

  16. Autoimmune hemolytic anemia during adalimumab treatment for plaque psoriasis.

    PubMed

    Harada, Yukinori; Yamamoto, Hiroaki; Sato, Midori; Kodaira, Mutsuki; Kono, Tsunesuke

    2015-01-01

    Adalimumab is commonly used to treat autoimmune diseases with few reported hematological adverse reactions. We herein describe the case of an 85-year-old Japanese man with plaque psoriasis who developed autoimmune hemolytic anemia (AIHA) after 3 years of adalimumab treatment. The patient suddenly developed hematuria and dyspnea on exertion while receiving adalimumab treatment. Laboratory data showed low hemoglobin levels and slightly increased reticulocyte counts, while direct and indirect antiglobulin tests were positive. The patient was diagnosed with AIHA which resolved after replacing the adalimumab treatment with prednisolone therapy. The findings from this case indicate that AIHA may be caused by long-term adalimumab treatment.

  17. Purification and characterization of moschins, arginine-glutamate-rich proteins with translation-inhibiting activity from brown pumpkin (Cucurbita moschata) seeds.

    PubMed

    Ng, T B; Parkash, A; Tso, W W

    2002-10-01

    From fresh brown pumpkin seeds, two proteins with a molecular mass of 12kDa and an N-terminal sequence rich in arginine and glutamate residues were obtained. The protein designated alpha-moschin closely resembled the fruitfly programmed-cell death gene product and the protein designated beta-moschin demonstrated striking similarity to prepro 2S albumin in N-terminal sequence. alpha- and beta-moschins inhibited translation in the rabbit reticulocyte lysate system with an IC(50) of 17 microM and 300nM, respectively.

  18. Isolation of cucurmoschin, a novel antifungal peptide abundant in arginine, glutamate and glycine residues from black pumpkin seeds.

    PubMed

    Wang, H X; Ng, T B

    2003-07-01

    A novel antifungal peptide, with a molecular mass of 8 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in gel filtration on Superdex 75 and designated cucurmoschin, was isolated from the seeds of the black pumpkin. The peptide was unadsorbed on DEAE-cellulose but adsorbed on Affi-gel blue gel. Cucurmoschin inhibited mycelial growth in the fungi Botrytis cinerea, Fusarium oxysporum and Mycosphaerella oxysporum. It inhibited translation in a cell-free rabbit reticulocyte lysate system with an IC50 of 1.2 microM. The N-terminal sequence of cucurmoschin was rich in arginine, glutamate and glycine residues.

  19. Isolation of ribosomes and polysomes.

    PubMed

    Rivera, Maria C; Maguire, Bruce; Lake, James A

    2015-03-02

    Here we describe a preparative differential centrifugation protocol for the isolation of ribosomes from a crude cell homogenate. The subcellular fraction obtained is enriched in ribosome monomers and polysomes. The protocol has been optimized for the homogenization and collection of the ribosomal fraction from prokaryotic cells, mammalian and plant tissues, reticulocytes, and chloroplasts. The quality of the ribosomal preparation is enhanced by the removal of the remaining cellular components and adsorbed proteins by pelleting through a sucrose cushion with a high concentration of monovalent salts, NH4Cl or KCl. The different components of the ribosomal fraction isolated using this protocol can be further purified by sucrose gradient centrifugation.

  20. Studies on the proteins from the seeds of Croton tiglium and of Jatropha curcas. Toxic properties and inhibition of protein synthesis in vitro.

    PubMed Central

    Stirpe, F; Pession-Brizzi, A; Lorenzoni, E; Strocchi, P; Montanaro, L; Sperti, S

    1976-01-01

    1. Proteins extracted from the seeds of the Euphorbiaceae croton tiglium and Jatropha curcas were separated into three major peaks (I,II,and III) by Sephadex chromatography. 2. The crude protein from both seeds and peaks I and II from Croton and peak I from Jatropha were toxic to mice, to different extents. 3. The crude protein and peak I and peak II from both seeds, inhibited protein synthesis by a reticulocyte lysate; maximum inhibition was exerted by peak II from both seeds. None of these preparations affected protein synthesis in vitro by Ehrlich ascites cells. PMID:942394

  1. Comparative analysis of eukaryotic cell-free expression systems.

    PubMed

    Hartsough, Emily M; Shah, Pankti; Larsen, Andrew C; Chaput, John C

    2015-09-01

    Cell-free protein synthesis (CFPS) allows researchers to rapidly generate functional proteins independent of cell culture. Although advances in eukaryotic lysates have increased the amount of protein that can be produced, the nuances of different translation systems lead to variability in protein production. To help overcome this problem, we have compared the relative yield and template requirements for three commonly used commercial cell-free translation systems: wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL). Our results provide a general guide for researchers interested in using cell-free translation to generate recombinant protein for biomedical applications.

  2. Reversible Vitamin B12 Deficiency Presenting with Acute Dementia, Paraparesis, and Normal Hemoglobin

    PubMed Central

    Mehdawi, Fahtima S.; Cheikh, Mohammed M.; Al-dhaheri, Fahmi; Aqeel, Abdullah Mahir

    2016-01-01

    Vitamin B12 is essential for neurological function and its deficiency is associated with many neuropsychiatric disorders. We report the case of a previously healthy 53-year-old male patient presenting with delirium and multiple neurological findings. Complete blood analysis indicated megaloblastic anemia. All infectious causes were excluded owing to negative cultures (blood and urine). Tests for human immunodeficiency virus, syphilis, and toxoplasma were also negative. Metabolic workup showed severe vitamin B12 deficiency, decreased reticulocyte count, and increased direct bilirubin and lactate dehydrogenase. Intramuscular injection of cobalamin was started, and the patient showed significant improvement. PMID:28070432

  3. Investigation of nonpoikilocytic normochromic normocytic anemia.

    PubMed

    Ward, P C

    1979-03-01

    When the mean corpuscular volume is normal in an anemic patient, examination of a blood smear becomes of paramount importance. When the RBCs are normochronic, normocytic, and nonpoikilocytic, the reticulocyte count determines the line of investigation. When the count is increased, either hemorrhage or hemolysis is present. When the count is not increased (ie, normal or low), the differential diagnosis includes anemias due to iron deficiency, chronic disease, renal disease, hemodilution, marrow infiltration, and marrow failure (aplastic anemia). Some morphologic clues to cause other than those pertaining to RBC morphology are discussed and illustrated. A working approach to the investigation of nonpoikilocytic normochromic normocytic anemia is suggested.

  4. Specific activities of poetam preparation (superlow-doses of antibodies to erythropoietin) and recombinant erythropoietin.

    PubMed

    Dygai, A M; Zhdanov, V V; Udut, E V; Simanina, E V; Gur'yantseva, L A; Khrichkova, T Yu; Epshtein, O I; Sergeeva, S A

    2006-09-01

    We compared the capacity of superlow-dose of antibodies to erythropoietin (Poetam) and recombinant erythropoietin (Recormon) to stimulate the recovery of adriamycin-suppressed erythropoiesis in mice. Both preparations exhibited high erythron activation capacity and considerably increased the content of erythrocytes and reticulocytes in the peripheral blood and content of erythrokaryocytes and erythroid precursors in the hemopoietic tissue of experimental animals. The effect of Recormon manifested immediately after injection, while the effect of Poetam was somewhat delayed, but more lasting (due to activation of host erythropoietin system).

  5. Hemolytic uremic syndrome associated with Plasmodium vivax malaria successfully treated with plasma exchange.

    PubMed

    Keskar, V S; Jamale, T E; Hase, N K

    2014-01-01

    We report a case of hemolytic uremic syndrome (HUS) in an adult patient with Plasmodium vivax malaria. The patient presented with worsening anemia, persistent thrombocytopenia and acute kidney injury. HUS was diagnosed based on the high serum lactate dehydrogenase, elevated reticulocyte count and presence of schistocytes on peripheral blood smear. Kidney biopsy showed features of thrombotic microangiopathy. Complete hematological remission was achieved after five sessions of therapeutic plasma exchange. Renal function partially recovered and stabilized at discharge. Vivax malaria, generally considered benign, may be rarely associated with HUS.

  6. Electrostimulation and morphologic study of the nerves to the bone marrow of the albino rat.

    PubMed

    DePace, D M; Webber, R H

    1975-01-01

    The innervation of the bone marrow of the albino rat was investigated by electrostimulation and morphological methods. Stimulation of the lumbar sympathetic trunks resulted in the release of reticulocytes and neutrophils into the circulating blood. The effects of stimulation on other cell types in the bone marrow could not be definitely established. It was concluded that the nerve fibers to the bone marrow were distributed to the arteries. It is postulated that the transmitter substance released at the autonomic nerve endings may have an effect upon the permeability of the venous sinusiods and the mobility of the blood cells in the marrow parenchyma resulting in their release into the circulating blood.

  7. Characterization of monoferric fragments obtained by tryptic cleavage of bovine transferrin.

    PubMed Central

    Brock, J H; Arzabe, F R; Richardson, N E; Deverson, E V

    1978-01-01

    1. The electrophoretically fast (F) and slow (S) fragments obtained by tryptic cleavage of bovine iron-saturated transferrin differed in carbohydrate content and peptide 'maps'. 2. A fragment capable of binding one Fe3+ ion per molecule was isolated after brief tryptic digestion of bovine apotransferrin and shown closely to resemble the S fragment obtained from the iron-saturated protein. 3. Fragments F and S are probably derived from the N- and C-terminal halves of the transferrin molecule respectively. 4. Bovine transferrin could donate iron to rabbit reticulocytes, but the monoferric fragments possessed little iron-donating ability. Images PLATE 1 PMID:646825

  8. Effects of ribosome-inactivating proteins on Escherichia coli and Agrobacterium tumefaciens translation systems.

    PubMed Central

    Girbés, T; Barbieri, L; Ferreras, M; Arias, F J; Rojo, M A; Iglesias, R; Alegre, C; Escarmis, C; Stirpe, F

    1993-01-01

    The effects of 30 type 1 and of 2 (ricin and volkensin) type 2 ribosome-inactivating proteins (RIPs) on Escherichia coli and Agrobacterium tumefaciens cell-free translation systems were compared with the effects on a rabbit reticulocyte translation system. The depurinating activity of RIPs on E. coli ribosomes was also evaluated. Only six type 1 RIPs inhibited endogenous mRNA-directed translational activity of E. coli lysates, with submicromolar 50% inhibitory concentrations. Four RIPs had similar activities on poly(U)-directed phenylalanine polymerization by E. coli ribosomes, and three RIPs inhibited poly(U)-directed polyphenylalanine synthesis by A. tumefaciens ribosomes, with submicromolar 50% inhibitory concentrations. Images PMID:8407849

  9. Rituximab-based immunosuppression for autoimmune haemolytic anaemia in infants.

    PubMed

    Svahn, Johanna; Fioredda, Francesca; Calvillo, Michaela; Molinari, Angelo C; Micalizzi, Concetta; Banov, Laura; Schmidt, Madalina; Caprino, Daniela; Marinelli, Doretta; Gallisai, Domenico; Dufour, Carlo

    2009-04-01

    We report a case series of four infants with severe autoimmune haemolytic anaemia (AIHA) who responded to treatment with rituximab and cyclosporine after having failed first line therapy with high-dose steroid (prednisolone 4-8 mg/kg/d). Rituximab was started at 11-90 d from onset due to continued haemolysis; three infants also received cyclosporine A. Three of four infants reached complete response, defined as normal haemoglobin, reticulocytes and negative indices of haemolysis, at 7-21 months from diagnosis. In long-term follow-up two infants remained disease-free with normal immunology, one had undefined immunodeficiency and one had autoimmune lymphoproliferative syndrome.

  10. AMPKalpha1 deletion shortens erythrocyte life span in mice: role of oxidative stress.

    PubMed

    Wang, Shaobin; Dale, George L; Song, Ping; Viollet, Benoit; Zou, Ming-Hui

    2010-06-25

    AMP-activated protein kinase (AMPK) is an energy sensor essential for maintaining cellular energy homeostasis. Here, we report that AMPKalpha1 is the predominant isoform of AMPK in murine erythrocytes and mice globally deficient in AMPKalpha1 (AMPKalpha1(-/-)), but not in those lacking AMPKalpha2, and the mice had markedly enlarged spleens with dramatically increased proportions of Ter119-positive erythroid cells. Blood tests revealed significantly decreased erythrocyte and hemoglobin levels with increased reticulocyte counts and elevated plasma erythropoietin concentrations in AMPKalpha1(-/-) mice. The life span of erythrocytes from AMPKalpha1(-/-) mice was less than that in wild-type littermates, and the levels of reactive oxygen species and oxidized proteins were significantly increased in AMPKalpha1(-/-) erythrocytes. In keeping with the elevated oxidative stress, treatment of AMPKalpha1(-/-) mice with the antioxidant, tempol, resulted in decreased reticulocyte counts and improved erythrocyte survival. Furthermore, the expression of Foxo3 and reactive oxygen species scavenging enzymes was significantly decreased in erythroblasts from AMPKalpha1(-/-) mice. Collectively, these results establish an essential role for AMPKalpha1 in regulating oxidative stress and life span in erythrocytes.

  11. Flow cytometric scoring of micronucleated erythrocytes: an efficient platform for assessing in vivo cytogenetic damage.

    PubMed

    Dertinger, Stephen D; Torous, Dorothea K; Hayashi, Makoto; MacGregor, James T

    2011-01-01

    The relative simplicity of the micronucleated erythrocyte endpoint has made it amenable to automated scoring approaches. Flow cytometry is one such scoring platform that has been employed successfully. This review describes the evolution and properties of flow cytometry-based scoring of micronucleated erythrocytes. The methodology has become widely applied to rodent blood specimens and the high throughput nature of the technology provides a number of advantages over manual microscopic scoring. For instance, the ability to efficiently survey many dose levels and many more cells per specimen relative to microscopy benefits studies that are designed to identify no observable effect levels or lowest observable effect levels. Furthermore, flow cytometry makes it practical to study species with low spontaneous reticulocyte (RET) counts and micronucleus (MN) frequencies, thereby facilitating integration of blood-based micronucleated reticulocyte (MN-RET) frequency measurements into experiments conducted across species of toxicological interest. This capability enhances genotoxicity assessments that have historically been made in dedicated MN tests performed in one species. Importantly, the feasibility of using MN-RET frequencies in blood from humans as an index of genetic damage in bone marrow opens a critical area of application that had not been practical previously. We conclude with recommendations for additional work that is needed to more fully realise the potential of flow cytometric in vivo MN scoring.

  12. Translation in cell-free systems

    SciTech Connect

    Jagus, R.

    1987-01-01

    The simplest, unambiguous identification of a particular mRNA is the identification of its protein product. This can be established by translation of the mRNA of interest in a cell-free protein-synthesizing system. Messenger RNA protein product identification is important in the isolation of a particular mRNA species for cDNA cloning and in the identification of positive cDNA clones. The two high-activity translation systems in common use are those prepared from rabbit reticulocytes and from wheat germ. Both systems are easy to prepare, and both are available commercially. Each has advantages and disadvantages over the other and a choice between the two will depend on the type of mRNAs to be translated, the prejudices of experience, and availability. The main disadvantage of the reticulocyte system is that it requires removal of endogenous mRNA. However, this is a relatively simple procedure. The wheat germ system does not require removal of endogenous mRNA and may translate weakly initiating mRNAs more efficiently. However, ionic optima for translation in the wheat germ system are more sensitive to the nature and concentration of mRNA and may need to be determined for each template. The biggest problem with the use of the wheat germ system is its tendency to produce incomplete translation products due to premature termination.

  13. Seasonal variations of haematological parameters in athletes.

    PubMed

    Banfi, Giuseppe; Lundby, Carsten; Robach, Paul; Lippi, Giuseppe

    2011-01-01

    The influence of training and competition workloads is crucial for evaluation of longitudinal haematological data in athletes. There are only a few papers on the variation of haematological parameters during long-lasting periods and, especially, during an entire competitive season. We summarized that some haematological parameters can be influenced by long-term training and competition periods. Haemoglobin (Hb) and haematocrit (Ht) are decreased during the more intense periods of training, throughout the season. In different sport disciplines, the decline of Hb ranges from 3 to 8% during the competition season, while the range of reticulocytes (Ret%) varies from 5 to 21%. Reticulocytes are also decreased after long periods of training and competitions, but their variation is not necessarily associated with that of Hb. The qualitative variations (trend of modifications) of haematological parameters are roughly independent of the sport discipline, but quantitatively (amount of modifications) dependent on sport discipline. The modifications are more evident in cycling, running, swimming than they are in football and rugby. The variations of haematological parameters within the same sport discipline are qualitatively concordant and quantitatively different among separate but consecutive competitive seasons. These findings are described in aerobic and team sports sportsmen. The definition of reliable reference ranges in sportsmen would only be possible by following the best laboratory practices. For antidoping purposes more studies investigating haematological modifications during the season are advisable.

  14. Erythroblastosis of the Donor Twin of Twin Anemia-Polycythemia Sequence.

    PubMed

    Takeuchi, Miharu; Maruyama, Hidehiko; Oura, Naoko; Kanazawa, Akane; Nakata, Yusei; Minami, Susumu; Kikkawa, Kiyoshi

    2016-08-01

    Twin anemia-polycythemia sequence (TAPS) is a group of disorders in monochorionic twins characterized by a large intertwin hemoglobin difference without amniotic fluid discordance. Reticulocyte count is used to diagnose this condition, but little is known about the role of erythroblasts, which are the prior stage of reticulocytes. In the present case of TAPS, the 25-yr-old Japanese mother showed no signs of oligohydramnios or polyhydramnios throughout gestation. The twins were born at 36 weeks and 6 days, weighing 2,648g and 1,994g. The intertwin hemoglobin difference in umbilical cord blood was (21.1-5.0=) 16.1g/dL and the donor twin showed signs of chronic anemia, including myocardial hypertrophy and pericardial effusion. Erythroblastosis of the donor twin was prolonged (53,088.5, 42,114.8 and 44,217.9/μL on days 0, 1 and 2, respectively). Erythroblastosis, which indicates chronic anemia, is also a good diagnostic indicator of TAPS.

  15. Tracking Resilience to Infections by Mapping Disease Space

    PubMed Central

    Thomas Tate, Ann; Rath, Poonam; Cumnock, Katherine; Schneider, David S.

    2016-01-01

    Infected hosts differ in their responses to pathogens; some hosts are resilient and recover their original health, whereas others follow a divergent path and die. To quantitate these differences, we propose mapping the routes infected individuals take through “disease space.” We find that when plotting physiological parameters against each other, many pairs have hysteretic relationships that identify the current location of the host and predict the future route of the infection. These maps can readily be constructed from experimental longitudinal data, and we provide two methods to generate the maps from the cross-sectional data that is commonly gathered in field trials. We hypothesize that resilient hosts tend to take small loops through disease space, whereas nonresilient individuals take large loops. We support this hypothesis with experimental data in mice infected with Plasmodium chabaudi, finding that dying mice trace a large arc in red blood cells (RBCs) by reticulocyte space as compared to surviving mice. We find that human malaria patients who are heterozygous for sickle cell hemoglobin occupy a small area of RBCs by reticulocyte space, suggesting this approach can be used to distinguish resilience in human populations. This technique should be broadly useful in describing the in-host dynamics of infections in both model hosts and patients at both population and individual levels. PMID:27088359

  16. Genetic variation in CD36, HBA, NOS3 and VCAM1 is associated with chronic haemolysis level in sickle cell anaemia: a longitudinal study.

    PubMed

    Coelho, Andreia; Dias, Alexandra; Morais, Anabela; Nunes, Baltazar; Ferreira, Emanuel; Picanço, Isabel; Faustino, Paula; Lavinha, João

    2014-03-01

    Chronic haemolysis stands out as one of the hallmarks of sickle cell anaemia, a clinically heterogeneous autosomal recessive monogenic anaemia. However, the genetic architecture of this sub-phenotype is still poorly understood. Here, we report the results of an association study between haemolysis biomarkers (serum LDH, total bilirubin and reticulocyte count) and the inheritance of 41 genetic variants of ten candidate genes in a series of 99 paediatric SS patients (median current age of 9.9 yr) followed up in two general hospitals in Greater Lisboa area (median follow-up per patient of 5.0 yr). Although in a large number of tests a seemingly significant (i.e. P < 0.05) association was observed, the following ones were confirmed upon correction for multiple comparisons: (i) an increased serum LDH level was associated with haplotype 7 within VCAM1 gene; (ii) a lower total bilirubin was associated with the 3.7-kb deletion at HBA gene, rs2070744_T allele at NOS3 gene, and haplotype 9 within VCAM1 promoter; and (iii) a diminished reticulocyte count was associated with the 3.7-kb deletion at HBA, whereas an increased count was associated with rs1984112_G allele at CD36 gene. On the whole, our findings suggest a complex genetic architecture for the sickle cell anaemia haemolysis process involving multiple pathways, namely control of vascular cell adhesion, NO synthesis and erythrocyte volume and haemoglobinisation.

  17. Changes in hematological profiles during winter field operations

    SciTech Connect

    Lopez, A.; Reed, L.; D'Alesandro, M. )

    1991-03-11

    The authors have previously shown that there are changes in hematological profiles during experimental cold acclimation. They now report on hematological changes in 9 military volunteers during a 12 week winter field operation and show results similar to those observed during experimental cold acclimation. Blood was collected before and after completion of winter field operations and analyzed in a paired fashion. Hematocrit (HCT) and erythrocyte counts (RBC) were decreased; mean corpuscular hemoglobin concentration (MCHC) and plasma volume (PV), which was calculated from hemoglobin (Hb) concentration and HCT, were increased. In addition, the reticulocyte count was increased from 1.37 {plus minus} 0.10% to 2.62 {plus minus} 0.24% after completion of field operations. There was a statistically significant inverse correlation between HCT and reticulocyte count, indicating the need for an enhanced rate of red cell production. Hemoglobin concentration, leukocyte count, and mean corpuscular volume were unchanged. The RBC population, to remain at steady state during periods of chronic cold exposure, shows alterations in the number of circulating cells, Hb concentration per cell and possibly cell turnover.

  18. HER-2 UPSTREAM OPEN READING FRAME EFFECTS ON THE USE OF DOWNSTREAM INITIATION CODONS

    PubMed Central

    Spevak, Christina C.; Park, Eun-Hee; Geballe, Adam P.; Pelletier, Jerry; Sachs, Matthew S.

    2006-01-01

    The her-2 (neu, erbB-2) oncogene encodes a 185-kDa transmembrane receptor tyrosine kinase. HER2 overexpression occurs in numerous primary human tumors and contributes to 25–30% of breast and ovarian carcinomas. Synthesis of HER2 is controlled in part by an upstream open reading frame (uORF) present in the transcript. We used synthetic capped and polyadenylated mRNAs containing sequences derived from the 5′ region of the her-2 transcript fused to firefly luciferase (LUC) reporter to examine this ORF’s effect on translation in cell-free systems derived from reticulocytes, wheat germ and Neurospora crassa, and in RNA-transfected HeLa cells. The uORF reduced translation of the downstream cistron in all systems. [35S]Met-labeling of in vitro translation products obtained indicated that the uORF also affected downstream start-site selection. Primer extension inhibition (toeprint) assays of ribosomes loaded at initiation codons in reticulocyte lysates indicated that the uORF affected the interaction of ribosomes with the primary her-2 AUG codon. PMID:17045969

  19. Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

    PubMed Central

    Maroto, F G; Sierra, J M

    1989-01-01

    A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos. Images PMID:2501660

  20. Blood volume changes. [weightlessness effects

    NASA Technical Reports Server (NTRS)

    Johnson, P. C.; Driscoll, T. B.; Leblance, A. D.

    1974-01-01

    Analysis of radionuclide volume determinations made for the crewmembers of selected Gemini and Apollo missions showed that orbital spaceflight has an effect on red cell mass. Because the methods and the protocol developed for earlier flights were used for the crews of the three Skylab missions, direct comparisons are possible. After each Skylab mission, decreases were found in crewmembers' red cell masses. The mean red cell mass decrease of 11 percent or 232 milliliters was approximately equal to the 10 percent mean red cell mass decrease of the Apollo 14 to 17 crewmembers. The red cell mass drop was greatest and the postrecovery reticulocyte response least for crewmembers of the 28-day Skylab 2 mission. Analyses of data from the red cell mass determinations indicate that the red cell mass drops occurred in the first 30 days of flight and that a gradual recovery of the red cell mass deficits began approximately 60 days after launch. The beginning of red cell mass regeneration during the Skylab 4 flight may explain the higher postmission reticulocyte counts.

  1. Clinical Applications of Hemolytic Markers in the Differential Diagnosis and Management of Hemolytic Anemia.

    PubMed

    Barcellini, W; Fattizzo, B

    2015-01-01

    Several hemolytic markers are available to guide the differential diagnosis and to monitor treatment of hemolytic conditions. They include increased reticulocytes, an indicator of marrow compensatory response, elevated lactate dehydrogenase, a marker of intravascular hemolysis, reduced haptoglobin, and unconjugated hyperbilirubinemia. The direct antiglobulin test is the cornerstone of autoimmune forms, and blood smear examination is fundamental in the diagnosis of congenital membrane defects and thrombotic microangiopathies. Marked increase of lactate dehydrogenase and hemosiderinuria are typical of intravascular hemolysis, as observed in paroxysmal nocturnal hemoglobinuria, and hyperferritinemia is associated with chronic hemolysis. Prosthetic valve replacement and stenting are also associated with intravascular and chronic hemolysis. Compensatory reticulocytosis may be inadequate/absent in case of marrow involvement, iron/vitamin deficiency, infections, or autoimmune reaction against bone marrow-precursors. Reticulocytopenia occurs in 20-40% of autoimmune hemolytic anemia cases and is a poor prognostic factor. Increased reticulocytes, lactate dehydrogenase, and bilirubin, as well as reduced haptoglobin, are observed in conditions other than hemolysis that may confound the clinical picture. Hemoglobin defines the clinical severity of hemolysis, and thrombocytopenia suggests a possible thrombotic microangiopathy or Evans' syndrome. A comprehensive clinical and laboratory evaluation is advisable for a correct diagnostic and therapeutic workup of the different hemolytic conditions.

  2. Human placental Na/sup +/, K/sup +/-ATPase. cap alpha. subunit: cDNA cloning, tissue expression, DNA polymorphism, and chromosomal localization

    SciTech Connect

    Chehab, F.F.; Kan, Y.W.; Law, M.L.; Hartz, J.; Kao, F.T.; Blostein, R.

    1987-11-01

    A 2.2-kilobase clone comprising a major portion of the coding sequence of the Na/sup +/, K/sup +/-ATPase ..cap alpha.. subunit was cloned from human placenta and its sequence was identical to that encoding the ..cap alpha.. subunit of human kidney and HeLa cells. Transfer blot analysis of the mRNA products of the Na/sup +/, K/sup +/-ATPase gene from various human tissues and cell lines revealed only one band (approx. = 4.7 kilobases) under low and high stringency washing conditions. The levels of expression in the tissues were intestine > placenta > liver > pancreas, and in the cell lines the levels were human erythroleukemia > butyrate-induced colon > colon > brain > HeLa cells. mRNA was undetectable in reticulocytes, consistent with the authors failure to detect positive clones in a size-selected ( > 2 kilobases) lambdagt11 reticulocyte cDNA library. DNA analysis revealed by a polymorphic EcoRI band and chromosome localization by flow sorting and in situ hybridization showed that the ..cap alpha.. subunit is on the short is on the short arm (band p11-p13) of chromosome 1.

  3. Spirulina platensis feeding inhibited the anemia- and leucopenia-induced lead and cadmium in rats.

    PubMed

    Simsek, Nejdet; Karadeniz, Ali; Kalkan, Yildiray; Keles, Osman N; Unal, Bünyami

    2009-05-30

    In the present investigation, the effect of Spirulina platensis (Sp) was undertaken on rats fed with lead and cadmium including diet by using physiological, enzymehistochemical and stereological methods. For this aim, 50 rats were equally divided into five groups as control (C), lead (Pb), Spirulina+lead (Sp+Pb), cadmium (Cd), and Spirulina+cadmium (Sp+Cd). Red blood cell (RBC) and white blood cell (WBC) counts, packed cell volume (PCV), and haemoglobine (Hb) concentrations were determined by haemocytometric methods in blood samples collected on 30th day. Population of T lymphocyte was counted by the alpha-naphthyl acetate esterase (ANAE) staining method, and reticulocytes were counted by stereological method. The counts of RBC, WBC, and ANAE positive T lymphocyte, and the values of Hb, PCV, and MCHC were decreased in the Pb and Cd groups compared to control group. Also, the number of reticulocytes (polychromatofilic erythrocyte) increased in the Pb groups, whereas it decreased in the Cd group. On the other hand, these values were ceased by S. platensis in the treated groups. These results suggest that S. platensis supplementation may be useful in adjuvant treatment of leukemia and anemia caused by lead and cadmium toxication.

  4. The familial Parkinson's disease gene DJ-1 (PARK7) is expressed in red cells and plays a role in protection against oxidative damage.

    PubMed

    Xu, Xiuling; Martin, Florent; Friedman, Jeffrey S

    2010-10-15

    The antioxidant enzyme manganese superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide. Impaired SOD2 activity in murine hematopoietic cells affects erythroid development, resulting in anemia characterized by intra-mitochondrial iron deposition, reticulocytosis and shortened red cell life span. Gene expression profiling of normal and SOD2 deficient erythroblasts identified the Parkinson's disease locus DJ-1 (Park7) as a differentially expressed transcript. To investigate the role of DJ-1 in hematopoietic cell development and protection against oxidative stress caused by Sod2 loss, we evaluated red cell parameters, reticulocyte count, red cell turnover and reactive oxygen species production in DJ-1 knockout animals and chimeric animals lacking both SOD2 and DJ-1 in hematopoietic cells generated by fetal liver transplantation. We also investigated DJ-1 protein expression in primary murine erythroid and erythroleukemia cells (MEL). Loss of DJ-1 exacerbates the phenotype of SOD2 deficiency, increasing reticulocyte count and decreasing red cell survival. Using MEL cells, we show that DJ-1 is up-regulated at the protein level during erythroid differentiation. These results indicate that DJ-1 plays a physiologic role in protection of erythroid cells from oxidant damage, a function unmasked in the context of oxidative stress.

  5. Delayed blood regeneration in lead exposure: An effect on reserve capacity

    SciTech Connect

    Grandjean, P.; Jensen, B.M.; Sando, S.H.; Jogensen, P.J.; Antonsen, S. )

    1989-10-01

    Twenty-five lead-exposed Danish battery production workers and 25-age-matched controls were examined to evaluate subclinical effects on blood formation. Blood lead levels averaged 2.14 mumol/L and 0.35 mumol/L in the two groups; the lead workers also showed high levels of erythrocyte protoporphyrin, as compared to the controls. Otherwise, the hematological parameters indicated an appropriate iron status and no other deviations. From all subjects, 0.45 L of blood was bled as part of a normal blood donation. Five and 11 days later, reticulocyte counts were significantly higher in the control group than in the lead-exposed workers. On day 15, the lead workers showed a significant delay in blood regeneration, as evidenced by lower hemoglobin concentration, and erythrocyte and reticulocyte counts. The lead exposure in the present study was within legal limits, and lead-induced anemia would be expected only at much higher exposure levels. Thus, despite the normal hematological findings in the initial examination, the lead exposure caused a decreased reserve capacity for blood formation, and this effect became evident only after the blood loss.

  6. Familial-skewed X-chromosome inactivation as a predisposing factor for late-onset X-linked sideroblastic anemia in carrier females.

    PubMed

    Cazzola, M; May, A; Bergamaschi, G; Cerani, P; Rosti, V; Bishop, D F

    2000-12-15

    X-linked sideroblastic anemia (XLSA) is caused by mutations in the erythroid-specific 5-aminolevulinic acid synthase (ALAS2) gene. An elderly woman who presented with an acquired sideroblastic anemia is studied. Molecular analysis revealed that she was heterozygous for a missense mutation in the ALAS2 gene, but she expressed only the mutated gene in reticulocytes. Her 2 daughters and a granddaughter were heterozygous for this mutation, had normal hemoglobin levels, and expressed the normal ALAS2 gene in reticulocytes. A grandson with a previous diagnosis of thalassemia intermedia was found to be hemizygous for the ALAS2 mutation. Treatment with pyridoxine completely corrected the anemia both in the proband and her grandson. All women who were analyzed in this family showed skewed X-chromosome inactivation in leukocytes, which indicated a hereditary condition associated with unbalanced lyonization. Because the preferentially active X chromosome carried the mutant ALAS2 allele, acquired skewing in the elderly likely worsened the genetic condition and abolished the normal ALAS2 allele expression in the proband. (Blood. 2000;96:4363-4365)

  7. Safety and mutagenicity evaluation of red mold dioscorea fermented from Monascus purpureus NTU 568.

    PubMed

    Hsu, Li-Chuan; Hsu, Ya-Wen; Hong, Chih-Chun; Pan, Tzu-Ming

    2014-05-01

    Monascus-fermented products, including red mold rice and red mold dioscorea, have been developed as functional foods with many health benefits. We performed safety and mutagenic evaluations on red mold dioscorea powder (RMDP) fermented from Monascus purpureus NTU 568. The results of Ames test using Salmonella typhimurium strains TA97a, TA98, TA100, TA102, and TA1535 showed that RMDP (⩽5 mg/plate) was not mutagenic. The mammalian chromosomal aberration test showed that the number of Chinese hamster ovary cells with abnormal chromosomes was <3% after RMDP treatment (maximum concentration: 5 mg/mL). Imprinting control region mice were used to estimate the genotoxicity of RMDP. Compared with the control, high-dose RMDP administration (2000 mg/kg) did not show significant differences in the number of reticulocytes or the occurrence of micronucleated reticulocytes. A 28-day oral toxicity assay in Sprague-Dawley rats was performed to investigate the no observed adverse effect level of RMDP. Compared with the control, high-dose RMDP administration (2000 mg/kg) caused no toxicological responses such as mortality, variation in body weight, or toxicopathologic lesions. Thus, RMDP from M. purpureus NTU 568 shows no significant mutagenic or toxic effects.

  8. Evaluation of Safety and Pharmacokinetics of Sodium 2,2 Dimethylbutyrate, a Novel Short Chain Fatty Acid Derivative, in a Phase 1, Double-Blind, Placebo-Controlled, Single- and Repeat-Dose Studies in Healthy Volunteers

    PubMed Central

    Perrine, Susan P.; Wargin, William A.; Boosalis, Michael S.; Wallis, Wayne J.; Case, Sally; Keefer, Jeffrey R.; Faller, Douglas V.; Welch, William C.; Berenson, Ronald J.

    2013-01-01

    Pharmacologic induction of fetal globin synthesis is an accepted therapeutic strategy for treatment of the beta hemoglobinopathies and thalassemias, as even small increases in hemoglobin F (HbF) levels reduce clinical severity in sickle cell disease and reduce anemia in beta thalassemia. Prior generation short chain fatty acid therapeutics, arginine butyrate and phenylbutyrate, increased fetal and total hemoglobin levels in patients, but were limited by high doses or intravenous infusion. A fetal globin-inducing therapeutic with convenient oral dosing would be an advance for these classic molecular diseases. Healthy adult human subjects were treated with a novel SCFA derivative, sodium 2,2 dimethylbutyrate (SDMB), or placebo, with one of four single dose levels (2, 5, 10 and 20 mg/kg) or daily doses (5, 10, or 15 mg/kg) over 14 days, and monitored for adverse clinical and laboratory events, drug levels, reticulocytes, and HbF assays. SDMB was well-tolerated with no clinically significant adverse events related to study medication. The terminal half-life ranged from 9–15 hours. Increases in mean absolute reticulocytes were observed at all dose levels in the 14-day study. The favorable PK profiles and safety findings indicate that SDMB warrants further investigation for treatment of anemic subjects with beta hemoglobinopathies. PMID:21422239

  9. Novel red cell indices indicating reduced availability of iron are associated with high erythropoietin concentration and low ph level in the venous cord blood of newborns.

    PubMed

    Ervasti, Mari; Sankilampi, Ulla; Heinonen, Seppo; Punnonen, Kari

    2008-08-01

    There is evidence that an elevated erythropoietin (EPO) concentration is associated with signs of iron deficient erythropoiesis. The aim of this study was to evaluate the iron status by means of novel cellular indices and serum iron markers and to determine whether these are associated with EPO and pH in the venous cord blood of 193 full-term newborns. There were positive correlations between EPO and the percentage of hypochromic red blood cells (%HYPOm) and reticulocytes (%HYPOr) [r = 0.45 (p < 0.001) and r = 0.56 (p < 0.001), respectively]. %HYPOm and %HYPOr also had negative correlations with pH [r = -0.53 (p = 0.001) and r = -0.46 (p = 0.001), respectively]. Newborns who had low pH (pH < or =7.15, n = 16) had significantly higher %HYPOm, %HYPOr, and serum transferrin receptor and transferrin concentrations in their cord blood than newborns with normal pH. Thus, in newborn cord blood, the higher number of red cells and reticulocytes with lower Hb content may have impaired the oxygen carrying capacity that has been a trigger for EPO production. Furthermore, signs of lower hemoglobinization of red cells are associated with low umbilical vein pH in the newborns, indicating an increased risk of birth asphyxia.

  10. Coefficient of variation of R-R intervals in electrocardiogram is a sensitive marker of anemia induced by autonomic neuropathy in type 1 diabetes.

    PubMed

    Saito, Takatoshi; Tojo, Katsuyoshi; Nishimura, Rimei; Kageyama, Shigeru; Tajima, Naoko

    2007-10-01

    The present study investigated the relationship between hemoglobin (Hb) levels and autonomic failure using a sensitive marker, coefficient of variation of R-R intervals in electrocardiogram (CVR-R) in order to clarify a cause of normocytic normochromic anemia in type 1 diabetic patients without overt nephropathy. We recruited 46 patients with type 1 diabetes and measured creatinine clearance (Ccr), HbA1c, albuminuria, Hb levels and CVR-R of all patients. In addition, the status of diabetic retinopathy and neuropathy were also evaluated. Serum erythropoietin (EPO), Fe, total iron binding capacity, lactate dehydrogenase, total bilirubin levels and number of reticulocytes and mean corpuscular volume were also measured to distinguish types of anemia. To survey the statistical correlation existing between Hb and body mass index (BMI), Ccr, HbA1c, albuminuria or retinopathy, multiple regression analysis was performed. Serum EPO, Fe, TIBC, LDH and TB levels and number of reticulocytes and MCV were within normal limits. Multiple regression analysis disclosed that HbA1c, nephropathy evaluated by albuminuria and Ccr, and retinopathy has no concern with Hb level. There is only significant relationship between Hb levels and CVR-R. Similar results were obtained even if we analyzed a group of male and female separately. We conclude that CVR-R has the strong relationship on anemia without overt nephropathy in type 1 diabetes, indicating that autonomic failure contributes on the progression of anemia via a poor response of EPO to anemia.

  11. Stress and body condition in a population of largemouth bass: implications for red-sore disease

    SciTech Connect

    Esch, G.W.; Hazen, T.C.

    1980-09-01

    The body conditions, K = 10/sup 5/(weight, g)/(standard length)/sup 3/, and various hematological characters were examined for largemouth bass (Micropterus salmoides) taken from Par Pond, a reservoir heated by effluent from a nuclear production reactor at the Savannah River Plant near Aiken, South Carolina. Largemouth bass with K less than 2.0 had significantly lower (P < 0.05) hematocrits, hemoglobin concentrations, total red blood cell counts, total white blood cell counts, and lymphocyte fractions, and significantly higher granulocyte fractions and cortisol concentrations, than those with K greater than 2.0; monocyte, thrombocyte, and reticulocyte fractions were not different between the two K-factor groupings. When data were pooled, all blood variables except the reticulocyte fraction were significantly correlated with K. Hematocrit, the lymphocyte fraction, and cortisol concentration account for 20.5% of the variation in K. These data support a previous hypothesis that elevated water temperature promotes stress. Stress within the Par Pond largemouth bass population may play an important role in the epizootiology of red-sore disease caused by the gram-negative bacterium, Aeromonas hydrophila.

  12. A systematic review of the literature for severity predictors in children with sickle cell anemia.

    PubMed

    Meier, Emily Riehm; Fasano, Ross M; Levett, Paul R

    2017-02-02

    All patients with HbSS (SCA) share the same genetic mutation but the clinical phenotype is variable and difficult to predict early in life. A reliable severity predictor would be invaluable toward directing therapeutic decisions in those patients at highest risk of SCA complications. A search of PubMed, Cochrane Clinical Trials Register, and Scopus was performed to determine which SCA severity predictors have been validated in pediatric patients. The full text of 94 of the 590 references identified was reviewed based on the title/abstract. Fifty-four articles were included in the analysis. Alpha globin gene number was the most commonly studied severity predictor, followed by fetal hemoglobin (HbF) and reticulocyte count. Alpha thalassemia trait was protective against overt stroke and abnormal transcranial Doppler (TCD) in all but one study, but not frequency of painful crisis or silent cerebral infarct. Two thirds of the HbF studies reported beneficial effects with increasing HbF levels; however, increased HbF levels were not associated with lower hospitalization or stroke rates in others. The ability to predict SCA complications was mixed for all variables, except TCD and absolute reticulocyte count. More reliable predictors are urgently needed to guide therapeutic decisions in children with SCA.

  13. Antioxidant effect of vitamin E in the treatment of nutritional iron deficiency anemia.

    PubMed

    Şimşek Orhon, Filiz; Öztürk, Gülyüz; Erbaş, Deniz; Hasanoğlu, Alev

    2006-03-05

    Oxidant status and antioxidants play important roles in anemias. The present study was conducted to investigate the oxidant-antioxidant status in iron deficiency anemia (IDA), and to evaluate the antioxidant effect of vitamin E in IDA treatment. Ten patients with IDA aged nine months were given only iron treatment, whereas another 10 patients were administered both iron and vitamin E. The complete blood count, plasma malonyldialdehyde (MDA) level, erythrocyte superoxide dismutase level, and the serum vitamin E level, both before and within the treatment phases were examined. The reticulocyte count at the first week of treatment was found lower in the vitamin E-treated group. The mean corpuscular volume (MCV) was found higher in the vitamin E-treated group at the end of therapy. The malonyldialdehyde levels of the group treated with vitamin E were found lower during treatment. These results suggest that iron administration in IDA treatment may stimulate lipid peroxidation, and that vitamin E supplied with iron may reduce the MDA production. The hematological indications of the findings of our study are that the reticulocyte response develops earlier and the microcytosis recovery occurs more rapidly in the vitamin E-administered group in comparison with the group treated with iron only.

  14. Neocytolysis on descent from altitude: a newly recognized mechanism for the control of red cell mass

    NASA Technical Reports Server (NTRS)

    Rice, L.; Ruiz, W.; Driscoll, T.; Whitley, C. E.; Tapia, R.; Hachey, D. L.; Gonzales, G. F.; Alfrey, C. P.

    2001-01-01

    BACKGROUND: Studies of space-flight anemia have uncovered a physiologic process, neocytolysis, by which young red blood cells are selectively hemolyzed, allowing rapid adaptation when red cell mass is excessive for a new environment. OBJECTIVES: 1) To confirm that neocytolysis occurs in another situation of acute plethora-when high-altitude dwellers with polycythemia descend to sea level; and 2) to clarify the role of erythropoietin suppression. DESIGN: Prospective observational and interventional study. SETTING: Cerro de Pasco (4380 m) and Lima (sea level), Peru. PARTICIPANTS: Nine volunteers with polycythemia. INTERVENTIONS: Volunteers were transported to sea level; three received low-dose erythropoietin. MEASUREMENTS: Changes in red cell mass, hematocrit, hemoglobin concentration, reticulocyte count, ferritin level, serum erythropoietin, and enrichment of administered(13)C in heme. RESULTS: In six participants, red cell mass decreased by 7% to 10% within a few days of descent; this decrease was mirrored by a rapid increase in serum ferritin level. Reticulocyte production did not decrease, a finding that establishes a hemolytic mechanism.(13)C changes in circulating heme were consistent with hemolysis of young cells. Erythropoietin was suppressed, and administration of exogenous erythropoietin prevented the changes in red cell mass, serum ferritin level, and(13)C-heme. CONCLUSIONS: Neocytolysis and the role of erythropoietin are confirmed in persons with polycythemia who descend from high altitude. This may have implications that extend beyond space and altitude medicine to renal disease and other situations of erythropoietin suppression, hemolysis, and polycythemia.

  15. Etiopathological mechanisms and clinical characteristics of hyperhemolysis syndrome in Spanish patients with thalassemia.

    PubMed

    Vagace, Jose Manuel; Cardesa, Rocío; Corbacho, Antonio; Vázquez, Teresa; de la Maya, Maria Dolores; Gonzalez, Fernando Ataulfo; Nieto, José Bartolomé; Urrutia, Emilia; Gómez, María Jesus; Pascual, Teresa; Aguinaco, Maria Reyes; Gervasini, Guillermo

    2016-09-01

    Hyperhemolysis syndrome (HHS) is characterized by severe intravascular hemolysis with a decrease in the reticulocyte count, which is triggered and aggravated by transfusion and cannot be explained by standard immunohematological studies. A nationwide study was conducted in order to retrospectively identify thalassemia patients with HHS in Spain in order to assess pre-disposing mechanisms for this syndrome. For this, the expression of adhesion (CD49, CD36) and complement-related molecules (C3a, CD59) and the levels of reticulocyte apoptosis and macrophage activation were measured in 4 thalassemia patients with HHS, 14 patients without HHS, and 10 healthy subjects. Five of the six thalassemia patients had δβ-thalassemia. The patients were not alloimmunized prior to the syndrome, which was developed after the first transfusion in all but one case. Patients with δβ-thalassemia did not respond to corticoids or immunoglobulins; only splenectomy was successful. The expression of CD49 (α4β1 integrin) was far higher in patients who had experienced HHS (85.07 ± 18.46 vs. 46.28 ± 24.31; p < 0.01), and the difference remained significant after correcting by the number of molecules analyzed (Bonferroni p < 0.05). In our population, δβ-thalassemia was the most common hemoglobinopathy in patients with HHS. Furthermore, the risk to develop this syndrome may be associated with an increased expression of α4β1 integrin.

  16. Hematological profile and martial status in rugby players during whole body cryostimulation.

    PubMed

    Lombardi, Giovanni; Lanteri, Patrizia; Porcelli, Simone; Mauri, Clara; Colombini, Alessandra; Grasso, Dalila; Zani, Viviana; Bonomi, Felice Giulio; Melegati, Gianluca; Banfi, Giuseppe

    2013-01-01

    Cold-based therapies are commonly applied to alleviate pain symptoms secondary to inflammatory diseases, but also to treat injuries or overuse, as done in sports rehabilitation. Whole body cryotherapy, a relatively new form of cold therapy, consists of short whole-body exposure to extremely cold air (-110°C to -140°C). Cryostimulation is gaining wider acceptance as an effective part of physical therapy to accelerate muscle recovery in rugby players. The aim of this study was to evaluate the effect of repeated cryostimulation sessions on the hematological profile and martial status markers in professional rugby players. Twenty-seven professional rugby players received 2 daily cryostimulation treatments for 7 consecutive days. Blood samples were collected before and after administration of the cryotherapic protocol and hematological profiles were obtained. No changes in the leukocyte count or composition were seen. There was a decrease in the values for erythrocytes, hematocrit, hemoglobin and mean corpuscular hemoglobin content, and an increase in mean corpuscular volume and red cell distribution width. Platelet count and mean volume remained unchanged. Serum transferrin and ferritin decreased, while soluble transferrin receptor increased. Serum iron and transferrin saturation were unchanged, as was reticulocyte count, whereas the immature reticulocyte fraction decreased substantially. In conclusion, in this sample of professional rugby players, cryostimulation modified the hematological profile, with a reduction in erythrocyte count and hemoglobinization paralleled by a change in martial status markers.

  17. High-Sensitivity Detection of PNH Red Blood Cells, Red Cell Precursors, and White Blood Cells.

    PubMed

    Sutherland, D Robert; Illingworth, Andrea; Keeney, Michael; Richards, Stephen J

    2015-04-01

    Flow cytometry is the method of choice to 'diagnose' paroxysmal nocturnal hemoglobinuria (PNH) and has led to improved patient management. Most laboratories have limited experience with PNH testing, and many different flow approaches are used. Careful selection and validation of antibody conjugates has allowed the development of reagent cocktails suitable for detection of PNH RBCs, CD71+ reticulocytes, and WBCs in clinical/sub-clinical PNH samples. A CD235a-FITC/CD59-PE assay was developed capable of detecting Type III PNH RBCs at 0.01% sensitivity. A protocol targeting immature CD71+ RBCs can detect PNH reticulocytes at similar sensitivity. Four-color FLAER-based neutrophil and monocyte assays were developed to detect PNH phenotypes at a level of 0.01% and 0.04% sensitivity, respectively. For instrumentation with five or more PMTs, a single-tube 5-color FLAER/CD157-based assay to simultaneously detect PNH neutrophils and monocytes is described. Using these standardized approaches, results have demonstrated good intra- and inter-laboratory performance characteristics even in laboratories with little prior experience performing PNH testing.

  18. Vitamin E levels during early iron supplementation in preterm infants.

    PubMed

    Arnon, Shmuel; Regev, Rivka H; Bauer, Sofia; Shainkin-Kestenbaum, Ruth; Shiff, Yakov; Bental, Yoram; Dolfin, Tzipora; Litmanovitz, Ita

    2009-05-01

    On the basis of preliminary data, this larger bi-institutional continuation trial evaluating the efficacy and safety of early iron supplementation in preterm infants calls attention to the levels of vitamin E, a marker of antioxidant activity, during iron treatment. A total of 116 preterm infants were randomly assigned to receive at 2 or 4 weeks of age ( N = 62, N = 54, respectively) 5 mg/kg/d of nonionic iron polymaltose complex concomitantly with a daily dose of 25 IU vitamin E (as dl-alpha-tocopherol acetate) from 2 weeks of age. Vitamin E (alpha-tocopherol) levels, iron, ferritin, hemoglobin concentration, and reticulocyte count were recorded from 2 to 8 weeks of age. The morbidities of prematurity associated with free radicals formation were also documented. A gradual increase of alpha-tocopherol levels within physiological range (0.8 to 3.5 mg/dL) was found in the 2-week and 4-week groups during the study period with no difference among the groups ( P > 0.05 for all comparisons). At 8 weeks of age, iron and ferritin levels, hemoglobin concentration, and reticulocyte count were higher in the 2-week group. No correlation was observed between timing of both iron and vitamin E supplement and hemolysis or morbidities associated with prematurity. Thus, treatment of iron with vitamin E supplement at 2 weeks of age is, in our experience, an efficacious and safe treatment for improving anemia in preterm infants.

  19. Time-course changes of hematology and clinical chemistry values in pregnant rats.

    PubMed

    Honda, Tatsuya; Honda, Katsuya; Kokubun, Chisato; Nishimura, Tomonari; Hasegawa, Mina; Nishida, Atsuyuki; Inui, Toshihide; Kitamura, Kazuyuki

    2008-08-01

    The aim of this study is to report how pregnancy alters hematology and clinical chemistry values in rats. Female and male Sprague-Dawley rats were mated; the day of copulation was designated as Day 0. Hematology and clinical chemistry measurements were conducted on Days 7, 14, 17 and 21 in pregnant rats. Measurements were also conducted in non-pregnant rats. Red blood cells (RBC), hemoglobin (Hb), hematocrit (Ht), total protein and albumin decreased on Days 7, 14, 17 and 21; sodium, chloride and glucose decreased on Days 14, 17 and 21; iron decreased on Days 17 and 21; hemoglobin content of reticulocytes (CHr), calcium, inorganic phosphorus and the albumin/globulin ratio decreased on Day 21; and total cholesterol, phospholipid and high-density lipoprotein cholesterol decreased on Day 14 in pregnant rats compared with non-pregnant rats. Reticulocyte increased on Days 7, 14 and 17; mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, neutrophil count and rate increased on Days 14, 17 and 21; platelets, fibrinogen, triglyceride and free fatty acid increased on Days 17 and 21; and activated partial thromboplastin time was prolonged on Days 17 and 21 in pregnant rats compared with non-pregnant rats. The decreased RBC, Hb, Ht, CHr and iron in pregnant rats indicated that they suffered from iron deficiency anemia. These data can be used as background information for effective evaluation in reproductive toxicology studies.

  20. Ricin - inhibitor design. Annual report, 15 April 1994-14 April 1995

    SciTech Connect

    Schramm, V.L.

    1995-05-14

    Substrates for ricin A-chain include short RNA stem-loop structures which have been synthesized with radioactive labels for ease of catalytic assay and for kinetic isotope effects. Ricin A-chain from several sources is incapable of completing multiple catalytic cycles using these substrates. A family of ricin substrate analogue molecules have been synthesized and tested which are specific for transition states with oxycarbonium character or for enzymatic mechanisms involving protonation of the adenine leaving group. Formycin analogues were incorporated into RNA oligomeric structures and tested for binding to ricin A-chain or as inhibitors of the ricin-inactivation of in vitro translation using rabbit reticulocyte lysates. Ribo-oxycarbonium ion analogues containing iminoribitol analogues of ribose were synthetically incorporated into RNA oligomeric structures. Neither formycin nor ribo-oxycarbonium analogues, either singly or in RNA oligomers caused significant inhibition of ricin A-chain when assayed in reticulocyte lysate translation assays. The results indicate a novel transition state mechanism for ricin A-chain, or a requirement for additional features of 28s rRNA to bind transition state analogues.

  1. Evaluations of the mutagenicity of a pigment extract from bulb culture of Hippeastrum reticulatum.

    PubMed

    Nitteranon, Viriya; Kittiwongwattana, Chokchai; Vuttipongchaikij, Supachai; Sakulkoo, Jenjira; Srijakkoat, Monthira; Chokratin, Pakawieng; Harinasut, Poontariga; Suputtitada, Saowanee; Apisitwanich, Somsak

    2014-07-01

    The use of anthocyanins in food products as colorants has been limited because of their instability toward alkaline pH and high temperature. This study aimed to determine color stability and mutagenicity of the anthocyanin-based pigment extract from bulb cultures of Hippeastrum (Hippeastrum reticulatum). The pigment extract retained its reddish-orange color under alkaline conditions (⩽pH 11) and was stable up to 6 h at 95 °C. The mutagenicity of the extract was evaluated in vitro and in vivo. Hippeastrum pigment extract up to 1.25 mg plate(-1) was found non-mutagenic in Ames test using Salmonella typhimurium strain TA98 and TA100. Chromosome aberrations were observed when human lymphocytes were treated with the extract up to 1.5 mg ml(-1). However, the extract up to 1.4 mg ml(-1) was found to exhibit relatively low or no mutagenicity in in vitro comet assays with human lymphocytes. In in vivo micronucleated reticulocyte assay, mice were treated orally with the extract up to 1 g kg(-1). No significant increase of the percentage of micronucleated peripheral reticulocytes compared to the negative control groups was found. Taken together, our study indicates that Hippeastrum pigment extract is potentially applicable as an additive colorant in the diet and related products.

  2. Erythrocyte Enrichment in Hematopoietic Progenitor Cell Cultures Based on Magnetic Susceptibility of the Hemoglobin

    PubMed Central

    Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V.; Moore, Lee R.; Chalmers, Jeffrey J.; Zborowski, Maciej

    2012-01-01

    Using novel media formulations, it has been demonstrated that human placenta and umbilical cord blood-derived CD34+ cells can be expanded and differentiated into erythroid cells with high efficiency. However, obtaining mature and functional erythrocytes from the immature cell cultures with high purity and in an efficient manner remains a significant challenge. A distinguishing feature of a reticulocyte and maturing erythrocyte is the increasing concentration of hemoglobin and decreasing cell volume that results in increased cell magnetophoretic mobility (MM) when exposed to high magnetic fields and gradients, under anoxic conditions. Taking advantage of these initial observations, we studied a noninvasive (label-free) magnetic separation and analysis process to enrich and identify cultured functional erythrocytes. In addition to the magnetic cell separation and cell motion analysis in the magnetic field, the cell cultures were characterized for cell sedimentation rate, cell volume distributions using differential interference microscopy, immunophenotyping (glycophorin A), hemoglobin concentration and shear-induced deformability (elongation index, EI, by ektacytometry) to test for mature erythrocyte attributes. A commercial, packed column high-gradient magnetic separator (HGMS) was used for magnetic separation. The magnetically enriched fraction comprised 80% of the maturing cells (predominantly reticulocytes) that showed near 70% overlap of EI with the reference cord blood-derived RBC and over 50% overlap with the adult donor RBCs. The results demonstrate feasibility of label-free magnetic enrichment of erythrocyte fraction of CD34+ progenitor-derived cultures based on the presence of paramagnetic hemoglobin in the maturing erythrocytes. PMID:22952572

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

    PubMed

    Jin, Xiaoxia; Abbot, Stewart; Zhang, Xiaokui; Kang, Lin; Voskinarian-Berse, Vanessa; Zhao, Rui; Kameneva, Marina V; Moore, Lee R; Chalmers, Jeffrey J; Zborowski, Maciej

    2012-01-01

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

  4. Alteration of the major phosphorylation site of eukaryotic protein synthesis initiation factor 4E prevents its association with the 48 S initiation complex.

    PubMed

    Joshi-Barve, S; Rychlik, W; Rhoads, R E

    1990-02-15

    Site-directed mutagenesis was used to replace the serine residue at the primary phosphorylation site of human eukaryotic initiation factor (eIF) 4E with an alanine residue. The mutated cDNA was transcribed in vitro, and the transcript was used to direct protein synthesis in a reticulocyte lysate system. The variant protein (eIF-4EAla) was retained on a 7-methylguanosine 5'-triphosphate (m7GTP)-Sepharose affinity column and was specifically eluted by m7GTP. Examination of eIF-4EAla by isoelectric focusing revealed two species which had the same pI values as the phosphorylated and nonphosphorylated forms of unaltered eIF-4E (here designated eIF-4ESer). However, conversion of unphosphorylated eIF-4EAla to the putative phosphorylated eIF-4EAla in the reticulocyte lysate system was slower than the corresponding conversion of eIF-4ESer. The possibility that the more acidic form of eIF-4EAla was due to NH2-terminal acetylation was ruled out by an experiment in which the acetyl-CoA pool of the reticulocyte lysate system was depleted with oxaloacetate and citrate synthase. The more acidic form of eIF-4EAla was, however, eliminated by treatment with calf intestine alkaline phosphatase, suggesting that it results from a second-site phosphorylation. When translation reaction mixtures were resolved on sucrose density gradients, the 35S-labeled eIF-4ESer was found on the 48 S initiation complex in the presence of guanylyl imidodiphosphate, as reported earlier (Hiremath, L.S., Hiremath, S.T., Rychlik, W., Joshi, S., Domier, L.L., and Rhoads, R.E. (1989) J. Biol. Chem. 264, 1132-1138). eIF-4EAla, by contrast, was not found on the 48 S complex, suggesting that phosphorylation of eIF-4E is necessary for it to carry out its role of transferring mRNA to the 48 S complex. Supporting this interpretation was the finding that eIF-4ESer isolated from 48 S initiation complexes consisted predominantly of the phosphorylated form.

  5. Comparison of male versus female responses in the Pig-a mutation assay

    PubMed Central

    Labash, Carson; Avlasevich, Svetlana L.; Carlson, Kristine; Torous, Dorothea K.; Berg, Ariel; Bemis, Jeffrey C.; MacGregor, James T.; Dertinger, Stephen D.

    2015-01-01

    Validation of the Pig-a gene mutation assay has been based mainly on studies in male rodents. To determine if the mutagen-induced responses of the X-linked Pig-a gene differ in females compared to males, 7- or 14-week old male and female Sprague Dawley rats were exposed to N-ethyl-N-nitrosourea (ENU). In the study with the 7-week old rats, exposure was to 0, 1, 5 or 25mg ENU/kg/day for three consecutive days (study Days 1–3). Pig-a mutant phenotype reticulocyte (RETCD59−) and mutant phenotype erythrocyte (RBCCD59−) frequencies were determined on study Days −4, 15, 29 and 46 using immunomagnetic separation in conjunction with flow cytometric analysis (In Vivo MutaFlow®). Additionally, blood samples collected on Day 4 were analysed for micronucleated reticulocyte (MN-RET) frequency (In Vivo MicroFlow®). The percentage of reticulocytes (%RET) was markedly higher in the 7-week old males compared to females through Day 15 (2.39-fold higher on Day −4). At 25mg/kg/day, ENU reduced Day 4 RET frequencies in both sexes, and the two highest dose levels resulted in elevated MN-RET frequencies, with no sex or treatment × sex interaction. The two highest dose levels significantly elevated the frequencies of mean RETCD59− and RBCCD59− in both sexes from Day 15 onward. RETCD59− and RBCCD59− frequencies were somewhat lower for females compared to males at the highest dose level studied, and differences in RETCD59− resulted in a statistically significant interaction effect of treatment × sex. In the study with 14-week old rats, treatment was for 3 days with 0 or 25mg ENU/kg/day. RET frequencies differed to a lesser degree between the sexes, and in this case there was no evidence of a treatment × sex interaction. These results suggest that the slightly higher response in younger males than in the younger females may be related to differences in erythropoiesis function at that age. In conclusion, while some quantitative differences were noted, there were no

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

    PubMed Central

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

    2015-01-01

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

  7. Changes in erythroid membrane proteins during erythropoietin-mediated terminal differentiation.

    PubMed

    Koury, M J; Bondurant, M C; Rana, S S

    1987-12-01

    Membrane and membrane skeleton proteins were examined in erythroid progenitor cells during terminal differentiation. The employed model system of erythroid differentiation was that in which proerythroblasts from mice infected with the anemia-inducing strain of Friend virus differentiate in vitro in response to erythropoietin (EP). With this system, developmentally homogeneous populations of cells can be examined morphologically and biochemically as they progress from proerythroblasts through enucleated reticulocytes. alpha and beta spectrins, the major proteins of the erythrocyte membrane skeleton, are synthesized in the erythroblasts both before and after EP exposure. At all times large portions of the newly synthesized spectrins exist in and are turned over in the cytoplasm. The remaining newly synthesized spectrin is found in a cellular fraction containing total membranes. Pulse-chase experiments show that little of the cytoplasmic spectrins become membrane associated, but that the proportion of newly synthesized spectrin which is membrane associated increases as maturation proceeds. A membrane fraction enriched in plasma membranes has significant differences in the stoichiometry of spectrin accumulation as compared to total cellular membranes. Synthesis of band 3 protein, the anion transporter, is induced only after EP addition to the erythroblasts. All of the newly synthesized band 3 is membrane associated. A two-dimensional gel survey was conducted of newly synthesized proteins in the plasma membrane enriched fraction of the erythroblasts as differentiation proceeded. A majority of the newly synthesized proteins remain in the same proportion to each other during maturation; however, a few newly synthesized proteins greatly increase following EP induction while others decrease markedly. Of the radiolabeled proteins observed in two dimensional gels, only the spectrins, band 3 and actin become major proteins of the mature erythrocyte membrane. Examination of

  8. Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal 'C-tag'.

    PubMed

    Jin, Jing; Hjerrild, Kathryn A; Silk, Sarah E; Brown, Rebecca E; Labbé, Geneviève M; Marshall, Jennifer M; Wright, Katherine E; Bezemer, Sandra; Clemmensen, Stine B; Biswas, Sumi; Li, Yuanyuan; El-Turabi, Aadil; Douglas, Alexander D; Hermans, Pim; Detmers, Frank J; de Jongh, Willem A; Higgins, Matthew K; Ashfield, Rebecca; Draper, Simon J

    2017-01-30

    Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS(2)Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 - currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as 'C-tag', composed of the four amino acids, glutamic acid - proline - glutamic acid - alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions

  9. [Iron deficiency in the elderly].

    PubMed

    Helsen, Tuur; Joosten, Etienne

    2016-06-01

    Anemia is a common diagnosis in the geriatric population, especially in institutionalized and hospitalized elderly. Most common etiologies for anemia in elderly people admitted to a geriatric ward are iron-deficiency anemia and anemia associated with chronic disease. Determination of serum ferritin is the most used assay in the differential diagnosis, despite low sensitivity and moderate specificity. New insights into iron homeostasis lead to new diagnostic assays such as serum hepcidin, serum transferrin receptor and reticulocyte hemoglobin equivalent.Importance of proper diagnosis and treatment for this population is large since there is a correlation between anemia and morbidity - mortality. Anemia is usually defined as hemoglobin less than 12 g/dl for women and less than 13 g/dl for men. There is no consensus for which hemoglobinvalue an investigation into underlying pathology is obligatory. This needs to be evaluated depending on functional condition of the patient.

  10. Purification and characterization of charantin, a napin-like ribosome-inactivating peptide from bitter gourd (Momordica charantia) seeds.

    PubMed

    Parkash, A; Ng, T B; Tso, W W

    2002-05-01

    A peptide designated charantin, with a molecular mass of 9.7 kDa, was isolated from bitter gourd seeds. The procedure comprised affinity chromatography on Affi-gel blue gel, ion-exchange chromatography on Mono S and gel filtration on Superdex 75. The N-terminal sequence of charantin exhibited marked similarity to that of the 7.8-kDa napin-like peptide previously isolated from bitter gourd seeds. Charantin inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 400 nm, a potency lower than that of the previously reported small ribosome-inactivating protein gamma-momorcharin (IC50 = 55 nm) which also exhibited an abundance of arginine and glutamate/glutamine residues. Charantin reacted positively in the N-glycosidase assay, yielding a band similar to that formed by the small ribosome-inactivating proteins gamma-momorcharin and luffin S.

  11. Hematologic changes in mice during and after exposure to severe hypobaric hypoxia

    NASA Technical Reports Server (NTRS)

    Huff, J. E.; Kaufman, G. E.; Ingram, M.

    1975-01-01

    Exposing mice to an atmospheric pressure of 300 mm Hg for 16 d caused a variety of hematologic effects. Hematocrit increased rapidly in the first 8 d of exposure and slowly in the second 8 d. Reticulocyte counts rose above normal, peaked on day 8, and then fell rapidly toward the control level. Macrocytic erythrocytes, formed during exposure, remained macrocytic after the termination of exposure and after the loss of their reticulum. The posthypoxic mice proved sensitive for erythropoietin bioassay. Mice injected with normal dog serum showed a significantly higher incorporation of Fe-59 than control mice injected with physiologic saline. A reduction of the duration of exposure to 10 d resulted in only a slight decrease in the sensitivity of the mouse bioassay system. However, a 16-d exposure at a pressure of 360 mm Hg resulted in considerably less sensitive bioassay animals.

  12. Elongation index of erythrocytes, study of activity of chosen erythrocyte enzymes, and the levels of glutathione, malonyldialdehyde in polycythemia vera (PV).

    PubMed

    Dąbrowski, Z; Dybowicz, A J; Marchewka, A; Teległów, A; Skotnicki, A; Zduńczyk, A; Aleksander, P; Filar-Mierzwa, K

    2011-01-01

    The principal aim of the study was to investigate rheological properties of erythrocytes obtained from patients admitted to the clinic, and diagnosed with polycythemia vera. The polycythemia vera diagnosis was based on the WHO criteria for polycythemia vera. Using a laser rheometer SSD Rheometer-Rheodyn, the elongation index of erythrocytes was determined, indicating an increased rigidity of the erythrocytes in this disease compared with the erythrocytes in healthy people. In order to explain (albeit partially) the reason for reduced elasticity, the erythrocytes of patients with polycythemia were studied for the activity of enzymes - glucose-6-phosphate dehydrogenase and acetylcholinesterase membrane enzyme, as well as the levels of glutathione and malonyldialdehyde. The elevated activities of these enzymes, the glutathione level, and elevated ‰ of reticulocytes, indicated an increased pool of juvenile erythrocyte forms; furthermore, the elevated value of malonyldialdehyde may suggest a lipid peroxidative damage in certain pool of the erythrocyte membrane in blood circulation.

  13. Red blood cell decreases of microgravity

    NASA Technical Reports Server (NTRS)

    Johnson, P. C.

    1985-01-01

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

  14. [Hemolytic anemias in adults].

    PubMed

    Müller, A; Zimmermann, R; Krause, S W

    2011-11-01

    The erythrocyte lifespan in haemolytic anemia is shortened while erythropoesis is increased. Important labaratory findings are increased reticulocytes, LDH, indirect bilirubin and a decreased haptoglobin level. The most important diagnostic tool for further work up of hemolytic anemia is the direct antiglobulin test (DAT, Coombs test) to differentiate autoimmune hemolytic anemia (AIHA) from other causes. Another important group are fragmentation syndroms (hemolytic uremic syndrome and thrombotic thrombocytopenic purpura). In these forms of haemolytic anemia fragmented red blood cells can be found in the blood smear together with thrombocytopenia. A severe problem in paroxysmal nocturnal hematuria is the incidence of thrombosis. The following review describes the most important forms of hemolytic anemia in the adult and the diagnostic and therapeutic strategies.

  15. Fatal Liver and Bone Marrow Toxicity by Combination Treatment of Dichloroacetate and Artesunate in a Glioblastoma Multiforme Patient: Case Report and Review of the Literature

    PubMed Central

    Uhl, Martin; Schwab, Stefan; Efferth, Thomas

    2016-01-01

    A 52-year-old male patient was treated with standard radiochemotherapy with temozolomide for glioblastoma multiforme (GBM). After worsening of his clinical condition, further tumor-specific treatment was unlikely to be successful, and the patient seeked help from an alternative practitioner, who administered a combination of dichloroacetate (DCA) and artesunate (ART). A few days later, the patient showed clinical and laboratory signs of liver damage and bone marrow toxicity (leukopenia, thrombocytopenia). Despite successful restoration of laboratory parameters upon symptomatic treatment, the patient died 10 days after the infusion. DCA bears a well-documented hepatotoxic risk, while ART can be considered as safe concerning hepatotoxicity. Bone marrow toxicity can appear upon ART application as reduced reticulocyte counts and disturbed erythropoiesis. It can be assumed that the simultaneous use of both drugs caused liver injury and bone marrow toxicity. The compassionate use of DCA/ART combination therapy outside of clinical trials cannot be recommended for GBM treatment. PMID:27774434

  16. New procedure for isolation of Rous sarcoma virus-specific RNA from infected cells.

    PubMed Central

    Bromley, P A; Spahr, P F; Darlix, J L

    1979-01-01

    The use of mercurated "strong stop" complementary DNA (complementary to the 5'-terminal 101 nucleotides of Rous sarcoma virus RNA) in the isolation of virus-specific RNA from infected chicken embryo fibroblasts is described. Strong stop Rous sarcoma virus complementary DNA was mercurated chemically, and, as a result of the low complexity of this DNA, short hybridization times (up to 15 min) and heating in the absence of formamide were found to be adequate conditions for the isolation of virus-specific RNA. The purity of the isolated RNA was demonstrated by analysis of labeled RNase T1-resistant oligonucleotides by two-dimensional polyacrylamide gel electrophoresis. The isolated RNA could be translated in the in vitro protein synthesis system derived from rabbit reticulocytes, and an analysis of polypeptides programmed by isolated RNA before and after immunoprecipitation further demonstrated both the purity of the isolated mRNA and the quantitative nature of the isolation procedure. Images PMID:228062

  17. Study of phosphorylation of translation elongation factor 2 (EF-2) from wheat germ.

    PubMed

    Smailov, S K; Lee, A V; Iskakov, B K

    1993-04-26

    Phosphorylation of elongation factor 2 (EF-2) by specific Ca2+/calmodulin-dependent kinase is considered as a possible mechanism of regulation of protein biosynthesis in animal cells at the level of polypeptide chain elongation. In this report we show that wheat germ EF-2 can be intensively phosphorylated by the rabbit reticulocyte EF-2 kinase. Phosphorylation results in inhibition of the activity of plant EF-2 in poly(U)-dependent cell-free translation system. Thus, the activity of EF-2 in plant cells can be potentially regulated by phosphorylation. However, we could not detect endogenous EF-2 kinase activity in wheat germ either in vitro or in vivo. Furthermore, EF-2 kinase activity is not displayed in different organs of wheat and other higher plants.

  18. cis-acting translational effects of the 5' noncoding region of c-myc mRNA

    SciTech Connect

    Parkin, N.; Darveau, A.; Nicholson, R.; Sonenberg, N.

    1988-07-01

    The authors previously shown that the 5' noncoding region of mouse c-myc mRNA has a negative effect on translational efficiency in a rabbit reticulocyte Iysate. They wanted to localize and characterize the inhibitory translational element(s) in the mRNA and to study its effect in other in vitro and in vivo systems. There they report that the restrictive element is confined to a 240-nucleotide sequence of the 5' noncoding region of mouse c-myc mRNA and that this sequence acts in cis to inhibit the translation of a heterologous mRNA. In addition, they report that the cis-inhibitory effect is also exhibited in microinjected Xenopus ooctyes and wheat-germ extracts but not in HeLa cell extracts. Transfection of corresponding plasmid DNA constructs into several established cell lines did not produce the cis-inhibitory effect. A model to explain these results is presented.

  19. Review: Ajwa date (Phoenix dactylifera)- an emerging plant in pharmacological research.

    PubMed

    Mallhi, Tauqeer Hussain; Qadir, Muhammad Imran; Ali, Muhammad; Ahmad, Bashir; Khan, Yusra Habib; Rehman, Attaur

    2014-05-01

    Date Fruits are consumed in Arab areas for a long time as a part of essential diet. Phoenix dactylifera belongs to family Arecaceae and its leaves, barks, pits, fruits and pollens have anticancer, antioxidant, hepatoprotective, antidiabetic, antihypertensive, antiulcertavie, anti-inflammatory, antiproliferative, antimutagenic, antidiarheal, antibacterial, antifungal and antiviral potential. Besides these, Dates also increase level of estrogen, testosterone, RBCs, Hb, PCV, reticulocytes and platelet counts. It can also cure lead induced heamotoxicity, side effects of methylprednisolon, male and female infertility. It has also cerebroprotective, neuroprotective and haemopoietic activity. Phoenix dactylifera can be used for number of complications if further evaluated and isolated. The present paper is an overview of pharmacological properties of Phoenix dactylifera reported in literature.

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

    SciTech Connect

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

    1983-03-30

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

  1. Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L. 1

    PubMed Central

    Matilla, Angel; Nicolás, Gregorio; Vicente, Oscar; Sierra, José Manuel

    1980-01-01

    Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m7 GTP and m7 GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA. PMID:16661345

  2. Diagnosis and treatment of anemia in patients with inflammatory bowel disease

    PubMed Central

    Mücke, Victoria; Mücke, Marcus M.; Raine, Tim; Bettenworth, Dominik

    2017-01-01

    Anemia represents one of the most frequent complications in inflammatory bowel disease (IBD) and severely impairs the quality of life of affected patients. The etiology of anemia in IBD patients can be multifactorial, often involving a combination of iron deficiency (ID) and anemia of chronic disease (ACD). Although current guidelines recommend screening for and treatment of anemia in IBD patients, current observational data suggest that it still remains underdiagnosed and undertreated. Besides basic laboratory parameters (e.g. mean corpuscular volume, reticulocyte count, serum ferritin, transferrin saturation, etc.), the concentration of soluble transferrin receptor (sTfR) and novel parameters such as the sTfR/log ferritin index can guide the challenging task of differentiating between ID and ACD. Once identified, causes of anemia should be treated accordingly. This review summarizes our current understanding of anemia in IBD patients, including the underlying pathology, diagnostic approaches and appropriate anemia treatment regimens. PMID:28042234

  3. Amiloride, protein synthesis, and activation of quiescent cells.

    PubMed

    Lubin, M; Cahn, F; Coutermarsh, B A

    1982-11-01

    Amiloride is known to inhibit both influx of sodium ions and activation of quiescent cells by growth factors. The coincidence of these effects has been cited to support the proposal that influx of sodium ions acts as a mitogenic signal. Although it was noted that amiloride inhibited protein synthesis, this was attributed to an action on transport of amino acids, particularly those coupled to sodium fluxes. We find, however, that amiloride directly inhibits polypeptide synthesis in a reticulocyte lysate. In Swiss 3T3 cells, concentrations of amiloride and of cycloheximide that are nearly matched in their degree of inhibition of protein synthesis, produce about the same degree of inhibition of transit of cells from G0 to S. Inhibition of protein synthesis is sufficient to explain the effect of amiloride on mitogenesis; the drug, therefore, is not suitable for testing the hypothesis that sodium influx is a mitogenic signal.

  4. [Clinical thinking and decision making in practice. The internist as a consultant for a patient with unexplained anemia in the cardiology department].

    PubMed

    Toorians, A W; Strack van Schijndel, R J

    2001-02-17

    A man aged 73, admitted because of unstable angina pectoris also had a anaemia with a haemoglobin concentration of 2.8 mmol/l. The department of Consultative Internal Medicine was asked to elucidate this anaemia. The anamnesis proved to contain extensive diagnostic tests concerning the anaemia which had had no results. In spite of a negative anamnesis, the low serum iron level had prompted an unsuccessful search for a source of haemorrhage in the proximal and distal parts of the digestive tract. The bone marrow had been examined three times without a clear diagnosis. It was also found that there had been a deviation from the classification of anaemia which should be guided by the size of the erythrocyte and the reticulocyte count. This had resulted in diagnostics that where inconvenient to the patient; also the patient had been treated without success with ferrofumarate. Ultimately, he proved to suffer from autoimmune hypothyroidism; the anaemia was resolved by substitution therapy.

  5. Chloroplast protein import inhibition by a soluble factor from wheat germ lysate.

    PubMed

    Schleiff, Enrico; Motzkus, Michael; Soll, Jürgen

    2002-09-01

    Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.

  6. [Pathophysiology, diagnosis and treatment of anemia].

    PubMed

    Ozawa, Keiya

    2008-03-01

    Anemia can result from deficient erythropoiesis [aplastic anemia, myelodysplastic syndromes (MDS), iron deficiency anemia, anemia of chronic disease (ACD), thalassemia, megaloblastic anemia, chronic renal failure, hematological malignancies, etc.], excessive RBC destruction [hereditary spherocytosis, inherited enzyme deficiency, hemoglobinopathies, autoimmune hemolytic anemia (AIHA), paroxysmal nocturnal hemoglobinuria (PNH), etc.], and blood loss. Based on the measured red cell size(MCV), anemia is classified as microcytic, normocytic, or macrocytic. Iron parameters (serum iron, serum ferritin, etc.), reticulocyte count, bone marrow examination, Coombs test, serum vitamin B12 level, and Ham test are also useful in the differential diagnosis of anemia. Novel treatment of anemia includes lenalidomide for 5q(-)MDS, azacitidine for high-risk MDS, and eculizumab for PNH. Oral iron chelator(deferasirox) developed for the treatment of transfusional iron overload is also very useful for the management of patients with bone marrow failure syndromes.

  7. Baicalein protects mice against radiation-induced DNA damages and genotoxicity.

    PubMed

    Gandhi, Nitin Motilal

    2013-07-01

    Baicalein is the major flavonoid extracted from the root of Scutellaria baicaleins. This flavonoid is used extensively in Chinese herbal medicine. In the present study baicalein is evaluated for its radioprotective properties. Human blood cells when exposed to the γ-radiation ex vivo in presence of baicalein underwent the reduced DNA damage compared to the control. Baicalein administration prior to the whole-body γ-radiation (4 Gy) exposure of mice resulted in protecting the damage to the DNA as measured in their blood cells by alkaline comet assay. Mice when exposed to the radiation (whole body; 1.7 Gy) resulted in damage to the bone marrow as measured by micronucleated reticulocyte (MNRET) formation. Baicalein pre-treatment reduces the radiation induced damage to the bone marrow cells, as there was decrease in the percentage MNRET formation. These findings indicate radio-protecting ability of baicalein.

  8. Glutathione peroxidase 4 prevents necroptosis in mouse erythroid precursors

    PubMed Central

    Canli, Özge; Alankuş, Yasemin B.; Grootjans, Sasker; Vegi, Naidu; Hültner, Lothar; Hoppe, Philipp S.; Schroeder, Timm; Vandenabeele, Peter; Bornkamm, Georg W.

    2016-01-01

    Maintaining cellular redox balance is vital for cell survival and tissue homoeostasis because imbalanced production of reactive oxygen species (ROS) may lead to oxidative stress and cell death. The antioxidant enzyme glutathione peroxidase 4 (Gpx4) is a key regulator of oxidative stress–induced cell death. We show that mice with deletion of Gpx4 in hematopoietic cells develop anemia and that Gpx4 is essential for preventing receptor-interacting protein 3 (RIP3)-dependent necroptosis in erythroid precursor cells. Absence of Gpx4 leads to functional inactivation of caspase 8 by glutathionylation, resulting in necroptosis, which occurs independently of tumor necrosis factor α activation. Although genetic ablation of Rip3 normalizes reticulocyte maturation and prevents anemia, ROS accumulation and lipid peroxidation in Gpx4-deficient cells remain high. Our results demonstrate that ROS and lipid hydroperoxides function as not-yet-recognized unconventional upstream signaling activators of RIP3-dependent necroptosis. PMID:26463424

  9. Hemolytic disease of the fetus and newborn due to anti-Ge3: combined antibody-dependent hemolysis and erythroid precursor cell growth inhibition.

    PubMed

    Blackall, Douglas P; Pesek, Gina D; Montgomery, Matthew M; Oza, Krishna K; Arndt, Patricia A; Garratty, George; Shahcheraghi, Ali; Denomme, Gregory A

    2008-10-01

    The Gerbich (Ge) antigens are a collection of high-incidence antigens carried on the red blood cell membrane glycoproteins, glycophorins C and D. Antibodies against these antigens are uncommon, and there have been only rare case reports of hemolytic disease of the fetus and newborn due to anti-Ge. In this case report, we present a neonate with severe anemia and hyperbilirubinemia due to anti-Ge3. Routine and special laboratory studies undertaken in this case suggested two mechanisms for the patient's hemolysis and persistent anemia. Antibody-dependent hemolysis was associated with early-onset hyperbilirubinemia, anemia, and a mild reticulocytosis, and inhibition of erythroid progenitor cell growth was associated with late anemia and normal bilirubin and reticulocyte values. Though rare, anti-Ge3 can be a dangerous antibody in pregnancy. Affected neonates may require intensive initial therapy and close follow-up for at least several weeks after delivery.

  10. Low doses of high-potency antithymocyte globulin (ATG) in severe aplastic anemia: experience with the Mexican ATG.

    PubMed

    Delgado-Lamas, J L; López-Karpovitch, X; Marín-López, A; Romero-García, F; Ruiz-Argüelles, G J; Ruiz-Gonzalez, D S; Taboada, C; Vázquez-Villegas, V; Elena Zarzosa, M

    1989-01-01

    Twenty patients with severe aplastic anemia (SAA) were treated with low doses (1-5 mg/kg/day) of a high-potency antithymocyte globulin (ATG) produced in Mexico, shown to have at least a 10-fold potency as compared with other globulins of commercial sources. Patients received ATG within a 10-day period, every other day (5 doses) at a dose of 1 mg/kg/day (4 courses), 2 mg/kg/day (12 courses) or 5 mg/kg/day (8 courses). Four patients received 2 consecutive courses of different doses of ATG. A response rate of 42% was recorded in the group, assessed by means of increases in reticulocytes, granulocytes or platelets. One patient showed a complete remission. The 570-day survival of the group was 51%. It is concluded that the domestically produced ATG is useful in the treatment of some patients with SAA in Mexico.

  11. Malaria invasion ligand RH5 and its prime candidacy in blood-stage malaria vaccine design

    PubMed Central

    Ord, Rosalynn L; Rodriguez, Marilis; Lobo, Cheryl A

    2015-01-01

    With drug resistance to available therapeutics continuing to develop against Plasmodium falciparum malaria, the development of an effective vaccine candidate remains a major research goal. Successful interruption of invasion of parasites into erythrocytes during the blood stage of infection will prevent the severe clinical symptoms and complications associated with malaria. Previously studied blood stage antigens have highlighted the hurdles that are inherent to this life-cycle stage, namely that highly immunogenic antigens are also globally diverse, resulting in protection only against the vaccine strain, or that naturally acquired immunity to blood stage antigens do not always correlate with actual protection. The blood stage antigen reticulocyte binding homolog RH5 is essential for parasite viability, has globally limited diversity, and is associated with protection from disease. Here we summarize available information on this invasion ligand and recent findings that highlight its candidacy for inclusion in a blood-stage malaria vaccine. PMID:25844685

  12. The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation

    PubMed Central

    Wölwer, Christina B.; Gödde, Nathan; Pase, Luke B.; Elsum, Imogen A.; Lim, Krystle Y. B.; Sacirbegovic, Faruk; Walkley, Carl R.; Ellis, Sarah; Ohno, Shigeo; Matsuzaki, Fumio; Russell, Sarah M.; Humbert, Patrick O.

    2017-01-01

    Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation. PMID:28095473

  13. Total versus partial splenectomy in pediatric hereditary spherocytosis: A systematic review and meta-analysis.

    PubMed

    Guizzetti, Leonardo

    2016-10-01

    To compare the clinical effectiveness of total splenectomy (TS) or partial splenectomy (PS) in pediatric hereditary spherocytosis, a systematic review and meta-analysis was performed (PROSPERO registration CRD42015030056). There were 14 observational studies comparing pre- and postoperative hematologic parameters. Secondary outcomes include in-hospital infections, surgical complications, symptomatic recurrence, and biliary disease. TS is more effective than PS to increase hemoglobin (3.6 g/dl vs. 2.2 g/dl) and reduce reticulocytes (12.5% vs. 6.5%) after 1 year; outcomes following PS are stable for at least 6 years. There were no cases of overwhelming postsplenectomy sepsis. A population-based patient registry is needed for long-term follow-up.

  14. [Familial pyridoxine-responsive sideroblastic anaemia. One case (author's transl)].

    PubMed

    Garbarz, M; Bernard, J F; Boivin, P

    1980-11-22

    In a 24-year old man presenting with hypochromic microcytic anaemia, low reticulocyte count, increased serum iron and bone marrow erythroid hyperplasia with numerous ringed sideroblasts, the diagnosis of sideroblastic anaemia was confirmed by radioisotope study and bone marrow electron microscopy. The hereditary nature of the disease was demonstrated by the presence of microcytosis and increased serum iron in the mother and two sisters of the patient. One of the two sisters also had anaemia and abnormal ringed sideroblasts in her bone marrow. Her haemoglobin values and those of the propositus returned to normal under pyridoxine treatment, but hypochromia and abnormal sideroblasts persisted. This case of familial pyridoxine-responsive sideroblastic anaemia is consisted with X-chromosome-mediated transmission. The incomplete effect of pyridoxine suggests a congenital deficiency of some pyridoxine metabolism enzyme or another, as yet unidentified mechanism.

  15. Chronic Restraint Stress Upregulates Erythropoiesis through Glucocorticoid Stimulation

    PubMed Central

    Voorhees, Jeffrey L.; Powell, Nicole D.; Moldovan, Leni; Mo, Xiaokui; Eubank, Timothy D.; Marsh, Clay B.

    2013-01-01

    In response to elevated glucocorticoid levels, erythroid progenitors rapidly expand to produce large numbers of young erythrocytes. Previous work demonstrates hematopoietic changes in rodents exposed to various physical and psychological stressors, however, the effects of chronic psychological stress on erythropoiesis has not be delineated. We employed laboratory, clinical and genomic analyses of a murine model of chronic restraint stress (RST) to examine the influence of psychological stress on erythropoiesis. Mice exposed to RST demonstrated markers of early erythroid expansion involving the glucocorticoid receptor. In addition, these RST-exposed mice had increased numbers of circulating reticulocytes and increased erythropoiesis in primary and secondary erythroid tissues. Mice also showed increases in erythroid progenitor populations and elevated expression of the erythroid transcription factor KLF1 in these cells. Together this work reports some of the first evidence of psychological stress affecting erythroid homeostasis through glucocorticoid stimulation. PMID:24205034

  16. Reading two bases twice: mammalian antizyme frameshifting in yeast.

    PubMed Central

    Matsufuji, S; Matsufuji, T; Wills, N M; Gesteland, R F; Atkins, J F

    1996-01-01

    Programmed translational frameshifting is essential for the expression of mammalian ornithine decarboxylase antizyme, a protein involved in the regulation of intracellular polyamines. A cassette containing antizyme frameshift signals is found to direct high-level (16%) frameshifting in yeast, Saccharomyces cerevisiae. In contrast to +1 frameshifting in the mammalian system, in yeast the same frame is reached by -2 frameshifting. Two bases are read twice. The -2 frameshifting is likely to be mediated by slippage of mRNA and re-pairing with the tRNA in the P-site. The downstream pseudoknot stimulates frameshifting by 30-fold compared with 2.5-fold in reticulocyte lysates. When the length of the spacer between the shift site and the pseudoknot is extended by three nucleotides, +1 and -2 frameshifting become equal. Images PMID:8635469

  17. The role of exosomes in hepatitis, liver cirrhosis and hepatocellular carcinoma.

    PubMed

    Shen, Jiliang; Huang, Chiung-Kuei; Yu, Hong; Shen, Bo; Zhang, Yaping; Liang, Yuelong; Li, Zheyong; Feng, Xu; Zhao, Jie; Duan, Lian; Cai, Xiujun

    2017-02-22

    Exosomes are small vesicles that were initially thought to be a mechanism for discarding unneeded membrane proteins from reticulocytes. Their mediation of intercellular communication appears to be associated with several biological functions. Current studies have shown that most mammalian cells undergo the process of exosome formation and utilize exosome-mediated cell communication. Exosomes contain various microRNAs, mRNAs and proteins. They have been reported to mediate multiple functions, such as antigen presentation, immune escape and tumour progression. This concise review highlights the findings regarding the roles of exosomes in liver diseases, particularly hepatitis B, hepatitis C, liver cirrhosis and hepatocellular carcinoma. However, further elucidation of the contributions of exosomes to intercellular information transmission is needed. The potential medical applications of exosomes in liver diseases seem practical and will depend on the ingenuity of future investigators and their insights into exosome-mediated biological processes.

  18. Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

    PubMed Central

    Ciechanover, A; Heller, H; Katz-Etzion, R; Hershko, A

    1981-01-01

    It had been shown previously that the heat-stable polypeptide of the ATP-dependent proteolytic system of reticulocytes, designated APF-1, forms covalent conjugates with protein substrates in an ATP-requiring process. We now describe an enzyme that carries out the activation by ATP of the polypeptide with pyrophosphate displacement. The formation of AMP-polypeptide and transfer of the polypeptide to a secondary acceptor are suggested by an APF-1 requirement for ATP-PPi and ATP-AMP exchange reactions, respectively. With radiolabeled polypeptide, an ATP-dependent labeling of the enzyme was shown to be by a linkage that is acid stable but is labile to treatment with mild alkali, hydroxylamine, borohydride, or mercuric salts. It therefore appears that the AMP-polypeptide undergoes attack by an -SH group of the enzyme to form a thiolester. PMID:6262770

  19. Normocytic normochromic anemia due to automatic neuropathy in type 2 diabetic patients without severe nephropathy: a possible role of microangiopathy.

    PubMed

    Saito, Takatoshi; Tojo, Katsuyoshi; Morimoto, Aya; Tajima, Naoko

    2005-12-01

    We describe here four male patients with long-term and poorly controlled type 2 diabetes mellitus. They shared many common characteristic complications, such as severe autonomic neuropathy, proliferative retinopathy and normocytic normochromic anemia without progressive renal failure and macroangiopathy. They also showed normal levels of erythropoietin and reticulocyte, which was considered relatively low. The coefficient of variation of R-R, a useful method to estimate autonomic failure, showed markedly advanced autonomic neuropathy in all four patients. Coronary angiography did not reveal stenosis, anomaly or collateral vessels, but left ventriclography showed diffuse or partial hypokinesis. Massive proteinuria, high urinary levels of N-acetyl-beta-D-glucosamidase (NAG) and beta2-microglobulin (beta2M) were detected, though creatinine clearance (Ccr) was not so deteriorated. Treatment with recombinant erythropoietin increased their hemoglobin and hematocrit levels. These common points have a possibility to be brought about by tubulointerstitial damage and microangiopathy may be involved in it.

  20. Refractory anemia in human immunodeficiency virus: Expect the unexpected.

    PubMed

    Mirgh, Sumeet Prakash; Mishra, Vikas A; Shah, Virti D; Sorabjee, Jehangir Soli

    2016-01-01

    Pure red cell aplasia (PRCA) is an uncommon hematological disorder affecting selectively the erythroid cell lines. PRCA is defined as anemia with normal leukocyte and platelet counts, a corrected reticulocyte count <1%, <5% erythroid precursors in the bone marrow and an absence of hemolysis. We describe a case of Zidovudine (AZT) induced PRCA causing severe anemia in a patient taking antiretroviral therapy (ART) after 4 months of starting therapy and in whom all other causes were excluded. The hematological abnormalities resolved after AZT was replaced with tenofovir and the patient remained transfusion independent thereafter. A slowly progressive normocytic-normochromic anemia and reticulocytopenia, without leukopenia and thrombocytopenia in a patient, should raise the suspicion of PRCA. Search for underlying diseases, infections and drugs may help in the diagnosis and etiology of acquired PRCA. Elimination of potentially causative factors may induce complete recovery. AZT is a well-known cause of anemia and thus should be used with caution in the initiation of ART.

  1. Clinico-biochemical studies on acute toxic nephropathy in goats due to uranyl nitrate

    SciTech Connect

    Dash, P.K.; Joshi, H.C.

    1989-02-01

    Acute toxic nephropathy was produced in 6 healthy goats by injecting intravenously 1% uranyl nitrate (UN) (15 mg/kg body weight). The early painful clinical signs simulating shock progressed with subnormal temperature, slow-shallow respiration and arrhythmic pulse followed by death due to respiratory failure within 96 to 120 hr. All the affected goats had normocytic normochromic anemia, leucocytosis, neutrophilia with left shift eosinopenia, decreased monocytes and presence of 1-2% reticulocytes in the peripheral blood smears. On blood chemical analysis, a uniform and continuous rise was seen in serum creatinine with a concomitant daily increase of serum urea and uric acid. Simultaneous analysis of urine indicated polyuria leading to oliguria, acidic pH, albuminuria, glycosuria with presence of neutrophils, RBC's, epithelial and fatty casts, increase of triple phosphate, and cystine crystals reflecting acute damage of kidneys in the affected goats.

  2. Coincidental Finding of Beta Thalassaemia Minor in a Patient of Lacquer Thinner Poisoning Presenting as Methaemoglobinemia

    PubMed Central

    Mohanpuria, Anisha; Buxi, Gurdeep; Kumar, Vijay

    2016-01-01

    Lacquer thinner, commonly used for removing household paints, is known to contain various aromatic hydrocarbons and naphtha; if ingested, may cause methaemoglobinemia. We report a case of 13-year-old girl who presented with episodes of vomiting, abdominal pain and numbness of limbs. Peripheral blood smear showed few fragmented erythrocytes and 10-12 nucleated red blood cells /100 white blood cells. Reticulocyte count was 15% with presence of Heinz bodies. There was a history of accidental ingestion of lacquer paint thinner. Levels of methaemoglobin were very high along with raised HbA2 levels. An impression of haemolytic anaemia, due to lacquer thinner paint poisoning, with methaemoglobinemia was given. Incidentally High Performance Liquid Chromatography (HPLC) revealed increased HbA2 levels i.e., heterozygous inheritance of beta thalassaemia minor. Patient responded well to methylene blue. Blood transfusion was performed and symptomatic treatment was given. PMID:28050380

  3. Age-related changes in haematology and serum chemistry of Weiser-Maples guineapigs (Cavia porcellus).

    PubMed

    Kitagaki, M; Yamaguchi, M; Nakamura, M; Sakurada, K; Suwa, T; Sasa, H

    2005-07-01

    Age-related changes in haematology and serum chemistry values were examined in male and female Weiser-Maples guineapigs (Cavia porcellus). Haematological changes that significantly (P<0.01) correlated with ageing were increased white blood cell and neutrophil counts in both sexes, decreased lymphocyte counts in both sexes, decreased reticulocyte and platelet counts in males, and decreased basophil counts in females. For serum chemistry, increases in total protein, triglycerides, blood urea nitrogen and creatinine were seen in both sexes, along with increases in total cholesterol in males and sodium in females. Decreased alkaline phosphatase in both sexes and decreased chloride in males were significantly (P<0.01) associated with age. These age-related changes are compared with the published literature.

  4. Iron-binding fragments from the carboxyl-terminal region of hen ovotransferrin.

    PubMed Central

    Williams, J

    1975-01-01

    1. When iron-saturated hen ovotransferrin was treated with subtilisin the N-terminal half was digested at a faster rate than the C-terminal half, allowing the latter to be isolated as a single-chain fragment of mol.wt 35000. 2. In mildly acid conditions iron-ovotransferrin loses iron preferentially from its N-terminal binding site. Trypsin digestion of the resulting monoferric ovotransferrin also gave rise to a C-terminal fragment. 3. Comparison of the N-terminal fragment with the C-terminal fragments shows differences in composition, peptide 'maps', CNBr-cleavage patterns and antigenic structures. The C-terminal fragments carry the carbohydrate group of ovotransferrin. 4. Both N-terminal and C-terminal fragments donate their bound iron to rabbit reticulocytes. Images PLATE 1 PLATE 2 PMID:811217

  5. Analyses of Interactions Between Heparin and the Apical Surface Proteins of Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Kobayashi, Kyousuke; Takano, Ryo; Takemae, Hitoshi; Sugi, Tatsuki; Ishiwa, Akiko; Gong, Haiyan; Recuenco, Frances C.; Iwanaga, Tatsuya; Horimoto, Taisuke; Akashi, Hiroomi; Kato, Kentaro

    2013-11-01

    Heparin, a sulfated glycoconjugate, reportedly inhibits the blood-stage growth of the malaria parasite Plasmodium falciparum. Elucidation of the inhibitory mechanism is valuable for developing novel invasion-blocking treatments based on heparin. Merozoite surface protein 1 has been reported as a candidate target of heparin; however, to better understand the molecular mechanisms involved, we characterized the molecules that bind to heparin during merozoite invasion. Here, we show that heparin binds only at the apical tip of the merozoite surface and that multiple heparin-binding proteins localize preferentially in the apical organelles. To identify heparin-binding proteins, parasite proteins were fractionated by means of heparin affinity chromatography and subjected to immunoblot analysis with ligand-specific antibodies. All tested members of the Duffy and reticulocyte binding-like families bound to heparin with diverse affinities. These findings suggest that heparin masks the apical surface of merozoites and blocks interaction with the erythrocyte membrane after initial attachment.

  6. Intrathoracic extramedullary hematopoiesis: appearance on /sup 99m/Tc sulfur colloid marrow scan

    SciTech Connect

    Bronn, L.J.; Paquelet, J.R.; Tetalman, M.R.

    1980-06-01

    Imaging of the bone marrow by radionuclide scanning was performed using colloids, which are phagocytized by the reticuloendothelial cells of the marrow, or radioiron, which is incorporated into reticulocytes. The use of the former radiopharmaceutical is based on the assumption, generally valid except in aplastic states or after irradiation, that the distribution of hematopoietic and reticuloendothelial tissue in the marrow is similar. Regardless of the method used, active adult marrow is normally distributed only in the axial skeleton and proximal humeri and femurs. Marrow imaging has been used in the evaluation of myeloproliferative disorders, leukemia, lymphoma, aplastic states, malignancy metastatic to marrow, and hemolytic anemia. We report a case of thalassemia major in which the diagnosis of intrathoracic extramedullary hematopoiesis was confirmed with the /sup 99m/Tc sulfur colloid bone marrow scan.

  7. Procedures for monitoring recombinant erythropoietin and analogues in doping control.

    PubMed

    Segura, Jordi; Pascual, José A; Gutiérrez-Gallego, Ricardo

    2007-08-01

    The present report summarizes the main analytical strategies developed to identify the presence of recombinant erythropoietin (EPO) administered as a doping agent. Indirect evidence is based on the analysis of blood parameters (haemoglobin, haematocrit, reticulocytes, macrocytes, etc.) and serum markers (concentration of EPO and serum transferrin receptors, etc.). The problem of intertechnique comparison for reliable results evaluation is emphasized, especially for serum markers. Charge differences between isoforms of recombinant EPO and native urinary EPO are the grounds for the isoelectric focusing-double blotting-chemiluminescence detection method presently approved for doping control. Works addressing its advantages and limitations are presented and commented on. The chemical bases of the differential detection are highlighted and some future approaches for detection are also presented. The appearance and detectability of EPO analogues and mimetics susceptible for abuse are also addressed.

  8. Solenin, a novel protein with translation-inhibiting activity from the traditional Chinese medicinal fish, the sea dragon Solenognathus hardwickii.

    PubMed

    Ng, T B; Lam, Y W

    2002-06-01

    A single-chained protein designated solenin was isolated from Solenognathus hardwickii, a fish used as traditional Chinese medicinal material. Solenin was capable of inhibiting translation in a cell-free rabbit reticulocyte lysate system with an IC(50) of 2 microM and expressing a ribonuclease activity of 0.8U/mg toward yeast transfer RNA, but it lacked N-glycosidase activity characteristic of ribosome inactivating proteins Solenin exhibited a molecular weight of 18kDa and possessed an N-terminal sequence AHDAEVNEVKAQVAA. The protein was adsorbed on three types of chromatographic media: Affi-gel blue gel, CM-Sepharose and Mono S. It was devoid of antifungal and lectin activities.

  9. Anti-Self Phosphatidylserine Antibodies Recognize Uninfected Erythrocytes Promoting Malarial Anemia.

    PubMed

    Fernandez-Arias, Cristina; Rivera-Correa, Juan; Gallego-Delgado, Julio; Rudlaff, Rachel; Fernandez, Clemente; Roussel, Camille; Götz, Anton; Gonzalez, Sandra; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel; Buffet, Pierre; Ndour, Papa Alioune; Rodriguez, Ana

    2016-02-10

    Plasmodium species, the parasitic agents of malaria, invade erythrocytes to reproduce, resulting in erythrocyte loss. However, a greater loss is caused by the elimination of uninfected erythrocytes, sometimes long after infection has been cleared. Using a mouse model, we found that Plasmodium infection induces the generation of anti-self antibodies that bind to the surface of uninfected erythrocytes from infected, but not uninfected, mice. These antibodies recognize phosphatidylserine, which is exposed on the surface of a fraction of uninfected erythrocytes during malaria. We find that phosphatidylserine-exposing erythrocytes are reticulocytes expressing high levels of CD47, a "do-not-eat-me" signal, but the binding of anti-phosphatidylserine antibodies mediates their phagocytosis, contributing to anemia. In human patients with late postmalarial anemia, we found a strong inverse correlation between the levels of anti-phosphatidylserine antibodies and plasma hemoglobin, suggesting a similar role in humans. Inhibition of this pathway may be exploited for treating malarial anemia.

  10. Effects of exercise on the secondary blood markers commonly used to suspect erythropoietin doping.

    PubMed

    Robinson, Neil; Saugy, Martial; Mangin, Patrice

    2003-01-01

    Numerous trials have reported that some haematological and biochemical parameters could be put together and be used to detect and fight recombinant erythropoietin doping. Unfortunately, none of the studies mentioned the necessity of taking pre-analytical precautions to avoid possible suspicious results coming from major plasma volume changes caused notably by dehydration. Therefore we studied the behaviour of the most common secondary blood markers before and after a strenuous physical activity to find out how reliable these parameters were. The soluble transferrin receptor and the haemoglobin concentrations as well as the haematocrit level increased significantly after effort, whereas the plasma EPO concentration and the reticulocyte count remained constant. On the other hand, if the values were corrected for haemoconcentration, the soluble transferrin receptor concentration remained stable.

  11. Genotoxic effects of bisphenol A on somatic cells of female mice, alone and in combination with X-rays.

    PubMed

    Gajowik, Aneta; Radzikowska, Joanna; Dobrzyńska, Małgorzata M

    2013-10-09

    Bisphenol A (BPA), a monomer used in the manufacture of epoxy, polycarbonate, and polystyrene resins, is a xenoestrogen present in many consumer products. We investigated the effects of 2-week exposure to BPA, either alone or in combination with X-rays, on the induction of DNA damage in somatic cells of female mice in vivo. The micronucleus and alkaline comet assays were used to evaluate genotoxicity. BPA induced DNA strand breaks in lung cells but not in bone marrow lymphocytes, liver, kidney, or spleen cells. Induction of micronuclei was observed only in polychromatic reticulocytes of peripheral blood. Levels of damage following combination exposure to ionizing radiation plus BPA depended on tissue, assay, and time.

  12. Association of acquired thrombotic thrombocytopaenic purpura in a patient with pernicious anaemia.

    PubMed

    Podder, Sidhertha; Cervates, Jose; Dey, Bimalangshu R

    2015-10-13

    Pernicious anaemia is an autoimmune disease caused by intrinsic factor antibody; it leads to vitamin B12 deficiency and is marked by ineffective erythropoiesis. Haematological features reveal macrocytosis, hyperchromasia and hypersegmented neutrophils. Schistocytes are typically seen in microangiopathy, such as in thrombotic thrombocytopaenic purpura (TTP)/haemolytic uraemic syndrome or disseminated intravascular haemolysis (DIC). We report a case of a patient with severe anaemia who presented to the emergency room. Peripheral smear revealed macrocytosis, hypersegmented neutrophils and marked schistocytosis. The patient also had high reticulocyte count with high serum lactate dehydrogenase, elevated D-dimer, low fibrinogen and low haptoglobin. Vitamin B12 level came back low and the presence of intrinsic factor antibody confirmed pernicious anaemia. ADAMTS13 level was noted to be mildly reduced, which raised the suspicion of the association of acquired TTP with pernicious anaemia. Acquired TTP is another autoimmune disorder and its association with pernicious anaemia needs further evaluation.

  13. The influence of space flight on erythrokinetics in man

    NASA Technical Reports Server (NTRS)

    Leach, C. S.

    1981-01-01

    An experiment is described the purpose of which is to measure specific factors relative to the control of erythrokinetics in man which might be altered during the first seven days of exposure to the weightless environment of space flight. It is anticipated that these data will provide information relative to the mechanism by which the red cell mass is reduced during space flight. Specifically, the experiment is designed to establish: (1) if there is a significant change in red cell mass and plasma volume during the initial period of exposure to weightlessness; (2) if the plasma concentration of erythropoietin is reduced; (3) if there is an increase in the plasma concentration of erythropoietin inhibitors; (4) if there is a change in the effectiveness of erythropoiesis; and (5) if the number and age distribution of reticulocytes are altered.

  14. Interaction of antibodies to synthetic peptides of proNGF with in vitro synthesized NGF precursors.

    PubMed

    Dicou, E

    1989-09-25

    Sera raised against three synthetic peptides that reproduce sequences of the pro-nerve growth factor (proNGF) protein were tested in immunoprecipitation experiments using in vitro translation products of SP6-directed NGF mRNA in a rabbit reticulocyte lysate. The interaction of these antibodies with bacterially synthesized chimeric preproNGF was also examined. Digestion of the translation products by the gamma-subunit generated the 22 and 18 kDa intermediates. A predominant 13 kDa intermediate was obtained after digestion of translation products in wheat germ extract. This is shown to be the N-terminal peptide by immunoprecipitation with an anti-peptide serum. These antibodies may be used to detect NGF precursor cleavage products in vivo.

  15. Reticulocytosis and anemia are associated with an increased risk of death and stroke in the newborn cohort of the Cooperative Study of Sickle Cell Disease.

    PubMed

    Meier, Emily Riehm; Wright, Elizabeth C; Miller, Jeffery L

    2014-09-01

    Prior analyses of the Cooperative Study of Sickle Cell Disease (CSSCD) newborn cohort identified elevated white blood cell (WBC) count, low baseline hemoglobin and dactylitis between the ages of 1 and 2 years as markers of severe disease. Reticulocytosis was also associated with severe disease. Here, we further analyzed data collected on enrolled CSSCD infants for the predictive value of those markers for stroke and death later in life. Three hundred fifty-four CSSCD subjects were identified who had absolute reticulocyte counts (ARC) measured during infancy (2 to 6 months of age). Infants with higher ARC had significantly increased risk of stroke or death during childhood; lower hemoglobin levels also increased the risk but to a lesser degree than ARC. WBC levels and dactylitis were not predictive of death or stroke. These data suggest that reticulocytosis among asymptomatic infants with sickle cell anemia is associated with an increased risk of death or stroke during childhood.

  16. Anti-M Antibody Induced Prolonged Anemia Following Hemolytic Disease of the Newborn Due to Erythropoietic Suppression in 2 Siblings.

    PubMed

    Ishida, Atsushi; Ohto, Hitoshi; Yasuda, Hiroyasu; Negishi, Yutaka; Tsuiki, Hideki; Arakawa, Takeshi; Yagi, Yoshihito; Uchimura, Daisuke; Miyazaki, Toru; Ohashi, Wataru; Takamoto, Shigeru

    2015-08-01

    Hemolytic disease of the newborn (HDN) arising from MNSs incompatibility is rare, with few reports of prolonged anemia and reticulocytopenia following HDN. We report the younger of 2 male siblings, both of whom had anti-M-induced HDN and anemia persisting for over a month. Peripheral reticulocytes remained inappropriately low for the degree of anemia, and they needed multiple red cell transfusions. Viral infections were ruled out. Corticosteroids were given for suspected pure red cell aplasia. Anemia and reticulocytopenia subsequently improved. Colony-forming unit erythroid assay revealed erythropoietic suppression of M antigen-positive erythroid precursor cells cultured with maternal or infant sera containing anti-M. In conclusion, maternal anti-M caused HDN and prolonged anemia by erythropoietic suppression in 2 siblings.

  17. Hematopoiesis in antiorthostatic, hypokinesic rats

    NASA Technical Reports Server (NTRS)

    Dunn, C. D. R.; Johnson, P. C.; Lange, R. D.

    1983-01-01

    Rats exposed to antiorthostatic, hypokinesia showed the following effects which are comparable to those seen in man during or after space flight: weight loss, reduced food and water consumption, transient increases in peripheral hematocrit and RBC count, decreasing MCV and reduced reticulocyte count. In addition, the hemoglobin P50 was shifted to the right. A significant shortening of RBC t1/2 was only seen after suspension. Changes in leukocyte and platelet numbers in suspended rats were also comparable to those in man during space flight, but leukocyte PHA sensitivity in rats showed no consistent alteration. The results demonstrate that this model reproduces many of the hematological effects of space flight and has potential as a tool in understanding the hematopoietic response to zero gravity.

  18. Regulation of hematopoiesis in rats exposed to antiorthostatic hypokinetic/hypodynamia. II - Mechanisms of the 'anemia'

    NASA Technical Reports Server (NTRS)

    Dunn, C. D. R.; Johnson, P. C.; Lange, R. D.

    1986-01-01

    The cause of the red cell mass (RCM) deficit, which occurs in rats during suspenion, is investigated. The experimental conditions and procedures, in which male Sprague-Dawley rats are subjected to antiorthostatic hyypokinetic/hypodynamia and changes in RCM are monitored, are described. The influences of stress, reduced food and water consumption, and antiorhostatic posture on RCM are analyzed. Changes in body weight, RCM, radioiron incorporation, red blood cells (RBC), and reticulocytes, for the rats after head-down suspension are graphically presented; only the changes in RBC are related to the antiorthostatic posture. The data reveal that anemia is primarily caused by reduced food and water consumption and secondly by restricted movements.

  19. Mildiomycin: a nucleoside antibiotic that inhibits protein synthesis.

    PubMed

    Feduchi, E; Cosín, M; Carrasco, L

    1985-03-01

    Mildiomycin, a new nucleoside antibiotic, selectively inhibits protein synthesis in HeLa cells, and is less active in the inhibition of RNA or DNA synthesis. An increased inhibition of translation by mildiomycin is observed in cultured HeLa cells when they are permeabilized by encephalomyocarditis virus. This observation suggests that this antibiotic does not easily pass through the cell membrane, as occurs with other nucleoside and aminoglycoside antibiotics. The inhibition of translation is also observed in cell-free systems, such as endogenous protein synthesis in a rabbit reticulocyte lysate or the synthesis of polyphenylalanine directed by poly (U). Finally the mode of action of mildiomycin was investigated and the results suggest that the compound blocks the peptidyl-transferase center.

  20. Transient Hemolytic Anemia after Transjugular Intrahepatic Portosystemic Stent Shunt

    PubMed Central

    Garcia-Rebollo, Sagrario; Santolaria-Fernández, Francisco; Diaz-Romero, Francisco; Rodriguez-Moreno, Fermin; Martinez-Riera, Antonio

    1996-01-01

    Management of variceal bleeding secondary to portal hypertension constitutes a challenging issue, particularly in child's C cirrhotic patients. Recently, transjugular placement of self-expanding metallic stents in the liver (TIPS), creating a shunt between the portal and hepatic branches has provided a safe and promising therapeutic approach in this clinical situation. We report here the case of a 66-year-old male cirrhotic patient who developed a moderately severe clinical picture of a Coombsnegative hemolytic anemia (serum hemoglobin, 93 g/l, serum bilirubin 160.74 umol/L (9.4 mg/dl), indirect 6.3 mg/dl (107.73 umol/L); serum LDH 1220 u/l, reticulocytes, 5.1%. serum ferritin, 1221 ug/1, schistocytes in peripheral blood smear) the week after undergoing a TIPS, suggesting the development ofa microangiopathic hemolytic anaemia secondary to red blood cell disruption by passing through the metallic network of the stent. PMID:8809588

  1. Practical Murine Hematopathology: A Comparative Review and Implications for Research

    PubMed Central

    O'Connell, Karyn E; Mikkola, Amy M; Stepanek, Aaron M; Vernet, Andyna; Hall, Christopher D; Sun, Chia C; Yildirim, Eda; Staropoli, John F; Lee, Jeannie T; Brown, Diane E

    2015-01-01

    Hematologic parameters are important markers of disease in human and veterinary medicine. Biomedical research has benefited from mouse models that recapitulate such disease, thus expanding knowledge of pathogenetic mechanisms and investigative therapies that translate across species. Mice in health have many notable hematologic differences from humans and other veterinary species, including smaller erythrocytes, higher percentage of circulating reticulocytes or polychromasia, lower peripheral blood neutrophil and higher peripheral blood and bone marrow lymphocyte percentages, variable leukocyte morphologies, physiologic splenic hematopoiesis and iron storage, and more numerous and shorter-lived erythrocytes and platelets. For accurate and complete hematologic analyses of disease and response to investigative therapeutic interventions, these differences and the unique features of murine hematopathology must be understood. Here we review murine hematology and hematopathology for practical application to translational investigation. PMID:25926395

  2. Some Biochemical Properties of an Acido-Thermophilic Archae-Bacterium Sulfolobus Acidocaldarius

    NASA Astrophysics Data System (ADS)

    Oshima, Tairo; Ohba, Masayuki; Wagaki, Takayoshi

    1984-12-01

    To elucidate the phylogenic status of archaebacteria, some basic cellular components of an acido-thermophilic archaebacterium,Sulfolobus acidocaldarius, were studied. Poly(A) containing RNA was present in the cells, and performed the role of mRNA in a cell-free extract of reticulocyte or the archaebacteria. Poly(A) containing RNA was also found in other archaebacterial cells. The absence of cap structure was suggested in these RNAs. The cell-free protein synthesis using the archaebacterial extract was inhibited by anisomycin, a specific inhibitor for eukaryotic ribosomes. Two unique membrane-bound ATPases were detected. Based on resistance to H+-ATPase inhibitors, these enzymes seemed not to be F0F1-ATPase.

  3. Purification of castamollin, a novel antifungal protein from Chinese chestnuts.

    PubMed

    Wang, H X; Ng, T B

    2003-11-01

    A novel antifungal protein, designated castamollin, was isolated from Chinese chestnut (Castanea mollisima) seeds with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-Sepharose and FPLC-gel filtration on Superdex 75. Castamollin possessed a novel N-terminal sequence demonstrating little similarity to N-terminal sequences of Castanea sativa chitinase. Castamollin exhibited a molecular mass of 37kDa in gel filtration and SDS-PAGE. It inhibited the activity of human immunodeficiency virus-1 reverse transcriptase with an IC(50) of 7microM and translation in a cell-free rabbit reticulocyte lysate system with an IC(50) of 2.7microM. Castamollin displayed antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola, Physalospora piricola, and Coprinus comatus but was devoid of lectin activity.

  4. Imerslund-Grasbeck syndrome in a 5-year-old Iranian boy

    PubMed Central

    Goudarzipour, K.; Zavvar, N.; Behnam, B.; Ahmadi, M. A.

    2016-01-01

    Imerslund-Grasbeck syndrome (IGS) is a rare syndrome characterized by clinical symptoms and signs of Vitamin B12 deficiency and proteinuria. Our patient was a 5-year-old boy with pallor, lack of appetite, and low weight gain. Laboratory studies showed severe macrocytic anemia, normal reticulocyte count, negative direct coombs test, normal osmotic fragility, and autohemolysis test. He has had intermittent proteinuria since 3 years ago despite normal creatinine level and absence of hematuria or hypertension. Finally, based on low level of serum B12 vitamin and normal folate level accompanied by asymptomatic proteinuria, the diagnosis of IGS was made. Furthermore, his sister has had laboratory abnormalities without any symptoms. IGS responded to B12 replacement therapy dramatically but intermittent proteinuria persisted even after appropriate therapy. PMID:27942180

  5. [The first pillar of patient blood management. Types of anemia and diagnostic parameters].

    PubMed

    Basora Macaya, M; Bisbe Vives, E

    2015-06-01

    Patient Blood Management (PBM) is the design of a personalized, multimodal multidisciplinary plan for minimizing transfusion and simultaneously achieving a positive impact on patient outcomes. The first pillar of PBM consists of optimizing the erythrocyte mass. The best chance for this step is offered by preoperative preparation. In most cases, a detailed medical history, physical examination and laboratory tests will identify the cause of anemia. A correct evaluation of parameters that assess the state and function of iron, such as ferritin levels, and the parameters that measure functional iron, such as transferrin saturation and soluble transferrin receptor levels, provide us with essential information for guiding the treatment with iron. The new blood count analyzers that measure hypochromia (% of hypochromic red blood cells and reticulocyte hemoglobin concentrations) provide us useful information for the diagnosis and follow-up of the response to iron treatment. Measuring serum folic acid and vitamin B12 levels is essential for treating deficiencies and thereby achieving better hemoglobin optimization.

  6. Primary Sjögren syndrome presenting with hemolytic anemia and pure red cell aplasia following delivery due to Coombs-negative autoimmune hemolytic anemia and hemophagocytosis.

    PubMed

    Komaru, Yohei; Higuchi, Takakazu; Koyamada, Ryosuke; Haji, Youichiro; Okada, Masato; Kamesaki, Toyomi; Okada, Sadamu

    2013-01-01

    A 36-year-old woman presented with hemolytic anemia without a reticulocyte response 38 days after delivery. A marked reduction in erythroid cells and an increase in macrophages with active hemophagocytosis were noted in the bone marrow. While conventional Coombs' tests were negative, the level of red blood cell (RBC)-bound immunoglobulin G (IgG) was increased. The patient was diagnosed with primary Sjögren syndrome (pSS) based on her symptoms, positive anti-SS-A antibodies, Coombs-negative autoimmune hemolytic anemia and pure red cell aplasia associated with RBC-bound IgG and hemophagocytosis. The unique presentation was considered to be a consequence of immunological derangement associated with pSS, pregnancy and delivery.

  7. A 2-month-old boy with hemolytic anemia and reticulocytopenia following intravenous immunoglobulin therapy for Kawasaki disease: a case report and literature review

    PubMed Central

    Kim, Na Yeon; Kim, Joon Hwan; Park, Jin Suk; Kim, Soo Hyun; Cho, Yeon Kyung; Cha, Dong Hyun; Kim, Ki Eun; Kang, Myung Suh; Lim, Kyung Ah

    2016-01-01

    Herein, we report a rare case of hemolytic anemia with reticulocytopenia following intravenous immunoglobulin therapy in a young infant treated for Kawasaki disease. A 2-month-old boy presented with fever lasting 3 days, conjunctival injection, strawberry tongue, erythematous edema of the hands, and macular rash, symptoms and signs suggestive of incomplete Kawasaki disease. His fever resolved 8 days after treatment with aspirin and high dose infusion of intravenous immunoglobulin. The hemoglobin and hematocrit decreased from 9.7 g/dL and 27.1% to 7.4 g/dL and 21.3%, respectively. The patient had normocytic hypochromic anemia with anisocytosis, poikilocytosis, immature neutrophils, and nucleated red blood cells. The direct antiglobulin test result was positive, and the reticulocyte count was 1.39%. The patient had an uneventful recovery. However, reticulocytopenia persisted 1 month after discharge. PMID:28018448

  8. Pure red cell aplasia following autoimmune hemolytic anemia: an enigma.

    PubMed

    Saha, M; Ray, S; Kundu, S; Chakrabarti, P

    2013-01-01

    A 26-year-old previously healthy female presented with a 6-month history of anemia. The laboratory findings revealed hemolytic anemia and direct antiglobulin test was positive. With a diagnosis of autoimmune hemolytic anemia (AIHA), prednisolone was started but was ineffective after 1 month of therapy. A bone marrow trephine biopsy revealed pure red cell aplasia (PRCA) showing severe erythroid hypoplasia. The case was considered PRCA following AIHA. This combination without clear underlying disease is rare. Human parvovirus B19 infection was not detected in the marrow aspirate during reticulocytopenia. The patient received azathioprine, and PRCA improved but significant hemolysis was once again documented with a high reticulocyte count. The short time interval between AIHA and PRCA phase suggested an increased possibility of the evolution of a single disease.

  9. Oral cyclophosphamide was effective for Coombs-negative autoimmune hemolytic anemia in CD16+CD56- chronic lymphoproliferative disorder of NK-cells.

    PubMed

    Sekiguchi, Nodoka; Nishina, Sayaka; Kawakami, Toru; Sakai, Hitoshi; Senoo, Noriko; Senoo, Yasushi; Ito, Toshiro; Saito, Hiroshi; Nakazawa, Hideyuki; Koizumi, Tomonobu; Ishida, Fumihiro

    2016-12-27

    An 84-year-old woman was referred to our hospital presenting anemia. Her hemoglobin level was 5.8 g/dL, and white blood cell count was 9400/μL, consisting of 82% lymphocytes. Given the lymphocyte phenotype (CD2+, CD3-, CD16+, and CD56-) and negative whole blood EBV viral load, we made a diagnosis of chronic lymphoproliferative disorder of NK cells (CLPD-NK). We suspected hemolytic anemia because of the high levels of reticulocytes in the peripheral blood and the low haptoglobin value. Although the direct Coombs test was negative and there was no cold agglutination, we examined her red-blood-cell-bound IgG (RBC-IgG), which was elevated. She was diagnosed as having as Coombs-negative autoimmune hemolytic anemia (AIHA). We report the effectiveness of oral cyclophosphamide for Coombs-negative autoimmune hemolytic anemia in CLPD-NK.

  10. Limenin, a defensin-like peptide with multiple exploitable activities from shelf beans.

    PubMed

    Wong, Jack H; Ng, T B

    2006-05-01

    From the seeds of the shelf bean, an antifungal peptide with a molecular mass of 6.5 kDa was isolated. The isolation procedure comprised affinity chromatography on Affi-gel blue gel, ion exchange chromatography on Mono S, and gel filtration on Superdex 75. The peptide was adsorbed on Affi-gel blue gel and Mono S. It potently suppressed mycelial growth in Botrytis cinerea, Fusarium oxysporum, and Mycosphaerella arachidicola with an IC(50) of 2.9, 2.1, and 0.34 microM, respectively. It exerted antibacterial activity toward several bacterial species with an IC(50) approximating 100 microM. [Methyl-(3)H]-thymidine incorporation into isolated mouse splenocytes was stimulated. [Methyl-(3)H]-thymidine incorporation into M1 (myeloma) and L1210 (leukemia) cells was inhibited. The peptide reduced the activity of HIV-1 reverse transcriptase and also inhibited translation in a cell-free rabbit reticulocyte lysate system.

  11. Methylthiodeoxynivalenol (MTD): insight into the chemistry, structure and toxicity of thia-Michael adducts of trichothecenes.

    PubMed

    Fruhmann, Philipp; Weigl-Pollack, Theresa; Mikula, Hannes; Wiesenberger, Gerlinde; Adam, Gerhard; Varga, Elisabeth; Berthiller, Franz; Krska, Rudolf; Hametner, Christian; Fröhlich, Johannes

    2014-07-28

    Methylthiodeoxynivalenol (MTD), a novel derivative of the trichothecene mycotoxin deoxynivalenol (DON), was prepared by applying a reliable procedure for the formal Michael addition of methanethiol to the conjugated double bond of DON. Structure elucidation revealed the preferred formation of the hemiketal form of MTD by intramolecular cyclisation between C8 and C15. Computational investigations showed a negative total reaction energy for the hemiketalisation step and its decrease in comparison with theoretical model compounds. Therefore, this structural behaviour seems to be a general characteristic of thia-Michael adducts of type B trichothecenes. MTD was shown to be less inhibitory for a reticulocyte lysate based in vitro translation system than the parent compound DON, which supports the hypothesis that trichothecenes are detoxified through thia-adduct formation during xenobiotic metabolism.

  12. Studies on the erythron and the ferrokinetic responses in beagles adapted to hypergravity

    NASA Technical Reports Server (NTRS)

    Beckman, D. A.; Evans, J. W.; Oyama, J.

    1978-01-01

    Red cell survival, ferrokinetics, and hematologic parameters were investigated in beagle dogs exposed to chronic hypergravity (2.6 Gx). Ineffective erythropoiesis, red cell mass, plasma volume, and Cr-51-elution were significantly increased; maximum Fe-59 incorporation was decreased; and there was no change in the mean erythrocyte life span following autologous injection of Cr-51-labeled red cells and Fe-59-labeled transferrin. Red cell count, F(cells), total body hemoglobin (Hb), susceptability to osmotic lysis, and differential reticulocyte count were increased. White blood cell count, venous blood %Hb, mean cell volume, mean cell Hb, mean cell Hb concentration, and serum iron were decreased. No changes were observed for body mass, mg Fe per g Hb, iron binding capacity, percent saturation of iron carrying capacity, or the electrophoretic mobility of purified Hb. This study indicated that chronic exposure to hypergravity induced changes in red cell size, volume, total mass, and membrane permeability.

  13. Clinical utility of flow cytometry in the study of erythropoiesis and nonclonal red cell disorders.

    PubMed

    Chesney, Alden; Good, David; Reis, Marciano

    2011-01-01

    Erythropoiesis involves proliferation and differentiation of small population of hematopoietic stem cells resident in the bone marrow into mature red blood cells. The determination of the cellular composition of the blood is a valuable tool in the diagnosis of diseases and monitoring of therapy. Flow cytometric analysis is increasingly being used to characterize the heterogeneous cell populations present in the blood and the hematopoietic cell differentiation and maturation pathways of the bone marrow. Here we discuss the role of flow cytometry in the study of erythropoiesis and nonclonal red blood cell disorders. First, we discuss flow cytometric analysis of reticulocytes. Next, we review salient quantitative methods that can be used for detection of fetal-maternal hemorrhage (FMH). We also discuss flow cytometric analysis of high hemoglobin F (HbF) in Sickle Cell Disease (SCD), hereditary spherocytosis (HS), red cell survival and red cell volume. We conclude by discussing cell cycle of erythroid cells.

  14. In vitro synthesis of thymosin beta 4 encoded by rat spleen mRNA.

    PubMed Central

    Filipowicz, A W; Horecker, B L

    1983-01-01

    Thymosin beta 4, containing 43 amino acids and acetylated at the NH2 terminus, is synthesized in vitro in a rabbit reticulocyte lysate or in a yeast protein-synthesis system in the presence of mRNA from rat spleen. The product formed was identified as beta 4 by immunoprecipitation by a specific anti-beta 4 antiserum, comigration with authentic beta 4 in NaDodSO4/polyacrylamide gel electrophoresis and in HPLC, and identity of peptide fragments. The immunoprecipitable product generated in the wheat germ protein-synthesizing system emerged slightly ahead of beta 4 in HPLC and appeared to lack the NH2-terminal acetyl group. There was no evidence for formation of a larger polypeptide precursor of beta 4 in any of the three systems used. In sucrose density gradient centrifugation, the mRNA coding for beta 4 was recovered in the 7-8S mRNA fraction. Images PMID:6572941

  15. Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes

    PubMed Central

    Shi, Jiahai; Kundrat, Lenka; Pishesha, Novalia; Bilate, Angelina; Theile, Chris; Maruyama, Takeshi; Dougan, Stephanie K.; Ploegh, Hidde L.; Lodish, Harvey F.

    2014-01-01

    We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes. PMID:24982154

  16. Engineered red blood cells as carriers for systemic delivery of a wide array of functional probes.

    PubMed

    Shi, Jiahai; Kundrat, Lenka; Pishesha, Novalia; Bilate, Angelina; Theile, Chris; Maruyama, Takeshi; Dougan, Stephanie K; Ploegh, Hidde L; Lodish, Harvey F

    2014-07-15

    We developed modified RBCs to serve as carriers for systemic delivery of a wide array of payloads. These RBCs contain modified proteins on their plasma membrane, which can be labeled in a sortase-catalyzed reaction under native conditions without inflicting damage to the target membrane or cell. Sortase accommodates a wide range of natural and synthetic payloads that allow modification of RBCs with substituents that cannot be encoded genetically. As proof of principle, we demonstrate site-specific conjugation of biotin to in vitro-differentiated mouse erythroblasts as well as to mature mouse RBCs. Thus modified, RBCs remain in the bloodstream for up to 28 d. A single domain antibody attached enzymatically to RBCs enables them to bind specifically to target cells that express the antibody target. We extend these experiments to human RBCs and demonstrate efficient sortase-mediated labeling of in vitro-differentiated human reticulocytes.

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

    PubMed

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

    2003-05-01

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

  18. Pharmacological inhibition of Polo Like Kinase 2 (PLK2) does not cause chromosomal damage or result in the formation of micronuclei

    SciTech Connect

    Fitzgerald, Kent; Bergeron, Marcelle; Willits, Christopher; Bowers, Simeon; Aubele, Danielle L.; Goldbach, Erich; Tonn, George; Ness, Daniel; Olaharski, Andrew

    2013-05-15

    Polo Like Kinase 2 (PLK2) phosphorylates α-synuclein and is considered a putative therapeutic target for Parkinson's disease. Several lines of evidence indicate that PLK2 is involved with proper centriole duplication and cell cycle regulation, inhibition of which could impact chromosomal integrity during mitosis. The objectives of the series of experiments presented herein were to assess whether specific inhibition of PLK2 is genotoxic and determine if PLK2 could be considered a tractable pharmacological target for Parkinson's disease. Several selective PLK2 inhibitors, ELN 582175 and ELN 582646, and their inactive enantiomers, ELN 582176 and ELN 582647, did not significantly increase the number of micronuclei in the in vitro micronucleus assay. ELN 582646 was administered to male Sprague Dawley rats in an exploratory 14-day study where flow cytometric analysis of peripheral blood identified a dose-dependent increase in the number of micronucleated reticulocytes. A follow-up investigative study demonstrated that ELN 582646 administered to PLK2 deficient and wildtype mice significantly increased the number of peripheral micronucleated reticulocytes in both genotypes, suggesting that ELN 582646-induced genotoxicity is not through the inhibition of PLK2. Furthermore, significant reduction of retinal phosphorylated α-synuclein levels was observed at three non-genotoxic doses, additional data to suggest that pharmacological inhibition of PLK2 is not the cause of the observed genotoxicity. These data, in aggregate, indicate that PLK2 inhibition is a tractable CNS pharmacological target that does not cause genotoxicity at doses and exposures that engage the target in the sensory retina. - Highlights: • Active and inactive enantiomers test negative in the in vitro micronucleus test. • ELN 582646 significantly increased micronuclei at 100 and 300 mg/kg/day doses. • ELN 582646 significantly increased micronuclei in PLK2 knockout mice. • ELN 582646 decreased

  19. Anticlastogenic and anticarcinogenic potential of Thai bitter gourd fruits.

    PubMed

    Kupradinun, Piengchai; Tepsuwan, Anong; Tantasi, Nopsaran; Meesiripun, Nuntana; Rungsipipat, Anudep; Kusamran, Wannee R

    2011-01-01

    Thai bitter gourd fruits (Momordica charantia Linn., TBG) has been previously demonstrated to possess phase II detoxificating enzymes inducing properties, as well as the ability to reduce phase I carcinogen activating enzyme activity in rat liver. In addition, it was partially inhibited 7,12-dimethylbenz(a)anthracene (DMBA)- induced mammary gland carcinogenesis in female Sprague-Dawley rats. In this study, we therefore examined the anticlastogenic and anticarcinogenic effect of TBG against clastogens, cyclophosphamide (CYP) and DMBA, in mice using the in vivo erythrocyte micronucleus assay and azoxymethane (AOM)-induced colon carcinogenesis in rats, respectively. For anticlastogenicity test, male mice were fed with modified AIN-76 diets containing 6.25% and 12.5% of ground freeze-dried TBG for 2 weeks prior to administration of clastogens till the end of experiment. Blood samples were collected and counted for reticulocytes by using the fluorescent microscope. For anticarcinogeicity test, male Wistar rats were fed with modified AIN-76 diets containing 5% and 10% ground freeze-dried TBG for 2 weeks prior to, during and 1 week after the completion of AOM administration (15 mg/kg once a week for 2 weeks). It was found that TBG at 6.25% resulted in a significant reduction in micronucleated peripheral reticulocytes (MNRETs) induced by only CYP. Study on anticarcinogenic potential demonstrated that rats fed with TBG diets at the concentration tested developed significantly higher incidence as well as the multiplicities of colon tumors than the control group. These results demonstrated that Thai bitter gourd fruits possesses anticlastogenic potential against clastogen in the mouse. Interestingly, it had no preventive potential against AOM-induced colon carcinogenesis in rat, rather increasing the incidence of colonic neoplasm when giving during the initiation stage.

  20. Neonatal CD71+ erythroid cells do not modify murine sepsis mortality

    PubMed Central

    Wynn, James L.; Scumpia, Philip O.; Stocks, Blair T.; Romano-Keeler, Joann; Alrifai, Mhd Wael; Liu, Jin-Hua; Kim, Annette S.; Alford, Catherine E.; Matta, Pranathi; Weitkamp, Jörn-Hendrik; Moore, Daniel J.

    2015-01-01

    Sepsis is a major cause of neonatal mortality and morbidity worldwide. A recent report suggested murine neonatal host defense against infection could be compromised by immunosuppressive CD71+ erythroid splenocytes. We examined the impact of CD71+ erythroid splenocytes on murine neonatal mortality to endotoxin challenge or polymicrobial sepsis and characterized circulating CD71+ erythroid (CD235a+) cells in human neonates. Adoptive transfer or antibody-mediated reduction of neonatal CD71+ erythroid splenocytes did not alter murine neonatal survival to endotoxin challenge or polymicrobial sepsis challenge. Ex vivo immunosuppression of stimulated adult CD11b+ cells was not limited to neonatal splenocytes as it also occurred with adult and neonatal bone marrow. Animals treated with anti-CD71 antibody showed reduced splenic bacterial load following bacterial challenge compared to isotype-treated mice. However, adoptive transfer of enriched CD71+ erythroid splenocytes to CD71+-reduced animals did not reduce bacterial clearance. Human CD71+CD235a+ cells were common among cord blood mononuclear cells and were shown to be reticulocytes. In summary, a lack of effect on murine survival to polymicrobial sepsis following adoptive transfer or diminution of CD71+ erythroid splenocytes under these experimental conditions suggests the impact of these cells on neonatal infection risk and progression may be limited. An unanticipated immune priming effect of anti-CD71 antibody treatment was likely responsible for the reported enhanced bacterial clearance, rather than a reduction of immunosuppressive CD71+ erythroid splenocytes. In humans, the well-described rapid decrease in circulating reticulocytes after birth suggests they may have a limited role in reducing inflammation secondary to microbial colonization. PMID:26101326

  1. Is organic farming safer to farmers’ health? A comparison between organic and traditional farming

    PubMed Central

    Costa, Carla; García-Lestón, Julia; Costa, Solange; Coelho, Patrícia; Silva, Susana; Valdiglesias, Vanessa; Mattei, Francesca; Dall’Armi, Valentina; Bonassi, Stefano; Laffon, Blanca; Snawder, John; Teixeira, João Paulo

    2015-01-01

    Background Exposure to pesticides is a major public health concern, because of the widespread distribution of these compounds and their possible long term effects. Recently, organic farming has been introduced as a consumer and environmental friendly agricultural system, although little is known about the effects on workers’ health. Objectives To evaluate genetic damage and immunological alterations in workers of both traditional and organic farming. Methods Eighty-five farmers exposed to several pesticides, thirty–six organic farmers and sixty-one controls took part in the study. Biomarkers of exposure (pyrethroids, organophosphates, carbamates, and thioethers in urine and butyrylcholinesterase activity in plasma), early effect (micronuclei in lymphocytes and reticulocytes, T-cell receptor mutation assay, chromosomal aberrations, comet assay and lymphocytes subpopulations) and susceptibility (genetic polymorphisms related to metabolism - EPHX1, GSTM1, GSTT1 and GSTP1 - and DNA repair – XRCC1 and XRCC2) were evaluated. Results When compared to controls and organic farmers, pesticide farmers presented a significant increase of micronuclei in lymphocytes (frequency ratio, FR=2.80) and reticulocytes (FR=1.89), chromosomal aberrations (FR=2.19), DNA damage assessed by comet assay (mean ratio, MR=1.71), and a significant decrease in the proportion of B lymphocytes (MR=0.88). Overall, organic farmers presented similar levels of genetic damage as controls, in some cases modulated by GSTT1 and GSTM1, GSTP1 105Ile/Ile and XRCC1 399Gln/Gln genotypes. Conclusions Results confirmed the increased presence of DNA damage in farmers exposed to pesticides, and showed as exposure conditions and genetic background influence observed effects. Findings from this study indicate that no evident genetic or immunologic damage can be observed in organic farmers. PMID:24576785

  2. Pharmacokinetics and pharmacodynamics of recombinant human erythropoietin in athletes. Blood sampling and doping control

    PubMed Central

    SOUILLARD, AGNES; AUDRAN, MICHEL; BRESSOLLE, FRANÇOISE; GAREAU, RAYNALD; DUVALLET, ALAIN; CHANAL, JEAN-LOUIS

    1996-01-01

    1The pharmacokinetics of recombinant human erythropoietin (rHuEpo) were initially determined in two healthy volunteers after a single subcutaneous dose (50 u kg−1). Twenty subjects then received repeated subcutaneous administrations of high dose (200 u kg−1) rHuEpo and 10 subjects received placebo. An immunoradiometric assay was used to measure the concentrations of erythropoietin (Epo) in serum and urine. 2Serum Epo concentration-time profiles were best described by a one-compartment open model with zero-order input. The mean elimination half-life (±s.d.) was 42.0±34.2 h. Clearance, uncorrected for bioavailability, was 0.05±0.01 l h−1 kg& minus;1. Erythropoietin concentrations returned to normal values in serum and urine, 7 and 4 days after the last administration, respectively. 3The recombinant hormone was well tolerated. Significant changes in reticulocytes and red blood cells, haemoglobin concentrations and haematocrit were observed after administration of rHuEpo. In the control group, these parameters remained unchanged. 4The change in reticulocytes was used as an index of the therapeutic effect of rHuEpo. The concentration-effect relationship was best described by an exponential model. 5These data show the limitations of the measurement of Epo concentrations in blood and urine samples, collected in athletes during competition, for antidoping control. Epo doping can be detected only during or within 4 to 7 days of ending, a course of rHuEpo. PMID:8877027

  3. Coxsackievirus B3 Infects the Bone Marrow and Diminishes the Restorative Capacity of Erythroid and Lymphoid Progenitors

    PubMed Central

    Althof, Nadine

    2013-01-01

    Coxsackievirus B3 (CVB3) is known to infect stem cells in the neonatal central nervous system. Here, we evaluated the effects of CVB3 infection on the major source and repository of stem cells, the bone marrow (BM). Viral genome was detectable in BM within 24 h of infection, and productive infection of BM cells was evident, peaking at 48 h postinfection (p.i.), when ∼1 to 2% of BM cells produced infectious virus particles. Beginning at 2 to 3 days p.i., a dramatic and persistent loss of immature erythroid cells, B and T lymphocytes, and neutrophils was observed in BM and, by day 3 to 4 p.i., the femoral BM stroma was largely destroyed. Analysis of peripheral blood revealed a modest neutrophilia, a loss of reticulocytes, and a massive lymphopenia. The abundance of multipotent progenitor cells (Lin−/c-kit+/Flt3+) in BM declined ∼10-fold during CVB3 infection and, consistent with a deficiency of primitive hematopoietic progenitors, serum levels of the hematopoietic growth factor Flt3 ligand were dramatically elevated. Therefore, we analyzed the regenerative capacity of BM from CVB3-infected mice. Granulocyte/macrophage progenitors displayed a relatively normal proliferative ability, consistent with the fact that the peripheral blood level of neutrophils—which are very short-lived cells—remained high throughout infection. However, erythroid and lymphoid hematopoietic progenitors in BM from CVB3-infected mice showed a markedly reduced colony-forming capacity, consonant with the observed loss of both lymphocytes and immature erythroid cells/reticulocytes from the BM and peripheral blood. In summary, CVB3 infects the BM and exerts differential effects on the various hematopoietic progenitor populations. PMID:23269810

  4. Translational recognition of the 5'-terminal 7-methylguanosine of globin messenger RNA as a function of ionic strength.

    PubMed

    Chu, L Y; Rhoads, R E

    1978-06-13

    The translation of rabbit globin mRNA in cell-free systems derived from either wheat germ or rabbit reticulocyte was studied in the presence of various analogues of the methylated 5' terminus (cap) as a function of ionic strength. Inhibition by these analogues was strongly enhanced by increasing concentrations of KCl, K(OAc), Na(OAc), or NH4(OAc). At appropriate concentrations of K(OAc), both cell-free systems were equally sensitive to inhibition by m7GTP. At 50 mM K(OAc), the reticulocyte system was not sensitive to m7GMP or m7GTP, but at higher concentrations up to 200 mM K(OAc), both nucleotides caused strong inhibition. The compound in m7G5'ppp5'Am was inhibitory at all concentrations of K(OAc) ranging from 50 to 200 mM, although more strongly so at the higher concentrations. Over the same range of nucleotide concentrations, the compounds GMP, GTP, and G5'ppp5'Am were not inhibitors. The mobility on sodium dodecyl sulfate-polyacrylamide electrophoresis of the translation product was that of globin at all K(OAc) concentrations in the presence of m7GTP. Globin mRNA from which the terminal m7GTP group had been removed by chemical treatment (periodate-cyclohexylamine-alkaline phosphatase) or enzymatic treatment (tobacco acid pyrophosphatase-alkaline phosphatase) was translated less efficiently than untreated globin mRNA at higher K(OAc) concentrations, but retained appreciable activity at low K(OAc) concentrations.

  5. Ubiquitin-dependent proteolytic pathway in wheat germ: Isolation of multiple forms of ubiquitin-activating enzyme, E1

    SciTech Connect

    Hatfield, P.M.; Vierstra, R.D. )

    1989-01-24

    Ubiquitin is a highly conserved protein involved in several important regulatory processes through its ATP-dependent, covalent ligation to a variety of eukaryotic target proteins. The authors describe here the characterization of ubiquitin conjugation in wheat germ extracts and the subsequent isolation of enzymes involved in conjugation. With {sup 125}I-ubiquitin as a substrate, wheat germ extracts form conjugates with either endogenous or added proteins. Ubiquitin-activating enzyme (E1) was purified from wheat germ extracts by using a modification of the covalent affinity chromatography procedure of Ciechanover et al. E1 from wheat germ, like that from rabbit reticulocytes, formed thiol ester intermediates with ubiquitin in the presence of ATP. Purified E1 preparations contained three polypeptides of apparent molecular masses of 117, 123, and 126 kDa after NaDodSO{sub 4}-PAGE. Under nondenaturing conditions, these proteins have native molecular masses of {approx}115 kDa, indicating that they exist as monomers. They concluded that all three species were E1 on the basis of their coelution with E1 activity, by immunorecognition by anti-human E1 antibodies, and by the similarity of their peptide maps. Furthermore, antibodies prepared against wheat germ E1's recognized E1 from rabbit reticulocytes. All three wheat germ E1's were detected in crude extracts prepared under conditions that minimized proteolysis, suggesting that the heterogeneity of the purified E1 preparations was not the result of posthomogenization breakdown. The immunological similarity of animal and plant E1's indicates that this conjugation enzyme, like ubiquitin, has been conserved through evolution.

  6. In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells

    SciTech Connect

    Fiorani, M.; Biagiarelli, B.; Vetrano, F.; Guidi, G.; Dacha, M.; Stocchi, V.

    1997-05-01

    The aim of this study was to investigate the effects of 50 Hz magnetic fields on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in the authors` laboratory showed that the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate induces hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. The authors also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work, they investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate that a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage in an oxidatively stressed erythrocyte system. In fact, exposure of intact erythrocytes incubated with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents.

  7. Exosome-like vesicles derived by Schistosoma japonicum adult worms mediates M1 type immune- activity of macrophage.

    PubMed

    Wang, Lifu; Li, Zhitao; Shen, Jia; Liu, Zhen; Liang, Jinyi; Wu, Xiaoying; Sun, Xi; Wu, Zhongdao

    2015-05-01

    Exosomes are 30-100-nm membrane vesicles of endocytic origin that are released into the extracellular space upon fusion of the multi-vesicular bodies (MVB) with the plasma membrane, while initial studies described that the role of exosomes was a reticulocyte cargo-disposal mechanism allowing remodeling of the plasma membrane during the maturation of reticulocytes to erythrocytes. Recent studies indicate that exosomes are secreted by most cells and pathogens and play an important role in intercellular signaling and exert regulatory function by carrying bioactive molecules. As numerous pathogens, adult worm of Schistosoma japonicum (S. japonicum) reside in mesenteric veins of definitive host including man and mammal animals. It was reported that the worms or the eggs also have specialized secretion systems to export effector proteins or other molecules into host target cells. However, the mechanisms involved remained unclear. This study investigated the isolation of the exosome-like vesicles secreted by S. japonicum adult worms and its immune activity on microphage in vitro. In this report, we identified exosome-based secretion as a new mechanism for protein secretion by S. japonicum. Electron microscopy tomography revealed the previously unidentified ultrastructural detail of exosome-like vesicles with high resolution; they were found to be typical spherical shape and to have a diverse population that varies in size of 30-100 nm. Exosome-like vesicles isolated from S. japonicum contained a significantly different protein compared with debris pelleted and the apoptosis body. We also demonstrate that macrophages were preferentially differentiated into the M1 subtype while being treated with S. japonicum exosome-like vesicles. This study reveals there are exosome-like vesicles derived by S. japonicum adult worms, and the exosome-like vesicles can mediate M1-type immune- activity of macrophage.

  8. Hydroxyurea and Growth in Young Children With Sickle Cell Disease

    PubMed Central

    Houston, Patricia E.; Wang, Winfred C.; Iyer, Rathi V.; Goldsmith, Jonathan; Casella, James F.; Reed, Caroline K.; Rogers, Zora R.; Waclawiw, Myron A.; Thompson, Bruce

    2014-01-01

    BACKGROUND: Growth impairment is a known complication of sickle cell disease. Effects of hydroxyurea (HU) on growth in very young children are not known. METHODS: Height, weight, BMI, and head circumference (HC) were compared with World Health Organization (WHO) standards in BABY HUG, a multicenter, randomized, double-blinded, placebo-controlled 2-year clinical trial of HU in 193 children 9 to 18 months of age. Anthropometric data were closely monitored and converted to z scores by using WHO standardized algorithms for descriptive analyses. The treatment and placebo groups were compared longitudinally by using a mixed model analysis. RESULTS: At entry, the z scores of BABY HUG children were higher than WHO norms. After 2 years of HU or placebo treatment, there were no significant differences between the groups, except for the mean HC z scores at study exit (HU: +0.8 versus placebo: +1.0, P = .05). Baseline z scores were the best predictors of z scores at study exit. The absolute neutrophil count, absolute reticulocyte count, and total white blood cell count had significant negative correlations with growth measures. CONCLUSIONS: Both groups had normal or near normal anthropometric measures during the study. The HC z scores at study entry and exit were slightly greater than WHO norms. Higher baseline white blood cell count, absolute reticulocyte count, and absolute neutrophil count were associated with poorer growth. The significance of the slightly lower HC in the treatment group at study exit is not clear. Trends toward normalization of weight and height and effects on HC will be monitored in ongoing BABY HUG follow-up studies. PMID:25157002

  9. Altered Turnover of Hypoxanthine Phosphoribosyltransferase in Erythroid Cells of Mice Expressing Hprt a and Hprt b Alleles

    PubMed Central

    Johnson, Gerald G.; Chapman, Verne M.

    1987-01-01

    We have previously shown that mice expressing Hprt a allele(s) have erythrocyte hypoxanthine phosphoribosyltransferase (HPRT) levels that are approximately 25-fold (Mus musculus castaneus) and 70-fold ( Mus spretus) higher than in mice that express the Hprt b allele (Mus musculus domesticus; C57BI/6J; C3H/HeHa), and that these differences in erythrocyte HPRT levels are due to differences in the turnover rates of the HPRT A and B proteins as reticulocytes mature to erythrocytes. We show here that: (1) the taxonomic subgroups of the genus Mus are essentially monomorphic for the occurrence of either the Hprt a or the Hprt b allele, with Hprt a being common in the aboriginal species (M. spretus, Mus hortulanus and Mus abbotti) and in several commensal species (Mus musculus musculus, M. m. castaneus, Mus musculus molossinus), while Hprt b is common in feral M. m. domesticus populations as well as in all inbred strains of mice tested; (2) in all these diverse Mus subgroups there is a strict association of Hprt a with high and Hprt b with low levels of erythrocyte HPRT; and, (3) the association between the occurrence of the Hprt a allele and elevated erythrocyte HPRT levels is retained following repeated backcrosses of wild-derived Hprt a allele(s) into the genetic background of inbred strains of mice with the Hprt b allele. Collectively, these observations indicate that the elevated and low levels of erythrocyte HPRT are specified by differences in the Hprt a and b structural genes. Since evidence indicates that Hprt a and b encode HPRT proteins which differ in primary structure, we infer that the structure of HPRT is an important factor in determining its sensitivity to turnover in mouse erythroid cells. Hprt a and b may provide a useful system of "normal" allelic gene products for identifying factors that participate in protein turnover during mouse reticulocyte maturation. PMID:3609725

  10. Understanding red blood cell parameters in the context of the frailty phenotype: interpretations of the FIBRA (Frailty in Brazilian Seniors) study.

    PubMed

    Silva, João Carlos; Moraes, Zélia Vieira de; Silva, Conceição; Mazon, Silvia de Barros; Guariento, Maria Elena; Neri, Anita Liberalesso; Fattori, André

    2014-01-01

    Frailty and anemia in the elderly appear to share a common pathophysiology associated with chronic inflammatory processes. This study uses an analytical, cross-sectional, population-based methodology to investigate the probable relationships between frailty, red blood cell parameters and inflammatory markers in 255 community-dwelling elders aged 65 years or older. The frailty phenotype was assessed by non-intentional weight loss, fatigue, low grip strength, low energy expenditure and reduced gait speed. Blood sample analyses were performed to determine hemoglobin level, hematocrit and reticulocyte count, as well as the inflammatory variables IL-6, IL-1ra and hsCRP. In the first multivariate analysis (model I), considering only the erythroid parameters, Hb concentration was a significant variable for both general frailty status and weight loss: a 1.0g/dL drop in serum Hb concentration represented a 2.02-fold increase (CI 1.12-3.63) in an individual's chance of being frail. In the second analysis (model II), which also included inflammatory cytokine levels, hsCRP was independently selected as a significant variable. Each additional year of age represented a 1.21-fold increase in the chance of being frail, and each 1-unit increase in serum hsCRP represented a 3.64-fold increase in the chance of having the frailty phenotype. In model II reticulocyte counts were associated with weight loss and reduced metabolic expenditure criteria. Our findings suggest that reduced Hb concentration, reduced RetAbs count and elevated serum hsCRP levels should be considered components of frailty, which in turn is correlated with sarcopenia, as evidenced by weight loss.

  11. Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis1[OPEN

    PubMed Central

    Law, Yee-Song; Zhang, Renshan; Guan, Xiaoqian; Cheng, Shifeng; Sun, Feng; Duncan, Owen; Murcha, Monika W.; Whelan, James; Lim, Boon Leong

    2015-01-01

    The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated. PMID:26304849

  12. Bioequivalence of Two Intravenous Artesunate Products with Its Active Metabolite Following Single and Multiple Injections

    PubMed Central

    Li, Qigui; Xie, Lisa; Melendez, Victor; Weina, Peter

    2011-01-01

    In animal species and humans, artesunate (AS) undergoes extensive and complex biotransformation to an active metabolite, dihydroartemisinin (DHA). The bioequivalence of two intravenous AS pharmaceutical products with 5% NaHCO3 (China Formulation) or 0.3 M PBS (WRAIR Formulation) was determined in rats in a two-formulation, two-period, and two-sequence crossover experimental design. Following single and multiple intravenous administrations, a series of blood samples was collected by using an automated blood sampler and drug concentrations were analyzed by LC-MS/MS. The 90% CI of the difference between the two intravenous formulations was contained within 80–125% of the geometric mean of pharmacokinetic parameters for AS and DHA in all animals dosed. Hematological effects were studied on days 1 and 3 after the final dosing, and a rapidly reversible hematological toxicity (significant reductions in reticulocyte levels) was seen in the peripheral blood of the rats treated with each formulation. The results showed that bioequivalence with the parent compound and active metabolite was fulfilled in the 82.3–117.7% ranges of all parameters (AUC0−t, Cmax, concentration average and degree of fluctuation) in the two-period and two-sequence crossover studies following single and repeated intravenous injections. For the metabolite, the equivalence was satisfied in most pharmacokinetic parameters tested due to the variability in the hydrolysis rate of AS to DHA. The WRAIR formulation of AS was considered to be bioequivalent to the Chinese formulation at steady-state according to the total drug exposure, in terms of both parent drug and active metabolite, rapidly reversal in reticulocyte decline, and extension of single and multiple administrations. Therefore, the parent drug and active metabolites should play similar important roles in the determination of efficacy and safety of the drug.

  13. Specific PABP effect on translation of YB-1 mRNA is neutralized by polyadenylation through a "mini-loop" at 3' UTR.

    PubMed

    Eliseeva, Irina A; Ovchinnikov, Lev P; Lyabin, Dmitry N

    2012-12-01

    YB-1 is a multifunctional cold shock domain containing protein that is involved virtually in all DNA- and mRNA-dependent cellular events. Its amount is regulated at the level of both transcription and translation. We showed previously that translation of poly A(-) YB-1 mRNA in vitro is selectively controlled by two proteins, YB-1 and PABP, through their specific and competitive binding to a regulatory element (RE) within 3' UTR of this mRNA. Here, we describe effects of these two proteins on translation of poly A(+) as compared with poly A(-) YB-1 mRNA in a rabbit reticulocyte cell-free translation system. We have found that YB-1 inhibits translation of both poly A(+) and poly A(-) YB-1 mRNAs at the same comparatively low YB-1/mRNA ratio. PABP has no positive effect on translation of poly A(+) YB-1 mRNA, although it has a stimulating effect on translation of poly A(-) YB-1 mRNA. A positive PABP effect on translation of poly A(+) YB-1 mRNA arose after removal of a portion of the sequence between RE and the poly(A) tail and disappeared after its replacement by another non-specific sequence of the same length. We also report that the RE fragment forms a complex with the poly(A) fragment in the presence of rabbit reticulocyte lysate (RRL) proteins. For its formation PABP is necessary but not sufficient. These results are in agreement with the proposed model implying formation of a mini-loop at 3' UTR of YB-1 mRNA that includes RE, RRL proteins and the poly(A) tail.

  14. EPO modulation in a 14-days undersea scuba dive.

    PubMed

    Revelli, L; Vagnoni, S; D'Amore, A; Di Stasio, E; Lombardi, C P; Storti, G; Proietti, R; Balestra, C; Ricerca, B M

    2013-10-01

    Erythropoiesis is affected during deep saturation dives. The mechanism should be related to a downregulation of serum Erythropoietin (s-EPO) concentration or to a toxic effect of the hyperbaric hyperoxia. We evaluated s-EPO and other haematological parameters in 6 scuba divers before, during and after a 14-days guinness saturation dive (8-10 m). Athletes were breathing air at 1.8-2 ATA, under the control of a team of physicians. Serum parameters were measured before diving (T0) and: 7 days (T1), 14 days (T2) after the beginning of the dive and 2 h (T3) and 24 h (T4) after resurfacing. Hgb, and many other haematological parameters did not change whereas Ht, s-EPO, the ratio between s-EPO predicted and that observed and reticulocytes (absolute, percent) declined progressively from T0 to T3. At T4 a significant rise in s-EPO was observed. Hgb did not vary but erythropoiesis seemed to be affected as s-EPO and reticulocyte counts showed. All these changes were statistically significant. The experiment, conducted in realistic conditions of dive length, oxygen concentration and pressure, allows us to formulate some hypotheses about the role of prolonged hyperbarism on erythropoiesis. The s-EPO rise, 24 h after resurfacing, is clearly documented and related to the "Normobaric Oxygen Paradox". This evidence suggests interesting hypotheses for new clinical applications such as modulation of s-EPO production and Hgb content triggered by appropriate O₂ administration in pre-surgical patients or in some anemic disease.

  15. Primary defect of congenital dyserythropoietic anemia type II. Failure in glycosylation of erythrocyte lactosaminoglycan proteins caused by lowered N-acetylglucosaminyltransferase II.

    PubMed

    Fukuda, M N; Dell, A; Scartezzini, P

    1987-05-25

    Congenital dyserythropoietic anemia type II or hereditary erythroblastic multinuclearity with positive acidified serum test (HEMPAS) is a genetic disease caused by membrane abnormality. Previously we have found that Band 3 and Band 4.5 are not glycosylated by lactosaminoglycans in HEMPAS erythrocytes, whereas normally these proteins have lactosaminoglycans (Fukuda, M. N., Papayannopoulou, T., Gordon-Smith, E. C., Rochant, H., and Testa, U. (1984) Br. J. Haematol. 56, 55-68). In order to find out where glycosylation of lactosaminoglycans stops, we have analyzed the carbohydrate structures of HEMPAS Band 3. By fast atom bombardment-mass spectrometry, methylation analysis, and hydrazinolysis followed by exoglycosidase treatments, the following structure was elucidated: (formula; see text) N-Linked glycopeptides synthesized in vitro by reticulocyte microsomes from HEMPAS were shown to be predominantly the above short oligosaccharide, whereas those from normal reticulocytes contain large molecular weight carbohydrates. The N-acetylglucosaminyltransferase II, which transfers N-acetylglucosamine to the C-2 position of the Man alpha 1----6Man beta 1----arm of the biantennary core structure, was therefore examined by using Man alpha 1----6(GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAcol as an acceptor. N-Acetylglucosaminyltransferase II activity was demonstrated in the lymphocyte microsome fraction from normal individuals. However, this enzyme activity was found to be decreased in those from HEMPAS patients. These results suggest that the primary defect of HEMPAS lies in the lowered activity of N-acetylglucosaminyltransferase II.

  16. Dose-response relationship of temozolomide, determined by the Pig-a, comet, and micronucleus assay.

    PubMed

    Guérard, M; Johnson, G; Dertinger, S; Duran-Pacheco, G; Funk, J; Zeller, A

    2017-02-15

    Temozolomide (TMZ), a monofunctional alkylating agent, was selected as a model compound to determine its quantitative genotoxic dose-response relationship in different tissues (blood, liver, and jejunum) and endpoints [Pig-a-, comet-, and micronucleus assay (MNT)] in male rats. TMZ was administered p.o. over 5 consecutive days (day 1-5), followed by a treatment-free period of 50 days (day 6-56) and a final administration prior to necropsy (day 57-59). TMZ showed a dose-dependent increase in DNA damage in all interrogated endpoints. A statistically significant increase in Pig-a mutant phenotypes was observed on day 44 starting at 7.5 mg/kg/day for mutant reticulocytes (for RET(CD59-)) and at 3.75 mg/kg/day for mutant red blood cells (RBC(CD59-)), respectively. In addition, a statistically significant increase in cytogenetic damage, as measured by micronucleated reticulocytes, was observed starting at 3.75 mg/kg/day on day 3 and 1.5 mg/kg/day on day 59. DNA strand breaks, as detected by the comet assay, showed a dose-dependent and statistically significant increase in liver, blood, and jejunum starting at doses of 3.75, 3.75, and 7.5 mg/kg/day, respectively. The dose-response relationships of the Pig-a, MNT, and comet data were analyzed for possible points of departure (PoD) using the benchmark-dose (BMD) software PROAST with different critical effect sizes (CES) (BMD0.1, BMD0.5, BMD1, and BMD1SD). Overall, PoD values show a high concordance between different tissues and endpoints, underlining the suitability of this experimental design to explore quantitative dose-response relationships in a variety of different tissues and endpoints, while minimizing animal use.

  17. Is organic farming safer to farmers' health? A comparison between organic and traditional farming.

    PubMed

    Costa, Carla; García-Lestón, Julia; Costa, Solange; Coelho, Patrícia; Silva, Susana; Pingarilho, Marta; Valdiglesias, Vanessa; Mattei, Francesca; Dall'Armi, Valentina; Bonassi, Stefano; Laffon, Blanca; Snawder, John; Teixeira, João Paulo

    2014-10-15

    Exposure to pesticides is a major public health concern, because of the widespread distribution of these compounds and their possible long term effects. Recently, organic farming has been introduced as a consumer and environmental friendly agricultural system, although little is known about the effects on workers' health. The aim of this work was to evaluate genetic damage and immunological alterations in workers of both traditional and organic farming. Eighty-five farmers exposed to several pesticides, thirty-six organic farmers and sixty-one controls took part in the study. Biomarkers of exposure (pyrethroids, organophosphates, carbamates, and thioethers in urine and butyrylcholinesterase activity in plasma), early effect (micronuclei in lymphocytes and reticulocytes, T-cell receptor mutation assay, chromosomal aberrations, comet assay and lymphocytes subpopulations) and susceptibility (genetic polymorphisms related to metabolism - EPHX1, GSTM1, GSTT1 and GSTP1 - and DNA repair-XRCC1 and XRCC2) were evaluated. When compared to controls and organic farmers, pesticide farmers presented a significant increase of micronuclei in lymphocytes (frequency ratio, FR=2.80) and reticulocytes (FR=1.89), chromosomal aberrations (FR=2.19), DNA damage assessed by comet assay (mean ratio, MR=1.71), and a significant decrease in the proportion of B lymphocytes (MR=0.88). Results were not consistent for organic farmers when compared to controls, with a 48% increase of micronuclei in lumphocytes frequency (p=0.016) contrasted by the significant decreases of TCR-Mf (p=0.001) and %T (p=0.001). Our data confirm the increased presence of DNA damage in farmers exposed to pesticides, and show as exposure conditions may influence observed effects. These results must be interpreted with caution due to the small size of the sample and the unbalanced distribution of individuals in the three study groups.

  18. Expression and characterization of human N-acetylglucosaminyltransferases and alpha2,3-sialyltransferase in insect cells for in vitro glycosylation of recombinant erythropoietin.

    PubMed

    Kim, Na Young; Kim, Hyung Gu; Kim, Yang Hyun; Chung, In Sik; Yang, Jai Myung

    2008-02-01

    The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennary-type complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of N-acetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using UDP-14C-Gal and CMP-14C-Sia as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the red blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.

  19. Metal nanoparticle-induced micronuclei and oxidative DNA damage in mice

    PubMed Central

    Song, Ming-Fen; Li, Yun-Shan; Kasai, Hiroshi; Kawai, Kazuaki

    2012-01-01

    Several mechanisms regarding the adverse health effects of nanomaterials have been proposed. Among them, oxidative stress is considered to be one of the most important. Many in vitro studies have shown that nanoparticles generate reactive oxygen species, deplete endogenous antioxidants, alter mitochondrial function and produce oxidative damage in DNA. 8-Hydroxy-2'-deoxyguanosine is a major type of oxidative DNA damage, and is often analyzed as a marker of oxidative stress in human and animal studies. In this study, we focused on the in vivo toxicity of metal oxide and silver nanoparticles. In particular, we analyzed the induction of micronucleated reticulocyte formation and oxidative stress in mice treated with nanoparticles (CuO, Fe3O4, Fe2O3, TiO2, Ag). For the micronucleus assay, peripheral blood was collected from the tail at 0, 24, 48 and 72 h after an i.p. injection of nanoparticles. Following the administration of nanoparticles by i.p. injection to mice, the urinary 8-hydroxy-2'-deoxyguanosine levels were analyzed by the HPLC-ECD method, to monitor the oxidative stress. The levels of 8-hydroxy-2'-deoxyguanosine in liver DNA were also measured. The results showed increases in the reticulocyte micronuclei formation in all nanoparticle-treated groups and in the urinary 8-hydroxy-2'-deoxyguanosine levels. The 8-hydroxy-2'-deoxyguanosine levels in the liver DNA of the CuO-treated group increased in a dose-dependent manner. In conclusion, the metal nanoparticles caused genotoxicity, and oxidative stress may be responsible for the toxicity of these metal nanoparticles. PMID:22573923

  20. Radioprotection conferred by dextran sulfate given before irradiation in mice

    SciTech Connect

    Ross, W.M.; Peeke, J.

    1986-02-01

    Dextran sulfate (DS) has been observed to cause mobilization (fivefold) of hemopoietic stem cells (HSC) and leukocytes, primarily lymphocytes, into the peripheral blood of mice within 2-3 h after intraperitoneal (i.p.) injection. This effect was dose dependent and was prolonged for several hours when the high-molecular-weight version DS500 (500,000 daltons) was used. When DS500 was given 1-3 days before irradiation, hemopoietic recovery was markedly enhanced. Postirradiation injection was ineffective. By ten days after irradiation (7.0 Gy), the number of endogenous spleen colonies (CFUs) and the splenic mass were much larger if DS pretreatment had been given. This effect was dependent on the dose of DS500 and on the time administered, 60 mg/kg producing a maximal effect when given three days before irradiation. DS500 caused a transient anaphylactoid shock, however, in most mice--mild at low doses but potentially lethal at doses above 40 mg/kg (10% mortality within 1-3 days after 60 mg/kg). The following results were obtained with 50 mg/kg, a compromise dose causing minimal mortality (3%) given three days before irradiation. Reticulocyte reappearance was earlier in irradiated mice given DS500, indicating earlier erythropoietic recovery. Some of these reticulocytes were resistant to lysing agents, so their appearance could be detected using the Coulter electronic cell counter, as well as in stained blood smears. The 30-day mortality due to bone marrow failure after irradiation was significantly decreased in DS-treated mice below 9.5 Gy, and the LD50/30 was increased by 0.5 Gy. This study shows that dextran sulfate exerts a radioprotective influence on the hemopoietic system and hence survival when administered prophylactically.

  1. Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis.

    PubMed

    Law, Yee-Song; Zhang, Renshan; Guan, Xiaoqian; Cheng, Shifeng; Sun, Feng; Duncan, Owen; Murcha, Monika W; Whelan, James; Lim, Boon Leong

    2015-10-01

    The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated.

  2. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

    SciTech Connect

    Devash, Y.; Reichman, M.; Sela, I.; Reichenbach, N.L.; Suhadolnik, R.J.

    1985-01-29

    An enzyme that converts (/sup 3/H, /sup 32/P)ATP, with a /sup 3/H:/sup 32/P ratio of 1:1, to oligoadenylates with the same /sup 3/H:/sup 32/P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of /sup 3/H:/sup 32/P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.

  3. Integration of Pig-a, micronucleus, chromosome aberration and comet assay endpoints in a 28-day rodent toxicity study with urethane

    PubMed Central

    Stankowski, Leon F.; Aardema, Marilyn J.; Lawlor, Timothy E.; Pant, Kamala; Roy, Shambhu; Xu, Yong; Elbekai, Reem

    2015-01-01

    As part of the international Pig-a validation trials, we examined the induction of Pig-a mutant reticulocytes and red blood cells (RETCD59− and RBCCD59−, respectively) in peripheral blood of male Sprague Dawley® rats treated with urethane (25, 100 and 250mg/kg/day) or saline by oral gavage for 29 days. Additional endpoints integrated into this study were: micronucleated reticulocytes (MN-RET) in peripheral blood; chromosome aberrations (CAb) and DNA damage (%tail intensity via the comet assay) in peripheral blood lymphocytes (PBL); micronucleated polychromatic erythrocytes (MN-PCE) in bone marrow; and DNA damage (comet) in various organs at termination (the 29th dose was added for the comet endpoint at sacrifice). Ethyl methanesulfonate (EMS; 200mg/kg/day on Days 3, 4, 13, 14, 15, 27, 28 and 29) was evaluated as the concurrent positive control (PC). All animals survived to termination and none exhibited overt toxicity, but there were significant differences in body weight and body weight gain in the 250-mg/kg/day urethane group, as compared with the saline control animals. Statistically significant, dose-dependent increases were observed for urethane for: RETCD59− and RBCCD59− (on Days 15 and 29); MN-RET (on Days 4, 15 and 29); and MN-PCE (on Day 29). The comet assay yielded positive results in PBL (Day 15) and liver (Day 29), but negative results for PBL (Days 4 and 29) and brain, kidney and lung (Day 29). No significant increases in PBL CAb were observed at any sample time. Except for PBL CAb (likely due to excessive cytotoxicity), EMS-induced significant increases in all endpoints/tissues. These results compare favorably with earlier in vivo observations and demonstrate the utility and sensitivity of the Pig-a in vivo gene mutation assay, and its ability to be easily integrated, along with other standard genotoxicity endpoints, into 28-day rodent toxicity studies. PMID:25934985

  4. Living high-training low: effect on erythropoiesis and aerobic performance in highly-trained swimmers.

    PubMed

    Robach, Paul; Schmitt, Laurent; Brugniaux, Julien V; Roels, Belle; Millet, Grégoire; Hellard, Philippe; Nicolet, Gérard; Duvallet, Alain; Fouillot, Jean-Pierre; Moutereau, Stéphane; Lasne, Françoise; Pialoux, Vincent; Olsen, Niels V; Richalet, Jean-Paul

    2006-03-01

    The "living high-training low" model (LHTL), i.e., training in normoxia but sleeping/living in hypoxia, is designed to improve the athletes performance. However, LHTL efficacy still remains controversial and also little is known about the duration of its potential benefit. This study tested whether LHTL enhances aerobic performance in athletes, and if any positive effect may last for up to 2 weeks after LHTL intervention. Eighteen swimmers trained for 13 days at 1,200 m while sleeping/living at 1,200 m in ambient air (control, n=9) or in hypoxic rooms (LHTL, n=9, 5 days at simulated altitude of 2,500 m followed by 8 days at simulated altitude of 3,000 m, 16 h day(-1)). Measures were done before 1-2 days (POST-1) and 2 weeks after intervention (POST-15). Aerobic performance was assessed from two swimming trials, exploring .VO(2max) and endurance performance (2,000-m time trial), respectively. Reticulocyte, serum EPO and soluble transferrin receptor responses were not altered by LHTL, whereas reticulocytes decreased in controls. In POST-1 (vs. before): red blood cell volume increased in LHTL only (+8.5%, P=0.03), .VO(2max) tended to increase more in LHTL (+8.1%, P=0.09) than in controls (+2.5%, P=0.21) without any difference between groups (P=0.42) and 2,000-m performance was unchanged with LHTL. In POST-15, both performance and hematological parameters were similar to initial levels. Our results indicate that LHTL may stimulate red cell production, without any concurrent amelioration of aerobic performance. The absence of any prolonged benefit after LHTL suggests that this LHTL model cannot be recommended for long-term purposes.

  5. In vitro effects of 50 Hz magnetic fields on oxidatively damaged rabbit red blood cells.

    PubMed

    Fiorani, M; Biagiarelli, B; Vetrano, F; Guidi, G; Dachà, M; Stocchi, V

    1997-01-01

    The aim of this study was to investigate the effects of 50 Hz magnetic fields (0.2-0.5 mT) on rabbit red blood cells (RBCs) that were exposed simultaneously to the action of an oxygen radical-generating system, Fe(II)/ascorbate. Previous data obtained in our laboratory showed at the exposure of rabbit erythrocytes or reticulocytes to Fe(II)/ascorbate hexokinase inactivation, whereas the other glycolytic enzymes do not show any decay. We also observed depletion of reduced glutathione (GSH) content with a concomitant intracellular and extracellular increase in oxidized glutathione (GSSG) and a decrease in energy charge. In this work we investigated whether 50 Hz magnetic fields could influence the intracellular impairments that occur when erythrocytes or reticulocytes are exposed to this oxidant system, namely, inactivation of hexokinase activity, GSH depletion, a change in energy charge, and hemoglobin oxidation. The results obtained indicate the a 0.5 mT magnetic field had no effect on intact RBCs, whereas it increased the damage with Fe(II)/ascorbate to a 0.5 mT magnetic field induced a significant further decay in hexokinase activity (about 20%) as well as a twofold increase in methemoglobin production compared with RBCs that were exposed to the oxidant system alone. Although further studies will be needed to determine the physiological implications of these data, the results reported in this study demonstrate that the effects of the magnetic fields investigated are able to potentiate the cellular damage induced in vitro by oxidizing agents.

  6. Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities.

    PubMed

    Devash, Y; Reichman, M; Sela, I; Reichenbach, N L; Suhadolnik, R J

    1985-01-29

    An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Molecular cloning of mouse erythrocyte protein 4.2: a membrane protein with strong homology with the transglutaminase supergene family.

    PubMed

    Rybicki, A C; Schwartz, R S; Qiu, J J; Gilman, J G

    1994-07-01

    We report the molecular cloning and characterization of mouse erythrocyte protein 4.2 (P4.2). Mouse erythrocyte P4.2 is a 691-amino-acid protein with a predicted MW of 77 kDa. Northern blot analysis detected a 2.2-kb transcript in mouse reticulocytes, compared with a 2.4- to 2.5-kb transcript in human reticulocytes, which is consistent with the absence of the 30-amino-acid splicing insert in mouse erythrocyte P4.2 that is found in the human protein (isoform I). Like the human erythrocyte P4.2, mouse erythrocyte P4.2 contains regions strikingly homologous with the transglutaminase (TGase) proteins although it too most likely lacks TGase crosslinking activity. Mouse P4.2 is on average 73% identical with human erythrocyte P4.2, although regional variations exist, with greatest conservation in the regions of the molecule that contain the TGase active site, the TGase calcium-binding site, and a band 3 binding site. Hydropathy analysis reveals a protein containing a series of hydrophobic domains, similar to the situation for human P4.2 and consistent with its tight binding to the membrane, although the mouse P4.2 is missing both the strongly hydrophilic region and adjacent highly charged region that are present in the human protein, suggesting that the two proteins could differ in their physical characteristics, binding associations, or functional properties. The availability of the complete mouse erythrocyte P4.2 cDNA should help in the design of P4.2-deficient animal models (for example, ribozyme or homologous recombinant "knockout" models) that should accelerate the understanding of P4.2 function in both erythroid and non-erythroid cells.

  8. Short-term hematological effects upon completion of a four-week simulated altitude camp.

    PubMed

    Pottgiesser, Torben; Garvican, Laura A; Martin, David T; Featonby, Jesse M; Gore, Christopher J; Schumacher, Yorck O

    2012-03-01

    Hemoglobin mass (tHb) is considered to be a main factor for sea-level performance after "live high-train low" (LHTL) altitude training, but little research has focused on the persistence of tHb following cessation of altitude exposure. The aim of the case study was to investigate short-term effects of various hematological measures including tHb upon completion of a simulated altitude camp. Five female cyclists spent 26 nights at simulated altitude (LHTL, 16.6 ± 0.4 h/d, 3000 m in an altitude house) where tHb was measured at baseline, at cessation of the camp, and 9 d thereafter. Venous blood measures (hemoglobin concentration, hematocrit, %reticulocytes, serum erythropoietin, ferritin, lactate dehydrogenase, and haptoglobin) were determined at baseline; on day 21 during LHTL; and at days 2, 5, and 9 after LHTL. Hemoglobin mass increased by 5.5% (90% confidence limits [CL] 2.5 to 8.5%, very likely) after the LHTL training camp. At day 9 after simulated LHTL, tHb decreased by 3.0% (90%CL -5.1 to -1.0%, likely). There was a substantial decrease in serum EPO (-34%, 90%CL -50 to -12%) at 2 d after return to sea level and a rise in ferritin (23%, 90%CL 3 to 46%) coupled with a decrease in %reticulocytes (-23%, 90%CL -34 to -9%) between day 5 and 9 after LHTL. Our findings show that following a hypoxic intervention with a beneficial tHb outcome, there may be a high probability of a rapid tHb decrease upon return to normoxic conditions. This highlights a rapid component in red-cell control and may have implications for the appropriate timing of altitude training in relation to competition.

  9. Genotoxicity of styrene-7,8-oxide and styrene in Fisher 344 rats: a 4-week inhalation study.

    PubMed

    Gaté, Laurent; Micillino, Jean-Claude; Sébillaud, Sylvie; Langlais, Cristina; Cosnier, Frédéric; Nunge, Hervé; Darne, Christian; Guichard, Yves; Binet, Stéphane

    2012-06-20

    The cytogenetic alterations in leukocytes and the increased risk for leukemia, lymphoma, or all lymphohematopoietic cancer observed in workers occupationally exposed to styrene have been associated with its hepatic metabolisation into styrene-7,8-oxide, an epoxide which can induce DNA damages. However, it has been observed that styrene-7,8-oxide was also found in the atmosphere of reinforced plastic industries where large amounts of styrene are used. Since the main route of exposure to these compounds is inhalation, in order to gain new insights regarding their systemic genotoxicity, Fisher 344 male rats were exposed in full-body inhalation chambers, 6 h/day, 5 days/week for 4 weeks to styrene-7,8-oxide (25, 50, and 75 ppm) or styrene (75, 300, and 1000 ppm). Then, the induction of micronuclei in circulating reticulocytes and DNA strand breaks in leukocytes using the comet assay was studied at the end of the 3rd and 20th days of exposure. Our results showed that neither styrene nor styrene-7,8-oxide induced a significant increase of the micronucleus frequency in reticulocytes or DNA strand breaks in white blood cells. However, in the presence of the formamidopyridine DNA glycosylase, an enzyme able to recognize and excise DNA at the level of some oxidized DNA bases, a significant increase of DNA damages was observed at the end of the 3rd day of treatment in leukocytes from rats exposed to styrene but not to styrene-7,8-oxide. This experimental design helped to gather new information regarding the systemic genotoxicity of these two chemicals and may be valuable for the risk assessment associated with an occupational exposure to these molecules.

  10. Synthesis and decay of calmodulin-ubiquitin conjugates in cell-free extracts of various rabbit tissues.

    PubMed

    Laub, M; Jennissen, H P

    1997-06-27

    Calmodulin is the natural substrate for ubiquitin-ligation by the enzyme ubiquitin-calmodulin ligase (uCaM-synthetase; EC 6.3.2.21). The activity of this ligase is regulated by the binding of the second messenger Ca2+ to the substrate calmodulin, which increases the activity ca. 10-fold. Up till now, two components of the ligase could be identified: uCaM Syn-F1 and uCaM Syn-F2, the first of which binds to ubiquitin and the second which binds to calmodulin. Since the physiological role of this enzyme is still unclear, this study was designed to examine whether the activity of uCaM-Synthetase in 40,000 x g tissue supernatants correlates with the calmodulin content in the various tissues. In reticulocytes, spleen, erythrocytes, testis and brain, which are rich in uCaM synthetase, the tissue contents calculated on the basis of activity measurements were between 4-80-fold higher than in red and white skeletal muscle. These activities did not correlate with the respective calmodulin contents of the tissues indicating that other factors were determining these enzyme levels. A second aim was to gain information on the role of the ATP-ubiquitin-dependent proteolytic pathway in those tissues displaying uCaM synthetase activity. In the reticulocyte system which contains the classical ATP-ubiquitin-dependent proteolytic pathway as measured with 125I-BSA, no ubiquitin-dependent degradation of calmodulin could be detected. We therefore examined the other tissues of the rabbit with the substrate 125I-BSA and succeeded in finding a ubiquitin-independent ATP-dependent proteolytic activity in every case but no ubiquitin-dependent activity. The ubiquitin-independent activity was highest in smooth muscle and red skeletal muscle being ca. 3-4-fold higher than in lung and testis. In 50% of the tissue crude extracts the time curve of calmodulin ubiquitylation progressed through a maximum indicating a dynamic steady state based on conjugate synthesis and decay. If a ubiquitylation pulse

  11. Effects of early parenteral iron combined erythropoietin in preterm infants

    PubMed Central

    Qiao, Linxia; Tang, Qingya; Zhu, Wenying; Zhang, Haiyan; Zhu, Yuefang; Wang, Hua

    2017-01-01

    Abstract Backgroud: The aim of the study was to evaluate the effect of early parenteral iron supplementation combined erythropoietin for prevention of anemia in preterm infants. Methods: In total, 96 preterm infants were randomly assigned to 3 groups: a control group receiving standard parenteral nutrition (group 1: n = 31), an iron-supplemented group (group 2: IS, n = 33), and an iron-supplemented combined erythropoietin group (group 3: IS+EPO, n = 32). The primary objective was to assess hemoglobin (Hb) levels. The secondary objectives included assessment of red blood cell counts (RBC), mean cell volume (MCV), serum iron, ferritin, percentages of reticulocyte (RET), total iron binding capacity (TIBC) and oxidative stress, which was assessed by measuring plasma levels of malondialdehyde and superoxide dismutase at baseline and at 2 weeks. The blood routine indices including Hb, RBC, MCV, and percentages of RET were measured at corrected age of 1 and 3 months. Results: At 2 weeks of life, the percentages of reticulocyte in group 2 and group 3 were significantly higher than those in group 1 (2.1±0.4, 2.5±0.3, and 1.7±0.3, respectively, P < 0.001, P<0.001), whereas TIBC were significantly lower than those in group 1 (36.7±4.6, 36.0±4.7, and 41.6 ± 5.2 respectively, P = 0.011, P = 0.006). There were no significant differences in RBC counts, the levels of hemoglobin, ferritin, malondialdehyde, and superoxide dismutase among the 3 groups at 2weeks of life. RBC, Hb, MCV, body weight, body length, and head circumference at a corrected age of 1 month did not differ among 3 groups. At corrected age of 3months, more infants in the control group had abnormal Hb and MCV levels (Hb levels: 114.3 ± 21.3, 123.7 ± 31.6, and 125.1 ± 21.2, P = 0.021, P = 0.034, respectively; MCV: 74.1 ± 3.5, 78.3 ± 4.7 and 79.1 ± 5.2, P = 0.017, P = 0.012, respectively), whereas cases of oral iron, cases of breastfeeding

  12. Biological impact of low dose-rate simulated solar particle event radiation in vivo.

    PubMed

    Chang, P Y; Doppalapudi, R; Bakke, J; Wang, A; Menda, S; Davis, Z

    2010-08-01

    C57Bl6-lacZ animals were exposed to a range of low dose-rate simulated solar particle event (sSPE) radiation at the NASA-sponsored Research Laboratory (NSRL) at Brookhaven National Laboratory (BNL). Peripheral blood was harvested from animals from 1 to 12 days after total body irradiation (TBI) to quantify the level of circulating reticulocytes (RET) and micronucleated reticulocytes (MN-RET) as an early indicator of radiation-induced genotoxicity. Bone marrow lymphocytes and hippocampal tissues from each animal were collected at 12 days and up to two months, to evaluate dose-dependent late effects after sSPE exposure. Early hematopoietic changes show that the % RET was reduced up to 3 days in response to radiation exposure but recovered at 12 days postirradiation. The % MN-RET in peripheral blood was temporally regulated and dependant on the total accumulated dose. Total chromosome aberrations in lymphocytes increased linearly with dose within a week after radiation and remained significantly higher than the control values at 4 weeks after exposure. The level of aberrations in the irradiated animals returned to control levels by 8 weeks postirradiation. Measurements of chromosome 2 and 8 specific aberrations indicate that, consistent with conventional giemsa-staining methods, the level of aberrations is also not significantly higher than in control animals at 8 weeks postirradiation. The hippocampus was surveyed for differential transcriptional regulation of genes known to be associated with neurogenesis. Our results showed differential expression of neurotrophin and their associated receptor genes within 1 week after sSPE exposure. Progressive changes in the profile of expressed genes known to be involved in neurogenic signaling pathways were dependent on the sSPE dose. Our results to date suggest that radiation-induced changes in the hematopoietic system, i.e., chromosome aberrations in lymphocytes, are transient and do not persist past 4 weeks after radiation

  13. Beta-Adrenergic Blockade Does not Prevent Polycythemia or Decrease in Plasma Volume in Men at 4300 m Altitude

    NASA Technical Reports Server (NTRS)

    Grover, R. F.; Selland, M. A.; McCullough, R. G.; Dahms, T. E.; Wolfel, E. E.; Butterfield, G. E.; Reeves, J. T.; Greenleaf, J. E.

    1998-01-01

    When humans ascend to high altitude (ALT) their plasma volume (PV) and total blood volume (BV) decrease during the first few days. With continued residence over several weeks, the hypoxia-induced stimulation of erythropoietin increases red cell production which tends to restore BV. Because hypoxia also activates the beta-adrenergic system, which stimulates red blood cell production, we investigated the effect of adrenergic beta-receptor inhibition with propranolol on fluid volumes and the polycythemic response in 11 healthy unacclimatized men (21-33 years old exposed to an ALT of 4300 m (barometric pressure 460 Torr) for 3 weeks on Pikes Peak, Colorado. PV was determined by the Evans blue dye method (PV(sub EB)), BV by the carbon monoxide method (BV(sub CO)), red cell volume (RCV)was calculated from hematocrit (Hct) and BV(sub CO), and serum erythropoietin concentration ([EPO]) and reticulocyte count, were also determined. All determinations were made at sea level and after 9-11 (ALT-10) and 9-20 (ALT-20) days at ALT. At sea level and ALT, six men received propranolol (pro, 240 mg/day), and five received a placebo (pla). Effective beta-blockade did not modify the mean (SE) maximal values of [EPO] [pla: 24.9 (3.5) vs pro: 24.5 (1.5) mU/ml] or reticulocyte count [pla: 2.7 (0.7) vs pro: 2.2 (0.5)%]; nor changes in PV(sub EB)[pla: -15.8 (3.8) vs pro: -19.9 (2.8)%], RCV(sub CO) [pla: +7.0 (6.7) vs pro: +10.1 (6.1)%], or BV(sub CO) [pla: -7.3 (2.3) vs pro: -7.1 (3.9)%]. In the absence of weight loss, a redistribution of body water with no net loss is implied. Hence, activation of the beta-adrenergic system did not appear to affect the hypovolemic or polycythemic responses that occurred during 3 weeks at 4300 m ALT in these subjects.

  14. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions

    PubMed Central

    Hostetler, Jessica B.; Sharma, Sumana; Bartholdson, S. Josefin; Wright, Gavin J.; Fairhurst, Rick M.; Rayner, Julian C.

    2015-01-01

    Background A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP) and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC), and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion. Methodology/Principal Findings We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further

  15. Hormonal and echocardiographic abnormalities in adult patients with sickle-cell anemia in Bahrain

    PubMed Central

    Garadah, Taysir S; Jaradat, Ahmed A; Alalawi, Mohammed E; Hassan, Adla B

    2016-01-01

    Background Adrenal, thyroid, and parathyroid gland hormonal changes are recognized in children with homozygous (HbSS) sickle-cell anemia (SCA), but are not clear in adult patients with SCA. Aim To assess the metabolic and endocrine abnormalities in adult patients with SCA and evaluate left ventricular (LV) systolic and diastolic functions compared with patients with no SCA and further study the relationship between serum levels of cortisol, free thyroxine (T4), and testosterone with serum ferritin. Materials and methods The study was conducted on 82 patients with adult HbSS SCA compared with a sex- and age-matched control group. The serum levels of cortisol, parathyroid hormone (PTH), testosterone, thyroid-stimulating hormone (TSH), and free T4 were compared. Blood levels of hemoglobin, reticulocyte count, lactate dehydrogenase (LDH), calcium, alkaline phosphatase (ALP), vitamin D3, and ferritin were also compared. Pulsed Doppler echo was performed to evaluate the LV mass, wall thickness, and cavity dimensions with diastolic filling velocities of early (E) and atria (A) waves. Biometric data were analyzed as mean ± standard deviation between the two groups. Multiple regression analysis was performed between serum levels of ferritin as independent variable and testosterone, cortisol, and thyroid hormones. Results A total of 82 adult patients with HbSS SCA were enrolled who had a mean age of 21±5.7 years, with 51 males (62%). Patients with SCA compared with the control group had significantly lower hemoglobin, body mass index, cortisol, vitamin D3, testosterone, and T4. Furthermore, there were significantly high levels of reticulocyte count, PTH, TSH, ferritin, LDH, ALP, and uric acid. The incidence of subclinical hypothyroidism and adrenal insufficiency was 7% and 4.8%, respectively, with hypogonadism 9.8% and vitamin D3 deficiency 61%. There were inverse relationships between ferritin as independent variable and serum levels of testosterone, T4, and cortisol

  16. Recovery of motor spontaneous activity after intranasal delivery of human recombinant erythropoietin in a focal brain hypoxia model induced by CoCl2 in rats.

    PubMed

    Merelli, Amalia; Caltana, Laura; Girimonti, Patricia; Ramos, Alberto Javier; Lazarowski, Alberto; Brusco, Alicia

    2011-08-01

    Stroke is a major human health problem inducing long-term disability without any efficient therapeutic option being currently available. Under hypoxia, hypoxia-inducible factor-1α (HIF-1α) activates several genes as erythropoietin receptor (Epo-R) related with O(2) supply, and the multidrug-resistance gene (MDR-1) related with drug-refractory phenotype. Brain cortical injection of CoCl(2) produces focal hypoxia-like lesion with neuronal and glial alterations, as well as HIF-1α stabilization and MDR-1 overexpression. Intranasal (IN) drug delivery can by-pass blood-brain barrier (BBB) where MDR-1 is normally expressed. We evaluated the effects of IN-rHu-Epo administration on spontaneous motor activity (SMA) and the brain pattern expression of HIF-1α, MDR-1, and Epo-R in our cobalt-induced hypoxia model. Adult male Wistar rats were injected by stereotaxic surgery in frontoparietal cortex, with CoCl(2) (2 μl-50 mM; n = 20) or saline (controls; n = 20). Ten rats of each group were treated with IN-rHu-Epo 24 U or IN-saline. In addition, erythropoietic stimulation was evaluated by reticulocytes (Ret) account during three consecutive days, after intraperitoneal (i.p.)-recombinant-human Epo (rHu-Epo) (950 U; n = 6) or IN-rHu-Epo (24 U; n = 6) administration. SMA was evaluated by open field and rotarod tests, before and after surgical procedures during five consecutive days. Histological and immunostaining studies of HIF-1α, MDR-1, and Epo-R were performed on brain slides. A significant difference in SMA was observed in the hypoxic rats of IN-rHu-Epo-administered group as compared with Co-Saline-treated subjects and controls (p < 0.001). HIF-1α, EPO-R, and MDR-1 were overexpressed in the hypoxic cortex areas, while in contralateral hemisphere or controls, they were negatives. Reticulocytes were only increased in intraperitoneal (i.p.)-rHu-Epo-administered group. In spite of MDR-1 overexpression being detected in neurons, the coexpression of Epo-R could

  17. Pain frequency, severity and QT dispersion in adult patients with sickle cell anemia: correlation with inflammatory markers

    PubMed Central

    Garadah, Taysir S; Jaradat, Ahmed A; AlAlawi, Mohammed E; Hassan, Adla B; Sequeira, Reginald P

    2016-01-01

    Background Inflammatory markers are increased during vaso-occlusive crisis (VOC) in adult patients with sickle cell anemia (SCA), but this is not clear in clinical steady state. Aim The present study aims to establish the frequency and intensity of bone pain episodes in adult patients with SCA in clinical steady state and to determine the correlation between different inflammatory markers, other variables including QT dispersion (QTd) and pain frequency and intensity in SCA. Patients and methods Patients were classified into two groups: group 1, those with more than three hospital admissions in the last 6 months, and group 2, those with no hospital admission. Pearson correlation between variables such as body mass index (BMI), level of tumor necrosis factor (TNF-α), interleukin-1 (IL-1), C-reactive protein (CRP), hemoglobin (Hb), reticulocyte count, white blood cell count (WBC), ferritin, lactate dehydrogenase (LDH), parathormone (PTH), vitamin D3 (25-OH cholecalciferol) and bone pain frequency with severity was evaluated. Results Forty-six patients were enrolled in this study with a mean age of 18.47±5.78 years, with 23 patients in each group. Vitamin D3 and Hb were lower (17.04±5.77 vs 37.59±4.83 ng/L, P<0.01 and 7.96±0.3 vs 8.44±0.27 g/dL, P<0.01, respectively); the inflammatory markers showed significantly higher level of TNF-α, IL-1 and CRP (56.52±5.43 pg/ml, 44.17±4.54 pg/ml and 3.20±0.72 mg/L, respectively, P<0.05); WBC, LDH and reticulocyte count were also significantly higher and the QTd was higher (45.0±2.22 vs 41.55±0.8 ms, P<0.05) in group 1 when compared with group 2. Pearson correlation coefficient showed significant positive correlation between serum level of TNF-α and bone pain frequency (r=0.414, P<0.005) and serum level of IL-1 (r=0.39, P<0.008). Conclusion There is a strong positive correlation between TNF-α, IL-1 and WBC and bone pain frequency in steady state in adult patients with SCA. CRP and low hemoglobin had weak positive

  18. Molecular cloning, expression and characterization of a ubiquitin conjugation enzyme (E2(17)kB) highly expressed in rat testis.

    PubMed Central

    Wing, S S; Jain, P

    1995-01-01

    Ubiquitin-conjugating enzymes (E2s) play a key role in ubiquitin-mediated proteolysis by catalysing the conjugation of ubiquitin to protein substrates. We have previously reported the cDNA cloning of a 14 kDa conjugating enzyme [E2(14)k; Wing, Dumas and Banville (1992) J. Biol. Chem. 267, 6495-6501] that efficiently supported ubiquitination and protein degradation in reticulocyte extracts. Surprisingly, the structure of this E2 was markedly more similar to the Saccharomyces cerevisiae DNA repair gene RAD6, than to the S. cerevisiae UBC4/UBC5 genes which are required for the degradation of short-lived proteins and support much of the ubiquitination of yeast proteins. This suggested that mammalian homologues of UBC4/UBC5 remained to be identified. Using oligonucleotides derived from the S. cerevisiae UBC4 sequence as primers in a PCR reaction with rat muscle cDNA as a template, a 390 bp DNA fragment was amplified which predicted an amino acid sequence that was 83% identical to yeast UBC4. Screening a rat testes cDNA library identified a family of cDNAs which predicted two very similar proteins with basic pIs and molecular masses of approx. 16,700 Da. Isoform 2E was expressed in Escherichia coli and purified to homogeneity. It supported ubiquitination to reticulocyte and testis proteins more rapidly in vitro and produced larger conjugates than E2(14)k. Examination of RNA from different tissues indicated that this type of E2 was expressed in a broad spectrum of tissues but at particularly high levels in the testis. Fractionation of a testis extract by anion-exchange chromatography identified several putative ubiquitin protein ligase activities with which this E2 could interact in promoting conjugation of ubiquitin to proteins. One of these activities supported conjugation of ubiquitin to histone H2A, a substrate degraded in the ubiquitin system by a non-N-end rule mechanism. This paper reports the first cloning of a apparent mammalian homologue of S. cerevisiae UBC4

  19. Detection of autologous blood transfusions in athletes: a historical perspective.

    PubMed

    Mørkeberg, Jakob

    2012-07-01

    Autologous blood transfusions (ABTs) has been used by athletes for approximately 4 decades to enhance their performance. Although the method was prohibited by the International Olympic Committee in the mid 1980s, no direct detection method has yet been developed and implemented by the World Anti-Doping Agency (WADA). Several indirect methods have been proposed with the majority relying on changes in erythropoiesis-sensitive blood markers. Compared with the first methods developed in 1987, the sensitivity of subsequent tests has not improved the detection of blood doping. Nevertheless, the use of sophisticated statistical algorithms has assured a higher level of specificity in subsequent detection models, which is a crucial aspect of antidoping testing particularly to avoid "false positives." Today, the testing markers with the best sensitivity/specificity ratio are the Hbmr model (an algorithm based on the total amount of circulating hemoglobin level [hemoglobin level mass] and percentage of reticulocytes, 4.51·ln(Hbmass)-√%ret) and the OFF-hr model (algorithm based on hemoglobin level concentration and percentage of reticulocytes, Hb(g/L)-60·√%ret). Only the OFF-hr model is currently approved by WADA. Recently, alternative indirect strategies for detecting blood doping have been proposed. One method is based upon a transfusion-induced immune-response resulting in specific changes in gene expression related to leukocytes such as T lymphocytes. Another method relies on detecting increased plasticizer metabolite levels in the urine caused by the leakage of plasticizers from the blood bags used during the blood storage. These methods need further development and validation across different types of transfusion regimes before they can be implemented. In addition, several research projects have been funded by WADA in recent years and are now under development including "Detection of Autologous Blood Transfusions Using Activated Red Blood Cells (the red blood cells

  20. The Ras-specific exchange factors mouse Sos1 (mSos1) and mSos2 are regulated differently: mSos2 contains ubiquitination signals absent in mSos1.

    PubMed Central

    Nielsen, K H; Papageorge, A G; Vass, W C; Willumsen, B M; Lowy, D R

    1997-01-01

    We have compared aspects of the mouse sos1 (msos1) and msos2 genes, which encode widely expressed, closely related Ras-specific exchange factors. Although an msos1 plasmid did not induce phenotypic changes in NIH 3T3 cells, addition of a 15-codon myristoylation signal to its 5' end enabled the resulting plasmid, myr-sos1, to induce approximately one-half as many foci of transformed cells as a v-H-ras control. By contrast, an isogenic myr-sos2 plasmid, which was made by fusing the first 102 codons from myr-sos1 at homologous sequences to an intact msos2 cDNA, did not induce focal transformation directly, although it could form foci in cooperation with c-H-ras. Pulse-chase experiments indicated that the half-life of Sos¿1 in NIH 3T3 cells was greater than 18 h, while that of Sos2 was less than 3 h. While in vitro-translated Sos1 was stable in a rabbit reticulocyte lysate, Sos2 was degraded in the lysate, as were each of two reciprocal chimeric Sos1-Sos2 proteins, albeit at a slower rate. In the lysate, Sos2 and the two chimeric proteins could be stabilized by ATPgammaS. Unlike Sos1, Sos2 was specifically immunoprecipitated by antiubiquitin antibodies. In a myristoylated version, the chimeric gene encoding Sos2 at its C terminus made a stable protein in NIH 3T3 cells and induced focal transformation almost as efficiently as myr-msos1, while the myristoylated protein encoded by the other chimera was unstable and defective in the transformation assay. We conclude that mSos2 is much less stable than mSos1 and is degraded by a ubiquitin-dependent process. A second mSos2 degradation signal, mapped to the C terminus in the reticulocyte lysate, does not seem to function under the growth conditions of the NIH 3T3 cells. PMID:9372945

  1. Fractionated exposure of high energy iron ions has a sparing effect in vivo

    NASA Astrophysics Data System (ADS)

    Chang, P. Y.; Bakke, J.; Puey, A.

    The radiation environment in deep space is complex and includes a broad spectrum of charged and highly energetic particle radiations. Exposure to these types of radiations may pose potential health risks in manned space missions. The detection of particle radiation-induced genomic alterations in vivo, particularly in slow or non-dividing tissues, is therefore important to provide relevant information in estimating risks. We are using a plasmid-based lacZ transgenic mouse model system to rapidly measure, in a statistically reliable way, the mutagenic potential of charged particle radiations relevant in the space environment. The lacZ transgenic mouse has been constructed so that every cell of the animal contains multiple copies of an integrated target reporter gene, allowing us to measure tissue-specific radiation-induced changes as a function of dosing regime. The nature of these mutations can also be characterized by restriction fragment length polymorphisms (RFLP). To examine the impact of dose protraction, animals were exposed to a single dose or daily fractions of 1 GeV/n iron ions. Cytotoxicity in the peripheral blood was measured by enumerating the frequency of circulating micronucleated reticulocytes (fMN-RET) in a time course from 24 h up to 1 week after completion of the radiation protocol. Brain and spleen tissues were harvested at 8 weeks after exposure and mutant frequencies (MF) in the transgene in these tissues were measured. Results from the fractionated protocol were compared to the responses obtained after the animals were exposed to the single dose treatment. We noted significantly lower levels of micronucleated reticulocytes in peripheral blood at 48 h after fractionated doses of iron ions when compared to the same total dose delivered in a single exposure demonstrating that protracted exposures of particle radiation resulted in an overall sparing effect in cytogenetic toxicity in the hematopoietic system in animals. Transgene mutation analysis

  2. Nutritive evaluation and effect of Moringa oleifera pod on clastogenic potential in the mouse.

    PubMed

    Promkum, Chadamas; Kupradinun, Piengchai; Tuntipopipat, Siriporn; Butryee, Chaniphun

    2010-01-01

    Moringa oleifera Lam (horseradish tree; tender pod or fruits) has been consumed as a vegetable and utilized as a major ingredient of healthy Thai cuisine. Previous studies have shown that M. oleifera pod extracts act as bifunctional inducers along with displaying antioxidant properties and also inhibiting skin papillomagenesis in mice. This study was aimed to determine the nutritive value, and clastogenic and anticlastogenic potentials of M. oleifera pod. The nutritive value was determined according to AOAC methods. The clastogenic and anticlastogenic potentials were determined using the in vivo erythrocyte micronucleus assay in the mouse. Eighty male mice were fed semi-purified diets containing 1.5%, 3.0% and 6.0% of ground freeze-dried boiled M. oleifera pod (bMO) for 2 weeks prior to administration of both direct-acting (mitomycin C, MMC) and indirect-acting (7, 12-dimethylbenz(a)anthracene, DMBA), clastogens. Blood samples were collected at 0, 24, 48 and 72 h, dropped on acridine orange-coated slides, and then counted for reticulocytes both with and without micronuclei by fluorescence microscopy. The nutritive value of 100 g bMO consisted of: moisture content, 8.2 g; protein, 19.2 g; fat, 3.9 g; carbohydrate (dietary fiber included), 60.5 g; dietary fiber, 37.5 g; ash, 8.1 g and energy, 354 kcal. Freeze-dried boiled M. oleifera had no clastogenic activity in the mouse while it possessed anticlastogenic activity against both direct and indirect-acting clastogens. Freeze-dried boiled M. oleifera pod at 1.5%, 3.0% and 6.0% in the diets decreased the number of micronucleated peripheral reticulocytes (MNRETs) induced by both MMC and DMBA. However, the effect was statistically significant in the dose dependent manner only in the MMC-treated group. In conclusion, the present study demonstrated that bMO has no clastogenicity and possesses anticlastogenic potential against clastogens, and particularly a direct-acting clastogen in the mouse.

  3. The Elav-like protein HuR exerts translational control of viral internal ribosome entry sites.

    PubMed

    Rivas-Aravena, Andrea; Ramdohr, Pablo; Vallejos, Maricarmen; Valiente-Echeverría, Fernando; Dormoy-Raclet, Virginie; Rodríguez, Felipe; Pino, Karla; Holzmann, Cristian; Huidobro-Toro, J Pablo; Gallouzi, Imed-Eddine; López-Lastra, Marcelo

    2009-09-30

    The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require direct binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.

  4. The 5'-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation.

    PubMed

    Vallejos, Maricarmen; Ramdohr, Pablo; Valiente-Echeverría, Fernando; Tapia, Karla; Rodriguez, Felipe E; Lowy, Fernando; Huidobro-Toro, J Pablo; Dangerfield, John A; López-Lastra, Marcelo

    2010-01-01

    In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5'-untranslated region (5'-UTR) of the mouse mammary tumor virus (MMTV). The 5'-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5'-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5'-UTR was resistant to the addition of m(7)GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5'-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function.

  5. The 5′-untranslated region of the mouse mammary tumor virus mRNA exhibits cap-independent translation initiation

    PubMed Central

    Vallejos, Maricarmen; Ramdohr, Pablo; Valiente-Echeverría, Fernando; Tapia, Karla; Rodriguez, Felipe E.; Lowy, Fernando; Huidobro-Toro, J. Pablo; Dangerfield, John A.; López-Lastra, Marcelo

    2010-01-01

    In this study, we demonstrate the identification of an internal ribosome entry site (IRES) within the 5′-untranslated region (5′-UTR) of the mouse mammary tumor virus (MMTV). The 5′-UTR of the full-length mRNA derived from the infectious, complete MMTV genome was cloned into a dual luciferase reporter construct containing an upstream Renilla luciferase gene (RLuc) and a downstream firefly luciferase gene (FLuc). In rabbit reticulocyte lysate, the MMTV 5′-UTR was capable of driving translation of the second cistron. In vitro translational activity from the MMTV 5′-UTR was resistant to the addition of m7GpppG cap-analog and cleavage of eIF4G by foot-and-mouth disease virus (FMDV) L-protease. IRES activity was also demonstrated in the Xenopus laevis oocyte by micro-injection of capped and polyadenylated bicistronic RNAs harboring the MMTV-5′-UTR. Finally, transfection assays showed that the MMTV-IRES exhibits cell type-dependent translational activity, suggesting a requirement for as yet unidentified cellular factors for its optimal function. PMID:19889724

  6. [C-reactive protein in the assessment of iron status in patients on hemodialysis].

    PubMed

    Rathaus, M

    2009-01-01

    Iron availability is a prerequisite for an efficient hematopoietic response to erythropoietin. Dynamic evaluation of iron status is difficult in hemodialysis patients and can be further complicated by the presence of an inflammatory state. Several cytokines, in particular interleukin 6 (IL-6), stimulate the production of hepcidin in the liver. This hormone is the main regulator of the extracellular iron concentration through its effect on the iron channel ferroportin, present in several cell types. IL-6 is also the major stimulus for the production of C-reactive protein (CRP), a nonspecific but sensitive marker of inflammation. Measurement of hepcidin is technically difficult and has so far been limited to research. On the other hand, measurement of CRP, which is both sensitive and easily measurable with automated techniques, might possibly be used as a surrogate measure of iron status in hemodialysis patients. Several studies have suggested the value of CRP in this context, but they dealt with small patient groups and single-time-point measurements. Even the definition of normal values of CRP in dialysis patients is uncertain. During the period between 2003 and 2007, we performed 8322 measurements of CRP in 401 hemodialysis patients followed for 3-60 months. All parameters of iron balance (serum iron, TSAT, percent hypochromic RBC and Hgb concentration in reticulocytes) were clearly affected by the presence of an inflammatory state. We believe that measurement of CRP must be part of the routine hematological assessment of hemodialyzed patients to allow the correct interpretation of data in anemia treatment.

  7. The heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays

    PubMed Central

    Duke-Cohan, Jonathan S.; Wollenick, Kristin; Witten, Elizabeth A.; Seaman, Michael S.; Baden, Lindsey R.; Dolin, Raphael; Reinherz, Ellis L.

    2009-01-01

    The renewed interest in strategies to combat infectious agents with epidemic potential has led to a re-examination of vaccination protocols against smallpox. To help define which antigens elucidate a human antibody response, we have targeted proteins known or predicted to be presented on the surface of the intracellular mature virion (IMV) or the extracellular enveloped virion (EEV). The predicted ectodomains were expressed in a mammalian in vitro coupled transcription/translation reaction using tRNAlys precharged with lysine-ε-biotin followed by solid phase immobilization on 384 well neutravidin-coated plates. The generated array is highly specific and sensitive in a microELISA format. By comparison of binding of vaccinia-immune sera to the reticulocyte lysate-produced proteins and to secreted post-translationally-modified proteins, we demonstrate that for several proteins including the EEV proteins B5 and A33, proper recognition is dependent upon appropriate folding, with little dependence upon glycosylation per se. We further demonstrate that the humoral immune response to vaccinia among different individuals is not uniform in specificity or strength, as different IMV and EEV targets predominate within the group of immunogenic proteins. This heterogeneity likely results from the diversity of HLA Class II alleles and CD4 T helper cell epitopes stimulating B cell antibody production. Our findings have important implications both for design of new recombinant subunit vaccines as well as for methods of assaying the human antibody response utilizing recombinant proteins produced in vitro. PMID:19146908

  8. Activity of core-modified 10-23 DNAzymes against HCV.

    PubMed

    Robaldo, Laura; Berzal-Herranz, Alfredo; Montserrat, Javier M; Iribarren, Adolfo M

    2014-09-01

    The highly conserved untranslated regions of the hepatitis C virus (HCV) play a fundamental role in viral translation and replication and are therefore attractive targets for drug development. A set of modified DNAzymes carrying (2'R)-, (2'S)-2'-deoxy-2'-C-methyl- and -2'-O-methylnucleosides at various positions of the catalytic core were assayed against the 5'-internal ribosome entry site element (5'-IRES) region of HCV. Intracellular stability studies showed that the highest stabilization effects were obtained when the DNAzymes' cores were jointly modified with 2'-C-methyl- and 2'-O-methylnucleosides, yielding an increase by up to fivefold in the total DNAzyme accumulation within the cell milieu within 48 h of transfection. Different regions of the HCV IRES were explored with unmodified 10-23 DNAzymes for accessibility. A subset of these positions was tested for DNAzyme activity using an HCV IRES-firefly luciferase translation-dependent RNA (IRES-FLuc) transcript, in the rabbit reticulocyte lysate system and in the Huh-7 human hepatocarcinoma cell line. Inhibition of IRES-dependent translation by up to 65 % was observed for DNAzymes targeting its 285 position, and it was also shown that the modified DNAzymes are as active as the unmodified one.

  9. Possible involvement of poly(A) in protein synthesis.

    PubMed Central

    Jacobson, A; Favreau, M

    1983-01-01

    The experiments of this paper have re-evaluated the possibility that poly(A) is involved in protein synthesis by testing whether purified poly(A) might competitively inhibit in vitro protein synthesis in rabbit reticulocyte extracts. We have found that poly(A) inhibits the rate of translation of many different poly(A)+ mRNAs and that comparable inhibition is not observed with other ribopolymers. Inhibition by poly(A) preferentially affects the translation of adenylated mRNAs and can be overcome by increased mRNA concentrations or by translating mRNPs instead of mRNA. The extent of inhibition is dependent on the size of the competitor poly(A) as well as on the translation activity which a lysate has for poly(A)+ RNA. In light of our results and numerous experiments in the literature, we propose that poly(A) has a function in protein synthesis and that any role in the determination of mRNA stability is indirect. Images PMID:6137807

  10. High-efficiency cloning of full-length cDNA.

    PubMed Central

    Okayama, H; Berg, P

    1982-01-01

    A widely recognized difficulty of presently used methods for cDNA cloning is obtaining cDNA segments that contain the entire nucleotide sequence of the corresponding mRNA. The cloning procedure described here mitigates this shortcoming. Of the 10(5) plasmid-cDNA recombinants obtained per microgram of rabbit reticulocyte mRNA, about 10% contained a complete alpha- of beta-globin mRNA sequence, and at least 30 to 50%, but very likely more, contained the entire globin coding regions. We attribute the high efficiency of cloning full- or nearly full-length cDNA to (i) the fact that the plasmid DNA vector itself serves as the primer for first- and second-strand cDNA synthesis, (ii) the lack of any nuclease treatment of the products, and (iii) the fact that one of the steps in the procedure results in preferential cloning of recombinants with full-length cDNA's over those with truncated cDNA's. Images PMID:6287227

  11. Oscillatory haematopoiesis in adults with sickle cell disease treated with hydroxycarbamide.

    PubMed

    Baird, John H; Minniti, Caterina P; Lee, Jung-Min; Tian, Xin; Wu, Colin; Jackson, Mary; Alam, Shoaib; Taylor, James G; Kato, Gregory J

    2015-03-01

    Hydroxycarbamide therapy has been associated with significant oscillations in peripheral blood counts from myeloid, lymphoid and erythroid lineages in patients with polycythaemia vera and chronic myeloid leukaemia. We retrospectively evaluated serial blood counts over an 8-year period from 44 adult patients with sickle cell disease receiving hydroxycarbamide. Platelet counts, leucocyte counts, haemoglobin values and reticulocyte counts, apportioned by hydroxycarbamide status, were analysed using a Lomb-Scargle periodogram algorithm. Significant periodicities were present in one or more counts in 38 patients receiving hydroxycarbamide for a mean duration of 4·81 years. Platelet and leucocyte counts oscillated in 56·8% and 52·3% of patients, respectively. These oscillations generally became detectable within days of initiating therapy. During hydroxycarbamide therapy, the predominant periods of oscillation were 27 ± 1 d for platelet counts and 15 ± 1 d for leucocyte counts. Despite an absolute decrease in leucocyte and platelet counts during hydroxycarbamide treatment, the amplitudes between nadirs and zeniths remained similar regardless of exposure. Our observations appear consistent with previously proposed models of cyclic haematopoiesis, and document that hydroxycarbamide-induced oscillations in blood counts are innocuous phenomena not limited to myeloproliferative disorders as described previously. We speculate the known cell cycle inhibitory properties of hydroxycarbamide may accentuate otherwise latent constitutive oscillatory haematopoiesis.

  12. Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2

    PubMed Central

    Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S.; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M. S. E.; Ackermann, Teresa F.; Föller, Michael; Schleicher, Erwin; Sheffield, William P.; Arthur, J. Simon C.; Lang, Florian; Qadri, Syed M.

    2015-01-01

    The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk−/−) and corresponding wild-type mice (msk+/+). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk−/− and msk+/+ mice, but reticulocyte count was significantly increased in msk−/− mice. Cell membrane PS exposure was similar in untreated msk−/− and msk+/+ erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk−/− erythrocytes than in msk+/+ erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk−/− erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk−/− mice. The spleens from msk−/− mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk+/+ mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice. PMID:26611568

  13. Accelerated apoptotic death and in vivo turnover of erythrocytes in mice lacking functional mitogen- and stress-activated kinase MSK1/2.

    PubMed

    Lang, Elisabeth; Bissinger, Rosi; Fajol, Abul; Salker, Madhuri S; Singh, Yogesh; Zelenak, Christine; Ghashghaeinia, Mehrdad; Gu, Shuchen; Jilani, Kashif; Lupescu, Adrian; Reyskens, Kathleen M S E; Ackermann, Teresa F; Föller, Michael; Schleicher, Erwin; Sheffield, William P; Arthur, J Simon C; Lang, Florian; Qadri, Syed M

    2015-11-27

    The mitogen- and stress-activated kinase MSK1/2 plays a decisive role in apoptosis. In analogy to apoptosis of nucleated cells, suicidal erythrocyte death called eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine (PS) externalization. Here, we explored whether MSK1/2 participates in the regulation of eryptosis. To this end, erythrocytes were isolated from mice lacking functional MSK1/2 (msk(-/-)) and corresponding wild-type mice (msk(+/+)). Blood count, hematocrit, hemoglobin concentration and mean erythrocyte volume were similar in both msk(-/-) and msk(+/+) mice, but reticulocyte count was significantly increased in msk(-/-) mice. Cell membrane PS exposure was similar in untreated msk(-/-) and msk(+/+) erythrocytes, but was enhanced by pathophysiological cell stressors ex vivo such as hyperosmotic shock or energy depletion to significantly higher levels in msk(-/-) erythrocytes than in msk(+/+) erythrocytes. Cell shrinkage following hyperosmotic shock and energy depletion, as well as hemolysis following decrease of extracellular osmolarity was more pronounced in msk(-/-) erythrocytes. The in vivo clearance of autologously-infused CFSE-labeled erythrocytes from circulating blood was faster in msk(-/-) mice. The spleens from msk(-/-) mice contained a significantly greater number of PS-exposing erythrocytes than spleens from msk(+/+) mice. The present observations point to accelerated eryptosis and subsequent clearance of erythrocytes leading to enhanced erythrocyte turnover in MSK1/2-deficient mice.

  14. [Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss].

    PubMed

    Maslova, M N; Kazennov, A M; Katiukhin, L N; Novozhilov, A V; Skverchinskaia, E A; Tavrovskaia, T V

    2007-01-01

    In experiments of Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activitities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12-15 % of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells - reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and their membrane preparations - ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed to optimization of blood circulation in large vessels and capillary network.

  15. Variation in use of erythrocyte invasion pathways by Plasmodium falciparum mediates evasion of human inhibitory antibodies

    PubMed Central

    Persson, Kristina E.M.; McCallum, Fiona J.; Reiling, Linda; Lister, Nicole A.; Stubbs, Janine; Cowman, Alan F.; Marsh, Kevin; Beeson, James G.

    2007-01-01

    Antibodies that inhibit Plasmodium falciparum invasion of erythrocytes are believed to be an important component of immunity against malaria. During blood-stage infection, P. falciparum can use different pathways for erythrocyte invasion by varying the expression and/or utilization of members of 2 invasion ligand families: the erythrocyte-binding antigens (EBAs) and reticulocyte-binding homologs (PfRhs). Invasion pathways can be broadly classified into 2 groups based on the use of sialic acid (SA) on the erythrocyte surface by parasite ligands. We found that inhibitory antibodies are acquired by malaria-exposed Kenyan children and adults against ligands of SA-dependent and SA-independent invasion pathways, and the ability of antibodies to inhibit erythrocyte invasion depended on the pathway used by P. falciparum isolates. Differential inhibition of P. falciparum lines that varied in their use of specific EBA and PfRh proteins pointed to these ligand families as major targets of inhibitory antibodies. Antibodies against recombinant EBA and PfRh proteins were acquired in an age-associated manner, and inhibitory antibodies against EBA175 appeared prominent among some individuals. These findings suggest that variation in invasion phenotype might have evolved as a mechanism that facilitates immune evasion by P. falciparum and that a broad inhibitory response against multiple ligands may be required for effective immunity. PMID:18064303

  16. Hydroxycarbamide alters erythroid gene expression in children with sickle cell anaemia.

    PubMed

    Flanagan, Jonathan M; Steward, Shirley; Howard, Thad A; Mortier, Nicole A; Kimble, Amy C; Aygun, Banu; Hankins, Jane S; Neale, Geoffrey A; Ware, Russell E

    2012-04-01

    Sickle cell anaemia (SCA) is a severe debilitating haematological disorder associated with a high degree of morbidity and mortality. The level of fetal haemoglobin (HbF) is well-recognized as a critical laboratory parameter: lower HbF is associated with a higher risk of vaso-occlusive complications, organ damage, and early death. Hydroxycarbamide treatment can induce HbF, improve laboratory parameters, and ameliorate clinical complications of SCA but its mechanisms of action remain incompletely defined and the HbF response is highly variable. To identify pathways of hydroxycarbamide activity, we performed microarray expression analyses of early reticulocyte RNA obtained from children with SCA enrolled in the HydroxyUrea Study of Long-term Effects (NCT00305175) and examined the effects of hydroxycarbamide exposure in vivo. Hydroxycarbamide affected a large number of erythroid genes, with significant decreases in the expression of genes involved in translation, ribosome assembly and chromosome organization, presumably reflecting the daily cytotoxic pulses of hydroxycarbamide. Hydroxycarbamide also affected expression of numerous genes associated with HbF including BCL11A, a key regulator of baseline HbF levels. Together, these data indicate that hydroxycarbamide treatment for SCA leads to substantial changes in erythroid gene expression, including BCL11A and other potential signalling pathways associated with HbF induction.

  17. Co-targeting the PI3K/mTOR and JAK2 signalling pathways produces synergistic activity against myeloproliferative neoplasms

    PubMed Central

    Bartalucci, Niccolò; Tozzi, Lorenzo; Bogani, Costanza; Martinelli, Serena; Rotunno, Giada; Villeval, Jean-Luc; Vannucchi, Alessandro M

    2013-01-01

    Aberrant JAK2 signalling plays a central role in myeloproliferative neoplasms (MPN). JAK2 inhibitors have proven to be clinically efficacious, however, they are not mutation-specific and competent enough to suppress neoplastic clonal haematopoiesis. We hypothesized that, by simultaneously targeting multiple activated signalling pathways, MPN could be more effectively treated. To this end we investigated the efficacy of BEZ235, a dual PI3K/mTOR inhibitor, alone and in combination with the JAK1/JAK2 inhibitor ruxolitinib, in different preclinical models of MPN. Single-agent BEZ235 inhibited the proliferation and induced cell cycle arrest and apoptosis of mouse and human JAK2V617F mutated cell lines at concentrations significantly lower than those required to inhibit the wild-type counterpart, and preferentially prevented colony formation from JAK2V617F knock-in mice and patients' progenitor cells compared with normal ones. Co-treatment of BEZ235 and ruxolitinib produced significant synergism in all these in-vitro models. Co-treatment was also more effective than single drugs in reducing the extent of disease and prolonging survival of immunodeficient mice injected with JAK2V617F-mutated Ba/F3-EPOR cells and in reducing spleen size, decreasing reticulocyte count and improving spleen histopathology in conditional JAK2V617F knock-in mice. In conclusion, combined inhibition of PI3K/mTOR and JAK2 signalling may represent a novel therapeutic strategy in MPN. PMID:24237791

  18. Biochemical and hematologic changes after short-term space flight

    NASA Technical Reports Server (NTRS)

    Leach, C. S.

    1992-01-01

    Clinical laboratory data from blood samples obtained from astronauts before and after 28 flights (average duration = 6 days) of the Space Shuttle were analyzed by the paired t-test and the Wilcoxon signed-rank test and compared with data from the Skylab flights (duration approximately 28, 59, and 84 days). Angiotensin I and aldosterone were elevated immediately after short-term space flights, but the response of angiotensin I was delayed after Skylab flights. Serum calcium was not elevated after Shuttle flights, but magnesium and uric acid decreased after both Shuttle and Skylab. Creatine phosphokinase in serum was reduced after Shuttle but not Skylab flights, probably because exercises to prevent deconditioning were not performed on the Shuttle. Total cholesterol was unchanged after Shuttle flights, but low density lipoprotein cholesterol increased and high density lipoprotein cholesterol decreased. The concentration of red blood cells was elevated after Shuttle flights and reduced after Skylab flights. Reticulocyte count was decreased after both short- and long-term flights, indicating that a reduction in red blood cell mass is probably more closely related to suppression of red cell production than to an increase in destruction of erythrocytes. Serum ferritin and number of platelets were also elevated after Shuttle flights. In determining the reasons for postflight differences between the shorter and longer flights, it is important to consider not only duration but also countermeasures, differences between spacecraft, and procedures for landing and egress.

  19. Clinical aspects and pathogenesis of congenital dyserythropoietic anemias: from morphology to molecular approach.

    PubMed

    Iolascon, Achille; Esposito, Maria Rosaria; Russo, Roberta

    2012-12-01

    Congenital dyserythropoietic anemias belong to a group of inherited conditions characterized by a maturation arrest during erythropoiesis with a reduced reticulocyte production in contrast with erythroid hyperplasia in bone marrow. The latter shows specific morphological abnormalities that allowed for a morphological classification of these conditions mainly represented by congenital dyserythropoietic anemias types I and II. The identification of their causative genes provided evidence that these conditions have different molecular mechanisms that induce abnormal cell maturation and division. Some altered proteins seem to be involved in the chromatin assembly, such as codanin-1 in congenital dyserythropoietic anemia I. The gene involved in congenital dyserythropoietic anemia II, the most frequent form, is SEC23B. This condition seems to belong to a group of diseases attributable to defects in the transport of newly synthesized proteins from endoplasmic reticulum to the Golgi. This review will analyze recent insights in congenital dyserythropoietic anemias types I and II. It will also attempt to clarify the relationship between mutations in causative genes and the clinical phenotype of these conditions.

  20. Subchronic Toxicity Study in Rats of Two New Ethyl-Carbamates with Ixodicidal Activity

    PubMed Central

    Prado-Ochoa, María Guadalupe; Abrego-Reyes, Víctor Hugo; Velázquez-Sánchez, Ana María; Muñoz-Guzmán, Marco Antonio; Ramírez-Noguera, Patricia; Angeles, Enrique; Alba-Hurtado, Fernando

    2014-01-01

    Female and male Wistar rats were used to determine the subchronic oral toxicities of two new ethyl-carbamates with ixodicidal activities (ethyl-4-bromphenyl-carbamate and ethyl-4-chlorphenyl-carbamate). The evaluated carbamates were administered in the drinking water (12.5, 25 and 50 mg/kg/day) for 90 days. Exposure to the evaluated carbamates did not cause mortality or clinical signs and did not affect food consumption or weight gain. However, exposure to these carbamates produced alterations in water consumption, hematocrit, percentages of reticulocytes, plasma proteins, some biochemical parameters (aspartate aminotransferase, gamma-glutamyl transpeptidase, cholinesterase, and creatinine activities), thiobarbituric acid reactive substances, and the relative weight of the spleen. Histologically, slight pathological alterations were found in the liver that were consistent with the observed biochemical alterations. The nonobserved adverse effect levels (NOAELs) of the evaluated carbamates were 12.5 mg/kg/day for both the female and male rats. The low severity and reversibility of the majority of the observed alterations suggest that the evaluated carbamates have low subchronic toxicity. PMID:24818142

  1. Gibberellin-induced changes in the populations of translatable mRNAs and accumulated polypeptides in dwarfs of maize and pea

    SciTech Connect

    Chory, J.; Voytas, D.F.; Olszewski, N.E.; Ausubel, F.M.

    1987-01-01

    Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize and pea lack endogenous gibberellin (GA/sub 1/) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of (/sup 35/S) methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs.

  2. Pernicious Anemia Associated Cobalamin Deficiency and Thrombotic Microangiopathy: Case Report and Review of the Literature

    PubMed Central

    Spinowitz, Bruce; Charytan, Chaim; Galler, Marilyn

    2017-01-01

    A 43-year-old Hispanic male without significant previous medical history was brought to emergency department for syncope following a blood draw to investigate a 40 lbs weight loss during the past 6 months associated with decreased appetite and progressive fatigue. The patient also reported a 1-month history of jaundice. On examination, he was hemodynamically stable and afebrile with pallor and diffuse jaundice but without skin rash or palpable purpura. Normal sensations and power in all extremities were evident on neurological exam. Presence of hemolytic anemia, schistocytosis, thrombocytopenia, and elevated lactate dehydrogenase (LDH) was suggestive of thrombotic thrombocytopenic purpura (TTP). However, presence of leukopenia, macrocytes, and an inadequate reticulocyte response to the degree of anemia served as initial clues to an alternative diagnosis. Two and one units of packed red blood cells were transfused on day 1 and day 3, respectively. In addition, one unit of platelets was transfused on day 2. Daily therapeutic plasma exchange (TPE) was initiated and continued until ADAMTS-13 result ruled out TTP. A low cobalamin (vitamin B12) level was evident at initial laboratory work-up and subsequent testing revealed positive intrinsic factor-blocking antibodies supporting a diagnosis of pernicious anemia with severe cobalamin deficiency. Hematological improvement was observed following vitamin B12 supplementation. The patient was discharged and markedly improved on day 9 with outpatient follow-up for cobalamin supplementation. PMID:28265287

  3. SAGE application in hematological research.

    PubMed

    Hashimoto, Shin-ichi; Matsushima, Kouji

    2008-10-01

    Blood cells perform many important functions within the body, including homeostasis and host defense against various invading stimuli such as viral infection, cancer and autoimmune diseases. The subsets of leukocytes interact with each other through various surface molecules such as cytokine receptors, co-stimulation molecules and adhesion molecules. Over the last several years, accumulation of cDNA and genome databases has led to the accelerated identification of the molecules responsible for cell-cell interaction, cell activation and cell differentiation. In addition, technologies used in functional genomics, such as DNA microarray and serial analysis of gene expression (SAGE), have allowed us to analyze the expression of thousands of genes. The comprehensive analysis of gene expression is very useful to elucidate the function of cells, because characteristics of each cell type depend on the genes selectively expressed at various stages. The SAGE method, which is very quantitative, can cover the number of expressed genes that are unequaled by any other mammalian DNA microarray systems yet available. In order to molecularly define the subset and function of blood cells, we and other groups have performed SAGE in various types of hematipoietic cells. Here, we review the SAGE data obtained from the gene expression libraries made from various differentiation and activation stages of a broad range of blood cells, including phagocytes, T cells, B cells, platelets, reticulocytes and NK cells.

  4. Cloning and molecular characterization of a human intracellular serine proteinase inhibitor.

    PubMed Central

    Coughlin, P; Sun, J; Cerruti, L; Salem, H H; Bird, P

    1993-01-01

    We describe a cDNA encoding a serine proteinase inhibitor present in placental tissue and the cytosolic fraction of K562 cells. On the basis of its interaction with thrombin, through which it was discovered, the inhibitor has been operationally named the placental thrombin inhibitor (PTI). Amino acid sequence comparisons suggest that its reactive center is located at Arg-341 and Cys-342, that it lacks a classical N-terminal signal sequence, and that it has the highest degree of similarity to intracellular serine proteinase inhibitors (serpins), such as the human monocyte/neutrophil elastase inhibitor and the equine leukocyte elastase inhibitor. PTI also resembles these inhibitors in that it contains oxidation-sensitive residues adjacent to the reactive site. The PTI cDNA was expressed in rabbit reticulocyte lysate and in COS-7 cells and a 42-kDa protein was produced. Recombinant PTI formed a 67-kDa complex when incubated with thrombin. The ability of native PTI to bind thrombin was destroyed by incubation with iodoacetamide. Analysis of human tissue mRNA indicated that PTI is expressed widely with the highest levels in cardiac and skeletal muscle and placenta. We conclude that PTI is a member of an emerging class of intracellular serpins. Images Fig. 2 Fig. 3 Fig. 4 PMID:8415716

  5. Isolation of an 18,000-dalton hypusine-containing protein from cultured mouse neuroblastoma cells

    SciTech Connect

    Dou, Q.P.; Chen, K.Y.

    1987-05-01

    An 18,000-dalton protein can be metabolically labeled by (TH)putrescine or spermidine in mammalian cells. The labeling is due to a post-translational conversion of a lysine residue to hypusine residue. Previous studies indicated that the labeling is growth-dependent and is greatly diminished in mouse neuroblastoma cells after differentiation. To further study the physiological functions of this protein in the differentiation of mouse neuroblastoma cells, they have developed a simple procedure to purify this protein from cultured NB-15 mouse neuroblastoma cells. The 4-steps procedure included a Cibacron-Blue column, an omega-diaminooctyl-agarose column, a Sephadex G-50 column, and a Mono Q column. The procedure resulted in a 500-fold purification and the preparation appeared to be homogenous as judged by SDS-PAGE. Peptide map analysis using V-8 protease digestion method indicated that the 18,000-dalton hypusine-containing protein from NB-15 cells was identical to eukaryotic initiation factor 4D isolated from rabbit reticulocytes. This purification scheme also enabled them to detect a very faintly labeled protein in NB-15 cells. This weakly labeled protein had an apparent molecular weight of 22,000-dalton and pI of 5.0.

  6. Echovirus 22 is an atypical enterovirus.

    PubMed Central

    Coller, B A; Chapman, N M; Beck, M A; Pallansch, M A; Gauntt, C J; Tracy, S M

    1990-01-01

    Although echovirus 22 (EV22) is classified as an enterovirus in the family Picornaviridae, it is atypical of the enterovirus paradigm, typified by the polioviruses and the coxsackie B viruses. cDNA reverse transcribed from coxsackievirus B3 (CVB3) RNA does not hybridize to genomic RNA of EV22, and conversely, cDNA made to EV22 does not hybridize to CVB3 genomic RNA or to molecular clones of CVB3 or poliovirus type 1. EV22 cDNA does not hybridize to viral RNA of encephalomyocarditis virus or to a molecular clone of Theiler's murine encephalomyelitis virus, members of the cardiovirus genus. The genomic RNA of EV22 cannot be detected by the polymerase chain reaction using generic enteroviral primers. EV22 does not shut off host cell protein synthesis, and the RNA of EV22 is efficiently translated in vitro in rabbit reticulocyte lysates. Murine enterovirus-immune T cells recognize and proliferate against EV22 as an antigen in vitro, demonstrating that EV22 shares an epitope(s) common to enteroviruses but not found among other picornaviruses. Images PMID:2159539

  7. Effect of phosphate supplementation on oxygen delivery at high altitude

    NASA Astrophysics Data System (ADS)

    Jain, S. C.; Singh, M. V.; Rawal, S. B.; Sharma, V. M.; Divekar, H. M.; Tyagi, A. K.; Panwar, M. R.; Swamy, Y. V.

    1987-09-01

    In the present communication, effect of low doses of phosphate supplementation on short-term high altitude adaptation has been examined. Studies were carried out in 36 healthy, male, sea-level residents divided in a double blind fashion into drug and placebo treated groups. 3.2 mmol of phosphate were given orally to each subject of the drug treated group once a day for 4 days on arrival at an altitude of 3,500 m. Sequential studies were done in the subjects in both groups on the 3rd, 7th, 14th and 21st day of their altitude stay. Haemoglobin, haematocrit, erythrocyte and reticulocyte counts increased to the similar extent in both groups. Blood pH, pO2 and adenosine tri-phosphate (ATP) did not differ between the two groups. On 3rd day of the altitude stay, inorganic phosphate and 2,3-diphosphoglycerate (2,3 DPG) levels in the drug treated group increased significantly as compared to the placebo group. No significant difference in inorganic phosphate and 2,3 DPG was observed later on in the two groups. Psychological and clinical tests also indicated that the drug treated subjects felt better as compared to the placebo treated subjects. The present study suggests that low doses of phosphate increases circulating 2,3-DPG concentration which in turn brings about beneficial effect towards short term high altitude adaptation.

  8. Clinical Outcomes of Splenectomy in Children: Report of the Splenectomy in Congenital Hemolytic Anemia (SICHA) Registry

    PubMed Central

    Rice, Henry E; Englum, Brian R; Rothman, Jennifer; Leonard, Sarah; Reiter, Audra; Thornburg, Courtney; Brindle, Mary; Wright, Nicola; Heeney, Matthew M; Smithers, Charles; Brown, Rebeccah L; Kalfa, Theodosia; Langer, Jacob C; Cada, Michaela; Oldham, Keith T; Scott, J Paul; St. Peter, Shawn; Sharma, Mukta; Davidoff, Andrew M.; Nottage, Kerri; Bernabe, Kathryn; Wilson, David B; Dutta, Sanjeev; Glader, Bertil; Crary, Shelley E; Dassinger, Melvin S; Dunbar, Levette; Islam, Saleem; Kumar, Manjusha; Rescorla, Fred; Bruch, Steve; Campbell, Andrew; Austin, Mary; Sidonio, Robert; Blakely, Martin L

    2014-01-01

    The outcomes of children with congenital hemolytic anemia (CHA) undergoing total splenectomy (TS) or partial splenectomy (PS) remain unclear. In this study, we collected data from 100 children with CHA who underwent TS or PS from 2005–2013 at 16 sites in the Splenectomy in Congenital Hemolytic Anemia (SICHA) consortium using a patient registry. We analyzed demographics and baseline clinical status, operative details, and outcomes at 4, 24, and 52 weeks after surgery. Results were summarized as hematologic outcomes, short-term adverse events (AEs) (≤ 30 days after surgery), and long-term AEs (31–365 days after surgery). For children with hereditary spherocytosis, after surgery there was an increase in hemoglobin (baseline 10.1 ± 1.8 gm/dl, 52 week 12.8 ± 1.6 gm/dl; mean ± SD), decrease in reticulocyte and bilirubin as well as control of symptoms. Children with sickle cell disease had control of clinical symptoms after surgery, but had no change in hematologic parameters. There was an 11% rate of short-term AEs and 11% rate of long-term AEs. As we accumulate more subjects and longer follow-up, use of a patient registry should enhance our capacity for clinical trials and engage all stakeholders in the decision-making process. PMID:25382665

  9. Farnesylation or geranylgeranylation? Efficient assays for testing protein prenylation in vitro and in vivo

    PubMed Central

    Benetka, Wolfgang; Koranda, Manfred; Maurer-Stroh, Sebastian; Pittner, Fritz; Eisenhaber, Frank

    2006-01-01

    Background Available in vitro and in vivo methods for verifying protein substrates for posttranslational modifications via farnesylation or geranylgeranylation (for example, autoradiography with 3H-labeled anchor precursors) are time consuming (weeks/months), laborious and suffer from low sensitivity. Results We describe a new technique for detecting prenyl anchors in N-terminally glutathione S-transferase (GST)-labeled constructs of target proteins expressed in vitro in rabbit reticulocyte lysate and incubated with 3H-labeled anchor precursors. Alternatively, hemagglutinin (HA)-labeled constructs expressed in vivo (in cell culture) can be used. For registration of the radioactive marker, we propose to use a thin layer chromatography (TLC) analyzer. As a control, the protein yield is tested by Western blotting with anti-GST- (or anti-HA-) antibodies on the same membrane that has been previously used for TLC-scanning. These protocols have been tested with Rap2A, v-Ki-Ras2 and RhoA (variant RhoA63L) including the necessary controls. We show directly that RasD2 is a farnesylation target. Conclusion Savings in time for experimentation and the higher sensitivity for detecting 3H-labeled lipid anchors recommend the TLC-scanning method with purified GST- (or HA-) tagged target proteins as the method of choice for analyzing their prenylation capabilities in vitro and in vivo and, possibly, also for studying the myristoyl and palmitoyl posttranslational modifications. PMID:16507103

  10. [Informative value of erythrocyte function in the assessment of ionizing radiation effect].

    PubMed

    Dlusskaia, I G; Ushakov, I B

    1998-01-01

    Proposed are new quantitative criteria of the functional state of erythrocytes based on automated recording parameters of the acid hemolysis erythrogram. Extended analysis of adaptation of the red blood system in rats exposed to ionizing radiation (60CO, 1.25 MeV, 0.5, 1.0, and 6.0 Gy) pointed out that the normally existing stimulating effect of diuretic products on accumulation of juvenile cellular forms was not changed by the dose of 0.5 Gy but ceased after exposure to higher doses. A distinct interrelation (r = 0.83 and 0.61 at p < 0.003, respectively) was revealed between the number of low-resistant erythrocytes (EZR) and subsequent (4 to 7 days later) amount of reticulocytes in the norm and following exposure to 0.5 Gy of ionizing radiation. After exposure to 1.0 Gy and 6.0 Gy r values were below 0.3 at p > 0.2. A "reference cascade" of regression equations on the order of Rt = a + b x EZR could be developed for individual evaluation of different stages of impairment of one of the normal regulatory mechanisms of hemopoiesis in humans systematically exposed to ionizing radiation.

  11. [Study of plant lectins from Viscum album using monoclonal antibodies].

    PubMed

    Tonevitskiĭ, A G; Rakhmanova, V A; Shamshiev, A T; Usachaeva, E A; Agapov, I I; Prokov'ev, S A; Denisenko, O N; Pfuller, U; Eifler, R

    1995-01-01

    Monoclonal antibodies (monAT) against both native (TA5, TB12) and denatured (TB33, TB35) plant toxin ML1 from Viscum album have been obtained. The interaction of monAT against native toxin with its isoforms ML2 and ML3 was investigated. It was shown that monAT TA5 to A-chain of ML1 toxin cross-reacted with ML2 and ML3 isoforms. TA5 did not inhibit enzyme activity of A-chain in cell-free rabbit reticulocyte system. It was shown that monAT TB12 reacted with galactose-binding site of B-subunit. Both monAT had no cross-reactions with plant toxin ricin. The binding constants for TA5 with ML1, ML2, ML3 respectively were 4.3.10(7) M-1, 1.2.10(7) M-1, and 0.3.10(7) M-1. The binding constants for TB12 were 2.10(7) M-1 with ML1 toxin, and more than 10(6) M-1 with ML2 and ML3. The nature of heterogeneity in ML toxin family is discussed. Test-systems for ML1 determination in different V. album extracts are suggested.

  12. tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products.

    PubMed

    Kurzchalia, T V; Wiedmann, M; Breter, H; Zimmermann, W; Bauschke, E; Rapoport, T A

    1988-03-15

    We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.

  13. Translational control of ferritin synthesis: a study of repression using natural and synthetic mRNAs

    SciTech Connect

    Dickey, L.F.; Wang, Y.H.; Wortman, I.; Shull, G.E.; Theil, E.C.

    1987-05-01

    Ferritin synthesis is a dramatic example of mRNA repression: excess iron causes recruitment of ferritin mRNA, increasing synthesis less than or equal to 40 x. Using the iron-storing tadpole red cell as a simple and accessible model, isolated poly(A+) RNA directed the synthesis of ferritin and globin in cell-free extracts from wheat germ (WG); in contrast, ferritin mRNA was specifically repressed (72%) in extracts from rabbit reticulocytes (RR) as it is in vivo. Translatable and hybridizable ferritin mRNA did not enter polysomes of RR in contrast to globin mRNA and to both ferritin and globin mRNA in WG. Single-sequence mRNA, uncapped, was prepared in vitro for both a ferritin M chain and a globin beta chain; both were translated with similar efficiency in RR (164 +/- 66) x 10/sup -3/ and (205 +/- 144) x 10/sup -3/ cpm (/sup 3/H)leucine/h/..mu..g RNA for ferritin and globin, respectively). Ferritin with the expected immunoreactivity and mobility in SDS gel electrophoresis was obtained. The results suggest that ferritin mRNA repression may require a cap or factors present in poly(A+) RNA and that repression can be released in WG but not in RR.

  14. Translocation of the potato 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase into isolated spinach chloroplasts

    SciTech Connect

    Zhao, Jianmin; Weaver, L.M.; Herrmann, K.M. )

    1990-05-01

    A cDNA for potato (Solanum tuberosum L.) 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase, the first enzyme of the shikimate pathway, encodes a 56 KD polypeptide whose amino terminus resembles a chloroplast transit sequence. The cDNA was placed downstream of the phage T7 polymerase recognition sequence in plasmid pGEM-3Z. DNA of the resulting plasmid pGEM-DWZ directed T7 polymerase to synthesize potato DAHP synthase mRNA in vitro. The mRNA was used in wheat germ and rabbit reticulocyte lysates for the synthesis of {sup 35}S-labeled pro-DAHP synthase. The predominant translation product is a 59 KD polypeptide that can be immunoprecipitated by rabbit polyclonal antibodies raised against the 53 KD DAHP synthase purified from potato tubers. Isolated spinach chloroplasts process the 59 KD pro-DAHP synthase to a 50 KD polypeptide. The processed polypeptide is protected from protease degradation, suggesting uptake of the enzyme into the cell organelle. Fractionation of reisolated chloroplasts after import of pro-DAHP synthase showed mature enzyme in the stroma. The uptake and processing of DAHP synthase is inhibited by antibodies raised against the mature enzyme. Our results are consistent with the assumption that potato contains a nuclear DNA encoded DAHP synthase that is synthesized as a proenzyme and whose mature form resides in the chloroplasts. Our data provide further evidence that green plants synthesize aromatic amino acids in plastids.

  15. Nuclear and nucleolar localization signals and their targeting function in phosphatidylinositol 4-kinase PI4K230

    SciTech Connect

    Kakuk, Annamaria; Friedlaender, Elza; Vereb, Gyoergy; Lisboa, Duarte; Bagossi, Peter; Toth, Gabor; Gergely, Pal; Vereb, Gyoergy

    2008-08-01

    PI4K230, an isoform of phosphatidylinositol 4-kinase, known primarily as a cytoplasmic membrane-bound enzyme, was detected recently also in the nucleolus of several cells. Here we provide mechanistic insight on the targeting function of its putative nuclear localization signal (NLS) sequences using molecular modeling, digitonin-permeabilized HeLa cells and binding to various importins. The synthetic sequence {sup 916}NFNHIHKRIRRVADKYLSG{sup 934} comprising a putative monopartite NLS (NLS1), targeted covalently bound fluorescent BSA to the nucleoplasm via classical importin {alpha}/{beta} mechanism employing importins {alpha}1 and {alpha}3 but not {alpha}5. This transport was inhibited by wheat germ agglutinin and GTP{gamma}S. The sequence {sup 1414}SKKTNRGSQLHKYYMKRRTL{sup 1433}, a putative bipartite NLS (NLS2) proved ineffective in nuclear targeting if conjugated to fluorescently labeled BSA. Nonetheless, NLS2 or either of its basic clusters directed to the nucleolus soybean trypsin inhibitor that can pass the nuclear pore complex passively; moreover, an expressed 58 kDa fragment of PI4K230 (AA1166-1667) comprising NLS2 was also imported into the nucleus by import factors of reticulocyte lysate or by importin {alpha}1/{beta} or {alpha}3/{beta} complexes and localized to the nucleolus. We conclude that the putative bipartite NLS itself is a nucleolar targeting signal, and for nuclear import PI4K230 requires a larger sequence around it or, alternatively, the monopartite NLS.

  16. Improvements in haemolysis and indicators of erythrocyte survival do not correlate with acute vaso-occlusive crises in patients with sickle cell disease: a phase III randomized, placebo-controlled, double-blind study of the Gardos channel blocker senicapoc (ICA-17043).

    PubMed

    Ataga, Kenneth I; Reid, Marvin; Ballas, Samir K; Yasin, Zahida; Bigelow, Carolyn; James, Luther St; Smith, Wally R; Galacteros, Frederic; Kutlar, Abdullah; Hull, James H; Stocker, Jonathan W

    2011-04-01

    Red blood cell (RBC) hydration is regulated in part by the Ca(2+) -activated K(+) efflux (Gardos) channel. Senicapoc selectively blocks potassium efflux through the Gardos channel, reducing RBC dehydration and haemolysis, and increasing haemoglobin levels in sickle cell disease (SCD). This randomized, placebo-controlled trial was designed to determine the safety and clinical efficacy of senicapoc in SCD patients. One hundred and forty-five patients were randomized to receive senicapoc and 144 patients to receive placebo for 52 weeks. Consistent with a previous study, patients in the senicapoc group had significantly increased haematocrit, haemoglobin, and decreased numbers of both dense erythrocytes and reticulocytes when compared to the placebo group. The unblinded Data Monitoring Committee terminated this study early due to a lack of efficacy when it determined that, despite improvements in anaemia and haemolysis, no significant improvement in the rate of sickle cell painful crises was observed in patients treated with senicapoc compared to those on placebo (0·38 vs. 0·31, respectively). Comparisons of the times to first, second and third crises between the senicapoc and placebo groups were not statistically significant. Nausea and urinary tract infections occurred more frequently in the senicapoc group than placebo. Serious adverse events were similar in the two groups.

  17. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-08-10

    Messenger RNAs for mouse embryonic globins were purified from yolk sac derived eyrthroid cells in mouse fetuses. Double stranded DNAs complementary to these messengers were synthesized and blunt end ligated to a EcoRI digested and DNA polymerase I repaired pBR322 plasmid. Of the ampicillin resistant transformants, one contained a plasmid with globin-specific cDNA. The inserted sequence is about 350 base pairs long. It contains one restriction site for EcoRI and one restriction site for HinfI about 170 and 80 base pairs from one end. The insert is not cleaved by HindIII, HindII, BamHI, PstI, SalI, AvaI, TaqI, HpaII, BglI. A mixture of purified messengers coding for alpha chains and for x, y and z embryonic chains was incubated with the recombinant plasmid and the hybridized messenger was translated in a mRNA depleted reticulocyte lysate protein synthesizing system. The product of translation was identified as a z chain by carboxymethylcellulose cromatography. The recombinant plasmid is named "pBR322-egz" after embryonic globin z.

  18. Ferulic acid inhibits gamma radiation-induced DNA strand breaks and enhances the survival of mice.

    PubMed

    Maurya, Dharmendra Kumar; Devasagayam, Thomas Paul Asir

    2013-02-01

    Ferulic acid (FA) is a monophenolic phenylpropanoid occurring in plant products such as rice bran, green tea, and coffee beans. It has been shown to have significant antioxidant effects in many studies. In the present study, we show that intraperitoneal administration of FA at a dose of 50 mg/kg body weight 1 hour prior to or immediately after whole-body γ-irradiation of mice with 4 Gy results in considerable reduction in the micronuclei formation in peripheral blood reticulocytes. Administration of the same amount of FA immediately after 4 Gy γ-irradiation showed significant decrease in the amount of DNA strand breaks in murine peripheral blood leukocytes and bone marrow cells as examined by comet assay. Further, immunostaining of mouse splenic lymphocytes for phspho-γH2AX was carried out, and it was observed that FA inhibits the γH2AX foci formation. Finally, the survival of mice upon 6, 8, and 10 Gy γ-ray exposure was monitored. FA enhances the survival of mice by a factor of 2.5 at a dose of 6 Gy γ-radiation but not at higher doses. In conclusion, FA has protective potential in both pre- and postirradiation exposure scenarios and enhances the survival of mice possibly by decreasing DNA damage as examined by γH2AX foci, micronuclei formation, and comet assay.

  19. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease

    PubMed Central

    ALAPAN, YUNUS; KIM, CEONNE; ADHIKARI, ANIMA; GRAY, KAYLA E.; GURKAN-CAVUSOGLU, EVREN; LITTLE, JANE A.; GURKAN, UMUT A.

    2016-01-01

    Sickle cell disease (SCD) afflicts millions of people worldwide and is associated with considerable morbidity and mortality. Chronic and acute vaso-occlusion are the clinical hallmarks of SCD and can result in pain crisis, widespread organ damage, and early movtality. Even though the molecular underpinnings of SCD were identified more than 60 years ago, there are no molecular or biophysical markers of disease severity that are feasibly measured in the clinic. Abnormal cellular adhesion to vascular endothelium is at the root of vaso-occlusion. However, cellular adhesion is not currently evaluated clinically. Here, we present a clinically applicable microfluidic device (SCD biochip) that allows serial quantitative evaluation of red blood cell (RBC) adhesion to endothelium-associated protein-immobilized microchannels, in a closed and preprocessing-free system. With the SCD biochip, we have analyzed blood samples from more than 100 subjects and have shown associations between the measured RBC adhesion to endothelium-associated proteins (fibronectin and laminin) and individual RBC characteristics, including hemoglobin content, fetal hemoglobin concentration, plasma lactate dehydrogenase level, and reticulocyte count. The SCD biochip is a functional adhesion assay, reflecting quantitative evaluation of RBC adhesion, which could be used at baseline, during crises, relative to various long-term complications, and before and after therapeutic interventions. PMID:27063958

  20. Interaction of HIV-1 gag and membranes in a cell-free system.

    PubMed

    Yang, Liuzhan; Ratner, Lee

    2002-10-10

    A coupled transcription-translation (TNT) reticulocyte lysate system was used to examine posttranslational alterations in HIV-1 Gag upon addition of Jurkat T cell membranes. Incubation of the Gag precursor protein, Pr55gag, with membranes resulted in a time-dependent alteration in Gag resulting in partial resistance to trypsin treatment. Treatment of membranes and TNT extract with apyrase or pretreatment of membranes with trypsin prevented this posttranslational alteration of Gag. In contrast, this activity was not disrupted by pretreatment of membranes with Triton X-100 at 4 degrees C, under conditions which do not solubilize raft-associated proteins. Flotation studies revealed that acquisition of trypsin-resistance was accompanied by Gag binding to membranes. The myristylation signal and nucleocapsid domain were found to mediate Gag binding to membranes. The posttranslational alteration of Gag accompanying membrane interaction may represent a conformational change, oligomerization, and/or association with or envelopment by membranes. These findings provide new clues to the stepwise process of HIV-1 assembly.

  1. Structural features of the Seneca Valley virus internal ribosome entry site (IRES) element: a picornavirus with a pestivirus-like IRES.

    PubMed

    Willcocks, Margaret M; Locker, Nicolas; Gomwalk, Zarmwa; Royall, Elizabeth; Bakhshesh, Mehran; Belsham, Graham J; Idamakanti, Neeraja; Burroughs, Kevin D; Reddy, P Seshidhar; Hallenbeck, Paul L; Roberts, Lisa O

    2011-05-01

    The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId(2) domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.

  2. Synthesis of a major integral membrane polypeptide of rat liver peroxisomes on free polysomes.

    PubMed Central

    Fujiki, Y; Rachubinski, R A; Lazarow, P B

    1984-01-01

    The manner of synthesis and assembly of the peroxisomal membrane proteins is unknown. Understanding these processes is essential to an understanding of the formation of the organelle. We have investigated the biogenesis of the previously identified major 21.7-kDa integral peroxisomal membrane polypeptide [Fujiki, Y., Fowler, S., Shio, H., Hubbard, A. L. & Lazarow, P. B. (1982) J. Cell Biol. 93, 103-110]. This protein was purified to apparent homogeneity and used to elicit a rabbit antiserum. In immunoblotting analysis, antibody bound only to the 22-kDa membrane polypeptide present exclusively in peroxisomal membranes. Total rat liver RNA was translated in a nuclease-treated rabbit reticulocyte cell-free protein-synthesizing system. The in vitro translation product, isolated by means of the antibody and Staphylococcus aureus cells, comigrated with the mature 22-kDa polypeptide in NaDodSO4/PAGE. Analysis of the translation products of RNAs from free and membrane-bound polysomes indicated that the mRNA for the 22-kDa membrane polypeptide is found predominantly in free polysomes. The results imply post-translational insertion of the membrane polypeptide into the peroxisomal membrane without proteolytic processing and suggest that peroxisomes, like mitochondria and chloroplasts, form by fission from preexisting organelles. Images PMID:6594687

  3. Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. I. Functional tests on rat liver microsomal subfractions

    PubMed Central

    1984-01-01

    A preparation of rat liver microsomes containing 70% of the total cellular endoplasmic reticulum (ER) membranes was subfractionated by isopycnic density centrifugation. Twelve subfractions of different ribosome content ranging in density from 1.06 to 1.29 were obtained and analyzed with respect to marker enzymes, RNA, and protein content, as well as the capacity of these membranes to bind 80S ribosomes in vitro. After removal of native polysomes from these microsomal subfractions by puromycin in a buffer of high ionic strength their capacity to rebind 80S ribosomes approached levels found in the corresponding native membranes before ribosome stripping. This indicates that in vitro rebinding of ribosomes occurs to the same sites occupied in the cell by membrane-bound polysomes. Microsomes in the microsomal subfractions were also tested for their capacity to effect the translocation of nascent secretory proteins into the microsomal lumen utilizing a rabbit reticulocyte translation system programmed with mRNA coding for the precursor of human placental lactogen. Membranes from microsomes with the higher isopycnic density and a high ribosome content showed the highest translocation activity, whereas membranes derived from smooth microsomes had only a very low translocation activity. These results indicate the membranes of the rough and smooth portions of the endoplasmic reticulum are functionally differentiated so that sites for ribosome binding and the translocation of nascent polypeptides are segregated to the rough domain of the organelle. PMID:6501423

  4. Neutralization of Plasmodium falciparum merozoites by antibodies against PfRH5

    PubMed Central

    Douglas, Alexander D.; Williams, Andrew R.; Knuepfer, Ellen; Illingworth, Joseph J.; Furze, Julie M.; Crosnier, Cécile; Choudhary, Prateek; Bustamante, Leyla Y.; Zakutansky, Sara E.; Awuah, Dennis K.; Alanine, Daniel G. W.; Theron, Michel; Worth, Andrew; Shimkets, Richard; Rayner, Julian C.; Holder, Anthony A.; Wright, Gavin J.; Draper, Simon J.

    2013-01-01

    There is intense interest in induction and characterization of strain-transcending neutralizing antibody against antigenically variable human pathogens. We have recently identified the human malaria parasite Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) as a target of broadly-neutralizing antibodies, but there is little information regarding the functional mechanism(s) of antibody-mediated neutralization. Here, we report that vaccine-induced polyclonal anti-PfRH5 antibodies inhibit the tight attachment of merozoites to erythrocytes, and are capable of blocking the interaction of PfRH5 with its receptor basigin. Furthermore, by developing anti-PfRH5 monoclonal antibodies (mAbs), we provide evidence that i) the ability to block the PfRH5-basigin interaction in vitro is predictive of functional activity, but absence of blockade does not predict absence of functional activity; ii) neutralizing mAbs bind spatially-related epitopes on the folded protein, involving at least two defined regions of the PfRH5 primary sequence; iii) a brief exposure window of PfRH5 is likely to necessitate rapid binding of antibody to neutralize parasites; and iv) intact bivalent IgG contributes to but is not necessary for parasite neutralization. These data provide important insight into the mechanisms of broadly-neutralizing anti-malaria antibodies and further encourage anti-PfRH5 based malaria prevention efforts. PMID:24293631

  5. Management of iron deficiency anemia in inflammatory bowel disease - a practical approach.

    PubMed

    Stein, Jürgen; Dignass, Axel U

    2013-01-01

    Although anemia is the most common systemic manifestation of inflammatory bowel disease (IBD), among the broad spectrum of extraintestinal disease complications encountered in IBD, including arthritis and osteopathy, it has generally received little consideration. However, not only in terms of frequency, but also with regard to its potential effect on hospitalization rates and on the quality of life and work, anemia is indeed a significant and costly complication of IBD. Anemia is multifactorial in nature, the most prevalent etiological forms being iron deficiency anemia (IDA) and anemia of chronic disease. In a condition associated with inflammation, such as IBD, the determination of iron status using common biochemical parameters alone is inadequate. A more accurate assessment may be attained using new iron indices including reticulocyte hemoglobin content, percentage of hypochromic red cells or zinc protoporphyrin. While oral iron supplementation has traditionally been a mainstay of IDA treatment, it has also been linked to extensive gastrointestinal side effects and possible disease exacerbation. However, many physicians are still reluctant to administer iron intravenously, despite the wide availability of a variety of new IV preparations with improved safety profiles, and despite the recommendations of international expert guidelines. This article discusses improved diagnostic and therapeutic strategies based on new clinical insights into the regulation of iron homeostasis.

  6. Effect of feeding green onions (Allium ascalonicum) to White Chinese geese (Threskiornis spinicollis).

    PubMed

    Crespo, Rocio; Chin, R P

    2004-07-01

    Sudden increase in mortality was observed in 2 different flocks of mature breeder geese fed green onions. At necropsy, birds had pale epicardium with random petechiation, sanguinous fluid accumulation in the pericardial sac, and mild swelling of the liver and spleen. Histologically, there was accumulation of hemosiderin in hepatocytes, Kupffer cells of the liver, macrophages, and renal tubules. There was also moderate to severe hepatic necrosis, vacuolation of hepatocytes, splenitis, and renal tubular nephrosis. To assess the effects of green onion ingestion, 2 feeding trials were carried out in 3 mature White Chinese geese. In the first trial, onions were thoroughly mixed with pellet maintenance ration. In the second trial, onions were offered in a separate trough from the pelleted diet. During the 21 days of experiments, the red blood cell count and hematocrit decreased, whereas the polychromasia and reticulocyte estimate increased. The blood changes were more marked in birds from the second feeding trial. Gross and histologic changes were similar in both trials. Mild swelling and severe darkening of the liver were the only significant findings at necropsy. Histologically, the liver looked similar to that seen from the field outbreak. The liver contained moderate amounts of hemosiderin in the hepatocytes and Kupffer cells, and had centrolobular necrosis and vacuolation of hepatocytes. This experimental study demonstrated that anemia and liver pathology could be caused by ingestion of onions. Furthermore, Heinz bodies are not a consistent finding in the blood of geese fed onions.

  7. Genetic modifiers of sickle cell anemia in the BABY HUG cohort: influence on laboratory and clinical phenotypes.

    PubMed

    Sheehan, Vivien A; Luo, Zhaoyu; Flanagan, Jonathan M; Howard, Thad A; Thompson, Bruce W; Wang, Winfred C; Kutlar, Abdullah; Ware, Russell E

    2013-07-01

    The recently completed BABY HUG trial investigated the safety and efficacy of hydroxyurea in infants with sickle cell anemia (SCA). To investigate the effects of known genetic modifiers, genomic DNA on 190 randomized subjects were analyzed for alpha thalassemia, beta-globin haplotype, polymorphisms affecting endogenous fetal hemoglobin (HbF) levels (XmnI, BCL11A, and HBS1L-MYB), UGT1A1 promoter polymorphisms, and the common G6PD A(-) mutation. At study entry, infants with alpha thalassemia trait had significantly lower mean corpuscular volume, total bilirubin, and absolute reticulocyte count. Beta-globin haplotypes associated with milder disease had significantly higher hemoglobin and %HbF. BCL11A and XmnI polymorphisms had significant effects on baseline HbF, while UGT1A1 promoter polymorphisms significantly influenced baseline serum bilirubin. At study exit, subjects randomized to placebo still exhibited laboratory effects of alpha thalassemia and other modifiers, while those assigned hydroxyurea had treatment effects that exceeded most genetic influences. The pain phenotype was influenced by HbF modifiers in both treatment groups. These data document that genetic polymorphisms do modify laboratory and clinical phenotypes even in very young patients with SCA. The hydroxyurea effects are more potent, however, indicating that treatment criteria should not be limited to certain genetic subsets, and supporting the use of hydroxyurea for all young patients with SCA.

  8. The Senegal DNA haplotype is associated with the amelioration of anemia in African-American sickle cell anemia patients.

    PubMed

    Nagel, R L; Erlingsson, S; Fabry, M E; Croizat, H; Susuka, S M; Lachman, H; Sutton, M; Driscoll, C; Bouhassira, E; Billett, H H

    1991-03-15

    We have previously determined that in African sickle cell anemia (SS) patients three different beta-like globin gene cluster haplotypes are associated with different percent G gamma (one of the two types of non-alpha chains comprising hemoglobin F [HbF]), mean percent HbF, and percent dense cells. We report now that in adult New York SS patients, the presence of at least one chromosome with the Senegal haplotype is associated with higher Hb levels (1.2 g/dL higher) than is found for any other non-Senegal haplotype (P less than .004). The percent reticulocytes and the serum bilirubin levels were lower in these patients. When the effect of alpha-gene number was analyzed by examining a sample of SS patients with concomitant alpha-thalassemia, the same results were obtained. Because the HbF level is significantly higher among the Senegal haplotype carriers in this sample, the inhibitory effect on sickling of this Hb variant may be one of the reasons for the haplotype effect. We conclude that the Senegal beta-like globin gene cluster haplotype is associated with an amelioration of the hemolytic anemia that characterizes sickle cell disease.

  9. Anti-KJ: a new antibody associated with the syndrome of polymyositis and interstitial lung disease.

    PubMed

    Targoff, I N; Arnett, F C; Berman, L; O'Brien, C; Reichlin, M

    1989-07-01

    Antibodies to aminoacyl-tRNA synthetases (anti-Jo-1, anti-PL-7, anti-PL-12) have been found in the serum of some patients with polymyositis (PM). Patients with these antibodies have an unusually high rate of interstitial lung disease (ILD) in association with their PM. Two patients (K.J. and B.T.) with severe ILD and PM were found to have antibodies to a cytoplasmic antigen, but tests to determine whether the antigen was an aminoacyl-tRNA synthetase were negative, including tests of KJ serum for inhibitory effects on the 20 synthetases. KJ immunoprecipitates did not contain tRNA, in contrast to antisynthetase sera. When IgG samples were added to a reticulocyte in vitro translation system at a concentration of 0.3 mg/ml, KJ IgG inhibited globin mRNA translation by 98%, while anti-Jo-1 IgG inhibited 62% and normal IgG had little effect. Thus, both anti-KJ and the antisynthetases are directed at antigens that are involved in translation and protein synthesis, and both are associated with the syndrome of lung disease and PM. This syndrome may be associated with antibodies to translation-related proteins in general, which may have implications for the link of PM and enteroviruses, which are mRNA viruses.

  10. Highly Sensitive Protein Translation Assay for Trichothecene Toxicity in Airborne Particulates: Comparison with Cytotoxicity Assays

    PubMed Central

    Yike, Iwona; Allan, Terry; Sorenson, William G.; Dearborn, Dorr G.

    1999-01-01

    Screening assays for environmental mycotoxins in bulk samples currently use cytotoxicity in cell cultures, but their application to air particulate samples often lacks sensitivity and specificity for fungal spores. An assay based on inhibition of protein synthesis using translation of firefly luciferase in a rabbit reticulocyte system has been developed for the detection of trichothecene mycotoxins found in the spores of toxigenic fungi. Ethanol extracts of air particulates trapped on polycarbonate filters are ultrafiltered and applied at several dilutions to a translation reaction mixture. The activity of translated luciferase is measured directly in a luminometer, eliminating the need for radioisotopes and time-consuming sample processing. Parallel standard curves using a commercially available trichothecene provide for expression of the results in T-2 toxin equivalents per cubic meter of air. The assay can be completed in 2 h and is readily applicable to multiple samples. Comparison to the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cytotoxicity assay indicates a 400-fold increase in sensitivity of trichothecene detection in addition to a much higher specificity for these toxins. Initial field testing indicates a strong correlation between the measured level of toxicity and the presence of toxigenic fungi detected with microbiological methods. In conclusion, this luciferase translation assay offers a rapid and highly sensitive and specific method for quantitative detection of trichothecene mycotoxin activity in air particulate samples. PMID:9872764

  11. Time Course and Variability of Polycythemic Response in Men at High Altitude

    NASA Technical Reports Server (NTRS)

    Grover, R. F.; Seiland, M.; McCullough, R. G.; Greenleaf, J. E.; Dahms, T. E.; Wolfel, E.; Reeves, J. T.

    2000-01-01

    Ten young men were exposed to 4,300 m (PB 460 Torr) for three weeks. Plasma volume (PV, Evans Blue dye). and blood volume (BV, carbon monoxide) measured simultaneously, and red cell volume (RCV) calculated from hematocrit, were determined twice at sea level and after 9-11 and 19-20 days at high altitude. After 19-20 days. half the subjects increased RCV +19.4 +/- 1.8% (p<0.001); the other 5 subjects had no significant change in RCV. All 10 subjects had a sustained decrease in PV (-16.2 +/- 1.9%, p<0.05) at altitude. Consequently, compared with sea level values, BV was unchanged (-3.1 +/- 1.8%) in the group with increased RCV, but BV decreased significantly (-12.2 +/- 1.4%, p<0.05) in the other group. Variability in RCV response was not explained by differences, in hypoxemic stimulus or the erythropoictin and reticulocyte responses. Since RCV reflects the balance between red cell. production and destruction, accelerated red cell destruction may have occurred in those individuals with no net change in RCV.

  12. Subcellular localization of RNAs in transfected cells: role of sequences at the 5' terminus.

    PubMed Central

    Alfonso-Pizarro, A; Carlson, D P; Ross, J

    1984-01-01

    We have investigated the intracellular location of RNAs transcribed from transfected DNA. COS cells transfected with a clone containing the human adult beta globin gene contain three classes of globin RNAs. Their 3' termini and splice sites are indistinguishable from those of mature reticulocyte beta globin mRNA, and they are polyadenylated. However, as determined by S1 mapping, their 5' sequences are different. The 5' terminus of one is the same as that of mature beta globin mRNA (+1, cap site). The presumed 5' terminus of the second is located 30 nucleotides downstream from the cap site (+30). The third class contains additional nucleotides transcribed from sequences located 5' to the cap site (5' upstream RNA). The 5' upstream RNA molecules are restricted to the nucleus and are more stable than heterogeneous nuclear RNA. The +30 and +1 RNAs are located primarily in the cytoplasm. The data support the notion that nucleotide sequences and/or secondary modifications in the 5' region determine if an RNA is to be transported. Images PMID:6209612

  13. Hsp90-Dependent Assembly of the DBC2/RhoBTB2-Cullin3 E3-Ligase Complex

    PubMed Central

    Manjarrez, Jacob R.; Sun, Liang; Prince, Thomas; Matts, Robert L.

    2014-01-01

    The expression of the wild-type tumor-suppressor gene DBC2 (Deleted-in-Breast Cancer 2, a.k.a RhoBTB2) is suppressed in many cancers, in addition to breast cancer. In a screen for Cdc37-associated proteins, DBC2 was identified to be a potential client protein of the 90 kDa heat shock protein (Hsp90) chaperone machine. Pull down assays of ectopically expressed DBC2 confirmed that DBC2 associated with Hsp90 and its co-chaperone components in reticulocyte lysate and MCF7 cells. Similar to other atypical Rho GTPases, DBC2 was found to have retained the capacity to bind GTP. The ability of DBC2 to bind GTP was modulated by the Hsp90 ATPase cycle, as demonstrated through the use of the Hsp90 chemical inhibitors, geldanamycin and molybdate. The binding of full length DBC2 to GTP was suppressed in the presence of geldanamycin, while it was enhanced in the presence of molybdate. Furthermore, assembly of DBC2-Cullin3-COP9 E3 ligase complexes was Hsp90-dependent. The data suggest a new paradigm for Hsp90-modulated assembly of a Cul3/DBC2 E3 ubiquitin ligase complex that may extend to other E3 ligase complexes. PMID:24608665

  14. Hemolysis induced by an extreme mountain ultra-marathon is not associated with a decrease in total red blood cell volume.

    PubMed

    Robach, P; Boisson, R-C; Vincent, L; Lundby, C; Moutereau, S; Gergelé, L; Michel, N; Duthil, E; Féasson, L; Millet, G Y

    2014-02-01

    Prolonged running is known to induce hemolysis. It has been suggested that hemolysis may lead to a significant loss of red blood cells; however, its actual impact on the erythrocyte pool is unknown. Here, we test the hypothesis that prolonged running with high hemolytic potential decreases total red blood cell volume (RCV). Hemolysis (n = 22) and RCV (n = 19) were quantified in ultra-marathon runners before and after a 166-km long mountain ultra-endurance marathon (RUN) with 9500 m of altitude gain/loss. Assessment of total hemoglobin mass (Hbmass) and RCV was performed using a carbon monoxide rebreathing technique. RUN induced a marked acute-phase response and promoted hemolysis, as shown by a decrease in serum haptoglobin (P < 0.05). Elevated serum erythropoietin concentration and reticulocyte count after RUN were indicative of erythropoietic stimulation. Following RUN, runners experienced hemodilution, mediated by a large plasma volume expansion and associated with a large increase in plasma aldosterone. However, neither Hbmass nor RCV were found to be altered after RUN. Our findings indicate that mechanical/physiological stress associated with RUN promotes hemolysis but this has no impact on total erythrocyte volume. We therefore suggest that exercise 'anemia' is entirely due to plasma volume expansion and not to a concomitant decrease in RCV.

  15. Severe hemolytic disease of the premature newborn due to RH1 incompatibility: a case report

    PubMed Central

    UWINGABIYE, JEAN; ZAHID, HAFID; LABRINI, FAYÇAL; EL KHAZRAJI, ABDELHAK; YAHYAOUI, ANASS; HADEF, RACHID; MESSAOUDI, NEZHA

    2016-01-01

    We report a case of dramatic outcome of severe hemolytic disease in a newborn due to RH1 incompatibility. A newborn with A RH1 blood group was admitted in the Mohammed V Military Teaching Hospital for the problem of hydrops fetalis associated with RH1 incompatibility. The blood group of his mother, aged 31, was AB RH1-negative and that of his 37 year old father was A RH1. The mother had a history of 4 term deliveries, 3 abortions, and 1 living child. There was no prevention by anti-D immunoglobulin postpartum. The mother’s irregular agglutinin test was positive and the pregnancy was poorly monitored. The laboratory tests of the newborn showed a high total serum bilirubin level (30 mg/L) and macrocytic regenerative anemia (Hemoglobin=4 g/dL, mean corpuscular volume = 183 fL, reticulocytes count =176600/m3). The blood smear showed 1256 erythroblasts per 100 leukocytes, Howell–Jolly bodies and many macrocytes. The direct antiglobulin test was positive. He was transfused with red blood cell concentrates and treated with conventional phototherapy. The evolution was unfavourable; he died three days after the death of his mother. The monitoring of these high-risk pregnancies requires specialized centers and a close collaboration between the gynaecologist and the blood transfusion specialist to strengthen the prevention, as well as clinico-biological monitoring in patients with a history of RH1 fetomaternal alloimunization. PMID:27857529

  16. Classification of anemia for gastroenterologists

    PubMed Central

    Moreno Chulilla, Jose Antonio; Romero Colás, Maria Soledad; Gutiérrez Martín, Martín

    2009-01-01

    Most anemia is related to the digestive system by dietary deficiency, malabsorption, or chronic bleeding. We review the World Health Organization definition of anemia, its morphological classification (microcytic, macrocytic and normocytic) and pathogenic classification (regenerative and hypo regenerative), and integration of these classifications. Interpretation of laboratory tests is included, from the simplest (blood count, routine biochemistry) to the more specific (iron metabolism, vitamin B12, folic acid, reticulocytes, erythropoietin, bone marrow examination and Schilling test). In the text and various algorithms, we propose a hierarchical and logical way to reach a diagnosis as quickly as possible, by properly managing the medical interview, physical examination, appropriate laboratory tests, bone marrow examination, and other complementary tests. The prevalence is emphasized in all sections so that the gastroenterologist can direct the diagnosis to the most common diseases, although the tables also include rare diseases. Digestive diseases potentially causing anemia have been studied in preference, but other causes of anemia have been included in the text and tables. Primitive hematological diseases that cause anemia are only listed, but are not discussed in depth. The last section is dedicated to simplifying all items discussed above, using practical rules to guide diagnosis and medical care with the greatest economy of resources and time. PMID:19787825

  17. Chromosomal localization and cDNA cloning of the genes (DDB1 and DDB2) for the p127 and p48 subunits of a human damage-specific DNA binding protein

    SciTech Connect

    Dualan, R.; Brody, T.; Keeney, S.

    1995-09-01

    DDB is a damage-specific DNA binding protein whose binding activity is absent from a minority of cell strains from individuals with xeroderma pigmentosum Group E, a human hereditary disease characterized by defective nucleotide excision DNA repair and an increased incidence of skin cancer. The binding activity from HeLa cells is associated with polypeptides of M{sub r} 124,000 and 41,000 as determined by SDS-polyacrylamide gels. This report describes the isolation of full-length human cDNAs encoding each polypeptide of DDB. The predicted peptide molecular masses based on open reading frames are 127,000 and 48,000. When expressed in an in vitro rabbit reticulocyte system, the p48 subunit migrates with an M{sub r} of 41 kDa on SDS-polyacrylamide gels, similarly to the peptide purified from HeLa cells. There is no significant homology between the derived p48 peptide sequence and any proteins in current databases, and the derived peptide sequence of p127 has homology only with the monkey DDB p127 (98% nucleotide identity and only one conserved amino acid substitution). Using a fluorescence in situ hybridization technique, the DDB p127 locus (DDB1) was assigned to the chromosomal location 11q12-q13, and the DDB p48 locus (DDB2) to 11p11-p12. 34 refs., 3 figs., 1 tab.

  18. Custom-Designed Molecular Scissors for Site-Specific Manipulation of the Plant and Mammalian Genomes

    NASA Astrophysics Data System (ADS)

    Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

    Zinc finger nucleases (ZFNs) are custom-designed molecular scissors, engineered to cut at specific DNA sequences. ZFNs combine the zinc finger proteins (ZFPs) with the nonspecific cleavage domain of the FokI restriction enzyme. The DNA-binding specificity of ZFNs can be easily altered experimentally. This easy manipulation of the ZFN recognition specificity enables one to deliver a targeted double-strand break (DSB) to a genome. The targeted DSB stimulates local gene targeting by several orders of magnitude at that specific cut site via homologous recombination (HR). Thus, ZFNs have become an important experimental tool to make site-specific and permanent alterations to genomes of not only plants and mammals but also of many other organisms. Engineering of custom ZFNs involves many steps. The first step is to identify a ZFN site at or near the chosen chromosomal target within the genome to which ZFNs will bind and cut. The second step is to design and/or select various ZFP combinations that will bind to the chosen target site with high specificity and affinity. The DNA coding sequence for the designed ZFPs are then assembled by polymerase chain reaction (PCR) using oligonucleotides. The third step is to fuse the ZFP constructs to the FokI cleavage domain. The ZFNs are then expressed as proteins by using the rabbit reticulocyte in vitro transcription/translation system and the protein products assayed for their DNA cleavage specificity.

  19. Inability to detect free cylindrospermopsin in spiked aquatic organism extracts plausibly suggests protein binding.

    PubMed

    Esterhuizen-Londt, Maranda; Pflugmacher, Stephan

    2016-11-01

    Even though the frequency and prevalence of cylindrospermopsin producing cyanobacteria are increasing, several publications have reported the absence of free cylindrospermopsin bioaccumulation in aquatic food chains. Cylindrospermopsin modification by protein binding has been suggested, however, only one publication has investigated this with eukaryotic reticulocyte lysate and concluded that cylindrospermopsin binds non-covalently to soluble proteins larger than 100 kDa associated with eukaryotic translation. With this as the extent of knowledge regarding cylindrospermopsin binding, the present study aimed to determine whether cylindrospermopsin binding also occurred with other proteins. In the present study, proteins from various organisms were extracted, incubated with cylindrospermopsin, and the amount of free cylindrospermopsin was determined by liquid chromatography tandem mass spectroscopy. Additionally, cylindrospermopsin binding to various ammonium sulfate precipitation fractions of Egeria densa protein, as well as with selected amino acids was investigated. We find that the percentage of free cylindrospermopsin varied with exposure to various animal and plant proteins as well as with various fractions of proteins but found no binding with single amino acids.

  20. Contribution of polyadenylate sequences to the translational efficiency of globin messenger RNAs.

    PubMed Central

    Parets Soler, A; Gozalbo, D; Zueco, J; Sentandreu, R

    1987-01-01

    mRNAs from reticulocyte polysomes were fractionated by chromatography on poly(U)-Sepharose and thermal elution. The molar ratio of alpha- to beta-globin mRNA was found to be 2:1 and 1:1 respectively in short- and long-poly(A) size classes. Translational analyses indicated that the globin mRNAs containing long poly(A) tracts (with a mean length of about 70 nucleotides) directed protein synthesis with higher rates than did mRNA containing short poly(A) tracts (15-35 nucleotides). Experiments performed with sub-saturating mRNA concentrations showed that the digestion with RNAase H induced a decrease in the translational capacity of both globin mRNAs and an increase in the alpha- to beta-globin synthesis ratio. No correlation was observed between the size of the poly(A) tail in mRNA and the optimal K+ requirement for translation. Images Fig. 1. PMID:3689323

  1. Increased Hematocrit During Sodium-Glucose Cotransporter 2 Inhibitor Therapy Indicates Recovery of Tubulointerstitial Function in Diabetic Kidneys

    PubMed Central

    Sano, Motoaki; Takei, Makoto; Shiraishi, Yasuyuki; Suzuki, Yoshihiko

    2016-01-01

    Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been attracting attention for cardiovascular as well as antidiabetic effects since the results of the Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients (EMPA-REG OUTCOME Trial) were reported. The hematocrit increases during treatment with SGLT2 inhibitors, which have a diuretic effect but do not cause sufficient hemoconcentration to increase the risk of cerebral infarction. Elevation of the hematocrit during SGLT2 inhibitor therapy is presumed to involve enhancement of erythropoiesis in addition to hemoconcentration. In patients with diabetes, the erythropoietin level increases after initiation of treatment with the SGLT2 inhibitor dapagliflozin and reaches a plateau in 2 - 4 weeks. The reticulocyte count increases simultaneously, followed by elevation of hemoglobin and hematocrit. In patients with diabetes, the proximal tubules are overtaxed by excessive glucose reabsorption and the increased oxygen requirement causes tubulointerstitial hypoxia. Consequently, erythropoietin production is impaired because “neural crest-derived” fibroblasts surrounding the damaged renal tubules undergo transformation into dysfunctional fibroblasts. SGLT2 inhibitors reduce the workload of the proximal tubules and improve tubulointerstitial hypoxia, allowing fibroblasts to resume normal erythropoietin production. These drugs represent a new class of diuretics that have a renoprotective effect by improving tubulointerstitial hypoxia, which is the final common pathway to end-stage renal disease. In patients with diabetes, elevation of hematocrit may be a surrogate marker for recovery from reversible tubulointerstitial injury. PMID:27829948

  2. Reference limits and behaviour of serum transferrin receptor in children 6-10 years of age.

    PubMed

    Danise, P; Maconi, M; Morelli, G; Di Palma, A; Rescigno, G; Esposito, C; Avino, D; Talento, B

    2008-08-01

    Serum transferrin receptor (sTfR) originates mostly from erythroblasts and lesser from reticulocytes. The usefulness of sTfR has been implicated in several clinical situations, mainly as a marker of accelerated erythropoiesis or iron deficiency. The assessment of sTfR may be useful in the period of rapid growth during infancy, childhood and adolescence. We evaluated sTfR and the other quantitative and qualitative parameters of the erythropoiesis (Hb, MCV, CHr, Ret-He) and of the iron storage (serum ferritin, sTfR/ferritin index) in a total of 916 children aged 6-10 years. Children were divided into three groups: (A) healthy children, (B) with storage iron deficiency (serum ferritin < 12 microg/l) and (C) Beta trait carriers (HbA2 > 3.3). We determined reference intervals by sex and by age in healthy children. sTfR showed a slight but statistically significant age related increase but did not show significant sex differences. We compared sTfR and the other parameters investigated in the three groups of children. sTfR is not a decisive parameter that can be utilized alone in discriminating the border-line situations between normal and pathologic ones but can help in completing the panel of tests in iron deficiency and in thalassaemia Beta trait carriers.

  3. Phosphorylation of the mRNA cap binding protein and eIF-4A by different protein kinases

    SciTech Connect

    Hagedorn, C.H.

    1987-05-01

    These studies were done to determine the identity of a protein kinase that phosphorylates the mRNA cap binding protein (CBP). Two chromatographic steps (dye and ligand and ion exchange HPLC) produced a 500x purification of an enzyme activity in rabbit reticulocytes that phosphorylated CBP at serine residues. Isoelectric focusing analysis of kinase treated CBP demonstrated 5 isoelectric species of which the 2 most anodic species were phosphorylated (contained /sup 32/P). This kinase activity phosphorylated CBP when it was isolated or in the eIF-4F complex. Purified protein kinase C, cAMP or cGMP dependent protein kinase, casein kinase I or II, myosin light chain kinase or insulin receptor kinase did not significantly phosphorylate isolated CBP or CBP in the eIF-4F complex. However, cAMP and cGMP dependent protein kinases and casein kinase II phosphorylated eIF-4A but did not phosphorylate the 46 kDa component of eIF-4F. cAMP dependent protein kinase phosphorylated a approx. 220 kDa protein doublet in eIF-4F preparations. These studies indicate that CBP kinase activity probably represents a previously unidentified protein kinase. In addition, eIF-4A appears to be phosphorylated by several protein kinases whereas the 46 kDa component of the eIF-4F complex was not.

  4. Control of protein synthesis in cell-free extracts of sea urchin embryos

    SciTech Connect

    Hansen, L.J.; Huang, W.I.; Jagus, R.

    1986-05-01

    Although the increase in protein synthesis that occurs after fertilization of sea urchin eggs results from increased utilization of stored maternal mRNA, the underlying mechanism is unknown. The authors have prepared cell-free extracts from S.purpuratus and A.puctulata unfertilized eggs and 2-cell embryos that retain the protein synthetic differences observed in vivo. The method is based on that of Dr. Alina Lopo. /sup 35/S methionine incorporation is linear during a 30 min incubation and is 10-20 fold higher in extracts from 2-cell embryos than unfertilized eggs. Addition of purified mRNA does not stimulate these systems, suggesting a regulatory mechanism other than mRNA masking. Addition of rabbit reticulocyte ribosomal salt wash stimulated protein synthesis in extracts from eggs but not embryos, suggesting deficiencies in translational components in unfertilized eggs. Mixing of egg and embryo lysates indicated the presence of a weak protein synthesis inhibitor in eggs. Translational control in developing sea urchin embryos thus appears to be complex, involving both stimulatory and inhibitory factors.

  5. Isolation and characterization of casein mRNAs from lactating ewe mammary glands.

    PubMed Central

    Gaye, P; Houdebine, L M

    1975-01-01

    RNA from bound polysomers of lactating ewe's mammary gland directs the synthesis of the three major milk proteins (alphas, beta and kappa-caseins) in a cell-free system derived from rabbit reticuyocytes. The "in vitro" product was identified by immunoprecipitation with specific antibodies and by electrophoresis in SDS polyacrylamide gel. Each of these messengers was purified from 20 to 25 fold from total membrane-bound polysomal RNA using poly U-Sepharose chromatography. This purified fraction assayed in a reticulocyte cell-free system is able to direct also the synthesis of 2 minor secretory proteins (beta-lactoglobulin and alpha-lactalbumin). The messenger RNAs purified by hybridization to poly U-Sepharose have a sedimentation coefficient of about 12 S and an apparent molecular weight of approximatively 3.5 s 10-5 daltons was observed by polyacrylamide gel electrophoresis under denaturing contitions. This value which correspond to about 900 nucleotides is significantly greater than the number expected for coding milk proteins. PMID:1144062

  6. Correlation of BACH1 and Hemoglobin E/Beta-Thalassemia Globin Expression

    PubMed Central

    Lee, Tze Yan; Muniandy, Logeswaran; Teh, Lai Kuan; Abdullah, Maha; George, Elizabeth; Sathar, Jameela; Lai, Mei I

    2016-01-01

    Objective: The diverse clinical phenotype of hemoglobin E (HbE)/β-thalassemia has not only confounded clinicians in matters of patient management but has also led scientists to investigate the complex mechanisms involved in maintaining the delicate red cell environment where, even with apparent similarities of α- and β-globin genotypes, the phenotype tells a different story. The BTB and CNC homology 1 (BACH1) protein is known to regulate α- and β-globin gene transcriptions during the terminal differentiation of erythroid cells. With the mutations involved in HbE/β-thalassemia disorder, we studied the role of BACH1 in compensating for the globin chain imbalance, albeit for fine-tuning purposes. Materials and Methods: A total of 47 HbE/β-thalassemia samples were analyzed using real-time quantitative polymerase chain reaction and correlated with age, sex, red blood cell parameters, globin gene expressions, and some clinical data. Results: The BACH1 expression among the β-thalassemia intermedia patients varied by up to 2-log differences and was positively correlated to age; α-, β-, and γ-globin gene expression level; and heme oxygenase 1 protein. BACH1 was also negatively correlated to reticulocyte level and had a significant correlation with splenectomy. Conclusion: This study indicates that the expression of BACH1 could be elevated as a compensatory mechanism to decrease the globin chain imbalance as well as to reduce the oxidative stress found in HbE/β-thalassemia. PMID:26377036

  7. Beta zero thalassemia caused by a base substitution that creates an alternative splice acceptor site in an intron.

    PubMed Central

    Metherall, J E; Collins, F S; Pan, J; Weissman, S M; Forget, B G

    1986-01-01

    A thalassemic beta-globin gene cloned from a haplotype I chromosome contains a T to G transversion at position 116 of IVS1 which results in the generation of an abnormal alternative acceptor splice site. Transient expression studies revealed a 4-fold decrease in the amount of RNA produced with greater than 99% of it being abnormally spliced despite preservation of the normal acceptor splice site at position 130. These results suggest that the mutation at IVS1 position 116 results in beta zero thalassemia. A closely related mutation at position 110 of IVS1 also generates a novel acceptor site and results in a similar decrease in total mRNA produced, but approximately 20% of the mRNA produced is normally spliced and thus the phenotype is that of beta + thalassemia. These observations suggest that short range position effects may play a dramatic role in the choice of potential splice acceptor sites. We demonstrate the presence of abnormally spliced mRNA in reticulocytes of affected individuals and show the mutation at IVS1 position 116 segregating from the mutation at IVS1 position 110 in a three generation pedigree. The mutation results in the creation of a MaeI restriction site, as do a number of other thalassemic mutations, and we demonstrate some difficulties that may arise in the differential diagnosis of these mutations. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3780671

  8. Hematinic effect of fruits of Opuntia elatior Mill. on phenylhydrazine-induced anemia in rats

    PubMed Central

    Chauhan, Sanjay P.; Sheth, Navin R.; Suhagia, Bhanubhai N.

    2015-01-01

    Introduction: The fruits of Opuntia elatior Mill. are known as prickly pear and folkloric use as hematinic, anti-inflammatory and antiasthmatic action. Previously, the fruit juice of prickly pear was evaluated in reversed anemia induced by HgCl2 in a dose dependant manner and present study revealed about its effect in acute hemolytic anemia. Aim: To evaluate the hematinic activity of fruits of Opuntia elatior Mill. Materials and Methods: The hematinic activity of an orally administered fruit juice was studied on phenylhydrazine (PHZ)-induced anemic rats. The hematological parameters such as hemoglobin (Hb) content, red blood cell (RBC), packed cell volume (PCV), and reticulocyte count were analyzed as indices of anemia. Results: PHZ altered the hematological parameters by hemolysis characterized by a decrease in Hb content, total RBC counts and PCV (P < 0.001) on day 3. The Hb content (g%) was significantly increased (P < 0.05) at day 7 in 10 and 15 ml/kg fruit juice treated rats, which was a good improvement compared to the standard. Conclusion: The speedy and progressive recovery of anemic rats responding to treatment of the O. elatior Mill. fruits may be due to increased erythropoiesis and/or antioxidant property of betacyanin. PMID:27011725

  9. A 90-day repeated dose oral (gavage) toxicity study of perfluorohexanoic acid (PFHxA) in rats (with functional observational battery and motor activity determinations).

    PubMed

    Chengelis, Christopher P; Kirkpatrick, Jeannie B; Radovsky, Ann; Shinohara, Motoki

    2009-06-01

    Possible toxic effects of perfluorohexanoic acid (PFHxA) were evaluated when administered orally by gavage to rats at levels up to 200mg/kg/day for 90 days. Lower body weight gains were noted in the 10, 50 and 200mg/kg/day group males (not dose-responsive) throughout dosing. Other changes included lower red blood cell parameters, higher reticulocyte counts and lower globulin in the 200mg/kg/day group males and females, higher liver enzymes in males at 50 and 200mg/kg/day, lower total protein and higher albumin/globulin ratio, and lower cholesterol, calcium in males at 200mg/kg/day. Minimal centrilobular hepatocellular hypertrophy was present in 200mg/kg/day group males and correlated with higher liver weights and slightly higher peroxisome beta oxidation activity at the end of the dosing period. Based on liver histopathology and liver weight changes, the no-observed-adverse-effect level (NOAEL) for oral administration was 50mg/kg/day for males and 200mg/kg/day for females.

  10. Molecular cloning and characterization of the mRNA for cyclin from sea urchin eggs.

    PubMed Central

    Pines, J; Hunt, T

    1987-01-01

    We have isolated a cDNA clone encoding sea urchin cyclin and determined its sequence. It contains a single open reading frame of 409 amino acids which shows homology with clam cyclins. RNA transcribed in vitro from this sequence was efficiently translated in reticulocyte lysates, yielding full-length cyclin. Injection of nanogram amounts of this synthetic mRNA into Xenopus oocytes caused them to mature more rapidly than with progesterone treatment. The sea urchin cyclin underwent two posttranslational modifications in the Xenopus oocytes during maturation. The first occurred at about the time that maturation became cycloheximide-resistant, when a small apparent increase in the molecular weight of cyclin was observed. The second modification involved destruction of the cyclin at about the time of white spot appearance, just as would have occurred at the metaphase/anaphase transition in the natural environment of a cleaving sea urchin embryo. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 7. Fig. 9. PMID:2826125

  11. Transition State Analogues Rescue Ribosomes from Saporin-L1 Ribosome Inactivating Protein†

    PubMed Central

    Sturm, Matthew B.; Tyler, Peter C.; Evans, Gary B.; Schramm, Vern L.

    2009-01-01

    Ribosome-inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves and demonstrate robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (Ki*) of 2.3 to 8.7 nM at physiological conditions and bind up to 40,000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1 linked immunotoxins. PMID:19764816

  12. Novel mechanism of elimination of malfunctioning mitochondria (mitoptosis): formation of mitoptotic bodies and extrusion of mitochondrial material from the cell.

    PubMed

    Lyamzaev, Konstantin G; Nepryakhina, Olga K; Saprunova, Valeria B; Bakeeva, Lora E; Pletjushkina, Olga Yu; Chernyak, Boris V; Skulachev, Vladimir P

    2008-01-01

    Energy catastrophe, when mitochondria hydrolyze glycolytic ATP instead of producing respiratory ATP, has been modeled. In highly glycolyzing HeLa cells, 30-50% of the population survived after inhibition of respiration and uncoupling of oxidative phosphorylation for 2-4 days. The survival was accompanied by selective elimination of mitochondria. This type of mitoptosis includes (i) fission of mitochondrial filaments, (ii) clustering of the resulting roundish mitochondria in the perinuclear area, (iii) occlusion of mitochondrial clusters by a membrane (formation of a "mitoptotic body"), (iv) decomposition of mitochondria inside this body to small membrane vesicles, (v) protrusion of the body from the cell, and (vi) disruption of the body boundary membrane. Autophagy was not involved in this mitoptotic program. Increased production of reactive oxygen species (ROS) was necessary for execution of the program, since antioxidants prevent mitoptosis and kill the cells treated with the mitochondrial poisons as if a ROS-linked mitoptosis serves for protection of the cells under conditions of severe mitochondrial stress. It is suggested that exocytosis of mitoptotic bodies may be involved in maturation of reticulocytes and lens fiber cells.

  13. Ricin and Ricinus communis agglutinin subunits are all derived from a single-size polypeptide precursor.

    PubMed

    Butterworth, A G; Lord, J M

    1983-12-01

    Antibodies have been raised in rabbits against the individually purified A and B subunits of the toxic castor bean lectin, ricin, and against the A' and B' subunits of Ricinus communis agglutinin type I. Each of the antisera recognised a single polypeptide species of Mr 60 500 when maturing castor bean endosperm mRNA was translated in vitro in a rabbit-reticulocyte-derived system. When dog pancreatic microsomal vesicles were included in the translational system, each subunit antiserum precipitated a group of 66 000-68 000-Mr core-glycosylated polypeptides which had been translocated into the lumen of the vesicles. The 60 500-Mr polypeptide appeared to be a common precursor to all four individual lectin subunits since (a) its glycosylated (66 000-68 000-Mr) forms were readily detected in the endoplasmic reticulum fraction isolated from maturing castor bean endosperm and (b) pulse-chase studies showed that the glycosylated precursors disappeared from the endoplasmic reticulum fraction with the concomittant appearance of authentic lectin subunits in a soluble protein fraction which included protein body matrix components. Antiserum prepared against whole R. communis agglutinin, type I, also precipitated the 65 000-Mr precursor in vitro and in vivo, but in addition precipitated a non-glycosylated 34 000-Mr polypeptide. This smaller protein is not a lectin subunit precursor, contradicting an earlier suggestion. It is most probably a precursor to the 2-S albumin storage proteins found in castor bean endosperm protein bodies.

  14. The haem-regulated eukaryotic initiation factor 2alpha kinase: a molecular indicator of lead-toxicity anaemia in rabbits.

    PubMed

    Anand, Sanjay; Pal, Jayanta K

    2002-08-01

    The haem-regulated eukaryotic initiation factor 2alpha kinase, also called the haem-regulated inhibitor (HRI), has been shown to increase in the peripheral blood cells as a function of drug-induced anaemia in rabbits, suggesting that it could be a molecular indicator of drug-induced anaemia [Anand and Pal (1997) J. Biosci. 22, 287-298]. In the present investigation, we have determined the expression of HRI during lead-induced anaemia in rabbits. The level of anaemia has been determined by routine procedures such as reticulocyte count, haemoglobin content and packed cell volume. These values were compared with the results obtained for a quantitative Western blot of HRI in the blood cell lysates of drug- and lead-induced anaemic rabbits. These results indicate that HRI could be used as a molecular marker for lead-induced anaemia since a progressive increase in HRI levels could be detected as a function of the time of lead exposure. In order to understand the role of stress proteins, heat-shock protein (Hsp) 70 and Hsp90, in inducing anaemia during lead exposure, levels of Hsp70 and Hsp90, and their interaction with HRI, have been determined. Increased levels of these proteins and their intermolecular complexes with HRI suggest their role in regulating protein synthesis during lead-induced anaemia. These observations further reiterate the use of HRI as a potential indicator for drug- and heavy-metal-induced anaemia in humans.

  15. Posttranslational incorporation of contractile proteins into myofibrils in a cell-free system

    PubMed Central

    1988-01-01

    The incorporation of newly synthesized protein into myofibrils has been examined in a cell-free system. Myofibrils were added to a reticulocyte lysate after the in vitro translation of muscle-specific poly(A)+RNA. Only a small number of the many synthesized proteins were found to associate with the exogenously added myofibrils. These proteins were all identified as sarcomeric components and had subunit mobilities (Mr) of 200, 140, 95, 86, 43, 38, 35, 25, 23, 20, and 18 kD. The association was rapid (t1/2 less than 15 min) and, for most of the proteins, relatively temperature insensitive. Except for a 43-kD polypeptide, tentatively identified as beta-actin, none of the proteins encoded by brain poly(A)+RNA associated with the myofibrils. When filaments made from purified myosin or actin were used as the "capture" substrates, only thick or thin filament proteins, respectively, were incorporated. Incorporation was substantially reduced when cross-linked myosin filaments were used. These results are compatible with a model in which proteins of the sarcomere are in kinetic equilibrium with homologous proteins in a soluble pool. PMID:3417763

  16. Deciphering the role of exosomes in tuberculosis.

    PubMed

    Kruh-Garcia, Nicole A; Wolfe, Lisa M; Dobos, Karen M

    2015-01-01

    Exosomes were originally described as small vesicles released from reticulocytes during the maturation process. These 40-200 nm microvesicles were hypothesized to be a mechanism for the removal of membrane proteins in lieu of intracellular degradation by Harding et al. (1984) and Johnstone et al. (1987) [1,2]. Exosomes can be distinguished from other extracellular vesicles (ectosomes, apoptotic blebs) based on their size and the protein indicators intercalated in their membrane (also, linking their derivation from the endocytic pathway) by Simpson (2012) [3]. The exact role which exosomes play in cell-to-cell communication and immune modulation is a topic of intense study. However, the focus of most reports has been directed towards discovering aberrations in exosomal protein and RNA content linked to disease onset and progression, and also primarily related to cancer. Nonetheless, exosomes are now documented to be released from a wide variety of cell types by Mathivanan et al. (2012), Simpson et al. (2012) and Kalra et al. (2012) [4-6] and have been isolated from all bodily fluids; thus, exosomes are an excellent source of biomarkers. Here we describe the discoveries related to the role exosomes play in tuberculosis disease, as well as translational work in vaccine development and how circulation of these dynamic vesicles can be harnessed for diagnostic purposes.

  17. Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata), and preparation of its immunotoxin against human melanoma cells.

    PubMed

    Xia, Heng Chuan; Li, Feng; Li, Zhen; Zhang, Zu Chuan

    2003-10-01

    A novel ribosome-inactivating protein designated Moschatin from the mature seeds of pumpkin (Cucurbita moschata) has been successively purified to homogeneity, using ammonium sulfate precipitation, CM-cellulose 52 column chromatography, Blue Sepharose CL-6B Affinity column chromatography and FPLC size-exclusion column chromatography. Moschatin is a type 1 RIP with a pI of 9.4 and molecular weight of approximately 29 kD. It is a rRNA N-glycosidase and potently blocked the protein synthesis in the rabbit reticulocyte lysate with a IC50 of 0.26 nM. Using the anti-human melanoma McAb Ng76, a novel immunotoxin Moschatin-Ng76 was prepared successfully and it efficiently inhibited the growth of targeted melanoma cells M21 with a IC50 of 0.04 nM, 1500 times lower than that of free Moschatin. The results implied that Moschatin could be used as a new potential anticancer agent.

  18. Anti-self phosphatidylserine antibodies recognize uninfected erythrocytes promoting malarial anemia

    PubMed Central

    Fernandez-Arias, Cristina; Rivera-Correa, Juan; Gallego-Delgado, Julio; Rudlaff, Rachel; Fernandez, Clemente; Roussel, Camille; Götz, Anton; Gonzalez, Sandra; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel; Buffet, Pierre; Ndour, Papa Alioune; Rodriguez, Ana

    2016-01-01

    Summary Plasmodium species, the parasitic agents of malaria, invade erythrocytes to reproduce resulting in erythrocyte loss. However, a greater loss is caused by the elimination of uninfected erythrocytes, sometimes long after infection has been cleared. Using a mouse model, we found that Plasmodium infection induces the generation of anti-self antibodies that bind to the surface of uninfected erythrocytes from infected, but not uninfected, mice. These antibodies recognize phosphatidylserine, which is exposed on the surface of a fraction of uninfected erythrocytes during malaria. We find that phosphatidylserine-exposing erythrocytes are reticulocytes expressing high levels of CD47, a ‘do-not-eat-me’ signal, but the binding of anti-phosphatidylserine antibodies mediates their phagocytosis, contributing to anemia. In human patients with late post-malarial anemia, we found a strong inverse correlation between the levels of anti-phosphatidylserine antibodies and plasma hemoglobin, suggesting a similar role in humans. Inhibition of this pathway may be exploited for treating malarial anemia. PMID:26867178

  19. Antigenotoxicity of artepillin C in vivo evaluated by the micronucleus and comet assays.

    PubMed

    de Azevedo Bentes Monteiro Neto, Moacir; de Souza Lima, Ildercílio Mota; Furtado, Ricardo Andrade; Bastos, Jairo Kenupp; da Silva Filho, Ademar Alves; Tavares, Denise Crispim

    2011-11-01

    Artepillin C (3,5-diprenyl-p-coumaric acid), a major compound found in Brazilian green propolis and Baccharis dracunculifolia, shows anti-inflammatory, antibacterial, antiviral, antioxidant and antitumoral activities, among others. The aim of this study was to evaluate the genotoxic potential of artepillin C and its ability to prevent the chemically induced chromosome breakage or loss and the primary DNA damage using the micronucleus and comet assays in male Swiss mice, respectively. The animals were treated by gavage with different doses of artepillin C (0.4, 0.8 and 1.6 mg kg(-1) b.w.). For the antigenotoxicity assays, the different doses of artepillin C were administered simultaneously to doxorubicin (DXR; micronucleus test; 15 mg kg(-1) b.w.) and to methyl methanesulfonate (MMS; comet assay; 40 mg kg(-1) b.w.). The results showed that artepillin C itself was not genotoxic in the mouse micronucleus and comet assays. In the animals treated with artepillin C and DXR, the number of micronucleated reticulocytes was significantly lower in comparison with the animals treated only with DXR. Regarding antigenotoxicity, artepillin C at the tested doses significantly reduced the extent of DNA damage in liver cells induced by MMS.

  20. Advances in laboratory diagnosis of hereditary spherocytosis.

    PubMed

    Farias, Mariela Granero

    2016-11-12

    Among the red cell membrane disorders, hereditary spherocytosis (HS) is one of the most common causes of inherited hemolytic anemia. HS results from the deficiency or dysfunction of red blood cell membrane proteins, such as α spectrin, β spectrin, ankyrin, anion channel protein (Band-3 protein), protein 4.1 and protein 4.2. Conventionally, HS diagnosis is established through a series of tests, which include spherocytes identification in peripheral smear, reticulocyte count, osmotic fragility, etc. Currently, different hematological analyzers provide erythrocyte indicators that estimate the presence of spherocytes and correlate that with HS, which can be useful for disease screening. The most traditional method is the osmotic fragility (OF) test, which is labor-intensive and time-consuming to perform and presents low sensitivity and specificity values. Thus, new methods have been developed for HS diagnosis, such as flow cytometry. Current guidelines recommend the use of flow cytometry as a screening test for HS diagnosis using the eosin-5'-maleimide (EMA) binding test. Thus, HS diagnosis is the result of a collaboration between clinicians and laboratories, who should take into account the family history and the exclusion of other causes of secondary spherocytosis.

  1. Swelling, NEM, and A23187 activate Cl(-)-dependent K+ transport in high-K+ sheep red cells

    SciTech Connect

    Fujise, H.; Lauf, P.K.

    1987-02-01

    In low K+ (LK) sheep red cells a significant fraction of the total ouabain-resistant (OR) K+ flux is inhibited when Cl- is replaced by other anions of the Hofmeister series except Br- (Cl(-)-dependent K+ flux). In contrast, high K+ (HK) sheep red cells in isosmotic media did not possess any significant OR Cl(-)-dependent K+ flux when Cl- was replaced by NO/sub 3/- or I-. However, exposure to hyposmotic solutions, treatment with the sulfhydryl (SH) group reagent N-ethylmaleimide (NEM) or with the bivalent metal ion (Me2+) ionophore A23187 in absence of external Me2+ caused a significant activation of Cl(-)-dependent K+ transport as measured with Rb+ as K+ congener. There was no Cl(-)-dependent Rb+ flux in A23187-treated cells when Mn2+, Mg2+, and Ca2+ were present at 1 mM concentrations, suggesting that cellular accumulation of these Me2+ is inhibitory. Similar to LK red cells, HK red cells failed to respond to A23187 when pretreated with NEM supporting the hypothesis proposed recently of a common mechanism of Cl(-)-dependent K+ transport activation. The magnitudes of the Cl(-)-dependent Rb+ fluxes in HK cells were much smaller than those elicited by identical treatments in LK red cells, and the effect of all interventions was not due to the presence of reticulocytes known to possess Cl(-)-dependent K+ transport.

  2. Experimental evidence for evolved tolerance to avian malaria in a wild population of low elevation Hawai'i 'Amakihi (Hemignathus virens).

    PubMed

    Atkinson, Carter T; Saili, Katerine S; Utzurrum, Ruth B; Jarvi, Susan I

    2013-12-01

    Introduced vector-borne diseases, particularly avian malaria (Plasmodium relictum) and avian pox virus (Avipoxvirus spp.), continue to play significant roles in the decline and extinction of native forest birds in the Hawaiian Islands. Hawaiian honeycreepers are particularly susceptible to avian malaria and have survived into this century largely because of persistence of high elevation refugia on Kaua'i, Maui, and Hawai'i Islands, where transmission is limited by cool temperatures. The long term stability of these refugia is increasingly threatened by warming trends associated with global climate change. Since cost effective and practical methods of vector control in many of these remote, rugged areas are lacking, adaptation through processes of natural selection may be the best long-term hope for recovery of many of these species. We document emergence of tolerance rather than resistance to avian malaria in a recent, rapidly expanding low elevation population of Hawai'i 'Amakihi (Hemignathus virens) on the island of Hawai'i. Experimentally infected low elevation birds had lower mortality, lower reticulocyte counts during recovery from acute infection, lower weight loss, and no declines in food consumption relative to experimentally infected high elevation Hawai'i 'Amakihi in spite of similar intensities of infection. Emergence of this population provides an exceptional opportunity for determining physiological mechanisms and genetic markers associated with malaria tolerance that can be used to evaluate whether other, more threatened species have the capacity to adapt to this disease.

  3. The impact of HFE mutations on haemoglobin and iron status in individuals experiencing repeated iron loss through blood donation*.

    PubMed

    Mast, Alan E; Lee, Tzong-Hae; Schlumpf, Karen S; Wright, David J; Johnson, Bryce; Carrick, Danielle M; Cable, Ritchard G; Kiss, Joseph E; Glynn, Simone A; Steele, Whitney R; Murphy, Edward L; Sacher, Ronald; Busch, Michael P

    2012-02-01

    Frequent blood donors become iron deficient. HFE mutations are present in over 30% of donors. A 24-month study of 888 first time/reactivated donors and 1537 frequent donors measured haemoglobin and iron status to assess how HFE mutations impact the development of iron deficiency erythropoiesis. Donors with two HFE mutations had increased baseline haemoglobin and iron stores as did those with one mutation, albeit to a lesser extent. Over multiple donations haemoglobin and iron status of donors with HFE mutations paralleled those lacking mutations. The prevalence of HFE mutations was not increased in higher intensity donors. Thus, in general, HFE mutations do not temper donation-induced changes in haemoglobin and iron status. However, in Black donors there was an increase of H63D carriers at baseline, from 3·7% in first time/reactivated donors to 15·8% in frequent donors, suggesting that the relative effects of HFE mutations on iron absorption may vary between racial/ethnic groups. In secondary analyses, venous haemoglobin decreased more slowly in donors with ferritin ≥12μg/l; and haemoglobin recovery time was shorter in donors with reticulocyte haemoglobin (CHr) ≥32·6pg, indicating that these biochemical measures are better indicators of a donor's response to phlebotomy than their HFE mutation status.

  4. A comprehensive panel of turn-on caspase biosensors for investigating caspase specificity and caspase activation pathways.

    PubMed

    Shekhawat, Sujan S; Campbell, Sean T; Ghosh, Indraneel

    2011-10-17

    Caspases play a central role in apoptosis, differentiation, and proliferation, and represent important therapeutic targets for treating cancer and inflammatory disorders. Toward the goal of developing new tools to probe caspase substrate cleavage specificity as well as to systematically interrogate caspase activation pathways, we have constructed and investigated a comprehensive panel of caspase biosensors with a split-luciferase enabled bioluminescent read out. We first interrogated the panel of caspase biosensors for substrate cleavage specificity of caspase 1-10 in widely utilized in vitro translation systems, namely, rabbit reticulocyte lysate (RRL) and wheat germ extract (WGE). Commercial RRL was found to be unsuitable for investigating caspase specificity, owing to surprising levels of endogenous caspase activity, while specificity profiles of the caspase sensors in WGE agree very well with traditional peptide probes. The full panel of biosensors was utilized for studying caspase activation and inhibition in several mammalian cytosolic extracts, clearly demonstrating that they can be utilized to directly monitor activation or inhibition of procaspase 3/7. Furthermore, the complete panel of caspase biosensors also provided new insights into caspase activation pathways wherein we surprisingly discovered the activation of procaspase 3/7 by caspase 4/5.

  5. Advances in genome-wide protein expression using the wheat germ cell-free system.

    PubMed

    Endo, Yaeta; Sawasaki, Tatsuya

    2005-01-01

    In the current post-genomic era, cell-free translation platforms are gaining importance in structural as well as functional genomics. They are based on extracts prepared from Escherichia coli cells, wheat germ, or rabbit reticulocytes, and when programmed with any mRNA in the presence of energy sources and amino acids, can synthesize the respective protein in vitro. Among the cell-free systems, the wheat germ-based translation system is of special interest due to its eukaryotic nature and robustness. This chapter outlines the existing protein production platforms and their limitations, and describes the basic concept of the wheat germ-based cell-free system. It also demonstrates how the conventional wheat germ system can be improved by eliminating endogenous inhibitors, by using an expression vector specially designed for this system and polymerase chain reaction-directed protein synthesis directly from cDNAs in a bi-layer translation system. Finally, a robotic procedure for translation based on the wheat germ extract and bi-layer cell-free translation is described.

  6. The Outcomes of Hypertransfusion in Major ABO Incompatible Allogeneic Stem Cell Transplantation

    PubMed Central

    Park, Se Hoon; Lee, Se Hoon; Lee, Kyung-Eun; Park, Jinny; Park, Joon Oh; Kim, Kihyun; Kim, Won Seog; Jung, Chul Won; Im, Young-Hyuk; Kang, Won Ki; Park, Keunchil; Kim, Seon Woo; Lee, Kyoo Hyung; Lee, Je Hwan

    2004-01-01

    Major ABO incompatibility may be potentially associated with immediate or delayed hemolysis and delayed onset of erythropoiesis in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). To determine if hemolysis can be prevented by the inhibition of graft erythropoiesis, we performed hypertransfusion and assessed red cell transfusion requirement and independence. Between October 1995 and December 2001, 28 consecutive patients receiving major ABO incompatible HSCT at Samsung Medical Center were hypertransfused to maintain their hemoglobin levels at 15 g/dL or more. We retrospectively compared the outcomes of these patients with those of 47 patients at Asan Medical Center whose target hemoglobin levels were 10 g/dL. Reticulocyte engraftment was significantly delayed in hypertransfused group (51 days vs. 23 days; p=.001). There was no significant difference in the total amount of red cells transfused within 90 days post-HSCT (25 units vs. 26 units; p=.631). No significant difference in the time to red cell transfusion independence was observed between the two groups (63 days vs. 56 days; p=.165). In conclusion, we failed to improve red cell transfusion requirement and independence in major ABO incompatible HSCT with hypertransfusion. PMID:14966346

  7. Venous incompetence, poverty and lactate dehydrogenase in Jamaica are important predictors of leg ulceration in sickle cell anaemia.

    PubMed

    Cumming, V; King, L; Fraser, R; Serjeant, G; Reid, M

    2008-07-01

    Clinical features and potential risk factors for chronic leg ulceration (duration >6 months) in homozygous sickle cell (SS) disease were examined in 225 subjects in the Jamaican Cohort Study. Potential risk factors included the number of HBA genes, steady state haematology, serum lactate dehydrogenase (LDH), venous incompetence, and socio-economic status. Chronic ulcers occurred in 53 subjects with the highest risk of ulcer development at 18 years. The prevalence was 29.5% and cumulative incidence 16.7%. Gender or alpha-thalassaemia trait did not affect the incidence of leg ulcer. Ulceration was associated with lower haemoglobin, red cell count, fetal haemoglobin, and socio-economic status and higher reticulocyte count, platelet count, serum LDH and venous incompetence in univariate analyses. Venous incompetence [Hazard Ratio (HR) 3.0-4.0] and socio-economic status (HR 0.8) were most consistently associated with leg ulceration on multivariate analysis. Regression models incorporating serum LDH suggested this to be a stronger predictor than haematological indices. The prevalence of ulcers at 30% is less than previous estimates in Jamaica, probably reflecting the lack of ascertainment bias in the Cohort Study, and also a real secular decline. In Jamaica, venous incompetence, low socio-economic status, and high serum LDH were the strongest predictors of chronic ulceration.

  8. Identification of mitochondrial proteins and some of their precursors in two-dimensional electrophoretic maps of human cells

    SciTech Connect

    Anderson, L.

    1981-04-01

    A set of at least 30 proteins disappears from the two-dimensional electrophoretic pattern of human lymphoid cells treated with various antimitochondrial agents. This set is similar to the set of proteins found in isolated mitochondria (except for the presence of actin in the latter group), indicating that the inhibitor effect stops production of a majority of mature mitochondrial proteins. Several proteins having the characteristics of precursors to the major cytoplasmically synthesized mitochondrial proteins can be observed in cells during fast-pulse experiments and in a reticulocyte lysate system fed with total lymphoid cell RNA. In the three major instances of mitochondrial precursor-product processing, the removal peptide is quite basic in each case, suggesting that a lysine- or arginine-rich terminal sequence may be necessary for initial recognition by the mitochondrial protein uptake apparatus. The inhibitor effect allows easy identification of a large set of mitochondrial proteins in two-dimensional maps of various cells, thereby specifying a particularly tractable and functionally distinctive subset of the cellular proteins. The nature and wide scope of the effect support the concept of energy-dependent vectorial processing and indicate that such a mechanism is generally applicable to the major class of cytoplasmically synthesized mitochondrial proteins in mammalian cells.

  9. X-linked macrocytic dyserythropoietic anemia in females with an ALAS2 mutation

    PubMed Central

    Sankaran, Vijay G.; Ulirsch, Jacob C.; Tchaikovskii, Vassili; Ludwig, Leif S.; Wakabayashi, Aoi; Kadirvel, Senkottuvelan; Lindsley, R. Coleman; Bejar, Rafael; Shi, Jiahai; Lovitch, Scott B.; Bishop, David F.; Steensma, David P.

    2015-01-01

    Macrocytic anemia with abnormal erythropoiesis is a common feature of megaloblastic anemias, congenital dyserythropoietic anemias, and myelodysplastic syndromes. Here, we characterized a family with multiple female individuals who have macrocytic anemia. The proband was noted to have dyserythropoiesis and iron overload. After an extensive diagnostic evaluation that did not provide insight into the cause of the disease, whole-exome sequencing of multiple family members revealed the presence of a mutation in the X chromosomal gene ALAS2, which encodes 5′-aminolevulinate synthase 2, in the affected females. We determined that this mutation (Y365C) impairs binding of the essential cofactor pyridoxal 5′-phosphate to ALAS2, resulting in destabilization of the enzyme and consequent loss of function. X inactivation was not highly skewed in wbc from the affected individuals. In contrast, and consistent with the severity of the ALAS2 mutation, there was a complete skewing toward expression of the WT allele in mRNA from reticulocytes that could be recapitulated in primary erythroid cultures. Together, the results of the X inactivation and mRNA studies illustrate how this X-linked dominant mutation in ALAS2 can perturb normal erythropoiesis through cell-nonautonomous effects. Moreover, our findings highlight the value of whole-exome sequencing in diagnostically challenging cases for the identification of disease etiology and extension of the known phenotypic spectrum of disease. PMID:25705881

  10. [Hereditary sideroblastic anemia: a rare diagnosis].

    PubMed

    Brahem-Jmili, N; Salem, N; Abdelkefi, S; Champ, B Grand; Bekri, S; Sboui, H; Mahjoub, T; Yacoub, S; Kortas, M

    2004-01-01

    Hereditary sideroblastic anemia is a very rare disease recessive and X-linked that affect heme biosynthesis by deficit or decreased of delta aminolevulinic acid synthase (ALAS) activity. We report a case of a six-month-old boy, admitted in the hospital for anemic syndrome. The hemogram showed anemia (hemoglobin: 4.5 g/dL), frankly hypochronic microcytic and a regenerated (mean corpuscular hemoglobin concentration: 26 g/dL, mean cell volume: 53 fl, reticulocytes: 10 x 10(9)/L) with red cells morphologic disorders in smears (anisopoikylocytosis) without attack of the other lineages; white blood cells: 11 x 10(9)/L (neutrophils: 64% and lymphocytes: 35%); platelets: 350 x 10(9)/L. Examination of bone marrow showed an important erythroid hyperplasia (about 69%) with dyserythropoiesis. Perls stain revealed intense siderosis with 90% of ringed sideroblasts and a large number of siderocytes. Exploration of ALAS2 and ABC7 genes on the DNA of the infant was not found abnormalities. Treatment with pyridoxine corrects moderately the anemia. By the way, we proposed to remind that iron deficiency, inflammatory syndrome and thalassemia are the common microcytic anemia. However, it's mandatory to explore other causes if diagnosis is not solved.

  11. cDNA isolated from a human T-cell library encodes a member of the protein-tyrosine-phosphatase family

    SciTech Connect

    Cool, D.E.; Tonks, N.K.; Charbonneau, H.; Walsh, K.A.; Fischer, E.H.; Krebs, E.G. )

    1989-07-01

    A human peripheral T-cell cDNA library was screened with two labeled synthetic oligonucleotides encoding regions of a human placenta protein-tyrosine-phosphatase. One positive clone was isolated and the nucleotide sequence was determined. It contained 1,305 base pairs of open reading frame followed by a TAA stop codon and 978 base pairs of 3{prime} untranslated end, although a poly(A){sup +} tail was not found. An initiator methionine residue was predicted at position 61, which would result in a protein of 415 amino acid residues. This was supported by the synthesis of a M{sub r} 48,000 protein in an in vitro reticulocyte lysate translation system using RNA transcribed from the cloned cDNA and T7 RNA polymerase. The deduced amino acid sequence was compared to other known proteins revealing 65% identity to the low M{sub r} PTPase 1B isolated from placenta. In view of the high degree of similarity, the T-cell cDNA likely encodes a newly discovered protein-tyrosine-phosphatase, thus expanding this family of genes.

  12. Association of adenylyl cyclase 6 rs3730070 polymorphism and hemolytic level in patients with sickle cell anemia.

    PubMed

    Cita, Kizzy-Clara; Ferdinand, Séverine; Connes, Philippe; Brudey, Laura; Tressières, Benoit; Etienne-Julan, Maryse; Lemonne, Nathalie; Tarer, Vanessa; Elion, Jacques; Romana, Marc

    2016-05-01

    A recent study suggested that adenosine signaling pathway could promote hemolysis in patients with sickle cell anemia (SCA). This signaling pathway involves several gene coding enzymes for which variants have been described. In this study, we analyzed the genotype-phenotype relationships between functional polymorphisms or polymorphisms associated with altered expression of adenosine pathway genes, namely adenosine deaminase (ada; rs73598374), adenosine A2b receptor (adora2b; rs7208480), adenylyl cyclase6 (adcy6; rs3730071, rs3730070, rs7300155), and hemolytic rate in SCA patients. One hundred and fifty SCA patients were genotyped for adcy6, ada, and adora2b variants as well as alpha-globin gene, a genetic factor known to modulate hemolytic rate. Hematological and biochemical data were obtained at steady-state. Lactate dehydrogenase, aspartate aminotransferase, reticulocytes and total bilirubin were used to calculate a hemolytic index. Genotype-phenotype relationships were investigated using parametric tests and multivariate analysis. SCA patients carrying at least one allele of adcy6 rs3730070-G exhibited lower hemolytic rate than non-carriers in univariate analysis (p=0.006). The presence of adcy6 rs3730070-G variant was associated with a decreased hemolytic rate in adjusted model for age and alpha-thalassemia (p=0.032). Our results support a protective effect of adcy6 rs3730070-G variant on hemolysis in SCA patients.

  13. Genotoxicity induced by Taenia solium and its reduction by immunization with calreticulin in a hamster model of taeniosis.

    PubMed

    Salazar, Ana María; Mendlovic, Fela; Cruz-Rivera, Mayra; Chávez-Talavera, Oscar; Sordo, Monserrat; Avila, Guillermina; Flisser, Ana; Ostrosky-Wegman, Patricia

    2013-06-01

    Genotoxicity induced by neurocysticercosis has been demonstrated in vitro and in vivo in humans. The adult stage of Taenia solium lodges in the small intestine and is the main risk factor to acquire neurocysticercosis, nevertheless its carcinogenic potential has not been evaluated. In this study, we determined the genotoxic effect of T. solium infection in the hamster model of taeniosis. In addition, we assessed the effect of oral immunization with recombinant T. solium calreticulin (rTsCRT) plus cholera toxin as adjuvant on micronuclei induction, as this protein has been shown to induce 33-44% protection in the hamster model of taeniosis. Blood samples were collected from the orbital venous plexus of noninfected and infected hamsters at different days postinfection, as well as from orally immunized animals, to evaluate the frequency of micronucleated reticulocytes as a measure of genotoxicity induced by parasite exposure and rTsCRT vaccination. Our results indicate that infection with T. solium caused time-dependent DNA damage in vivo and that rTsCRT immunization reduced the genotoxic damage induced by the presence of the tapeworms.

  14. Many disease-associated variants of hTERT retain high telomerase enzymatic activity

    PubMed Central

    Zaug, Arthur J.; Crary, Sharon M.; Jesse Fioravanti, Matthew; Campbell, Kristina; Cech, Thomas R.

    2013-01-01

    Mutations in the gene for telomerase reverse transcriptase (hTERT) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays. PMID:23901009

  15. Experimental evidence for evolved tolerance to avian malaria in a wild population of low elevation Hawai`i `Amakihi (Hemignathus virens)

    USGS Publications Warehouse

    Atkinson, Carter T.; Saili, Katerine S.; Utzurrum, Ruth B.; Jarvi, Susan I.

    2013-01-01

    Introduced vector-borne diseases, particularly avian malaria (Plasmodium relictum) and avian pox virus (Avipoxvirus spp.), continue to play significant roles in the decline and extinction of native forest birds in the Hawaiian Islands. Hawaiian honeycreepers are particularly susceptible to avian malaria and have survived into this century largely because of persistence of high elevation refugia on Kaua‘i, Maui, and Hawai‘i Islands, where transmission is limited by cool temperatures. The long term stability of these refugia is increasingly threatened by warming trends associated with global climate change. Since cost effective and practical methods of vector control in many of these remote, rugged areas are lacking, adaptation through processes of natural selection may be the best long-term hope for recovery of many of these species. We document emergence of tolerance rather than resistance to avian malaria in a recent, rapidly expanding low elevation population of Hawai‘i ‘Amakihi (Hemignathus virens) on the island of Hawai‘i. Experimentally infected low elevation birds had lower mortality, lower reticulocyte counts during recovery from acute infection, lower weight loss, and no declines in food consumption relative to experimentally infected high elevation Hawai‘i ‘Amakihi in spite of similar intensities of infection. Emergence of this population provides an exceptional opportunity for determining physiological mechanisms and genetic markers associated with malaria tolerance that can be used to evaluate whether other, more threatened species have the capacity to adapt to this disease.

  16. Human Cord Blood and Bone Marrow CD34+ Cells Generate Macrophages That Support Erythroid Islands

    PubMed Central

    Belay, Eyayu; Hayes, Brian J.; Blau, C. Anthony; Torok-Storb, Beverly

    2017-01-01

    Recently, we developed a small molecule responsive hyperactive Mpl-based Cell Growth Switch (CGS) that drives erythropoiesis associated with macrophages in the absence of exogenous cytokines. Here, we compare the physical, cellular and molecular interaction between the macrophages and erythroid cells in CGS expanded CD34+ cells harvested from cord blood, marrow or G-CSF-mobilized peripheral blood. Results indicated that macrophage based erythroid islands could be generated from cord blood and marrow CD34+ cells but not from G-CSF-mobilized CD34+ cells. Additional studies suggest that the deficiency resides with the G-CSF-mobilized CD34+ derived monocytes. Gene expression and proteomics studies of the in vitro generated erythroid islands detected the expression of erythroblast macrophage protein (EMP), intercellular adhesion molecule 4 (ICAM-4), CD163 and DNASE2. 78% of the erythroblasts in contact with macrophages reached the pre reticulocyte orthochromatic stage of differentiation within 14 days of culture. The addition of conditioned medium from cultures of CD146+ marrow fibroblasts resulted in a 700-fold increase in total cell number and a 90-fold increase in erythroid cell number. This novel CD34+ cell derived erythroid island may serve as a platform to explore the molecular basis of red cell maturation and production under normal, stress and pathological conditions. PMID:28135323

  17. Mechanisms and etiologies of thrombocytopenia in the intensive care unit: impact of extensive investigations

    PubMed Central

    2014-01-01

    Background Thrombocytopenia is common in the intensive care unit. Potential mechanisms and etiologies behind this phenomenon are multiple and often entangled. We assessed the effect of a systematic approach, using routinely available tests, on the proportion of patients in whom the mechanism (primary objective) and etiology (secondary objective) of thrombocytopenia in a mixed intensive care unit (ICU) could be identified. Methods Before-and-after study of all patients with thrombocytopenia was used. ‘Before’ group had no intervention. New standard operating procedures for thrombocytopenia management were introduced. In the ‘After’ group, bone marrow aspiration; determination of fibrinogen dosage, prothrombin time, factor V, D-dimers; assay of fibrin monomers, ferritin, triglycerides, lactic acid dehydrogenase, aspartate transaminase, alanine aminotransferase, vitamin B12, folates, reticulocytes, haptoglobin, and bilirubin were performed. Results In the Before group (n = 20), the mechanism (central, peripheral, or mixed) was identified in 10 % versus 83% in After group (n = 23) (p < 0.001) (48% peripheral, 35% mixed). Before intervention, ≥1 etiology was identified in 15% versus 95.7% in the After group (p < 0.001). Conclusions Systematic and extensive investigation using routine tests highlights the mechanisms and etiology of thrombocytopenia in most cases. PMID:25593741

  18. The value of the Thomas-plot in the diagnostic work up of anemic patients referred by general practitioners.

    PubMed

    Leers, M P G; Keuren, J F W; Oosterhuis, W P

    2010-12-01

    In patients with inflammatory conditions, diagnosing classic iron deficiency or anemia of chronic disease is challenging. In this study, we assessed the diagnostic value of the so-called Thomas'-plot [soluble transferrin receptor (sTfR)/log ferritin (sTfr/log Ferr) and the reticulocyte hemoglobin equivalent (Ret-HE)] in the anemia work up of patients referred by general practitioners. During July 2008-March 2009, 337 consecutive patients were included because of lowered Hb values. The laboratory results of the first 133 consecutive patients were used to determine the cut-off values for the diagnostic plot. The laboratory results of these patients were assessed and interpreted independently by two investigators, blinded from sTfR/log Ferr and Ret-HE values. The following 204 patients were used to test the plot in practice. In 32% of the first 133 patients, no indication of the cause of anemia could be found. However, when using the diagnostic plot in the following 204 patients, this fraction decreased to 14%. The 'Thomas'-plot is of diagnostic value for distinguishing functional iron deficiency from classic iron deficiency in a patient population referred by general practitioners.

  19. Clinical Predictors of Severe Malarial Anaemia in a Holoendemic Plasmodium falciparum Transmission Area

    PubMed Central

    Novelli, Enrico M.; Hittner, James B.; Davenport, Gregory C.; Ouma, Collins; Were, Tom; Obaro, Stephen; Kaplan, Sandra; Ong’echa, John M.; Perkins, Douglas J.

    2011-01-01

    SUMMARY Severe malarial anaemia (SMA) is a common complication of Plasmodium falciparum infections, resulting in mortality rates that may exceed 30% in paediatric populations residing in holoendemic transmission areas. One strategy for reducing the morbidity and mortality associated with SMA is to identify clinical predictors that can be readily recognized by caregivers for prompt therapeutic interventions. To determine clinical predictors of SMA, Kenyan children (3-36 mos., n=671) presenting with acute illness at a rural hospital in Siaya District were recruited. Demographic, clinical, laboratory and haematological parameters were measured upon enrolment. Since HIV-1 and bacteraemia promote reduced haemoglobin (Hb) concentrations, children with these infections were excluded from the analyses. Children with P. falciparum mono-infections (n=355) were stratified into three groups: uncomplicated malaria (Hb≥11.0 g/dL); non-SMA (6.0≤Hb<10.9), and SMA (Hb<6.0 g/dL). SMA was characterized by a younger age, monocytosis, thrombocytopaenia, reticulocytosis, reduced erythropoiesis, elevated pigment-containing monocytes (PCM), respiratory distress, conjunctival and palmar pallor, splenomegaly, signs of malnutrition, and protracted fever and emesis. Logistic regression analysis demonstrated that age, reticulocyte count, presence of PCM and conjunctival and palmar pallor were significant predictors of SMA. Recognition of these clinical signs in children residing in resource-poor settings may help guide the identification and management of SMA. PMID:20408849

  20. Structure-activity relationships among the kanamycin aminoglycosides: role of ring I hydroxyl and amino groups.

    PubMed

    Salian, Sumantha; Matt, Tanja; Akbergenov, Rashid; Harish, Shinde; Meyer, Martin; Duscha, Stefan; Shcherbakov, Dmitri; Bernet, Bruno B; Vasella, Andrea; Westhof, Eric; Böttger, Erik C

    2012-12-01

    The kanamycins form an important subgroup of the 4,6-disubstituted 2-deoxystreptamine aminoglycoside antibiotics, comprising kanamycin A, kanamycin B, tobramycin, and dibekacin. These compounds interfere with protein synthesis by targeting the ribosomal decoding A site, and they differ in the numbers and locations of amino and hydroxy groups of the glucopyranosyl moiety (ring I). We synthesized kanamycin analogues characterized by subtle variations of the 2' and 6' substituents of ring I. The functional activities of the kanamycins and the synthesized analogues were investigated (i) in cell-free translation assays on wild-type and mutant bacterial ribosomes to study drug-target interaction, (ii) in MIC assays to assess antibacterial activity, and (iii) in rabbit reticulocyte translation assays to determine activity on eukaryotic ribosomes. Position 2' forms an intramolecular H bond with O5 of ring II, helping the relative orientations of the two rings with respect to each other. This bond becomes critical for drug activity when a 6'-OH substituent is present.

  1. Characterization of antigens from Schistosoma mansoni and construction of a cDNA library for the study of schistosomiasis

    SciTech Connect

    Bugra, K.

    1986-01-01

    To examine the antigens of adult Schistosoma mansoni, /sup 35/S-methionine-labelled, detergent-extracted proteins were immunoprecipitated and analyzed on SDS-PAGE. Human infection serum immunoprecipitated 14 polypeptides with M/sub r/'s of 120, 105, 88, 86, 66, 64, 54, 48, 42, 38, 35, 32, 29, and 20 Kd. Upon digestion with endoglycosidase F polypeptides with M/sub r/'s of 120, 105, 54, 48, and 29 appeared to have carbohydrate moieties. Extracts of female and male S. mansoni were analyzed by immunoprecipitation and immunoblotting. Two polypeptides with M/sub r/'s of 86 Kd and 54 Kd were detected only in extracts of males. Polyadenylated RNA was extracted from S. mansoni and translated in rabbit reticulocyte lysates. Among the in vitro translation products, polypeptides with 120, 94, 64, 43, 37, 35, 30, 26 and 22 Kd apparent molecular weights were immunoprecipitated by human infection serum. When the translation products of female worms and male worms were compared, the polypeptides with M/sub r/'s of 94 and 64 Kd were only observed in males.

  2. Intestine-specific Disruption of Hypoxia-inducible Factor (HIF)-2α Improves Anemia in Sickle Cell Disease.

    PubMed

    Das, Nupur; Xie, Liwei; Ramakrishnan, Sadeesh K; Campbell, Andrew; Rivella, Stefano; Shah, Yatrik M

    2015-09-25

    Sickle cell disease (SCD) is caused by genetic defects in the β-globin chain. SCD is a frequently inherited blood disorder, and sickle cell anemia is a common type of hemoglobinopathy. During anemia, the hypoxic response via the transcription factor hypoxia-inducible factor (HIF)-2α is highly activated in the intestine and is essential in iron absorption. Intestinal disruption of HIF-2α protects against tissue iron accumulation in iron overload anemias. However, the role of intestinal HIF-2α in regulating anemia in SCD is currently not known. Here we show that in mouse models of SCD, disruption of intestinal HIF-2α significantly decreased tissue iron accumulation. This was attributed to a decrease in intestinal iron absorptive genes, which were highly induced in a mouse model of SCD. Interestingly, disruption of intestinal HIF-2α led to a robust improvement in anemia with an increase in RBC, hemoglobin, and hematocrit. This was attributed to improvement in RBC survival, hemolysis, and insufficient erythropoiesis, which is evident from a significant decrease in serum bilirubin, reticulocyte counts, and serum erythropoietin following intestinal HIF-2α disruption. These data suggest that targeting intestinal HIF-2α has a significant therapeutic potential in SCD pathophysiology.

  3. Interleukin-1β and interleukin-6 gene polymorphisms are associated with manifestations of sickle cell anemia.

    PubMed

    Vicari, Perla; Adegoke, Samuel A; Mazzotti, Diego Robles; Cançado, Rodolfo Delfini; Nogutti, Maria Aparecida Eiko; Figueiredo, Maria Stella

    2015-03-01

    Sickle cell anemia (SCA), a disorder characterized by both acute and chronic inflammation, exhibits substantial phenotypic variability. Interleukin-1 beta (IL-1β) and IL-6 are important in acute and chronic diseases, and their single nucleotide polymorphisms (SNPs) have been considered as predictors of prognosis in several inflammatory conditions. This study aims at exploring possible association of IL-1β and IL-6 SNPs as potential genetic modifiers and or predictors of SCA clinical and laboratory phenotypes. This cross-sectional study involved 107 SCA patients and 110 age, sex and ethnicity-matched healthy individuals. The SNPs were identified by PCR-RFLP for IL-1β (-511C>T and +3954C>T) and IL-6 (-597G>A and -174G>C) genes. Associations between these SNPs and the clinical and laboratory profiles of patients with SCA were then determined. Allelic and genotypic frequencies of IL-1β and IL-6 SNPs between patients with SCA and controls were similar and followed HWE. IL-1β +3954C>T SNP was associated with increased risk of osteonecrosis, elevated pulmonary arterial pressure and lower absolute reticulocyte count, while IL-6 -597G>A was associated with higher likelihood of retinopathy and leg ulcer. These data indicate that IL-1β and IL-6 gene SNPs are associated with SCA complications among Brazilian patients and may act as genetic predictors of SCA clinical heterogeneity.

  4. G6PD deficiency and absence of α-thalassemia increase the risk for cerebral vasculopathy in children with sickle cell anemia.

    PubMed

    Joly, Philippe; Garnier, Nathalie; Kebaili, Kamila; Renoux, Céline; Dony, Arthur; Cheikh, Nathalie; Renard, Cécile; Ceraulo, Antony; Cuzzubbo, Daniela; Pondarré, Corinne; Martin, Cyril; Pialoux, Vincent; Francina, Alain; Bertrand, Yves; Connes, Philippe

    2016-04-01

    The aim of this study was to test the association between hematological/genetic factors and cerebral vasculopathy in children with sickle cell anemia (SCA). A group with cerebral vasculopathy (VASC) was composed of children who had stroke (n = 6), silent infarct (n = 11), or an abnormal transcranial Doppler (n = 5). Eighty-four patients had neither positive history of stroke or silent infarct, nor abnormal transcranial Doppler (NORM group). An intermediate group (COND; n = 15) was composed of SCA children with a conditional transcranial Doppler. Biological analyses were performed on samples obtained at steady state and before the beginning of any chronic treatment. The comparisons of the three groups demonstrated a protective effect of α-thalassemia against cerebral vasculopathy through its effects on hemoglobin and reticulocyte levels. Moreover, we observed higher frequency of G6PD deficiency in the VASC group compared with the other groups. Our study confirms the key role of α-thalassemia and G6PD status in the pathophysiology of cerebral vasculopathy in SCA children.

  5. Mbh 1: a novel gelsolin/severin-related protein which binds actin in vitro and exhibits nuclear localization in vivo.

    PubMed Central

    Prendergast, G C; Ziff, E B

    1991-01-01

    We describe the characterization of a novel cDNA, mbh1 (myc basic motif homolog-1), which was found during a search for candidate factors which might interact with the c-Myc oncoprotein. Embedded within the amino acid sequence encoded by mbh1 is a region distantly related to the basic/helix-loop-helix (B/HLH) DNA-binding motif and a potential nuclear localization signal. Mbh1 encodes a polypeptide structurally similar to the actin-severing proteins gelsolin and severin. Translation of mbh1 RNA in rabbit reticulocyte extracts produces an approximately 45 kd protein capable of binding actin-coupled agarose beads in vitro in a Ca2(+)-dependent manner. Antiserum raised to a trpE/mbh1 bacterial fusion protein recognizes an approximately 45 kb protein in murine 3T3 fibroblasts, suggesting that the cDNA encodes the complete Mbh1 protein. Examination of Mbh1 localization in 3T3 fibroblasts by indirect immunofluorescence reveals a larger cell population showing diffuse staining, and a smaller population exhibiting a distinct nuclear stain. Western analysis corroborates this intracellular localization and indicates that total cellular levels and localization of Mbh1 are not affected by the cell growth state. The data suggest that Mbh1 may play a role in regulating cytoplasmic and/or nuclear architecture through potential interactions with actin. Images PMID:1849072

  6. Spontaneous Pathology and Routine Clinical Pathology Parameters in Aging Beagle Dogs: A Comparison With Adolescent and Young Adults.

    PubMed

    Barnes, J; Cotton, P; Robinson, S; Jacobsen, M

    2016-03-01

    AstraZeneca ran a bespoke study to generate age-matched clinical pathology and histopathology data from a cohort of Beagle dogs aged between 25 and 37 months to support the use of these older animals in routine preclinical toxicology studies. As the upper age range of Beagle dogs routinely used in toxicology studies does not normally exceed 24 months, there is an absence of appropriate age-matched historical control data. The generation of such data was crucial to understand whether age-related differences in spontaneous findings might confound the interpretation of toxicology study data. While the majority of the histopathology findings in all the older dogs occurred at a similar prevalence as those expected in young adult dogs (<24 months), a number of differences were observed in the thymus (involution), bone marrow (increased adiposity), testes (degenerative changes), and lung (fibrosis, pigment and alveolar hyperplasia) that could be misinterpreted as a test article effect. Minor differences in some clinical pathology values (hemoglobin, alkaline phosphatase, absolute reticulocytes) were of a small magnitude and considered unlikely to affect the interpretation of study data.

  7. Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion

    PubMed Central

    Sisquella, Xavier; Nebl, Thomas; Thompson, Jennifer K; Whitehead, Lachlan; Malpede, Brian M; Salinas, Nichole D; Rogers, Kelly; Tolia, Niraj H; Fleig, Andrea; O’Neill, Joseph; Tham, Wai-Hong; David Horgen, F; Cowman, Alan F

    2017-01-01

    The most lethal form of malaria in humans is caused by Plasmodium falciparum. These parasites invade erythrocytes, a complex process involving multiple ligand-receptor interactions. The parasite makes initial contact with the erythrocyte followed by dramatic deformations linked to the function of the Erythrocyte binding antigen family and P. falciparum reticulocyte binding-like families. We show EBA-175 mediates substantial changes in the deformability of erythrocytes by binding to glycophorin A and activating a phosphorylation cascade that includes erythrocyte cytoskeletal proteins resulting in changes in the viscoelastic properties of the host cell. TRPM7 kinase inhibitors FTY720 and waixenicin A block the changes in the deformability of erythrocytes and inhibit merozoite invasion by directly inhibiting the phosphorylation cascade. Therefore, binding of P. falciparum parasites to the erythrocyte directly activate a signaling pathway through a phosphorylation cascade and this alters the viscoelastic properties of the host membrane conditioning it for successful invasion. DOI: http://dx.doi.org/10.7554/eLife.21083.001 PMID:28226242

  8. Delayed hemolytic transfusion reaction due to anti-S antibody in patient with anti-Jk(a) autoantibody and multiple alloantibodies.

    PubMed

    Guastafierro, S; Sessa, F; Cuomo, C; Tirelli, A

    2004-05-01

    We describe the case of a 60-year-old woman with a delayed hemolytic transfusion reaction (DHTR). She had a history of an ulcerative colitis, blood transfusion because of rectal bleeding, and surgical removal of descendent and sigmoid colon. At admission, laboratory data showed Hb 6.3 g/dL, reticulocytes 120 x 10(9)/L, serum total bilirubin 1.2 mg/dL (direct bilirubin: 0.2 mg/dL). Pretransfusion antibody screening procedures were positive. A monospecific autoanti-Jk(a) and three alloantibodies (anti-c, -E, -K) were identified by immunohematologic studies. The patient received two units of crossmatch compatible concentrated red blood cells. Six days later biochemical serum values showed Hb 6.2 g/dL, LDH 975 I.U./L and total bilirubin 2.95 mg/dL (direct 0.35 mg/dL). Crossmatches with red cell suspension of transfused blood units and a post-transfusion serum were repeatedly positive. Laboratory tests showed the presence of anti-S alloantobody in the serum and eluate. Moreover, pre-transfusion serum of the patient was retrospectively retested: anti-S was not detected. These data suggested a DHTR. The present case is unusual and interesting because of the association of a rare autoanti-Jk(a), non responsible for anemia, and four alloantibodies of which anti-S involved in a DHTR.

  9. A fatal case of severe hemolytic disease of newborn associated with anti-Jk(b).

    PubMed

    Kim, Won Duck; Lee, Young Hwan

    2006-02-01

    The Kidd blood group is clinically significant since the Jk antibodies can cause acute and delayed transfusion reactions as well as hemolytic disease of newborn (HDN). In general, HDN due to anti-Jk(b) incompatibility is rare and it usually displays mild clinical symptoms with a favorable prognosis. Yet, we apparently experienced the second case of HDN due to anti-Jk(b) with severe clinical symptoms and a fatal outcome. A female patient having the AB, Rh(D)-positive blood type was admitted for jaundice on the fourth day after birth. At the time of admission, the patient was lethargic and exhibited high pitched crying. The laboratory data indicated a hemoglobin value of 11.4 mg/dL, a reticulocyte count of 14.9% and a total bilirubin of 46.1 mg/dL, a direct bilirubin of 1.1 mg/dL and a strong positive result (+++) on the direct Coomb's test. As a result of the identification of irregular antibody from the maternal serum, anti-Jk(b) was detected, which was also found in the eluate made from infant's blood. Despite the aggressive treatment with exchange transfusion and intensive phototherapy, the patient died of intractable seizure and acute renal failure on the fourth day of admission. Therefore, pediatricians should be aware of the clinical courses of hemolytic jaundice due to anti-Jk(b), and they should be ready to treat this disease with active therapeutic interventions.

  10. Novel alleles at the JK blood group locus explain the absence of the erythrocyte urea transporter in European families.

    PubMed

    Irshaid, Nidal M; Eicher, Nicole I; Hustinx, Hein; Poole, Joyce; Olsson, Martin L

    2002-02-01

    The Kidd (JK) blood group system is of importance in transfusion medicine. The Jk(null) phenotype is associated with absence of the urea transporter in erythrocytes and moderately reduced ability to concentrate urine. We and others recently reported different molecular alterations in the silenced Jkb-like alleles of Polynesians and Finns, populations with higher Jk(null) frequencies. Here we report novel molecular bases of this phenotype in Caucasians. Blood samples from a Swiss and an English family were investigated by serological methods, urea haemolysis test and JK genotyping. Genomic DNA and JK mRNA were sequenced. Genotyping showed homozygosity for Jka-like alleles. The Swiss Jk(null) alleles deviated from wild-type Jka sequence by a nonsense mutation in exon 7 causing an immediate stop codon (Tyr194stop). The English Jk(null) alleles revealed a genomic 1.6 kilobase pair deletion including exons 4 and 5, the former of which includes the translation start codon. Multiple mRNA splicing variants were detected in reticulocytes but exons 3-5 were absent in all transcripts analysed. Screening for these alleles was negative in random donors. Two novel molecular alterations at the JK locus were defined and a multiplex polymerase chain reaction method for detection of the five known silent Jk alleles was developed to complement JK genotyping in clinical transfusion medicine.

  11. ATP-dependent degradation of ubiquitin-protein conjugates.

    PubMed Central

    Hershko, A; Leshinsky, E; Ganoth, D; Heller, H

    1984-01-01

    Previous studies have indicated that the ATP-requiring conjugation of ubiquitin with proteins plays a role in the energy-dependent degradation of intracellular proteins. To examine whether such conjugates are indeed intermediates in protein breakdown, conjugates of 125I-labeled lysozyme with ubiquitin were isolated and incubated with a fraction of reticulocyte extract that lacks the enzymes that carry out ubiquitin-protein conjugation. ATP markedly stimulated degradation of the lysozyme moiety of ubiquitin conjugates to products soluble in trichloroacetic acid. By contrast, free 125I-labeled lysozyme was not degraded under these conditions, unless ubiquitin and the three enzymes required for ubiquitin conjugation were supplemented. Mg2+ was absolutely required for conjugate breakdown. Of various nucleotides, only CTP replaced ATP. Nonhydrolyzable analogs of ATP were not effective. In the absence of ATP, free lysozyme is released from ubiquitin-lysozyme conjugates by isopeptidases present in the extract. Thus, ATP is involved in both the formation and the breakdown of ubiquitin-protein conjugates. Images PMID:6324208

  12. Role of the alpha-amino group of protein in ubiquitin-mediated protein breakdown.

    PubMed Central

    Hershko, A; Heller, H; Eytan, E; Kaklij, G; Rose, I A

    1984-01-01

    Previous studies suggest that the conjugation of ubiquitin to NH2 groups of proteins is required for protein breakdown. We now show that the selective modification of NH2-terminal alpha-NH2 groups of globin and lysozyme prevents their degradation by the ubiquitin proteolytic system from reticulocytes. The conjugation by ubiquitin of epsilon-NH2 groups of lysine residues, usually seen in multiples, was also inhibited in alpha-NH2-blocked proteins. Naturally occurring N alpha-acetylated proteins are not degraded by the ubiquitin system at a significant rate, while their nonacetylated counterparts from other species are good substrates. This suggests that one function of N alpha-acetylation of cellular proteins is to prevent their degradation by the ubiquitin system. alpha-NH2-blocked proteins can have their activity as substrates for degradation increased by incorporation of alpha-NH2 groups through the introduction of polyalanine side chains. Proteins in which most epsilon-NH2 groups are blocked but the alpha-NH2 group is free are degraded by the ubiquitin system, but at a reduced rate. It is therefore suggested that the exposure of a free NH2 terminus of proteins is required for degradation and probably initiates the formation of ubiquitin conjugates committed for degradation. Images PMID:6095265

  13. The Elav-like protein HuR exerts translational control of viral internal ribosome entry sites

    SciTech Connect

    Rivas-Aravena, Andrea; Ramdohr, Pablo; Vallejos, Maricarmen; Valiente-Echeverria, Fernando; Dormoy-Raclet, Virginie; Rodriguez, Felipe; Pino, Karla; Holzmann, Cristian; Huidobro-Toro, J. Pablo; Gallouzi, Imed-Eddine; Lopez-Lastra, Marcelo

    2009-09-30

    The human embryonic-lethal abnormal vision (ELAV)-like protein, HuR, has been recently found to be involved in the regulation of protein synthesis. In this study we show that HuR participates in the translational control of the HIV-1 and HCV IRES elements. HuR functions as a repressor of HIV-1 IRES activity and acts as an activator of the HCV IRES. The effect of HuR was evaluated in three independent experimental systems, rabbit reticulocyte lysate, HeLa cells, and Xenopus laevis oocytes, using both overexpression and knockdown approaches. Furthermore, results suggest that HuR mediated regulation of HIV-1 and HCV IRESes does not require direct binding of the protein to the RNA nor does it need the nuclear translocation of the IRES-containing RNAs. Finally, we show that HuR has a negative impact on post-integration steps of the HIV-1 replication cycle. Thus, our observations yield novel insights into the role of HuR in the post-transcriptional regulation of HCV and HIV-1 gene expression.

  14. Trypanosoma evansi: hematologic changes in experimentally infected cats.

    PubMed

    Da Silva, Aleksandro Schafer; Costa, Márcio Machado; Wolkmer, Patrícia; Zanette, Régis Adriel; Faccio, Luciana; Gressler, Lucas Trevisan; Dorneles, Tagor Eduardo Andreolla; Santurio, Janio Morais; Lopes, Sonia Terezinha dos Anjos; Monteiro, Silvia Gonzalez

    2009-09-01

    This study aimed at evaluating hemogram and erythropoietic changes in cats experimentally infected with Trypanosoma evansi. Thirteen adult female non-breeding Felix catus were separated into two groups: seven animals were infected with 10(8) trypomastigotes each, and six animals were used as negative controls. Animals were kept in air-conditioned rooms and blood smears were performed daily for 49 days. Blood samples were collected from the jugular vein at days 0, 7, 21, 35 and 49 and stored in blood-collecting tubes containing anticoagulant. Bone marrow was collected from the proximal epiphysis of the right femur at days 14 and 42 post-inoculation (PI). Total erythrocyte count, hematocrit and hemoglobin showed statistical differences among groups from the seventh day PI onwards (P<0.05). The mean corpuscular volume and the mean corpuscular hemoglobin concentration remained normal, characterizing a normocytic-normochromic anemia. Reticulocyte count increased in the infected group from the 21st day onwards, but remained near normal values suggesting a mild regenerative anemia. Moreover, the myeloid:erythroid ratio significantly reduced at day 42 PI, evidencing a bone marrow hematopoietic response. Based on these results we conclude that cats infected with T. evansi have normocytic, normochromic, regenerative anemia.

  15. [Causes of anaemia in African children aged 5 to 10 years].

    PubMed

    Mbanya, Dora; Tagny, Claude Tayou; Akamba, Anastasie; Mekongo, Martin Ondoua; Tetanye, Ekoe

    2008-01-01

    This study was conducted among two groups of 105 children in a hospital in Yaoundé, Cameroon, to determine the causes of anaemia in children aged 5- 10-years-old. The study group included children presenting clinical evidence of anaemia while the control group was made of children without anaemia. Both sets of children were matched for neighbourhoods within the city of Yaoundé. Each child had a complete physical examination, full blood count, haemoglobin electrophoresis, reticulocyte counts, and stool samples examined for parasites and hemoparasites. The mean haemoglobin level was 7.77 +/- 1.54 g/dL in the study group and 12.37 +/- 1.23 in the control group. The anaemia was mainly normocytic, normochromic and regenerative in the study group, usually associated with evidence of haemolysis. Patients with homozygous sickle cell disease were found only in the study group (n = 13; 12.3%). Malaria parasites were noted in 89 children (84.7%) in the study group, compared with 34 (32.4%) in the control group (p = 0.0004). Prevalence of intestinal parasites was similar in both groups (50.8%). Protein-calorie malnutrition was rare and was not associated with anemia. We conclude that both malaria and sickle cell anaemia play an important role in anaemia in children aged 5-10 years.

  16. Congenital dyserythropoietic anaemia type II-like dysplastic anaemia preceding the development of non-Hodgkin lymphoma--a case report.

    PubMed

    Leong, C F; Zainina, S; Cheong, S K

    2005-06-01

    Anaemia is a frequent complication in patients with haematological malignancies and is caused by a variety of mechanisms including neoplastic cell infiltration into the bone marrow, haemolysis, nutritional deficiencies and defect in erythropoiesis or dysplastic anaemia as a result of the disease itself. However, acquired dysplastic anaemia which mimic congenital dyserythropoietic anaemia (CDA) type II morphology in the bone marrow is very rare. A 41-year-old Chinese man presented with refractory symptomatic anaemia in September 2001. He was clinically pale with no other significant physical finding. His initial peripheral blood picture showed normochromic normocytic anaemia with haemoglobin level of 26g/L, with no evidence of haemolysis and a poor reticulocyte response of 0.6%. Bone marrow aspiration was done and showed congenital dyserythropoietic anaemia (CDA) type II-like morphology. He was treated symptomatically with regular blood transfusions approximately every 3 weeks, until August 2002 when he developed multiple cervical lymphadenopathy with loss of appetite, loss of weight and low grade fever. Biopsy of the lymph node confirmed the diagnosis of small lymphocytic lymphoma. Staging with computed tomography and bone marrow aspirate revealed the infiltration of lymphoma cells into the marrow cavity consistent with the staging of IVB. This case report illustrates that CDA type II-like dysplastic anaemia can preceed the development of lymphoma.

  17. Purification and characterization of a novel type i ribosome inactivating protein, pachyerosin, from Pachyrhizus erosus seeds, and preparation of its immunotoxin against human hepatoma cells.

    PubMed

    Guo, Jin-Lin; Cheng, Yuan-Liu; Qiu, Yi; Shen, Cai-Hong; Yi, Bin; Peng, Cheng

    2014-07-01

    Pachyrhizus erosus seeds have a high protein content and are used in China due to their cytotoxic effect. Here we report the biological and pharmacological activity of the protein extracts from P. erosus seeds. A novel ribosome-inactivating protein, pachyerosin, from P. erosus seeds was successively purified to homogeneity using ammonium sulfate precipitation, DEAE-sepharose FF, and Sephacryl S-200. Pachyerosin showed to be a type I ribosome-inactivating protein with a molecular mass of 29 kDa and an isoelectric point of 9.19. It strongly inhibited protein synthesis of rabbit reticulocyte lysate with an IC50 of 0.37 ng/mL and showed N-glycosidase activity on rat liver ribosomes with an EC50 of 85.9 pM. The N-terminal 27 amino acids of pachyerosin revealed a 60.71% sequence identity with abrin A from the seeds of Abrus precatorius. With the aim of targeting the delivery of pachyerosin, immunotoxin was prepared by conjugating pachyerosin with anti-human AFP monoclonal antibodies SM0736. The immunotoxin pachyerosin-SM0736 efficiently inhibited the growth of the human hepatoma cell line HuH-7 with an IC50 of 0.050 ± 0.004 nM, 2360 times lower than that of pachyerosin and 430 times lower than that of the immunotoxin against human gastric cancer cell line SGC7901. These results imply that pachyerosin may be used as a new promising anticancer agent.

  18. Reliability of the dual-isotope Schilling test for the diagnosis of pernicious anemia or malabsorption syndrome

    SciTech Connect

    Domstad, P.A.; Choy, Y.C.; Kim, E.E.; DeLand, F.H.

    1981-05-01

    To evaluate the dual-isotope Schilling test for the diagnosis of pernicious anemia or malabsorption syndrome, 65 studies were selected for clinical correlation. Criteria for pernicious anemia included mean corpuscular volume greater than 100 cu micrometer, serum B12 greater than 100 ng/l, megaloblastic marrow, achlorhydria, reticulocytes greater than 5% on B12 therapy, atrophic gastritis, and elevated serum antibodies to parietal cells or intrinsic factor. Criteria for malabsorption syndrome included: decreased serum B12, folate, and carotene; increased fecal fat; abnormal D-xylose absorption; abnormal radiographic and biopsy findings. /sup 58/Co-cyanocobalamin and /sup 57/Co-cyanocobalamin bound to intrinsic factor were given orally to fasting patients; 1 mg of nonradioactive B12 was injected intramuscularly within two hours. Aliquots of 24-hour urine samples were counted. If the excretion of /sup 58/Co was less than 7% and the /sup 57/Co//sup 58/Co ratio was greater than 1.7, the test indicated pernicious anemia; a ratio less than 1.7 indicated malabsorption syndrome. Sensitivity, specificity, and accuracy of the dual-isotope Schilling test were 83%, 98%, and 94% for pernicious anemia, and 67%, 90%, and 86% for malabsorption syndrome, respectively.

  19. Isocitrate Treatment of Acute Anemia of Inflammation in a Mouse Model

    PubMed Central

    Kim, Airie; Fung, Eileen; Parikh, Sona G.; Gabayan, Victoria; Nemeth, Elizabeta; Ganz, Tomas

    2015-01-01

    Acute and severe anemia of inflammation (AI) is a common complication of various clinical syndromes, including fulminant infections, critical illness with multiorgan failure, and exacerbations of autoimmune diseases. Building on recent data showing beneficial results with isocitrate treatment for chronic low-grade AI in a rat model, we used a mouse model of acute and severe AI induced by intraperitoneal heat-killed Brucella abortus to determine if isocitrate would be effective in this more stringent application. Inflamed mice treated with isocitrate developed an early but transient improvement in hemoglobin compared to solvent-treated controls, with a robust improvement on day 7, and only a trend towards improvement by day 14. Reticulocyte counts were increased in treated mice transiently, with no significant difference by day 21. Serum erythropoietin (EPO) levels were similar in treated versus control mice, indicating that isocitrate increased sensitivity to EPO. Serum and tissue iron levels showed no significant differences between the treated and control mice, ruling out improved iron availability as the cause of the increased response to endogenous EPO. Compared to the milder rat model, much higher doses of isocitrate were required for a relatively modest benefit. PMID:26603720

  20. Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift in the synthesis of the putative viral replicase of potato leafroll luteovirus.

    PubMed Central

    Prüfer, D; Tacke, E; Schmitz, J; Kull, B; Kaufmann, A; Rohde, W

    1992-01-01

    The 5.8 kb RNA genome of potato leafroll luteovirus (PLRV) contains two overlapping open reading frames, ORF2a and ORF2b, which are characterized by helicase and RNA polymerase motifs, respectively, and possibly represent the viral replicase. Within the overlap, ORF2b lacks an AUG translational start codon and is therefore presumably translated by -1 ribosomal frameshifting as a transframe protein with ORF2a. This hypothesis was studied by introducing the putative frameshift region into an internal position of the beta-glucuronidase (GUS) gene and testing for the occurrence of frameshifting in vivo by transient expression of GUS activity in potato protoplasts as well as in vitro by translation in the reticulocyte system. Both experimental approaches demonstrate that a -1 frameshift occurs at a frequency of approximately 1%. Site-directed mutagenesis identified the frameshift region and the involvement of the novel heptanucleotide motif UUUAAAU in conjunction with an adjacent stem-loop structure. Part of this stem-loop encodes a basic region in the ORF2b moiety of the transframe protein which was shown by binding experiments with PLRV RNA to represent a nucleic acid-binding domain. These data support a possible biological significance of the frameshift to occur at this position of the large overlap by including the putative RNA template-binding site of the PLRV replicase in the ORF2a/ORF2b transframe protein. Images PMID:1547775

  1. Isolation and Characterization of Messenger RNAs for Seed Lectin and Kunitz Trypsin Inhibitor in Soybeans

    PubMed Central

    Vodkin, Lila O.

    1981-01-01

    The mRNAs for seed lectin and Kunitz trypsin inhibitor of soybean have been highly enriched by immunoadsorption of the polysomes synthesizing these proteins. Polysomes isolated from developing seed of variety Williams were incubated with monospecific rabbit antibodies produced against lectin subunits or trypsin inhibitor protein. The polysomal mixture was passed over a column containing goat anti-rabbit antibodies bound to Sepharose. Bound polysomes were eluted and the mRNA was selected by passage over oligo(dT)-cellulose. Lectin complementary DNA hybridized to an 1150-nucleotide message and trypsin inhibitor complementary DNA hybridized to a 770-nucleotide message in blotting experiments using total poly(A) RNA. Translation of soybean lectin mRNA using a rabbit reticulocyte lysate yielded a major polypeptide of 32,300 whereas the molecular weight for purified lectin subunits was 30,000. Trypsin inhibitor mRNA directed the synthesis of a 23,800-dalton polypeptide as compared to 21,500 daltons for trypsin inhibitor marker protein. Lectin specific polysomes could not be obtained from a soybean variety which lacks detectable lectin protein whereas trypsin inhibitor-specific polysomes were bound by immunoselection. These results confirmed the specificity of the immunoadsorption procedure and strongly indicated that the lectinless variety was deficient or substantially reduced in functional lectin mRNA. Images PMID:16661996

  2. [Infantile pyknocytosis: A cause of noenatal hemolytic anemia. Is recombinant erythropoietin an alternative to transfusion?].

    PubMed

    Bagou, M; Rolland, E; Gay, C; Patural, H

    2016-01-01

    Infantile pyknocytosis is a neonatal hemolytic disorder which causes anemia and icterus and is characterized by the presence of an increased number of distorted red blood cells called pyknocytes. Resolution spontaneously occurs in the first semester of life. It has been generally described as a rare entity, with an occasional family history. We report seven cases of infantile pyknocytosis observed in our hospital in 3 years. Most of the infants presented with hemolytic icterus and profound anemia that was reaching its peak by the 3rd week of life. Three neonates received one to three red blood cell transfusions, according to former recommendations. However, the following four received a treatment with recombinant erythropoietin administered subcutaneously. Only one of these four cases required a transfusion. All of them were free of hematological disease 2-3 months after completion of treatment. Infantile pyknocytosis is a recognized cause of neonatal hemolytic anemia, which requires careful examination of red cell morphology on a peripheral blood smear. The cause of this transient disorder remains unknown. Our observations show that recombinant erythropoietin therapy is effective in treating infantile pyknocytosis and increases the reticulocyte response, thus improving the hemoglobin level.

  3. Use of recombinant erythropoietin for the management of severe hemolytic disease of the newborn of a K0 phenotype mother.

    PubMed

    Manoura, Antonia; Korakaki, Eftychia; Hatzidaki, Eleftheria; Saitakis, Emmanuel; Maraka, Sofia; Papamastoraki, Isabella; Matalliotakis, Emmanuel; Foundouli, Kaliopi; Giannakopoulou, Christine

    2007-01-01

    Very few people do not express any Kell antigens on their red blood cells (K0 phenotype). They can be immunized by transfusion or pregnancy and develop antibodies against Kell system antigens. These maternal antibodies can cause severe hemolytic disease of the fetus/newborn, as a result of the suppression of erythropoiesis and hemolysis. Multiple intrauterine transfusions in the management of severe hemolytic disease have been shown to cause erythropoietic suppression as well. Recombinant erythropoietin has been successfully used in the management of late anemia of infants with Rh hemolytic disease and in 1 case of KEL1 (Kell)-associated hemolytic disease. The authors present the case of severe hemolytic disease of a newborn due to KEL5 (Ku) isoimmunization of his K0 phenotype mother. Regular intrauterine transfusions were performed to manage the severe fetal anemia (Hb 3 g/dL). A male infant was born at the 36th week of gestation having normal hemoglobin (15.8 g/dL) and developed only mild hyperbilirubinemia. On the 15th day of life, the infant's hematocrit had fallen to 27.3%, with low reticulocyte count and low erythropoietin level. The infant was managed successfully with recombinant erythropoietin.

  4. Haematological analysis conducted at the departure of the Tour de France 2001.

    PubMed

    Robinson, N; Schattenberg, L; Zorzoli, M; Mangin, P; Saugy, M

    2005-04-01

    Since the introduction of blood analysis performed before major cycling events in 1997, there have been discussions concerning the quality of the results. The aim of our study therefore was to measure blood samples and compare the results obtained on the field with those obtained in the laboratory. For this it was necessary to have blood samples analysed with different instruments to determine the exactness of the results and evaluate the performances of these instruments in order to validate the haematological testing used to reveal athletes abusing recombinant erythropoietin. We report on the haematological analysis of 177 professional cyclists who took part in the Tour de France 2001. All the blood samples were withdrawn in the morning between 7 and 9 am in Dunkerque (France) and were analysed immediately with a transportable analyser. Then the samples were flown to Lausanne (Switzerland) and were reanalysed in two independent ISO 17 025 accredited laboratories with three different analysers. The results confirmed that the most effective haematological follow-up should be performed under standardized pre-analytical conditions and with identical analysers of the same manufacturer to avoid too many variations notably on the haematocrit level and the reticulocyte count. Furthermore, this study suggests that analyses performed on the site are good and could enable the federations to perform a urinary test to detect rhEPO abuse right after the blood analysis. This time saving is essential to fight efficiently recombinant erythropoietin doping, because the half life of the hormone is very short.

  5. Time and temperature dependant changes in red blood cell analytes used for testing recombinant erythropoietin abuse in sports.

    PubMed

    Robinson, Neil; Mangin, Patrice; Saugy, Martial

    2004-01-01

    There has been a long debate since the introduction of blood analysis prior to major sports events, to find out whether blood samples should be analysed right away on the site of competition or whether they should be transported and analysed in an anti-doping laboratory. Therefore, it was necessary to measure blood samples and compare the results obtained right after the blood withdrawal with those obtained after a few hours delay. Furthermore, it was interesting to determine the effect of temperature on the possible deterioration of red blood cell analytes used for testing recombinant erythropoietin abuse. Healthy volunteers were asked to give two blood samples and one of these was kept at room temperature whereas the second one was put into a refrigerator. On a regular basis, the samples were rolled for homogenisation and temperature stabilisation and were analysed with the same haematological apparatus. The results confirmed that blood controls prior to competition should be performed as soon as possible with standardised pre-analytical conditions to avoid too many variations notably on the haematocrit and the reticulocyte count. These recommendations should ideally also be applied to the all the blood controls compulsory for the medical follow up, otherwise unexplainable values could be misinterpreted and could for instance lead to a period of incapacity.

  6. Insulin-like growth factor I stimulates erythropoiesis in hypophysectomized rats

    SciTech Connect

    Kurtz, A.; Zapf, J.; Eckardt, K.U.; Clemons, G.; Froesch, E.R.; Bauer, C. )

    1988-10-01

    Stimulation of erythropoiesis during growth is necessary to ensure proportionality between erythrocyte mass and body mass. However, the way by which erythrocyte formation is adapted to body growth is still unknown. Growth arrest in hypophysectomized rats is accompanied by decreased erythropoiesis. The authors have, therefore, examined whether insulin-like growth factor I (IGF-I), the mediator of growth hormone effects on body growth, is able to restore erythropoiesis in these animals. Subcutaneous infusions of 120 {mu}g of recombinant human IGF-I per day in hypophysectomized rats led to increases in body weight, {sup 59}Fe incorporation into erythrocytes, and the number of reticulocytes that were similar to increases caused by infusions of 28 milliunits of human growth hormone per day. Body weight gain and {sup 59}Fe incorporation were linearly correlated. Like growth hormone, IGF-I also caused a significant rise in serum erythropoietin concentrations. However, the stimulatory effect on erythropoiesis occurred before serum erythropoietin levels had risen. These results demonstrate that IGF-I mediates the stimulatory effect of growth hormone on erythropoiesis in vivo and thus further support the somatomedin concept. They also show that IGF-I can stimulate erythropoiesis in an endocrine manner, and they suggest two possible routes of action: a direct one and an indirect one by means of enhanced erythropoietin production.

  7. Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody.

    PubMed

    Favuzza, Paola; Guffart, Elena; Tamborrini, Marco; Scherer, Bianca; Dreyer, Anita M; Rufer, Arne C; Erny, Johannes; Hoernschemeyer, Joerg; Thoma, Ralf; Schmid, Georg; Gsell, Bernard; Lamelas, Araceli; Benz, Joerg; Joseph, Catherine; Matile, Hugues; Pluschke, Gerd; Rudolph, Markus G

    2017-02-14

    Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here we present the crystal structure of PfCyRPA and of its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. While PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, it possesses no sialidase activity, indicating that it fulfills a non-enzymatic function. Characterization of the epitope recognized by protective antibodies will facilitate design of peptidomimetics that focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines.

  8. Functional characterization of the Plasmodium falciparum and P. berghei homologues of macrophage migration inhibitory factor.

    PubMed

    Augustijn, Kevin D; Kleemann, Robert; Thompson, Joanne; Kooistra, Teake; Crawford, Carina E; Reece, Sarah E; Pain, Arnab; Siebum, Arjan H G; Janse, Chris J; Waters, Andrew P

    2007-03-01

    Macrophage migration inhibitory factor (MIF) is a mammalian cytokine that participates in innate and adaptive immune responses. Homologues of mammalian MIF have been discovered in parasite species infecting mammalian hosts (nematodes and malaria parasites), which suggests that the parasites express MIF to modulate the host immune response upon infection. Here we report the first biochemical and genetic characterization of a Plasmodium MIF (PMIF). Like human MIF, histidine-tagged purified recombinant PMIF shows tautomerase and oxidoreductase activities (although the activities are reduced compared to those of histidine-tagged human MIF) and efficiently inhibits AP-1 activity in human embryonic kidney cells. Furthermore, we found that Plasmodium berghei MIF is expressed in both a mammalian host and a mosquito vector and that, in blood stages, it is secreted into the infected erythrocytes and released upon schizont rupture. Mutant P. berghei parasites lacking PMIF were able to complete the entire life cycle and exhibited no significant changes in growth characteristics or virulence features during blood stage infection. However, rodent hosts infected with knockout parasites had significantly higher numbers of circulating reticulocytes. Our results suggest that PMIF is produced by the parasite to influence host immune responses and the course of anemia upon infection.

  9. External quality assessment scheme for haematology in Germany.

    PubMed

    Heller, S

    1995-01-01

    Quality control in haematology is performed in Germany for 20 years. Both cell count, haemoglobin measurement and differential count on smear with morphology exercise and probably diagnosis will be demanded by the participants. Until now this quality control is not mandatory, even efforts are done to change this circumstance, given by the main input of diagnostic value due to haematology results. So this regulation will be changed very soon, in order to submit haematological laboratories to governmental control, effected by the BAK (Bundesärztekammer), as already is done in clinical chemistry. For this EQA the participants cannot expected any financial support by any organization, nor public health, nor private assurance. The role of referee laboratories and reference values as well as difficulties for the adequate reference material are discussed. For the differential count other limits have to be established: recognition of pathological blood films is one of the most important point (in sense of morphological exercise) to ensure broad knowledge of "flag interpretation". Since the last year quality control for reticulocyte count and flow cytometry for immune status and leukemia-differentiation has been established.

  10. Assessment of the in vivo genotoxicity of new lead compounds to treat sickle cell disease.

    PubMed

    Dos Santos, Jean Leandro; Longhin Bosquesi, Priscila; Varanda, Eliana Aparecida; Moreira Lima, Lídia; Chung, Man Chin

    2011-04-06

    The compounds 1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)methyl nitrate (C1), (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)ethyl nitrate (C2), 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate (C3), 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-hydroxy-benzenesulfonamide (C4), 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)benzyl nitrate (C5), and 2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)phenyl]ethyl nitrate (C6) were evaluated with a micronucleus test using mouse peripheral blood to identify new candidate drugs for the treatment of sickle cell disease (SCD) that are safer than hydroxyurea. The compounds induced an average frequency of micronucleated reticulocytes (MNRET) of less than six per 1,000 cells at 12.5, 25, 50, and 100 mg/kg, whereas hydroxyurea induced an average MNRET frequency of 7.8, 9.8, 15, and 33.7 per 1000 cells respectively, at the same concentrations. Compounds C1-C6 are new non-genotoxic in vivo candidate drugs for the treatment of SCD symptoms.

  11. A Health Assessment of Technical Grade Dinitrotoluene ...

    EPA Pesticide Factsheets

    Technical grade dinitrotoluene (tgDNT) is a mixture of dinitrotoluene isomers. It is composed of 76% 2,4 DNT and 19% 2,6 DNT with the remaining 5% a combination of the other four dinitrotoluene isomers (2,3 , 2,5 , 3,4 , and 3,5 DNT). The toxicological effects of tgDNT were reviewed and the critical effects were identified by evaluating available human occupational and animal studies mainly via inhalation and oral exposure routes. In occupational studies of workers from DNT manufacturing plants, tgDNT exposure (predominately via the inhalation route) associates with clinical symptoms, adverse reproductive effects, and adverse effects on the cardiovascular system. However, these occupational studies are limited due to co-exposure to other known and/or unknown chemicals and lack of useful exposure information. Oral studies in F344 rats showed that tgDNT has a broad toxicity profile in both males and females including significantly increased reticulocytes and Heinz bodies, increased relative liver weight and kidney weight, spleen hemosiderin and extramedullary hematopoiesis, increased incidence of chronic interstitial nephritis, and hepatotoxicity characterized by increased incidences of hepatocyte necrosis, hyperbasophilic hepatocytes, and hepatocyte megalocytosis . The incidence of testicular degeneration in male rats is also significantly increased by tgDNT. In order to identify the most sensitive non-cancer effect(s), all toxicological endpoints from subchron

  12. DNA-dependent RNA polymerase subunits encoded within the vaccinia virus genome.

    PubMed Central

    Jones, E V; Puckett, C; Moss, B

    1987-01-01

    Antiserum to a multisubunit DNA-dependent RNA polymerase from vaccinia virions was prepared to carry out genetic studies. This antiserum selectively inhibited the activity of the viral polymerase but had no effect on calf thymus RNA polymerase II. The specificity of the antiserum was further demonstrated by immunoprecipitation of RNA polymerase subunits from dissociated virus particles. The presence in vaccinia virus-infected cells of mRNA that encodes the polymerase subunits was determined by in vitro translation. Immunoprecipitable polypeptides with Mrs of about 135,000, 128,000, 36,000, 34,000, 31,000, 23,000, 21,000, 20,000, and 17,000 were made when early mRNA was added to reticulocyte extracts. The subunits were encoded within the vaccinia virus genome, as demonstrated by translation of early mRNA that hybridized to vaccinia virus DNA. The locations of the subunit genes were determined initially by hybridization of RNA to a series of overlapping 40-kilobase-pair DNA fragments that were cloned in a cosmid vector. Further mapping was achieved with cloned HindIII restriction fragments. Results of these studies indicated that RNA polymerase subunit genes are transcribed early in infection and are distributed within the highly conserved central portion of the poxvirus genome in HindIII fragments E, J, H, D, and A. Images PMID:3033308

  13. Structure of the malaria vaccine candidate antigen CyRPA and its complex with a parasite invasion inhibitory antibody

    PubMed Central

    Favuzza, Paola; Guffart, Elena; Tamborrini, Marco; Scherer, Bianca; Dreyer, Anita M; Rufer, Arne C; Erny, Johannes; Hoernschemeyer, Joerg; Thoma, Ralf; Schmid, Georg; Gsell, Bernard; Lamelas, Araceli; Benz, Joerg; Joseph, Catherine; Matile, Hugues; Pluschke, Gerd; Rudolph, Markus G

    2017-01-01

    Invasion of erythrocytes by Plasmodial merozoites is a composite process involving the interplay of several proteins. Among them, the Plasmodium falciparum Cysteine-Rich Protective Antigen (PfCyRPA) is a crucial component of a ternary complex, including Reticulocyte binding-like Homologous protein 5 (PfRH5) and the RH5-interacting protein (PfRipr), essential for erythrocyte invasion. Here, we present the crystal structures of PfCyRPA and its complex with the antigen-binding fragment of a parasite growth inhibitory antibody. PfCyRPA adopts a 6-bladed β-propeller structure with similarity to the classic sialidase fold, but it has no sialidase activity and fulfills a purely non-enzymatic function. Characterization of the epitope recognized by protective antibodies may facilitate design of peptidomimetics to focus vaccine responses on protective epitopes. Both in vitro and in vivo anti-PfCyRPA and anti-PfRH5 antibodies showed more potent parasite growth inhibitory activity in combination than on their own, supporting a combined delivery of PfCyRPA and PfRH5 in vaccines. DOI: http://dx.doi.org/10.7554/eLife.20383.001 PMID:28195038

  14. The glial cell response is an essential component of hypoxia-induced erythropoiesis in mice

    PubMed Central

    Weidemann, Alexander; Kerdiles, Yann M.; Knaup, Karl X.; Rafie, Christopher A.; Boutin, Adam T.; Stockmann, Christian; Takeda, Norihiko; Scadeng, Miriam; Shih, Andy Y.; Haase, Volker H.; Simon, M. Celeste; Kleinfeld, David; Johnson, Randall S.

    2009-01-01

    A key adaptation to environmental hypoxia is an increase in erythropoiesis, driven by the hormone erythropoietin (EPO) through what is traditionally thought to be primarily a renal response. However, both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) also express EPO following ischemic injury, and this response is known to ameliorate damage to the brain. To investigate the role of glial cells as a component of the systemic response to hypoxia, we created astrocyte-specific deletions of the murine genes encoding the hypoxia-inducible transcription factors HIF-1α and HIF-2α and their negative regulator von Hippel–Lindau (VHL) as well as astrocyte-specific deletion of the HIF target gene Vegf. We found that loss of the hypoxic response in astrocytes does not cause anemia in mice but is necessary for approximately 50% of the acute erythropoietic response to hypoxic stress. In accord with this, erythroid progenitor cells and reticulocytes were substantially reduced in number in mice lacking HIF function in astrocytes following hypoxic stress. Thus, we have demonstrated that the glial component of the CNS is an essential component of hypoxia-induced erythropoiesis. PMID:19809162

  15. Clinical Course of Two Children with Unstable Hemoglobins: The Effect of Hydroxyurea Therapy.

    PubMed

    Loovers, Harriët M; Tamminga, Nienke; Mulder, André B; Tamminga, Rienk Y J

    2016-09-01

    Case reports on the effect of hydroxyurea (HU) therapy for unstable hemoglobins (Hbs) are sparse; only three adult cases have been reported. We report for the first time on the effect of HU therapy in children carrying unstable Hbs. The first case concerns a female child with a familial history of chronic hemolytic anemia. She was diagnosed with Hb Volga (HBB: c.83C>A) at the age of 7 months. At age 6, treatment options were reconsidered due to increasing fatigue and decreasing Hb concentration. The second case also concerns a female child with chronic hemolytic anemia and icterus since the age of 5. She was diagnosed with Hb Köln (HBB: c.295G>A) at the age of 9. At age 10, treatment options were reconsidered due to decreased general condition and poor school performance. Both children were started on HU therapy. The child with Hb Volga showed reduced clinical symptoms and increased average Hb concentrations. She has been on HU therapy for over 7 years at preparation of this manuscript. The child with Hb Köln showed decreasing Hb concentrations upon start of therapy; clinical symptoms did not improve. Therapy was discontinued after 3½ months. The Hb Volga case report suggests that HU therapy could improve clinical symptoms in some patients with unstable Hbs. Based on these and previously published cases, it was speculated that response can be predicted by the percentage of Hb F and reticulocyte counts.

  16. Cell-free synthesis of two proteins unique to RNA of transforming virions of Rous sarcoma virus.

    PubMed Central

    Kamine, J; Buchanan, J M

    1977-01-01

    We have utilized a reticulocyte lysate system to translate the 35S RNA of Rous sarcoma virus. Autoradiograms of the protein products separated on sodium dodecyl sulfate/polyacrylamide gels reveal a heterogeneous mixture of proteins of sizes ranging from 13,000 to 180,000 daltons. In comparing the translational products from 35S RNA of Prague B Rous sarcoma virus with those formed from the RNA of a transformation-defective deletion mutant derived from Prague B, we have found that two proteins, 25,000 and 18,000 daltons, are missing from the latter. Neither of these proteins is immunoprecipitated by monospecific antisera against the structural proteins of avian RNA tumor viruses. The combined atomic mass of 43,000 daltons corresponds to the amount of genetic coding capacity (40,000-50,000 daltons in terms of protein products) deleted from the RNA of the transformation-defective viruses. We propose that these proteins are coded for by the putative oncogene (onc) or sarc (src) gene and that one or both of them may be responsible for the oncogenic transformation caused by these viruses in infected cells. Images PMID:194246

  17. Mild increases in serum hepcidin and interleukin-6 concentrations impair iron incorporation in haemoglobin during an experimental human malaria infection.

    PubMed

    de Mast, Quirijn; van Dongen-Lases, Edmee C; Swinkels, Dorine W; Nieman, An-Emmie; Roestenberg, Meta; Druilhe, Pierre; Arens, Theo A; Luty, Adrian J; Hermsen, Cornelis C; Sauerwein, Robert W; van der Ven, Andre J

    2009-06-01

    The correct selection of individuals who will benefit from iron supplements in malaria-endemic regions requires improved insight in the effects of malaria on host iron homeostasis and innovative biomarkers. We assessed sequential changes in serum hepcidin and in traditional biochemical iron status indicators during an experimental Plasmodium falciparum malaria infection with five adult volunteers. The haemoglobin content of reticulocytes (Ret-H(e)) and of mature red blood cells (RBC-H(e)) represented iron incorporation into haemoglobin. Low-density parasitaemia and its treatment induced a mild increase in interleukin (IL)-6 and serum hepcidin concentrations. Despite this only mild increase, a marked hypoferraemia with a strong increase in serum ferritin concentrations developed, which was associated with a sharp fall in Ret-H(e), while RBC-H(e) remained unchanged. The ratio of soluble transferrin receptor (sTfR) to log ferritin concentrations decreased to an average nadir of 63% of the baseline value. We concluded that even mild increases in serum hepcidin and IL-6 concentrations result in a disturbed host iron homeostasis. Serum hepcidin, Ret-H(e) and Delta-H(e) (Ret-H(e) minus RBC-H(e)) are promising biomarkers to select those individuals who will benefit from iron supplements in malaria endemic regions, while the sTfR/log ferritin ratio should be used with caution to assess iron status during malaria.

  18. Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

    NASA Technical Reports Server (NTRS)

    Raghavan, V.

    1992-01-01

    Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.

  19. Characterization, biomarkers, and reversibility of a monoclonal antibody-induced immune complex disease in cynomolgus monkeys (Macaca fascicularis).

    PubMed

    Heyen, Jonathan R; Rojko, Jennifer; Evans, Mark; Brown, Tom P; Bobrowski, Walter F; Vitsky, Allison; Dalton, Shana; Tripathi, Niraj; Bollini, Sangeetha Subbarao; Johnson, Theodore; Lin, John C; Khan, Nasir; Han, Bora

    2014-06-01

    Two 6-month repeat-dose toxicity studies in cynomolgus monkeys illustrated immune complex-mediated adverse findings in individual monkeys and identified parameters that potentially signal the onset of immune complex-mediated reactions following administration of RN6G, a monoclonal antibody (mAb). In the first study, 3 monkeys exhibited nondose-dependent severe clinical signs accompanied by decreased erythrocytes with increased reticulocytes, neutrophilia, monocytosis, thrombocytopenia, coagulopathy, decreased albumin, azotemia, and increased serum levels of activated complement products, prompting unscheduled euthanasia. Histologically, immunohistochemical localization of RN6G was associated with monkey immunoglobulin and complement components in glomeruli and other tissues, attributable to immune complex disease (ICD). All 3 animals also had anti-RN6G antibodies and decreased plasma levels of RN6G. Subsequently, an investigational study was designed and conducted with regulatory agency input to detect early onset of ICD and assess reversibility to support further clinical development. Dosing of individual animals ceased when biomarkers of ICD indicated adverse findings. Of the 12 monkeys, 1 developed anti-RN6G antibodies and decreased RN6G exposure that preceded elevations in complement products, interleukin-6, and coagulation parameters and decreases in albumin and fibrinogen. All findings in this monkey, except for antidrug antibody (ADA), reversed after cessation of dosing without progressing to adverse sequelae typically associated with ICD.

  20. Use of a Mathematical Model as a Countermeasure to Prevent Spaceflight Anemia

    NASA Astrophysics Data System (ADS)

    Seth, R.; Coletta, E.; Trudel, G.

    2012-01-01

    Spaceflight results in the decrease of blood volume in astronauts, which then leads to a destruction of red blood cells causing space flight anemia upon return to a 1g environment. The mechanism for this destruction has not been clearly defined and measures to prevent red blood cell destruction do not currently exist. In this study, we investigate the possibility of using a mathematical model to determine the number of red blood cells destroyed as a result of being in a microgravity environment. We have developed a model using a linear piece wise function that incorporates normal red blood cell death and reticulocyte production. An astronaut, using this model, would be able compare their predicted earth normal (1g) red blood cell count with the actual 0g count and determine the number of red blood cells that have been destroyed as a result of being in a microgravity environment. This could then lead to the administration of erythropoietin to boost red blood cell production and restore the level to earth normal on the final leg of the trip back to earth to prevent space flight anemia.

  1. Regulation of hematopoiesis in the suspended rat as a model for space flight

    NASA Technical Reports Server (NTRS)

    Dunn, C. D. R.; Johnson, P. C.

    1984-01-01

    A series of studies was completed in which a variety of routine hematological and other parameters were obtained from sequential sampling of control and suspended rats. These data showed that, during suspension, the rats failed to gain weight at the same rate as the controls, ate and drank significantly less, demonstrated a transient increase in peripheral hematocrit and RBC count, a transient decrease in MCH, suppressed reticulocyte counts and a progressive decrease in MCV but no change in RBC shape. Leukocyte counts were variably decreased but no significant changes in platelet numbers were noted. Post-suspension, evidence of anemia was present from a reduced RBC count, hemoglobin, hematocrit, and MCV. A leukocytosis was also noted. Efforts directed to the collection of data aimed at understanding changes in blood volume during suspension are also discussed. As part of these studies the following parameters were investigated; RBC survival, in vitro leukocyte reactivity to PHA, bone marrow and spleen cellularity and morphology, ferrokinetics, and the hematopoietic inductive microenvironment.

  2. Red blood cell aquaporin-1 expression is decreased in hereditary spherocytosis.

    PubMed

    Crisp, Renée L; Maltaneri, Romina E; Vittori, Daniela C; Solari, Liliana; Gammella, Daniel; Schvartzman, Gabriel; García, Eliana; Rapetti, María C; Donato, Hugo; Nesse, Alcira

    2016-10-01

    Aquaporin-1 (AQP1) is the membrane water channel responsible for changes in erythrocyte volume in response to the tonicity of the medium. As the aberrant distribution of proteins in hereditary spherocytosis (HS) generates deficiencies of proteins other than those codified by the mutated gene, we postulated that AQP1 expression might be impaired in spherocytes. AQP1 expression was evaluated through flow cytometry in 5 normal controls, 1 autoimmune hemolytic anemia, 10 HS (2 mild, 3 moderate, 2 severe, and 3 splenectomized), and 3 silent carriers. The effect of AQP1 inhibitors was evaluated through water flow-based tests: osmotic fragility and hypertonic cryohemolysis. Serum osmolality was measured in 20 normal controls and 13 HS. The effect of erythropoietin (Epo) on AQP1 expression was determined in cultures of erythroleukemia UT-7 cells, dependent on Epo to survive. Independent of erythrocyte size, HS patients showed a lower content of AQP1 in erythrocyte membranes which correlated with the severity of the disease. Accordingly, red blood cells from HS subjects were less sensitive to cryohemolysis than normal erythrocytes after inhibition of the AQP1 water channel. A lower serum osmolality in HS with respect to normal controls suggests alterations during reticulocyte remodeling. The decreased AQP1 expression could contribute to explain variable degrees of anemia in hereditary spherocytosis. The finding of AQP1 expression induced by Epo in a model of erythroid cells may be interpreted as a mechanism to restore the balance of red cell water fluxes.

  3. Degradation of microinjected proteins: the role of substrate flexibility

    SciTech Connect

    Rote, K.V.

    1985-01-01

    RB-mediated microinjection was used to introduce radioiodinated proteins of similar structure, but diverse flexibilities, into HeLa cells. Rates of intracellular degradation were then measured by release of /sup 125/I-tyrosine into the media. Ribonuclease-A was much more stable to degradation by trypsin, pepsin, or papain than its relatively flexible derivatives ribonuclease-S and S-protein. Likewise, ribonuclease-S and S-protein were degraded more quickly in reticulocyte lysates than ribonuclease-A. In contrast, all three proteins displayed similar, if not identical, half-lives in vivo. Similarly, intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were in the same range whether they were measured in the free state or following complex formation, which drastically decreases flexibility. Trypsinogen, which contains a relatively flexible activation domain, was degraded more slowly than anhydrotrypsin. Nondenaturing agarose or polyacrylamide gel electrophoresis of microinjected cell lysates revealed that complexes of trypsin and its inhibitors remained intact following radioiodination and introduction into cells, and are therefore degraded as a unit. All microinjected proteins remained in their unbound, unprocessed forms prior to degradation.

  4. Molecular analysis of erythrocyte invasion in Plasmodium falciparum isolates from Senegal.

    PubMed

    Jennings, Cameron V; Ahouidi, Ambroise D; Zilversmit, Martine; Bei, Amy K; Rayner, Julian; Sarr, Ousmane; Ndir, Omar; Wirth, Dyann F; Mboup, Souleymane; Duraisingh, Manoj T

    2007-07-01

    The human malaria parasite, Plasmodium falciparum, utilizes multiple ligand-receptor interactions for the invasion of human erythrocytes. Members of the reticulocyte binding protein homolog (PfRh) family have been shown to be critical for directing parasites to alternative erythrocyte receptors that define invasion pathways. Recent studies have identified gene amplification, sequence polymorphism, and variant expression of PfRh paralogs as mechanisms underlying discrimination between pathways for invasion. In this study, we find considerable heterogeneity in the invasion profiles of clonal, uncultured P. falciparum parasite isolates from a low-transmission area in Senegal. Molecular analyses revealed minimal variation in protein expression levels of the PfRh ligands, PfRh1, PfRh2a, and PfRh2b, and an absence of gene amplification in these isolates. However, significant sequence polymorphism was found within repeat regions of PfRh1, PfRh2a, and PfRh2b. Furthermore, we identified a large sequence deletion ( approximately 0.58 kb) in the C-terminal region of the PfRh2b gene at a high prevalence in this population. In contrast to findings of earlier studies, we found no associations between specific sequence variants and distinct invasion pathways. Overall these data highlight the importance of region-specific elaborations in PfRh sequence and expression polymorphisms, which has important implications in our understanding of how the malaria parasite responds to polymorphisms in erythrocyte receptors and/or evades the immune system.

  5. Possible Involvement of the Double-Stranded RNA-Binding Core Protein ςA in the Resistance of Avian Reovirus to Interferon

    PubMed Central

    Martínez-Costas, José; González-López, Claudia; Vakharia, Vikram N.; Benavente, Javier

    2000-01-01

    Treatment of primary cultures of chicken embryo fibroblasts with a recombinant chicken alpha/beta interferon (rcIFN) induces an antiviral state that causes a strong inhibition of vaccinia virus and vesicular stomatitis virus replication but has no effect on avian reovirus S1133 replication. The fact that avian reovirus polypeptides are synthesized normally in rcIFN-treated cells prompted us to investigate whether this virus expresses factors that interfere with the activation and/or the activity of the IFN-induced, double-stranded RNA (dsRNA)-dependent enzymes. Our results demonstrate that extracts of avian-reovirus-infected cells, but not those of uninfected cells, are able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates, by blocking the activation of the dsRNA-dependent enzymes. In addition, our results show that protein ςA, an S1133 core polypeptide, binds to dsRNA in an irreversible manner and that clearing this protein from extracts of infected cells abolishes their protranslational capacity. Taken together, our results raise the interesting possibility that protein ςA antagonizes the IFN-induced cellular response against avian reovirus by blocking the intracellular activation of enzyme pathways dependent on dsRNA, as has been suggested for several other viral dsRNA-binding proteins. PMID:10627522

  6. [Clinical use of serum erythropoietin determination by the recombigen EPO RIA kit].

    PubMed

    Masaki, Y; Oka, N; Furuya, H; Ohguni, S; Takagi, C; Sato, T; Notsu, K; Kato, Y

    1992-06-01

    Serum erythropoietin (EPO) levels were determined by the recombigen EPO RIA kit (DPC) in normal subjects and patients with renal dysfunction, diabetes mellitus, hypothyroidism and a variety of hematological disorders. Mean (+/- SD) serum EPO levels were 18.6 +/- 5.6 mU/ml in 180 normal subjects and no sex difference was obtained. Serum EPO levels in older subjects were slightly greater than those in younger subjects. There was a negative correlation between serum EPO levels and Ht values in anemic patients with normal renal function, whereas serum EPO levels were within the normal range in anemic patients with renal disorders, suggesting that serum EPO levels were relatively low in patients with chronic renal failure. Serum EPO levels were rather increased in patients with diabetes mellitus and hypothyroidism. High serum EPO levels were obtained in patients with a variety of hematological disorders such as acute leukemia, multiple myeloma, myelodysplasia syndrome, aplastic anemia and pure red cell aplasia. In a patient with pure red cell aplasia treated with glucocorticoids, serum EPO levels were lowered before anemia was recovered and reticulocytes were increased. These findings indicate that measurement of serum EPO levels are useful for not only differential diagnosis of anemia but also clinical evaluation of the treatment.

  7. Hemolytic uremic syndrome associated with paraquat intoxication.

    PubMed

    Jang, Ha Nee; Bae, Eun Jin; Hwang, Kyungo; Kang, Yeojin; Yun, Seongeun; Cho, Hyun Seop; Chang, Se-Ho; Park, Dong Jun

    2014-06-01

    We report a case of a 66-year-old patient with paraquat intoxication resulting in the requirement for hemoperfusion, hemodialysis, and plasma exchange. His initial serum paraquat level was 0.24 µg/mL (0.0-0.1 µg/mL). Activated charcoal (50 g) was administered orally, and high-dose N-acetylcysteine (150 mg/kg) was administered intravenously. In addition, immediate 4 h hemoperfusion was also performed for three consecutive days after admission. Hemodialysis was started on the 4th day after admission because of uremia. On the 9th day after admission, laboratory findings demonstrated hemolytic uremic syndrome (HUS): microangiopathic hemolytic anemia (MAHA), thrombocytopenia, elevated reticulocyte count, and lactate dehydrogenase (LDH). Plasma exchange was performed three times consecutively. Anemia and thrombocytopenia were improved, and LDH was normalized after plasma exchange. Urine output increased to 2240 mL/day on the 18th day after admission, and hemodialysis was discontinued. He is currently being observed at our follow-up clinic without renal impairment or pulmonary dysfunction for 1.5 years since discharge. We should suspect paraquat-associated HUS when thrombocytopenia and anemia are maintained for a long time after paraquat intoxication.

  8. The 5' untranslated region and Gag product of Idefix, a long terminal repeat-retrotransposon from Drosophila melanogaster, act together to initiate a switch between translated and untranslated states of the genomic mRNA.

    PubMed

    Meignin, Carine; Bailly, Jean-Luc; Arnaud, Frédérick; Dastugue, Bernard; Vaury, Chantal

    2003-11-01

    Idefix is a long terminal repeat (LTR)-retrotransposon present in Drosophila melanogaster which shares similarities with vertebrates retroviruses both in its genomic arrangement and in the mechanism of transposition. Like in retroviruses, its two LTRs flank a long 5' untranslated region (5'UTR) and three open reading frames referred to as the gag, pol, and env genes. Here we report that its 5'UTR, located upstream of the gag gene, can fold into highly structured domains that are known to be incompatible with efficient translation by ribosome scanning. Using dicistronic plasmids analyzed by both (i) in vitro transcription and translation in rabbit reticulocyte or wheat germ lysates and (ii) in vivo expression in transgenic flies, we show that the 5'UTR of Idefix exhibits an internal ribosome entry site (IRES) activity that is able to promote translation of a downstream cistron in a cap-independent manner. The functional state of this novel IRES depends on eukaryotic factors that are independent of their host origin. However, in vivo, its function can be down-regulated by trans-acting factors specific to tissues or developmental stages of its host. We identify one of these trans-acting factors as the Gag protein encoded by Idefix itself. Our data support a model in which nascent Gag is able to block translation initiated from the viral mRNA and thus its own translation. These data highlight the fact that LTR-retrotransposons may autoregulate their replication cycle through their Gag production.

  9. Extremes of urine osmolality - Lack of effect on red blood cell survival

    NASA Technical Reports Server (NTRS)

    Leon, H. A.; Fleming, J. E.

    1980-01-01

    Rats were allowed a third of normal water intake for 20 days, and food consumption decreased. The reticulocyte count indicated a suppression of erythropoiesis. Urine osmolality increased from 2,000 mosmol/kg to 3,390 mosmol/kg. Random hemolysis and senescence of a cohort of red blood cell (RBC) previously labeled with (2-(C-14)) glycine was monitored via the production of (C-14)O. Neither hemolysis nor senescence was affected. Following water restriction, the polydipsic rats generated a hypotonic urine. Urine osmolality decreased to 1,300 mosmol/kg for at least 6 days; a reticulocytosis occurred, but RBC survival was unaffected. These results contradict those previously reported, which suggest that RBC survival is influenced by the osmotic stress imposed on the RBC by extremes of urine tonicity. This discrepancy, it is concluded, is due to differences in the methods employed for measuring RBC survival. The random-labeling technique employed previously assumes a steady state between RBC production and destruction. The cohort-labeling technique used here measures hemolysis and senescence independent of changes in RBC production, which is known to be depressed by fasting.

  10. beta zero thalassemia in Sardinia is caused by a nonsense mutation.

    PubMed Central

    Trecartin, R F; Liebhaber, S A; Chang, J C; Lee, K Y; Kan, Y W; Furbetta, M; Angius, A; Cao, A

    1981-01-01

    We report the characterization of a molecular lesion of beta thalassemia in Sardinia. Beta thalassemia in this area is predominantly the beta zero type with low levels of beta-globin mRNA. Translation assay of this messenger RNA in a cell-free system showed beta-globin chain synthesis only with the addition of an amber (UAG) suppressor transfer RNA. Double-stranded complementary DNA prepared from reticulocyte mRNA from a Sardinian patient was cloned in a bacterial plasmid and a beta-globin complementary DNA containing clone was isolated and sequenced. At the position corresponding to amino acid number 39, a single nucleotide mutation converted a glutamine codon (CAG) to an amber termination codon (UAG). We previously reported an amber nonsense mutation at amino acid 17 as a cause of Chinese beta zero thalassemia. Thus, beta zero thalassemia in Sardinia represents the second example of a nonsense mutation, and we predict that other beta zero thalassemias with mutations at various points along the beta-globin chain will be found to form a discrete subgroup of beta zero thalassemia. These experiments further illustrate the heterogeneity of lesions that lead to defective globin chain synthesis in beta thalassemia. Images PMID:6457059

  11. Glucose-6-phosphate dehydrogenase and red cell pyruvate kinase deficiency in neonatal jaundice cases in egypt.

    PubMed

    Abdel Fattah, Mohammed; Abdel Ghany, Eman; Adel, Alia; Mosallam, Dalia; Kamal, Shahira

    2010-05-01

    Glucose-6-phosphate dehydrogenase (G6PD) deficiency can lead to acute hemolytic anemia, chronic nonspherocytic hemolytic anemia, and neonatal jaundice. Neonatal red cell pyruvate kinase (PK) deficiency may cause clinical patterns, ranging from extremely severe hemolytic anemia to moderate jaundice. The authors aimed at studying the prevalence of G6PD and PK deficiency among Egyptian neonates with pathological indirect hyperbilirubinemia in Cairo. This case-series study included 69 newborns with unconjugated hyperbilirubinemia. All were subjected to clinical history, laboratory investigations, e.g., complete blood counts, reticulocytic counts, direct and indirect serum bilirubin levels, Coombs tests, qualitative assay of G6PD activity by methemoglobin reduction test, and measurement of erythrocytic PK levels. The study detected 10 neonates with G6PD deficiency, which means that the prevalence of G6PD deficiency among Egyptian neonates with hyperbilirubinemia is 14.4% (21.2% of males). G6PD deficiency was significantly higher in males than females (P = .01). The authors detected 2 cases with PK deficiency, making the prevalence of its deficiency 2.8%. These data demonstrate that G6PD deficiency is an important cause for neonatal jaundice in Egyptians. Neonatal screening for its deficiency is recommended. PK deficiency is not a common cause of neonatal jaundice. However, this needs further investigation on a larger scale.

  12. Epoetin-induced pure red-cell aplasia (PRCA): preliminary results from the research on adverse drug events and reports (RADAR) group.

    PubMed

    Evens, Andrew M; Bennett, Charles L; Luminari, Stefano

    2005-01-01

    In 2002, investigators from France reported 13 patients in whom pure red cell aplasia developed during treatment with recombinant human erythropoietin (epoetin). We reviewed 208 cases of this syndrome reported worldwide. Adverse event reports describing suspected and confirmed cases of epoetin-associated PRCA in websites maintained by the manufacturers and distributors of epoetin products and other publicly available sources were reviewed. Cases were reported from countries in Europe, North America, Asia, Australia and the United States (US). For >95% of the cases, EPREX had been administered subcutaneous to persons with chronic kidney disease (CKD) and anemia for a mean of nine months prior to diagnosis of PRCA. For 80% of persons with the syndrome, reversal of antibody production and recovery of reticulocytes occurred with discontinuation of epoetin and treatment with immunosuppressive agents. Patients with anemia of CKD who developed neutralizing anti-erythropoietin antibodies and pure red cell aplasia during treatment with epoetin have been identified in a number of countries. In non-US countries, switching renal dialysis patients from subcutaneous to intravenous administration of epoetin alpha and improved handling of the drug appear to have been successful strategies for reducing the occurrence of this toxicity. The decrease in cases occurred coincident with these varied changes, although it is difficult to prove causality. PRCA is a rare, but important side effect of epoetin therapy.

  13. Preconditioning with Hemin Decreases Plasmodium chabaudi adami Parasitemia and Inhibits Erythropoiesis in BALB/c Mice

    PubMed Central

    Dalko, Esther; Gaudreault, Véronique; Sanchez Dardon, Jaime; Moreau, Robert; Scorza, Tatiana

    2013-01-01

    Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during Plasmodium chabaudi adami infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and Th1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following P.c.adami infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology. PMID:23358441

  14. Hyporesponsiveness to anemia therapy--what are we doing wrong?

    PubMed

    MacDougall, I C

    2001-01-01

    Most patients receiving anemia therapy respond well, with a significant rise in hemoglobin concentration. However, approximately 5%-10% of patients fail to show a satisfactory response despite high doses of erythropoietin. The definition of hyporesponsiveness to anemia therapy is somewhat arbitrary, but it is generally regarded as a failure to achieve a hemoglobin concentration of 10-11 g/dL despite a dose of erythropoietin in excess of 200 U/kg weekly. The condition has many causes, the most important ones being iron deficiency, infection or inflammation, and underdialysis. Investigating a patient's poor response to erythropoietin should begin with a check for compliance, followed by screening for iron deficiency. If doubt exists about the presence of iron deficiency, then a trial of intravenous iron may be given. A reticulocyte count may be helpful. A significantly elevated count suggests the presence of blood loss or hemolysis. The level of C-reactive protein (CRP) may be useful as an indicator of underlying inflammation, and underdialysis may be corrected by increasing the dialysis prescription. If other, minor causes of hyporesponsiveness to erythropoietin have been excluded, then a bone marrow biopsy should be performed. Some patients may require higher doses of erythropoietin, and it is not unreasonable to increase the dose to 10,000 U thrice weekly. Some causes of hyporesponsiveness to erythropoietin, such as iron deficiency and underdialysis, are easily corrected; but others, such as primary bone marrow disorders and hemoglobinopathies, are not possible to overcome.

  15. From the Cradle to the Grave: The Role of Macrophages in Erythropoiesis and Erythrophagocytosis

    PubMed Central

    Klei, Thomas R. L.; Meinderts, Sanne M.; van den Berg, Timo K.; van Bruggen, Robin

    2017-01-01

    Erythropoiesis is a highly regulated process where sequential events ensure the proper differentiation of hematopoietic stem cells into, ultimately, red blood cells (RBCs). Macrophages in the bone marrow play an important role in hematopoiesis by providing signals that induce differentiation and proliferation of the earliest committed erythroid progenitors. Subsequent differentiation toward the erythroblast stage is accompanied by the formation of so-called erythroblastic islands where a central macrophage provides further cues to induce erythroblast differentiation, expansion, and hemoglobinization. Finally, erythroblasts extrude their nuclei that are phagocytosed by macrophages whereas the reticulocytes are released into the circulation. While in circulation, RBCs slowly accumulate damage that is repaired by macrophages of the spleen. Finally, after 120 days of circulation, senescent RBCs are removed from the circulation by splenic and liver macrophages. Macrophages are thus important for RBCs throughout their lifespan. Finally, in a range of diseases, the delicate interplay between macrophages and both developing and mature RBCs is disturbed. Here, we review the current knowledge on the contribution of macrophages to erythropoiesis and erythrophagocytosis in health and disease. PMID:28210260

  16. Iron deficiency anemia in inflammatory bowel disease

    PubMed Central

    Kaitha, Sindhu; Bashir, Muhammad; Ali, Tauseef

    2015-01-01

    Anemia is a common extraintestinal manifestation of inflammatory bowel disease (IBD) and is frequently overlooked as a complication. Patients with IBD are commonly found to have iron deficiency anemia (IDA) secondary to chronic blood loss, and impaired iron absorption due to tissue inflammation. Patients with iron deficiency may not always manifest with signs and symptoms; so, hemoglobin levels in patients with IBD must be regularly monitored for earlier detection of anemia. IDA in IBD is associated with poor quality of life, necessitating prompt diagnosis and appropriate treatment. IDA is often associated with inflammation in patients with IBD. Thus, commonly used laboratory parameters are inadequate to diagnose IDA, and newer iron indices, such as reticulocyte hemoglobin content or percentage of hypochromic red cells or zinc protoporphyrin, are required to differentiate IDA from anemia of chronic disease. Oral iron preparations are available and are used in patients with mild disease activity. These preparations are inexpensive and convenient, but can produce gastrointestinal side effects, such as abdominal pain and diarrhea, that limit their use and patient compliance. These preparations are partly absorbed due to inflammation. Non-absorbed iron can be toxic and worsen IBD disease activity. Although cost-effective intravenous iron formulations are widely available and have improved safety profiles, physicians are reluctant to use them. We present a review of the pathophysiologic mechanisms of IDA in IBD, improved diagnostic and therapeutic strategies, efficacy, and safety of iron replacement in IBD. PMID:26301120

  17. Safety and efficacy of hydroxyurea in children and adolescents with sickle/beta-thalassemia: two-year experience

    PubMed Central

    Papadopoulou, E; Teli, A; Theodoridou, S; Gompakis, N; Economou, M

    2015-01-01

    Background Hydroxyurea is a cytotoxic and myelosuppressive drug that has been used during recent years in the treatment of children with severe sickle cell disease. Nevertheless, questions remain regarding its role in young patients with no severe course, like sickle/beta-thalassemia (S/b-thal) patients often present. The aim of the present study was to evaluate the safety and efficacy of hydroxyurea in young patients with S/b-thal, which is the commonest form of the disease in Greece. Patients-Methods Hydroxyurea was given in thirteen children with S/b-thal for 24 months and for that period clinical and laboratory evaluation of the children was performed. Results A reduction in pain crises and rate of hospitalization was noted. None of the patients presented with a severe clinical event, related to the disease during the study period. A significant increase in hemoglobin, hemoglobin F, mean corpuscular volume, and mean corpuscular hemoglobin and a decrease in reticulocyte count, white blood cell and platelet count, and total bilirubin level was noted. With regards to adverse events, these were transient, short-term and dose-dependable. Conclusions To the best of our knowledge, this is the first study to specifically assess the effect of hydroxyurea therapy in young patients with S/b-thal and the results indicate is safe and efficacious in this patient cohort. Hippokratia 2015; 19 (2):172-175. PMID:27418768

  18. Inhibition of eukaryotic translation by tetratricopeptide-repeat proteins of Orientia tsutsugamushi.

    PubMed

    Bang, Sunyoung; Min, Chan-Ki; Ha, Na-Young; Choi, Myung-Sik; Kim, Ik-Sang; Kim, Yeon-Sook; Cho, Nam-Hyuk

    2016-02-01

    Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus. The genome of Orientia tsutsugamushi has revealed multiple ORFs encoding tetratricopeptide-repeat (TPR) proteins. The TPR protein family has been shown to be involved in a diverse spectrum of cellular functions such as cell cycle control, transcription, protein transport, and protein folding, especially in eukaryotic cells. However, little is known about the function of the TPR proteins in O. tsutsugamushi. To investigate the potential role of TPR proteins in host-pathogen interaction, two oriential TPR proteins were expressed in E. coli and applied for GSTpull down assay. DDX3, a DEAD-box containing RNA helicase, was identified as a specific eukaryotic target of the TPR proteins. Since the RNA helicase is involved in multiple RNA-modifying processes such as initiation of translation reaction, we performed in vitro translation assay in the presence of GST-TPR fusion proteins by using rabbit reticulocyte lysate system. The TPR proteins inhibited in vitro translation of a reporter luciferase in a dose dependent manner whereas the GST control proteins did not. These results suggested TPR proteins of O. tsutsugamushi might be involved in the modulation of eukaryotic translation through the interaction with DDX3 RNA helicase after secretion into host cytoplasm.

  19. Identification of amino acids inserted during suppression of UAA and UGA termination codons at the gag-pol junction of Moloney murine leukemia virus.

    PubMed Central

    Feng, Y X; Copeland, T D; Oroszlan, S; Rein, A; Levin, J G

    1990-01-01

    Expression of the murine leukemia virus pol gene occurs by translational readthrough of an in-frame UAG codon between the gag and pol coding regions. In a previous study, we mutated the UAG codon to UAA or UGA and demonstrated that both of these termination codons could be suppressed in reticulocyte lysates and in infected cells with the same efficiency as UAG. We now report the identity of the amino acids inserted in vitro in response to UAA and UGA in fusion products containing the gag-pol junction region. The results show that UAA, like UAG, directs the incorporation of glutamine, whereas UGA directs the incorporation of three amino acids, arginine, cysteine, and tryptophan. To our knowledge, this is the first report indicating misreading of UAA as glutamine and UGA as arginine and cysteine in higher eukaryotes. Interestingly, although our protein synthesis system presumably contains other known UAG and UGA suppressors, these tRNAs did not suppress the termination codons in our experiments. Thus, it seems possible that the sequence surrounding the gag-pol junction not only promotes suppression but also helps determine which tRNAs function in suppression. Images PMID:2247457

  20. Prognostic value of paroxysmal nocturnal haemoglobinuria clone presence in aplastic anaemia patients treated with combined immunosuppression: results of two-centre prospective study.

    PubMed

    Kulagin, Alexander; Lisukov, Igor; Ivanova, Maria; Golubovskaya, Irina; Kruchkova, Irina; Bondarenko, Sergey; Vavilov, Vladimir; Stancheva, Natalia; Babenko, Elena; Sipol, Alexandra; Pronkina, Natalia; Kozlov, Vladimir; Afanasyev, Boris

    2014-02-01

    Paroxysmal nocturnal haemoglobinuria (PNH) clones are frequently detected in patients with aplastic anaemia (AA). To evaluate the prognostic role of PNH clone presence we conducted a prospective study in 125 AA patients treated with combined immunosuppressive therapy (IST). Seventy-four patients (59%) had a PNH clone (PNH+ patients) at diagnosis, with a median clone size of 0·60% in granulocytes and 0·15% in red blood cells. The response rate at 6 months was higher in PNH+ patients than that in PNH- patients, both after first- and second-line IST: 68% vs. 45%, P = 0·0164 and 53% vs. 13%, P = 0·0502 respectively. Moreover, 42% of PNH+ patients achieved complete remission compared with only 16% of PNH- patients (P = 0·0029). In multivariate logistic regression analysis, PNH clone presence (odds ratio 2·56, P = 0·0180) and baseline absolute reticulocyte count (ARC) ≥30 × 10(9) /l (odds ratio 5·19, P = 0·0011) were independent predictors of response to treatment. Stratification according to PNH positivity and ARC ≥30 × 10(9) /l showed significant distinctions for cumulative incidence of response, overall and failure-free survival. The results of this prospective study confirmed the favourable prognostic value of PNH clone presence in the setting of IST for AA.

  1. Meta-analysis of Huangqi injection for the adjunctive therapy of aplastic anemia.

    PubMed

    Zhu, Changtai; Gao, Yulu; Jiang, Ting; Hao, Cao; Gao, Zongshuai; Sun, Yongning

    2015-01-01

    Aplastic anemia therapy remains difficult, due to lack of effective treatment regimens. In recent years, Huangqi injection for the adjunctive therapy of aplastic anemia has been reported in many clinical trials. Considering that Huangqi injection may be a novel approach to aplastic anemia treatment, we conducted a meta-analysis of clinical controlled trials to assess the clinical value of Huangqi injection in the treatment of aplastic anemia. We searched the Chinese Biomedical Literature Database (CBM), China National Knowledge Infrastructure (CNKI), Chinese Scientific Journals Full-text Database (VIP), Wanfang Database, PubMed and EMBASE database to collect the data about the trials of Huangqi injection combined with androgens for treating aplastic anemia. A total of ten studies involving 720 patients with aplastic anemia were included in this study. The meta-analysis showed significant increases in the pool effectiveness rate, white blood cells (WBC), haematoglobin (Hb), platelets (PLT), and reticulocytes (Ret) between the experimental group versus the control group. No severe side effects were found in this study. However, the lower Jadad scores and asymmetric funnel plot degrades the validity of the meta-analysis as the clinical evidence. Therefore, Huangqi injection may significantly enhance the efficacy of androgens for aplastic anemia, suggesting that the novel approach of Chinese traditional medicine combined with Western medicine is promising. The exact outcome required confirmation with rigorously well-designed multi-center trials.

  2. Genetic models in applied physiology: selected contribution: effects of spaceflight on immunity in the C57BL/6 mouse. II. Activation, cytokines, erythrocytes, and platelets

    NASA Technical Reports Server (NTRS)

    Gridley, Daila S.; Nelson, Gregory A.; Peters, Luanne L.; Kostenuik, Paul J.; Bateman, Ted A.; Morony, Sean; Stodieck, Louis S.; Lacey, David L.; Simske, Steven J.; Pecaut, Michael J.

    2003-01-01

    This portion of the study quantified the effects of a 12-day space shuttle mission (Space Transport System-108/UF-1) on body and lymphoid organ masses, activation marker expression, cytokine secretion, and erythrocyte and thrombocyte characteristics in C57BL/6 mice. Animals in flight (Flt group) had 10-12% lower body mass compared with ground controls housed either in animal enclosure modules or under standard vivarium conditions (P < 0.001) and the smallest thymus and spleen masses. Percentages of CD25(+) lymphocytes, CD3(+)/CD25(+) T cells, and NK1.1(+)/CD25(+) natural killer cells from Flt mice were higher compared with both controls (P < 0.05). In contrast, CD71 expression was depressed in the Flt and animal enclosure module control mice compared with vivarium control animals (P < 0.001). Secretion of interferon-gamma, IL-2, and IL-4, but not tumor necrosis factor-alpha and IL-5, by splenocytes from Flt mice was decreased relative to either one or both ground controls (P < 0.05). Flt mice also had high red blood cell and thrombocyte counts compared with both sets of controls; low red blood cell volume and distribution width, percentage of reticulocytes, and platelet volume were also noted (P < 0.05) and were consistent with dehydration. These data indicate that relatively short exposure to the spaceflight environment can induce profound changes that may become significant during long-term space missions.

  3. Identification of a frequent pseudodeficiency mutation in the fumarylacetoacetase gene, with implications for diagnosis of Tyrosinemia Type I

    SciTech Connect

    Rootwelt, H.; Brodtkorb, E.; Kvittingen, E.A.

    1994-12-01

    In healthy individuals, fumarylacetoacetase (FAH) activities close to the range found in hereditary tyrosinemia type 1 (HT1) patients indicated the existence of a pseudodeficiency allele. In an individual homozygous for pseudodeficiency of FAH and in three HT1 families also carrying the pseudodeficiency allele, western blotting of fibroblast extracts showed that the pseudodeficiency allele gave very little immunoreactive FAH protein, whereas northern analysis revealed a normal amount of FAH mRNA. Sequencing revealed an identical mutation, C{sup 1021} {yields} T (Arg341Trp), in all the pseudodeficiency alleles. Site-directed mutagenesis and expression in rabbit reticulocyte lysate system demonstrated that the C{sup 1021} {yields} T mutation gave reduced FAH activity and reduced amounts of the full-length protein. BsiEI restriction digestion of PCR products distinguished between the normal and the mutated sequences. Among 516 healthy volunteers of Norwegian origin, the C{sup 1021} {yields} T mutation was found in 2.2% of the alleles. Testing for the C{sup 1021} {yields} T mutation may solve the problem of prenatal diagnosis and carrier detection in families with compound heterozygote genotypes for HT1 and pseudodeficiency.

  4. An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells.

    PubMed

    Centrella, Michael; Porter, David L; McCarthy, Thomas L

    2011-08-15

    Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo.

  5. A General Map of Iron Metabolism and Tissue-specific Subnetworks

    PubMed Central

    Hower, Valerie; Mendes, Pedro; Torti, Frank M.; Laubenbacher, Reinhard; Akman, Steven; Shulaev, Vladmir; Torti, Suzy V.

    2009-01-01

    Iron is required for survival of mammalian cells. Recently, understanding of iron metabolism and trafficking has increased dramatically, revealing a complex, interacting network largely unknown just a few years ago. This provides an excellent model for systems biology development and analysis. The first step in such an analysis is the construction of a structural network of iron metabolism, which we present here. This network was created using CellDesigner version 3.5.2 and includes reactions occurring in mammalian cells of numerous tissue types. The iron metabolic network contains 151 chemical species and 107 reactions and transport steps. Starting from this general model, we construct iron networks for specific tissues and cells that are fundamental to maintaining body iron homeostasis. We include subnetworks for cells of the intestine and liver, tissues important in iron uptake and storage, respectively; as well as the reticulocyte and macrophage, key cells in iron utilization and recycling. The addition of kinetic information to our structural network will permit the simulation of iron metabolism in different tissues as well as in health and disease. PMID:19381358

  6. Iron Deficiency and Other Types of Anemia in Infants and Children.

    PubMed

    Wang, Mary

    2016-02-15

    Anemia, defined as a hemoglobin level two standard deviations below the mean for age, is prevalent in infants and children worldwide. The evaluation of a child with anemia should begin with a thorough history and risk assessment. Characterizing the anemia as microcytic, normocytic, or macrocytic based on the mean corpuscular volume will aid in the workup and management. Microcytic anemia due to iron deficiency is the most common type of anemia in children. The American Academy of Pediatrics and the World Health Organization recommend routine screening for anemia at 12 months of age; the U.S. Preventive Services Task Force found insufficient evidence to assess the benefits vs. harms of screening. Iron deficiency anemia, which can be associated with cognitive issues, is prevented and treated with iron supplements or increased intake of dietary iron. The U.S. Preventive Services Task Force found insufficient evidence to recommend screening or treating pregnant women for iron deficiency anemia to improve maternal or neonatal outcomes. Delayed cord clamping can improve iron status in infancy, especially for at-risk populations, such as those who are preterm or small for gestational age. Normocytic anemia may be caused by congenital membranopathies, hemoglobinopathies, enzymopathies, metabolic defects, and immune-mediated destruction. An initial reticulocyte count is needed to determine bone marrow function. Macrocytic anemia, which is uncommon in children, warrants subsequent evaluation for vitamin B12 and folate deficiencies, hypothyroidism, hepatic disease, and bone marrow disorders.

  7. In vitro translation of the full-length RNA transcript of figwort mosaic virus (Caulimovirus).

    PubMed

    Ranu, R S; Gowda, S; Scholthof, H; Wu, F C; Shepherd, R J

    1996-01-01

    The circular DNA genome of FMV consists of seven tandemly arranged genes placed successively on a full-length RNA transcript that spans the entire circular viral genome. This transcript is a tentative mRNA for at least five of the six major conserved genes of this virus (genes I-V) that are positioned on this transcript. The sixth major gene (gene VI) is expressed as a separate monocistronic transcript. A long 5'-nontranslated leader (598 nucleotides), a small nonconserved gene (VII), and a short intergenic region (57 nucleotides) precede the five major conserved genes (I through V) on the full-length transcript. A reporter gene (CAT), as a separate cistron or fused in-frame, to viral cistrons in various downstream positions in cloned versions of the viral genome was used in a transcription vector to generate artificial full-length transcripts of FMV. When these mRNAs were translated in vitro (rabbit reticulocyte lysate system), the reporter gene was translated efficiently in all positions. Translation of internal native viral gene positioned on the full-length transcript of FMV was also determined (the gene VI product). These observations suggest that the full-length FMV transcript functions as a polycistronic mRNA in plants. Results are best explained on the basis of translational coupling/relay race model.

  8. Effects of four-week feed restriction on toxicological parameters in beagle dogs

    PubMed Central

    TAKAMATSU, Kazuhiko; YAMASHITA, Hiroyuki; SATAKE, Shigeru; KAZUSA, Katsuyuki; TABATA, Hajime; NISHIKATA, Takahito

    2015-01-01

    This study was conducted to examine any changes caused by feed restriction in dogs to contribute to safety evaluation in toxicity studies. Two male 7-month-old beagle dogs/group were fed 300 (control), 150 (50% of control), or 70 g/animal of diet daily (23% of control) for 4 weeks. Effects of feed restriction, except for clinical signs, were noted depending on the feed dosage in almost all examinations. The principal outcomes were: decreased body weight and water consumption, ECG changes (decreased heart rate and prolonged QTc), and hematopoietic and lymphopoietic suppression (decreased reticulocyte ratio or white blood cell count in hematology, decreased nucleated cell count in bone marrow, decreased erythroid parameters in myelography, and hypocellularity of bone marrow and thymic atrophy in histopathology). In addition, some changes were noted in urinalysis (decreased urine volume and sodium and potassium excretion), blood chemistry (decreased ALP and inorganic phosphorus and increased creatinine), organ weights, and gastric histopathology. These results provide important reference data for distinguishing the primary effects of test compounds from secondary effects of decreased food consumption in toxicity studies in beagle dogs. PMID:25818481

  9. Effect of zinc deficiency of expression of specific mRNAs in rat liver

    SciTech Connect

    Chen, S.J.; Kimball, S.R.; Leure-duPree, A.E.; Jefferson, L.S. )

    1991-03-15

    Retinol is released from the liver bound to a specific transport protein, retinol binding protein (RBP), which binds to transthyretin (TTR) to transport retinol to the retinal pigment epithelium for use in the visual cycle. The synthesis of RBP as well as the transport of vitamin A from the liver is especially sensitive to zinc deficiency (ZD). Impaired hepatic synthesis of RBP has been reported in zinc-deficient rats. In the present study, the effect of ZD on the expression of mRNAs in the liver was examined by isolating total RNA from control, pair-fed, and zinc-deficient rats and translating the RNA in a messenger-dependent reticulocyte lysate. The radiolabeled translation products were analyzed by two-dimensional gel electrophoresis followed by autoradiography. The amounts of 12 of the approximately 200 radiolabeled translation products which could be distinguished were found to be altered in zinc-deficient compare to control samples. To investigate the expression of a specific mRNA, a cDNA to TTR was employed to probe the RNA samples. Slot blot analysis revealed that TTR mRNA was reduced to 57 {plus minus} 14% of the control in pair-fed rats to 29 {plus minus} 19% of control in zinc-deficient rats. The decrease in TTR mRNA is consistent with the observation that serum TTR is decreased during zinc deficiency caused by cirrhosis.

  10. The cytoskeletal binding domain of band 3 is required for multiprotein complex formation and retention during erythropoiesis

    PubMed Central

    Satchwell, Timothy J; Hawley, Bethan R; Bell, Amanda J; Ribeiro, M. Leticia; Toye, Ashley M

    2015-01-01

    Band 3 is the most abundant protein in the erythrocyte membrane and forms the core of a major multiprotein complex. The absence of band 3 in human erythrocytes has only been reported once, in the homozygous band 3 Coimbra patient. We used in vitro culture of erythroblasts derived from this patient, and separately short hairpin RNA-mediated depletion of band 3, to investigate the development of a band 3-deficient erythrocyte membrane and to specifically assess the stability and retention of band 3 dependent proteins in the absence of this core protein during terminal erythroid differentiation. Further, using lentiviral transduction of N-terminally green fluorescent protein-tagged band 3, we demonstrated the ability to restore expression of band 3 to normal levels and to rescue secondary deficiencies of key proteins including glycophorin A, protein 4.2, CD47 and Rh proteins arising from the absence of band 3 in this patient. By transducing band 3-deficient erythroblasts from this patient with band 3 mutants with absent or impaired ability to associate with the cytoskeleton we also demonstrated the importance of cytoskeletal connectivity for retention both of band 3 and of its associated dependent proteins within the reticulocyte membrane during the process of erythroblast enucleation. PMID:25344524

  11. Detection of EPO doping and blood doping: the haematological module of the Athlete Biological Passport.

    PubMed

    Schumacher, Yorck Olaf; Saugy, Martial; Pottgiesser, Torben; Robinson, Neil

    2012-11-01

    The increase of the body's capacity to transport oxygen is a prime target for doping athletes in all endurance sports. For this pupose, blood transfusions or erythropoiesis stimulating agents (ESA), such as erythropoietin, NESP, and CERA are used. As direct detection of such manipulations is difficult, biomarkers that are connected to the haematopoietic system (haemoglobin concentration, reticulocytes) are monitored over time (Athlete Biological Passport (ABP)) and analyzed using mathematical models to identify patterns suspicious of doping. With this information, athletes can either be sanctioned directly based on their profile or targeted with conventional doping tests. Key issues for the appropriate use of the ABP are correct targeting and use of all available information (e.g. whereabouts, cross sectional population data) in a forensic manner. Future developments of the passport include the correction of all concentration-based variables for shifts in plasma volume, which might considerably increase sensitivity. New passport markers from the genomic, proteomic, and metabolomic level might add further information, but need to be validated before integration into the passport procedure. A first assessment of blood data of federations that have implemented the passport show encouraging signs of a decreased blood-doping prevalence in their athletes, which adds scientific credibility to this innovative concept in the fight against ESA- and blood doping.

  12. Blood doping and its detection.

    PubMed

    Jelkmann, Wolfgang; Lundby, Carsten

    2011-09-01

    Hemoglobin mass is a key factor for maximal exercise capacity. Some athletes apply prohibited techniques and substances with intent to increase hemoglobin mass and physical performance, and this is often difficult to prove directly. Autologous red blood cell transfusion cannot be traced on reinfusion, and also recombinant erythropoietic proteins are detectable only within a certain timeframe. Novel erythropoietic substances, such as mimetics of erythropoietin (Epo) and activators of the Epo gene, may soon enter the sports scene. In addition, Epo gene transfer maneuvers are imaginable. Effective since December 2009, the World Anti-Doping Agency has therefore implemented "Athlete Biologic Passport Operating Guidelines," which are based on the monitoring of several parameters for mature red blood cells and reticulocytes. Blood doping may be assumed, when these parameters change in a nonphysiologic way. Hematologists should be familiar with blood doping practices as they may play an important role in evaluating blood profiles of athletes with respect to manipulations, as contrasted with the established diagnosis of clinical disorders and genetic variations.

  13. Effects of glycerol and creatine hyperhydration on doping-relevant blood parameters.

    PubMed

    Polyviou, Thelma P; Easton, Chris; Beis, Lukas; Malkova, Dalia; Takas, Pantazis; Hambly, Catherine; Speakman, John R; Koehler, Karsten; Pitsiladis, Yannis P

    2012-09-01

    Glycerol is prohibited as an ergogenic aid by the World Anti-Doping Agency (WADA) due to the potential for its plasma expansion properties to have masking effects. However, the scientific basis of the inclusion of Gly as a "masking agent" remains inconclusive. The purpose of this study was to determine the effects of a hyperhydrating supplement containing Gly on doping-relevant blood parameters. Nine trained males ingested a hyperhydrating mixture twice per day for 7 days containing 1.0 g·kg(-1) body mass (BM) of Gly, 10.0 g of creatine and 75.0 g of glucose. Blood samples were collected and total hemoglobin (Hb) mass determined using the optimized carbon monoxide (CO) rebreathing method pre- and post-supplementation. BM and total body water (TBW) increased significantly following supplementation by 1.1 ± 1.2 and 1.0 ± 1.2 L (BM, P < 0.01; TBW, P <0.01), respectively. This hyperhydration did not significantly alter plasma volume or any of the doping-relevant blood parameters (e.g., hematocrit, Hb, reticulocytes and total Hb-mass) even when Gly was clearly detectable in urine samples. In conclusion, this study shows that supplementation with hyperhydrating solution containing Gly for 7 days does not significantly alter doping-relevant blood parameters.

  14. Molecular characterization and expression of equine testicular cytochrome P450 aromatase.

    PubMed

    Seralini, Gilles Eric; Tomilin, Alexey; Auvray, Pierrïck; Nativelle-Serpentini, Celine; Sourdaine, Pascal; Moslemi, Safa

    2003-02-20

    We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androstenedione as substrate in a dose-dependent manner. The isolation of testicular aromatase by a new immunoaffinity method demonstrated that the protein could exist either glycosylated or not with a 2 kDa difference. All these results taken together allow new structural studies to progress in the understanding of this cytochrome P450.

  15. Analysis of gene expression on anodic porous alumina microarrays

    PubMed Central

    Nicolini, Claudio; Singh, Manjul; Spera, Rosanna; Felli, Lamberto

    2013-01-01

    This paper investigates the application of anodic porous alumina as an advancement on chip laboratory for gene expressions. The surface was prepared by a suitable electrolytic process to obtain a regular distribution of deep micrometric holes and printed bypen robot tips under standard conditions. The gene expression within the Nucleic Acid Programmable Protein Array (NAPPA) is realized in a confined environment of 16 spots, containing circular DNA plasmids expressed using rabbit reticulocyte lysate. Authors demonstrated the usefulness of APA in withholding the protein expression by detecting with a CCD microscope the photoluminescence signal emitted from the complex secondary antibody anchored to Cy3 and confined in the pores. Friction experiments proved the mechanical resistance under external stresses by the robot tip pens printing. So far, no attempts have been made to directly compare APA with any other surface/substrate; the rationale for pursuing APA as a potential surface coating is that it provides advantages over the simple functionalization of a glass slide, overcoming concerns about printing and its ability to generate viable arrays. PMID:23783000

  16. Polyurethane scaffolds seeded with CD34+ cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors

    PubMed Central

    Severn, Charlotte E.; Macedo, Hugo; Eagle, Mark J.; Rooney, Paul; Mantalaris, Athanasios; Toye, Ashley M.

    2016-01-01

    We describe a 3D erythroid culture system that utilises a porous polyurethane (PU) scaffold to mimic the compartmentalisation found in the bone marrow. PU scaffolds seeded with peripheral blood CD34+ cells exhibit a remarkable reproducibility of egress, with an increased output