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Sample records for reticulocytes

  1. Reticulocytes (image)

    MedlinePlus

    ... the body increases production of red blood cells (RBCs), and sends these cells into the bloodstream before ... Reticulocytes normally make up 1% of the total RBC count, but may exceed levels of 4% when ...

  2. [Renaissance of the reticulocyte].

    PubMed

    Nielsen, O J; Kjaersgaard, E; Karle, H

    1994-09-26

    Flowcytometric enumeration of reticulocytes using RNA-binding fluorochromes, a method both sensitive and reproducible, is rapidly gaining ground. It performs conspicuously better than the traditional manual method of reticulocyte counting, which is unacceptably inaccurate. The reticulocyte count is a valuable indicator of erythropoietic activity, of essential importance in discerning between the aregeneratory anaemias and anaemias accompanied by an adequate, partial or complete compensatory erythropoietic response. The reticulocyte count can be applied to the monitoring of the response to treatment including both allogeneic and autologous bone marrow transplantation, as well as to the evaluation of the response to recombinant human growth factors. The mean fluorescence intensity of reticulocytes enumerated by the flowcytometric analysis, is a variable possessing an independent clinical value for calculation of a reticulocyte production index and an erythropoiesis index. PMID:7985249

  3. 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 ...

  4. Phosphatidylinositol kinase from rabbit reticulocytes

    SciTech Connect

    Tuazon, P.T.; Heng, A.B.W.; Traugh, J.A.

    1986-05-01

    Phosphatidylinositol (PI) kinase was isolated from the postribosomal supernatant of rabbit reticulocytes. This activity was identified by the formation of a product that comigrated with phosphatidylinositol-4-phosphate (PIP) when purified PI was phosphorylated in the presence of (/sup 32/P)ATP and Mg/sup 2 +/. Three major peaks of PI kinase activity were resolved by chromatography on DEAE-cellulose. The first peak eluted at 50-100 mM NaCl together with several serine protein kinases, casein kinase (CK) I and protease activated kinase (PAK) I and II. The PI kinase was subsequently separated from the protein kinases by chromatography on phosphocellulose. The second peak eluted at 125-160 mM NaCl and contained another lipid kinase activity that produced a product which comigrated with phosphatidic acid on thin layer chromatography. The third peak, which eluted at 165-200 mM NaCl, partly comigrated with casein kinase (CK) II and an active protein kinase(s) which phosphorylated mixed histone and histone I. CK II and the histone kinase activities were also separated by chromatography on phosphocelluslose. The different forms of PI kinase were characterized and compared with respect to substrate and salt requirements.

  5. Interaction of human diferric transferrin with reticulocytes.

    PubMed Central

    Huebers, H; Csiba, E; Josephson, B; Huebers, E; Finch, C

    1981-01-01

    Methods have been devised for preparing human transferrin with a different isotope of iron selectively labeling each of the two iron binding sites and for determining the distribution of radioiron among transferrin molecules. When diferric human transferrin was exposed to human or animal reticulocytes, there was an equal contribution of radioiron from the acid-stable and acid-labile sites. In this delivery, both atoms of iron were removed simultaneously from the diferric transferrin molecule, converting it to apotransferrin. At similar iron concentrations the amount of iron delivered by diferric transferrin was twice that delivered by monoferric transferrin. PMID:6264452

  6. Reticulated platelets interfere with flow cytometric reticulocyte counts.

    PubMed

    Ivory, K; Sarria, B; Fairweather-Tait, S J; Hughes, D A

    2007-10-01

    As part of an iron absorption study, we needed to accurately count reticulocytes in the peripheral blood of healthy human volunteers before measuring their enrichment with stable iron isotopes given in an oral dose. Recent studies have suggested the usefulness of reticulocyte counting by flow cytometry, through a combination of differential light scatter and measurement of the stoichiometric binding of thiazole orange (TO) to RNA within the maturing erythrocyte. Using this method we set out to improve the precision of our quantitative analysis by counting more cells, as reticulocytes normally comprise <2% of the red cell population. To ensure exclusion of other cell types, we identified WBCs and platelets with CD16+CD45- allophycocyanin and CD61- phycoerythrin, respectively. After removal of CD16(+) CD45(+) TO(+) WBCs and CD61(+) TO(-) platelets from analysis, the remaining cells were a combination of CD61(-) TO(-) erythrocytes, CD61(-) TO(+) reticulocytes and CD61(+) TO(+) reticulated platelets. Reticulocyte counts were lower after exclusion of CD61(+) TO(+) cells from analysis. They were similarly lower when erythrocyte precursors were positively identified through their glycophorin A expression and TO uptake. We conclude that it is necessary to exclude reticulated platelets from flow cytometric reticulocyte analysis. PMID:17824916

  7. The mechanism of folate transport in rabbit reticulocytes

    PubMed Central

    Bobzien, William F.; Goldman, David

    1972-01-01

    Folate transport in phenylhydrazine-induced rabbit reticulocytes was studied with the non-metabolized folate-analog, methotrexate. The time-course of methotrexate uptake into a mixed population of reticulocytes and mature erythrocytes is a two-component process consisting of a small, but rapid, initial uptake phase followed by a much slower uptake component which remains essentially constant over the period of observation. The velocity of the latter uptake component is directly proportional to the per cent reticulocytes and appears to represent a unidirectional influx of methotrexate into these cells. Uptake of methotrexate into reticulocytes was found to have the following characteristics: (a) temperature sensitivity, Q10 of 4; (b) uptake velocity as a function of the extracellular methotrexate concentration approximated Michaelis-Menten kinetics with a maximum transport velocity of 48 pmoles/min per g dry wt; the extracellular methotrexate level at which the uptake velocity was one-half maximum was 1.4 μM; (c) 5-formyltetrahydrofolate markedly inhibited methotrexate uptake but pteroylglutamic acid inhibition was weak; (d) uptake was stimulated in cells preincubated with 5-formyltetrahydrofolate, indicative of hetero-exchange diffusion; (e) uptake was independent of extracellular sodium but was inhibited by anions including nitrate, phosphate, and glucose-6-phosphate; (f) uptake was enhanced by azide plus iodoacetate. These data indicate that folate transport in rabbit reticulocytes is mediated by a carrier mechanism which disappears with reticulocyte maturation. The mechanism of folate transport in rabbit reticulocytes is qualitatively similar to tumor cells previously studied; both appear to have an energy-dependent mechanism limiting folate uptake, and influx in both is inhibited by structurally unrelated inorganic and organic anions. These studies suggest that circulating pteroylglutamic acid is of little importance in meeting the folate requirements of

  8. Proteolipidic Composition of Exosomes Changes during Reticulocyte Maturation*

    PubMed Central

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

    2011-01-01

    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. PMID:21828046

  9. Evaluation of the Sysmex R-1000. An automated reticulocyte analyzer.

    PubMed

    Tichelli, A; Gratwohl, A; Driessen, A; Mathys, S; Pfefferkorn, E; Regenass, A; Schumacher, P; Stebler, C; Wernli, M; Nissen, C

    1990-01-01

    The new fully automated reticulocyte analyzer, Sysmex R-1000 (TOA Medical Electronics, Kobe, Japan), was evaluated for its routine use in the Hematological Laboratory at the University Hospital Basel, Switzerland. The operating characteristics, such as within-run precision, linearity, and carryover, fulfilled the manufacturer's specifications and are excellent. Correlation with the standard method, manual reticulocyte counting, is linear for normal and high values. For low reticulocyte counts the regression points show a deviation from their linearity. An absolute zero value is not obtained by the R-1000. The R-1000 measures total RNA content of each cell and expresses the value as low fluorescence ratio (LFR), medium fluorescence ratio (MFR), and high fluorescence ratio (HFR). The analysis of this ratio resolves the problem of zero reticulocytes: A fraction of less than 0.002 (0.2%) with an LFR of 100% represents aplasia; a shift of the intensity of fluorescence to HFR heralds regeneration. Results of samples stored at room temperature remain stable and within the range of the within-run precision for up to 12 hours, when stored at 5 degrees C for more than 48 hours. The authors conclude that the R-1000 is easy to operate, fulfills the criteria for accuracy and precision, and is highly suitable for daily routine use in a large central hematologic laboratory. PMID:2294704

  10. Effect of hydroxyurea on immature reticulocyte fraction in sickle cell anemia.

    PubMed

    Bagdasaryan, Robert; Glasser, Lewis; Quillen, Karen; Chaves, Fernando; Xu, Dongsheng

    2007-01-01

    Immature reticulocyte fraction (IRF) is a good indicator of bone marrow erythropoiesis in response to hemolysis or tissue hypoxia and is markedly increased in sickle cell disease (SS). We compared IRF changes in SS patients with those who were treated with hydroxyurea (SS-HU), and those who had concurrent alpha globin gene deletion (SS-(- deletion). Forty-two patients including 16 SS, 16 SS-HU, and 10 SS-alpha-deletion patients were studied. Significant decreases (P <.01) in reticulocyte indices including IRF, the reticulocyte percentage, and absolute reticulocyte count (ARC) were observed in SS-alpha-deletion compared to SS patients. On the other hand, although the reticulocyte percentage (P <.01) and ARC (P <.01) were significantly decreased in SS-HU compared with SS patients, the IRF was persistently elevated in both groups (P = .4), suggesting continuous bone marrow stimulation in SS-HU patients in response to tissue hypoxia. The possible underlying physiological mechanisms are discussed. PMID:17984040

  11. Characteristics of marrow production and reticulocyte maturation in normal man in response to anemia

    PubMed Central

    Hillman, Robert S.

    1969-01-01

    Erythropoiesis in normal man was studied during periods of phlebotomy-induced anemia of varying severity. This study permitted a comparison of marrow production measurements over a wide range of marrow production levels. As long as the serum iron remained above 50 μg/100 ml, measurements of plasma iron turnover provided an excellent index of marrow production at all levels of red cell production. In contrast, the absolute reticulocyte count demonstrated a poor correlation with the other measurements. This was shown to be the result of a prolongation of the time required for circulating reticulocytes to lose their reticulum, which correlated with the severity of the anemia. For the clinical application of the reticulocyte count as a measurement of marrow production, an adjustment must be made for this alteration in the circulating reticulocyte maturation time. PMID:5773082

  12. The ins and outs of reticulocyte maturation revisited: The role of autophagy 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

    2016-03-01

    Autophagy plays an important role in the removal of membrane bound organelles during the last stage of erythropoiesis as the enucleate reticulocyte matures into the erythrocyte. Autophagic vesicles are expelled from the reticulocyte as intact, inside-out, phosphatidylserine (PS) decorated vesicles and are subsequently removed during splenic passage. Failure to remove these vesicles causes the elevation in PS exposed red cells in Sickle Cell Disease. PMID:27046252

  13. The ins and outs of reticulocyte maturation revisited: The role of autophagy in sickle cell disease

    PubMed Central

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

    2016-01-01

    ABSTRACT Autophagy plays an important role in the removal of membrane bound organelles during the last stage of erythropoiesis as the enucleate reticulocyte matures into the erythrocyte. Autophagic vesicles are expelled from the reticulocyte as intact, inside-out, phosphatidylserine (PS) decorated vesicles and are subsequently removed during splenic passage. Failure to remove these vesicles causes the elevation in PS exposed red cells in Sickle Cell Disease. PMID:27046252

  14. Stress reticulocytes lose transferrin receptors by an extrinsic process involving spleen and macrophages.

    PubMed

    Rhodes, Melissa M; Koury, Stephen T; Kopsombut, Prapaporn; Alford, Catherine E; Price, James O; Koury, Mark J

    2016-09-01

    As they mature into erythrocytes during normal erythropoiesis, reticulocytes lose surface transferrin receptors before or concurrently with reticulin. Exosome release accounts for most of the loss of transferrin receptors from reticulocytes. During erythropoietic stress, reticulocytes are released early from hematopoietic tissues and have increased reticulin staining and transferrin receptors. Flow cytometry of dually stained erythrocytes of mice recovering from phlebotomy demonstrated delayed loss of reticulin and transferrin receptors during in vitro maturation compared to in vivo maturation, indicating that an in vivo process extrinsic to the reticulocytes facilitates their maturation. Splenectomy or macrophage depletion by liposomal clodronate inhibited in vivo maturation of reticulocytes and increased the numbers of reticulin-negative, transferrin receptor-positive cells during and after recovery from phlebotomy. This reticulin-negative, transferrin receptor-positive population was rarely found in normal mice. Transmission electron microscopy demonstrated that the reticulin-negative, transferrin receptor-positive cells were elongated and discoid erythrocytes, but they had intracellular and surface structures that appeared to be partially degraded organelles. The results indicate that maturation of circulating stress reticulocytes is enhanced by an extrinsic process that occurs in the spleen and involves macrophage activity. Complete loss of reticulin with incomplete loss of surface transferrin receptors in this process produces a reticulin-negative, transferrin receptor-positive erythrocyte population that has potential utility for detecting prior erythropoietic stresses including bleeding, hemolysis and erythropoietin administration, even after recovery has been completed. Am. J. Hematol. 91:875-882, 2016. © 2016 Wiley Periodicals, Inc. PMID:27194638

  15. Host Reticulocytes Provide Metabolic Reservoirs That Can Be Exploited by Malaria Parasites

    PubMed Central

    Srivastava, Anubhav; Creek, Darren J.; Evans, Krystal J.; De Souza, David; Schofield, Louis; Müller, Sylke; Barrett, Michael P.; McConville, Malcolm J.; Waters, Andrew P.

    2015-01-01

    Human malaria parasites proliferate in different erythroid cell types during infection. Whilst Plasmodium vivax exhibits a strong preference for immature reticulocytes, the more pathogenic P. falciparum primarily infects mature erythrocytes. In order to assess if these two cell types offer different growth conditions and relate them to parasite preference, we compared the metabolomes of human and rodent reticulocytes with those of their mature erythrocyte counterparts. Reticulocytes were found to have a more complex, enriched metabolic profile than mature erythrocytes and a higher level of metabolic overlap between reticulocyte resident parasite stages and their host cell. This redundancy was assessed by generating a panel of mutants of the rodent malaria parasite P. berghei with defects in intermediary carbon metabolism (ICM) and pyrimidine biosynthesis known to be important for P. falciparum growth and survival in vitro in mature erythrocytes. P. berghei ICM mutants (pbpepc-, phosphoenolpyruvate carboxylase and pbmdh-, malate dehydrogenase) multiplied in reticulocytes and committed to sexual development like wild type parasites. However, P. berghei pyrimidine biosynthesis mutants (pboprt-, orotate phosphoribosyltransferase and pbompdc-, orotidine 5′-monophosphate decarboxylase) were restricted to growth in the youngest forms of reticulocytes and had a severe slow growth phenotype in part resulting from reduced merozoite production. The pbpepc-, pboprt- and pbompdc- mutants retained virulence in mice implying that malaria parasites can partially salvage pyrimidines but failed to complete differentiation to various stages in mosquitoes. These findings suggest that species-specific differences in Plasmodium host cell tropism result in marked differences in the necessity for parasite intrinsic metabolism. These data have implications for drug design when targeting mature erythrocyte or reticulocyte resident parasites. PMID:26042734

  16. Free radicals promote in vitro intracellular decay of rabbit reticulocyte and erythrocyte hexokinase

    SciTech Connect

    Stocchi, V.; Biagiarelli, B.; Masat, L.; Palma, F.; Piccoli, G.; Cucchiarini, L. )

    1991-03-15

    The authors studied the behavior of enzymes involved in the glycolytic pathway incubating intact reticulocyte and erythrocyte at 37C in the presence of ascorbic acid and Fe{sup 2+}. The results obtained have shown evidence that among the glycolytic enzymes the hexokinase activity shows a pronounced decay. For this reason the authors have investigated how the chromatographic profile of hexokinase changes after exposure of reticulocytes and erythrocytes to the oxygen-radical generating system in trying to understand the molecular basis of this inactivation. The results obtained have shown a different effect of free radicals on the reticulocyte and erythrocyte hexokinase molecular forms. The analysis of the chromatographic profile was performed using a TSK Gel Toyopearl DEAE 650 S column which allows a complete resolution of the distinct forms of hexokinase together with a complete recovery of the enzyme activities. Concomitantly to the hexokinase decay, there is a fall in the GSH level when intact rabbit erythrocytes and reticulocytes are incubated in presence of iron and ascorbic acid. However, the fall of GSH is significantly higher in the erythrocytes where, after incubation of one hour, it reaches a mean value of 0.3 {mu}moles per ml of cells representing about 10% of the initial value. In the reticulocytes the GSH value, after the same treatment, remain as high as 1.6 {mu}moles per ml of cells. If we consider that the initial value of GSH is almost the same in the erythrocytes are reticulocytes the highest decay rate of hexokinase observed in these latter cells, cannot be related to the fall of the GSH level, but to a possible direct effect of free-radicals on the enzyme.

  17. 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. PMID:3398836

  18. Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance.

    PubMed

    Lin, Jing-Wen; Spaccapelo, Roberta; Schwarzer, Evelin; Sajid, Mohammed; Annoura, Takeshi; Deroost, Katrien; Ravelli, Raimond B G; Aime, Elena; Capuccini, Barbara; Mommaas-Kienhuis, Anna M; O'Toole, Tom; Prins, Frans; Franke-Fayard, Blandine M D; Ramesar, Jai; Chevalley-Maurel, Séverine; Kroeze, Hans; Koster, Abraham J; Tanke, Hans J; Crisanti, Andrea; Langhorne, Jean; Arese, Paolo; Van den Steen, Philippe E; Janse, Chris J; Khan, Shahid M

    2015-06-01

    Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes. PMID:25941254

  19. Replication of Plasmodium in reticulocytes can occur without hemozoin formation, resulting in chloroquine resistance

    PubMed Central

    Lin, Jing-wen; Spaccapelo, Roberta; Schwarzer, Evelin; Sajid, Mohammed; Annoura, Takeshi; Deroost, Katrien; Ravelli, Raimond B.G.; Aime, Elena; Capuccini, Barbara; Mommaas-Kienhuis, Anna M.; O’Toole, Tom; Prins, Frans; Franke-Fayard, Blandine M.D.; Ramesar, Jai; Chevalley-Maurel, Séverine; Kroeze, Hans; Koster, Abraham J.; Tanke, Hans J.; Crisanti, Andrea; Langhorne, Jean; Arese, Paolo; Van den Steen, Philippe E.; Janse, Chris J.

    2015-01-01

    Most studies on malaria-parasite digestion of hemoglobin (Hb) have been performed using P. falciparum maintained in mature erythrocytes, in vitro. In this study, we examine Plasmodium Hb degradation in vivo in mice, using the parasite P. berghei, and show that it is possible to create mutant parasites lacking enzymes involved in the initial steps of Hb proteolysis. These mutants only complete development in reticulocytes and mature into both schizonts and gametocytes. Hb degradation is severely impaired and large amounts of undigested Hb remains in the reticulocyte cytoplasm and in vesicles in the parasite. The mutants produce little or no hemozoin (Hz), the detoxification by-product of Hb degradation. Further, they are resistant to chloroquine, an antimalarial drug that interferes with Hz formation, but their sensitivity to artesunate, also thought to be dependent on Hb degradation, is retained. Survival in reticulocytes with reduced or absent Hb digestion may imply a novel mechanism of drug resistance. These findings have implications for drug development against human-malaria parasites, such as P. vivax and P. ovale, which develop inside reticulocytes. PMID:25941254

  20. Erythropoiesis activity, iron availability and reticulocyte hemoglobinization during treatment with hemodialysis and in subjects with uremia.

    PubMed

    Schoorl, Marianne; Schoorl, Margreet; Nubé, Menso J; Bartels, Piet C M

    2006-01-01

    In hemodialysis subjects correction of anemia is facilitated by combined supplementation of intravenous iron and recombinant human erythropoietin. Reticulocyte hemoglobin content (RET-He) is considered to be an actual indicator reflecting functional iron availability for erythropoiesis. In the present study, interdependence between biochemical analytes reflecting iron status and hemocytometric parameters indicating the degree of hemoglobinization of reticulocytes and red blood cells, respectively, is established. Participants of the study were reference subjects (n=75), subjects with iron deficiency anemia (n=52), subjects with uremia (n=19) and subjects undergoing hemodialysis treatment (n=43). If compared with the reference subjects the results for RBC counts and MCHC are statistically significantly decreased in case of subjects with hemodialysis and uremia, whereas increased results are established with regard to RDW-sd values. Significantly increased results for absolute reticulocyte counts and immature reticulocyte fractions (IRF) are also observed in case of subjects with hemodialysis and uremia. Slightly increased values for the ZPP/heme ratio in combination with elevated reticulocyte count reflect increased activity of erythropoiesis. At a definite MCV value, decreased levels for the hemoglobin content of reticulocytes (RET-He) and hemoglobin content of red blood cells (RBC-He) are observed in case of subjects treated with hemodialysis and in subjects with uremia if compared with identical MCV values of the group of reference subjects. For the ratio of RET-He and RBC-He obviously decreased results are demonstrated in case of subjects with iron deficiency anemia (1.02 +/- 0.08, mean +/- SD), hemodialysis (1.05 +/- 0.05) and uremia (1.02 +/- 0.10) if compared with the group of reference subjects (1.11 +/- 0.02). From the combined interpretation of the MCV values within the reference range and decreased values for RET-He and RET-He/RBC-He ratios, respectively

  1. 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

  2. DDX6 recruits translational silenced human reticulocyte 15-lipoxygenase mRNA to RNP granules

    PubMed Central

    Naarmann, Isabel S.; Harnisch, Christiane; Müller-Newen, Gerhard; Urlaub, Henning; Ostareck-Lederer, Antje; Ostareck, Dirk H.

    2010-01-01

    Erythroid precursor cells lose the capacity for mRNA synthesis due to exclusion of the nucleus during maturation. Therefore, the stability and translation of mRNAs that code for specific proteins, which function in late stages of maturation when reticulocytes become erythrocytes, are controlled tightly. Reticulocyte 15-lipoxygenase (r15-LOX) initiates the breakdown of mitochondria in mature reticulocytes. Through the temporal restriction of mRNA translation, the synthesis of r15-LOX is prevented in premature cells. The enzyme is synthesized only in mature reticulocytes, although r15-LOX mRNA is already present in erythroid precursor cells. Translation of r15-LOX mRNA is inhibited by hnRNP K and hnRNP E1, which bind to the differentiation control element (DICE) in its 3′ untranslated region (3′UTR). The hnRNP K/E1-DICE complex interferes with the joining of the 60S ribosomal subunit to the 40S subunit at the AUG. We took advantage of the inducible human erythroid K562 cell system that fully recapitulates this process to identify so far unknown factors, which are critical for DICE-dependent translational regulation. Applying RNA chromatography with the DICE as bait combined with hnRNP K immunoprecipitation, we specifically purified the DEAD-box RNA helicase 6 (DDX6) that interacts with hnRNP K and hnRNP E1 in a DICE-dependent manner. Employing RNA interference and fluorescence in situ hybridization, we show that DDX6 colocalizes with endogenous human (h)r15-LOX mRNA to P-body–like RNP granules, from which 60S ribosomal subunits are excluded. Our data suggest that in premature erythroid cells translational silencing of hr15-LOX mRNA is maintained by DDX6 mediated storage in these RNP granules. PMID:20884783

  3. Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain.

    PubMed

    Han, Jin-Hee; Lee, Seong-Kyun; Wang, Bo; Muh, Fauzi; Nyunt, Myat Htut; Na, Sunghun; Ha, Kwon-Soo; Hong, Seok-Ho; Park, Won Sun; Sattabongkot, Jetsumon; Tsuboi, Takafumi; Han, Eun-Taek

    2016-01-01

    The Plasmodium vivax reticulocyte-binding protein (RBP) family was identified based on the annotation of adhesive ligands in the P. vivax genome. Reticulocyte-specific interactions with the PvRBPs (PvRBP1 and PvRBP2) were previously reported. Plasmodium falciparum reticulocyte-binding protein homologue 4 (PfRh4, a homologue of PvRBP1) was observed to possess erythrocyte-binding activity via complement receptor 1 on the erythrocyte surface. However, the reticulocyte-binding mechanisms of P. vivax are unclear because of the large molecular mass of PvRBP1 (>326 kDa) and the difficulty associated with in vitro cultivation. In the present study, 34 kDa of PvRBP1a (PlasmoDB ID: PVX_098585) and 32 kDa of PvRBP1b (PVX_098582) were selected from a 30 kDa fragment of PfRh4 for reticulocyte-specific binding activity analysis. Both PvRBP1a and PvRBP1b were found to be localized at the microneme in the mature schizont-stage parasites. Naturally acquired immune responses against PvRBP1a-34 and PvRBP1b-32 were observed lower than PvDBP-RII. The reticulocyte-specific binding activities of PvRBP1a-34 and PvRBP1b-32 were significantly higher than normocyte binding activity and were significantly reduced by chymotrypsin treatment. PvRBP1a and 1b, bind to reticulocytes and that this suggests that these ligands may have an important role in P. vivax merozoite invasion. PMID:27244695

  4. Identification of a reticulocyte-specific binding domain of Plasmodium vivax reticulocyte-binding protein 1 that is homologous to the PfRh4 erythrocyte-binding domain

    PubMed Central

    Han, Jin-Hee; Lee, Seong-Kyun; Wang, Bo; Muh, Fauzi; Nyunt, Myat Htut; Na, Sunghun; Ha, Kwon-Soo; Hong, Seok-Ho; Park, Won Sun; Sattabongkot, Jetsumon; Tsuboi, Takafumi; Han, Eun-Taek

    2016-01-01

    The Plasmodium vivax reticulocyte-binding protein (RBP) family was identified based on the annotation of adhesive ligands in the P. vivax genome. Reticulocyte-specific interactions with the PvRBPs (PvRBP1 and PvRBP2) were previously reported. Plasmodium falciparum reticulocyte-binding protein homologue 4 (PfRh4, a homologue of PvRBP1) was observed to possess erythrocyte-binding activity via complement receptor 1 on the erythrocyte surface. However, the reticulocyte-binding mechanisms of P. vivax are unclear because of the large molecular mass of PvRBP1 (>326 kDa) and the difficulty associated with in vitro cultivation. In the present study, 34 kDa of PvRBP1a (PlasmoDB ID: PVX_098585) and 32 kDa of PvRBP1b (PVX_098582) were selected from a 30 kDa fragment of PfRh4 for reticulocyte-specific binding activity analysis. Both PvRBP1a and PvRBP1b were found to be localized at the microneme in the mature schizont-stage parasites. Naturally acquired immune responses against PvRBP1a-34 and PvRBP1b-32 were observed lower than PvDBP-RII. The reticulocyte-specific binding activities of PvRBP1a-34 and PvRBP1b-32 were significantly higher than normocyte binding activity and were significantly reduced by chymotrypsin treatment. PvRBP1a and 1b, bind to reticulocytes and that this suggests that these ligands may have an important role in P. vivax merozoite invasion. PMID:27244695

  5. 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. PMID:25868528

  6. Malnutrition and infection influence the peripheral blood reticulocyte micronuclei frequency in children.

    PubMed

    Cervantes-Ríos, Elsa; Ortiz-Muñiz, Rocío; Martínez-Hernández, Ana Lidia; Cabrera-Rojo, Lilián; Graniel-Guerrero, Jaime; Rodríguez-Cruz, Leonor

    2012-03-01

    Malnutrition is a serious public health problem that affects approximately one third of all children. Developing countries have the highest incidence of malnourished children, and approximately 60% of deaths that occur in children under five are directly related to malnutrition and associated diseases. The relationship between malnutrition and genetic damage has been widely studied in humans and animal models. The micronucleus (MN) assay is useful in detecting chromosome damage induced by several factors. The aim of this study was to evaluate the effects of infection and malnutrition on the frequency of MN in erythrocytes from the peripheral blood of well-nourished, uninfected (WN) and well-nourished, infected (WNI) children, and moderately malnourished (UNM) and severely malnourished (UNS) children, both with infection, using a flow cytometric analysis technique. The percentage of reticulocytes (RETs) was significantly higher (1.5-fold) in WNI children than well-nourished controls. In addition, the UNS group had a 2.2-fold increase in the percentage of RETs compared to the WNI group. The frequency of micronucleated reticulocytes (MN-RETs) was 2.5 times greater, in WNI group compared to the WN group. These frequencies were significantly higher (1.7- and 2.1-fold) in UNM and UNS, respectively, compared to the WNI group. The results suggest that infection and malnutrition induce DNA damage in children. PMID:22119781

  7. The effect of the iron saturation of transferrin on its binding and uptake by rabbit reticulocytes.

    PubMed Central

    Young, S P; Bomford, A; Williams, R

    1984-01-01

    Polyacrylamide-gel electrophoresis in urea was used to prepare the four molecular species of transferrin:diferric transferrin, apotransferrin and the two monoferric transferrins with either the C-terminal or the N-terminal metal-binding site occupied. The interaction of these 125I-labelled proteins with rabbit reticulocytes was investigated. At 4 degrees C the average value for the association constant for the binding of transferrin to reticulocytes was found to increase with increasing iron content of the protein. The association constant for apotransferrin binding was 4.6 X 10(6)M-1, for monoferric (C-terminal iron) 2.5 X 10(7)M-1, for monoferric (N-terminal iron) 2.8 X 10(7)M-1 and for diferric transferrin, 1.1 X 10(8)M-1. These differences in the association constants did not affect the processing of the transferrin species by the cells at 37 degrees C. Accessibility of the proteins to extracellular proteinase indicated that the transferrin was internalized by the cells regardless of the iron content of the protein, since in each case 70% was inaccessible. Cycling of the cellular receptors may also occur in the absence of bound transferrin. PMID:6743230

  8. 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

  9. Using the Hemoglobin Content of Reticulocytes (RET-He) to Evaluate Anemia in Patients With Cancer

    PubMed Central

    Peerschke, Ellinor I. B.; Pessin, Melissa S.; Maslak, Peter

    2016-01-01

    Objectives Evaluation of anemia, particularly iron deficiency, in patients with cancer is difficult. This study examined using the hemoglobin content of reticulocytes (RET-He) to rule out iron deficiency, as defined by serum iron studies (transferrin saturation <20%, serum iron <40 µg/ dL, and ferritin <100 ng/mL), in an unselected cancer patient population. Methods Patients were entered into the study based on the existence of concurrent laboratory test requests for CBC and serum iron studies. Results Using a threshold of 32 pg/cell, RET-He ruled out iron deficiency with a negative predictive value (NPV) of 98.5% and 100%, respectively, in the study population (n = 209) and in a subpopulation of patients with low reticulocyte counts (n = 19). In comparison, the NPV of traditional CBC parameters (hemoglobin, <11 g/dL; mean corpuscular volume, <80 fL) was only 88.5%. Conclusions These results support the use of RET-He in the evaluation of iron deficiency in a cancer care setting. PMID:25239418

  10. Enumeration of micronucleated CD71-positive human reticulocytes with a single-laser flow cytometer.

    PubMed

    Dertinger, Stephen D; Torous, Dorothea K; Hall, Nikki E; Murante, Francis G; Gleason, Sarah E; Miller, Richard K; Tometsko, Carol R

    2002-03-25

    The extreme rarity of micronucleated reticulocytes (RETs) in the peripheral blood of non-splenectomized humans has precluded facile enumeration of these cells, as well as evaluation of this endpoint as an index of cytogenetic damage. In this report, we describe a high-throughput, single-laser flow cytometric system for scoring the incidence of micronuclei (MN) in newly formed human RETs. The procedure is based on an immunochemical reagent that differentially labels the most immature fraction of RETs from mature erythrocytes based on the expression level of the transferrin receptor (also known as CD71). The resolution of four erythrocyte populations (young RETs and mature erythrocytes, with and without MN) was achieved for human blood cells treated with phycoerythrin-conjugated anti-CD71, RNase, and either SYTOX Green or SYBR Green I nucleic acid dyes. Anti-glycophorin A labeling of erythroid cells (CyChrome conjugate) was also incorporated into the staining procedure to ensure that debris or other potential artifacts did not adversely impact the analyses. Instrument calibration procedures utilizing malaria-infected rodent erythrocytes were also developed, and are described. Using this analytical system, blood samples from 10 healthy non-splenectomized human volunteers were analyzed for micronucleus frequencies with a single-laser flow cytometer. Average micronucleus frequencies in the mature and most immature fraction of RETs were 0.016 and 0.19%, respectively. Blood samples from three healthy splenectomized volunteers were also evaluated. As expected, these samples exhibited higher micronucleus frequencies in the mature subset of erythrocytes (range 0.03-0.18%). The resulting data suggest that MN can be quantified in human erythrocyte populations with a single-laser flow cytometer, and that the frequency of MN cells in the youngest reticulocyte population approaches values expected in the absence of splenic selection against MN-erythrocytes. This high throughput

  11. 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

  12. Ret-Y a measure of reticulocyte size: a sensitive indicator of iron deficiency anemia.

    PubMed

    Kickler, T S; Borowitz, M J; Thompson, R E; Charintranont, N; Law, R

    2004-12-01

    In this study the size of reticulocytes was measured, reticulocyte-Y (Ret-Y), to distinguish iron deficiency anemia from the anemia of chronic disease using a Sysmex XE2100 cell counter. We evaluated this parameter prospectively in 100 patients seen for the evaluation of anemia. A clinical diagnosis of iron deficiency anemia or anemia of chronic disease was made on the basis of a complete blood count, examination of the peripheral smear, and serum ferritin along with a history and physical examination. We analyzed the sensitivity and specificity of the Ret-Y in relationship to the clinical diagnosis. We also measured serum transferrin receptor levels to use as the gold standard laboratory test for iron deficiency against which we compared the Ret-Y. In 40 normal individuals with normal serum ferritin and transferrin receptor levels the mean Ret-Y was 1874 +/- 178 (1 SD). The mean Ret-Y in the anemia of chronic disease group (n=62) was 1722 +/- 162, not significantly different from normal. The mean Ret-Y value among iron-deficient patients (n=38), was 1407 +/- 136 (P <0.01 vs. the anemia of chronic disease group's Ret-Y value). Receiver operator curves showed that Ret-Y correlated closely to the serum transferrin receptor and was superior to the mean corpuscular volume, and ferritin level, in differentiating the type of anemia. The Ret-Y parameter has the highest overall sensitivity and specificity of the panel of tests routinely used in differentiating iron deficiency anemia from anemia of chronic disease. PMID:15596002

  13. In Vivo Evaluation of the Genotoxic Effects of Gonadotropins on Rat Reticulocytes

    PubMed Central

    Duran, Bulent; Koc, Onder; Ozdemirci, Safak; Topcuoglu, Ata; Ozdemir, Ozturk

    2011-01-01

    Background Gonadotropins, as ovulation-inducing drugs, have been used widely to treat infertility. An epidemiologic correlation between infertility therapy and ovarian cancer development has been reported. However, the effect of gonadotropins in the formation of reproductive tract cancers is controversial. Objective The aim of the study was to determine the in vivo genotoxic effects of gonadotropins on rat reticulocytes. Methods In this prospective, randomized, controlled study, rats were randomly assigned to 1 of 5 groups. The calculated rat doses of 0.65 human menopausal gonadotropin (hMG), 0.95 hMG, 0.65 follitropin beta (FB), 0.95 FB, or normal saline (control group) were injected, respectively. These calculated rat doses (U/g) are based on average human gonadotropin doses of 150 and 225 IU/d for a 70-kg woman given in 2-mL saline (the control group received 2 mL of saline). Injections were administered once per day for 5 days, followed by 5 days of rest. Each treatment was repeated for 6 estrus cycles in the rats for a total of 12 estrus cycles. Six months after the last day of the 12th cycle, the rats were euthanized. Bone marrow tissues were removed, and pluripotent reticulocyte cells with micronuclei, nuclear buds, and binuclear abnormalities were analyzed using an in situ micronuclei assay under light microscopy. The proportion of micronucleated cells, cells with anaphase bridge, nuclear buds, and other nuclear abnormalities were measured. Results The number of cells with nuclear buds and binuclear abnormalities in the hMG 225 and FB 225 groups was significantly higher (P < 0.05) than that from the hMG 150, FB 150, and control groups in the cytogenetic analysis of bone marrow stem cells. An increased rate of genotoxicity in all gonadotropin groups versus that of placebo was found. Conclusion In rats, the micronucleus genotoxicity assay suggests a dose-dependent gonadotropin effect on genomic instability in bone marrow stem cells in vivo. PMID:24648576

  14. Comparison of the Proteome of Adult and Cord Erythroid Cells, and Changes in the Proteome Following Reticulocyte Maturation.

    PubMed

    Wilson, Marieangela C; Trakarnsanga, Kongtana; Heesom, Kate J; Cogan, Nicola; Green, Carole; Toye, Ashley M; Parsons, Steve F; Anstee, David J; Frayne, Jan

    2016-06-01

    Cord blood stem cells are an attractive starting source for the production of red blood cells in vitro for therapy because of additional expansion potential compared with adult peripheral blood progenitors and cord blood banks usually being more representative of national populations than blood donors. Consequently, it is important to establish how similar cord RBCs are to adult cells. In this study, we used multiplex tandem mass tag labeling combined with nano-LC-MS/MS to compare the proteome of adult and cord RBCs and reticulocytes. 2838 unique proteins were identified, providing the most comprehensive compendium of RBC proteins to date. Using stringent criteria, 1674 proteins were quantified, and only a small number differed in amount between adult and cord RBC. We focused on proteins critical for RBC function. Of these, only the expected differences in globin subunits, along with higher levels of carbonic anhydrase 1 and 2 and aquaporin-1 in adult RBCs would be expected to have a phenotypic effect since they are associated with the differences in gaseous exchange between adults and neonates. Since the RBC and reticulocyte samples used were autologous, we catalogue the change in proteome following reticulocyte maturation. The majority of proteins (>60% of the 1671 quantified) reduced in abundance between 2- and 100-fold following maturation. However, ∼5% were at a higher level in RBCs, localized almost exclusively to cell membranes, in keeping with the known clearance of intracellular recycling pools during reticulocyte maturation. Overall, these data suggest that, with respect to the proteome, there is no barrier to the use of cord progenitors for the in vitro generation of RBCs for transfusion to adults other than the expression of fetal, not adult, hemoglobin. PMID:27006477

  15. 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. PMID:26323838

  16. Haem disorder in recombinant- and reticulocyte-derived haemoglobins: evidence for stereoselective haem insertion in eukaryotes.

    PubMed Central

    Mathews, A J; Brittain, T

    2001-01-01

    We have used NMR spectroscopy to measure haem disorder in adult human haemoglobin (HbA) obtained from mature erythrocyte cells and from yeast expressing recombinant HbA. Reticulocyte-derived HbA contained much higher levels of haem disorder (11% alpha- and 28% beta-subunit disorder) than observed for HbA from mature erythrocytes (1.5% alpha- and 8% beta-subunit disorder). Thus, unlike in vitro combination of haem and apoHb, biosynthetic haem insertion is not random with respect to orientation, but appears to show stereoselectivity. Recombinant HbA isolated from yeast showed 32% alpha- and 45% beta-subunit haem disorder. These levels relaxed to their equilibrium positions after incubating the Hb in the ferric form. Recombinant embryonic human Hbs showed less haem disorder than recombinant HbA. The levels of haem disorder in embryonic Hbs zeta(2)epsilon(2) and zeta(2)gamma(2) appear to have their equilibrium values. We propose that, in eukaryotes, in vivo haem insertion occurs via both co-translational mechanisms and insertion via semiHb-beta. PMID:11415464

  17. Retrospective study of reticulocyte indices as indicators of iron-restricted erythropoiesis in dogs with immune-mediated hemolytic anemia.

    PubMed

    Schaefer, Deanna M W; Stokol, Tracy

    2016-05-01

    Iron-restricted erythropoiesis can occur as a result of an absolute deficiency of iron stores, inflammation-mediated iron sequestration, or functional iron deficiency (in which release of stored iron is slower than the iron uptake by erythroid precursors during intense erythropoiesis). Reticulocyte indices are used to identify iron-restricted erythropoiesis, with the reticulocyte hemoglobin content (CHr) being the most commonly used index in human patients. Dogs with immune-mediated hemolytic anemia (IMHA) may have iron-restricted erythropoiesis caused by inflammation-mediated iron sequestration and/or functional iron deficiency, which could contribute to anemia severity and blunt the regenerative response in some dogs. To investigate this possibility, reticulocyte indices were examined retrospectively in 14 dogs (2-15 years of age; 9 spayed females, 1 intact female, 4 neutered males) with IMHA, and no clinical evidence of blood loss was found to suggest absolute iron deficiency. Five dogs (34%) had CHr below the preestablished lower reference limit (24.5 pg), and hematocrit was significantly lower in these dogs (p = 0.042, nonpaired t-test). Our results suggest that some dogs with IMHA may have iron-restricted erythropoiesis as a result of functional iron deficiency, inflammation-mediated iron sequestration, or (less likely) absolute iron deficiency. Further study is warranted to evaluate if dogs with IMHA may benefit from parenteral iron therapy. PMID:27034340

  18. Absolute Reticulocyte Count Acts as a Surrogate for Fetal Hemoglobin in Infants and Children with Sickle Cell Anemia

    PubMed Central

    Meier, Emily Riehm; Byrnes, Colleen; Weissman, Maxine; Lee, Y. Terry; Miller, Jeffery L.

    2015-01-01

    Hemoglobin switching is largely complete in humans by six months of age. Among infants with sickle cell anemia (HbSS, SCA), reticulocytosis begins early in life as fetal hemoglobin (HbF) is replaced by sickle hemoglobin (HbS). The objective of this study was to determine if absolute reticulocyte count (ARC) is related to HbF levels in a cohort of pediatric SCA patients. A convenience sample of 106 children with SCA between the ages of 1 month and 20 years who were not receiving hydroxyurea or monthly blood transfusions were enrolled in this observational study. Hematologic data, including ARC and HbF levels, were measured at steady state. F-cells were enumerated by flow cytometry. Initial studies compared infants with ARC greater than or equal to 200 K/μL (ARC ≥ 200) based upon the previously reported utility of this threshold as a predictive marker for SCA severity. Mean HbF and F-cell levels were significantly lower in the ARC ≥ 200 group when compared to the ARC < 200 group. Both HbF and F-cell percentages were negatively correlated to ARC in infants and in children between the ages of 1 and 9 years. However, the inverse relationship was lost after the age of 10 years. Overall, decreased expression and distribution of HbF during childhood SCA is well-correlated with increased reticulocyte production and release into the peripheral blood. As such, these data further support the clinical use of reticulocyte enumeration as a disease severity biomarker for childhood sickle cell anemia. PMID:26366562

  19. Effects of a 12-day "live high, train low" camp on reticulocyte production and haemoglobin mass in elite female road cyclists.

    PubMed

    Ashenden, M J; Gore, C J; Martin, D T; Dobson, G P; Hahn, A G

    1999-10-01

    The aim of this study was to document the effect of "living high, training low" on the red blood cell production of elite female cyclists. Six members of the Australian National Women's road cycling squad slept for 12 nights at a simulated altitude of 2650 m in normobaric hypoxia (HIGH), while 6 team-mates slept at an altitude of 600 m (CONTROL). HIGH and CONTROL subjects trained and raced as a group throughout the 70-day study. Baseline levels of reticulocyte parameters sensitive to changes in erythropoeisis were measured 21 days and 1 day prior to sleeping in hypoxia (D1 and D20, respectively). These measures were repeated after 7 nights (D27) and 12 nights (D34) of simulated altitude exposure, and again 15 days (D48) and 33 days (D67) after leaving the altitude house. There was no increase in reticulocyte production, nor any change in reticulocyte parameters in either the HIGH or CONTROL groups. This lack of haematological response was substantiated by total haemoglobin mass measures (CO-rebreathing), which did not change when measured on D1, D20, D34 or D67. We conclude that in elite female road cyclists, 12 nights of exposure to normobaric hypoxia (2650 m) is not sufficient to either stimulate reticulocyte production or increase haemoglobin mass. PMID:10502082

  20. 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

  1. Neurospora crassa glutamine synthetase. Translation of specific messenger ribonucleic acid in a cell-free system derived from rabbit reticulocytes.

    PubMed

    Palacios, R; Campomanes, M; Quinto, C

    1977-05-10

    The total reticulocyte lysate cell-free protein-synthesizing system was incubated in the presence of Neurospora crassa RNA. With the aid of an antibody directed against purified N. crassa glutamine synthetase, the synthesis of a specific protein was detected. This protein precipitates with antiglutamine synthetase using both direct and indirect procedures, migrates with the same molecular weight as the monomer of N. crassa glutamine synthetase when subjected to acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and chromatographs as N. crassa glutamine synthetase on anthranilate-bound Sepharose. These data indicate the translation of the mRNA that codes for N. crassa glutamine synthetase. This RNA behaves as poly(A)-containing material when fractionated on oly(U)-Sepha-rose. PMID:16013

  2. Validating high-throughput micronucleus analysis of peripheral reticulocytes for radiation biodosimetry: benchmark against dicentric and CBMN assays in a mouse model.

    PubMed

    Chen, Yuhchyau; Tsai, Ying; Nowak, Irena; Wang, Nancy; Hyrien, Ollivier; Wilkins, Ruth; Ferrarotto, Catherine; Sun, Hongliang; Dertinger, Stephen D

    2010-02-01

    Automation of radiation biodosimetry is one of the top priority tasks considered by the Office of Science and Technology Policy and the Homeland Security Council in preparation for the nation's readiness for a possible radionuclear terrorist attack. The Center for Biophysical Assessment and Risk Management Following Irradiation, a consortium of researchers and institutions centered at the University of Rochester, has been investigating automated scoring of radiation-induced micronucleus formation in reticulocytes for high-throughput radiation biodosimetry. The collaborative project is based on a commercially-available product by Litron Laboratories in Rochester, New York. The study was designed to validate the flow-cytometry based analysis of micronucleated reticulocyte expression for radiation biodosimetry by benchmarking against the standard lymphocyte-based biodosimetry methods in a mouse model. C57B1/6 mice and C3H mice were exposed to Cs total-body radiation from 0-3 Gy. Blood samples were subsequently analyzed for CD71+ micronucleated reticulocyte and reticulocyte frequencies by flow cytometry. Results showed a linear dose-response of MN-RET up to 1 Gy for C57B1/6 and 2 Gy for C3H mice. On the other hand, robust and good dose-response curves were obtained with lymphocyte-based dicentric assay and cytokinesis-block micronucleus assay up to 3 Gy. High-throughput, automated analyses of micronucleated reticulocytes is a sensitive and reproducible method for detecting recent radiation exposure. In mice, the dose range of detection is useful up to 1 Gy (C57Bl/6) and 2 Gy (C3H) but not reliable beyond these dose limits. The utilization of this automated analysis for human radiation biodosimetry is currently under investigation. PMID:20065686

  3. Genetic changes during laboratory propagation: copy number At the reticulocyte-binding protein 1 locus of Plasmodium falciparum.

    PubMed

    Nair, Shalini; Nkhoma, Standwell; Nosten, François; Mayxay, Mayfong; French, Neil; Whitworth, Jim; Anderson, Tim

    2010-08-01

    Comparative genomic hybridization studies have revealed elevated copy number (CN) at the reticulocyte-binding protein 1 gene (PfRh1) in fast growing lab-adapted parasites, while genetic manipulation demonstrates a causal link between cell invasion and PfRh1 CN. We therefore examined PfRh1 copy number variation (CNV) in 202 single clone parasite isolates from four countries to quantify the extent of CNV within natural populations. Surprisingly, we found that no natural parasite infections showed elevated CN. In contrast, 4/28 independent laboratory reference strains show elevated CN. One possibility is that amplification of PfRh1 (or neighboring loci) is selected during laboratory culture. In the case of FCR3 group of parasites, clone trees show that PfRh1 amplification arose in laboratory lines following establishment in culture. These data show that CNV at PfRh1 is rare or non-existent in natural populations, but can arise during laboratory propagation. We conclude that PfRh1 CNV is not an important determinant of gene expression, cell invasion or growth rate in natural parasite populations. PMID:20363264

  4. Absorption and induction of micronucleated peripheral reticulocytes in mice after oral administration of fragrant hydroxyfuranones generated in the Maillard reaction.

    PubMed

    Hiramoto, K; Kato, T; Takahashi, Y; Yugi, K; Kikugawa, K

    1998-07-01

    Fragrant hydroxyfuranone and dihydroxypyranone derivatives generated in the Maillard reaction of sugars and amino acids are detected in various processed foods and have been shown active to break DNA single-strand in the in vitro studies. In the present study, absorption of 2,5-dimethyl-4-hydroxy-3(2 H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2 H)-furanone (HEMF), both found in soy sauce, into plasma after a single intraperitoneal or oral administration at doses of 0.5-1.0 gkg-1 to mice was examined. Both compounds appeared in plasma 15 min after intraperitoneal administration and disappeared 2 h after the administration. They appeared in plasma 5 min after oral administration, reached maximum after 15-45 min, and gradually disappeared after 2 h, indicating that they are absorbed by the digestive tract. Both DMHF and HEMF induced micronucleated reticulocytes (MNRETs) in mouse peripheral blood in a dose-dependent manner after oral administration. The results indicate that DMHF and HEMF can cause genetic damage after oral administration. PMID:9711264

  5. Differences in erythrocyte receptor specificity of different parts of the Plasmodium falciparum reticulocyte binding protein homologue 2a.

    PubMed

    Gunalan, Karthigayan; Gao, Xiaohong; Liew, Kingsley J L; Preiser, Peter R

    2011-08-01

    The Plasmodium falciparum reticulocyte-binding-like protein homologue (RH) and erythrocyte binding-like (EBL) protein families play important roles during invasion, though their exact roles are not clear. Both EBL and RH proteins are thought to directly bind different receptors on the surface of the erythrocyte, and the binding properties for a number of EBLs and RHs have been described. While P. falciparum RH1 (PfRH1) and PfRH4 have been shown to act directly in two alternative invasion pathways used by merozoites, the functions of PfRH2a and PfRH2b during invasion are less defined. Here, using monoclonal antibodies raised against a unique region of PfRH2a, we show that PfRH2a moves from the rhoptry neck to the moving junction during merozoite invasion. The movement of PfRH2a to the junction is independent of the invasion pathway used by the merozoite, suggesting an additional function of the protein that is independent of receptor binding. We further show that PfRH2a is processed both in the schizont and during invasion, resulting in proteins with different erythrocyte binding properties. Our findings suggest that PfRH2a and, most likely, the other members of the RH family, depending on their processing stage, can engage different receptors at different stages of the invasion process. PMID:21628513

  6. A study of the alkaline hydrolysis of fractionated reticulocyte ribosomal ribonucleic acid and its relevance to secondary structure

    PubMed Central

    Cox, R. A.; Gould, Hannah J.; Kanagalingam, K.

    1968-01-01

    1. RNA isolated from the sub-units of rabbit reticulocyte ribosomes was hydrolysed by 0·4n-potassium hydroxide at 20°. The probability of main-chain scission was calculated from the number-average chain length, which was obtained from S25,w in 0·01m-phosphate buffer. 2. The fraction, f, of the original secondary structure that the fragments re-formed at neutral pH in 4m-guanidinium chloride, as well as in 0·01m- and 0·1m-phosphate buffer, was derived from changes in extinction over the range 220–310mμ on thermal denaturation. 3. The secondary structure of RNA is regarded as an assembly of hairpin loops each of 2N+b residues on average, where N is the number of base-paired residues and b is the number of unpaired residues. 4. If chain scission takes place at random then 2N+b=logf/log(1–p). 5. For RNA from the smaller sub-unit 2N+b was estimated as 25±5 residues, compared with 30±5 residues for the less stable species and 35±5 residues for the more stable species of hairpin loop of RNA from the larger sub-unit. PMID:5639928

  7. Protein synthesis in brine shrimp embryos and rabbit reticulocytes. The effect of Mg2+ on binary (eukaryotic initiation factor 2 X GDP) and ternary (eukaryotic initiation factor 2 X GTP X met-tRNAf) complex formation.

    PubMed

    Mehta, H B; Woodley, C L; Wahba, A J

    1983-03-25

    We have prepared eukaryotic initiation factor 2 (eIF-2) from rabbit reticulocytes and Artemia embryos and studied the effect of Mg2+ on binary (eIF-2 X GDP) and ternary (eIF-2 X GTP X Met-tRNAf) complex formation. Under conditions where Mg2+ inhibits Met-tRNAf binding to reticulocyte eIF-2, ternary complex formation with Artemia eIF-2 is not inhibited. Similarly, the formation of eIF-2 X GDP with Artemia eIF-2 is stimulated by Mg2+, whereas the corresponding reticulocyte binary complex is strongly inhibited. In the presence of 1 mM Mg2+, the isolated Artemia eIF-2 X GDP complex is stable in the absence of any added nucleotide, but readily exchanges bound GDP for free GTP. However, the reticulocyte eIF-2 X GDP complex is significantly more stable in the presence of GTP, and nucleotide exchange is dependent upon the addition of a factor isolated from either the postribosomal supernatant or the high salt wash of rabbit reticulocyte ribosomes. This factor also stimulates Met-tRNAf binding to both Artemia and reticulocyte eIF-2. PMID:6550599

  8. Phosphorylation of five aminoacyl-tRNA synthetases in reticulocytes and identification of the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver

    SciTech Connect

    Pendergast, A.M.; Traugh, J.A.

    1986-05-01

    Five aminoacyl-tRNA synthetases in the high molecular weight complex were phosphorylated in rabbit reticulocytes following labeling with /sup 32/P. The five synthetases phosphorylated were the glutamyl-, glutaminyl-, lysyl-, aspartyl- and methionyl-tRNA synthetases. In addition, a 37,000 dalton protein, associated with the synthetase complex and tentatively identified as casein kinase I, was also phosphorylated in intact cells. Phosphoamino acid analysis of the proteins indicated all of the phosphate was on seryl residues. Incubation of reticulocytes with /sup 32/P in the presence of 8-bromo-cAMP and o, the 3-isobutyl-1-methylxanthine resulted in a six-fold increase in phosphorylation of the glutaminyl-tRNA synthetase, a two-fold increase in phosphorylation of the aspartyl-tRNA synthetase, and a 50 to 60% decrease in phosphorylation of the glutamyl-, methionyl- and lysyl-tRNA synthetases and the M/sub r/ 37,000 protein. When the site(s) on the glutaminyl-tRNA synthetase phosphorylated in response to 8-bromo-cAMP was analyzed by two-dimensional tryptic phosphopeptide mapping, a single phosphopeptide was observed which was identical to that obtained in vitro upon phosphorylation with the cAMP-dependent protein kinase. Also, the authors identify here, the protein kinases phosphorylating threonyl-tRNA synthetase from rat liver. They are protease activated kinase I, the cAMP-dependent protein kinase and protein kinase C.

  9. The effect of in vivo resveratrol supplementation in irradiated mice on the induction of micronuclei in peripheral blood and bone marrow reticulocytes.

    PubMed

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

    2016-07-01

    The aim of the study was to investigate how coadministration of resveratrol (RSV) at different time after the start of irradiation influences the frequency of micronuclei (MN) in reticulocytes of bone marrow and peripheral blood, and if the RSV supplementation after termination of irradiation may influence the recovery process of damaged cells. Coadministration of RSV with 1-day delay after 1 Gy irradiation significantly decreased the levels of MN in bone marrow and in peripheral blood, whereas with 1-week delay, only in bone marrow reticulocytes. Above combined treatment did not improve the process of recovery. RSV supplementation with 1-day delay relatively to 0.5 Gy irradiation, significantly decreased the frequencies of MN, especially after coadministration with 28mg/kg bw of RSV. Coadministration of RSV since eighth day did not influence the frequencies of MN compared to irradiated cells. The recovery process in the presence of RSV proceeded faster. Supplementation of RSV following initiation of irradiation is beneficial in case of irradiation with lower doses. RSV should be supplemented as soon as possible. Supplementation of RSV after termination of irradiation significantly speed up the recovery. Current results confirmed the ability of RSV to mitigate the effect of irradiation. PMID:26681581

  10. betaMinor-globin messenger RNA accumulation in reticulocytes governs improved erythropoiesis in beta thalassemic mice after erythropoietin complementary DNA electrotransfer in muscles.

    PubMed

    Samakoglu, S; Fattori, E; Lamartina, S; Toniatti, C; Stockholm, D; Heard, J M; Bohl, D

    2001-04-15

    Mechanisms governing the induction of effective erythropoiesis in response to erythropoietin (Epo) oversecretion have been investigated in beta thalassemic C57Bl/6(Hbbth) mice. Naked DNA encoding an expression vector for mouse Epo was introduced into skeletal muscles by electrotransfer. A transient increase of serum Epo concentrations with a proportional augmentation of hematocrit values was observed. Various parameters relevant to beta thalassemia were surveyed in blood samples taken before treatment, at the peak of Epo secretion, and when the phenotype reverted to anemia. We measured globin messenger RNA (mRNA) levels in reticulocytes by real-time quantitative polymerase chain reaction, globin chain synthesis levels, and several indicators of erythrocyte membrane quality, including bound alpha chains, bound immunoglobulins, main protein components, and iron compartmentalization. Data indicated that high serum Epo levels primarily affect betaminor-globin mRNA accumulation in reticulocytes. Other changes subsequent to intense Epo stimulation, like increased betaminor/alpha-globin chain synthesis ratio, reduced levels of alpha chains and immunoglobulins bound to membranes, improved spectrin/band 3 ratio, increased red blood cell survival, and improved erythropoiesis appeared as consequences of increased betaminor-globin mRNA levels. This conclusion is consistent with models postulating that intense Epo stimulation induces the expansion and differentiation of erythroid progenitors committed to fetal erythropoiesis. Although phenotypic correction was partial in mice, and comparable achievements will probably be more difficult to obtain in humans, naked DNA electrotransfer may provide a safe and low-cost method for reassessing the potentials of Epo as an inducer of fetal erythropoiesis reactivation in patients with beta thalassemia. PMID:11290581

  11. Impaired renal endothelial nitric oxide synthase and reticulocyte production as modulators of hypertension induced by rHuEPO in the rat.

    PubMed

    Ribeiro, Sandra; Garrido, Patrícia; Fernandes, João; Vala, Helena; Rocha-Pereira, Petronila; Costa, Elísio; Belo, Luís; Reis, Flávio; Santos-Silva, Alice

    2016-04-15

    Our aim was to study the effect of a broad range of recombinant human erythropoietin (rHuEPO) doses on hematological and biochemical parameters, blood pressure (BP), renal function and damage in the rat, focusing on endothelial nitric oxide synthase (eNOS) and hypoxia-inducible factors (HIFs). Male Wistar rats were divided in 5 groups receiving different doses of rHuEPO (100, 200, 400 and 600IU/kg body weight (BW)/week) and saline solution (control), during 3weeks. Blood and 24h urine were collected to perform hematological and biochemical analysis. BP was measured by the tail-cuff method. Kidney tissue was collected to mRNA and protein expression assays and to characterize renal lesions. A dose-dependent increase in red blood cells count, hematocrit and hemoglobin levels was found with rHuEPO therapy, in rHuEPO200, rHuEPO400 and rHuEPO600 groups. Increased reticulocyte count was found in rHuEPO400 and rHuEPO600 groups. BP raised in all groups receiving rHuEPO. The rHuEPO200 and rHuEPO600 groups presented increased kidney protein levels of HIF2α, a reduction in kidney protein levels of eNOS, and the highest grade of vascular and tubular renal lesions. Our study showed that rHuEPO-induced hypertension is present before significant hematological changes occur and, therefore, might involve direct (renal) and indirect (hematological) effects, which varies according to the dose used. The presence of renal hypoxia reduces eNOS activity. Excessive erythrocytosis increases blood hyperviscosity, which can be modulated by an increase in reticulocytes. Hypertension leads to early renal damage without alterations in traditional markers of renal function, thus underestimating the serious adverse effects and risks. PMID:26924494

  12. A new purification scheme for elongation factor 1 from rabbit reticulocytes and investigation of the homology of the subunits with those of initiation factor 2.

    PubMed

    Moretti, S; Staehelin, T; Trachsel, H; Gordon, J

    1979-07-01

    The aim of this work was to compare the subunits of the elongation factor EF-1 and the initiation factor eIF-2 from rabbit reticulocytes. We devised a simple procedure for the purification of EF-1: stepwise chromatography on heparin-Sepharose, separation of the heavy form by sucrose gradient centrifugation, and a final step of stepwise chromatography on RNA-Sepharose. The heparin-Sepharose column also clearly separated EF-1 and EF-2 within one chromatographic step. The EF-1 was 350-fold puried and the yield was 10%. This preparation showed after electrophoresis on polyacylamide gels in the presence of sodium dodecyl sulfate three bands corresponding to those described by others as the subunits, with Mr of 54000, 49000 and 29200. An additional band of Mr 34000 was present but no others. The 49000-Mr and 34000-Mr bands corresponded exactly in molecular weight to two of three subunits of eIF-2. A more detailed comparison was therefore made of all subunits of EF-1 and eIF-2. This was done by examination of chymotryptic fingerprints on polyacrylamide gel electrophoresis. No evidence for homology between EF-1 and eIF-2 was found. However, the two larger subunits of eIF-2 had a majority of chymotryptic fragments in common, thus indicating some homology between these polypeptides. PMID:467435

  13. The uptake of iron by reticulocytes. The influence of purification of the ghosts on iron-containing components in the ghost suspension.

    PubMed

    von der Heul, C; Kroos, M J; de Jeu-Jaspars, C M; von Eijk, H G

    1980-10-01

    1. Rat reticulocytes previously incubated with ((59)Fe, (125)I)-labelled transferrin were hemolysed to yield labelled ghosts. 2. The solubilized ghosts can be fractionated, by gel filtration on Sepharose 2B and 6B, into several (59)Fe- and (125)I-containing compounds, classified as A, B(1) and B(2). 3. These fractions were prepared from ghosts which were obtained at different centrifugation rates and further purified by sucrose density gradient centrifugation in order to obtain membrane compounds purified from mitochondrial and lysosomal impurities. The influence of these purification steps on the appearance and the (59)Fe/(125)I activity of the three components was investigated. 4. The first Sepharose 2B fraction with high molecular mass, greater than 10(6) daltons, is an intracellular product of mitochondrial and lysosomal origin which precipitates with the membrane fractions during the preparation of the ghost. The first Sepharose 6B fraction, B(1), with M(r) approximately 10(6) seems to be a real membrane component. The second Sepharose 6B fraction, B(2), with M(r) approximately 230 000 represents a real membrane receptor transferrin. PMID:7417439

  14. Structural architecture and interplay of the nucleotide- and erythrocyte binding domain of the reticulocyte binding protein Py235 from Plasmodium yoelii.

    PubMed

    Grüber, Ardina; Manimekalai, Malathy S S; Preiser, Peter R; Grüber, Gerhard

    2012-11-01

    Human malaria is caused by the cyclical invasion of the host's red blood cells (RBCs) by the invasive form of the parasite, the merozoite. The invasion of the RBC involves a range of parasite ligand receptor interactions, a process which is under intensive investigation. Two protein families are known to be important in the recognition and invasion of the human erythrocyte, the erythrocyte-binding like (EBL) proteins and the reticulocyte binding like proteins, of which the Py235 family in Plasmodium yoelii is a member. Recently the nucleotide binding domain (NBD94), that plays a role in ATP sensing, and the erythrocyte binding domain (EBD) of Py235, called EBD(1-194), have been identified. Binding of ATP leads to conformational changes within Py235 from P. yoelli and results in enhanced binding of the protein to the RBC. Structural features of these domains have been obtained, providing the platform to discuss how the structural architecture creates the basis for an interplay of the sensing NBD and the EBD domain in Py235. In analogy to the receptor-mediated ligand-dimerization model of the EBL proteins PvDBP and PfEBA-175 from Plasmodium vivax and Plasmodium falciparum, respectively, we hypothesise that Py235 of P. yoelii binds via its EBD(1-194) domain to the RBC receptor, thereby inducing dimerization of the Py235-receptor complex. PMID:22878128

  15. Upregulation of expression of the reticulocyte homology gene 4 in the Plasmodium falciparum clone Dd2 is associated with a switch in the erythrocyte invasion pathway.

    PubMed

    Gaur, Deepak; Furuya, Tetsuya; Mu, Jianbing; Jiang, Lu-bin; Su, Xin-zhuan; Miller, Louis H

    2006-02-01

    The Plasmodium falciparum clone, Dd2, that requires sialic acid for invasion can switch to a sialic acid independent pathway, Dd2(NM). To elucidate the molecular basis of the switch in invasion phenotype of Dd2 to Dd2(NM), we performed expression profiling of the parasites using an oligonucleotide microarray and real-time RT-PCR. We found that four genes were upregulated in Dd2(NM) by microarray analysis, only two of which could be confirmed by real time RT-PCR. One gene, PfRH4, is a member of the reticulocyte homology family and the other, PEBL, is a pseudogene of the Duffy binding-like family. The two genes are contiguous but transcribed in opposite directions. The DNA sequence of these ORFs, their 5'-intergenic region and a 1.1kb region 3' to each ORF are identical between Dd2 and Dd2(NM), suggesting that their transcription upregulation relates to transactivating factors. The transcription upregulation of PfRH4 was reflected at the protein level as PfRH4 protein expression was detected in Dd2(NM) and not in Dd2. Other sialic acid independent and dependent clones of P. falciparum showed variable transcript levels of PfRH4 and PEBL, unrelated to their dependence on sialic acid for invasion, suggesting that different P. falciparum clones use different receptors for sialic acid independent invasion. As Dd2(NM) is a selected subclone of Dd2, the marked upregulation of PfRH4 expression in Dd2(NM) suggests its role in erythrocyte invasion through the sialic acid independent pathway of Dd2(NM). PMID:16289357

  16. Lack of DNA polymerase theta (POLQ) radiosensitizes bone marrow stromal cells in vitro and increases reticulocyte micronuclei after total-body irradiation.

    PubMed

    Goff, Julie P; Shields, Donna S; Seki, Mineaki; Choi, Serah; Epperly, Michael W; Dixon, Tracy; Wang, Hong; Bakkenist, Christopher J; Dertinger, Stephen D; Torous, Dorothea K; Wittschieben, John; Wood, Richard D; Greenberger, Joel S

    2009-08-01

    Abstract Mammalian POLQ (pol theta) is a specialized DNA polymerase with an unknown function in vivo. Roles have been proposed in chromosome stability, as a backup enzyme in DNA base excision repair, and in somatic hypermutation of immunoglobulin genes. The purified enzyme can bypass AP sites and thymine glycol. Mice defective in POLQ are viable and have been reported to have elevated spontaneous and radiation-induced frequencies of micronuclei in circulating red blood cells. To examine the potential roles of POLQ in hematopoiesis and in responses to oxidative stress responses, including ionizing radiation, bone marrow cultures and marrow stromal cell lines were established from Polq(+/+) and Polq(-/-) mice. Aging of bone marrow cultures was not altered, but Polq(-/-) cells were more sensitive to gamma radiation than were Polq(+/+) cells. The D(0) was 1.38 +/- 0.06 Gy for Polq(+/+) cells compared to 1.27 +/- 0.16 and 0.98 +/- 0.10 Gy (P = 0.032) for two Polq(-/-) clones. Polq(-/-) cells were moderately more sensitive to bleomycin than Polq(+/+) cells and were not hypersensitive to paraquat or hydrogen peroxide. ATM kinase activation appeared to be normal in gamma-irradiated Polq(-/-) cells. Inhibition of ATM kinase activity increased the radiosensitivity of Polq(+/+) cells slightly but did not affect Polq(-/-) cells. Polq(-/-) mice had more spontaneous and radiation-induced micronucleated reticulocytes than Polq+/+ and (+/-) mice. The sensitivity of POLQ-defective bone marrow stromal cells to ionizing radiation and bleomycin and the increase in micronuclei in red blood cells support a role for this DNA polymerase in cellular tolerance of DNA damage that can lead to double-strand DNA breaks. PMID:19630521

  17. Alphacoronavirus transmissible gastroenteritis virus nsp1 protein suppresses protein translation in mammalian cells and in cell-free HeLa cell extracts but not in rabbit reticulocyte lysate.

    PubMed

    Huang, Cheng; Lokugamage, Kumari G; Rozovics, Janet M; Narayanan, Krishna; Semler, Bert L; Makino, Shinji

    2011-01-01

    The nsp1 protein of transmissible gastroenteritis virus (TGEV), an alphacoronavirus, efficiently suppressed protein synthesis in mammalian cells. Unlike the nsp1 protein of severe acute respiratory syndrome coronavirus, a betacoronavirus, the TGEV nsp1 protein was unable to bind 40S ribosomal subunits or promote host mRNA degradation. TGEV nsp1 also suppressed protein translation in cell-free HeLa cell extract; however, it did not affect translation in rabbit reticulocyte lysate (RRL). Our data suggested that HeLa cell extracts and cultured host cells, but not RRL, contain a host factor(s) that is essential for TGEV nsp1-induced translational suppression. PMID:21047955

  18. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2. alpha. (eIF-2. alpha. ) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2. alpha. kinase

    SciTech Connect

    Chen, J.J.; Throop, M.S.; Kuo, I.; Pal, J.K.; Brodsky, M.; London, I.M. ); Gehrke, L. Harvard Medical School, Boston, MA )

    1991-09-01

    The authors have cloned the cDNA of the heme-regulated eIF-2{alpha} kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2{alpha} kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of {approx} 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2{alpha} kinase. This observation suggests that GCN2 protein kinase may be an eIF-2{alpha} kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

  19. Biochemical characterisation of cap–poly(A) synergy in rabbit reticulocyte lysates: the eIF4G–PABP interaction increases the functional affinity of eIF4E for the capped mRNA 5′-end

    PubMed Central

    Borman, Andrew M.; Michel, Yanne M.; Kean, Katherine M.

    2000-01-01

    The 5′ cap and 3′ poly(A) tail of eukaryotic mRNAs cooperate to synergistically stimulate translation initiation in vivo. We recently described mammalian cytoplasmic extracts which, following ultracentrifugation to partially deplete them of ribosomes and associated initiation factors, reproduce cap–poly(A) synergy in vitro. Using these systems, we demonstrate that synergy requires interaction between the poly(A)-binding protein (PABP) and the eukaryotic initiation factor (eIF) 4F holoenzyme complex, which recognises the 5′ cap. Here we further characterise the requirements and constraints of cap–poly(A) synergy in reticulocyte lysates by evaluating the effects of different parameters on synergy. The extent of extract depletion and the amounts of different initiation factors in depleted extracts were examined, as well as the effects of varying the concentrations of KCl, MgCl2 and programming mRNA and of adding a cap analogue. The results presented demonstrate that maximal cap–poly(A) synergy requires: (i) limiting concentrations of ribosome-associated initiation factors; (ii) precise ratios of mRNA to translation machinery (low concentrations of ribosome-associated initiation factors and low, non-saturating mRNA concentrations); (iii) physiological concentrations of added KCl and MgCl2. Additionally, we show that the eIF4G–PABP interaction on mRNAs which are capped and polyadenylated significantly increases the affinity of eIF4E for the 5′ cap. PMID:11058101

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

    PubMed

    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

  1. 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

  2. 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

  3. 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. PMID:25931013

  4. Hemolytic disease of the newborn

    MedlinePlus

    ... immature red blood cell (reticulocyte) count Bilirubin level Blood typing ... receiving extra fluids Light therapy (phototherapy) using ... Medicines to raise blood pressure if it drops too low In severe ...

  5. 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 ...

  6. A microfluidics cytometer for mice anemia detection.

    PubMed

    Ju, Yanrui; Song, Jian; Geng, Zhaoxin; Zhang, Hongze; Wang, Wei; Xie, Lide; Yao, Weijuan; Li, Zhihong

    2012-11-01

    The design and fabrication of a microfluidic cytometer system and its application for reticulocyte detection are described. This chip can count the target cells, which are focused at the detection window without sheath flow. This cytometer system based on optimized epifluoresence has a competitive advantage in the signal-to-noise ratio. Induced fluorescence from the reticulocyte binded with antibody is detected by the optical module and then transformed into the electronic signal by a photo multiplier tube. After signal processing, the results are automatically read out by a digital module and displayed on the system. To evaluate this microfluidic cytometer system, experiments employing polystyrene (PS) micro beads and induced reticulocyte of mice anemia are carried out, respectively, and the results illustrate that the microfluidic cytometer system is effective in detecting the reticulocyte. PMID:22907472

  7. 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.

  8. Reticulum vs Inclusions: A Learning Experience in Haemoglobin H Disease

    PubMed Central

    Sridevi, Hanaganahalli B; Hegde, Anupama; Balanthimogru, Prashantha; Khadilkar, Urmila N.

    2015-01-01

    Haemoglobin H disease, also known as the alpha-thalassaemia is characterized by the presence of HbH inclusions in red blood cells, detectable on supra-vital stain. We present a case of a previously asymptomatic 31-year-old male, who insidiously developed anaemia and had prominent splenomegaly. Peripheral smear examination revealed microcytic hypochromic anaemia with numerous spherocytes and moderate polychromasia. In reticulocyte preparation with Brilliant cresyl blue, HbH inclusions were mistakenly identified as granulofilamentous reticulum of reticulocytes, giving a spuriously high reticulocyte percentage. After the literature review, repeat assessment was performed and with the aid of high performance liquid chromatography result, it was possible to delineate the HbH inclusions. PMID:26557534

  9. Comparative study on the effects of five benzoylphenylurea insecticides on haematological parameters in rats.

    PubMed

    Tasheva, M; Hristeva, V

    1993-01-01

    Five benzoylphenylurea insecticides were administered to male Wistar rats for 28 days at oral doses of 100 mg kg-1 each. Elevation of methaemoglobin was found only in the diflubenzuron- and triflumuron treated groups. The number of reticulocytes was increased in all of the treated groups. PMID:8440876

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

    PubMed

    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

  11. Blood volume changes

    NASA Technical Reports Server (NTRS)

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

    1977-01-01

    Red cell mass measurements on Skylab crewmembers revealed a statistically significant decrease in circulating red cells. Low reticulocyte counts at crewmember recovery indicated increased splenic removal of reticulum or decreased bone marrow production rates. After the initial loss there is at least a 30-day delay before red cell mass begins to reconstitute itself.

  12. 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.

  13. 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

  14. Polychromatophilia

    MedlinePlus

    ... blood cells look under a microscope when the cells are stained with special dyes. It means there is more staining with certain dyes than normal. The extra staining is due to too many immature red blood cells (RBCs) called reticulocytes. These cells have a blue- ...

  15. Development of methods for characterizing fetal and adult somatic mutations detected in human erythroid precursor

    SciTech Connect

    Langlois, R.G.; Manchester, D.K.

    1994-12-31

    The glycophorin A (GPA) assay was developed to quantify somatic mutations in humans by measuring the frequency of peripheral erythrocytes with mutant phenotypes that are presumed to be progeny of mutated erythroid precursor cells. This assay has been used to identify GPA variant cells in unexposed individuals at a frequency of {approximately}10 per million erythrocytes, and to demonstrate significant increases in variant frequency after mutagenic exposures. Characterization of the mutations responsible for these variant cells requires that the assay be modified to allow flow analysis and sorting of variant erythroid precursor cells that contain nucleic acids. Cord blood samples contain low levels of both reticulocytes and nucleated erythrocytes. We have developed enrichment methods using centrifugation that yield samples containing up to 30% nucleated erythrocytes, and immunomagnetic separation methods that yield samples containing up to 90% reticulocytes. Enrichment methods for these two cell types are also being developed for adult bone marrow samples. We have confirmed that enrichment and labeling with a nucleic acid-specific dye are compatible with GPA analysis of erythrocytes, reticulocytes, and nucleated erythrocytes. Enriched samples have been successfully used for flow cytometric detection of GPA variant reticulocytes in cord blood. PCR-based analysis methods are being developed for molecular characterization of sorted variant cells at the mRNA level.

  16. Effects of low concentrations of benzene on mouse hematopoietic cells in vivo: A preliminary report

    SciTech Connect

    Farris, G.M.; Robinson, S.N.; Gaido, K.W.

    1996-12-01

    Evaluation of benzene-induced hematotoxicity following exposure to low concentrations is important for understanding mechanisms of toxicity and determining the dose response at benzene levels close to the current occupational exposure limit 0 ppm. Male B6C3F1 mice were exposed to 0, 1, 10, 100, or 200 ppm benzene by inhalation for 6 hr/day, 5 days/week for 1, 2, 4, or 8 weeks. At each sampling time, we evaluated primitive and committed progenitor cells, differentiating and maturing lineage-specific cells, and stromal cells in the bone marrow; T and B lymphocytes of the spleen and thymus; micronucleated reticulocytes and erythrocytes; and standard blood parameters. At 100 and 200 ppm benzene, there were rapid and significant reductions in number of reticulocytes in the blood, B lymphocytes in the bone marrow and spleen, and an increased frequency of micronucleated reticulocytes in the bone marrow. At 10 ppm, the only parameter affected was a transient reduction in the number of splenic B lymphocytes. There were no significant effects induced by 1 ppm benzene in this study. The present study suggests numbers of B lymphocytes and maturing erythrocytes, and frequency of micronucleated reticulocytes are sensitive indicators of benzene-induced hematotoxicity and will be useful in further investigation of the hematotoxicity induced by 10 to 100 ppm benzene. 7 refs., 2 figs.

  17. 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

  18. Host Erythrocyte Environment Influences the Localization of Exported Protein 2, an Essential Component of the Plasmodium Translocon

    PubMed Central

    Meibalan, Elamaran; Comunale, Mary Ann; Lopez, Ana M.; Bergman, Lawrence W.; Mehta, Anand; Vaidya, Akhil B.

    2015-01-01

    Malaria parasites replicating inside red blood cells (RBCs) export a large subset of proteins into the erythrocyte cytoplasm to facilitate parasite growth and survival. PTEX, the parasite-encoded translocon, mediates protein transport across the parasitophorous vacuolar membrane (PVM) in Plasmodium falciparum-infected erythrocytes. Proteins exported into the erythrocyte cytoplasm have been localized to membranous structures, such as Maurer's clefts, small vesicles, and a tubovesicular network. Comparable studies of protein trafficking in Plasmodium vivax-infected reticulocytes are limited. With Plasmodium yoelii-infected reticulocytes, we identified exported protein 2 (Exp2) in a proteomic screen of proteins putatively transported across the PVM. Immunofluorescence studies showed that P. yoelii Exp2 (PyExp2) was primarily localized to the PVM. Unexpectedly, PyExp2 was also associated with distinct, membrane-bound vesicles in the reticulocyte cytoplasm. This is in contrast to P. falciparum in mature RBCs, where P. falciparum Exp2 (PfExp2) is exclusively localized to the PVM. Two P. yoelii-exported proteins, PY04481 (encoded by a pyst-a gene) and PY06203 (PypAg-1), partially colocalized with these PyExp2-positive vesicles. Further analysis revealed that with P. yoelii, Plasmodium berghei, and P. falciparum, cytoplasmic Exp2-positive vesicles were primarily observed in CD71+ reticulocytes versus mature RBCs. In transgenic P. yoelii 17X parasites, the association of hemagglutinin-tagged PyExp2 with the PVM and cytoplasmic vesicles was retained, but the pyexp2 gene was refractory to deletion. These data suggest that the localization of Exp2 in mouse and human RBCs can be influenced by the host cell environment. Exp2 may function at multiple points in the pathway by which parasites traffic proteins into and through the reticulocyte cytoplasm. PMID:25662767

  19. Isolation and Characterization of a Type 1 Ribosome-Inactivating Protein from Fruiting Bodies of the Edible Mushroom (Volvariella volvacea).

    PubMed

    Yao; Yu; Ooi; Ng; Chang; Sun; Ooi

    1998-02-16

    A novel single-chained ribosome-inactivating protein (RIP) with a molecular weight of approximately 29 000 was purified from fruiting bodies of the edible mushroom Volvariellavolvacea with a procedure involving ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, and gel filtration on Superdex 75. The mushroom RIP, designated volvarin, exhibited a potent inhibitory action on protein synthesis in the rabbit reticulocyte lysate system with an IC(50) value of 0.5 nM. Like most plant RIPs, volvarin acted as an N-glycosidase that depurinated rRNA from rabbit reticulocyte lysate, releasing a characteristic RNA fragment after treatment with aniline. It also exerted a deoxyribonuclease activity on supercoiled SV-40 DNA and demonstrated a strong abortifacient effect in mice. PMID:10554316

  20. Eukaryotic cytosolic chaperonin contains t-complex polypeptide 1 and seven related subunits.

    PubMed Central

    Rommelaere, H; Van Troys, M; Gao, Y; Melki, R; Cowan, N J; Vandekerckhove, J; Ampe, C

    1993-01-01

    We have characterized the cytosolic chaperonin from both rabbit reticulocyte lysate and bovine testis. The heteromeric complex contains eight subunits. Partial amino acid sequence data reveal that one of these is t-complex polypeptide 1 (TCP-1), while the other seven are TCP-1-related polypeptides, implicating the existence of a multigene family of TCP-1 homologues. We provide evidence that TCP-1 ring complex from bovine testis can facilitate the folding of both actin and tubulin, although, as in the case of chaperonin from reticulocyte lysate, two cofactors are required for the generation of properly folded tubulin. An additional molecule of TCP-1 may associate with the chaperonin depending on the purification procedure used. We propose that a highly conserved region in these polypeptides and in other chaperonins of the cpn60 chaperone family participates in ATP binding. Images Fig. 1 Fig. 2 Fig. 3 PMID:7903455

  1. 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

    2014-06-01

    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. PMID:24759100

  2. Correlation between the presence of tRNA His GUG and the erythropoietic function in foetal sheep liver.

    PubMed Central

    Landin, R M; Boisnard, M; Petrissant, G

    1979-01-01

    Histidyl-tRNAs from foetal and adult sheep liver were compared to their reticulocyte counterparts. The combination of various techniques revealed the existence of two histidyl-tRNA species in reticulocytes, one of which was not retained on acetylated DBAE-cellulose columns and was guanylatable. Three histidyl-tRNA isoacceptors were identified in foetal liver. Two of these species were not adsorbed on acetylated DBAE-cellulose but only one was found to be guanylatable. An identical chromatographic behaviour on RPC-5 columns was observed for guanylated histidyl-tRNAs from both origins. These results suggest the occurrence of a GUG anticodon in these guanine-accepting tRNAs. In foetal liver the amount of guanylatable histidyl-tRNA was estimated to be 7% of the total tRNA population. This observation is in agreement with the erythropoietic function of liver during the foetal life. Images PMID:503863

  3. [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. PMID:25786319

  4. 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

  5. Characterizing the genetic diversity of the monkey malaria parasite Plasmodium cynomolgi.

    PubMed

    Sutton, Patrick L; Luo, Zunping; Divis, Paul C S; Friedrich, Volney K; Conway, David J; Singh, Balbir; Barnwell, John W; Carlton, Jane M; Sullivan, Steven A

    2016-06-01

    Plasmodium cynomolgi is a malaria parasite that typically infects Asian macaque monkeys, and humans on rare occasions. P. cynomolgi serves as a model system for the human malaria parasite Plasmodium vivax, with which it shares such important biological characteristics as formation of a dormant liver stage and a preference to invade reticulocytes. While genomes of three P. cynomolgi strains have been sequenced, genetic diversity of P. cynomolgi has not been widely investigated. To address this we developed the first panel of P. cynomolgi microsatellite markers to genotype eleven P. cynomolgi laboratory strains and 18 field isolates from Sarawak, Malaysian Borneo. We found diverse genotypes among most of the laboratory strains, though two nominally different strains were found to be genetically identical. We also investigated sequence polymorphism in two erythrocyte invasion gene families, the reticulocyte binding protein and Duffy binding protein genes, in these strains. We also observed copy number variation in rbp genes. PMID:26980604

  6. High speed flow cytometric detection of rare glycophorin A mutations in human blood cells

    SciTech Connect

    Langlois, R.G.; Engh, G. van den )

    1993-01-01

    The glycophorin A (GPA) assay utilizes immunofluorescent labeling and flow cytometry to measure the frequency of peripheral erythrocytes with mutant phenotypes, presumably due to mutations in erythroid precursor cells. Analysis of 5 [times] 10[sup 6] cells/assay is used to enumerate variant erythrocytes that occur at a frequency of 3-10 [times] 10[sup [minus]6] in unexposed donors. Extension of this assay to human reticulocytes requires detection of variants that occur at frequencies as low as 3 [times] 10[sup [minus]8]. The authors have used high speed data acquisition and cell classification electronics to perform 3-color analysis at rates up to 20,000 cells/s. High speed analysis of up to 10[sup 8] cells/assay has been used to enumerate GPA-variant reticulocytes in normal donors.

  7. 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. PMID:19202968

  8. Comprehensive Proteomic Analysis of Human Erythropoiesis.

    PubMed

    Gautier, Emilie-Fleur; Ducamp, Sarah; Leduc, Marjorie; Salnot, Virginie; Guillonneau, François; Dussiot, Michael; Hale, John; Giarratana, Marie-Catherine; Raimbault, Anna; Douay, Luc; Lacombe, Catherine; Mohandas, Narla; Verdier, Frédérique; Zermati, Yael; Mayeux, Patrick

    2016-08-01

    Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis. PMID:27452463

  9. Pure red cell aplasia in a simultaneous pancreas-kidney transplantation patient: inside the erythroblast

    PubMed Central

    Labbadia, Francesca; Salido-Fierréz, Eduardo; Majado-Martinez, Juliana; Cabañas-Perianes, Valentin; Moraleda, Jiménez José M.

    2012-01-01

    A case of pure red cell aplasia in a simultaneous kidney-pancreas transplant recipient on immunosuppressive therapy is reported here. The patient presented with anemia unresponsive to erythropoietin treatment. Bone marrow cytomorphology was highly suggestive of parvovirus pure red cell aplasia, which was confirmed with serology and polymerase chain reaction positive for parvovirus B19 DNA in peripheral blood. After the administration of intravenous immunoglobulin the anemia improved with a rising number of the reticulocytes. PMID:23087806

  10. Flow cytometric analysis of micronuclei in rat peripheral blood: An interlaboratory reproducibility study.

    PubMed

    Kasamoto, Sawako; Mukai, Daisuke; Masumori, Shoji; Suzuki, Kenichiro; Tanaka, Ryota; Torous, Dorothea K; Yamate, Jyoji; Hayashi, Makoto

    2014-03-01

    In anticipation of proposed OECD guideline changes that may include increasing the number of reticulocytes scored for micronuclei, an inter-laboratory reproducibility study of the rat peripheral blood micronucleus assay was performed using flow cytometry. In this experiment, male Sprague-Dawley (SD) rats were treated with the model clastogen cyclophosphamide (CP: 5, 10 or 15mg/kg) by a single oral administration. As controls, rats were treated with physiological saline (solvent) in the same manner as for the model clastogen. Peripheral blood was collected from each rat 48h after the treatment. The blood samples were prepared at the Public Interest Incorporated Foundation, BioSafety Research Center (BSRC) in duplicate using the rat MicroFlow(PLUS) Kit. After fixation, one replicate set of samples was shipped to Litron Laboratories, and each sample was analyzed by flow cytometry at the two laboratories. In addition, the frequency of micronucleated reticulocytes (MNRETs) was determined at the BSRC by microscopic analysis using supravital acridine orange (AO) staining. The reproducibility of micronucleated reticulocyte frequencies analyzed by microscopy and flow cytometry showed good correlation (r(2)=0.84). The frequencies of micronucleated reticulocytes analyzed by flow cytometry at the two independent laboratories showed good concordance (r(2)=0.97). The data indicate that the flow cytometric micronucleus analysis method is a good alternative to manual microscopic analysis. Flow cytometry allows groups to readily score 5000 or even 20,000 RETs in a matter of minutes compared to manual analysis. This results in increased reliability of the assay by achieving better statistical power. PMID:24548793

  11. Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier.

    PubMed

    Couch, Jessica A; Yu, Y Joy; Zhang, Yin; Tarrant, Jacqueline M; Fuji, Reina N; Meilandt, William J; Solanoy, Hilda; Tong, Raymond K; Hoyte, Kwame; Luk, Wilman; Lu, Yanmei; Gadkar, Kapil; Prabhu, Saileta; Ordonia, Benjamin A; Nguyen, Quyen; Lin, Yuwen; Lin, Zhonghua; Balazs, Mercedesz; Scearce-Levie, Kimberly; Ernst, James A; Dennis, Mark S; Watts, Ryan J

    2013-05-01

    Bispecific antibodies using the transferrin receptor (TfR) have shown promise for boosting antibody uptake in brain. Nevertheless, there are limited data on the therapeutic properties including safety liabilities that will enable successful development of TfR-based therapeutics. We evaluate TfR/BACE1 bispecific antibody variants in mouse and show that reducing TfR binding affinity improves not only brain uptake but also peripheral exposure and the safety profile of these antibodies. We identify and seek to address liabilities of targeting TfR with antibodies, namely, acute clinical signs and decreased circulating reticulocytes observed after dosing. By eliminating Fc effector function, we ameliorated the acute clinical signs and partially rescued a reduction in reticulocytes. Furthermore, we show that complement mediates a residual decrease in reticulocytes observed after Fc effector function is eliminated. These data raise important safety concerns and potential mitigation strategies for the development of TfR-based therapies that are designed to cross the blood-brain barrier. PMID:23636093

  12. Composition, assembly and activation of the avian progesterone receptor.

    PubMed

    Smith, D F; Toft, D O

    1992-03-01

    When isolated from chick oviduct cytosol by antibody adsorption, the inactive progesterone receptor is associated with the two heat shock proteins, hsp90 and hsp70, plus three additional proteins termed p54, p50, and p23 according to their molecular weights. While their functions remain unknown, all of these receptor associated proteins are dissociated upon receptor activation in intact cells. To better understand the assembly and activation mechanisms of progesterone receptor complexes, we have developed a cell-free system for studying receptor interactions with hsp90 and hsp70 and have used this system to examine requirements for hsp90 binding to the receptor. Purified receptor, free of hsp90 and immobilized on an antibody affinity resin, will rebind hsp90 in rabbit reticulocyte lysate when several conditions are met. These include: (1) absence of progesterone, (2) elevated temperature (30 degrees C), (3) presence of ATP, and (4) presence of Mg2+. We have obtained maximal hsp90 binding to receptor when lysate is supplemented with 3 mM MgCl2 and an ATP regenerating system. ATP depletion of lysate by dialysis or ATPase addition blocks hsp90 binding to the receptor. When progesterone is added to pre-formed receptor complexes in reticulocyte lysate it promotes activation and the dissociation of hsp90. This process is also dependent upon ATP. Thus, both the assembly, and activation of the progesterone receptor can be accomplished in the reticulocyte lysate system. PMID:1562503

  13. Variation of red blood cell distribution width and mean platelet volume after moderate endurance exercise.

    PubMed

    Lippi, Giuseppe; Salvagno, Gian Luca; Danese, Elisa; Tarperi, Cantor; Guidi, Gian Cesare; Schena, Federico

    2014-01-01

    Although physical exercise strongly influences several laboratory parameters, data about the hematological changes after medium distance running are scarce. We studied 31 middle-trained athletes (mean training regimen 217 ± 32 min/week) who performed a 21.1 km, half-marathon run. Blood samples were collected before the run, at the end, and 3 and 20 hours thereafter. The complete blood count was performed on Advia 2120 and included red blood cell (RBC), reticulocyte, and platelet counts; hemoglobin; mean corpuscular volume (MCV); mean corpuscular hemoglobin (MCH); reticulocyte haemoglobin content (Ret CHR); RBC distribution width (RDW), mean platelet volume (MPV). No significant variations were observed for MCH and Ret CHR. The RBC, reticulocyte, and hemoglobin values modestly decreased after the run. The MCV significantly increased at the end of running but returned to baseline 3 hours thereafter. The RDW constantly increased, reaching a peak 20 hours after the run. The platelet count and MPV both increased after the run and returned to baseline 3 hours thereafter. These results may have implications for definition of reference ranges and antidoping testing, and may also contribute to explaining the relationship between endurance exercise and mortality, since previous studies reported that RDW and MPV may be significantly associated with cardiovascular disease. PMID:25197280

  14. Hemoglobinization and functional availability of iron for erythropoiesis in case of thalassemia and iron deficiency anemia.

    PubMed

    Bartels, Piet C M; Schoorl, Margreet; Schoorl, Marianne

    2006-01-01

    Microcytic erythropoiesis in case of anemia is frequently due to iron deficiency or may be due to alpha- and beta- thalassemia trait as a result of increased activity of erythropoiesis. The aim of the present study was to evaluate alterations with regard to the degree of hemoglobinization in reticulocytes in comparison with mature erythrocytes. Iron availability in subjects with anemia resulting from iron deficiency and alpha- or beta- thalassemia was studied by application of conventional as well hemocytometric parameters that have recently become available. Participants of the study were reference subjects (n=75), subjects with iron deficiency anemia (IDA, n=52) and alpha- (n=26) or beta-thalassemia trait (n=24). If compared with the reference group obviously increased RBC counts together with decreased values for RDW-sd and MCHC were established in case of alpha- and beta- thalassemia subjects. Deviations were demonstrated to be more pronounced in case of beta- thalassemia. Accelerated erythropoiesis in the case of subjects with IDA and beta-thalassemia is manifested by detection of increased results for immature reticulocyte counts. In particular in case of beta- thalassemia, elevated reticulocyte counts combined with slightly increased values for ZPP/heme ratio reflect increased activity of erythropoiesis. In the case of subjects with beta-thalassemia serum transferrin concentrations revealed slightly decreased results, whereas serum ferritin and iron concentrations demonstrated a tendency towards higher values if compared with the group of reference subjects. At a definitive MCV level, the hemoglobin content of reticulocytes is decreased in the case of IDA if compared with the alpha- or beta- thalassemia trait. For the ratio of hemoglobin content of reticulocytes and erythrocytes, obviously decreased results are demonstrated in the case of subjects with iron deficiency anemia (1.02 +/- 0.08, mean +/- SD) and in the case of beta-thalassemia (1.06 +/- 0.04) if

  15. 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

  16. 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

    2015-06-01

    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

  17. 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

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

    PubMed Central

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

    2015-01-01

    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 500mg/kg/day) or EC (250mg/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. PMID:25833916

  19. 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. PMID:27583566

  20. MAT1 ('menage à trois') a new RING finger protein subunit stabilizing cyclin H-cdk7 complexes in starfish and Xenopus CAK.

    PubMed Central

    Devault, A; Martinez, A M; Fesquet, D; Labbé, J C; Morin, N; Tassan, J P; Nigg, E A; Cavadore, J C; Dorée, M

    1995-01-01

    The kinase responsible for Thr161-Thr160 phosphorylation and activation of cdc2/cdk2 (CAK:cdk-activating kinase) has been shown previously to comprise at least two subunits, cdk7 and cyclin H. An additional protein co-purified with CAK in starfish oocytes, but its sequencing did not reveal any similarity with any known protein. In the present work, a cDNA encoding this protein is cloned and sequenced in both starfish and Xenopus oocytes. It is shown to encode a new member of the RING finger family of proteins with a characteristic C3HC4 motif located in the N-terminal domain. We demonstrate that the RING finger protein (MAT1: 'menage à trois') is a new subunit of CAK in both vertebrate and invertebrates. However, CAK may also exist in oocytes as heterodimeric complexes between cyclin H and cdk7 only. Stable heterotrimeric CAK complexes were generated in reticulocyte lysates programmed with mRNAs encoding Xenopus cdk7, cyclin H and MAT1. In contrast, no heterodimeric cyclin H-cdk7 complex could be immunoprecipitated from reticulocyte lysates programmed with cdk7 and cyclin H mRNAs only. Stabilization of CAK complexes by MAT1 does not involve phosphorylation of Thr176, as the Thr176-->Ala mutant of Xenopus cdk7 could engage as efficiently as wild-type cdk7 in ternary complexes. Even though starfish MAT1 is almost identical to Xenopus MAT1 in the RING finger domain, the starfish subunit could not replace the Xenopus subunit and stabilize cyclin H-cdk7 in reticulocyte lysate, suggesting that the MAT1 subunit does not (or not only) interact with cyclin H-cdk7 through the RING finger domain. Images PMID:7588631

  1. Structurally conserved erythrocyte-binding domain in Plasmodium provides a versatile scaffold for alternate receptor engagement

    PubMed Central

    Gruszczyk, Jakub; Lim, Nicholas T. Y.; Arnott, Alicia; He, Wen-Qiang; Nguitragool, Wang; Roobsoong, Wanlapa; Mok, Yee-Foong; Murphy, James M.; Smith, Katherine R.; Lee, Stuart; Bahlo, Melanie; Mueller, Ivo; Barry, Alyssa E.

    2016-01-01

    Understanding how malaria parasites gain entry into human red blood cells is essential for developing strategies to stop blood stage infection. Plasmodium vivax preferentially invades reticulocytes, which are immature red blood cells. The organism has two erythrocyte-binding protein families: namely, the Duffy-binding protein (PvDBP) and the reticulocyte-binding protein (PvRBP) families. Several members of the PvRBP family bind reticulocytes, specifically suggesting a role in mediating host cell selectivity of P. vivax. Here, we present, to our knowledge, the first high-resolution crystal structure of an erythrocyte-binding domain from PvRBP2a, solved at 2.12 Å resolution. The monomeric molecule consists of 10 α-helices and one short β-hairpin, and, although the structural fold is similar to that of PfRh5—the essential invasion ligand in Plasmodium falciparum—its surface properties are distinct and provide a possible mechanism for recognition of alternate receptors. Sequence alignments of the crystallized fragment of PvRBP2a with other PvRBPs highlight the conserved placement of disulfide bonds. PvRBP2a binds mature red blood cells through recognition of an erythrocyte receptor that is neuraminidase- and chymotrypsin-resistant but trypsin-sensitive. By examining the patterns of sequence diversity within field isolates, we have identified and mapped polymorphic residues to the PvRBP2a structure. Using mutagenesis, we have also defined the critical residues required for erythrocyte binding. Characterization of the structural features that govern functional erythrocyte binding for the PvRBP family provides a framework for generating new tools that block P. vivax blood stage infection. PMID:26715754

  2. Synthesis and assembly of membrane skeletal proteins in mammalian red cell precursors

    SciTech Connect

    Hanspal, M.; Palek, J.

    1987-09-01

    The synthesis of membrane skeletal proteins in avian nucleated red cells has been the subject of extensive investigation, whereas little is known about skeletal protein synthesis in bone marrow erythroblasts and peripheral blood reticulocytes in mammals. To address this question, we have isolated nucleated red cell precursors and reticulocytes from spleens and from the peripheral blood, respectively, of rats with phenylhydrazine-induced hemolytic anemia and pulse-labeled them with (/sup 35/S)methionine. Pulse-labeling of nucleated red cell precursors shows that the newly synthesized alpha- and beta-spectrins are present in the cytosol, with a severalfold excess of alpha-spectrin over beta-spectrin. However, in the membrane-skeletal fraction, newly synthesized alpha- and beta-spectrins are assembled in stoichiometric amounts, suggesting that the association of alpha-spectrin with the membrane skeleton may- be rate-limited by the amount of beta-spectrin synthesized, as has been shown recently in avian erythroid cells. Pulse-chase experiments in the rat nucleated red cell precursors show that the newly synthesized alpha- and beta-spectrin of the cytosol turn over coordinately and extremely rapidly. In contrast, in the membrane-skeletal fraction, the newly synthesized polypeptides of spectrin are stable. In contrast to nucleated erythroid cells, in reticulocytes the synthesis of alpha- and beta-spectrins is markedly diminished compared with the synthesis and assembly of proteins comigrating with bands 2.1 and 4.1 on SDS gels. Thus, in nucleated red cell precursors, the newly synthesized spectrin may be attached to the plasma membrane before proteins 2.1 and 4.1 are completely synthesized and incorporated in the membrane.

  3. 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. PMID:17183843

  4. Sickle cell disease in Orissa State, India.

    PubMed

    Kar, B C; Satapathy, R K; Kulozik, A E; Kulozik, M; Sirr, S; Serjeant, B E; Serjeant, G R

    1986-11-22

    A study of 131 patients with homozygous sickle cell (SS) disease in Orissa State, India, indicated that, compared with Jamaican patients, Indian patients have higher frequencies of alpha thalassaemia, higher fetal haemoglobin, total haemoglobin, and red cell counts, and lower mean cell volume, mean cell haemoglobin concentration, and reticulocyte counts. Indian patients have a greater frequency and later peak incidence of splenomegaly, and hypersplenism is common. Painful crises and dactylitis are not uncommon in Indian patients but chronic leg ulceration is rare. Homozygous sickle cell disease in Orissa is similar to that in the Eastern Province of Saudi Arabia and is very different from that in populations of West African origin. PMID:2430154

  5. 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. PMID:26999424

  6. Globin mRNA contains a sequence complementary to double-stranded region of nuclear pre-mRNA.

    PubMed Central

    Ryskov, A P; Tokarskaya, O V; Georgiev, G P; Coutelle, C; Thiele, B

    1976-01-01

    Melted ds RNA isolated from rabbit bone marrow pre-mRNA was hybridized with excess of globin mRNA which was prepared from rabbit reticulocytes. 7-9% of ds sequences became RNAase-stable and about 30% of the sequences could be bound to poly(U)-Sepharose through poly (A) of mRNA. The size of RNAase-stable hybrid is about 30 nucleotides, that is one fourth of the length of one strand of the ds RNA. PMID:986644

  7. Erythropoietin May Improve Anemia in Patients with Autoimmune Hemolytic Anemia Associated with Reticulocytopenia

    PubMed Central

    Arbach, Olga; Funck, Robert; Seibt, Frank; Salama, Abdulgabar

    2012-01-01

    Background Management of patients with autoimmune hemolytic anemia (AIHA) and reticulocytopenia remains challenging. Case Reports Two patients with decompensated AIHA who were receiving immunosuppressive drugs were treated with erythropoietin (EPO). Administration of EPO increased reticulocyte counts and hemoglobin concentrations in both cases. One patient completely recovered following a short course of treatment. Hemolysis could be compensated in the second patient using only mild doses of immunosuppressive drugs in combination with EPO. Conclusion The administration of EPO should be considered in patients with therapy-refractory AIHA, particularly in the presence of reticulocytopenia. PMID:22851939

  8. Erythropoietin May Improve Anemia in Patients with Autoimmune Hemolytic Anemia Associated with Reticulocytopenia.

    PubMed

    Arbach, Olga; Funck, Robert; Seibt, Frank; Salama, Abdulgabar

    2012-06-01

    BACKGROUND: Management of patients with autoimmune hemolytic anemia (AIHA) and reticulocytopenia remains challenging. CASE REPORTS: Two patients with decompensated AIHA who were receiving immunosuppressive drugs were treated with erythropoietin (EPO). Administration of EPO increased reticulocyte counts and hemoglobin concentrations in both cases. One patient completely recovered following a short course of treatment. Hemolysis could be compensated in the second patient using only mild doses of immunosuppressive drugs in combination with EPO. CONCLUSION: The administration of EPO should be considered in patients with therapy-refractory AIHA, particularly in the presence of reticulocytopenia. PMID:22851939

  9. Comparative Studies on Biochemical Properties of Protein Synthesis of an Archae-Bacteria Thermoplasma-Sp

    NASA Astrophysics Data System (ADS)

    Ohba, Masayuki; Oshima, Tairo

    1982-12-01

    An acido-thermophillic archaebacteria,Thermoplasma strain KO-2, produced poly(A) containing RNA. The isolated poly(A)RNA showed the messenger activity in a cell-free extract of rabbit reticulocyte, indicating that the RNA is mRNA of the archaebacteria. 7-Methylgluanosine 5'-phosphate did not inhibit the reaction, suggesting that the cap structure is not present in the messenger. These results may suggest that poly(A) containing messenger arised at very early stage of evolution prior to the divergence between archaebacteria and eukaryotes.

  10. Development of a tRNA-dependent in vitro translation system.

    PubMed Central

    Jackson, R J; Napthine, S; Brierley, I

    2001-01-01

    A method is described for depleting rabbit reticulocyte lysates and wheat germ extracts of endogenous tRNAs by affinity chromatography using a matrix generated by coupling ethanolamine to epoxy-activated Sepharose 6B. Greater than 90% depletion of tRNA is achieved with the result that translation becomes in effect absolutely dependent on added tRNA. This depletion procedure should prove very useful for studying the influence of tRNA concentration, and the spectrum of the tRNA population, on recoding events such as programmed frameshifting and readthrough of termination codons. PMID:11350040

  11. 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

  12. Molecular cloning and expression of a chloride channel-associated protein pICln in human young red blood cells: association with actin.

    PubMed Central

    Schwartz, R S; Rybicki, A C; Nagel, R L

    1997-01-01

    We report the cloning and sequencing from human reticulocytes of cDNA coding for the Cl- channel-associated protein, pICln. Human reticulocyte pICln (HRpICln) cDNA encodes a protein (predicted molecular mass 26293Da) identical with human non-pigmented ciliary epithelial cell pICln. By using full-length HRpICln cDNA (approx. 1.2 kb) to probe human lymphocyte metaphase-chromosome spreads, the location of the human ICln gene was mapped to 11q13 by fluorescence in situ hybridization analysis. Polyclonal antibodies to recombinant HRpICln detected bands at approx. 43 kDa and approx. 37 kDa in both normal (AA) and sickle (SS) red blood cell (RBC) ghost membranes. In SS ghosts, and in ghosts from a patient with autoimmune haemolytic anaemia with 9.8% reticulocytes, the amount of HRpICln was increased compared with AA ghosts, suggesting that the expression or membrane assembly of HRpICln is cell age-dependent. Laser scanning confocal fluorescent microscopy immunolocalized HRpICln largely to the RBC membrane. The increased staining intensity of HRpICln in a reticulocyte-enriched AA RBC density-separated fraction is consistent with a dependence of HRpICln membrane content on cell age. HRpICln and beta-actin form stable complexes in vivo, demonstrated with the yeast two-hybrid system. Low-ionic-strength extraction of ghost membranes, which results in the extraction of the spectrin-actin cytoskeleton, also results in the extraction of HRpICln, consistent with the possibility for the association of these proteins in RBCs in vivo. The results presented here establish the presence of the Cl- channel-associated protein, pICln, in human RBCs, and raises the possibility that this protein has a role in RBC Cl- transport and volume regulation in young RBCs. Moreover the association of RBC pICln with actin offers a model in which to test interactions between RBC ion channels and the cytoskeleton. PMID:9359436

  13. Two types of receptors for iron on mitochondria.

    PubMed Central

    Weaver, J; Pollack, S

    1990-01-01

    ATP, the major ligand for Fe in the reticulocyte's low-Mr chelatable pool, transfers Fe to a mitochondrial receptor from which the Fe is incorporated into haem. However, the utilization of Fe bound to this receptor for haem synthesis is slow in comparison with that bound to a second receptor that does not accept Fe from ATP, but does accept it from AMP. The major pathway by which Fe is delivered for haem synthesis may be through hydrolysis of cytosolic ATP-Fe to AMP-Fe. PMID:2241925

  14. Detection of the Messenger RNA Encoding for the Ferredoxin-Dependent Glutamate Synthase in Maize Leaf

    PubMed Central

    Commere, Bernard; Vidal, Jean; Suzuki, Akira; Gadal, Pierre; Caboche, Michel

    1986-01-01

    Ferredoxin-dependent glutamate synthase (EC 1.4.7.1), glutamate oxoglutarate aminotransferase (glutamate synthase) (GOGAT) messenger RNA was extracted from maize (Zea mays L.) leaves and partially purified through oligo(dT)-cellulose chromatography and ultracentrifugation in a sucrose gradient. mRNA were translated in vitro using a reticulocyte system. The glutamate synthase subunit was characterized by immunoprecipitation with antibodies raised against the rice (Oryza sativa L.) ferredoxin-glutamate synthase. The in vitro synthesized protein and the 145 kilodaltons genuine maize leaf subunit of GOGAT were found to comigrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis experiments. Images Fig. 1 Fig. 2 Fig. 3 PMID:16664732

  15. 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.

  16. 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. PMID:27549671

  17. Hematologic responses to hypobaric hyperoxia.

    NASA Technical Reports Server (NTRS)

    Larkin, E. C.; Adams, J. D.; Williams, W. T.; Duncan, D. M.

    1972-01-01

    Study of the effects of hypoxia, activity, and G forces on human hematopoiesis in an attempt to elucidate these phenomena more precisely. Eight subjects were exposed to an atmosphere of 100% O2 at 258 mm Hg for 30 days, and thereafter immediately exposed to transverse G forces, simulating the Gemini flights' reentry profile. All subjects displayed a significant continuous decline in red cell mass during the exposure period, as measured by the carbon monoxide-dilution method. The Cr51 method also indicated a decline in red blood corpuscle mass. The decrease in red cell mass was due to suppression of erythropoiesis and to hemolysis. After exposure to hyperoxia, all subjects exhibited elevated plasma hemoglobin levels, decreased reticulocyte counts, and decreased red cell survivals. CO production rates and urine erythropoietin levels were unchanged. Two hours after termination of exposure to hyperoxia, all subjects exhibited increased reticulocyte counts which were sustained for longer than two weeks. The progressive decrease in red cell mass was promptly arrested on return to ground level atmospheres. Within 116 days after exposure to hyperoxia, the hematologic parameters of all eight subjects had returned to control levels.

  18. Mutational analysis of hsp90 binding to the progesterone receptor.

    PubMed

    Sullivan, W P; Toft, D O

    1993-09-25

    The 90-kDa heat shock protein, hsp90, is known to associate with steroid receptors that are in the inactive state. While the biochemical function of hsp90 is unclear, this association is believed to be significant because dissociation of hsp90 occurs when receptors are activated by hormone. Complexes between hsp90 and the progesterone receptor can be formed in vitro in rabbit reticulocyte lysate. This has been shown to be an ATP-dependent process, and dissociation of the complex occurs when progesterone is added to the system. We now show that hsp90 synthesized by in vitro translation in reticulocyte lysate can form complexes with progesterone receptor that are sensitive to hormone. This system was used to analyze several mutant forms of hsp90. A series of NH2-terminal deletions showed that amino acids 1-380 can be removed from hsp90 without substantial loss of receptor binding activity. However, several deletions in the COOH-terminal half of hsp90 resulted in a partial or complete loss of this activity. Two regions, amino acids 381-441 and 601-677, appear to be particularly important for receptor binding. These studies describe a convenient and reliable method for the initial screening of hsp90 mutants, and they provide important clues to the identification of domains on hsp90 that interact with other proteins. PMID:8376394

  19. Treatment of sickle cell anemia with 5-azacytidine results in increased fetal hemoglobin production and is associated with nonrandom hypomethylation of DNA around the gamma-delta-beta-globin gene complex.

    PubMed Central

    Charache, S; Dover, G; Smith, K; Talbot, C C; Moyer, M; Boyer, S

    1983-01-01

    Increased production of fetal hemoglobin (HbF) was observed in a patient with sickle cell anemia treated with 5-azacytidine. Each of four courses of therapy resulted in a rapid and prolonged increase in the percentage of HbF containing reticulocytes (F reticulocytes) and HbF containing erythrocytes (F cells). The percentage of HbF in peripheral blood rose from 1.8 to 8.9%. The rise in HbF production was accompanied by an increase in peripheral blood hemoglobin concentration from 8 to 12 g/dl and an increase in mean erythrocyte volume. Treatment with 5-azacytidine resulted in hypomethylation of total genomic and a Y-chromosome-specific DNA fragment isolated from both peripheral blood and bone marrow. Of 15 restriction enzyme sites around the gamma-delta-beta-globin gene complex, only 2 became hypomethylated: one 107 bases 5' to the gamma G and the other 107 bases 5' to the gamma A globin genes. Images PMID:6192443

  20. Phosphorylation of eukaryotic aminoacyl-tRNA synthetases

    SciTech Connect

    Pendergast, A.M.

    1986-01-01

    The phosphorylation of the highly purified aminoacyl-tRNA synthetase complex from rabbit reticulocytes was examined. The synthetase complex contained, in addition to eight aminoacyl-tRNA synthetases, three unidentified proteins and was free of endogenous protein kinase activity. Incubation of the complex with casein kinase I in the presence of ATP resulted in the phosphorylation of four synthetases, the glutamyl-, isoleucyl-, methionyl-, and lysyl-tRNA synthetases. Phosphorylation by casein kinase I altered binding to tRNA-Sepharose such that the phosphorylated complex eluted at 190 mM NaCl instead of the 275 mM salt observed for the nonphosphorylated form. Phosphorylation by casein kinase I resulted in a significant inhibition of aminoacylation with the four synthetases; the activities of the nonphosphorylated synthetases were unchanged. One of the unidentified proteins in the complex (M/sub r/ 37,000) was also an excellent substrate for casein kinase I. A comparison of the properties and two-dimensional phosphopeptide pattern of this protein with that of casein kinase I suggest that the 37,000 dalton protein in the synthetase complex is an inactive form of casein kinase I. Two other protein kinases were shown to phosphorylate aminoacyl-tRNA synthetases in the complex. The phosphorylation of threonyl-tRNA synthetase was also investigated. Five aminoacyl-tRNA synthetases in the high molecular weight complex were shown to be phosphorylated in rabbit reticulocytes following labeling with (/sup 32/P)orthophosphate.

  1. Seasonal changes in the red blood cell indices in Arabian brood mares and their foals.

    PubMed

    Gill, J; Kompanowska-Jezierska, E

    1986-01-01

    In 34 pure-bred Arabian horses, divided into four groups (Gr. I, 10 pregnant mares; Gr. II, seven barren mares; Gr. III, 10 foals born in 1981; Gr. IV, seven foals born in 1982), seasonal changes in haemoglobin level, haematocrit value, sedimentation rate, red blood cell number and diameter, percentage of erythroblasts and reticulocytes, and index F were studied. Seasonal cyclicity was found in all groups in the haemoglobin level, haematocrit value and RBC diameter. It was also found in the sedimentation rate (PCV) and in index F, but not for the youngest foals (Gr. IV). For the RBC number the cyclicity is given in both groups of foals, and the erythroblast and reticulocyte percentage only in the older foals (Gr. III). Out of eight indices studied cyclicity was found in all foals in Gr. III, in five of the mares in Groups I and II, and in four of the youngest foals in Gr. IV. There is no difference in the cyclicity of indices studied between pregnant and barren mares. PMID:2870857

  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. Biomarkers of Hypochromia: The Contemporary Assessment of Iron Status and Erythropoiesis

    PubMed Central

    Urrechaga, Eloísa; Borque, Luís; Escanero, Jesús F.

    2013-01-01

    Iron status is the result of the balance between the rate of erythropoiesis and the amount of the iron stores. Direct consequence of an imbalance between the erythroid marrow iron requirements and the actual supply is a reduction of red cell hemoglobin content, which causes hypochromic mature red cells and reticulocytes. The diagnosis of iron deficiency is particularly challenging in patients with acute or chronic inflammatory conditions because most of the biochemical markers for iron metabolism (serum ferritin and transferrin ) are affected by acute phase reaction. For these reasons, interest has been generated in the use of erythrocyte and reticulocyte parameters, available on the modern hematology analyzers. Reported during blood analysis routinely performed on the instrument, these parameters can assist in early detection of clinical conditions (iron deficiency, absolute, or functional; ineffective erythropoiesis, including iron restricted or thalassemia), without additional cost. Technological progress has meant that in recent years modern analyzers report new parameters that provide further information from the traditional count. Nevertheless these new parameters are exclusive of each manufacturer, and they are patented. This is an update of these new laboratory test biomarkers of hypochromia reported by different manufactures, their meaning, and clinical utility on daily practice. PMID:23555091

  4. The effect of anticoagulant, storage temperature and dilution on cord blood hematology parameters over time

    PubMed Central

    FREISE, K. J.; SCHMIDT, R. L.; GINGERICH, E. L.; VENG-PEDERSEN, P.; WIDNESS, J. A.

    2010-01-01

    SUMMARY The objective of the study was to determine whether selected hematologic parameters measured on umbilical cord blood samples using an automated hematology analyzer (Sysmex XE-2100) were affected by (i) anticoagulant (the specimens were collected in EDTA vs. sodium heparin), (ii) temperature (the specimens were maintained at 4° C vs. room temperature for up to 72 h) and (iii) 1 : 5 dilution vs. undiluted using the manufacturer's diluting solution. Use of heparin, instead of EDTA, had little effect on the hematologic results (n = 8) except for lower platelet and progenitor cell counts. Results were remarkably stable for 72 h at either room temperature or 4° C except for modest red blood cell swelling at 24 h. Specimens of blood diluted at 1 : 5 had an immediate small, but significant change on white cell count (+13.3%), reticulocyte count (−11.2%) and reticulocyte hemoglobin content (−19.6%). Diluted samples did not change further over 4 h at room temperature. With a 1 : 5 dilution, analysis of 40 μl of cord blood stored for 3 days at room temperature may provide useful hematologic information with little phlebotomy loss. PMID:18422712

  5. 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. PMID:27392662

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

    PubMed Central

    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. PMID:26819490

  7. Tracking Resilience to Infections by Mapping Disease Space.

    PubMed

    Torres, Brenda Y; Oliveira, Jose Henrique M; Thomas Tate, Ann; Rath, Poonam; Cumnock, Katherine; Schneider, David S

    2016-04-01

    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

  8. 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.

  9. Diagnosis and Management of Iron Deficiency in CKD: A Summary of the NICE Guideline Recommendations and Their Rationale.

    PubMed

    Ratcliffe, Laura E K; Thomas, Wayne; Glen, Jessica; Padhi, Smita; Pordes, Ben A J; Wonderling, David; Connell, Roy; Stephens, Suzanne; Mikhail, Ashraf I; Fogarty, Damian G; Cooper, Jan K; Dring, Belinda; Devonald, Mark A J; Brown, Chris; Thomas, Mark E

    2016-04-01

    The UK-based National Institute for Health and Care Excellence (NICE) has updated its guidance on iron deficiency and anemia management in chronic kidney disease. This report outlines the recommendations regarding iron deficiency and their rationale. Serum ferritin alone or transferrin saturation alone are no longer recommended as diagnostic tests to assess iron deficiency. Red blood cell markers (percentage hypochromic red blood cells, reticulocyte hemoglobin content, or reticulocyte hemoglobin equivalent) are better than ferritin level alone at predicting responsiveness to intravenous iron. When red blood cell markers are not available, a combination of transferrin saturation < 20% and ferritin level < 100ng/mL is an alternative. In comparisons of the cost-effectiveness of different iron status testing and treatment strategies, using percentage hypochromic red blood cells > 6% was the most cost-effective strategy for both hemodialysis and nonhemodialysis patients. A trial of oral iron replacement is recommended in people not receiving an erythropoiesis-stimulating agent (ESA) and not on hemodialysis therapy. For children receiving ESAs, but not treated by hemodialysis, oral iron should be considered. In adults and children receiving ESAs and/or on hemodialysis therapy, intravenous iron should be offered. When giving intravenous iron, high-dose low-frequency administration is recommended. For all children and for adults receiving in-center hemodialysis, low-dose high-frequency administration may be more appropriate. PMID:26763385

  10. Low shear red cell oxygen transport effectiveness is adversely affected by transfusion and further worsened by deoxygenation in sickle cell disease patients on chronic transfusion therapy

    PubMed Central

    Detterich, Jon; Alexy, Tamas; Rabai, Miklos; Dongelyan, Ani; Coates, Thomas; Wood, John; Meiselman, Herbert

    2012-01-01

    BACKGROUND Simple chronic transfusion therapy (CTT) is a mainstay for stroke prophylaxis in sickle cell anemia, but its effects on hemodynamics are poorly characterized. Transfusion improves oxygen carrying capacity, reducing demands for high cardiac output. While transfusion decreases factors associated with vaso-occlusion, including percent HbS, reticulocyte count and circulating cell-free hemoglobin, it increases blood viscosity, which reduces microvascular flow. The hematocrit to viscosity ratio (HVR) is an index of red cell oxygen transport effectiveness that varies with shear stress and balances the benefits of improved oxygen capacity to viscosity-mediated impairment of microvascular flow. We hypothesized that transfusion would improve HVR at high shear despite increased blood viscosity, but would decrease HVR at low shear. STUDY DESIGN AND METHODS To test this hypothesis, we examined oxygenated and deoxygenated blood samples from 15 sickle cell patients on CTT immediately pre-transfusion and again 12–120 hours post-transfusion. RESULTS Comparable changes in hemoglobin, hematocrit, reticulocyte count and hemoglobin S with transfusion were observed in all subjects. Viscosity, hematocrit and high-shear HVR increased with transfusion while low shear HVR decreased significantly. CONCLUSION Decreased low-shear HVR suggests impaired oxygen transport to low-flow regions and may explain why some complications of sickle cell anemia are ameliorated by chronic transfusion therapy and others may be made worse. PMID:22882132

  11. Changes in the activities of some membrane-associated enzymes during in vivo ageing of the normal human erythrocyte.

    PubMed

    Kadlubowski, M; Agutter, P S

    1977-09-01

    Human erythrocytes from healthy male donors were fractionated with respect to in vivo age by simple centrifugation in order to characterize changes in the functional integrity of the membrane during the life-span of the cell. The three enzymes, Na/K-ATPase, glyceraldehyde-3-phosphate dehydrogenase and NADH-ferricyanide reductase, were found not to change with age, but significant age-dependent decreases were observed in the cases of acetylcholinesterase, phosphoglycerate kinase, purine nucleoside phosphorylase, adenylate kinase, Mg-ATPase and alkaline phosphatase. The possibility that these changes were attributable to mechanisms other than age-related inactivation, such as reticulocyte contamination, differential resealing and crypticity, was investigated. Only the decrease in acetylcholinesterase could be explained wholly in terms of reticulocyte contamination. A decrease in membrane integrity on ageing was observed, which accounted for approximately half the change in alkaline phosphatase and may have contributed to the other enzyme activity changes. This membrane integrity effect masked a real decrease in the highly cryptic NADH-ferricyanide reductase, this decrease being apparent only after total disaggregation of the membrane with nonionic surfactant. PMID:145240

  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. 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.

  14. DNA strand break by 2,5-dimethyl-4-hydroxy-3(2H)-furanone, a fragrant compound in various foodstuffs.

    PubMed

    Hiramoto, K; Aso-o, R; Ni-iyama, H; Hikage, S; Kato, T; Kikugawa, K

    1996-01-16

    2,5-Dimethyl-4-hydroxy-3(2 H)-furanone (DMHF), produced by Maillard reaction of sugar/amino acid and found in various foodstuffs, showed mutagenicity to Salmonella typhimurium TA100 strain with and without S9 mix, and induced micronucleated mouse peripheral reticulocytes. DNA strand breaking activity of the compound at pH 7.4 increased with the increasing dose of the compound and with the increasing incubation time. The breaking activity was inhibited in the presence of superoxide dismutase, catalase, hydroxyl radical scavengers, spin trapping agents, thiol compounds and metal chelators, and also by removal of dissolved oxygen from the incubation mixture. Addition of Fe(III) ion to the incubation mixture enhanced the breaking activity. Incubation of DMHF with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) gave electron spin resonance signals characteristic to DMPO-OH adduct, indicating generation of hydroxyl radical. It was found that DMHF generated hydroxyl radical with an aid of a trace amount of metal ions, and induced DNA strand breaking. Mutagenicity and induction of micronucleated reticulocytes by DMHF may be caused as a result of DNA modification via hydroxyl radical. PMID:8569798

  15. The herpes simplex virus vhs protein induces endoribonucleolytic cleavage of target RNAs in cell extracts.

    PubMed

    Elgadi, M M; Hayes, C E; Smiley, J R

    1999-09-01

    The herpes simplex virus virion host shutoff (vhs) protein (UL41 gene product) is a component of the HSV virion tegument that triggers shutoff of host protein synthesis and accelerated mRNA degradation during the early stages of HSV infection. Previous studies have demonstrated that extracts from HSV-infected cells and partially purified HSV virions display vhs-dependent RNase activity and that vhs is sufficient to trigger accelerated RNA degradation when expressed as the only HSV protein in an in vitro translation system derived from rabbit reticulocytes. We have used the rabbit reticulocyte translation system to characterize the mode of vhs-induced RNA decay in more detail. We report here that vhs-dependent RNA decay proceeds through endoribonucleolytic cleavage, is not affected by the presence of a 5' cap or a 3' poly(A) tail in the RNA substrate, requires Mg(2+), and occurs in the absence of ribosomes. Intriguingly, sites of preferential initial cleavage were clustered over the 5' quadrant of one RNA substrate that was characterized in detail. The vhs homologue of pseudorabies virus also induced accelerated RNA decay in this in vitro system. PMID:10438802

  16. General RNA binding proteins render translation cap dependent.

    PubMed Central

    Svitkin, Y V; Ovchinnikov, L P; Dreyfuss, G; Sonenberg, N

    1996-01-01

    Translation in rabbit reticulocyte lysate is relatively independent of the presence of the mRNA m7G cap structure and the cap binding protein, eIF-4E. In addition, initiation occurs frequently at spurious internal sites. Here we show that a critical parameter which contributes to cap-dependent translation is the amount of general RNA binding proteins in the extract. Addition of several general RNA binding proteins, such as hnRNP A1, La autoantigen, pyrimidine tract binding protein (hnRNP I/PTB) and the major core protein of cytoplasmic mRNP (p50), rendered translation in a rabbit reticulocyte lysate cap dependent. These proteins drastically inhibited the translation of an uncapped mRNA, but had no effect on translation of a capped mRNA. Based on these and other results, we suggest that one function of general mRNA binding proteins in the cytoplasm is to promote ribosome binding by a 5' end, cap-mediated mechanism, and prevent spurious initiations at aberrant translation start sites. Images PMID:9003790

  17. Hepcidin/Ferritin Quotient Helps to Predict Spontaneous Recovery from Iron Loss following Blood Donation

    PubMed Central

    Lotfi, Ramin; Kroll, Christine; Plonné, Dietmar; Jahrsdörfer, Bernd; Schrezenmeier, Hubert

    2015-01-01

    Summary Background Iron supplementation is generally recommended for blood donors even though there are inter-individual differences in iron homeostasis. Methods Ferritin levels of repeat donors were compared with first-time donors, retrospectively. Prospectively, we tested 27 male repeat donors for the following parameters at the day of blood donation as well as 1, 3, 7, 10, and 56 days thereafter: ferritin, hepcidin, transferrin, transferrin receptor, hemoglobin, erythropoietin, reticulocytes, hemoglobin in reticulocyte, twisted gastrulation protein homolog 1, and growth differentiation factor-15. Results 56 days after blood donation, donors' average ferritin dropped to 55% (range 30-100%) compared to the initial value. Of all tested parameters hepcidin showed the highest and most significant changes beginning 1 day after donation and lasting for the whole period of 56 days. Along with ferritin, there was a high variation in hepcidin levels indicating inter-individual differences in hepcidin response to iron loss. Donors with a hepcidin/ferritin quotient < 0.3 regained 60% of their initial ferritin after 56 days, while those with a quotient ≥ 0.3 reached less than 50%. Conclusion As hepcidin appears to integrate erythropoietic and iron-loading signals, clinical measurement of hepcidin (together with the hepcidin-ferritin ratio) may become a useful indicator of erythropoiesis and iron kinetics. PMID:26733771

  18. 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.

  19. [THE METHODICAL APPROACHES TO DIAGNOSTIC OF NIGHT PAROXYSMAL HEMOGLOBINURIA].

    PubMed

    Plekhanova, O S; Naumova, E V; Lugovskaya, S A; Potchtar, M E; Bugrov, I Yu; Dolgov, V V

    2016-03-01

    The article presents diagnostic of night paroxysmal hemoglobinuria. The night paroxysmal hemoglobinuria is an orphan disease characterized by absence of GPI-anchor on blood cells as a result of mutation of PIG-A gene on the short arm of X-chromosome. The particular proteins bounded with GPI-anchor implement function of defense from activation of components of complement and development of membrane-attacking complex. The erythrocytes exposed to destruction in bloodstream are among the most impacted. Therefore, one of the main signs of night paroxysmal hemoglobinuria is complement-depending intravascular hemolysis which indicators for a long time played a key role in diagnostic of night paroxysmal hemoglobinuria. The actual technique of diagnostic of night paroxysmal hemoglobinuria is flow cytometry. The analysis of night paroxysmal hemoglobinuria clone is recommended to patients with hemolysis of unclear genesis, thrombosis of cerebral and abdominal veins, thrombocytopenia and macrocytosis and also patients with AA, myelodysplastic syndrome, myelofibrosis. The international protocol recommended by the International Society of Clinical Cytometry (2010) is implemented to diagnose night paroxysmal hemoglobinuria. The original technique of evaluation of reticulocytes was developed with purpose to detect night paroxysmal hemoglobinuria clone. The high correlation was substantiated between size of night paroxysmal hemoglobinuria clone measured among reticulocytes according to proposed mode and night paroxysmal hemoglobinuria clone measured among granulocytes and monocytes detected according international standardized approach. PMID:27506106

  20. Recombinant human erythropoietin in the prevention of late anemia in intrauterine transfused neonates with Rh-isoimmunization.

    PubMed

    Zuppa, Antonio Alberto; Alighieri, Giovanni; Calabrese, Valentina; Visintini, Federica; Cota, Francesco; Carducci, Chiara; Antichi, Eleonora; Noia, Giuseppe Antonio; Fortunato, Giuseppe; Romagnoli, Costantino

    2010-04-01

    The majority of neonates with Rh-isoimmunization develops late anemia between the second and the sixth week of life. We report the effectiveness of recombinant human erythropoietin (rHuEPO) in preventing late anemia in 25 intrauterine and nonintrauterine-transfused neonates. The neonates were treated from 11+/-4 days after birth to 26+/-14 days (400 U/kg/d of rHuEpo, administered subcutaneously). During rHuEpo therapy, vitamin E, calcium folinate, and iron maltose were administered intramuscularly on a daily basis. Hematocrit, platelet, and neutrophil counts did not differ significantly before and after 21-days therapy. However, average values for reticulocyte showed a significant increase. The hematocrit values in the non-intrauterine transfusion (IUT) group increased progressively from the beginning to the end of the treatment, whereas that in the IUT group remained stable. Reticulocyte count increased during treatment in both groups, but it was significantly elevated in the non-IUT group only. Moreover, we observed that only neonates transfused with IUTs needed transfusions before and after treatment. This study suggests the effectiveness of rHuEpo therapy in the treatment of neonates with Rh-isoimmunization and it highlights how IUTs decrease the neonatal response efficacy. Larger, better if multicentric, randomized controlled trial are needed to definitely state whether rHuEPO safely decreases the incidence of late onset anemia. PMID:20216236

  1. 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. PMID:24168396

  2. Safety and mutagenicity evaluation of Vigiis 101 powder made from Lactobacillus paracasei subsp. paracasei NTU 101.

    PubMed

    Tseng, Wei-Ting; Shih, Tsung-Wei; Liu, Shing-Hwa; Pan, Tzu-Ming

    2015-03-01

    The aim of the present work was to assess the genotoxic activity and the potential for toxicity upon repeated dosing of "Vigiis 101" powder, a probiotic consisting of dried bacteria Lactobacillus paracasei subsp. paracasei NTU 101. Results of the Ames test in Salmonella typhimurium strains TA1537, TA98, TA100, TA102, and TA1535 showed that Vigiis 101 (⩽5 mg per plate) was not mutagenic. We used experiments on ICR mice to evaluate the genotoxicity of Vigiis 101. Compared to the control, high-dose Vigiis 101 administration (16.72 g per kg of body weight) did not cause significant changes either in the number of reticulocytes or in the percentage (occurrence) of micronucleated reticulocytes. A mammalian chromosomal aberration test showed that the number of Chinese hamster ovary cells with abnormal chromosomes was <4% after Vigiis 101 treatment (maximal concentration was 5 mg/ml). A 28-day oral toxicity assay in Wistar rats was performed to assess the no-observed-adverse-effect level of Vigiis 101. Compared to the control, high-dose Vigiis 101 administration (5000 mg/kg/day) had no effects on mortality and body weight and did not cause toxicopathological lesions. Taken together, these results show that Vigiis 101 has no significant mutagenic or toxic effects. PMID:25481278

  3. Hematological Profile and Martial Status in Rugby Players during Whole Body Cryostimulation

    PubMed Central

    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. PMID:23383348

  4. Immunogenicity of a Synthetic Vaccine Based on Plasmodium vivax Duffy Binding Protein Region II

    PubMed Central

    Ntumngia, Francis B.; Barnes, Samantha J.; McHenry, Amy M.; George, Miriam T.; Schloegel, Jesse

    2014-01-01

    Molecules that play a role in Plasmodium merozoite invasion of host red blood cells represent attractive targets for blood-stage vaccine development against malaria. In Plasmodium vivax, merozoite invasion of reticulocytes is mediated by the Duffy binding protein (DBP), which interacts with its cognate receptor, the Duffy antigen receptor for chemokines, on the surface of reticulocytes. The DBP ligand domain, known as region II (DBPII), contains the critical residues for receptor recognition, making it a prime target for vaccine development against blood-stage vivax malaria. In natural infections, DBP is weakly immunogenic and DBPII allelic variation is associated with strain-specific immunity, which may compromise vaccine efficacy. In a previous study, a synthetic vaccine termed DEKnull that lacked an immunodominant variant epitope in DBPII induced functional antibodies to shared neutralizing epitopes on the native Sal1 allele. Anti-DEKnull antibody titers were lower than anti-Sal1 titers but produced more consistent, strain-transcending anti-DBPII inhibitory responses. In this study, we further characterized the immunogenicity of DEKnull, finding that immunization with recombinant DEKnull produced an immune response comparable to that obtained with native recombinant DBP alleles. Further investigation of DEKnull is necessary to enhance its immunogenicity and broaden its specificity. PMID:24964808

  5. 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. PMID:20842374

  6. 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.

  7. Hematological responses to arsine exposure: quantitation of exposure response in mice

    SciTech Connect

    Peterson, D.P.; Bhattacharyya, M.H.

    1985-01-01

    Hematological responses of the of mice to arsine exposures for 1 hr at 5 to 26 part per million volume (ppmv) are described. Exposure concentrations ranged from a no-effect level for the endpoints studied (5 ppmv) to a concentration lethal to all mice in 4 days (26 ppmv). Hematocrit values at 24 hr after exposure decreased linearly with increasing arsine concentration in the range 5 to 26 ppmv; the hematocrits of the 26-ppmv group reached 10.5% at 24 hr, compared to 48.4% for control mice. Hematocritis of mice from all surviving groups were at or slightly above control values by 11 days after exposure. Changes in numbers of erythrocytes paralleled changes in hematocrit. Significant increases in circulating reticulocytes occurred at 1 and 5 days after exposure ; reticulocyte values returned to control levels by 11 days after exposure. Changes in erythrocyte osmotic fragility were observed in mice exposed to 15 and 26 ppmv arsine. 22 references, 3 figures, 3 tables.

  8. Daily variation in radiosensitivity of circulating blood cells and bone marrow cell density in mice

    SciTech Connect

    Tabatabai, R.N.

    1984-01-01

    Mice on a 12/12 light/dark cycle were bled during a twenty-four hour period each week for eight weeks to establish daily values of circulating blood cells. No significant daily variation was found in total red blood cells, hematocrit, or percentage of reticulocytes. A significant (P < 0.001) daily variation was found in total white blood cells, with the minimum occurring at 8 PM and the maximum occurring during the daylight hours from 8 a.m. to 2 p.m. Mice were then exposed to 0 R, 20 R, 50 R, or 100 R of x-radiation to determine what dose significantly reduces the total white cell count in circulating blood. It was found that 100 R significantly (P < .05) reduces the total white cell count over a four week period post-exposure. To determine if circulating blood cells and bone marrow cells show a diurnal radiosensitivity, mice were exposed to 100 R or 200 R of x-radiation at noon or midnight. Hematocrits, reticulocyte and white blood cell counts, daily white blood cell rhythm, and bone marrow cell density indicate that these mice were more radiosensitive at night.

  9. 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.

  10. Adenosine 5'-triphosphate is required for the assembly of 11S seed proglobulins in vitro.

    PubMed Central

    Nam, Y W; Jung, R; Nielsen, N C

    1997-01-01

    Seed protein proglobulins were synthesized from cDNAs in reticulocyte lysates. Most proglobulins were recovered as trimers when translation rates were low, but mostly monomers were recovered at high translation rates. The prevalence of monomers was accompanied by elevated amounts of insoluble protein recovered at the bottom of sucrose density gradients. Apyrase treatment of translation mixtures after synthesis, but before significant assembly occurred, drastically reduced trimer assembly and increased the proportion of insoluble aggregate. These observations indicated that ATP is required for protein folding and/or trimer assembly. The appearance of insoluble aggregated protein when rates of synthesis were elevated or when ATP was absent suggested that protein misfolding had occurred. Trimer assembly was stimulated when wheat germ translation mixtures defective in supporting efficient trimer assembly were supplemented with fractions isolated from endoplasmic reticula of developing pea (Pisum sativum) seeds. Molecular chaperones are likely involved in folding and/or assembly of proglobulin trimers both in reticulocyte lysates and in seeds. Consistent with this hypothesis, trimer formation was reduced when carboxymethylated bovine albumin and alpha-casein, considered to mimic proteins with extended chain and molten globular conformations and thereby compete for Hsp70- and Hsp60-type molecular chaperones, respectively, were introduced into translation mixtures. PMID:9414566

  11. 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

  12. The CD14+CD16+ Inflammatory Monocyte Subset Displays Increased Mitochondrial Activity and Effector Function During Acute Plasmodium vivax Malaria

    PubMed Central

    Antonelli, Lis R. V.; Leoratti, Fabiana M. S.; Costa, Pedro A. C.; Rocha, Bruno C.; Diniz, Suelen Q.; Tada, Mauro S.; Pereira, Dhelio B.; Teixeira-Carvalho, Andrea; Golenbock, Douglas T.; Gonçalves, Ricardo; Gazzinelli, Ricardo T.

    2014-01-01

    Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax–infected patients display significant increase in circulating monocytes, which were defined as CD14+CD16− (classical), CD14+CD16+ (inflammatory), and CD14loCD16+ (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16+ cells, in particular the CD14+CD16+ monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14+ were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14+CD16+ monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14+CD16+ cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection. PMID:25233271

  13. The CD14+CD16+ inflammatory monocyte subset displays increased mitochondrial activity and effector function during acute Plasmodium vivax malaria.

    PubMed

    Antonelli, Lis R V; Leoratti, Fabiana M S; Costa, Pedro A C; Rocha, Bruno C; Diniz, Suelen Q; Tada, Mauro S; Pereira, Dhelio B; Teixeira-Carvalho, Andrea; Golenbock, Douglas T; Gonçalves, Ricardo; Gazzinelli, Ricardo T

    2014-09-01

    Infection with Plasmodium vivax results in strong activation of monocytes, which are important components of both the systemic inflammatory response and parasite control. The overall goal of this study was to define the role of monocytes during P. vivax malaria. Here, we demonstrate that P. vivax-infected patients display significant increase in circulating monocytes, which were defined as CD14(+)CD16- (classical), CD14(+)CD16(+) (inflammatory), and CD14loCD16(+) (patrolling) cells. While the classical and inflammatory monocytes were found to be the primary source of pro-inflammatory cytokines, the CD16(+) cells, in particular the CD14(+)CD16(+) monocytes, expressed the highest levels of activation markers, which included chemokine receptors and adhesion molecules. Morphologically, CD14(+) were distinguished from CD14lo monocytes by displaying larger and more active mitochondria. CD14(+)CD16(+) monocytes were more efficient in phagocytizing P. vivax-infected reticulocytes, which induced them to produce high levels of intracellular TNF-α and reactive oxygen species. Importantly, antibodies specific for ICAM-1, PECAM-1 or LFA-1 efficiently blocked the phagocytosis of infected reticulocytes by monocytes. Hence, our results provide key information on the mechanism by which CD14(+)CD16(+) cells control parasite burden, supporting the hypothesis that they play a role in resistance to P. vivax infection. PMID:25233271

  14. Plasmodium vivax trophozoite-stage proteomes

    PubMed Central

    Anderson, D.C.; Lapp, Stacey A.; Akinyi, Sheila; Meyer, Esmeralda V.S.; Barnwell, John W.; Korir-Morrison, Cindy; Galinski, Mary R.

    2015-01-01

    Plasmodium vivax is the causative infectious agent of 80–300 million annual cases of malaria. Many aspects of this parasite’s biology remain unknown. To further elucidate the interaction of P. vivax with its Saimiri boliviensis host, we obtained detailed proteomes of infected red blood cells, representing the trophozoite-enriched stage of development. Data from two of three biological replicate proteomes, emphasized here, were analyzed using five search engines, which enhanced identifications and resulted in the most comprehensive P. vivax proteomes to date, with 1375 P. vivax and 3209 S. boliviensis identified proteins. Ribosome subunit proteins were noted for both P. vivax and S. boliviensis, consistent with P. vivax’s known reticulocyte host–cell specificity. A majority of the host and pathogen proteins identified belong to specific functional categories, and several parasite gene families, while 33% of the P. vivax proteins have no reported function. Hemoglobin was significantly oxidized in both proteomes, and additional protein oxidation and nitration was detected in one of the two proteomes. Detailed analyses of these post-translational modifications are presented. The proteins identified here significantly expand the known P. vivax proteome and complexity of available host protein functionality underlying the host–parasite interactive biology, and reveal unsuspected oxidative modifications that may impact protein function. Biological significance Plasmodium vivax malaria is a serious neglected disease, causing an estimated 80 to 300 million cases annually in 95 countries. Infection can result in significant morbidity and possible death. P. vivax, unlike the much better-studied Plasmodium falciparum species, cannot be grown in long-term culture, has a dormant form in the liver called the hypnozoite stage, has a reticulocyte host–cell preference in the blood, and creates caveolae vesicle complexes at the surface of the infected reticulocyte

  15. Characterization of cell-free protein-synthesis systems from undeveloped and developing Artemia embryos.

    PubMed Central

    Moreno, A; Mendez, R; de Haro, C

    1991-01-01

    We have developed and characterized translationally active cell-free systems from Artemia embryos at different developmental times. The optimized lysates from 16 h-developed embryos incorporated radiolabelled amino acids into polypeptides for up to 120 min. The polypeptides synthesized ranged in Mr from 150,000 to 10,000, suggesting that the endogenous mRNA was capable of directing the synthesis of complete polypeptides. Similar results were obtained by using lysates from early developmental stages; even the cell-free system prepared from 1 h-developed embryos was partially active in protein synthesis. Furthermore, all these lysates were capable of re-initiation, as demonstrated by inhibition of initiation with the inhibitors edeine and 7-methylguanosine 5'-triphosphate. Because we found no endogenous protein-synthetic activity in the corresponding lysates from undeveloped embryos, we have used cell-free translation systems from 0 h- and 16 h-developed Artemia embryos to analyse the mechanisms limiting protein synthesis at very early developmental stages. Undeveloped-embryo lysates supplemented with nuclease-treated reticulocyte lysate were capable of translating endogenous mRNAs to give products with a wide spectrum of Mr values, but lysates of 16 h-developed embryos supplemented in this way were not further stimulated. The nuclease-treated lysate appeared to be unnecessary 5 h after resumption of development. These results suggested that a component(s) limiting translation in the undeveloped-embryo lysate was provided by the nuclease-treated reticulocyte lysate, and that this component(s) no longer limited protein synthesis after development. In view of these results, partially fractionated reticulocyte lysates were tested for restoration of protein-synthetic activity in the undeveloped embryo lysate. A high-salt ribosomal wash devoid of ribosomal subunits, which is considered a crude polypeptide-initiation-factor preparation, also restored translation activity

  16. 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

  17. alpha-Thalassemia caused by an unstable alpha-globin mutant.

    PubMed Central

    Liebhaber, S A; Kan, Y W

    1983-01-01

    In a previous study, molecular cloning of the alpha-globin genes from a patient with nondeletion Hb-H disease (genotype--/alpha alpha) showed that a single nucleotide mutation (CTG to CCG) in one of the genes resulted in a leucine to proline substitution. This paper describes the approach we used to detect the abnormal alpha-globin chain. The chain was identified using a cell-free translation system. It turned over rapidly both in vitro and in vivo in the patient's reticulocytes. The unusual feature of this unstable alpha-globin is that the alpha-globin deficiency causes alpha-thalassemia. Simple heterozygotes for this lesion (alpha Pro alpha/alpha alpha) resemble alpha-thalassemia carriers and do not exhibit the hemolytic anemia usually associated with unstable hemoglobins. Images PMID:6826718

  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. Innate Immune Activation during Salmonella Infection Initiates Extramedullary Erythropoiesis and Splenomegaly

    PubMed Central

    Jackson, Amy; Nanton, Minelva R.; O’Donnell, Hope; Akue, Adovi D.; McSorley, Stephen J.

    2011-01-01

    Systemic Salmonella infection commonly induces prolonged splenomegaly in murine or human hosts. Although this increase in splenic cellularity is often assumed to be due to the recruitment and expansion of leukocytes, the actual cause of splenomegaly remains unclear. We monitored spleen cell populations during Salmonella infection and found that the most prominent increase is found in the erythroid compartment. At the peak of infection, the majority of spleen cells are immature CD71−Ter119+ reticulocytes, indicating that massive erythropoiesis occurs in response to Salmonella infection. Indeed, this increase in RBC precursors corresponded with marked elevation of serum erythropoietin (EPO). Furthermore, the increase in RBC precursors and EPO production required innate immune signaling mediated by Myd88/TRIF. Neutralization of EPO substantially reduced the immature RBC population in the spleen and allowed a modest increase in host control of infection. These data indicate that early innate immunity to Salmonella initiates marked splenic erythropoiesis and may hinder bacterial clearance. PMID:20952675

  20. Effect of Tectona grandis on phenylhydrazine-induced anaemia in rats.

    PubMed

    Diallo, Aboudoulatif; Gbeassor, Messanvi; Vovor, Ahoefa; Eklu-Gadegbeku, Kwashie; Aklikokou, Kodjo; Agbonon, Amegnona; Abena, Ange A; de Souza, Comla; Akpagana, Koffi

    2008-07-01

    Traditional oral report indicates that Tectona grandis is used in the treatment of anaemia in Togo. For this purpose, the extract of T. grandis leaves is evaluated on anaemia model of rat induced by intraperitoneal injection of phenylhydrazine at 40 mg/kg for 2 days. Oral administration of T. grandis extract at 1 g/kg/day and 2 g/kg/day, to the rats previously treated with phenylhydrazine, increased the concentration of haemoglobin, red blood cells number, haematocrit and reticulocytes rate. Moreover, the extract of T. grandis enhanced the osmotic resistance of the red blood cells that confirm the important presence of young red blood cells. These results support partially the traditional use of T. grandis in the treatment of anaemia. PMID:18504074

  1. 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.

  2. 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

  3. alpha-Amanitin-insensitive transcription of mouse beta major-globin 5'-flanking and structural gene sequences correlates with mRNA expression.

    PubMed Central

    Carlson, D P; Ross, J

    1984-01-01

    A small proportion of the RNAs of mouse reticulocytes consists of beta major-globin mRNA sequences linked to sequences transcribed from the 5'-flanking region of the beta major-globin gene. These upstream RNAs are polyadenylylated and contain 700-800 nucleotides, and their 5' regions are heterogeneous. RNAs with similar or identical 5' regions are transcribed in cell-free extracts from a circular mouse beta major-globin gene template. Synthesis of most of the upstream RNAs in vitro is not inhibited by low levels (1 microgram/ml) of alpha-amanitin, indicating that they are transcribed by an enzyme(s) different from RNA polymerase II. During culture of mouse erythroleukemia cells with dimethyl sulfoxide, globin mRNA and upstream RNAs accumulate with similar kinetics. In contrast, upstream RNAs are not detected in hemin-treated cells. Images PMID:6595660

  4. Effect of long-term supplementation of folate on folate status in plasma and erythrocytes.

    PubMed

    Heseker, H; Schmitt, G

    1987-06-01

    Folate nutritional status was estimated by radioassay of folate levels in plasma and erythrocytes during and after a long-term supplementation of folic acid. A 1-mg dose of folic acid per day was administered orally to 6 healthy subjects for 17 weeks. After 4 weeks of supplementation the mean folate concentration in plasma reached 11 ng/ml and remained constant thereafter, but decreased exponentially after stopping the supplementation. However, the folate concentrations in reticulocytes and erythrocytes increased linearly in all subjects during the supplementation. These results suggest that folate-rich, young erythrocytes are mixed at a constant rate with circulating ripe ones, which have a lower folate content, during folate supplementation. PMID:3668697

  5. Laboratory Evaluation of Anemia

    PubMed Central

    Wallerstein, Ralph O.

    1987-01-01

    The laboratory evaluation of anemia begins with a complete blood count and reticulocyte count. The anemia is then categorized as microcytic, macrocytic or normocytic, with or without reticulocytosis. Examination of the peripheral smear and a small number of specific tests confirm the diagnosis. The serum iron level, total iron-binding capacity, serum ferritin level and hemoglobin electrophoresis generally separate the microcytic anemias. The erythrocyte size-distribution width may be particularly helpful in distinguishing iron deficiency from thalassemia minor. Significant changes have occurred in the laboratory evaluation of macrocytic anemia, and a new syndrome of nitrous oxide-induced megaloblastosis and neurologic dysfunction has been recognized. A suggested approach to the hemolytic anemias includes using the micro-Coombs' test and ektacytometry. Finally, a number of causes have been identified for normocytic anemia without reticulocytosis, including normocytic megaloblastic anemia and the acquired immunodeficiency syndrome. PMID:3577135

  6. Killing of human tumor cells in culture with adriamycin conjugates of human transferrin

    SciTech Connect

    Yeh, C.J.; Faulk, W.P.

    1984-07-01

    Receptors for human transferrin (Trf) in high density are found on reticulocytes and syncytiotrophoblast, but most unstimulated, normal adult cells do not bind Trf. In contrast, leukemia and breast adenocarcinoma cells have been shown to manifest Trf receptors, raising the possibility that these receptors might be employed to bind cytotoxic Trf conjugates. Trf was conjugated with adriamycin (Adr) and it was shown that the conjugates are bound by Trf receptors on plasma membranes of Daudi and HL-60 cells, following which Adr is identified in the nuclei of these cells. The biological effect of Adr is quantitated by the inhibition of tritiated thymidine uptake, and subsequent cell death is measured by trypan blue exclusion. The killing correlates directly with both the time of exposure and the amount of conjugate employed. These results suggest that such cytotoxic Trf conjugates hold promise for selective in vivo killing of some malignant cells.

  7. Depurination of ribosomal RNA and inhibition of viral RNA translation by an antiviral protein of Celosia cristata.

    PubMed

    Baranwal, V K; Tumer, Nilgun E; Kapoor, H C

    2002-10-01

    An antiviral protein (25 kD) isolated from leaves of Celosia cristata (CCP 25) was tested for depurination study on ribosomal RNA from yeast. Ribosomal RNA yielded 360 nucleotide base fragment after treatment with CCP 25 indicating that CCP 25 was a ribosome inactivating protein. CCP 25 also inhibited translation of brome mosaic virus (BMV) and pokeweed mosaic virus (PMV) RNAs in rabbit reticulocyte translation system. The radioactive assay showed that incorporation of [35S]-methionine was less in translation proteins of BMV nucleic acid when CCP 25 was added to translation system. This indicated that antiviral protein from Celosia cristata not only depurinated ribosomal RNA but also inhibited translation of viral RNA in vitro. PMID:12693705

  8. Sativin: a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon.

    PubMed

    Ye, X Y; Wang, H X; Ng, T B

    2000-07-01

    An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani. PMID:10968407

  9. Seasonal changes in gene expression represent cell-type composition in whole blood

    PubMed Central

    De Jong, Simone; Neeleman, Marjolein; Luykx, Jurjen J.; Ten Berg, Maarten J.; Strengman, Eric; Den Breeijen, Hanneke H.; Stijvers, Leon C.; Buizer-Voskamp, Jacobine E.; Bakker, Steven C.; Kahn, René S.; Horvath, Steve; Van Solinge, Wouter W.; Ophoff, Roel A.

    2014-01-01

    Seasonal patterns in behavior and biological parameters are widespread. Here, we examined seasonal changes in whole blood gene expression profiles of 233 healthy subjects. Using weighted gene co-expression network analysis, we identified three co-expression modules showing circannual patterns. Enrichment analysis suggested that this signal stems primarily from red blood cells and blood platelets. Indeed, a large clinical database with 51 142 observations of blood cell counts over 3 years confirmed a corresponding seasonal pattern of counts of red blood cells, reticulocytes and platelets. We found no direct evidence that these changes are linked to genes known to be key players in regulating immune function or circadian rhythm. It is likely, however, that these seasonal changes in cell counts and gene expression profiles in whole blood represent biological and clinical relevant phenomena. Moreover, our findings highlight possible confounding factors relevant to the study of gene expression profiles in subjects collected at geographical locations with disparaging seasonality patterns. PMID:24399446

  10. Isolation and characterization of a Xenopus laevis C protein cDNA: structure and expression of a heterogeneous nuclear ribonucleoprotein core protein.

    PubMed Central

    Preugschat, F; Wold, B

    1988-01-01

    The C proteins are major components of heterogeneous nuclear ribonucleoprotein complexes in nuclei of vertebrate cells. To begin to describe their structure, expression, and function we isolated and determined the DNA sequence of Xenopus laevis C protein cDNA clones. The protein predicted from the DNA sequence has a molecular mass of 30,916 kDa and is very similar to its human counterpart. Although mammalian genomes contain many copies of C protein sequence, the Xenopus genome contains few copies. When C protein RNA was synthesized in vitro and microinjected into stage-VI Xenopus oocytes, newly synthesized C proteins were efficiently localized in the nucleus. In vitro rabbit reticulocyte lysate and in vivo Xenopus oocyte translation systems both produce from a single mRNA two discrete polypeptide species that accumulate in a ratio similar to that of mammalian C1 and C2 proteins in vivo. Images PMID:2904678

  11. An inhibitory factor for cell-free protein synthesis from Salmonella enteritidis exhibits cytopathic activity against Chinese hamster ovary cells.

    PubMed

    Iwamaru, Y; Miyake, M; Arii, J; Tanabe, Y; Noda, M

    2001-12-01

    A factor inhibiting cell-free protein synthesis was purified from Salmonella enteritidis cell lysate by sequential ammonium sulfate precipitation, chromatography on anion exchange and hydrophobic interaction columns, and polyacrylamide disc gel electrophoresis. The purified factor, which was named SIPS (Salmonella inhibitor of protein synthesis), inhibited in vitro protein synthesis in rabbit reticulocyte lysate and had a molecular mass of 38 kDa, estimated by PAGE under denaturing conditions. SIPS was also cytopathic for Chinese hamster ovary cells. The N-terminal amino acid sequence (20 residues) of SIPS was found to be identical to that of mature L-asparaginase II of Escherichia coli. Indeed, the purified SIPS exhibited asparaginase activity, E. coli L-asparaginase II had cytopathic activity and inhibited in vitro protein synthesis. The results suggest that at least a part of cytotoxicity and inhibition of cell-free protein synthesis caused by S. enteritidis is a property of the bacterial L-asparaginase. PMID:11747376

  12. Developmental changes in translatable mRNAs for the cerebral enolase isozymes alphaalpha and gammagamma.

    PubMed Central

    Zeitoun, Y; Lamandé, N; Keller, A; Gros, F; Legault-Demare, L

    1983-01-01

    Using the rabbit reticulocyte cell-free translation system we have estimated during ontogenesis the proportions of in vitro translatable alpha and gamma brain enolase mRNAs, which are two minor mRNA species. No polypeptide precursor to these enzyme subunits appears to be synthesized during translation in vitro. During brain development, the changes in translatable alpha and gamma mRNA content seem to parallel those of the corresponding antigens. The proportion of each of the enolase mRNAs is highest in adult mouse brain. Mechanisms controlling alpha and gamma antigen expression are discussed. In order to prepare the specific cDNA probes, purification of alpha and gamma mRNAs was undertaken. Images Fig. 1. Fig. 2. Fig. 4. PMID:11892794

  13. [Favism presenting as an acute renal failure: report of one case].

    PubMed

    Torres C, Demetrio; Chandía C, Mauricio

    2012-08-01

    We report a 67-year-old man presenting with abdominal pain of acute onset, pallor, jaundice and behavioral changes after ingestion of fava beans. In the initial evaluation he appeared acutely ill and had resting dyspnea, edema and jaundice. His initial laboratory assessment disclosed azotemia, elevated lactate dehydrogenase levels, a low hemoglobin concentration (4.9 /dL) and a high corrected reticulocyte count (4,7%) with negative direct and indirect Coombs' test. The patient was transferred to the ICU, where he received support therapy with hemodialysis, mechanical ventilation, vasoactive drugs and transfusions of packed red cells. The evolution after 1 month was favorable and he was discharged without anemia and with normal renal function. Three months after discharge, the glucose-6-phosphate-dehydrogenase screening study did not demonstrate detectable enzymatic activity. PMID:23282778

  14. Finding the sweet spots of inhibition: understanding the targets of a functional antibody against Plasmodium vivax Duffy binding protein

    PubMed Central

    Ntumngia, Francis B.; King, Christopher L.; Adams, John H.

    2014-01-01

    Plasmodium vivax Duffy binding protein region II (DBPII) is an essential ligand for reticulocyte invasion, thereby making this molecule an attractive vaccine candidate against asexual blood-stage P. vivax. Similar to other Plasmodium blood-stage vaccine candidates, strain-specific immunity due to DBPII allelic variation may complicate vaccine efficacy. Targeting immune responses to more conserved epitopes that are potential targets of strain-transcending neutralizing immunity is necessary to avoid induction of strain-specific responses to dominant variant epitopes. In this article, we focus on different approaches to optimize the design of DBP immunogenicity to target conserved epitopes, which is important for developing a broadly effective vaccine against P. vivax. PMID:23068913

  15. 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.

  16. Production of ribosome-inactivating protein from hairy root cultures of Luffa cylindrica (L.) Roem.

    PubMed

    di Toppi, L S; Gorini, P; Properzi, G; Barbieri, L; Spanò, L

    1996-09-01

    Transformed root lines of Luffa cylindrica (L.) Roem. (Cucurbitaceae) were established by inoculation of in vitro grown plantlets with wild type Agrobacterium rhizogenes strain 1855. Cloned lines of hairy roots were tested for the presence of ribosome-inactivating proteins; crude extracts inhibited protein synthesis in a reaction mixture based on rabbit reticulocyte lysate. Inhibitory activity increased during culture period, reaching a maximum value in the stationary phase. No activity could be detected in the culture medium, nor in extracts from callus and/or suspension cultures. A ribosome-inactivating protein having specific activity of 62,100 U mg protein(-1) and a molecular mass of 26-28,000 Da was purified to homogeneity. The protein showed N-glycosidase activity on rat liver ribosomes. The results demonstrate that hairy root cultures can be successfully utilized for the in vitro production of ribosome-inactivating proteins. PMID:24178273

  17. [A case of myasthenia gravis with transient taste disorders followed by aplastic anemia after thymectomy].

    PubMed

    Osada, Osamu; Iwasaki, Akira; Nakahashi, Hirotaka; Kim, Yoshitora; Kaneko, Kou

    2016-01-01

    A 45-year-old man was admitted to our hospital because of taste disorders in March 2014. He exhibited cervical muscle weakness and left eye ptosis, which responded to Tensilon test, and was diagnosed with myasthenia gravis (MG). He developed aspiration pneumonia and myasthenic crisis, which was treated with intravenous immunoglobulin and steroid pulse therapy. All symptoms disappeared. Oral administration of prednisolone and tacrolimus was started. Chest CT revealed thymoma and extended thymectomy was performed in May 2014. In December 2014, seven months after the thymectomy, hematological examination showed pancytopenia including severe neutropenia. We diagnosed his illness as aplastic anemia (AA). Cyclosporine therapy with transfusion was administerd and led to reticulocyte count recovery. Since May 2015, hemoglobin recovery reached a blood transfusion free period. To our knowledge, this is the first case report with the patient supposed of relationship among taste disorders, AA and thymoma-associated MG. PMID:26876109

  18. 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.

  19. 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.

  20. Monovalent cation transport in irreversibly sickled cells.

    PubMed Central

    Clark, M R; Morrison, C E; Shohet, S B

    1978-01-01

    Using discontinuous density gradients of Stractan II, we have separated sickle cell blood into discrete subpopulations of reticulocytes, mature discoid cells, and irreversibly sickled cells (ISCs). We have measured active and passive fluxes of monovalent cations in mature discoid cells, ISCs, and normal control cells, also separated upon density gradients. These measurements revealed a decreased active cation transport in ISC-rich populations. However, parallel measurements of Na, K-ATPase activity showed normal ouabain-sensitive ATPase activity in ISCs. Passive permeability to external Rb was also normal in ISCs. The observation of depressed pump activity in intact ISCs, contrasted with normal ATPase activity in ISC membranes, suggests the presence of factors in the intact cell which inhibit the active transport of Na and K in ISCs. PMID:149799

  1. Hemolytic anemia with aortic stenosis resolved by urgent aortic valve replacement.

    PubMed

    Kawase, Isamu; Matsuo, Tatsuro; Sasayama, Koji; Suzuki, Hiroyuki; Nishikawa, Hideo

    2008-08-01

    A 78-year-old man with aortic stenosis complained of dark colored urine followed by recurrent chest pain and syncopal episodes. Echocardiography showed severely calcified aortic stenosis with the maximal pressure gradient of 125 mm Hg. Hemoglobin was 7.9 g/dL, lactate dehydrogenase was 2,295 IU/L, haptoglobin was less than 10 mg/dL, reticulocyte count was elevated, and Coombs' test was negative. We performed an urgent aortic valve replacement. After the surgery, the patient's urine became clear and his chest pain and syncope abated. All laboratory data returned to normal physiological values. In conclusion, the observed hemolysis was related to the aortic shear stress of a calcified aortic valve. PMID:18640351

  2. Deletion analysis of the 5' untranslated leader sequence of tobacco mosaic virus RNA.

    PubMed

    Takamatsu, N; Watanabe, Y; Iwasaki, T; Shiba, T; Meshi, T; Okada, Y

    1991-03-01

    To determine the sequences essential for viral multiplication in the 5' untranslated leader sequence of tobacco mosaic virus RNA, mutant TMV-L (a tomato strain) RNAs which carry several deletions in this 71-nucleotide sequence were constructed by an in vitro transcription system and their multiplication was analyzed by introducing mutant RNA into tobacco protoplasts by electroporation. Large deletions of the sequence from nucleotides 9 to 47 or 25 to 71 abolished viral multiplication; when about 10-nucleotide deletions were introduced throughout this 5' leader sequence, only deletion of the sequence from nucleotides 2 to 8 abolished detectable viral multiplication. This mutant RNA, however, directed the synthesis of the 130,000-molecular-weight protein in a rabbit reticulocyte lysate in vitro translation system, and consequently this 5'-proximal portion appears likely to be essential for replication. PMID:1995954

  3. Fast estimation of ATP/ADP ratio as a special step in pharmacological and toxicological studies using the cell-free translation systems.

    PubMed

    Kuznetsov, D A; Govorkov, A V; Zavijalov, N V; Sibileva, T M; Richter, V; Drawczek, J A

    1986-08-01

    We have developed a simple and effective reversed-phase HPLC procedure for rapid estimation of the ATP/ADP ratio in a cell-free translation system containing creatine kinase. Analysis of the acetone-extractable pool derived from a reticulocyte lysate cell-free system was carried out by automatic chromatography on S5CN-ODS stationary phase using a linear 10-65% pyridine elution gradient formed on the basis of methanol/water (9:1, v/v) mobile phase. This method was used to detect and characterize the inhibition of translation induced by considerable suppression of ATP resynthesis in vitro. It was shown that methyl mercury, unlike cycloheximide, pactamycin, CCl4 and barbituric acid, exerts inhibitory effect on the ATP regeneration in a cell-free translation system. PMID:3772019

  4. The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon.

    PubMed

    de Sousa, Taís Nóbrega; Kano, Flora Satiko; de Brito, Cristiana Ferreira Alves; Carvalho, Luzia Helena

    2014-08-01

    Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania. PMID:25185002

  5. The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon.

    PubMed

    Sousa, Taís Nóbrega de; Kano, Flora Satiko; Brito, Cristiana Ferreira Alves de; Carvalho, Luzia Helena

    2014-05-23

    Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania. PMID:24863975

  6. Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cell-free system.

    PubMed Central

    Maher, L J; Dolnick, B J

    1988-01-01

    Antisense oligonucleotides containing either anionic diester or neutral methylphosphonate internucleoside linkages were prepared by automated synthesis, and were compared for their ability to arrest translation of human dihydrofolate reductase (DHFR) mRNA in a nuclease treated rabbit reticulocyte lysate. In the case of oligodeoxyribonucleotides, tandem targeting of three 14-mers resulted in synergistic and complete selective inhibition of DHFR synthesis at a total oligomer concentration of 25 microM. Hybrid arrest by three or six tandem oligodeoxyribonucleoside methylphosphonates was dramatically less effective. This difference does not result from preferential recognition of hybrids involving oligodeoxyribonucleotides by endogenous RNaseH activity. A ribonuclease protection assay demonstrated that antisense oligodeoxyribonucleoside methylphosphonates bind selectively to target RNA sequences, but with 275 fold lower affinity than the corresponding oligodeoxyribonucleotides. This low binding affinity results in poor arrest of translation, and may be related to the stereochemistry of the methylphosphonate linkage. Images PMID:2836793

  7. Improved light microscopy counting method for accurately counting Plasmodium parasitemia and reticulocytemia.

    PubMed

    Lim, Caeul; Pereira, Ligia; Shardul, Pritish; Mascarenhas, Anjali; Maki, Jennifer; Rixon, Jordan; Shaw-Saliba, Kathryn; White, John; Silveira, Maria; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K; Duraisingh, Manoj T

    2016-08-01

    Even with the advances in molecular or automated methods for detection of red blood cells of interest (such as reticulocytes or parasitized cells), light microscopy continues to be the gold standard especially in laboratories with limited resources. The conventional method for determination of parasitemia and reticulocytemia uses a Miller reticle, a grid with squares of different sizes. However, this method is prone to errors if not used correctly and counts become inaccurate and highly time-consuming at low frequencies of target cells. In this report, we outline the correct guidelines to follow when using a reticle for counting, and present a new counting protocol that is a modified version of the conventional method for increased accuracy in the counting of low parasitemias and reticulocytemias. Am. J. Hematol. 91:852-855, 2016. © 2016 Wiley Periodicals, Inc. PMID:27074559

  8. Purification and properties of a new ribosome-inactivating protein with RNA N-glycosidase activity suitable for immunotoxin preparation from the seeds of Momordica cochinchinensis.

    PubMed

    Bolognesi, A; Barbieri, L; Carnicelli, D; Abbondanza, A; Cenini, P; Falasca, A I; Dinota, A; Stirpe, F

    1989-12-01

    A ribosome-inactivating protein similar to those already known (Stirpe and Barbieri (1986) FEBS Lett. 195, 1-8) was purified from the seeds of Momordica cochinchinensis. This protein, for which the name of momorcochin-S is proposed, is a glycoprotein, has an Mr of approx. 30,000, and an alkaline isoelectric point and can be considered as an iso-form of the previously purified momorcochin from the roots of M. cochinchinensis. Momorcochin-S inhibits protein synthesis by a rabbit-reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and alters rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Momorcochin-S was linked to a monoclonal antibody (8A) against human plasma cells, and the resulting immunotoxin was selectively toxic to target cells. PMID:2597699

  9. Internal 6-methyladenine residues increase the in vitro translation efficiency of dihydrofolate reductase messenger RNA.

    PubMed

    Heilman, K L; Leach, R A; Tuck, M T

    1996-07-01

    N6-Methyladenosine (m6A) is found internally in a number of mRNA molecules from higher eucaryotic cells. In these investigations, it was found that the presence of m6A residues increase the in vitro translation efficiency of capped T7 transcripts of mouse dihydrofolate reductase (DHFR) mRNA. Using an in vitro rabbit reticulocyte translation system, the formation of internal m6A residues in the DHFR transcripts resulted in a 1.5-fold increase in translated DHFR compared to transcripts void of internal m6A residues. Translation in a wheat germ system, however, resulted in no increase in translation efficiency upon m6A formation, suggesting that the mechanism may be species-specific. PMID:8925412

  10. 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. PMID:24903010

  11. 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

  12. 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.

  13. Purification and characterization of a toxin inhibiting protein synthesis from Croton mongue, a Madagascar Euphorbiaceae.

    PubMed

    Ralison, C; Creppy, E E; Boulanger, Y; Dirheimer, G

    1986-01-01

    Monguine, a thermostable toxic protein was extracted from the seeds of Croton mongue (Euphorbiaceae) and purified by ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-25 and G-15. Polyacrylamide gel electrophoresis of purified monguine in the presence of sodium dodecyl sulfate and after treatment with 2-mercaptoethanol showed one band corresponding to a molecular weight of 9000. The same molecular weight was determined by analytical centrifugation. Amino acid analysis revealed a high content in both aspartic and glutamic acids (or the corresponding amides). The LD50 (24 h) is 12 mg/kg of mouse body weight. Monguine inhibits protein synthesis in hepatoma tissue culture cells and globin synthesis in a rabbit reticulocyte lysate. PMID:3098307

  14. The mechanism of action of the cytotoxic lectin from Phoradendron californicum: the RNA N-glycosidase activity of the protein.

    PubMed

    Endo, Y; Oka, T; Tsurugi, K; Franz, H

    1989-05-01

    A toxic lectin from Phoradendron californicum (PCL) was found to inactivate catalytically 60 S ribosomal subunits of rabbit reticulocytes, resulting in the inhibition of protein synthesis. To study the mechanism of action of PCL, rat liver ribosomes were treated with the toxin and the extracted rRNA was treated with aniline. A fragment containing about 450 nucleotides was released from the 28 S rRNA. Analysis of the nucleotide sequence of the fragment revealed that the aniline-sensitive phosphodiester bond was between A4324 and G4325 of the 28 S rRNA. These results indicate that PCL inactivates the ribosomes by cleaving an N-glycosidic bond at A4324 of 28 S rRNA in the ribosomes as does ricin A-chain. PMID:2721668

  15. Therapy for aplastic anemia.

    PubMed

    DeZern, Amy E; Guinan, Eva C

    2011-01-01

    A 24-year-old man from Ecuador presents to your clinic with dyspnea on exertion, bruising, and petechiae. He is noted to be pancytopenic with ANC 430, hemoglobin 7.4 g/dL (reticulocyte count 0.9%), and platelets 18 000. His BM biopsy is hypocellular for age. Ultimately, he is diagnosed with severe aplastic anemia. He is the only child of 2 South American parents without any matches in the unrelated donor registry, including cord blood. He is red cell- and platelet transfusion-dependent. He has been recommended therapy with antithymocyte globulin and cyclosporine but declined it. He seeks recommendations about new alternatives to this regimen to improve his chance of response. PMID:22160016

  16. [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. PMID:27106490

  17. Self-Assembling Protein Microarrays

    NASA Astrophysics Data System (ADS)

    Ramachandran, Niroshan; Hainsworth, Eugenie; Bhullar, Bhupinder; Eisenstein, Samuel; Rosen, Benjamin; Lau, Albert Y.; C. Walter, Johannes; LaBaer, Joshua

    2004-07-01

    Protein microarrays provide a powerful tool for the study of protein function. However, they are not widely used, in part because of the challenges in producing proteins to spot on the arrays. We generated protein microarrays by printing complementary DNAs onto glass slides and then translating target proteins with mammalian reticulocyte lysate. Epitope tags fused to the proteins allowed them to be immobilized in situ. This obviated the need to purify proteins, avoided protein stability problems during storage, and captured sufficient protein for functional studies. We used the technology to map pairwise interactions among 29 human DNA replication initiation proteins, recapitulate the regulation of Cdt1 binding to select replication proteins, and map its geminin-binding domain.

  18. The Duffy binding protein as a key target for a Plasmodium vivax vaccine: lessons from the Brazilian Amazon

    PubMed Central

    de Sousa, Taís Nóbrega; Kano, Flora Satiko; de Brito, Cristiana Ferreira Alves; Carvalho, Luzia Helena

    2014-01-01

    Plasmodium vivax infects human erythrocytes through a major pathway that requires interaction between an apical parasite protein, the Duffy binding protein (PvDBP) and its receptor on reticulocytes, the Duffy antigen/receptor for chemokines (DARC). The importance of the interaction between PvDBP (region II, DBPII) and DARC to P. vivax infection has motivated our malaria research group at Oswaldo Cruz Foundation (state of Minas Gerais, Brazil) to conduct a number of immunoepidemiological studies to characterise the naturally acquired immunity to PvDBP in populations living in the Amazon rainforest. In this review, we provide an update on the immunology and molecular epidemiology of PvDBP in the Brazilian Amazon - an area of markedly unstable malaria transmission - and compare it with data from other parts of Latin America, as well as Asia and Oceania. PMID:25185002

  19. Dianthins, ribosome-damaging proteins with anti-viral properties from Dianthus caryophyllus L. (carnation).

    PubMed Central

    Stirpe, F; Williams, D G; Onyon, L J; Legg, R F; Stevens, W A

    1981-01-01

    1. Dianthin 30 and dianthin 32, two proteins isolated from the leaves of Diathus caryophyllus (carnation), were purified to homogeneity by chromatography on CM-cellulose. 2. The mol.wt. of dianthin 30 is 29 500 and that of dianthin 32 is 31 700. Both dianthins are glycoproteins containing mannose. 3. Dianthins inhibit protein synthesis in a lysate of rabbit reticulocytes, with an ID50 (concentration giving 50% inhibition) of 9.15 ng/ml (dianthin 30) and 3.6 ng/ml (dianthin 32). They act by damaging ribosomes in a less-than-equimolar ratio. Protein synthesis by intact cells is partially inhibited by dianthins at a concentration of 100 microgram/ml. 4. Dianthins mixed with tobacco-mosaic virus strongly decrease the number of local lesions on leaves of Nicotiana glutinosa. Images Fig. 2. PMID:7316958

  20. 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

  1. 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.

  2. Vulgarinin, a broad-spectrum antifungal peptide from haricot beans (Phaseolus vulgaris).

    PubMed

    Wong, Jack Ho; Ng, Tzi Bun

    2005-08-01

    From the seeds of haricot beans (Phaseolus vulgaris), an antifungal peptide with a molecular mass around 7 kDa was purified by using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. This peptide named vulgarinin manifested an antifungal activity toward fungal species such as Fusarium oxysporum, Mycosphaerella arachidicola, Physalospora piricola and Botrytis cinerea, and an antibacterial action on Mycobacterium phlei, Bacillus megaterium, Bacillus subtilis and Proteus vulgaris. It also inhibited proliferation in leukemia cell lines L1210 and M1 and breast cancer cell line MCF-7. This peptide could reduce the activity of HIV-1 reverse transcriptase and inhibited translation in a cell-free rabbit reticulocyte lysate system. Its antifungal activity was retained after incubation with trypsin. PMID:15896669

  3. Lunatusin, a trypsin-stable antimicrobial peptide from lima beans (Phaseolus lunatus L.).

    PubMed

    Wong, Jack Ho; Ng, Tzi Bun

    2005-11-01

    An anti-fungal peptide designated as lunatusin, with a molecular mass around 7kDa, was purified from the seeds of Chinese lima bean (Phaseolus lunatus L.). The peptide was isolated using a simple protocol consisting of affinity chromatography on Affi-gel blue gel and gel filtration on Superdex 75. Lunatusin exerted an anti-fungal activity toward fungal species such as Fusarium oxysporum, Mycosphaerella arachidicola and Botrytis cinerea, and an antibacterial action on, Bacillus megaterium, Bacillus subtilis, Proteus vulgaris and Mycobacterium phlei. It also inhibited proliferation in the breast cancer cell line MCF-7. Lunatusin reduced the activity of HIV-1 reverse transcriptase and it also inhibited translation in a cell-free rabbit reticulocyte lysate system. Its anti-fungal activity was retained after incubation with trypsin. Lunatusin elicited a mitogenic response from mouse splenocytes. PMID:16269344

  4. 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

    2013-10-01

    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. PMID:23954285

  5. Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii.

    PubMed

    da Silva, A M; da Costa Maia, J C; Juliani, M H

    1986-06-01

    Protein synthesis during sporulation in Blastocladiella emersonii is developmentally regulated as revealed using [35S]methionine pulse labeling and two-dimensional gel electrophoresis. A large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by two-dimensional gel electrophoresis of the polypeptides produced in a cell-free rabbit reticulocyte lysate primed with RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulation-specific messages are not present in the mature zoospores. PMID:3719699

  6. Use of protease sensitivity to probe the conformations of newly synthesised mutant forms of mitochondrial aspartate aminotransferase.

    PubMed

    Azzariti, A; Giannattasio, S; Doonan, S; Merafina, R S; Marra, E; Quagliariello, E

    1995-10-24

    Sensitivity to digestion with pronase has been used to show that the precursor form of mitochondrial aspartate aminotransferase, the form lacking the N-terminal presequence, that with a deletion of the first 9 residues and mutants of the mature enzyme in which residue Cys-166 is mutated to alanine or serine, all retain unfolded conformations after synthesis in a reticulocyte lysate. In the presence of lysed mitochondria the various forms of mitochondrial aspartate aminotransferase retained their susceptibilities to pronase in a way that mirrored the efficiencies with which they are imported into intact mitochondria. The results are interpreted as showing that the presequence of mitochondrial aspartate aminotransferase is not uniquely required for interaction with cytosolic factors required to maintain the newly synthesised protein in a form competent for interacting with, and being imported into, mitochondria. PMID:7488044

  7. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.

    PubMed

    Colombo, Marina; Raposo, Graça; Théry, Clotilde

    2014-01-01

    In the 1980s, exosomes were described as vesicles of endosomal origin secreted from reticulocytes. Interest increased around these extracellular vesicles, as they appeared to participate in several cellular processes. Exosomes bear proteins, lipids, and RNAs, mediating intercellular communication between different cell types in the body, and thus affecting normal and pathological conditions. Only recently, scientists acknowledged the difficulty of separating exosomes from other types of extracellular vesicles, which precludes a clear attribution of a particular function to the different types of secreted vesicles. To shed light into this complex but expanding field of science, this review focuses on the definition of exosomes and other secreted extracellular vesicles. Their biogenesis, their secretion, and their subsequent fate are discussed, as their functions rely on these important processes. PMID:25288114

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

    PubMed

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

    2015-01-01

    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. PMID:26464409

  9. Intravenous immunoglobulin-induced hemolytic anemia after thoracoscopic thymectomy for myasthenia gravis.

    PubMed

    Tsukada, Hisashi; Sunkara, Rajitha; Chi, Dorcas Doja; Keogh, Deirdre; Gaissert, Henning

    2014-06-01

    A 24-year-old woman underwent video-assisted thoracoscopic thymectomy for Osserman IIB myasthenia gravis (MG). In preparation for thymectomy, high-dose intravenous immunoglobulin (IVIG) was administered 1 week before the surgical procedure. After uneventful thoracoscopic thymectomy, the postoperative hemoglobin value decreased from 12.1 mg/dL to 8.2 mg/dL. A diagnosis of IVIG-associated hemolytic anemia was made based on a peripheral smear with numerous spherocytes, a positive direct antiglobulin test result, and increased reticulocyte count. Hemoglobin levels after IVIG administration should be monitored closely before and after elective surgical procedures to identify severe anemia. Transfusion of type-matched blood should be avoided and risk factors understood. PMID:24882299

  10. Biology of anemia, differential diagnosis, and treatment options in human immunodeficiency virus infection.

    PubMed

    Claster, Susan

    2002-05-15

    Anemia is the most common hematologic manifestation of human immunodeficiency virus (HIV) infection and acquired immunodeficiency syndrome. The causes of HIV-related anemia are multifactorial and include direct and indirect effects of HIV infection. HIV-related anemia generally is due to reduced red blood cell (RBC) production, secondary to a variety of causes, but it may also involve nutritional deficiencies, increased RBC destruction, or a combination of these problems. Evaluation of hemoglobin level, reticulocyte count, bilirubin, and mean corpuscular volume value and review of the peripheral blood smear are necessary for diagnosis. Treatment of HIV-related anemia should address the correctable underlying causes of this disorder, such as modifications of offending medications, nutritional deficiencies, and parvovirus infection. Patients with HIV infection have a blunted erythropoietin response to anemia. Therapeutic modalities for anemia that is not amenable to correction include blood transfusion and recombinant human erythropoietin (epoetin alfa). PMID:12001030

  11. 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.

  12. Bioactive Metabolites from Chaetomium aureum: Structure Elucidation and Inhibition of the Hsp90 Machine Chaperoning Activity

    PubMed Central

    Kabbaj, Fatima Zahra; Lu, Su; Faouzi, My El Abbés; Meddah, Bouchra; Proksch, Peter; Cherrah, Yahya; Altenbach, Hans-Josef; Aly, Amal H.; Chadli, Ahmed; Debbab, Abdessamad

    2014-01-01

    Chemical investigation of the EtOAc extract of the fungus Chaetomium aureum, an endophyte of the Moroccan medicinal plant Thymelaea lythroides, afforded one new resorcinol derivative named chaetorcinol, together with five known metabolites. The structures of the isolated compounds were determined on the basis of one- and two-dimensional NMR spectroscopy and high-resolution mass spectrometry as well as by comparison with the literature. All compounds were tested for their activity towards the Hsp90 chaperoning machine in vitro using the progesterone receptor (PR) and rabbit reticulocyte lysate (RRL). Among the isolated compounds, only sclerotiorin efficiently inhibited the Hsp90 machine chaperoning activity. However, sclerotiorin showed no cytotoxic effect on breast cancer Hs578T, MDA-MB-231 and prostate cancer LNCaP cell lines. Interestingly, deacetylation of sclerotiorin increased its cytotoxicity toward the tested cell lines over a period of 48h. PMID:25482429

  13. Mutagenic and Genotoxic Effect of Hydroxyurea

    PubMed Central

    Santos, Jean L.; Bosquesi, Priscila L.; Almeida, Adélia E.; Chin, Chung Man; Varanda, Eliana A.

    2011-01-01

    The hydroxyurea, a cytotoxic drug, is the mainly available therapeutical strategy for the treatment of sickle cell disease. This study aimed to evaluate the mutagenic and genotoxic potential of the hydroxyurea through the Salmonella/Microsome assay and micronucleus test in peripheral blood of mice. The doses were evaluated at 29.25-468 μmol/plate in Salmonella/Microsome assay in presence and absence of metabolic activation the drug. In the micronucleus test the doses were evaluated at 12.5; 25; 50; 75 and 100 mg/kg. The results show that hydroxyurea present mutagenic activity in TA98 and TA100 in doses above 117 μmol/plate and 234 μmol/plate respectively. The drug induced a significant increase in the frequency of micronuclei in reticulocytes of mice at concentrations of 50, 75 and 100 mg/kg, compared to negative control (water). These results demonstrated the mutagenic and genotoxic potential of hydroxyurea. PMID:23675245

  14. 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.

  15. Properties of volkensin, a toxic lectin from Adenia volkensii.

    PubMed

    Stirpe, F; Barbieri, L; Abbondanza, A; Falasca, A I; Brown, A N; Sandvig, K; Olsnes, S; Pihl, A

    1985-11-25

    Volkensin, a highly toxic protein from the roots of Adenia volkensii (kilyambiti, kinoria), was purified by affinity chromatography on acid-treated Sepharose 6B. The toxin is a glycoprotein (Mr 62,000, neutral sugar content 5.74%) consisting of an A subunit (Mr 29,000) and of a B subunit (Mr 36,000) linked by disulfide and noncovalent bond(s). The amino acid, amino sugar, and neutral sugar composition of the protein were determined. Volkensin is a galactose-specific lectin and is a potent inhibitor of eukaryotic protein synthesis in whole cells as well as in a cell-free system (a rabbit reticulocyte lysate). The inhibitory and the lectin activities are functions of the A and B subunits, respectively. Volkensin can be included amongst the ricin-like toxins and resembles most closely modeccin, the toxin of Adenia digitata. PMID:3932357

  16. Depressed eruption rate of the rat maxillary incisor in a drug-induced uncompensated hemolytic state model

    SciTech Connect

    Giglio, M.J.; Sanz, A.M.; Bozzini, C.E. )

    1990-03-01

    Female rats weighing about 180 g were separated into two groups. One group (A) received phenylhydrazine (PHZ) every other day during three weeks (for induction of an uncompensated hemolytic state), while the control group (C) received saline. The evidence for the establishment of the uncompensated hemolytic state was obtained by hematocrit value, reticulocyte count, and red-cell-volume-59Fe uptake. Body-weight gain (which is a measure of overall body growth rate), body-length gain (which is a measure of longitudinal skeletal growth rate), food intake, and maxillary incisor eruption rate (ER) were significantly depressed in rats of group A during the PHZ-injection period, in relation to rats of group C. These results indicate that anemia and/or associated factors depress ER, along with body growth and skeletal growth.

  17. 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.

  18. A coprecipitation-based validation methodology for interactions identified using protein microarrays.

    PubMed

    Marina, Ovidiu; Duke-Cohan, Jonathan S; Wu, Catherine J

    2011-01-01

    Candidate interactions identified by high-throughput protein microarray screening require rigorous -confirmation. Such validation is time-consuming and labor-intensive using conventional techniques. We describe a medium-throughput validation protocol based on coprecipitation of biotin-labeled -proteins synthesized in vitro using a rabbit reticulocyte lysate-coupled transcription and translation system. As our experimental system is based on screening for serum antibodies, we also present methods on purifying immunoglobulin from serum and quantifying the amount of coprecipitated (immunoprecipitated) target protein on Western blot. This technique provides a sensitive confirmatory test allowing for the rapid elimination of false positives prior to more extensive validation and analysis of target interactions in their native environment. PMID:21370070

  19. Dynamics of Erythropoiesis in Erythroblastic Islands in the Bone Marrow in Experimental Benzene-Induced Anemia.

    PubMed

    Tishevskaya, N V; Bolotov, A A; Lebedeva, Ya E

    2016-07-01

    In 14 days after triple subcutaneous injection of benzene to rats in a dose of 0.05 ml/100 g body weight, the number of erythroblastic islands in the bone marrow 2-fold decreased, de novo erythropoiesis ceased, de repeto erythropoiesis slowed down, and the number of lymphoid cells contacting with erythroblastic islands decreased. By day 56 of the experiment, proliferation of erythroid cells in the bone marrow start to recover due to erythropoiesis reconstitution, but the number of reticulocytes in the blood remained low. Analysis of phagocytic ability of macrophages in the erythroblastic islands showed that the intensity of phagocytosis of latex particles in mature islands decreased, which could affect denucleation of oxyphilic erythroblasts. PMID:27492403

  20. Identification of the vaccinia virus gene encoding nucleoside triphosphate phosphohydrolase I, a DNA-dependent ATPase.

    PubMed Central

    Broyles, S S; Moss, B

    1987-01-01

    Vaccinia virus encapsidates a DNA-dependent ATPase known as nucleoside triphosphate phosphohydrolase I (NPH I). A bacteriophage lambda gt11 expression library of poxvirus DNA was screened with antibodies specific for NPH I. Positive clones were used to probe restriction fragments of vaccinia virus genomic DNA to locate the NPH I gene. The identity of the open reading frame (ORF) was confirmed by placing it downstream of a bacteriophage T7 promoter, transcribing the ORF in vitro, and translating the RNA in a reticulocyte lysate. A polypeptide of the correct molecular weight, which was recognized by anti-NPH I antibody, was synthesized. Inspection of the deduced amino acid sequence of the NPH I ORF revealed consensus ATP-binding sites. Images PMID:2437324

  1. 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. PMID:27300151

  2. Intra-abdominal fluid aspirate from a dog.

    PubMed

    Crippa, Valentina; Ghisleni, Gabriele; Avallone, Giancarlo; Caniatti, Mario

    2016-02-01

    A 12-year-old, neutered female, Siberian husky, was presented with a 6-months history of progressive abdominal distension, anorexia, and weight loss. The dog appeared normal on physical examination except for marked abdominal distension. A fluid wave was balloted strongly suggesting an abdominal effusion. Ultrasound examination confirmed this clinical finding. Results of the CBC included mild nonregenerative anemia, with an RBC count of 4.9 × 10(6)/µL (reference interval 5.5-8.5 × 10(6)/µL), hemoglobin concentration of 12 g/dL (reference interval 12-18 g/dL), HCT of 36% (reference interval 37-55%), and reticulocytes <60,000/µL. No abnormalities in serum chemistry were detected. PMID:26668089

  3. 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. PMID:25236441

  4. 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. PMID:23134843

  5. Immunotoxicity of nitrobenzene in female B6C3F1 mice.

    PubMed

    Burns, L A; Bradley, S G; White, K L; McCay, J A; Fuchs, B A; Stern, M; Brown, R D; Musgrove, D L; Holsapple, M P; Luster, M I

    1994-01-01

    Nitrobenzene (NBZ) is primarily employed as an oxidizing agent in the synthesis of analine and benzene compounds. It produces myelotoxic effects and effects on erythrocytes in both animal models and man. Reported hepatosplenomegaly and effects on the bone marrow are indicators that NBZ may be immunotoxic. In these studies, female B6C3F1 mice were exposed to 30, 100 and 300 mg/kg of NBZ in corn oil by gavage for 14 consecutive days. To assess the immunotoxic potential of NBZ, body and organ weights were determined and selected immunologic and host resistance responses were studied. In these studies, the liver and spleen appeared to be the primary target organs. Both liver and spleen weights were dose dependently increased. Gross histopathologic examinations revealed significant changes in the spleen, consisting of severe congestion of the red pulp areas with erythrocytes and reticulocytes. Serum chemistry profiles showed increases in alanine aminotransferase and aspartate aminotransferase activities, indicating liver toxicity. Hematologic studies showed a decrease in erythrocyte number and a concomitant increase in mean corpuscular hemoglobin and mean corpuscular volume. A dose-dependent increase in peripheral reticulocytes was also seen. DNA synthesis was enhanced, as was the number of formed elements and the number of monocyte/granulocyte stem cells in the bone marrow of treated mice. IgM responses were decreased and the phagocytic activity of macrophages in the liver was dose dependently increased with a concomitant decrease in the activities in the spleen and lung. Other immunological parameters examined were unchanged. Host resistance to microbial or viral infection was not markedly altered by NBZ; however, there were trends towards increased susceptibility where T-cell function contributes to host defense. These data indicate that NBZ-induced hemolysis and liver injury are linked to the observed alterations in bone marrow activity. PMID:7988385

  6. Hyperglycaemic Environment: Contribution to the Anaemia Associated with Diabetes Mellitus in Rats Experimentally Induced with Alloxan

    PubMed Central

    Bashiru Shola, Oseni; Olatunde Olugbenga, Fakoya

    2015-01-01

    Background. Diabetes mellitus characterized by hyperglycaemia presents with various complications amongst which anaemia is common particularly in those with overt nephropathy or renal impairment. The present study has examined the contribution of the hyperglycaemic environment in diabetic rats to the anaemia associated with diabetes mellitus. Method. Sixty male albino rats weighing 175–250 g were selected for this study and divided equally into control and test groups. Hyperglycaemia was induced with 170 kgbwt−1 alloxan intraperitoneally in the test group while control group received sterile normal saline. Blood samples obtained from the control and test rats were assayed for packed cell volume (PCV), haemoglobin (Hb), red blood cell count (RBC), reticulocyte count, glucose, plasma haemoglobin, potassium, and bilirubin. Result. Significant reduction (P < 0.01) in PCV (24.40 ± 3.87 versus 40.45 ± 3.93) and haemoglobin (7.81 ± 1.45 versus 13.39 ± 0.40) with significant increase (P < 0.01) in reticulocyte count (12.4 ± 1.87 versus 3.69 ± 0.47), plasma haemoglobin (67.50 ± 10.85 versus 34.20 ± 3.83), and potassium (7.04 ± 0.75 versus 4.52 ± 0.63) was obtained in the test while plasma bilirubin showed nonsignificant increase (0.41 ± 0.04 versus 0.24 ± 0.06). Conclusion. The increased plasma haemoglobin and potassium levels indicate an intravascular haemolytic event while the nonsignificant increased bilirubin showed extravascular haemolysis. These play contributory roles in the anaemia associated with diabetes mellitus. PMID:26697217

  7. 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. PMID:16328191

  8. Expression of a human alpha-tubulin: properties of the isolated subunit.

    PubMed

    Yaffe, M B; Levison, B S; Szasz, J; Sternlicht, H

    1988-03-22

    We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer. PMID:2897863

  9. High-dose recombinant human erythropoietin for treatment of anemia in myelodysplastic syndromes and paroxysmal nocturnal hemoglobinuria: a pilot study.

    PubMed

    Stebler, C; Tichelli, A; Dazzi, H; Gratwohl, A; Nissen, C; Speck, B

    1990-12-01

    In a dose escalation study we tested the feasibility and tolerance of high-dose recombinant human erythropoietin (r-HuEPO) therapy in four patients with ineffective erythropoiesis due to myelodysplastic syndromes (MDS) or paroxysmal nocturnal hemoglobinuria (PNH). Recombinant human EPO was administered i.v. with an initial dose of 50 U/kg body weight (BW) three times per week. The dose was increased by steps of 25 or 50 U/kg bW with intervals of 1-4 weeks up to a maximum dose of 500 U/kg BW three times per week. All patients were treated as outpatients. Pre-study treatment with cyclosporin A and/or Danazol was continued in three patients. In one patient r-HuEPO was discontinued after 20 weeks because of relapse of severe aplastic anemia. No major side effects were observed even at the maximum dose. One patient with PNH showed an increase of hemoglobin from 89 to 139 g/liter that permitted monthly phlebotomies to reduce his iron overload. In one patient with MDS the reticulocyte count increased from 2.5 to 50 x 10(9)/liter, and the transfusion requirement decreased to 2 U every 3-4 weeks instead of every 2 weeks. Two patients did not complete the whole treatment period and showed no rise in reticulocyte count. We conclude that high dose r-HuEPO therapy is feasible in patients with anemia due to MDS or PNH. High-dose r-HuEPO appears to have some effect on anemia due to ineffective erythropoiesis in a subgroup of patients. Further studies are needed to identify potential responders and to define the optimal administration of r-HuEPO. PMID:2226680

  10. 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

  11. Persistence of cisplatin-induced mutagenicity in hematopoietic stem cells: implications for secondary cancer risk following chemotherapy.

    PubMed

    Dertinger, Stephen D; Avlasevich, Svetlana L; Torous, Dorothea K; Bemis, Jeffrey C; Phonethepswath, Souk; Labash, Carson; Carlson, Kristine; Mereness, Jared; Cottom, John; Palis, James; MacGregor, James T

    2014-08-01

    Cisplatin is a cytostatic agent used in the treatment of many types of cancer, but its use is associated with increased incidences of secondary leukemia. We evaluated cisplatin's in vivo genotoxic potential by analyzing peripheral blood for Pig-a mutant phenotype erythrocytes and for chromosomal damage in the form of micronuclei. Mutant phenotype reticuloyte and erythrocyte frequencies, based on anti-CD59 antibody labeling and flow cytometric analysis, were determined in male Sprague Dawley rats treated for 28 consecutive days (days 1-28) with up to 0.4 mg cisplatin/kg/day, and sampled on days -4, 15, 29, and 56. Vehicle and highest dose groups were evaluated at additional time points post-treatment up to 6 months. Day 4 and 29 blood samples were also analyzed for micronucleated reticulocyte frequency using flow cytometry and anti-CD71-based labeling. Mutant phenotype reticulocytes were significantly elevated at doses ≥0.1 mg/kg/day, and mutant phenotype erythrocytes were elevated at doses ≥0.05 mg/kg/day. In the 0.4 mg/kg/day group, these effects persisted for the 6 month observation period. Cisplatin also induced a modest but statistically significant increase in micronucleus frequency at the highest dose tested. The prolonged persistence in the production of mutant erythrocytes following cisplatin exposure suggests that this drug mutates hematopoietic stem cells and that this damage may ultimately contribute to the increased incidence of secondary leukemias seen in patients cured of primary malignancies with platinum-based regimens. PMID:24798381

  12. 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.

  13. Specific interaction between adenoviral 55-kDa E1B protein and in vivo produced p53 fusion proteins.

    PubMed

    Chumakov, A; Koeffler, H P

    1993-09-15

    Several protein fusion systems have been used in recent years to study protein-protein and DNA-protein interactions. Most of them use bacterially produced proteins which have several inherent disadvantages, notably, the absence of correct post-translational modifications and the frequent insolubility of recombinant proteins. We sought to develop a system to study proteins interacting with the nuclear phosphoprotein p53, which is believed to be a tumor suppressor. To prepare fusions of p53, we developed a convenient system that permits both in vivo and in vitro production and easy affinity purification of peptides and protein fragments as glutathione-transferase fusions. We placed the coding sequence of the Schistosoma japonica glutathione S-transferase (GST) under the control of the strong CMV/T7 promoter and SV40 splice and polyadenylation signals. An extensive polylinker (MCS) at the 3' end of the GST gene is preceded by the sequence encoding the cleavage site of the site-specific protease. We cloned the complete coding sequences of human wild-type p53, as well as p53 mutants representing all four mutational hotspots (codons 141, 175, 248, and 273), into our expression vector. In vitro transcription using the upstream T7 promoter and translation in reticulocyte lysates form an easy way to produce hybrid proteins; affinity purification on a glutathione-agarose column removes proteins that are present in reticulocyte lysates. We have also studied specific in vivo interactions of human p53 with the adenoviral 55-kDa E1B protein by transfecting expression constructs of GST-p53 fusions into human Ad5-transformed 293 cells. PMID:8406015

  14. Antifungal peptides, a heat shock protein-like peptide, and a serine-threonine kinase-like protein from Ceylon spinach seeds.

    PubMed

    Wang, Hexiang; Ng, Tzi Bun

    2004-07-01

    Two antifungal peptides (designated alpha- and beta-basrubrins) with molecular masses of 4-5 kDa and distinct N-terminal sequences, and a peptide and a protein with N-terminal sequences resembling heat shock protein (hsp) and serine-threonine kinase, respectively, were isolated from seeds of the Ceylon spinach Basella rubra. The purification procedure entailed saline extraction, (NH4)2SO4 precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose, and FPLC-gel filtration on a Superdex peptide column. alpha- and beta-basrubrins inhibited mycelial growth in Botrytis cirerea with an IC50 value of 7.5 and 14.7 microM, respectively, Mycosphaerella arachidicola with an IC50 of 12.4 and 6.9 microM, and Fusarium oxysporum with an IC50 of 5.8 and 6.2 microM. Neither alpha-basrubrin nor beta-basrubin exhibited DNase, RNase, lectin or protease activity, indicating that their antifungal action is not due to these activities. HIV-1 reverse transcriptase was inhibited by alpha- and beta-basrubrins with an IC50 of 246 and 370 microM, respectively. Translation in rabbit reticulocyte lysate was inhibited by alpha- and beta-basrubrins with an IC50 of 400 and 100 nM. The heat shock protein-like peptide and serine-threonine kinase-like protein exhibited a molecular mass of 3 and 30 kDa, respectively. They inhibited neither translation in a rabbit reticulocyte system at concentrations up to 50 microM nor HIV-1 reverse transcriptase activity at concentrations up to 400 microM. They did not exert antifungal activity toward B. cinerea, M. arachidicola, and F. oxysporum when tested up to 16 microg. None of the aforementioned proteins demonstrated DNase, RNase, protease or lectin activity. PMID:15245882

  15. Pig-a Mutation: Kinetics in Rat Erythrocytes Following Exposure to Five Prototypical Mutagens

    PubMed Central

    Phonethepswath, Souk; Franklin, Dean; Torous, Dorothea K.; Bryce, Steven M.; Bemis, Jeffrey C.; Raja, Sarojini; Avlasevich, Svetlana; Weller, Pamela; Hyrien, Ollivier; Palis, James; MacGregor, James T.; Dertinger, Stephen D.

    2010-01-01

    An in vivo mutation assay has been developed based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor–deficient rat erythrocytes. With this method, blood is incubated with anti-CD59-PE and SYTO 13 dye, and flow cytometry is used to score the frequency of CD59-negative erythrocytes. The experiments described herein were designed to define the kinetics of mutant erythrocyte appearance and disappearance from peripheral blood to support appropriate treatment and sampling designs for the assay. Wistar Han rats were treated with one of five prototypical mutagens: N-ethyl-N-nitrosourea (ENU); 7,12-dimethyl-1,2-benz[a]anthracene (DMBA); 4-nitroquinoline-1-oxide; benzo[a]pyrene; and N-methyl-N-nitrosourea. ENU and DMBA were also evaluated in Sprague Dawley rats. Animals were treated on three consecutive days (days 1–3) via oral gavage, and blood specimens were obtained on days −1, 4, 15, 30, 45, and 90 (and day 180 for ENU). A second endpoint of genotoxicity, the frequency of peripheral blood micronucleated reticulocytes, was measured on day 4. Each chemical induced micronuclei and the GPI anchor–deficient phenotype. Increased mutant cell frequencies were evident at day 15. Mutant reticulocyte frequencies remained relatively stable for some chemicals, but others peaked and then dropped significantly. The differences in kinetics observed are presumably related to the degree to which mutation occurs in hematopoietic stem cells versus more committed cells with limited self-renewal capacity. Collectively, the results suggest that enumerating GPI anchor–deficient erythrocytes is an efficient means of evaluating the in vivo mutagenic potential of chemicals. The kinetics and ease of scoring this blood-based endpoint suggest that integration into routine toxicology studies will be feasible. PMID:19965957

  16. Integration of Mutation and Chromosomal Damage Endpoints into 28-Day Repeat Dose Toxicology Studies

    PubMed Central

    Dertinger, Stephen D.; Phonethepswath, Souk; Franklin, Dean; Weller, Pamela; Torous, Dorothea K.; Bryce, Steven M.; Avlasevich, Svetlana; Bemis, Jeffrey C.; Hyrien, Ollivier; Palis, James; MacGregor, James T.

    2010-01-01

    Two endpoints of genetic toxicity, mutation at the X-linked Pig-a gene and chromosomal damage in the form of micronucleated reticulocytes (MN-RETs), were evaluated in blood samples obtained from 28-day repeat-dosing studies typical of those employed in toxicity evaluations. Male Wistar Han rats were treated at 24-h intervals on days 1 through 28 with one of five prototypical genotoxicants: N-ethyl-N-nitrosourea, 7,12-dimethyl-12-benz[a]anthracene, 4-nitroquinoline-1-oxide (4NQO), benzo(a)pyrene, and N-methyl-N-nitrosourea. Flow cytometric scoring of CD59-negative erythrocytes (indicative of glycosylphosphatidylinositol anchor deficiency and hence Pig-a mutation) was performed using blood specimens obtained on days −1, 15, 29, and 56. Blood specimens collected on days 4 and 29 were evaluated for MN-RET frequency using flow cytometry–based MicroFlow Kits. With the exception of 4NQO, each chemical induced significant increases in the frequency of MN-RETs on days 4 and 29. All five agents increased the frequency of mutant phenotype (CD59 negative) reticulocytes (RETs) and erythrocytes. Mutation responses in RETs occurred earlier than in erythrocytes and tended to peak, or nearly peak, at day 29. In contrast, the mutant phenotype erythrocyte responses were modest on day 29 and required additional time to reach their maximal value. The observed kinetics were expected based on the known turnover of RETs and erythrocytes. The data show that RETs can serve as an appropriate indicator cell population for 28-day studies. Collectively, these data suggest that blood-based genotoxicity endpoints can be effectively incorporated into routine toxicology studies, a strategy that would reduce animal usage while providing valuable genetic toxicity information within the context of other toxicological endpoints. PMID:20202993

  17. Persistence of Cisplatin-Induced Mutagenicity in Hematopoietic Stem Cells: Implications for Secondary Cancer Risk Following Chemotherapy

    PubMed Central

    Dertinger, Stephen D.; Avlasevich, Svetlana L.; Torous, Dorothea K.; Bemis, Jeffrey C.; Phonethepswath, Souk; Labash, Carson; Carlson, Kristine; Mereness, Jared; Cottom, John; Palis, James; MacGregor, James T.

    2014-01-01

    Cisplatin is a cytostatic agent used in the treatment of many types of cancer, but its use is associated with increased incidences of secondary leukemia. We evaluated cisplatin's in vivo genotoxic potential by analyzing peripheral blood for Pig-a mutant phenotype erythrocytes and for chromosomal damage in the form of micronuclei. Mutant phenotype reticuloyte and erythrocyte frequencies, based on anti-CD59 antibody labeling and flow cytometric analysis, were determined in male Sprague Dawley rats treated for 28 consecutive days (days 1–28) with up to 0.4 mg cisplatin/kg/day, and sampled on days −4, 15, 29, and 56. Vehicle and highest dose groups were evaluated at additional time points post-treatment up to 6 months. Day 4 and 29 blood samples were also analyzed for micronucleated reticulocyte frequency using flow cytometry and anti-CD71-based labeling. Mutant phenotype reticulocytes were significantly elevated at doses ≥0.1 mg/kg/day, and mutant phenotype erythrocytes were elevated at doses ≥0.05 mg/kg/day. In the 0.4 mg/kg/day group, these effects persisted for the 6 month observation period. Cisplatin also induced a modest but statistically significant increase in micronucleus frequency at the highest dose tested. The prolonged persistence in the production of mutant erythrocytes following cisplatin exposure suggests that this drug mutates hematopoietic stem cells and that this damage may ultimately contribute to the increased incidence of secondary leukemias seen in patients cured of primary malignancies with platinum-based regimens. PMID:24798381

  18. Decreased Globin Messenger RNA in Thalassemia Detected by Molecular Hybridization

    PubMed Central

    Kacian, D. L.; Gambino, R.; Dow, L. W.; Grossbard, E.; Natta, C.; Ramirez, F.; Spiegelman, S.; Marks, P. A.; Bank, A.

    1973-01-01

    In previous studies of patients with β thalassemia, mRNA extracted from reticulocytes in peripheral blood when added to cell-free systems reproduces the deficient β-chain synthesis characteristic of intact cells. The present studies with specific probes for α and β mRNA were designed to decide whether the decreased β mRNA activity is due to the presence of abnormal or reduced β globin mRNA in these cells. Purified α and β complementary DNAs (cDNAs) have been synthesized with RNA-instructed DNA polymerase; α and β mRNAs isolated from heavy (β-producing) and light (α-producing) polyribosomes of rabbit reticulocytes were used as templates. Each of the cDNAs is more than 80% pure by the criterion of biological activity. The α cDNA labeled with [32P]dCTP and the β cDNA labeled with [3H]dCTP have been added simultaneously to reaction mixtures containing various concentrations of mRNA from thalassemic and nonthalassemic subjects. The extent and rate of hybridization were determined, permitting a comparison of relative α and β mRNA content in the same annealing mixture. In six nonthalassemic patients, relatively equal amounts of hybridizable α and β mRNA appear to be present. In five of seven patients with β-thalassemia, significantly decreased amounts of β mRNA compared to α mRNA can be demonstrated. In two patients with Hemoglobin H disease, there is a decreased amount of α mRNA compared to β mRNA. PMID:4124307

  19. Clinical and haematological risk factors for cerebral macrovasculopathy in a sickle cell disease newborn cohort: a prospective study.

    PubMed

    Sommet, Julie; Alberti, Corinne; Couque, Nathalie; Verlhac, Suzanne; Haouari, Zinedine; Mohamed, Damir; François, Martine; Missud, Florence; Holvoet, Laurent; Elmaleh, Monique; Ithier, Ghislaine; Denjean, André; Elion, Jacques; Baruchel, André; Benkerrou, Malika

    2016-03-01

    Children with sickle cell disease (SCD) have a significant vascular morbidity, especially cerebral macrovasculopathy (CV), detectable by transcranial Doppler. This study aimed to identify risk factors for CV using longitudinal biological and clinical data in a SCD newborn cohort followed at the Robert Debre Reference centre (n = 375 SS/Sβ(0) ). Median follow-up was 6·8 years (2677 patient-years). Among the 59 children presenting with CV, seven had a stroke. Overall, the incidence of CV was 2·20/100 patient-years [95% confidence interval (95% CI): 1·64-2·76] and the incidence of stroke was 0·26/100 patient-years (95% CI: 0·07-0·46). The cumulative risk of CV by age 14 years was 26·0% (95% CI: 20·0-33·3%). Risk factors for CV were assessed by a Cox model encompassing linear multivariate modelling of longitudinal quantitative variables. Years per upper-airway obstruction [Hazard ratio (HR) = 1·47; 95% CI: 1·05-2·06] or bronchial obstruction (HR = 1·76; 95% CI: 1·49-2·08) and reticulocyte count (HR = 1·82 per 50 × 10(9) /l increase; 95% CI: 1·10-3·01) were independent risk factors whereas fetal haemoglobin level (HR = 0·68 per 5% increase; 95% CI: 0·48-0·96) was protective. Alpha-thalassaemia was not protective in multivariate analysis (ancillary analysis n = 209). Specific treatment for upper or lower-airway obstruction and indirect targeting of fetal haemoglobin and reticulocyte count by hydroxycarbamide could potentially reduce the risk of CV. PMID:26728571

  20. Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

    PubMed Central

    Konstantinidis, Diamantis G.; Pushkaran, Suvarnamala; Giger, Katie; Manganaris, Stefanos; Zheng, Yi; Kalfa, Theodosia A.

    2014-01-01

    Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis. We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells. PMID:24962543

  1. Coronavirus JHM: Cell-Free Synthesis of Structural Protein p60

    PubMed Central

    Siddell, Stuart G.; Wege, Helmut; Barthel, Andrea; ter Meulen, Volker

    1980-01-01

    Sac(-) cells infected with murine coronavirus strain JHM shut off host cell protein synthesis and synthesized polypeptides with molecular weights of 150,000, 60,000, and 23,000. The 60,000- and 23,000-molecular-weight polypeptides comigrated with virion structural proteins p60 and p23, and the 60,000-molecular-weight protein was identified as p60 by tryptic peptide fingerprinting. Polyadenylate-containing RNA [poly(A) RNA] extracted from the cytoplasm of infected cells directed the synthesis of both 60,000- and 23,000-molecular-weight polypeptides in messenger-dependent cell-free systems derived from mouse L-cells and rabbit reticulocytes. The reticulocyte system also synthesized a 120,000-molecular-weight polypeptide that was specifically immunoprecipitated by antiserum raised against JHM virions. The identity of the 60,000- and 23,000-molecular-weight in vitro products was established by comigration with virion proteins, immunoprecipitation, and in the case of p60, tryptic peptide fingerprinting. The cytoplasmic poly(A) RNAs which encoded p60 and p23 sedimented in sucroseformamide gradients at 17S and 19S, respectively, and were clearly separable. These RNAs were among the major poly(A) RNA species synthesized in the cytoplasm of actinomycin D-treated cells late in infection, and the in vitro translation of size-fractionated RNA released from polysomes confirmed that they represent physiological mRNA's. These results suggest that the expression of the coronavirus JHM genome involves more than one subgenomic mRNA. Images PMID:7365865

  2. Risk Factors of Pulmonary Hypertension in Brazilian Patients with Sickle Cell Anemia

    PubMed Central

    Lobo, Clarisse Lopes de Castro; do Nascimento, Emilia Matos; Abelha, Renato; Queiroz, Ana Maria Mach; Connes, Philippe; Cardoso, Gilberto Perez; Ballas, Samir K.

    2015-01-01

    This study was a prospective cross-sectional cohort study of 125 patients with sickle cell anemia (SS) between the ages of 16 to 60 years. Enrolled patients were followed-up prospectively for 15 months. Demographic, clinical, hematological and routine biochemical data were obtained on all patients. Six-minute walk test and Doppler Echocardiography were performed on all patients. A tricuspid regurgitant jet velocity (TRJV) < 2.5 m/sec was considered normal, 2.5 ≤ TRJV ≤ 3.0 was considered mild-moderate and > 3.0 m/sec, severe. Patients with abnormal TRJV were significantly older and more anemic, had significantly higher lactate dehydrogenase (LDH) levels, reticulocyte count and incidence of death. The logistic multimodal model implemented for the 125 patients indicated that age was the covariate that influenced the outcome of normal or abnormal TRJV with a cutoff age of thirty-two years. The survival rate for the group of patients with creatinine (Cr) > 1.0 mg/dL was lower than the group with Cr ≤ 1 and normal TRJV. A coefficient matrix showed that the LDH values were weakly correlated with the reticulocyte count but strongly correlated with hemoglobin suggesting that the TRJV values were not correlated with the hemolytic rate but with anemia. Ten patients died during the follow-up of whom 7 had TRJV > 2.5 m/sec. Acute chest syndrome was the most common cause of death followed by sepsis. In conclusion, this study shows that patients with SS older than thirty-two years with high LDH, elevated TRJV, severe anemia and Cr > 1 have poor prognosis and may be at risk of having pulmonary hypertension and should undergo RHC. PMID:26335226

  3. 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.

  4. Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays.

    PubMed

    Lent, Emily May; Crouse, Lee C B; Quinn, Michael J; Wallace, Shannon M

    2012-02-18

    Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer. PMID:22155124

  5. Diagnostic clues to megaloblastic anaemia without macrocytosis.

    PubMed

    Chan, C W J; Liu, S Y H; Kho, C S B; Lau, K H T; Liang, Y S; Chu, W R; Ma, S K E

    2007-06-01

    Masking of the macrocytic expression of megaloblastic anaemia (MA) by coexisting thalassaemia, iron deficiency and chronic illness has been widely reported. We described the haematological and clinical features of 20 Chinese patients with MA presenting with mean corpuscular volume (MCV) < or =99 fl, and analysed the steps leading to the final diagnosis of MA with concomitant thalassaemia trait (n = 11), thalassaemia trait and iron deficiency (n = 3), iron deficiency (n = 4) and chronic illness (n = 2). We also compared the haematological characteristics of this group of patients with a group of normocytic anaemic patients without vitamin B(12)/folate deficiency, and identified certain laboratory information useful for differentiating the two groups. Statistically significant parameters included the mean values of haemoglobin, MCV, red cell distribution width (RDW), reticulocyte index, platelet count and serum bilirubin. All provided clues to maturation disorders within the marrow. A decision flowchart for the diagnosis of MA without macrocytosis was proposed. In the studied population, by using the parameters of haemoglobin <10 g/dl, MCV 80-99 fl, RDW > or = 16% and reticulocyte index < or = 2% as indicators, there was a 58% chance that a patient had MA without macrocytosis if he/she had all the four indicators, and a 2.2% chance of having it if he/she did not have these indicators. We emphasized the importance of including peripheral blood smear examination in the diagnostic procedures for such patients, as well as the importance of paying attention to patients' medical history, racial background and previous MCV value. PMID:17474892

  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. 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. PMID:23670359

  8. Effects of Testosterone on Erythropoiesis in a Female Mouse Model of Anemia of Inflammation.

    PubMed

    Guo, Wen; Schmidt, Paul J; Fleming, Mark D; Bhasin, Shalender

    2016-07-01

    The anemia of inflammation is a common problem in inflammatory and autoimmune diseases. We characterized a mouse model of anemia of chronic inflammation induced by repeated injections of low doses of heat-killed Brucella abortus (HKBA), and determined the effects of T administration on erythropoiesis in this model. Female C57BL/6NCrl mice were injected weekly with HKBA for 10 wk. Weekly injections of T or vehicle oil were started 4 wk later. Control mice were injected with saline and vehicle oil in parallel. HKBA-injected mice had significantly lower hemoglobin, hematocrit, mean corpuscular volume, reticulocyte hemoglobin, transferrin saturation (TSAT), and tissue nonheme iron in liver and spleen, enlarged spleen, and up-regulated hepatic expression of inflammatory markers, serum amyloid A1, and TNFα, but down-regulated IL-6, bone morphogenic protein 6, and hepcidin compared with saline controls. HKBA also reduced serum hepcidin and increased serum erythropoietin. Bone marrow erythroid precursors were substantially reduced in HKBA-injected mice. Cotreatment with T increased the percentage of late-stage erythroid precursors in the bone marrow relative to HKBA-injected and saline controls and reversed HKBA-induced suppression of hemoglobin and hematocrit. T also normalized serum erythropoietin, TSAT, and reticulocyte hemoglobin without correcting the expression of the hepatic inflammation markers. Conclusions are that low-dose HKBA induces moderate anemia characterized by chronic inflammation, decreased iron stores, and suppression of erythroid precursors in the bone marrow. T administration reverses HKBA-induced anemia by stimulating erythropoiesis, which is associated with a shift toward accelerated maturation of erythroid precursors in the bone marrow. PMID:27074351

  9. HEME-DEPENDENT ACTIVATION OF NEURONAL NITRIC-OXIDE SYNTHASE BY CYTOSOL IS DUE TO AN HSP70-DEPENDENT, THIOREDOXIN-MEDIATED THIOL-DISULFIDE INTERCHANGE IN THE HEME/SUBSTRATE BINDING CLEFT†

    PubMed Central

    Morishima, Yoshihiro; Lau, Miranda; Peng, Hwei-Ming; Miyata, Yoshinari; Gestwicki, Jason E.; Pratt, William B.; Osawa, Yoichi

    2011-01-01

    We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20–30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-μ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination with either the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme. PMID:21755988

  10. Heme-dependent activation of neuronal nitric oxide synthase by cytosol is due to an Hsp70-dependent, thioredoxin-mediated thiol-disulfide interchange in the heme/substrate binding cleft.

    PubMed

    Morishima, Yoshihiro; Lau, Miranda; Peng, Hwei-Ming; Miyata, Yoshinari; Gestwicki, Jason E; Pratt, William B; Osawa, Yoichi

    2011-08-23

    We have reported that heme-dependent activation of apo-neuronal nitric oxide synthase (apo-nNOS) to the active holo-enzyme dimer is dependent upon factors present in reticulocyte lysate and other cytosols. Here, we find that both Hsp70 and thioredoxin are components of the activation system. The apo-nNOS activating activity of reticulocyte lysate is retained in a pool of fractions containing Hsp70 that elute from DE52 prior to Hsp90. All of the activating activity and 20-30% of the Hsp70 elute in the flow-through fraction upon subsequent ATP-agarose chromatography. Apo-nNOS activation by this flow-through fraction is inhibited by pifithrin-μ, a small molecule inhibitor of Hsp70, suggesting that a non-ATP-binding form of Hsp70 is involved in heme-dependent apo-nNOS activation. Previous work has shown that apo-nNOS can be activated by thiol-disulfide exchange, and we show substantial activation with a small molecule dithiol modeled on the active motifs of thioredoxin and protein disulfide isomerase. Further fractionation of the ATP-agarose flow-through on Sephacryl S-300 separates free thioredoxin from apo-nNOS activating activity, Hsp70, and a small amount of thioredoxin, all of which are eluted throughout the macromolecular peak. Incubation of apo-nNOS with the macromolecular fraction in combination either with the thioredoxin-containing fraction or with purified recombinant human thioredoxin restores full heme-dependent activating activity. This supports a model in which Hsp70 binding to apo-nNOS stabilizes an open state of the heme/substrate binding cleft to facilitate thioredoxin access to the active site cysteine that coordinates with heme iron, permitting heme binding and dimerization to the active enzyme. PMID:21755988

  11. 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

  12. 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. PMID:2953718

  13. 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

  14. Molecular cloning, primary structure, and expression of the human platelet/erythroleukemia cell 12-lipoxygenase.

    PubMed Central

    Funk, C D; Furci, L; FitzGerald, G A

    1990-01-01

    The major pathway of arachidonic acid metabolism in human platelets proceeds via a 12-lipoxygenase enzyme; however, the biological role of the product of this reaction, 12-hydro(pero)xyeicosatetraenoic acid [12-H(P)ETE], is unknown. Using a combination of the polymerase chain reaction and conventional screening procedures, we have isolated cDNA clones encoding the human platelet/human erythroleukemia (HEL) cell 12-lipoxygenase. From the deduced primary structure, human platelet/HEL 12-lipoxygenase would encode a Mr 75,000 protein consisting of 663 amino acids. The cDNA encoding the full-length protein (pCDNA-121x) under the control of the cytomegalovirus promoter was expressed in simian COS-M6 cells. Intact cells and lysed-cell supernatants were able to synthesize 12-H(P)ETE from arachidonic acid, whereas no 12-H(P)ETE synthesis was detected in mock-transfected cells. A single 2.4-kilobase mRNA was detected in erythroleukemia cells but not in several other tissues and cell lines evaluated by Northern blot analysis. Comparison of the human platelet/HEL 12-lipoxygenase sequence with that of porcine leukocyte 12-lipoxygenase and human reticulocyte 15-lipoxygenase revealed 65% amino acid identity to both enzymes. By contrast, the leukocyte 12-lipoxygenase is 86% identical to human reticulocyte 15-lipoxygenase. Sequence data and previously demonstrated immunochemical and biochemical evidence support the existence of distinct 12-lipoxygenase isoforms. The availability of cDNA probes for human platelet/HEL cell 12-lipoxygenase should facilitate elucidation of the biological role of this pathway. Images PMID:2377602

  15. Comparison of the hematological profile of elite road cyclists during the 2010 and 2012 GiroBio ten-day stage races and relationships with final ranking.

    PubMed

    Lombardi, Giovanni; Lanteri, Patrizia; Fiorella, Pier Luigi; Simonetto, Luigi; Impellizzeri, Franco M; Bonifazi, Marco; Banfi, Giuseppe; Locatelli, Massimo

    2013-01-01

    Cycling stage races are strenuous endurance events during which exercise-induced variations in hematological parameters are consistently observed. However, specific literature on such changes is scarce and published data have been derived from small samples of athletes. The aims of this study were: (1) to determine the hematological response to middle-term strenuous endurance; and (2) to determine whether a relationship exists between the athlete-specific hematological profile and final placement in a cycling stage race. The study population was male professional cyclists (n = 253) competing in the 2010 (n = 144) and 2012 (n = 109) GiroBio 10-day stage races. Blood draws taken before the start of the race, at mid-race, and at end-race were performed in strict compliance with academic and anti-doping pre-analytical warnings. Blood chemistry included white blood cell, red blood cell, hemoglobin concentration, hematocrit, mean corpuscular volume (MCV), mean hemoglobin content (MCH), mean corpuscular hemoglobin content (MCHC), platelets, and reticulocyte relative and absolute counts. Compared to baseline values, erythrocyte, hemoglobin, hematocrit, MCHC, platelet and reticulocyte counts were all consistently lower at mid-race, but returned to normal by race-end, while leukocytes were increased in the final phase. MCV increased during both events. MCH increased in the first part to then return to baseline in the 2012 race. The calculated OFF-score consistently decreased in the first half of the race before increasing, but remained lower than the baseline value. The trends of variation in hematological parameters were substantially similar in both events. There was an inverse, albeit weak, relationship between placement and erythrocyte, platelet, hemoglobin, hematocrit and OFF-score values in the 2010, but not in the 2012 race. In conclusion, the data confirm that, in this large series of elite road cyclists, the strenuous effort a rider sustains during a

  16. 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

  17. Effects of iron supplementation on red blood cell hemoglobin content in pregnancy

    PubMed Central

    Schoorl, Margreet; Schoorl, Marianne; van der Gaag, Derek; Bartels, Piet C. M.

    2012-01-01

    Although a mild degree of anemia is common in the third trimester of pregnancy, it remains a challenge to establish whether a decrease in hemoglobin (Hb) concentration is physiological or pathological. The World Health Organization suggested a Hb concentration of 110 g/L to discriminate anemia. Several European investigators recommended Hb cut-off values of between 101–110 g/L. The aim of this study was to establish short-term effects of iron supplementation on the hemoglobin content of reticulocytes (Ret-He) and red blood cells (RBC-He) in case of suspected iron deficient erythropoiesis (IDE) in the third trimester of pregnancy. Twenty-five subjects with suspected IDE during pregnancy (Hb ≤110g/L, Ret-He <29.6 pg, zinc protoporphyrin >75 mol/mol hem) participated in the study. After iron supplementation, reticulocyte counts increased from 0.061±0.015×1012/L to 0.079±0.026×1012/L and Ret-He increased from 23.6±2.8 pg to 28.3±2.6 pg (P=<0.001). RBC-He increased from 26.9±1.9 pg to 27.4±1.8 pg (not significant, NS) and Ret-He/RBC-He ratio increased from 0.97±0.06 towards 1.07±0.05 (P=<0.001). Hb concentrations demonstrated an obvious increase from 105±6 g/L towards 115±5 g/L (P≤0.001) after supplementation. An obvious increase in RBC distribution width was observed from 45.0±3.6 fL towards 52.3±7.0 fL (P≤0.001). We recommend that Ret-He and Ret-He/RBC-He ratio be integrated into the protocols for anemia screening and for monitoring effects of iron supplementation during pregnancy. In particular, the parameters should be considered in subjects with Hb results in the controversial range of 101–108 g/L. PMID:23355942

  18. 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

  19. The effect of intravenous iron on postoperative transfusion requirements in hip fracture patients: study protocol for a randomized controlled trial

    PubMed Central

    2013-01-01

    Background Anaemia following hip fracture is common. Approximately 30 to 45% of patients have haemoglobin concentrations below population norms on admission, and around 10% are severely anaemic. Anaemia on admission, and in the postoperative period, is associated with poor outcomes with regard to mobility, postoperative mortality and readmission. There is currently no clear consensus on the optimal method of managing perioperative anaemia in this group of frail patients with frequent comorbidity. Liberal red cell transfusion in the postoperative period does not appear to improve outcome, whereas tranexamic acid appears to reduce transfusion rate at the expense of increased cardiovascular morbidity. There are encouraging results from one centre with the use of agents to stimulate red cell production, including intravenous iron and erythropoietin. UK practice differs significantly from these patients and these studies, and it is not clear whether these promising results will translate to the UK population. Methods/Design This is a single-centre randomized controlled parallel group trial, in a British university hospital.Randomization is achieved using a website and computer-generated concealed tables. Participants are 80 patients 70 years or over with acute hip fracture undergoing operative repair. The intervention group receive three daily infusions of 200 mg iron sucrose, starting within 24 hours of admission. The control group receive standard hospital care at the discretion of the clinical team. Red cell transfusions for each group are given in accordance with standard clinical triggers. The primary outcome is an increase in mean reticulocyte count in the intervention group at day 7. Secondary outcome measures include haemoglobin concentrations, early and late transfusion rates, infectious and cardiovascular complications, mobility and 30-day mortality. Discussion This is a pilot study to demonstrate haematopoietic efficacy of intravenous iron in this setting

  20. Performance evaluation of Abbott CELL-DYN Ruby for routine use.

    PubMed

    Lehto, T; Hedberg, P

    2008-10-01

    CELL-DYN Ruby is a new automated hematology analyzer suitable for routine use in small laboratories and as a back-up or emergency analyzer in medium- to high-volume laboratories. The analyzer was evaluated by comparing the results from the CELL-DYN((R)) Ruby with the results obtained from CELL-DYN Sapphire . Precision, linearity, and carryover between patient samples were also assessed. Precision was good at all levels for the routine cell blood count (CBC) parameters, CV% being reticulocyte percentage was highest at the low level (10.4). In a comparative study, the CELL-DYN Ruby results showed a good correlation (R(2) >or= 0.98) with CELL-DYN Sapphire for the CBC parameters. For the absolute reticulocyte count, R(2) was 0.82. In the white blood cell (WBC) differentials, the between-days precision was good for all parameters (CV%: or= 0.97), and the correlation coefficient for absolute monocyte count and monocyte percentage were 0.91 and 0.87, respectively. For absolute basophil count and basophil percentage the correlations were weaker (R(2) = 0.46 and 0.34, respectively). Carryover was minimal for all the parameters studied. The linearities of WBC, red blood cell, PLTs, and hemoglobin were acceptable within the tested ranges. In conclusion, the results of the evaluation showed the performance of CELL-DYN Ruby to be good. PMID:19046315

  1. Harmonization of animal clinical pathology testing in toxicity and safety studies. The Joint Scientific Committee for International Harmonization of Clinical Pathology Testing.

    PubMed

    Weingand, K; Brown, G; Hall, R; Davies, D; Gossett, K; Neptun, D; Waner, T; Matsuzawa, T; Salemink, P; Froelke, W; Provost, J P; Dal Negro, G; Batchelor, J; Nomura, M; Groetsch, H; Boink, A; Kimball, J; Woodman, D; York, M; Fabianson-Johnson, E; Lupart, M; Melloni, E

    1996-02-01

    Ten scientific organizations formed a joint international committee to provide expert recommendations for clinical pathology testing of laboratory animal species used in regulated toxicity and safety studies. For repeated-dose studies in rodent species, clinical pathology testing is necessary at study termination. Interim study testing may not be necessary in long-duration studies provided that it has been done in short-duration studies using dose levels not substantially lower than those used in the long-duration studies. For repeated-dose studies in nonrodent species, clinical pathology testing is recommended at study termination and at least once at an earlier interval. For studies of 2 to 6 weeks in duration in nonrodent species, testing is also recommended within 7 days of initiation of dosing, unless it compromises the health of the animals. If a study contains recovery groups, clinical pathology testing at study termination is recommended. The core hematology tests recommended are total leukocyte (white blood cell) count, absolute differential leukocyte count, erythrocyte (red blood cell) count, evaluation of red blood cell morphology, platelet (thrombocyte) count, hemoglobin concentration, hematocrit (or packed cell volume), mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration. In the absence of automated reticulocyte counting capabilities, blood smears from each animal should be prepared for reticulocyte counts. Bone marrow cytology slides should be prepared from each animal at termination. Prothrombin time and activated partial thromboplastin time (or appropriate alternatives) and platelet count are the minimum recommended laboratory tests of hemostasis. The core clinical chemistry tests recommended are glucose, urea nitrogen, creatinine, total protein, albumin, calculated globulin, calcium, sodium, potassium, total cholesterol, and appropriate hepatocellular and hepatobiliary tests. For hepatocellular

  2. 8-Azido double-stranded RNA photoaffinity probes. Enzymatic synthesis, characterization, and biological properties of poly(I,8-azidoI).poly(C) and poly(I,8-azidoI).poly(C12U) with 2',5'-oligoadenylate synthetase and protein kinase.

    PubMed

    Li, S W; Moskow, J J; Suhadolnik, R J

    1990-04-01

    The technique of photoaffinity labeling has been applied to the double-stranded RNA (dsRNA)-dependent enzyme 2',5'-oligoadenylate (2-5A) synthetase to provide a means for the examination of RNA-protein interaction(s) in the dsRNA allosteric binding domain of this enzyme. The synthesis, characterization, and biological properties of the photoaffinity probe poly[( 32P]I,8-azidoI).poly(C) and its mismatched analog poly[( 32P]I,8-azidoI).poly(C12U), which mimic the parent molecules poly(I).poly(C) and poly(I).poly(C12U), are described. The efficacy of poly[( 32P]I,8-azidoI).poly(C) and poly[( 32P]I,8-azidoI).poly(C12U) as allosteric site-directed activators is demonstrated using highly purified 2-5A synthetase from rabbit reticulocyte lysates and from extracts of interferon-treated HeLa cells. The dsRNA photoprobes activate these two 2-5A synthetases. Saturation of 2-5A synthetase is observed at 6 x 10(-4) g/ml poly[( 32P]I,8-azidoI).poly(C) following photolysis for 20 s at 0 degrees C. The photoincorporation of poly[( 32P]I,8-azidoI).poly(C) is specific, as demonstrated by the prevention of photoincorporation by native poly(I).poly(C). DNA, poly(I), and poly(C) are not competitors of poly[( 32P]I,8-azidoI).poly(C). Following UV irradiation of 2-5A synthetase with poly[( 32P]I,8-azidoI).poly(C), the reaction mixture is treated with micrococcal nuclease to hydrolyze azido dsRNA that is not cross-linked to the enzyme. A radioactive band of 110 kDa (the same as that reported for native rabbit reticulocyte lysate 2-5A synthetase) is observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The specific photolabeling of the 2-5A synthetase suggests that the azido dsRNA is intrinsic to the allosteric binding domain. The utility of poly[( 32P]I,8-azidoI).poly(C) for the detection of dsRNA-dependent binding proteins and the isolation of peptides at or near the allosteric binding site is discussed. PMID:2318823

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

    PubMed Central

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

    1974-01-01

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

  4. Control of erythropoiesis after high altitude acclimatization.

    PubMed

    Savourey, Gustave; Launay, Jean-Claude; Besnard, Yves; Guinet, Angélique; Bourrilhon, Cyprien; Cabane, Damien; Martin, Serge; Caravel, Jean-Pierre; Péquignot, Jean-Marc; Cottet-Emard, Jean-Marie

    2004-10-01

    Erythropoiesis was studied in 11 subjects submitted to a 4-h hypoxia (HH) in a hypobaric chamber (4,500 m, barometric pressure 58.9 kPa) both before and after a 3-week sojourn in the Andes. On return to sea level, increased red blood cells (+3.27%), packed cell volume (+4.76%), haemoglobin (+6.55%) ( P<0.05), and increased arterial partial pressure of oxygen (+8.56%), arterial oxygen saturation (+7.40%) and arterial oxygen blood content ( C(a)O(2)) (+12.93%) at the end of HH ( P<0.05) attested high altitude acclimatization. Reticulocytes increased during HH after the sojourn only (+36.8% vs +17.9%, P<0.01) indicating a probable higher reticulocyte release and/or production despite decreased serum erythropoietin (EPO) concentrations (-46%, P<0.01). Hormones (thyroid, catecholamines and cortisol), iron status (serum iron, ferritin, transferrin and haptoglobin) and renal function (creatinine, renal, osmolar and free-water clearances) did not significantly vary (except for lower thyroid stimulating hormone at sea level, P<0.01). Levels of 2,3-diphosphoglycerate (2,3-DPG) increased throughout HH on return (+14.7%, P<0.05) and an inverse linear relationship was found between 2,3-DPG and EPO at the end of HH after the sojourn only ( r=-0.66, P<0.03). Inverse linear relationships were also found between C(a)O(2) and EPO at the end of HH before ( r=-0.63, P<0.05) and after the sojourn ( r=-0.60, P=0.05) with identical slopes but different ordinates at the origin, suggesting that the sensitivity but not the gain of the EPO response to hypoxia was modified by altitude acclimatization. Higher 2,3-DPG levels could partly explain this decreased sensitivity of the EPO response to hypoxia. In conclusion, we show that altitude acclimatization modifies the control of erythropoiesis not only at sea level, but also during a subsequent hypoxia. PMID:15248067

  5. INHIBITORY AND MECHANISTIC INVESTIGATIONS OF OXO-LIPIDS WITH HUMAN LIPOXYGENASE ISOZYMES

    PubMed Central

    Armstrong, Michelle M.; Diaz, Giovanni; Kenyon, Victor; Holman, Theodore R.

    2014-01-01

    Oxo-lipids, a large family of oxidized human lipoxygenase (hLOX) products, are of increasing interest to researchers due to their involvement in different inflammatory responses in the cell. Oxo-lipids are unique because they contain electrophilic sites that can potentially form covalent bonds through a Michael addition mechanism with nucleophilic residues in protein active sites and thus increase inhibitor potency. Due to the resemblance of oxo-lipids to LOX substrates, the inhibitor potency of 4 different oxo-lipids; 5-oxo-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-oxo-ETE), 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE), 12-oxo-5,8,10,14-(Z,Z,E,Z)-eicosatetraenoic acid (12-oxo-ETE), and 13-oxo-9,11-(Z,E)-octadecadienoic acid (13-oxo-ODE) were determined against a library of LOX isozymes; leukocyte 5-lipoxygenase (h5-LOX), human reticulocyte 15-lipoxygenase-1 (h15-LOX-1), human platelet 12-lipoxygenase (h12-LOX), human epithelial 15-lipoxygenase-2 (h15-LOX-2), soybean 15-lipoxygenase-1 (s15-LOX-1), and rabbit reticulocyte 15-LOX (r15-LOX). 15-oxo-ETE exhibited the highest potency against h12-LOX, with an IC50 = 1 ± 0.1 μM and was highly selective. Steady state inhibition kinetic experiments determined 15-oxo-ETE to be a mixed inhibitor against h12-LOX, with a Kic value of 0.087 ± 0.008 μM and a Kiu value of 2.10 ± 0.8 μM. Time-dependent studies demonstrated irreversible inhibition with 12-oxo-ETE and h15-LOX-1, however, the concentration of 12-oxo-ETE required (Ki = 36.8 ± 13.2 μM) and the time frame (k2 = 0.0019 ± .00032 s−1) were not biologically relevant. These data are the first observations that oxo-lipids can inhibit LOX isozymes and may be another mechanism in which LOX products regulate LOX activity. PMID:24924423

  6. 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. PMID:21039028

  7. 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

  8. 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.

  9. Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

    PubMed Central

    Fellous, A; Ginzburg, I; Littauer, U Z

    1982-01-01

    Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6964957

  10. A PfRH5-Based Vaccine Is Efficacious against Heterologous Strain Blood-Stage Plasmodium falciparum Infection in Aotus Monkeys

    PubMed Central

    Douglas, Alexander D.; Baldeviano, G. Christian; Lucas, Carmen M.; Lugo-Roman, Luis A.; Crosnier, Cécile; Bartholdson, S. Josefin; Diouf, Ababacar; Miura, Kazutoyo; Lambert, Lynn E.; Ventocilla, Julio A.; Leiva, Karina P.; Milne, Kathryn H.; Illingworth, Joseph J.; Spencer, Alexandra J.; Hjerrild, Kathryn A.; Alanine, Daniel G.W.; Turner, Alison V.; Moorhead, Jeromy T.; Edgel, Kimberly A.; Wu, Yimin; Long, Carole A.; Wright, Gavin J.; Lescano, Andrés G.; Draper, Simon J.

    2015-01-01

    Summary Antigenic diversity has posed a critical barrier to vaccine development against the pathogenic blood-stage infection of the human malaria parasite Plasmodium falciparum. To date, only strain-specific protection has been reported by trials of such vaccines in nonhuman primates. We recently showed that P. falciparum reticulocyte binding protein homolog 5 (PfRH5), a merozoite adhesin required for erythrocyte invasion, is highly susceptible to vaccine-inducible strain-transcending parasite-neutralizing antibody. In vivo efficacy of PfRH5-based vaccines has not previously been evaluated. Here, we demonstrate that PfRH5-based vaccines can protect Aotus monkeys against a virulent vaccine-heterologous P. falciparum challenge and show that such protection can be achieved by a human-compatible vaccine formulation. Protection was associated with anti-PfRH5 antibody concentration and in vitro parasite-neutralizing activity, supporting the use of this in vitro assay to predict the in vivo efficacy of future vaccine candidates. These data suggest that PfRH5-based vaccines have potential to achieve strain-transcending efficacy in humans. PMID:25590760

  11. Management of iron deficiency anemia in inflammatory bowel disease – a practical approach

    PubMed Central

    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. PMID:24714874

  12. 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

  13. 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. PMID:26926814

  14. Discovery of a Novel Dual Fungal CYP51/Human 5-Lipoxygenase Inhibitor: Implications for Anti-Fungal Therapy

    PubMed Central

    Hoobler, Eric K.; Rai, Ganesha; Warrilow, Andrew G. S.; Perry, Steven C.; Smyrniotis, Christopher J.; Jadhav, Ajit; Simeonov, Anton; Parker, Josie E.; Kelly, Diane E.; Maloney, David J.; Kelly, S. L.; Holman, Theodore R.

    2013-01-01

    We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11) and human 5-lipoxygenase (5-LOX) with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM) and CYP51 (IC50 = 43 nM) in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component. PMID:23826084

  15. [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. PMID:26563723

  16. Cloning and expression of codon-optimized recombinant darbepoetin alfa in Leishmania tarentolae T7-TR.

    PubMed

    Kianmehr, Anvarsadat; Golavar, Raziyeh; Rouintan, Mandana; Mahrooz, Abdolkarim; Fard-Esfahani, Pezhman; Oladnabi, Morteza; Khajeniazi, Safoura; Mostafavi, Seyede Samaneh; Omidinia, Eskandar

    2016-02-01

    Darbepoetin alfa is an engineered and hyperglycosylated analog of recombinant human erythropoietin (EPO) which is used as a drug in treating anemia in patients with chronic kidney failure and cancer. This study desribes the secretory expression of a codon-optimized recombinant form of darbepoetin alfa in Leishmania tarentolae T7-TR. Synthetic codon-optimized gene was amplified by PCR and cloned into the pLEXSY-I-blecherry3 vector. The resultant expression vector, pLEXSYDarbo, was purified, digested, and electroporated into the L. tarentolae. Expression of recombinant darbepoetin alfa was evaluated by ELISA, reverse-transcription PCR (RT-PCR), Western blotting, and biological activity. After codon optimization, codon adaptation index (CAI) of the gene raised from 0.50 to 0.99 and its GC% content changed from 56% to 58%. Expression analysis confirmed the presence of a protein band at 40 kDa. Furthermore, reticulocyte experiment results revealed that the activity of expressed darbepoetin alfa was similar to that of its equivalent expressed in Chinese hamster ovary (CHO) cells. These data suggested that the codon optimization and expression in L. tarentolae host provided an efficient approach for high level expression of darbepoetin alfa. PMID:26546410

  17. A novel and potent ribonuclease from fruiting bodies of the mushroom Pleurotus pulmonarius.

    PubMed

    Ye, X Y; Ng, T B

    2002-05-01

    A ribonuclease (RNase), with an N-terminal sequence different from those of ribonucleases from the mushrooms Irpex lacteus, Lentinus edodes, Pleurotus ostreatus, Pleurotus tuber-regium, and Volvariella volvacea, was purified from fruiting bodies of the edible mushroom Pleurotus pulmonarius. The N-terminal sequence of P. pulmonarius RNase manifested homology to a portion of the sequences of ribosome inactivating protein abrin-b, abrin-c, and abrin-d, and Bacillus subtilis transcriptional regulator. The ribonuclease was adsorbed on Affi-gel blue gel, CM-Sepharose, and Mono S. It displayed a molecular mass of 14.4 kDa in both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on Superdex 75. The ribonuclease exhibited an activity of 25 114 U/mg on yeast tRNA. The highest ribonucleolytic activity was demonstrated toward poly C, followed by poly A, and then by poly G. There was no activity toward poly U. The optimal pH for its activity was 7 and the optimal temperature was 55 degrees C. It inhibited cell-free translation in a rabbit reticulocyte lysate with an IC50 of 0.33 nM. PMID:12054550

  18. Polyurethane scaffolds seeded with CD34(+) cells maintain early stem cells whilst also facilitating prolonged egress of haematopoietic progenitors.

    PubMed

    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 when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34(+) cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36(+)GPA(-/low)). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded "CD34(+) cell" population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells. PMID:27573994

  19. An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells

    PubMed Central

    Centrella, Michael; Porter, David L.; McCarthy, Thomas L.

    2012-01-01

    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. PMID:21640800

  20. 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

  1. Tracing the history of the ubiquitin proteolytic system: the pioneering article.

    PubMed

    Ciechanover, Aaron

    2009-09-11

    A series of findings made by several researchers during a two-decade period between the mid-1950s and mid-1970s raised the suspicion that the lysosome might not be the organelle that degrades the bulk of cellular proteins under basal conditions. These findings predicted the existence of a nonlysosomal, adenosine triphosphate (ATP)-dependent proteolytic system. Yet, following the initial discovery of such activity in a crude cell extract, it was a single article published in this journal [A. Ciechanover, Y. Hod, A. Hershko, A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes, Biochem. Biophys. Res. Commun. 81 (1978) 1100-1105], my first study as a graduate student of Avram Hershko, that made it clear that the system that catalyzes the activity is novel and complex, and does not follow the paradigm in the field of proteolysis where a single protease typically cleaves its substrate; here at least two components were required to carry out this activity, and one of them was an unusual, small, and heat-stable protein later identified as ubiquitin. PMID:19539608

  2. 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

  3. Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

    PubMed Central

    Hershko, A; Ciechanover, A; Heller, H; Haas, A L; Rose, I A

    1980-01-01

    The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis. Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or alpha-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP. Images PMID:6990414

  4. 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

  5. Does intermittent hypoxia increase erythropoiesis in professional cyclists during a 3-week race?

    PubMed

    Villa, José G; Lucía, Alejandro; Marroyo, José A; Avila, Concepción; Jiménez, Fernando; Garcia-López, Juan; Earnest, Conrad P; Córdova, Alfredo

    2005-02-01

    In this study we examined the effects of intermittent hypoxia exposure (IHE) in a group of professional cyclists (n = 6; age 26 +/- 1 yr) competing in the 2001 Vuelta a España. After each daily stage, treated subjects received four 5-min bouts of normobaric IHE (mean O2 concentration of 12.6%, simulating a mean altitude of 4,000 m) interspersed with 5-min bouts of breathing hotel room air (normoxia) until completing a total IHE of 20-min duration. The primary outcome, compared to a control group of similar characteristics not receiving IHE (n = 5; age 25 +/- 1 yr), was the % increase in erythropoietin (Epo) from the beginning to the end of the Vuelta. Statistical analysis showed that Epo increase tended to be higher (p = 0.052) in the IHE group than in controls (37.4 +/- 5.8% vs. -4.4 +/- 19.5%, respectively). However IHE had no effect on reticulocytes or erythrocyte count (p > 0.05). PMID:15855683

  6. Heme binds to a short sequence that serves a regulatory function in diverse proteins.

    PubMed Central

    Zhang, L; Guarente, L

    1995-01-01

    Heme is a prosthetic group for numerous enzymes, cytochromes and globins, and it binds tightly, sometimes covalently, to these proteins. Interestingly, heme also potentiates binding of the yeast transcriptional activator HAP1 to DNA and inhibits mitochondrial import of the mammalian delta-aminolevulinate synthase (ALAS) and the catalytic activity of the reticulocyte kinase, HRI. All three of these proteins contain a short sequence, the heme regulatory motif (HRM), that occurs six times adjacent to the HAP1 DNA binding domain, twice in the leader targeting sequence of ALAS and twice near the catalytic domain of the HRI kinase. Here we show that a 10 amino acid peptide containing the HRM consensus binds to heme in the micromolar range, and shifts the heme absorption spectrum to a longer wavelength, a direction opposite to the change caused by cytochromes or globins. Further, we show that a single HRM regulates the acidic activation domains of HAP1 and GAL4 independently of regulation of DNA binding of the transcription factors. These findings thus establish a novel heme binding sequence which is structurally distinct from sequences in globins or cytochromes and which has a regulatory function. Images PMID:7835342

  7. Effect of magnesium deficiency on erythrocyte aging in rats.

    PubMed Central

    Elin, R. J.; Utter, A.; Tan, H. K.; Corash, L.

    1980-01-01

    Rats placed on a magnesium-deficient diet show decreased erythrocyte magnesium concentration, shortened erythrocyte survival, and erythrocyte membrane ultrastructure defects and become progressively anemic. Whether these pathologic processes are due to abnormal erythropoiesis or occur in the peripheral circulation is unknown. In the present study, magnesium and hemoglobin concentrations, reticulocyte count, erythrocyte pyrophosphatase, and pyruvate kinase activities were determined at weekly intervals for 6 weeks in whole blood and age-dependent erythrocyte fractions isolated from inbred Fisher rats fed a diet deficient in magnesium or the same diet with added magnesium. Freeze-fracture electron microscopic examinations were performed on age-dependent erythrocyte fractions to evaluate the membrane defect. The youngest red cells from magnesium-deficient rats were similar to those of control animals with respect to erythrocyte magnesium concentrations, pyrophosphatase activities, and membrane morphology. The older erythrocyte fractions from magnesium-deficient rats showed significant decreases in magnesium concentrations, pyrophosphatase activity, and the presence of membrane abnormalities. Thus, new erythrocytes produced in magnesium-deficient rats appear to be normal but rapidly develop biochemical and morphologic abnormalities with aging in a magnesium-deficient plasma environment. Images Figure 1 PMID:6106388

  8. 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. PMID:26296885

  9. Characterization of a protein cofactor that mediates protein kinase A regulation of the renal brush border membrane Na(+)-H+ exchanger.

    PubMed Central

    Weinman, E J; Steplock, D; Wang, Y; Shenolikar, S

    1995-01-01

    Activation of cAMP-dependent protein kinase A inhibits the renal proximal tubule brush border membrane Na(+)-H+ exchanger by a process involving participation of a regulatory cofactor (NHE-RF) that is distinct from the transporter itself. Recent studies from this laboratory reported a partial amino acid sequence of this putative cofactor (Weinman, E. J., D. H. Steplock, and S. Shenolikar. 1993. J. Clin. Invest. 92:1781-1786). The present experiments detail the structure of the NHE-RF protein as determined from molecular cloning studies. A codon-biased oligonucleotide probe to a portion of the amino acid sequence of the putative cofactor was used to isolate a 1.9-kb cDNA from a rabbit renal library. The encoded protein is 358 amino acids in length and is rich in proline residues. Search of existing data bases indicates that NHE-RF is a unique protein. Using a reticulocyte lysate, the cDNA translated a product of approximately 44 kD, which was recognized by an affinity-purified polyclonal antibody to NHE-RF. Potential phosphorylation sites for protein kinase A are present. The mRNA for the protein is expressed in kidney, proximal small intestine, and liver. Reverse transcription/PCR studies in the kidney indicate the presence of mRNA for NHE-RF in several distinct nephron segments including the proximal tubule. Images PMID:7738182

  10. 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. PMID:27465156

  11. Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis.

    PubMed

    Solomon, L R; Rubenstein, P A

    1987-08-15

    The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes. PMID:3301854

  12. The blood-stage malaria antigen PfRH5 is susceptible to vaccine-inducible cross-strain neutralizing antibody.

    PubMed

    Douglas, Alexander D; Williams, Andrew R; Illingworth, Joseph J; Kamuyu, Gathoni; Biswas, Sumi; Goodman, Anna L; Wyllie, David H; Crosnier, Cécile; Miura, Kazutoyo; Wright, Gavin J; Long, Carole A; Osier, Faith H; Marsh, Kevin; Turner, Alison V; Hill, Adrian V S; Draper, Simon J

    2011-01-01

    Current vaccine strategies against the asexual blood stage of Plasmodium falciparum are mostly focused on well-studied merozoite antigens that induce immune responses after natural exposure, but have yet to induce robust protection in any clinical trial. Here we compare human-compatible viral-vectored vaccines targeting ten different blood-stage antigens. We show that the full-length P. falciparum reticulocyte-binding protein homologue 5 (PfRH5) is highly susceptible to cross-strain neutralizing vaccine-induced antibodies, out-performing all other antigens delivered by the same vaccine platform. We find that, despite being susceptible to antibody, PfRH5 is unlikely to be under substantial immune selection pressure; there is minimal acquisition of anti-PfRH5 IgG antibodies in malaria-exposed Kenyans. These data challenge the widespread beliefs that any merozoite antigen that is highly susceptible to immune attack would be subject to significant levels of antigenic polymorphism, and that erythrocyte invasion by P. falciparum is a degenerate process involving a series of parallel redundant pathways. PMID:22186897

  13. Effects of Glycerol and Creatine Hyperhydration on Doping-Relevant Blood Parameters

    PubMed Central

    Polyviou, Thelma P.; Easton, Chris; Beis, Lukas; Malkova, Dalia; Takas, Pantazis; Hambly, Catherine; Speakman, John R.; Koehler, Karsten; Pitsiladis, Yannis P.

    2012-01-01

    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. PMID:23112907

  14. Effect of alpha-sarcin and ribosome-inactivating proteins on the interaction of elongation factors with ribosomes.

    PubMed

    Brigotti, M; Rambelli, F; Zamboni, M; Montanaro, L; Sperti, S

    1989-02-01

    alpha-Sarcin from Aspergillus giganteus and the ribosome-inactivating proteins (RIPs) from higher plants inactivate the 60 S ribosomal subunit. The former is an RNAase, whereas RIPs are N-glycosidases. The site of cleavage of RNA and that of N-glycosidic depurinization are at one nucleotide distance in 28 S rRNA [Endo & Tsurugi (1987) J. Biol. Chem. 262, 8128-8130]. The effect of alpha-sarcin and that of RIPs on the interaction of elongation factors with Artemia salina (brine shrimp) ribosomes have been investigated. alpha-Sarcin inhibits both the EF1 (elongation factor 1)-dependent binding of aminoacyl-tRNA and the GTP-dependent binding of EF2 (elongation factor 2) to ribosomes, whereas two of the RIPs tested, ricin from Ricinus communis (castor bean) and volkensin from Adenia volkensii (kilyambiti), inhibit only the latter reaction. EF2 protects ribosomes from inactivation by both alpha-sarcin and ricin. The EF1-binding site is affected only by alpha-sarcin. The sensitivity of this site to alpha-sarcin is increased by pretreatment of ribosomes with ricin. A. salina ribosomes were highly resistant to the third RIP tested, namely gelonin from Gelonium multiflorum. All four proteins tested have, however, a comparable activity on the rabbit reticulocyte-lysate system. PMID:2930482

  15. Changes in Protein Synthesis in Rapeseed (Brassica napus) Seedlings during a Low Temperature Treatment 1

    PubMed Central

    Meza-Basso, Luis; Alberdi, Miren; Raynal, Monique; Ferrero-Cadinanos, Maria-Luz; Delseny, Michel

    1986-01-01

    Changes induced by cold treatment in young rapeseed (Brassica napus) seedlings were investigated at the molecular level. Following germination at 18°C for 48 hours, one half of the seedlings was transferred to 0°C for another 48 hour period, the other half being kept at 18°C as a control. Newly synthesized proteins were labeled for the last 6 hours of incubation with [35S]methionine. The different polypeptides were separated by two-dimensional electrophoresis in polyacrylamide gels. Newly synthesized proteins were revealed by fluorography. Protein synthesis clearly continues at 0°C and some polypeptides preferentially accumulate at this temperature. On the other hand, synthesis of several others is repressed while many are insensitive to cold treatment. Similar changes are also observed when mRNA is prepared from cold treated seedlings, translated in vitro in a reticulocyte cell free system and compared with the products of mRNA extracted from control samples. Among the genes which are repressed we identified the small subunit of ribulose 1,6-bisphosphate carboxylase. These changes are also detectable after shorter treatments. Images Fig. 1 Fig. 2 Fig. 3 PMID:16665102

  16. Therapeutic fetal-globin inducers reduce transcriptional repression in hemoglobinopathy erythroid progenitors through distinct mechanisms.

    PubMed

    Dai, Yan; Sangerman, Jose; Luo, Hong Yuan; Fucharoen, Suthat; Chui, David H K; Faller, Douglas V; Perrine, Susan P

    2016-01-01

    Pharmacologic augmentation of γ-globin expression sufficient to reduce anemia and clinical severity in patients with diverse hemoglobinopathies has been challenging. In studies here, representative molecules from four chemical classes, representing several distinct primary mechanisms of action, were investigated for effects on γ-globin transcriptional repressors, including components of the NuRD complex (LSD1 and HDACs 2-3), and the downstream repressor BCL11A, in erythroid progenitors from hemoglobinopathy patients. Two HDAC inhibitors (MS-275 and SB939), a short-chain fatty acid derivative (sodium dimethylbutyrate [SDMB]), and an agent identified in high-throughput screening, Benserazide, were studied. These therapeutics induced γ-globin mRNA in progenitors above same subject controls up to 20-fold, and increased F-reticulocytes up to 20%. Cellular protein levels of BCL11A, LSD-1, and KLF1 were suppressed by the compounds. Chromatin immunoprecipitation assays demonstrated a 3.6-fold reduction in LSD1 and HDAC3 occupancy in the γ-globin gene promoter with Benserazide exposure, 3-fold reduction in LSD-1 and HDAC2 occupancy in the γ-globin gene promoter with SDMB exposure, while markers of gene activation (histone H3K9 acetylation and H3K4 demethylation), were enriched 5.7-fold. These findings identify clinical-stage oral therapeutics which inhibit or displace major co-repressors of γ-globin gene transcription and may suggest a rationale for combination therapy to produce enhanced efficacy. PMID:26603726

  17. Structural Analysis of the Synthetic Duffy Binding Protein (DBP) Antigen DEKnull Relevant for Plasmodium vivax Malaria Vaccine Design

    PubMed Central

    Chen, Edwin; Salinas, Nichole D.; Ntumngia, Francis B.; Adams, John H.; Tolia, Niraj H.

    2015-01-01

    The Plasmodium vivax vaccine candidate Duffy Binding Protein (DBP) is a protein necessary for P. vivax invasion of reticulocytes. The polymorphic nature of DBP induces strain-specific immune responses that pose unique challenges for vaccine development. DEKnull is a synthetic DBP based antigen that has been engineered through mutation to enhance induction of blocking inhibitory antibodies. We determined the x-ray crystal structure of DEKnull to identify if any conformational changes had occurred upon mutation. Computational and experimental analyses assessed immunogenicity differences between DBP and DEKnull epitopes. Functional binding assays with monoclonal antibodies were used to interrogate the available epitopes in DEKnull. We demonstrate that DEKnull is structurally similar to the parental Sal1 DBP. The DEKnull mutations do not cause peptide backbone shifts within the polymorphic loop, or at either the DBP dimerization interface or DARC receptor binding pockets, two important structurally conserved protective epitope motifs. All B-cell epitopes, except for the mutated DEK motif, are conserved between DEKnull and DBP. The DEKnull protein retains binding to conformationally dependent inhibitory antibodies. DEKnull is an iterative improvement of DBP as a vaccine candidate. DEKnull has reduced immunogenicity to polymorphic regions responsible for strain-specific immunity while retaining conserved protein folds necessary for induction of strain-transcending blocking inhibitory antibodies. PMID:25793371

  18. 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

  19. Hematologic Disorders: Anemia.

    PubMed

    Baltierra, David; Harper, Tiffany; Jones, Matthew Page; Nau, Konrad C

    2015-06-01

    Anemia occurs in up to 25% of the US population. Normal hemoglobin levels vary by race, sex, and age. Classification of anemia by mean corpuscular volume guides the differential diagnosis and evaluation. Iron studies, reticulocyte count, the red blood cell distribution width index, and blood test results are used to make the diagnosis. Iron deficiency anemia is the most common microcytic anemia and is managed with iron therapy. Parenteral iron is available when the oral route cannot be used. Patients who do not benefit from therapy should be evaluated for adherence, malabsorption, occult bleeding, systemic disease, or less common inherited disorders. A source of gastrointestinal bleeding is found in 60% to 70% of patients with iron deficiency anemia who are referred for endoscopy. Normocytic anemia has a broad differential, including nutritional deficiencies, blood loss, renal disease, malignancy (solid tumors or hematologic cancer), rheumatologic disorders, endocrine disorders, and other systemic diseases. Macrocytic anemias are seen with vitamin B12 and folate deficiency, alcohol use, thyroid disease, hydroxyurea, antiretroviral drugs, myelodysplastic syndromes, and myeloma. Oral vitamin B12 is underused, and can be as effective as intramuscular vitamin B12 in managing anemia due to vitamin B12 deficiency. PMID:26080453

  20. HMGB1 Mediates Anemia of Inflammation in Murine Sepsis Survivors

    PubMed Central

    Valdés-Ferrer, Sergio I; Papoin, Julien; Dancho, Meghan E; Olofsson, Peder S; Li, Jianhua; Lipton, Jeffrey M; Avancena, Patricia; Yang, Huan; Zou, Yong-Rui; Chavan, Sangeeta S; Volpe, Bruce T; Gardenghi, Sara; Rivella, Stefano; Diamond, Betty; Andersson, Ulf; Steinberg, Bettie M; Blanc, Lionel; Tracey, Kevin J

    2015-01-01

    Patients surviving sepsis develop anemia, but the molecular mechanism is unknown. Here we observed that mice surviving polymicrobial gram-negative sepsis develop hypochromic, microcytic anemia with reticulocytosis. The bone marrow of sepsis survivors accumulates polychromatophilic and orthochromatic erythroblasts. Compensatory extramedullary erythropoiesis in the spleen is defective during terminal differentiation. Circulating tumor necrosis factor (TNF) and interleukin (IL)-6 are elevated for 5 d after the onset of sepsis, and serum high-mobility group box 1 (HMGB1) levels are increased from d 7 until at least d 28. Administration of recombinant HMGB1 to healthy mice mediates anemia with extramedullary erythropoiesis and significantly elevated reticulocyte counts. Moreover, administration of anti-HMGB1 monoclonal antibodies after sepsis significantly ameliorates the development of anemia (hematocrit 48.5 ± 9.0% versus 37.4 ± 6.1%, p < 0.01; hemoglobin 14.0 ± 1.7 versus 11.7 ± 1.2 g/dL, p < 0.01). Together, these results indicate that HMGB1 mediates anemia by interfering with erythropoiesis, suggesting a potential therapeutic strategy for anemia in sepsis. PMID:26736178

  1. Evaluation of Neonatal Hemolytic Jaundice: Clinical and Laboratory Parameters

    PubMed Central

    Cherepnalkovski, Anet Papazovska; Krzelj, Vjekoslav; Zafirovska-Ivanovska, Beti; Gruev, Todor; Markic, Josko; Aluloska, Natasa; Zdraveska, Nikolina; Piperkovska, Katica

    2015-01-01

    BACKGROUND: Neonatal jaundice that occurs in ABO or Rhesus issoimunisation has been recognized as one of the major risk factors for development of severe hyperbilirubinemia and bilirubin neurotoxicity. AIM: Aim of our study was to investigate clinical and laboratory parameters associated with hemolytic jaundice due to Rh and ABO incompatibility and compare results with the group of unspecific jaundice. MATERIAL AND METHODS: One hundred sixty seven (167) neonatal hyperbilirubinemia cases were included in the study, 24.6% of which presented with ABO/Rhesus type hemolytic jaundice, and the rest with unspecific jaundice. Evaluation included: blood count, reticulocites, serum bilirubin, aminotransferases, blood grouping, and Coombs test, also the day of bilirubin peak, duration of the hyperbilirubinemia, and additional bilirubin measurements. RESULTS: We showed significantly lower mean values of hemoglobin, erythrocytes and hematocrit and significantly higher values of reticulocytes in the group of ABO/Rh incompatibility compared to the group of jaundice of unspecific etiology; also an earlier presentation and a higher-grade jaundice in this group. CONCLUSIONS: The laboratory profile in ABO/Rh isoimmunisation cases depicts hemolytic mechanism of jaundice. These cases carry a significant risk for early and severe hyperbilirubinemia and are eligible for neurodevelopmental follow-up. Hematological parameters and blood grouping are simple diagnostic methods that assist the etiological diagnosis of neonatal hyperbilirubinemia. PMID:27275310

  2. 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.

  3. A reconstituted cell-free assay for the evaluation of the intrinsic activity of purified human ribosomes.

    PubMed

    Penzo, Marianna; Carnicelli, Domenica; Montanaro, Lorenzo; Brigotti, Maurizio

    2016-07-01

    We describe a cell-free translation system for evaluating the activity of ribosomes stringently purified from human cells. This system is based on in vitro reconstitution of the cellular translation machinery, in which a ribosome-free rabbit reticulocyte lysate (RRL) is reassembled with human ribosomes and in vitro-transcribed reporter mRNAs. The protocol describes the preparation of the RRL-derived fractions, purification of ribosomes devoid of detectable nonribosomal-associated factors, and assembly of the reactions to evaluate ribosomal translational efficiency and fidelity using appropriate reporter transcripts. The whole procedure can be completed in ∼2.5 d (plus 2 weeks for RRL preparation and cell expansion time). This protocol can be applied to study intrinsic functional properties (cis-acting element-mediated translation initiation or translational fidelity) of ribosome populations from different sources (including nonhuman origin). It is therefore useful for the characterization of ribosomal function in ribosomopathies and cancer, and it will be applicable in the emerging fields of ribosome diversity and specialized ribosomes. PMID:27336708

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system.

    PubMed

    Hjerrild, Kathryn A; Jin, Jing; Wright, Katherine E; Brown, Rebecca E; Marshall, Jennifer M; Labbé, Geneviève M; Silk, Sarah E; Cherry, Catherine J; Clemmensen, Stine B; Jørgensen, Thomas; Illingworth, Joseph J; Alanine, Daniel G W; Milne, Kathryn H; Ashfield, Rebecca; de Jongh, Willem A; Douglas, Alexander D; Higgins, Matthew K; Draper, Simon J

    2016-01-01

    The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS(2), based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of 'difficult-to-make' proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. PMID:27457156

  9. Complex patterns of sequence variation and multiple 5' and 3' ends are found among transcripts of the erythroid ankyrin gene.

    PubMed

    Birkenmeier, C S; White, R A; Peters, L L; Hall, E J; Lux, S E; Barker, J E

    1993-05-01

    The structural protein ankyrin functions in red blood cells to link the spectrin-based membrane skeleton to the plasma membrane. Ankyrin proteins are now known to occur in most cell types, and two distinct ankyrin genes have been identified (erythroid (Ank-1) and brain (Ank-2)). We have characterized transcripts of the mouse erythroid ankyrin gene by cDNA cloning and DNA sequencing. Ank-1 transcripts of 7.5 and 9.0 kilobases are found in erythroid tissues, and a 9.0-kilobase transcript is found in cerebellum. RNA hybridization blot analysis of 13 additional mouse tissues has detected four novel Ank-1 transcripts (5.0, 3.5, 2.0, and 1.6 kilobases in size). Sequencing of Ank-1 cDNA clones isolated from mouse reticulocyte, spleen, and cerebellar libraries has identified (i) multiple 5' ends that indicate possible multiple promoters; (ii) alternative polyadenylation sites that probably account for the 7.5- and 9.0-kilobase size difference; (iii) a variety of small insertions and deletions that could produce transcripts (and ultimately proteins) of nearly identical size, but different functions; and (iv) clones with large deletions of coding sequence that account for the smaller transcripts seen in spleen, skeletal muscle, and heart. PMID:8486643

  10. The role of intravenous iron in the treatment of anemia in cancer patients

    PubMed Central

    2012-01-01

    Anemia is a major cause of morbidity in cancer patients resulting in poor physical performance, prognosis and therapy outcome. Initially, erythropoietin-stimulating agents (ESAs) were supposed to be the treatment of choice but about one third of patients turned out to be nonresponders and meta-analyses provided evidence of an increased risk of mortality if used excessively. This along with the successful use of intravenous iron for anemia in patients with chronic kidney disease prompted seven clinical studies evaluating the efficacy of intravenous iron as an adjunct to ESAs and four additional studies using intravenous iron only for anemia in cancer patients. These studies confirmed a superior response if ESAs are combined with intravenous iron and revealed iron only to be a useful option in patients with mild and absolute iron deficiency (AID). Currently, best treatment decisions for anemia in cancer might be based on measurements of serum ferritin (SF), transferrin saturation (TSAT), soluble transferrin receptor (sTfR), ferritin index (FI = sTfR/log SF), hypochromic reticulocytes (CHR) and C-reactive protein (CRP). However, there is still an urgent need for trials investigating diagnostic approaches to optimize therapy of anemia in cancer patients with iron and/or ESAs. PMID:23556124

  11. Hematological and Biochemical Markers of Iron Status in a Male, Young, Physically Active Population

    PubMed Central

    Nunes, Lázaro Alessandro Soares; Grotto, Helena Zerlotti W.; Brenzikofer, René; Macedo, Denise Vaz

    2014-01-01

    The aim of this study was to establish reference intervals (RIs) for the hemogram and iron status biomarkers in a physically active population. The study population included male volunteers (n = 150) with an average age of 19 ± 1 years who had participated in a regular and controlled exercise program for four months. Blood samples were collected to determine hematological parameters using a Sysmex XE-5000 analyzer (Sysmex, Kobe, Japan). Iron, total iron-binding capacity (TIBC), transferrin saturation and ferritin, and high-sensitivity C-reactive protein (CRP) concentrations in serum samples were measured using commercial kits (Roche Diagnostics, GmbH, Mannheim, Germany) and a Roche/Hitachi 902 analyzer. The RIs were established using the RefVal program 4.1b. The leucocyte count, TIBC, and CRP and ferritin concentrations exhibited higher RIs compared with those in a nonphysically active population. Thirty volunteers (outliers) were removed from the reference population due to blood abnormalities. Among the outliers, 46% exhibited higher CRP concentrations and lower concentrations of iron and reticulocyte hemoglobin compared with the nonphysically active population (P < 0.001). Our results showed that it is important to establish RIs for certain laboratory parameters in a physically active population, especially for tests related to the inflammatory response and iron metabolism. PMID:25045665

  12. 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.

  13. Truncated form of tenascin-X, XB-S, interacts with mitotic motor kinesin Eg5.

    PubMed

    Endo, Toshiya; Ariga, Hiroyoshi; Matsumoto, Ken-ichi

    2009-01-01

    XB-S is a protein with an amino-terminal-truncated form of tenascin-X (TNXB). However, the precise roles of XB-S in vivo are unknown. In this study, to determine the role of XB-S in vivo, we screened XB-S-binding proteins. FLAG-tagged XB-S was transiently introduced into 293T cells. Then its associated proteins were purified by immunoprecipitation using an anti-FLAG antibody and its components were identified by mass spectrometric analyses. Mitotic motor kinesin Eg5 was identified in the immunoprecipitates. XB-S and Eg5 proteins were co-localized in the cytoplasm in interphase and mitosis, but XB-S did not localize on mitotic spindle microtubules, on which Eg5 prominently localized in mitosis. As for Eg5 binding to XB-S, glutathione S-transferase-fused XB-S expressed in vitro directly bound to full-length Eg5 translated in reticulocyte lysate, and the XB-S-binding region was located in the motor domain of Eg5. Furthermore, during cell cycle progression XB-S showed a similar expression profile to that of Eg5. These results suggest possible involvement of XB-S in the function of Eg5. PMID:18679583

  14. Carcinogenesis response modulation induced by gelonin encapsulated in liposome.

    PubMed

    Alam, Anis; Nakhuru, K S; Singha, L I

    2008-08-01

    The effectiveness of gelonin to arrest protein synthesis, thereby limiting the growth of cancer cells was studied by encapsulating it into liposomes. The protein was extracted from the seeds of Indian plant Gelonium multiflorum by ammonium sulfate precipitation and purified using cation-exchange and gel-filtration chromatography. Biological activity of purified gelonin was determined using a rabbit reticulocyte lysate assay in the cell-free translational experiments. Gelonin was encapsulated in conventional liposomes prepared by the dry film method in order to retain biological activity of the entrapped protein. Carcinogenesis was induced in Swiss albino mice by intravenous administration of DBN (10 mg kg(-1) body weight) at weekly intervals. Marker enzyme assays (GGT, AChE, and GST), GSH levels, cell proliferation assay, hepatocyte DNA analysis, histological examination of micro sections of liver tissues were parameters used to monitor carcinogenesis induction, and regression in mice. From the in vitro experiments conducted, it was observed that gelonin upon its encapsulation into liposome, resulted in significant destruction of the transformed liver cells by its cytotoxic effects that arrest protein synthesis. Various parameters studied to monitor regression also suggested mass cell destruction to liver upon administration of liposomal gelonin in mice exposed to DBN. PMID:18500656

  15. Erythrocytosis associated with hemoglobin Rainier: oxygen equilibria and marrow regulation

    PubMed Central

    Adamson, John W.; Parer, Julian T.; Stamatoyannopoulos, George; Heinenberg, S.

    1969-01-01

    Hemoglobin Rainier (β145 tyrosine→histidine) is an abnormal hemoglobin associated with increased oxygen affinity, decreased heme-heme interaction, presence of a Bohr effect, and erythrocytosis, but without obvious clinical sequelae. Regulation of erythropoiesis was studied in affected members of families having either hemoglobin Rainier or Yakima, abnormal hemoglobins associated with erythrocytosis. Apart from the elevated but stable hemoglobin concentration and red cell mass, parameters of red cell production in the subjects were normal. Initially normal values of erythropoietin excretion were increased by phlebotomy indicating a significant hypoxic stress at an otherwise normal hematocrit. This stress led to increased reticulocyte production and an eventual return to the prephlebotomy hematocrit. The erythrocytosis in carriers of hemoglobins Rainer and Yakima appears to be secondary to the increased oxygen affinity and this, with the response to phlebotomy, is consistent with the postulate that the renal sensor tissue regulating erythropoietin production is primarily influenced by the oxygen tensions of venous rather than arterial blood. Images PMID:5796352

  16. Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli.

    PubMed

    Begam, Mehbuba; Kumar, Sushil; Roy, Sribash; Campanella, James J; Kapoor, H C

    2006-11-01

    A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV). PMID:16996549

  17. [Clinical application of blood matching with hemolytic test in vitro for transfusion treatment of crisis puerpera with acute hemolytic anemia].

    PubMed

    Yuan, Min; Tang, Cong-Hai; Gan, Wei-Wei; Wu, A-Yang; Yang, Hui-Cong; Zhang, Tian-Xin; Huang, Yan Xue; Qiu, Lu-Zhen; Chen, Hong-Pu; Lin, Feng-Li

    2014-08-01

    This study was aimed to establish the matching method of hemolytic test in vitro, and to guide the transfusion treatment for puerpera with acute hemolytic disease. The donor's erythrocytes were sensibilized by all the antibodies in plasma of patient in vitro and were added with complement, after incubation for 6.5 hours at 38 °C, the hemolysis or no hemolysis were observed. It is safe to transfuse if the hemolysis did not occur. The results showed that when the matching difficulty happened to puerpera with acute hemolytic disease, the compatible donor could be screened by hemolytic test in vitro. There were no untoward effects after transfusion of 6 U leukocyte-depleted erythrocyte suspension. The all hemoglobin, total bilirubins, indirect bilirubin, reticulocyte, D-dimex and so on were rapidly improved in patient after transfusion , showing obvious clinical efficacy of treatment. It is concluded that when the matching results can not judge accurately compatible or incompatible through the routine method of cross matching, the agglutinated and no-hemolytic erythrocytes can be screened by hemolytic test in vitro and can be transfused with good efficacy; the hemoglobin level can be promoted rapidly, and no untoward effects occur. PMID:25130835

  18. 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. PMID:24430825

  19. Haemolytic episode in G6 PD deficient workers exposed to TNT.

    PubMed Central

    Djerassi, L S; Vitany, L

    1975-01-01

    This is a report on three cases of acute haemolytic disease in glucose-6-phosphate dehydrogenase (G6PD) deficient workers exposed to trinitroluene (TNT). The courses of the haemolytic crises have several features in common, the most striking being the onset of the disease within two to four days after the start of exposure, and the fact that this has been the first and so far the only haemolytic episode in their lives in spite of detailed medical records of past diseases, injuries, and medications during 12 to 16 years preceding the haemolytic crisis and five to nine years following the disease. The lowest haemoglobin levels for the three patients were 4-0, 6-8, and 8-2 g/dl respectively; haematocrit values were 17 and 24%; reticulocytes rose in case 1 to 26-2%, in case 2 to 26%, and in case 3 to 10%. Indirect bilirubinaemia was increased in two patients (5-1 and 2-6 mg/100 ml) and stercobilinogen was as high as 2150 mg/24 hr in one patient. The presence of the metabolite monoamino 2-6 dinitrotoluene was proved in the urine of case 3 and reached 21 gamma%. The possibility of a dose-response relationship is briefly discussed and the risk of exposing G6 PD individuals in chemical processes is mentioned. PMID:1125128

  20. Safety of docosahexaenoic acid (DHA) administered as DHA ethyl ester in a 9-month toxicity study in dogs.

    PubMed

    Dahms, Irina; Beilstein, Paul; Bonnette, Kimberly; Salem, Norman

    2016-06-01

    DHA Ethyl Ester (DHA-EE) is a 90% concentrated ethyl ester of docosahexaenoic acid manufactured from the microalgal oil. The objective of the 9-month study was to evaluate safety of DHA-EE administered to beagle dogs at dose levels 150, 1000 and 2000 mg/kg bw/day by oral gavage and to determine reversibility of any findings after a 2-month recovery period. DHA-EE was well tolerated at all doses. There were observations of dry flaky skin with occasional reddened areas at doses ≥1000 mg/kg bw/day. These findings lacked any microscopic correlate and were no longer present after the recovery period. There were no toxicologically relevant findings in body weights, body weight gains, food consumption, ophthalmological examinations, and ECG measurements. Test article-related changes in hematology parameters were limited to decreases in reticulocyte count in the high-dose males and considered non-adverse. In clinical chemistry parameters, dose-related decreases in cholesterol and triglycerides levels were observed at all doses in males and females and attributed to the known lipid-lowering effects of DHA. There were no effects on other clinical chemistry, urinalysis or coagulation parameters. There were no abnormal histopathology findings attributed to test article. The No-Observable-Adverse-Effect Level of DHA-EE was established at 2000 mg/kg bw/day for both genders. PMID:27036332

  1. alpha 1-Fetoprotein mRNA of rat yolk sac and hepatoma.

    PubMed Central

    Chiu, J F; Decha-Umphai, W; Commer, P

    1979-01-01

    Rat alpha 1-fetoprotein mRNA was isolated and purified to apparent homogeneity by means of immunoadsorption and oligo (dT) cellulose affinity chromatography. Purified AFP mRNA migrated as a 21S peak in 2.5% SDS-polyacrylamide gels. The translation product of this mRNA in micrococcal nuclease treated reticulocyte lysate was identified as AFP by specific immunoprecipitation, SDS-gel electrophoresis and tryptic digestion analysis. DNA complimentary to AFP mRNA was synthesized with avian meyloblastosis virus RNA-dependent DNA polymerase. This AFP cDNA was used as a probe to quantitate AFP mRNA in the developing rat liver and to compare the complexity and diversity of AFP mRNA derived from the normal rat liver and Morris hepatoma 7777. We found that the amount of functional AFP mRNA is decreasing during liver development. There is very little, if any, AFP mRNA in the adult rat liver. A high degree of homology between the AFP mRNA sequences of yolk sac and hepatoma was also found. PMID:91159

  2. Clinical outcomes of splenectomy in children: report of the splenectomy in congenital hemolytic anemia registry.

    PubMed

    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

    2015-03-01

    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 to 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 g/dl, 52 week 12.8 ± 1.6 g/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

  3. Can Serum Gdf-15 be Associated with Functional Iron Deficiency in Hemodialysis Patients?

    PubMed

    Yilmaz, Hakki; Cakmak, Muzaffer; Darcin, Tahir; Inan, Osman; Bilgic, Mukadder Ayse; Bavbek, Nuket; Akcay, Ali

    2016-06-01

    Functional iron deficiency (FID) incidence is gradually increasing in hemodialysis (HD) patients. Recently, high levels of GDF-15 supressed the iron regulatory protein hepcidin and GDF-15 expression increased in iron-deficient patients. The relationship between FID, GDF-15, and hepcidin is currently unknown. The present study aimed to evaluate the association between GDF-15, hepcidin, and FID in chronic HD patients. Serum GDF-15 and hepcidin concentrations were measured in 105 HD patients and 40 controls. FID is defined as serum ferritin >800 ng/mL, TSAT <25 %, Hb levels <11 g/dL, and reticulocyte haemoglobin content (CHr) <29 pg. Serum GDF-15 and hepcidin levels were increased significantly in HD patients with FID, compared to HD patients without anemia and controls. GDF-15 correlated with ferritin, hepcidin, and CRP in the entire cohort. GDF-15 was related to ferritin and CRP in HD patients with FID. GDF-15 is better diagnostic marker than hepcidin for detection of FID [AUC = 0.982 (0.013) versus AUC = 0.921 (0.027); P = 0.0324]. GDF-15 appears to be a promising tool for detection of FID. High levels of ferritin and CRP correlated with GDF-15. Our results support GDF-15 as a new mediator of FID via hepcidin, chronic inflammation, or unknown pathways. PMID:27065587

  4. Regulation by nitrate of protein synthesis and translation of RNA in maize roots

    SciTech Connect

    McClure, P.R.; Bouthyette, P.Y.

    1986-04-01

    Roots of maize seedlings were exposed to /sup 35/S-methionine in the presence or absence of nitrate. Using SDS-PAGE, nitrate-induced changes in labeled polypeptides were noted in the soluble (at 92, 63 and 21kD) and organellar(at 14kD) fractions, as well as in a membrane fraction of putative tonoplast origin (at 31kD). No nitrate-induced changes were noted in a plasmamembrane-enriched fraction or in a membrane fraction of mixed origin. Total RNA from nitrate-treated and control roots was translated in a rabbit reticulocyte system. Five translation products (94, 63, 41, 39 and 21kD) were identified as nitrate-inducible by comparative gel electrophoresis. Changes in protein synthesis and translation of mRNA were apparent within 2-3 h after introduction of nitrate. Within 4-6 h after removal of nitrate, the level of nitrate-inducible translation products diminished to that of control roots. In contrast, the 31kD tonoplast polypeptide was still labeled 26 h after removal of external nitrate and /sup 35/S-methionine. The results will be discussed in relation to the nitrate induction of nitrate reductase, nitrite reductase, and the nitrate uptake system.

  5. 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

  6. Gender and response to antilymphocyte globulin (ALG) for severe aplastic anaemia.

    PubMed

    Nissen, C; Gratwohl, A; Tichelli, A; Stebler, C; Würsch, A; Moser, Y; Dalle Carbonare, V; Signer, E; Buser, M; Ritz, R

    1993-02-01

    We have evaluated the speed of haematological recovery in 103 severe aplastic anaemia (SAA) patients treated with antilymphocyte globulin (ALG) and followed at our institution for 3-15 years. We found that haemopoietic recovery was significantly delayed in six girls under the age of 10 years. This slow recovery in girls might be explained by their relative inability to release haemopoietic growth factors, granulocyte colony stimulating activity and burst promoting activity, compared to all other sex and age groups. This defect is not explained by disease severity at presentation and thus indicates a functional abnormality of monocytes/macrophages and T-lymphocytes in addition to the deficiency of haemopoietic stem cells. In a multivariate analysis, low factor production and low pretreatment reticulocyte counts turned out to be strong predictors of slow haemopoietic recovery. We conclude that young girls have a particular form of SAA characterized by low haemopoietic factor production and delayed recovery after ALG. They are preferential candidates for early bone marrow transplantation or, if they are not eligible, for treatment with recombinant human haemopoietic growth factors. PMID:7681318

  7. [Pure red cell aplasia and hypogammaglobulinemia after administration of Dioscorea rhizome and Poria cocos].

    PubMed

    Sato, Takayuki; Ueda, Yasunori

    2015-11-01

    A 56-year-old woman was referred to our department for detailed examination of anemia. She was diagnosed with pure red cell aplasia (PRCA) associated with severe reticulocytopenia based on blood testing and severe erythroblastopenia based on bone marrow aspiration. Blood tests revealed severe hypogammaglobulinemia, but monoclonal protein was not detected in either serum or urine by immunoelectrophoresis. Plasma cells were not increased in bone marrow aspirates or the biopsy specimen. Neither osteolytic lesions nor plasmacytoma was detected by computed tomography. We thus ruled out multiple myeloma. She had been treated with various Chinese herbal medicines prescribed at the referring hospital. We suspected PRCA induced by one of the Chinese herbal medicines and completely discontinued all of these herbal preparations. Hematologic testing revealed that the reticulocyte count and hemoglobin concentration began to recover on day 7 and the hemoglobin concentration and IgG levels had reached reference ranges on day 73 after discontinuation of the Chinese herbal medicines. We suspected Sanyaku (Dioscorea rhizome) or Bukuryou (Poria cocos) to have induced PRCA and hypogammaglobulinemia in this patient. To the best of our knowledge, this is the first report of PRCA and hypogammaglobulinemia induced by a Chinese herbal medicine. Clinicians must consider the possibility of drug-induced PRCA and hypogammaglobulinemia in patients taking Chinese herbal preparations. PMID:26666719

  8. Pure red-cell aplasia as the presenting feature of the carcionoid tumor of the thymus: case report.

    PubMed

    Petakov, S M; Suvajdzić, N; Petakov, D M; Sefer, D; Ognjanović, S; Macut, D; Durović, M; Isailović, T; Subotić, D; Stojsić, J; Todorović, V; Damjanović, S

    2010-03-01

    Acquired pure red-cell aplasia (PRCA) is an uncommon disorder of erythrocytopoiesis that can develop in association with thymic tumors. We present the very rare case of a severely anemic 62-year-old man with PRCA and a concurrent neuroendocrine carcinoid tumor of the thymus. The anterior mediastinal thymus tumor was completely excised, and following histological and immunohistochemical analyses (showing positive staining for cytokeratin, chromogranin A, synaptophysin, and neuron-specific enolase) the diagnosis of a (grade I; T(1)N(0)M(0)) typical carcinoid tumor of the thymus was made. Postoperatively the anemia persisted despite no signs of residual tumor on CT chest. A hematological work up found: normocellularity with <0.5% erythroblasts and preserved megakaryocytopoiesis and granulocytopoiesis in a trephine biopsy; reduced numbers of Colony Forming Unit Erythroid (CFU-E) and normal numbers of Burst-Forming Unit Erythroid (BFU-E) in bone marrow colony-forming assays; a markedly increased level of serum erythropoietin; normal T and B-cell numbers with a normal CD4/CD8 ratio; and no clonal T-cell receptor -gamma and -delta gene rearrangement) The patient responded favorably to a therapeutic trial of glucocorticoid immunosuppressive treatment (prednisone 1 mg/kg/day) with a normalization of the reticulocyte count and hematocrit, suggesting an immunologic mechanism for the PRCA. Though the exact mechanisms underlying the association between the PRCA and the carcinoid tumor of the thymus remain unknown. PMID:19224408

  9. 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.

  10. 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.

  11. 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.

  12. [MORPHOFUNCTIONAL STATE OF BLOOD CELLS AFTER CHRONIC EXPOSURE OF THE PROTEIN KINASES INHIBITOR MALEIMIDE DERIVATIVE].

    PubMed

    Byelinska, I V; Lynchak, O V; Tsyvinska, S M; Rybalchenko, V K

    2015-01-01

    The effect of the protein kinases inhibitor maleimide derivative (MI-1, 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione), inhibitor of VEGF-R1,2,3, FGF-R1, EGF-R(h), PDK1, Src(h), Syk(h), YES, ZAP70 et al. with antineoplastic activity, on blood cells parameters of rats after chronic exposure has been studied. Administration of MI-1 at doses 0.027 and 2.7 mg/kg (suppress colon carcinogenesis) for 20 and 26 weeks does not affect the morphofunctional state of red blood cells in healthy rats. This is confirmed by the lack of differences in the concentration of hemoglobin in blood, red blood cells count, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration, hematocrit and mean corpuscular volume, and the number of reticulocytes in blood after 20 and 26 weeks of exposure compared with the control group. MI-1 at indicated doses does not influence total leukocytes count and content (eosinophilic and neutrophilic granulocytes, lymphocytes, monocytes) and does not inhibit thrombocytopoiesis (platelet count remains unchanged). No negative effect of MI-1 on hematopoiesis is not limited (by the hemopoietic system) use of this compound as a potential antitumor drug PMID:26552308

  13. Uptake and release of iron from human transferrin.

    PubMed Central

    Huebers, H; Josephson, B; Huebers, E; Csiba, E; Finch, C

    1981-01-01

    Purified fractions of human apotransferrin, monoferric transferrins with iron on the acid-labile binding site and on the acid-stable binding site, and diferric transferrin have been prepared. The iron loading and unloading behavior of these preparations has been examined by isoelectric focusing. Iron release from the two monoferric transferrin preparations to human reticulocytes was of similar magnitude. In a mixture containing equal amounts of diferic and monoferric iron, approximately 4 times the amount of iron delivered by the monoferric species was delivered by the diferric species. Iron loading of transferrin in vitro showed a random distribution between monoferric and diferric transferrin. Among the monoferric transferrins, loading of the acid-labile binding sites was greater than that of the acid-stable binding sites. In vivo iron distribution in normal subjects, as evaluated by in vitro-added 50Fe, gave similar results. Absorption of a large dose of orally administered iron in iron-deficient subjects resulted in a somewhat greater amount of diferric transferrin at low saturation and a somewhat smaller amount of diferric transferrin at higher saturations than would have been anticipated by random loading. These data would indicate that in the human, iron loading of transferrin may be considered essentially random. Unloading from the two monoferric transferrin species is of similar magnitude but far less than that delivered by diferric transferrin. PMID:6941310

  14. 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 when directly compared to human bone scaffolds over 28 days. Immunofluorescence demonstrated the persistence of CD34+ cells within the scaffolds for the entirety of the culture. To characterise scaffold outputs, we designed a flow cytometry panel that utilises surface marker expression observed in standard 2D erythroid and megakaryocyte cultures. This showed that the egress population is comprised of haematopoietic progenitor cells (CD36+GPA−/low). Control cultures conducted in parallel but in the absence of a scaffold were also generally maintained for the longevity of the culture albeit with a higher level of cell death. The harvested scaffold egress can also be expanded and differentiated to the reticulocyte stage. In summary, PU scaffolds can behave as a subtractive compartmentalised culture system retaining and allowing maintenance of the seeded “CD34+ cell” population despite this population decreasing in amount as the culture progresses, whilst also facilitating egress of increasingly differentiated cells. PMID:27573994

  15. The heterogeneity of human antibody responses to vaccinia virus revealed through use of focused protein arrays.

    PubMed

    Duke-Cohan, Jonathan S; Wollenick, Kristin; Witten, Elizabeth A; Seaman, Michael S; Baden, Lindsey R; Dolin, Raphael; Reinherz, Ellis L

    2009-02-18

    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 elicit 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 tRNA(lys) precharged with lysine-epsilon-biotin followed by solid phase immobilization on 384-well neutravidin-coated plates. The generated array is highly specific and sensitive in a micro-ELISA 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

  16. 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.

  17. Nucleoside-Based Ultrasensitive Fluorescent Probe for the Dual-Mode Imaging of Microviscosity in Living Cells.

    PubMed

    Li, Jianping; Zhang, Yanyan; Zhang, Hua; Xuan, Xiaopeng; Xie, Mingsheng; Xia, Shuang; Qu, Guirong; Guo, Haiming

    2016-05-17

    Microviscosity changes of living cells have a far-reaching influence on diffusion and movement capacity of RNA and, more seriously, could modify RNA functions in living cells. Fluorescent rotor, whose fluorescence responds to different environmental viscosities, holds great potential for the imaging of viscosity in biosystem. Although many fluorescent rotors have been reported for viscosity, the fluorogenic rotor with ultrasensitivity for the determination of microviscosity (<10 cP) was rarely reported. Herein, we report a nucleoside-based two-photon fluorescent rotor (dABp-3) that can selectively and ultrasensitively image microviscosity in RNA region of living cells for the first time. 2'-Deoxyadenosine is selected as an electron donor to permit energy transfer via the acetylenic bond to acceptor, a typical boron dipyrromethene moiety. Another highlight, dABp-3 is based on 2'-deoxyadenosine, which result in its recognition capacity for RNA. dABp-3 with ultrasensitivity provides a varied linear response to the microrange viscosity (1.8-6.0 cP) in RNA region of living cells on dual-mode-two-photon ratio mode and fluorescence lifetime mode. After screening and optimization, advantageously, dABp-3 can be used to screen reticulocytes from mature blood cells of thrombosis models in vitro and in vivo because of targeting RNA, while simultaneously image microviscosity changes in these cells. So, dABp-3 as an analytical tool holds considerable promise for bioimaging and monitoring of microviscosity changes in complex biological systems. PMID:27118477

  18. 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.

  19. Invasion characteristics of a Plasmodium knowlesi line newly isolated from a human

    PubMed Central

    Amir, Amirah; Russell, Bruce; Liew, Jonathan Wee Kent; Moon, Robert W.; Fong, Mun Yik; Vythilingam, Indra; Subramaniam, Vellayan; Snounou, Georges; Lau, Yee Ling

    2016-01-01

    Plasmodium knowlesi is extensively used as an important malaria model and is now recognized as an important cause of human malaria in Malaysia. The strains of P. knowlesi currently used for research were isolated many decades ago, raising concerns that they might no longer be representative of contemporary parasite populations. We derived a new P. knowlesi line (University Malaya line, UM01), from a patient admitted in Kuala Lumpur, Malaysia, and compared it with a human-adapted laboratory line (A1-H.1) derived from the P. knowlesi H strain. The UM01 and A1-H.1 lines readily invade human and macaque (Macaca fascicularis) normocytes with a preference for reticulocytes. Whereas invasion of human red blood cells was dependent on the presence of the Duffy antigen/receptor for chemokines (DARC) for both parasite lines, this was not the case for macaque red blood cells. Nonetheless, differences in invasion efficiency, gametocyte production and the length of the asexual cycle were noted between the two lines. It would be judicious to isolate and characterise numerous P. knowlesi lines for use in future experimental investigations of this zoonotic species. PMID:27097521

  20. Short-term toxicity study of ST-20 (NSC-741804) by oral gavage in Sprague-Dawley rats.

    PubMed

    Terse, Pramod S; Johnson, Jerry D; Hawk, Michael A; Ritchie, Glenn D; Ryan, Michael J; Vasconcelos, Daphne Y; Contos, Denise A; Perrine, Susan P; Peggins, James O; Tomaszewski, Joseph E

    2011-06-01

    ST-20 (sodium 2,2-dimethylbutyrate) is a potential therapeutic agent for treatment of β-thalassemia and sickle cell disease. A subchronic oral toxicity study was conducted in Sprague-Dawley rats (10/sex/dose) at gavage dosages of 0 (vehicle control), 200, 600, or 1,000 mg/kg, once daily for up to 15 days followed by a 14-day recovery. Ataxia (females), rough coat/thin appearance (males), and decreased body weights were observed at 1,000 mg/kg. Functional observational battery (FOB) deficits were observed more frequently in females and included decreased body tone, rectal temperature, emotional reactivity, neuromotor-neuromuscular activity (as exhibited by a deficit in visual/tactile placing accuracy, ataxia, hind limb dragging, and decreased grip strength), and rearing. ST-20 caused a decrease in WBC/RBC counts and RBC parameters; increase in reticulocytes and red cell inclusion bodies; decrease in total protein, globulin, and glucose; and increase in AG ratio. Micronucleated polychromatic erythrocytes of the bone marrow increased significantly in males at 1,000 mg/kg. Mean liver and kidney weights increased, and hepatocellular hypertrophy was observed in males at 1,000 mg/kg. Toxicologic findings were fully recovered during the 14-day recovery period. In conclusion, the no-observed adverse effect level for FOB and general toxicity was 200 mg/kg following gavage administration of ST-20 for up to 15 consecutive days. PMID:21558467

  1. Rapid body mass loss affects erythropoiesis and hemolysis but does not impair aerobic performance in combat athletes.

    PubMed

    Reljic, D; Feist, J; Jost, J; Kieser, M; Friedmann-Bette, B

    2016-05-01

    Rapid body mass loss (RBML) before competition was found to decrease hemoglobin mass (Hbmass ) in elite boxers. This study aimed to investigate the underlying mechanisms of this observation. Fourteen well-trained combat athletes who reduced body mass before competitions (weight loss group, WLG) and 14 combat athletes who did not practice RBML (control group, CON) were tested during an ordinary training period (t-1), 1-2 days before an official competition (after 5-7 days RBML in WLG, t-2), and after a post-competition period (t-3). In WLG, body mass (-5.5%, range: 2.9-6.8 kg) and Hbmass (-4.1%) were significantly (P < 0.001) reduced after RBML and were still decreased by 1.6% (P < 0.05) and 2.6% (P < 0.001) at t-3 compared with t-1. After RBML, erythropoietin, reticulocytes, haptoglobin, triiodothyronine (FT3 ), and free androgen index (FAI) were decreased compared with t-1 and t-3. An increase occurred in ferritin and bilirubin. Peak treadmill-running performance and VO2peak did not change significantly, but performance at 4-mmol lactate threshold was higher after RBML (P < 0.05). In CON, no significant changes were found in any parameter. Apparently, the significant decrease in Hbmass after RBML in combat athletes was caused by impaired erythropoiesis and increased hemolysis without significant impact on aerobic performance capacity. PMID:25916419

  2. 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.

  3. Repression of the albumin gene in Novikoff hepatoma cells.

    PubMed Central

    Capetanaki, Y G; Flytzanis, C N; Alonso, A

    1982-01-01

    Novikoff hepatoma cells have lost their capacity to synthesize albumin. As a first approach to study the mechanisms underlying this event, in vitro translation in a reticulocyte system was performed using total polyadenylated mRNA from rat liver and Novikoff hepatoma cells. Immunoprecipitation of the in vitro translation products with albumin-specific antibody revealed a total lack of albumin synthesis in Novikoff hepatoma, suggesting the absence of functional albumin mRNA in these cells. Titration experiments using as probe albumin cDNA cloned in pBR322 plasmid demonstrated the absence of albumin-specific sequences in both polysomal and nuclear polyadenylated and total RNA from Novikoff cells. This albumin recombinant plasmid was obtained by screening a rat liver cDNA library with albumin [32P]cDNA reverse transcribed from immuno-precipitated mRNA. The presence of an albumin-specific gene insert was documented with translation assays as well as by restriction mapping. Repression of the albumin gene at the transcriptional level was further demonstrated by RNA blotting experiments using the cloned albumin cDNA probe. Genomic DNA blots using the cloned albumin cDNA as probe did not reveal any large-scale deletions, insertions, or rearrangements in the albumin gene, suggesting that the processes involved in the suppression of albumin mRNA synthesis do not involve extensive genomic rearrangements. Images PMID:6180302

  4. Cell-free translation of Drosophila C virus RNA: identification of a virus protease activity involved in capsid protein synthesis and further studies on in vitro processing of cricket paralysis virus specified proteins.

    PubMed

    Reavy, B; Moore, N F

    1983-01-01

    Drosophila C virus RNA acted as mRNA in rabbit reticulocyte lysates and directed the synthesis of at least one capsid protein and a number of higher molecular weight proteins. Kinetic analysis by pulse-chase experiments showed that a number of high molecular weight products acted as precursors to the capsid protein(s). Various dilution experiments were performed which showed that the virus specified a protease activity essential for the correct processing of precursors to give the capsid protein(s). A similar result was obtained with Cricket paralysis virus, and mixing experiments showed that the protease activity specified by one virus could perform some of the cleavages resulting in the production of the capsid proteins of the other virus. Some of the cleavages involving the highest molecular weight precursors could not be performed by the protease activity of the other virus. We could find no evidence for intramolecular cleavage of the capsid precursors of either of the viruses. PMID:6307220

  5. 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

  6. 15-Deoxyspergualin inhibits eukaryotic protein synthesis through eIF2α phosphorylation

    PubMed Central

    Ramya, T. N. C.; Surolia, Namita; Surolia, Avadhesha

    2006-01-01

    DSG (15-deoxyspergualin), an immunosuppressant with tumoricidal properties, binds potently to the regulatory C-terminal ‘EEVD’ motif of Hsps (heat-shock proteins). In the present study we demonstrate that DSG inhibits eukaryotic protein synthesis by sequestering Hsp70 which is required for maintaining HRI (haem-regulated inhibitor), a kinase of the eIF2α (eukaryotic initiation factor 2α), inactive. DSG stalled initiation of protein synthesis through phosphorylation of HRI and eIF2α. Addition of a recombinant eIF2α (S51A) protein, which lacks the phosphorylation site, lowered the inhibitory potential of DSG in reticulocyte lysate. The inhibitory effect of DSG was also attenuated in HRI knockdown cells. Moreover, exogenous addition of Hsp70 or the peptide ‘EEVD’ reversed the inhibitory effect of DSG. Interestingly, the inhibitory effect of DSG in different mammalian cancer cells was found to negatively correlate with the amount of Hsp70 expressed in the cells, emphasizing the link with Hsp70 in DSG inhibition of eukaryotic translation. PMID:16952278

  7. Effects of p38 MAP kinase inhibitors on the differentiation and maturation of erythroid progenitors.

    PubMed

    Dalmas, Deidre A; Tierney, Lauren A; Zhang, Cindy; Narayanan, Padma K; Boyce, Rogely W; Schwartz, Lester W; Frazier, Kendall S; Scicchitano, Marshall S

    2008-12-01

    In rodents, p38 MAP kinase inhibitors (p38is) induce bone marrow hypocellularity and reduce reticulocyte and erythrocyte counts. To identify target cell populations affected, a differentiating primary liquid erythroid culture system using sca-1(+)cells from mouse bone marrow was developed and challenged with p38is SB-203580, SB-226882, and SB-267030. Drug-related alterations in genes involved at different stages of erythropoiesis, cell-surface antigen expression (CSAE), burst-forming unit erythroid (BFU-E) colony formation, and cellular morphology (CM), growth (CG), and viability were evaluated. CSAE, CM, and decreases in BFU-E formation indicated delayed maturation, while CG and viability were unaffected. Terminal differentiation was delayed until day 14 versus day 7 in controls. CSAE demonstrated higher percentages of sca-1(+)cells after day 2 and reduced percentages of ter119(+) cells after day 7 in all treated cultures. Real-time reverse transcriptase polymerase chain reaction revealed a transient delay in expression of genes involved at early, intermediate, and late stages of erythropoiesis, followed by rebound expression at later time points. Results demonstrate p38is do not irreversibly inhibit erythrogenesis but induce a potency-dependent, transient delay in erythropoietic activity. The delay in activity is suggestive of effects on sca-1(+)bone marrow cells caused by alterations in expression of genes related to erythroid commitment and differentiation resulting in delayed maturation. PMID:19126791

  8. nm1054: a spontaneous, recessive, hypochromic, microcytic anemia mutation in the mouse.

    PubMed

    Ohgami, Robert S; Campagna, Dean R; Antiochos, Brendan; Wood, Emily B; Sharp, John J; Barker, Jane E; Fleming, Mark D

    2005-11-15

    Hypochromic, microcytic anemias are typically the result of inadequate hemoglobin production because of globin defects or iron deficiency. Here, we describe the phenotypic characteristics and pathogenesis of a new recessive, hypochromic, microcytic anemia mouse mutant, nm1054. Although the mutation nm1054 is pleiotropic, also resulting in sparse hair, male infertility, failure to thrive, and hydrocephaly, the anemia is the focus of this study. Hematologic analysis reveals a moderately severe, congenital, hypochromic, microcytic anemia, with an elevated red cell zinc protoporphyrin, consistent with functional erythroid iron deficiency. However, serum and tissue iron analyses show that nm1054 animals are not systemically iron deficient. From hematopoietic stem cell transplantation and iron uptake studies in nm1054 reticulocytes, we provide evidence that the nm1054 anemia is due to an intrinsic hematopoietic defect resulting in inefficient transferrin-dependent iron uptake by erythroid precursors. Linkage studies demonstrate that nm1054 maps to a genetic locus not previously implicated in microcytic anemia or iron phenotypes. PMID:15994289

  9. Altitude training causes haematological fluctuations with relevance for the Athlete Biological Passport.

    PubMed

    Bonne, Thomas Christian; Lundby, Carsten; Lundby, Anne Kristine; Sander, Mikael; Bejder, Jacob; Nordsborg, Nikolai Baastrup

    2015-08-01

    The impact of altitude training on haematological parameters and the Athlete Biological Passport (ABP) was evaluated in international-level elite athletes. One group of swimmers lived high and trained high (LHTH, n = 10) for three to four weeks at 2130 m or higher whereas a control group (n = 10) completed a three-week training camp at sea-level. Haematological parameters were determined weekly three times before and four times after the training camps. ABP thresholds for haemoglobin concentration ([Hb]), reticulocyte percentage (RET%), OFF score and the abnormal blood profile score (ABPS) were calculated using the Bayesian model. After altitude training, six swimmers exceeded the 99% ABP thresholds: two swimmers exceeded the OFF score thresholds at day +7; one swimmer exceeded the OFF score threshold at day +28; one swimmer exceeded the threshold for RET% at day +14; and one swimmer surpassed the ABPS threshold at day +14. In the control group, no values exceeded the individual ABP reference range. In conclusion, LHTH induces haematological changes in Olympic-level elite athletes which can exceed the individually generated references in the ABP. Training at altitude should be considered a confounding factor for ABP interpretation for up to four weeks after altitude exposure but does not consistently cause abnormal values in the ABP. PMID:25545030

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

    SciTech Connect

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

    1987-11-01

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

  11. 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.

  12. 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. PMID:493112

  13. Cochinin B, a novel ribosome-inactivating protein from the seeds of Momordica cochinchinensis.

    PubMed

    Chuethong, Juthamas; Oda, Kohei; Sakurai, Hiroaki; Saiki, Ikuo; Leelamanit, Wichet

    2007-03-01

    Cochinin B, a novel ribosome-inactivating protein (RIP) with a molecular weight of 28 kDa, was purified from the seeds of Momordica cochinchinensis (Cucurbitaceae). The isolation procedure entailed ammonium sulfate precipitation, cation-exchange chromatography on SP Sepharose column and size-exclusion chromatography on Superdex 75 column with a fast protein liquid chromatography (FPLC) system. The first twenty N-terminal amino acid residues of Cochinin B showed homology to type I RIPs from other Momordica species. The purified Cochinin B displayed a strong inhibitory activity on protein synthesis in the cell-free rabbit reticulocyte lysate system with IC50 of 0.36 nM. Furthermore, it exhibited N-glycosidase activity and cytotoxicity against Vero cell line with IC50 higher than 1540 nM. Interestingly, Cochinin B manifested strong anti-tumor activities on human cervical epithelial carcinoma (HeLa), human embryonic kidney (HEK293) and human small cell lung cancer (NCI-H187) cell lines with IC50 of 16.9, 114 and 574 nM, respectively. PMID:17329832

  14. 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.

  15. Modular domains of the Dicistroviridae intergenic internal ribosome entry site

    PubMed Central

    Jang, Christopher J.; Jan, Eric

    2010-01-01

    The intergenic region internal ribosome entry site (IGR IRES) of the Dicistroviridae viral family can directly assemble 80S ribosomes and initiate translation at a non-AUG codon from the ribosomal A-site. These functions are directed by two independently folded domains of the IGR IRES. One domain, composed of overlapping pseudoknots II and III (PKII/III), mediates ribosome recruitment. The second domain, composed of PKI, mimics a tRNA anticodon–codon interaction to position the ribosome at the ribosomal A-site. Although adopting a common secondary structure, the dicistrovirus IGR IRESs can be grouped into two classes based on distinct features within each domain. In this study, we report on the modularity of the IGR IRESs and show that the ribosome-binding domain and the tRNA anticodon mimicry domain are functionally interchangeable between the Type I and the Type II IGR IRESs. Using structural probing, ribosome-binding assays, and ribosome positioning analysis by toeprinting assays, we show that the chimeric IRESs fold properly, assemble 80S ribosomes, and can mediate IRES translation in rabbit reticulocyte lysates. We also demonstrate that the chimeric IRESs can stimulate the ribosome-dependent GTPase activity of eEF2, which suggests that the ribosome is primed for a step downstream from IRES binding. Overall, the results demonstrate that the dicistrovirus IGR IRESs are composed of two modular domains that work in concert to manipulate the ribosome and direct translation initiation. PMID:20423979

  16. Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system

    PubMed Central

    Hjerrild, Kathryn A.; Jin, Jing; Wright, Katherine E.; Brown, Rebecca E.; Marshall, Jennifer M.; Labbé, Geneviève M.; Silk, Sarah E.; Cherry, Catherine J.; Clemmensen, Stine B.; Jørgensen, Thomas; Illingworth, Joseph J.; Alanine, Daniel G. W.; Milne, Kathryn H.; Ashfield, Rebecca; de Jongh, Willem A.; Douglas, Alexander D.; Higgins, Matthew K.; Draper, Simon J.

    2016-01-01

    The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difficult-to-make’ proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. PMID:27457156

  17. 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.

  18. Hematotoxic effects of 3,5-dinitro-4-chloro-alpha,alpha,alpha-trifluorotoluene, a water contaminant

    SciTech Connect

    Guastadisegni, C.; Hall, D.; Macri, A.

    1986-10-01

    Three short-term studies of 7, 14, and 21 days, respectively, were made to investigate the nature of the anemia induced in rats by 3,5-dinitro-4-chloro-alpha,alpha,alpha-trifluorotoluene (DNCTT). This compound is an intermediate in the synthesis of dinitroaniline herbicides and was detected as a contaminant of a water-bearing stratum in northern Italy. DNCTT was mixed in a powdered rodent diet at a level of 2000 ppm and administered to Wistar-derived rats. DNCTT was shown to produce a hemolytic anemia of rapid onset; packed cell volume and hemoglobin concentration were decreased at all three treatment periods. Methemoglobin and reticulocyte count were increased in all the treated groups. The relative organ weights of the spleen and the liver were increased compared to those of the control groups. Spleen enlargement was also evident at the macroscopic examination, whereas the liver appearance was normal. Pearl's Prussian blue staining performed on the spleen and liver was highly positive in the spleen of treated rats, but no iron deposition was detected in the liver of treated rats.

  19. 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.

  20. Genotoxicity of intraperitoneal injection of lipoamphiphile CdSe/ZnS quantum dots in rats.

    PubMed

    Aye, Mélanie; Di Giorgio, Carole; Mekaouche, Mourad; Steinberg, Jean-Guillaume; Brerro-Saby, Christelle; Barthélémy, Philippe; De Méo, Michel; Jammes, Yves

    2013-12-12

    The main objective of the present in vivo rat study was to determine the genotoxicity of lipoamphiphile-coated CdSe/ZnS Quantum Dots (QDs), in several organs (brain, liver, kidneys, lungs and testicles). The second objective was to establish the correlations between the QDs genotoxic activity and the oxidative stress, the production of a proinflammatory cytokine (TNF-α), a stress-induced chaperone protein, the phosphorylated heat shock protein 70 (pHsp70), and an increase in the caspase-3 apoptosis factor. Four QDs doses were injected into the peritoneal cavity (5, 5×10(-1), 5×10(-2) and 5×10(-3)μg/kg). DNA lesions in the different organs were measured by the comet assay, and chromosome abnormalities were evaluated by the micronucleus assay on blood reticulocytes (MNRET). Twenty-four hours after the QDs injection, genotoxic effects were observed in the brain and liver and, only for the highest QDs concentration, in testicles. No genotoxic effect was seen in the kidney and lung. The MNRET test revealed a dose-response induction of micronuclei. In parallel, we did neither reveal oxidative stress nor significant variations of TNF-α, pHsp70, and caspase-3. In conclusion, the QDs exerted significant genotoxic effects in the brain and liver, even in the absence of any associated oxidative stress and inflammatory processes. PMID:24055877

  1. Sickle cell disease biochip: a functional red blood cell adhesion assay for monitoring sickle cell disease.

    PubMed

    Alapan, Yunus; Kim, Ceonne; Adhikari, Anima; Gray, Kayla E; Gurkan-Cavusoglu, Evren; Little, Jane A; Gurkan, Umut A

    2016-07-01

    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

  2. Identification of 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF) with DNA breaking activity in soy sauce.

    PubMed

    Li, X; Hiramoto, K; Yoshida, M; Kato, T; Kikugawa, K

    1998-04-01

    Components with DNA breaking activity in soy sauce were investigated. It was found that there were water soluble high molecular weight DNA breaking components in soy sauce. Two DNA breaking components in the ethyl acetate extract of soy sauce were identified as fragrant components, 2,5-dimethyl-4-hydroxy-3(2H)-furanone (DMHF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), in addition to the previously characterized DNA breaking fragrant component 4-hydroxy-5-methyl-3(2H)-furanone (HMF) (Hiramoto et al., 1996b). Characterization of DNA breaking activity of HEMF was performed, and the mechanisms for the breaking were considered. HEMF cleaved the single strands of supercoiled pBR 322 DNA at pH 7.4 dose dependently and time dependently. DNA breaking was inhibited by superoxide dismutase, catalase, hydroxyl radical scavengers, spin trapping agents and metal chelators, and enhanced by Fe(III) ion. Electron spin resonance-spin trapping technique revealed the generation of hydroxyl radical. Hence, active oxygen species derived from interaction of HEMF with metal ions and oxygen participated in the cleavage. HEMF exhibited mutagenicity to Salmonella typhimurium TA100 without metabolic activation and induced micronucleated mouse peripheral reticulocytes. PMID:9651047

  3. Disturbed iron metabolism among workers exposed to organic sulfides in a pulp plant.

    PubMed

    Klingberg, J; Beviz, A; Ohlson, C G; Tenhunen, R

    1988-02-01

    The aim of this study was to investigate a possible relationship between exposure to sulfides and disturbances of the synthesis of heme and the erythrocytes. Eighteen workers exposed to sulfides at a pulp and paper plant were examined and compared with individually matched referents from a thermomechanical pulp plant without such exposure. The exposure levels of methylmercaptan, dimethylsulfide, and dimethyldisulfide were low. However, five subjects were exposed to high levels of short duration, and their data were analyzed separately. The activity of the enzymes delta-aminolevulinic acid synthase and heme synthase in reticulocytes, characteristics of the erythrocytes, and the iron status were analyzed. A minor decrease, not statistically significant, was observed for the enzymes among the five highly exposed subjects. However, the concentrations of iron and transferrin were elevated and the concentration of ferritin was low in comparison to the corresponding levels of the referents. This combination will not occur spontaneously. A previous study indicated that sulfides may inhibit heme synthesis, and the present study suggests that they may also disturb iron metabolism. PMID:3353691

  4. Changes in haem synthesis associated with occupational exposure to organic and inorganic sulphides.

    PubMed

    Tenhunen, R; Savolainen, H; Jäppinen, P

    1983-02-01

    1. Analysis of reticulocytes for delta-amino-laevulinic acid synthase (AmLev synthase, EC 2.3.1.37) and haem synthase (EC 4.99.1.1) activity in 17 workers in pulp production with low-level hydrogen sulphide and methylmercaptan exposure showed decreased activities in eight and six cases respectively. 2. Erythrocyte protoporphyrin concentration was below the control range in seven cases. 3. Low AmLev synthase and haem synthase activities were found in one patient with hydrogen sulphide intoxication 1 week after the event. The activities had returned to the control levels 2 months later, though erythrocyte protoporphyrin remained abnormally low. 4. In vitro, hydrogen sulphide inhibited haem synthase with an apparent Ki of 3.4 mmol/l. Sulphide anion, on the other hand, inhibited AmLev synthase activity 85% at 10 mmol/l concentration. Thiosulphate anion inhibited AmLev synthase activity 18% (Ki 27 mmol/l) and haem synthase activity 43% at 10 mmol/l concentration. Selenite inhibited AmLev synthase (Ki 5.1 mmol/l) and haem synthase (Ki 9.0 mmol/l). 5. The assay of AmLev synthase and haem synthase could be a valuable addition to the assessment of workers' health in industries generating hydrogen sulphide or/and methylmercaptan, although the mechanism of the toxic effect remains speculative. PMID:6822055

  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. 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.

  7. The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice.

    PubMed

    Cui, Shuaiying; Lim, Kim-Chew; Shi, Lihong; Lee, Mary; Jearawiriyapaisarn, Natee; Myers, Greggory; Campbell, Andrew; Harro, David; Iwase, Shigeki; Trievel, Raymond C; Rivers, Angela; DeSimone, Joseph; Lavelle, Donald; Saunthararajah, Yogen; Engel, James Douglas

    2015-07-16

    Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD. PMID:26031919

  8. The genotoxicity of acrylamide and glycidamide in big blue rats.

    PubMed

    Mei, Nan; McDaniel, Lea P; Dobrovolsky, Vasily N; Guo, Xiaoqing; Shaddock, Joseph G; Mittelstaedt, Roberta A; Azuma, Mizuo; Shelton, Sharon D; McGarrity, Lynda J; Doerge, Daniel R; Heflich, Robert H

    2010-06-01

    Acrylamide (AA), a mutagen and rodent carcinogen, recently has been detected in fried and baked starchy foods, a finding that has prompted renewed interest in its potential for toxicity in humans. In the present study, we exposed Big Blue rats to the equivalent of approximately 5 and 10 mg/kg body weight/day of AA or its epoxide metabolite glycidamide (GA) via the drinking water, an AA treatment regimen comparable to those used to produce cancer in rats. After 2 months of dosing, the rats were euthanized and blood was taken for the micronucleus assay; spleens for the lymphocyte Hprt mutant assay; and liver, thyroid, bone marrow, testis (from males), and mammary gland (females) for the cII mutant assay. Neither AA nor GA increased the frequency of micronucleated reticulocytes. In contrast, both compounds produced small (approximately twofold to threefold above background) but significant increases in lymphocyte Hprt mutant frequency (MF, p < 0.05), with the increases having dose-related linear trends (p < 0.05 to p < 0.001). Neither compound increased the cII MF in testis, mammary gland (tumor target tissues), or liver (nontarget tissue), while both compounds induced weak positive increases in bone marrow (nontarget tissue) and thyroid (target tissue). Although the genotoxicity in tumor target tissue was weak, in combination with the responses in surrogate tissues, the results are consistent with AA being a gene mutagen in the rat via metabolism to GA. PMID:20200216

  9. 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. PMID:8882638

  10. Novel antifungal peptides from Ceylon spinach seeds.

    PubMed

    Wang, H; Ng, T B

    2001-11-01

    Two novel antifungal peptides, designated alpha- and beta-basrubrins, respectively, were isolated from seeds of the Ceylon spinach Basella rubra. The purification procedure involved saline extraction, (NH(4))(2)SO(4) precipitation, ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and FPLC-gel filtration on Superdex peptide column. alpha- and beta-basrubrins exhibited a molecular weight of 4.3 and 5 kDa, respectively. They inhibited translation in a rabbit reticulocyte system with an IC(50) value of 400 and 100 nM, respectively. alpha- and beta-basrubrin inhibited HIV-1 reverse transcriptase by (79.4 +/- 7.8)% and (54.6 +/- 3.6)%, respectively, at a concentration of 400 microM, and (10.56 +/- 0.92)% and (2.12 +/- 0.81)%, respectively, at a concentration of 40 microM. Both alpha- and beta-basrubrins exerted potent antifungal activity toward Botrytis cinerea, Mycosphaerella arachidicola, and Fusarium oxysporum. PMID:11688973

  11. Expression and subcellular localization of poliovirus VPg-precursor protein 3AB in eukaryotic cells: evidence for glycosylation in vitro.

    PubMed Central

    Datta, U; Dasgupta, A

    1994-01-01

    The poliovirus-encoded, membrane-associated VPg-precursor polypeptide 3AB has been implicated in the initiation of viral RNA synthesis. We have expressed 3AB and 3A polypeptides in eukaryotic cells and examined their localization using indirect immunofluorescence and a direct in vitro membrane-binding assay. Results presented here demonstrate that both 3AB and 3A are capable of localizing in the endoplasmic reticulum and the Golgi apparatus in transfected HeLa cells in the absence of any other poliovirus protein. We have also shown that the carboxy-terminal 18 amino acids of 3A that constitute an amphipathic domain are important in membrane binding of 3A and 3AB. Additionally, we demonstrate that a significant fraction of both 3A and 3AB can be glycosylated in a membrane-dependent fashion during in vitro translation in reticulocyte lysate. We demonstrate that 6-diazo-5-oxo-L-norleucine, an inhibitor of glycoprotein synthesis, significantly inhibits poliovirus RNA synthesis in vivo. The implications of glycosylation of 3AB (and 3A) in viral replication are discussed. Images PMID:8207820

  12. Characterization of trichobakin, a type I ribosome-inactivating protein from Trichosanthes sp. Bac Kan 8-98.

    PubMed

    Chi, P V; Truong, H Q; Ha, N T; Chung, W I; Binh, L T

    2001-10-01

    We have isolated a genomic clone encoding trichobakin (TBK), a type I ribosome-inactivating protein from the plant Trichosanthes sp. Bac Kan 8-98 (family Cucurbitaceae), by PCR using specific primers designed from the cDNA sequences of alpha-trichosanthin. The sequence encoding mature TBK was constructed in the pET-21d(+) vector for overexpression in Escherichia coli strain BL21(DE3). The recombinant protein was purified to homogeneity by CM-Sepharose chromatography on FPLC with a final yield of about 55 mg/l of culture. The protein has a molecular mass of about 27 kDa, as shown by SDS/PAGE and matrix-assisted laser-desorption ionization MS. It was found that the protein inhibited luciferase mRNA translation in the rabbit reticulocyte cell-free system with an IC(50) value (that which causes a 50% reduction of residual translational activity) of about 3.5 pM. The rRNA N-glycosidase activity of the protein was also proved at the above-mentioned concentration after rRNAs were treated with acid aniline. PMID:11592913

  13. Mammalian protein secretion without signal peptide removal. Biosynthesis of plasminogen activator inhibitor-2 in U-937 cells

    SciTech Connect

    Ye, R.D.; Wun, T.C.; Sadler, J.E.

    1988-04-05

    Plasminogen activator inhibitor-2 (PAI-2) is a serine protease inhibitor that regulates plasmin generation by inhibiting urokinase and tissue plasminogen activator. The primary structure of PAI-2 suggests that it may be secreted without cleavage of a single peptide. To confirm this hypothesis we have studied the glycosylation and secretion of PAI-2 in human monocytic U-937 cells by metabolic labeling, immunoprecipitation, glycosidase digestion, and protein sequencing. PAI-2 is variably glycosylated on asparagine residues to yield intracellular intermediates with zero, one, two, or three high mannose-type oligosaccharide units. Secretion of the N-glycosylated species began by 1 h of chase and the secreted molecules contained both complex-type N-linked and O-linked oligosaccharides. Enzymatically deglycosylated PAI-2 had an electrophoretic mobility identical to that of the nonglycosylated precursor and also to that of PAI-2 synthesized in vitro in a rabbit reticulocyte lysate from synthetic mRNA derived from full length PAI-2 cDNA. The amino-terminal protein sequence of secreted PAI-2 began with the initiator methionine residue. These results indicate that PAI-2 is glycosylated and secreted efficiently without the cleavage of a signal peptide. PAI-2 shares this property with its nearest homologue in the serine protease inhibitor family, chicken ovalbumin, and appears to be the first well characterized example of this phenomenon among natural mammalian proteins.

  14. The N-terminal region of the heme-regulated eIF2alpha kinase is an autonomous heme binding domain.

    PubMed

    Uma, S; Matts, R L; Guo, Y; White, S; Chen, J J

    2000-01-01

    The N-terminal domain (NTD) of the heme-regulated eukaryotic initiation factor (eIF)2alpha kinase (HRI) was aligned to sequences in the NCBI data base using ENTREZ and a PAM250 matrix. Significant similarity was found between amino acids 11-118 in the NTD of rabbit HRI and amino acids 16-120 in mammalian alpha-globins. Several conserved amino acid residues present in globins are conserved in the NTD of HRI. His83 of HRI was predicted to be equivalent to the proximal heme ligand (HisF8) that is conserved in all globins. Molecular modeling of the NTD indicated that its amino acid sequence was compatible with the globin fold. Recombinant NTD (residues 1-159) was expressed in Escherichia coli. Spectral analysis of affinity purified recombinant NTD indicated that the NTD contained stably bound hemin. Mutational analysis indicated that His83 played a critical structural role in the stable binding of heme to the NTD, and was required to stabilize full length HRI synthesized de novo in the rabbit reticulocyte lysate. These results indicate that the NTD of HRI is an autonomous heme-binding domain, with His83 possibly serving as the proximal heme binding ligand. PMID:10632719

  15. Early modification of sickle cell disease clinical course by UDP-glucuronosyltransferase 1A1 gene promoter polymorphism.

    PubMed

    Martins, Rute; Morais, Anabela; Dias, Alexandra; Soares, Isabel; Rolão, Cristiana; Ducla-Soares, J L; Braga, Lígia; Seixas, Teresa; Nunes, Baltazar; Olim, Gabriel; Romão, Luísa; Lavinha, João; Faustino, Paula

    2008-01-01

    Elevated erythrocyte destruction in sickle cell disease (SCD) results in chronic hyperbilirubinaemia and, in a subset of patients, cholelithiasis occurs. We investigated whether the (TA)n promoter polymorphism in the UDP-glucuronosyltransferase 1A1 gene (UGT1A1) may modify bilirubin metabolism, influencing bilirubinaemia, predisposition to cholelithiasis and subsequent cholecystectomy, in a group of 153 young SCD patients (mean age 12.0 +/- 9.0 years) predominantly of Bantu beta S haplotype. The concomitant effect of alpha thalassaemia was also analysed. Among the several UGT1A1 genotypes found, the most frequent were the (TA)6/(TA)6 (n = 37), (TA)6/(TA)7 (n = 60) and (TA)7/(TA)7 (n = 29). These groups of patients did not significantly differ in age, gender ratio and haemoglobin, foetal haemoglobin and reticulocyte levels. On the other hand, total bilirubin levels were significantly different between groups, with an increased (TA) repeat number being associated with higher bilirubinaemia. Furthermore, both cholelithiasis and cholecystectomy were more frequent in groups with higher (TA) repeat number, although the former association was not statistically significant. None of the mentioned parameters is statistically different within UGT1A1 groups with the presence of alpha thalassaemia. Thus, the UGT1A1 promoter polymorphism may represent an important nonglobin genetic modifier of Bantu SCD patients' clinical manifestations, even at a young age. PMID:18392554

  16. [Short-term curative effect of amifostine combined with rhbeta-EPO on patients with pure erythroid aplasia].

    PubMed

    Li, Su-Xia; Zhu, Hong-Li; Lu, Xue-Chun; Fan, Hui; Guo, Bo; Zhai, Bing

    2008-10-01

    The aim of this study was to investigate the curative effects of amifostine (AMF) combined with recombinant human beta-erythropoietin (rhbeta-EPO) on patients with pure erythroid aplasia (PEA). Two patients with PEA were treated with amifostine and rhbeta-EPO. The therapeutic regimen was adopted with AMF 0.4 g/day given by intravenous injection for 5 days first, then after a break of 2 days it went on for 3 weeks consecutively, that was considered as one treatment cycle. The rhbeta-EPO 6 000 U was used by subcutaneous injection for 3 times per week. The results showed that the red cell count, hemoglobin and reticulocyte count of two patients obviously increased after treatment. The erythroid ratio in bone marrow increased. Bone marrow biopsy showed that the erythroid proliferation improved. Intervals of red cell transfusions (RCT) in the two patients who live by red cell transfusion were prolonged after AMF treatments, and the amounts of each RCT was decreased obviously. The main side effect of amifostine was discomfort of digestive system, but was tolerated by all patients. In conclusion, amifostine plus rhbeta-EPO may be a new, effective and safety method especially for the elder PEA patients. The long-term curative effects and mechanism of amifostine still need further evaluation. PMID:18928605

  17. The acquired immunodeficiency syndrome: an ultrastructural study.

    PubMed

    Sidhu, G S; Stahl, R E; el-Sadr, W; Cassai, N D; Forrester, E M; Zolla-Pazner, S

    1985-04-01

    Blood and a variety of tissues from 97 patients with the acquired immunodeficiency syndrome (AIDS) and 25 with the AIDS prodrome were studied ultrastructurally. Tubuloreticular structures (TRS) were found in 85 per cent of the patients with AIDS and in 92 per cent of those with the prodrome. Test tube and ring-shaped forms (TRF), found in 41 per cent of the patients with AIDS and in 8 per cent of those with the prodrome, increased with disease progression. Among the patients with AIDS, as the number of sites examined per case increased, the incidence of TRS and TRF tended to approach 100 per cent, suggesting that they are present in all patients with AIDS. Other changes seen frequently were immunologic capping of blood lymphocytes, intramitochondrial iron in blood reticulocytes and marrow normoblasts, megakaryocytic immaturity and platelet phagocytosis, collections of membranous rings in hepatocytic cytoplasm, suggestive of non-A, non-B hepatitis, and proliferations and engorgement of hepatic Ito cells with lipid. The data suggest that TRS and TRF can be used as diagnostic and prognostic markers. PMID:3872253

  18. A premature termination codon within an alternative exon affecting only the metabolism of transcripts that retain this exon.

    PubMed

    Maillet, P; Dalla Venezia, N; Lorenzo, F; Morinière, M; Bozon, M; Noël, B; Delaunay, J; Baklouti, F

    1999-01-01

    Protein 4.1 pre-mRNA splicing is regulated in tissue- and development-specific manners. Exon 16, which encodes the N-terminal region of the spectrin/actin-binding domain, is one of the alternatively spliced sequence motifs. It is present in late differentiated erythroid cells but absent from early erythroblasts and from lymphoid cells. We describe a single nucleotide deletion of the erythroid protein 4.1 gene associated with hereditary elliptocytosis. The deletion located in exon 16 leads to a frameshift and a premature termination codon within the same exon. In an effort to examine the premature stop codon effect in relationship with exon 16 alternative splicing, we analyzed erythroid and lymphoid protein 4.1 mRNAs using the mutation and a linked downstream polymorphism as markers. We found that the premature stop codon does not affect the tissue-specific alternative splicing among the two cell types analyzed and that the resulting alteration of mRNA metabolism correlates with the retention of exon 16 in reticulocytes. Conversely, skipping of exon 16 in lymphoid cells converts the mutant mRNA to a normal lymphoid-specific mRNA isoform, hence bypassing the nonsense codon. Consistent with data obtained on constitutive nonsense exons, our observations argue in favor of a stop codon recognition mechanism that occurs after the regulated splicing status of the nonsense exon has been achieved. PMID:10425037

  19. Meta-analysis of Huangqi injection for the adjunctive therapy of aplastic anemia

    PubMed Central

    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. PMID:26379817

  20. The 5'-leader sequence of tobacco mosaic virus RNA enhances the expression of foreign gene transcripts in vitro and in vivo.

    PubMed Central

    Gallie, D R; Sleat, D E; Watts, J W; Turner, P C; Wilson, T M

    1987-01-01

    A 67-nucleotide portion of the non-coding, 5'-leader sequence of tobacco mosaic virus RNA [defined as omega' (Gr. omega prime)] has been shown to enhance the translation of contiguous foreign gene transcripts both in vitro and in vivo. Chemically-synthesized omega', containing convenient linker sequences, was inserted into derivatives of an in vitro transcription plasmid (pSP64) between the bacteriophage-SP6 promoter and sequences coding for either chloramphenicol acetyltransferase (CAT) or neomycin phosphotransferase (NPTII). Run-off in vitro transcripts, with or without a 5'-cap structure (G(5')ppp(5')G) and/or the omega' sequence, were tested in mRNA-dependent cell-free translation systems derived from rabbit reticulocyte lysate, wheat germ extract or Escherichia coli (MRE 600). In all cases, the presence of omega' increased the translational expression of both reporter genes, typically between 2- to 10-fold. Electroporation of isolated mesophyll protoplasts from Nicotiana tabacum cv. Xanthi, or microinjection of oocytes from Xenopus laevis, with SP6-transcripts containing the CAT-coding region confirmed and extended the value of omega' as a potential translational enhancer of gene expression in vivo. Images PMID:3575095

  1. Construction of a recombinant bacterial plasmid containing DNA sequences for a mouse embryonic globin chain.

    PubMed Central

    Fantoni, A; Bozzoni, I; Ullu, E; Farace, M G

    1979-01-01

    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. Images PMID:493112

  2. Erythroferrone contributes to recovery from anemia of inflammation.

    PubMed

    Kautz, Léon; Jung, Grace; Nemeth, Elizabeta; Ganz, Tomas

    2014-10-16

    Erythroferrone (ERFE) is an erythropoiesis-driven regulator of iron homeostasis. ERFE mediates the suppression of the iron-regulatory hormone hepcidin to increase iron absorption and mobilization of iron from stores. We examined the role of ERFE in the recovery from anemia of inflammation (AI) induced by injection of heat-killed Brucella abortus. B abortus-treated wild-type mice developed a moderate anemia and reached nadir hemoglobin 14 days after injection and partially recovered by 28 days. We observed that Erfe expression in the bone marrow and the spleen was greatly increased during anemia and peaked at 14 days after injection, a time course similar to serum erythropoietin. To determine whether ERFE facilitates the recovery from anemia, we analyzed Erfe-deficient mice injected with B abortus. Compared with wild-type mice, Erfe-deficient mice exhibited a more severe anemia, had higher hepcidin levels and consequently lower serum iron concentration on days 14 and 21, and manifested impaired mobilization of iron from stores (liver and spleen). Erfe(-/-) mice eventually compensated by further stimulating erythropoiesis and reticulocyte production. Thus, ERFE contributes to the recovery from AI by suppressing hepcidin and increasing iron availability. PMID:25193872

  3. Vitamin C supplementation does not improve hypoxia-induced erythropoiesis.

    PubMed

    Martinez-Bello, Vladimir E; Sanchis-Gomar, Fabian; Martinez-Bello, Daniel; Olaso-Gonzalez, Gloria; Gomez-Cabrera, Mari Carmen; Viña, Jose

    2012-12-01

    Hypoxia induces reactive oxygen species production. Supplements with antioxidant mixtures can compensate for the decline in red cell membrane stability following intermittent hypobaric hypoxia by decreasing protein and lipid oxidation. We aimed to determine whether supplementation with vitamin C is implicated in the regulation of erythropoiesis and in the oxygen-carrying capacity of the blood, and also whether antioxidant supplementation prevents the oxidative stress associated to intermittent hypoxia. Twenty-four male Wistar rats were randomly divided into four experimental groups: normoxia control (n=6), normoxia + vitamin C (n=6), hypoxia control (12 h pO(2) 12%/12 h pO(2) 21%) (n=6), and hypoxia + vitamin C (n=6). Animals were supplemented with vitamin C at a dose of 250 mg·kg(-1)·day(-1) for 21 days. Red blood cell count, hemoglobin, hematocrit, reticulocytes, erythropoietin, and oxidative stress parameters such as malondialdehyde and protein oxidation in plasma were analyzed at two different time points: basal sample (day zero) and final sample (day 21). Similar RBC, Hb, Hct, and Epo increments were observed in both hypoxic groups regardless of the vitamin C supplementation. There was no change on MDA levels after intermittent hypoxic exposure in any experimental group. However, we found an increase in plasma protein oxidation in both hypoxic groups. Vitamin C does not affect erythropoiesis and protein oxidation in rats submitted to intermittent hypoxic exposure. PMID:23270444

  4. Erythropoietin.

    PubMed

    Jelkmann, Wolfgang

    2016-01-01

    Total hemoglobin (Hb) mass is an important determinant of aerobic power. The glycoprotein erythropoietin (Epo) promotes the production of red blood cells (RBCs). The present article reviews the regulation of erythropoiesis and ways of its manipulation. The various Epos, e.g. recombinant human (rh)Epo and (epoetin), and their long-acting analogues can be misused by cheating athletes, but the drugs are detectable by chemical tests, because their glycan isoform structures differ from those of endogenous Epo. Still, anti-doping control has become more difficult, since additional erythropoiesis-stimulating agents have become available (Epo mimetics, activin inhibitors, and small-molecule chemical drugs activating EPO expression). A major problem is created by hypoxia-inducible factor (HIF) stabilizers (e.g. α-ketoglutarate competitors and Co2+ salt) which activate HIFs and thus increase EPO expression. Direct EPO transfer is theoretically also possible but medically little advanced. To overcome weaknesses of direct testing of biological fluids, the World Anti-Doping Agency has implemented the Athlete Biological Passport for continuous monitoring of RBC parameters of athletes. Blood doping is assumed when distinct parameters (blood Hb concentration and reticulocytes) change in a nonphysiological way. PMID:27348128

  5. Reticulocytopenia in severe autoimmune hemolytic anemia (AIHA) of the warm antibody type.

    PubMed

    Hauke, G; Fauser, A A; Weber, S; Maas, D

    1983-06-01

    A patient with severe AIHA of the warm antibody type, absence of reticulocytes and red cell hyperplasia of the bone marrow is described. In order to maintain a reasonable hemoglobin level 38 units of washed packed red cells were required within 24 days. The treatment with high doses of steroids showed no permanent beneficial effect. After splenectomy the red cell destruction was immediately reduced and the patient went into a remission. Bone marrow culture studies during the acute phase of the disease and at the time of complete hemato- and immunological remission, i.e. 4 months after splenectomy suggested a circulating autoantibody directed to early erythroid progenitors (BFU-E). The inhibitory activity in the patient's plasma did not influence granulocytic or mixed colony formation (CFU-GEMM). In addition to autoantibodies directed to erythroblasts and erythropoietin involved in the pathogenic mechanisms leading to red cell aplasia type I and II the culture studies suggest an unusual autoantibody that might cause the observed reticulocytopenia and erythropoietic hyperplasia of the bone marrow in AIHA. After the splenectomy the patient recovered, he required no further blood transfusions and his disease has not recurred. PMID:6850101

  6. 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.

  7. Purification of a new ribosome-inactivating protein from the seeds of Cinnamomum porrectum and characterization of the RNA N-glycosidase activity of the toxic protein.

    PubMed

    Li, X D; Liu, W Y; Niu, C L

    1996-12-01

    Porrectin, a new type II ribosome-inactivating protein (RIP), was purified from the seeds of the camphor tree (Cinnamomum porrectum) by affinity chromatography on acid-treated Sepharose 4B. Porrectin is a glycoprotein (M(r)64,500, sugar content 2.5%) consisting of an A-chain (M(r)30,500) and a B-chain (M(r)33,500) linked by the disulfide bond. The terminal sugar of glycan in porrectin B-chain is determined to be mannose. By non-denaturing polyacrylamide gel electrophoresis, porrectin displayed three isoforms that have different pl values with the same molecular weight. Porrectin is a potent inhibitor of eukaryotic protein synthesis in the rabbit reticulocyte lysate system. The molecular mechanism of action of porrectin on rat liver ribosomes is demonstrated to be specific for RNA N-glycosidase. The cleavage site is the adenosine at position 4324 (rat liver 28S rRNA) embedded in the highly conserved ricin/alpha-sarcin ('R/S') domain. PMID:8997493

  8. Inhibition of protein synthesis by the T cell receptor-inducible human TDAG51 gene product.

    PubMed

    Hinz, T; Flindt, S; Marx, A; Janssen, O; Kabelitz, D

    2001-05-01

    The T cell death associated gene 51 (TDAG51) was shown to be required for T cell receptor (TCR)-dependent induction of Fas/Apo1/CD95 expression in a murine T cell hybridoma. Despite the absence of a nuclear localization sequence and a nucleic acid binding domain, it was suggested to be localized in the nucleus and to function as a transcription factor regulating Fas-expression. However, we demonstrate that the human (h)TDAG51 protein is localized in the cytoplasm and the nucleoli, suggesting a role in ribosome biogenesis and/or translation regulation. Indeed, it strongly inhibited translation of a luciferase mRNA in a reticulocyte translational extract. Furthermore, cotransfection of hTDAG51 and the luciferase gene into 293T cells resulted in a strong inhibition of luciferase mRNA translation. Our findings were further strengthened by isolating in a yeast two-hybrid screen three proteins which are involved in the regulation of translation. We speculate that hTDAG51 couples TCR signaling to inhibition of protein biosynthesis in activated T lymphocytes. PMID:11369516

  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. Influence of transport and time on blood variables commonly measured for the athlete biological passport.

    PubMed

    Robinson, Neil; Giraud, Sylvain; Schumacher, Yorck Olaf; Saugy, Martial

    2016-02-01

    Some recent studies have characterized the stability of blood variables commonly measured for the Athlete Biological Passport. The aim of this study was to characterize the impact of different shipments conditions and the quality of the results returned by the haematological analyzer. Twenty-two healthy male subjects provided five EDTA tubes each. Four shipment conditions (24, 36, 48, 72 h) under refrigerated conditions were tested and compared to a set of samples left in the laboratory also under refrigerated conditions (group control). All measurements were conducted using two Sysmex XT-2000i analyzers. Haemoglobin concentration, reticulocytes percentage, and OFF-score numerical data were the same for samples analyzed just after collection and after a shipment under refrigerated conditions up to 72 h. Detailed information reported especially by the differential (DIFF) channel scatterplot of the Sysmex XT-2000i indicated that there were signs of blood deterioration, but were not of relevance for the variables used in the Athlete Biological Passport. As long as the cold chain is guaranteed, the time delay between the collection and the analyses of blood variables can be extended. PMID:25924812

  11. Morphological and morphometric characterization of agoutis' peripheral blood cells (Dasyprocta prymnolopha, Wagler, 1831) raised in captivity.

    PubMed

    Conde Júnior, Airton Mendes; De Moura Fortes, Eunice Anita; De Menezes, Danilo José Ayres; De Oliveira Lopes, Luana; De Carvalho, Maria Acelina Martins

    2012-03-01

    Thirty adult agoutis (Dasyprocta primnolopha) from the Nucleus of Study and Preservation of Wild Animals at the Federal University of Piauí were used. Blood scrubs of these animals were colored by the Leishman method and analyzed in light microscopy. The cells had been measured using programs that analyze images (Leica QWin - Image Processing and Analysis Software). Mature erythrocytes, basophil reticulocytes, lymphocytes, eosinophils, neutrophils, monocytes, and thrombocytes were identified. Agoutis' erythrocytes presented elliptical form, without nucleus with an average diameter of 5.64 micromeres ± 0.38. The lymphocytes are spherical cells with scarce cytoplasm, dense and with a very centralized rounded nucleus measuring an average diameter of 13.20 micromeres ± 0.35. The monocytes are slightly basophilic, with a spherical nucleus, central constriction, and an average diameter of 20.59 micromeres ± 0.32. The neutrophils are spherical, with a polymorphic lobulated nucleus, with an average diameter of 11.2 micromeres ± 0.20. The eosinophils are spherical with lobulated nucleus and with an average diameter of 14.25 micromeres ± 0.36. Only five basophils were observed, with abundance of cytoplasmic granules with 9.8 micrometers of diameter ± 0.30. Thrombocytopenic pleomorphism was frequent. There were similarities in the cellular constituents in peripheral blood of agoutis and of other rodents and humans. The cellular types from the peripheral blood, the morphology, and morphometry of the blood's cells did not vary according to sex. PMID:21898666

  12. Optimized high-throughput screen for Hepatitis C virus translation inhibitors

    PubMed Central

    Berry, Katherine E.; Peng, Betty; Koditek, David; Beeman, Douglas; Pagratis, Nikos; Perry, Jason K.; Parrish, Jay; Zhong, Weidong; Doudna, Jennifer A.; Shih, I-hung

    2011-01-01

    Hepatitis C virus (HCV) is a considerable global health problem for which new classes of therapeutics are needed. We developed a high-throughput assay to identify compounds that selectively block translation initiation from the HCV internal ribosome entry site (HCV IRES). Rabbit reticulocyte lysate conditions were optimized to faithfully report on authentic HCV IRES-dependent translation relative to a 5′ capped mRNA control. We screened a library of ~430,000 small molecules for IRES inhibition, leading to ~1,700 initial hits. After secondary counter screening the vast majority of hits proved to be luciferase and general translation inhibitors. Despite well-optimized in vitro translation conditions, in the end we found no selective HCV IRES inhibitors but did discover a new scaffold of general translation inhibitor. The analysis of these molecules, and the finding that a large fraction of false positives resulted from off-target effects, highlights the challenges inherent in screens for RNA-specific inhibitors. PMID:21297107

  13. The interaction of alpha-thalassemia and homozygous sickle-cell disease.

    PubMed

    Higgs, D R; Aldridge, B E; Lamb, J; Clegg, J B; Weatherall, D J; Hayes, R J; Grandison, Y; Lowrie, Y; Mason, K P; Serjeant, B E; Serjeant, G R

    1982-06-17

    Patients with homozygous sickle-cell disease may be homozygous for alpha-thalassemia 2 (alpha-/alpha-), may be heterozygous for alpha-thalassemia 2 (alpha-/alpha alpha), or may have a normal alpha-globin-gene complement (alpha alpha/alpha alpha). We compared the clinical and hematologic features of 44 patients who had sickle-cell disease and homozygous alpha-thalassemia 2 with those of controls with the two hematologic conditions. The patients with homozygous alpha-thalassemia 2 had significantly higher red-cell counts and levels of hemoglobin and hemoglobin A2, as well as significantly lower hemoglobin F, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, mean corpuscular volume, reticulocyte counts, irreversibly-sickled cell counts, and serum total bilirubin levels, than those with a normal alpha-globin-gene complement. Heterozygotes (alpha-/alpha alpha) had intermediate values. In the group with homozygous alpha-thalassemia 2, fewer patients had episodes of acute chest syndrome and chronic leg ulceration and more patients had splenomegaly, as compared with patients in other two subgroups. These data confirm previous suggestions that alpha-thalassemia inhibits in vivo sickling in homozygous sickle-cell disease and may be an important genetic determinant of its hematologic severity. PMID:6176865

  14. Metabolic indicators of oxidative stress correlate with haemichrome attachment to membrane, band 3 aggregation and erythrophagocytosis in beta-thalassaemia intermedia.

    PubMed

    Cappellini, M D; Tavazzi, D; Duca, L; Graziadei, G; Mannu, F; Turrini, F; Arese, P; Fiorelli, G

    1999-03-01

    Haematological data, genotype, transfusion requirements, metabolic indicators of oxidative stress (flux via hexose-monophosphate shunt (HMPS); steady state level of GSH and GSSG, NADPH and NADP; activity of anti-oxidant enzymes), parameters of membrane damage (aggregated band 3; membrane-bound haemichromes, autologous immunoglobulins (Igs) and C3 complement fragments) and erythrophagocytosis were measured in erythrocytes (RBC) of 15 beta-thalassaemia intermedia patients (nine splenectomized) with low, if any, transfusion requirements. Patients presented increased aggregated band 3, bound haemichromes, Igs and C3 complement fragments, and increased erythrophagocytosis. Bound haemichromes strongly correlated with aggregated band 3. Anti-band 3 Igs were predominantly associated with aggregated band 3. Erythrophagocytosis positively correlated with aggregated band 3, haemichromes and Igs, suggesting the involvement of haemichrome-induced band 3 aggregation in phagocytic removal of beta-thalassaemic RBC. Splenectomized patients showed higher degrees of membrane damage and phagocytosis, significantly higher numbers of circulating RBC precursors, and tendentially higher numbers of reticulocytes. Basal flux via HMPS was increased twofold, but HMPS stimulation by methylene blue was decreased, as was the glucose flux via HMPS. GSH was remarkably decreased, whereas NADPH was increased. Except for unchanged catalase and glutathione reductase, anti-oxidant enzymes had increased activity. Negative correlation between HMPS stimulation by methylene blue and bound haemichromes indicated that the ability to enhance HMPS may counteract haemichrome precipitation and limit consequent membrane damage leading to erythrophagocytosis. PMID:10086787

  15. Isolation and in vitro differentiation of human erythroid precursor cells.

    PubMed

    Kim, H C; Marks, P A; Rifking, R A; Maniatis, G M; Bank, A

    1976-05-01

    There is decreased beta-globin production in beta-thalassemic reticulocytes and nucleated erythroid cells. In this study, we have examined whether unbalanced globin synthesis is expressed at all stages of human erythroid cell maturation. In order to determine the pattern of globin synthesis in early erythroid cells during erythroid cell maturation, an in vitro culture system using human bone marrow erythroid precursor cells has been developed. Early erythroid precursor cells (proerythroblasts and basophilic erythroblasts) have been isolated from nonthalassemic and thalassemic human bone marrows by lysing more mature erythroid cells, using complement and a rabbit antiserum prepared against normal human red cells. In the presence of erythropoietin, differentiation and proliferation of erythroid cells in demonstrable in liquid suspension culture for 24-48 hr, as determined by morphological criteria and by an increase in globin synthesis. The ratio of alpha- to beta-globin chain synthesis in nonthalassemic cells in approximately 1 at all stages of erythroid cell differentiation during culture. In cells from four patients with homozygous beta- thalassemia there is decreased beta-globin synthesis compared to alpha-globin synthesis, both in early erythroid precursor cells and during their maturation in culture. These findings indicate that unbalanced globin chain synthesis is expressed at all stages of red cell maturation in homozygous beta-thalassemia. PMID:1260133

  16. Balsamin, a novel ribosome-inactivating protein from the seeds of Balsam apple Momordica balsamina.

    PubMed

    Kaur, Inderdeep; Yadav, Santosh K; Hariprasad, Gururao; Gupta, R C; Srinivasan, Alagiri; Batra, Janendra K; Puri, Munish

    2012-08-01

    Plant seeds, a rich source of proteins, are considered important for their application as functional ingredients in a food system. A novel ribosome-inactivating protein (RIP), balsamin was purified from the seeds of Balsam apple, Momordica balsamina. Balsamin was purified by ion exchange chromatography on CM Sepharose and gel filtration on superdex-75. It has a molecular weight of 28 kDa as shown by SDS-PAGE analysis. Balsamin inhibits protein synthesis in a rabbit reticulocyte lysate-based cell free translation assay with an IC(50) of 90.6 ng ml(-1). It has RNA N-glycosidase activity and releases a 400-base long fragment termed the Endo fragment from 28S rRNA in the same manner as does saporin-6 from Saponaria officinalis. The N-terminal sequence analysis of the first 12 amino acids of balsamin revealed that it shares 83% similarity with type I RIP α-MMC from Momordica charantia and 50% similarity with β-MMC (from Momordica charantia), bryodin I (from Bryonia dioica) and luffin a (from Luffa cylindrica). Balsamin was further characterized by mass spectrometry. CD spectroscopic studies indicate that secondary structure of balsamin contains helix (23.5%), β-strand (24.6%), turn (20%) and random coil (31.9%). Thus RIPs activity expressed in vegetables like Momordica sp. advocates its usage in diet. PMID:22120616

  17. 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. PMID:23112907

  18. Detecting ricin: sensitive luminescent assay for ricin A-chain ribosome depurination kinetics.

    PubMed

    Sturm, Matthew B; Schramm, Vern L

    2009-04-15

    Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of the elongation factor binding site resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Subpicomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 x 10(8) M(-1) s(-1), a diffusion limited reaction indicating catalytic perfection even with large reactants. PMID:19364139

  19. Changes in blood parameters in New Zealand White rabbits during pregnancy.

    PubMed

    Mizoguchi, Yasumoto; Matsuoka, Tetsuya; Mizuguchi, Hiroyasu; Endoh, Takako; Kamata, Ryo; Fukuda, Kazuya; Ishikawa, Tsutomu; Asano, Yuzo

    2010-01-01

    Changes in clinical pathology parameters, particularly those related to blood coagulation, were examined throughout the gestation period in New Zealand White rabbits. As compared with the non-pregnant group, the following major changes were observed in the pregnant group. For blood coagulation-related parameters, platelets increased progressively and fibrinogen increased slightly from organogenesis, prothrombin time was significantly prolonged during organogenesis and shortened in the late fetal growth stage, activated partial thromboplastin time was significantly prolonged during the fetal growth stage, and antithrombin III increased during and after late organogenesis. Such changes in blood coagulation-related parameters during the later stages of gestation seem to be physiological responses in preparation for protecting against excessive haemorrhage or haemostasis at parturition. For the other haematological and blood chemical parameters as well as progesterone, red blood cell counts, haemoglobin and haematocrit began to decrease during organogenesis and continued to decrease thereafter. Reticulocyte counts significantly increased during organogenesis and decreased thereafter. White blood cell parameters, except for neutrophils, showed significant decreases during the fetal growth stage. Serum progesterone concentration reached its highest level early in organogenesis and decreased thereafter. Total protein, albumin, glucose, cholesterol, calcium, blood urea nitrogen and creatinine decreased significantly during the middle and/or late periods of gestation. In conclusion, the data obtained from the present study can be used as background data for effective evaluation of reproductive toxicology in rabbits, and pregnant rabbits may serve as models of pregnant women in research pertaining to clinical pathology and gestation. PMID:19854754

  20. Background data for hematological and blood chemical examinations in juvenile beagles.

    PubMed

    Ishii, Toshiya; Hori, Hisako; Ishigami, Makoto; Mizuguchi, Hiroyasu; Watanabe, Dai

    2013-01-01

    As the first step to get historical background data for physiological examinations in juvenile dogs, hematology and blood chemistry data obtained from juvenile beagle dogs (less than 3 months of age) used in the control group of toxicity studies conducted in our laboratory were summarized and compared with those obtained from adult beagle dogs (6 months of age). In the hematological examination, growth of beagle dogs was shown to be associated with increases in erythrocyte parameters and with decreases in reticulocyte and leukocyte counts. In the blood chemical examination, growth of beagle dogs was shown to be associated with increases in aspartate aminotransferase, alanine aminotransferase, and creatinine and with decreases in creatine phosphokinase, glucose, total cholesterol, and calcium. The differential leukocyte ratio showed no age relation, but the actual count showed a tendency toward decrease. Alkaline phosphatase showed a tendency to increase from 0 months of age to 3 months of age, but it decreased at 6 months of age. The present results were roughly similar to those previously reported. PMID:23357940

  1. Reduction of Intramedullary Apoptosis after Stem Cell Transplantation in Black African Variant of Pediatric Sickle Cell Anemia

    PubMed Central

    Isgrò, Antonella; Sodani, Pietro; Marziali, Marco; Gaziev, Javid; Fraboni, Daniela; Paciaroni, Katia; Gallucci, Cristiano; De Angelis, Gioia; Alfieri, Cecilia; Ribersani, Michela; Armiento, Daniele; Roveda, Andrea; Andreani, Marco; Testi, Manuela; Lucarelli, Guido

    2014-01-01

    Background and Purpose Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative treatment for sickle cell anemia (SCA). We report our experience with transplantation in children with the Black African variant of SCA and the effects of transplant on erythroid compartment in bone marrow (BM). Patients and methods Twenty-seven consecutive patients who underwent BM transplantation from HLA-identical donors following a myeloablative conditioning regimen were included. Using both CD71 and FSC parameters, we obtained three erythroid populations: EryA–C. Ery A (CD71high FSChigh) are basophilic; Ery B (CD71high FSClow) are late basophilic and polychromatic; and Ery C (CD71low FSClow) are orthochromatic erythroblasts and reticulocytes. To analyze the effect of transplantation on intramedullary apoptosis, we studied Fas (CD95+) and caspase-3 expression in erythroblast subpopulations. Results All patients experienced sustained engraftment, and all surviving patients remained free of SCA-related events after transplantation. The erythroid population showed expansion in the BM at baseline. After transplant, levels decreased, especially of Ery C, in parallel to reduced Fas expression and an initial caspase 3 increase in erythroid population, similar to reported later steps of “normal” erythroid maturation. Conclusions The results suggest a good chance of cure for children with SCA, with an excellent survival rate. We also observed “normalization” of erythroid populations in parallel with a decreased intramedullary apoptosis rate, suggesting normal erythroid maturation in ex-SCA patients after HSCT. PMID:25045462

  2. 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

  3. Subchronic toxicity studies on 1,3,5-trinitrobenzene, 1,3-dinitrobenzene, and tetryl in rats. 14-day toxicity evaluation of n-methyl-n, 2,4,6-tetranitroaniline in Fischer 344 rats. Final report

    SciTech Connect

    Reddy, T.V.

    1994-05-01

    Subacute toxic effects of Tetryl in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of Tetryl (0, 500, 1250, 2000, 2500 and 5000 mg/kg diet) for fourteen days. The average daily Tetryl doses consumed were 32, 82, 130, 178 and 374 for females and 31, 80, 121, 170 and 350 for males. Food and water consumption were not significantly altered. Final body weight was reduced in only the high dose males while relative organ weights were significantly changed in this same dose group involving the liver (females), kidneys (males), and spleen (males). Hematology and clinical chemistry studies indicated increased values relating to reticulocytes (females) and methemoglobin (females and males) in high and mid dose groups while total protein and albumin were significantly increased in all groups except the 50 mg/kg female group. Alkaline phosphatase was decreased in these same female groups. The only histopathological change noted which was considered meaningful was a mild increase in hyaline droplet deposition in male kidneys in all dose groups.

  4. 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. PMID:25496995

  5. Overview of cell-free protein synthesis: historic landmarks, commercial systems, and expanding applications.

    PubMed

    Chong, Shaorong

    2014-01-01

    During the early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owing to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from "traditional" E. coli, rabbit reticulocyte lysate, and wheat germ extracts, to recent insect and human cell extracts, to defined systems reconstituted from purified recombinant components. Although each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This unit provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology. PMID:25271714

  6. 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.

  7. A first exon-encoded domain of E1A sufficient for posttranslational modification, nuclear-localization, and induction of adenovirus E3 promoter expression in Xenopus oocytes.

    PubMed Central

    Richter, J D; Young, P; Jones, N C; Krippl, B; Rosenberg, M; Ferguson, B

    1985-01-01

    The purified Escherichia coli-expressed human subgroup C adenovirus E1A 13S mRNA product induces expression from the adenovirus type 5 E3 promoter when injected into Xenopus oocytes. In the present communication, the E. coli-expressed E1A 13S and 12S mRNA products are shown to undergo a posttranslational modification in microinjected Xenopus oocytes, which causes a 2- to 4-kDa increase in apparent molecular size, identical to that occurring in HeLa cells expressing the E1A gene. The E. coli-expressed E1A proteins are similarly modified in vitro in a soluble rabbit reticulocyte lysate. The modified form of the E1A proteins preferentially localizes to the oocyte nucleus following cytoplasmic microinjection. The use of various deleted forms of E1A protein synthesized in E. coli shows that a first exon-encoded domain of E1A, residing between amino acid residues 23 and 120, is sufficient for the posttranslational modification and nuclear localization of E1A and also for the trans-activation of the E3 promoter by E1A in Xenopus oocytes. These results suggest that the posttranslational modification of E1A protein may be important for its function. Images PMID:2934733

  8. Induction of proteins and mRNAs after uv irradiation of human epidermal keratinocytes

    SciTech Connect

    Kartasova, T.; Ponec, M.; van de Putte, P.

    1988-02-01

    uv sensitivity of cultured human epidermal keratinocytes was analyzed at different growth conditions and compared with the sensitivity of dermal fibroblasts derived from the same skin specimen. No significant differences in survival curves were found between these two cell types, although keratinocytes grown under standard conditions were slightly more resistant to uv irradiation than fibroblasts. The extracellular concentration of calcium appeared to be critical not only in the regulation of keratinocyte proliferation and differentiation, but also in the uv sensitivity of these cells: keratinocytes grown under conditions which favor cell proliferation (low calcium concentration) are more resistant to uv irradiation than those grown under conditions favoring differentiation (high calcium concentration). Two-dimensional protein gel electrophoresis was used to detect a possible effect of uv irradiation on the accumulation of specific mRNAs in the cytoplasm and/or on the synthesis of specific proteins. Proteins were pulse labeled in vivo with (/sup 35/S)methionine or synthesized in vitro in rabbit reticulocyte lysates on mRNA isolated from keratinocytes that were irradiated with different uv doses at different periods of time prior to isolation. Alterations in expression were demonstrated for several proteins in both in vivo and in vitro experiments.

  9. Acylation of keratinocyte transglutaminase by palmitic and myristic acids in the membrane anchorage region

    SciTech Connect

    Chakravarty, R.; Rice, R.H.

    1989-01-05

    The membrane-bound form of keratinocyte transglutaminase was found to be labeled by addition of (/sup 3/H) acetic, (/sup 3/H)myristic, or (/sup 3/H)palmitic acids to the culture medium of human epidermal cells. Acid methanolysis and high performance liquid chromatography analysis of palmitate-labeled transglutaminase yielded only methyl palmitate. In contrast, analysis of the myristate-labeled protein yielded approximately 40% methyl myristate and 60% methyl palmitate. Incorporation of neither label was significantly affected by cycloheximide inhibition of protein synthesis. The importance of the fatty acid moiety for membrane anchorage was demonstrated in three ways. First, the enzyme was solubilized from the particulate fraction of cell extracts by treatment with neutral 1 M hydroxylamine, which was sufficient to release the fatty acid label. Second, solubilization of active enzyme from the particulate fraction upon mild trypsin treatment resulted in a reduction in size by approximately 10 kDa and removal of the fatty acid radiolabels. Third, the small fraction of soluble transglutaminase in cell extracts was found almost completely to lack fatty acid labeling. Keratinocyte transglutaminase translated from poly(A+) RNA in a reticulocyte cell-free system was indistinguishable in size from the native enzyme, suggesting anchorage requires only minor post-translational processing. Thus, the data are highly compatible with membrane anchorage by means of fatty acid acylation within 10 kDa of the NH/sub 2/ or COOH terminus.

  10. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    PubMed Central

    2011-01-01

    Background The surface glycoprotein (SU, gp120) of the human immunodeficiency virus (HIV) must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP) to bind the Duffy Antigen Receptor for Chemokines (DARC) and invade reticulocytes. Results Variable loop 3 (V3) of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket. PMID:21281498

  11. Cell-free synthesis of enzymically active tissue-type plasminogen activator. Protein folding determines the extent of N-linked glycosylation.

    PubMed Central

    Bulleid, N J; Bassel-Duby, R S; Freedman, R B; Sambrook, J F; Gething, M J

    1992-01-01

    Tissue-type plasminogen activator (t-PA) is synthesized in mammalian cells as a mixture of two forms that differ in their extent of N-linked glycosylation. We have investigated the mechanism underlying this variation in glycosylation, using a cell-free system that consists of a rabbit reticulocyte lysate optimized for the formation of disulphide bonds and supplemented with dog pancreas microsomal membranes. Molecules of human t-PA synthesized in vitro are enzymically active and responsive to natural activators and inhibitors, and are glycosylated in a pattern identical with that of the protein produced in vivo. This demonstrates that t-PA synthesized in vitro folds into the same conformation as the protein synthesized in vivo. We show that the extent of glycosylation of individual t-PA molecules is dependent on the state of folding of the polypeptide chain, since the probability of addition of an oligosaccharide side chain at Asn-184 is decreased under conditions that promote the formation of enzymically active molecules. This variation in glycosylation is independent of the rate of protein synthesis. Images Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:1520279

  12. 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

  13. Purification and partial characterization of another form of the antiviral protein from the seeds of Phytolacca americana L. (pokeweed).

    PubMed Central

    Barbieri, L; Aron, G M; Irvin, J D; Stirpe, F

    1982-01-01

    1. The pokeweed antiviral protein, previously identified in two forms (PAP and PAP II) in the leaves of Phytolacca americana (pokeweed) [Obrig. Irvin & Hardesty (1973) Arch. Biochem. Biophys. 155, 278-289; Irvin, Kelly & Robertus (1980) Arch. Biochem. Biophys. 200, 418-425] is a protein that prevents replication of several viruses and inactivates ribosomes, thus inhibiting protein synthesis. 2. PAP is present in several forms in the seeds of pokeweed. One of them, which we propose to call 'pokeweed antiviral protein from seeds' (PAP-S) was purified in high yield (180 mg per 100 g of seeds) by chromatography on CM-cellulose, has mol.wt. 30 000, and is similar to, but not identical with. PAP and PAP II. 3. PAP-S inhibits protein synthesis in a rabbit reticulocyte lysate with an ID50 (concentration giving 50% inhibition) of 1.1 ng/ml (3.6 x 10(-11) M), but has much less effect on protein synthesis by whole cells, with an ID50 of 1 mg/ml (3.3 x 10(-5) M), and inhibits replication of herpes simplex virus type 1. PMID:7103950

  14. Quantification of red blood cell fragmentation by the automated hematology analyzer XE-2100 in patients with living donor liver transplantation.

    PubMed

    Banno, S; Ito, Y; Tanaka, C; Hori, T; Fujimoto, K; Suzuki, T; Hashimoto, T; Ueda, R; Mizokami, M

    2005-10-01

    The fragmented red cell (FRC) is a useful index for diagnosing and determining the severity of thrombotic thrombocytopenic purpura (TTP), thrombotic microangiopathy (TMA) and other similar conditions, as it is found in peripheral blood in patients with these diseases. The FRC expression rate has conventionally been determined by manual methods using smear samples. However, it is difficult to attain accurate quantification by such methods as they are time consuming and prone to a great margin of error. With cases of living donor liver transplantation, the current study examined the possibility of using a multi-parameter automated hematology analyzer, the XE-2100 (Sysmex Corporation) for FRC quantification. While there was a notable correlation between the manual and automated measurements, the manual measurement resulted in higher values. This suggested remarkable variations in judgment by individuals. The FRC values had a significant correlation with the reticulocyte count, red blood cell distribution width (RDW), fibrin/fibrinogen degradation products (P-FDP) and lactate dehydrogenase (LDH) among the test parameters, and this finding was consistent with the clinical progression in patients. The automated method can offer precise measurements in a short time without inter-observer differences, meeting the requirement for standardization. The determination of FRC count (%) by the XE-2100 that enables early diagnoses and monitoring of TTP or TMA will be useful in the clinical field. PMID:16178907

  15. 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. PMID:23606168

  16. The Stringency of Start Codon Selection in the Filamentous Fungus Neurospora crassa*

    PubMed Central

    Wei, Jiajie; Zhang, Ying; Ivanov, Ivaylo P.; Sachs, Matthew S.

    2013-01-01

    In eukaryotic cells initiation may occur from near-cognate codons that differ from AUG by a single nucleotide. The stringency of start codon selection impacts the efficiency of initiation at near-cognate codons and the efficiency of initiation at AUG codons in different contexts. We used a codon-optimized firefly luciferase reporter initiated with AUG or each of the nine near-cognate codons in preferred context to examine the stringency of start codon selection in the model filamentous fungus Neurospora crassa. In vivo results indicated that the hierarchy of initiation at start codons in N. crassa (AUG ≫ CUG > GUG > ACG > AUA ≈ UUG > AUU > AUC) is similar to that in human cells. Similar results were obtained by translating mRNAs in a homologous N. crassa in vitro translation system or in rabbit reticulocyte lysate. We next examined the efficiency of initiation at AUG, CUG, and UUG codons in different contexts in vitro. The preferred context was more important for efficient initiation from near-cognate codons than from AUG. These studies demonstrated that near-cognate codons are used for initiation in N. crassa. Such events could provide additional coding capacity or have regulatory functions. Analyses of the 5′-leader regions in the N. crassa transcriptome revealed examples of highly conserved near-cognate codons in preferred contexts that could extend the N termini of the predicted polypeptides. PMID:23396971

  17. 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. PMID:26072930

  18. 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. PMID:27067484

  19. 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

    2015-01-01

    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

  20. Iron deficiency anemia in inflammatory bowel disease.

    PubMed

    Kaitha, Sindhu; Bashir, Muhammad; Ali, Tauseef

    2015-08-15

    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

  1. [Dietary fibers of secondary vegetable raw material for correction of carbohydrate and lipid metabolism in laboratory animals].

    PubMed

    Shchelkunov, L F

    2001-01-01

    Drop of consumption by the population of dietary fibers is one of the causes of increase of sickness rate by diabetes in particular, and violation of carbohydrate metabolism in general. The purpose of research is definition of a degree of effect of dietary fibers of by-products of grapes processing (grape seed-cakes and press cake of grape pyrenes in an mixture with sorbite) on a carbohydrate and lipide metabolism in laboratory animals. During examinations is detected, that the vegetable products based on dietary fibers do not have negative action on physical development of animals, their body weight. The parameters of an amount of reticulocytes, erythrocytes, lymphocytes, hematocrit were in normal range. In animal, receiving dietary fibers of secondary grape raw material in a ration, the concentration of a cholesterol in blood serum was lower on 2-3% (5.6-5.9 +/- 0.2 mmol/l) as against control (6.0 +/- 0.2 mmol/l). The basal level of a glucose has appeared equal for control: 6.1 +/- 0.5 mmol/l, and for others groups of rats, consuming a dietary fibers with sorbite from 5.6 +/- 0.4 up to 6.0 +/- 0.5 mmol/l. PMID:11517682

  2. Ribosome shunting in the cauliflower mosaic virus 35S RNA leader is a special case of reinitiation of translation functioning in plant and animal systems

    PubMed Central

    Ryabova, Lyubov A.; Hohn, Thomas

    2000-01-01

    The shunt model predicts that small ORFs (sORFs) within the cauliflower mosaic virus (CaMV) 35S RNA leader and downstream ORF VII are translated by different mechanisms, that is, scanning–reinitiation and shunting, respectively. Wheat germ extract (WGE) and rabbit reticulocyte lysate (RRL) in vitro translation systems were used to discriminate between these two processes and to study the mechanism of ribosomal shunt. In both systems, expression downstream of the leader occurred via ribosomal shunt under the control of a stable stem and a small ORF preceding it. Shunting ribosomes were also able to initiate quite efficiently at non-AUG start codons just downstream of the shunt landing site in WGE but not in RRL. The short sORF MAGDIS from the mammalian AdoMetDC RNA, which conditionally suppresses reinitiation at a downstream ORF, prevented shunting if placed at the position of sORF A, the 5′-proximal ORF of the CaMV leader. We have demonstrated directly that sORF A is translated and that proper termination of translation at the 5′-proximal ORF is absolutely required for both shunting and linear ribosome migration. These findings strongly indicate that shunting is a special case of reinitiation. PMID:10766738

  3. Subcutaneous recombinant human erythropoietin in children with renal anemia on continuous ambulatory peritoneal dialysis.

    PubMed

    Aufricht, C; Balzar, E; Steger, H; Lothaller, M A; Frenzel, K; Kohlhauser, C; Kiss, H; Khoss, A E; Kernova, T

    1993-11-01

    Subcutaneous recombinant human erythropoietin (rHuEpo) treatment of renal anemia was performed in four boys and eight girls on CAPD, aged 0.8-12.5 (mean 7.4) years. In contrast to previous studies, our therapeutic goal was not set with a hematocrit of 30% but with full correction of anemia. Following a maximum weekly rHuEpo dosage of median 120 (range 100-240) IU/kg body weight, hematocrit increased in 10 children from 24 (14-29)% within 12 (4-17) weeks to 40.1 (33.5-48.4)%. The weekly increase in hematocrit was 1.27 (0.5-3.1)%. The corrected reticulocyte count increased from 1.3 (0.7-1.8)% to 2.3 (1.4-3.9)% within 4 (2-6) weeks. Eight children fulfilled the protocol; six with an uncomplicated course were able to maintain a hematocrit of 37.1 (35.1-42.7)% with only one sc medication per week of approximately two-thirds of their highest weekly rHuEpo dosage. No serious adverse effect of rHuEpo therapy was observed. PMID:8111178

  4. Cross-protection in nonhuman primates against Argentine hemorrhagic fever.

    PubMed Central

    Weissenbacher, M C; Coto, C E; Calello, M A; Rondinone, S N; Damonte, E B; Frigerio, M J

    1982-01-01

    The susceptibility of the marmoset Callithrix jacchus to Tacaribe virus infection was investigated to perform cross-protection studies between Junin and Tacaribe viruses. Five marmosets inoculated with Tacaribe virus failed to show any signs of disease, any alterations in erythrocyte, leukocyte, reticulocyte, and platelet counts or any changes in hematocrit or hemoglobin values. No Tacaribe virus could be recovered from blood at any time postinfection. Anti-Tacaribe neutralizing antibodies appeared 3 weeks postinfection. The five Tacaribe-infected marmosets and four noninfected controls were challenged with the pathogenic strain of Junin virus on day 60 post-Tacaribe infection. The former group showed no signs of disease, no viremia, and no challenge virus replication, whereas the control group exhibited the typical symptoms of Argentine hemorrhagic fever, high viremia, and viral titers in organs. Soon after challenge, the Tacaribe-protected marmosets synthesized neutralizing antibodies against Junin virus. These results indicate that the marmoset C. jacchus can be considered an experimental model for protection studies with arenaviruses and that the Tacaribe virus could be considered as a potential vaccine against Junin virus. PMID:6276301

  5. Germiston virus transcriptase requires active 40S ribosomal subunits and utilizes capped cellular RNAs.

    PubMed Central

    Vialat, P; Bouloy, M

    1992-01-01

    The transcriptase associated with Germiston virus was assayed in an in vitro reaction in which transcription was coupled to translation by adding reticulocyte lysate under the appropriate salt conditions. When analyzed in polyacrylamide gels, the major transcripts migrated like authentic S mRNAs and possessed 12- to 18-base-long nontemplated 5' extensions similar to the 5' end of viral mRNAs. These transcripts were functional for the synthesis of at least proteins N and NSS. When translation was inhibited by adding protein synthesis inhibitors such as puromycin, cycloheximide, and anisomycin, a drastic inhibitory effect was observed on the synthesis of the complete S mRNA transcripts. However, initiation and part of the elongation process were still active, since short and incomplete RNA molecules with RNA primers at their 5' ends were synthesized. On the other hand, we found that edeine, another inhibitor of protein synthesis, stimulated not only synthesis of S mRNAs but also that of the full-length S cRNAs. Taking into account the mode of action of this antibiotic, we discuss the results, which emphasize the crucial role of active ribosomes during bunyavirus transcription and confirm the observations reported on La Crosse virions. Moreover, we showed that the RNA transcripts synthesized in a transcription-translation reaction were capped and that most of them have acquired the 5' terminal sequences of the alpha- or beta-globin mRNA. Images PMID:1731108

  6. Polymorphic CUG repeats in human mRNAs and their effects on gene expression.

    PubMed

    Tian, Bin; Mukhopadhyay, Rupa; Mathews, Michael B

    2005-01-01

    Expanded CUG repeats in the 3'-untranslated region (UTR) of the gene encoding myotonic dystrophy protein kinase (DMPK) cause myotonic dystrophy type 1 disease (DM1). The presence of such repeats has been found to impede gene expression at several levels in model systems. We took a bioinformatic approach to survey all human mRNA sequences for polymorphic CUG repeats. Our survey revealed that CUG repeats occur widely in various regions of mRNAs, with higher frequency in protein coding regions than 5'-UTRs or 3'-UTRs. About 30 genes were found to contain CUG repeats that are polymorphic in the number of repeats, suggesting the potential to expand or shrink. However, long polymorphic repeats were restricted to the 3'-UTR of the DMPK gene and the coding region of the ribosomal protein L14 gene. Using cell-free translation systems, we showed that extended CUG repeats can inhibit protein synthesis in vitro in the rabbit reticulocyte lysate, but not in wheat germ extracts, consistent with our previous finding of an interaction of CUG repeats with the protein kinase PKR. In transfected cells, CUG repeats can inhibit gene expression both in cis and in trans. However, observations with PKR-minus cells indicate that these effects are not primarily attributable to the interaction of extended CUG repeats with PKR. Northwestern blotting detected the presence in human cells of more CUG-binding proteins than are currently known. PMID:17114933

  7. Overview of Cell-Free Protein Synthesis: Historic Landmarks, Commercial Systems, and Expanding Applications

    PubMed Central

    Chong, Shaorong

    2014-01-01

    During early days of molecular biology, cell-free protein synthesis played an essential role in deciphering the genetic code and contributed to our understanding of translation of protein from messenger RNA. Owning to several decades of major and incremental improvements, modern cell-free systems have achieved higher protein synthesis yields at lower production costs. Commercial cell-free systems are now available from a variety of material sources, ranging from “traditional” E. coli, rabbit reticulocyte lysate and wheat germ extracts to recent insect and human cell extracts to defined systems reconstituted from purified recombinant components. Though each cell-free system has certain advantages and disadvantages, the diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology. This article provides a brief history of cell-free protein synthesis and describes key advances in modern cell-free systems, practical differences between widely used commercial cell-free systems, and applications of this important technology. PMID:25271714

  8. Compensatory erythropoiesis has no impact on the outcome of the in vivo Pig-a mutation assay in rats following treatment with the haemolytic agent 2-butoxyethanol.

    PubMed

    Kenyon, Michelle O; Coffing, Stephanie L; Ackerman, Joel I; Gunther, William C; Dertinger, Stephen D; Criswell, Kay; Dobo, Krista L

    2015-05-01

    The Pig-a assay has rapidly gained international interest as a useful tool for assessing the mutagenic potential of compounds in vivo. Although a large number of compounds, including both mutagens and non-mutagens, have been tested in the rat Pig-a assay in haematopoietic cells, there is limited understanding of how perturbations in haematopoiesis affect assay performance. Of particular concern is the possibility that regenerative haematopoiesis alone, without exposure to a genotoxic agent, could result in elevated Pig-a mutant cell frequencies. To address this concern, Wistar-Han rats were dosed by oral gavage with a non-genotoxic haemolytic agent, 2-butoxyethanol (2-BE). Dose levels ranging from 0 to 450 mg/kg were tested using both single administration and 28-day treatment regimens. Haematology parameters were assessed at minimum within the first 24h of treatment and 8 days after the final administration. Pig-a mutant frequencies were assessed on Days 15 and ~30 for both treatment protocols and also on Days 43 and 57 for the 28-day protocol. Even at doses of 2-BE that induced marked intravascular lysis and strong compensatory erythropoiesis, the average Pig-a mutant phenotype red blood cell and reticulocyte frequencies were within the historical vehicle control distribution. 2-BE therefore showed no evidence of in vivo mutagenicity in these studies. The data suggest that perturbations in haematopoiesis alone do not lead to an observation of increased mutant frequency in the Pig-a assay. PMID:25820171

  9. 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

  10. 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. PMID:25029173

  11. 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.

  12. A novel micronucleus in vitro assay utilizing human hematopoietic stem cells.

    PubMed

    Kotova, N; Hebert, N; Härnwall, E-L; Vare, D; Mazurier, C; Douay, L; Jenssen, D; Grawé, J

    2015-10-01

    The induction of micronucleated reticulocytes in the bone marrow is a sensitive indicator of chromosomal damage. Therefore, the micronucleus assay in rodents is widely used in genotoxicity and carcinogenicity testing. A test system based on cultured human primary cells could potentially provide better prediction compared to animal tests, increasing patient safety while also implementing the 3Rs principle, i.e. replace, reduce and refine. Hereby, we describe the development of an in vitro micronucleus assay based on animal-free ex vivo culture of human red blood cells from hematopoietic stem cells. To validate the method, five clastogens with direct action, three clastogens requiring metabolic activation, four aneugenic and three non-genotoxic compounds have been tested. Also, different metabolic systems have been applied. Flow cytometry was used for detection and enumeration of micronuclei. Altogether, the results were in agreement with the published data and indicated that a sensitive and cost effective in vitro assay to assess genotoxicity with a potential to high-throughput screening has been developed. PMID:26208286

  13. Erythroblastic Islands in the Bone Marrow of Patients with Immune-Related Pancytopenia

    PubMed Central

    Wang, Yi-Hao; Fu, Rong; Dong, Shu-Wen; Liu, Hui; Shao, Zong-Hong

    2014-01-01

    Background Immune-related pancytopenia (IRP) is characterized by pancytopenia caused by autoantibody-mediated bone marrow destruction or suppression. The bone marrows of IRP patients have remarkably increased erythroblastic islands (EIs). Methodology and Principal Findings We determined the immunoglobulin G (IgG) autoantibodies in some parts of EIs of IRP patients using immunofluorescence to investigate the biological function of EIs with IgG in the pathophysiology of IRP. The dominant class of autoantibodies detected in mononuclear cells was IgG (CD34 IgG, CD15 IgG, and GlycoA IgG), specifically IgG on GlycoA-positive cells (GlycoA IgG). Results show that extravascular hemolysis occurred in IRP through IgG autoantibodies in the EIs. These data included a high percentage of reticulocytes in the peripheral blood, hypererythrocytosis in the bone marrow, and high serum bilirubin. Furthermore, we examined the macrophages in the bone marrow of IRP patients. The results show that the number of activated macrophages relatively increased, and the phagocytic activity of macrophages significantly increased. Conclusions and Significance Increased EIs with IgG were the sites of erythroblast phagocytosis by the activated macrophages, rather than erythropoietic niches. The IgG autoantibodies in the EIs possibly functioned as adhesion molecules for a ring of erythroblasts around the macrophages, thereby forming morphologic EIs. PMID:24740145

  14. Translation of viral mRNAs that do not require eIF4E is blocked by the inhibitor 4EGI-1.

    PubMed

    Redondo, Natalia; García-Moreno, Manuel; Sanz, Miguel Angel; Carrasco, Luis

    2013-09-01

    High throughput screening has rendered new inhibitors of eukaryotic protein synthesis. One such molecule, 4EGI-1 has been reported to selectively block the initiation factor eIF4E. We have investigated the action of this inhibitor on translation directed by several viral mRNAs which, in principle, do not utilize eIF4E. We found that 4EGI-1 inhibits translation directed by poliovirus IRES, in rabbit reticulocyte lysates, to a similar extent as capped mRNA. Moreover, 4EGI-1 inhibits translation driven by poliovirus IRES, both in vitro and in cultured cells, despite cleavage of eIF4G by picornavirus proteases. Finally, translation of vesicular stomatitis virus mRNAs and Sindbis virus subgenomic mRNA is blocked by 4EGI-1 in infected cells to a similar extent as cellular mRNAs. These findings cast doubt on the selective action of this inhibitor, and suggest that this molecule may affect other steps in protein synthesis unrelated to cap recognition by eIF4E. PMID:23870416

  15. 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. PMID:21640800

  16. 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. PMID:26553522

  17. A novel gene family with a developmentally regulated expression in Xenopus laevis.

    PubMed

    Shim, S; Yoon, C S; Han, J K

    2000-01-19

    We have isolated a new maternal gene called 4G2. 4G2 cDNA encodes a predicted protein of 501 amino acids, and its apparent molecular mass of 61 kDa was determined by SDS-PAGE of 4G2 recombinant protein expressed in E. coli or in vitro translated in rabbit reticulocyte lysate. Amino acid analysis of 4G2 revealed the RGD and LDV motif with a potential cell attachment activity. The open reading frames (ORF) also contained a consensus bipartite nuclear localization signal (NLS). There were number of expressed tag sequences (ESTs) from Drosophila, zebrafish, chicken, mouse, and human origin that encode a high degree of identity to the predicted 4G2 protein, thereby suggesting that 4G2 may constitute a novel gene family whose function has not been elucidated. We also present evidence that 4G2 transcript is maternally synthesized in stage IV oocyte, localized to animal hemisphere of egg, and zygotically reactivated in mid-neurula stage. PMID:10631101

  18. mTOR Inhibition improves anaemia and reduces organ damage in a murine model of sickle cell disease.

    PubMed

    Wang, Jintao; Tran, Jennifer; Wang, Hui; Guo, Chiao; Harro, David; Campbell, Andrew D; Eitzman, Daniel T

    2016-08-01

    Mechanistic target of rapamycin (mTOR) has been shown to play an important role in red blood cell physiology, with inhibition of mTOR signalling leading to alterations in erythropoiesis. To determine if mTOR inhibition would improve anaemia in sickle cell disease (SCD), mice with SCD were treated with the dual mTORC1/2 inhibitor, INK128. One week after daily oral drug treatment, erythrocyte count, haemoglobin, and haematocrit were all significantly increased while reticulocyte counts were reduced. These parameters remained stable during 3 weeks of treatment. Similar effects were observed following oral treatment with the mTORC1 inhibitor, sirolimus. Sirolimus treatment prolonged the lifespan of sickle cell erythrocytes in circulation, reduced spleen size, and reduced renal and hepatic iron accumulation in SCD mice. Following middle cerebral artery occlusion, stroke size was reduced in SCD mice treated with sirolimus. In conclusion, mTOR inhibition is protective against anaemia and organ damage in a murine model of SCD. PMID:27030515

  19. Investigation of toxicological effects of Shuanghuanglian injection in Beagle dogs by metabonomic and traditional approaches.

    PubMed

    Yan, Guangyan; Zhao, Yinglan; Deng, Pengchi; Lv, Lei; Wang, Yanli; Bu, Qian; Liu, Bin; Hu, Chunyan; Zhuo, Yanqiang; Yang, Xunning; Wang, Li; Cen, Xiaobo

    2010-11-01

    In this study, clinical biochemistry, hematology, histopathology and (1)H nuclear magnetic resonance spectroscopy-based metabonomic approaches were applied to investigate the toxicological effects of Shuanghuanglian (SHL) injection after intravenous administration (dosed at 4, 12 and 36 mL stock/kg) in Beagle dogs for 30 d. Decreases in red blood cells, hemoglobin, mean cell volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration were observed in the high-dose group. Elevated reticulocytes, total bilirubin and direct bilirubin were also observed in this group. Moreover, significant hemosiderosis and Prussian blue positivity were detected in the liver, spleen and kidney from high-dose group animals, and transmission electron microscopy examination revealed an appreciable number of acanthrocytes in the liver. These results collectively indicate that SHL injection has the potential to cause hemolytic anemia. Metabonomic analysis showed increases in serum lactate, choline and phosphocholine but a decrease in taurine in treated groups and these findings may underlie the toxicological mechanism of SHL injection. In summary, SHL injection shows hemolytic effects in Beagle dogs; moreover, serum choline and phosphocholine as well as lactate and taurine may be the biomarkers for hemolytic anemia induced by SHL injection. PMID:20864460

  20. Peroxidase gene expression during tomato fruit ripening

    SciTech Connect

    Biggs, M.S.; Flurkey, W.H.; Handa, A.K.

    1987-04-01

    Auxin oxidation has been reported to play a critical role in the initiation of pear fruit ripening and a tomato fruit peroxidase (POD) has been shown to have IAA-oxidase activity. However, little is known about changes in the expression of POD mRNA in tomato fruit development. They are investigating the expression of POD mRNA during tomato fruit maturation. Fruit pericarp tissues from six stages of fruit development and ripening (immature green, mature green, breaker, turning, ripe, and red ripe fruits) were used to extract poly (A)/sup +/ RNAs. These RNAs were translated in vitro in a rabbit reticulocyte lysate system using L-/sup 35/S-methionine. The /sup 35/S-labeled products were immunoprecipitated with POD antibodies to determine the relative proportions of POD mRNA. High levels of POD mRNA were present in immature green and mature green pericarp, but declined greatly by the turning stage of fruit ripening. In addition, the distribution of POD mRNA on free vs bound polyribosomes will be presented, as well as the presence or absence of POD mRNA in other tomato tissues.

  1. 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

  2. Differential Modulation of Angiogenesis by Erythropoiesis-Stimulating Agents in a Mouse Model of Ischaemic Retinopathy

    PubMed Central

    McVicar, Carmel M.; Colhoun, Liza M.; Abrahams, Jodie L.; Kitson, Claire L.; Hamilton, Ross; Medina, Reinhold J.; Durga, Dash; Gardiner, Tom A.; Rudd, Pauline M.; Stitt, Alan W.

    2010-01-01

    Background Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models. Methodology/Principal Findings The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1–20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNFα and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001). Conclusions This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs. PMID:20686695

  3. Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

    PubMed

    Stirpe, F; Gasperi-Campani, A; Barbieri, L; Falasca, A; Abbondanza, A; Stevens, W A

    1983-12-15

    Ribosome-inactivating proteins, similar to those already known [Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520] were purified from the seeds of Saponaria officinalis (two proteins), of Agrostemma githago (three proteins), and of Asparagus officinalis (three proteins), and from the latex of Hura crepitans (one protein). The yield ranged from 8 to 400 mg/100 g of starting material. All proteins have an Mr of approx. 30000 and an alkaline isoelectric point. Their sugar content varies from 0 (proteins from S. officinalis) to 40% (protein from H. crepitans). The ribosome-inactivating proteins inhibit protein synthesis by rabbit reticulocyte lysate, the ID50 (concentration giving 50% inhibition) ranging from 1 ng/ml (a protein from S. officinalis) to 18 ng/ml (a protein from A. githago). Those which were tested (the proteins from S. officinalis and from A. githago) also inhibit polymerization of phenylalanine by isolated ribosomes, acting in an apparently catalytic manner. The protein from H. crepitans inhibited protein synthesis by HeLa cells, with an ID50 of 4 micrograms/ml, whereas the proteins from S. officinalis and from A. githago had an ID50 of more than 50-100 micrograms/ml. The ribosome-inactivating proteins from S. officinalis and from A. githago reduced the number of local lesions by tobacco-mosaic virus in the leaves of Nicotiana glutinosa. PMID:6667259

  4. Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony).

    PubMed

    Stirpe, F; Barbieri, L; Battelli, M G; Falasca, A I; Abbondanza, A; Lorenzoni, E; Stevens, W A

    1986-12-15

    Bryodin is a strongly basic (pI greater than or equal to 9.5) glycoprotein (neutral sugar content 6.3%) with Mr 30,000, purified from the roots of Bryonia dioica (white bryony). This protein inhibits protein synthesis by a rabbit reticulocyte lysate with and ID50 (concentration causing 50% inhibition) of 0.12 nM (3.6 ng/ml) and has much less effect on protein synthesis by whole cells, with ID50 values ranging from 46 nM to 2.27 microM (1.4-67 micrograms/ml). Bryodin acts by inactivating ribosomes, with a less-than-equimolar ratio, which suggests a catalytic action. Bryodin decreases the number of local lesions induced by tobacco mosaic virus in the leaves of Nicotiana glutinosa. From all its properties, bryodin can be considered to be a ribosome-inactivating protein, similar to those already known [reviews: Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520; Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8]. PMID:3827858

  5. Purification and properties of new ribosome-inactivating proteins with RNA N-glycosidase activity.

    PubMed

    Bolognesi, A; Barbieri, L; Abbondanza, A; Falasca, A I; Carnicelli, D; Battelli, M G; Stirpe, F

    1990-11-30

    Ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8) were purified from the seeds of Asparagus officinalis (two proteins, asparin 1 and 2), of Citrullus colocynthis (two proteins, colocin 1 and 2), of Lychnis chalcedonica (lychnin) and of Manihot palmata (mapalmin), from the roots of Phytolacca americana (pokeweed antiviral protein from roots, PAP-R) and from the leaves of Bryonia dioica (bryodin-L). The two latter proteins can be considered as isoforms, respectively, of previously purified PAP, from the leaves of P. americana, and of bryodin-R, from the roots of B. dioica. All proteins have an Mr at approx, 30,000, and an alkaline isoelectric point. Bryodin-L, colocins, lychnin and mapalmin are glycoproteins. All RIPs inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes and alter rRNA in a similar manner as the A-chain of ricin and related toxins (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). PMID:2248976

  6. Purification and partial characterization of single-chain ribosome-inactivating proteins from the seeds of Phytolacca dioica L.

    PubMed

    Parente, A; De Luca, P; Bolognesi, A; Barbieri, L; Battelli, M G; Abbondanza, A; Sande, M J; Gigliano, G S; Tazzari, P L; Stirpe, F

    1993-10-19

    Three ribosome-inactivating proteins (RIPs) similar to those already known (Stirpe et al. (1992) Bio/Technology 10, 405-412) were purified from the seeds of Phytolacca dioica. These proteins, called Phytolacca dioica RIPs (PD-S1, PD-S2 and PD-S3 RIPs), are glycoproteins, with M(r) approx. 30,000, inhibit protein synthesis by a rabbit reticulocyte lysate and phenylalanine polymerization by isolated ribosomes, and depurinate rat liver rRNA in an apparently identical manner as the A-chain of ricin and other RIPs (Endo et al. (1987) J. Biol. Chem. 262, 5908-5912). Part of the purified rat liver ribosomes appeared resistant to the action of PD-S RIPs. The most abundant protein, PD-S2 RIP, gave a weak or nil cross-reaction with sera against various other RIPs, including a pokeweed antiviral protein from the roots of Phytolacca americana. PD-S2 RIP was linked to a monoclonal antibody (Ber-H2) against the CD30 human lymphocyte antigen and the resulting immunotoxin was selectively toxic to the CD30 + Hodgkin's lymphoma-derived L540 cell line. PMID:8218414

  7. Bryodin, a ribosome-inactivating protein from the roots of Bryonia dioica L. (white bryony).

    PubMed Central

    Stirpe, F; Barbieri, L; Battelli, M G; Falasca, A I; Abbondanza, A; Lorenzoni, E; Stevens, W A

    1986-01-01

    Bryodin is a strongly basic (pI greater than or equal to 9.5) glycoprotein (neutral sugar content 6.3%) with Mr 30,000, purified from the roots of Bryonia dioica (white bryony). This protein inhibits protein synthesis by a rabbit reticulocyte lysate with and ID50 (concentration causing 50% inhibition) of 0.12 nM (3.6 ng/ml) and has much less effect on protein synthesis by whole cells, with ID50 values ranging from 46 nM to 2.27 microM (1.4-67 micrograms/ml). Bryodin acts by inactivating ribosomes, with a less-than-equimolar ratio, which suggests a catalytic action. Bryodin decreases the number of local lesions induced by tobacco mosaic virus in the leaves of Nicotiana glutinosa. From all its properties, bryodin can be considered to be a ribosome-inactivating protein, similar to those already known [reviews: Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520; Stirpe & Barbieri (1986) FEBS Lett. 195, 1-8]. PMID:3827858

  8. Ribosome-inactivating proteins from the seeds of Saponaria officinalis L. (soapwort), of Agrostemma githago L. (corn cockle) and of Asparagus officinalis L. (asparagus), and from the latex of Hura crepitans L. (sandbox tree).

    PubMed Central

    Stirpe, F; Gasperi-Campani, A; Barbieri, L; Falasca, A; Abbondanza, A; Stevens, W A

    1983-01-01

    Ribosome-inactivating proteins, similar to those already known [Barbieri & Stirpe (1982) Cancer Surveys 1, 489-520] were purified from the seeds of Saponaria officinalis (two proteins), of Agrostemma githago (three proteins), and of Asparagus officinalis (three proteins), and from the latex of Hura crepitans (one protein). The yield ranged from 8 to 400 mg/100 g of starting material. All proteins have an Mr of approx. 30000 and an alkaline isoelectric point. Their sugar content varies from 0 (proteins from S. officinalis) to 40% (protein from H. crepitans). The ribosome-inactivating proteins inhibit protein synthesis by rabbit reticulocyte lysate, the ID50 (concentration giving 50% inhibition) ranging from 1 ng/ml (a protein from S. officinalis) to 18 ng/ml (a protein from A. githago). Those which were tested (the proteins from S. officinalis and from A. githago) also inhibit polymerization of phenylalanine by isolated ribosomes, acting in an apparently catalytic manner. The protein from H. crepitans inhibited protein synthesis by HeLa cells, with an ID50 of 4 micrograms/ml, whereas the proteins from S. officinalis and from A. githago had an ID50 of more than 50-100 micrograms/ml. The ribosome-inactivating proteins from S. officinalis and from A. githago reduced the number of local lesions by tobacco-mosaic virus in the leaves of Nicotiana glutinosa. Images Fig. 2. PMID:6667259

  9. 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.

  10. 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.

  11. 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. PMID:21323872

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

    PubMed Central

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

    2015-01-01

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

  13. [Dyserythropoietic syndromes: incidence, diagnosis, therapy].

    PubMed

    Cacciola, E

    1990-10-01

    The nosography of the dyserythropoietic syndromes remains poorly defined in the field of clinical hematology. The prominent pathophysiologic feature lies in the "ineffective erythropoiesis" as expressed by bone marrow erythroid hyperplasia with dysplasia accompanied by a normal or only slightly increased reticulocyte count. Both erythrokinetics and ferrokinetics are impaired, as shown by either slight reduction of the red cell survival or marked increased rate of serum iron transport together with reduced cellular iron utilization. The dyserythropoietic syndromes can be classified as acquired, secondary or congenital. The acquired ones, especially the sideroblastic forms, belonging to the myelodysplastic syndromes, are typical of the elderly whereas the congenital are of childhood. Their treatment is still a matter of controversy. However, the employment of folic acid, Vit. B12, pyridoxine and androgens can be useful in selected cases. In case of severe anemia, blood transfusion are required in association with iron chelating agents. However, some biological molecules, such as erythropoietin, interleukins 3 and 4, hemopoietic growth factors (especially GM-CSF), could represent future prospects of treatment. PMID:2291009

  14. Robust microarray production of freshly expressed proteins in a human milieu

    PubMed Central

    Festa, Fernanda; Rollins, Sean M.; Vattem, Krishna; Hathaway, Margarita; Lorenz, Phillip; Mendoza, Eliseo Alejandro; Yu, Xiaobo; Qiu, Ji; Kilmer, Greg; Jensen, Penny; Webb, Brian; Ryan, Ed T.; LaBaer, Joshua

    2013-01-01

    Purpose In vitro transcription/translation (IVTT) systems are widely used in proteomics. For clinical applications, mammalian systems are preferred for protein folding and activity; however, the level of protein obtained is low. A new system extracted from human cells (1-Step Human Coupled IVT) has the potential to overcome this problem and deliver high yields of protein expressed in a human milieu. Experimental design Western blots and self-assembled protein microarrays were used to test the efficiency of protein synthesis by 1-Step Human Coupled IVT (HCIVT) compared to rabbit reticulocyte lysate (RRL). The arrays were also used to measure the immune response obtained from serum of patients exposed to pathogens or vaccine. Results HCIVT performed better than RRL in all experiments. The yield of protein synthesized in HCIVT is more than 10 times higher than RRL, in both western blot and protein microarrays. Moreover, HCIVT showed a robust lot-to-lot reproducibility. In immune assays, the signals of many antigens were detected only in HCIVT-expressed arrays, mainly due to the reduction in the background signal and the increased levels of protein on the array. Conclusion and clinical relevance HCIVT is a robust IVTT system that yields high levels of protein produced in a human milieu. It can be used in applications where protein expression in a mammalian system and high yields are needed. The increased immunogenic response of HCIVT-expressed proteins will be critical for biomarker discovery in many diseases, including cancer. PMID:23027544

  15. Adaptation to altitude as a vehicle for experiential learning of physiology by university undergraduates.

    PubMed

    Weigle, David S; Buben, Amelia; Burke, Caitlin C; Carroll, Nels D; Cook, Brett M; Davis, Benjamin S; Dubowitz, Gerald; Fisher, Rian E; Freeman, Timothy C; Gibbons, Stephen M; Hansen, Hale A; Heys, Kimberly A; Hopkins, Brittany; Jordan, Brittany L; McElwain, Katherine L; Powell, Frank L; Reinhart, Katherine E; Robbins, Charles D; Summers, Cameron C; Walker, Jennifer D; Weber, Steven S; Weinheimer, Caroline J

    2007-09-01

    In this article, an experiential learning activity is described in which 19 university undergraduates made experimental observations on each other to explore physiological adaptations to high altitude. Following 2 wk of didactic sessions and baseline data collection at sea level, the group ascended to a research station at 12,500-ft elevation. Here, teams of three to four students measured the maximal rate of oxygen uptake, cognitive function, hand and foot volume changes, reticulocyte count and hematocrit, urinary pH and 24-h urine volume, athletic performance, and nocturnal blood oxygen saturation. Their data allowed the students to quantify the effect of altitude on the oxygen cascade and to demonstrate the following altitude-related changes: 1) impaired performance on selected cognitive function tests, 2) mild peripheral edema, 3) rapid reticulocytosis, 4) urinary alkalinization and diuresis, 5) impaired aerobic but not anaerobic exercise performance, 6) inverse relationship between blood oxygen saturation and resting heart rate, and 7) regular periodic nocturnal oxygen desaturation events accompanied by heart rate accelerations. The students learned and applied basic statistical techniques to analyze their data, and each team summarized its results in the format of a scientific paper. The students were uniformly enthusiastic about the use of self-directed experimentation to explore the physiology of altitude adaptation and felt that they learned more from this course format than a control group of students felt that they learned from a physiology course taught by the same instructor in the standard classroom/laboratory format. PMID:17848594

  16. Development toxicity of cobalt in the rat

    SciTech Connect

    Paternain, J.L.; Domingo, J.L.; Corbella, J.

    1988-01-01

    To determine the potential developmental toxicity of cobalt, pregnant Sprague-Dawley rats were given by gavage a daily dose of 0, 25, 50, and 100 mg/kg cobalt(II) chloride on d 6-15 of gestation. Females were sacrificed on d 20. Maternal effects included significant reductions in weight gain and food consumption, particularly at 100 mk/kg x d. Hematocrit, hemoglobin concentration, mean corpuscular volume, mean corpuscular hemoglobin, and reticulocytes were increased significantly in the 100-mg/kg x d group. No treatment-related changes were recorded in the number of corpora lutea, total implants, resorptions, the number of live and dead fetuses, fetal size parameters, or fetal sex distribution data. Increased incidence of stunted fetuses per litter was the only adverse finding at 50 and 100 mg/kg x d group. However, this increase was not statistically significant. Examination of fetuses for gross external abnormalities, skeletal malformation, or ossification variations revealed that cobalt did not produce teratogenicity or significant fetotoxicity in the rat at doses as high as 100 mg/kg x d.

  17. Subchronic toxicity studies on 1,3,5-trinitrobenzene, 1,3-dinitrobenzene, and tetryl in rats. Subchronic toxicity evaluation of 1,3,5-trinitrobenzene in Fischer 344 rats. Final report

    SciTech Connect

    Reddy, T.V.; Daniel, F.B.; Robinson, M.; Olson, G.R.; Wiechman, B.

    1994-05-01

    Subchronic toxic effects of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 66.67, 400 and 800 mg/kg diet) so as to achieve a final target dose of 0, 5, 30 and 60 mg/kg b.w. for ninety days. Food intake in the 400 and 800 mg TNB dose groups of both sexes was reduced throughout the study and resulted in a significant decrease in absolute body weights. The calculated average TNB dosage was 4, 25 and 49 mg/kg/day for females and 4, 21 and 44 mg/kg/day for males. A decrease in testicular weight in males and increase in relative spleen weight of both sexes in the 400 and 800 mg TNB dose groups were noted. Also, the relative brain weight was increased in the male 400 and 800 mg TNB dose groups while the relative liver weight was increased in 800 mg TNB dose group of both sexes. Histopathological examinations suggested that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hemotopoiesis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in reticulocytes and increased methemoglobin concentration as compared to controls in both sexes.

  18. 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.

  19. SOD2 deficiency in hematopoietic cells in mice results in reduced red blood cell deformability and increased heme degradation

    PubMed Central

    Mohanty, Joy G.; Nagababu, Enika; Friedman, Jeffrey S.; Rifkind, Joseph M.

    2013-01-01

    Among the three types of super oxide dismutases (SODs) known, SOD2 deficiency is lethal in neonatal mice owing to cardiomyopathy caused by severe oxidative damage. SOD2 is found in red blood cell (RBC) precursors, but not in mature RBCs. To investigate the potential damage to mature RBCs resulting from SOD2 deficiency in precursor cells, we studied RBCs from mice in which fetal liver stem cells deficient in SOD2 were capable of efficiently rescuing lethally irradiated host animals. These transplanted animals lack SOD2 only in hematopoietically generated cells and live longer than SOD2 knockouts. In these mice, approximately 2.8% of their total RBCs in circulation are iron-laden reticulocytes, with numerous siderocytic granules and increased protein oxidation similar to that seen in sideroblastic anemia. We have studied the RBC deformability and oxidative stress in these animals and the control group by measuring them with a microfluidic ektacytometer and assaying fluorescent heme degradation products with a fluorimeter, respectively. In addition, the rate of hemoglobin oxidation in RBCs from these mice and the control group were measured spectrophotometrically. The results show that RBCs from these SOD2-deficient mice have reduced deformability, increased heme degradation products, and an increased rate of hemoglobin oxidation compared with control animals, indicative of increased RBC oxidative stress. PMID:23142655

  20. 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

  1. Performance evaluation of the latest fully automated hematology analyzers in a large, commercial laboratory setting: a 4-way, side-by-side study.

    PubMed

    Bourner, G; Dhaliwal, J; Sumner, J

    2005-01-01

    Gamma-Dynacare is a Canadian-based community laboratory partnership formed in the mid-1990s through the merger of 3 prominent Ontario medical diagnostic laboratories. Laboratory Corporation of America acquired an interest in the GD partnership in mid 2002. We service more than 10,000 community-based Canadian clinicians, hospital partners, and private clients with an integrated customer-focused system that includes specimen collection, transportation, and results reporting services. With more than 1,700 highly qualified medical, technical, and support staff and a network of laboratories, Gamma Dynacare aims to be at the forefront of technological innovation to better service the clinician base and ultimately deliver better patient care. We were looking for a hematology analyzer that would allow. (1) standardization throughout Ontario in our 4 largest sites and (2) better performance to effectively handle aged samples and minimize slide review. To select the best, most productive hematology analyzer for our environment, it was decided to perform a side-by-side comparison of the top hematology analyzers from Abbott (Cell-Dyn 3500), Beckman Coulter (LH 750), Bayer (Advia 120), and Sysmex (XE 2100), utilizing the same samples. CBC, differential and reticulocyte parameters were all evaluated according to CLSI (formerly NCCLS) and established hematology analyzer evaluation guidelines. We assessed each analyzer for precision, linearity, carryover, stability, differential capabilities, slide review rates, and throughput (clean bench studies). Two hundred samples were assessed for differential and morphology flagging on each analyzer using the reference 400 cell manual differential for comparison. Throughput was assessed by analyzing 700 consecutive samples representative of our workload mix. Stability studies at 24 hours showed that the Beckman Coulter LH 750 was least affected by EDTA, effect with minimal changes in the mean corpuscular volume (MCV) and hematocrit. Both

  2. [Metastatic prostate cancer complicated with chronic disseminated intravascular coagulopathy causing acute renal failure, mimicking thrombotic thrombocytopenic purpura and hemolytic uremic syndrome: pathomechanism, differential diagnosis and therapy related to a case].

    PubMed

    Deme, Dániel; Ragán, Márton; Kalmár, Katalin; Kovács, Lajos; Varga, Erzsébet; Varga, Tünde; Rakonczai, Ervin

    2010-12-01

    in the peripheral blood), normal INR, elevated lactate dehydrogenase (LDH) and ARF suggested TTP-HUS. Hemodialysis and six plasmaferesis (PF) were carried out. After the fifth PF, skin manifestations of thrombotic microangiopathy occurred on the feet. Clotting analysis revealed elevated D-dimer (>5 μg/mL), normal fibrinogen (3.2 g/L), a slightly raised INR (1.36) and activated partial prothrombin time (APTT) (45.8 sec), normal reticulocyte (57 G/L) and a slightly low platelet count (123 G/L), which proved to be chronic DIC. Therapeutic dose of low-molecular-weight heparin (LMWH) was started. Elevated prostate-specific antigen (PSA) (109.6 ng/mL) suggested prostatic carcinoma. Prostate biopsy revealed adenocarcinoma (Gleason: 4+4 for left lobe and 3+3 for right lobe). Elevated alkaline phosphatase suggested metastases in the bone, which were confirmed by bone scintigraphy. Combined androgen blockade (CAB) was started. After three months follow-up our patient's status is satisfactory. PSA is in the normal range (4.6 ng/mL). Thrombocytopenia of uncertain origin with normal or raised INR, APTT, elevated D-dimer, normal fibrinogen and reticulocyte count prove the diagnosis of chronic DIC. This process warrants searching for metastatic neoplasia. Due to the relatively low serum levels of circulating procoagulant factors (e.g. tissue factor), therapeutic dose of LMWH can be used with good efficiency in chronic DIC with low risk of bleeding. Severe DIC as a complication of metastatic prostate cancer can be treated by androgen deprivation therapy (ADT) or CAB in combination with ketokonazole and concomitant use of supportive treatment. Deme D, Ragán M, Kovács L, Kalmár K, Varga E, Varga T, Rakonczai E. Metastatic prostate cancer complicated with chronic disseminated intravascular coagulopathy causing acute renal failure mimicking thrombotic thrombocytopenic purpura and hemolytic uremic syndrome: pathomechanism, differential diagnosis and therapy related to a case. PMID

  3. Neonatal Hyperbilirubinemia in infants with G6PD c.563C > TVariant

    PubMed Central

    2012-01-01

    Background There is a strong correlation between glucose-6-phosphate dehydrogenase (G6PD) deficiency and neonatal hyperbilirubinemia with a rare but potential threat of devastating acute bilirubin encephalopathy. G6PD deficiency was observed in 4–14% of hospitalized icteric neonates in Pakistan. G6PD c.563C > T is the most frequently reported variant in this population. The present study was aimed at evaluating the time to onset of hyperbilirubinemia and the postnatal bilirubin trajectory in infants having G6PD c.563C > T. Methods This was a case–control study conducted at The Aga Khan University, Pakistan during the year 2008. We studied 216 icteric male neonates who were re-admitted for phototherapy during the study period. No selection was exercised. Medical records showed that 32 were G6PD deficient while 184 were G6PD normal. Each infant was studied for birth weight, gestational age, age at the time of presentation, presence of cephalhematoma, sepsis and neurological signs, peak bilirubin level, age at peak bilirubin level, days of hospitalization, whether phototherapy or exchange blood transfusion was initiated, and the outcome. During hospital stay, each baby was tested for complete blood count, reticulocyte count, ABO and Rh blood type, direct antiglobulin test and quantitative G6PD estimation [by kinetic determination of G6PDH]. G6PDgenotype was analyzed in 32 deficient infants through PCR-RFLP analysis and gene sequencing. Results G6PD variants c.563C > T and c.131 C > G were observed in 21 (65%) and three (9%) of the 32 G6PD deficient infants, respectively. DNA of eight (25%) newborns remained uncharacterized. In contrast to G6PD normal neonates, infants with c.563C > T variant had significantly lower enzyme activity (mean ± 1SD; 0.3 ± 0.2 U/gHb vs. 14.0 ± 4.5 U/gHb, p < 0.001) experienced higher peak levels of total serum bilirubin (mean ± 1SD; 16.8 ± 5.4 mg/dl vs. 13.8 ± 4.6 mg/dl, p = 0.008) which peaked earlier after

  4. Rapid CO breath test screening of drugs for protective effects on ribavirin-induced hemolysis in a rabbit model: a pilot study.

    PubMed

    Ma, Yong-Jian; Zhang, Hou-De; Wu, Chuang-Hong; Zhu, Guo-Liang; Ji, Yong-Qiang; Huang, Jia-Liang; Du, Li-Tao; Cao, Ping; Zang, De-Yue; Ji, Kun-Mei

    2016-01-01

    Hemolytic anemia is a major side effect of ribavirin antiviral treatment for chronic hepatitis C. Ribavirin dose reduction may compromise the antiviral response and erythropoietin can take several weeks to alleviate anemia. The purpose of the present study was to screen potentially protective drugs against ribavirin-induced hemolytic anemia in a rabbit model, using our modified CO breath test for measuring erythrocyte (RBC) lifespan, the gold standard diagnostic index of hemolysis. Fifteen rabbits were divided randomly into five groups (N  =  3/group): one vehicle control group, one ribavirin (only)-treated (RBV) group, and three groups initially treated with ribavirin only, followed by a combination of ribavirin with prednisone (RBV  +  Pred), polyene phosphatidyl choline (RBV  +  PPC), or reduced glutathione (RBV  +  GSH). RBC lifespan was calculated from accumulated CO measured in a closed rebreath apparatus, blood volume measured by the Evan's blue dye (EBD) dilution test, and hemoglobin concentration data. The RBC lifespan was normal in the vehicle control group (44-60 d), but reduced significantly in all of the ribavirin-treated groups before the addition of screened drugs (17-35 d). RBC lifespan rebounded significantly with the addition of glutathione, but not with the addition of prednisone or polyene phosphatidyl choline. A similar overall drug effect pattern was seen in the hemoglobin concentration and reticulocyte count data. In conclusion, the results of this pilot study indicate that reduced glutathione can attenuate ribavirin-induced hemolytic anemia, and that the RBC lifespan measured with our modified rapid CO breath test is feasible and reliable for use in animal studies. PMID:27506143

  5. Hyperhemolytic transfusion reaction attributable to anti-Fy3 in a patient with sickle cell disease.

    PubMed

    Reyes, M A; Illoh, O C

    2008-01-01

    A case of hyperhemolytic transfusion reaction attributable to anti-Fy3 in a 30-year-oldAfricanAmerican woman with a history of sickle cell disease is reported. The patient was admitted for vaso-occlusive sickle cell crisis and received 4 units of packed RBCs secondary to worsening symptomatic anemia (Hb 5.0 g/dL). On admission, the patient's antibody screen and identification showed anti-V and anti-E, and her antibody history included anti-E,-C,-Jk(b),-N, -V,-S,-Sl(a), and a cold agglutinin with possible anti-I specificity. A DAT performed on her RBCs was negative. RBC units that lacked E, C, Jk(b), N,V, and S were transfused. Posttransfusion Hb was 8.9 g/dL. On day 10 she developed a fever of 103 degrees F, and on day 11 her Hb decreased to 6.4 g/dL. She complained of severe back pain and dark urine. In addition, she became hypertensive, tachycardic, and jaundiced. The DAT indicated the presence of IgG on the patient's RBCs. Anti-Fy3 was identified in the serum and eluate. During the next 24 hours, her Hb decreased to 2.4 g/dL. The LDH level was 1687 U/L, and her reticulocyte count was 2.6%. A delayed hemolytic transfusion reaction with hyperhemolysis secondary to anti-Fy3 was suspected and was successfully treated with IVIG and high-dose prednisone. To the best of our knowledge, this is the first published case of hyperhemolysis in sickle cell disease attributable to anti-Fy3. PMID:19852130

  6. Biosynthesis and intracellular processing of carbonic anhydrase in Chlamydomonas reinhardtii.

    PubMed

    Toguri, T; Muto, S; Miyachi, S

    1986-08-01

    Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the CO2 concentration during photoautotrophic growth was decreased to air level. After decreasing the CO2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum. A 42-kDa polypeptide with isoelectric point (pI) of 7.1-7.3 was first synthesized. Within 5 min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pI of 6.1-6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis. PMID:2874027

  7. Anemia analyzer: algorithm and reflex testing in clinical practice leading to efficiency and cost savings.

    PubMed

    Haq, Samir M

    2009-01-01

    Anemia is a common disease affecting about 3.5 million people in the United States. In present day clinical practice, a clinician makes a diagnosis of anemia based on low hemoglobin levels discovered during a complete blood count (CBC) test. If the etiology of the anemia is not readily apparent, the clinician orders additional testing to discover the cause of the anemia. Which tests are ordered, in what order these tests are run, and how the information gathered from the tests is used is based primarily on the individual physician's knowledge and expertise. Using this system to determine the cause of anemia is not only labor and resource intensive but it carries a potential for morbidity and an occasional mortality. Utilizing previously published data, we created an algorithmic approach to analyze the cause of anemia in the majority of cases. The algorithm accepts as input three parameters from a CBC test: (1) mean corpuscular volume, (2) red cell distribution width, and (3) reticulocyte count. With these three parameters, the algorithm generates a probable etiology of the anemia. Additionally, the algorithm will automatically order reflex tests needed to confirm the diagnosis. These reflex tests can be modified depending on the policies of the institution using the algorithm, as different institutions may order different tests based on availability and costs. This is a simple algorithm that could be integrated into the CBC test output. When a low hemoglobin level is found, the algorithm suggests the probable etiology and orders reflex tests if they are desired. Such an approach would not only provide cost efficiency and time savings but would also elevate the level of every clinician ordering a CBC to that of an expert hematologist. PMID:19380908

  8. Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.

    PubMed

    Wilson, S A; Sieiro-Vazquez, C; Edwards, N J; Iourin, O; Byles, E D; Kotsopoulou, E; Adamson, C S; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    1999-08-15

    The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans. PMID:10432305

  9. Cloning and characterization of hIF2, a human homologue of bacterial translation initiation factor 2, and its interaction with HIV-1 matrix.

    PubMed Central

    Wilson, S A; Sieiro-Vazquez, C; Edwards, N J; Iourin, O; Byles, E D; Kotsopoulou, E; Adamson, C S; Kingsman, S M; Kingsman, A J; Martin-Rendon, E

    1999-01-01

    The cDNA for a human homologue (hIF2) of bacterial (bIF2) and yeast (yIF2) translation initiation factor two (IF2) has been identified during a screen for proteins which interact with HIV-1 matrix. The hIF2 cDNA encodes a 1220-amino-acid protein with a predicted relative molecular mass of 139 kDa, though endogeneous hIF2 migrates anomalously on SDS/PAGE at 180 kDa. hIF2 has an extended N-terminus compared with its homologues, although its central GTP-binding domain and C-terminus are highly conserved, with 58% sequence identity with yIF2. We have confirmed that hIF2 is required for general translation in human cells by generation of a point mutation in the P-loop of the GTP-binding domain. This mutant protein behaves in a transdominant manner in transient transfections and leads to a significant decrease in the translation of a reporter gene. hIF2 interacts directly with HIV-1 matrix and Gag in vitro, and the protein complex can be immunoprecipitated from human cells. This interaction appears to block hIF2 function, since purified matrix protein inhibits translation in a reticulocyte lysate. hIF2 does not correspond to any of the previously characterized translation initiation factors identified in mammals, but its essential role in translation appears to have been conserved from bacteria to humans. PMID:10432305

  10. Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase.

    PubMed

    Hüssy, P; Langen, H; Mous, J; Jacobsen, H

    1996-10-01

    The expression and processing of hepatitis C virus core protein was analyzed. Two protein bands, 21 kDa (P21), corresponding to the full-length core, and 19 kDa (P19), were detected as major products when core protein was expressed in the standard rabbit reticulocyte lysate system or in Sf9 insect cells. Core proteins with amino-terminal hexa-histidine tags were expressed which allowed the purification of the hexa-histidine P19 core with NI(2+)-NTA columns. With the help of mass spectrometry, the molecular weight of hexa-histidine-P19 was analyzed and its carboxy-terminus could be calculated. Fusion proteins of truncated core/core-E1 species fused to mouse dihydrofolate reductase (mDHFR) showed cleavage in the expected region. Cleavage sites could be determined by amino-terminal protein sequencing of the DHFR-fusion partner. Our data show that there are not one but two core products with an apparent molecular weight of about 19 kDa, ending either at amino acid leucine 179 or leucine 182, respectively. These cleavages in the hydrophobic, carboxy-terminal region of HCV core suggest processing by (a) recently proposed eucaryotic signal peptide peptidase(s) (F. Lyko et al. (1995) J. Biol. Chem. 270, 19873-19878). Furthermore, our results demonstrate that cleavage at these sites and the formation of the P19 species does not require previous processing at the signalase site (position 191/192) of the HCV-polyprotein. PMID:8862403

  11. Red Cell Properties after Different Modes of Blood Transportation

    PubMed Central

    Makhro, Asya; Huisjes, Rick; Verhagen, Liesbeth P.; Mañú-Pereira, María del Mar; Llaudet-Planas, Esther; Petkova-Kirova, Polina; Wang, Jue; Eichler, Hermann; Bogdanova, Anna; van Wijk, Richard; Vives-Corrons, Joan-Lluís; Kaestner, Lars

    2016-01-01

    Transportation of blood samples is unavoidable for assessment of specific parameters in blood of patients with rare anemias, blood doping testing, or for research purposes. Despite the awareness that shipment may substantially alter multiple parameters, no study of that extent has been performed to assess these changes and optimize shipment conditions to reduce transportation-related artifacts. Here we investigate the changes in multiple parameters in blood of healthy donors over 72 h of simulated shipment conditions. Three different anticoagulants (K3EDTA, Sodium Heparin, and citrate-based CPDA) for two temperatures (4°C and room temperature) were tested to define the optimal transportation conditions. Parameters measured cover common cytology and biochemistry parameters (complete blood count, hematocrit, morphological examination), red blood cell (RBC) volume, ion content and density, membrane properties and stability (hemolysis, osmotic fragility, membrane heat stability, patch-clamp investigations, and formation of micro vesicles), Ca2+ handling, RBC metabolism, activity of numerous enzymes, and O2 transport capacity. Our findings indicate that individual sets of parameters may require different shipment settings (anticoagulants, temperature). Most of the parameters except for ion (Na+, K+, Ca2+) handling and, possibly, reticulocytes counts, tend to favor transportation at 4°C. Whereas plasma and intraerythrocytic Ca2+ cannot be accurately measured in the presence of chelators such as citrate and EDTA, the majority of Ca2+-dependent parameters are stabilized in CPDA samples. Even in blood samples from healthy donors transported using an optimized shipment protocol, the majority of parameters were stable within 24 h, a condition that may not hold for the samples of patients with rare anemias. This implies for as short as possible shipping using fast courier services to the closest expert laboratory at reach. Mobile laboratories or the travel of the patients to

  12. Anaemia in Systemic Lupus Erythematosus Based on Iron Studies and Soluble Transferrin Receptor Levels

    PubMed Central

    Agarwal, Preeti; Wakhlu, Anupam; Kumar, Ashutosh; Mehrotra, Raj; Mittal, Saumya

    2016-01-01

    Introduction Haematological alterations such as anaemia, neutropenia and thrombocytopenia are frequent in Systemic Lupus Erythematosus (SLE). Ferritin being an acute phase reactant can be falsely elevated in lupus cases. Aim To evaluate the haematological alterations and to re-categorise the types of anemia by soluble transferrin receptor levels in diagnosed cases of SLE. Materials and Methods A sample of 30 newly diagnosed ANA positive SLE patients was taken. Complete blood counts, ESR, reticulocyte count, coagulation studies, diluted Russel Viper Venom Test (dRVVT), mixing studies, serological tests, high sensitivity CRP along with iron profile, transferrin saturation, soluble transferrin receptor (sol TFR) levels, anti-beta2 glycoprotein1, direct and indirect Coomb’s test were estimated in cases diagnosed as SLE. Clinical symptoms were co-related with and Systemic Lupus Erythaematosus Disease Activity Index (SLEDAI) was estimated. Results Anaemia was the most prevalent haematological alteration followed by thrombocytopenia. Further sub typing of anaemia was done by serum ferritin levels and using sol TFR assays. Ferritin is an acute phase reactant; it underestimated iron deficiency in patients of SLE. When sol TFR was used; patients with pure Anaemia of Chronic Disease (ACD) reduced from 68% to 26%, those with pure IDA reduced from 32% to 16% and a group with co-existing IDA & ACD (58%) was defined {Agreement=53%, p=0.09} by sol TFR which co-related with clinical response to Iron therapy in these patients. CRP was significantly raised in association with disease activity. Fever (p<0.0001), arthritis (p<0.03) were significantly related and CRP was elevated (p<0.04) in cases with high SLEDAI (severe flare). Conclusion Thus, in SLE, anaemia is the most frequent hematological alteration; iron deficiencies supercede in contrast to ACD and further autoimmune haemolytic anaemia. Sol TFR emerged as a better parameter to detect iron deficiency in patients of non

  13. Priming dose of phenylhydrazine protects against hemolytic and lethal effects of 2-butoxyethanol

    SciTech Connect

    Palkar, Prajakta S.; Philip, Binu K.; Reddy, Ramesh N.; Mehendale, Harihara M.

    2007-11-15

    Protection against a high dose of a toxicant by prior exposure to another toxicant is called heteroprotection. Our objective was to establish a heteroprotection model in RBCs. Female Sprague Dawley rats treated with an LD90 dose of 2-butoxyethanol (BE, 1500 mg/kg in water, 5 ml/kg po) 14 days after priming with 0.9% NaCl suffered 90% mortality by 15 days, whereas all rats receiving the LD90 dose of BE 14 days after priming with phenylhydrazine (PHZ, 125 mg/kg in 0.9% NaCl, 3 ml/kg po) survived. Hematocrit decreased from normal 45% to 24% by day 3 after PHZ priming and improved thereafter. Increasing the time interval between the priming and LD90 dose to 21 days abolished the heteroprotection. RBCs obtained on days 7 and 14 after PHZ priming unlike those on day 21 were resilient to the hemotoxic metabolite of BE, butoxyacetic acid (BAA). Unaltered hepatic alcohol and aldehyde dehydrogenase activities upon PHZ priming suggested that bioactivation of BE to BAA was unaffected. Lower renal (6 and 12 h) and hepatic (12 h) BAA levels and 3 fold higher excretion of BAA in PHZ-primed rat urine suggested a protective role of toxicokinetics. Higher erythropoietin, reticulocytes, and resiliency of PHZ-primed rat RBCs indicated that newly formed RBCs are resilient to hemolytic BAA. The antioxidant levels in the PHZ-primed rat RBCs did not indicate a protective role in heteroprotection. In conclusion, the resistance of PHZ-primed rats against BE-induced hemotoxicity and lethality is mediated by a combination of altered toxicokinetics, robust erythropoiesis, and resiliency of new RBCs.

  14. Inhibition of Mitogen-activated Protein Kinase (MAPK)-interacting Kinase (MNK) Preferentially Affects Translation of mRNAs Containing Both a 5'-Terminal Cap and Hairpin.

    PubMed

    Korneeva, Nadejda L; Song, Anren; Gram, Hermann; Edens, Mary Ann; Rhoads, Robert E

    2016-02-12

    The MAPK-interacting kinases 1 and 2 (MNK1 and MNK2) are activated by extracellular signal-regulated kinases 1 and 2 (ERK1/2) or p38 in response to cellular stress and extracellular stimuli that include growth factors, cytokines, and hormones. Modulation of MNK activity affects translation of mRNAs involved in the cell cycle, cancer progression, and cell survival. However, the mechanism by which MNK selectively affects translation of these mRNAs is not understood. MNK binds eukaryotic translation initiation factor 4G (eIF4G) and phosphorylates the cap-binding protein eIF4E. Using a cell-free translation system from rabbit reticulocytes programmed with mRNAs containing different 5'-ends, we show that an MNK inhibitor, CGP57380, affects translation of only those mRNAs that contain both a cap and a hairpin in the 5'-UTR. Similarly, a C-terminal fragment of human eIF4G-1, eIF4G(1357-1600), which prevents binding of MNK to intact eIF4G, reduces eIF4E phosphorylation and inhibits translation of only capped and hairpin-containing mRNAs. Analysis of proteins bound to m(7)GTP-Sepharose reveals that both CGP and eIF4G(1357-1600) decrease binding of eIF4E to eIF4G. These data suggest that MNK stimulates translation only of mRNAs containing both a cap and 5'-terminal RNA duplex via eIF4E phosphorylation, thereby enhancing the coupled cap-binding and RNA-unwinding activities of eIF4F. PMID:26668315

  15. Hereditary non-spherocytic haemolytic anaemia due to red blood cell glutathione synthetase deficiency in four unrelated patients from Spain: clinical and molecular studies.

    PubMed

    Corrons, J L; Alvarez, R; Pujades, A; Zarza, R; Oliva, E; Lasheras, G; Callis, M; Ribes, A; Gelbart, T; Beutler, E

    2001-02-01

    In four unrelated patients with chronic haemolysis and markedly reduced red blood cell (RBC) glutathione (49.5%, 12.6%, 11.5% and 15% of the normal concentration respectively), a severe glutathione synthetase (GSH-S, EC 6.3.2.3) deficiency was found. One case exhibited a neonatal haemolytic anaemia associated with oxoprolinuria, but without neurological manifestations. The family study revealed GSH-S activity in both parents to be around half the normal level, a finding consistent with the presumed autosomal recessive mode of inheritance of this enzymopathy. Two cases exhibited a well-compensated haemolytic syndrome without anaemia or splenomegaly at steady state. One of these cases was diagnosed after an episode of acute haemolytic anaemia after fava bean ingestion. The remaining patient suffered from moderate to severe chronic non-spherocytic haemolytic anaemia and splenomegaly, and required occasional blood transfusion for a haemolytic crisis associated with drug ingestion. In this patient, the anaemia was corrected by splenectomy. In addition to GSH-S, a panel of 16 other RBC enzyme activities was also studied in all the patients. Hexokinase, aldolase, glucose-6-phosphate dehydrogenase and pyruvate kinase activities all increased; these increases were to be expected, given the rise in the number of circulating reticulocytes. In two patients, the incubation of RBCs with hydrogen peroxide revealed an enhanced production of malonyldialdehyde. DNA analysis showed a homozygous state for 656 A-->G mutation in patients 2 and 3. The GSH-S gene of patient 1, studied elsewhere, revealed an 808 T-->C. The GSH-S gene of patient 4 was not available for study. The present study demonstrates that GSH-S deficiency is also present in Spain and further supports the molecular and clinical heterogeneity of this enzymopathy PMID:11167850

  16. Does platelet activity play a role in the pathogenesis of idiopathic ischemic priapism?

    PubMed Central

    Ufuk, Yavuz; Hasan, Yilmaz; Murat, Ustuner; Seyfettin, Ciftci; Kerem, Teke; Melih, Culha

    2016-01-01

    ABSTRACT Purpose Mean platelet volume (MPV) is used to measure platelet size and is defined as a potential marker of platelet reactivity. Higher MPV levels have been defined as a risk factor for increased incidence of intravascular thrombosis and its associated diseases. We aimed to determine whether a relationship exists between the MPV and veno-occlusive component of idiopathic ischemic priapism (IIP). Materials and methods Between 2010 and 2014, 38 subjects were analyzed in two groups. One was composed of 15 patients with diagnosis as IIP in our institute, and the other contained 23 healthy control subjects. Complete blood count reports were retrospectively evaluated in both groups. MPV, platelet count (PLT), platelet distribution width (PDW), white blood cells (WBC), red blood cells (RBC), hemoglobin (Hb), reticulocyte distribution width (RDW) were measured in both groups. : Results The mean ages were similar in IIP patients (45.86±15.82) and control subjects (47.65±10.99). The mean MPV values of IIP patients were significantly higher than control subjects (p<0.05). In contrast, also PLT counts were significantly lower in IIP patients, compared to control subjects (p<0.05). The mean hemoglobin and WBC values were significantly lower in control group (p<0.05). There was no significant difference of RBC, PDW and RDW values in both groups. Conclusions We found that the MPV was significantly higher in IIP patients compared to control subjects. The high MPV levels may have contributed to the veno-occlusive etiopathogenesis of IIP disease. We strongly suggest further prospective studies to recommend the use of MPV in routine practice. PMID:27136477

  17. Determinants of resting cerebral blood flow in sickle cell disease.

    PubMed

    Bush, Adam M; Borzage, Matthew T; Choi, Soyoung; Václavů, Lena; Tamrazi, Benita; Nederveen, Aart J; Coates, Thomas D; Wood, John C

    2016-09-01

    Stroke is common in children with sickle cell disease and results from an imbalance in oxygen supply and demand. Cerebral blood flow (CBF) is increased in patients with sickle cell disease to compensate for their anemia, but adequacy of their oxygen delivery has not been systematically demonstrated. This study examined the physiological determinants of CBF in 37 patients with sickle cell disease, 38 ethnicity matched control subjects and 16 patients with anemia of non-sickle origin. Cerebral blood flow was measured using phase contrast MRI of the carotid and vertebral arteries. CBF increased inversely to oxygen content (r(2)  = 0.69, P < 0.0001). Brain oxygen delivery, the product of CBF and oxygen content, was normal in all groups. Brain composition, specifically the relative amounts of grey and white matter, was the next strongest CBF predictor, presumably by influencing cerebral metabolic rate. Grey matter/white matter ratio and CBF declined monotonically until the age of 25 in all subjects, consistent with known maturational changes in brain composition. Further CBF reductions were observed with age in subjects older than 35 years of age, likely reflecting microvascular aging. On multivariate regression, CBF was independent of disease state, hemoglobin S, hemoglobin F, reticulocyte count and cell free hemoglobin, suggesting that it is regulated similarly in patients and control subjects. In conclusion, sickle cell disease patients had sufficient oxygen delivery at rest, but accomplish this only by marked increases in their resting CBF, potentially limiting their ability to further augment flow in response to stress. Am. J. Hematol. 91:912-917, 2016. © 2016 Wiley Periodicals, Inc. PMID:27263497

  18. Plasmodium falciparum aldolase and the C-terminal cytoplasmic domain of certain apical organellar proteins promote actin polymerization.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Grainger, Munira; Howell, Steven A; Green, Judith L; Holder, Anthony A

    2014-10-01

    The current model of Apicomplexan motility and host cell invasion is that both processes are driven by an actomyosin motor located beneath the plasma membrane, with the force transduced to the outside of the cell via coupling through aldolase and the cytoplasmic tail domains (CTDs) of certain type 1 membrane proteins. In Plasmodium falciparum (Pf), aldolase is thought to bind to the CTD of members of the thrombospondin-related anonymous protein (TRAP) family, which are micronemal proteins and represented by MTRAP in merozoites. Other type 1 membrane proteins including members of the erythrocyte binding antigen (EBA) and reticulocyte binding protein homologue (RH) protein families, which are also apical organellar proteins, have also been implicated in host cell binding in erythrocyte invasion. However, recent studies with Toxoplasma gondii have questioned the importance of aldolase in these processes. Using biolayer interferometry we show that Pf aldolase binds with high affinity to both rabbit and Pf actin, with a similar affinity for filamentous (F-) actin and globular (G-) actin. The interaction between Pf aldolase and merozoite actin was confirmed by co-sedimentation assays. Aldolase binding was shown to promote rabbit actin polymerization indicating that the interaction is more complicated than binding alone. The CTDs of some but not all type 1 membrane proteins also promoted actin polymerization in the absence of aldolase; MTRAP and RH1 CTDs promoted actin polymerization but EBA175 CTD did not. Direct actin polymerization mediated by membrane protein CTDs may contribute to actin recruitment, filament formation and stability during motor assembly, and actin-mediated movement, independent of aldolase. PMID:25261592

  19. A rapid and simple pipeline for synthesis of mRNA-ribosome-V(H)H complexes used in single-domain antibody ribosome display.

    PubMed

    Bencurova, Elena; Pulzova, Lucia; Flachbartova, Zuzana; Bhide, Mangesh

    2015-06-01

    The single-domain antibody (VHH) is a promising building block for a number of antibody-based applications. Ribosome display can successfully be used in the production of VHH. However, the construction of the expression cassette, confirmation of the translation and proper folding of the nascent chain, and the purification of the ribosome complexes, remain cumbersome tasks. Additionally, selection of the most suitable expression system can be challenging. We have designed primers that will amplify virtually all Camelidae VHH. With the help of a double-overlap extension (OE) polymerase chain reaction (PCR) we have fused VHH with the F1 fragment (T7 promoter and species-independent translation sequence) and the F2 fragment (mCherry, Myc-tag, tether, SecM arrest sequence and 3' stem loop) to generate a full-length DNA cassette. OE-PCR generated fragments were incubated directly with cell-free lysates (Leishmania torentolae, rabbit reticulocyte or E. coli) for the synthesis of mRNA-VHH-mCherry-ribosome complexes in vitro. Alternatively, the cassette was ligated in pQE-30 vector and transformed into E. coli to produce ribosome complexes in vivo. The results showed that the same expression cassette could be used to synthesize ribosome complexes with different expression systems. mCherry reporter served to confirm the synthesis and proper folding of the nascent chain, Myc-tag was useful in the rapid purification of ribosome complexes, and combination of the SecM sequence and 3' stem loop made the cassette universal, both for cells-free and E. coli in vivo. This rapid and universal pipeline can effectively be used in antibody ribosome display and VHH production. PMID:25902394

  20. Glycosylation is essential for translocation of carp retinol-binding protein across the endoplasmic reticulum membrane

    SciTech Connect

    Devirgiliis, Chiara; Gaetani, Sancia; Apreda, Marianna; Bellovino, Diana . E-mail: bellovino@inran.it

    2005-07-01

    Retinoid transport is well characterized in many vertebrates, while it is still largely unexplored in fish. To study the transport and utilization of vitamin A in these organisms, we have isolated from a carp liver cDNA library retinol-binding protein, its plasma carrier. The primary structure of carp retinol-binding protein is very conserved, but presents unique features compared to those of the correspondent proteins isolated and characterized so far in other species: it has an uncleavable signal peptide and two N-glycosylation sites in the NH{sub 2}-terminal region of the protein that are glycosylated in vivo. In this paper, we have investigated the function of the carbohydrate chains, by constructing three mutants deprived of the first, the second or both carbohydrates. The results of transient transfection of wild type and mutant retinol-binding protein in Cos cells followed by Western blotting and immunofluorescence analysis have shown that the absence of both carbohydrate moieties blocks secretion, while the presence of one carbohydrate group leads to an inefficient secretion. Experiments of carp RBP mRNA in vitro translation in a reticulocyte cell-free system in the presence of microsomes have demonstrated that N-glycosylation is necessary for efficient translocation across the endoplasmic reticulum membranes. Moreover, when Cos cells were transiently transfected with wild type and mutant retinol-binding protein (aa 1-67)-green fluorescent protein fusion constructs and semi-permeabilized with streptolysin O, immunofluorescence analysis with anti-green fluorescent protein antibody revealed that the double mutant is exposed to the cytosol, thus confirming the importance of glycan moieties in the translocation process.

  1. Evidence for the mode of action of the highly cytotoxic Streptomyces polyketide kendomycin.

    PubMed

    Elnakady, Yasser A; Rohde, Manfred; Sasse, Florenz; Backes, Christina; Keller, Andreas; Lenhof, Hans-Peter; Weissman, Kira J; Müller, Rolf

    2007-07-23

    The macrocyclic polyketide kendomycin exhibits antiosteoporotic and antibacterial activity, as well as strong cytotoxicity against multiple human tumor cell lines. Despite the promise of this compound in several therapeutic areas, the cellular target(s) of kendomycin have not been identified to date. We have used a number of approaches, including microscopy, proteomics, and bioinformatics, to investigate the mode of action of kendomycin in mammalian cell cultures. In response to kendomycin treatment, human U-937 tumor cells exhibit depolarization of the mitochondrial membrane, caspase 3 activation, and DNA laddering, consistent with induction of the intrinsic apoptotic pathway. To elucidate possible apoptotic triggers, DIGE and MALDI-TOF were used to identify proteins that are differently regulated in U-937 cells relative to controls. Statistical analysis of the proteomics data by the new web-based application GeneTrail highlighted several significant changes in protein expression, most notably among proteasomal regulatory subunits. Overall, the profile of altered expression closely matches that observed with other tumor cell lines in response to proteasome inhibition. Direct assay in vitro further shows that kendomycin inhibits the chymotrypsin-like activity of the rabbit reticulocyte proteasome, with comparable efficacy to the established inhibitor MG-132. We have also demonstrated that ubiquitinylated proteins accumulate in kendomycin-treated U-937 cells, while vacuolization of the endoplasmic reticulum and mitochondrial swelling are induced in a second cell line derived from kangaroo rat epithelial (PtK(2)) cells, phenotypes classically associated with inhibition of the proteasome. This study therefore provides evidence that kendomycin mediates its cytotoxic effects, at least in part, through proteasome inhibition. PMID:17592829

  2. Characterization of I-Ppo, an intron-encoded endonuclease that mediates homing of a group I intron in the ribosomal DNA of Physarum polycephalum.

    PubMed Central

    Muscarella, D E; Ellison, E L; Ruoff, B M; Vogt, V M

    1990-01-01

    A novel and only recently recognized class of enzymes is composed of the site-specific endonucleases encoded by some group I introns. We have characterized several aspects of I-Ppo, the endonuclease that mediates the mobility of intron 3 in the ribosomal DNA of Physarum polycephalum. This intron is unique among mobile group I introns in that it is located in nuclear DNA. We found that I-Ppo is encoded by an open reading frame in the 5' half of intron 3, upstream of the sequences required for self-splicing of group I introns. Either of two AUG initiation codons could start this reading frame, one near the beginning of the intron and the other in the upstream exon, leading to predicted polypeptides of 138 and 160 amino acid residues. The longer polypeptide was the major form translated in vitro in a reticulocyte extract. From nuclease assays of proteins synthesized in vitro with partially deleted DNAs, we conclude that both polypeptides possess endonuclease activity. We also have expressed I-Ppo in Escherichia coli, using a bacteriophage T7 RNA polymerase expression system. The longer polypeptide also was the predominant form made in this system. It showed enzymatic activity in bacteria in vivo, as demonstrated by the cleavage of a plasmid carrying the target site. Like several other intron-encoded endonucleases, I-Ppo makes a four-base staggered cut in its ribosomal DNA target sequence, very near the site where intron 3 becomes integrated in crosses of intron 3-containing and intron 3-lacking Physarum strains. Images PMID:2355911

  3. Molecular Design, Structural Analysis and Antifungal Activity of Derivatives of Peptide CGA-N46.

    PubMed

    Li, Rui-Fang; Lu, Zhi-Fang; Sun, Ya-Nan; Chen, Shi-Hua; Yi, Yan-Jie; Zhang, Hui-Ru; Yang, Shuo-Ye; Yu, Guang-Hai; Huang, Liang; Li, Chao-Nan

    2016-09-01

    Chromogranin A (CGA)-N46, a derived peptide of human chromogranin A, has antifungal activity. To further research the active domain of CGA-N46, a series of derivatives were designed by successively deleting amino acid from both terminus of CGA-N46, and the amino acid sequence of each derivative was analyzed by bioinformatic software. Based on the predicted physicochemical properties of the peptides, including half-life time in mammalian reticulocytes (in vitro), yeast (in vivo) and E. coli (in vivo), instability index, aliphatic index and grand average of hydropathicity (GRAVY), the secondary structure, net charge, the distribution of hydrophobic residues and hydrophilic residues, the final derivatives CGA-N15, CGA-N16, CGA-N12 and CGA-N8 were synthesized by solid-phase peptide synthesis. The results of bioinformatic analysis showed that CGA-N46 and its derivatives were α-helix, neutral or weak positive charge, hydrophilic, and CGA-N12 and CGA-N8 were more stable than the other derivatives. The results of circular dichroism confirmed that CGA-N46 and its derived peptides displayed α-helical structure in an aqueous solution and 30 mM sodium dodecylsulfate, but α-helical contents decreased in hydrophobic lipid vesicles. CGA-N15, CGA-N16, CGA-N12 and CGA-N8 had higher antifungal activities than their mother peptide CGA-N46. Among of the derived peptides, CGA-N12 showed the least hemolytic activity. In conclusion, we have successfully identified the active domain of CGA-N46 with strong antifungal activity and weak hemolytic activity, which provides the possibility to develop a new class of antibiotics. PMID:27165480

  4. Erythroid differentiation of human induced pluripotent stem cells is independent of donor cell type of origin

    PubMed Central

    Dorn, Isabel; Klich, Katharina; Arauzo-Bravo, Marcos J.; Radstaak, Martina; Santourlidis, Simeon; Ghanjati, Foued; Radke, Teja F.; Psathaki, Olympia E.; Hargus, Gunnar; Kramer, Jan; Einhaus, Martin; Kim, Jeong Beom; Kögler, Gesine; Wernet, Peter; Schöler, Hans R.; Schlenke, Peter; Zaehres, Holm

    2015-01-01

    Epigenetic memory in induced pluripotent stem cells, which is related to the somatic cell type of origin of the stem cells, might lead to variations in the differentiation capacities of the pluripotent stem cells. In this context, induced pluripotent stem cells from human CD34+ hematopoietic stem cells might be more suitable for hematopoietic differentiation than the commonly used fibroblast-derived induced pluripotent stem cells. To investigate the influence of an epigenetic memory on the ex vivo expansion of induced pluripotent stem cells into erythroid cells, we compared induced pluripotent stem cells from human neural stem cells and human cord blood-derived CD34+ hematopoietic stem cells and evaluated their potential for differentiation into hematopoietic progenitor and mature red blood cells. Although genome-wide DNA methylation profiling at all promoter regions demonstrates that the epigenetic memory of induced pluripotent stem cells is influenced by the somatic cell type of origin of the stem cells, we found a similar hematopoietic induction potential and erythroid differentiation pattern of induced pluripotent stem cells of different somatic cell origin. All human induced pluripotent stem cell lines showed terminal maturation into normoblasts and enucleated reticulocytes, producing predominantly fetal hemoglobin. Differences were only observed in the growth rate of erythroid cells, which was slightly higher in the induced pluripotent stem cells derived from CD34+ hematopoietic stem cells. More detailed methylation analysis of the hematopoietic and erythroid promoters identified similar CpG methylation levels in the induced pluripotent stem cell lines derived from CD34+ cells and those derived from neural stem cells, which confirms their comparable erythroid differentiation potential. PMID:25326431

  5. Ubiquitin-aldehyde: a general inhibitor of ubiquitin-recycling processes.

    PubMed Central

    Hershko, A; Rose, I A

    1987-01-01

    The generation and characterization of ubiquitin (Ub)-aldehyde, a potent inhibitor of Ub-C-terminal hydrolase, has previously been reported. We now examine the action of this compound on the Ub-mediated proteolytic pathway using the system derived from rabbit reticulocytes. Addition of Ub-aldehyde was found to strongly inhibit breakdown of added 125I-labeled lysozyme, but inhibition was overcome by increasing concentrations of Ub. The following evidence shows the effect of Ub-aldehyde on protein breakdown to be indirectly caused by its interference with the recycling of Ub, leading to exhaustion of the supply of free Ub: Ub-aldehyde markedly increased the accumulation of Ub-protein conjugates coincident with a much decreased rate of conjugate breakdown. release of Ub from isolated Ub-protein conjugates in the absence of ATP (and therefore not coupled to protein degradation) is markedly inhibited by Ub-aldehyde. On the other hand, the ATP-dependent degradation of the protein moiety of Ub conjugates, which is an integral part of the proteolytic process, is not inhibited by this agent. Direct measurement of levels of free Ub showed a rapid disappearance caused by the inhibitor. The Ub is found to be distributed in derivatives of a wide range of molecular weight classes. It thus seems that Ub-aldehyde, previously demonstrated to inhibit the hydrolysis of Ub conjugates of small molecules, also inhibits the activity of a series of enzymes that regenerate free Ub from adducts with proteins and intermediates in protein breakdown. Images PMID:3031653

  6. Effects of hydroxyurea treatment for patients with hemoglobin SC disease.

    PubMed

    Luchtman-Jones, Lori; Pressel, Sara; Hilliard, Lee; Brown, R Clark; Smith, Mary G; Thompson, Alexis A; Lee, Margaret T; Rothman, Jennifer; Rogers, Zora R; Owen, William; Imran, Hamayun; Thornburg, Courtney; Kwiatkowski, Janet L; Aygun, Banu; Nelson, Stephen; Roberts, Carla; Gauger, Cynthia; Piccone, Connie; Kalfa, Theodosia; Alvarez, Ofelia; Hassell, Kathryn; Davis, Barry R; Ware, Russell E

    2016-02-01

    Although hemoglobin SC (HbSC) disease is usually considered less severe than sickle cell anemia (SCA), which includes HbSS and HbS/β(0) -thalassemia genotypes, many patients with HbSC experience severe disease complications, including vaso-occlusive pain, acute chest syndrome, avascular necrosis, retinopathy, and poor quality of life. Fully 20 years after the clinical and laboratory efficacy of hydroxyurea was proven in adult SCA patients, the safety and utility of hydroxyurea treatment for HbSC patients remain unclear. Recent NHLBI evidence-based guidelines highlight this as a critical knowledge gap, noting HbSC accounts for ∼30% of sickle cell patients within the United States. To date, only 5 publications have reported short-term, incomplete, or conflicting laboratory and clinical outcomes of hydroxyurea treatment in a total of 71 adults and children with HbSC. We now report on a cohort of 133 adult and pediatric HbSC patients who received hydroxyurea, typically for recurrent vaso-occlusive pain. Hydroxyurea treatment was associated with a stable hemoglobin concentration; increased fetal hemoglobin (HbF) and mean corpuscular volume (MCV); and reduced white blood cell count (WBC), absolute neutrophil count (ANC), and absolute reticulocyte count (ARC). Reversible cytopenias occurred in 22% of patients, primarily neutropenia and thrombocytopenia. Painful events were reduced with hydroxyurea, more in patients >15 years old. These multicenter data support the safety and potentially salutary effects of hydroxyurea treatment for HbSC disease; however, a multicenter, placebo-controlled, Phase 3 clinical trial is needed to determine if hydroxyurea therapy has efficacy for patients with HbSC disease. PMID:26615793

  7. The influence of erythrocyte maturity on ion transport and membrane lipid composition in the rat.

    PubMed

    Vokurková, M; Rauchová, H; Dobešová, Z; Loukotová, J; Nováková, O; Kuneš, J; Zicha, J

    2016-01-01

    Significant relationships between ion transport and membrane lipid composition (cholesterol, total phospholipids and sphingomyelins) were found in erythrocytes of salt hypertensive Dahl rats. In these animals mean cellular hemoglobin content correlated negatively with Na(+)-K(+) pump activity and Na(+) leak but positively with Na(+)-K(+) cotransport activity. Immature erythrocytes exhibit lower mean cellular hemoglobin content (MCHC) than mature ones. The aim of the present study was to find a relationship between erythrocyte maturity, membrane lipid composition and ion transport activity in Wistar rats aged three months which were subjected to repeated hemorrhage (blood loss 2 ml/day for 6 days) to enrich circulating erythrocytes with immature forms. Immature and mature erythrocyte fractions in control and hemorrhaged rats were separated by repeated centrifugation. Hemorrhaged rats had increased number of reticulocytes but reduced hematocrit and MCHC compared to control rats. Immature erythrocytes of hemorrhaged rats differed from mature ones of control animals by elevated Na(+)-K(+) pump activity, reduced Na(+)-K(+) cotransport activity and increased Rb(+) leak. These ion transport changes in immature erythrocytes were accompanied by higher concentration of total phospholipids in their cell membranes. Membrane phospholipid content correlated positively with Na(+)-K(+) pump activity and cation leaks but negatively with Na(+)-K(+) cotransport activity. Moreover, they were also negatively related with MCHC which correlated negatively with Na(+)-K(+) pump activity and Rb(+) leak but positively with Na(+)-K(+) cotransport activity. Thus certain abnormalities of erythrocyte ion transport and membrane lipid composition detected in hypertensive animals might be caused by higher incidence of immature cells. PMID:26988297

  8. Arginase levels and their association with Th17-related cytokines, soluble adhesion molecules (sICAM-1 and sVCAM-1) and hemolysis markers among steady-state sickle cell anemia patients

    PubMed Central

    Vilas-Boas, Wendell; Cerqueira, Bruno A. V.; Zanette, Angela M. D.; Reis, Mitermayer G.; Barral-Netto, Manoel

    2010-01-01

    Sickle cell anemia (SCA) is characterized by a marked endothelial dysfunction, owing to many factors. Arginine metabolism can be related to the inflammatory chronic state presented by patients, playing a key role in their clinical outcome and vascular endothelium. We investigated the serum arginase levels in 50 SCA patients (22 men and 28 women, mean age of 17 ± 10.5 years) and 28 healthy controls. Serum arginase levels were associated with biochemical hemolysis markers and cytokines involved in Th17 response, as well as levels of soluble intercellular adhesion molecule-1 (sICAM-1) and vascular cell adhesion molecule-1 (sVCAM-1). Arginase concentrations were higher in SCA patients, compared with controls (p = 0.005), and were significantly and positively associated with total bilirubin (p = 0.004), indirect bilirubin (p = 0.04), and aspartate aminotransferase (AST; p = 0.039) in the SCA patient group. Moreover, arginase was significantly and positively associated with transforming growth factor-beta (TGF-beta; p = 0.008) among SCA patients. sICAM-1 was significantly and positively associated to reticulocytes (p = 0.014) and AST (p = 0.04). sVCAM-1 was likewise associated with lactate dehydrogenase (p = 0.03). These data suggest a new insight into arginase metabolism, as we show here a shift in arginine catabolism, where TGF-beta may induces the arginase pathway instead of the nitric oxide pathway and a possible involvement of the vascular activation and the serum arginase in chronic hemolysis among SCA patients. Additional studies should be carried out in order to investigate the mechanisms by which TGF-beta participates in the metabolism of arginase in SCA patients. PMID:20405289

  9. Enhanced eryptosis contributes to anemia in lung cancer patients

    PubMed Central

    Bissinger, Rosi; Schumacher, Carla; Qadri, Syed M.; Honisch, Sabina; Malik, Abaid; Götz, Friedrich; Kopp, Hans-Georg; Lang, Florian

    2016-01-01

    Objectives Anemia is a common complication of malignancy, which could result from either compromised erythropoiesis or decreased lifespan of circulating erythrocytes. Premature suicidal erythrocyte death, characterized by cell shrinkage and phosphatidylserine (PS) externalization, decreases erythrocyte lifespan and could thus cause anemia. Here, we explored whether accelerated eryptosis participates in the pathophysiology of anemia associated with lung cancer (LC) and its treatment. Methods Erythrocytes were drawn from healthy volunteers and LC patients with and without cytostatic treatment. PS exposure (annexin V-binding), cell volume (forward scatter), cytosolic Ca2+ (Fluo3 fluorescence), reactive oxygen species (ROS) production (DCFDA fluorescence) and ceramide formation (anti-ceramide antibody) were determined by flow cytometry. Results Hemoglobin concentration and hematocrit were significantly lower in LC patients as compared to healthy controls, even though reticulocyte number was higher in LC (3.0±0.6%) than in controls (1.4±0.2%). The percentage of PS-exposing erythrocytes was significantly higher in LC patients with (1.4±0.1%) and without (1.2±0.3%) cytostatic treatment as compared to healthy controls (0.6±0.1%). Erythrocyte ROS production and ceramide abundance, but not Fluo3 fluorescence, were significantly higher in freshly drawn erythrocytes from LC patients than in freshly drawn erythrocytes from healthy controls. PS exposure of erythrocytes drawn from healthy volunteers was significantly more pronounced following incubation in plasma from LC patients than following incubation in plasma from healthy controls. Conclusion Anemia in LC patients with and without cytostatic treatment is paralleled by increased eryptosis, which is triggered, at least in part, by increased oxidative stress and ceramide formation. PMID:26872376

  10. Plasmodium berghei and Plasmodium chabaudi: a neutral endopeptidase in parasite extracts and plasma of infected animals.

    PubMed

    Bernard, F; Mayer, R; Picard, I; Deguercy, A; Monsigny, M; Schrevel, J

    1987-08-01

    By using a sensitive fluorometric method with Val-Leu-Gly-Arg-3-amino-9-ethylcarbazole (VLGR-AEC) as a substrate, two endopeptidase activities were identified in two fractions of Sephacryl S-200 gel filtration from soluble P. berghei and P. chabaudi extracts. Controls with normal mouse erythrocytes, with leukocytes, and with reticulocyte enriched blood and different washing procedures during the preparation of soluble P. berghei extracts showed that the MW greater than 200 kDa fraction was a contaminant from erythrocytes and exhibited an optimal pH activity of 8.2. In contrast, the fraction 130 kDa was related to P. berghei and P. chabaudi and exhibited an optimal pH activity of 7.4. The two enzyme activities were compared with eight different substrates. The parasite endopeptidase showed a strong activity with Val-Leu-Gly-Lys-AEC (VLGK-AEC) and Ser-Gly-Lys-AEC (SGK-AEC) as substrates; in contrast, the mouse host endopeptidase poorly cleaved the VLGK-AEC and did not cleave SGK-AEC. Presence of the hydrophobic benzyl group on serine reduced the hydrolizing properties of P. berghei endopeptidase: the reverse was observed with host endopeptidase. The hydrolysis of the N-polyhydroxyalcanoyl-VLGK-AEC substrate by the parasite neutral endopeptidase strongly increased with the schizogonic stage, as shown with synchronized P. chabaudi in mice. By its physiological pH and specificity the release of this enzyme in mouse plasma during the infection could be of interest in a peptidyl-drug strategy. PMID:3301390

  11. Moderate malnutrition in rats induces somatic gene mutations.

    PubMed

    Pacheco-Martínez, M Monserrat; Cortés-Barberena, Edith; Cervantes-Ríos, Elsa; Del Carmen García-Rodríguez, María; Rodríguez-Cruz, Leonor; Ortiz-Muñiz, Rocío

    2016-07-01

    The relationship between malnutrition and genetic damage has been widely studied in human and animal models, leading to the observation that interactions between genotoxic exposure and micronutrient status appear to affect genomic stability. A new assay has been developed that uses the phosphatidylinositol glycan class A gene (Pig-a) as a reporter for measuring in vivo gene mutation. The Pig-a assay can be employed to evaluate mutant frequencies (MFs) in peripheral blood reticulocytes (RETs) and erythrocytes (RBCs) using flow cytometry. In the present study, we assessed the effects of malnutrition on mutagenic susceptibility by exposing undernourished (UN) and well-nourished (WN) rats to N-ethyl-N-nitrosourea (ENU) and measuring Pig-a MFs. Two week-old UN and WN male Han-Wistar rats were treated daily with 0, 20, or 40mg/kg ENU for 3 consecutive days. Blood was collected from the tail vein one day before ENU treatment (Day-1) and after ENU administration on Days 7, 14, 21, 28, 35, 42, 49, 56 and 63. Pig-a MFs were measured in RETs and RBCs as the RET(CD59-) and RBC(CD59-) frequencies. In the vehicle control groups, the frequencies of mutant RETs and RBCs were significantly higher in UN rats compared with WN rats at all sampling times. The ENU treatments increased RET and RBC MFs starting at Day 7. Although ENU-induced Pig-a MFs were consistently lower in UN rats than in WN rats, these differences were not significant. To understand these responses, further studies should use other mutagens and nucleated surrogate cells and examine the types of mutations induced in UN and WN rats. PMID:26994962

  12. Living high-training low: effect on erythropoiesis and maximal aerobic performance in elite Nordic skiers.

    PubMed

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

    2006-08-01

    The "living high-training low" model (Hi-Lo) may improve aerobic performance in athletes, and the main mechanism of this improvement is thought to be augmented erythropoiesis. A positive effect of Hi-Lo has been demonstrated previously by using altitudes of 2,000-3,000 m. Since the rate of erythropoiesis is altitude-dependent, we tested whether a higher altitude (3,500 m) during Hi-Lo increases erythropoiesis and maximal aerobic performance. Nordic skiers trained for 18 days at 1,200 m, while sleeping at 1,200 m in ambient air (control group, n = 5) or in hypoxic rooms (Hi-Lo, n = 6; 3 x 6 days at simulated altitudes of 2,500, 3,000 and finally 3,500 m, 11 h day(-1)). Measurements were done before, during (blood samples only) and 2 weeks after the intervention (POST). Maximal aerobic performance was examined from VO(2max) and time to exhaustion (T(exh)) at vVO(2max) (minimum speed associated with VO(2max)), respectively. Erythropoietin and soluble transferrin receptor responses were higher during Hi-Lo, whereas reticulocytes did not change. In POST (vs. before): hematological parameters were similar to basal levels, as well as red blood cell volume, being 2.68 +/- 0.83 l (vs. 2.64+/-0.54 l) in Hi-Lo and 2.62+/-0.57 l (vs. 2.87 +/- 0.59 l) in controls. At that time, neither VO(2max) nor T(exh) were improved by Hi-Lo, VO(2max) being non-significantly decreased by 2.0% (controls) and 3.7% (Hi-Lo). The present results suggest that increasing the altitude up to 3,500 m during Hi-Lo stimulates erythropoiesis but does not confer any advantage for maximal O2 transport. PMID:16786355

  13. Vitamin C Supplementation Does not Improve Hypoxia-Induced Erythropoiesis

    PubMed Central

    Sanchis-Gomar, Fabian; Martinez-Bello, Daniel; Olaso-Gonzalez, Gloria; Gomez-Cabrera, Mari Carmen; Viña, Jose

    2012-01-01

    Abstract Martinez-Bello,Vladimir E., Fabian Sanchis-Gomar, Daniel Martinez-Bello, Gloria Olaso-Gonzalez, Mari Carmen Gomez-Cabrera, and Jose Viña. Vitamin C Supplementation Does Not Improve Hypoxia-Induced Erythropoiesis. High Alt Med Biol 13:269–274, 2012.—Hypoxia induces reactive oxygen species production. Supplements with antioxidant mixtures can compensate for the decline in red cell membrane stability following intermittent hypobaric hypoxia by decreasing protein and lipid oxidation. We aimed to determine whether supplementation with vitamin C is implicated in the regulation of erythropoiesis and in the oxygen-carrying capacity of the blood, and also whether antioxidant supplementation prevents the oxidative stress associated to intermittent hypoxia. Twenty-four male Wistar rats were randomly divided into four experimental groups: normoxia control (n=6), normoxia + vitamin C (n=6), hypoxia control (12 h pO2 12%/12 h pO2 21%) (n=6), and hypoxia + vitamin C (n=6). Animals were supplemented with vitamin C at a dose of 250 mg·kg−1·day−1 for 21 days. Red blood cell count, hemoglobin, hematocrit, reticulocytes, erythropoietin, and oxidative stress parameters such as malondialdehyde and protein oxidation in plasma were analyzed at two different time points: basal sample (day zero) and final sample (day 21). Similar RBC, Hb, Hct, and Epo increments were observed in both hypoxic groups regardless of the vitamin C supplementation. There was no change on MDA levels after intermittent hypoxic exposure in any experimental group. However, we found an increase in plasma protein oxidation in both hypoxic groups. Vitamin C does not affect erythropoiesis and protein oxidation in rats submitted to intermittent hypoxic exposure. PMID:23270444

  14. Epoetin beta pegol, but not recombinant erythropoietin, retains its hematopoietic effect in vivo in the presence of the sialic acid-metabolizing enzyme sialidase.

    PubMed

    Aizawa, Ken; Kawasaki, Ryohei; Tashiro, Yoshihito; Hirata, Michinori; Endo, Koichi; Shimonaka, Yasushi

    2016-08-01

    Erythropoiesis-stimulating agents (ESAs) are widely used for treating chronic kidney disease (CKD)-associated anemia. The biological activity of ESAs is mainly regulated by the number of sialic acid-containing carbohydrates on the erythropoietin (EPO) peptide. Sialidase, a sialic acid-metabolizing enzyme that accumulates in CKD patients, is suspected of contributing to shortening the circulation half-life of ESAs. Epoetin beta pegol (continuous erythropoietin receptor activator; C.E.R.A.), is an EPO integrated with methoxypolyethylene glycol (PEG). It has been suggested that C.E.R.A. may exert a favorable therapeutic effect, even under conditions of elevated sialidase; however, no detailed investigation of the pharmacological profile of C.E.R.A. in the presence of sialidase has been reported. In the present study, we injected C.E.R.A. or EPO pre-incubated with sialidase into rats, and assessed the hematopoietic effect by reticulocyte count. The hematopoietic effect of C.E.R.A., but not EPO, was preserved after sialidase treatment, despite the removal of sialic acid. Proliferation of EPO-dependent leukemia cells (AS-E2) was significantly increased by desialylated C.E.R.A. and EPO compared to non-treated C.E.R.A. or EPO. In conclusion, we show that C.E.R.A. exerts a favorable hematopoietic effect even under conditions of elevated sialidase. Our findings may contribute to a better understanding of CKD and more effective therapeutic approaches based on a patient's profile of anemia. PMID:27084258

  15. The Binding of Plasmodium falciparum Adhesins and Erythrocyte Invasion Proteins to Aldolase Is Enhanced by Phosphorylation.

    PubMed

    Diaz, Suraya A; Martin, Stephen R; Howell, Steven A; Grainger, Munira; Moon, Robert W; Green, Judith L; Holder, Anthony A

    2016-01-01

    Aldolase has been implicated as a protein coupling the actomyosin motor and cell surface adhesins involved in motility and host cell invasion in the human malaria parasite Plasmodium falciparum. It binds to the cytoplasmic domain (CTD) of type 1 membrane proteins of the thrombospondin-related anonymous protein (TRAP) family. Other type 1 membrane proteins located in the apical organelles of merozoites, the form of the parasite that invades red blood cells, including apical membrane antigen 1 (AMA1) and members of the erythrocyte binding ligand (EBL) and reticulocyte binding homologue (RH) protein families have been implicated in host cell binding and invasion. Using a direct binding method we confirm that TRAP and merozoite TRAP (MTRAP) bind aldolase and show that the interaction is mediated by more than just the C-terminal six amino acid residues identified previously. Single amino acid substitutions in the MTRAP CTD abolished binding to aldolase. The CTDs of AMA1 and members of the EBL and RH protein families also bound to aldolase. MTRAP competed with AMA1 and RH4 for binding to aldolase, indicating overlapping binding sites. MTRAP CTD was phosphorylated in vitro by both calcium dependent kinase 1 (CDPK1) and protein kinase A, and this modification increased the affinity of binding to aldolase by ten-fold. Phosphorylation of the CTD of members of the EBL and RH protein families also increased their affinity for aldolase in some cases. To examine whether or not MTRAP expressed in asexual blood stage parasites is phosphorylated, it was tagged with GFP, purified and analysed, however no phosphorylation was detected. We propose that CTD binding to aldolase may be dynamically modulated by phosphorylation, and there may be competition for aldolase binding between different CTDs. The use and efficiency of alternate invasion pathways may be determined by the affinity of adhesins and cell invasion proteins for aldolase, in addition to their host ligand specificity. PMID

  16. Initial performance evaluation of the UniCel® DxH 800 Coulter® cellular analysis system

    PubMed Central

    HEDLEY, B D; KEENEY, M; CHIN-YEE, I; BROWN, W

    2011-01-01

    The Beckman Coulter UniCel® DxH 800 is a hematology analyzer incorporating new electronic and mechanical design with advanced algorithm technology to perform CBC, white blood cell (WBC) differential, nucleated red blood cell (NRBC), and reticulocyte analysis. Evaluation of this instrument was performed in our 800-bed tertiary care hospital and specifically centered upon the correlation of WBC, NRBC, and platelet (PLT) enumeration when compared to a predicate analyzer, the Coulter® LH 780, and flow cytometry (FCM) reference methods. Of particular interest were those samples with morphologically confirmed interference and extreme leukocytosis (evaluated with respect to red blood cell parameter correction). The sample set (n = 272) consisted of morphologically normal and hematologically abnormal patients. Correlation of the WBC, PLT, and NRBC showed r2 values of 0.994, 0.985, and 0.910 for the DxH 800 vs. FCM, respectively. The presence of interfering particles did not affect the accuracy of the DxH 800 with respect to WBC counts. The DxH 800 showed accurate PLT and NRBC counts in the clinically significant low range when compared to FCM. Compared to the LH 780, flagging rates were significantly reduced (NRBC flag), or equivalent (WBC, PLT flag) on the DxH 800. The DxH 800 demonstrated higher sensitivity and specificity for PLTs and NRBCs and achieved a lower NRBC false negative rate compared to the LH 780. The UniCel® DxH 800 represents a significant improvement to previous impedance analyzers in accurately detecting the presence of NRBCs at counts >1/100 WBC. Furthermore, it provides accurate PLT and WBC counts in the presence of interference and improved NRBC flagging efficiency when compared to the LH 780. Correction of red blood cell parameters is appropriate and accurate in cases of extreme leukocytosis. PMID:20491996

  17. Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter.

    PubMed Central

    Horisberger, M A; McMaster, G K; Zeller, H; Wathelet, M G; Dellis, J; Content, J

    1990-01-01

    The human protein p78 is induced and accumulated in cells treated with type I interferon or with some viruses. It is the human homolog of the mouse Mx protein involved in resistance to influenza virus. A full-length cDNA clone encoding the human p78 protein was cloned and sequenced. It contained an open reading frame of 662 amino acids, corresponding to a polypeptide with a predicted molecular weight of 75,500, in good agreement with the Mr of 78,000 determined on sodium dodecyl sulfate gels for the purified natural p78 protein. The cloned gene was expressed in vitro and corresponded in size, pI, antigenic determinant(s), and NH2 terminus sequence to the natural p78 protein. A second cDNA was cloned which encoded a 633-amino-acid protein sharing 63% homology with human p78. This p78-related protein was translated in reticulocyte lysates where it shared an antigenic determinant(s) with p78. A putative 5' regulatory region of 83 base pairs contained within the gene promoter region upstream of the presumed p78 mRNA cap site conferred human alpha interferon (IFN-alpha) inducibility to the cat reporter gene. The p78 protein accumulated to high levels in cells treated with IFN-alpha. In contrast, the p78-related protein was not expressed at detectable levels. The rate of decay of p78 levels in diploid cells after a 24-h treatment with IFN-alpha was much slower than the rate of decay of the antiviral state against influenza A virus and vesicular stomatitis virus, suggesting that the p78 protein is probably not involved in an antiviral mechanism. Furthermore, we showed that these proteins, as well as the homologous mouse Mx protein, possess three consensus elements in proper spacing, characteristic of GTP-binding proteins. Images PMID:2154602

  18. Measuring DNA Damage and Repair in Mouse Splenocytes After Chronic In Vivo Exposure to Very Low Doses of Beta- and Gamma-Radiation.

    PubMed

    Flegal, Matthew; Blimkie, Melinda S; Wyatt, Heather; Bugden, Michelle; Surette, Joel; Klokov, Dmitry

    2015-01-01

    Low dose radiation exposure may produce a variety of biological effects that are different in quantity and quality from the effects produced by high radiation doses. Addressing questions related to environmental, occupational and public health safety in a proper and scientifically justified manner heavily relies on the ability to accurately measure the biological effects of low dose pollutants, such as ionizing radiation and chemical substances. DNA damage and repair are the most important early indicators of health risks due to their potential long term consequences, such as cancer. Here we describe a protocol to study the effect of chronic in vivo exposure to low doses of γ- and β-radiation on DNA damage and repair in mouse spleen cells. Using a commonly accepted marker of DNA double-strand breaks, phosphorylated histone H2AX called γH2AX, we demonstrate how it can be used to evaluate not only the levels of DNA damage, but also changes in the DNA repair capacity potentially produced by low dose in vivo exposures. Flow cytometry allows fast, accurate and reliable measurement of immunofluorescently labeled γH2AX in a large number of samples. DNA double-strand break repair can be evaluated by exposing extracted splenocytes to a challenging dose of 2 Gy to produce a sufficient number of DNA breaks to trigger repair and by measuring the induced (1 hr post-irradiation) and residual DNA damage (24 hrs post-irradiation). Residual DNA damage would be indicative of incomplete repair and the risk of long-term genomic instability and cancer. Combined with other assays and end-points that can easily be measured in such in vivo studies (e.g., chromosomal aberrations, micronuclei frequencies in bone marrow reticulocytes, gene expression, etc.), this approach allows an accurate and contextual evaluation of the biological effects of low level stressors. PMID:26168333

  19. Genetic determinants of haemolysis in sickle cell anaemia

    PubMed Central

    Milton, Jacqueline N.; Rooks, Helen; Drasar, Emma; McCabe, Elizabeth L.; Baldwin, Clinton T.; Melista, Efi; Gordeuk, Victor R.; Nouraie, Mehdi; Kato, Gregory R.; Minniti, Caterina; Taylor, James; Campbell, Andrew; Luchtman-Jones, Lori; Rana, Sohail; Castro, Oswaldo; Zhang, Yingze; Thein, Swee Lay; Sebastiani, Paola; Gladwin, Mark T.; Steinberg, Martin H.

    2014-01-01

    Summary Haemolytic anaemia is variable among patients with sickle cell anaemia and can be estimated by reticulocyte count, lactate dehydrogenase, aspartate aminotransferase and bilirubin levels. Using principal component analysis of these measurements we computed a haemolytic score that we used as a subphenotype in a genome-wide association study. We identified in one cohort and replicated in two additional cohorts the association of a single nucleotide polymorphism in NPRL3 (rs7203560; chr16p13·3) (P = 6·04 × 10−07). This association was validated by targeted genotyping in a fourth independent cohort. The HBA1/HBA2 regulatory elements, hypersensitive sites (HS)-33, HS-40 and HS-48 are located in introns of NPRL3. Rs7203560 was in perfect linkage disequilibrium (LD) with rs9926112 (r2 = 1) and in strong LD with rs7197554 (r2 = 0·75) and rs13336641 (r2 = 0·77); the latter is located between HS-33 and HS-40 sites and next to a CTCF binding site. The minor allele for rs7203560 was associated with the −∝3·7thalassaemia gene deletion. When adjusting for HbF and ∝ thalassaemia, the association of NPRL3 with the haemolytic score was significant (P = 0·00375) and remained significant when examining only cases without gene deletion∝ thalassaemia (P = 0·02463). Perhaps by independently down-regulating expression of the HBA1/HBA2 genes, variants of the HBA1/HBA2 gene regulatory loci, tagged by rs7203560, reduce haemolysis in sickle cell anaemia. PMID:23406172

  20. Structure of glycosylated and unglycosylated gag and gag-pol precursor proteins of Moloney murine leukemia virus.

    PubMed Central

    Saris, C J; van Eenbergen, J; Liskamp, R M; Bloemers, H P

    1983-01-01

    Precursor polyproteins containing translational products of the gag gene of Moloney murine leukemia virus were purified by gel electrophoresis and cleaved into large fragments by hydroxylamine, mild acid hydrolysis, or cyanogen bromide. The hydroxylamine cleavage method (specific for asparagine-glycine bonds) was adapted especially for this study. The electrophoretic mobility and antigenicity of the fragments and, in some cases, the presence or absence of [35S]methionine revealed detailed information on the structure of Pr65gag, gPr80gag, and Pr75gag (the unglycosylated variant of gPr80gag formed in vivo in the presence of tunicamycin or in vitro in a reticulocyte cell-free system). When compared with Pr65gag, gPr80gag contains 7,000 daltons of additional amino acids, presumably as, or as part of, a leader sequence at or very close to its N terminus. We present evidence that this leader may have replaced part of the p15 sequence. Furthermore, gPr80gag contains three separate carbohydrate groups. One is attached to the presumed leader sequence or to the p15 domain, and two are attached to the p30 domain. Each of the Moloney murine leukemia virus gag precursor proteins Pr65gag, gPr80gag, and Pr75gag corresponds with a read-through product into the pol gene. We designated these products Pr180gag-pol, gPr200gag-pol, and Pr190gag-pol (the unglycosylated variant of gPr200gag-pol), respectively. gPr200gag-pol contains all of the extra amino acids and carbohydrate groups present in gPr80gag and at least one carbohydrate group in its pol sequences. Images PMID:6602220