scholarly journals A murine model for the switch from fetal to adult hemoglobin during ontogeny

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1100-1105 ◽  
Author(s):  
BP Alter ◽  
SC Goff
Keyword(s):  
Murine Model ◽  
Fetal Hemoglobin ◽  
Globin Chains ◽  
Adult Mice ◽  
Adult Hemoglobin ◽  
Globin Synthesis ◽  
Murine Erythroleukemia ◽  
Triton X ◽  
Total Beta

In murine erythroleukemia cells, the minor/major hemoglobin (Hb) ratio depends on the cell line and the inducing agent. To determine whether mouse minor hemoglobin is a “fetal” hemoglobin in vivo, globin chain composition and synthesis rates were determined in DBA/2 mice of various ages ranging from 14-day embryos to > 6-mo adults. Globin chains were separated by electrophoresis on polyacrylamide gels containing urea and Triton X-100. This method separates the embryonic (x,y,z) and the adult (alpha, beta ma, beta mi) globin chains. Fourteen day embryos had only 5%-10% adult globins but approximately 30% of the adult beta chains were beta mi. The % beta mi decreased with age and reached 20% in adult mice. Biosynthetic studies led to more pronounced differences: beta mi synthesis was 45% of total beta chain production in 14-day embryos and declined to 22% in adults. Thus beta minor/total beta globin synthesis declines during mouse ontogeny. This resembles qualitatively the human switch from fetal to adult hemoglobin and provides a murine model for studies of hemoglobin regulation.

scholarly journals A murine model for the switch from fetal to adult hemoglobin during ontogeny

Blood ◽  
1980 ◽  
Vol 56 (6) ◽  
pp. 1100-1105 ◽  
Author(s):  
BP Alter ◽  
SC Goff
Keyword(s):  
Murine Model ◽  
Fetal Hemoglobin ◽  
Globin Chains ◽  
Adult Mice ◽  
Adult Hemoglobin ◽  
Globin Synthesis ◽  
Murine Erythroleukemia ◽  
Triton X ◽  
Total Beta

Abstract In murine erythroleukemia cells, the minor/major hemoglobin (Hb) ratio depends on the cell line and the inducing agent. To determine whether mouse minor hemoglobin is a “fetal” hemoglobin in vivo, globin chain composition and synthesis rates were determined in DBA/2 mice of various ages ranging from 14-day embryos to > 6-mo adults. Globin chains were separated by electrophoresis on polyacrylamide gels containing urea and Triton X-100. This method separates the embryonic (x,y,z) and the adult (alpha, beta ma, beta mi) globin chains. Fourteen day embryos had only 5%-10% adult globins but approximately 30% of the adult beta chains were beta mi. The % beta mi decreased with age and reached 20% in adult mice. Biosynthetic studies led to more pronounced differences: beta mi synthesis was 45% of total beta chain production in 14-day embryos and declined to 22% in adults. Thus beta minor/total beta globin synthesis declines during mouse ontogeny. This resembles qualitatively the human switch from fetal to adult hemoglobin and provides a murine model for studies of hemoglobin regulation.

scholarly journals Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1278-1284 ◽  
Author(s):  
RS Weinberg ◽  
SE Antonarakis ◽  
HH Jr Kazazian ◽  
GJ Dover ◽  
SH Orkin ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Globin Chains ◽  
Hereditary Persistence ◽  
Enzyme Pattern ◽  
Globin Synthesis ◽  
The Individual ◽  
Globin Gene Expression

To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

scholarly journals Fetal hemoglobin synthesis in erythroid cultures in hereditary persistence of fetal hemoglobin and beta o-thalassemia

Blood ◽  
1984 ◽  
Vol 63 (6) ◽  
pp. 1278-1284 ◽  
Author(s):  
RS Weinberg ◽  
SE Antonarakis ◽  
HH Jr Kazazian ◽  
GJ Dover ◽  
SH Orkin ◽  
...  
Keyword(s):  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Globin ◽  
Globin Chains ◽  
Hereditary Persistence ◽  
Enzyme Pattern ◽  
Globin Synthesis ◽  
The Individual ◽  
Globin Gene Expression

Abstract To determine whether hemoglobin regulation is normal in diseases affecting beta-globin gene expression, globin synthesis was examined in members of a family of a patient with hereditary persistence of fetal hemoglobin/beta o-thalassemia (HPFH/beta o-thal). The HPFH defect is the Ghanian type II, with a deletion from psi beta 1 to at least 20 kb 3′ to beta. The beta o-thal gene has the haplotype II restriction enzyme pattern and has the beta 39 nonsense mutation. Erythroid colonies from blood BFU-E were radiolabeled, and globin chains were separated by gel electrophoresis. Colonies from the beta o-thal heterozygote had non-alpha/alpha ratios more balanced than in the reticulocytes. Gamma synthesis was 11% of non-alpha, which is higher than in reticulocytes, but within the range seen in normal adult colonies. Both HPFH heterozygotes produced 20%-30% gamma in erythroid colonies as well as reticulocytes, although non-alpha/alpha was more balanced in the colonies. The HPFH/beta o-thal patient produced 100% gamma in reticulocytes and in colonies. G gamma and gamma-synthetic proportions were not correlated at the individual colony level in the heterozygotes, suggesting that they had “adult” and not “fetal” progenitor cells. The Hb expression of these adult progenitors is presumably modulated normally in vivo in beta o-thal, but the normal decrease in HbF production does not occur in gene deletion HPFH.

scholarly journals Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3198-3204 ◽  
Author(s):  
G Stamatoyannopoulos ◽  
CA Blau ◽  
B Nakamoto ◽  
B Josephson ◽  
Q Li ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Sickle Cell Disease ◽  
Umbilical Cord Blood ◽  
Sodium Acetate ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Globin Synthesis

Abstract Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

Effective erythropoiesis and HbF reactivation induced by kit ligand in β-thalassemia

Blood ◽  
2008 ◽  
Vol 111 (1) ◽  
pp. 421-429 ◽  
Author(s):  
Marco Gabbianelli ◽  
Ornella Morsilli ◽  
Adriana Massa ◽  
Luca Pasquini ◽  
Paolo Cianciulli ◽  
...  
Keyword(s):  
Fetal Hemoglobin ◽  
Thalassemia Major ◽  
Globin Genes ◽  
Erythroid Progenitors ◽  
Ineffective Erythropoiesis ◽  
Kit Ligand ◽  
Globin Chains ◽  
In Vitro Response ◽  
Globin Synthesis

In human β-thalassemia, the imbalance between α- and non–α-globin chains causes ineffective erythropoiesis, hemolysis, and anemia: this condition is effectively treated by an enhanced level of fetal hemoglobin (HbF). In spite of extensive studies on pharmacologic induction of HbF synthesis, clinical trials based on HbF reactivation in β-thalassemia produced inconsistent results. Here, we investigated the in vitro response of β-thalassemic erythroid progenitors to kit ligand (KL) in terms of HbF reactivation, stimulation of effective erythropoiesis, and inhibition of apoptosis. In unilineage erythroid cultures of 20 patients with intermedia or major β-thalassemia, addition of KL, alone or combined with dexamethasone (Dex), remarkably stimulated cell proliferation (3-4 logs more than control cultures), while decreasing the percentage of apoptotic and dyserythropoietic cells (<5%). More important, in both thalassemic groups, addition of KL or KL plus Dex induced a marked increase of γ-globin synthesis, thus reaching HbF levels 3-fold higher than in con-trol cultures (eg, from 27% to 75% or 81%, respectively, in β-thalassemia major). These studies indicate that in β-thalassemia, KL, alone or combined with Dex, induces an expansion of effective erythropoiesis and the reactivation of γ-globin genes up to fetal levels and may hence be considered as a potential therapeutic agent for this disease.

Improvement of erythropoiesis in β-thalassemic mice by continuous erythropoietin delivery from muscle

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2793-2798 ◽  
Author(s):  
Delphine Bohl ◽  
Assumpció Bosch ◽  
Ana Cardona ◽  
Anna Salvetti ◽  
Jean Michel Heard
Keyword(s):  
Erythrocyte Membranes ◽  
Erythroid Cell ◽  
Suitable Model ◽  
Mouse Muscle ◽  
Membrane Function ◽  
Globin Chains ◽  
Long Term Treatment ◽  
Globin Synthesis

β-Thalassemias are highly prevalent genetic disorders that can cause severe hemolytic anemia. The main pathophysiologic feature of β-thalassemia is the accumulation of unpaired -globin chains in erythrocyte precursors and red blood cells (RBCs). This accumulation alters cell membrane function and results in early cell destruction and ineffective erythropoiesis. Correction of globin chain imbalance through the induction of fetal hemoglobin (HbF) synthesis is a tentative therapeutic approach for this class of diseases. In short-term in vitro or in vivo assays, recombinant human erythropoietin increases the frequency of erythroid precursors programmed to HbF in humans and to β-minor globin in mice. In contrast, long-term treatment of β-thalassemic patients did not induce HbF significantly. We took advantage of highly efficient adeno-associated virus–mediated (AAV-mediated) gene transfer into mouse muscle to induce a robust and sustained secretion of mouse erythropoietin in β-thalassemic mice, which represent a suitable model for human β-thalassemia intermedia. A 1-year follow-up of 12 treated animals showed a stable correction of anemia associated with improved RBC morphology, increased β-minor globin synthesis, and decreased amounts of -globin chains bound to erythrocyte membranes. More effective erythropoiesis probably accounted for a reduction of erythroid cell proliferation, as shown by decreased proportions of circulating reticulocytes and by reduced iron 59 (59Fe) incorporation into erythroid tissues. This study indicates that the continuous delivery of high amounts of autologous erythropoietin induced a sustained stimulation of β-minor globin synthesis and a stable improvement of erythropoiesis in the β-thalassemic mouse model.

Extracellular fetal hemoglobin induces increases in glomerular permeability: inhibition with α1-microglobulin and tempol

2014 ◽  
Vol 306 (4) ◽  
pp. F442-F448 ◽  
Author(s):  
Kristinn Sverrisson ◽  
Josefin Axelsson ◽  
Anna Rippe ◽  
Magnus Gram ◽  
Bo Åkerström ◽  
...  
Keyword(s):  
High Performance ◽  
Fetal Hemoglobin ◽  
Size Exclusion ◽  
Radical Scavenger ◽  
Glomerular Permeability ◽  
Exclusion Chromatography ◽  
Ros Scavenger ◽  
Adult Hemoglobin ◽  
Heme Binding Protein

Extracellular fetal hemoglobin (HbF) and adult hemoglobin (HbA) are proinflammatory and generate ROS. Increased plasma levels of extracellular HbF have recently been reported to occur in early preeclampsia. α1-Microglobulin (A1M) is a physiological heme-binding protein and radical scavenger that has been shown to counteract vascular permeability increases induced by HbA in the perfused placenta. The present study was performed to investigate whether HbF and HbA will increase glomerular permeability in vivo and to test whether A1M and tempol, a ROS scavenger, can prevent their effects. Anesthetized Wistar rats were continuously infused intravenously with either HbA, HbF, or cyano-inactivated HbF together with FITC-Ficoll-70/400, inulin, and 51Cr-labeled EDTA for 2 h. Plasma samples and urine samples (left ureter) were taken repeatedly and analyzed by high-performance size exclusion chromatography to assess glomerular sieving coefficients for Ficoll of radius 10–80 Å. In separate experiments, A1M or tempol was given before and during Hb infusions. Extracellular HbF caused rapid, transient increases in glomerular permeability to large Ficoll molecules (50–80Å), contrary to the effects of HbA and cyano-inactivated HbF. For HbF, glomerular sieving coefficients for Ficoll of radius 60Å increased from 3.85 ± 0.85 × 10−5 to 2.60 ± 0.96 × 10−4 at 15 min, changes that were abrogated by tempol and reduced by A1M. In conclusion, our data demonstrate that extracellular HbF, infused systemically, can acutely increase glomerular permeability through inducing oxidative stress.

scholarly journals The presence of N-terminal methionine on nascent protein of rat liver and rabbit reticulocytes and its cleavage during polypeptide-chain elongation

1973 ◽  
Vol 134 (4) ◽  
pp. 969-983 ◽  
Author(s):  
Anna Koffer-Gutmann ◽  
Henry R. V. Arnstein
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Chain Elongation ◽  
Amino Acid Residues ◽  
Free System ◽  
Methionine Residue ◽  
Globin Chains ◽  
N Terminus ◽  
Globin Synthesis

1. The size of nascent globin peptides from which the N-terminal methionine residue is cleaved has been investigated by comparing the proportion of N-terminal methionine and valine in short and long chains. Nascent chains were labelled in rabbit reticulocyte lysates, fractionated according to length by chromatography on Sephadex G-50, and analysed by the Edman degradation of selected pooled fractions. It was found that different peptide fractions contained either methionine or valine, but not both, as the N-terminal residue. Methionine was present at the N-terminus of globin chains containing up to approx. 50 amino acids whereas valine was found to be the N-terminal amino acid of longer peptides. 2. In similar experiments with nascent proteins of rat liver, labelled either in vivo or in a cell-free system containing microsomal material and cell sap, evidence was obtained for the presence of methionine at the N-terminus of nascent chains up to approx. 65 amino acid residues long. Thus protein synthesis in liver appears to be initiated also by methionine, but in this case cleavage takes place somewhat later during peptide elongation than in globin synthesis.

scholarly journals Fetal hemoglobin induction by acetate, a product of butyrate catabolism [see comments]

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 3198-3204 ◽  
Author(s):  
G Stamatoyannopoulos ◽  
CA Blau ◽  
B Nakamoto ◽  
B Josephson ◽  
Q Li ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Sickle Cell Disease ◽  
Umbilical Cord Blood ◽  
Sodium Acetate ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Gamma Globin ◽  
Hbf Induction ◽  
Globin Synthesis

Butyrate induces fetal hemoglobin (HbF) synthesis in cultures of erythroid progenitors, in primates, and in man. The mechanism by which this compound stimulates gamma-globin synthesis is unknown. In the course of butyrate catabolism, beta oxidation by mitochondrial enzymes results in the formation of two acetate molecules from each molecule of butyrate. Studies were performed to determine whether acetate itself induces HbF synthesis. In erythroid burst-forming unit (BFU-E) cultures from normal persons, and individuals with sickle cell disease and umbilical-cord blood, dose-dependent increases in gamma-globin protein and gamma mRNA were consistently observed in response to increasing acetate concentrations. In BFU-E cultures from normal adults and patients with sickle cell disease, the ratio of gamma/gamma + beta mRNA increased twofold to fivefold in response to acetate, whereas the percentage of BFU-E progeny staining with an anti-gamma monoclonal antibody (MoAb) increased approximately twofold. Acetate-induced increases in gamma-gene expression were also noted in the progeny of umbilical cord blood BFU-E, although the magnitude of change in response to acetate was less because of a higher baseline of gamma- chain production. The effect of acetate on HbF induction in vivo was evaluated using transgenic mouse and primate models. A transgenic mouse bearing a 2.5-kb mu locus control region (mu LCR) cassette linked to a 3.3-kb A gamma gene displayed a near twofold increase in gamma mRNA during a 10-day infusion of sodium acetate at a dose of 1.5 g/kg/d. Sodium acetate administration in baboons, in doses ranging from 1.5 to 6 g/kg/d by continuous intravenous infusion, also resulted in the stimulation of gamma-globin synthesis, with the percentage of HbF- containing reticulocytes (F reticulocytes) approaching 30%. Surprisingly, a dose-response effect of acetate on HbF induction was not observed in the baboons, and HbF induction was not sustained with prolonged acetate administration. These results suggest that both two- carbon fatty acids (acetate) and four-carbon fatty acids (butyrate) stimulate synthesis of HbF in vivo.

scholarly journals Monoclonal antibodies specific for globin chains

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 530-539 ◽  
Author(s):  
G Stamatoyannopoulos ◽  
M Farquhar ◽  
D Lindsley ◽  
M Brice ◽  
T Papayannopoulou ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Fetal Hemoglobin ◽  
Beta Subunits ◽  
Gamma Chain ◽  
Globin Chains ◽  
Gamma Subunits ◽  
Species Specific ◽  
Specific Reactions

Abstract Six monoclonal antibodies specific for human globin chains are described. They are produced by stable clones obtained by raising hybridomas using cells of mice immunized with either adult or fetal hemoglobin. Characterization of the antibodies included testing against tetrameric human and other animal hemoglobins, isolated hemoglobin chains, and when indicated, cyanogen bromide fragments. Monoclonals 16- 2 and 37–8 are beta-chain specific. Antibody 31–2 recognizes an antigenic determinant common to the alpha and beta subunits. Monoclonal 30–3 recognizes determinants best expressed in the alpha 2 beta 2 tetramer. Antibody 45–1 recognizes a determinant common to beta and gamma subunits, while antibody 51–7 is gamma-chain specific. None of the monoclonal antibodies recognizes mouse hemoglobin, and they display significant differences in binding to hemoglobins of various species. The species-specific reactions and the knowledge of the primary structures of globins allowed deductions about the antigenic sites recognized by two of the monoclonals (16–2 and 45–1). These antihemoglobin monoclonal antibodies will provide useful probes for studying hemoglobin expression in vivo and in vitro.

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