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Query: UMLS:C0039730 (thalassemia)
 10,305 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Hemoglobin synthesis was studied in culture of early erythroid precursors (BFU-E) from the blood of nine patients exhibiting sickle cell anemia and of 14 with various types of beta-thalassemia. The results concerning gamma gene expression and plating efficiency in heterozygotes for sickle cell anemia were similar to those of normal adults (gamma/alpha = 0.05; 65 BFU-E colonies/10(6) plated cells) while, in contrast, homozygotes for sickle cell disease exhibited average values higher than the controls (gamma/alpha = 0.18; 80 BFU-E colonies/10(6) plated cells). However, the results were very heterogeneous from one subject to another. In heterozygotes for beta-thalassemia, gamma gene expression and plating efficiency were both slightly higher than those for normal individuals (gamma/alpha = 0.095; 129 BFU-E colonies/10(6) plated cells). In patients homozygous for beta-thalassemia, a marked increase in plating efficiency and gamma-chain synthesis was constantly observed (gamma/alpha = 0.41; 221 BFU-E colonies/10(6) plated cells). The high proportion of gamma chain synthesis was not related to a positive selection of F cells, since the gamma/alpha ratio remained constant during the in vitro erythroid maturation. Furthermore, a major increase in free alpha chain proteolysis can be ruled out, since the beta/alpha ratio was of the same order of magnitude in culture and in freshly drawn cells. Thus, the increased Hb F synthesis in vitro was the consequence of a true stimulation of gamma gene expression, which permitted partial correction of the globin chain imbalance. Ultrastructural studies in two homozygotes for beta-thalassemia showed a marked decrease in the abnormalities of the erythroblasts derived from erythroid precursors in vitro in comparison to those from fresh bone marrow samples. In particular, Heinz bodies were much less numerous and a high frequency of mature erythroblasts and reticulocytes was observed in culture. These results support the view that, in sickle cell anemia and beta-thalassemia, a high potential for gamma gene expression exists and can be expressed in culture.
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PMID:Beta-thalassemia and sickle cell disease in culture of early erythroid precursors: hemoglobin synthesis and ultrastructural study. 718 47

The globin chains of human embryonic, fetal, and adult hemoglobins can be separated by electrophoresis on gels containing polyacrylamide, acid, urea, and Triton X-100. Whole hemolysates are used, and only microgram quantities are required. The order of the major human erythrocyte proteins, from anode to cathode, is zeta, epsilon, carbonic anhydrase, A gamma, delta and G gamma together, beta, and alpha. Protein composition can be measured on Coomassie blue-stained disc gels, and protein synthesis on fluorograms of slab gels containing 3H-leucine-labelled material. These gels have been used to examine the ratio of G gamma to A gamma in blood from fetuses and newborn infants, and to suggest that the switch from A gamma to G gamma during ontogeny may not be linked to the switch from gamma to beta production. beta/gamma synthetic ratios were determined in fetuses at risk for thalassemia. Embryonic and fetal globin synthesis ratios were measured in hemin-induced human erythroleukemia cells K562 in tissue culture. Fetal globin synthesis and the proportion that was of the "fetal" type (G gamma approximately 70%) was studied in erythroid colonies grown in plasma clot cultures from adult, newborn, and 6 month infant specimens. The gels provide a rapid, simple, and inexpensive approach to many problems of globin composition and synthesis.
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PMID:Gel electrophoretic separation of globin chains. 727 69

Much excess alpha chain is synthesized, but little accumulates in the erythroid cells of patients with homozygous beta thalassemia. To determine if the proteases known to exist in erythroid cells play a role in the destruction or alteration of any of this excess alpha chain, thalassemic and nonthalassemic erythroid cells were incubated for 90 minutes with 3H-leucine. The cells were then washed, and incubated twice for 15 minutes in 100 volumes of cold leucine-rich media, a procedure which eliminates almost all intracellular TCA soluble radioactivity. After these incubations levels of TCA soluble and TCA precipitable radioactivity in the cell lysates were determined, and the cells incubated for 120 minutes more in two volumes of leucine-rich media. At the end of this incubation, total TCA soluble and precipitable radioactivity was again determined in the cell lysate, and also in the two hour incubation media. The total increase in TCA soluble radioactivity in the cells and their media was divided by the 0 time TCA precipitable radioactivity, to determine the percent proteolysis labelled globin chains. In five control patients percent proteolysis ranged from 0 to 3.10 (mean = 1.50); in four severe and three mild thalassemia patients percent proteolysis ranged from 5.80 to 14.1 (mean = 11.0). The difference between the control and thalassemic groups was significant at a p of less than 0.001. This data is the first direct evidence that more proteolysis takes place in intact thalassemic cells than in non-thalassemic cells.
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PMID:Evidence for increased proteolysis in intact beta thalassemia erythroid cells. 731 26

We examined globin chain synthesis in erythroid bursts (BFU-E) of patients with heterozygous alpha or beta thalassaemia. BFU-E were cloned from circulating mononuclear cells, labelled with [3H]leucine and globin chains purified by gel filtration and column chromatography. In six patients heterozygous for beta thalassaemia, globin synthesis in BFU-E was nearly balanced, with an alpha/non alpha ratio of 1.05 +/- 0.12. These BFU-E produced 33.8 +/- 12.7% gamma globin chain, an amount similar (P > 0.05) to that found in 10 controls with sickle cell anaemia (25.6 +/- 6.7) but greater (P > 0.05) than that of five normal controls (17.2 +/- 2.2). The balanced globin synthesis appeared due to the large amounts of gamma chain made by BFU-E. In two alpha thalassaemia carriers, who also had sickle cell trait, the BFU-E alpha/non-alpha ratio was 0.67 and 0.79. These BFU-E produced 15% and 20% gamma chain and 39% and 45% betaS globin. The synthesis of betaS globin in BFU-E exceeded the erythrocyte levels of 20% and 29% HbS and indicated nearly equal expression of betaA and betaB globin genes in these proliferating erythroid precursors. This provides further evidence that the low levels of HbS in sickle cell carriers with alpha thalassaemia are due to post-translational events resulting from the differing affinity of betaS and betaA globin for alpha chain and the destruction of excessive betaS chain.
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PMID:Globin biosynthesis in erythroid bursts of heterozygous alpha or beta thalassaemia. 743 46

We have previously described a unique type of delta beta-thalassemia in a Chinese family characterized by increased expression of the G gamma and A gamma fetal globin genes in the absence of a large deletion in the beta-globlin gene cluster. Our earlier study of the beta-globin gene on this delta beta-thalassemia chromosome showed a promoter mutation in the TATA box. In this report, we describe the results of our study of the fetal globin domain of this delta beta-thalassemia chromosome. We have cloned a 13-kb DNA fragment that includes the G gamma and the A gamma genes and the 3' A gamma enhancer element of this delta beta-thalassemia chromosome. DNA sequence analysis of the G gamma and A gamma-globin genes including their promoters did not show any mutations, but analysis of the putative enhancer element downstream from the A gamma-globin gene showed a C to T substitution 2,401 nucleotides downstream from the A gamma cap site. We performed DNA linkage analysis to determine if this mutation is unique to this chromosome or represents a common polymorphism. Our linkage analysis showed that this mutation is not a common polymorphism and that it is also not an intrinsic part of the haplotype of the chromosome on which it was found. We also studied the interaction of nuclear proteins from erythroid and nonerythroid cells with the DNA sequences surrounding this mutation. We have shown by in vitro DNase I footprinting that this mutation falls within a region that is occupied by a novel DNA-binding protein that binds to this site in nuclear extracts from erythroid, but not nonerythroid cells. The binding of this nuclear protein to DNA appears to be dependent on GATA-1 binding to an adjacent GATA-1 site. We have also developed a new functional assay to compare the activity of the normal and mutant A gamma enhancer elements in erythroid cells. Analysis of the activity of the mutant enhancer shows that the mutation completely eliminates all enhancer activity in this assay. These findings suggest that this mutation of the A gamma enhancer on a chromosome that carries a partially inactivated beta-globin gene may be responsible for the increased expression of both gamma-globin genes seen in this condition.
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PMID:Increased expression of the G gamma and A gamma globin genes associated with a mutation in the A gamma enhancer. 751 19

Effective gene therapy for the severe hemoglobin (Hb) disorders, sickle-cell anemia and thalassemia, will require an efficient method to transfer, integrate, and express a globin gene in primary erythroid cells. To evaluate recombinant adeno-associated virus (rAAV) for this purpose, we constructed a rAAV vector encoding a human gamma-globin gene (pJM24/vHS432A gamma). Its 4725-nucleotide genome consists of two 180-bp AAV inverted terminal repeats flanking the core elements of hypersensitive sites 2, 3, and 4 from the locus control region of the beta-globin gene cluster, linked to a mutationally marked A gamma-globin gene (A gamma) containing native promoter and RNA processing signals. CD34+ human hematopoietic cells were exposed to rAAV particles at a multiplicity of infection of 500-1000 and cultured in semisolid medium containing several cytokines. A reverse transcriptase polymerase chain reaction assay distinguished mRNA signals derived from transduced and endogenous human gamma-globin genes. Twenty to 40% of human erythroid burst-forming unit-derived colonies expressed the rAAV-transduced A gamma-globin gene at levels 4-71% that of the endogenous gamma-globin genes. The HbF content of pooled control colonies was 26%, whereas HbF was 40% of the total in pooled colonies derived from rAAV transduced progenitors. These data establish that rAAV containing elements from the locus control region linked to a gamma-globin gene are capable of transferring and expressing that gene in primary human hematopoietic cells resulting in a substantial increase in HbF content.
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PMID:Recombinant adeno-associated virus (rAAV)-mediated expression of a human gamma-globin gene in human progenitor-derived erythroid cells. 752 85

The effects of heme, when added as the ferric chloride salt, hemin, on human erythroid cells grown in a two-phase liquid culture system were studied. When added together with erythropoietin, on initiation of the second phase of the culture, hemin greatly accelerated hemoglobin (Hb) accumulation in these cells. The effect was greater during their early stages of maturation, suggesting that heme availability is then a rate-limiting step for Hb synthesis. Hemin increased preferentially the production of fetal Hb (HbF) compared with adult Hb; this was associated with a selective twofold elevation in gamma-mRNA levels. Using succinylacetone, a potent inhibitor of heme synthesis, we showed that exogenously supplied hemin could be incorporated into the de novo formed Hb. Therefore, the mechanism of hemin action may be several fold, including effects on globin gene transcription and posttranslational events, eg, supplying the prosthetic group for Hb assembly. Hemin increased HbF of cells derived from patients with sickle cell anemia and beta-thalassemia as well as that of cells from normal donors. Moreover, when added in combination with other HbF-augmenting agents such as the cytotoxic drug, hydroxyurea, a synergistic effect was obtained, with considerably less cytotoxicity than with hydroxyurea alone. These results have clinical potential in light of the ameliorating effect that increased HbF has in patients with genetic diseases of the beta-globin chain and raise the possibility of combined treatment with hemin and other drugs now being used to treat these diseases.
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PMID:Hemin-induced acceleration of hemoglobin production in immature cultured erythroid cells: preferential enhancement of fetal hemoglobin. 753 86

An autosomally transmitted hypochromic microcytic mild anaemia with elevated haemoglobin (Hb) A2 and globin chain imbalance has been observed in a three-generation family of Portuguese origin. Extensive DNA analysis of the beta-globin gene cluster, including the complete sequencing of the beta-globin gene and flanking regions, failed to reveal any genetic alteration. The co-segregation of sickle-cell trait in this family enabled us to postulate a defective erythroid trans-acting factor was playing a role in the down-regulation of both beta A- and beta S-globin genes. Among the transcription factors that could possibly have caused the reported phenotype, NF-E2 is unlikely to be implicated, whereas Nrf1 and Nrf2 cannot be ruled out. Thus, this family carries a novel beta-thalassaemia autosomal determinant unlinked to the beta-globin gene. This observation reinforces the notion of the haemoglobinopathies as single gene disorders under polygenic regulation.
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PMID:Beta-thalassaemia unlinked to the beta-globin gene interacts with sickle-cell trait in a Portuguese family. 757 58

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta-thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha-globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.
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PMID:The instability of the membrane skeleton in thalassemic red blood cells. 757 65

The human alpha-globin-like embryonic zeta-globin chains are present in abundance during the first 5 to 6 weeks of gestation. Subsequently, zeta-globin chains are present in fetal blood at a very low level, which is supplanted by the expression of alpha-globin chains. Adult individuals who are carriers of the (--SEA/) alpha-thalassemia deletion, in contrast to normal adults, have low levels of embryonic zeta-globin chains in their circulating erythrocytes. In this investigation, we constructed stable mouse-human hybrid cells with murine erythroleukemia cells bearing human chromosome 16, with either the normal alpha-globin gene cluster (alpha alpha/) or the (--SEA/) type of alpha-thalassemia deletion. The results on the human zeta-globin gene expression in these hybrid cells indicate that murine adult erythroid transcription factors can induce the expression of human embryonic zeta-globin gene is cis to the (--SEA/) deletion, in parallel with the endogenous mouse alpha-globin gene expression. These data also show the importance of the DNA sequences within the (--SEA) deletion in regulating the expression of zeta-globin gene in cis during normal human hemoglobin ontogeny.
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PMID:Human embryonic zeta-globin gene expression in mouse-human hybrid erythroid cell lines. 762 Jan 74


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