Unique Pattern of Gene Expression in the Erythroid Precursor Cells

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU-E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt-genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt-genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU-E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt-genes may be induced after the stimulation of erythropoietin.     

Rubidium uptake in single cells

Summary Rubidium uptake was measured in single erythroid and myeloid cells of rabbit by means of X-ray microanalysis. It was found in the nucleated bone marrow cells that after incubation in rubidium the sums of potassium and rubidium concentrations were similar to the original potassium concentrations, indicating that there was one-to-one replacement of potassium by rubidium. Although the nuclear potassium and rubidium concentrations were higher than those in the cytoplasm, the nuclear and cytoplasmic ratios of K/Rb were similar. This implies that the potassium in both compartments exchanged freely with rubidium. In the erythroid line of cells there was a continuous reduction of potassium transport activity during the maturation process as indicated by the decrease in rubidium uptake rates. The uptake was measured in seven groups of cell types that could be distinguished on the basis of morphology and chemical composition. The order of the groups from high to low rubidium uptake were: esosinophilic myelocyte > early erythroblast and thinrimmed erythroblast > late erythroblast > early bone marrow red cell > late bone marrow red cell > peripheral blood red cell. Thus, there is a continuous decrease in rubidium transport as the erythroid cells mature.     

Enzymes of NAD+ catabolism in differentiating rabbit erythroid cells

A study was made of the hydrolysis of NAD+ in the process of rabbit erythroid cell differentiation. Activities of NAD-hydrolase and NAD-pyrophosphatase, examined during maturation of erythroid cells, demonstrated a more significant decrease in the course of erythroblast-reticulocyte transformation, than at the next step of erythropoesis (reticulocyte-erythrocyte transformation). Both enzymes are strongly associated with the stroma. Under discussion is a question of the possible role of NAD+ catabolism.     

KINETICS OF DEVELOPMENT OF EMBRYONIC ERYTHROID CELLS

Embryonic erythropoiesis is an intrinsically non-steady-state process. A method of non-steady-state analysis is employed to approximately determine the kinetics of maturation of embryonic erythroid cells during the hepatic phase of erythropoiesis in the mouse. It appears from this analysis that embryonic erythroid cells have significantly shorter maturation times than their adult counterparts. In the embryo, there is insufficient time for more than three divisions between the proerythroblast and the orthochromatic erythroblast.     

Expression of the erythroblast antigen on hematopoietic and nonhematopoietic mouse cells. 1. The flow cytofluorimetric analysis of stem, committed and differentiated bone marrow cells

Expression of the erythroblast antigen (AG-EB) on the membrane of the mouse hemopoietic cells was studied using a FACS II flow cytofluorimeter with monoclonal antibodies MAE15. It was shown that AG-EB was present on the surface of 14% of the late committed erythroid precursors (CFU-3) and of practically all more mature erythroid cells. The role of AG-EB in maturation of the erythroid cells is discussed.     

Down-regulation of Myc is essential for terminal erythroid maturation

Terminal differentiation of mammalian erythroid progenitors involves 4-5 cell divisions and induction of many erythroid important genes followed by chromatin and nuclear condensation and enucleation. The protein levels of c-Myc (Myc) are reduced dramatically during late stage erythroid maturation, coinciding with cell cycle arrest in G(1) phase and enucleation, suggesting possible roles for c-Myc in either or both of these processes. Here we demonstrate that ectopic Myc expression affects terminal erythroid maturation in a dose-dependent manner. Expression of Myc at physiological levels did not affect erythroid differentiation or cell cycle shutdown but specifically blocked erythroid nuclear condensation and enucleation. Continued Myc expression prevented deacetylation of several lysine residues in histones H3 and H4 that are normally deacetylated during erythroid maturation. The histone acetyltransferase Gcn5 was up-regulated by Myc, and ectopic Gcn5 expression partially blocked enucleation and inhibited the late stage erythroid nuclear condensation and histone deacetylation. When overexpressed at levels higher than the physiological range, Myc blocked erythroid differentiation, and the cells continued to proliferate in cytokine-free, serum-containing culture medium with an early erythroblast morphology. Gene expression analysis demonstrated the dysregulation of erythropoietin signaling pathway and the up-regulation of several positive regulators of G(1)-S cell cycle checkpoint by supraphysiological levels of Myc. These results reveal an important dose-dependent function of Myc in regulating terminal maturation in mammalian erythroid cells.     

Selective synthesis and modification of nuclear proteins during maturation of avian erythroid cells.

The synthesis of the nuclear proteins of duck erythroid cells at different stages of maturation has been investigated. Synthesis of histone fractions H1, H2a, H2b, H3, and H4 is restricted to the erythroblasts, while synthesis of H5 can be detected even at later stages of maturation after DNA synthesis has ceased. The synthesis of nonhistone nuclear proteins (NHNP), on the other hand, occurs in cells at all stages of maturation although their rates of synthesis decline as the cells mature. The same size classes of NHNP appear to be synthesized in erythroblasts and in early- and midpolychromatic erythrocytes. In late polychromatic erythrocytes the synthesis of a new group of NHNP of molecular weights ranging from 54,000 to 130,000 was observed. This group of proteins does not accumulate in the mature erythrocyte, indicating that their relative proportions are very small.Turnover of histone-bound phosphate was found to occur mainly at the erythroblast stage, except for histone H2a which was actively phosphorylated even at more advanced stages of maturation. Phosphorylation of most of the histones appears to be coupled to histone (and coordinate DNA) synthesis.Incorporation of radioactive acetate into histones occurs at all stages, but the rate of acetylation decreases four- to fivefold with maturation. Although the RNA synthetic activity of erythroid cells also decreases with age, experiments involving the use of RNA polymerase inhibitors suggest that the mechanisms that control RNA synthesis and histone acetylation are not tightly coupled.     

Differentiation of chick blood island erythroblasts into erythrocytes and stability in long-term culture

Clusters of 20-70 erythroblasts from blood islands of early chick blastoderm were cultured in serum-free chemically defined medium for a 3-month period. The erythroblast cluster produces erythroid cells and hemoglobins characteristic of the primitive and definitive erythroid cell lines. It seems there is a progenitor erythroid cell(s) in the erythroblast cluster which starts and/or continues maturing along various pathways of hemopoietic differentiation under simple culture conditions. The erythroid character of these cells is stable during the 3-month culture period.     

Cyclic AMP phosphodiesterase activity during differentiation of rabbit erythroid bone marrow cells.

Changes in the activity of cyclic AMP phosphodiesterase during differentiation of rabbit bone marrow erythroid cells were investigated. The cells were separated by velocity sedimentation at unit gravity into six fractions corresponding to different stages of development: proerythroblasts, basophilic cells, polychromatic cells, early orthochromatic and late orthochromatic cells and reticulocytes. Cyclic AMP phosphodiesterase was found to be very active in the most immature cells, the proerythroblasts, which also have the highest content of cyclic AMP. After differentiation into basophilic erythroblasts, a 4-fold decrease in cyclic AMP phosphodiesterase activity was observed. In these cells the amount of cyclic AMP was about 80% lower than that in proerythroblasts. In polychromatic cells a further drop in phosphodiesterase activity occurred. After the final cell division the enzyme activity was very low and the levels of cyclic AMP in the early and late orthochromatic cells remained constant. Kinetic studies demonstrated a heterogeneity of erythroid cell cyclic AMP phosphodiesterase: high affinity, low-Km (5.5 X 10(-6) M) and low affinity, high-Km (0.1 X 10(-3) M) enzymes were found. The phosphodiesterase activity was dependent on the presence of Mg2+ and was activated by Ca2+ at low Mg2+ concentrations (1 mM). The changes in cyclic AMP phosphodiesterase activity during differentiation and maturation of erythroid cells suggest the possible importance of this enzyme in the physiological control of cyclic AMP concentrations in developing erythroblasts. The loss of cyclic AMP phosphodiesterase activity after cessation of cell division supports the concept of the significance of the final cell division in erythroblast differentiation.     

Avian erythrocyte development: microtubules and the formation of the disk shape

The development of avian erythrocytes involves a spheroid to discoid transformation in shape. The disk shape of the young erythroid cells is dependent on the presence of microtubules in a marginal bundle in the early stages of postmitotic maturation. Disassembly of microtubules with colchicine, vincristine, sulfate or cold temperature produces the spheroidal shape. Erythrocytes which have acquired the flattened ellipsoidal shape do not alter their shape with disassembly of the microtubules. The number of microtubules decreases as cell maturation occurs. The correlation coefficient for the number of microtubular profiles in one end of erythrocytes and the concentration of ribosomes (cell age) is 0.88. Microtubules of immature erythrocytes disappear more rapidly at 0°C than do microtubules of mature cells.It is concluded that microtubules play little or no role in the maintenance of mature red cell shape; however, they play an important role in the development of the flat discoid shape of avian erythrocytes during maturation.     

Increase of 2,3-bisphosphoglycerate synthase/phosphatase during maturation of reticulocytes with high 2,3-bisphosphoglycerate content

In the rabbit and in the rat, which possess erythrocytes with high concentration of 2,3-bisphosphoglycerate, the 2,3-bisphosphoglycerate synthase activity increases more than two fold during reticulocyte maturation. Isolation of the enzymes with 2,3-bisphosphoglycerate synthase activity present in extracts of reticulocytes and mature erytrocytes by ion exchange fast liquid chromatography shows that the increase in the synthase activity is due to the accumulation of the bifunctional enzyme 2,3-bisphosphoglycerate synthase/phosphatase (EC 2.7.5.4/EC 3.1.3.13) which represents more than 80% of the synthase activity of the cell extracts. During reticulocyte maturation phosphoglycerate mutase (EC 5.4.2.1), which makes a small contribution to the 2,3-bisphosphoglycerate synthase activity in the erythroid cells, decreases in the rabbit and remains constant in the rat.     

Activity of the nucleolar organizers in cultured cells from the bone marrow erythroblastic islets

The erythroblast islands of the bone marrow are the morphofunctional units of erythropoiesis. In this work, the functional state of erythroblast islands' cells of the bone marrow for the first time was defined by estimation of the activity of the nucleolar organizers of erythroid cells in the erythroblast islands cultivated during 24 and 48 hours in presence of various doses of erythropoietin. The findings indicated that an increase in doses of erythropoietin was accompanied by a corresponding increase of the activity of nucleolar organizers in erythrokaryocytes of erythroblast islands. The nucleolar organizers of erythroid cells in cultures of erythroblast islands responded with activation to very small doses of erythropoietin; besides, a proliferative response of erythrokaryocytes was observed after activation of the nucleolar organizers.     

Patterns of expression of erythroblast non-globin mRNAs

Three classes of erythroblast non-globin mRNAs have been identified using cDNA recombinants isolated from a mouse foetal liver cDNA library. One erythroid cell-specific 12S mRNA coding for a 16-19,000 dalton protein has been identified using two independent cDNA recombinants (pFC5 and pFA6). The gene encoding this mRNA is unique in the genome and its organisation seems to be the same in both erythroid and non erythroid cell genomic DNA as judged by digestion with restriction enzymes and Southern blotting. Another erythroblast mRNA of size 16S represented by recombinant pFD12 is expressed in brain cells as well as a variety of haemopoietic cell types, but not in adult liver or fibroblasts. Two other erythroblast mRNAs of size 8S and 12S represented by recombinants pD6 and pA4 are expressed in many differentiated cell types with the exception of non-dividing cells such as reticulocytes, peripheral white blood cells, adult liver and brain cells. These mRNAs are, therefore, presumed to be involved ubiquitously in cell proliferation or general cell metabolism.     

Production of erythropoietic cells in vitro for continuous culture of Plasmodium vivax

Plasmodium vivax cannot be maintained in a continuous culture. To overcome this major obstacle to P. vivax research, we have developed an in vitro method to produce susceptible red blood cell (RBC) precursors from freshly isolated human cord hematopoietic stem cells (HSCs), which were activated with erythropoietin to differentiate into erythroid cells. Differentiation and maturation of erythroid cells were monitored using cell surface markers (CD71, CD36, GPA and Fy6). Duffy⁺ reticulocytes appeared after 10 days of erythroid cell culture and exponentially increased to high numbers on days 14–16. Beginning on day 10 these erythroid cells, referred to as growing RBCs (gRBCs), were co-cultured with P. vivax-infected blood directly isolated from patients. Parasite-infected gRBCs were detected by Giemsa staining and a P. vivax-specific immunofluorescence assay in 11 out of 14 P. vivax isolates. These P. vivax cultures were continuously maintained for more than 2 weeks by supplying fresh gRBCs; one was maintained for 85 days before discontinuing the culture. Our results demonstrate that gRBCs derived in vitro from HSCs can provide susceptible Duffy⁺ reticulocytes for continuous culture of P. vivax. Of particular interest, we discovered that parasites were able to invade nucleated erythroid cells or erythroblasts that are normally in the bone marrow. The possibility that P. vivax causes erythroblast destruction and hence inflammation in the bone marrow needs to be addressed.     

TRAIL promotes a pro-survival signal in erythropoietin-deprived human erythroblasts through the activation of an NF-kB/IkBalpha pathway

The biological activity of TNF-related apoptosis inducing ligand (TRAIL) was analyzed in primary human erythroblasts derived from mononuclear cells of blood donors, kept in culture in the presence of 20 percent foetal calf serum, growth factors (EPO, SCF, IL-3) and glucocorticoids (10-6 M dexamethasone, 10-6 M oestradiol) or under growth factor and serum starvation. In the presence of growth factors and serum, primary erythroblasts showed a differential expression of TRAIL-Receptors (Rs) at various degrees of maturation and responded to TRAIL treatment with a mild cytotoxicity. On the other hand, in the absence of serum and growth factors, TRAIL treatment unexpectedly up-regulated TRAIL-R4 decoy receptor and promoted erythroblast survival. The concomitant activation of NF-kB/IkB survival pathway was detected with Western blotting and immunofluorescence procedures and confirmed by experiments performed with SN50, a pharmacological inhibitor of the NF-kB/IkB pathway. Our study indicates that TRAIL has a twofold activity on erythroid lineages: it induces a mild erythroid cell cytotoxicity in the presence of serum and growth factors, while it promotes erythroid cell survival through the activation of the NF-kB/IkB pathway under starvation conditions.     

长吻鮠血细胞发生的研究

用Wright-Giemsa和PAS染色对长吻鮠头肾、肾脏、脾脏、肝脏等器官组织的涂片、印片染色观察发现,头肾、肾脏和脾脏是其主要造血器官。红细胞、粒细胞和淋巴细胞主要在肾脏和头肾中发生,其次是脾脏。单核细胞则主要在肾脏和脾脏中发生,头肾中也有少量单核细胞产生。肝脏中无原始型血细胞,可能不是其造血器官。红细胞的发育经历四个阶段,其胞体体积经历了由大到小,由小到大再变小的"两大两小"发育过程;粒细胞的发育经历五个阶段,其胞体体积均由大变小,双叶或多叶核的粒细胞可能是衰老的粒细胞亦即核的分叶是粒细胞衰老的标志;淋巴细胞和单核细胞的发育各经历了三个阶段,两者发育成熟过程中胞体体积均由大变小。巨噬细胞由单核细胞发育而来。原血细胞和部分早期幼稚血细胞可以进行有丝分裂,部分成熟红细胞和血栓细胞可以进行直接分裂。红细胞在整个发育过程中,PAS反应均呈阴性,各类白细胞的发育过程中,PAS反应由阴性到阳性并逐渐增强,这显示随着白细胞的逐渐发育成熟,细胞内糖原物质含量逐渐增多。     

Molecular changes in the membranes of mouse erythroid cells accompanying differentiation.

The development of the mouse erythroblast to a mature erythrocyte is accompanied by changes in the composition and properties of the plasma membranes of these cells. Using double fluorescence techniques, we have simultaneously determined the distribution of lectin receptors and spectrin on the membranes of these cells. The lateral mobility of the lectin receptors in the membranes decreases as differentiation proceeds, and this is accompanied by an increasing concentration of spectrin associated with the membranes. The most significant concentration of spectrin occurs, however, during the enucleation of the late erythroblast, where we observe a complete segregation of the spectrin to the incipient reticulocyte, as well as a previously observed enrichment of receptors for concanavalin A into the plasma membrane surrounding the extruding nucleus. On the basis of these and other observations, we explore the possible molecular mechanisms involved in erythroblast enucleation and the role of spectrin in the regulation of protein mobility in erythroid cell membranes.