Rate of synthesis and half-life of globin messenger ribonucleic acid. Rate of synthesis of globin messenger ribonucleic acid calculated from data of cell haemoglobin content

By the use of the favoured models defining mRNA synthesis and half-life from the preceding paper (Hunt, 1974) and the known content of globin in a reticulocyte it is possible to estimate the absolute rate of mRNA and globin synthesis and the mRNA and globin content in each type of erythroid cell. The best model requires an mRNA-synthetic rate of 3000 molecules per h/cell. This rate compares favourably with the estimated chain-extension rate of 43 nucleotides/s in Escherichia coli (Manor et al., 1969) provided that the four alpha- and beta-chain cistrons per cell are transcribed by polymerases spaced 50 nucleotide base pairs apart. Similar calculations can be made for erythropoiesis in the chick embryo, where cell times and relative globin content at each mitosis have been measured (Campbell et al., 1971), but where no reliable estimates of mRNA half-life have been made. In this case it was estimated that a constant rate of mRNA synthesis at 10000 molecules per h/cell through six cell divisions is necessary if the mRNA half-life is 15h; after the sixth mitosis the mRNA synthesis would stop and its half-life would increase to approx. 20h. If an mRNA half-life of 4.5h is used, the synthesis rate through the six mitoses would be 21000 molecules per h/cell, ceasing at the sixth mitosis, when the half-life would need to increase to 25h. The chain-elongation rate for the four alpha- and beta-globin cistrons per cell would be 1-2 times higher than in E. coli and would either require a greater rate, polymerases spaced between 25 and 50 nucleotide base pairs apart on the DNA, or limited gene replication. These possibilities are discussed in the light of the low values found for globin cistron multiplicity in ducks and mice.     

ELECTRON MICROSCOPY OF THE AMMONIACAL SILVER REACTION FOR HISTONES IN THE ERYTHROPOIETIC CELLS OF THE CHICK

The product of the postformalin ammoniacal silver reaction, which has been claimed to distinguish lysine-rich from arginine-rich histones with the light microscope on the basis of a color difference, was examined in developing erythroid cells of chick bone marrow with the electron microscope. Stem cells and early erythroblasts exhibit no, or little, ammoniacal silver reaction product, while small basophilic erythroblasts, polychromatophilic erythrocytes, and reticulocytes exhibit an increasing amount of reaction product as maturation proceeds. The reaction product is in the form of discrete electron-opaque particles associated with heterochromatin. The ammoniacal silver reaction in the erythroid cell series is interpreted as reflecting either the accumulation of newly synthesized arginine-rich histones or changes in the availability of reactive sites in preformed histones.     

Synthesis and expression of cell-surface glycoproteins during chick erythroid differentiation

Abstract. A panel of monoclonal antibodies to differentiation antigens on avian erythroid cells was used to study the reprogramming of protein synthesis during erythroid differentiation at the molecular level. This panel detected five distinct cell-surface glycoproteins on immature leukemic erythroblasts, all of which were initially synthesised as smaller intracellular precursors. Two distinct in vitro differentiation systems (erythroblasts transformed by ts mutants of the erb-B and sea retroviral oncogenes, in which the synchronous terminal differentiation of CFU-E-like precursors is induced by simple elevation of temperature) were used to study cell-surface expression and the biosynthesis of each protein during erythroid cell maturation. For four glycoproteins, both cell-surface expression and biosynthesis decreased between the erythroblast and erythrocyte stages, although with widely different time courses. The fifth glycoprotein, which is reticulocyte specific on normal erythroid progenitors and is aberrantly expressed in onco-gene-transformed erythroblasts, rapidly disappeared shortly after differentiation induction but was then re-expressed on reticulocytes with the same time course as that seen during normal erythroid differentiation. This indicates that ts erb-B- and ts sea -transformed erythroblasts revert to a normal precursor phenotype before undergoing temperature-induced differentiation.     

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.     

NADP+ catabolic enzymes in differentiating rabbit erythroid cells

NADP-glycohydrolase and NADP-pyrophosphates activities were examined during the rabbit erythroid cell differentiation. The former is high in erythroblast lysates, especially in the erythroblast nuclei. As erythroid cell maturation proceeds, the activity of NADP-glycohydrolase decreases. At the first step (erythroblast-reticulocyte transformation), this activity falls down more than by 20 times, whereas at the second step (reticulocyte-erythrocyte transformation) it decreases no more than twice. NADP-glycohydrolase is associated with the stroma of erythroid cells throughout their maturation, being bound with the nucleus in erythroblasts. NADP-pyrophosphatase activity has been detected in reticulocytes and mature cells only. The role of NADP- and NAD-glycohydrolases for characterization of the intracellular metabolic pools is discussed.     

Membrane-bound glycoconjugates of fetal mouse erythropoietic cells with special reference to phagocytosis by hepatic macrophages

Using lectin and colloidal iron (CI) stainings in combination with neuraminidase digestion, glycoconjugates on the surface of erythropoietic cells of the yolk sac and liver in fetal mice were examined. Fetal hepatic macrophages were capable of distinguishing between phagocytozed and non-phagocytozed erythroid elements as described in our previous study. Marked differences between these two elements could be ultrahistochemically detected on their cell surface. The phagocytozed elements, such as nuclei expelled from erythroblasts and degenerating primitive erythroblasts, faintly bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a weak peanut agglutinin (PNA) binding. In contrast, erythroblasts at various maturation stages, erythrocytes and normal primitive erythroblasts heavily bound neuraminidase-sensitive CI, and neuraminidase digestion imparted a moderate PNA binding. No differences in binding of either concanavalin agglutinin,Ricinus communis agglutinin-I or PNA were noted between phagocytozed and non-phagocytozed erythroid elements. Desialylation appears to be one of the most important signs for the recognition mechanism of fetal macrophage phagocytosis. During maturation of hepatic erythroblasts, sialic acid changes its affinity forLimax flavus agglutinin from strong to weak, and soybean agglutinin binding sites disappear at the basophilic erythroblast stage. Glycoconjugates on polychromatophilic erythroblasts acquire similar compositions to those of erythrocytes.     

Stimulation of adenylate cyclase activity by catecholamines and prostaglandins E during differentiation of rabbit bone marrow erythroid cells

After fractionation of rabbit bone marrow into erythroid cells at different developmental stages adenylate cyclase activity of membrane ghosts was assayed in the presence of sodium fluoride, catecholamines or prostaglandins E. Both basal and fluoride-stimulated adenylate cyclase decreased continuously during differentiation. Only catecholamines having beta 2-adrenergic activity stimulated adenylate cyclase and their effect was restricted to the most immature cells, the proerythroblasts and, to a lesser extent, the basophilic erythroblasts. Thus, uncoupling of beta-adrenergic receptors occurs early in erythroblast development and hormone responsiveness is lost before the final cell division. Prostaglandin E receptors and adenylate cyclase remain coupled throughout erythroid cell development.     

Heme oxygenase-1 deletion affects stress erythropoiesis

Background

Homeostatic erythropoiesis leads to the formation of mature red blood cells under non-stress conditions, and the production of new erythrocytes occurs as the need arises. In response to environmental stimuli, such as bone marrow transplantation, myelosuppression, or anemia, erythroid progenitors proliferate rapidly in a process referred to as stress erythropoiesis. We have previously demonstrated that heme oxygenase-1 (HO-1) deficiency leads to disrupted stress hematopoiesis. Here, we describe the specific effects of HO-1 deficiency on stress erythropoiesis.

Methodology/Principal Findings

We used a transplant model to induce stress conditions. In irradiated recipients that received hmox ^+/− or hmox ^+/+ bone marrow cells, we evaluated (i) the erythrocyte parameters in the peripheral blood; (ii) the staining intensity of CD71-, Ter119-, and CD49d-specific surface markers during erythroblast differentiation; (iii) the patterns of histological iron staining; and (iv) the number of Mac-1⁺-cells expressing TNF-α. In the spleens of mice that received hmox ^+/− cells, we show (i) decreases in the proerythroblast, basophilic, and polychromatophilic erythroblast populations; (ii) increases in the insoluble iron levels and decreases in the soluble iron levels; (iii) increased numbers of Mac-1⁺-cells expressing TNF-α; and (iv) decreased levels of CD49d expression in the basophilic and polychromatophilic erythroblast populations.

Conclusions/Significance

As reflected by effects on secreted and cell surface proteins, HO-1 deletion likely affects stress erythropoiesis through the retention of erythroblasts in the erythroblastic islands of the spleen. Thus, HO-1 may serve as a therapeutic target for controlling erythropoiesis, and the dysregulation of HO-1 may be a predisposing condition for hematologic diseases.     

Analysis of the ratio of alpha- to beta-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells.

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000--50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.     

The mechanism of inactivation of the bird erythrocyte genome. III. Pathways of terminal differentiation of erythrocytes]

Under the conditions of acute phenylhydrazine anemia in pigeons, early erythroblasts develop in reticulocytes with basophilic cytoplasm directly by passing the stages of polychromatophilic and ortochromic erythroblasts. During the maximum activity of bone marrow erythropoiesis stimulated by anemia over 80% of reticulocytes develop in such a way. A hypothesis is put forward to the effect that there exist in birds two alternative pathways of erythropoiesis: the main pathway which ensures the slow repopulation of blood by erythrocytes under normal conditions and the reserve pathway which opens for a short time under anemia and ensures rapid increase of the erythrocyte titer in blood up to its normal value.     

Differential regulatory and compensatory responses in hematopoiesis/erythropoiesis in alpha- and beta-globin hemizygous mice

Characterization of hematopoiesis/erythropoiesis in thalassemias from multipotent primitive cells to mature erythrocytes is of fundamental importance and clinical relevance. We investigated this process in alpha- and beta-globin hemizygous mice, lacking the two adult tandemly organized genes from either the alpha- or beta-globin locus. Although both mice backcrossed on a homogeneous background exhibited similar reduced red blood cell (RBC) survival, beta-globin hemizygous mice had less severe reticulocyte loss and globin chain imbalance, suggesting an apparently milder thalassemia than for alpha-globin hemizygous mice. In contrast, however, beta-globin hemizygous mice displayed a more marked perturbation of hematologic parameters. Quantification of erythroid precursor subpopulations in marrow and spleen of beta-globin hemizygous mice showed more severely impaired maturation from the basophilic to orthochromatophilic erythroblasts and substantial loss of these late precursors probably as a consequence of a greater susceptibility to an excess of free alpha-chain than beta-chain. Hence, only erythroid precursors exhibiting stochastically moderate chain imbalance would escape death and mature to reticulocyte/RBC stage, leading to survival and minimal loss of reticulocytes in the beta-globin hemizygous mice. Furthermore, in response to the ineffective erythropoiesis in beta-globin hemizygous mice, a dynamic compensatory hematopoiesis was observed at earlier differentiation stage as evidenced by a significant increase of erythroid progenitors (erythroid colony-forming units approximately 100-fold) as well as of multipotent primitive cells (day 12 spleen colony-forming units approximately 7-fold). This early compensatory mechanism was less pronounced in alpha-globin hemizygous mice. The expansion of multipotent primitive and potentially stem cell populations, taken together with ineffective erythropoiesis and increased reticulocyte/RBC destruction could confer major cumulative advantage for gene targeting/bone marrow transplantation. Therefore, this study not only corroborated the strong potential effectiveness of transplantation for thalassemic hematopoietic therapy but also demonstrated the existence of a differential regulatory response for alpha- and beta-thalassemia.     

Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.     

Globin-coding sequences in the nuclear 28S pre-mRNA and in the cytoplasmic RNA of pigeon erythroid cells

The steady-state content of globin-coding sequences in nuclear and cytoplasmic RNA of pigeon erythroid cells was estimated by hybridization in the excess of nuclear 28S RNA and cytoplasmic poly(A) + RNA with [3H]DNA, synthesized on globin mRNA. Sequences of 9S globin mRNA are found in 0.06% of molecules of non-ribosomal 28S nuclear RNA (pre-mRNA) of erythroblasts and in 0.5% of molecules of non-ribosomal 28S nuclear RNA of reticulocytes. The content of globin mRNA in erythroblast cytoplasm is, respectively lower than in that of reticulocytes.     

Cell proliferation in the erythroid compartment of guinea-pig bone marrow: studies with 3H-thymidine.

Single and multiple injections of 3H-TdR have been used for measuring the rate of proliferation in morphologically defined cell populations of guinea-pig bone marrow that are committed to erythroid differentiation. The conclusions are based on the analysis of absolute cell numbers in the maturational compartments, the labeling and mitotic indices, labeled mitotic curves, pulse and chase grain counts over dividing and interphase cells, and on the rate or labeling during multiple, repeated injections of 3H-TdR. The average duration of S and the rate of cycling is similar in all maturational compartments of the erythrom. The majority of cells progress to the next maturational compartment by the time they divide for the second time. All proerythroblasts and basophilic erythroblasts are in cycle. Polychromatic erythroblasts incapable of incorporating 3H-TdR reach the orthochromatic population in the span of 5-6 hr. The orthochromatic population is renewed every 20-24 hr. The number of divisions between the proerythroblast and orthochromatic erythroblast does not exceed four and some cells may undergo only two divisions during the maturation pathway. Cell input from a progenitor cell population contributes to the maintenance of the erythron. The kinetic behavior of progenitor cells is similar to that of proerythroblasts. By the time of their second division, progenitor cells may reach either the proerythroblast or basophilic erythroblast compartments. The kinetic behavior of basophilic transitional cells corresponds to the predicted behavior of the erythroblast progenitor cell pool. Several of the conclusions are based on the assumption that grain count halving is the result of cell division. In view of the evidence discussed, this assumption in the present studies seems justified.     

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.     

Stimulation of rabbit erythroblast multiplication and maturation in vitro by blood leukocytes: cytological observations. 3. Studies on erythropoiesis

Blood leukocytes incubated in vitro with rabbit-marrow cells induced a several-fold increase in basophilic erythroblasts and smaller increases in acidophils and reticulocytes. The main effect was nearly complete in one hour at 37°. Erythropoietin augmented the leukocyte effect; anti-erythropoietin inhibited it with or without erythropoietin. The erythroblast increase came entirely from the marrow cells; the precursor cell class has not been identified, except that division of pre-existing basophils appears to be excluded. Autologous and homologous leukocytes were about equally effective. A method is described of measuring on stained smears changes in relative concentrations of different classes of cells induced experimentally. A method of preparing highly concentrated peripheral blood leukocytes is described.     

Classification of erythroblastic islets of the bone marrow and the study of their cellular composition

Cell composition of erythroblastic islands (EI), normal and at the phase of stimulation and inhibition of erythropoiesis at modelling of heat adaptation has been studied on hemopoietic tissue of 108 rats. In the hemopoietic tissue, together with EI of the 1st, 2nd and 3d classes of maturity, that have in their composition correspondingly up to 8, from 9 up to 16 and above 16 nuclei-containing erythroid cells, there are involutive and reconstructing EI. The involutive EI are presented as nuclei-containing erythroid cells, that are not able to divide and are at late stages of differentiation: polychromatophilic and oxyphilic normoblasts, as well as reticulocytes. The reconstructing EI have in their composition both erythroid cells, that are not able to divide, and erythroid cells (pro-, erythroblasts and basophilic normoblasts), that are at early stages of differentiation. Application of the EI classification suggested, takes into account the rate, with which the erythroid cell-predecessors are drawn into erythropoiesis and intensity of erythroid differentiation. Therefore, it is possible to obtain earlier and more exact data on the state of hemopoiesis in comparison with traditional hematological methods.     

Hexokinase in developing rabbit erythroid cells

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits seperated by density centrifugation or by unit-gravity sedimentation. The specific activity of the enzyme was found to be about 150-fold higher in the basophilic erythroblasts as compared with the mature circulating erythrocytes. Mos of the falls in hexokinase activity take place whent the cell completes its final division and matures from the polychromatic stage to the orthochromatic stage. Concomitant with this strong decrease in enzyme activity, qualitative as well as quantitative changes in the hexokinase isozymic pattern become apparent. While in the basophilic and polychromatic erythroblasts the only hexokinase isozyme present is hexokinase type I, the orthochromatic cells also contain hexokinase Ib. This last isozymic form, which increases further at the reticulocyte stage, is also present in the circulating reticulocytes but not in mature red blood cells.