Fyn kinase is a novel modulator of erythropoietin signaling and stress erythropoiesis.

The signaling cascade induced by the interaction of erythropoietin (EPO) with its receptor (EPO-R) is a key event of erythropoiesis. We present here data indicating that Fyn, a Src-family-kinase, participates in the EPO signaling-pathway, since Fyn-/- mice exhibit reduced Tyr-phosphorylation of EPO-R and decreased STAT5-activity. The importance of Fyn in erythropoiesis is also supported by the blunted responsiveness of Fyn-/- mice to stress erythropoiesis. Fyn-/- mouse erythroblasts adapt to reactive oxygen species (ROS) by activating the redox-related-transcription-factor Nrf2. However, since Fyn is a physiologic repressor of Nrf2, absence of Fyn resulted in persistent-activation of Nrf2 and accumulation of nonfunctional proteins. ROS-induced over-activation of Jak2-Akt-mTOR-pathway and repression of autophagy with perturbation of lysosomal-clearance were also noted. Treatment with Rapamycin, a mTOR-inhibitor and autophagy activator, ameliorates Fyn-/- mouse baseline erythropoiesis and erythropoietic response to oxidative-stress. These findings identify a novel multimodal action of Fyn in the regulation of normal and stress erythropoiesis.

Erythropoietin regulates the expression of dimeric form of acetylcholinesterase during differentiation of erythroblast.

Acetylcholinesterase (AChE; EC 3.1.1.7) is known to hydrolyze acetylcholine at cholinergic synapses. In mammalian erythrocyte, AChE exists as a dimer (G2 ) and is proposed to play role in erythropoiesis. To reveal the regulation of AChE during differentiation of erythroblast, erythroblast-like cells (TF-1) were induced to differentiate by application of erythropoietin (EPO). The expression of AChE was increased in parallel to the stages of differentiation. Application of EPO in cultured TF-1 cells induced transcriptional activity of ACHE gene, as well as its protein product. This EPO-induced event was in parallel with erythrocytic proteins, for example, α- and ß-globins. The EPO-induced AChE expression was mediated by phosphorylations of Akt and GATA-1; because the application of Akt kinase inhibitor blocked the gene activation. Erythroid transcription factor also known as GATA-1, a downstream transcription factor of EPO signaling, was proposed here to account for regulation of AChE in TF-1 cell. A binding sequence of GATA-1 was identified in ACHE gene promoter, which was further confirmed by chromatin immunoprecipitation (ChIP) assay. Over-expression of GATA-1 in TF-1 cultures induced AChE expression, as well as activity of ACHE promoter tagged with luciferase gene (pAChE-Luc). The deletion of GATA-1 sequence on the ACHE promoter, pAChEΔGATA-1 -Luc, reduced the promoter activity during erythroblastic differentiation. On the contrary, the knock-down of AChE in TF-1 cultures could lead to a reduction in EPO-induced expression of erythrocytic proteins. These findings indicated specific regulation of AChE during maturation of erythroblast, which provided an insight into elucidating possible mechanisms in regulating erythropoiesis.

Residual pyruvate kinase activity in PKLR-deficient erythroid precursors of a patient suffering from severe haemolytic anaemia.

OBJECTIVE: Here, we present a 7-year-old patient suffering from severe haemolytic anaemia. The most common cause of chronic hereditary non-spherocytic haemolytic anaemia is red blood cell pyruvate kinase (PK-R) deficiency. Because red blood cells rely solely on glycolysis to generate ATP, PK-R deficiency can severely impact energy supply and cause reduction in red blood cell lifespan. We determined the underlying cause of the anaemia and investigated how erythroid precursors in the patient survive. METHODS: PK activity assays, Western blot and Sanger sequencing were employed to determine the underlying cause of the anaemia. Patient erythroblasts were cultured and reticulocytes were isolated to determine PK-R and PKM2 contribution to glycolytic activity during erythrocyte development. RESULTS: We found a novel homozygous mutation (c.583G>A) in the PK-R coding gene (PKLR). Although this mutation did not influence PKLR mRNA production, no PK-R protein could be detected in the red blood cells nor in its precursors. In spite of the absence of PK-R, the reticulocytes of the patient exhibited 20% PK activity compared with control. Western blotting revealed that patient erythroid precursors, like controls, express residual PKM2. CONCLUSIONS: We conclude that PKM2 rescues glycolysis in PK-R-deficient erythroid precursors.

Differential sensitivity to JAK inhibitory drugs by isogenic human erythroblasts and hematopoietic progenitors generated from patient-specific induced pluripotent stem cells.

Disease-specific induced pluripotent stem cells (iPSCs) provide an unprecedented opportunity to establish novel disease models and accelerate drug development using distinct tissue target cells generated from isogenic iPSC lines with and without disease-causing mutations. To realize the potential of iPSCs in modeling acquired diseases which are usually heterogeneous, we have generated multiple iPSC lines including two lines that are JAK2-wild-type and four lines homozygous for JAK2-V617F somatic mutation from a single polycythemia vera (PV) patient blood. In vitro differentiation of the same patient-derived iPSC lines have demonstrated the differential contributions of their parental hematopoietic clones to the abnormal erythropoiesis including the formation of endogenous erythroid colonies. This iPSC approach thus may provide unique and valuable insights into the genetic events responsible for disease development. To examine the potential of iPSCs in drug testing, we generated isogenic hematopoietic progenitors and erythroblasts from the same iPSC lines derived from PV patients and normal donors. Their response to three clinical JAK inhibitors, INCB018424 (Ruxolitinib), TG101348 (SAR302503), and the more recent CYT387 was evaluated. All three drugs similarly inhibited erythropoiesis from normal and PV iPSC lines containing the wild-type JAK2 genotype, as well as those containing a homozygous or heterozygous JAK2-V617F activating mutation that showed increased erythropoiesis without a JAK inhibitor. However, the JAK inhibitors had less inhibitory effect on the self-renewal of CD34+ hematopoietic progenitors. The iPSC-mediated disease modeling thus underlies the ineffectiveness of the current JAK inhibitors and provides a modeling system to develop better targeted therapies for the JAK2 mutated hematopoiesis.

Mammalian erythroblast enucleation requires PI3K-dependent cell polarization.

Enucleation, the final step in terminal differentiation of mammalian red blood cells, is an essential process in which the nucleus surrounded by the plasma membrane is budded off from the erythroblast to form a reticulocyte. Most molecular events in enucleation remain unclear. Here we show that enucleation requires establishment of cell polarization that is regulated by the microtubule-dependent local activation of phosphoinositide 3-kinase (PI3K). When the nucleus becomes displaced to one side of the cell, actin becomes restricted to the other side, where dynamic cytoplasmic contractions generate pressure that pushes the viscoelastic nucleus through a narrow constriction in the cell surface, forming a bud. The PI3K products PtdIns(3,4)P2 and PtdIns(3,4,5)P3 are highly localized at the cytoplasmic side of the plasma membrane. PI3K inhibition caused impaired cell polarization, leading to a severe delay in enucleation. Depolymerization of microtubules reduced PI3K activity, resulting in impaired cell polarization and enucleation. We propose that enucleation is regulated by microtubules and PI3K signaling in a manner mechanistically similar to directed cell locomotion.

Ultrastructural study of transcription factories in mouse erythroblasts.

RNA polymerase II (RNAPII) transcription has been proposed to occur at transcription factories; nuclear focal accumulations of the active, phosphorylated forms of RNAPII. The low ratio of transcription factories to active genes and transcription units suggests that genes must share factories. Our previous analyses using light microscopy have indicated that multiple genes could share the same factory. Furthermore, we found that a small number of specialized transcription factories containing high levels of the erythroid-specific transcription factor KLF1 preferentially transcribed a network of KLF1-regulated genes. Here we used correlative light microscopy in combination with energy filtering transmission electron microscopy (EFTEM) and electron microscopy in situ hybridization (EMISH) to analyse transcription factories, transcribing genes, and their nuclear environments at the ultrastructural level in ex vivo mouse foetal liver erythroblasts. We show that transcription factories in this tissue can be recognized as large nitrogen-rich structures with a mean diameter of 130 nm, which is considerably larger than that previously seen in transformed cultured cell lines. We show that KLF1-specialized factories are significantly larger, with the majority of measured factories occupying the upper 25th percentile of this distribution with an average diameter of 174 nm. In addition, we show that very highly transcribed genes associated with erythroid differentiation tend to occupy and share the largest factories with an average diameter of 198 nm. Our results suggest that individual factories are dynamically organized and able to respond to the increased transcriptional load imposed by multiple highly transcribed genes by significantly increasing in size.

Caspase activation is required for terminal erythroid differentiation.

The cysteine proteases known as caspases play a central role in most apoptotic pathways. Here, we show that caspase inhibitors arrest the maturation of human erythroid progenitors at early stages of differentiation, before nucleus and chromatin condensation. Effector caspases such as caspase-3 are transiently activated through the mitochondrial pathway during erythroblast differentiation and cleave proteins involved in nucleus integrity (lamin B) and chromatin condensation (acinus)without inducing cell death and cleavage of GATA-1. These observations indicate a new function for caspases as key proteases in the process of erythroid differentiation.

Rac1 and Rac2 GTPases are necessary for early erythropoietic expansion in the bone marrow but not in the spleen.

BACKGROUND: The small Rho GTPases Rac1 and Rac2 have both overlapping and distinct roles in actin organization, cell survival, and proliferation in various hematopoietic cell lineages. The role of these Rac GTPases in erythropoiesis has not yet been fully elucidated. DESIGN AND METHODS: Cre-recombinase-induced deletion of Rac1 genomic sequence was accomplished on a Rac2-null genetic background, in mouse hematopoietic cells in vivo. The erythroid progenitors and precursors in the bone marrow and spleen of these genetically engineered animals were evaluated by colony assays and flow cytometry. Apoptosis and proliferation of the different stages of erythroid progenitors and precursors were evaluated by flow cytometry. RESULTS: Erythropoiesis in Rac1(-/-);Rac2(-/-) mice is characterized by abnormal burst-forming unit-erythroid colony morphology and decreased numbers of megakaryocyte-erythrocyte progenitors, erythroid colony-forming units, and erythroblasts in the bone marrow. In contrast, splenic erythropoiesis is increased. Combined Rac1 and Rac2 deficiency compromises proliferation of the megakaryocyte-erythrocyte progenitor population in the bone marrow, while it allows increased survival and proliferation of megakaryocyte-erythrocyte progenitors in the spleen. Conclusions These data suggest that Rac1 and Rac2 GTPases are essential for normal bone marrow erythropoiesis but that they are dispensable for erythropoiesis in the spleen, implying different signaling pathways for homeostatic and stress erythropoiesis.

p-ERK1/2 is a predictive factor of response to erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes.

Serum erythropoietin level less than 100U/L and a transfusion requirement of less than 2 units per month are the best predictive factors for response to treatment by erythropoiesis-stimulating agents in low/int-1 myelodysplastic syndromes. To investigate the factors influencing the response to erythropoiesis-stimulating agents, we enrolled 127 low/int-1 myelodysplastic syndrome patients at diagnosis in a biological study of erythropoiesis. The 54 non-responders had a significantly lower number of burst-forming unit-erythroid and colony-forming unit-erythroid than responders. Erythropoietin-dependent proliferation and survival, and phospho (p)-ERK1/2 expression in steady state and after erythropoietin stimulation were defective in cultured erythroblasts. By flow cytometry, p-ERK1/2 was significantly lower in bone marrow CD45(-)/CD71(+)/GPA(-)cells from non-responders compared to responders or controls. Receiver Operator Characteristic curve analysis showed that this flow cytometry test was a sensitive biomarker for predicting the response to erythropoiesis-stimulating agents.

Studies of murine erythroid cell development. Synthesis of heme and hemoglobin.

Techniques of cell separation were used to isolate murine erythroid precursors at different states of maturation. Cells were studied before and after short-term incubation in the presence or absence of erythropoietin. Complementary results were obtained by direct examination of the cell fractions and by the short-term culture experiments. Indices of heme synthesis, including incorporation of 59Fe or [2-14C]glycine into heme and activity of delta-aminolevulinic acid synthetase, were already well developed in the least mature cells, chiefly pronormoblasts. Activity then rose moderately in the cell fractions consisting primarily of basophilic and polychromatophilic normoblasts, and fell off with further increases in cell maturity. On short-term culture in the presence of erythropoietin, activity declined with increasing cell maturation except in the least mature fraction where the original level of activity was maintained. By contrast, synthesis of labeled hemoglobin ([3H]leucine) was very low in the least mature cell fractions and rose progressively with increasing cell maturity. The rate of hemoglobin synthesis increase in cells at all stages of maturation when cultured in the presence of erythropoietin. Despite the different patterns observed for heme synthesis and hemoglobin synthesis, both synthetic activities were consistently higher in cells cultured with erythropoietin as compared to controls. These findings suggest that erythropoietin stimulates biochemical differentiation of erythroid precursors at various stages of maturation. They also demonstrate an asynchronism between heme synthesis and hemoglobin syhthesis; heme synthesis is already well developed in the least mature erythroid cells and begins to diminish as the capacity for hemoglobin synthesis continues to rise.

Regulation of CREB activation by p38 mitogen activated protein kinase during human primary erythroblast differentiation.

Among the molecular events underlying erythroid differentiation, we analyzed the signalling pathway leading to cAMP response element binding (CREB) nuclear transcription factor activation. Normal donor blood light density cells differentiated to pro-erythroblasts during the proliferative phase (10 days) of the human erythroblast massive amplification (HEMA) culture, and to orthochromatic erythroblasts, during the differentiation phase (4 additional days) of the culture. Since erythropoietin was present all over the culture, also pro-erythroblasts left in proliferative medium for 14 days continued their maturation without reaching the final steps of differentiation. p38 mitogen activated protein kinase (p38 MAPK) and CREB maximal activation occurred upon 4 days of differentiation induction, whereas a lower activation was detectable in the cells maintained in parallel in proliferative medium (14 days). Interestingly, when SB203580, a specific p38 MAPK inhibitor, was added to the culture the percentage of differentiated cells decreased along with p38 MAPK and CREB phosphorylation. All in all, our results evidence a role for p38 MAPK in activating CREB metabolic pathway in the events leading to erythroid differentiation.

Human erythroblasts with c-Kit activating mutations have reduced cell culture costs and remain capable of terminal maturation.

A major barrier to the in vitro production of red blood cells for transfusion therapy is the cost of culture components, with cytokines making up greater than half of the culture costs. Cell culture cytokines also represent a major expense for in vitro studies of human erythropoiesis. HUDEP-2 cells are an E6/E7 immortalized erythroblast line used for the in vitro study of human erythropoiesis. In contrast to other cell lines used to study human erythropoiesis, such as K562 cells, HUDEP-2 cells are capable of terminal maturation, including hemoglobin accumulation and chromatin condensation. As such, HUDEP-2 cells represent a valuable resource for studies not amenable to primary cell cultures; however, reliance on the cytokines stem cell factor (SCF) and erythropoietin (EPO) make HUDEP-2 cultures very expensive to maintain. To decrease culture costs, we used CRISPR/Cas9 genome editing to introduce a constitutively activating mutation into the SCF receptor gene KIT, with the goal of generating human erythroblasts capable of SCF-independent expansion. Three independent HUDEP-2 lines with unique KIT receptor genotypes were generated and characterized. All three lines were capable of robust expansion in the absence of SCF, decreasing culture costs by approximately half. Importantly, these lines remained capable of terminal maturation. Together, these data suggest that introduction of c-Kit activating mutations into human erythroblasts may help reduce the cost of erythroblast culture, making the in vitro study of erythropoiesis, and the eventual in vitro production of red blood cells, more economically feasible.

Biological and clinical relevance of matrix metalloproteinases 2 and 9 in acute myeloid leukaemias and myelodysplastic syndromes.

We analysed by immunocytochemistry metalloproteinase (MMP)-2 and MMP-9 expression in bone marrow cells from 54 acute myeloid leukaemia (AML) patients, 153 myelodysplastic syndrome (MDS) patients, and 52 non-haemopathic subjects, in order to evaluate whether MMP expression abnormalities were associated with relevant laboratory or clinical findings. In normal samples MMP-2 was detected in rare myeloid cells, MMP-9 in most maturing myeloid cells. In MDS MMP-2 myeloid levels were higher than in controls (P < 0.0001); MMP-2 and MMP-9 were often co-expressed. Also many erythroblasts expressed MMP-2. There was a positive correlation between MMP-2 erythroblast expression and erythroid dysplasia (P = 0.002) and an inverse correlation between MMP-2 or MMP-9 myeloid expression and blast cell percentage (P = 0.05 and P = 0.04 respectively). High MMP levels in myeloid cells were associated with longer overall survival (P = 0.03) and evolution-free survival (P = 0.04). In AML MMP-2 levels were lower than in MDS (P < 0.0001) and MMP-9 levels lower than in MDS and controls (P < 0.0001). MMP levels did not predict response to therapy. The release of active MMPs was detected by colorimetric analysis in cell cultures from representative MDS and AML cases. In conclusion, we have demonstrated an abnormal MMP expression in AML as well as in MDS. The production and release of these enzymes may influence haematopoietic cell behaviour. In MDS, the detection of MMP deregulated expression may be important also from the clinical point of view: it may provide a useful tool for diagnosis, prognosis and a possible target for experimental treatments.

Multiple enzymatic defects in mitochondria in hematological cells of patients with primary sideroblastic anemia.

Activities of mitochondrial enzymes in blood cells from 69 patients with primary sideroblastic anemia were determined to elucidate the pathogenesis of the disease. In erythroblasts of patients with primary acquired type the activities of both delta-aminolevulinic acid synthetase and mitochondrial serine protease were inevitably decreased. The susceptibility to the protease of apo-delta-aminolevulinic acid synthetase prepared from erythroblasts of patients with this type was within the normal range, in contrast to that of pyridoxine-responsive anemia. The activities of mitochondrial enzymes such as cytochrome oxidase, serine protease, and oligomycin-sensitive ATPase, except citrate synthetase, were usually decreased in mature granulocytes of the patients. Patients with hereditary sideroblastic anemia also had decreased delta-aminolevulinic acid synthetase activity in erythroblasts, and decreased serine protease activity in both erythroblasts and mature granulocytes. Mature granulocytes obtained from patients with pyridoxine-responsive anemia before therapy had decreased cytochrome oxidase activity, however, the activity increased to a normal level when the patients were in remission. The activities of other mitochondrial enzymes in mature granulocytes were within normal range in these patients before pyridoxine therapy. The activities of these mitochondrial enzymes in lymphocytes were within normal range in all groups of patients with primary sideroblastic anemia. We suggest that patients with primary acquired, and possibly also those with hereditary sideroblastic anemia have impaired mitochondrial function in both erythroblasts and granulocytes. That only anemia is observed in these patients is because a functional abnormality of mitochondria in erythroblasts is most important because of the role of mitochondria in the formation of heme in erythrocyte development. In contrast to these two types of sideroblastic anemia, only delta-aminolevulinic acid synthetase in both erythroblasts and granulocytes seems to be impaired in patients with pyridoxine-responsive anemia.

The Methyltransferase Setd8 Is Essential for Erythroblast Survival and Maturation.

Erythropoiesis is a highly regulated process that generates enucleate red blood cells from committed erythroid progenitors. Chromatin condensation culminating in enucleation is a defining feature of this process. Setd8 is the sole enzyme that can mono-methylate histone H4, lysine 20 and is highly expressed in erythroblasts compared to most other cell types. Erythroid Setd8 deletion results in embryonic lethality from severe anemia due to impaired erythroblast survival and proliferation. Setd8 protein levels are also uniquely regulated in erythroblasts, suggesting a cell-type-specific role for Setd8 during terminal maturation. Consistent with this hypothesis, Setd8 Δ/Δ erythroblasts have profound defects in transcriptional repression, chromatin condensation, and heterochromatin accumulation. Together, these results suggest that Setd8, used by most cells to promote mitotic chromatin condensation, is an essential aspect of the transcriptional repression and chromatin condensation that are hallmarks of terminal erythroid maturation.

Non-apoptotic functions of caspases in myeloid cell differentiation.

Subtle caspase activation is associated with the differentiation of several myeloid lineages. A tightly orchestrated dance between caspase-3 activation and the chaperone HSP70 that migrates to the nucleus to protect the master regulator GATA-1 from cleavage transiently occurs in basophilic erythroblasts and may prepare nucleus and organelle expel that occurs at the terminal phase of erythroid differentiation. A spatially restricted activation of caspase-3 occurs in maturing megakaryocytes to promote proplatelet maturation and platelet shedding in the bloodstream. In a situation of acute platelet need, caspase-3 could be activated in response to IL-1α and promote megakaryocyte rupture. In peripheral blood monocytes, colony-stimulating factor-1 provokes the formation of a molecular platform in which caspase-8 is activated, which downregulates nuclear factor-kappa B (NF-κB) activity and activates downstream caspases whose target fragments such as those generated by nucleophosmin (NPM1) cleavage contribute to the generation of resting macrophages. Human monocytes secrete mature IL-1ß in response to lipopolysaccharide through an alternative inflammasome activation that involves caspase-8, a pathway that does not lead to cell death. Finally, active caspase-3 is part of the proteases contained in secretory granules of mast cells. Many questions remain on how these proteases are activated in myeloid cell lineages, which target proteins are cleaved, whereas other are protected from proteolysis, the precise role of cleaved proteins in cell differentiation and functions, and the link between these non-apoptotic functions of caspases and the death of these diverse cell types. Better understanding of these functions may generate therapeutic strategies to control cytopenias or modulate myeloid cell functions in various pathological situations.

Hexokinase in developing rabbit erythroid cells.

The activity and isozyme distribution of hexokinase were studied in bone marrow cells from normal and anemic rabbits separated 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. Most of the falls in hexokinase activity take place when 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.

Arginine methyltransferase activity in chronic erythremic myelosis (Diguglielmo syndrome).

Activity of S-adenosylmethionine-dependent arginine methyltransferase was substantially higher in sonicated bone marrow samples from 6 patients with chronic erythremic myelosis than in bone marrow from 3 patients with untreated pernicious anemia, 2 patients with autoimmune hemolytic anemia, and 4 normal persons. Increased activity of this enzyme may be one of the factors contributing to the pathogenesis fo methylated arginines in histones of erythroblasts from patients with chronic erythremic myelosis.

Regulation of haem biosynthesis in normoblastic erythropoiesis: role of 5-aminolaevulinic acid synthase and ferrochelatase.

The development of haem biosynthetic enzyme activity during normoblastic human erythropoiesis was examined in seven patients. The first and last enzymes of the haem biosynthetic pathway, ALA synthase and ferrochelatase, were assayed by radiochemical/high performance liquid chromatographic (HPLC) methods. An assay for ferrochelatase activity in human bone marrow was developed. Enzyme substrates were protoporphyrin IX and 59Fe2+ ions. 59Fe-labelled haem was isolated by organic solvent extraction/sorbent extraction followed by reversed-phase HPLC. Optimal activity occurred at pH 7.3 in the presence of ascorbic acid, in darkness and under anaerobic conditions. Haem production was proportional to cell number and was linear with time to 30 min. The assay was sensitive to the picomolar range of haem production. ALA synthase and ferrochelatase activity was assayed in four highly purified age-matched erythroid cell populations. ALA synthase activity was maximal in the most immature erythroid cells and diminished as the cells matured with an overall five fold loss of activity from proerythroblast to late erythroblast development. Ferrochelatase activity was, however, more stable with less than a two fold change in activity observed during the same period of erythroid differentiation. Maximal activity occurred in erythroid fractions enriched with intermediate erythroblasts. These results support sequential rather than simultaneous appearance of these enzymes during normoblastic erythropoiesis. Quantitative analysis of relative enzyme activity however indicates that at all times during erythroid differentiation ferrochelatase activity is present in excess to that theoretically required relative to ALA synthase activity since ALA and haem are not produced in stoichiometric amounts. The lability of ALA synthase versus the stability and gross relative excess of ferrochelatase activity indicates a far greater role for ALA synthase in the regulation of erythroid haem biosynthesis than for ferrochelatase.

Intracellular distribution of ribonuclease activity during erythroid cell development.

Five ribonuclease activities, separable by polyacrylamide gel electrophoresis, have been detected in erythroid bone marrow cells from anaemic rabbits. Their intracellular distribution has been investigated and compared with that of the ribonucleases in reticulocytes. Both the acid and alkaline ribonuclease activities of reticulocytes are much lower (30--50 fold) than those of bone marrow erythroid cells. The most marked decrease in enzyme activity occurs in the fractions containing ribosomes and mitochondria plus lysosomes. In these subcellular organelles there was also a qualitative change in the ribonuclease electrophoretic pattern, whereas the cytosol enzymes of marrow erythroid cells and reticulocytes remained largely unchanged. Several ribonucleases released from reticulocyte membranes with urea were similar to those present in the lysosomal plus mitochondrial fraction, as shown by detection of enzyme activity after polyacrylamide gel electrophoresis. The decline in ribonuclease activity was found to begin in the orthochromatic cells, which have a highly condensed nucleus and are no longer active in DNA and RNA synthesis, and to coincide with a decrease in acid phosphatase activity and loss of lysosomes.