Comparative kinetic behaviour and regulation by fructose-1,6-bisphosphate and ATP of pyruvate kinase from erythrocytes, reticulocytes and bone marrow cells

1. Kinetic and regulatory properties of pyruvate kinase have been studied in haemolysates of erythrocytic populations from blood and bone marrow of rats. 2. Pyruvate kinase from normal rat erythrocytes showed sigmoidal kinetics vs phosphoenolpyruvate. In contrast, the enzyme from reticulocytes and erythroid-rich bone marrow cells behaved as hyperbolic. 3. The enzyme activities were always inhibited by ATP. Activation by fructose-1,6-bisphosphate was only observed in erythrocytes. 4. These kinetic differences suggest changes in pyruvate kinase isozymes in cells of the erythrocytic line of rats.     

Membrane differentiation of developing hemic cells of the bone marrow demonstrated by changes in concanavalin A surface labeling

The concanavalin A-gold labeled horseradish peroxidase (Con A-HRP-G) method has been employed in the ultrastructural localization of Con A surface receptor sites on glutaraldehyde-fixed normal human and guinea pig bone marrow cells. The number of gold particles per micron of cell surface was counted and data subjected to statistical analysis. All cells of the bone marrow exhibited Con A binding; however, the extent of surface labeling was dependent both on cell type and stage of differentiation. Distinctive modifications in mean surface density correlated with specific periods during the maturation of the erythrocytic, neutrophilic, eosinophilic and monocytic cell series. In several instances, the differentiative changes in surface Con A labeling proved to be species dependent. These observations are discussed in relationship to methodology and to potential changes in number and/or spatial arrangement of Con A receptor sites, primarily attributable to mannosyl and/or glucosyl residues associated with membrane glycoproteins and/or glycolipids of developing neutrophilic and erythrocytic cells.     

The effect of splenectomy on bone marrow haematopoiesis in mice.

Splenectomy was performed in strain H mice. Erythrocyte macrocytosis and an increase in the reticulocyte, leucocyte and thrombocyte count were found in the peripheral blood of splenectomized animals; only the erythrocyte count fell in the first 3 weeks after splenectomy. Changes in the myelogram during the first weeks after splenectomy were characterized by an increase in the proportion of cells of the erythrocytic and lymphocytic series. The stem cell count in the bone marrow (determined after Till and McCulloch) was slightly elevated on the 8th day after splenectomy, but in subsequent weeks was rather lower than the control group values. Whatever the post-splenectomy interval at which bone marrow was taken from splenectomized mice, there was no significant difference in the transplantation effect of bone marrow cells on white and thrombocyte haematopoiesis. Bone marrow transplantation was found have a stimulant effect only on the reticulocyte count and the sooner bone marrow was collected after splenectomy, the more pronounced the effect. Changes in the myelogram and splenogram of irradiated mice to which the bone marrow cells of splenectomized mice had been transplanted were relatively small.     

Haemoglobin types in peripheral blood and bone marrow cells in rats after a single blood loss.

The authors studied quantitative changes in the synthesis of haemoglobin types in rat peripheral erythrocytes and in the cells of the bone marrow erythrocytic series after a single blood loss. They demonstrated that raised synthesis of given haemoglobin types could be found in the peripheral cells 5-7 days after blood loss. These changes could already be detected in the bone marrow cells 24 hours after blood loss. The question of the probable site and level of the origin of altered haemoglobin synthesis is discussed.     

Solubility behaviour of phosphofructokinase from haemolysate of erythrocytes, reticulocytes and bone marrow cells in poly (ethylene glycol) solutions.

The precipitation profiles of phospofructokinase obtained by addition of poly(ethylene glycol) to haemolysates of erythrocytes, reticulocytes and bone marrow are displaced towards higher polymer concentrations when the pH decreases from 6 to 5 or increases from 6 to 8. In the pH range 5 to 8, the concentration of polymer required to provide any level of precipitation follows the order erythrocytic less than reticulocytic less than bone marrow phosphofructokinase. The precipitation of erythrocytic and reticulocytic phosphofructokinase is enhanced by the presence of F6P and ATP. No effect is observed for bone marrow phosphofructokinase. These results are consistent with an isoenzymatic variation of phosphofructokinase in erythrocytes, reticulocytes and bone marrow cells.     

Preferential expression of the c-fps protein in chicken macrophages and granulocytic cells.

We have studied the expression of the protein kinase activity of NCP98, the c-fps gene product, in several hemopoietic tissues of chickens as a function of the developmental stage of these organs. We found that in bone marrow, spleen, and bursa, maximum NCP98 kinase activity on a per-cell basis correlates with the peak of granulopoiesis in these organs. Furthermore, in a bovine serum albumin density gradient fractionation of bone marrow cells, granulocytic cells appeared to account for most of the NCP98 kinase activity. No correlation was found between the distribution of erythrocytic, lymphocytic, or thrombocytic cells and the distribution of the expression of NCP98 kinase activity. However, NCP98 protein and kinase activity were 10-fold higher in macrophages than in bone marrow. In addition, depletion by complement-mediated lysis of erythrocytic cells in bone marrow did not significantly reduce the total recovery of NCP98 kinase activity. These results argue for the specific expression of the c-fps gene product in granulocytic cells and macrophages.     

Heterologous antiserum to rat lymphohemopoietic precursor cells.

Lymphohemopoietic precursor cells in rat bone marrow are members of a subset of lymphocyte-like cells that bears the bone marrow lymphocyte antigen (BMLA) and that lacks antigens present on peripheral B and T cells. This was demonstrated by two experimental approaches. In the first, bone marrow cells with the potential to form hemopoietic colonies in spleen (CFU-S), to repopulate lumphoid tissues and blood, and to rescue lethally irradiated recipients were enriched approximately 10-fold by a fractionation procedure designed to isolate a "null" population of bone marro lymphocytes. In the second approach, the lymphohemopoietic precursor cell activity in bone marrow was completely abrogated by opsonization with rabbit antiserum (ALSBM) raised against this "null" population of bone marrow cells. Precursor cell activity was not affected by treatment with antiserum to T and B cells. Quantitative cross-absorption studies showed that the antigen detected by ALSBM on lymphohemopoietic precursor cells had the same cellular distribution as did the previously described bone marrow lymphocyte antigen. It is likely that this antigen is present both on pluripotent stem cells and on committed progenitors of the myelocytic, erythrocytic and lymphocytic series.     

Surface differentiation of hemopoietic cells demonstrated ultrastructurally with cationized ferritin

The ultrastructural cationized ferritin (CF) technique was employed as a probe of the surface binding characteristics of the various cell types present in normal human bone marrow. The number of CF particles per micron length of cell surface were counted and data subjected to statistical analysis. All cells of the bone marrow exhibited CF reactivity. The extent of labeling was cell specific and could be related to the stage of maturation of the cells in a given lineage. In the neutrophilic series, myeloblasts showed moderate labeling while promyelocytes and myelocytes revealed only minimal binding; CF binding increased sequentially in metamyelocytes, band and segmented neutrophils. Eosinophils and eosinophilic myelocytes showed similar membrane differnetiation patterns while basophils exhibited stronger CF labeling that other granulocytic cells. Lymphocytes were strongly reactive while monocytes and their precursors were moderately labeled with CF. Surface reactivity of developing nucleated erythrocytic cells was similar to that of the lymphocytes. Surface labeling from the proerythroblasts to early normoblasts stage was identical, CF binding increased in the late normoblasts stage and then decreased in the reticulocyte and mature erythrocyte stages. The extent of surface CF reactivity of the marrow cells was markedly different from that obtained with Thorotrast and colloidal iron. Thorotrast and colloidal iron stained the surface of all marrow cell intensely but failed to yield distinctive surface labeling patterns for the differing cell population in bone marrow.     

Various indicators of humoral immunity in patients with hematopoietic hypoplasia after bone marrow transplantation]

The authors examined 65 patients with hypoplasia of the hemopoiesis following bone marrow transplantation. To patients of the 1st group the bone marrow was chosen by erythrocytic antigens only, and to patients of the 2nd group both by erythrocytic and by leukocytic antigens. The data obtained pointed to the reduction of isoimmunization to the antigens of leukocytes and platelets in the patients with hypoplasia of the hemopoiesis following transplantation of the histocompatible bone marrow, in comparison with the patients in whom the bone marrow was chosen only by erythrocytic antigens alone.     

Localization of pyroantimonate-precipitable cation and surface coat anionic binding sites in developing erythrocytic cells and macrophages in normal human bone marrow

Summary The surfaces of developing erythrocytic cells and macrophages have been examined in normal human bone marrow by means of the pyroantimonate-osmium, ruthenium red and Thorotrast techniques for inorganic cations, surface glycoprotein-phospholipid complexes and surface anionic binding sites, respectively. No differences in the degree of surface coat reactivity were noted in the erythrocytic cells at different stages of maturation while pyroantimonate binding to the plasmalemma was not evident developmentally until the final stages of erythrocytic development. Rhopheocytotic invaginations proved to be chemically distinct from the remainder of the cell surface since they did not bind Thorotrast or pyroantimonate and gave more staining with ruthenium red. Pyroantimonate does not bind to the surface of macrophages and the binding of Thorotrast by these cells is less. Macrophage-erythrocytic cell contact zones did not stain with Thorotrast but stained with ruthenium red. The significance of these observations is discussed.Supported by Grant No. AM-HE-12084-14 from the National Institutes of Health, Bethesda, Maryland. — Appreciation is expressed to Anita Topson and Marjorie Griffith for their technical assistance and to Dr. Robert Hilberg for performing the bone marrow aspirations.     

Cytoenzymochemical changes in acute leukemia cells under cytostatic treatment

Cytomorphologic and cytoenzymochemical changes occurring in leukemic blastic cells of bone marrow and peripheral blood were studied concomitantly in 180 cases of acute leukemia treated with one or more cytostatics, in various association related to the main cytologic type. Cellular effects due to monochemotherapy in various types of acute leukemia varied depending on the cytostatic dose, duration of treatment, and sensitivity of blastic cells to the cytostat. The expression of cellular sensitivity was marked by megaloblastosis of myeloid elements, cell gigantism and intranuclear and cytoplasmic vacuolization. Resistance to cytostatics was demonstrated both by the morphologic aspect of blastic cells which remained unchanged and by their overloading with glycogen, lipids and peroxidases. The relationships between posttherapeutic cellular changes and clinical parameters are discussed.     

Hematopoietic tissue in malaria: facilitation of erythrocytic recycling by bone marrow in Plasmodium berghei-infected mice

In P. berghei murine malaria, parasitized erythrocytes and nonparasitized reticulocytes were concentrated in the lumen of venous sinuses of bone marrow, adherent to endothelium and to one another. Merozoites maturing in erythrocytes contained in venous sinuses are so positioned against adherent reticulocytes that when they break out they can penetrate adherent reticulocytes without significant extracellular exposure. Merozoites from cells not adjacent to reticulocytes would spill into the plasma and be opsonized and phagocytized. The vascular sinuses of bone marrow therefore appear to be sites where erythrocytic parasitization is secured and where the severity and chronicity of the erythrocytic phase of the disease is regulated in P. berghei and, perhaps, other reticulocyte-prone malarias. In P. chabaudi, where fully mature erythrocytes rather than reticulocytes are preferentially parasitized, parasitized erythrocytes show far less predilection for bone marrow.     

On artifacts appearing in the histochemical fixation of glycogen

1. Fixation artifacts associated with glycogen translocation are prevalent in tissues of parenchymatous type and scarce or non-existent in tissues of loose type. 2. Liver tissue treated with M/3 NaOH solution before fixation did not show an uneven distribution of glycogen. This was interpreted as indicating that the liver, a tissue of parenchymatous type, was changed, so to speak, into a loose type of tissue by alkali treatment. 3. The so called Alkohol-flucht of glycogen was produced in Yoshida's ascites tumor cells by a procedure which changed a loose type of tissue into a parenchymatous one, that is, by packing the tumor cells tightly. 4. The translocation of glycogen in cells appeared to occur when the fixatives penetrated the cells rapidly from a single direction, but failed to occur when the cells were attacked by the fixative from all directions. 5. In dried smears of Yoshida's ascites tumor cells and bone marrow cells, the glycogen particles are translocated to the peripheral regions of the cells, and coalesce there. The production of these artifacts is related in some way to the physicochemical properties of the protoplasm and plasma membrane of the cells.     

THE EFFECT OF POLYCYTHAEMIC SERUM ON THE PROLIFERATION OF RAT BONE MARROW CELLS IN VITRO

The effect of experimental polycythaemia on the rate of proliferation of erythrocytic precursor cells was investigated by means of an in vitro technique. The serum obtained from polycythaemic rats was found to inhibit significantly ³H-thymidine incorporation in normal rat bone marrow cells in vitro, as compared with normal serum. Autoradiographic analysis revealed that this inhibition resulted from a reduction in the number of labelled bone marrow cells. The inhibition proved to be specific to the erythrocyte precursor cells; the labelling index was reduced in the erythrocytic cell population by 21–50% (P < 0.001) at different incubation times, while the effect on the granulocytic cell population was negligible. It is deduced that an inhibitor substance responsible for the effects observed is present in polycythaemic serum. It is proposed that this factor is the ‘erythrocytic chalone'. The results support the general view that triggering of stem cells is not the only mode of regulation of erythropoiesis, but that the rate of proliferation of the precursor cells in the erythron is also regulated.     

Up-regulation of alpha2,6 sialylation during myeloid maturation: a potential role in myeloid cell release from the bone marrow

The mechanisms by which mature myeloid cells are released from the bone marrow into the peripheral blood are not clearly understood. Glycosylation is likely to play an important role, as has been shown in the homing of lymphocytes to lymph nodes and of neutrophils to inflamed endothelia. Cell surface sialylation is an important component of many cellular adhesive interactions, both as ligand-promoting interactions, as occurs in selectin and sialoadhesin-mediated adhesion, and for reducing cell adhesion as in some cancer cells. We have studied the expression of cell surface alpha2,6-linked sialic acid in the maturation of normal bone marrow myeloid cells, the expression of alpha2,6-sialyltransferase mRNA, and the role of sialylation in the adherence of myeloid cells to bone marrow stroma. Our data show that there is a dramatic increase in cell surface alpha2,6-sialylation during the late stage of maturation. This up-regulation is restricted to specific glycoproteins including CD11b and CD18. It is associated with a relative increase in the level of alpha2,6-sialyltransferase mRNA compared with alpha2,3-sialyltransferase mRNA. The changes in mature bone marrow myeloid cells are associated with reduced cell binding to fibronectin and cultured bone marrow stroma. Our data strongly suggest that alpha2,6-sialylation may be important in the interaction between maturing myeloid cells and bone marrow stroma and may govern the release of cells from the bone marrow into the peripheral blood.     

Pathophysiological Scenario of Hematopoietic Disorders: A Comparative Study of Aplastic Anemia,Myelodysplastic Syndrome and Leukemia in Experimental Animals

The conversion of physiology to pathophysiology in hematological disorders viz: aplastic anemia, myelodysplastic syndrome (MDS) and leukemia in murine models was the subject of study in the present programme. Peripheral blood hemogram, spleno-somatic index, bone marrow smear study, cytochemical staining of marrow, cell release kinetics study during marrow explants culture, hematopoietic niche assessment, chromosomal aberration study, plasma membrane stability study of marrow cells, lysosomal membrane and mitochondrial membrane stability study and innate immune parameters were performed in the aplastic anemia, leukemia and MDS mouse model. In bone marrow aplasia, peripheral blood pancytopenia, marrow hypocellularity, decreased marrow cellular viability, deterioration of bone marrow hematopoiesis as well as hematopoietic microenvironment and extramedullary hematopoiesis were noticed. In addition, disruption of mitochondrial and lysosomal membrane integrity along with reduction of innate immune parameters were found in the hematopoietic suppressed condition. Surprisingly, no noticeable chromosomal aberration was found in the aplastic condition. Ineffective marrow hematopoiesis together with the disruption of hematopoietic microenvironment was observed in MDS. Also, extramedullary hematopoiesis, increased marrow cellular death, chromosomal aberration and loss of innate immunity were the common events. During leukemia, the number of functionally and structurally immature cells in the peripheral blood and bone marrow was increased together with malignant conversion of hematopoietic cells in the presence of malignancy supportive stromal microenvironment. Chromosomal aberration, decrease of cell mediated immunity with least mitochondrial apoptotic damage were also found in leukemic condition as well.     

T-cell depletion of bone marrow does not inhibit in vitro hematopoiesis

One approach to overcome the problem of histoincompatibility in bone marrow transplantation is to use T cell depleted marrow from a haploidentical donor in an attempt to ameliorate graft-versus-host disease. Since the T cell requirements for normal hematopoiesis are uncertain, experiments were performed to study the effects of E rosette-T cell depletion on in vitro growth of hematopoietic progenitor cells. Marrow mononuclear cells were cultured in a modified CFU-GEMM assay before and after T cell depletion. The number of 7 day granulocytic and erythrocytic colonies, and 14 day granulocytic, erythrocytic and mixed colonies were enumerated and expressed in terms of colonies per 10(5) non T cells plated. T cell depletion did not result in decreased proliferation of any of these progenitors save possibly for 14 day granulocytic colonies in one of four experiments. In two cases, T cell depletion resulted in increased growth of progenitor cells. Three of four patients transplanted with T cell depleted haploidentical marrow cells engrafted. It is concluded that E rosette depletion of T cells from marrow does not decrease the potential of these cells to establish hematopoiesis in vitro or in vivo.     

Epigenetic and microenvironmental alterations in bone marrow associated with ROS in experimental aplastic anemia

Aplastic anemia or bone marrow failure often develops as an effect of chemotherapeutic drug application for the treatment of various pathophysiological conditions including cancer. The long-term bone marrow injury affects the basic hematopoietic population including hematopoietic stem/progenitor cells (HSPCs). The present study aimed in unearthing the underlying mechanisms of chemotherapeutics mediated bone marrow aplasia with special focus on altered redox status and associated effects on hematopoietic microenvironment and epigenetic status of hematopoietic cells. The study involves the development of busulfan and cyclophosphamide mediated mouse model for aplastic anemia, characterization of the disease with blood and marrow analysis, cytochemical examinations of bone marrow, flowcytometric analysis of hematopoietic population and microenvironmental components, determination of ROS generation, apoptosis profiling, expressional studies of Notch-1 signaling cascade molecules, investigation of epigenetic modifications including global CpG methylation of DNA, phosphorylation of histone-3 with their effects on bone marrow kinetics and expressional analysis of the anti-oxidative molecules viz; SOD-2 and Sdf-1. Severe hematopoietic catastrophic condition was observed during aplastic anemia which involved peripheral blood pancytopenia, marrow hypocellularity and decreased hematopoietic stem/progenitor population. Generation of ROS was found to play a central role in the cellular devastation in aplastic marrow which on one hand can be correlated with the destruction of hematopoiesis supportive niche components and alteration of vital Notch-1 signaling and on other hand was found to be associated with the epigenetic chromatin modifications viz; global DNA CpG hypo-methylation, histone-3 phosphorylation promoting cellular apoptosis. Decline of anti-oxidant components viz; Sdf-1 and SOD-2 hinted towards the irreversible nature of the oxidative damage during marrow aplasia. Collectively, the findings hinted towards the mechanistic correlation among ROS generation, microenvironmental impairment and epigenetic alterations that led to hematopoietic catastrophe under aplastic stress. The findings may potentiate successful therapeutic strategy development for the dreadful condition concerned.     

CONTROL OF GRANULOCYTE PRODUCTION: SEPARATION AND CHEMICAL IDENTIFICATION OF A SPECIFIC INHIBITOR (CHALONE)

Abstract. It has previously been shown by others that blood serum contains inhibitors of blood cell production acting on the proliferation of granulocy tic and erythrocytic precursor cells in the bone marrow. It is now shown that the active extract from calf blood serum can be further subfractionated into six different components, all of them exhibiting inhibitory effects on the proliferation of rat bone marrow cells in vitro. Ascitic fluid from rats treated intraperitoneally with polyvinylpyrrolidone contains inhibitors which apparently are the same as those found in calf serum.
It was further possible to demonstrate that only one of these inhibitors is contained in mature granulocytes where it is actively synthesized from amino acids and subsequently released into the surrounding medium. By chromatography on Sephadex G-25 of this conditioned medium the inhibiting substance could be obtained in relatively pure form being contaminated only by low amounts of two ninyhdrin-positive substances. the experiments allow the granulocytic inhibitor to be identified as a polypeptide with a molecular weight below 5000. the results suggest that this substance is the granulocytic chalone.     

Cellular changes in the bone marrow of Plasmodium berghei-infected mice: II. Blast transformation and phagocytosis

The present work is concerned with early cellular changes occurring during a malaria infection. Blast transformation by lymphoid cells and phagocytosis by adherent cells from the bone marrow was performed, using immune and nonimmune Balb/c mice. Nonadherent bone marrow cells from immune mice show an increased specific lymphoblast transformation. This increase was not observed during a lethal infection (PI). Adherent bone marrow cells were assayed for phagocytosis of parasitized (PE) or normal erythrocytes (NE). Cells from immune mice show an increase in phagocytosis of PE and NE. Cells from PI mice showed a decreased phagocytosis throughout the infection, beginning at Day 1 after challenge.