scholarly journals Acetylcholinesterase in murine erythroleukemia (Friend) cells: evidence for megakaryocyte-like expression and potential growth-regulatory role of enzyme activity

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2873-2879 ◽  
Author(s):  
F Paoletti ◽  
A Mocali ◽  
AM Vannucchi
Keyword(s):  
Ache Activity ◽  
Specific Activity ◽  
Soluble Form ◽  
Potential Growth ◽  
Erythroleukemia Cells ◽  
Friend Cells ◽  
Enzyme Specific Activity ◽  
Cellular Replication ◽  
Murine Erythroleukemia

Features of true acetylcholinesterase (AChE) regulation during growth and differentiation of Friend murine erythroleukemia cells (MELC) have been investigated with respect to other erythroid and nonerythroid murine elements. Enzyme levels of uninduced MELC were in between the very low AChE contents of erythroid cells and the huge amounts of activity exhibited by megakaryocytes and platelets. After MELC commitment to terminal division, the enzyme-specific activity increased largely, approaching values that were much closer to those of thrombocytic than of normal erythroid elements. The bulk of AChE activity in MELC, megakaryocytes, and platelets was found to be located in the cytosol as a free-soluble form. Moreover, during incubation, MELC actively released large amounts of AChE into the medium, like it occurs in murine thrombocytes. Conversely, the enzyme of the erythroid elements was mainly associated with the membranes and was not released extracellularly. Experiments with inducers showed that changes in AChE- specific activity of MELC correlated directly with the arrest of cell proliferation rather than with the activation of differentiated erythroid functions. The inverse relationship existing between MELC growth rates and AChE levels was further supported by the relative enzyme activities of the slow- and fast-growing subclones. We conclude that uninduced MELC potentially share properties of both the erythroid and megakaryoblastic phenotype. The latter might be revealed by typical regulation of AChE activity according to a thrombocytic-like program activated upon MELC commitment to terminal division. Eventually, the inhibition of MELC growth by exogenous pure bovine AChE suggested that the secreted murine enzyme might serve as a potential negative signal of cellular replication.

scholarly journals Acetylcholinesterase in murine erythroleukemia (Friend) cells: evidence for megakaryocyte-like expression and potential growth-regulatory role of enzyme activity

Blood ◽  
1992 ◽  
Vol 79 (11) ◽  
pp. 2873-2879 ◽  
Author(s):  
F Paoletti ◽  
A Mocali ◽  
AM Vannucchi
Keyword(s):  
Ache Activity ◽  
Specific Activity ◽  
Soluble Form ◽  
Potential Growth ◽  
Erythroleukemia Cells ◽  
Friend Cells ◽  
Enzyme Specific Activity ◽  
Cellular Replication ◽  
Murine Erythroleukemia

Abstract Features of true acetylcholinesterase (AChE) regulation during growth and differentiation of Friend murine erythroleukemia cells (MELC) have been investigated with respect to other erythroid and nonerythroid murine elements. Enzyme levels of uninduced MELC were in between the very low AChE contents of erythroid cells and the huge amounts of activity exhibited by megakaryocytes and platelets. After MELC commitment to terminal division, the enzyme-specific activity increased largely, approaching values that were much closer to those of thrombocytic than of normal erythroid elements. The bulk of AChE activity in MELC, megakaryocytes, and platelets was found to be located in the cytosol as a free-soluble form. Moreover, during incubation, MELC actively released large amounts of AChE into the medium, like it occurs in murine thrombocytes. Conversely, the enzyme of the erythroid elements was mainly associated with the membranes and was not released extracellularly. Experiments with inducers showed that changes in AChE- specific activity of MELC correlated directly with the arrest of cell proliferation rather than with the activation of differentiated erythroid functions. The inverse relationship existing between MELC growth rates and AChE levels was further supported by the relative enzyme activities of the slow- and fast-growing subclones. We conclude that uninduced MELC potentially share properties of both the erythroid and megakaryoblastic phenotype. The latter might be revealed by typical regulation of AChE activity according to a thrombocytic-like program activated upon MELC commitment to terminal division. Eventually, the inhibition of MELC growth by exogenous pure bovine AChE suggested that the secreted murine enzyme might serve as a potential negative signal of cellular replication.

scholarly journals A new appraisal of the endoglucanases of the fungus Trichoderma reesei

1985 ◽  
Vol 231 (1) ◽  
pp. 75-81 ◽  
Author(s):  
M L Niku-Paavola ◽  
A Lappalainen ◽  
T M Enari ◽  
M Nummi
Keyword(s):  
Trichoderma Reesei ◽  
Specific Activity ◽  
Relative Proportion ◽  
Culture Liquid ◽  
Enzymic Activity ◽  
Soluble Form ◽  
Immunological Methods ◽  
A Value ◽  
And Storage

The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble β-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form.

scholarly journals Calcium influx in induced differentiation of murine erythroleukemia cells

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 783-792 ◽  
Author(s):  
B Gillo ◽  
YS Ma ◽  
AR Marks
Keyword(s):  
Calcium Channel ◽  
Calcium Influx ◽  
Cdna Probe ◽  
Erythroid Differentiation ◽  
Early Event ◽  
Induced Differentiation ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Abstract Murine erythroleukemia cells (MELC) have served as a model for examining the regulation of erythroid differentiation. However, the role of Ca2+ in the signal transduction pathways regulating differentiation remains unclear. To begin to address this uncertainty we have characterized the regulation of cytoplasmic Ca2+ and the possible role of calcium channels during induced differentiation in MELC. MELC can be induced to terminal differentiation using the polar/apolar compound hexamethylene bisacetamide (HMBA). We found that HMBA stimulated Ca2+ influx within 3 to 6 minutes and that Ca2+ entry was required but not sufficient for MELC growth and differentiation. Nifedipine (1 to 10 mumol/L), a calcium channel antagonist, blocked HMBA-induced Ca2+ influx and inhibited differentiation by approximately 60%. Depolarization of the MELC membrane did not induce Ca2+ influx and whole-cell patch-clamp recordings failed to detect a voltage-activated Ca2+ current, suggesting that MELC do not express detectable levels of a functional voltage-dependent calcium channel (VDCC). However, a cDNA probe encoding a portion of the alpha 1 subunit of the cardiac VDCC detected an approximately 8-kb mRNA on Northern blots of total MELC RNA. Taken together, these data show that Ca2+ influx is an early event associated with HMBA-induced differentiation in MELC, blockade of this calcium influx inhibits induced differentiation, and a voltage- insensitive dihydropyridine-sensitive calcium channel may be involved in Ca2+ influx in MELC.

scholarly journals CRISPR/Cas9-mediated deletion of the Wiskott-Aldrich syndrome locus causes actin cytoskeleton disorganization in murine erythroleukemia cells

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6284
Author(s):  
Vanessa Fernández-Calleja ◽  
María-José Fernández-Nestosa ◽  
Pablo Hernández ◽  
Jorge B. Schvartzman ◽  
Dora B. Krimer
Keyword(s):  
Actin Cytoskeleton ◽  
Genomic Region ◽  
Loss Of Function ◽  
Gene Encoding ◽  
Wiskott Aldrich Syndrome ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Actin Protein ◽  
Murine Erythroleukemia Cells

Wiskott-Aldrich syndrome (WAS) is a recessive X-linked inmmunodeficiency caused by loss-of-function mutations in the gene encoding the WAS protein (WASp). WASp plays an important role in the polymerization of the actin cytoskeleton in hematopoietic cells through activation of the Arp2/3 complex. In a previous study, we found that actin cytoskeleton proteins, including WASp, were silenced in murine erythroleukemia cells defective in differentiation. Here, we designed a CRISPR/Cas9 strategy to delete a 9.5-kb genomic region encompassing theWasgene in the X chromosome of murine erythroleukemia (MEL) cells. We show thatWas-deficient MEL cells have a poor organization of the actin cytoskeleton that can be recovered by restoringWasexpression. We found that whereas the total amount of actin protein was similar between wild-type andWasknockout MEL cells, the latter exhibited an altered ratio of monomeric G-actin to polymeric F-actin. We also demonstrate thatWasoverexpression can mediate the activation of Bruton’s tyrosine kinase. Overall, these findings support the role of WASp as a key regulator of F-actin in erythroid cells.

scholarly journals Identification and conditions for selective expression of megakaryocytic markers in Friend erythroleukemia cells

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2624-2631 ◽  
Author(s):  
F Paoletti ◽  
AM Vannucchi ◽  
A Mocali ◽  
R Caporale ◽  
SA Burstein
Keyword(s):  
Specific Activity ◽  
Culture Media ◽  
Late Phase ◽  
Calf Serum ◽  
Erythroid Differentiation ◽  
High Serum ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

Increased Acetylcholinesterase Activity of Microparticles Derived from Red Cells (RMP) Compared to Platelets (PMP).

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3849-3849
Author(s):  
Lawrence L Horstman ◽  
Jacob Esquenazi ◽  
Wenche Jy ◽  
Yeon-Soong Ahn
Keyword(s):  
Flow Cytometry ◽  
Ache Activity ◽  
Specific Activity ◽  
Platelet Rich Plasma ◽  
Substrate Inhibition ◽  
Calcium Ionophore ◽  
Physiological Role ◽  
Natural Inhibitor ◽  
Free Plasma

Abstract INTRODUCTION. Cell-derived microparticles (MP) such as from platelets (PMP), endothelia (EMP) and leukocytes (LMP) are increasingly recognized as useful biomarker and important mediators of thrombosis and inflammation. However, little attention has been paid to the possible role of MP from RBC (RMP) in vascular disorders. RMP were identified by glycophorin (GPH) in flow cytometry in most studies. We reported heterogeneity of RMP in size and phenotypes and that GPH is expressed predominately in larger RMP, not in smaller RMP and that GPH+ RMP are more active than GPH- RMP in thrombin generation. Since acetylcholinesterase (AChE) activity has been measured on RMP, and was recently proposed as a marker of some inflammatory states, we investigated AChE activity of RMP compared to platelet-derived MP (PMP). AChE of PMP has not previously been reported. METHODS. RMP were prepared from intact washed RBC at 18% Ht exposed to calcium ionophore (4uM) in presence of calcium (2mM) for 30 min. PMP were prepared from 20 mL citrated blood, and exposing the platelet-rich plasma to 1 uM calcium ionophore (without added Ca2+) and collagen, 4ug/mL, for 20 min. AChE assay was based on Ellman’s method and reagent (DTNB), run in 96-well plates, 300uL. Substrate was acetylthiocholine iodide (1 mM f.c.). DTNB was used at 0.67 mM f.c. Tests were run +/− quinidine (Q) (1.2 uM) and some tests were in presence of saponin 0.01%. Q is known to inhibit AChE of plasma but RBC activity is insensitive. Activity is expressed in umols substrate cleaved /min per 108 MP, with provisos below. Flow cytometry using FITC labeled lectin, Ulex europaeus (Ulex) was used to quantitate RMP and PMP. RESULTS. As expected, Q inhibited AChE in plasma by >90% but not AChE of RMP. On contrary, RMP were consistently stimulated by Q, up to 150% activity +Q; some preparations of PMP were also stimulated. Saponin, which has been used in assay of RBC AChE, had little effect on PMP or RMP activity. In 12 experiments, AChE of PMP exhibited marked concentration-dependence. The apparent activity per mL of suspension was greater with lesser volumes, by as much as 3-fold between 2.5uL and 20uL added. This could not be explained by substrate inhibition since the effect varied in different preparations, was absent in particle-free plasma, and did not diminish in low substrate. This suggests the presence of a natural inhibitor. Calculation of specific activity of the MP was complicated by the dependence of apparent activity on volume assayed. However, when equal dilutions were compared, a representative experiment showed RMP had about 6-fold greater activity than PMP per 108 MP: 36.0 vs. 5.88 for 2.5uL suspension; and 29.0 vs. 3.9 for 20 uL assayed, in units above. CONCLUSIONS / DISCUSSION. The AChE activity of RMP is about 6-fold greater than PMP. Weaker activity on PMP is possibly attributed to a previously unreported natural inhibitor. Blood AChE activity has been shown to reflect inflammatory states. Since AChE is a GPI-anchored protein, it is preferentially depleted from cells on the MP shed off. Assay of this activity in patient cell-free plasma, +/− Q, may be a useful biomarker. It is well known that hemolytic anemia, where RMP are elevated, is often associated with thrombotic complications, whereas ITP, where PMP are frequently elevated, rarely is. Further study to characterize AChE in RMP and other MP, and to clarify the physiological role of MP- and cell-associated AChE in thrombosis, inflammation, and cardiovascular disease is in progress.

scholarly journals Identification and conditions for selective expression of megakaryocytic markers in Friend erythroleukemia cells

Blood ◽  
1995 ◽  
Vol 86 (7) ◽  
pp. 2624-2631 ◽  
Author(s):  
F Paoletti ◽  
AM Vannucchi ◽  
A Mocali ◽  
R Caporale ◽  
SA Burstein
Keyword(s):  
Specific Activity ◽  
Culture Media ◽  
Late Phase ◽  
Calf Serum ◽  
Erythroid Differentiation ◽  
High Serum ◽  
Hexamethylene Bisacetamide ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Abstract Friend murine erythroleukemia cells (MELCs) have been reevaluated in terms of their nature and potential pathways of differentiation. MELC induced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also found to exhibit traits of the megakaryocytic lineage. Erythroid differentiation was shown by the typical synthesis and accumulation of hemoglobin (Hb); megakaryoblastoid differentiation of MELCs upon induction was shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% fetal calf serum (FCS) (instead of the usual 5%), induced cells to selectively express high levels of AChE (up to approximately 170 mU/mg protein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the whole cell population and approached its maximum within 3 days of incubation with the inducer. Subsequently, MELCs become committed to terminal division, undergoing growth arrest and expression of the megakaryocytic phenotype even after the removal of HMBA. There were no appreciable changes of basal AChE levels in MELCs that were either made resistant to HMBA or treated with 0.1 mmol/L hemin that activated differentiated erythroid function without commitment. Phorbol 12-myristate 13-acetate (PMA), known to repress induced Hb synthesis in these cells, did not prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increase that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permitted only when MELCs were transferred in the late phase or at the end of commitment from low to high serum media. Variations of FCS content in culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either megakaryocytic or mixed erythromegakaryocytic phenotype. These findings suggested that MELC might be considered, at least, as a bipotential model of differentiation to be used for studies on regulation of either megakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE levels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-antimouse platelet monoclonal antibody 4A5.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Calcium influx in induced differentiation of murine erythroleukemia cells

Blood ◽  
1993 ◽  
Vol 81 (3) ◽  
pp. 783-792 ◽  
Author(s):  
B Gillo ◽  
YS Ma ◽  
AR Marks
Keyword(s):  
Calcium Channel ◽  
Calcium Influx ◽  
Cdna Probe ◽  
Erythroid Differentiation ◽  
Early Event ◽  
Induced Differentiation ◽  
Erythroleukemia Cells ◽  
Murine Erythroleukemia ◽  
Murine Erythroleukemia Cells

Murine erythroleukemia cells (MELC) have served as a model for examining the regulation of erythroid differentiation. However, the role of Ca2+ in the signal transduction pathways regulating differentiation remains unclear. To begin to address this uncertainty we have characterized the regulation of cytoplasmic Ca2+ and the possible role of calcium channels during induced differentiation in MELC. MELC can be induced to terminal differentiation using the polar/apolar compound hexamethylene bisacetamide (HMBA). We found that HMBA stimulated Ca2+ influx within 3 to 6 minutes and that Ca2+ entry was required but not sufficient for MELC growth and differentiation. Nifedipine (1 to 10 mumol/L), a calcium channel antagonist, blocked HMBA-induced Ca2+ influx and inhibited differentiation by approximately 60%. Depolarization of the MELC membrane did not induce Ca2+ influx and whole-cell patch-clamp recordings failed to detect a voltage-activated Ca2+ current, suggesting that MELC do not express detectable levels of a functional voltage-dependent calcium channel (VDCC). However, a cDNA probe encoding a portion of the alpha 1 subunit of the cardiac VDCC detected an approximately 8-kb mRNA on Northern blots of total MELC RNA. Taken together, these data show that Ca2+ influx is an early event associated with HMBA-induced differentiation in MELC, blockade of this calcium influx inhibits induced differentiation, and a voltage- insensitive dihydropyridine-sensitive calcium channel may be involved in Ca2+ influx in MELC.

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