Evidence for a striking increase in acetylcholinesterase activity in cultured human fibroblasts which are trisomic for chromosome two

Acetylcholinesterase (AchE) is reported to have a narrowly restricted distribution among human tissues. Three strains of human fibroblasts which are trisomic for chromosome 2 had an average level of AchE activity over 28 times higher than the average level in 19 control strains of human fibroblasts. In contrast, the mean pseudocholinesterase activity of the trisomy-2 strains did not differ appreciably, or significantly, from the mean for the control strains. The 19 control strains included 10 strains trisomic for autosomes other than 2, and 9 euploid strains. Our estimate of the mean AchE activity in the control strains did not differ significantly from zero and might, in any case, have originated from a minute amount of activity contributed to the cells by an esterase in our culture medium. Despite the striking elevation of AchE activity in fibroblasts trisomic for chromosome 2, extracts of these cells had only about 1.5% of the specific AchE activity (per microgram DNA) present in extracts of human cerebral cortex. None of the 22 strains studied had detectable activity for two other enzymes which, like AchE, have a restricted distribution among human tissues: xanthine oxidase and choline acetyltransferase.     

Effect of stress on the acetylcholinesterase activity of the hypothalamus-pituitary-adrenal axis in the rat

Acetylcholinesterase (AchE, EC 3.1.1.7) activity was determined in cerebral cortex, hypothalamus, adenohypophysis and adrenal gland in response to acute and chronic stress. Chronic exposure of animals to cold stress (at 4 degrees C for 7 days) resulted in significant decline of AchE activity in all tissues studied. Similar results were obtained when animals were exposed to acute immobilization and cold stress (at 4 degrees C) simultaneously. In another experiment, animals were treated with 2 mg/kg of corticosterone prior to AchE determination. Corticosterone administration resulted in a significant decline in AchE activity of the cortex, the hypothalamus and the adrenal but failed to affect the adenohypophysis AchE level. Exposing adrenalectomized animals to acute stress resulted in no significant changes in the cortex and the hypothalamus but caused a significant decline in AchE of the adenohypophysis. It was concluded from this study that corticosterone might mediate the stress effect on AchE activity.     

Clinical trials of bestatin for leukemia and solid tumors

Dissociated cells from 13- and 17-day-old embryonic rat mesencephali have grown in primary cultures in order to compare the early and late influences of different agents - insulin, dexamethasone and nerve growth factor (NGF) - on the expression of cholinergic maturation process. We have studied cholin acetyltransferase (ChAT) activity, which is regarded as a specific marker for cholinergic function of the brain, and a widely used differentiation marker, the acetyl-cholinesterase (AchE) enzyme. Biochemical maturation of increasing specific activity of ChAT in both younger and older cells was taken into consideration. During cultivation the AchE activity was slightly increased in younger cells, but a dramatic decrease could be noted in older ones. Insulin in concentration from 10 to 27 µg mL^–1 causes a significant inhibition in ChAT activity in comparison with the enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng), independently of embryos age. This polypeptide hormone is able to enhance AchE activity in the cultured cells, especially in older ones. With continuous treatment of the culture with dexamethasone, a synthetic glucocorticoid, the ChAT activity in younger cells reaches a maximum curve by day 9 (nine). At this time the AchE activity shows a slighter, no significant increase than at any other time during cultivation. In cell cultures taken from 17-day-old embryos however dexamethasone treatment evoked a significant decrease in ChAT activity with a concomitant increase of AchE activity which was compared to insulin treatment. In spite of the fact that the NGF is able to enhance the ChAT activity, no significant alteration in AchE activity can be measured in younger cell cultures. These results suggest an uneven expression of the enzymes in embryonic rat mesencephali in the presence of above agents depending on the age of cells.     

Quantitation of megakaryocytopoiesis in liquid culture by enzymatic determination of acetylcholinesterase

A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum +/- pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE-positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.     

Acetylcholinesterase activity in spiral ganglion cells in rats]

Acetylcholinesterase has been demonstrated in the spiral ganglion cells by the method of Karnovsky and Roots. After incubation for 24 h the content of AchE was measured with the help of a three-stage scale. 22% of the cells showed strong, 50% medium and 28% weak AchE activity. Within the spiral ganglion perikarya, the enzyme was equally distributed.     

The Role of Erythrocytic Acetylcholinesterase in Hormonal Regulation of Adaptation to Physical Exercise

Acetylcholinesterase (AchE) activity in erythrocytes and blood levels of cortisol and insulin were investigated in athletes training under different bioenergetic conditions (sprinters, middle-distance runners, and marathoners). The groups of sprinters and marathoners had a decreased enzyme activity compared to nonathletes (p < 0.05). In response to a standard exercise, load AchE activity increased in the groups of middle-distance runners and marathoners. A relationship was observed between the level of AchE activity and the cortisol-to-insulin ratio in the blood. This ratio is specific to the type of bioenergetic conditions and increases in the following order: controls, middle-distance runners, sprinters, and marathoners. In vitro experiments revealed an effect of insulin on AchE activity. This effect was significantly lower in sprinters than in the control group. A reduction in AchE activity and an increase in the cortisol-to-insulin ratio are considered as factors increasing metabolic turnover in athletes, mainly, lipid turnover. This mechanism ensures effective mobilization of substrates at the start of physical exercise and their recovery after. The observed relationship between the insulin level and the AchE activity may prove to be a mechanism of regulation of the insulin level. This relationship may change during adaptation to physical exercise, as in the case of sprinters, when the sensitivity of AchE to the inhibitory effect of insulin is decreased. A high blood level of cortisol and insulin is a distinctive feature of sprinters, which provides for a higher turnover of carbohydrates. In marathoners, low AchE activity leads to an increased effect of acetylcholine, which is manifested by an increased cortisol level and a decreased insulin level, thus providing for a higher lipid turnover.     

Development of myoplasm-enriched ascidian embryos

The fertilized ascidian egg is thought to be comprised of distinct regions of tissue-specific cytoplasmic determinants. This idea was tested by bisecting fertilized eggs into egg fragments and culturing them until the unoperated controls developed into larvae. Fertilized eggs were bisected using a microsurgical method in which part of the uncleaved zygote was extruded through a hole made in the follicular envelope and the cytoplasmic bridge between the two egg regions was severed. One egg fragment contained all of the egg myoplasm (termed myoplasm-enriched or ME fragment), while the other fragment lacked myoplasm. ME fragments consisting of 40-50% of the total egg volume in many cases cleaved normally and developed into larvae. In a few cases, ME larvae initiated metamorphosis and developed into normal juveniles. Triton-extraction of ME embryos and larvae showed that the myoplasm was redistributed into nonmuscle lineage cells at each stage of development. Despite the redistribution of myoplasm into many of the endoderm cells situated in the head region of ME larvae, the expression of the muscle-specific enzyme acetylcholinesterase (AchE) and a muscle-specific antigen (Mu-2) was restricted to the tail muscle cells. The endoderm cells situated in the head region of ME larvae expressed an endoderm-specific enzyme alkaline phosphatase (AP) as in the controls. Furthermore, cleavage-arrested four- and eight-cell ME embryos expressed AchE activity in the expected number of blastomeres. When a greater quantity of myoplasm was redistributed into cells that normally do not express AchE activity by producing 10-30% ME embryos, in a few cases more than the expected number of blastomeres expressed AchE activity. In conclusion, the main finding of the present investigation, based on the development of ME fragments comprising 40-50% of the total egg volume, is that ascidian embryos are capable of regulative development.     

Inhibition of spicule elongation in sea urchin embryos by the acetylcholinesterase inhibitor eserine

The activity of acetylcholinesterase (AchE) increases rapidly after the gastrula stage of sea urchin development. In this report, changes in activity and in the molecular differentiation of AchE were investigated. AchE activity increased slightly during gastrulation and rose sharply thereafter, and was dependent on new RNA synthesis. No activity of butyrylcholinesterase was found. Morphogenesis in sea urchin embryos was inhibited by the AchE inhibitor eserine, which specifically inhibited arm rod formation but not body rod formation. Spicule formation and enzyme activity in cultured micromeres were inhibited by eserine in a dose-dependent manner. During gastrulation, two molecular forms of AchE were detected with polyacrylamide gel electrophoresis. The appearance of an additional band on the gel was consistent with the occurrence of a remarkable increase in the enzyme activity. This additional band appeared as a larger molecular form in Anthocidaris crassispina, Hemicentrotus pulcherrimus, Stomopneustes variolaris, and Strongylocentrotus nudus, and as a smaller form in Clypeaster japonicus and Temnopleurus hardwicki. These results suggest that the change in the molecular form of AchE induced a change in enzymatic activity that in turn may play a role in spicule elongation in sea urchin embryos.     

The effects of ethanol on the structural stability of acetylcholine receptor and the activity of various molecular forms of acetylcholinesterase

The actions of ethanol on the structural stability of acetylcholine receptor (AchR)-enriched membrane vesicles and the activity of various molecular forms of acetylcholinesterase (AchE) were investigated, using the receptor and the enzyme isolated from the electric organ of Torpedo californica. In the presence of ethanol up to 200 mM, the thermogram of AchR-enriched membranes exhibited no significant decrease in the temperature (td) of receptor transition at 57 degrees C, but a decrease in the enthalpy change (delta Hd) indicated a slight ethanol-induced structural perturbation. The presence of 12.5 nmol alpha-bungarotoxin also caused a decrease in delta Hd. A complete loss of the receptor transition was observed at a higher concentration 500 nmol of alpha-bungarotoxin and no recovery of the transition was found with the addition of 200 mM ethanol. The results suggested a noncompetitive interaction of ethanol with the receptor. In the presence of 200-1000 mM ethanol, the activity of two soluble forms of AchE, a higher (117 S) aggregate and a lower (10 S) aggregate was not significantly affected. Comparing the activity of these two aggregates over a wide concentration range of ethanol (200-2000 mM) revealed no obvious difference in the level of ethanol effect between them. However, after removal of ethanol, the higher aggregate form of AchE exhibited a greater recoverability of the activity, suggesting a possible slightly greater structure-functional stability for it. Studies of soluble AchE and membrane-bound AchE showed that the presence of 200 or 600 mM ethanol caused a greater level of inhibition in membrane-bound enzyme than in soluble enzyme, possible due to a disruption of protein-lipid interaction needed to maintain the conformation of membrane-bound AchE. Interestingly, at a much higher concentration of ethanol (2.0 M), membrane-bound AchE became more resistant to ethanol than did the soluble forms of AchE. In this case, the effective concentration of ethanol felt by the enzyme was expected to be less for membrane-bound AchE, owing to ethanol's solubility in lipids.     

Changes in acetylcholinesterase during pupal development of Apis mellifera queen

Total, membrane, and soluble acetylcholinesterase (AchE, EC 3.1.1.7) activities increase during the pupal development of Apis mellifera queen to reach maximum values at emergence. Membrane and soluble AchE are inhibited by 10^-5 M eserine or BW284C51 except at Pr, Pdm, and Pdd stages in which soluble AchE presents eserine-sensitive and eserine-resistant fractions. At all pupal stages, AchE occurs in a major amphiphilic membrane form that represents about 98% of total AchE activity and whose sedimentation coefficient is about 5.7S, and in a minor hydrophilic form that represents about 2% of total AchE activity and whose sedimentation coefficient is about 7S. At all pupal stages, phosphatidylinositol-specific phospholipase C (PI-PLC) and glycosyl phosphatidylinositol-specific phospholipase D (GPI-PLD) convert the membrane form into soluble counterparts which electrophoretic mobilities differ from that of the soluble form. AchE exhibits a butyrylcholinesterase (BuChE) activity that represents about 14% of AchE activity. During pupal development, the BuChE/AChE ratio of the membrane fraction is relatively stable, whereas the BuChE/AChE ratio of the soluble fraction is subjected to significant variations. At early pupal stages (Pw–Pd), membrane AchE displayed a high K_m value, higher than 40 μM, that decreases to an intermediary value of about 30 μM at Pdl and Pdm stages, to reach finally about 20 μM at Pdd and emergence stages. Arch. Insect Biochem. Physiol. 36:69–84, 1997. © 1997 Wiley-Liss, Inc.     

Four Pairs of Neuroprotective Aryldihydronaphthalene-Type Lignanamide Enantiomers from the Herbs of Solanum lyratum

Four pairs of aryldihydronaphthalene-type lignanamide enantiomers were isolated from Solanum lyratum (Solanaceae). The enantiomeric separation was accomplished by chiral-phase HPLC, and five undescribed compounds were elucidated. Analysis by various spectroscopy and ECD calculations, the structures of undescribed compounds were illuminated. The neuroprotective effects of all compounds were evaluated using H₂O₂-induced human neuroblastoma SH-SY5Y cells and AchE inhibition activity. Among them, compound 4 a exhibited remarkable neuroprotective effects at high concentrations of 25 and 50 μmol/L comparable to Trolox. Compound 1 a showed the highest AchE inhibition with the IC₅₀ value of 3.06±2.40 μmol/L. Molecular docking of the three active compounds was performed and the linkage between the compounds and the active site of AchE was elucidated.     

快速检测蚊虫乙酰胆碱酯酶活性的方法

本研究采用微板法,根据乙酰硫代胆碱-二硫双硝基苯甲酸法的原理,检测了敏感品系和抗残杀威抗性品系淡色库故CulexpipienspallensCoguillett）单个蚊虫的乙酰胆碱酯酶（AchE）的活性。给果表明：敏感品系和抗性品系蚊虫的AchE活性有较大差别,敏感品系蚊虫的AchE可被一定量的杀虫剂抑制,而抗性品系的AchE则不被抑制,因此该方法能够区分敏感蚊虫和抗性蚊虫,用于抗性测定,具有简单易行,检测快速等优点。     

Clinico-biochemical use of serum acetylcholine esterase following treatment with synthetic pyrethroids, cypermethrin and fenvalerate, in cattle and buffalo experimentally infested with Boophilus microplus

Following treatment, cypermethrin and fenvalerate, were found to have inhibitory effect on serum acetylcholine esterase (AchE) activity of cattle and buffalo experimentally infested with B. microplus. The pattern of AchE activity in infested-pyrethroid-treated group was found to be significantly different from either healthy or tick-infested control. There was transient increase in the enzyme activity initially, followed by gradual decline and subsequent increase leading to normal level within 7 days of pyrethroid treatment. The enzyme activity was found to be low in buffalo than in cattle and the values remained below normal level up to day 7 in tick-infested group. The reversion of AchE activity to normal level in pyrethroid-treated group indicated that these compounds are prompt and safe ixodicides with least residual effect. The present investigation concludes that estimation of serum AchE might help in the clinico-biochemical diagnosis of tick toxin and pyrethroid toxicity in cattle and buffalo treated with these pyrethroids against tick infestation.     

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.
Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 × 10⁵ as determined by Sepharose CL–6B column chromatography.     

Disparate effects of interleukin 11 and thrombopoietin on megakaryocytopoiesis in vitro

The effects of interleukin-11 (IL-11) and thrombopoietin (TPO) on murine megakaryocytopoiesis were studied using a serum-free culture system. Acting alone, both IL-11 and TPO increased the number of acetylcholinesterase (AchE)(+)cells (megakaryocytes), the latter being more potent than the former. TPO, but not IL-11, increased the mean AchE activity per megakaryocyte (AchE activity/megakaryocyte). TPO increased both the number of megakaryocytes with high ploidy, and of those with low ploidy. In contrast, IL-11 increased only the number of megakaryocytes with high ploidy. The effect of TPO on megakaryocyte ploidy was stronger than that of IL-11. Both IL-11 and TPO increased the proportion of large megakaryocytes, but the latter was more potent than the former. While the stimulatory effects of IL-11 and TPO on the number of megakaryocytes were enhanced by IL-3 or stem cell factor (SCF), synergism of IL-11 or TPO with IL-3 or SCF in stimulating AchE activity/megakaryocyte was inconsistent. IL-11 and TPO stimulated the formation of colony-forming units of megakaryocyte in the presence of IL-3, but not alone, with similar maximum colony numbers for both cytokines. Our findings thus demonstrate that IL-11 principally stimulates megakaryocyte maturation rather than the proliferation of megakaryocytes, whereas TPO stimulates both.     

Separation of murine megakaryocytes and their progenitors on continuous gradients of Percoll

Murine bone marrow was separated on continuous Percoll density gradients to analyze the distribution of cells of the megakaryocyte lineage. Eighty-seven percent of the recovered megakaryocytes were found in fractions of density less than 1.058 g/cm3, with 63% of these cells found between 1.020 and 1.036 g/cm3. When megakaryocytes were classified according to size, 92% of the large (greater than or equal to 18 micron) acetylcholinesterase (AchE) positive cells were found in the least dense fractions (1.016-1.039 g/cm3), whereas 86% of the small (less than or equal to 10.6 micron) AchE positive cells were found in fractions of higher density (1.039-1.078 g/cm3). The distribution of enzymatic AchE activity of the separated fractions corresponded to the location of the histochemically positive cells. When ploidy measurements were made of various fractions, most of the high ploidy (32N and 64N) cells were found at low density (1.028-1.036 g/cm3), whereas no cells greater than 4N were found at density greater than 1.071 g/cm3. Thus, large AchE positive cells and the cells of highest ploidy were found at lower densities of Percoll, while small AchE positive cells and cells of low ploidy were found at higher densities. An exception to this inverse relationship was found in fractions of lowest density (less than 1.030 g/cm3) where an anomalous distribution of size and ploidy was found. The majority of megakaryocytic colony-forming cells (CFU-MK) were found at high density, as were the granulocyte-macrophage colony-forming cells (CFU-GM; approximately 1.074 g/cm3). The density distribution of the incorporation of tritiated thymidine into liquid marrow cultures was concordant with the high density distribution of colony-forming cells. The data show that megakaryocytic maturity and Percoll density varies inversely and that fractionation of marrow on continuous Percoll gradients may be a useful method for the separation and/or enrichment of megakaryocytes at different stages of differentiation.     

小脑电刺激对家鸽乙酰胆碱酯酶活力的影响

选用成年健康家鸽,对小脑进行连续电刺激后,分别抽取家鸽外周血清及全脑测定乙酰胆碱酯酶活力变化,以探讨鸟类小脑刺激与乙酰胆碱酯酶活力变化之间的相关性.结果 表明:在刺激小脑皮层后,家鸽外周血清中乙酰胆碱酯酶活力显著上升(P＜0.05);而在刺激小脑皮层后,脑组织中乙酰胆碱酯酶活力显著降低(P＜0.05).推测电刺激引起外周组织乙酰胆碱释放,从而引起肌肉强直,血清中胆碱酯酶的活力升高.而电刺激小脑使抑制性神经元功能兴奋,脑中胆碱能神经元功能减弱,乙酰胆碱的释放减少,脑组织中胆碱酯酶的活力降低.     

Ultrastructural and histochemical study of anural development in the ascidian Molgula pacifica (Huntsman)

Summary Tadpole development is eliminated in the life cycle of the ascidian Molgula pacifica. The elimination of a tailed larva is termed anural development, in contrast to urodele development which is exhibited by most ascidian species. In the present study, transmission electron microscopy and histochemistry were used to gain a better understanding of anural development in M. pacifica. The fine structure of M. pacifica oocytes and fertilized eggs was similar to urodele oocytes and eggs, except that a perivitelline space and test cells were absent. M. pacifica embryos exhibited the typical cleavage pattern of urodele embryos. Gastrulation was initiated at the vegetal pole, as in urodeles, and occurred at the same time as in two urodele species (Molgula manhattensis and Pyura haustor). However, changes in cell shapes and cell movements of the vegetal pole cells that participate in gastrulation were highly modified compared to commonly studied ascidians. The changes in shapes and movements of the vegetal pole cells were minimal and resulted in embryos having a very small archenteron and blastopore. The presence of large, yolky cells in the interior of the embryo likely restricted vegetal cell movements. Two ultrastructurally distinct types of epidermal cells were evident at the gastrula stage. When gastrulae were manually dechorionated from their surrounding mucous-follicular envelope layers, the embryos were already surrounded by a thin tunic. When day 1 juveniles in the process of hatching were sectioned along the anterior-posterior axis, regional differences in cell types were evident. Differentiated muscle cells in the posterior region were not evident. Day 1 M. pacifica juveniles, anural-developing M. provisionalis juveniles and tadpoles from three urodele species were tested for their abilities to express AchE activity. The highest levels of AchE activity were detected in the larval tail muscle cells of urodeles, low levels of activity were detected in the posterior region of M. provisionalis juveniles, whereas M. pacifica juveniles did not exhibit AchE activity. The results are discussed in terms of evolutionary mechanisms responsible for anural development in ascidians. Offprint requests to: W.R. Bates     

Acetylcholinesterase and nonspecific cholinesterase activities in rat liver: subcellular localization, molecular forms, and some extraction properties

Subcellular distribution and some extraction properties of acetylcholinesterase (AchE) (EC 3.1.1.7) and nonspecific cholinesterase (ChE) (EC 3.1.1.8) were studied in rat liver employing subcellular fractionation techniques. All purified subcellular fractions were enriched in total cholinesterase activity over the homogenate. Plasma membrane and Golgi fractions showed a significant enrichment in AchE activity, while ChE activity was enriched in both rough and smooth endoplasmic reticulum. Subcellular fractions were subjected to conditions that selectively release proteins having varying degrees of association to membranes. High-pH treatment (known to release peripheral and soluble proteins) extracted ChE activity, but more than 90% of AchE activity remained associated to the pellet. Solubility properties and molecular forms of AchE and ChE in this tissue were studied by extraction in high-salt medium with and without Triton X-100, followed by velocity sedimentation centrifugation. Most of AchE activity (88%) (41% G4 and 59% G2 + G1) was detergent soluble; 42% of ChE activity (detected only as G2 + G1) was high-salt soluble, whereas remaining ChE activity was detergent soluble. These results indicate not only a different subcellular location for both enzymes, but also point to a differential association to membranes. AchE behaves as an integral membrane protein and ChE behaves as a peripheral or a luminal soluble protein.     

Interleukin 3 promotes maturation of murine megakaryocytes in vitro

A fluorescence assay for the quantitation of acetylcholinesterase (AchE) has been adapted for measurement of megakaryocytic maturation in short-term serum-free cultures of murine marrow. When marrow cells were cultured for 3 days in the presence of pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) under serum-free conditions, AchE production was found to be related to the concentration of PWM-SCM. Interleukin 3 (IL3), a purified glycoprotein promoting the proliferation of several early hematopoietic progenitors including megakaryocytic colony-forming cells, also induced AchE production in a dose-responsive manner. The response to IL3 was linearly related to the number of cells cultured. When marrow was first subjected to plastic adherence and the nonadherent cells then separated on Percoll gradients, a small megakaryocyte-enriched population markedly depleted of colony-forming cells and large megakaryocytes, responded to IL3 in a similar dose-responsive manner. A significant amount of AchE was produced in the absence of any added factors. The data show that AchE production can be measured in 3-day serum-free cultures, and suggest that IL3, a factor promoting megakaryocytic proliferation in vitro, also promotes maturation.