Increased AChE Activity Research Articles

Inflammatory changes secondary to postoperative complications of Hirschsprung’s disease as a cause of histopathologic changes typical of intestinal neuronal dysplasia

Purpose The aim of this study was to clarify the pathogenesis of intestinal neuronal dysplasia (IND). Methods The bowel habits of 36 postoperative HD patients were assessed retrospectively. Twenty-five had no complaints. Seven had persistent enterocolitis and were the focus of our study. They were divided into group A (n = 2) if they were severe and had associated postoperative surgical complications, and group B (n = 5) if they were mild. The histological changes were assessed. Results The 7 patients who had persistent enterocolitis postoperatively had no AchE activity in the mucosa, and there was normal distribution of submucosal and myenteric ganglia in the proximal resection margin. Rectal biopsies performed postoperatively for investigation of persistent enterocolitis in group A showed inflammatory changes and typical histopathologic features of IND such as abundant acetylcholinesterase (AchE)-positive nerve fibers in the lamina propria associated with giant submucosal ganglia and hyperganglionosis, and in group B there was increased AchE activity without hyperganglionosis or giant ganglia. Conclusions This is the first report of histopathologic changes typical of IND occurring in response to persistent enterocolitis related to postoperative complications of surgery for HD.

Increased expression of an acetylcholinesterase gene may confer organophosphate resistance in the greenbug, Schizaphis graminum (Homoptera: Aphididae)

Acetylcholinesterases (AChE, EC 3.1.1.7) were purified from an organophosphate (OP)-susceptible (OSS) clone and an OP-resistant (OR-0) clone of the greenbug, Schizaphis graminum (Rondani). Enzyme inhibition kinetics showed that AChE from the OR-0 clone was 1.1- to 12.8-fold less sensitive to inhibition by chlorpyrifos oxon, paraoxon, methyl paraoxon, malaoxon, demeton- S-methyl and omethoate than AChE from the OSS clone based on their bimolecular rate constants ( k i ). Analyses of DNA sequences of PCR-amplified fragments from the AChE coding regions did not reveal any differences between the OSS and the three OP-resistant clones (OR-0, OR-1, and OR-2). Northern blot analysis, however, showed that the amount of AChE mRNA in the resistant clones was approximately 1.5-fold higher than that in the OSS clone. Southern blots did not provide any evidence of gene amplification for the increased mRNA. The increased AChE mRNA appears to be positively correlated with the amount of AChE in crude enzyme preparations. These results indicated that the increased AChE activity in OP-resistant clones was due to increased expression of the AChE gene. It is possible that an increased transcription rate and/or increased stability of the mRNA results in the increase of AChE mRNA in the OP-resistant clones of the greenbug.

Stability and Secretion of Acetylcholinesterase Forms in Skeletal Muscle Cells

Muscle cells express a distinct splice variant of acetylcholinesterase (AChE(T)), but the specific mechanisms governing this restricted expression remain unclear. In these cells, a fraction of AChE subunits is associated with a triple helical collagen, ColQ, each strand of which can recruit a tetramer of AChE(T). In the present study, we examined the expression of the various splice variants of AChE by transfection in the mouse C2C12 myogenic cells in vitro, as well as in vivo by injecting plasmid DNA directly into tibialis anterior muscles of mice and rats. Surprisingly, we found that transfection with an ACHE(H) cDNA, generating a glycophosphatidylinositol-anchored enzyme species, produced much more activity than transfection with AChE(T) cDNA in both C2C12 cells and in vivo. This indicates that the exclusive expression of AChE(T) in mature muscle is governed by specific splicing. Interaction of AChE(T) subunits with the complete collagen tail ColQ increased enzyme activity in cultured cells, as well as in muscle fibers in vivo. Truncated ColQ subunits, presenting more or less extensive C-terminal deletions, also increased AChE activity and secretion in C2C12 cells, although the triple helix could not form in the case of the larger deletion. This suggests that heteromeric associations are stabilized compared with isolated AChE(T) subunits. Coinjections of AChE(T) and ColQ resulted in the production and secretion of asymmetric forms, indicating that assembly, processing, and externalization of these molecules can occur outside the junctional region of muscle fibers and hence does not require the specialized junctional Golgi apparatus.

Area-specific modification of acetylcholinesterase activity following 3-mercaptopropionic acid-induced seizures.

Acetylcholinesterase activity (AChE) was assayed in rat CNS membrane fractions after administration of the convulsant 3-mercaptopropionic acid (150 mg/kg, ip). In comparison with saline-injected controls, total AChE activity decreased 12-20% in striatum and cerebellum during seizure and postseizure but failed to change in cerebral cortex. Specific AChE activity, assayed in the presence of 10(-4) M ethopropazine (a butyrylcholinesterase inhibitor), decreased 15-25% in striatum and cerebellum, increased 20-45% in hippocampus, but remained unchanged in cerebral cortex. Saline injection alone increased AChE activity in striatum (68%) and cerebellum (36%) but failed to modify enzyme activity in hippocampus and cerebral cortex. To conclude, AChE sensitivity to convulsant and saline administration is tissue-specific and not restricted to cholinergic areas.

Early and late hormonal modulation of cholinergic maturation in culture of embryonic mesencephali.

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 micrograms 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.

Effect of Recombinant Granulocyte-Macrophage Colony-Stimulating Factor on Murine Thrombocytopoiesis In Vitro and In Vivo

To investigate the effect of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) on murine megakaryocytopoiesis in vitro, the factor was added to both serum-free colony assays and liquid marrow cultures. GM-CSF had a significant megakaryocytic colony-stimulating activity. After 2 hours of preincubation with and without 10 ng/mL rGM-CSF, the percentage of megakaryocyte colony-forming cell (CFU-MK) in DNA synthesis was determined by tritiated-thymidine suicide using colony growth. The reduction of CFU-MK colony numbers in marrow culture was 47.5% +/- 9.9%, 20.9% +/- 5.2% (control), respectively, indicating that the factor affected cell cycle at CFU-MK levels. When acetylcholinesterase (AchE) production was measured fluorometrically after 4 days of liquid culture, rGM-CSF elicited an increase in AchE activity in a dose-dependent fashion. To determine if the hematopoietin acts directly on megakaryocytic differentiation, 2 ng/mL rGM-CSF was added to serum-free cultures of 295 single megakaryocytes isolated from CFU-MK colonies. An increase in size was observed in 65% of cells initially 10 to 20 microns in diameter, 71% of cells 20 to 30 microns, and 40% of cells greater than 30 microns. Conversely, in absence of GM-CSF, 17%, 31%, and 10% of cells in each group increased in diameter. These data suggest that rGM-CSF promotes murine megakaryocytopoiesis in vitro and that the response to the factor is direct. To determine if the factor influences megakaryocytic/thrombocytic lineage in vivo, 1 and 5 micrograms of rGM-CSF were administered intraperitoneally every 12 hours for 6 consecutive days. Although a two- to three-fold increase in peripheral granulocytes was observed, neither megakaryocytic progenitor cells or platelets changed. Histologic analysis of bone marrow megakaryocytes showed no increase in size and number. The in vivo studies demonstrated no effect of GM-CSF on thrombocytopoiesis. The discrepancies between the in vitro and in vivo effects of GM-CSF require additional investigations.

Effect of recombinant granulocyte-macrophage colony-stimulating factor on murine thrombocytopoiesis in vitro and in vivo

To investigate the effect of recombinant granulocyte-macrophage colony- stimulating factor (rGM-CSF) on murine megakaryocytopoiesis in vitro, the factor was added to both serum-free colony assays and liquid marrow cultures. GM-CSF had a significant megakaryocytic colony-stimulating activity. After 2 hours of preincubation with and without 10 ng/mL rGM- CSF, the percentage of megakaryocyte colony-forming cell (CFU-MK) in DNA synthesis was determined by tritiated-thymidine suicide using colony growth. The reduction of CFU-MK colony numbers in marrow culture was 47.5% +/- 9.9%, 20.9% +/- 5.2% (control), respectively, indicating that the factor affected cell cycle at CFU-MK levels. When acetylcholinesterase (AchE) production was measured fluorometrically after 4 days of liquid culture, rGM-CSF elicited an increase in AchE activity in a dose-dependent fashion. To determine if the hematopoietin acts directly on megakaryocytic differentiation, 2 ng/mL rGM-CSF was added to serum-free cultures of 295 single megakaryocytes isolated from CFU-MK colonies. An increase in size was observed in 65% of cells initially 10 to 20 microns in diameter, 71% of cells 20 to 30 microns, and 40% of cells greater than 30 microns. Conversely, in absence of GM- CSF, 17%, 31%, and 10% of cells in each group increased in diameter. These data suggest that rGM-CSF promotes murine megakaryocytopoiesis in vitro and that the response to the factor is direct. To determine if the factor influences megakaryocytic/thrombocytic lineage in vivo, 1 and 5 micrograms of rGM-CSF were administered intraperitoneally every 12 hours for 6 consecutive days. Although a two- to three-fold increase in peripheral granulocytes was observed, neither megakaryocytic progenitor cells or platelets changed. Histologic analysis of bone marrow megakaryocytes showed no increase in size and number. The in vivo studies demonstrated no effect of GM-CSF on thrombocytopoiesis. The discrepancies between the in vitro and in vivo effects of GM-CSF require additional investigations.

Morphometric study of neuromuscular junction in rabbits subjected to shock by superior mesenteric artery occlusion

The purpose of this study was to describe changes in the neuromuscular junction of rabbit sartorius muscle after shock by superior mesenteric artery occlusion in 18 rabbits. The results revealed that the numeric and volume densities of the acetylcholine (Ach) vesicles of the sartorius neuromuscular junction were decreased during shock when compared by electron microscopy with normal subjects (p less than .001). We also discovered that the release of lysosomes from hepatic cells, erythrocyte acetylcholinesterase (AchE) activity, and hepatic ATP values decreased during shock. Both scopolamine (0.023 mg/kg) and verapamil (0.74 mg/kg) blocked the release of Ach vesicles and lysosomes, and increased hepatic ATP values. Scopolamine also increased AchE activity. This study shows that the parasympathetic activation plays an important role in mesenteric artery occlusion shock.

Influence of triiodothyronine ( l-T3) on the morphological and biochemical development of fetal brain acetylcholinesterase-positive neurons cultured in a chemically defined medium

In cerebral hemisphere cultures initiated from 15-day-old rat embryos, the number of acetylcholinesterase-positive (AChE+) cells increased from 6.8 ± 1.6 cells/well on day 3 to 112 ± 16 cells/well on day 15. With time in culture, AChE+ cells increased both in size of the perikarya and neurite length. The addition of l-triiodothyronine ( l-T3) at a concentration of 3 × 10 −8 M at the initiation of the culture had no effect on the number of AChE+ cells but significantly increased the size and neurite length of AChE+ neurons after 5 days in vitro. These morphological effects are associated with biochemical effects. l-T3 increased AChE activity in both a dose- and time-dependent manner (the stimulatory effect of l-T3 becomes significant between day 8 and day 15). Since a major part of AChE+ cells may be cholinergic neurons, we have also measured the effect of l-T3 on ChAT activity. l-T3 also increased ChAT activity in a dose and time dependent manner. Furthermore, treatment of cultures with l-T3 at different times in culture demonstrated the presence of a critical period which occurs in vitro around day 20, since the stimulatory effect of l-T3 on ChAT activity is lost after 20 days in vitro. Studies of the time necessary for l-T3 to increase both ChAT and AChE activities show that 2 days and 15 days, respectively, are required for l-T3 to significantly stimulate both enzyme activities. This in vitro analysis demonstrated the morphological effect of l-T3 on the size and the neurite length of AChE+ cells. These effects are associated with biochemical effects on ChAT and AChE activities. Thus, it appears that thyroid hormones regulate several steps of neuronal maturation.

Human recombinant erythropoietin promotes differentiation of murine megakaryocytes in vitro.

To determine if erythropoietin affects megakaryocytopoiesis, we measured acetylcholinesterase (AchE) activity, a marker of the murine megakaryocytic lineage, after the addition of human recombinant erythropoietin to serumless murine bone marrow cultures. Erythropoietin increased AchE activity substantially. Moreover, when the hormone was added to serumless cultures of 426 isolated single megakaryocytes derived from megakaryocytic colonies, erythropoietin induced a significant increase in the diameters of these cells. From a Bayesian analysis of the likelihood that some megakaryocytes increased in DNA content during the culture period, we estimate that 61% of the cells increased in ploidy. These data indicate that the action of erythropoietin is not restricted to the erythroid lineage.

Changes in the acetylcholinesterase and choline acetyltransferase activities in the early development of the chick embryo

Acetylcholinesterase (AChE, EC 3.1.1.7) and choline acetyltransferase (CAT, EC 2.3.1.6) activities where studied in the early development of the chick embryo. A sharp increase in AChE activity occurred in the gastrulating embryo. The highest AChE activity was associated with hypoblast cells. By sucrose density gradient centrifugation three molecular forms of AChE with sedimentation coefficients 4.7 S, 6.8 S and 10.9 S were determined. During the gastrulation there was no remarkable change in the activity of CAT. A two-fold decrease in the CAT activity occurred at the end of gastrulation.

Interrelationship among the proliferating ability, morphological development and acetylcholin-esterase activity of the neural tube cells in early chick embryos.

Using new methods which perform 3H-thymidine autoradiography and AChE histochemistry on the same sections, the interrelationship among loss of proliferating ability, morphological development and increase of AChE activity during the course of differentiation of the neural tube cells were investigated in early chick embryos. The neural tube wall consisted of four kinds of cells: (1) AChE negative spindle shaped cells, (2) weakly positive spindle shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a few short profiles of r-ER, (3) moderately positive spindle shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a moderate number of r-ER profiles and (4) intensely positive large round cells showing AChE activity in the cisternae of the nuclear envelope and in a large number of r-ER profiles. Nuclei of the former three kinds of cells slet located in the ependymal layer, while the AChE intensely positive cells were in the mantle layer. The AChE negative and weakly positive cells were capable of proliferation and were regarded as undifferentiated neuroepithelial cells. The AChE moderately positive and intensely positive cells were, in contrast, no longer capable of proliferation and were regarded as neurons, especially the former as newly formed neurons. These findings indicate that the r-ER increases rapidly in number and in volume in newly formed neurons, soon after their final cell division, and that AChE activity increases in the neurons parallel to the development of the r-ER.

CSF acetylcholinesterase in dementia and in sequential samples of lumbar CSF

Acetylcholinesterase (AchE) activity (nmol/ml/min) was measured in lumbar CSF from 11 patients with dementia of the Alzheimer type (DAT), 8 patients with Korsakoff psychosis and 33 patients with low back pain who were undergoing myelography (controls). There was no significant difference in enzyme activity between the three groups. There was no significant correlation between age and AchE activity. AchE was also measured in 20 two-ml samples of CSF collected sequentially by lumbar puncture in two neurosurgical patients who had been recumbent for at least 8 hours. Variations in AchE between samples were small. In neither patient was there an increase in AchE activity with progressive sampling. These data indicate that (1) AchE is unchanged in Korsakoff psychosis (2) decreases in brain AchE which are found in DAT are not readily reflected in lumbar CSF (3) AchE in lumbar CSF has a diffuse origin including spinal cord (4) CSF AchE activity is unlikely to be a useful clinical marker for DAT.

Acetylcholine metabolism in rat spinal cord cultures: regulation by a factor involved in the determination of the neurotransmitter phenotype of sympathetic neurons

Acetylcholine metabolism has been studied in sister cultures of E13 rat spinal cord cells cultured for 1 to 3 weeks with or without conditioned medium (CM) from rat skeletal muscle cells. Spinal cord cells grown with CM synthesized and accumulated 3 to 4 times more [3H]ACh from [3H]choline than cultures grown without CM. This effect of CM was accompanied by a comparable increase in CAT activity and could not be mimicked by increasing the density of the spinal cord cultures. A 2- to 3-fold increase in AChE activity was also observed in 2- to 3-week-old CM cultures, whereas the activity of lactate dehydrogenase was identical in cultures grown with and without CM. We have compared the effects of CMs from various non-neuronal cell cultures on [3H]ACh synthesis and storage by spinal cord cultures and by sympathetic neuron cultures. CM by skeletal muscle greater than skin fibroblasts greater than rat heart muscle greater than C6 glioma cells were the most active on both types of neuron cultures, whereas CM from rat brain, L6 myoblasts, mouse 3T3, and PYT21 fibroblasts was inactive on spinal cord cultures and only weakly active on sympathetic neurons. Serum-free CM from skeletal muscle was inactive on both types of neuron cultures. The CM factor active on spinal cord cultures has been purified several thousand-fold by using a four-step fractionation scheme which has previously led to a partial purification of the CM factor involved in the regulation of CAT, AChE, and catecholamine-synthesizing enzymes in sympathetic neuron cultures ( Swerts , J. P., A. Le Van Thai, A. Vigny , and M. J. Weber (1983) Dev. Biol. 100: 1-11). Moreover, a comparison of dose-response curves established with this purified material showed that it exerted its effects on spinal cord and on sympathetic neuron cultures in the same range of concentration. Thus, these results suggest that the same macromolecule is involved in the regulation of neurotransmitter phenotype in both types of cultures despite their different embryological origins.

Effects of ambient temperature on brain acetylcholinesterase activity and protein content in three Egyptian vertebrates

1. 1. Exposure to cold caused an increase in AChE activity of the different brain regions of both Arvicanthis and Columba and a decrease in the enzyme activity of Scincus midbrain. 2. 2. Heat exposure provoked variable changes in AChE activity of the various brain regions of the three experimental species. 3. 3. The changes in AChE activities may be one of the mechanisms by which birds and mammals tend to acclimatize themselves to various forms of stress. 4. 4. In reptiles, AChE activity varied with changes in ambient temperature and this is probably due to the adaptive significance of thermally directed changes in enzyme substrate affinity.

Effect of gamma radiation on enzymatic activity and sulphydryl groups of human erythrocyte membrane.

The effect of ionizing radiation on human erythrocyte ghost membranes was studied by following changes in membrane -SH groups and activities of four membrane bound enzymes: Na+K+Mg2+ ATP-ase, Mg2+Ca2+ ATP-ase, Na+K+ATP-ase, and AChE. Irradiation up to 100 Gy gamma X-rays produced a significant decrease in the activity of ATP-ase and an increase in AChE activity. At higher radiation doses a marked decrease in the activities of all the enzymes was observed. A correlation between radiation-induced perturbations in enzyme activities and changes in membrane -SH groups was found.

Proliferating ability, morphological development and acetylcholinesterase activity of the neural tube cells in early chick embryos. An electron microscopic study.

Using 3H-thymidine autoradiography and AChE histochemistry at the electron microscopic level on the same sections, the interrelationships between loss of proliferating ability, morphological development and increase of AChE activity during the course of differentiation of the neural tube cells were investigated in early chick embryos. The neural tube wall consisted of spindle-shaped cells with no AChE activity, weakly positive spindle-shaped cells showing AChE activity in the cisternae of the nuclear envelope and in a few short profiles of r-ER, moderately positive spindle-shaped cells showing AChE activity in the nuclear envelope and in a moderate number of r-ER profiles and intensely positive large round cells showing AChE activity in the nuclear envelope and in a large number of r-ER profiles. Nuclei of the AChE-negative, weakly positive and moderately positive cells were located in the ependymal layer (matrix). The AChE-intensely positive cells were in the mantle layer. The AChE-negative and weakly positive cells were capable of proliferation and were regarded as undifferentiated neuroepithelial cells. In contrast, the moderately positive and intensely positive cells were no longer capable of proliferation and were considered to be neurons. These findings indicate that the r-ER increases rapidly in amount and volume in newly formed neurons soon after their final cell division, and that AChE increases in the neurons in parallel to the development of the r-ER.

Ultrastructural localization of acetylcholinesterase in cultured cells. III. DFP treated embryo muscle.

Brief treatment with 10(-4)M diisopropylfluorophosphate (DEP) irreversibly inactivates acetylcholinesterase (E.C.3.1.1.7; acetylcholine hydrolase) (AChE) activity in 10 day old chick embryonic muscle cultures. Electron microscopic cytochemistry was employed to follow the distribution of new AChE during recovery from DEP treatment. In normal 10 day cultures of embryo pectoralis muscles AChE is localized in the nuclear envelope, perinuclear sarcoplasm, sarcotubular system, subsurface vesicles and bound outside the cells. Immediately after DFP treatment AChE activity is absent in large myotubes. Within 15 min, activity is randomly present in small amounts in the sarcotubular system and nuclear envelope. There is a dramatic increase in activitv in the nuclear envelope during the 1st hr of recovery, and connections between the nuclear envelope and sarcotubular system are often seen. The next few hr of recovery show increased AChE activity. By 4 hr activity approaches that of controls. Six to 8 hr after treatment, AChE activity can be detected spectrophotometrically in the medium and can be seen bound outside the cells with the electron microscope. The spatial and temporal patterns of AChE activity demonstrate that the recovery of AChE and its mobilization and release from DFP-treated cells are not governed solely by the levels attained by the enzyme in the cultured embryo muscle.

Changes in the sedimentation properties of acetylcholinesterase during neuroblastoma differentiation

The adrenergic mouse neuroblastoma clone NIE 115 contains two species of AChE which sediment at 4S and 11S. These two species are found both in logarithmic growing phase cells (round neuroblast morphology) and in cells which have undergone morphological differentiation due to the elimination of serum. The 4S form is predominant in the growing cells (70 per cent) whereas the 11S form is more abundant (55 per cent to 65 per cent) in the cells with neurites. When protein synthesis is inhibited by cycloheximide, there is an increase states : we postulate that the 11S species is formed by a conversion of the 4S species. When cell division is blocked by sodium butyrate, there is an increase in AChE activity but no modification in the proportion of 4S/11S species as compared to the cells in growing phase. We were unable to associate temporally the increase in the 11S species with neurite outgrowth ; when cells were allowed to retract their neurites and were subsequently maintained in a GROWING state for 10 to 15 days, the 11s species still predominates. Although the presence of the 11S molecule cannot always be correlated with the state of morphological differentiation, the shift in sedimentation coefficient of AChE form 4S to 11S might eventually constitute a reference in the study of biochemical events leading to terminal differentiation in this system.

Effect of the Chemosterilant P,P′-Bias(1-aziridinyl)-N-(3-methoxypropyl) Phosphinothioic Acid on Esterases of the House Fly 12

Adult Musca domestica L. were chemosterilized by oral application of 5 μg/fly of P,P′ -bis(l-aziridinyl)-N-(3-methoxypropyl) phosphinothioic acid after they emerged. A considerable increase in the activity of acetylcholinesterase (AchE) during embryogenesis in the crude extract of the normal eggs was evident and was about double that present in the chemosterilized eggs. When the crude protein extract from chemosterilized eggs was purified by DEAE cellulose column chromatography, increased AchE activity was found. As shown by enzyme activity measurement and acrylamide gel electrophoresis patterns, AchE in ovaries, testes, and heads of chemosterilized flies appeared normal. During embryonic development period, the esterases showed a remarkable increase in the isoenzyme bands in the normal eggs over the chemosterilant eggs.