Biomedical subjects

H Soreq

Publications and source records attributed to H Soreq.

At least 73 records · Page 4Linked to original sources

PubMed1995

Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants.

To explore the molecular basis of the biochemical differences among acetylcholinesterase (AChE), butyrylcholinesterase (BuChE) and their alternative splicing and allelic variants, we investigated the acylation phase of cholinesterase catalysis, using phosphorylation as an analogous reaction. Rate constants for organophosphate (DFP) inactivation, as well as for oxime (PAM)-promoted reactivation, were calculated for antibody-immobilized human cholinesterases produced in Xenopus oocytes from natural and site-directed variants of the corresponding DNA constructs. BuChE displayed inactivation and reactivation rates 200- and 25-fold higher than either product of 3'-variable AChE DNAs, consistent with a putative in vivo function for BuChE as a detoxifier that protects AChE from inactivation. Chimeric substitution of active site gorge-lining residues in BuChE with the more anionic and aromatic residues of AChE, reduced inactivation 60-fold but reactivation only 4-fold, and the rate-limiting step of its catalysis appeared to be deacylation. In contrast, a positive charge at the acyl-binding site of BuChE decreased inactivation 8-fold and reactivation 30-fold. Finally, substitution of Asp70 by glycine, as in the natural 'atypical' BuChE variant, did not change the inactivation rate yet reduced reactivation 4-fold. Thus, a combination of electrostatic active site charges with aromatic residue differences at the gorge lining can explain the biochemical distinction between AChE and BuChE. Also, gorge-lining residues, including Asp70, appear to affect the deacylation step of catalysis by BuChE. Individuals carrying the 'atypical' BuChE allele may hence be unresponsive to oxime reactivation therapy following organophosphate poisoning.

Acetylcholinesterase↗

PubMed1995

Genetic predisposition to adverse consequences of anti-cholinesterases in 'atypical' BCHE carriers.

Normal butyrylcholinesterase (BuChE), but not several of its common genetic variants, serves as a scavenger for certain anti-cholinesterases (anti-ChEs). Consideration of this phenomenon becomes urgent in view of the large-scale prophylactic use of the anti-ChE, pyridostigmine, during the 1991 Persian Gulf War, in anticipation of nerve gas attack and of the anti-ChE, tacrine, for improving residual cholinergic neurotransmission in Alzheimer's disease patients. Adverse symptoms were reported for subjects in both groups, but have not been attributed to specific causes. Here, we report on an Israeli soldier, homozygous for 'atypical' BuChE, who suffered severe symptoms following pyridostigmine prophylaxis during the Persian Gulf War. His serum BuChE and recombinant 'atypical' BuChE were far less sensitive than normal BuChE to inhibition by pyridostigmine and several other carbamate anti-ChEs. Moreover, atypical BuChE demonstrated 1/200th the affinity for tacrine of normal BuChE or the related enzyme acetylcholinesterase (AChE). Genetic differences among BuChE variants may thus explain at least some of the adverse responses to anti-ChE therapies.

Acetylcholinesterase↗

PubMed1995

Synaptic and epidermal accumulations of human acetylcholinesterase are encoded by alternative 3'-terminal exons.

Tissue-specific heterogeneity among mammalian acetylcholinesterases (AChE) has been associated with 3' alternative splicing of the primary AChE gene transcript. We have previously demonstrated that human AChE DNA encoding the brain and muscle AChE form and bearing the 3' exon E6 (ACHE-E6) induces accumulation of catalytically active AChE in myotomes and neuromuscular junctions (NMJs) of 2- and 3-day-old Xenopus embryos. Here, we explore the possibility that the 3'-terminal exons of two alternative human AChE cDNA constructs include evolutionarily conserved tissue-recognizable elements. To this end, DNAs encoding alternative human AChE mRNAs were microinjected into cleaving embryos of Xenopus laevis. In contrast to the myotomal expression demonstrated by ACHE-E6, DNA carrying intron 14 and alternative exon E5 (ACHE-I4/E5) promoted punctuated staining of epidermal cells and secretion of AChE into the external medium. Moreover, ACHE-E6-injected embryos displayed enhanced NMJ development, whereas ACHE-I4/E5-derived enzyme was conspicuously absent from muscles and NMJs and its expression in embryos had no apparent effect on NMJ development. In addition, cell-associated AChE from embryos injected with ACHE-I4/E5 DNA was biochemically distinct from that encoded by the muscle-expressible ACHE-E6, displaying higher electrophoretic mobility and greater solubility in low-salt buffer. These findings suggest that alternative 3'-terminal exons dictate tissue-specific accumulation and a particular biological role(s) of AChE, associate the 3' exon E6 with NMJ development, and indicate the existence of a putative secretory AChE form derived from the alternative I4/E5 AChE mRNA.

Acetylcholinesterase↗

PubMed1994

Cholinotoxic effects on acetylcholinesterase gene expression are associated with brain-region specific alterations in G,C-rich transcripts.

To study the mechanisms underlying cholinotoxic brain damage, we examined ethylcholine aziridinium (AF64A) effects on cholinesterase genes. In vitro, AF64A hardly affected cholinesterase activities yet inhibited transcription of the G,C-rich AChE DNA encoding acetylcholinesterase (AChE) more than the A,T-rich butyrylcholinesterase (BChE) DNA. In vivo, intracerebroventricular injection of 2 nmol of AF64A decreased AChE mRNA in striatum and septum by 3- and 25-fold by day 7, with no change in BChE mRNA or AChE activity. In contrast, hippocampal AChE mRNA increased 10-fold by day 7 and BChE mRNA and AChE activity decreased 2-fold. By day 60 post-treatment, both AChE mRNA and AChE levels returned to normal in all regions except hippocampus, where AChE activity and BChE mRNA were decreased by 2-fold. Moreover, differential PCR displays revealed persistent induction, specific to the hippocampus of treated rats, of several unidentified G,C-rich transcripts, suggesting particular responsiveness of hippocampal G,C-rich genes to cholinotoxicity.

Acetylcholinesterase↗

PubMed1994

Transgenic engineering of neuromuscular junctions in Xenopus laevis embryos transiently overexpressing key cholinergic proteins.

To examine the role of key cholinergic proteins in the formation of neuromuscular junctions (NMJs), we expressed DNAs encoding the mouse muscle nicotinic acetylcholine receptor (nAChR) or human brain and muscle acetylcholinesterase (hAChE) in developing Xenopus laevis embryos. Acetylthiocholine hydrolysis and alpha-bungarotoxin binding in homogenates of transgenic embryos revealed transient overexpression of the respective proteins for at least 4 days postfertilization. Moreover, hAChE injection induced an approximately 2-fold increase in endogenous Xenopus nAChR. Electron microscopy coupled with cytochemical staining for AChE activity revealed that AChE-stained areas, which reached 0.17 microns2 in NMJs of control embryos raised at 21 degrees C, increased up to 0.53 and 0.60 microns2 in nAChR and hAChE transgenics, respectively. These increases coincided with the appearance of a class of large NMJs with average postsynaptic lengths up to 1.8-fold greater than controls. As much as 57% and 34% of the NMJs in animals transgenic for nAChR and hAChE, respectively, displayed AChE activity in nerve terminals in addition to muscle labeling, as compared with 10% nerve-labeled NMJs in control animals. Moreover, area, but not length values, were > 2-fold larger in hAChE-expressing NMJs labeled in their nerve terminals than in those labeled in muscle alone, reflecting a hAChE-induced increase in synaptic cleft width. These findings indicate that modulation of cholinergic neurotransmission in NMJs modifies the features of nerve-muscle connections.

Acetylcholinesterase↗

PubMed1994

Antisense oligonucleotide inhibition of acetylcholinesterase gene expression induces progenitor cell expansion and suppresses hematopoietic apoptosis ex vivo.

To examine the role of acetylcholinesterase (EC 3.1.1.7) in hematopoietic cell proliferation and differentiation, we administered a 15-mer phosphorothioate oligonucleotide, antisense to the corresponding ACHE gene (AS-ACHE), to primary mouse bone marrow cultures. Within 2 hr of AS-ACHE addition to the culture, ACHE mRNA levels dropped by approximately 90%, as compared with those in cells treated with the "sense" oligomer, S-ACHE. Four days after AS-ACHE treatment, ACHE mRNA increased to levels 10-fold higher than in S-ACHE cultures or in fresh bone marrow. At this later time point, differential PCR display revealed significant differences between cellular mRNA transcripts in bone marrow and those in AS-ACHE- or S-ACHE-treated cultures. These oligonucleotide-triggered effects underlay considerable alterations at the cellular level: AS-ACHE but not S-ACHE increased cell counts, reflecting enhanced proliferation. In the presence of erythropoietin it also enhanced colony counts, reflecting expansion of progenitors. AS-ACHE further suppressed apoptosis-related fragmentation of cellular DNA in the progeny cells, and it diverted hematopoiesis toward production of primitive blasts and macrophages in a dose-dependent manner promoted by erythropoietin. These findings suggest that the hematopoietic role of acetylcholinesterase, anticipated to be inverse to the observed antisense effects, is to reduce proliferation of the multipotent stem cells committed to erythropoiesis and megakaryocytopoiesis and macrophage production and to promote apoptosis in their progeny. Moreover, these findings may explain the tumorigenic association of perturbations in ACHE gene expression with leukemia.

Acetylcholinesterase↗

PubMed1994

Population diversity and distinct haplotype frequencies associated with ACHE and BCHE genes of Israeli Jews from trans-Caucasian Georgia and from Europe.

Variant alleles of the butyrylcholinesterase gene, BCHE, have often been used to trace the genetic histories of populations. The D70G substitution in BCHE causes prolonged postanesthesia apnea ("atypical" phenotype); H322N substitution in the closely related acetylcholinesterase gene, ACHE, is the basis of the mutually incompatible Yt blood groups. In both genes, additional point mutations were reported to be linked to these phenotypically evident ones. To examine whether the intragenic linkage reported for the ACHE and BCHE mutations in Americans is universal, we studied frequencies of these mutations in trans-Caucasian Georgian Jews, a population that has remained relatively isolated for 1500 years. To this end we employed PCR amplification followed by DNA sequencing and enzymatic restriction and compared the frequencies we found to corresponding reported phenotype data. Georgian Jews' N322 ACHE was a rather low 7.0% and was totally linked to a P446 mutation, in agreement with a recent report. In BCHE, however, G70 was a relatively high 5.8%, and the V497 and T539 mutations were not found, either in Georgian or in Ashkenazi Jews, in contrast to reported findings in Americans. Our findings reveal distinct displays of ACHE and BCHE haplotypes in Georgian Jews and suggest different founder effects, genetic drifts, and/or selection pressures in the evolution of each of these genes.

Acetylcholinesterase↗

PubMed1994

Expression of three alternative acetylcholinesterase messenger RNAs in human tumor cell lines of different tissue origins.

To study the molecular mechanisms underlying the intensive expression of acetylcholinesterase (AChE) in different tumor types, we characterized levels and composition of its messenger RNA (mRNA) sequences in heterologous tumor cell lines, primary tumor biopsies, and normal fetal and adult tissues and determined their exon-intron origin within the corresponding ACHE gene. Reverse transcription followed by polymerase chain reaction (RT-PCR) revealed three alternatively spliced ACHE mRNAs in NT2/D1 teratocarcinoma, NCI-N-592 small cell lung carcinoma, TE671 medulloblastoma, K-562 erythroleukemia, and 293 transformed embryonal kidney cells. The three ACHE mRNAs include the principal species expressed in brain and muscle and two additional transcripts containing insertions of 751 or 829 residues downstream from the exon 4 domain. The inserted region, which represents an intron in brain and muscle, is expressed in the tumor cell lines either as a "readthrough" form or with 78 residues deleted from its 5' end. A major band of 2.5 kb was labeled with ACHE cDNA in poly(A)+ RNA blots from medulloblastoma cells or brain tissue, whereas a PCR-amplified probe from the inserted domain labeled a 3.4-kb band but not the 2.5-kb band in poly(A)+ RNA from small cell lung carcinoma. The ACHE mRNAs including the alternative insertions were found only in cell lines with levels of the principal ACHE mRNA species equal to or higher than those in brain (1-10 molecules/cell), determined by following the kinetics of mRNA PCR amplification. Genomic DNA sequencing revealed that the inserted domains in the ACHE mRNAs expressed in the tumor cell lines encode C-terminal peptides of 40 and 14 residues. These include a free cysteine, terminate with the consensus HG element, and continue by a 29-residue-long C-terminal hydrophobic cleavable peptide, properties characteristic of precursors to phosphoinositide (PI)-linked proteins. In extension of the reported expression of PI-linked AChE in hemopoietic cells including K-562, our findings demonstrate the existence of ACHE mRNAs with the potential to encode one hydrophilic and two PI-linked forms of AChE in tumor cells from both hemopoietic and nonhemopoietic origins.

Acetylcholinesterase↗

PubMed1994

Antisense inhibition of butyrylcholinesterase gene expression predicts adverse hematopoietic consequences to cholinesterase inhibitors.

1. To investigate the possibility that cholinesterase inhibitors may cause adverse hematopoietic effects, we employed antisense oligodeoxynucleotides selectively inhibiting butyrylcholinesterase gene expression (AS-BCHE). Complementary sense (S) oligonucleotides served as controls. 2. In primary bone marrow cell cultures grown with interleukin 3 (IL-3), AS-BCHE but not S-BCHE reduced growth of megakaryocyte colony-forming units (CFU-MK) in a dose-dependent manner at the micromolar range. 3. In cultures grown with IL-3, transferrin, and erythropoietin (Epo), cell counts increased up to twofold, yet colony counts (CFU-GEMM) remained unchanged under AS-BCHE treatment. 4. Electrophoretic measurements of DNA ladder as an apoptotic index revealed that the above oligonucleotide effects were not due to nonspecific induction of programmed cell death. 5. Differential cell counts demonstrated increased myeloidogenesis and reduced levels of early megakaryocytes in CFU-GEMM under AS-BCHE, suggesting requirement of the BuChE protein for megakaryopoiesis. 6. In vivo injection of AS-BCHE reduced BCHE mRNA levels in both young and mature megakaryocytes for as long as 20 days, as shown by in situ hybridization. 7. Ex vivo growth of primary bone marrow cells revealed a twofold reduction in CFU-MK colonies grown from the AS-BCHE- but not the S-BCHE-injected mice, 15 days posttreatment. 8. These findings demonstrate that deficient butyrylcholinesterase expression, and hence interference with this enzyme's activity through treatment with or exposure to cholinesterase inhibitors, may cause hematopoietic differences in treated patients.

Animals↗

PubMed1994

Mutations and impaired expression in the ACHE and BCHE genes: neurological implications.

The acetylcholine hydrolysing cholinesterases control the termination of cholinergic signalling in multiple tissues and are targets for a variety of drugs, natural and man-made poisons and common insecticides. Molecular cloning and gene mapping studies revealed the primary structure of human acetyl- and butyrylcholinesterase and localized the corresponding ACHE and BCHE genes to the chromosomal positions 3q26-ter and 7q22, respectively. Several different point mutations in the coding region of BCHE were found to be particularly abundant in the Israeli population. Analytical expression studies in microinjected Xenopus oocytes have demonstrated that the biochemical properties of cholinesterases may be modified by rationalized site-directed mutagenesis and in chimeric ACHE/BCHE constructs. These properties are differently altered in the various allelic BCHE variants, conferring resistance to several anti-cholinesterases, which may explain the evolutionary emergence of these multiple alleles. At the clinical level, abnormal expression of both ACHE and BCHE and the in vivo amplification of the ACHE and BCHE genes has been variously associated with abnormal megakaryocytopoiesis, leukemias and brain and ovarian tumors. Moreover, antisense oligonucleotides blocking the expression of these genes were shown to interfere with hemocytopoiesis in culture, implicating these genes in cholinergic influence on cell growth and proliferation.

Acetylcholinesterase↗

PubMed1994

Site-directed mutagenesis of active site residues reveals plasticity of human butyrylcholinesterase in substrate and inhibitor interactions.

In search of the molecular mechanisms underlying the broad substrate and inhibitor specificities of butyrylcholinesterase (BuChE), we employed site-directed mutagenesis to modify the catalytic triad residue Ser198, the acyl pocket Leu286 and adjacent Phe329 residues, and Met437 and Tyr440 located near the choline binding site. Mutant proteins were produced in microinjected Xenopus oocytes, and Km values towards butyrylthiocholine and IC50 values for the organophosphates diisopropylfluorophosphonate (DFP), diethoxyphosphinylthiocholine iodide (echothiophate), and tetraisopropylpyrophosphoramide (iso-OMPA) were determined. Substitution of Ser198 by cysteine and Met437 by aspartate nearly abolished activity, and other mutations of Ser198 completely abolished it. Tyr440 and Leu286 mutants remained active, but with higher Km and IC50 values. Rates of inhibition by DFP were roughly parallel to IC50 values for several Leu286 mutants. Both Km and IC50 values increased for Leu286 mutants in the order Asp < Gln < Lys. In contrast, cysteine, leucine, and glutamine mutants of Phe329 displayed unmodified Km values toward butyrylthiocholine, but up to 10-fold decreased IC50 values for DFP, iso-OMPA, and echothiophate. These findings add Tyr440 and Phe329 to the list of residues interacting with substrate and ligands, demonstrate plasticity in the active site region of BuChE, and foreshadow the design of recombinant BuChEs with tailored scavenging properties.

Animals↗

PubMed1994

Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.

Formation of a functional neuromuscular junction (NMJ) involves the biosynthesis and transport of numerous muscle-specific proteins, among them the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE). To study the mechanisms underlying this process, we have expressed DNA encoding human AChE downstream of the cytomegalovirus promoter in oocytes and developing embryos of Xenopus laevis. Recombinant human AChE (rHAChE) produced in Xenopus was biochemically and immunochemically indistinguishable from native human AChE but clearly distinguished from the endogenous frog enzyme. In microinjected embryos, high levels of catalytically active rHAChE induced a transient state of over-expression that persisted for at least 4 days postfertilization. rHAChE appeared exclusively as nonassembled monomers in embryos at times when endogenous Xenopus AChE displayed complex oligomeric assembly. Nonetheless, cell-associated rHAChE accumulated in myotomes of 2- and 3-day-old embryos within the same subcellular compartments as native Xenopus AChE. NMJs from 3-day-old DNA-injected embryos displayed fourfold or greater overexpression of AChE, a 30% increase in postsynaptic membrane length, and increased folding of the postsynaptic membrane. These findings indicate that an evolutionarily conserved property directs the intracellular trafficking and synaptic targeting of AChE in muscle and support a role for AChE in vertebrate synaptogenesis.

Acetylcholinesterase↗

PubMed1994

Use of partially phosphorothioated "antisense" oligodeoxynucleotides for sequence-dependent modulation of hematopoiesis in culture.

To distinguish between sequence-dependent effects and non-specific cytotoxicity of phosphorothioate antisense oligonucleotides (AS-oligos), we introduced AS-oligos blocking expression of 2Hs, the Homo sapiens cell division controller cdc2 kinase, its hematopoietically expressed homolog CHED, and the acetylcholine-hydrolyzing enzyme butyrylcholinesterase (BCHE) into primary murine bone marrow (BM) culture. Antisense oligonucleotides were fully phosphorothioated (Ts) or prepared with three phosphorothioate groups at their 3' termini (S3). Each of these oligos could cause reductions in colony counts either as a result of its sequence-dependent biological capacity or due to sequence-independent cytotoxicity. The Ts and S3 forms of the matching sense oligo, S-BCHE, served for comparison. The S3 forms of AS-2Hs, AS-BCHE, and S-BCHE caused more limited drops in colony counts than their Ts counterparts, reflecting lower cytotoxicity. When incubated with electroblotted BM proteins, Ts but not S3 oligos intensively labeled two protein bands. Moreover, 5'-end 32P-labeled (Ts) S-BCHE labeled nuclear proteins in situ in small, mitotic cells, suggesting correlation between oligo-protein interactions and the sequence-independent cytotoxicity of Ts AS-oligos. Extension of the apparently nontoxic AS-CHED by two adenosine residues at the 3' end, creating a potential for intramolecular hydrogen bond formation, resulted in increased toxicity. These findings recommend the use of nonlooped, partially phosphorothioated oligos for the modulation of hematopoiesis.

Animals↗

PubMed1994

Testicular amplification and impaired transmission of human butyrylcholinesterase cDNA in transgenic mice.

Gene amplification occurs frequently in tumour tissues yet is, in general, non-inheritable. To study the molecular mechanisms conferring this restraint, we created transgenic mice carrying a human butyrylcholinesterase (BCHE) coding sequence, previously found to be amplified in a father and son. Blot hybridization of tail DNA samples revealed somatic transgene amplifications with variable restriction patterns and intensities, suggesting the occurrence of independent amplification events, in 31% (11/35) of mice from the FII generation but in only 3.5% (2/58) of the FIII and FIV generations. In contrast, > 10-fold amplifications of the BCHE transgene and the endogenous acetylcholinesterase and c-raf genes appeared in both testis and epididymis DNA from > 80% of FIII mice. Drastic, selective reductions in testis BCHEmRNA but not in actin mRNA were detected by the PCR amplification of testis cDNA from the transgenic mice, and apparently resulted in the limited transmission of amplified genes. The testicular amplification of the BCHE transgene may potentially represent a general phenomenon with clinical implications in human infertility.

Animals↗

PubMed1994

Antisense inhibition of acetylcholinesterase gene expression causes transient hematopoietic alterations in vivo.

Hematopoietic acetylcholinesterase (ACHE) gene expression and its implication for development were studied by in vivo administration to mice of an antisense phosphorothioate oligonucleotide targetted toward ACHE (AS-ACHE). Hematopoietic alterations were observed by differential cell counts and ACHE mRNA levels determined by quantified RNA polymerase chain reaction (RNA-PCR) and in situ hybridization analyses. In control mice, injected with phosphate-buffered saline and untreated, ACHE mRNA labeling with ACHE [35S]cRNA was about 10-fold higher on megakaryocytes (MK) compared with all other bone marrow cells and increased by 20-fold during MK development, similar to reports for MK actin mRNA. Drastic reductions occurred in the bone marrow lymphocyte and erythroid fractions 12 days following intraperitoneal injection of AS-ACHE (5 micrograms/g weight) into groups of four mice. RNA-PCR revealed over 1000-fold decreases in ACHE mRNA levels in lymph nodes and bone marrow at this time, while actin mRNA levels dropped by 10 and 100-fold in lymph nodes and bone marrow of AS-ACHE treated mice compared with controls. In view of the developmental increase in MK actin, this suggested arrest in MK development as well. By 20 days postinjection, bone marrow actin mRNA was fully restored and the sensitive in situ hybridization technique revealed that ACHE mRNA levels were also restored and reached levels only 2-3-fold lower than in controls in all bone marrow cells of AS-ACHE treated mice. Moreover, lymphocytes and erythroid cells repopulated to levels 25% above normal, and promegakaryocyte and mature MK fractions of the total MK were 3 and 2-fold higher, respectively, than in controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholinesterase↗

PubMed1993

Chimeric human cholinesterase. Identification of interaction sites responsible for recognition of acetyl- or butyrylcholinesterase-specific ligands.

Acetyl- and butyrylcholinesterases (AChE, BuChE) from various species differ in their substrate specificities and sensitivities to a wide range of inhibitors, yet display conserved sequence, structure and catalytic properties. To determine features that confer these selective properties, residues 58 through 133 of recombinant human BuChE were replaced with the corresponding sequence from human AChE. The replaced region (> 60% identity) spans the Asp70 residue, important for ligand interactions, and the choline binding site, and introduces differences of charge and hydrophobicity in the outer rim and on the surface of the active site gorge. Expressed in microinjected Xenopus laevis oocytes, the resultant chimera retained the catalytic activity, substrate specificity and the Km value toward butyrylthiocholine characteristic of BuChE. Further, it did not acquire substrate inhibition, which is unique to AChE, although it lost the property of substrate activation, characteristic of BuChE. Moreover, the chimera resembled BuChE in its sensitivity to succinylcholine and physostigmine, but acquired the AChE-like sensitivity to echothiophate and iso-OMPA, and displayed an intermediate pattern of inhibition, more similar to that of AChE than of BuChE, toward bambuterol, dibucaine and BW284C51. These findings demonstrate that the exchanged residues are involved in inhibitor recognition, but not in substrate distinction and in direct catalysis. Furthermore, substrate interaction with the exchanged domain may mediate structural changes leading to substrate activation in BuChE and inhibition in AChE. The two AChE-specific aromatic tyrosine residues positioned near Asp70 within this region are hence implicated in the peripheral anionic site of cholinesterases, which is involved in the recognition of various ligands.

Acetylcholinesterase↗

PubMed1993

Expression of a human acetylcholinesterase promoter-reporter construct in developing neuromuscular junctions of Xenopus embryos.

We have employed Xenopus embryos to express human acetylcholinesterase (AcChoEase; EC 3.1.1.7) in developing synapses. Transcription of AcChoEase mRNA was driven by a 2.2-kb sequence upstream from the initiator AUG in the ACHE gene encoding AcChoEase, with multiple potential sites for binding universal and tissue-specific transcription factors. These included clustered MyoD elements, E-box, SP1, EGR1, AP-2, and the development-related GAGA motif. A DNA construct composed of this sequence linked to a 2.1-kb sequence encoding human AcChoEase was designated human AcChoEase promoter-reporter (HpACHE). HpACHE but none of its several 5'-truncated derivatives was transcriptionally active in developing Xenopus embryos. Furthermore, PCR analysis using chimeric PCR primers revealed usage of the same 1.5-kb intron and 74-bp exon within the HpACHE sequence in microinjected embryos and various human tissues. Cytochemical staining revealed conspicuous accumulation of overexpressed AcChoEase in neuromuscular junctions and within muscle fibers of apparently normal 2-day Xenopus embryos injected with HpACHE. The same reporter driven by the cytomegalovirus promoter was similarly efficient in directing the heterologous human enzyme toward neuromuscular junctions, attributing the evolutionary conservation of AcChoEase targeting to the coding sequence. Our findings demonstrate that a short DNA sequence is sufficient to promote the exogenous transcription and faithful splicing of human AcChoEase mRNA in developing Xenopus embryos and foreshadow their use for integrative studies of cholinergic signaling and synapse formation.

Acetylcholinesterase↗

PubMed1993

Molecular dissection of cholinesterase domains responsible for carbamate toxicity.

Carbamate compounds marked for their cholinesterase (ChE) inhibition are widely used as therapeutics and as insecticides. Groups of closely related carbamate molecules provide an important tool in the understanding of the domains responsible for binding these ligands to ChEs. Comparative inhibition profiles were derived for five N-methyl carbamates, mostly carbofuran derivatives, differing in length and branching of their hydrocarbonic chain towards human erythrocyte acetylcholinesterase (H.AChE), human serum butyrylcholinesterase (H.BChE) in its normal form or in a mutant form containing the point mutation Asp70-->Gly, and Drosophila nervous system ChE. Carbofuran was more toxic to all three ChEs than any of the other derivatives, with IC50 values which differed by more than 1000-fold. Drosophila ChE appeared to be most sensitive to all of the examined carbamates, and H.AChE was consistently more sensitive than H.BChE. Moreover, inhibition efficiency for H.BChE decreased more effectively than it did for H.AChE with increased length and complexity of the side chain, indicating less flexible carbamate binding site in BChE as compared with AChE. The Asp70-->Gly mutation had no apparent effect on H.BChE inhibition by N-methyl carbamates, suggesting that the Asp70 domain localized near the rim of the active site groove is not important in carbamate binding. Comparison of the carbamate IC50 values with published LD50 values demonstrated correlation between the in vivo toxicity and inhibition of BChE by carbamates, suggesting a biological in addition to scavenging importance for BChE in mammals. Pinpointing different domains characteristic of carbamate binding in each member of the ChE family can thus shed light on the variable toxicity of these inhibitors to insects and mammals, predict the toxicity of yet untested inhibitor molecules and help in designing novel and improved ChE inhibitors.

Acetylcholinesterase↗