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H Zakut

Publications and source records attributed to H Zakut.

At least 37 records · Page 2Linked to original sources
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

Autologous umbilical cord blood transfusion.

The purpose of this study was to examine some aspects of umbilical cord blood collection for autologous transfusion in premature infants. All 120 microbacterial cultures (aerobic and anaerobic) of cord blood samples as well as 30 cultures of mycoplasma were treated. Cord prothrombin fragment (F 1 + 2) concentrations were quantified at one and 10 minutes after clamping of the cord. F 1 + 2 concentrations assessed on 25 newborn infants were similar and no linear association with time of clamping could be drawn. This means that cord blood thrombosis is not activated for at least 10 minutes following clamping of the cord. As far as is known, the first newborn infant to benefit from this method of transfusion is reported here. The premature infant received two portions of autologous blood (on days 5 and 7). No untoward effects were noted. Blood, collected from the umbilical cord, is a safe source for autotransfusion, provided that bacteriological testing has been carried out.

Blood Coagulation↗
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

Ureteric stent in severe hydronephrosis of pregnancy.

Eleven women (12 pregnancies) were included in the study. A double-pigtail ureteric stent was passed under cytoscopic vision in 8 patients. The mean gestational age on insertion of the stents was 29 weeks (range, 25-35 weeks). Delivery took place at a mean of 39 weeks (range, 35-41 weeks). Stents remained in situ for a mean of 9 weeks (range, 6-14 weeks) before delivery and were removed 4-5 weeks postpartum. Double pigtail ureteric stents did not expose the women to infection. Renal function tests remained within the normal limits. Ureteric stent is an effective method for internal drainage of severe hydronephrosis during pregnancy.

Adult↗
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

Carnitine palmitoyl transferase deficiency in pregnancy--a case report.

Carnitine palmitoyl transferase deficiency is one of the mitochondrial myopathies characterized by weakness, exercise intolerance, and myoglobinuria after prolonged exercise. This is the first case report concerning a pregnant woman with carnitine palmitoyl transferase deficiency that was also proved by the decrease of carnitine palmitoyl transferase activity in uterine myometrium biopsy.

Adult↗
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

Autologous cord blood transfusion.

Newborn piglets were exsanguinated (60% of blood volume) and retransfused 1 h later. One test group received adult pig red blood cells, the other piglet cord blood cells; controls were infused with plasma. While all controls died, satisfactory results were achieved in piglets transfused with either adult or foetal blood. The feasibility of collecting human cord blood for transfusion was assessed in 100 samples of human cord blood. Blood was collected aseptically and aerobic and anaerobic cultures set up. All samples of cord blood were sterile, and all were Mycoplasma negative. Coagulation parameters were analysed in eight cord plasma samples stored at -20 degrees C for 45 days. No significant abnormalities were found immediately after birth or after storage.

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

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↗
PubMed1993

Structure-function relationship studies in human cholinesterases reveal genomic origins for individual variations in cholinergic drug responses.

1. Due to their involvement in the termination of neurotransmission at cholinergic synapses and neuromuscular junctions, cholinesterases are the target proteins for numerous drugs of neuro-psychopharmacology importance. 2. In order to perform structure-function relationship studies on human cholinesterases with respect to such drugs, a set of expression vectors was engineered, all of which include cloned cDNA inserts encoding various forms of human acetyl- and butyrylcholinesterase. These vectors were designed to be transcribed in vitro into their corresponding mRNA products which, when microinjected into Xenopus oocytes, are efficiently translated to yield their catalytically active enzymes, each with its distinct substrate specificity and sensitivity to selective inhibitors. 3. A fully automated microtiter plate assay for evaluating the inhibition of said enzymes by tested cholinergic drugs and/or poisons has been developed, in conjunction with computerized data analysis, which offers prediction of such inhibition data on the authentic human enzymes and their natural or mutagenized variants. 4. Thus, it was found that asp70-->gly substitution renders butyrylcholinesterase succinylcholine insensitive and resistant to oxime reactivation while ser 425-->Pro with gly70 gives rise to the "atypical" butyrylcholinesterase phenotype, abolishing dibucaine binding. 5. Furthermore, differences in cholinesterase affinities to physostigmine, ecothiophate and bambuterol were shown in these natural variants. 6. Definition of key residues important for drug interactions may initiate rational design of more specific cholinesterase inhibitors, with fewer side effects. This, in turn, offers therapeutic potential in the treatment of clinical syndromes such as Alzheimer's and Parkinson's disease, glaucoma and myasthenia gravis.

Animals↗