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 and butyrylcholinesterase genes coamplify in primary ovarian carcinomas.

The genes for acetylcholinesterase (ACHE) and butyrylcholinesterase (CHE) are expressed in multiple tumor tissues, including ovarian carcinomas. Both CHE and ACHE genes coamplify in leukemias. To examine the relationship of gene amplification to the expression of these genes in tumors, ACHE and CHE genes and their expression were studied in primary ovarian carcinomas. DNA blot hybridization demonstrated a significant amplification and mutagenesis of both genes in 6 of 11 malignant tumors studied. This was greater or of the same order of magnitude as the amplification of the oncogenes c-rafi, v-sis, and c-fes in these tumors. No amplification was found in normal ovarian tissues or benign ovarian cysts. Xenopus oocyte microinjections, blot and in situ hybridizations, and immuno- and cytochemical staining revealed translatable CHEmRNA and its active protein product in discrete tumor foci. The frequent coamplification in ovarian carcinomas of ACHE and CHE genes implicates cholinesterases in neoplastic growth and/or proliferation.

Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.

Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.

Expression of alternatively terminated unusual human butyrylcholinesterase messenger RNA transcripts, mapping to chromosome 3q26-ter, in nervous system tumors.

To study the molecular origin of the altered regulation of butyrylcholinesterase (BuChE) in nervous system tumors, BuChE complementary DNA (cDNA) sequences from human glioblastoma and neuroblastoma cDNA libraries were compared with BuChE cDNAs from normal fetal and adult tissues. A single 2.6-kilobase BuChE cDNA sequence was found in all normal tissues, whereas an additional alternatively terminated BuChE cDNA clone was found in both tumor libraries. The tumor-specific cDNA contained a 3',0.7-kilobase nontranslatable extension, as well as several nucleotide alterations in the normal polyadenylation site. Single-base mutations in the coding region of this unusual BuChE cDNA infer two amino acid substitutions: Asp70----Gly and Ser425----Pro. The Asp70----Gly change has recently been implicated with "atypical" BuChE, which is deficient in its capacity to hydrolyze succinylcholine. The 3.6-kilobase mRNA was less abundant in RNA blot hybridization than the 2.6-kilobase mRNA, which is in agreement with the low ratios between the 3.6- and 2.6-kilobase BuChE cDNA clones in glioblastoma and neuroblastoma libraries. Furthermore, size fractionation and microinjection of glioblastoma polyadenylated RNA, followed by enzyme activity and selective inhibition measurements, demonstrated two peaks of functional BuChE mRNA, the heavier one probably reflecting the longer transcripts. Chromosomal mapping of the 0.7-kilobase 3' fragment by in situ hybridization localized it to a unique 3q26-ter position, where we recently found an inheritably amplified "silent" defective CHE gene in a family exposed to the cholinesterase inhibitor methyl parathion. Our findings confirm previous genetic linkage mapping of the functional CHE gene to the 3q26-ter position and demonstrate that extended functional mRNA transcripts encoding a BuChE form with two modified amino acids are produced from this gene in glioblastoma and neuroblastoma cells.

Gonadotropin-releasing hormone stimulates phosphatidylinositol labeling and prostaglandin E production in Leydig cells.

The direct effects of gonadotropin releasing hormone (GnRH) upon testicular function include a rapid (integral of 20 min) receptor-mediated increase in phosphatidylinositol (PI) turnover. Incubation of rat interstitial cells with the super-agonist [D-Ala6]desGly10-GnRH N ethylamide (GnRHa) resulted in increased incorporation of [32P]Pi into PI (2-fold at 20 min). The effect on phospholipid turnover was followed by increased prostaglandin E (PGE) and testosterone production (3 h; with ED50 values of 0.5 and 0.75 nM respectively). It is concluded that increased PI turnover and PGE production might be involved in mediating the direct testicular effects of GnRH and its agonists.

Stimulation of prostaglandin E and testosterone production in rat interstitial cells by a gonadotropin-releasing hormone agonist.

The early direct effects of a GnRH agonist analog [D-Ala6]des-Gly10-GnRH N ethylamide (GnRHa) on rat testicular interstitial cells include increased production of prostaglandin E (PGE) and testosterone (T) at 3 h (ED50 values of 0.5 and 0.75 nM, respectively). On the other hand, LH action on testicular function, which is mediated by increased cAMP, involves an increase in T production at 30 min followed by increased PGE formation at 3 h. GnRHa at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. The stimulatory effect of GnRHa on PGE, but not on T production, was abolished by the prostaglandin synthesis inhibitor indomethacin (1.5 microns). We conclude that cAMP does not play a role in mediating the direct testicular effects of GnRH on PGE and T production; that PGE is not involved in mediating GnRH-induced T production; and, finally, that increased PGE and T production might be involved in mediating the direct inhibitory and stimulatory testicular effects of GnRH and its agonist analogs.

Regulation of prostaglandin E, progesterone, and cyclic adenosine monophosphate production in ovarian granulosa cells by luteinizing hormone and gonadotropin-releasing hormone agonist: comparative studies.

The early direct effects of GnRH on the ovary were investigated using cultured granulosa cells from preovulatory rat follicles, and compared to the known stimulatory effects of LH. Stimulation of ovarian functions by a GnRH agonist include a rapid receptor-mediated phosphatidylinositol turnover (approximately 5 min). On the other hand, LH action on granulosa cells is initiated by increased cAMP production (approximately 10-15 min), consisting of an indomethacin-resistant and indomethacin-sensitive pools (40% and 60%, respectively). The GnRH agonist [D-Ala6] des-Gly10 N-ethylamide (GnRHa) at concentrations of 10(-12)-10(-8) M had no effect on basal or LH-stimulated cAMP production during a 4-h incubation test. Both LH and GnRHa increase progesterone formation (30 and 120 min, respectively) with ED50 values of 2.5 ng/ml and 10(-9) M, respectively and the stimulatory effect is not blocked by indomethacin. LH and GnRHa increase also prostaglandin E (PGE) formation (180 and 120 min, respectively) and the ED50 values were 0.1 microgram/ml and 10(-9) M, respectively. No inhibitory effect of GnRHa on LH actions was observed during 4 h of incubation. It is concluded that: 1) GnRH mimicks LH stimulation of ovarian PGE and progesterone production; 2) cAMP does not play a role in mediating the direct stimulatory effects of GnRH agonists on ovarian PGE and progesterone production; 3) PGE is not involved in mediating GnRH and LH stimulation of progesterone formation. 4) LH-induced cAMP production consists of indomethacin-sensitive and indomethacin-resistant pools.

Cholinoceptive properties of human primordial, preantral, and antral oocytes: in situ hybridization and biochemical evidence for expression of cholinesterase genes.

In addition to their well-known involvement in neuromuscular junctions and in brain cholinergic synapses, cholinergic mechanisms have been implicated in the growth and maturation of oocytes in various species. Functional acetylcholine receptors were electrophysiologically demonstrated in amphibian and mammalian oocyte membranes, and activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE), was biochemically measured in the exceptionally big oocytes of the frog Xenopus laevis. However, biochemical methods could not reveal whether AChE was produced within the oocytes themselves or in the surrounding follicle cells. Furthermore, this issue is particularly important for understanding growth and fertilization processes in the much smaller human oocytes, in which the sensitivity of AChE biochemical measurements is far too low to be employed. To resolve this question, a molecular biology approach was combined with biochemical measurements on ovarian extracts and sections. To directly determine whether the human cholinesterase (ChE) genes are transcriptionally active in oocytes, and, if so, at what stages in their development, the presence of ChE mRNA was pursued. For this purpose frozen ovarian sections were subjected to in situ hybridization using 35S-labeled human ChE cDNA. Highly pronounced hybridization signals were localized within oocytes in primordial, preantral, and antral follicles, but not in other ovarian cell types, demonstrating that within the human ovary ChE mRNA is selectively synthesized in viable oocytes at different developmental stages.(ABSTRACT TRUNCATED AT 250 WORDS)

Dissociation between the direct stimulatory and inhibitory effects of a gonadotropin-releasing hormone analog on ovarian functions.

The paradoxical effects of gonadotropin-releasing hormone (GnRH) on the ovary have hitherto been believed to result from different regimens of administration; an acute treatment was shown to stimulate the ovary while chronic administration of the hormone inhibited LH-induced responses. In the present report we demonstrate that a single injection of a GnRH analog (D-Ala6)des-Gly10-GnRH-N-ethylamide (GnRHa, 2 micrograms/rat) is sufficient to obtain a significant inhibition (75%) of hCG-induced ovulation in PMSG-primed, either intact or hypophysectomized, immature rats. Inhibition of ovarian development, in terms of growth and ovulation, by multiple injections with GnRHa (2 micrograms/rat, twice daily for 3 days) could be obtained only upon administration of the hormone at early stages of follicular development, i.e. concomitantly with the PMSG injection. When administered after PMSG, GnRHa could not inhibit the ovary but rather induced ovulation by itself in the absence of hCG. A 12-24 h delay in initiation of GnRHa treatment triggered 65% of the rats to ovulate while a delay of 48 h resulted in 100% ovulation. Under both regimes of GnRHa administration, either the inhibitory or the stimulatory, the oocytes of the treated rats were induced to resume meiotic maturation. Since under the inhibitory regime ovulation did not occur, maturation was followed by a massive degeneration of the oocytes trapped within their follicles. These findings demonstrate that the follicular stage of development rather than the dose and/or duration of GnRHa administration determines whether GnRHa inhibits ovarian growth and ovulation, while the competence of the oocytes to respond to the GnRHa stimulus and mature is independent of hormonal priming.

Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves' ophthalmopathy.

The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.

Electrical activity of the human uterus in the presence of intrauterine contraceptive device.

PIP: To determine the effects upon the electrophysiology of the female genital tract in the presence of an IUD and compare them with patterns obtained without an IUD, electrodes were implanted in volunteers and periodic recordings were taken. Much more electrical activity was recorded from the IUD electrode than from non-IUD-wearers. Most electrical activity was seen during menstruation which may be a reason that most IUD expulsions occur at that time. The increased electrical activity in IUD wearers may interfere with implantation of a fertilized ovum, thereby being a mode of IUD contraceptive action.^ieng

Incubation with sperm enhances in vitro maturation of the oocyte from the germinal vesicle to the M2 stage.

OBJECTIVE: To evaluate the effect of sperm in the culture medium on the rate of oocyte maturation in vitro from the germinal vesicle to the M2 stage. DESIGN: Prospective randomized controlled study. SETTING: The IVF Unit, Wolfson Medical Center, Holon, Israel. PATIENT(S): All women in whom oocytes were retrieved at the germinal vesicle stage between December 1995 and March 1996. INTERVENTION(S): Oocytes retrieved at the germinal vesicle stage were divided prospectively and randomly into four groups of incubation conditions: group 1, intact germinal vesicle with cumulus; group 2, intact germinal vesicle with sperm cells in the culture medium; group 3, stripped germinal vesicle; and group 4, stripped germinal vesicle with sperm cells. Oocytes were observed 24 hours after retrieval, and the stage of maturation was recorded. Oocytes that reached the M2 stage underwent the intracytoplasmic injection procedure, and the fertilization rate in each group was recorded at 48 hours. MAIN OUTCOME MEASURE(S): Maturation rate from the germinal vesicle to M2 stage and fertilization rate. RESULT(S): Each group contained 20 germinal vesicle oocytes. In groups 1 and 2, 2 (10%) and 9 (45%) oocytes, respectively, reached the M2 stage at 24 hours; at 48 hours, 1 (5%) and 8 (40%) embryos developed, respectively. The results in group 2 were significantly higher than in group 1. In groups 3 and 4, 6 (30%) and 16 (80%) oocytes, respectively, reached the M2 stage at 24 hours; at 48 hours, 5 (25%) and 14 (70%) embryos developed, respectively. Results in group 4 were significantly higher than those in groups 1, 2, and 3. CONCLUSION(S): Both methods of oocyte activation (i.e., addition of sperm to the culture medium or removal of the cumulus) enhance oocyte maturation in vitro, but the sperm-incubation method has a more pronounced effect. A combination of both methods leads to an exceptionally high rate of oocyte maturation, followed by a high fertilization rate.

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.

Lymphocyte subsets in habitual abortion.

Lymphocyte subpopulations were characterized by means of monoclonal antibodies in 25 women with habitual abortion and 21 multiparous normal women. Compared to nonpregnant women (N = 8), pregnant normal women were associated with significantly lower helper-to-suppressor ratios (1.71 +/- 0.41 versus 2.37 +/- 0.66). In contrast in pregnant women with habitual abortion (N = 13) the ratio remained high (2.32 +/- 0.73). Failure to increase the number of suppressors and a significant rise in helpers caused this increased ratio. We discuss the possible mechanisms and etiological importance of this finding in habitual abortion.

The incidence and significance of serum hCG and CEA in patients with gastrointestinal malignant tumors.

Beta-human chorionic gonadotropin (hCG) is normally produced and secreted by trophoblastic cells in pregnancy, by tumors arising from those cells and by a wide variety of tumors of nonendocrine origin. Gonadotropin is produced and secreted by various tissues (stomach, pancreas, ovary, etc.) and the incidence of ectopic secretion varies between 0 and 43%. Our report is an attempt to evaluate the incidence of high plasma beta hCG levels in 101 patients with gastrointestinal malignant tumors. The results revealed negative beta hCG in the control samples, while in the studied patients 41 were positive for beta hCG (44.4%). Three samples from oesophagus squamous cell carcinoma were positive. Twenty-five out of 69 with colorectal carcinoma had raised serum beta hCG (36.8%). Gastric carcinoma showed positive beta hCG in 52% of the patients. Among all the patients high beta hCG levels were far more common in those with positive lymphnodes (P less than 0.05). The beta hCG levels decreased with colorectal carcinoma tumor size and with smaller tumors there was the probability of increasing positive serum measurements (P less than 0.05). The patients with adenocarcinoma of the stomach showed good statistical correlation between stages of the disease at the operation time to beta hCG levels. In our opinion this serological assay will become one of the markers to be added to our armamentarium in the evaluation of patients with gastrointestinal malignant tumors.

Coincidence of down-regulation and desensitization in pituitary gonadotrophs stimulated by gonadotropin releasing hormone.

The dynamics of gonadotropin releasing hormone (GnRH) induced luteinizing hormone (LH) release was studied in vitro by superfusion of cultured pituitary cells. Continuous exposure of the cells to GnRH resulted in desensitization of the gonadotroph responsiveness to further stimulation by the hormone. The refractory state was achieved within 4 hr of hormone introduction (10(-7) M) and was accompanied by down-regulation of GnRH receptors (50%) assayed by equilibration with [125I]iodo-[D-Ala6]des-Gly10-GnRH N-ethylamide. The data indicate that GnRH can regulate the number of its own receptors, and that desensitization is accompanied by down-regulation.

Gonadotropin releasing hormone: regulation of phospholipid turnover and prostaglandin production in ovarian granulosa cells.

The direct effect of gonadotropin releasing hormone (GnRH) upon ovarian function, is initiated by a rapid receptor-mediated increase in phosphatidylinositol (PI) turnover (approximately 5 min) followed by prostaglandin E (PGE, 120 min) and progesterone (120 min) formation, oocyte maturation and induction of ovulation. In contrast, luteinizing hormone (LH) stimulation of oocyte maturation and induction of ovulation is mediated by increased adenosine 3',5'-monophosphate (cAMP, 15 min), progesterone (30 min) and PGE (180 min) production. Both LH and GnRH stimulation of oocyte maturation are inhibited by dibutyryl cAMP and 3-isobutyl-1-methylxanthine, whereas induction of ovulation by the two hormones is blocked by indomethacin. GnRH and LH differ, therefore, in the mechanism leading to PGE formation, but thereafter share a common mechanism responsible for oocyte maturation and independently for induction of ovulation.

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.

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.

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.