Tissue specific expression of the acetylcholinesterase gene in Drosophila melanogaster

Summary Acetylcholinesterase (AChE) is mainly membrane bound in the central nervous system (CNS) of larvae and in the head and thorax of adults of Drosophila melanogaster; it is mostly soluble in the larval carcass, the adult abdomen, similar to that of the embryos (Zador et al. 1986). The enzyme shows the same number of isozymes (four or five) in larvae and adults as in the head of the fly or in embryos (Zador et al. 1986). In the Df(3R)GE26/MKRS stock both the membrane bound and the soluble enzyme are at about half normal levels while in the Df(3R)Ace ^HD1/MKRS stock this is true only for the membrane bound AChE. Therefore the effect of the above deficiencies in larvae and adults is consistent with that in embryos (Zador et al. 1986). In heat-sensitive combinations of certain Ace mutant alleles both the membrane bound and the soluble enzyme has reduced activity.Abbreviations AChE acetylcholinesterase (acetylcholine acetyl hydrolase, EC 3.1.1.7) - BAP 1,5-bis(allyldimethylammonium-phenyl)-pentan-3-one dibromide - CNS central nervous system     

Modulation of Protein Phosphorylation by M r 25,000 Protein Partially Overlapping Phosvitin and Lipovitellin 2 in Xenopus laevis Vitellogenin B1 Protein

A phosphorylated protein with molecular mass of 25,000 (pp25) is a component of Xenopus laevis vitellogenin B1. In an attempt to elucidate the physiological role of pp25, its effect on protein phosphorylation was studied. In vitro phosphorylation of some endogenous proteins from the cytoplasm and membrane fraction of Xenopus oocytes by casein kinase II and protein kinase C (PKC) was inhibited by increasing the concentration of pp25. By Western blot analysis using an antibody against phospho-(Ser/Thr) PKC substrate, phosphorylation of some endogenous proteins, especially in the cytoplasm, of Xenopus embryos was seen to increase when pp25 disappeared during developmental stages 35–45. These results suggest that pp25 may have a role as an inhibitory modulator of some protein phosphorylation in Xenopus oocytes and embryos.     

Hormonal modulation of neuronal cells behaviour in vitro

In this study we have investigated the effect of insulin and/or of nerve growth factor (NGF) on enzyme activities of cholinergic neurotransmission, in cultured embryonic rat mesencephali. Our data show that choline-O-acetyltransferase (ChAT) and acetylcholinesterase (AChE) activity display a prominent change in the embryonic brain tissues as a function of time in vitro. The change depends on the age of embryos from which the brain cell cultures have been set up. Namely, ChAT activity increases in the cultures taken from 13-17-day-old embryos as a function of time in vitro. AChE activity shows a striking decrease if the cultures have been set up from the older embryos (17-day-old), while AChE activity increases in the cultures prepared from 13-day-old embryos continuously. Insulin (amount ranging 10-27 micrograms/ml) causes a significant inhibition in the ChAT activity in comparison with the increased enzyme activity measured in control cultures (insulin ranging from 1 to 100 ng). AChE activity of 13-day-old embryos was not influenced by insulin (20-27 micrograms/ml) but the same amount of insulin prevents the decrease of AChE activity in cultured brain cells originating from 17-day-old-embryos. Biochemical studies of NGF treated cultures (30 ng/ml) revealed that nerve growth factor resulted in 5-12-fold increase in specific activity of the cholinergic enzyme, choline acetyltransferase (ChAT). NGF did not influence the AChE activity in cultured brain cells (13-17-day-old).     

A novel function for KIF13B in germ cell migration

Primordial germ cell (PGC) development in Xenopus embryos relies on localised maternal determinants. We report on the identification and functional characterisation of such one novel activity, a germ plasm associated mRNA encoding for the Xenopus version of a kinesin termed KIF13B. Modulations of xKIF13B function result in germ cell mismigration and in reduced numbers of such cells. PGCs explanted from Xenopus embryos form bleb-like protrusions enriched in PIP3. Knockdown of xKIF13B results in inhibition of blebbing and PIP3 accumulation. Interference with PIP3 synthesis leads to PGC mismigration in vivo and in vitro. We propose that xKIF13B function is linked to polarized accumulation of PIP3 and directional migration of the PGCs in Xenopus embryos.     

Comparison of properties of native and lytic forms of acetylcholinesterase from Apis mellifera

The properties of purified native soluble AChE (sAChE) from Apis mellifera were compared with those of purified membrane AChE (mAChE), mAChE solubilized with phosphatidylinositol-specific phospholipase C (AChEPI-PLC), glycosyl phosphatidylinositol-specific phospholipase D (AChEGPI-PLD) and trypsin (AChETi), and other soluble derivative forms obtained from mAChE by autolysis (AChELyt) or limited digestions with proteinase K or chymotrypsin. Analysis by non-denaturing electrophoresis showed that the electrophoretic mobilities of all lytic soluble forms were higher than that of sAChE. sAChE had a K_m value of about 90 μM whereas mAChE, AChETi, AChELyt, AChEPI-PLC, and AChEGPI-PLD displayed a lower K_m value of about 20 μM. The sensitivity to organophosphates was lower for sAChE than for mAChE, AChETi, AChEPI-PLC, and AChEGPI-PLD and was due to higher K_d and lower k₂ values observed for sAChE. In Arrhenius plot analysis, mAChE, AChETi, AChEPI-PLC, and AChEGPI-PLD displayed a homogenous behaviour whereas sAChE exhibited a highly reproducible break at 18°C. Thermal stability studies at 52°C revealed that sAChE, AChEPI-PLC, and AChEGPI-PLD displayed the highest thermal stability with inactivation constants of about 0.020 min⁻¹. This high thermal stability contrasted with those of mAChE, AChELyt, and AChETi, which exhibited respective inactivation constants of 0.051, 0.074, and 0.171 min⁻¹. These results suggest that sAChE is not the mere cleavage product of mAChE by endogenous (glycosyl) phosphatidylinositol-specific phospholipase C/D or proteases. Arch. Insect Biochem. Physiol. 34:143–157, 1997. © 1997 Wiley-Liss, Inc.     

The induction of pyruvate kinase synthesis by heat shock in Xenopus laevis embryos

Heat-shocked Xenopus embryos have an unusually complex heat shock response. The dominant heat shock protein (Hsp) has a relative molecular mass (M_r) of 62,000 D (Hsp62). Affinity-purified IgGs against the glycolytic enzyme pyruvate kinase (PK; EC 2.7.1.40) specifically immunoprecipitated Hsp62 from extracts of embryos that had been heat-shocked at 37°C for 30 min. Thus, Hsp62 and pyruvate kinase are immunologically cross-reacting. Electrophoretic separation of PK isoforms suggests that heat-shocked Xenopus embryos increase synthesis of an isoform of PK. Thermal denaturation studies suggest that this isoform has enhanced thermal stability. The identification of PK as an Hsp is discussed within the context of a physiological requirement for elevated levels of anaerobic glycolysis in heatstressed cells as a vital component of the acquisition of thermotolerance. © 1993Wiley-Liss, Inc.     

Interactions of butane, but-2-ene or xylene-like linked bispyridinium para-aldoximes with native and tabun-inhibited human cholinesterases

Kinetic parameters were evaluated for inhibition of native and reactivation of tabun-inhibited human erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7) and human plasma butyrylcholinesterase (BChE, EC 3.1.1.8) by three bispyridinium para-aldoximes with butane (K074), but-2-ene (K075) or xylene-like linker (K114). Tested aldoximes reversibly inhibited both cholinesterases with the preference for binding to the native AChE. Both cholinesterases showed the highest affinity for K114 (K_i was 0.01 mM for AChE and 0.06 mM for BChE). The reactivation of tabun-inhibited AChE was efficient by K074 and K075. Their overall reactivation rate constants were around 2000 min⁻¹ M⁻¹, which is seven times higher than for the classical bispyridinium para-aldoxime TMB-4. The reactivation of tabun-inhibited AChE assisted by K114 was slow and reached 90% after 20 h. Since the aldoxime binding affinity of tabun-inhibited AChE was similar for all tested aldoximes (and corresponded to their K_i), the rate of the nucleophilic displacement of the phosphoryl-moiety from the active site serine was the limiting factor for AChE reactivation. On the other hand, none of the aldoximes displayed a significant reactivation of tabun-inhibited BChE. Even after 20 h, the reactivation maximum was 60% for 1 mM K074 and K075, and only 20% for 1 mM K114. However, lower BChE affinities for K074 and K075 compared to AChE suggest that the fast tabun-inhibited AChE reactivation by these compounds would not be obstructed by their interactions with BChE in vivo.     

Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs

Centrosomes are powerful and exclusive parthenogenetic agents in the Xenopus egg. We have previously shown that heterologous centrosomes from various vertebrate species were able to promote egg cleavage in Xenopus and that human centrosome activity was associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome. In this work, we have investigated the parthenogenetic capacity of more evolutionary distant centrosomes. We show that centrosomes devoid of centrioles, such as SPBs isolated from Saccharomyces cerevisiae, do not form asters of microtubules in cytoplasmic extracts from Xenopus eggs, and are inactive in the parthenogenetic test. We further show that Drosophila centrosomes which possess a typical centriole architecture, and are quite active to nucleate microtubules in Xenopus cytoplasmic extracts, are unable to trigger egg cleavage. This was observed both with centrosomes isolated from Drosophila syncytial embryos and nucleus-centrosome complexes from the Drosophila Kc23 cell line. We demonstrate that this inability could not be restored after pre-incubation of Drosophila centrosomes in the egg cytoplasm before injection. We conclude that the parthenogenetic activity of a centrosome is not directly linked to its capacity to nucleate microtubules from the egg tubulin, and that the evolutionary conserved nine-fold symmetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly.     

The preparation,in vitro screening and molecular docking of symmetrical bisquaternary cholinesterase inhibitors containing a but-(2E)-en-1,4-diyl connecting linkage

Carbamate inhibitors (e.g. pyridostigmine bromide) are used as a pre-treatment for the prevention of organophosphorus poisoning. They work by blocking the native function of acetylcholinesterases (AChE) and thus protect AChE against irreversible inhibition by organophosphorus compounds. However, carbamate inhibitors are known for their many undesirable side effects related to the carbamylation of AChE. In this paper, we describe 17 novel bisquaternary compounds and have analysed their effect on AChE inhibition. The newly prepared compounds were evaluated in vitro using both human erythrocyte AChE and human plasmatic butyrylcholinesterase. Their inhibitory ability was expressed as the half maximal inhibitory concentration (IC₅₀) and then compared to the standard carbamate drugs and two AChE reactivators. One of these novel compounds showed promising AChE inhibition in vitro (nM range) and was better than the currently used standards. Additionally, a kinetic assay confirmed the non-competitive inhibition of hAChE by this novel compound. Consequently, the docking results confirmed the apparent π-π or π-cationic interactions with the key amino acid residues of hAChE and the binding of the chosen compound at the enzyme catalytic site.     

Minireview: Genetic Manipulations of Cholinergic Communication Reveal Trans-Acting Control Mechanisms Over Acetylcholine Receptors

Abstract

Several approaches have been developed for genetic modulations of receptor expression. These initiated with gene cloning and heterologous expression in microinjected Xenopus oocytes, and proceeded through transgenic expression and genomic disruption of receptor genes in mice. In addition, antisense treatments have reduced receptor levels in a transient, reversible manner. Integration of foreign DNA with host genomic sequences yields both cis- and trans-acting responses. These may depend on the DNA integration site, host cells condition and, most importantly, the affected signal transduction circuit. For example, acetylcholinesterase (AChE) overexpression in microinjected Xenopus tadpoles has been shown to upregulate α-bungarotoxin binding levels, indicating trans-acting control conferring overproduction of muscle nicotinic acetylcholine receptors. In transgenic mice expressing human AChE, the hypothermic response to oxotremorine was suppressed, reflecting modified levels of brain muscarinic receptors. To dissociate the feedback processes occurring in transfected cells from responses related to DNA integration, we examined the endogenous expression of the α₇ neuronal nicotinic acetylcholine receptor in PC 12 cells transfected with DNA vectors carrying alternative splicing variants of human AChE mRNA. Our findings demonstrate suppression of α₇ receptor levels associated with the accumulation of foreign DNA in the transfected cells. Acetylcholine receptor levels thus depend on multiple elements, each of which should be considered when genetic interventions are employed.     

<Emphasis Type="Italic">Xenopus</Emphasis> importin beta validates human importin beta as a cell cycle negative regulator

Background  

Human importin beta has been used in all Xenopus laevis in vitro nuclear assembly and spindle assembly studies. This disconnect between species raised the question for us as to whether importin beta was an authentic negative regulator of cell cycle events, or a dominant negative regulator due to a difference between the human and Xenopus importin beta sequences. No Xenopus importin beta gene was yet identified at the time of those studies. Thus, we first cloned, identified, and tested the Xenopus importin beta gene to address this important mechanistic difference. If human importin beta is an authentic negative regulator then we would expect human and Xenopus importin beta to have identical negative regulatory effects on nuclear membrane fusion and pore assembly. If human importin beta acts instead as a dominant negative mutant inhibitor, we should then see no inhibitory effect when we added the Xenopus homologue.     

Design,synthesis, and characterization of novel,nonquaternary reactivators of GF-inhibited human acetylcholinesterase

The goal of this research was to identify structurally novel, non-quaternarypyridinium reactivators of GF (cyclosarin)-inhibited hAChE that possess the capacity to mediate in vitro reactivation of GF-inhibited human acetylcholinesterase (hAChE). New compounds were designed, synthesized and assessed in GF-inhibited hAChE assays. Structure activity relationships for AChE binding and reactivation of GF-inhibited hAChE were developed. Lead compounds from two different chemical series, represented by compounds 17 and 38, displayed proficient in vitro reactivation of GF-inhibited hAChE, while also possessing low inhibition of native enzyme.     

Cloning of noggin gene from hydra and analysis of its functional conservation using Xenopus laevis embryos

SUMMARY Hydra, a member of phylum Cnidaria that arose early in evolution, is endowed with a defined axis, organized nervous system, and active behavior. It is a powerful model system for the elucidation of evolution of developmental mechanisms in animals. Here, we describe the identification and cloning of noggin‐like gene from hydra. Noggin is a secreted protein involved at multiple stages of vertebrate embryonic development including neural induction and is known to exert its effects by inhibiting the bone morphogenetic protein (BMP)‐signaling pathway. Sequence analysis revealed that hydra Noggin shows considerable similarity with its orthologs at the amino acid level. When microinjected in the early Xenopus embryos, hydra noggin mRNA induced a secondary axis in 100% of the injected embryos, demonstrating functional conservation of hydra noggin in vertebrates. This was further confirmed by the partial rescue of Xenopus embryos by hydra noggin mRNA from UV‐induced ventralization. By using animal cap assay in Xenopus embryos, we demonstrate that these effects of hydra noggin in Xenopus embryos are because of inhibition of BMP signaling by Noggin. Our data indicate that BMP/Noggin antagonism predates the bilaterian divergence and is conserved during the evolution.     

Inhibition of Acetylcholinesterase by three New Pyridinium Compounds and their Effect on Phosphonylation of the Enzyme

Abstract

Three new mono-pyridinium compounds were prepared: 1-phenacyl-2-methylpyridinium chloride (1), 1-benzoylethylpyridinium chloride (2) and 1-benzoylethylpyridinium-4-aldoxime chloride (3) and assayed in vitro for their inhibitory effect on human blood acetylcholinesterase (EC 3.1.1.7, AChE). All the three compounds inhibited AChE reversibly; their binding affinity for the enzyme was compared with their protective effect (PI) on AChE phosphonylation by soman and VX. Compound 1 was found to bind to both the catalytic and the allosteric (substrate inhibition) sites of the enzyme with estimated dissociation constants of 6.9 μM (K_cat) and 27 μM (K_all), respectively. Compound 2 bound to the catalytic site with K_cat= 59 μM and compound 3 only to the allosteric site with K_all = 328 μM. PI was evaluated from phosphonylation measured in the absence and in presence of the compounds applied in a concentration corresponding to their K_cat or K_all value, and was also calculated from theoretical equations deduced from the reversible inhibition of the enzyme. Compounds 1 and 3 protected the enzyme from phosphonylation by soman and VX, whereas no protection was observed in the presence of compound 2 under the same conditions. Irrespective of the binding sites to AChE, PI for compounds 1 and 3 evaluated from phosphonylation agreed with PI calculated from reversible inhibition. Compound 3 was found to be a weak reactivator of methylphosphonylated AChE with k_r = 1.1 × 10²Lmol^-1 min^-1.     

Cellular polarity in cultured animal pole cells of Xenopus embryos

The expression of intracellular and surface polarity in cultured animal pole cells of Xenopus embryos (stages 6, 8, and 10) was examined morphologically and immunocytochemically. When control embryos reached stage 23, daughter cells derived from a single or a few animal pole cells formed aggregates. Outer cells of the aggregates displayed intracellular and surface polarity and expressed an epidermis-specific antigen (XEPI-1) on the apical surface circumference, while these characteristics had not yet been established in the animal pole cells at the time of isolation. However, inner cells of the aggregates did not display the cellular polarity along an outer-inner axis of the aggregates and displayed the antigen randomly within the aggregates. These results indicate that the expression of cellular polarity in epidermal differentiation of Xenopus embryos in vitro depends on the position within the aggregates formed by daughter cells derived from isolated animal pole cells.     

Fine structural localisation of acetylcholinesterase activity in the compound eye of the honeybee (Apis mellifica L.)

Summary Acetylcholinesterase (AChE) activity was demonstrated histochemically at the electron microscopic level in the compound eye of the worker bee (Apis mellifica L.) by use of the method of Lewis and Shute (1969).All photoreceptor axons (short and long visual fibres) display AChE activity. The reaction product is located in the axoplasm and at the plasma membrane. Substantial amounts of the reaction product can be detected in the intercellular spaces between the visual fibres. Along the visual fibres, the enzyme activity is unevenly distributed. High AChE activity is present in the distal parts of the axons, in contrast to lower enzyme levels in the lamina. However, AChE is also present in the proximal terminals of the visual fibres as well as in the intercellular spaces between visual fibre terminals and the postsynaptic neurones (monopolar cells). Intracellular enzyme activity is almost absent in the monopolars.The authors assume the high AChE activity in the visual fibres to be indicative of acetylcholine as the transmitter at the first synapse of the compound eye. This hypothesis is discussed in view of the results of autoradiographic, electrophysiological and pharmacological investigations of the compound eye and of the ocellus. Our data are at variance with results of studies on the eyes of Diptera.     

Subtilisin-like proprotein convertase activity is necessary for left–right axis determination in Xenopus neurula embryos

Signaling by members of TGF-β superfamily requires the activity of a family of site-specific endopeptidases, known as Subtilisin-like proprotein convertases (SPCs), which cleave these ligands into mature, active forms. To explore the role of SPCs in lateral plate mesoderm (LPM) differentiation in Xenopus, two SPC inhibitors, decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Dec-RVKR-CMK) and hexa-arginine, were injected into the left and right LPM of Xenopus neurulae. Left-side injection caused heart-specific left–right reversal, and this phenotype was rescued by co-injection of mature Nodal protein. In contrast, right-side injection caused left–right reversal of both the heart and gut. Tailbud embryos were less sensitive to SPC inhibitors than neurula embryos. Injection of inhibitors into either side of neurula embryos completely abolished expression of the left-LPM-specific genes, Xnr-1, antivin, and pitx2. SPC1 enzyme (Furin) was injected into the left or right LPM of mid-neurula embryos to determine the effect of enhancing SPC activity. Left-side injection of SPC1 did not cause a significant left–right reversal of the internal organs. However, right-side injection of SPC1 strongly induced the expression of Xnr-1 and pitx2 in the right LPM, and caused 100% left–right reversal of both the heart and gut. These results suggest that moderate level of SPC activity in the right LPM of the neurulae is necessary for proper left–right specification. Taken together, SPC enzymatic activity must be present in both LPMs for expression of the left-handed genes and left–right axis determination of the heart and gut in Xenopus embryos.     

Non-inhibition of Acetylcholinesterase by cyclosal Nucleotides

Abstract

An acetylcholinesterase (AChE) assay based on the Rappaport method was established to investigate the behaviour of several cycloSal nucleotides against AChE from electrophorus electricus and human sources (purified enzymes). AChE is a physiologically essential enzyme as it catalyzes the hydrolysis of the neurotransmitter acetylcholine. No inhibition was observed in any of the cases.