Reactivation of Cyclosarin-inhibited Rat Brain Acetylcholinesterase by Pyridinium–Oximes

Cyclohexyl methylphosphonofluoridate (cyclosarin, cyclosin, GF) is a highly toxic organophosphate, which is resistant to conventional oxime therapy. To gain insight into the reactivation kinetics, rat brain acetylcholinesterase (AChE) was inhibited in vitro by cyclosarin (pH 8.0, 25°C) and reactivated with 22 different pyridinium–oximes. Three compounds were shown to be superior to the other oximes: 4-carbamoyl-4′-[(hydroxyimino)methyl]-1,1′-(oxydimethylene)dipyridin-1-ium dichloride (HS-6), 4′-carbamoyl-2-[(hydroxyimino)methyl]-1,1′-(oxydimethylene)dipyridin-1-ium dichloride (HI-6), and 4′-carbamoyl-2-[(hydroxyimino)methyl]-1,1′-(but-2-ene-1,4-diyl)dipyridin-1-ium dichloride (BI-6).     

Effect of α-Bungarotoxin on Acetylcholinesterase bound to Mouse Diaphragm Endplates

IT has been shown that α-bungarotoxin (α-Bgt) irreversibly blocks cholinoreceptors^1–10 and that D-(+)tubocurarine (TC) protects these receptors from the toxin^4–7. On the other hand, it has been emphasized that α-Bgt has no effect on the catalytic activity of acetylcholinesterase (AChE)^4,6,9 and it was concluded that the cholinoreceptor and AChE must be two different macromolecules^8,9. Because typical cholinolytics, including TC and gallamine, characteristically influence the kinetics of a membrane-bound AChE^11,12, it seemed justified to reinvestigate whether α-Bgt really is an exception in this respect.     

Long Route or Shortcut? A Molecular Dynamics Study of Traffic of Thiocholine within the Active-Site Gorge of Acetylcholinesterase

The principal role of acetylcholinesterase is termination of nerve impulse transmission at cholinergic synapses, by rapid hydrolysis of the neurotransmitter acetylcholine to acetate and choline. Its active site is buried at the bottom of a deep and narrow gorge, at the rim of which is found a second anionic site, the peripheral anionic site. The fact that the active site is so deeply buried has raised cogent questions as to how rapid traffic of substrate and products occurs in such a confined environment. Various theoretical and experimental approaches have been used to solve this problem. Here, multiple conventional molecular dynamics simulations have been performed to investigate the clearance of the product, thiocholine, from the active-site gorge of acetylcholinesterase. Our results indicate that thiocholine is released from the peripheral anionic site via random pathways, while three exit routes appear to be favored for its release from the active site, namely, along the axis of the active-site gorge, and through putative back- and side-doors. The back-door pathway is that via which thiocholine exits most frequently. Our results are in good agreement with kinetic and kinetic-crystallography studies. We propose the use of multiple molecular dynamics simulations as a fast yet accurate complementary tool in structural studies of enzymatic trafficking.     

Acetylcholinesterase is not inhibited by pyridoxal 5′-phosphate

Acetylcholinesterase activity was assayed in the absence and presence of pyridoxal 5−phosphate. If substrate hydrolysis was measured by the pH-stat method, its rate was not significantly affected by pyridoxal 5′-phosphate. In the spectrophotometric assay, however, this compound led to an apparent decrease in rate. The discrepancy between the two assays is explained by stray-light artefacts produced by pyridoxal 5′-phosphate at the wavelenghts of the spectrophotometric assay.     

Synthesis and QSAR of Novel Ketoprofen–Chalcone-Amide Hybrides as Acetylcholinesterase (AChE) Inhibitors for Possible Treatment of Alzheimer Disease

Russian Journal of Bioorganic Chemistry - A new series of the anti-inflammatory drug ketoprofen derivatives bearing aryl chalcone-amide congeners were synthesized. The structures of the synthesized...     

Acetylcholinesterase: how is structure related to function?

In accordance with its biological role, termination of neurotransmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter, acetylcholine, acetylcholinesterase is one of nature's most efficient enzymes. Solution of its three-dimensional structure revealed that its active site is located at the bottom of a deep and narrow gorge. Such an architecture was unanticipated in view of its high turnover number. The present review examines how the highly specialized structure of acetylcholinesterase, with its sequestered active site, contributes to its catalytic efficacy, and discusses how the traffic of substrate and products to and from the active site is controlled.     

Effects In Vitro of Guanidinoacetate on Adenine Nucleotide Hydrolysis and Acetylcholinesterase Activity in Tissues from Adult Rats

Guanidinoacetate methyltransferase (GAMT) deficiency is a disorder of creatine metabolism characterized by low plasma creatine concentrations in combination with elevated guanidinoacetate (GAA) concentrations. The aim of this work was to investigate the in vitro effect of guanidinoacetate in NTPDase, 5′-nucleotidase and acetylcholinesterase activities in the synaptosomes, platelets and blood of rats. The results showed that in synaptosomes the NTPDase and 5′-nucleotidase activities were inhibited significantly in the presence of GAA at concentrations of 50, 100, 150 and 200 μM (P < 0.05). However, in platelets GAA at the same concentrations caused a significant increase in the activities of these two enzymes (P < 0.05). In relation to the acetylcholinesterase activity, GAA caused a significant inhibition in the activity of this enzyme in blood at concentrations of 150 and 200 μM (P < 0.05), but did not alter the acetylcholinesterase activity in synaptosomes from the cerebral cortex. Our results suggest that alterations caused by GAA in the activities of these enzymes may contribute to the understanding of the neurological dysfunction of GAMT-deficient patients.     

Kinetics and Molecular Docking Study of an Anti-diabetic Drug Glimepiride as Acetylcholinesterase Inhibitor: Implication for Alzheimer’s Disease-Diabetes Dual Therapy

Neurochemical Research - At the present time, treatment of two most common degenerative disorders of elderly population i.e., Type 2 Diabetes Mellitus (T2DM) and Alzheimer’s disease (AD) is a...     

Multi-target Design Strategies in the Context of Alzheimer’s Disease: Acetylcholinesterase Inhibition and NMDA Receptor Antagonism as the Driving Forces

In recent years, the multi-target-directed ligand concept has been used to design a variety of molecules hitting different biological targets for Alzheimer’s disease. We have sought to combine, in the same molecule, the neuroprotective action of N-methyl-d-aspartate receptor antagonism with the symptomatic relief offered by cholinergic activity through acetylcholinesterase inhibition. This strategy could potentially maintain the positive outcomes of memantine–acetylcholinesterase inhibitor combinations, but with the benefits of a single molecule therapy. Herein, we discuss selected examples of multifunctional compounds, which we rationally designed to simultaneously modulate these targets. We also examine the intertwined relationship between acetylcholinesterase, N-methyl-d-aspartate receptors, and other active players in the neurotoxic cascade.     

Acetylcholinesterase is associated with apoptosis in β cells and contributes to insulin-dependent diabetes mellitus pathogenesis

Acetylcholinesterase (AChE) expression is pivotal during apoptosis. Indeed, AChE inhibitors partially protect cells from apoptosis. Insulin-dependent diabetes mellitus (IDDM) is characterized in part by pancreatic β-cell apoptosis. Here, we investigated the role of AChE in the development of IDDM and analyzed protective effects of AChE inhibitors. Multiple low-dose streptozotocin (MLD-STZ) administration resulted in IDDM in a mouse model. Western blot analysis, cytochemical staining, and immunofluorescence staining were used to detect AChE expression in MIN6 cells, primary β cells, and apoptotic pancreatic β cells of MLD-STZ-treated mice. AChE inhibitors were administered intraperitoneally to the MLD-STZ mice for 30 days. Blood glucose, plasma insulin, and creatine levels were measured, and glucose tolerance tests were performed. The effects of AChE inhibitors on MIN6 cells were also evaluated. AChE expression was induced in the apoptotic MIN6 cells and primary β cells in vitro and pancreatic islets in vivo when treated with STZ. Induction and progressive accumulation of AChE in the pancreatic islets were associated with apoptotic β cells during IDDM development. The administration of AChE inhibitors effectively decreased hyperglycemia and incidence of diabetes, and restored plasma insulin levels and plasma creatine clearance in the MLD-STZ mice. AChE inhibitors partially protected MIN6 cells from the damage caused by STZ treatment. Induction and accumulation of AChE in pancreatic islets and the protective effects of AChE inhibitors on the onset and development of IDDM indicate a close relationship between AChE and IDDM.     

The Acetylcholinesterase Inhibitors Competitively Inhibited an Acetyl l-Carnitine Transport Through the Blood–Brain Barrier

We investigated the interaction of acetylcholinesterase (AChE) inhibitors with acetyl-L-carnitine (ALCAR) transporter at the blood-brain barrier (BBB). ALCAR uptake by conditionally immortalized rat brain capillary endothelial cell lines (TR-BBB cells), as an in vitro model of BBB, were characterized by cellular uptake study using [(3)H]ALCAR. In vivo brain uptake of [(3)H]ALCAR was determined by brain uptake index after carotid artery injection in rats. In results, the transport properties for [(3)H]ALCAR by TR-BBB cell were consistent with those of ALCAR transport by the organic cation/carnitine transporter 2 (OCTN2). Also, OCTN2 was confirmed to be expressed in the cells. The uptake of [(3)H]ALCAR by TR-BBB cells was inhibited by AChE inhibitors such as donepezil, tacrine, galantamine and rivastigmine, which IC(50) values are 45.3, 74.0, 459 and 800 μM, respectively. Especially, donepezil and galantamine inhibited the uptake of [(3)H]ALCAR competitively, but tacrine and rivastigmine inhibited noncompetitively. Furthermore, [(3)H]ALCAR uptake by the rat brain was found to be significantly decreased by quinidine, donepezil and galantamine. Our results suggest that transport of AChE inhibitors such as donepezil and galantamine through the BBB is at least partly mediated by OCTN2 which is involved in transport of ALCAR.     

Brain Acetylcholinesterase Promotes Amyloid-β-Peptide Aggregation but Does Not Hydrolyze Amyloid Precursor Protein Peptides

It has been suggested that acetylcholinesterase (AChE) has both a putative proteolytic activity against the amyloid precursor protein (APP), and a capacity to accelerate the assembly of amyloid--peptide (A) into Alzheimer's fibrils. Here, we have studied the ability of bovine brain AChE to share both activities. Results indicate that AChE purified through acridinium was able to process the APP peptides, however after further purification by an edrophonium column, the protease activity was lost. Under both conditions the capacity of the enzyme to promote amyloid formation was maintained. Kinetic studies of the A aggregation process using edrophonium-AChE, indicated that the lag phase of the aggregation process was smaller than the one observed with the esterase purified by acridinium alone. Considering that the total amount of amyloid formed, measured by thioflavine-T fluorescence, was similar for both AChE preparations, our results suggest that the edrophonium-AChE possesses an higher intrinsic capacity to stimulate the aggregation of A_1–40 peptide.     

Die Ultrastruktur der motorischen Endplatte im Zwerchfell der Ratte und Veränderungen nach Inhibierung der Acetylcholinesterase

Zusammenfassung Im Terminalaxon der motorischen Endplatte aus dem Zwerchfell der Albinoratte beobachteten wir Synapsenvesikel, Mitochondrien und in manchen Fällen Neurofilamente. Die fibrillären Bestandteile des Axoplasmas stehen möglicherweise mit den Synapsenvesikeln in Verbindung, die in unmittelbarer Nähe der präsynaptischen Membran lokalisiert sind. Vermutlich besteht zwischen Vesikeln und Neurofilamenten eine funktionelle Beziehung.Nach intraperitonealer Applikation von Soman (O-pinakolyl-methylphosphonsäurefluorid) erscheinen im Zwerchfell lokal begrenzte degenerative Veränderungen, die sich sowohl auf die neuromuskulären Verbindungen als auch auf die Muskelfasern erstrecken. Das Ausmaß der Schädigung ist in den einzelnen Nekrosebereichen verschieden, doch tritt in den meisten Fällen eine Auflösung des Sarcolemms im subneuralen Faltenapparat auf, wodurch vermutlich die Bildung von Acetylcholinesterase verhindert wird. In weiter fortgeschrittenen Stadien der Vergiftung werden auch die feinstrukturellen Bestandteile der Muskelfaser geschädigt. Anschwellung der Mitochondrien und Auflösung ihrer Cristae, Bildung von Myelinfiguren in den Mitochondrien, Pyknose der Zellkerne, Verschwinden der Querstreifung des Muskels und schließlich ein Zerfall der Myofilamente kennzeichnen eine durch Soman verursachte Zerstörung des Muskelgewebes.

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Summary In the terminal axon of the motor end-plate in the diaphragm of albino rat we observed synaptic vesicles, mitochondria, and in some cases neurofilaments. Possibly the fibrillar components of the axoplasm are connected with the synaptic vesicles, which are located near the praesynaptic membrane. Probably there exists a functional relation between vesicles and neurofilaments.In the diaphragm there appear degenerative changes when Soman (methyl-pinacolyloxy-phosphonylfluoride) has been applicated intraperitoneally. These are locally limited and extend to neuromuscular junctions as well as to muscle fibres. The extent of injury is very different in the various regions of necrosis, but in most cases there appears a dissolution of sarcolemma in the subneural infoldings probably preventing the production of acetylcholinesterase. In a more progressive state of poisoning the ultrastructural components of the muscle fiber are also injured. The destruction of muscle tissue caused by Soman is characterized by swelling of mitochondria, dissolving of its cristae, formation of myelin figures in mitochondria, pyknosis of nuclei, disappearing of the striation of muscle, and finally the decay of myofilaments.

     

Über die Synapsenformen und das Vorkommen von Acetylcholinesterase in der Epiphyse von Bombina variegata (L.), (Anura)

Zusammenfassung In der Epiphyse von Bombina kommen durch synaptic ribbons gekennzeichnete Synapsen und konventionelle Synapsen vor. Bei den ribbon-Synapsen handelt es sich um axodendritische und axosomatische Formen. Die axodendritischen ribbon-Synapsen lassen sich aufgrund der Zahl der Dendriten und der synaptic ribbons in 2 Typen gliedern. Es kommen Dendriten vor, die nacheinander in ribbon-Synapsen und konventionelle Synapsen einbezogen sind. Neben konventionellen und durch ribbons gekennzeichneten synaptischen Verbindungen finden sich weitere Kontakte zwischen Sinnes- und Nervenzellen und Interrezeptorkontakte, die jedoch beide nicht als echte Synapsen angesprochen werden können. Anhand der Befunde zur Synaptologie werden Probleme der neuronalen Schaltung der Epiphyse diskutiert.Beim Acetylcholinesterase-Nachweis findet sich das Reaktionsprodukt vor allem in den Neuropilzonen der Epiphyse. Eine eindeutige Zuordnung zu Fortsätzen bestimmter Zelltypen ist nicht möglich. Das Ergebnis des Acetylcholinesterase-Nachweises in der Epiphyse wird mit entsprechenden Befunden in anderen Bereichen des ZNS und in der Netzhaut verglichen.

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The forms of synapses and the occurrence of acetylcholinesterase in the Pineal Organ of Bombina variegata (L.), (Anura)

Summary Both conventional and ribbon synapses occur in the pineal organ of Bombina. Ribbon synapses are both axodendritic and axosomatic. Two axodendritic types can be distinguished on the basis of the number of dendrites and synaptic ribbons. Both conventional and ribbon synapses can be formed with the same dendrite. Other contacts, which cannot be classified as true synapses, are also found between sensory cells and nerve cells and likewise between sensory cells. The synaptology of the pineal organ permits a discussion of the problems of its neurocircuitry.Products of the acetylcholinesterase reaction occur mainly in the plexiform layer of the pineal organ. It is not possible to correlate the reaction products with definite cell types. The results of the acetylcholinesterase reaction in the pineal is compared with corresponding findings in other parts of the CNS and in the retina of the lateral eyes.

Herrn Prof. Dr. H. Altner danke ich für die Förderung der Arbeit und die kritische Durchsicht des Manuskripts.     

A Comparison of the Ability of a New Bispyridinium Oxime—1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane Dibromide and Currently used Oximes to Reactivate Nerve Agent-inhibited Rat Brain Acetylcholinesterase by In Vitro Methods

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.     

Alterations on Na+,K+-ATPase and Acetylcholinesterase Activities Induced by Amyloid-β Peptide in Rat Brain and GM1 Ganglioside Neuroprotective Action

Alzheimer’s disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-β peptide (Aβ). Aβ-induced toxicity is believed to involve alterations on as Na⁺,K⁺-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aβ1-42 infusion and GM1 treatment on recognition memory and on Na⁺,K⁺-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aβ1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aβ-induced cognitive deficit, corroborating its neuroprotective function. Aβ impaired Na⁺,K⁺-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aβ-induced alteration on Na⁺,K⁺-ATPase, though with no impact on AChE activity. Aβ caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aβ-triggered cognitive deficit involves region-specific alterations on Na⁺,K⁺-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na⁺,K⁺-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment.     

A comparative analysis of karyotypes of three species of Macleaya—producers of a complex of isoquinoline alkaloids

Using a set of methods (C-banding, DAPI-staining, fluorescence hybridization in situ (FISH) with probes of 26S and 5S rDNA, and analysis of meiosis), the first comparative cytogenetic study of three species of Macleaya, producers of complex isoquinoline alkaloids, cordate Macleaya cordata (Willd.) R. Br. (2n = 20), small-fruited Macleaya microcarpa (Maxim.) Fedde (2n = 20) and Macleaya kewensis Turrill (2n = 20), was first carried out. On the basis of morphometric analysis, formulas of karyotypes were made for each species. Species ideograms for M. cordata, M. microcarpa, and M. kewensis were constructed taking into account the polymorphic variants of the C-banding patterns and indicating the location of 26S and 5S rDNA sites. A comparative study revealed that the karyotypes of M. microcarpa and M. kewensis have more in common with each other than with M. cordata. Analysis of meiotic chromosomes suggests of genetic stability of Macleaya genomes. The results of chromosome analysis were used to confirm the close relationship of Macleaya and to clarify their phylogenetic relationships.     

Effect of γ-Irradiation on Development of Lachrymator of Onion

A thermosensitive uracil requiring mutant of Bacillus subtilis Marburg 168 thy trp₂ ts42 was examined as to the colony forming ability at the permissive and nonpermissive temperatures. The viability of the mutant cells decreased rapidly at the restrictive temperature in the modified Woese’s (MW) medium. However, the cells retained viability when sodium succinate or potassium chloride was added to the medium at that temperature although uracil deficiency was unchanged. A little but significant incorporation of adenine-8-¹⁴C into RNA still continued even after the incorporation of N-acetyl-³H-d-glucosamine into acid insoluble fraction of the cells terminated in the MW medium at 48°C. Both incorporations as well as increase of absorbance were slowed down in the presence of sodium succinate at 48°C. This mutant, ts42, was more sensitive to deoxycholate (DOC) than the parent strain. The restoration of colony forming ability after the temperature shift back from 48 to 37°C was suppressed by the addition of DOC to the medium. However, the cell became resistant to DOC when uracil was added to the medium prior to the temperature shift.