The characteristics of cholinesterase distribution in the development of the human and mammalian brains]

Histochemical studies have been made on the distribution of acetyl- and butyrylcholinesterases (ACHE and BCHE) in various parts of the human and rat brain. Statistical analysis showed that at the 8th week, the highest ACHE activity in the human foetus is observed in the intermediate and plexiform layers of the cerebral cortex. The highest BCHE activity was found in the ependymal layer of various cerebral regions. High BCHE and ACHE activities were noted in the dorsal thalamus and epithalamus. In 10-week human foetuses, total high level of ACHE and BCHE was revealed in various nuclei of the thalamus and subcortical structures of the forebrain (Meynert nucleus, nucleus caudatum). In rats, the highest ACHE activity at the 14th day of prenatal life was found only in subcortical structures of the forebrain. Accumulation of BCHE activity in some of the thalamic nuclei of rats begins at the 10-17th day of postnatal life.     

Molecular Forms of Acetylcholinesterase and Butyrylcholinesterase in the Aged Human Central Nervous System

The distribution of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) molecular forms and their solubility characteristics were examined, using density gradient centrifugation, in various regions of the postmortem human CNS. Total AChE activity varied extensively (50-fold) among the regions investigated, being highest in the telencephalic subcortical structures (caudate nucleus and nucleus of Meynert); intermediate in the substantia nigra, cerebellum, and spinal cord; and least in the fornix and cortical regions (hippocampus and temporal and parietal cortex). Total BChE activity was, in contrast, much more evenly distributed, with only a threefold variation between the regions studied. Although the patterns of molecular forms of each enzyme were broadly similar among the different areas, regional variations in the distribution and abundance of the various forms of AChE were much greater than those of BChE. Thus, although the tetrameric G4 form of AChE constituted the majority of the total AChE activity in all regions examined, the ratio of the G4 form to the monomeric G1 form, the latter of which constituted the majority of the remaining activity, varied markedly, ranging from 21 in the caudate nucleus to 1.7 in the temporal cortex. In addition to the G4 and G1 forms of AChE, the dimeric G2 form was observed in the nucleus of Meynert and a fast-sedimenting (16S) species was found in samples of both the parietal cortex and spinal cord. In contrast, the G4 and G1 forms of BChE were the only molecular species observed in the different areas and the G4:G1 ratio varied from 3.3 in the substantia nigra to 0.9 in the temporal cortex. Regarding the solubility characteristics of the individual AChE and BChE molecular forms, the majority of the G4 form of AChE was extractable only in the presence of detergent, indicating a predominantly membrane-bound localization of this species. The smaller AChE forms (G1 and G2) and both the G1 and G4 forms of BChE were all relatively evenly distributed between soluble and membrane-bound species. These findings are discussed in relation to neurochemical and neuroanatomical, particularly cholinergic, features of the regions examined.     

Comparison of methods used for the determination of cholinesterase activity in whole blood

Cholinesterases (ChEs) are classified as either acetylcholinesterase (AChE) or butyrylcholinesterase (BChE) based on their substrate and inhibitor specificity. Organophosphate and carbamate compounds commonly represented by herbicides, pesticides, and nerve gases irreversibly inhibit ChEs. Therefore, exposure to organophosphates and carbamates is normally assessed by measuring ChE activity in blood. There are two approaches for measuring AChE and BChE activity present in whole blood: (1) separating blood into erythrocytes, which contain only AChE, and plasma which contains only BChE, to measure their activity individually, or (2) use a BChE-specific inhibitor to measure the activity of AChE in whole blood. A number of studies have reported the use of different inhibitors for the simultaneous measurement of AChE and BChE activities. However, the inhibitors used for completely inhibiting BChE activity also inhibited AChE activity leading to errors in reported values. The goal of this study was to find the most accurate and simple method for the simultaneous determination of AChE and BChE activity in animal whole blood. Solutions containing human AChE and BChE in various proportions were prepared and AChE and BChE activities were measured using three reported methods. Results demonstrate that ethopropazine and (-) huperzine A appear to be the most specific ChE inhibitors. Preliminary results with human and animal whole blood suggest that 20muM ethopropazine and 500nM (-) huperzine A can be used for measuring AChE and BChE activities across species.     

Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse

We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.     

Acetylcholinesterase and butyrylcholinesterase activities in brain and plasma of freshwater teleosts: cross-species and cross-family differences

Brain and plasma acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) specific activities were assayed in 16 freshwater teleosts belonging to four families: Cyprinidae, Percidae, Esocidae and Lotidae. Brain AChE activity varied among fish species approximately 15-fold, ranging from 138 to 2011 micromol/g per h. All cyprinids had higher brain AChE activity than other fish families. Plasma AChE activity was on average 100-fold lower than that in brain, varying from 1.2 to 18.6 micromol/ml per h. Plasma BChE activity was found only in cyprinids with the exception of the common and crucian carp, and sabrefish. It varied from 26 to 1083 micromol/ml per h. In bream (Abramis brama) only 30% of specimens studied had BChE activity. The correlation coefficient values between activities of brain and plasma AChE, brain AChE and plasma BChE, plasma AChE and BChE were 0.67, 0.68 and 0.84, respectively. Cross-species and also cross-family differences in AChE and BChE activities among fish were demonstrated. Possible reasons for the differences are discussed.     

Localization of Butyrylcholinesterase at the Neuromuscular Junction of Normal and Acetylcholinesterase Knockout Mice

At the mouse neuromuscular junction (NMJ), there are two distinct cholinesterases (ChE): acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Until now, it has been difficult to determine the precise localization of BChE at the NMJ. In this study, we use a modification of Koelle''s method to stain AChE and BChE activity. This method does not interfere with fluorescent co-staining, which allows precise co-localization of ChE and other synaptic molecules at the NMJ. We demonstrate that AChE and BChE exhibit different localization patterns at the mouse NMJ. AChE activity is present both in the primary cleft and in the secondary folds, whereas BChE activity appears to be almost absent in the primary cleft and to be concentrated in subsynaptic folds. The same localization for BChE is observed in the AChE-knockout (KO) mouse NMJ. Collagenase treatment removed AChE from the primary cleft, but not from secondary folds in the wild-type mouse, whereas in the AChE-KO mouse, BChE remains in the secondary folds. After peripheral nerve injury and regeneration, BChE localization is not modified in either normal or KO mice. In conclusion, specific localization of BChE in the secondary folds of the NMJ suggests that this enzyme is not a strict surrogate of AChE and that the two enzymes have two different roles. (J Histochem Cytochem 58:1075–1082, 2010)     

Screening assays for cholinesterases resistant to inhibition by organophosphorus toxicants

Methods to measure resistance to inhibition by organophosphorus toxicants (OP) for mutants of butyrylcholinesterase (EC 3.1.1.8; BChE) and acetylcholinesterase (EC 3.1.1.7; AChE) enzymes were devised. Wild-type cholinesterases were completely inhibited by 0.1 mM echothiophate or 0.001 mM diisopropylfluorophosphate, but human BChE mutants G117H, G117D, L286H, and W231H and snake AChE mutant HFQT retained activity. Tissues containing a mixture of cholinesterases could be assayed for amount of G117H BChE. For example, the serum of transgenic mice expressing human G117H BChE contained 0.5 microg/ml human G117H BChE, 2 microg/ml wild-type mouse BChE, and 0.06 microg/ml wild-type mouse AChE. The oligomeric structure of G117H BChE in the serum of transgenic mice was determined by nondenaturing gel electrophoresis followed by staining for butyrylthiocholine hydrolysis activity in the presence of 0.1 mM echothiophate. Greater than 95% of the human G117H BChE in transgenic mouse serum was a tetramer. To visualize the distribution of G117H BChE in tissues of transgenic mice, sections of small intestine were treated with echothiophate and then stained for BChE activity. Both wild-type and G117H BChE were in the epithelial cells of the villi. These assays can be used to identify OP-resistant cholinesterases in culture medium and in animal tissues.     

Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development--a study of molecular forms.

The pattern of molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) separated by density gradient centrifugation was investigated in the brain and cerebrospinal fluid in Alzheimer's disease (AD), in human embryonic brain and in rat brain after experimental cholinergic deafferentation of the cerebral cortex. While a selective loss of the AChE G4 form was a rather constant finding in AD, a small but significant increase of G1 for both AChE and BChE was found in the most severely affected cases. Both in normal human brain and in AD a significant relationship could be established between the AChE G4/G1 ratio in different brain regions and the activity of choline acetyltransferase (ChAT). A similar decrease of the AChE G4 form as observed in AD can be induced in rat by experimental cholinergic deafferentation of the cerebral cortex. The increase in G1 of both AChE and BChE in different brain regions in AD is quantitatively related to the local density of neuritic plaques which are histochemically reactive for both enzymes. In human embryonic brain, a high abundance of G1 and a low G4/G1 ratio for both AChE and BChE was found resembling the pattern observed in AD. Furthermore, both in embryonic brain and in AD AChE shows no substrate inhibition which is a constant feature of the enzyme in the adult human brain. It is, therefore, concluded that the degeneration of the cholinergic cortical afferentation in AD as reflected by a decrease of AChE G4 is accompanied by the process of a neuritic sprouting response involved in plaque formation which is probably associated with the expression of a developmental form of the enzyme.     

Stereoselective inhibition of human, mouse, and horse cholinesterases by bambuterol enantiomers

Bambuterol is a chiral carbamate and a selective inhibitor of butyrylcholinesterase (BChE, EC 3.1.1.8). In order to relate bambuterol selectivity and stereoselectivity of BChE and acetylcholinesterase (AChE, EC 3.1.1.7) of different species, we studied the inhibition of human, mouse, and horse BChE, as well as AChE of human and mouse by (R)- and (S)-bambuterol. AChE and BChE of all studied species were progressively inhibited by both bambuterol enantiomers, with a preference for the (R)-bambuterol whose inhibition rate constants were about five times higher than that of (S)-bambuterol. We observed no significant difference between human and mouse in bambuterol enantiomer BChE inhibition. However, (R)-bambuterol inhibited horse BChE about 14 times slower than human and mouse BChE, and the inhibition rate for (S)-bambuterol was about 18 times slower. Although the primary structure of horse BChE differs from the other two species in 15 amino acids, we presumed that differences in inhibition rates could be attributed to threonine at position 69 located close to the peripheral site of BChE. Since BChE inhibition by bambuterol enantiomers was at least 8000 times faster than that of AChE, both bambuterol enantiomers proved to be selective BChE inhibitors, as was previously shown for racemate.     

Comparative study of acetylcholinesterase and butyrylcholinesterase in brain and serum of several freshwater fish: specific activities and in vitro inhibition by DDVP, an organophosphorus pesticide

A comparative study of specific activities and in vitro inhibition of brain and serum acetylcholinesterase (AChE; EC 3.1.1.7) and serum butyrylcholinesterase (BChE; EC 3.1.1.8) by DDVP, an organophosphorus pesticide, was conducted in 11 freshwater teleost species belonging to four families (Cyprinidae; common carp Cyprinus carpio, bream Abramis brama, blue bream A. ballerus, white bream Blicca bjoerkna, roach Rutilus rutilus, bleak Alburnus alburnus, ide Leuciscus idus; Percidae: perch Perca fluviatilis, pikeperch Stizostedion lucioperca; Esocidae: pike Esox lucius and Coregonidae: whitefish Coregonus albula). Specific AChE and BChE activities in brain and serum of fish were determined. Brain AChE activity varied among fish species approximately 10-fold, ranging from 192.6 to 1353.2 micromol g(-1) h(-1), respectively in perch and whitefish. All cyprinids had higher brain AChE activity than those of other fish families. Serum AChE activity was 100-fold lower than in brain. Serum BChE activity was found only in cyprinids with the exception of the common carp. It varied from 163.8 to 970.3 micromol g(-1) h(-1), respectively in roach and bleak. The bimolecular enzyme inhibition rate constants (kIIs) and pI50) values for DDVP were calculated. Sensitivity of fish AChEs both in brain and serum is similar to those of typical AChEs in mammals. The range of kIIs was 3.4-51.7 x 10(3) mol(-1) 1 min(-1) (pI50s were 5.3-6.5), respectively in white bream and ide. In contrast, fish serum BChE was more sensitive to inhibition than typical BChE and AChE in mammals. Values of kII for BChE were 1.0-2.5 x 10(7) mol(-1) 1 min(-1) (pI50 was 8.8-9.2), respectively in ide and bleak.     

Protection of red blood cell acetylcholinesterase by oral huperzine A against ex vivo soman exposure: next generation prophylaxis and sequestering of acetylcholinesterase over butyrylcholinesterase

As part of a phase Ib clinical trial to determine the tolerability and safety of the highly specific acetylcholinesterase (AChE) inhibitor huperzine A, twelve (12) healthy elderly individuals received an escalating dose regimen of huperzine A (100, 200, 300, and 400 microg doses, twice daily for a week at each dose), with three (3) individuals as controls receiving a placebo. Using the WRAIR whole blood cholinesterase assay, red blood cell AChE and plasma butyrylcholinesterase (BChE) were measured in unprocessed whole blood samples from the volunteers following each dose, and then for up to 48h following the final and highest (400 microg) dose to monitor the profile of inhibition and recovery of AChE. Significant inhibition of AChE was observed, ranging from 30-40% after 100 microg to >50% at 400 microg, and peaking 1.5h after the last dose. Gradual recovery of AChE activity then occurs, but even 48 h after the last dose red blood cell AChE was about 10% below control (pre-dose) values. Huperzine A levels in plasma peaked 1.5h after the final 400 microg dose (5.47+/-2.15 ng/mL). Plasma BChE was unaffected by huperzine A treatment (as expected). Aliquots of huperzine A-containing (from three individuals) and placebo blood samples were exposed ex vivo to the irreversible nerve agent soman (GD) for 10 min, followed by removal of unbound huperzine and soman from the blood by passing through a small C(18) reverse phase spin column. Eluted blood was diluted in buffer, and aliquots taken at various time intervals for AChE and BChE activity measurement to determine the time taken to achieve full return in activity of the free enzyme (dissociation from the active site of AChE by huperzine A), and thus the proportion of AChE that can be protected from soman exposure. Huperzine A-inhibited red blood cell (RBC) AChE activity was restored almost to the level that was initially inhibited by the drug. The increased doses of huperzine A used were well tolerated by these patients and in this ex vivo study sequestered more red blood cell AChE than has been previously demonstrated for pyridostigmine bromide (PB), indicating the potential improved prophylaxis against organophosphate (OP) poisoning.     

Aromatic amino-acid residues at the active and peripheral anionic sites control the binding of E2020 (Aricept) to cholinesterases.

E2020 (R,S)-1-benzyl-4-[(5,6-dimethoxy-1-indanon)-2-yl]methyl)piperidine hydrochloride is a piperidine-based acetylcholinesterase (AChE) inhibitor that was approved for the treatment of Alzheimer's disease in the United States. Structure-activity studies of this class of inhibitors have indicated that both the benzoyl containing functionality and the N-benzylpiperidine moiety are the key features for binding and inhibition of AChE. In the present study, the interaction of E2020 with cholinesterases (ChEs) with known sequence differences, was examined in more detail by measuring the inhibition constants with Torpedo AChE, fetal bovine serum AChE, human butyrylcholinesterase (BChE), and equine BChE. The basis for particular residues conferring selectivity was then confirmed by using site-specific mutants of the implicated residue in two template enzymes. Differences in the reactivity of E2020 toward AChE and BChE (200- to 400-fold) show that residues at the peripheral anionic site such as Asp74(72), Tyr72(70), Tyr124(121), and Trp286(279) in mammalian AChE may be important in the binding of E2020 to AChE. Site-directed mutagenesis studies using mouse AChE showed that these residues contribute to the stabilization energy for the AChE-E2020 complex. However, replacement of Ala277(Trp279) with Trp in human BChE does not affect the binding of E2020 to BChE. Molecular modeling studies suggest that E2020 interacts with the active-site and the peripheral anionic site in AChE, but in the case of BChE, as the gorge is larger, E2020 cannot simultaneously interact at both sites. The observation that the KI value for mutant AChE in which Ala replaced Trp286 is similar to that for wild-type BChE, further confirms our hypothesis.     

Butyrylcholinesterase and the control of synaptic responses in acetylcholinesterase knockout mice

At the neuromuscular junction (NMJ) acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) can hydrolyze acetylcholine (ACh). Released ACh quanta are known to diffuse rapidly across the narrow synaptic cleft and pairs of ACh molecules cooperate to open endplate channels. During their diffusion through the cleft, or after being released from muscle nicotinic ACh receptors (nAChRs), most ACh molecules are hydrolyzed by AChE highly concentrated at the NMJ. Advances in mouse genomics offered new approaches to assess the role of specific cholinesterases involved in synaptic transmission. AChE knockout mice (AChE-KO) provide a valuable tool for examining the complete abolition of AChE activity and the role of BChE. AChE-KO mice live to adulthood, and exhibit an increased sensitivity to BChE inhibitors, suggesting that BChE activity facilitated their survival and compensated for AChE function. Our results show that BChE is present at the endplate region of wild-type and AChE-KO mature muscles. The decay time constant of focally recorded miniature endplate currents was 1.04 +/- 0.06 ms in wild-type junctions and 5.4 ms +/- 0.3 ms in AChE-KO junctions, and remained unaffected by BChE-specific inhibitors, indicating that BChE is not limiting ACh duration on endplate nAChRs. Inhibition of BChE decreased evoked quantal ACh release in AChE-KO NMJs. This reduction in ACh release can explain the greatest sensitivity of AChE-KO mice to BChE inhibitors. BChE is known to be localized in perisynaptic Schwann cells, and our results strongly suggest that BChE's role at the NMJ is to protect nerve terminals from an excess of ACh.     

Cerebrospinal fluid gradients of acetylcholinesterase and butyrylcholinesterase activity in healthy aging

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were measured in 13 sequential 2 ml aliquots of cerebrospinal fluid (CSF) obtained by lumbar puncture from 7 young and 7 elderly healthy normal subjects. The slopes of the rostrocaudal gradients of AChE and BChE were calculated and compared to those of total protein concentration and the major dopaminergic metabolite homovanillic acid (HVA), for which a pronounced rostrocaudal gradient (with highest concentrations of HVA in more rostral CSF) is consistent with HVA originating primarily from the brain. AChE activity was higher in more caudal fractions of young, but not elderly subjects and there was a significant difference between the mean AChE gradient slopes in the young and old groups. These results suggest that the spinal cord makes an important contribution to AChE activity in lumbar CSF. Furthermore, the absence of a negative AChE gradient in elderly subjects may be the result of a greater rate of entry of cerebral AChE into CSF, possibly as a consequence of an increased ventricular surface area and shorter diffusion distances in atrophic elderly brains. In contrast to AChE, BChE activity and total protein concentrations were higher in more caudal CSF fractions of not only young but also old subjects. In addition, there was a significant correlation between the gradient slopes of BChE activity and total protein concentrations, suggesting that the majority of BChE activity in lumbar CSF derives from the same source as the majority of total protein, namely plasma. The diffuse (i.e. brain and spinal cord) origin of AChE in lumbar CSF would explain the relatively modest changes in lumbar CSF AChE activity in diseases involving certain central cholinergic systems, most notably Alzheimer's disease.     

On the distributive pattern of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in the medulla oblongata of the golden hamster (Mesocricetus auratus). A study on the topographical and functional linkages of the two enzymes

Histochemical study on the distributive patterns of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), amongst the constituents of the medulla oblongata of golden hamster (Mesocricetus auratus), viewed through the angle of the possible topographical and functional linkage of the two enzymes, have provided identical set-ups of AChE and BChE in many of the nuclei, fibers and tracts. In the case of AChE in many instances the results further point out that there is a balance of the enzymatic activity between the nuclei and the associated processes. On the other hand, in relation to BChE activity, the nuclei and their processes do not seem to be linked through such a histochemical factor. A detailed discussion on the significance of AChE relationship between the neurons and their processes from the functional point of view, against the data available in the literature, has been incorporated in the present study. Histoenzymologically, the ranges of the activities of AChE and BChE amongst the neurons and their processes are so closely related that such components are mirror images of each other in histochemical patterns. Naturally, such results have led to a detailed discussion in the contribution culminating in a proposition that AChE and BChE (amongst the locales studied) seems to be intimately linked in the neuro-physiological functioning undergoing in the regions of the medulla oblongata of golden hamster.     

Characterization and distribution of cholinesterase activity in mouse uterine horns: changes in estrous cycle

1. Both butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) are present in the mouse uterus, BChE being more abundant.2. Their molecular forms were sequentially solubilized by different extraction media obtaining three ChE fractions whose specific activity was different, depending on the stage of the estrous cycle: hydrosoluble (estrous: 75.5 ± 6.6 and diestrous: 47.9 ± 8.7 μU/mg prot); detergent-soluble or amphiphilic (estrous 26.6 ± 2.4 and diestrous 14.7 ± 3.3 mU/mg prot.), and high ionic strength-soluble (estrous: 18.7 ±4.2 and diestrous 12.8 ± 1.2 mU/mg prot.).3. Histochemical procedures demonstrated a different distribution for both ChE activities. AChE was found in nerves next to smooth muscle cells of the circular layer and blood vessels, while BChE was concentrated in the longitudinal stratum surrounding the smooth muscle cells. Under the predominance of progesterone, BChE was also found in the endometrial glands.4. Maximal contractions evoked by the addition of ACh to the isolated organ bath were concentration dependent and greater in estrous than in diestrous. Nevertheless the difference at the two stages of the estrous cycle disappeared when contractions were normalized to smooth muscle cross-sectional area.5. BChE but not AChE inhibition augmented maximal contractions elicited by ACh in longitudinal but not in circular smooth muscle.6. The effect of BChE inhibition on the contractile force developed was greater at lower concentrations of ACh and did not depend on the stage of the estrous cycle.     

Design and Synthesis of Selective Acetylcholinesterase Inhibitors: Arylisoxazole‐Phenylpiperazine Derivatives

In this work, a novel series of arylisoxazole‐phenylpiperazines were designed, synthesized, and evaluated toward acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Our results revealed that [5‐(2‐chlorophenyl)‐1,2‐oxazol‐3‐yl](4‐phenylpiperazin‐1‐yl)methanone ( 5c ) was the most potent AChE inhibitor with IC₅₀ of 21.85 μm . It should be noted that most of synthesized compounds showed no BChE inhibitory activity and [5‐(2‐fluorophenyl)‐1,2‐oxazol‐3‐yl](4‐phenylpiperazin‐1‐yl)methanone ( 5a ) was the most active anti‐BChE derivative (IC₅₀=51.66 μm ). Also, kinetic studies for the AChE and BChE inhibitory activity of compounds 5c and 5a confirmed that they have simultaneously bound to the catalytic site (CS) and peripheral anionic site (PAS) of both AChE and BChE. Furthermore, docking study of compound 5c showed desired interactions of that compound with amino acid residues located in the active and peripheral anionic sites. Compound 5c was also evaluated for its BACE1 inhibitory activity and demonstrated IC₅₀=76.78 μm . Finally, neuroprotectivity of compound 5c on Aβ‐treated neurotoxicity in PC12 cells depicted low activity.     

Expression and possible functions of the cholinergic system in a murine embryonic stem cell line

The expression of a cholinergic system during embryonic development is a widespread phenomenon. However, no precise function could be assigned to it during early pre-neural stages and there are only few studies that document when it precisely starts to be expressed. Here, we examined the expression of cholinergic components in a murine embryonic stem cell line by RT-PCR, histochemistry, and enzyme activity measurements; the acetylcholine (ACh) content was measured by HPLC. We have demonstrated that embryonic stem cells express ACh, acetylcholine receptors, choline acetyltransferase (ChAT), acetyl- and butyryl-cholinesterase (AChE and BChE). Butyryl-cholinesterase (BChE) expression was higher than AChE. The cholinesterase activity was down-regulated by adding specific inhibitors to culture medium. Inhibition of BChE led to a reduction of proliferation. This is the first demonstration that mouse embryonic stem cells express the full molecular equipment of a cholinergic system. Locally produced ACh might function as an intercellular signal, modulating the proliferation of stem cells.     

Evolution of acetylcholinesterase and butyrylcholinesterase in the vertebrates: an atypical butyrylcholinesterase from the Medaka Oryzias latipes

Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are thought to be the result of a gene duplication event early in vertebrate evolution. To learn more about the evolution of these enzymes, we expressed in vitro, characterized, and modeled a recombinant cholinesterase (ChE) from a teleost, the medaka Oryzias latipes. In addition to AChE, O. latipes has a ChE that is different from either vertebrate AChE or BChE, which we are classifying as an atypical BChE, and which may resemble a transitional form between the two. Of the fourteen aromatic amino acids in the catalytic gorge of vertebrate AChE, ten are conserved in the atypical BChE of O. latipes; by contrast, only eight are conserved in vertebrate BChE. Notably, the atypical BChE has one phenylalanine in its acyl pocket, while AChE has two and BChE none. These substitutions could account for the intermediate nature of this atypical BChE. Molecular modeling supports this proposal. The atypical BChE hydrolyzes acetylthiocholine (ATCh) and propionylthiocholine (PTCh) preferentially but butyrylthiocholine (BTCh) to a considerable extent, which is different from the substrate specificity of AChE or BChE. The enzyme shows substrate inhibition with the two smaller substrates but not with the larger substrate BTCh. In comparison, AChE exhibits substrate inhibition, while BChE does not, but may instead show substrate activation. The atypical BChE from O. latipes also shows a mixed pattern of inhibition. It is effectively inhibited by physostigmine, typical of all ChEs. However, although the atypical BChE is efficiently inhibited by the BChE-specific inhibitor ethopropazine, it is not by another BChE inhibitor, iso-OMPA, nor by the AChE-specific inhibitor BW284c51. The atypical BChE is found as a glycophosphatidylinositol-anchored (GPI-anchored) amphiphilic dimer (G(2) (a)), which is unusual for any BChE. We classify the enzyme as an atypical BChE and discuss its implications for the evolution of AChE and BChE and for ecotoxicology.