Tau phosphorylation by glycogen synthase kinase 3ß modulates enzyme acetylcholinesterase expression.

In Alzheimer's disease (AD), the enzyme acetylcholinesterase (AChE) co-localizes with hyperphosphorylated tau (P-tau) within neurofibrillary tangles. Having demonstrated that AChE expression is increased in the transgenic mouse model of tau Tg-VLW, here we examined whether modulating phosphorylated tau levels by over-expressing wild-type human tau and glycogen synthase kinase-3ß (GSK3ß) influences AChE expression. In SH-SY5Y neuroblastoma cells expressing higher levels of P-tau, AChE activity and protein increased by (20% ± 2%) and (440% ± 150%), respectively. Western blots and qPCR assays showed that this increment mostly corresponded to the cholinergic ACHE-T variant, for which the protein and transcript levels increased ~60% and ~23%, respectively. Moreover, in SH-SY5Y cells differentiated into neurons by exposure to retinoic acid (10 µM), over-expression of GSK3ß and tau provokes an imbalance in cholinergic activity with a decrease in the neurotransmitter acetylcholine in the cell (45 ± 10%). Finally, we obtained cerebrospinal fluid (CSF) from AD patients enrolled on a clinical trial of tideglusib, an irreversible GSK3ß inhibitor. In CSF of patients that received a placebo, there was an increase in AChE activity (35 ± 16%) respect to basal levels, probably because of their treatment with AChE inhibitors. However, this increase was not observed in tideglusib-treated patients. Moreover, CSF levels of P-tau at the beginning measured by commercially ELISA kits correlated with AChE activity. In conclusion, this study shows that P-tau can modulate AChE expression and it suggests that AChE may possibly increase in the initial phases of AD.

COLQ and ARHGAP15 are Associated with Diverticular Disease and are Expressed in the Colon.

BACKGROUND: Diverticular disease is a common but poorly understood disease of the gastrointestinal tract. Recent studies have identified several single nucleotide polymorphisms (SNPs) that are associated with diverticular disease. MATERIALS AND METHODS: The genotypes of three SNPs (rs4662344 in ARHGAP15, rs7609897 in COLQ, and rs67153654 in FAM155A) were identified by Taqman assay in 204 patients with diverticular disease. Clinical characteristics were obtained from the medical record to study association with genotype. To evaluate gene expression in colon tissue, qPCR was performed on 24 patients with diverticulitis, and COLQ was localized using immunohistochemistry. RESULTS: The ARHGAP15 and COLQ SNPs were significantly associated with both diverticular disease and specifically diverticulitis, while the FAM155A was not associated with either. No association was found with clinical disease characteristics. Heterozygous genotypes at the ARHGAP15 SNP was associated with lower ARHGAP15 expression in colon tissues. COLQ protein localized to the myenteric plexus in the colon. CONCLUSIONS: This study confirmed association of the ARHGAP15 and COLQ SNPs with diverticular disease in our patients but could not confirm FAM155A SNP association. Neither of these SNPs appeared to associate with more severe disease, but genotype at the ARHGAP15 SNP did impact expression of ARHGAP15 in the colon. Additionally, this study is the first to localize COLQ in the colon. Its presence in the myenteric nervous system suggests COLQ SNP variants may contribute to diverticular disease by altering motility.

Neuroprotective Effect of Bean Phosphatidylserine on TMT-Induced Memory Deficits in a Rat Model.

BACKGROUND: Trimethyltin (TMT) is a potent neurotoxin affecting various regions of the central nervous system, including the neocortex, the cerebellum, and the hippocampus. Phosphatidylserine (PS) is a membrane phospholipid, which is vital to brain cells. We analyzed the neuroprotective effects of soybean-derived phosphatidylserine (Bean-PS) on cognitive function, changes in the central cholinergic systems, and neural activity in TMT-induced memory deficits in a rat model. METHODS: The rats were randomly divided into an untreated normal group, a TMT group (injected with TMT + vehicle), and a group injected with TMT + Bean-PS. The rats were treated with 10% hexane (TMT group) or TMT + Bean-PS (50 mg·kg-1, oral administration (p.o.)) daily for 21 days, following a single injection of TMT (8.0 mg/kg, intraperitoneally (i.p.)). The cognitive function of Bean-PS was assessed using the Morris water maze (MWM) test and a passive avoidance task (PAT). The expression of acetylcholine transferase (ChAT) and acetylcholinesterase (AchE) in the hippocampus was assessed via immunohistochemistry. A positron emission tomography (PET) scan was used to measure the glucose uptake in the rat brain. RESULTS: Treatment with Bean-PS enhanced memory function in the Morris water maze (MWM) test. Consistent with the behavioral results, treatment with Bean-PS diminished the damage to cholinergic cells in the hippocampus, in contrast to those of the TMT group. The TMT+Bean-PS group showed elevated glucose uptake in the frontal lobe of the rat brain. CONCLUSION: These results demonstrate that Bean-PS protects against TMT-induced learning and memory impairment. As such, Bean-PS represents a potential treatment for neurodegenerative disorders, such as Alzheimer's disease.

Recombinant Acetylcholinesterase purification and its interaction with silver nanoparticle.

Although the use of silver nanoparticles (AgNPs) has substantial benefits, their entrance into the environment, food chain, and human body and their toxicity have come under serious scrutiny. Multiple noncovalent attractive forces between AgNPs and bio-macromolecules are responsible for immediate corona formation upon exposure to biological tissue. Here, the influence of AgNPs with neuro-enzyme Acetylcholinesterase (AChE) was investigated. AgNPs to enzyme ratio had an effect on the enzyme and features of the treated samples. It was also observed that time increments had a positive effect on the size of AgNPs and caused an increase in their initial size. In other words, smaller AgNPs resulted in size increments after interaction with enzymes, while the larger ones showed size decrements. The nano-crystalline AgNPs were identified in x-ray powder diffraction analyses before and after treatment with AChE. The (220) crystalline plane is related to the internal crystallinity of cubic Ag. The results show that the interaction between AChE and AgNPs could lead not only to a decrease in AChE activity, but also to a reduction in the crystallinity and stability of AgNPs. The circular dichroism demonstrates that the secondary structure of AChE also declined after 30 min of incubation with AgNPs at 37 °C.

Acetylcholine induces fibrogenic effects via M2/M3 acetylcholine receptors in non-alcoholic steatohepatitis and in primary human hepatic stellate cells.

BACKGROUND: The parasympathetic nervous system (PNS), via neurotransmitter acetylcholine (ACh), modulates fibrogenesis in animal models. However, the role of ACh in human hepatic fibrogenesis is unclear. AIMS: We aimed to determine the fibrogenic responses of human hepatic stellate cells (hHSC) to ACh and the relevance of the PNS in hepatic fibrosis in patients with non-alcoholic steatohepatitis (NASH). METHODS: Primary hHSC were analyzed for synthesis of endogenous ACh and acetylcholinesterase and gene expression of choline acetyltransferase and muscarinic ACh receptors (mAChR). Cell proliferation and fibrogenic markers were analyzed in hHSC exposed to ACh, atropine, mecamylamine, methoctramine, and 4-diphenylacetoxy-N-methylpiperidine methiodide. mAChR expression was analyzed in human NASH scored for fibrosis. RESULTS: We observed that hHSC synthesize ACh and acetylcholinesterase and express choline acetyltransferase and M1-M5 mAChR. We also show that M2 was increased during NASH progression, while both M2 and M3 were found upregulated in activated hHSC. Furthermore, endogenous ACh is required for hHSC basal growth. Exogenous ACh resulted in hHSC hyperproliferation via mAChR and phosphoinositide 3-kinase and Mitogen-activated protein kinase kinase (MEK) signaling pathways, as well as increased fibrogenic markers. CONCLUSION: We show that ACh regulates hHSC activation via M2 and M3 mAChR involving the phosphoinositide 3-kinase and MEK pathways in vitro. Finally, we provide evidence that the PNS may be involved in human NASH fibrosis.

Japanese Huperzia serrata extract and the constituent, huperzine A, ameliorate the scopolamine-induced cognitive impairment in mice.

Huperzia serrata has been used as a Chinese folk medicine for many years. It contains huperzine A, which has a protective effect against memory deficits in animal models; however, it is unclear if H. serrata extract exerts any effects in Alzheimer's disease (AD) models. We used H. serrata collected in Japan and determined its huperzine A content using HPLC. We determined its inhibitory effects on acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activity. H. serrata extract (30 mg/kg/day) and donepezil (10 mg/kg/day) were orally administrated for 7 days. After repeated administration, we performed the Y-maze and passive avoidance tests. H. serrata extract contained 0.5% huperzine A; H. serrata extract inhibited AChE, but not BuChE. H. serrata extract ameliorated cognitive function in mice. These results indicate that Japanese H. serrata extract ameliorates cognitive function deficits by inhibiting AChE. Therefore, H. serrata extract may be valuable for the prevention or treatment of dementia in AD.

[The effect of hypoxia on cholinesterase activity in rat sensorimotor cortex].

This study reports the dynamics of changes in postnatal ontogenesis of the activity of soluble and membrane-bound forms of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in sensorimotor cortex of rats as well as the pattern of their changes after prenatal hypoxia (E14, 7% O2, 3 h) or acute hypoxia in adult animals (4 months, 7% O2, 3 h). In normally developing rats the activity of the membrane-bound AChE form in the sensorimotor cortex gradually increased up to the end of the first month after birth and remained at this high level during all further postnatal ontogenesis, while the activity of the soluble form of AChE reached its maximum on the 10th day after birth and decreased significantly by the end of the first month. In animals exposed to prenatal hypoxia the activity both of the soluble and membrane bound forms of AChE during the first two weeks after birth was 20-25% lower, as compared to controls but increased by the end of the first month and even exceeded the control values remaining increased up to old age (1.5 years). The activity of both BChE forms in rat sensorimotor cortex at all stages of postnatal ontogenesis was significantly lower than of AChE, although the dynamics of their changes was similar to that of AChE. Prenatal hypoxia led to a decrease in the activity of the membrane-bound form of BChE, as compared to controls, practically at all developmental stages studied, but was higher at the end of the first month after birth. At the same time, the activity of the soluble form of BChE was decreased only on the 20th day of development, as compared to the control, but increased from the end of the first month of life onwards. Acute hypoxia in adult rats also led to a decrease in the activity of both forms of AChE and BChE in the sensorimotor cortex but the dynamics of these changes was different for each enzyme. Thus, insufficient oxygen supply to the nervous tissue at different stages of ontogenesis has a significant effect on the activity and ratio of various forms of cholinesterases exhibiting either growth factor or signaling properties. This may lead to changes in brain development and formation of behavioural reactions, including learning and memory, and also increase the risk of development of the sporadic form of Alzheimer's disease (AD)--one of the most common neurodegenerative diseases of advanced age. This study expands our knowledge of the properties of brain cholinesterases under normal and pathological conditions and may be useful for developing new approaches towards prevention and treatment of AD.

Taurine improves the spatial learning and memory ability impaired by sub-chronic manganese exposure.

BACKGROUND: Excessive manganese exposure induced cognitive deficit. Several lines of evidence have demonstrated that taurine improves cognitive impairment induced by numerous neurotoxins. However, the role of taurine on manganese-induced damages in learning and memory is still elusive. This goal of this study was to investigate the beneficial effect of taurine on learning and memory capacity impairment by manganese exposure in an animal model. RESULTS: The escape latency in the Morris Water Maze test was significantly longer in the rats injected with manganese than that in the rats received both taurine and manganese. Similarly, the probe trial showed that the annulus crossings were significantly greater in the taurine plus manganese treated rats than those in the manganese-treated rats. However, the blood level of manganese was not altered by the taurine treatment. Interestingly, the exposure of manganese led to a significant increase in the acetylcholinesterase activity and an evidently decrease in the choline acetyltransferase activity, which were partially restored by the addition of taurine. Additionally, we identified 9 differentially expressed proteins between the rat hippocampus treated by manganese and the control or the manganese plus taurine in the proteomic analysis using the 2-dimensional gel electrophoresis followed by the tandem mass spectrometry (MS/MS). Most of these proteins play a role in energy metabolism, oxidative stress, inflammation, and neuron synapse. CONCLUSIONS: In summary, taurine restores the activity of AChE and ChAT, which are critical for the regulation of acetylcholine. We have identified seven differentially expressed proteins specifically induced by manganese and two proteins induced by taurine from the rat hippocampus. Our results support that taurine improves the impaired learning and memory ability caused by excessive exposure of manganese.

Effects of chlorogenic acid, caffeine, and coffee on behavioral and biochemical parameters of diabetic rats.

Diabetes mellitus (DM) is associated with brain alterations that may contribute to cognitive dysfunctions. Chlorogenic acid (CGA) and caffeine (CA), abundant in coffee (CF), are natural compounds that have showed important actions in the brain. The present study aimed to evaluate the effect of CGA, CA, and CF on acetylcholinesterase (AChE), Na(+), K(+)-ATPase, aminolevulinate dehydratase (δ-ALA-D) activities and TBARS levels from cerebral cortex, as well as memory and anxiety in streptozotocin-induced diabetic rats. Animals were divided into eight groups (n = 5-10): control; control/CGA 5 mg/kg; control/CA 15 mg/kg; control/CF 0.5 g/kg; diabetic; diabetic/CGA 5 mg/kg; diabetic/CA 15 mg/kg; and diabetic/CF 0.5 g/kg. Our results demonstrated an increase in AChE activity and TBARS levels in cerebral cortex, while δ-ALA-D and Na(+), K(+)-ATPase activities were decreased in the diabetic rats when compared to control water group. Furthermore, a memory deficit and an increase in anxiety in diabetic rats were observed. The treatment with CGA and CA prevented the increase in AChE activity in diabetic rats when compared to the diabetic water group. CGA, CA, and CF intake partially prevented cerebral δ-ALA-D and Na(+), K(+)-ATPase activity decrease due to diabetes. Moreover, CGA prevented diabetes-induced TBARS production, improved memory, and decreased anxiety. In conclusion, among the compounds studied CGA proved to be a compound which acts better in the prevention of brain disorders promoted by DM.

Effects of pregnenolone intervention on the cholinergic system and synaptic protein 1 in aged rats.

OBJECTIVE: To observe the effect of pregnenolone (PREG) intervention on the cholinergic system function and the synaptic protein 1 (SYP1) expression in different brain regions of aged rats. METHOD: Twenty-four-month-old male Sprague Dawley rats intraperitoneally injected every other day for one month were divided into blank control group, solvent control group, PREG (0.5 mg/kg) intervention group and PREG (2.0 mg/kg) intervention group. The rats were sacrificed 2 d after the intervention and the corresponding regions of brain tissue were separated and cryopreserved. Western blot analysis was used to detect the expression level of choline acetyltransferase (ChAT), SYP1, serum PREG and the activity of ChAT and acetylcholinesterase (AChE) in different brain regions. In addition, the semiquantitative changes in the expression level of ChAT and SYP1 in frontal lobe and hippocampus were tested by immunohistochemistry. RESULT: Western blot and immunohistochemistry analysis showed that PREG (2.0 mg/kg) administration led to a significant increase of ChAT and SYP1 expressions in frontal lobe, temporal lobe, and hippocampus regions (p < 0.05). The result of enzyme-linked immunosorbent assay showed that PREG (2.0 mg/kg) administration significantly increased ChAT activity and serum PREG levels and caused a decrease in AChE activity (p < 0.05); while PREG (0.5 mg/kg) only elevated levels of serum PREG. CONCLUSION: PREG significantly improved the synaptic plasticity of memory-related brain areas of aged rats, significantly increased brain cholinergic activity and thus helps to improve learning and memory in aged rats.

Striatal cholinergic functional alterations in hypoxic neonatal rats: role of glucose, oxygen, and epinephrine resuscitation.

Molecular processes regulating cholinergic functions play an important role in the control of respiration under hypoxia. Cholinergic alterations and its further complications in respiration due to hypoxic insult in neonatal rats and the effect of glucose, oxygen, and epinephrine resuscitation was evaluated in the present study. Receptor binding and gene expression studies were done in the corpus striatum to analyse the changes in total muscarinic receptors, muscarinic M1, M2, M3 receptors, and the enzymes involved in acetylcholine metabolism, choline acetyltransferase and acetylcholinesterase. Neonatal hypoxia decreased total muscarinic receptors with reduced expression of muscarinic M1, M2, and M3 receptor genes. The reduction in acetylcholine metabolism is indicated by the downregulated choline acetyltransferase and upregulated acetyl cholinesterase expression. These cholinergic disturbances were reversed to near control in glucose-resuscitated hypoxic neonates. The adverse effects of immediate oxygenation and epinephrine administration are also reported. The present findings points to the cholinergic alterations due to neonatal hypoxic shock and suggests a proper resuscitation method to ameliorate these striatal changes.

Environmental enrichment upregulates micro-RNA-183 and alters acetylcholinesterase splice variants to reduce anxiety-like behavior in the little Indian field mouse (Mus booduga).

Environmental enrichment (EE) has an influential role in reducing behavioral reactivity to stress. We previously observed that EE reduces the anxiety-like behavior in the field mouse Mus booduga accompanied by a reduction in the expression of molecules involved in the stress pathway. In this study, we demonstrate the effect of different housing condition on regulation of micro-RNA-183-SC35-mediated splicing of acetylcholinesterase (AChE). Adult male M. booduga were captured from an agricultural field and housed under nonenriched standard conditions (SC) for 7 days and considered as directly from the wild (DW). On day 8, individuals were randomly assigned to three groups; DW, SC, and EE. The DW group's anxiety-like behavior was assessed in the elevated plus maze (EPM) and open field test (OFT). The SC and EE groups were transferred to their respective conditions and housed for another 30 days. The mice housed in EE showed less anxiety-like behavior on EPM and in OFT compared with DW and SC mice. Interestingly, miR-183 expression was increased following exposure to EPM in EE mice but not in SC mice. Subsequently, the upregulated miR-183 expression suppresses the SC35 expression and shifting of splicing from AChE-S (synaptic) to AChE-R (read-through) form, whereas standard housing condition downregulate miR-183 and induces the splicing of AChE. The upregulated AChE-R form possibly terminates ACh transmission, which is reflected in the level of anxiety-like behavior. Overall, the present study suggests that EE effectively regulates the miR-183 pathway to reduce anxiety-like behavior.

Thiamine deficiency induced neurochemical, neuroanatomical, and neuropsychological alterations: a reappraisal.

Nutritional deficiency can cause, mainly in chronic alcoholic subjects, the Wernicke encephalopathy and its chronic neurological sequela, the Wernicke-Korsakoff syndrome (WKS). Long-term chronic ethanol abuse results in hippocampal and cortical cell loss. Thiamine deficiency also alters principally hippocampal- and frontal cortical-dependent neurochemistry; moreover in WKS patients, important pathological damage to the diencephalon can occur. In fact, the amnesic syndrome typical for WKS is mainly due to the damage in the diencephalic-hippocampal circuitry, including thalamic nuclei and mammillary bodies. The loss of cholinergic cells in the basal forebrain region results in decreased cholinergic input to the hippocampus and the cortex and reduced choline acetyltransferase and acetylcholinesterase activities and function, as well as in acetylcholine receptor downregulation within these brain regions. In this narrative review, we will focus on the neurochemical, neuroanatomical, and neuropsychological studies shedding light on the effects of thiamine deficiency in experimental models and in humans.

Translational regulation of acetylcholinesterase by the RNA-binding protein Pumilio-2 at the neuromuscular synapse.

Acetylcholinesterase (AChE) is highly expressed at sites of nerve-muscle contact where it is regulated at both the transcriptional and post-transcriptional levels. Our understanding of the molecular mechanisms underlying its regulation is incomplete, but they appear to involve both translational and post-translational events as well. Here, we show that Pumilio-2 (PUM2), an RNA binding translational repressor, is highly localized at the neuromuscular junction where AChE mRNA concentrates. Immunoprecipitation of muscle cell extracts with a PUM2 specific antibody precipitated AChE mRNA, suggesting that PUM2 binds to the AChE transcripts in a complex. Gel shift assays using a bacterially expressed PUM2 RNA binding domain showed specific binding using wild type AChE 3'-UTR RNA segment that was abrogated by mutation of the consensus recognition site. Transfecting skeletal muscle cells with shRNAs specific for PUM2 up-regulated AChE expression, whereas overexpression of PUM2 decreased AChE activity. We conclude that PUM2 binds to AChE mRNA and regulates AChE expression translationally at the neuromuscular synapse. Finally, we found that PUM2 is regulated by the motor nerve suggesting a trans-synaptic mechanism for locally regulating translation of specific proteins involved in modulating synaptic transmission, analogous to CNS synapses.

Acetylcholinesterase deficiency contributes to neuromuscular junction dysfunction in type 1 diabetic neuropathy.

Diabetic neuropathy is associated with functional and morphological changes of the neuromuscular junction (NMJ) associated with muscle weakness. This study examines the effect of type 1 diabetes on NMJ function. Swiss Webster mice were made diabetic with three interdaily ip injections of streptozotocin (STZ). Mice were severely hyperglycemic within 7 days after the STZ treatment began. Whereas performance of mice on a rotating rod remained normal, the twitch tension response of the isolated extensor digitorum longus to nerve stimulation was reduced significantly at 4 wk after the onset of STZ-induced hyperglycemia. This mechanical alteration was associated with increased amplitude and prolonged duration of miniature end-plate currents (mEPCs). Prolongation of mEPCs was not due to expression of the embryonic acetylcholine receptor but to reduced muscle expression of acetylcholine esterase (AChE). Greater sensitivity of mEPC decay time to the selective butyrylcholinesterase (BChE) inhibitor PEC suggests that muscle attempts to compensate for reduced AChE levels by increasing expression of BChE. These alterations of AChE are attributed to STZ-induced hyperglycemia since similar mEPC prolongation and reduced AChE expression were found for db/db mice. The reduction of muscle end-plate AChE activity early during the onset of STZ-induced hyperglycemia may contribute to endplate pathology and subsequent muscle weakness during diabetes.

Acetylcholinesterase activity in the rat brain after pneumococcal meningitis.

Pneumococcal meningitis is a life-threatening disease characterized by acute purulent infection of the meninges causing neuronal injury, cortical necrosis and hippocampal apoptosis. Cholinergic neurons and their projections are extensively distributed throughout the central nervous system. The aim of this study was to assess acetylcholinesterase activity in the rat brain after pneumococcal meningitis. In the hippocampus, frontal cortex and cerebrospinal fluid, acetylcholinesterase activity was found to be increased at 6, 12, 24, 48 and 96 hr without antibiotic treatment, and at 48 and 96 hr with antibiotic treatment. Our data suggest that acetylcholinesterase activity could be related to neuronal damage induced by pneumococcal meningitis.

Impaired sweating in patients with cholinergic urticaria is linked to low expression of acetylcholine receptor CHRM3 and acetylcholine esterase in sweat glands.

Background: Cholinergic urticaria (CholU), a frequent form of chronic inducible urticaria, is characterized by itchy wheals and angioedema in response to sweating. As of now, the rate and pathophysiological relevance of impaired sweating in patients with CholU are ill-defined. Aim: To assess in CholU patients the rate and extent of impaired sweating and its links to clinical and pathophysiological features of CholU. Patients and methods: We assessed sweating in patients with CholU (n = 13) subjected to pulse-controlled ergometry (PCE) provocation testing. Pre- and post-PCE biopsies of lesional (L) and non-lesional (NL) skin were analyzed for the expression of acetylcholine receptor M3 (CHRM3) and acetylcholine esterase (ACh-E) by quantitative histomorphometry and compared to those of healthy control subjects (HCs). CholU patients were assessed for disease duration and severity as well as other clinical features. Results: Of the 13 patients with CholU, 10 showed reduced sweating in response to PCE provocation, and 3 had severely reduced sweating. Reduced sweating was linked to long disease duration and high disease severity. CholU patients with impaired sweating responses showed reduced sweat gland epithelial expression of CHRM3 and ACh-E. Conclusion: Reduced sweating is common in CholU patients, especially in those with long-standing and severe disease, and it can be severe. Reduced expression of CHRM3 and ACh-E may be the cause or consequence of CholU in patients with impaired sweating, and this should be explored by further studies.

The PRiMA-linked cholinesterase tetramers are assembled from homodimers: hybrid molecules composed of acetylcholinesterase and butyrylcholinesterase dimers are up-regulated during development of chicken brain.

Acetylcholinesterase (AChE) is anchored onto cell membranes by the transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric globular form that is prominently expressed in vertebrate brain. In parallel, the PRiMA-linked tetrameric butyrylcholinesterase (BChE) is also found in the brain. A single type of AChE-BChE hybrid tetramer was formed in cell cultures by co-transfection of cDNAs encoding AChE(T) and BChE(T) with proline-rich attachment domain-containing proteins, PRiMA I, PRiMA II, or a fragment of ColQ having a C-terminal GPI addition signal (Q(N-GPI)). Using AChE and BChE mutants, we showed that AChE-BChE hybrids linked with PRiMA or Q(N-GPI) always consist of AChE(T) and BChE(T) homodimers. The dimer formation of AChE(T) and BChE(T) depends on the catalytic domains, and the assembly of tetramers with a proline-rich attachment domain-containing protein requires the presence of C-terminal "t-peptides" in cholinesterase subunits. Our results indicate that PRiMA- or ColQ-linked cholinesterase tetramers are assembled from AChE(T) or BChE(T) homodimers. Moreover, the PRiMA-linked AChE-BChE hybrids occur naturally in chicken brain, and their expression increases during development, suggesting that they might play a role in cholinergic neurotransmission.

Induction of plasma acetylcholinesterase activity in mice challenged with organophosphorus poisons.

The restoration of plasma acetylcholinesterase activity in mice following inhibition by organophosphorus pesticides and nerve agents has been attributed to synthesis of new enzyme. It is generally assumed that activity levels return to normal, are stable and do not exceed the normal level. We have observed over the past 10 years that recovery of acetylcholinesterase activity levels in mice treated with organophosphorus agents (OP) exceeds pretreatment levels and remains elevated for up to 2 months. The most dramatic case was in mice treated with tri-cresyl phosphate and tri-ortho-cresyl phosphate, where plasma acetylcholinesterase activity rebounded to a level 250% higher than the pretreatment activity. The present report summarizes our observations on plasma acetylcholinesterase activity in mice treated with chlorpyrifos, chlorpyrifos oxon, diazinon, tri-ortho-cresyl phosphate, tri-cresyl phosphate, tabun thiocholine, parathion, dichlorvos, and diisopropylfluorophosphate. We have developed a hypothesis to explain the excess acetylcholinesterase activity, based on published observations. We hypothesize that acetylcholinesterase activity is induced when cells undergo apoptosis and that consequently there is a rise in the level of plasma acetylcholinesterase.

Expression and characterization of recombinant Locusta migratoria manilensis acetylcholinesterase 1 in Pichia pastoris.

The acetylcholinesterase 1 from Locusta migratoria manilensis (LmAChE1) was successfully expressed in methylotrophic yeast Pichia pastoris KM71. The maximum expression of recombinant LmAChE1 (reLmAChE1) was achieved after 9 days of induction at 2.5% methanol. The reLmAChE1 was first precipitated with ammonium sulfate (50% saturation) and then was purified with nickel affinity chromatography. The enzyme was purified 3.2×10(3)-fold with a yield of 68% and a specific activity of 8.1 U/mg. The purified reLmAChE1 exhibited highest activity at 30°C in 100 mM phosphate buffer (pH 7.4), and its activity could be inhibited by eserine sulfate and pentan-3-one-dibromide (BW284c51). Substrate specificity analysis showed that the purified reLmAChE1 preferred acetylthiocholine (ATC) and propionylthiocholine (BTC) rather than butyrylthiocholine (BTC). When ATC was used as substrate, the K(m) and V(max) values for the reLmAChE1 were 24.8 µM and 9.5 µmol/min/mg, respectively.