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Find video protocols related to scientific articles indexed in Pubmed.
Synergistic inhibition on acetylcholinesterase by the combination of berberine and palmatine originally isolated from Chinese medicinal herbs.
J. Mol. Neurosci.
PUBLISHED: 02-12-2014
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Alzheimer's disease is a multi-factorial neurodegenerative disorder devastatingly affecting the aged population worldwide. Previous studies have shown that medicinal herbs used in traditional Chinese medicine might be benefit to Alzheimer's disease patients. Berberine and palmatine, two isoquinoline alkaloids found in several medicinal herbs, were used for memory enhancement in China. In this study, the inhibitory effects of combined berberine and palmatine on acetylcholinesteras were evaluated using recombinant human acetylcholinesterase. Our results showed that the combination of berberine and palmatine inhibited acetylcholinesterase in a mixed competitive pattern. By the median-effect principle, the calculated combination index of the combination was less than 1, suggesting that berberine and plamatine inhibited acetylcholinesterase synergistically. Furthermore, the drug-reducing index of berberine and palmatine were 2.98 and 2.66, respectively. Taken together, the results showed that the combination of the two alkaloids might potentially be developed as a more effective therapeutic strategy for Alzheimer's disease patients.
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Quantification of the transcripts encoding different forms of AChE in various cell types: real-time PCR coupled with standards in revealing the copy number.
J. Mol. Neurosci.
PUBLISHED: 01-03-2014
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Acetylcholinesterase (AChE) is encoded by a single gene, and the alternative splicing at the 3' end produces different isoforms, including tailed (AChET), read-through (AChER), and hydrophobic (AChEH). Different forms of this enzyme exist in different cell types. Each AChE form has been proposed to have unique function, and all of them could be found in same cell type. Thus, the splicing process of different AChE forms remains unclear. Here, we aimed to establish a quantification method in measuring the absolute amount of each AChE splicing variants within a cell type. By using real-time PCR coupled with standard curves of defined copy of AChE variants, the copies of AChET transcript per 100 ng of total RNA were 5.7?×?10(4) in PC12 (rat neuronal cell), 1.3?×?10(4) in Caco-2 (human intestinal cell), 0.67?×?10(4) in TF-1 (human erythropoietic precursor), 133.3 in SH-SY5Y (human neuronal cell), and 56.7 in human umbilical vein endothelial cells (human endothelial cells). The copies of AChEH in these cell types were 0.3?×?10(4), 3.3?×?10(4), 2.7?×?10(4), 133.3, and 46.7, respectively, and AChER were 0.07?×?10(4), 0.13?×?10(4), 890, 3.3, and 2.7, respectively. Furthermore, PC12 and TF-1 cells were chosen for the analysis of AChE splicing pattern during differentiation. The results demonstrated a selective increase in AChET mRNA but not AChER or AChEH mRNAs in PC12 upon nerve growth factor-induced neuronal differentiation. PC12 cells could therefore act as a good cell model for the study on alternative splicing mechanism and regulation of AChET.
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Acetylcholinesterase Protein Level Is Preserved in the Alzheimers Brain.
J. Mol. Neurosci.
PUBLISHED: 10-18-2013
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Acetylcholinesterase (AChE) is a key enzyme in the cholinergic nervous system and is one of the most studied proteins in the field of Alzheimers disease (AD). Moreover, alternative functions of AChE unrelated with the hydrolysis of acetylcholine are suspected. Until now, the majority of investigations on AChE in AD pathology have been focused on the determination of its enzymatic activity level, which is depleted in the AD brain. Despite this overall decrease, AChE activity increases at the vicinity of the two hallmarks of AD, the amyloid plaques and the neurofibrillary tangles (NFT). In fact, AChE may directly interact with A? in a manner that increases the deposition of A? to form plaques. In the context of protein-protein interactions, we have recently reported that AChE can interact with presenilin-1, the catalytic component of ?-secretase, influencing its expression level and also its activity. However, the alteration of AChE protein in the AD brain has not been determined. Here, we demonstrated by Western blotting and immunohistochemistry that a prominent pool of enzymatically inactive AChE protein existed in the AD brain. The potential significance of these unexpected levels of inactive AChE protein in the AD brain was discussed, especially in the context of protein-protein interactions with ?-amyloid and presenilin-1.
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Characterizations of Cholinesterases in Golden Apple Snail (Pomacea canaliculata).
J. Mol. Neurosci.
PUBLISHED: 08-30-2013
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Cholinesterases (ChEs) have been identified in vertebrates and invertebrates. Inhibition of ChE activity in invertebrates, such as bivalve molluscs, has been used to evaluate the exposure of organophosphates, carbamate pesticides, and heavy metals in the marine system. The golden apple snail (Pomacea canaliculata) is considered as one of the worst invasive alien species harmful to rice and other crops. The ChE(s) in this animal, which has been found recently, but poorly characterized thus far, could serve as biomarker(s) for environmental surveillance as well as a potential target for the pest control. In this study, the tissue distribution, substrate preference, sensitivity to ChE inhibitors, and molecular species of ChEs in P. canaliculata were investigated. It was found that the activities of both AChE and BChE were present in all test tissues. The intestine had the most abundant ChE activities. Both enzymes had fair activities in the head, kidney, and gills. The BChE activity was more sensitive to tetra-isopropylpyrophosphoramide (iso-OMPA) than the AChE. Only one BChE molecular species, 5.8S, was found in the intestine and head, whereas two AChE species, 5.8S and 11.6S, were found there. We propose that intestine ChEs of this snail may be potential biomarkers for manipulating pollutions.
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Can Hedysari Radix replace Astragali Radix in Danggui Buxue Tang, a Chinese herbal decoction for woman aliment?
Phytomedicine
PUBLISHED: 01-25-2013
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Astragali Radix (AR) has been used for over 2000 years in China for the enrichment of "Qi". Hedysari Radix (HR), a herb having similar chemical composition with AR, has been commonly used as a substitute of AR in herbal decoction. In order to evaluate the possible replacement of HR for AR in Chinese herbal decoction, systematic comparison of AR and HR was done by chemical and biological assessments. The water extract of AR contained higher levels of calycosin, calycosin-glucoside, ononin, astragaloside III and astragaloside IV, while higher amount of formononetin was found in the HR extract. The estrogenic, erythropoetic and osteogenic effects were compared between the water extracts of AR and HR, and in all cases AR extract showed higher biological activities. Danggui Buxue Tang (DBT) is a very common herbal decoction for woman aliment, and which contains AR and Angelica Sinensis Radix. Here, we generated two forms of DBT having either AR or HR as the major herbs. Chemically, AR-contained DBT showed higher amounts of various active chemicals, except formononetin that was higher in HR-contained DBT. In parallel, the estrogenic, osteogenic and erythropoetic effects of DBT containing AR showed better activities than that of DBT having HR. Thus, AR and HR showed distinct differences in terms of chemical and biological properties. In order to achieve the best therapeutical effect, as well as to guarantee the safety, AR is recommended here to be used for making DBT.
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Molecular Assembly and Biosynthesis of Acetylcholinesterase in Brain and Muscle: the Roles of t-peptide, FHB Domain, and N-linked Glycosylation.
Front Mol Neurosci
PUBLISHED: 07-30-2011
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Acetylcholinesterase (AChE) is responsible for the hydrolysis of the neurotransmitter, acetylcholine, in the nervous system. The functional localization and oligomerization of AChE T variant are depending primarily on the association of their anchoring partners, either collagen tail (ColQ) or proline-rich membrane anchor (PRiMA). Complexes with ColQ represent the asymmetric forms (A(12)) in muscle, while complexes with PRiMA represent tetrameric globular forms (G(4)) mainly found in brain and muscle. Apart from these traditional molecular forms, a ColQ-linked asymmetric form and a PRiMA-linked globular form of hybrid cholinesterases (ChEs), having both AChE and BChE catalytic subunits, were revealed in chicken brain and muscle. The similarity of various molecular forms of AChE and BChE raises interesting question regarding to their possible relationship in enzyme assembly and localization. The focus of this review is to provide current findings about the biosynthesis of different forms of ChEs together with their anchoring proteins.
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The assembly of proline-rich membrane anchor (PRiMA)-linked acetylcholinesterase enzyme: glycosylation is required for enzymatic activity but not for oligomerization.
J. Biol. Chem.
PUBLISHED: 07-27-2011
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Acetylcholinesterase (AChE) anchors onto cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor) as a tetrameric form in vertebrate brain. The assembly of AChE tetramer with PRiMA requires the C-terminal "t-peptide" in AChE catalytic subunit (AChE(T)). Although mature AChE is well known N-glycosylated, the role of glycosylation in forming the physiologically active PRiMA-linked AChE tetramer has not been studied. Here, several lines of evidence indicate that the N-linked glycosylation of AChE(T) plays a major role for acquisition of AChE full enzymatic activity but does not affect its oligomerization. The expression of the AChE(T) mutant, in which all N-glycosylation sites were deleted, together with PRiMA in HEK293T cells produced a glycan-depleted PRiMA-linked AChE tetramer but with a much higher K(m) value as compared with the wild type. This glycan-depleted enzyme was assembled in endoplasmic reticulum but was not transported to Golgi apparatus or plasma membrane.
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Quality assessment of a formulated Chinese herbal decoction, Kaixinsan, by using rapid resolution liquid chromatography coupled with mass spectrometry: A chemical evaluation of different historical formulae.
J Sep Sci
PUBLISHED: 11-16-2010
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Kaixinsan is an ancient Chinese herbal decoction mainly prescribed for patients suffering from mental depression. This decoction was created by Sun Si-miao of Tang Dynasty (A.D. 600) in ancient China, and was composed of four herbs: Radix and Rhizome Ginseng, Radix Polygalae, Rhizoma Acori Tatarinowii and Poria. Historically, this decoction has three different formulations, each recorded at a different point in time. In this study, the chemical compositions of all three Kaixinsan formulae were analyzed. By using rapid resolution LC coupled with a diode-array detector and an ESI triple quadrupole tandem MS (QQQ-MS/MS), the Radix and Rhizome Ginseng-derived ginsenosides including Rb(1), Rd, Re, Rg(1), the Radix Polygalae-derived 3,6-disinapoyl sucrose, the Rhizoma Acori Tatarinowii-derived ?- and ?-asarone and the Poria-derived pachymic acid were compared among the three different formulations. The results showed variations in the solubility of different chemicals between one formula and the others. This systematic method developed could be used for the quality assessment of this herbal decoction.
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Expression of cAMP-responsive element binding proteins (CREBs) in fast- and slow-twitch muscles: a signaling pathway to account for the synaptic expression of collagen-tailed subunit (ColQ) of acetylcholinesterase at the rat neuromuscular junction.
Chem. Biol. Interact.
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The gene encoding the collagen-tailed subunit (ColQ) of acetylcholinesterase (AChE) contains two distinct promoters that drive the production of two ColQ mRNAs, ColQ-1 and ColQ-1a, in slow- and fast-twitch muscles, respectively. ColQ-1a is expressed at the neuromuscular junction (NMJ) in fast-twitch muscle, and this expression depends on trophic factors supplied by motor neurons signaling via a cAMP-dependent pathway in muscle. To further elucidate the molecular basis of ColQ-1as synaptic expression, here we investigated the expression and localization of cAMP-responsive element binding protein (CREB) at the synaptic and extra-synaptic regions of fast- and slow-twitch muscles from adult rats. The total amount of active, phosphorylated CREB (P-CREB) present in slow-twitch soleus muscle was higher than that in fast-twitch tibialis muscle, but P-CREB was predominantly expressed in the fast-twitch muscle at NMJs. In contrast, P-CREB was detected in both synaptic and extra-synaptic regions of slow-twitch muscle. These results reveal, for the first time, the differential distribution of P-CREB in fast- and slow-twitch muscles, which might support the crucial role of cAMP-dependent signaling in controlling the synapse-specific expression of ColQ-1a in fast-twitch muscles.
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Characterization of acetylcholinesterase in Hong Kong oyster (Crassostrea hongkongensis) from South China Sea.
Chem. Biol. Interact.
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Acetylcholinesterase (AChE) activity has been used to evaluate the exposure of mollusk bivalves to organophosphates, carbamate pesticides, and heavy metals. Crassostrea hongkongensis is a Hong Kong endemic oyster, and has a high commercial value along the coastal area of South China. The use of this species as a bio-indicator of the marine environment, and the use of AChE activity measurements in tissues of C. hongkongensis require prior characterization of AChE in this species. Here, we report that gill tissue contains the highest AChE activity in C. hongkongensis, and that the molecular form of AChE is most likely to be a dimeric form. In addition, the effect of the pesticide acephate on AChE activity in the gill of C. hongkongensis was analyzed, and the mean inhibition concentration (IC50) value was determined. This study suggests that AChE activity in the gill tissue of C. hongkongensis might be used as a biomarker in monitoring organophosphate contamination in the marine fauna of South China.
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N-linked glycosylation of dimeric acetylcholinesterase in erythrocytes is essential for enzyme maturation and membrane targeting.
FEBS J.
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Acetylcholinesterase (AChE) is well-known for its cholinergic functions in the nervous system; however, this enzyme is also found in other tissues where its function is still not understood. AChE is synthesized through alternative splicing as splicing variants, with isoforms including read-through (AChE(R)), tailed (AChE(T)) and hydrophobic (AChE(H)). In human erythrocytes, AChE(H) is a glycophosphatidylinositol-linked dimer on the plasma membrane. Three N-linked glycosylation sites have been identified in the catalytic domain of human AChE. Here, we investigate the roles of glycosylation in assembly and trafficking of human AChE(H). In transfected fibroblasts, expression of AChE(H) was able to mimic the function of the dimeric form of AChE on the erythrocyte membrane. A glycan-depleted form was constructed by site-directed mutagenesis. By comparison with the wild-type AChE(H), the mutant had a much lower enzymatic activity and a much higher K(m) value. In addition, the mutant was dimerized in the endoplasmic reticulum, but was not trafficked to the Golgi apparatus. The results suggest that the glycosylation may affect AChE(H) enzymatic activity and trafficking, but not dimer formation. The present findings indicate the significance of N-glycosylation in controlling the biosynthesis of the AChE(H) dimer form.
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N-linked glycosylation of proline-rich membrane anchor (PRiMA) is not required for assembly and trafficking of globular tetrameric acetylcholinesterase.
Neurosci. Lett.
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Acetylcholinesterase (AChE) is organized into globular tetramers (G(4)) by a structural protein called proline-rich membrane anchor (PRiMA), anchoring it into the cell membrane of neurons in the brain. The assembly of AChE tetramers with PRiMA requires the presence of a C-terminal "t-peptide" in the AChE catalytic subunit (AChE(T)). The glycosylation of AChE(T) is known to be required for its proper assembly and trafficking; however, the role of PRiMA glycosylation in the oligomer assembly has not been revealed. PRiMA is a glycoprotein containing two putative N-linked glycosylation sites. By using site-directed mutagenesis, the asparagine-43 was identified to be the N-linked glycosylation site of PRiMA. Abolishing glycosylation on mouse PRiMA appeared not to affect its assembly with AChE(T), the enzymatic properties of AChE, and the membrane trafficking of PRiMA-linked AChE tetramers. This result is contrary to the reports that glycosylation is essential for conformation and trafficking of membrane glycoproteins.
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