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Ferulic acid enhances the chemical and biological properties of astragali radix: a stimulator for danggui buxue tang, an ancient Chinese herbal decoction.
Planta Med.
PUBLISHED: 01-31-2014
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Danggui buxue tang, an ancient formula composed of astragali radix and Angelicae sinensis radix, has been used for treating menopausal irregularity in women for more than 800 years in China. In danggui buxue tang, the complete functions of astragali radix require the assistance of Angelicae sinensis radix, and both herbs have to work harmoniously in order to achieve the maximal therapeutic purposes. In order to analyze the relationship of the two herbs, the role of ferulic acid, a major chemical within Angelicae sinensis radix, in chemical and biological properties of astragali radix was determined. Using ferulic acid in the extraction of astragali radix, the amounts of astragaloside IV, calycosin, and formononetin were increased in the final extract; however, the astragali radix polysaccharide showed a minor increase. The chemical-enriched astragali radix extract showed robust induction in osteogenic and estrogenic activities in cultured osteosarcoma MG-63 and breast MCF-7 cells. However, ferulic acid itself did not show such biological responses. The current results strongly suggest that Angelicae sinensis radix-derived ferulic acid is a positive regulator for danggui buxue tang, which enhanced the solubilities of active ingredients derived from astragali radix, and which therefore increased the biological efficacies of danggui buxue tang.
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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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Yu Ping Feng San, an ancient Chinese herbal decoction, regulates the expression of inducible nitric oxide synthase and cyclooxygenase-2 and the activity of intestinal alkaline phosphatase in cultures.
PLoS ONE
PUBLISHED: 01-01-2014
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Yu Ping Feng San (YPFS), a Chinese herbal decoction comprising Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu), and Saposhnikoviae Radix (SR; Fangfeng), has been used clinically to treat inflammatory bowel diseases (IBD). Previously, we demonstrated a dual role of YPFS in regulating cytokine release in cultured macrophages. In this study, we elucidated the anti-inflammatory effect of YPFS that is mediated through modulating the expression of three key enzymes involved in IBD: inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and intestinal alkaline phosphatase (IALP). In a lipopolysaccharide (LPS)-induced chronic-inflammation model of cultured murine macrophages, YPFS treatment suppressed the activation of iNOS and COX-2 expression in a dose-dependent manner. Conversely, application of YPFS in cultured small intestinal enterocytes markedly induced the expression of IALP in a time-dependent manner, which might strengthen the intestinal detoxification system. A duality of YPFS in modulating the expression of iNOS and COX-2 was determined here. The expression of iNOS and COX-2 in macrophages was induced by YPFS, and this activation was partially blocked by the NF-?B-specific inhibitor BAY 11-7082, indicating a role of NF-?B signaling. These YPFS-induced changes in gene regulation strongly suggest that the anti-inflammatory effects of YPFS are mediated through the regulation of inflammatory enzymes.
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Flavonoids induce the expression of synaptic proteins, synaptotagmin, and postsynaptic density protein-95 in cultured rat cortical neuron.
Planta Med.
PUBLISHED: 11-15-2013
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Flavonoids, a family of phenolic compounds, are widely present in our daily diet and exist in traditional Chinese medicines, in which they act as the major active functional ingredients. Different lines of evidence indicate that flavonoids have positive impacts on human health. Here, different subclasses of flavonoids were analyzed for their inductive roles in promoting the expression of synaptic proteins, synaptotagmin, and post-synaptic density protein-95 in cultured rat cortical neurons. Among the screened 65 flavonoids, (-)-catechin, luteolin, and isorhamnetin, in micromolar concentration, were found to induce the expression of synaptic proteins in a dose-dependent manner: the induction values were from 2- to 8-fold that of the control. Similar results were revealed in the flavonoid-treated hippocampal neurons. The identification of these synapse-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimers disease and depression.
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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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Chemical and biological assessment of Ziziphus jujuba fruits from China: different geographical sources and developmental stages.
J. Agric. Food Chem.
PUBLISHED: 07-22-2013
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Chinese date, the fruit of Ziziphus jujuba Mill., has thousands of years cultivation history, and about 700 cultivars of dates in China. Two types of dates are commonly found in the market: (i) fresh immature dates consumed as fruits, and (ii) dried mature dates used as Chinese medicines. Here, chemical and biological properties of these dates were revealed. Different sources of dates showed similar chemical profiles; however, the amounts of identified chemicals showed a great variation. The amount of nucleotides, flavonoids and polysaccharides in dates could be affected by its maturity and drying process. In parallel, the antioxidative functions of their extracts were compared. The date extracts protected PC12 cells against tBHP-induced cytotoxicity, and which also stimulated the transcriptional activity of antioxidant response element. The antioxidative effects were varied among different dates. The current results suggested the optimization of sources and specific usage of different maturity dates.
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Dual-index evaluation of character changes in Panax ginseng C. A. Mey stored in different conditions.
J. Agric. Food Chem.
PUBLISHED: 06-25-2013
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Panax ginseng C. A. Mey has been used as a traditional medicine and functional food in Asia for thousands of years for its improvement of human immunity and metabolism and its antitumor and antifatigue activities. This study reports the impact of storage conditions and storage period on the quality of P. ginseng. The contents of four major ginsenosides in P. ginseng and phosphorylation activities of Akt of ginseng extracts were affected by both storage conditions and storage period. In contrast, the ATP generation capacity of ginseng extracts was affected by storage conditions, but not by storage period. The results showed that the quality of P. ginseng could be well maintained at a relative humidity between 70% and 90%, and dry conditions might decrease the quality of P. ginseng. Through dual-index evaluation, the present study extended our knowledge on the changes of ginsenosides and bioactivities in P. ginseng with respect to different storage conditions and storage periods.
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Kai-xin-san, a chinese herbal decoction containing ginseng radix et rhizoma, polygalae radix, acori tatarinowii rhizoma, and poria, stimulates the expression and secretion of neurotrophic factors in cultured astrocytes.
Evid Based Complement Alternat Med
PUBLISHED: 06-07-2013
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Kai-xin-san (KXS), a Chinese herbal decoction prescribed by Sun Simiao in Beiji Qianjin Yaofang about 1400 years ago, contains Ginseng Radix et Rhizoma, Polygalae Radix, Acori Tatarinowii Rhizoma, and Poria. In China, KXS has been used to treat stress-related psychiatric diseases with the symptoms of depression and forgetfulness. Although animal study has supported the antidepression function of KXS, the mechanism in cellular level is still unknown. Here, a chemically standardized water extract of KXS was applied onto cultured astrocytes in exploring the action mechanisms of KXS treatment, which significantly stimulated the expression and secretion of neurotrophic factors, including NGF, BDNF, and GDNF, in a dose-dependent manner: the stimulation was both in mRNA and protein levels. In addition, the water extracts of four individual herbs did not significantly stimulate the expression of neurotrophic factors, which could explain the optimized effect of KXS in a herbal decoction. The KXS-induced expression of neurotrophic factors did not depend on signaling mediated by estrogen receptor or protein kinase. The results suggested that the antidepressant-like action of KXS might be mediated by an increase of expression of neurotrophic factors in astrocytes, which fully supported the clinical usage of this decoction.
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Chemical changes of Angelicae Sinensis Radix and Chuanxiong Rhizoma by wine treatment: chemical profiling and marker selection by gas chromatography coupled with triple quadrupole mass spectrometry.
Chin Med
PUBLISHED: 05-26-2013
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Angelicae Sinensis Radix (ASR) and Chuanxiong Rhizoma (CR) can be treated with wine to promote their biological functions in Chinese medicine. Both ASR and CR contain similar volatile chemicals that could be altered after wine treatment. This study aims to identify the differential chemical profiles and to select marker chemicals of ASR and CR before and after wine treatment.
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Song bu li decoction, a traditional uyghur medicine, protects cell death by regulation of oxidative stress and differentiation in cultured PC12 cells.
Evid Based Complement Alternat Med
PUBLISHED: 05-21-2013
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Song Bu Li decoction (SBL) is a traditional Uyghur medicinal herbal preparation, containing Nardostachyos Radix et Rhizoma. Recently, SBL is being used to treat neurological disorders (insomnia and neurasthenia) and heart disorders (arrhythmia and palpitation). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness. Here, we aimed to evaluate the protective and differentiating activities of SBL in cultured PC12 cells. The pretreatment of SBL protected the cell against tBHP-induced cell death in a dose-dependent manner. In parallel, SBL suppressed intracellular reactive oxygen species (ROS) formation. The transcriptional activity of antioxidant response element (ARE), as well as the key antioxidative stress proteins, was induced in dose-dependent manner by SBL in the cultures. In cultured PC12 cells, the expression of neurofilament, a protein marker for neuronal differentiation, was markedly induced by applied herbal extract. Moreover, the nerve growth factor- (NGF-) induced neurite outgrowth in cultured PC12 cells was significantly potentiated by the cotreatment of SBL. In accord, the expression of neurofilament was increased in the treatment of SBL. These results therefore suggested a possible role of SBL by its effect on neuron differentiation and protection against oxidative stress.
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Synergistic Action of Flavonoids, Baicalein, and Daidzein in Estrogenic and Neuroprotective Effects: A Development of Potential Health Products and Therapeutic Drugs against Alzheimers Disease.
Evid Based Complement Alternat Med
PUBLISHED: 05-15-2013
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Despite the classical hormonal effect, estrogen has been reported to mediate neuroprotection in the brain, which leads to the searching of estrogen-like substances for treating neurodegenerative diseases. Flavonoids, a group of natural compounds, are well known to possess estrogenic effects and used to substitute estrogen, that is, phytoestrogen. Flavonoid serves as one of the potential targets for the development of natural supplements and therapeutic drugs against different diseases. The neuroprotection activity of flavonoids was chosen for a possible development of anti-Alzheimers drugs or food supplements. The estrogenic activity of two flavonoids, baicalein and daidzein, were demonstrated by their strong abilities in stimulating estrogen receptor phosphorylation and transcriptional activation of estrogen responsive element in MCF-7 breast cells. The neuroprotection effects of flavonoids against ? -amyloid (A ? ) were revealed by their inhibition effects on in vitro A ? aggregation and A ? -induced cytotoxicity in PC12 neuronal cells. More importantly, the estrogenic and neuroprotective activities of individual flavonoid could be further enhanced by the cotreatment in the cultures. Taken together, this synergistic effect of baicalein and daidzein might serve as a method to improve the therapeutic efficacy of different flavonoids against A ? , which might be crucial in developing those flavonoidsin treating Alzheimers disease in the future.
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Flavonoids induce the synthesis and secretion of neurotrophic factors in cultured rat astrocytes: a signaling response mediated by estrogen receptor.
Evid Based Complement Alternat Med
PUBLISHED: 04-23-2013
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Neurotrophic factors are playing vital roles in survival, growth, and function of neurons. Regulation of neurotrophic factors in the brain has been considered as one of the targets in developing drug or therapy against neuronal disorders. Flavonoids, a family of multifunctional natural compounds, are well known for their neuronal beneficial effects. Here, the effects of flavonoids on regulating neurotrophic factors were analyzed in cultured rat astrocytes. Astrocyte is a major secreting source of neurotrophic factors in the brain. Thirty-three flavonoids were screened in the cultures, and calycosin, isorhamnetin, luteolin, and genistein were identified to be highly active in inducing the synthesis and secretion of neurotrophic factors, including nerve growth factor (NGF), glial-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF). The inductions were in time- and dose-dependent manners. In cultured astrocytes, the phosphorylation of estrogen receptor was triggered by application of flavonoids. The phosphorylation was blocked by an inhibitor of estrogen receptor, which in parallel reduced the flavonoid-induced expression of neurotrophic factors. The results proposed the role of flavonoids in protecting brain diseases, and therefore these flavonoids could be developed for health food supplement for patients suffering from neurodegenerative diseases.
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Activation of UTP-sensitive P2Y2 receptor induces the expression of cholinergic genes in cultured cortical neurons: a signaling cascade triggered by Ca2+ mobilization and extracellular regulated kinase phosphorylation.
Mol. Pharmacol.
PUBLISHED: 04-16-2013
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ATP functions as an extracellular signaling molecule that is costored and coreleased with neurotransmitters at central and peripheral neuronal synapses. Stimulation by ATP upregulates the expression of synaptic genes in muscle-including the genes for nicotine acetylcholine receptor (?-, ?-, and ?-subunits) and acetylcholinesterase (AChE)-via the P2Y receptor (P2YR), but the trophic response of neurons to the activation of P2YRs is less well understood. We reported that cultured cortical neurons and the developing rat brain expressed different types of P2YRs, and among these the UTP-sensitive P2Y2R was the most abundant. P2Y2R was found to exist in membrane rafts and it colocalized with the postsynaptic protein PSD-95 in cortical neurons. Notably, agonist-dependent stimulation of P2Y2R elevated the neuronal expression of cholinergic genes encoding AChE, PRiMA (an anchor for the globular form AChE), and choline acetyltransferase, and this induction was mediated by a signaling cascade that involved Ca(2+) mobilization and extracellular regulated kinases 1/2 activation. The importance of P2Y2R action was further shown by the receptors synergistic effect with P2Y1R in enhancing cholinergic gene expression via the robust stimulation of Ca(2+) influx. Taken together our results revealed a developmental function of P2Y2R in promoting synaptic gene expression and demonstrated the influence of costimulation of P2Y1R and P2Y2R in neurons.
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Distribution and expression of Kirre, an IgSF molecule, during postnatal development of rat cerebellum.
Neurosci. Lett.
PUBLISHED: 03-11-2013
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Immunoglobulin superfamily (IgSF) molecules are actively involved in cell-cell adhesion, neuronal migration, axonal guidance and synapse formation in the nervous system. Kirre, as a member of this family, has been implicated in mammalian neuronal differentiation and development. Although the distribution of rKirre (a rat homologue of Drosophila Kirre) mRNA was previously analyzed in adult rat cerebellum by in situ hybridization, the expression levels of transcript and protein were not well studied. Here, we showed that the expressions of rKirre mRNA and protein significantly increased during postnatal development of rat cerebellum. rKirre mRNA was mainly expressed in the granular layers and Purkinje cell layer in the developing cerebellum, revealing a possible involvement of rKirre in granule cell migration and Purkinje cell development. An essential relationship between rKirre and Purkinje cells was implied by the co-localization of rKirre and NF-200 on the cell bodies of Purkinje cells. These results suggest that rKirre may play a potential role in postnatal developing rat cerebellum.
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Chemical and biological assessment of angelica roots from different cultivated regions in a chinese herbal decoction danggui buxue tang.
Evid Based Complement Alternat Med
PUBLISHED: 02-05-2013
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Roots of Angelica sinensis (Danggui) have been used in promoting blood circulation as herbal medicine for over 2000 years in China. Another species of Angelica roots called A. gigas is being used in Korea. To reveal the efficiency of different Angelica roots, the chemical and biological properties of Angelica roots from different cultivated regions were compared. Roots of A. sinensis contained higher levels of ferulic acid, Z-ligustilide, and senkyunolide A, while high amounts of butylphthalide and Z-butylenephthalide were found in A. gigas roots. The extracts deriving from A. gigas roots showed better effects in osteogenic and estrogenic properties than that of A. sinensis from China. However, this difference was markedly reduced when the Angelica roots were being prepared in a Chinese herbal decoction together with Astragali Radix as Danggui Buxue Tang. In contrast, the herbal decoction prepared from A. sinensis roots showed better responses in cell cultures. In addition, the extracts of A. gigas roots showed strong cell toxicity both as single herb and as Danggui Buxue Tang. This result revealed the distinct properties of Angelica roots from China and Korea suggesting the specific usage of herb in preparing a unique herbal decoction.
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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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Yu ping feng san, an ancient chinese herbal decoction containing astragali radix, atractylodis macrocephalae rhizoma and saposhnikoviae radix, regulates the release of cytokines in murine macrophages.
PLoS ONE
PUBLISHED: 01-01-2013
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Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1?2?1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-?B by enhancing I?B? degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.
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Fo Shou San, an ancient herbal decoction prepared from Angelicae Sinensis Radix and Chuanxiong Rhizoma, induces erythropoietin expression: a signaling mediated by the reduced degradation of hypoxia-inducible factor in cultured liver cells.
Planta Med.
PUBLISHED: 11-17-2011
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Fo Shou San (FSS) is an ancient herbal decoction composed of Angelicae Sinensis Radix (ASR; Danggui) and Chuanxiong Rhizoma (CR; Chuanxiong) in a ratio of 3:2. FSS is mainly prescribed for patients having a deficiency of blood supply, and it indeed has been shown to stimulate the production of erythropoietin (EPO) in cultured cells. In order to reveal the mechanism of this FSS-induced EPO gene expression, the upstream regulatory cascade, via hypoxia-induced signaling, was revealed here in cultured hepatocellular carcinoma cell line Hep3B. The induction of EPO gene expression, triggered by FSS, was revealed in cultured hepatocytes by: (i) the increase of EPO mRNA; and (ii) the activation of the hypoxia response element (HRE), an upstream regulator of the EPO gene. The FSS-induced EPO gene expression was triggered by an increased expression of hypoxia-inducible factor-1 ? (HIF-1 ?) protein; however, the mRNA expression of HIF-1 ? was not altered by the treatment of FSS. The increased HIF-1 ? was a result of reduced protein degradation after the FSS treatment. The current results therefore provide one of the molecular mechanisms of this ancient herbal decoction for its hematopoietic function.
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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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Effects of age, education and gender in the Consortium to Establish a Registry for the Alzheimers Disease (CERAD)-Neuropsychological Assessment Battery for Cantonese-speaking Chinese elders.
Int Psychogeriatr
PUBLISHED: 07-05-2011
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The Consortium to Establish a Registry for Alzheimers Disease Neuropsychological Assessment Battery (CERAD-NAB) offers information on the clinical diagnosis of Alzheimers disease (AD) and gives a profile of cognitive functioning. This study explores the effects of age, education and gender on participants performance on eight subtests in the Chinese-Cantonese version of the CERAD-NAB.
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Trillin, a steroidal saponin isolated from the rhizomes of Dioscorea nipponica, exerts protective effects against hyperlipidemia and oxidative stress.
J Ethnopharmacol
PUBLISHED: 06-10-2011
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Numerous efforts have been conducted in searching for effective agents against cardiovascular diseases, in particular from herbal medicines. The rhizome of Dioscorea nipponica (Dioscoreae Nipponicae Rhizoma) is a traditional Chinese herb being prescribed to improve the blood circulation. Here, we identified a steroidal saponin trillin from Dioscorea nipponica, which showed robust anti-hyperlipidemic effects.
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Baicalin, a flavone, induces the differentiation of cultured osteoblasts: an action via the Wnt/beta-catenin signaling pathway.
J. Biol. Chem.
PUBLISHED: 06-07-2011
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Flavonoids, a group of natural compounds found in a variety of vegetables and herbal medicines, have been intensively reported on regarding their estrogen-like activities and particularly their ability to affect bone metabolism. Here, different subclasses of flavonoids were screened for their osteogenic properties by measuring alkaline phosphatase activity in cultured rat osteoblasts. The flavone baicalin derived mainly from the roots of Scutellaria baicalensis showed the strongest induction of alkaline phosphatase activity. In cultured osteoblasts, application of baicalin increased significantly the osteoblastic mineralization and the levels of mRNAs encoding the bone differentiation markers, including osteonectin, osteocalcin, and collagen type 1?1. Interestingly, the osteogenic effect of baicalin was not mediated by its estrogenic activity. In contrast, baicalin promoted osteoblastic differentiation via the activation of the Wnt/?-catenin signaling pathway; the activation resulted in the phosphorylation of glycogen synthase kinase 3? and, subsequently, induced the nuclear accumulation of the ?-catenin, leading to the transcription activation of Wnt-targeted genes for osteogenesis. The baicalin-induced osteogenic effects were fully abolished by DKK-1, a blocker of Wnt/?-catenin receptor. Moreover, baicalin also enhanced the mRNA expression of osteoprotegerin, which could regulate indirectly the activation of osteoclasts. Taken together, our results suggested that baicalin could act via Wnt/?-catenin signaling to promote osteoblastic differentiation. The osteogenic flavonoids could be very useful in finding potential drugs, or food supplements, for treating post-menopausal osteoporosis.
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Chemical and biological assessment of Angelicae Sinensis Radix after processing with wine: an orthogonal array design to reveal the optimized conditions.
J. Agric. Food Chem.
PUBLISHED: 05-05-2011
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The roots of Angelica sinensis [Angelica Sinensis Radix (ASR)] have been used as a common health food supplement for womens care for thousands of years in China. According to Asian tradition, ASR could be processed with the treatment of wine, which subsequently promoted the biological functions of ASR. By chemical and biological assessments, an orthogonal array design was employed here to determine the roles of three variable parameters in the processing of ASR, including oven temperature, baking time, and flipping frequency. The results suggested that oven temperature and baking time were two significant factors, while flipping frequency was a subordinate factor. The optimized condition of processing with wine therefore was considered to be heating in an oven at 80 °C for 90 min with flipping twice per hour. Under the optimized processing conditions, the solubilities of ferulic acid and Z-ligustilide from ASR were markedly increased and decreased, respectively. In parallel, the biological functions of processed ASR were enhanced in both anti-platelet aggregation and estrogenic activation; these increased functions could be a result of the altered levels of ferulic acid and Z-ligustilide in wine-processed ASR. Thus, the chemical and biological assessment of the processed ASR was in full accordance with the Chinese old tradition.
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Flavonoids from Radix Astragali induce the expression of erythropoietin in cultured cells: a signaling mediated via the accumulation of hypoxia-inducible factor-1?.
J. Agric. Food Chem.
PUBLISHED: 02-10-2011
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Radix Astragali (RA) is commonly used as a health food supplement to reinforce the body vital energy. Flavonoids, including formononetin, ononin, calycosin, and calycosin-7-O-?-d-glucoside, are considered to be the major active ingredients within RA. Here, we provided different lines of evidence that the RA flavonoids stimulated the expression of erythropoietin (EPO), the central regulator of red blood cell mass, in cultured human embryonic kidney fibroblasts (HEK293T). A plasmid containing hypoxia response element (HRE), a critical regulator for EPO transcription, was tagged upstream of a firefly luciferase gene, namely, pHRE-Luc, which was being transfected into fibroblasts. The application of RA flavonoids onto the transfected cells induced the transcriptional activity of HRE. To account for the transcriptional activation after the treatment of flavonoids, the expression of hypoxia-inducible factor-1? (HIF-1?) was markedly increased: The increase was in both mRNA and protein levels. In addition, the degradation of HIF-1? was reduced under the effect of flavonoids. The regulation of HIF-1? therefore could account for the activation of EPO expression mediated by the RA flavonoids. The current results therefore reveal the function of this herb in enhancing hematopoietic functions.
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The establishment of a sensitive method in determining different neurotransmitters simultaneously in rat brains by using liquid chromatography-electrospray tandem mass spectrometry.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci.
PUBLISHED: 02-07-2011
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An effective way to determine the amount of different neurotransmitters is vital to the study of brain function. Here, a highly sensitive HPLC-MS/MS method was developed to simultaneously measure ?-aminobutyric acid, dopamine, epinephrine, norepinepherine, glutamate and serotonin in one sample. The quantification of the neurotransmitters was achieved by a tandem mass spectrometer using the selected reaction monitoring scan mode. The method validation included selectivity, linearity, accuracy, precision, stability, recovery and matrix effect. For the six neurotransmitters, the linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.991, and the limit of detection (LOD) and the limit of quantification (LOQ) were from 2.5 to 500 pg/mg and 7.5 to 1000 pg/mg, respectively. This method was employed here to reveal different types and amounts of neurotransmitters simultaneously in adult and embryonic rat brains. Here, the change of dopamine concentration in embryonic and adult brain was from 0.071 to 0.760 ng/mg of brain tissue, GABA was from 207.643 to 445.148 ng/mg, glutamate was from 679.535 to 1408.920 ng/mg, serotonin was from 0.058 to 0.485 ng/mg and norepinepherine was from 0.054 to 0.290 ng/mg. For epinephrine, it was only detected in embryonic stage but not in adult, with the concentration at 0.241 ng/mg.
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Expression of the IgSF protein Kirre in the rat central nervous system.
Life Sci.
PUBLISHED: 01-14-2011
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Immunoglobulin superfamily (IgSF) proteins play a critical role in development of the nervous system. Here, a new member of IgSF gene family was cloned from rat brain, which was subsequently identified as rat homolog of Drosophila Kirre. This new molecule was named as rat Kirre (rKirre). We aimed to reveal the developmental expression of rKirre, both at mRNA and protein levels, in the central nervous system. The deduced amino acid sequence of rKirre showed a putative PDZ binding motif at the C-terminus, which provided a rationale for analyzing the co-localization of rKirre and post-synaptic density protein 95 (PSD-95) in cultured rat cortical neurons.
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The extract of Rhodiolae Crenulatae Radix et Rhizoma induces the accumulation of HIF-1? via blocking the degradation pathway in cultured kidney fibroblasts.
Planta Med.
PUBLISHED: 12-14-2010
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Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance against hypoxia in mountain sickness. The mechanism of this adaptogenic property deriving from Rhodiola, however, has not been revealed. Erythropoietin (EPO) is an erythrocyte-specific hematopoietic hormone that increases the production of red blood cells: this hormone is a crucial factor in regulating the body balance in responding to hypoxia. In cultured kidney fibroblasts (HEK293T), application of water extract deriving from Rhodiola induced the expression of EPO both in mRNA and protein levels. The activation of the Hypoxia Response Element (HRE) located on the promoter region of the EPO gene is one of the mechanisms accounting for transcriptional activation. In addition, the Rhodiola-induced EPO expression was triggered by an increase of hypoxia-inducible factor-1 ? (HIF-1 ?) protein, via the reduction of HIF-1 ? degradation but not the induction of HIF-1 ? mRNA. Moreover, the same EPO induction effect by Rhodiola was also observed in cultured liver cells since liver is another vital organ to provide EPO regulation apart from the kidney. These results therefore elucidate one of the molecular mechanisms of this herb in mediating the anti-hypoxia function.
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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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ATP induces synaptic gene expressions in cortical neurons: transduction and transcription control via P2Y1 receptors.
Mol. Pharmacol.
PUBLISHED: 09-16-2010
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Studies in vertebrate neuromuscular synapses have revealed previously that ATP, via P2Y receptors, plays a critical role in regulating postsynaptic gene expressions. An equivalent regulatory role of ATP and its P2Y receptors would not necessarily be expected for the very different situation of the brain synapses, but we provide evidence here for a brain version of that role. In cultured cortical neurons, the expression of P2Y(1) receptors increased sharply during neuronal differentiation. Those receptors were found mainly colocalized with the postsynaptic scaffold postsynaptic density protein 95 (PSD-95). This arises through a direct interaction of a PDZ domain of PSD-95 with the C-terminal PDZ-binding motif, D-T-S-L of the P2Y(1) receptor, confirmed by the full suppression of the colocalization upon mutation of two amino acids therein. This interaction is effective in recruiting PSD-95 to the membrane. Specific activation of P2Y(1) (G-protein-coupled) receptors induced the elevation of intracellular Ca(2+) and activation of a mitogen-activated protein kinase/Raf-1 signaling cascade. This led to distinct up-regulation of the genes encoding acetylcholinesterase (AChE(T) variant), choline acetyltransferase, and the N-methyl-d-aspartate receptor subunit NR2A. This was confirmed, in the example of AChE, to arise from P2Y(1)-dependent stimulation of a human ACHE gene promoter. That involved activation of the transcription factor Elk-1; mutagenesis of the ACHE promoter revealed that Elk-1 binding at its specific responsive elements in that promoter was induced by P2Y(1) receptor activation. The combined findings reveal that ATP, via its P2Y(1) receptor, can act trophically in brain neurons to regulate the gene expression of direct effectors of synaptic transmission.
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The establishment of a highly sensitive method in detecting ketamine and norketamine simultaneously in human hairs by HPLC-Chip-MS/MS.
Forensic Sci. Int.
PUBLISHED: 08-12-2010
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An effective way to reveal the history of drug abuse is to determine the parental drug and its metabolites in hair. Here, a quantitative HPLC-Chip-MS/MS method was developed for simultaneous measurement of ketamine and its metabolite norketamine in human hair. Ketamine and norketamine were extracted from hair by acid hydrolysis, and then enriched by organic solvent extraction. The chromatographic separation was achieved in 15 min, with the drug identification and quantification by a tandem mass spectrometer. The linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.996. The limit of detection (LOD) and the limit of quantification (LOQ) for ketamine and norketamine were 0.5 and 1 pg/mg of hair, respectively. The standard curves were linear from the value of LOQ up to 100 pg/mg of hair. The validation parameters including selectivity, accuracy, precision, stability and matrix effect were also determined. In conclusion, this method was able to reveal the present of ketamine and norketamine with less hair from the drug abusers, and which had the sensitivity of ?1000-fold higher than the conventional method. In addition, the amount of ketamine and norketamine being detected in different hair segments would be useful in revealing the historical record of ketamine uptake in the drug abusers.
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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.
J. Biol. Chem.
PUBLISHED: 06-21-2010
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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.
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Antihyperlipidemic effect of protodioscin, an active ingredient isolated from the rhizomes of Dioscorea nipponica.
Planta Med.
PUBLISHED: 05-27-2010
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Developing drugs against metabolic-related disorders, including obesity and hyperlipidemia, is of importance for human health. Here, a bioactive phytochemical, protodioscin, isolated from the rhizomes of Dioscorea nipponica (Rhizoma Dioscoreae Nipponicae), was identified for its anti-hyperlipidemic effect. In hyperlipidemic rats, the post-administration of protodioscin significantly reduced the time of blood coagulation. In addition, the blood levels of triglyceride, cholesterol, low-density and high-density lipoproteins were also changed accordingly. Taken together, this was the first report of an antihyperlipidemic effect of protodioscin. Its great availability in various vegetables and medicinal plants will be useful in developing health food supplement(s) and/or drug(s) against hyperlipidemia.
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The expression of erythropoietin triggered by danggui buxue tang, a Chinese herbal decoction prepared from radix Astragali and radix Angelicae Sinensis, is mediated by the hypoxia-inducible factor in cultured HEK293T cells.
J Ethnopharmacol
PUBLISHED: 05-17-2010
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Danggui buxue tang (DBT), a Chinese medicinal decoction that is being commonly used as hematopoietic medicine to treating woman menopausal irregularity, contains two herbs: radix Astragali and radix Angelicae Sinensis. Pharmacological results indicate that DBT can stimulate the production of erythropoietin (EPO), a specific hematopoietic growth factor, in cultured cells.
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Galangin, a flavonol derived from Rhizoma Alpiniae Officinarum, inhibits acetylcholinesterase activity in vitro.
Chem. Biol. Interact.
PUBLISHED: 04-28-2010
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Acetylcholinesterase (AChE) inhibitors are widely used for the treatment of Alzheimers disease (AD). Several AChE inhibitors, e.g. rivastigmine, galantamine and huperzine are originating from plants, suggesting that herbs could potentially serve as sources for novel AChE inhibitors. Here, we searched potential AChE inhibitors from flavonoids, a group of naturally occurring compounds in plants or traditional Chinese medicines (TCM). Twenty-one flavonoids, covered different subclasses, were tested for their potential function in inhibiting AChE activity from the brain in vitro. Among all the tested flavonoids, galangin, a flavonol isolated from Rhizoma Alpiniae Officinarum, the rhizomes of Alpiniae officinarum (Hance.) showed an inhibitory effect on AChE activity with the highest inhibition by over 55% and an IC(50) of 120 microM and an enzyme-flavonoid inhibition constant (K(i)) of 74 microM. The results suggest that flavonoids could be potential candidates for further development of new drugs against AD.
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Anti-oxidative effects of the biennial flower of Panax notoginseng against H2O2-induced cytotoxicity in cultured PC12 cells.
Chin Med
PUBLISHED: 04-05-2010
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Radix notoginseng is used in Chinese medicine to improve blood circulation and clotting; however, the pharmacological activities of other parts of Panax notoginseng have yet to be explored. The present study reports the anti-oxidative effects of various parts of Panax notoginseng.
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Differential gene expression profiling on the muscle of acetylcholinesterase knockout mice: a preliminary analysis.
Chem. Biol. Interact.
PUBLISHED: 03-25-2010
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Acetylcholinesterase (AChE) (EC. 3.1.1.7) is the acetylcholine-hydrolyzing enzyme that plays an essential role on cholinergic neurotransmission at the synapses of the brain and at the neuromuscular junctions. In order to gain insight into the molecular mechanisms of neuromuscular dysfunction associated with AChE deficiency, we have compared the RNA expression profiles of the muscles of AChE knockout mice with those of the wild-type siblings. Total RNA from the leg muscle of the mice of the wild-type and the AChE nullizygous mice were subjected to microarray analyses with Affymetrix GeneChip((R)) Mouse Gene 1.0 ST Array. The pair-wise comparison of gene expression levels of the 28,853 mRNA transcripts showed that 303 genes were either up- or down-regulated by more than 2.0 folds in the AChE knockout mice. The interaction study of these differentially regulated genes indicated that some of these genes are clustered in biological functions that are related to lipid metabolism and the skeletal-muscular functions.
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Fo Shou San, an ancient herbal decoction prepared from Rhizoma Chuanxiong and Radix Angelicae Sinensis, stimulates the production of hemoglobin and erythropoietin in cultured cells.
Planta Med.
PUBLISHED: 03-22-2010
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Fo Shou San (FSS) is an ancient herbal decoction comprised of Rhizoma Chuanxiong (RC; Chuanxiong) and Radix Angelicae Sinensis (RAS; Danggui) in a ratio of 2?:?3. It is mainly prescribed for patients having a blood deficiency. This combination is considered the most popular herb pair among Chinese medicines; however, the rationale of having these two chemically similar herbs within the decoction has historically not been made clear. Here, we attempted to reveal the chemical and biological properties of this decoction as a means to deduce its mechanism of action. The effects of FSS were determined in different cell culture models. With respect to stimulation of blood circulation, FSS inhibited ADP-mediated platelet aggregation in a dose-dependent manner. In order to reveal the hematopoietic effect of this decoction, FSS was applied onto cultured K562 human leukemia cells and Hep3B human hepatocellular carcinoma cells. Application of FSS in cultured K562 cells inhibited cell proliferation and subsequently induced the production of hemoglobin. Additionally, the mRNA expression of erythropoietin (EPO) was induced in a dose-dependent manner when FSS was applied to Hep3B cells. The current results reveal the effects of FSS in different cell models, paving a direction for mechanistic studies.
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Targeting acetylcholinesterase to membrane rafts: a function mediated by the proline-rich membrane anchor (PRiMA) in neurons.
J. Biol. Chem.
PUBLISHED: 02-10-2010
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In the mammalian brain, acetylcholinesterase (AChE) is anchored in cell membranes by a transmembrane protein PRiMA (proline-rich membrane anchor). We present evidence that at least part of the PRiMA-linked AChE is integrated in membrane microdomains called rafts. A significant proportion of PRiMA-linked AChE tetramers from rat brain was recovered in raft fractions; this proportion was markedly higher at low rather than at high concentrations of cold Triton X-100. The detergent-resistant fraction increased during brain development. In NG108-15 neuroblastoma cells transfected with cDNAs encoding AChE(T) and PRiMA, PRiMA-linked G(4) AChE was found in membrane rafts and showed the same sensitivity to cold Triton X-100 extraction as in the brain. The association of PRiMA-linked AChE with rafts was weaker than that of glycosylphosphatidylinositol-anchored G(2) AChE or G(4) Q(N)-H(C)-linked AChE. It was found to depend on the presence of a cholesterol-binding motif, called CRAC (cholesterol recognition/interaction amino acid consensus), located at the junction of transmembrane and cytoplasmic domains of both PRiMA I and II isoforms. The cytoplasmic domain of PRiMA, which differs between PRiMA I and PRiMA II, appeared to play some role in stabilizing the raft localization of G(4) AChE, because the Triton X-100-resistant fraction was smaller with the shorter PRiMA II isoform than that with the longer PRiMA I isoform.
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PRiMA directs a restricted localization of tetrameric AChE at synapses.
Chem. Biol. Interact.
PUBLISHED: 02-08-2010
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Acetylcholinesterase (AChE), a highly polymorphic enzyme with various splicing variants and molecular isoforms, plays an essential role in the cholinergic neurotransmission by hydrolyzing acetylcholine into choline and acetate. The AChE(T) variant is expressed in the brain and muscle: this subunit forms non-amphiphilic tetramers with a collagen tail (ColQ) as asymmetric AChE (A(12) AChE) in muscle, and amphiphilic tetramers with a proline-rich membrane anchor (PRiMA) as globular AChE (G(4) AChE) in the brain and muscle. During the brain development, the expression of amphiphilic G(4) AChE is up regulated and becomes the predominant form of AChE there. This up-regulation of G(4) AChE can be attributed to the increased expressions of both AChE(T) and PRiMA. A significant portion of this membrane-bound G(4) AChE is localized at the membrane rafts of the cell membranes derived from the brain. This raft association could be directed by PRiMA via its CRAC (cholesterol recognition/interaction amino acid consensus) motif and C-terminus. In cultured cortical neurons and muscles, the PRiMA-linked AChE was clustered and partially co-localized with synaptic proteins. The restricted localizations suggest that the raft association of PRiMA-linked AChE could account for its synaptic localization and function.
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An induction effect of heat shock on the transcript of globular acetylcholinesterase in NG108-15 cells.
Chem. Biol. Interact.
PUBLISHED: 02-05-2010
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Heat shock response, an induced transcription of a set of genes in response to high temperature, occurs in all organisms. In neurons, the catalytic subunit of acetylcholinesterase (AChE(T)) interacts with proline-rich membrane anchor (PRiMA) to form a globular tetrameric form (G(4) form). In this study, we examined the effects of heat shock on the transcription and protein assembly of AChE(T) in cultured NG108-15 cells. The transcription of AChE(T) was rapidly induced by heat shock at 40 degrees C, reaching a 15-fold increase in 3h and decreasing thereafter. On the other hand, the level of PRiMA mRNA was not affected after the heat shock. In parallel with AChE(T) mRNA, the enzymatic activity of cellular AChE, in terms of G(1) and G(2) forms, was increased after heat shock; however, the PRiMA-linked G(4) remained unchanged. These results suggest that heat shock can induce the expression level of AChE(T) by the regulation of AChE(T) transcripts in NG108-15 cells.
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Ligustilide suppresses the biological properties of Danggui Buxue Tang: a Chinese herbal decoction composed of radix astragali and radix angelica sinensis.
Planta Med.
PUBLISHED: 10-21-2009
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Danggui Buxue Tang (DBT), a herbal decoction composed of Radix Astragali (RA) and Radix Angelica sinensis (RAS), has been used for treating menopausal irregularity in women for more than 800 years in China. According to the old tradition, RAS had to be processed with yellow wine before DBT preparation, which markedly reduced the amount of ligustilide in RAS and DBT, as well as enhanced the bioactivities of DBT. Here, we hypothesized that ligustilide would be an ingredient that possessed suppressive effects on DBTs functions. In the presence of ligustilide, the amount of astragaloside IV, calycosin, formononetin, and total polysaccharides extracted from RA were decreased. An increase of ligustilide caused a decrease of DBTs osteogenic activity in stimulating proliferation and differentiation of cultured bone cells. In addition, in the presence of a high level of ligustilide, DBT caused a side effect inducing the proliferation of breast MCF-7 cells. The current results strongly suggest that ligustilide is a negative regulator that hinders DBT to achieve its biological efficacy, which supports the traditional practice of preparing DBT using the ethanol-treated RAS.
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Expression and Localization of PRiMA-linked globular form acetylcholinesterase in vertebrate neuromuscular junctions.
J. Mol. Neurosci.
PUBLISHED: 07-06-2009
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Acetylcholinesterase (AChE) is well known to process different molecular forms via the distinct interacting partners. Proline-rich membrane anchor (PRiMA)-linked tetrameric globular AChE (G4 AChE) is mainly found in the vertebrate brain; however, recent studies from our laboratory have suggested its existence at neuromuscular junctions (nmjs). Both muscle and motor neuron express AChE at the nmjs. In muscle, the expression of PRiMA-linked AChE is down-regulated during myogenic differentiation and by motor neuron innervation. As compared with muscle, spinal cord possessed higher total AChE activity and contained PRiMA-linked AChE forms. The spinal cord expression of this form increased during development. More importantly, PRiMA-linked G4 AChE identified as aggregates localized at nmjs. These findings suggest that the restricted localization of PRiMA-linked G4 AChE at the nmjs could be contributed by the pre-synaptic motor neuron and/or the post-synaptic muscle fiber.
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Estrogenic and neuroprotective properties of scutellarin from Erigeron breviscapus: a drug against postmenopausal symptoms and Alzheimers disease.
Planta Med.
PUBLISHED: 06-16-2009
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Besides the classical hormonal effect, estrogen possesses neuroprotective effects in the brain, which leads to the searching of novel treatments for neurodegenerative diseases such as Alzheimers disease. Scutellarin is a major flavone derived from Herba Erigerontis, a Chinese medicine derived from Erigeron breviscapus, which has been shown here to possess both estrogenic and neuroprotective properties. Scutellarin showed the estrogenic effects by activating the estrogen responsive elements and phosphorylation of estrogen receptor alpha in cultured MCF-7 cells: the activation was in a dose-dependent manner. On the other hand, scutellarin inhibited the aggregation of beta-amyloid in vitro, and prevented the cell death mediated by beta-amyloid when applied to cultured neuronal PC12 cells. These results therefore suggested that Herba Erigerontis and its component scutellarin might have therapeutic effects against postmenopausal symptoms and Alzheimers disease.
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Restricted localization of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase at the neuromuscular junctions--contribution and expression from motor neurons.
FEBS J.
PUBLISHED: 04-20-2009
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The expression and localization of the proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G(4) AChE), were studied at vertebrate neuromuscular junctions. Both muscle and motor neuron contributed to this synaptic expression pattern. During the development of rat muscles, the expression of PRiMA and AChE(T) and the enzymatic activity increased dramatically; however, the proportion of G(4) AChE decreased. G(4) AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA. Using western blot and ELISA, both PRiMA protein and PRiMA-linked G(4) AChE were found to be present in large amounts in fast-twitch muscle (e.g. tibialis), but in relatively low abundance in slow-twitch muscle (e.g. soleus). These results indicate that the expression level of PRiMA-linked G(4) AChE depends on muscle fiber type. In parallel, the expression of PRiMA, AChE(T) and G(4) AChE also increased in the spinal cord during development. Such expression in motor neurons contributed to the synaptic localization of G(4) AChE. After denervation, the expression of PRiMA, AChE(T) and G(4) AChE decreased markedly in the spinal cord, and in fast- and slow-twitch muscles.
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Hibifolin, a flavonol glycoside, prevents beta-amyloid-induced neurotoxicity in cultured cortical neurons.
Neurosci. Lett.
PUBLISHED: 04-14-2009
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The toxicity of aggregated beta-amyloid (A beta) has been implicated as a critical cause in the development of Alzheimers disease (AD). Hibifolin, a flavonol glycoside derived from herbal plants, possessed a strong protective activity against cell death induced by aggregated A beta. Application of hibifolin in primary cortical neurons prevented the A beta-induced cell death in a dose-dependent manner. In cultured cortical neurons, the pre-treatment of hibifolin abolished A beta-induced Ca(2+) mobilization, and also reduced A beta-induced caspase-3 and caspase-7 activation. Moreover, DNA fragmentation induced by A beta could be suppressed by hibifolin. In addition to such protection mechanisms, hibifolin was able to induce Akt phosphorylation in cortical neurons, which could be another explanation for the neuroprotection activity. These results therefore provided the first evidence that hibifolin protected neurons against A beta-induced apoptosis and stimulated Akt activation, which would be useful in developing potential drugs or food supplements for treating AD.
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Stimulation of Apolipoprotein A-IV expression in Caco-2/TC7 enterocytes and reduction of triglyceride formation in 3T3-L1 adipocytes by potential anti-obesity Chinese herbal medicines.
Chin Med
PUBLISHED: 03-26-2009
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Chinese medicine has been proposed as a novel strategy for the prevention of metabolic disorders such as obesity. The present study tested 17 Chinese medicinal herbs were tested for their potential anti-obesity effects.
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Cordysinocan, a polysaccharide isolated from cultured Cordyceps, activates immune responses in cultured T-lymphocytes and macrophages: signaling cascade and induction of cytokines.
J Ethnopharmacol
PUBLISHED: 02-19-2009
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Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumor, anti-oxidation and stimulating the immune response; however, the identity of active component(s) is not determined. A strain of Cordyceps sinensis, namely UST 2000, has been isolated. By using activity-guided purification, a novel polysaccharide of molecular weight approximately 82 kDa was isolated from the conditioned medium of cultured Cordyceps. The isolated exo-polysaccharide, namely cordysinocan, contains glucose, mannose, galactose in a ratio of 2.4:2:1. In cultured T-lymphocytes, application of cordysinocan induced the cell proliferation and the secretion of interleukin-2, interleukin-6 and interleukin-8. In addition, the phosphorylation of extracellular signal-regulated kinases (ERK) was induced transiently by the treatment of cordysinocan. Moreover, application of cordysinocan in cultured macrophages increased the phagocytosis activity and the enzymatic activity of acid phosphatase. These results therefore verify the important role of Cordyceps polysaccharide in triggering such immune responses.
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Can Rhizoma Chuanxiong replace Radix Angelica sinensis in the traditional Chinese herbal decoction Danggui Buxue Tang?
Planta Med.
PUBLISHED: 02-09-2009
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Herein, we test the hypothesis that a member of a formulated Chinese herbal decoction cannot be replaced by another herb. Danggui Buxue Tang (DBT) is being used as an example for illustration: this is a traditional decoction containing Radix Astragali (RA) and Radix Angelicae Sinensis (RAS) in a weight ratio of 5 to 1. Rhizoma Chuanxiong (RC) and RAS are two chemically very similar herbs but with a distinct function. Following the preparation method of DBT, a herbal decoction, namely Chuanxiong Buxue Tang (CBT), was created, which contained RA and RC in a weight ratio of 5 to 1. The two decoctions, DBT and CBT, were compared in parallel regarding their chemical and biological properties. In all the tested parameters, DBT showed superior properties, both chemically and biologically, to that of CBT. The current results reveal the uniqueness of Chinese herbal decoctions that require a well-defined formulation, which is indispensable for its specific composition.
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Transcriptional regulation of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase in neuron: an inductive effect of neuron differentiation.
Brain Res.
PUBLISHED: 01-29-2009
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The transcriptional regulation of proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form of acetylcholinesterase (G(4) AChE), was revealed in cultured cortical neurons during differentiation. The level of AChE(T) protein, total enzymatic activity and the amount of G(4) AChE were dramatically increased during the neuron differentiation. RT-PCR analyses revealed that the transcript encoding PRiMA was significantly up-regulated in the differentiated neurons. To investigate the transcriptional mechanism on PRiMA regulation, a reporter construct of human PRiMA promoter-tagged luciferase was employed in this study. Upon the neuronal differentiation in cortical neurons, a mitogen-activated protein (MAP) kinase-dependent pathway was stimulated: this signaling cascade was shown to regulate the transcriptional activity of PRiMA. In addition, both PRiMA and AChE(T) transcripts were induced by the over expression of an active mutant of Raf in the cultured neurons. The treatment of a MAP kinase inhibitor (U0126) significantly blocked the expression of PRiMA transcript and promoter-driven luciferase activity as induced by the differentiation of cortical neurons. These results suggested that a MAP kinase signaling pathway served as one of the transcriptional regulators in controlling PRiMA gene expression during the neuronal differentiation process.
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A new variant of proline-rich membrane anchor (PRiMA) of acetylcholinesterase in chicken: expression in different muscle fiber types.
Neurosci. Lett.
PUBLISHED: 01-09-2009
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Proline-rich membrane anchor (PRiMA) is a molecule to organize acetylcholinesterase (AChE) into tetrameric globular form (G(4)) that anchors onto the plasma membrane in brain and muscle. In mammal, PRiMA is encoded by a single gene with two splicing variants, PRiMA I and PRiMA II: PRiMA II is different to PRiMA I by its absence of a C-terminal cytoplasmic domain. The existence of these isoforms has not been revealed in avian specie. By using RT-PCR and bioinformatic analyses, two splicing variants of PRiMA were identified in chicken cerebrum. One variant contains very similar domains as compared to mammalian PRiMA I. The other variant, named as PRiMA II, has a very distinct cytoplasmic C-terminus of having 26 amino acids. Both forms of chicken PRiMA were able to organize the formation of G(4) AChE when that was over expressed together with AChE(T) subunit in cultured cells. The level of PRiMA mRNA, mainly PRiMA I, was higher in slow-twitch muscle than that of in fast-twitch muscle of chicken. This finding suggests that the muscle fiber type-specific expression of G(4) AChE in chicken could be a result of the different expression pattern of PRiMA in fast- and slow-twitch muscles.
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Fo Shou San, an ancient Chinese herbal decoction, protects endothelial function through increasing endothelial nitric oxide synthase activity.
PLoS ONE
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Fo Shou San (FSS) is an ancient herbal decoction comprised of Chuanxiong Rhizoma (CR; Chuanxiong) and Angelicae Sinensis Radix (ASR; Danggui) in a ratio of 2:3. Previous studies indicate that FSS promotes blood circulation and dissipates blood stasis, thus which is being used widely to treat vascular diseases. Here, we aim to determine the cellular mechanism for the vascular benefit of FSS. The treatment of FSS reversed homocysteine-induced impairment of acetylcholine (ACh)-evoked endothelium-dependent relaxation in aortic rings, isolated from rats. Like radical oxygen species (ROS) scavenger tempol, FSS attenuated homocysteine-stimulated ROS generation in cultured human umbilical vein endothelial cells (HUVECs), and it also stimulated the production of nitric oxide (NO) as measured by fluorescence dye and biochemical assay. In addition, the phosphorylation levels of both Akt kinase and endothelial NO synthases (eNOS) were markedly increased by FSS treatment, which was abolished by an Akt inhibitor triciribine. Likewise, triciribine reversed FSS-induced NO production in HUVECs. Finally, FSS elevated intracellular Ca(2+) levels in HUVECs, and the Ca(2+) chelator BAPTA-AM inhibited the FSS-stimulated eNOS phosphorylation. The present results show that this ancient herbal decoction benefits endothelial function through increased activity of Akt kinase and eNOS; this effect is causally via a rise of intracellular Ca(2+) and a reduction of ROS.
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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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A standardized chinese herbal decoction, kai-xin-san, restores decreased levels of neurotransmitters and neurotrophic factors in the brain of chronic stress-induced depressive rats.
Evid Based Complement Alternat Med
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Kai-xin-san (KXS), a Chinese herbal decoction being prescribed by Sun Simiao in Beiji Qianjin Yaofang about 1400 years ago, contains Ginseng Radix et Rhizoma, Polygalae Radix, Acori tatarinowii Rhizoma, and Poria. KXS has been used to treat stress-related psychiatric disease with the symptoms of depression and forgetfulness in ancient China until today. However, the mechanism of its antidepression action is still unknown. Here, the chronic mild-stress-(CMS-) induced depressive rats were applied in exploring the action mechanisms of KXS treatment. Daily intragastric administration of KXS for four weeks significantly alleviated the CMS-induced depressive symptoms displayed by enhanced sucrose consumption. In addition, the expressions of those molecular bio-markers relating to depression in rat brains were altered by the treatment of KXS. These KXS-regulated brain biomarkers included: (i) the levels of dopamine, norepinephrine, and serotonin (ii) the transcript levels of proteins relating to neurotransmitter metabolism; (iii) the transcript levels of neurotrophic factors and their receptors. The results suggested that the anti-depressant-like action of KXS might be mediated by an increase of neurotransmitters and expression of neurotrophic factors and its corresponding receptors in the brain. Thus, KXS could serve as alternative medicine, or health food supplement, for patients suffering from depression.
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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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Isorhamnetin, A Flavonol Aglycone from Ginkgo biloba L., Induces Neuronal Differentiation of Cultured PC12 Cells: Potentiating the Effect of Nerve Growth Factor.
Evid Based Complement Alternat Med
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Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, share a chemical resemblance to estrogen, and indeed some of which have been used as estrogen substitutes. In searching for possible functions of flavonoids, the neuroprotective effect in brain could lead to novel treatment, or prevention, for neurodegenerative diseases. Here, different subclasses of flavonoids were analyzed for its inductive role in neurite outgrowth of cultured PC12 cells. Amongst the tested flavonoids, a flavonol aglycone, isorhamnetin that was isolated mainly from the leaves of Ginkgo biloba L. showed robust induction in the expression of neurofilament, a protein marker for neurite outgrowth, of cultured PC12 cells. Although isorhamnetin by itself did not show significant inductive effect on neurite outgrowth of cultured PC12 cells, the application of isorhamnetin potentiated the nerve growth factor- (NGF-)induced neurite outgrowth. In parallel, the expression of neurofilaments was markedly increased in the cotreatment of NGF and isorhamnetin in the cultures. The identification of these neurite-promoting flavonoids could be very useful in finding potential drugs, or food supplements, for treating various neurodegenerative diseases, including Alzheimers disease and depression.
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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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Er Zhi Wan, an ancient herbal decoction for woman menopausal syndrome, activates the estrogenic response in cultured MCF-7 cells: an evaluation of compatibility in defining the optimized preparation method.
J Ethnopharmacol
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Er Zhi Wan (EZW), a Chinese medicinal preparation, has been used clinically for treating menopausal syndrome for its kidney-invigorating function, which contains simply two herbs, Ecliptae Herba (EH) and Ligustri Lucidi Fructus (LLF). Although this herbal extract has been used for many years, there is no scientific basis about its effectiveness on menopausal symptom. Here, we aimed to evaluate the estrogenic activities of EZW and to study the compatibilities of two herbs including different processed-LLF in single and mixed preparation of EZW. Moreover, the weight ratio of EH to LLF in EZW was determined according to their estrogenic activities.
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The membrane permeability of Astragali Radix-derived formononetin and calycosin is increased by Angelicae Sinensis Radix in Caco-2 cells: a synergistic action of an ancient herbal decoction Danggui Buxue Tang.
J Pharm Biomed Anal
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Danggui Buxue Tang (DBT), a herbal decoction contains Astragali Radix (AR) and Angelicae Sinensis Radix (ASR), has been used as a health food supplement in treating menopausal irregularity in women for more than 800 years in China. Several lines of evidence indicate that the synergistic actions of AR and ASR in this herbal decoction leading to a better pharmacological effect of DBT. Here, the role of different herbs in directing the transport of active ingredients of DBT was determined. A validated RRLC-QQQ-MS/MS method was applied to determinate the permeability of ingredients across the Caco-2 cell monolayer. AR-derived chemicals, including astragaloside IV, calycosin and formononetin, as well as ASR-derived chemicals, including ferulic acid and ligustilide, were determined by RRLC-QQQ-MS/MS. The pharmacokinetic results showed that the membrane permeabilities of calycosin and formononetin, two of the major flavonoids in AR, could be markedly increased in the presence of ASR extract: this induction effect could be mediated by ferulic acid deriving from ASR. In contrast, the extract of AR showed no effect on the chemical permeability. The current results suggested that the ingredients of ASR (such as ferulic acid) could enhance the membrane permeability of AR-derived formononetin and calycosin in cultured Caco-2 cells. The possibility of herb-drug synergy within DBT was proposed here.
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Kaempferol as a flavonoid induces osteoblastic differentiation via estrogen receptor signaling.
Chin Med
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Flavonoids, a group of compounds mainly derived from vegetables and herbal medicines, chemically resemble estrogen and some have been used as estrogen substitutes. Kaempferol, a flavonol derived from the rhizome of Kaempferia galanga L., is a well-known phytoestrogen possessing osteogenic effects that is also found in a large number of plant foods.The herb K. galanga is a popular traditional aromatic medicinal plant that is widely used as food spice and in medicinal industries. In the present study, both the estrogenic and osteogenic properties of kaempferol are evaluated.
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Salidroside stimulates the accumulation of HIF-1? protein resulted in the induction of EPO expression: a signaling via blocking the degradation pathway in kidney and liver cells.
Eur. J. Pharmacol.
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Rhodiolae Crenulatae Radix et Rhizoma (Rhodiola), the root and rhizome of Rhodiola crenulata (Hook. f. et Thoms.) H. Ohba, has been used as a traditional Chinese medicine (TCM) to increase the body resistance to mountain sickness in preventing hypoxia; however, the functional ingredient responsible for this adaptogenic effect has not been revealed. Here, we have identified salidroside, a glycoside predominantly found in Rhodiola, is the chemical in providing such anti-hypoxia effect. Cultured human embryonic kidney fibroblast (HEK293T) and human hepatocellular carcinoma (HepG2) were used to reveal the mechanism of this hematopoietic function mediated by salidroside. The application of salidroside in cultures induced the expression of erythropoietin (EPO) mRNA from its transcription regulatory element hypoxia response element (HRE), located on EPO gene. The application of salidroside stimulated the accumulation of hypoxia-inducible factor-1? (HIF-1?) protein, but not HIF-2? protein: the salidroside-induced HIF-1? protein was via the reduction of HIF-1? degradation but not the mRNA induction. The increased HIF-1? could account for the activation of EPO gene. These results supported the notion that hematopoietic function of Rhodiola was triggered, at least partially, by salidroside.
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Microfluidic chip based nano liquid chromatography coupled to tandem mass spectrometry for the determination of abused drugs and metabolites in human hair.
Anal Bioanal Chem
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A microfluidic chip based nano-HPLC coupled to tandem mass spectrometry (nano-HPLC-Chip-MS/MS) has been developed for simultaneous measurement of abused drugs and metabolites: cocaine, benzoylecgonine, cocaethylene, norcocaine, morphine, codeine, 6-acetylmorphine, phencyclidine, amphetamine, methamphetamine, MDMA, MDA, MDEA, and methadone in the hair of drug abusers. The microfluidic chip was fabricated by laminating polyimide films and it integrated an enrichment column, an analytical column and a nanospray tip. Drugs were extracted from hairs by sonication, and the chromatographic separation was achieved in 15 min. The drug identification and quantification criteria were fulfilled by the triple quardropule tandem mass spectrometry. The linear regression analysis was calibrated by deuterated internal standards with all of the R(2) at least over 0.993. The limit of detection (LOD) and the limit of quantification (LOQ) were from 0.1 to 0.75 and 0.2 to 1.25 pg/mg, respectively. The validation parameters including selectivity, accuracy, precision, stability, and matrix effect were also evaluated here. In conclusion, the developed sample preparation method coupled with the nano-HPLC-Chip-MS/MS method was able to reveal the presence of drugs in hairs from the drug abusers, with the enhanced sensitivity, compared with the conventional HPLC-MS/MS.
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