Anxiety and depression with neurogenesis defects in exchange protein directly activated by cAMP 2-deficient mice are ameliorated by a selective serotonin reuptake inhibitor, Prozac.

Intracellular cAMP and serotonin are important modulators of anxiety and depression. Fluoxetine, a selective serotonin reuptake inhibitor (SSRI) also known as Prozac, is widely used against depression, potentially by activating cAMP response element-binding protein (CREB) and increasing brain-derived neurotrophic factor (BDNF) through protein kinase A (PKA). However, the role of Epac1 and Epac2 (Rap guanine nucleotide exchange factors, RAPGEF3 and RAPGEF4, respectively) as potential downstream targets of SSRI/cAMP in mood regulations is not yet clear. Here, we investigated the phenotypes of Epac1 (Epac1(-/-)) or Epac2 (Epac2(-/-)) knockout mice by comparing them with their wild-type counterparts. Surprisingly, Epac2(-/-) mice exhibited a wide range of mood disorders, including anxiety and depression with learning and memory deficits in contextual and cued fear-conditioning tests without affecting Epac1 expression or PKA activity. Interestingly, rs17746510, one of the three single-nucleotide polymorphisms (SNPs) in RAPGEF4 associated with cognitive decline in Chinese Alzheimer's disease (AD) patients, was significantly correlated with apathy and mood disturbance, whereas no significant association was observed between RAPGEF3 SNPs and the risk of AD or neuropsychiatric inventory scores. To further determine the detailed role of Epac2 in SSRI/serotonin/cAMP-involved mood disorders, we treated Epac2(-/-) mice with a SSRI, Prozac. The alteration in open field behavior and impaired hippocampal cell proliferation in Epac2(-/-) mice were alleviated by Prozac. Taken together, Epac2 gene polymorphism is a putative risk factor for mood disorders in AD patients in part by affecting the hippocampal neurogenesis.

Nerve growth factor-induced differentiation of PC12 cells is accompanied by elevated adenylyl cyclase activity.

Rat pheochromocytoma (PC12) cells characteristically undergo differentiation when cultured with nerve growth factor (NGF). Here we show that NGF dramatically increased the adenylyl cyclase-activating property of forskolin in PC12 cells. This effect of NGF was well maintained even when NGF was removed after 4 days, even though the morphological features of neuronal differentiation were rapidly lost on removal of NGF. The enhanced cAMP production in response to forskolin could be due to a synergistic interaction between forskolin and endogenously released agonists acting on G(s)-coupled receptors. However, responses to forskolin were not attenuated by antagonists of adenosine A2 receptors or pituitary adenylate cyclase-activating polypeptide (PACAP) receptors, suggesting that adenosine and PACAP were not involved. Adenylyl cyclases 3, 6 and 9 were the predominant isoforms expressed in PC12 cells, but we found no evidence for NGF-induced changes in expression levels of any of the 9 adenylyl cyclase isoforms, nor in the expression of Gα(s). These findings highlight that NGF has a subtle influence on adenylyl cyclase activity in PC12 cells which may influence more than the neurite extension process classically associated with neuronal differentiation.

In vitro estrogenic activity of formononetin by two bioassay systems.

AIM: To study the estrogenic activity of formononetin in vitro. METHODS: We have established a highly sensitive bioassay system by placing estrogen-responsive elements upstream of the luciferase reporter gene, and used this assay to determine the estrogenic activity of formononetin. Cell growth was measured by the MTT (3-(4,5-dimethylthioazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and MG-63 cell function was studied by measuring alkaline phosphatase activity. RESULTS: Formononetin activated expression of the estrogen-responsive reporter gene in human breast cell line MCF-7 in a concentration-dependent manner (0.5-500 microM), and this activation was inhibited by estrogen antagonist (ICI 182780 at 100 nM). Furthermore, it induced the proliferation of MCF-7 breast cancer cells and MG-63 osteosarcoma cells, and it also increased the alkaline phosphatase activity in MG-63 cells. CONCLUSION: Formononetin is a phytoestrogen that exhibits variable degrees of estrogen receptor agonism in different test systems.

Hypoglycemic activity of polysaccharide, with antioxidation, isolated from cultured Cordyceps mycelia.

Cordyceps sinensis, a well-known traditional Chinese medicine, possesses anti-tumor, immunostimulant and antioxidant activities; however, the identities of active components have not been determined. In our previous study using antioxidant activity-guided fractionation [Li et al., 2003. A polysaccharide isolated from Cordyceps sinensis, a traditional Chinese medicine, protects PC12 cells against hydrogen peroxide-induced injury. Life Sci. 73, 2503-2513], a polysaccharide of molecular weight approximately 210kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, named CSP-1, which has strong anti-oxidation activity, contains glucose, mannose and galactose in the ratio of 1:0.6:0.75. In the present study, we demonstrated the hypoglycemic effect of CSP-1 on normal and alloxan-diabetic mice and streptozotocin (STZ)-diabetic rats. The basal glucose level did not differ significantly among the normal mice. CSP-1 (at 200 and 400mg/kg body wt./day for 7 days, p.o.), however, significantly reduced the blood glucose level by 12.0+/-3.2% and 22.5+/-4.7% in normal mice, respectively (p<0.05). When administered at a dose of higher than 200mg/kg body wt. daily for 7 days, CSP-1 produced a significant drop in blood glucose level in both STZ-induced diabetic rats and alloxan-induced diabetic mice. The serum insulin levels in diabetic animals were also increased by administration of CSP-1 (p<0.05). CSP-1 with hypoglycemic properties increased circulating insulin level in diabetic animals, which suggests that CSP-1 may stimulate pancreatic release of insulin and/or reduce insulin metabolism.

Ginsenoside Re attenuate beta-amyloid and serum-free induced neurotoxicity in PC12 cells.

AIM: To study the effect of ginsenoside Re on PC12 cell damage induced by serum deprivation and beta-amyloid peptide. METHODS: PC 12 cell survival was measured by MTT and lactate dehydrogenase (LDH) assay. Results Serum-free medium and beta-amyloid peptide (10-100 microM) induced cytotoxicity in PC 12 cells. Ginsenoside Re (0.1-100 microM) attenuated the cytotoxic effects of serum-free medium and beta-amyloid peptide (50 microM) in a concentration-dependent manner. CONCLUSION: Ginsenoside Re prevented PC 12 cells from lesion induced by serum-free medium and beta-amyloid peptide.

Orthogonal array design in optimizing the extraction efficiency of active constituents from roots of Panax notoginseng.

The root of Panax notoginseng (Radix Notoginseng, Sanqi) is a commonly used traditional Chinese medicine, which is mainly cultivated in Wenshan of Yunnan China. The identified active constituents in Radix Notoginseng include saponin, ssavonoid and polysaccharide; however, the levels of these active constituents vary greatly with different extraction processes. This variation causes a serious problem in standardizing the herbal extract. By using HPLC and spectrophotometry, the contents of notoginsenoside R(1), ginsenoside R(g1), R(b1), R(d), and ssavonoids were determined in the extracts of Radix Notoginseng that were derived from different processes of extraction according to an orthogonal array experimental design having three variable parameters: nature of extraction solvent, extraction volume and extraction time. The nature of extraction solvent and extraction volume were two distinct factors in obtaining those active constituents, while the time of extraction was a subordinate factor. The optimized condition of extraction therefore is considered to be 20 volumes of water and extracted for 24 h. In good agreement with the amount of active constituents, the activity of anti-platelet aggregation was found to be the highest in the extract that contained a better yield of the active constituents. The current results provide an optimized extraction method for the quality control of Radix Notoginseng.

Distinction of water-soluble constituents between natural and cultured Cordyceps by capillary electrophoresis.

Cordyceps is an expensive traditional Chinese medicine, which has anti-tumor activity and significant effects on the immune system. In Southeast Asia, Cordyceps is commonly sold in capsule form as a health food product. Most of these products are derived from cultured Cordyceps mycelia. Because of the price difference, some manufacturers claim their products are from natural Cordyceps. In order to distinguish among various types of Cordyceps in the market, the profiles of water-soluble constituents derived from different sources of Cordyceps were determined by capillary electrophoresis (CE). Both natural and cultured Cordyceps showed three peak clusters migrated at 5-7, 9-11 and 12-13 min, and the height and resolution of these peak clusters were rather distinct. Peak cluster at 9-11 min was identified as adenosine, guanosine and uridine, and shared a similarity between natural and cultured products. In contrast, the peak cluster at 5-7 min was characteristic of natural Cordyceps, regardless of hosts and sources. By using the peak characteristics of CE profiles of different Cordyceps samples, hierarchical clustering analysis was performed. The result shows that those samples of natural Cordyceps were grouped together distinct from the cultured and commercial products. Thus, the CE profiles could serve as fingerprints for the quality control of Cordyceps.

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis.

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.

Genetic distinction of radix adenophorae from its adulterants by the DNA sequence of 5S-rRNA spacer domains.

Radix Adenophorae (Shashen), a traditional Chinese medicine commonly used as an antitussive and expectorant, is derived from roots of Adenophora stricta Miq. and Adenophora tetraphylla (Thunb.) Fisch. Twelve species and varieties of Adenophora and Glehnia, however, could act as substitutes or adulterants of Radix Adenophorae on the commercial markets in South East Asia, and roots of Adenophora hunanensis Nannf. and Glihnia littoralis F. Schmidt ex Miq. are the most common examples. The authentic identification of dried roots of A. stricta and A. tetraphylla, however, is difficult on the basis of appearance and morphology. A molecular genetic approach was developed here to identify the species of Radix Adenophorae. The 5S-rRNA spacer domains (approximately 250 bp) were amplified by the polymerase chain reaction (PCR) from genomic DNAs isolated from A. stricta, A. tetraphylla, A. hunanensis and G. littoralis, and subsequently, the nucleotide sequences were determined. Diversity in DNA sequence and restriction enzyme mapping among various species were found in their 5S-rRNA spacer domains, which could serve as markers for authentic identification of Radix Adenophorae.

The fruiting body and its caterpillar host of Cordyceps sinensis show close resemblance in main constituents and anti-oxidation activity.

Cordyceps (summer-grass, winter-worm), one of the most valued traditional Chinese medicines, is used commonly for the replenishment of body health. It consists of the dried fungus Cordyceps sinensis growing on caterpillar larvae. For medication, the fruiting body (fungus) and the worm (caterpillar) are used together. However, the pharmacological efficiency and the main constituents of the individual parts have not been determined. In the present study the water extracts from the fruiting body and worm of natural Cordyceps were analyzed for their content of nucleosides and polysaccharides; the results showed that the worm had chemical composition similar to the fruiting body. In addition, both the fruiting body and worm of Cordyceps showed similar potency in their anti-oxidation activities in the xanthine oxidase assay, the induction of hemolysis assay and the lipid-peroxidation assay. These results suggest that the function of the worm in Cordyceps is to provide a growth medium for the fruiting body, and that eventually, the worm is totally invaded by C. sinensis mycelia.

Expression of the P2Y1 nucleotide receptor in chick muscle: its functional role in the regulation of acetylcholinesterase and acetylcholine receptor.

In vertebrate neuromuscular junctions, ATP is stored at the motor nerve terminals and is co-released with acetylcholine during neural stimulation. Here, we provide several lines of evidence that the synaptic ATP can act as a synapse-organizing factor to induce the expression of acetylcholinesterase (AChE) and acetylcholine receptor (AChR) in muscles, mediated by a metabotropic ATP receptor subtype, the P2Y(1) receptor. The activation of the P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphates and intracellular Ca(2+) mobilization in cultured chick myotubes. P2Y(1) receptor mRNA in chicken muscle is very abundant before hatching and again increases in the adult. The P2Y(1) receptor protein is shown to be restricted to the neuromuscular junctions and colocalized with AChRs in adult muscle (chicken, Xenopus, and rat) but not in the chick embryo. In chicks after hatching, this P2Y(1) localization develops over approximately 3 weeks. Denervation or crush of the motor nerve (in chicken or rat) caused up to 90% decrease in the muscle P2Y(1) transcript, which was restored on regeneration, whereas the AChR mRNA greatly increased. Last, mRNAs encoding the AChE catalytic subunit and the AChR alpha-subunit were induced when the P2Y(1) receptors were activated by specific agonists or by overexpression of P2Y(1) receptors in cultured myotubes; those agonists likewise induced the activity in the myotubes of promoter-reporter gene constructs for those subunits, actions that were blocked by a P2Y(1)-specific antagonist. These results provide evidence for a novel function of ATP in regulating the gene expression of those two postsynaptic effectors.

Chips and Qi: microcomponent-based analysis in traditional Chinese medicine.

Over the last 50 years or so Traditional Chinese medicine (TCM) has been subject to intensive basic and clinical research. Although the effectiveness and remarkable safety of TCM have been documented after controlled clinical studies, there are several herbal and animal parts that are toxic or difficult to identify. DNA polymorphism-based assays have recently been developed for the identification of herbal medicines. In this approach, small amounts of DNA are amplified by the polymerase chain reaction and the reactions products are analyzed by gel electrophoresis, sequencing, or hybridization with species-specific probes. With the DNA based identification of TCM materials as an example, chip-based analytical micro devices were developed with the goal of fabricating an integrated device that will enable sample preparation, amplification, and analysis on a single microchip-based device ("lab-on-a-chip"). The application of a silicon-based polymerase chain reaction microreactor and a DNA microarray for the DNA sequence-based identification of toxic medicinal plants is reported here.

Anti-oxidation activity of different types of natural Cordyceps sinensis and cultured Cordyceps mycelia.

Cordyceps, one of the well-known traditional Chinese medicines, consists of the dried fungus Cordyceps sinensis growing on the larva of the caterpillar. It is commonly used for the replenishment of body health. One of the known pharmacological effects is its anti-oxidation activity. However, there is a great variation of the quality in different sources of Cordyceps. Here, the water extracts of various sources of natural C. sinensis and cultured Cordyceps mycelia were analyzed for their anti-oxidation activity by using three different assay methods such as the xanthine oxidase assay, the induction of hemolysis assay and the lipid peroxidation assay. The results showed that Cordyceps, in general, possesses a strong anti-oxidation activity in all assays tested. However, both natural and cultured Cordyceps showed the lowest inhibition in the lipid peroxidation when compared with the other two assay methods. The cultured Cordyceps mycelia had equally strong anti-oxidation activity as compared to the natural Cordyceps. Besides, the anti-oxidation activities were increased to 10-30 folds in the partially purified polysaccharide fractions from the cultured Cordyceps mycelia, which suggested that the activity could be derived partly from Cordyceps polysaccharides.

The cyclic AMP-mediated expression of acetylcholinesterase in myotubes shows contrasting activation and repression between avian and mammalian enzymes.

Cyclic adenosine 3',5'-monophosphate (cAMP)-dependent signalling pathway has been proposed to regulate acetylcholinesterase (AChE) expression in chick muscle; however, its role in mammalian enzyme is not known. We provide several lines of evidence to suggest that the cAMP-mediated AChE expression in myotube is oppositely regulated between avian and mammalian enzymes. Human AChE promoter was tagged with luciferase, namely Hp-Luc, which was transfected into cultured chick myotubes. Application of cAMP and forskolin induced the expression of chick AChE but reduced human AChE promoter-driven luciferase activity. Transfection of cDNAs encoding active mutants of G proteins altered the intracellular cAMP level in myotubes as well as the expression of chick and human AChE. When the constitutively active forms of Activating Transcription Factor-1 (EWS/ATF-1 oncogene) were over expressed in Hp-Luc transfected myotubes, the expression of chick AChE transcript and protein increased from approximately 1.8- to approximately 2.5-fold, but the luciferase activity was decreased by over 60%. Overexpression of cAMP-responsive element binding protein (CREB) in Hp-Luc transfected myotubes markedly enhanced the cAMP-mediated AChE expression in up- and downregulated chick and human enzymes, respectively. In addition, CREB bound the CRE sequence of human AChE promoter. Mutation on the CRE site markedly enhanced the expression of the promoter-driven luciferase; however, its response to cAMP inhibition in cultured myotubes was still retained. These findings suggest that a cAMP-dependent pathway is contrasting activation and repression of AChE expression in chick and human muscles.

Authentic identification of stigma Croci (stigma of Crocus sativus) from its adulterants by molecular genetic analysis.

Stigma Croci, stigma of Crocus sativus L., is a precious traditional Chinese medicine, which is commonly used to activate blood circulation and to dissipate blood stasis. Three plant species, Carthamus tinctorius L., Hemerocallis fulva (L.) L. and Hemerocallis citrina Baroni, could carry the name Stigma Croci in the commercial markets of South East Asia. However, C. sativus is the only one that has proven its effectiveness, while the others could act as adulterants. The authentic identification of C. sativus on the market is difficult. By using molecular genetic method, the spacer domains of 5S-rRNA were cloned from the genomic DNAs that were isolated from C. sativus, C. tinctorius, H. fulva and H. citrina. The cDNAs encoding the spacer domains, about 300 to 500 bp, were sequenced. The nucleotide sequences of these four species showed great diversity, which could serve as markers for authentic identification of Stigma Croci to distinguish from its substitution and counterfeit.

Cdk5 is involved in neuregulin-induced AChR expression at the neuromuscular junction.

Here we describe an important involvement of Cdk5/p35 in regulating the gene expression of acetylcholine receptor (AChR) at the neuromuscular synapse. Cdk5 and p35 were prominently expressed in embryonic muscle, and concentrated at the neuromuscular junction in adulthood. Neuregulin increased the p35-associated Cdk5 kinase activity in the membrane fraction of cultured C2C12 myotubes. Co-immunoprecipitation studies revealed the association between Cdk5, p35 and ErbB receptors in muscle and cultured myotubes. Inhibition of Cdk5 activity not only blocked the NRG-induced AChR transcription, but also attenuated ErbB activation in cultured myotubes. In light of our finding that overexpression of p35 alone led to an increase in AChR promoter activity in muscle, Cdk5 activation is sufficient to mediate the up-regulation of AChR gene expression. Taken together, these results reveal the unexpected involvement of Cdk5/p35 in neuregulin signaling at the neuromuscular synapse.

Determination of nucleosides in natural Cordyceps sinensis and cultured Cordyceps mycelia by capillary electrophoresis.

Cordyceps sinensis is a well-known traditional Chinese medicine, and some of the active components are nucleosides. The analysis of nucleosides in Cordyceps material has been performed by reversed-phase high-performance liquid chromatography (HPLC) with gradient elution or by spectrometry. Here, we have explored the possibility of using capillary electrophoresis to determine the content of three major nucleosides (adenosine, guanosine and uridine) in Cordyceps. Capillary electrophoresis needs no gradients, and it provides a better separation due to its higher efficiency. In order to optimize the resolution, the separation of adenosine, guanosine and uridine was determined in Cordyceps with respect to the variation of buffer concentration, pH, temperature, and voltage. By using the calibrated electrophoresis system, the separation was achieved for the three nucleosides in less than 10 min with a background electrolyte consisting of 0.2 M boric acid-sodium hydroxide buffer, pH 8.5. The nucleoside contents of various types of natural Cordyceps and cultured Cordyceps mycelia were determined and compared. There was a great variation of nucleoside content in different sources of Cordyceps; the cultured Cordyceps mycelia, however, contains a much higher concentration than the natural Cordyceps.

[The contents and their change of nucleosides from natural Cordyceps sinensis and cultured cordyceps mycelia].

AIM: To compare the content of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia, and the effect of humidity and heat on the contents of nucleosides. METHODS: The contents of nucleosides were determined using high performance capillary electrophoresis (HPCE). Beckman P/ACE System 5010 apparatus equipped with a UV detector and a Beckman untreated fused-silica capillary (57 cm x 75 microns, 50 cm effective length) were used. Before sample injection, the capillary was rinsed with 1 mol.L-1 sodium hydroxide and running buffer for 5 minutes, respectively. 20 kV was applied during separation. Pressure injection was 586 kPa for 6 seconds, and the wavelength of detector was 254 nm. The running time was 20 minutes at 20 degrees C. The effect of humidity and heat on the content of nucleosides from natural Cordyceps sinensis and cultured Cordyceps mycelia was observed for 1, 3, 5 and 10 days at temperature 40 degrees C, and relative humidity 75%. RESULTS: The contents of nucleosides from cultured Cordyceps mycelia were higher than that of those from natural Cordyceps sinensis. The contents of nucleosides from freshly collected natural Cordyceps sinensis, were under the detection limit. The contents of nucleosides from natural Cordyceps sinensis were significantly increased by effect of humidity and heat, the phenomenon was not observed in cultured Cordyceps mycelia. CONCLUSION: There are difference in nucleosides content between natural Cordyceps sinensis and cultured Cordyceps mycelia. The nucleosides in natural Cordyceps sinensis may be derived from the degradation of macro-molecular nucleic acids. That means it is probably controversial that adenosine was used for quality control of natural Cordyceps sinensis.

Immunoglobulin G1 Fc fragment-tagged human opioid receptor-like receptor retains the ability to inhibit cAMP accumulation.

The human opioid receptor-like (ORL(1)) receptor was tagged with the immunoglobulin G1 Fc fragment at the carboxy-terminus and expressed in human embryonic kidney 293 cells. Expression of the ORL(1)-Fc receptor was confirmed by immunohistochemical staining. The fusion protein was enriched by affinity chromatography and then verified by immunodetection. The function of the ORL(1)-Fc receptor was determined by examining nociceptin/OFQ-induced inhibition of cAMP accumulation. The ORL(1)-Fc receptor inhibited the forskolin-stimulated cAMP accumulation. The inhibitory response was selectively induced by nociceptin/OFQ in a dose-dependent and pertussis toxin-sensitive manner. Our results indicate that the carboxy-terminal Fc-tagged ORL(1) receptor retained the ability to interact with G(i) proteins to inhibit adenylyl cyclase.

Species identification of Radix Astragali (Huangqi) by DNA sequence of its 5S-rRNA spacer domain.

About 300 species and varieties of Astragalus are identified in China, making the identification of the origin of a particular Astragalus species on the consumer market difficult. A molecular genetic approach was developed to identify various species of Astragalus. Although the 5S-rRNA coding sequence is conserved in higher eukaryotes, the spacer domain of the 5S-rRNA gene has great diversity among different species. The 5S-rRNA spacer domain was amplified by polymerase chain reaction (PCR) from the isolated genomic DNA, and the PCR products (approximately 300 bp) covering the 5S-rRNA spacer domain were sequenced. The nucleotide sequences of Astragalus membranaceus, A. membranaceus var. mongholicus, A. lehmannianus, A. hoantchy, and of one closely related species Hedysarum polybotrys (Hongqi), were determined. Diversity in DNA sequence and restriction enzyme mapping among various species was found in their 5S-rRNA spacer domains. This is the first report on the detection of 5S-rRNA spacer region sequence of Astragalus, and the results could be used for genetic identification of Huangqi.