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.

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.