RESUMEN
Cordyceps sinensis, a well-known traditional Chinese medicine, possesses activities in anti-tumour, anti-oxidation and stimulating the immune system; however, the identity of active component(s) is not determined. By using anti-oxidation activity-guided fractionation, a polysaccharide of molecular weight approximately 210 kDa was isolated from cultured Cordyceps mycelia by ion-exchange and sizing chromatography. The isolated polysaccharide, having strong anti-oxidation activity, contains glucose, mannose and galactose in a ratio of 1 : 0.6 : 0.75. The pre-treatment of isolated polysaccharide on the cultured rat pheochromocytoma PC12 cells shows strong protective effect against hydrogen peroxide (H(2)O(2))-induced insult. Treatment of the cells with the isolated polysaccharide at 100 microg/ml prior to H(2)O(2) exposure significantly elevated the survival of PC12 cells in culture by over 60%. In parallel, the H(2)O(2)-induced production of malondialdehyde in cultured cells was markedly reduced by the polysaccharide treatment. Moreover, the pre-treatment of the isolated polysaccharide significantly attenuated the changes of glutathione peroxidase and superoxide dismutase activities in H(2)O(2)-treated cells in a dose-dependent manner. This is the first report in identifying a polysaccharide from Cordyceps, which protects against the free radical-induced neuronal cell toxicity.
Asunto(s)
Cordyceps/química , Peróxido de Hidrógeno/toxicidad , Medicina Tradicional China , Polisacáridos/farmacología , Sustancias Protectoras/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Electroforesis Capilar , Glutatión Peroxidasa/metabolismo , Malondialdehído/metabolismo , Células PC12 , Polisacáridos/aislamiento & purificación , Sustancias Protectoras/aislamiento & purificación , Ratas , Superóxido Dismutasa/metabolismoRESUMEN
A cDNA encoding P2Y(1) receptor was isolated by cross-hybridization with chicken homolog. The deduced amino acid sequence of P2Y(1) receptor with 361 amino residues is 80-85% identical to human, rodent and avian homologs. When the cDNA was expressed in mammalian cells, the activation of P2Y(1) receptor by adenine nucleotides stimulated the accumulation of inositol phosphate, and adenosine 3',5'-bismonophosphate (A3P5P) or other antagonists blocked its action; these pharmacological properties showed resemblance of P2Y(1) receptor family in higher vertebrate. A transcript encoding P2Y(1) receptor at approximately 3.2 kb was revealed in the brain, spinal cord and muscle of adult, and it is strongly expressed in developing brain, spinal cord and myotomal muscles of the embryos by hybridization. P2Y(1) receptor was shown to be restricted to the neuromuscular junctions and co-localized with AChRs in adult muscle. These results support the notion that ATP and its P2Y(1) receptor subtype are effectors in organizing the post-synaptic apparatus.
Asunto(s)
ADN Complementario/genética , Unión Neuromuscular/metabolismo , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Xenopus/genética , Xenopus/metabolismo , Secuencia de Aminoácidos/genética , Animales , ADN Complementario/aislamiento & purificación , Inmunohistoquímica/métodos , Hibridación in Situ , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Receptores Purinérgicos P2Y1 , Coloración y EtiquetadoRESUMEN
The expression of acetylcholinesterase (AChE) is markedly increased during myogenic differentiation of C2C12 myoblasts to myotubes; the expression is mediated by intrinsic factor(s) during muscle differentiation. In order to analyze the molecular mechanisms regulating AChE expression during myogenic differentiation, a approximately 2.2-kb human AChE promoter tagged with a luciferase reporter gene, namely pAChE-Luc, was stably transfected into C2C12 cells. The profile of promoter-driven luciferase activity during myogenic differentiation of C2C12 myotubes was found to be similar to that of endogenous expression of AChE catalytic subunit. The increase of AChE expression was reciprocally regulated by a cAMP-dependent signaling pathway. The level of intracellular cAMP, the activity of cAMP-dependent protein kinase, the phosphorylation of cAMP-responsive element binding protein and the activity of cAMP- responsive element (CRE) were down-regulated during the myotube formation. Mutating the CRE site of human AChE promoter altered the original myogenic profile of the promoter activity and its suppressive response to cAMP. In addition, the suppressive effect of the CRE site is dependent on its location on the promoter. Therefore, our results suggest that a cAMP-dependent signaling pathway serves as a suppressive element in regulating the expression of AChE during early myogenesis.