Protective effect of the hooks and stems of Uncaria sinensis against nitric oxide donor-induced neuronal death in cultured cerebellar granule cells

We have previously shown that an aqueous extract of the hooks and stems of Uncaria sinensis (O_.) H_., Uncariae Uncus Cum Ramulus, protects against glutamate-induced neuronal death in vitro. Nitric oxide (NO) free radicals are also implicated in the process of neuronal death. In this study, we investigated the protective effects of Uncaria sinensis extract (USE) and its phenolic and alkaloid fractions against NO donors, sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), -induced neuronal death in cultured rat cerebellar granule cells. MTT assay showed cell viability to be significantly increased by the addition of USE (10, 30 and 100 μg/ml) compared with exposure (6, 12 and 24 h) to SNP (30 μM) only, and by the addition of USE (10 and 30 μg/mi) compared with exposure (6, 12 and 24 h) to SIN-1 (300μM) only. Phenolic fraction of USE (10 and 30μg/ml) significantly protected against SNP (30 μM, 24 hr)-induced cell death, and 3 and 10 μg/ml of this fraction significantly protected against SIN-1 (300 μM, 24 hr)-induced cell death. Alkaloid fraction of USE (30 and 100 μg/ml) significantly protected against SNP (30 μM, 24 hr) and SIN-1 (300 μM, 24 hr)-induced cell death. These results appear to indicate that Uncaria sinensis has a protective effect against NO-mediated neuronal death in cultured cerebellar granule cells and that its active components are included in phenolic and alkaloid fractions. 培養ラット小脳顆粒細胞を用いて,NO donor誘導神経細胞死に対する釣藤鈎エキスの保護作用を検討した。NO donorにはsodium nitroprusside(SNP) と 3-morpholinosydnonimine (SIN-1)を用い,細胞生存率の評価にはMTT法を用いた。釣藤鈎エキス(10,30,100μg/ml)は,SNP(30μM)6,12,24時間添加による神経細胞死を有意に抑制した。釣藤鈎エキス(10,30μg/ml)は,SIN-1(300μM)6,12,24時間添加による神経細胞死を有意に抑制した。釣藤鈎エキスのフェノール画分は,10,30μg/mlの濃度でSNP(30μM,24時間)添加による神経細胞死を有意に抑制し,同じく3,10μg/mlの濃度でSIN-1(300μM,24時間)添加による神経細胞死を有意に抑制した。釣藤鈎エキスのアルカロイド画分(30,100g/ml)は,SNP(30μM,24時間)およびSIN-1(300μM,24時間)添加による神経細胞死を有意に抑制した。以上より,釣藤鈎エキスはNO donorによって誘導される神経細胞死に対して保護作用を有し,その活性は釣藤鈎のフェノール画分およびアルカロイド画分にあることが示唆された

Characterization of a sperm factor for egg activation at fertilization of the newt Cynops pyrrhogaster

AbstractEggs of the newt, Cynops pyrrhogaster, arrested at the second meiotic metaphase are activated by sperm at fertilization and then complete meiosis to initiate development. We highly purified a sperm factor for egg activation from a sperm extract with several chromatographies. The purified fraction containing only a 45 kDa protein induced egg activation accompanied by an intracellular Ca2+ increase when injected into unfertilized eggs. Although injection of mouse phospholipase C (PLC) ζ-mRNA caused a Ca2+ increase and egg activation, partial amino acid sequences of the 45 kDa protein were homologous to those of Xenopus citrate synthase, but not to PLCs. An anti-porcine citrate synthase antibody recognized the 45 kDa protein both in the purified fraction and in the sperm extract. Treatment with the anti-citrate synthase antibody reduced the egg-activation activity in the sperm extract. Injection of porcine citrate synthase or mRNA of Xenopus citrate synthase induced a Ca2+ increase and caused egg activation. A large amount of the 45 kDa protein was localized in two lines elongated from the neck to the middle piece of sperm. These results indicate that the 45 kDa protein is a major component of the sperm factor for egg activation at newt fertilization