目的：通过观察低氧刺激对培养SY5Y神经母细胞瘤细胞内nPKCε和nPKCθ膜转位激活的影响，以探讨两者是否参与细胞低氧预适应的发生。方法：利用本室建立的体外培养SY5Y细胞低氧刺激模型，并应用SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）、蛋白印记（Western-Blot）等方法，半定量分析SY5Y细胞内nPKCε、nPKCθ的膜转位水平。结果：SY5Y细胞经低氧刺激后，随刺激时间（0.5、2、4、6、8、12和24h）的延长，nPKCε在胞浆内的含量逐渐减少，而胞膜成分中的含量则明显增加，且于低氧刺激2h后的增高水平具有统计学显著意义（P<0.001）；然而nPKCθ在正常情况下只存在于胞膜成分内，且其含量不随低氧刺激时间的增加而改变（P>0.01）结论：nPKCε可能参与了细胞低氧耐受的发生。

FUSE-binding protein (FBP) binds the single-stranded far upstream element of active c-myc genes, pos- sesses potent transcription activation and repression domains, and is necessary for c-myc expression. A novel 60 kDa protein, the FBP interacting repressor (FIR), blocked activator-dependent, but not basal, transcription through TFIIH. Recruited through FBP' s nucleic acid-binding domain, FIR formed a ternary complex with FBP and FUSE. FIR repressed a c-myc reporter via the FUSE. The amino terminus of FIR con-tained an activator-selective repression domain capable of acting in cis or even in trans in vivo and in vitro. The repression domain of FIR targeted only TFIIH' s p89/XPB helicase, required at several stages in tran-scription, but not factors required for promoter selection. Thus, FIR locks TFIIH in an activation-resistant configuration that still supports basal transcription.

Neurogranin (Ng) is a brain-specific, postsynaptically located protein kinase C (PKC) substrate, highly expressed in the cortex, hippocampus, striatum, and amygdala. This protein is a Ca2-sensitive calmodulin (CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. To investigate the role of Ng in neural function, a strain of Ng knockout mouse (KO) was generated. Previously we reported (Pak, J. H., Huang, F. L., Li, J., Balschun, D., Reymann, K. G., Chiang, C., Westphal, H., and Huang, K. -P. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11232- 11237) that these KO mice displayed no obvious neuroanatomical abnormality, but exhibited deficits in learning and memory and activation of Ca2/CaM-dependent protein kinase II. In this report, we analyzed several downstream phosphorylation targets in phorbol 12-myristate 13-acetate-and forskolin-treated hippocampal slices from wild type (WT) and KO mice. Phorbol 12-myristate 13-acetate caused phosphorylation of Ng in WT mice and promoted the translocation of PKC from the cytosolic to the particulate fractions of both the WT and KO mice, albeit to a lesser extent in the latter. Phosphorylation of downstream targets, including mitogenactivated protein kinases, 90-kDa ribosomal S6 kinase, and the cAMP response element binding protein (CREB) was significantly attenuated in KO mice. Stimulation of hippocampal slices with forskolin also caused greater stimulation of protein kinase A (PKA) in the WT as compared with those of the KO mice. Again, phosphorylation of the downstream targets of PKA was attenuated in the KO mice. These results suggest that Ng plays a pivotal role in regulating both PKC-and PKA-mediated signaling pathways, and that the deficits in learning and memory of spatial tasks detected in the KO mice may be the result of defects in the signaling pathways leading to the phosphorylation of CREB.

Neurogranin/RC3 (Ng), a postsynaptic neuronal protein kinase C (PKC) substrate, binds calmodulin (CaM) at low level of Ca21. In vitro, rat brain Ng can be oxidized by nitric oxide (NO) donors and by oxidants to form an intramolecular disulfide bond with resulting downward mobility shift on nonreducing SDS-polyacrylamide gel electrophoresis. The oxidized Ng, as compared with the reduced form, is a poorer substrate of PKC but like the PKC-phosphorylated Ng has a lower affinity for CaM than the reduced form. To investigate the physiological relevance of Ng oxidation, we tested the effects of neurotransmitter, N-methyl-D-aspartate (NMDA), NO donors, and other oxidants such as hydrogen peroxide and oxidized glutathione on the oxidation of this protein in rat brain slices. Western blot analysis showed that the NMDA-induced oxidation of Ng was rapid and transient, it reached maximum within 3-5 min and declined to base line in 30min. The response was dose-dependent (EC50 ;100mm) and could be blocked by NMDA-receptor antagonist 2-amino-5-phosphonovaleric acid and by NO synthase inhibitor NG-nitro-L-arginine methyl ester and NG-monomethyl-L-arginine. Ng was oxidized by NO donors, sodium nitroprusside, S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione, and H2O2 at concentrations less than 0.5mm. Oxidation of Ng in brain slices induced by sodium nitroprusside could be reversed by dithiothreitol, ascorbic acid, or reduced glutathione. Reversible oxidation and reduction of Ng were also observed in rat brain extracts, in which oxidation was enhanced by Ca21 and the oxidized Ng could be reduced by NADPH or reduced glutathione. These results suggest that redox of Ng is involved in the NMDA-mediated signaling pathway and that there are enzymes catalyzing the oxidation and reduction of Ng in the brain. We speculate that the redox state of Ng, similar to the state of phosphorylation of this protein, may regulate the level of CaM, which in turn modulates the activities of CaM-dependent enzymes in the neurons.

NeurograninyRC3 is a neural-specific Ca21-sensitive calmodulin CaM)-binding protein whose CaM-binding affinity is modulated by phosphorylation and oxidation. Here we show that deletion of the Ng gene in mice did not result in obvious developmental or neuroanatomical abnormalities but caused an impairment of spatial learning and changes in hippocampal short- and long-term plasticity (paired-pulse depression, synaptic fatigue, long-term potentiation induction). These deficits were accompanied by a decreased basal level of the activated Ca21yCaM-dependent kinase II (CaMKII) ('60% of wild type). Furthermore, hippocampal slices of the mutant mice displayed a reduced ability to generate activated CaMKII after stimulation of protein phosphorylation and oxidation by treatments with okadaic acid and sodium nitroprusside, respectively. These results indicate a central role of Ng in the regulation of CaMKII activity with decisive influences on synaptic plasticity and spatial learning.