探讨cPKCs特定亚型在脑低氧预适应形成过程中的作用，借助已建立的小鼠整体低氧预适应模型，应用SDS-PAGE和Western bolt等生化技术，并结合Gel Doc成像系统，半定量检测脑组织内cPKCα和γ的膜转位水平和蛋白表达量。结果发现，随低氧暴露次数（低氧2-4次）增加，在小鼠海马和皮层组织内cPKCγ膜转位水平增高显著（p<0.05，n=6）；然而，cPKCα的膜转位水平和cPKCα及γ的蛋白表达量的变化均不显著。提示，cPKCγ的激活可能参与了脑低氧预适应的形成过程。

Neurogranin (NG) binding of calmodulin (CaM) at its IQ domain is sensitive to Ca2+ concentration and to modifications by protein kinase C (PKC) and oxidants. The PKC phosphorylation site of NG is within the IQ domain whereas the four oxidant-sensitive Cys residues are outside this region. These Cys residues were oxidized forming two pairs of intramolecular disulfides, and could also be glutathiolated by S-nitrosoglutathione resulting in the incorporation of four glutathiones per NG. Circular dichroism (CD) showed that modification of NG by phosphorylation, oxidation forming intramolecular disulfides, or glutathiolation did not affect the R-helical content of this protein. Mutation of the four Cys residues [Cys (-)-NG] to Gly and Ser did not affect the R-helical content either. Interaction of CaM with the reduced (red), glutathiolated (GS)-, or Cys(-)-NG in the Ca2+-free solution resulted in an increase in the R-helicity determined by their CD spectra, but relatively little change was seen with the oxidized NG (ox-NG) or phosphorylated NG (PO4-NG). The binding affinities between the various modified forms of NG and CaM were determined by CD spectrometry and sedimentation equilibrium: their affinities were Cys (-)-NG > red-NG, GS-NG > ox-NG > PO4-NG. Unlike Cys (-)-, red-, and GS-NG, neither ox-nor PO4-NG bound to a CaM-affinity column. Thus, both oxidation of NG to form intramolecular disulfides and phosphorylation of NG by PKC are effective in modulating the intracellular level of CaM. These results indicate that modification of NG to form intramolecular disulfides outside the IQ domain provides an alternative mechanism for regulation of its binding affinity to CaM.

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.

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。

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.