叶酸缺乏对小鼠胚胎干细胞Nespas DMR 区甲基化修饰的影响

目的：探讨叶酸缺乏对小鼠胚胎干细胞（ESCs）中Nespas 差异甲基化区域（Differentially Methylated Region, DMR）甲基化修 饰的影响以及叶酸浓度与甲基化水平的关系。方法：多种不同浓度叶酸处理小鼠ESCs,化学发光免疫分析法检测ESCs 细胞内叶 酸浓度。利用MassARRAY 技术平台检测三种不同叶酸浓度处理后的ESCs中Nespas DMR 启动子区,外显子区和内含子区甲基 化修饰状态,并且分析Nespas DMR 启动子区,外显子区和内含子区甲基化水平与叶酸浓度之间的关系。结果：无叶酸组（FF）小鼠 ESCs 细胞内叶酸浓度显著低于低叶酸组（FD）与正常叶酸组（FN）（P<0.05）。Nespas DMR 中启动子区、外显子区以及内含子区甲 基化水平在FF组显著低于FD 和FN 组（P<0.05）,并且Nespas DMR中启动子区以及内含子区甲基化水平与叶酸浓度存在显著 的正相关（P<0.05）。结论：叶酸缺乏影响小鼠ESCs 中Nespas DMR 区甲基化修饰的建立。     

Impulsive choices in mice lacking imprinted Nesp55

Genomic imprinting is the process whereby germline epigenetic events lead to parent‐of‐origin specific monallelic expression of a number of key mammalian genes. The imprinted gene Nesp is expressed from the maternal allele only and encodes for Nesp55 protein. In the brain, Nesp55 is found predominately in discrete areas of the hypothalamus and midbrain. Previously, we have shown that loss of Nesp55 gives rise to alterations in novelty‐related behaviour. Here, we extend these findings and demonstrate, using the Nesp^m/+ mouse model, that loss of Nesp55 leads to impulsive choices as measured by a delayed‐reinforcement task, whereby Nesp^m/+ mice were less willing to wait for a delayed, larger reward, preferring instead to choose an immediate, smaller reward. These effects were highly specific as performance in another component of impulsive behaviour, the ability to stop a response once started as assayed in the stop‐signal reaction time task, was equivalent to controls. We also showed changes in the serotonin system, a key neurotransmitter pathway mediating impulsive behaviour. First, we demonstrated that Nesp55 is co‐localized with serotonin and then went on to show that in midbrain regions there were reductions in mRNA expression of the serotonin‐specific genes Tph2 and Slc6a4, but not the dopamine‐specific gene Th in Nesp^m/+ mice; suggesting an altered serotonergic system could contribute, in part, to the changes in impulsive behaviour. These data provide a novel mode of action for genomic imprinting in the brain and may have implications for pathological conditions characterized by maladaptive response control.     

叶酸缺乏对小鼠胚胎干细胞Nespas DMR区甲基化修饰的影响

目的：探讨叶酸缺乏对小鼠胚胎干细胞（ESCs）中Nespas差异甲基化区域（Differentially Methylated Region,DMR）甲基化修饰的影响以及叶酸浓度与甲基化水平的关系。方法：多种不同浓度叶酸处理小鼠ESCs,化学发光免疫分析法检测ESCs细胞内叶酸浓度。利用MassARRAY技术平台检测三种不同叶酸浓度处理后的ESCs中Nespas DMR启动子区,外显子区和内含子区甲基化修饰状态,并且分析Nespas DMR启动子区,外显子区和内含子区甲基化水平与叶酸浓度之间的关系。结果：无叶酸组（FF）小鼠ESCs细胞内叶酸浓度显著低于低叶酸组（FD）与正常叶酸组（FN）（P〈0.05）。Nespas DMR中启动子区、外显子区以及内含子区甲基化水平在FF组显著低于FD和FN组（P〈0.05）,并且Nespas DMR中启动子区以及内含子区甲基化水平与叶酸浓度存在显著的正相关（P〈0.05）。结论：叶酸缺乏影响小鼠ESCs中Nespas DMR区甲基化修饰的建立。     

Mammalian microtubule P-body dynamics are mediated by nesprin-1

Nesprins are a multi-isomeric family of spectrin-repeat (SR) proteins, predominantly known as nuclear envelope scaffolds. However, isoforms that function beyond the nuclear envelope remain poorly examined. Here, we characterize p50^Nesp1, a 50-kD isoform that localizes to processing bodies (PBs), where it acts as a microtubule-associated protein capable of linking mRNP complexes to microtubules. Overexpression of dominant-negative p50^Nesp1 caused Rck/p54, but not GW182, displacement from microtubules, resulting in reduced PB movement and cross talk with stress granules (SGs). These cells disassembled canonical SGs induced by sodium arsenite, but not those induced by hydrogen peroxide, leading to cell death and revealing PB–microtubule attachment is required for hydrogen peroxide-induced SG anti-apoptotic functions. Furthermore, p50^Nesp1 was required for miRNA-mediated silencing and interacted with core miRISC silencers Ago2 and Rck/p54 in an RNA-dependent manner and with GW182 in a microtubule-dependent manner. These data identify p50^Nesp1 as a multi-functional PB component and microtubule scaffold necessary for RNA granule dynamics and provides evidence for PB and SG micro-heterogeneity.     

Identification of imprinted loci by methylation-sensitive representational difference analysis: application to mouse distal chromosome 2

Imprinted genes are distinguished by different patterns of methylation on their parental alleles, a property by which imprinted loci could be identified systematically. Here, representational difference analysis (RDA) is used to clone HpaII fragments with methylation differences on the maternal and paternal copies of distal chromosome (Chr) 2 in the mouse. Uniparental inheritance for this region causes imprinting phenotypes whose molecular basis is only partially understood. RDA led to the recovery of multiple differentially methylated HpaII fragments at two major sites of imprinted methylation: paternal-specific methylation at the Nesp locus and maternal-specific methylation at the Gnasxl locus. Nesp and Gnasxl represent oppositely imprinted promoters of the Gnas gene, which encodes the G-protein subunit, Gsalpha. The organization of the Nesp-Gnasxl-Gnas region was determined: Nesp and Gnasxl were found to be 15 kb apart, and Gnasxl was found to be 30 kb upstream of Gnas. Sites of imprinted methylation were also detected at the loci for neuronatin on Chr 2 and for M-cadherin on Chr 8. RDA was highly effective at identifying imprinted methylation, and its potential applications to imprinting studies are discussed.     

Identification of a methylation imprint mark within the mouse Gnas locus

The imprinted mouse gene Gnas produces the G protein alpha-subunit G(S)alpha and several other gene products by using alternative promoters and first exons. G(S)alpha is maternally expressed in some tissues and biallelically expressed in most other tissues, while the gene products NESP55 and XLalphas are maternally and paternally expressed, respectively. We investigated the mechanisms of Gnas imprinting. The G(S)alpha promoter and first exon are not methylated on either allele. A further upstream region (approximately from positions -3400 to -939 relative to the G(S)alpha translational start site) is methylated only on the maternal allele in all adult somatic tissues and in early postimplantation development. Within this region lies a fourth promoter and first exon (exon 1A) that generates paternal-specific mRNAs of unknown function. Exon 1A and G(S)alpha mRNAs have similar expression patterns, making competition between their promoters unlikely. Differential methylation in this region is established during gametogenesis, being present in oocytes and absent in spermatozoa, and is maintained in preimplantation E3. 5d blastocysts. Therefore, this region is a methylation imprint mark. In contrast, differential methylation of the NESP55 and XLalphas promoter regions (Nesp and Gnasxl) is not established during gametogenesis. The methylation imprint mark that we identified may be important for the tissue-specific imprinting of G(S)alpha.     

The effect of methionine and 5-azacytidine on fragile X expression.

The cellular mechanism for the expression of the fragile site at Xq28 is unknown. We tested the effect of 5-azacytidine and methionine on fragile X expression in lymphocytes and lymphoblastoid cells in an attempt to determine if DNA methylation was involved. We were unable to demonstrate a consistent dosage effect of methionine on fragile X expression. While 5-azacytidine was found to inhibit the fragile X in both males and females, it did so only at relatively high concentrations. We conclude that the role, if any, of DNA methylation in fragile X expression is likely to be secondary, the primary effect being due to thymidylate depletion.     

Comparative Analysis of Proteome and Transcriptome in Human Hepatocellular Carcinoma using 2D-DIGE and SAGE

Proteome analysis of human hepatocellular carcinoma was conducted using two-dimensional difference gel electrophoresis, and the protein expression profiles were compared to the mRNA expression profiles made from serial analysis of gene expression (SAGE) in identical samples from a single patient. Image-to-image analysis of protein abundances together with protein identification by peptide mass fingerprinting yielded the protein expression profiles. A total of 188 proteins were identified, and the expression profiles of 164 proteins which had the corresponding SAGE data were compared to the mRNA expression profiles. Among them, 40 proteins showed significant differences in the mRNA expression levels between non HCC and HCC. We compared expression changes of proteins with those of mRNAs. We found that the expression tendency of 24 proteins were similar to that of mRNA, whereas 16 proteins showed different or opposite tendency to the mRNA expression.     

人膀胱癌中某些癌基因的研究

本实验应用Northern和斑点印渍杂交技术探测了人膀胱癌细胞株中c-myc、c-fos、erbB等癌基因的表达,以及TPA对这些癌基因表达的调控,发现BIU-87细胞有这些癌基因的表达,并能被TPA所增强,同时也发现人膀胱癌组织有c-myc、c-fos、erbB、N-ras基因的高表达。提示蛋白激酶C的激活可以诱导某些癌基因的表达。多种癌基因的表达异常可能在膀胱癌中起重要作用。