黄芪水提取物促进小鼠胚胎干细胞向血管平滑肌细胞分化的实验研究

目的：研究黄芪水提取物（Astragalus radix extract, ARE）对小鼠胚胎干细胞（Embryonic stem cells, ESCs）来源的血管平滑肌 细胞（Vascular smooth muscle cells, VSMCs）分化的影响。方法：将ESCs直接接种到IV 型胶原包被的培养皿中建立VSMC 分化 模型，向培养液中添加不同剂量ARE（对照组不添加ARE，ARE 低剂量组60 g/mL ARE, ARE 高剂量组（120 g/mL ARE），将3 组 细胞连续培养14 天。分别应用免疫荧光染色、实时定量PCR 及Western Blot 技术检测各组VSMC 分化标志物表达情况。应用胆 碱能受体激动剂卡巴可刺激各组细胞检测收缩反应。应用实时定量PCR技术检测调控VSMC 分化的重要因子的变化。结果：与 对照组相比，ARE 处理后VSMC 分化标志物表达显著增加，且呈剂量依赖性。ARE 处理可提高VSMCs 的收缩能力。此外，ARE 处理后，转化生长因子（Transforming growth factor ）的表达显著增加。结论：黄芪水提取物可能通过激活TGF- 促进ESCs向VS MCs分化。     

Nuclear factor Nrf2 and antioxidant response element regulate NRH:quinone oxidoreductase 2 (NQO2) gene expression and antioxidant induction

Human NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic protein that catalyzes the metabolic reduction of quinones and provides protection against myelogenous hyperplasia and chemical carcinogenesis. NQO2 gene expression is induced in response to antioxidant tert-butylhydroquinone (tBHQ). Sequence analysis revealed six putative antioxidant response elements (ARE1 through 6) in the human NQO2 gene promoter. Deletion mutagenesis and transfection studies suggested that the ARE region between nucleotides -1433 and -1424 is essential for basal expression and antioxidant induction of NQO2 gene expression. Mutation of this ARE from 3.8 kb NQO2 gene promoter significantly repressed expression and abrogated the induction in response to antioxidant in transfected cells. Band shift, supershift, and chromatin immunoprecipitation (ChIP) assays demonstrated binding of nuclear factors Nrf2 and JunD with human NQO2 gene ARE. Coimmunoprecipitation experiments revealed an association between Nrf2 and JunD. Overexpression of Nrf2 upregulated and overexpression of Nrf2 dominant-negative mutant downregulated ARE-mediated NQO2 gene expression. The treatment of Hep-G2 cells with Nrf2-specific RNAi significantly reduced Nrf2 and NQO2 gene expression and tBHQ induction. The results combined demonstrated that Nrf2 associates with JunD, binds to ARE at nucleotide -1433, and regulates human NQO2 gene expression and induction in response to antioxidants.     

Chaperone Hsp27 modulates AUF1 proteolysis and AU-rich element-mediated mRNA degradation

AUF1 is an AU-rich element (ARE)-binding protein that recruits translation initiation factors, molecular chaperones, and mRNA degradation enzymes to the ARE for mRNA destruction. We recently found chaperone Hsp27 to be an AUF1-associated ARE-binding protein required for tumor necrosis factor alpha (TNF-α) mRNA degradation in monocytes. Hsp27 is a multifunctional protein that participates in ubiquitination of proteins for their degradation by proteasomes. A variety of extracellular stimuli promote Hsp27 phosphorylation on three serine residues--Ser(15), Ser(78), and Ser(82)-by a number of kinases, including the mitogen-activated protein (MAP) pathway kinases p38 and MK2. Activating either kinase stabilizes ARE mRNAs. Likewise, ectopic expression of phosphomimetic mutant forms of Hsp27 stabilizes reporter ARE mRNAs. Here, we continued to examine the contributions of Hsp27 to mRNA degradation. As AUF1 is ubiquitinated and degraded by proteasomes, we addressed the hypothesis that Hsp27 phosphorylation controls AUF1 levels to modulate ARE mRNA degradation. Indeed, selected phosphomimetic mutants of Hsp27 promote proteolysis of AUF1 in a proteasome-dependent fashion and render ARE mRNAs more stable. Our results suggest that the p38 MAP kinase (MAPK)-MK2-Hsp27 signaling axis may target AUF1 destruction by proteasomes, thereby promoting ARE mRNA stabilization.     

Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5' to the ARE.

The 3' noncoding region (NCR) AU-rich element (ARE) selectively confers rapid degradation on many mRNAs via a process requiring translation of the message. The role of cotranslation in destabilization of ARE mRNAs was examined by insertion of translation-blocking stable secondary structure at different sites in test mRNAs containing either the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE or a control sequence. A strong (-80 kcal/mol [1 kcal = 4.184 kJ]) but not a moderate (-30 kcal/mol) secondary structure prevented destabilization of mRNAs when inserted at any position upstream of the ARE, including in the 3' NCR. Surprisingly, a strong secondary structure did not block rapid mRNA decay when placed immediately downstream of the ARE. Studies are also presented showing that the turnover of mRNAs containing control or ARE sequences is not altered by insertion of long (1,000-nucleotide) intervening segments between the stop codon and the ARE or between the ARE and poly(A) tail. Characterization of ARE-containing mRNAs in polyadenylated and whole cytoplasmic RNA fractions failed to find evidence for decay intermediates degraded to the site of strong secondary structure from either the 5' or 3' end. From these and other data presented, this study demonstrates that complete translation of the coding region is essential for activation of rapid mRNA decay controlled by the GM-CSF ARE and that the structure of the 3' NCR can strongly influence activation. The results are consistent with activation of ARE-mediated decay by possible entry of translation-linked decay factors into the 3' NCR or translation-coupled changes in 3' NCR ribonucleoprotein structure or composition.     

Roles of Climate,Vegetation and Soil in Regulating the Spatial Variations in Ecosystem Carbon Dioxide Fluxes in the Northern Hemisphere

Climate, vegetation, and soil characteristics play important roles in regulating the spatial variation in carbon dioxide fluxes, but their relative influence is still uncertain. In this study, we compiled data from 241 eddy covariance flux sites in the Northern Hemisphere and used Classification and Regression Trees and Redundancy Analysis to assess how climate, vegetation, and soil affect the spatial variations in three carbon dioxide fluxes (annual gross primary production (AGPP), annual ecosystem respiration (ARE), and annual net ecosystem production (ANEP)). Our results showed that the spatial variations in AGPP, ARE, and ANEP were significantly related to the climate and vegetation factors (correlation coefficients, R = 0.22 to 0.69, P < 0.01) while they were not related to the soil factors (R = -0.11 to 0.14, P > 0.05) in the Northern Hemisphere. The climate and vegetation together explained 60 % and 58 % of the spatial variations in AGPP and ARE, respectively. Climate factors (mean annual temperature and precipitation) could account for 45 - 47 % of the spatial variations in AGPP and ARE, but the climate constraint on the vegetation index explained approximately 75 %. Our findings suggest that climate factors affect the spatial variations in AGPP and ARE mainly by regulating vegetation properties, while soil factors exert a minor effect. To more accurately assess global carbon balance and predict ecosystem responses to climate change, these discrepant roles of climate, vegetation, and soil are required to be fully considered in the future land surface models. Moreover, our results showed that climate and vegetation factors failed to capture the spatial variation in ANEP and suggest that to reveal the underlying mechanism for variation in ANEP, taking into account the effects of other factors (such as climate change and disturbances) is necessary.     

水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

水稻RNA结合蛋白C3H12与RNA结合位点的鉴定

CCH型锌指蛋白质C3H12是进化上保守的RNA结合蛋白质,它含有5个串联的CCCH锌指结构域ZnF1-5,形成2个紧密的锌指簇ZnF1-3和ZnF4-5。早期的研究发现,C3H12可能通过与mRNA结合的方式在转录后水平调控基因的表达。然而,与C3H12结合的mRNA类型和他们的结合模式,并未通过实验得到证明。本文表达纯化了一系列C3H12截短及全长蛋白质,并合成了一些潜在RNA底物ARE9、ARE19及对照Random21。通过等温滴定量热法 (isothermal titration calorimetry, ITC) 确定了C3H12与富含腺嘌呤尿嘧啶单元 (AU-rich element, ARE) mRNA底物的结合,并揭示了互作核心区域和热力学性质。通过荧光光谱分析和微型热泳动 (microscale thermophoresis, MST)技术对ITC的结果进一步佐证。结果表明：(1) C3H12与ARE底物的相互作用是焓驱动的能量有利的 (ΔG<0) 特异性结合,结合比为1:1。(2) C3H12与ARE19的亲和力较ARE9更高（约2倍）。(3) C3H12中ZnF1-3在与ARE类底物的结合活性中发挥主导作用。(4) C3H12结构中的141个氨基酸残基的接头不直接参与和ARE底物的相互作用。本研究揭示的CCCH型锌指蛋白质C3H12与ARE底物结合模式,将为进一步在分子结构水平阐明C3H12与ARE底物结合的机制奠定基础。     

An acyl-CoA:cholesterol acyltransferase (ACAT)-related gene is involved in the accumulation of triacylglycerols in Saccharomyces cerevisiae

The major route for the synthesis of triacylglycerol (TAG) in yeast as well as in all TAG-accumulating organisms has been suggested to occur via the acylation of diacylglycerol (DAG) by acyl-CoA:diacylglycerol acyltransferase (DAGAT). Genes encoding DAGAT have been identified in both plant and animal tissues. These genes show strong sequence similarities to genes encoding acyl-CoA:cholesterol acyltransferase (ACAT). So far no Saccharomyces cerevisiae DAGAT gene has been published; however, two ACAT-like genes, ARE1 and ARE2, are present in the yeast genome. Both these genes have been suggested to be involved in the synthesis of sterol esters. We have now shown that the ARE1 gene in yeast also is involved in the synthesis of TAG, whereas the ARE2 gene is more specifically involved in the synthesis of sterol esters.