大肠杆菌ptsHI-crr基因的敲除及其对苯丙氨酸生产的影响

L-苯丙氨酸(L-phenylalanine)是重要的食品和医药中间体。利用大肠杆菌发酵葡萄糖生产苯丙氨酸时,对葡萄糖转运起重要作用的磷酸烯醇丙酮酸糖磷酸转移酶系统(PTS)对苯丙氨酸产量有很大影响。由ptsHI-crr操纵子编码的磷酸组氨酸载体蛋白(HPr),酶I(EI)和酶IIAGlc是PTS的必要组分,通过敲除ptsHI-crr得到PTS缺陷菌株,可以使葡萄糖代谢更多地流向苯丙氨酸生物合成。采用Red同源重组技术将大肠杆菌染色体上的ptsHI-crr基因替换为四环素抗性基因,得到PTS缺陷菌株。该菌株在以葡萄糖为惟一碳源的培养基中摇瓶培养,菌密度为对照菌株的2.7倍,苯丙氨酸产量为对照菌株的6.3倍。     

LC-MS/MS determination of helicid in human plasma and its application in pharmacokinetic studies

Helicid is a traditional Chinese medicine used to treat headache and insomnia with definite effects. To facilitate pharmacokinetic studies of helicid in man, a sensitive and specific LC-MS/MS method for the quantitative detection of helicid in human plasma was developed and validated. The method involved the addition of bergeninum as the internal standard (IS), protein precipitation, HPLC separation, and quantification by MS/MS system using negative electrospray ionization in the multiple reaction monitoring mode (MRM). The precursor→product ion transitions were monitored at m/z 282.8→120.9 for helicid and m/z 326.9→192.2 for the IS, respectively. The lower limit of quantification (LLOQ) was 0.2 μg/L. The calibration curves for helicid was linear over a concentration range of 0.2-20 μg/L. The intra- and inter-batch analyses of QC samples at 0.4, 2, 20 μg/L indicated good precision (%R.S.D. between 2.69 and 5.47%) and accuracy (between 96.15 and 105.05%). The helicid was stable in human plasma stored at room temperature for at least 24h, 4°C for at least 24h, -20°C for at least 1 month, and for routine three freeze-thaw cycles. This accurate and specific assay provides a useful method for evaluating the pharmacokinetic profile of helicid in humans.     

Progesterone and DNA damage encourage uterine cell proliferation and decidualization through up-regulating ribonucleotide reductase 2 expression during early pregnancy in mice

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.     

小鼠pαMHC-EGFP胚胎干细胞株的构建

应用电穿孔方法将含有α肌球蛋白重链启动子的pαMHC-EGFP载体转染到D3系小鼠胚胎千细胞,应用200μg／ml新霉素进行药物选择。采用悬浮培养法,体外诱导分化心肌细胞。荧光显微镜下,观察到第7天和第8天拟胚体中出现“跳动”的心肌细胞并同时有绿色荧光蛋白的表达。同时与D3系小鼠胚胎干细胞比较心肌细胞分化率的变化无显著差异（P〉0．05）。该细胞株在分化心肌细胞的同时,具有绿色荧光蛋白的标记,因而利于对心肌细胞的识别和纯化。     

Cell Surface Beta 1, 4-galactosyltransferase 1 promotes apoptosis by inhibiting epidermal growth factor receptor pathway

Our previous studies have shown that overexpression of β1,4-galactosyltransferase1 (β1,4GT1) leads to increased apoptosis induced by cycloheximide (CHX) in SMMC-7721 human hepatocarcinoma cells. However, the role of β1,4GT1 in apoptosis remains unclear. Here we demonstrated that cell surface β1,4GT1 inhibited the autophosphorylation of epidermal growth factor receptor (EGFR) especially at Try 1068. The phosphorylation of protein kinase B (PKB/Akt) and extracellular signal-regulated protein kinase1/2 (ERK1/2), which are downstream molecules of EGFR, were also reduced in cell surface β1,4GT1-overexpressing cells. Furthermore, the translocations of Bad and Bax that are regulated by PKB/Akt and ERK1/2 were also increased in these cells. As a result, the release of cytochrome c from mitochondria to cytosol was increased and caspase-3 was activated. In contrast, RNAi-mediated knockdown of β1,4GT1 increased the autophosphorylation of EGFR. These results demonstrated that cell surface β1,4GT1 may negatively regulate cell survival possibly through inhibiting and modulating EGFR signaling pathway.     

不同生态环境对蒲公英超氧物歧化酶（SOD）的影响

选择干生,湿生,阳生,阴生四种生境中生长的蒲公英Taraxacum mongolicum Hand.-Mazz分根,叶,花分别测定了SOD活性和比活性,并比较了同工酶谱的变化情况,结果表明不同生境未能改变SOD的同工酶条带数,但对其活性大小有影响,这种影响尤其表现在根上,湿生和阴生根的SOD活性明显高于干生和阳生根的SOD活性,显示出蒲公英的不同器官适应不同的环境效应。