Salicylic-Acid-Induced Self-excision of the Marker Gene <Emphasis Type="Italic">npt</Emphasis>II from Transgenic Tomato Using the Cre–<Emphasis Type="Italic">loxP</Emphasis> System

The presence of antibiotic-resistant genes in genetically engineered crops together with the target gene has generated a number of environmental and consumer concerns. In order to alleviate public concerns over the safety of food derived from transgenic crops, marker gene elimination is desirable. Marker-free transgenic tomato plants were obtained by using a salicylic-acid-regulated Cre–loxP-mediated site-specific DNA recombination system in which the selectable marker neomycin phosphotransferase nptII and cre genes were flanked by two directly oriented loxP sites. Upon induction by salicylic acid, the cre gene produced a recombinase that eliminated sequences encoding nptII and cre genes, sandwiched by two loxP sites from the tomato genome. Regenerant plants with the Cre–loxP system were obtained by selection on kanamycin media and polymerase chain reaction (PCR) screening. Transgenic plants were screened for excision by PCR using nptII, cre, and PR-1a promoter primers following treatment with salicylic acid. The footprint of the excision was determined by sequencing the T-DNA borders after a perfect recombination event. The excision efficiency was 38.7%. A new plant transformation vector, pBLNSC (Genbank accession number EU327497), was developed, containing six cloning sites and the self-excision system. This provided an effective approach to eliminate the selectable marker gene from transgenic tomato, thus expediting public acceptance of genetically modified tomato.     

孕激素诱导cyclin G1在小鼠子宫内膜上皮细胞的表达及其对细胞增殖的影响

本文在体外培养条件下研究卵巢激素诱导小鼠子宫内膜上皮细胞cyclin G1的表达及细胞增殖和细胞周期进程的变化,以探讨孕激素依赖的细胞周期调控因子cyclin G1对子宫内膜上皮细胞增殖的负调控作用.原代培养小鼠子宫内膜上皮细胞,待其生长汇合后分为4组:对照组(C组)、雌激素组(E组)、孕激素组(P组)、雌、孕激素共同作用组(EP组).加入相应激素作用24 h后,用细胞免疫化学方法检测各组细胞cyclin G1的表达水平:四甲基偶氮唑蓝(MTT)比色法检测各组细胞活力,间接观察子宫内膜上皮细胞的增殖情况;用流式细胞仪检测分布在细胞周期各时相的子宫内膜上皮细胞所占百分数.细胞免疫化学结果显示,cyclin G1在C组和E组子宫内膜上皮细胞上无明显表达,而在P组和EP组子宫内膜上皮细胞中表达明显,且定位于细胞核内.MTT法结果显示,与C组相比,E组细胞活力明显增高,而P组和EP组的细胞活力均明显下降,表明雌激素能促进子宫内膜上皮细胞增殖,而孕激素则具有抑制子宫内膜上皮细胞增殖的作用.流式细胞术检测显示,与C组相比,E组中处于S期的子宫内膜上皮细胞百分数增多;P组与EP组中处于S期的子宫内膜上皮细胞百分数明显减少,而处于G1期的细胞百分数和G2/M期的细胞百分数则明显增加.上述结果提示,孕激素依赖的cyclin G1可能通过阻滞细胞周期进程来参与孕激素对子宫内膜上皮细胞增殖的负调控作用.     

热休克蛋白60与细胞凋亡

热休克蛋白60(heat shock protein 60, HSP60)是主要存在于线粒体内的分子伴侣蛋白,对于维持线粒体蛋白的正常结构和功能不可或缺.线粒体中的HSP60可作用于凋亡相关因子而抑制线粒体凋亡通路的激活,并且能够减少线粒体产生氧自由基;胞浆中的少量HSP60亦可通过与凋亡相关因子的相互作用等途径抑制细胞凋亡.相反,在某些刺激因素作用下或者HSP60细胞定位异常时,HSP60可产生促凋亡效应.HSP60在细胞凋亡中的双重作用及其对于肿瘤等疾病诊治的意义已引起高度关注.     

层析法去除重组人血清白蛋白干扰素α2b融合蛋白的内毒素

用柱层析方法在生产重组人血清白蛋白干扰素α2b融合蛋白过程中去除产品中的内毒素.所用柱层析组合为Blue-sepharose亲和柱层析、SOURCE 15 ISO疏水柱层析、Q Sepharose F.F.离子交换柱层析、Sephadex G25 Coarse凝胶过滤柱.采用鲎试剂法检测柱层析各阶段得到蛋白中的内毒素含量;并用RP-HPLC方法测定柱层析各阶段得到蛋白的纯度与浓度,求出每毫克蛋白的内毒素含量.结果为每步柱层析过程式均有去除内毒素的作用,最终所得蛋白的内毒素含量降为1 EU/mg,去除率达到99.9%.因而生产重组人血清白蛋白干扰素α2b融合蛋白所用分离纯化柱层析技术在纯化蛋白的同时能有效的去除产品中内毒素,所得产品内毒素的含量远低于药典对注射剂的内毒素含量要求.     

Identification of tyrosine 972 as a novel site of Jak2 tyrosine kinase phosphorylation and its role in Jak2 activation

Jak2 is a 130 kDa tyrosine kinase that is important in a number of cellular signaling pathways. Its function is intrinsically regulated by the phosphorylation of a handful of its 49 tyrosines. Here, we report that tyrosine 972 (Y972) is a novel site of Jak2 phosphorylation and, hence, autoregulation. Specifically, we found that Y972 is phosphorylated and confirmed that this residue resides on the surface of the protein. Using expression plasmids that expressed either wild-type Jak2 or a full-length Jak2 cDNA containing a single Y972F substitution mutation, we investigated the consequences of losing Y972 phosphorylation on Jak2 function. We determined that the loss of Y972 phosphorylation significantly reduced the levels of both Jak2 total tyrosine phosphorylation and phosphorylation of Y1007/Y1008. Additionally, Y972 phosphorylation was shown to be important for maximal kinase function. Interestingly, in response to classical cytokine activation, the Jak2 Y972F mutant exhibited a moderately impaired level of activation when compared to the wild-type protein. However, when Jak2 was activated via a GPCR ligand, the ability of the Y972F mutant to be activated was completely lost, therefore suggesting a differential role of Y972 in Jak2 activation. Finally, we found that phosphorylation of Y972 enhances Jak2 kinase function via a mechanism that appears to stabilize the active conformation of the protein. Collectively, our results suggest that Y972 is a novel site of Jak2 phosphorylation and plays an important differential role in ligand-dependent Jak2 activation via a mechanism that involves stabilization of the Jak2 active conformation.     

Position-specific residue preference features around the ends of helices and strands and a novel strategy for the prediction of secondary structures

It has been many years since position-specific residue preference around the ends of a helix was revealed. However, all the existing secondary structure prediction methods did not exploit this preference feature, resulting in low accuracy in predicting the ends of secondary structures. In this study, we collected a relatively large data set consisting of 1860 high-resolution, non-homology proteins from the PDB, and further analyzed the residue distributions around the ends of regular secondary structures. It was found that there exist position-specific residue preferences (PSRP) around the ends of not only helices but also strands. Based on the unique features, we proposed a novel strategy and developed a tool named E-SSpred that treats the secondary structure as a whole and builds models to predict entire secondary structure segments directly by integrating relevant features. In E-SSpred, the support vector machine (SVM) method is adopted to model and predict the ends of helices and strands according to the unique residue distributions around them. A simple linear discriminate analysis method is applied to model and predict entire secondary structure segments by integrating end-prediction results, tri-peptide composition, and length distribution features of secondary structures, as well as the prediction results of the most famous program PSIPRED. The results of fivefold cross-validation on a widely used data set demonstrate that the accuracy of E-SSpred in predicting ends of secondary structures is about 10% higher than PSIPRED, and the overall prediction accuracy (Q(3) value) of E-SSpred (82.2%) is also better than PSIPRED (80.3%). The E-SSpred web server is available at http://bioinfo.hust.edu.cn/bio/tools/E-SSpred/index.html.     

Refractoriness of the sheep superior vena cava myocardial sleeve

The cardiomyocytes in the superior vena cava (SVC) myocardial sleeve have distinct action potentials and ionic current profiles, but the refractoriness of these cells has not been reported. Using standard intracellular microelectrode techniques, we demonstrated in sheep that the effective refractory period (ERP) of the cardiomyocytes in the SVC (114.7 +/- 6.5 ms) is shorter than that in the inferior vena cava (IVC) (166.7 +/- 6.2 ms), right atrial free wall (RAFW) (201.0 +/- 6.0 ms) and right atrial appendage (RAA) (203.1 +/- 5.8 ms) (P < 0.05). The right atrial cardiomyocyte ERP was heterogeneously shortened by acetylcholine, a muscarinic type 2 receptor (M(2)R) agonist. After perfusion with 15 microM acetylcholine, the shortest ERP occurred in the SVC (the ERP in the SVC, IVC, RAFW and RAA was 53.6 +/- 2.7, 98.9 +/- 2.2, 121.8 +/- 6.0 and 109.7 +/- 5.1 ms, respectively; P < 0.05). Carbachol (1 microM), another M(2)R agonist, produced a similar effect as acetylcholine. Furthermore, we used methoctramine, a M(2)R blocker, 4-DAMP, a muscarinic type 3 receptor (M(3)R) blocker, and tropicamide, a muscarinic type 4 receptor (M(4)R) blocker to inhibit the acetylcholine-induced ERP shortening of SVC cardiomyocytes, and found that the 50% inhibitory concentration for methoctramine, 4-DAMP and tropicamide was 5.91, 45.72 and 80.34 nM, respectively. Therefore, we conclude that the sheep SVC myocardial sleeve is a unique electrophysiological region of the right atrium with the shortest ERP both under physiological condition and under cholinergic agonist stimulation. M(2)R might play a major role in the response of the SVC myocardial sleeve to parasympathetic nerve tone. The association between the distinct refractoriness in SVC and atrial fibrillation originating from the region deserves further investigation.