重组大肠杆菌表达铜绿假单胞菌溶血性磷脂酶C

[目的]构建产溶血性磷脂酶C (Hemolytic Phospholipase C,PLCH)的重组大肠杆菌(Escherich coli菌株,并初步优化其发酵条件.[方法]首先利用卵黄硼砂平板分离法筛选到一株产磷脂酶C(Phospholipase C,PLC)活性较高的菌株,命名为铜绿假单胞菌(Pseudomonas aeruginosa)41;进一步以P.aeruginosa 41基因组DNA为模板设计引物,PCR扩增获得溶血性磷脂酶C(PLCH)基因,构建重组大肠杆菌表达质粒并转化大肠杆菌E.coli BL21 (DE3);筛选转化子并检测PLC活性和溶血能力,并初步优化其发酵条件.[结果]成功构建了重组大肠杆菌E.coli BL21(DE3) /pET28a-plcH;在硼砂卵黄平板上对重组菌进行PLC活性测定,显示重组菌有明显的磷脂酶C活性;在哥伦比亚血琼脂平板上对重组菌进行溶血性试验,表明PLCH具有较强的溶血活性;初步优化摇瓶发酵条件为:5％转接量,37℃、200 r/min下培养4h添加IPTG至终浓度为0.9 mmol/L,转为25℃、150 r/min诱导培养14 h;优化后重组菌的酶活可达到722.89±0.47 U/mL.[结论]本文成功构建了一株产溶血性磷脂酶C活性较高的重组大肠杆菌菌株,并通过优化发酵条件使其酶活达到了722.89±0.47 U/mL,本实验在国内首次实现了铜绿假单胞菌来源的溶血性磷脂酶C基因在大肠杆菌的胞内表达,该研究为研究磷脂酶C产业化奠定了一定的基础.     

Positive Selection of CAG Repeats of the ATXN2 Gene in Chinese Ethnic Groups

The ataxin-2 （ATXN2） gene is located on human chromo-some 12q24.1. In normal individuals, the coding region in exon 1 of this gene has fewer than 31 CAG repeats （Yu et al., 2005： Laffita-Mesa et al., 2012）. However, an abnormal expansion of CAG trinucleotide repeats results in the aggre-gation of polyglutamine （polyQ）, which causes spinocer-ebellar ataxia type 2 （SCA2） （Pulst et al., 1996）. The expanded alleles have more than 32 repeats in the affected individuals, and generally there is an inverse correlation between CAG repeat length and age of onset （Pulst et al., 1996）. SCA2 is an autosomal dominant inheritance neurodegenerative disease, whose major clinical feature is progressive cerebellar ataxia. Atrophies of the brainstem and frontal lobe have been frequently detected by magnetic resonance imaging （MRI） （Yamamoto-Watanabe et al., 2010）. This disease has the strong effect on sensory and motor control.     

The pKa of the protonated Schiff bases of gecko cone and octopus visual pigments.

A visual pigment is composed of retinal bound to its apoprotein by a protonated Schiff base linkage. Light isomerizes the chromophore and eventually causes the deprotonation of this Schiff base linkage at the meta II stage of the bleaching cycle. The meta II intermediate of the visual pigment is the active form of the pigment that binds to and activates the G protein transducin, starting the visual cascade. The deprotonation of the Schiff base is mandatory for the formation of meta II intermediate. We studied the proton binding affinity, pKa, of the Schiff base of both octopus rhodopsin and the gecko cone pigment P521 by spectral titration. Several fluorinated retinal analogs have strong electron withdrawing character around the Schiff base region and lower the Schiff base pKa in model compounds. We regenerated octopus and gecko visual pigments with these fluorinated and other retinal analogs. Experiments on these artificial pigments showed that the spectral changes seen upon raising the pH indeed reflected the pKa of the Schiff base and not the denaturation of the pigment or the deprotonation of some other group in the pigment. The Schiff base pKa is 10.4 for octopus rhodopsin and 9.9 for the gecko cone pigment. We also showed that although the removal of Cl- ions causes considerable blue-shift in the gecko cone pigment P521, it affects the Schiff base pKa very little, indicating that the lambda max of visual pigment and its Schiff base pKa are not tightly coupled.     

奇异果甜蛋白及其基因工程

奇异果甜蛋白(thaumatin)是迄今为止最甜的物质之一,对其研究具有很重要的意义。奇异果甜蛋白的生化性质基本清楚,基因序列和氨基酸序列都已测定。它的甜味可能是由奇异果甜蛋白上特定基团和受体结合引起的。对奇异果甜蛋白的生理功能知之甚少。近二十年来,在奇异果甜蛋白的基因工程上取得了一定进展,但仍然存在许多困难。 Abstract:Thaumatin is one of the sweetest substances known to date,it is important to study the thaumatin.The biochemical properties of thaumatin have been clarified clearly.Thaumatin had been isolated and sequenced.The mechanism of the sweetness of thaumatin may be due to the combination of some special groups and the receptors.The exact function of thaumatin is still not clear.Although gene engineering of thaumatin has been carried out for 20 years,there are still some difficulties to be solved for using in the market.     

靶向AXL克服肺腺癌EGFR-TKIs获得性耐药

目的:探索AXL在肺腺癌细胞(Lung adenocarcinoma cell, LAC)EGFR-TKIs获得性耐药中的作用,为肺癌临床治疗和新型药物的研发提供实验依据。方法:构建EGFR-TKIs获得性耐药的肺腺癌模型并通过CCK-8法检测耐药株对肺腺癌靶向治疗药物吉非替尼(Gefitinib)、厄洛替尼(Erlotinib)和奥希替尼(Osimertinib)的敏感性。基于基因组学分析筛选出潜在的克服耐药的靶点AXL,通过Western blot和qRT-PCR技术检测AXL的表达情况,并同时检测上皮-间质转化(Epithelial-mesenchymal transition,EMT)分子标志物。R428是AXL的小分子抑制剂,通过CCK-8法、Transwell以及划痕实验等探究靶向AXL对肺腺癌亲本及耐药株增殖和迁移能力的影响。结果:AXL在构建的耐药株中显著高表达,其蛋白表达水平上调15-20倍(P0.001),m RNA水平上调2-5倍(P0.01);EGFR-TKIs耐药株发生上皮间质转化(EMT);靶向AXL选择性抑制耐药株的增殖能力并且恢复了耐药株对EGFR-TKIs的敏感性(P0.001);靶向AXL显著抑制耐药株增强的迁移能力,与亲本株相比最高抑制率可达80%左右(P0.001)。结论:用遗传学和药理学手段靶向AXL可以显著逆转肺腺癌对EGFR-TKIs耐药,逆转耐药株所增强的迁移等肿瘤生物学特征,对克服EGFR-TKIs获得性耐药有着重要的临床治疗价值以及转化医学前景。     

沙地樟子松带状混交林土壤碳氮磷化学计量特征

探究不同沙地樟子松混交林土壤碳氮磷化学计量变化规律,为其混交经营提供科学依据.以沙地樟子松纯林为对照,在沙地樟子松×榆树及沙地樟子松×怀槐带状混交林中,沿沙地樟子松和伴生树种两个方向在距离混交中心0、1、2、3和4 m处的不同土层采集土样,分析土壤有机C、全N、全P、速效N、速效P含量及其计量比的特征.结果表明: 沙地樟子松混交林土壤有机C、全N和速效N含量高于纯林, 沙地樟子松与榆树混交主要增加了深层土壤有机C和全N含量,以及C/N和C/P, 沙地樟子松与怀槐混交提高了土壤N含量, 降低了P含量.随着远离混交林中心,沙地樟子松×榆树混交林中沙地樟子松林带的土壤C/N先升高后降低,全P和速效P含量下降,N/P增加;榆树林带土壤C/N下降,速效P含量先升高后降低.沙地樟子松×怀槐混交林土壤全N含量在沙地樟子松林带先降低后升高,在怀槐林带先升高后降低.沙地樟子松混交林提高了土壤C、N储量,沙地樟子松应与榆树行间混交,与怀槐间隔2行混交.     

Moderate mechanical stimulation rescues degenerative annulus fibrosus by suppressing caveolin-1 mediated pro-inflammatory signaling pathway

Mechanical loading can induce or antagonize the extracellular matrix (ECM) synthesis, proliferation, migration, and inflammatory responses of annulus fibrosus cells (AFCs), depending on the loading mode and level. Caveolin-1 (Cav1), the core protein of caveolae, plays an important role in cellular mechanotransduction and inflammatory responses. In the present study, we presented that AFCs demonstrated different behaviors when subjected to cyclic tensile strain (CTS) for 24 h at a magnitude of 0%, 2%, 5% and 12%, respectively. It was found that 5% CTS had positive effects on cell proliferation, migration and anabolism, while 12% CTS had the opposite effects. Besides, cells exposed to interleukin-1β stimulus exhibited an increase expression in inflammatory genes, and the expression of these genes decreased after exposure to moderate mechanical loading with 5% CTS. In addition, 5% CTS decreased the level of Cav1 and integrin β1 and exhibited anti-inflammatory effects. Moreover, the expression of integrin β1 and p-p65 increased in AFCs transfected with Cav1 plasmids. In vivo results revealed that moderate mechanical stimulation could recover the water content and morphology of the discs. In conclusion, moderate mechanical stimulation restrained Cav1-mediated signaling pathway and exhibited anti-inflammatory effects on AFCs. Together with in vivo results, this study expounds the underlying molecular mechanisms on the effect of moderate mechanical stimulation on intervertebral discs (IVDs) and may provide a new therapeutic strategy for the treatment of IVD degeneration.     

Development of a homogeneous time-resolved fluorescence leukotriene B4 assay for determining the activity of leukotriene A4 hydrolase

Leukotriene A4 (LTA4) hydrolase catalyzes a rate-limiting final biosynthetic step of leukotriene B4 (LTB4), a potent lipid chemotactic agent and proinflammatory mediator. LTB4 has been implicated in the pathogenesis of various acute and chronic inflammatory diseases, and thus LTA4 hydrolase is regarded as an attractive therapeutic target for anti-inflammation. To facilitate identification and optimization of LTA4 hydrolase inhibitors, a specific and efficient assay to quantify LTB4 is essential. This article describes the development of a novel 384-well homogeneous time-resolved fluorescence assay for LTB4 (LTB4 HTRF assay) and its application to establish an HTRF-based LTA4 hydrolase assay for lead optimization. This LTB4 HTRF assay is based on competitive inhibition and was established by optimizing the reagent concentration, buffer composition, incubation time, and assay miniaturization. The optimized assay is sensitive, selective, and robust, with a Z' factor of 0.89 and a subnanomolar detection limit for LTB4. By coupling this LTB4 HTRF assay to the LTA4 hydrolase reaction, an HTRF-based LTA4 hydrolase assay was established and validated. Using a test set of 16 LTA4 hydrolase inhibitors, a good correlation was found between the IC50 values obtained using LTB4 HTRF with those determined using the LTB enzyme-linked immunoassay (R = 0.84). The HTRF-based LTA4 hydrolase assay was shown to be an efficient and suitable assay for determining compound potency and library screening to guide the development of potent inhibitors of LTA4 hydrolase.