Nitric oxide reverses drug resistance by inhibiting ATPase activity of p-glycoprotein in human multi-drug resistant cancer cells

Background

Development of resistance to chemotherapy drugs is a significant problem in treating human malignancies in the clinic. Overexpression of drug efflux proteins, including P-170 glycoprotein (P-gp), an ATP-dependent efflux protein, is one of the main mechanisms responsible for multi-drug resistance (MDR). Because our previous studies have shown that nitric oxide (^˙NO) or its related species inhibit the ATPase activities of topoisomerase II, we hypothesized that ^˙NO should also inhibit the ATPase activity of P-gp and increase drug accumulation in MDR cells, causing a reversal of drug resistance.

榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因的克隆与表达

牻牛儿基牻牛儿基焦磷酸合酶是产紫杉醇内生真菌紫杉醇合成下游途径中的关键步骤之一,在榛子产紫杉醇内生真菌中进行紫杉醇生物合成研究时首先要确认GGPP合酶的存在。该研究通过RT-PCR方法克隆得到了榛子产紫杉醇内生真菌Penicillium aurantiogriseum中GGPP合酶基因Pa GGPPS(Gen Bank登录号为KM881430)的c DNA开放阅读框(Open reading frame,ORF)。利用生物信息学方法,我们分析了该基因的序列,并对其编码的氨基酸序列进行了预测。结果发现该基因ORF长度为1 113 bp,预计蛋白分子量为40.98k D,等电点为6.168,表明该蛋白呈酸性。对其进行亲疏水性分析发现肽链整体呈现为亲水性。Pa GGPPS的主要二级结构元件为α-螺旋,并且包含一个异戊二烯类化合物合酶功能域。对榛子产紫杉醇内生真菌与其他物种的GGPPS进行氨基酸同源性分析,发现其与娄地青霉、曲霉菌和费氏新萨托菌的一致性较高,分别为94%、76%和76%。进化树分析表明来自动物、真菌和酵母的GGPPS聚为一类,来自植物的GGPPS聚为一类,其中榛子产紫杉醇内生真菌的GGPPS和青霉菌的进化关系最近,与植物的GGPPS的进化关系最远。对其进行基础生物信息学分析后,我们构建了原核表达载体,成功诱导其表达并得到可溶性蛋白。该研究结果为下一步深入研究GGPPS基因在内生真菌Penicillium aurantiogriseum紫杉醇合成途径中的作用及和构建高产紫杉醇基因工程菌株奠定了一定的基础。     

欧洲红豆杉AP2/ERF转录因子基因序列分析

以东北红豆杉AP2转录因子氨基酸序列为查询序列,搜索比对欧洲红豆杉基因组草图数据得到一个AP2转录因子全长序列,并命名为TbAP2。同时,利用生物信息学方法对TbAP2基因序列及其编码蛋白进行了特征分析和功能预测,发现该基因全长1 817bp,编码一个217个氨基酸的ORF;TbAP2相对分子量为23.68×103,等电点为4.92,亚细胞定位分析推测该转录因子定位在细胞核中,保守结构域分析发现其含有一个典型的AP2结构域,除与东北红豆杉具有99%的高一致性外,还分别与杨树、苜蓿和大豆的AP2蛋白具有81%、80%和78%的高一致性,进化树分析表明,相近科属植物的AP2转录因子归为一类,红豆杉AP2转录因子与玉米的AP2转录因子亲缘关系最近。本研究获得了TbAP2基因电子序列,为更好地利用分子生物学技术调控紫杉醇的生物合成提供理论基础。     

Genomic diversity within Taxus cuspidata var. nana revealed by random amplified polymorphic DNA markers

Taxus cuspidata var. nana is a cultivated variety of Taxus cuspidata and contains taxol, a valuable secondary metabolite of medical importance, both in their stems and leaves. In this paper, random amplified polymorphic DNA (RAPD) markers were used to assess the genomic diversity of individual plants within T. cuspidata population. Seventy-four randomly selected plants were analyzed by 29 selected primers among which 25 primers produced polymorphic banding patterns. The coefficient of similarity among the plants ranged from 0.30 to 1.00 with a mean of 0.605. Our results showed that a surprisingly high level of genomic diversity existed within T. cuspidata, and RAPD markers were effective in revealing the diversity. Cluster analysis divided the plants into two groups. This data, when taken together with earlier findings showing variation in taxol content within a natural population of T. cuspidata, suggests a tantalizing possibility for selecting genomically homogeneous T. cuspidata plant lines with elevated and stable taxol content. Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 5, pp. 771–776. The text was submitted by the authors in English.     

ggpps 5'-侧翼序列的克隆及其启动子功能验证

目的:研究牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl diphosphate synthase,GGPPs)基因启动子的活性;方法:从曼地亚红豆杉细胞中克隆ggpps基因5'-侧翼序列,并将该侧翼序列代替pBI121质粒上的CaMV35S启动子,以Gus基因作为报告基因构建植物表达载体,并进一步导入农杆菌LBA4404中获得阳性转化子,然后用叶盘转化法验证该侧翼序列的启动子活性;结果:本研究从曼地亚红豆杉细胞中成功克隆了ggpps基因的5'-侧翼序列,并且验证了该侧翼序列具有启动子活性;结论:ggpps基因的5'-侧翼序列的测序结果表明本实验成功克隆了该侧翼序列,启动子功能验证结果表明ggpps 5'-侧翼序列具有启动子活性,这些结果为进一步的通过缺失法进行ggpps基因启动子功能研究奠定了基础.     

Separation of a tungus producing taxol

An endogenous filariform fungus has been separated from a treenamed Taxus growing in the Aba region, Sichuan, China. The fungus is fermented in fluid medium for 3 weeks at 25℃, then the HPLC and MALDI-TOF analysis of the zymotic fluid show that the zymotic fluid contains taxol. So it is a fungus which can produce taxol. It is named Taxomyces sp. temporarily.     

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone‐induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone‐triggered ABA generation and assayed the effect of fluridone on ozone‐induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone‐triggered ABA generation but also blocked the ozone‐induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose‐dependent. Interestingly, the suppression of fluridone on ozone‐induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone‐induced Taxol production of T. chinensis suspension cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

一株产紫杉醇内生真菌液体发酵工艺的优化

紫杉醇是一种高效、低毒、广谱的天然抗癌药物,可以有效地治疗乳腺癌、子宫癌等。近年来的研究发现,从植物内生真菌中发酵生产紫杉醇被证明是解决药源问题的有效途径。从分离到的420株内生真菌中筛选到一株产紫杉醇的内生真菌XC1-07为实验材料进行发酵条件的初步优化。结果表明：最适碳源、氮源分别是麦芽糖和NH4NO3;在含10g/L NH4NO3、90g/L麦芽糖、1.0g/L MgSO4、pH6的优化培养基中培养13d,紫杉醇的产量为1,124.34μg/L。为目前报道的植物内生真菌发酵生产紫杉醇最高的产量。