RSK1 vs. RSK2 Inhibitory Activity of the Marine β-Carboline Alkaloid Manzamine A: A Biochemical,Cervical Cancer Protein Expression,and Computational Study

Manzamines are complex polycyclic marine-derived β-carboline alkaloids with reported anticancer, immunostimulatory, anti-inflammatory, antibacterial, antiviral, antimalarial, neuritogenic, hyperlipidemia, and atherosclerosis suppression bioactivities, putatively associated with inhibition of glycogen synthase kinase-3, cyclin-dependent kinase 5, SIX1, and vacuolar ATPases. We hypothesized that additional, yet undiscovered molecular targets might be associated with Manzamine A’s (MZA) reported pharmacological properties. We report here, for the first time, that MZA selectively inhibited a 90 kDa ribosomal protein kinase S6 (RSK1) when screened against a panel of 30 protein kinases, while in vitro RSK kinase assays demonstrated a 10-fold selectivity in the potency of MZA against RSK1 versus RSK2. The effect of MZA on inhibiting cellular RSK1 and RSK2 protein expression was validated in SiHa and CaSki human cervical carcinoma cell lines. MZA’s differential binding and selectivity toward the two isoforms was also supported by computational docking experiments. Specifically, the RSK1-MZA (N- and C-termini) complexes appear to have stronger interactions and preferable energetics contrary to the RSK2–MZA ones. In addition, our computational strategy suggests that MZA binds to the N-terminal kinase domain of RSK1 rather than the C-terminal domain. RSK is a vertebrate family of cytosolic serine-threonine kinases that act downstream of the ras-ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway, which phosphorylates substrates shown to regulate several cellular processes, including growth, survival, and proliferation. Consequently, our findings have led us to hypothesize that MZA and the currently known manzamine-type alkaloids isolated from several sponge genera may have novel pharmacological properties with unique molecular targets, and MZA provides a new tool for chemical-biology studies involving RSK1.     

抗小麦黄矮病相关蛋白激酶TiDPK1与BYDV外壳蛋白的互作

小麦黄矮病是由大麦黄矮病毒(Barley yellow dwarf virus, BYDV)引起的小麦重要病毒病。分离于小麦–中间偃麦草易位系的蛋白激酶编码基因TiDPK1, 是一个抗小麦黄矮病相关基因。本文报道利用酵母双杂交技术和双分子荧光互补技术对TiDPK1与BYDV外壳蛋白(coat protein, CP)互作的研究结果。酵母双杂交分析结果表明, TiDPK1能够与BYDV-GAV、-PAV株系的CP互作, 双分子荧光互补分析结果进一步表明, TiDPK1可与BYDV的CP互作、产生双分子荧光互补信号, 说明TiDPK1确可与BYDV CP相互作用, 该结果对了解TiDPK1在小麦抗BYDV反应机制具有一定意义。     

Novel Adociaquinone Derivatives from the Indonesian Sponge Xestospongia sp.

Seven new adociaquinone derivatives, xestoadociaquinones A (1a), B (1b), 14-carboxy-xestoquinol sulfate (2) and xestoadociaminals A–D (3a, 3c, 4a, 4c), together with seven known compounds (5–11) were isolated from an Indonesian marine sponge Xestospongia sp. Their structures were elucidated by extensive 1D and 2D NMR and mass spectrometric data. All the compounds were evaluated for their potential inhibitory activity against eight different protein kinases involved in cell proliferation, cancer, diabetes and neurodegenerative disorders as well as for their antioxidant and antibacterial activities.     

猪细胞周期蛋白依赖性激酶2多克隆抗体的制备与鉴定

为研究猪细胞周期蛋白依赖性激酶2(pCDK2)的生物学功能,本试验构建重组原核表达载体pET28a-pCdk2,转化大肠杆菌BL21 (DE3)受体菌,用IPTG诱导重组蛋白(rpCDK2)表达并进行SDS-PAGE和Western blotting鉴定,蛋白经纯化及与弗氏佐剂乳化后免疫家兔制备多克隆抗体。分别用Western blotting和免疫过氧化物酶单层细胞染色法(IPMA)检测抗体的免疫活性。结果显示,成功表达了rpCDK2蛋白,该蛋白以可溶性和包涵体两种形式存在,可溶性rpCDK2蛋白分子质量约为38 ku,包涵体rpCDK2蛋白在38~43 ku间有3种不同分子质量形式;IPMA检测结果显示,所制备的pCDK2蛋白多克隆抗体与猪肾细胞(PK-15)和猪睾丸细胞(ST)免疫染色呈特异性反应,且Western blotting分析显示,多克隆抗体与这两种细胞的总蛋白反应出现4条特异性条带(分子质量在34~50 ku之间),可能跟pCDK2的多种磷酸化形式有关。本试验利用原核表达系统成功制备了rpCDK2蛋白,并制备了免疫活性和特异性良好的pCDK2蛋白多克隆抗体,为该蛋白生物学功能及相关疾病研究提供了基础材料。     

鸭瘟病毒胸苷激酶基因的原核表达及其蛋白间接ELISA检测方法的建立

本研究旨在以表达纯化的鸭瘟病毒胸苷激酶(thymidine kinase,TK)重组蛋白作为包被抗原,建立检测鸭瘟病毒抗体的间接ELISA方法。根据GenBank上收录的鸭瘟病毒TK基因序列设计出1对引物,采用PCR扩增出鸭瘟病毒TK基因。将TK基因与原核表达载体pET-32a连接,通过PCR、双酶切和测序鉴定,将阳性重组质粒转化至大肠杆菌BL21(DE3)工程菌,IPTG诱导表达。采用切胶纯化的方法纯化蛋白,Western blotting分析鉴定表达产物。用纯化的TK重组蛋白作为包被抗原,并优化其他条件,最终建立检测鸭瘟病毒抗体的间接ELISA方法。结果显示,重组质粒构建成功,经Western blotting检测得到目的蛋白,TK重组蛋白能被阳性血清识别,说明该重组蛋白具有较好的抗原性。确定了最佳反应条件:酶标二抗的最佳稀释度为1:200,最佳抗原、抗体的稀释度分别为1:400和1:200,抗原抗体作用时间为60 min,最佳封闭液为5% BSA,最佳封闭时间为60 min,最佳显色时间为10 min。结果表明,该方法具有良好的稳定性及较高的敏感性,为鸭瘟的检疫、监测和防制工作提供了一种有效的手段。     

茶树鲜叶中维生素B6代谢酶PLK的活性分析

吡哆醛激酶（Pyridoxal Kinase, PLK）是维生素B₆（VB₆）代谢关键酶。从茶鲜叶中直接提取含PLK的粗酶液,经过硫酸铵沉淀、透析处理后,采用苯肼衍生化方法检测定PLK活性,并对其基本酶学性质进行分析。结果表明,提取时加入100%茶鲜重的PVPP,效果最佳。纯化后的茶鲜叶PLK比活力为0.737 nmol/(min·mg),纯化倍数为4.0;当有二价金属离子存在时,PLK才具有催化活性,其中Zn²⁺对其催化作用最强。该酶的最适反应温度为60℃;在pH6.0左右酶活力最高。在最适反应条件下,测得反应底物ATP和PL的Km值分别为19.3 μmol/L和53 μmol/L。     

象耳豆根结线虫Me-mapk1基因的克隆及RNAi分析

促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)是线虫体内信号转导的重要组分,在生长发育过程中具有重要作用。本研究从象耳豆根结线虫中克隆了1个促分裂原活化蛋白激酶基因Me-mapk1c DNA序列,该序列全长1 369 bp,包含1个1 185 bp的完整开放阅读框,推导编码394个氨基酸,蛋白质分子量为45.39 ku。同源性搜索比对结果发现,该基因编码的蛋白序列与南方根结线虫MI-MAPK1具有99%的一致性。通过构建原核表达载体p ET-32a-MAPK,在IPTG的诱导下得到70 ku左右的特异融合蛋白(包括大小约26 ku的标签序列)。利用RNAi技术对象耳豆根结线虫二龄幼虫Me-mapk1基因进行沉默,线虫诱导番茄根结形成的数量显著减少,推测Me-mapk1基因的下调表达有可能影响象耳豆根结线虫二龄幼虫(J2)的生长发育。     

通过酵母双杂交筛选水稻类受体激酶CR4的胞外结合蛋白（英文）

[目的]寻找CR4的胞外结合蛋白。[方法]采用OsCR4的胞外域作为诱饵蛋白,酵母双杂交筛选14d的小苗cDNA文库。[结果]得到了许多蛋白,包括1个过氧化物B（D26484）、甲硫氨酸硫氧还原酶（ABF96078）和1个未知功能的蛋白。[结论]为研究CR4的信号转导机制提供了理论依据。