一株红豆杉内生真菌Xylariales sp.次级代谢产物的研究

对一株药用植物南方红豆杉内生真菌Xylariales sp.的次级代谢产物进行了分离纯化、结构鉴定和抗菌活性测定。用反相(RP-18)中压液相柱层析、凝胶(Sephadex LH-20)柱层析和硅胶柱层析等方法从该菌株固体发酵产物的乙酸乙酯相中初步分离得到了4个化合物。运用现代波谱技术结合文献数据对照,鉴定其结构分别是:10,13-Octadecadienoic acid(1),Naphthalenemethanol,1,4,4a,5,6,7,8,8a-octahydro-2,5,5,8a-tetramethyl-(2),4H-Phenaleno[1,2-b]furan-4,6(5H)-dione,8,9-dihydro-3,5,7-trihydroxy-1,8,8,9-tetramethyl-5-(2-oxopropyl)-(3),Rousselianone A'(4)。化合物3与化合物4表现出了针对枯草芽孢杆菌专一的生长抑制活性,四个化合物在浓度为10μg/ml对He La细胞无毒性。宁夏大学自然科学基金(No.ZR1324

姜黄素对酵母细胞影响的荧光分析

以最简单的真核生物——酿酒酵母为材料,利用姜黄素的荧光性质,借助荧光光谱法和荧光显微镜研究了姜黄素在酵母细胞中的定位及其对酵母生长的影响.结果表明,姜黄素可以进入酵母细胞中并抑制酵母生长,初步得到了姜黄素对酵母作用的量效关系和时间效应结果,证明了姜黄素可以与酵母细胞线粒体结合.本文结果为以酵母为模型研究姜黄素通过线粒体影响细胞代谢及其与细胞凋亡的关系奠定了基础.国家自然科学基金(批准号:81373296);;厦门大学细胞应激生物学国家重点实验室开放课题(批准号:SKLCSB2017KF003)资助~

南方红豆杉内生真菌的初步研究

从共生理论出发,对采自福建省龙岩山区的药用植物南方红豆杉的内生真菌进行分离。从南方红豆杉的树叶、短茎和树皮中共分离得到107株内生真菌。抗菌、抗氧化和抗肿瘤活性检测结果显示,其中有8株菌对1种或者几种指示菌有一定的抗菌活性,占供测菌株的7.48%;有40株菌对HElA细胞有较强的细胞毒活性,占供测菌株的37.38%;有2株菌对dPPH·有机自由基有较强清除活性,占供测菌株的1.87%。从南方红豆杉内生真菌XylArIAlES SP.的固体发酵产物中初步分离并鉴定出化合物10,11-dIHydrOXynErOlIdOl,该化合物表现出了一定的抗氧化作用。说明南方红豆杉植物中含有丰富的菌种资源,具有开发生物活性物质的潜力

姜黄素衍生物C1209抑制慢性粒细胞白血病细胞增殖与阻断Hsp90伴侣功能的关系

目的研究姜黄素衍生物C1209抑制慢性粒细胞白血病细胞增殖与阻断Hsp90伴侣功能的关系。方法采用荧光光谱法,研究不同浓度C1209与Hsp90、其N端片段（NHsp90）、M端片段（MHsp90）、C端片段（CHsp90）的相互作用,以及不同温度（293 K、303 K、310 K）下,C1209与Hsp90的相互作用;实验选取280 nm为激发波长,290510 nm的波长范围内进行荧光光谱扫描。采用孔雀绿磷钼酸铵-无机磷检测法,研究C1209对Hsp90-ATPase活性的抑制。四甲基偶氮唑蓝（MTT）法和羟基荧光素二醋酸盐琥珀酰亚胺脂（CFSE）染色法体外检测C1209对白血病细胞K562及耐伊马替尼（IM）的白血病细胞K562/G01细胞的增殖抑制作用;应用蛋白免疫印迹法检测Hsp90客户蛋白及其下游蛋白、Hsp90分子伴侣的表达。结果C1209解离常数为（14.733±0.713）μmol·L-1;C1209与Hsp90之间的主要作用力为静电作用力;C1209与Hsp90的C端片段结合能力最强。当ATP为1 mmol·L-1时,C1209作用于Hsp90的IC50值为11.4μmol·L-1。C1209呈剂量依赖性地抑制K562和K562/G01细胞的增殖,作用24 h的IC50为1.14μmol·L-1和0.56μmol·L-1;C1209影响Hsp90的分子伴侣功能,且下调K562和K562/G01细胞中Bcr-Abl、Akt、MEK、ERK、c-Raf及其相应磷酸化蛋白p-Akt、p-MEK、p-ERK等Hsp90客户蛋白及其下游蛋白的表达。结论姜黄素衍生物C1209是Hsp90抑制剂,对K562和耐IM的白血病细胞K562/G01具有明显的增殖抑制作用,可能与其抑制Hsp90的分子伴侣功能、下调Hsp90客户蛋白有关。福建省自然科学基金资助项目(No 2017J01821);;福建省卫计委青年项目(No 2015-1-72);;国家自然科学基金资助项目(No 81173096);;国家科技部“重大新药创制”科技重大专项(No 2012ZX09103-101-028);;福建省高校产学合作项目(No 2016Y4005

Preparation and identification of polyclonal antibody raised against heat shock protein 90 of fission yeast

作者简介:李文珠(1983－),女,博士研究生,主要研究方向:细胞生物学 通信联系人：靳全文，教授，主要研究方向：细胞周期调控及表观遗传学， [email protected][中文文摘]为制备兔抗裂殖酵母Hsp90多克隆抗体,在通过PCR获得了裂殖酵母Hsp90(Swo1)基因后,构建了pMALc2x-Swo1表达载体,可用于表达编码正确氨基酸序列的目的基因。转化大肠杆菌BL21(DE3),IPTG诱导表达,Amylose Resin柱纯化。目的蛋白表达量占菌体总蛋白的30%以上。纯化后,蛋白纯度达95%以上。纯化后的MBP-Swo1融合蛋白抗原加福氏完全佐剂背部皮内注射首次免疫新西兰大白兔,第28天用MBP-Swo1融合抗原加福氏不完全佐剂同样剂量加强免疫,第35天时再次免疫。第49天心脏采血。收集血清后,用免疫印迹(westernblot)检测Swo1多克隆抗体的特异性。免疫印迹检测结果显示该抗体能够特异性识别内源性的裂殖酵母Hsp90/Swo1蛋白,但并不识别人类细胞中的Hsp90α/β。[英文文摘]To prepare the polyclonal antibody of Hsp90(Swo1) of fission yeast Schizosaccharomyces pombe,the encoding gene swo1+was amplified from the yeast genome and then inserted into expression vector pMALc2x.The resulted plasmid pMALc2x-Swo1 was transformed and expressed in E.coli BL21(DE3).The expressed fusion protein was purified through Amylose Resin column.The proteins were expressed mainly as secretion with the yield of more than 30% of total bacterial proteins.After purification,the purity of the proteins was about 95%. The New Zealand white rabbits were immunized with Freund’s complete adjuvant plus purified MBP-Swo1 fusion antigen through the back skin intradermal injection for the first time. The same dose of Freund’s incomplete adjuvant plus MBP-Swo1 was injected to strengthen the immunity after 28 and the 35 days respectively. After 49 days, blood sample was collected from heart, and antisera were extracted. The specificity of Swo1 polyclonal antibody was examined by Western blot. It showed that the antibody obtained had a high specificity to detect endogeneous Swo1 from fission yeast, but it could not recognize Hsp90α/β in human cells. Key words: fission yeast；Hsp90/Swo1；polyclonal antibody.高等学校博士学科点专项科研基金资助项目(20100121110003);国家自然科学基金资助项目(30871376);教育部科学技术研究重点项目(108076

姜黄素对食管癌KYSE410细胞的生长抑制与氧化调节

通过四氮唑蓝盐化合物（MTS）和重要氧化还原酶及其代谢物的试剂盒检测等方法,考察了姜黄素对食管癌KYSE410细胞生长以及对细胞氧化还原状态和代谢的影响.结果表明,姜黄素对KYSE410细胞具有较强的抑制作用,其IC50=17. 9μmol/L.进一步研究发现,姜黄素可引起细胞培养上层清液中超氧化物歧化酶（SOD）和丙二醛（MDA）水平发生变化.当姜黄素浓度为40μmol/L时,MDA水平比对照组提高了125. 1%,而SOD水平则降低了43. 2%;同时,乳酸水平降低了44. 4%,乳酸脱氢酶的活性下降了58. 2%,丙酮酸激酶的活性升高了216. 7%.姜黄素可能通过干扰氧化还原途径,致使发生脂质过氧化反应,并抑制肿瘤细胞糖酵解作用,进而抑制食管癌KYSE410细胞增殖.国家自然科学基金(批准号:81373296);;\n细胞应激生物学国家重点实验室(厦门大学)开放课题基金(批准号:SKLCSB2017KF003);;\n厦门大学大学生创新创业训练计划项目资助~

Study on the Interaction of Gliotoxin with BSA

应用荧光、圆二色和紫外—可见吸收等波谱法研究胶毒素与牛血清白蛋白(bSA)的相互作用。荧光光谱实验结果表明胶毒素主要靠疏水作用与bSA结合,而对其内源荧光产生猝灭作用,其淬灭方式为静态猝灭,胶毒素与bSA的结合常数为7.2x103l/MOl。圆二色光谱检测发现,随着胶毒素浓度的增加,bSA的α-螺旋数量也增加,当胶毒素浓度为bSA浓度的100倍时,bSA的α-螺旋增加40.1%,表明胶毒素与bSA的结合改变了bSA的空间构象。The interaction between Gliotoxin and bovine serum albumin (BSA) was studied by the fluo-rescence, Circular Dichroism (CD) and ultraviolet visible (UV-Vis) techniques.The fluorescent experiment showed that the intrinsic fluorescence of BSA was quenched by the binding of gliotoxin in a static quenching procedure, with an association constant of 7.2×103 L/mol and in hydropobic forces.And the CD spectrum revealed that gliotoxin effected the conformation of BSA by increased the mass of α-helix.国家863计划项目(No.2006AA09Z410

The Isolation and Activity Analysis of Endogenetic Bacteria from Echinus' Spawn

海胆卵中含有丰富的唾液酸聚糖蛋白,作者从共生的理论出发,采用涂布法两次从海胆卵内共分离到83株菌,通过形态学的方法初步鉴定大部分为细菌.采用液体发酵法进行发酵,83株菌的发酵液均对唾液酸的特征显色反应呈阳性,唾液酸含量测定表明有1株菌唾液酸的含量达到5.8μg/mL,有7.23%的菌株唾液酸含量达到或接近3μg/mL.通过对其抗菌、抗氧化和抗肿瘤等生物活性测定表明,有8株菌具有不同程度的抗菌活性,占被检测菌株总数的19.05%;有2株菌对肺癌细胞具有明显的抑制作用.上述结果表明海洋动物海胆中含有丰富的菌种资源,具备开发生物活性物质的潜力.Echinus' spawn contains plenty of sialoglycoprotein.Eighty-three strains were isolated from echinus' spawn based on the theory of intergrowth,which presented positive result according to the special color reaction of sialic acid,and were identified as bacterium.The testing of sialic acid contents proved that one of the endophyte's fermentations reached 5.8 μg/mL,and 7.23% of the endophytes achieved about 3 μg/mL.Their bioactivities were also primarily studied.Ruselts indicated: eight strains of those endophytes had antimicrobial activities against one or more microbial,forty-two stains with antioxidant activities were screened out,and two strains of those endophyte bacteria presented obvious anti-tumor activity against zr-75-30 cell line.The results suggest that marine animal echinus is rich of endophyte and is one of the potential resources for exploiting biological active substance.国家863计划项目(2006AA09Z410);; 福建省自然科学基金(2006J0378)资

Study on Bioactivity of Endophytic Fungi from Ephedra

从内蒙古赤峰等地的中草药麻黄中分离得到141株内生真菌,体外抗氧化、抗肿瘤等活性测定表明,有11株内生真菌对H2O2具有较好的清除活性(LD50均大于50,LD50为抑制率为50%时的样品稀释倍数),占菌株总数的7.80%;有27株内生真菌对Raji和HepG-2具有肿瘤细胞毒活性(LD50大于50),占菌株总数的19.15%,其中以抑制悬浮Raji细胞为主.此外,在141株菌中,有74株对一种或二种以上的指示菌显示出抑菌活性,占菌株总数的52.48%.本实验结果表明,麻黄中含有丰富的内生真菌,是抗氧化、抗菌及抗肿瘤等活性物质的重要来源.为进一步从中草药植物中分离筛选新药先导化合物提供了科学依据.One hundred and forty-one endophytic fungi strains were isolated from ephedra that were from Chifeng et al places in Inner Mongolia,and their bioactivities were primarily studied.Results indicated: all of the samples,eleven fungi strains had the ability of scavenging H2O2(with the LD50 above 50),accounting for 7.80% of the strains tested,twenty-one fungi strains with high cytotoxic activity(with the LD50 above 50) were screened out,accounting for 19.15% of the total strains,and this ability focused on inhibition of the growth of Raji cells,accounting for 17.73% of all the strains.Furthermore,seventy-four fungi strains had antimicrobial activities against one or more microbial,accounting for 52.48% of the strains tested.This study could provide scientific knowledge of screening new-leading compounds of medicine from Chinese herbs.福建省自然科学基金(2006J0378)资

Interactions between curcumin derivatives C085 and various constructs of Hsp90 and effects of C085 on Hsp90 ATPase activity

目的研究姜黄素衍生物C085与HSP90的结合作用,及其对HSP90-ATPASE活性抑制作用。方法采用荧光光谱法,研究不同浓度C085与HSP90、nHSP90、MHSP90、CHSP90的相互作用以及293k、303k和310k下,C085与HSP90的相互作用;实验选取280nM为激发波长,290~510 nM的波长范围内进行荧光光谱扫描。采用孔雀绿磷钼酸铵-无机磷检测法,研究C085对HSP90-ATPASE活性的抑制。结果 C085解离常数为(11.163±0.316)μMOl·l-1;C085与HSP90之间的主要作用力为静电作用力;C085与HSP90的C端片段结合能力最强。当ATP为1 MMOl·l-1时,C085作用于HSP90的IC50值为6.04μMOl·l-1。结论经过荧光光谱分析,可以确定C085与HSP90的结合机制,且C085能抑制HSP90-ATPASE活性;Aim To estimate the affinity between C085 and Hsp90 and the inhibitory effects of C085 on the activity of Hsp90 ATPase.Methods The fluorescence spectrum experiment was applied to examine the affinity between different C085 concentrations and Hsp90,NHsp90,MHsp90,CHsp90; fluorescence intensities were recorded in the range of 290- 510 nm at293 K,303 K and 310 K,respectively; a colorimetric assay for inorganic phosphate based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green was used to examine the inhibitory effects of C085 on the activity of Hsp90 ATPase.Results The dissociation constant KDvalue of C085 was( 11.163 ± 0.316) μmol·L- 1.The interaction between C085 and Hsp90 was driven mainly by electrostatic interaction.C085 showed strongest affinity with CHsp90.When the concentration of ATP was 1 mmol·L- 1,the inhibition of Hsp90 ATPase activity of C085 with the IC50 value was 6.04 μmol·L- 1.Conclusions The interaction mechanism between C085 and Hsp90 can be analyzed by fluorescence spectrum.C085 shows strong inhibition ATPase activity of Hsp90.国家自然科学基金资助项目(No81173096); 科技部“重大新药创制”科技重大专项(No2012ZX09103-101-028