Structure-activity relationship at the proximal phenyl group in a series of non-peptidyl N-type calcium channel antagonists.

Selective N-Type Voltage Sensitive Calcium Channel (VSCC) antagonists have shown utility in several models of pain and ischemia. We report the structure-activity relationship at the proximal phenyl group in a series of non-peptidyl VSCC blockers.     

不同预处理对大麦花药3种内源激素含量和过氧化物酶活性的影响

研究了大麦（Ｈｏｒｄｅｕｍ ｖｕｌｇａｒｅ Ｌ．）花药－花粉培养中不同预处理对花药内源激素（ＡＢＡ,ＩＡＡ,ＩＰＡ）含量和过氧化物酶活性的影响。结果表明：１． 经甘露醇预处理不同天数,同一种基因型的３种内源激素的变化和不同基因型的同一种内源激素的变化规律十分相似,均是在预处理初期含量急剧增加,最高值在０．５或１ ｄ 处。以后开始逐渐下降;最后,保持在一定的水平上。２． 在甘露醇预处理过程中,两种基因型花药过氧化物酶活性的变化规律也十分相似。在预处理前期（从开始到第３天）活性呈直线上升,第３天达到最大值。从第３天到第５天活性减弱。最后,活性又开始增强。３． 在低温预处理过程的初期（２～５ ｄ） 两种基因型花药过氧化物酶活性都出现第一个小峰。在第１４天（“Ｈａｒｒｉｎｇｔｏｎ”）或第２１天（“Ｉｇｒｉ”）又出现第二个峰值,但后者较高。在预处理的后期（从第２８天到第３５天）两种基因型花药过氧化物酶的活性又呈现上升的趋势。同甘露醇预处理后期变化一致     

祁连山西段草地土壤温度、水分变化特征

研究土壤水热要素对合理预测祁连山气候变化特征及建立气候预警系统有重要意义。采用18套土壤温、湿度自动记录仪(HOBOU30)对祁连山西段草地进行3a连续定位监测,旨在探索分析该区土壤水热变化特征,阐明土壤水热互作效应及耦合机制。表明:1)冷期(1—3、11—12月)占全年的42%,暖期(4—10月)占58%;7月前土壤均温随月份增大而增大,月份增大1月土壤均温增大3.53℃,水分增大3.23%;7月后随月份增大而减小,月份增大1月土壤均温降低4.73℃,水分下降2.55%。2)日变化16:00前土壤均温为7.45℃,水分为16.26%,16:00后均温为9.1℃,水分为16.79%,16:00后比前均温高1.65℃,水分高0.54%。3)土温、水分与海拔均呈线性正相关,前者差异显著(P0.05),后者不显著(P0.05)。4)土温逐层(0—120 cm)平均递增0.07℃,水分递减0.58%;土温与土层深度呈显著线性正相关(P0.05,R2=0.99),水分呈显著线性负相关(P0.05,R2=0.97)。5)该区土温与水分呈显著线性负相关。因此,祁连山西段草地土温变幅较小,水分变幅大,两者在空间上变化均呈二次函数,时间上均呈一次函数。     

Trans‐3,5,4´‐trimethoxystilbene reduced gefitinib resistance in NSCLCs via suppressing MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345 and miR‐498

Despite initial dramatic efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR‐TKIs) in EGFR‐mutant lung cancer patients, subsequent emergence of acquired resistance is almost inevitable. Resveratrol and its derivatives have been found to exert some effects on EGFR‐TKI resistance in non‐small cell lung cancer (NSCLC), but the underlying mechanisms remain unclear. We screened several NSCLC cell lines with gefitinib resistance by MTT assay and analysed the miR‐345/miR‐498 expression levels. NSCLC cells were pre‐treated with a resveratrol derivative, trans‐3,5,4‐trimethoxystilbene (TMS) and subsequently challenged with gefitinib treatment. The changes in apoptosis and miR‐345/miR‐498 expression were analysed by flow cytometry and q‐PCR respectively. The functions of miR‐345/miR‐498 were verified by CCK‐8 assay, cell cycle analysis, dual‐luciferase reporter gene assay and immunoblotting analysis. Our results showed that the expression of miR‐345 and miR‐498 significantly decreased in gefitinib resistant NSCLC cells. TMS pre‐treatment significantly upregulated the expression of miR‐345 and miR‐498 increasing the sensitivity of NSCLC cells to gefitinib and inducing apoptosis. MiR‐345 and miR‐498 were verified to inhibit proliferation by cell cycle arrest and regulate the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways by directly targeting MAPK1 and PIK3R1 respectively. The combination of TMS and gefitinib promoted apoptosis also by miR‐345 and miR‐498 targeting the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways. Our study demonstrated that TMS reduced gefitinib resistance in NSCLCs via suppression of the MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345/498. These findings would lay the theoretical basis for the future study of TMS for the treatment of EGFR‐TKI resistance in NSCLCs.     

Differential catalytic efficiency of allelic variants of human glutathione S-transferase Pi in catalyzing the glutathione conjugation of thiotepa.

Alkylating agents are extensively used in the treatment of cancer. The clinical usefulness of this class of anticancer drugs, however, is often limited by the emergence of drug-resistant tumor cells. Increased glutathione (GSH) conjugation through catalysis by GSH S-transferases (GSTs) is believed to be an important mechanism in tumor cell resistance to alkylating agents. In the present study, we report that the allelic variants of human Pi class GST (hGSTP1-1), which differ in their primary structures at amino acids in positions 104 and/or 113, exhibit significant differences in their activity in the GSH conjugation of alkylating anticancer drug thiotepa. Mass spectrometry revealed that the major product of the reaction between thiotepa and GSH was the monoglutathionyl-thiotepa conjugate. While nonenzymatic formation of monoglutathionyl-thiotepa was negligible, the formation of this conjugate was increased significantly in the presence of hGSTP1-1 protein. The hGSTP1-1-catalyzed GSH conjugation of thiotepa was time and protein dependent and followed Michaelis-Menten kinetics. The catalytic efficiency of hGSTP1-1(I104, A113) variant was approximately 1.9- and 2.6-fold higher compared with hGSTP1-1(V104,A113) and hGSTP1-1(V104,V113) isoforms, respectively. The results of the present study indicate that the hGSTP1-1 polymorphism may be an important factor in GST-mediated tumor cell resistance to thiotepa, and that subjects homozygous for the hGSTP1-1(I104,A113) allele, which is most frequent in human populations, are likely to be at a greater risk for developing GST-mediated resistance to thiotepa than heterozygotes or homozygotes with valine 104 background.     

HOPS复合体蛋白在细胞自噬过程中的功能研究

自噬(autophagy)是一种溶酶体依赖性的细胞内降解途径,其主要功能是将生物大分子(蛋白质、多糖等)或细胞器(线粒体等)回收至溶酶体中并将其降解为单糖、氨基酸等小分子以重复利用。发现HOPS复合体中的两个基因vps39和vps41的缺失会导致酵母内GFP-ATG8大量积累。进一步研究表明,积累的原因是GFP-ATG8与液泡不能发生融合。而在HOPS复合体中的另外两个基因vps16和vps18缺失的情况下,自噬融合没有受到影响;在vps16和vps18双敲除的菌株中,自噬融合同样没有受到影响。该实验结果为理解HOPS复合体的功能和自噬体与液泡融合的过程提供了新的线索。     

喀斯特小流域栓皮栎林土壤螨类群落结构特征

为比较石漠化环境与喀斯特森林土壤螨类的群落结构差异,对贵州喀斯特地区朝营小流域栓皮栎林的土壤螨类群落结构本底进行了研究,经2014年各季节的4次调查,共发现土壤螨类3目54科83属.对螨类属数、个体数量、个体密度、Shannon多样性指数(H)、Margalef丰富度指数(SR)、Pielou均匀性指数(J)、捕食性螨类成熟度指数(MI)、甲螨MGP类群和甲螨营养结构等进行了分析.结果表明:土壤螨类在类群属数和个体数量上均以甲螨亚目的属占优势,夏季和秋季具有丰富的属数、较高的个体密度与多样性,春季和秋季具有丰富的个体数量,群落分布具有明显的表聚性.捕食性螨类夏季生态类群以K选择型为主,其他季节以r选择型为主;甲螨生态类群主要为P型和O型,缝甲螨属、异珠足甲螨属和合若甲螨属等属构成了栓皮栎林土壤螨类的营养功能集团.研究表明,该区山毛榉林与其他地区山毛榉林、其他不同类型森林的土壤螨类主要类群存在差异,其中含丰富属组成的派伦螨科、厉螨科、奥甲螨科和单翼甲螨科以及多奥甲螨属、派伦满属、菌甲螨属和单翼甲螨属等数量上占优势的类群属可作为山毛榉林土壤环境的生物指示.     

抗菌肽VIP在毕赤酵母中的高效表达及鉴定

基于人抗菌肽VIP(Vasoactive intestinal peptide)基因序列,按照毕赤酵母密码子偏好性设计引物;用SOE-PCR法扩增目的基因;然后将目的基因克隆至毕赤酵母分泌型表达载体pPICZαA上,构建VIP分泌表达菌株GS115-p PICZαA-vip。用甲醇诱导96 h收集上清,用质谱进行鉴定,结果显示分泌表达产物与人抗菌肽VIP理论值(3 326.82 Da)完全一致,表明人抗菌肽VIP成功得到分泌表达。琼脂糖凝胶扩散法实验结果显示,重组VIP对大肠杆菌Escherichia coli ATCC25922和金黄色葡萄球菌Staphylococcus aureus ATCC25923都有很强的抗菌活性,MIC(Minimal inhibitory concentration)分别为8 mmol/L和16 mmol/L。进一步细胞毒性和溶血性实验结果显示,重组VIP对正常细胞NCM460和IPEC-J2没有毒性,其对SD大鼠红细胞不具有溶血活性。通过透射电镜观察了VIP的抗菌机制,结果显示VIP主要通过破坏细胞膜的方式抑杀细菌。本研究为人抗菌肽VIP的开发应用和大量生产奠定了基础。     

巴氏杆菌糖酵解酶的黏附作用研究

巴氏杆菌（主要是多杀性巴氏杆菌）可以引起多种动物疫病（巴氏杆菌病）,同时也引起人类感染发病。[目的] 研究巴氏杆菌糖酵解酶对宿主细胞（兔肾细胞）和两种常见分子[纤连蛋白（fibronectin,Fn）和血浆纤维蛋白溶解酶原（plasminogen,Plg）]的黏附作用。[方法] 采用原核表达系统对多杀性巴氏杆菌的糖酵解酶进行表达并纯化及制备多克隆抗体,通过菌体表面蛋白定位检测、黏附与黏附抑制等实验探究巴氏杆菌糖酵解酶的黏附作用。[结果] 菌体表面蛋白检测结果显示除烯醇化酶和丙酮酸激酶外的7个糖酵解酶在多杀性巴氏杆菌表面存在。这7个糖酵解酶均能黏附兔肾细胞,但仅有磷酸葡萄糖异构酶的多克隆抗体能对多杀性巴氏杆菌黏附宿主细胞产生抑制作用。Far Western blotting结果显示9个糖酵解酶均能结合宿主Fn和Plg。招募抑制实验结果显示磷酸葡萄糖异构酶、醛缩酶、磷酸甘油酸变位酶的抗体对多杀性巴氏杆菌结合Fn和Plg都有抑制作用,磷酸果糖激酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶抗体仅对多杀性巴氏杆菌结合Fn或Plg有抑制作用。[结论] 多杀性巴氏杆菌糖酵解酶成员葡萄糖异构酶、磷酸果糖激酶、醛缩酶、丙糖磷酸异构酶、甘油醛-3-磷酸脱氢酶、磷酸甘油激酶、磷酸甘油酸变位酶在多杀性巴氏杆菌黏附宿主细胞或分子过程中发挥作用。该研究的完成将加深巴氏杆菌病分子发病机制的认识,并为巴氏杆菌病的诊断标识筛选、新型疫苗创制和药物靶标筛选等提供基础数据。     

Induction of defence gene expression by oligogalacturonic acid requires increases in both cytosolic calcium and hydrogen peroxide in Arabidopsis thaliana

Responses to oligogalacturonic acid (OGA) were determined in transgenic Arabidopsis thaliana seedlings express-ing the calcium reporter protein aequorin. OGA stimulated a rapid, substantial and transient increase in the concentration of cytosolic calcium ([Ca^2 ]cyt) that peaked after ca. 15 s. This increase was dose-dependent, saturating at ca. 50 μg Gal equiv/ml of OGA. OGA also stimulated a rapid generation of H202. A small, rapid increase in H2O2 content was followed by a much larger oxidative burst, with H2O2 content peaking after ca. 60 min and declining thereafter. Induction of the oxidative burst by OGA was also dose-dependent, with a maximum response again being achieved at ca. 50 μg Gal equiv/mL. Inhibitors of calcium fluxes inhibited both increases in [Ca^2 ]cyt and [H2O2], whereas inhibitors of NADPH oxidase blocked only the oxidative burst. OGA increased strongly the expression of the defence-related genes CHS,GST, PAL and PR-1. This induction was suppressed by inhibitors of calcium flux or NADPH oxidase, indicating that increases in both cytosolic calcium and H2O2 are required for OGA-induced gene expression.