Synthesis,in vitro stability,and antiproliferative effect of d‐cysteine modified GnRH‐doxorubicin conjugates

Overexpression of gonadotropin‐releasing hormone (GnRH) receptor in many tumors but not in normal tissues makes it possible to use GnRH analogs as targeting peptides for selective delivery of cytotoxic agents, which may help to enhance the uptake of anticancer drugs by cancer cells and reduce toxicity to normal cells. The GnRH analogs [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH, [d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH, and [d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH were conjugated with doxorubicin (Dox), respectively, through N‐succinimidyl‐3‐maleimidopropionate as a linker to afford three new GnRH‐Dox conjugates. The metabolic stability of these conjugates in human serum was determined by RP‐HPLC. The antiproliferative activity of the conjugates was examined in GnRH receptor‐positive MCF‐7 human breast cancer cell line by MTT assay. The three GnRH‐Dox conjugates showed improved metabolic stability in human serum in comparison with AN‐152. The antiproliferative effect of conjugate II ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NHEt]‐GnRH‐Dox) on MCF‐7 cells was higher than that of conjugate I ([d ‐Cys⁶, desGly¹⁰, Pro⁹‐NH₂]‐GnRH‐Dox) and conjugate III ([d ‐Cys⁶, α‐aza‐Gly¹⁰‐NH₂]‐GnRH‐Dox), and the cytotoxicity of conjugate II against GnRH receptor‐negative 3T3 mouse embryo fibroblast cells was decreased in comparison with free Dox. GnRH receptor inhibition test suggested that the antiproliferative activity of conjugate II might be due to the cellular uptake mediated by the targeting binding of [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH to GnRH receptors. Our study indicates that targeting delivery of conjugate II mediated by [d ‐Cys⁶‐des‐Gly¹⁰‐Pro⁹‐NHEt]‐GnRH is a promising strategy for chemotherapy of tumors that overexpress GnRH receptors.     

中国县域碳汇时空格局及影响因素

以全国1300个县级行政单位作为研究对象，利用全国县域尺度土地利用数据和社会经济数据，核算了1990-2015年间中国县域碳汇总量，并结合标准差椭圆、空间自相关、冷热点分析和地理加权回归分析方法，探析中国县域碳汇的时空分异特征及影响因素，旨在为优化国土空间开发格局，实施差异化减排路径及推动生态文明建设提供参考。研究表明：（1）时空变化上，1990-2015年中国碳汇总量呈波动下降趋势，由13307.79×10⁴t下降至13198.27×10⁴t；林地为主要碳汇类型，其余碳汇类型比例结构基本不变；在空间分布上，中国县域碳汇呈现"西部 > 东北 > 南部 > 中部"的"西高东低"格局。（2）在空间分异和聚集上，碳汇空间分布中心向西南移动，分布范围呈收缩态势，西南地区对整体碳汇空间格局影响作用加强；1990-2015年中国县域碳汇总量的冷热点集聚程度呈现波动稳定特征，空间集聚程度呈现高值与低值聚集，高-低区域零星分布的特征。（3）从影响因素分析来看，2015年经济发展、产业结构、土地利用程度对碳汇产生影响并存在空间异质性。建议通过合理规划县域非建设用地的土地利用方式、差异性制定各区域内策略、控制建设用地规模等方式达到县域的低碳发展。     

Quantitative proteomics study of breast cancer cell lines isolated from a single patient: Discovery of TIMM17A as a marker for breast cancer

The proteins involved in breast cancer initiation and progression are still largely elusive. To gain insights into these processes, we conducted quantitative proteomic analyses with 21T series of breast cell lines, which include a normal, primary tumor and a metastatic tumor that were isolated from a single patient. Stable isotope labeling of amino acid in cell culture followed by LC‐MS/MS analysis was performed and deregulated proteins were identified using statistical analysis. Gene ontology analysis revealed that proteins involved in metabolic processes were the most deregulated in both tumorigenesis and metastasis. Interaction network analysis indicated that ERBB2 signaling played a critical role in tumorigenesis. In addition to known markers such as ERBB2 and E‐cadherin, novel markers, including BRP44L, MTHFD2 and TIMM17A, were found to be overexpressed in 21T breast cancer cells and verified in additional breast cell lines. mRNA expression analysis as well as immunohistochemistry analysis in breast cancer tissues indicated that expression level of TIMM17A was directly correlated with tumor progression, and survival analysis suggested that TIMM17A was a powerful prognosis factor in breast cancer. More interestingly, overexpression and siRNA knockdown experiments indicated an oncogenic activity of TIMM17A in breast cancer. Our study provides a list of potential novel markers for breast cancer tumorigenesis and metastasis using a unique cell model. Further studies on TIMM17A as well as other markers on the list may reveal mechanisms that result in more effective therapeutics for cancer treatment.     

人DFF45蛋白多克隆抗体制备及其初步应用

目的：表达、纯化带GST标签的DNA裂解因子45（DNA fragmentation factor 45 ,DFF45）融合蛋白并制备多克隆抗体。方法：构建pGEX-5X-1/DFF45原核表达质粒,转化大肠杆菌BL21,用IPTG诱导融合蛋白表达,经纯化后免疫日本大耳白兔得到多克隆抗体并用CNBr-activated Sepharose? 4B进行纯化。用间接ELISA法检测抗体效价,Western blot鉴定抗体特异性,同时用免疫荧光染色鉴定抗体特异性并观察DFF45的细胞定位。进一步检测DFF45在人类几种细胞系的表达差异情况。结果：成功构建原核表达质粒,表达、纯化DFF45蛋白并免疫动物后得到多克隆抗体。间接ELISA法显示抗体效价达1:20 000,Western blot确定抗体具有高度特异性。应用该抗体检测发现DFF45在人类几种细胞系中表达存在差异并观察到其细胞定位。结论：DFF45多克隆抗体的成功制备及其在人类细胞系的差异表达,为进一步研究DFF45基因与肿瘤及其相关疾病的关系奠定了基础。     

滇产与日产松茸的IGS1-RFLP比较分析

应用Cfr13I限制性内切酶对采自于云南省16个市县的127个松茸子实体进行了IGS1-RFLP比较分析,发现126个松茸子实体属于A类型,来自大理白族自治州剑川县的1例(TF89)为C类型。通过对A类型的IGS1序列分析发现产自云南的松茸有一个CTTT的简单重复,滇产松茸IGS1序列差异不明显。滇产松茸的IGS1-RFLP与日产松茸的主要类型十分相似,两地松茸可能是同源的。     

乌勇布拉克盐湖嗜盐古菌絮凝效果筛选及活性检测

【目的】从嗜盐古菌中筛选可产生生物絮凝剂的菌株,对发酵液、上清液、菌悬液、胞外聚合物的絮凝作用进行检测,筛选能够适应高盐废水处理,且具有广谱盐度及pH作用范围的微生物絮凝剂。【方法】以新疆乌勇布拉克干盐湖沉积物为研究对象,利用纯培养方法对嗜盐古菌进行分离,对絮凝菌株进行初筛及16S rRNA基因测序,构建系统进化树,初步判断菌株分类地位;复筛检测不同生物材料的絮凝效果;选择絮凝效果较好的生物材料,检测其盐度、pH的絮凝效果稳定性。【结果】采用纯培养方法共分离到28株嗜盐古菌,絮凝初筛共筛选出16株嗜盐古菌,分布于碱线菌属(Natrinema)、盐缓长菌属(Halopiger)和盐土生菌属(Haloterrigena)。菌株发酵液、上清液、菌悬液、胞外聚合物具有不同程度的絮凝效果。菌株A279-1、A133、RP33、NGA0064、RM-152、A389的发酵液、上清液的絮凝效果较好,其中菌株A389的发酵液絮凝率为61.06%,上清液为67.92%。所有菌株菌悬液的絮凝率达到80%以上。菌株所产胞外聚合物表现出较好的絮凝效果,菌株RM-152所产胞外聚合物的絮凝率最高,达89.86%,其次是A389 (81.53%)。菌株A389所产胞外聚合物的产量最大,达12.53 g/L,具有广泛的盐度和pH适应性。【结论】乌勇布拉克干盐湖沉积物中蕴含丰富的可产生微生物絮凝剂的嗜盐古菌资源。嗜盐古菌菌株发酵液、上清液、菌悬液及胞外聚合物均具有良好的絮凝作用,尤其是胞外聚合物表现出较好的絮凝效果,具有广谱的盐度和pH耐受性。嗜盐古菌所产生物絮凝剂的发现对于后续高盐废水功能材料开发具有重要应用价值。     

用超微细胞化学定位技术揭示ATP酶在紫茎泽兰高温适应性中的作用

【背景】外来人侵植物紫茎泽兰自然演化出耐高温种群,其适应机制与各种生理代谢有关。【方法】本文从超微细胞化学水平,对紫茎泽兰抗高温种群、敏感种群ATP酶活性定位,明确其在高温适应性中的作用,试图阐明该草的生态适应机制。【结果】正常情况下,紫茎泽兰ATP酶主要定位于细胞壁及细胞间隙周围的细胞壁表面;经40℃高温处理后,在不同的处理时间下,抗性、敏感种群之间ATP酶的活性表现出明显差异,其中以处理12h时差异最大,具体表现为抗高温种群的ATP酶活性明显高于敏感种群,ATP酶的定位点除细胞壁外,在细胞膜上也呈现大量的分布,而敏感种群在处理12h时的酶活性明显降低,只在细胞壁上有零星的分布。处理24h时,敏感种群叶片已完全萎蔫,细胞结构毁坏,细胞膜破损;而抗高温种群叶片仍然完好,细胞膜上仍有ATP酶分布。【结论与意义】经40℃高温处理后,紫茎泽兰抗高温种群ATP酶活性明显高于敏感种群,初步认为紫茎泽兰对高温的适应性与ATP酶活性相关。本研究为进一步阐明与紫茎泽兰适应性相关的入侵机理提供了资料。     

Numerical Simulation of Dynamic Electro-Mechanical Response of Ionic Polymer-Metal Composites

Ionic Polymer-Metal Composites (IPMC) is an emerging class of Electro-Active Polymer (EAP) materials. IPMC has attractive features, such as high sensitivity and light weight, which are useful for developing novel designs in the fields of bionic actuators, artificial muscles and dynamic sensors. A Finite Element (FE) model was developed for simulating the dynamic electro-mechanical response of an IPMC structure under an external voltage input. A lumped Resistor-Capacitor (RC) model was used to describe the voltage-to-current relationship of a Nafion IPMC film for the computation of electric field intensity. Moreover, the viscoelastic property of the IPMC film was considered in the model and the non-uniform bending behavior was also taken into account. Based on the proposed model and the assumption that the thicknesses of the two electrodes are the same and uniform, the optimal coating thickness of the IPMC electrode was determined. It was demonstrated that the dynamic electro-mechanical response of the IPMC structure can be predicted by the proposed FE model, and the simulation results were in good agreement with the experimental findings.     

S100A1 enhances the L-type Ca2+ current in embryonic mouse and neonatal rat ventricular cardiomyocytes

S100A1 is an EF-hand type Ca2+-binding protein with a muscle-specific expression pattern. The highest S100A1 protein levels are found in cardiomyocytes, and it is expressed already at day 8 in the heart during embryonic development. Since S100A1 is known to be involved in the regulation of Ca2+ homeostasis, we tested whether extracellular S100A1 plays a role in regulating the L-type Ca2+ current (I(Ca)) in ventricular cardiomyocytes. Murine embryonic (day 16.5 postcoitum) ventricular cardiomyocytes were incubated with S100A1 (0.001-10 microM) for different time periods (20 min to 48 h). I(Ca) density was found to be significantly increased as early as 20 min (from -10.8 +/- 1 pA/pF, n = 18, to -22.9 +/- 1.4 pA/pF; +112.5 +/- 13%, n = 9, p < 0.001) after the addition of S100A1 (1 microM). S100A1 also enhanced I(Ca) current density in neonatal rat cardiomyocytes. Fluorescence and capacitance measurements evidenced a fast translocation of rhodamine-coupled S100A1 from the extracellular space into cardiomyocytes. S100A1 treatment did not affect cAMP levels. However, protein kinase inhibitor, a blocker of cAMP-dependent protein kinase A (PKA), abolished the S100A1-induced enhancement of I(Ca). Accordingly, measurements of PKA activity yielded a significant increase in S100A1-treated cardiomyocytes. In vitro reconstitution assays further demonstrated that S100A1 enhanced PKA activity. We conclude that the Ca2+-binding protein S100A1 augments transsarcolemmal Ca2+ influx via an increase of PKA activity in ventricular cardiomyocytes and hence represents an important regulator of cardiac function.