6aR,12aR,12a-羟基紫穗槐苷元上调Bim诱导肝癌细胞SMMC-7721发生凋亡的机制

肝细胞癌(HCC)是一种高度恶性的肿瘤。化疗是临床上一种重要的治疗方法,但由于化疗药物的毒副作用和耐药效应,现有的化疗药物或多或少有一定的局限性。因此,寻找毒副作用小且有效的化疗药物一直是肝癌患者的迫切需求。本研究从紫穗槐种子中分离纯化出一种天然产物6aR,12aR,12a-羟基紫穗槐苷元,采用CCK-8法检测了其对人肝癌SMMC-7721细胞的杀伤作用,应用流式细胞术检测了其对肝癌细胞的凋亡诱导情况,通过免疫印迹法检测了6aR,12aR,12a-羟基紫穗槐苷元上调Bim蛋白表达进而诱导肝癌细胞凋亡的作用机制。结果表明,6aR,12aR,12a-羟基紫穗槐苷元可呈浓度依赖性和时间依赖性抑制人肝癌SMMC-7721细胞的活力,24 h、48 h和72 h的半数抑制浓度(IC50)分别为21.77μmol/L、5.65μmol/L、5.0μmol/L;同时可浓度依赖性地提高细胞凋亡率;可通过下调抑凋亡蛋白Survivin、Mcl-1、Bcl-XL、Bcl-2,上调促凋亡蛋白Bak、Bim、Bax、Bid的表达,进而促进PARP和caspase-3的活化,从而诱导细胞发生凋亡;进一步地研究发现:6aR,12aR,12a-羟基紫穗槐苷元通过延长Bim的半衰期而增加Bim的积累,且具有和MG132类似的生物学功能,能够阻断蛋白的降解途径来抑制Bim降解。上述研究表明,6aR,12aR,12a-羟基紫穗槐苷元可通过抑制蛋白降解从而上调细胞中Bim蛋白的水平,进而诱导肝癌细胞SMMC-7721发生凋亡。     

细胞和分子技术在甘薯茎线虫病抗病育种中的应用

甘薯茎线虫病是我国北方甘薯薯区的重要病害之一。为进一步研究甘薯茎线虫病育种中线虫和甘薯的抗感关系,利用细胞培养技术建立发根农杆菌转化甘薯发根,用该发根培养马铃薯腐烂线虫,构建植物寄生线虫培养和抗性鉴定的技术平台;用聚合酶链式反应(polymerase chain reaction,PCR)分子标记技术筛选甘薯茎线虫病抗感品种的分子标记,并对发根培养马铃薯腐烂线虫体系和分子标记辅助选择抗甘薯茎线虫病品种技术进行了展望。     

甘薯、胡萝卜发根单寄主培养体系繁殖马铃薯腐烂线虫的研究

利用发根农杆菌A4转化甘薯品种徐薯18和胡萝卜品种天红2号的发根,建立甘薯茎线虫病病原线虫(马铃薯腐烂线虫)的单寄主培养体系。通过该体系对马铃薯腐烂线虫的行为进行观察以及繁殖情况进行调查。结果表明:(1)马铃薯腐烂线虫在甘薯和胡萝卜发根上都能正常发育和繁殖,完成其生活周期;(2)培养4周和8周后,在甘薯发根上线虫繁殖倍数分别为2.6和50.6倍;在胡萝卜发根上线虫繁殖倍数分别为1.7和9.9倍;相同培养时间内,线虫在甘薯发根上的繁殖数极显著高于在胡萝卜发根上的繁殖数。(3)利用发根系统繁殖马铃薯腐烂线虫,便于研究其行为,在显微镜下可以直接观察到线虫在发根上活动情况,这对研究发根和线虫相互关系十分有利。基于上述结果,初步证实构建甘薯发根单寄主培养体系繁殖马铃薯腐烂线虫是可行的,且优于胡萝卜发根繁殖马铃薯腐烂线虫体系。     

C3aR过度活化与糖尿病肾病肾组织损伤的关系

C3aR是补体C3裂解产物C3a的受体.最近的一些研究提示C3aR通路可能参与了糖尿病肾病(DN)的病理过程,但有关C3aR通路在DN中的确切病理作用及有关机制远未清楚.需要特别指出的是,现有的有关C3aR参与DN肾组织损伤的证据主要来自一些动物模型的研究,临床上尚缺乏较为系统全面的对DN患者肾组织C3aR通路与肾组织损伤关系的观察分析.为此,本文首次以较大的样本量分析了不同病理时期DN患者肾组织C3aR和C3a的表达变化情况及其与DN患者肾组织损伤的相关性.在此基础上,进而利用体外细胞模型,对高糖环境下C3aR活化致肾小球足细胞损伤的作用及机制进行了探讨.结果显示:a.与正常对照组相比,DN患者肾组织C3a和C3aR的表达水平随DN的进展而升高,C3aR在DN患者肾组织中的表达上调主要见于肾小管上皮细胞和肾小球足细胞;b.DN患者肾组织C3aR和C3a水平与患者肾组织损伤程度,特别是小管和小管间质损伤程度、肾小球足细胞损伤程度具有显著相关性;c.外加C3a激活C3aR可使高糖环境中的足细胞的细胞骨架发生明显改变、足细胞标记分子表达下调、足细胞通透性增加.这些结果说明:a.DN患者肾组织中确实存在C3a/C3aR轴过度活化的现象;b.C3a/C3aR轴的过度活化很可能在DN患者肾组织损伤,特别是小管和小管间质损伤、肾小球足细胞损伤中具有重要作用;c.可能通过破坏成熟足细胞特有的细胞骨架,改变足细胞标记分子表达,增加足细胞的通透性,C3a/C3aR轴过度活化参与DN足细胞损伤过程.本文不仅为C3a/C3aR通路参与DN病理过程提供了新的必不可少的临床证据,也增加了对C3a/C3aR通路过度活化致DN患者肾组织损伤机制,特别是肾小球足细胞损伤机制的了解,这对于拓展对DN病理机制的认识,发展DN防治新思路,无疑都是有益的.     

黄花木的异黄酮类成分研究(英文)

黄花木(Piptanthus concolor Harrow)茎枝85%乙醇提取物采用多种色谱技术分离纯化,从中分离到11个异黄酮类化合物,经波谱分析鉴定为美迪紫檀素(1),山槐素(2),阿弗洛莫生(3),11b-羟基-11b,1-二氢美迪紫檀素(4),多花紫藤苷(5),trifolirhizin(6),wighteone(7),3’-甲氧基-大豆苷元(8),红车轴草素(9),毛蕊异黄酮(10),erythrinin C(11)。其中,化合物1～9和11为首次从该植物中分离得到。     

缺氧通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR表达

C3aR是补体C3a的受体．在肾脏,已发现C3aR表达于包括肾小管上皮细胞在内的多种细胞．在特定的病理情况下,C3aR表达上调并参与多种肾脏疾病的病理过程,但有关C3aR在肾脏细胞中的表达调控机制仍不清楚．小管间质缺氧是肾脏疾病中的一种常见现象,也是一种重要致病因素．为了探讨缺氧对C3aR的表达调控作用,本文利用体外缺氧模型,对模型条件下C3aR在肾小管上皮细胞中的表达变化情况进行了分析,同时检测了HIF-1α和NF-κB的表达变化及活化情况,以及HIF-1α和NF-κB抑制剂对C3aR的表达影响情况．结果发现缺氧可诱导C3aR mRNA及蛋白质水平的表达上调、HIF-1α和NF-κB的核转移．HIF-1α和NF-κB抑制剂可阻断缺氧对C3aR的诱导作用,且HIF-1α抑制剂可抑制NF-κB的核转移．这些结果说明缺氧可通过HIF-1α/NF-κB通路诱导肾小管上皮细胞C3aR的表达．考虑到C3aR活化可促进肾小管的损伤,　我们推测C3aR通路可能参与了缺氧和NF-κB诱导的肾小管损伤过程,可能是防治缺氧和NF-κB诱导肾组织损伤的一个新靶标．     

过敏毒素受体(C3aR)在db/db糖尿病肾病小鼠肾脏中的表达及病理意义分析

利用半定量RT-PCR、免疫组化和Western blotting的方法,同时从mRNA水平和蛋白质水平对过敏毒素受体(C3aR)在不同病理阶段的2型糖尿病肾病模型小鼠——db/db小鼠肾脏中的表达情况进行了较为系统的分析．结果发现：a．在糖尿病前的db/db小鼠(4周龄的db/db小鼠),C3aR与作为正常对照的db/m小鼠相比没有明显差异．随着肥胖的加剧,高血糖、蛋白尿的发生和发展,C3aR在db/db小鼠肾脏中的表达显著升高．b．免疫组化分析显示,C3aR广泛地表达于db/m和db/db小鼠肾脏的皮质和髓质,分布于肾脏的上皮细胞中(包括肾小管上皮细胞、肾小球中的脏层上皮细胞(足细胞)和壁层上皮细胞)．从部位来看,皮髓交界处的肾小管中C3aR表达量明显要比其他部位的多．在肾小球,C3aR特异地存在于足细胞部位．在db/m小鼠,不同周龄小鼠肾脏中C3aR的表达量并没有明显变化,但在db/db小鼠,从8周龄开始,分布在db/db小鼠肾小管上皮细胞和小球足细胞中的C3aR均随小鼠周龄的增加而增加,至少在时间上,与小鼠糖尿病肾病的发生发展相关,其中尤以足细胞中和皮髓交界处肾小管上皮细胞中的变化最为明显． c．在糖尿病肾病小鼠中高表达C3aR的肾小管上皮细胞常有空泡变性的情况．上述工作印证了先前对2型糖尿病肾病患者肾小球基因表达谱的分析结果,更加明确了C3aR与糖尿病肾病的相关性,同时揭示了C3aR在正常小鼠和糖尿病肾病小鼠肾脏中的表达、分布和变化规律,有利于进一步揭示C3aR的功能及其在糖尿病肾病发生、发展过程中的可能作用,探讨糖尿病肾病的分子机制．     

高表达C3aR的肥大细胞在糖尿病肾病患者肾组织中的分布及病理意义分析

选取糖尿病肾病(diabetic nephropathy,DN)早期、中期、晚期以及正常移植供肾各15例,分别作为DN早期组、中期组、晚期组和正常对照组,利用各个病例的肾穿刺组织标本进行C3aR免疫组化染色,分析各组中高表达C3aR浸润细胞的数量和分布特点,及其与DN发生、发展的关系,利用连续切片、免疫荧光双套色染色、免疫电镜、及甲苯胺蓝染色等方法对DN患者肾组织中高表达C3aR的浸润细胞进行细胞类型鉴定.结果表明:a.光镜和免疫电镜的形态学分析显示DN患者肾组织中高表达C3aR的浸润细胞在形态上具有肥大细胞的特点,免疫荧光双套色染色的分析显示,C3aR浸润细胞CD45阴性、CD68和类胰蛋白酶阳性,与肥大细胞的情况一致,甲苯胺蓝特殊染色进一步证实这是一种肥大细胞.b.正常移植供肾组织中虽有肥大细胞分布,但数量很少;从DN早期组到DN中期组,再到DN晚期组,肾组织中肥大细胞的数量有一种不断增加的趋势;DN患者肾组织肥大细胞的数量与24 h尿蛋白和血肌酐水平均具有很好的线性相关性.上述工作不仅揭示了在DN发生发展过程中肥大细胞在DN患者肾组织中的数量及分布情况,提示肥大细胞很可能参与了DN的发生发展过程,同时,DN患者肾组织肥大细胞高表达过敏毒素C3a受体C3aR的发现,提示C3a/C3aR通路很可能在DN患者肥大细胞的招募和激活过程中有重要作用.     

基于MaxEnt模型的腐烂茎线虫在内蒙古地区适生区预测

【目的】为保障马铃薯产业健康发展,加强腐烂茎线虫的检疫防控工作,对其在内蒙古地区的适生区进行预测分析。【方法】根据腐烂茎线虫在我国最新的分布和环境数据,通过MaxEnt生态位模型和ArcGIS软件,预测当前和未来气候条件下腐烂茎线虫在内蒙古地区的适生区。【结果】MaxEnt模型的AUC平均值为0.929,预测结果准确可靠。预测结果显示:当前气候条件下腐烂茎线虫在内蒙古地区的适生区面积为41.8万km²,占全区面积的35.31%。研究表明,在2021—2040年和2041—2060年,腐烂茎线虫在内蒙古地区的总适生区面积将不断扩大;其中在SSP1_2.6气候情景下,在赤峰市、通辽市和兴安盟将出现腐烂茎线虫的高适生区;在SSP3_7.0气候情景下,在鄂尔多斯市、赤峰市、通辽市将出现腐烂茎线虫高适生区。利用Jackknife刀切法计算得到影响腐烂茎线虫分布的主导环境因子为温度季节性变化方差、最湿月降雨量、最冷季平均温、昼夜温差月均温、最湿季平均温。【结论】腐烂茎线虫在内蒙古地区有进一步扩散的风险,建议在未来气候情景下出现腐烂茎线虫高度适生区的盟市,应加强调运检疫和疫情监测,防止腐烂茎线虫进一步扩散。     

越南槐根的抗HIV活性成分研究

为研究越南槐(Sophora tonkinensis)根的抗HIV蛋白酶活性成分及其分子对接机制,采用硅胶、MCI、Sephadex LH-20等多种色谱分离方法,对越南槐根的化学成分进行分离,采用HIV蛋白酶对化合物进行体外抗HIV活性筛选,运用分子对接手段初步探究活性化合物与HIV-1蛋白酶的结合机制。结果表明,从越南槐中共分离得到8个化合物,根据波谱数据分别鉴定为三叶豆紫檀苷(1)、苦参碱(2)、N-acetylnicotinamide (3)、2′-O-甲基腺苷(4)、毛蕊异黄酮苷(5)、玫瑰花苷(6)、环广豆根素(7)、芒柄花苷(8),此外还分离得到塑化剂衍生物邻苯二甲酸二(2-乙基)己酯(9)和邻苯二甲酸二异丁酯(10)。抗HIV蛋白酶活性测试显示化合物1和2的IC50分别为13.2和38.6μg/m L,分子对接表明其与HIV蛋白酶有一定的结合作用。化合物3～5为首次从该植物中分离得到,化合物1和2显示中等的抗HIV蛋白酶活性。     

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or β-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a–C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.     

Structure and characterization of a high affinity C5a monoclonal antibody that blocks binding to C5aR1 and C5aR2 receptors

C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a–C5aR1 receptor are well defined, whereas C5a–C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement–mediated bacterial cell killing. Unlike other anti–C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a–C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia–reperfusion injury.     

Discovery of human antibodies against the C5aR target using phage display technology

Phage display technologies have been increasingly utilized for the generation of therapeutic, imaging and purification reagents for a number of biological targets. Using a variety of different approaches, we have developed antibodies with high specificity and affinity for various targets ranging from small peptides to large proteins, soluble or membrane-associated as well as to activated forms of enzymes. We have applied this approach to G-protein coupled receptors (GPCRs), often considered difficult targets for antibody therapeutics and targeting. Here we demonstrate the use of this technology for the identification of human antibodies targeting C5aR, the chemoattractant GPCR receptor for anaphylatoxin C5a. The N-terminal region (residues 1-31) of C5aR, one of the ligand binding sites, was synthesized, biotinylated and used as the target for selection. Three rounds of selection with our proprietary human Fab phage display library were performed. Screening of 768 isolates by phage ELISA identified 374 positive clones. Based on sequence alignment analysis, the positive clones were divided into 22 groups. Representative Fab clones from each group were reformatted into IgGs and tested for binding to C5aR-expressing cells, the differentiated U-937 cells. Flow cytometric analysis demonstrated that nine out of 16 reformatted IgGs bound to cells. Competition with a C5aR monoclonal antibody S5/1 which recognizes the same N-terminal region showed that S5/1 blocked the binding of positive cell binders to the peptide used for selections, indicating that the identified cell binding IgGs were specific to C5aR. These antibody binders represent viable candidates as therapeutic or imaging agents, illustrating that phage display technology provides a rapid means for developing antibodies to a difficult class of targets such as GPCRs.     

Pterocarpans and Isoflavans from Astragalus membranaceus (Fisch.) Bunge

A new pterocarpan, (6aR, llaR)-3, 9-dimethoxy-l0-hydroxypterocarpan with four known compounds, (6aR,11aR)-3,9,10-tri-methoxypterocarpan,9,l0-di-methoxypterocarpan-3-O-β-D-glucopyranoside, (3R)-2'-hydroxy-7, 3', 4'-trimethoxy-isoflavan, 2'-hydroxy-3', 4'- dimethoxyisoflavan-7-O-β-D-glucopyranoside, were isolated from ethanolic extract of the root of Astragalus membranaceus (Fisch.) Bunge. Their structures were elucidated by means of spectral analysis and chemical conversions.     

A new prenylisoflavone from Ulex jussiaei

A new naturally occurring isoflavone, derrone, was isolated from Ulex jussiaei (Leguminosae) together with the isoflavones ulexins A-C, lupalbigenin, isolupalbigenin, 7-O-methylso-lupalbigenin, isoderrone, ulexone A and isochandalone, the pterocarpans (6aR,11aR)-(-)-maackiain, (6aR,11aR)-(-)-2-methoxymaackiain and (6aR,11aR)-(-)-4-methoxymaackiain, the chalcone 4-hydroxylonchocarpine and the dihydrochalcone crotaramosmine. The antifungal activity of the new compound was tested by a bioautographic method against Cladosporium cucumerinum, and as expected from structural features it proved to have no activity.     

Structural role of bacterioruberin in the trimeric structure of archaerhodopsin-2

Archaerhodopsin-2 (aR2), a retinal protein-carotenoid complex found in the claret membrane of Halorubrum sp. aus-2, functions as a light-driven proton pump. In this study, the membrane fusion method was utilized to prepare trigonal P321 crystals (a = b = 98.2 Å, c = 56.2 Å) and hexagonal P6₃ crystals (a = b = 108.8 Å, c = 220.7 Å). The trigonal crystal is made up of stacked membranes in which the aR2 trimers are arranged on a honeycomb lattice. Similar membranous structures are found in the hexagonal crystal, but four membrane layers with different orientations are contained in the unit cell. In these crystals, the carotenoid bacterioruberin [5,32-bis(2-hydroxypropan-2-yl)-2,8,12,16,21,25,29,35-octamethylhexatriaconta-6,8,10,12,14,16,18,20,22,24,26,28,30-tridecaene-2,35-diol] binds to crevices between the subunits of the trimer. Its polyene chain is inclined from the membrane normal by an angle of about 20° and, on the cytoplasmic side, it is surrounded by helices AB and DE of neighbouring subunits. This peculiar binding mode suggests that bacterioruberin plays a striking structural role for the trimerization of aR2. When compared with the aR2 structure in another crystal form containing no bacterioruberin, the proton release channel takes a more closed conformation in the P321 or P6₃ crystal; i.e., the native conformation of protein is stabilized in the trimeric protein-bacterioruberin complex. Interestingly, most residues participating in the trimerization are not conserved in bacteriorhodopsin, a homologous protein capable of forming a trimeric structure in the absence of bacterioruberin. Despite a large alteration in the amino acid sequence, the shape of the intratrimer hydrophobic space filled by lipids is highly conserved between aR2 and bacteriorhodopsin. Since a transmembrane helix facing this space undergoes a large conformational change during the proton pumping cycle, it is feasible that trimerization is an important strategy to capture special lipid components that are relevant to the protein activity.     

Estrogenic constituents of the heartwood of Dalbergia parviflora

From the heartwood of Dalbergia parviflora, five compounds, dalparvin A (1), B (2), C (3), dalparvinol C (4), and neokhriol A (5), along with 11 known compounds, kenusanone G (6), cajanin (7), sophorol (8), alpinetin (9), hesperetin (10), 3'-O-methylorobol, odoratin, (2R)(3R)-2,3-trans 7-hydroxy-5-methoxydihydroflavonol, (6aR, 11aR)-3,8-dihydroxy-9-methoxypterocarpan, (6aR, 11aR)- vesticarpan, and methyl-3,4-dihydroxy-2-methoxybenzoate were isolated and characterized. Isolates were evaluated for their cell proliferation stimulatory activity against MCF-7, T-47D, and BT20 human breast cancer cell lines. Along with 7-10, two compounds 2 and 3 stimulated not only MCF-7, but also T-47D human breast cancer cell proliferation. Compound 6 had activity only against MCF-7 cells, and the activity of 7 was more than equivalent to that of daidzein. On the other hand, none of the isolates had any significant effects on BT20 cell proliferation, and these results indicated that the stimulative activity of these compounds was not general to any cell proliferations. Furthermore, these compounds were tested in the estrogen-responsive transient luciferase reporter assay.