多肽抗体检测原发性肝癌血清标志物ELISA方法的建立及应用

血清多肽是癌症诊断信息的重要来源,建立、优化了检测多肽标志物的直接ELISA法,并应用于肝癌血清中的多肽标志物的检测。制备及纯化针对多肽标志物Pep5的单克隆抗体并进行辣根过氧化物酶标记,用其建立检测相应抗原的直接ELISA法。方法线性范围为1.5-20 ng/mL,检测限为1.24 ng/mL;标准品批内及批间CV分别小于3.66%及4.89%,血清样本批内及批间CV分别小于11.69%及18.18%;线性范围内(9、12和15 ng/mL)的回收率分别为98.98%,99.61%和101.58%。应用该方法共检测160例正常血清、104例肝硬化及156例肝癌患者血清,正常组与肝硬化组及肝癌组间差异显著(P<0.001),Pep5诊断肝癌的敏感性和特异性分别为80.8%和96.2%。同时检测94例HCC血清中的AFP和Pep5,AFP检出率为63.8%,Pep5检出率为90.4%,AFP联合Pep5检测时,能将HCC的检出率提高至94.7%。     

整合素αvβ3对MDA-MB-231BO乳腺癌细胞AKT信号通路的调控

旨在探究整合素αvβ3的单克隆抗体LM609在BSP不同表达水平的乳腺癌细胞中对AKT(蛋白激酶B)信号通路的影响。利用免疫细胞化学法检测BSP不同表达水平的乳腺癌细胞中整合素αvβ3的表达量。BSP基因沉默乳腺癌MDA-MB-231BO细胞,Western blotting在蛋白水平检测磷酸化AKT的表达,MTT试验和细胞划痕试验分别检测细胞增殖、迁移能力的变化。结果显示,与231BO-Scrambled细胞相比,231BO-BSP27细胞中BSP蛋白水平明显降低,抑制率达到(59.43±1.71)%;LM609分别处理两株细胞后,与对照组231BO-Scrambled细胞相比,BSP基因沉默组21BO-BSP27细胞中AKT磷酸化水平下调明显,为(33.78±1.51)%(P<0.01);231BO-BSP27细胞和对照组231BO-Scrambled中细胞的增殖和迁移能力均有不同程度的下降(P<0.05)。LM609能够抑制胞内整合素αvβ3功能的表达,进而对AKT信号通路进行调控,并影响细胞增殖和迁移的发生。     

草莓MDHAR及AO基因的克隆及序列分析

从新鲜幼嫩‘丰香’草莓(Fragaria×ananassa cv.Toyonaka)果实中提取分离总RNA,反转录成cDNA,根据已报道的其他植物单脱氢抗坏血酸还原酶(MDHAR)及抗坏血酸氧化酶(AO)基因的保守区分别设计 一对引物,通过PCR扩增均得到目的条带.序列分析发现:mdhar基因片段长372bp,与刺梨同源性最高达96％,该片段编码123个氨基酸,推导的氨基酸序列与苹果属植物同源性为91％,与其他植物该基因编码的氨基酸序列也有较高相似性;a基因片段长842 bp,编码280个氨基酸,该片段与其他多种植物的ao基因均具有较高同源性,与甜瓜和黄瓜ao基因的同源性最高,均为70％,编码的氨基酸序列与其他多种植物均具有70％左右的相似性.     

一株芝麻香型白酒高温大曲的施莱格尔氏菌的分离与鉴定

从芝麻香型白酒高温大曲中分离到一株革兰氏阴性高温细菌Zm91,并对其进行形态学、生理生化代谢特征以及细胞代谢酶活性和药物敏感性分析,结合16S rRNA基因序列分析结果,Zm91鉴定为水生施莱格尔氏菌(Schlegelella aquatica).该菌株是首次从我国芝麻香型白酒高温大曲中分离得到的施莱格尔氏菌,其G+C mol％含量为67.5％,具有碱性磷酸盐酶、酯酶(C4)、脂酯酶(C8)、白氨酸芳胺酶、颉氨酸芳胺酶、胰凝乳蛋白酶、萘酚-AS-BI-磷酸水解酶及α葡萄糖苷酶活性,对青霉素、氨苄西林、链霉素、利福平敏感.     

高梁抗蚜研究进展

高粱(Sorghum bicolour)是世界上最重要的粮食、饲料、酿造和能源作物之一,也是C4植物研究的模式植物.蚜虫是农业生产上的重要害虫,几乎危害所有的栽培作物.危害高粱的蚜虫主要包括高粱蚜(Melanaphis sacchan)、麦二叉蚜(Schizaphis graminum)和玉米蚜(Rhopalosiphum maidis).高粱的抗蚜资源尚不丰富且缺乏深入系统的研究.目前研究较多的是麦二叉蚜的抗性遗传方面,已定位20个抗性QTLs,单一QTL对抗性差异贡献率最高可达80.3％,对高粱蚜和玉米蚜的研究尚需进一步加强.高粱的理化特性与其抗蚜性能相关,故可与育种实践相结合.高粱和蚜虫(Acyrthosiphon pisum)的全基因组测序工作已经完成,这将有助于蚜虫-植物间的相互作用关系及植物对蚜虫的抗性机制研究.目前已克隆到2个抗蚜基因,且多个抗蚜基因(位点)已被定位在染色体上.该文重点综述了上述研究成果并对高粱抗蚜的研究前景进行了展望.     

基于不同零模型的点格局分析

在种群空间格局研究中,定量分析格局及其形成过程已成为生态学家的主要目标。在量化分析的众多方法中,点格局分析是最常用的方法,而在选择零模型时,完全空间随机模型以外的复杂零模型很少使用,实际上,这些零模型可能有助于认识格局的内在特征。为此,我们在研究实例中,选择完全空间随机模型(complete spatial randomness)、泊松聚块模型(Poisson cluster process)和嵌套双聚块模型(nested double-cluster process)对典型草原处于不同恢复演替阶段的羊草(Leymus chinensis)种群空间格局进行了分析。结果发现:完全空间随机模型仅能检测种群在不同尺度下的格局类型;而通过泊松聚块模型和嵌套双聚块模型检验表明,在恢复演替的初期阶段,羊草种群在小尺度范围内偏离泊松聚块模型,而在整个取样范围内完全符合嵌套双聚块模型;随着恢复演替时间的推移,在恢复演替的后期,在整个取样尺度上,羊草种群与泊松聚块模型相吻合。这是很有意义的生态学现象。这一实例表明在应用点格局分析种群空间格局时,仅通过完全空间随机模型的检验来分析格局特征,或许很难论证复杂的生态过程,而选择一些完全空间随机模型以外的较复杂的零模型,可能发现一些有价值的生态学现象,对揭示格局掩盖下的内在机制有所裨益。     

Feasibility and Safety of Transthoracic Echocardiography-Guided Transcatheter Closure of Atrial Septal Defects with Deficient Superior-Anterior Rims

Although previous studies showed that transthoracic echocardiography (TTE) can be used to guide transcatheter closure of atrial septal defect (ASD), whether TTE can be used to guide transcatheter closure of secundum ASD with a deficient superior-anterior rim is unknown and this critical issue was addressed in the present study. A total of 280 patients with secundum ASD who underwent transcatheter ASD closure were recruited and divided into groups A and B depending on ASD superior-anterior rim>4 mm (n = 118) or ≤4 mm (n = 162). TTE was used to guide Amplatzer-type septal occluder (ASO) positioning and assess residual shunt. Procedure success was defined as no, trivial and small residual shunt immediately after the procedure as assessed by color Doppler flow imaging. Group A and group B did not differ in complication rate (8.55% vs.7.55%), procedure success rate (98.3% vs. 95.0%) or complete closure rate immediately after the procedure (89.7% vs. 89.3%) or at 6-month follow-up (98.3% vs. 96.8%). The mean procedure and fluoroscopy time in group B were much longer than those in group A. In conclusion, the absence of a sufficient superior-anterior rim in patients undergoing percutaneous closure of secundum-type ASDs using fluoroscopic and TTE guidance is associated with slightly greater device malposition and migration as well as increased procedural and fluoroscopic times, but the overall complication rate did not differ with TTE guidance when compared to historical controls that used TEE guidance.     

Preferential Release of Newly Synthesized Insulin Assessed by a Multi-Label Reporter System Using Pancreatic β-Cell Line MIN6

Newly synthesized hormones have been suggested to be preferentially secreted by various neuroendocrine cells. This observation indicates that there is a distinct population of secretory granules containing new and old hormones. Recent development of fluorescent timer proteins used in bovine adrenal chromaffin cells revealed that secretory vesicles segregate into distinct age-dependent populations. Here, we verify the preferential release of newly synthesized insulin in the pancreatic β-cell line, MIN6, using a combination of multi-labeling reporter systems with both fluorescent and biochemical procedures. This system allows hormones or granules of any age to be labeled, in contrast to the timer proteins, which require fluorescence shift time. Pulse-chase labeling with different color probes distinguishes insulin secretory granules by age, with younger granules having a predominantly intracellular localization rather than at the cell periphery.     

Molecular basis of recognition of human osteopontin by 23C3, a potential therapeutic antibody for treatment of rheumatoid arthritis

Osteopontin plays an important role in the development and perpetuation of rheumatoid arthritis (RA). Antibodies targeting osteopontin have shown promising therapeutic benefits against this disease. We have previously reported a novel anti-RA monoclonal antibody, namely, 23C3, and shown it capable of alleviating the symptoms of RA in a murine collagen-induced arthritis model, restoring the cytokine production profile in joint tissues, and reducing T-cell recall responses to collagen type II. We describe here the crystal structure of 23C3 in complex with its epitope peptide. Analyses of the complex structure reveal the molecular mechanism of osteopontin recognition by 23C3. The peptide folds into two tandem β-turns, and two key residues of the peptide are identified to be critical for the recognition by 23C3: TrpP43 is deeply embedded into a hydrophobic pocket formed by AlaL34, TyrL36, LeuL46, TyrL49, PheL91, and MetH102 and therefore has extensive hydrophobic interactions with 23C3, while AspP47 has a network of hydrophilic interactions with residues ArgH50, ArgH52, SerH53, and AsnH56 of the antibody. Besides the complementarity-determining region loops, the framework region L2 of 23C3 is also shown to interact with the epitope peptide, which is not common in the antibody-antigen interactions and thus could be exploited in the engineering of 23C3. These results not only provide valuable information for further improvement of 23C3 such as chimerization or humanization for its therapeutic application, but also reveal the features of this specific epitope of osteopontin that may be useful for the development of new antibody drugs against RA.     

Diabetes reduces aortic endothelial gap junctions in ApoE-deficient mice: simvastatin exacerbates the reduction.

We examined the endothelial gap junctions in diabetic hyperlipidemic mice. Male apolipoprotein E (apoE)-deficient mice were made diabetic by streptozotocin. Three weeks later, the animals were treated with simvastatin for 2 weeks. The expression of aortic gap junctions in the non-diabetic (n=10), untreated diabetic (n=10), and simvastatin-treated diabetic animals (n=6) was analyzed. There was a >4-fold increase in serum cholesterol level and >50% increase in plaque areas in the diabetic mice, regardless of simvastatin treatment. Western blotting of aortae showed reduced expression of connexin37 (Cx37) and Cx40 in the diabetic mice, which were further decreased in the simvastatin-treated diabetic mice. Immunoconfocal microscopy showed that endothelial gap junctions made of Cx37 and Cx40 were both reduced in the untreated diabetic mice compared with the non-diabetic mice (decrease: Cx37, 41%; Cx40, 42%; both p<0.01). The reduction was greater in the simvastatin-treated mice (decrease in treated diabetic vs non-diabetic: Cx37, 61%; Cx40, 79%; both p<0.01; decrease in treated diabetic vs untreated diabetic: Cx37, 34%; Cx40, 63%; both p<0.01). Cx37 and Cx40 were decreased in the endothelium of plaque surface. Cx43 appeared in the medial layer and inner layer of the intima. All three connexins were rarely expressed in monocytes/macrophages inside the plaques. In conclusion, in apoE-deficient mice, streptozotocin-induced diabetes is associated with downregulation of endothelial Cx37 and Cx40 gap junctions. Short-term treatment with simvastatin exacerbates the downregulation.