微乳体系中11β-羟基甲羟孕酮的C1,2生物脱氢

为改善过程传质,提高甾类药物中间体11β-羟基甲羟孕酮C1,2生物脱氢转化率,采用简单节杆菌Arthrobacter simplex UR016菌株在Tween-80/乙醇/食油/水构成的微乳体系中进行生物脱氢,并考察了微乳体系组成、转化温度、投料浓度对脱氢反应的影响。结果表明:以菌体培养液作为水相,食油作为油相构建微乳体系,食油最适加量为10g/L,表面活性剂Tween-80加量为4g/L;底物经醇溶后水析投料,乙醇最适加量为发酵液体积的7%(V/V);最适转化温度为33oC;当底物浓度为4g/L时,在构建的微乳体系中转化46h,脱氢转化率达88.6%,与水相转化工艺相比提高了66.2%。在该体系中疏水性11β-羟基甲羟孕酮底物得到了有效的增溶和扩散,生物脱氢转化率明显提高。     

上海部分地区戊型肝炎病毒(HEV)基因型的分析

为了解戊型肝炎病毒(HEV)在上海部分地区流行的基因型,采用RT-nPCR的方法检验35例急性散发性戊型肝炎患者中HEV RNA,并对阳性产物进行克隆测序,然后对其基因型进行分析.结果显示在35例急性散发性戊型肝炎患者中PCR阳性为9例,测序证实8例为HEV的基因序列;其中1例为HEV 1型,7例为HEV 4型.提示在上海部分地区的急性散发性戊型肝炎中以HEV 4型感染为主.     

一株广谱中和抗原性出血热病毒株的发现

一株分离自杭州市褐家鼠的出血热病毒Gou_3株的免疫血清对10株I型病毒的中和滴度除二株为160外均为320,而对4株Ⅱ型病毒的滴度为320—640,说明Gou_3株免疫血清对两型毒株中和效价大多数无差异或只差2倍,是一株中和抗原广谱的毒株。用I型和Ⅱ型毒株免疫血清对Gou_3株进行型别检定结果表明Gou_3株是Ⅱ型病毒。     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Homology modeling threedimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis

AnxB1,a novel annexin previously isolated from Cysticercus cellulose,shows high thrombi affinity and anticoagulant activity in vivo.In order to investigate the relationship between structure and biological function,a predicted three-dimensional(3D)model of AnxB1 was generated by homology modeling.This model contains four homologous internal-domains and the Cα trace of domain Ⅰ,Ⅱ and IV shows high similarity.Based on the structure characterization,four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E.coli.Two of the mutants,GST-M3 and GST-M4 reserved high anticoagulant activity(p＜0.01 vs.GST).Furthermore,compared with the wild type GST-AnxB1,the immunogenicity of GST-M3 and GST-M4 was reduced significantly(p＜0.01)and the molecular weight was lowered to 27 kD and 34 kD,respectively.These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.     

刺五加皂苷合成关键酶基因表达的半定量RT-PCR分析

根据已克隆的刺五加鲨烯合酶(squalene synthase,SS)、鲨烯环氧酶(squalene epoxidase,SE)和β-香树酯醇合成酶(β-amyrin synthase,bAS)基因序列信息设计引物,通过半定量RT-PCR分析了SS、SE和bAS基因在刺五加不同生长发育时期和不同器官中表达量的变化.结果表明,SS、SE和bAS基因在各生长发育时期和各器官中均有表达,但表达量差异显著(P<0.05),三者均在盛花期表达量最高,之后降低,进入果实成熟期后SS和bAS的表达量迅速回升,SE无显著变化.SS和bAS在叶片和根中的表达量较高,SE表达量的最大值出现在叶片和幼茎中.刺五加SS、SE和bAS基因的表达间存在显著的正相关关系(P<0.05).研究结果为进一步分析关键酶基因对刺五加三萜皂苷生物合成的影响奠定了基础.     

A sensitive signal-on assay for MTase activity based on methylation-responsive hairpin-capture DNA probe

This work develops a simple, sensitive and signal-on electrochemical sensor for methyltransferase (MTase) activity analysis. The sensor is composed of a methylene blue-modi?ed "signaling DNA probe" and a "capture DNA probe" tethered methylation-responsive hairpin DNA (hairpin-capture DNA probe). The thiol- modified hairpin-capture DNA probe at 5' end was firstly self-assembled on gold electrode via Au-S bonding. Methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the "capture DNA probe" section on the gold electrode. Subsequently, the remained "capture DNA probe" on the gold electrode can hybridize with the methylene blue-modi?ed "signaling DNA probe", mediating methylene blue onto the gold electrode surface to generate redox current. It was eT on state. The developed facile signal-on electrochemical sensing system showed a linear response to concentration of Dam MTase range from 0.1 to 1.0 U/mL. The detection limit of Dam MTase activity was determined to be 0.07 U/mL and the total detection time is 7h. The sensor also has the ability to provide information about the dynamics of methylation process. Furthermore, we demonstrated that this sensor could be utilized to screen inhibitors or drugs for Dam MTase.     

Choline Transporters in Human Lung Adenocarcinoma： Expression and Functional Implications

Choline is an essential nutrient for cell survival and proliferation, however, the expression and function of choline transporters have not been well identified in cancer. In this study, we detected the mRNA and protein expression of organic cation transporter OCT3, carnitine/cation transporters OCTN 1 and OCTN2, and choline transporter-like protein CTL1 in human lung adenocarcinoma cell lines A549, H 1299 and SPC-A-1. Their expression pattern was further confirmed in 25 human primary adenocarcinoma tissues. The choline uptake in these cell lines was significantly blocked by CTL1 inhibitor, but only partially inhibited by OCT or OCTN inhibitors. The efficacy of these inhibitors on cell proliferation is closely correlated with their abilities to block choline transport. Under the native expression of these transporters, the total choline uptake was notably blocked by specific PI3K/AKT inhibitors. These results describe the expression of choline transporters and their relevant function in cell proliferation of human lung adenocarcinoma, thus providing a potential ＂choline-starvation＂ strategy of cancer interference through targeting choline transporters, especially CTL1.     

不同加热方式对嗜肺军团菌环介导等温扩增检测法的影响

目的:研究不同的加热方式对嗜肺军团菌环介导等温扩增检测法的影响方法:用已知的13株嗜肺军团菌样本,采用空气浴、水浴和PCR仪同时进行环介导等温扩增,观察沉淀反应、荧光反应以及产物电泳结果。结果:水浴和PCR仪加热LAMP反应的沉淀产物较多,荧光反应较强,电泳检测结果较为明显。空气浴的3种检测结果均较弱。结论:采用水浴和PCR仪进行环介导等温扩增反应的效果较好,从仪器设备的成本及实验条件考虑,采用水浴是环介导等温扩增反应首选的加热方式。     

Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.