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.     

Aromatic‐interaction‐mediated inhibition of β‐amyloid assembly structures and cytotoxicity

Abnormal aggregation of β‐amyloid (Aβ) peptide plays an important role in the onset and progress of Alzheimer's disease (AD); hence, targeting Aβ aggregation is considered as an effective therapeutic strategy. Here, we studied the aromatic‐interaction‐mediated inhibitory effect of oligomeric polypeptides (K8Y8, K4Y8, K8W8) on Aβ42 fibrillization process. The polypeptides containing lysine as well as representative aromatic amino acids of tryptophan or tyrosine were found to greatly suppress the aggregation as evaluated by thioflavin T assay. Circular dichroism spectra showed that the β‐sheet formation of Aβ42 peptides decreased with the polypeptide additives. Molecular docking studies revealed that the oligomeric polypeptides could preferentially bind to Aβ42 through π–π stacking between aromatic amino acids and Phe19, together with hydrogen bonding. The cell viability assay confirmed that the toxicity of Aβ42 to SH‐SY5Y cells was markedly reduced in the presence of polypeptides. This study could be beneficial for developing peptide‐based inhibitory agents for amyloidoses. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Pro-Thyrotropin-Releasing Hormone Processing by Recombinant PC1

Abstract: Pro-thyrotropin-releasing hormone (proTRH) is the precursor to thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH₂), the hypothalamic releasing factor that stimulates synthesis and release of thyrotropin from the pituitary gland. Five copies of the TRH progenitor sequence (Gln-His-Pro-Gly) and seven cryptic peptides are formed following posttranslational proteolytic cleavage of the 26-kDa rat proTRH precursor. The endopeptidase(s) responsible for the physiological conversion of proTRH to the TRH progenitor form is currently unknown. We examined the in vitro processing of [³H]leucine-labeled or unlabeled proTRH by partially purified recombinant PC1. Recombinant PC1 processed the 26-kDa TRH precursor by initially cleaving the prohormone after the basic amino acid at either position 153 or 159. Based on the use of our well-established antibodies, we propose that the initial cleavage gave rise to the formation of a 15-kDa N-terminal peptide (preproTRH_25–152 or preproTRH_25–158) and a 10-kDa C-terminal peptide (preproTRH_154–255 or preproTRH_160–255). Some initial cleavage occurred after amino acid 108 to generate a 16.5-kDa C-terminal peptide. The 15-kDa N-terminal intermediate was further processed to a 6-kDa peptide (preproTRH_25–76 or preproTRH_25–82) and a 3.8-kDa peptide (preproTRH_83–108), whereas the 10-kDa C-terminal intermediate was processed to a 5.4-kDa peptide (preproTRH_206–255). The optimal pH for these cleavages was 5.5. ZnCl₂, EDTA, EGTA, and the omission of Ca²⁺ inhibited the formation of pYE₂₇ (preproTRH_25–50), one of the proTRH N-terminal products, by 48, 82, 72, and 45%, respectively. This study provides evidence, for the first time, that recombinant PC 1 enzyme can process proTRH to its predicted peptide intermediates.     

Determination of genetic relationships among five indigenous Chinese goat breeds with six microsatellite markers

Microsatellite variation was analyzed in five Chinese indigenous goat breeds, which include four Cashmere breeds (Tibetan, Neimonggol, Liaoning, Taihang) and one Hubei local breed (Matou) used for meat production. Five ovine and one bovine microsatellites, selected from eight ovine microsatellites and five bovine microsatellites were suitable for use in this study. With these six microsatellites, allele frequencies, heterozygosity, polymorphism information content (PIC) and effective allele number were calculated. A neighbor-joining tree was constructed using Nei's standard genetic distance (1978). In the tree, Neimonggol and Liaoning were grouped together, then with Taihang; while Tibetan and Matou individually had their own branch. The genetic relationship of five breeds corresponds to their history and geographic origins.     

微生态制剂在胃肠道疾病中的临床应用

微生态制剂(microbial ecological agents, MEA)是利用益生菌及其代谢产物而制成的一种药物制剂。MEA主要是通过补充有益的微生物来重建人体肠道内的菌群平衡,以治疗多种胃肠道疾病。现就近年来MEA在胃肠道中的作用机制,以及在防治炎症性肠病、与抗生素相关的腹泻、幽门螺杆菌感染和慢性肝病等疾病中的临床应用作一概述,为更好地开发和利用MEA治疗疾病奠定基础。     

苦瓜MAP30蛋白基因克隆、表达及其抗肿瘤活性研究

通过PCR技术,从苦瓜总DNA中扩增出编码MAP30成熟蛋白的基因,经测序鉴定后亚克隆到原核表达载体pET30a中,构建成带有N端6Histag的融合表达载体。表达载体用CaCl2介导的化学转化法转化E.coliBL21(DE3),然后利用PCR筛选阳性克隆。工程菌经1mmol/LIPTG诱导4h实现高效表达,而且在30℃时融合蛋白表达量最高,约占菌体总蛋白56%。可溶性分析表明,该融合蛋白在大肠杆菌中主要以可溶的形式存在。重组蛋白通过Ni2+鏊合亲和层析进行纯化,纯化蛋白占上清总蛋白37.2%,发酵液产率为250mg/L。Westernblot分析表明,重组蛋白可与兔抗histag多克隆抗体发生特异性反应。利用MTT法分析重组MAP30的细胞毒性,结果表明其对小鼠3T3和S180肿瘤细胞株具有明显的抑制作用,ID50分别约为50μg/ml和30mg/ml,而对人正常胚肺二倍体WI38细胞株的毒性极小。     

Environmental Drivers and Spatial Prediction of the Critically Endangered Species <i>Thuja sutchuenensis</i> in Sichuan-Chongqing,China

Identifying the ecological environment suitable for the growth of Thuja sutchuenensis and predicting other potential distribution areas are essential to protect this endangered species. After selecting 24 environmental factors that could affect the distribution of T. sutchuenensis, including climate, topography, soil and Normalized Difference Vegetation Index (NDVI), we adopted the Random Forest-MaxEnt integrated model to analyze our data. Based on the Random Forest study, the contribution of the mean temperature of the warmest quarter, mean temperature of the coldest quarter, annual mean temperature and mean temperature of the driest quarter was large. Based on MaxEnt model prediction outputs, the potential distribution map not only identified areas that originally recorded T. sutchuenensis, such as Xuanhan County, Kai County and Chengkou County, but also identified highly suitable distribution areas where T. sutchuenensis may exist, including Wanyuan County, Sichuan Province, and the junction of Chongqing and Hubei Province. This provides a more explicit geographic range for ex situ conservation and reintroduction of T. sutchuenensis. Our results also indicate that, in addition to climate factors, topography and soil factors are also important environmental factors that affect distribution. This provides a theoretical basis for subsequent laboratory construction to simulate the indoor growth of T. sutchuenensis.     

MD Simulations and first principles to evaluate the role of binary Fe–V alloys layer on the radiation resistance in the alpha-iron

Radiation damage in reactor materials caused by the collision of the fast neutrons has a great impact on the reliability and safety of nuclear reactors. The element vanadium has attracted interest in many fields due to its advantageous properties in alloys. Thus, molecular dynamics simulation (MD) and first-principles calculation have been executed here to explore the radiation-resistant properties of five materials adding a layer in the bulk (pure iron and four types of Fe–V alloys containing 10%-40% V). The following results were inferred from these simulations. Firstly, the number of Frenkel pairs (FPs) at the stable quenching stage in the bulk decreases when the Fe–V alloy is added as an anti-radiation layer to the bulk. These benefits are evident for the Fe₈₀V₂₀ and alloy layers with more vanadium. The main reason is that the Fe–V binding energy is greater than the Fe-Fe binding energy, which can make the Primary Knock-On atom (PKA) lose more energy at the Fe–V alloy layer. Secondly, the average value of point-defect, cluster and defect clustered fractions in the bulk of Fe–V alloy is smaller than that in the pure iron at the stable quenching stage, especially for the Fe₈₀V₂₀ alloy.     

TLR4全长及其截断体重组腺病毒的制备和功能鉴定

制备脂多糖 (LPS)Toll样受体 4 (TLR4 )全长及其胞内段缺失的TLR4截断体 (ΔTLR4 )的绿色荧光蛋白重组腺病毒并鉴定其功能 .用PCR方法扩增TLR4及ΔTLR4基因片段 ,酶切后亚克隆至腺病毒穿梭质粒中 ,形成带有目的基因的穿梭载体pAdTrack TLR4和pAdTrack ΔTLR4 .用BJ5 1 83细菌同源重组法将目的基因重组于腺病毒骨架载体中 ;将重组腺病毒质粒用PacⅠ酶切线性化后 ,用脂质体法转染HEK 2 93细胞进行腺病毒的包装扩增 .将重组腺病毒感染CHO K1细胞 ,采用荧光毒酶报告基因方法检测其对LPS诱导NF κB激活的影响 .酶切及测序表明 ,TLR4全长及其截断体ΔTLR4的重组腺病毒载体构建正确 .荧光素酶报告基因检测结果表明 ,TLR4全长及其截断体的重组腺病毒感染细胞对LPS诱导的反应具有不同的影响 ,Ad ΔTLR4明显抑制了LPS引起的NF κB激活 (P <0 0 5 ) ,Ad TLR4则使LPS引起的NF κB活性增强 (P <0 0 5 ) .LPS对细胞的激活作用依赖于TLR4的结构完整性     

白芨的组培快繁(简报)

本文扼要地阐述了以白芨种子为培养材料,经筛选的培养基诱导、分化、生长,最终可获得苗质好、性状均一的白芨;为进一步研究快速繁殖白芨提供参考.