一种来源于大菱鲆的热敏型尿嘧啶DNA糖苷酶的克隆表达及酶学性质鉴定

目的:尿嘧啶DNA糖苷酶UDGase是一种广泛应用于q PCR、二代测序等领域的工具酶,由于其应用特性,只有热敏性UDGase才具有较大开发利用潜力。目前全球热敏性UDGase工具酶仅有2个物种来源,均有专利保护且价格昂贵,亟待开发新来源且具有优良热敏特性的UDGase。方法:根据前人研究及序列分析推测大菱鲆(Scophthalmus maximus)具有热敏性UDGase。经验证,大菱鲆肝脏匀浆呈现UDGase活性。从大菱鲆肝脏匀浆克隆得到大菱鲆UDGase基因SmUDGase,并使用大肠杆菌工程菌株实现重组表达,分离纯化后进行活力表征检测。结果:序列比对结果表明,SmUDGase基因克隆成功。重组表达并经亲和层析、离子交换层析分离纯化,获得纯酶纯度约95%,产率1. 51mg/L,比活力2 295. 08U/mg。Sm UDGase具有热敏性,在40℃时酶活即开始迅速降低。其他酶学性质,如pH适应范围、金属离子依赖性和对抑制剂的敏感性均与当前商业化UDGase一致。结论:成功克隆并鉴定来自大菱鲆的新来源SmUDGase,该酶具有热敏感性,酶学特性接近目前商业化UDGase。并探索该酶的重组表达和纯化工艺,所得纯酶基本达到商业化生产水平,为该类型生物工具酶的开发提供了理论参考和技术储备。     

产广谱细菌素乳酸菌的筛选和鉴定

以大肠杆菌、金黄色葡萄球菌、藤黄微球菌、铜绿假单胞杆菌和枯草芽孢杆菌为指示菌,从分离自四川传统发酵食品中的267株乳酸菌中,采用平板打孔法初筛、牛津杯法复筛（排除酸、过氧化氢干扰以及胰蛋白酶和木瓜蛋白酶处理）,筛选出1株分离自醪糟的具有较强抑菌作用的产广谱细菌素的乳杆菌菌株P158,结合形态学、生理生化特性和16S rDNA序列同源性分析,该菌株被鉴定为植物乳杆菌（Lactobacillus plantarum）。     

Immunological and structural properties of a pectic polymer from Glinus oppositifolius

The aim of this paper was to further elucidate the structure and the immunomodulating properties of the pectic polymer GOA2, previously isolated from Glinus oppositifolius. Enzymatic treatment of GOA2 by endo-alpha-d-(1 --> 4)-polygalacturonase led to the isolation of three pectic subunits, GOA2-I, GOA2-II, and GOA2-III, in addition to oligogalacturonides. GOA2-I was shown to consist of 1,2-linked Rhap and 1,4-linked GalpA in an approximately 1:1 ratio, and NMR-analysis showed that the monomers were linked together in a strictly alternating manner. The galactose units in GOA2-I were found as terminal-, 1,3-, 1,6-, 1,4-, 1,3,4-, and 1,3,6-linked residues, while the arabinofuranosyl existed mainly as terminal- and 1,5-linked units. A rhamnogalacturonan-I type structure was suggested being the predominant part of GOA2-I. According to linkage analysis GOA2-II and GOA2-III contained glycosidic linkages characteristic for rhamnogalacturonan-II type structures. GOA2 was shown by sedimentation velocity in the analytical ultracentrifuge, to have a broad degree of polydispersity with a mode s(20,w) value of approximately 1.9 S, results reinforced by atomic force microscopy measurements. The polydispersity, as manifested by the proportion of material with s(20,w) > 3 S, decreased significantly with enzyme treatment. The abilities of GOA2, GOA2-I, GOA2-II, and GOA2-III to induce the proliferation of B cells, and to exhibit complement fixing activities were tested. In both test systems, GOA2-I showed significantly greater effects compared to its native pectin GOA2. GOA2-I was in addition shown to exhibit a more potent intestinal immune stimulating activity compared to GOA2. The ability of GOA2 to induce secretion of proinflammatory cytokines was examined. Marked upregulations in mRNA for IL-1beta from rat macrophages and IFN-gamma from NK cells were found.     

广西巴马小型猪PGC-1α基因克隆与组织表达分析

目的克隆广西巴马小型猪PGC-1α基因编码区（CDS）序列,利用RT-PCR和QRT-PCR方法分析PGC-1αmRNA组织表达情况。方法本实验以广西巴马小型猪背最长肌cDNA为模版,PCR扩增PGC-1α基因CDS序列,将其连接至pEASY-T5载体,转染细菌、验证和序列测定;通过RT-PCR半定量和QRT-PCR实时荧光定量检测PGC-1α基因在小型猪多个组织中的表达情况。结果克隆获得广西巴马小型猪PGC-1α基因CDS序列,全长2391 bp,编码796个氨基酸,与参考序列的同源性为99.9%,两处碱基发生同义突变,分别是C-A1105和GA1524;PGC-1α基因在广西巴马小型猪心脏和肾脏中的表达丰度最高,其次是肝脏、皮下脂肪和背最长肌,而在胰腺中未检测到其表达。结论成功克隆了广西巴马小型猪PGC-1α基因编码区序列并进行了多种组织表达分析,为后续研究PGC-1α在小型猪2型糖尿病发生过程中作用途径打下基础。     

低剂量胸部CT 自动管电流调节技术在超重体型患者检查中的 可行性研究

摘要目的：探讨16 层螺旋CT自动管电流调节技术（CARE Dose 4D）在超重体型患者检查中的可行性。方法：收集行胸部CT 检 查的超重体型患者（BMI值于24-27.9 之间）100 例,扫描分低剂量组（A 组）和常规剂量组（B 组）,其中A 组扫描运用CARE Dose 4D技术自动调节管电流;B 组扫描运用常规剂量管电流为70 mAs 扫描。扫描完成后记录加权CT 剂量指数(CTDlvol)、有效mAs 值、剂量长度乘积（DLP）,计算出有效剂量（ED）及剂量减低比值（DR）,并比较两组的剂量及图像质量。结果：与B 组相比,A 组常 规扫描辐射剂量显著降低,差异有显著统计学意义（P<0.01）,A 组剂量减低比值（DR）降低约20.84%,而图像质量无明显下降,不 影响诊断,在主动脉弓上4 cm（肺尖）层面A 组图像质量优于B 组,差异有统计学意义(X2=8.442,P=0.015)。结论：自动管电流调节 技术既可以减少患者的辐射剂量,对待超重体型患者个体化、人性化,又不影响影像诊断,是一项有价值的检查方法。     

重症显微镜下多血管炎1例报告并文献复习

目的:显微镜下多血管炎(microscopic polyangiitis,MPA)通常易侵犯肾脏,很少累及神经系统,而同时累及肺及中枢神经系统的重症病例较少见。本文旨在探讨重症显微镜下多血管炎(MPA)的临床表现、诊断与治疗,为临床提供借鉴。方法:回顾性分析重症显微镜下多血管炎(MPA)的文献资料,结合本中心病例,54岁女性,尿检异常6年,咳血3年因肾功能衰竭合并心衰入院。结合实验室检查及影像学改变,诊断为重症AASV,考虑MPA可能性大。结果:入院后即给予激素联合环磷酰胺冲击治疗,病情明显好转;3周后出现病情反复,再次激素冲击治疗,效果不如前。继而出现脑出血,病情进一步恶化,最终因肾、肺、脑等多器官功能衰竭于入院5周后死亡。结论:对MPA患者关键在于早发现早治疗;而对缓解重症MPA患者病情,激素联合环磷酰胺冲击治疗在初始阶段有效。     

Experimental Evolution of an Oncolytic Vesicular Stomatitis Virus with Increased Selectivity for p53-Deficient Cells

Experimental evolution has been used for various biotechnological applications including protein and microbial cell engineering, but less commonly in the field of oncolytic virotherapy. Here, we sought to adapt a rapidly evolving RNA virus to cells deficient for the tumor suppressor gene p53, a hallmark of cancer cells. To achieve this goal, we established four independent evolution lines of the vesicular stomatitis virus (VSV) in p53-knockout mouse embryonic fibroblasts (p53−/− MEFs) under conditions favoring the action of natural selection. We found that some evolved viruses showed increased fitness and cytotoxicity in p53−/− cells but not in isogenic p53+/+ cells, indicating gene-specific adaptation. However, full-length sequencing revealed no obvious or previously described genetic changes associated with oncolytic activity. Half-maximal effective dose (EC₅₀) assays in mouse p53-positive colon cancer (CT26) and p53-deficient breast cancer (4T1) cells indicated that the evolved viruses were more effective against 4T1 cells than the parental virus or a reference oncolytic VSV (MΔ51), but showed no increased efficacy against CT26 cells. In vivo assays using 4T1 syngeneic tumor models showed that one of the evolved lines significantly delayed tumor growth compared to mice treated with the parental virus or untreated controls, and was able to induce transient tumor suppression. Our results show that RNA viruses can be specifically adapted typical cancer features such as p53 inactivation, and illustrate the usefulness of experimental evolution for oncolytic virotherapy.     

Redox-related gene expression and sugar accumulation patterns are altered in the edible inflorescence produced by the cultivated form of pacaya palm (Chamaedorea tepejilote)

Background and AimsThe pacaya palm is a dioecious neotropical palm species that is exploited in Latin America for its male inflorescence, which is edible when immature. It is cultivated, in a non-intensive manner, in Guatemala, where a morphotype occurs that produces much larger, more highly branched inflorescences compared with wild palms. We sought to identify molecular factors underlying this phenotypic divergence, which is likely to be a product of domestication.MethodsWe performed RNA-seq-based studies on immature pacaya palm male inflorescences in order to identify genes that might be directly or indirectly affected in their expression in relation to domestication. We also measured the accumulation of a range of soluble sugar molecules to provide information on the biochemical status of the two different types of material.Key ResultsA total of 408 genes were found to display significantly different expression levels between the wild and cultivated morphotypes. Three different functional categories were found to be enriched in the gene set that was upregulated in the cultivated morphotype: redox balance; secondary metabolism; and transport. Several sugars were found to accumulate at higher levels in inflorescences of the cultivated morphotype, in particular myo-inositol, fructose and glucose.ConclusionsThe observed upregulation of redox-related genes in the cultivated morphotype is corroborated by the observation of higher myo-inositol accumulation, which has been shown to be associated with enhanced scavenging of reactive oxygen species in other plants and which may affect meristem activity.