油菜BnHMGB2基因的克隆及表达分析

通过RACE技术从陇油6号油菜中克隆得到了一种新的BnHMGB2基因的cDNA,全长823 bp,其中包括438 bp的开放阅读框,131 bp的5′非翻译区（5′UTR）,253 bp的3′非翻译区（3′UTR）。与拟南芥AtHMGB2的同源性达到了87.4%,因此命名为BnHMGB2(GenBank登录号：JN807314)。该基因编码145个氨基酸的蛋白质,分子量15.9 KDa,等电点为5.63。实时荧光定量PCR结果表明该基因在油菜根、茎、叶、下胚轴均有表达,根中表达量最高。同时,该基因的表达受低温胁迫的诱导,表明该基因在油菜适应低温胁迫的过程中发挥作用。     

Combinatorial network of transcriptional regulation and microRNA regulation in human cancer

Background

In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model??s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes.

Results

We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF) with grouping reaction (GR) constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via ??GR constraints??. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation.

Conclusions

FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm was validated through the experiments on the enhanced production of putrescine in E. coli, in addition to the comparison with the previously reported experimental data. The FVSEOF strategy with GR constraints will be generally useful for developing industrially important microbial strains having enhanced capabilities of producing chemicals of interest.     

MicroRNA-29a-5p Is a Novel Predictor for Early Recurrence of Hepatitis B Virus-Related Hepatocellular Carcinoma after Surgical Resection

It is still difficult to predict the probability of tumor recurrence after resection of hepatocellular carcinoma (HCC). In this study, we set out to identify specific microRNA (miRNA) in microdissected hepatitis B virus (HBV)-related HCC tissue from formalin-fixed paraffin-embedded (FFPE) samples which might be used in predicting early recurrence after HCC resection. Taqman low density arrays were used to detect the 667 miRNA profiles in both the microdissected tumorous and adjacent non-tumorous liver tissues from 20 HCC patients (discovery set) including 10 patients with early tumor recurrence and 10 without early tumor recurrence and to identify the differentially expressed miRNAs related to HCC recurrence. Then quantitative real-time PCR (qRT-PCR) was used to verify the findings in 106 patients (training set), and to develop a predictive assay. The identified miRNAs were further validated in an independent cohort of 112 patients (validation set). Thirty seven miRNAs were identified from 20 HCC patients and validated in 106 HCC patients using qRT-PCR. A significant association was found between miR-29a-5p level in HCC tissues and early tumor recurrence (P = 0.0002). This association was further confirmed in the independent validation set of 112 patients (P = 0.0154). MiR-29a-5p level was significantly associated with both time to tumor recurrence (TTR) (P = 0.0015) and overall survival (OS) (P = 0.0079) in validation set. In the multivariate analyses, miR-29a-5p was identified as an independent factor for TTR, particularly for those patients with early stage of HCC. The sensitivity and specificity of miR-29a-5p for the prediction of early HCC recurrence of BCLC 0/A stage HCC were 74.2% and 68.2%, respectively. These suggest that miR-29a-5p might be a useful marker for the prediction of early tumor recurrence after HCC resection, especially in BCLC 0/A stage HCCs.     

强磁重力环境对人成骨细胞发动蛋白-2表达和定位的影响

研究强磁重力环境对人成骨细胞MG-63中发动蛋白-2表达和分布的影响.采用大梯度超导磁体模拟空间重力环境,该超导磁体可以提供3种不同的强磁重力环境(high magnetic gravi-tational environment,HMGE),即0 g(12 T),1 g(16 T)和2 g(12 T).将MG-63细胞分别在0 g(12 T)、1 g(16 T)、2 g(12 T)及对照环境(1 g,地磁)中培养24 h后,采用Real Time PCR、Western印迹以及激光扫描共聚焦显微术等方法检测HMGE对发动蛋白-2的表达及定位的影响.结果显示,与对照组相比,0 g(12 T)、1 g(16 T),2 g(12T)环境处理组MG-63细胞中发动蛋白-2在mRNA上的表达量分别上调6.43、15.57、1.29倍;在蛋白质水平上,0 g(12 T)、1 g(16 T)环境处理组细胞发动蛋白-2分别上调89%和7%,而2 g(12 T)环境处理组则下调41%;在对照组中发动蛋白-2弥散分布于细胞质中,而0 g(12 T)处理组发动蛋白-2则有从细胞边缘向核周集中的趋势.上述结果说明强磁重力环境影响了发动蛋白-2在MG-63细胞中的表达和定位.     

Enzymatic activity and substrate specificity of mitogen-activated protein kinase p38alpha in different phosphorylation states

The mitogen-activated protein (MAP) kinases are essential signaling molecules that mediate many cellular effects of growth factors, cytokines, and stress stimuli. Full activation of the MAP kinases requires dual phosphorylation of the Thr and Tyr residues in the TXY motif of the activation loop by MAP kinase kinases. Down-regulation of MAP kinase activity can be initiated by multiple serine/threonine phosphatases, tyrosine-specific phosphatases, and dual specificity phosphatases (MAP kinase phosphatases). This would inevitably lead to the formation of monophosphorylated MAP kinases. However, the biological functions of these monophosphorylated MAP kinases are currently not clear. In this study, we have prepared MAP kinase p38alpha, a member of the MAP kinase family, in all phosphorylated forms and characterized their biochemical properties. Our results indicated the following: (i) p38alpha phosphorylated at both Thr-180 and Tyr-182 was 10-20-fold more active than p38alpha phosphorylated at Thr-180 only, whereas p38alpha phosphorylated at Tyr-182 alone was inactive; (ii) the dual-specific MKP5, the tyrosine-specific hematopoietic protein-tyrosine phosphatase, and the serine/threonine-specific PP2Calpha are all highly specific for the dephosphorylation of p38alpha, and the dephosphorylation rates were significantly affected by different phosphorylated states of p38alpha; (iii) the N-terminal domain of MPK5 has no effect on enzyme catalysis, whereas deletion of the MAP kinase-binding domain in MKP5 leads to a 370-fold decrease in k(cat)/K(m) for the dephosphorylation of p38alpha. This study has thus revealed the quantitative contributions of phosphorylation of Thr, Tyr, or both to the activation of p38alpha and to the substrate specificity for various phosphatases.     

中华绒螯蟹受精卵、流产卵氨基酸及脂肪酸组成的比较研究

采用气相色谱仪和氨基酸分析仪测定中华绒螯蟹不同种群 (太湖种群和温州种群 )受精卵和温州种群流产卵脂肪酸及氨基酸的组成。结果表明 ,中华绒螯蟹太湖种群受精卵、温州种群受精卵和温州种群流产卵在必需氨基酸和半必需氨基酸的含量上无显著差异 ,而谷氨酸 (Glu)、胱氨酸 (Cys)和丝氨酸 (Ser)三种非必需氨基酸的含量差异显著。太湖种群受精卵、温州种群受精卵及温州种群流产卵的脂肪酸种类分别为 16、13和 14种。饱和脂肪酸(SFA)占脂肪酸总量的 18 96 %— 2 3 0 9% ,单烯酸 (MUFA)占 5 1 76 %— 6 2 6 5 % ,多不饱和脂肪酸 (PUFA)占14 2 7%— 2 8 96 % ,其中最主要的脂肪酸为C16∶0、C16∶1和C18∶1。除C2 0∶1外 ,其他脂肪酸含量在三者之间都有显著差异。太湖种群受精卵的C18∶2、C18∶3显著高于温州种群受精卵 ,而温州种群流产卵的DHA要显著高于太湖种群受精卵。温州种群受精卵和流产卵在C16以下的脂肪酸 ,其含量均无显著差异。此外 ,本文还讨论了脂肪酸及氨基酸含量与流产的关系及脂肪酸组成对出苗率的影响。     

中国昆虫病原线虫一新纪录

采用分子生物学和形态学相结合的方法,对采自广东省翁源县的编号为GDa71的昆虫病原线虫进行分类鉴定.该线虫各虫期的主要形态特征、测量值以及基于ITS1和部分28S rDNA两段序列,分别构建系统进化树均显示它与印度异小杆线虫Heterorhabditis indica相似,是后者的一个品系,为中国新纪录.     

谷氨酸脱羧酶放射测量法的改良及应用

用NaOH代替苯乙胺作为¹⁴CO₂的吸附剂,改进谷氨酸脱羧酶（GAD）活性的放射测定方法,结果发现NaOH为吸附剂组内变异系数为9.6%, 以苯乙胺为吸附剂组内变异系数为31.9%;以NaOH为吸附剂72 h后测量其放射活性仍稳定不变,以苯乙胺为吸附剂者1 h后放射性活性即下降47%,6 h后已降低至本底水平;¹⁴CO₂重吸收实验亦证明以苯乙胺为吸附剂吸附的¹⁴CO₂ 6 h内已有80%以上重新被NaOH吸附;以NaOH作为吸附剂测定GAD的活性,在0.39～17.8 mg脑组织样品范围内GAD量与¹⁴CO₂生成量之间有线性关系.NaOH代替苯乙胺作为¹⁴CO₂的吸附剂测定GAD的活性其灵敏度提高1.66倍.用此方法测定组织和细胞内GAD活性证明其具有良好的重复性和稳定性,值得推广应用.     

High allozymic diversity in natural populations of Mycoheterotrophic Orchid Gastrodia elata,an endangered medicinal plant in China

Gastrodia elata Blume, a mycoheterotrophic orchid native to the Far East, is an endangered medicinal plant in China. Genetic variation among 19 natural populations of G. elata was examined in central China by using allozyme polymorphism (16 loci in six enzymatic systems). The species exhibited high level of genetic diversity (P = 56.3%, A = 2.2 and H_E = 0.221), which was mainly attributed to its perennial habit and mixed reproduction system (both sexual and asexual). Evident genetic differentiation in G. elata natural populations was suggested by F_ST = 0.241. AMOVA analysis showed 31.3% of the total molecular variation was attributed to inter-population differentiation. Obvious genetic structure and genetic depauperation of some populations indicate forest fragmentation and over-collection have affected genetic variation of G. elata. A conservation strategy, which is conserving populations with great genetic distinction or high level of genetic variation from four management units, is recommended.     

依赖于微环境的TGFβ信号

转化生长因子β（transforming growth factor β，TGFβ）家族成员是一类分泌的细胞因子，在胚胎发育、免疫调节、伤口修复、细胞增殖和抑制等过程中发挥重要作用。其中,TGFβ1、TGFβ2和TGFβ3在进化上最晚出现，并以潜伏态分泌。有别于家族中的大多数成员，TGFβ1-3的信号具有时空区域性，并依赖于其所处的微环境。阐明依赖于微环境的TGFβ信号的多层次调控机制对于理解TGFβ信号在免疫、癌症等生理、病理条件下的功能，以及开发临床治疗策略具有重要意义。本文从TGFβ前体复合物的结构入手，并从TGFβ的激活，配体-受体识别，跨膜信号传导及转录调控等方面，论述依赖于微环境的TGFβ信号的调控机制，同时讨论肿瘤微环境中多效的TGFβ信号，进而对今后的研究方向进行展望。