Mutation of Trp84 and Asp313 of the dopamine transporter reveals similar mode of binding interaction for GBR12909 and benztropine as opposed to cocaine

The different psychomotor-stimulant effects of cocaine, GBR12909, and benztropine may partially stem from their different molecular actions on the dopamine transporter (DAT). To explore this possibility, we examined binding of these inhibitors to mutated DATs with altered Na(+) dependence of DAT activities and with enhanced binding of a cocaine analog, [(3)H]2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT). In [(3)H]CFT competition assays with intact cells, the mutation-induced change in the ability of Na(+) to enhance the apparent affinity of CFT, cocaine, GBR12909, and benztropine was inhibitor-independent. Thus, for the four inhibitors, the curve of [Na(+)] versus apparent ligand affinity was steeper at W84L compared with wild type, shallower at D313N, and flat at W84LD313N. At each mutant, the apparent affinity of CFT and cocaine was enhanced regardless of whether Na(+) was present. However, the apparent affinity of GBR12909 and benztropine for W84L was reduced in the absence of Na(+) but near normal in the presence of 130 mm Na(+), and that for D313N and W84LD313N was barely changed. At the single mutants, the alterations in Na(+) dependence and apparent affinity of the four inhibitors were comparable between [(3)H]CFT competition assays and [(3)H]dopamine uptake inhibition assays. These results demonstrate that DAT inhibitors producing different behavioral profiles can respond in an opposite way when residues of the DAT protein are mutated. For GBR12909 and benztropine, their cocaine-like changes in Na(+) dependence suggest that they prefer a DAT state similar to that for cocaine. However, their cocaine-unlike changes in apparent affinity argue that they, likely via their diphenylmethoxy moiety, share DAT binding epitopes that are different from those for cocaine.     

植物发育与microRNA

microRNA为动、植物的一类内源、非编码小分子RNA,其长度约为23nt,对动、植物的发育具有非常重要的调节作用.microRNA作为调节类的核酸分子,通过识别和负调控靶基因来行使其生物学功能,多数microRNA的表达具有时空特异性,它们对植物的发育有多向性调节,在发育的多个层面具有重要的功能,有些microRNA的调节作用还要受到外因的诱导.该文对近年来国内外有关microRNA在植物发育过程中的功能及其研究进展进行综述.     

A well-characterized polycistronic-like gene expression system in yeast

Efficient expression of multiple genes is critical to yeast metabolic engineering for the bioproduction of bulk and fine chemicals. A yeast polycistronic expression system is of particular interest because one promoter can drive the expression of multiple genes. 2A viral peptides enable the cotranslation of multiple proteins from a single mRNA by ribosomal skipping. However, the wide adaptation of 2A viral peptides for polycistronic-like gene expression in yeast awaits in-depth characterizations. Additionally, a one-step assembly of such a polycistronic-like system is highly desirable. To this end, we have developed a modular cloning (MoClo) compatible 2A peptide-based polycistronic-like system capable of expressing multiple genes from a single promoter in yeast. Characterizing the bi-, tri-, and quad-cistronic expression of fluorescent proteins showed high cleavage efficiencies of three 2A peptides: E2A from equine rhinitis B virus, P2A from porcine teschovirus-1, and O2A from Operophtera brumata cypovirus-18. Applying the polycistronic-like system to produce geraniol, a valuable industrial compound, resulted in comparable or higher titers than using conventional monocistronic constructs. In summary, this highly-characterized polycistronic-like gene expression system is another tool to facilitate multigene expression for metabolic engineering in yeast.     

Systematics of Mukdenia and Oresitrophe (Saxifragaceae): Insights from genome skimming data

Oresitrophe and Mukdenia (Saxifragaceae) are epilithic sister genera used in traditional Chinese medicine. The taxonomy of Mukdenia, especially of M. acanthifolia, has been controversial. To address this, we produced plastid and mitochondrial data using genome skimming for Mukdenia acanthifolia and Mukdenia rossii, including three individuals of each species. We assembled complete plastomes, mitochondrial CDS and nuclear ribosomal ETS/ITS sequences using these data. Comparative analysis shows that the plastomes of Mukdenia and Oresitrophe are relatively conservative in terms of genome size, structure, gene content, RNA editing sites and codon usage. Five plastid regions that represent hotspots of change (trnH-psbA, psbC-trnS, trnM-atpE, petA-psbJ and ccsA-ndhD) are identified within Mukdenia, and six regions (trnH-psbA, petN-psbM, trnM-atpE, rps16-trnQ, ycf1 and ndhF) contain a higher number of species-specific parsimony-informative sites that may serve as potential DNA barcodes for species identification. To infer phylogenetic relationships between Mukdenia and Oresitrophe, we combined our data with published data based on three different datasets. The monophyly of each species (Oresitrophe rupifraga, M. acanthifolia and M. rossii) and the inferred topology ((M. rossii, M. acanthifolia), O. rupifraga) are well supported in trees reconstructed using the complete plastome sequences, but M. acanthifolia and M. rossii did not form a separate clade in the trees based on ETS + ITS data, while the mitochondrial CDS trees are not well-resolved. We found low recovery of genes in the Angiosperms353 target enrichment panel from our unenriched genome skimming data. Hybridization or incomplete lineage sorting may be the cause of discordance between trees reconstructed from organellar and nuclear data. Considering its morphological distinctiveness and our molecular phylogenetic results, we strongly recommend that M. acanthifolia be treated as a distinct species.     

Carbon uptake in Eurasian boreal forests dominates the high-latitude net ecosystem carbon budget

Arctic-boreal landscapes are experiencing profound warming, along with changes in ecosystem moisture status and disturbance from fire. This region is of global importance in terms of carbon feedbacks to climate, yet the sign (sink or source) and magnitude of the Arctic-boreal carbon budget within recent years remains highly uncertain. Here, we provide new estimates of recent (2003–2015) vegetation gross primary productivity (GPP), ecosystem respiration (R_eco), net ecosystem CO₂ exchange (NEE; R_eco − GPP), and terrestrial methane (CH₄) emissions for the Arctic-boreal zone using a satellite data-driven process-model for northern ecosystems (TCFM-Arctic), calibrated and evaluated using measurements from >60 tower eddy covariance (EC) sites. We used TCFM-Arctic to obtain daily 1-km² flux estimates and annual carbon budgets for the pan-Arctic-boreal region. Across the domain, the model indicated an overall average NEE sink of −850 Tg CO₂-C year⁻¹. Eurasian boreal zones, especially those in Siberia, contributed to a majority of the net sink. In contrast, the tundra biome was relatively carbon neutral (ranging from small sink to source). Regional CH₄ emissions from tundra and boreal wetlands (not accounting for aquatic CH₄) were estimated at 35 Tg CH₄-C year⁻¹. Accounting for additional emissions from open water aquatic bodies and from fire, using available estimates from the literature, reduced the total regional NEE sink by 21% and shifted many far northern tundra landscapes, and some boreal forests, to a net carbon source. This assessment, based on in situ observations and models, improves our understanding of the high-latitude carbon status and also indicates a continued need for integrated site-to-regional assessments to monitor the vulnerability of these ecosystems to climate change.     

酸角研究进展

从酸角的地理分布,生物生态学特性,类型划分,酸角的开发利用,酸角的栽培管理技术等方面,对酸角的研究进展进行了综述;探讨了酸角研究存在的问题及对策,以期为酸角的进一步研究提供参考。     

Theoretical insights into the effects of molar ratios on stabilities,mechanical properties,and detonation performance of CL-20/HMX cocrystal explosives by molecular dynamics simulation

To research and estimate the effects of molar ratios on structures, stabilities, mechanical properties, and detonation properties of CL-20/HMX cocrystal explosive, the CL-20/HMX cocrystal explosive models with different molar ratios were established in Materials Studio (MS). The crystal parameters, structures, stabilities, mechanical properties, and some detonation parameters of different cocrystal explosives were obtained and compared. The molecular dynamics (MD) simulation results illustrate that the molar ratios of CL-20/HMX have a direct influence on the comprehensive performance of cocrystal explosive. The hardness and rigidity of the 1:1 cocrystal explosive was the poorest, while the plastic property and ductibility were the best, thus implying that the explosive has the best mechanical properties. Besides, it has the highest binding energy, so the stability and compatibility is the best. The cocrystal explosive has better detonation performance than HMX. In a word, the 1:1 cocrystal explosive is worth more attention and further research. This paper could offer some theoretical instructions and technological support, which could help in the design of the CL-20 cocrystal explosive.     

The first intron of rice EPSP synthase enhances expression of foreign gene

The first intron (EPI) of rice 5-enolpyruvylshikimate 3-phosphate synthase gene was isolated by PCR from one clone with genomic EPSP synthase gene. Sequence analysis showed that the first intron is 704 bp in length with 36.2% G+C content. To investigate its effect on expression of foreign gene, we inserted the first intron between CaMV35S promoter and β-glucuronidase (GUS) gene. The transient expression results showed that GUS could be expressed effectively with EPI. The GUS activity in transgenic tobacco shows that the EPI can greatly enhance the expression level of β-glucuronidase (P < 0.01) compared with transgenic tobacco without the first intron, and 3-to 6-fold increase in GUS activity in some transgenic tobaccos. Northern blot indicated the first intron was spliced from GUS pre-mRNA, and the steady-state mRNA levels of GUS with EPI in transgenic tobaccos were higher than that in transgenic tobacco without EPI, which suggested that the first intron of EPSP was a non-translated intron.     

肿瘤坏死因子相关的凋亡诱导配体(TRAIL)cDNA的克隆、表达和活性测定

肿瘤坏死因子相关的凋亡诱导配体 (TRAIL)能选择性诱导肿瘤细胞凋亡 .为利用基因工程技术获得重组TRAIL蛋白可溶性片段 (sTRAIL) ,设计 1对引物 .利用PCR技术特异性扩增出sTRAIL的cDNA ,克隆于质粒pGEM 3Zf( )的EcoRⅠ和PstⅠ位点 .经测序证明序列正确后克隆于表达质粒pBV2 2 0的EcoRⅠ和PstⅠ位点 ,转化大肠杆菌DH5α .转化菌株经温度诱导 ,SDS PAGE检测和Western印迹鉴定 ,获得重组sTRAIL的高水平非融合表达菌株 .表达量占菌体总蛋白的 2 0 % .对其表达产物进行了初步纯化 ,SDS PAGE结果显示纯度可达 90 %以上 .用L92 9细胞测定其生物学活性表明 ,重组蛋白在体外能明显诱导肿瘤细胞凋亡