Crystal structure of the VHS and FYVE tandem domains of Hrs, a protein involved in membrane trafficking and signal transduction

We have determined the 2 A X-ray structure of the 219-residue N-terminal VHS and FYVE tandem domain unit of Drosophila Hrs. The unit assumes a pyramidal structure in which the much larger VHS domain (residues 1-153) forms a rectangular base and the FYVE domain occupies the apical end. The VHS domain is comprised of an unusual "superhelix" of eight alpha helices, and the FYVE domain is mainly built of loops, two double-stranded antiparallel sheets, and a helix stabilized by two tetrahedrally coordinated zinc atoms. The two-domain structure forms an exact 2-fold-related homodimer through antiparallel association of mainly FYVE domains. Dimerization creates two identical pockets designed for binding ligands with multiple negative charges such as citrate or phosphatidylinositol 3-phosphate.     

Low sink-induced stomatal closure alters photosynthetic rates of source leaves in beans as dependent on H2O2 and ABA accumulation in guard cells

Low sink demand provided by pod removal and stem girdling of beans (Vicia faba, cv. Daqingshan) (-Sink) induced a significantly lower net photosynthetic rate (P _n), stomatal conductance (g _s), internal CO₂ concentration (C _i), and transpiration rate (E) compared with pod and root sink retention (CK). This depression in P _n was due to stomatal limitation. Low sink demand of -Sink plants resulted in a higher leaf sucrose content, but a lower sucrose content in guard cells. Moreover, the significant accumulation of H₂O₂ and ABA were observed in both leaves and guard cells of -Sink plants. The most intensive electron dense deposit of cerium perhydroxides, produced by H₂O₂ reaction with cerium chloride, was present in the cell walls, especially the dorsal walls of guard cells. Immunogold electron-microscopy localization of ABA showed that ABA was distributed in ventral walls of guard cells and the intercellular space of mesophyll cells of -Sink leaves in contrast to CK plants. Application of exogenous sucrose to isolated bean leaves increased H₂O₂ and ABA contents. H₂O₂ and ABA in leaves was likely generated by two independently regulated pathways, each affected by the high sucrose concentration induced by low sink demand. Increased sucrose in leaves in response to low sink demand may have caused the increase of H₂O₂ and ABA, and their accumulation in mesophyll cells and guard cells was likely the primary cause for stomatal closure under low sink demand.     

Polyploidization and sexual dimorphism of floral traits in a subdioecious population of Dasiphora glabra

银露梅亚雌雄异株种群的多倍化和花特征性二态 性二态是性别分离植物的常见性状,对植物的雌性和雄性功能可能产生不同的影响。本文以青藏高原特有植物银露梅(Dasiphora glabra)为研究对象,利用银露梅同时存在两性、雌性和雄性植株的种群,探讨银露梅与传粉相关的花特征性二态现象及其对传粉者访问、花粉流和结实的影响。另外,本文还利用流式细胞仪检查了两性植株、雄性植株和雌性植株之间基因组大小的差异。研究结果表明：银露梅雌性植株和雄性植株数量约占种群中植株总数量的40%,表明银露梅两性植株数量多于雄性和雌性的植株数量。两性植株的花数量显著多于雄性和雌性植株的花数量。雄花的花大小显著大于雌花和两性花的花大小。雄花的花粉数量与两性花的花粉数量没有差异,但是雌花的胚珠数量显著高于两性花的胚珠数量。双翅目的蝇类是银露梅最主要的访花者,尽管访花者偏好访问更大的花,但是在花间连续访问对大花没有表现出偏好。模拟花粉流实验表明,两性植株和雄性植株的有效花粉转移都很低,从而导致较低的结实率。雌性植株和雄性植株的DNA含量约为两性植株的4倍,并且单性植株和两性植株之间相互授粉不能结实。以上结果表明,与两性花相比,雌花和雄花在与传粉相关的花特征二态性上分别在雌性和雄性功能上表现出优势,但这种优势在很大程度上被低效的花粉转移所掩盖。银露梅的雌性植株和雄性植株经历了多倍化事件,导致与两性植株之间存在繁殖隔离,从而维持了3种性别植株的共存。     

Selective electrochemical detection of cysteine in complex serum by graphene nanoribbon

Selective detection of cysteine in serum samples was achieved on a graphene nanoribbon (GNR) and Nafion nanocomposite modified electrode with high precision. The superior conductivity and abundant amount of active chemical oxygen groups on the edge of GNR led to extremely highly electrocatalytic activity of GNR towards the electrochemical oxidation of cysteine at +0.025 V. The electrocatalytic behavior was further used for sensitive detection of cysteine by differential pulse voltammetry. Under optimized conditions, the calibration curve was linear in the range from 25 nM to 500 μM. The electrochemical sensor showed strong antifouling ability, good stability and selectivity. It could effectively exclude the interferences from other kinds of biothiols and the biological relevant species, thus had great perspective for in vivo analysis of biological samples.     

弱激光对改善血红蛋白携氧功能的机理研究

本文用群论分析了卟啉的对称结构,并用量子力学导出激光－铁卟啉相互作用系统的自由能,改进了文献「２」的工作,对激光血管内照射能增强血液携氧能力的现象进行诠释。     

丙型肝炎病毒核心蛋白基因真核表达质粒的构建和表达

目的:构建丙型肝炎病毒(HCV)核心蛋白真核表达质粒,并在HepG2细胞中稳定表达.方法:采用RT-RCR方法从丙型肝炎患者血清中得到HCVC区的cDNA序列,然后克隆入pcDNA3.0载体,构建真核表达质粒pcDNA-HCV,并进行酶切鉴定和测序鉴定;采用脂质体转染技术将pcDNA-HCV稳定转染于HepG2细胞系,并用G-418进行筛选;采用Western Blotting方法和免疫细胞化学方法检测HepG2细胞中HCV核心蛋白表达情况.结果:从HCV感染者血清中扩增得到的503bpHCV cDNA序列.属于HCV 1型基因C区,并成功构建了真核表达质粒pcDNA-HCV.经Western blotting和免疫细胞化学检测,稳定转染的HepG2细胞中有HCV核心蛋白的表达.结论:成功构建HCV核心蛋白真核表达质粒pcDNA-HCV,并可以在真核细胞HepG2中稳定表达.     

Transcriptional activation of microRNA-34a by NF-kappa B in human esophageal cancer cells

Background

RNA ligases are essential reagents for many methods in molecular biology including NextGen RNA sequencing. To prevent ligation of RNA to itself, ATP independent mutant ligases, defective in self-adenylation, are often used in combination with activated pre-adenylated linkers. It is important that these ligases not have de-adenylation activity, which can result in activation of RNA and formation of background ligation products. An additional useful feature is for the ligase to be active at elevated temperatures. This has the advantage or reducing preferences caused by structures of single-stranded substrates and linkers.

Results

To create an RNA ligase with these desirable properties we performed mutational analysis of the archaeal thermophilic RNA ligase from Methanobacterium thermoautotrophicum. We identified amino acids essential for ATP binding and reactivity but dispensable for phosphodiester bond formation with 5’ pre-adenylated donor substrate. The motif V lysine mutant (K246A) showed reduced activity in the first two steps of ligation reaction. The mutant has full ligation activity with pre-adenylated substrates but retained the undesirable activity of deadenylation, which is the reverse of step 2 adenylation. A second mutant, an alanine substitution for the catalytic lysine in motif I (K97A) abolished activity in the first two steps of the ligation reaction, but preserved wild type ligation activity in step 3. The activity of the K97A mutant is similar with either pre-adenylated RNA or single-stranded DNA (ssDNA) as donor substrates but we observed two-fold preference for RNA as an acceptor substrate compared to ssDNA with an identical sequence. In contrast, truncated T4 RNA ligase 2, the commercial enzyme used in these applications, is significantly more active using pre-adenylated RNA as a donor compared to pre-adenylated ssDNA. However, the T4 RNA ligases are ineffective in ligating ssDNA acceptors.

Conclusions

Mutational analysis of the heat stable RNA ligase from Methanobacterium thermoautotrophicum resulted in the creation of an ATP independent ligase. The K97A mutant is defective in the first two steps of ligation but retains full activity in ligation of either RNA or ssDNA to a pre-adenylated linker. The ability of the ligase to function at 65°C should reduce the constraints of RNA secondary structure in RNA ligation experiments.