Oligomerization state-dependent hyperlipidemic effect of angiopoietin-like protein 4

Angiopoietin-like protein 4 (Angptl4) is the second member of the angiopoietin-like family of proteins previously shown to increase plasma triglyceride (TG) levels in vivo. We recently reported that Angptl4 is a variable-sized oligomer formed by intermolecular disulfide bonds and undergoes regulated proteolytic processing upon secretion. We now show that adenoviral overexpression of Angptl4 potently increases plasma TG levels by a mechanism independent of food intake or hepatic VLDL secretion. We determined that cysteine residues at positions 76 and 80 of Angptl4, conserved among mouse, rat, and human, are required to form higher order structures. By generating adenoviral expression vectors of Angptl4 containing different epitope tags at both N and C termini, we show that loss of oligomerization results in decreased stability of the N-terminal coiled-coil domain of Angptl4 as well as decreased ability to increase plasma TG levels, suggesting that intermolecular disulfide bond formation plays important roles in determining the magnitude of the hyperlipidemic effect of Angptl4. Because Angptl4 is more potent than Angptl3 in increasing plasma TG levels in mice, inappropriate oligomerization of Angptl4 could be associated with disorders of lipid metabolism in vivo.     

siRNA沉默ERp57对细胞中CRT表达与定位的影响

探讨ERp57基因表达沉默对人小细胞肺癌A549细胞中CRT表达和定位的影响。利用siRNA技术获得ERp57基因表达沉默的人A549肺癌细胞株,分析该细胞株中ERp57基因以及CRT基因的蛋白表达水平,免疫荧光法检测细胞中CRT的表达和亚细胞定位,荧光法检测细胞凋亡。成功获得ERp57基因表达沉默的人A549肺癌细胞株。在该细胞中,CRT表达上调但仍定位于内质网中。用米托蒽醌处理对照细胞14 h后,可使CRT大量转移到细胞膜表面并发生簇集,但在ERp57表达沉默的细胞中,CRT的膜转移和簇集现象不明显。细胞凋亡分析显示,米托蒽醌处理细胞48 h后,所有细胞均出现凋亡细胞典型细胞核固缩、分裂现象。试验证明抑制ERp57蛋白表达会增加A549肺癌细胞中CRT的含量,但同时也阻断蒽环类药物诱导的CRT膜转移,提示ERp57也是介导肿瘤细胞免疫原性凋亡的重要因子。     

Molecular mechanism of ERK dephosphorylation by striatal‐enriched protein tyrosine phosphatase

Striatal‐enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal‐regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho‐ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho‐ERK by STEP is not known. Therefore, we examined STEP activity toward para‐nitrophenyl phosphate, phospho‐tyrosine‐containing peptides, and the full‐length phospho‐ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N‐terminal regulatory region and key residues in its active site. Specifically, both kinase interaction motif (KIM) and kinase‐specific sequence of STEP were required for ERK interaction. In addition to the N‐terminal kinase‐specific sequence region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho‐ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho‐ERK peptide sequence through its active site, and the contact of STEP F311 with phospho‐ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP‐ERK recognition, which could serve as a potential therapy for neurological disorders.

     

啮齿动物及凋落物存留对天山野果林野生樱桃李种子命运的影响

2010年9月至2013年4月通过设置人工土壤种子库,研究了野生樱桃李土壤种子库的动态及啮齿动物和凋落物覆盖对种子库中种子命运的影响.结果表明: 在有取食动物扰动下,48.3%的种子萌发输出为幼苗,50%的种子被动物搬运或当场取食,4%的种子腐烂.在排除了取食动物干扰的条件下,樱桃李种子形成了短期持久的土壤种子库,3年后依然有部分种子萌发并输出为幼苗.凋落物覆盖和对照处理中,被搬运和当场取食种子的比例均显著低于清除凋落物的裸地.地表凋落物存留能减少动物搬运、取食,但不足以导致新生幼苗的大量出现,而啮齿类动物的搬运或取食是影响野生樱桃李种子命运和种子库动态的主要因素.
     

Efficiency Enhancement of Organic Solar Cells Using Hydrophobic Antireflective Inverted Moth‐Eye Nanopatterned PDMS Films

Poly‐dimethylsiloxane (PDMS) films with 2D periodic inverted moth‐eye nanopatterns on one surface are implemented as antireflection (AR) layers on a glass substrate for efficient light capture in encapsulated organic solar cells (OSCs). The inverted moth‐eye nanopatterned PDMS (IMN PDMS) films are fabricated by a soft imprint lithographic method using conical subwavelength grating patterns formed by laser interference lithography/dry etching. Their optical characteristics, together with theoretical analysis using rigorous coupled‐wave analysis simulation, and wetting behaviors are investigated. For a period of 380 nm, IMN PDMS films laminated on glass substrates exhibit a hydrophobic surface with a water contact angle (θ_CA) of ≈120° and solar weighted transmittance (SWT) of ≈94.2%, both significantly higher than those (θ_CA≈ 36° and SWT ≈ 90.3%) of bare glass substrates. By employing IMN PDMS films with a period of 380 nm on glass substrates for OSCs, an enhanced power conversion efficiency (PCE) of 6.19% is obtained mainly due to the increased short‐circuit current density (J_sc) of 19.74 mA cm^‐2 compared to the OSCs with the bare glass substrates (PCE = 5.16% and J_sc = 17.25 mA cm^‐2). For the OSCs, the device stability is also studied.     

A real-time fluorescence polarization activity assay to screen for inhibitors of bacterial ribonuclease P

Ribonuclease P (RNase P) is an essential endonuclease that catalyzes the 5′ end maturation of precursor tRNA (pre-tRNA). Bacterial RNase P is an attractive potential antibacterial target because it is essential for cell survival and has a distinct subunit composition compared to the eukaryal counterparts. To accelerate both structure-function studies and discovery of inhibitors of RNase P, we developed the first real-time RNase P activity assay using fluorescence polarization/anisotropy (FP/FA) with a 5′ end fluorescein-labeled pre-tRNA^Asp substrate. This FP/FA assay also detects binding of small molecules to pre-tRNA. Neomycin B and kanamycin B bind to pre-tRNA^Asp with a K_d value that is comparable to their IC₅₀ value for inhibition of RNase P, suggesting that binding of these antibiotics to the pre-tRNA substrate contributes to the inhibitory activity. This assay was optimized for high-throughput screening (HTS) to identify specific inhibitors of RNase P from a 2880 compound library. A natural product derivative, iriginol hexaacetate, was identified as a new inhibitor of Bacillus subtilis RNase P. The FP/FA methodology and inhibitors reported here will further our understanding of RNase P molecular recognition and facilitate discovery of antibacterial compounds that target RNase P.     

A Proteomic Analysis of Placental Trophoblastic Cells in Preeclampsia–Eclampsia

To explore the proteomic changes of placental trophoblastic cells in preeclampsia–eclampsia (PE), placental trophoblastic cells from normally pregnant women and women with hypertension during gestational period were prepared by laser capture microdissection (LCM), and proteins isolated from these cells were subjected to labeling and proteolysis with isotope-coded affinity tag reagent. A qualitative and quantitative analysis of the proteome expression of placental trophoblastic cells was made using two-dimensional liquid chromatography tandem mass spectrometry (2D LC–MS/MS). A total of 831 proteins in placental trophoblastic cells were identified by combined use of LCM technique and 2D LC–MS/MS. The result was superior to that of conventional two-dimensional electrophoresis method. There were marked differences in 169 proteins of placental trophoblastic cells between normally pregnant women and women with PE. Of 70 (41.4 %) proteins with more than twofold differences, 31 proteins were down-regulated, and 39 were up-regulated in placental trophoblastic cells of the woman with PE. Laminin expression in placenta trophoblastic cells of women with PE was significantly down-regulated as confirmed by Western blot analysis. These findings provide insights into the proteomic changes in placental trophoblastic cells in response to PE and may identify novel protein targets associated with the pathogenesis of PE.     

靶向铜绿假单胞菌(Pseudomonas aeruginosa)粘肽合成酶PBP3的抗菌先导化合物的虚拟筛选及活性研究

【目的】开发出与铜绿假单胞菌粘肽合成酶PBP3有高亲和力,具有全新结构的先导化合物。【方法】以铜绿假单胞菌粘肽合成酶PBP3为靶点,通过分子对接软件DOCK6.5,对含有104万个小分子化合物的数据库进行了大规模虚拟筛选,选取结构相对简单的中选化合物进行合成,得到先导化合物,并验证其抑菌活性。【结果】通过grid score进行第一轮初筛,筛选出grid score分值小于–30 kcal/mol的6万个化合物,接着以amber score进行第二轮筛选,筛出amber score分值小于–20 kcal/mol的化合物约200个。最终,经过观察分析,从中挑选出4种打分高并且结构新颖的小分子化合物作为先导化合物。合成出的先导化合物及其衍生物对铜绿假单胞菌等常见微生物的最小抑菌浓度(MIC)在175–275μg/m L之间,对革兰氏阴性菌和阳性菌均有效,MIC值比作为阳性对照的磺胺嘧啶更低,说明先导化合物具有较好的抗菌活性。【结论】这些先导化合物可以进一步开发为新型抗菌药物,用于解决铜绿假单胞菌的耐药性问题。     

The recruitment of Fas-associated death domain/caspase-8 in Ras-induced apoptosis.

Oncogenic Ras induces cells to undergo apoptosis after inhibition of protein kinase C (PKC) activity. The integration of differential signaling pathways is required for full execution of apoptosis. In this study, we used Jurkat as well as Fas/FADD-defective cell lines expressing v-ras to determine the upstream elements required for activation of the caspase cascade in PKC/Ras-mediated apoptosis. During this Ras-induced apoptotic process, caspase-8 was activated, possibly through its binding to Fas-associated death domain (FADD), in Jurkat/ras and Jurkat/Fas(m)/ras cells but not in Jurkat/FADD(m)/ras cells. c-Jun NH(2)-terminal kinase (JNK) was activated in all three cell lines expressing ras in response to apoptotic stimulation. Suppression of JNK by dn-JNK1 blocked the interaction of FADD and caspase-8 and partially protected Jurkat/ras and Jurkat/Fas(m)/ras cells from apoptosis. However, dn-JNK1 had no effect on PKC/Ras-induced apoptosis in Jurkat/FADD(m)/ras cells. The results indicate that FADD/caspase-8 signaling is involved in PKC/Ras-mediated apoptosis, and JNK may be an upstream effector of caspase activation.     

Analysis of Gliadin Polymorphism in aTriticum speltaL. Collection

Using gliadins, endorperm storage proteins of kernels, as markers, the genetic diversity of 170 samples from the Triticum speltaL. collection of the Vavilov Institute of Plant Industry was studied. High intraspecific polymorphism of the gliadin electrophoretic patterns was revealed. On the basis of similarity of the gliadin electrophoretic patterns, groups of samples were isolated, and the genetic stucturization of the collection was performed.