Feedback regulation of endothelial cell surface plasmin generation by PKC-dependent phosphorylation of annexin A2

In response to blood vessel injury, hemostasis is initiated by platelet activation, advanced by thrombin generation, and tempered by fibrinolysis. The primary fibrinolytic protease, plasmin, can be activated either on a fibrin-containing thrombus or on cells. Annexin A2 (A2) heterotetramer (A2·p11)(2) is a key profibrinolytic complex that assembles plasminogen and tissue plasminogen activator and promotes plasmin generation. We now report that, in endothelial cells, plasmin specifically induces activation of conventional PKC, which phosphorylates serine 11 and serine 25 of A2, triggering dissociation of the (A2·p11)(2) tetramer. The resulting free p11 undergoes ubiquitin-mediated proteasomal degradation, thus preventing further translocation of A2 to the cell surface. In vivo, pretreatment of A2(+/+) but not A2(-/-) mice with a conventional PKC inhibitor significantly reduced thrombosis in a carotid artery injury model. These results indicate that augmentation of fibrinolytic vascular surveillance by blockade of serine phosphorylation is A2-dependent. We also demonstrate that plasmin-induced phosphorylation of A2 requires both cleavage of A2 and activation of Toll-like receptor 4 on the cell surface. We propose that plasmin can limit its own generation by triggering a finely tuned "feedback" mechanism whereby A2 becomes serine-phosphorylated, dissociates from p11, and fails to translocate to the cell surface.     

Overexpression of the Arabidopsis ��-expansin gene AtEXPA1 accelerates stomatal opening by decreasing the volumetric elastic modulus

Guard cell walls of stomata are highly specialized in plants. Previous research focused on the structure and anatomy of guard cell walls, but little is known about guard cell regulation during stomata movement. In this work, we investigate the possible biological role of the Arabidopsis expansin gene AtEXPA1 in stomatal opening. The AtEXPA1 promoter drove the expression of the GUS reporter gene specifically in guard cells. Light-induced stomatal opening was accelerated in 35S::AtEXPA1 lines, whereas the anti-AtEXPA1 antibody decelerated light-induced stomatal opening. The inhibition of the anti-AtEXPA1 antibody on stomatal opening was largely dependent on the environmental pH. The volumetric elastic modulus (ε) was measured as an indicator of changes in the cell wall. The ε value of guard cells in 35S::AtEXPA1 lines was smaller than in the wild types. The putative role of AtEXPA1 as controller of stomatal opening rate and its regulation are discussed.     

Sample Collection Protocol Effects on Quantification of Gene Expression in Potato Leaf Tissue

New platforms allow quantification of gene expression from large, replicated experiments but current sampling protocols for plant tissue using immediate flash freezing in liquid nitrogen are a barrier to these high-throughput studies. In this study, we compared four sampling methods for RNA extraction for gene expression analysis: (1) the standard sampling method of flash freezing whole leaves in liquid nitrogen immediately upon removal from the plant; (2) incubation of excised leaf disks for 2 min at field temperature followed by flash freezing; (3) incubation of excised leaf disks for 1 h on ice followed by flash freezing; and (4) incubation of excised leaf disks for 1 h at field temperature followed by flash freezing. Gene expression analysis was done for 23 genes using nCounter, and normalization of the data was done using the geometric mean of five housekeeping genes. Quality of RNA was highest for protocol A and lowest for protocol D. Despite some differences in RNA quality, gene expression was not significantly different among protocols A, B, and C for any of the 23 genes. Expression of some genes was significantly different between protocol D and the other protocols. This study demonstrates that when sampling leaf disks for gene expression analysis, the time between tissue removal from the plant and flash freezing in liquid nitrogen can be extended. This increase in time allowable during sampling provides greater flexibility in sampling large replicated field experiments for statistical analysis of gene expression data.     

SV40 large T antigen hexamer structure: domain organization and DNA-induced conformational changes

Simian Virus 40 replication requires only one viral protein, the Large T antigen (T-ag), which acts as both an initiator of replication and as a replicative helicase (reviewed in ). We used electron microscopy to generate a three-dimensional reconstruction of the T-ag hexameric ring in the presence and absence of a synthetic replication fork to locate the T-ag domains, to examine structural changes in the T-ag hexamer associated with DNA binding, and to analyze the formation of double hexamers on and off DNA. We found that binding DNA to the T-ag hexamer induces large conformational changes in the N- and C-terminal domains of T-ag. Additionally, we observed a significant increase in density throughout the central channel of the hexameric ring upon DNA binding. We conclude that conformational changes in the T-ag hexamer are required to accommodate DNA and that the mode of DNA binding may be similar to that suggested for some other ring helicases. We also identified two conformations of T-ag double hexamers formed in the presence of forked DNA: with N-terminal hexamer-hexamer contacts, similar to those formed on origin DNA, or with C-terminal contacts, which are unlike any T-ag double hexamers reported previously.     

Synthesis and cytotoxicity evaluation of novel indolylpyrimidines and indolylpyrazines as potential antitumor agents

Novel indolylpyrimidines and indolylpyrazines have been synthesized as potential antitumor agents. They were screened in a panel of 60 human tumor cell lines in vitro. Compounds 7, 9, 10, 15, 21 exhibited efficiently cytotoxic activities with GI(50) values in the low micromolar range against a variety of human cancer cell lines. 2,4-Bis(3'-indolyl)pyrimidine 8 displayed selective cytotoxic activity against IGROV1 tumor cell line with the GI(50) value below 0.01 microM.     

Salvia petrophila sp. nov. (Lamiaceae) from north Guangxi and south Guizhou,China

Salvia petrophila G. X. Hu, E. D. Liu & Yan Liu, a new species from the Chinese provinces of north Guangxi and south Guizhou, is described and illustrated. The new species is similar to S. miltiorrhiza, but can easily be distinguished by its shorter, unbranched stem, subsucculent simple leaf, longer pedicel and corolla tube, oblong‐entire middle lobe of lower corolla lip and larger oblate pollen. It is included in S. sect. Drymosphace on the basis of its falcate‐compressed upper corolla lip and connivent posterior connectives. Trichome and pollen micromorphology of the new species and similar species are described and compared. Additionally, assessment of the conservation status, current geographical distribution and notes on ecology of the new species are given.     

Identification of novel knockout and up-regulated targets for improving isoprenoid production in E. coli

Discovery of novel potential genetic targets to increase the supply of isoprenoid precursors, isopentyl/dimethylallyl diphosphate, is of importance for microbial production of isoprenoids. Here, to improve isoprenoid precursor supply, a flux distribution comparison analysis, based on the genome-scale model, was utilized to simultaneously predict the knockout, down- and up-regulated targets in Escherichia coli. 51 targets were in silico discovered. All knockout and up-regulated targets were experimentally tested to enhance lycopene production. Five knockout targets (deoB, yhfw, yahI, pta and eutD) and four up-regulated targets (ompN, ompE, ndk and cmk) led to 10–45 % increases of lycopene yield, respectively, which had not been uncovered in previous studies. When engineering of the five most significant targets gdhA, eutD, tpiA, ompE and ompN, were combined the lycopene titer improved by 174 % in shake-flask and 81 % in bioreactor fermentations with a maximum yield of 454 mg l^?1.     

Changes in the microbial community structure of filaments and floc formers in response to various carbon sources and feeding patterns

Filamentous bulking is a complicated problem in wastewater treatment plants treating various wastewaters, leading to the deterioration of the settling properties and the effluent quality. This study systematically investigated long-term effects of various carbon sources and feeding patterns on the growth of filamentous bacteria, in order to reveal the mechanism of filamentous bulking. Sludge volume index (SVI), microscopic observations, staining (Gram and Neisser staining), scan electron microscopic, and fluorescent in situ hybridization (FISH) were used to monitor the bulking and track the changes of microbial morphology and community structure of activated sludge in six lab-scale sequencing batch reactors (SBRs) fed with different carbon sources. Filamentous bulking was not observed in all SBRs under anoxic feeding pattern with a short fill time, in which SVI remained below 150 mL/g. In contrast, serious bulking (SVI?>?500 mL/g) occurred under aerobic feeding pattern when fed with ethanol, propionate, acetate, and glucose, in which Thiothrix and Sphaerotilus natans proliferated as dominant filaments. Compared to glucose-fed reactor, relatively light bulking was caused in starch-fed reactor with the growth of Nostocoida limicola II. In addition, flocs in starch-fed reactor were more open and fluffy than flocs formed on readily biodegradable substrates. Finally, a framework integrating kinetic selection, diffusion selection, storage selection, and protozoa capture mechanism was proposed to explain filamentous bulking.     

LATS1过表达对肺癌A549细胞增殖及周期的影响

通过过表达手段上调大肿瘤抑制因子1（1arge tumor suppressor gene 1,LATS1）基因在A549细胞中的表达,研究LATS1对A549细胞生长和细胞周期调控的作用。构建过表达LATS1基因的慢病毒载体,转染A549N胞株,采用RT-PCR和蛋白质印迹法检测转染后A549细胞中LATS1、YAPmRNA和蛋白的表达效率;流式细胞术检测细胞凋亡、周期情况：CCK-8检测细胞的增殖水平变化。结果发现,过表达LATS1慢病毒载体转染A549细胞株后,LATS1mRNA及蛋白表达水平高于未处理组及转染空载体组,YAPmRNA及蛋白表达水平低于未处理组及转染空载体组;过表达LATS1慢病毒转染后,A549细胞增殖率从第五天开始低于对照组（P〈0．05）,过表达组细胞G1期比例明显增高（P〈0．05）,凋亡率明显增加（P〈0．05）,差异均有统计学意义。以上结果提示,LATS1可通过下调YAP的表达水平促进A549细胞的凋亡,诱导G1期阻滞,降低细胞的增殖能力。