Sonneratia ovata Backer–A genetically depauperate mangrove species

It is expected that geographically widespread and outcrossing tree species would have high level of genetic variation. Three chloroplast regions and six nuclear genes were sequenced to assess the genetic variation of a species of mangroves, Sonneratia ovata, from China and Thailand. No nucleotide polymorphism was found in the three chloroplast regions and in the six nuclear genes from all of the four populations examined. The depauperation of polymorphism in S. ovata in comparison to moderate polymorphisms of other congeneric species is surprising, particularly considering high level of polymorphism in the past and relatively wide geographic distribution of the species. Since multiple independent loci were surveyed in this study, the most plausible explanation for our observation is that S. ovata has experienced severe demographic bottlenecks in the Pleistocene glaciation, followed by subsequent recolonization by sea currents in China and Southeast Asia regions. The lack of polymorphism may also be attributable to its small population size, despite its wide geographic distribution.     

正交试验法优选薯蓣皂苷元萃取条件研究

目的:为明确薯蓣皂苷元的最佳提取工艺,我们采用超临界CO2萃取技术,通过正交试验优化穿山龙中薯蓣皂苷元的提取工艺。方法:探讨萃取压力、萃取温度、分离Ⅰ压力、分离Ⅰ温度等因素对薯蓣皂苷元收率的影响。确定了薯蓣皂苷元的最佳萃取条件。结果:穿山龙中薯蓣皂苷元超临界萃取的最佳条件为萃取压力30 MPa,萃取温度50℃,分离Ⅰ压力13 MPa,分离Ⅰ温度40℃。结论:超临界CO2萃取法提取薯蓣皂苷元工艺简单,安全有效。     

A Model of Disturbed Flow-Induced Atherosclerosis in Mouse Carotid Artery by Partial Ligation and a Simple Method of RNA Isolation from Carotid Endothelium

Despite the well-known close association, direct evidence linking disturbed flow to atherogenesis has been lacking. We have recently used a modified version of carotid partial ligation methods [1,2] to show that it acutely induces low and oscillatory flow conditions, two key characteristics of disturbed flow, in the mouse common carotid artery. Using this model, we have provided direct evidence that disturbed flow indeed leads to rapid and robust atherosclerosis development in Apolipoprotein E knockout mouse [3]. We also developed a method of endothelial RNA preparation with high purity from the mouse carotid intima [3]. Using this mouse model and method, we found that partial ligation causes endothelial dysfunction in a week, followed by robust and rapid atheroma formation in two weeks in a hyperlipidemic mouse model along with features of complex lesion formation such as intraplaque neovascularization by four weeks. This rapid in vivo model and the endothelial RNA preparation method could be used to determine molecular mechanisms underlying flow-dependent regulation of vascular biology and diseases. Also, it could be used to test various therapeutic interventions targeting endothelial dysfunction and atherosclerosis in considerably reduced study duration.Download video file.^{(125M, mp4)}     

Preparation and characterization of recombinant murine p65/L-plastin expressed in Escherichia coli and high-titer antibodies against the protein

We previously identified a 65-kDa protein (p65) that was phosphorylated in activated macrophages. It has turned out to be a murine homologue of human L-plastin, which was identified as a novel protein in human cancer cells. p65/L-plastin is characterized by a series of Ca(2+)-, calmodulin-, and actin-binding domains, and is thought to play a crucial role in leukocytes and cancer cells. We have expressed a recombinant (r) p65/L-plastin in Escherichia coli that binds to beta-actin and prepared high-titer antibodies using large amounts of the protein as immunogen. Anti-rp65/L-plastin antibodies recognize native p65/L-plastin as well as rp65/L-plastin and have enabled us to detect the fine structures of intracellular p65/L-plastin, and it was found that its localization was extensively changed by stimulation with bacterial components. We further developed an enzyme-linked immunosorbent assay system and a flow cytometry method using these reagents, which made it possible to measure antibodies, including autoantibodies, against p65/L-plastin and to evaluate the maturation-dependent expression of the protein in leukocytes.     

Silencing of antiapoptotic survivin gene by multiple approaches of RNA interference technology

Silencing of mammalian gene expression by RNA interference (RNAi) technology can be achieved using small interfering RNA (siRNA) or short hairpin RNA (shRNA). However, the relative effectiveness of these two approaches is not known. It is also not clear whether gene-specific shRNA transcribed from an RNA polymerase II (Pol II)-directed promoter in a fusion form can disrupt the targeted gene expression. Here, we report that using both luciferase and antiapoptotic survivin genes as targets, both siRNA and shRNA approaches significantly silenced the targeted gene expression in cancer cells. We further demonstrated that shRNAs transcribed from an RNA Pol II-mediated promoter in a green fluorescent protein (GFP) fusion form at the 3'-untranslated region silenced luciferase and survivin expression as well, suggesting that the extra RNA sequence outside of the shRNA hairpin does not disrupt shRNA function. We also showed that silencing of survivin expression selectively induces apoptosis in transfected cells. Together, we have validated multiple approaches of RNAi technology using both survivin and luciferase genes as targets and demonstrated for the first time that GFP-shRNAs transcribed from an RNA Pol II-mediated promoter could mediate gene silencing, which may lead to new directions for the application of RNAi technology.     

Persistent protease-activated receptor 4 signaling mediates thrombin-induced microglial activation

We have previously reported that thrombin, the ultimate serine protease in the coagulation cascades, is a proinflammatory agent that causes proliferation and activation of brain microglial cells. However, participation of its principal receptor, the protease-activated receptor 1 (PAR1) appears to be limited to promoting microglial proliferation and not induction of inflammatory mediators. In the present study, we now report that thrombin action in promoting inflammatory mediators from brain microglia is mediated through another thrombin receptor, PAR4. Here we show that the PAR4 agonist peptide (PAR4AP, GYPGKF), but not the PAR1AP (TRAP, SFLLRN), induced tumor necrosis factor-alpha (TNF-alpha) production not only in cultured murine microglial cells in vitro but also in rat cortex in vivo. Down-regulation of PAR4 expression in microglial cultures by a specific antisense, but not a sense, oligonucleotide reduced PAR4AP-induced TNF-alpha. Mechanistic studies indicated that, in comparison with PAR1 signaling, prolonged increase of [Ca2+]i and phosphorylation of p44/42 mitogen-activated protein kinases, as well as NFkappaB activation may be responsible for PAR4AP-induced TNF-alpha production in microglia. Taken together, these results demonstrate that PAR4 activation mediates the potentially detrimental effects of thrombin on microglia, implying that perspectives of exploiting PAR1 as a potential anti-inflammatory target should be shifted toward PAR4 as a much more specific therapeutic target in brain inflammatory conditions associated with neurotrauma and neurodegenerations.     

Tandem heterocyclization activity of the multidomain 230 kDa HMWP2 subunit of Yersinia pestis yersiniabactin synthetase: interaction of the 1-1382 and 1383-2035 fragments.

The six-domain, 2035-amino acid subunit high-molecular weight protein 2 (HMWP2) activates salicylate and two cysteines and loads them covalently on its three carrier protein domains during assembly of the iron-chelating virulence factor, yersiniabactin of the plague bacterium Yersinia pestis. The 1-1382 fragment of HMWP2 (ArCP-Cy1-A), overproduced in Escherichia coli, contains the first three domains: the aryl carrier protein (ArCP) domain, the cysteine specific adenylation domain (A), and the first condensation/cyclization domain (Cy1). The ArCP can be posttranslationally phosphopantetheinylated on Ser52 and then loaded with a salicyl group on the phosphopantetheine (Ppant) thiol by action of the YbtE, a salicyl-AMP ligase. The HMWP2 1-1382 fragment can activate L-cysteine as Cys-AMP. The HMWP2 1383-2035 fragment contains the remaining three domains: two peptidyl carrier proteins (PCP1 and PCP2) separated by a second condensation/cyclization domain (Cy2). Phosphopantetheinylation of the HMWP2 1383-2035 fragment at Ser1439 (PCP1) and Ser1977 (PCP2) facilitates cysteinylation of both thiols by HMWP2 1-1382. When the holo 1-1382 and bis-holo 1383-2035 protein fragments are mixed with ATP, salicylate, and cysteine, four products are slowly released [salicylcysteine (Sal-Cys), (hydroxyphenylthiazolinyl)cysteine (HPT-Cys), HPT-Cys-Cys, and the bisheterocyclic HPTT-Cys], reflecting thiolytic rerouting by cysteine in solution of elongating acyl-S-enzyme intermediates tethered at ArCP, PCP1, and PCP2 carrier protein domains, respectively. Conducting the in trans reconstitution with the S1439A mutant of HMWP2 1383-2035 releases only Sal-Cys, while the S1977A mutant leads to HPT-Cys formation but not HPT-Cys-Cys or HPTT-Cys. These results suggest localization of particular acyl-S-enzyme intermediates to each of the three carrier protein regions and also establish the sequential action of Cy1 and Cy2, with the latter producing the tandem 4,2-bisheterocyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) moiety characteristic of this class of siderophores.