虫害马尾松(Pinus massoniana Lamb)邻枝针叶挥发物及其内源茉莉酸甲酯的快速变化

对盆栽马尾松针叶进行接虫咬食危害处理后,通过TCT-GC/MS分析了同一株受害枝相邻的健康枝针叶挥发物的成分及相对含量的时序变化。结果表明：萜烯类化合物是邻枝针叶挥发物的主要成分,其次是含氧化合物、含氮化合物等。与对照相比,多数挥发物的相对含量1h略高于对照,2h维持较高水平。同时,用GC/MS分析了邻枝针叶不同时间序列中茉莉酸甲酯的含量,结果表明：虫害马尾松邻枝针叶1h茉莉酸甲酯含量就有所升高,2h显著高于对照,是对照的近1倍。证明马尾松受到虫害后,启动了体内的防御系统,并诱导邻枝产生抗性。     

Protein Kinase D Promotes Airway Epithelial Barrier Dysfunction and Permeability through Down-regulation of Claudin-1

At the interface between host and external environment, the airway epithelium serves as a major protective barrier. In the present study we show that protein kinase D (PKD) plays an important role in the formation and integrity of the airway epithelial barrier. Either inhibition of PKD activity or silencing of PKD increased transepithelial electrical resistance (TEER), resulting in a tighter epithelial barrier. Among the three PKD isoforms, PKD3 knockdown was the most efficient one to increase TEER in polarized airway epithelial monolayers. In contrast, overexpression of PKD3 wild type, but not PKD3 kinase-inactive mutant, disrupted the formation of apical intercellular junctions and their reassembly, impaired the development of TEER, and increased paracellular permeability to sodium fluorescein in airway epithelial monolayers. We further found that overexpression of PKD, in particular PKD3, markedly suppressed the mRNA and protein levels of claudin-1 but had only minor effects on the expression of other tight junctional proteins (claudin-3, claudin-4, claudin-5, occludin, and ZO-1) and adherent junctional proteins (E-cadherin and β-catenin). Immunofluorescence study revealed that claudin-1 level was markedly reduced and almost disappeared from intercellular contacts in PKD3-overexpressed epithelial monolayers and that claudin-4 was also restricted from intercellular contacts and tended to accumulate in the cell cytosolic compartments. Last, we found that claudin-1 knockdown prevented TEER elevation by PKD inhibition or silencing in airway epithelial monolayers. These novel findings indicate that PKD negatively regulates human airway epithelial barrier formation and integrity through down-regulation of claudin-1, which is a key component of tight junctions.     

Xanthomonas campestris sensor kinase HpaS co-opts the orphan response regulator VemR to form a branched two-component system that regulates motility

Xanthomonas campestris pv. campestris (Xcc) controls virulence and plant infection mechanisms via the activity of the sensor kinase and response regulator pair HpaS/hypersensitive response and pathogenicity G (HrpG). Detailed analysis of the regulatory role of HpaS has suggested the occurrence of further regulators besides HrpG. Here we used in vitro and in vivo approaches to identify the orphan response regulator VemR as another partner of HpaS and to characterize relevant interactions between components of this signalling system. Bacterial two-hybrid and protein pull-down assays revealed that HpaS physically interacts with VemR. Phos-tag SDS-PAGE analysis showed that mutation in hpaS reduced markedly the phosphorylation of VemR in vivo. Mutation analysis reveals that HpaS and VemR contribute to the regulation of motility and this relationship appears to be epistatic. Additionally, we show that VemR control of Xcc motility is due in part to its ability to interact and bind to the flagellum rotor protein FliM. Taken together, the findings describe the unrecognized regulatory role of sensor kinase HpaS and orphan response regulator VemR in the control of motility in Xcc and contribute to the understanding of the complex regulatory mechanisms used by Xcc during plant infection.     

苜蓿红豆草属间体细胞杂种的分子生物学鉴定

通过原生质体融合和培养获得苜蓿红豆草属间体细胞杂种植株。采用一种简便方法从杂种组织再生植株叶片、红豆草羟脯氨酸抗性系再生植株叶片和苜蓿根癌农杆菌702转化系愈伤组织提取DNA用于RAPD和Southern杂交分析。随机引物扩增结果显示两种亲本的RAPD多态具有明显差异。在所用20种随机引物中,6种产生较多的DNA片段。杂种组织具有两种亲本特有的DNA片段,但倾向于排除红豆草亲本的染色体,表明该杂种为非对称杂种,两种亲本染色体之间可能发生了重组。由于红豆草DNA的介入,杂种组织表现出较强的分化能力。分别利用RAPD扩增得到的OPA141000bp红豆草羟脯氨酸抗性系特异产物和OPA141600bp苜蓿根癌农杆菌702转化系特异产物为探针进行Southern分子杂交,证明杂种组织同时具有这两种DNA片段的同源序列。     

Phosphorylation screening identifies translational initiation factor 4GII as an intracellular target of Ca(2+)/calmodulin-dependent protein kinase I

CaMKI is a Ca2+/calmodulin-dependent protein kinase that is widely expressed in eukaryotic cells and tissues but for which few, if any, physiological substrates are known. We screened a human lung cDNA expression library for potential CaMKI substrates by solid phase in situ phosphorylation ("phosphorylation screening"). Multiple overlapping partial length cDNAs encoding three proteins were detected. Two of these proteins are known: 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase and eukaryotic translation initiation factor (eIF) 4GII. To determine whether CaMKI substrates identified by phosphorylation screening represent authentic physiological targets, we examined the potential for [Ca2+]i- and CaMKI-dependent phosphorylation of eIF4GII in vitro and in vivo. Endogenous eIF4GII immunoprecipitated from HEK293T cells was phosphorylated by CaMKI, in vitro as was a recombinant fragment of eIF4GII encompassing the central and C-terminal regions. The latter phosphorylation occurred with favorable kinetics (Km = 1 microm; kcat = 1.8 s-1) at a single site, Ser1156, located in a segment of eIF4GII aligning with the phosphoregion of eIF4GI. Phosphopeptide mapping and back phosphorylation experiments revealed [Ca2+]i-dependent, CaMKI site-specific, eIF4GII phosphorylation in vivo. This phosphorylation was blocked by kinase-negative CaMKI consistent with a requirement for endogenous CaMKI for in vivo eIF4GII phosphorylation. We conclude that phosphorylation screening is an effective method for searching for intracellular targets of CaMKI and may have identified a new role of Ca2+ signaling to the translation apparatus.     

Regulation of alternative splicing of Bcl-x by IL-6, GM-CSF and TPA

The splicing of many alternative exons in the precursor messenger RNA (pre-mRNA) is regulated by extracellular factors but the underlying molecular bases remain unclear. Here we report the differential regulation of Bcl-x pre-mRNA splicing by extracellular factors and their distinct requirements for pre-mRNA elements. In K562 leukemia cells, treatment with interleukin-6 (IL-6) or granulocyte-macrophage colony stimulating factor (GM-CSF) reduced the proportion of the Bcl-xL variant mRNA while treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) had no effect. In U251 glioma cells, however, TPA efficiently increased the Bcl-xL level. These regulations were also seen for a transfected splicing reporter mini-gene. Further analyses of deletion mutants indicate that nucleotides 1-176 of the downstream intron are required for the IL-6 effect, whereas additional nucleotides 177-284 are essential for the GM-CSF effect. As for the TPA effect, only nucleotides 1-76 are required in the downstream intron. Thus, IL-6, GM-CSF and TPA differentially regulate Bcl-x splicing and require specific intronic pre-mRNA sequences for their respective effects.