Crystal and EM Structures of Human Phosphoribosyl Pyrophosphate Synthase I (PRS1) Provide Novel Insights into the Disease-Associated Mutations

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.     

A CysB regulator positively regulates cysteine synthesis,expression of type III secretion system genes,and pathogenicity in Ralstonia solanacearum

A syringe-like type III secretion system (T3SS) plays essential roles in the pathogenicity of Ralstonia solanacearum, which is a causal agent of bacterial wilt disease on many plant species worldwide. Here, we characterized functional roles of a CysB regulator (RSc2427) in R. solanacearum OE1-1 that was demonstrated to be responsible for cysteine synthesis, expression of the T3SS genes, and pathogenicity of R. solanacearum. The cysB mutants were cysteine auxotrophs that failed to grow in minimal medium but grew slightly in host plants. Supplementary cysteine substantially restored the impaired growth of cysB mutants both in minimal medium and inside host plants. Genes of cysU and cysI regulons have been annotated to function for R. solanacearum cysteine synthesis; CysB positively regulated expression of these genes. Moreover, CysB positively regulated expression of the T3SS genes both in vitro and in planta through the PrhG to HrpB pathway, whilst impaired expression of the T3SS genes in cysB mutants was independent of growth deficiency under nutrient-limited conditions. CysB was also demonstrated to be required for exopolysaccharide production and swimming motility, which contribute jointly to the host colonization and infection process of R. solanacearum. Thus, CysB was identified here as a novel regulator on the T3SS expression in R. solanacearum. These results provide novel insights into understanding of various biological functions of CysB regulators and complex regulatory networks on the T3SS in R. solanacearum.     

A nodule-specific plant cysteine proteinase, AsNODF32, is involved in nodule senescence and nitrogen fixation activity of the green manure legume Astragalus sinicus

Asnodf32, encoding a nodule-specific cysteine proteinase in Astragalus sinicus, is probably involved in nodule senescence. To obtain direct evidence of its role in nodule senescence, Agrobacterium rhizogenes-mediated RNA interference was applied to A. sinicus hairy roots. Real-time qRT-PCR was used to estimate the efficiency of suppression. The senescent phenotype of transgenic nodules was examined with paraffin-embedded slides, TUNEL (TdT-mediated dUTP nick-end labeling) assay, and transmission electron microscopy, and the bacteroid nitrogen fixation activity was also measured. It was found that silencing of Asnodf32 delayed root nodule and bacteroid senescence. The period of bacteroid active nitrogen fixation was significantly extended. Interestingly, nodules enlarged in length were also observed on Asnodf32-silenced hairy roots. The results reported here indicate that Asnodf32 plays an important role in the regulation of root nodule senescence.     

黄土高原子午岭林区两种天然次生林植物叶片-凋落叶-土壤生态化学计量特征

了解我国黄土高原子午岭林区两种天然林下植物叶片-凋落叶-土壤生态化学计量特征,有助于人们更深入地认识黄土高原子午岭天然次生林生态系统养分循环规律和系统稳定机制。结果表明:(1)辽东栎和白桦两种植物叶片碳、氮、磷平均含量为468.6、17.1、2.1 g/kg;凋落叶碳、氮、磷平均含量为457.3、12.5、1.6 g/kg;土壤碳、氮、磷平均含量分别为17.6、1.4、0.5 g/kg。(2)白桦叶片N、P含量之间II类线性回归斜率大于1(P=0.07),表明白桦叶片建成过程中存在N、P元素按比例投入的依赖。白桦凋落叶N、P含量之间的II类线性回归斜率显著小于1(P0.05),两种天然次生林凋落叶整体N、P含量之间的II类线性回归斜率也显著小于1(P0.05),反映了凋落叶中单位P含量与单位N含量间不存在等速损耗关系。(3)黄土高原子午岭两种天然次生林凋落叶氮含量与土壤氮含量具有显著相关性(P0.01),表明凋落叶分解对土壤氮库有增加作用。相比于凋落叶,植物叶片磷含量与土壤磷含量具有较紧密的关系,表现为高的土壤P含量则植物叶片也具有较高的P含量。黄土高原子午岭林区两种天然次生林下土壤有机质具有较快的矿化作用。(4)辽东栎作为植被演替到顶极群落的优势物种,其凋落叶C∶N值为26.7远低于白桦凋落叶C∶N值44.9(P0.05),有利于微生物对凋落叶的分解。两种天然次生林的植物叶片N∶P均值为7.9714,低于全国和全球尺度的其他研究结果,表明这两种天然次生林主要受N限制。     

子午岭典型植被凋落叶-土壤养分与酶活性特征

对黄土高原子午岭任家台林区内刺槐、油松、侧柏等3种人工林以及桦树、辽东栎等两种天然次生林的凋落叶C、N、P含量、林下土壤基本理化性质和碱性磷酸酶、脲酶、蔗糖酶3种酶的活性进行分析,并研究凋落叶C、N、P含量与土壤C、N、P含量之间的相关关系,以及土壤基本理化性质与酶活性之间的相关关系,为该区植被恢复效果评价提供科学依据与参考。结果发现:刺槐、辽东栎凋落叶碳氮比值显著低于其他植被,凋落叶分解速率相对较快;辽东栎土壤有机碳、全氮含量最高,分别为19.18、1.60g/kg,刺槐土壤全磷含量最高(0.61g/kg);土壤酶活性主要受土壤有机碳、全氮、容重及p H影响,与土壤全磷相关性不显著;人工林中,侧柏土壤中3种酶活性均高于其他植被,且侧柏凋落叶碳氮比值相对较低,分解速率较快,相比于刺槐作为造林树种更占优势。     

Real-time monitoring full length bid interacting with Bax during TNF-α-induced apoptosis

Bid, a member of the pro-apoptotic Bcl-2 protein family, is activated through caspase-8-mediated cleavage into a truncated form (p15 tBid) during TNF-α(tumor necrosis factor α)-induced apoptosis. Activated tBid can induce Bax oligomerization and translocation to mitochondria, triggering the release of cytochrome c, caspase-3 activation and cell apoptosis. However, it is debatable that whether Bid and tBid can interact directly with Bax in living cells. In this study, we used confocal fluorescence microscope, combined with both FRET (fluorescence resonance energy transfer) and acceptor photobleaching techniques, to study the dynamic interaction between Bid and Bax during TNF-α-induced apoptosis in single living cell. In ASTC-a-1 cells, full length Bid induced Bax translocation to mitochondria by directly interacting with Bax transiently in response to TNF-α treatment before cell shrinkage. Next, we demonstrated that, in both ASTC-a-1 and HeLa cells, Bid was not cleaved before cell shrinkage even under the condition that caspase-8 had been activated, but in MCF-7 cells Bid was cleaved. In addition, in ASTC-a-1 cells, caspase-3 activation was a biphasic process and Bid was cleaved after the second activation of caspase-3. In summary, these findings indicate that, FL-Bid (full length-Bid) directly regulated the activation of Bax during TNF-α-induced apoptosis in ASTC-a-1 cells and that the cleavage of Bid occurred in advanced apoptosis.     

Mammalian Target of Rapamycin Complex 2 Signaling Pathway Regulates Transient Receptor Potential Cation Channel 6 in Podocytes

Transient receptor potential cation channel 6 (TRPC6) is a nonselective cation channel, and abnormal expression and gain of function of TRPC6 are involved in the pathogenesis of hereditary and nonhereditary forms of renal disease. Although the molecular mechanisms underlying these diseases remain poorly understood, recent investigations revealed that many signaling pathways are involved in regulating TRPC6. We aimed to examine the effect of the mammalian target of rapamycin (mTOR) complex (mTOR complex 1 [mTORC1] or mTOR complex 2 [mTORC2]) signaling pathways on TRPC6 in podocytes, which are highly terminally differentiated renal epithelial cells that are critically required for the maintenance of the glomerular filtration barrier. We applied both pharmacological inhibitors of mTOR and specific siRNAs against mTOR components to explore which mTOR signaling pathway is involved in the regulation of TRPC6 in podocytes. The podocytes were exposed to rapamycin, an inhibitor of mTORC1, and ku0063794, a dual inhibitor of mTORC1 and mTORC2. In addition, specific siRNA-mediated knockdown of the mTORC1 component raptor and the mTORC2 component rictor was employed. The TRPC6 mRNA and protein expression levels were examined via real-time quantitative PCR and Western blot, respectively. Additionally, fluorescence calcium imaging was performed to evaluate the function of TRPC6 in podocytes. Rapamycin displayed no effect on the TRPC6 mRNA or protein expression levels or TRPC6-dependent calcium influx in podocytes. However, ku0063794 down-regulated the TRPC6 mRNA and protein levels and suppressed TRPC6-dependent calcium influx in podocytes. Furthermore, knockdown of raptor did not affect TRPC6 expression or function, whereas rictor knockdown suppressed TRPC6 protein expression and TRPC6-dependent calcium influx in podocytes. These findings indicate that the mTORC2 signaling pathway regulates TRPC6 in podocytes but that the mTORC1 signaling pathway does not appear to exert an effect on TRPC6.     

山鹧鸪属鸟类线粒体基因组的比较及系统发育研究

海南山鹧鸪(Arborophila ardens)对生境选择比较严格,种群数量稀少,属于濒危物种。为进一步研究山鹧鸪属的进化和系统发育关系,文章利用Illumina Hiseq2000高通量测序技术获得了海南山鹧鸪线粒体全基因组序列,从比较基因组学角度分析了4种山鹧鸪鸟类的线粒体基因组特征,并探讨了山鹧鸪属鸟类的系统发育地位。研究结果表明：(1) 海南山鹧鸪线粒体基因组长度为16 730 bp,编码13个蛋白质编码基因、2个核糖体RNA基因、22个转运RNA基因以及1个控制区;(2) 山鹧鸪属物种受到了纯化选择的作用,且在进化过程中积累了更多的非同义替换;(3) 山鹧鸪属位于雉科鸟类系统树的基部位置,其中白眉山鹧鸪与红喉山鹧鸪互为姐妹群,海南山鹧鸪位于山鹧鸪属的基部位置,与其他3种山鹧鸪鸟类的亲缘关系较远。     

Low-power laser irradiation activates Src tyrosine kinase through reactive oxygen species-mediated signaling pathway

Low-power laser therapy in medicine is widespread but the mechanisms are not fully understood. It has been suggested that low-power laser irradiation (LPLI) could induce photochemical reaction and activate several intracellular signaling pathways. Reactive oxygen species (ROS) are considered to be the key secondary messengers produced by LPLI. Here, we studied the signaling pathway mediated by ROS upon the stimulation of LPLI. Src tyrosine kinases are well-known targets of ROS and can be activated by oxidative events. Using a Src reporter based on fluorescence resonance energy transfer (FRET) and confocal laser scanning microscope, we visualized the dynamic Src activation in Hela cells immediately after LPLI. Moreover, Src activation by LPLI was in a dose-dependent manner. The increase of Src phosphorylation at Tyr416 was detected by Western blotting. In the presence of vitamin C, catalase alone, or the combination of catalase and superoxide dismutase (SOD), the activation of Src by LPLI is significantly abolished. In contrast, G?6983 loading, a PKC inhibitor, did not affect this response. Treatment of Hela cells with exogenous H(2)O(2) also resulted in a concentration-dependent activation of Src. These results demonstrated that it was ROS that mediated Src activation by LPLI. Cellular viability assay revealed that laser irradiation of low doses (相似文献