Lpa-miR-nov-66拮抗IGF1促羊驼黑色素细胞黑色素生成及细胞迁移作用

胰岛素样生长因子1（IGF1）能够促进细胞迁移。我们最近的研究发现,一种新发现的miRNA（lpa-miR-nov-66）可通过靶向抑制可溶性腺苷酸环化酶（soluble guanylate cyclase,sGC）抑制黑色素细胞产生黑色素。然而,当二者联合作用于黑色素细胞时,究竟如何影响黑色素细胞的黑色素产生及细胞迁移尚未见报道。本研究证明,lpa-miR-nov-66可拮抗IGF1的促黑色素细胞的黑色素生成及促细胞迁移作用。ELISA法检测结果揭示,与单纯IGF1处理比较,过表达lpa-miR-nov-66或过表达lpa-miR-nov-66联合IGF1处理可明显降低IGF1诱导羊驼黑色素细胞的cAMP生成水平。实时定量PCR和Western印迹证明,与单纯IGF1处理比较,过表达lpa-miR-nov-66或联合处理可明显降低毛色生成相关基因--MITF、TYR、和TYRP1在黑色素细胞的表达。此外,细胞增殖和细胞划痕实验显示,与单纯IGF1处理比较,过表达lpa-miR-nov-66或联合处理可显著抑制黑色素细胞的迁移。上述结果表明,lpa-miR-nov-66可以减少cAMP产生,抑制IGF1的促黑色素细胞产生黑色素的作用,并抑制IGF1对黑色素细胞增殖和迁移的促进作用。     

Construction and validation of a neutrally-marked strain of Pseudomonas fluorescens SBW25

Neutrally marked bacterial strains are useful in many experimental evolution and molecular ecology studies to assess the relative fitness of a given strain. Here we describe the construction and validation of a neutral marker for the model organism Pseudomonas fluorescens SBW25. The marked strain, called SBW25-lacZ, was created by integrating a promoterless 'lacZ into the defective prophage locus of the SBW25 chromosome. Fitness assays conducted in various laboratory media and in planta revealed that the fitness levels of SBW25-lacZ were comparable with the wild-type ancestor.     

The Common Nodulation Genes of Astragalus sinicus Rhizobia Are Conserved despite Chromosomal Diversity

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2.0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.     

Behavior of plasmid pJB5JI in Rhizobium huakuii under free-living and symbiotic conditions

The Rhizobium leguminosarum biovar viceae host-range plasmid pJB5JI was transferred into Rhizobium huakuii strains, both wild-type 7653R and its sym plasmid-cured mutant 7653R-1. Transconjugant 7653R-1 (pJB5JI) acquired the ability to form ineffective nodules on pea plants, whereas transconjugant 7653R (pJB5JI) could not do so, indicating that the indigenous symbiotic plasmid could restrict the functional expression of pJB5JI. On the other hand, transconjugant 7653R (pJB5JI) showed higher nitrogenase activity on A. sinicus and higher shoot dry weight than the recipient strain 7653R. The alien plasmid pJB5JI in both kinds of transconjugants remained stable during frequent transfer on culture media, but in part of the isolates from nodules formed by them the pJB5JI was not visualized on gel by the Eckhardt procedure. Southern hybridization with Tn5 and nod gene probes showed that these isolates still reserved, at least in part, DNA of pJB5JI, which was probably intergrated onto the chromosome of cells.     

Genetic characterization of <Emphasis Type="Italic">psp</Emphasis> encoding the DING protein in <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> SBW25

Background  

DING proteins constitute a conserved and broadly distributed set of proteins found in bacteria, fungi, plants and animals (including humans). Characterization of DING proteins from animal and plant tissues indicated ligand-binding ability suggesting a role for DING proteins in cell signaling and biomineralization. Surprisingly, the genes encoding DING proteins in eukaryotes have not been identified in the eukaryotic genome or EST databases. Recent discovery of a DING homologue (named Psp here) in the genome of Pseudomonas fluorescens SBW25 provided a unique opportunity to investigate the physiological roles of DING proteins. P. fluorescens SBW25 is a model bacterium that can efficiently colonize plant surfaces and enhance plant health. In this report we genetically characterize Psp with a focus on conditions under which psp is expressed and the protein exported.     

Potato Y Potyvirus Helper Component Proteinase Enhances Long-distance Movement of Potato X Potexvirus

To mutagenize two conserved CCCT and PTK motifs in the central domain of Chinese strain of potato Y potyvirus (PVY-C) helper component proteinase (HC-Pro), four mutants of HC-Pro gene were obtained by PCR and site-directed mutagenesis, and then were inserted into the constitutive expression vector pBin438. Leaves from tobacco ( Nicotiana tabacum L. cv. K326) were transformed with these four plant expression plasmids by Agrobacterium -mediated transformation, respectively. Southern and Western blotting analyses showed that these four mutants were integrated into tobacco genomic DNA and could express the corresponding proteins in most of the transgenic plants. The challenge of transgenic plants with potato X potexvirus (PVX) revealed that the expression products of PVY-C HC-Pro mutants in transgenic plants greatly abolished functions of HC-Pro in enhancing the accumulation and pathogenicity of PVX, indicating that CCCT and PTK motifs of HC-Pro were required for PVX/PVY synergism. Meanwhile, the results demonstrated that PVY-C HC-Pro had a function in accelerating the long-distance movement of PVX in these transgenic plants for the first time.     

脂肪酸影响草鱼肝细胞脂质蓄积状态及诱导其凋亡的离体研究

为探究外源脂肪酸对草鱼肝细胞脂质代谢及健康状况的影响及其机理,体外培养草鱼肝细胞,并采用不同浓度（0-1 mmol/L）油酸（Oleic acid）进行细胞孵育,噻唑兰比色法（Methyl thiazolte trazoliu,MTT）和油红O染色提取法检测肝细胞活力及脂质蓄积状况,BODIBY和DAPI染色法观察肝细胞脂滴及细胞核情况,流式细胞术检测肝细胞凋亡率变化,Real-time qPCR检测脂质合成标志基因过氧化物酶体增殖物激活受体γ（Peroxidase proliferation activated receptor,PPARγ）和CCAAT/增强子结合蛋白α（CCAAT/enhancer binding protein alpha,C/EBPα）、凋亡相关基因Caspase家族等的表达情况。结果显示,随着油酸处理浓度的增加,肝细胞活力和细胞内脂质积累呈现先上升后下降的趋势,分别在0.4和0.6 mmol/L时达到最大值（P < 0.05）;肝细胞凋亡率则先下降后上升,在0.4 mmol/L油酸处理时最低,1 mmol/L油酸处理时最高（P < 0.05）;此外,0.4 mmol/L油酸处理抑制了肝细胞Caspase-3b和Caspase-9基因的表达,上调Bcl-2/Bax mRNA比值（P < 0.05）,而0.8 mmol/L油酸处理显著促进Caspase-3b、Caspase-8、Caspase-9及凋亡诱导因子（Apoptosis inducing factor,AIF）基因的表达,下调Bcl-2/Bax的mRNA比值（P < 0.05）。研究表明,一定浓度的脂肪酸可增强草鱼肝细胞活力,促进胞内脂质积累,抑制细胞凋亡,而脂肪酸浓度过高则抑制肝细胞活力并诱导肝细胞凋亡,其作用与脂肪酸影响脂质代谢及凋亡基因的表达有关。