A B Functional Gene Cloned from Lily Encodes an Ortholog of <Emphasis Type="BoldItalic">Arabidopsis PISTILLATA</Emphasis> (<Emphasis Type="BoldItalic">PI</Emphasis>)

The classical ABC model proposed for flower development in Arabidopsis and Antirrhinum appropriately sheds light on the biological process of flower development and differentiation and serves in manipulating the floral structure of other important ornamental plants. In this study, LLGLO1, a B functional gene from Lilium longiflorum was isolated and characterized. RT-PCR analysis elucidated that temporal and spatial expression pattern of LLGLO1. This putative gene was strongly expressed in 1, 2, and 3 whorl organs, i.e., outer whorl tepals, inner whorl tepals, and stamens. Genetic effect of LLGLO1 was assayed by ectopic expression in model plant Arabidopsis. Transformed plants showed homeotic transformation of sepals into petaloid sepals in the first whorl, which is similar to the transgenic plants of 35S::PI. So LLGLO1 was one member of GLO/PI sub-family gene to function in flower development.     

A ligation-independent cloning method using nicking DNA endonuclease

Using nicking DNA endonuclease (NiDE), we developed a novel technique to clone DNA fragments into plasmids. We created a NiDE cassette consisting of two inverted NiDE substrate sites sandwiching an asymmetric four-base sequence, and NiDE cleavage resulted in 14-base single-stranded termini at both ends of the vector and insert. This method can therefore be used as a ligation-independent cloning strategy to generate recombinant constructs rapidly. In addition, we designed and constructed a simple and specific vector from an Escherichia coli plasmid back-bone to complement this cloning method. By cloning cDNAs into this modified vector, we confirmed the predicted feasibility and applicability of this cloning method.     

Complexity and Specificity of Precursor microRNAs Driven by Transposable Elements in Rice

In eukaryotes, small noncoding RNA molecules of 16–29 nucleotides in length play crucial roles in the regulation of gene expression. Some 377 sequences representing rice pseudo-microRNAs (miRNAs) are available in release 13.0 of the miRBase sequence database () and are grouped into 143 families. Most newly deposited miRNA sequences are likely to be species-specific. To understand the relationship between miRNAs and transposable elements (TEs) in rice, the RepeatMasker application () was used to screen single-stranded precursor miRNA (pre-miRNA) sequences. This analysis revealed that 33.1% of miRNAs and 36.4% of miRNA families are associated with interspersed repeats, and most of them are species-specific. Furthermore, multiple miRNA families can be encoded by the same TE class. Alignment analysis revealed that miR439 originated from an MuDR4-OS TE, which amplified and diversified in the genome as an inverted repeat of the core sequence followed by multiple repeats. Multiple copies of miR445 and its complexity originate from and are driven by the DNA/Tourist TE class. These results provide an important contribution to the elucidation of TE-driven mechanisms that regulate the species specificity and complexity of rice miRNAs.     

Efficient expression of human soluble guanylate cyclase in Escherichia coli and its signaling-related interaction with nitric oxide

Soluble guanylate cyclase (sGC), as a nitric oxide (NO) sensor, is a critical heme-containing enzyme in NO-signaling pathway of eukaryotes. Human sGC is a heterodimeric hemoprotein, composed of a α-subunit (690 AA) and a heme-binding β-subunit (619 AA). Upon NO binding, sGC catalyzes the conversion of guanosine 5′-triphosphate (GTP) to 3′,5′-cyclic guanosine monophosphate (cGMP). cGMP is a second messenger and initiates the nitric oxide signaling, triggering vasodilatation, smooth muscle relaxation, platelet aggregation, and neuronal transmission etc. The breakthrough of the bottle neck problem for sGC-mediated NO singling was made in this study. The recombinant human sGC β1 subunit (HsGCβ619) and its truncated N-terminal fragments (HsGCβ195 and HsGCβ384) were efficiently expressed in Escherichia coli and purified successfully in quantities. The three proteins in different forms (ferric, ferrous, NO-bound, CO-bound) were characterized by UV–vis and EPR spectroscopy. The homology structure model of the human sGC heme domain was constructed, and the mechanism for NO binding to sGC was proposed. The EPR spectra showed a characteristic of five-coordinated heme-nitrosyl species with triplet hyperfine splitting of NO. The interaction between NO and sGC was investigated and the schematic mechanism was proposed. This study provides new insights into the structure and NO-binding of human sGC. Furthermore, the efficient expression system of E. coli will be beneficial to the further studies on structure and activation mechanism of human sGC.     

Antioxidant and antiproliferative activities of hydroxyl-substituted Schiff bases

Eight hydroxyl-substituted Schiff bases with the different number and position of hydroxyl group on the two asymmetric aromatic rings (A and B rings) were prepared by the reaction between the corresponding aromatic aldehyde and aniline. Their antioxidant effects against the stable galvinoxyl radical (GO) in ethyl acetate and methanol, and 2,2′-azobis(2-amidinopropane hydrochloride) (AAPH)-induced DNA strand breakage, and their antiproliferative effects on human hepatoma HepG2 cells, were investigated. Structure–activity relationship analysis demonstrates that o-dihydroxyl groups on the aromatic A ring and 4-hydroxyl group attached to the aromatic B ring contribute critically to the antioxidant and antiproliferative activities.     

Stereochemistry–activity relationship of orally active tetralin S1P agonist prodrugs

Modifying FTY720, an immunosuppressant modulator, led to a new series of well phosphorylated tetralin analogs as potent S1P1 receptor agonists. The stereochemistry effect of tetralin ring was probed, and (?)-(R)-2-amino-2-((S)-6-octyl-1,2,3,4-tetrahydronaphthalen-2-yl)propan-1-ol was identified as a good SphK2 substrate and potent S1P1 agonist with good oral bioavailability.     

Isoform-selective inhibition of chrysin towards human cytochrome P450 1A2. Kinetics analysis,molecular docking,and molecular dynamics simulations

Our kinetics studies demonstrated that the nature product chrysin exhibited a high inhibitory affinity of 54 nM towards human cytochrome P450 1A2 and was comparable to α-naphthoflavone (49 nM), whereas it represented a moderate affinity of 5225 nM against human cytochrome P450 2C9. However, it remains unclear how this inhibitor selectively binds 1A2. To better understand the isoform selectivity of chrysin, molecular docking and molecular dynamics simulations were performed. Chrysin formed a strong H-bond with Asp313 of 1A2. The stacking interactions with Phe226 also contributed to its tight binding to 1A2. The larger and much more open active site architectures of 2C9 may explain the weaker inhibitory affinity of chrysin towards 2C9. The predicted binding free energies suggest that chrysin preferred 1A2 (ΔG_{bind, pred} = ?23.11 kcal/mol) to 2C9 (?20.41 kcal/mol). Additionally, the present work revealed that 7-hydroxy-flavone bound to 1A2 in a similar pattern as chrysin and represented a slightly less negative predicted binding free energy, which was further validated by our kinetics analysis (IC₅₀ = 240 nM). Results of the study can provide insight for designing novel isoform-selective 1A2 inhibitors.     

展示生长抑素的猪细小病毒样颗粒构建及其免疫原性

为了获得既可预防猪细小病毒感染又能促进生长的嵌合病毒样颗粒疫苗,以PPV NJ-a株基因组DNA为模板扩增VP2基因片段,在VP2基因N端融合人工合成的4拷贝生长抑素基因,构建杆状病毒转移载体pFast-SS4-VP2。通过转化DH10Bac感受态细胞,pFast-SS4-VP2与穿梭载体Bacmid重组,获得重组Bacmid,命名为rBacmid-SS4-VP2。rBacmid-SS4-VP2转染Sf-9细胞,获得重组病毒rBac-SS4-VP2。SDS-PAGE与Western blotting鉴定可见约68 kDa的rSS4-VP2条带;rBac-SS4-VP2感染细胞IFA检测产生很强的特异性绿色荧光;感染细胞超薄切片电镜观察到大量特征性病毒样颗粒。将重组蛋白分别辅以铝胶、IMS和白油不同佐剂免疫小鼠,通过检测免疫小鼠VP2特异性ELISA抗体、PPV特异性中和抗体、生长抑素的抗体水平及生长激素水平来评价嵌合病毒样颗粒的免疫原性。结果表明,辅以铝胶与IMS佐剂重组蛋白组均产生了与PPV全毒组相似的ELISA抗体与中和抗体反应;重组蛋白免疫组均产生较好的针对生长抑素的抗体反应;免疫小鼠体内生长激素的水平明显升高;其中以铝胶佐剂组产生的各抗体水平最高,白油佐剂组各抗体水平最低。为以后生产安全、有效的颗粒化亚单位疫苗提供了一个新的设计思路,又为应用病毒样颗粒递呈外源肽,从而生产多联亚单位疫苗奠定了基础。     

一种定量检测人血清高敏C反应蛋白的化学发光免疫方法

旨在建立一种可定量检测人血清高敏CRP的化学发光检测方法 (High-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay,hs-CRP CLIA)。首先利用亲和层析和离子交换层析技术从肝硬化病人腹水中纯化出高纯度的天然CRP作为免疫原制备了22株CRP单克隆抗体 (单抗),其中13株单抗在磷酸胆碱配体捕获ELISA中呈阳性,然后利用方正滴定法筛选出单抗10C5和10C11建立了hs-CRP CLIA。试剂盒评估结果显示：该方法对血清中干扰物质IgG、血红蛋白、甘油三酯等无非特异性反应;该方法检测灵敏度高,在0.04~20.38 mg/L范围内定量检测人血清CRP标准品呈良好线性关系 (R2＞0.993);该方法准确性高、可重复性好,平均回收率为99％,批内差为4.2％~5.8％,批间差为9.0％~11.5％;该方法与进口商品化高敏CRP ELISA试剂盒平行比较检测90份血清标本,结果显示两者有良好的可比性 (r=0.968)。综上,建立的hs-CRP CLIA是一种准确、可靠、可定量的高灵敏C反应蛋白检测方法,该方法的临床应用,有利于改善我国心脏病风险评估及肠炎性疾病预后判断。