Structural Basis for the Interaction of 14-3-3<i>β</i>with Tricarboxylic Acid Cycle Intermediate Malate

The protein family of 14-3-3(s) has risen to a position of higher importance as an adaptor protein in cell biology. The seven highly conserved human 14-3-3 proteins coordinate diverse cellular processes including apoptosis, DNA damage response, protein trafficking, and others. In liver hepatocytes, 14-3-3β binds to Ser196-phosphorilated glucose-responsive carbohydrate response element-binding protein (ChREBP) to inhibit converting excess carbohydrate to fat by regulating the nuclear/cytosol trafficking of ChREBP. Here, we report X-ray crystal structures of homodimeric mammalian 14-3-3β in its apo, Malate-bound forms. The determined apo structure was captured with one monomer in the closed state, whereas the other one had an open conformation. Strikingly, 14-3-3β binds Malate dynamically with a double-closed state, which is distinct from all previously characterized 14-3-3(s) and target ligand-binding modes. Malate docks into a first-time observed cofactor pocket located at the concaved interface of 14-3-3β helices α2, α3, α4 through mainly electrostatic and hydrogen interactions. Such a Tricarboxylic Acid Cycle intermediate Malate bond model might offer a new approach to further analyze insulin-independent 14-3-3/ChREBP pathway of de novo fat synthesis in the liver.

Synthesis and Crystal Structure of 4-(4,6-dimethoxyl -pyrimidin-2-yl)-3-thiourea Carboxylic Acid Methyl Ester

The title compound 4-（4,6-dimethoxylpyrimidin-2-yl）-3-thiourea carboxylic acid methyl ester was synthesized by the reaction of 2-amino-4,6-dimethoxyl pyrimidine, potassium thiocyanate and methyl chloroformate in ethyl acetate. Single crystals suitable for X-ray measurement were obtained by recrystallization with the solvent of dimethyl formamide at the room temperature. The structure was characterized by elemental analysis and IR and determined by X-ray diffraction analysis＇ Crystallographic data： C9H12N4O4S, Mr = 272.29, monoclinic, space group C2/m with a = 1.6672（3）, b = 0.66383（12）, c = 1.1617（2） nm, β = 109.275（2）°, V = 1.2136（4） nm^3, Dc = 1.490 g/cm^3,μ = 0.281 mm^-1, F（000） = 568, Z = 4, R1 = 0.0341and wR2 = 0.1042.

Synthesis and Crystal Structure of 4-(4,6-Dimethoxylpyrimidin-2-yl)-3-thiourea Carboxylic Acid Ethyl Ester

4-（4,6-Dimethoxyl-pyrimidin-2-yl）-3-thiourea carboxylic acid ethyl ester was synthesized by the reaction of 2-amino-4,6-dimethoxyl pyrimidine, potassium thiocyanate and methyl chloroformate in ethyl acetate. Single crystals suitable for X-ray measurement were obtained by recrystallization with the solvent of dimethyl formamide at room temperature. The crystal structure was determined by X-ray diffraction analysis. Crystallographic data： C10H14N4O4S, M, = 286.31, monoclinic, space group C2/c with a = 2.5309（3）, b = 0.67682（6）, c = 1.74237（19） nm, β = 114.744（3）°, V= 2.7106（5） nm3, Dc = 1.403 g/cm3, p = 0.225 mm-1, F（000） = 1200, Z= 8, R= 0.0514 and wR= 0.1529.

2-十三烷基苯并咪唑的合成工艺研究

苯并咪唑是一类存在于多种药物中的重要结构单元,在生物体中起着非常重要的作用。合成具有苯并咪唑结构的化合物对新药的开发和生物化学的研究都有着十分重要的意义。本实验以邻苯二胺和十四酸为原料,在二甲苯介质中加热回流得到2-十三烷基苯并咪唑,考查了原料配比、温度、反应时间等条件对产率的影响。结果表明:胺酸物质的摩尔比为1.2:1、酰化反应温度160℃、酰化反应时间6.0h、环合反应温度210℃、环合反应时间8.0h是最适宜的反应条件,其产率可达90.0%。

Apoptotic effect and mechanisms of AHPN on human skin malignant melanoma cell A375

Objective： To study apoptotic effects of synthetic retinoic acid 6-[3-（1-adamantyl）-4-hydroxyphenyl]-2-naphthalene carboxylic acid（AHPN） on human skin malignant melanoma A375 cells in comparison with the natural ligand all-trans-retinoic acid（ATRA） in vitro and the mechanisms related to the actions of AHPN. Methods：MTT assay was used to determine the anti-proliferative effects of AHPN and ATRA on A375 cells. Flow cytometry was performed to investigate the influence of AHPN and ATRA on cell cycle and cell apoptosis. In addition, transfection and luciferase activity assays were employed to explore the mechanisms of how AHPN executes its proapoptotic function. Results：Firstly, AHPN promoted apoptosis and GI arrest in A375 cells compared with ATRA. Secondly, the activity of NF-K B in A375 cells treated with AHPN increased 2-3 times compared with solvent DMSO treatment. Conclusion：AHPN, in comparison with ATRA, is a more effective alternative for therapy of malignant melanoma. The potentially proapoptotic function of AHPN requires activation of NF-K B.

UPLC-Q/TOF法同时定性定量分析滇芹药材中主要化学成分

目的建立UPLC-Q/TOF法同时定性、定量分析滇芹中主要化学成分。方法采用超高效液相色谱串联飞行时间质谱法进行分析,色谱柱为Agilent Poroshell 120 Hilic,流动相为乙腈-0.1%甲酸水溶液,梯度洗脱,体积流量0.3 m L/min;定性、定量采用Q-TOF正负离子全扫描+IDA(信息关联采集)模式。结果利用已建立的筛查数据库将满足质量误差小于5×10-6、同位素分布正确且含有二级碎片的离子作为分析目标化合物,结合软件Formula Finder、Mass Calculators等功能、在线数据库(Human Metabolome Database、Pub Chem、Chemical Book等)及二级碎片裂解规律,共鉴定出23个化合物,滇芹首次发现4个化合物。确定阿魏酸为定量分析指标性成分,其定量、定性离子分别为178和149;阿魏酸检测质量浓度线性范围为1.14~1 140 ng/m L(r=1.000),检测限、定量限分别为0.87、2.91 ng/m L,精密度、稳定性、重复性试验的RSD<2%,加样回收率为98.13%~101.25%(RSD=1.37%,n=5)。结论该方法灵敏度高、重现性好,分析快速、准确、可靠,可用于同时定性、定量检测滇芹药材中主要化学成分;云南滇芹中阿魏酸含量要高于西藏滇芹。

Calcium-dependent activation of CPK12 facilitates its cytoplasm-to-nucleus translocation to potentiate plant hypoxia sensing by phosphorylating ERF-Ⅶ transcription factors

Calcium-dependent protein kinases(CDPKs/CPKs)are key regulators of plant stress signaling that translate calcium signals into cellular responses by phosphorylating diverse substrate proteins.However,the molecular mechanism by which plant cells relay calcium signals in response to hypoxia remains elusive.Here,we show that one member of the CDPK family in Arabidopsis thaliana,CPK12,is rapidly activated during hypoxia through calcium-dependent phosphorylation of its Ser-186 residue.Phosphorylated CPK12 shuttles from the cytoplasm to the nucleus,where it interacts with and phosphorylates the group Ⅶ ethylene-responsive transcription factors(ERF-Ⅶ)that are core regulators of plant hypoxia sensing,to enhance their stabilities.Consistently,CPK12 knockdown lines show attenuated tolerance of hypoxia,whereas transgenic plants overexpressing CPK12 display improved hypoxia tolerance.Nonethelss,loss of function of five ERF-Ⅶ proteins in an erf-vii pentuple mutant could partially suppress the enhanced hypoxia-tolerance phenotype of CPK12-overexpressing lines.Moreover,we also discovered that phosphatidic acid and 14-3-3κ protein serve as positive and negative modulators of the CPK12 cytoplasm-to-nucleus translocation,respectively.Taken together,these findings uncover a CPK12-ERF-Ⅶ regulatory module that is key to transducing calcium signals from the cytoplasm into the nucleus to potentiate hypoxia sensing in plants.

二妙丸HPLC指纹图谱及UPLC-LTQ-Orbitrap成分分析

目的建立二妙丸的HPLC指纹图谱质量评价方法,并对二妙丸中的化学成分进行UPLC-LTQ-Orbitrap定性分析。方法指纹图谱采用HPLC法,色谱柱为Shiseido C18柱(250 mm×4.6 mm,5μm),乙腈-0.1%甲酸水溶液为流动相进行梯度洗脱,体积流量为1.0 m L/min,检测波长为330 nm,以黄柏、苍术混合对照药材生成的色谱图为标准对照图谱,采用"中国药典委员会相似度评价软件(2012A)"对样品进行相似度评价;采用UPLC-LTQ-Orbitrap技术对二妙丸中化学成分进行定性分析,色谱柱为Thermo Scientific Syncronis C18柱(100 mm×2.1 mm,1.9μm),乙腈-0.1%甲酸水溶液梯度洗脱,经DAD检测器后采用LTQ-Orbitrap Elite质谱正离子模式采集,各成分的一级全扫描离子在离子阱内进行CID多级质谱、FT检测高分辨质谱数据分析。结果所建立的指纹图谱有10个共有色谱峰,20批样品的相似度在0.869~0.992。通过对照品指认、文献比对以及高分辨质谱数据解析,鉴定了21个化学成分,分别为neo-chlorogenic acid、木兰箭毒碱、竹叶椒碱、木兰花碱、3-O-feruloylquinic acid、蝙蝠葛碱、demethyleneberberine、oxyberberine、非洲防己碱、药根碱、小檗红碱、巴马汀、小檗碱、丁香酸、咖啡酸、(E)-4-(3-hydroxyprop-1-en1yl)-2-methoxyphenol、白术内酯II、acetosyringone、白术内酯I、selina-4(14),7(11)-dien-8-one、苍术素。结论建立的二妙丸HPLC指纹图谱专属性强,重现性好,结合液质联用技术的定性分析,可全面评价二妙丸的内在质量。