Relationship between the berrant hypermethylation profile of 14-3-3 sigma and its reduced transcription levels in Chinese women sporadic breast carcinogenesis

Objective: To study the relationship between 14-3-3 sigma gene promoter hypermethylation and its transcrip-tion levels in sporadic breast carcinogenesis. Methods: Hypermethylation of 14-3-3 sigma gene was detected by sensitive MSP assay in carcinous, non-cancerious and normal tissue, and its mRNA was also detected by real-time PCR based on SYBR Green 1. Results: The hypermethylation frequencies of 14-3-3 sigma were 90% in 68 cases of sporadic breast cancer patients. Hypermethylation was presented in portions (2/13,18%) of hyperplastic samples, and no hypermethylation was presented in normal tissue. The hypermethylation change of 14-3-3 sigma gene was markedly related with various types, grades and lymph node metastases (P < 0.05), and no significant differences in methylation frequencies were seen between premenopause and postmenopause (P > 0.05). The hypermethylation of 14-3-3 sigma showed reverse relationship with its mRNA transcription (P < 0.05). Only lymph node metastases was strongly associated with poor outcome (P = 0.02). Whether 14-3-3 sigma promoter methylation or not did not affect the 5-year survival rate of sporadic breast cancer (P > 0.05). Conclu-sion: Epigenetics alterations of the 14-3-3 sigma can contribute to reducing or losing the expression of 14-3-3 sigma protein, which plays an important role in the development of sporadic breast carcinomas including various types, grades and lymph node metastases.

利用Red同源重组系统构建兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶基因打靶载体

利用EL350基因工程菌进行同源重组,成功进行基因敲除已有报道,但利用该系统进行兔次黄嘌呤-鸟嘌呤磷酸核糖转移酶(hypoxanthine guanine phosphoribosyl transferase,HPRT)基因突变和基因打靶方面的研究还没有报道。实验首先在已经筛选到含有兔全长HPRT基因BAC克隆(LBNL1-304M19)的基础上,利用Red重组系统,通过Gap-Repair方式从此克隆上将一段47kb无启动子的HPRT基因组片段(不含有第1个外显子)克隆到pBACLinkSp质粒上,产生pBACLinkSp-rHPRT质粒。然后基于pBACLinkSp-rHPRT质粒,设计不同的同源臂,从而删除了HPRT基因的不同编码区,成功构建了三个不同的HPRT基因打靶载体。同时对利用同源重组技术敲除不同大小的DNA片段的效率进行了研究。基于实验所构建的三个不同的兔HPRT基因打靶载体,为探索兔成纤维细胞和胚胎干细胞基因打靶的适宜条件,及进一步获得兔HPRT基因敲除动物疾病模型奠定了基础。

三萜类化合物Bardoxolone methyl对急性呼吸窘迫综合征大鼠氧化应激信号通路Nrf2-ARE/HO-1的影响

目的观察三萜类化合物Bardoxolone methyl(BARD)对急性呼吸窘迫综合征(ARDS)大鼠氧化应激信号通路核转录因子相关因子2(Nrf2-ARE)/血红素氧合酶-1(HO-1)的影响。方法100只大鼠按随机数字法分为药物BARD组及生理盐水组,喂养一周后两组按是否建立ARDS模型再各分为两亚组,产生四组:ARDS模型+BARD干预组(A1组),ARDS模型+生理盐水组(A2组),正常+BARD干预组(B1组),正常+生理盐水组(B2组)。各组大鼠再喂养72h后处死留取肺组织及血液标本。病理观察肺组织结构变化。免疫组织化学检测肺组织Nrf2、HO-1蛋白表达情况。反转录-多聚酶链反应(RT-PCR)检测肺组织Nrf2 mRNA、HO-1 mRNA表达情况。结果肺组织病理结构变化:A2组肺组织损伤程度最重,病理评分最高。其次A1组,B1及B2组结构大致正常。Nrf2、HO-1蛋白表达情况:A1组(3.78±0.37,2.86±0.16)及B1组(3.01±0.34,2.47±0.19)高于A2(1.82±0.31,2.01±0.21)及B2组(0.92±0.13,1.41±0.17),A1组高于B1组,A2组高于B2组(P均<0.05)。Nrf2mRNA、HO-1mRNA表达情况:A1组(2.16±0.17,1.45±0.12)最为明显,B1组(1.79±0.14,1.18±0.05)高于B2组(0.42±0.06,0.68±0.11),A2组(1.18±0.19,1.01±0.07)高于B2组(0.42±0.06,0.68±0.11,P均<0.05)。结论三萜类化合物Bardoxolone methyl能够明显上调肺组织Nrf2-ARE/HO-1信号通路相关蛋白的表达,从而发挥抗氧化应激作用,减轻ARDS时肺组织的损伤。

利用Red系统快速改构含鼠β-酪蛋白基因的细菌人工染色体

采用Red系统介导的同源重组方法对含有鼠β-酪蛋白基因的RPCI23-440C1BAC进行快速改构。首先通过PCR方法,获得两端带有鼠β-酪蛋白基因同源序列的tPAm-Zeo同源重组片段,然后将此同源重组片段电击转化至已含有编码Red重组酶质粒的RPCI23-440C1BAC菌中,在λRed重组系统的帮助下,通过同源重组片段两端与RPCI23-440C1中β-酪蛋白同源的序列在菌体内与β-酪蛋白基因发生同源重组,将其置换。最后利用Zeocin抗性基因两侧的FRT位点,通过FLP位点专一性重组将抗性基因剔除。经Southern blot和序列分析鉴定表明,获得了重组正确且无编码两种重组酶质粒的tPAm-RPCI23-440C1BAC克隆。

The Popularization and Application of Cold Storage Red Blood Cells or Whole Blood at -80 ℃ of the Rh(D) Negative Patients in Surgical Operation

Summary: The efficiency of cold storage red blood cells (CSRBC) or whole blood at -80 ℃ used in 27 Rh(D) negative patients during surgical operation was reported. The Rh(D) negative patients received the transfusion of CSRBC or whole blood stored at -80 ℃ for 180 to 360 days. The changes in the indexes, such as blood TB, DB, K +, Na +, BUN, Cr, urine protein (URPO), UOB, Hb, HCT, serum total protein, relative to hemolytic reaction and blood volume before and after transfusion were observed. The results showed that after transfusion of CSRBC or whole blood 27 cases were negative for urine protein and UOB, and the levels of BUN and Cr were normal (P>0.05). Blood TB, DB, Hb, and HCT were increased, while pH, blood K + and blood Na + was normal with the difference being not significant before and after operation (P>0.05). Plasma protein was decreased, but there was no significant difference before and after operation (P>0.05). It was suggested that CSRBC or whole blood at -80 ℃ could be safely infused to the Rh(D) negative patients without side effects during the surgical operation.

Synthesis,Crystal Structure and Fungicidal Activity of(E)-2-[(4-tert-Butyl-5-(4-methoxybenzyl)thiazol-2-ylimino)methyl]phenol

The title compound （E）-2-[（4-tert-butyl-5-（4-methoxybenzyl）thiazol-2-ylimino）methyl]phenol was synthesized by the reaction of 5-（4-methoxybenzyl）-4-tert- butylthiazol-2-amine with salicylaldehyde, and its crystal structure was determined by single-crystal X-ray diffraction. The crystal belongs to the monoclinic system, space group P21/c with a = 5.9362（8）, b = 11.5070（15）, c = 29.460（4）A, β= 97.326（3）°, V = 1995.9（5） A^3, Z = 4, F（000） = 808, C22H24N2O2S, Mr= 380.49, De= 1.266 g/cm^3, S = 1.031,μ = 0.181 mm^-1, the final R = 0.0474 and wR = 0.1441 for 4327 observed reflections （I 〉 2σ（I））. Intramolecular O-H…N hydrogen bond is observed in the crystal. The preliminary bioassay showed that the title compound exhibits 95% inhibition rate against Rhizoctonia solani at the test concentration of 500 mg/L.

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 (Z)-Methyl-3-methoxy-2-{2-[(4-(E-3-p-tolylacryloyl)phenoxy)methyl]phenyl}acrylate

The title compound,(Z)-methyl-3-methoxy-2-{2-[(4-(E-3-p-tolylacryloyl)phenoxy)-methyl]phenyl}acrylate,was synthesized and determined by X-ray single-crystal diffraction.The crystal belongs to the triclinic system,space group P1 with a=8.0157(8),b=12.5748(13),c=13.3768(14)Å,α=64.770(2),β=75.720(2),γ=89.784(2)°,μ=0.085 mm^(-1),Mr=442.49,V=1174.1(2)Å3,Z=2,Dc=1.252 g/cm^(3),F(000)=468,T=294(2)K,R=0.0603 and wR=0.1498 for 2644 observed reflections with I〉2σ(I).X-ray diffraction analysis reveals that the single crystal contains strong non-classical hydrogen bonds.The preliminary bioassay showed that the title compound exhibits inhibitory activity against the Pseudoperoniospora cubensis and Rhizoctonia solani at the test concentration of 200 mg/L.

Synergistic Effects of a Night Temperature Shift and Methyl Jasmonate on the Production of Anthocyanin in Red Leaf Lettuce

The production of a secondary metabolite such as anthocyanin is coordinately regulated by plant intrinsic factors and influenced by multiple environmental factors. In red leaf lettuce, the red pigment component anthocyanin is important for the commercial value of the crop, but its synchronous regulation by multiple factors is not well understood. Here, we examined the synergistic effects of a night temperature shift and methyl jasmonate (MJ) on the production of anthocyanin in red leaf lettuce. Low or high night temperature treatment for 3 days just before harvesting induced the production of anthocyanin without affecting plant biomass. Temperature-dependent activation of anthocyanin accumulation was accelerated by treating with MJ. Night temperature shifts and MJ triggered oxidative stresses in leaves, as indicated by hydrogen peroxide accumulation and lipid peroxidation. Interestingly, these oxidative stresses were more evident in leaves simultaneously treated with both a high night temperature and MJ. The activity of the superoxide dismutase (SOD) was increased alongside the elevation of oxidative stress. Taken together, these results indicate that the combined treatment of a night temperature shift with MJ may accelerate anthocyanin production by increasing the levels of oxidative stress to the leaves of red leaf lettuce.

Trace Level Arsenic Quantification through Methyl Red Bromination

A simple protocol has been developed for the quantification of trace level arsenic through methyl red bromination. The proposed method is based on the oxidation of arsenic(III) to arsenic(V) by the bromine and the residual bromine’s reaction with methyl red to form colorless bromo methyl red. As the concentration of arsenic increases, the bleaching of the dye decreases due to bromine consumption. Measuring the intensity of the unreacted methyl red at 515 nm forms the basis of arsenic quantification. The molar absorptivity of this method has been found to be 2.25 × 103 L/mol/cm. The method obeys Beer’s law in the concentration range 0 - 0.25 μg/mL. The Sandell sensitivity and the limit of detection (LOD) were found to be 0.03 μg/mL/cm2 and 0.03 μg/mL respectively. The relative standard deviation has been found to be 0.35% at 1.0 μg/mL. The reaction conditions have been optimized and the interference due to various common cations and anions were studied. The proposed method has been successfully applied to the determination of trace level arsenic in various environmental samples like water, soil and vegetable samples.

Pseudo Constants for Methyl Red Sorption: A Rate Study of Received and Derived Activated Carbon

This effluent remediation research on discoloration tends to disagree with the use of commercial activated carbon as received from manufacturers. Product specification and authentication is a key task to chemists and scientist. Here, Batch kinetic studies via pseudo approximations treatments was adopted to to investigate the rate of Methyl Red (MR) dye solution uptake onto carbon animalis earlier received as Granulated Activated Carbon (GAC) and later formulated as Powdered Activated Carbon (PAC). The rate of dye uptake was studied with data fitted in to the Lagergren’s pseudo first and second order kinetic models. Justification by the R2 values (0.984) for GACgreater than 0.865 for PAC, low statistical error (SSE%) range of 1.065 - 2.310 and closeness between the experimented and calculated qe values all favored the second order kinetic model. The deviation of the line from the origin further showed that intra-particle transport is not the only rate limiting step. Generally the research supported the manufacturer’s choice of particle size (as GAC and not PAC) for the chosen adsorbate.

Synthesis, Structure and Quantum Mechanical Calculations of Methyl 2-(5-((Quinolin-8-yloxy)-methyl)-1,3,4-oxadiazol-2-ylthio)-acetate

The title compound was synthesized by the base catalyzed reaction of 5-（（quinolin- 8-yloxy）methyl）-1,3,4-oxadiazole-2（3H）-thione with methyl chloroacetate. The structure was supported by the spectroscopic data and unambiguously confirmed by single-crystal X-ray diffraction studies. It crystallizes from a methanol solution in the triclinic space group Pi with unit cell dimensions a = 7.4509（9）, b = 10.2389（12）, c = 12.2299（15）A, a = 74.771（2）, β = 77.956（2）, 7 = 69.263（2）°, V = 834.98（17） A3 and Z = 2. In order to gain some valuable insights into the molecular structure, the quantum mechanical calculations were performed using both HF and time-dependent density functional theory at the B3LYP/6-31G（d,p） level. The molecular geometry from X-ray determination of the title compound in the ground state has been compared using the Hartree-Fock （HF） and density functional theory （DFT） with the 6-31G（d） basis set. The calculated results show that the DFT and HF can well reproduce the structure of the title compound. The energetic behavior of the title compound was examined using the B3LYP method with the 6-31G（d） basis set. The harmonic vibrational frequencies calculated have been compared with the experimental FTIR and FT-Raman spectra. The restricted Hartree-Fock and density functional theory-based nuclear magnetic resonance （NMR） calculation procedure was also performed, and it was used for assigning the 13C and 1H NMR chemical shifts of the title compound. Moreover, molecular electrostatic potential and thermodynamic parameters of the title compound were investigated by theoretical calculations.

Stereoselective Synthesis of (+)-△~5-Dehydrosugiyl Methyl Ether

A stereoselective synthetic route to △~5-dehydrosugiyl methyl ether was developed from (S)-(-)-α-cyclocitral, DDQ as a better oxidant for enone was used.

用甲基橙与溴甲酚绿-甲基红作指示剂标定盐酸溶液的差异分析

实验发现在使用无水碳酸钠标定盐酸溶液时,用甲基橙与溴甲酚绿-甲基红作为指示剂标定的盐酸溶液浓度存在一定差异。本文用以上2种指示剂,标定0.3、0.5、0.8、1.0 mol/L盐酸溶液浓度结果的比对分析,探索其结果差异及对甲基丙烯酸丁酯纯度分析结果的影响。

Synthesis and Crystal Structure of Methyl 3-(5-Bromo-1-ethyl-1H-indole-3-carbonyl)aminopropionate

Methyl 3-（5-bromo-l-ethyl-lH-indole-3-carbonyl）aminopropionate has been synthesized by the acylation of 5-bromo-3-trichloroacetylindole with β-alanine methyl ester, followed by alkylation with ethyl iodide, in 82.6% yield. Its crystal structure was gotten and determined by X-ray diffraction method. The crystal is of monoclinic, space group P2/c with a = 11.7927（8）, b = 14.9342（8）, c = 9.0060（5） A, β = 101.558（6）°, V = 1553.93（16） A3, Z = 4, Dc= 1.510 g/cm3, 2 = 0.71073 A,μ（MoKa） = 2.656 mm-1, Mr = 353.22 and F（000） = 720. The structure was refined to R = 0.0401 and wR = 0.0825 for 1704 observed reflections with I 〉 2σ（I）. In the crystal structure, intermolecular N（2）-H（2）...O（1） hydrogen bond and weak intermolecular bonds （C（1）-H（1）...O（1） and C（10）-H（10B）-O（2）） are formed, and π-π stacking also exists.

Rapid and Continuous Extraction of Methyl Red from Wastewater Using Counter Current Chromatography

A new prototype and simple low speed counter current chromatography (LSCCC) has been fabricated and a laboratory work has been carried out for rapid and continuous removal of methyl red from wastewater using xylene as an extractant. The distribution ratio (D) and percentage of recoveries were calculated. The optimum concentration for extraction of methyl red was in the range of 2 × 10-4 to 8 × 10-4 mol·L-1. Maximum extraction achieved at pH range of 2 to 4.5. Various dye concentrations and solvent effects were studied to optimized conditions. It was shown that when the ratio of the organic phase to aqueous phase increased to more than 3:1, the striping efficiency decreased sharply. The experiments were carried out for 5 times and the highest extraction achieved was 99.8 per cent by two-solvent system LSCCC in just 5 minutes. Real wastewater samples were analyzed and the efficiency of the technique was compared with liquid-liquid extraction (LLE). In practice it was shown that although both techniques are rapid, the efficiency of the LSCCC is much better than LLE.

Synthesis and Crystal Structure of 2-[(4-Methoxy- 6-methylthio-2-pyrimidinyl)aminocarbonyl- aminosulfonyl] Benzoic Acid Methyl Ester

The title compound 2-[(4-methoxy-6-methylthio-2-pyrimidinyl)aminocarbonyl- aminosulfonyl]benzoic acid methyl ester (C15H16N4O6S2, Mr = 412.44) was obtained by the reaction of (4-methoxy-6-methylthio-2-pyrimidinyl)amine with 2-methoxylcarbonylbenzene-sulfonylisocya- nate. The crystal is of monoclinic, space group P21/c with a =11.169(3), b = 9.508(3), c = 17.690(5) ? b = 91.593(5), Z = 4, V = 1877.9(10) 3, Dc = 1.459 g/cm3, F(000) = 856, m(MoKa) = 0.324 mm-1, R = 0.0690 and wR = 0.1368 for 3301 observed reflections (I > 2s(I)). The N(1)H…N(3) and N(2)H…O(4) hydrogen bonds can be observed. In the molecule the phenyl plane(I), pyrimi- din-2-yl-urea bridge plane(Ⅱ) and ester plane(Ⅲ) form three conjugated systems.

Synthesis and Crystal Structure of Methyl 2-(Diphenylamino)-4-phenyl-1,3-thiazole-5-carboxylate

The title compound, methyl 2-（diphenylamino）-4-phenyl-1,3-thiazole-5-carboxylate, was synthesized and studied by single-crystal X-ray diffraction method. The structure of the product was confirmed by IR, 1H- and 13C-NMR spectroscopy and elemental analysis. These experimental studies were supported by quantum mechanical calculations. The structure was solved in monoclinic, space group P21/c with a = 9.573（3）, b = 19.533（7）, c = 9.876（3）, β = 92.35（4）°, V = 1845.2（10）3, T = 85（2） K, Z = 4, R = 0.040 and wR = 0.089 for 6424 observed reflections with I2σ（I）.

HELIX SENSE-SELECTIVE COPOLYMERIZATION OF TRIPHENYLMETHYL METHACRYLATE WITH CHIRAL ANIONIC INITIATORS

Optically active copolymers of pairs of three monomers, triphenyl (methyl methacrylate)and one or two pyridyl substituted methyl methacrylate homologues, were obtained by helix-sense-selective copolymerization using complexes of organolithium with chiral ligand as anionicinitiators in toluene at low temperature. The copolymers obtained with (-)-sparteine (Sp) and(S,S)-(+)-and (R, R)-(-)-2, 3-dimethoxy-1, 4-bis (dimethylamino) butanes((+)-and (-)-DDB) complexes of organolithium showed low optical activity, but PMP complex with N, N-diphenylethyleneamine monolithium amide (PMP-DPEDA-Li) was effective in synthesizingcopolymers of high optical rotation ([α]_D^(25) about+320～1370°)which were comparable to thoseof relative homopolymers with one-handed helical structure.

Syntheses and Crystal Structures of Methyl 2-(4,5-Dibromo-1-methylpyrrole-2-carbonylamino)-3-phenylpropanoate and 4,5-Dibromo-1-methyl-2- trichloroacetylpyrrole

4,5-Dibromo-1-methyl-2-trichloroacetylpyrrole Ⅰ, prepared by the reaction of 1-methyl-2-trichloroacetylpyrrole with Br2 in 98.6 % yield, was condensed with methyl L-2-amino-3- phenylpropanoate to afford methyl 2-（4,5-dibromo-1-methylpyrrole-2-carboxmido）-3-phenylpropanoate Ⅱ in 90.8% yield. Crystal data for Ⅰ： monoclinic system, space group P21/c with a = 8.595（3）, b = 10.900（4）, c = 12.321（5） ,A°,β = 92.292（7）°, V = 1153.4（8）A°^3, Dc = 2.213 g/cm^3, F（000） = 728, CTH4Br2Cl3NO, Mr = 384.28, λ = 0.71073 A°, μ（MoKa） = 7.688 mm^-1, Z = 4, R = 0.0338 and wR2 = 0.0840 for 1963 observed reflections with I 〉 2a（I）. Crystal data for Ⅱ： monoclinic system, space group P21 with a = 4.886（2）, b = 15.921（8）, c = 11.635（6） A°,β = 101.803（9）°, V = 885.9（7） A°^3, Dc = 1.665 g/cm^3, F（000） = 440, C16H16Br2N2O3, Mr = 444.13, λ = 0.71073 A°, μ（MoKa） = 4.590 mm^-1, Z = 2, R = 0.0335 and wR2 = 0.0837 for 3191 observed reflections with I 〉 2σ（I）. The crystal structure reveals that compound Ⅱ forms the one-dimensional chain structure via the intermolecular hydrogen bonds of N（2）-H…O（1）.