[B10Glu,B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

采用PCR方法,将人胰岛素分子B链B10位His突变为Glu,在B24和B25位之间插入Asp,构建了[B10Glu,B24-Asp-B25]胰岛素原基因.利用通用型质粒pBV220构建表达载体,在大肠杆菌DH5α中表达,表达蛋白为包含体形式,约占菌体总蛋白的20%～30%.经过复性和凝胶过滤得到胰岛素原融合蛋白.用胰蛋白酶和羧肽酶B酶切,经DEAE离子交换和RP-HPLC纯化得胰岛素突变体类似物.用凝胶过滤法测定了蛋白质分子自身的缔合性质,用圆二色谱测定了构象变化.放射性免疫活性及受体结合活性测定结果表明,突变体分子缔合性明显下降,放免活性和受体结合活性分别约为人胰岛素的73.6%和146%.     

[Bl0Glu，B24-Asp-B25]人胰岛素类似物的制备、鉴定与性质研究

采用PCR方法，将人胰岛素分子B链B10位His突变为Glu，在B24和B25位之间插入Asp．构建了[B10Glu，B24-Asp-B25]胰岛素原基因、利用通用型质粒pBV220构建表达载体，在大肠杆菌DH5a中表达．表达蛋白为包含体形式．约占菌体总蛋白的20％～30％．经过复性和凝胶过滤得到胰岛素原融合蛋白、用胰蛋白酶和羧肽酶B酶切．经DEAE离子交换和RP-HPLC纯化得胰岛素突变体类似物．用凝胶过滤法测定了蛋白质分子自身的缔合性质．用圆二色谱测定了构象变化、放射性免疫活性及受体结合活性测定结果表明，突变体分子缔合性明显下降．放免活性和受体结合活性分别约为人胰岛素的73．6％和146％。     

胰岛素分子与其受体结合的可能机制

以最小二乘叠合技术和图象显示技术对DPI、二锌胰岛素及其他一些衍生物结构进行了深入的比较和分析,确认与受体分子的结合相互作用是发生在胰岛素分子的一个两性表面上,其中部是由许多疏水残基构成的面积约150~2的疏水表面,而一些极性基团在其周围构成亲水带区。疏水表面通常被运动性很大的B链羧端肽段所覆盖,不受极性溶剂分子的干扰。两性表面在方位上与分子在二体中的缔合面构成约20°的夹角。Al-L-Trp胰岛素及Al-D-Trp胰岛素结构详细的比较结果,既很好地解释了两者在生物活性上的显著差异,又进一步确认了我们所提出的胰岛素分子与其受体结合的作用模型。     

1.9分辨率(D-Ala)~(B_0)猪胰岛素晶体结构的研究

本文应用差值Fourier技术和制约立体化学参数最小二乘技术以二锌猪胰岛素模型为起点测定了1.9分辨率的(D-Ala)~(B0)猪胰岛素晶体结构,R因子为0.211,键长均方差为0.057。B_0残基的电子密度表现清晰。B_0的引入降低了B链N端肽段构象的运动性,使分子在晶体中的堆积更加紧密。(D-Ala)~(B0)猪胰岛素和二锌猪胰岛素结构的主要差异在于分子可能的受体结合表面中部分极性基团的构象和分子IA链两段螺旋的组装。(D-Ala)~(B0)猪胰岛素B链N端晶体学微环境与(Trp)~(B1)猪胰岛素和去B_1牛胰岛素有很大的差异。本文还简单地讨论了胰岛素分子结构与免疫功能的关系。     

神经介素B受体与拮抗剂、激动剂的分子模拟研究

大鼠神经介素B受体(rat neuromedin B receptor, rNMBR)属于G蛋白偶联受体(G-protein coupled receptor, GPCR) A家族的成员. GPCR的结构特征和在信号传导中的重要作用决定了其可以作为很好的药物靶标. 关于rNMBR与内源性激动剂神经介素B (neuromedin B, NMB)以及与非肽类拮抗剂pd168368作用机制的研究对于合理设计受体药物分子有重要的指导意义. 在这一研究中, 我们使用同源模建, 构建受体的三维结构, 进行分子对接和分子动力学的计算. 基于受体三维结构, 通过10 ns的空载受体、激动剂-受体、拮抗剂-受体的分子动力学模拟, 探讨受体与激动剂与拮抗剂的作用机制. 研究表明rNMB-R中跨膜(transmembrane, TM)螺旋3, 5, 6, 7参与配体的结合. NMB与受体的结合, 使受体转变为活性构象, 而受体同拮抗剂pd168368恰好相反.     

多壁碳纳米管活化A549细胞核转录子-κB的机制

探讨多壁碳纳米管对人肺上皮细胞A549核转录因子-κB(NF-κB)活性的影响及其活化机制.不同浓度的多壁碳纳米管作用于A549细胞后,用活性氧(ROS)敏感探针2′,7′-二氯荧光素二乙酸酯结合流式细胞仪检测细胞内氧化应激状态;用凝胶电泳迁移率改变这一分析技术检测A549细胞NF-κB DNA结合活性;用蛋白印迹检测A549细胞NF-κB p65蛋白和IκBα蛋白表达;用免疫荧光结合共聚焦显微镜观察A549细胞NF-κB p65蛋白的核转位情况.结果表明,多壁碳纳米管诱导A549细胞内ROS过量产生和NF-κB DNA结合活性;同时伴有p65蛋白核移位和IκBα蛋白胞浆降解.抗氧化剂N-乙酰半胱氨酸(NAC)可抑制多壁碳纳米管诱导的A549细胞内ROS产生、NF-κB DNA结合活性、p65蛋白核移位以及IκBα蛋白降解.结果表明,多壁碳纳米管可以通过诱导A549细胞氧化应激机制从而活化核转录因子NF-κB活性.     

去B链羧端七肽胰岛素晶体结构的测定

去B链羧端七肽胰岛素(DHPI)晶体中不对称单位合二个分子,空间群P2_12_12_1。运用Patterson搜索技术确定了二个分子在晶胞中的取向,联合运用平移函数和R因子搜索测定了两个分子各自在晶胞中的位置。运用生物大分子刚体最小二乘修正技术和能量极小化最小二乘制约修正精化分子的取向和位置后,在6分辨率晶体学R因子下降到0.384。初始Fourier图显示,与天然胰岛素分子相较,DHPI分子的B链N端(B1—B8)和C端(B20—B23)肽段的构象有剧列变化,但A,B链的三段螺旋及其相对配置大体保持。     

胰岛素单体的结构特征及其运动方式

运用最小二乘叠合技术及图象显示技术对已测定的不同种属和不同晶型的胰岛素及各种胰岛素衍生物空间结构进行了广泛的构象比较.研究结果表明:二锌胰岛素分子Ⅰ更接近于天然单体的结构;二体内两个分子之间的构象差异发生在二体形成过程中,并在六体形成及六体堆积过程中完善和稳定;通过一些铰链肽A10,B4,B8,B24,B20及B23,胰岛素分子内各结构片段之间具有较灵活的相对运动,羧端残基可能移动大于10A;由于所处的环境不同,各残基侧链的运动性差别很大.     

分子密堆积法及其在B链羧端去六肽(B25—30)胰岛素结构测定中的应用

基于分子密堆积原理我们定义了一个能够表达在单位晶胞中对称相关分子之间堆积相容性的密堆积函数,并在计算机上模拟晶格中的分子密堆积,同时计算该函数在分子的每个旋转及平移位置的值,使分子密堆积定量化。这一方法不仅可以从多个旋转函数或平移函数峰中判断出一个正确的解,而且可能独立地、定量地和快速地解决一些特殊的旋转及平移问题。用已知结构B链羧端去五肽胰岛素作为例子检验了分子密堆积法及其程序的有效性,并用分子密堆积法独立地解决了未知结构B链羧端去六肽胰岛素的平移问题.R因子搜索等方法证实了密堆积法的解的正确性。分子密堆积法可以作为一种既相对独立于分子置换法又与之相辅相承的方法。     

重组人胰岛素注射液中B3和B3iso脱氨人胰岛素的分析与鉴定

建立了有效分离重组人胰岛素注射液中B3和B3iso脱氨人胰岛素的反相高效液相色谱(RP-HPLC)法,并采用高效液相色谱-四极杆-飞行时间质谱联用技术(HPLC-MS/MS)对其进行氨基酸序列的鉴定。在《中国药典》2010版重组人胰岛素注射液有关物质检查分析方法的基础上,优化流动相p H值,以0.2mol/L硫酸盐缓冲液(p H 3.6)-乙腈(90∶10)作为A相,乙腈-水(50∶50)为B相,梯度洗脱,分别收集杂质峰1和杂质峰2,脱盐浓缩后,用丝氨酸蛋白酶(V8酶)酶切,取部分酶切溶液经三(2-羧乙基)膦盐酸盐(TCEP)还原,分别得到还原和非还原肽段溶液,利用LC-MS/MS鉴定氨基酸序列,未知杂质分别为重组人胰岛素B3位发生脱氨和异构化的产物,即B3和B3iso脱氨人胰岛素。将优化后的方法与《中国药典》方法对市售重组人胰岛素注射液进行有关物质的对比分析,已上市厂家的重组人胰岛素注射液均有不同程度的B3和B3iso脱氨人胰岛素存在。该方法不影响重组人胰岛素其它有关物质的检测,可用于监控人胰岛素注射液产品储存过程中脱氨类杂质的增长趋势。     

Structure and stability of B6, B,and B clusters

We investigated various isomers of B₆, B, and B clusters with ab initio [Hartree–Fock (HF), MP2)] and density functional theory (DFT) methods. Ten B₆ isomers, 6 B isomers, and 6 B isomers are determined to be local minima on their potential energy hypersurfaces by the HF, B3LYP, B3PW91, and MP2 methods. Fourteen of these structures are first reported. The most stable neutral B₆ cluster is the capped pentagonal pyramid (C_5v), in agreement with the results reported previously. Hexagon B (C_2h) isomer and fan‐shaped B (C_2v) isomer are found to be the most stable on the cationic and anionic energy hypersurfaces, respectively. Natural bond orbital analysis suggests that there are three‐centered bonds in the most stable B₆ neutral and ionic clusters. The multicentered bonds are responsible for the special stability of the lowest‐energy isomer. © 2003 Wiley Periodicals, Inc. Int J Quantum Chem 94: 269–278, 2003     

B,B, B-Trimethyl-N,N, N-triäthylborazin- und B,B, B-Triäthyl-N,N, N-trimethylborazin-molybdäntricarbonyl

The title compounds have been prepared in low yields starting with cis-(CH₃CN)₃-Mo(CO)₃ or Mo(CO)₆ (photochemical route). The new complexes are generally less stable than the corresponding hexaalkylborazine-chromium compounds and react very smoothly with Lewis bases by cleavage of the borazine-metal bond. The IR.-spectra indicate the similarity of type of bonding in hexaalkylborazine-molybdenum and -chromium complexes.     

Quantum chemistry study on B14, B,and B14H

The structure of B₁₄, B, and B₁₄H in octahedral symmetry has been investigated by ab initio calculations at the STO-3G and 4–31G levels. The relationship of molecular orbitals among them has been analyzed and it can be found that the number of valence bonding orbitals of high borane obeys the Wade rule. The similarities and difference between boron clusters and carbon clusters are also discussed. © 1994 John Wiley & Sons, Inc.     

Roles of vitamins B5, B8, B9, B12 and molybdenum cofactor at cellular and organismal levels

Many efforts have been made in recent decades to understand how coenzymes, including vitamins, are synthesised in organisms. In the present review, we describe the most recent findings about the biological roles of five coenzymes: folate (vitamin B9), pantothenate (vitamin B5), cobalamin (vitamin B12), biotin (vitamin B8) and molybdenum cofactor (Moco). In the first part, we will emphasise their biological functions, including the specific roles found in some organisms. In the second part we will present some nutritional aspects and potential strategies to enhance the cofactor contents in organisms of interest.     

Quantitative Assessment of B−B−B,B−Hb−B,and B−Ht Bonds: From BH3 to B12H122−

We report the thermodynamic stabilities and the intrinsic strengths of three-center-two-electron B−B−B and B−H_b−B bonds ( : bridging hydrogen), and two-center-two-electron B−H_t bonds ( : terminal hydrogen) which can be served as a new, effective tool to determine the decisive role of the intermediates of hydrogenation/dehydrogenation reactions of borohydride. The calculated heats of formation were obtained with the G4 composite method and the intrinsic strengths of B−B−B, B−H_b−B, and B−H_t bonds were derived from local stretching force constants obtained at the B3LYP-D2/cc-pVTZ level of theory for 21 boron-hydrogen compounds, including 19 intermediates. The Quantum Theory of Atoms in Molecules (QTAIM) was used to deepen the inside into the nature of B−B−B, B−H_b−B, and B−H_t bonds. We found that all of the experimentally identified intermediates hindering the reversibility of the decomposition reactions are thermodynamically stable and possess strong B−B−B, B−H_b−B, and B−H_t bonds. This proves that thermodynamic data and intrinsic B−B−B, B−H_b−B, and B−H_t bond strengths form a new, effective tool to characterize new (potential) intermediates and to predict their role for the reversibility of the hydrogenation/dehydrogenation reactions.     

Concise, asymmetric, stereocontrolled total synthesis of stephacidins A, B and notoamide B

Concise asymmetric total syntheses of the fungal metabolites (-)-stephacidin A, (+)-stephacidin B, and (+)-notoamide B are described. Key features of these total syntheses include (1) a facile synthesis of (R)-allyl proline methyl ester, (2) a revised route toward the pyranoindole ring system, (3) a novel cross-metathesis strategy for the introduction of important functional groups, and (4) an SN2' cyclization to form the [2.2.2] bridged bicyclic ring system. Furthermore, our synthesis has taken advantage of microwave heating to shorten reaction times as well as increase yields for the preparation of vital intermediates.     

Enantioselective Synthesis of Glutarimide Alkaloids Cordiarimides A,B, Crotonimides A,B, and Julocrotine

Five glutarimide alkaloids cordiarimide A ( 5 ), cordiarimide B ( 6 ), crotonimide A ( 3 ), crotonimide B ( 4 ), and julocrotine ( 2 ) have been synthesized starting from Boc‐L‐glutamine ( 7 ). The benzylic alcohol chiral centre of cordiarimides B ( 6 ) has been established in 6:1 diastereoselectivity by catalytic asymmetric hydrogenation using Zhou's catalytic system Pd(CF₃CO₂)₂/(R,R)‐Me‐DuPhos.     

Total synthesis of 16-desmethylepothilone B, epothilone B10, epothilone F, and related side chain modified epothilone B analogues

The macrolactonization-based strategy for the total synthesis of epothilones has been streamlined and improved to a high level of efficiency and stereoselectivity. This strategy has been applied to the construction of vinyl iodide 19 which served as a common intermediate for the synthesis of a series of natural and designed epothilones including an epothilone B10 (3), epothilone F (5), 16-desmethylepothilone B (14), pyridine epothilones 57a-57g, dimeric epothilones 59 and 61, and benzenoid epothilones 63a-63g.     

高精度定量毛细管电泳法同时测定复合维生素B片中B1、B2、B6、烟酰胺及泛酸钙

建立了高精度定量毛细管电泳法同时测定复合维生素B片中维生素B₁、B₂、B₆、烟酰胺和泛酸钙的方法。样品经乙腈-水（20∶80，v/v）超声提取后，采用全自动高精度定量毛细管电泳仪，以高精度进样阀定量进样，以40 mmol/L硼砂-硼酸缓冲液（pH 9.0）为背景电解质溶液，以工作电压为-10 kV电泳分离。维生素B₁、B₂、B₆、烟酰胺的检测波长为280 nm，然后切换至检测波长210 nm检测泛酸钙。结果表明，各组分之间均得到良好分离，峰面积日内重复性（RSD）为1.3%~1.9%，显著优于普通毛细管电泳。维生素B₁、B₂、B₆、烟酰胺及泛酸钙的浓度在各自线性范围内的相关系数（r）为0.9968~0.9998，检出限2.5~36.0 mg/L，平均回收率为94.1%~98.9%。该法准确可靠，可用于实际复合维生素B片中维生素B₁、B₂、B₆、烟酰胺和泛酸钙含量的同时测定。     

The crystal structure of LaIr4B4, ThOs4B4, ThIr4B4 (NdCo4B4-type) and URu4B4, UOs4B4 (LuRu4B4-type)

The crystal structure of LaIr₄B₄ has been refined from single crystal counter data. LaIr₄B₄ is tetragonal,P4₂/n,Z=2, isotypic with NdCo₄B₄, |F|/|F _o|=0.039 for 312 independent reflections [|F _o|>2 (F _o)]. ThIr₄B₄ and ThOs₄B₄ also belong to the NdCo₄B₄-type structure. URu₄B₄ and UOs₄B₄ were found to crystallize with LuRu₄B₄-type structure. The crystal chemistry of (RE)T ₄B₄-phases is discussed and simple geometric relations are shown to exist between them.Dedicated to Prof.B. T. Matthias in celebration of his 60th birthday.