中药三七、茯苓中克百威和抗蚜威的测定

[目的]获得中药三七、茯苓中克百威和抗蚜威的检测方法.[方法]通过液相色谱法对中药三七和茯苓中的克百威和抗蚜威的检测方法进行研究.[结果]确定了较优液相色谱检测条件为柱温30℃,流速1 mL/min,检测波长210nm,流动相水-乙腈(体积比61.5:38.5).获得抗蚜威和克百威分别在1.99×10-4～9.95×10-4 μg(r=0.9987)和1.97×10-4～9.85×10-4μg(r=0.9947)范围内线性良好.克百威和抗蚜威的RSD值分别为13.2％和7.1％.[结论]获得了克百威和抗蚜威的检测方法,并且2种中药样品中不含有克百威和抗蚜威.该方法出峰快,能避开受试中药中基质的干扰.     

抗蚜威原药及其杂质的GC-MS分析研究

采用气相色谱-质谱联用法对抗蚜威原药进行分离鉴定,使用HP-5MS(30 m×0.25 mm,0.25μm)毛细管柱,EI离子源,得到抗蚜威及其4个杂质的质谱图,根据标准谱库推断出这些谱图对应的杂质成分,根据合成路线探讨了杂质来源可能,并讨论了抗蚜威的质谱裂解规律。     

液相色谱-串联质谱法测定进出口人参、西洋参中蚜灭磷等6种农药

建立了人参、西洋参中凝胶色谱-固相萃取-液相色谱-串联质谱法测定蚜灭磷、吡虫啉、涕灭威、甲基内吸磷、甲硫威、辛硫磷6种农药的分析方法。样品经丙酮超声提取,凝胶色谱(GPC)净化后,固相萃取(SPE)净化,在HPLC/MS/MS多反应监测(MRM)模式下进行了定性和定量分析方法。在上述检测方法条件下,所测的6种农药的最低检出限为0.01 mg/kg,回收率范围为79.2%~107.3%,线性范围为0.01~0.2 mg/kg。     

血中非那西丁的GC/MS检测

建立了非那西丁的气相色谱/质谱/质谱(GC/MS/MS)联用检测血中去痛片的主要代谢物非那西丁的新方法。采用液液提取法,用乙醚提取血中非那西丁,提取物水浴浓缩后进行GC/MS和GC/MS/MS分析,检测总离子流色谱(TIC)和一级质谱,并以非那西丁一级质谱基峰为母离子的测定其二级质谱,体内药物用GC/MS全扫描定性分析即用二级质谱结合其总离子流色谱峰的保留时间定性;选择离子扫描定量分析,采用内标曲线法,根据非那西丁和内标SKF525A二级质谱的TIC峰面积比进行定量分析。     

MSPD-HPLC同时测定枸杞中3种氨基甲酸酯类农药

[目的]建立基质固相分散净化、高效液相色谱测定枸杞中速灭威、克百威、抗蚜威的残留量。[方法]用乙腈提取,C18基质固相分散净化,甲醇-二氯甲烷(体积比1颐1)洗脱,Agilent TC-C18色谱柱,流动相为甲醇-水梯度洗脱,流速1.0 m L/min,柱温30℃,进样量20滋L。[结果]速灭威和抗蚜威质量浓度1.0~100 mg/L、克百威质量浓度0.20~20 mg/L时,线性关系良好;添加水平为10~60滋g时,速灭威、克百威、抗蚜威的加标回收率分别为95.5%、92.4%、96.3%,RSD分别为2.34%、2.28%、3.46%。[结论]该方法准确、快速、简便,符合农药残留分析要求。     

血中非那西丁的GC/MS检测

建立了非那西丁的气相色谱/质谱/质谱(GC/MS/MS)联用检测血中去痛片的主要代谢物非那西丁的新方法。采用液液提取法,用乙醚提取血中非那西丁,提取物水浴浓缩后进行GC/MS和GC/MS/MS分析,检测总离子流色谱(TIC)和一级质谱,并以非那西丁一级质谱基峰为母离子的测定其二级质谱,体内药物用GC/MS全扫描定性分析即用二级质谱结合其总离子流色谱峰的保留时间定性;选择离子扫描定量分析,采用内标曲线法,根据非那西丁和内标SKF525A二级质谱的TIC峰面积比进行定量分析。     

MSPD-HPLC测定大米中痕量西维因和抗蚜威残留

研究了应用基质固相分散(MSPD)——高效液相色谱法测定大米中痕量西维因和抗蚜威残留物的新方法.并讨论了应用MSPD技术进行前处理与传统残留分析的区别.试验将MSPD技术用于大米中西维因和抗蚜威残留物的高效液色谱分析。并对定量检测条件作了详细的研究.     

新疆黑山煤液化重质油340-360℃馏分离及分析

用气相色谱/质谱联用仪(GUMS)分析馏程为340-360℃的新疆黑山煤液化重质油,检测出的主要成分为稠环芳烃及其衍生物,此外还有正构烷烃和少量含氧化合物.在超声辅助条件下,对液化重质油进行甲醇萃取,所得第一次甲醇萃取物用硅胶柱层析法分离.各洗脱馏分用GC/MS分析,其可测成分与直接检测相比更丰富,表明合适的预分离对提高GC/MS分析的灵敏度非常重要.     

番泻叶萃取物的气相色谱/质谱分析

分别以正己烷和无水乙醇作为溶剂,在微波辐射条件下对番泻叶进行萃取,用气相色谱/质谱联用仪(GC/MS)对萃取物进行了分析。结果表明,角鲨烯是GC/MS可检测的主要成分,正己烷对番泻叶中角鲨烯的萃取具有较好的选择性。     

甲霜灵试纸实地检测与液相色谱-质谱联用比对结果分析

报道了甲霜灵胶体检测试纸实地应用的验证研究成果. 甲霜灵胶体金检测试纸的实地检测与液相色谱-质谱联用法(LC/MS -MS)比对试验结果显示两种方法得出的检测结果相一致. 检测试纸可作为蔬菜采摘前农药甲霜灵残留自我检测手段的应用,从而实现对甲霜灵农药残留的源头把关.     

欧盟公布染料化学名称

该文根据欧盟2000年公布的第五版和2002年公布的第六版《欧洲化学物质申报清单》，选择一些重要的染料品种，介绍了他们的化学名称，以及可供参考的结构式。     

An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases

Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT(0)-ACP(0) was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT(0)-ACP(0) fragments, which were used to replace sacB by lambda-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.     

Effect of protein fusion on the transition temperature of an environmentally responsive elastin-like polypeptide: a role for surface hydrophobicity?

The limited throughput, scalability and high cost of protein purification by chromatography provide motivation for the development of non-chromatographic protein purification technologies that are cheaper and easier to implement in a high-throughput format for proteomics applications and to scale up for industrial bioprocessing. We have shown that genetic fusion of a recombinant protein to an elastin-like polypeptide (ELP) imparts the environmentally sensitive solubility property of the ELP to the fusion protein, and thereby allows selective separation of the fusion protein from Escherichia coli lysate by aggregation above a critical temperature (T(t)). Further development of ELP fusion proteins as widely applicable purification tools necessitates a quantitative understanding of how fused proteins perturb the ELP T(t) such that purification conditions (T(t)) may be predicted a priori for new recombinant proteins. We report here the effect that fusing six different proteins has on the T(t) of an ELP. A negative correlation between T(t) and the fraction hydrophobic surface area on the fused proteins was observed, which was determined from computer modeling of the available three-dimensional structure. The thermally triggered aggregation behavior of ELP-coated, functionalized gold colloids as well as ligand binding to the tendamistat-ELP fusion protein support the hypothesis that hydrophobic surfaces in molecular proximity to ELPs depress the ELP T(t) by a mechanism analogous to hydrophobic residue substitution in the ELP repeat, Val-Pro-Gly-Xaa-Gly.     

Determination of Algerian Hassi-Messaoud asphaltene aromaticity with different solid-state NMR sequences

Algerian oil well deposit derived asphaltene fraction was characterized by different MAS/NMR sequences to investigate asphaltene aromaticity and the best cross-polarization contact time. The aromaticity was estimated by single pulse sequence (SP), Hahn-echo (HE), cross-polarization (CP) and variable cross-polarization (VACP) sequences. The values found ranging from 0.58 to 0.48 are of the same order of magnitude as ones published in the literature. The discrepancies between the values are thought to be relevant to both the specificity of each sequence and the asphaltene structure. Spectra band de-convolution enables us the determination of the average number of carbon atoms per side chain according to each sequence. The obtained values spanning from 3 to 7 are also sequence nature dependent.     

A novel emulsion mixture for in vitro compartmentalization of transcription and translation in the rabbit reticulocyte system

Emulsion formulations used for in vitro compartmentalization (IVC) methods were found to be incompatible with protein expression in the rabbit reticulocyte (RRL) system, causing rapid discoloration and translation shutdown. Here we identify possible causes and describe a novel water-in-oil emulsion which abolished discoloration and allowed high-level in-emulsion expression of active luciferase and human telomerase using the RRL. This novel emulsion greatly expands the range of potential protein targets for IVC.     

Conservation and covariance in PH domain sequences: physicochemical profile and information theoretical analysis of XLA-causing mutations in the Btk PH domain

Mutations that cause X-linked agammaglobulinemia (XLA) appear throughout the Bruton tyrosine kinase (Btk) sequence, including the pleckstrin homology (PH) domain. To analyze the basis of this disease with respect to protein structure, we studied the relationships between PH domain sequences and structures by comparing sequence-based profiles of physicochemical properties and solvent accessibility profiles. The diversity of the distribution of amino acids was measured by calculating entropies for sequences containing mutations at different positions in multiple sequence alignments. Mutual information was calculated to quantify positional covariation. Eight conserved extrema were apparent in all profiles. The majority of the XLA disease-causing mutations in the Btk PH domain were found at positions having significant mutual information, indicating that there are covariant constraints for both structure and function. Together with additional structural analyses, all the XLA mutations that were analyzed could be explained at the molecular level. The method developed here is applicable to the design of mutations for protein engineering.     

Identification of potent human anti-IL-1RI antagonist antibodies

Interleukin-1 (IL-1) blockade by IL-1 receptor antagonist benefits some arthritis patients by reducing joint damage. This fact inspired us to develop antagonist human therapeutic antibodies against IL-1R(I) using phage libraries that display single-chain variable fragment (scFv) antibody fragments. Panning libraries against human IL-1R(I) generated 39 unique scFv-phage whose binding to IL-1R(I) was competed by IL-1 ligands. Fifteen of these scFv-phage, identified using IL-1R(I)-binding assays and dissociation rate ranking, were reformatted as scFv-Fc and IgG(4) molecules. The ease of producing antibodies in the scFv-Fc format permitted rapid identification of four lead clones (C10, C13, C14, C15) that inhibit NF-kappaB nuclear translocation induced by IL-1. Reformatting these clones as IgG(4) molecules increased their inhibition potency by 相似文献   

Fusion of the antiferritin antibody VL domain to barnase results in enhanced solubility and altered pH stability

Chimeric immunotoxins that combine antigen recognition domains of antibodies and cytotoxic RNases have attracted much attention in recent years as potential targeted agents for cancer immunotherapy. In an attempt to obtain a structurally minimized immunofusion for folding/stability studies, we constructed the chimeric protein VL-barnase. The chimera comprises a small cytotoxic enzyme barnase, ribonuclease from Bacillus amyloliquefaciens, fused to the C-terminus of the light chain variable domain (VL) of the anti-human ferritin monoclonal antibody F11. While the individual VL domain was expressed in Escherichia coli as insoluble protein packed into inclusion bodies, its fusion to barnase resulted in a significant ( approximately 70%) fraction of soluble protein, with only a minor insoluble fraction ( approximately 30%) packed into inclusion bodies. The in vivo solubilizing effect of barnase was also observed in vitro and suggests a chaperone-like role that barnase exerted with regard to the N-terminal VL domain. Cytoplasmic VL-barnase was analyzed for structural and functional properties. The dimeric state of the chimeric protein was demonstrated by size-exclusion chromatography, thus indicating that fusion to barnase did not abrogate the intrinsic dimerization propensity of the VL domain. Ferritin-binding affinity and specificity in terms of constants of association with isoferritins were identical for the isolated VL domain and its barnase fusion, and RNase activity remained unchanged after the fusion. Intrinsic fluorescence spectra showed a fully compact tertiary structure of the fusion protein. However, significantly altered pH stability of the fusion protein versus individual VL and barnase was shown by the pH-induced changes in both intrinsic fluorescence and binding of ANS. Together, the results indicate that VL-barnase retained the antigen-binding affinity, specificity and RNase activity pertinent to the two individual constituents, and that their fusion into a single-chain chimeric protein resulted in an altered tertiary fold and pH stability.     

A cell-based screen for function of the four-helix bundle protein Rop: a new tool for combinatorial experiments in biophysics

Combinatorial methodologies have revolutionized studies in biomolecular function, but they have so far proven less useful for understanding macromolecular structure and stability. This is largely because of the difficulty of screening libraries of molecules for biophysical properties, and the difficulty of interpreting structural effects in complicated molecules. Here, we report a novel, robust, cell-based screen for function of the four-helix bundle protein, Rop. By expression of green fluorescent protein from a ColE1 plasmid, the screen reports the copy number of the plasmid, which is modulated in Escherichia coli by Rop. We have engineered the screen so that the fluorescent phenotype can correspond to either Rop activity or lack thereof. We have used the screen to demonstrate with systematically constructed Rop core variants that not all molecules that bind small stem-loop RNAs in vitro are active in vivo. Rop is well understood from structural work and systematic mutations, which makes it possible to construct rational, targeted libraries. This screen makes it possible to rapidly interrogate such libraries effectively for proper protein folding and stability. In addition to its intended utility for combinatorial experiments in biophysics, the screen will allow further dissection of the mechanism of Rop-mediated plasmid copy number regulation in vivo.