重金属残留的快速检测

环境及农畜产品中的重金属残留已对人类安全构成严重威胁,急需快速、高效的重金属残留检测方法。该文总结了国内外有关采用生物、化学传感器、免疫学方法等快速检测环境及农畜产品中重金属残留的研究进展,并对其发展前景及市场化作了预测及展望。     

Impact of calcium on N1 influenza neuraminidase dynamics and binding free energy

The highly pathogenic influenza strains H5N1 and H1N1 are currently treated with inhibitors of the viral surface protein neuraminidase (N1). Crystal structures of N1 indicate a conserved, high affinity calcium binding site located near the active site. The specific role of this calcium in the enzyme mechanism is unknown, though it has been shown to be important for enzymatic activity and thermostability. We report molecular dynamics (MD) simulations of calcium‐bound and calcium‐free N1 complexes with the inhibitor oseltamivir (marketed as the drug Tamiflu), independently using both the AMBER FF99SB and GROMOS96 force fields, to give structural insight into calcium stabilization of key framework residues. Y347, which demonstrates similar sampling patterns in the simulations of both force fields, is implicated as an important N1 residue that can “clamp” the ligand into a favorable binding pose. Free energy perturbation and thermodynamic integration calculations, using two different force fields, support the importance of Y347 and indicate a +3 to +5 kcal/mol change in the binding free energy of oseltamivir in the absence of calcium. With the important role of structure‐based drug design for neuraminidase inhibitors and the growing literature on emerging strains and subtypes, inclusion of this calcium for active site stability is particularly crucial for computational efforts such as homology modeling, virtual screening, and free energy methods. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Lead stress in seedlings of Avicennia marina, a common mangrove species in South China, with and without cotyledons

The effects of lead (Pb; 0-1000 mg L⁻¹) stress on the growth and biochemical responses of seedlings of Avicennia marina were examined, with and without cotyledons. After 50 days exposure to Pb, the growth of A. marina was not affected at low concentrations (0-50 mg L⁻¹ Pb). Roots tolerated to high Pb concentrations, with a significant reduction in biomass only at 1000 mg L⁻¹ Pb. In leaves and stems, 500 mg L⁻¹ Pb already caused a significant decline in biomass (0.6-fold). Accumulation of Pb occurred mainly in roots, with some accumulation in cotyledons but very little in leaves. Pb concentrations in both roots and cotyledons were proportional to the Pb levels in the substrate (y = 25.945x − 4281, r² = 0.67, P = 0.001 for roots, and y = 0.249x + 45.636, r² = 0.879, P < 0.001 for leaves). In treatments with 500 and 1000 mg L⁻¹ Pb, nitrogen concentrations in cotyledons were higher, while the carbon to nitrogen ratios were significantly lower than in the control without Pb. The Pb levels had significant positive effects on sugar content, MDA concentration and POD activity in both roots and leaves, while the removal of cotyledons significantly decreased the POD activity and MDA content in roots A. marina seedlings according to the two-way multivariate analysis of variance test. The sugar content in the cotyledon of Pb-treated seedlings was significantly lower than that in the control (without Pb), suggesting that more carbohydrate reserves (e.g., sugar) stored in cotyledons had been mobilized to leaves and even roots under Pb treatment.     

Categoric prediction of metal ion mechanisms in the active sites of 17 select type II restriction endonucleases

Recently determined crystal structures of type II restriction endonucleases have produced a plethora of information on the basis for target site sequence selectivity. The positioning and role of metal ions in DNA recognition sites might reflect important properties of protein-DNA interaction. Although acidic and basic groups in the active sites can be identified, and in some cases divalent-metal binding sites delineated, a convincing picture clarifying the way in which the attacking hydroxide ion is generated, and the leaving group stabilized, has not been elucidated for any of the enzymes. We have examined the interatomic distances between metal ions and proposed key catalytic residues in the binding sites of seventeen type II restriction endonucleases whose crystal structures are documented in literature. The summary and critical evaluation of structural assignments and predictions made earlier have been useful to group these enzymes. All the enzymes used for this study have been categorized on the basis of the number of metal ions identified in their crystal structures. Among 17 experimentally characterized (not putative) type II REases, whose apparently full-length sequences are available in REBASE, we predict 8 (47%) to follow the single metal ion mechanism, 5 to follow the two metal ion mechanism, 2, the three metal ion mechanism, 1, the four metal ion mechanism and 1 the six metal ion mechanism.     

Synthesis of LeLe oligosaccharide fragments and efficient one-step deprotection

We describe here the synthesis of two oligosaccharide fragments of the tumor associated carbohydrate antigen Le^aLe^x. While the linear lacto-N-triose I: β-d-Galp-(1→4)-β-d-GlcNAcp-(1→3)-β-d-Galp-OMe is a known compound, this is the first reported preparation of the branched tetrasaccharide β-d-GlcNAcp-(1→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-β-d-GlcNAcp-OMe. Our synthetic schemes involved using an N-trichloroacetylated trichloroacetimidate glucosaminyl donor activated with excess TMSOTf at 0 °C for glycosylation at O-3 of galactosyl residues and that of trichloroacetimidate galactosyl donors activated with excess BF₃·OEt₂ to glycosylate either O-3 or O-4 of glucosamine residues. The fucosylation at O-3 of the glucosamine acceptor was accomplished using a thiofucoside donor activated with copper(II) bromide and tetrabutylammonium bromide. Thus, syntheses of the protected tri- and tetrasaccharides were achieved easily and efficiently using known building blocks. Of particular interest, we also report that these protected oligosaccharides were submitted to dissolving metal conditions (Na-NH₃) to provide in one single step the corresponding deprotected compounds. Under these conditions all protecting groups (O-acyl, benzylidene, benzyl, and N-trichloroacetyl) were efficiently cleaved. The work-up procedure for such reactions usually involves quenching with excess methanol and then neutralization with acetic acid. In our work the neutralization was carried out using acetic anhydride rather than acetic acid to ensure N-acetylation of the glucosamine residue. Both fully deprotected compounds were then simply purified and desalted by gel permeation chromatography on a Biogel P2 column eluted with water.     

Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins

The rate at which X-ray structures of membrane proteins are solved is on a par with that of soluble proteins in the late 1970s. There are still many obstacles facing the membrane protein structural community. Recently, there have been several technical achievements in the field that have started to dramatically accelerate structural studies. Here, we summarize these so-called ‘tricks-of-the-trade’ and include case studies of several mammalian transporters.     

脊髓损伤修复相关10号蛋白在COS7细胞中的表达、纯化与鉴定

目的:在COS7细胞中表达脊髓损伤修复相关10号蛋白(SCIRR10),并对表达产物进行纯化和鉴定。方法:在实验室前期研究基础上,用PCR方法扩增SCIRR10基因,将其克隆入pcDNA3.1/myc-HisA穿梭质粒中,转染COS7细胞进行表达;对表达产物用金属螯合层析方法纯化,并进行SDS-PAGE和Western印迹检测。结果及结论:构建了含有SCIRR10基因的穿梭质粒pcDNA3.1/myc-His-SCIRR10,并使其在COS7细胞中得到表达,纯化后的重组蛋白SCIRR10-His-myc的纯度在80%以上,可用于下一步的实验研究。     

浙江雪胆几种金属元素分布及其与土壤养分之间的关系

对浙江雪胆各器官主要金属元素分布及其与土壤养分之间的关系进行了研究,结果表明,1)浙江雪胆原生土壤中Fe元素的含量最高,其次是K元素,Mn和Zn含量较低;2)植物的各器官中,均以K元素含量最高,其次是Ca和Mg,微量元素Fe、Mn和Zn含量均相对较低;3)Fe、Mn和Zn 3种微量元素均以根中含量为最高,Mg和Ca元素均以块茎中较高,K则以叶中最高;4)同一器官元素分布均呈正相关,而不同器官之间的元素分布整体上呈负相关;5)根中Mg、K和Ca均与土壤元素含量整体上呈负相关,块茎中元素均与土壤元素含量均呈正相关,叶中除Fe元素外,其他元素均与土壤元素含量均呈负相关;6)各器官大量元素的富集系数较高。     

A metal‐chelate affinity reverse micellar system for protein extraction

A new nonionic reverse micellar system is developed by blending two nonionic surfactants, Triton X‐45 and Span 80. At total surfactant concentrations lower than 60 mmol/L and molar fractions of Triton X‐45 less than 0.6, thermodynamically stable reverse micelles of water content (W₀) up to 30 are formed. Di(2‐ethylhexyl) phosphoric acid (HDEHP; 1–2 mmol/L) is introduced into the system for chelating transition metal ions that have binding affinity for histidine‐rich proteins. HDEHP exists in a dimeric form in organic solvents and a dimer associated with one transition metal ion, including copper, zinc, and nickel. The copper‐chelate reverse micelles (Cu‐RM) are characterized for their W₀, hydrodynamic radius (R_h), and aggregation number (N_ag). Similar with reverse micelles of bis‐2‐ethylhexyl sodium sulfosuccinate (AOT), R_h of the Cu‐RM is also linearly related to W₀. However, N_ag is determined to be 30–90 at W₀ of 5–30, only quarter to half of the AOT reverse micelles. Then, selective metal‐chelate extraction of histidine‐rich protein (myoglobin) by the Cu‐RM is successfully performed with pure and mixed protein systems (myoglobin and lysozyme). The solubilized protein can be recovered by stripping with imidazole or ethylinediaminetetraacetic acid (EDTA) solution. Because various transition metal ions can be chelated to the reverse micelles, it is convinced that the system would be useful for application in protein purification as well as simultaneous isolation and refolding of recombinant histidine‐tagged proteins expressed as inclusion bodies. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

New metal complexes of N2S2 tetradentate ligands: Synthesis and spectral studies

New tetradentate ligands 2-(2-mercaptoethylthio)-N-(pyridin-2-ylmethyl)acetamide H₂L¹ and 2-chloro-2-(2-mercaptoethylthio)-N-(pyridin-2-ylmethyl)acetamide H₂L² were synthesised from the reaction of 2-aminomethanepyridine with 1,4-dithian-2-one and 3-chloro-1,4-dithian-2-one, respectively. Monomeric complexes of these ligands, of general formulae K[Cr^III(Lⁿ)Cl₂], K₂[Mn^II(Lⁿ)Cl₂] and [M(Lⁿ)] (M = Fe(II), Co(II), Ni(II), Cu(II), Zn(II), Cd(II) or Hg(II); n = 1, 2) are reported. The mode of bonding and overall geometry of the complexes were determined through IR, UV-Vis, NMR and mass spectral studies, magnetic moment measurements, elemental analysis, metal content and conductance. These studies revealed octahedral geometries for the Cr(III), Mn(II) complexes, square planar for Ni(II) and Cu(II) complexes and tetrahedral for the Fe(II), Co(II), Zn(II), Cd(II) and Hg(II) complexes. The study of complex formation via molar ratio in DMF solution has been investigated and results were consistent to those found in the solid complexes with a ratio of (M:L) as (1:1).