A general and direct method for the solid phase synthesis of intramolecularly quenched fluorogenic protease substrates using a bifunctional coumarin derivative

A general method for the solid phase preparation offluorogenic peptide substrates or intramolecularly quenchedones (IQFS) is presented, using the highly fluorescentbifunctional coumarin derivative 7-amino-4-coumarinyl-acetic acid. The key feature of this method is theconjugation of H–Aca–OH through its carboxyl group on theresin, followed by the development of the peptide chainthrough its amino group, using standard Fmoc-derived solidphase peptide synthesis methodology. The 2,4-dinitrophenylgroup was used as quencher and introduced directly to theresin-bound peptides. The IQFSDnp–Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (2) andfour Dnp–X-Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (3–6), where X = Val, Lys, Ser and Glu at P₆ position,potential substrates for cathepsin D, were synthesized forproving the utility of the method. The compoundsH–Ile–Lys–Leu–Aca–OH (7),H–Lys–Pro–Ile–Cys–Phe–Ile–Lys–Leu–Aca–OH (8),H–Leu–Aca–OH (9), Dnp–Leu–Aca–OH (10) and Dnp-Leu-OH (11) were also synthesized for comparisonpurposes. The fluorescence properties of compounds 9and 10 were measured.     

Functional cartography of the ectodomain of the type I interferon receptor subunit ifnar1

Ligand-induced cross-linking of the type I interferon (IFN) receptor subunits ifnar1 and ifnar2 induces a pleiotrophic cellular response. Several studies have suggested differential signal activation by flexible recruitment of the accessory receptor subunit ifnar1. We have characterized the roles of the four Ig-like sub-domains (SDs) of the extracellular domain of ifnar1 (ifnar1-EC) for ligand recognition and receptor assembling. Various sub-fragments of ifnar1-EC were expressed in insect cells and purified to homogeneity. Solid phase binding assays with the ligands IFN(alpha)2 and IFN(beta) revealed that all three N-terminal SDs were required and sufficient for ligand binding, and that IFN(alpha)2 and IFN(beta) compete for this binding site. Cellular binding assays with different fragments, however, highlighted the key role of the membrane-proximal SD for the formation of an in situ IFN-receptor complex. Even substitution with the corresponding SD from homologous cytokine receptors did not restore high-affinity ligand binding. Receptor assembling analysis on supported lipid bilayers in vitro revealed that the membrane-proximal SD controls appropriate orientation of the receptor on the membrane, which is required for efficient association of ifnar1 into the ternary complex.     

Ligand-induced assembling of the type I interferon receptor on supported lipid bilayers

Type I interferons (IFNs) elicit antiviral, antiproliferative and immuno-modulatory responses through binding to a shared receptor consisting of the transmembrane proteins ifnar1 and ifnar2. Differential signaling by different interferons, in particular IFNalphas and IFNbeta, suggests different modes of receptor engagement. Using reflectometric interference spectroscopy (RIfS), we studied kinetics and affinities of the interactions between IFNs and the extracellular receptor domains of ifnar1 (ifnar1-EC) and ifnar2 (ifnar2-EC). For IFNalpha2, we determined a K(D) value of 3 nM and 5 microM for the interaction with ifnar2-EC and ifnar1-EC, respectively. As compared to IFNalpha2, IFNbeta formed complexes with ifnar2-EC as well as ifnar1-EC with substantially higher affinity. For neither IFNalpha2 nor IFNbeta was stabilization of the complex with ifnar1-EC in the presence of soluble ifnar2-EC observed. We investigated ligand-induced complex formation with ifnar1-EC and ifnar2-EC being tethered onto solid-supported, fluid lipid bilayers by RIfS and total internal reflection fluorescence spectroscopy. We observed very stable binding of IFNalpha2 at high receptor surface concentrations with an apparent k(d) value approximately 200 times lower than that for ifnar2-EC alone. The apparent k(d) value was strongly dependent on the surface concentration of the receptor components, suggesting kinetic stabilization. This was corroborated by the fast exchange of labeled IFNalpha2 bound to the receptor by unlabeled IFNalpha2. Taken together, our results indicate that IFN first binds to ifnar2 and subsequently recruits ifnar1 in a transient fashion. In particular, this second step is much more efficient for IFNbeta than for IFNalpha2, which could explain differential activities observed for these IFNs.     

Visualizing the assembly of human Rad51 filaments on double-stranded DNA

Rad51 is the core component of the eukaryotic homologous recombination machinery and assembles into extended nucleoprotein filaments on DNA. To study the dynamic behavior of Rad51 we have developed a single-molecule assay that relies on a combination of hydrodynamic force and microscale diffusion barriers to align individual DNA molecules on the surface of a microfluidic sample chamber that is coated with a lipid bilayer. When visualized with total internal reflection fluorescence microscopy (TIRFM), these "molecular curtains" allow for the direct visualization of hundreds of individual DNA molecules. Using this approach, we have analyzed the binding of human Rad51 to single molecules of double-stranded DNA under a variety of different reaction conditions by monitoring the extension of the fluorescently labeled DNA, which coincides with assembly of the nucleoprotein filament. We have also generated several mutants in conserved regions of Rad51 implicated in DNA binding, and tested them for their ability to assemble into extended filaments. We show that proteins with mutations within the DNA-binding surface located on the N-terminal domain still retain the ability to form extended nucleoprotein filaments. Mutations in the L1 loop, which projects towards the central axis of the filament, completely abolish assembly of extended filaments. In contrast, most mutations within or near the L2 DNA-binding loop, which is also located near the central axis of the filament, do not affect the ability of the protein to assemble into extended filaments on double-stranded (ds)DNA. Taken together, these results demonstrate that the L1-loop plays a crucial role in the assembly of extended nucleoprotein filaments on dsDNA, but the N-terminal domain and the L2 DNA-binding loop have significantly less impact on this process. The results presented here also provide an important initial framework for beginning to study the biochemical behaviors of Rad51 nucleoprotein filaments using our novel experimental system.     

A method of reversible biomolecular immobilization for the surface plasmon resonance quantitative analysis of interacting biological macromolecules

This article presents a new procedure for the immobilization of macromolecules on gold surfaces, with the purpose of studying macromolecular interactions by simple optical configurations rendering surface plasmon resonance. Gold surfaces were covered by a three-layer structure composed of poly-L-lysine irreversibly bound to gold, followed by a second layer of heparin and a third layer of polylysine. The three-layer structure of polylysine-heparin-polylysine remains irreversibly bound to gold, it prevents biomolecules from coming into direct contact with the metal surface, and it allows the irreversible binding of different proteins and polynucleotides. After binding of a macromolecule to the three-layer structure, the interaction with a second macromolecule can be studied, and then the complex formed by the two interacting macromolecules, together with the second heparin layer and the third polylysine layer, can be broken down just by treatment with an alkaline solution having a pH value above the pK value of the amino groups of polylysine. The first polylysine layer remains irreversibly bound to gold, ready to form a new three-layer structure and, therefore, to support a new macromolecular interaction on the same regenerated surface. Polynucleotide interactions, the proteolytic action of chymotrypsin, and the interaction between the component subunits of a heterotetrameric enzyme are described as examples of macromolecular interactions studied by using this system. The method may be especially suitable for developing of low-cost systems aimed to look for surface resonance signals, and it offers the advantage of allowing calculation of parameters related to the size and stoichiometry of the interacting macromolecules, in addition to the kinetic and equilibrium properties of the interaction.     

G蛋白偶联受体与G蛋白相互作用的最新研究进展

传统观念认为,在激动剂作用下,G蛋白偶联受体(GPCRs)能够激活G蛋白的α亚基,从而使Gα亚基与Gβγ亚基分离,被激活的Gα亚基通过信号转导进一步参与细胞的生理过程。但是,最新研究发现GPCRs和G蛋白存在多种偶联关系,GPCRs不仅能够激活Gα亚基,还可以与Gβγ亚基相互靠近,甚至会使G蛋白亚基构象发生重排而不分离,这对于疾病发病机制的研究及新的药物靶点的发现具有重要意义。就GPCRs与G蛋白之间的相互作用以及最新研究技术作一简要综述。     

A new method for multilayered,site-directed immobilization of antibody on polystyrene surface

Polystyrene is a common substrate material for protein adsorption in biosensors and bioassays. Here, we present a new method for multilayered, site-directed immobilization of antibody on polystyrene surface through the linkage of a genetically engineered ligand and the assembly of staphylococcal protein A (SPA) with immunoglobulin G (IgG). In this method, antibodies were stacked on polystyrene surface layer by layer in a potential three-dimensional way and exposed the analyte-binding sites well. Enzyme-linked immunosorbent assay (ELISA) revealed that the new method showed a 32-fold higher detection sensitivity compared with the conventional one. Pull-down assay and Western blot analysis further confirmed that it is different from the ones of monolayer adsorption according to the comparison of adsorption capacity. The differentiated introduction of functional ligands, which is the key of this method, might offer a unique idea as a way to interfere with the dynamic behavior of a protein complex during the process of adsorption.     

A modified method of quantitative colorimetric DNA-DNA hybridization on membrane filters for bacterial identification

Based on conventional membrane filter dot hybridization protocols, a modified method for the quantitative measurement of DNA-DNA reassociation is described. Labeled DNA probes are prepared with Photobiotin and hybridized with immobilized target DNAs on nitrocellulose filters. The extent of hybridization is detected by an enzyme linked assay in microtiter plates using streptavidine-alkaline phosphatase conjugates as reporter enzymes and nitrophenylphosphate as the colorimetric substrate. The procedure is non-destructive and allows the re-use of the filter holding the target DNAs. The results of the membrane filter hybridizations have been compared to spectroscopic DNA-DNA hybridizations and the limits and the applicability of the method for bacterial taxonomy and bacterial identification are discussed.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.     

Determination of the distribution coefficient for rifabutin in a liposome-water system by fluorescence measurements

Fluorescence quenching was used to determine the distribution coefficient K _d for a tuberculostatic rifabutin in a liposome-water system at pH 6.4 and 7.4. Liposomes were large unilamellar vesicles composed of phosphatidylcholine or its mixtures with cholesterol or cardiolipin and containing a fluorescent label (anthryl phosphatidylcholine with the fluorophore in the hydrophobic region). The K _d values calculated in the Stern-Volmer model are comparable for phosphatidylcholine and phosphatidylcholine/cholesterol at both pH, and testify to rifabutin hydrophobicity (logK _d ≈ 2.4–2.6). Inclusion of negatively charged cardiolipin increases the K _d by more than an order of magnitude at pH 6.4, and ionization of the second phosphate at pH 7.4 produces an additional increase. These results demonstrate the large contribution of electrostatic forces into the interaction of rifabutin with model membranes.     

An accurate method for the quantitation of Fmoc-derivatized solid phase supports

Summary By performing the Fmoc resin loading determination with DBU instead of piperidine, highly reproducible results were obtained that showed excellent correlation with data obtained by independent analytical methods.     

A solid phase adsorption method for preparation of bovine serum albumin-bovine hemoglobin conjugate

A solid phase adsorption method was proposed to prepare well-defined bovine serum albumin–bovine hemoglobin (Hb) conjugate. After adsorption by the solid phase, Q Sepharose Fast Flow media, bovine serum albumin (BSA) molecules were allowed to react with glutaraldehyde. The spacing out of BSA molecules on the solid phase was assumed to limit polymerization of BSA molecules, except some molecules bound closely on the solid phase resulting in minor dimer formation. Following the elution procedure, the activated monomeric BSA was separated from the dimers by gel filtration chromatography on Superdex 200 and then reacted with bovine Hb at 4 °C and pH 9.5. The 1:1 (BSA:Hb) conjugate was obtained with the yield of 64%. The P₅₀ values of the conjugates, prepared under anaerobic and aerobic conditions, were 19.1 and 14.2 mmHg, respectively. The dependence of the P₅₀ on chloride ions for the conjugate was slightly diminished, presumably due to covalent attachment of BSA to bovine Hb.     

A method for quantitative analysis of spatially variable physiological processes across leaf surfaces

Many physiological processes are spatially variable across leaf surfaces. While maps of photosynthesis, stomatal conductance, gene expression, water transport, and the production of reactive oxygen species (ROS) for individual leaves are readily obtained, analytical methods for quantifying spatial heterogeneity and combining information gathered from the same leaf but with different instruments are not widely used. We present a novel application of tools from the field of geographical imaging to the multivariate analysis of physiological images. Procedures for registration and resampling, cluster analysis, and classification provide a general framework for the analysis of spatially resolved physiological data. Two experiments were conducted to illustrate the utility of this approach. Quantitative analysis of images of chlorophyll fluorescence and the production of ROS following simultaneous exposure of soybean leaves to atmospheric O₃ and soybean mosaic virus revealed that areas of the leaf where the operating quantum efficiency of PSII was depressed also experienced an accumulation of ROS. This correlation suggests a causal relationship between oxidative stress and inhibition of photosynthesis. Overlaying maps of leaf surface temperature and chlorophyll fluorescence following a photoinhibition treatment indicated that areas with low operating quantum efficiency of PSII also experienced reduced stomatal conductance (high temperature). While each of these experiments explored the covariance of two processes by overlaying independent images gathered with different instruments, the same procedures can be used to analyze the covariance of information from multiple images. The application of tools from geographic image analysis to physiological processes occurring over small spatial scales will help reveal the mechanisms generating spatial variation across leaves.     

Convenient solid phase synthesis method for the preparation of cysteine C-terminally derivatized peptides

Summary This paper describes a novel solid phase peptide synthesis method for the systematic C-terminal modification of cysteine-containing peptides. In this method, cysteine is linked to chloromethylated polystyrene resin by its thiol functionality, followed by protection of the N-terminus and derivatization of the carboxylic acid to esters or amides. We report here on examples of the methodology and its application to the synthesis of Ac-Asp-cyclo(Cys-Gly-Pro-Cys)-NHBzl, a cyclic peptide amide. The method has been applied to the synthesis of complex esters as well as amides.Abbreviations Ac acetyl - AcN acetonitrile - Ac₂O acetic anhydride - AcOH acetic acid - Boc t-butyloxycarbonyl - BOP benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate - Bzl benzyl - cHex cyclohexyl - DBU 1,8-diazabicyclo[5.4.0]-undec-7-ene - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA diisopropylethylamine - DMF dimethylformamide - DMS dimethylsulfide - HOB 1-hydroxybenzotriazole - MBzl 4-methyl benzyl - MeOH methanol - TEA triethylamine - TEAP triethylammonium phosphate - TFA trifluoroacetic acid     

临床皮炎外瓶霉荧光定量PCR检测方法的建立

目的 建立基于TaqMan探针技术的皮炎外瓶霉荧光定量PCR检测方法.方法 通过对皮炎外瓶霉ITS区域基因组序列（GenBank：JN675373.1）进行分析,设计合成特异性引物和荧光标记探针,优化荧光定量PCR反应条件.以临床标本中分离的皮炎外瓶霉为阳性菌株,及其他种类真菌和细菌作为阴性对照菌株,从特异性、敏感性及重复性方面对该方法检测效果进行评价.结果 该研究设计的引物和探针能扩增皮炎外瓶霉特异性序列.临床分离得到的皮炎外瓶霉在反应中有明显扩增曲线,而甄氏外瓶霉、棘状外瓶霉、烟曲霉、白色念珠菌、新生隐球菌、马内菲青霉等20株菌在CT值≤38范围内均未有扩增;利用基因重组构建的标准品完成了标准曲线的绘制,在1.0×103～1.0×107拷贝数（Cp）内具有良好的线性关系（R2=1.000）,最低可检出量为10 Cp/μL.结论 成功建立了荧光定量PCR检测皮炎外瓶霉方法,该法特异度强、敏感度高、重复性好,将有助于临床皮炎外瓶霉感染的早期诊断和针对性治疗.     

A novel non-contact bioassay method for quantitative evaluation of vapour phase toxicity of insecticides against mosquitoes

The recent advancement in new generation fluorinated pyrethroids (e.g., transfluthrin, metofluthrin etc.), the use of semi-volatile vapour phase insecticides for control of mosquitoes and other domestic pests rises. Enabling the examination of the vapour toxicity profiles of these molecules and many other similar new generation molecules will provide new avenues for researchers for understanding the bio-potency in the spatial killing of pests. Hence, it is critical to establish a well-controlled portable vapour-phase bioassay method that can provide the desired precision, accuracy, linearity and robustness. In this respect, we have designed a vapour-toxicity apparatus comprising glass assemblies and developed a novel bioassay method. We found that KT₅₀ and percentage knockdown at 60?min reflect the concentration dependency. This validates and confirms that the method is sensitive enough to distinguish between concentrations and suitable for concentration-response experiments. We found that KT₅₀ and percentage knockdown at 60?min at a given concentration does not differ significantly between experiments. Hence, the method has repeatability and precision. Percentage mortality and total KT₅₀ against Culex quinquefasciatus shows that percentage mortality increases and KT₅₀ decreases linearly with the increasing concentration. This method provides an easy to operate tool to test the vapour toxicity profiles of any vapour phase insecticide molecules against mosquitoes and flying insects.