Analysis of Ras:RasGEF interactions by phage display and static multi-angle light scattering

Molecular switches such as small GTPases of the Ras family cycle between inactive GDP-bound and active GTP-bound states. Their essential role in controlling development and cell homeostasis requires mechanisms which determine amplitude and timing of activation. This is achieved in part by the action of guanine nucleotide exchange factors, which function as highly controlled enzymes whose activity relies on spatial segregation and intra- and intermolecular regulation. Here, we describe two experimental methodologies that permit the identification and characterization of GTPase binding sites on activators by assaying complex formation within a broad range of affinities. In the first assay system, proteins presented on the surface of filamentous phage are used to probe affinity determinants of protein-protein interactions. In this application, a protein-displayed phage library is generated by random mutagenesis and a plate-based selection is performed to identify mutations that confer higher binding affinity to an immobilized target. The second method uses light scattering as a tool for measuring the molecular weight, stoichiometry, and polydispersity of protein complexes in solution. In this application, conventional gel filtration chromatography provides initial fractionation, and in-line light scattering measurements allow accurate determination of molar masses of the eluent. This technique also provides information about conformational homogeneity which can be used as a quality     

Quantification of microbial productivity via multi-angle light scattering and supervised learning

This article describes the use of chemometric methods for prediction of biological parameters of cell suspensions on the basis of their light scattering profiles. Laser light is directed into a vial or flow cell containing media from the suspension. The intensity of the scattered light is recorded at 18 angles. Supervised learning methods are then used to calibrate a model relating the parameter of interest to the intensity values. Using such models opens up the possibility of estimating the biological properties of fermentor broths extremely rapidly (typically every 4 sec), and, using the flow cell, without user interaction. Our work has demonstrated the usefulness of this approach for estimation of yeast cell counts over a wide range of values (10(5)-10(9) cells mL-1), although it was less successful in predicting cell viability in such suspensions.     

Aggregation of amphiphilic pullulan derivatives evidenced by on-line flow field flow fractionation/multi-angle laser light scattering

Size-exclusion chromatography (SEC) is a useful steric separation technique for the analysis of water-soluble polysaccharides in aqueous solution. However, in the case of amphiphilic derivatives, the usefulness is limited because of interactions between hydrophobic segments and the stationary phase. Alkyl-bearing pullulans differing from the extent and the length of alkyl groups were characterized using flow-field flow fractionation with on-line coupling multi-angle laser light scattering (F⁴/MALLS). Comparison of SEC and F⁴ is presented and the interest of F⁴ in the field of amphiphilic derivatives is demonstrated.     

A new purification procedure and molecular properties of Pseudomonas cytochrome oxidase

A procedure has been developed for purification of the cytochrome oxidase from Pseudomonas aeruginosa (EC 1.9.3.2) using DEAE- and CM-cellulose chromatography, gel filtration and crystallization. The final preparation was found to be homogeneous according to ultracentrifugal and disc electrophoretic criteria. The crystalline preparation also exhibited nitrite reductase activity. The spectrum of the enzyme characterizes it as cytochrome cd. At 280 nm E^{1 %}_{1 cm} was 18.5 after dry weight analysis.

The molecular weight of the cytochrome oxidase was calculated to be 119000 based on a sedimentation coefficient s° _20,w = 7.36 S, diffusion coefficient D _20,w = 5.36×10⁻⁷ cm²×s⁻¹ and partial specific volume of 0.72 ml/g. The iron content of the enzyme (0.166 %) indicates that this entity contains four iron atoms per molecule. Succinylation of the enzyme produced two probably identical subunits containing both hemes c and d, having a sedimentation coefficient s° _20,w = 4.30 S and an approximate molecular weight of 65000. In dodecylsulphate-acrylamide gel electrophoresis the cytochrome oxidase also dissociates into two subunits with molecular weight of 63000.     

Self-association of long-acting insulin analogues studied by size exclusion chromatography coupled to multi-angle light scattering

Two structurally very different insulin analogues analysed here, belong to a class of analogues of which two have been reported to have a protracted action through self-assembly to high molar mass in subcutis. The process of self-association of insulin analogues Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin and Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin was investigated using size exclusion chromatography (SEC) in connection with multi-angle light-scattering. Self-assembly to high molar mass was obtained by exchanging the formulation containing phenolic preservatives with an isotonic eluent during SEC. It was shown that increasing amounts of zinc in the formulations of the two analogues increased the size of the self assemblies formed during gel filtration. The addition of 0.2 mM phenol to the elution buffer slowed down the self-association process of zinc containing formulations and shed light on the initial association process. The results indicated that a dihexamer is a possible building block during self-association of Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin. Surprisingly, in the absence of zinc the two analogues behaved very differently. Lys(B29) (N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin was in equilibrium between oligomers smaller than a hexamer, whereas Lys(B29) (N(ε)-lithocholyl) des(B30) human insulin self-associated and formed even larger complexes than in the presence of zinc.     

Facile purification of mono-PEGylated interleukin-1 receptor antagonist and its characterization with multi-angle laser light scattering

Recombinant human interleukin-1 receptor antagonist (rhIL-1ra) was chemically conjugated with succinimidyl carbonate monomethoxyl polyethylene glycols of 5 kDa (SC-PEG_5k) and 10 kDa (SC-PEG_10k) molecular weight. A facile purification of the conjugates was achieved by one-step cationic exchange chromatography. The purity of mono-PEGylated protein was greater than 95%. The purified conjugate was characterized by multi-angle laser light scattering (MALLS) for determining the apparent gyration radius (r_g) and hydrodynamic radius (r_h). MALLS results showed that the conjugation of PEG markedly enhanced r_g and r_h of parent protein (r_g: from 15.7 to 48.2 nm for the PEG_5k and 81.9 nm for the PEG_10k; r_h: from 4.2 to 58.4 nm for the PEG_5k and 102.3 nm for the PEG_10k). The PEGylated rhIL-1ra retained 44.6% of binding activities to the cell receptor for PEG_5k and 40.2% for PEG_10k, compared to the original protein.     

乳杆菌对致病假单胞菌的抑制作用研究进展*

假单胞菌污染事件在临床就医和日常饮食中频发,屡次产生致病、致死等恶劣后果,有效抑制致病假单胞菌并降低其耐药性作为解决该问题的关键手段,是目前的研究重点。相关研究表明益生菌等天然活性成分对假单胞菌产生多方面影响,以应用范围最广的益生菌——乳杆菌为例,综合国内外最新研究进展,论述了乳杆菌对假单胞菌的生物膜结构、生长活性、生物毒性、黏附细胞表面能力及被假单胞菌感染后的小鼠等产生的影响。深入挖掘乳杆菌等益生菌及其代谢产物成分的作用机制,是防治假单胞菌等微生物污染和感染的关键。     

The exopolysaccharides produced by Streptococcus thermophilus Rs and Sts have the same repeating unit but differ in viscosity of their milk cultures

The polysaccharides produced by Streptococcus thermophilus Rs and Sts in skimmed milk consist of -Gal and -Rha in a molar ratio of 5:2. Linkage analysis and 1D/2D NMR (¹H and ¹³C) studies revealed that both polysaccharides have the same branched heptasaccharide repeating unit:

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Remarkably, the two strains differ in their effects on the viscosity of stirred milk cultures. The milk culture of S. thermophilus Rs is non-ropy and affords 135 mg/L polysaccharide with an average molecular mass of 2.6×10³ kDa. In contrast, the milk culture of S. thermophilus Sts is ropy and produces 127 mg/L polysaccharide with an average molecular mass of 3.7×10³ kDa. Permeability measurements of non-stirred milk cultures of both strains suggest that both strains have a similar effect on the protein–polysaccharide network. Therefore, the only clear difference between both strains, which may cause the difference in ropiness of the milk cultures, is the difference in molecular mass of the polysaccharide.     

Measurement of inherent particle properties by dynamic light scattering: introducing electrorotational light scattering.

Common dynamic light scattering (DLS) methods determine the size and zeta-potential of particles by analyzing the motion resulting from thermal noise or electrophoretic force. Dielectric particle spectroscopy by common microscopic electrorotation (ER) measures the frequency dependence of field-induced rotation of single particles to analyze their inherent dielectric structure. We propose a new technique, electrorotational light scattering (ERLS). It measures ER in a particle ensemble by a homodyne DLS setup. ER-induced particle rotation is extracted from the initial decorrelation of the intensity autocorrelation function (ACF) by a simple optical particle model. Human red blood cells were used as test particles, and changes of the characteristic frequency of membrane dispersion induced by the ionophore nystatin were monitored by ERLS. For untreated control cells, a rotation frequency of 2 s-1 was induced at the membrane peak frequency of 150 kHz and a field strength of 12 kV/m. This rotation led to a decorrelation of the ACF about 10 times steeper than that of the field free control. For deduction of ERLS frequency spectra, different criteria are discussed. Particle shape and additional field-induced motions like dielectrophoresis and particle-particle attraction do not significantly influence the criteria. For nystatin-treated cells, recalculation of dielectric cell properties revealed an ionophore-induced decrease in the internal conductivity. Although the absolute rotation speed and the rotation sense are not yet directly accessible, ERLS eliminates the tedious microscopic measurements. It offers computerized, statistically significant measurements of dielectric particle properties that are especially suitable for nonbiological applications, e.g., the study of colloidal particles.     

Genetic and molecular characterization of the Pseudomonas plasmid pVS1

A restriction map of the 30-kb nonconjugative Pseudomonas plasmid pVS1 was constructed. Derivatives of pVS1 obtained in vitro by successive deletions were used to localize on the physical map the determinant for resistance to mercuric ions (carried by transposon Tn501), the gene(s) encoding sulfonamide resistance, a 1.6-kb region affecting plasmid stability and establishment in P. fluorescens ATCC 13525, and a segment required for mobilization of pVS1 by plasmid RP1. The sulfonamide resistance determinant of pVS1 appeared to be closely related to that of transposon Tn21. A mini-pVS1 replicon, pME259, consisting of an essential 1.55-kb segment (designated rep and thought to carry the origin of replication) and a mercury resistance determinant was able to replicate P. aeruginosa PAO but selective pressure was needed for plasmid maintenance. The copy number of pVS1 derivatives was estimated to be 6-8 per chromosome equivalent. Plasmids possessing the essential rep segment plus the adjacent stability region could be established in strains of P. aeruginosa, P. putida, P. fluorescens, P. acidovorans, P. cepacia, P. mendocina, P. stutzeri, P. syringae, Agrobacterium tumefaciens, and Rhizobium leguminosarum.     

Characterization of 1,3-regiospecific lipases from new Pseudomonas and Bacillus isolates

Lipase producing ability of 120 bacterial isolates was examined qualitatively, resulting in 32 lipase producers, which were further screened for 1,3-regiospecificity. Three Bacillus (GK-8, GK-31 and GK-42) and one Pseudomonas (GK-80) were found to produce 1,3-regiospecific lipases. These lipases were alkaline in nature as they showed pH optima of 9.0 and high stability in the alkaline pH range of 8.0–11.0. The lipases from three Bacillus isolates, viz. GK-8, GK-31 and GK-42 showed temperature optima of 37 °C, whereas the Pseudomonas (GK-80) lipase showed optimum activity at 50 °C. The lipase of GK-8 was highly stable and showed enhanced activity in different organic solvents like petroleum ether (172%), diethyl ether (143%) and acetone (135%).     

Extracellular adsorption of nickel by a strain of Pseudomonas sp.

A strain of Pseudomonas has been studied for its nickel accumulation capacity. Most assays were carried out exposing cells to the metal in a resting state. Results indicate an extracellular, metabolically independent adsorption that is decreased by the presence of acetate. A possible exchange of nickel for magnesium from the outer membrane is suggested. Nickel tolerance of this strain in minimal medium is limited.     

Liposome fractionation and size analysis by asymmetrical flow field-flow fractionation/multi-angle light scattering: influence of ionic strength and osmotic pressure of the carrier liquid

Asymmetrical flow field-flow fractionation (AsFlFFF)/multi-angle light scattering (MALS) was employed for studying filter-extruded liposomes in carrier solutions with different ionic strength and osmolarity. By dilution of preformed liposome suspensions with different media, only the ionic strength in the external free aqueous phase was changed. Under such conditions the liposomes were found to elute at almost identical elution times, which is in contrast to earlier studies. This may be explained by two opposing effects: (a) modulation of inter-particulate and particle-wall-repulsion effects and (b) osmotic stress-induced changes in vesicle size. The latter effect was demonstrated when analysing liposomes upon dilution in media of constant ionic strength, but varying osmotic pressure (with or without 150 mmol L^?1 sucrose supplement). The osmotic stress-induced change in liposome size was found to be size dependent. Larger liposomes appeared to both shrink and swell when exposed to hyper- or hypoosmotic media, respectively. Smaller liposomes appeared to shrink but not to swell. The potential causes of this effect are discussed.     

Determination of the second virial coefficient of bovine serum albumin under varying pH and ionic strength by composition-gradient multi-angle static light scattering

Composition-gradient multi-angle static light scattering (CG-MALS) is an emerging technique for the determination of intermolecular interactions via the second virial coefficient B₂₂. With CG-MALS, detailed studies of the second virial coefficient can be carried out more accurately and effectively than with traditional methods. In addition, automated mixing, delivery and measurement enable high speed, continuous, fluctuation-free sample delivery and accurate results. Using CG-MALS we measure the second virial coefficient of bovine serum albumin (BSA) in aqueous solutions at various values of pH and ionic strength of a univalent salt (NaCl). The systematic variation of the second virial coefficient as a function of pH and NaCl strength reveals the net charge change and the isoelectric point of BSA under different solution conditions. The magnitude of the second virial coefficient decreases to 1.13 x 10⁻⁵ ml*mol/g² near the isoelectric point of pH 4.6 and 25 mM NaCl. These results illuminate the role of fundamental long-range electrostatic and van der Waals forces in protein-protein interactions, specifically their dependence on pH and ionic strength. Electronic supplementary material The online version of this article (doi:10.1007/s10867-014-9367-7) contains supplementary material, which is available to authorized users.     

Reducing the allelopathic effect of Partheniumhysterophorus L. on wheat (Triticumaestivum L.) by Pseudomonasputida

The present study evaluates the role of Pseudomonas putida NBRIC19 in alleviating biotic stress of Parthenium hysterophorus (Parthenium) in Triticum aestivum. Due to presence of Parthenium there was 43.76, 53.08 and 78.65% inhibition in root length, shoot length and dry weight of wheat respectively. This inhibition was recovered when P. putida NBRIC19 treatment resulted in 52.29, 28.73 and 76.31% increase in root length, shoot length and dry weight respectively as compared to control. P. putida NBRIC19 was able to form more biofilm under toxic environment of allelochemicals and enhanced expression of stress responsive genes in wheat. Inoculated wheat plants showed lower activity of catalase and ascorbate peroxidase under biotic stress of Parthenium indicating that inoculated plants felt less stress as compared to uninoculated plants. Microbial community structure in bacterized and nonbacterized wheat rhizosphere in presence and absence of Parthenium, was investigated using Biolog. There was significant increase in microbial diversity in P. putida NBRIC19 bacterized wheat rhizosphere. Functional microbial diversity revealed that P. putida NBRIC19 had shifted the microflora in such a manner that utilization of phytotoxic allelochemicals increased to lessen its toxic effect and finally it resulted in better growth of wheat in presence of Parthenium. Principal component analysis showed that microbial community function in nonbacterized wheat rhizosphere in presence (WPC) and absence (WC) of Parthenium is totally different from each other but due to P. putida NBRIC19 treatment there was close clustering of WPT and WT indicating a total shift in microbial community structure.     

Resolution of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase

Lipase from Pseudomonas sp. (PSL) was immobilized on SBA-15 (a highly ordered hexagonal array mesoporous silica molecular sieve) through physical adsorption and the immobilized PSL was used in resolution of (R,S)-2-octanol with vinyl acetate as acyl donor. Enhanced activity and enantioselectivity were observed for the immobilized PSL compared with those of the free one. The effects of reaction conditions, such as solvents, temperature, water activity and substrate ratio were investigated. Under the optimum conditions, the residual (S)-2-octanol was recovered with 99% enantiomeric excess at 52% conversion. The results also indicated that the immobilized PSL maintained 90% of its initial activity even after reusing it five times.     

应用高效体积排阻色谱偶联多角度激光散射鉴定猪圆环病毒2型灭活疫苗及病毒样颗粒疫苗抗原

本文旨在建立基于高效体积排阻色谱(high-performance size-exclusion chromatography,HPSEC)偶联多角度激光散射仪(multi-angle laser light scattering,MALLS)的猪圆环病毒2型(porcine circovirus type 2,PCV2)疫苗抗原检测方法。以纯化的PCV2灭活病毒及病毒样颗粒(virus-like particles,VLP)为参照,对4家生产企业的2种PCV2灭活病毒疫苗(a、b)及VLP疫苗(c、d)破乳后进行HPSEC-MALLS检测及分子量分析;结合PCV2抗原检测卡、Western blotting和透射电子显微镜(transmission electron microscope,TEM),鉴定了特征色谱峰;考察了方法的重复性和检测线性。结果表明,两家企业生产的PCV2灭活病毒疫苗破乳液水相经HPSEC分离,在保留时间约13.3 min处出现抗原特征峰;MALLS计算该色谱峰分子量分别为2.61×10⁶(±4.34%) Da和2.40×10⁶(±2.51%) Da。两种VLP疫苗也在13.3 min处出现抗原特征峰,分子量分别为2.09×10⁶(±2.94%) Da和2.88×10⁶(±11.85%) Da,接近PCV2的理论分子量;同时在保留时间约11.4 min处也出现色谱峰,经检测分子量为4.37×10⁶(±0.42%) Da,TEM表征显示为VLP二聚体。取疫苗d和PCV2 VLP纯品进行重复检测,抗原色谱峰面积的RSD(n=3)均小于1.5%,重复性好;将PCV2 VLP纯品梯度稀释检测,VLP及其多聚体的色谱峰面积与浓度均呈良好的线性关系,R²分别为0.999及0.997,能够满足定量及多聚体含量分析。该方法有望成为一种准确、高效的PCV2疫苗的体外评价方法,用于质量评价与提升。     

Characterisation of the polysaccharide produced by Acetobacter xylinum strain CR1/4 by light scattering and atomic force microscopy

The molecular weight of the extracellular polysaccharide (CR1/4) produced by Acetobacter xylinum strain CR1/4 has been shown to be dependent upon growth conditions. Under normal growth conditions a high molecular weight polysaccharide (>1×10⁶ Da) is produced. Maintaining the pH at 5 results in an order of magnitude increase in the total yield of polysaccharide, but also an order of magnitude decrease in molecular weight. Analysis of the CR1/4 polysaccharides by the techniques of atomic force microscopy and static light scattering suggests that they are double helices. In solution the molecules behave as stiff coils with a Kuhn statistical segment length of 325 nm.     

Production of substituted catechols from substituted benzenes by a Pseudomonas sp.

In the presence of a halobenzene or benzonitrile, Pseudomonas T-12 can produce substituted catechols from the corresponding substituted benzenes. A variety of monosubstituted benzenes with substituents containing up to four carbons, and some meta- and para- disubstituted benzenes, can serve as catechol precursors.     

高效液相尺寸排阻色谱-多角度激光散射定量检测灭活禽流感病毒抗原中完整病毒颗粒

本研究旨在建立一种灭活禽流感病毒原料液中完整病毒颗粒准确定量的方法。针对直接采用高效液相尺寸排阻色谱法（high performance size exclusion chromatography,HPSEC）检测灭活禽流感病毒原液存在杂质干扰的问题,首先以H5N8型抗原为对象,分别考察了聚乙二醇（polyethylene glycol,PEG）沉淀和离子交换色谱法（ion exchange chromatography,IEC）进行预处理。在优化条件下,经DEAE FF阴离子交换层析纯化预处理,杂蛋白去除率为86.87%,病毒血凝回收率为100%。HPSEC分析预处理后的样品,8.5–10.0 min处色谱峰样品电泳检测显示主要为H5N8病毒蛋白,动态光散射分析平均粒径为127.7 nm,推测为完整病毒特征峰;在IEC预处理后的样品中加入抗体进行HPSEC检测,8.5-10.0 min处特征峰消失,显示IEC预处理有效去除了杂质干扰。通过将HPSEC与多角度激光散射技术（multi-angle laser scattering technique,MALLS）联用,可以准确获得样品中完整病毒颗粒的数量,且病毒颗粒数与色谱峰面积具有良好线性关系（R²=0.997）。建立的IEC预处理-HPSEC-MALLS检测方法应用于其他亚型（H7N9）、批次和浓度的病毒原液中完整病毒颗粒数的准确检测,均具有良好适用性,且重复性好,相对标准偏差（relative standard deviation,RSD）<5%,n=3。