Assembly of amyloid protofibrils via critical oligomers--a novel pathway of amyloid formation

The amyloid formation of phosphoglycerate kinase (PGK) was investigated by static and dynamic light-scattering. The time-course of the scattering intensity and the hydrodynamic radius scale with initial monomer concentration in a linear fashion over a range of about 50 in concentration. This sets limits on theories for aggregation kinetics that can be used, and points towards irreversible, cascade type models. In addition, circular dichroism (CD) was used to monitor the transition between a predominantly alpha-helical spectrum to a beta-sheet enriched one. The time-course of the CD also proves to scale linearly with initial monomer concentration. Electron microscopy shows that small oligomers as well as protofibrils are present during aggregation. The found coupling between growth of intermediates and acquisition of beta-sheet structure is interpreted in terms of a generalized diffusion-collision model, where stabilization of beta-strands takes place by intermolecular interactions.     

Electrostatic properties of cells estimated by absorption and fluorescence spectroscopy

A colloid titration method was used to determine the surface charge of cells of a human colon adenocarcinoma cell line WiDr; 6.2±0.8×10⁸ charges per cell were found. The apparent surface charge density was calculated using the cell surface area estimated by a Coulter counter. Alternatively, the lower limit of the cell surface area was estimated by visible microscopy. The same procedure was applied for human skin fibroblasts, resulting in the value 9.4±1.1×10⁸ charges per cell. This is significantly higher (p<0.05) than that of WiDr cells, presumably because of the different size of the cells. According to the estimations using the Coulter counter, the median diameter was higher in the case of skin fibroblasts. Fluorimetric titration of the fluorescent probe U-6 was used to estimate the interfacial potential of the WiDr cells. A shift of the titration curve of the U-6 probe toward higher pH values compared to that in pure buffer solutions was found in the presence of the WiDr cells. From the displacement of the midpoints of the titration curves, the interfacial potential of the WiDr cells was found to be about−35.8 mV. Incubation of the cells at two different pH values (7.4 and 6.8) did not result in any significant modification of the electrostatic properties of the cells under the experimental conditions of the present study. Electron microscopy revealed a distinct difference in the surface morphology of the WiDr cells compared to human skin fibroblasts. Numerous microvilli present on the surface of WiDr cells indicated marked uncertainties in cell surface area estimations. This gives large uncertainties in the real surface charge densities of cells.     

Update on biochemical properties of recombinant Pseudomonas diminuta phosphotriesterase

Phosphotriesterase from Pseudomonas diminuta (PTE; EC 3.1.8.1) hydrolyzes organophosphate insecticides and chemical warfare agents. The two zinc cations in the active center can be substituted. Co²⁺-containing PTE is the most efficient but least stable isoform. Gel filtration showed that PTE is monomeric at the submicromolar concentrations used in kinetic assays. The analysis of the recombinant enzyme by X-ray fluorescence spectrometry and CCT-ICP-MS, confirms that recombinant Zn-PTE contains only Zn²⁺ whereas Co-PTE has Zn²⁺ and Co²⁺ in equimolar amount, with Co²⁺ most likely in the reported labile β-site. We noted that recombinant PTE is unstable at low concentrations and must be stabilized by a protein environment. We tested the effect of excess of various metal cofactors on PTE-catalyzed hydrolysis of paraoxon. We notably observed that ZnCl₂ induces a non-competitive partial inhibition of Zn²⁺- and Co²⁺-PTE at pH 8.5 (apparent Ki=155 μM and 52 μM, respectively). Inhibition results from interactions with colloidal Zn(OH)₂ formed in alkaline buffer that alters the catalytic machinery. NiCl₂ caused a similar effect at higher concentrations (apparent Ki=3 mM). We observed that mutating His123, a surface residue close to an alleged allosteric site, dramatically altered the bacterial expression yield of Co²⁺-PTE, Ki for Zn(OH)₂ inhibition, k_cat (up to 60 fold) for paraoxon hydrolysis, but not K_M. Issues addressed in this work are important for future biotechnological developments of PTE as a detoxifying enzyme.     

两种典型土壤胶体对镉的生物有效性的影响

采用黑麦草盆栽试验,研究了人工Cd污染(10.91mg·kg-1)黄棕壤和红壤(简称原土)及其胶体组分(简称胶体)和去胶后组分(简称去胶)Cd的生物有效性,并研究了EDTA对Cd解吸和生物有效性的影响.结果表明:(1)各处理黑麦草株高、地上部干重、根干重、总生物量都表现为胶体＞原土＞去胶,胶体上总生物量分别是原土和去胶处理的(1.31±0.02)倍和(1.82±0 21)倍.(2)黑麦草体内Cd浓度、及其对Cd的富集系数都表现为胶体＜原土＜去胶,表明胶体中Cd的生物有效性＜原土＜去胶.(3)黄棕壤各组分Cd的解吸率分别表现为胶体和原土约为0,去胶组分为(10.5±3.5)%,红壤各组分平均为(20.8±1 9)%,但加入EDTA则明显增加了Cd的解吸,导致黑麦草体内Cd浓度显著增加,黑麦草地上部干重、根干重、总生物量降低.EDTA对Cd的活化作用表现为去胶＞原土＞胶体,黄棕壤＞红壤,EDTA对各处理植株Cd总量的影响与此吻合.这说明,土壤镉的生物有效性受土壤胶体及其pH等的强烈影响.     

Contact patterns in concanavalin A agglutinated erythrocytes

Agglutination of human erythrocytes by the lectin concanavalin A is enhanced when the erythrocytes are pretreated with neuraminidase, which removes sialic acids, or with pronase, which degrades both the glycophorins and band 3 protein. In the present work transmission electron microscopy of the enzymatically pretreated erythrocytes shows a regular pattern of interruption of contact between interacting plasma membranes. The lengths characteristic of the pattern were 0.66 and 0.50 μm for pronase- and neuraminidase-pretreated cells, respectively. Agglutination of normal erythrocytes and of neuraminidase-pretreated erythrocytes can be fully reversed by exposure to the competitive inhibitor methyl α-D-mannopyranoside. Complete reversal of contact does not occur with pronase-pretreated cells. The comparatively greater tenacity of contact between cells that were treated with pronase before exposure to lectin argues for an involvement of nonspecific interactions in the agglutination process. The results are compared with previously published studies of spatially periodic contact patterns induced by a range of other polymers.     

Dielectric studies on water in solutions of purified lecithin vesicles

The complex permittivity of sonicated aqueous solutions of purified dimyristoylphosphatidylcholine has been measured as a function of frequency between 3 kHz and 40 GHz. The dielectric spectrum of the samples shows two dispersion/absorption regions, one centered at about 80 MHz the other at about 20.GHz (30°C). Otherwise than in previous studies no additional dispersion/absorption process has been found at frequencies below 10 MHz.The complex dielectric spectrum of the samples is discussed with respect to the dynamical state of solvent water in solutions of single-bilayer vesicles. The main relaxation time of the solvent water, τ₁ ((2πτ₁)^?1 ≈ 20 GHz), is smaller than that of pure water, τ_W, at the same temperature. This effect results from the action of internal depolarizing fields which obviously overcompensate and enhancement of τ₁ due to specific solute/solvent interactions (hydration) as had been previously found with micellar solutions of lysolecithins.It cannot be excluded, that some solvent water shows unusual dynamical behaviour. If there exists a substantial amount of such motionally perturbed water, however, it must be characterized by a relaxation time close to that of the phosphorylcholine zwitterions, τ₂ ((2πτ₂)^?1 ≈ 80 MHz).     

草酸对土壤胶体与矿物表面酶的吸附及活性影响

采用平衡批处理法,研究了模拟根系分泌物--草酸溶液的浓度、pH对酸性磷酸酶在针铁矿、高岭石及黄棕壤和砖红壤胶体(＜2μm)上的吸附及比活的影响.结果表明,针铁矿对磷酸酶的吸附量受草酸浓度的影响较小,其它供试胶体对蛋白的吸附量随草酸浓度的升高,一般表现为先急剧降低(0～5mmol·L^-1),之后逐渐升高到与对照相当或略低.这与草酸在土壤胶体和矿物表面的配位形态及其对载体表面的电荷改变、溶解有关.草酸体系中,供试胶体对磷酸酶的吸附顺序为针铁矿>黄棕壤＞高岭石＞砖红壤.酶在草酸体系中的最大吸附点位一般出现在蛋白的等电点(IEP)和供试胶体的PZC之间,而酶在草酸体系中被固定到供试胶体上之后,其最适比活点随胶体类型的不同而没有变化或有所高移.     

金溶胶基底的 SERS 增强效果的实验研究

本文以结晶紫作为探针分子,研究了以金溶胶膜、pH ＝6以及 pH ＝13的金溶胶溶液为活性基底的表面增强拉曼光谱的增强效果。采用化学还原法制备金溶胶,加入氢氧化钠改变其 pH 值,并以自组装法制备金溶胶膜。通过比较金溶胶膜、pH ＝6及 pH ＝13时金溶胶溶液的增强因子以及在这三种金溶胶基底上结晶紫的检测限,分析不同活性基底增强效果的差异。三种活性基底的增强因子分别可达到5．9×103、1．5×105、2．3×107,pH ＝13的金溶胶溶液有最佳的增强效果。以这三种金溶胶为基底对结晶紫进行表面增强拉曼光谱探测,可得到检测限为70．7 nmol／L、9．6 nmol／L、1．8 nmol／L。结果表明,金溶胶溶液的增强效果明显优于金溶胶膜,而通过改变金溶胶体系的 pH 值可以改变金纳米颗粒的聚合程度及对探测物的吸附特性从而获得更高灵敏度的活性基底。     

Preparation and Friction Force Microscopy Measurements of Immiscible,Opposing Polymer Brushes

Solvated polymer brushes are well known to lubricate high-pressure contacts, because they can sustain a positive normal load while maintaining low friction at the interface. Nevertheless, these systems can be sensitive to wear due to interdigitation of the opposing brushes. In a recent publication, we have shown via molecular dynamics simulations and atomic force microscopy experiments, that using an immiscible polymer brush system terminating the substrate and the slider surfaces, respectively, can eliminate such interdigitation. As a consequence, wear in the contacts is reduced. Moreover, the friction force is two orders of magnitude lower compared to traditional miscible polymer brush systems. This newly proposed system therefore holds great potential for application in industry. Here, the methodology to construct an immiscible polymer brush system of two different brushes each solvated by their own preferred solvent is presented. The procedure how to graft poly(N-isopropylacrylamide) (PNIPAM) from a flat surface and poly(methyl methacrylate) (PMMA) from an atomic force microscopy (AFM) colloidal probe is described. PNIPAM is solvated in water and PMMA in acetophenone. Via friction force AFM measurements, it is shown that the friction for this system is indeed reduced by two orders of magnitude compared to the miscible system of PMMA on PMMA solvated in acetophenone.     

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two dimensional (2D) structures. These technologies are mature, offer high precision and many of them can be implemented in a high-throughput manner. We leverage the advantages of planar lithography and combine them with self-folding methods^1-20 wherein physical forces derived from surface tension or residual stress, are used to curve or fold planar structures into three dimensional (3D) structures. In doing so, we make it possible to mass produce precisely patterned static and reconfigurable particles that are challenging to synthesize.In this paper, we detail visualized experimental protocols to create patterned particles, notably, (a) permanently bonded, hollow, polyhedra that self-assemble and self-seal due to the minimization of surface energy of liquefied hinges^21-23 and (b) grippers that self-fold due to residual stress powered hinges^24,25. The specific protocol described can be used to create particles with overall sizes ranging from the micrometer to the centimeter length scales. Further, arbitrary patterns can be defined on the surfaces of the particles of importance in colloidal science, electronics, optics and medicine. More generally, the concept of self-assembling mechanically rigid particles with self-sealing hinges is applicable, with some process modifications, to the creation of particles at even smaller, 100 nm length scales^{22, 26} and with a range of materials including metals²¹, semiconductors⁹ and polymers²⁷. With respect to residual stress powered actuation of reconfigurable grasping devices, our specific protocol utilizes chromium hinges of relevance to devices with sizes ranging from 100 μm to 2.5 mm. However, more generally, the concept of such tether-free residual stress powered actuation can be used with alternate high-stress materials such as heteroepitaxially deposited semiconductor films^5,7 to possibly create even smaller nanoscale grasping devices.