The Effect of Protein Concentration on Electrophoretic Mobility

A theory of sedimentation in a concentrated suspension of spherical soft particles (i.e., polyelectrolyte-coated particles) is developed to obtain general expressions for sedimentation velocity of soft particles and sedimentation potential in the suspension. An Onsager relation between sedimentation potential and electrophoretic mobility of spherical soft particles in concentrated suspensions is derived for the case of low potentials and nonoverlapping electrical double layers of adjacent particles. Copyright 2000 Academic Press.     

Electrophoretic Mobility of a Polyelectrolyte Capsule

The electrophoretic motion of a polyelectrolyte capsule has been considered in a uniform electric field. The capsule carries a uniformly distributed charge and is permeable to ions of different natures. An electrolyte identical to a dispersion medium is located inside the capsule. The flow in the porous layer of the capsule has been described by the Brinkman equations taking into account the effect of electrostatic forces. The distribution of ions in the vicinity of the capsule has been determined, and its electrophoretic mobility has been found in a linear approximation. The mobility of the capsule has been studied as depending on its geometric characteristics, permeability, and charge density. In particular, a complex extremal character of variations in the mobility as depending on the solid phase fraction in the capsule has been revealed at different ratios between the thicknesses of the electrical double layer and the Brinkman filtration layer.     

Dynamic Electrophoretic Mobility of a Soft Particle

A theory of the dynamic electrophoretic mobility of a spherical soft particle (that is, a polyelectrolyte-coated spherical particle) in an oscillating electric field is presented. In the absence of the polyelectrolyte layer a spherical soft particle becomes a spherical hard particle, while in the absence of the particle core it tends to a spherical polyelectrolyte. The present theory thus covers two extreme cases, that is, dynamic electrophoresis of hard particles and that of spherical polyelectrolytes. Simple analytic mobility expressions are derived. It is shown how the dynamic electrophoretic mobility of a soft particle depends on the volume charge density distributed in the polyelectrolyte layer, on the frictional coefficient characterizing the frictional forces exerted by the polymer segments on the liquid flow in the polyelectrolyte layer, on the particle size, and on the frequency of the applied oscillating electric field. Copyright 2001 Academic Press.     

Liposomal FRET Assay Identifies Potent Drug-Like Inhibitors of the Ceramide Transport Protein (CERT)

Ceramide transfer protein (CERT) mediates non-vesicular transfer of ceramide from endoplasmic reticulum to Golgi apparatus and thus catalyzes the rate-limiting step of sphingomyelin biosynthesis. Usually, CERT ligands are evaluated in tedious binding assays or non-homogenous transfer assays using radiolabeled ceramides. Herein, a facile and sensitive assay for CERT, based on Förster resonance energy transfer (FRET), is presented. To this end, we mixed donor and acceptor vesicles, each containing a different fluorescent ceramide species. By CERT-mediated transfer of fluorescent ceramide, a FRET system was established, which allows readout in 96-well plate format, despite the high hydrophobicity of the components. Screening of a 2 000 compound library resulted in two new potent CERT inhibitors. One is approved for use in humans and one is approved for use in animals. Evaluation of cellular activity by quantitative mass spectrometry and confocal microscopy showed inhibition of ceramide trafficking and sphingomyelin biosynthesis.     

Image-Based Morphological Profiling Identifies a Lysosomotropic,Iron-Sequestering Autophagy Inhibitor

Chemical proteomics is widely applied in small-molecule target identification. However, in general it does not identify non-protein small-molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image-based morphological profiling in the cell painting assay. A compound-induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe²⁺ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.     

微流控芯片上的红细胞电泳淌度研究

以单细胞成像法分别测量了 3组无农药含水质净化剂、有农药无水质净化剂及其有农药有水质净化剂处理后的杂交鲤鱼红细胞在塑料 ( polymethalmethacrylate ,PMMA)微流控芯片上的电泳淌度。其值分别为 1.138× 10 -4、0 .12 79× 10 -4和 - 0 .85 2 0× 10 -4cm2 v-1s-1。从电泳淌度的差别可以看出功夫菊酯和水质条件恶化均可使鲤鱼红细胞表面的电荷密度发生变化 ,从而引起电泳淌度的变化。同时还初步考察了细胞电泳淌度作为参数进行细胞分类的可行性 ,显示这种淌度差别有可能作为细胞分类的一项依据     

Electrophoretic Mobility of Soft Particles in Concentrated Suspensions

A general theory is developed for the electrophoretic mobility of spherical soft particles (i.e., spherical hard colloidal particles of radius a coated with a layer of polyelectrolytes of thickness d) in concentrated suspensions in an electrolyte solution as a function of the particle volume fraction φ on the basis of Kuwabara's cell model. In the limit d-->0, the mobility expression obtained tends to that for spherical hard particles in concentrated suspensions, whereas in the limit a-->0, it becomes that for spherical polyelectrolytes (charged porous spheres with no particle core). Simple approximate analytic mobility expressions are derived for the case where relaxation effect is negligible. It is found that in practical cases, the φ dependence of the mobility is negligible for da, the mobility strongly decreases with increasing φ. Copyright 2000 Academic Press.     

On the General Expression for the Electrophoretic Mobility of a Soft Particle

Electrokinetic equations for electrophoresis of a soft particle (that is, a hard particle covered with a layer of polyelectrolytes) have been solved previously under the conditions that the net force acting on the soft particle as a whole (the particle core plus the polyelectrolyte layer) must be zero and that the electrical force acting on the polymer segment is balanced with a frictional force exerted by the liquid flow (J. Colloid Interface Sci. 163, 474 (1994)). In the present work we replaced the latter condition by the alternative and more appropriate condition that pressure is continuous at the boundary between the surface layer and the surrounding electrolyte solution to solve the electrokinetic equations and obtained the general mobility expression for the electrophoretic mobility of a spherical soft particle. It is found that the general mobility expression thus obtained reproduces all of the approximate mobility expressions derived previously and, in addition, that the continuous pressure condition leads to the correct limiting behavior of the electrophoretic mobility in the case where the frictional coefficient tends to zero (this behavior cannot be derived from the force balance condition for the polyelectrolyte layer). Copyright 2000 Academic Press.     

A Computer Method for Predicting the Electrophoretic Mobility of Peptides

A computer model is presented for the prediction of the electrophoretic mobilities of peptides from physical constants derived from their amino acid sequences. The model assumes that the electrophoretic mobility can be represented by a product of four functions according to the relation:  l_comp= l(L)w(W)q(Q)c(CC), where L (a length parameter) is represented by the number of amino acid residues of the peptide, W (a width parameter) is represented by the average residue mass, Q = the charge of the peptide, and CC = the position of the center of charge relative to the center of mass. The model was used to calculate the electrophoretic mobilities of peptides in a 50 mM phosphate buffer at pH 2.5. Sixty-four test peptides ranging in size from 2 to 39 amino acid residues were used for this study. The calculated mobilities show excellent correlation with experimental measurements with a correlation coefficient greater than 0.98.     

非样品手性配体交换毛细管电泳测定手性多巴

以L-多巴为测定目标, 以天然芳香氨基酸为替代样品, 建立了以非样品α-羟基酸为手性配体, 以铜和镍等二价金属为中心离子的手性配体交换毛细管电泳新方法. 当弱配位的α-羟基酸与中心离子的摩尔比大于6∶1时, 所建立的方法即可实现对D,L-氨基酸和D,L-多巴的基线分离, 可用于口服多巴药片含量的测定, 检测出质量分数为0.5%的异构体杂质, 其定量的线性范围是0.025~2.5 mmol/L, 回收率为96.3%. 据此方法测得某商品药片的实际含量(质量分数)为标称值的(97.33±0.70)%.     

A Designed Inhibitor of a CLC Antiporter Blocks Function through a Unique Binding Mode

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Highlights? OADS is a known small-molecule inhibitor of a CLC antiporter ? OADS specifically inhibits the ClC-ec1 antiporter but not the ClC-1 channel ? Photoaffinity labeling and mass spectrometry have localized OADS binding to two discrete sites ? The unique binding mode and lipid dependence of OADS suggest potential mechanisms of action     

缓蚀剂在阳极电泳涂装中的应用

在电泳涂装工艺中使用缓蚀剂，可提高阳极电泳涂层的防腐蚀性，实验表明，沥青电泳漆与缓蚀剂的电泳共沉积涂层对基底金属具有较高的防护性能，探讨了共沉积涂层中缓蚀剂的缓蚀机理。     

Photoswitchable Red Fluorescent Protein with a Large Stokes Shift

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Image‐Based Morphological Profiling Identifies a Lysosomotropic,Iron‐Sequestering Autophagy Inhibitor

Chemical proteomics is widely applied in small‐molecule target identification. However, in general it does not identify non‐protein small‐molecule targets, and thus, alternative methods for target identification are in high demand. We report the discovery of the autophagy inhibitor autoquin and the identification of its molecular mode of action using image‐based morphological profiling in the cell painting assay. A compound‐induced fingerprint representing changes in 579 cellular parameters revealed that autoquin accumulates in lysosomes and inhibits their fusion with autophagosomes. In addition, autoquin sequesters Fe²⁺ in lysosomes, resulting in an increase of lysosomal reactive oxygen species and ultimately cell death. Such a mechanism of action would have been challenging to unravel by current methods. This work demonstrates the potential of the cell painting assay to deconvolute modes of action of small molecules, warranting wider application in chemical biology.     

The Electrophoretic Mobility and Electric Conductivity of a Concentrated Suspension of Colloidal Spheres with Arbitrary Double-Layer Thickness

The electrophoresis in a monodisperse suspension of dielectric spheres with an arbitrary thickness of the electric double layers is analytically studied. The effects of particle interactions are taken into account by employing a unit cell model, and the overlap of the double layers of adjacent particles is allowed. The electrokinetic equations, which govern the ionic concentration distributions, the electric potential profile, and the fluid flow field in the electrolyte solution surrounding the charged sphere in a unit cell, are linearized assuming that the system is only slightly distorted from equilibrium. Using a perturbation method, these linearized equations are solved with the surface charge density (or zeta potential) of the particle as the small perturbation parameter. Analytical expressions for the electrophoretic mobility of the colloidal sphere in closed form correct to O(zeta) are obtained. Based on the solution of the electrokinetic equations in a cell, a closed-form formula for the electric conductivity of the suspension up to O(zeta(2)) is derived from the average electric current density. Comparisons of the results of the cell model with different conditions at the outer boundary of the cell are made for both the electrophoretic mobility and the electric conductivity. Copyright 2001 Academic Press.     

Low-Zeta-Potential Analytic Solution for the Electrophoretic Mobility of a Spherical Colloidal Particle in an Oscillating Electric Field

The equations developed by C. S. Mangelsdorf and L. R. White (J. Chem. Soc. Faraday Trans. 88, 3567 (1992)) to calculate the electrophoretic mobility of a solid, spherical colloidal particle subjected to an oscillating electric field are solved analytically for low zeta potential, ζ, to obtain the electrophoretic mobility correct to (eζ/k_BT). Due to severe numerical cancellation of the exponential integrals, two forms of the analytic solution are presented which are numerically stable for different regions of κa (where a is the particle radius and κ^-1 is the Debye screening length). This low-ζ analytic solution is valid for all frequencies, particle sizes, and electrolyte concentrations, and agrees to at least two significant figures with the "exact" results obtained by Mangelsdorf and White at eζ/k_BT = 1 (ζ ≈ 25 mV). A program implementing this low-zeta analytic formula for the electrophoretic mobility is available from the authors.     

Synthesis of the seco-Limonoid BCD Ring System Identifies a Hsp90 Chaperon Machinery (p23) Inhibitor

D-Ring-seco-limonoids (tetranortriterpenoids), such as gedunin and xylogranin B display anti-cancer activity, acting via inhibition of Hsp90 and/or associated chaperon machinery (e.g., p23). Despite this, these natural products have received relatively little attention, both in terms of an enabling synthetic approach (which would allow access to derivatives), and as a consequence their structure–activity relationship (SAR). Disclosed herein is a generally applicable synthetic route to the BCD ring system of the seco-D-ring double bond containing limonoids. Furthermore, cell based assays revealed the first skeletal fragment that exhibited inhibition of the p23 enzyme at a level which was equipotent to that of gedunin, despite being much less structurally complex.     

Rapid Detection of Irreversible Acetylcholineasterase Inhibitor by Mass Spectrometry Assay

Here we developed a rapid method to detect acetylcholinesterase (AChE) activity by matrix‐assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI‐FTMS) for screening irreversible AChE inhibitors. Due to its good salt‐tolerance and low sample consumption, MALDI‐FTMS could facilitate rapid detection, especially detection in real application. AChE activity was determined through calculating abundance of substrate and product in mass spectrometry. By this approach, we investigated the relation of organophosphorous (OP) concentrations and AChE inhibition. Shown in different inhibition curves from different OP pesticides, enzyme inhibitions still kept good correlation with concentration of OPs. Finally, this AChE‐inhibited method was applied to screen whole bloods of four decedents and discuss their death reason. In contrast to healthy persons, three of decedents showed low AChE activity, and probably died for irreversible AChE inhibitors. Through the following detecting in GC‐MS/MS, the possible death reason of these three decedents was confirmed, and another decedent actually died for sumicidin, a non‐AChE inhibitor. It demonstrated that screening irreversible AChE inhibitors by detecting enzyme activity in MALDI‐FTMS provided fast and accurate analysis results and excluded another toxicants not functioning on AChE. This method offered alternative choices for indicating the existence of enzyme inhibitors.     

A Time-Resolved FRET Assay Identifies a Small Molecule that Inhibits the Essential Bacterial Cell Wall Polymerase FtsW

The peptidoglycan cell wall is essential for bacterial survival. To form the cell wall, peptidoglycan glycosyltransferases (PGTs) polymerize Lipid II to make glycan strands and then those strands are crosslinked by transpeptidases (TPs). Recently, the SEDS (for shape, elongation, division, and sporulation) proteins were identified as a new class of PGTs. The SEDS protein FtsW, which produces septal peptidoglycan during cell division, is an attractive target for novel antibiotics because it is essential in virtually all bacteria. Here, we developed a time-resolved Förster resonance energy transfer (TR-FRET) assay to monitor PGT activity and screened a Staphylococcus aureus lethal compound library for FtsW inhibitors. We identified a compound that inhibits S. aureus FtsW in vitro. Using a non-polymerizable Lipid II derivative, we showed that this compound competes with Lipid II for binding to FtsW. The assays described here will be useful for discovering and characterizing other PGT inhibitors.