Enzymatic activity and chromatographic characteristics of the cell membrane immobilized on silica surface

A new cell membrane preparation. silica carrier cell membrane (CCM), has been presented, in which cell membrane was immobilized on silica which acted as carrier by the absorption. The surface characteristics of the silica CCM have been tested by a scanning electron microscope and surface energy spectrometer. Comparison has been made of the changeable rule of enzymatic activity of rabbit red cell membrane in three states: the silica CCM, the suspension and the precipitation cell membranes, with temperature and time. The result showed that the silica CCM still keeps the same enzymatic activity as the other two states of cell membranes. The interactions between calcium antagonists and rabbi red and cardiac muscle cell membranes, and the stereoscopic interactions between dihydropridines and rabbit cerebellar cell membrane have been investigated by the chromatographic system of the silica CCM as a stationary phase. From the foregoing results, the chromatographic parameters obtained from the system would be expected to indicate the special interaction and enantioselectivity between drugs and membrane receptorsin vivo.     

Silica-Based Solid Phase Extraction of DNA on a Microchip

Micro total analysis systems for chemical and biological analysis have attracted much attention. However, microchips for sample preparation and especially DNA purification are still underdeveloped. This work describes a solid phase extraction chip for purifying DNA from biological samples based on the adsorption of DNA on bare silica beads prepacked in a microchannel. The chip was fabricated with polydimethylsiloxane. The silica beads were packed in the channel on the chip with a tapered microchannel to form the packed bed. Fluorescence detection was used to evaluate the DNA adsorbing efficiency of the solid phase. The polymerase chain reaction was used to evaluate the quality of the purified DNA for further use. The extraction efficiency for the DNA extraction chip is approximately 50% with a 150-nL extraction volume, Successful amplification of DNA extracted from human whole blood indicates that this method is compatible with the polymerase chain reaction.     

Basement membrane polarizes lectin binding sites of Drosophila larval fat body cells

Vertebrate epithelial cells in monolayers are asymmetrical in that their apical and basal membranes differ in morphology and function. That this cell polarity depends on the presence of tight junctions can be demonstrated by labelling one surface of a cell monolayer in culture with fluorescent lectins and lipid probes, and subsequently observing whether the labels disperse to the opposite cell surfaces. Here we report on a differential distribution of binding sites for the lectin wheat germ agglutinin (WGA) on the cells of Drosophila melanogaster larval fat body, and show that the pattern is correlated with the structural association between the cell surfaces and their overlying basement membrane.     

Interaction of C-reactive protein with artificial phosphatidylcholine bilayers.

C-Reactive protein (CRP), the most characteristic of the 'acute phase proteins' (ref. 1) is thought to participate in the mediation and/or modulation of acute inflammatory processes, but its exact function is unknown. CRP has a Ca2+-dependent binding specificity for phosphorylcholine, the polar head group of two widely distributed lipids, lecithin (phosphatidylcholine, PC) and sphingomyelin (SM). A number of observations suggest that at least some of the biological activities of CRP depend on its interaction with phospholipids of cell membranes. In addition, interaction of CRP with PC- and SM-containing lipid dispersions and with PC-containing liposomes can activate the complement system. We report here that binding of CRP to model membranes of PC requires the incorporation into the bilayer of lysophosphatidylcholine (LPC). Thus, a disturbance of the molecular organisation of the bilayer appears to be necessary for binding of CRP. These findings provide a possible biochemical explanation for binding of CRP to damaged but not intact cell membranes and might be relevant to its biological function.     

The p21 ras C-terminus is required for transformation and membrane association

The Harvey murine sarcoma virus (Ha-MuSV) transforming gene, v-rasH, encodes a 21,000 molecular weight protein (p21) that is closely related to the p21 proteins encoded by the cellular transforming genes of the ras gene family. The primary translation product (prop21), which is found in the cytosol, undergoes posttranslational modification and the mature protein subsequently becomes associated with the inner surface of the plasma membrane and binds lipid tightly. The p21 proteins have the capacity to bind guanine nucleotides non-covalently in vitro. To assess the biological relevance of these biochemical features of the protein, we have now studied a series of deletion mutants located at or near the C-terminus of the viral p21 protein. Our tissue culture studies indicate that amino acids located at or near the C-terminus are required for cellular transformation, membrane association and lipid binding.     

Phospholipids and the origin of cationic gating charges in voltage sensors

Cells communicate with their external environment through physical and chemical processes that take place in the cell-surrounding membrane. The membrane serves as a barrier as well as a special environment in which membrane proteins are able to carry out important processes. Certain membrane proteins have the ability to detect the membrane voltage and regulate ion conduction or enzyme activity. Such voltage-dependent processes rely on the action of protein domains known as voltage sensors, which are embedded inside the cell membrane and contain an excess of positively charged amino acids, which react to an electric field. How does the membrane create an environment suitable for voltage sensors? Here we show under a variety of conditions that the function of a voltage-dependent K+ channel is dependent on the negatively charged phosphodiester of phospholipid molecules. A non-voltage-dependent K+ channel does not exhibit the same dependence. The data lead us to propose that the phospholipid membrane, by providing stabilizing interactions between positively charged voltage-sensor arginine residues and negatively charged lipid phosphodiester groups, provides an appropriate environment for the energetic stability and operation of the voltage-sensing machinery. We suggest that the usage of arginine residues in voltage sensors is an adaptation to the phospholipid composition of cell membranes.     

The appearance of single-ion channels in unmodified lipid bilayer membranes at the phase transition temperature

Ion-specific channels in artificial membranes have been formed by the addition of gramicidin A, alamethicin, polyene antibiotics and some proteins to the solution surrounding the bilayer lipid membrane. Until now there have been no reports of single-ion channels in unmodified lipid membranes. We have now studied the electrical conductance of planar lipid bilayers membranes made of synthetic distearoylphosphorylcholine (DSPC). Current fluctuations of amplitude approximately 1pA and duration approximately 1 s have been discovered at phase transition temperature, which shows that the appearance of ionic channels may be the result of lipid domain interactions. This would explain the dramatic increase in ion permeability observed in liposomes during phase transition. We suggest that these channels could conduct the transmembrane ionic current in biological membranes without the involvement of peptides and proteins.     

Nanoscopic channel lattices with controlled anisotropic wetting

Engineered microscopic surface structures allow local control of physical surface properties such as adhesion, friction and wettability. These properties are related both to molecular interactions and the surface topology--for example, selective adsorption and molecular recognition capabilities require controlled anisotropy in the surface properties. Chemistry with extremely small amounts of material has become possible using liquid-guiding channels of sub-micrometre dimensions. Laterally structured surfaces with differing wettabilities may be produced using various techniques, such as microcontact printing, micromachining, photolithography and vapour deposition. Another strategy for introducing anisotropic texture is based on the use of the intrinsic material properties of stretched ultrathin polymer coatings. Here we present a fast and simple method to generate extended patterned surfaces with controlled wetting properties on the nanometre scale, without any lithographic processes. The technique utilizes wetting instabilities that occur when monomolecular layers are transferred onto a solid substrate. The modified surfaces can be used as templates for patterning a wide variety of molecules and nanoclusters into approximately parallel channels, with a spatial density of up to 20,000 cm(-1). We demonstrate the transport properties of these channels for attolitre quantities of liquid.     

TiO_2粒子对膜吸收过程中近膜壁面处溶质传质行为的影响

采用纯CO2-去离子水为实验体系,在板式膜器中研究了孔隙率对膜吸收过程传质效果的影响,并考察了吸收剂搅拌与否对传质性能的影响。结果表明,吸收剂静止时,孔隙率对传质效果影响很小。当吸收剂处于搅拌状态时,传质得到强化,但由于膜壁面处溶质浓度分布情况不同使得传质效果随孔隙率的增加而增大。为改变近膜壁面处溶质浓度的分布情况,利用固体粒子将动量引入液相边界层内,使膜壁面处溶质浓度分布趋于均匀。固相粒子的加入减小了不同孔隙率膜之间的传质效果差异,同时还强化了传质,传质效果提高40%以上,且孔隙率越小强化效果越大。此外,随固含率的增加,强化效果增强,固含率的继续增大,强化效果的增强变得平缓。随着搅拌转速的增加,强化效果减弱。     

Surface characterization of polyimide and polyethylene terephthalate membranes toward plasma and UV treatments

This study is to bring an overview on various methods regarding the surface treatment performed on different polymeric membranes in relation to a deference between the treatment mechanism and their chemical structure. Herein, plasma and UV surface treatments (PST and UST) were utilized to treat the surfaces of two commercial membranes, i.e., aromatic polyimide and polyethylene terephthalate for various durations using a discharge electrode and an UV LED curing system, respectively. Based on the effects of PST and UST processes on the surface of these membranes, their surface properties have been characterized to surface morphology and hydrophilicity (or wettability), at same time the changes have probably occurred on their surface during the PST and UST processes. As a result, their hydrophilic behavior was significantly enhanced after conducting these treatment processes based on the investigation of water contact angle. Moreover, Fourier-transform infrared and Raman spectroscopy manifested the changes in the chemical bonds, interactions and orientations of molecules, which confirmed the apparition and insertion of polar functional groups after the surface treatments, and accordingly, their wettability was enhanced significantly. Also, optical microscopy images showed direct impactions on their surface and the evidence of slight surface aging/damage during the treatment processes based on the presentation of the strange streaks on the treated surfaces. In particular, the peel forces exhibited an increase comparing to the initial membranes, which is characteristically for enhancing their adhesion behavior. Hence, these results are needful for improving the surface properties of a material, and obtaining better adhesion behavior on the membrane-like materials and recycling polymer materials.     

改性金属膜膜萃取萃铜速度影响因素考察

针对有机中空纤维膜材料在膜萃取过程中出现膜孔溶胀的问题,采用聚四氟乙烯涂敷在不锈钢纤维烧结膜表面制备一种有机／无机复合膜并用作膜萃取的支撑材料,在平板式膜器中进行萃铜研究．考察了影响铜的萃取及反萃过程传质速度的主要因素,包括料液浓度、水相及有机相流速等,并根据试验结果初步探讨了膜萃取过程传质阻力的分布情况,结果表明有机相边界层阻力大于水相边界层阻力．     

C_(60)修饰的s-BLM离子传感器的研究与应用

研究了以C60镶嵌物修饰的固体支撑双层类脂膜(s-BLM)体系对I-的电化学传感行为。结果表明,在s-BLM膜中镶嵌C60,体系对I-的检测灵敏度达到10-7mol L。通过对s-BLM膜中镶嵌物的选择,可获得更高稳定性和检出限的离子传感器。     

Using of the surface plasmon resonance cytosensor for real-time and non-invasive monitoring of cellular effects in living C6 cells induced by PMA

By cell and molecular biology, the traditional life science research has been improved into modern ex-perimental science. Because cell is the basic structural and functional unit of the mysterious life, the study of life phenomena will be ultimately embodied into the study of cell biology. How to acquire a dynamic knowledge of the intricate biomolecular reactivity and process in living cells has become one of the hotspots in recent biological researches. For the complexity of cellular effects,…     

Single-molecule fluorescence imaging of membrane-bound proteins for studies of cell signal transduction

Fluorescence imaging of single molecules is becoming a powerful tool to examine biological processes at the molecular level.Using total internal reflection fluorescence microscopy (TIRFM),it has been possible to study the dynamic behavior of single molecules on living cell membranes.Herein,we briefly review the application of TIRFM-based single-molecule imaging in studies of membrane receptors involved in signal transduction.Furthermore,we discuss several examples of our own research on growth factor receptors,including TGF-β receptors,HER2,and EGFR,and speculate possible applications of this technique to investigate other cellular events occurring on or near the plasma-membrane.     

Phosphoinositides in cell regulation and membrane dynamics

Inositol phospholipids have long been known to have an important regulatory role in cell physiology. The repertoire of cellular processes known to be directly or indirectly controlled by this class of lipids has now dramatically expanded. Through interactions mediated by their headgroups, which can be reversibly phosphorylated to generate seven species, phosphoinositides play a fundamental part in controlling membrane-cytosol interfaces. These lipids mediate acute responses, but also act as constitutive signals that help define organelle identity. Their functions, besides classical signal transduction at the cell surface, include regulation of membrane traffic, the cytoskeleton, nuclear events and the permeability and transport functions of membranes.     

Phosphatidylinositol is the membrane-anchoring domain of the Thy-1 glycoprotein

Glycoproteins exposed on cell surfaces are commonly anchored in the membrane via hydrophobic peptide domains which penetrate the lipid bilayer. However, it has recently been appreciated that there are exceptions to this generalization and certain cell-surface proteins appear to be anchored via a specific association with phosphatidylinositol. Thy-1 glycoprotein may also be attached to cell membranes by a non-protein hydrophobic domain located at the C-terminus and although the chemical nature of this moiety has not been determined, it was postulated that it might be a lipid. On the other hand, amino-acid sequences predicted from nucleotide sequence analyses suggest that a C-terminal hydrophobic peptide segment not found in the purified, detergent-solubilized Thy-1 glycoprotein may be responsible for attachment. We report here that a highly purified phospholipase C specific for phosphatidylinositol selectively released Thy-1 from viable normal or malignant T lymphocytes. This result supports the proposed lipid nature of the Thy-1 anchoring domain and further suggests that this lipid is, or is closely related to, phosphatidylinositol.     

微波（2450MHz）对整体小白鼠红细胞膜的损伤效应及药物防护

本文采用２４５０ＭＨｚ微波慢性辐射整体昆明小白鼠，从分子水平研究微波对小白鼠红细胞膜的影响。选择功率密度０ｍＷ／ｃｍ２、５ｍＷ／ｃｍ２（ＳＡＲ＝５．５ｍＷ／ｇ）、１０ｍＷ／ｃｍ２（ＳＡＲ＝１１ｍＷ／ｇ）的微波连续辐射小白鼠４０天，每天６小时。发现１０ｍＷ／ｃｍ２的微波对小白鼠红细胞膜的损伤较为明显。表现为小白鼠红细胞的渗透脆性增加，膜脂流动性下降，血浆丙二醛（ＭＤＡ）及红细胞膜ＭＤＡ均为明显升高，但红细胞溶血度未增加，红细胞ＳＯＤ、红细胞膜ＡｃｈＥ活动和膜蛋白组份的百分含量也未见明显变化。此外，本文还从自由基角度初步探讨了微波辐射损伤的机理。实验表明，喂服维生素Ｃ的小白鼠有抗微波损伤的作用，这启示一定剂量的维生素Ｃ对微波损伤有防护作用。     

用密度泛函理论预测带电半透膜性质

从分子水平上研究半透膜的性质在生命科学、化学以及化学工程中有着重要的作用。该文采用密度泛函理论研究了带电半透膜附近各种离子的分布情况,其中除静电作用外,膜与其附近的离子之间的色散能用方阱势能函数来表示。研究表明用密度泛函理论预测的不同膜面电荷密度和不同离子直径比情况下离子在半透膜附近密度分布与M on teC arlo模拟数据符合得很好。密度泛函理论预测结果表明,膜面电荷密度越大,被吸附到膜中的离子越多,膜面电荷对大离子密度分布的影响要大于小离子。     

The principle of gating charge movement in a voltage-dependent K+ channel

The steep dependence of channel opening on membrane voltage allows voltage-dependent K+ channels to turn on almost like a switch. Opening is driven by the movement of gating charges that originate from arginine residues on helical S4 segments of the protein. Each S4 segment forms half of a 'voltage-sensor paddle' on the channel's outer perimeter. Here we show that the voltage-sensor paddles are positioned inside the membrane, near the intracellular surface, when the channel is closed, and that the paddles move a large distance across the membrane from inside to outside when the channel opens. KvAP channels were reconstituted into planar lipid membranes and studied using monoclonal Fab fragments, a voltage-sensor toxin, and avidin binding to tethered biotin. Our findings lead us to conclude that the voltage-sensor paddles operate somewhat like hydrophobic cations attached to levers, enabling the membrane electric field to open and close the pore.     

Cell surface clustering of Cadherin adhesion complex induced by antibody coated beads

Cadherin receptors mediate cell-cell adhesion, signal transduction and assembly of cytoskeletons. How a single transmembrane molecule Cadherin can be involved in multiple functions through modulating its binding activities with many membrane adhesion molecules and cytoskeletal components is an unanswered question which can be elucidated by clues from bead experiments. Human lung cells expressing N-Cadherin were examined. After co-incubation with anti-N-Cadherin monoclonal antibody coated beads, cell surface clustering of N-Cadherin was induced. Immunofluorescent detection demonstrated that in addition to Cadherin, β-Catenin, α-Catenin, α-Actinin and Actin fluorescence also aggregated respectively at the membrane site of bead attachment. Myosin heavy chain (MHC), another major component of Actin cytoskeleton, did not aggregate at the membrane site of bead attachment. Adhesion unrelated protein Con A and polylysine conjugated beads did not induce the clustering of adhesion molecules. It is indicated that the Cadherin/Catenins/α-Actinin/Actin complex is formed at Cadherin mediated cell adherens junction; occupancy and cell surface clustering of Cadherin is crucial for the formation of Cadherin adhesion protein complexes.