Detection of molecular interactions at membrane surfaces through colloid phase transitions

The molecular architecture of-and biochemical processes within--cell membranes play important roles in all living organisms, with many drugs and infectious disease agents targeting membranes. Experimental studies of biochemical reactions on membrane surfaces are challenging, as they require a membrane environment that is fluid (like cell membranes) but nevertheless allows for the efficient detection and characterization of molecular interactions. One approach uses lipid membranes supported on solid substrates such as silica or polymers: although the membrane is trapped near the solid interface, it retains natural fluidity and biological functionality and can be implanted with membrane proteins for functional studies. But the detection of molecular interactions involving membrane-bound species generally requires elaborate techniques, such as surface plasmon resonance or total internal reflection fluorescence microscopy. Here we demonstrate that colloidal phase transitions of membrane-coated silica beads provide a simple and label-free method for monitoring molecular interactions on lipid membrane surfaces. By adjusting the lipid membrane composition and hence the pair interaction potential between the membrane-supporting silica beads, we poise our system near a phase transition so that small perturbations on the membrane surface induce dramatic changes in the macroscopic organization of the colloid. We expect that this approach, used here to probe with high sensitivity protein binding events at membrane surfaces, can be applied to study a broad range of cell membrane processes.     

Investigation of biomembrane-drug interactions of different biomembranes by capillary electrophoresis

Biomembrane-coated capillaries are prepared by coating different kinds of biomembranes including mice red cell membrane, human red cell membrane and S-180 cell membrane on the inner surface of the capillary, respectively. Effects of immobilized biomembrane amount, pH and biomembrane species on the interactions between drugs and biomembranes have been evaluated. The results showed that the chromatographic retention factor for each drug was essentially related to the amount of immobilized biomembrane, the pH and the type of biomembrane.     

Establishment of the model of vascular endothelial cell membrane chromatography and its preliminary application

A model of vascular endothelial cell membrane chromatography was established by using an ECV304 cell membrane stationary phase (ECV304 CMSP) prepared by immobilizing the ECV304 cell membrane onto the surface of silica carrier. The surface and chromatographic characteristics of ECV304 CMSP were studied. The active component from Caulophyllum robustum was screened by using the model of vascular endothelial cell membrane chromatography. The interaction between the active component and membrane receptor was determined by using a replace experiments. The effect of the active component was tested by using tube formation of ECV304 cell. The results indicated that the model of ECV304 cell membrane chromatograph (ECV304 CMC) can stimulate the interaction between drug and receptor in vitro and the retention characteristics of taspine as active component was similar to that of model molecule in the model of ECV304 CMC. And therefore, taspine acted on VEGFR2 and inhibited the tube formation of ECV304 cell induced by VEGF. This model can be used to screen definite active component as a screening model.     

Development of the model of pancreatic β-cell membrane chromatography and its chromatographic characteristics

A new model of pancreatic β-cell membrane chromatography has been established by using a β-cell membrane stationary phase （β-CMSP） prepared by immobilizing the β-cell membrane onto the surface of silica carrier. The protein level and K^＋, Na^＋-ATP enzymatic activity of the β-CMSP were detected respectively. The surface characteristics of the β-CMSP were tested by using the scanning electron microscope and surface energy spectrometer. In this model, the column （10 mm × 2 mm, I.D.） packed with β-CMSP, 25 mmol/ammonium sulfate buffer solution （pH 7.4） as mobile phase with the flow rate of 0.2 mL/min at 37 ± 0.5℃ were used in the following studies. The retention characteristics of the sulfonylureas （gliquidone, glibenclamide, gliclazide and glipizide） were investigated under the chromatographic conditions above. The affinities of the sulfonylureas on β-cell membrane and receptor will be expressed by using the logk＇ values （the logarithm of capacity factor of a solute） in the model. The correlation of the affinity with the pharmacological effect of the sulfonylureas was analyzed also.     

基于多巴胺的杂化膜界面优化及其气体分离性能强化

采用仿生黏合剂多巴胺改性二氧化硅纳米颗粒（PDA@SiO₂）并与聚乙烯醇（PVA）高分子基质共混，再将获得的PVA-PDA@SiO₂杂化材料涂覆到聚砜（PSf）中空纤维支撑层表面制备中空纤维复合膜用于丙烯气体除湿，研究了杂化膜的微观形貌、晶态结构、高分子链运动性、自由体积特性等物理化学性质。实验结果表明，当填充剂PDA@SiO₂含量（质量分数，下同）为1%时，在298 K、原料气水含量为0.5%的条件下，PVA-PDA@SiO₂/PSf膜分离因子为24 837，是PVA/PSf膜的12.7倍，渗透系数为904 GPU[1 GPU=7.501×10^-10 cm³/（cm²·s·Pa）]，是PVA/PSf膜的3.6倍。仿生黏合剂多巴胺的多重相互作用可改善PVA与填充剂SiO₂的界面相容性，进而提升杂化膜的分离性能。     

Cyclic nucleotides may mediate taste transduction

Taste stimulus adsorption is believed to occur at the taste cell microvillous membrane. But due to technical difficulties of inserting glass electrodes into the mammalian taste cell, little is known about the mechanisms of taste transduction. Reliable intracellular recordings are necessary to determine the characteristics of taste cells. This has been accomplished previously in the mouse and is reported here. Recent experiments indicated that cyclic nucleotides can act on the inner surface of the membranes of a variety of cells to alter their ion-channel activity, and these substances might act as intracellular transmitters in taste cells. But tight junctions found at the apical membrane of mammalian taste cells do not allow stimuli to enter the taste bud, making it difficult to alter the environment of the taste cell by perfusing with chemical solutions. Here we report that cyclic AMP, cyclic GMP, EGTA or tetraethyl-ammonium electrophoretically injected into the mouse taste cell induce membrane depolarization and increased membrane resistance. These results suggest that a cyclic nucleotide enzymatic cascade, modulated by calcium ions, may mediate the potassium permeability that controls taste, in a way analogous to visual and olfactory transduction.     

Preparation of high selectivity silicalite-1 membranes by two-step <Emphasis Type="Italic">in situ</Emphasis> hydrothermal synthesis

High selectivity silicalite-1 membranes were synthesized on silica tubes by in situ hydrothermal synthesis. Using a two-step synthesis, a membrane with a separation factor of 99 was prepared for separating an ethanol/water mixture at 60°C. The average (n = 4) flux and separation factor of the membranes were 0.47 kg m-2 h-1 and 89, respectively. The membranes exhibited high reproducibility, and the relative standard deviation of the average (n = 4) flux and separation factor were only 5.3% and 9.2%, respectively. These results suggest that silica is a suitable support for synthesis of high-performance silicalite-1 membranes.     

Control of malarial invasion by phosphorylation of the host cell membrane cytoskeleton

It has been shown that the entry of the malaria parasite into the red blood cell requires the presence of ATP in the host cell cytoplasm. In red blood cell ghosts that contain no ATP the receptor on the extracellular surface remains in place and parasites will bind to the membrane, but will not enter. ATP is thus necessary for one of the steps in the invasion sequence that follows recognition and attachment. The process of entry appears to involve the active participation of the host cell membrane cytoskeleton. We have suggested that the function of the intracellular ATP may be to regulate phosphorylation of the cytoskeleton. We now present evidence that the activity of the membrane-associated cyclic AMP-independent kinase of the red blood cell is inseparable from invasion; the active substrate may be spectrin.     

Ca2+-activated ATPase and ATP-dependent calmodulin-stimulated Ca2+ transport in islet cell plasma membrane

Calcium is known to play an essential part in the regulation of insulin secretion in the pancreatic beta cell. Calcium influx/efflux studies indicate that glucose promotes an accumulation of calcium by the beta cell. However, interpretation of such data is particularly difficult due to the complex compartmentalization of calcium within the cell. Although indirect evidence using chlorotetracycline suggests that control of calcium homeostasis at the plasma membrane may be central to insulin secretion, the mechanism by which secretagogues influence the handling of calcium remains unknown. Despite its continuous diffusive entry, intracellular calcium is maintained in the submicromolar range by energy-dependent mechanisms. One such process which has been well characterized in erythrocytes is a plasma membrane calcium extrusion pump whose enzymatic basis is a high affinity (Ca+2 + Mg+2)ATPase. A similar mechanism regulated by insulin has recently been identified in adipocyte plasma membranes. We report here the presence of a high affinity (Ca+2 + Mg+2)ATPase and ATP-dependent calmodulin-stimulated calcium transport system in rat pancreatic islet cell plasma membranes.     

β2-Adrenoceptor affinity chromatography and its application in the screening of the active compounds from Semen Armeniacae Amarum

β2-Adrenoceptor （β2-AR） was purified from the rabbit lung tissue by sepherose-salbutamol affinity chromatographic column. To prepare the β2-AR stationery phase, β2-AR was evenly immobilized on the surface of macro-pore silica with e mild chemical coupling method through covalent bond. The retention properties of β2-AR stationery phase were characterized by four ligends, selbutamol sulfate, noredreneline bitartrete, adrenaline hydrochloride end proprenolol hydrochloride, to establish the β2-AR affinity chromatography. Then, the method was used to screen the active compounds from the total extracts of Semen Armeniacae Amarum. The results showed that β2-AR on the surface of the stationary phase could keep its original bioectivity end selectivity. Amygdalin retained in the chromatographic column was proved to be the active compound of the total extracts of Semen Armeniacae Amarum. Compared with the existing chromatographic screening approaches, this method showed e good stability end high selectivity. The active compounds which could interact with β2-AR in traditional Chinese medicine （TCM） could be screened efficiently by this method, providing e new way to screen the active compounds in complicated samples such as TCM.     

Teratogenic drugs inhibit tumour cell attachment to lectin-coated surfaces

Interactions between embryonic cells are generally thought to have a central role in the control of development. When these morphogenic interactions are interrupted by either physical intervention or genetic defects, normal development is impaired. In accord with these experiments, specific interactions between embryonic cells have been demonstrated in several in vitro systems. Many investigators have described homotypic aggregation of chick embryo cells, and heterotypic specificity has been described. Because of the importance of morphogenic cell-cell interactions in development it follows that agents that interfere with these interactions, regardless of the interference mechanism, are potential teratogens. Here we have used a simple in vitro cell to surface recognition system in an attempt to screen for potential teratogens. We have found a very high correlation between inhibitory activity in the in vitro assay and reported teratogenic activity in human or animal studies. This suggests that many teratogenic agents may act by interfering, in an as yet unknown way, in normal cell to cell interactions.     

Galactosyltransferase and membrane glycoprotein abnormality in human platelets from Tn-syndrome donors.

Early studies on the analysis of membranes isolated from the erythrocytes of Tn-patients by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a severe reduction in the staining capacity of glycophorin with the periodate-Schiff (PAS) reaction. A low sialic acid and galactose (Gal) content of the polyagglutinable red cells was confirmed while it was reported that the abnormal red cells of Tn-patients contained little or no UDPGal: GalNAc-beta-3-D-galactosyltransferase (T-transferase) activity. The glycoprotein (GP) abnormality in Tn-erythrocytes appeared to be due to incomplete synthesis of the alkali-labile oligosaccharide chaims of glycophorin. We now report studies on the membrane GP composition and the T-transferase activity of platelets isolated from there Tn-syndrome patients whose red cell membranes contain GP abnormalities which are typical of those found in this rare clinical condition.     

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.     

Altered Gs and adenylate cyclase activity in human GH-secreting pituitary adenomas

Gs and Gi are guanine nucleotide-binding, heterotrimer proteins that regulate the activity of adenylate cyclase, and are responsible for transferring stimulatory and inhibitory hormonal signals, respectively, from cell surface receptors to the enzyme catalytic unit. These proteins can be directly activated by agents such as GTP and analogues, fluoride and magnesium. Decreased amounts of Gs and Gi, and even the absence of Gs, have been described, whereas an altered Gs has been reported in a cultured cell line (UNC variant of S49 lymphoma cells), but has never been observed in human disease states. We have found a profoundly altered Gs protein in a group of human growth hormone-secreting pituitary adenomas, characterized by high secretory activity and intracellular cyclic AMP levels. In the membranes from these tumours no stimulation of adenylate cyclase activity by growth hormone-releasing hormone, by GTP or by fluoride was observed. Indeed, the last two agents caused an inhibition, probably mediated by Gi. In contrast, adenylate cyclase stimulation by Mg2+ was enormously increased. This altered pattern of adenylate cyclase regulation was reproduced when a cholate extract of the tumour membranes (which contains G proteins) was reconstituted with Gs-free, cyc- S49 cell membranes. Inasmuch as secretion from somatotrophic cells is known to be a cAMP-dependent function, the alteration of Gs could be the direct cause of the high secretory activity of the tumours in which it occurs.     

中药方剂对家兔注射FMD疫苗部分生理指标影响试验

对家兔注射FMD疫苗前后,服用中草药制剂,观察其对部分生理指标影响。试验选取健康家兔16只,随机分为4组;A组:阴性对照组;B组:每只家兔肌注苗0.5mL,不口服中药制剂;C组:每兔注苗0.5mL,30min后口服中药剂量5mL;D组:每只家兔注苗0.5mL,注射前1h口服10mL中药制剂,观察不同时间段家兔血液部分生理指标的变化。实验结果表明:注射FMD疫苗能引起家兔的白细胞、红细胞、淋巴细胞、粒细胞数量显著增加;心率、血压下降。使用僵蚕、蚕蜕等中药,可抑制试验组家兔的白细胞、粒细胞、淋巴细胞数量;心率、血压有所上升。     

聚乙烯醇静电纺丝法固定葡萄糖氧化酶

利用静电纺丝纳米纤维具有高比表面积和多孔疏松结构的优势固定葡萄糖氧化酶，以提高酶电极的性能．通过聚乙烯醇（PVA）和葡萄糖氧化酶（GOD）共同静电纺丝的方法在金电极表面获得了固定化酶膜，用于构筑安培型葡萄糖生物传感器，膜的红外光谱、紫外-可见光谱和扫描电镜的分析均表明酶成功固定在静电纺丝形成的纳米纤维膜中．循环伏安测试表明固定化酶在静电纺丝纳米纤维中保持了活性，采用PVA静电纺丝法固定COD比利用浇铸膜法所得到的酶修饰电极对葡萄糖有更好的电流响应特性，通过在静电纺丝溶液中加入纳米金进一步提高了酶电极的电化学响应特性．     

乳酸菌内化小鼠小肠派伊尔结特性及其影响因素

为了探索乳酸菌表面物理化学性质和红细胞凝集性与其内化派伊尔结(PP)之间的关系,分别用细菌黏附正十六烷法(BATH)、ε电位仪、接触角和液体表面张力测定疏水性、细胞表面电荷和表面自由能;并用兔红细胞凝集活性评价乳酸菌表面凝集素表达性质;3H标记的乳酸菌,分析其在PP内化的能力,并在体内分析了3H标记的三株乳杆菌体内内化于PP的性质。体外试验证明,22株乳酸菌体外内化PP的性质具有显著的菌株特异性;细菌的疏水性和内化PP的能力没有相关性;而细菌的表面自由能和内化PP的能力呈负相关趋势。选用的4株乳酸菌在体内内化PP的能力也和体外的结果一致。乳酸菌内化PP的能力具有菌种依赖性。细菌表面电荷、疏水性和红细胞凝集能力与内化能力之间没有相关关系,但细菌的表面自由能与内化能力呈负相关趋势。     

Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension

Lipid bilayer membranes--ubiquitous in biological systems and closely associated with cell function--exhibit rich shape-transition behaviour, including bud formation and vesicle fission. Membranes formed from multiple lipid components can laterally separate into coexisting liquid phases, or domains, with distinct compositions. This process, which may resemble raft formation in cell membranes, has been directly observed in giant unilamellar vesicles. Detailed theoretical frameworks link the elasticity of domains and their boundary properties to the shape adopted by membranes and the formation of particular domain patterns, but it has been difficult to experimentally probe and validate these theories. Here we show that high-resolution fluorescence imaging using two dyes preferentially labelling different fluid phases directly provides a correlation between domain composition and local membrane curvature. Using freely suspended membranes of giant unilamellar vesicles, we are able to optically resolve curvature and line tension interactions of circular, stripe and ring domains. We observe long-range domain ordering in the form of locally parallel stripes and hexagonal arrays of circular domains, curvature-dependent domain sorting, and membrane fission into separate vesicles at domain boundaries. By analysing our observations using available membrane theory, we are able to provide experimental estimates of boundary tension between fluid bilayer domains.     

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.     

A novel nonviral nanoparticle gene vector: Poly-L-lysine-silica nanoparticles

DNA delivery is a core technology for gene structure and function research as well as clinical settings. The ability to safely and efficiently targeted transfer foreign DNA into cells is a fundamental goal in biotechnology. With the development of nanobiotechnology, nanoparticle gene vectors brought about new hope to reach the goal. In our research, silica nanoparticles (SiNP) were synthesized first in a microemulsion system polyoxyethylene nonylphenyl ether (OP-10)/cyclohexane/ammonium hydroxide, at the same time the effects of SiNP size and its distribution were elucidated by orthogonal analysis; then poly-L-lysine (PLL) was linked on the surface of SiNP by nanoparticle surface energy and electrostatically binding; lastly a novel complex nanomateial—poly-L-lysine-silica nanoparticles (PLL-SiNP) was prepared. The analysis of plasmid DNA binding and DNase I enzymatic degradation discovered that PLL-SiNP could bind DNA, and protect it against enzymatic degradation. Cell transfection showed that PLL-SiNP could efficiently transfer PEGFPC-2 plasmid DNA into HNE1 cell line. These results indicated that PLL-SiNP was a novel nonviral nanoparticle gene vector, and would probably play an important role in gene structure and function research as well as gene therapy.