Application of heparinized cellulose matrices for substrate-mediated bFGF peptide and transgene delivery to stimulate cellular proliferation

We investigated the use of heparinized cellulose matrices (H-CM) as affinity substrates for binding of basic fibroblast growth factor (bFGF), a heparin-binding peptide, to facilitate cellular proliferation and substrate-mediated transgene delivery. Using human HT-1080 fibroblasts and Saos-2 osteoblasts as cellular models, we showed that H-CM was a friendly substrate for cellular adhesion. Once adhered, cells received stimulation from the bound bFGF, leading to enhanced proliferation. Furthermore, taking advantage of the negative zeta potential of H-CM, we applied electrostatic adsorption to immobilize cationic poly-ethylenimine/DNA polyplexes onto the surface for transgene delivery upon cellular adhesion. Because bFGF stimulated cellular proliferation, we observed a significant increase in transfection efficiency in comparison to transfection on H-CM without the bFGF binding. We showed that H-CM was capable of mediating both bFGF peptide and bFGF transgene delivery to induce a synergistic stimulation of cellular proliferation, thus offering a useful device for fabrication of tissue scaffolds.     

两种固定化金属螯合复合亲和膜色谱介质制备

以纤维素滤纸为基质,通过碱处理、环氧活化、偶联亚氨基二乙酸二钠、固定化Ｃｕ＾２＋后制得了大孔纤维素亲和膜。另外,在活化后的膜上通过共价交联覆盖上琼脂糖,制得了具有类似“三明治”结构且性能优于的复合亲和膜,装柱后分别制得固定化金属螯合亲和膜色谱柱。对两种亲和膜进行牛血清白蛋白等温吸附测定显示,两者的最大吸附量分别为１．１７ｍｇ／ｃｍ＾２和１．３０ｍｇ／ｃｍ＾２,与传统的琼脂糖凝胶类介质吸附量相当,表     

Affinity enrichment of plasma membrane for proteomics analysis

Proteomics analysis of plasma membranes from cells exposed to different extracellular environments is potentially a powerful approach for the identification of membrane-associated proteins responding to these environments. Preparation of high concentration plasma membrane fractions with low contamination from cellular organelles is essential for such studies. Here, we describe an affinity enrichment method, which combines cell surface biotinylation with affinity enrichment by immobilized streptavidin beads, for the isolation of plasma membranes. This method results in a 400-fold enrichment of plasma membrane relative to endoplasmic reticulum, a major contaminant in standard plasma membrane preparations, and dramatically reduces contamination from other cellular organelles. The biotinylation reaction did not interfere with ligand-dependent activation of receptor tyrosine kinases or G-protein coupled receptors, suggesting cell-surface signal transduction machinery remains functional. Membrane fractions prepared by this method should provide excellent starting materials for membrane proteomics analysis such as studies of dynamic trafficking and regulation of signaling molecules or identification of disease-specific membrane markers.     

等离子体聚合法制备膜蒸馏用疏水微孔复合膜

本文以八氟环丁烷为单体,采用等离子体聚合法将亲水性硝酸纤维素微孔膜改性,制得疏水硝酸纤维素微孔复合膜。所得疏水微孔复合膜可用于膜蒸馏,并具有优良的膜蒸馏性能,其通量达到反渗透水平。利用扫描电镜、X-射线显微分析和XPS等分析手段研究了聚合条件对所得复合膜结构性能的影响.     

Visualization of the turkey erythrocyte beta-adrenergic receptor

We have recently described the affinity chromatography purification of the turkey erythrocyte beta-adrenergic receptor. The minute amounts obtained initially precluded extensive biochemical characterization. To improve the yield of the receptor, the erythrocyte membranes have been prepared by a new method. This procedure resulted in a 10-fold higher receptor density in comparison with the membrane preparation used previously. The new membranes also contained a catecholamine-sensitive guanine triphosphatase and an adenylate cyclase sensitive to Gpp(NH)p and l-epinephrine. Solubilization by a double digitonin extraction resulted in a preparation containing 4-6 pmoles of 3H-dihydroalprenolol binding sites per mg of membrane protein. A single step of affinity chromatography on alprenolol-sepharose of the soluble digitonin extract resulted in an additional 1,000-fold purification of the receptor. The overall purification factor was 20,000 relative to the binding activity of the crude membrane preparations. Electrophoresis is SDS-polyacrylamide of iodinated purified beta-receptors revealed, after autoradiography, the presence of four major components. Three of these, corresponding to molecular weights of 170,000, 33,000, and 30,000, respectively, were not affected by reduction with beta-mercaptoethanol and were not observed when the digitonin extracts were loaded on the affinity gel in the presence of an excess of l-propranolol. A fourth 52,000-dalton component (60,000 daltons after reduction with beta-mercaptoethanol) remained apparent even when affinity purification was prevented by addition of l-propranolol. Our results suggest that the beta-adrenergic receptor is composed of at least three subunits that interact by noncovalent bonds.     

Structure and microporous formation of cellulose/silk fibroin blend membranes: Part II. Effect of post-treatment by alkali

Blend membranes (RCF1) were prepared from mixture solution of cellulose and silk fibroin (SF) in cuoxam by coagulating with acetone–acetic acid (4:1 by volume). The blend membranes were subjected to post-treatment with 10% NaOH aqueous solution, and their structure and properties were characterized by FT-IR, X-ray diffraction, DSC, SEM and DMTA. In previous work, cellulose/SF blend membranes (RCF2) prepared by coagulating with 10% NaOH aqueous solution formed a microporous structure, in which the SF as a pore former was almost completely removed from the membrane. However, when the blend membranes RCF1 were immersed in 10% NaOH aqueous solution for post-treatment, a strong hydrogen bonding between cellulose and SF inhibited the removal of SF. Although alkali is a good solvent for SF, the blend membranes RCF1 such obtained from cellulose and SF were alkali resistant. The crystallinity and the mean pore size of the blend membranes slightly decreased with increasing post-treatment time. This work provided a cellulose/silk blend membrane, which can be used under alkaline medium.     

用新型尼龙亲和膜纯化人血浆蛋白

 以微孔尼龙膜为基质 ,分别采用三氯三嗪和 1,4 丁二醇二甘油醚活化法制备了一系列亲和膜。首次使用平板亲和膜方法经一步操作即从人血浆中纯化得到高纯度的γ 球蛋白、纤溶酶原。电泳分析显示 ,纯化得到的人血浆γ 球蛋白 ,纯度在 83%以上 ;纯化得到的γ 球蛋白和纤溶酶原的纯度均高于市售同类产品 ;另外 ,初步探索了人血浆凝血酶的一步纯化法。该法纯化得到的凝血酶比活为 42 0NIH单位 /mg～ 42 5NIH单位 /mg 。     

Synthesis of an artificial cell surface receptor that enables oligohistidine affinity tags to function as metal-dependent cell-penetrating peptides

Cell-penetrating peptides and proteins (CPPs) are important tools for the delivery of impermeable molecules into living mammalian cells. To enable these cells to internalize proteins fused to common oligohistidine affinity tags, we synthesized an artificial cell surface receptor comprising an N-alkyl derivative of 3beta-cholesterylamine linked to the metal chelator nitrilotriacetic acid (NTA). This synthetic receptor inserts into cellular plasma membranes, projects NTA headgroups from the cell surface, and rapidly cycles between the plasma membrane and intracellular endosomes. Jurkat lymphocytes treated with the synthetic receptor (10 microM) for 1 h displayed approximately 8,400,000 [corrected]NTA groups on the cell surface. Subsequent addition of the green fluorescent protein AcGFP fused to hexahistidine or decahistidine peptides (3 microM) and Ni(OAc)(2) (100 microM) enhanced the endocytosis of AcGFP by 150-fold (hexahistidine fusion protein) or 600-fold (decahistidine fusion protein) within 4 h at 37 degrees C. No adverse effects on cellular proliferation or morphology were observed under these conditions. By enabling common oligohistidine affinity tags to function as cell-penetrating peptides, this metal-chelating cell surface receptor provides a useful tool for studies of cellular biology [corrected]     

Activation, exposure and penetration of virally encoded, membrane-active polypeptides during non-enveloped virus entry

Host cell entry by influenza and other enveloped viruses is well characterized, however, the manner in which non-enveloped viruses deliver their genome across host cell membranes in the absence of membrane fusion remains unresolved. The discovery of short, membrane altering, amphipathic or hydrophobic sequences in several non-enveloped virus capsid proteins such as the gamma (gamma) peptide of nodaviruses and tetraviruses, VP4 and the N-terminal region of VP1 of picornaviruses, micro1N of reoviruses, and protein VI of adenoviruses suggests that these small peptides facilitate breaching of the host membrane and the delivery of the viral genome into the host cell. In spite of conspicuous differences in entry among non-enveloped virions, the short stretches of membrane active regions are associated with similar, entry-related events including: I) proteolytic cleavage of a precursor capsid protein resulting in increased dynamic character and/or accessibility of these peptides; II) structural changes in the virus capsid triggered by receptor binding and/or low pH in entry compartments, resulting in peptide exposure; III) externalized peptides interact with host membranes and disrupt them, facilitating delivery of the viral genome inside the host cell. Here we discuss the membrane alteration activity in non-enveloped viruses with reference to the gamma peptide of nodaviruses. Virtually all of the characteristics of gamma are shared by analogous peptides in other non-enveloped viruses, making it a simple prototype for comparative purposes.     

金属螯合亲和膜吸附分离与纯化溶菌酶的研究(Ⅱ)──不同金属螯合亲和膜对溶菌酶的吸附性能

采用低温氧或氨等离子体法改性聚丙烯微孔膜,基于等离子体改性膜的活化、偶联及螯合过程的机理,制备了Fe³⁺,Ni²⁺,Cu²⁺和Zn²⁺等金属离子螯合亲和膜,并用于溶菌酶的吸-脱附实验.实验结果表明,Ni²⁺和Cu²⁺离子螯合亲和膜对溶菌酶具有较高的吸附量,螯合过程中采用氯化物盐溶液制得的膜对溶菌酶吸附量比采用硫酸盐溶液制得的膜的吸附量高.两种膜的重复吸-脱附性能相近,而Fe³⁺螯合亲和膜基本上不能用于重复吸-脱附实验.采用补充金属螯合离子,能部分恢复亲和膜对溶菌酶的吸附量,是实现亲和膜重复使用的有效方法.     

Preparation,properties, and application of polypropylene micro/nanofiber membranes

Nanofiber membranes have huge potential applications in many areas due to their unique properties. However, the thermoplastic micro/nanofiber membranes were rarely reported. In this paper, polypropylene (PP) nanofibers were prepared by melt extrusion of immiscible blends of PP, cellulose acetate butyrate (CAB), and subsequent removal of the CAB matrix. The wet‐laid application was used to make PP nanofiber membranes and PP‐g‐MAH/nonwoven micro/nanofiber membrane. The properties of membranes including morphology, apparent density, porosity, contact‐angle, pore size distribution, and water flux were characterized. The results showed that the consequent membranes were provided with optimistic porosity and pore size distribution. Moreover, they were all with high pure water fluxes, which were superior to that of PP microporous membrane. They performed an excellent separation performance of TiO₂ suspension and dyeing wastewater. The work revealed this method could be an efficient one to make thermoplastic polymer micro/nanofiber membranes, and they would have a brilliant potential application for water treatment. Copyright © 2010 John Wiley & Sons, Ltd.     

Intermolecular Communication on a Liposomal Membrane: Enzymatic Amplification of a Photonic Signal with a Gemini Peptide Lipid as a Membrane‐Bound Artificial Receptor

A supramolecular system that can activate an enzyme through photo‐isomerization was constructed by using a liposomal membrane scaffold. The design of the system was inspired by natural signal transduction systems, in which enzymes amplify external signals to control signal transduction pathways. The liposomal membrane, which provided a scaffold for the system, was prepared by self‐assembly of a photoresponsive receptor and a cationic synthetic lipid. NADH‐dependent L ‐lactate dehydrogenase, the signal amplifier, was immobilized on the liposomal surface by electrostatic interactions. Recognition of photonic signals by the membrane‐bound receptor induced photo‐isomerization, which significantly altered the receptor’s metal‐binding affinity. The response to the photonic signal was transmitted to the enzyme by Cu²⁺ ions. The enzyme amplified the chemical information through a catalytic reaction to generate the intended output signal.     

Crosslinked chitosan composite membrane for the pervaporation dehydration of alcohol mixtures and enhancement of structural stability of chitosan/polysulfone composite membranes

Chitosan composite membranes having a microporous polysulfone substrate were prepared and tested for the pervaporation dehydration of aqueous isopropanol mixtures. When the composite membrane experienced excessive swelling at the feed mixture of high water content, the composite membranes were found to be segregated in structure due to the opposite characteristics to water of chitosan and polysulfone. Efforts to enhance the structural stability under various pervaporation operational conditions were made. The polysulfone substrate was immersed into hydrophilic binding polymer solutions such as polyvinyl alcohol, polyacrylic acid, and hydroxyethylcellulose before the casting of chitosan layer to increase the affinity between the thin chitosan layer and porous polysulfone layer which resulted in increased geometrical stability of the chitosan/polysulfone composite membranes. The chitosan layer was crosslinked with glutaraldehyde and H₂SO₄ in acetone solution to control the permselectivity.     

Estrogen-derived steroidal metal complexes: agents for cellular delivery of metal centers to estrogen receptor-positive cells

Targeted cellular delivery of drugs to specific tissues is an important goal in biomedical chemistry. Achieving this requires harnessing and applying molecular-level recognition events prevalent in (or specific to) the desired tissue type. Tissues rich in estrogen receptors (ERs), which include many types of breast cancer, accumulate molecules that have high binding affinities for these receptors. Therefore, molecules that (i) bind to the ER, (ii) have favorable cellular transport properties, and (iii) contain a second functionality (such as a center that may be used for diagnostic imaging or medical therapy) are exciting synthetic targets in the field of drug delivery. To this end, we have prepared a range of metallo-estrogens based on 17alpha-ethynylestradiol and examined their binding to the ER both as isolated receptor and in whole cell assays (ER positive MCF-7 cells). Estrogens functionalized with metal binding units are prepared by palladium-catalyzed cross-coupling reactions and a wide range of metal centers introduced readily. All the compounds prepared and tested exhibit effective binding to the estrogen receptor and are delivered across the cell membrane into MCF-7 cells. In the whole cell assays, despite their monocationic nature, the palladium and platinum complexes prepared exhibit similar (and even enhanced) receptor binding affinities compared to their corresponding neutral free ligands. It is unprecedented for a higher ER binding affinity to be observed for a cationic complex than for its metal-free ligand.     

Enhanced stability designs of cell membrane chromatography for screening drug leads

Cell membrane chromatography is an effective method for screening bioactive components acting on specific receptors in complex systems, which maintains the biological activity of the membrane receptors and improves screening efficiency. However, traditional cell membrane chromatography suffers from poor stability, resulting in a limited life span and low reproducibility, greatly limiting the application of this method. To address this problem, cyanuric chloride-decorated silica gel was used for the covalent immobilization of the cell membranes. Cyanuric chloride reacts with amino groups on the cell membranes and membrane receptors to form covalent bonds. In this way, the cell membranes are not easy to fall off. The column life of the cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography column was extended to more than 8 days, whereas the column life of the normal cell membrane chromatography column dropped sharply in the first 3 days. A cyanuric chloride-decorated epidermal growth factor receptor/cell membrane chromatography online HPLC-IT-TOF-MSn system was applied for screening drug leads from Trifolium pratense L. One potential drug lead, formononetin, which acts on the epidermal growth factor receptor, was screened. Our strategy of covalently immobilizing cell membrane receptors also improved the stability of cell membrane chromatography.     

Fractionation of human plasma proteins by hydrophobic interaction membrane chromatography

This paper discusses the fractionation of human plasma proteins HSA and HIgG by hydrophobic interaction membrane chromatography. A type of microporous polyvinylidine fluoride (PVDF) membrane having 0.1 μm pore size was identified as being suitable for carrying out this separation. This membrane bound HIgG at 1.5 M ammonium sulphate concentration, a condition at which HSA did not. Based on this selective binding resulting from the selective pressure induced by the high anti-chaotropic salt concentration, these human plasma proteins were fractionated. The HIgG binding capacity of the PVDF membrane examined in this study was 42.8 mg/ml at a feed concentration of 0.45 mg/ml. Separation of simulated HSA/HIgG mixtures were carried out in the pulse and step input modes and the HSA and HIgG fractions thus obtained were analysed for purity using affinity chromatography and SDS-PAGE. HSA and HIgG purities were typically in excess of 97–98%.     

Synthesis of Pd particle-deposited microporous silica membranes via a vacuum-impregnation method and their gas permeation behavior

Pd particle-deposited microporous silica membranes were synthesized to improve hydrogen permselectivity of the microporous silica membrane and to overcome high cost of palladium and crack formation through hydrogen embrittlement. Pd particles below 400 nm in diameter were readily deposited on the microporous silica membrane via a vacuum-impregnation method by using a Pd(C(3)H(5))(C(5)H(5)) precursor. After deposition of Pd particles on the microporous silica membrane, hydrogen permselectivity over nitrogen considerably increased from 11-28 to 30-115 in a permeation temperature range of 25-350 degrees C due to plugging membrane defects and hydrogen adsorption diffusion through the interface between the Pd and silica layer. The activation energy of the Pd-deposited silica membrane (6.32 kJ mol(-1)) was higher than that of the microporous silica membrane (4.22 kJ mol(-1)). In addition, the Pd-particle deposition led to an increase in the permselectivity of He and CO(2) with little chemical affinity for the Pd particles, which indicates that Pd-particle deposition gives the effect of plugging defects such as pinholes or cracks, which could be formed during the membrane preparation. Therefore it is demonstrated that Pd-particle deposition on the silica membrane is effective for induction of the hydrogen adsorption diffusion and plugging membrane defects.     

Membrane-based receptor affinity chromatography.

Membrane-based receptor affinity chromatography (MRAC), which utilizes the molecular recognition between an immobilized receptor and its soluble protein ligand, has been developed for the purification of human interleukin-2 and related biomolecules. The multi-purpose affinity membrane used in this study consisted of a soluble form of interleukin-2 receptor (IL-2R) chemically bonded to hollow-fiber membranes in an oriented fashion. A model system involving anti-Tac-H (a humanized monoclonal antibody to IL-2R) was used to study the important factors influencing the performance of MRAC, including support morphology, mass transfer rate and adsorption kinetics. All three are shown to be highly efficient. MRAC has been successfully applied to the purification of anti-Tac-H, recombinant human interleukin-2 (rIL-2) and interleukin 2-Pseudomonas exotoxin fusion protein (IL2-PE40). Overall, MRAC was found to be a viable, scalable and extremely productive affinity purification method.     

Characterization of a mini membrane inlet mass spectrometer for on-site detection of contaminants in both aqueous and liquid organic samples

A mini membrane inlet mass spectrometer (mini-MIMS) of a total weight of 12 kg was constructed using a miniature Multipole mass spectrometer, a small vacuum system and a flexible flat sheet membrane inlet, where the exposed membrane area can be changed by a factor of 80. The variable membrane area together with the possibility of operating the Multipole at pressures up to 1 x 10(-3) Torr made it possible to test the system with three microporous membranes (cellulose, polyether sulfone and polypropylene) normally not compatible with standard electron ionization MIMS systems and a standard non-porous polydimethylsiloxane membrane. We found that the hydrophilic cellulose and polyether sulfone membranes had selectivity characteristics opposite to those of the standard silicone membrane. They demonstrated preferential detection of hydrophilic compounds in hydrophobic organic solvents, whereas the silicone membrane preferentially detects hydrophobic organic compounds in aqueous solution. Using the cellulose membrane, organic contaminants and water could be detected in organic solvents at 10-100 ppm levels by weight, the relative high detection limits primarily caused by interference from a high chemical background from the solvent. When being used with the standard silicone membrane the mini-MIMS behaved just like most standard MIMS systems with detection limits of volatile organic compounds in water at concentrations just below 1 ppm. The hydrophobic microporous polypropylene membrane was not found to be useful with the mini-MIMS.