EL转染试剂转染食管鳞癌细胞的条件优化

该文探讨了关于EL转染试剂转染Hsa-miR-6743质粒至食管鳞癌细胞转染效果的影响因素.以食管鳞癌细胞株Eca-109、TE-1和Eca-9706为研究对象,GFP标记的Hsa-miR-6743为报告基因,通过倒置荧光显微镜检测荧光信号优化转染试剂和质粒比值.结果表明,食管鳞癌细胞的种类影响EL转染试剂的转染效果,...     

Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels

Ralstonia eutropha grows on and produces polyhydroxyalkanoates (PHAs) from fermentation acids. Acetic acid, one major organic acid from acidogenesis of organic wastes, has an inhibitory effect on the bacterium at slightly alkaline pH (6 g HAc/L at pH 8). The tolerance of R. eutropha to acetate, however, was increased significantly up to 15 g/L at the slightly alkaline pH level with high cell mass concentration. A metabolic cell model with five fluxes is proposed to depict the detoxification mechanism including mass transfer and acetyl-CoA formation of acetic acid and the formation of three final metabolic products, polyhydroxybutyrate (PHB), active biomass, and CO(2). The fluxes were measured under different conditions such as cell mass concentration, acetic acid concentration, and medium composition. The experimental results indicate that the acetate detoxification by high cell mass concentration is attributed to the increased fluxes at high extracellular acetate concentrations. The fluxes could be doubled to reduce and hence detoxify the accumulated intracellular acetate anions.     

Preparation, stability, and in vitro performance of vesicles made with diblock copolymers

Vesicles made completely from diblock copolymers-polymersomes-can be stably prepared by a wide range of techniques common to liposomes. Processes such as film rehydration, sonication, and extrusion can generate many-micron giants as well as monodisperse, approximately 100 nm vesicles of PEO-PEE (polyethyleneoxide-polyethylethylene) or PEO-PBD (polyethyleneoxide-polybutadiene). These thick-walled vesicles of polymer can encapsulate macromolecules just as liposomes can but, unlike many pure liposome systems, these polymersomes exhibit no in-surface thermal transitions and a subpopulation even survive autoclaving. Suspension in blood plasma has no immediate ill-effect on vesicle stability, and neither adhesion nor stimulation of phagocytes are apparent when giant polymersomes are held in direct, protracted contact. Proliferating cells, in addition, are unaffected when cultured for an extended time with an excess of polymersomes. The effects are consistent with the steric stabilization that PEG-lipid can impart to liposomes, but the present single-component polymersomes are far more stable mechanically and are not limited by PEG-driven micellization. The results potentiate a broad new class of technologically useful, polymer-based vesicles.     

Purification of Lac repressor protein using polymer displacement and immobilization of the protein

Lac repressor protein was purified from E. coli BMH8117 harboring plasmid pWB1000 and E. coli K₁₂BMH 71-18 strains. Displacement of the protein with poly(ethyleneimine) (PEI) from phosphocellulose cation exchange column was shown to be an effective elution strategy. It resulted in better recoveries and sharper elution profiles than traditional salt elution without effecting the purity of the protein. The elution is assumed to proceed via displacement of bound protein by PEI when the polymer binds to the ion exchanger. The minor impurities in the protein solution were finally removed by chromatography on immobilized metal affinity column. The repressor protein undergoes distinct conformational changes upon addition of specific inducer isopropyl--D-thiogalactoside (IPTG), which is evidenced by changes in ultraviolet absorption spectrum. The protein was immobilized covalently to the Sepharose matrix. The intact biological activity of the protein after immobilization was shown by binding of genomic DNA and lac operator plasmid DNA from E. coli to the immobilized lac repressor.     

Molecular Orbital Study of the Magnetic Properties of Pyrazine- and Pyrimidine-Bridged Copper(II) Complexes

Magnetic interactions in the three copper(II)-complex polymers, [Cu(PZ)(NO₃)₂]_n, [Cu(PM)(NO₃)₂(H₂O)₂]_n, and [Cu(PM)₂(NO₃)₂]_n are discussed on the basis of extended Hückel calculations inthe formulas PZ and PM stand for pyrazine and pyrimidine, respectively. Interactions between the Cu-3d orbitals and the lone-pair orbitals of pyrazine and pyrimidine are analyzed from the viewpoint of `through-space' and `through-bond' interactions using binuclear complexes to model the three copper(II) polymers. Three conclusions can be drawn from the orbital interaction analysis: (1) in the first polymer, a superexchange pathway is formed with the bond of Cu–-N and the through-bond interaction between the lone pairs of the nitrogen atoms of pyrazine will lead to an antiferromagnet state; (2) in the second polymer a superexchange pathway is formed with the bond of Cu–-N and the through-space interaction between the lone pairs of the nitrogen atoms of pyrimidine, and as a result an antiferromagnetic state will be preferred; and (3) in the third polymer., there is no effective pathway in respect of overlap interaction and the HOMO and the LUMO are actually degenerate, and thus a ferromagnetic state will arise. The band structures are analyzed to characterize the magnetic properties of the antiferromagnetic polymers, [Cu(PZ)(NO₃)₂]_n and [Cu(PM)(NO₃)₂(H₂O)₂], and the ferromagnetic polymer, [Cu(PM)₂(NO₃)₂]_n.     

Complex between α-Chymotrypsin and poly(ethylene glycol) catalytically active in organic media

-Chymotrypsin and poly(ethylene glycol) were mixed in an aqueous phosphate buffer solution and lyophilized. The preparation was readily soluble in anhydrous isooctane. The polymer/enzyme complex, i.e., poly(ethylene glycol)/-chymotrypsin, was catalytically active in anhydrous isooctane even when the molecular ratio of them was unity.     

Screening combinatorial libraries of molecularly imprinted polymer films casted on membranes in single-use membrane modules

A new and fast technique for screening combinatorial libraries of molecularly imprinted polymers is presented. The procedure is based on commercially available membrane modules which are rinsed with pre-polymerization imprinting mixtures. After the in situ polymerization and generation of MIP films on the PTFE membranes within the modules, the membranes are evaluated in terms of affinity towards the target molecule (template) R-(-)-phenylbutyric acid. Therefore, after template extraction from the freshly produced membranes a solution of this target molecule is flushed through the module. By analyzing the remaining analyte concentration in the permeate, the amount of analyte adsorbed on the membrane can be calculated and related to specific interactions with the molecular imprints. By this means, optimized recipes in terms of cross-linker to template ratios could be obtained in combination with the optimal porogen, when screening p-divinylbenzene or ethylene glycol dimethacrylate as cross-linker and porogens like acetonitrile, dimethylsulfoxide and methanol.     

Homology modeling and molecular dynamics simulations of the N-terminal domain of wheat high molecular weight glutenin subunit 10

High molecular weight glutenin subunits (HMW-GS) are of a particular interest because of their biomechanical properties, which are important in many food systems such as breadmaking. Using fold-recognition techniques, we identified a fold compatible with the N-terminal domain of HMW-GS Dy10. This fold corresponds to the one adopted by proteins belonging to the cereal inhibitor family. Starting from three known protein structures of this family as templates, we built three models for the N-terminal domain of HMW-GS Dy10. We analyzed these models, and we propose a number of hypotheses regarding the N-terminal domain properties that can be tested experimentally. In particular, we discuss two possible ways of interaction between the N-terminal domains of the y-type HMW glutenin subunits. The first way consists in the creation of interchain disulfide bridges. According to our models, we propose two plausible scenarios: (1) the existence of an intrachain disulfide bridge between cysteines 22 and 44, leaving the three other cysteines free of engaging in intermolecular bonds; and (2) the creation of two intrachain disulfide bridges (involving cysteines 22-44 and cysteines 10-55), leaving a single cysteine (45) for creating an intermolecular disulfide bridge. We discuss these scenarios in relation to contradictory experimental results. The second way, although less likely, is nevertheless worth considering. There might exist a possibility for the N-terminal domain of Dy10, Nt-Dy10, to create oligomers, because homologous cereal inhibitor proteins are known to exist as monomers, homodimers, and heterooligomers. We also discuss, in relation to the function of the cereal inhibitor proteins, the possibility that this N-terminal domain has retained similar inhibitory functions.     

Computational simulation of the statistical properties of unfolded proteins

A simple Monte Carlo method was used to generate ensembles of simulated polypeptide conformations that are restricted only by steric repulsion. The models used for these simulations were based on the sequences of four real proteins, ranging in size from 26 to 268 amino acid residues, and included all non-hydrogen atoms. Two sets of calculations were performed, one that included only intra-residue steric repulsion terms and those between adjacent residues, and one that included repulsion terms between all possible atom pairs, so as to explicitly account for the excluded volume effect. Excluded volume was found to increase the average radius of gyration of the chains by 20-40%, with the expansion factor increasing with chain length. Contrary to recent suggestions, however, the excluded volume effect did not greatly restrict the distribution of dihedral angles or favor native-like topologies. The average dimensions of the ensembles calculated with excluded volume were consistent with those measured experimentally for unfolded proteins of similar sizes under denaturing conditions, without introducing any adjustable scaling factor. The simulations also reproduced experimentally determined effective concentrations for the formation of disulfide bonds in reduced and unfolded proteins. The statistically generated ensembles included significant numbers of conformations that were nearly as compact as the corresponding native proteins, as well as many that were as accessible to solvent as a fully extended chain. On the other hand, conformations with as much buried surface area as the native proteins were very rare, as were highly extended conformations. These results suggest that the overall properties of unfolded proteins can be usefully described by a random coil model and that an unfolded polypeptide can undergo significant collapse while losing only a relatively small fraction of its conformational entropy.     

Actomyosin motility on nanostructured surfaces

We have here, for the first time, used nanofabrication techniques to reproduce aspects of the ordered actomyosin arrangement in a muscle cell. The adsorption of functional heavy meromyosin (HMM) to five different resist polymers was first assessed. One group of resists (MRL-6000.1XP and ZEP-520) consistently exhibited high quality motility of actin filaments after incubation with HMM. A second group (PMMA-200, PMMA-950, and MRI-9030) generally gave low quality of motility with only few smoothly moving filaments. Based on these findings electron beam lithography was applied to a bi-layer resist system with PMMA-950 on top of MRL-6000.1XP. Grooves (100-200nm wide) in the PMMA layer were created to expose the MRL-6000.1XP surface for adsorption of HMM and guidance of actin filament motility. This guidance was quite efficient allowing no U-turns of the filaments and approximately 20 times higher density of moving filaments in the grooves than on the surrounding PMMA.