聚乙二醇修饰牛血清白蛋白的反应与分析

采用N,N′-羰基二咪唑活化法活化单甲氧基聚乙二醇5000分子一端的羟基,对活化后的单甲氧基聚乙二醇分子进行了元素分析。用该活化产物对牛血清白蛋白的赖氨酸侧链氨基进行化学修饰。应用毛细管电泳对聚乙二醇修饰后的产物进行了分析,并与高效液相色谱分析结果作了对照研究,表明毛细管电泳对修饰后的牛血清白蛋白有更好的分析效果。     

A radioimmunoassay of plasma estradiol-17beta with the use of polyethylene glycol to separate free and antibody-bound hormone

A radioimmunoassay for plasma estradiol-17β was developed using polyethylene glycol to separate free from antibody-bound hormone. Specificity for estradiol-17β was achieved by a modified celite microcolunm procedure in which estradiol was.separated from interfering estrogens, including estrone. Using trace ³H-estradiol to monitor procedural losses, the method was shown to be sensitive and accurate. Intra- and inter-assay coefficient of variation of the method was 8.7 and 10.6%, respectively. Polyethylene glycol used for antibody precipitation appears to be a generally applicable method for steroid hormone radioimmunoassays. The simplicity, precision and rapid analysis, coupled with its lack of time dependence and ease in automation, makes this a convenient and practical method.     

Hyaluronic acid salt — a mechanoelectrical transducer

Hyaluronic acid transduces a very gentle pressure into an electrical potential. Such pressure, depending on its direction, changes the optical rotary dispersion properties of the salt, either increasing the rotation in the direction already shown by the unpressured salt or changing and increasing the rotation in the opposite direction. These findings have implications for understanding the funtion of the cochlear and vestibular fluids, renal function, and the approximation to frictionless motion of normal joints.     

Enzymatic and Hybridization Properties of Oligonucleotide Analogs Containing Novel Phosphoramidate Internucleotide Linkages

In line with the paradigm, that antisense oligonucleotides should contain minimal structural modifications, in order to minimize the risk of toxicity and antigenicity, we describe here the preparation and the properties of oligonucleotides modified to contain, in addition to phosphodiester bonds, a small number of phosphoramidate internucleotide linkages substituted with aminoethoxyethyl groups in order to convey protection against exo‐ and endonucleases. Prolonged stability was, in fact, found in model experiments with respective enzymes, as well as in studies done in human blood serum. Regardless of number and position of phosphoramidate linkages, the modified oligonucleotides showed only a slight decrease of T_m in hybridization studies with complementary oligonucleotides.     

Density Gradient Multilayered Polymerization (DGMP): A Novel Technique for Creating Multi-compartment,Customizable Scaffolds for Tissue Engineering

Complex tissue culture matrices, in which types and concentrations of biological stimuli (e.g. growth factors, inhibitors, or small molecules) or matrix structure (e.g. composition, concentration, or stiffness of the matrix) vary over space, would enable a wide range of investigations concerning how these variables affect cell differentiation, migration, and other phenomena. The major challenge in creating layered matrices is maintaining the structural integrity of layer interfaces without diffusion of individual components from each layer¹. Current methodologies to achieve this include photopatterning^2-3, lithography⁴, sequential functionalization5, freeze drying⁶, microfluidics⁷, or centrifugation⁸, many of which require sophisticated instrumentation and technical skills. Others rely on sequential attachment of individual layers, which may lead to delamination of layers⁹. DGMP overcomes these issues by using an inert density modifier such as iodixanol to create layers of varying densities¹⁰. Since the density modifier can be mixed with any prepolymer or bioactive molecule, DGMP allows each scaffold layer to be customized. Simply varying the concentration of the density modifier prevents mixing of adjacent layers while they remain aqueous. Subsequent single step polymerization gives rise to a structurally continuous multilayered scaffold, in which each layer has distinct chemical and mechanical properties. The density modifier can be easily removed with sufficient rinsing without perturbation of the individual layers or their components. This technique is therefore well suited for creating hydrogels of various sizes, shapes, and materials.A protocol for fabricating a 2D-polyethylene glycol (PEG) gel, in which alternating layers incorporate RGDS-350, is outlined below. We use PEG because it is biocompatible and inert. RGDS, a cell adhesion peptide¹¹, is used to demonstrate spatial restriction of a biological cue, and the conjugation of a fluorophore (Alexa Fluor 350) enables us to visually distinguish various layers. This procedure can be adapted for other materials (e.g. collagen, hyaluronan, etc.) and can be extended to fabricate 3D gels with some modifications¹⁰.     

Mimicking the germinal center reaction in hybridoma cells to isolate temperature-selective anti-PEG antibodies

Modification of antibody class and binding properties typically requires cloning of antibody genes, antibody library construction, phage or yeast display and recombinant antibody expression. Here, we describe an alternative “cloning-free” approach to generate antibodies with altered antigen-binding and heavy chain isotype by mimicking the germinal center reaction in antibody-secreting hybridoma cells. This was accomplished by lentiviral transduction and controllable expression of activation-induced cytidine deaminase (AID) to generate somatic hypermutation and class switch recombination in antibody genes coupled with high-throughput fluorescence-activated cell sorting (FACS) of hybridoma cells to detect altered antibody binding properties. Starting from a single established hybridoma clone, we isolated mutated antibodies that bind to a low-temperature structure of polyethylene glycol (PEG), a polymer widely used in nanotechnology, biotechnology and pharmaceuticals. FACS of AID-infected hybridoma cells also facilitated rapid identification of class switched variants of monoclonal IgM to monoclonal IgG. Mimicking the germinal center reaction in hybridoma cells may offer a general method to identify and isolate antibodies with altered binding properties and class-switched heavy chains without the need to carry out DNA library construction, antibody engineering and recombinant protein expression.     

Modified Method for Purifying Rat Liver Branched-Chain α-Ketoacid Dehydrogenase Complex

Branched-chain α-ketoacid dehydrogenase complex (BCKDC) is a rate-limiting enzyme in the branched-chain amino acid catabolic pathway. We have developed a method of BCKDC purification from rat liver using hydrophobic interaction column chromatography (Shimomura et al., Arch. Biochem. Biophys., 283, 293–299 (1990)). Here we report a modification of the method designed to obtain the purified enzyme with high reproducibility.     

Intermolecular interactions during ultrafiltration of pegylated proteins

Recent studies have demonstrated the feasibility of using membrane ultrafiltration for the purification of pegylated proteins; however, the separations have all been performed at relatively low protein concentrations where intermolecular interactions are unimportant. The objective of this study was to examine the behavior at higher PEG concentrations and to develop an appropriate theoretical framework to describe the effects of intermolecular interactions. Ultrafiltration experiments were performed using pegylated α‐lactalbumin as a model protein with both neutral and charged composite regenerated cellulose membranes. The transmission of the pegylated α‐lactalbumin, PEG, and α‐lactalbumin all increase with increasing PEG concentration due to the increase in the solute partition coefficient arising from unfavorable intermolecular interactions in the bulk solution. The experimental results were in good agreement with a simple model that accounts for the change in Gibbs free energy associated with these intermolecular interactions, including the effects of concentration polarization on the local solute concentrations upstream of the membrane. These intermolecular interactions are shown to cause a greater than expected loss of pegylated product in a batch ultrafiltration system, and they alter the yield and purification factor that can be achieved during a diafiltration process to remove unreacted PEG. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:655–663, 2013     

New insights into the binding mode of pyridine-3-carboxamide inhibitors of E. coli DNA gyrase

Previously we have reported on a series of pyridine-3-carboxamide inhibitors of DNA gyrase and DNA topoisomerase IV that were designed using a computational de novo design approach and which showed promising antibacterial properties. Herein we describe the synthesis of additional examples from this series aimed specifically at DNA gyrase, along with crystal structures confirming the predicted mode of binding and in vitro ADME data which describe the drug-likeness of these compounds.     

Escherichia coli AspP activity is enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars

Escherichia coli ADP-sugar pyrophosphatase (AspP) is a "Nudix" hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to glycogen biosynthesis. Moderate increases of AspP activity in the cell are accompanied by significant reductions of the glycogen content. In vitro analyses showed that AspP activity is strongly enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars, providing a first set of indicative evidences that AspP is a highly regulated enzyme. To our knowledge, AspP is the sole bacterial enzyme described to date which is activated by both G1,6P(2) and nucleotide-sugars.