Application of polyethyleneimine cellulose for the class separation of steroidal carboxylic acids from neutral steroids and pigments in urine

Metabolites of corticosteroids that contain the 21-oic acid moiety are found in human urine. The acids from neutral steroids and urinary pigments have been separated by passing the mixture through a column of polyethyleneimine cellulose. The acids adhering to the column are quantitatively eluted with dilute formic acid. The purified preparation is suitable for derivatization and chromatographic analysis.     

Restriction endonuclease EcoO109 from Escherichia coli H709c with heptanucleotide recognition site 5'-PuG/GNCCPy

A new restriction endonuclease, EcoO109, has been isolated from Escherichia coli H709c by polyethyleneimine (PEI) precipitation, DEAE-cellulose chromatography and heparin agarose chromatography. The yield was high, more than 3000 units/g of wet cells. The EcoO109 endonuclease recognizes and cleaves a nucleotide sequence of (formula: see text), in the presence of 10 mM Mg2+. The enzyme will be useful for structural analysis and molecular cloning of DNA because of the stability, high yield and easy handling of the producer strain.     

Alg-g-PEI/pVEGF复合物体内促血管生成作用

研究氧化海藻酸——低分子量聚乙烯亚胺两性刷型共聚物 (Algin-a-te-graft-PEI, Alg-g-PEI) 与血管内皮生长因子质粒 (pVEGF) 复合后对体内血管生成的影响。采用细胞和斑马鱼实验检测了Alg-g-PEI/pVEGF复合物的毒性;通过凝胶电泳实验评价了Alg-g-PEI对DNA的保护作用;利用鸡胚绒毛尿囊膜 (CAM) 和斑马鱼模型,选用PEI 25K/pVEGF作为阳性对照、生理盐水为空白对照,观察复合物对血管新生的促进作用。结果发现：Alg-g-PEI对质粒有较好的保护作用;Alg-g-PEI/pVEGF复合物具有较低的细胞、斑马鱼毒性,能显著促进CAM和斑马鱼的血管生成,且具有剂量依赖性,当pVEGF用量等于2.4 μg/CAM时,复合物对CAM血管生成的促进作用最明显,血管面积和CAM面积比 (VA/CAM) 为44.04％,高于阳性对照组 (35.90％) 和空白对照组 (24.03％) (**P＜0.01)。复合物对斑马鱼血管新生的促进作用随氮磷比 (N/P) 的增加而增强,其中N/P=110时,血管新生最明显：肠下血管总长度和面积分别为1.11 mm和1.70×103像素,高于空白对照组 (0.69 mm和0.95×103像素) (**P＜0.01) 和阳性对照组 (0.82 mm和1.11×103像素) (**P＜0.01) 。综上所述,Alg-g-PEI/pVEGF能于体内促进血管生成,该载体有望用于临床缺血性疾病的治疗。     

Polyethyleneimine-based immunopolyplex for targeted gene transfer in human lymphoma cell lines

Background

Specific and efficient delivery of genes into targeted cells is a priority objective in non‐viral gene therapy. Polyethyleneimine‐based polyplexes have been reported to be good non‐viral transfection reagents. However, polyplex‐mediated DNA delivery occurs through a non‐specific mechanism. This article reports the construction of an immunopolyplex, a targeted non‐viral vector based on a polyplex backbone, and its application in gene transfer over human lymphoma cell lines.

Methods

Targeting elements (biotin‐labeled antibodies), which should recognize a specific element of the target cell membrane and promote nucleic acid entry into the cell, were attached to the polyplex backbone through a bridge protein (streptavidin). Immunopolyplex transfection activity was studied in several hematological cell lines [Jurkat (CD3+/CD19?), Granta 519 (CD3?/ CD19+), and J.RT3‐T3.5 (CD3?/CD19?)] using the EGFP gene as a reporter gene and anti‐CD3 and anti‐CD19 antibodies as targeting elements. Transfection activity was evaluated via green fluorescence per cell and the percentage of positive cells determined by flow cytometry.

Results

A significant selectivity of gene delivery was observed, since the anti‐CD3 immunopolyplex worked only in Jurkat cells while the anti‐CD19 immunopolyplex worked only in the Granta cell line. Moreover, transfection of a CD3+/CD3? cell mixture with anti‐CD3 immunopolyplexes showed up to 16‐fold more transfection in CD3+ than in CD3? cells. Several non‐specific transfection reagents showed poor or no transfection activity.

Conclusion

It is concluded that immunopolyplex is a good non‐viral vector for specific and selective nucleic acid delivery. Immunopolyplex design allows easy replacement of the targeting element (antibody) – the streptavidin–polyplex backbone remaining intact – thereby conferring high versatility. Copyright © 2002 John Wiley & Sons, Ltd.

     

Immobilization and stabilization of cephalosporin C acylase on aminated support by crosslinking with glutaraldehyde and further modifying with aminated macromolecules

In this work, cephalosporin C acylase (CA), a heterodimeric enzyme of industrial potential in direct hydrolysis of cephalosporin C (CPC) to 7‐aminocephalosporanic acid (7‐ACA), was covalently immobilized on the aminated support LX1000‐HA (HA) with two different protocols. The stability of CA adsorbed onto the HA support followed by crosslinking with glutaraldehyde (HA–CA–glut) was better than that of the CA covalently immobilized on the glutaraldehyde preactivated HA support (HA–glut–CA). The thermostabilization factors (compared with the free enzyme) of these two immobilized enzymes were 11.2‐fold and 2.2‐fold, respectively. In order to improve the stability of HA–CA–glut, a novel strategy based on postimmobilization modifying with aminated molecules was developed to take advantage of the glutaraldehyde moieties left on the enzyme and support. The macromolecules, such as polyethyleneimine (PEI) and chitosan, had larger effects than small molecules on the thermal stability of the immobilized enzyme perhaps due to crosslinking of the enzymes and support with each other. The quaternary structure of the CA could be much stabilized by this novel approach including physical adsorption on aminated support, glutaraldehyde treatment, and macromolecule modification. The HA–CA–glut–PEI20000 (the HA–CA–glut postmodified with PEI M_w = 20,000) had a thermostabilization factor of 20‐fold, and its substrate affinity (K_m = 14.3 mM) was better than that of HA–CA–glut (K_m = 33.4 mM). The half‐life of the immobilized enzymes HA–CA–glut–PEI20000 under the CPC‐catalyzing conditions could reach 28 cycles, a higher value than that of HA–CA–glut (21 cycles). © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:387–395, 2015     

Transient production of recombinant proteins by Chinese hamster ovary cells using polyethyleneimine/DNA complexes in combination with microtubule disrupting anti-mitotic agents

We have developed a simple and robust transient expression system utilizing the 25 kDa branched cationic polymer polyethylenimine (PEI) as a vehicle to deliver plasmid DNA into suspension-adapted Chinese hamster ovary cells synchronized in G2/M phase of the cell cycle by anti-mitotic microtubule disrupting agents. The PEI-mediated transfection process was optimized with respect to PEI nitrogen to DNA phosphate molar ratio and the plasmid DNA mass to cell ratio using a reporter construct encoding firefly luciferase. Optimal production of luciferase was observed at a PEI N to DNA P ratio of 10:1 and 5 mug DNA 10(6) cells(-1). To manipulate transgene expression at mitosis, we arrested cells in G2/M phase of the cell cycle using the microtubule depolymerizing agent nocodazole. Using secreted human alkaline phosphatase (SEAP) and enhanced green fluorescent protein (eGFP) as reporters we showed that continued inclusion of nocodazole in cell culture medium significantly increased both transfection efficiency and reporter protein production. In the presence of nocodazole, greater than 90% of cells were eGFP positive 24 h post-transfection and qSEAP was increased almost fivefold, doubling total SEAP production. Under optimal conditions for PEI-mediated transfection, transient production of a recombinant chimeric IgG4 encoded on a single vector was enhanced twofold by nocodazole, a final yield of approximately 5 microg mL(-1) achieved at an initial viable cell density of 1 x 10(6) cells mL(-1). The glycosylation of the recombinant antibody at Asn297 was not significantly affected by nocodazole during transient production by this method.     

A Fibrin-Specific Monoclonal Antibody from a Designed Phage Display Library Inhibits Clot Formation and Localizes to Tumors In Vivo

Fibrin formation from fibrinogen is a rare process in the healthy organism but is a pathological feature of thrombotic events, cancer and a wide range of inflammatory conditions. We have designed and constructed an antibody phage display library (containing 13 billion clones) for the selective recognition of the N-terminal peptide of fibrin alpha chain. The key structural feature for selective fibrin binding was a K94E mutation in the VH domain. From this library, an antibody was isolated (termed AP2), which recognizes the five N-terminal amino acids of fibrin with high affinity (K_d = 44 nM), but does not bind to fibrinogen. The AP2 antibody could be expressed in various formats (scFv, small immune protein and IgG) and inhibited fibrin clot formation in a concentration-dependent manner. Moreover, the AP2 antibody stained the fibrin-rich provisional stroma in solid tumors but did not exhibit any detectable staining toward normal tissues. Using a radioiodinated antibody preparation and quantitative biodistribution studies in tumor-bearing mice, AP2 was shown to selectively localize to fibrin-rich F9 murine teratocarcinomas, but not to SKRC-52 human kidney cancer xenografts. Collectively, the experiments indicate that the AP2 antibody recognizes fibrin in vitro and in vivo. The antibody may facilitate the development of fibrin-specific therapeutic agents.     

A simple assay of cyclic-AMP phosphodiesterase in the presence of interfering enzymes in Vibrio harveyi

PEI-cellulose thin layer chromatography is used to separate the reaction products formed from cyclic-AMP initiated by phosphodiesterase in crude extracts of bacteria.