Precipitation of nucleic acids with polyethyleneimine and the chromatography of nucleic acids and proteins on immobilised polyethyleneimine.
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Trapping of chemical carcinogens with magnetic polyethyleneimine microcapsules: I. Microcapsule preparation and in vitro reactivity of encapsulated nucleophiles.
In this paper we describe the synthesis and characterization of magnetic microcapsules, intended for use in vivo, and which contain polyethyleneimine nucleophilic targets capable of trapping electrophilic carcinogens. The microcapsules, 15-50 microns in diameter, consist of a semipermeable cross-linked nylon membrane surrounding core polyethyleneimine and magnetite. These microcapsules can be readily manipulated and extracted from aqueous suspensions by magnetic fields. Core polyethyleneimine was released after membrane rupture by sonication. Magnetic hemoglobin microcapsules were also prepared but were unsuitable due to precipitation of hemoglobin within the core. Treatment by proteolytic enzymes that are present in the gastrointestinal tract caused microcapsule damage resulting in protein release, whereas polyethyleneimine microcapsules remained unaffected. After incubation with N-[methyl-14C]-N-nitrosourea, (1) the microcapsules retained covalently bound radiolabel, both in core polyethyleneimine and the microcapsule membrane. The efficiency of the binding of 1 was investigated by varying the polymer concentration during microcapsule manufacture. These type of microcapsules appear to have the desired properties for investigating carcinogen exposure in the mammalian gastrointestinal tract. They can be prepared easily and reproducibly, contain sufficient magnetite to allow their facile recovery from aqueous suspensions, are easily broken to release soluble core polyethyleneimine, and are stable to hydrolytic enzymes (trypsin) in vitro.
Carcinogens↗
The ultrastructure of anionic sites in rat articular cartilage as revealed by different preparation methods and polyethyleneimine staining.
The ultrastructure of anionic sites in the middle layer of rat articular cartilages was studied by two methods, the quick-freezing and deep-etching method, and the quick-freezing and freeze-substitution method. The anionic sites were visualized with a cationic tracer, polyethyleneimine. They were also compared with those revealed in tissues subjected to conventional fixation, such as pre-embedding or post-embedding. With the deep-etching method, three-dimensional meshwork structures were observed more clearly in the extracellular matrix compared with those seen in conventional ultrathin sections. In combination with polyethyleneimine staining, in which no chemical contrast was needed for visualization of anionic sites, numerous stained particles were detected around filaments in the extracellular matrix, indicating that they were anionic sites consisting mainly of proteoglycans. With the pre-embedding method and polyethyleneimine staining, the shapes of aggregated stained particles varied with different preparation procedures, including chemical fixation and contrasting. The fine meshworks were also observed with the post-embedding method and polyethyleneimine staining. It is suggested that such images of anionic sites, as revealed by the deep-etching method and the post-embedding polyethyleneimine-staining method with low-temperature dehydration, are probably closer to native states than those revealed by the conventional pre-embedding polyethyleneimine-staining method.
Animals↗
Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor.
Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.
Animals↗
GI pharmacology of polyethyleneimine I: effects on gastric emptying in rats.
Experiments were conducted to determine the effect of ingested polyethyleneimine upon gastric emptying of the fasted rat. Emptying was evaluated by the phenolsulfonphthalein and resin bead methods. The two techniques gave comparable results; both showed that this agent inhibited gastric emptying. A delay in gastric emptying could be detected within 15 min of intubation. The effect was dose related, quite long lasting (approximately 4 hr), and reversible. Commercially available, branched polyethyleneimines were highly active, but the linear polyethyleneimine was without observable effect. A branched polyethyleneimine derivative with all primary amine sites selectively acetylated also was inactive.
Animals↗
GI pharmacology of polyethyleneimine II: motor activity in anesthetized dogs.
The effects of orally and intravenously administered doses of polyethyleneimine were observed in 18 chloralose-urethan-anesthetized dogs. Polyethyleneimine produced an initial augmentation of rhythmic segmenting gastric antral contractions, a copious flow of gastric mucus, increased segmenting and propulsive activities of the small and large intestines, and occasional micturition and defecation. The gastric corpus and fundic regions became relaxed and enlarged. These events were associated with the prompt appearance of retching. The retching response to oral administration could only be abolished by bilateral vagotomy or bilateral sympathectomy. The skeletal muscle component of retching was blocked by tubocurarine. Intravenous administration of chlorpromazine blocked the retching response and gastric corporal atonia to either intravenous or oral doses of polyethyleneimine. Either oral or intravenous administration of polyethyleneimine produced no detectable changes in the lead II ECG but was associated with marked transient reductions in both mean and pulsatile arterial blood pressures. These depressor effects showed clear tachyphylaxis. In all cases where GI effects were noted, respiration was augmented and erratic in a manner associated with the retching responses.
Animals↗
Visualization of anionic sites using polyethyleneimine-metal complexes.
17 Polyethyleneimine-metal complexes were synthesized and 3 of them were tested cytochemically for visualization of negative tissue charges. The demonstration of the anionic sites was carried out on rat cerebral cortex and on frog cutaneous pectoral muscle. As controls, neuraminidase digestion, methylation, and omission of osmium-postfixation were used. The osmiophilic properties of polyethyleneimine, polyethyleneimine salts, and polyethyleneimine-metal complexes were discussed.
Animals↗
Demonstration of negative tissue charges by means of polyethyleneimine-metal complexes.
Three polyethyleneimine-metal complexes were synthesized and cytochemically tested for demonstration of negatively charged sites. For this purpose rat cerebral cortex synaptosomes were used. It was established that both types of polyethyleneimine-copper complexes labeled the synaptosomal membrane and synaptic vesicles with electron-dense granules, whereas the polyethyleneimine-lead complex marked the anionic sites with amorphous electron-dense material.
Animals↗
Histological study of articular cartilage in experimental rat knee arthritis induced by intracapsular injection of cationic polyethyleneimine.
Degeneration of proteoglycans and abnormalities in their metabolism have been assumed to be critical at an early stage of osteoarthritis. To clarify a trigger mechanism of osteoarthritis, anionic sites of knee articular cartilage were blocked by intracapsular injection of cationic polyethyleneimine of 2000 or 70,000 molecular weight (MW) into rat knee joints. Experimental or normal rats were examined by gross observation, roentgenography, cytokine interleukin-1beta (IL-1beta) assay, and light or electron microscopy. In both groups with the polyethyleneimine injection, the rats showed temporarily limping and swelling of their knee joints. By light or electron microscopic studies, knee articular cartilage was found to be degenerated at an early stage, but IL-1beta was within normal levels. At 2, 4, or 6 months, the deformity of the knee joints was obvious by roentgenography. Degeneration and hypofunction of proteoglycans might be related to the ultrastructural changes of articular cartilage at an early stage, though the inflammatory cytokine was not involved. At later stages the pathophysiology of the knee joints advanced chronically to typical osteoarthritis, which was more radically induced by the large molecular polyethyleneimine (70,000 MW). The functional impairment of anionic sites is a key point for elucidating the mechanism of osteoarthritic development in this animal model.
Animals↗
Preparation of coenzymic activity of soluble polyethyleneimine-bound NADP+ derivatives.
Alkylation at N-1 of the NADP+ adenine ring with 3,4-epoxybutanoic acid gave 1-(2-hydroxy-3-carboxypropyl)-NADP+. Enzymic reduction of the latter, followed by alkaline Dimroth rearrangement and enzymic reoxidation, gave N6-(2-hydroxy-3-carboxypropyl)-NADP+. On the other hand, bromination at C-8 of the NADP+ adenine ring, followed by reaction with the disodium salt of 3-mercaptroproionic acid, gave 8-(2-carboxyethylthio)-NADP+. Carbodimide coupling of the three carboxylic NADP+ derivatives to polyethyleneimine afforded the corresponding macromolecular NADP+ analogues. The carboxylic and the polyethyleneimine derivatives synthesized have been shown to be co-enzymically active with yeast glucose-6-phosphate dehydrogenase, liver glutamate dehydrogenase and yeast aldehyde dehydrogenase. The degree of efficiency relative to NADP+ with the three enzymes ranged from 17% to 100% for the carboxylic derivatives and from 1% to 36% for the polyethyleneimine analogues. On comparing the efficiences with the three enzymes of the N-1 derivatives to the one of the corresponding N6 anc C-8 analogues, the order of activity was N-1 greater than N6 greater C-8, except in the case of the carboxylic compounds with glutamate dehydrogenase, where this order was inverted. None of these modified cofactors were active with pig heart isocitrate dehydrogenase.
Aldehyde Oxidoreductases↗
Immobilization of aminoacylase in polyethyleneimine stabilized calcium alginate beads for L-phenylalanine production.
Aminoacylase I (E.C.3.5.1.14) was immobilized by entrapment in calcium alginate beads coated with polyethyleneimine for the production of L-phenylalanine by the hydrolysis of a racemic mixture of N-acetyl-DL-phenylalanine. The operational stability in terms of batch operation and continuous reaction in packed-bed bioreactor were studied. Kinetic constants, Km and Vmax values of free and immobilized enzymes were studied. Polyethyleneimine treatment was found to enhance the operational stability of the enzyme though its activity was substantially reduced. When polyethyleneimine-coated calcium alginate beads were packed into packed bed bioreactor, it was stable for at least 25 days under continuous operation without appreciable loss of activity.
Alginates↗
Axonal microtubules are stained and cross-linked by highly cationic polyethyleneimine.
Highly cationic polyethyleneimine was used as an electron microscopic tracer for anionic sites in the axoplasm of rat sciatic and optic nerve fibres. Microtubules showed a markedly increased electron density and aggregated to form large groups, mostly located in the immediate proximity of membranous axoplasmic organelles. Walls of adjacent microtubules were fused; there was also fusion of microtubules with the membranes of smooth axoplasmic reticulum and with axolemma. In contrast, neurofilaments had unaltered electron density and axoplasmic distribution. Staining and clustering of microtubules were interpreted as electrostatic binding of cationic polyethyleneimine to acidic tubulin. These findings may be relevant to the role of microtubules in fast axonal transport.
Animals↗
Assay of UDP, GDP, CDP, and ADP reductase activities by column chromatography on polyethyleneimine cellulose.
Deoxyribonucleosides were separated from ribonucleosides by chromatography on polyethyleneimine cellulose columns (Pasteur pipettes. The deoxyribonucleosides were quantitatively eluted with 25 mM boric acid in less than 10 ml while the ribonucleosides were retained. The ribonucleosides were eluted with 1 M NaCl. This method was utilized to assay for GDP, UDP, ADP, and CDP reductase activities after hydrolysis of the substrate and product nucleotides to the corresponding nucleosides. All four reductase activities were assayed using identical conditions of column size, eluting solution (25 mM boric acid), and elution volume. The use of polyethyleneimine cellulose columns with boric acid can be adapted to other enzyme assays such as purine nucleoside phosphorylase and for the isolation of deoxyribonucleotides from cellular extracts.
Cellulose↗
A filtration assay for solubilized prolactin receptors using polyethyleneimine-treated membrane filters.
Free 125I-labeled ovine prolactin can be separated from detergent-solubilized prolactin-receptor complex by filtration on triacetate membrane filters pretreated with polyethyleneimine. Up to 98% of the total 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilized prolactin-receptor complexes from rat liver bound to polyethyleneimine-treated membranes. This simple and rapid technique can be used to quantitate solubilized prolactin-receptor complexes.
Animals↗
Polyethyleneimine-bonded phases in the separation of proteins by capillary electrophoresis.
A hydrophilic, positively charged, durable coating has been developed for capillary electrophoresis of macromolecules. Polyethyleneimine is adsorbed to the inner wall of fused silica capillaries and the adsorbed coating cross-linked into a stable layer. Capillaries of polyethyleneimine-coated silica gave unique separations owing to the reversal of electro-osmotic flow caused by the positively charged coating. The resulting coating was stable from pH 2-12 and could be used over a wide pH range without substantial change in electro-osmotic flow. High-molecular-weight polymers were needed to give thick coatings which mask silanol groups on the wall. Proteins were resolved quickly and efficiently with good recovery using capillaries of 50 cm in length.
Animals↗
A simple staining method for dna and rna blotted on a membrane using a polyethyleneimine-enzyme conjugate.
We describe a new method for the detection of nucleic acids fixed on a membrane involving enzyme-conjugated polyethyleneimine. This method is based on the strong ionic interaction of the cationic polymer, polyethyleneimine (aziridine), with nucleic acids. Sensitivities of 10 pg for DNA and 1 ng for rRNA were achieved in slot-spot blotting experiments. DNA fragments blotted onto a membrane by Southern transfer can be stained after hybridization with a labeled nucleic acid probe. By combining this method with a non-isotopic detection system, the positions of probe hybridized and non-hybridized signals were visualized as distinct colors on the same membrane without superimposing any other image. This technique should have wide application for the detection of nucleic acids fixed on membranes in blot-hybridization experiments.
Alkaline Phosphatase↗
New isoelectric buffers for capillary electrophoresis: N-carboxymethylated polyethyleneimine as a macromolecular isoelectric buffer.
Isoelectric buffers are attractive for electrophoresis because of their low conductivity, and their compatibility with indirect photometric detection in capillary electrophoresis (CE) where they do not interfere with the detection by exhibiting competitive displacement of the UV-absorbing probe ion. N-carboxymethylated polyethyleneimine (CMPEI) was prepared by introducing a half molar equivalent of carboxylate groups onto a polyethyleneimine backbone. Its isoelectric point determined by conductometric titration and from the pH of its dilute aqueous solution is approx. 6.8, which allows isoelectric buffering at a lower pH compared to histidine (pI7.7). Although the isoelectric point is somewhat diffuse, as expected for a polymeric compound, it exhibits a buffering capacity at a pI point of about twice that of histidine. Studies of electroosmotic flow (EOF) profile at various pH values in fused silica capillaries showed that CMPEI adsorbs onto the fused silica wall and reverses the EOF at pH < 6.5. CMPEI was applied as a buffer in an electrolyte containing 0.5 mM of the anionic dye tartrazine used as the probe for indirect detection of anions. The separation system exhibited a stable baseline, no system peaks, separation efficiencies of up to 195,000 theoretical plates, and detection limits down to 0.2 microM or 2 amol of injected analyte.
Journal Article↗
The behaviour of oligodeoxynucleotides on thin-layer chromatography on polyethyleneimine-cellulose and ion-exchange paper electrophoresis. Applications in fractionating and sequencing terminally labelled oligodeoxynucleotides.
The electrophoretic behaviour of oligodeoxynucleotides on ion-exchange papers was studied systematically with particular attention to applications in sequence analysis. Complex mixtures of larger oligonucleotides were fractionated by electrophoresis on cellulose acetate followed by t.l.c. on polyethyleneimine-cellulose. The behaviour of the oligonucleotides on polyethyleneimine-cellulose and on the two-dimensional system can provide a useful guide to their sequence. Detailed results from some of these experiments have been deposited as Supplementary Publication SUP 50031 (13 pages) at the British Library (Lending Division) (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973), 131, 5.
Adenosine Triphosphate↗