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[Structure of the B-form of DNA using the homopolymer poly(dA) . poly(dT) as an example. Refinement in accordance with x-ray and energy criteria].

The refinement of the B-form DNA structure was made using homopolymer poly(dA) (dT) as a model system. In the course of refinement 9 conformational and 22 independent experimental parameters (20 intensities of reflections and 2 helical parameters) were used. After the goal-function, including R-factor, helical and conformational parameters, was optimised, the structures with the values of potential energy and R-factor close correspondingly to the optimal values were selected. "The best" conformations obtained according to energy criteria have the value of an axial projection per one nucleotide pair of about 2,9 A that is equal to the corresponding value in the recently discovered relaxed form of DNA in solution. The most probable conformations according to combine energy and structural criteria have better value of the R-factor in comparison with those derived on the basis of X-ray only, although they are slightly less satisfactory according to the energy criteria. The conformation of DNA which have the values of an axial projection per one nucleotide pair--3,29 A; of the angle of rotation of neighboring nucleotide pair relative each other--36 degrees and of the angle of nucleotide pair with an axis of the helix -13,7 degrees seems to be the most accurate description of the doublehelical structure of poly(dA) . poly(dT). The atomic coordinates of the new model may be used in analysis of stereochemistry of the complexes of nucleic acids with polypeptide and proteins, that is in consideration of the problem of protein-nucleic acid recognition.

DNA↗

[Termination of DNA transcription by a poly(dA).poly(dT) fragment inserted into pBR325 plasmid].

A recombinant DNA was constructed by inserting polynucleotide (dA).(dT) of 80-100 base pairs long into EcoRI site of the pBR325 plasmid DNA. Transcription of this DNA was studied in E. coli RNA polymerase system in vitro. Some transcripts obtained with the recombinant plasmid were shown to have poly(U) clusters at the 3'-ends. Obtained data indicate that poly(dA).poly(dT) sequence acts as a terminator of RNA synthesis. Orientation of this sequence in recombinant DNA was also established.

Base Composition↗

Hybridization of poly(A)-containing ribonucleic acid transported in vitro to cyclic deoxyribonucleic acid-cellulose transcribed from cytoplasmic poly(A)-containing ribonucleic acid.

Isolated nuclei have been employed to study nucleocytoplasmic RNA transport in vitro; however, the specificity of the in vitro process remains largely undefined. To examine this specificity we have employed a hybridization technique that allows recovery of undegraded RNA after hybridization under stringent conditions. Cytoplasmic poly(A)-containing RNA was isolated from rat liver and used as a template for synthesis of cDNA-cellulose. Rat liver nuclei were isolated and incubated in vitro, and the released poly(A)-containing RNA was hybridized to the cDNA-cellulose transcribed from authentic cytoplasmic RNA. A significant portion hybridized (18.5 +/- 4.4%), comparable to the portion of homologous cytoplasmic poly(A)-containing RNA that hybridized (20.4 +/- 2.3%), indicating a considerable sequence homology between these populations. Similar results were obtained when cDNA was transcribed from RNA which was transported in vitro in 45S ribonucleoprotein (21.6%). Control nuclear RNA hybridized to a significantly smaller extent (9.1 +/- 2.7%), and the percentage hybridizing decreased markedly under conditions that allowed nuclear RNA processing but not transport (to 4.3 +/- 2.1%), showing that the hybridization was not due to nonspecific leakage of nuclear RNA. This technique should prove valuable for following in vitro processing of specific probes and for enrichment of sequences differentially transported in vitro following acute injury or carcinogen treatment of rats.

Animals↗

[Interferon-inducing activity of poly(I)-poly(C) and its complexes with polycations and their resistance to enzymatic degradation].

The insoluble complexes of poly (I) . poly (C) with histones and poly-L-lysin have been obtained. These complexes are insoluble in isotonic solutions and have no admixtures of polycations. Resistance to enzymatic degradation, the melting point and sorption on the cells of the complexes obtained increase with the growth of the polycation amount in the complexes, while the interferon-inducing capacity reduces. Based on the obtained and reported data the conclusion is made that resistance to enzymatic degradation, the high melting point and the high sorption of the inductor on the cells are necessary but insufficient conditions for the induction of interferon synthesis. It is assumed that the inductor ability to make complexes with cellular proteins is the characteristics responsible for the interferon-inducing capacity of the inductor.

Animals↗

[Interferon induction in monkeys by poly I-C complex with poly-L-lysine].

The spectrum characteristics, the stability to pancreatic RN-ase and hydrolases of the monkey and mouse blood as well as interferon induction in mice and monkeys by poly I: poly C and its complex with poly-l-lysine were studied. The complex was found to be more stable to the treatments tested as well as markedly interferonogenically active in Macaca rhesus monkeys.

Animals↗

E1A-induced enhancer activity of the poly(dG-dT).poly(dA-dC) element (GT element) and interactions with a GT-specific nuclear factor.

The alternating sequence poly(dG-dT).poly(dA-dC) is a highly repeated sequence in the eucaryotic genome. We have examined the effect of trans-acting early viral proteins on the ability of the GT element to stimulate transcription of the adenovirus major late promoter (MLP). We find that the GT element alone does not activate expression from the MLP in either the presence or absence of another enhancer element. However, in the presence of the E1A gene products of either adenovirus type 5 or 2, the GT element activated expression from the MLP. The stimulatory activity of the GT element in the presence of E1A had the properties of an enhancer element, and the trans-activating effect on the GT element was additive in conjunction with the E1A-responsive BK virus enhancer. We also have demonstrated that a specific nuclear factor(s) binds to the GT element. However, the E1A protein(s) do not affect the initial factor interaction(s) with the GT element. Overall, our data demonstrate that trans modulation of promoter activity can be mediated through the GT element.

Adenovirus Early Proteins↗

The molecular electrostatic potential of the B-DNA helix. VI. The regions of the base pairs in poly (dG.dC) and poly (dA.dT).

The evaluation of the electrostatic molecular potential at important nucleophilic sites of the purine and pyrimidine bases in poly (dG.dC) and poly (dA.dT) and of the evolution of the potential through the series free bases-nucleosides-nucleotides-single polynucleotide helices-double helices enables the interpretation of the evolution of the corresponding reactivity of the bases towards a series of electrophilic carcinogenic and mutagenic reactants.

Base Composition↗

Reactions of nucleic acid bases with alpha-acetylenic esters. Chemical modification of poly(A) and poly(C).

The reaction of chlorotetrolic (4-chloro-2-butynoic) esters with adenine and cytosine derivatives, in which a new heterocycle bearing an alkylating chloromethyl side chain is fused to the purine or pyrimidine ring, was extended to poly(A) and poly(C) used as models of nucleic acids. The derivatization proceeds under mild conditions and its extent can be controlled by the reaction time. The additional rings can exist in two isomeric forms and the nature of the isomer formed depends on steric factors in the vicinity of the reacting base. The reaction with chlorotetrolic esters discriminates between the single-stranded (reactive) and double-stranded (unreactive) forms, between the exposed an hidden adenine and cytosine bases and even between the exposed and sterically hindered fragments of the base moiety and thus allows structural investigations of these nucleic acids. The chloromethyl group of the derivatized nucleobases can be used to bind the modified polymers to other molecules.

Alkaline Phosphatase↗

Nucleic acid hybridization in plasma: method for the quantitation of poly(I).poly(C12, U) in plasma of cancer patients.

Using a new method for the direct measurement of the double-stranded RNA (dsRNA) molecule poly(I).poly(C12, U) in plasma, levels of 100 X 10(-9) g of drug were routinely quantified. The samples were digested by proteinase K in a buffered solution containing 0.1% of Brij-35 and deoxycholate detergents. The digestions were terminated after 1 h by the addition of Brij-58 and boiling saturated NaI (1.67 g/ml). Serially diluted samples were filtered onto nitrocellulose and the filters washed and hybridized. Levels of the hybridized-radioactive probe, synthesized de novo in an RNA dependent DNA transcription system, were determined by liquid scintillation spectrophotometry and quantified by comparison to a standard curve. The efficiency of hybridization declined when the plasma concentration in the reaction fell below 1.0 mg/ml. Incubation and denaturation temperatures significantly altered the amount of radioactive probe hybridized; results varied in the extent of hybridization and in the concentration range of dsRNA showing a linear response. Elevated temperature during proteinase K digestion showed reduced hybridization efficiencies: 100% at 25, 80% at 37, 35% at 45, and 25% at 55 degrees C. Incubation at elevated temperatures, prior to the addition of NaI, caused a decline in the amount of radioactivity hybridized, but did not have an effect during hybridization.

Humans↗

Direct evidence for a bimorphic structure of a DNA-RNA hybrid, poly(rA).poly(dT), at high relative humidity.

31P solid state nuclear magnetic resonances of highly oriented poly(rA).poly(dT) fibers demonstrated that this hybrid undergoes a conformational transition induced by hydration at about 90% relative humidity, namely, the hybrid has a single backbone conformation of the A family below 87% and two distinct conformations above 92% relative humidity. The structural model (of the B-like form) proposed on the basis of the x-ray diffraction data of this hybrid by Zimmerman and Pheiffer (Zimmerman, S.B., and Pheiffer, B.H. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 78-82) was proved to be fully consistent with our NMR data.

DNA↗

Controlled delivery of taxol from poly(ethylene glycol)-coated poly(lactic acid) microspheres.

The development of injectable microspheres for sustained drug delivery to the arterial wall is a major challenge. We demonstrated the possibility of entrapping an antiproliferative agent, taxol, in poly(ethylene glycol) (PEG)-coated biodegradable poly(lactic acid) (PLA) microspheres with a mean diameter of 2-6 microm. A solution of taxol and PLA dissolved in an acetone/dichloromethane mixture was poured into an aqueous solution of PEG [or poly(vinyl alcohol) (PVA] with stirring with a high-speed homogenizer for the formation of microspheres. Taxol recovery in PLA-PEG microspheres was higher (61.2 +/- 2.3%) than with PVA-based (41.6 +/- 1.8%) preparations. An analysis by diffuse reflectance infrared Fourier transform spectroscopy revealed that PEG was incorporated well on the PLA microsphere surface. Scanning electron microscopy revealed that the PEG-coated PLA microspheres were spherical in shape and had a smooth surface texture like those of PVA-based preparations. The amount of drug release was much higher initially (25-30%); this was followed by a constant slow-release profile for a 30-day period of study. This PEG-coated PLA microsphere formulation may have potential for the targeted delivery of antiproliferative agents to treat restenosis.

Biocompatible Materials↗

Liposomes as immunological adjuvants in eliciting antibodies specific to the synthetic polypeptide poly(LTyr, LGlu)-poly(DLAla)--(LLys) with high frequency of site-associated idiotypic determinants.

The antibody response to the synthetic polypeptide, poly(LTyr, LGlu)-poly(DLAla)--poly(LLys), [(T,G)-A--L], injected entrapped in liposomes which served as adjuvant has been analyzed. The liposomes used were composed of phosphatidylcholine, cholesterol, dicetylphosphate and DL alpha-tocopherol (molar ratios as 4:3:0.1:0.5) and therefore, were negatively charged. Since the (T,G)-A--L is also negatively charged, no free complexes were formed. The (T,G)-A--L was found to be entrapped inside the enclosed volume of the liposomes, and no (T,G)-A--L antigenic determinants could be detected on the liposomal membranes. Injection of high-responder C3H.SW (H-2b) mice with (T,G)-A--L-bearing liposomes demonstrated that the i.p. and the i.v. routes of immunization were efficient in eliciting (T, G)-A--L specific antibodies, whereas the i.d. injection led to poor antibody responses. The latter route of immunization is the most effective when (T,G)-A--L is injected in complete Freund's adjuvant (CFA). When low doses (0.1 and 1 microgram) of (T, G)-A--L were used for immunization, the liposomes were better adjuvants than CFA. The effectiveness of the liposomes as immunological adjuvants was also shown in their ability to induce high-potential, primed memory cells. The pattern of low (H-2k,a) and high (H-2b) responsiveness to (T,G)-A--L was retained following immunization with (T,G)-A--L entrapped in liposomes, as tested in two pairs of congenic strains. (T,G)-A--L-specific antibodies induced by injection with 1 microgram antigen entrapped in liposomes bear the (T,G)-A--L site-related idiotypic markers of C3H.SW (Igh-1a) mice in a significantly higher frequency than the homologous idiotypes, namely the antibodies elicited in this strain against (T,G)-A--L in CFA. Thus, liposomes may serve as adjuvants for the production of relatively restricted (T,G)-A--L-specific antibodies of high quality.

Adjuvants, Immunologic↗

Synthesis and adsorption of a poly(N-acetylethyleneimine)-polyethyleneoxide-poly (N-acetylethyleneimine) triblock-copolymer at a silica/solution interface. Influence of its preadsorption on platelet adhesion and fibrinogen adsorption.

The synthesis of a triblock copolymer poly-(N-acetylethyleneimine)-polyethylenoxide-poly(N-acet ylethyleneimine) includes two successive steps: the first is the functionalization of a poly(ethyleneglycol) precursor by creating sulfonic esters at its chain ends, the second uses these esters to initiate the cationic polymerization of 2-methyl-2-oxazoline. Homopolymers appear in the raw product; hence successive selective extractions of the copolymer with benzene and dioxane are necessary. The final yield in pure copolymer was 11%. The copolymer was characterized by UV and 1H-NMR spectrometry and light scattering. Adsorption isotherms were determined on silica, for varying pH and salt concentration. Optimum conditions for coating silica with the polymer were determined. The efficiency of this precoating to reduce the adsorption of fibrinogen was very high (99.2% reduction with respect to bare silica). Steric exclusion chromatography of a variety of proteins gave a satisfactory calibration curve. Platelet accumulation on copolymer precoated glass was reduced to 10-20% of its value on bare glass, a result superior to that obtained by albumin passivation of the same glass surface.

Adsorption↗

Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA).

The tissue response and in vivo molecular stability of injection-molded polyhydroxyacids--polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA, 5-22% VA content)--were studied. Polymers were implanted subcutaneously in mice and extirpated at 1, 3, and 6 months in order to study tissue response and polymer degradation. All polymers were well tolerated by the tissue. No acute inflammation, abscess formation, or tissue necrosis was observed in tissues adjacent to the implanted materials. Furthermore, no tissue reactivity or cellular mobilization was evident remote from the implant site. Mononuclear macrophages, proliferating fibroblasts, and mature vascularized fibrous capsules were typical of the tissue response. Degradation of the polymers was accompanied by an increase in collagen deposition. For the polylactide series, the inflammatory response after 1 month of implantation was less for materials containing the D-unit in the polymer chain, whereas in the case of the polyhydroxybutyrate/valerates, the number of inflammatory cells increased with increasing content of the valerate unit in the polymer chain. Between 1-3 months, there was slightly more tissue response to the PHB and PHB/VA polymers than to PLA. This response is attributed to the presence of leachable impurities and a low molecular weight soluble component in the polyhydroxybutyrate/valerates. At 6 months, the extent of tissue reaction was similar for both types of polymers. All polylactides degraded significantly (56-99%) by 6 months. For a poly(L-lactide) series, degradation rate in vivo decreased with increasing initial molecular weight of the injection-molded polymer. Several samples showed pronounced bimodal molecular weight distributions (MWD), which may be due to differences in degradation rate, resulting from variability in distribution of crystalline and amorphous regions within the samples. This may also be the result of two different mechanisms, i.e., nonenzymatic and enzymatic, which are involved in the degradation process, the latter being more extensive at the later stage of partially hydrolyzed polymer. The PHB and PHB/VA polymers degraded less (15-43%) than the polylactides following 6 months of implantation. Generally, the polymer with higher valerate content (19%, 22%) degraded most. The decrease in molecular weight was accompanied by a narrowing of the MWD for PHB and copolymers; there was no evidence of a bimodal MWD, possibly indicating that the critical molecular weight that would permit enzyme/polymer interaction had not been reached. Weight loss during implantation ranged from 0-50% for the polylactides, whereas for the PHB polymers weight loss ranged from 0-1.6%.

Absorption↗

Improvement of retroviral vectors by coating with poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL).

BACKGROUND: Although some cationic reagents, such as polybrene, improve gene transduction in vitro, their use in vivo is prohibited due to their toxicity to the exposed cells. This paper demonstrates that a new cationic reagent, poly(ethylene glycol)-poly(L-lysine) block copolymer (PEG-PLL), improves gene transduction with retroviral vectors without increasing cell toxicity. METHODS: A retroviral vector derived from the Moloney leukemia virus, containing the lacZ gene, was modified with PEG-PLL prior to transduction into NIH3T3, Lewis lung carcinoma, and primary cultured mouse brain cells. LacZ transduction efficacy was evaluated by counting the number of X-Gal-positive cells. RESULTS: We have demonstrated that PEG-PLL is able to stably modify the viral particle surface due to the affinity of the PEG moiety to the biomembrane, and neutralizes negative charges by the cationic nature of the poly-lysine residue. Thus, PEG-PLL increased the gene transduction efficiency and minimized cell toxicity because free PEG-PLL was removable by centrifugation. We have shown that PEG-PLL increased the viral gene transduction efficiency 3- to 7-fold with NIH3T3 or Lewis lung carcinoma cell lines without increasing cytotoxicity. It improved retroviral gene transduction efficacy even against labile cells, such as primary cultured brain cells. CONCLUSIONS: PEG-PLL is a novel reagent that improves retroviral gene transduction efficacy without increasing cytotoxicity.

3T3 Cells↗

Bioengineering Functional Copolymers. IX. Poly[(maleic anhydride-co-hexene-1)-g-poly(ethylene oxide)].

Amphiphilic bioengineering copolymers having a combination of hydrophilic/hydrophobic linkages and polyelectrolyte behavior, along with an ability to interact with biomacromolecules, in particular with the invertase enzyme, have been synthesized by (a) complex-radical copolymerization of maleic anhydride (MA, the acceptor) and hexene-1 (H-1, the donor) monomers with benzoyl peroxide as the initiator in 1,4-dioxane at 65 degrees C under high-conversion conditions and (b) subsequent grafting (polyesterification) of synthesized poly(MA-alt-H-1) with alpha-methoxy-omega-hydroxy-poly(ethylene oxide) (PEO). Copolymerizations were also carried out in the steady state, in order to essentially reduce the effect of copolymer composition drift. The values of the monomer reactivity ratios (r(1) and r(2)) determined by using the known terminal models of Fineman-Ross (FR) and Kelen-Tüdös (KT), as well as by nonlinear regression (NLR) analysis, are: r(1) = 0.16 and r(2) = 0.30 (FR), r(1) = 0.14 and r(2) = 0.27 (KT), and r(1) = 0.15 and r(2) = 0.29 (NLR), respectively. All the copolymers and graft copolymers were characterized by FTIR spectroscopy, (1)H{(13)C} NMR spectroscopy, viscometric measurements, and chemical (acid number), thermal (DSC and TGA), and X-ray diffraction analyses. Unlike poly(MA-alt-H-1)s, PEO macrobranched graft copolymers exhibit expressed polyelectrolyte and swelling behavior in diluted and concentrated dioxane solutions, respectively. The copolymer and its PEO hyperbranched derivatives can be used as carriers for enzyme immobilization.

Enzymes, Immobilized↗

Preparation and characterization of poly(N-isopropylacrylamide)-modified poly(2-hydroxyethyl acrylate) hydrogels by interpenetrating polymer networks for sustained drug release.

In order to investigate the influence of hydrophobic moieties formed by poly(N-isopropylacrylamide) (PNIPAm) in a hydrogel matrix on the release behavior of the hydrogel, a series of poly(N-isopropylacrylamide) (PNIPAm)-modified poly(2-hydroxyethyl acrylate-co-2-hydroxyethyl 2-hydroxyethyl methacrylate) (P(HEA-co-HEMA)) via interpenetrating polymer networks (IPNs) were prepared by a sequential UV solution polymerization. Interestingly, it was found that P(HEA-co-HEMA)/PNIPAm IPN indicated a single glass transition temperature (T(g)) and the T(g)s of the IPNs increased with an increase in the PNIPAm component. This phenomenon may be attributed to hydrogen bonding between the two polymer networks, but the hydrogen bonding exerts less influence on the swelling behavior of the IPNs, due to the fact that IPNs can respond to changes in temperature like PNIPAm. Using 2-[(diphenylmethyl)sulphiny]acetamide (modafinil, MOD) and p-hydroxybenzoic acid (HBA) as model drug compounds, the release behavior of the IPNs was studied at body temperature, and it was found that the presence of PNIPAm could retard drug release regardless of the solubility of the drugs. Release profiles of HBA from the IPNs and their component samples as a function of time at 37 degrees C.

Acrylates↗

Complexation between poly(maleic acid/octyl vinyl ether) and poly(vinyl caprolactam) in aqueous solution and at the alumina/water interface.

Solution and interfacial properties of binary polymer mixtures of poly(maleic acid/octyl vinyl ether) (PMAOVE) and poly(vinyl caprolactam) (PVCAP) have been studied for the alumina/water system. To test the hydrophobic effect, mixtures of poly(maleic acid/methyl vinyl ether) (PMAMVE) and PVCAP are also investigated and compared to the behavior of PMAOVE/PVCAP. At low pH, both polymer mixtures become turbid upon mixing. The turbidity increases at low mixing ratios of PVCAP to the vinyl ether component, reaches a maximum, and then decreases at higher mixing ratios. Upon shifting the pH to the alkaline range, i.e., pH 7.5 and above, the turbid solution becomes clear for both the polymer mixtures. Cloud point measurements indicate the absence of complexation of PVCAP with PMAMVE under the alkaline conditions, but strong interaction with PMAOVE. This is attributed to the different forces involved in the complexation among the polymers: H bonding for PVCAP/PMAMVE and both H bonding and hydrophobic effects for PVCAP/PMAOVE. At the alumina/water interface, the normally nonadsorbing PVCAP is triggered to adsorb by PMAOVE, attributed to the hydrophobic complexation between the two. However, the adsorption of PVCAP shows a maximum as a function of the concentration of PMAOVE. At concentrations of PMAOVE above the onset of its own plateau adsorption, the amount of PVCAP triggered to adsorb is reduced possibly due to the polymer complex formation in solution.

Journal Article↗