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[Effect of synthetic polynucleotides and RNA on poly(C)-dependent poly(G) polymerase activity of Q beta replicase].

The effect of synthetic polynucleotides and phage RNA on poly(C)-dependent synthesis of poly (G) by Qbeta replicase is studied. It is shown that single stranded poly(U) and poly (dT) are strong inhibitors whereas structured polynucleotides poly(A), MS2 RNA as well as double stranded complexes poly(A)-poly(U) and poly(A)-poly(dT) do not affect the synthesis of poly(G). It is suggested that contact region of template with enzyme has single stranded unhelical structure and affinity of polynucleotides to Qbeta replicase is determined by degree of their secondary structure.

Kinetics↗

Immobilization of the enzyme beta-lactamase on biotin-derivatized poly(L-lysine)-g-poly(ethylene glycol)-coated sensor chips: a study on oriented attachment and surface activity by enzyme kinetics and in situ optical sensing.

Understanding the conformation, orientation, and specific activity of proteins bound to surfaces is crucial for the development and optimization of highly specific and sensitive biosensors. In this study, the very efficient enzyme beta-lactamase is used as a model protein. The wild-type form was genetically engineered by site-directed mutagenesis to introduce single cysteine residues on the surface of the enzyme. The cysteine thiol group is subsequently biotinylated with a dithiothreitol (DTT)-cleavable biotinylation reagent. beta-Lactamase is then immobilized site-specifically via the biotin group on neutral avidin-covered surfaces with the aim to control the orientation of the enzyme molecule at the surface and study its effect on enzymatic activity using Nitrocefin as the substrate. The DTT-cleavable spacer allows the release of the specifically bound enzyme from the surface. Immobilization of the enzyme is performed on a monolayer of the polycationic, biotinylated polymer PLL-g-PEG/PEG-biotin assembled on niobium oxide (Nb2O5) surfaces via neutral avidin as the docking site. Two different assembly protocols, the sequential adsorption of avidin and biotinylated beta-lactamase and the immobilization of preformed complexes of beta-lactamase and avidin, are compared in terms of immobilization efficiency. In situ optical waveguide lightmode spectroscopy and colorimetric analysis of enzymatic activity were used to distinguish between specific and unspecific enzyme adsorption, to sense quantitatively the amount of immobilized enzyme, and to determine Michaelis-Menten kinetics. All tested enzyme variants turned out to be active upon immobilization at the polymeric surface. However, the efficiency of immobilized enzymes relative to the soluble enzymes was reduced about sevenfold, mainly because of impaired substrate (Nitrocefin) diffusion or restricted accessibility of the active site. No significant effect of different enzyme orientations could be detected, probably because the enzymes were attached to the surface through long, flexible PEG chain linkers.

Adsorption↗

Double-stranded complex of polyguanylic and polycytidylic acids and its antiviral activity in tissue culture.

The antiviral activity and conditions of formation of the most active double-stranded complexes of synthetic homopolynucleotides, polyriboguanylic and polyribocytidylic acids, were studied on the model of primary trypsinized chick embryo cells and RNA-containing viruses. The (poly G).(poly C) complex was very active against the viruses tested; their replication in cell cultures was inhibited completely. The antiviral activity of the (poly G).(poly C) complex increased markedly in the presence of diethylaminoethyl- (DEAE-) dextran. After treatment with 1 mug/ml of (poly G). (poly C) for 1 hour in the presence of 100 mug/ml DEAE-dextran, the cell sheet remained protected for 5-7 days. Preparations of (poly G).(poly C) obtained under optimal conditions were as active as (poly I).(poly C) complexes and exceeded them markedly in the level of the therapeutic index which under the present experimental conditions was 5-10 times 10(3) for (poly G).(poly C). Highly purified homopolymers with sufficiently high molecular weight must be used for production of active and stable (poly G).(poly C) complexes.

Animals↗

Preparation and characterization of intelligent core-shell nanoparticles based on poly(D,L-lactide)-g-poly(N-isopropyl acrylamide-co-methacrylic acid).

New thermo-responsive, pH-responsive, and biodegradable nanoparticles comprised of poly(D,L-lactide)-graft-poly(N-isopropyl acrylamide-co-methacrylic acid) (PLA-g-P(NIPAm-co-MAA)) were developed by grafting biodegradable poly(D,L-lactide) onto N-isopropyl acrylamide and methacrylic acid. A core-shell type nano-structure was formed with a hydrophilic outer shell and a hydrophobic inner core, which exhibited a phase transition temperature above 37 degrees C suitable for biomedical application. Upon heating above the phase transition temperature, PLA-g-P(NIPAm-co-MAA) nanoparticle showed a polarity increase of pyrene in either buffer solution or intra-hepato-carcinoma cells as determined by fluorescence measurement, indicating that the structure of nanoparticles caused leakages from outer shell copolymers aggregation and collapsed. The drug loading level of 5-fluorouracil (5-FU) encapsulated in the PLA-g-P(NIPAm-co-MAA) nanoparticles can be as high as 20%. The release of 5-FU from nanoparticles was strongly controlled by the pH in the aqueous solution. Based on these results, PLA-g-P(NIPAm-co-MAA) nanoparticles can be used as a drug carrier for intracellular delivery of anti-cancer drug.

Acrylamides↗

Trypsin inhibition, calcium and zinc ion binding of starch-g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures for peroral peptide drug delivery.

Newly synthesised starch-g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures were evaluated for their in vitro inhibition potency towards the proteolytic enzyme trypsin. Their Ca2+ and Zn2+ binding capacity was measured. Carbopol 934P was used as reference polymer. Starch-g-poly(acrylic acid) copolymers were prepared by chemical grafting and 60Co irradiation, the starch/poly(acrylic acid) mixtures by freeze-drying. The influence of preparation method, the ratio starch:acrylic acid, the neutralisation degree and for the freeze-dried polymers the influence of heat treatment after freeze-drying was investigated. All freeze-dried polymers showed a higher inhibition factor (IF) than the chemically grafted and 60Co irradiated starches, which all showed significantly lower IF than Carbopol 934P. The heat treated freeze-dried polymer Amioca/poly(acrylic acid) (1:1) showed a significantly higher IF than the reference polymer (Mann-Whitney test, p<0.05). The Ca2+ and Zn2+ binding capacity of all chemically grafted starches was much lower than for Carbopol 934P. Only the 60Co irradiated starches and freeze-dried polymers with ratio 1:3 approached the binding capacity of the reference polymer. The freeze-dried polymers showed the highest proteolytic enzyme inhibition potency. Freeze-drying and 60Co irradiation could result in the highest ion binding capacity. This combination of proteolytic enzyme inhibition activity and ion binding capacity makes these polymers hopeful excipients for successful oral peptide delivery.

Acrylic Resins↗

Variation of gonococcal lipooligosaccharide structure is due to alterations in poly-G tracts in lgt genes encoding glycosyl transferases.

The lipooligosaccharide (LOS) expressed by gonococci spontaneously varies its structure at high frequency, but the underlying genetic mechanism has not been described. We have previously reported that the genes encoding the glycosyl transferases responsible for the biosynthesis of the variable alpha chain of the LOS of Neisseria gonorrhoeae are located in a locus containing five genes, lgtA, lgtB, lgtC, lgtD, and lgtE. Sequence analysis showed that lgtA, lgtC, and lgtD contained poly-G tracts within the coding frames, leading to the hypothesis that shifts in the number of guanosine residues in the poly-G tracts might be responsible for the high frequency variation in structure of gonococcal LOS. We now provide experimental evidence confirming this hypothesis.

Base Sequence↗

Interactions of quercetin and its lanthane complex with double stranded DNA/RNA and single stranded RNA: spectrophotometric sensing of poly G.

Spectrophotometric titrations revealed that stability of the quercetin/double stranded (ds) DNA or double stranded (ds) RNA non-covalent complexes is significantly higher compared to the quercetin/ss-RNA complexes. This observation can easily be correlated with the significantly larger aromatic surface of base pairs compared to single nucleobases, and it is in good agreement with other experimental data pointing toward intercalative binding mode of quercetin. Fluorescence increase of quercetin induced by ds-RNA is significantly stronger than observed for ds-DNA, offering usage of quercetin as the ds-RNA selective fluorescent probe. Also, addition of poly G yielded more than order of magnitude stronger changes in UV/visible and fluorescence spectrum of quercetin compared to the changes upon addition of poly A and poly U revealing possible usage of quercetin as a powerful spectroscopic probe for poly G sequences. Stability and stoichiometry of lanthane(III)/quercetin complexes in physiologically relevant aqueous media was determined. The interactions of (LaQ)(3+) with double stranded DNA and RNA were significantly different compared to the free quercetin, revealing increase of complex stability and thus significant impact of La(III) in binding of (LaQ)(3+) to polynucleotides. Similar results were observed for interactions of (LaQ)(3+) with single stranded RNA.

Binding Sites↗

PEG-g-poly(GdDTPA-co-L-cystine): a biodegradable macromolecular blood pool contrast agent for MR imaging.

Biodegradable PEGylated Gd-DTPA l-cystine copolymers, PEG-g-poly(GdDTPA-co-l-cystine), were prepared and tested as a blood pool contrast agent in mice. The biodegradable macromolecular agent was designed to be broken down into smaller Gd complexes by endogenous thiols via the disulfide-thiol exchange reaction to facilitate the clearance of Gd complexes after the contrast-enhanced MRI examination. Gd-DTPA l-cystine copolymers were synthesized by condensation polymerization of l-cystine and DTPA-dianhydride in water followed by chelating with Gd(OAc)(3). MPEG-NH(2) (MW = 2000) was then conjugated to the polymeric backbone in different ratios. The macromolecular contrast agent was readily degraded with the incubation of l-cysteine. It also demonstrated superior contrast enhancement in the heart and blood vessels as compared to a low molecular weight control agent, Gd-(DTPA-BMA). At 1 h postcontrast, the PEGylated macromolecular agent still showed prominent enhancement, while little contrast enhancement was detectable in the blood pool by the control agent. PEG-g-poly(GdDTPA-co-l-cystine) shows promise as an MR blood pool imaging agent.

Animals↗

Crosslinked carboxymethylchitosan-g-poly(acrylic acid) copolymer as a novel superabsorbent polymer.

A novel carboxymethylchitosan-g-poly(acrylic acid) (CMCTS-g-PAA) superabsorbent polymer was prepared through graft polymerization of acrylic acid onto the chain of carboxymethylchitosan and subsequent crosslinking. It was demonstrated by FTIR spectroscopy that acrylic acid had been graft polymerized with carboxymethylchitosan. The thermal stability of the polymer was characterized by thermogravimetric analysis. By studying the swelling ratio of the polymer synthesized under different conditions, optimization conditions were found for a polymer with the highest swelling ratio. The rate of water absorption of the polymer was high, and the swelling of the polymer fitted the process of first dynamics. The swelling ratio of the polymer was pH-dependent.

Absorption↗

Ex vivo bioadhesion and in vivo testosterone bioavailability study of different bioadhesive formulations based on starch-g-poly(acrylic acid) copolymers and starch/poly(acrylic acid) mixtures.

Starch-g-poly(acrylic acid) copolymers or grafted starches synthesized by 60Co irradiation or chemical modification and co-freeze-dried starch/poly(acrylic acid) mixtures were evaluated on their ex vivo bioadhesion capacity. The buccal absorption of testosterone from a bioadhesive tablet formulated with the grafted starches or starch/poly(acrylic acid) mixtures was investigated. The results were compared to a reference formulation (physical mixture of 5% Carbopol 974P and 95% Drum Dried Waxy Maize). Rice starch-based irradiated grafted starches showed the best bioadhesion results. Partial neutralization of the acrylic acid with Ca(2+) ions resulted in significantly higher bioadhesion values compared to the reference. Ca(2+) and Mg(2+) partially neutralized maltodextrin-based irradiated grafted starches showed significantly higher bioadhesion values compared to the reference formulation. The chemically modified grafted starches showed significantly higher adhesion force values than for the reference tablet. None of the co-freeze-dried starch/poly(acrylic acid) mixtures showed significantly higher bioadhesion results than the reference (Bonferroni test, P<0.05). A chemically modified grafted starch could sustain the 3 ng/ml plasma testosterone target concentration during +/- 8 h (T(>3 ng/ml)). By lyophilization of a partially neutralized irradiated grafted starch, the in vivo adhesion time (22.0 +/- 7.2 h) and the T(>3 ng/ml) (13.5 +/- 1.3 h) could be increased. The absolute bioavailability of the lyophilized formulation approached the reference formulation. Some of the grafted starches showed to be promising buccal bioadhesive drug carriers for systemic delivery.

Adhesives↗

A kinetic model for the B-Z transition of poly[d(G-C)].poly[d(G-C)] and poly[d(G-m5C)].poly[d(G-m5C)].

The analysis of the kinetic data of the B-Z conformational changes induced by salt in sized double-stranded poly[d(G-C)].poly[d(G-C)] and poly[d(G-m5C)].poly[d(G-m5C)] polymers indicated that there exists a salt threshold which reveals some largely, as yet, unrecognised characteristics of the transition. It was observed that there is a direct correlation between the length of the polymer and the rate of the B-Z transition when the salt concentration in the polymer solution is lower than the salt threshold. The correlation is inverse when the salt concentration is higher than the salt threshold. Thus, the molecular mechanism of the B- to Z-DNA transition varies depending on whether the salt concentration is higher or lower than the threshold. In this context, we have found that the contrasting results reported in the literature describing the rate of the B-Z transition are not contradictory but complementary. The finding of a salt threshold leads to the establishment of a relationship between the cooperativity index of the B-Z transition and the polymer chain length. That relationship is dependent on the chemical structure of the polymer but is temperature independent.

DNA↗

Reovirus protein lambda 3 is a poly(C)-dependent poly(G) polymerase.

Reovirus protein lambda 3 has been isolated from cells infected with two recombinant vaccinia viruses into the TK gene of which the reovirus serotype3 strain Dearing L1 genome segment under the control of the bacteriophage T7 RNA polymerase promoter, or the T7 polymerase gene itself, had been cloned. Highly purified protein lambda 3 does not transcribe double-stranded reovirus RNA into single-stranded RNA, or plus-stranded reovirus RNA into minus-stranded RNA, but it does transcribe poly(C) into poly(G). It prefers Mn2+ to Mg2+. A polymer consisting of poly(C) linked linearly to poly(U) provided template activity only for its poly(C) moiety. Protein lambda 3 forms complexes with protein lambda 1, as well as with protein lambda 2, and with both lambda 1 and lambda 2, which are sufficiently stable to be precipitated by monospecific antisera. None of these complexes are capable of transcribing either ds- or ssRNA.

Base Sequence↗

[Effect of different polyribonucleotide interferonogens on acute and latent viral infections in mice].

Poly(G) . poly(C) and poly(I) . poly(C) complexes administered soon after the viral challenge induced a high survival rate in mice with experimental tick-borne encephalitis. The protective effect was still noted when the treatment was given 24 hours after the infection. If the therapy was conducted at the end of the incubation period, at the peak of the virus reproduction in the mouse brain, poly(I) . poly(C) intensified the infection development and increased the animal death rate, while poly(G) . poly(C) had no such effect. Poly(I) . poly(C) injected 12 hours after the peak of the virus-induced interferonogenesis led to death of 80% animals inoculated with non-pathogenous Newcastle disease virus. The action of various samples of poly(I) . poly(C) was diverse. Poly(G) . poly(C) failed to effect the outcome of latent viral infection. The death of infected mice induced by polyribonucleotide complexes was not connected with their anti-viral interferonogenous activity, but correlated with the level of their toxicity for the intact animals. The results of the study have confirmed the risk of using poly(I) . poly(C) for the therapy of viral infections, especially during their clinical manifestation, and proved the safety of application of poly(G) . poly(C) and of some other polyribonucleotide interferonogens.

Animals↗

Single-strand regions of poly(G) act as templates for oligo(C) synthesis.

Oligonucleotide primers consisting of a sequence of four or more deoxycytidylate residues terminated by a single ribocytidylate residue are extended by reaction with cytidine 5'-phosphoro(2-methyl)imidazolide using polyguanylic acid as a template. The efficiency of the reaction decreases as the length of the primer increases. The reaction does not seem to depend on the dissociation of poly(G) tetrahelices but uses as templates single-stranded segments that are already present in enzymatically synthesized polyguanylic acid.

Base Sequence↗

Identification of poly G bound to thymidylate synthase.

Thymidylate synthase activity is increased in some methotrexate-resistant strains of Streptococcus faecium. The purified enzyme is associated with a polynucleotide which is not removed by dialysis. This polynucleotide contains one mole each of purine ribose and phosphate per mole base. Phosphate analyses after incubation with digestive enzymes indicate a tetranucleotide with one terminal phosphate. The constituent nucleosides are recovered quantitatively in a specific assay for guanosine. On HPLC, they are inseparable from authentic guanosine and the UV spectrum after HPLC is identical to that of guanosine. We conclude that poly G (GpGpGpGp) is bound to thymidylate synthase.

Chromatography, High Pressure Liquid↗

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↗

[Interaction of ethidium bromide with synthetic double-stranded polyribonucleotides].

The interaction of ethidium bromide (EtBr) with double helical synthetic polyribonucleotides poly(G).poly(C), poly(A).poly(U) and poly(I).poly(C) has been investigated by the method of isothermal microcalorimetry and according to the character of changes on the spectra of circular dichroism, absorption and fluorescence at binding. The calculations showed that at binding of EtBr with poly(A).poly(U) the saturation stechiometry was one EtBr molecule per 2 base pairs with binding constant (2.5 +/- 0.5).10(6) M-1 at 30 degrees C and 0.1 M. NaCl. In the case of binding of EtBr with poly(G).poly(C) and poly(I).poly(C) the saturation stechiometry was not less than 0.2 EtBr molecule per 1 base pair with binding constant (4 +/- 1).10(3) M-1 and (1.5 +/- 0.3).10(4) M-1 respectively, at 25 degrees C and 0.1 M NaCl. The binding enthalpies of EtBr with poly(A).poly(U) and poly(G).poly(C) have been determined to be (-7.5 +/- 0.5) Kcal per 1 mol of bound EtBr in average for both polymers. It has been shown that the observed strong selectivity of EtBr binding with polyribonucleotides is of entropic origin.

Circular Dichroism↗