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PubMedDate not supplied

Effects of polynucleotides on production and activity of streptolysin S.

Activity of various natural and synthetic polynucleotides as a carrier for streptolysin S was tested in a resting cell system. As the carrier, intact molecules of MS2 RNA, E coli tRNA or rat liver RNA were almost inactive, whereas RNase I core of these RNAs, especially the core fractions eluted from DEAE-cellulose column at higher NaCl concentrations, effectively induced production of the extracellular hemolysin. The carrier activity of yeast RNA was significantly enhanced by simultaneous addition of RNase I, to the streptococcal suspension. Preincubation of yeast RNA with growing streptococci or a protein fraction from the bacterial culture supernatant increased its carrier activity. Evidences were obtained suggesting involvement of streptococcal nuclease in the enhancement of the carrier effect. Production of the streptococcal hemolysin was markedly promoted by polyguanylic acid but neither by polyadenylic acid, polycytidylic acid nor by polyuridylic acid. Like trypan blue, polyguanylic acid exerted potent inhibitory effect on hemolytic activity of streptolysin S complex. Similar but less marked effect was observed with certain RNase I core fractions of rat liver RNA. Some data concerning effect of polynucleotides on stability of the hemolysin were also presented.

Animals↗
PubMed1991

The effects of polyamines on the immunogenicity of polynucleotides.

Polyamines--putrescine, spermidine, and spermine--are small organic cations that are present in all living cells. Recent studies revealed that polyamines could provoke a left-handed Z-DNA conformation in poly(dA-dC).poly(dG-dT) and related alternating purine-pyrimidine sequences. In order to examine whether polyamine-induced Z-DNA conformation of poly(dA-dC).poly(dG-dT) is capable of eliciting anti-Z-DNA antibodies, we immunized rabbits with poly(dA-dC).poly(dG-dT) in the presence and absence of spermidine and spermine. Rabbits immunized with the polynucleotide alone produced antibodies reacting toward poly(dA-dC).poly(dG-dT) and heat-denatured calf thymus DNA (ssDNA). In contrast, immunization with poly(dA-dC).poly(dG-dT) complexed with spermidine or spermine produced antibodies reacting with Z-DNA in addition to those binding toward poly(dA-dC).poly(dG-dT) and ssDNA. Antibodies elicited by polynucleotide.polyamine complexes had no reactivity toward polyamines. Solution inhibition studies suggested that anti-poly(dA-dC).poly(dG-dT), anti-ssDNA and anti-Z-DNA antibodies are distinct populations that favor each one of these antigens. Our results suggest that natural polyamines are capable of altering the immunogenicity of polynucleotides by mechanisms involving the stabilization of Z-DNA conformation. This result may have implications in the recent findings of high levels of polyamines and anti-Z-DNA antibodies in the sera of lupus patients and autoimmune mice.

Animals↗
PubMed1976

Effects of synthetic single- and multistranded polynucleotides on human monocyte IgG receptor activity in vitro.

Human peripheral monocytes demonstrated a higher IgG receptor activity in vitro after incubation with multistranded polynucleotides (poly(I:C) and poly(I:C:U)) after 6-hr cultivation periods. These changes were most pronounced using SRBC coated with low anti-Forssman antibody concentrations (EA, 1:1600 and 1:6400). Polyinosinic and polycytidylic acids failed to show changes in receptor activity. These findings suggest monocyte activation in vitro by multistranded polynucleotides and may in part explain their antitumor activity.

Cell Membrane↗
PubMed1999

Modification of proteins and polynucleotides by peroxynitrite.

Varied intensities of nitrotyrosine immunoreactivity were detected by Western blots after the reaction of proteins or enzymes with peroxynitrite (PN), a strong oxidant derived from nitric oxide. Intense immunoreactivity of cAMP-dependent protein kinase, calmodulin and most histones may depend on greater access to tyrosine residues in the reaction, whereas the absence of immunoreactivity of caspase-3, ubiquitin and S-100 proteins may reflect lack of accessibility. In addition, the changes in UV/visible absorbency were observed after PN-treatment of polynucleotides, polypeptides or proteins. Brief PN-treatment of invertase increased its enzymatic activity. Furthermore, PN-treatment of rabbit IgG decreased its recognition by anti-IgG. The results suggest that PN may chemically modify polypeptides, proteins and polynucleotides and may subsequently alter their biological activity.

Animals↗
PubMed1976

Effects of some polynucleotides on DNA synthesis in human lymphocytes.

The effect of several single-and double-stranded synthetic polyribonucleotides on the 3H-thymidine incorporation in intact and PHA-stimulated human lymphocyte cultures was investigated. Only polyuridilic and polyguanilic acids were found to stimulate DNA synthesis in the intact cells. All other polynucleotides tested had much weaker or had no stimulating activity. PHA-activated lymphocytes responded to addition of polynucleotides by the decrease of 3H-thymidine uptake. The mostly profound inhibition of lymphocyte response to PHA was observed with polyguanilic acid.

Adjuvants, Immunologic↗
PubMed1990

Oligonucleotides with pyrene fluorophore at the sugar fragment: synthesis and properties in binding to complementary polynucleotide.

Oligonucleotides with 1-pyrenylmethyl substituent at the designated sugar residue were synthesized by using 5'-dimethoxytrityl 2'-(1-pyrenylmethyl)uridine 3'-phosphorobisdiethylamidite 1. It was shown that the pyrene-oligonucleotides have enhanced affinity in binding to the complementary polynucleotide sequence. The fluorescence yield and life time of the pyrene-oligonucleotide was drastically enhanced when bound by the complementary polynucleotide.

Base Sequence↗
PubMed1990

[Stepwise synthesis of oligonucleotides. XXXV. Native and immobilized polynucleotide phosphorylase from Thermus thermophilus in oligoribonucleotide synthesis].

A polynucleotide phosphorylase was isolated from the Thermus thermophilus protein fractions, obtained at different steps of purification of elongation factors, and immobilized on agarose activated with cyanogen bromide and macroporous glass modified with (3,3-diethoxypropyl)triethoxysilane. The preparations of the native and immobilized enzyme catalyzed rather efficiently the addition of adenylyl and guanylyl residues to oligonucleotide primers, in contrast to the E. coli and M. luteus polynucleotide phosphorylases. Tri-, tetra- and pentanucleotides with 3'-terminal guanosine and adenosine were obtained including structural analogues of the anticodon fragment 34-37 of yeast tRNA(Phe).

Catalysis↗
PubMed1985

[General method of isolation and analysis of polynucleotide fragments cross-linked with proteins].

A fragment of 16S RNA, cross-linked to S7 protein by UV irradiation of the 30S subunit of E. coli ribosome, was obtained by the action of T1 ribonuclease on the irradiated nucleoprotein. The digest was treated with polynucleotide kinase in the presence of [gamma-32P]ATP and the S7-cross-linked oligonucleotides were isolated. An individual oligonucleotide attached to S7 protein was obtained after proteinase treatment of the respective spot followed by electrophoresis. Sequencing of this oligonucleotide established its structure as 1233-1240 fragment of 16S RNA, the U1239 residue being the site of the S7 cross-linking. The developed general approach can be used for localizing protein - cross-linked residues in polynucleotides, whatever is the procedure employed for cross-linking.

Base Sequence↗
PubMedDate not supplied

[Antisense polynucleotides and prospects for their use in fighting viruses].

Natural or synthetic anti-sense (as) polynucleotides complementary to distinct functional regions of mRNA (asRNA or asDNA) are able to inhibit the expression of any target gene. If certain viral mRNAs important for virus replication are targeted the inhibition of viral infection by asRNA or asDNA takes place. Inhibitory effects of complementary polynucleotides on gene activity in eukaryotic cells is due to the disturbance of translation of corresponding mRNAs as well as to the impairment of their splicing or transportation from the nuclei to cytoplasm. In prokaryotic cells, obviously, only the first factor is operating. The recombinant genes programming anti-viral asRNA can confer the resistance to the infection by other virus to the transformed cells. The resistance to viral infection observed in transgenic animals, expressing asRNA genes, may be considered as a new unnatural form of informational immunity.

Base Sequence↗
PubMedDate not supplied

[Possibility of spectrophotometric registration of conformation changes of polynucleotides in the system poly(A:U)-pyronine G].

The spectrophotometric study of poly(A:U) (90:10)-pyronine G (PG) complex formation has shown that the interaction of PG with polyriboadenylic acid pH = 6.3 is not changed despite the presence of 10% of pyrimidine bases in the single-stranded polynucleotide chain. Analysis of the dependence of complex formation versus acidity increase in the visible spectral range suggests that there are two forms of protonated double-stranded helix of poly(A:U) (90:10), as well as poly(A). PG dye does not interact with the more strongly protonated form by means of the aggregation. There is a strong resemblance between the interaction of PG with the less strongly protonated form and with the neutral single-stranded form of poly(A:U) (90:10). One can determine pH-transition ranges of the two poly(A:U) (90:10) protonated forms using the difference in their interaction with PG at low concentrations of polynucleotide.

Hydrogen-Ion Concentration↗
PubMed1977

Purification and characterization of polynucleotide phosphorylase from Escherichia coli. Probe for the analysis of 3' sequences of RNA.

A simple procedure for purifying polynucleotide phosphorylase from Escherichia coli cells by means of affinity chromatography on an RNA-Sepharose column is described. The purified enzyme preparation has a specific activity 3500-fold that of the crude extract and is essentially homogeneous, as determined by ultracentrifugation, polyacrylamide gel electrophoresis under denaturing conditions, isoelectric focusing and serological assays. It is virtually free of nuclease contamination, a property which permits its use in the synchronous phosphorolysis of RNA chains. The enzyme molecule is composed of three identical subunits of Mr = 84,000. Each subunit contains three cysteine residues, one of which reacts with 5,5'-dithiobis(2-nitrobenzoic acid) whereas the two other groups are only exposed on denaturation of the protein. All three enzyme subunits participate in the processive phosphorolysis of the poly(A) tail of each globin mRNA chain. An advantageous method was developed for synchronous phosphorolysis of RNA molecules using a molar excess of polynucleotide phosphorylase immobilized onto Sepharose.

Chromatography, Affinity↗
PubMed1986

Neutral reactions of haloacetaldehydes with polynucleotides: mechanisms, monomer and polymer products.

The generally accepted mechanism for the formation of etheno derivatives upon reaction of adenosine or cytidine with haloacetaldehydes involves two intermediates. The first, a primary addition to the exocyclic amino group, has not been experimentally verified. The second, a cyclic form of the first intermediate, has been described in monomers but presumed to be too unstable to exist in polynucleotides since such derivatives would be readily dehydrated to other derivatives at pHs below neutrality. We have found that the cyclic intermediates of adenosine and cytidine are the predominant products in polynucleotides, even upon extensive reaction with chloroacetaldehyde at neutrality. The hydrated compounds have half-lives at pH 7, 37 degrees C, of 1.4 h and 13 h for adenosine and cytidine, respectively. Two types of evidence are presented for the existence of the first intermediate, a (1-hydroxy-2-chloroethyl)-substituted exocyclic amino group. Firstly, poly d[A-T] cannot form etheno derivatives (except when denatured) and the observed cross-linking is therefore attributed to alkylation by the chlorinated sidechain of the adenine residue (A), acting on the N6 of A on the opposite strand. Secondly, our results show that blocking of the acceptor nitrogen, needed for cyclization, leads to the formation of relatively stable derivatives of adenosine and cytidine. Guanosine, as a monomer, is modified extensively, but in synthetic polymers no reaction was detected, possibly due to secondary structure.

Acetaldehyde↗
PubMed1987

Transfer of recA protein from one polynucleotide to another. Kinetic evidence for a ternary intermediate during the transfer reaction.

We have analyzed the transfer kinetics of recA protein from one polynucleotide to another by monitoring the change in fluorescence of a modified single-stranded M13 DNA, referred to as etheno M13 DNA, that accompanies recA protein dissociation. The observed rate of transfer is dependent on the concentration of competitor polynucleotide, polythymidylic acid (poly(dT]; increasing the poly(dT) concentration increases the rate of transfer. These data are inconsistent with the rate-limiting step in the transfer mechanism occurring by a simple dissociation process. Under certain conditions, the apparent rate constant displays plateau behavior at high poly(dT) concentrations. This result is indicative of transfer occurring through a ternary intermediate including etheno M13 DNA and poly(dT). The transfer reaction was found to occur through two kinetically distinct species of transferring recA protein X DNA complexes. The relative amounts of these two species was affected by both the MgCl2 and protein concentration, suggesting that the two kinetic components reflect different aggregation states of the recA protein X DNA complex. Because etheno M13 DNA and poly(dT) contain no complementary sequences, we have concluded that recA protein has the intrinsic ability to form a kinetic ternary intermediate with two separate single-stranded DNA molecules in the absence of homology.

Adenosine Diphosphate↗
PubMedDate not supplied

[Interaction of oligophosphates of pyridoxal with certain enzymes of polynucleotide synthesis].

The interaction of pyridoxal, pyridoxal-5'-mono-, di- and triphosphate with certain enzymes of polynucleotide synthesis (DNA-dependent RNA polymerase, DNA-dependent DNA polymerase I and polynucleotide phosphorylase from Escherichia coli and terminal deoxyribonucleotide transferase from calf thymus) was studied. All compounds tested was found to be reversible and competitive inhibitors of these enzymes. The reduction of the enzyme-inhibitor complex with NaBH4 gives rise to the complete irreversible inhibition of the enzymes under study. The comparison of the inhibition constants for pyridoxal and its phosphorylated derivatives with those for mono-, di- and triphosphates of nucleosides was carried out for the enzymes. The results obtained suggest that the modified epsilon-amino-group of lysine residue should be localized at the catalytic site in the vicinity of the pyrophosphate binding area of an enzyme.

Animals↗
PubMed1985

In vivo augmentation of rat lung natural killer cell activity and inhibition of experimental metastases by double-stranded polynucleotides.

The in vivo effect on natural killer cell activity of the compound rln X r(C12 X U)m, a mismatched nontoxic analogue of polyinosinic-polycytidylic acid [rln X rCn], was tested in inbred Wistar AG rats by measuring the natural killer cell activity of lymphocytes freshly isolated from lung capillaries. Lysis was measured in a 4-h chromium release assay using YAC-1 or syngeneic P77 lung tumor cells as target. Treatment of an animal with rln X r(C12 X U)n substantially enhanced natural killer cell activity to an extent comparable to the increase produced by rln X rCn. This boosting of natural killer activity was correlated closely with a decrease in the incidence of experimental pulmonary metastasis. However, the beneficial effect lasted for only a short period after stimulation, due to the transient nature of double stranded polynucleotide enhancement and to the concomitantly rapid extravasation of tumor cells into the lung parenchyma. It is concluded that natural killer cells are important in host defense against circulating tumor cells and can be boosted by nontoxic mismatched double stranded polynucleotides.

Animals↗
PubMedDate not supplied

Immunological recognition of three polynucleotide structures associating adenylic and uridylic acids.

The reactions of antibodies directed against the two double-stranded synthetic polyribonucleotide complexes poly(A).poly(U) or poly(I). poly(C) with three polynucleotide structures associating equimolar amounts of adenylic and uridylic acids were studied by immunodiffusion, quantitative precipitation and readioimmunoassay. The three sequence isomers of the same base composition but different nucleotide distribution were: the double helical complex poly(A).poly(U) containing two homopolymeric strands; the copolynucleotide poly(A-U) composed of a strictly repeating riboadenylic acid and ribouridylic acid sequence in both strands; and the copolymer poly(A,U) where the two strands contain both residues but in a random distribution. The three antigens reacted with anti-poly(A).poly(U) and antipoly(I).poly(C) antisera but not to the same extent. The random copolymer poly(A,U) reacted poorly with sera of both specificity. In contrast the reactivity of the alternating copolnucleotide poly(A-U) was widely different according to the specificity of the sera used. Whereas it was recognized by the anti-poly(A).poly(U) antibodies almost to the same extent than the homologous complex, its activity was much lower with anti-poly(I).poly(C) antisera. In addition, the relative efficiency of poly(A-U) was approximately 50 times lower than that of poly(A).poly(U) when tested with anti-poly(I).poly(C) antibody, thereby indicating recognition of different antigenic determinants in both duplexes. Antibodies directed against defined conformational determinants are therefore able to distinguish between three polynucleotide structures of the same base composition but different sequence.

Animals↗
PubMedDate not supplied

[Use of a TLC method for the simultaneous determination of an enzyme complex of nucleic acid metabolism during the isolation of polynucleotide phosphorylase].

For the simultaneous determining of several enzymes of nucleic acid metabolism during polynucleotide phosphorylase isolation TLC was used. It was found that using TLC one can simultaneously detect six and more enzymes, e. g. polynucleotide phosphorylase, 5'-nucleotidase, exoribonuclease together with nucleosidediphosphatase, desaminase etc. The method is simple and accessible.

5'-Nucleotidase↗