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PubMed1981

Partial characterization of ribonuclease (RNase) activity from the isolated and solubilized brush border of Hymenolepis diminuta.

The isolated brush border membrane of Hymenolepis diminuta contained ribonuclease (RNase) activity which was demonstrable using yeast RNA or synthetic homopolymers of adenylic, cytidylic, inosinic, or uridylic acids as substrates. Polyguanylic acid was not hydrolyzed by worm RNase. RNase activity was inhibited by EDTA and divalent cations as well as sulfhydryl blocking and reducing agents. Polyguanylic acid and DNA were also inhibitors of RNase activity; these compounds were not hydrolyzed, but inhibited the hydrolysis of other substrates, possibly by nonproductive substrate binding. Data suggested that RNase (endonuclease) was probably the major enzyme activity in the degradation of long chain polyribonucleotides at the work's surface, while phosphodiesterase (exonuclease) activity did not contribute significantly to the hydrolysis of these compounds.

Animals↗
PubMed1983

Results of space experiment program "Interferon". I. Production of interferon in vitro by human lymphocytes aboard space laboratory Solyut-6 ("Interferon I") and influence of space flight on lymphocyte functions of cosmonauts ("Interferon III").

The results of the biological space experiment "Interferon" performed by two international cosmonaut crews aboard the space laboratory Solyut-6 are reported. Human lymphocytes separated from the blood of healthy donors and placed into "Interferon I" equipment could be kept for 7 days in suspension culture under spaceflight conditions. Interferon production could be induced in human lymphocytes by preparations of different origin, such as virus, synthetic polyribonucleotides, bacterial protein and plant pigment. An increased lymphocyte interferon production was observed in the space laboratory as compared to the ground control. A decrease of induced interferon production and natural killer cell activity was observed in the cosmonauts' lymphocytes on the 1st day on Earth after 7 days spaceflight.

Cell Membrane Permeability↗
PubMedDate not supplied

[alpha-Interferon indices in systemic lupus erythematosus and the effect of interferon therapy].

The effect of interferon inducers of viral and nonviral nature as well as of exogenous leukocyte interferon on interferon response of leukocytes (IRL) was studied in patients with different rheumatic diseases, mostly in patients with systemic lupus erythematosus (SLE). Studies in vitro showed that synthetic polyribonucleotide poly(1) X poly(C) induced in SLE patients on the average 3 times as high IRL indices as the viral interferon inducer Newcastle disease virus. A two-week course of treatment of some SLE patients with exogenous leukocyte interferon in various dosages (10(6), 3 X 10(5), and 10(4) units) resulted in a 2--16-fold increase of IRL values in the majority of the patients.

Adolescent↗
PubMed1984

Conformation of ribooligonucleotide duplexes containing an alternating C-G sequence which show an unusual circular dichroism spectrum.

The poly[r(G-C)] duplex shows an unusually large negative band in the long wavelength region of the CD spectrum. In order to elucidate this phenomenon, r(C-G-C-G) and r(C-G-C-G-C-G) were synthesized chemically and their properties were examined by UV and CD, and 1H and 31P NMR spectroscopy. These ribooligomers form a self-complementary duplex at low temperature, the CD spectrum of which shows a negative band at around 290 nm and a positive band at around 265 nm with almost equal magnitudes. The proton resonances in the 1H NMR spectra of the oligo[r(C-G)] duplexes were assigned by nuclear Overhauser effect experiments. The chemical shift-temperature profiles of the base proton signals and the sharp singlets observed for all H1' protons are consistent with a normal A-RNA structure but not with a Z-DNA like structure. Moreover, a 500-MHz two-dimensional nuclear Overhauser effect experiment recorded for r(C-G-C-G-C-G) shows that all guanine bases adopt the normal anti-conformation. CD-temperature profiles and 31P NMR spectra of oligo[r(C-G)]s support this conclusion. These results indicate that duplexes of oligo- and polyribonucleotides containing alternating C-G sequences can give an unusually large negative CD band in the long wavelength region despite their right-handed helical structure.

Base Sequence↗
PubMed1983

Deoxyribonuclease A of chick embryo. Partial purification and characterization of the enzyme.

A deoxyribonuclease has been purified 570-fold from the 14-day-old chick embryos. The purified enzyme requires Mg2+ or Mn2+ ions for maximum activity. The optimum pH is 9.0 in 20 mM Tris-HCl buffer. Its isoelectric point is 6.7. NaCl and N-ethylmaleimide strongly inhibit the reaction. An apparent molecular weight of 45,000 is determined by sedimentation in a glycerol density gradient. The enzyme hydrolyzes denatured DNA 50 to 100 times more rapidly than duplex DNA. RNA and synthetic polyribonucleotides are not substrate for the enzyme. DNase A catalyzes the endonucleolytic and exonucleolytic cleavages of single-stranded DNA. The enzyme produces DNA fragments having 70 to 100 nucleotides long at early time of reaction and then degrades these DNA fragments to acid-soluble materials, of which more than 70% is mononucleotides. In the exonucleolytic attack, the enzyme initiates hydrolysis of a single-stranded DNA from 5' to 3' direction. Chick embryo DNA-binding protein gives an intensive effect on the DNase A reaction by inhibiting the endonuclease activity rather than exonuclease activity under the standard assay conditions.

Animals↗
PubMed1981

Enhancement by immunostimulants of the production by mouse spleen cells of factor(s) stimulating differentiation of mouse myeloid leukemic cells.

Mouse myeloid leukemic Ml cells can be induced to differentiate into macrophages and granulocytes in vitro by a factor(s) stimulating differentiation of the cells (D-factor) or by various chemical compounds. Spleen lymphocytes and spleen macrophages have been shown to produce D-factor when treated with various mitogens including lipopolysaccharide or synthetic double-stranded polyribonucleotide, poly(I) . poly(C). Several immunostimulants from microorganisms were tested for ability to induce differentiation of Ml cells and to stimulate production of D-factor by spleen cells. Mycobacterium bovis BCG (500 micrograms/ml) and the cell wall skeleton of Nocardia rubra (50 micrograms/ml) induced differentiation of Ml cells; but Corynebacterium parvum CN6134 and the cell wall skeleton of Propionibacterium acnes C7 had no effect on Ml cells. On the other hand, all immunostimulants tested at concentrations of more than 10 micrograms/ml were found to stimulate production of D-factor by both spleen lymphocytes and spleen macrophages. Synthetic derivatives of N-acetylmuramyl dipeptide(MDP), the minimal adjuvant-active subunit of the bacterial cell wall, had no direct effect on the differentiation of Ml cells and only slightly stimulated the production of D-factor by spleen cells. Therefore, the structure of microorganisms required for induction of differentiation of Ml cells and for stimulation of production of D-factor are suggested to be different from that of MDP.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1980

[Production of directed mutations in T7 phage by transfecting Escherichia coli using locally alkylated T7 DNA].

Modified polyribonucleotides carrying a number of alkylating groups were used for alkylation of complementary sites of DNA to induce gene-directed mutations. Polyfunctional alkylating mustard, N,N,N'-tri-(beta-chloroethyl)-N'-(p-formylphenyl)-propylene diamine-1,3, moieties were attached to T7 phage early transcripts. The transcripts modified to 4-5% were hybridized with T7 DNA to form R-loops, and RNA molecules were covalently bound to the complementary DNA sites as a result of the activation of RNA carried alkylating groups. T7 DNA molecules alkylated by modified RNAs transcribed from 0.3 and 1.1 T7 phage genes were used for transfection of Escherichia coli. In the progeny of phages generated by the transfection of 24 negative colonies, 3 contained double amber mutants in amounts of 2-10%. Complementation tests permitted to assign all mutations to the same complementation group. Recombination analysis established that one of the mutations concerns gene 0.3. The results of complementation studies indicate that the second mutation, with high probability, concerns gene 1.1.

Alkylation↗
PubMed1978

Glycosylation of interferons. Effects of tunicamycin on human immune interferon.

Human immune interferon, induced in leukocytes by phytohemagglutinin, was prepared in the absence and presence of tunicamycin, an antibiotic which selectively inhibits the glycosylation of newly synthesized glycoproteins. Interferon preparations, produced in the absence of the antibiotic, displayed a considerable chromatographic heterogeneity on: (a) concanavalin A-agarose, (b) phenyl-agarose, (c) Cibacron Blue F3GA-agarose, and (d) polyuridylic acid-agarose. This heterogeneity was completely eliminated when tunicamycin (2 microgram/ml) was present during induction of interferon; all activity was then recovered in the breakthrough fractions from all sorbents. The level of interferon activity in leukocyte culture fluid was not affected by tunicamycin within the range of concentration 0.05 to 2.0 microgram/ml. These data indicate that (a) human immune interferon undergoes glycosylation, and tunicamycin is an effective inhibitor of this process. Thus, it appears that (b) at least some of the carbohydrates of human immune interferon are N-glycosidically linked. Moreover, it seems that (c) glycosylation is not necessary for an interferon molecule to either be secreted by the cell or (d) to express its antiviral function. Such properties of human immune interferon as (e) the apparent hydrophobicity and (f) an affinity for a polyribonucleotide are conferred only when its glycosylation is unimpaired.

Glucosamine↗
PubMed1982

Purification and properties of a mammalian tRNA pseudouridine synthase.

A tRNA pseudouridine synthase has been extensively purified from steer thymus extracts, using undermodified tRNA from hisT- mutants of Salmonella typhimurium as a substrate. The enzyme synthesizes a group of psi residues in the anticodon region of various hisT- isoacceptors and behaves like a eukaryotic homologue of Salmonella tRNA psi synthase I. The thymus enzyme requires a thiol and a monovalent cation (NH4+ or K+) for optimum activity; no energy sources or cofactors are required. The activity is inhibited by single tRNAs or bulk tRNA from all sources tested, and by ribosomal RNAs, various polyribonucleotides, and DNA. The enzyme modifies the two hisT- tRNAPhe isoacceptors, both tRNATyr acceptors and at least five of the tRNALeu isoacceptors to products that coelute with the respective wild type species on reverse-phase columns. With pure hisT- tRNA2Phe as substrate, the enzyme specifically converts residue U39 to psi. Interestingly, a psi residue is still present at position 32, in the anticodon loop of hisT- tRNA2Phe, indicating the existence of other uncharacterized pseudouridylation enzymes in S. typhimurium. These composite results show that the thymus enzyme can form psi at residues 38, 39, and 40 in the anticodon region of appropriate hisT- isoacceptors. During the enzyme purification, a second activity is partially resolved, which releases 3H from wild type S. typhimurium [pyrimidine-5-3H]tRNA. This activity may be associated with an enzyme that pseudouridylates sites that are uniquely modified in eukaryotic tRNAs, but not in Salmonella tRNAs. Our observations support the view that the psi residues in tRNA are synthesized by a family of enzymes, whose members act on uridine residues in specific regions of the molecule.

Amino Acyl-tRNA Synthetases↗
PubMed1978

[Specificity of the action of the RNA released by Ehrlich carcinoma cells on the transplantability and growth of a homologous tumor].

A comparative study was undertaken on the effect of different RNA preparations on the transplatability and growth of Ehrlich carcinoma. The RNA isolated from Ehrlich carcinoma ascites fluid was found to render the specific stimulating action on the transplantability and growth of a homologous tumor, while total RNA from bovine liver, tRNA from rabbit liver and synthetic polyribonucleotides show no stimulating effect. The RNA from Ehrlich carcinoma ascites fluid is formed as a result of its release from intact tumor cells, and it seems to be one of the factors responsible for the interaction between the tumor and the organism.

Animals↗
PubMed1981

Immunochemical studies of DNA modified by cis-dichlorodiammineplatinum(II) in vivo and in vitro.

Two rabbits were immunized with native DNA modified in vitro by cis-dichlorodiammineplatinum(II) (cis-Pt). The interactions between the antiserum and several natural and synthetic nucleic acids were studied primarily by radioimmunoassays. Native DNA's modified by platinum compounds with labile groups in the cis position are recognized by the antiserum. No cross-reaction is found with native DNA's substituted by other platinum compounds [trans-dichlorodiammineplatinum(II), cis-diamminotetrachloroplatinum(IV), and chlorodiethylenetriamminoplatinum(II)] and with natural and synthetic modified polyribonucleotides. cis-Pt-modified oligodeoxyribonucleotides and enzymatically hydrolyzed cis-Pt-modified native DNA do not bind to the antiserum. cis-Pt-modified double-stranded synthetic polydeoxyribonucleotides are either hardly recognized or not recognized at all. The antiserum recognized a modified double-stranded helix in DNA which is not formed in several modified synthetic DNA's and RNA's. At least two different antigenic determinants are present in cis-Pt-modified DNA at a ratio of cis-Pt to bases equal to 0.05. Finally, the antiserum does not react with in vivo cis-Pt-modified DNA.

Animals↗
PubMed1980

Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei.

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.

Cell Nucleus↗
PubMed1994

Polyinosinic:polycytidylic acid is a potent activator of endothelial cells.

Polyinosinic:polycytidylic acid (poly I:C) is a synthetic double-stranded polyribonucleotide that elicits immune responses analogous to those observed during viral infection. It is also known to modulate the expression of certain autoimmune disorders including diabetes mellitus in the BB rat and NOD mouse. The mechanism underlying these immunomodulatory effects is not known, but it could involve activation of vascular endothelium. We now report that parenteral poly I:C induces rat pancreatic endothelium to hyperexpress intercellular adhesion molecule 1 (CD54). This is accompanied by a perivascular recruitment of mononuclear cells to the exocrine pancreas. Corollary in vitro studies demonstrated that poly I:C is a potent activator of both rat and human endothelial cells in culture. It upregulates endothelial expression of several leukocyte adhesion molecules, stimulates the release of interleukin-6 and interleukin-8, and antagonizes interferon-gamma induction of major histocompatibility complex class II expression. We conclude that poly I:C activates endothelial cells to express surface molecules and cytokines in a pattern classically associated with leukocyte recruitment. These effects may in part contribute to the immunomodulatory effects of poly I:C in animal models of autoimmunity.

Animals↗
PubMedDate not supplied

[Spectrophotometric complexes of nucleic acids with pyronine G dyes as a test for radiation damage of DNA].

The spectrophotometric titration curves of the complexes of polynucleotide matrix with pyronine G are highly specific depending on the type and conformation of polyelectrolyte (native and denatured DNA, polyribonucleotides, tRNA). It is found that DNA from the liver of experimental animals of different age, permanent inhabitants of the Chernobyl zone, forms complexes with RNA. A method for testing the defects in a DNA secondary structure have been developed based on comparative analysis of the spectra for the complexes of different nucleic acids with dyes and relative curves of spectrophotometric titration.

Animals↗
PubMed1995

[Endogenous cellular reverse transcriptase in the rat brain. A comparative analysis of the authentic enzyme and recombinant reverse transcriptase coded by a mobile genetic element of the LINE class].

A purified preparation of endogenous RNA-dependent DNA-polymerase (reverse transcriptase) earlier identified in rat brain (Ivanov, V.A., Pakhotin, P.I., Bobkova, N.V., and Ilyin, Yu.V. parallel Dokl. RAN (1992). V. 323, P. 173-177) has been obtained. A comparative analysis of the enzyme and recombinant reverse transcriptase coded by the mobile genome element jockey earlier expressed in a heterological cell system (Ivanov V.V., Melnikov A.A., Siunov A.V., Fodor I.I., Ilyin, Yu.V. parallel EMBO J. (1991), V. 10, P. 2489-2495) has been carried out. Like retroviral RNA-dependent DNA-polymerases, these enzymes show preference for polyribonucleotides and can use poly(rCm) as template. Besides they are inhibited by SH-reagents and require bivalent cations (Mg2+ or Mn2+) and detergent and/or KCl as ionic strength carrier. The enzymes differ drastically from retrovirus reverse transcriptases by a number of catalytic properties (low optima of concentration of requisite cations and ionic strength, strong preference for Mn2+, highly efficiency in using poly(rCm), lack of associated RNase H activity) but exhibit a high degree of similarity among themselves with regard to the above properties. It is suggested that endogenous reverse transcriptase from rat brain is a product of expression of the mobile genome element of the LINE family.

Animals↗
PubMedDate not supplied

[Adaptive response in vivo: reasons for increase of radioresistance in CFU-S].

The data concerning the reasons of CFU-S radioresistance increase after low dose irradiation of mice were obtained; the reparation processes in CFU-S adapted by low dose irradiation were shown to be more active than in intact ones. Colony formation increase was demonstrated to be more pronounced in adapted animals when they had been injected by 50 mcg of synthetic polyribonucleotide PolyI-PolyC two days before irradiation in challenge dose. The thymogen immunomodulator administration to adapted and intact donor animals leads to the increase of colony yield in equal extent.

Adaptation, Physiological↗
PubMed1993

Mammalian poly(A)-binding protein II. Physical properties and binding to polynucleotides.

The 49-kDa poly(A)-binding protein II (PAB II) was purified to homogeneity from calf thymus. The 70-kDa poly(A)-binding protein I (PAB I) was obtained in different fractions of the same preparation. Whereas PAB II stimulated poly(A) polymerase, PAB I was an inhibitor. In analytical ultracentrifugation, the predominant form of PAB II was a monomer of 50.3 kDa. A sedimentation constant of only 2.2 S indicated a distinctly non-spherical shape. Binding was specific for single-stranded purine polyribonucleotides. The dependence of the dissociation constant on the length of oligoriboadenylate indicated a binding site size of 12 nucleotides. A single site was bound with a KD of 2 x 10(-9) M, as determined by nitrocellulose filter binding assays. From fluorescence quenching and gel retardation experiments, the packing ratio on poly(A) was estimated as 23 nucleotides/protein monomer.

Animals↗
PubMed1995

[The interferonogenic properties of yeast RNA-tilorone complexes in a cell culture].

The use of molecular complexes of yeast RNA with thyloron as inductors of type I interferons (alpha/beta-interferons) in the culture of cells L929 is described. It was shown that the complexes induced the interferon synthesis in the cells at the level comparable to that of the standard inductors of the polyribonucleotide nature i.e. larifan and poly(I)-poly(C). In the experimental doses the complexes proved to be nontoxic. It was concluded that the use of the yeast RNA and thyloron complexes as inductors in large-scale production of type I interferons was promising.

Animals↗