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PubMed2005

Complex-formation of Ethidium Bromide with poly[d(A-T)].poly[d(A-T)].

The interaction of Ethidium Bromide (EtBr) with double-stranded (ds-) and single-stranded (ss-) poly[d(A-T)] was studied in different ionic strengths solutions. Optical spectroscopy and Scatchard analysis results indicate that the ligand interacts to both helix and coiled structures of the polynucleotide by "strong" and "weak" binding modes. The association parameters (binding constant -K- and the number of nucleotides corresponding to a binding site -n) of the strong type of interaction were found to be independent of Na+ concentration. Weak interaction occurs at low ionic strength and/or high EtBr concentration. Estimated binding parameters of EtBr with ss- and ds-polynucleotide are in good agreement with those for EtBr-B-DNA complexes. Data obtained provided an evidence for a stacking interaction of EtBr with single stranded poly[d(A-T)].

Ethidium↗
PubMed1985

Induction of human interferon gamma in nylon-column nonadherent human lymphocyte cells by heat-treated poly I:poly C.

Human peripheral blood mononuclear leukocytes (PBML) were separated by Ficoll-hypaque gradient from human peripheral whole blood cells. The PBML were further separated by nylon-column. When nylon-column nonadherent cells (NNA cells) were induced by poly I:poly C which was prepared by heat-treatment, high levels of acid- and heat-labile interferon (IFN) were obtained. The IFN was not neutralized with human IFN-alpha or -beta anti-serum, but was completely neutralized with human IFN-gamma anti-serum, indicating that the IFN was human IFN-gamma.

Cell Adhesion↗
PubMed1986

Production and characterization of poly(I):poly(C)-induced rat interferons in vitro.

The synthetic polyribonucleotide polyinosinate:polycytidylate [poly(I):poly(C)] was used to induce interferon (IFN) production in a rat leiomyosarcoma cell line. Three separate IFN species were distinguished by antiviral activity on homologous and heterologous cells, interaction with Blue Sepharose CL-6B chromatography matrix, and antibody neutralization studies. Two IFN species, one with a weakly ionic and the other with a hydrophobic interaction with Blue Sepharose, exhibited heterologous antiviral activity, and were acid- and heat-labile. Both had a molecular weight of 30,000 daltons by HPLC-based molecular exclusion chromatography. The weakly ionic species was neutralized only by antiserum to L-cell IFN, whereas the hydrophobic species was neutralized by antiserum to L-cell IFN and antiserum to human leukocyte IFN. The remaining IFN species, with a weakly ionic interaction with Blue Sepharose, was acid- and heat-stable, exhibited no heterologous cell antiviral activity, and was not neutralized by any antisera to IFN. This species had a molecular weight of 39,000 daltons by HPLC-based molecular exclusion chromatography and 34,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These rat fibroblast IFNs possess greater heterogeneity than that reported or observed for murine or human fibroblast IFNs. These IFNs could thus be classified as follows: one species of rat IFN-beta and two different species of unique acid-labile rat IFNs.

Animals↗
PubMed1997

Chemotherapeutic purine analogs alter the level of interferon-beta mRNA induced by poly I-poly C in cultured osteosarcoma cells.

The purine nucleoside analogs fludarabine, 2-chlorodeoxyadenosine, and 2'-deoxycoformycin exhibit impressive activity in lymphoproliferative malignancies of adults and children. Their mechanism of action is not clear. Studies have suggested that their use is associated with significant myelosuppression, immunosuppression, and in some circumstances, increased infection with viral and opportunistic pathogens. Because interferons (IFNs) are known to have immunomodulatory activity as well as potent antiproliferative and antiviral activity, we examined whether the chemotherapeutic purine nucleoside analogs alter interferon-beta (IFN-B) gene expression in MG63 in human osteosarcoma cells. Northern blot analysis showed a dose-dependent inhibition of IFN-B mRNA accumulation in response to a known inducer (Poly I-Poly C) all three purine analogs. Hybridization analysis also revealed that inhibition of IFN-beta mRNA accumulation by the purine analogs is not a result of decreased mRNA stability. Further analysis of gene expression by PCR differential display indicated that the effect of the purine analogs was restricted to only a limited number of inducible genes. The data suggest that these molecules alter the signaling process involved in regulating the expression of specific genes, including IFN-beta. These findings predict that the use of purine nucleoside analogs may reduce IFN production in vivo and thereby abrogate host defenses against infectious pathogens.

Antineoplastic Agents↗
PubMed1982

Conformational transitions of poly(dA-dT)poly(dA-dT) in ethanolic solutions.

Examination of circular dichroic and phosphorus nuclear magnetic resonance spectra showed that poly(dA-dT)-poly(dA-dT) exhibited an ethanol-induced transition to the A form in an Na+ containing medium like natural DNAs. A mere replacement of the Na+ by Cs+ counterions meant that the polynucleotide was with a little cooperativity transformed into a novel conformation displaying a deep negative band in the long wavelength part of the CD spectrum. The presence of very low concentration of Cs2+ shifted the midpoint of the transition to a lower content of ethanol.

Circular Dichroism↗
PubMed1984

Structure of a pleiomeric form of poly d(AT):poly d(AT).

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.

Base Sequence↗
PubMed1985

Oligomerization of activated derivatives of 3'-amino-3'-deoxyguanosine on poly(C) and poly(dC) templates.

3'-amino-3'-deoxyuridine reacts with the nucleoside 5'-phosphorimidazolides in aqueous solution to give dinucleoside phosphoramidates. The reactions are one to two orders of magnitude faster than the corresponding reactions of uridine. In the presence of poly(C) or poly(dC) it is known that guanosine-5'-phosphorimidazolide does not condense efficiently or regiospecifically. However, the introduction of a methyl group at the 2-position of the imidazole ring leads to efficient synthesis of long 3'-5'-linked oligomers. The corresponding imidazole derivatives of 3'-amino-3'-deoxyguanosine-5'-phosphate both condense on these templates to give virtually identical families of products. Our results suggest that the intrinsically greater nucleophilicity of the amine groups will permit a much wider range of efficient template-directed syntheses with 3'-amino-3'-deoxynucleoside derivatives than with the corresponding derivatives of the parent nucleosides.

Chromatography, High Pressure Liquid↗
PubMed1976

Toxicity of poly(rI).poly(rC) for interferon-treated cells. An ultrastructural evaluation.

The cytotoxic effect of poly(rI).poly(rC) for interferon-treated cells is preceded by discrete ultrastructural alterations in (1) the mitochondria: swelling of the cristae and increased density of their matrix, (2) the nucleus: disorganization of the structure, and (3) the endoplasmic reticulum: slight dilatation with disorganization and depletion of the filament bundles. Our results do not allow us to conclude whether any of these alterations is a primary effect of the treatment. Possibly they are secondary to some, as yet unknown, functional defect, and only reflect incipient cell death.

Animals↗
PubMed1979

Priming increases the amount of interferon mRNA in poly(rI).poly(rC)-treated L cells.

Priming by mouse interferon pre-treatment resulted in an accumulation of interferon mRNA in poly(rI).poly(rC)-treated L cells, starting early in the period of interferon synthesis. On electrophoresis, the priming activity of an interferon preparation co-migrated with the antiviral activity, which suggests identity of the functional principle(s) for these activities.

Animals↗
PubMed1992

Transition from rapid processive to slow nonprocessive polyadenylation by vaccinia virus poly(A) polymerase catalytic subunit is regulated by the net length of the poly(A) tail.

The mRNA of vaccinia virus, like that of eukaryotes, possesses a poly(A) tail. VP55, the catalytic subunit of the heterodimeric vaccinia virus poly(A) polymerase, was overexpressed and purified to near homogeneity. VP55 polyadenylated a 30-mer primer representing the 3' end of a vaccinia virus mRNA bimodally: 30-35 adenylates were added in a rapid, processive, initial burst, after which polyadenylation decelerated dramatically and became nonprocessive. Polyadenylation of variants of the 30-mer primer, which contained preformed 3'-oligo(A) extensions, showed that the transition between the two modes of polyadenylation was regulated by the net length of the 3'-oligo(A) tail rather than the number of adenylate additions catalyzed by VP55. Primers comprising oligo(A) alone were polyadenylated only if they were greater than 34 nucleotides in length and, then, only in the slow nonprocessive mode. These data support a dynamic model whereby the mode of polyadenylation by VP55 is regulated by sequences within the 3' 30-35 nucleotides of the mRNA: Polyadenylation is rapid and processive until a net 3'-oligo(A) length of 30-35 nucleotides is achieved. Consistent with this, excess oligo(A) did not compete with the 30-mer primer for rapid processive polyadenylation. The primer specificity of VP55 may contribute to the selective polyadenylation of newly formed mRNA.

Adenosine Monophosphate↗
PubMed2005

Interaction of anti-proliferative protein Tob with poly(A)-binding protein and inducible poly(A)-binding protein: implication of Tob in translational control.

Tob is a member of an emerging family of anti-proliferative proteins that suppress cell growth when over-expressed. tob mRNA is highly expressed in anergic T cells and over-expression of Tob suppresses transcription of interleukin-2 (IL-2) through its interaction with Smads. Here, we identified two types of cDNA clones coding for poly(A)-binding protein (PABP) and inducible PABP (iPABP) by screening an expression cDNA library with the GST-Tob probe. Co-immunoprecipitation and GST-pull down experiments showed that Tob associated with the carboxyl-terminal region of iPABP. We then found that iPABP, like PABP, was involved in regulation of translation: iPABP enhanced translation of IL-2 mRNA in vitro. The enhanced translation of IL-2 mRNA required the 3'UTR and poly(A) sequences. Tob abrogated the enhancement of translation through its interaction with carboxyl-terminal region of iPABP in vitro. Consistently, over-expression of Tob in NIH3T3 cells, in which exogenous iPABP was stably expressed, resulted in suppression of IL-2 production from the simultaneously transfected IL-2 expression plasmid. Finally, Tob, whose expression was induced by anergic stimulation, was co-immunoprecipitated with iPABP in human T cells. These findings suggest that Tob is involved in the translational suppression of IL-2 mRNA in anergic T cells through its interaction with iPABP.

3' Untranslated Regions↗
PubMed1991

Post-transcriptional effect of ultraviolet light on gene expression in human cells. Stabilization of cytokine-induced and poly(I).poly(C)-induced messenger RNA.

It is well established that ultraviolet light modulates gene expression in mammalian cells, particularly at transcriptional and post-translational levels. The present study was undertaken to investigate whether the fate of mRNA is also altered in ultraviolet-light-irradiated human cells. In order to facilitate distinction between transcriptional and post-transcriptional effects, this analysis has focused on six genes whose transcription is conditional on the supply of exogenous inducers, interferon-alpha, interleukin-1 alpha or the double-stranded RNA, poly(I).poly(C). Human cells induced to express these genes were found to retain a significantly higher concentration of corresponding transcripts when irradiated with ultraviolet light at the end of the inducing treatment. This stimulation was due to dose-dependent ultraviolet-light stabilization of preformed mRNA, as shown by run-on and pulse/chase experiments. This work uncovers a new facet of the cellular response to genotoxic stresses, i.e. extension of the life-span of transcription products. Whether this stabilizing effect contributes to cell recovery by promoting gene expression remains to be determined.

Blotting, Northern↗
PubMed1991

Excited-state properties of the alternating polynucleotide poly(dA-dT).poly(dA-dT).

Measurements of the steady-state fluorescence spectrum and anisotropy, r, of the alternating polynucleotide poly(dA-dT).poly(dA-dT) were carried out in order to characterize its photophysical properties at room temperature. The shape of the fluorescence spectrum depends on the excitation wavelength, namely, the relative fluorescence intensity of the short-wavelength peak decreases for excitation at short wavelengths. When monitoring the emission at short wavelengths, r is 0.18 and independent of the excitation wavelength. When monitoring the emission at long wavelengths, however, r is very low, about 0.03. These results suggest that: (i) the short-wavelength emission stems from thymine; and (ii) the long-wavelength emission stems from an excited-state complex (excimer), with the same one being formed regardless of whether thymine or adenine is excited. The corresponding fluorescence spectra have been resolved. The occurrence of transfer of electronic energy is discussed.

Fluorescence Polarization↗
PubMed1978

Properties of poly(ADP-ribose) synthetase from rat pancreas and poly(ADP-ribosylation) of basic nuclear proteins.

The isolated nuclei of rat pancreas contain an enzyme system that will incorporate 3H-labeled NAD into an acid-insoluble product, which is shown to be poly(ADP-ribose). The enzyme has an optimum pH of 7.8 and the optimum temperature is between 20 and 30 degrees C. Optimum Mg2+ concentration is 8 mM and dithiothreitol also stimulates the enzyme at a concentration of 8 mM. Under standard conditions, the Km value for the reaction is 0.25 mM and an inhibition by the substrate is observed at high substrate concentrations. It has also been found that only one basic nuclear protein, that is, histone H1, is modified by the synthetase. An average chain length of 5.0 is found in the nuclei and of 4.5 on histone H1. Radioautographic studies show that poly(ADP-ribose) is closely associated with chromatin.

Animals↗
PubMed1996

Binding of anti-double stranded (ds) DNA-positive sera to denatured (d) DNA and synthetic poly[dA-dT] x poly[dA-dT] double stranded copolymer in an ELISA format.

Using an ELISA assay anti-nuclear antibody-positive sera from 300 patients with various immune-related diseases and 64 anti-nuclear antibody-negative sera were analysed for binding to S1-nuclease-treated double stranded (ds) DNA. In addition, the pattern of reactivity of 50 selected anti-dsDNA-positive sera was established using denatured (d) DNA and poly[dA-dT] X poly[dA-dT] double-stranded alternating copolymer (dAT) as additional DNA antigens. None of the 64 anti-nuclear antibody-negative sera and 76 of the 300 anti-nuclear antibody-positive sera (25%) were anti-dsDNA-positive. Of the anti-nuclear antibody-positive and anti-dsDNA-positive sera, 48 (63%) were from systemic lupus erythematosus patients, and 7 (9%) from rheumatoid arthritis patients, whereas 21 patients (27.6%) suffered from various immune and non-immune related diseases. Anti-dsDNA-positive reactivity was highly correlated with dDNA and dAT reactivity (r = 0.906, p < 0.0001 and r = 0.93, p < 0.0001, respectively). Although the majority of the 50 selected (37 systemic lupus erythematosus and 13 non-systemic lupus erythematosus) anti-dsDNA-positive sera concomitantly bound to both additional antigens, 7 of these (14%) did not bind to dAT, and 2 (4%) did not bind to dDNA. Anti-dsDNA-positive sera (n = 37) showed a similar pattern, in which 8.1% and 2.7% of sera did not bind to dAT and to dDNA, respectively. In contrast, anti-dsDNA-negative sera from various immune-related diseases bound either ssDNA (12.5%) or dDNA and dAT (12.5%). These data suggest that dsDNA and dAT-based assays detect similar but not identical specificities in the sera of patients suffering from systemic lupus erythematosus and in a proportion of non-systemic lupus erythematosus patients.

Antibodies, Antinuclear↗