Induction of differentiation of mouse myeloid leukemia cells by poly(ADP-ribose).

The effects of poly(ADP-Rib) on the differentiation of mouse myeloid leukemia cells were studied. The myeloid leukemia cells differentiated into cells with phagocytic activity, Fc receptors, and lysozyme activity on treatment with poly(ADP-Rib). Cells with morphological characteristics of macrophages and granulocytes also appeared on incubation with poly(ADP-Rib). Dextran sulfate and polyvinylsulfate were also effective for the induction of phagocytic cells, but poly(A), poly(U), poly(C), poly(I), poly(I) · poly(C), and poly(A) · poly(U) were not. The uptake of poly(ADP-Rib) by the myeloid leukemia cells is discussed in relation to their differentiation.     

Induction of interferon by polyribonucleotides containing thiopyrimidines.

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).     

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.     

Differential susceptibilities of DNA polymerases-alpha and -beta to polyanions.

The effects of various polyanions including synthetic polynucleotides on DNApolymerases-alpha and -beta from blastulae of the sea urchin Hemicentrotus pulcherrimus and HeLa cells were studied. Only DNA polymerase-alpha was inhibited by polyanions, such as polyvinyl sufate, dextran sulfate, heparin, poly(G), poly(I), poly(U) and poly(ADP-Rib). Of the various polynucleotides tested, poly(G) and poly(I) were the strongest inhibitors. Kinetic studies showed that the Ki value for poly(G) was 0.3 microgram/ml and that poly(G) had 20-fold higher affinity than activated DNA for the template-primer site of DNA polymerase-alpha. Poly(U) and poly(ADP-Rib) were also inhibitory, but they were one hundredth as inhibitory as poly(G) or poly(I). Poly(A), poly(C), poly(A).poly(U) AND POLY(I).poly(C) were not inhibitory to DNA polymerase-alpha. In contrast, DNA olymerase-beta was not affected at all by these polyanions under the same conditions.     

Hybridization of polymers of antibiotic C-nucleoside phosphates, poly(formycin phosphate) and poly(laurusin phosphate)

The ability of complex formation of poly-(formycin phosphate), poly(F), and poly(laurusin phosphate), poly(L), with the polymers of natural polynucleotides was examined mainly by mixing experiments in 0.1 M NaCl-0.05 M sodium cascodylate buffer (pH 7.0) at 2 degrees. Poly(F) formed complexes with poly(U) and poly(I) in the ratio of 1:1 and 1:2, respectively. Poly(L) formed complexes with poly(A) in 2:1 ration and poly(C) in 1:2 and 2:1 ratios in addition to a self-complex. Poly(F) and poly(L) also formed a 1:2 complex between them. Some of these complexes were assumed to contain novel types of base pairings using the 7-NH group. Thus it was concluded that poly(L) could form complexes with both, the oligomer of cycloadenylic acid (?cn-120 degrees) and polymers of natural nucleotides (?cn0degrees), showing flexibility of the torsion angle of the laurusin residue.     

Toll-like receptor 3 activation promotes desensitization of histamine response in human gingival fibroblasts: Poly (I:C) induces histamine receptor desensitization in human gingival fibroblasts

Viruses are associated with the development of periodontal disease, particularly during periods of suppressed cellular immunity. For this reason, we evaluated the hypothesis that viral components regulate the actions of histamine, an important mediator of immune responses. We assessed the effect of Poly (I:C) on histamine-mediated intracellular calcium mobilization in human gingival fibroblasts. Our results show that histamine induces an increase in intracellular calcium concentrations in a dose-dependent manner. This response was blocked when cells were incubated in the presence of Poly (I:C). In addition, phorbol esters, a diacylglycerol analog, mimics the inhibitory actions of Poly (I:C) in response to histamine. The effect of Poly (I:C) was reversed by Stuarosporine (1 μM), GÖ6983 (7 μM), Bisindolylmaleimide (1 μM) [a protein inhibitor (PKC)], and SB 203580 (3 μM) (a p38-MAPK inhibitor). These findings suggest that Poly (I:C) regulates histamine-induced calcium mobilization through activation of PKC and p38.     

Heavy metals and spermatozoan motility. II. Turbidity changes induced by divalent cations and adenosinetriphosphate in sea urchin sperm flagella

The initial binding of synthetic polyribonucleotides and different RNAs to Ehrlich ascites cells has been studied using cell electrophoresis. When cells were pre-incubated in the presence of polylysine significant differences in the binding of different polyribonucleotides were observed. For example, Poly (A):Poly (U) caused a greater change in electrophoretic mobility than Poly (I):Poly (C). The increased electrophoretically detectable change after treatment with Ehrlich ascites RNA compared with E. coli tRNA could be correlated with increased uptakes of labelled Ehrlich ascites RNA compared with tRNA. An interesting observation was that when Poly (A) was added before Poly (U), a greater effect on electrophoretic mobility was found compared with the effects when the order of addition was reversed. It is suggested that copolymer formation from the homopolymers can occur at the cell surface.     

Polynucleotides. XLV Synthesis and properties of poly(2''-azido-2''-deoxyinosinic acid).

Poly (2'-azido-2'-deoxyinosinic acid), [poly (Iz)], was synthesized from 2'-azido-2'-deoxyinosine diphosphate by the action of polynucleotide phosphorylase. Poly (Iz) has UV absorption properties similar to poly (I) and hypochromicity of 11% at 0.15M Na+ and neutrality. In solutions of high Na+ ion concentration, poly (Iz) forms a multi-stranded complex and its Tm at 1.0M Na+ ion concentration was 43 degrees. Upon mixing with poly (C), poly (Iz) forms a 1:1 complex having a Tm lower than that of poly (I)-poly (C) complex in the same conditions. The effect of substitution at the 2'-position of the poly (I) strand was discussed in relation to the interferon-inducing activity.     

A kinetic and structural characterization of adenosine-5'-triphosphate: ribonucleic acid adenylyltransferase from Pseudomonas putida.

A catalytic and structural study of ATP:RNA adenylyltransferase (EC 2.7.7.19) from the particulate fraction of Pseudomonas putida was made. During the large-scale purification of this enzyme, designated adenylyltransferase B, a previously undetected ATP-incorporating activity, designated adenylyltransferase A, was observed. Adenylyltransferases A and B were indistinguishable catalytically; however, they differed in their chromatographic and sedimentation properties. Adenylyltransferases A and B were resolved by phosphocellulose, by poly (U)-Sepharose and by Bio-Gel P-100 chromatographies. Adenylytransferase A was determined to have a sedimentation coefficient (S020,w) of 9.3 S and B of 4.3 S. The molecular weight of adenylyltransferase A was estimated to be 185000 and that of adenylyltransferase B to be 50000-60000. Apparently, adenylyltransferase A was generated from adenylyltransferase B during the purification. The AMP incorporation catalyzed by adenylyltransferases A and B was inhibited by two derivatives of the antibiotic rifamycin, AF/013 (50% at 5 mug/ml) and AF/DNFI (50% at 10 mug/ml). The 5'-triphosphate derivative (3'-dATP) of the drug cordycepin (3'-deoxyadenosine/ was a competitive inhibitor with ATP for both adenylyltransferases. The Ki for 3'-deoxyadenosine 5'-triphosphate was 6 - 10(-4)--10 - 10(-4) M, while the Km for ATP was 1 - 10(-4)--2 - 10(-4) M. Several other anaolgs of ATP, 2'-deoxyadenosine 5' triphosphate, 2'-O-methyl ATP, or the fluorescent 3-beta-D-ribofuranosylimidazo [2,1-i] purien 5'-triphosphate did not affect the activity of adenylyltransferase A or B. Poly(U) and poly(dT) were competitive inhibitors of the ribosomal RNA-primed polymerization reaction. The Ki for poly(U) or poly(dT), in terms of nucleotide phosphate, was 4 - 10-6)--10 - 10(-6) M for adenylyltransferases A and B, compared to 2 - 10(-4)--4 - 10(-4) M for the Km of ribosomal RNA. The inhibition was a result of the competition between the non-priming poly(U), or poly(dT), and ribosomal RNA for the primer binding site on the enzyme.     

Probing the binding of two sugar bearing anticancer agents aristololactam-β-(D)-glucoside and daunomycin to double stranded RNA polynucleotides: a combined spectroscopic and calorimetric study

The plant alkaloid aristololactam-β-d-glucoside and the anticancer chemotherapy drug daunomycin are two sugar bearing DNA binding antibiotics. The binding of these molecules to three double stranded ribonucleic acids, poly(A)·poly(U), poly(I)·poly(C) and poly(C)·poly(G), was studied using various biophysical techniques. Absorbance and fluorescence studies revealed that these molecules bound non-cooperatively to these ds RNAs with the binding affinities of the order 10(6) for daunomycin and 10(5) M(-1) for aristololactam-β-d-glucoside. Fluorescence quenching and viscosity studies gave evidence for intercalative binding. The binding enhanced the melting temperature of poly(A)·poly(U) and poly(I)·poly(C) and the binding affinity values evaluated from the melting data were in agreement with that obtained from other techniques. Circular dichroism results suggested minor conformational perturbations of the RNA structures. The binding was characterized by negative enthalpy and positive entropy changes and the affinity constants derived from calorimetry were in agreement with that obtained from spectroscopic data. Daunomycin bound all the three RNAs stronger than aristololactam-β-d-glucoside and the binding affinity varied as poly(A)·poly(U) > poly(I)·poly(C) > poly(C)·poly(G). The temperature dependence of the enthalpy changes yielded negative values of heat capacity changes for the complexation suggesting substantial hydrophobic contribution to the binding process. Furthermore, an enthalpy-entropy compensation behavior was also seen in all systems. These results provide new insights into binding of these small molecule drugs to double stranded RNA sequences.     

Effect of double-stranded RNA and interferon on the antiviral activity of Atlantic salmon cells against infectious salmon anemia virus and infectious pancreatic necrosis virus

Type I interferons (IFN) establish an antiviral state in vertebrate cells by inducing expression of Mx and other antiviral proteins. We have studied the effect of Atlantic salmon interferon-like activity (AS-IFN) and poly I:C on the Mx protein expression and antiviral activity against infectious salmon anaemia virus (ISAV) and infectious pancreatic necrosis virus (IPNV) in the Atlantic salmon cell lines SHK-1 and TO. The double-stranded RNA poly I:C is an inducer of type I IFN in vertebrates. A cell cytotoxicity assay and measurements of virus yield were used to measure protection of cells against virus infection. Maximal induction of Mx protein in TO and SHK-1 cells occurred 48 h after poly I:C stimulation and 24 h after AS-IFN stimulation. TO cells pretreated with AS-IFN or poly I:C were protected from infection with IPNV 24 to 96 h after stimulation. Poly I:C or AS-IFN induced a minor protection against ISAV infection in SHK-1 cells, but no protection was induced against ISAV in TO cells. Western blot analysis showed that ISAV induced expression of Mx protein in TO and SHK-1 cells whereas IPNV did not induce Mx protein expression. These results suggest that ISAV and IPNV have very different sensitivities to IFN-induced antiviral activity and have developed different strategies to avoid the IFN-system of Atlantic salmon. Moreover, Atlantic salmon Mx protein appears not to inhibit replication of ISAV.     

TLR3 is essential for the induction of protective immunity against Punta Toro Virus infection by the double-stranded RNA (dsRNA), poly(I:C12U), but not Poly(I:C): differential recognition of synthetic dsRNA molecules

In the wake of RNA virus infections, dsRNA intermediates are often generated. These viral pathogen-associated molecular patterns can be sensed by a growing number of host cell cytosolic proteins and TLR3, which contribute to the induction of antiviral defenses. Recent evidence indicates that melanoma differentiation-associated gene-5 is the prominent host component mediating IFN production after exposure to the dsRNA analog, poly(I:C). We have previously reported that Punta Toro virus (PTV) infection in mice is exquisitely sensitive to treatment with poly(I:C(12)U), a dsRNA analog that has a superior safety profile while maintaining the beneficial activity of the parental poly(I:C) in the induction of innate immune responses. The precise host factor(s) mediating protective immunity following its administration remain to be elucidated. To assess the role of TLR3 in this process, mice lacking the receptor were used to investigate the induction of protective immunity, type I IFNs, and IL-6 following treatment. Unlike wild-type mice, those lacking TLR3 were not protected against PTV infection following poly(I:C(12)U) therapy and failed to produce IFN-alpha, IFN-beta, and IL-6. In contrast, poly(I:C) treatment significantly protected TLR3(-/-) mice from lethal challenge despite some deficiencies in cytokine induction. There was no indication that the lack of protection was due to the fact that TLR3-deficient mice had a reduced capacity to fight infection because they were not found to be more susceptible to PTV. We conclude that TLR3 is essential to the induction of antiviral activity elicited by poly(I:C(12)U), which does not appear to be recognized by the cytosolic sensor of poly(I:C), melanoma differentiation-associated gene-5.     

Polynucleotides. XL. Synthesis and properties of poly 2''-azido-2''-deoxyadenylic acid.

Poly 2'-azido-2'-deoxyadenylic acid (Poly Az) was synthesized from 2'-azido-2'-deoxyadenosine diphosphate by polynucleotide phosphorylase. Poly (Az) has U.V. absorption properties similar to poly (A) and hypochromicity of 40% at 0.1 M Na+ and neutrality. CD curve also resembled to that of poly (A), but has smaller ellipticity. Titration of poly (Az) with HCl gave a transition at pH 5.5, but exact structure of the acid-form complex was not elucidated. Upon mixing with poly (U), poly (Az) forms a 1:1 and 1:2 complexes having Tm's somewhat higher than that of poly (A)- poly (U) complex in the same condition.     

Interferon induction by poly(inosinic acid).poly(cytidylic acid) segmented by spin-labels

Poly(inosinic acid).poly(cytidylic acid) [(I)n.(C)n] duplexes of which the (C)n strand was modified to various degrees chemically or enzymatically with nitroxide radicals (spin-labels) were evaluated for interferon-inducing activity. Upon annealing of the chemically modified (C)n, (1C,Cx)n (X = 1000 or 16), with (I)n, the interferon-inducing activity was similar to that of (I)n.(C)n in PRK cell cultures. However, to overcome hydrolysis of the spin-label linkage in (1C,Cx)n, and enzymatic approach was taken to synthesize (1S(4)U,Cx)n copolymers with x = 100, 38, 16, and 8. The (1s(4)U,Cx)n copolymers were chemically stable, and upon annealing with (I)n the correlation time of the nitroxide moiety in (I)n. (1s(4)U,Cx)n was determined. A comparison of this correlation time with that measured for (RUGT,U100)n.(A)n, which contains the nitroxide moiety in position 5 of the U moiety, suggests that the 1s(4)U residue is in a nonintrahelical conformation and partitions the duplex into double-helical segments of varying size. The interferon-inducing activity of (I)n. (1s(4)U,Cx)n was evaluated in primary rabbit kidney, human skin fibroblast (strain VGS), and mouse L-929 cell cultures as well as in rabbits. The 1s(4)U residue did not cause a significant change in the interferon induction as compared to (I)n.(C)n in most systems tested unless x less than 16. These findings indicate that double-helical segments of approximately 16 base pairs partitioned by nonintrahelical 1s(4)U residues suffice to trigger the interferon response in all systems studied.     

Modified nucleotide polymers as inhibitors of DNA polymerases.

We have compared the relative inhibitory activity of poly (A) with its analogues poly N6-isopentenyl adenylic acid (poly(i6 A)) and poly N6-benzyl adenylic acid (poly(bzl6A)), and of poly (U) with its analogue poly 2'-fluoro-2'-deoxyuridylic acid (poly(dUfl)), against DNA polymerase, alpha, beta and gamma and terminal deoxynucleotidyl transferase from human cells and two oncorna virus DNA polymerases. Although poly (A) and its analogues were equally inhibitory against endogenous RNA-directed DNA polymerases of murine and feline leukemia viruses, the analogues in contrast to poly (A) were strongly inhibitory against all four cellular enzymes. Poly (dUfl), on the other hand, was up to 100-fold more potent than poly (U) against both viral and cellular enzymes. Since poly (U) at 100 mug/ml and poly (dUfl) at 1 mug/ml had no effect on terminal deoxynucleotidyl transferase while inhibiting other enzymes by 80--100 per cent these polymers could be useful in the characterization and assay of terminal deoxynucleotidyl transferase. In addition, the polymers such as poly (igA) and poly (bzl5A) which were strongly inhibitory to all cellular enzymes, could be useful in cancer chemotherapy if taken up preferentially by the malignant calls due to their high pinocytic activity. The results also demonstrate potential for large variation in inhibitory activity of polyribonucleotides as related to their chemical composition.     

Effects on protein synthesis of injecting synthetic polyribonucleotides into living cells

Micro-injection into the oocytes and eggs of Xenopus laevis was used to ascertain the effects of synthetic polyribonucleotides on protein synthesis in living cells. Poly(U) and poly(A) were not translated detectably, nor did they change the rate of endogenous protein synthesis. The same was true of poly(G,U), poly(A,G,U), poly(A,C,G,U), G-U-G-(U)(n), A-(U)(n) and AUG. In contrast, A-U-G-(U)(n) was a potent inhibitor of protein synthesis in the cell. This might be because it is initiated normally but lacks a termination codon, or because it inhibits the translation of other molecules in some way not dependent on its normal initiation. Poly(G,U), poly(A,G,U) and poly(A,C,G,U) inhibited haemoglobin synthesis when they were injected into the oocyte with haemoglobin mRNA. The synthetic polyribonucleotides did not inhibit the translation of the natural mRNA when the two sorts of molecules were injected at different times. It is suggested that the synthetic RNA molecules compete with the natural mRNA for a pre-initiation factor in limited supply.     

Demonstrations of the production of specific antibodies to poly(I).poly(C) in rabbits

The rabbit antiserum against poly(I).poly(C) purified by hydroxyapatite column chromatography contained three distinct antibodies. They were fractionated into three antibody populations by a series of precipitations (with poly(A).poly(U), poly(I), and poly(I).poly(C)) and their specificities were examined by quantitative complement fixation, double diffusion tests and radioimmunoassay. The first population was common to the double helical structure of double-stranded RNAs. The second was specific for poly(I) and the third was specific for poly(I).poly(C). These studies demonstrated that specific antibodies exclusively reactive with poly(I).poly(C) existed in the rabbit antiserum against poly(I).poly(C).