Polyribocytidylic Acid Research Articles

Voltammetric studies of the adsorption and conformation of polyribocytidylic acid at the mercury electrode surface

The adsorption and interfacial behaviour of polyribocytidylic acid (poly-C) was studied by phase sensitive ac voltammetry and single-sweep voltammetry. The two helical conformations of poly-C were investigated as a function of various parameters such as pH, adsorption potential, temperature and the nature of the anions of supporting electrolyte. The adsorbed acid form of poly-C undergoes an opening of the double helix because the electric field at the charged interface renders some of cytosine moieties accessible for reduction. The limiting value of the surface concentration of poly-C indicates that the adsorption occurs via the base residues attached to the nucleotide chain. The results are compared with the similar adsorption behaviour of polyriboadenylic acid (poly-A) and DNA.

Endogenous pyrogen formation by human blood monocytes stimulated by polyriboinosinic acid:polyribocytidylic acid.

The pyrogenic response to supernatants from human blood monocytes stimulated with polyriboinosinic acid:polyribocytidylic acid (poly I:C) was characteristic of a response to endogenous pyrogen in that it was brief and monophasic, and was destroyed by heating the supernatants at 70 degrees C for 30 min. Pyrogen production was unimpaired when the incubations were carried out in the presence of cycloheximide (50 micrograms/ml; an inhibitor of protein synthesis) or indomethacin (50 micrograms/ml; an inhibitor of prostaglandin synthesis). Also, neither interferon, interleukins, tumor necrosis factor nor prostaglandin E2 were detectable in the supernatants from the poly I:C-stimulated human monocytes.

Alpha-methyl-p-tyrosine reduces poly I:C-induced augmenting interferon production and splenic natural killer cell activity in mice.

The present investigation was designed to extend our previous observations that alpha-methyl-p-tyrosine (AMPT; an inhibitor of norepinephrine synthesis) reduced splenic natural killer (NK) cell activity and to test whether NK augmentation or augmenting interferon (IFN) production induced by polyriboinosinic acid:polyribocytidylic acid (poly I:C; an IFN inducer) can be attenuated by AMPT in mice. Therefore, in the current study, the effects of AMPT (300 mg/kg i.p.) on splenic NK activity or plasma titers of IFN-alpha+beta were assessed in inbred C57BL/6 mice. It was found that treatment with AMPT reduced both poly I:C induced serum IFN and splenic NK activity in a dose- and time-dependent manner. These results suggest that AMPT inhibits IFN production and decreases splenic NK activity in vivo.

The effects of an interferon inducer, polyriboinosinic polyribocytidylic acid on cytochrome P-450 dependent hepatic progesterone metabolism

A time course study on the effects of an interferon inducing agent, polyriboinosinic polyribocytidylic acid (poly rIrC) on the hepatic cytochrome P-450 dependent progesterone metabolism was performed. Administration of a single dose of poly rIrC (10 mg/kg i.p.) to adult male Wistar rats caused a time dependent effect on liver weight, microsomal protein and total cytochrome P-450 levels, as well as the 16α and 6β hydroxylation of progesterone. The response was multiphasic, with a maximal depression of both hydroxylase activity 48 hours post-injection, followed by enhanced activity at 72 hours and subsequent return to control activity twenty-four hours later. A second less dramatic rise in the activities followed, bringing the 16α and 6β hydroxylase activity to 159% and 141% of their respective control values by 336 hours, at which point of time, the trend appeared to be still on the rise. The enhanced activity at 72 hours was preceded by an increase in serum cortisol and corticosterone levels, the ability of which to enhance the activity of 6β hydroxylation of steroids may partly explain the phenomenon.

The role of double-stranded RNA in induction of the acute-phase response in an abortive influenza virus infection model.

The inducer of the acute-phase response in "flu-like" viral infections is not defined. The hypothesis that virus-associated double-stranded (ds) RNA serves this function was investigated by comparison of several acute-phase responses (fever and sleep patterns, white and nucleated red blood cell levels, serum antiviral activity and ceruloplasmin) induced by the synthetic dsRNA polyriboinosinic:polyribocytidylic acid (poly[rI:rC]) with those induced by influenza virus in rabbits. The capacity of either dsRNA or influenza virus to induce hyporesponsiveness with respect to these acute-phase parameters upon rechallenge with the same agent or cross-challenge 24 h later was also examined. Poly(rI:rC) induced only minimal hyporesponsiveness to itself but was a potent inducer of hyporesponsiveness to virus with respect to fever, sleep, leukograms, and antiviral activity. Therefore, poly(rI:rC) can substitute for virus in terms of induction of acute-phase hyporesponsiveness, suggesting that dsRNA of viral origin triggers the acute-phase response in this model of influenza.

Interferon inducer, polyriboguanylic · polyribocytidylic acid, inhibits experimental hepatic metastases in mice

The antitumour activity of an interferon inducer,the double-stranded complex of polyriboguanylic · polyribocytidylic acid was studied on murine lymphosarcoma LS/BL. The antitumour effect was determined with the aid of an experimental liver-colony model and compared to that exhibited by another synthetic RNA, polyriboinosinic · polyribocytidylic acid. We found that both polynucleotide complexes decreased the number of liver colonies and prolonged the survival of the tumour-beaing mice. This effect was onlyobserved if the complexes were applied in an appropriate dose schedule which included the administration of the drug prior to tumour cell inoculation and subsequent continuous tretment. We have also verified that the polynucleotide complexes did not exert their antitumour effect by a direct action on tumour cells.

Cellular and humoral factors associated with polyriboinosinic-polyribocytidylic acid (poly I:C) induced resistance to banzi virus encephalitis : D.C. Barnhart, E.P. Mayer, A. Ghaffar, and J.D. Gangemi, Department of Microbiology and Immunology, USC School of Medicine, Columbia, SC, 29208, USA

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte–endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-β, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.

Avian myeloblastosis virus reverse transcriptase inhibition by nalidixic acid

Nalidixic acid, a very specific inhibitor of bacterial DNA synthesis, has been studied for its action on the avian myeloblastosis virus reverse transcriptase activity. The drug inhibited the DNA synthesis reaction catalyzed by the viral enzyme in the presence of different template-primers. The inhibitory effect by nalidixic acid was higher with polyriboadenylic acid than with polyribocytidylic acid as a synthetic template. With activated DNA as a template nalidixic acid preferentially inhibited the TMP incorporation when compared with the dAMP incorporation. Both these results showed the importance of the presence of adenine in the templates for a more efficient inhibition by nalidixic acid. The inhibition for this drug was also shown in the presence of Mn2+ instead of Mg2+ as the divalent cation, and with a 2'-fluorinated analogue of polyriboadenylic acid as the template. Kinetic data showed a non-competitive inhibition by nalidixic acid in relation to polyriboadenylic acid and to TTP in the reaction catalyzed by reverse transcriptase.

Poly I:C-induced anti-herpes simplex virus type 1 activity in inflammatory macrophages is mediated by induction of interferon-beta.

We examined the mechanism by which Polyriboinosinic:Polyribocytidylic acid (Poly I:C) augments resistance of thioglycolate-elicited inflammatory macrophages to infection with herpes simplex virus type 1 (HSV-1). We show that Poly I:C-induced antiviral activity is completely abrogated by antibodies to interferon-beta (IFN-beta) whereas antibodies to other interferons or to other cytokines have no effect. Furthermore, treatment of inflammatory macrophages with exogenous IFN renders them resistant to HSV-1, whereas treatment with other cytokines does not. In addition, we demonstrate that supernatants from macrophages treated with Poly I:C contain IFN-beta but not IFN-alpha. Taken together these data indicate that the antiviral effects of Poly I:C in inflammatory macrophages are mediated solely by IFN-beta, which acts in an autocrine manner to induce resistance to HSV-1.

Biophysical studies of the complexes of polyriboguanylic acid with brominated and chlorinated polyribocytidylic acids

Conformation of double-stranded complexes of polyriboguanylic acid with halogenated polyribocytidylic acid [poly(C)] was studied with the aid of differential pulse polarography, terbium fluorescence and circular dichroism spectrometry. It was shown that halogenation at C(5) of cytosine residues in poly(C) disturbed the ordered structure of the double-helical complex. In addition, this halogenation does not improve antiviral activity of the polynucleotide complex studied in the system of vesicular stomatitis virus and the cell culture of chicken embryos. It was concluded that the regularity of the secondary structure of synthetic RNAs might play an important role in the mechanism of biological activity of these biomacromolecules.

The Fever Induced by Polyriboinosinic:Polyribocytidylic Acid Is Not Related to Interferon Synthesis in the Rabbit's Hypothalamus

We previously demonstrated that direct administration of interferon (IFN) or its inducer polyriboinosinic acid:polyribocytidylic acid [poly(I:C)] into the hypothalamus caused dose-dependent fever in rabbits. It was not clear whether the fever induced by intrahypothalamic injection of poly(I:C) was due to stimulation of IFN or prostaglandin E-2 synthesis in the hypothalamic tissues. Therefore, in the current experiments, we used an established model in which rabbit hypothalamic minces or brain cells were incubated in vitro with poly(I:C) to test for the ability of poly(I:C) to stimulate IFN or PGE-2 synthesis. The results showed that poly(I:C) stimulated PGE-2, but not IFN, synthesis in the hypothalamus. Thus, it appears that the fever induced by poly(I:C) is not related to IFN synthesis in the hypothalamus.

Suppression of P450IIC12 gene expression and elevation of actin messenger ribonucleic acid levels in the livers of female rats after injection of the interferon inducer poly rI·poly rC

Interferons and interferon inducers cause a reduction in hepatic microsomal cytochrome P450 [AH, reduced-flavoprotein:oxygen oxidoreductase (RH hydroxylating), EC 1.14.14.1] content and drug-metabolizing activities in experimental animals. In the present study, the acute effects of administration of the interferon inducer polyriboinosinic acid · polyribocytidylic acid (poly rI·polyrC) to female rats on expression of the microsomal apoprotein and hepatic mRNA for P450IIC12, a constitutive enzyme comprising a significant fraction of the total P450 in untreated female rats, were examined. Poly rI·poly rC treatment (10mg/kg, i.p.) caused a suppression of P450IIC12 apoprotein that was of greatest magnitude (47% of control levels) 24 hr after injection. P450IIC12 was still suppressed significantly (P<0.05) 48 hr after treatment. The time course of suppression and recovery of P450IIC12 protein, as well as the magnitude of the effect, were similar to those of total microsomal P450 measured spectrophotometrically. P450IIC12 mRNA levels were also suppressed by the poly rI·poly rC treatment, reaching 29% of control values within 24 hr. Comparison of the kinetics of suppression of the P450IIC12 mRNA and apoprotein indicated that at least part of the suppression of the protein is mediated pretranslationally. However, the existence of a posttranslational component could not be excluded. Concomitant with the suppression of P450IIC12, actin mRNA content was found to be elevated by at least 3.6-fold in the livers of poly rI·poly rC-treated female rats, with the maximum effect occuring 12 hr after injection of the drug. This effect of poly rI·poly rC on expression of actin mRNA appeared to be at least partially sex-specific, since in a previous study [Morgan ET and Norman CA, Drug Metab Dispos 18: 649–653, 1990] a significant effect of the interferon inducer on actin expression was not observed in livers of male rats.

Vaccine-induced alterations in hepatic drug metabolism

Administration of Diphtheria and Tetanus Toxoids and Pertussis Vaccine Adsorbed (DTP) vaccine to mice causes dose- and time-dependent alterations in hepatic drug metabolism as determined by hexobarbital-induced sleep time and several direct measurements of soluble and microsomal enzyme activities. Vaccines containing only tetanus and/or diphtheria toxoids did not alter hepatic drug metabolism, implicating the pertussis component as the cause of the observed changes. Other pyrogenic whole cell vaccines such as typhoid and cholera also had no effect on drug metabolizing capacity. However, polyriboinosinic polyribocytidylic acid (poly I:C), a compound thought to exert its effects through the induction of interferon, induced changes comparable to those seen with DTP vaccine. The similarities in the effects following administration of DTP vaccine and poly I:C suggest that vaccine-induced alterations of drug metabolism may be mediated by immunomodulatory agents such as interferons and interleukins. Studies with purified cytokines are planned to address this question.

Biophysical studies of the modification of poly(rG) · poly(rC) by cisplatin. Relations to the biological activity of the complex

The integrity of the double-stranded complex polyriboguanylic•polyribocytidylic acid [poly(rG)•poly(rC)] modified by antitumour cis-diamminedichloroplatinum(II) ( cis-DDP) was studied with the aid of differential pulse polarography and terbium fluorescence measurement. The modification was made to the level corresponding to r b = 0.05 ( r b is defined as the number of platinum atoms covalently bound per one nucleotide residue). Two modes of the modification of the polynucleotide complex were employed: The action of cis-DDP on poly(G) before formation of the complex with poly(C) and on the complex already formed from non-modified polynucleotides. It was shown that in the latter case modification disordered the intergrity of the complex only negligibly, while in the former case the modification resulted in a noticeably more extensive disturbance of the double-stranded polynucleotide complex. Moreover, the modification of the complex (after its formation) at r b = 0.02 led to improved interferon-inducing and antiviral activity of poly(rG)•poly(rC) tested on mice infected by influenza virus. It was suggested that the combined effects of interferon-inducing and antiviral activities of poly(rG)•poly(rC) and antiviral activity of cis-DDP may result in an increased effect over and above what may be expected from the actions of the two modalities separately.

Paramedian reticular nucleus: a thermolytic area in the rat medulla oblongata

The effects of stimulation or ablation of the paramedian reticular nucleus (PRN) of the rat medulla oblongata on the thermal responses induced by ambient temperature changes, a pyrogen, or a hypothermic substance were assessed. Electrical stimulation of the PRN elicited thermolytic reactions (including decreased metabolism, cutaneous vasodilation and hypothermia) which could be mimicked by micro-injection of kainic acid (an excitotoxic amino acid) into the same region. Bilateral electrolytic lesions in the PRN prevented the animals from responding to heat stress (35 degrees C for 30 min) to some extent, but did not prevent responses to cold stress (4 degrees C for 60 min). In addition, the thermogenic reactions induced by intrahypothalamic injection of polyriboinosinic acid: polyribocytidylic acid (a pyrogenic substance), or the thermolytic reactions induced by intraperitoneal administration of chlorpromazine (a tranquilizer), were antagonized respectively by activation or ablation of the PRN. This suggests that the PRN of the caudal medulla may function as a thermolytic area.

Purification and Initial Characterization of Human Placental Trophoblast Interferon Induced by Polyriboinosinic {middle dot} Polyribocytidylic Acid

Human placental trophoblast interferon (tro-IFN), induced in trophoblast cultures by a superinduction procedure, was purified to a homogeneous product with retention of biological activity. The problems associated with isolation from serum-containing medium were overcome by a combination of Blue Sepharose affinity chromatography and reversed-phase HPLC (RP-HPLC) on Separon SGX C-18. This two-step purification procedure yielded tro-IFN with a specific activity of 3.4 x 10(7) international units/mg of protein. The overall recovery of interferon activity was 66.7%. The purified tro-IFN was shown to be a glycoprotein with an Mr of 24K on native and SDS-PAGE. Its antiviral activity was stable at pH 2.0 at 37 degrees C but was sensitive to heat at 56 degrees C for 1 h and was neutralized by antibodies to human IFN-beta.

In vitro translational activity of messenger-RNA isolated from mice treated with the interferon inducer, polyriboinosinic acid·polyribocytidylic acid

Treatment of mice with interferon and interferon inducers causes down regulation of a number of hepatic proteins. In a previous publication it was demonstrated that these treatments depress hepatic protein synthesis and increase protein degradation, particularly of the endoplasmic reticulum Gooderham NJ and Mannering GJ, Arch Biochem Biophys 250: 418–425, 1986. In the present study the effects of polyriboinosinic acid-polyribocytidylic acid (poly IC) treatment on mouse hepatic RNA levels and the translation of this RNA in a cell-free system were examined. Poly IC treatment of mice increased hepatic poly (A +)RNA levels. The translation of isolated poly(A +)RNA was evaluated at various intervals after the administration of poly IC. Translation was marginally increased at 3–6 hr after treatment and depressed after 12–18 hr. Antibodies were employed to examine the effects of poly IC treatment on specific polypeptides in order to evaluate the in vitro translation of mRNAs for tyrosine aminotransferase and albumin; translation of these proteins was biphasic with pronounced depression. These studies indicate that in vivo interferon may regulate gene expression by altering levels of hepatic proteins via increased transcription and decreased translation.

Febrile effects of polyriboinosinic acid: polyribocytidylic acid and interferon: relationship to somatostatin in rat hypothalamus

The changes in thermoregulatory effectors produced by an injection of polyriboinosinic acid: polyribocytidylic acid (Poly I:C) or interferon were assessed and compared in control rats, in rats with hypothalamic somatostatin (SS) receptor blockade and in rats with hypothalamic SS depletion. Intrahypothalamic (i.h., 0.05-0.50 microgram) or intraperitoneal (i.p., 100-600 micrograms) administration of Poly I:C caused a dose-related rise in colon temperature in control rats at all ambient temperatures (Ta) studied. A Poly I:C-induced fever was produced by increased metabolism at a Ta of 8 degrees C, whereas at 30 degrees C, it was caused by cutaneous vasoconstriction. At a Ta of 22 degrees C, the fever was caused by increased metabolism and cutaneous vasoconstriction. On the other hand, i.h. administration of SS-14 antagonist (0.1-0.5 ng) caused a dose-related fall in colon temperature at Ta of 8 degrees C or 22 degrees C. At a Ta of 8 degrees C, the hypothermia was caused by decreased metabolism, whereas at 22 degrees C, it was caused by decreased metabolism and cutaneous vasodilation. At a Ta of 30 degrees C, the thermoregulatory effectors were not affected by SS-14 antagonist treatment. Furthermore, the fever induced by Poly I:C or interferon was significantly reduced by pretreatment of rats with an i.p. dose of cysteamine (30 mg. kg-1) or an i.h. dose of SS-14 antagonist (0.1 ng). The results indicate that a somatostatinergic pathway in rat hypothalamus may mediate the fever induced by interferon or its inducer Poly I:C.

Independent induction of interleukin 6 and prostaglandin E2 by interleukin 1 in human articular chondrocytes

Interleukin 6 is a cytokine with growth and differentiation activities on a number of cell types. Human articular chondrocytes produce interleukin 6 and this production appears to be constitutive but can be stimulated in a dose-dependent manner by interleukin 1. Other stimulators of interleukin 6 production in chondrocytes include tumour necrosis factor-α, polyriboinosinic: polyribocytidylic acid and bacterial lipopolysaccharide. Interleukin 6 production is not inhibited by prostaglandin E2 but may be partially dependent on prostaglandin E2 production. Using an antiserum to interleukin 6 we have demonstrated that the production of prostaglandin E2 under basal conditions and in response to interleukin 1 is probably not mediated by interleukin 6.

Depletion of hypothalamic norepinephrine reduces the fever induced by polyriboinosinic acid: polyribocytidylic acid (Poly I:Poly C) in rats.

Administration of either Poly I:Poly C (0.05-0.50 micrograms) or norepinephrine (2-8 micrograms) into the anterior hypothalamic area produced a dose-related fever in rats. The fever induced by Poly I:Poly C was attenuated after selective depletion of norepinephrine in the hypothalamus. However, selective depletion of hypothalamic norepinephrine did not affect the fever induced by intrahypothalamic norepinephrine. The data indicate that Poly I:Poly C may act to induce fever through the endogenous release of norepinephrine from the rat's hypothalamus.