Potentiation of the antiviral activity of poly r(A-U) by xanthene dyes

Ten xanthene dyes (XAN) are evaluated for their ability to potentiate the antiviral activity of poly r(A-U) using a human foreskin fibroblast-vesicular stomatitis virus bioassay in which the XAN is combined with 0.2 mM poly r(A-U) to produce a XAN/ribonucleotide ratio of 1/4. Four of the ten XANs tested in this study, rhodamine 123, rhodamine B, rhodamine 6G and sulforhodamine B, enhance the antiviral activity of poly r(A-U) 8- to 15-fold. The interferon-inducing activity of the four active XAN/poly r(A-U) combinations is equal to the sum of the activities of their constituents. These four XANs appear to potentiate the antiviral activity of the poly r(A-U) without superinduction of interferon. The direct viral inactivation study demonstrates that the XANs, poly r(A-U) and the XAN/poly r(A-U) combinations do not inactivate the VSV at concentrations near the 50% effective dose.     

Enhancement of the antiviral and interferon-inducing activities of poly r(A-U) by carminic acid

Experiments have been designed to systematically examine the effects of carminic acid (CAR) on the antiviral/interferon-inducing activity of poly r(A-U), using the human foreskin fibroblast-vesicular stomatitis virus bioassay system. Modulation of the antiviral/interferon-inducing activity of poly r(A-U) by carminic acid was examined at fixed poly r(A-U) concentrations of 0.05 mM or 0.2 mM while varying the carminic acid concentrations to produce variable CAR/ribonucleotide ratios ranging from 1:16 to 2:1. Carminic acid and poly r(A-U) were tested individually at the concentrations employed in the CAR/poly r(A-U) combinations. Neither the carminic acid alone nor poly r(A-U) alone were effective antiviral agents/interferon inducers. The antiviral/interferon-inducing activity of poly r(A-U) was potentiated twelve-fold at CAR/ribonucleotide ratios in the region of 1/6 to 1/4. These results suggest a synergism between the poly r(A-U) and the carminic acid at the concentrations employed in this study.     

In vitro antiviral activity of poly (A-U) and ellipticines.

The role of N2-methyl-9-hydroxy-ellipticine (NMHE) and N2,N6-dimethyl-9-hydroxy-ellipticine (DMHE) in modulating the antiviral activity of poly (A-U) was examined using a human foreskin fibroblast-vesicular stomatitis virus (HSF-VSV) bioassay in which the concentration of poly (A-U) was fixed at 0.05 mM or 0.2 mM while the NMHE or DMHE concentration was varied to produce variable NMHE (or DMHE)/ribonucleotide ratios ranging from 1:16 to 2:1. Poly (A-U), NMHE and DMHE tested individually were not efficacious antiviral agents. When the poly (A-U) was combined with the NMHE or DMHE, the antiviral activity of the poly (A-U) was potentiated 16- to 20-fold a NMHE (or DMHE)/ribonucleotide ratios in the region of 1/4. Poly (A-U), NMHE and DMHE induce beta-IFN. The interferon-inducing activity of the NMHE (or DMHE)/poly (A-U) combination was equal to the sum of the interferon-inducing activity of the poly (A-U) alone and the NMHE (or DMHE) alone. The direct viral inactivation study demonstrated that NMHE, DMHE, poly (A-U) and the NMHE (or DMHE)/poly (A-U) combinations did not inactivate VSV at concentrations near the 50% viral inhibitory dose. Photomicrographs of HSF cells incubated with NMHE alone or with a NMHE/poly (A-U) combination suggest that poly (A-U) affects the subcellular distribution of the NMHE by steering the NMHE to the nucleolus. These observations suggest that modulation of a nuclear process may be responsible for the enhanced antiviral activity.     

Effect of enthidium on the morphology, antiviral activity and subcellular distribution of Poly r(A-U)

When ethidium bromide (EB) is combined with poly r(A-U) at an EB/ribonucleotide ratio of 1/4, the antiviral activity of the EB increases 22-fold. The increased antiviral activity is not due to increased interferon induction, direct viral inactivation or host cell cytotoxicity. Phase contrast, confocal and fluorescence microscopic observations reveal an increase in the nucleolar accumulation of the EB and/or the poly r(A-U) in the EB/poly r(A-U)-treated fibroblasts. Ultrastructure of negatively stained and replica preparations demonstrated that EB-induced condensation of poly r(A-U). These results suggest the elevated antiviral activity may be related to the altered uptake and subcellular distribution of the EB/poly r(A-U) complex.     

Potentiation of the antiviral activity of poly r(A-U) by riboflavin, FAD and FMN

The role of riboflavin (RFN), FAD or FMN in modulating the antiviral activity of poly r(A-U) was examined by the human foreskin fibroblast-vesicular stomatitis virus bioassay in which the concentrations of poly r(A-U) was fixed at 0.1 mM or 0.2 mM while the riboflavin, FAD or FMN concentration was varied to produce variable RFN (or FAD or FMN)/ribonucleotide ratios ranging from 1/16 to 2/1. Riboflavin, FAD and FMN tested individually did not exhibit any antiviral activity, while poly r(A-U) alone exhibited antiviral activity. When poly r(A-U) was combined with riboflavin, FAD or FMN, the antiviral activity was potentiated seven- to twelve-fold at RFN (or FAD or FMN)/ribonucleotide ratios in the region of 1/4.     

Subcellular localization and antiviral activity of carminic acid/poly r(A-U) combinations

Carminic acid (CAR) enhances the antiviral activity of poly r(A-U) twelve-fold without increasing interferon induction, inactivating the vesicular stomatitis virus or inducing host cell cytotoxicity. Phase contrast photomicrographs of human foreskin fibroblasts (HSF) incubated with CAR alone, poly r(A-U) alone or with a CAR/poly r(A-U) combination illustrate that the CAR/poly r(A-U) combinations display altered subcellular distribution with the CAR being localized in the nucleoli and chromatin. Phase contrast and fluorescence photomicrographs of adriamycin (ADR)-treated and ADR/poly r(A-U)-treated HSF cells corroborate these findings. These results suggest that modulation of one or more nucleolar processes may be responsible for the enhanced antiviral activity.     

Enhanced antiviral activity and altered subcellular distribution of magnesium/poly r(A-U) combinations.

When Mg2+ or ethidium bromide (EB) were combined with poly r(A-U) at a ligand/ribonucleotide ratio of 1/4, the antiviral activity of the Mg2+ and EB increased 136-fold and 154-fold. Eriochrome Blue SE was employed to visualize the subcellular distribution of Mg2+ following co-incubation of Human Foreskin Fibroblasts (HSF) with Mg2+ alone or with the Mg2+/poly r(A-U) combination. Phase contrast micrographs of these Mg(2+)-treated HSF cells as well as phase contrast and fluorescence micrographs of EB-treated or EB/poly r(A-U)-treated HSF cells illustrated that the Mg2+ (or EB)/poly r(A-U) combinations display altered subcellular distribution with the Mg2+ and EB being localized in the nucleoli and chromatin of the HSF cells. These results suggest that modulation of nuclear processes may be responsible for the enhanced antiviral activity.     

Antiviral activity of magnesium and magnesium/poly r(A-U) combinations against two RNA viruses.

Magnesium (Mg2+) potentiated the anti-vesicular stomatitis virus (VSV) activity of poly r(A-U) or poly r(G-C) and the anti-HIV-1 activity of poly r(A-U). Mg2+ did not affect the anti-VSV activity of poly (rI).poly (rC), poly (dA-dT).poly (dA-dT) or poly (dG-dC).poly (dG-dC). Modulation of one or more nuclear (nucleolar) processes of the host cell may be responsible for the synergistic antiviral activity.     

Antiviral Activity of Magnesium and Magnesium/poly r(A-U) Combinations Against two RNA Viruses

Abstract

Magnesium (Mg²⁺) potentiated the anti-vesicular stomatitis virus (VSV) activity of poly r(A-U) or poly r(G-C) and the anti-HIV-1 activity of poly r(A-U). Mg²⁺ did not affect the anti-VSV activity of poly (rI) ? poly (rC), poly (dA-dT) ? poly (dA-dT) or poly (dG-dC) ? poly (dG-dC). Modulation of one or more nuclear (nucleolar) processes of the host cell may be responsible for the synergistic antiviral activity.     

A.U and G.C base pairs in synthetic RNAS have characteristic vacuum UV CD bands.

The vacuum UV CD spectra of GpC, CpG, GpG, poly[r(A)], poly[r(C)], poly[r(U)], poly[r(A-U)], poly[r(G).r(C)], poly[r(A).r(U)], and poly[r(A-U).r(A-U)] were measured down to at least 174 nm. These spectra, together with the published spectra of poly[r(G-C).r(G-C)], CMP, and GMP, were sufficient to estimate the CD changes upon base pairing for four double-stranded RNAs. The vacuum UV CD bands of poly[r(A)], poly[r(C)], and the dinucleotides GpC and CpG were temperature dependent, suggesting that they were due to intrastrand base stacking. The dinucleotide sequence isomers GpC and CpG had very different vacuum UV CD bands, indicating that the sequence can play a role in the vacuum UV CD of single-stranded RNA. The vacuum UV CD bands of the double-stranded (G.C)-containing RNAs, poly[r(G).r(C)] and poly[r(G-C).r(G-C)], were larger than the measured or estimated vacuum UV CD bands of their constituent single-stranded RNAs and were similar in having an exceptionally large positive band at about 185 nm and negative bands near 176 and 209 nm. These similarities were enhanced in difference-CD spectra, obtained by subtracting the CD spectra of the single strands from the CD spectra of the corresponding double strands. The (A.U)-containing double-stranded RNAs poly[r(A).r(U)] and poly[r(A-U).r(A-U)] were similar only in that their vacuum UV CD spectra had a large positive band at 177 nm. The spectrum of poly[r(A).r(U)] had a shoulder at 188 nm and a negative band at 206 nm, whereas the spectrum of poly[r(A-U).r(A-U)] had a positive band at 201 nm. On the other hand, difference spectra of both of the (A.U)-containing polymers had positive bands at about 177 and 201 nm. Thus, the difference-CD spectra revealed CD bands characteristic of A.U and G.C base pairing. (ABSTRACT TRUNCATED AT 250 WORDS)     

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 level corresponding to rb = 0.05 (rb 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 integrity 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 rb = 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.     

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.     

Modification of duplex poly(G).poly(C) by platinum (II) compounds.

The modification of the double-stranded poly(G).poly(C) complex by cis-diamminedichloroplatinum(II) was studied by two modes: the action of cis-DDP on poly(G) before formation of the duplex with poly(C) and that on the prepared duplex. It was shown that in the latter case modification disordered the integrity of the duplex only negligibly at rb less than or equal to 0.05 and led to improved interferon-inducing and antiviral activity tested on mice infected by Influenza and Herpes viruses.     

Bromination of alkyl quinolones by Microbulbifer sp. HZ11, a marine Gammaproteobacterium,modulates their antibacterial activity

Alkyl quinolones (AQs) are multifunctional bacterial secondary metabolites generally known for their antibacterial and algicidal properties. Certain representatives are also employed as signalling molecules of Burkholderia strains and Pseudomonas aeruginosa. The marine Gammaproteobacterium Microbulbifer sp. HZ11 harbours an AQ biosynthetic gene cluster with unusual topology but does not produce any AQ-type metabolites under laboratory conditions. In this study, we demonstrate the potential of strain HZ11 for AQ production by analysing intermediates and key enzymes of the pathway. Moreover, we demonstrate that exogenously added AQs such as 2-heptyl-1(H)-quinolin-4-one (referred to as HHQ) or 2-heptyl-1-hydroxyquinolin-4-one (referred to as HQNO) are brominated by a vanadium-dependent haloperoxidase (V-HPO_HZ11), which preferably is active towards AQs with C5–C9 alkyl side chains. Bromination was specific for the third position and led to 3-bromo-2-heptyl-1(H)-quinolin-4-one (BrHHQ) and 3-bromo-2-heptyl-1-hydroxyquinolin-4-one (BrHQNO), both of which were less toxic for strain HZ11 than the respective parental compounds. In contrast, BrHQNO showed increased antibiotic activity against Staphylococcus aureus and marine isolates. Therefore, bromination of AQs by V-HPO_HZ11 can have divergent consequences, eliciting a detoxifying effect for strain HZ11 while simultaneously enhancing antibiotic activity against other bacteria.     

Improved anthraquinone accumulation in cell cultures of Cinchona `Robusta' by feeding of biosynthetic precursors and inhibitors

The effects of feeding of biosynthetic precursors and pathway specific inhibitors on anthraquinone (AQ) accumulation in fungal elicited cell cultures of Cinchona`Robusta' were studied. Addition of glyceraldehyde (1 mM), the initial precursor in the methyl-d-erythritol 4-phosphate (MEP) pathway, did not increase AQ accumulation, suggesting that the endogenous level of this precursor is not a limiting factor of AQ flux. It is proposed that AQs in Cinchona might be derived from the phenylpropanoid pathway, e.g. from caffeic acid. Addition of ferulic acid (1 mM) did not stimulate AQ accumulation, while addition of caffeic acid increased AQ accumulation by 48% compared to the control. The stimulating effect of feeding caffeic acid on AQ accumulation might be due to activation of other pathways. Addition of tectoquinone (2-methyl-anthraquinone) did not change the AQ patterns nor the shifts between AQs in control and tectoquinone-treated cell cultures. Addition of lovastatin, a specific inhibitor of the mevalonic acid pathway, did not inhibit the AQ accumulation. Clomazone, an inhibitor in the MEP pathway, inhibited the AQ accumulation, however. The simultaneous addition of lovastatin and clomazone inhibited both cell growth and AQ accumulation. These results further support the finding that isopentenyl diphosphate, which constitutes ring C of AQs in Cinchona `Robusta', is derived from the MEP pathway, and not from the mevalonic acid pathway.     

Relationship between the Molecular Size of Poly I·Poly C and Its Biological Activity1

Seven polyinosinic·polycytidylic acid (poly I·poly C) preparations, ranging from 4.2 S to 21.2 S, prepared from various sizes of polyinosinate and polycytidylate, were examined for toxicity and interferon-inducing activity in mice. The increase in size of poly I·poly C was accompanied by increases both in the maximal amount of interferon produced and in the length of persistence of a high level of interferon in plasma. Toxicity of poly I·poly C was proportional to the molecular size within the range of 8 S to 16 S. The amount of interferon induced by 1/5 LD₅₀ of poly I·poly C depended on the size of the inducer, being increasingly lower with progressively smaller sizes. Next, activities of poly I·poly C in culture cells were examined. The resistance-inducing activity of poly I·poly C in primary chick embryo cells (CEC) increased with the size of the inducer (4.2 S to 11.6 S), whereas the activity in L cells was not so markedly dependent upon its molecular size as in CEC. In the presence of calf serum during induction of resistance the activity was lowered. The activities of preparations with small molecular sizes were affected by calf serum more markedly than those of large molecular sizes. The interferon-inducing activity in RK13 was not appreciably influenced by the size of poly I·poly C, especially in the presence of DEAE-dextran, while the activity in L cells was markedly dependent upon the size of the inducer. These results suggest that the influence of the molecular size of poly I·poly C upon the resistance-inducing and interferon-inducing activities varies among different kinds of cells, and alters in the presence of serum or DEAE-dextran.     

Heterogenous effects of anthraquinones on drug-metabolizing enzymes in the liver and small intestine of rat

The induction of a variety of drug-metabolizing enzymes by six anthraquinones (AQs) has been investigated in the liver and small intestine of rat. In the liver, the intragastric administration for 3 days of 100 mg/kg 9,10-anthraquinone (9,10-AQ). 1-hydroxy-AQ, 1,4-dihydroxy-AQ, but not 1,2-dihydroxy-AQ and 2-carboxy-AQ, resulted in a significant induction of the UDP-GT, DT-diaphorase, P450 1A-linked monooxygenase activities and in particular the methoxyresorufin-O-demethylase (MEROD), an activity dependent on P450 1A2. Immunoblot analysis indicated that 1-hydroxy-AQ and 1,4-dihydroxy-AQ induced P450 1A2 but not 1A1 and 9,10-AQ induced both P4501A2 and P4502B. Northern blotanalysis, using a cDNA probe for CYP 1A1 and CYP 1A2, confirmed that the AQs induce CYP 1A2 but not 1A1 mRNA. In the mucosa of small intestine, none of the above-mentioned enzymatic activities were enhanced following AQ administration. The induction mechanism of the hepatic enzymes by AQs is not known and it deserves a further study as it might be independent from the activation of the Ah-receptor as reported for other tricyclic compounds. The results from inhibition experiments showed that the hydroxylated AQs were strong inhibitors of P450 1A2-dependent monooxygenases. This suggests that long-term ingestion of certain AQs, may affect the toxicity of other components present in the diet through the hepatic induction or inhibition of P450 1A2.     

Determination of base pairing in ribonucleic acid by Fourier-transform infrared spectrometry: yeast ribosomal 5S ribonucleic acid

Fourier-transform infrared (FT-IR) spectra of yeast ribosomal 5S RNA have been acquired at several temperatures between 30 and 90 degrees C. The difference spectrum between 90 (bases unstacked) and 30 degrees C (bases stacked) provides a measure of base stacking in the RNA. Calibration difference spectra corresponding to stacking of G-C or A-U pairs are obtained from "reference" FT-IR spectra of poly(rG) X poly(rC) minus 5'-GMP and 5'-CMP or poly(rA) X poly(rU) minus 5'-AMP and 5'-UMP. The best fit linear combination of the calibration G-C and A-U difference spectra to the 5S RNA (90-30 degrees C) difference spectrum leads to a total of 25 +/- 3 base pairs (17 G-C pairs + 8 A-U pairs) for the native yeast 5S RNA in the absence of Mg2+. In the presence of Mg2+, an additional six base pairs are detected by FT-IR (one G-C and five A-U). FT-IR melting curve midpoints show that A-U and G-C pairs melt together (65 and 63 degrees C) in the presence of Mg2+ but A-U pairs melt before G-C pairs (47 vs. 54 degrees C) in the absence of Mg2+.