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PubMed1982

Effects of olivacine on the metabolism of proteins and nucleic acids in Escherichia coli.

Olivacine, a semi-synthetic isomer of ellipticine, interferes with the growth of Escherichia coli. Investigation of the effects of graded concentration shows that, in vivo, the synthesis of overall proteins is markedly more sensitive to low concentrations than is the synthesis of RNA and DNA, respectively. Furthermore, at moderate concentrations, olivacine is much more inhibitory to the synthesis of cytoplasmic proteins than to that of envelope proteins. At lethal concentration (10-4 M) all macromolecular syntheses are inhibited, DNA synthesis, however, to a lower extent. Olivacine blocks the synthesis of polyribonucleotide and polypeptide chains at the elongation step. The resulting nascent products of both transcription and translation are unstable, whereas completed RNA molecules are partially stabilized in the presence of the drug.

Antineoplastic Agents, Phytogenic↗
PubMed1993

Activation of the murine monocyte/macrophage cell line, J774A1 by poly(A).poly(U): I. Binding of poly(A).poly(U) and induction of oligo-2',5'-adenylate synthetase.

Binding and internalization of the synthetic double-stranded complex poly(A).poly(U) were studied on a murine monocyte/macrophage cell line J774A1. Poly(A).poly(U) increased in a dose-dependent fashion the oligo-2',5'-adenylate synthetase demonstrating that those cells were responsive to this agonist. Binding of [32P]poly(A).[32P]poly(U) to the cells reached an apparent kinetic equilibrium within 4 h and was saturable (apparent Kd = 9.99 +/- 0.09.10(-2) g/l and Bmax 13.3 +/- 5.3.10(-3) g/l per 10(6) cells) and temperature-dependent. The binding of poly(A).poly(U) was competitively inhibited by various polynucleotides but not by other structurally unrelated compounds. Analysis of cell-associated [32P]poly(A).[32P]poly(U) demonstrated a minimal degradation of this polyribonucleotide over a 4-h incubation period. Autoradiography of cells incubated with [3H]poly(A).[3H]poly(U) revealed that poly(A).poly(U) was internalized and migrated to cell nuclei. These results suggest that poly(A).poly(U) is internalized in J774A1 cells via an endocytotic process.

2',5'-Oligoadenylate Synthetase↗
PubMed1988

Comparative study of various immunomodulators for macrophage and natural killer cell activation and antiviral efficacy against exotic RNA viruses.

Several immunomodulators were compared for immunomodulatory and antiviral activity in B6C3F1 female mice. Our results demonstrate that murine recombinant gamma interferon (rIFN-G), human recombinant alpha A/D interferon (rIFN-A), ampligen (a polyribonucleotide) and CL246,738 modulate nonspecific immunity and are effective antiviral agents in vivo. Administration of each of these agents 1 day before cell harvest induced high levels of splenic natural killer (NK) cell activity against YAC-1 target cells. rIFN-G was also a potent activator of peritoneal macrophages (M phi), as evidenced by high levels of antitumor activity and changes in ectoenzyme phenotype that is characteristic of tumoricidal M phi. rIFN-A, ampligen and CL246,738 induced moderate to low levels of M phi activation by these criteria. In vivo protection experiments showed that repeated therapeutic treatment with rIFN-A protected mice against i.p. infection with Venezuelan equine encephalitis (an alpha togavirus, VEE), Banzi (a flavivirus) and herpes simplex virus type 2 (HSV-2). Similar treatment with rIFN-G was effective against VEE and HSV-2, but ineffective against Banzi virus. A single prophylactic i.p. dose of ampligen 1 day before virus challenge was very effective against Banzi virus, moderately effective against HSV-2, and ineffective against VEE and Caraparu (a bunyavirus) infection. A single prophylactic oral dose of CL246,738 provided almost complete protection of mice against VEE, Banzi, and HSV-2, and also increased the mean survival time for Caraparu infected mice. Collectively, these results indicate that rIFN-A, r-IFN-G, ampligen and CL246,738 may be useful in prophylactic or early therapeutic treatment of several serious virus infections. Since these agents stimulate NK cells and M phi, their antiviral activity may result, in part, from the alterations they induce in the natural immune system.

Adjuvants, Immunologic↗
PubMed1975

Ionic strength dependence of the hysteresis in the polyriboadenylate-polyribouridylate system.

The hysteresis observed in cyclic acid-base titrations of the three-standed polyribonucleotide helix poly (A)-2 POLY (U) strongly depends on ionic strength. For NaCl and at 25 degrees C, hysteresis occurs in the limited concentration range between 0.03 M and 1.0 M(NaCl). The transition points associated with the cyclic conversions between the triple helix and the poly (A)-poly (A) double helix and (free) poly (U) constitute a (pH ionic strength) phase diagram covering the ranges of stability and metastability of the hysteresis system. Variations with NaCl concentration of some hysteresis parameters can be quantitatively described in terms of polyelectrolyte theories based on the cylinder-cell model for rodlike polyions. The results of this analysis suggest that the metastability is predominantly due to dlectrostatic energy barriers preventing the equilibrium transition of the partially protonated triple helix above a critical pH value. Ultraviolet absorbance and potentiometric titration data of poly (A)in the acidic pH range can be analyzed in terms of two types of double-helical structures. Spectrophotometric titrations reveal isosbestic wavelengths for structural transitions of poly (A). "Time effects" commonly observed in poly (A) titrations are suggested to reflect helix transitions between the two acidic structures.

Adenine Nucleotides↗
PubMed1977

Effects of polyamines and polyanions on a cyclic nucleotide-independent and a cyclic AMP-dependent protein kinase.

The phosphorylation of phosvitin in vitro by a cyclic nucleotide-independent protein kinase (phosvitin kinase) derived from rooster liver is markedly stimulated by the divalent cation, Mg2+. In addition, the activity is further stimulated by low concentrations of the polyamines putrescine, spermidine and spermine leading to higher rates of phosphate incorporation than could be obtained at any concentration of Mg2+. Spermine is inhibitory at higher concentrations. The polyamines shift the Mg2+ requirement for maximal activity to lower concentrations. The activity of a cyclic AMP-dependent histone kinase from beef heart is not altered by the presence of polyamines. Heparin is a potent inhibitor of phosvitin kinase but has no effect on histone kinase. Polyribonucleotides (polyadenylic acid and transfer RNA) inhibit both types of kinases, but the degree of inhibition of phosvitin kinase is variable and depends upon the type of the polyanion present. Sermidine and spermine, but not Mg2+, efficiently counteract the inhibitory action of heparin and tRNA. The results suggest that, also in vivo, naturally occurring polyamines and polyanions such as tRNA may have a regulatory function on protein kinases.

Animals↗
PubMed2004

An assessment of immunostimulants as Mx inducers in Atlantic salmon (Salmo salar L.) parr and the effect of temperature on the kinetics of Mx responses.

The aims of this study were (i) to identify alternative Mx stimulatory compounds in Atlantic salmon (Salmo salar L.) and to characterise the kinetics and intensity of the stimulated responses and (ii) to investigate the effect of temperature on such responses by semi-quantitative RT-PCR. Mx transcripts were measured in Atlantic salmon parr kept at 14 degrees C and injected with either LPS, the synthetic double-stranded polyribonucleotide poly I:C, Vibrio anguillarum serotypes I and II-ordalii bacterin, beta-glucan, whole yeast cells or yeast RNA. Sampling periods lasted until transcripts were undetectable or up to three weeks after immunisation. The effect of temperature on poly I:C-induced Mx response was studied by injecting parr kept at 6 degrees C. Newly hatched salmon fry were immersed once, twice or three times in the Vibrio bacterin diluted five or 10 times and sampled for three weeks. None of the yeast compounds induced Mx expression in Atlantic salmon parr. LPS induced a very low Mx response 2 and 3 days after injection. The Vibrio bacterin administered by injection in parr (but not by immersion in fry) resulted in strong Mx induction on days 2 and 3, disappearing by day 6. Poly I: C-induced Mx responses that were more intense and longer lasting than those induced by the bacterin, peaking on day 3 and lasting over 6 days, disappearing by day 9 at 14 degrees C. Lower temperature caused a longer lasting Mx response to poly I:C (at least 21 days), which peaked on days 7-14, with a similar intensity and no delayed onset as compared with the response at 14 degrees C. However, some toxicity of the poly I:C was indicated in treatments at 6 degrees C.

Adjuvants, Immunologic↗
PubMed2004

Nicotinic stimulation modulates tyrosine hydroxylase mRNA half-life and protein binding to the 3'UTR in a manner that requires transcription.

Tyrosine hydroxylase (TH) expression increases in adrenal chromaffin cells treated with the nicotinic agonist, dimethylphenylpiperazinium (DMPP; 1 microM). We are using this response as a model of the changes in TH level that occur during increased cholinergic neural activity. Here we report a 4-fold increase in TH mRNA half-life in DMPP-treated cells chromaffin cells that is apparent when using a pulse-chase analysis to measure TH mRNA half-life. No increase is apparent using actinomycin D to measure half-life, indicating a requirement for ongoing transcription. Characterization of protein binding to the TH 3'UTR responsible for stabilization using labeled TH 3'UTR probes and electro-mobility shift assays shows the presence of two complexes both of which are increased by DMPP-treatment. The faster migrating complex (FMC) increases 2.5-fold and the slower migrating complex (SMC) increases 1.5-fold. Both changes are prevented by actinomycin D. Characterization of the protein binding to the TH UTR probes indicates SMC is disrupted by polyribonucleotides, poly (A) and poly (U), while binding to FMC is reduced by poly (CU). Separation of UV crosslinked RNA-protein complexes on SDS polyacrylamide gels shows FMC to contain a single protein whereas SMC contains three proteins. Northwesterns yielded similar results. Comparison of DMPP-induced protein binding with the poly C binding protein (PCBP) involved in hypoxia induced rat PC12 TH mRNA stability indicates none of the bovine UTR binding proteins are the PCBP. Thus, nicotinic stimulation produces a transcription-dependent increase in TH mRNA half-life that is mediated by previously unrecognized TH mRNA binding proteins.

3' Untranslated Regions↗
PubMed2004

Lead toxicity through the leadzyme.

Lead is one of the most dangerous toxic agents for all living organisms. In humans, elevated levels of lead have been linked to a number of disorders for which various molecular mechanisms have been proposed. However, none of them has been fully understood. It has also been known for several years that at micromolar concentrations lead can bind a unique RNA motif and catalyze a site-specific hydrolysis of the polyribonucleotide chain. This motif, called leadzyme, may be one of the major targets for lead within the cell, and it can cleave various cellular RNAs. A search of GenBank revealed the sequences that can potentially fold into the structure containing the leadzyme motif and that they are rather common in eukaryotic genomes. We found that the domain occurs with a high frequency in human mRNA sequences. Thus, the leadzyme nucleolytic properties should be considered as a possible mechanism for destruction of RNA within a cell. In particular, targeting of the RNA scaffold of ribosomes or spliceosomes may explain lead-mediated toxicity leading to cell death.

Humans↗
PubMed1996

Theory, design, and characterization of a microdialysis flow cell for Raman spectroscopy.

The theory, design, and application of a dialysis flow cell for Raman spectroscopy are described. The flow cell permits rapid collection of Raman spectra concurrent with the efflux of small solute molecules or ions into a solution of macromolecules and is well suited to acquisition of data during hydrogen-isotope exchange reactions of biological molecules. Kinetic parameters of the device are described by a diffusion model, which accounts satisfactorily for the observed rates of efflux of deuterium oxide (K2H = 0.30 min-1), calcium ions (KCa = 0.10 min-1) and EGTA (KEGTA = 0.07 min-1). Application to the kinetics of glutamate protonation in a peptide copolymer [poly(Glu, Lys, Tyr)] shows that pH-titration rates as high as 3.3 pH units/min can be monitored. It is also shown that one can extract first-order hydrogen-isotope exchange rate constants from measured second-order exchanges by taking into account the rate of entry of 2H2O effluent into the bulk H2O solution. Deuterium exchanges of the single-stranded polyribonucleotides poly(rA) and poly(rU) and of the double-stranded RNA genome from bacteriophage phi 6 have been investigated. The measured nucleotide base exchange rates are comparable with those determined previously by other methods. The results indicate that base exchanges as fast as approximately 2 min-1 can be determined reliably with the present design. Application of the Raman flow cell to hydrogen-isotope exchange of the basic pancreatic trypsin inhibitor confirms consistency with results obtained previously on this protein by tritiation and NMR techniques.

Calcium↗
PubMed2002

Omnipotent RNA.

The capability of polyribonucleotide chains to form unique, compactly folded structures is considered the basis for diverse non-genetic functions of RNA, including the function of recognition of various ligands and the catalytic function. Together with well-known genetic functions of RNA - coding and complementary replication - this has led to the concept of the functional omnipotence of RNA and the hypothesis that an ancient RNA world supposedly preceded the contemporary DNA-RNA-protein life. It is proposed that the Woese universal precursor in the ancient RNA world could be a cell-free community of mixed RNA colonies growing and multiplying on solid surfaces.

Catalysis↗
PubMed1998

Template-dependent biosynthesis of poly(G) x poly (C) and its antiviral activity in vitro and in vivo.

Experimental conditions for poly(G) synthesis from GTP on a poly(C) template with the aid of Escherichia coli DNA-dependent RNA polymerase were investigated. The reaction product was purified without the use of RNase. On the basis of spectral data, gel permeation chromatography, affinity adsorption and electron microscopic visualization, the poly(G) x poly(C) product was assumed to possess a high degree of structural regularity. Its in vitro and in vivo antiviral activities were compared with those of traditional poly(G) x poly(C) and poly(I) x poly(C). Template-dependent poly(G) x poly(C) was similar in its in vitro activity to poly(I) x poly(C) or even surpassed it, whereas the 'traditional' poly(G) x poly(C) was only slightly active in vitro. However, 'traditional' poly(G) x poly(C) and poly(I) x poly(C) had similar activity in vivo, whereas template-dependent poly(G) x poly(C) was much less active in vivo. The role of intramolecular structural regularity in the in vitro and in vivo antiviral activity of polyribonucleotide duplexes is discussed.

Animals↗
PubMed2003

Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).

Human papilloma virus (HPV) found in 99.7% of cervical cancers represents an attractive immunotherapeutic target for novel adjuvant dendritic cell (DC) immunotherapy. DC primed with HPV antigens have been shown to be capable of inducing CTL responses powerful enough to eradicate established murine tumours expressing HPV16 antigen. The use of tumour lysate has been found to be an effective means of priming DC with tumour associated antigens in animal models and in clinical trials leading to significant anti-tumour responses. Autologous DC primed with sonicated HPV expressing tumour lysate have been shown to be capable of inducing HPV specific classes I and II T-cell immunity in a pilot clinical study.Synthetic double stranded polyribonucleotides are effective in vitro activation/maturation agents capable of inducing a stable mature DC phenotype producing high levels of IL12. However, the prototype polymer poly [I]:poly [G] has proved to be clinically toxic. Preliminary in vitro data have demonstrated that a novel clinically non-toxic analogue polymer poly [I]:poly [C(12)U] (Ampligen R) can effectively induce in vitro maturation of human monocyte derived DC with sustained bioactive IL12 production. Human monocyte derived DC primed with tumour lysate and matured with synthetic dsRNA may therefore offer an effective way of optimising Th1 specific anti-cancer T-cell responses in cancer patients. This strategy is currently being tested in a clinical trial in patients with cervical cancer.

Clinical Trials as Topic↗
PubMed1976

Varied effects of polyadenylic-polyuridylic acid on immune responses.

The effect of the double-stranded synthetic polynucleotide, poly(A).poly(U), on the immune response of inbred mouse strains to multichain synthetic polypeptides was studied. Poly(A).poly(U) did not affect immune responses controlled by H-2 linked genes. Thus, when either (T,G)-A- -L or (Phe,G)-A--L were injected into high or low responder mice followed by administration of poly(A).poly(U) 24 h after immunization, no increase in the antibody titers was observed. In contrast, poly(A).poly(U) increased significantly the response to polyproline, which is controlled by a non H-2 linked gene, in low responder mice. However, the polyribonucleotide had no effect on the antibody titers of the SJL mice, the high responders to multichain polyproline. When poly(A).poly(U) was injected into DBA/1 mice following immunization with (Phe,G)-Pro- -L, the polynucleotide enhanced the low response to the Pro- -l region at the expense of the anti (Phe,G) response which is normally high in this mouse strain. In this case poly(A).poly(U) caused an intramolecular antigenic competition. The general conclusion of this study is that the chemical nature of the antigenic determinant plays an important role in determining the type of influence exerted by poly(A).poly(U).

Animals↗
PubMed1978

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.

Animals↗
PubMed1990

The formation of covalent adducts between benzo[a]pyrenediol epoxide and RNA: structural analysis by mass spectrometry.

Racemic 7-r,8-t-dihydroxy-9-t,10-t-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene was reacted with yeast RNA. Modified nucleosides were isolated and resolved by high-performance liquid chromatography; nine adduct peaks were collected for analysis. The bases in these adducts were identified by comparing their retention times with those of adducts from poly(G), poly(A), and poly(C). These samples gave two major and two minor Guo adducts, four major Ado adducts, and at least four Cyd adducts. The relative efficiencies of adduct formation with the polyribonucleotides were poly(G) greater than yeast RNA greater than poly(A) greater than poly(C). Fluorescence measurements show that emission from Guo adducts is strongly quenched relative to that from Ado adducts. Liquid secondary ion mass spectrometry (LSIMS) of underivatized samples and electron-impact mass spectrometry (EIMS) of permethyl derivatives were used to confirm the base identities and establish the alkylation sites of the RNA adducts. Unique nitrogen-containing hydrocarbon fragments that were observed with all samples by EIMS establish that in each adduct analyzed the C-10 position of the hydrocarbon is linked to the exocyclic amino group of the base. This suggested that the multiple adducts formed with each base are diastereomers derived from cis/trans epoxide ring opening of the (+) and (-) enantiomers of the carcinogen. Several adducts exhibited molecular ions by both LSIMS and EIMS. Large fragments observed by EIMS usually resulted from the loss of CH3OH, CH3O., CH2O, CH3., and H. from the molecular ion. Major fragmentation pathways also resulted in formation of nucleoside, base, ribose, hydrocarbon, and base-hydrocarbon ions. Each of these major ions in turn resulted in further characteristic fragmentation patterns.

Animals↗
PubMed1990

Structure calculations for single-stranded DNA complexed with the single-stranded DNA binding protein GP32 of bacteriophage T4: a remarkable DNA structure.

In this study it is established by calculation which regular conformations single-stranded DNA and RNA can adopt in the complex with the single-stranded DNA binding protein GP32 of bacteriophage T4. In order to do so, information from previous experiments about base orientations and the length and diameter of the complexes is used together with knowledge about bond lengths and valence angles between chemical bonds. It turns out that there is only a limited set of similar conformations which are in agreement with experimental data. The arrangement of neighboring bases is such that there is ample space for aromatic residues of the protein to partly intercalate between the bases, which is in agreement with a previously proposed model for the binding domain of the protein [Prigodich, R. V., Shamoo, Y., Williams, K. R., Chase, J. W., Konigsberg, W. H., & Coleman, J. E. (1986) Biochemistry 25, 3666-3671]. Both C2'endo and C3'endo sugar conformations lead to calculated DNA conformations that are consistent with experimental data. The orientation of the O2' atoms of the sugars in RNA can explain why the binding affinity of GP32 for polyribonucleotides is lower than for polydeoxyribonucleotides.

Binding Sites↗
PubMed1990

Self-cleavage of hepatitis delta virus genomic strand RNA is enhanced under partially denaturing conditions.

Self-cleavage of a polyribonucleotide containing an autocleaving sequence from the genomic strand of hepatitis delta virus was enhanced by conditions that destabilized RNA structure. Self-cleavage of the transcripts used in this study required Mg2+ (or another divalent cation), and in the absence of denaturants, maximum cleavage was observed at very low Mg2+ concentrations (0.05-0.1 mM). However, at 37 degrees C and in the presence of 2-10 mM Mg2+ the rate of cleavage was increased as much as 50-fold with the addition of urea to 5 M or formamide to 10 M. Cleavage was prevented by higher concentrations of the same reagents (9.5 M urea or 22.5 M formamide), presumably because a structure required for self-cleavage is disrupted by strongly denaturing conditions. In contrast to a previous report [Wu, H.-N., & Lai, M. M. C. (1989) Science 243, 652-654], we find that chelating Mg2+ with EDTA terminates the cleavage reaction without promoting measurable amounts of ligation of the cleavage products. The ability of denaturants to promote rapid self-cleavage in vitro raises the possibility that an unidentified factor could have a similar effect in vivo.

Base Sequence↗
PubMed1982

Purification and characterization of the messenger ribonucleic acid capping enzyme GTP:RNA guanylyltransferase from wheat germ.

A GTP:RNA guanylyltransferase or capping enzyme has been purified approximately 2000-fold from wheat germ. The enzyme catalyzes the transfer of the GMP residue from GTP to the 5' end of RNA or synthetic polyribonucleotides. Diphosphate-ended polymers were capped more efficiently than molecules with triphosphate ends, and molecules with monophosphate ends were not capped at all. There appears to be little specificity since RNAs with purine or pyrimidine ends served as acceptors. Other features of the wheat germ RNA guanylyltransferase include relatively low Km values for GTP (2.7 microM) and ppA (pA)n (14.2 nM), a divalent cation requirement satisfied by low (0.5 mM) concentrations of MnCl2 or higher (5 mM) concentrations of MgCl2, and a pH optimum around neutrality.

Kinetics↗