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P F Torrence

Publications and source records attributed to P F Torrence.

At least 73 records · Page 4Linked to original sources
PubMed1986

Synthesis and biological activities of oligo(8-bromoadenylates) as analogues of 5'-O-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine.

The 8-bromoadenosine analogue of 5'-O-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine (2-5A) and its derivatives were synthesized, and their biological activity was evaluated in mouse L cell extracts. All compounds, except 5'-dephosphorylated "cores" bound to the 2-5A-dependent endonuclease with a relative activity, depending on the derivative, of 1 to 0.035 of that of 2-5A trimer. 8-Bromoadenylate trimer 5'-mono-, -di, and -triphosphates inhibited protein synthesis with a relative activity of 0.0023, 0.050, and 0.015 compared to 2-5A. Tetramer 5'-monophosphate also inhibited protein synthesis (relative activity 0.0033). The corresponding pentamer 5'-monophosphate did not; however, the pentamer 5'-diphosphate was able to inhibit translation (relative activity 0.0092). All compounds that possessed inhibitory activity in the protein synthesis inhibition assay gave ribosomal RNA cleavage patterns characteristic of the action of 2-5A-dependent endonuclease. Thus, 8-bromination of the all of the adenine rings of 2-5A leads to 20- to 70-fold reduction in the biological activity of the corresponding 5'-di- and -triphosphate, respectively; however, this same alteration of the three adenine moieties gives rise to a 5'-monophosphate with much enhanced translational inhibitory activity compared to the parent 2-5A trimer 5'-monophosphate.

Adenine Nucleotides↗
PubMed1986

Two molecular weight forms of human 2',5'-oligoadenylate synthetase have different activation requirements.

Ion-exchange and gel filtration chromatography were used to purify partially a 33,000-dalton (33 kD) 2',5'-oligoadenylate synthetase and a 110,000-dalton (110 kD) 2',5'-oligoadenylate synthetase from HeLa cells treated with alpha-interferon (IFN-alpha). The 33-kD enzyme was optimally activated only when double-stranded RNA was added in 100 to 1000-fold excess of the concentration activating the 110-kD enzyme. Certain double- or triple-stranded RNAs, which were observed to activate the 110-kD enzyme, failed to activate the 33-kD enzyme, even when added at high concentration. The 110-kD enzyme manifested an alkaline pH optimum of 7.5 or more and the 33-kD enzyme an acidic pH optimum of 6.0 or less. The results suggest that intracellular activation of the two forms of 2',5'-oligoadenylate synthetase may occur under markedly different conditions.

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Antagonism of 2-5 A-mediated inhibition of protein synthesis in intact cells by 2',5'-(pA)3.

In vitro studies have shown that the translational inhibitory activity of 2-5 A can be blocked by the oligoribonucleotide 2',5'-(pA)3. We have examined the effect of simultaneous introduction of inhibitor and antagonist into intact mouse cells using calcium phosphate coprecipitation. Upon introduction of 10(-4) M 2',5'-(pA)3 and 10(-6) M 2-5 A, inhibition of protein synthesis was prevented. Efficiency of calcium phosphate precipitation of 2-5 A in the presence or absence of 2',5'-(pA)3 was comparable. Introduction of 2',5'-(pA)3 analogs showed that nucleotides which do not bind well to the 2-5 A dependent endonuclease do not prevent 2-5 A inhibitory activity. Thus, 2',5'-(pA)3 functions as an antagonist of 2-5 A in vivo.

Adenine Nucleotides↗
PubMed1985

Antibody-nucleic acid interactions. Monoclonal antibodies define different antigenic domains in 2',5'-oligoadenylates.

To define the epitopes involved in binding anti-oligonucleotide antibodies, several hybridomas producing monoclonal antibodies directed against 2',5'-oligoadenylate were established. A solid-phase enzyme-linked immunoassay that employed microtiter wells coated with Ficoll-2',5'-oligoadenylate conjugates proved useful in screening and characterizing hybridoma supernatants. Control experiments demonstrated that the conjugates were irreversibly adsorbed to polystyrene wells under the conditions employed in the assay. Reactivity of monoclonal antibodies with numerous analogues of 2',5'-oligoadenylate was measured by using a competition assay. Several monoclonal antibodies originating from different mice immunized with the same or different immunogens possessed distinctive fine specificities. At least one 2',5'-phosphodiester bond was important in forming each epitope, suggesting that the ribose phosphate backbone is a critical element in defining an antigenic domain of an oligonucleotide. The purine bases were also important, and modification of the bases had varied effects on the extent of antibody recognition. The length of the oligonucleotide and the nature of the termini were also of some importance. In several instances the modification created by linkage of 2',5'-oligoadenylate to carrier protein also contributed to the determinant. The monoclonal antibody most specific for 2',5'-oligoadenylates was relatively insensitive to ionic strength. In contrast, a monoclonal antibody with a 2',5'-oligopurine specificity appeared to bind 2',5'-oligoadenylate through one ion pair, whereas the binding of a monoclonal antibody with a low degree of base specificity appeared to bind through two ion pairs. The results demonstrated that 2',5'-linked oligoadenylate-protein complexes possess at least three distinct oligonucleotide-related antigenic surfaces that can be recognized with high apparent affinity by monoclonal antibodies. A model for the three epitopes is presented.

Adenine Nucleotides↗
PubMed1985

Cordycepin analogs of ppp5'A2'p5'A2'p5'A (2-5A) inhibit protein synthesis through activation of the 2-5A-dependent endonuclease.

Analogs of the triphosphate 2'-5'-linked adenylate trimer (ppp5'A2'p5'A2'p5'A, called 2-5A) which contain 3'-deoxyadenosine (cordycepin) instead of adenosine either in positions one and two, or in all three positions, are 10-100-fold less potent than is parent 2-5A in inhibition of protein synthesis in intact cells, when utilizing calcium co-precipitation techniques to introduce the 5'-triphosphate oligonucleotides into the cells. That the inhibition of protein synthesis was a consequence of activation of the 2-5A-dependent endonuclease by the 3'-deoxyadenosine analogs of 2-5A was demonstrated in obtaining the ribosomal RNA cleavage pattern that is characteristic of endonuclease activation by parent 2-5A. Additional results (i.e. lack of activity by the dimer species ppp5'(3'dA)2'p5'-(3'dA) or the monomer 3'dA) as well as kinetic analysis both in intact cells and in cell-free extracts provided further evidence that the inhibition of protein synthesis observed with these 3'-deoxyadenosine 2-5A analogs was not due to their degradation to the antimetabolite monomer unit 3'-deoxyadenosine.

Animals↗
PubMed1985

Respective role of each of the purine N-6 amino groups of 5'-O-triphosphoryladenylyl(2'----5')adenylyl(2----5')adenosine in binding to and activation of RNase L.

We have synthesized a series of 2-5A (ppp5'-A2'p5'A2'p5'A) analogs in which each adenosine residue has been sequentially replaced by inosine: viz., ppp5'I2'p5'A2'p5'A, ppp5'A2'p5'I2'p5'A, and ppp5'A2'p5'A2'p5'I. These transformations enabled us to delineate the role of each of the three purine N-6 amino groups of 2-5A in determining oligonucleotide binding to and activation of the 2-5A-dependent endoribonuclease, RNase L. With the RNase L activity of both mouse L cells and human Daudi lymphoblastoid cells, we found that the N-6 amino group of the first adenosine nucleotide residue (from the 5'-terminus) is of crucial importance in determining binding to the endonuclease; however, removal of the N-6 amino moieties of the second or third adenosine nucleotide residues resulted in only a minimal decrease in binding to the endonuclease. On the other hand, conversion of the third adenosine residue to inosine effected a dramatic (10,000-fold compared to 2-5A) loss in ability to activate the nuclease; however, execution of the same N-6 amino group conversion at either the first or second adenosine residue did not cause a major change in nuclease activation ability when the accompanying decreased endonuclease binding was considered. These results clearly demonstrate that the N-6 amino group of the first adenosine residue of 2-5A is critical in RNase L binding whereas the N-6 amino function of the third adenosine residue of 2-5A is crucial for the activation of RNase L.

Adenine Nucleotides↗
PubMed1985

Phosphorylation of 2-5A core 5'-diphosphate to 2-5A in mouse L cell extracts.

When added to extracts of mouse L cells containing ATP and an energy regenerating system, the 5'-diphosphate of 2-5A core, pp5'A2'p5'A2'p5'A, as well as a bromoadenylate analog, pp5' (br8A)2'p5'(br8A)2'p5'(br8A), can be phosphorylated to the corresponding 5'-triphosphate, ppp5'A2'p5'A2'p5'A and ppp5'(br8A)2'p5'(br8A)2'p5(br8A), respectively. The extent of this conversion was about 0.5% when the concentration of 5'-diphosphate was about 10(-4) M. Thus, although previous studies have shown that the 5'diphosphate, pp5'A2'p5'A2'p5'A, can activate the 2-5A-dependent endonuclease, this may be related to a phosphorylation reaction in the crude cell extracts employed in these studies and may not represent a true ability of such a 5'-diphosphate to activate directly the endonuclease.

Adenine Nucleotides↗
PubMed1985

Replacement of the ribofuranose oxygen of 2-5A derivatives by methylene: synthesis of an aristeromycin analogue of 2-5A core 5'-monophosphate (5'-O-phosphoryladenylyl)(2'----5')adenyl(2'----5')adenosine.

The ribofuranose oxygens of the three adenosine residues of the 5'-monophosphate of the 2-5A core [adenylyl(2'----5')adenylyl(2'----5')adenosine] were replaced by methylenes through the synthesis of an aristeromycin [9-[(1R,2S,3R,4R)-2,3-dihydroxy-4-(hydroxymethyl)cyclopentyl]adenosine] analogue. In the synthetic approach, the chlorophosphite triester procedure was employed together with the use of dimethoxytrityl and tert-butyldimethylsilyl protecting groups. The final product 14 was bound to the 2-5A-dependent endonuclease of mouse, rabbit, or human cells 100-300 times less effectively than parent p5'A2'p5'A2'p5'A. In extracts of human Daudi cells where the monophosphate p5'A2'p5'A2'p5'A was able to effect ribosomal RNA cleavage at 2 X 10(-7) M, 14 required a concentration of 2 X 10(-5) M to bring about discernible rRNA cleavage.

Adenine Nucleotides↗
PubMed1985

Immunochemistry, physical chemistry and biology of 2',5'-oligoadenylates.

Monoclonal antibodies directed against 2-5A were developed and characterized. 2',5'-Oligoadenylate-protein complexes possess at least three distinct antigenic surfaces defined primarily by the ribose-phosphate backbone. A schematic model for the three epitopes is presented. Antibodies directed against 2-5A, in conjunction with other techniques, were employed to quantify 2-5A in various tissues of pathogen-free mice. Levels of 2-5A were in the range of 400-800 fmole/gm. Mice injected with poly(I).poly(c) or encephalomyocarditis virus (EMCV) showed elevated levels of 2-5A. Administration of poly(I).poly(C) or EMCV increased the level of 2-5A in different tissues to different extents. Raman spectroscopy was employed to explore the conformation of 2-5A. Distinct differences were observed in bands arising from the backbone portion of 2-5A relative to those of 3-5A. The most striking finding was the appearance of a strong, sharp band at 1460 cm-1 in spectra of 5'-monophosphorylated 2-5A's. This band was barely detectable in core and triphosphorylated 2-5A. Its presence suggests that 5'-monophosphorylated 2-5A's possess a unique conformational feature that distinguish them from cores and 5'-triphosphorylated forms.

Adenine Nucleotides↗
PubMed1985

Sequence-specific 2'5'-oligonucleotides in the molecular dissection of the biological activity of 2-5A.

Recently developed chemical synthetic methodology for facile preparation of sequence-specific 2'5'-oligonucleotides has allowed more exacting questions to be asked regarding the biological role of each of the nucleotide residues of 2-5A. We have found, employing analogs in which each adenosine residue has been sequentially replaced by adenosine (viz, ppp5'I2'p5'A2'p5'I, ppp5'A2'p5'I2'p5'A, ppp5'A2'p5'A2'p5'I) that the N6 amino group of the first (or 5'-terminal) adenosine residue of 2-5A trimer is critical in RNase L binding whereas the N6 amino moiety of the third (or 2'-terminal) adenosine residue of 2-5A is crucial for the activation of RNase L. The second or middle adenosine unit of 2-5A does not seem to be critical for either binding or activation. Similarly, in studies on sequence-specific tubercidin analogs of 2-5A, activation of but not binding to mouse RNase L was dependent on the presence of the purine N7 atoms of the first and third adenosine residues of 2-5A, but, as with the N6 amino group, the N7 moiety of the second adenosine residue of 2-5A was not essential for either binding to or activation of the 2-5A-dependent endonuclease. Finally, sequence-specific purine 8-bromination provided analogs of dramatically varying biological properties, and provided a 5'-monophosphate, p5'A2'p5'(br8A)2'p5'(br8A), which possessed 8% of the translational inhibitory action of 2-5A itself. This latter result may represent an important impetus toward obtaining a 2-5A derivative with biological activity in an intact cell.

Adenine Nucleotides↗
PubMed1984

Synthesis and biological activity of tubercidin analogues of ppp5'A2'p(5'A2'p)n5'A.

A series of tubercidin (7-deazaadenosine) analogues of 2-5A of the general formula p5'(c7A)2'p[5'(c7A)-2'p]n5'(c7A) (n = 0-5) were prepared by lead ion catalyzed polymerization of the 5'-phosphoroimidazolidate of tubercidin. Through the corresponding imidazolidates, these oligonucleotide 5'-monophosphates were converted to the 5'-triphosphates. All reported structures were corroborated by enzyme digestion and 1H or 31P nuclear magnetic resonance. When evaluated for its ability to bind to the 2-5 A-dependent endonuclease of mouse L cells, the tubercidin analogue of trimeric 2-5A, namely, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), and the corresponding tetramer were bound as effectively as 2-5A itself; nonetheless, it and the corresponding tetramer, ppp5'-(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), failed to stimulate the 2-5A-dependent endonuclease as judged by its inability to inhibit translation in extracts of mouse L cells programmed with encephalomyocarditis virus RNA and to give rise to ribosomal RNA cleavage in the same cell system under conditions where 2-5A showed activity at 10(-9) M. The trimer, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A), was an antagonist of 2-5A action in the L cell extract. In the lysed rabbit reticulocyte system, both the trimeric and tetrameric tubercidin 2-5A analogues were bound to the 2-5A-dependent endonuclease as well as 2-5A, but in this case, the tetramer triphosphate, ppp5'(c7A)2'p5'(c7A)2'p5'(c7A)2'p5'(c7A), was just as potent an inhibitor of translation as 2-5A tetramer triphosphate. Moreover, this inhibition was prevented by the established 2-5A antagonist p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1984

Synthesis and biological activity of 5'-capped derivatives of 5'-triphosphoadenylyl(2'----5')adenylyl(2'----5')adenosine.

The oligonucleotides A5'pp5'A2'p5'A2'p5'A and A5'ppp5'A2'p5'A2'p5'A were prepared by reaction of AMP or ADP, respectively, with the 5'-(phosphoimidazolidate) of A2'p5'A2'p5'A. A5'pppp5'A2'(p5'A)n (n = 1-3) were synthesized by reaction of p5'A2'(p5'A)n (n = 1-3) with adenosine 5'-trimetaphosphate. All structures were confirmed by enzyme digestion and 1H and 31P nuclear magnetic resonance (NMR). The products A5'pppp5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were found to be identical with two of the products of the 2-5A synthetase catalyzed reaction of Ap4A with ATP, thus confirming the structural assignments made by earlier investigators. In extracts of mouse L cells programmed with encephalomyocarditis virus RNA, A5'pppp5'A2'p5'A2'p5'A2'p5'A and A5'pppp5'A2'p5'A2'p5'A were equipotent with 2-5A itself as inhibitors of translation. The oligomers A5'ppp5'A2'p5'A2'p5'A and A2'pppp5'A2'p5'A were about 100 times less active than 2-5A, and A5'pp5'A2'p5'A2'p5'A was without translational inhibitory activity. When affinity for the 2-5A-dependent endonuclease was determined (by displacement of 2-5A[32P]pCp from endonuclease), all of the analogues, as well as 2-5A itself, had similar affinities for the endonuclease except for A5'pppp5'A2'p5'A, which was bound approximately 100 times less effectively. Under conditions of the radiobinding assay, A5'pppp5'A2'p5'A2'p5'A was degraded (t1/2 = 2 h) to ATP, ADP, AMP, ppp5'A2'p5'A2'p5'A, and p5'A2'p5'A2'p5'A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine Nucleotides↗
PubMed1984

Preparation and biological properties of a highly active poly(G) X poly(C) inducer of interferon.

Experimental conditions necessary for the full expression of interferon-inducing activity by a complex of polyguanylic acid and polycytidylic acid included: (i) a sufficiently high molecular size of each homopolymer; (ii) annealing conditions which insured complete denaturation of polyguanylic acid self-structure; and (iii) the specific biological assay employed to assay interferon-inducing potency. The complex of polyguanylic acid and polycytidylic acid possessed several properties that suggested it may be an atypical polynucleotide interferon inducer. For instance, it was inactive in primary rabbit kidney cell cultures, usually exquisitely sensitive to polynucleotide interferon inducers, unless it was incubated on the cell cultures for prolonged times or in the presence of DEAE-dextran. Polyguanylic acid X polycytidylic acid could induce interferon in rabbits and mice but gave a more protracted response than did poly(I) X poly(C). Finally, poly(G) X poly(C) was, without any modification, resistant to degradation by serum nucleases.

Animals↗
PubMed1984

Oligonucleotide structural parameters that influence binding of 5'-O-triphosphoadenylyl-(2'----5')-adenylyl-(2'----5')-adenosine to the 5'-O-triphosphoadenylyl-(2'----5')-adenylyl-(2'----5')-adenosine dependent endoribonuclease: chain length, phosphorylation state, and heterocyclic base.

A number of 2',5'-linked oligoadenylates and their analogues were prepared and evaluated for their ability to interact with the 5'-O- triphosphoadenylyl -(2'----5')-adenylyl-(2'----5')-adenosine (2-5A) dependent endoribonuclease of mouse L cells. The oligonucleotides were assayed for their ability to antagonize the action of 2-5A, to displace a radiolabeled probe from the 2-5A-dependent nuclease, or to inhibit translation in a cell-free system. These experiments demonstrated the following: (1) Three AMP residues in a 5'-phosphorylated oligonucleotide were needed for maximum interaction with the endonuclease, and higher oligomers (greater than or equal to 4 AMP residues) did not show significantly higher binding. (2) The third (2'-terminal) adenosine residue was required for optimal binding activity. (3) 5'-Phosphorylation of the oligonucleotide was necessary for maximum binding to the endonuclease, but the first (from the 5' terminus) internucleotide phosphate of higher unphosphorylated or core oligomers, such as A2'p5'A2'p5'A2'p5'A, may partly replace the requirement for a 5'-monophosphate moiety; in agreement with this, the 5'-methyl ester of 5'pA2'p5'A2'p5'A, i.e., Me-p5'A2'p5'A2'p5'A, was bound to the endonuclease as well as or better than the higher core oligomers but approximately 100 times more effectively than the trimer core, A2'p5'A2'p5'A. (4) Base-modified analogues, such as p5'C2'p5'C2'p5'C, p5'U2'p5'U2'p5'U, or p5'I2'p5'I2'p5'I, were at least 2000 times less effectively bound to the endonuclease than p5'A2'p5'A2'p5'A. (5) The triphosphate ppp5 'I2'p5'I2'p5'I was 10 000 times less active than 2-5A as an inhibitor of translation. These latter two points implied the critical role of the adenine N1-nitrogen and/or exocyclic amino group in the binding of 2-5A to the endonuclease.

Adenine Nucleotides↗
PubMed1984

Chemical synthesis and biological activities of partially inosine-substituted 2',5'-oligoadenylates: functional discrimination of three adenine amino groups on 2-5A for the binding to and activation of 2-5A-dependent endonuclease.

Binding and activation efficacies to the 2-5A-dependent endonucease by chemically synthesized partially inosine-substituted 2-5A analogs, namely, pppI2'p5'A2'p5'A, pppA2'p5'I2'p5'A and pppA2'p5'A2'p5'I were compared with that of native 2-5A in mouse L cell and human lymphoblastoid cell extracts. The results obtained in this study indicated that the first adenine amino group from the 5' terminus of 2-5A molecule plays critical role in binding to the endonuclease, whereas the third adenine amino group has a function for the activation of this enzyme.

Adenine Nucleotides↗
PubMed1983

A sensitive immunoenzymometric assay for 2',5'-oligoadenylate. Detection of elevated 2',5'-oligoadenylate synthetase in human peripheral mononuclear cells.

A competition immunoenzymometric assay for 2',5'-oligoadenylate was developed and employed to measure the interferon-inducible enzyme 2',5'-oligoadenylate synthetase in cell extracts. Microtiter plates coated with a novel conjugate of p5'A2'p5'A2'p5'A and N-(2-aminoethyl)-carbamylmethylated-Ficoll (AECM-Ficoll) bound rabbit polyclonal or mouse monoclonal antibody directed against 2',5'-oligoadenylate. Binding was inhibited by soluble 2',5'-oligoadenylate. Estimates of 2',5'-oligoadenylate concentrations based on inhibition of antibody binding compared favorably with those obtained using a protein synthesis inhibition assay. Low concentrations of 2',5'-oligoadenylate synthesized in vitro by extracts of human peripheral mononuclear cells were conveniently estimated using less than or equal to 10(6) cells. Virtually identical results were obtained when either total extract or synthetase bound to poly(I) . poly(C)-agarose was used for the in vitro incubation. When peripheral mononuclear cells were incubated in vitro with interferon, the normally low levels of 2',5'-oligo(A) synthetase rose dramatically. The assay was employed to measure synthetase levels in peripheral mononuclear cells isolated from patients with systemic lupus erythematosus. Some of these patients were found to have elevated levels of 2',5'-oligoadenylate synthetase.

2',5'-Oligoadenylate Synthetase↗
PubMed1983

Biological activities of phosphodiester linkage isomers of 2-5A.

To determine the relative importance of the 2',5'-phosphodiester bond of 2-5A in its binding to and activation of the 2-5A-dependent ribonuclease (RNase L, RNase F), a number of phosphodiester linkage isomers of 2-5A were prepared. These isomers were obtained either by lead ion-catalyzed polymerization of adenosine 5'-phosphorimidazolidate or by T4 polynucleotide kinase-catalyzed 5'-phosphorylation of adenylyl(3' leads to 5')adenylyl(3' leads to 5')adenosine followed by reaction of the corresponding phosphorimidazolidates with tri(n-butylammonium)pyrophosphate. The following 2-5A isomers thus were prepared: ppp5'A2'p5'A3'p5'A, ppp5'A3'p5'A2'p5'A, ppp5'A3'p5'A3'p5'A("3-5A"), ppp5'A2'p5'A3'p5'A2'p5'A,and ppp5'A3'p5'A2'p5'-A2'p5'A. The ability of these isomeric 2-5As to interact with the 2-5A-dependent endonuclease was ascertained by three different criteria: (i) ability to prevent the protein synthesis inhibitory effects of 2-5A, (ii) activity as an inhibitor of translation in encephalomyocarditis RNA-programmed L cell extracts, and (iii) ability to prevent binding of the radiolabeled probe, ppp5'A2'p5'A2'p5'A2'p5'A3'[32P]p5'Cp, to the endonuclease of L cell extracts. In certain experiments, degradation of oligonucleotide was minimized or eliminated by altering assay conditions, providing alternate phosphodiesterase substrates, or by using purified endoribonuclease of Ehrlich ascites cells. By all criteria, replacement of 2',5'-bond by a 3',5'-bond led to a substantial decrease in biological activity. Generally, replacement of just one 2',5'-phosphodiester bond with a 3',5'-linkage led to at least a one order of magnitude loss of activity. In accord with this trend, ppp5'A3'p5'A3'p5'A(3-5A) was greater than 10,000 less active than 2-5A in binding to the endonuclease or as an inhibitor of protein synthesis.

Adenine Nucleotides↗