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P F Torrence

Publications and source records attributed to P F Torrence.

At least 91 records · Page 5Linked to original sources
PubMed1983

Assay of 2',5'-oligoadenylate phosphodiesterase activity in mouse L-cell extracts.

A rapid and convenient assay for adenylyl(2' leads to 5')adenosine(A2'p5'A) or adenylyl(3' leads to 5')adenosine(A3'p5'A) phosphodiesterase activities is described. The dinucleotides A3'p5'A and A2'p5'A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [3H]A3'p5'A or [3H]A2'p5'A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2'p5'A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2'p5'A as extracts from interferon pretreated cells.

Adenosine Monophosphate↗
PubMed1983

Cordycepin analogues of 2-5A and its derivatives. Chemical synthesis and biological activity.

Cordycepin (3'-deoxyadenosine) analogues of 2-5A were prepared by dicyclohexylcarbodiimide-induced polymerization of cordycepin 5'-monophosphate. A series of oligomers of the general formula p5'(3'dA)-2'[p5'(3'dA)]n (n = 1-5) was obtained. Cordycepin trimer 5'-monophosphate (n = 2) and cordycepin tetramer 5'-monophosphate (n = 3) were converted to the corresponding 5'-di- and triphosphates via the phosphorimidazolide method. Confirmation of assigned structures was provided through enzyme digestions, 1H and 31P NMR, and comparison with material which was synthesized by a completely independent route. Neither the cordycepin trimer triphosphate, ppp5'(3'dA)-2'p5'(3'dA)2'p5'(3'dA), nor the cordycepin tetramer triphosphate, ppp5'(3'dA)2'p5'(3'dA)2'p5'(3'dA)2'p5'-(3'dA), were inhibitors of translation in cell-free extracts of mouse L-cells programmed with encephalomyocarditis virus. In rabbit reticulocyte lysates, the cordycepin trimer triphosphate was devoid of activity, whereas the cordycepin tetramer triphosphate had approximately 1/100th the activity of 2-5A tetramer triphosphate, ppp5'A2'p5'A2'p5'A2'p5'A. Even though the cordycepin analogues were unable to activate the 2-5A-dependent endoribonuclease, they were able to bind to the endonuclease, thereby antagonizing the translational inhibitory effects of 2-5A. Thus, replacement of the 3'-hydroxyl groups of all the ribose moieties of 2-5A results in an analogue which can bind to the 2-5A-dependent endoribonuclease but is incapable of activating the enzyme. The 3'-hydroxyl groups of 2-5A, therefore, are critical for activation of the 2-5A-dependent endonuclease.

Animals↗
PubMed1983

Double-stranded RNA-dependent phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha does not correlate with protein synthesis inhibition in a cell-free system from interferon-treated mouse L cells.

The double-stranded RNAs (I)n X (C)n and (A)n X (dUfl)n (dUfl is 2'-fluoro-2'-deoxyuridylic acid) have been compared as inhibitors of translation in cell-free systems from interferon-treated mouse L cells and from rabbit reticulocytes. In the interferon-treated mouse L-cell system, both double-stranded RNAs stimulated kinase activity, leading to phosphorylation of protein P1 and eukaryotic initiation factor 2 alpha (eIF-2 alpha), but only (1)n X (C)n activated the (2'-5')-oligoadenylate synthetase. Moreover, in this system, (I)n X (C)n, but not (A)n X (dUfl)n, inhibited translation. Both (A)n X (dUfl)n and (I)n X (C)n also activated the rabbit reticulocyte kinase to phosphorylate protein P1 and eIF-2 alpha, but, in contrast to mouse L-cell systems, both (A)n X (dUfl)n and (I)n X (C)n were potent inhibitors of translation in reticulocyte lysates. These results indicate that protein P1 and eIF-2 alpha phosphorylation are not sufficient to cause inhibition of protein synthesis in interferon-treated mouse L-cell extracts. They further suggest that protein synthesis inhibition by (I)n X (C)n in extracts of interferon-treated L cells correlates better with activation of (2'-5')-oligoadenylate synthetase than with activation of the protein P1 and eIF-2 alpha kinase.

Animals↗
PubMed1982

5-Substituted 2'-deoxyuridines: correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase.

A large variety of 5-substituted 2'deoxyuridines (dUrds) and 2'-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their Ki/Km values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP greater than 5-formyl-dUMP greater than 5-fluoro-dUMP greater than 5-oxime of 5-formyl-dUMP greater than 5-azidomethyl-dUMP greater than (E)-5-(2-bromovinyl)-dUMP. The ki/Km values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37 degrees). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5'-monophosphates followed by an inhibition of dTMP synthetase.

Animals↗
PubMed1982

Chemical modification potentiates the biological activities of 2-5A and its congeners.

Chemical modification of p5'A2'(p5'A2')np5'A by a periodate oxidation/Schiff base formation/borohydride reduction cycle gave a series of 2-5A analogues in which the ribose of the 2'-terminal nucleotide was transformed to an N-substituted morpholine (azahexapyranose). 2',5'-Oligoriboadenylate 5'-monophosphates bearing this modification were 5-10 times more potent as antagonists of the action of 2-5A or poly(I).poly(C) than was unmodified p5'A2'p5'A2'p5'A. Application of this modification to the tetramer triphosphate ppp5'A2'p5'A2'p5'A2'p5'A resulted in an analogue with 10 times the activity of ppp5'A2'p5'A2'p5'A (2-5A trimer triphosphate) as an inhibitor of protein synthesis or activator of the 2-5A-dependent endoribonuclease. This activator of the 2-5A-dependent endoribonuclease. This new 2-5A analogue, the most potent 2-5A derivative reported to date, inhibited translation in extracts of mouse L-cells programmed with encephalomyocarditis virus RNA at a concentration of 10(-10) M (concentration for half-maximal inhibition). All such N-substituted morpholine modified 2',5'-oligoriboadenylates were found to be extremely resistant to degradation by L-cell extracts under conditions where unmodified 2-5A or its derivatives were quickly destroyed. These data demonstrate the necessity for an intact terminal ribose ring for the action of the 2-5A phosphodiesterase. Thus, extensive chemical modification of 2' terminus of 2-5A may be possible without adversely affecting its biological activity while endowing it with other favorable properties such as resistance to degradation.

Adenine Nucleotides↗
PubMed1982

Role of thymidine kinase in the inhibitory activity of 5-substituted-2'-deoxyuridines on the growth of human and murine tumor cell lines.

Twenty-four 5-substituted 2'-deoxyuridines have been evaluated for their inhibitory effects on the growth of three human lymphoblast cell lines (Namalva, RAji and TK- (thymidine kinase deficient) Raji) and these inhibitory effects were compared to those for two murine leukemia cell lines (L1210/0 and L1210/BdUrd). The latter was selected from the parental L1210/0 cell line by its ability to grow at high concentrations of 5-bromo-dUrd and could also be considered as TK-. There was a close correlation between the inhibitory effects of the deoxyuridine analogs on Namalva, Raji and L1210 cells: the correlation coefficient (r) for log ID50 (median inhibitory dose) for L1210 cell growth, on the one hand, and log ID50 for Namalva or Raji cell growth, on the other hand, was 0.902 and 0.929, respectively. There was also a strong correlation (r = 0.936) between the log ID50 values for the two human lymphoblast cell lines. However, there was no significant correlation (r less than 0.40) either between the log ID50 for the TK- Raji cells and the parental TK+ Raji cells, or between the log ID50 for the TK- L1210/BdUrd cells and the parental TK+ L1210/0 cells. We may conclude therefore, that (i) the murine leukemia L1210 cell system is predictive for the growth-inhibitory effects of 5-substituted 2'-deoxyuridines on human lymphoblast cell lines, and (ii) the antitumor cell activity of the 5-substituted 2'-deoxyuridines is, to a large extent, dependent on the thymidine kinase activity of the tumor cells.

Animals↗
PubMed1982

Antiviral, antimetabolic, and cytotoxic activities of 5-substituted 2'-deoxycytidines.

Various 5-substituted 2'-deoxycytidines, including 5-bromo-dCyd, 5-iodo-dCyd, 5-nitro-dCyd, 5-ethynyl-dCyd, 5-propyl-dCyd, (E)-5-(2-bromovinyl)-dCyd, and (E)-5-(2-iodovinyl)-dCyd, were evaluated for their antiviral and antimetabolic properties in primary rabbit kidney (PRK) cell cultures and for their inhibitory effects on murine L1210 cell proliferation. All dCyd analogues proved to be selective inhibitors of herpes simplex virus (HSV) replication: 5-bromo-dCyd, 5-iodo-dCyd, 5-nitro-dCyd, and 5-ethynyl-dCyd were more selective in their anti-HSV activity than were the corresponding 5-substituted 2'-deoxyuridines, whereas 5-propyl-dCyd, (E)-5-(2-bromovinyl)-dCyd, and (E)-5-(2-iodovinyl)-dCyd were as selective as their dUrd counterparts. The dCyd analogues were also less cytotoxic (for both PRK and L1210 cells), as could be monitored by inhibition of either cell proliferation or host-cell DNA synthesis (incorporation of radiolabeled precursors). Of all 5-substituted 2'-deoxycytidines tested, the (E)-5-(2-halogenovinyl) derivatives emerged as the most potent and most selective inhibitors of HSV (Type 1) replication.

Animals↗
PubMed1982

Oligonucleotide structural features involved in binding to and activation of the 2-5A-dependent endoribonuclease of L cells.

Several analogues of 2-5A were prepared and evaluated for their ability to bind to and/or activate the 2-5A dependent endonuclease from murine L cells. Replacement of one of the two 2',5'-phosphodiester bonds of ppp5'A2'p5'A2'p5'A with 3',5'-phosphodiester bonds gave analogues 40-50 X less active than 2-5A in binding to or activation of the 2-5A-activated endonuclease whereas replacement of both 2',5'-linkages with 3',5'-linkage resulted in a compound (3-5A) which was 10,000 X less effective in binding to the endonuclease and devoid of activity as an inhibitor of protein synthesis. The sequence of periodate oxidation/Schiff base formation/borohydride reduction gave a derivative of ppp5'A2'p5'A2'p5'A2'p5'A in which the 2'-terminal ribose was replaced with a N-hexyl morpholine ring. This material was 10 X more active than 2-5A an a inhibitor of translation. One possible explanation for this increased activity is that the 2'-terminally modified oligomer is resistant to degradation by the 2',5'-phosphodiesterase responsible for the degradation of 2-5A in cell extracts.

Adenine Nucleotides↗
PubMed1981

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

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

Animals↗