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R J Suhadolnik

Publications and source records attributed to R J Suhadolnik.

At least 55 records · Page 3Linked to original sources
PubMed1987

Biosynthesis of 2'-deoxycoformycin: evidence for ring expansion of the adenine moiety of adenosine to a tetrahydroimidazo[4,5-d][1,3]diazepine system.

2'-Deoxycoformycin (2'-dCF), a nucleoside antitumor agent produced in trace quantities by Streptomyces antibioticus, has been shown in earlier work to originate from the intact carbon-nitrogen framework of adenosine. Additional experiments using 13C and two-dimensional Fourier transform NMR techniques, together with radiolabeling studies, identify the C-1 of D-ribose, and not the tetrahydrofolate "C-1 pool", as the source of the C-7 carbon in the aglycon of 2'-dCF. These results show that the adenine portion of adenosine (or a nucleotide thereof) undergoes a unique ring expansion, by insertion of a -CH2- unit between the N-1 and C-6 of the adenine ring, to furnish the 1,3-diazepine portion of 2'-dCF.

Adenosine↗
PubMed1985

Differential antiproliferative actions of 2',5' oligo A trimer core and its cordycepin analogue on human tumor cells.

The antiproliferative effect of 2',5' A3 core and 2',5'-3'dA3 (cordycepin trimer) core was measured in 8 human tumor cell lines. Cells were treated in a dose-response manner for 72 hr and the concentration of drug necessary to inhibit cell growth 50% (GI50) was determined. A wide range of sensitivities to these drugs was found, even among tumors of the same histological type. The cell lines showed different sensitivities and dose-response curves to the 2',5'A3 and 2',5'-3'dA3 cores. Uptake studies of the 2',5'A3 and 2',5'-3'dA3 cores, using high-pressure liquid chromatography, demonstrated that both cores were rapidly degraded in the tissue culture medium and taken up as adenosine or cordycepin, respectively. There was a direct correlation between the uptake of cordycepin and the antiproliferative effect. In contrast, there was no correlation between cell sensitivity and the uptake of the 2',5'A3 core degradation products. Analysis of intracellular nucleosides and nucleotides indicated that differences in intracellular metabolism of adenosine might explain the different sensitivities of the various cell lines to 2',5'A3 core. Molar equivalent concentrations of adenosine and cordycepin inhibited cell growth; however, equimolar concentrations of these nucleosides were not effective. In addition, the antiproliferative effect of both core compounds and their corresponding nucleosides could be potentiated by the addition of the adenosine deaminase inhibitor, deoxycoformycin. The results indicate that these cores act as prodrugs and that the active metabolites are their corresponding nucleosides.

Adenine Nucleotides↗
PubMed1985

2',5'-Oligoadenylates chiral at phosphorus: enzymatic synthesis, properties, and biological activities of 2',5'-phosphorothioate trimer and tetramer analogues synthesized from (SP)-ATP alpha S.

The enzymatic synthesis and characterization of (RP)-2',5'-AMPS trimer and tetramer (SP)-5'-O-(1-thiotriphosphates) from chirally substituted (SP)-[alpha-35S]ATP alpha S by 2',5'-oligoadenylate synthetase from interferon-treated L cell extracts are described. The (RP)-ATP alpha S isomer is not a substrate for the synthetase. The identification of the trimer and tetramer analogues (molar ratio 70:30) was accomplished by high-performance liquid chromatography and subsequent separation by charge using DEAE-cellulose thin-layer chromatography. The digestion of the analogue by snake venom phosphodiesterase I (SVPD) to [alpha-35S]ATP alpha S and [35S]AMPS but not by T2 RNase demonstrated the presence of the 2',5' linkage. The assignment of RP configuration of the 2',5'-phosphorothiodiester linkage was based on the highly specific stereoselectivity of SVPD for RP diastereomers [Burgers, P. M. J., & Eckstein, F. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4978-4800; Bryant, F. R., & Benkovic, S. J. (1979) Biochemistry 18, 2825-2828; Nelson, P. S., Bach, C. T., & Verheyden, J. P. H. (1984) J. Org. Chem. 49, 2314-2317]. This suggests that the synthesis of the phosphorothioate analogues proceeded via inversion of configuration at the chiral phosphorus of (SP)-ATP alpha S. The putative (RP)-2',5'-AMPS tetramer (SP)-5'-O-(1-thiotriphosphate) displaced the 2',5'-p3A4[32P]pCp analogue from 2',5'-oligoadenylate-dependent endonuclease 5 times more efficiently than did equimolar concentrations of authentic 2',5'-adenylate tetramer triphosphate. Furthermore, in studies using the calcium phosphate coprecipitation technique, the 2',5'-phosphorothioate trimer and tetramer analogues inhibited protein synthesis better than did 2',5'-adenylate trimer and tetramer triphosphates.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗
PubMed1985

Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities.

An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)

2',5'-Oligoadenylate Synthetase↗
PubMed1984

Accumulation of low molecular weight DNA and changes in chromatin structure in HeLa cells treated with human fibroblast interferon.

The addition of human fibroblast interferon (IFN-beta) (100 units/ml) at the S/G2 boundary of the cell cycle of synchronously grown HeLa cells is characterized by the accumulation of newly synthesized low molecular weight DNA and changes in chromatin assembly. In addition, there is a 3-fold stimulation in the incorporation of tracer amounts of [3H]thymidine, but not [3H] deoxyguanosine, into DNA and a 2-fold increase in the incorporation of [3H]dTTP into the DNA of isolated nuclei. Fluorescence-activated cell sorting by laser flow cytometry revealed that IFN-beta-treated cells were delayed in entering and passing through the S phase. The inhibition of proliferation of HeLa cells treated with IFN-beta is characterized by a 3-fold accumulation of newly synthesized DNA of Mr less than 56 X 10(6) compared to untreated cells as determined by alkaline sucrose gradient centrifugation. The newly synthesized DNA in IFN-beta-treated cells was replicative and not repair DNA. The observation that IFN-beta inhibits the processing of newly synthesized low molecular weight DNA into normal DNA might be explained by the intracellular accumulation of S-adenosylhomocysteine in IFN-beta-treated HeLa cells (de Ferra, F., and Baglioni, C. (1983) J. Biol. Chem. 258, 2118-2121) which could change the soluble ribonucleotide and deoxyribonucleotide pool and ultimately affect DNA processing. Interferon may also affect processing of DNA by interfering with normal chromatin assembly. Evidence for the effect of IFN-beta on chromatin assembly is provided; we have observed a more condensed structure in IFN-beta treated cells by circular dichroism spectroscopy. Simultaneous with the affect on chromatin assembly, there is a 70% decrease in poly(ADP-ribosylation) of either histone and/or non-histone proteins. The loss of coordination between the pool size for DNA synthesis, decreased postsynthetic modifications of chromatin, and normal chromatin formation may explain the inability of the cell to differentiate and to continue cell division.

Cell Cycle↗
PubMed1984

5'-dephosphorylated 2',5'-adenylate trimer and its analogs. Inhibition of tobacco mosaic virus replication in tobacco mosaic virus-infected leaf discs, protoplasts, and intact tobacco plants.

The effect of the 5'-dephosphorylated 2',5'-adenylate trimer and its 2',5'-trimer core analogs on the inhibition of tobacco mosaic virus (TMV) replication was determined in tobacco leaf discs, protoplasts, and whole tobacco plants, using infectivity tests and enzyme-linked immunosorbent assays. A structure-activity-metabolic stability-toxicity analysis of the 2',5'-adenylate trimer core molecule in TMV-infected Nicotiana glutinosa was determined. Modification at either the 6-amino position of the adenylate residues (i.e. inosinate trimer core) or at the 2' terminus (i.e. A-A-ara-A or A-A-Tu) inhibited replication of TMV. Modification of the 3'-hydroxyl group of the adenylate residues to 3-deoxyribose (i.e. the 2',5'-cordycepin trimer core) inhibited TMV replication better than the 2',5'-adenylate trimer core molecule. With enzyme-linked immunosorbent assays, there was complete inhibition of TMV replication by 200 nM 2',5'-adenylate trimer core for 60 h and by 200 nM 2',5'-cordycepin trimer core for 96 h. The amount of 2',5'-oligonucleotides associated with the leaves was determined using 2',5'-[3H]cordycepin trimer core; 1 X 10(-12) mol/cm2 of plant leaves inhibited TMV replication by 99%. No 2',5'-phosphodiesterase activity was detected in TMV-infected and noninfected leaf extracts. Therefore, the 2',5'-trimer cores were potent inhibitors of TMV replication at nanomolar concentrations, i.e. at 1000-fold lower concentration than that required in mammalian systems.

Adenosine↗
PubMed1984

Evidence for the conversion of adenosine to 2'-deoxycoformycin by Streptomyces antibioticus.

The incorporation and distribution of 14C in 2'-deoxycoformycin, elaborated by Streptomyces antibioticus, were studied with [U-14C]glycine, [U-14C]adenosine and [U-14C]adenine. Similar ratios of 14C in the aglycon and carbohydrate portions of 2'-deoxycoformycin, ara-A, and adenosine isolated from the RNA indicated that [U-14C]adenosine was incorporated into 2'-deoxycoformycin without cleavage of the N-glycosylic bond. Following the addition of [U-14C]adenine, 98% of the 14C isolated from [14C]-2'-deoxycoformycin resided in the aglycon. 2'-Deoxycoformycin biosynthesis may not require the de novo purine biosynthetic pathway as evidenced by the failure to detect incorporation of [U-14C]glycine into 2'-deoxycoformycin. These data suggest that the biosynthesis of 2'-deoxycoformycin involves the incorporation of the carbon-nitrogen skeleton of an intact purine nucleoside or nucleotide, thereby implying that a purine ring is opened enzymatically between C-6 and N-1 and a one-carbon unit is added to form the 1,3-diazepine ring of 2'-deoxycoformycin.

Adenine↗
PubMed1984

Increased 2',5'-oligoadenylate synthetase activity in blood mononuclear leukocytes from patients with advanced cutaneous T-cell lymphoma.

The activity of the interferon-induced enzyme 2',5'-oligoadenylate synthetase (2',5' An synthetase) was found to be increased in the lysates of peripheral blood mononuclear leukocytes obtained from 10 of 25 (40%) patients with cutaneous T-cell lymphoma (mycosis fungoides and Sézary syndrome). A positive association was found between the level of mean total synthetase activity and extent of involvement (stage) of disease. However, the leukocytes from two patients with relatively high proportions of neoplastic T-cells in the blood (Sézary syndrome) had negligible enzyme activity. It is speculated that increased interferon production in vivo may account for the observed enzyme changes in normal blood leukocytes from these patients, and that the neoplastic T-cell population is the most likely source of the presumed interferon production. Possible mechanisms and biologic consequences of high levels of endogenous interferon in patients with cutaneous T-cell lymphoma are discussed.

2',5'-Oligoadenylate Synthetase↗
PubMed1984

2',5'-Oligoadenylate trimer core and the cordycepin analog augment the tumoricidal activity of human natural killer cells.

Interferon (IFN) augments the lytic activity of natural killer (NK) cells, inhibits the transformation of human peripheral blood lymphocytes (PBL) by Epstein Barr virus (EBV), and induces a 2',5'-oligoadenylate (2',5'-An) synthetase. Exogenous 2',5'-An by itself can inhibit the transformation of human PBL by EBV. The present studies report that 2',5'-An and its cordycepin analog also augmented the tumoricidal activity of human NK cells. Incubation of nylon wool-passed PBL for 1 to 2 hr with the 5'-dephosphorylated core trimer of 2',5'-An boosted natural killing of tumor target cells modestly, but consistently. The cordycepin analog (3'-deoxyadenylate) also augmented NK activity. The optimal concentration both of 2',5'-A3 core and of 2',5'-3'dA3 core was 50 microM, and the optimal time for this effect was 2 hr of treatment. Kinetic analysis revealed that 2',5'-A3 core increased the lytic rate of NK cells by about one-third. This increase was due to an even greater increase (about 50%) in the lytic activity of individual NK cells, coupled with a slight decrease in the number of actual NK effector cells. In contrast, 3',5'-A3 core did not increase NK activity even at 300 microM, at which point it was toxic. In addition, to rule out a pro-drug effect as the basis for the boosting of NK activity by 2',5'-A3 core and by 2',5'-3'dA3 core, the effect of adenosine and cordycepin monomers on NK activity was tested. Neither adenosine nor cordycepin, tested at 150 microM (three times the optimal concentration of the trimer cores), boosted NK activity. The addition of 2'-deoxycoformycin (2 microM) had no effect on the actions of adenosine and cordycepin monomers. The data presented here demonstrate that 2',5'-A3 core and its analog 2',5'-3'dA3 core have another IFN-like action, augmentation of NK activity, in addition to inhibiting EBV-induced transformation.

Adenine Nucleotides↗
PubMed1983

Some properties of a partially purified inhibitor(s) of protein synthesis from rat-liver mitochondria.

We have previously described an inactive inhibitor of protein synthesis from rat liver mitochondria and its activation by brief incubation with N-ethylmaleimide [Wu, J. M. and Ibrahim, N.G. (1980) FEBS Lett. 119, 25-28]. To study the mode of action of the mitochondrial translational inhibitor (MTI), the relative distribution of monosomes and polysomes in rabbit reticulocyte lysates has been analysed by sucrose gradient centrifugation. These studies show that MTI causes a significant decrease in the amount of polysomes with marginal effect on the polysome profile. Under identical experimental conditions, addition of partially purified heme-regulated inhibitor results in a complete disaggregation of polysomes. Studies with micrococcal-nuclease-treated rabbit reticulocyte lysates suggest that the primary target of MTI is the inactivation of globin mRNA with relatively little effect on other components of the translational machinery. The inhibitor also degrades poly(U), vesicular stomatis virus mRNA, reovirus mRNA, but appears to be inactive against poly(A), Escherichia coli 16S rRNA, HeLa cell rRNA or chick embryo DNA. Chromatography of MTI on heparin-agarose results in the resolution of at least two inhibitory activities. The first inhibitory activity (eluted with 250 mM KCl) can be reversed (50-70%) with high concentrations of glucose 6-phosphate or MgGTP (0.7 mM or 3.3 mM) whereas the second inhibitory activity can only be partially reversed with poly(A)-rich RNA.

Animals↗
PubMed1983

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells.

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.

Animals↗