Visualization of drug-nucleic acid interactions at atomic resolution. IV. Structure of an aminoacridine--dinucleoside monophosphate crystalline complex, 9-aminoacridine--5-iodocytidylyl (3'--5') guanosine

9-Aminoacridine forms a crystalline complex with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine (iodoCpG). These crystals are monoclinic, space group P2₁ with $\text{a = 13.98}\text{A}\text{?}\text{, b = 30.58}\text{A}\text{?}\text{, c = 22.47}\text{A}\text{?}$ and β = 113.9 °. The structure has been solved to atomic resolution by Patterson and Fourier methods, and refined by a combination of Fourier and sum-function Fourier methods. The asymmetric unit contains four 9-aminoacridine molecules, four iodoCpG molecules and 21 water molecules, a total of 245 atoms. 9-Aminoacridine demonstrates two different intercalative binding modes and, along with these, two slightly different intercalative geometries in this model system.The first of these is very nearly symmetric, the 9-amino group lying in the narrow groove of the intercalated base-paired nucleotide structure. The second shows grossly asymmetric binding to the dinucleotide, the 9-amino group lying in the wide groove of the structure. Associated with these two different intercalative binding modes is a difference in geometries in the structures. Although both structures demonstrate C3′ endo (3′–5′) C2′ endo mixed sugar puckering patterns (i.e. both cytidine residues have C3′ endo sugar conformations, while both guanosine residues have C2′ endo sugar conformations), with corresponding twist angles between base-pairs of about 10 °, they differ in the magnitude of the helical screw axis dislocation accompanying intercalation (Sobell et al., 1977a,b). In the pseudosymmetric intercalative structure, this value is about +0.5 Å, whereas in the asymmetric intercalative structure this value is about +2.7 Å. These conformational differences can be best described as a “sliding” of base-pairs on the intercalated acridine molecule.Although the pseudosymmetric intercalative structure can be used in 9-aminoacridine-DNA binding, the asymmetric intercalative structure cannot since this poses stereochemical difficulties in connecting neighboring sugar-phosphate chains to the intercalated dinucleotide. It is possible, however, that the asymmetric binding mode is related to the mechanism of 9-aminoacridine-induced frameshift mutagenesis (Sakore et al., 1977), and we discuss this possibility here in further detail.     

Adaptation of the cells ofEscherichia coli to the presence of hydroxyurea increases the level of inorganic pyrophosphatase activity

Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, completely arrested the net synthesis of DNA for 3–4 h, when it was added in 30 mM concentration to growing cultures ofEscherichia coli K12. Thereafter the net synthesis of DNA started again, although slowly, and simultaneously with it the formation of inorganic pyrophosphatase activity was stimulated leading to a 2-fold increase in the specific activity of the enzyme in 2–3 h. Subsequently cell division began again. In this way a new steady state, stable in the presence of hydroxyurea, was reached. This new state was characterized by the high specific activity of inorganic pyrophosphatase, a small but constant amount of DNA/cell mass (1/4 of the normal value), and large elongated cells. All these changes were slowly reversed during 5–6 h, when the cells were transferred into a drug-free medium.The activity of isoleucyl-tRNA synthetase, assayed as a control, did not change significantly in the presence of hydroxyurea.Hydroxyurea had no effect on the activity of inorganic pyrophosphatase in vitro.     

Stability of (2'-5')oligoriboadenylates in various sera

Avian and mammalian sera were found to contain an enzyme activity degrading 2-5A oligonucleotides. The most extensive degradation of the A2' p5' A was observed in chicken serum. Degradation of this compound is not affected by the presence of cAMP, dsRNA, Mg2+, but is significantly inhibited by EDTA. The enzyme activity described is not inactivated by heating to 56 degrees C for 30 min. The 5-mU3' p5' A has also been degraded in chicken serum.     

Catabolism of capped (3'-5')- and (2'-5')-adenylates in rat liver nuclei

We describe studies concerning the ability of a nuclear dinucleoside triphosphatase to act as a decapping enzyme in RNA catabolism. The enzymatic release of GMP from the Gp3A moiety was determined in the capped RNA model compounds Gp3A3'pA, Gp3A3'pA-isoprop and Gp3A2'pA in isolated rat liver nuclei; i.e., in the environment in which the dinucleoside triphosphatase operates in vivo. The Gp3A cap moiety is hydrolyzed in (3'-5') linked nucleotides only, whereas an extension of the Gp3A in the 2'-direction prevents the nuclear triphosphatase to operate.     

Partial characterization of 5′(3′)-ribonucleotide and 3′-ribonucleotide phosphohydrolases from Tradescantia albiflora leaves

Two acid phosphomonoesterases, 5′(3′)-ribonucleotide phosphohydrolase and 3′-ribonucleotide phosphohydrolase, were isolated from Tradescantia albiflora leaf tissue and purified by ammonium sulphate precipitation, gel filtration on Sephadex G-200 and repeated chromatography on DEAE-cellulose. The enzymes differed in their sensitivity to dialysis against 1 mM EDTA; the activity of 5′(3′)-ribonucleotide phosphohydrolase was unaffected, while 3′-ribonucleotide phosphohydrolase showed an increase of 60–90%. Both enzymes were rapidly inactivated above 50°. Their ion sensitivity was identical: 1 m M Zn²⁺ and Fe²⁺ were inhibitors for both by 20–80%; while Mg²⁺, Ca²⁺, Co²⁺, K⁺, Na⁺ at 1–10 mM had no significant effect on the activity of either enzyme. Inorganic phosphate inhibited both enzymes almost completely. EDTA (1 mM) did not inhibit either enzyme; none of the divalent cations tested were enzyme activators. 3′-Ribonucleotide phosphohydrolase hydrolysed both 3′- and 5′-nucleoside monophosphates (3′-AMP, 3′-CMP, 3′-GMP, 3′-UMP, 5′-AMP, 5′-CMP, 5′-GMP, 5′-UMP). 5′(3′)-Ribonucleotide phosphohydrolase showed a preference for the 3′-nucleoside monophosphates. Adenosine 3′,5′-cyclic monophosphate, purine and pyrimidine 2′,3′-cyclic mononucleotides at 0.1–1.OmM did not inhibit the enzymes.     

Evidence for two distinct pyruvate kinase genes in Escherichia coli K-12

A strain of Escherichia coli K-12 defective in pyruvate kinase F has been produced. The existence of this mutant, in conjunction with earlier results, strongly suggests that the two pyruvate kinases in this bacterium are distinct forms and not interconvertible. Either form of pyruvate kinase appeared to be equally effective in the glycolytic conversion of phosphoenolpyruvate to pyruvate. Genes specifying pyruvate kinase A and pyruvate kinase F were present on the small F-prime F506 and the locus for pyruvate kinase F was found to be at minute 36.5 on the E. coli genetic map.     

The stability of methyl and ethyl phosphotriesters in DNA in vivo

C57BL male mice were injected with N-methyl-N-nitrosourea (MNUA) or N-ethyl-N-nitrosourea (ENUA) and the concentration of alkyl phosphotriesters in the DNA of lung, liver, brain, kidney, spleen and thymus determined from the extent of degradation induced in isolated DNA by alkali. The same total dose of reagent was given either as a single injection (i.p.) or by weekly injections carried out over 5-20 weeks. Methyl phosphotriesters induced in liver, lung and kidney by the single injection were lost with a half-life of about 7 days, in brain the loss was more rapid, t1/2 = 2-3 days. During the multiple injections the observed t1/2 was 16 days. Ethyl phosphotriesters formed in the DNA of lung, liver, kidney and brain were much more stable than the methyl derivatives, t1/2 = 10-15 weeks. Phosphotriesters formed in the DNA of spleen and thymus disappeared very quickly after the single injection presumably as a result of dilution due to DNA replication. No accumulation of phosphotriesters occurred in the DNA of these tissues during the multiple injections. The general pattern of the results suggests that phosphotriesters are not excised by cellular repair systems.     

Reactions of adenosine 5′-phosphorimidazolide with adenosine analogs on a polyuridylic acid template: The uniqueness of the 2′-3′-unsubstituted β-ribosyl system

We have studied the reactions between adenosine 5′-phosphorimidazolide and various adenosine analogs on a poly(U) template. The nucleosides were adenosine (I), 2′-deoxyadenosine (II), 3′-deoxyadenosine (III), 2′-O-methyladenosine (IV), 3′-O-methyladenosine (V), 9-β-d-xylofuranosyladenine (VI), and 9-β-d-arabinofuranosyladenine (VII). We find that the various analogs form triple helices with poly(U) which are of comparable stability, but that only the β-riboside takes part in an efficient template-directed condensation.     

Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues – 2′-deoxyguanosine-5′-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5′-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3′-5′)-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3′-5′)-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.     

Effects on N6, O2′-dibutyryladenosine-3′,5′ cyclic monophosphate on calcium accumulation by mouse mastocytoma cells

The accumulation of ⁴⁵Ca²⁺ by intact mouse mastocytoma cells was examined before and after treatment of the cells with N⁶,O^2′-dibutyryladenosine 3′,5′, cyclic monophosphate and theophylline to inhibit growth. In the presence of phosphate either glycolysis, respiration or ATP supported ⁴⁵Ca²⁺ uptake by the cells and in each case the accumulated ⁴⁵Ca²⁺ appeared to be retained by mitochondria. Inhibition of growth by drug treatment for 20h increased subsequent ⁴⁵Ca²⁺ accumulation when cells were incubated with ⁴⁵CaCl₂, succinate and phosphate. Since prior drug treatment did not increase ⁴⁵Ca²⁺ accumulation with glucose, ATP or malate the drugs appeared to increase ⁴⁵Ca²⁺ accumulation by affecting succinate metabolism.     

Stimulation by ATP of [3H]dopamine binding to synaptic membrane fractions of canine caudate nucleus

The rate of [³H]dopamine binding to crude synaptic membranes from canine caudate nucleus was considerably increased by 2 mM ATP, 5′-adenylylimidodiphosphate and GTP or by 1 mM 5′-guanylyl-imidodiphosphate, while strongly inhibited by 2 mM ADP and GDP. Half maximal concentrations of [³H]dopamine to bind to the membranes were 1.11 × 10^?7M and 8.75 × 10^?6M in the absence of 4 mM ATP, indicating a negative cooperativity of the dopamine receptor, and 9.25 × 10^?7 M in its presence. Hill coefficient was increased from 0.70 to 1.04 by addition of 4 mM ATP. The optimal concentration of ATP for [³H]dopamine binding was in the range of 0.5 to 5 mM.     

Antagonistic effect of methadone on steroidogenesis and the adenylate cyclase system in isolated rat adrenocortical cells

Methadone exhibits an antagonistic effect toward steroidogenesis which lies prior to progesterone in the biosynthetic pathway in isolated rat adrenal cells. Levels of adenosine cyclic 3′–5′ monophosphate are depressed in a dose dependent fashion in ACTH stimulated cells as is steroidogenesis in cells stimulated with N⁶O²-dibutyryl adenosine cyclic 3′–5′ monophosphate. Stimulation produced by the ACTH analog, O-nitrophenyl sulfenyl ACTH, is also inhibited by methadone. The participation of adenosine cyclic 3′–5′ monophosphate as an obligatory messenger in ACTH stimulated steroidogenesis is discussed with respect to the pharmacological properties of methadone in this system.     

Isoflavones from Dipteryx odorata

Five isoflavones have been isolated from the heartwood of Dipteryx odorata: retusin, retusin 8-methyl ether, 3′-hydroxyretusin 8-methyl ether, odoratin (7,3′-dihydroxy-6,4′-dimethoxyisoflavone) and dipteryxin (7,8-dihydroxy-6,4′-dimethoxyisoflavone).     

Metabolism to methionine and growth stimulation by 5'-methylthioadenosine and 5'-methylthioinosine in mammalian cells

Viable human and murine lymphoblasts, and normal human tissue extracts, converted the thioether nucleosides 5'-methylthioadenosine (MeSAdo) and 5'-methylthioinosine (MeSIno) to methionine. Both MeSAdo and MeSIno, but not homocysteine, supported the short-term growth of human or murine lymphoblasts in methionine deficient medium. However, MeSAdo at concentrations greater than 25 microM inhibited cell growth. MeSIno was non-toxic at concentrations up to 200 microM, and supported the long-term growth of lymphoblasts in methionine-free medium.