Synthesis of inosine 6-phosphate diesters via phosphitylation of the carbonyl oxygen.

Inosine derivatives bearing a phosphodiester group at the O(6)-position of the nucleobase were synthesized via phosphitylation of the carbonyl oxygen using phosphoramidites activated by non-nucleophilic acidic activators.

Identification of a major enzyme for the synthesis and hydrolysis of cyclic ADP-ribose in amphibian cells and evolutional conservation of the enzyme from human to invertebrate.

Cyclic ADP-ribose (cADPR), a metabolite of NAD(+), is known to function as a second messenger for intracellular Ca(2+) mobilization in various vertebrate and invertebrate tissues. In this study, we isolated two Xenopus laevis cDNAs (frog cd38 and cd157 cDNAs) homologous to the one encoding the human cADPR-metabolizing enzyme CD38. Frog CD38 and CD157 are 298-amino acid proteins with 35.9 and 27.2 % identity to human CD38 and CD157, respectively. Transfection of expression vectors for frog CD38 and CD157 into COS-7 cells revealed that frog CD38 had NAD(+) glycohydrolase, ADP-ribosyl cyclase (ARC), and cADPR hydrolase activities, and that frog CD157 had no enzymatic activity under physiological conditions. In addition, when recombinant CD38 and frog brain homogenate were electrophoresed on an SDS-polyacrylamide gel, ARC of the brain homogenate migrated to the same position in the gel as that of frog CD38, suggesting that frog CD38 is the major enzyme responsible for cADPR metabolism in amphibian cells. The frog cd38 gene consists of eight exons and is ubiquitously expressed in various tissues. These findings provide evidence for the existence of the CD38-cADPR signaling system in frog cells and suggest that the CD38-cADPR signaling system is conserved during vertebrate evolution.

Molecular characterization of adenosine 5'-monophosphate deaminase--the key enzyme responsible for the umami taste of nori (Porphyra yezoensis Ueda, Rhodophyta).

The enzyme adenosine 5'-monophosphate deaminase (AMPD, EC 3.5.4.6) catalyzes the conversion of adenosine 5'-monophosphate to inosine 5'-mononucleotide (IMP). IMP is generally known as the compound responsible for the umami taste of the edible red alga Porphyra yezoensis Ueda that is known in Japan as nori. Therefore, we suspect that AMPD plays a key role in providing a favorable nori taste. In this study, we undertake the molecular characterization of nori-derived AMPD. The nori AMPD protein has a molecular mass of 55 kDa as estimated from both gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The calculated molecular mass from the amino acid sequence deduced from cDNA is 57.1 kDa. The isoelectric point is 5.71. The coding region of AMPD consists of 1,566 bp encoding 522 amino acids and possesses a transmembrane domain and two N-glycosylation sites. The sequence identity of nori AMPD in human and yeast AMPDs was found to be less than 50% and 20% in DNA and amino acid sequences, respectively. Proline in the conserved motif of [SA]-[LIVM]-[NGS]-[STA]-D-D-P was found to be converted to glutamate. These results indicate that nori AMPD is a novel type of AMPD.

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion.

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.

8-Bromo-cyclic inosine diphosphoribose: towards a selective cyclic ADP-ribose agonist.

cADPR (cyclic ADP-ribose) is a universal Ca(2+) mobilizing second messenger. In T-cells cADPR is involved in sustained Ca(2+) release and also in Ca(2+) entry. Potential mechanisms for the latter include either capacitative Ca(2+) entry, secondary to store depletion by cADPR, or direct activation of the non-selective cation channel TRPM2 (transient receptor potential cation channel, subfamily melastatin, member 2). Here we characterize the molecular target of the newly-described membrane-permeant cADPR agonist 8-Br-N(1)-cIDPR (8-bromo-cyclic IDP-ribose). 8-Br-N(1)-cIDPR evoked Ca(2+) signalling in the human T-lymphoma cell line Jurkat and in primary rat T-lymphocytes. Ca(2+) signalling induced by 8-Br-N(1)-cIDPR consisted of Ca(2+) release and Ca(2+) entry. Whereas Ca(2+) release was sensitive to both the RyR (ryanodine receptor) blocker RuRed (Ruthenium Red) and the cADPR antagonist 8-Br-cADPR (8-bromo-cyclic ADP-ribose), Ca(2+) entry was inhibited by the Ca(2+) entry blockers Gd(3+) (gadolinium ion) and SKF-96365, as well as by 8-Br-cADPR. To unravel a potential role for TRPM2 in sustained Ca(2+) entry evoked by 8-Br-N(1)-cIDPR, TRPM2 was overexpressed in HEK (human embryonic kidney)-293 cells. However, though activation by H(2)O(2) was enhanced dramatically in those cells, Ca(2+) signalling induced by 8-Br-N(1)-cIDPR was almost unaffected. Similarly, direct analysis of TRPM2 currents did not reveal activation or co-activation of TRPM2 by 8-Br-N(1)-cIDPR. In summary, the sensitivity to the Ca(2+) entry blockers Gd(3+) and SKF-96365 is in favour of the concept of capacitative Ca(2+) entry, secondary to store depletion by 8-Br-N(1)-cIDPR. Taken together, 8-Br-N(1)-cIDPR appears to be the first cADPR agonist affecting Ca(2+) release and secondary Ca(2+) entry, but without effect on TRPM2.

Synthesis of N-1-alkyl analogues of cyclic inosine diphosphate ribose (cIDPR) by a new solid phase approach.

Herein we report an efficient solid-phase synthesis of some N-1-alkyl-substituted analogs of cyclic inosine-diphosphate-ribose (cIDPR), a mimic of cyclic ADP-ribose (cADPR) which has been described as an agonist of the cADPR/Ca(2+) signalling system. The proposed synthetic strategy uses a polystyrene support bearing inosine by a 2',3'-acetal linkage which is converted into several N-1-alkylinosine-bis-phosphate derivatives which in turn were cyclized by a solid-phase pyrophosphate bond formation.

Synthesis of a new ribose modified analogue of cyclic inosine diphosphate ribose.

A new analogue of cyclic inosine diphosphate ribose (cIDPR), in which the N-1 and N-9 ribosyl moieties were substituted by an alkyl moiety and an hydroxy-alkyl chain, has been synthesized and characterized.

Cellular effects and metabolic stability of N1-cyclic inosine diphosphoribose and its derivatives.

BACKGROUND AND PURPOSE: Recently, a number of mimics of the second messenger cyclic ADP-ribose (cADPR) with replacement of adenosine by inosine were introduced. In addition, various alterations in the molecule ranging from substitutions at C8 of the base up to full replacement of the ribose moieties still retained biological activity. However, nothing is known about the metabolic stability and cellular effects of these novel analogues. EXPERIMENTAL APPROACH: cADPR and the inosine-based analogues were incubated with CD38, ADP-ribosyl cyclase and NAD-glycohydrolase and metabolism was analysed by RP-HPLC. Furthermore, the effect of the analogues on cytokine expression and proliferation was investigated in primary T-lymphocytes and T-lymphoma cells. KEY RESULTS: Incubation of cADPR with CD38 resulted in degradation to adenosine diphosphoribose. ADP-ribosyl cyclase weakly catabolised cADPR whereas NAD-glycohydrolase showed no such activity. In contrast, N1-cyclic inosine 5'-diphosphoribose (N1-cIDPR) was not hydrolyzed by CD38. Three additional N1-cIDPR analogues showed a similar stability. Proliferation of Jurkat T-lymphoma cells was inhibited by N1-cIDPR, N1-[(phosphoryl-O-ethoxy)-methyl]-N9-[(phosphoryl-O-ethoxy)-methyl]-hypoxanthine-cyclic pyrophosphate (N1-cIDP-DE) and N1-ethoxymethyl-cIDPR (N1-cIDPRE). In contrast, in primary T cells neither proliferation nor cytokine expression was affected by these compounds. CONCLUSIONS AND IMPLICATIONS: The metabolic stability of N1-cIDPR and its analogues provides an advantage for the development of novel pharmaceutical compounds interfering with cADPR mediated Ca2+ signalling pathways. The differential effects of N1-cIDPR and N1-cIDPRE on proliferation and cytokine expression in primary T cells versus T-lymphoma cells may constitute a starting point for novel anti-tumor drugs.

Ligand binding distributions in nucleic acids.

We illustrate a new method for the determination of the complete binding polynomial for nucleic acids based on experimental titration data with respect to ligand concentration. From the binding polynomial, one can then calculate the distribution function for the number of ligands bound at any ligand concentration. The method is based on the use of a finite set of moments of the binding distribution function, which are obtained from the titration curve. Using the maximum-entropy method, the moments are then used to construct good approximations to the binding distribution function. Given the distribution functions at different ligand concentrations, one can calculate all of the coefficients in the binding polynomial no matter how many binding sites a molecule has. Knowledge of the complete binding polynomial in turn yields the thermodynamics of binding. This method gives all of the information that can be obtained from binding isotherms without the assumption of any specific molecular model for the nature of the binding. Examples are given for the binding of Mn(2+) and Mg(2+) to t-RNA and for the binding of Mg(2+) and I(6) to poly-C using literature data.

Synthetic study on carbocyclic analogs of cyclic ADP-ribose, a novel second messenger: an efficient synthesis of cyclic IDP-carbocyclic-ribose.

An efficient synthesis of cyclic IDP-carbocyclic-ribose, as a stable mimic for cyclic ADP-ribose, was achieved. 8-Bromo-N1-carbocyclic-ribosylinosine derivative 10, prepared from N1-(2,4-dinitrophenyl)inosine derivative 5 and an optically active carbocyclic amine 6, was converted to 8-bromo-N1-carbocyclic-ribosylinosine bisphosphate derivative 15. Treatment of 15 with I2 in the presence of molecular sieves in pyridine gave the desired cyclic product 16 quantitatively, which was deprotected and reductively debrominated to give the target cyclic IDP-carbocyclic-ribose (3).

Template-directed synthesis using the heterogeneous templates produced by montmorillonite catalysis. A possible bridge between the prebiotic and RNA worlds.

The synthesis of oligoguanylates [oligo(G)s] is catalyzed by a template of oligocytidylates [oligo(C)s] containing 2',5'- and 3',5'-linked phosphodiester bonds with and without incorporated C5'ppC groupings. An oligo(C) template containing exclusively 2',5'-phosphodiester bonds also serves as a template for the synthesis of complementary oligo(G)s. The oligo(C) template was prepared by the condensation of the 5'-phosphorimidazolide of cytidine on montmorillonite clay. These studies establish that RNA oligomers prepared by mineral catalysis, or other routes on the primitive earth, did not have to be exclusively 3',5'-linked to catalyze template-directed synthesis, since oligo(C)s containing a variety of linkage isomers serve as templates for the formation of complementary oligo(G)s. These findings support the postulate that origin of the RNA world was initiated by the RNA oligomers produced by polymerization of activated monomers formed by prebiotic processes.

[Sequence specific conjugates of minor groove ligands with inosine containing oligodeoxyribonucleotides]. / Sikvensspetsifichnye kon''iugaty maloborozdochnogo liganda s inozinosoderzhashchimi oligodezoksiribonukleotidami.

Conjugates of the distamycin tetrapyrrole analogue containing four pyrrolecaboxamide fragments (MGB) with inosine-containing oligodeoxyribonucleotides were synthesized. The stability of duplexes formed by these conjugates depends on the composition of inosine-containing pairs and decreases in the order: IC > IA > > IT. For the duplexes (d(TTTATATA)p(MGB))2, (d(TTCICICI)p(MGB))2, and (d(TTAIAIAI)p(MGB))2, the melting temperatures are 68, 54, and 35-45 degrees C, respectively; (d(TTTITITI)p(MGB))2 forms no duplexes at temperatures above 4 degrees C. The binding was shown to be highly specific: in the duplex d(GGCATCTA)p(MGB).d(GGTAIATI)p(MGB), the substitution of only one A.T pair by A.A decreases the binding constant by almost three orders of magnitude.