X-Ray Structure Of A Ternary Metal-Complex - Copper(Ii) Inosine 5'-Monophosphate Imidazole - Metal-Binding To The N(7) Atom Of The Nucleotide In A Mixed-Ligand System Containing An Aromatic Amine

A ternary metal-nucleotide complex, Na2[Cu(5’-IMP)2(im)o,8(H20)l,2(H20)2h]as~ 1be2e.n4 pHr2ep0a,r ed and its structure analyzed by X-ray diffraction (5’-IMP = inosine 5’-monophos hate; im = imidazole). The complex crystallizes in space group C222, with a = 8.733 (4) A, b = 23.213 (5) A, c = 21.489 (6) 1, and Z = 4. The structure was solved by the heavy-atom method and refined by full-matrix least-squares technique on the basis of 2008 observed reflections to a final R value of 0.087. Symmetry-related 5’-IMP anions coordinate in cis geometry through the N(7) atoms of the bases. The other cis positions of the coordination plane are statistically occupied by nitrogen atoms of disordered im groups and water oxygens with occupancies 0.4 and 0.6, respectively. Water oxygens in axial positions complete the octahedral coordination of Cu(I1). The complex is isostructural with C~S-[P~(S’-IMP),(NH~)~a] m”,o del proposed for Pt(I1) binding to DNA. The base binding observed in the present case is different from the typical ”phosphate only” binding shown from earlier studies on metal-nucleotide complexes containing various other ?r-aromatic amines.

Interaction of metal ions with 6-oxopurine nucleotides. Part 1. X-Ray structures of ternary cobalt(III) complexes with inosine 5'-monophosphate or guanosine 5'-monophosphate

The crystal structures of two ternary metal nucleotide complexes of cobalt, [Co(en)2(H2O)2]-[Co(5?-IMP)2(H2O)4]Cl2·4H2O (1) and [Co(en)2(H2O)2][Co(5?-GMP)2(H2O)4]Cl2·4H2O (2), have been analysed by X-ray diffraction (en = ethylenediamine, 5?-IMP = inosine 5?-monophosphate, and 5?-GMP = guanosine 5?-monophosphate). Both complexes crystallize in the orthorhombic space group C2221 with a= 8.725(1), b= 25.891(5), c= 21.212(5)Å, Z= 4 for (1) and a= 8.733(2), b= 26.169(4), c= 21.288(4)Å, Z= 4 for (2). The structure of (1) was solved by the heavy-atom method, while that of (2) was deduced from (1). The structures were refined to R values of 0.09 and 0.10 for 1 546 and 1 572 reflections for (1) and (2) respectively. The two structures are isomorphous. A novel feature is that the chelate ligand en and the nucleotide are not co-ordinated to the same metal ion. One of the metal ions lying on the two-fold a axis is octahedrally co-ordinated by two chelating en molecules and two water oxygens, while the other on the two-fold b axis is octahedrally co-ordinated by two N(7) atoms of symmetry-related nucleotides in a cis position and four water oxygens. The conformations of the nucleotides are C(2?)-endo, anti, and gauche�gauche. In both (1) and (2) the charge-neutralising chloride ions are disordered in the vacant space between the molecules. These structures bear similarities to the mode of nucleotide co-ordination to PtII complexes of 6-oxopurine nucleotides, which are the proposed models for intrastrand cross-linking in DNA by a metal complex.

Conformational Variability in Modified Nucleosides. Crystal and Molecular Structure of 2',3'-O-Isopropylidene Inosine

The crystal structure of 2',3'-O-isopropylidene inosine shows a number of interesting features. The four independent molecules in the asymmetric unit exhibit significant conformational variations. Ribose puckers fall in the O(4')-exo region, unfavourable in unsubstituted nucleosides. Hypoxanthine bases show base-pairing (I.I) in a manner analogous to the guanine self pairs (G.G) in 2',3'-O-isopropylidene guanosine but with a C(2)-H…O(6) hydrogen bond instead of N(2)-H…O(6).

Anodic voltammetric behavior of inosine on a glassy carbon electrode

The anodic voltammetric behavior of inosine (I) was investigated by linar-sweep voltammetry, differential-pulse voltammetry and cyclic voltammetry at a glassy carbon electrode. In a medium of 0.1 mol/L N2HPO4, inosine showed a well defined anodic peak. The peak potential was about 1.42 V (vs. Ag/AgCl). A linear relationship held between the peak current and the concentration of inosine in the rang of 5 x 10(-4) similar to 8 x 10(-2) g/L. The peak potential decreased with the decrease of the acidity of the solution. The four anodic peaks of inosine with hypoxanthine, xanthine and uric acid were obtained. Their peak potentials were about at 1.42, 1.07, 0.72 and 0.26 Vt vs. Ag/AgCl). The method has been used for the direct determination of inosine in injections. Recoveries of inosine in urine samples were about 85%. Experimental result proved that the electrode reaction was diffusion-controlled and irreversible.

Monitoring guanine photo-oxidation by enantiomerically resolved Ru(II) dipyridophenazine complexes using inosine-substituted oligonucleotides

The intercalating [Ru(TAP)2(dppz)]2+ complex can photo-oxidise guanine in DNA, although in mixed-sequence DNA it can be difficult to understand the precise mechanism due to uncertainties in where and how the complex is bound. Replacement of guanine with the less oxidisable inosine (I) base can be used to understand the mechanism of electron transfer (ET). Here the ET has been compared for both L- and D-enantiomers of [Ru(TAP)2(dppz)]2+ in a set of sequences where guanines in the readily oxidisable GG step in {TCGGCGCCGA}2 have been replaced with I. The ET has been monitored using picosecond and nanosecond transient absorption and ps-time-resolved IR spectroscopy. In both cases inosine replacement leads to a diminished yield, but the trends are strikingly different for L- and D-complexes.

Structures of human purine nucleoside phosphorylase complexed with inosine and ddI

Human purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effect on B-cell function. PNP is highly specific for 6-oxopurine nucleosides and exhibits negligible activity for 6-aminopurine nucleosides. The catalytic efficiency for inosine is 350,000-fold greater than for adenosine. Adenine nucleosides and nucleotides are deaminated by adenosine deaminase and AMP deaminase to their corresponding inosine derivatives which, in turn, may be further degraded. Here we report the crystal structures of human PNP in complex with inosine and 2',3'-dideoxymosine, refined to 2.8 Angstrom resolution using synchrotron radiation. The present structures provide explanation for ligand binding, refine the purine-binding site, and can be used for future inhibitor design. (C) 2003 Elsevier B.V. All rights reserved.

Inosine stimulates extensive axon collateral growth in the rat corticospinal tract after injury

The purine nucleoside inosine has been shown to induce axon outgrowth from primary neurons in culture through a direct intracellular mechanism. For this study, we investigated the effects of inosine in vivo by examining whether it would stimulate axon growth after a unilateral transection of the corticospinal tract. Inosine applied with a minipump to the rat sensorimotor cortex stimulated intact pyramidal cells to undergo extensive sprouting of their axons into the denervated spinal cord white matter and adjacent neuropil. Axon growth was visualized by anterograde tracing with biotinylated dextran amine and by immunohistochemistry with antibodies to GAP-43. Thus, inosine, a naturally occurring metabolite without known side effects, might help to restore essential circuitry after injury to the central nervous system.

Inosine-5′-Monophosphate Dehydrogenase Is a Rate-determining Factor for p53-dependent Growth Regulation

We have proposed that reduced activity of inosine-5′-monophosphate dehydrogenase (IMPD; IMP:NAD oxidoreductase, EC 1.2.1.14), the rate-limiting enzyme for guanine nucleotide biosynthesis, in response to wild-type p53 expression, is essential for p53-dependent growth suppression. A gene transfer strategy was used to demonstrate that under physiological conditions constitutive IMPD expression prevents p53-dependent growth suppression. In these studies, expression of bax and waf1, genes implicated in p53-dependent growth suppression in response to DNA damage, remains elevated in response to p53. These findings indicate that under physiological conditions IMPD is a rate-determining factor for p53-dependent growth regulation. In addition, they suggest that the impd gene may be epistatic to bax and waf1 in growth suppression. Because of the role of IMPD in the production and balance of GTP and ATP, essential nucleotides for signal transduction, these results suggest that p53 controls cell division signals by regulating purine ribonucleotide metabolism.

Long RNA hairpins that contain inosine are present in Caenorhabditis elegans poly(A)+ RNA

Adenosine deaminases that act on RNA (ADARs) are RNA-editing enzymes that convert adenosine to inosine within double-stranded RNA. In the 12 years since the discovery of ADARs only a few natural substrates have been identified. These substrates were found by chance, when genomically encoded adenosines were identified as guanosines in cDNAs. To advance our understanding of the biological roles of ADARs, we developed a method for systematically identifying ADAR substrates. In our first application of the method, we identified five additional substrates in Caenorhabditis elegans. Four of those substrates are mRNAs edited in untranslated regions, and one is a noncoding RNA edited throughout its length. The edited regions are predicted to form long hairpin structures, and one of the RNAs encodes POP-1, a protein involved in cell fate decisions.

Insulin and Oleate Promote Translocation of Inosine-5' Monophosphate Dehydrogenase to Lipid Bodies

In the present study we identify inosine-5' monophosphate dehydrogenase (IMPDH), a key enzyme in de novo guanine nucleotide biosynthesis, as a novel lipid body-associated protein. To identify new targets of insulin we performed a comprehensive 2-DE analysis of P-32-labelled proteins isolated from 3T3-L1 adipocytes (Hill et al. J Biol Chem 2000; 275: 24313-24320). IMPDH was identified by liquid chromatography/tandem mass spectrometry as a protein which was phosphorylated in a phosphatidylinositol (PI) 3-kinase-dependent manner upon insulin treatment. Although insulin had no significant effect on IMPDH activity, we observed translocation of IMPDH to lipid bodies following insulin treatment. Induction of lipid body formation with oleic acid promoted dramatic redistribution of IMPDH to lipid bodies, which appeared to be in contact with the endoplasmic reticulum, the site of lipid body synthesis and recycling. Inhibition of PI 3-kinase blocked insulin- and oleate-induced translocation of IMPDH and reduced oleate-induced lipid accumulation. However, we found no evidence of oleate-induced IMPDH phosphorylation, suggesting phosphorylation and translocation may not be coupled events. These data support a role for IMPDH in the dynamic regulation of lipid bodies and fatty acid metabolism and regulation of its activity by subcellular redistribution in response to extracellular factors that modify lipid metabolism.

Human milk xanthine oxidase and neonatal salivary nucleotide precursors generate hydrogen peroxide; a novel pathway with potential role in regulating oral microflora

Background: Xanthine oxidase (XO) is a complex molybdeno-flavoprotein occurring with high activity in the milk fat globule membrane (MFGM) in all mammalian milk and is involved in the final stage of degradation of purine nucleotides. It catalyzes the sequential oxidation of hypoxanthine to xanthine and uric acid, accompanied by production of hydrogen peroxide and superoxide anion. Human saliva has been extensively described for its composition of proteins, electrolytes, cortisol, melatonin and some metabolites such as amino acids, but little is known about nucleotide metabolites. Method: Saliva was collected with swabs from babies; at full-term 1-4 days, 6-weeks, 6-months and 12-months. Unstimulated fasting (morning) saliva samples were collected directly from 77 adults. Breast milk was collected from 24 new mothers. Saliva was extracted from swabs and ultra-filtered. Nucleotide metabolites were analyzed by RP-HPLC with UV-photodiode array and ESI-MS/MS. XO activity was measured as peroxide production from hypoxanthine. Bacterial inhibition over time was assessed using CFU/mL or OD. Results: Median concentrations (μmol/L) of salivary nucleobases and nucleosides for neonates/6-weeks/6-months/12-months/adult respectively were: uracil 5.3/0.8/1.4/0.7/0.8, hypoxanthine 27/7.0/1.1/0.8/2.0, xanthine 19/7.0/2.0/2.0/2.0, adenosine 12/7.0/0.9/0.8/0.1, inosine 11/5.0/0.3/0.4/0.2, guanosine 7.0/6.0/0.5/0.4/0.1, uridine 12/0.8/0.3/0.9/0.4. Deoxynucleosides and dihydropyrimidines concentrations were essentially negligible. XO activity (Vmax:mean ± SD) in breast milk was 8.9 ± 6.2 μmol/min/L and endogenous peroxide was 27 ± 12 μmol/L; mixing breast milk with neonate saliva generated ~40 μmol/L peroxide,which inhibited Staphylococcus aureus. Conclusions: Salivary metabolites, particularly xanthine/hypoxanthine, are high in neonates, transitioning to low adult levels between 6-weeks to 6-months (p < 0.001). Peroxide occurs in breast milk and is boosted during suckling as an antibacterial system.

Nucleotide Degradation Profiles of Iced Barramundi (Lates calcarifer) and Nile Perch (Lates niloticus) Muscle

Adenine nucleotides and their related compounds were determined in muscle extracts from two species of fish that were stored in ice after thawing. The fish were the closely related species, Australian barramundi (Lates calcarifer ) and Kenyan Nile perch (Lates niloticus ) which had different process histories. For all samples, adenine nucleotides did not exceed 6% of the total nucleotide pool. Inosine monophosphate (IMP) decreased steadily with storage. Hypoxanthine (Hx) was the major product of adenosine triphosphate (ATP) degradation in both barramundi and Nile perch, showing a steady increase with days of iced storage. The Hx level did not reach a maximum during the 9d storage period. The K-value also increased regularly with time of storage and for the later stages (i.e., 7 and 9d) and was significantly different (P < 0.01) for the two species. The iced storage life of these typical samples of barramundi and Nile perch was estimated to be 3d after thawing using a K-value of < 30% to indicate excellent quality. Despite the differences in process history the nucleotide profiles were remarkably similar during storage. This precludes the use of nucleotide levels as a means of differentiating between these species.

Phylogenetic analysis of human Chlamydia pneumoniae strains reveals a distinct Australian indigenous clade that predates European exploration of the continent

Background The obligate intracellular bacterium Chlamydia pneumoniae is a common respiratory pathogen, which has been found in a range of hosts including humans, marsupials and amphibians. Whole genome comparisons of human C. pneumoniae have previously highlighted a highly conserved nucleotide sequence, with minor but key polymorphisms and additional coding capacity when human and animal strains are compared. Results In this study, we sequenced three Australian human C. pneumoniae strains, two of which were isolated from patients in remote indigenous communities, and compared them to all available C. pneumoniae genomes. Our study demonstrated a phylogenetically distinct human C. pneumoniae clade containing the two indigenous Australian strains, with estimates that the most recent common ancestor of these strains predates the arrival of European settlers to Australia. We describe several polymorphisms characteristic to these strains, some of which are similar in sequence to animal C. pneumoniae strains, as well as evidence to suggest that several recombination events have shaped these distinct strains. Conclusions Our study reveals a greater sequence diversity amongst both human and animal C. pneumoniae strains, and suggests that a wider range of strains may be circulating in the human population than current sampling indicates.

An Insight to the Dynamics of Conserved Water Molecular Triad in IMPDH II (Human): Recognition of Cofactor and Substrate to Catalytic Arg 322

Inosine 5' monophosphate dehydrogenase (IMPDH II) is a key enzyme involved in the de novo biosynthesis pathway of purine nucleotides and is also considered to be an excellent target for cancer inhibitor design. The conserve R 322 residue (in human) is thought to play some role in the recognition of inhibitor and cofactor through the catalytic D 364 and N 303. The 15 ns simulation and the water dynamics of the three different PDB structures (1B3O, 1NF7, and 1NFB) of human IMPDH by CHARMM force field have clearly indicated the involvement of three conserved water molecules (W-L, W-M, and W-C) in the recognition of catalytic residues (R 322, D 364, and N 303) to inhibitor and cofactor. Both the guanidine nitrogen atoms (NH1 and NH 2) of the R 322 have anchored the di- and mono-nucleotide (cofactor and inhibitor) binding domains via the conserved W-C and W-L water molecules. Another conserved water molecule W-M seems to bridge the two domains including the R 322 and also the W-C and W-L through seven centers H-bonding coordination. The conserved water molecular triad (W-C - W-M - W-L) in the protein complex may thought to play some important role in the recognition of inhibitor and cofactor to the protein through R 322 residue.