Changes in purines concentration in the cerebrospinal fluid of patients experiencing pain: A case-control study

This study analyzes the relationship between extracellular purines and pain perception in humans. Cerebrospinal fluid (CSF) levels of purines and their metabolites were compared between patients displaying acute and/or chronic pain syndromes and control subjects. The CSF levels of IMP, inosine, guanosine and uric acid were significantly increased in the chronic pain group and correlated with pain severity (P<0.05). Patients displaying both chronic and acute pain presented similar changes in the CSF purines concentration (P<0.05). However, in the acute pain group, only CSF inosine and uric acid levels were significantly increased (P<0.05). These findings suggest that purines, in special inosine, guanosine and uric acid, are associated with the spinal mechanisms underlying nociception. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Nonrandom Distribution of Cryptic Repeating Triplets of Purines and Pyrimidines (RNY)(n) in gp120 of HIV Type1.

Abstract We have analyzed purine (R) and pyrimidine (Y) codon patterns in variable and constant regions of HIV-1 gp120 in seven patients infected with different HIV-1 subtypes and naive to antiretroviral therapy. We have calculated the relative frequency of each in-frame codon RNY, YNR, RNR, and YNY (N=any nucleotide) in variable and constant regions of gp120, in the sequence within indels and at indels' flanking sites. Our data show that hypervariable regions V1, V2, V4, and V5 are characterized by the presence of long stretches of RNY codons constituting the majority of the sequence portion within insertions/deletions. In full-length gp120 and within inserted/deleted fragments the number of AVT (V=A, C, G) codons did not exceed 50% of the total RNY codons. RNY strings in variable regions spanned up to 21 codons and were always in frame. In contrast, RNY strings in constant regions were mostly out of frame and their length was limited to five codons. The frequency of the codon RNY was found to be significantly higher in variable regions (p<0.0001; t-test), within indels, and at indels' flanking sites (p<0.0001; χ(2) test). Analysis of the distribution of RNY strings equal to or longer than five codons in the full genome of HXB2 also shows that these sequences are mostly out of frame, unless they contain a potential N-glycosylation site or an asparagine. These data suggest that cryptic repeats of RNY may play a role in the genesis of multiple base insertions and deletions in hypervariable regions of gp120.

Biochemical effects of cadmium, salinity and temperature on the catabolism of proteins and purines in oreochromis mossambicus (Peters)

In the present investigation, three important stressors: cadmium ion (Cd++), salinity and temperature were selected to study their effects on protein and purine catabolism of O. mossambicus. Cadmium (Cd) is a biologically nonessential metal that can be toxic to aquatic animals. Cadmium is a trace element which is a common constituent of industrial effluents. It is a non-nutrient metal and toxic to fish even at low concentrations. Cadmium ions accumulate in sensitive organs like gills, liver, and kidney of fish in an unregulated manner . Thus; the toxic effects of cadmium are related to changes in natural physiological and biochemical processes in organism. The mechanics of osmoregulation (i.e. total solute and water regulation) are reasonably well understood (Evans, 1984, 1993), and most researchers agree that salinities that differ from the internal osmotic concentration of the fish must impose energetic regulatory costs for active ion transport. There is limited information on protein and purine catabolism of euryhaline fish during salinity adaptation. Within a range of non-lethal temperatures, fishes are generally able to cope with gradual temperature changes that are common in natural systems. However, rapid increases or decreases in ambient temperature may result in sub lethal physiological and behavioral responses. The catabolic pathways of proteins and purines are important biochemical processes. The results obtained signifies that O. mossambicus when exposed to different levels of cadmium ion, salinity and temperature show great variation in the catabolism of proteins and purines. The organism is trying to attain homeostasis in the presence of stressors by increasing or decreasing the activity of certain enzymes. The present study revealed that the protein and purine catabolism in O. mossambicus is sensitive to environmental stressors.

Purines, creatine, defective methylation and their biochemical and clinical relationship

Marinus Duran; Isabel G. Tavares de Almeida; Helena P. Gaspar Tomás

The electrochemical reduction of the purines guanine and adenine at platinum electrodes in several room temperature ionic liquids

The reduction of guanine was studied by microelectrode voltammetry in the room temperature ionic liquids (RTILs) N-hexyltriethylammonium bis (trifluoromethanesulfonyl) imide [N6,2,2,2][N(Tf)2], 1-butyl-3-methylimidazolium hexafluorosphosphate [C4mim][PF6], N-butyl-N-methyl-pyrrolidinium bis(trifluoromethanesulfonyl)imide [C4mpyrr][N(Tf)2], 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide [C4mim][N(Tf)2], N-butyl-N-methyl-pyrrolidinium dicyanamide [C4mpyrr][N(NC)2] and tris(P-hexyl)-tetradecylphosphonium trifluorotris(pentafluoroethyl)phosphate [P14,6,6,6][FAP] on a platinum microelectrode. In [N6,2,2,2][NTf2] and [P14,6,6,6][FAP], but not in the other ionic liquids studied, guanine reduction involves a one-electron, diffusion-controlled process at very negative potential to produce an unstable radical anion, which is thought to undergo a dimerization reaction, probably after proton abstraction from the cation of the ionic liquid. The rate of this subsequent reaction depends on the nature of the ionic liquid, and it is faster in the ionic liquid [P14,6,6,6][FAP], in which the formation of the resulting dimer can be voltammetrically monitored at less negative potentials than required for the reduction of the parent molecule. Adenine showed similar behaviour to guanine but the pyrimidines thymine and cytosine did not; thymine was not reduced at potentials less negative than required for solvent (RTIL) decomposition while only a poorly defined wave was seen for cytosine. The possibility for proton abstraction from the cation in [N6,2,2,2][NTf2] and [P14,6,6,6][FAP] is noted and this is thought to aid the electrochemical dimerization process. The resulting rapid reaction is thought to shift the reduction potentials for guanine and adenine to lower values than observed in RTILs where the scope for proton abstraction is not present. Such shifts are characteristic of so-called EC processes where reversible electron transfer is followed by a chemical reaction. © 2009 Elsevier B.V.

Leishmania Metacyclogenesis Is Promoted in the Absence of Purines

Leishmania parasites, the causative agent of leishmaniasis, are transmitted through the bite of an infected sand fly. Leishmania parasites present two basic forms known as promastigote and amastigote which, respectively, parasitizes the vector and the mammalian hosts. Infection of the vertebrate host is dependent on the development, in the vector, of metacyclic promastigotes, however, little is known about the factors that trigger metacyclogenesis in Leishmania parasites. It has been generally stated that "stressful conditions" will lead to development of metacyclic forms, and with the exception of a few studies no detailed analysis of the molecular nature of the stress factor has been performed. Here we show that presence/absence of nucleosides, especially adenosine, controls metacyclogenesis both in vitro and in vivo. We found that addition of an adenosine-receptor antagonist to in vitro cultures of Leishmania amazonensis significantly increases metacyclogenesis, an effect that can be reversed by the presence of specific purine nucleosides or nucleobases. Furthermore, our results show that proliferation and metacyclogenesis are independently regulated and that addition of adenosine to culture medium is sufficient to recover proliferative characteristics for purified metacyclic promastigotes. More importantly, we show that metacyclogenesis was inhibited in sand flies infected with Leishmania infantum chagasi that were fed a mixture of sucrose and adenosine. Our results fill a gap in the life cycle of Leishmania parasites by demonstrating how metacyclogenesis, a key point in the propagation of the parasite to the mammalian host, can be controlled by the presence of specific purines.

Radiation metabolomics. 2. Dose- and time-dependent urinary excretion of deaminated purines and pyrimidines after sublethal gamma-radiation exposure in mice

Gamma-radiation exposure of humans is a major public health concern as the threat of terrorism and potential hostile use of radiological devices increases worldwide. We report here the effects of sublethal gamma-radiation exposure on the mouse urinary metabolome determined using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry-based metabolomics. Five urinary biomarkers of sublethal radiation exposure that were statistically significantly elevated during the first 24 h after exposure to doses ranging from 1 to 3 Gy were unequivocally identified by tandem mass spectrometry. These are deaminated purine and pyrimidine derivatives, namely, thymidine, 2'-deoxyuridine, 2'-deoxyxanthosine, xanthine and xanthosine. Furthermore, the aminopyrimidine 2'-deoxycytidine appeared to display reduced urinary excretion at 2 and 3 Gy. The elevated biomarkers displayed a time-dependent excretion, peaking in urine at 8-12 h but returning to baseline by 36 h after exposure. It is proposed that 2'-deoxyuridine and 2'-deoxyxanthosine arise as a result of gamma irradiation by nitrosative deamination of 2'-deoxycytidine and 2'-deoxyguanosine, respectively, and that this further leads to increased synthesis of thymidine, xanthine and xanthosine. The urinary excretion of deaminated purines and pyrimidines, at the expense of aminopurines and aminopyrimidines, appears to form the core of the urinary radiation metabolomic signature of mice exposed to sublethal doses of ionizing radiation.

Fosa de purines vs separación in situ de heces y orina en porcino de cebo: producción de purín y de estiércol

El objetivo de este estudio fue comparar, en un alojamiento de cebo de porcino con enrejillado parcial (Laboratorio de Bienestar Porcino), la producción de residuos con un sistema tradicional de fosa de purines frente a un sistema de separación in situ con cinta plana. La fosa se vació cada cinco semanas, y la cinta se vació dos veces al día a lo largo del periodo de cebo. Ambos sistemas tuvieron el mismo tipo de ventilación por extracción bajo suelo. La cantidad de residuos generados con la separación in situ fue el 70,50% de la correspondiente al sistema tradicional, que, a su vez, fue sensiblemente menor que la habida en las explotaciones comerciales. El sistema de ventilación por extracción bajo suelo fue muy eficiente para la evaporación de agua de las deyecciones y la consecuente reducción de la cantidad de residuos generados; asimismo, los resultados obtenidos ponen de manifiesto que la separación in situ permitió una notable reducción adicional de dicha cantidad, en comparación con el sistema tradicional.

New 9-Terpenyl(7-Terpenyl)Purines: synthesis and cytotoxicity

The purine ring system is one of the most widely distributed N-heterocycles in Nature [1] and many structurally modified purine nucleosides and nucleotides have activities ranging from antineoplastic and antiviral to antihypertensive, antiasthmatic, antituberculosis, etc [2]. Among the purine derivatives, we have put our attention on natural N-alkylpurines such as the asmarines or agelasimines, a group of secondary metabolites isolated from marine sponges with very interesting biological properties [3]. They have a diterpenoid moiety attached to the N-7 nitrogen atom of an adenine and are usually isolated in very small quantities, which limited their structure-activity relationship studies. Our research group has been involved for years in the design, synthesis and biological evaluation of cytotoxic compounds related to natural products, including the chemoinduction of bioactivity on inactive terpenoids [4]. These diterpenoid include compounds such as communic or cupressic acids that bear decaline moieties very close to those present in the above-mentioned marine natural products. These facts prompted us to design and prepare new terpenylpurine derivatives starting from natural monoterpenoids and diterpenoids, commercially available or isolated from their natural sources and transformed into appropriate alkylated agents. Thus, we have prepared purines alkylated at N-7 and N-9 positions with isoprenoids, monoterpenoids and diterpenoids, using two different synthetic approaches: from 6-chloropurine or from 4,5-diamine-6-chloropyrimidine. The structure of the synthesized purines are shown in the following figure. The purine analogues synthesized have been evaluated for their cytotoxicity against four tumour human cell lines (breast, non-small lung, cervical and hepatocellular carcinoma) and non-tumour cells (porcine liver primary cells). The most cytotoxic derivatives were those with a diterpenoid rest on the purine. The results obtained allowed to draw conclusions on the structure-activity relationship of the compounds in order to evaluate the influence of the terpenyl size on their cytotoxic properties.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

Complete Genome Sequence of Mycoplasma suis and Insights into Its Biology and Adaption to an Erythrocyte Niche

Mycoplasma suis, the causative agent of porcine infectious anemia, has never been cultured in vitro and mechanisms by which it causes disease are poorly understood. Thus, the objective herein was to use whole genome sequencing and analysis of M. suis to define pathogenicity mechanisms and biochemical pathways. M. suis was harvested from the blood of an experimentally infected pig. Following DNA extraction and construction of a paired end library, whole-genome sequencing was performed using GS-FLX (454) and Titanium chemistry. Reads on paired-end constructs were assembled using GS De Novo Assembler and gaps closed by primer walking; assembly was validated by PFGE. Glimmer and Manatee Annotation Engine were used to predict and annotate protein-coding sequences (CDS). The M. suis genome consists of a single, 742,431 bp chromosome with low G+C content of 31.1%. A total of 844 CDS, 3 single copies, unlinked rRNA genes and 32 tRNAs were identified. Gene homologies and GC skew graph show that M. suis has a typical Mollicutes oriC. The predicted metabolic pathway is concise, showing evidence of adaptation to blood environment. M. suis is a glycolytic species, obtaining energy through sugars fermentation and ATP-synthase. The pentose-phosphate pathway, metabolism of cofactors and vitamins, pyruvate dehydrogenase and NAD(+) kinase are missing. Thus, ribose, NADH, NADPH and coenzyme A are possibly essential for its growth. M. suis can generate purines from hypoxanthine, which is secreted by RBCs, and cytidine nucleotides from uracil. Toxins orthologs were not identified. We suggest that M. suis may cause disease by scavenging and competing for host nutrients, leading to decreased life-span of RBCs. In summary, genome analysis shows that M. suis is dependent on host cell metabolism and this characteristic is likely to be linked to its pathogenicity. The prediction of essential nutrients will aid the development of in vitro cultivation systems.

Adenosine binding to low-molecular-weight purine nucleoside phosphorylase: the structural basis for recognition based on its complex with the enzyme from Schistosoma mansoni

Schistosomes are unable to synthesize purines de novo and depend exclusively on the salvage pathway for their purine requirements. It has been suggested that blockage of this pathway could lead to parasite death. The enzyme purine nucleoside phosphorylase (PNP) is one of its key components and molecules designed to inhibit the low-molecular-weight (LMW) PNPs, which include both the human and schistosome enzymes, are typically analogues of the natural substrates inosine and guanosine. Here, it is shown that adenosine both binds to Schistosoma mansoni PNP and behaves as a weak micromolar inhibitor of inosine phosphorolysis. Furthermore, the first crystal structures of complexes of an LMW PNP with adenosine and adenine are reported, together with those with inosine and hypoxanthine. These are used to propose a structural explanation for the selective binding of adenosine to some LMW PNPs but not to others. The results indicate that transition-state analogues based on adenosine or other 6-amino nucleosides should not be discounted as potential starting points for alternative inhibitors.

Structural complexes of human adenine phosphoribosyltransferase reveal novel features of the APRT catalytic mechanism

Adenine phosphoribosyltransferase (APRT) is an important enzyme component of the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida are unable to synthesize purines de novo and use the salvage pathway for the synthesis of purine bases rendering this biosynthetic pathway an attractive target for antiparasitic drug design. The recombinant human adenine phosphoribosyltransferase (hAPRT) structure was resolved in the presence of AMP in the active site to 1.76 angstrom resolution and with the substrates PRPP and adenine simultaneously bound to the catalytic site to 1.83 angstrom resolution. An additional structure was solved containing one subunit of the dimer in the apo-form to 2.10 angstrom resolution. Comparisons of these three hAPRT structures with other `type I` PRTases revealed several important features of this class of enzymes. Our data indicate that the flexible loop structure adopts an open conformation before and after binding of both substrates adenine and PRPR Comparative analyses presented here provide structural evidence to propose the role of Glu 104 as the residue that abstracts the proton of adenine N9 atom before its nucleophilic attack on the PRPP anomeric carbon. This work leads to new insights to the understanding of the APRT catalytic mechanism.

Calcium signaling and the novel anti-proliferative effect of the UTP-sensitive P2Y11 receptor in rat cardiac myofibroblasts

During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and -smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca2+]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca2+]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca2+ in the extracellular fluid. The biphasic [Ca2+]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca2+]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.

Synthesis and anticancer activity of N3-substituted-6,8-diaminopurines

[Excerpt] Cancer is the second most common cause of death in developed countries appearing just after cardiovascular diseases. The treatment of cancer remains a major medical challenge and the development of new anticancer drugs is an emerging topic for the scientific community. During the past three decades several chemical classes of anticancer drugs have been identified. In particular, 2,6-diamino purines proved to be important candidates as new anti-cancer agents.