The Regulation of Brain Nucleoside Utilization

The homeostatic regulation of intracellular purine and pyrimidine pools has long been studied at the level of de novo nucleotide synthesis. However, brain maintains the proper qualitative and quantitative nucleotide balance by salvaging preformed nucleosides, imported from blood stream, rather than by de novo synthesis from simple precursors. The main salvage enzymes are the nucleoside-kinases, catalyzing the ATP mediated phosphorylation of nucleosides in their 5’-position. Salvaged nucleoside-monophosphates are then either further phosphorylated, or converted back to nucleosides by a set of 5’-nucleotidases. This poses the following problem: why are nucleosides produced from nucleosidemonophosphates, to be converted back to the same compounds at the expense of ATP? As discussed in this article, the quantitative and qualitative intracellular balance of brain purine and pyrimidine compounds is maintained i) by the intracellular interplay between the rates of nucleoside-kinases and 5’-nucleotidases, ii) by the relative rates of the inward and outward nucleoside transport through equilibrative and concentrative transport systems, iii) by the metabolic cross-talk between extracellularly exported nucleoside-triphosphate breakdown and the intracellular process of nucleoside-triphosphate salvage synthesis

role of pentose phosphates in nucleoside catabolism and interconversion

Purine and pyrimidine are the basic constituents of the polynucleotides DNA and RNA; they are considered of predominant importance also as information molecules, energy transducers, antioxidants and they have also important roles in cellular signalling processes. Reports suggest protective roles for purines and pyrimidines in various pathological conditions ranging from cancer, to ischemia-associated injury, traumatic tissue damage, bone resorption, stress and haemorrhagic shock. Some papers have shown that adenosine and inosine protect neural cells during hypoxia/ischemia in vivo and in vitro. While in some cases the action of adenosine is receptor-mediated, to explain the effect of its deamination product, inosine, the contribution of hitherto unknown specific receptors has been invoked. On the other hand, several papers report a receptor-independent mechanism of nucleoside action, which indicates that nucleosides donate their ribose moiety to the pentose phosphate pathway, where it is converted to intermediates for entry into the glycolytic pathway for anaerobic production of ATP. To shed light on the mechanism underlying the protective role of purine and pyrimidine during ischemia or brain insults, we have used a human astrocytoma cell line which has been subjected to metabolic stress conditions by exclusion of glucose and pre-incubation with oligomycin. This treatment brings a significant decrease of the adenylate energy charge. The presence of purine nucleosides in the culture medium preserves the adenylate energy charge, and improves cell viability. Besides purine nucleosides, also pyrimidine nucleosides, such as uridine and, to a lesser extent, cytidine, are able to preserve the ATP pool. The determination of lactate in the incubation medium indicates that nucleosides can preserve the ATP pool through anaerobic glycolysis, thus pointing to a relevant role of the phosphorolytic cleavage of the N-glycosidic bond of nucleosides which generates, without energy expense, the phosphorylated pentose, which through the pentose phosphate pathway and glycolysis can be converted to energetic intermediates also in the absence of oxygen. Additionally, the phosphorylated ribose moiety of nucleosides may be used itself for the salvage of pyrimidine nucleosides or can be converted by 5-phosphoribosyl-1-pyrophoshate (PRPP) synthetase, into PRPP which is an essential compound for the salvage pathway of purines. In fact, adenine and hypoxanthine, in the presence of PRPP, are substrates of adenine phosphoribosyltransferase and hypoxanthine-guanine phosphoribosyltransferase, which catalyze the formation of adenosine and inosine monophosphate, respectively. On the other hand, uridine can be both phosphorolytically cleaved by uridine phosphorylase (UPase) to ribose-1-phosphate (Rib-1-P) and the uracil base, or can be converted by uridine kinase (UK) into uridine nucleotides. It has been shown that disruption of uridine homeostasis by deletion of rat UPase gene leads to disorders of both pyrimidine and purine nucleotide synthesis, thus suggesting a linkage between the two metabolic processes. We have hypothesized that the UPase–UK enzyme system, which maintains uridine homeostasis, regulates the processes of both purine and pyrimidine salvage. Exogenous uridine and, to a lesser extent inosine, activate the salvage of exogenous adenine in human astrocytoma cells in a concentration-dependent manner. Moreover, uridine is also able to activate the salvage of exogenous hypoxanthine. When uridine and inosine are present, more Rib-1-P becomes available, through the action of UPase and purine nucleoside phosphorylase, for the PRPP-mediated adenine and hypoxanthine salvage. Moreover, exogenous inosine favours not only uracil salvage but also 5-FU activation through a Rib-1-P-mediated process. The pre-treatment of the cells with cytidine brings about an inhibition of the pyrimidine salvage and an activation of the purine salvage in the presence of uridine as ribose phosphate donor. In fact, cytidine enters the cells and is converted into CTP which inhibits UK and this inhibition causes a shift of the equilibrium of the reversible UPase reaction towards uridine phosphorolysis. The Rib-1-P formed is then converted into PRPP, which is used for the purine salvage synthesis. Conversely, when the concentration of CTP is relatively low, the fully active UK, which catalyzes a virtual irreversible reaction, drives uridine towards uridine nucleotide formation, thus lowering the rate of purine synthesis. Therefore, the ribose phosphate stemming from the phosphorolysis of purine and pyrimidine nucleosides not only can be converted into energetic intermediates in order to restore the ATP pool during cellular stress but it can be considered a link between the purine and pyrimidine salvage and these two processes are regulated at the level of UPase–UK enzyme system by the relative pyrimidine nucleoside triphosphate concentration

Thiol oxidase ability of copper ion is specifically retained upon chelation by aldose reductase

Bovine lens aldose reductase is susceptible to a copper-mediated oxidation, leading to the generation of a disulfide bridge with the concomitant incorporation of two equivalents of the metal and inactivation of the enzyme. The metal complexed by the protein remains redox active, being able to catalyse the oxidation of different physiological thiol compounds. The thiol oxidase activity displayed by the enzymatic form carrying one equivalent of copper ion (Cu1-AR) has been characterized. The efficacy of Cu1-AR in catalysing thiol oxidation is essentially comparable to the free copper in terms of both thiol concentration and pH effect. On the contrary, the two catalysts are differently affected by temperature. The specificity of the AR-bound copper towards thiols is highlighted with Cu1-AR being completely ineffective in promoting the oxidation of both low-density lipoprotein and ascorbic acid

How the chemical features of molecules may have addressed the settlement of metabolic steps

Introduction: While the evolutionary adaptation of enzymes to their own substrates is a well assessed and rationalized field, how molecules have been originally selected in order to initiate and assemble convenient metabolic pathways is a fascinating, but still debated argument. Objectives: Aim of the present study is to give a rationale for the preferential selection of specific molecules to generate metabolic pathways. Methods: The comparison of structural features of molecules, through an inductive methodological approach, offer a reading key to cautiously propose a determining factor for their metabolic recruitment. Results: Starting with some commonplaces occurring in the structural representation of relevant carbohydrates, such as glucose, fructose and ribose, arguments are presented in associating stable structural determinants of these molecules and their peculiar occurrence in metabolic pathways. Conclusions: Among other possible factors, the reliability of the structural asset of a molecule may be relevant or its selection among structurally and, a priori, functionally similar molecules

Experience in implementing a Document Delivery Service

In this paper we propose an integration between electronic mail and web services for people such as library operators who need to send large files to Internet users. The proposed solution permits librarians to continue using the e-mail service to send large documents, but at the same time overcomes problems that users can encounter downloading large size files with e-mail agents. The library operator sends the document as an attachment to the destination address, on fly the e-mail server extracts and saves the attachments in a web-server disk file and substitutes then with a new message part that includes the URL pointing to the saved document. The receiver can download these large objects using user-friendly browser

Models of enzyme inhibition and apparent dissociation constants from kinetic analysis to study the differential inhibition of aldose reductase

In order to explain the negative slope of K-app(M)/(app)k(cat) versus inhibitor concentration observed in the study of epigallocatechin gallate acting as an inhibitor of aldose reductase, a kinetic analysis was performed to rationalise the phenomenon. Classical and non-classical models of complete and incomplete enzyme inhibition were devised and analysed to obtain rate equations suitable for the interpretation of experimental data. The results obtained from the different approaches were discussed in terms of the meaning of the emerging kinetic constants. A decrease of K-app(M)/(app)k(cat) versus the inhibitor concentration was revealed to be a valuable indication of the occurrence of an incomplete inhibition. This indication, which is univocal in the case of an uncompetitive inhibition, may be especially useful when the residual activity resulting from inhibition is rather low

Apparent cooperativity and apparent hyperbolic behavior of enzyme mixtures acting on the same substrate

It is well known that a negative cooperative behavior displayed by a monomeric enzyme may be associated with the simultaneous presence of two enzymes acting on the same substrate. In this paper, emphasis is given to the effect exerted by a rapid equilibrium between the enzyme forms in leading to a hyperbolic behavior, thus masking the presence of multiple enzyme forms

The use of dimethylsulfoxide as a solvent in enzyme inhibition studies: the case of aldose reductase

Aldose reductase (AR) is an enzyme devoted to cell detoxification and at the same time is strongly involved in the aetiology of secondary diabetic complications and the amplification of inflammatory phenomena. AR is subjected to intense inhibition studies and dimethyl sulfoxide (DMSO) is often present in the assay mixture to keep the inhibitors in solution. DMSO was revealed to act as a weak but well detectable AR differential inhibitor, acting as a competitive inhibitor of the L-idose reduction, as a mixed type of non-competitive inhibitor of HNE reduction and being inactive towards 3-glutathionyl-4-hydroxynonanal transformation. A kinetic model of DMSO action with respect to differently acting inhibitors was analysed. Three AR inhibitors, namely the flavonoids neohesperidin dihydrochalcone, rutin and phloretin, were used to evaluate the effects of DMSO on the inhibition studies on the reduction of L-idose and HNE

Edible vegetables as a source of aldose reductase differential inhibitors

The hyperactivity of aldose reductase (AR) on glucose in diabetic conditions or on glutathionyl-hydroxynonenal in oxidative stress conditions, the source of cell damage and inflammation, appear to be balanced by the detoxifying action exerted by the enzyme. This detoxification acts on cytotoxic hydrophobic aldehydes deriving from membrane peroxidative processes. This may contribute to the failure in drug development for humans to favorably intervene in diabetic complications and inflammation, despite the specificity and high efficiency of several available aldose reductase inhibitors. This paper presents additional features to a previously proposed approach, on inhibiting the enzyme through molecules able to preferentially inhibit the enzyme depending on the substrate the enzyme is working on. These differential inhibitors (ARDIs) should act on glucose reduction catalyzed by AR without little or no effect on the reduction of alkenals or alkanals. The reasons why AR may be an eligible enzyme for differential inhibition are considered. These mainly refer to the evidence that, although AR is an unspecific enzyme that recognizes different substrates such as aldoses and hydrophobic aldehydes, it nevertheless displays a certain degree of specificity among substrates of the same class. After screening on edible vegetables, indications of the presence of molecules potentially acting as ARDIs are reported