Francesco Balestri

Purine and pyrimidine nucleosides preserve human astrocytoma cell adenylate energy charge under ischemic conditions.

The brain depends on both glycolysis and mitochondrial oxidative phosphorylation for maintenance of ATP pools. Astrocytes play an integral role in brain functions providing trophic supports and energy substrates for neurons. In this paper, we report that human astrocytoma cells (ADF) undergoing ischemic conditions may use both purine and pyrimidine nucleosides as energy source to slow down cellular damage. The cells are subjected to metabolic stress conditions by exclusion of glucose and incubation with oligomycin (an inhibitor of oxidative phosphorylation). This treatment brings about a depletion of the ATP pool, with a concomitant increase in the AMP levels, which results in 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. In fact, ADF cells possess both purine nucleoside phosphorylase and uridine phosphorylase activities.

A New Approach to Control the Enigmatic Activity of Aldose Reductase

Aldose reductase (AR) is an NADPH-dependent reductase, which acts on a variety of hydrophilic as well as hydrophobic aldehydes. It is currently defined as the first enzyme in the so-called polyol pathway, in which glucose is transformed into sorbitol by AR and then to fructose by an NAD+-dependent dehydrogenase. An exaggerated flux of glucose through the polyol pathway (as can occur in diabetes) with the subsequent accumulation of sorbitol, was originally proposed as the basic event in the aethiology of secondary diabetic complications. For decades this has meant targeting the enzyme for a specific and strong inhibition. However, the ability of AR to reduce toxic alkenals and alkanals, which are products of oxidative stress, poses the question of whether AR might be better classified as a detoxifying enzyme, thus raising doubts as to the unequivocal advantages of inhibiting the enzyme. This paper provides evidence of the possibility for an effective intervention on AR activity through an intra-site differential inhibition. Examples of a new generation of aldose reductase “differential” inhibitors (ARDIs) are presented, which can preferentially inhibit the reduction of either hydrophilic or hydrophobic substrates. Some selected inhibitors are shown to preferentially inhibit enzyme activity on glucose or glyceraldehyde and 3-glutathionyl-4-hydroxy-nonanal, but are less effective in reducing 4-hydroxy-2-nonenal. We question the efficacy of D, L-glyceraldehyde, the substrate commonly used in in vitro inhibition AR studies, as an in vitro reference AR substrate when the aim of the investigation is to impair glucose reduction.

Key role of uridine kinase and uridine phosphorylase in the homeostatic regulation of purine and pyrimidine salvage in brain

Uridine, the major circulating pyrimidine nucleoside, participating in the regulation of a number of physiological processes, is readily uptaken into mammalian cells. The balance between anabolism and catabolism of intracellular uridine is maintained by uridine kinase, catalyzing the first step of UTP and CTP salvage synthesis, and uridine phosphorylase, catalyzing the first step of uridine degradation to beta-alanine in liver. In the present study we report that the two enzymes have an additional role in the homeostatic regulation of purine and pyrimidine metabolism in brain, which relies on the salvage synthesis of nucleotides from preformed nucleosides and nucleobases, rather than on the de novo synthesis from simple precursors. The experiments were performed in rat brain extracts and cultured human astrocytoma cells. The rationale of the reciprocal regulation of purine and pyrimidine salvage synthesis in brain stands (i) on the inhibition exerted by UTP and CTP, the final products of the pyrimidine salvage pathway, on uridine kinase and (ii) on the widely accepted idea that pyrimidine salvage occurs at the nucleoside level (mostly uridine), while purine salvage is a 5-phosphoribosyl-1-pyrophosphate (PRPP)-mediated process, occurring at the nucleobase level. Thus, at relatively low UTP and CTP level, uptaken uridine is mainly anabolized to uridine nucleotides. On the contrary, at relatively high UTP and CTP levels the inhibition of uridine kinase channels uridine towards phosphorolysis. The ribose-1-phosphate is then transformed into PRPP, which is used for purine salvage synthesis.

Metabolic interplay between intra- and extra-cellular uridine metabolism via an ATP driven uridine–UTP cycle in brain

Uridine, a pyrimidine nucleoside essential for the synthesis of RNA and biomembranes, has several trophic functions in the central nervous system, that involve a physiological regulation of pyrimidine nucleotides and phospholipids content, and a maintenance of brain metabolism under ischemia, or pathological situations. The understanding of uridine production in the brain is therefore of fundamental importance. Brain has a limited capacity to synthesize ex novo the pyrimidine ring, and a reasonable source of brain uridine is UTP. The kinetics of UTP breakdown, as catalysed by post-mitochondrial brain extracts and membrane preparations reported herein suggests that in normoxic conditions uridine is locally generated in brain exclusively in the extracellular space, and that any uptaken uridine is salvaged to UTP. It is now well established that cytosolic UTP can be released to interact with a subset of P2Y receptors, inducing a variety of molecular and cellular effects, leading to neuroprotection, while uridine is uptaken via an equilibrative or a Na(+)-dependent transport system, to exert its trophic effects in the cytosol. An ATP driven uridine-UTP cycle can be envisaged, based on the strictly compartmentalized processes of uridine salvage to UTP and uridine generation from UTP, in which uptaken uridine is anabolised to UTP in the cytosol, and converted back to uridine in extracellular space.

Rapid colorimetric determination of reduced and oxidized glutathione using an end point coupled enzymatic assay

AbstractA simple and rapid colorimetric coupled enzymatic assay for the determination of glutathione is described. The proposed method is based on the specific reaction catalyzed by γ-glutamyltransferase, which transfers the γ-glutamyl moiety from glutahione to an acceptor, with the formation of the γ-glutamyl derivative of the acceptor and cysteinylglycine. The latter dipeptide is a substrate of leucyl aminopeptidase, which hydrolyzes cysteinylglycine to glycine and cysteine that can be easily measured spectrophotometrically. The proposed method was used to measure the content of glutathione in acid extracts of bovine lens, to follow the NADPH-dependent reduction of glutathione disulfide (GSSG) to reduced glutathione (GSH) catalyzed by the enzyme glutathione reductase and to determine the glutathione content in human astrocytoma ADF cells subjected to oxidative stress. The results obtained showed that the method can be suitably used for the determination of GSH and GSSG in different biological samples and to monitor tissue or cell redox status under different conditions. It is also applicable for following reactions involving GSH and/or GSSG. FigColorimetric method for the specific measurement of glutathione. γ-glutamyltransferase (γ-GT) transfers the γ-glutamyl moiety from glutathione to an acceptor (Gly-Gly), with the formation of γ-glutamyl-Gly-Gly and Cys-Gly. The latter dipeptide is hydrolized by leucyl-aminopeptidase (LAP) to form cysteine, which can be easily measured using a colorimetric assay at 560 nm

Molecular mechanisms of nucleoside recycling in the brain.

A major role of plasma membrane bound ectonucleotidases is the modulation of ATP, ADP, adenosine (the purinergic agonists), UTP, and UDP (the pyrimidinergic agonists) availability in the extracellular space at their respective receptors. We have recently shown that an ATP driven uridine-UTP cycle is operative in the brain, based on the strictly compartmentalized processes of uridine salvage to UTP and uridine generation from UTP, in which uptaken uridine is anabolized to UTP in the cytosol, and converted back to uridine in the extracellular space by the action of ectonucleotidases (Ipata et al. Int J Biochem Cell Biol 2010;42:932-7). In this paper we show that a similar cytidine-CTP cycle exists in rat brain. Since (i) brain relies on imported preformed nucleosides for the synthesis of nucleotides, RNA, nuclear and mitochondrial DNA, coenzymes, pyrimidine sugar- and lipid-conjugates and (ii) no specific pyrimidinergic receptors have been identified for cytidine and their nucleotides, our results, taken together with previous studies on the intra- and extracellular metabolic network of ATP, GTP, UTP, and their nucleosides in the brain (Barsotti and Ipata. Int J Biochem Cell Biol 2004;36:2214-25; Balestri et al. Neurochem Int 2007;50:517-23), strongly suggest that, apart from the modulation of ligand availability, ectonucleotidases may serve the process of local nucleoside recycling in the brain.

Modulation of aldose reductase activity by aldose hemiacetals

BACKGROUND Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate. METHODS The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR. RESULTS The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose. CONCLUSIONS Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates. GENERAL SIGNIFICANCE The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications.

l-Idose: an attractive substrate alternative to d-glucose for measuring aldose reductase activity

Although glucose is one of the most important physio-pathological substrates of aldose reductase, it is not an easy molecule for in vitro investigation into the enzyme. In many cases alternative aldoses have been used for kinetic characterization and inhibition studies. However these molecules do not completely match the structural features of glucose, thus possibly leading to results that are not fully applicable to glucose. We show how aldose reductase is able to act efficiently on L-idose, the C-5 epimer of D-glucose. This is verified using both the bovine lens and the human recombinant enzymes. While the kcat values obtained are essentially identical to those measured for D-glucose, a significant decrease in KM was observed. This can be due to the significantly higher level of the free aldehyde form present in L-idose compared to D-glucose. We believe that L-idose is the best alternative to D-glucose in studies on aldose reductase.

Human carbonyl reductase 1 as efficient catalyst for the reduction of glutathionylated aldehydes derived from lipid peroxidation

Human recombinant carbonyl reductase 1 (E.C. 1.1.1.184, hCBR1) is shown to efficiently act as aldehyde reductase on glutathionylated alkanals, namely 3-glutathionyl-4-hydroxynonanal (GSHNE), 3-glutathionyl-nonanal, 3-glutathionyl-hexanal and 3-glutathionyl-propanal. The presence of the glutathionyl moiety appears as a necessary requirement for the susceptibility of these compounds to the NADPH-dependent reduction by hCBR1. In fact the corresponding alkanals and alkenals, and the cysteinyl and γ-glutamyl-cysteinyl alkanals adducts were either ineffective or very poorly active as CBR1 substrates. Mass spectrometry analysis reveals the ability of hCBR1 to reduce GSHNE to the corresponding GS-dihydroxynonane (GSDHN) and at the same time to catalyze the oxidation of the hemiacetal form of GSHNE, generating the 3-glutathionylnonanoic-δ-lactone. These data are indicative of the ability of the enzyme to catalyze a disproportion reaction of the substrate through the redox recycle of the pyridine cofactor. A rationale for the observed preferential activity of hCBR1 on different GSHNE diastereoisomers is given by molecular modelling. These results evidence the potential of hCBR1 acting on GSHNE to accomplish a dual role, both in terms of HNE detoxification and, through the production of GSDHN, in terms of involvement into the signalling cascade of the cellular inflammatory response.

Zolfino landrace (Phaseolus vulgaris L.) from Pratomagno: general and specific features of a functional food

Background The Zolfino bean is a variety of Phaseolus vulgaris, which is cultivated in a limited area of Tuscany, Italy, and is widely appreciated for its flavor and culinary uses. Objectives A yellow Zolfino landrace cultivated in the Leccio-Reggello area was characterized and compared with three other varieties of Phaseolus vulgaris (i.e. the Borlotto, Cannellino, and Corona beans) in terms of its general features and potential as an antioxidant/anti-inflammatory agent. Design The length, width, thickness, equatorial section surface, weight, volume, and seed coat section were measured in all the beans. The seed surface area was also estimated by an original empirical method. The ability of the different beans to interfere with the enzymes of the polyol pathway (that is, aldose reductase (AR) and sorbitol dehydrogenase) was tested using the supernatant after soaking the beans at room temperature and after thermal treatment, which simulated the bean-cooking process in a controlled fashion. Results Concerning the general features, Zolfino was comparable with other beans, except Corona, in terms of surface–volume ratio, which possesses the lowest tegument thickness. Moreover, Zolfino appears the most effective in inhibiting AR activity. The inhibitory ability is unaffected by thermal treatment and appears to be associated with compound(s) present in the coat of the bean. Conclusions The ability of Zolfino to inhibit AR, thus reducing the flux of glucose through the polyol pathway, highlights the features of Zolfino as a functional food, potentially useful in treating the dysfunctions linked to the hyperactivity of AR, such as diabetic complications or inflammatory responses.