Biomarkers mapping of neuropathic pain in a nerve chronic constriction injury mice model.

Neuropathic pain is caused by a lesion or disease of the somatosensory nervous system and has a considerable impact on the quality of life. Neuropathic pain has a dynamic and complex aetiology and gives heterogeneous symptoms across patients; therefore, it represents an important clinical challenge. Current pharmacological treatment includes tricyclic antidepressant serotonin-noradrenaline uptake inhibitors such as duloxetine, pregabalin, and gabapentin. However, these drugs do not show efficacy in all patients suffering from neuropathic pain. In this work we used a nerve chronic constriction injury mice model based on the ligation of sciatic nerve to analyse, by two-dimensional electrophoresis and mass spectrometry, blood proteins significantly altered by neuropathic pain one-week after surgery. A sham-ligated group of mice acting as control and a group of ligated mice treated with gabapentin were also analysed. The results indicated that four haptoglobin isoforms were significantly more expressed, while transthyretin and alpha-2-macroglobulin expression decreased in the serum of the murine neuropathic pain model with respect to the control mice. Interestingly, the treatment with the gabapentin reversed these conditions. The outcomes of this study can provide a further understanding of the pathophysiological meaning of the biomarkers involved in neuropathic pain.

Association between ACP(1) genetic polymorphism and favism.

An association between favism (a hemolytic reaction to consumption of fava beans), glucose-6-phosphate dehydrogenase deficiency (G6PD(-)) and acid phosphatase locus 1 (ACP(1)) phenotypes has been reported; the frequency of carriers of the p(a) and p(c) ACP(1) alleles was found to be significantly higher in G6PD(-) individuals showing favism than in the general population. Here, we investigated the hypothesis that favism is caused by toxic Vicia faba substances, which in some ACP(1) phenotypes cause increased phosphorylation and consequently increased glycolysis, with strong reduction in reduced glutathione production, resulting in hemolysis. It has been demonstrated that ACP(1) f isoforms have physiological functions different from those of s isoforms and are responsible for most of the phosphatase activity, in addition to being less stable in the presence of oxidizing molecules. Thus, the C, CA and A phenotypes, characterized by lower concentrations of f isoforms, could be more susceptible to damage by oxidative events compared to the other phenotypes. To test this hypothesis, the (f+s) enzymatic activity of different ACP(1) phenotypes with and without added V. faba extract was analyzed. Enzymatic activities of ACP(1) A, -CA, -C groups (low activity) and -B, -BA, -CB groups (high activity) were significantly different after addition of V. faba extract. Phenotypes A, CA and C had extremely low enzymatic activity levels, which would lead to low levels of reduced glutathione and bring about erythrocyte lysis.

Modulation of human cytidine deaminase by specific aminoacids involved in the intersubunit interactions.

An investigation was made of the role exerted by some residues supposed to be involved in the intersubunit interaction and also in the catalytic site of homotetrameric human cytidine deaminase (T-CDA). Attention was focused on Y33, Y60, R68, and F137 residues that are a part of a conserved region in most T-CDAs. Hence, a series of site-directed mutagenesis experiments was set up obtaining seven mutants: Y60G, Y33G, Y33F Y33S, F137A, R68G, and R68Q. Each active purified mutant protein was characterized kinetically, with a series of substrates and inhibitors, and the effect of temperature on enzyme activity and stability was also investigated. Circular dichroism (CD) experiments at different temperatures and in presence of small amounts of sodium dodecyl sulphate (SDS) were performed in all the soluble mutant CDAs. The results obtained by site-directed mutagenesis studies were compared to the crystallographic data of B. subtilis CDA and E. coli CDA and to molecular modeling studies previously performed on human CDA. The mutation of Y60 to glycine produced an enzyme with a more compact quaternary structure with respect to the wild-type; this mutation did not have a dramatic effect on cytidine deamination, but it slightly affected the binding with the substrate. None of the mutant CDAs in Y33 showed enzymatic activity; they existed only as monomers, indicating that this residue, located at the intersubunit interface, may be responsible for the correct folding of human CDA. The insertion of an alanine instead of phenylalanine at position 137 led to a soluble but completely inactive enzyme unable to form a tetramer, suggesting that F137 residue may be important for the assembling of the tetramer and also for the arrangement of the CDA active site. Finally, R68G and R68Q mutations revealed that the presence of the amino group seems to be important for the catalytic process but not for substrate binding, as already shown in B. subtilis CDA. The quaternary structure of R68Q was not affected by the mutation, as shown by the SDS-induced dissociation experiments and CD studies, whereas R68G dissociated very easily in presence of small amounts of SDS. These experiments indicated that in the human CDA, the side chain of arginine 68 involved in the catalytic process in one subunit active site might come from another subunit. The data obtained from these studies confirmed the presence of a complicated set of intersubunit interactions in the active site of human CDA, as shown in other T-CDAs.

Studies on thermal stability of human cytidine deaminase.

The thermal stability of human cytidine deaminase (CDA), an enzyme involved in pyrimidine metabolism was investigated. With this in view, the residues R68 and Y60, supposed to be involved in the intersubunit interactions and in the catalytic site of CDA, were mutated to glutamine and glycine, respectively. Thermal stability experiments were performed on the purified mutants by means of circular dichroism and enzymatic assays. The results obtained should be useful for designing more efficient cytidine based drugs for chemotherapy.

NAD+ metabolism in lymphocytes of patients affected by B-cell chronic lymphocytic leukaemia.

In the present work we have quantified some enzymatic activities, related to NAD+ metabolism, in lymphocytes of healthy donors and of patients affected by B-cell chronic lymphocytic leukemia (B-CLL): NADase activity for NAD+ degradation and NMN adenylyltransferase (NMNAT) activity for NAD+ biosynthesis. Most of the samples under investigation (12 B-CLL patients and 12 healthy donors) presented the enzymatic activities assayed. No significant differences in terms of specific activity have been found between ill and healthy subjects. Nevertheless by expressing the activity as mU/10(9) cells (nmol min(-1) 10(9) cells) we observed significantly higher values of both enzymatic activities, involved in the NAD+ metabolism, in healthy donors with respect to the B-CLL patients.

Isoenzymatic forms of human cytidine deaminase.

Cytidine deaminase (CDA) purified from human placenta revealed the presence of five isoenzymatic forms that differ only in their isoelectric point. Since human cytidine deaminase exists in two variants (CDA 1 and CDA 2) with a non-conservative amino acid substitution at codon 27, in this work we demonstrate that these two variants may combine together in vitro, giving five CDA isoforms as observed in vivo from human placenta. For this purpose, each of the two forms of CDA was purified close to homogeneity and dissociated into monomers in the presence of a small amount of sodium dodecyl sulfate as a dissociating agent. The monomers were mixed together and subjected to anion-exchange chromatography and to chromatofocusing analysis in order to visualize the formation of the five isoforms. Furthermore, for both CDA 1 and CDA 2 some substrates and inhibitors of CDA were assayed, with the aim of demonstrating different kinetic behavior between the two natural variants.

Apoptosis in sea bream Sparus aurata eggs.

In the present study, the sea-bream Sparus aurata, a pelagic egg spawner, was used as experimental model, in order to establish the occurrence of apoptosis in vertebrates with external reproduction. The same female ovulates floating and nonfloating eggs, but only the former, after fertilization, proceed to embryo development. The eggs were divided into floating and nonfloating and both were analyzed for the presence of several apoptosis markers. The results here reported provide evidence that the nonfloating cells present severe shrinkage and highly express both FAS receptor and FAS ligand on their surface. Furthermore, DNA fragmentation and mitochondria swelling were found, suggesting that the nonfloating eggs were cells programmed to die.

NAD+ catabolism in pheochromocytoma rat cells.

The catabolic pathway of nicotinamide adenin dinucleotide (NAD) in cultured pheochromocytoma rat cells (PC12) was investigated. The first evidence obtained in these studies was that, despite inducing cell differentiation, NGF treatment did not modify NAD catabolism. Following incubation of PC12 homogenate with NAD, ADP-ribose, AMP, IMP, and HYP was produced. The catabolic fate of AMP and ADPR so obtained was followed by monitoring to a final production of inosine and hypoxanthine through several enzymatic steps. When intact PC12 cells were incubated with NAD in the culture medium AMP, IMP and HYP were found but, no ADPR and cADPR were present in the growth medium. "Nucleotides analyses" carried out on the homogenate obtained from these cells, confirmed the absence of cADPR and an increase of intracellular ADPR. These results led us to believe that in PC12 cells the ADP ribosyl cyclase activity is absent and that NADase is an ecto-enzyme able to transfer the ADPR, produced from NAD catabolism, inside the cells.

NAD(P)(+)-glycohydrolase from human spleen: a multicatalytic enzyme.

NAD(P)(+)-glycohydrolase (NADase, EC 3.2.2.6) was partially purified from microsomal membranes of human spleen after solubilization with Triton X-100. In addition to NAD+ and NADP+, the enzyme catalyzed the hydrolysis of several NAD+ analogues and the pyridine base exchange reaction with conversion of NAD+ into 3-acetylpyridine adenine dinucleotide. The enzyme also catalyzed the synthesis of cyclic ADP-ribose (cADPR) from NAD+ and the hydrolysis of cADPR to adenosine diphosphoribose (ADPR). Therefore, this enzyme is a new member of multicatalytic NADases recently identified from mammals, involved in the regulation of intracellular cADPR concentration. Human spleen NADase showed a subunit molecular mass of 45 kDa, a pI of 4.9 and a Km value for NAD+ of 26 microM. High activation of ADPR cyclase activity was observed in the presence of Ag+ ions, corresponding to NADase inhibition.

NMN adenylyltransferase from bull testis: purification and properties.

The purification procedure of NMN adenylyltransferase from bull testis presented here consists of a heat step and an acidic precipitation followed by four chromatographic steps, including dye ligand, adsorption and hydrophobic chromatography. The final enzyme preparation subjected to non-denaturing and denaturating PAGE with silver nitrate staining exhibited a single band. At this step the enzyme appeared to be homogeneous. The M(r) value of the native enzyme calculated by gel filtration was about 133,000. The protein appeared to possess a quaternary structure with four subunits of apparent M(r) 33,000 without disulphide interchain bonds. Isoelectric experiments gave a pI of 6.2, and pH studies showed the possible presence of an acidic group in the active site having a pKa of 4.9. Analysis of the amino acid composition showed the presence of more acidic residues than basic ones, according to the pI value calculated by Mono P FPLC. The Ea calculated by Arrhenius plot gave an apparent value of 55.7 kJ/mol. The Km values for NMN, ATP, NAD+ and PPi were 0.11, 0.023, 0.37 and 0.16 nM respectively. The polyclonal antiserum produced against the NMN adenylyltransferase reacted with the purified enzyme at different dilutions and recognized the enzyme in the homogenate as well.

Assay methods for nicotinamide mononucleotide adenylyltransferase of wide applicability.

NMN adenylyltransferase (NMNAT) reversibly catalyzes the synthesis of NAD+ or NaAD+ from ATP and NMN or NaMN. In this work, we describe a continuous coupled spectrophotometric assay that can be rapidly and routinely used in place of the previous cumbersome two-step assay. The reaction rates measured with the coupled assay display a linear dependence with respect to enzyme concentration over the range investigated. The method yields accurate and reliable estimates of the enzyme activity in the direction of NAD+ synthesis. Furthermore, we developed an HPLC-based method suitable for the assay activity both in the forward and reverse directions of the enzymatic reaction. The method appears particularly useful for measuring the NMNAT activity when the product is not NAD+ (e.g., in studies using alternative substrates), and offers the possibility of monitoring simultaneously both the NMNAT-catalyzed reaction and interfering side reactions. This is achieved through the HPLC identification and quantitation of metabolites and derivatives produced in the reaction mixture during the assay. The two methods described here should cover most needs for the assay of NMNAT activity.