[MALDI-TOF and SELDI-TOF analysis: "tandem" techniques to identify potential biomarker in fibromyalgia]. / MALDI-TOF e SELDI-TOF: due tecniche "tandem" per l'individuazione di potenziali biomarker nella fibromialgia.

OBJECTIVE: Fibromyalgia (FM) is characterized by the presence of chronic widespread pain throughout the musculoskeletal system and diffuse tenderness. Unfortunately, no laboratory tests have been appropriately validated for FM and correlated with the subsets and activity. The aim of this study was to apply a proteomic technique in saliva of FM patients: the Surface Enhance Laser Desorption/Ionization Time-of-Flight (SELDI-TOF). METHODS: For this study, 57 FM patients and 35 HC patients were enrolled. The proteomic analysis of saliva was carried out using SELDI-TOF. The analysis was performed using different chip arrays with different characteristics of binding. The statistical analysis was performed using cluster analysis and the difference between two groups was underlined using Student's t-test. RESULTS: Spectra analysis highlighted the presence of several peaks differently expressed in FM patients compared with controls. The preliminary results obtained by SELDI-TOF analysis were compared with those obtained in our previous study performed on whole saliva of FM patients by using electrophoresis. The m/z of two peaks, increased in FM patients, seem to overlap well with the molecular weight of calgranulin A and C and Rho GDP-dissociation inhibitor 2, which we had found up-regulated in our previous study. CONCLUSION: These preliminary results showed the possibility of identifying potential salivary biomarker through salivary proteomic analysis with MALDI-TOF and SELDI-TOF in FM patients. The peaks observed allow us to focus on some of the particular pathogenic aspects of FM, the oxidative stress which contradistinguishes this condition, the involvement of proteins related to the cytoskeletal arrangements, and central sensibilization.

Proteomic analysis of human thyroid fine needle aspiration fluid.

The aim of this study was to determine the protein pattern of human thyroid fine needle aspiration fluid (FNA) using a proteomic approach. FNA proteins were separated using 2-dimensional gel electrophoresis (2DE), digested and then analyzed by peptide mass fingerprinting. For the first time, we provided an image of the protein components of the FNA, in which approximately 220 protein spots can be identified. The proteome analysis revealed a specific fingerprint of FNA with proteins appertaining to various functional systems. Our preliminary results of FNA protein pattern could be a starting point in studying the presence of potential markers implicated in thyroid diseases.

A Novel Panel of Serum Biomarkers for MPM Diagnosis.

Exposure to asbestos is the main cause of malignant pleural mesothelioma (MPM), a highly aggressive cancer of the pleura. Since the only tools for early detection are based on radiological tests, some authors focused on serum markers (i.e., mesothelin). The aim of this study was the evaluation of new serum biomarkers to be used individually or in combination, in order to improve the outcome of patients whose disease would be diagnosed at an earlier stage. Serum and plasma were available from 43 subjects previously exposed to asbestos and 27 MPM patients, all being epithelioid type. All the new markers found differentially expressed in MPM and healthy subjects, by proteomic and genomic approaches, have been validated in the serum by the use of specific ELISA. The combined approach, using tools of genomics and proteomics, is found to be highly innovative for this type of disease and led to the identification of new serum markers in the diagnosis of MPM. These results, if confirmed in a larger series, may have a strong impact in this area, because early detection of this cancer in people at high risk could significantly improve the course of the disease and the clinical approach to an individualized therapy.

Peripheral benzodiazepine receptors on platelets of fibromyalgic patients.

OBJECTIVE: The aim of the present study was to analyze if alterations of peripheral-type benzodiazepine receptor (PBR) characteristics occurred in platelet membranes of patients affected by primary fibromyalgia (FM). DESIGN AND METHODS: Platelets were obtained from 30 patients with FM. Evaluation of kinetic parameters of PBR was performed using [(3)H] PK11195 as specific radioligand compared with 16 healthy volunteers. RESULTS: The results showed a significant increase of PBR binding sites value in platelet membranes from FM patients (B(max) was 5366+/-188 fmol/mg vs. controls, 4193+/-341 fmol/mg, mean+/-SEM) (**p<0.01) but not for affinity value (K(d) was 4.90+/-0.39 nM vs. controls, 4.74+/-0.39 nM, mean+/-SEM) (p>0.05). Symptom severity scores (pain and tiredness) were positively correlated with B(max). CONCLUSIONS: Our results showed an up-regulation of PBR in platelets of FM patients, and this seems to be related to the severity of fibromyalgic symptoms.

Bottom-up proteomics suggests an association between differential expression of mitochondrial proteins and chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a debilitating and complex disorder characterized by unexplained fatigue not improved by rest. An area of investigation is the likely connection of CFS with defective mitochondrial function. In a previous work, we investigated the proteomic salivary profile in a couple of monozygotic twins discordant for CFS. Following this work, we analyzed mitochondrial proteins in the same couple of twins. Nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-MS) was used to study the mitochondria extracted from platelets of the twins. Subsequently, we selected three proteins that were validated using western blot analysis in a big cohort of subjects (n=45 CFS; n=45 healthy), using whole saliva (WS). The selected proteins were as follows: aconitate hydratase (ACON), ATP synthase subunit beta (ATPB) and malate dehydrogenase (MDHM). Results for ATPB and ACON confirmed their upregulation in CFS. However, the MDHM alteration was not confirmed. Thereafter, seeing the great variability of clinical features of CFS patients, we decided to analyze the expression of our proteins after splitting patients according to clinical parameters. For each marker, the values were actually higher in the group of patients who had clinical features similar to the ill twin. In conclusion, these results suggest that our potential markers could be one of the criteria to be taken into account for helping in diagnosis. Furthermore, the identification of biomarkers present in particular subgroups of CFS patients may help in shedding light upon the complex entity of CFS. Moreover, it could help in developing tailored treatments.

Solubilization of rat kidney benzodiazepine binding sites.

The high-affinity binding site for [3H]Ro 5-4864 has been solubilized from rat kidney using 1% Triton X-100. After lowering the concentration of detergent and using a poly(ethylene glycol) gamma-globulin assay, it has been possible to demonstrate solubilization of about 90% of the binding sites. A single soluble class of binding sites with a Kd of 1.8 nM is found. The order of potency of benzodiazepines is identical for the solubilized receptor and the membrane-bound form. Gel filtration revealed a major peak of binding activity with apparent molecular weight of 215000 and a Stokes' radius of 5.03 nm.

Chemical modifications of striatal A2A adenosine receptors: a possible role for tyrosine at the ligand binding sites.

A2A adenosine receptors were examined in bovine striatal membranes following exposure to tetranitromethane (TNM) which modifies tyrosine and cysteine residues. TNM (0.05-0.5 mM) treatment caused an irreversible, concentration-dependent decrease in the binding activity of the selective A2A agonist [3H]CGS 21680. Protection studies showed that TNM inactivation could be prevented by the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) and by the antagonist xanthine amine congener (XAC), suggesting that TNM modified residues at the ligand-binding sites. Scatchard analysis of the binding data showed that 0.15 mM TNM decreased the [3H]CGS 21680 Bmax value from 447 +/- 39 to 273 +/- 21 fmol/mg of proteins without any significant change in the Kd values (13.5 +/- 1.4 and 14.7 +/- 1.5 for control and treated membranes, respectively). We carried out a series of successive chemical modifications with the reducing agent dithiothreitol (DTT), which indicated that the residues modified by TNM, under our experimental conditions, are tyrosine residues and not cysteine residues.

Regulation of agonist binding to A2A adenosine receptors: effects of guanine nucleotides (GDP[S] and GTP[S]) and Mg2+ ion.

Adenosine acts as a neuromodulator through at least two receptor subtypes, A1 and A2. A2 receptors have been further divided into A2A (high agonist affinity) and A2B (low agonist affinity) receptors. Both A1 and A2 receptors belong to the superfamily of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. A Gs protein couples the A2A receptor to the activation of adenylyl cyclase. In order to elucidate the mechanism of coupling between the A2A receptor and Gs, we studied the modulation by guanine nucleotides and divalent cations of agonist binding to the A2A receptor in rat striatal membranes, using [3H]CGS 21680 as a selective high-affinity agonist. We demonstrated that in rat striatal membranes agonist binding to A2A receptors was modulated by guanine nucleotides. Both GDP and GTP inhibited [3H]CGS 21680 binding to rat striatal membranes with about equal potency. The nonhydrolyzable analogs, GDP[S] and GTP[S], were equipotent inhibitors and approx. 100-times more potent than GDP and GTP. Data from competition studies with labeled and unlabeled CGS 21680 when analyzed by nonlinear regression demonstrated the presence of two binding sites in rat striatal membranes with mean values for KD of 5.6 and 343 nM and Bmax of 200 and 942 fmol/mg protein. The high-affinity binding site has the characteristics of the A2A receptor. In the presence both of (0.1 mM) GDP[S] and GTP[S], the KD values for the high-affinity site were increased severalfold, whereas the low-affinity site was no longer detected in filtration assays. Dissociation studies revealed monophasic dissociation curves both in the absence and presence of 0.1 mM GDP[S]. However the K-1 value increased in the presence of guanine nucleotide. We also showed that in bovine striatal membranes agonist binding to A2A receptors was modestly modulated by guanine nucleotides, suggesting differences of receptor Gs-protein-coupling a mechanism in different species. Divalent cations often increase agonist binding to different receptors, whereas Mg2+ ions play a role in regulating the initial steps of G-protein activation. We investigated the effects of divalent cations on [3H]CGS 21680 binding to the A2A receptor and determined the requirement of these cations to obtain the modulation of binding by guanine nucleotides. We found that millimolar concentrations of divalent cations were required to obtain an effective interaction between the A2A receptor and Gs. The high-affinity binding of [3H]CGS 21680 to the A2A receptor in rat striatal membranes was dependent on the presence of Mg2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Affinity labeling of histidine and lysine residue in the adenosine deaminase substrate binding site.

1. Adenosine deaminase was inactivated by 9-(4-bromoacetamidobenzyl)-adenine (I) and 9-(2-bromoacetamidobenzyl)adenine (II), two affinity labels. 2. The stoichiometry of the reaction with reagent II is reported: 1 mol reagent is bound per mol inactive enzyme. Amino acid analysis of the 6 N HCl hydrolyzate of the inactive enzyme identified CM-histidine as the main alkylation product. This is the first evidence of the presence of a histidine in the active site region. 3. The alkylation rate and involved amino acid residues were studied for both reagents I and II, at pH 8 and 5.5. The particular reactivity of a lysine near or in the active site is discussed.

Role of cysteine residues of rat A2a adenosine receptors in agonist binding.

In the present study, we investigated the role of disulfide bridges and sulfhydryl groups in A2a adenosine receptor binding of the agonist 2-p-(2-carboxyethyl)phenylethylamino)-5'-N-ethylcarboxamidoadenosi ne (CGS 21680). To evaluate the presence of essential disulfide bridges, rat striatal membranes were incubated with [3H]CGS 21680 in the presence of dithiothreitol and binding of the agonist to membranes was measured. The amount of [3H]CGS 21680 which specifically bound, decreased progressively upon pretreatment of membranes with increasing concentrations of dithiothreitol. Pretreatment of rat striatal membranes with 12.5 mM dithiothreitol for 15 min at 25 degrees C resulted in a 2-fold decrease of A2a adenosine receptor affinity for [3H]CGS 21680, and a reduction in the maximal number of binding sites. The presence of agonist or antagonist ligands protected the A2a adenosine receptor sites from the effect of dithiothreitol. We also examined the susceptibility of A2a adenosine receptors to inactivation by the sulfhydryl alkylating reagent, N-ethylmaleimide. When rat striatal membranes were pretreated with N-ethylmaleimide for 30 minutes at 37 degrees C, a decrease in specific [3H]CGS 21680 binding was observed. Pretreatment of membranes with 1 mM N-ethylmaleimide also resulted in a 2-fold reduction of A2a adenosine receptor affinity for [3H]CGS 21680, as well as a slight decrease in the maximal number of binding sites. Neither agonist nor antagonist ligands were effective in protecting the receptor sites from inactivation by N-ethylmaleimide. In contrast, addition of 100 microM guanosine-5'-O-(3-thiotriphosphate) or 5'-guanylylimidodiphosphate were both effective in protecting the receptor sites from inactivation by N-ethylmaleimide. This protective effect was significant but not complete. Our data suggest that disulfide bridges play a role in the structural integrity of the A2a adenosine receptor, furthermore, reduced sulfhydryl groups appear to be important but we do not yet know if they are on the receptor or on the Gs alpha subunit.

Biochemical and pharmacological characterization of periodate-oxidized adenosine analogues at adenosine A1 receptors.

Periodate oxidation of eight N6-substituted adenosine derivatives was performed with the aim of oxidizing the vicinal 2' and 3' hydroxyl groups of the ribose moiety. A thermodynamical and pharmacological characterization of the products of this transformation allowed us to verify that oxidized adenosine analogues act as agonists at adenosine A1 receptors. The dependence of their association constants on temperature indicates that their binding is entropy driven, a feature typical of adenosine A1 receptor agonists; moreover all synthesized compounds were able to fully inhibit the forskolin induced c-AMP accumulation in rat isolated adipocytes. This is the first report suggesting that the presence of an intact ribose moiety is not necessary for agonistic activity at adenosine A1 receptor. In fact periodate oxidation of the ribose moiety yields a dialdehyde and it is recognized that nucleoside dialdehydes are complex equilibrium mixtures of cyclic and acyclic hydrates and hemiacetals.

Regulation of the platelet serotonin transporter by protein kinase C in the young and elderly.

BACKGROUND: Some data show that different factors may influence the serotonin (5-HT) uptake rate. Our study aimed at evaluating the possible role of a protein kinase C (PKC) activator, i.e., 4-beta-12-tetradecanoylphorbol-13-acetate (beta-TPA) on the platelet 5-HT uptake of young and elderly subjects, through the measurement of the 5-HT uptake itself and 3H-paroxetine ([3H]PAR) binding sites, which correspond to the transporter protein. METHODS: Human platelets and 5-HT uptake were evaluated according to the method of Arora and Meltzer, while [3H]PAR binding was performed following the Marazziti et al method. RESULTS: The results showed that beta-TPA reduced significantly the maximal velocity (Vmax) of 5-HT uptake, with no change in the Michaelis constant or in [3H]PAR binding parameters, in platelets of both young and elderly subjects. Although this last group of subjects had a significantly lower Vmax than the other, the degree of inhibition was almost the same (75%) in both. CONCLUSIONS: These findings indicate that PKC decreases the 5-HT uptake rate by modifying the phosphorylation state of the transporter and with no change in the number of [3H]PAR binding sites. The responsiveness of this pathway is identical in both young and elderly subjects.

Presence and characterization of the serotonin transporter in human resting lymphocytes.

Although evidence exists of the presence of a serotonin (5-HT) reuptake system in lymphocytes, no information is available on the pharmacological characterization of this structure. Our study aimed to investigate this matter, therefore, by means of the binding of [3H]-paroxetine ([3H]PAR), a selective 5-HT reuptake inhibitor (SSRI), which is considered the ligand of choice for binding studies. Lymphocytes were obtained from a pool of 20 healthy subjects who volunteered for the study. The results showed the presence of a specific and saturable [3H]PAR binding to lymphocyte membranes, with a Hill number close to unity indicative of the presence of one site only. The most potent drugs inhibiting [3H]PAR binding were SSRIs (paroxetine, fluoxetine, citalopram) followed by clomipramine, imipramine, and 5-HT, whereas haloperidol, mazindol, and nomifensine had a negligible effect. These findings suggest that [3H]-PAR in human resting lymphocytes specifically labels the 5-HT transporter.

Specific inhibition of benzodiazepine receptor binding by some N-(indol-3-ylglyoxylyl)amino acid derivatives: stereoselective interactions.

Several optically active N-(indol-3-ylglyoxylyl)amino acid derivatives were synthesized and tested for [3H]flunitrazepam displacing activity in bovine brain membranes. IC50 values were measured and revealed that the D form of the amino acid moiety of the compounds was more potent than both the L form and racemic form, suggesting a key role of the amino acid stereochemistry on the affinity to the benzodiazepine receptors. GABA ratio and proconvulsant/convulsant data reported for the most active compounds reveal they behave as inverse agonists at the benzodiazepine receptor.

3-[(2-ethoxyphenoxy)methyl]piperidine derivatives. Synthesis and antidepressant activity.

The 3-[(2-ethoxyphenoxy)methyl]piperidine derivatives 3-5 were synthesized and screened as potential antidepressant agents by the reserpine interaction test in mice and the evaluation of reuptake inhibition of biogenic amines in pig brain synaptosomal fractions. In addition, their anticonvulsant activity, tested by pentyleneetrazole antagonism, and approximate acute toxicity were evaluated. In vivo and in vitro tests showed that compounds 3 and 5 possess a biological activity comparable to that of the antidepressant drug viloxazine.

Tricyclic heteroaromatic systems. Synthesis and A1 and A2a adenosine binding activities of some 1-aryl-1,4-dihydro-3-methyl[1]benzopyrano[2,3-c] pyrazol-4-ones, 1-aryl-4,9-dihydro-3-methyl-1H-pyrazolo[3,4-b]quinolin-4- ones, and 1-aryl-1H-imidazo[4,5-b]quinoxalines.

The syntheses and A1 and A2a adenosine binding activities of some new 1-aryl-1,4-dihydro-3-methyl[1]benzopyrano[2,3-c]pyrazol-4-ones, 1-aryl-4,9-dihydro-3-methyl-1H-pyrazolo[3,4-b]-quinolin-4-ones, and 1-aryl-1H-imidazo[4,5-b]quinoxalines are reported. Some compounds show A1 adenosine receptor affinity and selectivity. Structure-activity relationships on these new classes of adenosine receptor ligands are defined.

Synthesis of some 2-aryl-1,2,4-triazolo[1,5-c][1,3]benzoxazin-5-ones as tools to define the essential pharmacophoric descriptors of a benzodiazepine receptor ligand.

The synthesis and benzodiazepine receptor (BZR) affinity of some 1,2,4-triazolo[1,5-c][1,3]benzoxazin-5-ones, 2-22, are reported. Compounds 2-22 are devoid of the proton donor group, which according to a BZR schematic model was one of the pharmacophoric descriptors for receptor-ligand interaction. The binding data show that 2-(2-fluorophenyl)-9-chloro-1,2,4-triazolo[1,5-c][1,3]benzoxazin-5 -one (12) and some other compounds display nanomolar BZR affinity, indicating that the hydrogen donor group is not essential for the anchoring of 6,6,5-tricyclic systems to the BZR but only affects the potency of a ligand.

Structure-activity relationships of 1,2,4-triazolo[1,5-a] quinoxalines and their 1-deaza analogues imidazo[1,2-a]quinoxalines at the benzodiazepine receptor.

The synthesis, BZR binding activity, and GABA ratio of some 1,2,4-triazolo[1,5-a]quinoxalines and imidazo[1,2-a]quinoxalines are reported. Both series of compounds displayed similar affinities while their efficacies were different. The structure-activity relationships have provided the opportunity to localize on the BZR accessory areas which are able to enhance the affinity and evaluate the importance of the presence or absence of a proton acceptor atom to determine different trends of efficacy.

8-Azaxanthine derivatives as antagonists of adenosine receptors.

A series of 1,3-dimethyl- and 1,3-dipropyl-8-azaxanthines, substituted at the N8 or N7 position with substituents which usually increase the affinity of the xanthines for the adenosine receptors, was synthesized and studied in radioligand binding experiments. The substitution of CH with N at the 8-position of both theophylline and caffeine dramatically reduced the affinity, as demonstrated by the fact that 8-azatheophylline and 8-azacaffeine were inert. The introduction of a methyl group at 8-position of 8-azatheophylline restored the antagonistic activity at A2 receptors, while a 8-cycloalkyl substituent increased the affinity for both receptor subtypes. A more favorable effect on affinity was produced by the substitution of the 7-methyl group in 8-azacaffeine with cycloalkyl groups. 7-Cyclopentyl-1,3-dimethyl-8-azaxanthine was 3 times more potent than caffeine at A1 receptors and 6 times less active at A2 receptors. On the contrary, the 7-cyclohexyl-1,3-dimethyl-8-azaxanthine was more potent than caffeine at A2 receptors. The substitution of 1- and 3-methyl groups with propyl in both 7- and 8-substituted 8-azatheophylline increased remarkably the affinity for A1 receptors. The 7-cyclopentyl-1,3-dipropyl-8-azaxanthine appears to be one of the most potent and selective among 7-alkyl-substituted xanthines at A1 receptors so far known. Because the 8-aza analogues of 8-substituted 1,3-dialkylxanthine were in any case less active than the corresponding xanthine derivatives, it was confirmed that the hydrogen atom at the 7-position of xanthines plays an important role in the binding to adenosine receptors.

Pyrazolo[4,5-c]quinolines. 2. Synthesis and specific inhibition of benzodiazepine receptor binding.

A series of 1-aryl-3,5-dimethyl-4,5-dihydro-1H-pyrazolo[4,5-c]quinolin-4-ones (2a-e) and 1-aryl-3-methyl-1H-pyrazolo[4,5-c]quinolines (3-7a-e) bearing different substituents at position 4 were prepared and tested for their ability to displace specific [3H]flunitrazepam binding from bovine brain membranes. The 5-N-methyl derivatives 2a-c,e were the compounds that bound with the highest affinity within this class. The replacement of the carbonyl group with other substituents and the resulting aromatization of the pyridine moiety greatly decreased the binding affinity. From a Lineweaver-Burk analysis on the most active compound 2b, it appears that the inhibition is a competitive one.