Gino Giannaccini

Fine-needle aspiration of thyroid nodules: Proteomic analysis to identify cancer biomarkers

At present, the clinical and pathological analysis used in the diagnosis of papillary thyroid cancer (PTC) are insufficient to discern tumor behavior, and new diagnostic and prognostic markers need to be identified. In this study, we performed a comparative proteome analysis to examine the global changes of fine needle aspiration fluid (FNA) protein patterns of two variants of malignant PTC (classical variant PTC (cPTC) and tall cell variant PTC (TCV)) with respect to the controls. Changes in protein expression were identified using two-dimensional electrophoresis (2DE) and peptide mass fingerprinting via MALDI-TOF mass spectrometry (MS), as well as Western blot analysis. A statistical significant up-regulation of 17 protein spots in cPTC and/or TCV with respect to controls was demonstrated. These proteins included transthyretin precursor (TTR), ferritin light chain (FLC), proteasome activator complex subunit 1 and 2, alpha-1-antitrypsin precursor, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase chain B (LDH-B), apolipoprotein A1 precursor (Apo-A1), annexin A1, DJ-1 protein and cofilin-1. In addition, 12 protein spots were found exclusively in cPTC and three exclusively in TCV. These latter proteins (ferritin heavy chain (FHC), peroxiredoxin 1 (PRX1) and 6-phosphogluconate dehydrogenase (6-PDGH)) correspond to stress response proteins and, until now, had not been described in thyroid tumors. These findings illustrate the potential use of FNA proteomics to identify protein changes associated with thyroid cancer and to advance potential protein biomarkers in the diagnostic classification of the disease.

Decreased Platelet 3H-Paroxetine Binding in Obsessive-Compulsive Patients

The similarities between the serotonin (5-HT) transporter in both human platelets and human brain permit us to investigate this structure in patients with different psychiatric disorders. Several reports have shown abnormalities of the 5-HT transporter, by means of the measurement of the 5-HT uptake or of the 3H-imipramine binding, in platelets of patients with obsessive-compulsive disorder (OCD). The availability of the ligand 3H-paroxetine, a selective 5-HT reuptake inhibitor, to label the 5-HT transporter, promoted us to evaluate the binding of 3H-paroxetine in platelets of 18 drug-free patients with OCD. The results, showing that the patients had a lower number of 3H-paroxetine sites, which is inversely correlated with the Yale Brown Obsessive-Compulsive Scale total score, than a similar group of controls, add supporting evidence to the involvement of 5-HT in OCD. In addition, the decreased functionality of the 5-HT transporter seems to be linked to the severity of OC symptoms.

Platelet serotonin transporter in patients with diarrhea-predominant irritable bowel syndrome both before and after treatment with alosetron

Platelet serotonin transporter in patients with diarrhea-predominant irritable bowel syndrome both before and after treatment with alosetron

Alteration of serotonin transporter density and activity in fibromyalgia

The aim of the study was to evaluate the kinetic parameters of a specific serotonin transporter (SERT) and serotonin uptake in a mentally healthy subset of patients with fibromyalgia. Platelets were obtained from 40 patients and 38 healthy controls. SERT expression and functionality were evaluated through the measurement of [3H]paroxetine binding and the [3H]serotonin uptake itself. The values of maximal membrane binding capacity (Bmax) were statistically lower in the patients than in the healthy volunteers, whereas the dissociation constant (Kd) did not show any statistically significant variations. Moreover, a decrease in the maximal uptake rate of SERT (Vmax) was demonstrated in the platelets of patients, whereas the Michaelis constant (Km) did not show any statistically significant variations. Symptom severity score (tiredness, tender points index and Fibromyalgia Impact Questionnaire) were negatively correlated with Bmax and with Vmax, and positively correlated with Km. A change in SERT seems to occur in fibromyalgic patients, and it seems to be related to the severity of fibromyalgic symptoms.

New 1,2,3-triazolo[1,5-a]quinoxalines: synthesis and binding to benzodiazepine and adenosine receptors. II.

On pursuing research about 1,2,3-triazolo[1,5-a]quinoxalines, in this paper we report synthesis and binding assays toward the benzodiazepine and A(1) and A(2A) adenosine receptors, of a new series of derivatives, bearing some structural changes (introduction of fluorine and trifluoromethyl in the seventh position, amino substituents in the fourth position, benzyl group in the fifth position and aroyl substituents in the third position). The biological tests have shown that only the 7-fluorosubstituted compounds 3a and 4a and the N-benzyl derivative 7 have a good affinity toward the benzodiazepine receptors, while only the 7-trifluoromethyl substituted compound 3b presents a moderate affinity with low selectivity toward the A(1) adenosine receptors. The other structural modifications strongly decreased biological activity.

Distribution and characterization of [3H]mesulergine binding in human brain postmortem

Much interest is currently being directed towards serotonin (5-HT) receptors of type 2C (5-HT2C) because of their possible involvement in the control of different activities, such as the composition of the cerebrospinal fluid, locomotion, feeding, neuronal excitability and anxiety. The limited information regarding their distribution in the human brain prompted us to investigate, and to characterize the binding of [3H]mesulergine, a HT2C antagonist, in autopsy samples from 24 subjects. The results showed that the [3H]mesulergine binding represented 95% of the total binding and equilibrium saturation binding experiments resulted in a single straight line, consistent with the presence of one site only. The area with the highest density of [3H]mesulergine binding was the choroid plexus, followed at a significantly lower level by the hippocampus, substantia nigra, basal ganglia, amygdala, hypothalamus and prefrontal cortex. The pharmacological profile of the [3H]mesulergine binding was consistent with that of 5-HT2C receptors, since the most effective displacers were ritanserin, mesulergine and mianserine, followed by clozapine, ketanserine and m-CPP, while other compounds had a negligible or no effect. These findings, showing a wide distribution of [3H]mesulergine binding sites in the human brain, could provide anatomical bases for the different functions attributable to 5-HT2C receptors in humans.

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.

The trace element content of top-soil and wild edible mushroom samples collected in Tuscany, Italy

The amount of the trace elements As, Ba, Cd, Cr, Cu, Hg, Li, Mn, Ni, Pb, Rb, Se, Sr, and Zn was measured in top soils and edible mushrooms, Boletus edulis, Macrolepiota procera, collected at five distinct green microhabitats inside the Lucca province, North-Central Italy (years 2008–2009). Results showed a top soil element content within the Italian statutory limits. Concerning the amount of mushroom elements, we observed significant species-differences obtaining higher levels of Ni, Rb, and Se in B. edulis or As, Pb, Cu in M. procera. Bioaccumulation factors (BCFs: element in mushroom/element in soil) resulted species-dependent and element-selective: in particular, B. edulis preferentially accumulated Se (BCFs varying from 14 to 153), while M. procera mainly concentrated Cu (BCFs varying from 5 to 15). As well, both species displayed between-site BCF differences. By a multivariate principal component approach, cluster analysis (CA), we could resolve two main clusters of soil element composition, corresponding to the most ecologically divergent sites. Besides, CA showed no cluster relating to element contents of B. edulis at the different collection sites, while a separation in groups was found for M. procera composition with respect to harvesting locations, suggesting uptake systems, in this saprotrophic species, sensitive to microhabitat. Regarding consumer safety, Cd, Hg, Pb levels resulted sometime relevant in present samples, never reaching values from current literature on mushrooms collected in urban-polluted areas. Our findings encourage a deeper assessment of the molecular mechanisms of metal intake by edible mushrooms, encompassing genetic biochemical and geo-ecological variables, with particular awareness to element bioavailability in soils and fungi.

SUBSTITUTED 1,2,3-TRIAZOLO[1,5-A]QUINAZOLINES: SYNTHESIS AND BINDING TO BENZODIAZEPINE AND ADENOSINE RECEPTORS

This paper reports the synthesis and evaluation of the biological affinity towards benzodiazepine and A(1) and A(2A) adenosine receptors of some 3-ethoxycarbonyl or 3-phenyl-substituted 1,2, 3-triazolo[1,5-a]quinazolines. Starting from the appropriate chloro-substituted phenylazides, the series of 7 or 8 chloro-substituted triazoloquinazolines were prepared. Nitration reactions of the triazoloquinazoline ring and chlorination reactions of the hydroxyl group in the 5 position of the same ring are also reported. By nucleophilic displacement of halogen, the corresponding 5-amino derivatives and some analogous derivatives bearing cyclohexylamino and p-toluidino substituents were obtained. The binding assays showed a generalized decrease in the affinity towards the benzodiazepine receptors and confirmed a moderate affinity towards the A(1) adenosine receptors in comparison with the previously studied triazoloquinazoline derivatives.

Changes in peripheral benzodiazepine receptors in patients with panic disorder and obsessive-compulsive disorder

Peripheral benzodiazepine (BDZ) receptors were investigated through the binding of the specific ligand 3H-PK-11195 to platelet membranes, in 17 patients suffering from panic disorder (PD) and in 16 patients affected by obsessive-compulsive disorder (OCD). The results, showing that the density (Bmax) of peripheral BDZ receptors was significantly lower in patients with PD than in controls or OC patients, suggest that the number of platelet BDZ receptors varies with different anxiety disorders and that perhaps this marker may be beneficial in differentiating some subtypes of these disorders.