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.

Characterization of thyroglobulin epitopes in patients with autoimmune and non-autoimmune thyroid diseases using recombinant human monoclonal thyroglobulin autoantibodies.

CONTEXT: Thyroglobulin (Tg) epitopes of serum Tg autoantibodies (TgAb) have been characterized using inhibition of Tg binding by human monoclonal TgAb in autoimmune thyroid diseases (AITD) [Hashimoto's thyroiditis (HT) and Graves' disease (GD)] but not in non-AITD [nontoxic multinodular goiter (NTMG) and papillary thyroid carcinoma (PTC)]. OBJECTIVE: Our objective was to compare Tg epitopes of serum TgAb from patients with AITD, non-AITD, and PTC associated with histological thyroiditis (PTC-T) using inhibition of Tg binding by four recombinant human TgAb-Fab (epitopic regions A-D). DESIGN: Inhibition of Tg binding of 24 HT, 25 GD, 19 NTMG, 15 PTC, and 25 PTC-T TgAb-positive sera by each TgAb-Fab was evaluated in ELISA. Inhibition by the pool of the four TgAb-Fab was evaluated using labeled Tg. RESULTS: Levels of inhibition were different for TgAb-Fab regions A (P = 0.001), B (0.007), and D (0.011). Inhibition by region A TgAb-Fab was significantly higher in HT, GD, and PTC-T than in NTMG and PTC patients. Inhibition levels by region B TgAb-Fab were significantly higher in HT compared with NTMG and PTC patients and in GD compared with NTMG patients. Inhibition by D region TgAb-Fab was significantly lower in NTMG than in the other groups. Inhibition by the pool ranged from 44% (NTMG) to 72% (GD). CONCLUSIONS: The pattern of Tg recognition is similar when HT patients are compared to GD and NTMG to PTC patients and differs when AITD are compared with non-AITD patients. In PTC-T patients, it is similar to that of AITD patients.

Short-term effects of 3,4-methylen-dioxy-metamphetamine (MDMA) on 5-HT(1A) receptors in the rat hippocampus.

The first effects of 3,4-methylen-dioxy-metamphetamine (MDMA, "ecstasy"), on serotonin 1A (5-HT(1A)) receptors in rat hippocampus were determined by means of [(3)H]-8-hydroxy-dipropylamino-tetralin ([(3)H]-8-OH-DPAT) and 5'guanosine-(gamma-[(35)S]-thio)triphosphate ([(35)S]-GTPgammaS) binding as well as inhibition of forskolin (FK)-stimulated adenylyl cyclase (AC) activity. The study was completed by [(35)S]-GTPgammaS functional autoradiography experiments carried out in frontal sections of rat brain, including the hippocampal region. Results showed that MDMA was either able to displace [(3)H]-8-OH-DPAT binding (K(i) congruent with 500 nM) or to reduce the number of specific sites (B(max)) without affecting K(d). The drug also failed to change the [(35)S]-GTPgammaS binding or to inhibit AC velocity, underlying its behavior as a non-competitive 5-HT(1A) receptor antagonist. Further, MDMA (1 or 100 microM), partially antagonized either [(35)S]-GTPgammaS binding stimulation of the agonists 5CT and 8-OH-DPAT or the AC inhibition induced by 5CT and DP-5CT. However, in contrast to binding studies, in AC assays the amphetamine displayed an effect also on EC(50), always being less potent than the reference antagonist WAY100,635. In functional autoradiography, MDMA behaved either as a partial 5-HT(1A) antagonist in limbic areas or, added alone, as an agonist, increasing the coupling signal presumably through 5-HT release from synapses. Interestingly, the selective 5-HT re-uptake inhibitor (SSRI) fluoxetine had no effect on MDMA [(35)S]-GTPgammaS binding activation. This latter finding indicates that the amphetamine can release 5-HT via alternative mechanisms to 5-HT transporter binding, probably via membrane synaptic receptors or vesicular transporters. The release of other transmitters is not excluded. Therefore, our results encourage at extending the study of MDMA biochemical profiles, in the attempt to elucidate those amphetamine-induced pathways with a potential for neurotoxicity or psycho-stimulant activity.

Tubby protein in human lymphocytes from normal weight and obese subjects.

OBJECTIVES: The presence of tubby protein in human lymphocytes was investigated and their electrophoretic mobility property between normal weight and obese subjects was compared. DESIGN AND METHODS: 2-DE proteome map of lymphocytes has been generated and western blot analysis was conducted using anti-tub polyclonal antibody. RESULTS AND CONCLUSIONS: We found the presence of tubby protein both in normal weight and in obese subjects; however in the latter an isoelectric point shift toward the acidic end was observed.

Autoradiographic localization and binding study of benzodiazepines receptor sites in carp brain (Cyprinus carpio L.).

This study demonstrates, for the first time, by both autoradiography and binding assay that [3H]Ro 15-1788 binds to carp brain with a high degree of anatomical selectivity. Saturation binding of the radioligand was determined in seven anatomically defined regions and suggested the presence of one class of binding sites (Type I-lke). In general, there was a good correlation between the autoradiographic and the binding data. By far, the optic tectum and the vagal, facial, and glossopharyngeal lobes showed the majority of [3H]Ro 15-1788 binding sites. Low to negative concentration of binding sites was detected in the cerebellum. The location of [3H]Ro 15-1788 binding sites in particular brain regions, indicates that benzodiazepine receptors could be associated with pathways involved in the control of basic central functions as spatial learning acquisition and retention, and feeding behaviour.

[3H]PK11195 binding sites in human neutrophils: effect of fMLP stimulation and modulation in rheumatic diseases.

OBJECTIVE: The objectives of this study were to evaluate the [3H]PK11195 binding parameters in a model of acute inflammation, the N-formylmethionine-leucine-phenylalanine (fMLP)-stimulated neutrophil cell membranes, and to analyze if alterations of peripheral-type benzodiazepine receptor (PBR) characteristics occurred in neutrophil cell membranes of patients affected by osteoarthritis (OA), rheumatoid arthritis (RA), and psoriasic arthritis (PA). DESIGN AND METHODS: Neutrophils were obtained from 15 patients with OA, 15 patients with RA, and 15 patients with PA. fMLP stimulation was performed to aliquots of neutrophils from six healthy individuals. Evaluation of kinetic parameters of PBR was performed using [3H]PK11195, as specific radioligand compared with 15 healthy volunteers. RESULTS: The results showed a significant decrease of Kd and Bmax in fMLP-stimulated neutrophil membranes. Moreover, an increase of PBR binding sites and affinity value was observed in neutrophils membranes from PA patients. CONCLUSIONS: Our data suggested a fMLP modulation on [3H]PK11195 binding in human neutrophils. Moreover, our results showed an up-regulation of PBR in neutrophils of PA patients.

Altered amino acid homeostasis in subjects affected by fibromyalgia.

OBJECTIVES: To evaluate plasma amino acid (AA) concentrations in patients affected by fibromyalgia (FM) and to study the relationships between their levels and FM clinical parameters. DESIGN AND METHODS: 20 AAs were assessed in 34 FM patients and in 18 healthy volunteers by means of a modified version of the Waters picotag method. RESULTS: Significant lower plasma taurine, alanine, tyrosine (Tyr), valine, methionine, phenylalanine and threonine concentrations, and the sum of essential AAs were observed in FM patients vs healthy controls (P<0.05). Tyr CAA' ratio and the sum of AAs competing with tryptophan for brain uptake were significantly reduced in FM (P<0.05). A significant correlation was found between FM clinical parameters and certain AAs. CONCLUSIONS: Our results suggest probable defects of gut malabsorption of certain AAs in FM patients. Moreover, given the reduced Tyr CAA' ratio in FM patients, a possible impairment of the cathecolaminergic system in the FM syndrome may be suggested.

Species comparison of adenosine receptor subtypes in brain and testis.

We aimed at comparing the binding characteristics of adenosine A(1) and A(2A) receptors (A(1)Rs and A(2A)Rs) in high-expressing cerebral areas, the cortex and striatum respectively, of human, bovine and rat brain. Adenosine A(3) receptor (A(3)R) binding was studied in rat and bovine testis. Results confirmed species differences in AR saturation-displacement binding parameters. To investigate A(3)Rs in CNS, we carried out immunoblot in human brain, resolving two signals, a 52 KDa band with the highest density in hippocampus and a 48 KDa one, slightly more expressed in cortex. Subsequently, A(3)R binding was performed by [(125)I]-4-aminobenzyl-5'-N-methylcarboxamidoadenosine ([(125)I]-AB-MECA) in human hippocampus, revealing an high affinity population of sites and another non saturable component. [(125)I]-AB-MECA first site displacement by N(6 )(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 1,3-dipropyl-8-cyclopenthyl-xanthine (DPCPX) distinguished two affinity sites, being only in part identified as A(3)Rs. Therefore, A(3)Rs result clearly expressed by Western blot in human brain, but their full CNS characterization needs further investigation.

ATP, calcium and magnesium levels in platelets of patients with primary fibromyalgia.

OBJECTIVES: To evaluate the intracellular levels of the high energy adenosine triphosphate nucleotide ATP and essential divalent cations, calcium and magnesium, in platelets of patients affected by primary fibromyalgia syndrome (FMs). DESIGN AND METHOD: Platelet ATP and cation concentrations were measured in 25 patients affected by FMs and 25 healthy volunteers through a chemiluminescent and a fluorimetric assay, respectively. RESULTS: Significant lower ATP levels were observed inside platelets of FM patients (fmol ATP/plt: 0.0169+/-0.0012 vs. healthy controls, fmol ATP/plt: 0.0306+/-0.0023, mean+/-SEM) (*** P<0.0001). A trend towards higher calcium concentrations (P=0.06) together with significant increased magnesium levels were also reported in platelets of patients by comparison with controls (P=0.02). CONCLUSIONS: This preliminary study suggests that disturbances in the homeostasis of platelet ATP metabolism-signaling and calcium-magnesium flows might have a relevance in the pathogenesis of FMs.

Proteome analysis of whole saliva: a new tool for rheumatic diseases--the example of Sjögren's syndrome.

Sjögren's syndrome (pSS) is a systemic disease that affects salivary glands directly, and is therefore expected to influence the composition of human whole saliva (WS) fluid. The aim of this study was to characterize the WS proteins of pSS patients using a proteomic approach to assess a valid procedure to examine the global changes of the salivary protein profiles in connective tissue disorders. The WS proteins expressed in patients affected by pSS and healthy volunteers were analyzed using the 2-DE technique. The WS protein pattern was altered in pSS patients compared to controls, with a decrease in some of the typical salivary proteins. Particularly, a remarkable alteration of carbonic anhydrase VI was observed. Moreover, a comparison of WS protein profile of pSS patients with the one obtained from controls revealed a set of differentially expressed proteins. These proteins were related to acute and chronic inflammation while some others were involved in oxidative stress injury. These findings are in line with the systemic immuno-inflammatory aspects of pSS and open the possibility for a systematic search of diagnostic biomarkers and targets for therapeutic intervention in pSS.

[3H] muscimol receptors sites in the carp (Cyprinus carpio L.) brain: binding assay and autoradiographic distribution.

Autoradiographic and binding techniques were used to study the presence of [(3)H]muscimol receptors sites in the carp brain. The radioligand was distributed with an high degree of anatomical selectivity. We found abundant labelling in the cerebellum, in the nucleus diffusus lobi inferioris, and in the torus longitudinalis. No labelling was detected within the epithalamus, thalamus and hypothalamus, while the telencephalon and the rhombencephalon displayed a low density of [(3)H]muscimol receptors sites. Binding assay showed the highest concentration of receptor sites in the nucleus diffusus lobi inferioris and the lowest in the medulla oblongata. Presence of [(3)H]muscimol binding sites within the visceral sensory area was noted. The rank order of displacement efficacy of unlabelled ligands observed, suggested that in brain membranes of carp the receptor binding of [(3)H]muscimol has the same pharmacological specificity previously reported in a large number of experiments with tissue homogenates. A general agreement in binding and autoradiography was observed. The present findings suggested that muscimol receptor could be involved in neuronal pathway controlling basic central actions like gustatory signal processing or spatial learning acquisition and retention.

Specific proteins identified in whole saliva from patients with diffuse systemic sclerosis.

OBJECTIVE: To evaluate the global changes of salivary protein profiles in patients with systemic sclerosis (SSc) using a proteomic approach. METHODS: Whole saliva (WS) was collected from 15 patients with diffuse SSc and 15 healthy volunteers. Protein expression profiles for each sample were generated by 2-dimensional gel electrophoresis, and protein spots of interest were identified using peptide mass fingerprinting. RESULTS: The level of all the most representative salivary proteins except keratin 6L remained unchanged and only qualitative differences were observed between control subjects and patients with SSc. A total of 19 spots were found in SSc that were not matched with the controls. Fourteen out of a total of 19 spots were identified by mass analysis and were found to collapse into 9 unique proteins. These spots were identified to be cyclophilin A, calgranulin B, psoriasin, beta2-microglobulin, calgranulin A, glyceraldehyde-3-phosphate dehydrogenase, triose phosphate isomerase (TPI), actin-related protein 2/3 complex subunit 2 (Arp2/3 complex), and cystatin B. CONCLUSION: Our study is the first reporting the WS protein pattern of patients with SSc and comparing the differences between WS of patients with SSc and WS of healthy subjects. Both previously identified and newly identified proteins were detected in WS using a proteomic approach. Some of these proteins, like keratin 6L, psoriasin, TPI, and Arp2/3 complex, might have a pathological significance for SSc. It is possible that some of them can be defined as new therapeutic targets or diagnostic markers for SSc disease.

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.

Autoradiographic distribution of neuropeptide Y binding sites in the brain of the carp Cyprinus carpio L. (Cyprinidae, Teleostei).

The present study reports the distribution of neuropeptide Y (NPY)-binding sites in the brain of the adult carp Cyprinus carpio L. Radioiodinated NPY was used as tracer in the autoradiographic procedure. The NPY-binding sites (NPY-bs) were widely distributed in the carp brain. Generally, a good match was observed between the distribution of NPY-bs and the distribution of NPY-immunoreactive (NPY-ir) elements previously reported in the forebrain of the carp. Low to moderate concentration of NPY-bs were found in the telencephalon, this finding indicates that NPY may play a role in the processing of olfactory inputs and in more complex behaviours like spatial learning acquisition and retention, whose importance could correlated with similar results obtained in mammals. Moreover, in the rhombencephalon, the presence of NPY-bs at level of lobus vagus and the lobus facialis suggests that NPY may be implicated in food-seeking behaviour and swallowing reflex.