Taste sensitivity to PROP varies greatly among individuals and is associated with polymorphisms in the bitter receptor gene TAS2R38, and with differences in fungiform papilla density on the anterior tongue surface. Recently we showed that the PROP non-taster phenotype is strongly associated with the G variant of polymorphism rs2274333 (A/G) of the gene that controls the salivary trophic factor, gustin. The aims of this study were 1) to investigate the role of gustin gene polymorphism rs2274333 (A/G), in PROP sensitivity and fungiform papilla density and morphology, and 2) to investigate the effect of this gustin gene polymorphism on cell proliferation and metabolic activity. Sixty-four subjects were genotyped for both genes by PCR techniques, their PROP sensitivity was assessed by scaling and threshold methods, and their fungiform papilla density, diameter and morphology were determined. In vitro experiments examined cell proliferation and metabolic activity, following treatment with saliva of individuals with and without the gustin gene mutation, and with isolated protein, in the two iso-forms. Gustin and TAS2R38 genotypes were associated with PROP threshold (p=0.0001 and p=0.0042), but bitterness intensity was mostly determined by TAS2R38 genotypes (p<0.000001). Fungiform papillae densities were associated with both genotypes (p<0.014) (with a stronger effect for gustin; p=0.0006), but papilla morphology was a function of gustin alone (p<0.0012). Treatment of isolated cells with saliva from individuals with the AA form of gustin or direct application of the active iso-form of gustin protein increased cell proliferation and metabolic activity (p<0.0135). These novel findings suggest that the rs2274333 polymorphism of the gustin gene affects PROP sensitivity by acting on fungiform papilla development and maintenance, and could provide the first mechanistic explanation for why PROP super-tasters are more responsive to a broad range of oral stimuli.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor ␣ (TNF␣)-inducible NO synthesis, because by affecting NF-B activation it inhibits inducible Ca 2؉ -independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/ TNF␣-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF␣ induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/ TNF␣-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF␣-elicited NF-B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-B availability. Nitric oxide (NO)1 is a major messenger molecule playing key roles in many physiological and pathological processes (1). NO production is catalyzed by at least two major forms of the NO synthase (NOS) enzyme: a constitutive Ca 2ϩ -dependent NOS isoform (cNOS) and an inducible Ca 2ϩ -independent NOS isoform (iNOS), which is expressed after stimulation with Escherichia coli lipopolysaccharide (LPS) and cytokines. Recently, we have demonstrated that LPS and/or TNF␣ are able to induce iNOS in human ramified microglia leading to a high NO output (2). On the other hand, NO release from mouse microglia is thought to play an important role in neuronal cell death (3-5). In a recent work, Meda et al. (6) suggested a possible involvement of NO produced by rat microglia after activation with ␤-amyloid protein and IFN-␥ in the pathogenesis of neuronal degradation occurring with age and in Alzheimer's disease.Preserving iNOS gene from its undesirable induction may be important for neuronal survival. Down-regulation of iNOS expression was reported to be achieved by some factors such as dexamethasone, interleukin-4, transforming growth factor-␤, and basic fibroblast growth factor (7-9). Recently, Griscavage et al. However, little is known about the regulatory effects on the mechanism by the variable low concentrations of the available NO before iNOS induction. Recently, we have observed that sodium nitroprusside (SNP), a well known NO donor, elicited inhibition of LPS-induced iNOS expression in rat neutrophils, suggesting a possible suppressive effect on iNOS gene expression by exogenous NO (12). The promoter region of human iNOS gene in vascular smooth muscle cells was shown to contain the consensus sequences for the binding of NFB, a nuclear transcriptional factor (13), and iNOS transc...

Various cells types, including stem and progenitor cells, can exchange complex information via plasma membrane-derived vesicles, which can carry signals both in their limiting membrane and lumen. Astrocytes, traditionally regarded as mere supportive cells, play previously unrecognized functions in neuronal modulation and are capable of releasing signalling molecules of different functional significance. In the present study, we provide direct evidence that human fetal astrocytes in culture, expressing the same feature as immature and reactive astrocytes, release membrane vesicles larger than the microvesicles described up to now. We found that these large vesicles, ranging from 1-5 to 8 μm in diameter and expressing on their surface β1-integrin proteins, contain mitochondria and lipid droplets together with ATP. We documented vesicle content with fluorescent-specific dyes and with the immunocytochemistry technique we confirmed that mitochondria and lipid droplets were co-localized in the same vesicle. Scanning electron microscopy and transmission electron microscopy confirmed that astrocytes shed from surface membrane vesicles of the same size as the ones detected by fluorescence microscopy. Our results report for the first time that cultured astrocytes, activated by repetitive stimulation of ATP released from neighboring cells, shed from their surface large membrane vesicles containing mitochondria and lipid droplets.

TAR deoxyribonucleic acid-binding protein 43 (TDP-43) is a key protein in the pathogenesis of amyoptrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Recent studies suggest that mutations in the TDP-43 coding gene, TARDBP, as well as variations in TDP-43 protein expression levels may disrupt the dynamics of stress granules (SGs). However, it remains unclear whether the pathogenetic effect of the TDP-43 protein is exerted at the cytoplasmic level, through direct participation to SG composition, or at nuclear level, through control of proteins essential to SG assembly. To clarify this point, we investigated the dynamics of SG formation in primary skin fibroblast cultures from the patients with ALS together with the A382T mutation and the patients with ALS and healthy controls with wild-type TDP-43. Under stress conditions induced by sodium arsenite, we found that in human fibroblasts TDP-43 did not translocate to the SGs but instead contributed to the SG formation through a regulatory effect on the G3BP1 core protein. We found that the A382T mutation caused a significant reduction in the number of SGs per cell (P < 0.01) as well as the percentage of cells that form SGs (P < 0.00001). Following stress stimuli, a significant decrease of viability was observed for cells with the TDP-43 A382T mutation (P < 0.0005). We can therefore conclude that the A382T mutation caused a reduction in the ability of cells to respond to stress through loss of TDP-43 function in SG nucleation. The pathogenetic action revealed in our study model does not seem to be mediated by changes in the localization of the TDP-43 protein.

Mesoporous silica nanoparticles (MSNs), based on the MCM-41 matrix, were functionalized with amino groups, and then with hyaluronic acid (HA) or chitosan (CHIT) to fabricate bioactive conjugates. The role of the functional groups toward cytotoxicity and cellular uptake was investigated using 3T3 mouse fibroblast cells. A very high biocompatibility of MSN-NH2, MSN-HA and MSN-CHIT matrices was assessed through the MTS biological assay and Coulter counter evaluation. No significant differences in cytotoxicity data arise from the presence of different functional groups in the investigated MSNs. Fluorescence microscopy experiments performed using fluorescein isothiocyanate-conjugated MSN-NH2, MSN-HA, and MSN-CHIT, and transmission electron microscopy experiments performed on slices of the investigated systems embedded in epoxy resins give evidence of significant differences due to type of functionalization in terms of cellular uptake and stability of the particles in the biological medium. MSN-NH2 and MSN-HA conjugates are easily internalized, the uptake of the HA-functionalized MSNs being much higher than that of the -NH2-functionalized MSNs. Differently, MSN-CHIT conjugates tend to give large aggregates dispersed in the medium or localized at the external surface of the cell membranes. Both fluorescence microscopy and TEM images show that the MSNs are distributed in the cytoplasm of the cells in the case of MSN-NH2 and MSN-HA, whereas only a few particles are internalized in the case of MSN-CHIT. Flow cytometry experiments confirmed quantitatively the selectively high cellular uptake of MSN-HA particles.