Development of a molecular method for the rapid screening and identification of the three functionally relevant polymorphisms in the human TAS2R38 receptor gene in studies of sensitivity to the bitter taste of PROP

The objective of this work was to develop a rapid screening method to identify the three single nucleotide polymorphisms (SNPs) in the TAS2R38 gene, with the aim of providing a significant contribution to studies designed to assess sensitivity to the bitter taste of 6-n-propylthiouracil (PROP). Specifically, the objective of this study was to characterize the TAS2R38 gene haplotypes in a group of 60 subjects with variable sensitivity to PROP and preliminarily genotyped for the rs2274333 allele (A/G) of carbonic anhydrase isoform VI gene (CA6). The molecular characterization of the TAS2R38 gene was conducted using the PCR-restriction fragment length polymorphism technique after creating artificial restriction sites upstream or downstream of the SNPs, as none of the three polymorphisms contributes to the formation of a restriction site for a specific endonuclease. The results indicate that the method described in this paper could be a valid and simple experimental strategy to identify genetic differences related to taste sensitivity to bitter taste, and could be applied as a nutrigenetics test in studies aimed at understanding people’s eating behaviors

The gustin (CA6) gene polymorphism, rs2274333 (A/G), as a mechanistic link between PROP tasting and fungiform taste papilla density and maintenance

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 stimul

molecular analysis of a copper and zinc containing superoxide dismutase gene isolated from the latex of euphorbia characias another piece in the molecular puzzle of euphorbiaceae latex proteins

A copper- and zinc-containing superoxide dismutase (Cu/Zn-SOD) cDNA was isolated from the Euphorbia characias latex (Elx) using consensus degenerate hybrid oligonucleotide primer (CODEHOP) design and RT-PCR strategy. Both 3'and 5'untraslated regions (UTR) were isolated by rapid amplification of cDNA ends (RACE) method. Analysis of the nucleotide sequence revealed that the Cu/Zn-SOD cDNA contains an open reading frame encoding a protein of 152 amino acids. Bioinformatic analyses of Elx SOD gene revealed a high identity rate with a large number of plant Cu/Zn-SODs in the deduced amino acid sequence. Since isoenzymes may be generated through the multiplicity of SOD genes or result from post-trascriptional events, genomic Southern blot in conjunction with northern blot experiments were also performed. The genomic analysis showed that the E. characias genome contains a single SOD gene. Northern blot analyses confirmed the presence of a single SOD mRNA demonstrating that alternative splicing does not occur. Quantita- tive real time-PCR (qRT-PCR) experiments showed that SOD gene expression in latex reaches maximum levels during the summer. These results suggest that the Cu/Zn-SOD of Euphorbia characias latex probably may be involved in the antioxid- ative process triggered by oxidative stress induced by the conditions of environmental change in which the plant lives

Ceruloplasmin Protects Against Rotenone-Induced Oxidative Stress and Neurotoxicity

To clarify the neuroprotective property of ceruloplasmin and the pathogenesis of aceruloplasminemia, we generated ceruloplasmin-deficient (CP−/−) mice on the C57BL/10 genetic background and further treated them with a mitochondrial complex I inhibitor, rotenone. There was no iron accumulation in the brains of CP−/− mice at least up to 60 weeks of age. Without rotenone treatment, CP−/− mice showed slight motor dysfunction compared with CP+/+ mice, but there were no detectable differences in the levels of oxidative stress markers between these two groups. A low dose of rotenone did not affect the mitochondrial complex I activity in our mice, however, it caused a significant change in motor behavior, neuropathology, or the levels of oxidative stress markers in CP−/− mice, but not in CP+/+ mice. Our data support that ceruloplasmin protects against rotenone-induced oxidative stress and neurotoxicity, probably through its antioxidant properties independently of its function of iron metabolism

Protective Effect of Tetrahydroxystilbene Glucoside on 6-OHDA-Induced Apoptosis in PC12 Cells through the ROS-NO Pathway

Oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, such as Parkinson's disease. The molecule, 2,3,5,4′-tetrahydr- oxystilbene-2-O-β-D-glucoside (TSG), is a potent antioxidant derived from the Chinese herb, Polygonum multiflorum Thunb. In this study, we investigated the protective effect of TSG against 6-hydroxydopamine-induced apoptosis in rat adrenal pheochromocytoma PC12 cells and the possible mechanisms. Our data demonstrated that TSG significantly reversed the 6-hydroxydopamine-induced decrease in cell viability, prevented 6-hydroxydopamine-induced changes in condensed nuclei and decreased the percentage of apoptotic cells in a dose-dependent manner. In addition, TSG slowed the accumulation of intracellular reactive oxygen species and nitric oxide, counteracted the overexpression of inducible nitric oxide syntheses as well as neuronal nitric oxide syntheses, and also reduced the level of protein-bound 3-nitrotyrosine. These results demonstrate that the protective effects of TSG on rat adrenal pheochromocytoma PC12 cells are mediated, at least in part, by the ROS-NO pathway. Our results indicate that TSG may be effective in providing protection against neurodegenerative diseases associated with oxidative stress