Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation

Grape berries of Muscat cultivars (Vitis vinifera L.) contain high levels of monoterpenols and exhibit a distinct aroma related to this composition of volatiles. A structural gene of the plastidial methyl-erythritol-phosphate (MEP) pathway, 1-deoxy-D-xylulose 5-phosphate synthase (VvDXS), was recently suggested as a candidate gene for this trait, having been co-localized with a major quantitative trait locus for linalool, nerol, and geraniol concentrations in berries. In addition, a structured association study discovered a putative causal single nucleotide polymorphism (SNP) responsible for the substitution of a lysine with an asparagine at position 284 of the VvDXS protein, and this SNP was significantly associated with Muscat-flavoured varieties. The significance of this nucleotide difference was investigated by comparing the monoterpene profiles with the expression of VvDXS alleles throughout berry development in Moscato Bianco, a cultivar heterozygous for the SNP mutation. Although correlation was detected between the VvDXS transcript profile and the accumulation of free monoterpenol odorants, the modulation of VvDXS expression during berry development appears to be independent of nucleotide variation in the coding sequence. In order to assess how the non-synonymous mutation may enhance Muscat flavour, an in vitro characterization of enzyme isoforms was performed followed by in vivo overexpression of each VvDXS allele in tobacco. The results showed that the amino acid non-neutral substitution influences the enzyme kinetics by increasing the catalytic efficiency and also dramatically affects monoterpene levels in transgenic lines. These findings confirm a functional effect of the VvDXS gene polymorphism and may pave the way for metabolic engineering of terpenoid contents in grapevine

Cooperative Induction of a Tolerogenic Dendritic Cell Phenotype by Cytokines Secreted by Pancreatic Carcinoma Cells

AbstractAg presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123− myeloid DC (MDC)) or immunosuppressive T cell development (CD11c−,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-γ. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-β, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses

Antagonistic interactions between gemcitabine and 5-fluorouracil in the human pancreatic carcinoma cell line Capan-2.

Although the recently-developed Gemcitabine (GEM) has renewed interest in clinical research in pancreatic carcinoma, it offers modest improvement of tumor-related symptoms and marginal survival advantage, even when combined with other currently-available chemotherapeutic agents such as 5-Fluorouracil (5-FU). We hypothesized that this disappointing result could be due to an interaction between the two drugs affecting cytotoxic activity. We measured in-vitro growth inhibition, cell cycle distribution, gene and protein expression of apoptosis regulators bcl-2, bcl-x and survivin, NFkappaB and telomerase activities of human pancreatic carcinoma cell line Capan-2 following exposure to GEM and 5-FU singly or combined, by MTT assay and median effect analysis, flow cytometry, real-time RT-PCR, Western blotting, electrophoretic mobility shift assay (EMSA) and telomeric repeat amplification protocol (TRAP) assay, respectively. We found cell growth to be inhibited by both drugs, decreasing the percentage of cells in S and G2/M phases and inducing apoptosis, dependent on the levels of bcl-2, bcl-xL and survivin expression in the case of 5-FU, but not for GEM. Moreover, while telomerase activity was reduced equally by both drugs, 5-FU but not GEM effectively downregulated NFkappaB binding activity. Intriguingly, a substantial antagonistic effect was noticed when GEM was combined with 5-FU in the concentration range tested, with the exception of the TRAP assay. These indications of an antagonistic interaction between GEM and 5-FU in some pancreatic cancer context urge further investigation of both genetic and non-genetic differences to identify the variables most relevant for optimal selection and dosing of treatment for the individual patient