High-refined carbohydrate diet consumption induces neuroinflammation and anxiety-like behavior in mice.

Consumption of poor nutrients diets is associated with fat tissue expansion and with a central and peripheral low-grade inflammation. In this sense, the microglial cells in the central nervous system are activated and release pro-inflammatory cytokines that up-regulate the inducible nitric oxide synthase (iNOS), promoting Nitric Oxide (NO) production. The excess of NO has been proposed to facilitate anxious states in humans and rodents. We evaluated whether consumption of a high-refined carbohydrate-containing diet (HC) in mice induced anxiety-like behavior in the Novelty Suppressed Feeding Test (NFST) trough facilitation of NO, in the prefrontal cortex (PFC) and hippocampus (HIP). We also verified if HC diet induces activation of microglial cells, alterations in cytokine and leptin levels in such regions. Male BALB/c mice received a standard diet or a HC diet for 3 days or 12 weeks. The chronic consumption of HC diet, but not acute, induced an anxiogenic-like effect in the NSF test and an increase in the nitrite levels in the PFC and HIP. The preferential iNOS inhibitor, aminoguanidine (50 mg/kg, i.p.), attenuated such effects. Moreover, microglial cells in the HIP and PFC were activated after chronic consumption of HC diet. Finally, the expression of iNOS in the PFC and TNF, IL6 and leptin levels in HIP were higher in chronically HC fed mice. Taken together, our data reinforce the notion that diets containing high-refined carbohydrate facilitate anxiety-like behavior, mainly after a long period of consumption. The mechanisms involve, at least in part, the augmentation of neuroinflammatory processes in brain areas responsible for anxiety control.

Selection and validation of reference genes for quantitative gene expression analyses in various tissues and seeds at different developmental stages in Bixa orellana L.

Bixa orellana L., popularly known as annatto, produces several secondary metabolites of pharmaceutical and industrial interest, including bixin, whose molecular basis of biosynthesis remain to be determined. Gene expression analysis by quantitative real-time PCR (qPCR) is an important tool to advance such knowledge. However, correct interpretation of qPCR data requires the use of suitable reference genes in order to reduce experimental variations. In the present study, we have selected four different candidates for reference genes in B. orellana, coding for 40S ribosomal protein S9 (RPS9), histone H4 (H4), 60S ribosomal protein L38 (RPL38) and 18S ribosomal RNA (18SrRNA). Their expression stabilities in different tissues (e.g. flower buds, flowers, leaves and seeds at different developmental stages) were analyzed using five statistical tools (NormFinder, geNorm, BestKeeper, ΔCt method and RefFinder). The results indicated that RPL38 is the most stable gene in different tissues and stages of seed development and 18SrRNA is the most unstable among the analyzed genes. In order to validate the candidate reference genes, we have analyzed the relative expression of a target gene coding for carotenoid cleavage dioxygenase 1 (CCD1) using the stable RPL38 and the least stable gene, 18SrRNA, for normalization of the qPCR data. The results demonstrated significant differences in the interpretation of the CCD1 gene expression data, depending on the reference gene used, reinforcing the importance of the correct selection of reference genes for normalization.

Unraveling new genes associated with seed development and metabolism in Bixa orellana L. by expressed sequence tag (EST) analysis.

The tropical tree Bixa orellana L. produces a range of secondary metabolites which biochemical and molecular biosynthesis basis are not well understood. In this work we have characterized a set of ESTs from a non-normalized cDNA library of B. orellana seeds to obtain information about the main developmental and metabolic processes taking place in developing seeds and their associated genes. After sequencing a set of randomly selected clones, most of the sequences were assigned with putative functions based on similarity, GO annotations and protein domains. The most abundant transcripts encoded proteins associated with cell wall (prolyl 4-hydroxylase), fatty acid (acyl carrier protein), and hormone/flavonoid (2OG-Fe oxygenase) synthesis, germination (MADS FLC-like protein) and embryo development (AP2/ERF transcription factor) regulation, photosynthesis (chlorophyll a-b binding protein), cell elongation (MAP65-1a), and stress responses (metallothionein- and thaumatin-like proteins). Enzymes were assigned to 16 different metabolic pathways related to both primary and secondary metabolisms. Characterization of two candidate genes of the bixin biosynthetic pathway, BoCCD and BoOMT, showed that they belong, respectively, to the carotenoid-cleavage dioxygenase 4 (CCD4) and caffeic acid O-methyltransferase (COMT) families, and are up-regulated during seed development. It indicates their involvement in the synthesis of this commercially important carotenoid pigment in seeds of B. orellana. Most of the genes identified here are the first representatives of their gene families in B. orellana.

Isolation and purification of RNA from tissues rich in polyphenols, polysaccharides, and pigments of annatto (Bixa orellana L.).

The tropical plant Bixa orellana L. (annatto) produces an array of natural products, including the pigment bixin used in the food and cosmetics industries. In order to understand the biochemical and molecular basis of the biosynthesis of these natural products, a reliable method for isolating high yields of high-quality RNA is required. Here we described a successful and reproducible method for isolation and purification of high-quantity and high-quality RNA from different tissues of annatto. This protocol overcomes the usual problems associated with large amounts of polyphenols, polysaccharides, pigments, and other secondary metabolites that are not easily removed by conventional extraction procedures. Furthermore, the proposed protocol can be easily carried out in any laboratory and it could also be extended to isolate RNA from other plant species showing similar abundance of compounds that interfere with RNA extractions. The yield and quality of the RNA were monitored by spectrophotometric analysis, separation on agarose gel, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), and construction of a cDNA library.