Talía del Pozo

Enhancement in liver SREBP-1c/PPAR-α ratio and steatosis in obese patients: Correlations with insulin resistance and n-3 long-chain polyunsaturated fatty acid depletion

Sterol receptor element-binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor-alpha (PPAR-alpha) mRNA expression was assessed in liver as signaling mechanisms associated with steatosis in obese patients. Liver SREBP-1c and PPAR-alpha mRNA (RT-PCR), fatty acid synthase (FAS) and carnitine palmitoyltransferase-1a (CPT-1a) mRNA (real-time RT-PCR), and n-3 long-chain polyunsaturated fatty acid (LCPUFA)(GLC) contents, plasma adiponectin levels (RIA), and insulin resistance (IR) evolution (HOMA) were evaluated in 11 obese patients who underwent subtotal gastrectomy with gastro-jejunal anastomosis in Roux-en-Y and 8 non-obese subjects who underwent laparoscopic cholecystectomy (controls). Liver SREBP-1c and FAS mRNA levels were 33% and 70% higher than control values (P<0.05), respectively, whereas those of PPAR-alpha and CPT-1a were 16% and 65% lower (P<0.05), respectively, with a significant 62% enhancement in the SREBP-1c/PPAR-alpha ratio. Liver n-3 LCPUFA levels were 53% lower in obese patients who also showed IR and hipoadiponectinemia over controls (P<0.05). IR negatively correlated with both the hepatic content of n-3 LCPUFA (r=-0.55; P<0.01) and the plasma levels of adiponectin (r=-0.62; P<0.005). Liver SREBP-1c/PPAR-alpha ratio and n-3 LCPUFA showed a negative correlation (r=-0.48; P<0.02) and positive associations with either HOMA (r=0.75; P<0.0001) or serum insulin levels (r=0.69; P<0.001). In conclusion, liver up-regulation of SREBP-1c and down-regulation of PPAR-alpha occur in obese patients, with enhancement in the SREBP-1c/PPAR-alpha ratio associated with n-3 LCPUFA depletion and IR, a condition that may favor lipogenesis over FA oxidation thereby leading to steatosis.

Gene expression profiling analysis of copper homeostasis in Arabidopsis thaliana

As a result of copper essentiality for life, plants and most other organisms have developed a conserved and complex network of proteins to handling Cu in order to prevent its deficit and to avoid its potentially toxic effects. To better understand regulation of Cu homeostasis in plants, we use adult plant of Arabidopsis thaliana to provide an integrated view of how Cu status affects the expression of genes involved in cellular Cu homoeostasis. In doing so, we use real-time RT-PCR to compare shoot and roots transcriptional responses to Cu. We measure changes in the abundance of transcripts encoding transporters, chaperones and P-type ATPases and correlated those changes with variation of Cu content in both tissues. Our results indicated that in both tissues transcript levels of COPT2, 4, and ZIP2 transporters and CCH chaperone were significantly down-regulated comparing to controls plants in response to Cu excess. In contrast, Cu chaperones ATX1, CCS, COX17-1 including two putative mitochondrial chaperones (At3g08950; At1g02410) were up-regulated under similar conditions. Regarding P-type ATPases, a reduction of HMA1, PAA1, PAA2, and RAN1 transcript levels in shoot after Cu exposure was observed, while HMA5 transcripts increased exclusively in roots. In plants growing under Cu-deficient conditions, COPT2, ZIP2, HMA1, andPAA2, were significantly up-regulated in shoots. Thus, our results indicated a common transcriptional regulation pattern of transporters and chaperone components, in particular transcriptional changes of COPT2, ZIP2, and CCH showed an inverse relation with Cu content suggesting that these proteins are required to avoid excess and deficit of Cu.

Alteration of Gene Expression Profile in Niemann-Pick Type C Mice Correlates with Tissue Damage and Oxidative Stress

Background Niemann-Pick type C disease (NPC) is a neurovisceral lipid storage disorder mainly characterized by unesterified cholesterol accumulation in lysosomal/late endosomal compartments, although there is also an important storage for several other kind of lipids. The main tissues affected by the disease are the liver and the cerebellum. Oxidative stress has been described in various NPC cells and tissues, such as liver and cerebellum. Although considerable alterations occur in the liver, the pathological mechanisms involved in hepatocyte damage and death have not been clearly defined. Here, we assessed hepatic tissue integrity, biochemical and oxidative stress parameters of wild-type control (Npc1 +/+; WT) and homozygous-mutant (Npc1 −/−; NPC) mice. In addition, the mRNA abundance of genes encoding proteins associated with oxidative stress, copper metabolism, fibrosis, inflammation and cholesterol metabolism were analyzed in livers and cerebella of WT and NPC mice. Methodology/Principal Findings We analyzed various oxidative stress parameters in the liver and hepatic and cerebellum gene expression in 7-week-old NPC1-deficient mice compared with control animals. We found signs of inflammation and fibrosis in NPC livers upon histological examination. These signs were correlated with increased levels of carbonylated proteins, diminished total glutathione content and significantly increased total copper levels in liver tissue. Finally, we analyzed liver and cerebellum gene expression patterns by qPCR and microarray assays. We found a correlation between fibrotic tissue and differential expression of hepatic as well as cerebellar genes associated with oxidative stress, fibrosis and inflammation in NPC mice. Conclusions/Significance In NPC mice, liver disease is characterized by an increase in fibrosis and in markers associated with oxidative stress. NPC is also correlated with altered gene expression, mainly of genes involved in oxidative stress and fibrosis. These findings correlate with similar parameters in cerebellum, as has been previously reported in the NPC mice model.

Identification of reference genes for quantitative real-time PCR studies in human cell lines under copper and zinc exposure

Accurate quantification depends on normalization of the measured gene expression data. In particular, gene expression studies with exposure to metals are challenging due their toxicity and redox-active properties. Here, we assessed the stability of potential reference genes in three cell lines commonly used to study metal cell metabolism: Caco-2 (colon), HepG2 (liver) and THP-1 (peripheral blood) under copper (Cu) or zinc (Zn) exposure. We used combined statistical tools to identify the best reference genes from a set of eleven candidates, which included traditional “housekeeping” genes such as GAPDH and B-ACTIN, in cell lines exposed to high and low, Zn and Cu concentrations. The expression stabilities of ATP5B (ATP synthase) and CYC1 (subunits of the cytochrome) were the highest considering the effect of Zn and Cu treatments whereas SDHA (succinate dehydrogenase) was found to be the most unstable gene. Even though the transcriptional effect of Zn and Cu is very different in term of redox properties, the same best reference genes were identified when Zn or Cu treatments were analyzed together. Our results indicate that ATP5B/CYC1 are the best candidates for reference genes after metal exposure, which can be used as a suitable starting point to evaluate gene expression with other metals or in different cell types in human models.

The complete mitochondrial genome of the Chilean endemic frog Telmatobius chusmisensis Formas, Cuevas & Nuñez, 2006 (Anura, Telmatobiidae)

Abstract Telmatobius chusmisensis is an endemic frog of northern Chile that is only known at its type locality, Chusmisa. In this study, the complete mitochondrial genome of Telmatobius chusmisensis was assembled using high-throughput sequencing data, yielding a circular genome of 19 312 bp with a nucleotide composition of A = 30.8%, C = 24.4%, G = 13.6% and T = 31.2%. Its gene composition and structure were similar to other anuran genomes available: 13 protein-coding genes, two rRNA genes, 22 tRNA genes and the D-loop region. Phylogenetic analysis using Bayesian inference (BI) and maximum likelihood (ML) showed that Telmatobius chusmisensis, T. bolivianus and T. vellardi are a highly supported monophyletic group. This genome information will allow us to gain a better understanding into phylogenetic and phylogeographic relationships in the genus Telmatobius.