Validation of endogenous control genes in human adipose tissue: relevance to obesity and obesity-associated type 2 diabetes mellitus.

The aim of the present study was to test the influence of obesity and the presence of type 2 diabetes mellitus (T2DM) on the expression of ten housekeeping genes and of the 18S rRNA in a group of human adipose tissue samples from the omental and subcutaneous depot. Adipose tissue biopsies were obtained by laparoscopic surgery from lean and obese patients. After the extraction, mRNA levels in adipose tissue samples were quantified by real-time PCR using the commercial HUMAN ENDOGENOUS CONTROL PLATES. From the genes analyzed, 18S rRNA exhibited the most stable expression levels in both depots regardless of the pathophysiological conditions of obesity and obesity-associated T2DM. Contrarily, GAPD was the gene with the highest variation in its expression levels, being upregulated (8.0-fold) in the obese group and downregulated (3.5-fold) in obesity-associated T2DM. Our results show that 18S rRNA may be the most suitable gene for normalization in expression studies performed in human adipose tissue samples obtained from patients suffering from obesity and/or obesity-associated T2DM, whereas GAPD is less appropriate for comparison purposes under these circumstances.     

Microzooplankton grazing of phytoplankton in a tropical upwelling region

We measured grazing by herbivorous zooplankton (<200 μm fraction) in coastal and slope regions of the South Brazil Bight. Using the dilution technique, we performed nine experiments during the austral summer, when nutrient-rich South Atlantic Central Water is present on the shelf, and five during winter. These experiments provide the first estimates of microzooplankton grazing in the western South Atlantic Ocean. Model II regression showed a strong relationship between phytoplankton intrinsic growth rates and grazing, with a slope of 0.64 (±0.28; 95% confidence interval) indicating that microzooplankton grazing could account for the majority of phytoplankton mortality. Both phytoplankton growth and microzooplankton grazing were higher during the summer upwelling season, compared to winter. For the two experiments that were conducted in oligotrophic slope water, grazing accounted for >80% of phytoplankton production. A comparison of incubations with and without added inorganic nutrients showed no consistent stimulation of phytoplankton growth (slope of enriched versus unenriched treatments not significantly different from 1). Estimates from microscopic counts of heterotrophic organisms >10 μm indicated that copepod nauplii comprised the largest share of the microzooplankton biomass (mean 62.4 ± 5.8% SE). Grazing estimates were not correlated with microzooplankton biomass, whether or not nauplii were included, suggesting that most of the grazing was done by nano-sized zooplankton. Electronic Supplementary Material Electronic supplementary material is available in the online version of this article at and is accessible for authorized users. Handling editor: S. Wellekens     

Electron transport chain of Saccharomyces cerevisiae mitochondria is inhibited by H2O2 at succinate-cytochrome c oxidoreductase level without lipid peroxidation involvement

The deleterious effects of H₂O₂ on the electron transport chain of yeast mitochondria and on mitochondrial lipid peroxidation were evaluated. Exposure to H₂O₂ resulted in inhibition of the oxygen consumption in the uncoupled and phosphorylating states to 69% and 65%, respectively. The effect of H₂O₂ on the respiratory rate was associated with an inhibition of succinate-ubiquinone and succinate-DCIP oxidoreductase activities. Inhibitory effect of H₂O₂ on respiratory complexes was almost completely recovered by β-mercaptoethanol treatment. H₂O₂ treatment resulted in full resistance to Q_O site inhibitor myxothiazol and thus it is suggested that the quinol oxidase site (Q_O) of complex III is the target for H₂O₂. H₂O₂ did not modify basal levels of lipid peroxidation in yeast mitochondria. However, H₂O₂ addition to rat brain and liver mitochondria induced an increase in lipid peroxidation. These results are discussed in terms of the known physiological differences between mammalian and yeast mitochondria.     

Cooperative propagation of local stability changes from low-stability and high-stability regions in a SH3 domain

Site-directed mutagenesis has been used to produce local stability changes at two regions of the binding site surface of the alpha-spectrin SH3 domain (Spc-SH3) differing in their intrinsic stability. Mutations were made at residue 56, located at the solvent-exposed side of the short 3(10) helix, and at residue 21 in the tip of the flexible RT-loop. NMR chemical-shift analysis and X-ray crystallography indicated negligible changes produced by the mutations in the native structure limited to subtle rearrangements near the mutated residue and at flexible loops. Additionally, mutations do not alter importantly the SH3 binding site structure, although produce significant changes in its affinity for a proline-rich decapeptide. The changes in global stability measured by differential scanning calorimetry are consistent the local energy changes predicted by theoretical models, with the most significant effects observed for the Ala-Gly mutations. Propagation of the local stability changes throughout the domain structure has been studied at a per-residue level of resolution by NMR-detected amide hydrogen-deuterium exchange (HX). Stability propagation is remarkably efficient in this small domain, apparently due to its intrinsically low stability. Nevertheless, the HX-core of the domain is not fully cooperative, indicating the existence of co-operative subunits within the core, which is markedly polarized. An equilibrium phi-analysis of the changes in the apparent Gibbs energies of HX per residue produced by the mutations has allowed us to characterize structurally the conformational states leading to HX. Some of these states resemble notably the folding transition state of the Spc-SH3 domain, suggesting a great potential of this approach to explore the folding energy landscape of proteins. An energy perturbation propagates more effectively from a flexible region to the core than in the opposite direction, because the former affects a broader region of the energy landscape than the latter. This might be of importance in understanding the special thermodynamic signature of the SH3-peptide interaction and the relevance of the dual character of SH3 binding sites.     

Self-assembly of human latexin into amyloid-like oligomers

Background  

In conformational disorders, it is not evident which amyloid aggregates affect specific molecular mechanisms or cellular pathways, which cause disease because of their quantity and mechanical features and which states in aggregate formation are pathogenic. Due to the increasing consensus that prefibrillar oligomers play a major role in conformational diseases, there is a growing interest in understanding the characteristics of metastable polypeptide associations.     

Pallial origin of basal forebrain cholinergic neurons in the nucleus basalis of Meynert and horizontal limb of the diagonal band nucleus

The majority of the cortical cholinergic innervation implicated in attention and memory originates in the nucleus basalis of Meynert and in the horizontal limb of the diagonal band nucleus of the basal prosencephalon. Functional alterations in this system give rise to neuropsychiatric disorders as well as to the cognitive alterations described in Parkinson and Alzheimer's diseases. Despite the functional importance of these basal forebrain cholinergic neurons very little is known about their origin and development. Previous studies suggest that they originate in the medial ganglionic eminence of the telencephalic subpallium; however, our results identified Tbr1-expressing, reelin-positive neurons migrating from the ventral pallium to the subpallium that differentiate into cholinergic neurons in the basal forebrain nuclei projecting to the cortex. Experiments with Tbr1 knockout mice, which lack ventropallial structures, confirmed the pallial origin of cholinergic neurons in Meynert and horizontal diagonal band nuclei. Also, we demonstrate that Fgf8 signaling in the telencephalic midline attracts these neurons from the pallium to follow a tangential migratory route towards the basal forebrain.     

Up-regulation of the novel proinflammatory adipokines lipocalin-2, chitinase-3 like-1 and osteopontin as well as angiogenic-related factors in visceral adipose tissue of patients with colon cancer

Background

Obesity is widely recognised as an important risk factor for colorectal cancer (CC).

Aim

The study aimed to evaluate the effect of CC on circulating concentrations and gene expression levels of inflammatory and angiogenesis-related factors in human visceral adipose tissue (VAT).

Methods

VAT biopsies were obtained from 18 healthy individuals and 11 patients with CC. Real-time polymerase chain reactions were performed to quantify gene expression levels and zymographic analyses were used to determine the activity of matrix metalloproteinases (MMPs).

Results

Patients with CC exhibited increased mRNA expression levels of lipocalin-2 (P=.014), osteopontin (P=.027), tumor necrosis factor-α (TNF-α) (P=.016) and chitinase-3 like-1 (P=.006) compared to control subjects in VAT. Gene expression levels of hypoxia-inducible factor-1 α, vascular endothelial growth factor and MMP-2 were significantly higher (P<.05) in VAT of patients with CC. The expression of insulin-like growth factor I, insulin growth factor binding protein 3 and MMP-9 followed the same trend, although no significant differences were reached. The enzymatic activity of MMP-9 was increased (P<.001) in patients with CC. Furthermore, individuals with CC showed increased (P<.05) circulating concentrations of the inflammatory markers interleukin-6, tumour necrosis factor α and hepatocyte growth factor, whereas levels of the anti-inflammatory adipokine adiponectin were decreased (P<.01).

Conclusion

These findings represent the first observation that mRNA levels of the novel inflammatory factors lipocalin-2, chitinase-3 like-1 and osteopontin are increased in human VAT of subjects with CC. This observation together with the up-regulation of angiogenic factors suggests that adipokines secreted by VAT may be involved in the development of colon cancer.     

Unusual viral ligand with alternative interactions is presented by HLA-Cw4 in human respiratory syncytial virus-infected cells

Short viral antigens bound to human major histocompatibility complex (HLA) class I molecules are presented on infected cells. Vaccine development frequently relies on synthetic peptides to identify optimal HLA class I ligands. However, when natural peptides are analyzed, more complex mixtures are found. By immunoproteomics analysis, we identify in this study a physiologically processed HLA ligand derived from the human respiratory syncytial virus matrix protein that is very different from what was expected from studies with synthetic peptides. This natural HLA-Cw4 class I ligand uses alternative interactions to the anchor motifs previously described for its presenting HLA-Cw4 class I molecule. Finally, this octameric peptide shares its C-terminal core with the H-2D(b) nonamer ligand previously identified in the mouse model. These data have implications for the identification of antiviral cytotoxic T lymphocyte responses and for vaccine development.