Overexpression of delta-12 desaturase in the yeast Schwanniomyces occidentalis enhances the production of linoleic acid.

The oleaginous yeast Schwanniomyces occidentalis was previously isolated because of its excellent suitability to convert lignocellulosic hydrolysates into triacyl glycerides: it is able to use a broad range of sugars and is able to tolerate high concentrations of lignocellulosic hydrolysate inhibitors. Compared to other oleaginous yeasts S. occidentalis however produces a low content of unsaturated fatty acids. We show here that the linoleic acid content can be significantly improved by (over)expression Δ12-desaturases derived from S. occidentalis and Fusarium moniliforme. Expression was stable for the homologous expression but decreased during heterologous expression. Both homologous and heterologous expression of mCherry-Δ12-desaturase led to a 4-fold increase in linoleic acid from 0.02 g/g biomass to 0.08 g/g biomass resulting in the production of 2.23 g/L and 2.05 g/L of linoleic acid.

Selection of oleaginous yeasts for fatty acid production.

BACKGROUND: Oleaginous yeast species are an alternative for the production of lipids or triacylglycerides (TAGs). These yeasts are usually non-pathogenic and able to store TAGs ranging from 20 % to 70 % of their cell mass depending on culture conditions. TAGs originating from oleaginous yeasts can be used as the so-called second generation biofuels, which are based on non-food competing "waste carbon sources". RESULTS: In this study the selection of potentially new interesting oleaginous yeast strains is described. Important selection criteria were: a broad maximum temperature and pH range for growth (robustness of the strain), a broad spectrum of carbon sources that can be metabolized (preferably including C-5 sugars), a high total fatty acid content in combination with a low glycogen content and genetic accessibility. CONCLUSIONS: Based on these selection criteria, among 24 screened species, Schwanniomyces occidentalis (Debaromyces occidentalis) CBS2864 was selected as a promising strain for the production of high amounts of lipids.

Balance between angiopoietin-1 and angiopoietin-2 is in favor of angiopoietin-2 in atherosclerotic plaques with high microvessel density.

INTRODUCTION: Atherosclerotic plaque microvessels are associated with plaque hemorrhage and rupture. The mechanisms underlying plaque angiogenesis are largely unknown. Angiopoietin (Ang)-1 and -2 are ligands of the endothelial receptor Tie-2. Ang-1 induces formation of stable vessels, whereas Ang-2 destabilizes the interaction between endothelial cells and their support cells. We studied the expression patterns of Ang-1 and -2 in relation to plaque microvessels. METHODS AND RESULTS: Carotid endarterectomy specimens were studied (n = 100). Microvessel density (MVD) was correlated with the presence of macrophages and with a (fibro)atheromatous plaque phenotype. A negative correlation was observed between Ang-1 expression and MVD. A positive correlation was observed between the ratio of Ang-2/Ang-1 and MVD. Ang-2 expression was correlated with matrix metalloproteinase-2 (MMP-2) activity. Immunohistochemical staining of Ang-1 was observed in smooth muscle cells, whereas Ang-2 was detected in endothelial cells, smooth muscle cells and macrophages. CONCLUSIONS: In plaques with high MVD, the local balance between Ang-1 and Ang-2 is in favor of Ang-2. Plaque Ang-2 levels are associated with MMP-2 activity. Ang-2-induced MMP-2 activity might play a role in the development of (unstable) plaque microvessels.

HIF-1 alpha expression is associated with an atheromatous inflammatory plaque phenotype and upregulated in activated macrophages.

OBJECTIVE: Angiogenesis and inflammation are important features in atherosclerotic plaque destabilization. The transcription factor hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of angiogenesis and is also involved in inflammatory reactions. We studied HIF-1 alpha expression in different atherosclerotic plaque phenotypes. METHODS AND RESULTS: HIF-1 alpha expression was observed in 18/37 (49%) carotid and in 9/15 (60%) femoral endarterectomy specimens. Expression of HIF-1 alpha was associated with the presence of a large extracellular lipid core (P=0.03) and macrophages (P=0.02). HIF-1 alpha co-localized with vascular endothelial growth factor (VEGF), an important downstream target of HIF-1 alpha. In addition, a strong association was observed between expression levels of HIF-1 alpha and VEGF (P=0.001). The average number of plaque microvessels was higher in plaques with no or minor HIF-1 alpha staining than in plaques with moderate or heavy HIF-1 alpha staining (P=0.03). In human macrophages, lipopolysaccharide activation induced HIF-1 alpha expression. In embryonic fibroblasts derived from wild-type mice, lipopolysaccharide activation induced an increase in HIF-1 alpha mRNA, whereas in Toll-like receptor 4 defective embryonic fibroblasts no effect was observed after lipopolysaccharide stimulation. CONCLUSIONS: In atherosclerotic plaque, the transcription factor HIF-1 alpha is associated with an atheromatous inflammatory plaque phenotype and with VEGF expression. HIF-1 alpha expression is upregulated in activated macrophages under normoxic conditions.

Functional expression of the epithelial Ca(2+) channels (TRPV5 and TRPV6) requires association of the S100A10-annexin 2 complex.

TRPV5 and TRPV6 constitute the Ca(2+) influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca(2+) channels using yeast two-hybrid and GST pull-down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C-terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull-down and co-immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5-S100A10 complex. Furthermore, the S100A10-annexin 2 pair colocalizes with the Ca(2+) channels in TRPV5-expressing renal tubules and TRPV6-expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2-specific small interfering RNA inhibited TRPV5 and TRPV6-mediated currents in transfected HEK293 cells. In conclusion, the S100A10-annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.