The finding that SE levels were dramatically decreased in cells grown on oleate was striking and a novel effect of oleate in yeast. However, the reason for this down-regulation of sterol esterification was not obvious. Previously, it was reported that genes encoding proteins of ergosterol synthesis (ERG1 and ERG3) were down-regulated by oleate (), and knock-out of the acyltransferases decreased expression of genes involved in ergosterol formation (ERG3, ERG4, and ERG5) (). Therefore, we presumed that lack of substrates, ergosterol and/or free fatty acid, might cause the decrease in sterol esterification. To prove or disprove this hypothesis, we analyzed amounts of free ergosterol (unesterified) and FFA in cells grown on oleate and or glucose (). We found that the level of free ergosterol was increased in all mutant strains compared with wild type (A). This effect was observed with cells grown on YPD or YPO. Consequently, free ergosterol was not limiting when cells were grown on oleate.
Our group has been actively involved in the synthesis of QDs and magnetic QDs (MQDs) [-]. We have successfully demonstrated the magnetic and fluorescent properties of Fe2O3-CdSe MQDs, silica -coated QDs or MQDs and their application in cell labeling (Figure ) . The silanization using aminopropyl triethoxysilane (APS) in a reverse microemulsion produced thin silica coating on bare CdSe QDs or Fe2O3-CdSe MQD with surface NH2 groups. The methoxy groups of APS were hydrolyzed and condensed with another APS, exposing surface amine groups on the silanized QDs (SiO2/QDs) for conjugation with oleyl-O-poly(ethyleneglycol)-succinyl-N-hydroxysuccinimidyl ester, denoted as bio-anchored membrane (BAM). The reaction between the amine group and NHS ester resulted in a covalent amide bond formation, leaving the exposed oleyl group for the effective targeting of cell membrane. The labeling of live cell membranes (HepG2 human liver cancer cells and NIH-3T3 mouse fibroblast cells) using confocal laser scanning microscopy (CLSM) indicated the successful conjugation of silica-coated QDs or MQDs with BAM.
Esters Products - Custom Synthesis
Finally, we investigated whether FFA had an impact on SE synthesis in vitro. For this purpose, we performed assays with homogenates and purified ER from wild type cells grown on glucose in the absence or presence of oleate and palmitate, respectively. We found that both FFA decreased the esterification of ergosterol in vitro (A), but the effect of oleate was much stronger than of palmitate. This effect was not only seen when radiolabeled oleoyl-CoA was used as a co-substrate for the reaction but also with palmitoyl-CoA (). We also ruled out the possibility that oleoyl-CoA instead of free oleate caused the decreased SE synthase activity by substrate inhibition. We performed control experiments using increasing amounts of oleoyl-CoA in the assays, but no indication of inhibition was observed (). Moreover, we tested PL species containing different fatty acids for their influence on the enzyme activity, but they also did not exhibit an inhibitory effect on the esterification of sterols (). Finally, we wished to exclude an unspecific and more general effect of oleate as detergent or inhibitor of acyltransferases. Therefore, we performed DAG acyltransferase (Dga1p) assays as a control. As can bee seen from B, free oleate did not affect the activity of this enzyme but specifically decreased sterol esterification by Are2p. To further elucidate whether our measurements specifically accounted for inhibition of Are2p activity, we investigated sterol esterification in ER fractions isolated from either wild type, TM ARE1, or TM ARE2 (C). As can be seen from these results, hardly any activity was found in the strain with only Are1p active (TM ARE1), which is in line with previous findings that Are1p activity was found only in cells grown anaerobically (). Inhibition of SE synthesis by oleate was found almost identical in wild type and TM ARE2, indicating that Are2p is the target for oleate under conditions tested here. We conclude from these results that the inhibition of SE synthase by oleate is specific and accounts for SE depletion in cells grown on oleate.