In this investigation, we examined the effects of different unsaturated fatty acid compositions of Saccharomyces cerevisiae on the growth-inhibiting effects of ethanol. The unsaturated fatty acid (UFA) composition of S. cerevisiae is relatively simple, consisting almost exclusively of the mono-UFAs palmitoleic acid (⌬ 9 Z-C 16:1 ) and oleic acid (⌬ 9 Z-C 18:1 ), with the former predominating. Both UFAs are formed in S. cerevisiae by the oxygen-and NADH-dependent desaturation of palmitic acid (C 16:0 ) and stearic acid (C 18:0 ), respectively, catalyzed by a single integral membrane desaturase encoded by the OLE1 gene. We systematically altered the UFA composition of yeast cells in a uniform genetic background (i) by genetic complementation of a desaturase-deficient ole1 knockout strain with cDNA expression constructs encoding insect desaturases with distinct regioselectivities (i.e., ⌬ 9 and ⌬ 11 ) and substrate chain-length preferences (i.e., C 16:0 and C 18:0 ); and, (ii) by supplementation of the same strain with synthetic mono-UFAs. Both experimental approaches demonstrated that oleic acid is the most efficacious UFA in overcoming the toxic effects of ethanol in growing yeast cells. Furthermore, the only other UFA tested that conferred a nominal degree of ethanol tolerance is cis-vaccenic acid (⌬ 11 Z-C 18:1 ), whereas neither ⌬ 11 Z-C 16:1 nor palmitoleic acid (⌬ 9 Z-C 16:1 ) conferred any ethanol tolerance. We also showed that the most ethanol-tolerant transformant, which expresses the insect desaturase TniNPVE, produces twice as much oleic acid as palmitoleic acid in the absence of ethanol and undergoes a fourfold increase in the ratio of oleic acid to palmitoleic acid in response to exposure to 5% ethanol. These findings are consistent with the hypothesis that ethanol tolerance in yeast results from incorporation of oleic acid into lipid membranes, effecting a compensatory decrease in membrane fluidity that counteracts the fluidizing effects of ethanol.

Desaturation of coenzyme-A esters of saturated fatty acids is a common feature of sex pheromone biosynthetic pathways in the Lepidoptera. The enzymes that catalyze this step share several biochemical properties with the ubiquitous acyl-CoA Δ 9 -desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acyl-CoA Δ 9 -desaturases, pheromone desaturases have evolved unusual regio- and stereoselective activities that contribute to the remarkable diversity of chemical structures used as pheromones in this large taxonomic group. In this report, we describe the isolation of a cDNA encoding a pheromone gland desaturase from the cabbage looper moth, Trichoplusia ni , a species in which all unsaturated pheromone products are produced via a Δ 11 Z-desaturation mechanism. The largest ORF of the ≈1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn Δ 11 Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast Δ 9 -desaturases, suggesting conserved transmembrane topology. A 182-aa core domain delimited by conserved histidine-rich motifs implicated in iron-binding and catalysis has 72 and 58% similarity (including conservative substitutions) to acyl-CoA Δ 9 Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an ≈1,250-nt PDesat-Tn Δ 11 Z mRNA that is consistent with the spatial and temporal distribution of Δ 11 -desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn Δ 11 Z resulted in complementation of the strain’s fatty acid auxotrophy and the production of Δ 11 Z-unsaturated fatty acids.

Tissue factor (TF) is a low molecular weight glycoprotein that initiates the clotting cascade and is considered to be a major regulator of coagulation, hemostasis, and thrombosis. TF is not expressed in the intima or media of normal adult blood vessels. Accordingly, it has been hypothesized that the initiation of intravascular coagulation may require the "induced" expression of TF in the vessel wall. We report that TF mRNA and protein are rapidly and markedly induced in early and late passaged vascular smooth muscle cells (VSMC) by growth factors (serum, platelet-derived growth factor, epidermal growth factor), vasoactive agonists (angiotensin II), and a clotting factor (a-thrombin). The induction of TF mRNA by these agents is dependent upon mobilization of intracellular Ca2" and is blocked by Ca2" chelation. In contrast to other growth factorresponsive genes, such as KC and c-fos, downregulation of protein kinase C activity by prolonged treatment with phorbol esters fails to block agonist-mediated TF induction. This raises the possibility that protein kinase C activation may not be necessary for TF mRNA induction in VSMC. VSMC may play a role in the generation or propagation of thrombus through the induction of TF, particularly in settings, such as those associated with acute vessel injury, where the endothelium is denuded and the VSMC are exposed to circulating blood. (J. Clin. Invest.