Search

Stearoyl-CoA desaturase-1 (SCD-1) is the rate-limiting enzyme in the biosynthesis of monounsaturated fatty acids (MUFA), which are required for efficient neutral lipid esterification. In the present investigation, we demonstrate that loss of SCD-1 activity increases free cholesterol (FC) content and induces Xbp-1 splicing. We assessed the small molecule SCD-1 inhibitor A939572 on [[sup.14]C]stearate incorporation into neutral lipids and found its incorporation into triglyceride was unaffected, whereas labeled cholesteryl ester (CE) content was notably diminished. Using either A939572 or liver knockout mice (LKO), we show that loss of SCD-1 activity increases FC levels and activates the liver X receptor (LXR) pathway. Using adenoviral delivery of an active form of X-box binding protein-1 (Xbp-1; Xbp-1s), we show increased sterol synthesis only when cells lack the ability to generate MUFA. The results of the cell-based model were confirmed in LKO mice where fasting-refeeding decreased CE, increased FC, and increased Xbp-1s. On the basis of the present data, we conclude that SCD-1 activity is required for efficient cholesterol esterification to MUFA and that loss of its activity increases Xbp-1s-mediated FC synthesis. It is likely that the accumulation of FC enhances Xbp-1 splicing, induces LXR transcriptional activity, and increases ABCA1 (ATP-binding cassette transporter A1) expression to maintain cholesterol homeostasis. cell stress; fasting-refeeding; cholesterol metabolism; acyl-coenzyme A:cholesterol acyltransferase-1; X-box binding protein-1; caveolin-1 doi: 10.1152/ajpendo.00388.2010.

A sucrose-rich diet (SRD), compared with a starch diet, induces time-dependent metabolic disorders and insulin resistance with hypertriglyceridemia, similar to type 2 diabetes. In this study, we examined the effect of SRD, after 8 too, on nuclear receptors peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]), and liver X receptor-[alpha] (LXR[alpha]), stearoyl-CoA desaturase-1 (SCD-1), and [DELTA]6 and [DELTA]5 desaturases mRNA and activity, hepatic enzymes involved in lipid metabolism, and fatty acid (FA) composition as well as the reversal produced by cod liver oil. SRD induced triglyceride increase in plasma and liver, increasing the anabolic FA synthase, malic enzyme, and glucose-6-phosphate dehydrogenase, but not the prooxidative enzymes FA oxidase and carnitine palmitoyltransferase I, and correspondingly decreased PPAR[alpha] and increased LXR[alpha] expressions. Results suggest a contribution of both nuclear receptors' interaction on these enzymatic activities. SRD depressed SCD-1 without altering oleic acid proportion and increased [DELTA]6 and [DELTA]5 desaturases and the proportion of n-6 arachidonic acid. Therefore, the data do not support that SRD hypertriglyceridemia is produced by increased SCD-1-dependent oleic acid biosynthesis. The administration of 7% cod liver oil for 2 mo depressed LXR[alpha], enhancing PPAR[alpha] in control and SRD-fed rats, reversing the activity of the hepatic enzymes involved in lipid metabolism and therefore the hyperlipidemia produced by the SRD. Fish oil increased n-3 PUFA and depressed n-6 PUFA of liver lipids without altering the 18:1/18:0 ratio, suggesting that its effects were produced mainly by competition of dietary n-6 and n-3 FA and not through desaturase activity modification. type 2 diabetes similar model; peroxisome proliferator-activated receptor-[alpha]; liver X receptor-[alpha]; fatty acid biosynthesis; n-3 vs. n-6 polyunsaturated fatty acid doi:10.1152/ajpendo.00513.2009.

Three cases having acyl-CoA dehydrogenase 9 (ACAD9) deficiency with episodic liver dysfunction during otherwise mild illness or cardiomyopathy, along with chronic neurologic dysfunction, are reported. The studies of the tissue distribution and gene regulation of ACAD9 and very-long-chain acyl-CoA dehydrogenase have identified the presence of two independently regulated functional pathways for long-chain fat metabolism, showing that these two enzymes might be involved in different physiological functions.

Protection of poikilothermic animals from seasonal cold is widely regarded as being causally linked to changes in the unsaturation of membrane phospholipids, yet in animals this proposition remains formally untested. We have now achieved this by the genetic manipulation of lipid biosynthesis of Caenorhabditis elegans independent of temperature. Worms transferred from 25[degree]C to 10 [degree]C develop over several days a much-increased tolerance of lethal cold (0[degree]C) and also an increased phospholipid unsaturation, as in higher animal models. Of the three C. elegans [DELTA]9-desaturases, transcript levels of fat-7 only were up-regulated by cold transfer. RNAi suppression of fat-7 caused the induction of fat-5 desaturase, so to control desaturase expression we combined RNAi of fat-7 with a fat-5 knockout. These fat-5/fat-7 manipulated worms displayed the expected negative linear relationship between lipid saturation and cold tolerance at 0[degree]C, an outcome confirmed by dietary rescue. However, this change in lipid saturation explains just 16% of the observed difference between cold tolerance of animals held at 25[degree]C and 10[degree]C. Thus, although the manipulated lipid saturation affects the tolerable thermal window, and altered [DELTA]9-desaturase expression accounts for cold-induced lipid adjustments, the effect is relatively small and none of the lipid manipulations were sufficient to convert worms between fully cold-sensitive and fully cold-tolerant states. Critically, transfer of 10[degree]C-acclimated worms back to 25[degree]C led to them restoring the usual cold-sensitive phenotype within 24 h despite retaining a lipid profile characteristic of 10[degree]C worms. Other nonlipid mechanisms of acquired cold protection clearly dominate inducible cold tolerance. [DELTA]9-acyl desaturase | lipid composition | RNAi

The biosynthesis of female moth sex pheromone blends is controlled by a number of different enzymes, many of which are encoded by members of multigene families. One such multigene family, the acyl-CoA desaturases, is composed of certain genes that function as key players in moth sex pheromone biosynthesis. Although much is known regarding the function of some of these genes, very little is known regarding how novel genes have evolved within this family and how this might impact the establishment of new sex pheromone blends within a species. We have discovered that several cryptic [DELTA]11 and [DELTA]14 desaturase genes exist in the genomes of the European and Asian corn borers (Ostrinia nubilalis and Ostrinia furnacalis, respectively). Furthermore, an entirely novel class of desaturase gene has arisen in the Ostrinia lineage and is derived from duplication of the [DELTA]11 desaturase gene and subsequent fusion with a retroposon. Interestingly, the genes have been maintained over relatively long evolutionary time periods in corn borer genomes, and they have not been recognizably pseudogenized, suggesting that they maintain functional integrity. The existence of cryptic desaturase genes in moth genomes indicates that the evolution of moth sex pheromone desaturases in general is much more complex than previously recognized. Ostrinia | phylogeny | pseudogene | biosynthesis | evolution

Patient cells, minigenes, and in vitro assays are used to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene has caused exon skipping by inactivating a crucial exonic splicing enhancer (ESE). Results reveal a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and have shown that the deleterious effects of mutations on splicing should be evaluated in the context of relevant haplotype.

Acute exercise can reverse muscle insulin resistance, but the mechanism(s) of action are unknown. With the use of a hindlimb perfusion model, we have found that acute contraction restores insulin-stimulated glucose uptake in muscle of obese Zucker rats to levels witnessed in lean controls. Previous reports have suggested that obesity-related insulin resistance stems from lipid oversupply and tissue accumulation of toxic lipid intermediates that impair insulin signaling. We reasoned that contraction might activate hydrolysis and oxidation of intramuscular lipids, thus alleviating 'lipotoxicity' and priming the muscle for enhanced insulin action. Indeed, analysis of mitochondrial-derived acyl-carnitine esters suggested that contraction caused robust increases in [beta]-oxidative flux and mitochondrial oxidation. As predicted, contraction decreased intramuscular triacylglycerol content; however, diacylglycerol and long chain acyl-CoAs, lipid intermediates presumed to trigger insulin resistance, were either unchanged or increased. In muscles from obese animals, insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 remained impaired after contraction, whereas phosphorylation of the downstream signaling protein, AS160, was partially restored. These results suggest that acute exercise enables diabetic muscle to circumvent upstream defects in insulin signal transduction via mechanisms that are more tightly coupled to increased mitochondrial energy metabolism than the lowering of diacylglycerol and long chain acyl-CoA. skeletal muscle; intramuscular lipids; signaling; exercise

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of triacylglycerol. It has been well recognized that mammals possess multiple enzymatically distinct proteins with GPAT activity. Although the mitochondrial-associated GPAT has been cloned and extensively characterized, the molecular identity of the endoplasmic reticulum (ER)-associated GPAT, which accounts for the majority of total GPAT activity in most tissues, has remained elusive. Here we report the identification of genes encoding human and mouse ER-associated GPAT (termed GPAT3). GPAT3 is a member of the acyltransferase family predominantly expressed in tissues characterized by active lipid metabolism, such as adipose tissue, small intestine, kidney, and heart. Ectopic expression of GPAT3 leads to a significant increase in N-ethylmaleimide-sensitive GPAT activity, whereas acyltransferase activity toward a variety of other lysophospholipids, as well as neutral lipid substrates, is not altered. Overexpression of GPAT3 in mammalian cells results in increased triacylglycerol, but not phospholipid, formation. GPAT3 is localized to the ER when over-expressed in COS-7 cells. GPAT3 mRNA is dramatically up-regulated during adipocyte differentiation, is reciprocally regulated in adipose tissue and liver of ob/ob mice, and is up-regulated in mice treated with a peroxisome proliferator-activated receptor [gamma], (PPAR[gamma]) agonist. A substantial loss of GPAT activity in 3T3-L1 adipocytes was achieved by reducing GPAT3 mRNA levels through GPAT3-specific siRNA knockdown. These findings identify GPAT3 as a previously uncharacterized triacylglycerol biosynthetic enzyme. Similar to other lipogenic enzymes, GPAT3 may be useful as a target for the treatment of obesity. bioinformatics | endoplasmic reticulum | lysophosphatidic acid | regulation

Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long-chain acyl-CoAs (LC-CoA) by the enzyme acyl-CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1, -3, -4, -5, -6), which vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity > 10-fold compared with controls after 24 h. HepG2 cells overexpressing ACSL1 had a 40% higher triglyceride (TG) content (93 [+ or -] 3 vs. 67 [+ or -] 2 nmol/mg protein in controls, P < 0.05) after 24-h exposure to 1 mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCA-CoA content (160 [+ or -] 6 vs. 100 [+ or -] 6 nmol/g protein in controls, P < 0.05) and increased [[sup.14]C]oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than fivefold over controls at 4 days postinfection. ACSL1 overexpression caused a twofold increase in TG content in mouse liver (39 [+ or -] 4 vs. 20 [+ or -] 2 [micro]mol/g wet wt in controls, P < 0.05), and overexpression in rat liver increased [1-[sup.14]C]palmitate clearance into liver TG. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver. acyl-coenzyme A synthetase; hepatic triglyceride synthesis; adenovirus; fatty acid metabolism

Mice lacking acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), an enzyme that catalyzes the terminal step in triacylglycerol synthesis, have enhanced insulin sensitivity and are protected from obesity, a result of increased energy expenditure. In these mice, factors derived from white adipose tissue (WAT) contribute to the systemic changes in metabolism. One such factor, adiponectin, increases fatty acid oxidation and enhances insulin sensitivity. To test the hypothesis that adiponectin is required for the altered energy and glucose metabolism in DGAT1-deficient mice, we generated adiponectin-deficient mice and introduced adiponectin deficiency into DGAT1-deficient mice by genetic crosses. Although adiponectin-deficient mice fed a high-fat diet were heavier, exhibited worse glucose tolerance, and had more hepatic triacylglycerol accumulation than wild-type controls, mice lacking both DGAT1 and adiponectin, like DGAT1-deficient mice, were protected from diet-induced obesity, glucose intolerance, and hepatic steatosis. These findings indicate that adiponectin is required for normal energy, glucose, and lipid metabolism but that the metabolic changes induced by DGAT1-deficient WAT are independent of adiponectin and are likely due to other WAT-derived factors. Our findings also suggest that the pharmacological inhibition of DGAT1 may be useful for treating human obesity and insulin resistance associated with low circulating adiponectin levels. triglyceride; adipose tissue; gene knockout; acyl-coenzyme A:diacylglycerol acyltransferase 1 doi:10.1152/ajpendo.00621.2005

Glucose infusion in rats for 1-4 days results in insulin resistance and increased triglyceride, whole long-chain fatty acyl-CoA (LCA-CoA), and malonyl-CoA content in red skeletal muscle. Despite this, the relation between these alterations and the onset of insulin resistance has not been defined. We aimed to 1) identify whether the changes in these lipids and of diacylglycerol (DAG) precede or accompany the onset of insulin resistance in glucose-infused rats, 2) determine whether the insulin resistance is associated with alterations in AMP-activated protein kinase (AMPK), and 3) assess whether similar changes occur in liver and in muscle. Hyperglycemia (17-18 mM) was maintained by intravenous glucose infusion in rats for 3 or 5 h; then euglycemia was restored and a 2-h hyperinsulinemic clamp was performed. Significant (P < 0.01) muscle and liver insulin resistance first appeared in red quadriceps and liver of the glucose-infused group at 5 h and was associated with a twofold increase in DAG and malonyl-CoA content and a 50% decrease in AMPK and acetyl-CoA carboxylase (ACC) phosphorylation and AMPK activity. White quadriceps showed qualitatively similar changes but without decreases in AMPK or ACC phosphorylation. Triglyceride mass was increased at 5 h only in liver, and whole tissue LCA-CoA content was not increased in liver or either muscle type. We conclude that the onset of insulin resistance induced by glucose oversupply correlates temporally with increases in malonyl-CoA and DAG content in all three tissues and with reduced AMPK phosphorylation and activity in red muscle and liver. In contrast, it was not associated with increased whole tissue LCA-CoA content in any tissue or triglyceride in muscle, although both are observed at later times. adenosine monophosphate-activated protein kinase; hyperglycemia; glucose clamp; triglyceride; long-chain acyl-coenzyme A; glucotoxicity

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet [beta]-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 [micro]M) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action. acyl-coenzyme A-binding protein; free fatty acid; hormone-sensitive lipase; lipid signaling

Activation of the AMP-activated protein kinase (AMPK) results in acute changes in cellular metabolism and transcriptional events that make the cell more robust when encountering an energy challenge. AMPK is thought to be inhibited by glycogen, the major storage form of intracellular carbohydrate. We hypothesized that long-chain acyl-CoA esters (LCACEs) might also inhibit AMPK signaling. Cytosolic LCACEs are available for immediate transport and oxidation within the mitochondria and accordingly may be representative of the lipid energy charge of the cell. We found that LCACEs inhibited phosphorylation of AMPK by the recombinant AMPK kinase (AMPKK) LKB1/STRAD/MO25 in a concentration-dependent manner. Palmitoyl-CoA (PCoA) did not affect the activity of phosphothreonine-172 AMPK. PCoA potently inhibited AMPKK purified from liver. Conversely, PCoA stimulated the kinase activity of LKB1/STRAD/MO25 toward the peptide substrate LKB tide. Octanoyl-CoA, palmitate, and palmitoylcarnitine did not inhibit AMPKK activity. Removal of AMP from the reaction mixture resulted in reduced AMPKK activity in the presence of PCoA. In conclusion, these results demonstrate that the AMPKK activity of LKB1/STRAD/MO25 is substrate specific and distinct from the kinase activity of LKB 1/STRAD/MO25 toward the peptide substrate LKB1tide. They also demonstrate that LCACEs inhibit the AMPKK activity of LKB1/STRAD/MO25 in a specific manner with a dependence on both a long fatty chain and a CoA moiety. These results suggest that the AMPK signaling cascade may directly sense and respond to the lipid energy charge of the cell. adenosine 5'-monophosphate-activated protein kinase; adenosine 5'monophosphate-activated protein kinase kinase; diabetes; fatty acid oxidation; STK 11

Results reveal that the G185S variant enzyme of short chain acyl-CoA dehydrogenase exhibits impaired kinetics although the G185S substitution is outside of the substrate-binding pocket. Further, the tertiary conformation of the variant enzyme shows reduced flexibility resulting in an impaired charge-transfer substrate-enzyme intermediate complex, which causes catalytic dysfunction.

Rat liver microsomes were incubated with 1:1 mixtures of substrate to determine intermolecular deuteriumisotope effects on C-H bond cleavage steps involved in dihydroceramide delta4 desaturation.

The importance of cholesterol ester synthesis by acyl CoA:cholesterol acyltransferase (ACAT) enzymes in intestinal and hepatic cholesterol metabolism has been unclear. We now demonstrate that ACAT2 is the major ACAT in mouse small intestine and liver, and suggest that ACAT2 deficiency has profound effects on cholesterol metabolism in mice fed a cholesterol-rich diet, including complete resistance to diet-induced hypercholesterolemia and cholesterol gallstone formation. The underlying mechanism involves the lack of cholesterol ester synthesis in the intestine and a resultant reduced capacity to absorb cholesterol. Our results indicate that ACAT2 has an important role in the response to dietary cholesterol, and suggest that ACAT2 inhibition may be a useful strategy for treating hypercholesterolemia or cholesterol gallstones., Author(s): Kimberly K. Buhman [1, 2, 6]; Michel Accad [1, 2, 5, 6]; Sabine Novak [1, 6]; Rebekah S. Choi [1]; Jinny S. Wong [2]; Robert L. Hamilton [2, 3]; [...]

The fatty acid desaturase produced by the fat-1 gene of Caenorhabditis elegans has the regioselectivity of an omega-3 desaturase. Desaturation begins when a C-H bond at a carbon close to the C-terminus is cleaved.

Research has been conducted on the dienoyl-coenzyme A thiolesters. The investigation of the stereochemical course of these thiolesters' reduction is discussed.

Results demonstrate that desaturase mRNA level is temperature regulated and desaturase enzyme is the only component of the desaturation system that is temperature controlled in Bacillus subtilis.

Stearoyl acyl protein (delta)9 desaturase requires a diiron center to catalyze the NADPH- and O2-dependent desaturation of stearoyl acycl carrier protein (ACP) to form oleoyl-ACP. The steady-state kinetics of this reaction were investigated using (delta)9 desaturase from recombinant Ricinus communis. Results indicated that hydrophobic binding energy was used to enhance catalysis since experiments showed that the binding of shorter acyl chains apparently did not provide sufficient activation energy for efficient desaturase catalysis.

The authors discuss the characterization of Pseudomonas aeruginosa fabD-fabG-acpP-fabF gene cluster. Description of acyl carrier protein (ACP) and malonyl-coenzyme A:ACP transacylase (FabD) purification and characterization is presented.

Research conducted on Pseudomonas aeruginosa fabI gene and its product to demonstrate the presence of enoyl-acyl carrier protein reductase in this bacterium is presented. Experiment indicate the role of FabI in N-butyryl-L-homoserine lactone synthesis.

This investigation addressed the hypothesis that stearoyl coenzyme A desaturase (SCD) gene expression would serve as a postnatal marker of adipocyte differentiation in bovine s.c. adipose tissue. Samples of tailhead s.c. adipose tissue were obtained by biopsy from preweaning steer calves 2.5 wk, 5 mo, and 7.5 mo of age and from yearling steers 12 mo of age. Samples also were obtained at slaughter when the steers were 18 mo of age. The steers sampled as yearlings were fed native pasture from weaning until 12 mo of age, and the steers sampled at slaughter were fed a high-concentrate diet from 12 to 18 mo of age. Major peak adipocyte volumes for the 2.5-wk-, 5-mo-, and 7.5-mo-old steers were 14, 270, and 700 pL, respectively (P < .001). The steers did not gain weight during pasture feeding, and at 12 mo of age peak adipocyte volume had decreased (P = .009) to 270 pL. At this time, a second, smaller population of adipocytes had appeared with a peak volume of 115 pL. At slaughter, adjusted fat thickness of the steers was 1.60 [+ or -] .13 cm, the USDA yield grade of the carcasses was 3.51 [+ or -] .31, and peak adipocyte volume had increased (P = .01) to over 2,500 pL. The number of adipocytes per 100 mg of adipose tissue doubled (P = .006) between 2.5 wk and 5 mo of age, concurrent with the nearly 20-fold increase in peak adipocyte volume, indicating that this was a period of apparent adipocyte hyperplasia. Uncoupling protein mRNA was undetectable at all stages of postnatal growth, indicating that differentiating tailhead s.c. adipocytes do not acquire brown adipocyte characteristics postnatally. Lipogenesis expressed on a cellular basis was low in all preweaning samples and increased significantly above preweaning values only in the 18-mo-old steers. Stearoyl coenzyme A desaturase mRNA concentration also was low in all preweaning samples, but it peaked (P = .07) at 12 mo of age. Because the peak in SCD mRNA concentration preceded a significant rise in lipogenesis and lipid filling, we conclude that the level SCD gene expression may be indicative of the extent of terminal differentiation in bovine tailhead s.c. adipose tissue. Key Words: Adipocytes, Differentiation, Acyl-CoA Desaturase, Protein, Genes, Cattle

Abnormalities of fatty acid metabolism are recognized to play a significant role in human disease, but the mechanisms remain poorly understood. Long-chain acyl-CoA dehydrogenase (LCAD) catalyzes the initial step in mitochondrial fatty acid oxidation (FAO). We produced a mouse model of LCAD deficiency with severely impaired FAO. Matings between LCAD +/- mice yielded an abnormally low number of LCAD +/- and -/- offspring, indicating frequent gestational loss. LCAD -/- mice that reached birth appeared normal, but had severely reduced fasting tolerance with hepatic and cardiac lipidosis, hypoglycemia, elevated serum free fatty acids, and nonketotic dicarboxylic aciduria. Approximately 10% of adult LCAD -/- males developed cardiomyopathy, and sudden death was observed in 4 of 75 LCAD -/- mice. These results demonstrate the crucial roles of mitochondrial FAO and LCAD in vivo.

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 [[Delta].sup.9]-desaturases of animals and fungi, suggesting a common ancestral origin. Unlike metabolic acylCoA [[Delta].sup.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 [[Delta].sup.11]Z-desaturation mechanism. The largest ORF of the [approximately equal to]1,250-bp cDNA encodes a 349-aa apoprotein (PDesat-Tn [[Delta].sup.11]Z) with a predicted molecular mass of 40,240 Da. Its hydrophobicity profile is similar overall to those of rat and yeast [[Delta].sup.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 [[Delta].sup.9]Z-desaturases of rat and yeast, respectively. Northern blot analysis revealed an [approximately equal to]1,250-nt PDesat-Tn [[Delta].sup.11]Z mRNA that is consistent with the spatial and temporal distribution of [[Delta]11]-desaturase enzyme activity. Genetic transformation of a desaturase-deficient strain of the yeast Saccharomyces cerevisiae with an expression plasmid encoding PDesat-Tn [[Delta].sup.11]Z resulted in complementation of the strain's fatty acid auxotrophy and the production of [[Delta].sup.11]Z-unsaturated fatty acids.

Optical and resonance Raman spectroscopies were conducted to determine the intermediate conformation of stearoyl-acyl carrier protein Delta9 desaturase. Results reveal that the addition of four electrons converts the native conformation to a peroxodiferric intermediate similar to ribonucleotide reductase. Decay involves the release of two electrons during the reversion to the diferric state. The stoichiometry of the desaturase reaction corresponds to that of an oxidase reaction.

Research was conducted to examine the hypothesis that the positional specificity of lecithin-cholesterol acyltransferase is dictated by the structure of both the acyl groups and not just the sn-2-acyl group. A series of synthetic PC substrates containing either 18:0 or 20:4 at the sn-2 position and several acyl groups differing in chain length and unsaturation at the sn-1 position were tested. Results indicate that the structure of the sn-1-acyl group substantially modifies the positional specificity of lecithin-cholesterol acyltransferase.

Research was conducted to examine the oxidase activity of the acyl-CoA dehydrogenases. A second chromophoric substrate of the medium chain enzyme was used and another representative of the acyl-CoA dehydrogenase family was tested. Results indicate that oxygen interacts not only with the free enzyme but also with several reduced enzyme species. These results underscore the complexity of an apparently simple reaction as the reoxidation of the acyl-CoA dehydrogenase by molecular oxygen.

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

A general method to synthesize an acyl coenzyme A (CoA) analogue synthon with a reversed thioester orientation is described. A triortho ester was utilized as a protected thioester in the aminolysis reaction to prepare the analogue. The acyl coenzyme analogue formed an unnatural acyl-enzyme where the CoA moiety was covalently linked to an active site cysteine. The reversed succinate thioester together with the hydroxamate and the hydrazide analogues were synthesized to show the role of the acyl CoA as an analogue synthon of CoA.