A diversity of putative carboxylesterases are expressed in the antennae of the noctuid moth Spodoptera littoralis

Recent studies have suggested that pheromone‐degrading enzymes belonging to the carboxylesterase family could play a role in the dynamics of the olfactory response to acetate sex pheromones in insects. Bioinformatic analyses of a male antennal expressed sequence tag library allowed the identification of 19 putative esterase genes expressed in the antennae of the moth Spodoptera littoralis. Phylogenetic analysis revealed that these genes belong to different insect esterase clades, defined by their putative cellular localization and substrate preferences. Interestingly, two of the 19 genes appeared to be antennal specific, suggesting a specific role in olfactory processing. This high esterase diversity suggested that the antennae are the location for intense esterase‐based metabolism, against potentially a large range of exogenous and endogenous molecules.     

Relationship between unusual hepatic acyl coenzyme A profiles and the pathogenesis of Reye syndrome.

This study examines the relationship between impaired fatty acid oxidation and the pathogenesis of Reye syndrome. We present a hypothesis proposing that many clinical signs of this childhood disease are caused by accumulation of unusual acyl CoA esters, precursors to deacylated metabolites found in the patients' blood and urine. A new method was developed to measure acyl CoA compounds in small human liver biopsy samples, offering several advantages over previous techniques. A major finding was an accumulation in Reye syndrome patients of short- and medium-chain acyl CoA intermediates of fatty acid and branched-chain amino acid oxidation. These metabolites included octanoyl, isovaleryl, butyryl, isobutyryl, propionyl, and methylmalonyl CoA esters. The findings were explained in a model of hepatic fatty acid oxidation involving three interrelated pathways: mitochondrial beta-oxidation, peroxisomal beta-oxidation, and omega-oxidation in the endoplasmic reticulum. The results suggest that pathogenesis in Reye syndrome stems from generalized mitochondrial damage resulting in accumulation of acyl CoA esters. High levels of these compounds lead to inhibition of mitochondrial pathways for ureogenesis, gluconeogenesis, and fatty acid oxidation. The inhibited pathways, in turn, could cause the hyperammonemia, hypoglycemia, and hypoketonemia observed in patients. The model also explains underlying biochemical differences between patients with Reye syndrome and medium-chain acyl CoA dehydrogenase deficiency, another disorder of fatty acid metabolism. Acetyl CoA levels, in the latter disease, were dramatically decreased, compared with both human controls and Reye syndrome patients.     

Resveratrol inhibits fatty acid and triacylglycerol synthesis in rat hepatocytes

Background The putative role of resveratrol, a polyphenol present in grapes and other plants, in modulating dislypidemia, thus preventing cardiovascular diseases, is generally based on proliferating cell lines and in vivo studies in different pathological conditions. The aim of the present study was to investigate whether resveratrol plays a role on lipid biosynthesis in rat hepatocytes. Materials and methods The effect of resveratrol on total rate of fatty acid, cholesterol and complex lipid synthesis, assayed by the incorporation of [1‐¹⁴C]acetate into these lipid fractions, was investigated in rat hepatocyte suspensions. Enzyme activities of acetyl‐CoA carboxylase (ACC) and fatty acid synthase (FAS) as well as 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase (HMG‐CoA‐R), pace‐setting steps of de novo fatty acid and cholesterol synthesis, respectively, were in situ measured in digitonin‐permeabilized hepatocytes. Results Resveratrol‐treated hepatocytes exhibited a short‐term (30 min) inhibition (IC₅₀ ~25 µm ) of total fatty acid synthesis from [1‐¹⁴C]acetate. Among neosynthesized fatty acids, palmitic acid formation was mainly reduced, thus suggesting that enzymatic step(s) of de novo fatty acid synthesis was affected by resveratrol. In digitonin‐permeabilized hepatocytes, only ACC activity was noticeably reduced, while no change in FAS activity was observed. A noticeable resveratrol‐induced reduction of label incorporation into triacylglycerols was also detected. Conversely, cholesterol synthesis and HMG‐CoA‐R activity were unaffected by resveratrol. Conclusion Results here reported show that in isolated hepatocytes from normal rats a resveratrol‐induced short‐term inhibition of fatty acid and triacylglycerol synthesis occurs. This finding may represent a potential mechanism contributing to the reported hypolipidemic effect of resveratrol.     

Fatty Acid Biosynthesis in Human Leukocytes

Extracts from human leukocytes have been examined for the enzymes of de novo fatty acid biosynthesis. These extracts do not catalyze the synthesis of long-chain fatty acids because they lack acetyl CoA carboxylase, the first enzyme unique to the fatty acid synthesis pathway.Since these cells cannot form malonyl CoA, they are unable to synthesize long-chain fatty acids. This inability can be corrected by addition of either purified acetyl CoA carboxylase from rat liver or malonyl CoA to leukocyte extracts. The incorporation of acetate-1-(14)C into fatty acids by intact leukocytes is shown to represent chain elongation of preformed fatty acids rather than de novo synthesis by the fact that 60-100% of the label incorporated resides in the carboxyl carbon of the fatty acids formed.Both mature leukocytes and erythrocytes are unable to synthesize fatty acids because of a lack of acetyl CoA carboxylase even though both contain the other enzymes of fatty acid synthesis. It is possible that a precursor hematopoietic cell may have the capacity to synthesize fatty acids de novo. This hypothesis is supported by the finding of acetyl CoA carboxylase activity in extracts from human leukemic blast cells.The leukocyte fatty acid synthetase activity from malonyl CoA of a number of normal volunteers and of patients with a variety of hematologic diseases is reported.     

Fatty acid binding protein. Role in esterification of absorbed long chain fatty acid in rat intestine.

Fatty acid binding protein (FABP) is a protein of 12,000 mol wt found in cytosol of intestinal mucosa and other tissues, which exhibits high affinity for long chain fatty acids. It has been suggested that FABP (which may comprise a group of closely related proteins of 12,000 mol wt) participates in cellular fatty acid transport and metabolism. Although earlier findings were consistent with this concept, the present studies were designed to examine its physiological function more directly. Everted jejunal sacs were incubated in mixed fatty acid-monoglyceride-bile acid micelles, in the presence or absence of equimolar concentrations of either of two compounds which inhibit oleate binding to FABP:flavaspidic acid-N-methyl-glucaminate and alpha-bromopalmitate. Oleate uptake, mucosal morphology, and oxidation of [14C]acetate remained unaffected by these agents, but oleate incorporation into triglyceride was inhibited by 62-64% after 4 min. The inhibition by flavaspidic acid was reversible with higher oleate concentrations. The effect of these compounds on enzymes of triglyceride biosynthesis was examined in intestinal microsomes. Neither flavaspidic acid nor alpha-bromopalmitate inhibited acyl CoA:monoglyceride acyl-transferase. Fatty acid:coenzyme A ligase activity was significantly enhanced in the presence of partially purified FABP, probably reflecting a physical effect on the fatty acid substrate or on the formation of the enzyme-substrate complex. Activity of the enzyme in the presence of 0.1 mM oleate was only modestly inhibited by equimolar flavaspidic acid and alpha-bromopalmitate, and this effect was blunted or prevented by FABP. We conclude that in everted gut sacs, inhibition of triglyceride synthesis by flavaspidic acid and alpha-bromopalmitate could not be explained as an effect on fatty acid uptake or on esterifying enzymes in the endoplasmic reticulum but rather can be interpreted as reflecting inhibition of fatty acid binding to FABP. These findings lend further support to the concept that FABP participates in cellular fatty acid transport and metabolism. It is also possible that FABP, by effecting an intracellular compartmentalization of fatty acids and acyl CoA, may play a broader role in cellular lipid metabolism.     

Aldehyde reductase activity in the antennae of Helicoverpa armigera

In the present study, we identified two aldehyde reductase activities in the antennae of Helicoverpa species, NADH and NADPH‐dependent activity. We expressed one of these proteins of H. armigera, aldo‐keto reductase (AKR), which bears 56% identity to bovine aldose reductase, displays a NADPH‐dependent activity and is mainly expressed in the antennae of adults. Whole‐mount immunostaining showed that the enzyme is concentrated in the cells at the base of chemosensilla and in the nerves. The enzyme activity of H. armigera AKR is markedly different from those of mammalian enzymes. The best substrates are linear aliphatic aldehydes of 8–10 carbon atoms, but not hydroxyaldehydes. Both pheromone components of H. armigera, which are unsaturated aldehydes of 16 carbons, are very poor substrates. Unlike mammalian AKRs, the H. armigera enzyme is weakly affected by common inhibitors and exhibits a different behaviour from the action of thiols. A model of the enzyme suggests that the four cysteines are in their reduced form, as are the seven cysteines of mammalian enzymes. The occurrence of orthologous proteins in other insect species, that do not use aldehydes as pheromones, excludes the possibility of classifying this enzyme among the pheromone‐degrading enzymes, as has been previously described in other insect species.     

Calcineurin is required for male sex pheromone biosynthesis and female acceptance

Lepidoptera sex pheromone biosynthesis is regulated by pheromone biosynthesis activating neuropeptide (PBAN). PBAN regulates not only female sex pheromone biosynthesis but also male sex pheromone biosynthesis. Previous research has confirmed that PBAN regulates sex pheromone biosynthesis using Ca²⁺ as a secondary messenger in all examined species to date. However, the downstream signal of Ca²⁺ has remained elusive. In the present study, calcineurin A (CNA), a downstream signal of Ca²⁺, was discovered in Helicoverpa armigera male hairpencil and named HaCNA. Sequence analysis demonstrated that the open reading frame of HaCNA contains 1488 nucleotides encoding 495 amino acid residues. A homology search revealed that HaCNA shares a high amino acid identity with the CNA of other insects. Developmental and spatial expression analyses revealed that the mRNA levels of HaCNA peaked at 24 h after emergence and that HaCNA expression was ubiquitous in all examined tissues. Activity analysis revealed that PBAN activates HaCNA, and a Ca²⁺ inhibitor, Lacl₃, attenuated the effect of PBAN by decreasing HaCNA activity. Pharmacological inhibitor and RNA interference‐mediated knockdown assays revealed that both activity inhibition and decreased mRNA levels of HaCNA led to a significant decrease in the production of the male sex pheromone components [octadecanol and (Z)‐11 hexadecanol)] and in the efficacy of female mating acceptance. Our results demonstrate that HaCNA acts as downstream signal of PBAN/Ca²⁺ and plays an important role in PBAN‐induced male sex pheromone biosynthesis and female mating acceptance.     

Activation of cholesterol synthesis in preference to fatty acid synthesis in liver and adipose tissue of transgenic mice overproducing sterol regulatory element-binding protein-2.

We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNA for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase and HMG CoA reductase were especially marked (13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acid synthesis, as measured by intraperitoneal injection of [3H]water. These results contrast with previously reported effects of dominant-positive SREBP-1a, which activated fatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs for cholesterol biosynthetic enzymes were elevated, but the mRNAs for fatty acid biosynthetic enzymes were not. We conclude that SREBP-2 is a relatively selective activator of cholesterol synthesis, as opposed to fatty acid synthesis, in liver and adipose tissue of mice.     

Identification and characterization of the pyrokinin/pheromone biosynthesis activating neuropeptide family of G protein‐coupled receptors from Ostrinia nubilalis

Insects have two closely related G protein‐coupled receptors belonging to the pyrokinin/pheromone biosynthesis activating neuropeptide (pyrokinin/PBAN) family, one with the ligand PBAN or pyrokinin‐2 and another with diapause hormone or pyrokinin‐1 as a ligand. A related receptor is activated by products of the capa gene, periviscerokinins. Here we characterized the PBAN receptor and the diapause hormone receptor from the European corn borer, Ostrinia nubilalis. We also identified a partial sequence for the periviscerokinin receptor. Quantitative PCR of mRNA for all three receptors indicated differential expression in various life stages and tissues. All three splice variants of the PBAN receptor were identified with all variants found in pheromone gland tissue. Immunohistochemistry of V5 tags of expressed receptors indicated that all three variants and the diapause hormone receptor were expressed at similar levels in Spodoptera frugiperda 9 (Sf9) cells. However, the A‐ and B‐variants were not active in our functional assay, which confirms studies from other moths. Functional expression of the C‐variant indicated that it is has a 44 nM half effective concentration for activation by PBAN. The diapause hormone receptor was activated by diapause hormone with a 150 nM half effective concentration.     

A cytochrome P450 gene plays a role in the recognition of sex pheromones in the tobacco cutworm,Spodoptera litura

Cytochrome P450 (P450 or CYP) genes are involved in fundamental physiological functions, and might be also associated with the olfactory recognition of sex pheromones in beetles and moths. A P450 gene, Spodoptera litura CYP4L4 (SlituCYP4L4), was cloned for the first time from the antennae of S. litura. SlituCYP4L4 was almost exclusively expressed in the adult stage and predominantly expressed in the adult antennae. In situ hybridization showed that SlituCYP4L4 localized mainly at the base of the long sensilla trichoidea, which responds to sex pheromone components. Pretreatment with an S. litura sex pheromone significantly reduced the expression levels of SlituCYP4L4, consistent with other genes involved in sex pheromone recognition. The expression level of SlituCYP4L4 was different in moths collected with different ratios of sex pheromone lures and collected in different geographical locations. After gene knockdown of SlituCYP4L4 in the antennae, the electroantennogram (EAG) responses of male and female moths to (9Z,11E)‐tetradecadienyl acetate or (9Z,12E)‐tetradecadienyl acetate were significantly decreased. In contrast, EAG responses to plant volatiles and sex pheromones of other moth species were not significantly influenced in these moths. SlituCYP4L4 was also expressed in the gustatory tissues and sensilla, which suggests that SlituCYP4L4 may have other functions in the chemosensory system. Our results have shown for the first time the function of a CYP gene with appendage‐specific expression in insect sex pheromone recognition, especially in adult moths.     

The sensory neurone membrane protein SNMP1 contributes to the sensitivity of a pheromone detection system

Male moths detect female‐released sex pheromones with extraordinary sensitivity. The remarkable sensory ability is based on a cooperative interplay of pheromone binding proteins in the lymph of hair‐like sensilla trichodea and pheromone receptors in the dendrites of sensory neurones. Here we examined whether in Heliothis virescens the so‐called ‘sensory neurone membrane protein 1’ (SNMP1) may contribute to responsiveness to the pheromone component, (Z)‐11‐hexadecenal (Z11‐16:Ald). By means of immunohistochemistry and in situ hybridization we demonstrated that SNMP1 is in fact present in cells expressing the Z11‐16:Ald receptor HR13 and the dendrites of sensory neurones. To assess a possible function of SNMP1 we monitored the responsiveness of cell lines that expressed HR13 alone or the combination SNMP1/HR13 to stimulation with Z11‐16:Ald by calcium imaging. It was found that SNMP1/HR13 cells were 1000‐fold more sensitive to pheromone stimulation compared with HR13 cells. In contrast, cells that expressed HR13 and the non‐neuronal SNMP2‐type showed no change in pheromone sensitivity. Overall, our reconstitution experiments demonstrate that the presence of SNMP1 significantly increases the HR13‐based responsiveness of cells to Z11‐16:Ald, suggesting that SNMP1 also contributes to the response of the antennal neurones and thus to the remarkable sensitivity of the pheromone detection system.     

Purification to homogeneity and characterization of acyl coenzyme A:6-aminopenicillanic acid acyltransferase of Penicillium chrysogenum.

The acyl coenzyme A (CoA):6-aminopenicillanic acid (6-APA) acyltransferase of Penicillium chrysogenum AS-P-78 was purified to homogeneity, as concluded by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The enzyme is a monomer with a molecular weight of 30,000 +/- 1,000 and a pI of about 5.5. The optimal pH and temperature were 8.0 and 25 degrees C, respectively. This enzyme converts 6-APA into penicillin by using phenylacetyl CoA or phenoxyacetyl CoA as acyl donors. The pure enzyme showed a high specificity and affinity for 6-APA and did not accept benzylpenicillin, 7-aminocephalosporanic acid, cephalosporin C, or isocephalosporin C as substrates. The enzyme converted isopenicillin N into penicillin G, although with a lower efficiency than when 6-APA was used as the substrate. It did not show penicillin G acylase activity. The acyl CoA:6-APA acyltransferase required dithiothreitol or other thiol-containing compounds, and it was protected by thiol-containing reagents against thermal inactivation. The acyltransferase was inhibited by several divalent and trivalent cations and by p-chloromercuribenzoate and N-ethylmaleimide. The activity was absent in four different mutants that were blocked in penicillin biosynthesis.     

Deficient acetyl CoA carboxylase activity in multiple carboxylase deficiency

Multiple carboxylase deficiency has previously been characterized by deficient activity of three biotin-dependent enzymes: propionyl CoA carboxylase, pyruvate carboxylase and beta-methylcrotonyl CoA carboxylase. We have demonstrated that the activity of a fourth carboxylase, acetyl CoA carboxylase (ACC), is also deficient in fibroblasts from two patients with this disorder. Furthermore, ACC activity increased six- to eight-fold when cells from these patients were incubated in culture medium containing supplemental biotin. If the primary defect in multiple carboxylase deficiency is due to deficient activity of holocarboxylase synthetase, our results would indicate that there may be a common holocarboxylase synthetase, or at least a common subunit, for all the carboxylases. Finally, since ACC catalyzes the initial step in fatty acid biosynthesis, our results further suggest the importance of dietary supplementation with fatty acids in addition to treating these patients with pharmacologic doses of biotin.     

In vitro studies of maleidride-forming enzymes

In vitro assays of enzymes involved in the biosynthesis of maleidrides from polyketides in fungi were performed. The results show that the enzymes are closely related to primary metabolism enzymes of the citric acid cycle in terms of stereochemical preferences, but with an expanded substrate selectivity. A key citrate synthase can react both saturated and unsaturated acyl CoA substrates to give solely anti substituted citrates. This undergoes anti-dehydration to afford an unsaturated precursor which is cyclised in vitro by ketosteroid-isomerase-like enzymes to give byssochlamic acid.

In vitro synthesis of byssochkamic acid 12 was achieved from hexenoyl CoA 14dvia anhydride 1.