Hyperammonemia is a frequent finding in various organic acidemias. One possible mechanism involves the inhibition of the enzyme N-acetylglutamate synthase (NAGS), by short-chain acyl-CoAs which accumulate due to defective catabolism of amino acids and/or fatty acids in the cell. The aim of this study was to investigate the effect of various acyl-CoAs on the activity of NAGS in conjunction with the formation of glutamate esters. NAGS activity was measured in vitro using a sensitive enzyme assay with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) product analysis. Propionyl-CoA and butyryl-CoA proved to be the most powerful inhibitors of N-acetylglutamate (NAG) formation. Branched-chain amino acid related CoAs (isovaleryl-CoA, 3-methylcrotonyl-CoA, isobutyryl-CoA) showed less pronounced inhibition of NAGS whereas the dicarboxylic short-chain acyl-CoAs (methylmalonyl-CoA, succinyl-CoA, glutaryl-CoA) had the least inhibitory effect. Subsequent work showed that the most powerful inhibitors also proved to be the best substrates in the formation of N-acylglutamates. Furthermore, we identified N-isovalerylglutamate, N-3-methylcrotonylglutamate and N-isobutyrylglutamate (the latter two in trace amounts), in the urines of patients with different organic acidemias. Collectively, these findings explain one of the contributing factors to secondary hyperammonemia, which lead to the reduced in vivo flux through the urea cycle in organic acidemias and result in the inadequate elimination of ammonia.

The posttranslational modifications induced on PKC isozymes as result of their activation were investigated. Reciprocal immunoprecipitations followed by Western blot analysis demonstrated that all PKC isozymes expressed in rat hepatocytes are modified by tyrosine nitration and tyrosine phosphorylation in different ways upon exposure of cells to a direct PKC activator (TPA), or to an extracellular ligand known to activate PKC-dependent pathways (epinephrine). Our data demonstrate for the first time that all PKC isozymes are also dynamically modified by O-linked beta-N-acetylglucosamine (O-GlcNAc); the presence of this modification was confirmed in part by FT-ICR mass spectrometry analysis. Interestingly, the O-GlcNAc modified Ser or Thr were mapped at similar positions in several PKC isozymes. The biochemical meaning of these posttranslational modifications was investigated for PKC alpha and delta. It was found that the PKC phosphorylation status of both isozymes in tyrosine and serine residues seems to regulate directly the enzyme activity since catalytic inactivation correlate with dephosphorylation of Ser at the C-terminus autophosphorylation sites of each PKC isozyme, and with an increase in the level of tyrosine phosphorylation. Whereas none of the other posttranslational modifications showed per se a direct effect in PKC delta activity, increased tyrosine nitration and O-GlcNAc modifications correlate negatively with PKCalpha activity.

Fatty acid ethyl esters (FAEE) are non-oxidative products of ethanol metabolism that have been proposed to mediate pathological changes in various organs and tissues resulting from excessive ethanol consumption. Evidence supporting this proposal is scant, however, mainly because of the lack of adequate methods with which to solubilize the highly hydrophobic FAEE in aqueous medium for testing under physiological conditions. In this report we describe a simple and practical method for solubilizing FAEE in aqueous medium by binding them to albumin. We also report that the albumin-bound FAEE are readily taken up by rat alveolar macrophages in culture. The availability of FAEE bound to albumin, their main physiological carrier in vivo, will facilitate the investigation of the role that these metabolites may have in mediating pathological changes associated with excess ethanol consumption.

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.

The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca(2+)-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c-jun and c-fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB, PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.

Plasma cholesterol from low- and high-density lipoproteins (LDL and HDL) are cleared from the circulation by specific receptors that either totally degrade lipoproteins as the LDL receptor or selectively take up their cholesteryl esters (CE) like the scavenger receptor class B type I (SR-BI). The aim of the present study was to define the effect of apoC-I on the uptake of LDL and HDL(3) by HepG2 cells. In experiments conducted with exogenously added purified apoC-I, no significant effect was observed on lipoprotein-protein association and degradation; however, LDL- and HDL(3)-CE selective uptake was significantly reduced in a dose-dependent manner. This study also shows that apoC-I has the ability to associate with HepG2 cells and with LDL and HDL(3). Moreover, pre-incubation of HepG2 cells with apoC-I reduces HDL(3)-CE selective uptake and pre-incubation of LDL and HDL(3) with apoC-I decreases their CE selective uptake by HepG2 cells. Thus, apoC-I can accomplish its inhibitory effect on SR-BI activity by either binding to SR-BI or lipoproteins. We conclude that by reducing hepatic lipoprotein-CE selective uptake, apoC-I has an atherogenic character.

Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-β-D-glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates.

In the present study we have investigated the effect of partially purified retinal fatty acid binding protein (FABP) against nonenzymatic lipid peroxidation stimulated by hydroperoxides derived from fatty acids on rod outer segment (ROS) membranes. Linoleic acid hydroperoxide (LHP), arachidonic acid hydroperoxide (AHP) and docosahexaenoic acid hydroperoxide (DHP) were prepared from linoleic acid, arachidonic acid and docosahexaenoic acid, respectively, by means of lipoxidase. ROS membranes were peroxidized using an ascorbate-Fe(+2) experimental system. The effect on the peroxidation of ROS containing different amounts of lipid hydroperoxides (LOOH) was studied; ROS deprived of exogenously added LOOH was utilized as control. The degradative process was measured simultaneously by determining chemiluminescence and fatty acid composition of total lipids isolated from ROS. The addition of hydroperoxides to ROS produced a marked increase in light emission. This increase was hydroperoxide concentration-dependent. The highest value of activation was produced by DHP. The decrease percentage of the more polyunsaturated fatty acids (PUFAs) (20:4 n6 and 22:6 n3) was used to evaluate the fatty acid alterations observed during the process. We have compared the fatty acid composition of total lipids isolated from native ROS and peroxidized ROS that were incubated with and without hydroperoxides. The major difference in the fatty acid composition was found in the docosahexaenoic acid content, which decreased by 45.51+/-1.07% in the peroxidized group compared to native ROS; the decrease was even higher, 81.38+/-1.11%, when the lipid peroxidation was stimulated by DHP. Retinal FABP was partially purified from retinal cytosol. Afterwards, we measured its effect on the reaction of lipid peroxidation induced by LOOH. As a result, we observed a decrease of chemiluminescence (inhibition of lipid peroxidation) when adding increasing amounts (0.2 to 0.6 mg) of retinal FABP to ROS. The inhibitory effect reaches its highest value in the presence of DHP (41.81+/-10.18%). Under these conditions, bovine serum albumin (BSA) produces a smaller inhibitory effect (20.2+/-7.06%) than FABP.

We have recently reported that dietary intake of soybean isoflavone phytoestrogens resulted in increased oxidation resistance of isolated low density lipoprotein (LDL). In order to explore the underlying mechanisms we designed two types of in vitro experiments. First, we prepared several different isoflavone fatty acid esters to increase their lipid solubility and studied their incorporation into LDL. Second, the oxidation resistance of the isoflavone-containing LDLs was investigated with Esterbauer's 'conjugated diene' method using Cu2+ as prooxidant. Unesterified daidzein and genistein as well as genistein stearic acid esters were incorporated into LDL to a relatively small extent (0.33 molecules per LDL particle, or less) and they did not significantly influence oxidation resistance. The oleic acid esters of isoflavones were incorporated more effectively, reaching a level of 2.19 molecules per LDL particle or more, and the 4',7-O-dioleates of daidzein and genistein exhibited prolongations of lag times by 46% (P<0.05) and 202% (P<0.01), respectively. A smaller but significant increase in lag time (20.5%, P<0.01) was caused by daidzein 7-mono-oleate. In summary, esterification of soybean isoflavones daidzein and genistein with fatty acids at different hydroxyl groups provided lipophilicity needed for incorporation into LDL. Some isoflavone oleic acid esters increased oxidation resistance of LDL following their incorporation.

The relationship between cholesterol and atherosclerosis has gained wide credence and red wine polyphenols have been shown to have an anti-atherogenic activity. In the present in vitro studies, we have evaluated and compared the effects of resveratrol, an active compound of red wine, and of a whole red wine polyphenolic extract (RWE) on the pancreatic bile salt-dependent lipase (BSDL). BSDL is involved in the duodenal hydrolysis of lipid esters and in part of cholesteryl esters thus favoring the bioavailability of free cholesterol. Resveratrol and RWE decrease the human and rat enzyme activities. Resveratrol and RWE also impaired the secretion of BSDL by the rat pancreatic AR4-2J cells used as secreting model. This effect is reversed by the removal of resveratrol or RWE from the cell culture medium. Further, resveratrol (but not RWE) affects the transcription of the gene encoding BSDL and dramatically diminishes the quantity of the enzyme that is expressed and secreted by AR4-2J cells. Results suggest that the hypolipemic effects of red wine polyphenols could partly originate from the inhibition of BSDL activity and secretion in the duodenum. In vivo, these effects could decrease the hydrolysis of dietary lipid esters and likely the absorption of free cholesterol.

Lipolysis of stored triacylglycerols provides lipid precursors for the assembly of apolipoprotein B (apoB) lipoproteins in hepatocytes. Abhydrolase domain containing 5 (ABHD5) is expressed in liver and facilitates the lipolysis of triacylglycerols. To study the function of ABHD5 in lipoprotein secretion, we silenced the expression of ABHD5 in McA RH7777 cells using RNA interference and studied the metabolism of lipids and secretion of apoB lipoproteins. McA RH7777 cells deficient in ABHD5 secreted reduced amounts of apoB, triacylglycerols, and cholesterol esters. Detailed analysis of liquid chromatography-mass spectrometry data for the molecular species of secreted triacylglycerols revealed that deficiency of ABHD5 significantly reduced secretion of triacylglycerols containing oleate, even when oleate was supplied in the culture medium; the ABHD5-deficient cells partially compensated by secreting higher levels of triacylglycerols containing saturated fatty acids. In experiments tracking the metabolism of [(14)C]oleate, silencing of ABHD5 reduced lipolysis of cellular triacylglycerols and incorporation of intermediates derived from stored lipids into secreted triacylglycerols and cholesterol esters. In contrast, the incorporation of exogenous oleate into secreted triacylglycerols and cholesterol esters was unaffected by deficiency of ABHD5. These findings suggest that ABHD5 facilitates the use of lipid intermediates derived from lipolysis of stored triacylglycerols for the assembly of lipoproteins.

Some recent studies have reported that low-density lipoprotein (LDL) isolated from estrogen-treated postmenopausal women exhibited increased oxidation resistance ex vivo. However, the underlying mechanisms responsible for this effect are not clear. We explored the possibility that lipophilic derivatives of 17beta-estradiol (E(2)) could be incorporated into LDL and high-density lipoprotein (HDL) particles inhibiting lipoprotein oxidation. Introduction of small amounts of esterified E(2) into lipoproteins by means of incubation of free E(2) and E(2) 17-stearate in plasma did not result in any antioxidant effect. Using an artificial transfer system (Celite dispersion), larger amounts of E(2) esters could be incorporated into lipoproteins. Concentrations ranging between 0.27 and 1.38 molecules/LDL particle for E(2) 17-stearate and between 0.36 and 1.93 molecules/LDL particle for E(2) 17-oleate resulted in increased Cu(2+)-induced oxidation resistance of LDL as indicated by statistically significant lag time prolongations. Significant prolongations of lag times were also observed for HDL following incorporation of E(2) esters using Celite as transfer system. Our results suggest that free E(2) can be esterified and incorporated into lipoproteins during incubation in plasma. However, incorporation of supraphysiologic concentrations of E(2) esters into lipoproteins by means of the artificial transfer system was required in order to reduce their oxidation susceptibility.

Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.

Streptococcus pneumoniae is a major pathogen responsible of important diseases worldwide such as pneumonia and meningitis. An increasing resistance level hampers the use of currently available antibiotics to treat pneumococcal diseases. Consequently, it is desirable to find new targets for the development of novel antimicrobial drugs to treat pneumococcal infections. Surface choline-binding proteins (CBPs) are essential in bacterial physiology and infectivity. In this sense, esters of bicyclic amines (EBAs) such as atropine and ipratropium have been previously described to act as choline analogs and effectively compete with teichoic acids on binding to CBPs, consequently preventing in vitro pneumococcal growth, altering cell morphology and reducing cell viability.

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes. Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.

beta2-Chimaerin, an intracellular receptor for the second messenger diacylglycerol and phorbol esters, is a GTPase-activating protein (GAP) specific for Rac. beta2-Chimaerin negatively controls many Rac-dependent pathophysiological events including tumor development. However, the regulatory mechanism of beta2-chimaerin remains largely unknown. Here we report that beta2-chimaerin is tyrosine-phosphorylated by Src-family kinases (SFKs) upon cell stimulation with epidermal growth factor (EGF). Mutational analysis identified Tyr-21 in the N-terminal regulatory region as a major phosphorylation site. Intriguingly, the addition of SFK inhibitor and the replacement of Tyr-21 with Phe (Y21F) markedly enhanced Rac-GAP activity of beta2-chimaerin in EGF-treated cells. Moreover, the Y21F mutant inhibited integrin-dependent cell spreading, in which Rac1 plays a critical role, more strongly than wild-type beta2-chimaerin. These results suggest Tyr-21 phosphorylation as a novel, SFK-dependent mechanism that negatively regulates beta2-chimaerin Rac-GAP activity.

We have investigated the mechanistic basis of our recent observation that exposing young mice to an industrial surfactant potentiates the inhibition of fatty-acid beta-oxidation that occurs with subsequent virus infection (Murphy et al., Biochim. Biophys. Acta 1315, 208-216, 1996). In our mouse model for acute hepatic encephalopathy (AHE), neonatal mice were painted on their abdomens from birth to postnatal day 12 with nontoxic amounts of the industrial surfactant, Toximul MP8 (Tox), and then infected with a sublethal dose (LD30) of mouse-adapted human Influenza B (Lee) virus (FluB). Mortality in mice treated with Tox + FluB was significantly higher than that in mice treated with FluB alone. In vitro assays of hepatic beta-oxidation of [1-(14)C]palmitic and [1-(14)C]octanoic acids in the presence or absence of exogenous coenzyme A (CoA) indicated that Tox-mediated inhibition of oxidation was masked when CoA was added to the assays. FluB also inhibited beta-oxidation by 20-30%, however this effect was independent of exogenous CoA which suggested that it involved a different mechanism. Tox-mediated potentiation of the inhibitory effect was most obvious (> 80% inhibition) when assays were done without added CoA. Analysis of hepatic CoA and its esters indicated that levels of both free CoA and acetyl-CoA were significantly lower in mice that were painted with Tox for 12 days. Tox-dependent reductions of acetyl-CoA were transient and returned to normal values after cessation of painting, whereas those of CoA persisted. FluB infection alone significantly reduced hepatic acetyl-CoA and the magnitude of this reduction (> 30%) was not affected by pre-exposing the mice to Tox. Relative to control mice, levels of acid insoluble acyl-CoA esters were elevated significantly in FluB and Tox + FluB treated mice. Activation of both [1-(14)C]palmitic and [1-(14)C]octanoic acids was reduced in Tox-exposed mice at experimental day 12, but only when exogenous CoA was not included in the assay media; this effect appeared to persist after cessation of painting. Collectively, these data support the concept that Tox and FluB have independent effects on hepatic CoA metabolism that are associated with abnormalities in fatty-acid beta-oxidation. However, these do not fully explain the synergistic effect of the virus and chemical on beta-oxidation inhibition, which is a candidate co-mechanism for potentiation of mortality in this mouse model of AHE.

It is known that protein kinase C (PKC) signal transduction is enhanced in a diabetic state, and that PKC activator phorbol esters increase the gene expression of MDR1 in human tumor cells. To clarify the expression of the liver transporters under diabetic conditions and the roles of PKCalpha and the transcription factor NF-kappaB, we investigated the expression levels of Mdr1a, Mdr1b, Mdr2, Mrp2, Bcrp, Bsep, Oct1, Oat2, and Oat3 transporters, PKCalpha, IkappaB, and NF-kappaB in the liver of rats with STZ-induced hyperglycemia. A selective increase in the gene expression of Mdr1b was detected by RT-PCR. Western blotting with C219 antibody revealed an increase in P-glycoprotein. Although the mRNA level of PKCalpha was not affected, translocation of PKCalpha to the microsomal fraction was detected. NF-kappaB p65, IkappaBalpha and IkappaBbeta mRNA levels were increased as was the level of nuclear NF-kappaB p65. From these findings, it was suggested that STZ-induced hyperglycemia caused the upregulation of Mdr1b P-gp expression through the activation of PKCalpha and NF-kappaB.

Nannochloropsis gaditana belongs to Eustigmatophyceae, a class of eukaryotic algae resulting from a secondary endosymbiotic event. Species of this class have been poorly characterized thus far but are now raising increasing interest in the scientific community because of their possible application in biofuel production. Nannochloropsis species have a peculiar photosynthetic apparatus characterized by the presence of only chlorophyll a, with violaxanthin and vaucheriaxanthin esters as the most abundant carotenoids. In this study, the photosynthetic apparatus of this species was analyzed by purifying the thylakoids and isolating the different pigment-binding complexes upon mild solubilization. The results from the biochemical and spectroscopic characterization showed that the photosystem II antenna is loosely bound to the reaction center, whereas the association is stronger in photosystem I, with the antenna-reaction center super-complexes surviving purification. Such a supramolecular organization was found to be conserved in photosystem I from several other photosynthetic eukaryotes, even though these taxa are evolutionarily distant. A hypothesis on the possible selective advantage of different associations of the antenna complexes of photosystems I and II is discussed.