Vorinostat in the acute neuroinflammatory form of X-linked adrenoleukodystrophy.

OBJECTIVE: To identify a pharmacological compound targeting macrophages, the most affected immune cells in inflammatory X-linked adrenoleukodystrophy (cerebral X-ALD) caused by ABCD1 mutations and involved in the success of hematopoietic stem cell transplantation and gene therapy. METHODS: A comparative database analysis elucidated the epigenetic repressing mechanism of the related ABCD2 gene in macrophages and identified the histone deacetylase (HDAC) inhibitor Vorinostat as a compound to induce ABCD2 in these cells to compensate for ABCD1 deficiency. In these cells, we investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFAs) and their differentiation status. We investigated ABCD2 and pro-inflammatory gene expression, restoration of defective peroxisomal ß-oxidation activity, accumulation of very long-chain fatty acids (VLCFA) and differentiation status. Three advanced cerebral X-ALD patients received Vorinostat and CSF and MRI diagnostics was carried out in one patient after 80 days of treatment. RESULTS: Vorinostat improved the metabolic defects in X-ALD macrophages by stimulating ABCD2 expression, peroxisomal ß-oxidation, and ameliorating VLCFA accumulation. Vorinostat interfered with pro-inflammatory skewing of X-ALD macrophages by correcting IL12B expression and further reducing monocyte differentiation. Vorinostat normalized the albumin and immunoglobulin CSF-serum ratios, but not gadolinium enhancement upon 80 days of treatment. INTERPRETATION: The beneficial effects of HDAC inhibitors on macrophages in X-ALD and the improvement of the blood-CSF/blood-brain barrier are encouraging for future investigations. In contrast with Vorinostat, less toxic macrophage-specific HDAC inhibitors might improve also the clinical state of X-ALD patients with advanced inflammatory demyelination.

Valnoctamide, a non-teratogenic amide derivative of valproic acid, inhibits arachidonic acid activation in vitro by recombinant acyl-CoA synthetase-4.

OBJECTIVE: Valproic acid (VPA), a mood stabilizer used for treating bipolar disorder (BD), uncompetitively inhibits acylation of arachidonic acid (AA) by recombinant AA-selective acyl-CoA synthetase 4 (Acsl4) at an enzyme inhibition constant (Ki ) of 25 mM. Inhibition may account for VPA's ability to reduce AA turnover in brain phospholipids of unanesthetized rats and to be therapeutic in BD. However, VPA is teratogenic. We tested whether valnoctamide (VCD), a non-teratogenic amide derivative of a VPA chiral isomer, which had antimanic potency in a phase III BD trial, also inhibits recombinant Acsl4. METHODS: Rat Acsl4-flag protein was expressed in Escherichia coli. We used Michaelis-Menten kinetics to characterize and quantify the ability of VCD to inhibit conversion of AA to AA-CoA by recombinant Acsl4 in vitro. RESULTS: Acsl4-mediated activation of AA to AA-CoA by Acsl4 was inhibited uncompetitively by VCD, with a Ki of 6.38 mM. CONCLUSIONS: VCD's ability to uncompetitively inhibit AA activation to AA-CoA by Acsl4, at a lower Ki than VPA, suggests that, like VPA, VCD may reduce AA turnover in rat brain phospholipids. If so, VCD and other non-teratogenic Acsl4 inhibitors might be considered further for treating BD.

Long chain fatty acyl-CoA synthetase 5 expression is induced by insulin and glucose: involvement of sterol regulatory element-binding protein-1c.

In a screen for sterol regulatory element-binding protein (SREBP)-1c target genes in the liver, we identified long chain fatty acyl-CoA synthetase 5 (ACS-5). Hepatic ACS-5 mRNA is poorly expressed during fasting and diabetes and strongly induced by carbohydrate refeeding and insulin treatment. In cultured hepatocytes, insulin and a high glucose concentration induce ACS-5 mRNA. Adenoviral overexpression of a nuclear form of SREBP-1c in liver of diabetic mice or in cultured hepatocytes mimics the effect of insulin to induce ACS-5. By contrast, a dominant negative form of SREBP-1c abolishes the effect of insulin on ACS-5 expression. The dietary and SREBP-1c-mediated insulin regulation of ACS-5 expression indicate that ACS-5 is involved in the anabolic fate of fatty acids.

Eicosapentaenoic acid-induced apoptosis depends on acyl CoA-synthetase.

Marine n-3 FA are known to inhibit proliferation or induce cell death in several cancer cell lines. We have previously reported that EPA promotes apoptosis in the lymphoma cell line Ramos, whereas the U-698 cell line is insensitive to EPA. Furthermore, acyl-CoA synthetase (ACS) is expressed to a higher extent in Ramos cells compared to U-698 cells. To investigate the importance of ACS in EPA-induced apoptosis, we incubated Ramos cells with triacsin C, an inhibitor of ACS. This caused a 70% reduction in the amount of cell-associated EPA and diminished activation of EPA. In addition, triacsin C caused a 90% reduction in EPA-induced apoptosis. Several different approaches were tried to overexpress ACS4 in EPA-insensitive lymphoma cell lines, but we did not obtain viable cells with high expression of acyl-CoA activation. However, we show that overexpression of ACS4 in the more robust COS-1 cells caused up to a fivefold increase in activation of EPA and a 67% increase in the amount of cell-associated radiolabeled EPA. Furthermore, we observed 28% elevated cellular level of TAG in EPA-incubated COS-1 cells overexpressing ACS4. The present study provides new information about ACS as an important enzyme for EPA-induced apoptosis in Ramos cells. Our data offer a potential mechanism that may explain the effect of dietary marine n-3 PUFA on growth of certain malignant cells.

The effects of a high-fat or high-sucrose diet on serum lipid profiles, hepatic acyl-CoA synthetase, carnitine palmitoyltransferase-I, and the acetyl-CoA carboxylase mRNA levels in rats.

The purpose of this study was to investigate the effects of altering relative intakes of fat and carbohydrates on serum lipid profiles, hepatic acyl-CoA synthetase (ACS), carnitine palmitoyltransferase-I (CPT-I), and the acetyl-CoA carboxlyase (ACC) mRNA level in Sprague-Dawley rats. For four weeks the rats were fed either an AIN-76 diet or one of its modified diets that were supplemented with 20% beef tallow (high-fat diet, HF) and 66.3% sucrose (high-sucrose diet, HS). The HS group had significantly higher serum triglyceride and total cholesterol concentrations when compared with the other groups. Serum LDL-cholesterol concentrations in the HS and HF groups were significantly higher when compared to the normal diet (ND) group. Serum HDL-cholesterol levels of the ND and HS groups were significantly higher than those of the HF group. The hepatic total lipid level of the HF group was significantly higher than those of other groups; triglyceride levels of the HS and HF groups were significantly higher than those of the ND group. Hepatic ACS mRNA levels of the HF group were significantly higher than those of the ND group. Hepatic CPT-I mRNA levels were higher in the HF group than other groups. Also, ACC mRNA levels in the liver increased in the HF group. In conclusion, changes in the composition of dietary fat and carbohydrates could affect the hepatic ACS, CPT-I, and ACC mRNA levels. These results facilitate our understanding of the coordinated regulation of the ACS, CPT-I, and ACC mRNA levels and will serve to enhance our understanding of the molecular mechanisms that underlie the regulation of fatty acid metabolism.

Benzoic acid biosynthesis in cell cultures of Hypericum androsaemum.

Biosynthesis of benzoic acid from cinnamic acid has been studied in cell cultures of Hypericum androsaemum L. The mechanism underlying side-chain shortening is CoA-dependent and non-beta-oxidative. The enzymes involved are cinnamate:CoA ligase, cinnamoyl-CoA hydratase/lyase and benzaldehyde dehydrogenase. Cinnamate:CoA ligase was separated from benzoate:CoA ligase and 4-coumarate:CoA ligase, which belong to xanthone biosynthesis and general phenylpropanoid metabolism, respectively. Cinnamoyl-CoA hydratase/lyase catalyzes hydration and cleavage of cinnamoyl-CoA to benzaldehyde and acetyl-CoA. Benzaldehyde dehydrogenase finally supplies benzoic acid. In cell cultures of H. androsaemum, benzoic acid is a precursor of xanthones, which accumulate during cell culture growth and after methyl jasmonate treatment. Both the constitutive and the induced accumulations of xanthones were preceded by increases in the activities of all benzoic acid biosynthetic enzymes. Similar changes in activity were observed for phenylalanine ammonia-lyase and the xanthone biosynthetic enzymes benzoate:CoA ligase and benzophenone synthase.

HbaR, a 4-hydroxybenzoate sensor and FNR-CRP superfamily member, regulates anaerobic 4-hydroxybenzoate degradation by Rhodopseudomonas palustris.

Under anaerobic conditions, structurally diverse aromatic compounds are catabolized by bacteria to form benzoyl-coenzyme A (benzoyl-CoA), the starting compound for a central reductive pathway for aromatic ring degradation. The structural genes required for the conversion of 4-hydroxybenzoate (4-HBA) to benzoyl-CoA by Rhodopseudomonas palustris have been identified. Here we describe a regulatory gene, hbaR, that is part of the 4-HBA degradation gene cluster. An hbaR mutant that was constructed was unable to grow anaerobically on 4-HBA. However, the mutant retained the ability to grow aerobically on 4-HBA by an oxygen-requiring pathway distinct from the anaerobic route of 4-HBA degradation. The effect of the HbaR protein on expression of hbaA encoding 4-HBA-CoA ligase, the first enzyme for 4-HBA degradation, was investigated by using hbaA::'lacZ transcriptional fusions. HbaR was required for a 20-fold induction of beta-galactosidase activity that was observed with a chromosomal hbaA::'lacZ fusion when cells grown anaerobically on succinate were switched to anaerobic growth on succinate and 4-HBA. HbaR also activated expression from a plasmid-borne hbaA-'lacZ fusion when it was expressed in aerobically grown Pseudomonas aeruginosa cells, indicating that the activity of this regulator is not sensitive to oxygen. The deduced amino acid sequence of HbaR indicates that it is a member of the FNR-CRP superfamily of regulatory proteins. It is most closely related to transcriptional activators that are involved in regulating nitrate reduction. Previously, it has been shown that R. palustris has an FNR homologue, called AadR, that is also required for 4-HBA degradation. Our evidence indicates that AadR activates expression of hbaR in response to anaerobiosis and that HbaR, in turn, activates expression of 4-HBA degradation in response to 4-HBA as an effector molecule.

Cholesterol-lowering effects of NTE-122, a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, on cholesterol diet-fed rats and rabbits.

Pharmacological characterization of NTE-122 (trans-1,4-bis[[1-cyclohexyl-3-(4-dimethylamino phenyl)ureido]methyl]cyclohexane), a novel acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, was performed with both in vitro and in vivo assay systems. NTE-122 inhibited microsomal ACAT activities of various tissues (liver of rabbit and rat, small intestine of rabbit and rat, and aorta of rabbit) and cultured cells (HepG2 and CaCo-2), with IC50 values from 1.2 to 9.6 nM. The inhibition mode of NTE-122 was competitive for HepG2 ACAT. NTE-122 had no effect on other lipid metabolizing enzymes, such as 3-hydroxy-3-methylglutaryl-CoA reductase, acyl-CoA synthetase, cholesterol esterase, lecithin:cholesterol acyltransferase, acyl-CoA:sn-glycerol-3-phosphate acyltransferase and cholesterol 7alpha-hydroxylase up to 10 microM. When NTE-122 was administered to the cholesterol diet-fed rats, serum and liver cholesterol levels were markedly reduced with an ED50 of 0.12 and 0.44 mg/kg/day, respectively. In the cholesterol diet-fed rabbits, NTE-122 significantly lowered plasma and liver cholesterol levels at more than 2 mg/kg/day. These results indicate that NTE-122 is a potent, selective and competitive inhibitor of ACAT, making it a worth while therapeutic agent for hypercholesterolemia and atherosclerosis.

Characterization of Aquamicrobium defluvii gen. nov. sp. nov., a thiophene-2-carboxylate-metabolizing bacterium from activated sludge.

A gram-negative bacterium was isolated from activated sewage sludge with thiophene-2-carboxylate as the sole source of carbon and with nitrate as an electron acceptor. The isolate, strain NKK, was a motile, oxidase- and catalase-positive, rod-like bacterium with a G+C content of 61.7 mol%. Besides nitrate, oxygen could serve as a terminal electron acceptor. Among many carbon sources tested, only a few sugars, fatty acids, and thiophene-2-carboxylate supported growth. Other heterocyclic compounds were not used. The sulfur atom of thiophene-2-carboxylate was oxidized to thiosulfate when cells were grown aerobically, or to elemental sulfur when cells were grown anaerobically with nitrate. Nitrate was reduced to nitrite. Growth on thiophene-2-carboxylate was dependent on the addition of molybdate to the medium. Tungstate, a specific antagonist of molybdate, inhibited growth on thiophene-2-carboxylate at concentrations > 10(-7) M. Three inducible enzymes involved in the metabolism of thiophene-2-carboxylate were detected: an ATP-, CoA-, thiophene-2-carboxylate- and Mg2+-dependent thiophene-2-carboxyl-CoA ligase (AMP-forming), a molybdenum-containing thiophene-2-carboxyl-CoA dehydrogenase, and a thiophene-2-carboxyl-CoA thioesterase. The sequence of the 16S rRNA gene suggested a classification of strain NKK within the alpha-subgroup of the Proteobacteria as a new genus and species, Aquamicrobium defluvii gen. nov. sp. nov. (DSM 11603), closely related to Mesorhizobium sp. and Phyllobacterium sp., but representing a distinct lineage equal in depth to those of the two mentioned genera. Aquamicrobium defluvii can be distinguished from both genera by a distinct spectrum of substrates, the maximal growth temperature, and a different salt tolerance.

Detection of acyl-CoA synthetase, acyl-CoA:lysophospholipid acyltransferase and phospholipase A2 activities in non-pregnant and pregnant guinea-pig uterine tissues.

Acyl-CoA synthetase (ACS), acyl-CoA:lysophospholipid acyltransferase (ACLAT) and phospholipase (PL) A2 activities were detected in guinea-pig endometrium on days 7 and 15 of the cycle, and on days 15, 29 and 36 of pregnancy. Ovariectomy of non-pregnant animals resulted in an increase in the apparent activities of these three enzymes which was reversed by treatment with oestradiol and/or progesterone. ACS, ACLAT and PLA2 activities were detected in day 15 conceptuses, and in the placenta, sub-placenta, chorion and amnion on days 29 and 36 of pregnancy. Apparent activities of the enzymes were generally higher in the fetal membranes than in the placental tissue. This study has established that the enzymes involved in turnover of arachidonic acid in phospholipids are present in tissues in the non-pregnant and pregnant guinea-pig uterus. The higher apparent activities of enzymes (ACS and ACLAT) involved in arachidonic acid uptake compared to the enzyme (PLA2) involved in arachidonic acid release is in agreement with there being very low concentrations of free arachidonic acid in tissues.

Modulation of methylnitrosourea-induced breast cancer in Sprague Dawley rats by dehydroepiandrosterone: dose-dependent inhibition, effects of limited exposure, effects on peroxisomal enzymes, and lack of effects on levels of Ha-Ras mutations.

Dehydroepiandrosterone (DHEA), the major steroid precursor of androgens and estrogens produced in peripheral tissues in primates, is an effective chemopreventive agent in the N-methyl-N-nitrosourea (MNU)-induced rat mammary tumor model. Dietary DHEA (5-600 ppm; 600 mg/kg diet) was administered beginning 1 week before MNU and administered continually throughout the duration of the experiment. The highest dose of DHEA (600 ppm) significantly decreased tumor incidence from 95 to 45% and increased tumor latency and decreased tumor multiplicity from 4.1 to 0.5 tumors/rat. Lower doses of DHEA (5, 24, and 120 ppm) were also effective, decreasing tumor multiplicity by 28, 40, and 55%, respectively, increasing tumor latency in a dose-dependent manner but only minimally affecting final tumor incidence. DHEA in the diet caused a dose-dependent increase in serum levels of DHEA. The 120-ppm dietary dose of DHEA resulted in serum levels of DHEA of approximately 42 pmol/ml levels, similar to those seen in young humans. When we examined whole mounts of mammary glands derived from rats exposed to higher levels of DHEA (600 ppm), we observed a striking increase in lobular development. The doses of DHEA used in these studies (< or =600 ppm) had minimal effects on the induction of fatty acid CoA synthetase, a peroxisome-associated enzyme. In contrast, a dose of 2000 ppm substantially increased levels of peroxisome-associated fatty acid CoA synthetase. The varied and striking efficacy of DHEA was achieved in the absence of any significant effect on body weight gain in the treated rats. Furthermore, tumors from rats treated with MNU alone or rats treated with MNU plus DHEA were examined for the presence of mutations in the Ha-Ras oncogene. There was a slight decrease in the percentage of tumors bearing Ha-Ras mutations in tumors derived from MNU-control rats as contrasted with tumors from MNU-DHEA (120 and 600 ppm)-treated rats. Based on the striking chemopreventive efficacy of continual exposure to DHEA, we examined the effects of more limited exposure to DHEA. Rats were treated with DHEA for a period of 7 weeks immediately before and after MNU injection. Rats were then placed on the control diet for the ensuing 15 weeks. Even this limited exposure to DHEA for a period of 7 weeks profoundly decreased final tumor incidence and multiplicity. Additionally, we examined the effects of intermittent dosing with DHEA. Rats were treated alternatively at 3-week intervals either with diet containing DHEA or with control diet. It was found that this intermittent dosing with DHEA also substantially inhibited the formation of mammary tumors.

Comparison of long-chain fatty acyl-CoA synthetases from rabbit heart and liver: substrate preferences and effects of Mg2+.

Rabbit heart has a single, non-specific, fatty acyl-CoA synthetase (HP1) which is dependent on Mg2+, apart from the requirement for MgATP2-. Two long-chain fatty acyl-CoA synthetase activities (LP1 and LP2) can be resolved by hydroxyapatite chromatography of liver preparations; the Mg2+ requirement for these enzymes is undefined. These experiments were done to define the Mg2+ requirements of the liver enzymes and to compare them with the heart enzyme. For all three sources of enzyme and for arachidonic, oleic and palmitic acid substrates, the overall velocity of the reaction increased as [Mg2+] increased. Depending on the substrate and the source of enzyme, the increase in overall velocity could be attributed to changes in affinity or maximal velocity or both. The substrate preference of the HP1 enzyme for arachidonic acid (AA) was fifth or sixth of eight substrates regardless of the concentration of Mg2+. In contrast, increasing [Mg2+] shifted the relative substrate preference of both liver enzymes for AA. At low [Mg2+], AA was ranked seventh or eighth (least preferred) of eight substrates, whereas at high [Mg2+], AA was ranked as fifth or sixth. Hill plots of competition studies were consistent with Mg2+-induced positive co-operativity in LP1, but not in HP1 or LP2. Although enzymes from the three sources exhibit substantial kinetic differences, it is uncertain whether they are three different enzymes.

Species differences in the intracellular distribution of ciprofibroyl-CoA hydrolase. Implications for peroxisome proliferation.

Peroxisomal proliferators (HPP), such as ciprofibrate and clofibric acid, are species-specific drugs. Since HPP-coenzyme A derivatives might be involved in their action, we studied the subcellular distribution of liver ciprofibroyl-CoA hydrolase in rat and in two HPP-unresponsive species, humans and guinea pig. Total activity was similar in the three species and was not induced by clofibric acid treatment. In guinea pig, as in humans, the enzyme is localized in the mitochondrial and soluble fractions and no changes are observed after drug treatment. In the rat, the enzyme has a microsomal localization, but upon clofibric acid treatment it changes to a mitochondrial and soluble distribution, as in unresponsive species. These results raise the possibility that drug-induced hydrolases in rats might be normally expressed in humans and guinea pigs.

Phytanic acid oxidation: topographical localization of phytanoyl-CoA ligase and transport of phytanic acid into human peroxisomes.

To understand the possible role of phytanoyl-CoA ligase, present in the membrane, in the oxidation of phytanic acid in the matrix of peroxisomes (Pahan, K. and I. Singh. 1993. FEBS Lett. 333: 154-158) we examined the transport of phytanic acid/phytanoyl-CoA into peroxisomes and the topology of the active site of phytanoyl-CoA ligase in the peroxisomal membrane. The increase in lignoceroyl-CoA ligase as compared to no change in the activities of palmitoyl-CoA and phytanoyl-CoA ligases when peroxisomes were disrupted with detergent or sonication and inhibition of the activities of both palmitoyl-CoA and phytanoyl-CoA ligase by impermeable inhibitor of acyl-CoA ligases (mercury-dextran) and trypsin treatment in the intact peroxisomes. On the other hand, the lignoceroyl-CoA ligase activity was inhibited by mercury-dextran and trypsin only in the disrupted peroxisomes. Taken together, these studies support the conclusion that the enzymatic site of phytanoyl-CoA ligase is on the cytoplasmic surface of peroxisomal membrane. This implies that phytanoyl-CoA is synthesized on the cytoplasmic surface of peroxisomal membrane and is translocated through the membrane for its alpha-oxidation to pristanic acid in the matrix of peroxisomes. To delineate the transport for phytanic acid through the peroxisomal membrane, we examined cofactors and energy requirements for its transport into peroxisomes. The similar rates of transport of phytanoyl-CoA and phytanic acid under conditions favorable for fatty acid activation (presence of ATP, CoASH, and MgCl2) and the lack of transport of phytanic acid when ATP and/or CoASH were removed or replaced with their inactive analogues (ATP and/or CoASH) from assay medium clearly demonstrates that the transport of phytanic acid requires prior synthesis of phytanoyl-CoA by phytanoyl-CoA ligase. The prerequisite activation of phytanic acid to phytanoyl-CoA for its alpha-oxidation only in intact peroxisomes, and oxidation of free phytanic acid in digitonin-permealized peroxisomes or isolated matrix, suggests that phytanoyl-CoA ligase (in peroxisomal membrane) regulates the oxidation of phytanic acid in peroxisomes by providing phytanoyl-CoA for its transport into peroxisomes.

Effects of tetradecylthiopropionic acid and tetradecylthioacrylic acid on rat liver lipid metabolism.

Studies of effects of 4-thia-substituted fatty acid analogues on rat liver lipid metabolism are described. With isolated hepatocytes tetradecylthiopropionate was shown to divert [1-14C]oleate from beta-oxidation into esterification, the total amount of [1-14C]oleate metabolized remaining unchanged. Tetradecylthiopropionyl-CoA was a good substrate for mitochondrial carnitine palmitoyltransferases I and II (EC 2.3.1.21), acyl-CoA oxidase (EC 1.3.3.6), for the microsomal (but not mitochondrial) glycerophosphate acyltransferase (EC 2.3.1.15), and for long-chain acyl-CoA dehydrogenase (EC 1.3.99.3). In isolated hepatocytes, its 4-thia-trans-2-enoic derivative, tetradecylthioacrylate, inhibits both beta-oxidation of, and incorporation of, [1-14C]oleate into lipids. In rat liver mitochondria tetradecylthiocrylate inhibited beta-oxidation. The degree of inhibition was not markedly increased by preincubation with tetradecylthioacrylate. Tetradecylthioacrylyl-CoA was a poor substrate for carnitine palmitoyltransferase I, and inhibited carnitine palmitoyltransferase II, microsomal glycerophosphate acyltransferase and acyl-CoA oxidase. It is concluded that the inhibitory effects of tetradecylthiopropionyl-CoA are expressed intramitochondrially, whereas primary sites of inhibition by tetradecylthioacrylyl-CoA are extramitochondrial.

Fatty acid uptake in Escherichia coli: regulation by recruitment of fatty acyl-CoA synthetase to the plasma membrane.

Fatty acid uptake in Escherichia coli has been shown to be inhibited by starvation and to be reversed by a short preincubation of the starved cells with D- or L-lactate, succinate, and acetate; these effects on oleate uptake were due to regulation of the rate-limiting step which involves fatty acyl-CoA synthetase. Investigation into the mechanism of regulation of fatty acyl-CoA synthetase showed that D-lactate did not affect the activity of the enzyme directly. Fatty acyl-CoA synthetase was found to be activated by about 20-fold by Triton X-100 and by another 4-fold by the addition of bacterial membranes. D-Lactate treatment was shown to result in coisolation of fatty acyl-CoA synthetase with the plasma membrane; these results are consistent with the interpretation that recruitment of the enzyme to the plasma membrane by D-lactate results in its activation and consequently in the increased level of fatty acid uptake.

Mechanism of action of atpenin B on Raji cells.

Atpenin B, a new antifungal antibiotic produced by Penicillium sp. FO-125, inhibited the growth of Raji cells (IC50, 30 microM). The incorporation of [14C]leucine and [3H]thymidine into Raji cells was inhibited by atpenin B with IC50 values of 0.10 and 0.12 microM, respectively. The incorporation of [14C]palmitate into the cells was not inhibited but its incorporation into lipid fractions was inhibited by atpenin B (IC50, 0.13-0.24 microM). Studies on the site of atpenin B action demonstrated that atpenin B decreases the cellular adenosine 5'-triphosphate (ATP) level in Raji cells with IC50 value of 0.020 microM, suggesting the inhibition of ATP-generating system by atpenin B.