BBA - Molecular and Cell Biology of Lipids (v.1484, #2-3)

Glucosylcaldarchaetidylglycerol, a minor phosphoglycolipid from Thermoplasma acidophilum by Ikuko Uda; Akihiko Sugai; Akio Shimizu; Yuko H. Itoh; Toshihiro Itoh (83-86).
A novel phosphoglycolipid (GPL-K) was isolated from Thermoplasma acidophilum (ATCC 27658). The chemical components of GPL-K were analyzed by gas liquid chromatography and GC-MS. The sugar moiety of GPL-K and its anomeric region were analyzed by NMR assignment. The core lipid of GPL-K was caldarchaeol, and its main hydrocarbon chains were acyclic and monocyclic C40 biphytanyl. The polar head groups were α-glucose and glycerophosphate. The negative FAB-MS spectrum of GPL-K confirmed that the lipid peak of m/z 1614 consists of a caldarchaeol (including one cyclopentane ring), a hexose sugar, and a glycerophosphate. We have proposed the tentative structure of GPL-K.
Keywords: Ether lipid; Phosphoglycolipid; Archaea; Thermoplasma acidophilum;

The change in leukotrienes and lipoxins in activated mouse peritoneal macrophages by Lido Calorini; Antonella Mannini; Francesca Bianchini; Gabriele Mugnai; Salvatore Ruggieri (87-92).
The aim of this study was to investigate to what extent the generation of leukotrienes (LTs) and lipoxins (LXs) was affected by the expression of definite levels of macrophage activation. We used a system of murine peritoneal macrophages at different states of activation consisting in resident macrophages and FCS-, thioglycollate- or Corynebacterium parvum-elicited macrophages. The profile of lipoxygenase metabolites in resident macrophages was characterized by the presence of high levels of 12-HETE, followed by 15-HETE, 5-HETE, LTB4 and 6-trans-LTB4, 6-trans-12-epi-LTB4. A comparable pattern was also found in FCS-elicited macrophages which appeared not to be responsive to the challenge with interferon γ plus LPS, as measured by the generation of NO and tumor necrosis factor α. Resident as well as FCS-elicited macrophages also generated appreciable quantities of LXs (A4 and B4). Thioglycollate-elicited macrophages, which expressed a state of ‘responsive’ macrophages, showed a block of the LT and LX synthesis. This block was also present in C. parvum-elicited macrophages which expressed a fully ‘activated’ phenotype, reflected by their capacity of releasing NO and tumor necrosis factor α even though they were not challenged. These results provide the first evidence that the level of ‘responsive’ as well as ‘activated’ macrophages was associated with of a simultaneous block of LTB4 and LXs.
Keywords: Resident macrophage; Fetal calf serum-elicited macrophage; Thioglycollate-elicited macrophage; Corynebacterium parvum-elicited macrophage; Prostaglandin; Leukotriene; Lipoxin; 15-Hydroxy-eicosatetraenoic acid; 12-Hydroxy-eicosatetraenoic acid; 5-Hydroxy-eicosatetraenoic acid;

Post-translational modification of proteins with isoprenoids was first recognized as a general phenomenon in 1984. In recent years, our understanding, including mechanistic studies, of the enzymatic reactions associated with these modifications and their physiological functions has increased dramatically. Of particular functional interest is the role of prenylation in facilitating protein–protein interactions and membrane-associated protein trafficking. The loss of proper localization of Ras proteins when their farnesylation is inhibited has also permitted a new target for anti-malignancy pharmaceuticals. Recent advances in the enzymology and function of protein prenylation are reviewed in this article.
Keywords: Prenylation; G Protein; Ras; Trafficking; Farnesyltransferase inhibitors;

Functions of a new family of sphingosine-1-phosphate receptors by Sarah Spiegel; Sheldon Milstien (107-116).
Keywords: Sphingosine 1-phosphate; EDG; G Protein-coupled receptor; Signal transduction;

Living organisms are exposed to a number of different fatty acids and their various derivatives arising either via endogenous synthesis or from exogenous sources. These hydrophobic compounds can play specific metabolic, structural or endocrinic functions in the organisms before their elimination, which can be metabolism to CO2 or to more polar lipid metabolites allowing their excretion. Quantitatively, one of the major pathways metabolizing fatty acids is β-oxidation, which consists of a set of four reactions operating at the carbons 2 or 3 of acyl-CoA esters and shortening of the acyl-chain. To allow the β-oxidation of acyl groups with various steric variants to proceed, different strategies have been developed. These strategies include evolution of β-oxidation enzymes as paralogues showing specificity with respect to either chain-length or modified acyl-chain, metabolic compartmentalization in eukaryotic cells, controlling of substrate transport across membranes, development of auxiliary enzyme systems, acquisition of enzymes with adaptive active sites and recruiting and optimizing enzymes from non-homologous sources allowing them to catalyze a parallel set of reactions with different substrate specificities.
Keywords: Fatty acid; Mitochondrion; Peroxisome; Metabolic compartmentalization; Auxiliary enzyme; Paralogue;

There is ample evidence from experimental models and human metabolic disorders indicating that cholesterol and sphingomyelin (SM) levels are coordinately regulated. Generally it has been observed that altering the cellular content of sphingomyelin or cholesterol results in corresponding changes in mass and/or synthesis of the other lipid. In the case of cholesterol synthesis and trafficking, SM regulates the capacity of membranes to absorb cholesterol and thereby controls sterol flux between the plasma membrane and regulatory pathways in the endoplasmic reticulum. This relationship exemplifies the importance of cholesterol/sphingolipid-rich domains in cholesterol homeostasis, as well as other aspects of cell signaling and transport. Evidence for regulation of sphingomyelin metabolism by cholesterol is less convincing and dependent on the model system under study. Sphingomyelin biosynthetic rates are not dramatically affected by alterations in cholesterol balance suggesting that sphingomyelin or its metabolites serve other indispensable functions in the cell. A notable exception is the robust and specific regulation of both SM and cholesterol synthesis by 25-hydroxycholesterol. This finding is reviewed in the context of the role of oxysterol binding protein and its putative role in cholesterol and SM trafficking between the plasma membrane and Golgi apparatus.

Macrophage-derived apoE, produced in the vessel wall, may have important effects during atherogenesis. The production of apoE by macrophages can be regulated at a transcriptional level by cellular differentiation state, cytokines and sterol loading. In addition, there are post-transcriptional and post-translational loci for regulation. We have recently identified an intermediate density cell membrane fraction in which the degradation of apoE can be modulated by sterols. Suppressing degradation of apoE in this fraction by pre-incubating cells in sterols led to enhanced apoE secretion. In this report we demonstrate that the suppressive effect of sterols on the degradation of newly synthesized apoE in this fraction depends on the presence on its C-terminal domain, by studying a macrophage cell line transfected to express a mutant form of apoE in which amino acids beyond amino acid 202 were deleted. In addition, two modulators of cellular sterol transport, progesterone and U1866A, inhibited the degradation of full-length apoE. In contrast, incubation of cells in the acyl-CoA:cholesterol acyltransferase inhibitor S58035 did not influence apoE degradation. As would be predicted based on the results of degradation assays, U1866A, but not S58035, increased the secretion of apoE from a cell line transfected to constitutively express full-length apoE cDNA. The effect of U1866A on apoE degradation, like the effect of sterol, required the presence of the apoE C-terminal domain. Our results indicate that alteration of intracellular sterol homeostasis by pre-incubation in sterols or by drugs that modify the subcellular transport of sterol, modulates the susceptibility of apoE to degradation and that this modulation requires the presence of C-terminal lipid binding domains.
Keywords: Macrophage; Apolipoprotein E; Atherosclerosis; Cell sterol transport;

Membrane perturbation by mastoparan 7 elicits a broad alteration in lipid composition of L1210 cells by Heung Soon Park; Sang Yoon Lee; Young Hwan Kim; Jin Young Kim; Soo Jae Lee; Myung-Un Choi (151-162).
Mastoparan 7 (Mas-7), an amphiphilic peptide possessing membrane perturbing activity, has been known to selectively stimulate some lipases. To examine changes in the lipid composition induced by Mas-7, we carried out systemic lipid analysis of L1210 cells after Mas-7 treatment. The total lipid was determined by HPLC, gas-liquid chromatography, and electrospray ionization mass spectrometry in conjunction with differential radiolabelling with [32P]orthophosphate, [3H]myristic acid, and [3H]arachidonic acid. The lipid analysis revealed multiple changes in more than 10 lipid classes. Free fatty acids (FFAs) and phosphatidylethanol (PEt), the phospholipase D product in the presence of ethanol, were increased significantly and phosphatidylcholine (PC) was decreased. Digitonin, a membrane permeabilizing reagent, similarly affected the lipid composition of L1210. The FFA released showed a very broad distribution of saturated, monounsaturated, and polyunsaturated fatty acids, implying that phospholipase A2 alone could not account for all of the FFAs released. By comparing the molecular species of PEt with those of endogenous PC, we showed that phospholipase D in L1210 cells appeared to act selectively on diacyl-PC. The perturbation-induced alterations in the lipid composition brought about by Mas-7 might play a crucial role in the physiology of the affected cells.
Keywords: Membrane perturbation; Lipid composition; Mastoparan 7; L1210 cell; Fatty acid; Phospholipase D;

Susceptibility of S49 lymphoma cell membranes to hydrolysis by secretory phospholipase A2 during early phase of apoptosis by Kelli H. Nielson; Cari A. Olsen; Darin V. Allred; Kim L. O’Neill; Gregory F. Burton; John D. Bell (163-174).
During cell death, plasma membranes of cells become vulnerable to attack by extracellular secretory phospholipase A2. The purpose of this study was to identify the timing of this phenomenon relative to other events that occur during the process of cell death. Death was induced in S49 murine lymphoma cells by treatment with dexamethasone, dibutyryl cAMP, ionomycin, thapsigargin, or heat shock (1 h at 43°C). The appearance of membrane susceptibility to secretory phospholipase A2 was compared to the following apoptotic events: loss of mitochondrial membrane potential, phosphatidylserine exposure in the outer leaflet of the cell membrane, early DNA damage assessed by the comet assay, and changes in cell size and internal complexity assessed by flow cytometry. Each inducer of death was distinct in the time course of events produced. Although dead cells were susceptible to the action of phospholipase A2, live cells (impermeable to propidium iodide) also became vulnerable to the enzyme during characteristic time courses after exposure to each inducer. In fact, susceptibility to sPLA2 was observed in each case prior to or concurrent with the earliest of the markers of apoptosis. These results demonstrate that the onset of susceptibility to sPLA2 is an early event in apoptosis suggesting that changes in membrane structure may be relevant to initial aspects of the apoptotic process.
Keywords: Phosphatidylserine exposure; Comet assay; Cell death; Phospholipid hydrolysis;

Identification and chromosomal localisation by fluorescence in situ hybridisation of human gene of phosphoinositide-specific phospholipase C β1 by Daniela Peruzzi; Giuseppe Calabrese; Irene Faenza; Lucia Manzoli; Alessandro Matteucci; Fernando Gianfrancesco; Anna Maria Billi; Liborio Stuppia; Giandomenico Palka; Lucio Cocco (175-182).
Members of phosphoinositide-specific phospholipase C (PLC) families are central intermediary in signal transduction in response to the occupancy of receptors by many growth factors. Among PLC isoforms, the type β1 is of particular interest because of its reported nuclear localisation in addition to its presence at the plasma membrane. It has been previously shown that both the stimulation and the inhibition of the nuclear PLCβ1 under different stimuli implicate PLCβ1 as an important enzyme for mitogen-activated cell growth as well as for murine erythroleukaemia cell differentiation. The above findings hinting at a direct involvement of PLCβ1 in controlling the cell cycle in rodent cells, and the previously reported mapping of its gene in rat chromosome band 3q35–36, a region frequently rearranged in rat tumours induced by chemical carcinogenesis, prompted us to identify its human homologue. By screening a human foetal brain cDNA library with the rat PLCβ1 cDNA probe, we have identified a clone homologous to a sequence in gene bank called KIAA 0581, which encodes a large part of the human PLCβ1. By using this human cDNA in fluorescence in situ hybridisation on human metaphases, it has been possible to map human PLCβ1 on chromosome 20p12, confirming the synteny between rat chromosome 3 and human chromosome 20 and providing a novel locus of homology between bands q35–36 in rat and p12 in man. Since band 20p12 has been recently reported amplified and/or deleted in several solid tumours, the identification and chromosome mapping of human PLCβ1 could pave the way for further investigations on the role exerted both in normal human cells and in human tumours by PLCβ1, which has been shown to behave as a key signalling intermediate in the control of the cell cycle.
Keywords: Human phospholipase Cβ1; Chromosome; Gene; Nucleus;

Accumulation of unsaturated lipids in monocytes during early phase pyrogen tolerance by Malgorzata Szewczenko-Pawlikowski; Wieslaw Kozak (183-194).
This paper presents data that inspired a new explanation for the mechanism of early phase endotoxin tolerance. Rabbits injected intravenously with LPS from Salmonella abortus developed a two-phase fever (6 h) and monophasic hyperlipidemia of very low density lipoproteins (two consecutive days). If during these days rabbits were injected with the same dose of LPS at 24-h intervals, the second phase of fever disappeared, i.e. early phase pyrogenic tolerance was obtained. This was correlated with a decrease of lipoprotein hyperlipidemia (measured 1.5 h after LPS injection) and an accumulation of lipids rich in double bonds in monocytes (measured 3.5 h after LPS injection). Results showed that the degree of unsaturation of acyl chains (AC) in monocytes (AC/DB, DB=double bonds) is negatively correlated (r=−0.72) with fever response (fever index). The authors maintain that a gradual increase in monocyte membrane fluidity is an adaptation to repeated exposure of monocytes to lipid A and is responsible for the progressive desensitization of monocytes to endotoxin. It is suggested that disorders of this mechanism lead to an accumulation of abnormal quantities of saturated lipids and cholesterol within macrophages, which, as foam cells, are the starting point for atherosclerosis pathology.
Keywords: Endotoxin; Fever; Tolerance; Hyperlipidemia; Monocyte; Lipoprotein;

The ability of human group IIa secreted phospholipase A2 (human sPLA2) to hydrolyse the phospholipid membrane of whole cell suspensions of Gram-positive bacteria is demonstrated in real time using a continuous fluorescence displacement assay. Micrococcus luteus is used as a model system and demonstrates an almost absolute specificity for this human enzyme compared with porcine pancreatic and Naja naja venom sPLA2s. This specificity is due to selective penetration of the highly cationic human sPLA2 through the highly anionic bacterial cell wall. Disruption of the peptidoglycan cell wall by treatment with lysozyme allows all three enzymes to express similar hydrolytic activity against the anionic bacterial cell membrane. Extensive (>50%) phospholipid hydrolysis was observed and this was confirmed by electrospray mass spectrometry that allowed the identification of several molecular species of phosphatidylglycerol as the targets for hydrolysis. However, the bactericidal activity of the human enzyme under these assay conditions was low, highlighting the capacity of the organism to survive a major phospholipid insult. In addition to pure enzyme, the human sPLA2 activity in tears was demonstrated using M. luteus as substrate. In comparison to M. luteus, cell suspensions of Staphylococcus aureus were highly resistant to hydrolysis by human sPLA2 as well as to the pancreatic and venom enzymes. Treatment of this organism with the specific cell wall protease lysostaphin resulted in a dramatic enhancement in cell membrane phospholipid hydrolysis by all three sPLA2s. Overall, the results highlight the potential of the human sPLA2 as a selective antimicrobial agent against Gram-positive bacteria in vivo because this enzyme is essentially inactive against mammalian plasma membranes. However, the enzyme will be most effective in combination with other antimicrobial agents that enhance the permeability of the bacterial cell wall and where potentiation of the effectiveness of other antibiotics would be expected.
Keywords: Secreted phospholipase A2; Membrane hydrolysis; Antimicrobial; Antibacterial; Lysostaphin; Lysozyme; Micrococcus luteus; Staphylococcus aureus;

Subcellular localization and PKC-dependent regulation of the human lysophospholipase A/acyl-protein thioesterase in WISH cells by Aijun Wang; Christina A. Johnson; Ying Jones; Mark H. Ellisman; Edward A. Dennis (207-214).
Lysophospholipases play essential roles in keeping their multi-functional substrates, the lysophospholipids, at safe levels. Recently, a 25 kDa human lysophospholipase A (hLysoPLA I) that is highly conserved among rat, mouse, human and rabbit has been cloned, expressed and characterized and appears to hydrolyze only lysophospholipids among the various lipid substrates. Interestingly, this enzyme also displays acyl-protein thioesterase activity towards a G protein α subunit. To target the subcellular location of this hLysoPLA I, we have carried out immunocytochemical studies and report here that hLysoPLA I appears to be associated with the endoplasmic reticulum (ER) and nuclear envelope in human amnionic WISH cells and not the plasma membrane. In addition, we found that the hLysoPLA I can be up-regulated by phorbol 12-myristate 13-acetate (PMA) stimulation, a process in which phospholipase A2 is activated and lysophospholipids are generated in WISH cells. Furthermore, the PMA-induced hLysoPLA I expression can be blocked by the protein kinase C (PKC) inhibitor Gö6976. The regulated expression of the LysoPLA/acyl-protein thioesterase by PKC may have important implications for signal transduction processes.
Keywords: Lysophospholipase; Acyl-protein thioesterase; WISH cell; Immunocytochemistry; Phorbol 12-myristate 13-acetate; Regulation;

Regulation of the expression of group IIA and group V secretory phospholipases A2 in rat mesangial cells by H.A. van der Helm; A.J. Aarsman; M.J.W. Janssen; F.W. Neys; H. van den Bosch (215-224).
Rat mesangial cells synthesize and secrete a secretory phospholipase A2 upon stimulation of the cells with cytokines, like IL-1β and TNF and with cAMP elevating agents like forskolin. This enzyme was previously characterized to belong to group IIA sPLA2. The discovery of several other low molecular weight phospholipases, like group IIC in murine testis and group V in human and rat heart, prompted investigations on the presence of group IIC and group V sPLA2 in rat mesangial cells. This was done by isolating the RNA from stimulated cells and performing RT-PCR, using primers specific for group IIC and V sPLA2. The results indicate that rat mesangial cells upon stimulation express next to group IIA also group V sPLA2. No indications were obtained for the expression of group IIC sPLA2. The regulation of the expression of group V sPLA2 at the mRNA level was further investigated by examining the time-dependent expression, the influence of dexamethasone and the signaling route of the IL-1β stimulation. The results show that the IL-1β induced expression of group V sPLA2 mRNA was time dependent and, similar to that of group IIA sPLA2 mRNA, involves activation of NF-κB. However, in contrast to the group IIA sPLA2, the expression of group V sPLA2 was not influenced by the presence of dexamethasone. The expression of both phospholipases was also examined at the protein level in stimulated mesangial cells. Western blot analysis shows that stimulated mesangial cells synthesize both group IIA and group V sPLA2 protein but the expression of group V is lower compared to that of group IIA sPLA2. In addition, the extent of secretion into the medium appears to be considerably higher for group IIA than for group V sPLA2.
Keywords: Group IIA sPLA2; Group IIC sPLA2; Group V sPLA2; Kidney;

The polar-lipid composition of the sphingolipid-producing bacterium Flectobacillus major by Stanislav G. Batrakov; Anatolii E. Mosezhnyi; Alexander O. Ruzhitsky; Vladimir I. Sheichenko; Denis I. Nikitin (225-240).
Polar lipids comprise about 90% of the total chloroform-methanol extractable lipids of the Gram-negative, fresh-water, ring-forming bacterium Flectobacillus major FM and consist of at least 10 constituents. These are aminophosphosphingolipids, 2-N-(2′-D-hydroxy-13′-methyltetradecanoyl)-15-methyl-4(E)-hexadecasphingenyl-1-phosphoethanolamine (36.8% of the total polar lipids) and its 2′-deoxy derivative (3.7%); sulfonic-acid analogues of ceramide, 2-D-(2′-D-hydroxy-13′-methyltetradecanoyl)amino-3-D-hydroxy-15-methylhexadecane-1-sulfonic acid (18.1%) and its 2′-deoxy derivative (3.5%); a lipoamino acid, N-[3-D-(15′-methylhexadecanoyloxy)-15-methylhexadecanoyl]-glycine (3.7%); a lipodipeptide, N-{N′-[3″-D-(15‴-methylhexadecanoyloxy)-15″-methylhexadecanoyl]glycyl}-L-serine (7.8%); 1,2-diacyl-sn-glycero-3-phosphoethanolamine (7.7%), 1,2-diacyl-3-α-D-galactopyranosyl-sn-glycerol (2.9%); ceramide phospho-myo-inositol (4.9%), and a previously described unusual glycosphingolipid, 7-deoxy-7-amino-D-manno-heptulosonopyranosyl (1-hydroxycarbonyl-6-deoxy-6-amino-α-D-mannopyranosyl) ceramide (10.9%); the last two lipids contain only 15-methyl-4(E)-hexadecasphingenine as a long-chain base. The sole structural type of amide-bound fatty acids in the sphingolipids, including the sulfonic-acid analogues, is iso-15:0, either non-hydroxylated or hydroxylated at 2-C, whereas 15-methylhexadecanoic acid is the major ester-bound fatty acid in the remaining lipids.
Keywords: Ceramide phosphoethanolamine; Ceramide phospho-myo-inositol; α-D-Galactopyranosyldiacylglycerol; Lipoamino acid; Lipodipeptide; Sulfonic-acid analogue of ceramide; Flectobacillus major;

Insulin secretion by pancreatic islet β-cells is impaired in diabetes mellitus, and normal β-cells are enriched in phospholipids with arachidonate as sn-2 substituent. Such molecules may play structural roles in exocytotic membrane fusion or serve as substrates for phospholipases activated by insulin secretagogues. INS-1 insulinoma cells respond to secretagogues and permit the study of effects of culture with free fatty acids on phospholipid composition and secretion. INS-1 cell glycerophosphocholine (GPC) and glycerophosphoethanolamine (GPE) lipids are demonstrated here by electrospray ionization mass spectrometry to contain a lower fraction of molecules with arachidonate and a higher fraction with oleate as sn-2 substituent than native islets. Palmitic acid supplementation induces little change in these INS-1 cell lipids, but supplementation with linoleate or arachidonate induces a large rise in the fraction of INS-1 cell GPC species with polyunsaturated sn-2 substituents and a fall in oleate-containing species to yield a GPC profile similar to native islets. The fraction of GPE lipids comprised of plasmenylethanolamine species with polyunsaturated sn-2 substituents in early-passage INS-1 cells is similar to that of islets, but declines on serial passage. Such molecules might participate in exocytotic membrane fusion, and late-passage INS-1 cells have reduced insulin secretory responses. Arachidonate supplementation induces a rise in the fraction of INS-1 cell GPE lipids with polyunsaturated sn-2 substituents and partially restores responses to insulin secretagogues by late-passage INS-1 cells, but does not further amplify secretion by early-passage cells. Effects of extracellular free fatty acids on β-cell phospholipid composition and secretory responses could be involved in changes in β-cell function during the period of hyper-free fatty acidemia that precedes diabetes mellitus.
Keywords: Arachidonic acid; Plasmenylethanolamine; Insulin secretagogue;

Function of human brain short chain L-3-hydroxyacyl coenzyme A dehydrogenase in androgen metabolism by Xue-Ying He; George Merz; Ying-Zi Yang; Raju Pullakart; Pankaj Mehta; Horst Schulz; Song-Yu Yang (267-277).
Human brain short chain L-3-hydroxyacyl-CoA dehydrogenase (SCHAD) has been demonstrated to be a unique 3α-hydroxysteroid dehydrogenase (HSD) that can convert 5α-androstane-3α,17β-diol (3α-adiol) to dihydrotestosterone (DHT), whose affinity to the androgen receptor is 105-fold higher than that of 3α-adiol. The catalytic efficiency of human SCHAD for this oxidative 3α-HSD reaction was estimated to be 164 min−1 mM−1, about 10-fold higher than that measured for the backward reaction. Thus, human brain SCHAD may function in androgen metabolism as a new kind of 3α-HSD by counteracting all other known 3α-HSDs, which would unidirectionally catalyze the reduction of DHT to the almost inactive 3α-adiol. Human SCHAD is identical to an amyloid-β binding protein (ERAB) involved in Alzheimer’s disease, which was previously reported to be associated with the endoplasmic reticulum. This protein is, in fact, localized in mitochondria, not endoplasmic reticulum, as evidenced by immunocytochemical studies and its noncleavable mitochondrial targeting sequence and lack of endoplasmic reticulum targeting signals or transmembrane segments. These results prompt the suggestion that the mitochondrion plays not only an essential role in the initial step of steroidogenesis, but also important roles in the intracellular homeostasis of sex steroid hormones. Northern blot analysis revealed that the human SCHAD gene is expressed in both gonadal and peripheral tissues including the prostate whose growth notably requires DHT, the most potent androgen. This study represents the first report of a 3α-HSD that could act to generate DHT from 3α-adiol and thereby maintain intracellular DHT levels. We propose that inhibitors of the 3α-HSD activity of human brain SCHAD could be useful for the treatment of benign prostatic hyperplasia and other disorders involving DHT metabolism, in combination with known inhibitors of steroid 5α-reductases.
Keywords: 3α-Hydroxysteroid dehydrogenase; Dihydrotestosterone; Benign prostatic hyperplasia; Prostatic cancer; Aromatase inhibitor; Alzheimer’s disease;

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 μM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 μM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K d of 3.5 μM. Oleate competed the fluorescent probe from the protein with a K d of 1.4 μM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (ΔS=+231.6 J mol−1 deg−1). The presence of oleate significantly increased the K ATP m from 0.47 mM to 0.89 mM while the K glucose m in the presence of the FA (0.026±0.003 mM) was not significantly different from the control (0.014±0.004 mM). A decrease in V max values in the presence of oleate indicated that a mixed allosteric inhibition was operating.
Keywords: Hexokinase; Glycolysis; Fatty acid; Lipid metabolism; Inhibition; Activation;

Genetic evidence for a multi-subunit complex in the O-methyltransferase steps of coenzyme Q biosynthesis by Adam Y. Hsu; Thai Q. Do; Peter T. Lee; Catherine F. Clarke (287-297).
Coq3 O-methyltransferase carries out both O-methylation steps in coenzyme Q (ubiquinone) biosynthesis. The degree to which Coq3 O-methyltransferase activity and expression are dependent on the other seven COQ gene products has been investigated. A panel of yeast mutant strains harboring null mutations in each of the genes required for coenzyme Q biosynthesis (COQ1COQ8) have been prepared. Mitochondria have been isolated from each member of the yeast coq mutant collection, from the wild-type parental strains and from respiratory deficient mutants harboring deletions in ATP2 or COR1 genes. These latter strains constitute Q-replete, respiratory deficient controls. Each of these mitochondrial preparations has been analyzed for COQ3-encoded O-methyltransferase activity and steady state levels of Coq3 polypeptide. The findings indicate that the presence of the other COQ gene products is required to observe normal levels of O-methyltransferase activity and the Coq3 polypeptide. However, COQ3 steady state RNA levels are not decreased in any of the coq mutants, relative to either wild-type or respiratory deficient control strains, suggesting either a decreased rate of translation or a decreased stability of the Coq3 polypeptide. These data are consistent with the involvement of the Coq polypeptides (or the Q-intermediates formed by the Coq polypeptides) in a multi-subunit complex. It is our hypothesis that a deficiency in any one of the COQ gene products results in a defective complex in which the Coq3 polypeptide is rendered unstable.
Keywords: Coenzyme Q; S-Adenosyl-L-methionine dependent methyltransferase; Ubiquinone; Yeast; Saccharomyces cerevisiae;

15-Lipoxygenation of leukotriene A4 by Susanne Tornhamre; Annika Elmqvist; Jan Åke Lindgren (298-306).
The unstable epoxide leukotriene (LT) A4 is a key intermediate in leukotriene biosynthesis, but may also be transformed to lipoxins via a second lipoxygenation at C-15. The capacity of various 12- and 15-lipoxygenases, including porcine leukocyte 12-lipoxygenase, a human recombinant platelet 12-lipoxygenase preparation, human platelet cytosolic fraction, rabbit reticulocyte 15-lipoxygenase, soybean 15-lipoxygenase and human eosinophil cytosolic fraction, to catalyze conversion of LTA4 to lipoxins was investigated and standardized against the ability of the enzymes to transform arachidonic acid to 12- or 15-hydroxyeicosatetraenoic acids (HETE), respectively. The highest ratio between the capacity to produce lipoxins and HETE (LX/HETE ratio) was obtained for porcine leukocyte 12-lipoxygenase with an LX/HETE ratio of 0.3. In addition, the human platelet 100 000×g supernatant 12-lipoxygenase preparation and the human platelet recombinant 12-lipoxygenase and human eosinophil 100 000×g supernatant 15-lipoxygenase preparation possessed considerable capacity to produce lipoxins (ratio 0.07, 0.01 and 0.02 respectively). In contrast, lipoxin formation by the rabbit reticulocyte and soybean 15-lipoxygenases was much less pronounced (LX/HETE ratios <0.002). Kinetic studies of the human lipoxygenases revealed lower apparent K m for LTA4 (9–27 μM), as compared to the other lipoxygenases tested (58–83 μM). The recombinant human 12-lipoxygenase demonstrated the lowest K m value for LTA4 (9 μM) whereas the porcine leukocyte 12-lipoxygenase had the highest V max. The profile of products was identical, irrespective of the lipoxygenase used. Thus, LXA4 and 6S-LXA4 together with the all-trans LXA4 and LXB4 isomers were isolated. Production of LXB4 was not observed with any of the lipoxygenases. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate was considerably more efficient to inhibit conversion of LTA4 to lipoxins, as compared to the inhibitory effect on 12-HETE formation from arachidonic acid (IC50 1 and 50 μM, respectively) in the human platelet cytosolic fraction.
Keywords: Lipoxygenation; Lipoxin synthesis; Leukotriene A4; Platelet 12-lipoxygenase; Eosinophil 15-lipoxygenase;

Low-density lipoproteins (LDL) are taken up by LDL receptor (LDLr)-dependent and -independent pathways; the role and importance of the latest being less well defined. We analyzed the importance of these pathways in the mouse by comparing LDL binding to primary cultures of hepatocytes from LDLr knockout (LDLr KO) and normal C57BL/6J mice. Saturation curve analysis shows that 125I-LDL bind specifically to normal and LDLr KO mouse hepatocytes with similar dissociation constants (K d) (31.2 and 22.9 μg LDL-protein/ml, respectively). The maximal binding capacity (B max) is, however, reduced by 48% in LDLr KO mouse hepatocytes in comparison to normal hepatocytes. Conducting the assay in the presence of a 200-fold excess of high-density lipoprotein-3 (HDL3) reduced by 39% the binding of 125I-LDL to normal hepatocytes and abolished the binding to the LDLr KO mouse hepatocytes. These data indicate that in normal mouse hepatocytes, the LDLr is responsible for approximately half of the LDL binding while a lipoprotein binding site (LBS), interacting with both LDL and HDL3, is responsible for the other half. It can also be deduced that both receptors/sites have a similar affinity for LDL. The metabolism of LDL-protein and cholesteryl esters (CE) was analyzed in both types of cells. 125I-LDL-protein degradation was reduced by 95% in LDLr KO hepatocytes compared to normal hepatocytes. Comparing the association of 125I-LDL and 3H-CE-LDL revealed a CE-selective uptake of 35.6- and 22-fold for normal and LDLr KO mouse hepatocytes, respectively. Adding a 200-fold excess of HDL3 in the assay reduced by 71% the CE-selective uptake in LDLr KO hepatocytes and by 96% in normal hepatocytes. This indicates that mouse hepatocytes are able to selectively take up CE from LDL by the LBS. The comparison of LDL-CE association also showed that the LBS pathway provides 5-fold more LDL-CE to the cell than the LDLr. Overall, our results indicate that in mouse hepatocytes, LDLr is almost completely responsible for LDL-protein degradation while the LBS is responsible for the major part of LDL-CE entry by a CE-selective uptake pathway.
Keywords: Low-density lipoprotein; hepatic cell; LDLr knockout mice;

Endogenously produced glycosaminoglycans affecting the release of lipoprotein lipase from macrophages and the interaction with lipoproteins by Robert Zimmermann; Peter Sartipy; Rudolf Winkler; Rudolf Zechner; Eva Hurt-Camejo; Gert M Kostner (316-324).
Macrophages are intimately involved in the pathogenesis of atherosclerotic diseases. A key feature of this process is their uptake of various lipoproteins and subsequent transformation to foam cells. Since lipoprotein lipase (LPL) is believed to play a role in foam cell formation, we investigated if endogenously produced proteoglycans (PGs) affect the release of this enzyme from macrophages. The human leukaemic cell line THP-1 which differentiates into macrophages by treatment with phorbol ester (phorbol 12-myristate 13-acetate) served as a model. The differentiation of THP-1 macrophages promoted the release of PGs into the cell medium which caused the detachment of LPL activity from the cell surface, and prevented LPL re-uptake and inactivation. These PGs were mainly composed of chondroitin sulfate type and exerted a heparin-like effect on LPL release. LPL is known to increase the cell association of lipoproteins by the well known bridging function. Exogenous bovine LPL at a concentration of 1 μg/ml enhanced low density lipoprotein (LDL)-binding 10-fold. Endogenously produced PGs reduced LPL-mediated binding of LDL. It is proposed that the differentiation-dependent increase in the release of PGs interferes with binding of LPL and reduces lipoprotein-binding to macrophages.
Keywords: Atherosclerosis; Macrophage; Proteoglycan; Heparin; Low density lipoprotein;