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BBA - Molecular and Cell Biology of Lipids (v.1735, #3)
Lipid kinase activity of antibodies from milk of clinically healthy human mothers by Dmitrii V. Gorbunov; Nataliya A. Karataeva; Valentina N. Buneva; Georgy A. Nevinsky (pp. 153-166).
We have shown recently that polyclonal human milk sIgA contains a subfraction of antibodies (Abs) tightly bound to unusual minor milk lipids containing sialic acid. Here, we show that a small subfraction of milk IgG is tightly bound to the similar or the same minor lipids. The ability of small fractions of sIgA and IgG from human milk to phosphorylate selectively two minor lipids in the presence of [γ-32P]nucleoside triphosphates was shown here for the first time to be an intrinsic property of these antibodies. In contrast to known kinases, antibodies with lipid kinase activity can transfer phosphoryl group to lipids not only from ATP but also from other different nucleotides (dATP, GTP, dGTP, UTP, TTP) with comparable efficiencies (30–100%). To our knowledge, there are no examples of enzymes using orthophosphate as a substrate of phosphorylation reactions. An extremely unusual property of lipid kinase Abs is their high affinity for orthophosphate ( Km=1.6–5.6 μM) and capability to phosphorylate minor lipids using [32P] orthophosphate as donor of phosphate group. The relative specific activity and affinity of abzymes for orthophosphate and ATP depend significantly on donor milk. However, the levels of Ab-dependent phosphorylation of lipids for all Abs in the case of ATP (100%) and orthophosphate (60–80%) as substrates are comparable. The first example of natural abzymes with synthetic activity was milk sIgA with protein kinase activity. Most probably, lipid kinase sIgA and IgG of human milk are the second example of Abs with synthetic activity.
Keywords: Abbreviations; Ab; antibodies; Abz; abzymes; PAGE; polyacrylamide gel electrophoresis; SLE; systemic lupus erythematosus; AI; autoimmuneCatalytic antibody; Phosphorylation of minor lipid; Human milk
Cholic acid as key regulator of cholesterol synthesis, intestinal absorption and hepatic storage in mice by Charlotte Murphy; Paolo Parini; Jin Wang; Ingemar Björkhem; Gösta Eggertsen; Mats Gåfvels (pp. 167-175).
To study the effects of cholic acid (CA) feeding on hepatic cholesterol metabolism, male sterol 12α-hydroxylase (CYP8B1) knockout (−/−) mice and wildtype controls (+/+) were fed either a control diet or the same diet supplemented with CA (0.1% or 0.5% w/w) or cholesterol (1% w/w). During feeding of the control diet, cholesterol synthesis was increased in CYP8B1−/− compared to +/+ mice. Both cholesterol and CA feeding down regulated mRNA expression of cholesterogenic genes and hepatic de novo cholesterol synthesis as also reflected by a concomitant decrease in the nuclear factor SREBP-2 precursor protein and increased hepatic free cholesterol levels. Mice with an intact CYP8B1 gene (CYP8B1+/+ and C57Bl/6 mice) accumulated higher concentrations of cholesteryl esters (24- and 25-fold, respectively) in their livers compared to CYP8B1−/− mice (8-fold). Feeding of CA increased intestinal cholesterol absorption in CYP8B1+/+ mice by 23% and in CYP8B1−/− mice by 50%. While plasma cholesterol did not differ between CYP8B1+/+ and −/− mice under control conditions and cholesterol feeding a decrease was seen in CYP8B1−/− but not CYP8B1+/+ mice fed CA. This study indicates that CA is an important determinant for intestinal cholesterol absorption and that the levels of the transcription factor SREBP-2 in the liver are dependent upon the combined effect of CA on intestinal cholesterol absorption and CYP7A1. The possibility is discussed that inhibition of CYP8B1 and thus CA synthesis may be beneficial for the treatment of hyperlipidemic disorders.
Keywords: Cholesterol; Cholic acid; Sterol 12α-hydroxylase
Trans10, cis12-conjugated linoleic acid induces mitochondria-related apoptosis and lysosomal destabilization in rat hepatoma cells by Masao Yamasaki; Yumi Miyamoto; Hitomi Chujo; Kazuo Nishiyama; Hirofumi Tachibana; Koji Yamada (pp. 176-184).
Conjugated linoleic acid (CLA) is a powerful anti-carcinogenic fatty acid. Previously, we showed that 10 trans 12 cis (10 t, 12 c) CLA induced apoptotic cell death in rat hepatoma. Here, we demonstrated significant cytotoxic effects of 1 μM 10 t, 12 c-CLA, but not 9 c, 11 t-CLA, on dRLh-84 rat hepatoma cells. 9 t, 11 t and 9 c, 11 c-CLA also showed low levels of cytotoxic activity. 10 t, 12 c-CLA activated caspase-3, 9 followed by cytochrome c release from mitochondria into the cytosol. Inhibitors of caspase-3, 9 blocked the cytotoxicity of 10 t, 12 c-CLA. 10 t, 12 c-CLA also induced translocation of Bax protein into the mitochondrial membrane and cleavage of Bid protein. Lysosomal destabilization induced by 10 t, 12 c-CLA was observed by monitoring the re-localization of Acridine Orange and the leakage of β-hexosaminidase from lysosomes. 10 t, 12 c-CLA directly degraded the isolated lysosomes from the rat liver. Our observations indicate that 10 t, 12 c-CLA induces mitochondria-related apoptosis accompanied by lysosomal destabilization in rat hepatoma cells.
Keywords: Conjugated linoleic acid; Caspase; Apoptosis; Hepatoma; Lysosome
Culture age and carbamoylcholine increase the incorporation of endogenously synthesized linoleic acid in lipids of Trypanosoma cruzi epimastigotes by A.L. Villasuso; M. Aveldaño; A. Vicario; E.E. Machado-Domenech; M. Garcia de Lema (pp. 185-191).
Physiological and cellular adaptations to environmental changes are known to be related to modifications in membrane lipids. This work provides metabolic and compositional evidence that Trypanosoma cruzi epimastigotes are able to synthesize and desaturate fatty acids, to incorporate them into their lipids, and to modify this incorporation when carbamoylcholine is present in the medium. The fatty acids formed from [2-14C]acetate in the period from 2 to 9 days were mostly (70%) incorporated in phospholipids, the remainder 30% being recovered in neutral lipids, such as triacylglycerols (TAG) and diacylglycerols (DAG). The main fatty acids formed from [2-14C]acetate were saturates (16:0, 18:0), monoenes (16:1, 18:1) and dienes (mostly 18:2). The ratios between labelled unsaturated and saturated fatty acids increased continuously with growth, consistent with a precursor–product relationship between the main fatty acids, and with the occurrence in T. cruzi of Δ9- and Δ12-desaturases. From days 2 to 5, [14C]18:2 was the main fatty acid produced. Accordingly, the fatty acid profiles showed a significant increase in the percentage of 18:2 in all lipids in the period under study, especially in the first 2 to 5 days. In the presence of carbamoylcholine, the labelling of DAG and TAG with [14C]18:2 augmented. The results indicate that T cruzi is able to synthesize the main types of fatty acids required to form its membrane lipids, and to exchange them actively in response to environmental stimuli.
Candida yeast long chain fatty alcohol oxidase is a c-type haemoprotein and plays an important role in long chain fatty acid metabolism by Qi Cheng; Dominique Sanglard; Sipo Vanhanen; Huan Ting Liu; Paolo Bombelli; Alison Smith; Antoni R. Slabas (pp. 192-203).
The industrial yeasts Candida tropicalis or Candida cloacae are able to grow on a variety of long chain alkanes and fatty acids as the sole carbon source. The complete oxidation of these substrates involves two sequential oxidative pathways: ω-oxidation, comprising the P450 alkane oxidase, a flavin-dependent membrane-bound long chain fatty alcohol oxidase [FAO] and a possible separate aldehyde oxidase [F.M. Dickinson, C. Wadforth, Purification and some properties of alcohol oxidase from alkane-grown Candida tropicalis, Biochem. J. 282 (1992) 325–331], and the β-oxidation pathway, which utilises acylCoA substrates. We recently purified the membrane-bound long chain fatty alcohol oxidase FAO1 and confirmed it is also a c-type haemoprotein. Multiple isoforms may exist for many of these long chain fatty alcohol oxidases and the in vivo requirements for individual genes with respect to specific substrates are still being elucidated. In vitro reconstitution experiments have demonstrated that in Candida maltosa, the cytochrome P450 52A3 gene product can completely oxidise alkanes to dicarboxylic acids [U. Scheller, T. Zimmer, D. Becher, F. Schauer, W. Schunck, Oxygenation Cascade in Conversion of n-Alkanes to, -Dioic Acids Catalyzed by Cytochrome P450 52A3, J. Biol. Chem. 273 (1998) 32528–32534], potentially obviating requirements for a long chain alcohol oxidase. Here, we directly determine in vivo the role of the long chain alcohol oxidase (FAOT) in C. tropicalis, grown on a variety of substrates, followed by gene deletion. The faot double knockout has no detectable faot activity and is incapable of growth on octadecane, but it grows well on oleic acid, palmitic acid and shorter chain alkanes/fatty acids. A spontaneous mutation[s] may have occurred in the faot double gene knockout of C. tropicalis resulting in its inability to grow on oleic acid and hexadecane. The mutations demonstrate that different pathways of octadecane, hexadecane, oleic acid and palmitic acid utilisation exist in C. tropicalis.
Keywords: Abbreviations; FAO; long chain fatty alcohol oxidase; ALK; alkane; YNB; yeast nitrogen base; TE; Tris–HCl; EDTA; buffer; UTR; untranslated region; CDS; coding sequence; PCR; polymerase chain reaction; TLC; thin layer chromatography; GC-MS; gas chromatography-mass spectroscopy; TMS; trimethylsilyl; ABTS; 2, 2′-azino-bis(3-ethylbenzo-6-thiazoline-sulfonic acid); HRP; horseradish peroxidase; BSTFA/TMCS; N; ,; N; -bis(trimethylsilyl trifluoroacedamid)/trimethylchlorosilane Candida; Homologous recombination; Omega-oxidation; n; -alkane assimilation; Fatty acid; Alcohol oxidase; Lipid degradation
Analysis of the contribution of the β-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9- cis-11- trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae by Heique Bogdawa; Syndie Delessert; Yves Poirier (pp. 204-213).
β-Oxidation of the conjugated linoleic acid 9- cis,11- trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the β-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10- trans,12- cis-octadecadienoic acid, oleic acid or 10- cis-heptadecenoic acid. Furthermore, the degradation of 10- cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Δ3,5,Δ2,4-dienoyl-CoA isomerase but instead relied on the presence of Δ3,Δ2-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Δ3,Δ2-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Δ3,Δ2-enoyl-CoA isomerase, along with the enzyme of the core β-oxidation cycle.
Keywords: Conjugated linoleic acid; Peroxisome; S; accharomyces cerevisiae; Dienoyl-CoA isomerase; Enoyl-CoA isomerase; Dienoyl-CoA reductase; Rumenic acid
Yolk sac cholesteryl ester secretion rates can be manipulated in the Golden Syrian hamster: Effect of yolk sac cholesterol concentrations by M. Hayden Lichtenberg; Catherine S. Wilke; Julie A. McConihay; Norman A. Granholm; Laura A. Woollett (pp. 214-221).
The yolk sac is one of two extra-embryonic fetal tissues that separates the fetal and maternal circulations. The yolk sac can secrete lipoprotein particles to the vitelline vessels, which supply yolk sac-derived nutrients to the embryo. The amount and composition of lipoproteins secreted from the rat yolk sac can be manipulated by fatty acid content and gestational age. The goals of the current studies were to determine, first, if tissue cholesterol concentration could mediate cholesterol secretion rate from the yolk sac and, second, if some of the secreted cholesterol could be derived from the maternal circulation. Golden Syrian hamsters were fed 2% added cholesterol to increase the yolk sac cholesterol concentration. Yolk sac explants secreted similar amounts of triglyceride and apolipoproteins B and E into the media regardless of yolk sac cholesterol concentration. In contrast, yolk sacs with greater cholesterol concentrations secreted 2.3-fold more cholesterol into the media as compared to control yolk sacs; the increase was found mostly as cholesteryl ester. At least part of the secreted cholesterol was maternally derived. These data demonstrate that yolk sac cholesterol concentration influences cholesterol secretion rates, and that at least some of the cholesterol secreted originates from the maternal circulation.
Keywords: Abbreviations; Smith–Lemli–Opitz syndrome; SLOS; 3β-hydroxysteroid-delta; 7; -reductase; Δ; 7; reductase; apolipoprotein; apo; Eagle's Basal Media; EBM; lactate dehydrogenase; LDH; Sonic hedgehog; ShhFetus; Lipoprotein; Smith–Lemli–Opitz syndrome; Visceral endoderm
Identification and molecular cloning of a gene encoding Phospholipase A2 ( plaA) from Aspergillus nidulans by SaHyun Hong; Hiroyuki Horiuchi; Akinori Ohta (pp. 222-229).
The plaA gene encoding a protein that contains the cytosolic Phospholipase A2 (cPLA2) motif is cloned for the first time from the filamentous fungus, Aspergillus nidulans. The translated 837 amino acid protein product of plaA comprises conserved lipase regions that are present in most mammalian cPLA2 homologs. High expression of plaA was observed in glucose–lactose medium by Northern blot analyses. Deletion mutants of plaA grew and formed conidia similar to the wild-type strain, but showed decreased PLA2 activity. Expression of the N-terminal truncated form of plaA in yeast cells resulted in increased Ca2+-dependent PLA2 activity with14C-labeled phosphatidylcholine (PC) and phosphatidylethanolamine (PE) as substrates, compared with vector-transformed cells. In conclusion, we have identified and cloned a phospholipid-hydrolyzing novel cPLA2 protein from A. nidulans for the first time.
Keywords: Abbreviation; cPLA; 2; α; cytosolic phospholipase A; 2; αPhospholipase A; 2; Aspergillus nidulans; plaA; Fungi; Phospholipid
Characterization of transcription factors and cis-acting elements that regulate human CTP: Phosphoethanolamine cytidylyltransferase (Pcyt2) by Christa M. Johnson; Zongfei Yuan; Marica Bakovic (pp. 230-235).
CTP: phosphoethanolamine cytidylyltransferase (Pcyt2) promoter was isolated from human breast cancer MCF-7 cells and its activity delineated by luciferase reporter assays and gel-shift analysis. The Pcyt2 promoter is driven by a functional CAAT box (−90/−73) and by negative (−385/−255) and positive regulatory elements (−255/−153) in the upstream regions.
Keywords: Abbreviations; PE; phosphatidylethanolamine; DAG; diacylglycerols; AAG; alkylacylglycerolsPcyt2; Transcription; Promoter; Regulation