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BBA - Molecular and Cell Biology of Lipids (v.1733, #2-3)
Sphingosine kinase 1 is involved in dibutyryl cyclic AMP-induced granulocytic differentiation through the upregulation of extracellular signal-regulated kinase, but not p38 MAP kinase, in HL60 cells by Masahiro Koda; Takashi Murate; Shulin Wang; Kenji Ohguchi; Sayaka Sobue; Mika Ikeda; Keiko Tamiya-Koizumi; Yasuyuki Igarashi; Yoshinori Nozawa; Yoshiko Banno (pp. 101-110).
The role of sphingosine kinase (SPHK) in the dibutyryl cyclic AMP (dbcAMP)-induced granulocytic differentiation of HL60 cells was investigated. During differentiation, SPHK activity was increased, as were mRNA and protein levels of SPHK1, but not of SPHK2. Pretreatment of HL60 cells with N, N-dimethylsphingosine (DMS), a potent SPHK inhibitor, completely blocked dbcAMP-induced differentiation. The phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK was also increased during dbcAMP-induced differentiation. Pretreatment of HL60 cells with the MEK inhibitor, U0126, but not the p38 MAPK inhibitor, SB203580, completely suppressed dbcAMP-induced ERK1/2 activation and granulocytic differentiation, but did not affect the increase in SPHK activity. DMS inhibited dbcAMP-induced ERK1/2 activation, but had little effect on p38 MAPK activation. DMS had no effect on the dbcAMP-induced membrane translocation of protein kinase C (PKC) isozymes, and PKC inhibitors had no significant effect on ERK activation. The overexpression of wild-type SPHK1, but not dominant negative SPHK1, resulted in high basal levels of ERK1/2 phosphorylation and stimulated granulocytic differentiation in HL60 cells. These data show that SPHK1 participates in the dbcAMP-induced differentiation of HL60 cells by activating the MEK/ERK pathway.
Keywords: Abbreviations; SPHK; sphingosine kinase; S1P; sphingosine-1-phosphate; DMS; N; ,; N; -dimethylsphingosine; dbcAMP; dibutyryl cyclic AMP; NBT; nitroblue tetrazolium; fMLP; N; -formyl-methionyl-leucyl-phenylalanine; ERK; extracellular regulating protein kinase; MAPK; mitogen activated protein kinase; PKC; protein kinase C; PTX; pertussis toxinSphingosine kinase; ERK; p38 MAP kinase; Differentiation; HL60 cell
Detection of the steroidogenic acute regulatory protein, StAR, in human liver cells by E.A. Hall; S. Ren; P.B. Hylemon; D. Rodriguez-Agudo; K. Redford; D. Marques; D. Kang; G. Gil; W.M. Pandak (pp. 111-119).
Overexpressing StAR (a mitochondrial cholesterol transporter) increases (>5-fold) the rate of 27-hydroxylation of cholesterol and the rates of bile acid synthesis in primary rat hepatocytes; suggesting that the transport of cholesterol into mitochondria is rate-limiting for bile acid biosynthesis via the CYP27A1 initiated ‘acidic’ pathway. Our objective was to determine the level of StAR expression in human liver and whether changes in StAR would correlate with changes in CYP27A1 activity/bile acid synthesis rates in human liver tissues. StAR mRNA and protein were detected in primary human hepatocytes and HepG2 cells by RT-PCR/Northern analysis and by Western analysis, respectively. In immunocompetition assays, liver StAR was competed away with the addition of purified human adrenal StAR. Overexpressing CYP27A1 in both cell types led to >2-fold increases in liver StAR concentration. StAR protein levels also increased ∼2-fold with the addition of 27-hydroxycholesterol to HepG2 cell culture medium. Overexpressing StAR increased the rates of 27-hydroxylation of cholesterol/bile acid synthesis in both cell lines and increased intracellular levels of 27-hydroxycholesterol. In conclusion, human liver cells contain regulable StAR protein whose level of expression appears capable of regulating cellular cholesterol homeostasis, representing a potential therapeutic target in the management of hyperlipidemia.
Keywords: Steroidogenic acute regulatory protein; Regulation; Lipid transport; Cholesterol; Bile acid
Stress-induced platelet-activating factor synthesis in human neutrophils by John S. Owen; Paul R.S. Baker; Joseph T. O'Flaherty; Michael J. Thomas; Michael P. Samuel; Rhonda E. Wooten; Robert L. Wykle (pp. 120-129).
Platelet-activating factor (1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine; PAF) is a potent inflammatory mediator produced by cells in response to physical or chemical stress. The mechanisms linking cell injury to PAF synthesis are unknown. We used liquid chromatography-tandem mass spectrometry to investigate stress-induced PAF synthesis in human neutrophils. PAF synthesis induced by extracellular pH 5.4 correlated with the activation of a stress-activated kinase, p38 mitogen-activated protein kinase (MAPK), and was blocked by the p38 MAPK inhibitor SB 203580. A key enzyme of PAF synthesis, acetyl-CoA:lysoPAF acetyltransferase, which we have previously shown is a target of p38 MAPK, was also activated in an SB 203580-sensitive fashion. Another MAPK pathway, extracellular signal-regulated kinase-1/2 (ERK-1/2), was also activated. Surprisingly, the pharmacological blockade of the ERK-1/2 pathway with PD 98059 did not block, but rather enhanced, PAF accumulation. Two unexpected actions of PD 98059 may underlie this phenomenon: an augmentation of stress-induced p38 MAPK phosphorylation and an inhibition of PAF catabolism. The latter effect did not appear to be due to a direct inhibition of PAF acetylhydrolase. Finally, similar results were obtained using another form of cellular stress, hypertonic sodium chloride. These data are consistent with a model in which stress-induced PAF accumulation is regulated positively by p38 MAPK and negatively by ERK-1/2. Such a model contrasts with the PAF accumulation induced by other forms of stimulation, which we and others have found is up-regulated by both p38 MAPK and ERK-1/2.
Keywords: Abbreviations; GPC; sn; -glycero-3-phosphocholine; PAF; platelet-activating factor; d; 3; -PAF; 1-; O; -hexadecyl-2-[; 2; H; 3; ]acetyl-GPC; 16:0 PAF; 1-; O; -hexadecyl-2-acetyl-GPC; 18:0 PAF; 1-; O; -octadecyl-2-acetyl-GPC; 18:1 PAF; 1-; O; -octadec-9′-; cis; -enyl-2-acetyl-GPC; [; 3; H]lysoPAF; 1-; O; -[1′, 2′-; 3; H; 2; ]hexadecyl-2-lyso-GPC; PLA; 2; phospholipase A; 2; lysoPAF acetyltransferase; acetyl-coenzyme A:1-; O-; alkyl-2-lyso-GPC acetyltransferase; MAPK; mitogen-activated protein kinase; ERK-1/2; extracellular signal-regulated kinase-1 and -2; HBSS; modified Hanks's Balanced Salt Solution with 24 mM Tris, pH 7.4, 1.4 mM CaCl; 2; , and no Mg; 2+; PBS; Dulbecco's phosphate-buffered saline with 1.4 mM CaCl; 2; and 0.5 mM MgCl; 2; MS/MS; tandem mass spectrometry; LC-MS/MS; liquid chromatography coupled to tandem mass spectrometryPlatelet-activating factor; Stress; p38 MAPK; Mitogen-activated protein kinase; Acetyltransferase; Neutrophil
Diversity of SREBP-1 gene expression in rat adipose tissue depots in response to refeeding after food restriction by Ewa Stelmanska; Elzbieta Sucajtys-Szulc; Justyna Korczynska; Krystian Adrych; Julian Swierczynski (pp. 130-136).
The SREBP-1c mRNA level and precursor (microsomal) form of SREBP-1 abundance were significantly higher in epididymal and perirenal than in subcutaneous white adipose tissue of control rats. Moreover, the SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were significantly higher in the epididymal and perirenal white adipose tissue of rats maintained on restricted diet and refed ad libitum for 48 h as compared to the control animals. No significant effects of food restriction/refeeding on SREBP-1c mRNA level and an amount of precursor form of SREBP-1 were found in subcutaneous white adipose tissue. The mature (nuclear) form of SREBP-1 was significantly increased in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed animals. The activity, protein level and the mRNA abundance of malic enzyme (one of the target genes for SREBP-1) increased significantly in the epididymal, perirenal and subcutaneous white adipose tissue of the food restricted/refed rats as compared to the control animals, however the increase in perirenal and epididymal was higher than in the subcutaneous white adipose tissue.The results presented suggest that SREBP-1c is differently expressed in various rat white adipose tissue depots both under basal (control) and dieting conditions.
Keywords: Sterol regulatory element binding protein-1; Malic enzyme; Restricted diet; White adipose tissue; Rat
Gene expression profile of isolated rat adipocytes treated with anthocyanins by Takanori Tsuda; Yuki Ueno; Hitoshi Kojo; Toshikazu Yoshikawa; Toshihiko Osawa (pp. 137-147).
Adipocyte dysfunction is strongly associated with the development of obesity and insulin resistance. It is accepted that the regulation of adipocytokine secretion or the adipocyte specific gene expression is one of the most important targets for the prevention of obesity and amelioration of insulin sensitivity. Recently, we demonstrated that anthocyanins, which are pigments widespread in the plant kingdom, have the potency of anti-obesity in mice and the enhancement adipocytokine secretion and adipocyte gene expression in adipocytes. In this study, we have shown for the first time the gene expression profile in isolated rat adipocytes treated with anthocyanins (cyanidin 3-glucoside; C3G or cyanidin; Cy). The rat adipocytes were treated with 100 μM C3G, Cy or vehicle for 24 h. The total RNA from the adipocytes was isolated and carried out GeneChip microarray analysis. A total of 633 or 427 genes was up-regulated (>1.5-fold) by the treatment of adipocytes with C3G or Cy, respectively. The up-regulated genes include lipid metabolism and signal transduction-related genes, however, the altered genes were partly different between the C3G- and Cy-treated groups. Based on the gene expression profile, we demonstrated the up-regulation of hormone sensitive lipase and enhancement of the lipolytic activity by the treatment of adipocytes with C3G or Cy. These data have provided an overview of the gene expression profiles in adipocytes treated with anthocyanins and identified new responsive genes with potentially important functions in adipocytes related with obesity and diabetes that merit further investigation.
Keywords: Abbreviations; BSA; bovine serum albumin; C/EBP; CCAAT/enhancer binding protein; C3G; cyanidin 3-glucoside; Cy; cyanidin; DMEM; Dulbecco's modified Eagle's medium; FFA; free fatty acid; HSL; hormone sensitive lipase; KRBH; Krebs-Ringer bicarbonate buffer; PPAR; peroxisome proliferator-activated receptor; RT-PCR; reverse transcription-polymerase chain reaction; TZD; thiazolidinediones; UCP; uncoupling proteinAnthocyanin; Cyanidin; Adipocyte; Gene expression profile; Nutrigenomics; DNA microarray
Nuclear phospholipase C-β1b activation during G2/M and late G1 phase in nocodazole-synchronized HL-60 cells by Vesna Lukinoviƈ-Å kudar; Lana Ä?onlagiƈ; Hrvoje BanfÃƈ; Dora ViÅ¡njiƈ (pp. 148-156).
In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G2/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-β1. Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-β1 in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-β1b was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G1 into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-β1b on serine, occur at the G2/M and late G1 phase and are necessary for the progression of the cells through the cell cycle.
Keywords: Abbreviations; PLC; phospholipase C; PtdIns(4,5)P; 2; phosphatidylinositol 4,5-bisphosphate; DAG; diacylglycerol; Ins(1,4,5)P; 3; inositol(1,4,5)P; 3; PKC; protein kinase C; PI3K; phosphatidylinositol 3-kinase; MAPK; mitogen-activated protein kinase; MEK; MAP kinase kinasePhospholipase C-β; 1b; Nuclei; Nocodazole; Cell cycle; HL-60 cell
Statins potentiate the IFN-γ-induced upregulation of group IIA phospholipase A2 in human aortic smooth muscle cells and HepG2 hepatoma cells by Mario Menschikowski; Albert Hagelgans; Ben Heyne; Ute Hempel; Volker Neumeister; Peter Goez; Werner Jaross; Gabriele Siegert (pp. 157-171).
The present study shows that the incubation of human aortic smooth muscle cells (HASMC) and HepG2 cells with atorvastatin and mevastatin as HMG-CoA reductase inhibitors potentiated the interferon-γ (INF-γ)-induced group IIA phospholipase A2 (sPLA2-IIA) expression in a dose- and time-dependent manner. The effect of statins on sPLA2-IIA expression was reduced by mevalonate, farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Inversely, inhibitors of the farnesyl transferase and geranylgeranyl transferase-I mimicked the effects of statins. Clostridium difficile toxin B (TcdB), Y-27632 and H-1152, functioning as inhibitors of Rho proteins and Rho-associated kinase, also augmented the sPLA2-IIA expression in combination with IFN-γ. The same effects were observed when inhibitors of mitogen-activated/extracellular response protein kinase kinase (MEK), PD98059 or U0126 were used. Further, the Janus kinase-2 (Jak2)-specific inhibitor, AG-490 and inhibitors of nuclear factor-κB (NFκB) abrogated the sPLA2-IIA elevating effects of statins, TcdB and PD98059 in the presence of IFN-γ. This cytokine alone increased the NFκB p65 and CAAT-enhancer-binding protein-β (C/EBP-β) activity in HASMC nuclear extract, but only C/EBP-β was further augmented when the cells were incubated in addition to IFN-γ with atorvastatin, H-1152, PD98059 or U0126. Moreover, after the incubation of cells with atorvastatin and IFN-γ the stability of sPLA-2IIA mRNA significantly increased in comparison to those after incubation with IFN-γ alone. In conclusion, the obtained data suggest that (i) the expression of sPLA2-IIA is negatively regulated by RhoA/Rho-associated kinase and MEK/ERK signaling pathways and (ii) statins, because of their ability to down-regulate these pathways, can potentiate the IFN-γ-induced sPLA2-II expression at transcriptional and post-transcriptional levels.
Keywords: Abbreviations; C/EBP-β; CAAT-enhancer-binding protein-β; cPLA; 2; cytosolic phospholipase A; 2; DRB; 5,6-Dichloro-1-β-; d; -ribofuranosylbenzimidazole; ELISA; enzyme linked immunosorbent assay; ERK; extracellular signal-regulated kinase; FCS; fetal calf serum; FPP; farnesyl pyrophosphate; FTI; farnesyl transferase inhibitor; GAPDH; glycerine aldehyde-3-phosphate dehydrogenase; GGPP; geranylgeranyl pyrophosphate; GGTI; geranylgeranyl transferase-I inhibitor; HASMC; human aortic smooth muscle cells; HDL; high-density lipoproteins; HMG-CoA; 3-hydroxy-3-methylglutaryl-coenzyme A; IFN-γ; interferon-γ; IL-1β; interleukin-1β; IL-6; interleukin-6; Jak; Janus kinase; MEK; mitogen-activated/extracellular response protein kinase kinase; NFκB; nuclear factor-κB; PBS; phosphate-buffered saline; PSI; proteasome inhibitor; RT-PCR; reverse transcription polymerase chain reaction; sPLA; 2; -IIA; secretory phospholipase A; 2; of group IIA; STAT; signal transducer and activator of transcription; TcdB; Clostridium difficile; toxin B; TNF-α; tumor necrosis factor-αStatin; HMG-CoA reductase inhibitor; Secretory phospholipase A; 2; Inflammation; HASMC; HepG2 cells
MAPKs mediate the activation of cytosolic phospholipase A2 by amyloid β(25–35) peptide in bovine retina pericytes by Ambra Nicotra; Gabriella Lupo; Giovanni Giurdanella; Carmelina D. Anfuso; Nicolò Ragusa; Cataldo Tirolo; Bianca Marchetti; Mario Alberghina (pp. 172-186).
We have previously shown that, in bovine retina pericytes, amyloid β(1–42) and its truncated form containing amino acids 25–35, after 24 h treatment, stimulate arachidonic acid (AA) release and phosphatidylcholine hydrolysis, by activation of both cytosolic (cPLA2) and Ca2+-independent (iPLA2) phospholipase A2. A putative role for MAP kinases in this process emerged. Here we studied the role of the MAP-kinase family as well as both cPLA2 and iPLA2 mRNA expression by a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) in the same sublethal model of amyloid-β (Aβ) damage to pericytes in vitro. Aβ(25–35) peptide evoked AA release as well as stimulated phosphorylation of ERK1/2, p38 MAPKs and cPLA2, but not c- Jun N-terminal kinase (JNK/SAPK). PD98059, an inhibitor of ERK-activating kinase MEK-1, and SB203580, an inhibitor of p38 protein kinase, abolished the stimulation of AA release and MAPK activities. In cells stimulated by Aβ(25–35) peptide, Western blotting and confocal microscopy analyses confirmed either an increase in the phosphorylated form of ERKs and p38 or their nuclear translocation. A complete inhibition of MAPK activation and AA release was also observed when pericytes were treated with GF109203X, a general PKC inhibitor, indicating the important role of both PKC and the two MAPKs in mediating the Aβ peptide response. Compared with samples untreated or treated with reverse Aβ(35–25) peptide, pretreatment with 50 μM Aβ(25–35) for 24 h significantly increased the level of constitutively expressed iPLA2 mRNA by 25%, which seems to depend on the activation of kinases. By contrast, the level of cPLA2 mRNA remained unchanged. Together, these data link either the stimulation of PKC-ERK-p38 cascades or PLA2 activity by Aβ peptide to prooxidant mechanism induced by amyloid, which may initially stimulate the cell reaction as well as metabolic repair, such as during inflammation.
Keywords: Amyloid β(25–35); Pericyte; Cytosolic phospholipase A; 2; MAPK; ERK; p38; mRNA
Anti-inflammatory effects of phospholipid transfer protein (PLTP) deficiency in mice by Axel Schlitt; Jin Liu; Daoguang Yan; Mary Mondragon-Escorpizo; Allen J. Norin; Xian-Cheng Jiang (pp. 187-191).
We previously reported that phospholipid transfer protein-deficient (PLTP KO) mice exhibit a lower rate of atherosclerosis. We proposed two possible mechanisms: a reduction in hepatic apoB secretion (Nat Med 7 (2001) 847) and induction of lipoprotein anti-oxidation activity (J Biol Chem 277 (2002) 31850). We now hypothesized that PLTP KO mice may exhibit an anti-inflammatory state per se. First, we found that PLTP KO mice have significantly lower IL-6 levels than wild type (WT) mice. Secondly, we found that IL-6 treatment increased plasma TNFα levels in WT mice, but not in PLTP KO mice. Thirdly, we used flow cytometric analyses to measure the mean fluorescence intensity of I-Ab, a MHC-class II molecule, on peripheral monocytes and found that IL-6 treatment significantly increased the I-Ab-positive cell levels in WT mice, whereas no changes were observed in the cell levels in PLTP KO mice. The results of our experiments demonstrated an anti-inflammatory effect of PLTP deficiency as a further aspect of its proatherogenic potency.
Turpentine-induced inflammation reduces the hepatic expression of the multiple drug resistance gene, the plasma cholesterol concentration and the development of atherosclerosis in apolipoprotein E deficient mice by Mònica Tous; Vicent Ribas; Natàlia Ferré; Joan Carles Escolà-Gil; Francisco Blanco-Vaca; Carlos Alonso-Villaverde; Blai Coll; Jordi Camps; Jorge Joven (pp. 192-198).
We aimed to investigate the effect of turpentine-induced inflammation in an atherosclerosis-prone murine model. We have induced a chronic aseptic inflammation in apolipoprotein E-deficient mice, with or without a dietary supplement of aspirin ( n=10, each), by the injection of a mixture (1:1) of turpentine and olive oil in the hind limb twice weekly for a period of 12 weeks. Control animals were injected with olive oil alone ( n=10). The control mice did show any alteration neither in plasma nor at the site of injection. Turpentine-treated mice showed a significant increase in plasma TNF-α and SAA concentrations which indicated a systemic inflammatory response that was not substantially affected by aspirin. Also, turpentine injections significantly reduced the plasma cholesterol concentration, probably decreasing intestinal cholesterol re-absorption, and attenuated the size of atherosclerotic lesion. Both effects were minimally influenced by aspirin. The burden of atherosclerosis correlated with plasma lipid levels but not with plasma inflammatory markers. Finally, there was a concomitant decrease in the expression of the hepatic mdr1b gene that correlated with the decrease in plasma cholesterol concentration. Therefore, we conclude that mdr1 is an additional factor to consider in the complexity of alterations in cholesterol metabolism that occur in this model.
Keywords: Apo-E-deficient mice; Absorption; Arteriosclerosis; Cholesterol; Inflammation; mdr1b
The phospholipid scramblase PLSCR1 increases UV induced apoptosis primarily through the augmentation of the intrinsic apoptotic pathway and independent of direct phosphorylation by protein kinase C δ by Kendra Bailey; Harold W. Cook; Christopher R. McMaster (pp. 199-209).
Cell death by apoptosis can be caused by the DNA mutagen UV light whose exposure causes the direct activation of both the caspase 9 regulated cell damage intrinsic pathway and the caspase 8 regulated plasma membrane extrinsic pathway. We determined that increased activity of the plasma membrane phospholipid scramblase, PLSCR1, amplified UV mediated apoptosis primarily through the activation of the intrinsic apoptotic pathway. The caspase 8 inhibitor z-IETD-fmk was not as effective an inhibitor of PLSCR1 augmented UV induced apoptosis compared to treatment with caspase 3, caspase 9, or pan-caspase inhibitors. The inability of the caspase 8 inhibitor to decrease UV induced apoptosis was dependent on PLSCR1, as UV induced apoptosis was decreased by a similar amount in the control cells in the presence of inhibitors of caspase 8, caspase 9, caspase 3, or the pan-caspase inhibitor. PKC-δ directly phosphorylates human PLSCR1 resulting in increased PLSCR1 scramblase activity. PKC-δ can also be activated by caspase mediated cleavage resulting in the release of a constitutively active kinase domain. We observed that replacing the PKC-δ phosphorylation site of PLSCR1 with an alanine did not affect the ability of PLSCR1 to enhance UV induced apoptosis implying that PKC-δ does not directly phosphorylate PLSCR1 to increase plasma membrane scramblase activity during apoptosis. Cells transfected with a PLSCR1 mutant that contained an alanine substitution at its known PKC-δ phosphorylation site underwent UV induced apoptosis at a level similar to those transfected with wild type PLSCR1. The combined results indicate that UV exposure in cells possessing PLSCR1 increases apoptosis primarily by enhancement of the intrinsic apoptotic pathway, and also imply that the increased apoptosis observed upon exposure to UV light is not through direct phosphorylation of PLSCR1 by PKC-δ.
Keywords: Abbreviations; PtdSer; phosphatidylserine; CHO; Chinese hamster ovary; DMEM; Dulbecco's modified Eagle's medium; FBS; fetal bovine serum; TBS; Tris buffered saline; PLSCR; phospholipid scramblase; PKC; protein kinase C; PVDF; polyvinylidene difluoride; GFP; green fluorescent protein; TBST; Tris buffered saline plus Tween-20; HRP; horse radish peroxidasePhosphatidylserine; Apoptosis; UV light; Scramblase; Protein kinase C; PLSCR1
Time-resolved fluoroimmunoassays of the complete set of secreted phospholipases A2 in human serum by Timo J. Nevalainen; Leena I. Eerola; Esa Rintala; V. Jukka O. Laine; Gérard Lambeau; Michael H. Gelb (pp. 210-223).
Time-resolved fluoroimmunoassays (TR-FIA) were developed for all human secreted phospholipases A2 (PLA2), viz. group (G) IB, GIIA, GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA2 and the GXIIB PLA2-like protein. Antibodies were raised in rabbits against recombinant human PLA2 proteins and used in sandwich-type TR-FIAs as both catching and detecting antibodies, the latter after labeling with Europium. The antibodies were non-cross-reactive. The analytical sensitivities were 1 μg/L for the TR-FIA for GIB PLA2, 1 μg/L (GIIA), 35 μg/L (GIID), 3 μg/L (GIIE), 4 μg/L (GIIF), 14 μg/L (GIII), 11 μg/L (GV), 2 μg/L (GX), 92 μg/L (GXIIA) and 242 μg/L (GXIIB). All secreted PLA2s were assayed by these TR-FIAs in serum samples from 34 patients (23 men and 11 women, mean age 53.2 years) treated in an intensive care unit for septic infections, and in control samples from 28 volunteer blood donors (14 men and 14 women, mean age 57.0 years). Five serum samples (3 in the sepsis group and 2 in the blood donor group) gave high TR-FIA signals that were reduced to background (blank) levels by the addition of non-immune rabbit IgG to the sera. This reactivity was assumed to be due to the presence of heterophilic antibodies in these subjects. In all other subjects, including septic patients and healthy blood donors, the TR-FIA signals for GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA2 and the GXIIB PLA2-like protein were at background (blank) levels. Four patients in the sepsis group had pancreatic involvement and elevated concentration of GIB PLA2 in serum (median 19.0 μg/L, range 13.1–33.7 μg/L, n=4) as compared to the healthy blood donors (median 1.8 μg/L, range 0.8–3.4 μg/L, n=28, P<0.0001). The concentration of GIIA PLA2 in the sera of septic patients (median 315.7 μg/L, range 15.9–979.6 μg/L, n=34) was highly elevated as compared to that of the blood donors (median 1.8 μg/L, range 0.8–5.8 μg/L, n=28, P<0.0001). Our current results confirmed elevated concentrations of GIB and GIIA PLA2 in the sera of patients suffering from acute pancreatitis or septic infections, respectively, as compared to healthy subjects. However, in the same serum samples, the concentrations of the other secreted PLA2s, viz. GIID, GIIE, GIIF, GIII, GV, GX and GXIIA PLA2 and the GXIIB PLA2-like protein were below the respective analytical sensitivities of the TR-FIAs. It is concluded that generalized bacterial infections do not lead to elevated serum levels of GIIE, GIIF, GIII, GV and GX PLA2s above the detection limits of the current TR-FIAs.
Keywords: Blood donor; Immunoassay; Infection; Inflammation; Intensive care; Sepsis
Supplementation with α-linolenic acid-rich diacylglycerol suppresses fatty liver formation accompanied by an up-regulation of β-oxidation in Zucker fatty rats by Takatoshi Murase; Masafumi Aoki; Ichiro Tokimitsu (pp. 224-231).
Insulin resistance-related obesity and diabetes mellitus are the predominant causes of fatty liver disease. Here we examine the effects of dietary diacylglycerol (DG), which is a minor component of plant oils, on lipid accumulation and the expression of genes involved in lipid metabolism in the liver. The animals were fed diets containing either 10% triacylglycerol (TG), 10% TG+4% α-linolenic acid-rich TG (ALATG) or 10% TG+4% α-linolenic acid-rich diacylglycerol (ALADG) for a period of 1 month. Supplementation with ALADG significantly inhibited hepatic triglyceride accumulation; this was accompanied by the up-regulation of β-oxidation activity, and acyl-CoA oxidase (ACO) and medium-chain acyl-CoA dehydrogenase (MCAD) mRNA levels. By contrast, no significant changes were observed in the levels of peroxisome proliferator-activated receptor-α (PPARα) and sterol regulatory element-binding protein-1 (SREBP-1) mRNAs. These results indicate that ALADG might be useful in the prevention of fatty liver formation; this effect could be closely related to the stimulation of lipid catabolism in the liver. In addition, our findings suggest that both acylglycerol structure (that is, the structural difference between TG and DG) and fatty-acid species affect the nutritional behaviour of dietary lipids.
Keywords: Abbreviations; ACAT; acyl-CoA carnitine acyltransferase; ACO; acyl-CoA oxidase; ALA; α-linolenic acid; ALADG; α-linolenic acid-rich diacylglycerol; ALATG; α-linolenic acid-rich triacylglycerol; DG; diacylglycerol; DGAT; diacylglycerol acyltransferase; DHA; docosahexaenoic acid; EPA; eicosapentaenoic acid; γ-GPT; γ-glutamate pyruvate transaminase; GOT; glutamate oxaloacetate transaminase; MCAD; medium-chain acyl-CoA dehydrogenase; NAFLD; non-alcoholic fatty liver disease; NASH; non-alcoholic steatohepatitis; NEFAs; Non-esterified fatty acids; NIDDM; non-insulin-dependent diabetes mellitus; PPAR; peroxisome proliferator-activated receptor; RT-PCR; reverse transcription-polymerase chain reaction; SREBP; sterol regulatory element-binding protein; TG; triacylglycerol; WATs; white adipose tissuesα-Linolenic acid; β-Oxidation; Diacylglycerol; Fatty liver; Obesity
The activities of monocyte lysophosphatidylcholine acyltransferase and coenzyme A-independent transacylase are changed by the inflammatory cytokines tumor necrosis factor alpha and interferon gamma by Nicola T. Neville; Joan Parton; John L. Harwood; Simon K. Jackson (pp. 232-238).
Alteration of membrane phospholipid fatty acid compositions has been shown to be important for leukocyte inflammatory responses. Such modification of the molecular species of these lipid classes requires deacylation and reacylation reactions and for phosphatidylcholines, lysophosphatidylcholine acyltransferase (LPCAT) and a coenzyme A-independent transacylase (CoAIT) can each be involved. Since previous studies have shown a significant IFNγ- and TNFα-induced modification of phosphatidylcholine species, we have examined whether these inflammatory cytokines alter the activity of reacylation enzymes in the human monocyte cell line MonoMac 6 (MM6). IFN-γ caused a significant increase in the activity of the LPCAT and CoAIT enzymes in the microsomal fraction at concentrations and over a time-course consistent with an important role for these enzymes in the sensitization (priming) of monocytes. In contrast, TNFα was found to significantly increase the activity of the CoAIT by 50% over controls in MM6 cells after 30 min incubation with the cytokine, but decreased LPCAT activity by 65% after 24 h incubation. Such data imply that CoAIT is important for the remodelling of phospholipid composition, which is seen during the acute response of cells to TNFα. The results provide further information to emphasise the role of acyltransferases as part of the molecular mechanism underlying inflammation.
Keywords: Abbreviations; BSA; bovine serum albumin; CoAIT; CoA-independent transacylase; DAG; diacylglycerol; HBS; Hepes buffered saline; IFNγ; interferon gamma; LPCAT; lysophosphatidylcholine acyltransferase; LPS; lipopolysaccharide; MM6; Mono Mac 6; PAF; platelet activating factor; PUFA; polyunsaturated fatty acids; RPMI; Rosswell Park Memorial Institute; TNFα; tumor necrosis factor alpha; PtdCho; phosphatidylcholine; PtdOH; Phosphatidic acidAcyltransferase; Transacylase; Phosphatidylcholine; TNFα, IFN-γ