H. van den Bosch

Cytokine-stimulated secretion of group II phospholipase A2 by rat mesangial cells. Its contribution to arachidonic acid release and prostaglandin synthesis by cultured rat glomerular cells.

Potent pro-inflammatory cytokines, such as interleukin 1 (IL-1) or tumor necrosis factor (TNF) alpha have been found to increase group II phospholipase A2 (PLA2) synthesis and secretion by mesangial cells. In all cases 85-90% of the enzyme is secreted from the cells and a parallel increase in prostaglandin (PG)E2 synthesis is observed. We report here that co-incubation with a monoclonal antibody that specifically binds and neutralizes rat group II PLA2 attenuates IL-1 beta and TNF alpha-stimulated PGE2 production by 45% and 52%, respectively. CGP43182, a specific inhibitor of group II PLA2, potently blocks mesangial cell group II PLA2 in vitro with a half-maximal inhibitory concentration (IC50) of 1.5 microM, while only slightly affecting mesangial cell high molecular weight PLA2. CGP 43182 markedly attenuates IL-1 beta- and TNF alpha-stimulated PGE2 synthesis in intact mesangial cells with IC50s of 1.3 and 1.0 microM, respectively. PLA2 secreted from cytokine-stimulated mesangial cells was purified to homogeneity. Addition of the purified enzyme to unstimulated mesangial cells causes a marked release of arachidonic acid and a subsequent increased synthesis of PGE2. Moreover, addition of purified PLA2 to a cloned rat glomerular epithelial cell line and cultured bovine glomerular endothelial cells augmented both arachidonic acid release and PGE2 synthesis, with the endothelial cells being especially sensitive. Thus, cytokine-triggered synthesis and secretion of group II PLA2 by mesangial cells contributes, at least in part, to the observed synthesis of PGE2 that occurs in parallel to the enzyme secretion. Furthermore, extracellular PLA2 secreted by mesangial cells is able to stimulate arachidonic acid release and PGE2 synthesis by the adjacent endothelial and epithelial cells. These data suggest that expression and secretion of group II PLA2 triggered by pro-inflammatory cytokines may crucially participate in the pathogenesis of inflammatory processes within the glomerulus.

Deficiency of acyl-CoA: Dihydroxyacetone phosphate acyltransferase in patients with Zellweger (cerebro-hepato-renal) syndrome

We have recently reported on plasmalogen deficiency in tissues and fibroblasts from patients with Zellweger syndrome. In this paper we have analyzed the activity of the first enzyme in the pathway leading to plasmalogen biosynthesis, i.e. acyl-CoA:dihydroxyacetone phosphate acyltransferase in liver, brain and cultured skin fibroblasts from Zellweger patients and controls. The results indicate a severe deficiency of this enzyme in Zellweger patients. Thus, the Zellweger syndrome constitutes the first inborn error of metabolism with a deficiency in an enzyme involved in phospholipid biosynthesis. Cultured amniotic fluid cells contained an enzymatic activity comparable to that of control fibroblasts. These findings suggest a method for prenatal diagnosis of this disease.

Peroxisomal disorders in neurology

Although peroxisomes were initially believed to play only a minor role in mammalian metabolism, it is now clear that they catalyse essential reactions in a number of different metabolic pathways and thus play an indispensable role in intermediary metabolism. The metabolic pathways in which peroxisomes are involved include the biosynthesis of ether phospholipids and bile acids, the oxidation of very long chain fatty acids, prostaglandins and unsaturated long chain fatty acids and the catabolism of phytanate and (in man) pipecolate and glyoxylate. The importance of peroxisomes in cellular metabolism is stressed by the existence of a group of inherited diseases, the peroxisomal disorders, caused by an impairment in one or more peroxisomal functions. In the last decade our knowledge about peroxisomes and peroxisomal disorders has progressed enormously and has been the subject of several reviews. New developments include the identification of several additional peroxisomal disorders, the discovery of the primary defect in several of these peroxisomal disorders, the recognition of novel peroxisomal functions and the application of complementation analysis to obtain information on the genetic relationship between the different peroxisomal disorders. The peroxisomal disorders recognized at present comprise 12 different diseases, with neurological involvement in 10 of them. These diseases include: (1) those in which peroxisomes are virtually absent leading to a generalized impairment of peroxisomal functions (the cerebro-hepato-renal syndrome of Zellweger, neonatal adrenoleukodystrophy, infantile Refsum disease and hyperpipecolic acidaemia); (2) those in which peroxisomes are present and several peroxisomal functions are impaired (the rhizomelic form of chondrodysplasia punctata, combined peroxisomal beta-oxidation enzyme protein deficiency); and (3) those in which peroxisomes are present and only a single peroxisomal function is impaired (X-linked adrenoleukodystrophy, peroxisomal thiolase deficiency (pseudo-Zellweger syndrome), acyl-CoA oxidase deficiency (pseudo-neonatal adrenoleukodystrophy) and probably, the classic form of Refsum disease.

Phosphatidylcholine mobility in liver microsomal membranes.

Abstract Purified phosphatidylcholine exchange protein from bovine liver was used to exchange rat liver microsomal phosphatidylcholine for egg phosphatidylcholine. It was found that at 25 and 37°C rat liver microsomal phosphatidylcholine was completely and rapidly available for replacement by egg phosphatidylcholine. In contrast, phosphatidylcholine in vesicles prepared from total microsomal lipids could only be exchanged for about 60%. At 8 and 0°C complex exchange kinetics were observed for phosphatidylcholine in rat liver microsomes. The exchange process had neither effect on the permeability of the microsomal membrane to mannose 6-phosphate, nor on the permeability of the phosphatidylcholine vesicles to neodymium (III) cations. Purified phospholipase A 2 from Naja naja could hydrolyze some 55–60% of microsomal phosphatidylcholine at 0°C, but 70–80% at 37°C. Microsomal phosphatidylcholine, remaining after phospholipase treatment at 37°C, could be exchanged for egg phosphatidylcholine at 37°C, but at a slower rate than with intact microsomes. Microsomal phosphatidylcholine remaining after phospholipase treatment at 0 and 37°C had a lower content of arachidonic acid than the original phosphatidylcholine. These results are discussed with respect to the localization and transmembrane movement of phosphatidylcholine in liver microsomes.

Biosynthesis of cardiolipin in liver mitochondria

Abstract Diphosphatidylglycerol (cardiolipin) biosynthesis from CDP-diglyceride and sn-glycerol-3-phosphate has been demonstrated in a preparation of mitochondria from rat liver in a reaction requiring Mg2+. The identity of the diphosphatidylglycerol was established by thin-layer chromatography and mild alkaline hydrolysis. Diphosphatidyl-glycerol formation from [3H]phosphatidylglycerol was shown to occur only in the presence of CDP-diglyceride. The rate of diphosphatidylglycerol formation appears to be quite slow in mitochondria and may explain why it was not previously observed. Evidence is presented which eliminates the possibility of a bacterial contribution to the biosynthetic process observed.

Chemical structure and biochemical significance of lysolecithins from rat liver

Abstract 1. 1. Synthetic lecithins containing in 2-position a [14C]fatty acid constituent were found to be hydrolysed by rat-liver homogenates so as to form both 1-acyl-glycero-3-phosphorylcholine and 2-acyl-glycero-3-phosphorylcholine. 2. 2. A comparison of the fatty acid pattern of lysolecithin obtained from rat liver by thin-layer chromatography with the fatty acid composition of 1-acyl-glycero-3-phosphorylcholine prepared by breakdown of liver lecithin with snake venom phospholipase A (EC 3.1.1.4) suggested that two structurally isomeric lysolecithins occur. 3. 3. Phospholipase A degradation of lysolecithin from rat liver previously labelled with 32P demonstrated that, apart from 1-acyl-glycero-3-phosphorylcholine, 2-acyl-glycero-3-phosphorylcholine was also present. Hydrolysis of lysolecithin with phospholipase C (EC 3.1.4.3) and separation of the monoglycerides formed confirmed that rat-liver lysolecithin consists of two structural isomers. 4. 4. A combination of these findings with the results obtained by Lands and co-workers on the selective enzymic transacylation of isomeric lysophosphoglycerides allows a monoacyl-diacyl phospholipid cycle to be formulated. This mechanism is postulated to play a part in maintaining the liquid-crystalline state of membraneous lipids.

Studies on lysophospholipases: I. Purification and some properties of a lysophospholipase from beef pancreas

Abstract 1. 1. Lysophospholipase was purified from homogenates of fresh beef pancreas by acid treatment, (NH 4 ) 2 SO 4 precipitation in the presence of n -butanol and ion-exchange chromatography using SE-Sephadex C-50 and DEAE-Sephadex A-50. 2. 2. The molecular weight of the nearly homogeneous enzyme was estimated to be 65000 from sodium dodecyl sulfate disc electrophoresis and Sephadex G-200 filtration. 3. 3. The products of the hydrolysis of 1-acyl-glycerylphosphorylcholine containing long chain acyl groups were shown to be free fatty acid and glycerylphosphorylcholine, whereas long chain phosphatidylcholine was not attacked at all. 4. 4. The enzyme did not require bivalent metal ions and was unaffected by SH-group reagents. Diisopropylfluorophosphate in I mM concentration completely abolished enzymatic activity.

SUBCELLULAR AND SUBMITOCHONDRIAL LOCALIZATION OF THE BIOSYNTHESIS OF CARDIOLIPIN AND RELATED PHOSPHOLIPIDS IN RAT LIVER

Abstract 1. 1. Intact mitochondria, inner and outer mitochondrial membranes and microsomes were isolated from rat liver and their purity determined with specific marker enzymes. 2. 2. Diphosphatidylglycerol synthesis was found exclusively in the inner mitochondrial membrane. Phosphatidylglycerol was predominantly synthesized in theinner membrane although a small contribution was noted in the outer membrane. CDP and dCDP diglyceride synthesis occurred primarily in the microsomal fraction. 3. 3. Diphosphatidylglycerol synthesis from phosphatidylglycerol was shown to require CDP diglyceride or dCDP diglyceride and a divalent cation (either Mg24, Mn2+ or Co2+). The reaction was strongly inhibited by nonionic detergents such as Triton X-100

Lipocortin inhibition of extracellular and intracellular phospholipases A2 is substrate concentration dependent

Hydrolysis of Escherichia coli membrane phospholipids by pancreatic phospholipase A2 was inhibited by lipocortin from human monocytes in a substrate dependent manner. Inhibition was completely overcome at substrate concentrations above 250 μM. Lipocortin also inhibited partially purified preparations of two intracellular phospholipases A2, isolated from rat liver mitochondria and rat platelets when these enzymes were assayed at low micromolar concentrations of phosphatidylethanolamine. Inhibition gradually decreased with increasing substrate concentrations both for pancreatic and platelet phospholipase A2 and became completely abolished above 15 and 50 μM phosphatidylethanolamine, respectively.

Evidence for isotropic motion of phospholipids in liver microsomal membranes. A 31P NMR study

1. The motional properties of phospholipids in bovine and rat liver microsomes and aqueous dispersions of the extracted lipids have been investigated employing 31 P NMR techniques. 2. The 31P NMR spectra obtained from the microsomes indicate that a considerable portion of the constituent phospholipids experience isotropic motion on the NMR timescale (10(-5) s). This is in strong contrast to the spectra obtained from aqueous dispersions of the extracted lipids, which display the characteristic lineshape associated with liquid crystalline phospholipids in (large) bilayer structures, which experience restricted anisotropic motion. 3. Evidence is presented which strongly suggests that the isotropic motion of microsomal phospholipids does not arise from tumbling of the microsomal vesicles or from lateral diffusion of phospholipids around these vesicles. 4. These results are discussed in terms of possible transitory formation of intramembrane non-bilayer lipid configurations, with which the bulk (bilayer) phospholipids are in rapid exchange.