Henk van den Bosch

Peroxisomal disorders: a newly recognised group of genetic diseases

A new group of genetic diseases, in which peroxisomal functions are impaired, has recently been recognised. Although peroxisomes are subcellular organelles that are universally found in animal cells (except mature erythrocytes), their function in mammalian metabolism remained unclear until recently. Now, however, it is evident that peroxisomes play an essential role in several metabolic processes and that dysfunction of these organelles can have serious clinical consequences. Genetic diseases involving peroxisomes can be divided into two classes: firstly, those in which only a single peroxisomal function is impaired (acatalasaemia, X-linked adrenoleukodystrophy, the adult form of Refsum disease); secondly, those in which there is an impairment of more than one peroxisomal function (cerebro-hepato-renal (Zellweger) syndrome, the infantile form of Refsum disease, the neonatal form of adrenoleukodystrophy, the rhizomelic type of chondrodysplasia punctata and hyperpipecolic acidaemia). Whether genetic diseases like intrahepatic bile duct hypoplasia with elevation of trihydroxycoprostanoic acid and cerebrotendinous xanthomatosis can also be classified as peroxisomal disorders remains to be established. The aim of this paper is to summarise the rapid developments that have occurred during the last 3 years with regard to our knowledge of peroxisomal disorders and to describe briefly the clinical, morphological and biochemical characteristics of this new group of genetic disorders.

A role for secretory phospholipase A2 and C-reactive protein in the removal of injured cells

The acute phase response is initiated in response to infection or physical trauma and is characterized by an increase in the levels of some plasma proteins. Here, Erik Hack and colleagues suggest that the combined actions of two of these acute phase proteins, secretory phospholipase A2 and C-reactive protein, may serve to promote phagocytosis of injured cells and tissue debris, thereby enhancing inflammation and tissue damage.

Peroxisomal β-oxidation enzyme proteins in the Zellweger syndrome

Abstract The absence of peroxisomes in patients with the cerebro-hepato-renal (Zellweger) syndrome is accompanied by a number of biochemical abnormalities, including an accumulation of very longchain fatty acids. We show by immunoblotting that there is a marked deficiency in livers from patients with the Zellweger syndrome of the peroxisomal β-oxidation enzyme proteins acyl-CoA oxidase, the bifunctional protein with enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities and 3-oxoacyl-CoA thiolase. Using anti-(acyl-CoA oxidase), increased amounts of cross-reactive material of low M r were seen in the patients. With anti-(oxoacyl-CoA thiolase), high M r cross-reactive material, presumably representing precursor forms of 3-oxoacyl-CoA thiolase, was detected in the patients. Catalase protein was not deficient, in accordance with the finding that catalase activity is not diminished in the patients. Thus in contrast to the situation with catalase functional peroxisomes are required for the stability and normal activity of peroxisomal β-oxidation enzymes.

Interleukin-1β, tumor necrosis factor and forskolin stimulate the synthesis and secretion of group II phospholipase A2 in rat mesangial cells

Abstract Treatment of rat glomerular mesangial cells with interleukin-1β, tumor necrosis factor or forskolin resulted in the release of phospholipase A2 activity in the culture medium. Essentially all of this phospholipase A2 activity was bound to immobilized monoclonal antibodies raised against rat liver mitochondrial 14 kDa group II phospholipase A2. Gelfiltration confirmed the absence of higher molecular weight phospholipases A2 in the culture medium. Immunoblot experiments showed the virtual absence of this 14 kDa group II phospholipase A2 in unstimulated mesangial cells. The time-dependent increase of phospholipase A2 activity in both cells and culture medium upon stimulation with interleukin-1β plus forskolin is accompanied with elevated 14 kDa phospholipase A2 protein levels. These results indicate that the increased phospholipase A2 activity upon treatment of mesangial cells with these stimulators is due to increased synthesis of group II phospholipase A2. Over 85 % of this newly synthesized phospholipase A2 appears to be secreted from the cells.

Cholesterol in the year 2000.

Cholesterol research was one of the key areas of scientific investigation in the 20th century. Little was known about the structure of cholesterol until the pioneering research of A. Windaus and H. Wieland in the first part of the century. The structure of cholesterol was completely elucidated in 1932. With the development of isotopic tracers in the 1930s studies on cholesterol biosynthesis were initiated. In 1942 K. Bloch and D. Rittenberg showed that deuterium-labeled acetate was incorporated into the ring structure and side chain of cholesterol. Another important discovery from Blochs laboratory was that squalene was a precursor of cholesterol. In 1956, the main elements of the biosynthetic pathway became known when isopentenyl pyrophosphate was discovered as a precursor. In 1966, J. Cornforth and G. Popjak predicted that there were 16234 possible stereochemical pathways by which mevalonate could be converted into squalene. They subsequently showed which of these pathways was correct. In the 1970s and 1980s K. Bloch was able to provide intriguing evidence for an evolutionary advantage of cholesterol over lanosterol or some of the intermediates in the conversion of lanosterol to cholesterol. The last quarter of the 20th century was when M. Brown and J. Goldstein showed that the low density lipoprotein receptor was a key regulator of cholesterol homeostasis. They have also demonstrated that cholesterol balance in the cell is transcriptionally regulated via the sterol regulatory element binding protein. In the later part of the 20th century drugs were developed that effectively lower plasma cholesterol and lessen the risk of atherosclerosis and cardiovascular disease.

Deficiency of plasmalogens in the cerebro-hepato-renal (Zellweger) syndrome.

We have analyzed the phospholipid composition of various organs of patients with the cerebro-hepato-renal (Zellweger) syndrome. The phospholipid composition of tissues from controls and patients was very similar except for their plasmalogen contents. In controls about 50% of the phosphatidylethanolamine fraction of brain, heart, kidney and skeletal muscle and about 10% of that fraction in control liver tissue was found to consist of plasmalogen. In control heart muscle, but not in other control tissues about 25% of the phosphatidylcholine fraction consist of plasmalogens. In contrast, plasmalogens were nearly absent in the corresponding tissues of Zellweger patients.The amount of phosphatidylethanolamine plasmalogens in both erythrocytes and fibroblasts of Zellweger patients is lowered significantly compared to control erythrocytes and control fibroblasts respectively, although this reduction is not as dramatic as in brain, heart, kidney, skeletal muscle and liver of patients. Phosphatidylcholine-plasmalogens are only present in low amounts in both controls, heterozygotes and patients.In recent years considerable evidence has accumulated to show that peroxisomes are involved in cellular lipid metabolism. Notably, the key enzymes of ether lipid (plasmalogen) biosynthesis in rodents were recently found to be located in peroxisomes. Since electronmicroscopic studies have shown that peroxisomes are absent in liver and kidney of patients with the cerebro-hepato-renal syndrome, our results suggest that an inability to integrate these key enzymes in a functional peroxisome leads to a severe disturbance in plasmalogen biosynthesis.We propose that the multiple clinical and biochemical defects in Zellweger patients are secondary to a deficiency in peroxisomal function.

Regulation of the cellular expression of secretory and cytosolic phospholipases A2, and cyclooxygenase-2 by peptide growth factors.

Secretory group II (sPLA2) and cytosolic (cPLA2) phospholipases A2 and cyclooxygenase-2 (Cox-2) play a pivotal role in release of proinflammatory eicosanoids. Excessive activity of sPLA2 per se can also propagate inflammation. Endogenous control of the above enzymes has not been completely elucidated. We investigated the combined impact of promoting cytokines and inhibitory peptide growth factors on the expression of mRNA of the above enzymes, on protein content and extracellular release of sPLA2 and on PGE2 production in osteoblasts (FRCO). The synthesis and release of sPLA2 were enhanced by about 20-fold by 0.5 ng/ml IL-1beta or by 50 ng/ml of TNFalpha. Coaddition of both cytokines resulted in synergistic 150-fold increase in the release of sPLA2 implying the existence of two paths of induction. IL-1beta and TNFalpha markedly enhanced the transcription of sPLA2 mRNA. Kinetic study showed that IL-1/TNF initiated sPLA2 release after 12 h, reaching maximum at 48 h. IL-1alpha was a weak stimulator of sPLA2 release, whereas IL-6, IL-8, IGF, IFN-gamma, growth hormone, insulin and GM-CSF were not stimulatory. Peptide growth hormones TGFbeta, PDGF-BB, EGF and bFGF markedly inhibited the extracellular release of sPLA2. TGFbeta and PDGF-BB significantly reduced the level of sPLA2 mRNA, thus acting upon transcription whereas EGF and bFGF were not inhibitory, acting rather upon the translational or posttranslational steps. IL-1/TNF and growth factors had no significant effect on cPLA2 mRNA expression. Cox-2 mRNA expression was markedly enhanced by IL-1/TNF and suppressed by all growth factors tested. Cytokines enhanced the extracellular release of PGE2 and further enhancement was induced by growth factors with the exception of TGFbeta. Cycloheximide abolished completely the release of sPLA2 and markedly reduced the release of PGE2 from cytokine-stimulated FRCO, regardless of whether growth factors were present or not. NS-398, a specific inhibitor of Cox-2 abolished almost completely the release of PGE2 from cytokine-stimulated cells, regardless of the presence of growth factors. Thus, different signalling mechanisms are involved in the impact of growth factors on mRNA expression of sPLA2, cPLA2 and Cox-2. The differences between the impact on FRCO sPLA2 and that reported in other cells, imply that endogenous control of arachidonic acid cascade is cell-specific.

Nucleotide sequence of human alkyl-dihydroxyacetonephosphate synthase cDNA reveals the presence of a peroxisomal targeting signal 2

Abstract Two overlapping clones were isolated from a human liver cDNA library in lambda gt11 that coded for human alkyl-dihydroxyacetonephosphate synthase using guinea pig and PCR-derived human cDNA probes. The open reading frame encodes a protein of 658 amino acids that shows a homology of 92% with the guinea pig homolog and a similarity of 98%. The peroxisomal targeting signal 2 that was recently identified in the presequence of the guinea pig enzyme appeared to be completely preserved in the human enzyme. Supportive confirmation for parts of the sequence of the mature protein was obtained from the Expressed Sequence Tags database of the National Center for Biotechnology Information. This database contained nine cDNA sequences, derived from seven independent clones, that correspond exactly to parts of the cDNA of human alkyl-dihydroxyacetonephosphate synthase. One of these clones most likely represents a not fully processed RNA with a putative intron containing an Alu sequence. An unexpected homology with d -lactate dehydrogenase (cytochrome C) precursor from Saccharomyces cerevisiae and with glycolate oxidase subunit D from Escherichia coli was also revealed.

Cytokine-and forskolin-induced synthesis of group II phospholipase A2 and prostaglandin E2 in rat mesangial cells is prevented by dexamethasone

We have previously described that treatment of rat glomerular mesangial cells with interleukin-1 beta, tumor necrosis factor or forskolin stimulates the synthesis and secretion of prostaglandin E2 and group II phospholipase A2. We now report that pretreatment of the mesangial cells with dexamethasone dose-dependently suppresses the cytokines- and forskolin-induced synthesis of prostaglandin E2 as well as the induced synthesis and secretion of group II phospholipase A2. These observations implicate that the inhibition of the cellular or secreted phospholipase A2 activity by dexamethasone in rat mesangial cells is not due to induced synthesis of phospholipase A2 inhibitory proteins but caused by direct inhibition of phospholipase A2 protein expression.

Extracellular nucleotides activate the p38‐stress‐activated protein kinase cascade in glomerular mesangial cells

Extracellular ATP and UTP have been reported to activate a nucleotide receptor (P2Y2‐receptor) that mediates arachidonic acid release with subsequent prostaglandin formation, a reaction critically depending on the activity of a cytosolic phospholipase A2. In addition, extracellular nucleotides trigger activation of the classical mitogen‐activated protein kinase (MAPK) cascade and cell proliferation as well as of the stress‐activated protein kinase (SAPK) cascade. In this study, we report that ATP and UTP are also able to activate the p38‐MAPK pathway as measured by phosphorylation of the p38‐MAPK and its upstream activators MKK3/6, as well as phosphorylation of the transcription factor ATF2 in a immunocomplex‐kinase assay. Time courses reveal that ATP and UTP induce a rapid and transient activation of the p38‐MAPK activity with a maximal activation after 5 min of stimulation which declined to control levels over the next 20 min. A series of ATP and UPT analogues were tested for their ability to stimulate p38‐MAPK activity. UTP and ATP were very effective analogues to activate p38‐MAPK, whereas ADP and γ‐thio‐ATP had only moderate activating effects. 2‐Methyl‐thio‐ATP, βγ‐imido‐ATP, AMP, adenosine and UDP had no significant effects of p38‐MAPK activity. In addition, the extracellular nucleotide‐mediated effect on p38‐MAPK was almost completely blocked by 1 mM of suramin, a putative P2‐purinoceptor antagonist. In summary, these results demonstrate for the first time that extracellular nucleotides are able to activate the MKK3/6‐ p38‐MAPK cascade most likely via the P2Y2‐receptor. Moreover, this finding implies that all three MAPK subtypes are signalling candidates for extracellular nucleotide‐stimulated cell responses.