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R J Wanders

Publications and source records attributed to R J Wanders.

At least 253 records · Page 14Linked to original sources
PubMed1995

Fatty acid beta-oxidation in peroxisomes and mitochondria: the first, unequivocal evidence for the involvement of carnitine in shuttling propionyl-CoA from peroxisomes to mitochondria.

We have investigated how [1-14C]propionyl-CoA, which is the first product of the peroxisomal beta-oxidation of [1-14C] pristanic acid, is transported to mitochondria for further oxidation in human skin fibroblasts from patients with a defect in the mitochondrial carnitine/acylcarnitine translocase and carnitine-palmitoyltransferase II (CPT II) (EC 2.3.1.21), respectively. Oxidation of pristanic acid was found to be partially deficient in both types of mutant cells. More important, 14CO2 production was completely deficient in the carnitine/acylcarnitine translocase deficient cells but not in the carnitine-palmitoyltransferase II deficient cells. These results strongly suggest that formation of 14CO2 in the Krebs cycle from [1-14C]propionyl-CoA as generated in peroxisomes requires the active participation of the mitochondrial carnitine/acylcarnitine translocase. The results described in this paper provide the first evidence suggesting that propionyl-CoA leaves the peroxisome as a carnitine ester and strongly suggest that the commonly accepted concept that peroxisomal beta-oxidation is not dependent on carnitine is incorrect.

Acyl Coenzyme A↗
PubMed1995

Inhibition of oxidative phosphorylation by palmitoyl-CoA in digitonin permeabilized fibroblasts: implications for long-chain fatty acid beta-oxidation disorders.

Long-chain fatty acid oxidation deficient patients present early in life with more severe features than patients with a medium-chain fatty acid oxidation deficiency. This may be related to the more toxic effect of long-chain fatty acid derivatives. In this paper we have studied the effect of different acyl-CoA esters, and palmitoyl-CoA in particular, on succinate-driven oxidative phosphorylation, using digitonin permeabilized human fibroblasts. Palmitoyl-CoA was found to inhibit the succinate-driven oxidative phosphorylation in a concentration dependent manner. If the inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions this might explain some of the abnormalities found in patients with defects in long-chain fatty acid beta-oxidation.

2,4-Dinitrophenol↗
PubMed1995

Peroxisomal and mitochondrial carnitine acetyltransferases of Saccharomyces cerevisiae are encoded by a single gene.

Carnitine acetyltransferase (CAT) is present in mitochondria and peroxisomes of oleate-grown Saccharomyces cerevisiae. Both proteins are encoded by the same gene, YCAT, which encodes a protein with a mitochondrial targeting signal (MTS) at the N-terminus, and a peroxisomal targeting signal type 1 (PTS-1) at the C-terminus. Deletion of both motifs revealed the presence of an additional internal targeting sequence. Import of CAT via this internal signal was shown to be dependent on PAS10, a protein which is required for the import of PTS-1 containing proteins. An interaction of PAS10 with this internal targeting signal was demonstrated using the yeast two-hybrid technique. Expression of the YCAT gene behind a heterologous promoter resulted in loss of peroxisomal targeting, indicating that differential targeting is controlled at transcriptional or translational level. Determination of the 5'-ends of YCAT mRNAs revealed that YCAT transcripts initiating after the first AUG were present in oleate-grown cells. These transcripts were virtually absent in acetate- or glycerol-grown cells. We propose that in response to oleate, shorter transcripts are produced from which the peroxisomal form of CAT is translated, resulting in a CAT protein without a MTS, which can be targeted to peroxisomes.

Base Sequence↗
PubMed1995

The membrane of peroxisomes in Saccharomyces cerevisiae is impermeable to NAD(H) and acetyl-CoA under in vivo conditions.

We investigated how NADH generated during peroxisomal beta-oxidation is reoxidized to NAD+ and how the end product of beta-oxidation, acetyl-CoA, is transported from peroxisomes to mitochondria in Saccharomyces cerevisiae. Disruption of the peroxisomal malate dehydrogenase 3 gene (MDH3) resulted in impaired beta-oxidation capacity as measured in intact cells, whereas beta-oxidation was perfectly normal in cell lysates. In addition, mdh3-disrupted cells were unable to grow on oleate whereas growth on other non-fermentable carbon sources was normal, suggesting that MDH3 is involved in the reoxidation of NADH generated during fatty acid beta-oxidation rather than functioning as part of the glyoxylate cycle. To study the transport of acetyl units from peroxisomes, we disrupted the peroxisomal citrate synthase gene (CIT2). The lack of phenotype of the cit2 mutant indicated the presence of an alternative pathway for transport of acetyl units, formed by the carnitine acetyltransferase protein (YCAT). Disruption of both the CIT2 and YCAT gene blocked the beta-oxidation in intact cells, but not in lysates. Our data strongly suggest that the peroxisomal membrane is impermeable to NAD(H) and acetyl-CoA in vivo, and predict the existence of metabolite carriers in the peroxisomal membrane to shuttle metabolites from peroxisomes to cytoplasm and vice versa.

Acetyl Coenzyme A↗
PubMed1995

Altered kinetics of cytochrome c oxidase in a patient with severe mitochondrial encephalomyopathy.

Deficiency of cytochrome c oxidase activity was established in a girl born to consanguineous parents. She showed symptoms of dysmaturity, generalized hypotonia, myoclonic seizures and progressive respiratory failure, leading to death on the seventh day of life. Structural abnormalities of the central nervous system consisted of severe cerebellar hypoplasia and optic nerve atrophy. Biochemical analysis of a muscle biopsy specimen demonstrated deficiency of cytochrome c oxidase activity. Cultured fibroblasts from this patient also showed a selective decrease in the activity of cytochrome c oxidase, excluding a muscle-specific type of deficiency. Further investigations in cultured fibroblasts revealed that synthesis, assembly and stability of both the mitochondrial and the nuclear subunits of the enzyme were entirely normal. The steady-state concentration of cytochrome c oxidase in the fibroblasts of the patient was also normal, suggesting that the kinetic properties of the enzyme were altered. Analysis of the kinetic parameters of cytochrome c oxidase demonstrated an aberrant interaction between cytochrome c oxidase and its substrate, cytochrome c, most likely because of a mutation in one of the nuclear subunits of the enzyme.

Cells, Cultured↗
PubMed1995

Meta-iodobenzylguanidine inhibits complex I and III of the respiratory chain in the human cell line Molt-4.

In this paper we report the effects of meta-iodobenzylguanidine (MIBG), a structural analogue of norepinephrine, on cell proliferation and several parameters related to mitochondrial respiration in Molt-4 cells. In micromolar concentrations, MIBG completely inhibited the proliferation of Molt-4 cells. In intact Molt-4 cells, a progressive increase in the lactate to pyruvate ratio was observed after incubation of these cells with glucose and increasing concentrations of MIBG. In Molt-4 cells permeabilized with digitonin, MIBG inhibited mitochondrial ATP synthesis when malate was used as a substrate. Succinate-driven synthesis of ATP was also inhibited by MIBG, although higher concentrations were required. These results indicate that apart from inhibition of complex I, MIBG inhibits at least one other complex of the respiratory chain. Measurement of the activities of the individual enzyme complexes in the presence of MIBG revealed that complex III is the other enzyme complex susceptible to inhibition by MIBG. Although maximal inhibition of ATP synthesis was observed at a concentration of 10 microM, maximal inhibition of cell proliferation was observed at a concentration of 50 microM of MIBG. This suggests that MIBG also influences other cellular processes apart from mitochondrial oxidative phosphorylation, resulting in additional inhibition of cell proliferation.

3-Iodobenzylguanidine↗
PubMed1995

A misdiagnosis of X-linked adrenoleukodystrophy in cultured chorionic villus cells by the measurement of very long chain fatty acids.

A case is reported of a male fetus at risk of X-linked adrenoleucodystrophy who showed a normal cultured chorionic villus cell very long chain fatty acid (VLCFA) profile but at birth exhibited grossly abnormal plasma and cultured fibroblast VLCFAs. Maternal contamination or a sample mix-up was excluded by chromosome analysis and analysis of polymorphic markers. This is the second report of a fetus affected with this disorder who showed normal cultured chorionic villus cell VLCFAs. It highlights the importance of a proper audit of all prenatal diagnoses to evaluate method reliability.

Adrenoleukodystrophy↗
PubMed1995

Measurement of dihydroxyacetone-phosphate acyltransferase (DHAPAT) in chorionic villous samples, blood cells and cultured cells.

Dihydroxyacetone-phosphate acyltransferase (DHAPAT) is a peroxisomal enzyme catalysing the first step in ether-phospholipid biosynthesis. DHAPAT is deficient in cells from patients suffering from a variety of peroxisomal disorders. Accurate measurement of the activity of this enzyme is of great importance, especially since it is a central parameter in the prenatal diagnosis of the disorders of peroxisome biogenesis, rhizomelic chondrodysplasia punctata and DHAPAT-deficiency. We describe a straightforward and accurate assay allowing the activity of DHAPAT to be measured reliably in chorionic villus samples, blood cells, cultured skin fibroblasts, cultured chorionic villus fibroblasts and cultured amniocytes.

Acyltransferases↗
PubMed1995

Immunoblot analysis of peroxisomal proteins in liver and fibroblasts from patients.

Identification of a patient as suffering from a peroxisomal disorder usually starts by the finding of elevated very long-chain fatty acids in plasma and/or serum. This is followed by more detailed studies in blood, fibroblasts and tissues, including immunoblot analysis. Indeed, immunoblot analysis has become a valuable tool in the correct diagnosis and assignment of individual patients, except for X-linked adrenoleukodystrophy (X-ALD). We describe a simple immunoblotting procedure applicable to liver and fibroblast homo-genates using antibodies raised against catalase and the three beta-oxidation enzyme proteins acyl-CoA oxidase I, bifunctional protein and peroxisomal thiolase. The same procedure can also be used for chorionic villus biopsy specimens and has now become the method of choice for the prenatal diagnosis of Zellweger syndrome (and other disorders of peroxisome biogenesis) and rhizomelic chondrodysplasia punctata.

Alkaline Phosphatase↗
PubMed1995

Measurement of peroxisomal fatty acid beta-oxidation in cultured human skin fibroblasts.

One of the main functions of mammalian peroxisomes is the beta-oxidation of a variety of fatty acids and fatty acid derivatives, including very long-chain fatty acids. Oxidation of these fatty acids is deficient in a number of different peroxisomal disorders, including the disorders of peroxisome biogenesis (Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease), X-linked adrenoleukodystrophy and a number of other disorders of peroxisomal beta-oxidation of known and unknown aetiology. Accurate measurement of peroxisomal fatty acid oxidation is of utmost importance for correct postnatal and prenatal diagnosis of these disorders. In this paper we describe a straightforward and accurate assay method to measure the beta-oxidation of palmitic acid (C16:0), hexacosanoic acid (C26:0) and pristanic acid in intact fibroblasts.

Cell Adhesion↗
PubMed1995

Meta-iodobenzylguanidine (MIBG) inhibits malate and succinate driven mitochondrial ATP synthesis in the human neuroblastoma cell line SK-N-BE(2c).

In this paper, we report on our studies of the effects of MIBG, a structural analogue of norepinephrine, on SK-N-BE(2c) cells. In micromolar concentrations, MIBG caused almost complete inhibition of the proliferation of SK-N-BE(2c) cells. In intact SK-N-BE(2c) cells, addition of MIBG led to a decrease of the ATP to ADP ratio. A progressive increase of the lactate to pyruvate ratio (due to increased lactate production) was observed after incubation of the cells with glucose and increasing concentrations of MIBG. In cells treated with digitonin, MIBG inhibited malate driven ATP synthesis. Comparable inhibition of ATP synthesis with succinate as a substrate required higher concentrations of MIBG. These results indicate that, apart from inhibition of complex I, MIBG was capable of inhibiting at least one other complex of the respiratory chain. Although maximal inhibition of ATP synthesis was observed at a concentration of 10 microM, optimal inhibition of cell proliferation occurred at a MIBG concentration > 25 microM. This suggests that MIBG also influences other cellular processes apart from mitochondrial ATP synthesis, resulting in additional inhibition of cell proliferation.

3-Iodobenzylguanidine↗