Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions.

The uptake as well as the export of citric acid by Aspergillus niger occur by active, deltapH-driven, H(+)-symport dependent systems. They are inhibited by nonmetabolizable tricarboxylic acid analogues and phthalic acid, and by several other mono-, di- and tribasic organic acids. However, citrate export could only be demonstrated in a mycelium cultivated under manganese-deficient growth conditions, whereas the uptake of citrate from the medium was only detectable upon precultivation of A. niger in a medium supplemented with Mn2+ ions. In addition, the uptake of citrate was dependent on the presence of Mn2+ ions in the assay, and inhibited by EDTA. This requirement for Mn2+ could also be partially fulfilled by Mg2+, Fe2+ or Zn2+, whereas Cu2+ ions inhibited citrate transport. The observed divergent effects of manganese ions on citrate uptake and citrate export may be a major reason for the well documented requirement for manganese deficiency of citric acid accumulation.

Rat adrenoleukodystrophy-related (ALDR) gene: full-length cDNA sequence and new insight in expression.

X-linked adrenoleukodystrophy (X-ALD) is an inherited demyelinating disorder due to mutations in the ALD gene, which encodes a peroxisomal ABC half-transporter (ALDP). It has been suggested that ALDP assembles with ALDRP (adrenoleukodystrophy-related protein), a close homologous half-transporter, to form a functional heterodimer. For the first time full-length ALDRP cDNA (5.5 kb) was cloned, and 5' and 3' RACE analysis revealed that alternative usage of polyadenylation sites generates the two transcripts of 3.0 and 5.5 kb observed in the rat in Northern blot analysis. Southern blotting and chromosomal mapping demonstrated one ALDR locus in the rat genome. Characterisation of the 3' flanking region suggested that an ID sequence might be responsible for high expression of the 5.5 kb ALDRP transcript in rat brain. ALDR gene expression was found to be high in the liver of rats before weaning and very low in adult rats; the reverse developmental regulation was observed in the brain. Fenofibrate, which is a potent inducer of the ALDR gene in the liver of adult rats, could not induce the ALDR gene in suckling rats. The exact significance of this result with regard to development of an efficient pharmacological gene therapy for X-ALD is discussed.

Rolipram does not normalize very long-chain fatty acid levels in adrenoleukodystrophy protein-deficient fibroblasts and mice.

In its severe form, X-linked adrenoleukodystrophy (X-ALD) is a lethal neurodegenerative disorder with inflammatory demyelination, in which defective peroxisomal beta-oxidation causes accumulation of very long-chain fatty acids (VLCFA) in tissues and plasma, in particular in the nervous system and adrenal glands. Recently, several drugs have been reported to reduce VLCFA in cultured human fibroblasts of X-ALD patients, and therefore to be potential candidates for novel therapeutic treatments in X-ALD. Among the most promising of these substances is the antidepressant rolipram, because of favourable adverse event profile in clinical studies and its additionally reported anti-inflammatory action. To further elucidate the effects of rolipram on peroxisomal beta-oxidation and VLCFA accumulation, we administered rolipram orally in the diet to ALD protein-deficient mice and ALD protein-deficient cultured human and mouse fibroblasts and assayed the accumulation of VLCFA. In contrast to the previously reported reduction of VLCFA, our data did not demonstrate a decrease in VLCFA content either in vivo or in vitro. NMR spectroscopic analysis verified the structural integrity and purity of the rolipram used here, thus excluding inauthenticity as a reason for the discrepancy. We therefore suggest that rolipram should be excluded from the current list of potential therapeutic agents for X-ALD.

Adrenoleukodystrophy-related protein can compensate functionally for adrenoleukodystrophy protein deficiency (X-ALD): implications for therapy.

Inherited defects in the peroxisomal ATP-binding cassette (ABC) transporter adrenoleukodystrophy protein (ALDP) lead to the lethal peroxisomal disorder X-linked adrenoleukodystrophy (X-ALD), for which no efficient treatment has been established so far. Three other peroxisomal ABC transporters currently are known: adrenoleukodystrophy-related protein (ALDRP), 70 kDa peroxisomal membrane protein (PMP70) and PMP70- related protein. By using transient and stable overexpression of human cDNAs encoding ALDP and its closest relative ALDRP, we could restore the impaired peroxisomal beta-oxidation in fibroblasts of X-ALD patients. The pathognomonic accumulation of very long chain fatty acids could also be prevented by overexpression of ALDRP in immortalized X-ALD cells. Immunofluorescence analysis demonstrated that the functional replacement of ALDP by ALDRP was not due to stabilization of the mutated ALDP itself. Moreover, we were able to restore the peroxisomal beta-oxidation defect in the liver of ALDP-deficient mice by stimulation of ALDRP and PMP70 gene expression through a dietary treatment with the peroxisome proliferator fenofibrate. These results suggest that a correction of the biochemical defect in X-ALD could be possible by drug-induced overexpression or ectopic expression of ALDRP.

The four murine peroxisomal ABC-transporter genes differ in constitutive, inducible and developmental expression.

Four ATP-binding cassette (ABC) half-transporters have been identified in mammalian peroxisomes: adrenoleukodystrophy protein (ALDP), adrenoleukodystrophy-related protein (ALDRP), 70-kDa peroxisomal membrane protein (PMP70) and PMP70-related protein (P70R). Inherited defects in ALDP cause the neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). By comparative Northern blot analyses we found each of the four murine peroxisomal ABC transporter mRNA species at maximum abundance only in a few tissues, which differed for each family member. The four genes were also regulated differentially during mouse brain development: ALDP mRNA was most abundant in embryonic brain and gradually decreased during maturation; ALDRP and P70R mRNA accumulated in the early postnatal period; and the amount of PMP70 transcript increased slightly during the second and third postnatal week. The different expression patterns could explain why beta-oxidation is defective in X-ALD, although ALDRP and PMP70 can replace ALDP functionally in fibroblasts. Dietary fenofibrate had no effect on the ALD and P70R genes, but strongly increased expression of the ALDR and PMP70 genes in mouse liver. However, in P-glycoprotein Mdr1a-deficient mice fenofibrate treatment increased ALDR gene expression also in the brain, suggesting that the multidrug-transporter P-glycoprotein restricts entry of fenofibrate to the brain at the blood-brain barrier. Analysis of the promoter sequences revealed a cryptic nuclear hormone receptor response element of the DR+4 type in the ALDR promoter and a novel 18-bp sequence motif present only in the 5' flanking DNA of the ALDR and PMP70 genes. The mouse ALDR gene uses a single transcription start site but alternative polyadenylation sites. These data are of importance for the use of ALDP-deficient mice as a model in pharmacological gene therapy studies.