Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralardeficient (Aralar ؊/؊ ) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar ؊/؊ mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar ؊/؊ mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar ؊/؊ mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.
Classical citrullinemia (CTLN1), a rare autosomal recessive disorder, is caused by mutations of the argininosuccinate synthetase (ASS) gene, localized on chromosome 9q34.1. ASS functions as a rate-limiting enzyme in the urea cycle. Previously, we identified 32 mutations in the ASS gene of CTLN1 patients mainly in Japan and the United States, and to date 34 different mutations have been described in 50 families worldwide. In the present study, we report ASS mutations detected in 35 additional CTLN1 families from 11 countries. By analyzing the entire coding sequence and the intron-exon boundaries of the ASS gene using RT-PCR and/or genomic DNA-PCR, we have identified 16 novel mutations (two different 1-bp deletions, a 67-bp insertion, and 13 missense) and have detected 12 known mutations. Altogether, 50 different mutations (seven deletion, three splice site, one duplication, two nonsense, and 37 missense) in 85 CTLN1 families were identified. On the basis of primary sequence comparisons with the crystal structure of E. coli ASS protein, it may be concluded that any of the 37 missense mutations found at 30 different positions led to structural and functional impairments of the human ASS protein. It has been found that three mutations are particularly frequent: IVS6-2A>G in 23 families (Japan: 20 and Korea: three), G390R in 18 families (Turkey: six, U.S.: five, Spain: three, Israel: one, Austria: one, Canada: one, and Bolivia: one), and R304W in 10 families (Japan: nine and Turkey: one). Most mutations of the ASS gene are "private" and are distributed throughout the gene, except for exons 5 and 12-14. It seems that the clinical course of the patients with truncated mutations or the G390R mutation is early-onset/severe. The phenotype of the patients with certain missense mutations (G362V or W179R) is more late-onset/mild. Eight patients with R86H, A118T, R265H, or K310R mutations were adult/late-onset and four of them showed severe symptoms during pregnancy or postpartum. However, it is still difficult to prove the genotype-phenotype correlation, because many patients were compound heterozygotes (with two different mutations), lived in different environments at the time of diagnosis, and/or had several treatment regimes or various knowledge of the disease.
Macroscopic and neurohistological observations of the red, gray and tree kangaroo and wombat's tongues are described. On the dorsum, three circumvallate papillae, foliate papillae (gland-duct type), fungiform papillae and filiform papillae are observed. In the wombat, the vallate papillae are shaped like a "lotusnut." Its summit is broadened, rough, and reaches the level of the dorsal surface, The papillae of the kangaroo are shaped like a "walnut" and are situated below the dorsal surface of the tongue. The vallate papilla is occupied with abundant nerves, thin non-myelinated and thick myelinated fibers, and ganglion cells, multipolar and unipolar. In the upper area of the wombat's papilla, however, there is a demarcated thin layer of non-innervated connective tissue. Therefore, taste buds are located in nearly the whole wall of the papilla in the kangaroo, but only in the lateral wall in the wombat, closely associated with the subgemmal nerve plexus. Foliate papillae
In the present study, the dihydrolipoamide succinyltransferase gene of the 2-oxoglutarate dehydrogenase complex was isolated from a human genomic DNA library and its entire nucleotide sequence was determined. This gene was approximately 23 kbp in size with 15 exons and 14 introns. All of the donor and acceptor splice sites of this gene conformed to the GT/AG rule. A quanine residue 43 bases upstreams of the ATG initiating translation codon was the transcription initiation site of the human dihydrolipoamide succinyltransferase mRNA. Sequence analysis of the promoterregulatory region showed the presence of a CAAT-box-like sequence but the presence of a TATAbox-like sequence was not evidenced. Also located in this region were sequences resembling glucocorticoid-responsive and CAMP-responsive elements, and an Spl binding site. No nucleotide sequence corresponding to the E3-binding and/or El-binding domain was found in any region of the gene. Therefore, the exon coding for the E3-binding and/or El-binding domain may have been lost from the gene during evolution. Moreover, a processed pseudogene of dihydrolipoamide succinyltransferase was isolated and sequenced. The nucleotide sequence of the pseudogene is 93 % similar to the sequence of the human dihydrolipoamide succinyltransferase cDNA, but the pseudogene is not functional for base changes, deletions and insertions of the pseudogene. Southern-blot analysis showed the presence of a single copy of this gene and a single copy of a pseudogene in the human genome. In addition, a possible relationship between dihydrolipoamide succinyltransferase and familial Alzheimer's disease is discussed The 2-oxoglutarate dehydrogenase complex belongs to a family of 2-oxoacid dehydrogenase complexes, which includes the pyruvate dehydrogenase complex and branchedchain 2-oxoacid dehydrogenase complex [l -61. These complexes are composed of three different enzymes, 2-oxoacid decarboxylase (El), dihydrolipoamide acyltransferase (E2) and dihydrolipoamide dehydrogenase (E3) [l -61. In mammals, the three complexes are localized in mitochondria and catalyze the oxidative decarboxylation of 2-oxoacids [l -61.Correspondence to S. Matuda, Department of Biology, Kanoya National Institute of Fitness and Sports, Kanoya, Kagoshima 891-23 JapanAbbreviations. E l , 2-oxoglutarate decarboxylase (or 2-oxoacid decarboxylase); E2, dihydrolipoamide succinyltransferase (or dihydrolipoamide acyltransferase); E3, dihydrolipoamide dehydrogenase ; E2 cDNA, human dihydrolipoamide succinyltransferase cDNA; CD4, a T cell cell-surface glycoprotein.Enzymes. 2-Oxoglutarate decarboxylase (EC 1.2.4.2) ; dihydrolipoamide succinyltransferase (EC 2.3.1.61) ; dihydrolipoamide dehydrogenase (EC 1.8.1.4).Note. The novel nucleotide sequence data published here have been submitted to the GSDB, DDBJ, EMBL and NCBl sequence data bank(s) and are available under accession number(s) D26535 and D29970.The skeletal structure of 2-oxoglutarate dehydrogenase complex is formed by the self-assembly of multiple copies of dihydrolipoamid...
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