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T Orii

Publications and source records attributed to T Orii.

At least 127 records · Page 7Linked to original sources
PubMed1996

Immunotitration analysis of cytosolic acetoacetyl-coenzyme A thiolase activity in human fibroblasts.

There are five known thiolases in human fibroblasts, and all but mitochondrial trifunctional protein (TFP) have thiolase activity toward acetoacetyl-CoA (AACoA). We investigated the contribution of mitochondrial acetoacetyl-CoA thiolase (AACoAT) (T2), cytosolic AACoAT (CT), and mitochondrial 3-ketoacyl-CoA thiolase (T1) to the total AACoAT activity in control human fibroblasts. Immunotitration of AACoAT activity with antibodies against T2, CT, or T1 was carried out in control fibroblasts, with the following results. In the case of AACoAT activity in the absence of potassium ion, 26-38%, 40-47%, and 11-20% of the total activity derived from CT, T1, and T2, respectively. The residual 6-9% total activity was not immunotitrated when three antibodies were used in combination. Hence, the contribution of peroxisomal 3-ketoacyl-CoA thiolase to the total AACoAT activity in the absence of potassium ion was at least less than 6-9%. Because the normal range of total AACoAT activity is relatively wide, it is difficult to evaluate CT defects based on a decrease of total AACoAT activity. Immunotitration with anti-CT antibody in six control fibroblasts revealed that CT activity ranges between 1.3 and 2.4 nmol/min/mg of protein. Immunotitration proved to be an accurate method to evaluate CT activity. The two cell lines from patients with CT deficiency have become extinct.

Acetyl-CoA C-Acetyltransferase↗
PubMed1996

Mucopolysaccharidosis IVA: four new exonic mutations in patients with N-acetylgalactosamine-6-sulfate sulfatase deficiency.

We report four new mutations in Japanese patients with mucopolysaccharidosis IVA (MPSIVA) who were heterozygous for a common double gene deletion. A nonsense mutation of CAG to TAG at codon 148 in exon 4 was identified, resulting in a change of Q to a stop codon and three missense mutations. V (GTC) to A (GCC) at codon 138 in exon 4, P (CCC) to S (TCC) at codon 151 in exon 5, and P (CCC) to L (CTC) at codon 151 in exon 5. Introduction of these mutations into the normal GALNS cDNA and transient expression in cultured fibroblasts resulted in a significant decrease in the enzyme activity. V138A and Q148X mutations result in changes of restriction site, which were analyzed by restriction-enzyme assay. P151S and P151L mutations that did not alter the restriction site were detected by direct sequencing or allele specific oligohybridization. Detection of the double gene deletion was initially done using Southern blots and was confirmed by PCR. Haplotypes were determined using seven polymorphisms to the GALNS locus in families with the double gene deletion. Haplotype analysis showed that the common double gene deletion occurred on a single haplotype, except for some variation in a VNTR-like polymorphism. This finding is consistent with a common founder for all individuals with this mutation.

Adolescent↗
PubMed1996

Human peroxisome assembly factor-2 (PAF-2): a gene responsible for group C peroxisome biogenesis disorder in humans.

Peroxisome-biogenesis disorders (PBD) are genetically heterogeneous and can be classified into at least ten complementation groups. We recently isolated the cDNA for rat peroxisome assembly factor-2 (PAF-2) by functional complementation using the peroxisome-deficient Chinese-hamster-ovary cell mutant, ZP92. To clarify the novel pathogenic gene of PBD, we cloned the full-length human PAF-2 cDNA that morphologically and biochemically restores peroxisomes of group C Zellweger fibroblasts (the same as group 4 in the Kennedy-Krieger Institute) and identified two pathogenic mutations in the PAF-2 gene in two patients with group C Zellweger syndrome. The 2,940-bp open reading frame of the human PAF-2 cDNA encodes a 980-amino-acid protein that shows 87.1% identity with rat PAF-2 and also restored the peroxisome assembly after gene transfer to fibroblasts of group C patients. Direct sequencing of the PAF-2 gene revealed a homozygous 1-bp insertion at nucleotide 511 (511 insT) in one patient with group C Zellweger syndrome (ZS), which introduces a premature termination codon in the PAF-2 gene, and, in the second patient, revealed a splice-site mutation in intron 3 (IVS3+1G-->A), which skipped exon 3, an event that leads to peroxisome deficiency. Chromosome mapping utilizing FISH indicates that PAF-2 is located on chromosome 6p21.1. These results confirm that human PAF-2 cDNA restores peroxisome of group C cells and that defects in the PAF-2 produce peroxisome deficiency of group C PBD.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1995

Genomic DNA organization of human mitochondrial very-long-chain acyl-CoA dehydrogenase and mutation analysis.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is a major enzyme catalyzing long-chain fatty acids in the first step of mitochondrial beta-oxidation system. Inborn error of this enzyme can cause sudden infant death syndrome and hypertrophic cardiomyopathy is present at a significantly high frequency. To investigate VLCAD deficiency at the genomic DNA level, we cloned the VLCAD gene and analyzed the structure. The gene is about 5.4 kb long and contains 20 exons. We performed mutation analysis in two patients, both having a 105 bp deletion encompassing bases 1078-1182 in cDNA. A point mutation (GT-->AT) at 5' splice site of intron 11 was identified in both patients. This mutation seems to cause skipping of exon 11 corresponding to the 105 bp deletion. This is the first documentation of aberrant splicing in the VLCAD gene.

Acyl-CoA Dehydrogenase, Long-Chain↗
PubMed1995

Identification of the D-enantiomer of 2-hydroxyglutaric acid in glutaric aciduria type II.

We determined the optical isomer of the 2-hydroxyglutaric acid (2HG) that was elevated in the urine of five Japanese children with a mild form of glutaric aciduria type II (GA2), caused by a deficiency of electron transfer flavoprotein (ETF) or ETF-ubiquinone oxidoreductase (ETF-QO). The D- and L-enantiomers of 2HG were separated by capillary gas chromatography with a combination of (S)-(+)-2-octanol derivatization and chromatography on a DB-1 column. The isomer that was elevated in GA2 patients was predominantly the D-enantiomer, an observation that may serve as an additional marker for the biochemical diagnosis of GA2. D-2HG dehydrogenation, but not L-2HG dehydrogenation is apparently blocked in GA2. A specific D-2HG dehydrogenase or D-2HG-CoA dehydrogenase may be metabolically linked to ETF and ETF-QO in the mitochondria.

Biomarkers↗
PubMed1995

Adrenoleukodystrophy: the restoration of peroxisomal beta-oxidation by transfection of normal cDNA.

In order to elucidate the function of ALDP [a protein encoded by the gene responsible for adrenoleukodystrophy (ALD)], normal ALDP cDNA, inserted in an expression vector driven by chicken beta-actin promotor, was transfected into ALD fibroblasts. In a transient expression system, the fatty acid composition did not change even though the ALDP was newly synthesized based on the findings of a western blot analysis. In a stable expression system, 3 cell lines were strongly positive for ALDP. In these cells the level of very long chain fatty acid (C26:0) turned out to be as low as those of the control, while the activities of C24 beta-oxidation, as checked by two different methods, became normal. From these results, it is concluded that ALDP is indispensable for the function of peroxisomal beta-oxidation, and thus the treatment of ALD may be possible by the supplementation of ALDP.

ATP Binding Cassette Transporter, Subfamily D, Mem↗
PubMed1995

Molecular, biochemical, and clinical characterization of mitochondrial acetoacetyl-coenzyme A thiolase deficiency in two further patients.

The molecular basis of mitochondrial acetoacetyl-CoA thiolase (T2) deficiency was studied in two patients (GK11 and GK16). Fibroblasts from each patient had detectable immunoreactive T2 polypeptide (CRM). In pulse-chase experiments, fibroblasts from GK11 had two types of CRM: one (type I CRM) disappeared after a 24-hr chase and migrated more slowly than that of the normal control; the other (type II CRM) was detected with a small amount even after a 72-hr chase and had normal electrophoretic mobility. GK16's fibroblasts had a CRM (type III) which was also detectable even after a 72-hr chase and showed a slower mobility than type I CRM. By analyzing amplified cDNA and genomic fragments, we showed that both patients are genetic compounds; GK11 for the mutations N158D and T297M, and GK16 for the mutations A301P and IVS8 (+1). Expression analyses confirmed that mutant T2 subunits with N158D, T297M, and A301P correspond to type I, II, and III CRM, respectively. Among them, only the mutant T2 polypeptide with T297M appeared to have a detectable residual activity, in spite of its instability. Cotransfection of two cDNAs containing N158D and T297M suggested that heterotetramer formation reduces residual activity in GK11 cells.

Acetyl-CoA C-Acetyltransferase↗
PubMed1995

Molecular basis of beta-ketothiolase deficiency: mutations and polymorphisms in the human mitochondrial acetoacetyl-coenzyme A thiolase gene.

beta-Ketothiolase deficiency is a deficiency in mitochondrial acetoacetyl-CoA thiolase (T2). We present here an update on mutations and polymorphisms in the human T2 gene. No large deletion or insertion has been observed in Southern blot analysis. Seventeen mutations were identified in 13 T2-deficient patients: nine missense, one nonsense, and five splice-site mutations, and two small deletions. Two polymorphic base substitutions were also detected. A common mutation in T2 deficiency has not been detected but 4 mutations (N158D, Q272X, 828 + 1, 1163 + 2) were identified in two independent families. Eleven of 25 mutant alleles identified caused aberrant splicing. In vivo expression analysis of 13 mutant cDNAs using a Lipofectin reagent suggested that T297M, A301P, A380T mutant alleles retain 5-10% normal T2 activity. A correlation between clinical phenotype and genotype in T2 deficiency seems unlikely.

Acetyl-CoA C-Acetyltransferase↗
PubMed1995

Mucopolysaccharidosis type II (Hunter disease): identification and characterization of eight point mutations in the iduronate-2-sulfatase gene in Japanese patients.

Mucopolysaccharidosis type II (Hunter disease) is a lysosomal storage disorder caused by a deficiency of the enzyme iduronate-2-sulfatase. Varied clinical phenotypes of this disease have been described. To identify mutations in individual patients and to examine possible correlations between mutations and clinical phenotypes, we analyzed the iduronate-2-sulfatase gene in Japanese patients with different clinical phenotypes. Five missense mutations, S333L (severe), R468Q (severe), R468L (severe), W337R (intermediate), R48P (mild), and three nonsense mutations, W345X (severe), R443X (intermediate), Q531X (mild), were identified by the RT-PCR method. Transient expression in the enzyme-deficient fibroblasts revealed that all five missense mutant enzymes were synthesized as the normal-size precursor (73 kD), and the nonsense mutant enzymes were synthesized as truncated ones (W345X:54 kD, R443X:59 kD, and Q531X:69 kD), although stable mature enzymes (45-56 kD) were not detected by Western blot analysis. Furthermore, expression of the eight mutant cDNAs resulted in severe reductions of iduronate-2-sulfatase enzyme activity in comparison with a normal cDNA.

Adolescent↗
PubMed1995

Prenatal diagnosis in a family with mitochondrial acetoacetyl-coenzyme A thiolase deficiency with the use of the polymerase chain reaction followed by the heteroduplex detection method.

Mitochondrial acetoacetyl-coenzyme A (CoA) thiolase deficiency is an organic aciduria which affects isoleucine and ketone body catabolism. GK16 (the index patient) was affected with this disorder and previous studies had revealed that GK16 was a compound heterozygote with IVS8(+1) gt to tt and A301P mutations. In a subsequent pregnancy, prenatal diagnosis was performed and the fetus's amniocytes were analysed by the polymerase chain reaction (PCR) followed by the heteroduplex detection method on a Mutation Detection Enhancement gel. The fetus was identified as a carrier of the IVS8(+1) mutation. We confirmed the diagnosis by immunoblot analysis of extracted amniocytes and gene analysis with blood filter paper after delivery. This is the first report of prenatal diagnosis of this disorder at the gene level.

Acetyl-CoA C-Acetyltransferase↗
PubMed1995

Immunohistochemistry for a bifunctional protein in patients with peroxisomal disorders.

Immunohistochemical studies using antisera against bifunctional protein, a beta-oxidation enzyme, were performed on liver, kidney, and brain tissue specimens from patients with peroxisomal disorders and from controls to investigate the distribution and development of peroxisomes. Bifunctional protein-positive granules were not found in patients with Zellweger syndrome or neonatal adrenoleukodystrophy, whereas positive immunoreactivity was observed from 8 and 6 weeks gestation in the liver and kidney, respectively, and in the brain, from 23-25 weeks in the brainstem neurons and from 12-14 weeks in the white matter glia, in controls. Bifunctional protein immunoreactivity then increased with gestation in the brain. These results suggest that bifunctional protein immunohistochemistry is useful for the detection of peroxisomes, which are closely related to neuronal maturation and gliogenesis in premyelination in human brain development.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1995

Peroxisomal assembly defects: clinical, pathologic, and biochemical findings in two patients in a newly identified complementation group.

We describe the clinical, pathologic, and biochemical findings for two peroxisome-deficient patients in a newly identified complementation group. Both patients had biochemical findings typical of patients with peroxisome biogenesis disorders. However, whereas one patient had the typical clinicopathologic features of Zellweger syndrome, the other patient's phenotype was atypical.

Catalase↗
PubMed1995

Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant.

Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1995

Four novel mutations in mucopolysaccharidosis type VII including a unique base substitution in exon 10 of the beta-glucuronidase gene that creates a novel 5'-splice site.

Mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, is a lysosomal storage disorder caused by a deficiency in the enzyme beta-glucuronidase. Various clinical phenotypes of this autosomal recessively inherited disease have been described. Recent isolation and characterization of human beta-glucuronidase cDNA and the genomic sequences facilitate analysis of molecular defects underlying the different phenotypes, and eight mutations in the beta-glucuronidase gene have been described. This report summarizes studies characterizing four new mutations in two Caucasian patients with a severe form of MPS VII. Three are point mutations, resulting in two missense and one nonsense change, and one is a 38 bp deletion. The first patient was a compound heterozygote having P148S and Y495C alleles. The second patient was a compound heterozygote of W507X and a 38 bp deletion at position 1642-1679 in exon 10(1642 delta 38nt). The 38 bp deletion was caused by a single base change mutation in exon 10 that generates a new, premature 5' splice site. Expression of mutant cDNAs encoding each of the four mutations showed that all four resulted in a severe reduction of beta-glucuronidase activity, indicating that these mutations are responsible for the reduced enzyme activity in patient cells. These four previously undescribed mutations provide further evidence for the broad molecular heterogeneity in Sly syndrome.

Base Sequence↗