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Biomedical subjects

S Miyabayashi

Publications and source records attributed to S Miyabayashi.

At least 19 recordsLinked to original sources
PubMed2000

Heterogeneous mutations in the glucose-6-phosphatase gene in Japanese patients with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by glucose-6-phosphatase (G6Pase) deficiency. It is characterized by short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. Various mutations have been reported in the G6Pase gene (G6PC). However, in Japanese patients, a g727t substitution was found to be the major cause of GSD-Ia, accounting for 20 of 22 mutant alleles [Kajihara et al., 1995], and no other mutations have been found in this population. We analyzed four Japanese GSD-Ia patients and identified three other mutations in addition to the g727t. They included two missense mutations (R83H and P257L) and one nonsense mutation (R170X). Each of the three mutations exhibited markedly decreased G6Pase activity when expressed in COS7 cells. A patient homozygous for R170X showed multiple episodes of profound hypoglycemia associated with convulsions, while P257L was associated with a mild clinical phenotype. The presence of R170X in three unrelated families may implicate that it is another important mutation in the etiology of GSD-Ia in Japanese patients. Thus, the detection of non-g727t mutations is also important in establishing the DNA-based diagnosis of GSD-Ia in this population.

Amino Acid Substitution↗
PubMed2000

Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1g<a, Gly122-to-Asp (G122D) and His179-to-Pro (H179P), were identified. We were able to detect "ectopically" transcribed G6Pase-mRNA in Epstein-Barr virus-transformed lymphoblastoid cells and observed aberrant mRNA splicing associated with the g727t and IVS1-1g<a mutations. To our knowledge, this is the first report that ectopic expression can be utilized for the characterization of GSD-Ia mutations. Our findings suggest that a screening for the g727t, R170X, and R83H mutations by simple DNA-based diagnostic methods can detect 95% of the G6Pase mutant alleles in Japanese patients with GSD-Ia, and remaining mutations can be identified and characterized by the direct sequencing of genomic DNA and/or the analysis of ectopically expressed mRNA. The noninvasive molecular diagnosis for GSD-Ia may ultimately replace the conventional means of enzymatic diagnosis that requires liver biopsy.

Alleles↗
PubMed2000

Complete repopulation of mouse mitochondrial DNA-less cells with rat mitochondrial DNA restores mitochondrial translation but not mitochondrial respiratory function.

By the fusion of mtDNA-less (rho(0)) cells of Mus musculus domesticus with platelets from different species, mtDNA repopulated cybrids were obtained for finding the mtDNA species that could induce mitochondrial abnormalities. Expression of mitochondrial dysfunction might be expected in these cybrids due to incompatibility between nuclear and mitochondrial genomes from different species. The results showed that mouse rho(0) cells could receive mtDNA from a different mouse species, M. spretus, or even mtDNA from the rat, Rattus norvegicus, and that the introduced rat mtDNA, but not M. spretus mtDNA, caused mitochondrial dysfunction, even though rat mtDNA could restore normal mitochondrial translation in the cybrids. Considering that mitochondrial respiratory complexes consist of nuclear DNA- and mtDNA-coded polypeptides, these observations suggest that the nuclear and mitochondrial interactions required for replication, transcription, and translation of introduced rat mtDNA must be less stringently controlled than those required for formation of normal respiratory complexes. As no procedure for introduction of mutagenized mouse mtDNA into living cells has yet been established, these findings provide important insights into generating mtDNA-knockout mice.

Adenosine Triphosphatases↗
PubMed1999

Apple chlorotic leaf spot virus 50 kDa protein is targeted to plasmodesmata and accumulates in sieve elements in transgenic plant leaves.

We investigated the in situ localization of the 50 kDa protein encoded by ORF2 of Apple chlorotic leaf spot virus (ACLSV) genome which is thought to be a movement protein. In immunogold electron microscopy of ACLSV-infected Chenopodium quinoa leaves, the 50 kDa protein was localized on plasmodesmata and nearby cytoplasm. Observation of transgenic Nicotiana occidentalis leaves expressing the 50 kDa protein fused to enhanced green fluorescent protein (EGFP) by fluorescence and confocal laser scanning microscopes revealed that green fluorescence was observed as spots on the cell wall or strands passing through the cell wall of several cell types, i.e., epidermal, palisade and spongy mesophyll and collenchyma cells. In transverse and longitudinal sections of leaf veins of transgenic plants showed that the 50K-EGFP fusion accumulated in sieve elements and formed an extensive interconnecting network of threadlike structure. These results indicated that ACLSV 50 kDa protein can target plasmodesmata and traffic into sieve elements.

Chenopodiaceae↗
PubMed1999

Molecular analysis of methylmalonyl-CoA mutase deficiency: identification of three missense mutations in mut0 patients.

Genetic defects in the methylmalonyl-CoA mutase (MCM) gene cause methylmalonic acidemia (MMA). Only three mutations have been reported among Oriental patients to date. We studied fibroblast cell lines established from three Japanese patients with MCM deficiency. Enzymatic study showed that these patients had the muttype of MMA. Nucleotide sequencing of MCM cDNAs identified three missense mutations: a T to A change at nucleotide position 2082, which results in an amino acid substitution of Glu669 for valine (V669E); a T to A change at position 1179 with the corresponding amino acid substitution of Asp368 for valine (V368D); and a G to A change at position 1182 with the corresponding amino acid substitution of His369 for arginine (R369H). Each of the three missense mutations abolished MCM activity according to a transient expression study. Alignment of these mutations with a recently reported homology model of human MCM allowed us to speculate on the effect of these nonconservative amino acid substitutions on MCM activity: V368D and R369H affected residues in the beta/alpha-(TIM-) barrel domain, on one of the two alpha-helices that form the dimer interface, while V669E altered a residue in the adenosylcobalamin-binding domain in the C terminus.

Amino Acid Metabolism, Inborn Errors↗
PubMed1999

Adult leigh syndrome: treatment with intravenous soybean oil for acute central respiratory failure.

This study reports a 38-year-old woman with adult Leigh syndrome associated with partial deficiency of the pyruvate dehydrogenase complex. The patient had intermittent diplopia, loss of vision, dystonia, central respiratory failure and unconsciousness with lactic acidosis. Treatment with an intravenous ketogenic emulsion resulted in rapid clinical and biochemical improvement. In patients with acute respiratory failure under these circumstances, intravenous ketogenic emulsion therapy is worth consideration.

Acute Disease↗
PubMed1998

Mutation and polymorphic marker analyses of 65K- and 67K-glutamate decarboxylase genes in two families with pyridoxine-dependent epilepsy.

Pyridoxine-dependent epilepsy is a disease inherited as an autosomal recessive trait, characterized by rapid response to pharmacological dosages of pyridoxine. The defect has been suggested to reside in glutamate decarboxylase (GAD), since a mutant GAD with an abnormally high Km for a cofactor, pyridoxal phosphate, could not synthesize an adequate amount of gamma-amino butyric acid [Scriver and Whelan (1969) Ann NY Acad Sci 166: 83]. To test this hypothesis, we studied two affected families by screening for mutations in the GAD mRNA and by analyzing a polymorphic marker in the GAD gene. Since two forms of GAD, GAD65 and GAD67, have been identified in human brain, we analyzed both forms. To overcome the limited accessibility of brain tissues, we utilized the minute amounts of GAD mRNAs ectopically transcribed in lymphoblasts. The ectopic GAD transcripts were amplified by reverse-transcription-mediated, nested polymerase chain reaction for mutation analysis. Two and three base substitutions were found in GAD65 and GAD67 cDNAs, respectively. All of them were, however, polymorphisms that were also found in control subjects. We then examined a (CA) repeat polymorphism in the GAD65 gene and found that different maternal alleles were transmitted to two affected sibs in one family. Thus, an etiological mechanism other than a K(m) mutant GAD is responsible for pyridoxine-dependent epilepsy.

DNA Mutational Analysis↗
PubMed1998

Mitochondrial deoxyribonucleic acid 3256C-T mutation in a Japanese family with noninsulin-dependent diabetes mellitus.

Accumulating reports indicate a relationship between mitochondrial DNA mutation and impaired glucose-induced insulin secretion leading to a subtype of noninsulin-dependent diabetes mellitus. DNA from a 45-yr-old Japanese woman with noninsulin-dependent diabetes mellitus and muscle atrophy was isolated and studied for mitochondrial DNA mutations. We identified a mitochondrial DNA C-T heteroplasmic mutation at nucleotide position 3256. The mutation was located in the transfer ribonucleic acidLeu in a region conserved in evolution. Eight other members of her family were examined for the mutation. Six of them had the same mutation together with noninsulin-dependent diabetes mellitus, and one teenage boy had the mutation and impaired glucose tolerance. The other family member who did not have this mutation had normal glucose tolerance. The enzyme activity of the mitochondrial oxidative phosphorylation pathway in the muscle of the proband was measured. The enzyme activity was decreased in the proband, especially in complex I. This mutation might be responsible for the abnormal glucose metabolism.

Adolescent↗
PubMed1997

Identification of inheritance modes of mitochondrial diseases by introduction of pure nuclei from mtDNA-less HeLa cells to patient-derived fibroblasts.

A nuclear genome delivery system was developed to deduce the modes of inheritance of the clinical phenotypes observed in patients with mitochondrial diseases by transfer of pure nuclei from normal cells to fibroblasts from the patients. The problem of possible contamination of the nuclei with a small amount of mtDNA was overcome by using mtDNA-less (rho0) human cells as nuclear donors. In this study, intercellular transfer of pure nuclei was carried out by simple fusion of rho0 HeLa cells with 533 fibroblasts from a patient with a fatal mitochondrial disease, which were deficient in cytochrome c oxidase and succinate dehydrogenase activities. The results showed that the cytochrome c oxidase and succinate dehydrogenase activities were restored by the introduction of pure HeLa nuclei, suggesting that the observed phenotypes of mitochondrial dysfunction were not due to mtDNA mutations but to nuclear, recessive mutations. Thus, our nuclear transfer system is effective for determining whether a mitochondrial or nuclear genome of a patient is responsible for a disease and whether deficiency of mitochondrial enzymes, including enzymes exclusively encoded by nuclear genomes, is transmitted in a nuclear recessive or nuclear dominant way, providing the parents of the patients with valuable information for genetic counseling on the risk of mitochondrial diseases in their next babies.

Adenosine Triphosphatases↗
PubMed1996

Enzymatic diagnosis of holocarboxylase synthetase deficiency using apo-carboxyl carrier protein as a substrate.

We developed a simple and sensitive method for assessing holocarboxylase synthetase (HCS) activity that is based on measuring incorporation of [3H]biotin into apo-carboxyl carrier protein, a subunit of acetyl-CoA carboxylase from E. coli. Kinetic analysis of HCS from normal fibroblasts showed that the Km for biotin was 260 +/- 94 nmol/l (mean +/- S.D.; n = 5). In contrast, the Km values of HCS from two cell lines derived from patients with HCS deficiency were 7200 and 3700, clearly distinguishable from the control value. The sensitivity of this assay was so high that we were able to characterize a mutant enzyme whose activity had not been previously detected. Our method is useful for enzymatic diagnosis of HCS deficiency and characterization of HCS.

Acetyl-CoA Carboxylase↗
PubMed1996

[Leigh syndrome: serial CT and MR imaging findings].

Serial CT and MRI findings in five patients (two boys and three girls) with Leigh syndrome were retrospectively reviewed in a follow-up period lasting from six months to 10 years. The two boys were found to have cytochrome c oxidase deficiency and one of the girls to have mitochondrial DNA mutation, while the remaining two girls had no detectable enzyme deficiency. CT and MRI revealed symmetrical involvement of the brain bilaterally in all cases. The focal lesions were found most frequently in putamina and caudate nuclei (four cases each), followed by thalami (three cases), globi pallidi, and midbrain (two cases each). In addition, diffuse white matter and/or cortical lesions were disclosed in three cases. MRI and CT at an early stage of the disease revealed swollen, symmetrical lesions which showed shrinkage in size accompanied by the adjacent brain atrophy on later images. Some lesions were detected only in the early stage and were not shown in later images. Thus, careful reading of sequential changes appears to be required for accurate diagnosis of Leigh syndrome.

Brain↗
PubMed1995

Molecular analysis of holocarboxylase synthetase deficiency: a missense mutation and a single base deletion are predominant in Japanese patients.

Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients' families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.

Amino Acid Sequence↗
PubMed1994

Functional and morphological abnormalities of mitochondria in human cells containing mitochondrial DNA with pathogenic point mutations in tRNA genes.

mtDNA with a point mutation in the tRNA(Ile) gene at nucleotide position 4269 found in a patient with fatal cardiomyopathy and mtDNA with a point mutation in the tRNA(Arg) gene at 10410 found in a patient with Alpers disease were transferred cytoplasmically to rho zero HeLa cells (HeLa cells lacking mtDNA) to determine whether these novel mtDNA mutations in the tRNA genes are responsible for the defects in mitochondrial respiration function observed in these diseases. Cybrid clones (clones of rho zero HeLa cells with mtDNA from the patients) were isolated, and respiratory function and morphology of the mitochondria of the cybrid clones containing wild-type mtDNA and mutant mtDNA predominantly were compared. The results showed that accumulation of mutant mtDNA at 4269 alone without defects in the nuclear genome was sufficient to produce a disease phenotype, while mutant mtDNA at 10410 was not related to pathogenesis and reflected one of the rare polymorphic sites of human mtDNA. Moreover, we found that mitochondria in living cells were significantly swollen only when they contained predominantly the pathogenic mutant mtDNA, suggesting that the functional abnormality of mitochondria induced by pathogenic mtDNA mutations in tRNA genes is always associated with their swollen structure.

Cardiomyopathies↗
PubMed1994

Nuclear but not mitochondrial genome involvement in human age-related mitochondrial dysfunction. Functional integrity of mitochondrial DNA from aged subjects.

The role of mtDNA and nuclear genome in human aging was examined by their intercellular transfer using skin fibroblasts and mtDNA-less HeLa cells (rho o-HeLa cells). We found in vivo age-related reductions in the activity of cytochrome c oxidase in human skin fibroblasts obtained from 16 donors of various ages (0-97 years). The abnormality in mitochondria of the aged donors was not attributable to either decrease in the copy number of mtDNA molecules or increase in the copy number of deletion mutant mtDNA molecules, but to significant decrease in overall polypeptide synthesis in the mitochondria. However, intercellular mtDNA transfer experiments showed that fibroblast mtDNA from elderly donors is functionally intact. By contrast, intercellular transfer of HeLa nuclei to fibroblasts from aged donors restored cytochrome c oxidase activity, suggesting that the age-related phenotype was nuclear recessive. However, during subsequent cultivation of these hybrids, the activity gradually reduced again, associated with gradual chromosome loss. These observations support the idea that accumulation of nuclear recessive somatic mutations, but not mtDNA mutations, is responsible for the in vivo age-related mitochondrial dysfunction observed in human skin fibroblasts.

Aged↗
PubMed1994

Fetal liver biopsy for prenatal diagnosis of carbamoyl phosphate synthetase deficiency.

Carbamoyl phosphate synthetase deficiency is a type of an inborn error of metabolism with a poor prognosis. Carbamoyl phosphate synthetase is a liver-specific enzyme, and its deficiency can only be diagnosed by enzyme assay using liver biopsy specimens. A pregnant woman at risk for carbamoyl phosphate synthetase deficiency was evaluated for the purpose of prenatal diagnosis of the condition. Fetal liver biopsy was performed at the 22nd week of gestation. The results of enzyme assays on fetal liver biopsy revealed normal enzymatic activity and the diagnosis of carbamoyl phosphate synthetase deficiency was ruled out prenatally. Methods and safety of fetal liver biopsy are discussed. Measurements of activity of liver-specific enzymes were evaluated with respect to methodology and manner of assessment. Prenatal diagnosis of deficiencies in liver-specific enzymes, which have hitherto been considered difficult to detect before birth, has now become possible.

Adult↗