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Biomedical subjects

F Endo

Publications and source records attributed to F Endo.

At least 91 records · Page 5Linked to original sources
PubMed1995

Excess copper and ceruloplasmin biosynthesis in long-term cultured hepatocytes from Long-Evans Cinnamon (LEC) rats, a model of Wilson disease.

Immortalized hepatic cell lines obtained from laboratory animals or patients with defects in copper metabolism in the liver provide new approaches to examine related metabolism and toxicity. We established a series of hepatic cell lines from the liver of Long-Evans Cinnamon (LEC) rats, using recombinant adenovirus which expresses SV40 large T. Cells from the LEC rats were cultured and accumulated larger amounts of copper than did control cells, when the concentrations of copper in the culture medium exceeded 5 microM. The secretion of ceruloplasmin (CP) from the cultured cells was not reduced in hepatocytes from LEC cells, as compared with the control cells. As accumulation of copper did not affect CP secretion, CP production was not likely to be affected by the accumulation of copper in LEC rat hepatocytes. The production of holo-CP was further investigated by transfection of human CP cDNA and detection of human holo-CP by immunological procedures and use of a monoclonal antibody (mAb CP60) which recognizes human holo-CP but not human apo-CP and rat CP. Hepatocytes from the LEC rats processed and secreted holo-CP into the medium, even with excess copper present in the medium. These observations suggest that the genetic defect in LEC rats did not alter biosynthetic and secretory pathways of CP and that the intracellular copper concentration did not regulate the synthesis and processing of CP in the cultured hepatocytes. Low ceruloplasmin levels are observed in most, but not all, patients with Wilson disease, as well as in LEC rats. Our results do suggest that the copper transporting ATPase encoded in the Wilson disease gene is not a integral part of the biochemical mechanism of copper incorporation into apoprotein. The cell lines and immunological procedures we used are expected to add to information on biologically important process related to copper metabolism and to CP biosynthesis.

Adenoviruses, Human↗
PubMed1995

A nonsense mutation in the 4-hydroxyphenylpyruvic acid dioxygenase gene (Hpd) causes skipping of the constitutive exon and hypertyrosinemia in mouse strain III.

4-Hydroxyphenylpyruvic acid dioxygenase (HPD; EC 1.13.11.27) is an important enzyme in tyrosine catabolism in most organisms. Decreased activity of 4-hydroxyphenylpyruvic acid dioxygenase in the liver of mouse strain III is associated with tyrosinemia. We report a nucleotide substitution that generates a termination codon in exon 7 of the 4-hydroxyphenylpyruvic acid dioxygenase gene in III mice. This mutation is associated with partial exon skipping, and most of the mRNA lacks sequences corresponding to exon 7. The partial exon skipping apparently is the result of a nonsense mutation in the exon. Mouse strain III is a model for human tyrosinemia type 3 (McKusick 276710), and this strain together with recently established models for tyrosinemia type 1 will facilitate studies of hereditary tyrosinemias.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1995

Identification of two new aberrant splicings in the ornithine carbamoyltransferase (OCT) gene in two patients with early and late onset OCT deficiency.

Ornithine carbamoyltransferase (OCT) is a liver-specific enzyme located in the mitochondrial matrix. OCT deficiency is an X-linked disease with a heterogeneous phenotype, even in affected males. We studied two male patients (K.M., K.G.) with early and late onset, respectively. OCT activity was zero in the autopsied liver of patient K.M. and was 6% of control in the biopsied liver of K.G. Sequencing of OCT cDNAs revealed exon 5 skipping in K.M., resulting from a T-to-C transition of the initial dinucleotide of the 5' splicing donor site of intron 5, and a G-to-T transversion at position +45 in exon 9 (L304F) in K.G., providing three OCT mRNAs of different lengths: a normally spliced transcript, 23 bp insertion of intron 8 and the first 50bp missing within exon 9. Exon 5 skipping and two other aberrant splicings produced stop codons early downstream in mature OCT mRNAs. Expression study of a missense allele, L304F, transfected to cultured Cos 1 cells revealed a 34.4% value of the control. The difference of OCT activities between the patient liver and transfected cells (6% vs. 34%) can be explained by this splicing abnormality.

Amino Acid Metabolism, Inborn Errors↗
PubMed1995

Splicing mutations in the COL4A5 gene in Alport's syndrome: different mRNA expression between leukocytes and fibroblasts.

The COL4A5 gene from 40 patients with Alport's syndrome was examined using single-strand conformation substitution at the acceptor site (-2) of intron 50 and a G-to-C substitution at the donor site (+1) of intron 47, respectively. The transcript in peripheral leukocytes from the former had a 10-nucleotide deletion. This shortened transcript was derived from abnormal splicing in a cryptic acceptor site within exon 51. This could be translated into a protein with an alteration of three amino acids followed by premature termination, which eliminated 23 amino acids from the carboxyl end. Gene tracking revealed that the mother and a brother carried the mutant allele. In the latter, the transcript in leukocytes was normal, but that in cultured skin fibroblasts showed skipping of exon 47, the result being that 71 amino acids were absent. Glomerular basement membrane from the patient did not react with the anti-alpha 5(IV) antibody. His maternal grandmother, mother, and a sister, all with abnormal urinalysis, carried the mutant allele. Thus, the appearance of exons of the COL4A5 gene in leukocytes may differ from that in fibroblasts. If kidney mRNA is not available, mRNAs from cultured skin fibroblasts, in addition to leukocytes, can be used for gene analysis in subjects with Alport's syndrome.

Adolescent↗
PubMed1995

Clinical and molecular analysis of a Japanese boy with Morquio B disease.

Morquio B disease was found in a 15-year-old Japanese boy who presented with progressive generalized skeletal dysplasia without neurological manifestations. Mild keratan sulfaturia was found, and beta-galactosidase was deficient in fibroblasts. Gene analysis revealed two mutant alleles, 83Tyr-->His (Y83H) and 482Arg-->Cys (R482C). The former expressed a low enzyme activity (2-5% of normal), and the latter expressed no detectable enzyme activity.

Adolescent↗
PubMed1994

Structural organization and analysis of the human fumarylacetoacetate hydrolase gene in tyrosinemia type I.

Fumarylacetoacetate hydrolase (FAH) is a metabolic enzyme functioning at the last step of tyrosine catabolism. Deficiency in this enzyme activity is associated with tyrosinemia type I, characterized by hypertyrosinemia, liver dysfunction, renal tubular dysfunction, liver cirrhosis, and hepatic tumors. We isolated from a human gene library a chromosomal gene related to FAH. The human FAH gene is 30 kilobases long and is split into 14 exons. All of the splice donor and acceptor sites conform to the GT/AG rule. We also analyzed findings in a patient with tyrosinemia type I with respect to the mutation responsible for defects in the enzyme. A nucleotide change from T to G was found in the exon 2 of the gene and this change was accompanied by an amino acid substitution (Phe62Cys). Transfection and expression analysis of the cDNA in cultured BMT-10 cells with the nucleotide substitution demonstrated that the substitution was indeed responsible for the decreased activity of the enzyme in the patient. These results confirmed that the T to G mutation was one of the causes of tyrosinemia type I. Structure of the FAH gene and tests for expression of the mutant FAH will facilitate further understanding of various aspects of FAH.

Base Sequence↗
PubMed1994

Deficiency of the E1 beta subunit in the branched-chain alpha-keto acid dehydrogenase complex due to a single base substitution of the intron 5, resulting in two alternatively spliced mRNAs in a patient with maple syrup urine disease.

A patient with maple syrup urine disease (MSUD) associated with a E1 beta subunit deficiency of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex was investigated at the molecular level. The defect responsible for the deficiency of the E1 beta subunit protein was identified by analysis of cDNA and genomic DNA by polymerase chain reaction. Total RNA isolated from lymphoblastoid cells was transcribed into cDNA and amplified using a set of primers located within exon 3 and exon 9 of the E1 beta gene. Agarose gel electrophoresis of cDNA amplification products revealed two shortened bands as well as a faint band of normal size. Nucleotide sequencing of the shortened cDNA amplification products showed that sequences corresponding to exon 5 and both exons 5 and 6 were absent. Nucleotide sequencing of the proband's amplified genomic DNA corresponding to this region of the E1 beta gene revealed a single base substitution from G to T of the invariant GT dinucleotides at 5' splice site of the intron 5. Analysis of family members using primer-specified restriction map modification showed that the patient is homozygous for this mutation. We postulate that this mutation leads to the skipping of either exon 5 or both exons 5 and 6, thus producing two shortened E1 beta mRNA. The percentage of normal and two shortened transcripts was estimated to be 9, 71 and 20%, respectively. To our best knowledge, this is the first documented example of exon skipping in the E1 beta gene as the cause of MSUD and the novel mutation of the invariant G at the 5' splice site which results in two alternatively spliced mRNA due to the skipping of the preceding exon as well as both preceding and following exon.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1994

Structure of the human 4-hydroxyphenylpyruvic acid dioxygenase gene (HPD).

4-Hydroxyphenylpyruvic acid dioxygenase (HPD) is an important enzyme in tyrosine catabolism in most organisms. The activity of this enzyme is expressed mainly in the liver and developmentally regulated in mammals, and a genetic deficiency in this enzyme in humans and mice leads to hereditary tyrosinemia type 3. Using human HPD cDNA as a probe, a chromosomal gene related to HPD was isolated from human gene libraries. The human HPD gene is over 30 kb long and is split into 14 exons. The extract size and boundaries of exon blocks were determined, and all of the splice donor and acceptor sites conformed to the GT/AG rule. Analysis of the 5' flanking sequence of the gene suggests that expression of the gene is regulated by hepatocyte-specific and liver-enriched transcription factors, as well as by hormones. These features of the 5' flanking region of the gene are similar to those of other genes that are specifically expressed in hepatocytes and that are developmentally regulated.

4-Hydroxyphenylpyruvate Dioxygenase↗
PubMed1994

Expression of four mutant human ornithine transcarbamylase genes in cultured Cos 1 cells relates to clinical phenotypes.

Ornithine transcarbamylase (OTC) deficiency is an X-linked disease with a heterogeneous phenotype, even in affected males. To detect mutations in the OTC gene using genomic DNA, we have developed a method in which all exons and adjacent introns are amplified and sequenced. Although this approach detected mutations in many cases, the relationship between a mutation and the OTC phenotype was not firmly established. Therefore, we investigated the issue by expression analysis of mutant OTC cDNA in cultured cells. Four mutant OTC cDNAs were constructed, based on the reported cases, using our newly developed method. The normal (wild-type) human OTC cDNA was reproducibly expressed at high levels in these Cos 1 cells. Predicted OTC activities of mutant OTC cDNAs ranged from 0% to 8.9% of the normal level together with variable amounts of the enzyme protein. The predicted enzyme activities account for the clinical phenotype of the disease. Our observations confirm that these mutations are responsible for OTC deficiency in these patients.

Amino Acid Metabolism, Inborn Errors↗
PubMed1994

A randomized trial of intrahepatic arterial infusion of 4'-epidoxorubicin with Lipiodol versus 4'-epidoxorubicin alone in the treatment of hepatocellular carcinoma.

We conducted a prospective randomized trial to evaluate the efficacy of Lipiodol in intrahepatic arterial infusion chemotherapy for patients with hepatocellular carcinoma (HCC). A total of 38 patients with unresectable HCCs and underlying cirrhosis were entered in this trial, and 36 of them were evaluable. Every 4 weeks, 17 patients received 70 mg of 4'-epidoxorubicin (epirubicin) alone (group A), whereas 19 patients received a Lipiodol emulsion containing the same dose of epirubicin (group B) through the hepatic artery. A tumor response (CR+PR) was observed in 12% of group A patients and in 42% of group B patients. The group B patients showed a significantly higher response rate than the group A patients. There was a tendency for an increased duration of survival (P = 0.09) in the group B patients. These results suggested that the infusion of the Lipiodol emulsion with epirubicin was more effective than epirubicin alone for the treatment of these patients with HCC.

Aged↗
PubMed1994

Characterization of a point mutation in the pyruvate dehydrogenase E1 alpha gene from two boys with primary lactic acidaemia.

We report here a novel mutation in the codon for amino acid 263 resulting in the change from arginine to glutamine in the pyruvate dehydrogenase (PDH) E1 alpha gene, in two boys with primary lactic acidaemia, from independent families. The mutation changes an amino acid located between the two serine residues which are the sites of phosphorylation of the subunit protein. In one family, the mutation was de novo and in the other it was transmitted from mother to son. The amino acid substitution may affect function of the PDH complex via phosphorylation and dephosphorylation of the E1 alpha subunit. Derangement in the regulation of activity of the PDH complex may explain the primary lactic acidaemia in the patients.

Acidosis, Lactic↗
PubMed1994

Mutations in the COL4A5 gene in Alport syndrome: a possible mutation in primordial germ cells.

Using a combination of gene amplification with single strand conformation polymorphisms analysis and sequencing, we examined the COL4A5 gene in 37 patients with Alport syndrome. In patient A8, a single base insertion was noted at codon 1,597 tyrosine in exon 49. The premature terminal signal appeared and 89 amino acids (approximately one-third) of the non-collagenous domain were lost. The mutation was present in the mother, hence she is heterozygous. In patient A12, the nucleotide changed from C to T at codon 1,679 glutamine in exon 51, which created a termination codon, and 7 amino acids at the carboxyl terminus were lost. Gene tracking using peripheral leukocytes revealed that the parents did not carry the mutant allele, while the sister was heterozygous. DNA samples from hair roots and skin fibroblasts of the mother were normal and immunological examination of the epidermis of the mother indicated that the alpha 5(IV) chain was normally expressed. As these results suggest that somatic cells of the mother do not carry the mutant allele, the primordial germ cells possibly carry a fresh mutation in the mother of patient A12.

Adolescent↗
PubMed1993

Heterogeneity of mutations in maple syrup urine disease (MSUD): screening and identification of affected E1 alpha and E1 beta subunits of the branched-chain alpha-keto-acid dehydrogenase multienzyme complex.

Maple syrup urine disease (MSUD) is an autosomal recessive disease caused by a deficiency in subunits of the branched-chain alpha-keto-acid dehydrogenase complex (BCKDH). To characterize the mutations present in five patients with MSUD (four classic and one intermediate), three-step analyses were established: (1), identification of the involved subunit by complementation analysis using three different cell lines derived from homozygotes having E1 alpha, E1 beta or the E2 mutant gene; (2), screening for a mutation site in cDNA of the corresponding subunit by RT-PCR-SSCP and (3), mutant analysis by sequencing the amplified cDNA fragment. Four single-base missense mutations, R115W, Q146K [corrected], A209T and I282T, were detected in the E1 alpha subunit. A single-base missense mutation H156R and three frame-shift mutations to generate stop codons downstream, including an 11-bp deletion of the tandem repeat in exon 1, a single-base (T) deletion and a single-base (G) insertion, were identified in the E1 beta subunit gene. All except one (11-bp deletion in E1 beta (Nobukuni, Y., Mitsubuchi, H., Akaboshi, I., Indo, Y., Endo, F., Yoshioka, A. and Matsuda, I. (1991) J. Clin. Invest. 87, 1862-1866)) were novel mutations. The sites of amino-acid substitution were all conserved in other species. Thus, mutations causing MSUD are heterogenous.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1993

Identification of a single base insertion in the COL4A5 gene in Alport syndrome.

We identified a novel mutation in the COL4A5 gene of a Japanese patient with Alport syndrome. A combination of in vitro amplification of the exons with single strand conformation polymorphisms (SSCP) analysis suggested the presence of a mutation in exon 48. Sequencing of the amplified DNA revealed a single base (T) insertion which was between nucleotides T 4750 and G 4751 within the methionine 1516. This mutation caused a shift in the reading frame of nine amino acids and introduced a premature termination signal that would be expected to lack about two-thirds of the noncollagenous (NC1) domain. This mutation may interfere with type IV collagen assembly leading to increased permeability and play a causative role in the glomerular basement membrane abnormality of this patient with typical Alport syndrome. Gene tracking by restriction enzyme NlaIII digestion revealed that the patient's mother is heterozygous whereas the patient's brother and one sister are normal, albeit they have hematuria and proteinuria. Without gene analysis, they would have been misdiagnosed. We propose that the diagnosis of Alport syndrome should be made on the basis of both clinical phenotypes and molecular defects.

Adolescent↗