[Peroxisomes and neurological disorders].
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Bile Acids and Salts↗
Biomedical subjects
T Orii
Publications and source records attributed to T Orii.
[Molecular basis of mucopolysaccaridosis type VII (beta-glucuronidase deficiency)].
We have identified several mutations causing beta-glucuronidase (beta Gl) deficiency in three cases with mucopolysaccaridosis type VII (MPS VII). Enzyme assay of lysates of these lymphocytes or cultured fibroblasts showed little residual activity and that the beta Gl-specific mRNA levels were normal, as revealed by Northern blot analysis. Mutated cDNA clones including the entire coding sequencing were isolated from a library in case 1 and PCR (polymerase chain reaction) products in case 2 and 3 derived from cultured fibroblasts. Sequencing of the full-length mutated cDNA revealed C----T transitions, an event causing a single Ala619----Val change (cases 1 and 2) and Arg382----Cys and Pro649----Leu changes (case 3). The former change is detected by loss of the cleavage site for the enzyme Fnu 4 HI in the mutated cDNA. On the basis of the loss of Fnu 4 HI restriction site, the patients (cases 1 and 2) were shown to be a homozygote with this mutation and the parents and brother in case 1 were heterozygotes. The Ala619----Val and Arg382----Cys mutations disrupt a functional domain consisting of a region of sequence highly conserved among human, rat and bacterial beta Gl's, and they lower the enzyme activity, as tested by transfection of COS cells with expression vectors harboring the mutated cDNA. However the Pro649----Leu mutation does not lower the enzyme activity.
Amino Acid Sequence↗
[Clinical, pathological and biochemical abnormality in peroxisomal disorders].
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Brain↗
The organization and the complete nucleotide sequence of the human NADH-cytochrome b5 reductase gene.
The organization and the complete nucleotide (nt) sequence of the b5R gene encoding human NADH-cytochrome b5 reductase (b5R; EC 1.6.2.2) have been determined by a combination of restriction mapping and nt sequence analysis of overlapping genomic DNA clones. The entire gene is about 31 kb in length and contains nine exons and eight introns. Exon 2 contains the junction of the membrane-binding domain and the catalytic domain of b5R, indicating that two forms of b5R, a soluble and a membrane-bound form, are generated by post-translational processing. The 5' portion of the b5R gene lacks the canonical 5' transcriptional regulatory elements, but contains five copies of the GC box sequence G-G-G-C-G-G. While the average G + C content of the b5R gene is 55%, that of the 5' portion of the gene is extraordinarily high (86%). The CpG dinucleotide sequence was found at a very high frequency in this G + C-rich region. These structural features are very similar to those of the regulatory regions of constitutively expressed 'housekeeping' genes. Several transcription start points were identified by the primer extension experiment. Seventeen complete and twelve incomplete Alu family sequences were found in introns. An uncanonical polyadenylation signal was detected in the 3'-untranslated region of the gene as A-G-T-A-A-A instead of A-A-T-A-A-A.
Amino Acid Sequence↗
Diagnosis of subtypes of GM1 gangliosidosis in vitro and in vivo--using urinary oligosaccharides as substrates.
In order to delineate clinical subtypes of GM1 gangliosidosis enzymologically, we prepared galactosyl oligosaccharides from the urine of patients, as substrates, and established the method of the galactosyl oligosaccharide beta-galactosidase assay. Galactosyl oligosaccharides beta-galactosidase activities (nmol/mg protein/20 h) in vitro, using substrates without repeating structures were; type 1, 1.0 +/- 0.5 (n = 6), type 2A, 2.1, type 2B, 3.4 +/- 0.7 (n = 5), type 3, 4.9 +/- 0.2 (n = 2). The activities in vitro using substrates with repeating structures were: type 1, 0.3 +/- 0.2 (n = 5), type 2A, 1.2, type 2B, 2.2 +/- 0.5 (n = 4), type 3, 4.2 +/- 0.3 (n = 2). The activities using substrates with and without repeating structures were affected in the fibroblasts of patients, and the residual activities in each subtype were reduced progressively with the increasing severity of the clinical features. The ratio between activities using substrates without repeating structures and activities using substrates with repeating structures indicated that beta-galactosidase activities toward Gal beta 1- of repeating structures were reduced progressively with the increasing severity of the clinical features. The activities in vivo (pmol/mg protein per 24 h) were: type 1, 11.8 +/- 1.8 (n = 2), type 2A, 24.8, type 2B, 40.0 +/- 9.7 (n = 2), type 3, 63.2. The activities in vivo were affected in the fibroblasts of patients and the residual activities were reduced in proportion to the severity of the clinical features. These differences of residual activities among each subtype make it possible to delineate clinical subtypes enzymologically.
Child↗
Severity of GM1 gangliosidosis and urinary oligosaccharide excretion.
Urinary oligosaccharides of GM1 gangliosidosis patients (type 2A, 4 cases; type 2B, 2 cases) were investigated using the Bio Gel system. The levels of urinary oligosaccharide excreted (nmol/mg creatinine) by the type 2A patients were 4.1 times the levels of the type 2B patients. Patients of type 2A excreted high molecular weight oligosaccharides which were not detected in the urine of type 2B, and excreted oligosaccharides with long linkages of repeating structures. Thus, type 2A apparently has biochemically different characteristics from type 2B related to urinary oligosaccharide. Differentiation of type 2A from type 2B can thus be made biochemically. The structures of 5 different kinds of oligosaccharides not reported previously were confirmed.
Adolescent↗
Gene assignment of Zellweger syndrome to 7q11.23: report of the second case associated with a pericentric inversion of chromosome 7.
The case of a newborn girl with Zellweger syndrome and a pericentric inversion of chromosome 7, 46,XX, inv(7)(p12q11.23), is reported. The diagnosis was confirmed by marked deficiency of peroxisomal beta-oxidation enzymes in hepatic cells from autopsy samples. This is the second case of Zellweger syndrome associated with a rearrangement of chromosome 7, the tentative gene assignment to 7q11 being further supported; the gene is probably confiend to 7q11.23.
Acetyl-CoA C-Acyltransferase↗
3-Ketothiolase deficiency: heterogeneity in a defect of mitochondrial acetoacetyl-CoA thiolase biosynthesis in fibroblasts from four patients.
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Acetyl-CoA C-Acetyltransferase↗
Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat mitochondrial acetoacetyl-CoA thiolase.
cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1,460 bases and contained a 1,272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44,694 and 41,364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.
Acetyl-CoA C-Acetyltransferase↗
Restriction fragment length polymorphism analysis in healthy Japanese individuals and Japanese families with Gaucher disease.
We analysed the restriction fragment length polymorphism (RFLP) of 30 unrelated, healthy Japanese individuals and the members of seven Japanese families with Gaucher disease (5 Type I, 2 Type III) with the cDNA for human glucocerebrosidase, to investigate the frequency of the RFLP among healthy Japanese individuals and to determine the feasibility of the use of this probe DNA for carrier detection and prenatal diagnosis in Japanese families with Gaucher disease. In the Pvu II polymorphism, the common allele reported by Sorge et al was the rare allele for healthy Japanese individuals. The frequency of the common allele (+) in Japanese was 0.75 and the rare allele (-) was 0.25. The RFLP study was informative for carrier detection in the family in which both parents were heterozygous for the Pvu II polymorphism. Even in the family in which only one parent was heterozygous, carrier detection was feasible by combining the clinical history and the RFLP, although prenatal diagnosis was not feasible. The Kpn I polymorphism was not detected. It is necessary for precise carrier detection and prenatal diagnosis to investigate other restriction endonucleases which reveal the RFLP.
DNA↗
Heterogeneity of defects in mitochondrial acetoacetyl-CoA thiolase biosynthesis in fibroblasts from four patients with 3-ketothiolase deficiency.
Deficient mitochondrial acetoacetyl-CoA thiolase in fibroblasts from four patients with 3-ketothiolase deficiency was studied using immunochemical methods. We also examined fibroblasts from two heterozygotes, the mother and the brother of the case 1 patient, identified on the basis of the results of the enzyme activity measurements, using 2-methylacetoacetyl-CoA as substrate. The results were as follows: 1) in fibroblasts from all four patients, the thiolase activity using acetoacetyl-CoA was not activated by K+, although that of the controls and the heterozygotes was activated about 2-fold. 2) by immunoblot analyses, mitochondrial acetoacetyl-CoA thiolase was not detectable in fibroblasts from cases 2 and 3, although a very faint band was seen in tissues from cases 1 and 4. However, the band of mitochondrial 3-ketoacyl-CoA thiolase was clearly detected in all patients to the same extent as in the controls. 3) mitochondrial acetoacetyl-CoA thiolase was observed after pulse labeling for 1-h and a 72-h chase period in three cell lines (cases 1, 2, and 4), but was fainter compared to the controls. In another cell line (case 3), a fluorographic band at the same position was detected following a 1-h pulse, but disappeared following a 6-h chase. These results demonstrate heterogeneity in the enzyme defect resulting in a deficiency of mitochondrial acetoacetyl-CoA thiolase in fibroblasts from patients with 3-ketothiolase deficiency.
Acetyl-CoA C-Acetyltransferase↗
Major peroxisomal membrane polypeptides are synthesized in cultured skin fibroblasts from patients with Zellweger syndrome.
Biosynthesis of three major peroxisomal membrane polypeptides (70, 26, and 22 kD) were investigated in an attempt to account for the absence of peroxisomes in patients with Zellweger syndrome. Immunoblot analysis using membrane fractions of autopsied livers revealed that 70- and 22-kD polypeptides were deficient in three Japanese patients with this syndrome. However, pulse-labeling and chase experiments using cultured skin fibroblasts demonstrated that 70-, 26, and 22-kD polypeptides were synthesized in these patients at levels similar to those in the controls. No significant difference in the degradation rates of these polypeptides was noted between the patients and the controls. Subcellular fractionation after pulse-chase experiments revealed 70-kD polypeptides present in a membrane fraction of the cells. These observations indicate that at least 70-, 26-, and 22-kD peroxisomal membrane polypeptides are normally synthesized and considered to be transported into the disorganized peroxisomal membrane in patients with Zellweger syndrome. Molecular and functional analyses of these polypeptides are underway to clarify whether they contribute to membrane integrity or to the transport of peroxisomal matrix enzymes.
Cells, Cultured↗
[Inherited storage disease associated mononuclear phagocytic system].
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Gangliosidoses↗
B cell suppressing and CD8+ T cell enhancing effects of photosensitive dye platonin in humans.
Platonin is one of the photosensitive dyes of trithiazole pentamethine cyanine. It is used as an effective medicine for rheumatoid arthritis. In our study, platonin suppressed the immunoglobulin (Ig) production of human peripheral blood lymphocytes (PBL) stimulated by Staphylococcus aureus Cowan I (SAC) or pokeweed mitogen (PWM). It also suppressed the PWM induced Ig production of B cells when T cells and/or B cells were pretreated with platonin, respectively. The percentage of CD8+ T cells was increased by platonin. Our results suggest that platonin suppresses Ig production through suppressing B cells and enhancing CD8+ (suppressor/cytotoxic) T cells.
Antigens, Differentiation, T-Lymphocyte↗
Immunohistochemical demonstration of complement components in formalin-fixed and paraffin-embedded renal tissues.
Although there are many articles describing the detection of complement components in formalin-fixed and paraffin-embedded renal tissues, it is still difficult to obtain reproducible results. The authors determined the optimal conditions for detecting complement components in routine paraffin sections by the avidin-biotin-peroxidase complex method and concluded that proteolytic digestion of the deparaffinized sections was crucial to detect complement and to preserve tissue structures. The optimal proteolytic digestion was a 15-minute incubation at 37 degrees C in trypsin solution, 0.5 mg/ml, pH 7.6. Under these conditions, all of the complement components so far studied (C1q, C1s, C4, C3c, C3b, C5, C6, C9, and properdin) were detected without significant destruction of tissue structures in 52 renal biopsy specimens from patients with various forms of glomerulonephritis. Immunohistochemistry using the avidin-biotin-peroxidase complex method was equivalent to indirect immunofluorescence and superior to direct immunofluorescence in utility and was useful in the diagnosis of glomerular diseases.
Complement System Proteins↗
Changes of serum apo A-IV levels in a newborn with chylothorax treated with medium-chain triglyceride milk.
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Apolipoprotein B-100↗
Diagnosis of Zellweger syndrome by rectal biopsy: immunoblot of peroxisomal beta-oxidation enzyme and activity of dihydroxyacetone phosphate acyltransferase in rectal mucosa.
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Acetyl-CoA C-Acyltransferase↗
Molecular analysis of peroxisomal beta-oxidation enzymes in infants with Zellweger syndrome and Zellweger-like syndrome: further heterogeneity of the peroxisomal disorder.
The biosynthesis of enzymes of peroxisomal beta-oxidation was investigated in an attempt to elucidate the mechanism of deficiencies of proteins of these enzymes in 3 infants with Zellweger syndrome and in a baby with Zellweger-like syndrome with clinical and biochemical findings consistent with Zellweger syndrome except that the peroxisomes were detected electronmicroscopically. Enzyme proteins of peroxisomal beta-oxidation, acyl-CoA oxidase, bifunctional protein and 3-ketoacyl-CoA thiolase were hardly detectable, in both syndromes. Total hepatic RNA extracted from the liver of one patient with each syndrome and three controls was translated in a rabbit reticulocyte lysate protein-synthesizing system in the presence of [35S]methionine. Translatable mRNAs for all of the peroxisomal beta-oxidation enzymes were detected in both patients at much the same levels seen in the controls. Pulse labelling and chase experiments of fibroblasts from the control revealed that the 72 kDa subunit of acyl-CoA oxidase was first synthesized, after which the 52 kDa and 21 kDa subunits were processed from the 72 kDa subunit. In the patient with Zellweger syndrome, little of the 52 kDa and 21 kDa subunits of acyl-CoA oxidase were synthesized. The mature form of peroxisomal 3-ketoacyl-CoA thiolase was also not processed from its precursor form, in this patient. We consider that Zellweger-like syndrome is a new variant form of a peroxisomal disorder in which biogenesis of peroxisomes is intact, while in contrast, the biogenesis of peroxisome is considered to be defective in those with Zellweger syndrome. Multiple defects of enzymes of beta-oxidation in Zellweger-like syndrome are assumed to be caused by a defect of transport or localization of these enzymes. Our molecular analyses indicate that the enzymes of peroxisomal beta-oxidation are synthesized in patients with Zellweger and Zellweger-like syndrome but that these enzymes are not processed normally and are degraded rapidly.
Abnormalities, Multiple↗