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Biomedical subjects

Y Fujiki

Publications and source records attributed to Y Fujiki.

At least 73 records · Page 4Linked to original sources
PubMed1998

Mutation in PEX16 is causal in the peroxisome-deficient Zellweger syndrome of complementation group D.

Peroxisome-biogenesis disorders (PBDs), including Zellweger syndrome (ZS), are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as by a malfunction of peroxisomes, among which>10 genotypes have been identified. We have isolated a human PEX16 cDNA (HsPEX16) by performing an expressed-sequence-tag homology search on a human DNA database, by using yeast PEX16 from Yarrowia lipolytica and then screening the human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex16p) made up of 336 amino acids. Among 13 peroxisome-deficiency complementation groups (CGs), HsPEX16 expression morphologically and biochemically restored peroxisome biogenesis only in fibroblasts from a CG-D patient with ZS in Japan (the same group as CG-IX in the United States). Pex16p was localized to peroxisomes through expression study of epitope-tagged Pex16p. One patient (PBDD-01) possessed a homozygous, inactivating nonsense mutation, C-->T at position 526 in a codon (CGA) for 176Arg, that resulted in a termination codon (TGA). This implies that the C-terminal half is required for the biological function of Pex16p. PBDD-01-derived PEX16 cDNA was defective in peroxisome-restoring activity when expressed in the patient's fibroblasts. These results demonstrate that mutation in PEX16 is the genetic cause of CG-D PBDs.

Amino Acid Sequence↗
PubMed1998

Temperature-sensitive mutation in PEX1 moderates the phenotypes of peroxisome deficiency disorders.

The peroxisome biogenesis disorders (PBDs), including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD), are autosomal recessive diseases caused by deficiency of peroxisome assembly as well as malfunction of peroxisomes, where >10 genotypes have been reported. ZS patients manifest the most severe clinical and biochemical abnormalities, while those with NALD and IRD show the least severity and the mildest features, respectively. PEX1 is the causative gene for PBDs of complementation group I (CG1), the highest incidence PBD, and encodes the peroxin, Pex1p, a member of the AAA ATPase family. In the present work, we found that peroxisomes were morphologically and biochemically formed at 30 but not 37 degrees C, in the fibroblasts from all CG1 IRD patients examined, whereas almost no peroxisomes were seen in ZS and NALD cells, even at 30 degrees C. A point missense mutation, G843D, was identified in the PEX1 allele of most CG1 IRD patients. The mutant PEX1, termed HsPEX1G843D, gave rise to the same temperature-sensitive phenotype on CG1 CHO cell mutants upon transfection. Collectively, these results demonstrate temperature-sensitive peroxisome assembly to be responsible for the mildness of the clinical features of PEX1 -defective IRD of CG1.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1998

Mutations in PEX10 is the cause of Zellweger peroxisome deficiency syndrome of complementation group B.

Peroxisome biogenesis disorders (PBD), such as Zellweger syndrome, are autosomal recessive diseases caused by a deficiency in peroxisome assembly as well as a malfunction of the peroxisomes, where at least 10 genotypes have been reported. We have isolated a human PEX10 cDNA (HsPEX10) by an expressed sequence tag homology search on a human DNA database using yeast PEX10 from Hansenula polymorpha, followed by screening of a human liver cDNA library. This cDNA encodes a peroxisomal protein (a peroxin Pex10p) comprising 326 amino acids, with two putative transmembrane segments and a C3HC4zinc finger RING motif. Both the N- and C-terminal regions of Pex10p are exposed to the cytosol, as assessed by an expression study of epitope-tagged Pex10p. HsPEX10 expression morphologically and biochemically restored peroxisome biogenesis in fibroblasts from Zellweger patients of complementation group B in Japan (complementation group VII in the USA). One patient (PBDB-01) possessed a homozygous, inactivating mutation, a 2 bp deletion immediately upstream of the RING motif, which resulted in a frameshift, altering 65 amino acids from the normal. This implies that the C-terminal part, including the RING finger, is required for biological function of Pex10p. PEX10 cDNA derived from patient PBDB-01 was defective in peroxisome-restoring activity when expressed in patient fibroblasts. These results demonstrate that mutation in PEX10 is the genetic cause of complementation group B PBD.

Amino Acid Sequence↗
PubMed1998

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.

Amino Acid Sequence↗
PubMed1998

PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p.

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of peroxisome-deficient Chinese hamster ovary cell mutants representing human complementation group III.

We made use of the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) method and isolated peroxisome-deficient mutant cells. TKa cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1) were used to avoid frequent isolation of the Z65-type, PEX2-defective mutants. P9OH/UV-resistant cell colonies were examined for the intracellular location of catalase, a peroxisomal matrix enzyme, by immunofluorescence microscopy and using anti-catalase antibody. As six mutant cell clones showed cytosolic catalase, there was likely to be a deficiency in peroxisome assembly. These mutants also showed the typical peroxisome assembly-defective phenotype, including significant decrease of dihydroxyacetonephosphate acyltransferase, the first step key enzyme in plasmalogen synthesis, and loss of resistance to 12-(1'-pyrene)dodecanoic acid/UV treatment. By transfection of Pex2p and Pex6p (formerly PAF-2) cDNAs and cell fusion analysis between the CHO cell mutants, two mutants, ZP104 and ZP109, were found to belong to a novel complementation group. Further complementation analysis using fibroblasts from patients with peroxisome biogenesis disorders revealed that the mutants belonged to human complementation group III. Taken together, ZP104 and ZP109 are in a newly identified fifth complementation group in CHO mutants reported to date and represent the human complementation group III.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1997

Isolation of a new peroxisome-deficient CHO cell mutant defective in peroxisome targeting signal-1 receptor.

For the study of mechanism of peroxisome biogenesis, we attempted to isolate CHO cell mutants deficient in peroxisome biogenesis. We used as the parent strain a stable CHO transformant of rat PEX2 (formerly named peroxisome assembly factor-1) cDNA, to avoid unusually frequent isolation of Pex2 mutants. Among the three peroxisome-deficient mutants obtained, ZP102 was a new CHO mutant of complementation group 2, and was restored for peroxisome assembly by the transfection of human PEX5 (formerly called PXR1 or PTS1R) cDNA. This approach would facilitate the isolation of new complementation gorups of peroxisome-deficient CHO mutants and the identification of essential genes for peroxisome biogenesis.

Animals↗
PubMed1997

Two-dimensional arrangement of a functional protein by cysteine-gold interaction: enzyme activity and characterization of a protein monolayer on a gold substrate.

We have characterized the functional protein, myosin subfragment 1 (S1), attached to a gold substrate by the sulfhydryl groups of cysteine in proteins. The amino groups of the regulatory light chain (RLC) isolated from myosin were labeled with a radioisotope (125I), and the labeled RLC was incorporated into S1 from which the RLC had been removed. The radiation from 125I showed that S1 molecules had attached to the gold and, through the interference effect of the monochromatic radiation from 125I, provided information about the position of labeled RLC sites in the S1 monolayer. The interference fringes showed that the RLC was located close to the gold surface and that all of the adsorbed S1 molecules had the same orientation. We confirmed that the motor function of S1 on the gold surface is maintained by observing sliding movement at low ionic strength and by observing the detachment at high ionic strength of fluorescent actin filaments in the presence of ATP. We also found that the adsorbed S1 molecules were not removed from the Au surface by a reducing agent. Thus the Au-S bond is more stable than the S-S bond.

Actins↗
PubMed1997

Risk factors for sensitisation to methyltetrahydrophthalic anhydride.

OBJECTIVES: To examine an association between specific IgE to methyltetrahydrophthalic anhydride (MTHPA) and exposure time, atopic history, smoking habits, and total IgE concentrations. METHODS: A cross sectional survey was carried out on a population of 148 workers from two condenser plants using epoxy resin with MTHPA, an acid anhydride curing agent known to cause allergy. RESULTS: Using a Pharmacia CAP system with a MTHPA human serum albumin conjugate, specific IgE antibody was detected in serum from 97 (66%) out of the 148 workers exposed to MTHPA. Stepwise multiple linear regression analysis showed a striking relation between log concentrations of specific and total IgE (P < 0.0001). Furthermore, when the workers were divided into two groups according to a cut-off point (100 IU/ml) between low and high total IgE, current smoking was significantly (P = 0.025) associated with specific IgE production only in the group with low total IgE (< 100 IU/ml). CONCLUSIONS: Smoking is the most significant risk factor for raising specific IgE to MTHPA in the group with low total IgE concentrations.

Adult↗
PubMed1997

Specific antibodies against methyltetrahydrophthalic anhydride and risk factors for sensitization in occupationally exposed subjects.

OBJECTIVES: Occupational exposure to methyltetrahydrophthalic anhydride (MTHPA) from an epoxy resin was studied to evaluate the nature of reported ocular and nasal complaints and some risk factors for sensitization. METHODS: Seventy-three current and 22 former workers underwent a questionnaire survey and serologic investigations. Total and MTHPA-specific immunoglobulin (Ig)E levels were measured by means of a Pharmacia CAP system, and MTHPA-specific immunoglobulin (Ig)E levels were measured by enzyme immunoassay. RESULTS: Forty-six (63%) of the currently exposed workers has positive MTHPA-specific IgE, and no significant difference was found between those continuously or intermittently exposed (58% and 71%, respectively). Work-related ocular or nasal symptoms were significantly associated with specific IgE but not with specific IgG4. This finding indicates that there is an IgE-mediated mechanism in most cases of work-related symptoms associated with MTHPA exposure. The total IgE levels were significantly (P < 0.005) higher in the specific IgE-positive workers than in the specific IgE-negative workers (geometric mean 101 IU/ml and 44.8 IU/ml, respectively). Multiple logistic regression analysis also revealed that the group with high total IgE levels (> or = 80 IU/ml) had a significant relative risk (RR 4.7) of producing MTHPA-specific IgE as compared with the group with low total IgE levels (< 80 IU/ml). CONCLUSIONS: These results show that MTHPA has a high sensitizing ability and that a high total IgE level is the most significant risk factor for workers exposed to MTHPA. However, to evaluate conclusively the effect of high total IgE levels on sensitization, further prospective studies are necessary.

Adult↗
PubMed1997

Newly identified Chinese hamster ovary cell mutants defective in peroxisome biogenesis represent two novel complementation groups in mammals.

We isolated peroxisome biogenesis mutants from Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/ UV) method and wild-type CHO-K1 cells that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1). Three mutant cell clones, ZP110, ZP111, and ZP114, showed cytosolic localization of catalase, thereby indicating a defect in peroxisome biogenesis, whereas ZP112 and ZP113 contained fewer but larger catalase-positive particles. Mutant ZP115 displayed an aberrant, tubular structure immunoreactive to anti-catalase antibody. Mutants lacking morphologically recognizable peroxisomes also showed the typical peroxisome assembly-defective phenotype such as severe loss of catalase latency and resistance to 12-(1'-pyrene)dodecanoic acid (P12)/UV treatment. ZP110 and ZP111, and ZP114 were found to belong to two novel complementation groups, respectively, by complementation group analysis with cDNA transfection and cell fusion. Cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome revealed that ZP110 and ZP114 could not be classified to any of human complementation groups. Thus, ZP110/ZP111 and ZP114 are the first, two peroxisome-deficient cell mutants of newly identified complementation groups distinct from the ten mammalian groups previously characterized.

Acetyl-CoA C-Acyltransferase↗
PubMed1996

Echogenic fetal bowel in the third trimester associated with trisomy 18.

We present the first case of trisomy 18 in which echogenic fetal bowel was detected in the third trimester after normal echogenicity was documented during the second trimester. Fetal karyotyping should be considered in cases of increased echogenicity of the fetal lower abdomen in the second as well as the third trimester.

Abnormalities, Multiple↗
PubMed1996

Correction by gene expression of biochemical abnormalities in fibroblasts from Zellweger patients.

Zellweger syndrome is a prototype of peroxisomal biogenesis disorders and a fatal autosomal recessive disease with no effective therapy. We identified nine genetic complementation groups of these disorders, and mutations in peroxisome assembly factor-1 (PAF-1) and the 70-kD peroxisomal membrane protein (PMP70) genes have been detected by our group F and Roscher's group 1, respectively. We now describe permanent recovery from generalized peroxisomal abnormalities in fibroblasts of a Zellweger patient from group F, such as biochemical defects of peroxisomal beta-oxidation, plasmalogen biosynthesis, and morphologic absence of peroxisomes, by stable transfection of human cDNA encoding PAF-1. In the light of these observations, we designed a gene expression system using fibroblasts from patients with peroxisomal biogenesis disorders. In Zellweger fibroblasts obtained from Roscher's group 1 and transfected with human cDNA encoding PMP70, peroxisomes were not morphologically identifiable, and peroxisomal function did not normalize.

ATP-Binding Cassette Transporters↗