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Biomedical subjects

Y Fujiki

Publications and source records attributed to Y Fujiki.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1996

[Awareness of health and welfare planning for elderly and methods to increase awareness: based on social marketing].

To clarify the socio-psychological factors affecting awareness of health and welfare planning for the elderly, a survey of community residents was performed in the Tokyo metropolitan area. The results obtained were as follows: 1. There were three factors which affected awareness concern about health and welfare services for the elderly as a social issue: concern about the local community; and concern about the local government. 2. Two other factors which did not affect the level of awareness were: anxiety about the health and welfare services for the elderly as a personal issue; and having personal experience of nursing care. From these results, a method to increase awareness was studied based on social marketing methods. Two primary target groups for increasing awareness were identified. One target was people who have the socio-psychological factors described above. Another target was people who have the potential need for health and welfare services but who were not aware of it. That is, they have relatively high anxiety about health and welfare services for the elderly as a personal issue and have personally experienced nursing care. The method of approach for these targets were studied. For the first group, the amount of the information available seemed to be important because these persons are ready to recognize the need for planning. Therefore, efficient information channels should be selected. For the second group, approaches that generate greater consumer participation by presenting this as an efficient method for solution of the problem should be adopted.

Adult↗
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

Multiple genes, including a member of the AAA family, are essential for degradation of unassembled subunit 2 of cytochrome c oxidase in yeast mitochondria.

Cytochrome c oxidase consists of three mitochondrion- and several nucleus-encoded subunits. We previously found that in a mutant of Saccharomyces cerevisiae lacking nucleus-encoded subunit 4 of this enzyme (CoxIV), subunits 2 and 3 (CoxII and CoxIII), both encoded by the mitochondrial DNA, were unstable and rapidly degraded in mitochondria, presumably because the subunits cannot assemble normally. To analyze the molecular machinery involved in this proteolytic pathway, we obtained four mutants defective in the degradation of unassembled CoxII (osd mutants) by screening CoxIV-deficient cells for the accumulation of CoxII. All of the mutants were recessive and were classified into three different complementation groups. Tetrad analyses revealed that the phenotype of each mutant was caused by a single nuclear mutation. These results suggest strongly that at least three nuclear genes (the OSD genes) are required for this degradation system. Interestingly, degradation of CoxIII was not affected in the mutants, implying that the two subunits are degraded by distinct pathways. We also cloned the OSD1 gene by complementation of the temperature sensitivity of osd1-1 mutants with a COXIV+ genetic background on a nonfermentable glycerol medium. We found it to encode a member of a family (the AAA family) of putative ATPases, which proved to be identical to recently described YME1 and YTA11. Immunological analyses revealed that Osd1 protein is localized to the mitochondrial inner membrane. Disruption of the predicted ATP-binding cassette by site-directed mutagenesis eliminated biological activities, thereby underscoring the importance of ATP for function.

ATP-Dependent Proteases↗
PubMed1994

Molecular cloning of the aminopeptidase Y gene of Saccharomyces cerevisiae. Sequence analysis and gene disruption of a new aminopeptidase.

A yeast genomic DNA encoding a new vacuolar aminopeptidase, aminopeptidase Y, was isolated by using a cDNA fragment obtained by screening the lambda gt11 yeast cDNA library with anti-aminopeptidase Y antibody. The DNA sequence encodes 537 amino acids. The "mature" protein, whose NH2-terminal sequence was determined previously by analysis of the purified enzyme, consists of 481 amino acids, and the calculated molecular weight (52,900) coincides with the value obtained by SDS-polyacrylamide gel electrophoresis of the enzyme after removal of sugar chains, 53 kDa. The 56-residue preprosequence was divided into two parts by putative processing sites for signal peptidase and conversion to the mature form; the 21-residue presequence has a hydrophobic stretch which may function as the signal sequence for transit through the endoplasmic reticulum, and the 35-residue prosequence (4013 Da) accounts for the 4-kDa difference between proaminopeptidase Y in the vacuolar proteases-deleted ABYS1 mutant and wild-type mature enzyme. The aminopeptidase Y gene was localized on chromosome II by genetic mapping. A deletion mutant was constructed by disrupting the aminopeptidase Y gene. Vacuolar aminopeptidase activities toward Ala-4-methylcoumaryl-7-amide (MCA) and Lys-MCA were 13 and 20% of wild-type, and those in the presence of Co2+ were 2.2 and 2.8%, respectively. Mutant cells showed no ability to hydrolyze Lys-Ala-MCA to Lys and Ala-MCA, although vacuolar carboxypeptidase Y activity was similar to that in wild-type cells.

Amino Acid Sequence↗
PubMed1994

Characterization of the signal peptide at the amino terminus of the rat peroxisomal 3-ketoacyl-CoA thiolase precursor.

The amino-terminal presequences of rat peroxisomal 3-ketoacyl-CoA thiolase precursors (types A and B) were reported to be cleavable signal peptides for peroxisomal protein translocation. In the present study, this was proven by immunoelectron microscopy of the cultured Chinese hamster ovary cells stably expressing fusion proteins of the amino-terminal sequences of the thiolase precursor and Escherichia coli dihydrofolate reductase. The fusion proteins were processed into mature forms of the apparently correct sizes. Site-directed mutagenesis studies of the charged residues in the B-type presequence (26 amino acid residues) revealed that arginine at position -24 and histidine at position -17 were both indispensable. Even replacement of these residues with other basic amino acids abolished the import activity. Both Arg-24 and His-17 were also required in a longer presequence (36 amino acid residues) of the thiolase A, thereby suggesting that the signal can function in an internal position. When glutamic acid at position -11 was changed to amino acids other than aspartic acid, the signal peptide became apparently effective in both peroxisomal and mitochondrial targeting. All of these data indicate that the thiolase signal peptide is a newly defined type of peroxisomal targeting signal recognized by a mechanism presumably different from that for a known peroxisomal signal, the carboxy-terminal Ser-Lys-Leu-COOH motif.

Acetyl-CoA C-Acyltransferase↗
PubMed1994

Post-translational import of 3-ketoacyl-CoA thiolase into rat liver peroxisomes in vitro.

Cell-free translation products of hepatic free polysomal RNA from a clofibrate-treated rat were incubated at 26 degrees C for 0-60 min with a post-heavy mitochondrial supernatant fraction from normal rat liver. Exogenously added proteinase K-resistant precursor and mature forms of peroxisomal 3-ketoacyl-CoA thiolase were recovered in a particulate fraction and increased with time. Both forms of thiolase cosedimented with peroxisomes, when the proteinase K-treated import reaction mixture was centrifuged in a sucrose density gradient. The in vitro import and processing of thiolase precursors, types A and B, was likewise reproduced with highly purified peroxisomes. These results strongly suggest that the precursor form of 3-ketoacyl-CoA thiolase is translocated into peroxisomes, apparently without tight coupling with proteolytic processing to the mature protein.

Acetyl-CoA C-Acyltransferase↗
PubMed1994

Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis.

A cDNA encoding 35-kDa peroxisome assembly factor 1 (PAF-1), a peroxisomal integral membrane protein, was cloned from Chinese hamster ovary (CHO) cells and sequenced. The CHO PAF-1 comprised 304 amino acids, one residue shorter than rat or human PAF-1, and showed high homology to rat and human PAF-1: 90 and 86% at the nucleotide sequence level and 92 and 90% in amino acid sequence, respectively. PAF-1 from these three species contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family. PAF-1 cDNA from a peroxisome-deficient CHO cell mutant, Z65 (T. Tsukamoto, S. Yokota, and Y. Fujiki, J. Cell Biol. 110:651-660, 1990), contained a nonsense mutation at the codon for Trp-114, resulting in premature termination. Truncation in PAF-1 of either 19 amino acids from the N terminus or 92 residues from the C terminus maintained the peroxisome assembly-restoring activity when tested in both the Z65 mutant and the fibroblasts from a Zellweger patient. In contrast, deletion of 27 or 102 residues from the N or C terminus eliminated the activity. PAF-1 is encoded by free polysomal RNA, consistent with a general rule for biogenesis of peroxisomal proteins, including membrane polypeptides, implying the posttranslational transport and integration of PAF-1 into peroxisomal membrane.

Amino Acid Sequence↗
PubMed1994

A glucan having reticuloendothelial system-potentiating and anti-complementary activities from the tuber of Pinellia ternata.

A glucan, called pinellian G, was isolated from the tuber of Pinellia ternata BREIT. It was homogenous on electrophoresis and gel chromatography, and its molecular mass was estimated to be 1.5 x 10(4). It is composed solely of D-glucose, in addition to a few O-acetyl groups. Methylation analysis, nuclear magnetic resonance and enzymic degradation studies indicated that it is a branched glucan mainly composed of alpha-1,4-linked D-glucopyranose residues with partially alpha-1,3-linked units and 4,6-branching points. The glucan showed significant reticuloendothelial system-potentiating activity in a carbon clearance test, as well as pronounced anti-complementary activity.

Acetylation↗
PubMed1994

[Human peroxisome-deficient disorders and pathogenic gene].

Peroxisome is a model organelle to investigate the mechanism of protein translocation and organelle assembly. Human autosomal recessive peroxisomal disorders are of clinical consequence and a model system to study the biogenesis and physiological significance of peroxisomes. In patients with generalized peroxisomal disease such as cerebrohepatorenal Zellweger syndrome where peroxisomes are morphologically absent, all peroxisomal proteins appear to be normally synthesized but assembly of peroxisomes is impaired. Thus far, nine complementation groups have been reported for these peroxisome-deficient disorders including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. To investigate the molecular mechanism of peroxisome biogenesis and the primary defect of peroxisomal disorders, we have thus far isolated three different complementation groups of Chinese hamster ovary (CHO) cell mutants defective in biogenesis of peroxisomes. By genetic functional complementation analysis following the transfection of cDNA library to one of these cell mutants, Z65, we identified 35-kDa peroxisome assembly factor-1 (PAF-1) essential for peroxisome assembly. Moreover, we delineated the primary defect in a Zellweger patient who belonged to the same complementation group as Z65. The cause of this syndrome was a homozygous nonsense point mutation at 119Arg in PAF-1 gene. Comparison of PAF-1 sequences from rat, human, and Chinese hamster revealed that PAF-1 is highly conserved through the evolution and contains a novel cysteine-rich zinc finger, RING finger motif.

Adrenoleukodystrophy↗
PubMed1993

Microsomal aldehyde dehydrogenase or its cross-reacting protein exists in outer mitochondrial membranes and peroxisomal membranes in rat liver.

We investigated the subcellular distribution of microsomal aldehyde dehydrogenase (msALDH) in rat liver and revealed by the immunoblotting method that msALDH or a cross-reacting 54-kDa protein(s) exists in the outer mitochondrial membranes and peroxisomes. Anti-msALDH antibody markedly inhibited the decanal aldehyde dehydrogenase activity of the outer mitochondrial membranes as well as that of the microsomes. Immunogold electron microscopic observations showed that gold particles are localized over the ER, outer mitochondrial membranes and peroxisomal membranes. These results suggest that msALDH or its cross-reacting related protein is distributed not only in the ER membranes but also in the mitochondrial outer membranes and peroxisomal membranes.

Aldehyde Dehydrogenase↗