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Y Fujiki

Publications and source records attributed to Y Fujiki.

At least 37 records · Page 2Linked to original sources
PubMed2000

Isolation and characterization of cDNA clones for the e1beta and E2 subunits of the branched-chain alpha-ketoacid dehydrogenase complex in Arabidopsis.

Branched-chain alpha-ketoacid dehydrogenase (BCKDH) has been known in mammals to be a key enzyme of the catabolic pathway of branched-chain amino acids. We have isolated two cDNA clones encoding the E1beta and E2 subunits of BCKDH, respectively, from Arabidopsis thaliana. Proteins encoded in these cDNA sequences had putative mitochondrial targeting sequences and conserved domains reported for their mammalian counterparts. Northern blot and immunoblot analyses showed that transcripts from the respective genes and E2 protein markedly accumulated in leaves kept in the dark. We found that the activity of BCKDH in the leaf extracts also increased when plants were placed in the dark. Addition of sucrose to detached leaves inhibited the accumulation of transcripts, whereas application of a photosynthesis inhibitor strongly induced the expression of these genes even under light illumination. These observations indicate that the cellular sugar level is likely responsible for the dark-induced expression of these genes. The transcript levels of these genes were also high in senescing leaves, in which photosynthetic activity is low and free amino acids from degraded protein are likely to serve as an alternative energy source.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

Molecular mechanism of detectable catalase-containing particles, peroxisomes, in fibroblasts from a PEX2-defective patient.

Patients with peroxisome biogenesis disorders (PBD) can be identified by detection of peroxisomes in their fibroblasts, by means of immunocytochemical staining using an anti-catalase antibody. We report here data on three PBD patients with newly identified mutations (del550C and del642G) in the PEX2 gene which encodes a 35-kDa peroxisomal membrane protein containing two membrane-spanning and a C-terminal cysteine-rich region. Some of the fibroblasts from the patient with the del642G mutation contained numerous catalase-containing particles, whereas no fibroblasts containing such particles were found in the patient with the del550C mutation. We confirmed that the del642G mutation caused a partial defect in peroxisome synthesis and import by expression of the mutated PEX2 into PEX2-defective CHO mutant cells. We propose that the two putative membrane-spanning segments in Pex2p are important domains for peroxisome assembly and import and that a defect in one of these domains severely affects PBD patients. Furthermore, a defect in the C-terminal portion of Pex2p exposed to the cytosol containing a RING finger motif caused the mild phenotype, residual enzyme activities, and mosaic detectable peroxisomes in fibroblasts from the patient.

Amino Acid Sequence↗
PubMed2000

Dominant expansion of human T cells in non-obese diabetic/severe combined immunodeficiency mice implanted with human bone fragments.

OBJECTIVE: To establish an in vivo animal model in which human T cells develop and function normally, a step toward developing new vaccines or chemical compounds that modulate immune functions and toward understanding T-cell immunity in humans. MATERIALS AND METHODS: Human bone fragments were implanted into non-obese diabetes/severe combined immunodeficiency (NOD/SCID) mice. The presence of human blood cells in the peripheral blood of these mice was monitored periodically by immunostaining and fluorescence-activated cell sorting. RESULTS: After implantation of bone fragments, dominant expansion of human T lymphocytes, rather than myeloid and B cells, was observed over a 3-month period. In some cases, the proportion of human T cells rose to 40% of the peripheral blood mononuclear cells. These T cells showed CD4/CD8 ratios similar to those observed in human peripheral blood lymphocytes and had a broad repertoire of rearranged T-cell receptor genes. Graft-versus-host reaction was not noted in any organ analyzed. To assess the suitability of NOD/SCID mice implanted with human bone fragments (hu-bone-NOD/SCID mice) as an in vivo model for HIV infection, the mice were infected with a T-lymphotropic strain of HIV-1 (NL4-3) at 7 weeks posttransplant. Serum p24 gag was detected at 2 weeks after inoculation, after which total CD4-positive cell numbers declined, as seen clinically in patients infected with HIV. CONCLUSION: Although the precise mechanism is yet to be determined by which predominant expansion of human T cells occurs in hu-bone-NOD/SCID mice, such mice appear likely to serve as a useful and versatile model for studies involving human T-cell immunity.

Animals↗
PubMed2000

Restoration of biochemical function of the peroxisome in the temperature-sensitive mild forms of peroxisome biogenesis disorder in humans.

We have found that peroxisome assembly is temperature-sensitive (ts) in mild forms of peroxisome biogenesis disorders (PBDs), that is all infantile Refsum disease (IRD) patients and a few neonatal adrenoleukodystrophy patients of several complementation groups. The number of peroxisomes increased daily in incubation at 30 degrees C in the ts cells. Oxidation of very long-chain fatty acids, processing of acyl-CoA oxidase and dihydroxyacetonephosphate acyltransferase activity also improved after 8 days incubation at 30 degrees C in the IRD fibroblasts. These biochemical functions of the peroxisome did not change at 30 degrees C in Zellweger fibroblasts. Number of peroxisomes gradually decreased after 4 days when the temperature shifted from 30 to 37 degrees C in the ts cells. These results indicate that the biochemical functions of peroxisome are also restored by incubation at 30 degrees C in the mild and ts phenotype of PBDs, and the results will aid to predict the severity and the prognosis of affected children.

Animals↗
PubMed2000

The peroxin pex3p initiates membrane assembly in peroxisome biogenesis.

Rat cDNA encoding a 372-amino-acid peroxin was isolated, primarily by functional complementation screening, using a peroxisome-deficient Chinese hamster ovary cell mutant, ZPG208, of complementation group 17. The deduced primary sequence showed approximately 25% amino acid identity with the yeast Pex3p, thereby we termed this cDNA rat PEX3 (RnPEX3). Human and Chinese hamster Pex3p showed 96 and 94% identity to rat Pex3p and had 373 amino acids. Pex3p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. A homozygous, inactivating missense mutation, G to A at position413, in a codon (GGA) for Gly(138) and resulting in a codon (GAA) for Glu was the genetic cause of peroxisome deficiency of complementation group 17 ZPG208. The peroxisome-restoring activity apparently required the full length of Pex3p, whereas its N-terminal part from residues 1 to 40 was sufficient to target a fusion protein to peroxisomes. We also demonstrated that Pex3p binds the farnesylated peroxisomal membrane protein Pex19p. Moreover, upon expression of PEX3 in ZPG208, peroxisomal membrane vesicles were assembled before the import of soluble proteins such as PTS2-tagged green fluorescent protein. Thus, Pex3p assembles membrane vesicles before the matrix proteins are translocated.

ATP-Binding Cassette Transporters↗
PubMed2000

Multiple signaling pathways in gene expression during sugar starvation. Pharmacological analysis of din gene expression in suspension-cultured cells of Arabidopsis.

We have identified many dark-inducible (din) genes that are expressed in Arabidopsis leaves kept in the dark. In the present study we addressed the question of how plant cells sense the depletion of sugars, and how sugar starvation triggers din gene expression in suspension-cultured cells of Arabidopsis. Depletion of sucrose in the medium triggered marked accumulation of din transcripts. Suppression of din gene expression by 2-deoxy-Glc, and a non-suppressive effect exerted by 3-O-methyl-Glc, suggested that sugar-repressible expression of din genes is mediated through the phosphorylation of hexose by hexokinase, as exemplified in the repression of photosynthetic genes by sugars. We have further shown that the signaling triggered by sugar starvation involves protein phosphorylation and dephosphorylation events, and have provided the first evidence that multiple pathways of protein dephosphorylation exist in sugar starvation-induced gene expression. An inhibitor of serine/threonine protein kinase, K-252a, inhibited din gene expression in sugar-depleted cells. Okadaic acid, which may preferentially inhibit type 2A protein phosphatases over type 1, enhanced the transcript levels of all din genes, except din6 and din10, under sugar starvation. Conversely, a more potent inhibitor of type 1 and 2A protein phosphatases, calyculin A, increased transcripts from din2 and din9, but decreased those from other din genes, in sugar-depleted cells. On the other hand, calyculin A, but not okadaic acid, completely inhibited the gene expression of chlorophyll a/b-binding protein under sugar starvation. These results indicate that multiple signaling pathways, mediated by different types of protein phosphatases, regulate gene expression during sugar starvation.

Arabidopsis↗
PubMed2000

Temperature-sensitive mutation of PEX6 in peroxisome biogenesis disorders in complementation group C (CG-C): comparative study of PEX6 and PEX1.

Peroxisome biogenesis disorders (PBD), including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease, are a group of genetically heterogeneous autosomal-recessive diseases caused by mutations in PEX genes that encode peroxins, proteins required for peroxisome biogenesis. Zellweger syndrome patients present the most severe phenotype, whereas neonatal adrenoleukodystrophy patients are intermediate and infantile Refsum disease patients have the mildest features. PEX6 is a causative gene for PBD of complementation group C (CG-C) and encodes the peroxin Pex6p, one of the ATPases associated with diverse cellular activities and a member of the same family of proteins as Pex1p, a causative protein for PBD of CG-E (CG1). Here, we identified the temperature sensitivity of peroxisomes in the fibroblasts of a patient with neonatal adrenoleukodystrophy in CG-C. Peroxisomes were morphologically and biochemically formed at 30 degrees C but not at 37 degrees C. This patient was homozygous for a missense mutation, T-->C at nucleotide 170 resulting in a change from leucine to proline at amino acid 57 (L57P) in Pex6p. CG-C cell mutants (ZP92) in the Chinese hamster ovary transfected with L57P in HsPEX6 revealed the same temperature-sensitive phenotype. However, PEX1-deficient Chinese hamster ovary cell mutants (ZP101) transfected with L111P in PEX1, the counterpart to L57P in PEX6, showed no temperature sensitivity. In addition, ZP92 transfected with G708D in PEX6, the counterpart to the temperature-sensitive mutation G843D in PEX1, revealed no temperature-sensitive phenotype. These results indicate that L57P in Pex6p is a temperature-sensitive mutation causing the milder phenotype in a patient with PBD in CG-C. They also indicate that the amino acid residues responsible for temperature sensitivity do not seem to be conserved between Pex6p and Pex1p.

ATPases Associated with Diverse Cellular Activitie↗
PubMed2000

Peroxisome biogenesis and molecular defects in peroxisome assembly disorders.

Peroxisome assembly in mammals requires more than 14 genes. So far, we have isolated seven complementation groups (CGs) of peroxisome biogenesis-defective Chinese hamster ovary (CHO) cell mutants, Z65, Z24/ZP107, ZP92, ZP105/ZP139, ZP109, ZP110, ZP114. Two peroxin cDNAs, PEX2 and PEX6, were first cloned by genetic phenotype-complementation assay using Z65 and ZP92, respectively, and were shown to be responsible for peroxisome biogenesis disorders (PBD) such as Zellweger syndrome, of CG-F (the same as CG-X in U.S.A.) and CG-C (the same as CG-IV), respectively. Pex2p is a RING zinc finger membrane protein of peroxisomes and Pex6p is a member of the AAA ATPase family. We likewise isolated PEX12 encoding a peroxisomal integral membrane protein in the RING family, by functional complementation of ZP109, demonstrating PEX12 to be responsible for CG-III PBD. We also cloned PEX1 by screening of human liver cDNA library, using ZP107. PEX1 mutation was delineated to be the genetic cause of PBD in the most highest incidence group, CG-E (the same as CG-I). Moreover, we recently found that Pex5p is involved in transport of not only PTS1- but also PTS2-protein, distinct from yeast Pex5p, using PEX5-defective ZP105 and ZP139. Thus, CHO cell mutants defective in peroxisome biogenesis are indeed shown to be very useful for the studies of peroxisome assembly and delineating pathogenic genes in PBD. Furthermore, we have isolated novel CGs of CHO mutants, ZP119 and ZP126.

Gene Expression Regulation↗
PubMed1999

Estimating amounts of toluene inhaled by workers with protective mask using biological indicators of toluene.

Personal air samplers were attached to workers wearing protective masks to determine the levels of toluene vapor in the breathing zone. Concentrations of toluene in exhaled air, blood and urine; and hippuric acid and o-cresol concentrations in the urine of the workers were determined. Subsequently, toluene concentrations in the air inhaled by workers with and without gas masks were estimated by single and multiple regression equations. Analysis of single regression equations revealed that, compared with toluene concentrations in air, masks decreased the concentrations of the four biological exposure indicators: toluene in exhaled air, urinary toluene, urinary hippuric acid and urinary o-cresol by about 29% in average. Analysis by multiple regression equations showed a decrease of 38% in four biological indicators. Since average exposure to toluene in the shop was relatively low, the workers wore the masks only during high concentrations of toluene; they were, however, exposed to direct inhalation when the masks were removed in lower concentrations.

Adult↗
PubMed1999

Functional heterogeneity of C-terminal peroxisome targeting signal 1 in PEX5-defective patients.

To investigate mechanisms related to functions of the peroxisome targeting signal (PTS) 1 receptor, Pex5p, we analyzed peroxisome matrix protein import in fibroblasts from three patients with peroxisome biogenesis disorders, all with different mutations in the PEX5 gene. The patients 2-01 (Zellweger syndrome) and 2-05 (neonatal adrenoleukodystrophy) have the reported mutations, R390X and N489K, and patient 2-03 (infantile Refsum disease) has a newly identified mutation, S563W. Fibroblasts from 2-03 (S563W) were detected in both PTS1 and PTS2 imports despite the PEX5 defect, findings in contrast with fibroblasts from 2-05 (N489K) severely defective in PTS1 import and those from 2-01 (R390X) severely defective in both PTS1 and PTS2. The PTS1 receptor in 2-03 is functional for only the C-terminal -SKL sequence (acyl-CoA oxidase) and had little or no function for C-terminal -AKL (D-bifunctional protein and sterol carrier protein 2) and -KANL (catalase) sequences, respectively. After transfection of these mutated PEX5 cDNA into the PEX5-defective CHO mutant, transformants of ZP102 revealed that each mutation was responsible for each dysfunction of the PTS1 import. It seems apparent that -AKL and -KANL are poorer variants of PTS1 and are likely to be more susceptible to effects of mutation of its receptor, Pex5p.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Newly identified Chinese hamster ovary cell mutants defective in peroxisome assembly represent complementation group A of human peroxisome biogenesis disorders and one novel group in mammals.

We isolated peroxisome biogenesis-defective mutants from rat PEX2-transformed Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet method. A total of 18 mutant cell clones showing cytosolic localization of catalase were isolated. By complementation group (CG) analysis by means of PEX cDNA transfection and cell fusion, cell mutants, ZP124 and ZP126, were found to belong to two novel CGs of CHO mutants. Mutants, ZP135 and ZP167, were also classified to the same CG as ZP124. Further cell fusion analysis using 12 CGs fibroblasts from patients with peroxisome deficiency disorders such as Zellweger syndrome revealed that ZP124 belonged to human CG-A, the same group as CG-VIII in the United States. ZP126 could not be classified to any of human and CHO CGs. These mutants also showed typical peroxisome assembly-defective phenotypes such as severe loss of catalase latency and impaired biogenesis of peroxisomal enzymes. Collectively, ZP124 represents CG-A, and ZP126 is in a newly identified CG distinct from the 14 mammalian CGs previously characterized.

Animals↗
PubMed1999

Isolation and characterization of novel peroxisome biogenesis-defective Chinese hamster ovary cell mutants using green fluorescent protein.

We developed an improved method for isolation of peroxisome biogenesis-defective somatic animal cell mutants, using a combination of green fluorescent protein (GFP) expression and the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/UV) selection method. We used TKaG1 and TKaG2 cells, the wild-type Chinese hamster ovary (CHO) cells, CHO-K1, that had been stably transfected with cDNAs each encoding rat Pex2p as well as GFP tagged at the C-terminus with peroxisome targeting signal type 1 (PTS1) or N-terminally PTS2-tagged GFP. P9OH/UV-resistant cell colonies were examined for intracellular location of GFP on unfixed cells, by fluorescence microscopy. Seven each of the mutant cell clones isolated from TKaG1 and TKaG2 showed cytosolic GFP-PTS1 and PTS2-GFP, respectively, indicating the defect in peroxisome assembly. By transfection of PEX2, PEX5, PEX6, and PEX12 cDNAs and cell fusion analysis between the CHO cell mutants, five different complementation groups (CGs) were identified. Two mutant clones, ZPG207 and ZPG208, belonged to novel CGs. Further CG analysis using fibroblasts from patients with peroxisome biogenesis disorders, including rhizomelic chondrodysplasia punctata (RCDP), revealed that ZPG208 belonged to none of human CGs. ZPG207 was classified into the same CG as RCDP. Taken together, ZPG208 is in a newly identified, the 12th, CG in peroxisome-deficient CHO mutants reported to date and represents a novel mammalian CG.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

The peroxin Pex14p. cDNA cloning by functional complementation on a Chinese hamster ovary cell mutant, characterization, and functional analysis.

Rat cDNA encoding a 376-amino acid peroxin was isolated by functional complementation of a peroxisome-deficient Chinese hamster ovary cell mutant, ZP110, of complementation group 14 (CG14). The primary sequence showed 28 and 24% amino acid identity with the yeast Pex14p from Hansenula polymorpha and Saccharomyces cerevisiae, respectively; therefore, we termed this cDNA rat PEX14 (RnPEX14). Human and Chinese hamster Pex14p showed 96 and 94% identity to rat Pex14p, except that both Pex14p comprised 377 amino acids. Pex14p was characterized as an integral membrane protein of peroxisomes, exposing its N- and C-terminal parts to the cytosol. Pex14p interacts with both Pex5p and Pex7p, the receptors for peroxisome targeting signal type 1 (PTS1) and PTS2, respectively, together with the receptors' cargoes, PTS1 and PTS2 proteins. Mutation in PEX14 from ZP161, the same CG as ZP110, was determined by reverse transcription-PCR as follows. A 133-base pair deletion at nucleotide residues 37-169 in one allele created a termination codon at 40-42; in addition to this mutation, 103 base pairs were deleted at positions 385-487, resulting in the second termination immediately downstream the second deletion site in the other allele. Neither of these two mutant forms of Pex14p restored peroxisome biogenesis in ZP110 and ZP161, thereby demonstrating PEX14 to be responsible for peroxisome deficiency in CG14.

Amino Acid Sequence↗
PubMed1999

Accumulation of glycolipids in mutant Chinese hamster ovary cells (Z65) with defective peroxisomal assembly and comparison of the metabolic rate of glycosphingolipids between Z65 cells and wild-type CHO-K1 cells.

The influence of peroxisomal dysfunction on glycosphingolipid metabolism was investigated using mutant Chinese hamster ovary (CHO) cells (Z65) with defective assembly of the peroxisomal membranes. In accordance with previous observations, the concentration of very long chain fatty acid (C24:0) was shown to be higher in Z65 cells than in control cells. We then compared the composition of glycolipids in Z65 cells with that in CHO-K1 cells, which are wild-type Chinese hamster ovary cells with intact peroxisomes, and found significantly increased concentrations of ceramide monohexoside (CMH) and ganglioside GM3 in Z65 cells. However, there were no differences in the concentrations of glycerophospholipids, triglycerides, free fatty acids and cholesterol between Z65 and CHO-K1 cells. Further, to investigate the metabolic rate of the major lipids, Z65 and CHO-K1 cells were pulse-labeled with [3-14C]serine. [3-14C]Serine was incorporated into phosphatidylserine, phosphatidylethanolamine and sphingomyelin more quickly in CHO-K1 than in Z65 cells. However, after 48 h, the radioactivity incorporated into those lipids, including CMH, was greater in Z65 cells than in CHO-K1 cells. Thus, the altered metabolism of glycosphingolipids, probably due to peroxisomal dysfunction, was thought to be responsible for the change in glycosphingolipid composition in Z65 cells.

Animals↗
PubMed1999

Human PEX19: cDNA cloning by functional complementation, mutation analysis in a patient with Zellweger syndrome, and potential role in peroxisomal membrane assembly.

At least 11 complementation groups (CGs) have been identified for the peroxisome biogenesis disorders (PBDs) such as Zellweger syndrome, for which seven pathogenic genes have been elucidated. We have isolated a human PEX19 cDNA (HsPEX19) by functional complementation of peroxisome deficiency of a mutant Chinese hamster ovary cell line, ZP119, defective in import of both matrix and membrane proteins. This cDNA encodes a hydrophilic protein (Pex19p) comprising 299 amino acids, with a prenylation motif, CAAX box, at the C terminus. Farnesylated Pex19p is partly, if not all, anchored in the peroxisomal membrane, exposing its N-terminal part to the cytosol. A stable transformant of ZP119 with HsPEX19 was morphologically and biochemically restored for peroxisome biogenesis. HsPEX19 expression also restored peroxisomal protein import in fibroblasts from a patient (PBDJ-01) with Zellweger syndrome of CG-J. This patient (PBDJ-01) possessed a homozygous, inactivating mutation: a 1-base insertion, A764, in a codon for Met255, resulted in a frameshift, inducing a 24-aa sequence entirely distinct from normal Pex19p. These results demonstrate that PEX19 is the causative gene for CG-J PBD and suggest that the C-terminal part, including the CAAX homology box, is required for the biological function of Pex19p. Moreover, Pex19p is apparently involved at the initial stage in peroxisome membrane assembly, before the import of matrix protein.

Amino Acid Sequence↗
PubMed1999

Genomic structure and identification of 11 novel mutations of the PEX6 (peroxisome assembly factor-2) gene in patients with peroxisome biogenesis disorders.

The PEX6 (peroxisome assembly factor-2, PAF-2) gene encodes a member of the AAA protein (ATPases associated with diverse cellular activities) family and restores peroxisome assembly in fibroblasts from peroxisome biogenesis disorder patients belonging to complementation group C (group 4 in the United States). We have now clarified the genomic DNA structure of human PEX6 and identified mutations in patients from various ethnic groups. The human PEX6 gene consists of 17 exons and 16 introns, spanning about 14kb. The largest exon, exon 1, has at least 952 bp nucleotides. Eleven novel mutations (18 alleles) were identified by direct sequencing of the PEX6 cDNA from 10 patients. All these mutations have been confirmed in the corresponding genomic DNA. There was no common mutation, but an exon skip was identified in two unrelated Japanese patients. Most of the mutations led to premature termination or large deletions of the PEX6 protein and resulted in the most severe peroxisome biogenesis disorder phenotype of Zellweger syndrome. A patient with an atypical Zellweger syndrome had a missense mutation that was shown to disrupt the cell's ability to form peroxisomes. This mutation analysis will aid in understanding the functions of the PEX6 protein in peroxisomal biogenesis. Hum Mutat 13:487-496, 1999.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1999

Comparison of hematopoietic activities of human bone marrow and umbilical cord blood CD34 positive and negative cells.

Although the hematopoietic activities of human CD34+ bone marrow (BM) and cord blood (CB) cells have been well characterized, the phenotype of nonobese-diabetic severe combined immunodeficient (NOD/SCID) mice repopulating cells (SRCs) in CB and BM has not yet been fully examined. To address this issue, various hematopoietic activities were compared in terms of total and CD34+ CB and BM cells. Clonal culture of fluorescence-activated cell sorter (FACS) CD34+ CB and BM cells revealed a higher incidence of colony-forming cells with greater proliferation capacity in CB over BM CD34+ cells. CB CD34+ cells also demonstrated higher secondary plating efficiency over BM cells. In addition, we demonstrated that mice transplanted with CB mononuclear cells (MNCs) showed significantly higher levels of chimerism than those transplanted with BM MNCs. However, recipients of FACS-sorted CD34+ CB cells showed significantly lower levels of chimerism than those that received total CB MNCs, suggesting a role of facilitating cells in the CD34- cell population. To further analyze the role of CD34- cells, the NOD/SCID repopulating ability of FACS-sorted CB CD34-c-kit+Lin- and CD34-c-kit-Lin- cells were examined. However, SRCs were not detected in those cells. Taken together, these data suggest that CB is a better source of hematopoietic stem cells and that there are cells in the CD34- fraction that facilitate repopulation of hematopoiesis in the NOD/SCID environment.

Animals↗