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Biomedical subjects

N Kondo

Publications and source records attributed to N Kondo.

At least 145 records · Page 8Linked to original sources
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

Genes for the human mitochondrial trifunctional protein alpha- and beta-subunits are divergently transcribed from a common promoter region.

Human HADHA and HADHB genes encode the subunits of an enzyme complex, the trifunctional protein, involved in mitochondrial beta-oxidation of fatty acids. Both genes are located in the same region of chromosome 2p23. We isolated genomic clones, including 5' flanking regions, for HADHA and HADHB. Sequencing revealed that both of these genes are linked in a head-to-head arrangement on opposite strands and have in common a 350-bp 5' flanking region. The 5' flanking region has bidirectional promoter activity within this region; two cis elements proved critical for the activity. Transcription factor Sp1 functions as an activator for the bidirectional promoter by binding to both elements. Therefore, expression of trifunctional protein subunits are probably coordinately regulated by a common promoter and by Sp1.

Base Sequence↗
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

Modulation of the thermoregulatory sweating response to mild hyperthermia during activation of the muscle metaboreflex in humans.

1. To investigate the effect of the muscle metaboreflex on the thermoregulatory sweating response in humans, eight healthy male subjects performed sustained isometric handgrip exercise in an environmental chamber (35 C and 50 % relative humidity) at 30 or 45 % maximal voluntary contraction (MVC), at the end of which the blood circulation to the forearm was occluded for 120 s. The environmental conditions were such as to produce sweating by increase in skin temperature without a marked change in oesophageal temperature. 2. During circulatory occlusion after handgrip exercise at 30 % MVC for 120 s or at 45 % MVC for 60 s, the sweating rate (SR) on the chest and forearm (hairy regions), and the mean arterial blood pressure were significantly above baseline values (P < 0.05). There were no changes from baseline values in the oesophageal temperature, mean skin temperature, or SR on the palm (hairless regions). 3. During the occlusion after handgrip exercise at 30 % MVC for 60 s and during the occlusion alone, none of the measured parameters differed from baseline values. 4. It is concluded that, under mildly hyperthermic conditions, the thermoregulatory sweating response on the hairy regions is modulated by afferent signals from muscle metaboreceptors.

Adult↗
PubMed1999

Sequence analysis of an 800-kb genomic DNA region on chromosome 8q21 that contains the Nijmegen breakage syndrome gene, NBS1.

An 800-kb region on chromosome 8q21, which complements the phenotype of cells from Nijmegen breakage syndrome patients, is a candidate for the locus of the underlying gene, termed NBS1. The sequence of this 800-kb region of DNA indicated that the size of this segment is 755,832 bp with an additional 36-kb gap. From this region, we identified four genes including NBS1, a gene coding for a 27-kDa vitamin D-dependent calcium-binding protein (27-kDa calbindin), the mitochondrial 2,4-dienoyl-CoA reductase gene, and a novel gene, C8orf1/hT41. All four genes were aligned in a 250-kb centromeric portion of the region, and no gene was found in the remaining telomeric portion containing 500 kb. The genomic organization of the C8orf1/hT41 and NBS1 genes has been analyzed using the computer programs GRAIL 2 and GENSCAN. They predicted and successfully found more than 93% of the exons, even a small 54-bp exon, indicating that one or more exons in any gene can be identified by these programs. GENSCAN was more efficient at locating the four genes than GRAIL 2 and identified 15 of the 16 exons of the NBS1 gene. This 800-kb region contained repetitive sequences, including 179 copies of the Alu sequence (1 copy/4.2 kb), 123 copies of the L1 sequence (1 copy/6.1 kb), 107 copies of the LTR sequence (1 copy/7.1 kb), and 63 copies of the MER sequence (1 copy/12 kb). There was a slight but not significant difference in the repetitive content of the gene-rich region and the remaining noncoding region. Our results indicate that computer-assisted methods are useful and powerful for identifying exons of both known and novel genes.

Abnormalities, Multiple↗
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

A novel nonsense mutation of the PEX7 gene in a patient with rhizomelic chondrodysplasia punctata.

Mutations in the PEX7 gene encoding a peroxisome targeting signal 2 (PTS2) were identified in two patients with rhizomelic chondrodysplasia punctata (RCDP). A 7-year-old girl, the first Japanese individual to be diagnosed biochemically as a case of RCDP, had a novel nonsense mutation, R232ter, in the PEX7 gene, which had been inherited from her consanguineous parents. Another patient, a Chilean boy with RCDP, had compound heterozygous mutations of PEX7, L292ter and A218V, both of which have been documented. R232ter, which deletes all of the last two WP40 repeats in the PEX7 gene, is sufficient to inactivate functions of the PEX7 gene.

Amino Acid Sequence↗
PubMed1999

Prenatal diagnosis of peroxisomal D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein deficiency.

The prenatal diagnosis of peroxisomal D-3-hydroxyacyl-coenzyme A (CoA) dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP) deficiency was performed by peroxisomal beta-oxidation assay, indirect immunofluorescence staining, immunoblot analysis, and gene analysis of cultured amniocytes obtained from a fetus at 16 weeks' gestational age. beta-Oxidation activity, measured by [1-14C] lignoceric acid oxidation, was markedly decreased compared with the controls. Large peroxisomes were readily identified by immunofluorescence staining with anti-human catalase, as was found in the reported patients. Immunoreactive D-BP material was absent on immunoblot analysis and immunofluorescence staining with anti-human D-BP. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the presence of the same 237-bp deletion in the cDNA as that detected in a sibling (the proband). The autopsied fetus showed the characteristic facial appearance and D-BP was deficient on immunoblot and immunohistopathological studies of the fetal tissues. No neuronal migration disorder was identified. This seems to be the first prenatal diagnosis of D-BP deficiency.

17-Hydroxysteroid Dehydrogenases↗
PubMed1999

Differences of genetic effects for the development of allergic diseases in two cities of Japan.

BACKGROUND: Differences in the effects of genetic factors on allergic diseases in two areas of the same race within the same country have not been studied with multiple logistic regression. OBJECTIVE: To determine whether the effects of genetic factors on allergic diseases differ between two areas. METHODS: A questionnaire provided information about family histories of allergic diseases and environmental factors was distributed to children attending kindergarten, elementary, or junior high school in two Japanese cities: Gifu, with a temperate climate, and Itoman, with a subtropical one. The number of subjects analyzed were 1,243 in Gifu and 1,953 in Itoman. Multiple logistic regression analysis was performed with SAS. RESULTS: Multiple logistic regression analysis showed that in both cities children of families with allergic histories have significantly higher risk of contracting allergic disease and atopic dermatitis, even after being controlled for environmental factors. In Gifu, in families where both parents suffered from allergy, there was a higher incidence of allergic diseases than when only one of the parents was suffering from it. On the other hand, in Itoman there were no differences of relative risk between paternal history and maternal history. CONCLUSIONS: We speculated that there are some differences of genetic factors between different areas of the same country, and these differences of genetic effects may influence on the difference of the prevalence of allergic diseases as well as the environmental factors.

Adolescent↗
PubMed1999

Near-infrared monitoring of tissue oxygenation during application of lower body pressure at rest and during dynamical exercise in humans.

During the application of a wide range of graded lower body pressures (LBP) (-50 to 50 mmHg), we examined how (1) the tissue oxygenation in the lower and upper parts of the body changes at rest, and (2) how tissue oxygenation changes in the lower extremities during dynamical leg exercise. We used near-infrared spectroscopy (NIRS) to measure the changes induced by LBP in total Hb content and Hb oxygenation in seven subjects. At rest, total Hb increased and Hb oxygenation decreased in the thigh muscles during -25 and -50 mmHg LBP, while both decreased during +25 and +50 mmHg LBP. However, in the forearm muscles during graded LBP, the pattern of change in total Hb was the reverse of that in the thigh. Measurements from the forehead showed changes only during +50 mmHg LBP. These results demonstrated that the pattern of change in total Hb and Hb oxygenation differed between upper and lower parts with graded LBP at rest. During dynamical leg exercise, total Hb and Hb oxygenation in the thigh muscles decreased during stepwise increases in LBP above -25 mmHg, Hb oxygenation decreasing markedly during +50 mmHg LBP. These results suggest that during dynamical exercise (i) LBP at +25 mmHg or more causes a graded decline in blood volume and/or flow in the thigh muscles, and (ii) especially at +50 mmHg LBP, the O2 content may decrease markedly in active muscles. Our results suggest that NIRS can be used to monitor in a non-invasive and continuous fashion the changes in oxygenation occurring in human skeletal muscles and head during the graded changes in blood flow and/or volume caused by changes in external pressure and secondary reflexes both at rest and during dynamical exercise.

Adolescent↗
PubMed1999

Relationship between skin blood flow and sweating rate in prepubertal boys and young men.

We sought the mechanisms in the maturation-related change of skin blood flow to heat stress. Eight prepubertal boys (7-11 years) and 11 young men (21-25 years) were exposed to a mild passive heating [by placing the lower legs and feet in a 42 degrees C water bath for 60 min while sitting in a neutral air condition (25 degrees C, 45% relative humidity)]. No age-related differences were observed for the increase in rectal temperature [0.61 +/- 0.05 (SEM) vs. 0.62 +/- 0.09 degrees C in the boys and men at the end of the passive heating, respectively], mean skin temperature (34.51 +/- 0.28 vs. 34.81 +/- 0.27 degrees C) or metabolic heat production (68 +/- 4 vs. 60 +/- 3 W m-2) during the passive heating. During the heating age-related differences in skin blood flow by laser Doppler flowmetry (LDF) and local sweating rate (m(sw)) varied by site; the boys had greater LDF on the chest and back, similar LDF on the forearm and thigh, lower m(sw) on the chest and thigh, similar (m)sw on the back, and greater m(sw) on the forearm, compared with the men. The relationship between LDF and m(sw) during the heat exposure was divided into three temporal phases: (a) an increase of LDF without an increase in m(sw), (b) an increase of m(sw)without the secondary increase of LDF, (c) a proportional increase of LDF and m(sw) increase of LDF in phase (a) and the slopes of the regression lines between the LDF and m(sw) in phase (c) were significantly greater on the chest and back for the boys (P < 0.05), compared with the men, but not on the forearm and thigh. These results suggest that the greater LDF observed on the trunk in the boys may be owing to a greater withdrawal of vasoconstrictor tone and a greater active vasodilation. Regional differences may exist in the maturation-related alterations in vasoconstriction and vasodilation.

Adult↗
PubMed1999

Cytolytic mechanisms involved in non-MHC-restricted cytotoxicity in Chediak-Higashi syndrome.

To determine the mechanisms responsible for the impaired lymphocyte-mediated cytotoxicity in Chediak-Higashi syndrome (CHS), we investigated the killing ability of peripheral blood lymphocytes (PBL) from three patients with CHS using several kinds of target cells that were sensitive to perforin, Fas ligand (FasL), and/or tumour necrosis factor-alpha (TNF-alpha). Freshly isolated CHS PBL did not kill K562 target cells, killing of which by normal PBL was perforin-dependent, as demonstrated by complete inhibition by concanamycin A (CMA), an inhibitor of perforin-based cytotoxicity. In contrast, the CHS PBL exhibited substantial cytotoxicity against Jurkat cells, which was only partially inhibited by CMA treatment but not by the addition of neutralizing anti-FasL or anti-TNF-alpha antibodies. IL-2-activated CHS PBL exhibited substantial levels of cytotoxicity against K562 and Jurkat cells, the levels being 74% and 83% of the respective normal control values, respectively. CMA treatment showed that while the cytotoxicity of IL-2-activated CHS PBL against K562 was largely dependent on perforin, that against Jurkat was largely not. IL-2-activated CHS PBL expressed FasL mRNA, and killed Fas transfectants. These findings indicate that CHS PBL have an ability to kill some target cells via a perforin-mediated pathway, especially when they are activated by IL-2. It was also demonstrated that CHS PBL can exert cytotoxicity against certain target cells by utilizing FasL and an undefined effector molecule other than perforin, FasL, or TNF-alpha.

Adult↗
PubMed1999

Expression of the BLM gene in human haematopoietic cells.

Bloom's syndrome (BS) is a rare autosomal recessive disorder characterized by stunted growth, sun-sensitive erythema and immunodeficiency. Chromosomal abnormalities are often observed. Patients with BS are highly predisposed to cancers. The causative gene for BS has been identified as BLM. The former encodes a protein, which is a homologue of the RecQ DNA helicase family, a family which includes helicases such as Esherichia coli RecQ, yeast Sgs1, and human WRN. WRN is encoded by the gene that when mutated causes Werner's syndrome. The function of BLM in DNA replication and repair has not yet been determined, however. To understand the function of BLM in haematopoietic cells and the cause of immunodeficiency in BS, expression of the BLM gene in various human tissues and haematopoietic cell lines was analysed and the involvement of BLM in immunoglobulin rearrangement examined. In contrast to WRN, BLM was expressed strongly in the testis and thymus. B, T, myelomonocytic and megakaryocytic cell lines also expressed BLM. All of the examined sequences at the junction of the variable (V), diversity (D) and joining (J) regions of the immunoglobulin heavy-chain genes were in-frame, and N-region insertions were also present. The frequency of abnormal rearrangements of the T cell receptor was slightly elevated in the peripheral T cells of patients with BS compared with healthy individuals, whereas a higher frequency of abnormal rearrangements was observed in the cells of patients with ataxia-telangiectasia (A-T). In DND39 cell lines, the induction of sterile transcription, which is required for class switching of immunoglobulin heavy-chain constant genes, was correlated with the induction of the BLM gene. Taking into consideration all these results, BLM may not be directly involved in VDJ recombination, but is apparently involved in the maintenance of the stability of DNA.

Adenosine Triphosphatases↗
PubMed1999

Nonsense and temperature-sensitive mutations in PEX13 are the cause of complementation group H of peroxisome biogenesis disorders.

Peroxisome biogenesis disorders, including Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease, are lethal hereditary diseases caused by abnormalities in peroxisomal assembly. To date, 12 genotypes have been identified. We now have evidence that the complete human cDNA encoding Pex13p, an SH3 protein of a docking factor for the peroxisome targeting signal 1 receptor (Pex5p), rescues peroxisomal matrix protein import and its assembly in fibroblasts from PBD patients of complementation group H. In addition, we detected mutations on the human PEX13 cDNA in two patients of group H. A severe phenotype of a ZS patient (H-02) was homozygous for a nonsense mutation, W234ter, which results in the loss of not only the SH3 domain but also the putative transmembrane domain of Pex13p. A more mildly affected NALD patient (H-01), whose fibroblasts showed the temperature-sensitive (TS) phenotype, was homozygous for a missense mutation in the SH3 domain of Pex13p, I326T. This mutant PEX13 cDNA expression in a PEX13-defective CHO mutant showed I326T to be a TS mutation and thus suggested that Pex13p with the I326T mutation in the SH3 domain is stable at 30 degrees C but is somewhat unstable at 37 degrees C.

Amino Acid Sequence↗