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T Fukao

Publications and source records attributed to T Fukao.

At least 55 records · Page 3Linked to original sources
PubMed2000

Characterization of the nuclear localization signal in the DNA helicase responsible for Bloom syndrome.

Bloom syndrome (BS) is a rare genetic disorder characterized by small body size, photosensitivity, immunodeficiency and a high predisposition to various types of cancer. BLM was identified as the causative gene for BS. The BLM protein is homologous to DNA helicase and has two basic amino acid clusters in its C-terminal region. Previously, we reported that the distal arm of these basic amino acids clusters in the BLM protein functioned as the nuclear localization signal (NLS) of the protein. In this study, we generated plasmid constructs for expression of enhanced green fluorescent protein (EGFP) fused with various BLM protein variants having a mutation with deletions or substitutions in the basic amino acid and analyzed the subcellular localization of the expressed proteins. The EGFP-fused protein containing the basic amino acid cluster region proximal to the C-terminus of BLM helicase was localized exclusively in the nucleus. However, the EGFP-BLM proteins that lacked either Arg1344 or Lys1346 distributed in both the cytoplasm and the nucleus equally. Deletion of Arg1347 also resulted in localization in both the nucleus and cytoplasm, and substitution of Arg1344, Lys1346, Arg1347 or Arg1348 with non-basic amino acids reduced the nuclear localization of BLM protein. Mouse BLM protein which also migrate to the nucleus has two basic amino acid clusters in the C-terminus and the basic amino acids (Lys1346-Pro1347-Lys1348-Arg1349-Arg1350) proximal to the C-terminus are conserved between mouse and human. These findings suggest that the Arg1344-Ser1345-Lys1346-Arg1347 sequence at the C-terminus of the human BLM protein is essential for nuclear localization of this protein.

Adenosine Triphosphatases↗
PubMed2000

Combined effect of hop resins and sodium hexametaphosphate against certain strains of Escherichia coli.

The combined antimicrobial effects of hop resins with sodium hexametaphosphate, glycerol monocaprate, and lysozyme were investigated aiming to make an effective agent against Escherichia coli. When they are used separately, the antimicrobial activity against E. coli was minimal. However, the combination of hop resins with sodium hexametaphosphate exhibited strong antimicrobial activity against E. coli, but no effect was found in combinations of hop resins with the other agents. The activity was strongest when the combination was added at the beginning of growth of the bacteria, resulting in a prolonged lag phase. However, when the antimicrobials were added during the log phase, growth was depressed considerably. By addition of these materials, cell components with absorbance near 260 nm were leaked out. This possibly may have resulted from damage to the cell membranes of the bacteria. The combined effect was also detected in model food systems such as mashed potatos. The use of hop resins and sodium hexametaphosphate in combination may thus be useful for controlling E. coli.

Animals↗
PubMed1999

Mutations of the IL-12 receptor beta2 chain gene in atopic subjects.

Interleukin-12(IL-12) promotes cell-mediated Th1 responses and production of IFN-gamma that downregulates IgE production. The signal of IL-12 is transduced through the IL-12 receptor (IL-12R) and Stat4. Twenty-four of 75 atopic individuals with high levels of IgE showed insufficient IFN-gamma production by peripheral blood mononuclear cells (PBMCs) following stimulation with IL-12 but not that with phytohemagglutinin (PHA). Interestingly, 10 of the above 24 subjects were found to be heterozygous for truncated (2496 del 91) or missense (1577 A to G and 2799 A to G) mutations of IL-12R beta2 chain gene (IL-12R beta2). Insufficient phosphorylation of Stat4 was also demonstrated in these 10 individuals. This is the first report showing that reduced IFN-gamma production following IL-12 stimulation is associated with the heterozygous IL-12R beta2 mutations in atopic subjects.

Adolescent↗
PubMed1999

ATM is upregulated during the mitogenic response in peripheral blood mononuclear cells.

Patients with the human genetic disorder ataxia-telangiectasia (A-T) are characterized by immunodeficiency and a predisposition to develop lymphoid malignancies. The gene mutated in A-T patients, ATM, codes for a high molecular weight protein that is implicated in DNA damage recognition and cell cycle control. The ATM protein does not change in amount or cellular distribution throughout the cell cycle or in response to DNA damaging agents. Because peripheral blood mononuclear cells (PBMCs) are largely in a state of quiescence and can be readily stimulated to enter a proliferative phase and because A-T cells exhibit growth abnormalities and senescence, indicative of a general intracellular defect in signalling, we chose PBMCs to examine the relationship of ATM to the proliferative status of the cell. We show here that ATM protein is present at low levels in freshly isolated PBMCs and increases approximately 6-fold to 10-fold in response to a mitogenic stimulus, reaching a maximum after 3 to 4 days. A similar, but delayed response, was evident in the presence of serum only. This increase in ATM protein was accompanied by an increase in ATM kinase activity. While expression of ATM protein increased during proliferation, ATM mRNA expression was unchanged in stimulated and unstimulated cells and there was no evidence for increased ATM protein stability in the phytohemagglutinin (PHA)-treated cells. In keeping with the reduced levels of ATM in quiescent cells, the extent of radiation-induction of the p53 pathway was significantly lower than in mitogen-stimulated cells. Basal levels of p21 were elevated in quiescent cells, and the response to radiation was negligible or reduced compared with proliferating cells over a 2-hour period. Overall, the data suggest that the increase in ATM protein in proliferating cells is due to posttranscriptional regulation and points to a role for ATM in more general signalling.

Ataxia Telangiectasia↗
PubMed1999

Interleukin 12-dependent interferon gamma production by CD8alpha+ lymphoid dendritic cells.

We investigated the role of antigen-presenting cells in early interferon (IFN)-gamma production in normal and recombinase activating gene 2-deficient (Rag-2(-/-)) mice in response to Listeria monocytogenes (LM) infection and interleukin (IL)-12 administration. Levels of serum IFN-gamma in Rag-2(-/-) mice were comparable to those of normal mice upon either LM infection or IL-12 injection. Depletion of natural killer (NK) cells by administration of anti-asialoGM1 antibodies had little effect on IFN-gamma levels in the sera of Rag-2(-/-) mice after LM infection or IL-12 injection. Incubation of splenocytes from NK cell-depleted Rag-2(-/-) mice with LM resulted in the production of IFN-gamma that was completely blocked by addition of anti-IL-12 antibodies. Both dendritic cells (DCs) and monocytes purified from splenocytes were capable of producing IFN-gamma when cultured in the presence of IL-12. Intracellular immunofluorescence analysis confirmed the IFN-gamma production from DCs. It was further shown that IFN-gamma was produced predominantly by CD8alpha+ lymphoid DCs rather than CD8alpha- myeloid DCs. Collectively, our data indicated that DCs are potent in producing IFN-gamma in response to IL-12 produced by bacterial infection and play an important role in innate immunity and subsequent T helper cell type 1 development in vivo.

Animals↗
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

Mental health problems after stroke.

We investigated the mental health of 47 subjects (30 men, mean age 63.8+/-7.7; 17 women, mean age 68.9+/-8.7) with the 60-item General Health Questionnaire (GHQ). All the subjects lived at home in a Japanese rural community and were examined from 2 to 3 years after suffering a stroke. Among the subjects, 18 (38.3%) had GHQ scores of 17 or more, which indicated a mental health problem (MHP). The following variables were included in multiple logistic regression analysis: age, sex (men/women), grade of motor paralysis (no/slight/moderate/severe), side of motor paralysis (no/left side/right side/both sides: in analysis, we used dummy variables), paresthesia (no/yes), rehabilitation (need no rehabilitation or participate in rehabilitation/fail to participate in rehabilitation), social support (not needed or sufficient/insufficient) and overall physical recovery (1/2/3: 1 = 67-100, 2 = 34-66, and 3 = 0-33 on a visual analog scale 100 mm long, 100 meaning full recovery). In univariate analysis all variables except age and sex showed statistically significant associations with MHP. In multivariate analysis, only one variable, overall physical recovery', had a statistically independent association with the status of MHP (Odds ratio 4.39, 95% confidence interval 1.46-13.19). The results of logistic regression analysis indicate that the presence of an MHP is more strongly dependent upon subjective assessment about overall physical recovery after stroke than upon physical impairments and the other psychosocial variables. Therefore, in the community setting, the visual analog scale of overall physical recovery is considered to be a simple, valid method for assessing MHP following stroke.

Aged↗
PubMed1999

[Development and evaluation of parent-related quality of life questionnaires for asthmatic children and their parents or caregivers--evaluation of treatment process with sustained-release theophylline dry syrup].

A quality of life (QOL) questionnaire for asthmatic children and their parents or caregivers was shown to exhibit reliability, factorial validity and possibility for application study in our previous report. The aim of this study was to examine longitudinal study of the questionnaires, including changes of QOL, responsibility to asthmatic symptoms, consistency with conventional asthma scores and clinical outcomes during treatment with theophylline dry syrup for about three months. Overall QOL scores increased significantly (p < 0.01) with improvement in clinical outcomes. Longitudinal changes of QOL were compared with differences (after-before) and changes of ratio [(after-before/before)] in QOL by correlation analysis between differences of asthma scores. Changes of ratio in QOL were reflected by the clinical outcomes. Results of investigation of correlation between differences in asthma scores and changes of ratio in QOL domains were considered as follows. Physical domain was correlated with asthma symptoms, given the moderate correlation (rho = 0.497). Social and family domains showed fair correlation (rho = 0.382 - 0.384 respectively), both domains were hard to correlate with differences in asthma scores. Emotional domain showed fair correlation (rho = 0.264) for reflecting family's emotion. Poor correlation (rho = 0.071) was observed in individual growth domain which was necessary to evaluate for longer term. Changes of ratio in QOL responded significantly (p < 0.01) to improved and worst-unchanged groups which were categorized by the differences in asthma scores. In conclusion, QOL questionnaires showed longitudinal validity for clinical outcomes and responsibility to asthmatic symptoms. It yields useful information for understanding how asthmatic symptoms affect parents or caregivers and investigating what treatments should be prescribed for the patients with asthma.

Administration, Oral↗
PubMed1999

[Development of parent-rated quality of life questionnaire for asthmatic children and their parents or caregivers--reliability and factorial validity].

We developed a questionnaire form on quality of life (QOL) designed for data entry by parents or caregivers of asthmatic children, which consisted of five domains: physical, social, family, emotional, and individual growth domains. QOL scores were determined according to the five-point scale by which one point was given for maximal impairment and five points for no impairment. The form included 23 questions for children under the age of four and 31 questions for children over the age of four. This study examined reproducibility, reliability, and factorial validity of the form. The numbers of asthmatic children, whose symptoms of asthma attacks had been stable for the 2 weeks before this study, were as follows: eight children under the age of four; and 26 children over the age of four. The intra-class correlation coefficients for these two categories of children were 0.879 and 0.793, respectively; therefore, reproducibility was shown. The values of Cronbach's alfa for the total scores of QOL were 0.6329 to 0.8829 in 102 asthmatic children, 40 healthy children, and 12 non-asthmatic children, e.g., children affected by cold. Therefore, internal consistency was shown. The rank-orders of the replies to the questions associated with the physical, social, family, and emotional domains other than the individual growth domain decreased significantly (p < 0.05) in asthmatic children as compared with healthy or non-asthmatic children. Factor analysis confirmed the five established domains and each facets therein at the cumulative variance of 60% or higher. In conclusion, this questionnaire form on QOL of asthmatic children was considered to have reproducibility, constitutional reliability, and factorial validity, to be applicable at the time of medication, and to be useful in investigating approaches for the management of bronchial asthma and the targeting of treatment.

Asthma↗
PubMed1998

Molecular basis of selective IgG2 deficiency. The mutated membrane-bound form of gamma2 heavy chain caused complete IGG2 deficiency in two Japanese siblings.

Patients with IgG2 deficiency have recurrent sinopulmonary infections caused by Pneumococcus and Hemophilus. Hereditary and selective IgG2 deficiency was suspected in two Japanese siblings whose serum IgG2 levels were under detection limits, while other serum levels of immunoglobulin subclasses were within normal ranges. Expression level of spontaneous germline Cgamma2 transcript was normal, but that of the spontaneous mature Cgamma2 transcript was greatly decreased in the patients' PBMCs, suggesting the presence of a defect at or after the class switch to Cgamma2. We sequenced the Cgamma2 gene region, and in both patients a homozygous one-base insertion (1793insG) was present in exon 4 of the Cgamma2 gene, just upstream from the alternative splice site for M exons. The mutant membrane-bound gamma2 heavy chain loses the transmembrane domain and the evolutionarily conserved cytoplasmic domain. Considering several lines of evidence showing that intact expression of the membrane-bound heavy chain is essential for a normal response of B cells and production of secreted immunoglobulin in mice, we concluded that 1793insG is responsible for selective and complete IgG2 deficiency in these two siblings. This is the first documentation of a mutation in human selective IgG2 deficiency.

Amino Acid Sequence↗
PubMed1998

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency: two pathogenic mutations, V133E and C456F, in Japanese siblings.

Succinyl-CoA:3-ketoacid CoA transferase (SCOT; EC 2.8.3.5; locus symbol OXCT) is the key enzyme of ketone body utilization. Hereditary SCOT deficiency (MIM 245050) causes episodes of severe ketoacidosis. We developed a transient expression system for mutant SCOT cDNAs, using immortalized SCOT-deficient fibroblasts. This paper describes and characterizes three missense mutations in two SCOT-deficient siblings from Japan. They are genetic compounds who inherited the mutation C456F (c1367 G-->T) from their mother. Their paternal allele contains two mutations in cis, T58M (c173 C-->T) and V133E (c398T-->A). Expression of SCOT cDNAs containing either V133E or C456F produces no detectable SCOT activity, whereas T58M is functionally neutral. T58M is a rare sequence variant not detected in 100 control Japanese alleles. In fibroblasts from the proband (GS02), in whom immunoblot demonstrated no detectable SCOT peptide, we measured an apparent residual SCOT activity of 20-35%. We hypothesize that the high residual SCOT activity in homogenates may be an artifact caused by use of the substrate, acetoacetyl-CoA by other enzymes. Expression of mutant SCOT cDNAs more accurately reflects the residual activity of SCOT than do currently available assays in cell or tissue homogenates.

Cell Line, Transformed↗
PubMed1998

Characterization of N93S, I312T, and A333P missense mutations in two Japanese families with mitochondrial acetoacetyl-CoA thiolase deficiency.

Mitochondrial acetoacetyl-CoA thiolase (T2) deficiency is an inborn error of ketone body and isoleucine catabolisms. Japanese patients, GK01 and GK19, were found to be compound heterozygotes of 149delC and A333P, and N93S and I312T, respectively. The latter three missense mutations were individually characterized by analyses of transient expression of the cDNAs and heat stability. A333P and I312T subunits showed aberrant electrophoretic mobility on SDS-PAGE. T2 protein was destabilized by A333P and existed as an insoluble form in the mitochondria. I312T mutation also destabilized T2 protein; however, some T2 protein was retained in soluble form and reduced residual activity was apparent. N93S mutation did not change the heat stability of T2 activity and the reduced residual activity was retained, however a considerable amount was observed in an insoluble form. The effects of mutations were interpreted based on a tertiary structural model of a subunit of the human T2. This model was constructed from the X-ray crystal structure of the homologous peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae. On the basis of this model, the positions of Ala333 and Ile312 were far from the active site and the mutations would be expected to destabilize the tertiary structure of T2 subunit. By contrast, Asn93 is located near the active site and may function to maintain a local loop structure. The mutation of Asn93 could directly disrupt disposition of the active site.

Acetyl-CoA C-Acetyltransferase↗
PubMed1998

Ataxia-telangiectasia in the Japanese population: identification of R1917X, W2491R, R2909G, IVS33+2T-->A, and 7883del5, the latter two being relatively common mutations.

We analyzed the data regarding six Japanese ataxia-telangiectasia (A-T) patients from four unrelated families, at the DNA level, to search for possible common mutations in the Japanese population. Among eight mutant alleles in the four families, c. 4612del165 (exon 33 skipping) was identified in two alleles, and c. 5749A to T (R1917X), c. 7471T to C (W2491R), c.7883de15, and c. 8725A to G (R2909G) were identified in one allele each. We found no mutations in the other two alleles. The IVS33 + 2T-->A mutation was identified at the genomic level as the cause of exon 33 skipping. We also identified the IVS33 + 2T-->A mutation in a Japanese patient ATL105 who was previously found to be a homozygote of c. 4612del165. W2491R and R2909G mutations were not detected in more than 100 control Japanese alleles. The latter is located in a highly conserved PI-3 kinase domain and is a completely conserved residue among ATM-related proteins. Taken together with previously documented mutations in five other Japanese A-T patients, IVS33 + 2T-->A and 7883del5 were identified in four and five alleles, respectively, in a total of 18 mutant alleles of Japanese A-T patients. These results suggest that these two mutations are relatively common mutations in the Japanese population.

Adolescent↗
PubMed1998

Mutation analysis in the iduronate-2-sulphatase gene in 43 Japanese patients with mucopolysaccharidosis type II (Hunter disease).

Our series of studies on Hunter disease in Japanese patients showed allelic heterogeneity of IDS gene mutations, genotype/phenotype correlation and racial differences in distribution of mutations. Twenty-five different small mutations have been characterized. Small mutations in the Japanese population are widely distributed through the IDS gene, although some mutations were unevenly concentrated on exon 5 (28%) and on exon 9 (24%). Mutations were seen at the same codon 468 in exon 9 in 5 patients. These findings are in good agreement with data on other ethnic groups. Two unique mutations linked to a severe phenotype were apparently associated with aberrant splicings; one was a point mutation within exon 3 (P86L), partially activating a cryptic splice acceptor site at 28 bp downstream from the mutation site within exon 3 and producing a 44-base truncated mRNA, and the other was a point mutation at the consensus sequence of the splice donor site of intron 2, causing exon 2 skipping.

Base Sequence↗